2. Minutes of Immunobiology Working Committee at Tandem :20 pm (M Fernandez-Vina) (Attachment 1)

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1 Not for publication or presentation A G E N D A CIBMTR WORKING COMMITTEE FOR IMMUNOBIOLOGY San Diego, California Wednesday, February 1, 2012, 12:15 pm 4:45 pm Co-Chair: Co-Chair: Co-Chair: Statisticians: Co-Scientific Dir: Co-Scientific Dir: Carlheinz Müller, MD, PhD, German National Bone Marrow Donor Registry Telephone: ; Fax: ; carlheinz.mueller@zkrd.de David Miklos, MD, PhD, Stanford University Telephone: ; Fax: ; dmiklos@stanford.edu Marcelo Fernandez-Vina, PhD, Stanford University Telephone: ; Fax: ; marcelof@stanford.edu Michael Haagenson, MS, CIBMTR Statistical Center Telephone: ; Fax: ; mhaagens@nmdp.org Tanya Pedersen, MPH, CIBMTR Statistical Center Telephone: ; Fax: ; tpederse@nmdp.org John Klein, PhD, CIBMTR Statistical Center Telephone: ; Fax: ; klein@mcw.edu Tao Wang, PhD, CIBMTR Statistical Center Telephone: ; Fax: ; taowang@mcw.edu Stephanie Lee, MD, MPH, Fred Hutchinson Cancer Research Center Telephone: ; Fax: ; sjlee@fhcrc.org Stephen Spellman, MBS, CIBMTR Immunobiology Research Telephone: ; Fax: ; sspellma@nmdp.org 1. Welcome and introduction (M Fernandez-Vina) 12:15 pm 2. Minutes of Immunobiology Working Committee at Tandem :20 pm (M Fernandez-Vina) (Attachment 1) 3. Completed project summary (published or submitted work) 12:25 pm a. IB06-03 Valcárcel D, Sierra J, Wang T, Kan F, Gupta V, Hale GA, Marks D, McCarthy PL, Oudshoorn M, Petersdorf EW, Ringdén O, Setterholm M, Spellman SR, Waller EK, Gajewski JL, Marino SR, Senitzer D, Lee SJ. One antigen mismatched related vs. HLAmatched unrelated donor hematopoietic transplantation in adults with acute leukemia: CIBMTR results in the era of molecular typing. Published. Biol Blood Marrow Transplant, Vol. 17, Issue 5, Pages May b. IB07-01 Woolfrey A, Klein JP, Haagenson M, Spellman SR, Petersdorf E, Oudshoorn M, Gajewski J, Hale GA, Horan J, Battiwalla M, Marino SR, Setterholm M, Ringden O, Hurley CK, Flomenberg N, Anasetti C, Fernandez-Vina M and Lee SJ. HLA-C Antigen mismatches are associated with worse outcomes in unrelated donor peripheral blood stem cell transplantation. Published. Biol Blood Marrow Transplant, Vol. 17, Issue 6, Pages , June

2 Not for publication or presentation c. IB06-04 Dong L, Wu T, Gao ZY, Zhang MJ, Kan F, Spellman SR, Tan XY, Zhao YL, Wang JB, Lu DP, Miklos D, Petersdorf E, Fernandez-Vina M and Lee SJ. The outcomes of family haploidentical hematopoietic stem cell transplantation in haematological malignancies are not associated with patient age. Published. Biol Blood Marrow Transplant, Vol. 17, Issue 8, Pages August d. IB07-02 Marino SR, Lin S, Maiers M, Haagenson M, Spellman S, Klein JP, Binkowski TA, Lee SJ, and van Besien K. Identification by random forest method of HLA class I amino acid substitutions associated with lower survival at day 100 in unrelated donor hematopoietic cell transplantation. In press. Bone Marrow Transplantation. Published online: DOI: /bmt March e. IB10-05 Spellman S, Klein JP, Haagenson M, Askar M, Baxter-Lowe LA, He J, Hsu S, Blasczyk R, Hurley CK. Scoring HLA Class I Mismatches by HistoCheck Does Not Predict Clinical Outcome in Unrelated Hematopoietic Stem Cell Transplantation. In press. Biol Blood Marrow Transplant, Uncorrected proof: 29 September f. IB05-03s Shamim Z, Faucher S, Spellman S, Decker W, Haagenson M, Wang T, Lee SJ, Ryder LP, and Muller K. Polymorphism in the genes encoding human interleukin-7 receptor-alpha and outcomes after allogeneic hematopoietic cell transplantation with matched unrelated donor. Submitted. g. IB06-11s Rocha V, Spellman S, Zhang MJ, Ruggeri A, Purtill D, Brady C, Altamuro D, Baxter-Lowe LA, Baudoux E, Beddard RL, Bergamaschi P, Chow R, Freed B, Koegler G, Kurtzberg J, Larghero J, Lecchi L, Mrowiec Z, Nagler A, Navarette C, Prasad V, Prasath A, Price T, Pouthier F, Ratanatharathorn V, Sander J, Sender L, van Rood JJ, Horowitz MM, Gluckman E, Eapen M. Effect of HLA-matching recipients to donor non-inherited maternal antigens on outcomes after mismatched umbilical cord blood transplantation for hematologic malignancies. Submitted. h. IB07-09 Pearce KF, Lee SJ, Haagenson M, Petersdorf EW, Norden J, Collin MP, Klein JP, Spellman SR, Lowerson SA, Davies S, Dickinson AM. Analysis of non-hla genomic risk factors in HLA-matched unrelated donor hematopoietic cell transplantation for chronic myeloid leukemia. Submitted. i. IB07-06 Fleischhauer K, Shaw B, Gooley T, Malkki M, Bardy P, Bignon JD, Horowitz M, Madrigal A, Morishima Y, Spellman S, Velardi A, Zino E, Petersdorf E. Non-permissive HLA-DPB1 T cell epitope mismatches increase mortality after unrelated donor hematopoietic cell transplantation. Submitted. j. R04-74s Venstrom JM, Pittari G, Gooley TA, Chewning J, Spellman S, Haagenson M, Gallagher MM, Malkki M, Petersdorf E, Dupont B, Hsu KC. Donor activating KIR2DS1 protects against acute myeloid leukemia relapse in an HLA-dependent manner. Submitted. 4. Research Repository update and accrual tables (S Spellman) (Attachment 2) 12:25 pm 5. Proposed studies and discussion for Immunobiology Working Committee 12:35 pm a. Voting guidelines (C Müller) b. PROP Is a well-matched HLA-identical unrelated male donor (10/10) an alternative to a female HLA-identical sibling donor for a male recipient in need of hematopoietic stem cell transplantation? (O Ringden/T Erkers/J Torlen) (Attachment 3) 2

3 Not for publication or presentation c. PROP Are major HLA-antigens targets for the graft-versus-leukemia effect? (T Erkers/O Ringden/J Torlen) (Attachment 4) d. PROP Impact of unrelated donor HLA-mismatch in reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation outcomes (J Koreth) (Attachment 5) e. PROP Impact of HLA mismatch and patient/donor non-hla variables on transplantation outcome: An updated analysis utilizing NMDP high-resolution typing project data (J Pidala/C Anasetti) (Attachment 6) f. PROP Determining the effects of HLA-C KIR ligand expression on outcomes of unrelated hematopoietic stem cell transplantation (J Venstrom) (Attachment 7) g. PROP Plasma YKL-40 and CHI3L1 genotype to predict mortality after allogeneic hematopoietic cell transplantation (HCT) (B Kornblit/P Garred/L Vindelov) (Attachment 8) h. PROP Natural killer cell genomics and outcomes after allogeneic transplantation for lymphoma (V Bachanova/J Miller/D Weisdorf/L Burns) (Attachment 9) i. PROP Effect of genetic ancestry matching on HSCT outcomes. (A Madbouly/M Maiers/N Majhail) (Attachment 10) j. Proposal voting 6. BREAK 30 minutes 2:15 pm th International Histocompatibility and Immunogenetics Workshop 2:45 pm Collaboration updates (E Petersdorf) 8. Studies in progress (Attachment 11) 3:05 pm MINOR HISTOCOMPATIBILITY ANTIGENS No updates. 3:05 pm SENSITIZATION/TOLERANCE 3:05 pm (Chair: D Miklos) a. R03-65s HY antigen (D Miklos) - update Manuscript preparation b. IB11-01 Analysis of the NIMA effect on the outcome of Typing unrelated PBSC/BM transplantation (G Ehninger/JJ van Rood/ A Schmidt) (Attachment 12) update c. IB11-07 Effect of Pretransplant Rituximab upon ABO Manuscript preparation Mismatch Hematopoietic Cell Transplantation (D Miklos/A Logan) (Attachment 13) update d. IB06-09s Detection of HLA antibody to the mismatched Manuscript preparation antigen in single antigen HLA-mismatched unrelated donor transplants: Is it a predictor of graft-versus-host disease outcome? (S Arai/D Miklos) no update e. IB09-08 A retrospective study on impact of donor and Manuscript preparation recipient birth order on outcome of HLA-identical sibling stem cell transplantation (SCT) in hematological malignancies reported to the CIBMTR (C Dobbelstein) (Attachment 14) no update f. IB10-02 Development of GVHD prevention diagnostic test (R Somogyi/L Greller) no update Analysis 3

4 Not for publication or presentation OTHER GENES (Chair: C Müller) a. IB08-08 Genome-Wide Association in Unrelated Donor Transplant Recipients and Donors: A Pilot Study (R Goyal) - (Attachment 15) - update b. IB09-04s Association of donor and recipient gene polymorphisms of drug metabolisms [GSTP, GSTT, GSTM and UGT (2B17, 2B7, 2B28)] and innate immune response [CD14, TIRAP, and NALPs (1 and 3)] with outcomes after allele matched unrelated hematopoietic stem cell transplantation (V Rocha) - no update c. IB09-06s/RT09-04s Genetic polymorphisms and HCT related mortality Re: Pre-HCT conditioning in matched unrelated donor HCT (T Hahn) no update d. IB10-01 Donor and Recipient Telomere Length as Predictors of Outcomes after Hematopoietic Stem Cell Transplant in Patients with Acquired Severe Aplastic Anemia (S Gadalla) no update e. IB10-03 TLR and HMGB1 gene polymorphisms in unrelated haematopoietic stem cell transplantation (K Müller/B Kornblit) no update f. IB10-04s A validation study of the role of base excision repair pathway as a predictor of outcome after hematopoietic stem cell transplant (B Thyagrajan /M Arora) (Attachment 16) no update Typing Analysis Typing Typing Analysis Analysis 3:25 pm NK/KIR (Chair: M Fernandez-Vina) a. R02-40s/R03-63s KIR Program Project/NK receptor acquisition (J Miller/E Trachtenberg) (Attachment 17) - update b. R04-74s KIR functional significance (IHWG) (B Dupont/K Hsu/J Venstrom) (Attachment 18) - update c. IB07-03 Analysis of Killer Immunoglobulin-like Receptor (KIR) ligands in reduced intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (HSCT) (R Sobecks/K Hsu/M Askar) (Attachment 19) update d. IB08-06 Analysis of Killer Immunoglobulin-Like Receptor (KIR) ligands in umbilical cord blood transplantation (R Sobecks) - no update e. IB11-05s KIR genotyping and immune function in MDS patients prior to unrelated donor transplantation (E Warlick/J Miller) - no update CYTOKINE/CHEMOKINE (Chair: M Fernandez-Vina) a. IB08-04s Immune response gene polymorphisms in unrelated donor stem cell transplantation in children (K Müller) no update 3:30 pm Ongoing Manuscript preparation Data file preparation Protocol development Protocol development 3:50 pm Protocol development 4

5 Not for publication or presentation HLA GENES CLASSICAL MATCHING (Chair: D Miklos) a. IB11-03 Evaluation of the impact of allele homozygosity at HLA loci on outcome (C Hurley/A Woolfrey/M Maiers) (Attachment 20) - update b. IB11-04 Impact of amino acid substitutions at peptide binding pockets of HLA class I molecules on hematopoietic cell transplantation (HCT) outcomes (J Pidala/C Anasetti) (Attachment 21) - update c. IB11-06 Evaluation of the impact of potentially nonimmunogenic HLA-C allele level mismatch (M Fernandez- Vina/M Setterholm) (Attachment 22) - update d. IB08-02 Evaluation of HLA matching requirements in unrelated hematopoietic stem cell transplantation for nonmalignant disorders (J Horan/A Woolfrey) (Attachment 23) no update e. IB09-02 Non-permissive HLA-DPB1 disparities based on T cell alloreactivity (K Fleischhauer) (Attachment 24) no update f. IB10-07 Use of HLA Structure and Function Parameters to Understand the Relationship between HLA Disparity and Transplant Outcomes (LA Baxter-Lowe) no update g. IB06-02 Mismatching for low expression HLA loci in matched unrelated donor transplants (M Fernandez-Vina) - no update 3:50 pm Analysis Analysis Analysis Manuscript preparation Manuscript preparation Protocol development Manuscript preparation 8. Deferred studies pending accrual/funding 4:10 pm a. R04-80s HLA matching in unrelated cord blood transplants (S Rodriguez-Marino) - no update b. IB06-10 Evaluation of the impact of the exposure to NIMA during fetal life and breast feeding and to the IPA during pregnancy on the clinical outcome of HSCT from haploidentical family members (J van Rood) no update c. IB06-13 HLA disparity in unrelated cord blood transplantation: Delineation of factors contributing to transplant outcomes (L Baxter-Lowe) - no update d. IB08-05s Evaluation of lymphotoxin alpha (LTA) alleles in relation to relapse in AML and CML (P Posch) - no update e. IB10-06 Identification of Common, Clinically Significant, Minor Histocompatibility Antigens through Stem Cell Transplant Donor/Patient Polymorphism Disparities (P Armistead) no update f. IB11-02s Impact of CTLA4 single nucleotide polymorphisms on outcome after unrelated donor transplant (M Jagasia/W Clark/B Savani/S Sengsayadeth) no update Data collection Data collection Data collection Data collection Awaiting funding Awaiting funding 9. Closing remarks (C Müller) 4:15 pm 5

6 Not for publication or presentation Attachment 1 MINUTES CIBMTR WORKING COMMITTEE FOR IMMUNOBIOLOGY Honolulu, Hawaii Thursday, February 17, 2011, 12:15 pm 4:45 pm Co-Chair: Co-Chair: Co-Chair: Statisticians: Co-Scientific Dir: Co-Scientific Dir: Carlheinz Müller, MD, PhD, German National Bone Marrow Donor Registry Telephone: ; Fax: ; carlheinz.mueller@zkrd.de David Miklos, MD, PhD, Stanford University Telephone: ; Fax: ; dmiklos@stanford.edu Marcelo Fernandez-Vina, PhD, Stanford University Telephone: ; Fax: ; marcelof@stanford.edu Michael Haagenson, MS, CIBMTR Statistical Center Telephone: ; Fax: ; mhaagens@nmdp.org Fiona Kan, MS, MA, CIBMTR Statistical Center Telephone: ; Fax: ; fkan@nmdp.org John Klein, PhD, CIBMTR Statistical Center Telephone: ; Fax: ; klein@mcw.edu Tao Wang, PhD, CIBMTR Statistical Center Telephone: ; Fax: ; taowang@mcw.edu Stephanie Lee, MD, MPH, Fred Hutchinson Cancer Research Center Telephone: ; Fax: ; sjlee@fhcrc.org Stephen Spellman, MBS, CIBMTR Immunobiology Research Telephone: ; Fax: ; sspellma@nmdp.org 1. Welcome and introduction Dr. Marcelo Fernandez-Vina began the meeting at 12:20 pm. Drs. Carlheinz Müller and Fernandez-Vina led the meeting. 2. Minutes of Immunobiology Working Committee at Tandem 2010 The minutes were approved as written. 3. Completed project summary (published or submitted work) a. R03-70s McDermott DH, Conway SE, Wang T, Ricklefs SM, Agovi M, Porcella SF, Tran HTB, Milford E, Spellman S and Abdi R. Donor and recipient chemokine receptor CCR5 genotype is associated with survival after bone marrow transplantation. Blood, March 2010; 115: b. R04-74s Venstrom J, Gooley TA, Spellman SR, Pring J, Malkki M, Dupont B, Petersdorf E, Hsu KC. Donor activating KIR3DS1 is associated with decreased acute GVHD in unrelated allogeneic hematopoietic stem cell transplantation. Blood, April 2010; 115: c. R04-98s Spellman S, Bray R, Rosen-Bronson S, Haagenson M, Klein JP, Flesch S, Vierra- Green C and Anasetti C. The detection of donor-directed, HLA-specific alloantibodies in recipients of unrelated hematopoietic cell transplantation is predictive of graft failure. 6

7 Not for publication or presentation Attachment 1 Blood, April 2010; 115: d. IB06-07s Nguyen Y, Al-Lehibi A, Gorbe E, Li E, Haagenson M, Wang T, Spellman S, Lee SJ and Davidson NO. Insufficient evidence for association of NOD2/CARD15 or other inflammatory bowel disease-associated markers on GVHD incidence or other adverse outcomes in T-replete, unrelated donor transplantation. Blood, April 2010; 115: e. IB06-03 Valcárcel D, Sierra J, Wang T, Kan F, Gupta V, Hale GA, Marks D, McCarthy PL, Oudshoorn M, Petersdorf EW, Ringdén O, Setterholm M, Spellman SR, Waller EK, Gajewski JL, Marino SR, Senitzer D, Lee SJ. One antigen mismatched related vs. HLAmatched unrelated donor hematopoietic transplantation in adults with acute leukemia: CIBMTR results in the era of molecular typing. In press. Biol Blood Marrow Transplant., Published online: DOI: /j.bbmt f. IB07-01 Woolfrey A, Klein JP, Haagenson M, Spellman SR, Petersdorf E, Oudshoorn M, Gajewski J, Hale GA, Horan J, Battiwalla M, Marino SR, Setterholm M, Ringden O, Hurley CK, Flomenberg N, Anasetti C, Fernandez-Vina M and Lee SJ. HLA-C Antigen mismatches are associated with worse outcomes in unrelated donor peripheral blood stem cell transplantation. In press. Biol Blood Marrow Transplant, Published online: DOI: /j.bbmt g. R02-40s Cooley S, Weisdorf DJ, Guethlein LA, Klein JP, Wang T, Le CT, Marsh SGE, Geraghty D, Spellman S, Haagenson MD, Ladner M, Trachtenberg E, Parham P and Miller JS. Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia. Blood, October 2010; 116: h. IB06-06 Shaw P, Kan F, Ahn KW, Spellman SR, Aljurf M, Ayas M, Burke M, Cairo MS, Chen AR, Davies SM, Frangoul H, Gajewski J, Gale RP, Godder K, Hale GA, Heemskerk MBA, Horan J, Kamani N, Kasow KA, Chan KW, MD 18 ; Lee SJ, Leung WH, Lewis VA, Miklos D, Oudshoorn M, Petersdorf EW, Ringdén O, Sanders J, Schultz KR, Seber A, Setterholm M, Wall DA, Yu L and Pulsipher MA. Outcomes of pediatric bone marrow transplantation for leukemia and myelodysplasia using matched sibling, mismatched related or matched unrelated donors. Blood. November 2010; 116: i. IB06-04 Dong L, Wu T, Gao ZY, Zhang MJ, Kan F, Spellman SR, Tan XY, Zhao YL, Wang JB, Lu DP, Miklos D, Petersdorf E, Fernandez-Vina M and Lee SJ. The outcomes of family haploidentical hematopoietic stem cell transplantation in haematological malignancies are not associated with patient age. In press. Biol Blood Marrow Transplant, Published online: DOI: /j.bbmt j. IB07-02 Marino SR, Lin S, Maiers M, Haagenson M, Spellman S, Klein JP, Binkowski TA, Lee SJ, and van Besien K. Identification by random forest method of HLA class I amino acid substitutions associated with lower survival at day 100 in unrelated donor hematopoietic cell transplantation. In press. Bone Marrow Transplantation. k. IB05-03s Shamim Z, Faucher S, Spellman S, Decker W, Haagenson M, Wang T, Lee SJ, Ryder LP, and Muller K. Polymorphism in the genes encoding human interleukin-7 receptro-alpha and outcomes after allogeneic hematopoietic cell transplantation with matched unrelated donor. Submitted. 7

8 Not for publication or presentation Attachment 1 4. Research Repository update and accrual tables Steve Spellman provided an overview of the Research Repository and accrual tables. Many unrelated donors, cord blood and transplant recipients pre-transplant/pre-conditioning have samples from NMDP facilitated transplants. There are also related donors/recipient samples in the Research Repository from the SCTOD, which has been expanded to BMT-CTN core sites with 39 sites participating. In 2010, over 20,000 sample aliquots were distributed. Currently, sample types for the Repository are 10-20ml of whole blood from donors and recipients, or any source of DNA from infused CBUs, which are stored as frozen aliquots (-80 o C and LN) and whole blood spotted on filter paper. In the summer of 2008, ACD-A plasma collection started. Prior to 2002, the sample types were B-LCL, PBMC, granulocytes and serum. The unrelated HSCT inventory includes more than 21,700 donor/recipient pairs with more than 13,500 from first transplants, most with high-resolution HLA typing. The umbilical cord blood inventory includes 748 single UCBT pairs and 357 double UCBT. For the related cases, the sample inventory is 682 paired samples, 802 related recipients, and 743 related donors. One note to keep in mind is data submission lags behind sample submission, so we only have demographics on 385 pairs. The diseases for these 385 cases are mostly AML (N=100), ALL (N=60), MDS (N=56) and NHL (N=69). Other demographics will be available online at under 2011 BMT Tandem Meetings Working committees. The shipping and handling costs vary, depending on sample type and whether the requester has industry funding or an academic affiliation. Filter paper spots are $9.05 for academia and $35 for industry, while non-viable samples at -80 C are $35 for academia and $120 for industry, and viable samples stored in LN are $50 for academia and $300 for industry. There is financial support to defer sample costs through the NMDP Immunobiology Grants and through CIBMTR for NIH/other grant submissions. Investigators are required to submit the interpreted results of all testing to the NMDP/CIBMTR. The data will then be made available to the HSCT research community to eliminate duplicative testing and preserve resources/sample inventory. Data is captured in NMDP Immunobiology database and is linked to sample inventory. The Repository is overseen by the NMDP Histocompatibility Advisory Group members, who are experts in histocompatibility, immunology and transplantation. They provide recommendations for study prioritization and Immunobiology scientific agenda, approval of sample usage for studies, oversight of Repository operations including sample collection/storage methodologies and monitoring sample submission rates. 5. Proposed studies and discussion for Immunobiology Working Committee a. Voting guidelines Dr. Müller discussed the voting guidelines for the proposals. The assessment should be based on the following considerations: study objectives, study feasibility, study innovation, and the impact of the study on practice in the transplant community. b. PROP Impact of amino acid substitution at peptide binding pockets of HLA class I molecules on hematopoietic cell transplantation (HCT) outcome (J Pidala/C Anasetti): Dr. Joseph Pidala presented this proposal. The hypothesis is that amino acid residue substitution at peptide binding pockets of the HLA class I molecule adversely affects 8

9 Not for publication or presentation Attachment 1 transplantation outcome. Dr. Pidala plans to examine the impact of amino acid substitution at key (9, 77, 99, 116, and 156) residues on grade III-IV agvhd, NRM and overall survival among unrelated donor-recipient pairs in the existing NMDP database. The study population will include first transplants of AML, ALL, CML and MDS patients from with no restrictions on age, conditioning regimen or graft source. In the frequency table, there are 7573 cases available for analysis. The amino acid substitution (AAS) frequencies are still being determined but the protocol contains an estimated frequency projected based on other studies. One advantage of preparing a data set for this study is that the data could be used for several other studies. Chronic GvHD is not yet in the protocol, but that can be added. The comparisons for this study will consider high risk AAS yes vs. no, and they should be 7/8 with high risk AAS vs. 7/8 without high risk AAS vs. 8/8 matched. Dr. Pidala also plans to evaluate if there is a dosage effect of high risk AAS c. PROP Analysis of the NIMA effect on the outcome of unrelated PBSC/BM transplantation (G Ehninger): Dr. Alexander Schmidt presented this study in Dr. Gerhard Ehninger s absence. The hypothesis is that there is a lifelong tolerance of maternal antigens and that noninherited maternal HLA antigens (NIMA) matches reduce the negative impact of mismatch in unrelated HSCT. Dr. Jon van Rood et al had a publication in PNAS in 2009 which showed that for cord blood transplantation, TRM for a single HLA mismatch and NIMA matched is lower than the other HLA mismatched and NIMA matching groups. This conclusion, however, was based on 79 NIMA matches out of 1,059 mismatched donor/recipient pairs where the minimum patient age was 10 years old. In the current proposal, DKMS donors with at least one mismatch to their recipients would be contacted to try to obtain buccal swab samples and informed consent from their mothers. CIBMTR is being asked to provide patient clinical and follow-up information. One of the main concerns with this protocol is whether sufficient maternal HLA data can be collected to identify enough NIMA matches. A pilot study was carried out where 150 donors were contacted and 68 provided HLA samples, 6 mothers were on the DKMS file and 76 had no HLA information (so HLA data were obtainable from 49% of mothers). Of the ones providing HLA samples, 4 had a NIMA match and 64 had no NIMA match. Of the mothers on the DKMS file, all had no NIMA match. NIMA matching will be on the allele-level for HLA-A, B, C and DRB1. The study population for this proposal is still being adjusted to include Transplant Essential Data forms (TED data). d. PROP Impact of CLTA4 single nucleotide polymorphisms on outcome after unrelated donor transplant (M Jagasia): Dr. Madan Jagasia presented this proposal. The hypotheses for this study are 1) CTLA4 single nucleotide polymorphisms (SNPs) will predict relapse incidence in patients undergoing URD transplant for AML, 2) CTLA4 SNPs will predict acute and chronic GVHD; and 3) CTLA4 SNPs will identify a subset of patients at higher risk of relapse after thymoglobulin based preparative regimens. The study population will be AML in first or second complete remission with graft sources of bone marrow or PBSC from with 7/8 or 8/8 matched donors. Any preparative regimen will be considered. The sample size for this study is based on the SNP rs where the population frequency of minor allele is 12%-26%, but the assumption was 12%, so GG (1.44%), AG (21.1%) and AA (77.4%) were the assumed frequencies. The calculated ratio of AA to 9

10 Not for publication or presentation Attachment 1 (AG+GG) is 3.43, but the assumption will be 3. The sample size needed is 288 patients, but since regimen intensity is a potential variable impacting relapse, it is recommended to have 288 patients from each regimen intensity group. Discussion was held about sample size, stating that the study will likely need more samples since this calculation was based on a univariate analysis and a single SNP. The frequency table in the document online states 1172 cases are available, with 757 for myeloablative, 274 for reduced intensity, 117 for nonmyeloablative and 24 yet to be determined. e. PROP Evaluation of the impact of allele homozygosity at HLA loci on outcome (C Hurley/A Woolfrey/M Maiers): Steve Spellman presented this study in Dr. Ann Woolfrey s absence. The hypothesis is that 7/8 HLA matched cases have similar outcomes, whether they are homozygous or heterozygous. The comparison will be outcomes of 7/8 homozygous mm (HVG and GVH) vs. 7/8 heterozygous mismatches vs. 6/8 mismatches vs. 8/8 homozygous matched transplants with the 8/8 homozygous being the baseline. The primary outcomes will be overall survival, disease free survival and engraftment. The secondary outcome will be acute GvHD, both grades II-IV and III-IV. There were too few of cases (N=3) where the recipient and donor were homozygous and the matching was 7/8 (or 6/8 by some designations). There are 69 7/8 with an HvG mismatch, 98 7/8 with a GvH mismatch, /8 mismatched in both directions and 633 6/8 mismatched in both directions. The 8/8 homozygous data has not been determined yet. There is no Crossmatch data available. ATG may have an effect on outcome, so that variable will be added to the list. It was suggested that the study consider the presence of donor specific HLA antibodies for any graft failure cases if serum is available in the Repository. f. PROP KIR genotyping and immune function in MDS patients prior to unrelated donor transplantation (E Warlick/J Miller): Dr. Erica Warlick presented this proposal. The hypothesis is MDS patients requiring transplant will have KIR-A/A haplotype because-kir-b haplotypes are protective against disease progression due to presence of activating receptors. The study aims are to determine whether advanced MDS patients requiring transplant have KIR genotype distributions that deviate from normal controls, to determine function of NK cells and suppressor cells (T regulatory cells and myeloid derived suppressor cells) in subtypes of MDS patients requiring transplant, and to analyze pre-transplant cell frequencies and function for ability to predict post transplant outcomes such as DFS, relapse and OS. The study population will be MDS patients with RA, RARS, RAEB1&2, or RAEBT from De novo or tmds will be included as will any ages for recipients. The donor source will be unrelated bone marrow, PBSC or cord blood. Data will be restricted to patients with viable cell and DNA samples available. There are n=971 DNA samples available. One aliquot of viable cells collected pretransplant is needed for 1) NK Cell Functional Studies and 2) T regulatory cells and MDSCs. The proposed plan is to do a pilot study of about 120 samples, assuming good data from 2/3 of samples, to better project goals and sample sizes for viable cells. g. PROP Evaluation of the impact of potentially non-immunogenic HLA-C allele level mismatch (M Fernandez-Vina/M Setterholm): Dr. Fernandez-Vina presented this study. Specific aims are to compare: 1) the impact of a single mismatch in the alleles C*03:03/C*03:04 with other single antigen and/or allele level mismatches in HLA-C, 2) a single mismatch in the alleles C*03:03/C*03:04 with other single antigen or allele level mismatches in HLA-C or HLA-A, B or DRB1, 3) a 10

11 Not for publication or presentation Attachment 1 single mismatch in the allele C*03:03/C*03:04 versus fully matched in HLA-A, B, C or DRB1 loci, 4) one allele or antigen mismatch in HLA-A, B, C and DRB1 with an additional mismatch in the alleles C*03:03/C*03:04 versus two mismatches in alleles or antigens of HLA-A, B, C or DRB1 loci excluding mismatches in C*03:03/C*03:04 versus a single antigen or allele level mismatches HLA-A, B, C or DRB1 other than C*03:03/C*03:04. The proposed comparisons may prove that the mismatch in the alleles HLA- C*03:03/C*03:04 is tolerable. If this was the case, then the availability of well matched unrelated donors may be expanded for significant number of patients, in particular those carrying HLA-B*15:01 (present in > 12.5 percent of European subjects) and B*40:02 (present in > 6.0 percent of Asian subjects). h. PROP Effect of Rituximab and ABO mismatch (D Miklos): Dr. Aaron Logan presented this proposal in Dr. David Miklos s absence. This study would look at an existing data set of lymphoma cases and evaluate the effect of ABO mismatch and Rituximab. ABO mismatching is categorized as minor when the graft contains pre-sensitized anti-abo B cells, major when the recipient contains presensitized anti-abo B cells and bi-directional when both the graft and recipient contain pre-sensitized anti-abo B cells. In an analysis of Stanford patients (n=1594), minor ABO mismatch has worse survival that ABO matched (p=0.012). ABO minor mismatches also have significantly higher NRM than ABO matched cases. However, the overall survival results were only true for the diseases of AML, ALL, CML and MDS. NHL and CLL did not show this difference. The question of this study would be Does pre-transplant Rituximab ameliorate risk of ABO minor mismatch? The proposed study would reanalyze a lymphoma dataset to see if ABO mismatch is associated with overall survival, non-relapse mortality, and acute GVHD. The hypothesis is that such an association will be seen in the absence of rituximab, but that rituximab given within 6 months prior to HCT will abrogate the association. The Ns for this proposal are 265 for ABO matched, 78 for Major ABO Mismatch, 74 for Minor ABO Mismatch and 17 Bidirectional ABO Mismatch. The analysis would include a bone marrow vs. PBSC adjustment. In IBWC discussion, questions were raised about conducting this study in the lymphoma population, since the previous Stanford study did not see an effect in this population. i. Proposal voting Five minutes was taken to finish the voting on these proposals at 1:50 pm. A twenty minute break was then taken. 6. International Histocompatibility and Immunogenetics Workshop Collaboration update a. R04-75s CGP and post-transplant complication (IHWG) (E Petersdorf) b. R04-76s Identification of functional SNPs (IHWG) (E Petersdorf) c. IB05-02s The effect of a single MHC class I mismatch with numerous sequence differences on the clinical outcome of unrelated HSCT (M Heemskerk) d. IB06-05 Use of high-resolution HLA data from the National Marrow Donor Program for the IHWG in hematopoietic cell transplantation (E Petersdorf) e. IB07-04 Employing advanced bioinformatic methods for predicting peptide specificities of HLA molecules in the characterization of permissible mismatches in hematopoietic cell transfer (IHWG) (S Buus) f. IB07-05 Impact of donor-recipient ethnicity on risk of acute GVHD among HLA-A, B, C, DRB1, 11

12 Not for publication or presentation Attachment 1 DQB1, DPB1 matched unrelated transplants (IHWG) (Y Morishima) g. IB07-06 HLA DP epitope study (IHWG) (B Shaw) h. IB07-07 HLA-DR15 and Transplant Outcome (IHWG) (A Gratwohl) i. IB09-01s Clinical importance of MHC haplotypes in umbilical cord blood transplantation (E Petersdorf) j. IB09-03s Clinical relevance of cytokine/immune response gene polymorphisms in umbilical cord blood transplantation (E Petersdorf) k. IB09-05s Identification of functional SNPs in umbilical cord blood transplantation (E Petersdorf) l. IB09-07s Clinical Significance of Genome-wide Variation in Unrelated Hematopoietic Cell Transplantation (E Petersdorf) Dr. Effie Petersdorf presented the summary of active studies within the IHWG. The first study for discussion was Dr. Bronwen Shaw and Dr. Katharina Fleischhauer s study titled Permissive HLA-DPB1 mismatching compared to a non-permissive mismatching significantly improves overall survival following allogeneic transplantation in patients with both 10/10 and 9/10 matched unrelated donors. This study categorizes DPB1 matching based on T cell epitopes into 3 groups: fully matched (12/12, 20% of the population), TCE3 permissive (50% of the population) and TCE3 non-permissive (30% of the population). There were /10 HLA matched cases and /10 HLA-matched cases. 10/10 TCE permissive patients were considered the baseline. Acute GvHD is lower in the 12/12 (DPB1 fully matched) and TCE3 permissive is lower than the TCE3 nonpermissive group. Relapse is higher in the 12/12 when compared to the TCE3 permissive group. Non-relapse mortality is lowest in the 12/12, and TCE3 permissive group had significantly lower NRM than the TCE3 nonpermissive group. Overall mortality was significantly higher for the TCE3 nonpermissive group than the TCE3 permissive group. In the 9/10 groups, DPB1 matched (11/12) had significantly higher GvHD than the TCE3 permissive group while TCE3 nonpermissive group had significantly higher GvHD than the TCE3 permissive group. NRM and overall mortality were significantly higher in the TCE3 nonpermissive group when compared to the TCE3 permissive group. Dr. Petersdorf next discussed a proposal to the IHWG about synergism between minor and major histocompatibility antigens by Dr. Eric Spierings and Dr. Alois Gratwohl. The hypotheses of this proposal are: 1) a synergistic effect of mismatching for minor and major histocompatibility antigens in SCT leads to an excessive risk of GvHD in patients transplanted from a donor mismatched at both major and minor histocompatibility antigens, and 2) sex-mismatching and GvHD is not an issue in HLA identical sibling transplantations, but is a serious problem in MUD transplantations where HLA mismatches exist. The study population includes the DPB1 matched cases where 54 F F, 59 F M, 71 M F and 124 M M from the NMDP, and the DPB1 mismatched cases where 313 F F, 291 F M, 398 M F and 694 M M from the NMDP, for a total of 1984 NMDP cases. The study population includes a total of 5874 when including non-nmdp transplants. Dr. Petersdorf then discussed the three cord blood studies, where the goal is to define the clinical importance of MHC resident variation in CBT. The first study Determine the impact of HLA mismatching (IB09-01s) has the hypothesis that transplant outcome is shaped by MHC-resident variation which is ethno-geographically distinct. The approach to this study is to determine the importance of HLA-C and DQB1 matching; and define the role of high resolution matching for HLA-A, B and DRB1. The second study is Identify non-permissible HLA mismatches (IB09-01s) where the hypothesis is that non-permissible HLA mismatches are defined by substitutions at specific residues, the taxonomic history of the mismatched HLA alleles, and the structure of the molecules. The approach will be to examine HLA coding residues, SNP sequence taxonomy and 12

13 Not for publication or presentation Attachment 1 structure, and relate these to outcome. The third study is called Identify novel genes within the MHC that are clinically relevant (IB09-01s) where the hypothesis is MHC variation is functionally significant. The approach will be to define MHC region variation (SNPs) in cord blood recipients and unit(s) and to identify SNPs that are associated with outcome. In these studies, study samples will come from all recipients of UCBT for the treatment of malignant and non-malignant disorders; there will be no restriction to HLA match grade or conditioning regimen; and single and double unit UCBT will be included. There are 3700 anticipated CBTs of which 700 are currently available in the NMDP Repository (80% single, 20% double CBTs). The IHWG expects diverse DNA sources, such as 1) DNA extracted from PBMC/ PBL by commercial kits, or salting out method which produce more intact DNA, 2) EBV-transformed cell lines, or 3) Fibroblasts. Dr. Petersdorf then discussed IB09-05s which is Identification of functional SNPs in UCBT. The hypotheses are 1) recipients and CBU differ for SNPs and 2) SNPs mapping is a robust approach for identifying putative UCBT determinants. The methods would include 1) SNP genotyping in commercial platforms (Illumina and/or Affymetrix ), 2) define SNP frequencies for UCBU and recipients and LD with HLA, 3) define the frequency of SNP (mis)matching and between recipient and CBU(s), 4) identify SNPs associated with the engrafting CBU, 5) identify SNPs associated with post-transplant complications, 6) compare results in UCBT with those from unrelated transplant population (CIBMTR study # R04-76s). The data analysis would include a Genotype model where hazard ratios for the AB and BB genotypes will be compared with the AA genotype, the designated reference group, where A represents the more frequent (major) allele and B represents the less frequent (minor) allele as well as a mismatch model where a pair will be defined as SNP mismatched if the recipient encodes a SNP genotype not shared by the UCBU, and/or the UCBU encodes a genotype not shared by the recipient. 7. Studies in progress HLA GENES CLASSICAL MATCHING a. IB08-02 Evaluation of HLA matching requirements in unrelated hematopoietic stem cell transplantation for nonmalignant disorders (J Horan/A Woolfrey): Dr. Stephanie Lee presented this study in the absence of Dr. Ann Woolfrey. The objective of this study is to examine effect of HLA-A, -B, -C, -DRB1, DQ or DP mismatching on risk for overall mortality, GvHD and graft rejection in nonmalignant diseases. The analyses were adjusted for clinical characteristics. The study population had 667 non-malignant cases and severe aplastic anemia was the largest disease group (N=357). The transplants were performed from Overall survival was significantly different between the 8/8 Matched group (65%), the 7/8 Matched group (57%) and the 6/8 Matched group (47%), with an increased risk of mortality with each additional HLA-mismatch. HLA-DQ and DP did not show a significant association with mortality. There was an increased risk of graft failure with each additional HLA-mismatch where primary or secondary graft failure was 6% for 8/8, 11% for 7/8 and 18% for 6/8. Other conclusions include 1) isolated class I allele level mismatches are not associated with lower survival, but small numbers, and 2) lower survival in HLA-mismatched recipients cannot be explained by an increase in risk for GVHD. The committee suggested including an evaluation ofthe number of transfusions in the cases of graft failure in the analysis. b. IB09-02 Non-permissive HLA-DPB1 disparities based on T cell alloreactivity (K Fleischhauer): Dr. Fernandez-Vina presented this study in the absence of Dr. Katharina Fleischhauer. The specific aims of this study are 1) to validate, in an independent NMDP unrelated transplant cohort, the updated algorithm for definition of non-permissive HLA-DPB1 T cell epitope (TCE) disparities predictive of outcome in an Italian Registry Study, and 2) to analyze the role of HLA- 13

14 Not for publication or presentation Attachment 1 DPA1 in determining the immunogenic TCE relevant for non-permissive HLA-DPB1 disparities. The study population included 1281 donor/recipient pairs with AML, CML or MDS who received myeloablative conditioning regimen. All of these pairs were 10/10 and had DPB1 and DPA1 typing available. There were 239 HLA-matched (12/12), 585 TCE3 permissive, 226 GvH TCE3 non-permissive and 231 HvG TCE3 non-permissive. For TCE4, there were the same 239 HLA-matched, but there were 367 TCE4 permissive, 333 GvH TCE4 non-permissive and 342 TCE4 non-permissive. Outcomes included overall mortality, TRM, relapse, acute and chronic GvHD. A Brier Score comparison at 1, 3 and 5 years of survival was used. The multivariate models adjusted for all major clinical variables. Study aim 2 used the same 1281 NMDP 10/10 transplants as for Aim 1, the same clinical outcomes and statistical methods, the same TCE3 or TCE4 scoring system for DPB1, but with additional inclusion of DPA1 as follows: 1) Non- Permissive GvH or HvG possible only if DPA1*02 is present in the patient (GvH) or in the donor (HvG), 2) Non-Permissive GvH or HvG possible only if donor and recipient are mismatched for DPA1 alleles (any mismatch). The conclusions are 1) In 10/10 matched NMDP transplants, nonpermissive TCE disparities are associated with significantly increased NRM and, when present in the GvH vector, with a significant protection from disease relapse; 2) association with mortality and agvhd was less marked, possibly due to competing impacts. A post-hoc analysis suggests that a 5x larger cohort is needed to evaluate mortality, 3) the inclusion of DPA1 in the scoring system did not have an impact on the results and for several endpoints abrogated the associations observed with DPB1 scoring alone, 4) this is in line with results from molecular modeling suggesting that the polymorphic amino acid residues in the DP alpha chain are not in critical contact with most of the polymorphic sites in the DP beta chain, 5) these data will be presented as oral presentation at EBMT (#1115) and at EFI (evaluation pending). c. IB10-05 Evaluation of a Scoring System for HLA Mismatching: HistoCheck (R Blaszcyk/ C Hurley): Dr. Lee Ann Baxter-Lowe presented this study in the absence of Dr. Carolyn Hurley. Histocheck looks at dissimilarities of HLA molecules and assigns a score to each mismatch. There are major and minor mismatches, where the major mismatches are at peptide binding groove or at T-cell receptor (TCR) contact. The analysis compared outcomes of single HLA-A, B or C mismatches based on 1) scores assigned using HistoCheck algorithm (Dissimilarity Score (DSS)) where increasing score indicates more disparity. DSS analyzed as quartiles and as a continuous variable, and the DSS quartiles were group 1: , group 2: > , group 3: > and group 4: > Multivariate analyses using the proportional hazards model were used to compare the DSS groups. The study population included patients receiving first myeloablative unrelated hematopoietic cell transplantation (HCT) for AML, ALL, CML and MDS from with 92% being bone marrow. Retrospective high resolution HLA typing was completed for HLA-A, B, C, DRB1, DQB1 and DPB1. GVHD prophylaxis was 77% calcineurin inhibitor-based and 23% T-cell depletion. The conclusions of the analysis are that DSS scores assigned by HistoCheck do not correlate with transplant outcome and that HistoCheck does not provide an effective strategy for ranking HLA mismatches and choosing the best mismatched donor. d. IB10-07 Use of HLA Structure and Function Parameters to Understand the Relationship between HLA Disparity and Transplant Outcomes (LA Baxter-Lowe): Dr. Baxter-Lowe presented this study, which considers amino acid substitutions in an HLA molecule. Productive TCR interaction is generally restricted to self HLA molecules. Dr. Baxter-Lowe is currently working on a scoring system with a dataset received in January. Once she scores the specific mismatches, we will analyze whether the scores are associated with transplant outcomes. 14

