Donor CD3 þ lymphocyte infusion after reduced intensity conditioning allogeneic stem cell transplantation: Single-center experience

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1 Experimental Hematology 2013;41:17 27 Donor CD3 þ lymphocyte infusion after reduced intensity conditioning allogeneic stem cell transplantation: Single-center experience Jean El-Cheikh a,b, Roberto Crocchiolo a,b, Sabine Furst a,b, Patrick Ladaique c, Luca Castagna a,b, Catherine Faucher a,b, Boris Calmels c, Claire Oudin a,b, Claude Lemarie c, Angela Granata a,b, Raynier Devillier a,b, Norbert Vey b, Reda Bouabdallah b, Christian Chabannon c, and Didier Blaise a,b a Unite de Transplantation et de Therapie Cellulaire (U2T), Institut Paoli-Calmettes, Marseille, France; b Departement d Onco-Hematologie, Institut Paoli-Calmettes, Marseille, France; c Centre de Therapie Cellulaire, Institut Paoli-Calmettes, Marseille, France (Received 29 June 2012; revised 3 September 2012; accepted 20 September 2012) Donor lymphocyte infusions (DLI) can induce remission in patients with hematologic malignancies who relapse after allogeneic stem cell transplantation. However, graft-vs-host disease (GVHD) remains a major complication of this strategy. We have used escalating doses of DLI for many years, and wanted to assess the risk factors for GVHD and transplant-related mortality as well as disease outcomes according to the reason for DLI. We analyzed 65 patients who received a total of 111 DLI for different reasons and at different intervals after transplantation. Median number of DLI was 2 (range, 1 4), median interval between transplantation and DLI was 9 months (range, 1 41 months) and median number of infused CD3 + cells/kg recipient body weight was (range, ). Reasons for DLI were relapse or progression in 37 patients (57%), residual disease in 15 patients (23%), and persistence of mixed chimerism in 13 patients (20%). Seven patients (11%) developed acute GVHD grade II to IV and 5 patients (8%) developed extensive chronic GVHD. In univariate analysis, we could identify a transplantation DLI interval #6 months, the dose of DLI ($ ), and DLI number as predictive factors of GVHD. In multivariate analysis, these results were confirmed only for the transplantation DLI interval (hazard ratio [ 19.48; ; p [ 0.007). Our findings indicate that this form of adoptive immunotherapy is well tolerated and induces a low incidence of GVHD and transplant-related mortality, supporting further investigation as an upfront modality to enhance the graft-vstumor response in high-risk patients. Ó 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. Donor lymphocyte infusions (DLI) can induce remission in patients with hematologic malignancies who relapse after allogeneic stem cell transplantation (allo-sct). DLI is the most commonly used adoptive immunotherapy after allo- SCT. However, graft-vs-host disease (GVHD) remains a major complication of this strategy. The initial use of DLI in patients with chronic myeloid leukemia (CML) produced optimism that DLI alone would be a successful therapy for relapsed hematologic malignancies because of the high (þ70%) complete remission (CR) rates and achieving good results with a high incidence of durable cytogenetic and molecular remission [1 3]. Offprint requests to: Jean El-Cheikh, M.D., Unite de Transplantation et de Therapie Cellulaire (U2T), Institut Paoli-Calmettes, 232 Boulevard Ste Marguerite, Marseille Cedex 09, France; elcheikhj@ipc. unicancer.fr Subsequently, DLI were used in other malignant disorders, and for different reasons, such as progression of disease, molecular relapse, or mixed chimerism, and also as prophylactic immunotherapy for patients with a high risk of relapse. Their efficacy under these conditions is much less well documented [4 6]. DLI studies in patients with acute myelogenous leukemia (AML), with the majority receiving AML-specific reinduction chemotherapy before DLI, yield CR rates ranging from 28% to 65% [7,8]. The discrepant results between CML and AML/myelodysplastic syndrome responses are potentially related to the underlying biology and kinetics of disease or the differential responsiveness to a graft-vs-leukemia effect between chronic and acute leukemias [9,10]. In lymphoid malignancies, CR rates after DLI range from 42% to 76%, depending on the underlying disease, but most reports describe less consistent use of pre-dli chemotherapy [11,12]. In X/$ - see front matter. Copyright Ó 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

2 18 J. El-Cheikh et al./ Experimental Hematology 2013;41:17 27 multiple myeloma (MM), the relapse rate after reducedintensity conditioning (RIC) allo-sct remains considerable; DLI was given for disease progression and only a few reports as relapse prophylaxis [13,14]. Recently, several new antimyeloma drugs, such as lenalidomide or thalidomide and bortezomib, were associated with DLI [15 17]. Recently, DLI have been increasingly given empirically in the post-transplantation setting as a pre-emptive strike against relapse or on the assumption that they may eliminate undetectable minimal residual disease (MRD) [14,17,18]. The monitoring of chimerism after RIC allo-sct is standard and is used to assess for graft rejection, relapse, and/or the need and the timing of DLI [19]. There seems to be no doubt that the evaluation of chimerism in the early posttransplantation period (within the first 6 months) is helpful for the prediction of the relapse risk in patients with AML. Some investigators have given DLI pre-emptively (as part of the protocol), whereas others have reserved them for disease progression or persistent mixed chimerism (MC). However, it remains controversial whether a finding of MC alone justifies initiation of therapeutic interventions in the post-transplantation period, considering, for example, the risk of acute or chronic GVHD in case of DLI application or the hematotoxicity of demethylating agents. RIC allo-sct confronts physicians with a number of new clinical situations in which decisions must be made rapidly. Unfortunately, there are no published data defining the optimal cell dose and the timing of these infusions. As a result, it is not possible to advise individual patients accurately about the chance of success. Similarly, data on the probability of severe toxicities, such as graft hypoplasia and severe acute and chronic GVHD, are scarce and may become clear only with large-scale studies [20 22]. We have been using escalating doses of DLI for many years in our center, and wanted to assess the risk factors for GVHD and transplant-related mortality (TRM) as well as disease outcomes according to the reason for DLI in patients who received DLI after RIC allo-sct. Methods We retrospectively analyzed 65 patients who received a total of 111 DLI in our center between 2000 and 2011 for different reasons and at different intervals after transplantation. Median patient age was 48 years (range, years); the patient cohort included 39 males (60%) and 26 females (40%). Patients evaluated in this study were adult patients with multiple myeloma (n 5 22), non- Hodgkin lymphoma (n 5 13), acute myeloid leukemia (n 5 12), chronic myeloid leukemia (n 5 4), Hodgkin lymphoma (n 5 4), chronic lymphocytic leukemia (n 5 3), primary myelofibrosis (n 5 3), myelodysplastic syndrome (n 5 2), and severe aplastic anemia (n 5 2). Fifty-six (86%) and nine patients (14%) had sibling donors and unrelated donors (URD), respectively. The stem cell source was peripheral blood stem cells in 57 patients (88%) and the other 8 patients (12%) received marrow. Fifty-one patients (78%) were treated with an RIC based on fludarabine (30 mg/m 2 /d), busulfan (3.2 mg/kg/d intravenously over 2 to 3 days) and anti-thymoglobulin (2.5 mg/kg/d), while 12 patients (19%) received fludarabine (25 mg/kg/d for 3 days) and 2 Gy total body irradiation. Post-graft immunosuppression consisted of cyclosporine alone in 51 patients (78%) or cyclosporine and mycophenolate mofetil in 14 patients (22%). Transplant characteristics and details are shown in Table 1. Chimerism and residual disease Chimerism was performed in all cases between days 30 and 120 post-transplantation. Chimerism was investigated by quantitative real-time PCR (q-pcr) from peripheral blood or bone marrow for several genetic polymorphisms or Y-chromosome specific sequences in case of sex-mismatched allo-sct. These methods guarantee sensitivities of Full donor chimerism meant a O99.9% proportion of donor alleles. MC was defined as donor and recipient allele proportions being detectable in at least 1% each. Loss of donor chimerism was defined as!1% of donor alleles. MRD assessment on days þ100 after transplantation allowed identification of patients at risk of relapse. MRD monitoring by flow cytometry and/or reverse transcription PCR analysis was performed in all patients with acute and chronic leukemia. Table 1. Patient characteristics (n 5 65) Characteristic Age (y), median (range) 48 (20 67) Sex, n, male/female 39/26 (60/40) Sibling/unrelated donor, n (%) 56/9 (86/14) Disease, n (%) Multiple myeloma 22 (34) Non-Hodgkin lymphoma 13 (20) Acute myeloblastic leukemia 12 (18) Chronic myeloid leukemia 4 (6) Hodgkin lymphoma 4 (6) Primary myelofibrosis 3 (5) Chronic lymphoid leukemia 3 (5) Myelodysplastic syndrome 2 (3) Other 2 (3) Stem cell source, n (%) Marrow 8 (12) PBSC 57 (88) Conditioning regimen, n (%) F þ Bx þ ATG 51 (78) F þ TBI (2 Gy) 12 (18) Other 2 (4) Days of ATG, n (%) 1 18 (28) 2 30 (46) 3 3 (5) Days of busulfan, n (%) 2 22 (34) 3 10 (15) Prophylaxis of GVHD, n (%) CSA 51 (78) CSA þ MMF 14 (22) ATG 5 anti-thymoglobulin; Bx 5 busulfan IV (busilvex); CSA 5 cyclosporine; F 5 fludarabine; MMF 5 mycophenolate mofetil; PBSC 5 peripheral blood stem cells; TBI 5 total body irradiation.

