Xiao-Jun Huang & Yu Wang & Dai-Hong Liu & Lan-Ping Xu & Huan Chen & Yu-Hong Chen & Wei Han & Hong-Xia Shi & Kai-Yan Liu

J Clin Immunol (2008) 28:390 397 DOI 10.1007/s10875-008-9193-4 Modified Donor Lymphocyte Infusion (DLI) for the Prophylaxis of Leukemia Relapse after Hematopoietic Stem Cell Transplantation in Patients with Advanced Leukemia Feasibility and Safety Study Xiao-Jun Huang & Yu Wang & Dai-Hong Liu & Lan-Ping Xu & Huan Chen & Yu-Hong Chen & Wei Han & Hong-Xia Shi & Kai-Yan Liu Received: 24 January 2008 / Accepted: 26 February 2008 / Published online: 18 March 2008 # Springer Science + Business Media, LLC 2008 Abstract Purpose We retrospectively evaluated the feasibility and safety of a modified prophylactic donor lymphocytes infusion (DLI) approach in advanced leukemia. Materials and methods Thirty-three patients with advanced leukemia received modified prophylactic DLI; that is, granulocyte colony-stimulating factor-primed peripheral blood progenitor cells instead of steady-donor lymphocyte harvests were used, and a short-term immunosuppressive agent (cyclosporine A or methotrexate 10 mg once per week for 2 to 4 weeks) was used for prevention of DLIassociated graft versus host disease (GVHD) after human leukocyte antigen-identical sibling hematopoietic stem cell transplantation. Results Thirty-nine infusions were performed in 33 patients. The mononuclear cells and median CD3+ cells infused for DLI were 1 2 10 8 and 0.93 10 6 per kilogram, respectively. Six patients experienced II IV-grade acute GVHD. Twenty patients developed chronic GVHD. No GVHD-related death or transfusion-related pancytopenia was observed. With an 18-month median follow-up, 16 patients were in disease-free survival, and overall survival at 1 and 1.5 years was 69.0% and 50.2%, respectively. Conclusion The modified prophylactic DLI strategy might represent a step forward in the treatment of advanced leukemia. X.-J. Huang (*) : Y. Wang : D.-H. Liu : L.-P. Xu : H. Chen : Y.-H. Chen : W. Han : H.-X. Shi : K.-Y. Liu Peking University Institute of Hematology, People s Hospital, Beijing 100044, People s Republic of China e-mail: xjhrm@medmail.com.cn Keywords Advanced leukemia. donor lymphocytes infusion. granulocyte colony-stimulating factor. hematopoietic stem cell transplantation Introduction Adult patients with advanced hematologic malignancies after allogeneic hematopoietic stem cell transplantation (allo-hsct) have a poor prognosis because of a high rate of relapse and transplant-related mortality. DLI exhibits definite antileukemia effects in patients with highly aggressive hematological malignancies [1 3]. It is also known that DLI could be followed by a high rate of severe graft versus host disease (GVHD) and, sometimes, pancytopenia and infection [3]. Many studies have demonstrated that in vivo granulocyte colony-stimulating factor (G-CSF) could modify the function of lymphocytes in peripheral blood (PB) harvests [4 5]. In addition, we recently reported that the infusion of donor G-CSF-mobilized PB progenitor cells (GPBPCs) instead of steady-donor lymphocytes demonstrated super antileukemia effects, as this approach may accompany a decrease in infusion-related GVHD and is rarely complicated by pancytopenia [1]. Recently, our group has introduced a sequential allo- HSCT followed by prophylactic GPBPC infusion in patients with hematological malignancies who have a high risk of relapse. Preliminary subgroup analysis of this trial suggested that patients with advanced leukemia may particularly benefit from the procedure, especially by decreasing the incidence of relapse [2]. The current study was initiated to retrospectively assess the feasibility and

J Clin Immunol (2008) 28:390 397 391 safety of the modified prophylactic donor lymphocyte infusion in human leukocyte antigen (HLA) identical sibling stem cell transplantation (SCT). Patients and Methods Eligibility Criteria From September of 2002 to June of 2007, 33 patients with advanced leukemia who received modified prophylactic DLI consecutively after HLA identical sibling HSCT were included in this study. Ten of these 33 patients were reported in 2006 [2], and these patients were further followed up in this study. Advanced leukemia is defined as patients who fulfilled at least one of the following criteria [6]: (1) acute leukemia in the first complete remission (CR1) with unfavorable cytogenetic abnormality with mainly positive Ph chromosome, (2) acute leukemia in more than CR1 status or in the nonremission state, (3) chronic myelogenous leukemia (CML) in accelerated phase (AP) and blast phase (BP), and (4) other myeloproliferative disease with unfavorable cytogenetic abnormalities such as 8p11. Protocol of Modified DLI The protocol included two elements: (1) GPBPC instead of steady-donor lymphocyte harvests were used; (2) a short term immunosuppressive agent was used for prevention of DLI-associated GVHD. Lymphocytes were obtained from cryopreserved G-CSF-mobilized PB (G-PB) or from fresh G-PB (mentioned above in the section Stem Cell Collection ). The Peking University Review Board approved the cryopreservation, remobilization, and infusion of the G-PB. All donors and patients signed a consent form. Subsequent DLI was planned from days 45 to 120 after transplantation in patients when no early death, recurrence of leukemia, or GVHD occurred or GVHD was controlled. Before DLI, serious infection has to be cleared, and no serious organ failure is present. The initial cell dose for DLI was 1 10 8 mononuclear cells (MNCs) per kilogram; For patients receiving DLI before day 90 after transplantation, original cyclosporine A (CsA) was continued for another 2 weeks after the infusion, then tapered and stopped within 4 weeks if no DLI-associated GVHD occurred. For patients receiving DLI after day 90, all immunosuppressive agents should be stopped for at least 2 weeks before the infusion, and no active GVHD present. These patients took oral CsA or methotrexate (MTX) 10 mg once per week for 2 to 4 weeks after DLI for the prevention of DLI-associated GVHD. Conditioning Regimen All patients received myeloablative conditioning with total body irradiation (TBI) and cyclophosphomide (Cy) regimen, which consisted of total body irradiation with 7.7 Gy and Cy 1.8 g m 2 day 1 2 days or the modified busulfan (Bu) Cy regimen (mbucy), which consisted Ara-C 2 g m 2 day 1 1 day, Cy 1.8 g m 2 day 1 2 days, Bu 4 mg kg 1 day 1 3 day, and 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea 250 mg kg 1 day 1 1 day. Our data showed no difference between these two regimens on relapse and the disease-free survival, and CR rate were higher in the mbucy group than those in TBI group for high-risk patients [7]; furthermore, it is more convenient for the mbucy regimen. Prophylaxis of GVHD All patients who received CsA or short-term MTX as prophylaxis of DLI-associated GVHD received CsA, mycophenolate mofetil (MMF), and short-term MTX to prevent GVHD after transplantation. MTX was administered at 15 mg/m 2 intravenously on day 1 after transplantation and 10 mg/m 2 intravenously on days 3 and 6 after transplantation. MMF was administered 0.5g, q12h, orally from day 9 before transplantation to the day of engraftment after transplantation. The dose of CsA was 2.5 mg kg 1 day 1, administered intravenously from day 9 before transplantation until the patient s bowel function was normal, then CsA was taken orally. Whole-blood CsA concentration was monitored weekly using a fluorescence polarization immunoassay. Stem Cell Collection Donors were mobilized with recombinant human G-CSF (Filgrastim,Kirin, Japan) at 5 μg/kg daily injected subcutaneously for 5 consecutive days. Stem cells from bone marrow (BM) were collected on the fourth day of mobilization and PB stem cells (PBSCs) were collected on the fifth day in case of BM transplantation (BMT) combined with PBSC transplantation (PBSCT). In the case of PBSCT, G-CSF-mobilized PBSC was collected on the fourth and fifth days. Greater than 6 10 8 MNCs per kilogram or 4 10 6 CD34+ cells per kilogram were planned for harvest. The target numbers of cells of greater than 4 10 8 MNCs per kilogram or 2 10 6 CD34+ cells per

392 J Clin Immunol (2008) 28:390 397 kilogram were planned for transplantation. BM was infused in patients within 3 h after collection, and the PBSC product was infused fresh and unmanipulated. The extra harvested cells were cryopreserved with dimethyl sulfoxide in a nitrogen tank. Automated counter instrument was used to count MNCs. The numbers of CD þ 34,CDþ 3,CDþ 4, and CD þ 8 cells contained in the collected product were counted by flow cytometry. In case of ABO major blood group incompatibility, red cells in the marrow were removed by hydroxyethyl sediment manipulation. The source of stem cells was G-CSF-mobilized PBSCs for 18 patients and the mixture of G-CSF-mobilized BM cells and G-CSF-mobilized PBSCs for the other 15 patients. HLA Typing Standard serologic typing was used for HLA A, B, and C; the latter antigen only typed in 75% of the patients. Highresolution molecular typing using polymerase chain reaction (PCR) in the sampled deoxyribonucleic acid with sequence-specific primers was performed for class II alleles (HLA DRB1 and DQB1). Supportive Care HSCT recipients were nursed in the isolation rooms with laminar airflow systems. Prophylactic trimethoprim/sulfamethoxazole, acyclovir, and fluconazole were taken to prevent from infection of pneumocystis carinii, herpes simplex, and fungi. Blood products were infused to patients with hematocrit levels less than 25% and/or platelet counts less than 20 10 9 /L. All blood products were irradiated prior to use. Therapy of GPBPC Infusion-associated GVHD For acute GVHD (agvhd) after MTX alone or MTX in combination with CsA as prophylaxis regimens, methylprednisolone (MP) is the best initial therapy. Prednisone and CsA are considered as the first-line therapy for patients with chronic GVHD (cgvhd). Other therapeutic options for agvhd or cgvhd are MMF, tacrolimus (FK506), azathioprine, thalidomide, monoclonal antibodies directed against CD3 and CD25, and MTX. Evaluations and Definitions During hospitalization at a laminar air flow unit, clinical status, adverse events, and hematologic and clinical chemistry parameters were monitored daily. Following discharge, patients were seen in the outpatient clinic once to twice a week up to day +100 and at gradually increasing intervals thereafter. agvhd and cgvhd were graded as described [8]. Minimal residual disease (MRD) was monitored using flow cemetery or reverse transcriptase- PCR for the bcr/abl gene. Primary induction failure (PIF) is defined as nonremission after two or more cycles of chemotherapy. Date of neutrophil recovery was defined as the first of 3 consecutive days with an absolute neutrophil count within the PB exceeding 500/μL. Date of platelet engraftment was defined as the first of 7 days with an absolute platelet count exceeding 20 g/l without transfusion support. At day +30, disease response and chimerism were assessed in PB and BM. Because thrombocyte regeneration could be postponed by factors other than leukemia and cytotoxic therapy (i.e., GVHD, virus infection, drugs), CR was defined as less than 5% blasts without evidence of dysplasia in BM and more than 1,500 neutrophils per microliter in PB. Donor chimerism in unfractionated BM was compared before and after prophylactic GPBPC infusion, using fluorescence in situ hybridization [9] in sex-mismatched and short-tandem repeats (STRs) analysis [10] in sex-matched transplantations. Hematologic relapse was defined by reappearance of blasts in the PB, by any manifestation of leukemia outside the hematopoietic system, or by BM infiltration by greater than 5% of blasts in a representative smear. Death from leukemia was defined as death with refractory disease after transplantation or as death from any cause following post-transplantation relapse. Nonrelapse mortality (NRM) was defined as death from any cause other than refractory disease or relapse, including death from preexisting comorbidities. Statistical Analysis Results were analyzed as of December 1, 2007. Overall survival (OS) at 1.5 years from transplantation was the primary end point of the study. Secondary end points included leukemia-free survival (LFS; defined as being alive and free of disease), NRM, and relapse incidence, as well as incidence and severity of agvhd and cgvhd. The day of stem cell transfusion was counted as day 0, and all intervals were calculated based on this date. Numeric variables were analyzed as categories considering their value below or above the median of the entire cohort, as indicated in Results. A landmark analysis was performed at 60 days after DLI to evaluate GVHD. OS and LFS were estimated using the Kaplan Meier method. Logrank test and a Cox proportional hazard regression model

J Clin Immunol (2008) 28:390 397 393 were used for analysis of risk factors for time-to-event variables. Probabilities of NRM and death from leukemia were calculated using reciprocal cumulative incidence estimates to account for competing risks. SPSS software packages were used for data analysis. Results Patient and Donor Characteristics From September of 2002 to June of 2007, 50 consecutive patients with advanced leukemia received HLA-identical sibling HSCT. Seventeen of these 50 patients did not receive prophylactic DLI because of GVHD (n=4), infection (n=2), graft failure (n=2), and early relapse (n= 9). The other 33 out of these 50 patients with advanced leukemia who received modified prophylactic DLI consecutively were included in this study. The median patient age at transplantation was 37 years (range, 5 51 years). Characteristics of patients are displayed in Table I. Engraftment, Cell Count, and Chimerism After SCT, all patients were engrafted. No primary graft failure was observed. The median time to neutrophil and thrombocyte engraftment was 17 (range, 12 23 days) and 15 days (range, 7 45 days), respectively. All patients achieved complete allogeneic engraftment before and after DLI as confirmed by STR study of chimerism status or by chromosome examination. All patients received prophylactic DLI after allo-sct. Although DLI was planned from days +45 to +120, it had to be ahead of schedule because of signs of MRD for four patients or postponed in five patients because of infection. As shown in the table, the prophylactic DLI was administered between 20 and 314 days (median 70 days). A total of 39 prophylactic infusions were performed, of which 27 were before +90 day post-sct. The MNCs and CD3 + cells infused for prophylactic DLI were 1 2 10 8 and 0.93 10 6 per kilogram (range 0.20 2.12 10 6 per kilogram), respectively. Twenty-nine patients received once and two patients received twice the prophylactic DLI, and two patients received three times the prophylactic infusion in escalating doses. Reasons for giving more than one DLI were that persistent MRD existed after DLI without signs of GVHD. Graft-versus-Host Disease/Pancytopenia Before prophylactic DLI, agvhd developed in 6 of 33 patients, reaching grade I in one, grade II in three, grade III in two, and there were no grade IV patients. The median time of onset was day +42 (range, 29 49). Skin, liver, and Table I Patients Characteristics Patient number Sex Age Diagnosis Disease status Conditioning 1 M 39 CML BP BuCy 2 M 36 CML BP Cy/TBI 3 M 26 CML BP BuCy 4 M 38 AML PIF BuCy 5 M 36 AML PIF BuCy 6 M 37 AML PIF BuCy 7 M 29 AML PIF BuCy 8 M 22 MPD REL2 BuCy 9 M 28 AML PIF BuCy 10 M 42 AML CR3 BuCy 11 M 43 AML REL BuCy 12 M 36 AML PIF BuCy 13 M 48 AML REL BuCy 14 F 43 AML REL BuCy 15 F 40 AML PIF BuCy 16 F 36 ALL PIF BuCy 17 M 15 ALL REL3 BuCy 18 M 40 ALL Ph+ PIF BuCy 19 M 39 ALL Ph+ PIF BuCy 20 F 37 ALL Ph+ PIF BuCy 21 M 20 ALL CR3 Cy/TBI 22 M 15 ALL PIF Cy/TBI 23 M 51 CML AP BuCy 24 M 12 CML AP BuCy 25 M 42 AML PIF BuCy 26 M 20 ALL CR3 BuCy 27 M 5 ALL PIF BuCy 28 F 48 AML PIF BuCy 29 M 41 ALL CR2 BuCy 30 F 48 AML CR2 BuCy 31 M 34 AML REL BuCy 32 F 38 ALL PIF Cy/TBI 33 F 45 AML REL BuCy gut were affected in five, one, and three patients, respectively. For these six patients, GVHD was soon controlled. Before GPBPC infusion, all immunosuppressive agents was stopped for at least 2 weeks and no active GVHD was present again. Therefore, prophylactic DLI was administered between days +64 and +122 for these six patients. After prophylactic DLI, DLI-associated agvhd developed in six of 39 infusions, reaching grade II in three, grade III in two, and grade IV in one patient. The respective time of onset was days +1, +30, +1, +45, +60, and +47 after DLI. The median time was day +38 (range, 1 60) after DLI. Skin, liver, and gut were affected in five, one, and six patients, respectively. At 60 days after DLI, agvhd occurred in 6 out of 32 patients (16.7%, one patient died 7 days after DLI because of infection). Among the six patients, for four patients, immunosuppressive agent was tapered or stopped ahead of schedule

394 J Clin Immunol (2008) 28:390 397 due to advancement of leukemia. One patient (patient 13) received prophylactic DLI on day 20 after transplantation, then CsA was stopped 2 weeks later, and she developed grade IV GVHD 30 days after DLI, 16 days after CsA was stopped; one patient (patient 25) received prophylactic DLI on day 314; then, MTX was used only twice, and he developed grade III GVHD 45 days after DLI; 35 days after, MTX was stopped. One patient (patient 24) received prophylactic DLI on day 47 after transplantation; CsA was not adjusted to effective concentration, and she developed grade II GVHD 1 day after DLI; the other patient (patient 26) received prophylactic DLI on day 60; then, CsA was tapered 1 week later, and he developed grade II GVHD 60 days after DLI. For another two patients, the immunosuppressive agent was used as scheduled. For these two patients, one patient (patient 11) received prophylactic DLI on day 39 after transplantation, and he developed grade III GVHD 1 day after DLI; the other patient (patient 28) received prophylactic DLI on day 44, and she developed grade II GVHD 47 days after DLI. All six patients who developed agvhd after DLI (only one occurred when the prophylactic DLI was administered after day 90 post-transplant) were controlled after CsA was adjusted to an effective concentration or changed to FK506 plus MP or monoclonal antibodies against CD25 (patient 11) or MTX (patients 13 and 24). There was no GVHDrelated death. In total, agvhd developed in 11 (33.3%) of 33 patients after transplantation (including before and after DLI). None died of agvhd. cgvhd developed in 20 (62.5%) of 32 patients (patient 20 died of infection 7 days after DLI) following prophylactic DLI. The median interval from DLI to onset of cgvhd was 60 days (range, 38 150 days). Seven patients had limited disease; 13 had extensive disease. No patients had profound and lasting pancytopenia after modified prophylactic DLI. Relapse As of Dec. 1, 2007, after a median follow-up of 18 months (range of 3 62 months), 15 patients experienced leukemia relapse at a median of 3 months (range of 1.3 14 months) after transplantation. The median interval from first and last DLI to onset of relapse was 50 (range of 11 318 days) and 35 days (range of 6 241 days), respectively. There was hematologic relapse in 13 cases and extramedullary relapse in two cases. Fourteen cases (93%) of relapse occurred within the first year after transplantation. Nine of these patients died during reinduction therapy; one achieved a secondary CR after reinduction therapy; two achieved a secondary CR following adoptive immunotherapy, but one of these two patients died from infection; three patients received a second transplant, and one is in hematologic remission, while the other two died from a second relapse. Among the 15 patients, six patients had acute myeloid leukemia (AML), two were transplanted with PIF, four were relapsed patients, who were in untreated relapse including one chronic myelomonocytic leukemiatransformed AML, six were acute lymphoblastic leukemia (ALL), three were transplanted with PIF including two Philadelphia chromosome-positive (Ph+) ALL, three were relapsed patients, one in refractory relapse and two in second or third CR at the time of transplantation including one patient with T cell non-hodgkin s lymphoma-all, two patients were CML in AP and BP including one AP and one BP, and one patient had myeloproliferative disease with 8p11 in refractory relapse. Extramedullary disease, such as central nervous system (CNS) and soft tissue infiltration, was present before the start of conditioning in three patients. None of the myelodysplastic syndrome (MDS) patients relapsed. In terms of predictors for cumulative incidences of relapse rate, multivariate analysis showed that the significant factors associated with lower relapse rate were cgvhd (P=0.003), remission status before transplantation (P=0.024), non-all (P=0.034), and younger recipients (P=0.040) but not with sex (P=0.195), CNS leukemia (P= 0.203), conditioning regimen (P=0.793), and the source of stem cells (P=0.460). Five of 20 patients suffering from cgvhd experienced leukemic relapse, three of whom relapsed after cgvhd occurred, including two Ph+ ALL and one AML with PIF, and the other two relapsed before GVHD occurred, including one CML-BP and one relapsed AML (Table II). Patients 19 (Ph+ ALL) and 22 (ALL with CNS involvement) received a second prophylactic GPBPC infusion, and patients 15 (AML in the nonremission state with CNS involvement) and 24 (CML-AP) received a third prophylactic GPBPC infusion. Patient 22 showed no signs of GVHD after the first prophylactic infusion and were diagnosed with cgvhd after the second prophylactic infusion and kept in disease-free survival after follow-up of 1,825 days. Patient 19 had limited cgvhd after the second prophylactic infusion and died of disease recurrence on day 407 after transplantation. Patients 15 and 24 never showed any signs of GVHD and died of disease recurrence on day 365 and 547 after transplantation, respectively. Follow-up and Outcome As of Dec. 1, 2007, 11 patients (33.3%) had died from relapsed leukemia at a median time of 12 months (range of 5 18.2 months) from transplantation. Estimated cumulative incidence of death from leukemia at 1 and 1.5 years from transplantation was 20.3% (95% CI, 12.9%-27.7%) and

J Clin Immunol (2008) 28:390 397 395 Table II Clinical Results of Modified DLI Patients Patient number DLI (day) GVHD after GPBSCI T to R (day) a Follow-up (day) Status Acute (day) Chronic (day) 1 43 0 Extensive (60) No 1,768 Alive 2 122 0 Extensive (60) No 1,351 Alive 3 30 0 Extensive (60) 60 150 Relapse 4 82 0 Limited (60) No 1,865 Alive 5 196 0 Limited (38) No 1,528 Alive 6 76 0 Limited (82) No 827 Alive 7 120 0 Limited (150) No 1,157 Alive 8 41 0 0 91 543 Alive 9 116 0 Extensive (60) No 1,514 Alive 10 228 0 Extensive (40) No 1,524 Alive 11 39 III (1) Extensive (51) No 540 Infection 12 40 0 0 No 137 Infection 13 20 IV (30) Extensive (100) 40 180 Relapse 14 40 0 0 90 396 Relapse 15 47/56/74 0 0 80 365 Relapse 16 180 0 0 420 850 Alive 17 31 0 0 49 434 Infection 18 84 0 Extensive (38) 240 520 Relapse 19 160/273 0 Limited (48) 339 407 Relapse 20 77 0 0 No 84 Infection 21 78 0 0 180 180 Relapse 22 76/90 0 Limited (60) No 1,825 Alive 23 98 0 Extensive (60) No 1,609 Alive 24 47/280/330 II (1)0 0 365 547 Relapse 25 314 III (45) 0 No 1,718 Alive 26 60 II (60) Extensive (100) No 1,514 Alive 27 120 0 Limited (60) No 1,318 Alive 28 44 II (47) Extensive (80) 285 368 Relapse 29 46 0 0 75 268 Relapse 30 90 0 Extensive (60) No 210 Infection 31 64 0 0 75 234 Relapse 32 38 0 Extensive (120) No 298 Hemorrhage 33 27 0 0 58 162 Alive a T to R means time from transplant to relapse 39.3% (95% CI, 29.9 48.7%), respectively. Six patients (18.2%) died of causes other than leukemia, between days 84 and +540 (median, day +254). Causes of death were infections (n=4), GVHD in combination with infections (n=1), and hemorrhage (n=1). Estimated NRM rate at 100 days, 1 year, and 1.5 years from transplantation was 3.0% (95% CI, 0 6.0%), 16.3% (95% CI, 9.6 23%), and 21.2% (95% CI, 13.3 29.1%), respectively. As of Dec. 1, 2007, 16 of 33 patients were alive without disease recurrence. Median survival of the entire cohort was 33.8 months (95% CI, 25.6 42 months). Twelve out of 16 patients that are alive are still suffering from cgvhd (six patients have limited and the other six patients have extensive GVHD). At 1 and 1.5 years after transplantation, the estimated OS was 69.0% and 50.2%, respectively. The LFS rates were also 69.0% and 50.2%, respectively. In comparison to the entire cohort, patients who were not affected by ALL before SCT (n=23) including AML, MDS refractory anemia with excess of blasts in transformation, and CML-AP or BP showed a better outcome, achieving a 1- and 1.5-year LFS of 71.8% and 57.4%, but has no statistical significance. Discussion Allogeneic SCT is the recommended treatment for patients with advanced leukemia [11 13]. However, results are limited by a high relapse incidence and NRM [14 16]. Mengarelli et al. [6] reported that OS rates were 25% and relapse incidence was 58% for high-risk acute leukemia patients undergoing HLA-identical HSCT.

396 J Clin Immunol (2008) 28:390 397 In addition to the antileukemic effects of the conditioning regimen, the therapeutic efficacy of allogeneic SCT mainly relies on the GvL reaction. Previous studies demonstrate that treatment with conventional chemotherapy followed by DLI is feasible and effective in patients with relapse after allogeneic BMT [1, 17 20]. These results suggest that the strategy against disease recurrence may convert from a therapeutic DLI to prophylactic DLI approach for those with advanced leukemia. The main obstacle for the application of prophylactic DLI is infusionassociated GVHD, pancytopenia, and consequently, infection [3], of which there have been few studies to date. So far, no regular sequential prophylactic donor lymphocyte transfusion (pdlt) has been reported in high-risk patients with advanced leukemia. Our recent study found that when GPBPCs instead of steady-donor lymphocyte harvests were infused in patients with advanced leukemia, the incidence of GVHD was lowered, and the GvL effect was not weakened but even increased [1]. In HLA-mismatched DLI, our primary data indicate that when combined with short-term CsA or MTX for GVHD prophylaxis, the incidence of GVHD after GPBPC infusion was further reduced [21]. Preliminary subgroup analysis suggested that this modified DLI can be prophylactically used in patients with advanced leukemia to decrease the incidence of leukemia relapse after HLAidentical sibling donor transplantation [2]. The current study was initiated to assess the long-term feasibility and efficacy of the approach. In this report, agvhd occurred in 6 of 39 infusions, of which four developed agvhd due to the fact that a prophylactic immunosuppressive agent was stopped ahead of schedule and there was no GVHD-related death. The relative low incidence of the modified DLIrelated GVHD may be due to several reasons. Firstly, G- CSF-mobilized PBPC instead of steady-donor lymphocytes was used. The effect of in vivo G-CSF application on T cell function has been extensively explored. Several reports including our data indicate that in vivo G-CSF application indirectly induced a decrease in T cell proliferation and type II helper T cell polarization of the cytokine profile. Furthermore, T cell hyporesponsiveness in BM allografts induced by in vivo G-CSF may be related to a selective decrease in DC1 and the downregulation of CD28/B7 costimulatory signals. This may contribute to the antigenspecific hyporesponsiveness of T cells in G-CSF-mobilized harvests [4 5]. The second reason may be the use of CsA or MTX for prophylaxis of GVHD. In our own therapeutic GPBPC infusion study for HLA-mismatched HSCT, the incidence of severe GVHD was significantly reduced by GVHD prophylaxis (5 of 9 versus 1 of 11, P=0.013) [21]. Whether severe GVHD related to the modified prophylactic DLI could be mostly relieved by the short time period of the immunosuppressive agent and how long the immunosuppressive agent should be given needs to be answered in the future. No profound, lasting pancytopenia was observed after prophylactic GPBPC infusion and is in accordance with the observations of Sohn et al. [22]. It may be because there are relatively high percentages of CD34 + cells [23]. Hence, this retrospective study showed that modified prophylactic DLI in high-risk leukemia is safe and feasible in terms of GVHD and pancytopenia. It needs a further prospective study to verify the safety and feasibility of this approach. In our cohort, 14 patients relapsed within the first year after transplantation, and the cumulative 1-year relapse incidence was 42%, lower than the 51% to 63% reported from the studies using standard conditioning regimens [6]. However, the relapse rate was still high. The disease category and status of our patients and the time of infusion may be related to the high relapse rate. Our data showed remission status before transplantation (P=0.024), and non- ALLs (P=0.034) were factors associated with a lower relapse rate. Our patients are comprised of one third (11 of 33) of highly advanced refractory ALL including Ph+ ALL, 70% (23 of 33) in the nonremission state. As we all know, ALL such as Ph+ ALL seems insensitive to DLI [17]. Our data showed that the most significant factor associated with a lower relapse rate was cgvhd, while prophylaxis of infusion-related GVHD or agvhd was not the predictor, and we observed that some patients relapsed soon after DLI, which indicates that earlier prophylactic DLI may further decrease the relapse rate. Other measures such as MRD are needed to be explored to combine with modified DLI to further decrease the relapse rate. The estimated OS at 1 and 1.5 years in our cohort after transplantation was 69.0% and 50.2%, respectively, which was much higher than previous studies on the treatment of refractory AML using standard conditioning regimens for SCT, wherein OS rates from 20% to 30% have been reported [24 26]. In our earlier publication, we reported that from May 2001 to March 2004, 9 out of 12 patients who received modified prophylactic DLI were free of relapse (1 year disease-free survival, 74%) while 8 out of 12 patients who did not receive modified prophylactic DLI relapsed between days 37 and 120 after transplantation (P=0.018) [2]. Schmid et al. also reported the clinical results obtained after pdlt in a subgroup of a refractory AML study population (n=17, from day +120) with a 2-year OS of 87% [27]. All these data indicated that patients with advanced leukemia may benefit from prophylactic DLI after HSCT, although the favorable results may be partly due to the fact that we have selected a more favorable subset of patients (being alive and in remission at day +45, without GVHD, off immunosuppressive therapy, and free of severe infections). In conclusion, this modified DLI approach is safe in terms of GVHD/pancytopenia and can be used for prophylaxis of

J Clin Immunol (2008) 28:390 397 397 leukemia relapse in advanced patients and may represent a step forward in the treatment of advanced leukemia. More cases and more stem cell transplant units are needed to prospectively participate in this clinical setting to confirm the results. Acknowledgment This article is supported in part from Hi-tech Research and Development Program of China, Innovation Team Program of Ministry of Education of China, National Outstanding Young Talent Natural Science Foundation, Leading Program of Clinical Faculty accredited by the Ministry of Health of China. References 1. Huang XJ, Guo NL, Ren HY. An improved anti-leukemic effect achieved with donor progenitor cell infusion for relapse patients after allogeneic bone marrow transplantation. Chin Med J. 2003;116:736 41. 2. Huang XJ, Liu DH, Xu LP. 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