15 Not for publication or presentation Attachment 1 e. IB06-02 Mismatching for low expression HLA loci in matched unrelated donor transplants (M Fernandez-Vina): Dr. Fernandez-Vina presented this study, which was presented at ASH There were some discrepant cases (about 17 or 18 cases) in the initial assignments of low expression loci, so the typings were reanalyzed. The re-analysis did not find a significant effect of multiple mismatches in the low expression loci in overall survival. The association with TRM remained significant. CYTOKINE/CHEMOKINE a. IB05-03s Genetic polymorphisms in the genes encoding human interleukin-7 receptor- : Prognostic significance in allogeneic stem cell transplantation (K Müller): The manuscript for this study has been submitted. Otherwise, no update was given. b. IB08-04s Immune response gene polymorphisms in unrelated donor stem cell transplantation in children (K Müller): No update was given. NK/KIR a. R02-40s/R03-63s KIR Program Project/NK receptor acquisition (J Miller/E Trachtenberg): Dr. Sarah Cooley presented this study, which considers KIR gene organization on chromosome 19. Four centromeric and two telomeric gene-content motifs combine to form the seven sequenced gene-content haplotypes. There are classifications into A and B for these seven haplotypes, broken down by centromeric (Cen) and telomeric (Tel) positions. The analysis in AML showed that KIR Cen-B/B donors are associated with the lowest relapse risk and the best relapse-free survival. The multivariate analysis of relapse showed that more B is better, with the relative risks being less for each increase of B in the group. When the B content is 0 or 1 (N=727), then the relapse rate is considered neutral. When the B content is 2 but not Cen B/B (N=213), then the rate is lower, but when the B content is Cen B/B (N=114), the relapse rate is lowest. This has led to a prospective trial of unrelated donor selection for AML incorporating KIR genotyping, which is in collaboration with the NMDP/CIBMTR and the top 20 AML URD centers in the US. The goal is to demonstrate decreased relapse and improved RFS in AML associated with higher frequency of donors with favorable KIR genotypes. KIR genotyping will be completed at confirmatory typing and the KIR best/better/neutral assignment provided to the transplant center to identify the best KIR genotype among the best HLA matched donors. b. R04-74s KIR functional significance (IHWG) (B Dupont/K Hsu/J Venstrom): Dr. Katharine Hsu presented this study which will be an oral presentation Friday evening. The study population is 1245 AML patients from that are 10/10 or 9/10 HLA-allele matched donors, where the HLA high-resolution genotype was verified through the NMDP. Donor KIR genotyping was performed using PCR with sequence-specific primers for 13 individual KIR genes. Cox regression was used to examine the association between donor KIR and HCT outcome. The multivariate analysis showed that donor 3DS1 is associated with lower NRM and higher OS in AML and that donor KIR2DS1 is associated with lower AML relapse and higher overall survival. The analysis also showed that 2DS1+ NK cells from C1/C1 and C1/C2 donors into C2 recipients are most protective against relapse and that 2DS1 educates C1 NK cells leading to protection from relapse. KIR2DS1-educated NK donors confer a survival benefit, and the impact of KIR2DS1 on AML relapse is stronger in an HLA-C mismatched HCT. However, in this analysis, there was no statistically significant effect of donor cenbb, like there was with R02-40s/R03-63s (p= ). The conclusions are that there is a role for KIR genotyping in 15

16 Not for publication or presentation Attachment 1 allogeneic HCT, that the greatest donor KIR effects in HCT are seen in AML, that activating KIR3DS1 impacts TRM and OS, that activating KIR2DS1 impacts AML relapse in manner consistent with in vitro findings of NK education (C1) and tolerance (C2C2), and that in an HLA- C mismatched HCT, KIR2DS1-mediated NK alloreactivity augments GvL without increasing agvhd or NRM. c. IB07-03 Analysis of Killer Immunoglobulin-like Receptor (KIR) ligands in reduced intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (HSCT) (R Sobecks): No update was given. d. IB08-06 Analysis of Killer Immunoglobulin-Like Receptor (KIR) ligands in umbilical cord blood transplantation (R Sobecks): No update was given. SENSITIZATION/TOLERANCE a. IB06-11s The effect of NIMA in cord blood transplantation (V Prasad/L Baxter-Lowe/ J Kurtzberg): Steve Spellman presented this study in Dr. Mary Eapen s and Dr. Vanderson Rocha s absence. Dr. Jon van Rood et al. showed lower relapse and non-relapse mortality (NRM) for hematologic malignancies after NIMA matched (NIMA+) UCBT in his publication in PNAS 2009). This study is a cooperation study between CIBMTR, EBMT and Eurocord, examining the effect of NIMA+ on UCBT in a separate cohort. Donor maternal typing was available for 508 donor-recipient pairs, and all transplants were 5/6 or 4/6 matched. Most patients had acute leukemia. N = 52 were NIMA+ (10% frequency), so there was a low frequency of NIMA+ transplants, and a matchedpair analysis was done. Variables for matching were determined by multivariate analysis for NRM mismatched (NIMA -) in 508 patients. Prognostic factors for NRM were 1) HLA match 5/6 vs. 4/6, 2) age ( 16 vs. >16 years), 3) disease status (CR1 vs. CR2/CR3 vs. not in remission at HCT), and 4) transplant conditioning regimen (MA +TBI vs. MA without TBI vs. RIC). The matching for HLA was done by number of matches, regardless of locus. The analysis also matched on disease and TNC (infused 3 vs. >3 x107/kg), which was a PhD statistician recommendation. The final data set included 48 NIMA+ matched to 116 NIMA- controls. The results showed no differences between NIMA+ and NIMA- UCBT for engraftment and GVHD. NRM after NIMA+ UCBT was lower but did not reach statistical significance (HR 0.45, p=0.12), relapse was lower after NIMA+ UCBT (HR 0.43, p=0.05). Treatment failure (inverse of LFS) was lower after NIMA+ UCBT (HR 0.42, p=0.03) and overall mortality was lower after NIMA+ UCBT (HR 0.46, p=0.05). The conclusions of this study are 1) re-exposure of cord blood to NIMA improves overall survival in patients receiving UCBTx for hematologic malignancies, 2) relapse was lower and 3) TRM appears to be lower but is not significant. The additive effect of lower TRM and relapse resulted in higher disease-free and overall survival, and in mismatched UCBT selection, an equivalently sized/matched (5/6 or 4/6) NIMA matched UCB may be preferable to a NIMA mismatched UCB. b. IB09-08 A retrospective study on impact of donor and recipient birth order on outcome of HLAidentical sibling stem cell transplantation (SCT) in hematological malignancies reported to the CIBMTR (C Dobbelstein): Dr. Stephanie Lee presented this study in Dr. Christiane Dobbelstein s absence. Previously, several studies have shown better outcomes when the recipient is older than the donor with higher survival, less acute GVHD II-IV and lower relapse. The proposed mechanism is microchimerism due to fetomaternal and transmaternal sibling cell trafficking where the donor is exposed to recipient antigens in utero. The effect has been most prominent in myeloid diseases. This study 16

17 Not for publication or presentation Attachment 1 looked at HLA-identical sibling donor transplants where the patients had AML, ALL, MDS or CML of all ages from Cases were excluded when the donor and recipient were > 15 years apart or when less than 1 year apart because we want to rule out fraternal siblings. Endpoints include relapse, OS, LFS, TRM, acute and chronic GVHD. There were 6089 cases where the matched sibling donors were younger than the recipient, and there were 5788 cases where the matched sibling donors were older than the recipient. In the multivariate analysis, overall survival showed an interaction with age where better survival was observed if the recipient was older in the youngest (< 10) age group while there was better survival in donor older for the year old group. TRM was lower if donor older which was opposite from hypothesized effect. No birth order effect seen in LFS, relapse, acute or chronic GVHD. The overall conclusion is that we did not see the hypothesized positive effect of having a donor younger than the recipient, compared to pairs where the donor is older than the recipient. c. R03-65s HY antigen (D Miklos): No update was given. d. GV04-01 Non-identical twin transplant for leukemia (J Barrett): No update was given. e. IB06-09s Detection of HLA antibody to the mismatched antigen in single antigen HLAmismatched unrelated donor transplants: Is it a predictor of graft-versus-host disease outcome? (S Arai/D Miklos): No update was given. f. IB06-10 Evaluation of the impact of the exposure to NIMA during fetal life and breast feeding and to the IPA during pregnancy on the clinical outcome of HSCT from haploidentical family members (J van Rood): No update was given. g. IB10-02 Development of GVHD prevention diagnostic test (R Somogyi/L Greller): No update was given. OTHER GENES a. IB08-08 Genome-Wide Association in Unrelated Donor Transplant Recipients and Donors: A Pilot Study (R Goyal): Dr. Rakesh Goyal presented this study which is currently in sample typing. The study population contains 10/10 allele-matched for HLA-A, -B, -C, -DRB1, and -DQB1 loci with European- American ancestry. The cases are first transplants receiving standard ablative pretransplant conditioning. No T-cell manipulation of transplant graft was allowed. There are a total of 579 recipient samples, of which 291 have grade III-IV acute GvHD and 288 do not have acute GvHD. There are also 656 donor samples, of which 315 came from the cases where the patient experienced grade III-IV acute GvHD and 341 came from cases without acute GvHD. The study will analyze the groups of grade III-IV agvhd vs. no agvhd using GWAS. Dr. Goyal has just finished analyzing the GWAS data. He has identified19 recipient markers and 3 donor markers which appear promising. b. IB10-01 Donor and Recipient Telomere Length as Predictors of Outcomes after Hematopoietic Stem Cell Transplant in Patients with Acquired Severe Aplastic Anemia (S Gadalla/S Savage): Dr. Shahinaz Gadalla presented this study. The study aims are 1) to determine pre-transplant blood telomere length in patients who received HSCT for SAA compared with age-matched 17

18 Not for publication or presentation Attachment 1 controls and patients with dyskeratosis congenita; 2) to assess the relationships between transplant telomere length and post-transplant outcomes including telomere length for the donor, for the recipient, and for early change after transplantation, and including the outcomes of death, engraftment, GVHD, malignancy and organ fibrosis; 3) to identify factors that modify the association between recipient and/or donor telomere length, and post-transplant outcomes in patients with SAA. The study population includes 388 recipients where 60 have both pre and post-transplant samples, 388 donors, 200 healthy controls and 40 DC patients. Telomere length assessment and telomere gene sequencing is in process. The next step will be to merge the clinical data and do the analysis. c. IB07-08 SNPs in the P53 pathway (P53, MDM2, ATM AND P21/WAF1) and transplant outcome after unrelated hematopoietic stem cell transplantation (B Dupont): No update was given. d. IB07-09 To develop and test a prognostic index for survival in CML MUD cohorts (A Dickinson): No update was given. e. IB09-04s Association of donor and recipient gene polymorphisms of drug metabolisms [GSTP, GSTT, GSTM and UGT (2B17, 2B7, 2B28)] and innate immune response [CD14, TIRAP, and NALPs (1 and 3)] with outcomes after allele matched unrelated hematopoietic stem cell transplantation (V Rocha): No update was given. f. IB09-06s/RT09-04s Genetic polymorphisms and HCT related mortality Re: Pre-HCT conditioning in matched unrelated donor HCT (T Hahn): No update was given. g. IB10-03 TLR and HMGB1 gene polymorphisms in unrelated haematopoietic stem cell transplantation (K Müller/B Kornblit): No update was given. h. IB10-04 A validation study of the role of base excision repair pathway as a predictor of outcome after hematopoietic stem cell transplant (B Thyagrajan /M Arora): No update was given. MINOR HISTOCOMPATIBILITY ANTIGENS a. IB10-06 Identification of Common, Clinically Significant, Minor Histocompatibility Antigens through Stem Cell Transplant Donor/Patient Polymorphism Disparities (P Armistead): No update was given. 8. Deferred studies pending accrual No update was given on the following studies because they are all in data collection and are deferred. a. R04-80s HLA matching in unrelated cord blood transplants (S Rodriguez-Marino) b. IB06-13 HLA disparity in unrelated cord blood transplantation: Delineation of factors contributing to transplant outcomes (L Baxter-Lowe) c. IB08-05s Evaluation of lymphotoxin alpha (LTA) alleles in relation to relapse in AML and CML (P Posch) 18

19 Not for publication or presentation Attachment 1 9. Feedback from Committee There was no feedback from the committee. 10. Closing remarks The Chairs thanked the committee members for their participation, and the meeting was adjourned at 4:05 pm. 19

20 Not for publication or presentation Attachment 2 Accrual Summary for Immunobiology Working Committee Characteristics of recipients of first transplants reported to the CIBMTR and NMDP CIBMTR HLA-identical Sibling CIBMTR Alternative Related CIBMTR Unrelated (non-us) CIBMTR Unrelated (US) Variable N (%) N (%) N (%) N (%) Number of patients Number of centers Age, median (range), years 30 (<1-82) 21 (<1-82) 27 (<1-75) 33 (<1-80) Age at transplant < 10 y 5761 (14) 1835 (29) 1669 (22) 4769 (19) y 7144 (17) 1169 (19) 1224 (16) 3448 (13) y 7575 (18) 956 (15) 1184 (16) 3282 (13) y 8152 (20) 883 (14) 1320 (18) 3640 (14) y 7253 (18) 786 (12) 1123 (15) 4423 (17) 50 y 5428 (13) 685 (11) 950 (13) 6206 (24) Male sex (58) 3796 (60) 4455 (60) (58) Karnofsky prior to transplant > 90% (73) 3913 (67) 5164 (72) (71) HLA-A,B,DRB1 groups low resolution 6/ (100) 1463 (25) 746 (10) (68) 5/ (15) 266 ( 4) 5055 (20) 4/ (16) 72 ( 1) 1732 ( 7) 3/6 0 N/A 0 49 (<1) Other/ < 3/6 /Unknown/TBD (45) 6386 (85) 1427 ( 6) High resolution available 5/8 N/A N/A 29 ( 5) 2225 (12) 6/8 N/A N/A 52 ( 8) 1961 (11) 7/8 N/A N/A 100 (16) 4254 (16) 8/8 N/A N/A 182 (29) (23) HLA high-res. typed/audited (out of 8) N/A N/A 373 ( 5) (49) Graft type Bone marrow (73) 4668 (74) 4698 (63) (52) Peripheral blood (26) 1549 (25) 1665 (22) 8322 (32) Cord blood 177 (<1) 31 (<1) 1085 (15) 3918 (15) Other 167 (<1) 64 ( 1) 8 (<1) 171 ( 1) Conditioning regimen Myeloablative (82) 5145 (81) 5560 (74) (72) Reduced intensity 3584 ( 9) 615 (10) 1089 (15) 4283 (17) Nonmyeloablative 1442 ( 3) 251 ( 4) 505 ( 7) 2196 ( 9) Other/To Be Determined 2322 ( 6) 306 ( 5) 316 ( 4) 799 ( 3) Donor age, median (range), years 30 (<1-93) 33 (<1-80) 35 (<1-68) 34 (<1-61) Donor age < 10 (including UCB Tx) 4947 (12) 442 ( 7) 749 (11) 1847 ( 9) (18) 773 (13) 53 ( 1) 215 ( 1) (19) 1270 (21) 1476 (21) 5083 (25) (20) 1554 (25) 2061 (30) 6717 (33) (17) 1137 (18) 1475 (21) 4644 (23) (14) 996 (16) 1123 (16) 1850 ( 9) 20

21 Not for publication or presentation Attachment 2 Accrual Summary for Immunobiology Working Committee Characteristics of recipients of first transplants reported to the CIBMTR and NMDP CIBMTR HLA-identical Sibling CIBMTR Alternative Related CIBMTR Unrelated (non-us) CIBMTR Unrelated (US) Variable N (%) N (%) N (%) N (%) Disease at transplant AML (27) 1526 (24) 1767 (24) 7960 (31) ALL 6696 (16) 1211 (19) 1583 (21) 4683 (18) Other leukemia 1025 ( 3) 154 ( 3) 196 ( 3) 1141 ( 4) CML 7967 (19) 971 (15) 1691 (23) 3954 (15) MDS/MPS 2643 ( 6) 332 ( 5) 753 (10) 3072 (12) Non-Hodgkin s lymphoma 2602 ( 6) 364 ( 6) 203 ( 3) 1541 ( 6) HD-Hodgkin s lymphoma 267 ( 1) 48 ( 1) 10 (<1) 92 (<1) MYE-plasma cell disorder, MM 973 ( 2) 139 ( 2) 39 ( 1) 131 ( 1) Other malignancies 327 ( 1) 57 ( 1) 29 (<1) 68 (<1) Breast cancer 63 (<1) 22 (<1) 0 3 (<1) Severe aplastic anemia 4218 (10) 469 ( 7) 425 ( 6) 988 ( 4) Inherited ab erythro. diff-funct ( 6) 259 ( 4) 216 ( 3) 426 ( 2) SCID & other immune deficienc. 544 ( 1) 555 ( 9) 254 ( 3) 651 ( 3) Inherited abnormal. of platelets 19 (<1) 7 (<1) 11 (<1) 43 (<1) Inherited disorder of metabolism 263 ( 1) 151 ( 2) 186 ( 2) 618 ( 2) Histiocytic disorders 102 (<1) 40 ( 1) 89 ( 1) 314 ( 1) Autoimmune diseases 17 (<1) 4 (<1) 2 (<1) 7 (<1) Other 24 (<1) 5 (<1) 5 (<1) 37 (<1) Disease status at transplant Early (53) 2726 (43) 4089 (55) (53) Intermediate 1118 ( 3) 232 ( 4) 373 ( 5) 1288 ( 5) Advanced 5139 (12) 1093 (17) 1249 (17) 4225 (16) Non-malignant disease/other (32) 2266 (36) 1759 (24) 6691 (26) GVHD prophylaxis FK506 + (MTX or MMF or Steroids) ± Other 3304 ( 8) 385 ( 6) 509 ( 7) 9634 (37) FK506 ± other 458 ( 1) 60 ( 1) 46 ( 1) 906 ( 4) CsA + MTX ± other (50) 2069 (33) 4474 (60) 7175 (28) CsA ± other 9822 (24) 1075 (17) 1772 (24) 4282 (17) MMF ± other 42 (<1) 6 (<1) 19 (<1) 75 (<1) MTX ± other 2949 ( 7) 259 ( 4) 29 (<1) 141 ( 1) T-cell depletion 2886 ( 7) 1294 (20) 432 ( 6) 2824 (11) Other / To be determined 1086 ( 3) 1169 (19) 189 ( 3) 737 ( 3) Donor/recipient sex match Male/Male (32) 2047 (35) 2298 (39) 568 (32) Male/Female 8385 (22) 963 (17) 1308 (22) 349 (20) Female/Male 9899 (26) 1425 (25) 1245 (21) 494 (28) Female/Female 7505 (20) 1337 (23) 1113 (19) 362 (20) Donor/recipient CMV match Negative/Negative 9289 (22) 1417 (22) 1801 (24) 5990 (23) Negative/Positive 5995 (15) 837 (13) 1686 (23) 5782 (22) Positive/Negative 3741 ( 9) 773 (12) 954 (13) 2649 (10) Positive/Positive (37) 1965 (31) 1829 (24) 3753 (15) Unknown 7007 (17) 1325 (21) 1200 (16) 7600 (29) 21

22 Not for publication or presentation Attachment 2 Accrual Summary for Immunobiology Working Committee Characteristics of recipients of first transplants reported to the CIBMTR and NMDP CIBMTR HLA-identical Sibling CIBMTR Alternative Related CIBMTR Unrelated (non-us) CIBMTR Unrelated (US) Variable N (%) N (%) N (%) N (%) Year of transplant (12) 886 (14) 35 (<1) 13 (<1) ( 3) 257 ( 4) 14 (<1) 18 (<1) ( 4) 248 ( 4) 31 (<1) 34 (<1) ( 4) 244 ( 4) 53 ( 1) 96 (<1) ( 4) 258 ( 4) 100 ( 1) 179 ( 1) ( 5) 315 ( 5) 136 ( 2) 289 ( 1) ( 5) 249 ( 4) 171 ( 2) 408 ( 2) ( 5) 271 ( 4) 227 ( 3) 472 ( 2) ( 5) 273 ( 4) 236 ( 3) 577 ( 2) ( 4) 254 ( 4) 247 ( 3) 710 ( 3) ( 5) 317 ( 5) 323 ( 4) 855 ( 3) ( 5) 311 ( 5) 405 ( 5) 991 ( 4) ( 4) 294 ( 5) 375 ( 5) 1052 ( 4) ( 4) 216 ( 3) 421 ( 6) 1065 ( 4) ( 3) 199 ( 3) 417 ( 6) 1118 ( 4) ( 3) 206 ( 3) 433 ( 6) 1145 ( 4) ( 3) 221 ( 3) 443 ( 6) 1194 ( 5) ( 3) 185 ( 3) 441 ( 6) 1266 ( 5) ( 3) 160 ( 3) 456 ( 6) 1445 ( 6) ( 3) 142 ( 2) 580 ( 8) 1620 ( 6) ( 3) 169 ( 3) 544 ( 7) 1765 ( 7) ( 3) 141 ( 2) 463 ( 6) 1994 ( 8) ( 2) 80 ( 1) 272 ( 4) 1958 ( 8) ( 2) 218 ( 3) 272 ( 4) 1975 ( 8) ( 2) 142 ( 2) 240 ( 3) 1878 ( 7) ( 1) 47 ( 1) 100 ( 1) 1230 ( 5) (<1) 14 (<1) 35 (<1) 427 ( 2) Median follow-up of recipients, mos 95 (<1-465) 91 (1-443) 61 (<1-259) 63 (<1-287) 22

23 Not for publication or presentation Attachment 2 Accrual Summary for First Transplants with Samples Available for Recipient and/or Donor through the NMDP for Adult Unrelated Donor Transplants Samples Available for Recipient and Donor Samples Available for Recipient Only Samples Available for Donor Only Variable N Eval N (%) N Eval N (%) N Eval N (%) Number of cases Number of centers Age, median (range), years (<1-78) (<1-79) (<1-75) Age at transplant < 10 y 1790 (12) 799 (14) 454 (17) y 1881 (13) 803 (14) 368 (14) y 2060 (14) 905 (16) 374 (14) y 2296 (15) 1089 (19) 399 (15) y 2851 (19) 1203 (21) 485 (18) 50 y 4134 (28) 964 (17) 643 (24) Male sex (58) (60) (60) Karnofsky prior to transplant > 90% (71) (72) (71) HLA-A, B, DRB1 groups high-res /6-2/6 33 (<1) 8 (<1) 0 3/6 159 ( 1) 35 ( 1) 1 (<1) 4/6 668 ( 5) 178 ( 6) 20 ( 2) 5/ (22) 706 (23) 249 (20) 6/ (72) 2178 (70) 978 (78) HLA high-res. typed and audited (99) (98) (98) Disease status at transplant Early 7754 (52) 3313 (57) 1340 (49) Intermediate 716 ( 5) 363 ( 6) 140 ( 5) Advanced 2541 (17) 931 (16) 490 (18) Non-malignant disease/other 4002 (27) 1156 (20) 754 (28) Graft type Bone marrow 8630 (58) 4724 (82) 1668 (61) Peripheral blood 6197 (41) 990 (17) 1031 (38) Cord blood Other 156 ( 1) 45 ( 1) 19 ( 1) Conditioning regimen Myeloablative 10331(69) 4747 (82) 1878 (69) Reduced intensity 2581 (17) 624 (11) 460 (17) Nonmyeloablative 1296 ( 9) 256 ( 4) 215 ( 8) Other/To be determined 805 ( 5) 136 ( 2) 171 ( 6) Donor age, median (range), years (18-61) (18-61) (18-59) Donor age < ( 1) 47 ( 1) 25 ( 1) (22) 1506 (26) 627 (23) (30) 2063 (36) 838 (31) (21) 1355 (24) 606 (22) ( 6) 379 ( 7) 205 ( 8) Unknown/TBD 2897 (19) 413 ( 7) 423 (16) 23

24 Not for publication or presentation Attachment 2 Accrual Summary for First Transplants with Samples Available for Recipient and/or Donor through the NMDP for Adult Unrelated Donor Transplants Samples Available for Recipient and Donor Samples Available for Recipient Only Samples Available for Donor Only Variable N Eval N (%) N Eval N (%) N Eval N (%) Disease at transplant AML 4605 (31) 1425 (25) 854 (31) ALL 2566 (17) 1005 (17) 517 (19) Other leukemia 761 ( 5) 203 ( 4) 126 ( 5) CML 2503 (17) 1707 (30) 409 (15) MDS/MPS 1921 (13) 601 (10) 317 (12) Non-Hodgkin s lymphoma 1049 ( 7) 266 ( 5) 156 ( 6) HD-Hodgkin s lymphoma 62 (<1) 16 (<1) 10 (<1) MYE-plasma cell disorder, MM 93 ( 1) 46 ( 1) 14 ( 1) Other malignancies 37 (<1) 7 (<1) 9 (<1) Breast cancer 4 (<1) 0 0 Severe aplastic anemia 598 ( 4) 199 ( 3) 114 ( 4) Inherited ab erythro. diff-funct. 219 ( 1) 60 ( 1) 38 ( 1) SCID & other immune deficienc. 248 ( 2) 85 ( 1) 55 ( 2) Inherited abnormal. of platelets 17 (<1) 6 (<1) 5 (<1) Inherited disorder of metabolism 183 ( 1) 97 ( 2) 46 ( 2) Histiocytic disorders 123 ( 1) 38 ( 1) 36 ( 1) Autoimmune disease 1 (<1) 0 0 Other 13 (<1) 2 (<1) 8 (<1) GVHD prophylaxis FK506 ± MMF ± MTX ± Steroids ± other 6190 (41) 1381 (24) 950 (35) FK506 ± other 520 ( 3) 104 ( 2) 78 ( 3) CsA + MTX ± other 4721 (31) 2732 (47) 957 (35) CsA ± other 1347 ( 9) 344 ( 6) 299 (11) MMF ± other 46 (<1) 12 (<1) 7 (<1) MTX ± other 101 ( 1) 44 ( 1) 9 (<1) T-cell depletion 1751 (12) 1079 (19) 333 (12) Other / To be determined 337 ( 2) 67 ( 1) 91 ( 3) Donor/recipient sex match Male/Male 5710 (39) 2286 (40) 1040 (40) Male/Female 3572 (24) 1277 (22) 558 (21) Female/Male 2843 (19) 1149 (20) 531 (20) Female/Female 2634 (18) 1019 (18) 487 (19) Donor/recipient CMV match Negative/Negative 3952 (26) 1815 (31) 701 (26) Negative/Positive 3964 (26) 1587 (28) 781 (29) Positive/Negative 1785 (12) 839 (15) 319 (12) Positive/Positive 2588 (17) 1049 (18) 509 (19) Unknown 2724 (18) 473 ( 8) 414 (15) 24

25 Not for publication or presentation Attachment 2 Accrual Summary for First Transplants with Samples Available for Recipient and/or Donor through the NMDP for Adult Unrelated Donor Transplants Samples Available for Recipient and Donor Samples Available for Recipient Only Samples Available for Donor Only N Eval N N Variable N (%) Eval N (%) Eval N (%) Year of transplant (<1) (<1) 43 ( 1) 6 (<1) ( 1) 60 ( 1) 5 (<1) ( 1) 108 ( 2) 23 ( 1) ( 2) 169 ( 3) 48 ( 2) ( 2) 161 ( 3) 77 ( 3) ( 2) 256 ( 4) 105 ( 4) ( 3) 371 ( 6) 86 ( 3) ( 3) 468 ( 8) 99 ( 4) ( 3) 541 ( 9) 109 ( 4) ( 4) 587 (10) 115 ( 4) ( 4) 651 (11) 117 ( 4) ( 4) 567 (10) 106 ( 4) ( 5) 162 ( 3) 111 ( 4) ( 5) 130 ( 2) 113 ( 4) ( 4) 116 ( 2) 304 (11) ( 5) 150 ( 3) 285 (10) ( 7) 255 ( 4) 190 ( 7) ( 8) 205 ( 4) 162 ( 6) ( 9) 216 ( 4) 168 ( 6) ( 9) 152 ( 3) 158 ( 6) ( 8) 141 ( 2) 128 ( 5) ( 7) 161 ( 3) 131 ( 5) ( 4) 91 ( 2) 75 ( 3) (<1) 2 (<1) 2 (<1) Median follow-up of recipients, mo (<1-270) (2-270) (<1-248) 25

26 Not for publication or presentation Attachment 2 Accrual Summary for First Transplants with Samples Available for Recipient and/or Cord Blood Unit(s) through the NMDP for Cord Blood Transplants Samples Available - Recipient and UCB Unit(s) Samples Available - Recipient Only Samples Available - UCB Unit(s) Only Variable N (%) N (%) N (%) Number of cases Number of centers Number of cord blood units in transplant One 775 (86) 743 (82) 244 (82) Two 128 (14) 163 (18) 54 (18) Three 1 (<1) 2 (<1) 0 Age, median (range), years 13 (<1-79) 17 (<1-73) 16 (<1-68) Age at transplant, by decade < 10 y 385 (43) 338 (37) 121 (41) y 135 (15) 151 (17) 39 (13) y 89 (10) 87 (10) 31 (10) y 55 ( 6) 62 ( 7) 25 ( 8) y 83 ( 9) 84 ( 9) 30 (10) 50 y 157 (17) 186 (20) 52 (17) Male sex 506 (56) 490 (54) 174 (58) Karnofsky prior to transplant > 90% 591 (74) 598 (74) 196 (78) HLA-A, B, DRB1 groups high-res 0/6-2/6 26 ( 3) 32 ( 4) 13 ( 6) 3/6 104 (13) 131 (17) 32 (14) 4/6 349 (44) 302 (40) 104 (45) 5/6 218 (27) 219 (29) 61 (26) 6/6 96 (12) 70 ( 9) 21 ( 9) HLA high resolution typed and audited 227 (25) 126 (14) 1 (<1) Disease status at transplant Early 454 (50) 458 (50) 136 (46) Intermediate 43 ( 5) 34 ( 4) 16 ( 5) Advanced 101 (11) 110 (12) 39 (13) Non-malignant disease/other 306 (34) 306 (34) 107 (36) Conditioning regimen Myeloablative 557 (62) 552 (61) 174 (58) Reduced intensity 134 (15) 159 (18) 44 (15) Nonmyeloablative 132 (15) 153 (17) 50 (17) Other/To be determined 81 ( 9) 44 ( 5) 30 (10) Donor/recipient CMV match Negative/Negative 1 (<1) 3 (<1) 2 ( 1) Negative/Positive 6 ( 1) 4 (<1) 4 ( 1) Positive/Negative 1 (<1) 0 3 ( 1) Positive/Positive 7 ( 1) 12 ( 1) 4 ( 1) Unknown 889 (98) 889 (98) 285 (96) 26

27 Not for publication or presentation Attachment 2 Accrual Summary for First Transplants with Samples Available for Recipient and/or Cord Blood Unit(s) through the NMDP for Cord Blood Transplants Samples Available - Recipient and UCB Unit(s) Samples Available - Recipient Only Samples Available - UCB Unit(s) Only Variable N (%) N (%) N (%) Disease at transplant AML 309 (34) 354 (39) 91 (31) ALL 227 (25) 185 (20) 69 (23) Other leukemia 45 ( 5) 43 ( 4) 14 ( 5) CML 29 ( 3) 31 ( 3) 6 ( 2) MDS/MPS 22 ( 2) 19 ( 2) 6 ( 2) Non-Hodgkin s lymphoma 32 ( 4) 35 ( 4) 13 ( 4) HD-Hodgkin s lymphoma 2 (<1) 5 ( 1) 0 Other malignancies 1 (<1) 3 (<1) 0 Severe aplastic anemia 25 ( 3) 30 ( 3) 8 ( 3) Inherited ab erythro. diff-funct. 19 ( 2) 26 ( 3) 7 ( 2) SCID & other immune deficienc. 50 ( 6) 45 ( 5) 24 ( 8) Inherited abnormal. of platelets 2 (<1) 3 (<1) 1 (<1) Inherited disorder of metabolism 58 ( 6) 52 ( 6) 19 ( 6) Histiocytic disorders 31 ( 3) 28 ( 3) 11 ( 4) Autoimmune diseases 3 (<1) 0 0 Other 4 (<1) 3 (<1) 2 ( 1) GVHD prophylaxis FK506 ± MMF ± MTX ± Steroids ± other 291 (32) 308 (34) 92 (31) FK506 ± other 35 ( 4) 31 ( 3) 16 ( 5) CsA + MTX ± other 31 ( 3) 45 ( 5) 14 ( 5) CsA ± other 486 (54) 481 (53) 147 (49) MMF ± other 3 (<1) 2 (<1) 2 ( 1) MTX ± other 3 (<1) 2 (<1) 0 T-cell depletion 2 (<1) 0 0 Other / To be determined 53 ( 6) 39 ( 4) 27 ( 9) Year of transplant (<1) 2 (<1) 4 ( 1) ( 1) 15 ( 2) 6 ( 2) (<1) 10 ( 1) ( 1) 35 ( 4) 1 (<1) ( 1) 32 ( 4) 6 ( 2) ( 4) 73 ( 8) 13 ( 4) ( 7) 185 (20) 16 ( 5) (26) 40 ( 4) 58 (19) (29) 36 ( 4) 91 (31) (23) 225 (25) 65 (22) ( 9) 253 (28) 38 (13) (<1) 0 Median follow-up of recipients, mo 24 (3-96) 14 (1-96) 24 (1-121) 27

28 Not for publication or presentation Attachment 2 Accrual Summary for First Transplants with Samples Available for Recipient and/or Donor through the NMDP for Adult Related Donor Transplants Samples Available for Recipient and Donor Samples Available for Recipient Only Samples Available for Donor Only Variable N Eval N (%) N Eval N (%) N Eval N (%) Number of cases Number of centers Age, median (range), years (<1-73) (6-74) (16-74) Age at transplant < 10 y 25 ( 5) 2 ( 4) y 30 ( 6) 3 ( 7) 2 (15) y 42 ( 9) 2 ( 4) y 47 (10) 5 (11) 2 (15) y 86 (18) 12 (26) 3 (23) 50 y 245 (52) 22 (48) 6 (46) Male sex (61) (61) 13 9 (69) Karnofsky prior to transplant > 90% (71) 4 4 (100) 0 N/A Disease status at transplant Early 30 ( 6) 2 ( 4) 0 Intermediate 3 ( 1) 1 ( 2) 0 Advanced 7 ( 1) 1 ( 2) 0 Non-malignant disease/other 435 (92) 42 (91) 13 (100) Graft type Bone marrow 68 (14) 8 (17) 3 (23) Peripheral blood 401 (85) 36 (78) 10 (77) Cord blood 1 (<1) 1 ( 2) 0 Other 2 (<1) 1 ( 2) 0 Conditioning regimen Myeloablative 81 (17) 6 (13) 0 Reduced intensity 18 ( 4) 0 0 Nonmyeloablative 14 ( 3) 0 0 Other/To be determined 362 (76) 40 (87) 13 (100) Donor age, median (range), years (<1-71) 4 37 (12-49) 0 N/A Donor age < ( 2) 1 ( 2) ( 1) 1 ( 2) ( 2) ( 2) 2 ( 4) ( 9) 0 0 Unknown/TBD 395 (83) 42 (91) 13 (100) 28

29 Not for publication or presentation Attachment 2 Accrual Summary for First Transplants with Samples Available for Recipient and/or Donor through the NMDP for Adult Related Donor Transplants Samples Available for Recipient and Donor Samples Available for Recipient Only Samples Available for Donor Only Variable N Eval N (%) N Eval N (%) N Eval N (%) Disease at transplant AML 130 (27) 14 (30) 2 (15) ALL 82 (17) 9 (20) 2 (15) Other leukemia 34 ( 7) 5 (11) 0 CML 24 ( 5) 0 0 MDS/MPS 73 (15) 11 (24) 6 (46) Non-Hodgkin s lymphoma 81 (17) 4 ( 9) 1 ( 8) HD-Hodgkin s lymphoma 8 ( 2) 1 ( 2) 1 ( 8) MYE-plasma cell disorder, MM 10 ( 2) 1 ( 2) 0 Other malignancies 2 (<1) 0 0 Breast cancer 1 (<1) 0 0 Severe aplastic anemia 17 ( 4) 0 0 Inherited ab erythro. diff-funct. 9 ( 2) 1 ( 2) 1 ( 8) SCID & other immune deficienc. 2 (<1) 0 0 Histiocytic disorders 1 (<1) 0 0 Other 1 (<1) 0 0 GVHD prophylaxis FK506 ± MMF ± MTX ± Steroids ± other 330 (69) 18 (39) 8 (62) FK506 ± other 66 (14) 20 (43) 2 (15) CsA + MTX ± other 30 ( 6) 2 ( 4) 1 ( 8) CsA ± other 8 ( 2) 1 ( 2) 1 ( 8) MMF ± other MTX ± other 1 (<1) 0 0 T-cell depletion 1 (<1) 0 0 Other / To be determined 39 ( 8) 5 (11) 1 ( 8) Donor/recipient sex match Male/Male 164 (36) 15 (34) 5 (38) Male/Female 79 (17) 7 (16) 3 (23) Female/Male 110 (24) 12 (27) 4 (31) Female/Female 104 (23) 10 (23) 1 ( 8) Donor/recipient CMV match Negative/Negative 77 (16) 2 ( 4) 0 Negative/Positive 97 (20) 8 (17) 2 (15) Positive/Negative 42 ( 9) 2 ( 4) 1 ( 8) Positive/Positive 209 (44) 28 (61) 9 (69) Unknown 50 (11) 6 (13) 1 ( 8) 29

30 Not for publication or presentation Attachment 2 Accrual Summary for First Transplants with Samples Available for Recipient and/or Donor through the NMDP for Adult Related Donor Transplants Samples Available for Recipient and Donor Samples Available for Recipient Only Samples Available for Donor Only N Eval N Eval N Eval Variable N (%) N (%) N (%) Year of transplant ( 1) 1 ( 2) (20) 5 (11) 1 ( 8) (29) 10 (22) 3 (23) (39) 23 (50) 6 (46) (11) 7 (15) 3 (23) Median follow-up of recipients, mo (2-47) 32 9 (2-37) 10 6 (2-23) 30

31 Not for publication or presentation Attachment 3 Study Proposal Study Title: Is a well-matched HLA-identical unrelated male donor (10/10) an alternative to a female HLA-identical sibling donor for a male recipient in need of hematopoietic stem cell transplantation? Olle Ringdén, MD, PhD, Karolinska Institutet, Stockholm, Sweden Tom Erkers, MSc, Karolinska Institutet, Stockholm, Sweden Johan Törlén, MD, Karolinska University Hospital, Stockholm, Sweden Study Objectives: To compare outcomes after HSCT using a well-matched unrelated male donor (10/10 match) to a male recipient compared to a female HLA-identical sibling donor. The following outcome measures will be compared: 1. Acute GVHD grades II-IV, III-IV 2. Treatment-related mortality 3. Chronic GVHD 4. Relapse probability 5. Overall survival 6. Relapse-free survival Scientific Justification: A female donor to a male recipient is associated with an increased risk of acute GVHD. 1, 2 In a study from the CIBMTR, a female donor to a male recipient was the only risk-factor for acute GVHD. A female donor to a male recipient also increases the risk for chronic GVHD as found in studies by the CIBMTR and NMDP. 3,4 It has been suggested that human minor histocompatibility antigens encoded on Y chromosome contribute to the alloreactivity by female donor cells in a male recipient. 5-7 Several minor histocompatibility antigens have been identified to elicit T-cell responses from female donors. Several studies have also shown that a female donor to a male recipient results in worse survival and leukemiafree survival, compared to other gender combinations Unrelated donors are increasingly used in HSCT Using genomic typing, outcome following unrelated donor transplantation was improved compared to serologic typing.17,18 Using genomically wellmatched donors, the outcome is similar as using HLA-identical sibling donors Therefore, a wellmatched unrelated donor may be as good as an HLA-identical sibling donor for transplantation. Because a female donor to a male recipient is correlated to worse outcome, it is possible that under certain circumstances a male unrelated donor may be superior. For instance, a male donor with an elder female sibling donor may be better off selecting a young unrelated male donor, because young donor age and a high cell dose is also correlated to improved outcome. 4,23 A CIBMTR study found that the graft-versus-leukemia effect was the same using well-matched unrelated donors as using HLA-identical sibling donors. 24 In this analysis, it may also be possible to analyze if there is a graft-versus-leukemia effect associated with the Y chromosome. It was also demonstrated that female donors contribute to a selective graft-versus-leukemia effect in male recipients. 25 A reduced risk of GVHD and TRM using a male donor may be offset by an increased risk of relapse. The purpose of this analysis is to select the optimal donor to a male recipient with leukemia. Study Population: - Patients with ALL, AML, CML, transplanted between 1995 and 2011, using myeloablative or reduced intensity conditioning. - The study population includes male recipients with either an HLA-identical female sibling donor, or a well-matched (10/10) identical male unrelated donor. 31

32 Not for publication or presentation Attachment 3 - (Male patients with a male HLA-identical sibling donor or an unrelated female donor may be included as controls.) Cord blood transplants will be excluded. Variables to be Analyzed: Patient-related data: - Age at transplant - Karnofsky score pre transplant: <90% vs. 90% Disease-related data: - Disease: AML vs. ALL vs. CML - Disease status at time of transplant: Early vs. intermediate vs. advanced - Acute leukemia: - AML: FAB subtype - ALL: Immunophenotype - WBC at diagnosis: <25 x 10 9 /L, x 10 9 /L, x 10 9 /L, >100 x10 9 /L - Duration of CR1 (for patients beyond CR1): <6 months, 6-12 months, >12 months - Time from remission to transplant (for patients in CR1): <3 months, 3-6 months, >6 months. - Cytogenetics: - AML: good vs. intermediate vs. poor prognosis vs. no abnormalities - ALL: No abnormalities vs. hyperdiploid vs. hypodiploid/ t(9;22)/ t(4;11)/ t(8;14) vs. other abnormalities - CML: time from diagnosis to transplant: <12 months, months, >24 months Transplant-related: - Source of stem cells: bone marrow vs. peripheral stem cells - Donor age: continuous and group by 10 year increments - Donor-recipient CMV status (-/- vs. -/+ vs. +/- vs. +/+) - Conditioning regimens: categories will be determined by the most common regimens used, for instance Bu/Cy vs. Cy/TBI vs. various RIC and NMA protocols - Immunosuppression: categories will be determined by the most commonly used, for instance CyA + MTX, tacrolimus + MTX, CyA + MMF, etc. - Year of transplant. Supplemental data is not required. Sample Requirements: No samples are required. Study Design: The objective of this study is to evaluate whether a well-matched unrelated male donor will have comparable or improved leukemia-free survival in male recipients when compared to grafts from HLAidentical female donors. Secondary objectives include comparison between acute and chronic GVHD, transplant-related mortality, relapse rates and overall survival between these two groups. Patient-, disease- and transplant-related factors will be compared between the two groups using the chisquare test for categorical variables and the Wilcoxon two-sample test for continuous variables. The product-limit estimater proposed by Kaplan-Meier will be used to estimate the median and range of the follow-up time. Probabilities of acute and chronic GVHD, treatment-related mortality and relapse will be calculated using cumulative incidence to accommodate for competing risks. Univariate probabilities of overall and leukemia-free survival will be calculated using the Kaplan-Meier estimator. 95% confidence intervals for all probabilities and p-values of pairwise comparisons will be derived from pointwise estimates and calculated using the standard techniques. Multivariate analysis models for acute and chronic 32

33 Not for publication or presentation Attachment 3 GVHD, TRM, relapse, overall survival and leukemia-free survival will be done to test the main effect covariates and additional co-variates will be fitted in the model in a forward stepwise approach with a p- value of <0.05 to indicate a significance. References: 1. Atkinson K, Farrell C, Chapman G, Downs K, Penny R, Biggs J. Female marrow donors increase the risk of acute graft-versus-host disease: effect of donor age and parity and analysis of cell subpopulations in the donor marrow inoculum. Br J Haematol. 1986;63(2): Gale RP, Bortin MM, van Bekkum DW, Biggs JC, Dicke KA, Gluckman E, et al. Risk factors for acute graft-versus-host disease. Br J Haematol. 1987;67(4): Atkinson K, Horowitz MM, Gale RP, van Bekkum DW, Gluckman E, Good RA, et al. Risk factors for chronic graft-versus-host disease after HLA-identical sibling bone marrow transplantation. Blood. 1990;75(12): Kollman C, Howe CW, Anasetti C, Antin JH, Davies SM, Filipovich AH, et al. Donor characteristics as risk factors in recipients after transplantation of bone marrow from unrelated donors: the effect of donor age. Blood. 2001;98(7): Meadows L, Wang W, den Haan JM, Blokland E, Reinhardus C, Drijfhout JW, et al. The HLA-A*0201-restricted H-Y antigen contains a posttranslationally modified cysteine that significantly affects T cell recognition. Immunity. 1997;6(3): Epub 1997/03/ Warren EH, Gavin MA, Simpson E, Chandler P, Page DC, Disteche C, et al. The human UTY gene encodes a novel HLA-B8-restricted H-Y antigen. J Immunol. 2000;164(5): Epub 2000/02/ Vogt MH, van den Muijsenberg JW, Goulmy E, Spierings E, Kluck P, Kester MG, et al. The DBY gene codes for an HLA-DQ5-restricted human male-specific minor histocompatibility antigen involved in graft-versus-host disease. Blood. 2002;99(8): Epub 2002/04/ Aschan J, Ringden O. Prognostic factors for long-term survival in leukemic marrow recipients with special emphasis on age and prophylaxis for graft-versus-host disease. Clin Transplant. 1994;8(3 Pt 1): Gahrton G. Risk assessment in haematopoietic stem cell transplantation: impact of donor-recipient sex combination in allogeneic transplantation. Best practice & research Clinical haematology. 2007;20(2): Epub 2007/04/ Loren AW, Bunin GR, Boudreau C, Champlin RE, Cnaan A, Horowitz MM, et al. Impact of donor and recipient sex and parity on outcomes of HLA-identical sibling allogeneic hematopoietic stem cell transplantation. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation. 2006;12(7): Epub 2006/06/ Nannya Y, Kataoka K, Hangaishi A, Imai Y, Takahashi T, Kurokawa M. The negative impact of female donor/male recipient combination in allogeneic hematopoietic stem cell transplantation depends on disease risk. Transplant international : official journal of the European Society for Organ Transplantation. 2011;24(5): Epub 2011/02/ Anasetti C, Etzioni R, Petersdorf EW, Martin PJ, Hansen JA. Marrow transplantation from unrelated volunteer donors. Annual review of medicine. 1995;46: Epub 1995/01/01. 33

34 Not for publication or presentation Attachment Madrigal JA, Arguello R, Scott I, Avakian H. Molecular histocompatibility typing in unrelated donor bone marrow transplantation. Blood reviews. 1997;11(2): Epub 1997/06/ Flomenberg N, Baxter-Lowe LA, Confer D, Fernandez-Vina M, Filipovich A, Horowitz M, et al. Impact of HLA class I and class II high-resolution matching on outcomes of unrelated donor bone marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplantation outcome. Blood. 2004;104(7): Petersdorf EW, Anasetti C, Martin PJ, Hansen JA. Tissue typing in support of unrelated hematopoietic cell transplantation. Tissue Antigens. 2003;61(1): Shaw BE, Gooley TA, Malkki M, Madrigal JA, Begovich AB, Horowitz MM, et al. The importance of HLA-DPB1 in unrelated donor hematopoietic cell transplantation. Blood. 2007;110(13): Lee SJ, Klein J, Haagenson M, Baxter-Lowe LA, Confer DL, Eapen M, et al. Highresolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood. 2007;110(13): Epub 2007/09/ Nademanee A, Schmidt GM, Parker P, Dagis AC, Stein A, Snyder DS, et al. The outcome of matched unrelated donor bone marrow transplantation in patients with hematologic malignancies using molecular typing for donor selection and graft-versushost disease prophylaxis regimen of cyclosporine, methotrexate, and prednisone. Blood. 1995;86(3): Kiehl MG, Kraut L, Schwerdtfeger R, Hertenstein B, Remberger M, Kroeger N, et al. Outcome of allogeneic hematopoietic stem-cell transplantation in adult patients with acute lymphoblastic leukemia: no difference in related compared with unrelated transplant in first complete remission. J Clin Oncol. 2004;22(14): Ringden O, Remberger M, Persson U, Ljungman P, Aldener A, Andstrom E, et al. Similar incidence of graft-versus-host disease using HLA-A, -B and -DR identical unrelated bone marrow donors as with HLA-identical siblings. Bone Marrow Transplant. 1995;15(4): Saarinen-Pihkala UM, Gustafsson G, Ringden O, Heilmann C, Glomstein A, Lonnerholm G, et al. No disadvantage in outcome of using matched unrelated donors as compared with matched sibling donors for bone marrow transplantation in children with acute lymphoblastic leukemia in second remission. J Clin Oncol. 2001;19(14): Yakoub-Agha I, Mesnil F, Kuentz M, Boiron JM, Ifrah N, Milpied N, et al. Allogeneic marrow stem-cell transplantation from human leukocyte antigen-identical siblings versus human leukocyte antigen-allelic-matched unrelated donors (10/10) in patients with standard-risk hematologic malignancy: a prospective study from the French Society of Bone Marrow Transplantation and Cell Therapy. J Clin Oncol. 2006;24(36): Epub 2006/11/ Sierra J, Storer B, Hansen JA, Bjerke JW, Martin PJ, Petersdorf EW, et al. Transplantation of marrow cells from unrelated donors for treatment of high-risk acute leukemia: the effect of leukemic burden, donor HLA-matching, and marrow cell dose. Blood. 1997;89(11):

35 Not for publication or presentation Attachment Ringden O, Pavletic SZ, Anasetti C, Barrett AJ, Wang T, Wang D, et al. The graftversus-leukemia effect using matched unrelated donors is not superior to HLA-identical siblings for hematopoietic stem cell transplantation. Blood. 2009;113(13): Randolph SS, Gooley TA, Warren EH, Appelbaum FR, Riddell SR. Female donors contribute to a selective graft-versus-leukemia effect in male recipients of HLAmatched, related hematopoietic stem cell transplants. Blood. 2004;103(1): Epub 2003/09/13. 35

36 Not for publication or presentation Attachment 3 Table 1: Characteristics of first transplants for male patients with AML, ALL and CML between 1995 and 2011 by female HLA-identical sibling donors vs. male 10/10 unrelated NMDP donors a Female HLA-identical donors 10/10 Male unrelated donors Characteristics of patients N Eval N (%) N Eval N (%) Number of patients Number of centers Age, median (range), years (<1-75) (<1-73) Age at transplant y 223 ( 9) 101 ( 7) y 405 (16) 168 (11) y 377 (15) 255 (17) y 489 (19) 225 (15) y 513 (20) 276 (19) 50 and older 546 (21) 461 (31) Recipient race Caucasian 1782 (70) 1360 (92) African American 91 ( 4) 20 ( 1) Asian/Pacific Islander 295 (12) 12 ( 1) Hispanic 216 ( 8) 69 ( 5) Native American 6 (<1) 4 (<1) Other/Multiple/Declined/Unknown 163 ( 6) 21 ( 1) Male sex (100) (100) Karnofsky prior to transplant > (76) (72) Disease at transplant AML 1177 (46) 736 (50) ALL 676 (26) 415 (28) CML 700 (27) 335 (23) Disease status at transplant Early 2098 (82) 1145 (77) Intermediate 107 ( 4) 76 ( 5) Advanced 348 (14) 265 (18) Graft type Bone marrow 1203 (47) 813 (55) Peripheral blood 1350 (53) 673 (45) a - Data is not CAP-modeled. 36

37 Not for publication or presentation Attachment 3 Table 1. Continued. Characteristics of patients N Eval N (%) Conditioning regimen Myeloablative 2195 (86) 1189 (80) Reduced intensity 259 (10) 221 (15) Nonmyeloablative 99 ( 4) 76 ( 5) GVHD prophylaxis FK506 + (MTX or MMF or Steroids) other 424 (17) 737 (50) FK506 other 51 ( 2) 53 ( 4) CsA + MTX other 1561 (61) 469 (32) CsA other (No MTX) 352 (14) 87 ( 6) MMF other 3 (<1) 3 (<1) MTX other (No CsA) 24 ( 1) 8 ( 1) T-cell depletion 59 ( 2) 107 ( 7) Other 79 ( 3) 22 ( 1) In vivo T-cell depletion No 2358 (92) 1122 (76) Yes 195 ( 8) 364 (24) Donor/recipient sex match Male/Male (100) Female/Male 2553 (100) 0 Donor/recipient CMV match Negative/Negative 624 (24) 473 (32) Negative/Positive 356 (14) 392 (26) Positive/Negative 306 (12) 176 (12) Positive/Positive 1140 (45) 204 (14) Unknown 127 ( 5) 241 (16) Donor age, median (range), years (<1-81) (19-61) Donor age ( 9) (15) 15 ( 1) (16) 375 (25) (19) 493 (33) (20) 312 (21) 50 and older 533 (21) 75 ( 5) To Be Determined 24 ( 1) 216 (15) 37

38 Not for publication or presentation Attachment 3 Table 1. Continued. Characteristics of patients N Eval N (%) Year of transplant (11) 57 ( 4) (12) 55 ( 4) ( 8) 68 ( 5) ( 7) 64 ( 4) ( 7) 68 ( 5) ( 7) 86 ( 6) ( 5) 75 ( 5) ( 6) 77 ( 5) ( 5) 87 ( 6) ( 7) 147 (10) ( 7) 147 (10) ( 5) 185 (12) ( 3) 165 (11) ( 5) 122 ( 8) ( 3) 83 ( 6) ( 3) (<1) 0 Median follow-up of survivors, mo (range) (1-193) (5-193) 38

39 Not for publication or presentation Attachment 4 Study Proposal Study Title: Are major HLA-antigens targets for the graft-versus-leukemia effect? Tom Erkers, MSc, Karolinska Institutet, Stockholm, Sweden Olle Ringdén, MD, PhD, Karolinska Institutet, Stockholm, Sweden Johan Törlén, MD, Karolinska Institutet, Stockholm, Sweden Study Objectives: 1. Compare the cumulative incidence of relapse in patients undergoing HLA one or two antigen mismatched related transplants, as opposed to HLA-identical sibling donor transplantation. Cord blood transplants with HLA identity will be compared to one, two or three antigens mismatched cord blood transplants. 2. Evaluate the effect of acute and chronic graft-versus-host disease on risk of relapse after HLAmismatched transplant, versus HLA-identical sibling transplantation. This will also be evaluated in the cord blood setting. 3. To determine whether other risk-factors influence the impact of graft-versus-leukemia effect after HLA-mismatched or HLA-identical sibling donor transplantation. Graft-versus-leukemia will also be analyzed in the cord blood transplants. Scientific Justification: It is well known from experimental animals and also clinical hematopoietic stem cell transplantation that the immune system may control cancer. 1-5 The Seattle group reported a decreased risk of relapse in patients with GVHD, especially chronic GVHD, as opposed to patients without GVHD. In addition, twins who undergo hematopoietic stem cell transplantation (HSCT) and recipients of T-cell depleted grafts have an increased risk of relaps. 4,6-8 From all these studies, it is evident that GVHD and especially chronic GVHD improve long-term survival in patients undergoing HSCT for leukemia. A recent CIBMTR study compared the graft-versus-leukemia effect in patients undergoing myeloablative conditioning and stem cell transplantation, using well-matched unrelated donors. 9 That study showed that risk of relapse was the same between unrelated donor transplants and HLA-identical sibling transplants for ALL and CML, but was worse using unrelated donor transplants for AML. This suggests that the graft-versus-leukemia effect is not better using unrelated donors, compared to HLA-identical sibling transplants. Because this was a retrospective registry analysis, the data have to be interpreted with caution. Anyhow, these data may raise the question regarding the relative importance of minor histocompatibility antigens as targets for a graft-versus-leukemia effect. It is expected that minor histocompatibility antigens may be more common using an unrelated donor, compared to an HLAidentical sibling donor. In the light of that study, it may be important to study the outcome in HLA-mismatched related transplants, compared to HLA-identical sibling transplants. Such as study would address if major HLAantigens are targets for a graft-versus-leukemia effect. The hypothesis is that this would be the case and that recipients of HLA-mismatched related transplants or HLA-mismatched cord blood transplants would have a lower probability of relapse, when adjusted for the graft-versus-leukemia effect by acute and chronic GVHD. 39

40 Not for publication or presentation Attachment 4 Study Population: Patient eligibility selection criteria include: - Patients with ALL, AML, or CML transplanted between 1995 and 2011, using myeloablative or reduced intensity conditioning. - HLA related one or two antigen mismatched donors, umbilical cord blood transplants with sero, 1 or 2 antigens mismatch and a control group of HLA-identical sibling transplants. - Patients receiving T-cell depleted grafts will be excluded. Variables to be Analyzed: Patient-related data: - Age at transplant - Gender: male vs. female - Karnofsky score pre transplant: <90% vs. 90% Disease-related data: - Disease: AML vs. ALL vs. CML - Disease status at time of transplant: Early vs. intermediate vs. advanced - Acute leukemia: - AML: FAB subtype - ALL: Immunophenotype - WBC at diagnosis: <25 x 10 9 /L, x 10 9 /L, x 10 9 /L, >100 x10 9 /L - Duration of CR1 (for patients beyond CR1): <6 months, 6-12 months, >12 months - Time from remission to transplant (for patients in CR1): <3 months, 3-6 months, >6 months. - Cytogenetics: - AML: good vs. intermediate vs. poor prognosis vs. no abnormalities - ALL: No abnormalities vs. hyperdiploid vs. hypodiploid/ t(9;22)/ t(4;11)/ t(8;14) vs. other abnormalities - CML: time from diagnosis to transplant: <12 months, months, >24 months Transplant-related: - Source of stem cells: bone marrow vs. peripheral stem cells vs. cord blood - Donor age: continuous and group by 10 year increments - Donor-recipient gender match: F to M vs. M to F vs. M to M vs. F to F - Donor-recipient CMV status (-/- vs. -/+ vs. +/- vs. +/+) - Conditioning regimens: categories will be determined by the most common regimens used. - Bu/Cy vs. Cy/TBI vs. various RIC and NMA protocols - Immunosuppression: categories will be determined by the most commonly used, for instance CyA + MTX, tacrolimus + MTX, CyA + MMF, etc.. - Year of transplant. Acute GVHD grade II-IV: - Yes vs. no Chronic GVHD: - Yes vs. no Supplemental data is not required. Sample Requirements: No samples are required. Study Design: To summarize the characteristics of the dataset, descriptive tables of patient, disease and transplant related variables will be reported. For discrete factors, the number of cases and their respective percentages will be calculated. For continuous factors, the median and range will be calculated. 40

41 Not for publication or presentation Attachment 4 The main objective of this study is to compare the cumulative incidence of relapse between recipients of HLA one or two antigens mismatched related grafts, and HLA-identical sibling donor transplantation, using myeloablative and non-myeloablative conditioning. Cord blood transplants will be analyzed separately comparing HLA-identical cord blood transplants with one, two or three antigens mismatched cord blood transplants. Secondary objectives include comparison of transplant-related mortality and disease-free survival between HLA-mismatched and HLA-identical sibling donor transplantation. If sufficient data, cord blood transplants will also be analyzed. Relapse and TRM will be calculated according to the cumulative incidence. In multivariate analysis, cumulative incidence estimates of relapse and TRM will be estimated using the pseudo-value approach of Klein with a logistic link function. 10 A forward stepwise regression model using a generalized linear model for the pseudo-values will be employed. The influence of acute graft-versus-host disease and chronic graft-versus-host disease on development of relapse in different donor sources will be evaluated by treating occurrence of the graft-versus-host as a time-dependent variable. Probability of disease-free survival will be calculated using the Kaplan-Meier estimator with variance estimated by Greenwood s formula. Comparison of survival curves will be done using the log-rank test. To study the effect of other risk-factors on disease-free survival, proportional hazards model will be performed with acute and chronic graft-versus-host disease treated as time dependent variables to evaluate their effects on outcomes. The proportional hazards assumption will be assessed for each variable using time-dependent or graphical approaches. Forward stepwise regression will be used to build models for the prognostic factors under consideration. Two-way interactions will be checked between all significant variables in the model. SAS will be used in all analyses. References: 1. Truitt RL, Johnson BD, McCabe CM, Weiler MB. Graft-versus-leukemia. In: Ferrara JLM, Deeg HJ, Burakoff S, editors. Graft vs Host Disease, Second Edition. 2 ed. New York: Marcel Dekker, Inc.; p Weiden PL, Flournoy N, Thomas ED, Prentice R, Fefer A, Buckner CD, et al. Antileukemic effect of graft-versus-host disease in human recipients of allogeneic-marrow grafts. N Engl J Med. 1979;300(19): Weiden PL, Sullivan KM, Flournoy N, Storb R, Thomas ED. Antileukemic effect of chronic graft-versus-host disease: contribution to improved survival after allogeneic marrow transplantation. N Engl J Med. 1981;304(25): Horowitz MM, Gale RP, Sondel PM, Goldman JM, Kersey J, Kolb HJ, et al. Graft-versusleukemia reactions after bone marrow transplantation. Blood. 1990;75(3): Ringden O, Karlsson H, Olsson R, Omazic B, Uhlin M. The allogeneic graft-versus-cancer effect. Br J Haematol. 2009;147(5): Gale RP, Horowitz MM, Ash RC, Champlin RE, Goldman JM, Rimm AA, et al. Identical-twin bone marrow transplants for leukemia. Ann Intern Med. 1994;120(8): Goldman JM, Gale RP, Horowitz MM, Biggs JC, Champlin RE, Gluckman E, et al. Bone marrow transplantation for chronic myelogenous leukemia in chronic phase. Increased risk for relapse associated with T-cell depletion. Ann Intern Med. 1988;108(6): Marmont AM, Horowitz MM, Gale RP, Sobocinski K, Ash RC, van Bekkum DW, et al. T-cell depletion of HLA-identical transplants in leukemia. Blood. 1991;78(8):

42 Not for publication or presentation Attachment 4 9. Ringden O, Pavletic SZ, Anasetti C, Barrett AJ, Wang T, Wang D, et al. The graft-versusleukemia effect using matched unrelated donors is not superior to HLA-identical siblings for hematopoietic stem cell transplantation. Blood. 2009;113(13): Klein JP. Modelling competing risks in cancer studies. Statistics in medicine. 2006;25(6): Epub 2006/01/26. 42

43 Not for publication or presentation Attachment 4 Characteristics of first transplants for patients with AML, ALL and CML between 1995 and 2011 by stem cell source a Mismatched Single Unit Cord related donors Blood (Unrelated) Characteristics of patients N (%) N (%) Number of patients Number of centers Age, median (range), years 33 (<1-73) 8 (<1-72) Age at transplant 0-9 y 67 (12) 372 (57) y 94 (16) 148 (23) y 92 (16) 30 ( 5) y 111 (19) 29 ( 4) y 112 (19) 34 ( 5) 50 and older 103 (18) 44 ( 7) Recipient race Caucasian 386 (67) 383 (58) African American 39 ( 7) 81 (12) Asian/Pacific Islander 70 (12) 27 ( 4) Hispanic 52 ( 9) 149 (23) Native American 2 (<1) 4 ( 1) Other/Multiple/Declined/Unknown 30 ( 5) 13 ( 2) Male sex 347 (60) 338 (51) Karnofsky prior to transplant > (71) 488 (78) Disease at transplant AML 282 (49) 346 (53) ALL 163 (28) 299 (46) CML 134 (23) 12 ( 2) Disease status at transplant Early 403 (70) 499 (76) Intermediate 36 ( 6) 53 ( 8) Advanced 140 (24) 105 (16) Graft type Bone marrow 317 (55) 0 Peripheral blood 262 (45) 0 Umbilical cord blood (100) a - Data is not CAP-modeled. 43

44 Not for publication or presentation Attachment 4 Continued. Mismatched related donors Single Unit Cord Blood (Unrelated) Characteristics of patients N (%) N (%) Number of patients Conditioning regimen Myeloablative 461 (80) 576 (88) Reduced intensity 61 (11) 39 ( 6) Nonmyeloablative 57 (10) 42 ( 6) GVHD prophylaxis FK506 + (MTX or MMF or Steroids) other 113 (20) 189 (29) FK506 other 16 ( 3) 31 ( 5) CsA + MTX other 259 (45) 54 ( 8) CsA other (No MTX) 94 (16) 361 (55) MMF other 0 0 MTX other (No CsA) 0 3 (<1) T-cell depletion 69 (12) 0 Other 28 ( 5) 19 ( 3) In vivo T-cell depletion No 353 (61) 272 (41) Yes 226 (39) 385 (59) Donor/recipient sex match Male/Male 193 (33) 165 (27) Male/Female 123 (21) 162 (26) Female/Male 153 (26) 156 (25) Female/Female 109 (19) 138 (22) Donor/recipient CMV match Negative/Negative 158 (27) 2 (<1) Negative/Positive 115 (20) 4 ( 1) Positive/Negative 53 ( 9) 0 Positive/Positive 220 (38) 3 (<1) Unknown 33 ( 6) 648 (99) Donor age, median (range), years 33 (<1-79) N/A Donor age (10) N/A (13) N/A (18) N/A (23) N/A (21) N/A 50 and older 84 (15) N/A To Be Determined 4 ( 1) N/A 44

45 Not for publication or presentation Attachment 4 Continued. Mismatched related donors Single Unit Cord Blood (Unrelated) Characteristics of patients N (%) N (%) Number of patients HLA Matching for HLA-A, -B and DRB1 6/ (16) 5/6 336 (58) 287 (44) 4/6 159 (27) 260 (39) 3/6 0 5 ( 1) TBD 84 (15) 0 Year of transplant (13) (11) (10) ( 5) ( 7) ( 6) 4 ( 1) ( 7) 29 ( 4) ( 4) 23 ( 4) ( 5) 44 ( 7) ( 4) 44 ( 7) ( 6) 66 (10) ( 5) 97 (15) ( 2) 83 (13) ( 8) 80 (12) ( 5) 103 (16) ( 1) 77 (12) ( 1) Median follow-up of survivors, mo (range) 59 (1-181) 38 (2-122) 45

46 Not for publication or presentation Attachment 5 Study Proposal Study Title: Impact of Unrelated Donor HLA-Mismatch in Reduced-Intensity Conditioning Allogeneic Hematopoietic Stem Cell Transplantation Outcomes John Koreth, MBBS, PhD, Dana-Farber Cancer Institute, Boston, MA Study Objectives: 1. Outcomes (OS, DFS, NRM, acute GVHD) of HLA-matched versus HLA-mismatched unrelated donor RIC HSCT. 2. Impact of individual locus mismatch at HLA-A, -B, -C, -DRB1 in unrelated donor RIC HSCT. 3. Interaction between disease-risk and HLA-mismatch on RIC HSCT survival. Scientific Justification: The impact of HLA mismatch has been defined in myeloablative allogeneic hematopoietic stem cell transplantation, but data on reduced intensity conditioning transplantation remains limited, and is suitable for a dedicated CIBMTR analysis. In myeloablative bone marrow (BM) transplantation, the Japan Marrow Donor Program (JMDP) reported higher mortality with allele or antigen-level mismatch at HLA-A and/or -B, but not HLA-C or -DRB1, 1,2 though its applicability to non-japanese populations remained in question. A retrospective study from the CIBMTR compared HLA-mismatched (6/8, 7/8) versus 8/8 HLA-matched (HLA-A, -B, -C, -DRB1) unrelated donor transplantation, and documented worse non-relapse mortality (NRM), acute graft-versushost disease (GVHD), and overall and disease-free survival (OS, DFS) with increasing degree of antigen and/or allele-level HLA-mismatch. 3 French BM registry data reported that mismatch at HLA-DQB1 was also associated with significant decrement in survival. 4 Reports of the International Histocompatibility Working Group (IHWG) identified impaired survival of HLA-DQB1 mismatch as well, while HLA- DPB1 mismatch was associated with increased acute GVHD (p<0.001) but reduced relapse risk (p=0.01). 5,6 Disease-risk appears a modifier, with greater survival impairment of an HLA-mismatch seen in low- versus intermediate- or high-risk disease (p<0.0001; p=0.02; p=0.43, respectively). 5 Similar outcome impairment is reported for 1-antigen HLA-mismatched (HLA-A, -B, -C, -DRB1) unrelated donor peripheral blood stem cell (PBSC) transplantation. 7 BM and PBSC HLA-mismatch both appear to have a similar deleterious impact on HSCT. 8 The impact of HLA-mismatch in reduced-intensity conditioning (RIC) transplantation is less well defined. Limited data document impaired outcomes in 1-2 locus HLA-mismatched RIC transplantation, with high rates of acute GVHD, NRM and impaired survival. In a retrospective report of the Dana-Farber Cancer Institute, HLA-C allele/antigen disparity was associated with increased grade II-IV and III-IV acute GVHD, (46 vs. 26%, p=0.04; 33 vs. 12%, p=0.01), increased NRM (48 vs. 16%; p=0.0001), and worse 2-year OS (30 vs. 51%, p=0.008). 9 A prospective phase I/II study of T-replete 1-2 locus HLAmismatched RIC HSCT utilizing PBSC grafts and calcineurin-inhibitor (CNI) based GVHD prophylaxis from the Fred Hutchinson Cancer Research Center described similar poor outcomes, with grade II-IV acute GVHD of 69%, grade III-IV acute GVHD of 26%, NRM of 47% and 2-year OS of 29%. 10 A CIBMTR analysis of HLA-mismatch in unrelated donor PBSC grafting undertaken between included a subset of 673 RIC transplantations, and documented impaired survival of HLA-C antigen mismatch compared to 8/8 HLA-matched RIC (RR 1.40, 95% CI , p=0.04). 7 However, allele mismatch and other loci were not identified as relevant, possibly due to limited sample size. The interaction of HLA-mismatch with disease-risk in RIC transplantation outcomes, if any, remains to be determined. 46

47 Not for publication or presentation Attachment 5 Quantifying the impact of HLA-mismatch in unrelated donor RIC transplantation would help in optimizing donor selection, especially if a differential impact is noted for HLA-mismatch in high-risk versus low-risk disease survival. It would also constitute a baseline comparator for novel regimens in HLA-mismatched RIC transplantation. Given that ~1100 unrelated donor RIC transplants have accrued annually at CIBMTR post-2006 (CIBMTR summary slides 2010), a larger analysis focused solely on RIC HSCT appears feasible, relevant and timely. Study Population: - RIC HSCT recipients - Hematologic malignancy diagnosis restricted to: AML, ALL, MDS, CML (could consider all hematologic malignancies, but may add heterogeneity) - Transplantation in Unrelated donors with high-resolution typing at HLA-A, -B, -C, -DRB1 (additional loci: -DP and -DQ requested, but not required) - PBSC or BM grafts - T-replete grafts (ATG/Campath etc. conditioning acceptable). Variables to be Analyzed: Patient variables: - Age - Sex - Performance status - Donor/recipient sex match - Donor/recipient CMV status - Date of transplant - Date of last follow up or death - Disease status at last follow up Disease variables: - Diagnosis - Disease-risk - High-risk: acute leukemia or CML beyond CR1/CP1, MDS other than de-novo RA/RARS at transplant - Low-risk: the remainder HLA-match variables: - High resolution typing at HLA-A, -B, -C, -DRB1 (-DPB1 and -DQB1/A1 if available) RIC regimen variables: - Conditioning (TBI-based, other) - GVHD prophylaxis (CNI-based, other) - T-cell depletion (in-vivo with ATG/Campath etc, none) Outcome variables: - NRM - Relapse/progression - Acute GVHD (grade II-IV, III-IV) - OS - DFS Sample Requirements: No samples are required. 47

48 Not for publication or presentation Attachment 5 Study Design: This is proposed as a retrospective analysis of RIC HSCT outcomes stratified by HLA-mismatch. Probabilities for OS and PFS will be calculated via the Kaplan-Meier estimator. Survival curves will be compared by the log-rank test. Death will be considered a competing risk for all endpoints except OS and PFS. Relapse and NRM will be considered competing risks. The association between number and type of HLA mismatches would be evaluated by multivariable models adjusting for significant clinical covariates. Covariates tested would include disease, disease risk (low- vs. high-risk), performance status, patient age, donor age, donor/recipient sex match (F/M vs. other), donor/recipient CMV match, graft source (PBSC vs. BM), T-cell depletion (ATG/Campath vs. none), GVHD regimen (CNI-based vs. other), and year of transplantation. Similar to the 2007 CIBMTR/NMDP survey 3, the analysis would compare subgroups of HLA-mismatched pairs with 8/8 HLA-matched pairs. P values would be adjusted for multiple comparisons. References: 1. Sasazuki T, Juji T, Morishima Y, et al. Effect of matching of class I HLA alleles on clinical outcome after transplantation of hematopoietic stem cells from an unrelated donor. Japan Marrow Donor Program. N Engl J Med. Oct ;339(17): Morishima Y, Sasazuki T, Inoko H, et al. The clinical significance of human leukocyte antigen (HLA) allele compatibility in patients receiving a marrow transplant from serologically HLA- A, HLA-B, and HLA-DR matched unrelated donors. Blood. Jun ;99(11): Lee SJ, Klein J, Haagenson M, et al. High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood. Dec ;110(13): Loiseau P, Busson M, Balere ML, et al. HLA Association with hematopoietic stem cell transplantation outcome: the number of mismatches at HLA-A, -B, -C, -DRB1, or -DQB1 is strongly associated with overall survival. Biol Blood Marrow Transplant. Aug 2007;13(8): Petersdorf EW, Gooley T, Malkki M, Horowitz M. Clinical significance of donor-recipient HLA matching on survival after myeloablative hematopoietic cell transplantation from unrelated donors. Tissue Antigens. Apr 2007;69 Suppl 1: Shaw BE, Gooley TA, Malkki M, et al. The importance of HLA-DPB1 in unrelated donor hematopoietic cell transplantation. Blood. Dec ;110(13): Woolfrey A, Klein JP, Haagenson M, et al. HLA-C antigen mismatch is associated with worse outcome in unrelated donor peripheral blood stem cell transplantation. Biol Blood Marrow Transplant. Jun 2011;17(6): Eapen M, Rocha V, Sanz G, et al. Effect of graft source on unrelated donor haemopoietic stemcell transplantation in adults with acute leukaemia: a retrospective analysis. Lancet Oncol. Jul 2010;11(7): Ho VT, Kim HT, Liney D, et al. HLA-C mismatch is associated with inferior survival after unrelated donor non-myeloablative hematopoietic stem cell transplantation. Bone Marrow Transplant. May 2006;37(9): Nakamae H, Storer BE, Storb R, et al. Low-dose total body irradiation and fludarabine conditioning for HLA class I-mismatched donor stem cell transplantation and immunologic recovery in patients with hematologic malignancies: a multicenter trial. Biol Blood Marrow Transplant. Mar 2010;16(3):

49 Not for publication or presentation Attachment 5 Characteristics of first transplants for patients with AML, ALL, CML and MDS and are highresolution typed unrelated NMDP donor cases receiving reduced intensity or nonmyeloablative conditioning regimens a Characteristics of patients N Eval N (%) Number of patients 3380 CRF patients 2387 (71) TED patients 993 (29) Number of centers 172 Age, median (range), years (<1-80) Age at transplant y 37 ( 1) y 100 ( 3) y 153 ( 5) y 206 ( 6) y 389 (12) 50 and older 2495 (74) Recipient race 3380 Caucasian 3016 (89) African American 105 ( 3) Asian/Pacific Islander 52 ( 2) Hispanic 130 ( 4) Native American 8 (<1) Other/Multiple/Declined/Unknown 69 ( 2) Male sex (58) Karnofsky prior to transplant > 90 (CRF only; TED TBD) (61) Disease at transplant 3380 AML 1909 (56) ALL 272 ( 8) CML 226 ( 7) MDS 973 (29) Disease status at transplant 3380 Early 1644 (49) Intermediate 76 ( 2) Advanced 550 (16) Other/TED TBD 1110 (33) Graft type 3380 Bone marrow 545 (16) Peripheral blood 2835 (84) a - Data is not CAP-modeled. 49

50 Not for publication or presentation Attachment 5 Continued. Characteristics of patients N Eval N (%) HLA matching for HLA-A, -B, -C and DRB / (76) 7/8 710 (21) 6/8 106 ( 3) Conditioning regimen 3380 Reduced intensity 1887 (56) Nonmyeloablative 727 (22) TBD/TED (Claim to be RIC/nonmyeloablative) 766 (22) GVHD prophylaxis 3380 FK506 + (MTX or MMF or Steroids) other 1986 (59) FK506 other 282 ( 8) CsA + MTX other 277 ( 8) CsA other (No MTX) 687 (20) MMF other 37 ( 1) MTX other (No CsA) 14 (<1) T-cell depletion 0 Other 97 ( 3) In vivo T-cell depletion 3380 No 1974 (58) Yes 1406 (42) Donor/recipient sex match 3289 Male/Male 1351 (41) Male/Female 845 (26) Female/Male 544 (17) Female/Female 549 (17) Donor/recipient CMV match 3380 Negative/Negative 682 (20) Negative/Positive 907 (27) Positive/Negative 281 ( 8) Positive/Positive 633 (19) Unknown/TBD 877 (26) Donor age, median (range), years (18-61) Donor age ( 1) (14) (16) (12) 50 and older 135 ( 4) To Be Determined/(TED does not give donor age) 1811 (54) 50

51 Not for publication or presentation Attachment 5 Continued. Characteristics of patients N Eval N (%) High-resolution HLA matching for HLA-DQB Fully allele-level matched 2908 (91) Single mismatch (allele or antigen) 287 ( 9) Double mismatch (allele and/or antigen) 8 (<1) High-resolution HLA matching for HLA-DPB1 700 Fully allele-level matched 107 (15) Single mismatch (allele or antigen) 397 (57) Double mismatch (allele and/or antigen) 196 (28) Year of transplant ( 1) ( 2) ( 2) ( 2) ( 5) ( 7) ( 8) (10) (12) (12) (16) (16) ( 9) Median follow-up of survivors, mo (range) ( ) 51

52 Not for publication or presentation Attachment 5 High-resolution typed HLA distribution of mismatching for AML, ALL, CML or MDS 8/8 7/8 6/8 Characteristics of patients N (%) N (%) N (%) HLA matching for HLA-A, -B, -C and DRB1 Fully matched 2564 (100) 0 0 Single MM at HLA-A (33) 0 Single MM at HLA-B (14) 0 Single MM at HLA-C (40) 0 Single MM at HLA-DRB (13) 0 1 MM at A, 1 MM at B ( 5) 1 MM at A, 1 MM at C (17) 1 MM at A, 1 MM at DRB ( 3) 1 MM at B, 1 MM at C (45) 1 MM at B, 1 MM at DRB ( 8) 2 MM at C (11) 1 MM at C, 1 MM at DRB (10) 2 MM at DRB ( 1) 52

53 Not for publication or presentation Attachment 5 Characteristics of first transplants for patients with Lymphoma or CLL and are high-resolution typed unrelated NMDP donor cases receiving reduced intensity or nonmyeloablative conditioning regimens a Characteristics of patients N Eval N (%) Number of patients 1110 CRF patients 799 (72) TED patients 311 (28) Number of centers 123 Age, median (range), years (8-75) Age at transplant y 1 (<1) y 16 ( 1) y 41 ( 4) y 84 ( 8) y 190 (17) 50 and older 778 (70) Recipient race 1110 Caucasian 992 (89) African American 39 ( 4) Asian/Pacific Islander 11 ( 1) Hispanic 45 ( 4) Native American 0 Other/Multiple/Declined/Unknown 23 ( 2) Male sex (67) Karnofsky prior to transplant > 90 (CRF Only/ TED TBD) (67) Disease at transplant 1110 NHL 764 (69) Hodgkin s Lymphoma 47 ( 4) CLL 299 (27) Graft type 1110 Bone marrow 176 (16) Peripheral blood 934 (84) a - Data is not CAP-modeled. 53

54 Not for publication or presentation Attachment 5 Continued. Characteristics of patients N Eval N (%) HLA matching for HLA-A, -B, -C and DRB /8 858 (77) 7/8 218 (20) 6/8 34 ( 3) Conditioning regimen 1110 Reduced intensity 461 (42) Nonmyeloablative 413 (37) TBD/TED cases 236 (21) GVHD prophylaxis 1110 FK506 + (MTX or MMF or Steroids) other 695 (63) FK506 other 100 ( 9) CsA + MTX other 53 ( 5) CsA other (No MTX) 221 (20) MMF other 15 ( 1) MTX other (No CsA) 3 (<1) T-cell depletion 0 Other 23 ( 2) In vivo T-cell depletion 1110 No 640 (58) Yes 470 (42) Donor/recipient sex match 1094 Male/Male 530 (48) Male/Female 230 (21) Female/Male 204 (19) Female/Female 130 (12) Donor/recipient CMV match 1110 Negative/Negative 296 (27) Negative/Positive 317 (29) Positive/Negative 104 ( 9) Positive/Positive 228 (21) Unknown/TBD 165 (15) Donor age, median (range), years (18-60) Donor age ( 1) (19) (22) (14) 50 and older 45 ( 4) To Be Determined/(TED cases do not have donor age) 453 (41) 54

55 Not for publication or presentation Attachment 5 Continued. Characteristics of patients N Eval N (%) High-resolution HLA matching for HLA-DQB Fully allele-level matched 987 (92) Single mismatch (allele or antigen) 78 ( 7) Double mismatch (allele and/or antigen) 4 (<1) High-resolution HLA matching for HLA-DPB1 270 Fully allele-level matched 33 (12) Single mismatch (allele or antigen) 147 (54) Double mismatch (allele and/or antigen) 90 (33) Year of transplant ( 1) ( 3) ( 3) ( 3) ( 8) ( 7) ( 9) (13) (13) ( 9) (13) (11) ( 6) Median follow-up of survivors, mo (range) ( ) 55

56 Not for publication or presentation Attachment 5 High-resolution typed HLA distribution of mismatching for Lymphoma or CLL 8/8 7/8 6/8 Characteristics of patients N (%) N (%) N (%) HLA matching for HLA-A, -B, -C and DRB1 Fully matched 858 (100) 0 0 Single MM at HLA-A 0 69 (32) 0 Single MM at HLA-B 0 31 (14) 0 Single MM at HLA-C 0 93 (43) 0 Single MM at HLA-DRB (11) 0 1 MM at A, 1 MM at B ( 6) 1 MM at A, 1 MM at C (24) 1 MM at A, 1 MM at DRB ( 3) 1 MM at B, 1 MM at C (47) 1 MM at B, 1 MM at DRB ( 6) 2 MM at C (12) 1 MM at C, 1 MM at DRB ( 3) 56

57 Not for publication or presentation Attachment 6 Study Proposal Study Title: Impact of HLA mismatch and patient/donor non-hla variables on transplantation outcome: An updated analysis utilizing NMDP high-resolution typing project data Joseph Pidala, MD, MS, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, USA Claudio Anasetti, MD, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, USA Study Objectives: 1. Determine the impact of allele and antigen-level HLA mismatch among donor-recipient pairs among the current NMDP high-resolution typing project on major outcomes including overall survival, disease-free survival, treatment related mortality (TRM), primary disease relapse, and incidence of acute and chronic graft vs. host disease (GVHD) 2. Study the impact of donor and recipient non-hla variables on transplantation outcome Scientific Justification: As the majority of potential HCT recipients will not have an HLA identical sibling, unrelated volunteer donors serve a vital role in allowing successful HCT. In the conduct of an unrelated donor search in the US, the identification of potential unrelated donors is facilitated by the National Marrow Donor Program (NMDP). 1 Several studies have demonstrated that high resolution molecular typing identifies allele level disparities in donor-recipient pairs that are otherwise considered completely matched. In addition, large unrelated donor registry studies have demonstrated that HLA loci allelic disparities between donor and recipient predict inferior transplantation outcome. 2-8 In the largest of these studies to date, Lee, et al conducted an analysis of NMDP data from 3,857 donorrecipient pairs typed with high-resolution DNA matching at HLA-A, -B, -C, -DRB1, -DQB1, -DQA1, - DPB1, and -DPA1 alleles. Single allele or antigen-level mismatches were associated with greater risk of agvhd, mortality, and worsened overall survival, and multiple loci mismatches further increased these risks. 4 This work has demonstrated the adverse impact of HLA loci allele disparity, as well as the relative importance of disparity at specific loci. A number of factors specific to this analysis suggest the importance of a follow-up analysis utilizing the currently available total data set of the NMDP high-resolution HLA typing project: First, this analysis encompassed data between 1988 and 2003, with a study population of n = Additional data accrued since that time would provide additional power for analyses on the impact of HLA mismatch, as well as other patient and donor factors on transplantation outcome. Several findings with small, but nonsignificant impact on outcomes in this analysis (e.g. impact of HLA-B allele or antigen level mismatch on survival and disease-free survival; impact of additional HLA-DQ or HLA-DP mismatch among 6-7/8 matched pairs; impact of HLA-DP mismatch on treatment related mortality (TRM) and relapse) may be afforded clarity through an analysis with larger total dataset. Second, the majority (94%) of transplants represented in this data set were bone marrow; incorporation of more current data would facilitate greater understanding of the impact of stem cell source on outcome, given increased utilization of peripheral blood mobilized products. Next, greater than 80% of conditioning regimens in this analysis were total body irradiation based; thus, there is limited representation of chemotherapy-only myeloablative conditioning regimens increasingly utilized in current transplantation practice. As well, while this analysis confirmed the effect of non-hla factors including disease, disease status, patient age and race, and donor/recipient CMV match, there was no detected impact of donor age on survival, in contradistinction to prior reports; 9 additional power afforded by a larger data set may bring clarity on this point. 57

58 Not for publication or presentation Attachment 6 Study Population: The study population will include all adult and pediatric patients who underwent a myeloablative first unrelated bone marrow or peripheral blood stem cell HCT for hematological malignancy (AML, ALL, CML and MDS) between 1988 and the 2011, provided minimum one year follow up time at time of analysis. Patient and donor will have complete high resolution typing for HLA-A, B, C, DRB1, DQA1, DQB1, DPA1, and DPB1. Primary outcomes: - Overall survival: Time to death from any cause. Events will be summarized by a survival curve. Cases will be analyzed at the time of last follow-up. - Treatment-related mortality: Death in continuous remission of primary disease. Events will be summarized by the cumulative incidence estimate with relapse as a competing risk. - Relapse: Development of clinical relapse of the primary disease as defined by the CIBMTR. The event will be summarized by the cumulative incidence estimate and patients analyzed at last follow-up. Death is a competing risk. - Acute GVHD: Development of Grades II-IV, and III-IV acute GVHD using the Glucksberg or Consensus system. Event will be summarized by the cumulative incidence estimate. Cases will be analyzed at time of last follow-up. Death is a competing risk. Second transplant is a censoring event. - Chronic GVHD: Development of limited or extensive chronic GVHD. Events will be summarized by the cumulative incidence estimate. Cases will be analyzed at time of last followup. Death is a competing risk. Second transplant is a censoring event. Variables to be Analyzed: Primary effect to be tested: - Impact of single allele or antigen-level mismatch on HCT outcomes - Study outcome according to 8/8 vs. 7/8 vs. 6/8 Secondary effects: - Impact of mismatch at each HLA locus on outcomes - Comparison of allele vs. antigen-level mismatch - Impact of multiple mismatches on outcome Other Variables: Patient-related (at time of transplant): - Age: in decades (< 10, 10-19, 20-29, 30-39, 40-49, 50) - Race (White vs. Hispanic vs. Black vs. Other) - Karnofsky score at transplant: < 90 vs Disease-Related: - Disease at transplant: AML, ALL, CML and MDS - Disease stage at transplant: early vs. intermediate vs. advanced vs. unknown - Time from diagnosis to transplant for CML and MDS Transplant-Related: - Source of stem cells: marrow (BM) vs. peripheral blood stem cells (PB) - Donor age: in decades (18-29, 30-39, 40-49, 50) - Donor parity (male or not parous vs. parous) - Year of transplant - Gender match: M-M vs. M-F vs. F-M vs. F-F - Donor/recipient CMV status: -/- vs. -/+ vs. +/- vs. +/+ vs. Unknown - Conditioning regimen: TBI vs. others - GVHD prophylaxis - in vivo T cell depletion (ATG or Campath) - Transplant center 58

59 Not for publication or presentation Attachment 6 Sample Requirements: No samples are required. Study Design: We will examine the independent impact of HLA disparity on the outcomes of overall survival, diseasefree survival, treatment related mortality, primary disease relapse, acute and chronic GVHD. To summarize the characteristics of the dataset, descriptive tables of patient-, disease and transplant-related factors will be reported. 1. For discrete factors, the number of cases and their respective percentages will be calculated. Chi- Square tests will be used to compare discrete factors between the HLA matched vs. mismatched groups. 2. For continuous factors, the median and ranges will be calculated. The Kruskal-Wallis test will be used to compare the continuous factors between the HLA matched vs. mismatched groups. Probabilities for overall survival and disease-free survival will be calculated using the Kaplan-Meier estimator with variance estimated by Greenwood's formula. Comparison of survival curves will be done using the log-rank test. Values for other outcomes will be calculated according to cumulative incidence using a linear approximation to estimate the variance. A p-value < 0.01 will be considered significant. Multivariate analyses will be performed using the proportional hazards model. Comparison will be between pairs mismatched at specific HLA loci to HLA-matched pairs. All clinical variables will be tested for the affirmation of the proportional hazards assumption. Factors violating the proportional hazards assumption will be adjusted through stratification. Then a stepwise model building approach will be used in developing models for the primary outcomes. Interactions among HLA disparity and the clinical variables will be tested. References: 1. Karanes C, Nelson GO, Chitphakdithai P, Agura E, Ballen KK, Bolan CD, Porter DL, Uberti JP, King RJ, Confer DL. Twenty years of unrelated donor hematopoietic cell transplantation for adult recipients facilitated by the National Marrow Donor Program. Biol Blood Marrow Transplant. 2008;14: Davies SM, Kollman C, Anasetti C, Antin JH, Gajewski J, Casper JT, Nademanee A, Noreen H, King R, Confer D, Kernan NA. Engraftment and survival after unrelated-donor bone marrow transplantation: a report from the national marrow donor program. Blood. 2000;96: Flomenberg N, Baxter-Lowe LA, Confer D, Fernandez-Vina M, Filipovich A, Horowitz M, Hurley C, Kollman C, Anasetti C, Noreen H, Begovich A, Hildebrand W, Petersdorf E, Schmeckpeper B, Setterholm M, Trachtenberg E, Williams T, Yunis E, Weisdorf D. Impact of HLA class I and class II high-resolution matching on outcomes of unrelated donor bone marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplantation outcome. Blood. 2004;104: Lee SJ, Klein J, Haagenson M, Baxter-Lowe LA, Confer DL, Eapen M, Fernandez-Vina M, Flomenberg N, Horowitz M, Hurley CK, Noreen H, Oudshoorn M, Petersdorf E, Setterholm M, Spellman S, Weisdorf D, Williams TM, Anasetti C. High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood. 2007;110: Morishima Y, Sasazuki T, Inoko H, Juji T, Akaza T, Yamamoto K, Ishikawa Y, Kato S, Sao H, Sakamaki H, Kawa K, Hamajima N, Asano S, Kodera Y. The clinical significance of human leukocyte antigen (HLA) allele compatibility in patients receiving a marrow transplant from 59

60 Not for publication or presentation Attachment 6 serologically HLA-A, HLA-B, and HLA-DR matched unrelated donors. Blood. 2002;99: Petersdorf EW, Longton GM, Anasetti C, Mickelson EM, McKinney SK, Smith AG, Martin PJ, Hansen JA. Association of HLA-C disparity with graft failure after marrow transplantation from unrelated donors. Blood. 1997;89: Sasazuki T, Juji T, Morishima Y, Kinukawa N, Kashiwabara H, Inoko H, Yoshida T, Kimura A, Akaza T, Kamikawaji N, Kodera Y, Takaku F. Effect of matching of class I HLA alleles on clinical outcome after transplantation of hematopoietic stem cells from an unrelated donor. Japan Marrow Donor Program. N Engl J Med. 1998;339: Speiser DE, Tiercy JM, Rufer N, Grundschober C, Gratwohl A, Chapuis B, Helg C, Loliger CC, Siren MK, Roosnek E, Jeannet M. High resolution HLA matching associated with decreased mortality after unrelated bone marrow transplantation. Blood. 1996;87: Kollman C, Howe CW, Anasetti C, Antin JH, Davies SM, Filipovich AH, Hegland J, Kamani N, Kernan NA, King R, Ratanatharathorn V, Weisdorf D, Confer DL. Donor characteristics as risk factors in recipients after transplantation of bone marrow from unrelated donors: the effect of donor age. Blood. 2001;98:

61 Not for publication or presentation Attachment 6 Characteristics of first transplants for patients with AML, ALL, CML and MDS and are highresolution typed for HLA-A, -B, -C and DRB1 with unrelated NMDP donor cases receiving myeloablative conditioning regimens a Characteristics of patients N Eval N (%) Number of patients CRF patients 9503 (76) TED patients 3060 (24) Number of centers 228 Age, median (range), years (<1-74) Age at transplant y 1234 (10) y 1616 (13) y 1966 (16) y 2223 (18) y 2619 (21) 50 and older 2905 (23) Recipient race Caucasian (83) African American 619 ( 5) Asian/Pacific Islander 297 ( 2) Hispanic 990 ( 8) Native American 51 (<1) Other/Multiple/Declined/Unknown 228 ( 2) Male sex (56) Karnofsky prior to transplant > (72) Disease at transplant AML 4973 (40) ALL 3209 (26) CML 2433 (19) MDS 1948 (16) Disease status at transplant (CRF Only) 9503 Early 6575 (69) Intermediate 598 ( 6) Advanced 2089 (22) Other 241 ( 3) Graft type Bone marrow 6968 (55) Peripheral blood 5595 (45) a - Data is not CAP-modeled. 61

62 Not for publication or presentation Attachment 6 Continued. Characteristics of patients N Eval N (%) HLA matching for HLA-A, -B, -C and DRB / (62) 7/ (25) 6/ ( 9) 1/8-5/8 580 ( 5) GVHD prophylaxis FK506 + (MTX or MMF or Steroids) other 5659 (45) FK506 other 499 ( 4) CsA + MTX other 4189 (33) CsA other (No MTX) 464 ( 4) MMF other 15 (<1) MTX other (No CsA) 58 (<1) T-cell depletion 1338 (11) Other 341 ( 3) In vivo T-cell depletion No 8942 (71) Yes 3621 (29) Donor/recipient sex match Male/Male 4655 (38) Male/Female 3168 (26) Female/Male 2304 (19) Female/Female 2281 (18) Donor/recipient CMV match Negative/Negative 3441 (27) Negative/Positive 3618 (29) Positive/Negative 1522 (12) Positive/Positive 2405 (19) Unknown/TBD 1577 (13) Donor age, median (range), years (18-61) Donor age ( 1) (18) (24) (17) 50 and older 642 ( 5) To Be Determined/(TED does not give donor age) 4404 (35) 62

63 Not for publication or presentation Attachment 6 Continued. Characteristics of patients N Eval N (%) High-resolution HLA matching for HLA-DQB Fully allele-level matched (88) Single mismatch (allele or antigen) 1390 (11) Double mismatch (allele and/or antigen) 63 ( 1) High-resolution HLA matching for HLA-DPB Fully allele-level matched 834 (14) Single mismatch (allele or antigen) 3151 (54) Double mismatch (allele and/or antigen) 1880 (32) Year of transplant (<1) (<1) ( 1) ( 1) ( 2) ( 2) ( 3) ( 3) ( 3) ( 4) ( 3) ( 4) ( 4) ( 4) ( 3) ( 4) ( 6) ( 7) ( 8) ( 7) ( 8) ( 9) ( 8) ( 5) Median follow-up of survivors, mo (range) (<1-265) 63

64 Not for publication or presentation Attachment 6 High-resolution typed HLA distribution of mismatching for AML, ALL, CML or MDS 8/8 7/8 6/8 1/8-5/8 Characteristics of patients N (%) N (%) N (%) N (%) HLA matching for HLA-A, -B, -C and DRB1 Fully matched 7741 (100) Single MM at HLA-A (33) 0 0 Single MM at HLA-B (14) 0 0 Single MM at HLA-C (40) 0 0 Single MM at HLA-DRB (13) MM at A ( 2) 0 1 MM at A, 1 MM at B ( 7) 0 1 MM at A, 1 MM at C (22) 0 1 MM at A, 1 MM at DRB ( 3) 0 2 MM at B ( 1) 0 1 MM at B, 1 MM at C (43) 0 1 MM at B, 1 MM at DRB ( 4) 0 2 MM at C ( 7) 0 1 MM at C, 1 MM at DRB (10) 0 2 MM at DRB ( 1) 0 5/ (69) 4/ (23) 3/ ( 6) 2/ ( 2) 1/ (<1) 64

65 Not for publication or presentation Attachment 7 Study Proposal Study Title: Determining the Effects of HLA-C KIR Ligand Expression on Outcomes of Unrelated Hematopoietic Stem Cell Transplantation Jeffrey M. Venstrom, MD, University of California, San Francisco, CA Study Objectives: 1. To determine the influence of recipient high expression and low expression HLA-C encoded KIR ligands on outcome of unrelated hematopoietic cell transplantation. 2. To determine the influence of donor high expression and low expression HLA-C encoded KIR ligands on outcome of unrelated hematopoietic cell transplantation. Scientific Justification: The HLA-C KIR ligands C1 and C2 have been identified as major factors affecting clinical outcomes in HCT, however the results are conflicting and based largely on small single center studies. 1-3 In a large cohort of AML patients receiving an unrelated HLA-compatible HCT, we recently observed that recipients homozygous for HLA-C2 had an increased risk of relapse compared to patients who are HLA- C1/C1 or HLA-C1/C2 [HR 1.35 ( ), p=.03], suggesting a gene-dose effect of HLA-C encoded KIR ligands on relapse following unrelated HCT. We hypothesize that this gene dose effect of the HLA-C KIR ligand is a biomarker for cell surface expression of the HLA-C protein, and that grouping HLA-C alleles based on predicted cell surface expression is a rational strategy for testing the influence of HLA-C KIR ligands on HCT outcomes. We predict that this new grouping strategy, based on cell surface expression, may also dictate permissive HLA-C mismatches. We aim to study this model in a larger cohort of HLA-mismatched patient-donor pairs. We will test these hypotheses using high resolution HLA genotyping of HCT donor/recipient pairs verified through the NMDP retrospective HLA typing program with clinical outcomes provided by the CIBMTR, and dividing patient/donor pairs based into one of four groups (table 1) based on the HLA-C KIR binding epitope (C1 or C2) and the predicted HLA-C allelic expression (C1-high, C1-low, C2-high, C2-low). 4 We aim to identify a donor selection strategy, based on high resolution donor-recipient HLA typing, that will optimize NK alloreactivity, identify permissive HLA-C mismatches, and improve clinical outcomes for URD HCT. Table 1. HLA-C alleles grouped according to KIR ligand assignment & cell surface expression HLA-C1 KIR ligand (Ser 77, Asn 80) High surface expression* *0801, *0802, *0804, *1202, *1203, *1601, *1604 Low surface expression* *0102, *0302, *0303, *0304, *0701, *0702, *0704, *1402 HLA-C2 KIR ligand (Asn 77, Lys 80) *0202, *0501, *0602, *1502, *1505, *1506, *1602 *0401, *1701 * predicted HLA-C expression based on a single base pair insertion/deletion at position 263 within the 3' untranslated region of HLA-C, the binding site for microrna hsa-mir-148a/b 4 65

66 Not for publication or presentation Attachment 7 Study Population: The study population will consist of patients receiving his/her first peripheral blood stem cell unrelated donor transplant for AML, ALL, CML or MDS. Transplant pairs must have high resolution genotyping for HLA-A, B, C, and DRB1 through the NMDP retrospective high resolution typing program. The population will exclude syngeneic transplants, and second transplants. The population will include any age, HLA-matched and mismatched, and non-myeloabaltive conditioning. Outcomes: - Overall survival: time to death from any cause. - Acute GVHD: grades II-IV and III-IV acute GVHD. - Chronic GVHD: limited, extensive, mild, moderate, severe chronic GVHD. - Disease relapse: disease recurrence is considered an event. - Disease-free survival: Time to relapse or death from any cause other than relapse. - Non-relapse mortality: death in continuous complete remission of primary disease. - Donor chimerism: percent donor/host chimerism. - Cause of death (if available). Variables to be Analyzed: Patient-related: - High resolution HLA genotyping - Age - Gender - Karnofsky performance score - Race - CMV status Disease-related: - Disease - Disease stage at transplant - Disease status prior to transplant Treatment-related: - Year of transplant - Donor age - Donor race - Donor gender - Donor CMV status - Conditioning regimen - Graft manipulation - GVHD prophylaxis - Time from diagnosis to transplant Sample Requirements: No biologic samples from the NMDP Repository are required. Study Design: Patients and donors will be divided into groups based on the algorithm in table 1. We will estimate overall and disease-free survival using the Kaplan-Meier method. Cases will be analyzed at the time of last follow-up, and for patients surviving in continuous complete remission, patients will be censored at date of last contact. Second transplant is a censoring event. We will use cumulative incidence estimates to summarize the probability of relapse, and we will consider death without relapse as a competing risk. Cox regression models will be used to examine the association of the different genetic groups with the hazard 66

67 Not for publication or presentation Attachment 7 of failure for the time-to-event outcomes (mortality, relapse, NRM). Logistic regression will be used to assess the association of genotype group with the probability of acute GvHD. Models will be adjusted for patient age, disease severity (low, intermediate, or high), patient cytomegalovirus (CMV) serostatus, T- cell depletion, conditioning [ablative with total body irradiation (TBI), ablative without TBI, or nonablative], HLA mismatch (10/10 vs. 9/10), and cytogenetics (no abnormalities, good risk, intermediate risk, or poor risk). The primary outcome for the study is overall survival, defined as time to death from any cause. The secondary outcomes include disease relapse, acute GVHD (grades II-IV and III-IV), chronic GVHD (mild, moderate, severe, limited, extensive), disease-free survival (time to relapse or death from any cause other than relapse), non-relapse mortality (death in continuous complete remission of primary disease, donor chimerism (where available, as percent donor/host). HLA-C alleles with unknown or indeterminate cell surface expression will be excluded from the initial analysis, with additional analyses planned to include additional HLA-C alleles using extrapolation of HLA-C expression from published data. HLAmatched and -mismatched donor-recipient pairs will be aggregated in the initial analysis, but divided based on HLA-matching in subsequent analyses. References: 1. Cook MA, Milligan DW, Fegan CD, et al: The impact of donor KIR and patient HLA-C genotypes on outcome following HLA-identical sibling hematopoietic stem cell transplantation for myeloid leukemia. Blood 103: , Clausen J, Wolf D, Petzer A, et al: Impact of natural killer cell dose and donor killer-cell immunoglobulin-like receptor (KIR) genotype on outcome following human leucocyte antigen identical haematopoietic stem cell transplantation. Clin Exp Immunol 148: , Schellekens J, Rozemuller EH, Petersen EJ, et al: Activating KIRs exert a crucial role on relapse and overall survival after HLA-identical sibling transplantation. Molecular Immunology 45: , Kulkarni S, Savan R, Qi Y, et al: Differential microrna regulation of HLA-C expression and its association with HIV control. Nature 472: ,

68 Not for publication or presentation Attachment 7 Characteristics of first transplants for patients with AML, ALL, CML and MDS and are high-resolution typed unrelated NMDP donor cases a Characteristics of patients N Eval N (%) Number of patients 9588 Number of centers 184 Age, median (range), years (<1-74) Age at transplant y 881 ( 9) y 1227 (13) y 1476 (15) y 1672 (17) y 1939 (20) 50 and older 2393 (25) Recipient race 9588 Caucasian 8173 (85) African American 446 ( 5) Asian/Pacific Islander 171 ( 2) Hispanic 660 ( 7) Native American 31 (<1) Other/Multiple/Declined/Unknown 107 ( 1) Male sex (57) Karnofsky prior to transplant > (71) Disease at transplant 9588 AML 3597 (38) ALL 2201 (23) CML 2279 (24) MDS 1511 (16) Disease status at transplant 9588 Early 6537 (68) Intermediate 618 ( 6) Advanced 2198 (23) Other 235 ( 2) Graft type 9588 Bone marrow 6036 (63) Peripheral blood 3552 (37) a - Data is not CAP-modeled. 68

69 Not for publication or presentation Attachment 7 Continued. Characteristics of patients N Eval N (%) HLA matching for HLA-A, -B, -C and DRB / (57) 7/ (25) 6/ (11) 1/8-5/8 599 ( 6) HLA-C matching 9588 Allele-level matched 7073 (74) Single mismatch (allele or antigen) 2245 (23) Double mismatch (allele and/or antigen) 270 ( 3) Conditioning regimen 9588 Myeloablative 7780 (81) Reduced intensity 1325 (14) Nonmyeloablative 483 ( 5) GVHD prophylaxis 9588 FK506 + (MTX or MMF or Steroids) other 3695 (39) FK506 other 284 ( 3) CsA + MTX other 3554 (37) CsA other (No MTX) 649 ( 7) MMF other 17 (<1) MTX other (No CsA) 58 ( 1) T-cell depletion 1201 (13) Other 130 ( 1) In-vivo T-cell depletion No 7069 (74) Yes 2519 (26) Donor/recipient sex match 9497 Male/Male 3561 (37) Male/Female 2297 (24) Female/Male 1836 (19) Female/Female 1803 (19) Donor/recipient CMV match 9588 Negative/Negative 2739 (29) Negative/Positive 2757 (29) Positive/Negative 1295 (14) Positive/Positive 1756 (18) Unknown 1041 (11) Donor age, median (range), years (18-61) 69

70 Not for publication or presentation Attachment 7 Continued. Characteristics of patients N Eval N (%) Donor age ( 1) (25) (34) (25) 50 and older 666 ( 7) To Be Determined 842 ( 9) Year of transplant (<1) ( 1) ( 1) ( 2) ( 3) ( 3) ( 3) ( 4) ( 4) ( 5) ( 4) ( 5) ( 6) ( 6) ( 5) ( 6) ( 7) ( 9) ( 9) ( 9) ( 5) ( 3) Median follow-up of survivors, mo (range) (2-269) 70

71 Not for publication or presentation Attachment 8 Study Proposal Study Title: Plasma YKL-40 and CHI3L1 genotype to predict mortality after allogeneic hematopoietic cell transplantation (HCT) Brian Kornblit, MD, PhD, Rigshospitalet, University of Copenhagen, Denmark. Peter Garred, MD, DMSc, Rigshospitalet, University of Copenhagen, Denmark. Lars Vindeløv, MD, DMSc, Rigshospitalet, University of Copenhagen, Denmark. Study Objectives: 1. Describe the variation in plasma YKL-40 levels in recipients and donors pre-transplant and pre- G-CSF/bone marrow harvest. 2. Describe the variation in pretransplant and pre-g-csf/bone marrow harvest plasma YKL-40 levels according to CHI3L1 genotype (rs ) 3. Assess the prognostic value of recipient pre-transplantation plasma YKL-40 levels on overall survival (OS), progression free survival (PFS), relapse related mortality (RRM), nonrelapse mortality (NRM) and acute and chronic graft versus host disease (GVHD). 4. Assess the prognostic value of donor pre-g-csf/pre-bone marrow harvest plasma YKL-40 levels on OS, PFS, RRM, NRM and acute and chronic GVHD. 5. Assess the prognostic value of recipient and donor genetic variation in promoter SNP (rs ) in the CHI3L1 gene, on OS, PFS, RRM, NRM and acute and chronic GVHD. Scientific Justification: With the introduction of reduced intensity conditioning and better supportive care, allogeneic HCT has become a curative treatment option for a variety of malignant hematologic diseases in older and medically infirm patient. However complications such as GVHD, infections and relapse are still major causes of morbidity and mortality. Prognostic factors such as relapse risk 1 and the HCT comorbidity index 2,3 have proven useful in predicting RRM and NRM. However, to appropriately balance the likelihood of disease control against the risk of debilitating complications, still more accurate pretransplant tools are necessary to predict outcome. Inflammatory biomarkers hold promise as prognostic factors in a variety of disease settings. In allogeneic HCT, biomarkers such as C reactive protein (CRP), interleukin (IL)-6 and tumor necrosis factor (TNF)-α are predictive of treatment related toxicity in the posttransplantation period. 4-7 In the pretransplant period increased concentrations of CRP have been shown to associate with increased incidence of infectious complications, acute GVHD and NRM. 8,9 YKL-40, also called chitinase-3-like-1 (CHI3L1) protein is a member of the family of mammalian chitinase-like proteins, and is regarded as an acute phase reactant. Increased levels of YKL-40 protein and messenger RNA have been observed in cancer and in a wide variety of inflammatory diseases of infectious and non infectious aetiology (reviewed by Johansen et al. 10 ). In solid tumors such as adenocarcinomas, squamous cell carcinomas, and astrocytomas, pre-treatment plasma YKL-40 levels have been associated with disease severity and treatment outcome. In hematological malignancies such as acute myeloid leukemia, multiple myeloma and Hodgkins lymphoma, increased pre-treatment plasma YKL-40 levels where associated with poorer prognosis YKL-40 is secreted by cancer cells, 14 vascular smooth muscle cells, 15 connective tissue cells, 16 neutrophile granulocytes and macrophages, 17,18 and although its exact biological function is unknown, YKL-40 regulates VEGF 19,20 and is involved in processes in cell proliferation and differentiation, 21 angiogenesis, 22 matrix re modelling and inflammation. 14 Secretion of YKL-40 is induced by INF

72 Not for publication or presentation Attachment 8 and IL It has also been suggested that YKL-40 can function as an opsonin and activate innate immune responses. 14 Compared to another acute phase reactant such as CRP, YKL-40 has a different tempero-spatial secretion profile and therefore only a weak correlation to CRP. 25,26 Furthermore, YKL-40 may reflect disease activity more accurately, as it originates from cancer cells and inflammatory cells directly involved in the pathological process, while CRP is secreted by hepatocytes as a response to IL We have previously investigated the prognostic value of pre-transplant plasma concentrations of YKL-40 and CHI3L1 genotype in a cohort of 149 patients treated with related (n=86) or unrelated (n=63) allogeneic HCT after nonmyeloablative conditioning. 27 Recipients with pre-transplant plasma YKL-40 concentrations above the age adjusted 95 th percentile (high) had higher RRM (33% versus 18%, P=0.036; adjusted hazard ratio (HR) 4.41, P=0.012), lower PFS (38% versus 64%, P=0.001; adjusted HR 2.84, P=0.005) and OS (42% versus 69%, P=0.001; adjusted HR 3.09, P=0.006). Recipients transplanted with donors with high plasma YKL-40 concentrations had an increased probability and risk of grade 2-4 acute GVHD (93% versus 62%, P=0.001; adjusted HR 2.25, P=0.017). Four different promoter SNP s in CHI3L1 were investigated in a healthy control cohort. The major and minor allele frequencies were between and , respectively. Due to perfect linkage disequilibrium (LD) between rs and rs only 3 SNP s were investigated in the transplant cohort (rs , rs and rs ). Pair-wise LD was tight between the 3 SNP s in the transplant cohort with no difference in genotype distribution compared to the healthy controls or donors. All SNP s displayed gene-dose-dependent associations with pretransplant/pre-g-csf plasma YKL-40 levels. Homozygosity for the major alleles was associated with high levels of plasma YKL-40, while heterozygosity and homozygosity for the minor alleles were associated with intermediate and low levels, respectively. The only association that was observed between genotype and outcome was that patients homozygous or recipients of grafts from donors homozygous for the minor alleles all died. This is in contrast to the general consensus that low YKL-40 levels are associated with better survival in most investigated disease settings. 14 The number of patients and donors homozygous for the minor alleles were only 4, suggesting that the finding could be an artefact. However, in a recent study of 463 patients admitted to an intensive care unit with systemic inflammatory response syndrome, the same phenomenon was observed (unpublished data, Brian Kornblit). Despite an association between high plasma concentrations YKL-40 at time of admission and mortality, patients homozygous for the minor allele (n=15) had low plasma YKL-40 levels at time of admission and lower survival. Although it is not known whether the associations between plasma concentrations of YKL-40 and outcome are causal or just bystander effects, an inflammatory biomarker, such as YKL-40, that defines relapse risk and possibly treatment related toxicity could be a valuable tool complementing clinical risk scores in HCT. Furthermore, as increased plasma concentrations of YKL-40 are regarded as a measure of ongoing inflammation, donor plasma YKL-40 levels could possibly identify a high-risk proinflammatory donor population. Lastly, recipient and donor genotype would also be investigated to, assess whether minor allele homozygosity truly does define a high risk population of recipients and donors. Based on these finding from our institution 27 we propose a confirmatory study in a CIBMTR cohort. Study Population: Eligibility Criteria: - Adults 18 years or older who received an allogeneic HCT between 2001 and 2009 for AML, MDS and non-hodgkin lymphomas. - AML patients in remission at the time of transplantation. 72

73 Not for publication or presentation Attachment 8 - MDS patients with a blast count < 5% at the time of transplantation. - Pretransplant or pre-gcsf/pre bone marrow harvest cryopreserved plasma available from recipients and donors, respectively. - Nonmyeloablative, reduced intensity and high dose conditioning and peripheral blood stem cell and bone marrow grafts will be allowed, as there is no biological reason for the effect of the proposed plasma proteins to be limited to certain subgroups. Exclusion: Cord blood transplants, syngeneic donors, and patients having a prior autologous or allograft will be excluded. The CIBMTR will provide a list of donor/recipient pairs that meet the eligibility criteria and with available cryopreserved plasma. Data Requirements: The information requested is routinely collected on the data submission forms. Variables to be collected include age, recipient/donor sex, Karnofsky PS, disease, disease status, time from diagnosis to HCT, year of transplant, donor relation, HLA match, PBSC or BM, Ablative versus reduced intensity/non-ablative, T-cell depletion, Recipient/donor CMV status, prior autologous transplant, GVHD prophylaxis. Sample Requirements: YKL-40 ELISA will be performed using a commercial kit according to manufacturer s protocols (Quidel, Santa Clara, CA). The ELISAs will be carried out at the Departments of Medicine and Oncology, Herlev Hospital, University of Copenhagen, Denmark under the supervision of Dr. Julia S. Johansen. J.S.J. has extensive experience with YKL-40 ELISA, and has published extensively on the subject (a small selection is included in the reference list) ,17,21,24,25,27-33 Plasma prepared from EDTA-anticoagulated blood should be stored at -80 C until YKL-40 analysis. ELISA s will be performed in duplicates and maximal sample volume needed is 100 µl. Plasma YKL-40 is stable at room temperature in EDTA-anticoagulated blood for up to 8 hours, thereafter small increases in plasma YKL-40 levels are observed. 28 This is not affected by sampling in ACDA which is the standard anticoagulant used by the CIBMTR. Genotyping: Genotyping of Recipient and Donor DNA will be performed at the Laboratory of Molecular Medicine, Department of Clinical Immunology, Rigshospitalet, University of Copenhagen, Denmark, under the supervision of PI s BK and PG. BK is currently working as a post doctoral fellow in the Laboratory of Molecular Medicine and The Allogeneic Hematopoietic cell Transplantation Laboratory. He has been working with genetics and genotyping since 2006 and has obtained a PhD degree based on studies of genetic variation in HMGB1. On the subject of genetic variation BK has published 7 papers, whereof 1 deals with YKL-40 (CHI3L1) in the setting of allogeneic HCT. 27,34-39 PG, Professor at the University of Copenhagen, head of the Laboratory of Molecular Medicine and consultant at the Department of Clinical Immunology. He has extensive experience in genetics and has over 200 publications on the subject. Most recently in the New England Journal of Medicine. 40 Genotyping for rs will performed on pre-transplant genomic DNA from recipients and donors by pyrosequencing according to manufacturer s protocol. The method has already been established in our laboratory. 27 Required sample volume is 1 µl DNA. 73

74 Not for publication or presentation Attachment 8 Variables to be Analyzed: Variables to be analyzed in multivariate analyses: Main effect: - Age adjusted recipient and donor plasma YKL-40 levels: high vs. normal - CHI3L1 genotype (rs ): CC vs. CG vs. GG Patient related: - Gender - Age - Karnofsky PS at transplant: 90 vs HCT-CI Disease related: - Disease at transplant: AML vs. MDS vs. NHL - Disease stage at transplant: CR, PR or progressive disease - Prior autologous transplant Transplant related: - Year of transplantation - Donor age - Gender mismatch: female -> male vs. other - Donor/recipient CMV status: -/- vs. +/- vs.-/+ vs. +/+ - T-cell depletion - Conditioning regimen: high dose vs. reduced intensity vs. nonmyeloablative - GVHD prophylaxis - Graft source: PBSC vs. bone marrow Study Design: The study will investigate the association between recipient and donor plasma YKL-40 and CHI3L1 genotype (rs ) and transplantation outcome. Plasma and DNA will be obtained pretransplant from recipients and pre-gcsf/bone marrow harvest from donors. The reference group for plasma concentrations of YKL-40 will be a cohort of 8899 Danish subjects, aged years, from the Danish general population. 31 Plasma YKL-40 concentrations increase exponentially with increasing age in healthy subjects. As it is important to account for this, the cohort in the current study will be divided into patients with normal plasma YKL-40 concentrations, that is, below an age adjusted 95 th percentile, and into subjects with high plasma YKL-40 concentrations, that is, equal to or above the age-adjusted 95 th percentile. 41 Associations between clinical outcomes and plasma concentrations of YKL-40 will be performed according to whether patients or donors are high or normal in relation to the age-adjusted plasma YKL-40 concentration. Genotyping will only be performed for the CHI3L1 SNP, rs , as it has been associated with reduced transcription in luciferase reporter assays. 42 Furthermore pair-wise LD s between rs and the other three SNPs investigated in the previous study were high (r 2 = ). The minor allele frequency of rs in a cohort of 100 healthy controls was The reference groups for the CHI3L1 genotype (rs ) will be a cohort of 100 Caucasian healthy blood donors. 27 Genotype will be tested for adherence to the Hardy-Weinberg equilibrium by using gene frequencies obtained by simple gene-counting and the chi-square test. Correlation analyses between plasma YKL-40 levels and CHI3L1 genotype (rs ) and age will be performed. Association analyses between YKL-40 levels and disease (AML, MDS or NHL) and disease stage will also be performed. 74

75 Not for publication or presentation Attachment 8 The effect of recipient and donor age-adjusted plasma YKL-40 levels and CHI3L1 genotype (rs ) will be investigated in relation to the following outcomes: OS, PFS, RRM, NRM, grade II-IV and III-IV acute GHVD and limited and extensive chronic GVHD. Multivariate analyses will be performed using proportional hazards models. These analyses will fit models to determine which risk factors may be related to a given outcome. All variables will first be examined to assure that they comply with the proportional hazards assumption. A stepwise building approach will then be used to develop models for clinical outcomes. References: 1. Kahl C, Storer BE, Sandmaier BM, Mielcarek M, Maris MB, Blume KG, et al. Relapse risk in patients with malignant diseases given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 2007 Oct 1;110(7): Sorror ML, Maris MB, Storb R, Baron F, Sandmaier BM, Maloney DG, et al. Hematopoietic cell transplantation (HCT)-specific comorbidity index: a new tool for risk assessment before allogeneic HCT. Blood 2005 Oct 15;106(8): Sorror ML, Giralt S, Sandmaier BM, De LM, Shahjahan M, Maloney DG, et al. Hematopoietic cell transplantation specific comorbidity index as an outcome predictor for patients with acute myeloid leukemia in first remission: combined FHCRC and MDACC experiences. Blood 2007 Dec 15;110(13): Pihusch M, Pihusch R, Fraunberger P, Pihusch V, Andreesen R, Kolb HJ, et al. Evaluation of C-reactive protein, interleukin-6, and procalcitonin levels in allogeneic hematopoietic stem cell recipients. Eur J Haematol 2006 Feb;76(2): Fuji S, Kim SW, Fukuda T, Mori S, Yamasaki S, Morita-Hoshi Y, et al. Preengraftment serum C-reactive protein (CRP) value may predict acute graft-versus-host disease and nonrelapse mortality after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2008 May;14(5): Schots R, Kaufman L, Van R, I, Ben OT, De WM, Van CB, et al. Proinflammatory cytokines and their role in the development of major transplant-related complications in the early phase after allogeneic bone marrow transplantation. Leukemia 2003 Jun;17(6): Min CK, Lee WY, Min DJ, Lee DG, Kim YJ, Park YH, et al. The kinetics of circulating cytokines including IL-6, TNF-alpha, IL-8 and IL-10 following allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 2001 Nov;28(10): Artz AS, Wickrema A, Dinner S, Godley LA, Kocherginsky M, Odenike O, et al. Pretreatment C-reactive protein is a predictor for outcomes after reduced-intensity allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2008 Nov;14(11): Kanda J, Mizumoto C, Ichinohe T, Kawabata H, Saito T, Yamashita K, et al. Pretransplant serum ferritin and C-reactive protein as predictive factors for early bacterial infection after allogeneic hematopoietic cell transplantation. Bone Marrow Transplant 2011 Feb;46(2): Johansen JS, Schultz NA, Jensen BV. Plasma YKL-40: a potential new cancer biomarker? Future Oncol 2009 Sep;5(7): Bergmann OJ, Johansen JS, Klausen TW, Mylin AK, Kristensen JS, Kjeldsen E, et al. High serum concentration of YKL-40 is associated with short survival in patients with acute myeloid leukemia. Clin Cancer Res 2005 Dec 15;11(24 Pt 1): Mylin AK, Rasmussen T, Johansen JS, Knudsen LM, Norgaard PH, Lenhoff S, et al. Serum YKL-40 concentrations in newly diagnosed multiple myeloma patients and YKL-40 expression in malignant plasma cells. Eur J Haematol 2006 Nov;77(5): Biggar RJ, Johansen JS, Smedby KE, Rostgaard K, Chang ET, Adami HO, et al. Serum YKL- 40 and interleukin 6 levels in Hodgkin lymphoma. Clin Cancer Res 2008 Nov 1;14(21):

76 Not for publication or presentation Attachment Johansen JS. Studies on serum YKL-40 as a biomarker in diseases with inflammation, tissue remodelling, fibroses and cancer. Dan Med Bull 2006 May;53(2): Malinda KM, Ponce L, Kleinman HK, Shackelton LM, Millis AJ. Gp38k, a protein synthesized by vascular smooth muscle cells, stimulates directional migration of human umbilical vein endothelial cells. Exp Cell Res 1999 Jul 10;250(1): Hakala BE, White C, Recklies AD. Human cartilage gp-39, a major secretory product of articular chondrocytes and synovial cells, is a mammalian member of a chitinase protein family. J Biol Chem 1993 Dec 5;268(34): Volck B, Price PA, Johansen JS, Sorensen O, Benfield TL, Nielsen HJ, et al. YKL-40, a mammalian member of the chitinase family, is a matrix protein of specific granules in human neutrophils. Proc Assoc Am Physicians 1998 Jul;110(4): Rehli M, Krause SW, Andreesen R. Molecular characterization of the gene for human cartilage gp-39 (CHI3L1), a member of the chitinase protein family and marker for late stages of macrophage differentiation. Genomics 1997 Jul 15;43(2): Francescone RA, Scully S, Faibish M, Taylor SL, Oh D, Moral L, et al. Role of YKL-40 in the angiogenesis, radioresistance, and progression of glioblastoma. J Biol Chem 2011 Apr 29;286(17): Faibish M, Francescone R, Bentley B, Yan W, Shao R. A YKL-40-neutralizing antibody blocks tumor angiogenesis and progression: a potential therapeutic agent in cancers. Mol Cancer Ther 2011 May;10(5): Johansen JS, Hoyer PE, Larsen LA, Price PA, Mollgard K. YKL-40 protein expression in the early developing human musculoskeletal system. J Histochem Cytochem 2007 Dec;55(12): Nishikawa KC, Millis AJ. gp38k (CHI3L1) is a novel adhesion and migration factor for vascular cells. Exp Cell Res 2003 Jul 1;287(1): Kzhyshkowska J, Mamidi S, Gratchev A, Kremmer E, Schmuttermaier C, Krusell L, et al. Novel stabilin-1 interacting chitinase-like protein (SI-CLP) is up-regulated in alternatively activated macrophages and secreted via lysosomal pathway. Blood 2006 Apr 15;107(8): Nielsen AR, Plomgaard P, Krabbe KS, Johansen JS, Pedersen BK. IL-6, but not TNF-alpha, increases plasma YKL-40 in human subjects. Cytokine 2011 Jul;55(1): Johansen JS, Stoltenberg M, Hansen M, Florescu A, Horslev-Petersen K, Lorenzen I, et al. Serum YKL-40 concentrations in patients with rheumatoid arthritis: relation to disease activity. Rheumatology (Oxford) 1999 Jul;38(7): Johansen JS, Krabbe KS, Moller K, Pedersen BK. Circulating YKL-40 levels during human endotoxaemia. Clin Exp Immunol 2005 May;140(2): Morup AM, Kornblit B, Johansen JS, Masmas TN, Madsen HO, Vindelov L, et al. The prognostic value of YKL-40 concentrations in nonmyeloablative conditioning allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2011 Sep;17(9): Hogdall EV, Johansen JS, Kjaer SK, Price PA, Blaakjaer J, Hogdall CK. Stability of YKL-40 concentration in blood samples. Scand J Clin Lab Invest 2000 Jul;60(4): Johansen JS, Krabbe KS, Moller K, Pedersen BK. Circulating YKL-40 levels during human endotoxaemia. Clin Exp Immunol 2005 May;140(2): Johansen JS, Krabbe KS, Moller K, Pedersen BK. Circulating YKL-40 levels during human endotoxaemia. Clin Exp Immunol 2005 May;140(2): Johansen JS, Bojesen SE, Tybjaerg-Hansen A, Mylin AK, Price PA, Nordestgaard BG. Plasma YKL-40 and total and disease-specific mortality in the general population. Clin Chem 2010 Oct;56(10): Ostergaard C, Johansen JS, Benfield T, Price PA, Lundgren JD. YKL-40 is elevated in cerebrospinal fluid from patients with purulent meningitis. Clin Diagn Lab Immunol 2002 May;9(3):

77 Not for publication or presentation Attachment Vind I, Johansen JS, Price PA, Munkholm P. Serum YKL-40, a potential new marker of disease activity in patients with inflammatory bowel disease. Scand J Gastroenterol 2003 Jun;38(6): Kornblit B, Masmas T, Petersen SL, Madsen HO, Heilmann C, Schejbel L, et al. Association of HMGB1 polymorphisms with outcome after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2010 Feb;16(2): Kornblit B, Munthe-Fog L, Madsen HO, Strom J, Vindelov L, Garred P. Association of HMGB1 polymorphisms with outcome in patients with systemic inflammatory response syndrome. Crit Care 2008;12(3):R Kornblit B, Munthe-Fog L, Petersen SL, Madsen HO, Vindelov L, Garred P. The genetic variation of the human HMGB1 gene. Tissue Antigens 2007 Aug;70(2): Kornblit B, Hagve TA, Taaning P, Birgens H. Phenotypic presentation and underlying mutations in carriers of beta-thalassaemia and alpha-thalassaemia in the Danish immigrant population. Scand J Clin Lab Invest 2007;67(1): Kornblit B, Taaning P, Birgens H. Beta-thalassemia due to a novel nonsense mutation at codon 37 (TGG-->TAG) found in an Afghanistani family. Hemoglobin 2005;29(3): Larsen ME, Kornblit B, Larsen MV, Masmas TN, Nielsen M, Thiim M, et al. Degree of predicted minor histocompatibility antigen mismatch correlates with poorer clinical outcomes in nonmyeloablative allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2010 Oct;16(10): Munthe-Fog L, Hummelshoj T, Honore C, Madsen HO, Permin H, Garred P. Immunodeficiency associated with FCN3 mutation and ficolin-3 deficiency. N Engl J Med 2009 Jun 18;360(25): Bojesen SE, Johansen JS, Nordestgaard BG. Plasma YKL-40 levels in healthy subjects from the general population. Clin Chim Acta 2011 Apr 11;412(9-10): Zhao X, Tang R, Gao B, Shi Y, Zhou J, Guo S, et al. Functional variants in the promoter region of Chitinase 3-like 1 (CHI3L1) and susceptibility to schizophrenia. Am J Hum Genet 2007 Jan;80(1):

78 Not for publication or presentation Attachment 8 Characteristics of patients with plasma samples in the NMDP repository who received an allogeneic HCT for AML or MDS between 2008 and 2010 Characteristics of patients N% Number of patients 510 Number of centers 89 Age, median (range), years 49 (18-74) ( 2) (11) (11) (21) (30) >60 83 (16) Missing 47 ( 9) Sex Male 257 (50) Female 253 (50) Karnofsky score <90% 168 (33) >= 90% 322 (63) missing 20 ( 4) Disease AML 447 (88) MDS 63 (12) Disease status AML early 317 (62) AML intermediate 130 (25) AML missing disease status 2 (<1) MDS early 6 ( 1) Missing 55 (11) Time from DX to TX, median (range), months 6 (<1-117) Year of transplant (20) (49) (30) 78

79 Not for publication or presentation Attachment 8 Continued. Characteristics of patients N% Donor/recipient HLA match HLA-identical sib 4 (<1) Other related 5 (<1) Well-matched URD 386 (76) Partially matched URD 107 (21) Mismatched URD 8 ( 2) Graftype BM 91 (18) PB 419 (82) Donor/recipient sex match male/male 4 (<1) male/female 5 (<1) female/male 9 ( 2) Missing 492 (96) Donor/recipient CMV match Negative/Negative 11 ( 2) Positive/Positive 7 ( 1) Positive/Negative 3 (<1) Negative/Positive 10 ( 2) unknown 479 (94) Conditioning regimen Flu+Bu+/-other 190 (37) Flu+Mel+/-other 24 ( 5) Flu+TBI+/-other 20 ( 4) Flu+Cy+/-other 6 ( 1) Bu+Cy+/-other 127 (25) Cy+TBI+/-other 109 (21) Flu+Cy+TBI 3 (<1) TBI+/-other 3 (<1) TBI+Bu+Flu 8 ( 2) Bu+/-other 5 (<1) Mel+/-other 1 (<1) Flu+Bu 2 (<1) Flu+BU+TBI 2 (<1) Bu+TBI+/-other 1 (<1) Bu+Flu+/-other 2 (<1) TBD 7 ( 1) 79

80 Not for publication or presentation Attachment 8 Continued. Characteristics of patients N% GVHD prophylaxis T-cell depletion 1 (<1) CSA+MMF 22 ( 4) CSA+MTX 22 ( 4) CSA+MTX+other 3 (<1) CSA+Rapa 1 (<1) CSA+other 4 (<1) Post-HCT Cy 2 (<1) Tacro+MMF 44 ( 9) Tacro+MMF+other 7 ( 1) Tacro+MTX 271 (53) Tacro+MTX+Rapa 12 ( 2) Tacro+MTX+other 33 ( 6) Tacro+Rapa 19 ( 4) Tacro+other 15 ( 3) Other 54 (11) Median follow-up of survivors, range, months 13 (3-30) 80

81 Not for publication or presentation Attachment 9 Study Proposal Study Title: Natural killer cell genomics and outcomes after allogeneic transplantation for lymphoma Veronika Bachanova, MD, PhD, University of Minnesota, Minneapolis, MN, USA Jeffrey Miller, MD, University of Minnesota, Minneapolis, MN, USA Daniel Weisdorf, MD, University of Minnesota, Minneapolis, MN, USA Linda Burns, MD, University of Minnesota, Minneapolis, MN, USA Study Objectives: Our proposal is based on recent advances linking natural killer (NK) cell function to NK cell genetics. We hypothesize that the number and content of genes regulating donor-derived NK cell function may influence the graft-versus-lymphoma effect and subsequent relapse and survival of patients with lymphoma undergoing allogeneic hematopoietic cell transplantation (HCT). To test this hypothesis, we propose the analysis of donor NK cell genetics in a large cohort of B-cell non- Hodgkin lymphoma patients. We plan to investigate the following specific aims: 1. To evaluate whether donor NK cells killer immunoglobulin receptor (KIR) genes content affects relapse rate and survival in non-hodgkin lymphoma patients treated with allogeneic donor transplantation 2. To evaluate whether the absence of a recipient KIR ligand for one or more donor KIR receptors affects relapse rate, disease-free survival, and overall survival in non-hodgkin lymphoma patients treated with allogeneic donor transplantation 3. To examine the prognostic significance of donor NK cell immunogenetics in multivariate analyses examining patient, disease, and transplant characteristics Scientific Justification: Allogeneic donor transplantation can cure non-hodgkin lymphoma by inducing a graft-versus-lymphoma effect. A promising 5-year survival rate of 60-70% has been reported for relapsed follicular lymphoma and mantle cell lymphoma; and approximately 30-40% patients with recurrent diffuse large B-cell lymphoma and T cell lymphomas survive long-term. 1-6 To improve these outcomes, a multi-pronged approach is needed to identify which unique patients, disease and transplant determinants can be manipulated to most effectively prevent post-transplant relapse. We propose a study focusing on genetic donor-derived factors that are distinct from human leukocyte antigens (HLA). We plan to analyze whether genetic polymorphisms associated with donor-derived native immunity affects the relapse and survival of lymphoma patients post-transplant. Rationale: NK cell biology. NK cells are the critical component of native immunity. They were discovered by their capacity to kill malignant cells. 7 NK cell lysing activity directed at other cells is partially regulated by inhibitory killer immunoglobulin receptors (KIR) that limit autologous NK cell-induced cell lysis by engagement of self HLA class 1 alleles. NK cells recognize and kill target cells as a result of the balance of signaling by both inhibitory and activating NK cell receptors. 7 KIR genes and HLA genes segregate independently on different chromosomes and therefore even fully HLA-matched allogeneic donors likely have a higher frequency of NK cells mismatched between KIR and cognate HLA class 1 ligands. These donor-derived NK cells permit potent alloreactivity toward tumor cells and play potentially a significant role in eradicating residual disease following allogeneic HCT. 8 81

82 Not for publication or presentation Attachment 9 KIR genetics. KIR genes are closely linked and inherited as a haplotype. Individuals vary in the number of KIR genes contained in their genome and can be simply organized into 2 specific haplotypes: A or B. The main difference between group A and B haplotypes is that group B contains variable number of NK cell-activating receptor groups, while group A has fixed gene content and no activating receptors. About 30% of the population has haplotype B. KIR locus can be further organized by presence of particular B- specific genes in centromeric (Cen) and telomeric (Tel) parts of locus. 9 Clinical relevance in allogeneic HCT. NK cell-mediated protection from relapse was first clinically demonstrated by Ruggieri et al in T cell-depleted, HLA-haploidentical HCT for acute myelogenous leukemia (AML). 8 Results of the antileukemic effects against AML after mismatched transplantation, when the KIR ligand incompatibility existed in the direction of graft-versus-host, have been mixed More recently, the importance of KIR genetics was clearly demonstrated in a CIMBTR-supported study led by Miller et al. In a cohort of AML patients who underwent unrelated donor HCT, Miller and colleagues found that the relapse rate for KIR haplotype B donor transplants was 50% lower than that for KIR haplotype A donor transplants. 9 The rationale to study NK cell genetics in lymphoma: The effect of NK cells on lymphoma is supported by the following laboratory evidence: 1. Activated NK cells are capable of killing lymphoma cells in vitro and in vivo Missing-selfreactivity can be mimicked by blocking self-specific inhibitory receptors on NK cells. In a mouse model, an inhibitory receptor blockade in vivo caused rejection of syngeneic lymphoma cells. The selective rejection of tumor cells was NK cell-dependent and specifically induced by Ly49C/I (the mouse analogue of KIR) blockade. 13 Using a human cell line and gene transfer, Binyani demonstrated that a KIR blockade (KIR3DL1) promoted NK cell cytotoxicity only against the targets that expressed cognate ligand (HLA Bw4 epitope). 14 Similarly, among different B-lymphoblast cell lines, the density of HLA expression, but not CD20 expression, correlated with NK-cell activity against rituximab-labeled targets. Antibody-dependent cellular cytotoxicity was increased significantly following HLA shielding or KIR activation by anti-kir antibodies, respectively Significance of NK cell-activated receptors on tumors. In an in vivo c-myc transgenic B-cell lymphomagenesis model, the survival of tumor-bearing mice was significantly prolonged if tumor cells had low MHC class I expression and also expressed activating NKG2D ligands (NKG2D- L). The survival was totally abrogated by NK cell depletion, suggesting that the presence of both signals rendered tumors more immunogenic and susceptible to NK cell mediated killing. 12 Similarly, in a mouse model examining second hit in lymphomagenesis, NK and T cellmediated killing of BCL-2-expressing lymphoma cells was significantly increased compared to those not expressing BCL-2. Increased immunogenicity was associated with higher expression of the NKG2D ligand MULT1 (a ligand for an NK cell-activating receptor). 15 In aggregate, these data clearly suggest that NK cell-mediated killing of lymphoma cells can be manipulated by HLA and NK cell receptor interactions. Significance: There is a critical need for novel biologic and genetic markers to identify patients likely to respond to allogeneic HCT. For adults with non-hodgkin lymphoma, predicting outcomes following allogeneic HCT would allow clinicians to individualize therapy, optimize long-term survival, and prevent unnecessary toxicities. Large studies of AML, 9 and more recently myeloma patients, 16 showed that KIR on NK cells and HLA gene polymorphisms governing NK cell function influence disease progression and survival for patients treated with allogeneic HCT. Choosing a more favorable donor on the basis of the KIR genotype may more accurately model host-tumor interactions and better predict survival. A better understanding of lymphoma immunogenicity can be achieved by studying the expression of ligands for activating NK receptors in different types of lymphomas. Our data may open up new possibilities for 82

83 Not for publication or presentation Attachment 9 optimizing allogeneic transplantation for patients with lymphoma by using a unique immune systembased genetic donor choice algorithm. Study Population: - All ages - Diagnosis - Follicular lymphoma (FL) - Diffuse large B cell lymphoma (DLBCL) - Mantle cell lymphoma (MCL) - T cell replete allogeneic HCT using one of these donor types: - Matched (any match) - Unrelated (any match) adult and umbilical cord blood - Transplanted between Available donor DNA sample Outcomes: - Overall survival: time to death from any cause. - Acute GVHD: grades II-IV and III-IV acute GVHD. - Chronic GVHD: limited, extensive, mild, moderate, severe chronic GVHD. - Disease relapse: disease recurrence is considered an event. - Disease-free survival: Time to relapse or death from any cause other than relapse. - Non-relapse mortality: death in continuous complete remission of primary disease. - Donor chimerism: percent donor/host chimerism. - Cause of death (if available). Variables to be Analyzed: We plan to extract data on the following patient, disease, and transplant characteristics: Patient-related characteristics: - Age - Gender - Karnofsky performance status (KPS) or Lansky performance status (LPS) - HCT comorbidity index (HCT-CI) Disease characteristics: - Histopathology (FL, MCL, DLBCL) - Stage at diagnosis - Disease status at transplant - Chemo sensitivity Transplant characteristics: - Graft source (marrow, peripheral blood, cord blood) - Type of donor (sibling, unrelated adult, cord blood) - HLA matching, donor/recipient CMV match - Conditioning regimen - Time from diagnosis to transplant - Year of transplant - GVHD prophylaxis HLA-Matching data: - Complete high resolution HLA matching data at HLA A, B, C, DRB1, DQB1 Sample Requirements: We request a sample of donor DNA (approximately mcg) for KIR genotyping from Research Sample Repository of the NMDP. 83

84 Not for publication or presentation Attachment 9 Study Design: Transplant donors will be typed for the presence and absence of individual KIR genes We will analyze 15 KIR genes (KIR3DL3, KIR2DS2, KIR2DL2, KIR2DL3, KIE2DL5A/B, KIR2DS3/2DS5, KIR2DP1, KIR2DL1, KIR3DP1, KIR2DL4, KIR3DL1, KIR3DS1, KIR2DS1, KIR2DS4 and KIR2DL2) by using high-throughput analysis of single nucleotide polymorphisms by mass spectrometry as described. 9 From the genotypes we will determine whether each donor is of A/A or B/x genotype. For the B/x donors we will further determine whether their B haplotype genes are in the centromeric or telomeric part of the KIR locus, or in both. We will define the KIR B content score for each donor's KIR genotype as the number of centromeric and telomeric gene-content motifs containing B haplotype defining genes. Permissible values for the KIR B content score are 0, 1, 2, 3, and 4. The laboratory of Dr. Jeff Miller has extensive experience performing this assay and recently published the results of KIR genotyping in a cohort of 1409 acute leukemia patients. 9 We plan to analyze the KIR genetics at several different levels [similarly as published by Cooley et al: 9 1. Haplotype AA vs Bx 2. Cen AA, AB, BB 3. Tel AA, AB, BB 4. B content score (1,2 vs 3,4) 5. Neutral, better, best B content score as in the AML model Our goals are to determine the following transplant outcomes: 1. treatment-related mortality (TRM) 2. cumulative incidence of relapse 3. lymphoma-free survival (LFS) 4. overall survival (OS) 5. incidence of II-IV acute GVHD 6. incidence of chronic GVHD Kaplan-Meier curves will be used to estimate the probability of OS and LFS. Cumulative incidence will be used to estimate relapse rate, TRM, and incidence of acute and chronic GVHD. Relapses will be summarized by cumulative incidence estimated with TRM as a competing risk. LFS and OS patients will be censored at the time of last follow-up. An unadjusted comparison between KIR genotypes will be made using a log rank test on either hazard rates for OS and LFS or the crude hazard rates for relapse, TRM, and GVHD. Cox proportional hazard models will be used to adjust for important clinical factors including KIR genotype, HLA match, time from diagnosis to transplant, histopathology group, graft source, patient age, race, and KPS. Forward stepwise regression modeling will be used to determine which factors require adjustment in each model on the basis of a significance level of 5%. All models will include the A/B KIR genotype. The model for LFS will also include disease status. We will look at significant interaction between KIR genotype groups and other covariates. References: 1. Shea T, Johnson J, Westervelt P, Farag S, McCarty J, Bashey A, et al. Reduced-Intensity Allogeneic Transplantation Provides High Event-Free and Overall Survival in Patients with Advanced Indolent B Cell Malignancies: CALGB Biol Blood Marrow Transplant 2011 Sep;17(9):

85 Not for publication or presentation Attachment 9 2. Dietrich S, Tielesch B, Rieger M, Nickelsen M, Pott C, Witzens-Harig M, et al. Patterns and outcome of relapse after autologous stem cell transplantation for mantle cell lymphoma. Cancer 2011 May 1;117(9): Khouri IF, McLaughlin P, Saliba RM, Hosing C, Korbling M, Lee MS, et al. Eight-year experience with allogeneic stem cell transplantation for relapsed follicular lymphoma after nonmyeloablative conditioning with fludarabine, cyclophosphamide, and rituximab. Blood 2008 Jun 15;111(12): Maloney DG. Graft-vs.-lymphoma effect in various histologies of non-hodgkin's lymphoma. Leuk Lymphoma 2003;44 Suppl 3:S Brunstein CG, Cantero S, Cao Q, Majhail N, McClune B, Burns LJ, et al. Promising progression-free survival for patients low and intermediate grade lymphoid malignancies after nonmyeloablative umbilical cord blood transplantation. Biol Blood Marrow Transplant 2009 Feb;15(2): Tomblyn M, Brunstein C, Burns LJ, Miller JS, MacMillan M, DeFor TE, et al. Similar and promising outcomes in lymphoma patients treated with myeloablative or nonmyeloablative conditioning and allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2008 May;14(5): Lanier LL. Natural killer cells: roundup. Immunol Rev 2006 Dec;214: Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002 Mar 15;295(5562): Cooley S, Weisdorf DJ, Guethlein LA, Klein JP, Wang T, Le CT, et al. Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia. Blood 2010 Oct 7;116(14): Davies SM, Ruggieri L, DeFor T, Wagner JE, Weisdorf DJ, Miller JS, et al. Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Killer immunoglobulin-like receptor. Blood 2002 Nov 15;100(10): Brunstein CG, Wagner JE, Weisdorf DJ, Cooley S, Noreen H, Barker JN, et al. Negative effect of KIR alloreactivity in recipients of umbilical cord blood transplant depends on transplantation conditioning intensity. Blood 2009 May 28;113(22): Brenner CD, King S, Przewoznik M, Wolters I, Adam C, Bornkamm GW, et al. Requirements for control of B-cell lymphoma by NK cells. Eur J Immunol 2010 Feb;40(2): Vahlne G, Lindholm K, Meier A, Wickstrom S, Lakshmikanth T, Brennan F, et al. In vivo tumor cell rejection induced by NK cell inhibitory receptor blockade: maintained tolerance to normal cells even in the presence of IL-2. Eur J Immunol 2010 Mar;40(3): Binyamin L, Alpaugh RK, Hughes TL, Lutz CT, Campbell KS, Weiner LM. Blocking NK cell inhibitory self-recognition promotes antibody-dependent cellular cytotoxicity in a model of antilymphoma therapy. J Immunol 2008 May 1;180(9): Schuster C, Berger A, Hoelzl MA, Putz EM, Frenzel A, Simma O, et al. The cooperating mutation or "second hit" determines the immunological visibility towards MYC-induced murine lymphomas. Blood 2011 Aug Kröger N, Zabelina T, Berger J, Duske H, Klyuchnikov E, Binder T, Stübig T, Hilde-brandt Y, Atanackovic D, Alchalby H, Ayuk F, Zander AR, Bacher U, Eiermann T. Donor KIR haplotype B improves progression-free and overall survival after allogeneic hematopoietic stem cell transplantation for multiple myeloma. Leukemia Oct;25(10): Epub 2011 Jun 7 85

86 Not for publication or presentation Attachment 9 Table 1: Characteristics of patients receiving first transplants for either NHL or Hodgkin s Lymphoma and have donor and recipient samples available by type of recipient samples available. Pre-TX only Pre and Post TX Characteristics of patients N Eval N (%) N Eval N (%) Number of patients Number of centers Age, median (range), years (2-75) (4-54) Age at transplant y 18 ( 2) 5 (10) y 51 ( 6) 2 ( 4) y 84 (10) 12 (24) y 117 (14) 8 (16) y 196 (24) 16 (32) 50 and older 351 (43) 7 (14) Recipient race Caucasian 731 (89) 46 (92) African American 30 ( 4) 3 ( 6) Asian/Pacific Islander 5 ( 1) 0 Hispanic 41 ( 5) 1 ( 2) Other 10 ( 1) 0 Male sex (65) (68) Karnofsky prior to transplant > (68) (70) Disease at transplant Non-Hodgkin s Lymphoma 767 (94) 47 (94) Hodgkin s Lymphoma 50 ( 6) 3 ( 6) Histological Diagnosis NHL, Follicular 185 (23) 9 (18) NHL, Diffuse large B-cell 118 (14) 7 (14) NHL, Mantle Cell 122 (15) 1 ( 2) NHL, Burkitt s 22 ( 3) 0 NHL, Other 320 (39) 30 (60) Hodgkin s Lymphoma 50 ( 6) 3 ( 6) 86

87 Not for publication or presentation Attachment 9 Table 1. Continued. Pre-TX only Pre and Post TX Characteristics of patients N Eval N (%) N Eval N (%) Disease status at transplant PIF-unk sens 5 ( 1) 0 1 PIFRES 95 (12) 0 2 PIF-sensitive 130 (16) 1 ( 2) 3 CR1 First compl.remission 78 (10) 0 4 CR2 2nd compl.remission 87 (11) 2 ( 4) 5 CR3+ 3rd or subseq CR 34 ( 4) 0 6 REL1-untreated 3 (<1) 0 7 REL1-resistant 62 ( 8) 2 ( 4) 8 REL1-sensitive 66 ( 8) 1 ( 2) 9 REL2-untreated 6 ( 1) 0 10 REL2-resistant 28 ( 4) 0 11 REL2-sensitive 41 ( 5) 0 12 REL3+ untreated 7 ( 1) 0 13 REL3+ resistant 30 ( 4) 0 14 REL3+ sensitive 30 ( 4) 0 31 CRU1 First compl.remission(h2018) 5 ( 1) 0 32 CRU2 2nd compl.remission undetermined(h2018) 2 (<1) 0 33 CRU3+ 3rd or subseq CR undetermined(h2018) 1 (<1) 0 40 PIF-untreated 1 (<1) 0 41 REL1-unk sens 6 ( 1) 0 42 REL2-unk sens 4 ( 1) 0 43 REL3+ unk sens 4 ( 1) 1 ( 2) 44 CR 5 ( 1) 6 (13) 45 CR undetermined 4 ( 1) 3 ( 6) 46 Partial remission 20 ( 3) 5 (11) 47 No response/progressive disease 28 ( 4) 16 (34) 48 Not evaluable 1 (<1) 1 ( 2).A Missing 15 ( 2) 9 (19) Graft type Bone marrow 319 (39) 50 (100) Peripheral blood 498 (61) 0 Conditioning regimen Myeloablative 391 (48) 43 (86) Reduced Intensity 259 (32) 6 (12) Nonmyeloablative 167 (20) 1 ( 2) 87

88 Not for publication or presentation Attachment 9 Table 1. Continued. Pre-TX only Pre and Post TX Characteristics of patients N Eval N (%) N Eval N (%) GVHD prophylaxis FK506+(MTX or MMF or Steroids) other 453 (55) 11 (22) FK506 other 54 ( 7) 0 CsA + MTX other 153 (19) 26 (52) CsA other (No MTX) 90 (11) 0 MMF other 5 ( 1) 0 MTX other 2 (<1) 0 T-cell depletion 50 ( 6) 13 (26) Other 10 ( 1) 0 HLA Matching out of 8 HLA-A, -B, -C and DRB /8 564 (69) 26 (52) 7/8 185 (23) 14 (28) 6/8 50 ( 6) 5 (10) 5/8 13 ( 2) 5 (10) 4/8 3 (<1) 0 3/8 2 (<1) 0 Donor/recipient sex match Male/Male 377 (47) 25 (50) Male/Female 188 (23) 9 (18) Female/Male 147 (18) 9 (18) Female/Female 93 (12) 7 (14) Donor/recipient CMV match Negative/Negative 269 (33) 15 (30) Negative/Positive 235 (29) 21 (42) Positive/Negative 80 (10) 8 (16) Positive/Positive 142 (17) 5 (10) Unknown 91 (11) 1 ( 2) Donor age, median (range), years (18-59) (20-51) Donor age yrs 9 ( 1) yrs 212 (26) 11 (22) yrs 276 (34) 23 (46) yrs 198 (24) 15 (30) 50 and older 41 ( 5) 1 ( 2) Unknown/TBD 81 (10) 0 88

89 Not for publication or presentation Attachment 9 Table 1. Continued. Pre-TX only Pre and Post TX Characteristics of patients N Eval N (%) N Eval N (%) Year of transplant ( 2) (<1) ( 1) 5 (10) (<1) 1 ( 2) (<1) 4 ( 8) (<1) 4 ( 8) ( 1) 3 ( 6) ( 1) 9 (18) ( 2) 7 (14) ( 2) 7 (14) ( 4) 9 (18) ( 6) ( 6) ( 5) (11) ( 9) (13) (16) (14) ( 5) ( 3) 0 Median follow-up of survivors, mo (range) ( ) (82-239) 89

90 Not for publication or presentation Attachment 10 Study Proposal Study Title: Effect of genetic ancestry matching on HSCT outcomes Abeer Madbouly, PhD, National Marrow Donor Program, Minneapolis, MN, USA Martin Maiers, BS, National Marrow Donor Program, Minneapolis, MN, USA Navneet Majhail, MD, MS, National Marrow Donor Program, Minneapolis, MN, USA Hypotheses: 1. Differences in genetic ancestry detected by ancestry informative Single Nucleotide Polymorphisms (SNPs) for HLA matched unrelated donors and recipients can identify potential transplantation determinants. 2. Recipient non-hla genetic ancestry can impact transplant outcomes. 3. Donor non-hla genetic ancestry can impact transplant outcomes 4. HLA matched European American recipients and unrelated donors differ in regional European ancestry (reflected in their background genotype) which could potentially impact transplant outcomes. Study Objectives: The general goal of this study is to advance unrelated HSCT outcomes by exploring the impact of non- MHC genetic ancestry. 1. We will use a panel of SNP ancestry informative markers (AIMs) that identifies continental as well as European ancestral substructure within a patient/donor cohort. The majority of individuals will have primarily European ancestry, but individuals of different continental ancestry may also be included. 2. In Phase 1, we will conduct a pilot study of a small sample of patient-donor pairs to identify appropriate analyses, get a preliminary estimate of the sample size for adequate statistical power and evaluate several patient/donor proximity measures to associate ancestry with the relevant transplant outcomes. We will also test multiple published AIMs panels 1-3 to identify continental as well as sub-european ancestral structures. 3. In Phase 2, we will type 10 of 10 HLA-matched donors and recipients for the AIMs panel of choice and analyze the effect on the corresponding transplant outcomes: a. We will analyze outcome with respect to differences in genetic ancestry between donors and recipients using appropriate distance measures defined in Phase 1. b. We will analyze outcome with respect to different continental ancestry and European ancestry substructure identified in patients. 4. Primary outcomes measures: a. Overall Survival b. Treatment-related mortality 5. Secondary outcome measures: a. Acute and chronic GVHD b. Neutrophil recovery c. Relapse d. Disease-free survival Scientific Justification: Survival after unrelated donor (URD) hematopoietic stem cell transplantation (HSCT) is dependent on HLA matching between donor and recipient, donor and recipient ethnicity and numerous other factors, most of which are still unknown. Disparities exist between the outcomes of HSCTs using an HLAmatched sibling donor (of similar genetic ancestry as the patient) and those with unrelated donors that are 90

91 Not for publication or presentation Attachment 10 fully HLA matched. While the search for an HLA matched unrelated donor typically begins with individuals with the same self identified race/ethnicity (SIRE) as the patient (due to increased likelihood of an HLA match within the same SIRE group), it is not clear whether matched race/ancestral background specifically results in better survival. While the majority of HSCTs are performed on patients and donors with European ancestry, these individuals usually fall under one race category: white. Previous studies have addressed racial disparities in outcome for HLA matched related and unrelated donor or CBU HCT. 3-7 However, there are no studies to date analyzing outcome with respect to differences in genetic ancestry between donor and recipient. This study will test the hypothesis that for patient/donor pairs that are matched at the five primary HLA loci (HLA-A, -C, -B, -DRB1 and DQB1), genetic ancestry affects transplant outcome. We will test several published AIMs panels 1-3 to infer continental ancestry as well as European substructure in the donor-recipient cohort. Unsupervised clustering techniques 9 and principal component analysis (PCA) 10 will be used to identify ancestral clusters of individuals, and distance measures will be applied within and between these clusters for each donor-recipient pair, and analyzed with respect to transplant outcome. In addition, patient ancestry will be analyzed with respect to outcome. Study Population: Patients receiving a first 10/10 matched (verified through retrospective typing) unrelated donor myeloablative transplantation for hematological malignancy (AML, ALL, CML and MDS). HLA-DPB1 data and samples available. Variables to be Analyzed: Primary effect: Association of genetic ancestry with HCT outcomes - Impact of genetic ancestry distance between donor and recipient - Genetic ancestry of donor - Genetic ancestry of recipient Patient-related (at time of transplant): - Age: in decades (0-9, 10-19, 20-29, 30-39, 40-49, 50 and older). - Gender: female vs. male - Karnofsky score at transplant: < 90 vs Disease-related: - Disease at transplant: ALL, AML, CML and MDS - Disease status prior to transplant: early vs. intermediate vs. advanced Transplant-related: - Source of stem cells: marrow (BM) vs. peripheral blood stem cells (PB) - Donor age: in decades (18-29, 30-39, 40-49, 50 and older) - Year of transplant: ( ) - Race match - Gender match: M-M vs. M-F vs. F-M vs. F-F - Donor/recipient CMV status: -/- vs. -/+ vs. +/- vs. +/+ vs. Unknown - Conditioning regimen: Myeloablative vs. reduced intensity or non-myeloablative - GvHD prophylaxis: Tacrolimus +/-others vs. CSA +/-others vs. TCD vs. others [Note: Consider CSA/Tac prophylaxis as one group] - ATG use: Yes vs. no Study Design: The study will proceed as follows: 1. Pilot study and preliminary analysis. 2. SNP typing of CIBMTR samples. 91

92 Not for publication or presentation Attachment Genetic ancestry analysis on study cohort. 4. Data analysis with respect to outcome. 1. Pilot study In this phase we will test multiple published AIMs panels 1-3 to identify continental as well as sub- European ancestral structures. Two hundred to three hundred randomly chosen PD pairs will be genotyped using the chosen AIMs panel. All patient/donor pairs will be fully (10/10) HLA matched. A preliminary genetic ancestry analysis (see details in phase 3) will be conducted and several distance measures assessed in order to identify the most effective measures of patient/donor ancestry proximity. The primary purpose of the pilot study is to allow an estimate of necessary sample size for Phase 2 based on the likely proportions of genetic distances in the study cohort. 2. SNP typing of full cohort 3. Analysis of genetic ancestry in study cohort Unsupervised clustering methods 9 will be applied to individuals in the study cohort according to AIMs genotypes. Clustering will be augmented by principal components analysis (PCA) as implemented in the software EIGNESTRAT. 10 Self identified race and ethnicity will be examined with respect to the principal clusters. The initial aim is to confirm clear separation among the main continental groups: European, African, Asian and Hispanic. Since the majority of individuals will be of European descent, we will delineate three primary substructures within this group of European- American individuals: North-Western Europe, South-Eastern Europe and Ashkenazi Jews, as well as finer structures as the data permits. The patient/donor ancestry proximity measures identified in the pilot study will be applied to each donor-recipient pair. 4. Data analysis and association with outcomes Donor-recipient pairs that are matched for genetic ancestry will be compared to pairs with mismatched genetic ancestry. The definition of genetic ancestry matching will be determined with the assistance of the CIBMTR Statistical Center. Comparisons will also be made based on the genetic ancestry clustering of the recipients alone without regard for the donor s ancestry to investigate differences in outcome related to the recipient genetic background. To summarize the characteristics of the dataset, descriptive tables of patient-, disease and transplantrelated factors will be reported. For discrete factors, the number of cases and their respective percentages will be calculated. Chi-Square tests will be used to compare discrete factors between the HLA matched vs. mismatched groups. For continuous factors, the median and ranges will be calculated. The Kruskal-Wallis test will be used to compare the continuous factors between the genetic ancestry matched vs. disparate groups. Probabilities for overall survival and disease-free survival will be calculated using the Kaplan-Meier estimator with variance estimated by Greenwood's formula. Comparison of survival curves will be done using the log-rank test. Values for other listed outcomes will be calculated according to cumulative incidence using a Taylor series linear approximation to estimate the variance. Multivariate analyses will be performed using the proportional hazards model to compare the genetic ancestry matched and mismatched groups. Models will be fitted to determine which risk factors may be related to a given outcome. All variables will be tested for the affirmation of the proportional hazards assumption. Factors violating the proportional hazards assumption will be adjusted first before the stepwise model building approach is used in developing models for the primary and secondary outcomes. 92

93 Not for publication or presentation Attachment 10 References: 1. Price AL, Butler J, Patterson N, et al. Discerning the ancestry of European Americans in genetic association studies. PLoS Genet. 2008;4(1): e236. doi: /journal.pgen Paschou P, Drineas P, Lewis J, et al. Tracing Sub-Structure in the European American Population with PCA-Informative Markers. PLoS Genet. 2008;4(7): e doi: /journal.pgen Tian C, Plenge RM, Ransom M, et al. Analysis and Application of European Genetic Substructure Using 300 K SNP Information. PLoS Genet. 2008;4(1): e4. doi: /journal.pgen Davies SM, Kollman C, Anasetti C, et al. Engraftment and survival after unrelated-donor bone marrow transplantation: a report from the National Marrow Donor Program. Blood. 2000;96: Serna DS, Lee SJ, Zhang MJ, et al. Trends in survival rates after allogeneic hematopoietic stem-cell transplantation for acute and chronic leukemia by ethnicity in the United States and Canada. J. Clin Oncol. 2003;21: Oh H, Loberiza FR Jr, Zhang MJ, et al. Comparison of graft versus-host-disease and survival after HLA-identical sibling bone marrow transplantation in ethnic populations. Blood. 2005;105: Baker KS, Davies SM, Majhail NS, et al. Race and socioeconomic status influence outcomes of unrelated donor hematopoietic cell transplantation. Biol Blood Marrow Transplant. 2009;15: Ballen KK, Klein JP, Pedersen TL, et al. Relationship of Race/Ethnicity and Survival after Single Umbilical Cord Blood Transplantation for Adults and Children with Leukemia and Myelodysplastic Syndromes, Biol Blood Marrow Transplant Nov (in press) 9. Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000;155: Price A, Patterson N, Plenge R, et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet. 2006;38:

94 Not for publication or presentation Attachment 10 Characteristics of first transplants for patients with AML, ALL, CML and MDS and are 10/10 high-resolution matched using unrelated NMDP donors ab Characteristics of patients N Eval N (%) Number of patients 2692 Number of centers 150 Age, median (range), years (<1-74) Age at transplant y 209 ( 8) y 288 (11) y 436 (16) y 557 (21) y 617 (23) 50 and older 585 (22) Recipient race 2692 Caucasian 2498 (93) African American 44 ( 2) Asian/Pacific Islander 29 ( 1) Hispanic 96 ( 4) Native American 5 (<1) Other/Multiple/Declined/Unknown 20 ( 1) Race matching - Yes (91) Male sex (58) Karnofsky prior to transplant > (72) Disease at transplant 2692 AML 890 (33) ALL 568 (21) CML 845 (31) MDS 389 (14) Disease status at transplant 2692 Early 1903 (71) Intermediate 181 ( 7) Advanced 608 (23) Graft type 2692 Bone marrow 2070 (77) Peripheral blood 622 (23) a - Data is not CAP-modeled. b Sample availability has not been checked. 94

95 Not for publication or presentation Attachment 10 Continued. Characteristics of patients N Eval N (%) Conditioning regimen 2692 Myeloablative 2312 (86) Reduced intensity 253 ( 9) Nonmyeloablative 127 ( 5) GVHD prophylaxis 2692 FK506 + (MTX or MMF or Steroids) other 846 (31) FK506 other 35 ( 1) CsA + MTX other 1257 (47) CsA other (No MTX) 165 ( 6) MMF other 3 (<1) MTX other (No CsA) 17 ( 1) T-cell depletion 344 (13) Other 25 ( 1) GVHD prophylaxis 2692 FK506 + MTX other 726 (27) FK506 + (MMF or Steroids) other 120 ( 4) FK506 other 35 ( 1) CsA + MTX other 1257 (47) CsA other (No MTX) 165 ( 6) MTX other (No CsA) 17 ( 1) T-cell depletion 344 (13) Other 28 ( 1) ATG given 2692 No 2181 (81) Yes 511 (19) Donor/recipient sex match 2677 Male/Male 1119 (42) Male/Female 642 (24) Female/Male 445 (17) Female/Female 471 (18) 95

96 Not for publication or presentation Attachment 10 Continued. Characteristics of patients N Eval N (%) Donor/recipient CMV match 2692 Negative/Negative 829 (31) Negative/Positive 684 (25) Positive/Negative 366 (14) Positive/Positive 409 (15) Unknown 404 (15) Donor age, median (range), years (18-60) Donor age (24) (34) (24) 50 and older 168 ( 6) To Be Determined 339 (13) Year of transplant (<1) ( 1) ( 2) ( 3) ( 3) ( 3) ( 5) ( 5) ( 6) ( 7) ( 7) ( 7) ( 9) ( 9) ( 8) ( 1) (<1) ( 2) ( 3) ( 4) ( 5) ( 7) Median follow-up of survivors, mo (range) (3-265) 96

97 Not for publication or presentation Attachment 11 TO: Immunobiology Working Committee Members FROM: Steve Spellman, MBS; Co-Scientific Director for the Immunobiology WC Stephanie Lee, MD, MPH; Co-Scientific Director for the Immunobiology WC RE: Studies in Progress Summary HLA GENES IB05-02s: Effect of single Class I mismatching on unrelated donor hematopoietic stem cell transplantation (HCT) (MBA Heemskerk): This analysis will assess whether success of HCT with HLA class I mismatched unrelated donors differs depending on the number of amino acid sequence differences in the -helices and -sheet of the molecule. Results may help identification of potentially permissible mismatches. This is a collaborative study with the International Histocompatibility Working Group (IHWG). Analysis is underway. IB06-02: Impact of mismatches in low expression HLA loci on the outcome of unrelated donor transplantation (HCT) (M Fernandez-Vina): This study investigates the role of incompatibilities in the HLA DRB3/4/5, DQ and DP (low expression) loci (LEL) on the outcome of unrelated HCT. The hypothesis is that the effects of these loci are weak, cumulative and only demonstrable in combination with mismatches in other loci. A draft manuscript is underway and it is expected to be submitted in IB06-05: Use of high-resolution HLA data from the National Marrow Donor Program for the International Histocompatibility Working Group (IHWG) in hematopoietic cell transplantation (HCT) (E Petersdorf): The goal of the study is to to define the clinical importance of mismatching at specific HLA loci and at class I and II amino acid residues. This is a collaborative study with the International Histocompatibility Working Group (IHWG). Analysis is underway. IB07-04: Employing advanced bioinformatic methods for predicting peptide specificities of HLA molecules in the characterization of permissible mismatches in hematopoietic cell transplantation (HCT) (S Buus): This study proposes to identify a way to use a bioinformatic tool, MHCNetPan (M. Nielsen, et al., PLoS ONE2, e796, 2007) to define the distances for each pairwise donor-recipient HLA class I and II allele mismatch that are most strongly associated with post-transplant risks of acute GVHD and mortality. This is a collaborative study with the IHWG. Analysis is underway. IB07-05: Impact of donor-recipient ethnicity on risk of acute GVHD (GVHD) among HLA -A, B, C, DRB1, DQB1, DPB1 matched unrelated donor transplants (Y Morishima): The objective of the study is to evaluate the role of recipient and donor race on clinical outcomes after unrelated donor HCT. The analysis found that Japanese recipients experienced lower risks of acute GVHD, relapse and mortality compared to other ethnic groups. An abstract was presented at ASH This is a collaborative study with the IHWG. A draft manuscript is underway. IB07-06: Human leukocyte antigen: DP epitope study (B Shaw): This study proposes to determine the clinical importance of permissible and non-permissible T-cell epitopes defined HLA-DPB1 mismatches. This is a collaborative study with the IHWG. An oral abstract was presented at ASH 2010 and the manuscript was submitted to Lancet Oncology. 97

98 Not for publication or presentation Attachment 11 IB07-07: HLA -DR15 and hematopoietic stem cell transplantation (HCT) outcome (A Gratwohl): This study tests the hypothesis that the presence of HLA-DR15 influences clinical outcome after unrelated donor HCT. Results presented at EBMT 2009 suggest that DR15 does not confer a special susceptibility to alloimmune effects. This is a collaborative study with the IHWG. A draft manuscript is underway. IB08-02: HLA matching for unrelated hematopoietic cell transplantation (HCT) for non-malignant disorders (J Horan / A Woolfrey): This study will evaluate the impact of high resolution HLA-A, B, C, DRB1, DQB1 and DPB1 matching on the risks for mortality, GVHD and relapse after unrelated donor HCT for non-malignant disease. An oral abstract was presented at Tandem A draft manuscript has been circulated to the writing committee. IB09-02s: Non-permissive HLA-DPB1 disparities based on T-cell alloreactivity (K Fleischhauer): This study will validate the previous finding that HLA-DPB1 disparities in unrelated donor HCT can be classified as permissive and non-permissive according to T cell alloreactivity patterns and determine whether HLA-DPA1*0201 contributes to alloreactivity. Non-permissive HLA-DPB1 disparities were associated with increased risk of NRM and with protection from disease relapse in non-permissive pairs in the GVHD direction. The inclusion of HLA-DPA1 in the scoring scheme did not improve the assignments. An oral presentation was presented at EBMT 2011 as well as a poster presentation at EFI A draft manuscript is underway. IB11-03: Evaluation of the impact of allele homozygosity at HLA loci on outcome (CK Hurley/A Woolfrey/M Maiers): This study will evaluate the impact of HLA homozygosity at mismatched HLA loci in unrelated donor HCT. Previous studies have found an increased risk of graft failure in HCT where the mismatch is only in the host versus graft direction (recipient homozygous for the mismatched locus). Analysis is underway. IB11-04: Impact of amino acid substitution at peptide binding pockets of HLA class I molecules on HCT outcome (J Pidala/C Anasetti): The goal of the study is to examine the impact of specific amino acid substitutions at HLA class I peptide binding pockets on mismatched unrelated donor HCT. Analysis is underway. IB11-06: Evaluation of the impact of potentially non-immunogenic HLA-C allele level mismatch (M Fernandez-Vina/M Setterholm): This study will analyze the effect of an HLA0-C*03:03/C*03:04 (a putative non-immunogenic HLA mismatch) on the outcome of unrelated donor HCT. Analysis is underway. CYTOKINE/CHEMOKINE R04-75s: Functional significance of cytokine gene polymorphisms in modulating risk of post-transplant complications (E Petersdorf): This study is designed to identify immune response gene variants that are associated with risks of acute GVHD, relapse and mortality after unrelated donor HCT. This is a collaborative study with the IHWG. A draft manuscript is underway. IB05-03s: Genetic polymorphisms in the genes encoding human IL-7 Receptor-a: Prognostic significance in allogeneic stem cell transplantation (HCT) (K Muller): This study is designed to validate previous results from a single center analysis that suggested that donors carrying the +1237G variant in the alpha chain of IL-7R are associated with increased treatment related mortality and acute GVHD. The manuscript was submitted and rejected by Genes and Immunity. Revisions are underway and resubmission is planned for early

99 Not for publication or presentation Attachment 11 NK/KIR R02-40s/R03-63s: Choosing donors with favorable KIR B genotypes for unrelated hematopoietic cell transplantation (HCT) results in superior relapse protection and better relapse-free survival for patients with acute myeloid leukemia (AML) (J Miller): This is an ongoing study in support of Dr. Miller s NK Biology program project grant. R04-74s: Functional significance of Killer-IG-ligand genes in HLA-matched and mismatched unrelated hematopoietic cell transplantation (HCT) (K Hsu / B DuPont): The purpose of this study is to determine the influence of donor KIR genotypes and haplotypes on HCT for leukemia. This is a collaborative study with the IHWG and is an ongoing effort. An oral abstract was presented at Tandem 2011 and a manuscript was submitted to the New England Journal of Medicine. IB07-03: Analysis of KIR ligands in reduced intensity conditioning allogeneic hematopoietic stem cell transplantation (HCT) (R Sobecks): The objectives of this study are to evaluate the clinical effects of KIR ligand absence in recipients of HLA matched and mismatched unrelated donor RIC HCT for myeloid malignancies. Retrospective donor KIR genotyping to determine the relevance of recipient KIR ligand absence was recently completed. Analysis is underway. IB11-05: KIR genotyping and Immune function in MDS patients prior to unrelated donor transplantation (E Warlick/J Miller): The goal of this study is assess the correlation of NK cell function with disease progression in MDS and potentially develop therapeutic immune strategies to control MDS progression. The retrospective cohort provided by the CIBMTR will be compared to a prospective cohort of newly diagnosed MDS patients from the University of Minnesota. This is an ongoing study in support of Dr. Miller s NK Biology program project grant. OTHER GENES R04-76s: Identification of functional single nucleotide polymorphisms (SNPs) in unrelated hematopoietic cell transplantation (HCT) (E Petersdorf): This study proposes to identify novel major histocompatibility complex resident SNPs of clinical importance. HLA matched pairs were genotyped for 1120 MHC SNPs and correlation with outcome is underway. This is a collaborative study with the IHWG. Analysis is underway. IB07-08: Single nucleotide polymorphisms in the P53 Pathway (P53, MDM2, ATM AND P21/WAF1) and transplant outcome after unrelated haematopoietic stem cell transplantation (HCT) (B DuPont): The goal of the study is to determine if tumor suppressor p53 Arg72Pro polymorphism affects posttransplantation survival. The study found that recipient polymorphism in p53 may be a risk factor for worse long-term survival. The study was expanded to include RIC regimens and HLA mismatched cases. The committee provided a letter of support and contributed text describing the sample resources available to the study for an R01 grant proposal. This is a collaborative study with the IHWG. Testing is underway. IB07-09: To develop and test a prognostic index for survival in chronic myelogenous leukemia matched unrelated donor cohorts (A Dickinson): The purpose of the study is to validate previous work in HLAmatched sibling transplantation showing that cytokine SNPs were associated with decreased survival and improved prognostic accuracy when combined with the European Group for Blood and Marrow Transplantation (EBMT) risk score in CML patients. The results of the analysis were negative. The manuscript was submitted to Haematologica in IB08-08: Genome-wide association in unrelated donor hematopoietic cell transplant (HCT) recipients and donors (R Goyal): This study hypothesizes that an unbiased recipient-donor genome-wide 99

100 Not for publication or presentation Attachment 11 association (GWA) study will identify genes associated with risk of acute graft versus host disease (agvhd) after HLA-matched unrelated donor HCT. Testing is underway. IB09-04s: Donor/recipient gene polymorphisms of drug metabolism and in innate immune response post allele-matched unrelated donor hematopoietic stem cell transplantation (HCT) (V Rocha): This study is designed to validate associations between polymorphisms in drug metabolism and innate immune response genes and outcomes previously identified in matched sibling donor HCT in the HLA-matched unrelated donor HCT setting. Testing is underway. IB09-06s/RT09-04*: Genetic susceptibility to transplant-related mortality after matched unrelated stem cell transplant (T Hahn): This is a joint study with the Regimen Related Toxicity working committee and is supported by an R01 grant to Drs. Hahn and Sucheston. This study will test for a genetic association with transplant-related and overall mortality in recipients of myeloablative and reduced intensity conditioning matched unrelated donor HCT. The committee provided a letter of support and contributed text describing the sample resources available to the study for the R01 grant proposal. Testing is underway. IB09-07s: Clinical significance of genome-wide variation in unrelated donor hematopoietic stem cell transplantation (HCT) (E Petersdorf): This study is designed to assess the impact of genome-wide variation between donors and recepients in HLA matched unrelated donor HCT. This is a collaborative study with the IHWG. The committee provided a letter of support and contributed text describing the sample resources available to the study for a U01 grant proposal. Testing is underway. IB10-01s: Donor and recipient telomere length as predictors of outcomes after hematopoietic stem cell transplant in patients with acquired severe aplastic anemia (S Gadalla): More than one-third of patients with acquired severe aplastic anemia (SAA) have short telomeres. Telomere shortening in peripheral blood is associated with increased risk of malignancies, pulmonary and liver fibrosis, and other complications. This study will explore the role that telomere length plays in outcomes after HCT for SAA. Testing is underway. IB10-03: TLR and HMGB1 gene polymorphisms in unrelated haematopoietic stem cell transplantation (K Müller/ B Kornblit): The objective of this study is to validate single center findings of a correlation between High Mobility Group Box 1 (HMGB1) polymorphism in the recipient and relapse rates following HLA matched unrelated donor HCT. Analysis is underway. IB10-04: A validation study of the role of base excision repair pathway as a predictor of outcome after hematopoietic stem cell transplant (M Arora): This study is designed to validate findings from a single center that noted a correlation between SNPs in the DNA repair genes and relapse and TRM after HLA matched unrelated donor HCT. The committee provided a letter of support and contributed text describing the sample resources available to the study for an R01 grant proposal. Analysis is underway. IB11-02s: Impact of CTLA4 single nucleotide polymorphisms on outcome after unrelated donor transplant (M Jagasia/W Clark/B Savani/S Sengsayadeth): The aim of the study is to validate findings from a single center analysis that found an association between CTLA4 single nucleotide polymorphisms and relapse, acute and chronic GVHD in unrelated donor HCT for hematological malignancies. A draft protocol is complete and external funding is being pursued to complete the sample testing. 100

101 Not for publication or presentation Attachment 11 SENSITIZATION and TOLERANCE R03-65s: Detection of H-Y antibodies in healthy female donors: Does H-Y presensitization predict male hematopoietic stem cell transplantation (HCT) outcome (D Miklos): The testing methodology has been refined and sample testing will be completed in IB06-09s: Detection of HLA antibody to the mismatched antigen in single antigen HLA-mismatched unrelated donor transplantation (HCT): Is it associated with GVHD outcome? (S Arai): Draft manuscript is underway and expected to be submitted in IB06-10: Evaluation of the impact of exposure to non-inherited maternal antigens (NIMA) during fetal life and breast feeding and to the inherited paternal antigens during pregnancy on the clinical outcome of hematopoietic cell transplantation (HCT) from haploidentical family members (J Van Rood): This study will evaluate the impact of NIMA on haploidentical related HCT for hematological malignancies. The study requires HLA typing on the patient, donor and patient parents to assign the presence or absence of NIMA matches. A comprehensive review of the available HLA data was completed August Planning is underway for a combined study with EBMT. IB06-11s: Effect of non-inherited maternal antigens (NIMA) in cord blood transplantation (VK Prasad / LA Baxter-Lowe): This study will test the hypothesis that matching for NIMA in the selection of cord blood units will favorably influence outcomes. The U.S. study population was combined with a similar cohort from Eurocord to augment the power for analysis. A matched case-control analysis was completed and found that NIMA matching had a positive effect on treatment related mortality and overall survival. An oral abstract was presented at EBMT 2011 and manuscript was submitted to Blood. IB09-08: Donor/recipient birth order in matched sibling hematopoietic stem cell transplantation (HCT) (C Dobbelstein): The study proposes to validate findings from a single center study that found matched sibling HCT utilizing donors born after the recipient yielded superior survival with lower incidences of acute GVHD and relapse. The results of the analysis did not support the hypothesis. A draft manuscript is underway. An oral abstract was presented at EHA IB11-01: Analysis of the NIMA effect on the outcome of unrelated PBSC/BM transplantation (G Ehninger/J van Rood/A Schmidt): The goal of this study is to determine whether matching for NIMA in the selection of unrelated donors can lead to better outcomes. The study will be restricted to transplants where the donors were supplied by the DKMS. The DKMS team will attempt to collect samples from each donor s mother for HLA typing and assignment of NIMA matching. Testing is underway. IB11-07: Effect of Rituximab and ABO mismatch (D Miklos/A Logan): The aim of the study is to validate the finding of a single center analysis that found an association between ABO minor mismatch and increased acute GVHD. The study did not validate the acute GVHD findings, but did find a negative impact of minor ABO mismatch on overall survival, treatment related mortality and treatment failure compared to ABO matched transplantation. A draft manuscript is underway. MINOR HISTOCOMPATIBILITY ANTIGENS IB11-08: Synergism between minor and major histocompatibility antigens (E Spierings): Approved studies not yet initiated: HLA GENES IB10-07: Use of HLA structure and function parameters to understand the relationship between HLA disparity and transplant outcomes (LA Baxter-Lowe): This study is designed to develop an HLA disparity scoring system based on the molecular structure and alloreactive function of the mismatched 101

102 Not for publication or presentation Attachment 11 amino acids. The system will then be evaluated for correlation with survival, disease-free survival, relapse, GVHD and engraftment following HLA mismatched unrelated donor HCT. A protocol will be available March R04-80s: Impact of HLA matching on outcome in pediatric patients undergoing unrelated umbilical cord blood transplantation (SR Marino): Study deferred pending data collection. IB06-13: HLA disparity among unrelated umbilical cord blood transplants (LA Baxter-Lowe): Study deferred pending data collection. IB09-01s: Clinical importance of major histocompatibility complex haplotypes in umbilical cord blood transplantation (E Petersdorf): Study deferred pending data and sample collection. CYTOKINE/CHEMOKINE IB08-05/LK08-04*: Lymphotoxin alpha alleles in acute myelogenous leukemia (AML) relapse (P Posch): This is a joint study with the Acute Leukemia Working Committee which proposes to determine whether LTA alleles correlate with relapse in AML and CML and to determine if the correlation is associated with high or low LTA production. Study deferred pending in vitro assessment of LTA polymorphism. IB08-04: Immune response gene polymorphisms in unrelated donor hematopoietic cell transplantation (HCT) in children (K Muller): Study awaiting completion of IB05-03s by the same study team. IB09-03s: Clinical relevance of cytokine/immune response genes in umbilical cord blood transplant (E Petersdorf): Study deferred pending data and sample collection. NK/KIR IB08-06: KIR ligands in umbilical cord blood hematopoietic cell transplantation (HCT) (R Sobecks): Study deferred pending data and sample collection. OTHER GENES IB09-05s: Identification of functional single nucleotide polymorphisms (SNPs) in umbilical cord blood transplant (E Petersdorf): Study deferred pending data and sample collection. MINOR HISTOCOMPATIBILITY ANTIGENS IB10-06: Identification of common, clinically significant, minor histocompatibility antigens through stem cell transplant donor/patient polymorphism disparities (P Armistead): The central hypothesis of this research project is that many donor/patient mhag mismatches result from non-synonymous (i.e. encoding a different amino acid) single nucleotide polymorphisms in the coding regions of genes (csnps) and that cytotoxic T lymphocytes (CTLs) recognizing these mhags can cause GvHD and/or GvL. These two conditions may be separable based upon the tissue specificity of the individual mhags. The study will proceed following receipt of funding. 102

103 Not for publication or presentation Attachment 12 CIBMTR IB11-01 ANALYSIS OF THE NIMA EFFECT ON THE OUTCOME OF UNRELATED PBSC/BM TRANSPLANTATION DRAFT PROTOCOL Study Chair: Study Co-Chairs: Gerhard Ehninger, MD University Hospital Carl Gustav Carus Fetscherstrasse Dresden, Germany Telephone: Fax: Jon J. van Rood, MD Leiden University Medical Center Albinusdreef 2, 2333 ZA Leiden, The Netherlands Telephone: Fax: Alexander H. Schmidt, MD, PhD DKMS German Bone Marrow Donor Center Kressbach 1, Tuebingen, Germany Telephone: Fax: Study Statistician: Scientific Directors: TBD Stephanie Lee, MD, MPH Fred Hutchinson Cancer Research Center P.O. Box 19024, D Fairview Avenue North Seattle, WA USA Telephone: Fax:

104 Not for publication or presentation Attachment 12 Scientific Directors: Working Committee Chairs: Stephen Spellman, MS CIBMTR National Marrow Donor Program 3001 Broadway Street NE, Suite 100 Minneapolis, MN USA Telephone: Fax: David Miklos, MD, PhD Stanford University Department of Medicine; BMT Division CCSR, Room # West Campus Drive Stanford, CA USA Telephone: Fax: Marcelo Fernandez-Vina, PhD Professor Department of Pathology/Blood Center Stanford University School of Medicine 3373 Hillview Avenue Palo Alto, CA USA Telephone: Fax: Carlheinz Mueller, MD, PhD Director German National Bone Marrow Donor Registry ZKRD Helmholtzstrabe 10, Ulm, Germany Telephone: Fax:

105 Not for publication or presentation Attachment 12 OBJECTIVES: 1.0 Analyze the NIMA effect for unrelated adult donor PBSC/BM transplantation for hematological malignancies, including myelodysplasia. Primary study end point: Transplant-related mortality (TRM) Secondary study end points: Neutrophil engraftment (ANC 500/mm 3 for 3 consecutive lab values on different days) Platelet engraftment ( 20 x 10 9 /l, 50 x 10 9 /l) Incidence of grade II-IV and III-IV agvhd Incidence of cgvhd Relapse Overall mortality Treatment failure (relapse or death) 2.0 SCIENTIFIC JUSTIFICATION: The relevance of the NIMA effect, i.e., the lifelong modulating impact of non-inherited maternal antigens on the immune response of the child, for the outcome of allogeneic stem cell transplantation is under scientific debate for many decades. In haploidentical sibling transplantation, a reduction of GVHD risk after transplantation through the NIMA effect could be observed (van Rood et al., 2002). Especially for older patients, unrelated NIMA-matched cord blood transplantation resulted in lower transplant-related mortality, overall mortality, and treatment failure (van Rood et al., 2009). No studies so far have analyzed the NIMA effect for unrelated adult donor stem cell transplantation. The main problem in this setting lies in the general unavailability of maternal HLA phenotype data. In the proposed study, we will ask stem cell donors to provide maternal samples from buccal swabs for HLA typing. A verification of the NIMA effect for unrelated adult donor stem cell transplantation could have considerable impact on unrelated donor search. For example, a maternal sample could routinely be requested on the CT level. 3.0 STUDY POPULATION: - Patients with AML, ALL, CML, NHL, or MDS who have undergone a T-cell repleted unrelated bone marrow (BM) or peripheral blood stem cell (PBSC) donation. - HLA typing result of patients and donors must be available at high resolution at least for the HLA loci A, B, C, and DRB1. - Patient s donor must be from DKMS German Bone Marrow Donor Center. - For donors with at least one mismatch: Sample of the donor s mother must be available. (Sample and HLA typing of the donor s mother will be provided by DKMS.) 4.0 OUTCOMES: Primary: Transplant-related mortality: Death without evidence of disease recurrence. Event will be summarized by the cumulative incidence estimate with relapse as competing risk. 105

106 Not for publication or presentation Attachment 12 Secondary: Neutrophil engraftment: Achievement of ANC 500/mm 3 for 3 consecutive lab values on different days. Event will be summarized by the cumulative incidence estimate with death as competing risk. Platelet engraftment ( 20 x 10 9 /l, 50 x 10 9 /l): Achievements of platelets 20 x 10 9 /l and platelets 50 x 10 9 /l. Events will be summarized by the cumulative incidence estimate with death as competing risk. Incidence of grade II-IV and III-IV agvhd: Development of grades II-IV and III-IV acute GVHD (Glucksberg system). Events will be summarized by the cumulative incidence estimate with death as competing risk. Incidence of cgvhd: Development of symptoms in any organ system fulfilling the criteria of chronic GVHD. Event will be summarized by the cumulative incidence estimate with death as competing risk. Relapse: Recurrence of disease. Event will be summarized by the cumulative incidence estimate with TRM as competing risk. Overall mortality: Time to death from any cause. Event will be summarized by a survival curve. Cases will be censored at the time of last follow-up. Treatment failure (relapse or death): Time to death or relapse. Event will be summarized by a survival curve. Cases will be censored at the time of last follow-up. 5.0 VARIABLES TO BE ANALYZED: Patient-related: - Age: in decades - Gender: female vs. male - Karnofsky score at transplant: < 90 vs Disease-related: - Disease at transplant: AML, ALL, CML, NHL and MDS - Disease stage at transplant: early vs. intermediate vs. advanced Donor- and transplant-related: - Stem cell source: BM vs. PBSC - Conditioning regimen: myeloablative vs. non-myeloablative - GVHD prophylaxis: CSA + MTX +/- others vs. CSA + others (no MTX) vs. MTX + others (no CSA) vs. FK506 +/- others vs. MMF +/- others vs. others - Time from diagnosis to transplant: 1 year vs. > 1 year - HLA match: no MM vs. 1 MM + NIMA match vs. 1 MM + NIMA MM vs. 2 MM + NIMA match vs. 2MM + 1NIMA MM vs. 2MM + 2NIMA MM - Gender match: M-M vs. M-F vs. F-M vs. F-F - Donor recipient CMV status: -/- vs. -/+ vs. +/- vs. +/+ - Donor age: in decades - Year of transplant: ATG: yes vs. no 6.0 STUDY DESIGN: To summarize the characteristics of the data set, descriptive tables of patient-, disease-, and donor-/transplant-related variables will be reported. 106

107 Not for publication or presentation Attachment 12 - For discrete variables, the number of cases and their respective percentages will be calculated. X 2 tests will be used to compare discrete variables between the various groups according to with the 1 MM + NIMA MM group as reference. - For continuous variables, the median and ranges will be calculated. The Kruskal-Wallis test will be used to compare continuous variables between the various groups according to with the 1 MM + NIMA MM group as reference. Probabilities for overall mortality (5.2.6) or treatment failure (5.2.7) will be calculated using the Kaplan-Meier estimator with variance estimated by Greenwood s formula. Values for other outcomes given in 5.1 and 5.2 will be calculated according to cumulative incidence using a Taylor series linear approximation to estimate the variance. Multivariate analysis will be performed using the proportional hazards model to compare the various groups according to with the 1 MM + NIMA MM group as reference. Additional Information: Based on actual donor-recipient pairs and allele frequencies of the donor population, we estimate about 5% of all mismatches to be NIMA matches. We expect to observe about 50% of the NIMA matches for the C locus. A pilot study was carried out in order to estimate the feasibility of collecting maternal DNA samples. Within this pilot, 150 donors were contacted and asked to forward information material and buccal swabs to their mothers. 68 mothers provided samples for HLA high-resolution typing of the mismatched locus of the original transplantation. 6 additional mothers provided their DKMS donor IDs and declared informed consent for scientific evaluation of their HLA data within the project, leading to a total return rate of 49.3% (74/150).Within the pilot, 3 NIMA matches were observed on the allele level (4 on the antigen level). Assumptions for power calculation: TRM for the 1 MM + NIMA MM group: 45%; TRM for the 1 MM + NIMA match group: 30%; 5% of MM are NIMA matches; type I error =0.05; study power 1- =0.8. From these data we calculated a required sample size of 1,804 donor-recipient pairs (including 90 with NIMA matches) using Fisher s exact test. Based on the return rate of 49.3% that was derived from the pilot study, 3,657 donors have to be contacted to reach this sample size. 7.0 REFERENCES: 1. Van Rood JJ, Loberiza FR, Zhang MJ, et al. Effect of tolerance to noninherited maternal antigens on the occurrence of graft-versus-host disease after bone marrow transplantation from a parent or an HLA-haploidentical sibling. Blood 2002; 99(5): Van Rood JJ, Stevens CE, Smits J, et al. Reexposure of cord blood to noninherited maternal HLA antigens improves transplant outcome in hematological malignancies. Proc Natl Acad Sci USA. 2009; 106:

108 Not for publication or presentation Attachment 12 Table 1. Characteristics of recipients receiving first t-replete transplants for AML, ALL, CML, MDS or NHL and are high-resolution typed for HLA-A, -B, -C and DRB1 * Variable N Eval N (%) Number of patients 1381 Number of centers 115 Recipient age, median (range), years (<1-74) Age at transplant y 51 ( 4) y 100 ( 7) y 177 (13) y 182 (13) y 296 (21) Over 50 y 574 (42) Male sex (57) Karnofsky prior to transplant > (64) Disease at transplant 1381 AML 630 (46) ALL 237 (17) CML 135 (10) MDS 229 (17) NHL 150 (11) Disease status at transplant 1381 Early 474 (34) Intermediate 282 (20) Advanced 279 (20) Other 346 (25) Graft type 1381 Bone marrow 301 (22) PBSC 1080 (78) HLA matching for HLA-A, -B, -C and -DRB Well matched 1028 (75) Partially matched 294 (21) Mismatched 59 ( 4) High-resolution typing for HLA-A,-B,-C and -DRB /8 allele-matched 1028 (74) 7/8 single mismatch 294 (21) 6/8 two mismatches 49 ( 4) 5/8 three mismatches 7 ( 1) 4/8 four mismatches 3 (<1) * Data has not been CAP-modeled. 108

109 Not for publication or presentation Attachment 12 Table 1. Continued. Variable N Eval N (%) Conditioning regimen 1381 Myeloablative 895 (65) Reduced intensity 282 (20) Nonmyeloablative 133 (10) Other/To be determined 71 ( 5) GVHD prophylaxis 1381 FK506 + (MTX or MMF or Steroids) other 912 (66) FK506 other 61 ( 4) CsA + MTX other 233 (17) CsA other (no MTX) 106 ( 8) MMF other 3 (<1) MTX other (no CsA) 6 (<1) Other/To be determined 60 ( 4) Donor/recipient sex match 964 Male/Male 416 (43) Male/Female 255 (26) Female/Male 135 (14) Female/Female 158 (16) Donor/recipient CMV match 1381 Negative/Negative 356 (26) Negative/Positive 335 (24) Positive/Negative 100 ( 7) Positive/Positive 147 (11) Unknown 443 (32) Donor age, median (range), years ( ) Donor age ( 2) (27) (26) (12) 50 and older 26 ( 2) Unknown 430 (31) 109

110 Not for publication or presentation Attachment 12 Table 1. Continued. Variable N Eval N (%) Year of transplant (<1) (<1) (<1) ( 1) ( 2) ( 1) ( 2) ( 5) ( 9) (12) (17) (19) (14) (14) ( 2) Median follow-up of recipients, mo (range) (1-149) 110

111 Not for publication or presentation Attachment 13 CIBMTR IB11-07 EFFECT OF PRETRANSPLANT RITUXIMAB UPON ABO MISMATCH HEMATOPOIETIC CELL TRANSPLANTATION DRAFT PROTOCOL Study Chairs: Study Co-Chair: Study Statistician: Scientific Directors: David Miklos, MD, PhD Stanford University School of Medicine Department of Blood and Marrow Transplant 875 Blake Wilbur Dr, Rm 2315 MC 5825 Stanford, CA Telephone: Fax: Aaron C. Logan, MD, PhD Stanford University School of Medicine Department of Blood and Marrow Transplant 875 Blake Wilbur Dr, Rm 2315 MC 5825 Stanford, CA Telephone: Fax: Michael D. Haagenson, MS Center for International Blood and Marrow Transplant Research 3001 Broadway Street NE, Suite 100 Minneapolis, MN USA Telephone: Fax: Stephanie J. Lee, MD, MPH Fred Hutchinson Cancer Research Center 1100 Fairview Ave North, D5-290 Seattle, WA Telephone: Fax:

112 Not for publication or presentation Attachment 13 Stephen Spellman, MBS CIBMTR National Marrow Donor Program 3001 Broadway Street NE, Suite 100 Minneapolis, MN USA Telephone: Fax: Working Committee Chairs: David Miklos, MD, PhD Stanford University Department of Medicine; BMT Division CCSR, Room # West Campus Drive Stanford, CA USA Telephone: Fax: Marcelo Fernandez-Vina, PhD Professor Department of Pathology/Blood Center Stanford University School of Medicine 3373 Hillview Avenue Palo Alto, CA USA Telephone: Fax: Carlheinz Mueller, MD, PhD Director German National Bone Marrow Donor Registry ZKRD Helmholtzstrabe 10, Ulm, Germany Telephone: Fax:

113 Not for publication or presentation Attachment OBJECTIVES: Reanalyze the already available 435 patient Rituximab vs. No Rituximab cohort focusing on donor and patient ABO status. 1.1 Determine if minor ABO mismatch is associated with increased acute GVHD and NRM in the absence of prior rituximab therapy. 1.2 Determine if rituximab within 6 months of allo-hct prevents acute GVHD and NRM differences in minor ABO mismatch compared to match 2.0 SCIENTIFIC JUSTIFICATION: A previous CIBMTR study showed rituximab infusion within 6 months prior to allogeneic hematopoietic cell transplantation (allo-hct) was associated with decreased acute GVHD and improved overall survival. 1 Minor ABO mismatch has been associated with increased acute GVHD, 2 and Stanford institutional analysis shows minor ABO mismatch is associated with increased non-relapse mortality and may be mitigated by rituximab therapy prior to allo-hct. 3 The CIBMTR study did not analyze donor and recipient ABO impact. The clinical impact of ABO blood group mismatching between hematopoietic cell transplant (HCT) patients and their donors remains controversial. HCT recipients with antibodies against ABO antigens expressed by donor are ABO major mismatches (ex. Rec: O DNR: A) while minor ABO minor mismatch describes the converse of donor plasma containing antibodies against recipient expressed ABO antigen. Seebach et al. BBMT 2005; CIBMTR ABO Mismatch Analysis ABO impact varies by conditioning regimen, graft source, donor engraftment chimerism, and institution transfusion practices. 4-7 Recently Ludajic et al. demonstrated minor ABO mismatch is associated with significant risk of acute GVHD with an estimated risk increase of almost 3 fold (hazard ratio =2.92 P=0.003 suggesting ABO may play a role in acute GVHD pathophysiology. 2 We recently analyzed clinical outcomes for 1955 allogeneic transplants performed at Stanford University since 1986 as a function of donor and recipient ABO types, and found increased nonrelapse mortality in minor ABO mismatched patients. 3 From January 1986 thru January 2010, 1955 patients underwent allogeneic HCT with ages ranging 3-74 (median 46 years). Myeloablative conditioning including total body irradiation high-dose chemotherapy alone was used for 1443 transplants. Nonmyeloablative conditioning (n=512) included single 200TBI (n=193), and total lymphoid irradiation antithymoglobulin (TLI-ATG; n=319). Overall, donor and recipient ABO typing identified 63% ABO-matched, 15% ABO-major mm, 15% ABO-minor 113

114 Not for publication or presentation Attachment 13 Figure 1. mm, and 7% ABO-bidirectional mm. ABO mismatch distribution did not differ by donor or recipient age, conditioning regimen, or disease. Considering all 1955 Stanford patients, Figure 1a shows ABO minor mm had decreased overall survival compared to ABO matched transplants (p<0.05). This mortality deficit developed by 100 days after HCT (p<0.01) and was predominately due to non-relapse mortality (Figure 1b) and acute GVHD (Figure 1d). In contrast, ABO-major mm and bidirectional mm were similar to ABO matched transplants through 2 years. Univariate analysis showed decreased OS and 100 day survival in ABO minor mm associated with bone marrow graft source (figure 1c), and 2) non rituximab treated diseases including AML, ALL, MDS, CML (Figure 3). Figure 1a. Overall survival by ABO match ABO MATCHED MINOR MISMATCHED MAJOR MISMATCHED BIDIRECTIONAL MM 1b. NRM by ABO match ABO MATCHED MINOR MISMATCHED 1c. Overall survival in patients receiving bone marrow grafts 1d. Grade 2 4 agvhd by ABO ABO MATCHED, n=436 MINOR MISMATCHED, n=111 ABO MATCHED MINOR MISMATCHED We have subsequently gone on to score for non-hodgkin lymphoma patients who received rituximab prior to allo-hct and show that ABO minor mismatch and ABO matched patients have similar OS (Figure 3b). Ludajic et al. studied 154 patients with unrelated donors mixing T cell depletion, graft source (BM v PBSC), and diseases, but report ABO minor mismatch 114

115 Not for publication or presentation Attachment 13 provides a RR=4 [CI: ; p=0.002]. The authors suggest ABO minor mismatches may play a role in agvhd pathophysiology, and recommend ABO matching. 2 Ratanatharathorn et al reported the 2009 CIBMTR analysis of 435 B cell lymphoma patients who received allo-hct from Rituximab was received within 6 months of allo-hct in 179 subjects while 256 did not receive rituximab within 6 months of allo-hct. Figure 1 is reproduced from their publication and label Figure 2a-d in this proposal. This CIBMTR analysis showed significantly lower incidence of transplant related mortality (TRM) [RR=0.68, ; p=0.05] with lower acute GVHD III-IV RR=0.55, ]. But importantly, ABO mismatch was not evaluated (Confirmed with S. Spellman). Figure 2 reproduces Figure 1 from Ratanatharathorn et al. British Journal of Haematology 2009, CIBMTR analysis of Rituximab before HCT. 115

116 Not for publication or presentation Attachment 13 Figure 3. Minor ABO mismatch has decreased overall survival in AML, MDS, CML patients transplanted at Stanford that never received rituximab compared to B cell NHL patients receiving rituximab pretransplant. 3.0 STUDY POPULATION: 3.1 Included: - The same 435 B cell lymphoma patient cohort studied by Ratanatharathorn et al. 1 will be studied. - All grafts were peripheral blood stem cell grafts. - Allo-HCT occurred between 1999 and patients received rituximab as part of their conditioning regimen and were included in the 179 rituximabcohort (RTX) 3.2 Excluded: - Patients that had received prior therapy with anti-cd52 or anti-t cell antibodies - Recipients of marrow or cord blood grafts - Recipients of T-depleted graft from any source - Recipients of mismatched sibling graft 4.0 OUTCOMES: 4.1 Primary: Transplant-related mortality: as a function of minor ABO mismatch vs. ABO matched in 256 subjects who did not receive rituximab within 6 months of HCT (No-RTX cohort) Transplant-related mortality: as a function of minor ABO mismatch vs. ABO matched in 179 subjects who did receive rituximab within 6 months of HCT (RTX cohort). 4.2 Secondary (end points related to ABO status): Incidence of grade II-IV and III-IV agvhd 116

117 Not for publication or presentation Attachment Incidence of cgvhd Relapse Overall mortality 5.0 VARIABLES TO BE ANALYZED: 5.1 Patient-related variables: - Age at transplant (21-40, 41-50, and years) - Lymphoma histology (small lymphocytic and follicular vs. diffuse large B cell vs. mantle cell lymphoma) - Disease status prior to transplantation 5.2 Transplant-related variables: - Donor type Related vs. unrelated - Time from last dose of rituximab to transplantation (>6 months vs. < 6 months). - Gender: female vs. male - Karnofsky score at transplant: <90 vs Conditioning regimen myeloablative vs. non-myeloablative - Year of transplant ( vs ) 6.0 STUDY DESIGN: We will reanalyze the rituximab vs. no rituximab CIBMTR already available data set for impact of donor and recipient ABO effect, especially ABO minor mismatch impact on TRM, acute GVHD, chronic GVHD, relapse, and overall survival using the same clinical outcomes analysis previously reported For discrete variables, the number of cases and their respective percentages will be calculated. χ2 tests will be used to compare discrete variables between the various groups. 6.2 For continuous variables, the median and ranges will be calculated. The Kruskal-Wallis test will be used to compare continuous variables between the various groups. 6.3 Probabilities for overall mortality or treatment failure will be calculated using the Kaplan-Meier estimator with variance estimated by Greenwood s formula. Values for other outcomes will be calculated according to cumulative incidence using a Taylor series linear approximation to estimate the variance. Multivariate analysis will be performed using the proportional hazards model to compare the various groups. Ongoing Correlative studies at Stanford: - Measuring ABO specific B cells using Jerne Plaque Technique 14, 28, and 56 days after allo- HCT in ABO mismatched and matched recipients - Developing B cell specific A and B tetramer FACS reagent. 7.0 REFERENCES: 1. Ratanatharathorn V, Logan B, Wang D, et al. Prior rituximab correlates with less acute graftversus-host disease and better survival in B-cell lymphoma patients who received allogeneic peripheral blood stem cell transplantation. Br J Haematol. 2009;145:

118 Not for publication or presentation Attachment Ludajic K, Balavarca Y, Bickeboller H, et al. Minor ABO-mismatches are risk factors for acute graft-versus-host disease in hematopoietic stem cell transplant patients. Biol Blood Marrow Transplant. 2009;15: Alimoghaddam K, Logan AC, Wong R, et al. Minor ABO mismatch is associated with increased non-relapse mortality after allogeneic hematopoietic cell transplantation. American Socitey of Blood and Marrow Transplantation Honolulu, HI; Wang Z, Sorror ML, Leisenring W, et al. The impact of donor type and ABO incompatibility on transfusion requirements after nonmyeloablative haematopoietic cell transplantation. Br J Haematol;149: Benjamin RJ, McGurk S, Ralston MS, Churchill WH, Antin JH. ABO incompatibility as an adverse risk factor for survival after allogeneic bone marrow transplantation. Transfusion. 1999;39: Seebach JD, Stussi G, Passweg JR, et al. ABO blood group barrier in allogeneic bone marrow transplantation revisited. Biol Blood Marrow Transplant. 2005;11: Blin N, Traineau R, Houssin S, et al. Impact of donor-recipient major ABO mismatch on allogeneic transplantation outcome according to stem cell source. Biol Blood Marrow Transplant;16:

119 Not for publication or presentation Attachment 13 Table 1. Characteristics of patients receiving HLA-identical sibling or unrelated donor peripheral blood transplants for Non-Hodgkin s Lymphoma between 1999 and 2004, reported to CIBMTR Characteristics of patients No Rituximab Rituximab N (%) N (%) P-value d Number of patients Age, median (range), years 50 (22-70) 50 (22-67) 0.42 Age at transplant (17) 34 (19) (31) 60 (34) (52) 85 (47) Male sex 169 (66) 121 (68) 0.71 Performance score 0.32 < (35) 54 (30) (64) 119 (66) Unknown 4 ( 2) 6 ( 3) Histology of lymphoma 0.07 Small cell 18 ( 7) 4 ( 2) Follicular 133 (52) 92 (51) Diffuse large cell 45 (18) 46 (26) Mantle cell 53 (21) 32 (18) Other a 7 ( 3) 5 ( 3) Disease status prior to transplant 0.07 PR partial remission 90 (35) 63 (35) CR complete remission 55 (21) 47 (26) Rel sensitive 58 (23) 50 (28) Rel resistant/untreated/unknown/progressive disease 51 (20) 18 (10) Missing 2 ( 1) 1 ( 1) Donor HLA-identical siblings 208 (81) 122 (68) Unrelated donor 48 (19) 57 (32) Number of chemotherapy regimen 0.69 < 3 lines 95 (37) 72 (40) 3-6 lines 153 (60) 100 (56) > 6 lines 8 ( 3) 7 ( 4) Radiation therapy, yes 54 (21) 40 (22) 0.75 Conditioning regimen 0.84 Myeloblative 117 (46) 80 (45) Non-myeloblative 139 (54) 99 (55) GVHD prophylaxis <0.001 CSA + MTX ± other 103 (40) 46 (26) CSA ± other 84 (33) 45 (25) FK506 + MTX ± other 40 (16) 63 (35) FK506 ± other 25 (10) 22 (12) Other b 4 ( 2) 3 ( 2) 119

120 Not for publication or presentation Attachment 13 Table 1. Continued. Characteristics of patients No Rituximab Rituximab N (%) N (%) P-value d Donor/Recipient sex match 0.76 Male / Male 92 (36) 75 (42) Male / Female 46 (18) 32 (18) Female / Male 77 (30) 46 (26) Female / Female 39 (15) 25 (14) Unknown 2 ( 1) 1 ( 1) Donor pregnancy 0.37 Male donor 138 (54) 107 (60) Female, no pregnancy 17 ( 7) 14 ( 8) 1 or more pregnancies 60 (23) 30 (17) Unknown 41 (16) 28 (16) Donor/Recipient CMV match 0.05 D(-)/R(-) 66 (26) 57 (32) D(-)/R(+) 51 (20) 43 (24) D(+)/R(-) 32 (13) 30 (17) D(+)/R(+) 92 (36) 42 (23) Unknown 15 ( 6) 7 ( 4) Year of transplant < (34) 24 (13) (66) 155 (87) Number of doses of rituxan received N/A 1 dose (46) 2 doses (36) 3 doses (11) 4 doses -- 9 ( 5) 5 doses -- 3 ( 2) Time from last dose of rituxan to transplant N/A < 3 months (67) 3-6 months (33) ABO Matching for recipient/donor 0.87 ABO matched 152 (59) 104 (58) Minor ABO mismatch 47 (18) 30 (17) Major ABO mismatch 44 (17) 33 (18) Bidirectional mismatch 12 ( 5) 10 ( 6) Unknown blood type for recipient and/or donor 1 (<1) 2 ( 1) HLA-matching 0.01 HLA-identical sibling 208 (81) 122 (68) Well-matched 38 (15) 42 (23) Partially matched 8 ( 3) 10 ( 6) Mismatched 2 ( 1) 5 ( 3) Median (range) time from diagnosis to transplant, months 25 (2-194) 22 (4-196) 0.04 Median follow-up of survivors, months 39 (3-88) 30 (3-82) Abbreviations: CSA = Cyclosporine, MTX = Methotrexate, FK506 = Tacrolimus. 120

121 Not for publication or presentation Attachment EUROPEAN HEMATOLOGY ASSOCIATION MEETINGS ABSTRACT Birth Order is not a Major Factor Influencing Transplant Outcome in HLA-Identical Sibling SCT an Analysis on Behalf of the CIBMTR Christiane Dobbelstein 1, Michael Haagenson 2, Kwang Woo Ahn 3, Stephen Spellman 2, Marcelo Fernandez-Vina 4, Matthias Eder 1, Arnold Ganser 1, Stephanie Lee 5 1 Hannover Medical School, Hannover, Germany 2 Center for International Blood and Marrow Transplant Research, Minneapolis, MN, USA 3 Medical College of Wisconsin, Milwaukee, WI, USA 4 M. D. Anderson Cancer Center, Houston, TX, USA 5 Fred Hutchinson Cancer Research Center, Seattle, WA, USA Background: Recent single-center studies have found a birth order effect in HLA-identical sibling myeloablative stem cell transplantation (SCT): superior outcome (e. g. overall survival (OS), less relapse, lower relapse mortality) when the recipient is older than the donor. The proposed mechanism is microchimerism (MC) due to fetomaternal and transmaternal sibling cell trafficking as the donor is exposed to recipient antigens in utero. Aim: The aim of this study was to validate single-center data in a multicenter patient cohort. Methods: This is a retrospective analysis from the CIBMTR dataset. Patients with a diagnosis of a hematologic malignancy (AML, ALL, MDS, CML), at any age (adults and pediatric patients), receiving an allogeneic SCT from HLA-identical sibling donors (recipient and donor of different ages at the time of transplant, but no more than 15 years apart) since 1990 up to December 2007 were included. Outcome was analysed in terms of OS, relapse rate and relapse mortality, leukemia free survival (LFS), treatment related mortality (TRM), acute and chronic graft versus host disease (GvHD). Results: A total of patients (6089 with a younger sibling donor; 5788 with an older donor) have been identified. The median age of the patient was 35 years (range 2-75y.) in the younger donor group compared to older donor group with 31 years (range < 1-72y.; p< 0,0001). In univariate analysis, patients with a younger sibling donor have a higher TRM which was confirmed in multivariate analysis. In multivariate analysis for survival, there was an interaction between age and birth order. A better survival was observed in donor older in the youngest recipient age group compared to a superior OS in recipients with younger donor in the age period of years. Further, there was no main effect on LFS, relapse rate, acute or chronic GVHD. Conclusions: The hypothesized positive effect of having the donor younger than the recipient compared to pairs where the donor is older than the recipient was not observed in this study. We are testing whether these conclusions are confirmed in distinct subsets (e. g. only adult patients, myeloid diseases, stem cell source, T-cell replete graft) examined in previous studies. 121

122 Not for publication or presentation Attachment 15 CIBMTR IB08-08 GENOME-WIDE ASSOCIATION IN UNRELATED DONOR TRANSPLANT RECIPIENTS AND DONORS: A PILOT STUDY REVISED PROTOCOL Study Chair: Study Co-chairs: Rakesh K. Goyal, MD Pediatric Hematology-Oncology and BMT Room 328, 4B DeSoto Children s Hospital of Pittsburgh 3705 Fifth Avenue Pittsburgh, PA Telephone: Fax: rakesh.goyal@chp.edu Robert E. Ferrell, PhD Graduate School of Public Health A304, Crabtree Hall, 130 DeSoto Street Pittsburgh, PA Telephone: Fax: rferrell@hgen.pitt.edu Bernard J. Devlin, PhD, Robert E. Ferrell, PhD Computational Genetics Program Western Psychiatric Institute & Clinic 3811 O'Hara Street Pittsburgh, PA Telephone: devlinbj@upmc.edu Study Statistician: Michael Haagenson, MS CIBMTR 3001 Broadway Street, N.E., Suite 110 Minneapolis, MN USA Telephone: Fax: mhaagens@nmdp.org 122

123 Not for publication or presentation Attachment 15 Scientific Directors: Stephanie Lee, MD, MPH Fred Hutchinson Cancer Research Center P.O. Box 19024, D Fairview Avenue North Seattle, WA USA Telephone: Fax: Stephen R. Spellman, MBS CIBMTR National Marrow Donor Program 3001 Broadway St. N.E. Minneapolis, MN USA Telephone: Fax: Working Committee Chairs: Carlheinz Mueller, MD, PhD Director German National Bone Marrow Donor Registry ZKRD Helmholtzstrabe 10, Ulm, Germany Telephone: Fax: David Miklos, MD, PhD Stanford University Department of Medicine; BMT Division CCSR, Room # West Campus Drive Stanford, CA USA Telephone: Fax: Marcelo Fernandez-Vina, PhD Laboratory Medicine M. D. Anderson Cancer Center 1515 Holcombe Blvd., Box 423 Houston, TX USA Telephone: Fax:

124 Not for publication or presentation Attachment HYPOTHESIS: We hypothesize that an unbiased recipient-donor genome-wide association (GWA) study will identify genes associated with risk of acute graft versus host disease (agvhd) after HLAmatched unrelated donor BMT. The study will examine: 1.1 The genetic differences between recipients who experience grade III-IV agvhd (G+) versus those who get no agvhd (G-) 1.2 The genetic differences between donors in the G+ and G- cohorts, and 1.3 Recipient-donor genetic mismatches between the two groups 2.0 OBJECTIVES: Contrast frequencies of single nucleotide polymorphisms (SNPs) in G+ versus G- recipient genomes Contrast frequencies of SNPs in G+ versus G- donor genomes, and Evaluate recipient-donor SNP mismatches 3.0 SCIENTIFIC JUSTIFICATION: Donor-recipient matching on HLA has its limit in improving patient survival and identification of additional loci that impact agvhd is important to further progress. Efforts to date have focused on a small number of genes almost all related to inflammation, in BMT recipients. While these are rational candidate genes, they assume that we have an understanding of the mechanisms of agvhd. Our proposal will globally assess recipient and donor genomes to identify genes associated with risk of agvhd. Improved understanding of multiple genomic variants and their interactions should help identify recipients at the highest risk of GvHD, develop individualized immunosuppressive strategies, and ultimately improve outcome for allo-bmt recipients. 4.0 STUDY POPULATION: Unrelated donor transplant recipients who develop grade III-IV agvhd (G+) will serve as cases and those with no agvhd (G-) by day 100 posttransplant will serve as controls. Both cohorts will be drawn from the same patient population. HLA disparity is the major genetic determinant of agvhd. Other risk factors include recipient age, type and status of underlying disease, graft source, conditioning intensity, use of radiation, agvhd prophylaxis and use of anti-thymocyte globulin (ATG). The study population will be selected to ensure a similar distribution of risk factors in the case and control arms. For this discovery phase of GWA study, to maximize the probability of identifying highly significantly associated variants, we would focus on a homogeneous group of patients. Recipient-donor pairs are allele matched for HLA-A, -B, -C, -DRB1, and -DQB1 loci Recipient-donor pairs are of self-identified European-American ancestry First transplant Standard ablative pretransplant conditioning. Excludes reduced-intensity (mini) transplants No T-cell manipulation of transplant graft 124

125 Not for publication or presentation Attachment OUTCOMES: Primary Outcome used to assign cases (G+) and controls (G-): Acute GVHD: Development of Grades III-IV acute GVHD using Glucksberg system which grades GVHD based on the pattern and severity of abnormalities in skin, gastrointestinal and liver. 6.0 VARIABLES TO BE ANALYZED: The frequency of single nucleotide polymorphisms (SNPs) in G+ versus G- recipient genomes. 7.0 STUDY DESIGN: 7.1 Sample Requirements: One-time aliquots of recipient and donor sample pairs of nonviable cells will be requested for DNA extraction and sample preparation. Ideally we would like to get few micrograms yield of high quality DNA from individual subject s frozen peripheral blood aliquot or EBV transformed B-cell line. 7.2 GWAS Experiments: A pooling strategy will be used for the initial discovery phase of GWAS. To illustrate the study design, a sample size of 500 recipient-donor pairs with grade III-IV acute GvHD and 500 recipient-donor pairs with no acute GvHD has been used. Grade III-IV agvhd (G+) No agvhd (G-) Recipients R1 R2 Donors D1 D2 All sample processing, DNA isolation and quantitation, pooling of DNA specimens and hybridization to the Human Quad V1 arrays, will be carried out in the Human Genomics and Proteomics Core Laboratory of the University of Pittsburgh ( GWA scan approach is outlined for R1 cohort in the following schematic. A similar approach will be used for R2, D1 and D2 cohorts. 125

126 Not for publication or presentation Attachment Statistical Analyses 8.0 REFERENCES: Aim (1): Hypothesis 1: A handful of variants in recipients genomes exert detectable influence on G+ versus G- status. Estimate and contrast allele frequencies between R1 and R2. Rank the SNPs according to the P values from the contrast. Cutoff P value for follow-up genotyping of SNPs, using an individual samples from the pooled experiment as well as new, independent samples, will be determined by statistical theory and the data Aim (2): Hypothesis 2: A handful of variants in donors genomes exert detectable influence on G+ versus G- status. The same approach outlined in Aim (1) will be used to compare donor groups D1 and D Aim (3): Hypothesis 3: In addition to known variation, G+ versus G- status is determined by degree of matching of donor and recipient genotypes. Two subhypotheses are possible H3a: Donor and recipient matching for a handful of detectable novel variants is critical for good outcome of the transplant H3b: Donor and recipient matching for myriad novel loci, each of small effect, is critical for good outcome of the transplant. We can test H3b with good power while generating pilot data useful for H3a and their validation will require independent funding. We will also randomly choose 40 G+ and G- pairs from R1, R2, D1 and D2 groups for individual genotyping on Illumina HapMap610 Arrays. Results from this experiment will be used to discover covariates to refine estimated, pooled allele frequencies.cutoff P value for follow-up genotyping of SNPs will be determined by statistical theory and the data. Single-marker allelic tests will be performed and SNPs with greatest significance and of those that remain significant at the 0.05 level after corrections for multiple comparisons will be chosen for replication studies in using individual samples from this pooled experiment as well as different independent cohorts of unrelated donor transplants. 1. Mullighan CG, Petersdorf EW. Genomic polymorphism and allogeneic hematopoietic transplantation outcome. Biol Blood Marrow Transplant. 2006;12: Dickinson AM, Middleton PG, Rocha V, Gluckman E, Holler E. Genetic polymorphisms predicting the outcome of bone marrow transplants. Br J Haematol. 2004;127: Goyal R, Lin Y, Livote E, et al. TNF-alpha and TNF Receptor Superfamily Member 1B Polymorphisms Predict Risk of Acute GVHD Following Matched Unrelated Donor BMT in Children. BMT Tandem Meetings. San Diego, CA; 2008:Abstract # Pearson TA, Manolio TA. How to interpret a genome-wide association study. JAMA. 2008;299: Pearson JV, Huentelman MJ, Halperin RF, et al. Identification of the genetic basis for complex disorders by use of pooling-based genome wide single-nucleotide-polymorphism association studies. Am J Hum Genet. 2007;80:

127 Not for publication or presentation Attachment Macgregor S, Zhao ZZ, Henders A, Nicholas MG, Montgomery GW, Visscher PM. Highly cost-efficient genome-wide association studies using DNA pools and dense SNP arrays. Nucleic Acids Res. 2008;36:e Craig DW, Huentelman MJ, Hu-Lince D, et al. Identification of disease causing loci using an array-based genotyping approach on pooled DNA. BMC Genomics. 2005;6: Benjamini Y, Drai D, Elmer G, Kafkafi N, Golani I. Controlling the false discovery rate in behavior genetics research. Behav Brain Res. 2001;125: Chowdari KV, Northup A, Pless L, et al. DNA pooling: a comprehensive, multi-stage association analysis of ACSL6 and SIRT5 polymorphisms in schizophrenia. Genes Brain Behav. 2007;6: Tzeng JY, Devlin B, Wasserman L, Roeder K. On the identification of disease mutations by the analysis of haplotype similarity and goodness of fit. Am J Hum Genet. 2003;72: Patterson N, Price AL, Reich D. Population structure and eigenanalysis. PLoS Genet. 2006;2:e

128 Not for publication or presentation Attachment 15 Table 1. Characteristics of patients receiving a T-replete, first transplant for any disease where recipient received a myeloablative conditioning regimen and the donor/recipient pairs are Caucasian and are high resolution matched for HLA-A, -B, -C and -DRB1 through the NMDP by Grades acute GvHD 3-4 vs. no acute GvHD by Day 100 post-transplant a No agvhd by Day 100 Grades 3-4 agvhd by Day 100 Variable N eval N (%) N eval N (%) Number of patients Number of centers Age at transplant, median (range), years (<1-66) (<1-67) Age at transplant in decades < 10 y 37 ( 8) 25 ( 6) y 39 ( 9) 28 ( 7) y 95 (21) 87 (21) y 87 (19) 71 (17) y 108 (24) 107 (26) Over 50 y 87 (19) 93 (23) Male sex (57) (60) Karnofsky prior to transplant > (77) (76) Disease at transplant AML 145 (32) 103 (25) ALL 92 (20) 69 (17) Other leukemia 13 ( 3) 18 ( 4) CML 89 (20) 137 (33) MDS 82 (18) 66 (16) NHL 32 ( 7) 18 ( 4) Disease status at transplant Early 168 (37) 168 (41) Intermediate 103 (23) 86 (21) Advanced 94 (21) 96 (23) Other 88 (19) 61 (15) Stem cell source Bone marrow 266 (59) 241 (59) Peripheral blood stem cells (PBSC) 187 (41) 170 (41) GVHD prophylaxis FK506 MTX MMF Steroids other 224 (49) 184 (45) FK506 other 20 ( 4) 13 ( 3) CsA + MTX other 184 (41) 195 (47) CsA other (no MTX) 12 ( 3) 17 ( 4) MTX other (no CSA) 1 (<1) 1 (<1) Other/To be determined 12 ( 3) 1 (<1) HLA matching at HLA-DQB Allele-matched 425 (94) 377 (92) One allele mismatch 28 ( 6) 34 ( 8) 128

129 Not for publication or presentation Attachment 15 Table 1. Continued. No agvhd by Day 100 Grades 3-4 agvhd by Day 100 Variable N eval N (%) N eval N (%) Donor/Recipient sex match Male/Male 199 (44) 155 (38) Male/Female 122 (27) 99 (24) Female/Male 59 (13) 91 (22) Female/Female 73 (16) 66 (16) Donor/Recipient CMV match Negative/Negative 169 (37) 150 (36) Negative/Positive 137 (30) 131 (32) Positive/Negative 60 (13) 51 (12) Positive/Positive 78 (17) 66 (16) Unknown 9 ( 2) 13 ( 3) Donor age, median (range), years (18-60) (19-58) Donor age in decades (<1) 2 (<1) (38) 120 (29) (34) 166 (40) (22) 99 (24) 50 and older 26 ( 6) 24 ( 6) Time from dx to tx, overall, median (range) months ( ) ( ) Year of transplant (<1) (<1) ( 2) (<1) 14 ( 3) ( 1) 15 ( 4) ( 2) 12 ( 3) ( 3) 12 ( 3) ( 3) 13 ( 3) ( 3) 13 ( 3) ( 3) 11 ( 3) ( 2) 17 ( 4) ( 4) 15 ( 4) ( 5) 25 ( 6) ( 7) 9 ( 2) ( 5) 20 ( 5) (10) 32 ( 8) (17) 45 (11) (16) 62 (15) (14) 63 (15) ( 6) 22 ( 5) Median follow-up of survivors, months (3-168) (2-205) a Data is not adjusted for the NMDP corrective action plan. Notes: The cumulative incidence rate for Grades 3-4 acute GvHD is 30 (28-32)%. Abbreviations: HLA - Human leukocyte antigens, SCID - Severe combined immunodeficiency, FK506 - Tacrolimus, MTX - Methotrexate, MMF - Mycophenolate mofetil, CsA - Cyclosporine, CMV - Cytomegalovirus, dx - diagnosis, tx - transplant. 129

130 Not for publication or presentation Attachment 16 CIBMTR IB10-04s A VALIDATION STUDY OF THE ROLE OF BASE EXCISION REPAIR PATHWAY AS A PREDICTOR OF OUTCOME AFTER HEMATOPOIETIC STEM CELL TRANSPLANT DRAFT PROTOCOL Study Chair: Study Co-Chairs: Bharat Thyagarajan, PhD, MD Mayo Mail Code Delaware St S.E. Minneapolis, MN Telephone: Fax: thya0003@umn.edu Daniel Weisdorf, MD Masonic Cancer Center, University of Minnesota Mayo Mail Code Delaware Street S.E. Minneapolis, MN Telephone: weisd001@umn.edu Mukta Arora, MD Masonic Cancer Center, University of Minnesota Mayo Mail Code Delaware Street S.E. Minneapolis, MN Telephone: arora005@umn.edu Study Statistician: JohnBosco Umejiego, MPH CIBMTR 3001 Broadway Street, N.E., Suite 100 Minneapolis, MN USA Telephone: Fax: jumejieg@nmdp.org 130

131 Not for publication or presentation Attachment 16 Scientific Directors: Stephanie Lee, MD, MPH Fred Hutchinson Cancer Research Center P.O. Box 19024, D Fairview Avenue North Seattle, WA USA Telephone: Fax: Stephen Spellman, MS CIBMTR National Marrow Donor Program 3001 Broadway Street NE, Suite 100 Minneapolis, MN USA Telephone: Fax: Working Committee Chairs: David Miklos, MD, PhD Stanford University Department of Medicine; BMT Division CCSR, Room # West Campus Drive Stanford, CA USA Telephone: Fax: Marcelo Fernandez-Vina, PhD Professor Department of Pathology/Blood Center Stanford University School of Medicine 3373 Hillview Avenue Palo Alto, CA USA Telephone: Fax: Carlheinz Mueller, MD, PhD Director German National Bone Marrow Donor Registry ZKRD Helmholtzstrabe 10, Ulm, Germany Telephone: Fax:

132 Not for publication or presentation Attachment OBJECTIVES: To validate the associations between SNPs in the base excision repair pathway and TRM detected in patients undergoing allogeneic myeloablative HSCT in a single center study, using an independent sample set obtained from the Center for Blood and Marrow Transplant Research (CIBMTR). 2.0 SCIENTIFIC JUSTIFICATION: Hematopoietic stem cell transplant (HSCT) is a known curative treatment for several life threatening malignancies. However, the procedure continues to be associated with a high morbidity and mortality with transplant related mortality (TRM) ranging from 20% to as high as 50%. 1-3 In those who survive the acute toxicity, disease relapse remains a major cause of death. 3 The primary hypothesis of this study is that single nucleotide polymorphisms (SNPs) in DNA repair genes can influence DNA repair capacity and will be associated with TRM and disease relapse among patients undergoing allogeneic HSCT. Despite rapid advances in HSCT techniques, many patients die due to complications of the transplant procedure while others die due to disease relapse, suggesting that standard therapeutic regimens may be excessive for some patients but unable to eradicate the underlying disease in others. High doses of ionizing radiation and alkylating agents such as cyclophosphamide and mephalan, are commonly used during HSCT. 3 All these agents induce significant DNA damage that result in cell death and is the main mode of action of these agents. 4 Inter-individual variation in ability to repair DNA damage can impact the efficacy of various therapeutic modalities used in HSCT. Decades of research into basic mechanisms of DNA repair have significantly enhanced our understanding of DNA repair at the molecular level. A detailed catalog of all the genes involved in various types of DNA repair is readily available. 5 One DNA repair pathway, the base excision repair (BER) is involved in the repair caused by commonly used chemotherapeutic agents in HSCT. 4 Numerous studies, using various functional assays to estimate DNA repair activity, have shown 30%-40% variation in DNA repair activity among individuals without any apparent disease. 6,7 Resequencing of BER genes among 90 individuals through the National Institute of Environmental Health Sciences (NIEHS) SNPs project has revealed significant genetic variation in almost all genes involved in the BER suggesting that genetic variation in BER is common in the general population. 8 Currently, there has been no systematic evaluation of the role of SNPs in BER genes in predicting treatment response after HSCT. Despite rapid improvements in our understanding of DNA repair mechanisms, the translation of these laboratory findings into clinically useful applications has been limited. This proposal intends to translate our understanding of genetic variants in BER genes into clinically useful information that can be potentially used to individualize therapeutic options for patients undergoing treatment of malignancies. If genetic variants in BER genes are shown to be associated with TRM or disease relapse and the biological significance of these associations are understood, then HSCT conditioning regimens could be tailored such that patients with low DNA repair could receive lower doses of radiation and alkylating agents to minimize the side effects of therapy while patients with high DNA repair may require higher doses of radiation and chemotherapy to obtain optimal benefit. Thus results from this study have great potential for translational application and patient benefit since we anticipate that individualizing treatment regimens will not only reduce mortality related to HSCT but also significantly reduce morbidity associated with HSCT. 132

133 Not for publication or presentation Attachment 16 Previous studies: Single nucleotide polymorphisms in the DNA repair genes, including BER genes, have been associated with increased risk of a variety of cancer, which include (but not limited to) breast, pancreatic, colon and lung cancers and some of these SNPs have also been associated with 6, 9-11 differences in DNA repair activity. However, only one study has shown an association between two polymorphisms in DNA repair genes and treatment outcome in patients undergoing autologous transplants for treatment of multiple myeloma. 12 Thus, overall the role of the genetic variants in the DNA repair pathway remains understudied without evaluation of the entire pathway in conjunction with relevant clinical factors. Preliminary data: We conducted a preliminary single center study at the University of Minnesota to evaluate the association between SNPs in the BER pathway, and treatment outcome after allogeneic HSCT. Study Design: We conducted a retrospective cohort study at the University of Minnesota of 483 pediatric and adult patients who underwent allogeneic HSCT for treatment of hematological malignancies between 1998 and Material and Methods: Pre-transplant blood samples available at the Molecular Diagnostics Laboratory were used to extract DNA samples for these patients and were used to genotype 28 genes involved in the base excision repair pathway. TagSNPs with at least 5% allele frequency in Caucasian population in all 28 genes were selected for genotyping. Genotyping for these samples was performed using the Sequenom iplex genotyping platform. Blinded duplicates samples indicated that genotyping concordance was >90% for all the SNPs genotyped. Four hundred and seventy three samples were successfully genotyped for 220 tagsnps. Statistical Analysis: All statistical analyses were performed using R statistical software package and PLINK. We evaluated linkage disequilibrium (LD) among all the SNPs genotyped in this study and also calculated allele frequencies for all SNPs. We assumed a dominant model in which the heterozygote and the homozygous minor for each SNP were combined into a single category and compared with the homozygous major genotype for the particular SNP. We evaluated the association between each of these SNPs individually with treatment related mortality (TRM) at 1 year after transplant using a proportional hazards model with competing risks as described by Fine and Gray. Alternative methods, such as an additive model to evaluate the SNP-TRM association showed results similar to the dominant model and are not presented in the preliminary analyses section. Results: Sixteen SNPs that were associated with 1 year TRM in the univariate analyses had minor allele frequencies similar to that observed in the Caucasian population. In addition, SNPs in 3 genes (TDG, MUTYH and POLE) were in strong LD (r2>0.9). After excluding 4 SNPs in strong LD and adjustment for potential confounders and independent risk factors such as age at transplant, donor type, clinical diagnosis, disease status at transplant, conditioning regimen, gender mismatch at time of transplant, and mismatch in CMV status at time of transplant we identified 12 SNPs that were significantly associated with 1 year TRM at the 0.05 level (Table 1, Model 1). Most of these associations remained significant when we restricted the analyses to myeloablative transplants (Table 1, Model 2). Limitations and Future Directions: The findings from this preliminary study support the hypothesis that SNPs in the DNA repair genes can predict treatment response in patients 133

134 Not for publication or presentation Attachment 16 undergoing HSCT. However, the small sample size of this study limits our statistical power to detect associations while controlling for multiple comparisons. Since false positive associations are a major limitation of genetic association studies, these findings will need to be confirmed in a larger independent sample of patients undergoing allogeneic HSCT. 3.0 STUDY POPULATION: Adult (>18 years) Caucasian patients with acute leukemia (AML, ALL) or chronic myeloid leukemia (CML) transplanted between 1995 and 2005 using myeloablative conditioning 8/8 matched URD transplants (High resolution matching available at HLA-A, B, C and DRB1) T-cell depleted transplants will be excluded. Patients with advanced stage disease (relapse or primary induction failure, or blast crisis) at time of transplant will be excluded. 4.0 OUTCOMES: Relapse: time to onset of leukemia recurrence. Event will be summarized by cumulative incidence estimate with treatment related mortality as a competing risk. Treatment related mortality: time to death without evidence of leukemia recurrence. This will be calculated as cumulative incidence estimate with relapse as competing risk. 5.0 VARIABLES TO BE ANALYZED: 5.1 Patient-related: - Age at transplant - Karnofsky score pre transplant 5.2 Disease-related: - Disease: AML vs. ALL vs. CML - Disease status at time of transplant - AML: FAB subtype - ALL: Immunophenotype - WBC at diagnosis - Duration of CR1 (for patients beyond CR1) - Time from Remission to transplant (for patients in CR1) - Cytogenetics - AML: good vs. intermediate vs. poor prognosis vs. no abnormalities. - ALL: No abnormalities vs. hyperdiploid vs. hypodiploid/ t(9;22)/ t(4;11)/ t(8;14) vs. other abnormalities. - CML: time from diagnosis to transplant 5.3 Transplant-related: - Source of Stem cell: Bone marrow vs. peripheral blood stem cells - Donor age 134

135 Not for publication or presentation Attachment 16 - Donor recipient gender match: F-M vs. M-F vs. M-M vs. F-F - Donor-recipient CMV status (-/- vs. -/+ vs. +/- vs. +/+) - Conditioning regimen - GVHD prophylaxis - Year of transplant - Acute GVHD Grade II-IV - Chronic GVHD 6.0 STUDY DESIGN: The proposed study will evaluate the association between SNPs in BER genes and their association with treatment outcome after myeloablative allogeneic HSCT. We propose to conduct a retrospective cohort study of 1000 adult Caucasian patients who underwent myeloablative allogeneic HSCT from an unrelated matched (8/8 HLA matched at HLA-A, B, C and DRB1 alleles) donor registered with the National Marrow Donor Program (NMDP) between January 1995 and December 2005 for treatment of specific hematological malignancies (Acute Leukemia (AML/ALL) and chronic myeloid leukemia (CML). We will evaluate the association between 12 tagsnps in BER genes with treatment related mortality and relapse following unrelated matched myeloablative HSCT. Laboratory Methods: DNA will be extracted from whole blood or buffy coats using the Flexigene DNA extraction method (Qiagen, Inc). SNPs will be genotyped using the Taqman genotyping platform. We request for 1 aliquot (1ml of whole blood or corresponding PBMC) of donor and recipient samples for the study. Statistical Analysis: The primary outcomes of this study are cumulative incidence of treatment related mortality (TRM) at 1 year and disease relapse. A proportional hazards model with competing risks described by Fine and Gray 13 will be used to examine the association of each SNP (assuming a dominant genetic model) with development of TRM or disease relapse. The estimated statistical power for this study was calculated using PS Power ver The estimated power to detect an association between individual SNPs and TRM at 1 year is shown in Table 2. This table shows the estimated power for a range of minor allele frequencies (5% - 50%). Assuming the distribution of genotypes for all SNPs will follow Hardy Weinberg proportions and that the presence of one or two minor alleles will have the same effect (i.e.) dominant model, we estimated the proportion of the population that will have the genotypes of interest (exposed proportion). Since patients who underwent a myeloablative allogeneic HSCT between 1995 and 2005 will be eligible for this study, we estimated an accrual interval of 10 years, and additional follow-up after the accrual interval of 1 year for TRM and up to 3 years for relapse. We assumed the median survival time for people with the homozygous major genotype will be 1 year. Assuming we will be able to obtain DNA samples and clinical data on 1000 patients, Table 2 provides an estimate of power of this study for a range of hazard ratios from 1.3 to 1.5 using log rank tests. The Type I error probability associated with this test of this null hypothesis is

136 Not for publication or presentation Attachment REFERENCES: 1. Bredeson CN, Zhang MJ, Agovi MA, Bacigalupo A, Bahlis NJ, Ballen K, et al. Outcomes following HSCT using fludarabine, busulfan, and thymoglobulin: a matched comparison to allogeneic transplants conditioned with busulfan and cyclophosphamide. Biol Blood Marrow Transplant 2008;14: Canals C, Martino R, Sureda A, Altes A, Briones J, Subira M, et al. Strategies to reduce transplant-related mortality after allogeneic stem cell transplantation in elderly patients: Comparison of reduced-intensity conditioning and unmanipulated peripheral blood stem cells vs a myeloablative regimen and CD34+ cell selection. Exp Hematol 2003;31: Schattenberg AV, Levenga TH. Differences between the different conditioning regimens for allogeneic stem cell transplantation. Curr Opin Oncol 2006;18: Lieberman HB. DNA damage repair and response proteins as targets for cancer therapy. Curr Med Chem 2008;15: Wood RD, Mitchell M, Lindahl T. Human DNA repair genes, Mutat Res 2005;577: Wu X, Zhao H, Wei Q, Amos CI, Zhang K, Guo Z, et al. XPA polymorphism associated with reduced lung cancer risk and a modulating effect on nucleotide excision repair capacity. Carcinogenesis 2003;24: Thyagarajan B, Anderson KE, Lessard CJ, Veltri G, Jacobs DR, Folsom AR, Gross MD. Alkaline unwinding flow cytometry assay to measure nucleotide excision repair. Mutagenesis 2007;22: (Accessed.) 9. Allan JM, Shorto J, Adlard J, Bury J, Coggins R, George R, et al. MLH1-93G>A promoter polymorphism and risk of mismatch repair deficient colorectal cancer. Int J Cancer 2008;123: Li D, Suzuki H, Liu B, Morris J, Liu J, Okazaki T, et al. DNA repair gene polymorphisms and risk of pancreatic cancer. Clin Cancer Res 2009;15: Manuguerra M, Saletta F, Karagas MR, Berwick M, Veglia F, Vineis P, Matullo G. XRCC3 and XPD/ERCC2 single nucleotide polymorphisms and the risk of cancer: a HuGE review. Am J Epidemiol 2006;164: Vangsted A, Gimsing P, Klausen TW, Nexo BA, Wallin H, Andersen P, et al. Polymorphisms in the genes ERCC2, XRCC3 and CD3EAP influence treatment outcome in multiple myeloma patients undergoing autologous bone marrow transplantation. Int J Cancer 2007;120: Fine J, Gray R. A proportional hazards model for the subdistribution of a competing risk. Journal of The American Statistical Association 1999;94: Dupont WD, Plummer WD, Jr. Power and sample size calculations for studies involving linear regression. Control Clin Trials 1998;19:

137 Not for publication or presentation Attachment 16 Table 1. Association between tagsnps in BER pathway and TRM at 1 year. SNPs Minor allele Frequency Hazard Ratio (95% CI) p-value, Sample size (n) Model 1 + (n=473) Model 2 + (n=380) *SNP (1.02, 2.15);p= (0.88, 2.00);p=0.18 *SNP (0.26, 0.80);p= (0.29, 0.96);p=0.04 *SNP (0.43, 0.92);p= (0.46, 1.03);p=0.07 *SNP (0.14, 0.86);p= (0.16, 1.01);p=0.05 *SNP (1.26, 2.87);p= (1.09, 2.78);p=0.02 *SNP (1.13, 2.33);p= (1.15, 2.56);p=0.008 *SNP (1.21, 2.60);p= (1.19, 2.76);p=0.006 *SNP (0.47, 0.98);p= (0.47, 1.04);p=0.08 *SNP (0.98, 2.18);p= (1.06, 2.61);p=0.03 *SNP (0.42, 0.89);p= (0.44, 1.02);p=0.06 *SNP (1.14, 4.20);p= (1.19, 4.78);p=0.02 *SNP (1.04, 2.23);p= (1.20, 2.90);p= Both models are adjusted for age at transplant, type of donor and conditioning. *Significant SNPs were detected in the following genes: OGG1, TDG, LIG3, MUTYH, POLE, RFC5, SMUG1 Table 2. Estimated power to detect SNP-TRM associations Minor Allele Frequency (MAF) Exposed Proportion (Heterozygotes + Homozygous for minor allele) Hazard ratio (LR)=1.3 Hazard ratio (LR)=1.4 Hazard ratio (LR)= % % % % % %

138 Not for publication or presentation Attachment 16 Table 3. Characteristics of adult patients with AML, ALL or CML in early or intermediate disease stage from that received a myeloablative conditioning regimen for 10/10 matched unrelated donors from the NMDP ab Variable N Eval N (%) Number of patients 928 Number of centers 104 Recipient age, median (range), years (18-67) Age at transplant y 34 ( 4) y 215 (23) y 209 (23) y 280 (30) 50 and over 190 (20) Male sex (55) Karnofsky prior to transplant > (78) Disease at transplant 928 AML 418 (45) ALL 218 (23) CML 292 (31) WBC count - median (range) ( ) Disease status at transplant 928 Early 569 (61) Intermediate 359 (39) Graft type 928 Bone marrow 454 (49) PBSC 474 (51) GVHD prophylaxis 928 FK506 MTX MMF Steroids other 474 (51) FK506 other 31 ( 3) CsA + MTX other 328 (35) CsA other (No MTX) 22 ( 2) MTX other (No CsA) 4 (<1) Other 69 ( 7) a - Data has been CAP-modeled. b - Sample availability has been determined. 138

139 Not for publication or presentation Attachment 16 Table 3. Continued. Variable N Eval N (%) Donor/recipient sex match 928 Male/Male 376 (41) Male/Female 262 (28) Female/Male 132 (14) Female/Female 158 (17) Donor/recipient CMV match 928 Negative/Negative 309 (33) Negative/Positive 316 (34) Positive/Negative 119 (13) Positive/Positive 164 (18) Unknown 20 ( 2) Donor age - median (range), years (18-60) Donor age ( 1) (34) (37) (22) 50 and older 58 ( 6) Year of transplant ( 4) ( 6) ( 5) ( 6) ( 5) ( 4) ( 8) (12) (18) (19) (14) Median follow-up of survivors, mo (range) (<1-135) Cytogenetics for AML 418 No abnormalities 117 (28) Good risk 39 ( 9) Intermediate risk 167 (40) Poor risk 32 ( 8) Unknown 63 (15) 139

140 Not for publication or presentation Attachment 16 Table 3. Continued. Variable N Eval N (%) Cytogenetics for ALL 218 Normal (No abnormalities) 51 (23) Hypodiploid/ t(9;22)/ t(4;11)/ t(8;14) 73 (33) Other 46 (21) Missing 48 (22) Acute GVHD by IBMTR grading (36) A 78 ( 8) B 257 (28) C 187 (20) D 73 ( 8) Chronic GvHD (55) Sub-disease 928 AML or ANLL, not otherwise specified 58 ( 6) AML M1 Myeloblastic 56 ( 6) AML M2 Myelocytic 88 ( 9) AML M3 Promyelocytic (APML) 6 ( 1) AML M4 Myelomonocytic 74 ( 8) AML M5, monocytic 53 ( 6) AML M6, Erythroblastic 12 ( 1) AML M7, Megakaryoblastic 8 ( 1) AML Granulocytic sarcoma 5 ( 1) AML, Other specify 33 ( 4) ALL, Not otherwise specified 6 ( 1) ALL, Mature B-cell (L3) 4 (<1) ALL T-cell 30 ( 3) ALL Null cell (Early Pre-B) 11 ( 1) ALL calla (including Pre-B) 96 (10) ALL, B-lineage, Not otherwise specified 36 ( 4) ALL, Other specify 35 ( 4) M0, Stem cell 25 ( 3) CML Ph unknown, BCR/ABL unknown 1 (<1) CML Ph+, BCR/ABL+ 178 (19) CML Ph+, BCR/ABL- 9 ( 1) CML Ph+, BCR/ABL unknown 96 (10) CML Ph-, BCR/ABL+ 4 (<1) CML Ph-, BCR/ABL- 2 (<1) CML Ph unknown, BCR/ABL+ 2 (<1) 140

141 Not for publication or presentation Attachment 16 Table 4. Characteristics of adult patients with AML, ALL or CML in early or intermediate disease stage from that received a reduced intensity or nonmyeloablative conditioning regimen for 10/10 matched unrelated donors from the NMDP ab Variable N Eval N (%) Number of patients 283 Number of centers 68 Recipient age, median (range), years (18-74) Age at transplant y 5 ( 2) y 12 ( 4) y 6 ( 2) y 32 (11) 50 and over 228 (81) Male sex (52) Karnofsky prior to transplant > (68) Disease at transplant 283 AML 218 (77) ALL 24 ( 8) CML 41 (14) WBC count - median (range) ( ) Disease status at transplant 283 Early 192 (68) Intermediate 91 (32) Graft type 283 Bone marrow 44 (16) PBSC 239 (84) GVHD prophylaxis 283 FK506 MTX MMF Steroids other 155 (55) FK506 other 17 ( 6) CsA + MTX other 18 ( 6) CsA other (No MTX) 76 (27) MMF other 1 (<1) Other 16 ( 6) a - Data has been CAP-modeled. b - Sample availability has been determined. 141

142 Not for publication or presentation Attachment 16 Table 4. Continued. Variable N Eval N (%) Donor/recipient sex match 283 Male/Male 106 (37) Male/Female 76 (27) Female/Male 42 (15) Female/Female 59 (21) Donor/recipient CMV match 283 Negative/Negative 86 (30) Negative/Positive 109 (39) Positive/Negative 28 (10) Positive/Positive 50 (18) Unknown 10 ( 4) Donor age - median (range), years (18-60) Donor age ( 2) (36) (30) (27) 50 and older 14 ( 5) Year of transplant ( 1) ( 2) ( 1) ( 3) (10) (19) (17) (24) (23) Median follow-up of survivors, mo (range) (3-88) Cytogenetics for AML 218 No abnormalities 80 (37) Good risk 10 ( 5) Intermediate risk 74 (34) Poor risk 23 (11) Unknown 31 (14) 142

143 Not for publication or presentation Attachment 16 Table 4. Continued. Variable N Eval N (%) Cytogenetics for ALL 24 Normal (No abnormalities) 6 (25) Hypodiploid/ t(9;22)/ t(4;11)/ t(8;14) 12 (50) Other 2 ( 8) Missing 4 (17) Acute GVHD by IBMTR grading (43) A 24 ( 8) B 69 (24) C 51 (18) D 16 ( 6) Chronic GvHD (46) Sub-disease 283 AML or ANLL, not otherwise specified 38 (13) AML M1 Myeloblastic 35 (12) AML M2 Myelocytic 41 (14) AML M3 Promyelocytic (APML) 4 ( 1) AML M4 Myelomonocytic 41 (14) AML M5, monocytic 22 ( 8) AML M6, Erythroblastic 9 ( 3) AML M7, Megakaryoblastic 5 ( 2) AML Granulocytic sarcoma 1 (<1) AML, Other specify 18 ( 6) ALL, Not otherwise specified 1 (<1) ALL T-cell 2 ( 1) ALL Null cell (Early Pre-B) 1 (<1) ALL calla (including Pre-B) 11 ( 4) ALL, B-lineage, Not otherwise specified 7 ( 2) ALL, Other specify 2 ( 1) M0, Stem cell 4 ( 1) CML Ph+, BCR/ABL+ 35 (12) CML Ph+, BCR/ABL- 1 (<1) CML Ph+, BCR/ABL unknown 4 ( 1) CML Ph-, BCR/ABL- 1 (<1) 143

144 Not for publication or presentation Attachment 17 AMERICAN SOCIETY OF HEMATOLOGY (ASH) ABSTRACT 2009 Selection of Donors with Favorable KIR B Genotypes for Unrelated Hematopoietic Cell Transplantation Results in Superior Relapse Protection and Better Relapse-Free Survival for Patients with AML Sarah Cooley 1, Lisbeth Guethlein, PhD 2*, Elizabeth Trachtenberg, PhD 3*, Martha Ladner 3*, Xianghua Luo 4*, Chap T Le 5*, Tao Wang, PhD 6*, John P. Klein, PhD 6*, Steven G.E. Marsh, PhD 7*, Stephen Spellman 8*, Michael D Haagenson, MS 9*, Daniel J. Weisdorf, MD 1*, Peter Parham 2* and Jeffrey S. Miller 10 1 Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN 2 Structural Biology, Standford University, Stanford, CA 3 Children's Hospital & Research Center Oakland, Oakland, CA 4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 5 Biostatistics, University of Minnesota, Minneapolis, MN 6 Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, WI 7 Anthony Nolan Research Institute, London 8 National Marrow Donor Program, Minneapolis, MN 9 Center for International Blood and Marrow Transplant Research, Minneapolis, MN 10 University of Minnesota, Minneapolis, MN Unrelated donor (URD) transplants from donors with KIR group B/x genotypes (vs. A/A) confer a significant relapse-free survival (RFS) benefit for patients with acute myeloid leukemia (AML) (RR: 0.70 [95% CI: ]; P =.002; Blood 2009; 113[3]). This new analysis was designed to investigate the beneficial effect of KIR B donors and to develop a donor selection strategy to improve clinical outcomes after hematopoietic cell transplantation (HCT) for leukemia. Based on an analysis of 27 unique KIR haplotype sequences we identified common centromeric and telomeric gene content motifs. KIR A haplotypes contain a Cen-A motif (defined by the presence of the inhibitory KIR gene 2DL3) and a Tel-A motif (defined by the presence of the activating gene 2DS4). The B haplotypes were defined as containing Cen-B (presence of 2DS2 and/or 2DL2) and/or Tel-B (presence of 2DS1), with further subdivisions possible at the allelic level. Thus, based on gene content alone, donor KIR genotypes can be classified as homogyzous A/A or defined by the type (Cen-B or Tel-B) or number (0, 1, 2 or 3; B domain content score) of B domains. Multivariate models were used to evaluate the effect of donor KIR genotypes on clinical outcomes after URD transplants facilitated by the National Marrow Donor Program for AML (n=1086) and acute lymphoblastic leukemia (ALL: n=334) between 1988 and The improved RFS associated with donor KIR B genotypes (Cen-A/B, Cen-B/B, Tel-A/B or Tel-B/B) vs. KIR A genotypes (Cen-A/A or Tel-A/A) in AML was most evident in the 115 donors (10.6%) who were homogyzous for the Cen-B motif (RR 0.72 [95% CI ]; p=0.014). Likewise, Cen-B/B donors conferred significant protection against relapse (RR 0.34 [95% CI ]; p < ); with absolute relapse rates of only 10% (Cen-B/B) vs. ~31% (A/A). Similarly, multivariate models demonstrated that compared to KIR A/A donors, donors with higher KIR B domain content scores resulted in improved RFS (2B motifs: (RR 0.78 [95% CI ]; p=0.013; n=244) or 3 B motifs: (RR 0.76 [95% CI ]; p=0.07; n=84) and 144

145 Not for publication or presentation Attachment 17 less relapse (2B motifs: (RR 0.54 [95% CI ]; p=0.0001) or 3 B motifs: (RR 0.45 [95% CI ]; p=0.0017). Donor KIR genotype had no effect on rates of graft vs. host disease or treatment related mortality. Importantly, the use of KIR B donors of any type was not associated with any improvement in clinical outcomes for patients with ALL. These data suggest that AML blasts may be particularly sensitive to killing by NK cells and raises the possibility that activating genes present in the donor KIR B haplotypes may uniquely recognize ligands on AML blasts. The KIR B genotype effects were not affected by the degree of HLA matching. Therefore, these data support the consideration of KIR genotyping with HLA typing into the unrelated donor search criteria for patients with AML. To capture the benefit of Cen-B and/or higher B domain content scores we propose that the ~30% of donors who have 2 B domains (includes all Cen-B/B donors) be given preference over donors with 0 or 1 KIR B domains. In this large retrospective dataset, assignment by that rule resulted in significant improvements in RFS (RR 0.80 [95% CI ]; p=0.0063) and relapse (RR 0.53 [95% CI ]; p<0.0001). A prospective trial to test this strategy is planned. 145

146 Not for publication or presentation Attachment BMT TANDEM MEETINGS ABSTRACT Donor KIR2DS1 and KIR3DS1 are associated with improved outcomes following unrelated allogeneic stem cell transplantation for acute myeloid leukemia Jeffrey M. Venstrom 1, Gianfranco Pittari 1, Joseph Chewning 1, Ted A. Gooley 2, Stephen Spellman 3, Michael Haagenson 3, Meighan M. Gallagher 1, Mari Malkki 2, Effie Petersdorf 2,, Bo Dupont 1, Katharine C. Hsu 1 1 Memorial Sloan-Kettering Cancer Center; New York, New York 2 Fred Hutchinson Cancer Research Center; Seattle, Washington 3 Center for International Blood and Marrow Transplant Research; Minneapolis, Minnesota Background: Current concepts of NK alloreactivity are based on donor/recipient KIR ligand mismatching and on donor KIR genotype variants, while little is known about the role of specific activating KIR genes. Specifically, the importance of the telomeric KIR2DS1 and KIR3DS1genes, and the centromeric KIR2DS2 gene has not been studied in detail. We tested the hypothesis that individual donor activating KIR genes would influence the outcome of AML patients undergoing unrelated allogeneic HCT. Methods: Donor KIR genotyping was performed for 1229 AML patients receiving transplants between 1989 and 2008 from 9/10 or 10/10 HLA-matched unrelated donors with outcome data provided by the CIBMTR. HLA genotyping was verified through the NMDP retrospective typing program. Cox regression was used to examine the association between donor KIR genotype and HCT outcome. Models were adjusted for age, disease severity, donor/patient gender, T-cell depletion, conditioning, and HLA mismatch. Results: 422 donors were KIR2DS1-positive, 429 donors were KIR3DS1-positive (n=355 KIR2DS1pos, KIR3DS1pos), and 630 donors were KIR2DS2-positive. The presence of donor KIR2DS1 was associated with lower relapse [HR 0.76 ( ), p=0.02], even after adjusting for presence of KIR2DS2 [adjusted HR: 0.77 ( ), p=0.02] and presence of donor KIR3DS1, with which it shares strong positive LD [adjusted HR 0.71 ( ), p=0.04]. Donor KIR3DS1 was associated with reduced nonrelapse mortality [HR 0.68 ( ), p=0.01] and decreased overall mortality [HR 0.79 ( ), p= 0.03] even after adjusting for donor KIR2DS1. Interestingly, after adjusting for KIR3DS1, KIR2DS1 positivity had little association with overall mortality [HR 1.00 ( , p=.98)]. There was no statistically significant association of donor KIR2DS2 on survival, relapse, or non-relapse mortality. Furthermore, the protective effect of donor centromeric KIR B-haplotype homozygosity on relapse was not statistically significant [CenBB vs. CenAA, HR 0.8 ( ), p=0.23]. Conclusion: Individual activating KIR may mediate independent effects in unrelated allogeneic HCT for AML, with KIR2DS1 protecting from relapse and KIR3DS1 protecting from non-relapse mortality. 146

147 Not for publication or presentation Attachment 19 CIBMTR IB07-03 ANALYSIS OF KILLER IMMUNOGLOBULIN-LIKE RECEPTOR (KIR) LIGANDS IN REDUCED INTENSITY CONDITIONING ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION REVISED PROTOCOL Study Chairs: Ronald M. Sobecks, MD The Cleveland Clinic 9500 Euclid Avenue Mail Code R35 Cleveland, OH USA Telephone: Fax: Katharine Hsu, MD, PhD Allogeneic Marrow Transplantation Service Memorial Sloan-Kettering Cancer Center 1275 York Avenue New York, NY USA Telephone: Fax: Medhat Askar, MD, PhD The Cleveland Clinic 9500 Euclid Avenue Mail Code C100 Cleveland, OH USA Telephone: Fax: Study Statistician: Michael Haagenson, MS CIBMTR 3001 Broadway Street, N.E., Suite 100 Minneapolis, MN USA Telephone: Fax:

148 Not for publication or presentation Attachment 19 Scientific Directors: Stephanie Lee, MD, MPH Fred Hutchinson Cancer Research Center 1100 Fairview Ave. North, D5-290 PO Box Seattle, WA Phone : Fax : sjlee@fhcrc.org Stephen Spellman, MS CIBMTR National Marrow Donor Program 3001 Broadway Street NE, Suite 100 Minneapolis, MN USA Telephone: Fax: sspellma@nmdp.org Working Committee Chairs: David Miklos, MD, PhD Stanford University Department of Medicine; BMT Division CCSR, Room # West Campus Drive Stanford, CA Phone: Fax: dmiklos@stanford.edu Marcelo Fernandez-Vina, PhD Professor Department of Pathology/Blood Center Stanford University School of Medicine 3373 Hillview Avenue Palo Alto, CA USA Telephone: Fax: marcelof@stanford.edu Carlheinz Mueller, MD, PhD Director German National Bone Marrow Donor Registry ZKRD Helmholtzstrabe 10, Ulm, Germany Telephone: Fax: carlheinz.mueller@zkrd.de 148

149 Not for publication or presentation Attachment OBJECTIVES The objectives of this study are: 1.1 To evaluate the clinical effects of donor KIR genotype and KIR ligand absence in recipients of matched and mismatched unrelated donor RIC allogeneic HSCT for myeloid malignancies including CMV reactivation (if data available) graft-versus-host disease (GVHD), relapse, disease-free survival, transplant-related mortality and overall survival. 1.2 To assess the effects of donor KIR ligand absence in matched and mismatched unrelated donor RIC allogeneic HSCT for myeloid malignancies including graft rejection and for those patients with data available the achievement of T-cell complete donor chimerism (CDC). 2.0 SCIENTIFIC JUSTIFICATION: Disease relapse continues to be a significant cause of treatment failure after allogeneic HSCT. In particular with RIC approaches, the graft-versus-leukemia (GVL) effect is critical for successful outcomes in patients with advanced myeloid malignancies. As such, further strategies to optimize conditions for achievement of a GVL effect are needed. The GVL effect has been attributed to donor-derived alloreactive immune cells including T- lymphocytes and natural killer (NK) cells 1-4. The reactivity of NK cells and some T- lymphocyte subsets is regulated by the interaction of KIRs with target cell HLA-class I molecules 4. KIR interactions have been suggested to influence outcomes of haploidentical 3, 5, matched unrelated donor 6-8 and HLA-matched related donor (MRD) allogeneic HSCT 9, 10, particularly for AML patients. However, in RIC allogeneic HSCT when both donor and recipient hematopoiesis may coexist, the effect of KIR interactions on outcomes is not well known. Donor activating KIR genotype has been implicated as a contributory factor for CMV reactivation after myeloablative and reduced-intensity conditioning allogeneic HSCT Activating KIR genotype has also been reported to influence other posttransplant outcomes including grade 2-4 acute GVHD, transplant-related mortality, relapse-free survival and overall survival Group A and B KIR haplotypes have distinctive centromeric (cen) and telomeric (tel) gene-content motifs. Cen and tel B motifs have been reported to have less relapse and improved survival compared to A haplotype motifs in AML patients undergoing unrelated donor HSCT 18. This effect was most notable for cen-b homozygosity. We have analyzed 51 patients who received related donor RIC allogeneic HSCT and have observed that KIR matching influenced the achievement of T-cell (CD3+) CDC 19, 20. Recipient KIR genotype and donor HLA KIR ligands (HLA-A3/11, -Bw4, -C group 1 and C group 2) were used to generate an inhibitory KIR score for patients from 1 to 4 corresponding to the potential number of recipient inhibitory KIRs available for engagement with donor HLA KIR ligands. As compared to patients with an inhibitory KIR score of greater than 1, those with a score of 1 were less likely to achieve CDC. With increasing inhibitory KIR score, patients tended to be more likely to achieve CDC. This difference could not be attributed to distinct diagnostic subsets or to CD3+ or CD34+ cell doses infused. Thus, patients with lower inhibitory KIR scores may have more active anti-donor effector cells (NK cells and T cell subsets) that may reduce donor cell chimerism. Conversely, those with higher inhibitory KIR scores may have less active populations and be more likely to achieve CDC. 149

150 Not for publication or presentation Attachment STUDY POPULATION: The study population will include all patients reported to the CIBMTR who had received a RIC allogeneic HSCT for AML, MDS, and CML from 1999 to 2007 from an unrelated donor. Subjects who received campath were excluded from the population. 4.0 OUTCOMES: 4.1 Overall survival Time to death from any cause. Event will be summarized by Kaplan- Meier estimate. Cases will be analyzed at the time of last follow-up. There are no competing risks. 4.2 Disease-free survival Time to disease relapse/progression or death. Event will be summarized by Kaplan-Meier estimate. Cases will be analyzed at the time of last followup. There are no competing risks. 4.3 Transplant-related mortality - Death in continuous remission of primary disease. Event will be summarized by the cumulative incidence estimate with relapse as a competing risk and second transplant as a censoring event. 4.4 Acute GVHD - Development of Grades II-IV and III IV acute GVHD using the Consensus Criteria system which grades GVHD based on the pattern and severity of abnormalities in skin, gastrointestinal and liver. Event will be summarized by the cumulative incidence estimate. Cases will be analyzed at time of last follow-up. Death is a competing risk. Second transplant is a censoring event. 4.5 Chronic GVHD - Development of symptoms in any organ system fulfilling the criteria of extensive chronic GVHD. The event will be summarized by the cumulative incidence estimate. Patients will be analyzed at last follow-up. Death is a competing risk and second transplant is a censoring event. 4.6 Disease relapse Development of clinical relapse of the primary disease as defined by the CIBMTR. The event will be summarized by the cumulative incidence estimate and patients analyzed at last follow-up. Death is a competing risk and second transplant is a censoring event. 4.7 Graft rejection - Primary graft rejection is defined as failure to ever achieve any donorderived hematopoiesis defined as > 5% donor chimerism before day 100. This endpoint will be expressed as a binary outcome at day 100. Patients who die before a chimerism test is performed or who do not have a chimerism result recorded before day 100 are excluded from the analysis of primary graft rejection. Secondary graft rejection is defined as the loss of donor-derived hematopoiesis (donor chimerism < 5%) after complete or mixed chimerism was achieved (documentation of > 5% donor chimerism). The event will be summarized by the cumulative incidence estimate. Patients will be analyzed at last follow-up. Death is a competing risk and second transplant is a censoring event. Patients who do not ever achieve chimerism > 5% are excluded from this analysis. 4.8 T-cell complete donor chimerism - Defined as achievement of >95% donor chimerism. The event will be summarized by the cumulative incidence estimate and patients analyzed at last follow-up. Death and second transplants are competing risks. 150

151 Not for publication or presentation Attachment 19 Patients who die before a chimerism test is performed are excluded from the analysis. 5.0 VARIABLES TO BE ANALYZED: 5.1 Main effect: Missing KIR Ligand Model - Recipient HLA KIR Ligands: HLA-Bw4, HLA-C group 1, HLA-C group 2 (present or absent) Quantify the number of missing ligands (none vs. >1; and 0 vs. 1 vs. 2 vs. 3 vs. 4)HLA-A3/11 (present or absent) - Donor HLA KIR Ligands: HLA-Bw4, HLA-C group 1, HLA-C group 2 (present or absent) Quantify the number of missing ligands (none vs. >1; and 0 vs. 1 vs. 2 vs. 3 vs. 4) HLA-A3/11 (present or absent) Missing Self Model - Donor: 0 = self conserved for all ligands 1 = missing self for HLA-C group 1 alone (2DL2/2DL3: present or absent) 2 = missing self for HLA-C group 2 alone (2DL1: present or absent) 3 = missing self for HLA-Bw4 alone (3DL1: present or absent) 4 = missing self for HLA-Bw4 and C1 or C2 Quantify the number missing (none vs. >1; and 0 vs. 1 vs. 2 vs. 3 vs. 4*) (*Missing self for Bw 4 and C1 or C2) Assessment of individual activating KIRs (for donor) - 2DS1 (present or absent) - 2DS2 (present or absent) - 2DS3 (present or absent) - 2DS4 (present or absent) - 2DS5 (present or absent) - 3DS1 (present or absent) Examine the effects of KIR 2DS1 with each of the KIR ligand groups Donor KIR genotype will then be assessed regarding the centromeric-telomeric content hypothesis - haplotype AA (2DL3+ only, telaa) - cenab (2DS2 and/or 2DL2 positive, 2DL3 positive) - cen BB (2DS2 and/or 2DL2 positive, 2DL3 negative) - cenaa/telb (haplotype AB or BB, 2DL3+ only) 151

152 Not for publication or presentation Attachment Patient related: - Age: vs. >50 yrs - Gender - Karnofsky score at transplant: <70 vs vs Disease related: - Disease at transplant: AML, MDS, and CML - Group assessments: AML + MDS + CML AML + MDS AML MDS CML - Disease stage at transplant: Early vs. Intermediate vs. Advanced AML + MDS + CML AML + MDS AML MDS CML 5.4 Transplant related: - HLA Matching (HLA disparities: HLA-A, -B, -C, -DR, -DQ, -DP or none) - Conditioning regimen (Reduced intensity, non-myeloablative) - Low-dose TBI (200 cgy), Flu/TBI, Flu/Cy, Flu/Cy/idarubicin, Mel/Flu - Thiotepa/Cy, Thiotepa/Mel/ATG, Bu/Flu/ATG. Subset analysis of those who had ATG/T-cell depletion vs. no ATG/TCD - GvHD prophylaxis (calcineurin inhibitor + MMF, calcineurin inhibitor + MTX, calcineurin inhibitor ± other) - Stem cell source (BM vs. PBSC) - Hematopoietic stem cell dose: CD3, CD34 and TNC - T-cell chimerism analysis: molecular methods (e.g., PCR-based STR, VNTR, RFLP) vs. other (e.g., FISH, standard cytogenetics) - Donor-recipient sex: M-M vs. M-F vs. F-M vs. F-F - ABO mismatch between donor and recipient - Year of transplant 6.0 STUDY DESIGN: The presence or absence of HLA KIR ligands (Bw4, C1 and C2) will be determined for each recipient from high resolution HLA class I typing. Patients will then be grouped according to homozygosity for HLA-Bw6, C1 and C2, corresponding to a lack of ligand for donor inhibitory KIR3DL1, KIR2DLI and KIR2DL2/3, respectively. The presence of absence of HLA-A antigens/alleles sharing a Bw4 epitope (A23, A24 and A32) will be assessed for each recipient and will be included as KIR3DL1 ligands. Patients with all KIR ligands present will then be compared with those missing 1 or more KIR ligands (0 vs. >1; and 0 vs. 1 vs. 2 vs. 3 vs. 4; as above in section 5.0) with regards to the development of CMV reactivation (if data available), acute and chronic GVHD, relapse, disease-free survival and overall survival, taking into consideration whether they have HLA matched vs. mismatched donors. The HLA matched patients will be analyzed independently from the HLA mismatched patients. An independent 152

153 Not for publication or presentation Attachment 19 analysis will also assess the presence or absence of recipient HLA-A3/A11 KIR ligands. Patients will then be grouped according to homozygosity for non-hla A3/11 type, corresponding to a lack of ligand for donor inhibitory KIR3DL2. The presence or absence of HLA KIR ligands (Bw4, C1 and C2) will then be determined for each donor from high resolution HLA class I typing. Donors will also be grouped according to homozygosity for HLA-Bw6, C1 and C2, corresponding to a lack of ligand for donor inhibitory KIR3DL1, KIR2DLI and KIR2DL2/3, respectively. The presence of absence of HLA-A antigens/alleles sharing a Bw4 epitope (A23, A24 and A32) will be assessed for each donor and will be included as KIR3DL1 ligands in the main analysis, but subset analyses that exclude these ligands may be performed. Donors with all KIR ligands present will then be compared with those missing 1 or more KIR ligands (0 vs. >1; and 0 vs. 1 vs. 2 vs. 3) with regards to the development of graft rejection and achievement of T-cell CDC. The HLA matched patients will be analyzed independently from the HLA mismatched patients. An independent analysis will also assess the presence or absence of donor HLA-A3/A11 KIR ligands. Donors will then be grouped according to homozygosity for non-hla A3/11 type, corresponding to a lack of ligand for donor inhibitory KIR3DL2. For the subset of patients with T-cell chimerism data available the number of patients who achieved CDC will be determined at the following posttransplant time points: days +28, +56, +100, +180, A windowing algorithm will be used to capture T-cell chimerism data for those patients whose data is available but not specifically on one of these defined time points. This will allow the data to be categorized within the nearest defined post-transplant time point. At each of these time points the median percentage of donor T-cell chimerism will be assessed as well as the number of patients with graft falure (< 5% donor chimerism). The patients whose T-cell chimerism was based upon molecular methods will be analyzed independently from those performed with other methods. The missing self model will then be tested for the different combinations defined in section 5.0. This will be performed for the graft-vs.-host vector only (since no KIR data is available for recipients) in which there will be a comparison of the number of missing self groups (none vs. >1; and 0 vs. 1 vs. 2 vs. 3 vs. 4) with regards to the development of graft failure (and achievement of T-cell CDC if possible). The individual donor activating KIRs (2DS1, 2DS2, 2DS3, 2DS4, 2DS5 and 3DS1) will be assessed and compared between those present and absent with regards to the development CMV reactivation (if data available), acute and chronic GVHD, relapse, disease-free survival and overall survival, taking into consideration whether they have HLA matched vs. mismatched donors. Analyses to test donor KIR2DS1-mediated activity will be performed to examine its effect with each of the KIR ligand groups. Donor KIR genotype will then be assessed regarding the centromeric-telomeric content hypothesis. The following outcomes that will be evaluated include CMV reactivation (if data available), acute and chronic GVHD, relapse, disease-free survival and overall survival, taking into consideration whether they have HLA matched vs. mismatched donors. The analysis will initially examine all diagnoses combined (AML, MDS and CML) and then focus on the following diagnostic subgroups: AML + MDS; AML alone, MDS alone and CML alone. Cox s proportional hazards regression analyses will then be used to analyze the 153

154 Not for publication or presentation Attachment 19 association of homozygosity for HLA-Bw6, C1 and C2 with regards to each of the post-transplant outcomes. This approach may then be used to analyze the association of homozygosity for non- HLA A3/11 type with regards to each of the post-transplant outcomes. Because there are 7 endpoints, a p-value of 0.01 will be considered statistically significant. 7.0 REFERENCES: 1. Molldrem JJ, Lee PP, Wang C, et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nat Med 2000; 6: Hercend T, Takvorian T, Nowill A, et al. Characterization of natural killer cells with antileukemia activity following allogeneic bone marrow transplantation. Blood 1986; 67: Ruggeri L, Capanni M, Urbani E, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002; 295: Farag S, Fehniger T, Ruggeri L, et al. Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect. Blood 2002; 100: Ruggeri L, Capanni M, Casucci M, et al. Role of natural killer cell alloreactivity in HLAmismatched hematopoietic stem cell transplantation. Blood 1999; 94: Giebel S, Locatelli FW, Lamparelli T, et al. Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors. Blood 2003; 102: Hsu K, Gooley T, Malkki M, et al. KIR ligands and prediction of relapse after unrelated donor hematopoietic cell transplantation for hematologic malignancy. Biol Blood Marrow Transplant 2006; 12: Miller J, Cooley S, Parham P, et al. KIR ligand absence in recipients of unrelated donor (URD) allogeneic hematopoietic cell transplantation (HCT) is associated with less relapse and increased graft versus host disease (GVHD). Blood 2006; 108:55a (abstr # 171). 9. Cook MA, Milligan DW, Fegan CD, et al. The impact of donor KIR and patient HLA-C genotypes on outcome following HLA-identical sibling hematopoietic stem cell transplantation for myeloid leukemia. Blood 2004; 103: Hsu KC, Keever-Taylor CA, Wilton A, et al. Improved outcome in HLA-identical sibling hematopoietic stem cell transplantation for acute myelogenous leukemia (AML) predicted by KIR and HLA genotypes. Blood 2005; 105: Cook M, Briggs D, Craddock C, et al. Donor KIR genotype has a major influence on the rate of cytomegalovirus reactivation following T-cell replete stem cell transplantation. Blood 2006 Feb 1;107(3): Chen C, Busson M, Rocha V, et al. Activating KIR genes are associated with CMV reactivation and survival after non-t-cell depleted HLA-identical sibling bone marrow transplantation for malignant disorders. Bone Marrow Transplant 2006 Sep;38(6): Zaia JA, Sun JY, Gallez-Hawkins GM, et al. The effect of single and combined activating killer immunoglobulin-like receptor genotypes on cytomegalovirus infection and immunity after hematopoietic cell transplantation. Biol Blood Marrow Transplant 2009 Mar;15(3): Sobecks RM, Askar M, Thomas D, et al. Cytomegalovirus reactivation after matched sibling donor reduced-intensity conditioning allogeneic hematopoietic stem cell transplant correlates with donor killer immunoglobulin-like receptor genotype. Exper Clin Transplant 2011 Feb; 9(1):

155 Not for publication or presentation Attachment Venstrom JM, Gooley TA, Spellman S et al. Donor activating KIR3DS1 is associated with decreased acute GVHD in unrelated allogeneic hematopoietic stem cell transplantation. Blood 2010 Apr 15;115(15): Epub 2010 Feb Cooley S, Trachtenberg E, Bergemann TL et al. Donors with group B KIR haplotypes improve relapse-free survival after unrelated hematopoietic cell transplantation for acute myelogenous leukemia. Blood Jan 15;113(3): Epub 2008 Oct Pende D, Marcenaro S, Falco M, et al. Anti-leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for pediatric patients: evaluation of the functional role of activating KIR and redefinition of inhibitory KIR specificity. Blood Mar 26;113(13): Epub 2008 Oct Cooley S, Weisdorf DJ, Guethlein LA, et al. Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia. Blood Oct 7;116(14): Epub 2010 Jun Sobecks R, Ball E, Askar M, et al. Influence of Killer Immunoglobulin-like Receptor (KIR) matching on achieving T cell (CD3+) complete donor chimerism (CDC) in related donor nonmyeloablative allogeneic hematopoietic stem cell transplantation (NMHSCT). Blood 2006; 108:854a (abstr # 3012). 20. Sobecks RM, Ball EJ, Askar M, et al. Influence of Killer Immunoglobulin-Like Receptor/HLA Ligand Matching on Achievement of T-cell Complete Donor Chimerism in Related Donor Nonmyeloablative Allogeneic Hematopoietic Stem Cell Transplantation. Bone Marrow Transplant 2008 Apr;41(8): Epub 2008 Jan

156 Not for publication or presentation Attachment 19 Table 1. Characteristics of subjects reported to CIBMTR receiving a RIC allogeneic HSCT for AML, CML, or MDS from 1990 to 2007 from an unrelated donor, stratified by recipient ligand count >= 1 Ligand Missing All Ligands Present Variable N (%) N (%) Number of Patients Age, median (range), years 53 (2-74) 53 (2-72) Age at transplant ( 1) 5 ( 1) ( 4) 13 ( 3) ( 9) 30 ( 8) (10) 43 (11) (18) 61 (16) (32) 122 (32) (26) 105 (28) Donor age, median (range), years 36 (18-61) 35 (19-59) Donor age ( 2) 5 ( 1) (29) 107 (28) (38) 142 (37) (25) 90 (24) ( 7) 35 ( 9) Sex Male 486 (56) 214 (56) Female 388 (44) 165 (44) Karnofsky score prior to transplant < (32) 107 (28) >= (58) 229 (60) Missing 86 (10) 43 (11) HLA matching <=7/10 37 ( 4) 23 ( 6) 8/10 89 (10) 39 (10) 9/ (25) 97 (26) 10/ (60) 220 (58) Disease at transplant AML 506 (58) 204 (54) CML 121 (14) 76 (20) MDS 247 (28) 99 (26) 156

157 Not for publication or presentation Attachment 19 Table 1. Continued. >= 1 Ligand Missing N (%) All Ligands Present N (%) Variable Disease status at transplant Early 418 (48) 165 (44) Intermediate 197 (23) 85 (22) Advanced 256 (29) 129 (34) Missing 3 ( 0) 0 ( 0) Conditioning regimen TBI 200 cgy 20 ( 2) 10 ( 3) Fludarabine + TBI 138 (16) 61 (16) Fludarabine + Cy 103 (12) 40 (11) Fludarabine + Melphalan 214 (24) 82 (22) Fludarabine + Busulfan + ATG 189 (22) 75 (20) Fludarabine + Busulfan (No ATG) 132 (15) 67 (18) Thiotepa + Cy + ATG 78 ( 9) 44 (12) ATG Use No 469 (54) 205 (54) Yes 405 (46) 174 (46) GvHD prophylaxis CsA + MTX 108 (12) 54 (14) CsA + MMF 175 (20) 64 (17) CsA ± other (No MTX and no MMF) 36 ( 4) 16 ( 4) FK506 + MTX 307 (35) 136 (36) FK506 + MMF 124 (14) 63 (17) FK506 ± other (No MTX and no MMF) 46 ( 5) 15 ( 4) MMF ± other (No CsA and no FK506) 7 ( 1) 1 ( 0) MTX ± other (No CsA and no FK506) 2 ( 0) 0 ( 0) T-cell depletion 45 ( 5) 24 ( 6) Other 24 ( 3) 6 ( 2) Stem cell source Bone marrow 274 (31) 120 (32) PBSC 600 (69) 259 (68) Donor/recipient sex match Male -> Male 331 (38) 143 (38) Male -> Female 224 (26) 81 (21) Female -> Male 155 (18) 71 (19) Female -> Female 164 (19) 84 (22) 157

158 Not for publication or presentation Attachment 19 Table 1. Continued. >= 1 Ligand Missing N (%) All Ligands Present N (%) Variable Donor/recipient CMV match Negative/Negative 235 (27) 108 (28) Negative/Positive 308 (35) 130 (34) Positive/Negative 109 (12) 43 (11) Positive/Positive 203 (23) 89 (23) Unknown 19 ( 2) 9 ( 2) Year of transplant ( 1) 5 ( 1) ( 0) 2 ( 1) ( 1) 5 ( 1) ( 1) 5 ( 1) ( 0) 1 ( 0) ( 2) 8 ( 2) ( 1) 2 ( 1) ( 1) 6 ( 2) ( 1) 6 ( 2) ( 2) 9 ( 2) ( 5) 15 ( 4) ( 7) 30 ( 8) ( 6) 23 ( 6) ( 9) 26 ( 7) (14) 45 (12) (18) 69 (18) (21) 77 (20) (11) 45 (12) Median follow-up of survivors, months 15 (0-198) 23 (0-160) 158

159 Not for publication or presentation Attachment 19 Table 2: Characteristics of subjects reported to CIBMTR receiving a RIC allogeneic HSCT for AML, CML, or MDS from 1990 to 2007 from an unrelated donor, stratified by donor ligand count >= 1 Ligand Missing All Ligands Present Variable N (%) N (%) Number of Patients Age, median (range), years 53 (2-74) 54 (2-72) Age at transplant ( 2) 4 ( 1) ( 4) 13 ( 3) ( 8) 34 ( 9) (10) 41 (11) (19) 54 (14) (33) 115 (31) (24) 113 (30) Donor age, median (range), years 36 (18-61) 35 (19-59) Donor age ( 2) 5 ( 1) (29) 103 (28) (37) 144 (39) (25) 85 (23) ( 7) 37 (10) Sex Male 494 (56) 206 (55) Female 385 (44) 168 (45) Karnofsky score prior to transplant < (31) 114 (30) >= (59) 216 (58) Missing 85 (10) 44 (12) HLA matching <=7/10 40 ( 5) 20 ( 5) 8/10 98 (11) 30 ( 8) 9/ (24) 104 (28) 10/ (60) 220 (59) Disease at transplant AML 501 (57) 209 (56) CML 127 (14) 70 (19) MDS 251 (29) 95 (25) 159

160 Not for publication or presentation Attachment 19 Table 2. Continued. >= 1 Ligand Missing N (%) All Ligands Present N (%) Variable Disease status at transplant Early 423 (48) 160 (43) Intermediate 197 (22) 85 (23) Advanced 257 (29) 128 (34) Missing 2 ( 0) 1 ( 0) Conditioning regimen TBI 200 cgy 18 ( 2) 12 ( 3) Fludarabine + TBI 136 (15) 63 (17) Fludarabine + Cy 106 (12) 37 (10) Fludarabine + Melphalan 210 (24) 86 (23) Fludarabine + Busulfan + ATG 187 (21) 77 (21) Fludarabine + Busulfan (No ATG) 135 (15) 64 (17) Thiotepa + Cy + ATG 87 (10) 35 ( 9) ATG Use No 470 (53) 204 (55) Yes 409 (47) 170 (45) GvHD prophylaxis CsA + MTX 111 (13) 51 (14) CsA + MMF 175 (20) 64 (17) CsA ± other (No MTX and no MMF) 34 ( 4) 18 ( 5) FK506 + MTX 305 (35) 138 (37) FK506 + MMF 129 (15) 58 (16) FK506 ± other (No MTX and no MMF) 46 ( 5) 15 ( 4) MMF ± other (No CsA and no FK506) 7 ( 1) 1 ( 0) MTX ± other (No CsA and no FK506) 2 ( 0) 0 ( 0) T-cell depletion 48 ( 5) 21 ( 6) Other 22 ( 3) 8 ( 2) Stem cell source Bone marrow 287 (33) 107 (29) PBSC 592 (67) 267 (71) Donor/recipient sex match Male -> Male 335 (38) 139 (37) Male -> Female 224 (25) 81 (22) Female -> Male 159 (18) 67 (18) Female -> Female 161 (18) 87 (23) 160

161 Not for publication or presentation Attachment 19 Table 2. Continued. >= 1 Ligand Missing N (%) All Ligands Present N (%) Variable Donor/recipient CMV match Negative/Negative 239 (27) 104 (28) Negative/Positive 313 (36) 125 (33) Positive/Negative 112 (13) 40 (11) Positive/Positive 196 (22) 96 (26) Unknown 19 ( 2) 9 ( 2) Year of transplant ( 1) 7 ( 2) ( 0) 2 ( 1) ( 1) 4 ( 1) ( 1) 4 ( 1) ( 0) 1 ( 0) ( 2) 7 ( 2) ( 1) 1 ( 0) ( 1) 5 ( 1) ( 1) 4 ( 1) ( 2) 7 ( 2) ( 5) 15 ( 4) ( 7) 29 ( 8) ( 6) 25 ( 7) ( 9) 27 ( 7) (14) 46 (12) (18) 65 (17) (20) 80 (21) (11) 45 (12) Median follow-up of survivors, months 16 (0-198) 21 (0-146) 161

162 Not for publication or presentation Attachment 20 CIBMTR IB11-03 EVALUATION OF THE IMPACT OF ALLELE HOMOZYGOSITY AT HLA LOCI ON OUTCOME REVISED PROTOCOL Study Chairs: Carolyn Hurley, PhD Georgetown University Medical Center E404 Research Building 3970 Reservoir Road NW Washington, DC Telephone: Fax: Ann Woolfrey, MD Fred Hutchinson Cancer Research Center 1100 Fairview Avenue North D5-280 Seattle, WA Telephone: Fax: Martin Maiers, MS National Marrow Donor Program 3001 Broadway Street N. E. Suite 100, Minneapolis, MN Telephone: Study Statisticians: Tao Wang, PhD CIBMTR Statistical Center Medical College of Wisconsin 9200 W. Wisconsin Ave. CLCC, Suite C5500 Milwaukee, WI USA Telephone: Fax:

163 Not for publication or presentation Attachment 20 JohnBosco Umejiego, MPH CIBMTR National Marrow Donor Program 3001 Broadway Street NE, Suite 100 Minneapolis, MN USA Telephone: Fax: Scientific Directors: Stephen Spellman, MBS CIBMTR National Marrow Donor Program 3001 Broadway Street NE, Suite 100 Minneapolis, MN USA Telephone: Fax: Stephanie Lee, MD, MPH Fred Hutchinson Cancer Research Center P.O. Box 19024, D Fairview Avenue North Seattle, WA USA Telephone: Fax: Working Committee Chairs: David Miklos, MD, PhD Stanford University Department of Medicine; BMT Division CCSR, Room # West Campus Drive Stanford, CA USA Telephone: Fax: Marcelo Fernandez-Vina, PhD Professor Department of Pathology/Blood Center Stanford University School of Medicine 3373 Hillview Avenue Palo Alto, CA USA Telephone: Fax:

164 Not for publication or presentation Attachment 20 Working Committee Chairs: Carlheinz Mueller, MD, PhD Director German National Bone Marrow Donor Registry ZKRD Helmholtzstrabe 10, Ulm, Germany Telephone: Fax:

165 Not for publication or presentation Attachment OBJECTIVES: To compare the outcome of unrelated donor transplants in which the recipient and/or the donor is homozygous at a single mismatched HLA locus (HLA-A or B or C or DRB1) to: 1.1 7/8 matches in which the donor and the recipient are heterozygous for the mismatched locus 1.2 6/8 matches in which the donor and the recipient are each heterozygous and differ for 2 alleles at the same locus or multiple loci. [Potentially dividing this group into mismatches at the same locus and mismatches at two loci.] 1.3 8/8 matches in which the donor and the recipient are homozygous for one of the loci To determine if transplants mismatched in the HVG direction only are associated with graft failure. To determine if transplants mismatched in the GVH direction only are associated with graft versus host disease. Mismatches in which either the donor or the recipient is homozygous for the mismatched locus will have outcome indistinguishable from a 7/8 mismatched transplant, will exhibit better outcome than a 6/8 mismatched transplant, will exhibit worse outcomes than a 8/8 matched transplant. 2.0 SCIENTIFIC JUSTIFICATION: There is no consensus among transplant centers as to how homozygosity should be handled in determining the degree of mismatch between a patient and a potential donor. In an article published in 2001 (1), the Seattle transplant group suggested that risk of graft failure was increased if the recipient of a bone marrow graft was HLA homozygous at the mismatched class I locus. 471 donor-recipient pairs were studied; primary graft failure was observed in 26 patients and secondary failure in two. 280 pairs were matched for HLA-A, B, C with 66 of those also matched for DRB1, DQB1, DPB pairs were mismatched for one allele/antigen with variable matching at class II loci. Of the 7 recipients homozygous at the mismatched locus, 5 exhibited graft failure compared to 7 of the 98 heterozygous recipients (p-value<0.001). An increased percent of homozygous recipients with multiple class I mismatches also showed more frequent graft failure compared to heterozygous recipients with multiple mismatches. Based on these data, the level of mismatch for homozygous recipients with a mismatch at that locus is counted as 2 mismatches in Seattle s current matching criteria (A. Woolfrey, personal communication). However, this count of mismatches is not standard among transplant centers. A recent report by Stevens et al. (5) evaluated the outcomes of 1,202 cord blood transplantations. 98 pairs had only unidirectional mismatches. Engraftment was faster in patients with a unidirectional mismatch in the GVH direction compared to bidirectional mismatches. Patients with hematologic malignancies given unidirectional GVH direction mismatches had lower mortality with outcome similar to matched grafts. Unidirectional mismatched grafts in the HVG direction had slower engraftment, higher rates of graft failure, and higher relapse rates. The authors recommended prioritizing grafts with unidirectional mismatches in the GVH direction and avoiding unidirectional mismatched grafts in the HVG direction. Registries also vary in the design of their search algorithm regarding the tally of mismatches when donor and/or recipient are homozygous. In 2009, the World Marrow Donor Association 165

166 Not for publication or presentation Attachment 20 published a generalized discussion of a registry s matching algorithm (2). In this document, it was recommended that If an individual is homozygous (or being treated as homozygous), the single Ag/ allele is only considered once in counting the mismatches (that is, A*01:02, patient and A*03:02, donor would have one allelic mismatch, not two). A table in supplemental material gives examples of homozygous recipient (AA) vs. heterozygous donor (AB) being counted as a single mismatch in the HvG vector and heterozygous recipient (AB) vs. homozygous donor (BB) being counted as a single mismatch in the GvH vector. An option to consider AA vs. BB as a total of two mismatches was also described. Since counting of mismatches in homozygotes is not standard, this may result in a different selection of potential matches when an international registry search is performed. Unfortunately the evaluation of AA vs BB in terms of impact cannot be evaluated in this study due to the low number of cases (3 total). 3.0 STUDY POPULATION: The study population consists of patients receiving their first marrow or peripheral blood stem cell unrelated donor transplantation for the treatment of AML, ALL, CML or MDS. Transplant pairs must be high resolution typed for HLA-A, B, C, DRB1 through the NMDP retrospective high resolution typing program. Categories of HLA matching to be included in the study (examples only): Donor Recipient Match (A B C DRB1 (3)) Comment A*0201, A*0201 Homozygous A*0201, A*0301 Heterozygous A*0201, A*0201 Homozygous A*0201, A*0201 Homozygous A*0101, A*0201 Heterozygous A*0201, A*0301 Heterozygous 4.0 OUTCOMES: A*0201, A*0201 Homozygous A*0201, A*0201 Homozygous A*0201, A*0301 Heterozygous A*0301, A*0301 Homozygous A*0301, A*0201 Heterozygous A*0205, A*2301 Heterozygous 8/8 7/8 Vector: HvG 7/8 Vector: GvH 7/8 (may be called 6/8) Vector: Both 7/8 Vector: Both 6/8 Vector: Both Suggested by WG Comparison group Pairs of interest Pairs of interest Insufficient cases so exclude Comparison group Comparison group, mismatches at same or different loci Primary: 4.1 Overall survival (OS) Time to death from any cause. Event will be summarized by a survival curve. Cases will be analyzed at the time of last follow-up. There are no competing risks. 4.2 Disease-free survival (DFS) Survival without recurrence of primary disease. Events are disease relapse or death. Cases will be analyzed at the time of last follow-up. There are no competing risks. 166

167 Not for publication or presentation Attachment 20 Secondary: Rejection/graft failure: 4.3 Neutrophil engraftment: Achievement of ANC 500 for 3 consecutive lab values on different days. Event will be summarized by the cumulative incidence estimate with death as a competing risk. 4.4 Secondary graft failure: Loss of graft following achievement of ANC 500 for 3 consecutive lab values on different days. Death and relapse are competing risk and a second transplant is a censoring event. Analysis will be restricted to recipients that engraft. Graft versus host disease/ Graft versus leukemia: 4.5 Acute GVHD: Development of Grades II-IV and III-IV acute GVHD using the Glucksberg or Consensus system. Events will be summarized by the cumulative incidence estimate. Cases will be analyzed at time of last follow-up. Death is a competing risk. Second transplant is a censoring event. 4.6 Chronic GVHD: Development of limited or extensive chronic GVHD. Events will be summarized by the cumulative incidence estimate. Cases will be analyzed at time of last follow-up. Death is a competing risk. Second transplant is a censoring event. 4.7 Disease relapse: Events are disease recurrence. Events will be summarized by the cumulative incidence estimate with death in continuous complete remission as a competing risk. For patients surviving in continuous complete remission, patients will be censored at date of last contact. 4.8 Treatment-related Mortality: Death in continuous complete remission of primary disease. Events will be summarized by the cumulative incidence estimate with relapse as a competing risk. 5.0 VARIABLES TO BE ANALYZED: [Note that small co-variate groups will be combined as necessary for the multivariate modelling.] 5.1 Main Effect to be tested: Impact for donors and/or recipients who are homozygous at a single mismatched locus to pairs heterozygous for the mismatched locus with 7/8 or 6/8 match grade scores and to pairs that are 8/8 matches with homozygosity at one of the loci 5.2 Patient-related (at time of transplant): - Age: in decades (0-9, 10-19, 20-29, 30-39, 40-49, 50 and older). - Gender: female vs. male - Karnofsky score at transplant: < 90 vs Disease-related: - Disease at transplant: ALL, AML, CML and MDS - Disease status prior to transplant: early vs. intermediate vs. advanced 167

168 Not for publication or presentation Attachment Transplant-related: - Source of stem cells: marrow (BM) vs. peripheral blood stem cells (PB) - Donor age: in decades (18-29, 30-39, 40-49, 50 and older) - Year of transplant: ( ) - Race match - Gender match: M-M vs. M-F vs. F-M vs. F-F - Donor/recipient CMV status: -/- vs. -/+ vs. +/- vs. +/+ vs. Unknown - Conditioning regimen: Myeloablative vs. reduced intensity or non-myeloablative - GvHD prophylaxis: Tacrolimus +/-others vs. CSA +/-others vs. TCD vs. others [Note: Consider CSA/Tac prophylaxis as one group] - ATG use: Yes vs. no 6.0 STUDY DESIGN: Donor-recipient pairs who are typed for HLA alleles at HLA-A,-B, -C, -DRB1 where either the donor and/or the recipient are homozygous at the single mismatched locus will be compared to 7/8 and 6/8 matched pairs in which the mismatched locus is not homozygous in both the recipient and the donor. A comparison will also be made to 8/8 matched pairs in which the locus in question is homozygous. Mismatches will be treated in the GvH (graft vs. host disease), HvG (graft failure) or both directions (survival). To summarize the characteristics of the dataset, descriptive tables of patient-, disease and transplant-related factors will be reported. For discrete factors, the number of cases and their respective percentages will be calculated. Chi-Square tests will be used to compare discrete factors between the HLA matched vs. mismatched groups. For continuous factors, the median and ranges will be calculated. The Kruskal-Wallis test will be used to compare the continuous factors between the HLA matched vs. mismatched groups. Probabilities for overall survival and diseasefree survival will be calculated using the Kaplan-Meier estimator with variance estimated by Greenwood's formula. Comparison of survival curves will be done using the log-rank test. Values for other outcomes listed in section 5 will be calculated according to cumulative incidence using a Taylor series linear approximation to estimate the variance. Multivariate analyses will be performed using the proportional hazards model to compare the homozygous locus mismatched vs. heterozygous mismatched groups. Models will be fit to determine which risk factors may be related to a given outcome. All variables will be tested for the affirmation of the proportional hazards assumption. Factors violating the proportional hazards assumption will be adjusted for first before the stepwise model building approach will be used in developing models for the primary and secondary outcomes. If graft failure associated with recipient homozygosity is noted and if resources and a testing laboratory can be identified, recipient serum samples will be requested from the repository. These samples will be tested for the presence of antibodies specific for the donor s mismatched HLA type in order to evaluate a potential cause of the graft failure (4). 168

169 Not for publication or presentation Attachment REFERENCES: 1. Petersdorf EW, Hansen JA, Martin PJ, Woolfrey A, Malkki M, Gooley T et al. Major-histocompatibility-complex class I alleles and antigens in hematopoieticcell transplantation. N Engl J Med 2001; 345(25): Bochtler W, Maiers M, Bakker JN, Oudshoorn M, Marsh SG, Baier D et al. World Marrow Donor Association framework for the implementation of HLA matching programs in hematopoietic stem cell donor registries and cord blood banks. Bone Marrow Transplant Lee SJ, Klein J, Haagenson M, Baxter-Lowe LA, Confer DL, Eapen M et al. High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood 2007; 110(13): Spellman S, Bray R, Rosen-Bronson S, Haagenson M, Klein J, Flesch S et al. The detection of donor-directed, HLA-specific alloantibodies in recipients of unrelated hematopoietic cell transplantation is predictive of graft failure. Blood 2010; 115(13): Cladd E. Stevens, Carmelita Carrier, Carol Carpenter, Dorothy Sung, and Andromachi Scaradavou. HLA mismatch direction in cord blood transplantation: impact on outcome and implications for cord blood unit selection Blood 2011; published ahead of print July 12, 2011, doi: /blood

170 Not for publication or presentation Attachment 20 Table 1. Characteristics of recipients receiving myeloablative first transplants for AML, ALL, CML or MDS that are high-resolution typed for HLA-A, -B, -C and -DRB1 by HLA categories * 8/8 Homo 7/8 HVG 7/8 GVH 7/8 Both 6/8 Both P value Variable zygous MM MM Directions Directions N (%) N (%) N (%) N (%) N (%) Number of patients Number of centers Recipient age, median (range), years 38(<1-70) 35(4-65) 32(<1-66) 33(<1-69) 30(<1-60) <.0001 Age at transplant < y 84 ( 7) 8 ( 7) 18 (14) 151 (10) 70 (14) y 111 (10) 21 (17) 16 (13) 220 (15) 92 (18) y 189 (17) 17 (14) 24 (19) 264 (18) 90 (18) y 214 (19) 35 (29) 23 (18) 297 (20) 117 (23) y 287 (25) 21 (17) 23 (18) 337 (22) 99 (20) 50 y and older 241 (21) 19 (16) 21 (17) 228 (15) 32 ( 6) Recipient race <.0001 Caucasian 1012(90) 96(79) 109(87) 1207(81) 329(79) African-American 35(3) 11(9) 5(4) 97(6) 30(6) Asian/Pacific Islander 22(2) 6(5) 4(3) 29(2) 18(4) Hispanic 44(4) 7(6) 7(6) 143(10) 50(10) Native American 3(<1) 0 0 6(<1) 5(1) Other/Unknown 10(<1) 1(<1) 0 15(<1) 0 Male sex 646 (57) 68 (56) 62 (50) 829 (55) 274 (55) 0.50 Karnofsky prior to transplant > (72) 80 (71) 78 (67) 1004 (71) 356 (73) 0.73 Disease at transplant <.0001 AML 418 (37) 47 (39) 46 (37) 531 (35) 141 (28) ALL 243 (22) 27 (22) 35 (28) 406 (27) 133 (27) CML 274 (24) 34 (28) 25 (20) 374 (25) 181 (36) MDS 191 (17) 13 (11) 19 (15) 186(13) 45 ( 9) Disease status at transplant Early 800 (71) 88 (73) 88 (70) 1046 (70) 328 (66) 0.21 Intermediate 61 ( 5) 8 ( 7) 7 ( 6) 91 ( 6) 46 ( 9) Advanced 265 (24) 25 (21) 30 (24) 360 (24) 126 (25) Graft type <.0001 Bone marrow 689 (61) 78 (64) 86 (69) 1000 (67) 463 (93) PBSC 437 (39) 43 (36) 39 (31) 497 (33) 37 ( 7) In vivo T-cell depletion 0.03 No 891 (79) 82 (68) 94 (75) 1130 (76) 387 (77) Yes 235 (21) 39 (32) 31 (25) 367 (24) 113 (23) 170

171 Not for publication or presentation Attachment 20 Table 1. Continued. 8/8 Homo 7/8 HVG 7/8 GVH 7/8 Both 6/8 Both Variable zygous MM MM Directions Directions P-value N (%) N (%) N (%) N (%) N (%) Number of patients GVHD prophylaxis <.0001 FK506 ± other 564 (50) 55 (45) 51 (41) 635 (42) 100 (20) CsA ± other 517 (46) 58 (48) 69 (55) 782 (52) 358 (72) T-cell depletion 45 ( 4) 8 ( 7) 5 ( 4) 80 ( 5) 42 ( 8) Donor/Recipient sex match Male/Male 464 (41) 37 (31) 41 (33) 518 (35) 153 (31) Male/Female 282 (25) 25 (21) 35 (28) 352 (23) 117 (23) Female/Male 182 (16) 31 (26) 21 (17) 311 (21) 121 (24) Female/Female 198 (18) 28 (23) 28 (22) 316 (21) 109 (22) Donor/Recipient CMV match Negative/Negative 376 (33) 43 (36) 44 (35) 455 (30) 166 (33) Negative/Positive 354 (31) 34 (28) 39 (31) 423 (28) 146 (29) Positive/Negative 142 (13) 17 (14) 24 (19) 230 (15) 82 (16) Positive/Positive 200 (18) 22 (18) 16 (13) 341 (23) 104 (21) Unknown 54 (5) 5 (4) 2 (2) 48 (3) 2 (0) Interval from diagnosis to transplant 10 (<1-317) 10 (<1-249) 10 (<1-99) 12 (<1-309) 14 (<1-161) <.0001 HLA matching HLA-A,B,C and DRB1 8/8 Matched 1126(100) HLA-A one mismatch (MM) 0 39(32) 51(41) 442(30) 0 HLA-B one MM 0 7(6) 10(8) 237(16) 0 HLA-C one MM 0 59(99) 59(47) 663(44) 0 HLA-DRB1 one MM 0 16(13) 5(4) 155(10) 0 HLA-A two MMs (3) HLA-A one MM, -B one MM (7) HLA-A one MM, -C one MM (18) HLA-A one MM, -DRB1 one MM (4) HLA-B two MMs (<1) HLA-B one MM, -C one MM (45) HLA-B one MM, -DRB1 one MM (5) HLA-C two MMs (7) HLA-C one MM, -DRB1 one MM (10) HLA-DRB1 two MMs (<1) N/A Donor age, median (range), years 35(19_59) 36(20-56) 38(20-57) 37(19-61) 37(19-60) <

172 Not for publication or presentation Attachment 20 Table 1. Continued. 8/8 Homo 7/8 HVG 7/8 GVH 7/8 Both 6/8 Both Variable zygous MM MM Directions Directions P-value N (%) N (%) N (%) N (%) N (%) Number of patients Donor age < (33) 33(27) 25(20) 376(25) 135(27) (40) 41(34) 50(40) 551(37) 175(35) (22) 41(34) 39(31) 425(28) 147(29) 50 and older 58(5) 6(5) 11(8) 145(10) 43(9) Donor race Caucasian 982(87) 90(74) 103(82) 1142 (76) 378(76) African-American 21(2) 7(6) 7(6) 95(6) 23(5) Asian/Pacific Islander 22(2) 6(5) 6(5) 35(2) 21(4) Hispanic 42(4) 7(6) 1(<1) 133(9) 44(9) Native American 7(<1) 1(<1) 0 17(1) 5(1) Other/Unknown 30(2) 4(3) 3(2) 46(3) 9(2) <.0001 Year of transplant < (<1) 1 ( 1) 0 3 (<1) 1 (<1) (<1) 0 1 ( 1) 2 (<1) 2 (<1) ( 1) 2 ( 2) 2 ( 2) 15 ( 1) 11 ( 2) ( 2) 1 ( 1) 0 23 ( 2) 24 ( 5) ( 2) 4 ( 3) 5 ( 4) 24 ( 2) 26 ( 5) ( 2) 3 ( 2) 3 ( 2) 40 ( 3) 34 ( 7) ( 3) 1 ( 1) 6 ( 5) 55 ( 4) 40 ( 8) ( 3) 7 ( 6) 3 ( 2) 72 ( 5) 40 ( 8) ( 3) 2 ( 2) 6 ( 5) 71 ( 5) 50 (10) ( 4) 6 ( 5) 9 ( 7) 73 ( 5) 41 ( 8) ( 4) 7 ( 6) 7 ( 6) 72 ( 5) 47 ( 9) ( 5) 6 ( 5) 7 ( 6) 91 ( 6) 37 ( 7) ( 6) 5 ( 4) 9 ( 7) 91 ( 6) 55 (11) ( 4) 4 ( 3) 9 ( 7) 127 ( 8) 48 (10) ( 5) 8 ( 7) 7 ( 6) 61 ( 4) 31 ( 6) ( 7) 11 ( 9) 7 ( 6) 101 ( 7) 8 ( 2) ( 7) 15 (12) 10 ( 8) 128 ( 9) (10) 11 ( 9) 8 ( 6) 125 ( 8) 1 (<1) (12) 12 (10) 11 ( 9) 126 ( 8) (10) 9 ( 7) 8 ( 6) 111 ( 8) 2 (<1) ( 5) 4 ( 3) 6 ( 5) 66 ( 4) 1 (<1) ( 4) 2 ( 2) 1 ( 1) 20 ( 1) 1 (<1) Median follow-up of recipients, mo (range) 66(3-244) 20(206) 56( (3-240) 132 ( ) * Data has been CAP-modeled to adjust for over-sampling of deceased patients due to a lack of consent for NMDP cases prior to *Completeness index % with 1 year follow-up: overall: 99%. % with 3 year follow-up: overall: 95% % with 5 year follow-up: overall: 89% <

173 Not for publication or presentation Attachment 21 CIBMTR IB11-04 IMPACT OF AMINO ACID SUBSTITUTION AT PEPTIDE BINDING POCKETS OF HLA CLASS I MOLECULES ON HEMATOPOIETIC CELL TRANSPLANTATION (HCT) OUTCOME REVISED PROTOCOL Study Chair: Study Co-Chair: Study Statisticians: Joseph Pidala, MD, MS, H. Lee Moffitt Cancer Center & Research Institute Magnolia Drive Tampa, FL 33612, FOB 3308 Telephone: Fax: joseph.pidala@moffitt.org Claudio Anasetti, MD, H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, USA Telephone: Fax: claudio.anasetti@moffitt.org Michael Haagenson CIBMTR-Minneapolis 3001 Broadway Street NE, Suite 100 Minneapolis, MN USA Telephone: Fax: mhaagens@nmdp.org Tao Wang, PhD Associate Professor, Division of Biostatistics Medical College of Wisconsin 8701 Watertown Plank Road Milwaukee, WI USA Telephone: Fax: taowang@mcw.edu 173

174 Not for publication or presentation Attachment 21 Scientific Directors: Stephanie Lee, MD, MPH Fred Hutchinson Cancer Research Center P.O. Box 19024, D Fairview Avenue North Seattle, WA USA Telephone: Fax: Stephen Spellman, MS CIBMTR National Marrow Donor Program 3001 Broadway Street NE, Suite 500 Minneapolis, MN USA Telephone: Fax: Working Committee Chairs: David Miklos, MD, PhD Stanford University Department of Medicine; BMT Division CCSR, Room # West Campus Drive Stanford, CA USA Telephone: Fax: Marcelo Fernandez-Vina, PhD Professor Department of Pathology/Blood Center Stanford University School of Medicine 3373 Hillview Avenue Palo Alto, CA USA Telephone: Fax: Carlheinz Mueller, MD, PhD Director German National Bone Marrow Donor Registry ZKRD Helmholtzstrabe 10, Ulm, Germany Telephone: Fax:

175 Not for publication or presentation Attachment OBJECTIVES: Examine the impact of amino acid substitution (AAS) at HLA class I peptide binding pockets on grade III-IV agvhd, chronic GvHD, treatment-related mortality, relapse and overall survival following unrelated donor allogeneic hematopoietic cell transplantation. 2.0 SCIENTIFIC JUSTIFICATION: Donor-recipient disparity at HLA loci is associated with greater risk for severe acute graft-versus-host disease as well as inferior survival after unrelated donor allogeneic hematopoietic cell transplantation. The impact of AAS at peptide binding pockets of the HLA molecule is incompletely understood. A systematic examination of this problem would both provide mechanistic insights into donor-recipient immunoreactivity, and also provide an enhanced method for the informed selection of unrelated volunteer donors. Given the limited likelihood of finding a HLA identical sibling donor for potential candidates for allogeneic hematopoietic cell transplantation, there is an important need for the identification of suitably matched unrelated volunteer donors. Molecular typing methods have allowed the definition of allelic disparity between potential donors and recipients not possible with serologic typing methods. Seminal work has characterized the importance of allelic disparity at HLA loci in determining transplantation outcomes including severe acute graft-versus-host disease (agvhd), non-relapse mortality, and overall survival. 1-7 Lee, et al conducted an analysis of NMDP data from 3,857 donor-recipient pairs typed with high-resolution DNA matching at HLA-A, -B, -C, -DRB1, -DQB1, -DQA1, -DPB1, and -DPA1 alleles. Single allele or antigen-level mismatches were associated with greater risk of agvhd, mortality, and worsened overall survival, and multiple loci mismatches further increased these risks. 3 This work has demonstrated the adverse impact of HLA loci allele disparity, as well as the relative importance of disparity at specific loci. These considerations guide the selection of potential unrelated donors. These studies, however, have not defined the importance of specific donor-recipient allele mismatch combinations. In contrast, emerging data has begun to elucidate the impact of specific non-permissive allele mismatches on transplantation outcome. Several reports have demonstrated the relationship between specific HLA-DPB1 non-permissive allele mismatches and graft rejection, severe agvhd, and mortality In the largest report to date, Kawase, et al have examined the impact of donor-recipient HLA allele mismatch combinations in an analysis of 5,210 donor-recipient pairs from the Japan Marrow Donor Program. 12 In multivariable analysis, 15 non-permissive (defined by their independent prediction of grade III-IV agvhd) HLA allele mismatch combinations were identified. These included 4 in HLA-A, 1 in HLA-B, 7 in HLA-C, 1 in HLA-DRB1, and 2 in HLA-DPB1. Those with one or greater non-permissive allele mismatches suffered a significantly increased hazard for grade III-IV agvhd, as well as inferior overall survival. Interestingly, those without non-permissive allele mismatches, but with other allele mismatches had similar severe agvhd and survival compared to those who were completely matched at all loci. These data have provided insight into the impact of specific allele mismatch combinations on transplantation outcome. In this analysis of 5,210 donor-recipient pairs from the Japan Marrow Donor Program, Kawase, et al also demonstrated that AAS at position 9 and 116 of the HLA-A molecule, as well as those at positions 9, 77, 80, 99, 116, and 156 in the HLA-C molecule significantly predicted grade III-IV agvhd. 12 With the exception of positions 77 and 80 (associated with KIR2DL ligand in HLA-C), these amino acid positions reside within important peptide binding pockets in the HLA class I molecule. These positions of interest 175

176 Not for publication or presentation Attachment 21 are supported by allied investigation Substitution at such amino acid residues of the HLA class I molecule would be expected to alter peptide binding and presentation of antigen to T cells, and thus have importance in determining donor-recipient alloreactivity. The investigation proposed here would examine a focused question of the impact of AAS at these key positions on HCT outcome utilizing NMDP/CIBMTR data. 3.0 STUDY POPULATION: The study population will include all adult and pediatric patients who underwent a myeloablative or reduced intensity/non-myeloablative first unrelated bone marrow or peripheral blood stem cell HCT for hematological malignancy (AML, ALL, CML and MDS) between 1988 and Patient and donor will have complete high resolution typing for HLA-A, B, C, and DRB1. All donors and patients will be either fully high resolution matched for HLA-A, B, C, and DRB1 (8/8) or have a single mismatch at HLA-A, B or C (7/8). Cases will be categorized based on 8/8, 7/8 with AAS at positions 9, 77, 99, 116 or 156 (in any combination) and 7/8 with AAS only at other positions. 5.0 OUTCOMES: Primary outcomes: 5.1 Overall survival Time to death from any cause. Events will be summarized by a survival curve. Cases will be analyzed at the time of last follow-up. 5.2 Treatment-related mortality - Death in continuous remission of primary disease. Events will be summarized by the cumulative incidence estimate with relapse as a competing risk. 5.3 Acute GVHD - Development of Grades III-IV acute GVHD using the Glucksberg or Consensus system. Event will be summarized by the cumulative incidence estimate. Cases will be analyzed at time of last follow-up. Death is a competing risk. Second transplant is a censoring event. 5.4 Chronic GVHD: Development of limited or extensive chronic GVHD. Events will be summarized by the cumulative incidence estimate. Cases will be analyzed at time of last follow-up. Death is a competing risk. Second transplant is a censoring event. 5.5 Relapse - Development of clinical relapse of the primary disease as defined by the CIBMTR. The event will be summarized by the cumulative incidence estimate and patients analyzed at last follow-up. Death is a competing risk. 6.0 VARIABLES TO BE ANALYZED: 6.1 Main effect to be tested: Impact of mismatching at AAS positions 9, 77, 99, 116 and 156 vs. other AAS mismatches vs. fully (8/8) matched 6.2 Secondary effects: - Compare mismatching at 0, 1, 2, 3, 4 or 5 AAS at positions 9, 77, 99, 116, Evaluate specific combinations of AAS at positions 9, 77, 99, 116, 156 vs. matching, where there are sufficient numbers of transplants to be analyzed (i.e, 30, as utilized in Lee et al, 2007). 176

177 Not for publication or presentation Attachment 21 Other Variables: - Evaluate the specific mismatches identified by Kawase as statistically associated with either relapse or acute GVHD vs. 8/8 matched pairs 6.3 Patient-related (at time of transplant): - Age: in decades (< 10, 10-19, 20-29, 30-39, 40-49, 50) - Race (White vs. Hispanic vs. Black vs. Other) - Karnofsky score at transplant: < 90 vs Disease-Related: - Disease at transplant: AML, ALL, CML and MDS - Disease stage at transplant: early vs. intermediate vs. advanced vs. unknown - Time from diagnosis to transplant for CML and MDS 6.5 Transplant-Related: - Source of stem cells: marrow (BM) vs. peripheral blood stem cells (PB) - Donor age: in decades (18-29, 30-39, 40-49, 50) - Year of transplant - Gender match: M-M vs. M-F vs. F-M vs. F-F - Donor/recipient CMV status: -/- vs. -/+ vs. +/- vs. +/+ vs. Unknown - Conditioning regimen: myeloablative vs. reduced intensity/non-myeloablative - GvHD prophylaxis - Use of in vivo T cell depletion (ATG or Campath) - Transplant center 7.0 STUDY DESIGN: We will examine the independent impact of AAS at key (9, 77, 99, 116, and 156) residues on the outcomes of grade III-IV agvhd, chronic GvHD, treatment-related mortality, relapse and overall survival among unrelated donor-recipient pairs. The frequencies of the AAS within the population of mismatched cases are shown in table 1. To summarize the characteristics of the dataset, descriptive tables of patient-, disease and transplant-related factors will be reported. - For discrete factors, the number of cases and their respective percentages will be calculated. Chi- Square tests will be used to compare discrete factors between the HLA matched vs. mismatched groups. - For continuous factors, the median and ranges will be calculated. The Kruskal-Wallis test will be used to compare the continuous factors between the HLA matched vs. mismatched groups. Probabilities for overall survival and disease-free survival will be calculated using the Kaplan-Meier estimator with variance estimated by Greenwood's formula. Comparison of survival curves will be done using the log-rank test. Values for other outcomes listed in section 5 will be calculated according to cumulative incidence using a linear approximation to estimate the variance. A p-value < 0.01 will be considered significant. 177

178 Not for publication or presentation Attachment 21 Multivariate analyses will be performed using the proportional hazards model. All clinical variables will be tested for the affirmation of the proportional hazards assumption. Factors violating the proportional hazards assumption will be adjusted through stratification. Then a stepwise model building approach will be used in developing models for the primary outcomes. Based on these models, the main AAS variables will be tested by forcing them into the models. Interactions among the significant AAS variables and the clinical variables will be tested. For both the primary and the secondary endpoints, an overall trend test for the 0-5 substitutions and pairwise comparisons will first be tested. An overall p-value < 0.01 will be considered significant. Specific AA mismatch at a particular position with more than 30 cases will also be compared with 8/8 matched pairs. These tests for specific residue positions will be performed for each position separately and the p-value will be adjusted for multiple testing using Bonferroni criterion. A multivariate analysis for the amino acid mismatches will be performed by treating the AAS at each position as an independent variable. Then a stepwise model selection procedure will be adopted to identify the amino acid substitution sites that are most associated with the outcomes, controlling for other mismatches presented in the model. Finally, the specific mismatch combinations identified by Kawase as associated with either relapse or acute GVHD will be tested in this dataset. Pairs with at least one of the important Kawase mismatches will be compared with the 8/8 matched pairs. Since the mismatches associated with relapse and acute GVHD were primarily different, a separate analysis will be performed for each endpoint. Relapse:

179 Not for publication or presentation Attachment 21 Acute GVHD III-IV: REFERENCES: 1. Davies SM, Kollman C, Anasetti C, et al. Engraftment and survival after unrelated-donor bone marrow transplantation: a report from the national marrow donor program. Blood. 2000;96: Flomenberg N, Baxter-Lowe LA, Confer D, et al. Impact of HLA class I and class II highresolution matching on outcomes of unrelated donor bone marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplantation outcome. Blood. 2004;104: Lee SJ, Klein J, Haagenson M, et al. High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood. 2007;110: Morishima Y, Sasazuki T, Inoko H, et al. The clinical significance of human leukocyte antigen (HLA) allele compatibility in patients receiving a marrow transplant from serologically HLA-A, HLA-B, and HLA-DR matched unrelated donors. Blood. 2002;99: Petersdorf EW, Longton GM, Anasetti C, et al. Association of HLA-C disparity with graft failure after marrow transplantation from unrelated donors. Blood. 1997;89: Sasazuki T, Juji T, Morishima Y, et al. Effect of matching of class I HLA alleles on clinical outcome after transplantation of hematopoietic stem cells from an unrelated donor. Japan Marrow Donor Program. N Engl J Med. 1998;339: Speiser DE, Tiercy JM, Rufer N, et al. High resolution HLA matching associated with decreased mortality after unrelated bone marrow transplantation. Blood. 1996;87: Crocchiolo R, Zino E, Vago L, et al. Nonpermissive HLA-DPB1 disparity is a significant independent risk factor for mortality after unrelated hematopoietic stem cell transplantation. Blood. 2009;114: