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

Transcription

1 Not for publication or presentation A G E N D A CIBMTR WORKING COMMITTEE FOR IMMUNOBIOLOGY Honolulu, Hawaii Thursday, February 17, 2011, 12:15 pm 4:45 pm Co-Chair: Co-Chair: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, M. D. Anderson Cancer Center Telephone: ; Fax: ; mfernand@mdanderson.org 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 (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. 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. Blood, April 2010; 115:

2 Not for publication or presentation d. IB06-07s Nguyen Y, Al-Lehibi A, Gorbe E, Li E, Haagenson M, Wang T, Spellman S, Lee SJ and Davidson NO.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. 2

3 Not for publication or presentation 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 Impact of amino acid substitution at peptide binding pockets of HLA class I molecules on hematopoietic cell transplantation (HCT) outcome (J Pidala/C Anasetti) (Attachment 3) c. PROP Analysis of the NIMA effect on the outcome of unrelated PBSC/BM transplantation (G Ehninger) (Attachment 4) d. PROP Impact of CLTA4 single nucleotide polymorphisms on outcome after unrelated donor transplant (M Jagasia) (Attachment 5) e. PROP Evaluation of the impact of allele homozygosity at HLA loci on outcome (C Hurley/A Woolfrey/M Maiers) (Attachment 6) f. PROP KIR genotyping and immune function in MDS patients prior to unrelated donor transplantation (E Warlick/J Miller) (Attachment 7) g. PROP Evaluation of the impact of potentially non-immunogenic HLA-C allele level mismatch (M Fernandez-Vina/M Setterholm) (Attachment 8) h. PROP Effect of Rituximab and ABO mismatch (D Miklos) (Attachment 9) i. Proposal voting 6. International Histocompatibility and Immunogenetics Workshop 2:00 pm Collaboration update (E Petersdorf) a. R04-75s CGP and post-transplant complication (IHWG) Manuscript preparation (E Petersdorf) b. R04-76s Identification of functional SNPs (IHWG) (E Manuscript preparation Petersdorf) c. IB05-02s The effect of a single MHC class I mismatch with Manuscript preparation numerous sequence differences on the clinical outcome of unrelated HSCT (M Heemskerk) d. IB06-05 Use of high-resolution HLA data from the National Manuscript preparation Marrow Donor Program for the IHWG in hematopoietic cell transplantation (E Petersdorf) e. IB07-04 Employing advanced bioinformatic methods for Analysis 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 Manuscript preparation GVHD among HLA-A, B, C, DRB1, DQB1, DPB1 matched unrelated transplants (IHWG) (Y Morishima) (Attachment 10) g. IB07-06 HLA DP epitope study (IHWG) (B Shaw) Manuscript preparation (Attachment 11) h. IB07-07 HLA-DR15 and Transplant Outcome (IHWG) (A Manuscript preparation Gratwohl) i. IB09-01s Clinical importance of MHC haplotypes in umbilical Data collection cord blood transplantation (E Petersdorf) - no update j. IB09-03s Clinical relevance of cytokine/immune response Data collection gene polymorphisms in umbilical cord blood transplantation (E Petersdorf) - no update k. IB09-05s Identification of functional SNPs in umbilical cord blood transplantation (E Petersdorf) - no update Data collection 3

4 Not for publication or presentation l. IB09-07s Clinical Significance of Genome-wide Variation in Unrelated Hematopoietic Cell Transplantation (E Petersdorf) - no update BREAK 15 minutes 7. Studies in progress (Attachment 12) HLA GENES CLASSICAL MATCHING (C Müller) a. IB08-02 Evaluation of HLA matching requirements in unrelated hematopoietic stem cell transplantation for nonmalignant disorders (J Horan/A Woolfrey) (Attachment 13) - update b. IB09-02 Non-permissive HLA-DPB1 disparities based on T cell alloreactivity (K Fleischhauer) (Attachment 14) - update c. IB10-05 Evaluation of a Scoring System for HLA Mismatching: HistoCheck (R Blaszcyk/ C Hurley) (Attachment 15) - update d. IB10-07 Use of HLA Structure and Function Parameters to Understand the Relationship between HLA Disparity and Transplant Outcomes (LA Baxter-Lowe) (Attachment 16) - update e. IB06-02 Mismatching for low expression HLA loci in matched unrelated donor transplants (M Fernandez-Vina) - no update CYTOKINE/CHEMOKINE (Chair: M Fernandez-Vina) a. IB05-03s Genetic polymorphisms in the genes encoding human interleukin-7 receptor-α: Prognostic significance in allogeneic stem cell transplantation (K Müller) - no update b. IB08-04s Immune response gene polymorphisms in unrelated donor stem cell transplantation in children (K Müller) no update 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) no update d. IB08-06 Analysis of Killer Immunoglobulin-Like Receptor (KIR) ligands in umbilical cord blood transplantation (R Sobecks) - no update Protocol development 2:20 pm 2:35 pm Analysis Manuscript preparation Manuscript preparation Data file preparation Manuscript preparation 3:00 pm Manuscript preparation Protocol development 3:00 pm Ongoing Manuscript preparation Data file preparation Protocol development 4

5 Not for publication or presentation SENSITIZATION/TOLERANCE 3:15 pm (Chair: M Fernandez-Vina) a. IB06-11s The effect of NIMA in cord blood transplantation (V Manuscript preparation Prasad/L Baxter-Lowe/J Kurtzberg) - (Attachment 19) - update b. 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 20) - update c. R03-65s HY antigen (D Miklos) - no update Manuscript preparation d. GV04-01 Non-identical twin transplant for leukemia Protocol development (J Barrett) (Attachment 21) no update e. 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 f. IB06-10 Evaluation of the impact of the exposure to NIMA Protocol development 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) (Attachment 16) - no update g. IB10-02 Development of GVHD prevention diagnostic test (R Somogyi/L Greller) - no update Typing OTHER GENES (Chair: C Müller) a. IB08-08 Genome-Wide Association in Unrelated Donor Transplant Recipients and Donors: A Pilot Study (R Goyal) - (Attachment 22) - update 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) (Attachment 23) - update 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 d. IB07-09 To develop and test a prognostic index for survival in CML MUD cohorts (A Dickinson) - no update 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 f. IB09-06s/RT09-04s Genetic polymorphisms and HCT related mortality Re: Pre-HCT conditioning in matched unrelated donor HCT (T Hahn) no update 3:30 pm Typing Typing Manuscript preparation Manuscript preparation Typing Protocol development 5

6 Not for publication or presentation g. IB10-03 TLR and HMGB1 gene polymorphisms in unrelated haematopoietic stem cell transplantation (K Müller/B Kornblit) no update 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 MINOR HISTOCOMPATIBILITY ANTIGENS (Chair: C Müller) a. IB10-06 Identification of Common, Clinically Significant, Minor Histocompatibility Antigens through Stem Cell Transplant Donor/Patient Polymorphism Disparities (P Armistead) no update Typing Typing Deferred 3:45 pm 8. Deferred studies pending accrual 3:45 pm a. R04-80s HLA matching in unrelated cord blood transplants Data collection (S Rodriguez-Marino) - no update b. IB06-13 HLA disparity in unrelated cord blood Data collection transplantation: Delineation of factors contributing to transplant outcomes (L Baxter-Lowe) - no update c. IB08-05s Evaluation of lymphotoxin alpha (LTA) alleles in relation to relapse in AML and CML (P Posch) - no update Data collection 9. Feedback from Committee (C Müller) 3:50 pm 10. Closing remarks (Chair: C Müller) 4:00 pm 6

7 Not for publication or presentation Attachment 1 MINUTES CIBMTR WORKING COMMITTEE FOR IMMUNOBIOLOGY Orlando, Florida Wednesday, February 24, 2010, 12:15 pm 4:45 pm Co-Chair: Co-Chair: Co-Chair: Statisticians: Co-Scientific Dir: Co-Scientific Dir: Effie Petersdorf, MD, Fred Hutchinson Cancer Research Center Telephone: ; Fax: ; epetersd@fhcrc.org David Miklos, MD, PhD, Stanford University Telephone: ; Fax: ; dmiklos@stanford.edu Marcelo Fernandez-Vina, PhD, M. D. Anderson Cancer Center Telephone: ; Fax: ; mfernand@mdanderson.org 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, National Marrow Donor Program Telephone: ; Fax: ; sspellma@nmdp.org 1. Welcome and Introduction Dr. Effie Petersdorf called the meeting to order at 12:20 pm. She introduced the Immunobiology Working Committee (IBWC) leadership along with the newly appointed chair Carlheinz Mueller, MD, PhD, from the Zentrales Knochenmarkspender-Register Deutschland (ZKRD). Mr. Spellman thanked Dr. Petersdorf for her contributions as a co-chair to the IBWC over the past five years and presented her with a small gift from the CIBMTR for her service. 2. Minutes of Immunobiology Working Committee at Tandem 2009 The minutes were approved as written. 3. Accrual Tables Mr. Spellman referred the group to the accrual table summaries in the agenda. 4. Proposed Studies and Discussion for Immunobiology Working Committee a. Voting guidelines for proposals were discussed by Dr. David Miklos. He reviewed the guidelines and discussed the purpose of evaluating the proposals. He reminded committee members to consider what proposals best represent the Immunobiology Working Committee (IBWC) purpose, and to consider whether data are available at the NMDP or CIBMTR to support the proposal. IBWC members were encouraged to write comments and to vote the extremes, where 0=low priority, 1=medium priority and 2=high priority. 7

8 Not for publication or presentation Attachment 1 b. Discuss proposals to evaluate donor specific anti-hla antibodies (DSA) in cord blood transplants (CBT) Mr. Spellman informed the group that the IBWC received three proposals focused on evaluating the impact of DSA in CBT. He explained that the analyses require pre-transplant recipient plasma and that the NMDP Repository initiated plasma processing and storage in July There are currently 208 single-cord blood unit (CBU) transplants and 186 multi- CBU transplants in the inventory. A power calculation based on published DSA rates in CBT from the Japanese Red Cross suggests that ~350 single CBU transplants would be required to detect a significant difference (5% level) in engraftment with 80% power. Annual accrual rates suggest the study could proceed within 1-2 years. The study is further complicated by the need for high resolution HLA typing data on the CBU and recipient and the impact of DSA screening practices at Transplant Centers on the selection of specific cords. NMDP is prioritizing retrospective high resolution testing of CBT pairs to address the first issue. A survey of IBWC members suggests that half of the centers represented screen for potential DSA prior to CBT, and consider this information in cord selection. The final study population will likely need to be restricted to non-screening centers. c. PROP TLR and HMGB1 gene polymorphisms in unrelated haematopoietic stem cell transplantation Dr. Brian Kornblit presented this proposal which would validate the Danish group s previous findings of associations between TLR and HMGB1 gene polymorphisms in patients and donors and overall survival, progression-free survival and transplant related mortality after allogeneic HCT. Drs. Kornblit and Muller intend to use the cohort of 850 Caucasian cases from Dr. Muller s previous study IB05-03s. The genotyping methods are already established in the laboratory and the DNA is ready to go. They will evaluate SNPs with minor allele frequencies greater than 1 percent. The Committee raised concerns about the sample size and suggested possibly expanding the number of pairs following the initial analysis. d. PROP A validation study of the role of base excision repair pathway as a predictor of outcome after hematopoietic stem cell transplant Dr. Mukta Arora presented this proposal, which intends to validate the results of a study performed at the University of Minnesota where they found correlation between polymorphisms in the Base Excision Repair pathway and incidence of TRM and relapse. This will be a study on an NMDP cohort of approximately 1000 adult Caucasian patients who underwent myeloablative allogeneic HCT from an unrelated matched (8/8 HLA matched at HLA-A, B, C and DRB1 alleles) donor for treatment of acute leukemias or CML. The Committee noted that the study may need to control for the influence of DP mismatching. e. PROP Evaluation of a scoring system for HLA mismatching: HistoCheck Dr. Carolyn Hurley presented this proposal as co-principal investigator with Dr. Rainer Blasczyk. This study will evaluate the scoring system for HLA mismatches called Histocheck. Histocheck is readily accessible on the internet and is being used by some transplant centers to prioritize HLA mismatches for donor selection. A previous analysis by the International Histocompatibility Workshop Group (IHWG) found no correlation between the Histocheck score and transplant outcome, but these results have not been published. This study would use a data set already prepared by the CIBMTR/NMDP for the evaluation of HLA mismatch scoring, e.g. Dr. Rene Duquesnoy s MatchMaker data set, to minimize the dataset preparation efforts at the Statistical Center. The Committee strongly felt that the results of the analysis should be published regardless of the findings. There was a suggestion 8

9 Not for publication or presentation Attachment 1 to include cord blood transplants in this study but others felt that numbers are small, cords are not fully typed, and inclusion of cords would complicate the analysis. f. PROP Identification of common, clinically significant, minor histocompatibility antigens through stem cell transplant donor/patient polymorphism disparities Dr. Paul Armistead presented this proposal which has the central hypothesis that GvHD is mediated by immunodominant minor Histocompatibility Antigens (mha) presented in the context of specific HLA molecules. Some of these mha occur at high frequency in patients undergoing allogeneic SCT. These high frequency mha can be identified by genomic screening methods. It has been shown that mha mediate acute GvHD. This study would evaluate and type 612 donor/recipient pairs, which would give 80% power to find an odds ratio of 2.0. This proposal would focus on potential mha that could be presented in the context of HLA-A*02 and would look at 10/10 matched unrelated donor transplants for AML, ALL, CML and MDS. This study will require samples. The Committee suggested Dr. Armistead not include cases from MD Anderson, since they were included in his preliminary analysis and restrict to HLA-A*0201 positive cases. g. PROP Use of HLA structure and function parameters to understand the relationship between HLA disparity and transplant outcomes Dr. Marcelo Fernandez-Vina presented this proposal in Dr. Lee Ann Baxter-Lowe s absence. This proposal would evaluate an HLA mismatch scoring algorithm based on the location of mismatched residues. Factors incorporated into the scoring system for allorecognition potential include high and low frequency allorecognition factors. Some of the high frequency allorecognition factors include high TCR docking potential, such as identical docking amino acids and conservation of surrounding amino acids, as well as peptide differences, such as the sequence and orientation of the peptides. The low frequency allorecognition factors include TCR docking disruption without differences in peptide binding or orientation. The hypothesis is that TCR docking is critical for allorecognition. The goal is to look at HLA-A and -B mismatches first and then consider HLA-C and -DRB1 separately. This study would use a data set already prepared by the CIBMTR/NMDP for the evaluation of HLA mismatch scoring, e.g. Dr. Rene Duquesnoy s MatchMaker data set, to minimize the dataset preparation efforts at the Statistical Center. The Committee suggested that the study population be restricted to T cell replete transplants. h. Proposal voting was completed at this time. 5. Discussion about Advisory Committee recommendations and response to them about ways to improve the Immunobiology Working Committee (IBWC) Dr. Stephanie Lee presented a summary of the Advisory Board recommendations for the IBWC and IBWC leadership response to these suggestions. She solicited feedback from the group. Dr. John Hansen noted the collaboration with the BMTCTN Biomarker committee and that a proposal for comprehensive sample collection was under preparation. The main concerns with collection of samples through BMTCTN were the limited sample size available for accrual through the BMTCTN trials and limitations on phenotype data collection. Break - A break was taken from 2:00 pm - 2:15 pm. 9

10 Not for publication or presentation Attachment 1 6. Studies in Progress OTHER GENES a. IB08-08 Genome-wide association in unrelated donor transplant recipients and donors: a pilot study Dr. Rakesh Goyal presented an update. This study examines the genetic differences between recipients who experience grade III-IV acute GvHD (G+) vs. those who experience no acute GvHD (G-). The study population consists of 10/10 allele-matched Caucasians receiving their first transplant with standard ablative conditioning and no T-cell manipulation. The study is currently in the typing stage. b. IB09-06s/RT09-04s Genetic polymorphisms and HCT related mortality re: pre-hct conditioning in matched unrelated donor HCT Dr. Theresa Hahn presented an update to her study with the co-principal investigator Dr. Lara Sucheston in the audience. Specific aims of this study include 1) To undertake a Genome Wide Association Study (GWAS) to map the independent and joint effects of recipient and donor genetic variation associated with TRM after HLA-matched unrelated donor BMT; 2) To determine the modifying effects of conditioning regimens on associations between recipient and/or donor genetic variants and TRM; and 3) To replicate the top genetic associations in an independent cohort of high resolution 10/10 HLA-matched BMT recipientunrelated donor pairs. The NHLBI initial review requested to add donors to the cohort. The grant was resubmitted in November 2009 and received a score of 15 and has a percentile rank of 2%. Dr. Hahn expects to hear the final decision in May and will most likely receive the grant in July c. IB10-01 Donor and recipient telomere length as predictors of outcomes after hematopoietic stem cell transplant in patients with acquired severe aplastic anemia Dr. Shahinaz Gadalla presented an update to her study. The specific aims of this study are to 1) determine pre-transplant blood telomere length in SAA who received unrelated HSCT compared with age-matched controls and patients with dyskeratosis congenita; 2) assess the relationships between telomere length, and post-transplant outcomes in patients who received unrelated donor HSCT for SAA; and 3) identify factors that modify the association between recipient and/or donor telomere length, and post-transplant outcomes in those individuals. The study population consists of patients less than 40 years of age. There are currently 395 cases with samples. This study currently has funding, and the PIs are preparing for DNA extraction. d. IB07-08 SNPs in the P53 pathway (P53, MDM2, ATM AND P21/WAF1) and transplant outcome after unrelated hematopoietic stem cell transplantation No update was given. e. IB07-09 To develop and test a prognostic index for survival in CML MUD cohorts No update was given. f. 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 No update was given. SENSITIZATION/TOLERANCE a. IB06-11s The effect of NIMA in cord blood transplantation Dr. Vinod Prasad presented this study which looks at the influence of HLA mismatching in non-inherited maternal antigens (NIMA) on the relationship between HLA disparity and outcomes of cord blood transplants. This study tests the hypotheses that NIMA tolerance favorably influences outcomes of cord blood transplants. The COBLT study contained 102 transplants that have NIMA typing available. A preliminary 80% power calculation showed 10

11 Not for publication or presentation Attachment 1 that the survival rates of 50% vs. 30% for NIMA vs. non-nima groups would require patients if 3/6, 4/6, and 5/6 were combined, so additional cases are needed. The study population will consist of transplants with a single-cord unit. The main effect variable will be NIMA matches vs. NIMA mismatches. Standard outcomes will be evaluated during this study. The NMDP/CIBMTR has identified approximately 600 single cord transplant cases treated for malignancy for inclusion in the study. In addition, Eurocord has expressed interest in collaborating on the project. The NMDP will support the collection and HLA testing of maternal samples from the NMDP Cord Blood Bank Network to facilitate the analysis. Due to the anticipated low frequency of NIMA mismatches the study will likely employ a matched case control strategy to maximize power to detect a difference between NIMA matched cases versus mismatched controls. b. IB09-08 A retrospective study on impact of donor and recipient birth order on outcome of HLA-identical sibling stem cell transplantation (SCT) in hematological malignancies reported to the CIBMTR Dr. Christiane Dobbelstein presented her study which evaluates outcome after HLA-identical sibling or matched unrelated donor (MUD) transplants when considering birth order of the recipient and donor. There are 11,745 HLA-identical siblings and 2052 MUD transplants in the current selection. Specific aims include the primary objective of evaluating relapse rate after HLA-identical sibling stem cell transplantation in hematological malignancies depending on birth order of donor and recipient. Secondary objectives include evaluating disease-free-survival (DFS), overall survival (OS), non-relapse-mortality, acute and chronic GvHD after HLA-identical sibling stem cell transplantation in hematological malignancies depending on birth order of donor and recipient and to check if there is a similar association between relapse rate, DFS, OS, NRM, and acute and chronic GvHD in HLA-identical MUD SCT depending on relative ages of the donor and recipient as a surrogate for birth order. The study is currently in protocol development and will soon be distributed to the whole working committee to form a writing committee. There will also be planned subset analyses. c. R03-65s HY antigen No update was given. d. 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? No update was given. e. 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 No update was given. NK/KIR a. R02-40s/R03-63s KIR Program Project/NK receptor acquisition Dr. Jeffrey Miller presented this ongoing project which has recently looked at KIR genotype assignments and the effect of KIR genotypes on outcome. Donor DNA samples were typed for 15 individual KIR genes by Dr. Elizabeth Trachtenberg using a validated single nucleotide polymorphism (SNP)-based MALDI-TOF assay. KIR genotypes were assigned as haplotypes of A/A or B/x where the B haplotype checks for the presence of 2DS2, 2DL5, 2DS3/S5, 3DS1, 2DS. Further definitions include Centromeric/Telomeric Segments: Cen/Tel A/A, A/B, B/B with a B-content score between 0 and 4. Multivariate analysis shows that KIR Cen-B/B donors are associated with the lowest relapse risk. Multivariate analysis also shows that donors with >2 KIR B-gene motifs decrease relapse and improve survival. Additional analyses showed that donors with > 2 KIR-B gene motifs protect against relapse in HLA matched or HLA mismatched transplants. Donor KIR genotype does not affect outcomes for 11

12 Not for publication or presentation Attachment 1 ALL. This is an ongoing project with more analysis planned in the near future. b. R04-74s KIR functional significance (IHWG) Dr. Katharine Hsu presented this study which analyzed an NMDP cohort of samples. The study looks at KIR genotyping of donors. The analysis showed that there is increased overall survival, less transplant-related mortality for donor KIR3DS1 homozygous compared to other KIR3DS1 groups but no significant difference in relapse. Upon further subset analysis, the effects appeared mostly in AML. The IHWG found reduced relapse with donors who have KIR2DS5 & KIR3DS1. KIR2DS2 in AML showed no significant difference. c. IB07-03 Analysis of Killer Immunoglobulin-like Receptor (KIR) ligands in reduced intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (HSCT) Dr. Ronald Sobecks presented this study which has gone through some changes since the protocol was sent out a few months ago. First, this study may incorporate genotyping. Second, comments from the writing committee are being incorporated into the protocol. The study population will change to The number of missing KIR ligands will likely be quantified as 0 vs. 1 vs. 2 vs. 3 instead of present vs. absent. The study design will incorporate recipient and donor KIR ligands. The outcomes will be acute and chronic GvHD, overall survival, relapse and disease-free survival. Samples are available on the whole cohort. Typing will either use the IHWG typing or will go through the NMDP retrospective typing project. d. IB08-06 Analysis of Killer Immunoglobulin-Like Receptor (KIR) ligands in umbilical cord blood transplantation No update was given. CYTOKINE/CHEMOKINE a. IB05-03s Genetic polymorphisms in the genes encoding human interleukin-7 receptor-α: Prognostic significance in allogeneic stem cell transplantation No update was given. b. IB08-04s Immune response gene polymorphisms in unrelated donor stem cell transplantation in children No update was given. HLA GENES CLASSICAL MATCHING a. IB06-04 Effect of age on outcome in patients undergoing related HLA-mismatched/ haploidentical stem cell transplantation Dr. Stephanie Lee gave an update on the study in Dr. Lujia Dong s absence. The study has a draft manuscript in progress and has a poster presentation at the Tandem BMT Meetings this year. The study was limited by numbers, and no significant age effect was found. b. IB08-02 Evaluation of HLA matching requirements in unrelated hematopoietic stem cell transplantation for nonmalignant disorders Dr. John Horan presented this study in Dr. Ann Woolfrey s absence. The hypothesis of this study is that the effect of HLA matching in unrelated hematopoietic stem cell transplantation in patients with non-malignant disorders differs from its effect in patients with malignant diseases. Some reasons for this are that 1) by reducing the risk for relapse, GVHD may be beneficial in patients with malignancies. In patients with non-malignant disorders, on the other hand, GVHD is not beneficial. 2) Patients with non-malignant disorders tend to be younger, and, therefore may be less apt to develop and die from GVHD. 3) Patients with many non-malignant disorders are prone to graft failure. Patients are eligible if they received an unrelated BM or PB graft for a nonmalignant disease, including severe aplastic anemia, immunodeficiency disorders, inherited disorders, and autoimmune disorders between 1988 and They are also eligible if they received reduced intensity, non-myeloablative, nontraditional ablative or standard myeloablative regimens. Patients are ineligible if either they received an unrelated cord blood graft or they or their donor lack high resolution typing of 12

13 Not for publication or presentation Attachment 1 HLA-A, B, C and DRB1. Outcomes include survival, acute and chronic GVHD, transplantrelated mortality and graft failure. Main effect will be the impact of allele and antigen mismatching at the A, B, C, DRB1 and DQB1 loci (available for all pairs). The effect of DPB1 will be estimated if the numbers are sufficient. Analysis will likely only include the 0 mismatches, 1 mismatch and the 2 mismatches groups. There are only 56 patients with three or more mismatches. c. IB09-02 Non-permissive HLA-DPB1 disparities based on T cell alloreactivity Dr. Marcelo Fernandez-Vina presented this study in Dr. Katharina Fleischhauer s absence. This study intends to validate the findings from the Italian group. Specific aims of this study are 1) to validate in vitro an algorithm for definition of non-permissive HLA-DPB1 disparities predictive of mortality after unrelated HSCT, and 2) to analyze the role of HLA- DPA1 in determining the immunogenic T cell epitope (TCE) relevant for non-permissive HLA-DP disparities. The study population consists of 1281 myeloablative transplants that are 10/10 allele matched, DPB1 mismatched and DPA1 typed pairs available in the NMDP database. An analysis will be performed looking at TCE3 and TCE4, which are grouped as matched, permissive, non-permissive and potentially permissive. The primary endpoint is overall survival with secondary endpoints of TRM, acute GvHD and relapse. There will also be a Brier Score comparison at 1, 3 and 5 years of survival for both TCE3 and TCE4. The protocol has been distributed to the whole working committee and the writing committee is being formed. d. IB06-02 Mismatching for low expression HLA loci in matched unrelated donor transplants No update was given. 7. International Histocompatibility and Immunogenetics Workshop Group (IHWG) Collaboration Update Dr. Effie Petersdorf gave a summary of the IHWG studies that showed progress over the past year. a. R04-75s CGP and post-transplant complication No update was given. b. R04-76s Identification of functional SNPs SNPs Genotyping includes MHC exon centric Illumina SNP panels genotyped for HLA 10/10 for 3,310 transplants; HLA 9/10 for 1,622 transplants; and HLA 8/10 or less for 457 transplants (which is the ethnicity panel). Currently, the analyses are ongoing. Comparisons of recipient and donor minor allele frequencies (MAF) show little difference between them. Recipient and donor SNP mismatching has a low percentage around HLA-A, -B, -C, -DRB1 and -DQB1 c. IB07-05 Impact of donor-recipient ethnicity on risk of acute GVHD among HLA-A, B, C, DRB1, DQB1, DPB1 matched unrelated transplants Dr. Petersdorf presented this update for Dr. Yasuo Morishima. The purpose of this study is to compare the incidence of acute GVHD between ethnic groups based on the same background. This is a large scale IHWG HCT database of 5555 pairs looking at 10/10 HLA allele matched transplants. The GVHD prophylaxis includes T cell replete stem cell sources and leukemia and MDS patients. Results obtained from this analysis will become basic data for further international analysis of HLA mismatched unrelated HSCT and for donor exchange of unrelated donor. There were 2062 Asian & Pacific/Asian & Pacific pairs, 2419 Caucasian/Caucasian pairs, 39 African American/African American pairs, 21 Hispanic/Hispanic pairs, 2 Native American/Native American pairs, 268 mismatched race pairs (in non-jmdp) and 744 unknown donor ethnicity. Multivariate analysis showed that after adjusting for clinical factors, Caucasian pairs and mismatched race pairs have more acute GvHD (both grades II-IV and grades III-IV) than Asian/Pacific pairs. African American 13

14 Not for publication or presentation Attachment 1 pairs also have more grades III-IV acute GvHD than the Asian/Pacific pairs. Multivariate analysis also shows that after adjusting for clinical factors, Caucasian, Hispanic, African American and mismatched race pairs all have a higher overall mortality rate than Asian/Pacific pairs. d. IB07-06 HLA DP epitope study Dr. Petersdorf provided the update for Dr. Bronwen Shaw. The hypothesis is that mismatching for T cell epitopes (TCE) in the HLA-DP molecule may be as important or more important as allele-matching for predicting outcome of unrelated donor HSCT. The study evaluated group-specific matching rather than allele-specific DPB1 matching where the groups were TCE-disparate as Non-Permissive and TCE-matched as all others. The study population included /10 (HLA-A,-B,-C,-DRB1,-DQB1) allele-level matched pairs. The analysis considered groups of 1) DPB1 allele-mismatched (n=4490) or 12/12 (n=1348) or TCE disparate (TCED) in GvH or HvG, or TCE matched (TCEM). The multivariate results were adjusted for classical variables with the TCEM and DPB1 allele mismatched serving as the reference group. The multivariate results show that the 10/10 TCE disparate group has significantly higher TRM, grades III-IV acute GvHD and overall mortality than the TCE matched DPB1 allele-mismatched group. Multivariate results also show that the 12/12 DPB1 matched group has lower TRM, grades III-IV acute GvHD and higher relapse rates than the TCE matched DPB1 allele-mismatched group. Ongoing work will include defining the impact of the TCE in the 9/10 HLA matched transplants (N=3315) as well as completing an analysis of particular diseases (B cell malignancies) and determining the role of hypervariable region (HvR) disparity in defining non-permissive mismatches. The Committee questioned whether the selection for permissible vs. non-permissive DP mismatches was feasible in the general transplant population. e. IB05-02s The effect of a single MHC class I mismatch with numerous sequence differences on the clinical outcome of unrelated HSCT Dr. Petersdorf presented the update for this study. The hypothesis is that HCT mismatched for a single MHC class I allele that has many amino acid differences on the α helices and β sheet of the molecule, leads to a more successful outcome compared to HCT with other single class I HLA mismatches. There are 654 recipient-donor pairs with a single class I mismatch (196 HLA-A; 88 HLA-B; 370 HLA-C) in the study population. Some current discussion points for this study include highly divergent HLA mismatches do not lead to superior survival results when compared to other single class I mismatches. The difference with the previous study is that in the Dutch study, the transplants were mainly performed with T-cell depleted grafts and in the IHWG/CIBMTR group; they were performed with T-cell replete grafts. The next steps include repeating the study with patients who received T-cell depleted grafts. An analysis of 2,324 T-repleted and 534 T-depleted transplants is currently ongoing. Future plans are to include functional similarity or dissimilarity of substituted amino acids in the analysis. f. IB06-05 Use of high-resolution HLA data from the National Marrow Donor Program for the IHWG in hematopoietic cell transplantation Dr. Petersdorf presented the update for this study. The hypothesis is that the risks associated with HLA mismatched transplantation are influenced by 1) the number of mismatched residues within α1 and α2 of class I and DRB and DQB, 2) the location of the amino acid mismatch, and 3) the nature of the mismatched amino acids. The study population is restricted to single mismatches (a set of 2572 cases). The global p-values for the HLA loci are as follows: HLA-A p=0.27, HLA-B p=0.05, HLA-C p=0.01, HLA-DRB1 p=0.97, and HLA- DQB1 p=0.04. The study plans to look at the specific residues at HLA-B, -C, and -DQB1, but perhaps not at HLA-A or -DRB1, based on the global p-values. 14

15 Not for publication or presentation Attachment 1 g. IB07-04 Employing advanced bioinformatic methods for predicting peptide specificities of HLA molecules in the characterization of permissible mismatches in hematopoietic cell transfer No update was given. h. IB07-07 HLA-DR15 and transplant outcome No update was given. i. IB09-01s Clinical importance of MHC haplotypes in umbilical cord blood transplantation No update was given. j. IB09-03s Clinical relevance of cytokine/immune response gene polymorphisms in umbilical cord blood transplantation No update was given. k. IB09-05s Identification of functional SNPs in umbilical cord blood transplantation No update was given. l. IB09-07s Clinical significance of genome-wide variation in unrelated hematopoietic cell transplantation No update was given. 8. Deferred studies pending accrual a. GV04-01 Non-identical twin transplant for leukemia No update was given. b. IB06-13 HLA disparity in unrelated cord blood transplantation: Delineation of factors contributing to transplant outcomes No update was given. c. IB08-05s Evaluation of lymphotoxin alpha (LTA) alleles in relation to relapse in AML and CML No update was given. d. R04-80s HLA matching in unrelated cord blood transplants No update was given. 9. Feedback from Committee The Immunobiology working committee leadership asked for comments from the whole group as to what could be done differently for the meeting or for the committee in general. No comments were made. 10. Completed project summary (published or submitted work) a. R02-40s/R03-63s Cooley S, Trachtenberg E, Bergemann TL, Saeteurn K, Klein J, Le C, Marsh SGE, Guethlein LA, Parham P, Miller JS, Weisdorf DJ. Donors with group B KIR haplotypes improve relapse-free survival after unrelated hematopoietic cell transplantation for acute myelogenous leukemia. Blood, January 2009; b. R03-57s Shah R, Selby ST, Yokley B, Slack RS, Hurley CK, Posch PE. TNF, LTA and TGFB1 genotype distributions among acute graft versus host disease (agvhd) subsets after HLAmatched unrelated hematopoietic stem cell transplantation: a pilot study. Tissue Antigens, July 2009, 74(1): c. IB05-01s Spellman S, Warden MB, Haagenson M, Pietz BC, Goulmy E, Warren EH, Wang T, Ellis TM. Effects of mismatching for minor histocompatibility antigens on clinical outcomes in HLA-matched, unrelated hematopoietic stem cell transplants. Biol Blood Marrow Transplant, July 2009, 15(7):

16 Not for publication or presentation Attachment 1 d. R01-60 Baxter-Lowe L, Maiers M, Spellman S, Haagenson M, Wang T, Fernandez-Vina M, Marsh SGE, Horowitz M, Hurley CK. HLA-A disparities illustrate challenges for ranking the impact of HLA mismatches on bone marrow transplant outcomes in the United States. Biol Blood Marrow Transplant, August 2009, 15(8): e. IB06-08 Anderson E, Grzywacz B, Wang H, Wang T, Haagenson M, Spellman S, Blazar BR, Miller JS, and Verneris MR. Limited role of MHC class I chain related gene A (MICA) typing in assessing graft-versus-host disease risk after fully human leukocyte antigen matched unrelated donor transplantation. Blood, November 2009, 114: f. 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. In press. Blood, prepublished online January g. 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. In press. Blood, prepublished online January h. 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. Submitted. i. 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. HLA-matched unrelated donor hematopoietic transplantation in adults with acute leukemia: CIBMTR results in the era of molecular typing. Submitted. j. IB07-02 Marino SR, Lin S, Maiers M, Haagenson M, Spellman S, Lee SJ, Klein JP, Binkowski TA, and van Besien K. Mismatched unrelated donor stem cell transplantation: identification of HLA class I amino acid substitutions associated with survival at day 100. Submitted. k. 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. Submitted. l. 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. Submitted. 11. Closing Remarks Dr. Petersdorf thanked the committee members for extending their efforts in getting the studies submitted and published in the last year. She also thanked everyone for their participation. The meeting was adjourned at 4:15 pm. 16

17 Not for publication or presentation Attachment 1 Based on the voting results, the priority scores for this year s proposals is as follows: Proposal # and PI Average Priority Score (0-2)* Assigned Hours for July June 2011 PROP (Müller/Kornblit) PROP (Armistead) PROP (Arora) PROP (Baxter-Lowe) PROP (Blasczyk/Hurley) * The final score is the weighted average. 17

18 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 5767 (14) 1841 (29) 1626 (22) 4496 (19) y 7192 (17) 1186 (19) 1230 (16) 3320 (14) y 7585 (18) 961 (15) 1195 (16) 3124 (13) y 8153 (20) 878 (14) 1338 (18) 3448 (14) y 7185 (17) 776 (12) 1128 (15) 4218 (17) 50 y 5240 (13) 641 (10) 962 (13) 5676 (23) Male sex (58) 3777 (60) 4444 (59) (58) Karnofsky prior to transplant > 90% (73) 3892 (67) 5186 (72) (71) HLA-A,B,DRB1 groups low resolution 6/ (100) 1458 (23) 3771 (50) (71) 5/ (17) 754 (10) 3746 (15) 4/ (15) 367 ( 5) 990 ( 4) 3/6 0 N/A 31 (<1) 9 (<1) Other / < 3/6 / unknown / TBD (45) 2557 (34) 2302 ( 9) High resolution available 3/6 N/A N/A 20 (<1) 547 ( 2) 4/6 N/A N/A 59 ( 1) 1467 ( 6) 5/6 N/A N/A 174 ( 2) 3934 (16) 6/6 N/A N/A 359 ( 5) (47) HLA high-res. typed/audited (out of 8) N/A N/A 371 ( 5) (51) Graft type Bone marrow (74) 4635 (74) 4726 (63) (54) Peripheral blood (26) 1540 (25) 1710 (23) 7741 (32) Cord blood 183 (<1) 31 (<1) 1017 (14) 3338 (14) Other 193 (<1) 72 ( 1) 23 (<1) 202 ( 1) Conditioning regimen Myeloablative (82) 5159 (82) 5590 (75) (72) Reduced intensity 3545 ( 9) 603 (10) 1096 (15) 3954 (16) Non-myeloablative 1424 ( 3) 226 ( 4) 484 ( 6) 1987 ( 8) Other/To be determined 2428 ( 6) 299 ( 5) 310 ( 4) 779 ( 3) Donor age, median (range), years 30 (<1-93) 33 (<1-80) 33 (<1-68) 34 (<1-61) 18

19 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 (%) Donor age < 10 (including UCB tx) 4966 (12) 444 ( 7) 778 (11) 3446 (17) (18) 772 (13) 53 ( 1) 189 ( 1) (20) 1267 (21) 1503 (22) 4640 (23) (20) 1552 (25) 2065 (30) 6037 (30) (17) 1134 (18) 1457 (21) 4177 (21) (13) 965 (16) 1121 (16) 1725 ( 9) Disease at transplant AML (26) 1517 (24) 1762 (24) 7340 (30) ALL 6679 (16) 1220 (19) 1597 (21) 4461 (18) Other leukemia 1022 ( 3) 155 ( 3) 194 ( 3) 1091 ( 4) CML 7945 (19) 969 (15) 1698 (23) 3888 (16) MDS/MPS 2553 ( 6) 323 ( 5) 760 (10) 2809 (12) Non-Hodgkin s lymphoma 2581 ( 6) 343 ( 5) 206 ( 3) 1469 ( 6) HD-Hodgkin s lymphoma 269 ( 1) 48 ( 1) 10 (<1) 94 (<1) MYE-plasma cell disorder, MM 975 ( 2) 133 ( 2) 40 ( 1) 128 ( 1) Other malignancies 328 ( 1) 57 ( 1) 30 (<1) 69 (<1) Breast cancer 63 (<1) 22 (<1) 0 4 (<1) Severe aplastic anemia 4274 (10) 473 ( 8) 438 ( 6) 943 ( 4) Inherited ab erythro. diff-funct ( 6) 264 ( 4) 213 ( 3) 407 ( 2) SCID & other immune deficienc. 545 ( 1) 551 ( 9) 242 ( 3) 599 ( 2) Inherited abnormal. of platelets 19 (<1) 7 (<1) 11 (<1) 39 (<1) Inherited disorder of metabolism 264 ( 1) 150 ( 2) 179 ( 2) 592 ( 2) Histiocytic disorders 101 (<1) 41 ( 1) 85 ( 1) 297 ( 1) Autoimmune diseases 17 (<1) 4 (<1) 2 (<1) 8 (<1) Other 26 (<1) 5 (<1) 5 (<1) 33 (<1) Disease status at transplant Early (38) 1525 (24) 2496 (33) 6900 (28) Intermediate 5730 (14) 1135 (18) 1649 (22) 5373 (22) Advanced 5369 (13) 1184 (19) 1251 (17) 4277 (18) Non-malignant disease / Other (35) 2443 (39) 2084 (28) 7738 (32) GVHD prophylaxis FK506 + (MTX or MMF or steroids) 3059 ( 7) 394 ( 6) 544 ( 7) 9037 (37) ± other FK506 ± other 418 ( 1) 56 ( 1) 50 ( 1) 870 ( 4) CsA + MTX ± other (51) 2065 (33) 4415 (59) 7216 (30) CsA ± other 9739 (24) 893 (14) 1732 (23) 4754 (20) MMF ± other 44 (<1) 12 (<1) 20 (<1) 68 (<1) MTX ± other 2952 ( 7) 257 ( 4) 28 (<1) 172 ( 1) T-cell depletion 2858 ( 7) 1397 (22) 470 ( 6) 1194 ( 5) Other / To be determined 1192 ( 3) 1213 (19) 221 ( 3) 977 ( 4) 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 (%) Donor/Recipient sex match Male/Male (32) 2045 (35) 2353 (38) 2237 (36) Male/Female 8383 (22) 959 (17) 1351 (22) 1308 (21) Female/Male 9893 (26) 1426 (25) 1300 (21) 1394 (23) Female/Female 7503 (20) 1335 (23) 1150 (19) 1195 (19) Donor/Recipient CMV match Negative/Negative 9161 (22) 1392 (22) 1809 (24) 5976 (25) Negative/Positive 5866 (14) 832 (13) 1715 (23) 5762 (24) Positive/Negative 3673 ( 9) 763 (12) 970 (13) 2637 (11) Positive/Positive (37) 1968 (31) 1846 (25) 3730 (15) Unknown 7180 (17) 1332 (21) 1140 (15) 6183 (25) Year of transplant (12) 884 (14) 35 (<1) 11 (<1) ( 3) 257 ( 4) 14 (<1) 18 (<1) ( 4) 248 ( 4) 31 (<1) 34 (<1) ( 4) 244 ( 4) 53 ( 1) 95 (<1) ( 4) 258 ( 4) 100 ( 1) 178 ( 1) ( 5) 316 ( 5) 136 ( 2) 288 ( 1) ( 5) 250 ( 4) 171 ( 2) 410 ( 2) ( 5) 271 ( 4) 226 ( 3) 468 ( 2) ( 5) 273 ( 4) 235 ( 3) 576 ( 2) ( 4) 254 ( 4) 247 ( 3) 709 ( 3) ( 5) 316 ( 5) 324 ( 4) 852 ( 4) ( 5) 312 ( 5) 411 ( 5) 986 ( 4) ( 4) 295 ( 5) 381 ( 5) 1040 ( 4) ( 4) 216 ( 3) 423 ( 6) 1057 ( 4) ( 3) 199 ( 3) 416 ( 6) 1112 ( 5) ( 3) 205 ( 3) 435 ( 6) 1142 ( 5) ( 3) 218 ( 3) 445 ( 6) 1194 ( 5) ( 3) 184 ( 3) 444 ( 6) 1264 ( 5) ( 3) 159 ( 3) 454 ( 6) 1443 ( 6) ( 3) 139 ( 2) 581 ( 8) 1616 ( 7) ( 3) 169 ( 3) 546 ( 7) 1762 ( 7) ( 3) 139 ( 2) 465 ( 6) 1992 ( 8) ( 2) 79 ( 1) 275 ( 4) 1904 ( 8) ( 3) 238 ( 4) 309 ( 4) 1826 ( 8) ( 2) 145 ( 2) 255 ( 3) 1730 ( 7) ( 1) 19 (<1) 68 ( 1) 581 ( 2) Median follow-up of recipients, mos 90 (<1-465) 85 (1-443) 57 (<1-252) 59 (<1-275) 20

21 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 Donor Transplants Samples Available for Recipient and Donor Samples Available for Recipient Only Samples Available for Donor Only Variable N N N N (%) N (%) Eval Eval Eval N (%) Number of cases Number of centers Age, median (range), years (<1-78) (<1-79) (<1-75) Age at transplant < 10 y 1608 (12) 605 (17) 451 (17) y 1708 (13) 516 (14) 379 (14) y 1912 (14) 499 (14) 380 (14) y 2140 (16) 543 (15) 406 (15) y 2629 (20) 662 (18) 480 (18) 50 y 3472 (26) 788 (22) 561 (21) Male sex (58) (58) (60) Karnofsky prior to transplant > (70) (70) (71) 90% HLA-A, B, DRB1 groups highres /6-2/6 33 (<1) 20 ( 1) 0 3/6 156 ( 1) 99 ( 6) 1 (<1) 4/6 650 ( 5) 231 (14) 16 ( 2) 5/ (23) 396 (24) 210 (20) 6/ (71) 915 (55) 808 (78) HLA high-res. typed and audited (99) (98) (96) Disease status at transplant Early 4133 (31) 1130 (31) 753 (28) Intermediate 2949 (22) 883 (24) 617 (23) Advanced 2553 (19) 652 (18) 536 (20) Non-malignant disease / Other 3834 (28) 948 (26) 753 (28) Graft type Bone marrow 8151 (61) 2118 (59) 1770 (67) Peripheral blood 5210 (39) 848 (23) 873 (33) Cord blood (17) 0 Other 108 ( 1) 23 (<1) 16 ( 1) Conditioning regimen Myeloablative 9881 (73) 2763 (76) 1983 (75) Reduced intensity 2327 (17) 545 (15) 444 (17) Non-myeloablative 1065 ( 8) 265 ( 7) 183 ( 7) Other/To be determined 196 ( 1) 40 ( 1) 49 ( 2) Donor age, median (range), years (18-61) (18-61) (18-59) Donor age < (11) 638 (20) 284 (11) (25) 787 (25) 613 (25) (33) 944 (30) 811 (33) (24) 607 (19) 598 (24) ( 7) 159 ( 5) 185 ( 7) 21

22 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 Donor Transplants Samples Available for Recipient and Donor Samples Available for Recipient Only Samples Available for Donor Only Variable N N N N (%) N (%) Eval Eval Eval N (%) Disease at transplant AML 4153 (31) 1163 (32) 793 (30) ALL 2405 (18) 680 (19) 536 (20) Other leukemia 569 ( 4) 133 ( 4) 107 ( 4) CML 2459 (18) 666 (18) 475 (18) MDS/MPS 1668 (12) 401 (11) 298 (11) Non-Hodgkin s lymphoma 920 ( 7) 200 ( 6) 142 ( 5) HD-Hodgkin s lymphoma 59 (<1) 14 (<1) 9 (<1) MYE-plasma cell disorder, MM 84 ( 1) 23 ( 1) 13 (<1) Other malignancies 34 (<1) 10 (<1) 7 (<1) Breast cancer 4 (<1) 0 0 Severe aplastic anemia 465 ( 3) 111 ( 3) 109 ( 4) Inherited ab erythro. diff-funct. 162 ( 1) 52 ( 1) 32 ( 1) SCID & other immune deficienc. 194 ( 1) 59 ( 2) 58 ( 2) Inherited abnormal. of platelets 12 (<1) 4 (<1) 3 (<1) Inherited disorder of metabolism 169 ( 1) 61 ( 2) 41 ( 2) Histiocytic disorders 95 ( 1) 33 ( 1) 28 ( 1) Other 12 (<1) 3 (<1) 6 (<1) GVHD prophylaxis FK506 ± MMF ± MTX ± steroids ± other 5329 (40) 1206 (33) 868 (33) FK506 ± other 474 ( 4) 121 ( 3) 75 ( 3) CsA + MTX ± other 4649 (35) 1228 (34) 1035 (39) CsA ± other 1727 (13) 731 (20) 428 (16) MMF ± other 36 (<1) 12 (<1) 5 (<1) MTX ± other 110 ( 1) 20 ( 1) 13 (<1) T-cell depletion 607 ( 5) 170 ( 5) 148 ( 6) Other / To be determined 537 ( 4) 125 ( 3) 87 ( 3) Donor/Recipient sex match Male/Male 1110 (38) 321 (40) 220 (35) Male/Female 649 (22) 168 (21) 137 (22) Female/Male 598 (20) 181 (23) 134 (21) Female/Female 569 (19) 129 (16) 146 (23) Donor/Recipient CMV match Negative/Negative 3832 (28) 882 (24) 771 (29) Negative/Positive 3779 (28) 851 (24) 810 (30) Positive/Negative 1732 (13) 422 (12) 345 (13) Positive/Positive 2413 (18) 582 (16) 513 (19) Unknown 1713 (13) 876 (24) 220 ( 8) 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 Donor Transplants Samples Available for Recipient and Donor Samples Available for Recipient Only Samples Available for Donor Only Variable N N N N (%) N (%) Eval Eval Eval N (%) Year of transplant (<1) (<1) 9 (<1) 8 (<1) ( 1) 6 (<1) 9 (<1) ( 1) 20 ( 1) 30 ( 1) ( 2) 50 ( 1) 60 ( 2) ( 2) 38 ( 1) 90 ( 3) ( 2) 68 ( 2) 125 ( 5) ( 3) 109 ( 3) 111 ( 4) ( 3) 185 ( 5) 115 ( 4) ( 4) 227 ( 6) 125 ( 5) ( 4) 222 ( 6) 138 ( 5) ( 4) 259 ( 7) 137 ( 5) ( 5) 217 ( 6) 130 ( 5) ( 6) 157 ( 4) 109 ( 4) ( 6) 159 ( 4) 106 ( 4) ( 4) 127 ( 4) 289 (11) ( 6) 188 ( 5) 273 (10) ( 7) 288 ( 8) 184 ( 7) ( 9) 282 ( 8) 162 ( 6) (10) 392 (11) 158 ( 6) ( 9) 196 ( 5) 143 ( 5) ( 5) 113 ( 3) 73 ( 3) ( 4) 231 ( 6) 69 ( 3) ( 1) 70 ( 2) 13 (<1) Median follow-up of recipients, mo (<1-257) (1-231) (3-248) 23

24 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 2 or 3 UCB Units Only Samples Available - Single UCB Unit Only Variable N (%) N (%) N (%) N (%) Number of cases Number of centers Number of cord blood units in transplant One 748 (68) 449 (48) (100) Two 357 (32) 359 (38) 74 (96) 0 Three 2 (<1) 2 (<1) 3 ( 4) 0 Unknown (14) 0 0 Age, median (range), years 15 (<1-80) 19 (<1-73) 40 (1-73) 6 (<1-68) Age at transplant, by decade < 10 y 438 (40) 323 (34) 10 (13) 107 (63) y 204 (18) 155 (17) 4 ( 5) 25 (15) y 89 ( 8) 117 (12) 7 ( 9) 10 ( 6) y 79 ( 7) 77 ( 8) 16 (21) 3 ( 2) y 95 ( 9) 88 ( 9) 14 (18) 8 ( 5) 50 y 202 (18) 178 (19) 26 (34) 16 ( 9) Male sex 597 (54) 530 (57) 50 (65) 98 (58) Karnofsky prior to transplant > 90% 733 (73) 614 (73) 56 (79) 112 (77) HLA-A, B, DRB1 groups high-res 0/6-2/6 27 ( 3) 28 ( 4) 6 ( 9) 2 ( 2) 3/6 131 (14) 131 (20) 11 (18) 17 (14) 4/6 394 (43) 281 (43) 34 (55) 42 (35) 5/6 253 (28) 174 (26) 11 (18) 41 (34) 6/6 112 (12) 47 ( 7) 0 17 (14) HLA high resolution typed and audited 204 (18) 125 (13) 0 1 ( 1) Disease status at transplant Early 260 (23) 238 (25) 16 (21) 28 (17) Intermediate 268 (24) 231 (25) 19 (25) 35 (21) Advanced 112 (10) 115 (12) 12 (16) 12 ( 7) Non-malignant disease / Other 467 (42) 354 (38) 30 (39) 94 (56) Conditioning regimen Myeloablative 693 (63) 582 (62) 31 (40) 108 (64) Reduced intensity 158 (14) 145 (15) 19 (25) 25 (15) Non-myeloablative 179 (16) 155 (17) 23 (30) 22 (13) Other/To be determined 77 ( 7) 56 ( 6) 4 ( 5) 14 ( 8) Donor/recipient CMV match Negative/Negative 438 (40) 361 (38) 21 (27) 63 (37) Negative/Positive 572 (52) 517 (55) 48 (62) 87 (51) Positive/Negative Positive/Positive Unknown 97 ( 9) 60 ( 6) 8 (10) 19 (11) 24

25 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 2 or 3 UCB Units Only Samples Available - Single UCB Unit Only Variable N (%) N (%) N (%) N (%) Disease at transplant AML 379 (34) 346 (37) 36 (47) 29 (17) ALL 257 (23) 224 (24) 15 (19) 40 (24) Other leukemia 34 ( 3) 36 ( 4) 0 3 ( 2) CML 22 ( 2) 27 ( 3) 4 ( 5) 3 ( 2) MDS/MPS 97 ( 9) 77 ( 8) 8 (10) 16 ( 9) Non-Hodgkin s lymphoma 25 ( 2) 13 ( 1) 0 5 ( 3) HD-Hodgkin s lymphoma 46 ( 4) 38 ( 4) 6 ( 8) 5 ( 3) MYE-plasma cell disorder, MM 4 (<1) 4 (<1) 0 1 ( 1) Other malignancies 3 (<1) 3 (<1) 0 0 Breast cancer 31 ( 3) 29 ( 3) 4 ( 5) 3 ( 2) Severe aplastic anemia 24 ( 2) 25 ( 3) 0 9 ( 5) Inherited ab erythro. diff-funct. 65 ( 6) 41 ( 4) 0 23 (14) SCID & other immune deficienc. 2 (<1) 3 (<1) 0 1 ( 1) Inherited abnormal. of platelets 75 ( 7) 42 ( 4) 3 ( 4) 21 (12) Inherited disorder of metabolism 36 ( 3) 23 ( 2) 0 7 ( 4) Histiocytic disorders 2 (<1) 5 ( 1) 0 0 Other 4 (<1) 2 (<1) 1 ( 1) 3 ( 2) GVHD prophylaxis FK506 ± MMF ± MTX ± steroids ± 349 (32) 335 (36) 25 (32) 39 (23) other FK506 ± other 33 ( 3) 40 ( 4) 8 (10) 12 ( 7) CsA + MTX ± other 33 ( 3) 44 ( 5) 0 13 ( 8) CsA ± other 625 (56) 479 (51) 36 (47) 90 (53) MMF ± other 6 ( 1) ( 1) MTX ± other 3 (<1) 2 (<1) 0 0 Other / To be determined 58 ( 5) 38 ( 4) 8 (10) 13 ( 8) Year of transplant (<1) 1 (<1) 0 4 ( 2) ( 1) 15 ( 2) 0 6 ( 4) (<1) 11 ( 1) ( 1) 35 ( 4) 0 1 ( 1) ( 1) 32 ( 3) 0 7 ( 4) ( 3) 73 ( 8) 4 ( 5) 9 ( 5) ( 4) 197 (21) 2 ( 3) 10 ( 6) (21) 78 ( 8) 12 (16) 34 (20) (27) 138 (15) 29 (38) 46 (27) (28) 257 (27) 18 (23) 35 (21) (13) 101 (11) 12 (16) 17 (10) Median follow-up of recipients, mo 21 (1-96) 24 (1-96) 12 (1-36) 21 (1-121) 25

26 Not for publication or presentation Attachment 3 Study Proposal Study Title: Impact of amino acid substitution at peptide binding pockets of HLA class I molecules on hematopoietic cell transplantation (HCT) outcome 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 Introduction: 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 amino acid substitution 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. Hypothesis: Amino acid residue substitution at peptide binding pockets of the HLA class I molecule adversely affects transplantation outcome. Specific Aim: Examine the impact of amino acid substitution at HLA class I peptide binding pockets on grade III-IV agvhd, non-relapse mortality and overall survival following unrelated donor allogeneic hematopoietic cell transplantation. Scientific Justification: 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 26

27 Not for publication or presentation Attachment 3 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 amino acid substitution 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 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 amino acid substitution at these key positions on HCT outcome utilizing NMDP/CIBMTR data. Patient Eligibility Population: 1. First allogeneic hematopoietic cell transplant recipient and unrelated donor pairs 2. HCT date from 1988 to 2003 (this time frame is selected based on date range in existing NMDP retrospective high-resolution typing project data set; if possible to extend further, would consider a range of 1988 to 2009, for a one year minimum follow up) 3. Complete high resolution HLA typing performed 4. Diagnoses: a. Acute myelogenous leukemia b. Acute lymphoblastic leukemia c. Chronic myelogenous leukemia d. Myelodysplastic syndrome 5. Age: no age range limitation; will consider all ages available in data set, and address the impact of age in multivariable modeling. 6. Conditioning regimen: no restrictions; will consider both myeloablative and reduced-intensity regimens in analysis. 7. Graft source: will include both bone marrow and peripheral blood mobilized stem cells Data Requirements: Data collection forms required: 1. Pre-transplant essential data (2400) 2. Confirmation of HLA typing (2500) 3. Post-transplant essential data (2450) Variables to gather from existing CIBMTR data collection forms: Outcomes of interest: 1. Overall survival (defined as time from date of transplant until death or last follow up) 2. Cumulative incidence of non-relapse mortality 3. Cumulative incidence of grade III-IV agvhd Variables of interest: 1. Year of HCT 2. Donor and recipient age 3. Donor and recipient sex 4. Recipient race 5. Donor and recipient CMV serostatus 6. Karnofsky performance status prior to HCT 7. Disease, and remission status 27

28 Not for publication or presentation Attachment 3 8. CIBMTR disease risk category 9. Graft type (peripheral blood, bone marrow) 10. Conditioning regimen (myeloablative, reduced intensity) 11. AGVHD prophylaxis 12. Additional agents received (ATG, campath, rituximab) Sample Requirements (if study will use biologic samples from the NMDP Research Sample Repository): No NMDP samples are requested in this protocol, only high-resolution HLA typing project data. Study Design (Scientific Plan): We propose examining the independent impact of amino acid substitution at these key (9, 77, 99, 116, and 156) residues on the outcomes of grade III-IV agvhd, non-relapse mortality and overall survival among unrelated donor-recipient pairs in the existing NMDP database. To estimate the feasibility of this study, we have determined the frequency of amino acid substitution at these residues from existing data (Petersdorf, EW, et al), 21 and estimated projected numbers (see table on following page) for the NMDP data (Lee, SJ, et al). 3 In the planned analysis, multivariable modeling will examine the association between specific amino acid substitution at these residues and the above outcomes. Analysis will be performed for each residue separately, with comparison for each made to donor-recipient pairs matched at these residues. Another potential approach would be to perform this analysis according to the number of these residues mismatched. In all analyses, patient, disease, and transplant variables will be considered as covariates in multivariable models. 28

29 Not for publication or presentation Attachment 3 Estimated frequency of amino acid substitution at residues of interest in peptide binding region of HLA molecule Petersdorf (observed) Lee (expected) Only other substitutions Total Zero mismatch One Two Three Four 3 23 Five * 0 defined as no amino acid substitution at any residues within the peptide binding region; only other substitutions defined as amino acid substitution at residues other than those of interest (i.e. 9, 77, 99, 116, 156) within the peptide binding region. * Substitution at additional amino acid residues present for each of the categories of residues of interest (i.e. 9, 77, 99, 116, 156) 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:

30 Not for publication or presentation Attachment 3 5. 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: Fleischhauer K, Locatelli F, Zecca M, et al. Graft rejection after unrelated donor hematopoietic stem cell transplantation for thalassemia is associated with nonpermissive HLA-DPB1 disparity in host-versus-graft direction. Blood. 2006;107: Ludajic K, Balavarca Y, Bickeboller H, et al. Impact of HLA-DPB1 allelic and single amino acid mismatches on HSCT. Br J Haematol. 2008;142: Zino E, Frumento G, Marktel S, et al. A T-cell epitope encoded by a subset of HLA-DPB1 alleles determines nonpermissive mismatches for hematologic stem cell transplantation. Blood. 2004;103: Kawase T, Morishima Y, Matsuo K, et al. High-risk HLA allele mismatch combinations responsible for severe acute graft-versus-host disease and implication for its molecular mechanism. Blood. 2007;110: Ferrara GB, Bacigalupo A, Lamparelli T, et al. Bone marrow transplantation from unrelated donors: the impact of mismatches with substitutions at position 116 of the human leukocyte antigen class I heavy chain. Blood. 2001;98: Hogan KT, Clayberger C, Bernhard EJ, et al. Identification by site-directed mutagenesis of amino acid residues contributing to serologic and CTL-defined epitope differences between HLA-A2.1 and HLA-A2.3. J Immunol. 1988;141: Macdonald WA, Purcell AW, Mifsud NA, et al. A naturally selected dimorphism within the HLA-B44 supertype alters class I structure, peptide repertoire, and T cell recognition. J Exp Med. 2003;198: Madden DR. The three-dimensional structure of peptide-mhc complexes. Annu Rev Immunol. 1995;13: Marsh S, Parham, P, Barber, L. The HLA Facts Book: Academic Press; Mattson DH, Shimojo N, Cowan EP, et al. Differential effects of amino acid substitutions in the beta-sheet floor and alpha-2 helix of HLA-A2 on recognition by alloreactive viral peptidespecific cytotoxic T lymphocytes. J Immunol. 1989;143: Shimojo N, Cowan EP, Engelhard VH, Maloy WL, Coligan JE, Biddison WE. A single amino acid substitution in HLA-A2 can alter the selection of the cytotoxic T lymphocyte repertoire that responds to influenza virus matrix peptide J Immunol. 1989;143: Tynan FE, Elhassen D, Purcell AW, et al. The immunogenicity of a viral cytotoxic T cell epitope is controlled by its MHC-bound conformation. J Exp Med. 2005;202: Petersdorf EW, Hansen JA, Martin PJ, et al. Major-histocompatibility-complex class I alleles and antigens in hematopoietic-cell transplantation. N Engl J Med. 2001;345:

31 Not for publication or presentation Attachment 3 Characteristics of recipients receiving first transplants for AML, ALL, CML or MDS that are highresolution typed for HLA-A, -B, -C and DRB1 and are 8/8 allele-matched or 7/8 allelematched with single mismatch at Class I locus a Characteristics of patients N Eval N (%) Number of patients 7573 Number of centers 176 Recipient age, median (range), years (<1-74) Age at transplant y 607 ( 8) y 856 (11) y 1106 (15) y 1258 (17) y 1578 (21) Over (29) Male sex (56) Karnofsky prior to transplant > (70) Disease at transplant 7573 AML 2989 (39) ALL 1624 (21) CML 1649 (22) MDS 1311 (17) Disease status at transplant 7573 Early 2956 (39) Intermediate 1994 (26) Advanced 1825 (24) Other 798 (11) Graft type 7573 Bone marrow 4370 (58) PBSC 3203 (42) Conditioning regimen 7573 Traditional myeloablative 5054 (67) Reduced intensity 1139 (15) Non-myeloablative 397 ( 5) Non-traditional myeloablative 849 (11) To be determined/other 134 ( 2) GVHD prophylaxis 7573 FK506 + (MTX or MMF or steroids) ± other 3249 (43) FK506 ± other 264 ( 3) CsA + MTX ± other 2690 (36) CsA ± other (no MTX) 780 (10) MMF ± other (no CsA) 18 (<1) MTX ± other (no CsA) 44 ( 1) T-cell depletion 279 ( 4) Other 249 ( 3) 31

32 Not for publication or presentation Attachment 3 Continued. Characteristics of patients N Eval N (%) HLA Matching for HLA-A, -B, -C and -DRB /8 allele matched 5435 (72) Single MM at HLA-A 727 (10) Single MM at HLA-B 339 ( 4) Single MM at HLA-C 1072 (14) Donor/Recipient sex match 6912 Male/Male 2647 (38) Male/Female 1706 (25) Female/Male 1246 (18) Female/Female 1313 (19) Donor/Recipient CMV match 7573 Negative/Negative 2218 (29) Negative/Positive 2209 (29) Positive/Negative 979 (13) Positive/Positive 1355 (18) Unknown 812 (11) Donor age, median (range), years ( ) Donor age ( 1) (26) (34) (24) 50 and older 473 ( 6) Unknown 700 ( 9) Year of transplant (<1) ( 1) ( 1) ( 1) ( 2) ( 2) ( 3) ( 3) ( 3) ( 4) ( 4) ( 5) ( 6) ( 6) ( 5) ( 6) ( 8) (10) (11) (11) ( 6) ( 3) Median follow-up of recipients, mo (range) (2-252) a - Data has not been CAP-modeled. 32

33 Not for publication or presentation Attachment 4 Study Proposal Study Title: Analysis of the NIMA effect on the outcome of unrelated PBSC/BM transplantation Gerhard Ehninger, University Hospital Carl Gustav Carus, Dresden, Germany Jon J. van Rood, Leiden University Medical Center, Leiden, The Netherlands Alexander H. Schmidt, DKMS German Bone Marrow Donor Center, Tuebingen, Germany Specific Aims: Analyze the NIMA effect for unrelated adult donor PBSC/BM transplantation for hematological malignancies, including myelodysplasia. Primary study end point: 1. Transplant-related mortality (TRM) Secondary study end points: 1. Neutrophil engraftment (ANC 500/mm 3 for 3 consecutive lab values on different days) 2. Platelet engraftment ( 20 x 10 9 /l, 50 x 10 9 /l) 3. Incidence of grade II-IV and III-IV agvhd 4. Incidence of cgvhd 5. Relapse 6. Overall mortality 7. Treatment failure (relapse or death) 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. Patient eligibility population: 1. 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. 2. HLA typing result of patients and donors must be available at high resolution at least for the HLA loci A, B, C, and DRB1. 3. Patient s donor must be from DKMS German Bone Marrow Donor Center. 4. 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.) Data requirements: Primary outcome: - Transplant-related mortality: Death without evidence of disease recurrence. Event will be summarized by the cumulative incidence estimate with relapse as competing risk. 33

34 Not for publication or presentation Attachment 4 Secondary outcomes: - 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. 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 Sample Requirements: No repository samples are needed. Study Design: - To summarize the characteristics of the data set, descriptive tables of patient-, disease-, and donor-/transplant-related variables will be reported. - 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. 34

35 Not for publication or presentation Attachment 4 - 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 highresolution 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 α=0.05; study power 1-β= 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. 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:

36 Not for publication or presentation Attachment 4 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 * Characteristics of patients 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) 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) 36

37 Not for publication or presentation Attachment 4 Continued. Characteristics of patients N Eval N (%) 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) 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) * Data has not been CAP-modeled. 37

38 Not for publication or presentation Attachment 5 Study Proposal Study Title: Impact of CTLA4 single nucleotide polymorphisms on outcome after unrelated donor transplant Madan Jagasia, MBBS, MS, Vanderbilt-Ingram Cancer Center, Department of Medicine, Nashville, TN USA William Clark, MD, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA USA Bipin N Savani, MD, Vanderbilt-Ingram Cancer Center, Department of Medicine, Nashville, TN USA Specific Aims: 1. To study the impact of CTLA4 SNPS on cumulative incidence of relapse after unrelated donor transplant for hematological malignancies 2. To study the impact of CTLA4 SNPS on cumulative incidence of acute and chronic GVHD after unrelated donor transplant for hematological malignancies 3. To study the interaction of CTLA4 SNPs and dose of thymoglobulin/atg in cumulative incidence of relapse and GVHD (both acute and chronic) after unrelated donor transplant for hematological malignancies Scientific Justification: Allogeneic hematopoietic cell transplantation (allo-hct) is a potentially curative treatment modality for a variety of hematological malignancies. Graft-versus-host disease (GVHD) and relapse of the underlying malignancies remain an important problem. Although a variety of clinical factors, both pre and posttransplant have been identified to predict these events, true biologic prediction and individualization of outcomes has eluded us. Cytotoxic T-Lymphocyte Antigen 4 (CTLA4): The fundamental event in the cascade of both GVHD and graft-versus-leukemia (GVL) effect remain the initial interaction between the antigen presenting cell (APC) and donor T cell. In addition to the cognate T-cell receptor (TCR)-Human Leukocyte Antigen (HLA) recognition, engagement of the co-receptors on APC and T cells is required for the occurrence of a productive immune response. These-co-stimulatory signals are mediated via CD80/CD86 molecules on APCs and CD 28 or CTLA4 receptors on T cells, leading to positive or negative regulation of T-cell activation. CTLA4 (or CD152) is a T-cell surface molecule that negatively regulates T-cell activation. It also plays a crucial role in maintenance of T cell homeostasis, balance of T0helper cell subsets, T cell differentiation and is establishment of central and peripheral T cell tolerance. Multiple investigators have studied various SNPs of CTLA4 gene and the association with clinical outcomes. Results are discordant due to inhomogeneous datasets, and differing ethnic populations. Most studies have shown an association with GVHD outcomes. 1-4 Pilot Data from Vanderbilt University Medical Center (ASBMT 2011): Adult patients undergoing allo-hct (cord transplant excluded) at a single center ( ), with a minimum survival of 120 days, and with the availability of pre-transplant recipient and donor germline DNA samples were included (n=164). Ten tagsnps of the CTLA-4 gene (rs231775, rs231779, rs , rs231777, rs , rs , rs231725, rs , rs , rs ) were identified using previously published criteria. All SNPs analyzed passed quality control [test of Hardy- Weinberg equilibrium (HWE) P > 0.001, MAF > 0.10, SNP call rate > 0.95, and pair-wise r2 value less than 0.8]. In univariate analyses, the donor SNP rs A/A genotype was associated with improved RFS (P=0.019) and OS (P=0.033) compared to the A/G or G/G genotype. Multivariable analysis, adjusted for 38

39 Not for publication or presentation Attachment 5 stem cell source (peripheral blood vs. bone marrow), GVHD subtype (classic/overlap vs. acute subtypes/none), and disease risk (low vs. intermediate vs. high) were performed to test the association of the significant SNPs with RFS and OS. rs A/A genotype (HR 2.44, P=0.015), classic/overlap GVHD (HR 2.98, P=0.002) and low/intermediate disease risk (HR 2.16, P=0.03) were independent predictors of superior OS. rs A/A genotype (HR 2.39, P=0.016), and classic/overlap GVHD (HR 3.84, P=0.001) were independent predictors of superior RFS. Our study confirms reports that genetic variation in donor CTLA-4 is associated with outcome after allo- HCT and may allow for identification of patient subsets that may benefit from pre-emptive modulation of immunosuppressive therapy. Our study contrasts with the other published studies, as the number of SNPs anlayzed is more extensive. rs is in the promoter region of CTLA4 and has been associated with response to ipilumamab (CTLA4-antibody) in melanoma. 5 Thymoglobulin/ATG and Unrelated Donor Transplant: The role of thymoglobulin/atg in unrelated transplant continues to evolve. The dosing of thymoglobulin/atg remains controversial with data suggesting a range of effective doses The dosing of thymoglobulin/atg is weight based, while the target of thymoglobulin/atg i.e. donor T cells and host APCs are not a function of weight. Our hypothesis is that patients with CTLA4 SNPs that render the donor T-cells hyporesponsive will be at a higher risk of ATG/Thymoglobulin related negative events (i.e. relapse) and will have a significantly decreased chance of acute and chronic GVHD. In order to do such an analysis, a large dataset of patients with a uniform diagnosis and preparative regimen are needed. Although it would be ideal to study only one type of regimen intensity, most studies have shown similar outcome data irrespective of regimen intensity, suggesting that efficacy of GVL in effecting a cure is independent of regimen intensity. The efficacy of GVT is a dependent on tumor type, thus we would propose to limit the analysis to only AML, as relapse after allo-sct continues to remain a major challenge in this diagnostic category. CIBMTR is well suited and poised to conduct this study. DNA samples will be needed form the NMDP biorepository. The SNP analysis can be conducted at Vanderbilt University Medical Center. If our hypothesis is correct, it will allow us to individualize the use of thymoglobulin in recipients of unrelated allo-hct. Patient Eligibility Population: Adult ( 18 years) recipients of first unrelated allo-hct (excluding cord transplants) AML in CR1 or CR2 HLA 8/8, 7/8. Haplo-identical donors excluded Transplanted between Any preparative regimen (myeloablative, reduced intensity, non-myeloablative) Any GVHD prophylaxis (except in vitro T cell depletion, or use of in vivo/in vitro alemtuzumab) Availability of adequate donor DNA samples for SNP analyses Outcomes to be Studied: 1. Acute GVHD: Occurrence of grade II-IV GVHD will be analyzed based on Glucksberg s staging of skin, gastrointestinal and liver disease. 2. Chronic GVHD: Occurrence of symptoms in any organ system fulfilling the criteria of chronic GVHD and classified as limited or extensive. 3. Relapse: time to onset of recurrence either in BM or extramedullary. This event will be summarized by cumulative incidence estimate with TRM as the competing risk. 39

40 Not for publication or presentation Attachment 5 4. Transplant-related mortality: time to death without evidence of disease relapse. This event will be summarized as cumulative incidence estimate with relapse as the competing risk. 5. Leukemia-free survival: will be defined as time to relapse or death from any cause. Patients are censored at last follow-up. 6. Overall survival: Time to death, patients censored at last follow-up. Variables: Patient-related (at the time of transplant): Age at transplant: (continuous and <20 vs vs. >40) Gender: female vs. male Karnofsky performance status: <90% vs. 90% Disease-related: CIBMTR risk status at transplant Transplant-related: Conditioning regimen (TBI vs. non-tbi) Myeloablative versus other (reduced intensity plus non-myeloablative) Donor age vs vs vs. > 40y Donor-recipient gender: M-M vs. M-F vs. F-M vs. F-F HLA-matching and degree of match (8/8; 7/8; 6/8) CMV status of donor and recipient: +/+ vs. +/- vs. -/- Source of stem cells: BM vs. PBSC CD34 cell dose, x 10 6 /kg: BM, above median vs. below median; PBSC, above median vs. below median CD3 cell dose, x 10 6 /kg: BM, above median vs. below median; PBSC above median vs. below median Year of transplant GVHD prophylaxis: CSA+/-other (not MTX) vs. CSA+MTX+/-other vs. FK506+/-other vs. T- cell depletion T-cell depletion: Thymoglobulin or ATG, dose of thymoglobulin/atg, number of days of administration, time before stem cell infusion. Cohorts will be divided as thymoglobulin <5 mg/kg (ATG equivalent < 40 mg/kg); mg/kg (40-60 mg/kg); >7.5 mg/kg (>60 mg/kg) Post-transplant: Acute GVHD: none vs. II-IV, I-II vs. III-IV GVHD beyond day 100: late acute, chronic GVHD (limited or extensive) Relapse: yes/no Chimerism: (for reduced intensity and non-ablative only) CD3 and CD33 at day 30, day 100 CTLA4 SNPs: Donor SNPs Study Design: Clinical: Descriptive tables of patient-, disease-, and transplant-related factors will be prepared. The tables will list median and range for continuous variables and percent of total for categorical variables. Characteristics of patients will be compared using the chi-square test for categorical variables and the Wilcoxon two-sample test for continuous variables. Comparing the outcomes in the different transplant groups will require adjustment for two sources of bias: differences in time to transplant and differences in the baseline patient characteristics. The date of achieving CR will be the starting point for both the groups. To adjust for differences in the baseline characteristics, Cox proportional hazards regression will be used. Probability of LFS and OS will be calculated using Kaplan-Meier estimator. Comparison of survival curves will be done using the log-rank test. 40

41 Not for publication or presentation Attachment 5 Genetic: Ten tagsnps of the CTLA-4 gene (rs231775, rs231779, rs , rs231777, rs , rs , rs231725, rs , rs , rs ) will be analyzed. Univariate logistic regression will performed to test for association between cumulative incidence of relapse (with TRM as competing risk), cumulative incidence of acute GVHD, and chronic GVHD and each SNP using an unadjusted, additive genetic model. P-values will be adjusted using false discovery rate (FDR). Multivariate analyses incorporating significant univariate clinical factors and significant SNPs (identified from univariate analyses) will be built. Interaction of CTLA4 SNPs and thymoglobulin use and dose will be specifically analyzed. Sample Size: The population frequencies of the CTLA4 SNPs are known. In our pilot study, rs was an independent predictor of relapse adjusted for other important pre-transplant predictors. Sample size estimate is based on the assumption of finding this SNP as a significant predictor in the proposed study. Based on data from CIBMTR, there are 1172 cases of AML that meet our eligibility criteria. The minor allele of rs is G and has a population frequency of 12%-26.3%. Assuming a conservative estimate of 12%, the population frequency of the 3 genotypes GG, AG and AA are 1.44%, 21.1% and 77.4%. Thus the ratio of AA to AG+ GG is 3.43 (77.5%/22.5%). We assumed ratio of AA to AG+ GG as 3:1 for purpose of sample size calculation. Assuming an effect size of effect size of 1.5, accrual period of 7 years, follow up of 3 years, alpha of </= 0.01, power of 0.99, we will need 288 patients. As the regimen intensity is a known variable in impacting relapse, we would like to use a random subset of 288 patients from each regimen intensity (i.e., ablative, reduced intensity, and non-ablative). This strategy will minimize the impact of regimen intensity being a potential confounder. References: 1. Perez-Garcia A, De la Camara R, Roman-Gomez J et al. CTLA-4 polymorphisms and clinical outcome after allogeneic stem cell transplantation from HLA-identical sibling donors. Blood 2007;110: Vannucchi AM, Guidi S, Guglielmelli P et al. Significance of CTLA-4 and CD14 genetic polymorphisms in clinical outcome after allogeneic stem cell transplantation. Bone Marrow Transplant. 2007;40: Azarian M, Busson M, Lepage V et al. Donor CTLA A/G*GG genotype is associated with chronic GVHD after HLA-identical haematopoietic stem-cell transplantations. Blood 2007;110: Sellami MH, Bani M, Torjemane L et al. Effect of donor CTLA-4 alleles and haplotypes on graftversus-host disease occurrence in Tunisian patients receiving a human leukocyte antigen-identical sibling hematopoietic stem cell transplant. Hum.Immunol Breunis WB, Tarazona-Santos E, Chen R et al. Influence of cytotoxic T lymphocyte-associated antigen 4 (CTLA4) common polymorphisms on outcome in treatment of melanoma patients with CTLA-4 blockade. J.Immunother. 2008;31: Bacigalupo A, Lamparelli T, Bruzzi P et al. Antithymocyte globulin for graft-versus-host disease prophylaxis in transplants from unrelated donors: 2 randomized studies from Gruppo Italiano Trapianti Midollo Osseo (GITMO). Blood 2001;98: Bacigalupo A, Lamparelli T, Barisione G et al. Thymoglobulin prevents chronic graft-versus-host disease, chronic lung dysfunction, and late transplant-related mortality: long-term follow-up of a randomized trial in patients undergoing unrelated donor transplantation. Biol.Blood Marrow Transplant. 2006;12:

42 Not for publication or presentation Attachment 5 8. Finke J, Schmoor C, Lang H, Potthoff K, Bertz H. Matched and mismatched allogeneic stem-cell transplantation from unrelated donors using combined graft-versus-host disease prophylaxis including rabbit anti-t lymphocyte globulin. J.Clin.Oncol. 2003;21: Finke J, Bethge WA, Schmoor C et al. Standard graft-versus-host disease prophylaxis with or without anti-t-cell globulin in haematopoietic cell transplantation from matched unrelated donors: a randomised, open-label, multicentre phase 3 trial. Lancet Oncol. 2009;10: Schleuning M, Gunther W, Tischer J, Ledderose G, Kolb HJ. Dose-dependent effects of in vivo antithymocyte globulin during conditioning for allogeneic bone marrow transplantation from unrelated donors in patients with chronic phase CML. Bone Marrow Transplant. 2003;32: Zander AR, Kroger N, Schleuning M et al. ATG as part of the conditioning regimen reduces transplant-related mortality (TRM) and improves overall survival after unrelated stem cell transplantation in patients with chronic myelogenous leukemia (CML). Bone Marrow Transplant. 2003;32:

43 Not for publication or presentation Attachment 5 Characteristics of adult recipients with AML in CR1 or CR2 receiving first T-replete transplants that are high-resolution typed and 7/8 or 8/8 for HLA-A, -B, -C and DRB1 * Characteristics of patients N Eval N (%) Number of patients 1172 Number of centers 119 Recipient age, median (range), years (18-74) Age at transplant y 35 ( 3) y 178 (15) y 162 (14) y 282 (24) Over 50 y 515 (44) Male sex (51) Karnofsky prior to transplant > (73) Disease status at transplant st complete remission (Early) 755 (64) 2 nd complete remission (Intermediate) 417 (36) Graft type 1172 Bone marrow 349 (30) PBSC 823 (70) Conditioning regimen 1172 Myeloablative 757 (65) Reduced intensity 274 (23) Non-myeloablative 117 (10) To Be Determined/Other 24 ( 2) GVHD prophylaxis 1172 FK506 + (MTX or MMF or steroids) ± other 700 (60) FK506 ± other 45 ( 4) CsA + MTX ± other 234 (20) CsA ± other (no MTX) 150 (13) MMF ± other (no CsA) 4 (<1) MTX ± other (no CsA) 2 (<1) Other/To be determined 37 ( 3) HLA matching out of 8 (high-resolution) /8 293 (25) 8/8 879 (75) Donor/recipient sex match 1144 Male/Male 406 (35) Male/Female 326 (28) Female/Male 181 (16) Female/Female 231 (20) Donor/Recipient CMV match 1172 Negative/Negative 330 (28) Negative/Positive 401 (34) Positive/Negative 138 (12) Positive/Positive 246 (21) Unknown 57 ( 5) 43

44 Not for publication or presentation Attachment 5 Continued. Characteristics of patients N Eval N (%) Donor age, median (range), years ( ) Donor age ( 1) (31) (36) (24) 50 and older 78 ( 7) Unknown 22 ( 2) Year of transplant ( 6) ( 7) ( 6) ( 8) (13) (17) (21) (22) Median follow-up of recipients, mo (range) (3-125) * Data has not been CAP-modeled. 44

45 Not for publication or presentation Attachment 6 Study Proposal Study Title: Evaluation of the impact of allele homozygosity at HLA loci on outcome NMDP Histocompatibility Advisory Group Carolyn Hurley, Georgetown University Medical Center, Washington, DC USA Ann Woolfrey, Fred Hutchinson Cancer Research Center, Seattle, WA USA Martin Maiers, National Marrow Donor Program, Minneapolis, MN USA Hypothesis: 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 and exhibit better outcome than a 6/8 mismatched transplant. Primary outcomes for the study are: Overall survival (OS) Disease-free survival (DFS) Graft failure (Neutrophil engraftment) Secondary outcomes for the study are: Acute graft versus host disease grades II-IV and III-IV Specific Aims: To compare the outcome of unrelated donor transplants in which the recipient and/or the donor is homozygous at a single mismatched HLA locus to: (1) 7/8 matches in which the donor and the recipient are heterozygous for the mismatched locus and (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. Scientific Justification: There is no consensus among transplant centers as to how homozygosity should be handled in determined 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. 202 pairs were matched for HLA-A, B, C with 66 of those also matched for DRB1, DQB1, DPB1. 76 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. In a recent discussion among registries, it was realized that registries varied in the design of their search algorithm regarding the tally of mismatches when donor and/or recipient were homozygous. In 2009, the World Marrow Donor Association 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 45

46 Not for publication or presentation Attachment 6 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. Study Population: The study population consists of patients receiving their first myeloablative 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, DQB1 and DPB1 through the NMDP retrospective high resolution typing program. Categories of HLA matching to be included in the study: 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 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*0101, 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 Outcomes: Primary outcomes: 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. 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. 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. Secondary outcomes: Acute GVHD: Development of grades II-IV and III-IV acute GVHD using the Glucksberg system. Events will be summarized by the cumulative incidence Variables to be Analyzed: 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 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 46

47 Not for publication or presentation Attachment 6 Subanalysis by each disease: ALL, AML, CML and MDS Disease status prior to transplant: early vs. intermediate vs. advanced vs. others Subanalysis by disease stage: early, intermediate and 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: ( ) Gender match: M-M vs. M-F vs. F-M vs. F-F Donor/recipient CMV status: -/- vs. -/+ vs. +/- vs. +/+ vs. Unknown Conditioning regimen: Traditional Myeloablative vs. non-traditional myeloablative GvHD prophylaxis: Tacrolimus +/-others vs. CSA +/-others vs. TCD vs. others ATG use: Yes vs. no 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 both 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 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 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 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. References: 1. Petersdorf EW, Hansen JA, Martin PJ, Woolfrey A, Malkki M, Gooley T, Storer B, Mickelson E, Smith A, Anasetti C. Major-histocompatibility-complex class I alleles and antigens in hematopoietic-cell transplantation. N Engl J Med 2001: 345: Bochtler W, Maiers M, Bakker JN, Oudshoorn M, Marsh SG, Baier D, Hurley CK, Muller CR. 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. 2010: 3. 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:

48 Not for publication or presentation Attachment 6 Characteristics of recipients receiving first transplants for AML, ALL, CML or MDS with a myeloablative conditioning regimen and high-resolution typed for HLA-A, -B, -C, -DRB1, -DQB1 and DPB1 by homozygosity groups * Characteristics of patients 7/8 HVG MM 7/8 GVH MM 7/8 Both Directions N (%) 6/8 Both Directions N (%) N (%) N (%) Number of patients Number of centers Recipient age, median (range), years 34 (<1-60) 27 (<1-61) 32 (<1-65) 28 (<1-60) Age at transplant 10 y 8 (12) 16 (16) 131 (12) 92 (15) y 8 (12) 13 (13) 170 (16) 132 (21) y 8 (12) 24 (24) 185 (17) 114 (18) y 23 (33) 16 (16) 229 (21) 138 (22) y 13 (19) 19 (19) 256 (24) 118 (19) Over 50 y 9 (13) 10 (10) 113 (10) 39 ( 6) Male sex 38 (55) 48 (49) 590 (54) 358 (57) Karnofsky prior to transplant > (73) 65 (69) 763 (72) 459 (74) Disease at transplant AML 19 (28) 29 (30) 332 (31) 167 (26) ALL 17 (25) 34 (35) 271 (25) 187 (30) CML 25 (36) 24 (24) 354 (33) 219 (35) MDS 8 (12) 11 (11) 127 (12) 60 ( 9) Disease status at transplant Early 28 (41) 34 (35) 383 (35) 240 (38) Intermediate 16 (23) 35 (36) 335 (31) 205 (32) Advanced 17 (25) 19 (19) 278 (26) 156 (25) Other 8 (12) 10 (10) 88 ( 8) 32 ( 5) Graft type Bone marrow 63 (91) 86 (88) 964 (89) 595 (94) PBSC 6 ( 9) 12 (12) 120 (11) 38 ( 6) Number of patients GVHD prophylaxis FK506 + (MTX or MMF or steroids) ± 18 (26) 24 (24) 252 (23) 105 (17) other FK506 ± other 2 ( 3) 0 15 ( 1) 6 ( 1) CsA + MTX ± other 34 (49) 55 (56) 559 (52) 346 (55) CsA ± other (no MTX) 9 (13) 11 (11) 102 ( 9) 70 (11) MTX ± other (no CsA) ( 1) 7 ( 1) T-cell depletion 4 ( 6) 5 ( 5) 108 (10) 61 (10) Other/To be determined 2 ( 3) 3 ( 3) 41 ( 4) 38 ( 6) Donor/Recipient sex match Male/Male 18 (28) 33 (35) 346 (33) 197 (31) Male/Female 17 (26) 26 (27) 247 (24) 142 (23) Female/Male 16 (25) 15 (16) 223 (21) 160 (25) Female/Female 14 (22) 21 (22) 230 (22) 131 (21) Donor/Recipient CMV match Negative/Negative 24 (35) 33 (34) 326 (30) 205 (32) Negative/Positive 22 (32) 28 (29) 300 (28) 182 (29) Positive/Negative 10 (14) 16 (16) 171 (16) 101 (16) Positive/Positive 8 (12) 16 (16) 227 (21) 126 (20) Unknown 5 ( 7) 5 ( 5) 60 ( 6) 19 ( 3) 48

49 Not for publication or presentation Attachment 6 Continued. Characteristics of patients 7/8 HVG MM 7/8 GVH MM 7/8 Both Directions N (%) 6/8 Both Directions N (%) N (%) N (%) Donor age, median (range), years 34 (20-54) 37 (19-57) 36 (19-59) 37 (18-59) Donor age ( 2) 6 ( 1) 6 ( 1) (28) 18 (18) 247 (23) 159 (25) (35) 38 (39) 397 (37) 226 (36) (28) 30 (31) 297 (27) 194 (31) 50 and older 3 ( 4) 7 ( 7) 95 ( 9) 43 ( 7) Unknown 4 ( 6) 3 ( 3) 42 ( 4) 5 ( 1) Number of patients Year of transplant ( 1) 0 3 (<1) 1 (<1) ( 1) 3 (<1) 3 (<1) ( 4) 3 ( 3) 19 ( 2) 16 ( 3) ( 1) 0 35 ( 3) 35 ( 6) ( 6) 7 ( 7) 41 ( 4) 39 ( 6) ( 6) 5 ( 5) 50 ( 5) 44 ( 7) ( 1) 6 ( 6) 71 ( 7) 49 ( 8) ( 9) 4 ( 4) 93 ( 9) 56 ( 9) ( 4) 6 ( 6) 86 ( 8) 61 (10) ( 9) 10 (10) 88 ( 8) 55 ( 9) (10) 8 ( 8) 101 ( 9) 57 ( 9) ( 9) 10 (10) 112 (10) 46 ( 7) (10) 10 (10) 107 (10) 65 (10) ( 7) 14 (14) 133 (12) 59 ( 9) (13) 7 ( 7) 68 ( 6) 34 ( 5) ( 3) 1 ( 1) 18 ( 2) 8 ( 1) (<1) ( 2) 11 ( 1) ( 1) 5 (<1) 2 (<1) ( 4) 2 ( 2) 17 ( 2) 2 (<1) ( 1) 1 ( 1) 20 ( 2) 1 (<1) Median follow-up of recipients, mo (range) 107 (22-234) 120 (12-158) 115 (3-240) 132 (22-225) * Data has not been CAP-modeled. 49

50 Not for publication or presentation Attachment 7 Study Proposal Study Title: KIR genotyping and Immune function in MDS Patients Prior to Unrelated Donor Transplantation Erica Warlick, MD, University of Minnesota, Minneapolis, MN USA Daniel Weisdorf, MD, University of Minnesota, Minneapolis, MN USA Julie Ross, PhD, University of Minnesota, Minneapolis, MN USA David Lagarspada, PhD, University of Minnesota, Minneapolis, MN USA Jeffrey Miller, MD, University of Minnesota, Minneapolis, MN USA Specific Aims: The overarching hypothesis it that NK cell function correlates with disease progression in MDS and could lead to therapeutic immune strategies to control MDS. In the cohort selected here, we presume that all patients have advanced disease based on their need for transplantation. These results will be compared to a separate analysis on a prospective study on newly diagnosed MDS patients and those undergoing therapy. The following specific aims for this proposal support our hypothesis: 1. Determine whether advanced MDS patients requiring transplant have a KIR genotype that deviates from a cohort of normal controls 2. Determine function of NK cells and suppressor cells (T regulatory cells and myeloid derived suppressor cells) in MDS patients requiring transplant 3. Analyze pre-transplant frequencies and function for ability to predict post transplant outcomes Scientific Justification: Immune dysregulation has long been implicated for a role in the pathogenesis of myelodysplastic syndromes as evident by the clinical responses to immune modulating drugs (cyclosporine and ATG) as well as the potential curative potential with allogeneic bone marrow transplantation. Much of the attention on immune dysregulation in the past has been focused on B and T cell changes noted in MDS patients citing correlations with autoimmune disorders especially in lower risk MDS 1, 2 and notable findings of V- beta and J region skewing in oligoclonal T cell populations 3-5 as potential contributors to disease. We summarize here the current literature regarding immune dysregulation in MDS with respect to NK cells, T cell/t regulatory cells, myeloid suppressor cells, alterations of this immune profile in response to therapy and propose clinical interventions based on these data. Natural Killer Cell Function in MDS: Alterations in NK cell cytotoxicity has been noted in MDS patients. In brief review of NK cell biology, a number of both activating and inhibitory receptors are present on NK cells and mediate their cytotoxicity. Natural cytotoxicity receptors (NCRs) NKp46, NKp30 as well as activating receptor NKG2D are constitutively active activating receptors involved in NK-mediated cytotoxicty 6, 7. Few published reports evaluating NK cell characteristics in MDS patients, and the three most detailed studies reveal contradictory results. Kaledjian and colleagues showed that despite normal numbers of NK cells and activating receptors NKp46, NKp30, and NKG2D, cytolytic function and response to IL-2 stimulation was decreased. 8 Based on these data they questioned the possibility of soluble/humoral factors impacting NK cell cytotoxicity. Kiladjian s data showed that a representative population of the NK cells (25-44%) in these MDS patients were clonal harboring the same clone as the MDS cells the existence of a dual population of NK cells suggesting the possible contribution of the cytogenetic abnormalities to the NK cell dysfunction 8. Conversely, Epling-Burnette and colleagues found that NK cells fell into two categories of cytotoxicity: low function and normal function. The low function NK cell group was most notably present in the high risk MDS patients: 1) INT2 or high risk by IPSS, 2) those with abnormal cytogenetics, and 3) those in 50

51 Not for publication or presentation Attachment 7 RAEB-1, RAEB-2, and AML WHO categories. Additionally, they found decreased activating receptors NKp30 and NKG2D in MDS patients with only NKG2D presence correlated with level of cytotoxicity. The importance of the various receptors in terms of cytotoxicty was tested with antibody assay blocking of the various receptors, and NKG2D was identified as the primary receptor responsible for the killing. Epling s group addressed the question of a soluble factor contributing to the decreased NK cytotoxicity by evaluating for soluble MICA and MICB (the receptor ligands for NKG2D) and found no evidence for this. Additionally, in contrast to Kiladjian s reports, Epling found that IL-2 treatment was able to increase the presence of the activating receptors as well as increase NK cell mediated cytotoxicity 9. In contrast to the above two groups that evaluated peripheral blood NK cell function in MDS patients, Carlsten s group recently published their findings regarding bone marrow NK cell function. Their studies focused on the CD56 dim cytotoxic NK cells assessing presence of activating and inhibitory receptors, assessing actual cytotoxicity, and performing blast analysis. They noted down-regulation of both DNAM- 1 (DNAX-accessory molecule 1) and NKG2D on bone marrow NK cells with an inverse correlation with increased blasts and noted a more central role of DNAM-1 compared with NGK2D. They found no significant changes in NCRs. The loss of DNAM-1 was more pronounced in those with blast percentage > 5% possibly consistent with clonal expansion in the absence of functional cytotoxic NK cells. The peripheral blood NK cell evaluation interestingly revealed no down-regulation of DNAM-1, NKG2D, or the NCRs NKp30 or NKp46. NK cell degranulation was assessed and both bone marrow and peripheral blood NK cells showed decreased degranulation after coincubation with K562, despite the presence of normal receptor expression in the peripheral blood NK cells. Analysis of CD34+ blasts interestingly showed ongoing expression of the DNAM-1 Ligands CD155 and CD112 but rare expression of the NKG2D ligands MIC and ULBP. Assessment of direct killing of autologous blasts by the BM NK cells showed that these autologous NK cells were not able to kill the blasts 10. In summary, the data for NK cell alterations in MDS reveal discrepant results citing either soluble/humoral factors decreasing peripheral blood NK cell mediated cytotoxicity 8 versus decreased activating receptors (NKp30, NKp46, NKG2D) with NKG2D the primary effector 9 versus decreased expression of bone marrow NK cell activating receptor DNAM-1 contributing to the NK cell dysfunction 10. The above studies include relatively small sample sizes raising the need for NK cell profile analysis on a larger scale. T Cell Alterations in MDS: Given the association with autoimmune disorders, the aberrant inflammatory milieu with altered levels of TNF-alpha,etc, and the impact of immunosuppressive therapy in MDS, there is extensive literature on T cell alterations found in those patients with MDS. Characterization of the T cell repertoire in MDS patients has demonstrated T cell expansion with recurrent patterns of V-Beta and J region skewing suggestive of an ongoing antigenic immune response 3,5,11. Specific in vitro work by Smith et al. investigated the possibility of auto-reactive T cells and noted the presence of inhibitory T cells in patients with MDS 11 with subsequent clinical studies showing in vivo success using anti T-cell therapies (antithymocyte globulin (ATG) 4,12 and cyclosporine-a (CSA)) 13. Subsequent investigations revealed an abnormal T cell repertoire and T cell-mediated inhibition of bone marrow CFU-GM 4 as a potential mechanism to the response to ATG. More recent data has suggested that this clonal T cell expansion of effector cells (CD8+/CD57+/CD28-) is not limited to MDS with features historically associated with response to immunosuppressive therapy such as marrow cellularity, age, IPSS category, etc and is likely common to most MDS subgroups 14. The influence of T regulatory cells (Tregs), defined as CD4+, CD25 high, Foxp3+, on immune surveillance and development of autoimmunity or suppression of host immune response has been widely researched. Numerous studies in solid tumors and now hematologic malignancies are citing increased numbers of Tregs in those patients with active malignancies and progressive disease. The variable course of disease in 51

52 Not for publication or presentation Attachment 7 MDS with early/low grade MDS often mimicking an autoimmune type disorder with increase inflammatory milieu, increased apoptosis and response to immunosuppressive therapies and later/higher grade MDS showing more of a rapid progression to leukemia and a more proliferative nature raises the question of Treg impact on MDS disease progression and pathophysiology. A number of studies to date have evaluated the impact of Tregs in MDS. Kordasti et al showed a correlation of increased peripheral blood T regulatory cells in more advanced MDS with higher percentage of cells in those with more aggressive WHO classification (5q/RA < RCMD< RAEB < MDS/MPD), higher blast percentage, higher IPSS score, and more complex cytogenetics. The Tregs were found to be polyclonal by spectratyping suggesting that they may arise by peripheral expansion 15. Hamdi et all found a similar increase Tregs in MDS patients but slightly different pattern with higher levels in both RA/RARS and RAEB compared with RCMD 16. Kotsiandis et al went on to assess the kinetics, function and bone marrow trafficking of Tregs in MDS and analyzed both peripheral blood and bone marrow. They found Treg expansion in both PB and BM in later stage MDS compared with normal controls/early MDS. Treg frequency and number showed a direct correlation with disease progression or response to therapy with increases at times of disease progression, stability with disease stability, and decrease with response to therapy 17. Functionally, the T regulatory cells retained their function in late stage MDS, theoretically consistent with their impact on suppressing immune attack on the underlying disease, and in early stage MDS were less efficient at suppression of polyclonal T cell proliferative responses again theoretically consistent with the higher degree of autoimmunity in early stage disease. Alfinito et al investigated the immune profile in MDS patients as well and confirmed the increased Treg frequency in INT-2/High risk patients and lower numbers in Low/INT-1 patients. They also noted increased CD8 cells and B cells in the BM of low/int-1 patients. Combined these data can support the role of Tregs in leukemic progression INT-2/High risk MDS and a more autoimmune milieu in the low/int-1 patients 18. In summary, these data would suggest that Tregs play a key role in immune modulation in MDS and have a significant role in progression of disease but further larger scale analysis is warranted. Patient Eligibility Population: Myelodysplastic Syndromes (MDS) including the following subtypes (FAB): - RA - RARS - RAEB1/2 - RAEB t 1st Allogeneic HSCT De novo and Treatment related MDS will both be included Age : All Donor: Related or Unrelated Stem Cell source: BM, PB, or UCB Those patients with viable call and DNA samples available Data Requirements: (Data to Be Collected) Patient-related: Age Gender KPS and HCT-CI prior to transplant Disease-related: FAB subtype of MDS at diagnosis (RA/RARS/RAEB 1 or 2/RAEBt) IPSS Score at Diagnosis Percentage blasts at diagnosis Blood counts at diagnosis: Hgb, WBC, platelet, ANC Cytogenetics at diagnosis Transfusion dependence: 52

53 Not for publication or presentation Attachment 7 - PRBC: yes or no - Platelets: yes or no Therapy Prior to transplant - Conventional Induction Type chemotherapy: yes or no - Hypomethylating Agents (azacitidine or decitabine): yes or no - Immunomodulatory Medications (thalidomide/lenalidomide/atg+csa): yes or no - Prior Erythropoietin Use: Yes or No - No Therapy: yes or no Time from Diagnosis to Transplant Disease Status at the time of transplant - % Blasts: 0-4%;< 5% - Advanced Disease (RAEB1/2) vs. Less Advanced Disease (RA/RARS) - CR vs. untreated vs. treated with <5% blasts vs. treated with > 5% blasts Transplant-related: Donor Type (related vs. unrelated) and HLA matching Graft Source ( BM, PB, cord blood) GVHD Prophylaxis Conditioning Intensity (Myeloablative vs. RIC) Year of transplant ( , , ) Outcomes: KIR genotype comparison between MDS subsets and normal controls (we already have transplant donor control (n~2500) data from KIR genotyped cohorts performed as a part of J. Miller s NCI P01) Comparison of NK cell, T regulatory cell and myeloid derived suppressor cell immune phenotype between various subsets of MDS patients Assessment for correlation between Immune phenotype and post transplant outcomes of DFS, relapse, OS Sample Requirements: DNA sample for KIR genotyping (n=971, excluding samples already analyzed in the initial P01 donor/recipient cohort of 209 already received and analyzed): One aliquot of viable cells collected pre-transplant should be sufficient for these studies - NK Cell Functional Analysis - T regulatory and myeloid derived suppressor cell number We would like to pilot this functional data with an initial request of 120 samples, assuming that 50% will yield interpretable results (samples need to be both viable and have enough NK cells for analysis). This will allow a better assessment of assay variability and projected endpoints. We hypothesize that this initial assessment will tell us if the cohort will stratify into a high and low function group compared to normal subjects. If the cohort exhibits uniform low function, then we could conclude that patients eligible for unrelated transplant are advanced and beyond the early phases of hypothesized intact immune integrity, important in disease progression. However, if a dichotomy of immune function is found (i.e. patients stratify into a high and low group), additional samples will be requested to allow correlation between immune function and MDS characteristics to better understand this biology. Study Design: DNA samples from all MDS patients will be used for KIR genotyping by the laboratory of Jeffrey Miller. KIR genotype profile will be compared with normal controls (available through Dr. Miller s lab) to assess for any meaningful deviation between the two groups NK cell and T regulatory (Treg) cell and myeloid derived suppressor numbers and functional analysis will be performed by Dr. Miller s laboratory. Initial studies will be descriptive to 53

54 Not for publication or presentation Attachment 7 assess for meaningful patterns of immune dysregulation observed in the various MDS subsets. Methods for high-resolution dual function 9-color flow cytometry will evaluate CD107a degranulation (as a surrogate marker of NK cell killing) and interferon gamma production in addition to surface markers CD56, CD3, CD45, NKG2A, CD158a, CD158b, CD158e. Thawed samples will be tested after resting overnight and then stimulation with IL- 12/IL-18 and class I negative K562 targets. The Miller lab has extensive experience with this assay and it has been validated with frozen samples. Treg assays have been developed and validated and the assay for myeloid derived suppressor cells is in progress. As immune profile patterns are identified across the MDS subsets, correlation with transplant outcomes will be studied Statistical Support: The work proposed here falls within the scope of Miller s NK P01 and will be supported by the biostatistical core of that grant either through the U of MN site (Dr, Chap Le and colleagues) or the MCOW (Dr. John Klein and colleagues). Clinical outcome data will be needed from the NMDP/CIBMTR including survival, DFS, relapse, TRM, acute GVHD, and chronic GVHD for the MDS patients included here. References: 1. Marisavljevic D, Kraguljac N, Rolovic Z. Immunologic abnormalities in myelodysplastic syndromes: clinical features and characteristics of the lymphoid population. Med Oncol. 2006;23: Billstrom R, Johansson H, Johansson B, Mitelman F. Immune-mediated complications in patients with myelodysplastic syndromes--clinical and cytogenetic features. Eur J Haematol. 1995;55: Epperson DE, Nakamura R, Saunthararajah Y, Melenhorst J, Barrett AJ. Oligoclonal T cell expansion in myelodysplastic syndrome: evidence for an autoimmune process. Leuk Res. 2001;25: Molldrem JJ, Jiang YZ, Stetler-Stevenson M, Mavroudis D, Hensel N, Barrett AJ. Haematological response of patients with myelodysplastic syndrome to antithymocyte globulin is associated with a loss of lymphocyte-mediated inhibition of CFU-GM and alterations in T-cell receptor Vbeta profiles. Br J Haematol. 1998;102: Melenhorst JJ, Eniafe R, Follmann D, Nakamura R, Kirby M, Barrett AJ. Molecular and flow cytometric characterization of the CD4 and CD8 T-cell repertoire in patients with myelodysplastic syndrome. Br J Haematol. 2002;119: Miller JS. Biology of natural killer cells in cancer and infection. Cancer Invest. 2002;20: Wu J and Lanier LL. Natural killer cells and cancer. Adv Cancer Res. 2003;90: Kiladjian JJ, Bourgeois E, Lobe I, et al. Cytolytic function and survival of natural killer cells are severely altered in myelodysplastic syndromes. Leukemia. 2006;20: Epling-Burnette PK, Bai F, Painter JS, et al. Reduced natural killer (NK) function associated with high-risk myelodysplastic syndrome (MDS) and reduced expression of activating NK receptors. Blood. 2007;109: Carlsten M, Baumann BC, Simonsson M, et al. Reduced DNAM-1 expression on bone marrow NK cells associated with impaired killing of CD34(+) blasts in myelodysplastic syndrome. Leukemia. 2010;24: Smith MA and Smith JG. The occurrence subtype and significance of haemopoietic inhibitory T cells (HIT cells) in myelodysplasia: an in vitro study. Leuk Res. 1991;15: Molldrem JJ, Caples M, Mavroudis D, Plante M, Young NS, Barrett AJ. Antithymocyte globulin for patients with myelodysplastic syndrome. Br J Haematol. 1997;99:

55 Not for publication or presentation Attachment Jonasova A, Neuwirtova R, Cermak J, et al. Cyclosporin A therapy in hypoplastic MDS patients and certain refractory anaemias without hypoplastic bone marrow. Br J Haematol. 1998;100: Epling-Burnette PK, Painter JS, Rollison DE, et al. Prevalence and clinical association of clonal T-cell expansions in Myelodysplastic Syndrome. Leukemia. 2007;21: Kordasti SY, Ingram W, Hayden J, et al. CD4+CD25high Foxp3+ regulatory T cells in myelodysplastic syndrome (MDS). Blood. 2007;110: Hamdi W, Ogawara H, Handa H, Tsukamoto N, Nojima Y, Murakami H. Clinical significance of regulatory T cells in patients with myelodysplastic syndrome. Eur J Haematol. 2009;82: Kotsianidis I, Bouchliou I, Nakou E, et al. Kinetics, function and bone marrow trafficking of CD4+CD25+FOXP3+ regulatory T cells in myelodysplastic syndromes (MDS). Leukemia. 2009;23: Alfinito F, Sica M, Luciano L, et al. Immune dysregulation and dyserythropoiesis in the myelodysplastic syndromes. Br J Haematol. 2010;148:

56 Not for publication or presentation Attachment 7 Characteristics of recipients receiving first transplants for MDS where DNA and viable cells are available for the proposed study * Characteristics of patients N Eval N (%) Number of patients 971 Number of centers 116 Recipient age, median (range), years (<1-73) Age at transplant y 81 ( 8) y 94 (10) y 85 ( 9) y 152 (16) y 232 (24) Over (34) Male sex (57) Karnofsky prior to transplant > (64) Disease status at transplant 971 Early 233 (24) Advanced 454 (47) Other 284 (29) Graft type 971 Bone marrow 666 (69) PBSC 305 (31) Conditioning regimen 971 Traditional myeloablative 688 (71) Reduced intensity 132 (14) Non-myeloablative 71 ( 7) Non-traditional myeloablative 74 ( 8) To Be Determined/Other 6 ( 1) Donor/recipient sex match 971 Male/Male 363 (37) Male/Female 222 (23) Female/Male 192 (20) Female/Female 194 (20) GVHD prophylaxis 971 FK506 + (MTX or MMF or steroids) ± other 256 (26) FK506 ± other 20 ( 2) CsA + MTX ± other 448 (46) CsA ± other (no MTX) 99 (10) MMF ± other (no CsA) 3 (<1) MTX ± other (no CsA) 6 ( 1) T-cell depletion 131 (13) Other 8 ( 1) HLA Matching for HLA-A, -B, -C and -DRB1 971 Well-matched 501 (52) Partially matched 302 (31) Mismatched 168 (17) 56

57 Not for publication or presentation Attachment 7 Continued. Characteristics of patients N Eval N (%) Donor/Recipient CMV match 971 Negative/Negative 320 (33) Negative/Positive 290 (30) Positive/Negative 146 (15) Positive/Positive 199 (20) Unknown 16 ( 2) Donor age, median (range), years (18-60) Donor age ( 1) (25) (41) (26) 50 and older 70 ( 7) Year of transplant (<1) ( 1) ( 2) ( 2) ( 2) ( 3) ( 4) ( 5) ( 5) ( 7) ( 7) ( 6) ( 8) ( 9) ( 7) (12) (15) ( 7) Median follow-up of recipients, mo (range) (5-222) * - Data has not been CAP-modeled. 57

58 Not for publication or presentation Attachment 8 Study Proposal Study Title: Evaluation of the impact of potentially non-immunogenic HLA-C allele level mismatch Marcelo Fernandez-Viña, PhD, MD Anderson Cancer Center, Houston, TX USA Michelle Setterholm, National Marrow Donor Program, Minneapolis, MN USA Specific Aims: 1. To determine the effect of a putative non-immunogenic HLA mismatches on the outcome of unrelated donor transplantation. 2. To compare the impact of the single mismatch in the alleles C*03:03/C*03:04 with other single antigen and/or allele level mismatches in HLA-C on the outcome of unrelated donor transplantation. 3. To compare the impact of the 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 on the outcome of unrelated donor transplantation. 4. To compare the impact of the single mismatch in the allele C*03:03/C*03:04 on the outcome of unrelated donor transplantation with the outcome of transplants in which the patient and the unrelated donors are fully matched in HLA-A, B, C or DRB1 loci. 5. To compare the outcome of transplants with 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 with the outcome of transplants in which the patient and the unrelated donors present two mismatches in alleles or antigens of HLA-A, B, C or DRB1 loci excluding mismatches in C*03:03/C*03: To compare the outcome of transplants with 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 with the outcome of transplants in which the patient and the donor present a single antigen or allele level mismatches HLA-A, B, C or DRB1 other than C*03:03/C*03:04. Scientific Justification: A study evaluating the cytotoxic T-lymphocyte precursor (CTLp) frequencies directed against incompatibilities at the HLA-A, -B, and -C locus in donor-recipient pairs (1) showed a significant correlation between HLA class I incompatibilities (p < 0.001) and high CTLp frequencies. The analysis of HLA amino acid sequences in the HLA-C allele mismatched group showed that mismatches involving alleles with amino acid differences at five polymorphic positions 97, 99, 113, 114, and 116 situated at the peptide binding groove always resulted in the high CTLp frequency group, while the low/absent CTLp frequency group included mainly pairs with the mismatch in the alleles C*03:03/C*03:04 that differ only by one amino acid replacement at residue 91. This residue is not a contact site with the T-cell receptor is not part of any of the pockets that accommodate the peptide side chains in the antigen recognition site. Two CIBMTR outcome studies (2-3) including marrow and PBSC as source of allogeneic HSC from unrelated donors have shown that the isolated allele level mismatch in HLA-C do no not associate with detrimental effects in any outcome. These two alleles (C*03:03 and C*03:04) associate frequently with B*15:01 in European populations and with B*40:02 in Asian populations; in unrelated HSC transplantation many of the donor/recipient pairs that match in HLA-B and carry B*15:01 turn presenting an allele level mismatch in these subtypes of HLA-C*03. It appears that in most populations no other allele of HLA-C associates often with two alleles of the same HLA-C group. We propose that the most predominant allele level mismatch in HLA-C in these transplant cohorts included the pair of alleles C*03:03/C*03:04. Therefore, we propose the apparent lack of impact in outcome of the HLA-C allele level mismatch results from the lack of immunogenicity of 58

59 Not for publication or presentation Attachment 8 the mismatch C*03:03/C*03:04 that was demonstrated in the in-vitro experiments (1). It is conceivable that other allele level mismatches in HLA-C have a negative impact in outcome. The proposed comparisons may prove that the mismatch in the alleles HLA-C*03:03/C*03:04 is tolerable; if this was to be 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). Patient Eligibility 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, DQB1 and DPB1 through the NMDP retrospective high resolution typing program. Outcomes to be Studied: 1. Overall survival 2. Acute GVHD 3. Chronic GVHD 4. Relapse 5. DFS 6. TRM Variables to be Analyzed: Main effect to be tested: C*03:03 vs. C*03:04 single mismatches (7/8) vs. any other HLA-C locus mismatches vs. any other single HLA mismatches (7/8) vs. 8/8 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 - Subanalysis by each disease: ALL, AML, CML and MDS Disease status prior to transplant: early vs. intermediate vs. advanced vs. others - Subanalysis by disease stage: early, intermediate and 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: ( ) Gender match: M-M vs. M-F vs. F-M vs. F-F Donor/recipient CMV status: -/- vs. -/+ vs. +/- vs. +/+ vs. Unknown Conditioning regimen: Traditional Myeloablative vs. reduced intensity GvHD prophylaxis: Tacrolimus +/-others vs. CSA +/-others vs. others Study Design: To compare the impact of the single mismatch in the alleles C*03:03/C*03:04 with: Other single antigen and/or allele level mismatches in HLA-C Other single antigen or allele level mismatches in HLA-C or HLA-A, B or DRB1 Transplants fully matched in HLA-A, B, C or DRB1 loci Transplants in which the patient and the unrelated donors present two mismatches in alleles or antigens of HLA-A, B, C or DRB1 loci excluding mismatches in C*03:03/C*03:04 59

60 Not for publication or presentation Attachment 8 Transplants in which the patient and the donor present a single antigen or allele level mismatches HLA-A, B, C or DRB1 other than C*03:03/C*03:04. 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 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 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. References: 1. Oudshoorn M, Doxiadis II, van den Berg-Loonen PM, Voorter CE, Verduyn W, Claas FH. Functional versus structural matching: can the CTLp test be replaced by HLA allele typing? Hum Immunol Mar;63(3): 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 Dec 15;110(13): Woolfrey A, Klein JP, Haagenson M, Spellman S, Petersdorf E, Oudshoorn M, Gajewski J, Hale GA, Horan J, Battiwalla M, Marino SR, Setterholm M, Ringden O, Hurley C, Flomenberg N, Anasetti C, Fernandez-Vina M, Lee SJ. HLA-C Antigen Mismatch Is Associated with Worse Outcome in Unrelated Donor Peripheral Blood. Stem Cell Transplantation. Biol Blood Marrow Transplant Sep

61 Not for publication or presentation Attachment 8 Characteristics of recipients receiving first transplants for AML, ALL, CML or MDS with highresolution HLA typing for HLA-A, -B, -C, -DRB1, and DQB1 by HLA matching groups * Characteristics of patients C*03:03/ C*03:04 N (%) 7/8 HLA-C MM N (%) 7/8 HLA- Other MM N (%) 8/8 Matched N (%) Number of patients Number of centers Recipient age, median (range), years 34 (<1-68) 37 (<1-72) 34 (<1-74) 41 (<1-74) Age at transplant 10 y 19 (13) 74 ( 8) 144 (11) 401 ( 7) y 20 (14) 127 (14) 186 (14) 554 (10) y 25 (17) 136 (15) 224 (17) 775 (14) y 23 (16) 168 (18) 234 (17) 899 (16) y 35 (24) 205 (22) 259 (19) 1138 (21) Over 50 y 24 (16) 217 (23) 298 (22) 1683 (31) Male sex 76 (52) 532 (57) 730 (54) 3094 (57) Karnofsky prior to transplant > (68) 627 (73) 864 (68) 3509 (70) Disease at transplant AML 54 (37) 354 (38) 513 (38) 2173 (40) ALL 35 (24) 217 (23) 342 (25) 1104 (20) CML 37 (25) 211 (23) 299 (22) 1181 (22) MDS 20 (14) 145 (16) 191 (14) 993 (18) Disease status at transplant Early 56 (38) 317 (34) 498 (37) 2203 (40) Intermediate 41 (28) 280 (30) 392 (29) 1370 (25) Advanced 37 (25) 253 (27) 310 (23) 1287 (24) Other 12 ( 8) 77 ( 8) 145 (11) 591 (11) Conditioning regimen Myeloablative 125 (86) 747 (81) 1098 (82) 4158 (76) Reduced intensity 16 (11) 124 (13) 170 (13) 874 (16) Non-myeloablative 5 ( 3) 39 ( 4) 51 ( 4) 319 ( 6) Other/TBD 0 17 ( 2) 26 ( 2) 100 ( 2) Graft type Bone marrow 95 (65) 591 (64) 820 (61) 3045 (56) PBSC 51 (35) 336 (36) 525 (39) 2406 (44) HLA-DQB1 matching Allele-matched 124 (85) 812 (88) 1097 (82) 4991 (92) Single allele mismatch 22 (15) 111 (12) 238 (18) 449 ( 8) Double allele mismatch 0 4 (<1) 10 ( 1) 11 (<1) HLA-DPB1 matching Allele-matched 9 ( 6) 68 ( 7) 93 ( 7) 415 ( 8) Single allele mismatch 55 (38) 316 (34) 405 (30) 1501 (28) Double allele mismatch 23 (16) 196 (21) 250 (19) 815 (15) HLA-DPB1 data missing 59 (40) 347 (37) 597 (44) 2720 (50) 61

62 Not for publication or presentation Attachment 8 Continued. Characteristics of patients GVHD prophylaxis FK506 + (MTX or MMF or steroids) ± other C*03:03/ C*03:04 N (%) 7/8 HLA-C MM N (%) 7/8 HLA- Other MM N (%) 8/8 Matched N (%) 53 (36) 346 (37) 522 (39) 2442 (45) FK506 ± other 2 ( 1) 28 ( 3) 49 ( 4) 196 ( 4) CsA + MTX ± other 65 (45) 363 (39) 477 (35) 1866 (34) CsA ± other (no MTX) 14 (10) 97 (10) 150 (11) 557 (10) MMF ± other 0 3 (<1) 2 (<1) 14 (<1) MTX ± other (no CsA) 2 ( 1) 4 (<1) 9 ( 1) 32 ( 1) T-cell depletion 8 ( 5) 47 ( 5) 76 ( 6) 171 ( 3) Other/To be determined 2 ( 1) 39 ( 4) 60 ( 4) 173 ( 3) Donor/Recipient sex match Male/Male 51 (36) 304 (35) 405 (33) 1966 (40) Male/Female 40 (29) 196 (23) 291 (24) 1234 (25) Female/Male 21 (15) 198 (23) 256 (21) 828 (17) Female/Female 28 (20) 171 (20) 267 (22) 901 (18) Donor/Recipient CMV match Negative/Negative 54 (37) 271 (29) 352 (26) 1615 (30) Negative/Positive 41 (28) 267 (29) 361 (27) 1619 (30) Positive/Negative 24 (16) 126 (14) 197 (15) 669 (12) Positive/Positive 21 (14) 189 (20) 273 (20) 912 (17) Unknown 6 ( 4) 74 ( 8) 162 (12) 636 (12) Donor age, median (range), years 37 (19-56) 36 (18-61) 37 (19-60) 35 (18-61) Donor age ( 1) 5 ( 1) 6 (<1) 68 ( 1) (23) 205 (22) 285 (21) 1486 (27) (29) 350 (38) 446 (33) 1811 (33) (32) 242 (26) 360 (27) 1235 (23) 50 and older 16 (11) 64 ( 7) 117 ( 9) 298 ( 5) Unknown 6 ( 4) 61 ( 7) 131 (10) 553 (10) 62

63 Not for publication or presentation Attachment 8 Continued. Characteristics of patients C*03:03/ C*03:04 N (%) 7/8 HLA-C MM N (%) 7/8 HLA- Other MM N (%) 8/8 Matched N (%) Year of transplant ( 1) 12 (<1) ( 1) 6 ( 1) 12 ( 1) 39 ( 1) ( 3) 7 ( 1) 20 ( 1) 54 ( 1) ( 3) 12 ( 1) 28 ( 2) 81 ( 1) ( 2) 20 ( 2) 34 ( 3) 100 ( 2) ( 3) 23 ( 2) 38 ( 3) 106 ( 2) ( 3) 33 ( 4) 42 ( 3) 161 ( 3) ( 8) 44 ( 5) 50 ( 4) 160 ( 3) ( 5) 47 ( 5) 42 ( 3) 175 ( 3) ( 4) 49 ( 5) 49 ( 4) 204 ( 4) ( 5) 46 ( 5) 64 ( 5) 197 ( 4) ( 6) 61 ( 7) 64 ( 5) 222 ( 4) ( 5) 66 ( 7) 76 ( 6) 283 ( 5) ( 3) 72 ( 8) 92 ( 7) 276 ( 5) ( 5) 53 ( 6) 40 ( 3) 275 ( 5) ( 5) 52 ( 6) 95 ( 7) 317 ( 6) (11) 85 ( 9) 91 ( 7) 457 ( 8) ( 9) 71 ( 8) 130 (10) 564 (10) ( 8) 69 ( 7) 126 ( 9) 641 (12) ( 6) 55 ( 6) 127 ( 9) 627 (12) ( 1) 40 ( 4) 85 ( 6) 324 ( 6) ( 1) 16 ( 2) 33 ( 2) 176 ( 3) Median follow-up of recipients, mo (range) 84 (8-234) 82 (3-229) 64 (2-240) 60 (3-252) * Data has not been CAP-modeled. 63

64 Not for publication or presentation Attachment 9 Study proposal Study Title: Effect of Rituximab and ABO Mismatch David Miklos, MD, PhD, Stanford University, Stanford, CA USA Specific Aims: Reanalyze the same data set for impact of donor and recipient ABO effect, especially ABO minor mismatch impact on OS, III-IV acute GVHD, and NRM using the usual CIBMTR clinical outcomes analysis. If the CIBMTR review of the 435 lymphoma patients shows ABO effect in relation to rituximab then EBMT abstract (Miklos planning to attend already). Ongoing correlative studies at Stanford: 1. Measuring ABO specific B cells using Jerne Plaque Technique 14, 28, and 56 days after allo- HCT in ABO mismatched and matched recipients 2. Developing B cell specific A and B tetramer FACS reagent. Significance: 1. Avoid ABO minor mismatch 2. Consider CTN clinical trial randomizing rituximab treatment for ABO mismatch patients. Scientific Justification: Introduction: 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. ABO impact varies by conditioning regimen, graft source, donor engraftment chimerism, and institution transfusion practices. We recently analyzed clinical outcomes for all allogeneic transplants performed at Stanford University since 1986 as a function of donor and recipient ABO types. Seebach et al. BBMT 2005; CIBMTR ABO Mismatch Analysis Material and Methods: 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 mm, and 7% ABO-bidirectional mm. ABO mismatch distribution did not differ by donor or recipient age, conditioning regimen, or disease. HLA-matched related donors accounted for 57.8% of grafts, and their sibling recipients were HLA matched ##% (p=sig compared to URD). 64

65 Not for publication or presentation Attachment 9 Results: Considering all 1955 patients, 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 nonrelapse mortality. In contrast, ABO-major mm and bidirectional mm were similar to ABO matched transplants through 2 years. Univariate analysis decreased OS and 100 day survival in ABO minor mm associated with 1) bone marrow graft source, and 2) non rituximab treated diseases (AML, ALL, MDS, CML). ABO matched 63% Overall Survival is Worst n= 1955 ABO Major Mismatch ABO Minor Mismatch ABO Bidirectional Mismatch 15% 15% 7% ABO Minor Mismatch p=0.012 vs ABO Match OS Following Myeloablative Conditioning n=1443 OS Following Nonmyeloablative Conditioning n=512 ABO Minor Mismatch p=0.012 vs ABO Match ABO Minor Mismatch p<0.05 vs ABO Match 65

66 Not for publication or presentation Attachment 9 Bone Marrow Grafts Only n= 833 ABO matched ABO minor mismatch P<0.01 vs ABO Match ABO Minor Mismatch Effect is Absent in NHL and CLL Patients Following Myeloablative Conditioning AML, ALL, MDS, and CML NHL and CLL ABO matched ABO minor mismatch ABO matched ABO minor mismatch P<0.05 We have subsequently gone on to score for patients who received rituximab within 6 months of allo-hct and show that ABO minor mismatch and ABO matched patients have similar OS. 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 provides a RR=4 [CI: ; p=0.002] The authors suggest ABO minor 66

67 Not for publication or presentation Attachment 9 mismatches may play a role in agvhd pathophysiology, and recommend ABO matching (Ludajic et al. BBMT 2010). We hypothesize: ABO is an important immune response target after allohct for donor B cells that can be overcome by in vivo B cell depletion. Ratanatharathorn et al for CIBMTR GVHD Committee Brit Journal of HAem. 145: The 435 lymphoma patient CIBMTR analysis shows 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). We are preparing the Stanford ABO minor mismatch experience for ASBMT. 67