3 J. El-Cheikh et al./ Experimental Hematology 2013;41: Furthermore, patients with lymphoid diseases (MM or non- Hodgkin lymphoma or Hodgkin lymphoma) with full donor chimerism but not achieving CR after the immunosuppression withdrawing of the cyclosporine A were considered in partial remission or residual disease. DLI Before DLI, most patients showed a Karnofsky performance status of $90%. The infusion was always given at the day hospital and without an overnight stay in the hospital. The first dose of DLI was given directly after receiving the fresh donor cells; the remaining cells were cryopreserved in different bags according to the dosage plan indicated in our institutional protocol. Cryopreservation was performed according to local standards. DLI was given to patients without immunosuppressive therapy for at least 1 month, and if the patient was without clinical signs of GVHD after O30 days without immunosuppressive therapy, without active infection and without a history of higher than grade II acute GVHD. Independently of the cause of DLI, the dose was the same in the three groups. The dose escalation was as follows: /kg of body weight (BW) of CD3 þ cells for the first DLI and /kg of BW of CD3 þ cells for the second and third DLI in the case of cells collected from MRD; or /kg of BW of CD3 þ cells for the first DLI and /kg of BW of CD3 þ cells for the second and third DLI in case of cells collected from URD. Weekly examination, in particular for signs of GVHD or infection, was performed for 6 weeks after the last DLI. The cause of DLI was the presence of relapse or progression in 37 patients (57%), residual disease in 15 patients (23%) and presence of MC in 13 patients (20%). Twenty patients (31%) received treatment before DLI, 13 patients (20%) for relapse or progression, and 7 patients (11%) for residual disease. Disease characteristics and details are shown in Tables 2 and 3. GVHD Acute GVHD was graded according to the Glucksberg criteria and chronic GVHD was classified as limited or extensive [23,24]. None of our patients had already experienced acute GVHD after the initial allo-sct, and none had minimal signs of chronic GVHD at the moment of DLI. In the case of at least grade II acute GVHD or extensive chronic GVHD after DLI, initial therapy with 2 mg/kg prednisolone for acute GVHD and 1 mg/kg for chronic GVHD was started immediately to avoid unwanted allogeneic immune reactions. Statistical analysis Factors studied for an association between GVHD and TRM were donor type (MRD/URD), year of DLI (!/O2006), maximum dose of DLI (!/O ), recipient age (#/O50 years), number of DLI (!/O1), transplant DLI interval (!/O6 months), cause of DLI (relapse vs mixed chimerism vs residual disease), disease type, and recipient sex. Uni- and multivariate regression analyses were used for acute and chronic GVHD (logistic) and TRM (Cox). The p value was significant if!0.05. The probabilities of overall survival (OS) and progression-free survival (PFS) were calculated from the day of transplantation with the Kaplan-Meier method and log-rank test for comparisons between groups. The probability of TRM and relapse were estimated using cumulative incidence analysis with competing risk [25,26]. Table 2. DLI characteristics and outcomes Characteristic Treatment before DLI, n (%) 20 (31) Relapse or progression, n (%) 13 (20) MRD, n (%) 7 (11) Cause of DLI, n (%) Relapse or progression 37 (57) MRD 15 (23) Mixed chimerism 13 (20) No. of DLI, median (range) 2 (1 4) No. of DLI (n 5 111), n (%) 65 patients 1 31 (48) 2 23 (35) 3 10 (15) 4 1 (2) Median dose of DLI CD3 þ 10 7 /kg 2.5 ( ) Interval allo-sct and DLI, mos, 10 (1 41) median (range) Interval first and second DLI, mos, 4 (1 22) median (range) Interval second and third DLI, mos, 6 (2 18) median (range) Interval third and fourth DLI, mos, 3 median (range) Follow-up, mos, median (range) 43 (9 132) Acute GVHD, n (%) 7 (11) Grade (II III) 6 (9) Grade (IV) 1 (2) Chronic GVHD, n (%) 5 (8) Limited 0 Extensive 5 (8) Status at last follow-up, n (%) Alive 34 (52) Dead 31 (48) Cause of death, n (%) TRM 3 (5) GVHD 1 Infection 2 Disease 28 (43) Disease status at last follow-up, n (%) CR 21 (32) PR 4 (6) Progression or refractory 9 (14) MRD 5 minimum residual disease; PR 5 partial remission. Results Median number of DLI was 2 (range, 1 4), the median interval between transplant and DLI was 10 months (range, 1 41 months), median number of infused CD3 þ cells 10 6 /kg of recipient body weight was ( ). Details are given in Table 2. Seven patients (11%) developed acute grade II to IV GVHD and five patients (8%) developed extensive chronic GVHD. The median time to acute GVHD manifestation was 46 days (range, 6 74 days) and 37 days (range, 8 76 days) for chronic GVHD after DLI administration. Furthermore, none of our patients presented graft hypoplasia after DLI. At a median follow-up from transplantation interval of 43 months (range, months), 34 patients were still

4 20 J. El-Cheikh et al./ Experimental Hematology 2013;41:17 27 Table 3. Outcomes for 65 patients after DLI No. Sex Age (y) Disease Interval HSCT DLI (mos) No. of DLI Cause of DLI Treatment before DLI agvhd/cgvhd after DLI Grade of GVHD Cause of death Follow-up (mos) Status 1 M 50 NHL 10 2 R/PD Yes No PD 14 Dead 2 M 63 NHL 4 1 R/PD No No PD 7 Dead 3 M 25 NHL 3 1 R/PD Yes Yes agvhd (grade I) PD 4 Dead 4 F 29 HL 5 2 R/PD No Yes cgvhd (ext) PD 23 Dead 5 F 48 MM 35 1 R/PD Yes No 38 Alive 6 M 61 MM 7 2 R/PD Yes No 17 Alive 7 M 47 NHL 3 2 R/PD No No Infection 12 Dead 8 M 55 CML 4 3 R/PD No No 84 Alive 9 M 53 NHL 5 1 R/PD No No PD 7 Dead 10 M 60 MPN 7 2 R/PD No No PD 57 Dead 11 F 60 AML 36 2 R/PD Yes No 39 Alive 12 F 44 NHL 8 2 R/PD No No PD 14 Dead 13 F 58 MM 14 4 R/PD No No PD 93 Dead 14 M 43 CML 1 2 R/PD No No PD 11 Dead 15 M 61 AML 25 2 R/PD No No PD 39 Dead 16 F 27 MM 11 2 R/PD No No 80 Alive 17 F 20 HL 14 2 R/PD No No 51 Alive 18 F 35 CLL 34 2 R/PD Yes No 63 Alive 19 F 30 HL 4 1 R/PD No No PD 11 Dead 20 M 60 MM 7 2 R/PD No No PD 18 Dead 21 F 61 AML 4 1 R/PD No No PD 7 Dead 22 M 62 NHL 9 1 R/PD Yes No 12 Alive 23 F 52 MM 6 3 R/PD No No PD 32 Dead 24 M 38 NHL 9 1 R/PD No No 34 Alive 25 M 46 NHL 3 1 R/PD No No PD 6 Dead 26 M 41 AML 3 1 R/PD No Yes agvhd (grade II) PD 23 Dead 27 F 52 AML 12 1 R/PD No No PD 14 Dead 28 F 56 MM 13 3 R/PD No No PD 77 Dead 29 M 47 MM 40 3 R/PD Yes No 47 Alive 30 M 47 MM 22 1 R/PD Yes No PD 67 Dead 31 F 50 AML 3 1 R/PD No Yes agvhd (grade III) 132 Alive 32 M 63 MM 31 1 R/PD Yes No PD 34 Dead 33 F 52 MM 5 3 R/PD No No PD 88 Dead 34 M 46 MM 2 1 R/PD No No PD 3 Dead 35 F 52 MM 12 3 R/PD Yes No 18 Alive 36 M 37 MM 12 2 R/PD Yes No PD 61 Dead 37 M 43 MM 5 2 R/PD No Yes agvhd (grade III) PD 9 Dead 38 F 47 MDS 16 1 R/PD Yes Yes agvhd (grade II) 27 Alive 39 M 51 CLL 10 2 RD No No 24 Alive 40 M 48 NHL 8 1 RD Yes No 16 Alive 41 M 51 MM 19 3 RD Yes No 22 Alive 42 F 46 MM 9 3 RD Yes No 25 Alive 43 F 45 MM 7 2 RD Yes No PD 20 Dead 44 M 24 NHL 4 2 RD No No 74 Alive 45 F 24 HL 6 1 RD No No 61 Alive 46 M 48 MM 7 2 RD No No 46 Alive 47 F 38 CML 3 2 RD No No PD 38 Dead 48 F 66 MM 5 1 RD No Yes agvhd (grade II) Infection 31 Dead 49 F 39 NHL 5 1 RD No Yes cgvhd (ext) 27 Alive 50 M 59 MM 8 3 RD Yes No 19 Alive 51 F 65 MM 6 3 RD Yes No 43 Alive 52 M 55 NHL 6 2 RD Yes No 15 Alive 53 M 51 AML 9 1 MC No No 15 Alive 54 M 62 AML 10 1 MC No No 13 Alive 55 M 54 CMML 4 1 MC No Yes cgvhd (ext) 82 Alive 56 M 42 CLL 5 1 MC No Yes cgvhd (ext) 40 Alive 57 M 21 AA 3 2 MC No No 122 Alive 58 F 41 AML 6 2 MC No No 131 Alive 59 M 59 MPN 6 1 MC No No 9 Alive (continued)

5 J. El-Cheikh et al./ Experimental Hematology 2013;41: Table 3. (continued ) No. Sex Age (y) Disease Interval HSCT DLI (mos) No. of DLI Cause of DLI Treatment before DLI agvhd/cgvhd after DLI Grade of GVHD Cause of death Follow-up (mos) Status 60 M 60 AML 5 1 MC No Yes agvhd (grade IV) GVHDþ 6 Dead infection 61 F 67 AML 6 1 MC No No PD 9 Dead 62 M 38 CML 5 1 MC No Yes cgvhd (ext) 95 Alive 63 M 48 AML 4 1 MC No Yes agvhd (grade I) 87 Alive 64 M 57 MDS 3 1 MC No Yes agvhd (grade II) 93 Alive 65 M 63 MPN 5 1 MC No No PD 18 Dead agvhd 5 acute graft-vs-host disease; cgvhd 5 chronic graft-vs-host disease; CLL 5 chronic lymphoblastic leukemia; CML 5 chronic myeloid leukemia; CMML 5 chronic myelomonocytic leukemia; ext 5 extensive; F 5 female; HL 5 Hodgkin lymphoma; HSCT 5 hematopoietic stem cell transplantation; M 5 male; MDS 5 myelodysplastic syndrome; MM 5 multiple myeloma; MPN 5 myeloproliferative neoplasms; NHL 5 non-hodgkin lymphoma; MPD 5 progressive disease; MC 5 mixed chimerism; R 5 relapse; RD 5 residual disease. alive (52%). The OS at 3 and 5 years was 61% (48 74%) and 53% (95% CI, 39 67%), respectively; the PFS at 3 and 5 years was 27% (95% CI, 15 39%) and 21% (95% CI, 9 33%), respectively (Figs. 1 and 2). The primary cause of death was relapse of the original disease in 28 patients (43%), whereas 3 patients died of TRM (5%). The TRM at 1 and 2 years was 3% (95% CI, 0 7%) and 5% (95% CI, 0 10%), respectively, whereas the relapse or progression rate at 1 and 2 years was 46% (95% CI, 34 58%) and 58% (95% CI, 46 71%), respectively (Fig. 3). The OS after DLI significantly differed according to the reason for DLI. The OS at 3 and 5 years was 73% (95% CI, 48 98%) for patients receiving DLI for mixed chimerism (MC); 76% (95% CI, %) and 61% (95% CI, 26 96%) for patients receiving DLI for MRD and 50% (95% CI, 34 66%) and 42% (95% CI, 24 60%) for patients with relapse or progression at 3 and 5 years, respectively (p ) (Fig. 4). In the univariate analysis, we could identify a transplant-dli interval of #6 months, the dose of DLI ($1 Figure 1. Overall survival.

6 22 J. El-Cheikh et al./ Experimental Hematology 2013;41:17 27 Figure 2. Progression-free survival ) and DLI number as a predictors factors of GVHD. Age, source of stem cells, donor type (MRD vs URD), and the year of DLI (before or after 2006) were not identified as predictor factors for GVHD or TRM. In the multivariate analysis, these results were confirmed for the transplantation DLI interval only (hazard ratio ; 95% CI, ; p ). Furthermore, according to treatment before DLI, we did not observe any difference in OS in the group receiving DLI after re-induction treatment vs the patients without previous treatment before DLI at 3 (72% vs 56%) and 5 years (72% vs 47%) (p ; Fig. 5). We did not observe any impact of the disease type on the outcomes after DLI. The OS and the PFS at 5 years were not statistically significant in the different hematological entities (AML, n 5 13; myelodysplastic syndrome/mpn, n 5 9; non-hodgkin lymphoma/hodgkin lymphoma, n 5 21; and MM n 5 22) (Fig. 6). Discussion RIC allo-sct has been piloted in a number of centers and a proportion of patients who may not be eligible for conventional intensity allo-sct can become long-term disease-free survivors. However, the published data describe a very heterogeneous group of patients and do not provide a sound evidence base for future investigations. RIC allo-sct has the advantage of being less toxic initially, but also carries the potential disadvantages of being less directly tumoricidal and having a higher chance of resulting in incomplete donor chimerism [2,27 30]. In 2002, a multicentric study in the United Kingdom described the toxicity and efficacy of DLI after RIC allo- SCT in 81 patients. The incidence of acute GVHD and chronic GVHD occurred in 28% and 33% of patients, respectively, similar to previous studies that indicated a substantial overlap between a GVL effect and GVHD [22,31]. In our study, the incidence of acute GVHD and chronic GVHD occurred in 11% and 8% of patients, respectively. Our analysis is supported by its homogenous unicentric population, the relatively long follow-up of 43 months vs 16 months in the British study, and the fact that, in our cohort, none of our patients had GVHD before the first DLI or were on immunosuppressive therapy at the time of DLI vs 14 patients (17%) and 10 patients, respectively, in the British study [22]. The incidence of both acute and chronic GVHD appears to be less than that reported after RIC or conventional allo-sct, but many patients in previous studies did not receive a program of incremental doses of DLI [22,29]. In addition, it is also of interest that the RIC regimen included campath-1h in 77% of patients in the British study and none of them received anti-thymoglobulin, in contrast to our cohort where 78% of patients received

7 J. El-Cheikh et al./ Experimental Hematology 2013;41: Figure 3. Transplant-related mortality and relapse. anti-thymoglobulin and none of them received campath-1h; this could explain the low incidence of acute and chronic GVHD in our study [22,28,32]. However, additional studies are required to determine the impact of, and correlation with, these drugs and GVHD post-dli. A major practical difficulty for clinicians was that there are few data concerning the toxicity of early DLI and certainly no convincing published evidence that giving DLI is effective. In our study, early DLI after an interval #6 months post allo-sct was identified as a predictive factor of acute GVHD in univariate and multivariate analysis. An American phase I study, using full-intensity allografts, examined the relationship between CD3 þ cell dose and toxicity [3]. In our study, the dose of DLI ($ ) and DLI number were identified as a predictive factors of acute GVHD (p and p , respectively). Although, in the multicentric British study, DLI given in the first 6 months after transplantation had a O50% chance of resulting in acute or chronic GVHD, the investigators were unable to demonstrate an association between the maximum CD3 þ cell dose given and the development of GVHD [22]. In contrast, in our study, the cell dose of DLI ($ ) was correlated with acute GVHD (p ). However, this latter finding should be interpreted with caution because of the relatively small number of patients at each dose level. It is harder to be definite about efficacy; DLI after RIC allo- SCT are associated with a significant response rate, but the median response duration of complete responders of only 7 months and relapse after DLI-induced remission is well documented [29,33]. It is encouraging that in the 34 living patients at the last follow-up, 21 (32%) have been in continuous CR, which also might be evidence of the efficacy of DLI. This group included five patients with AML, four patients with MM, four patients with non-hodgkin lymphoma, two patients with CML, two patients with myelodysplastic syndrome, and three patients with other diseases (Table 3). In some studies, the results of DLI are certainly very promising for follicular non-hodgkin lymphoma and CML [6,11,32,34,35]. Our 65 patients comprised eight main disease types with small numbers in each group, and the disease type was not identified as an important factor for GVHD or the response rate. Similar systematic studies need to be performed for each specific disease because the efficacy of DLI can depend on the disease being treated. The cause of DLI was not identified as a predictive factor for GVHD or TRM. However, 15 patients (23%) were given pre-emptive DLI for MC, 9 (60%) of which converted from MC to full donor chimerism. It is also of interest that those nine patients (60%) were long-term survivors and in CR. In our study, OS after DLI significantly differed in patients receiving DLI for MC

8 24 J. El-Cheikh et al./ Experimental Hematology 2013;41:17 27 Figure 4. Overall survival according to DLI cause. or MRD from patients with relapse or progression of disease (p ) (Fig. 4). However, specific appropriate studies are required to determine if it is necessary to give DLI to patients with persistent MC or whether they should be reserved for patients with overt disease. Patients with persistent MC can experience GVHD (and therefore may benefit from a graft-vs-tumor effect), but they may also be at risk of graft Figure 5. Overall survival according to treatment before DLI.

9 J. El-Cheikh et al./ Experimental Hematology 2013;41: Figure 6. Overall survival and PFS according to disease type. rejection, although stable MC is seen in some preclinical models [36 38]. In addition, the role of pre-emptive therapy in patients with MC is considered in those with high risk of relapse, which requires further evaluation. Taking into account the small number of patients, this factor does not allow for any firm conclusions regarding the efficacy of this approach. In our study, the primary cause of death was relapse or progression of the original disease in 43% of patients, whereas death from TRM occurred in only 5% (Fig. 3). Although, we did not observe an impact of the administration of DLI after re-induction treatment in terms of OS at 3 and 5 years in patients with relapse or progression compared with patients receiving DLI without earlier reinduction treatment (p ) (Fig. 5). We did not observe an impact of the disease type on the outcomes after DLI in terms of OS and PFS (Fig. 6). Finally, the incidence of relapse or progression seems to be higher than usually reported after allo-sct, it has to be noticed that our population presents unfavorable characteristics, requiring the use of DLI as a therapeutic option. The limitations of our study include the small sample size, which prevents us from drawing any significant conclusions or about future directions for development of DLI after RIC allo-sct. Conclusions This series of consecutive DLI provides important information on the role of DLI after RIC allo-sct. The risk of GVHD and TRM was relatively low, and we identified only a transplantation DLI interval of!6 months as a significant predictor of acute GVHD. At the preliminary analysis, DLI seemed to show efficacy when administered

10 26 J. El-Cheikh et al./ Experimental Hematology 2013;41:17 27 for MC and residual disease, but not for overt relapse in those patients receiving re-induction treatment. Funding disclosure We would like to thank the Association pour la Recherche sur le Cancer (ARC) (Pole ARECA) for their generous support of our research. Our group is supported by several grants from the French Ministry of Health as part of the Programme Hospitalier de Recherche Clinique (PHRC). Acknowledgments We thank the nursing staff for providing excellent care for our patients and the physicians of the Haematology Department at the Institut Paoli-Calmettes for their important study contributions and dedicated patient care. Author contributions: Jean El-Cheikh conceived and designed the study, collected and analyzed data, performed statistical analysis, provided clinical care, wrote and revised the manuscript; Roberto Crocchiolo, collected and analyzed data, performed statistical analysis, provided clinical care, and revised the manuscript. Sabine Furst, Claire Oudin, Angela Granata, Catherine Faucher, Norbert Vey, and Reda Bouabdallah provided clinical care and commented on the manuscript. Christian Chabannon, Patrick Ladaique, Boris Calmels, and Claude Le Marie are in charge of the cell therapy facility that collected and delivered allogeneic blood cell grafts infused to patients included in this analysis, and commented on the manuscript. Didier Blaise recruited patients, provided clinical care, and commented on the manuscript. Conflict of interest disclosure No financial interest/relationships with financial interest relating to the topic of this article have been declared. References 1. Dazzi F, Szydlo RM, Goldman JM. Donor lymphocyte infusions for relapse of chronic myeloid leukemia after allogeneic stem cell transplant: where we now stand. Exp Hematol. 1999;27: Dazzi F, Szydlo RM, Craddock C, et al. Comparison of single-dose and escalating-dose regimens of donor lymphocyte infusion for relapse after allografting for chronic myeloid leukemia. Blood. 2000;95: Mackinnon S, Papadopoulos EB, Carabasi MH, et al. Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus-host disease. Blood. 1995;86: Hongeng S, Pakakasama S, Benjaponpitak S, Kamchaisatian W, Chaisiripoomkere W, Direkwatanachai C. Donor lymphocyte infusion can eliminate mixed chimerism in nonmyeloablative stem cell transplantation for correction of hyper-igm syndrome. Acta Haematol. 2005;114: Lokhorst HM, Schattenberg A, Cornelissen JJ, et al. Donor lymphocyte infusions for relapsed multiple myeloma after allogeneic stemcell transplantation: predictive factors for response and long-term outcome. J Clin Oncol. 2000;18: Porter DL, Collins RH Jr, Hardy C, et al. Treatment of relapsed leukemia after unrelated donor marrow transplantation with unrelated donor leukocyte infusions. Blood. 2000;95: Levine JE, Braun T, Penza SL, et al. Prospective trial of chemotherapy and donor leukocyte infusions for relapse of advanced myeloid malignancies after allogeneic stem-cell transplantation. J Clin Oncol. 2002; 20: Schmid C, Labopin M, Nagler A, et al. Donor lymphocyte infusion in the treatment of first hematological relapse after allogeneic stem-cell transplantation in adults with acute myeloid leukemia: a retrospective risk factors analysis and comparison with other strategies by the EBMT Acute Leukemia Working Party. J Clin Oncol. 2007;25: Kurosawa S, Fukuda T, Tajima K, et al. Outcome of 93 patients with relapse or progression following allogeneic hematopoietic cell transplantation. Am J Hematol. 2009;84: Campregher PV, Gooley T, Scott BL, et al. Results of donor lymphocyte infusions for relapsed myelodysplastic syndrome after hematopoietic cell transplantation. Bone Marrow Transplant. 2007;40: De Rosa G, Pezzullo L, Scarpato N, Selleri C, Lucania A, Rotoli B. Donor lymphocyte infusion for post-transplant relapse of Hodgkin s lymphoma. Haematologica. 2000;85: Ito T, Makishima H, Nakazawa H, Senoo Y, Senoo N, Ishida F. Donor lymphocyte infusion for extranodal NK/T cell lymphoma, nasal type, relapsed after allogeneic hematopoietic SCT. Bone Marrow Transplant. 2011;46: Ayuk F, Shimoni A, Nagler A, et al. Efficacy and toxicity of low-dose escalating donor lymphocyte infusion given after reduced intensity conditioning allograft for multiple myeloma. Leukemia. 2004;18: Bellucci R, Alyea EP, Weller E, et al. Immunologic effects of prophylactic donor lymphocyte infusion after allogeneic marrow transplantation for multiple myeloma. Blood. 2002;99: El-Cheikh J, Crocchiolo R, Furst S, et al. Lenalidomide plus donorlymphocytes infusion after allogeneic stem-cell transplantation with reduced-intensity conditioning in patients with high-risk multiple myeloma. Exp Hematol. 2012;40: Kroger N, Shimoni A, Zagrivnaja M, et al. Low-dose thalidomide and donor lymphocyte infusion as adoptive immunotherapy after allogeneic stem cell transplantation in patients with multiple myeloma. Blood. 2004;104: Kroger N, Badbaran A, Lioznov M, et al. Post-transplant immunotherapy with donor-lymphocyte infusion and novel agents to upgrade partial into complete and molecular remission in allografted patients with multiple myeloma. Exp Hematol. 2009;37: Huang XJ, Wang Y, Liu DH, et al. 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. J Clin Immunol. 2008;28: El-Cheikh J, Vazquez A, Crocchiolo R, et al. Acute GVHD is a strong predictor of full donor CD3þ T cell chimerism after reduced intensity conditioning allogeneic stem cell transplantation. Am J Hematol Aug 2; [Epub ahead of print]. 20. Garicochea B, van Rhee F, Spencer A, et al. Aplasia after donor lymphocyte infusion (DLI) for CML in relapse after sex-mismatched BMT: recovery of donor-type haemopoiesis predicted by nonisotopic in situ hybridization (ISH). Br J Haematol. 1994;88: Kolb HJ, Schattenberg A, Goldman JM, et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood. 1995;86: Marks DI, Lush R, Cavenagh J, et al. The toxicity and efficacy of donor lymphocyte infusions given after reduced-intensity conditioning allogeneic stem cell transplantation. Blood. 2002;100:

11 J. El-Cheikh et al./ Experimental Hematology 2013;41: Glucksberg H, Storb R, Fefer A, et al. Clinical manifestations of graftversus-host disease in human recipients of marrow from HL-Amatched sibling donors. Transplantation. 1974;18: Shulman HM, Sullivan KM, Weiden PL, et al. Chronic graft-versushost syndrome in man. A long-term clinicopathologic study of 20 Seattle patients. Am J Med. 1980;69: Gray RJ. A class of K-sample tests for comparing the cumulative incidence of a competing risk. Ann Stat. 1988;16: Kaplan EL, Meier P. Non-parametric estimation from incomplete observations. J Am Stat Assoc. 1958;53: Bacigalupo A, Ballen K, Rizzo D, et al. Defining the intensity of conditioning regimens: working definitions. Biol Blood Marrow Transplant. 2009;15: Blaise D, Farnault L, Faucher C, et al. Reduced-intensity conditioning with Fludarabin, oral Busulfan, and thymoglobulin allows long-term disease control and low transplant-related mortality in patients with hematological malignancies. Exp Hematol. 2010;38: Collins RH Jr, Shpilberg O, Drobyski WR, et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol. 1997;15: Mohty M, De Lavallade H, El-Cheikh J, et al. Reduced intensity conditioning allogeneic stem cell transplantation for patients with acute myeloid leukemia: long term results of a donor versus no donor comparison. Leukemia. 2009;23: Mattsson J, Uzunel M, Brune M, et al. Mixed chimaerism is common at the time of acute graft-versus-host disease and disease response in patients receiving non-myeloablative conditioning and allogeneic stem cell transplantation. Br J Haematol. 2001;115: Lush RJ, Haynes AP, Byrne J, et al. Allogeneic stem-cell transplantation for lymphoproliferative disorders using BEAM-CAMPATH (þ/ fludarabine) conditioning combined with post-transplant donorlymphocyte infusion. Cytotherapy. 2001;3: Dazzi F, Szydlo RM, Cross NC, et al. Durability of responses following donor lymphocyte infusions for patients who relapse after allogeneic stem cell transplantation for chronic myeloid leukemia. Blood. 2000;96: Orsini E, Alyea EP, Chillemi A, et al. Conversion to full donor chimerism following donor lymphocyte infusion is associated with disease response in patients with multiple myeloma. Biol Blood Marrow Transplant. 2000;6: Salama M, Nevill T, Marcellus D, et al. Donor leukocyte infusions for multiple myeloma. Bone Marrow Transplant. 2000;26: Mapara MY, Kim YM, Wang SP, Bronson R, Sachs DH, Sykes M. Donor lymphocyte infusions mediate superior graft-versus-leukemia effects in mixed compared to fully allogeneic chimeras: a critical role for host antigen-presenting cells. Blood. 2002;100: Mapara MY, Kim YM, Marx J, Sykes M. Donor lymphocyte infusionmediated graft-versus-leukemia effects in mixed chimeras established with a nonmyeloablative conditioning regimen: extinction of graftversus-leukemia effects after conversion to full donor chimerism. Transplantation. 2003;76: McSweeney PA, Storb R. Mixed chimerism: preclinical studies and clinical applications. Biol Blood Marrow Transplant. 1999;5: