CMV Serostatus Research Articles

Imprint Of Early CMV Reactivation On The Reconstituting T-Lymphocyte Compartment One and Two Year After Hematopoietic Stem Cell Transplantation

BackgroundCytomegalovirus (CMV) reactivation frequently occurs during early immune reconstitution after hematopoietic stem cell transplantation (HSCT). Although CMV is associated with accelerated immune ageing in healthy individuals, the impact of early CMV reactivation on T-cell immunity long term post HSCT is unknown. In this study, we report the impact of early CMV reactivations on the reconstitution and composition of the T-lymphocyte compartment one to two year after HSCT in a large cohort of pediatric HSCT recipients. MethodsWe analyzed the lymphocyte compartment one and two year after transplantation in 131 consecutive (2002 - 2011) pediatric HSCT recipients that were eligible for follow up one year post HSCT. Viral infections were routinely monitored by weekly serum viral DNA PCR in the first 100 days. Peripheral blood mononuclear cells were routinely analyzed by multicolor flow cytometry. Six patients with early CMV reactivation and an HLA type for which CMV-tetramers were available were analyzed for the presence and phenotype of CMV-specific CD8+ T-lymphocytes. ResultsOne year post HSCT, patients with early CMV reactivation (n = 46, PCR ≥ 1x ≥ 200 copies / mL) had significantly higher lymphocyte counts compared to patients without CMV reactivation (n = 85, PCR always < 200 copies / mL). This could be attributed to a significant increase of CD3+ T-lymphocytes while NK- and B-cell numbers did not differ. Within the T-cell compartment, a three-fold expansion of CD8+ T-cells (median 1323 vs. 424 cells / μL, p <0.0001, Mann-Whitney) and an increase in gamma-delta T-cells (median 104 vs. 47 cells / μL, p = 0.0005) was observed, while absolute numbers of CD4+ T-cells did not differ between the groups. This effect was not observed in relation to Epstein-Barr virus (EBV) or Adenovirus (HAdV) reactivation. In multivariate analysis, CD8+ T-cell numbers one year post HSCT were highly influenced by CMV reactivation (p <0.0001, linear regression) but not affected by pre-transplant CMV serostatus of donor (p = 0.22) or recipient (p = 0.14).In a representative subcohort (n = 53, 2008 - 2010), we more closely analyzed the differentiation stages of T-cells based on expression of CD45RA and CCR7. In both the CD4+ and CD8+T-cell subset, the proportion of Effector Memory (EM) and EMRA T-cells was enlarged in patients with (n = 18) compared to patients without (n = 35) early CMV reactivation. In the CD8+ T-cell compartment, this was caused by a major expansion of CD8+ EM and EMRA T-cells (median 485 vs. 141, p <0.0001 and 509 vs. 114 cells / μL, p <0.0001, Figure A), while the Naive (median 105 vs. 169 cells / μL, p = 0.17) and Central Memory (CM) compartment did not differ. In the CD4+ compartment, a non-significant increase of EM and EMRA cells was accompanied by a non-significant decrease of N and CM cells. CMV-specific CD8+ T-cells (median 4.1, range 0.3 - 25.6% of CD8+ T-cells) were detected within the EM and EMRA compartment.Two year after HSCT, data were available for 76 patients. Both in patients with (lymphocyte subsets: n = 34 / T-cell differentiation: n = 12) and without (n = 42 / n = 19) early CMV reactivation, a further reconstitution of CD4+ and CD8+ T-cells was observed, reflected by an expansion of Naive and CM cells (Figure B). However, the total number of CD8+ T-cells decreased in patients with early CMV reactivation, caused by a contraction of late differentiated CD8+ EM and EMRA cells (median 825 to 618, p = 0.0068 and 555 to 378 cells / μL, p = 0.0342, Wilcoxon). ConclusionEarly CMV reactivation leaves a virus-specific and dynamic imprint on the reconstituting immune system 1 and 2 year after HSCT. The marked expansion of CD8 + EM and EMRA T-cells was not seen in patients with early EBV or HAdV reactivation and did not compromise the reconstitution of the Naive and CM compartment available to respond to neo- and recall antigens. Pediatric HSCT recipients differed from solid organ transplantation recipients in which CMV has been correlated to an accelerated and ongoing accumulation of late differentiated T-cells. The dynamic contraction of the CD8+ late differentiated memory T-cell compartment in the second year after HSCT implies that an ongoing process of immune-regulation and further reconstitution is modeling the cellular immune system after discontinuation of immunosuppressive medication.▪ Disclosures:No relevant conflicts of interest to declare.

Human herpesvirus-6 DNAemia is a sign of impending primary CMV infection in CMV sero-discordant renal transplantations

BackgroundHuman herpesvirus-6 (HHV-6) frequently reactivates in immunocompromized individuals. Most commonly HHV-6 DNA is detected without organ localized disease. This HHV-6 DNAemia usually occurs in patients who also have CMV reactivations. The question if reactivation of one virus causes reactivation of the other, or whether both viruses reactivate independently, cannot be answered in populations with high seroprevalence for both viruses. Our study is the first in which 35 patients have been included who were CMV seronegative prior to transplantation. ObjectiveWe investigated the occurrence of HHV-6 reactivations in relation to the CMV-serostatus of renal transplantation donor and recipient. Study design9 consecutive patients receiving a renal transplantation were included. All available stored whole blood samples were tested for HHV-6 DNA by quantitative PCR. Details including CMV serostatus of donor and recipient were recorded. ResultsCMV-seropositive recipients have a 68% chance of developing HHV-6 DNAemia if the kidney came from a CMV-seropositive donor, compared to 26% if the kidney came from a CMV-seronegative donor. CMV-seronegative recipients, who are bound to undergo primary CMV infections following transplantation with a renal graft from a CMV-seropositive donor, have 88% chance of developing HHV-6 DNAemia. If they receive a graft from a CMV-seronegative donor the chance of developing HHV-6 DNAemia is 22%. ConclusionReceiving a renal transplant from a CMV-seropositive donor increases the chance of developing HHV-6 DNAemia. HHV-6 DNAemia is a sign of impending primary CMV infections following sero-discordant renal transplantations.

Cytomegalovirus prophylaxis in pediatric kidney transplantation: The Dutch experience

Many children receiving a kidney transplant are seronegative for CMV and therefore, highly susceptible to a primary CMV infection. This study aims at evaluating incidence, time of occurrence, and severity of CMV infection in the first year post-transplantation in relation to different types of CMV prophylaxis. Transplantations in three centers in the Netherlands between 1999 and 2010 were included. Retrospective, observational, multicenter study. Clinical data and PCR measurements of CMV were collected. Prophylaxis in high-risk patients (CMV serostatus D+R-) consisted of (val)ganciclovir during threemonths, or acyclovir plus CMV immunoglobulin at a former stage. Intermediate-risk patients (R+) received (val)acyclovir, or acyclovir plus CMV immunoglobulin at a former stage. Low-risk patients (D-R-) did not receive prophylaxis. Infection was defined as CMV PCR above 50geq/mL plasma or whole blood, a clinically relevant infection above 1000geq/mL. One hundred and fifty-nine transplantations were included. CMV infection was documented for 41% of high-risk, 24% of intermediate-risk, and 13% of low-risk patients, in the latter two groups typically during the first threemonths. The infection rate was highest in the high-risk group after cessation of valganciclovir prophylaxis. Valganciclovir provided better protection than did acyclovir+CMV immunoglobulin. Adding an IL2-receptor blocker to the immunosuppressive regimen did not affect the infection rate. Acute graft rejection was not related with CMV infection. Valganciclovir prophylaxis effectively prevents CMV infection in high-risk pediatric kidney recipients, but only during prophylaxis. Valacyclovir prophylaxis in intermediate-risk patients is less effective.

NKG2C+CD57+ CD56dim NK cells are enriched following Epstein-Barr virus transformed lymphoblastoid cell line co-culture with peripheral blood mononuclear cells from cytomegalovirus+ donors (P4359)

Abstract Cytomegalovirus (CMV) is a persistent herpesvirus that promotes NKG2C+ Natural Killer (NK) cell differentiation. Highly mature CD57+ NKG2C+ NK cells have been proposed as the equivalent of mouse memory-like NK cells, which have features as long-term persistence and enhanced recall responses. AIM: To examine if co-infection with another common herpesvirus, Epstein-Barr virus (EBV), affects the NKG2C+ CD56dim NK cell population. METHODS: PBMCs from 45 five-year old children with known EBV and CMV serostatus were phenotyped. Further, PBMCs from 6 CMV sero+ EBV sero- infants were used for co-culture with EBV+ lymphoblastoid cell-lines (LCLs) during 72 hours. EBV- LCLs (Ramos) served as controls. Anti CD3, CD14, CD19, CD56, CD57, NKG2C and ki-67 antibodies were used for flow cytometric analysis. RESULTS: EBV and CMV co-infection was related to higher proportions of NKG2C+ and NKG2C+CD57+ CD56dim NK cells compared to infection with CMV or EBV alone, or seronegativity. In contrast to co-culture with Ramos, PBMC co-culture with EBV+ LCLs generally led to enrichment of NKG2C+CD57+ NK cells (stimulation index median 1.23, range 0.87-1.30), which was not related with higher NK cell proliferation. CONCLUSIONS: EBV co-infection seems to promote the existence of higher proportions of memory-like NK cells. Our data herein indicate that this is not due to enhanced proliferation of these cells; rather it suggests that memory-like NK cells may be maintained by EBV co-infection.

IMPACT OF PRETRANSPLANT DONOR AND RECIPIENT CYTOMEGALOVIRUS SEROSTATUS ON OUTCOME FOR MULTIPLE MYELOMA PATIENTS UNDERGOING REDUCED INTENSITY CONDITIONING ALLOGENEIC STEM CELL TRANSPLANTATION.

Scope of the study was to investigate the impact of pre-transplant CMV serostatus of the donor and/or recipient on the outcome of patients undergoing allogeneic hematopoietic stem cell transplantation (Allo-SCT) for Multiple Myeloma (MM). To our knowledge no data are available in the literature about this issue.We retrospectively followed 99 consecutive patients who underwent reduced-intensity conditioning (RIC) Allo-SCT for MM in our cancer center at Marseille between January 2000 and January 2012. Based upon CMV serostatus, patients were classified as low risk (donor [D]−/recipient [R] −) 17 patients (17.1%), intermediate risk (D+/R) 14 patients (14.1%), or high risk – either (D−/R+) 31 patients (31.3%) or (D+/R+), 37 patients (37.3%).Cumulative incidence of CMV reactivation was 39% with a median time of 61 days (26–318). Three patients (3%) developed CMV disease. Two factors were associated with CMV reactivation: CMV serostatus group (low: 0% vs. intermediate: 29% vs. high: 50%; p=0.001) and the presence of grade II–IV acute GvHD (Hazard Ratio: HR=2.1 [1.1–3.9]). Thirty-six of the 39 patients (92%) with CMV reactivation did not present positive detection of CMV after a 21-day median duration preemptive treatment with ganciclovir. Cumulative incidence of day 100 grade II–IV acute GvHD, 1-year chronic GvHD and day 100 transplantation related mortality (TRM) were 37%, 36% and 9%, respectively. CMV reactivation and serostatus were not associated with increased GvHD and TRM or short survival. Only the presence of acute GvHD as a time dependent variable was significantly associated with increased TRM (p=0.005). Two-year overall and progression free survival were 56% and 34%, respectively.Donor and recipient CMV serostatus and acute GvHD are independent factors for increased CMV reactivation in high-risk MM patients undergoing RIC Allo-SCT. However, we did not find any influence of CMV reactivation on post transplantation outcome. CMV monitoring and pre-emptive treatment strategy could in part explain these results. Novel prophylactic measures such as immunotherapy and drug prophylaxis need to be considered in this group of patients, warranting further prospective studies.

Large granular lymphocytosis and its impact on long-term clinical outcomes following allo-SCT

A total of 418 patients receiving hematopoietic SCT and surviving beyond day 100 were examined for the occurrence of large granular lymphocytes (LGLs). LGL lymphocytosis was defined as the presence of at least two of the following criteria: (1) sustained lymphocytosis above 3.0 × 10(9)/L observed in at least three consecutive determinations over a time frame of 2-3 months, (2) predominance (>30%) of LGLs in peripheral blood, (3) confirmation of monoclonality by T-cell receptor analysis using PCR 77 patients developed LGL lymphocytosis during their post-transplant course with a median onset of 312 days from transplant. The cumulative incidence at 1-, 2- and 3-years was 12.3±1.8, 20.8±2.4 and 23.6±2.7%. Patients with LGL lymphocytosis showed an OS advantage (86.2 vs 53.8%, P<0.001), lower non-relapse mortality (NRM; 3.2 vs 27.3%, P<0.001) and lower relapse incidence (9.6 vs 29.4%, P<0.001). Three clinical factors were associated with the development of LGL lymphocytosis: (1) CMV seropositive recipients (CMV-R(+)) compared with CMV seronegative recipients (CMV-R(-); P<0.001) regardless of CMV serostatus of donor; (2) CMV reactivation (P<0.001); (3) chronic GVHD (P=0.007). In conclusion, the incidence of LGL lymphocytosis following allogeneic hematopoietic SCT was detected in ~20% of recipients and is associated with favorable outcomes.

Outcomes After Double Cord Blood Transplantation Compared to Single Cord Blood Transplantation in Adults with Acute Leukemia Given a Reduced Intensity Conditioning Regimen

Abstract Abstract 232 Unrelated cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cell transplantation (HSCT) for adults with acute leukemia lacking a HLA matched donor. Double cord blood unit (dUCBT) has been increasingly used over single CB unit (sUCBT) after reduced intensity conditioning regimen (RIC). Since there is an increased relapse incidence (RI) using RIC-HSCT compared to myeloablative conditioning regimen, we have driven the hypothesis that RI may be decreased and leukemia-free survival (LFS) rate increased after dUCBT compared to sUCBT, due to probably increased graft-versus leukemia (GVL) effect. With this aim, we analyzed 360 adults (>18 years) with ALL (n= 77) or AML (n=238) in CR1 (n=212) and in CR2 (n=148) transplanted with a sUCBT (n=131) or a dUCBT (n=229) after a RIC. Only patients transplanted with a single unit containing more than 2.5×107/kg total nucleated cells (TNC) were included. Patients were transplanted from 2005–2011 in EBMT centers. Comparing the two groups of patients receiving a sUCBT or a dUCBT in CR1, there were no statistical differences according to age, diagnosis (AML or ALL), weight, CMV serostatus, cytogenetics risk, number of HLA incompatibilities. However, dUCBT were performed more recently (2009 vs 2008), the time from CR1 to transplantation was longer (142 days vs 121 days), more frequently transplanted with CY+FLU+TBI2Gy (87% vs 68%), lower frequency of ATG use (21% vs 35%) and finally, dUCBT recipients received higher number of TNC collected (5×107/kg vs 3.9×107/kg) or infused (4×107/kg vs 3.1×107/kg). Median follow-up was 23 months in both groups. Cumulative incidence (CI) of 60 days neutrophil recovery was 82±3% after dUCBT and 76±2% after sUCBT (p=0.86) and frequency of full donor chimerism at day 100, was not statistically different between dUCBT (81%) and sUCBT (86%). At day 100, CI of acute GVHD (grade II-IV) was 35% in both groups, however there was a trend of increased incidence of grade III-IV after sUCBT (19%) compared to dUCBT (10%, p=0.06) but increased incidence of grade II aGVHD after dUCBT (28%) compared to 17% after sUCBT (p=0.05). CI of chronic GvHD at 2 years was 21±4% after dUCBT and it was 12±5% after sUCBT (p=0.15). At 2 years, CI of non relapse mortality (NRM) after dUCBT was 28±4% and it was 30±6% after sUCBT (p=0.87). However, CI of 2y relapse was 21±4% after dUCBT whereas it was 38±2% after sUCBT (p=0.03). In a multivariate analysis adjusting for the differences between the 2 groups, dUCBT was associated with lower incidence of relapse compared to sUCBT (HR=0.74, p=0.01). Therefore, there was an improved 2-y LFS after dUCBT (51±5%) compared to sUCBT (32±3%; p=0.03). This was confirmed in a multivariate analysis (HR=0.64, p=0.04). Concerning patients transplanted in CR2 (n=148), there were no statistically differences of outcomes after dUCBT (n=93) or sUCBT (n=55). At 2y, LFS was 40±6% after dUCBT and 48±3% after sUCBT (p=0.32). In a subgroup analysis of dUCBT (n=118) and sUCBT (n=51) recipients using the same conditioning regimen (CY+FLU+TBI2Gy), 2 y LFS were 54±5% and 33±7% respectively (p=0.05). Conclusion: In this retrospective comparative based registry analysis, in AL patients transplanted in CR1, neutrophil recovery, GVHD and NRM were not statistically different after RIC-dUCBT or RIC-sUCBT, however, dUCBT recipients had decreased relapse incidence and improved LFS. For AL patients transplanted in CR2, there was no benefit of using dUCBT when compared to sUCBT recipients. Disclosures: No relevant conflicts of interest to declare.

16th IHIW: Immunogenetics of Aging

Ageing is a process characterised by progressive loss of function in multiple different organ systems, such as the nervous, endocrine and immune systems. Current data showing that ageing processes may be associated with alterations in the immune system suggest that some of the genetic determinants of senescence might be polymorphic genes that regulate immune responses. The 'Immunogenetics of Aging' programme was a component introduced in the 14th International HLA and Immunogenetics Workshop (IHIWS) and developed further within the 15th and 16th. The aim of this component was to determine the contribution of immune genes to successful ageing and an increased capacity to reach the extreme limits of lifespan. Within the 16th IHIWS, new populations were included, and the number of samples analysed was increased. Analysis was focused on innate immunity genes (KIR and MBL2) and their correlation with CMV serostatus. Collaborative studies suggested that both activating and inhibitory KIR and functionally relevant MBL2 haplotypes are important factors for control of CMV infection in the elderly and therefore for chronic low-grade inflammation. Results showed that these genes might be predictive biomarkers in ageing and longevity. Prevalence of MBL2 haplotypes determining absence of the protein (LYPB, LYQC and HYPD) was observed in elderly people with a higher CMV antibody titre. The high CMV titre was also associated with a decreased frequency of the activatory KIR2DS5 and A1B10 haplotypes in elderly. Due to the role of KIR and low or deficient MBL haplotypes in viral infections, these genetic markers could be considered as indicators of a need for CMV prophylaxis at younger age and therefore increased probability of longer lifespan.

CMV Serostatus of Donor-Recipient Pairs Influences the Risk of CMV Infection/Reactivation in HSCT Patients

CMV donor/recipient serostatus was analyzed in 200 patients allografted in our institution from unrelated (122 patients) donors and 78 sibling donors in the years 2002–2011 in relation to posttransplant complications. On a group basis independently of the CMV serostatus of donor-recipient pairs sibling transplantations and those from unrelated donors that matched 10/10 at allele level had a similar rate of CMV reactivation (17/78 versus 19/71, P = ns). The rate of CMV reactivation/infection was higher in patients grafted from donors accepted at the lower level of matching than 10/10 (18/38 versus 36/149, P = 0.008). The incidence of aGvHD followed frequencies of CMV reactivation in the tested groups, being 40/156 and 25/44 in patients grafted from sibling or unrelated donors that 10/10 matched and in those grafted from donors taht HLA mismatched, respectively (P = 0.001). Regarding the rate of reactivation in both groups seropositive patients receiving a transplant from seronegative donors had more frequently CMV reactivation as compared to those with another donor-recipient matching CMV serostatus constellation (22/43 versus 32/143, P = 0 < 0.001). Multivariate analysis revealed that seropositivity of recipients with concomitant seronegativity of donors plays an independent role in the CMV reactivation/infection (OR = 2.669, P = 0.037; OR = 5.322, P = 0.078; OR = 23.034, P = 0.023 for optimally matched and mismatched patients and the whole group of patients, resp.).

Impact of Pretransplant Donor and Recipient Cytomegalovirus Serostatus On Outcome for Multiple Myeloma Patients Undergoing Reduced Intensity Conditioning Allogeneic Stem Cell Transplantation

AbstractAbstract 4496 Purpose:To investigate the impact of pre-transplant CMV serostatus of donor or recipient on outcome of patients undergoing allogeneic hematopoietic stem cell transplantation (Allo-SCT) for Multiple Myeloma (MM). Patients and methods:We retrospectively followed 99 consecutive patients who underwent reduced-intensity conditioning (RIC) Allo-SCT for MM in our cancer centre at Marseille between January 2000 and January 2012. Based upon CMV serostatus, patients were classified as low risk (donor [D]-/recipient [R]-) 17 patients (17.1%), intermediate risk (D+/R) 14 patients (14.1%), or high risk – either (D-/R+) 31 patients (31.3%) or (D+/R+), 37 patients (37.3%). Results:Cumulative incidence of CMV reactivation was 39% with a median time of 61 days (26–318). Three patients (3%) developed CMV disease. Two factors were associated with CMV reactivation: CMV serostatus group (low: 0% vs intermediate: 29% vs high: 50%; p=0.001) and the presence of grade II–IV acute GvHD (Hazard Ratio: HR=2.1 [1.1–3.9]). Thirty-six of the 39 patients (92%) with CMV reactivation did not present positive detection of CMV after a 21-day median duration preemptive treatment with ganciclovir. Cumulative incidence of day 100 grade II–IV acute GvHD, 1-year chronic GvHD and day 100 transplantation related mortality (TRM) were 37%, 36% and 9%, respectively. CMV reactivation and serostatus were not associated with increased GvHD and TRM or short survival. Only the presence of acute GvHD as a time dependent variable was significantly associated with increased TRM (p=0.005). Two-year overall and progression free survival were 56% and 34%, respectively. Conclusion:Donor and recipient CMV serostatus and acute GvHD are independent factors for increased CMV reactivation in high-risk MM patients undergoing RIC Allo-SCT. However, we did not find any influence of CMV reactivation on post transplantation outcome. CMV monitoring and pre-emptive treatment strategy could in part explain these results. Novel prophylactic measures such as immunotherapy and drug prophylaxis need to be considered in this specific group of patients, warranting further prospective studies.Table 1Patient and transplantation characteristicsPatient and transplantation characteristicsn = 99 (%)Patients Age (median) [range]53 years [27–67]Male gender59 (60)Myeloma-subtypeIgG52 (53)IgA23 (23)Light Chain12 (12)Bence jones8 (8)Other4 (4)Cytogenetics at diagnosisNormal14 (14)Del(13) or Del (17) or t(4;14)24 (24)NA61 (62)Median number of prior chemotherapies before Allo-SCT [range]2 [1–5]1 line27 (27)2 lines46 (47)3 lines15 (15)>3 lines11 (11)Median number of prior Auto-SCT [range]2 [1–4]157 (58)231 (31)> 28 (8)Status of Myeloma at Allo-SCTCR12 (12)VGPR10 (10)PR/SD66 (67)PD11 (11)Median interval between Auto- and Allo-SCT months [range]19 [1–89]Donor typeMRD73 (74)URD26 (26)Donor/recipient sex mismatch48 (48)ABO compatibilityYes63 (64)No36 (36)Donor/recipient CMV serostatusD−/R− (low risk)17 (17)D+/R− (intermediate risk)14 (14)D+/R+ (high risk)31 (31)D−/R+ (high risk)37 (37)Donor Median age years (range)46 (20–71)Conditioning regimenFlu + Bu + ATG68 (69)Flu + TBI25 (25)Other RIC6 (6)GvHD prophylaxisCSA56 (57)CSA+MMF41 (41)MMF2 (2)Stem cell sourcePeripheral Blood88 (89)Bone Marrow9 (9)Cord blood2 (2)Stem cell dose median [range]CD34+ × 106/kg5.41 [0.16–12.8]CD3+ × 106/kg299 [5–745] Legend:CR, complete remission; VGPR, very good partial remission; PR, partial remission, PD, progressive disease; Flu, fludarabine; Bu, busulfan; ATG, antithymocyte globulin; CR, complete remission; VGPR, very good partial remission; PR, partial remission, PD, progressive disease; MRD, matched related donor; URD, unrelated donor; GVHD, graft versus host disease; CSA, cyclosporine A; MMF, mycophenolate mofetyl.Table 2CMV reactivationCumulative incidence of CMV reactivationpAll patients (n=99)39%CMV serostatusLow risk (n=17)0%0.001Intermediate risk (n=14)29%High risk (n=68)50%Donor type*MRD (n=72)38%0.427URD (n=24)46%Graft source*PBSC (n=88)38%0.215Bone marrow (n=8)63%Transplantation period<2006 (n=42)33%0.368>=2006 (n=57)43%Conditioning regimen with ATGYes (n=68)43%0.164No (n=31)29%CMV detection methodpp65 (n=65)38%0.819PCR (n=34)39%Acute GVHD$HR=2.1 [1.1–3.9]0.032Chronic GVHD$HR=0.8 [0.1–8.6]0.837$The occurrence of GVHD was analysed as a time dependent variable Disclosures:No relevant conflicts of interest to declare.

Early Reconstitution of T-Cell Immunity to CMV After HLA-Haploidentical Hematopoietic Stem Cell Transplantation Is a Strong Surrogate Biomarker for Lower Non-Relapse Mortality Rates

Abstract 4191 Introduction:HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a readily available therapeutic option for patients suffering from high-risk hematological malignancies who lack an HLA-compatible donor or for whom there is no time to find one. To avoid GVHD, haplo-HSCT has been classically performed under conditions of strict T-cell depletion, in the form of CD34-selected (“naked”) grafts. Unfortunately, naked haplo-HSCT is followed by a profound state of immune incompetence, which associates with high non-relapse mortality (NRM) rates, mainly due to opportunistic infections. Over the last decade, different strategies have been developed in order to speed-up immune reconstitution after haplo-HSCT, including the delayed infusion of donor T cells (DLI), the selective depletion of T-cell subsets from the graft or the use of an unmanipulated graft followed by novel strategies of immune suppression. Aim:To analyze in depth the early phenomena of immune reconstitution following haplo-HSCT in order to find early surrogate biomarkers of NRM. Results:From 2004 to 2010, we prospectively studied multiple parameters of T-cell immune reconstitution in 89 pts treated at our Center with haplo-HSCT. Time-points of analysis were pre-transplant, at day 30, day 90 and day 180 post-transplant. Underlying diseases included: high-risk AML 53 pts (60%), ALL 11 pts (12%), CLL 2 pts (2%), tyrosine kinase inhibitors-resistant CML 4 pts (5%), Hodgkin lymphoma 6 pts (7%), NHL 2 pts (2%), MDS with high International Prognostic Scoring System 7 pts (7%), other 4 pts (4%). Eighteen patients (20%) were given prophylactic suicide gene-modified DLI starting at day 30 post-transplant, while 64 patients (80%) received an unmanipulated graft followed by rapamycin until day 100. Overall, the incidence of grade III-IV acute GvHD was as low as 12%. Chronic extensive GvHD developed in 28% of pts. Compared with historical naked haplo-HSCT, the recovery of T-cell counts was accelerated: at day 90, median CD3+ cells were 378 per μL (0–2817), CD4+ 127 (0–804), CD8+ 173 (0–1922). Higher T-cell counts, however, did not clearly associate with lower NRM rates. After the initial expansion of effector memory cells (CD45RA−/CD62L−) in patients given DLI (P<0.01) and of central memory cells (CD45RA−/CD62+) in patients receiving rapamycin (P<0.05), there was a progressive normalization of the memory differentiation phenotype with late appearance of naïve cells (CD45RA+/CD62L+), which displayed a TCR spectratyping complexity score similar to pre-transplant values. Nevertheless, none of these biomarkers performed enough to be considered for surrogating lower NRM rates. In this series at high risk for CMV reactivation (CMV serostatus: H+/D+ 68%, H+/D- 27%) the event was observed in 46 pts (52%) and CMV disease in 8 pts (9%), all treated according to guidelines. By using Receiver Operating Characteristics (ROC) curve analysis of CMV-specific IFN-γ ELISPOT results at day 30–90, we found that cut-off values of 1000 spots/mL allowed to discriminate with high specificity (>95%) pts that did not reactivate the virus at later time points. Strikingly, while in pts with <1000 spots/mL, the 2-yrs NRM rate was 32%, in those with >1000 spots/mL, this was 0% (P<0.05). Interestingly, when comparing NRM rates in pts that achieved or not a CD4+cell value of 200 per μL, the result was not significant (21% vs 30%, P=0.8). Conclusions:These results clearly indicate that the early reconstitution of T-cell immunity to CMV after haplo-HSCT does not only protect from subsequent viral reactivation, but also surrogates for lower NRM rates. Moreover, they warrant the investigation of a CMV-specific IFN-γ ELISPOT cut-off value of 1000 spots/mL as a predictive biomarker in larger, multicenter series. Disclosures:No relevant conflicts of interest to declare.

Allele Level HLA Matching On Outcomes After Double Umbilical Cord Blood (dUCB) Transplantation for Hematological Malignancies: Stronger Graft Vs. Leukemia with HLA Mismatch

AbstractAbstract 1976Multiple single center and registry reports have documented the critical impact of donor-recipient HLA match on engraftment, transplant-related mortality (TRM) and survival after umbilical cord blood (UCB) transplantation. However, nearly all reports have only considered HLA A and B at antigen level and HLA DRB1 at allele level typing without consideration of HLA C or DQ. Therefore, we retrospectively performed allele level HLA typing for HLA-A, B, C, DRB1, DQB1 for UCB donor-recipient pairs in order to assess the importance of high resolution HLA typing on transplant outcomes. After 2002, most patients received a dUCB transplant in order to achieve the desired cell doses of ≥3, ≥4 and ≥5 × 10e7 NC/kg for grafts that were HLA 6/6, 5/6 and 4/6 matched by original typing resolution, respectively. Therefore, the analysis was limited to 275 recipients of dUCBT for hematological malignancy and whom DNA from both units was available. The effect of HLA match was based on the HLA type of the predominant long term engrafting unit. The median recipient age and weight was 44 years (range, 0.6–69) and 76.9 kg (range, 7.1–148), respectively. Conditioning was myeloablative (40%) consisting of cyclophosphamide (CY) 120 mg/kg, fludarabine (FLU) 75 mg/m2 and total body irradiation (TBI) 1320 cGy, or non-myeloablative (60%) consisting of CY 50 mg/kg, FLU 200 mg/m2, TBI 200 cGy with 95% receiving cyclosporine A (CsA) and mycophenolate mofetil (MMF) immunosuppression. Patients had acute leukemia (62%), standard risk disease (62%), cytomegalovirus seropositive (59%), and received at least one UCB unit that was sex mismatched to the recipient (78%). Results reported are based on the long-term predominant UCB unit. Notably, survival was not adversely affected by HLA mismatch. The probability of survival at 5 years was 46% (95%CI, 33–58%), 47% (95%CI, 38–54%) and 29% (95%CI, 13–47%) in patients engrafting with a 3–5/10, 6–8/10 and 9–10/10 HLA-matched UCB grafts, respectively (p=.47). In multivariable analysis after adjusting for disease risk, CMV serostatus, and KPS, there was similar risk of overall mortality for all groups regardless of HLA matching level. All other transplant outcomes including the incidence of acute and chronic GVHD were similar for all HLA-matching groups (data not shown). In the subset with acute leukemia (n=174), however, greater HLA mismatch was associated with a significantly lower risk of relapse without a deleterious effect on risk of TRM, resulting in a benefit in LFS (inverse of treatment failure) as shown below.Transplant Outcome for Acute LeukemiaHLA 3–5/10 match (n=84) RR (95%CI), p-valueHLA 6–8/10 match (n=168) RR (95%CI), p-valueHLA 9–10/10 match (n=34) RR (95%CI), p-valueTRM at 2 years1.01.1 (0.6–2.4) P=.731.1 (0.6–2.4) P=.73Leukemia Relapse1.01.9 (0.9–4.3) P=.113.5 (1.3–9.5) P=.01Treatment failure1.01.4 (0.8–2.4) P=.192.2 (1.1–4.2) P=.02Together these data indicate that UCB units with greater HLA mismatch may confer greater GVL effect without greater TRM compared to HLA better-matched UCB grafts. These results suggest importance of evaluating allele level HLA typing in the setting of dUCB transplantation. If confirmed, these results could have major implications not only on graft selection (ie avoidance of HLA matched units), but also the target size of the international UCB banking inventory. Disclosures:No relevant conflicts of interest to declare.

Myeloablative Umbilical Cord Blood Transplantation for Hematologic Malignancies Is Comparable to Unrelated Donor Transplantation: A Retrospective Single-Center Study

AbstractAbstract 1995 Background:The number of cord blood transplants (CBT) is rapidly increasing with suggestion of outcomes comparable to those obtained after unrelated donor transplantation (URD). We conducted, a retrospective analysis comparing post-transplant outcomes between myeloablative CBT and myeloablative URD at our Institution. Methods:Between January 2006 and December 2011 a total of 488 patients received either a CBT (n=88) or URD (n=400). Of these 400, 358 (90%) received a 10/10 HLA-matched (MURD) and 42 (10%) a ≤9/10 HLA-mismatched (MMURD) graft. All patients received a double CB graft except for 12 patients (13%) who received a single CB unit. In addition, 25 (28%) patients received an ex vivo expanded graft as part of either a single or double CBT. Mycophenolate mofetil and cyclosporine were used for graft-versus-host disease (GVHD) prophylaxis in all CBT recipients, while FK506 + methotrexate was preferentially used among URD patients (n=339, 84%). Conditioning regimens for both groups are summarized in Table 1. Time-to-event outcomes were compared between groups using Cox regression, and logistic regression was used for acute GvHD. All models were adjusted for age, disease risk and CMV serostatus. Results:Patient characteristics are shown in Table 1. Differences between groups included higher median age in URD recipients and a higher proportion of non-caucasian and CMV seropositivity in CB recipients. Disease risk was similar between the 2 groups. Peripheral blood stem cells (PBSC) was used for the majority of URD grafts (61%). The median time to neutrophil [URD 19 days vs CBT 23 days; hazard ratio (HR) 1.91 (1.46–2.51, p<0.0001)] and platelet recovery [URD 19 days vs CBT 45 days; HR=2.76 (2.05–3.71, p<0.0001)] was significantly shorter for URD recipients. In multivariate analysis, the risk of mortality was similar in URD vs CBT (HR=1.11 (0.71–1.73, p=0.64)). When HLA-match status was considered in the URD group, the risk of death was higher in the MMURD group compared to CBT, although the difference was not statistically significant (HR=1.37 (0.83–2.26, p=0.22)). The risk of relapse was suggestively higher in the URD group overall relative to CBT (HR=1.90 (0.94–3.84, p=0.07)), and this difference was enhanced when HLA matching and source of stem cells in URD were considered. In particular, recipients of unrelated (matched or mismatched) PBSC had a higher risk of relapse relative to CBT (HR=2.33 (1.11–4.91, p=0.03)), as did the MMURD (BM or PBSC) group (HR=2.34 (1.07–5.11, p=0.03)). Furthermore, unrelated recipients of matched or mismatched PBSC each had a higher risk of relapse relative to CBT (matched PBSC: HR=2.44 (1.11–5.38, p=0.03); mismatched PBSC: HR=3.89 (1.63–9.30, p=0.002)). The combined results for mortality and relapse led to an increased risk of relapse-free survival (RFS) failure (earliest of relapse or death) for patients receiving PBSC from a mismatched URD (HR=1.88 (1.08–3.27, p=0.03)); the risk of failure was also increased for PBSC recipients from a matched URD, but the difference was not statistically significant (HR=1.42 (0.87–2.32, p=0.16)). The risk of non-relapse mortality (NRM) was similar between URD and CBT (HR=0.89 (0.52–1.53, p=0.67)), and while there was less chronic GvHD in the URD group, the difference was not statistically significant, and this slight reduction was largely due to the effect in BM recipients (URD BM vs CBT, HR=0.59 (0.35–0.98, p=0.04); URD PBSC vs CBT, HR=1.01 (0.62–1.66, p=0.95)). The risks of grades 2–4 and 3–4 acute GvHD were similar between URD and CBT groups (odds ratio (OR) 1.10 (0.57–2.11, p=0.78)), and (OR=0.70 (0.38–1.31, p=0.26)), respectively. Conclusions:Our data suggest that OS, RFS and NRM after CBT are not inferior to those observed after URD transplantation, and OS and RFS might be higher when the URD group is restricted to recipients of PBSC, particularly those who are mismatched with their unrelated donor. Relapse occurred less frequently for CBT recipients especially when compared to MMURD or URD with PBSC. The retrospective nature of this study and the heterogeneity of the population do not allow us to draw definitive conclusions, however, our results reinforce the need for a randomized study to definitively address these comparisons. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Outcomes After Double Cord Blood Transplantation Compared to Single Cord Blood Transplantation in Adults with Acute Leukemia Given a Reduced Intensity Conditioning Regimen

AbstractAbstract 232▪▪This icon denotes a clinically relevant abstractUnrelated cord blood (UCB) is an alternative source of allogeneic hematopoietic stem cell transplantation (HSCT) for adults with acute leukemia lacking a HLA matched donor. Double cord blood unit (dUCBT) has been increasingly used over single CB unit (sUCBT) after reduced intensity conditioning regimen (RIC). Since there is an increased relapse incidence (RI) using RIC-HSCT compared to myeloablative conditioning regimen, we have driven the hypothesis that RI may be decreased and leukemia-free survival (LFS) rate increased after dUCBT compared to sUCBT, due to probably increased graft-versus leukemia (GVL) effect. With this aim, we analyzed 360 adults (>18 years) with ALL (n= 77) or AML (n=238) in CR1 (n=212) and in CR2 (n=148) transplanted with a sUCBT (n=131) or a dUCBT (n=229) after a RIC. Only patients transplanted with a single unit containing more than 2.5×107/kg total nucleated cells (TNC) were included. Patients were transplanted from 2005–2011 in EBMT centers. Comparing the two groups of patients receiving a sUCBT or a dUCBT in CR1, there were no statistical differences according to age, diagnosis (AML or ALL), weight, CMV serostatus, cytogenetics risk, number of HLA incompatibilities. However, dUCBT were performed more recently (2009 vs 2008), the time from CR1 to transplantation was longer (142 days vs 121 days), more frequently transplanted with CY+FLU+TBI2Gy (87% vs 68%), lower frequency of ATG use (21% vs 35%) and finally, dUCBT recipients received higher number of TNC collected (5×107/kg vs 3.9×107/kg) or infused (4×107/kg vs 3.1×107/kg). Median follow-up was 23 months in both groups.Cumulative incidence (CI) of 60 days neutrophil recovery was 82±3% after dUCBT and 76±2% after sUCBT (p=0.86) and frequency of full donor chimerism at day 100, was not statistically different between dUCBT (81%) and sUCBT (86%). At day 100, CI of acute GVHD (grade II-IV) was 35% in both groups, however there was a trend of increased incidence of grade III-IV after sUCBT (19%) compared to dUCBT (10%, p=0.06) but increased incidence of grade II aGVHD after dUCBT (28%) compared to 17% after sUCBT (p=0.05). CI of chronic GvHD at 2 years was 21±4% after dUCBT and it was 12±5% after sUCBT (p=0.15). At 2 years, CI of non relapse mortality (NRM) after dUCBT was 28±4% and it was 30±6% after sUCBT (p=0.87). However, CI of 2y relapse was 21±4% after dUCBT whereas it was 38±2% after sUCBT (p=0.03). In a multivariate analysis adjusting for the differences between the 2 groups, dUCBT was associated with lower incidence of relapse compared to sUCBT (HR=0.74, p=0.01). Therefore, there was an improved 2-y LFS after dUCBT (51±5%) compared to sUCBT (32±3%; p=0.03). This was confirmed in a multivariate analysis (HR=0.64, p=0.04).Concerning patients transplanted in CR2 (n=148), there were no statistically differences of outcomes after dUCBT (n=93) or sUCBT (n=55). At 2y, LFS was 40±6% after dUCBT and 48±3% after sUCBT (p=0.32). In a subgroup analysis of dUCBT (n=118) and sUCBT (n=51) recipients using the same conditioning regimen (CY+FLU+TBI2Gy), 2 y LFS were 54±5% and 33±7% respectively (p=0.05). Conclusion:In this retrospective comparative based registry analysis, in AL patients transplanted in CR1, neutrophil recovery, GVHD and NRM were not statistically different after RIC-dUCBT or RIC-sUCBT, however, dUCBT recipients had decreased relapse incidence and improved LFS. For AL patients transplanted in CR2, there was no benefit of using dUCBT when compared to sUCBT recipients. Disclosures:No relevant conflicts of interest to declare.

HLA-DPA1 Polymorphism Is a Risk Factor for Graft-Versus-Host Disease Following Unrelated Hematopoietic Stem Cell Transplantation (HSCT)

AbstractAbstract 4181 Background:The HLA-DP molecule has been shown to be a target of allograft rejection and graft-versus-host-disease (GVHD) in HSCT. Nevertheless the way in which HLA-DP matching in HSCT should be approached remains unclear. Studies point out that some mismatched allele combinations result in a weaker alloreactive response than others, suggesting that epitope matching should be considered rather than allele matching. Most studies focus on the highly polymorphic beta chain of the DP molecule that is considered to play a dominant role in T cell interaction. Several groups have tried to identify immunogenic epitopes in the peptide binding groove of the beta chain to unravel DP immunogenicity, but there is no consensus on epitopes and amino acid residues. The alpha chain of the DP molecule is characterized by low polymorphism and therefore is considered less relevant in T cell recognition. Nonetheless, modeling studies identified amino acid position 31 in the peptide binding groove of the alpha chain as a potential immunogenic residue that influences peptide binding by the polymorphic residue Methionine (M) vs. Glutamine (Q). We hypothesized that HLA-DPA1 polymorphism at position 31 may impact patient outcomes after unrelated HSCT. [Display omitted] Methods:Engraftment, GVHD, graft failure, relapse, and mortality had been analyzed in 81 patients who received HSCT in a single institute from 8/2003-12/2010 with a 10/10 matched unrelated donor. The median age was 47 years, and 58% were males. 30% had reduced-intensity conditioning and 70% were myeloablative. HLA-DPA1 polymorphism was determined using Luminex PCR-SSO method. Prognostic factors for outcomes were identified using Cox proportional hazards analysis. Variables analyzed included age, gender, co-morbidity, diagnosis, conditioning, cell source, donor characteristics, CMV serostatus, DPB1 mismatching, DPA1 polymorphism, and hematopoietic stem cell dose. Multivariable prognostic factors for each outcome were assessed using stepwise Cox proportional hazards. Results:Neither allele DPB1 mismatching nor recipient DPA1 allele polymorphism was associated with any of the outcome variables. Interestingly, in uni- and multivariable analysis homozygous M at position 31 (31M) of DPA1 in the donor was associated with a lower risk of acute GVHD compared to homozygous Q & heterozygous M/Q at position 31 (HR 0.54, P=0.031). Also, 31M donors, had a trend towards a lower risk of chronic GHVD (HR 0.52, P=0.08) and all-cause mortality (HR 0.63, P=0.11). Conclusion:With the limitation of the small population size, this is the first clinical study describing a potential role of amino acid polymorphism in the alpha chain of the HLA-DP molecule in unrelated HSCT. These results show an increased risk of donor DPA polymorphism for aGVHD and warrant further investigation in a larger cohort. Disclosures:No relevant conflicts of interest to declare.

Equivalent Outcome Between Reduced Intensity Versus Conventional Myeloablative Conditioning Hematopoietic Stem Cell Transplantation for Patients Older Than 35 Years with Acute Myeloid Leukemia.

AbstractAbstract 3103▪▪This icon denotes a clinically relevant abstract Introduction:Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) provides the best chance of long-term survival for patients with intermediate or high-risk acute myeloid leukemia (AML). The major limitation of this procedure is the risk of treatment related mortality (TRM). Use of reduced intensity conditioning (RIC) regimen has become standard practice among older candidates with comorbidities. Although RIC regimen have been used for over a decade in older patients, the benefit of this approach in younger patients with AML compared with the risk of toxicity of standard regimen (MAC) is still discussed. We compared the outcomes for patients with AML over 35 years using RIC or MAC HSCT. Patients, methods, and transplantation characteristics:From January 2000 to December 2010, 132 consecutive patients older than 35 years with AML (18 secondary AML) received HSCT in our center, either from siblings (n=87) or HLA 10/10 allele-matched donors (n=45). MAC (n=72) and RIC (n=60) regimens were defined as previously described (Bacigalupo, 2009). Seventy-three patients were in first complete remission (CR1); 30% of patients had poor risk cytogenetics (MRC classification). Karnofsky performance status was scored at time of HSCT. Engraftment, acute and chronic graft-versus-host disease (GvHD), transplantation-related mortality (TRM), relapse rate as well as overall survival (OS) at 4 years were compared according to the intensity of the conditioning regimen. First a classical multivariable Cox analysis was conducted. In a second step, baseline confounding factors were adjusted for using inverse probability-of-treatment weighting (IPTW). Results of the comparison:Patient characteristics according to the intensity of the conditioning regimen were similar for AML type (de novo versus secondary), gender, karnofsky performance status, CR#, donor type and number of CD34+ infused. Particularly, cytogenetic risks were comparable in both groups. Patients were younger in the MAC group (median age 44 years [range 35 to 56 years] vs 54[37 to 66] for RIC, p<0,0001), received mainly bone marrow as source of stem cells (54% versus 2% for RIC, p<0,0001) and GvHD prophylaxis using cyclosporine plus methotrexate (89% versus 5% for RIC, p<0,0001). Moreover, ATG in the conditioning regimen (more ATG in RIC: 51 vs. 14%, p<0.0001), donor age (older for RIC: 49 vs. 39 years, p=0.002) and number of nucleated cells infused (higher in RIC: 11 vs. 4 × 108/kg, p<0.0001) were also different. The median follow-up was 47 months (10 to 134), and 25% of patients had a follow-up of at least 74 months. During evolution, all patients engrafted. The cumulative incidence (CIf) of acute GVHD grade II-IV was 49% (35% after RIC vs 61% after MAC, p=0.001). The 5-year CIf of chronic GVHD was 37% (40% after RIC vs 30% after MAC, p=0.32). During FU, 71 patients died. The 5-year CIf of TRM was 21% (13% after RIC vs 28% after MAC, p=0.009). Adjusting for cytogenetic risk, gender donor/recipient mismatch and infused nucleated cells, no difference was observed between RIC and MAC (HR 0.9, p=0.16). The 5-year CIf of relapse was 42% (51% after RIC vs 35% after MAC (p=0.22)). Adjusting for gender donor/recipient mismatch, donor/recipient CMV serostatus and infused CD34+ cells, no marked difference was observed between RIC and MAC (HR 0.8, 95%CI 0.4–1.5, p=0.50). The 5-year OS was 39% (50% after RIC vs 34% after MAC, p=0.38). Using both Cox regression and IPTW to account for imbalance in patients characteristics, similar OS was found after RIC and MAC (Figure 1), with adjusted HRs for MAC vs RIC of 0.9 (95%CI 0.4–1.8, p=0.68) with Cox regression and 0.9 (95%CI 0.4–1.8, p=0.76) with IPTW. Conclusion:In patients with AML over 35 years, MAC regimen lead to a non significant higher rate of treatment related mortality with no benefit in terms of relapse when compared with RIC regimen. Until prospective trials are completed, this study supports the use of a RIC regimen for patients with AML older than 35 years who are transplanted either from siblings or matched unrelated donors. [Display omitted] Disclosures:Fenaux:Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Prognostic Effect of Mutations in the Splicing Gene Machinery in 339 Patients with MDS or Secondary AML Following MDS After Allogeneic Hematopoietic Stem Cell Transplantation

AbstractAbstract ▪357▪This icon denotes a clinically relevant abstract Introduction:Molecular predictors for treatment outcome after allogeneic hematopoietic stem cell transplantation (HSCT) of MDS and AML patients are limited. Recently, mutations in the splicing gene machinery have been described as frequent aberrations in MDS. The aim of this study was to investigate the prognostic impact of mutations in the splicing genes U2AF1, SRSF2 and SF3B1 in a large cohort of patients with high risk MDS or secondary AML following MDS (sAML) undergoing allogeneic HSCT. Patients and Methods:Patients (n=339) with a diagnosis of MDS (50.1%) or sAML (49.9%) who received allogeneic HSCT at four German university medical centers (Dresden, Düsseldorf, Hamburg and Hannover) between 1996 and 2011 and for whom genomic DNA was available from a time when the disease was active, were evaluated for the presence of mutations in the splicing genes U2AF1, SRSF2, and SF3B1 by direct sequencing. Results:Median follow up from time of transplantation was 3.27 years. Median patient age at time of HSCT was 58 years (range 19–74). 74 patients (21.8%) were in complete remission and 265 patients (78.2%) had active disease before transplantation. Low, intermediate, and high risk cytogenetics according to IPSS were found in 204 (60.2%), 42 (12.4%), and 76 (22.4%) patients, respectively (in 5% cytogenetic information was not available). Related donor HSCT was performed in 82 patients (24.2%), and unrelated donor HSCT in 257 patients (75.8%). Myeloablative preparative regimens were used in 51 patients (15%), and a non-myeloablative regimen was given to 288 patients (85%).Mutations in U2AF1, SRSF2 and SF3B1 were detected in 14 (4.1%), 32 (9.4%) and 18 (5.3%) patients, respectively. SRSF2 and SF3B1 mutations co-occured in two patients, while the other patients had not more than one mutation in the investigated genes. Baseline characteristics were similarly distributed between U2AF1, SRSF2, or SF3B1 mutated and wildtype patients, respectively (sex, MDS vs sAML, cytogenetics, CMV status of patient, type of previous treatment, and remission status prior to transplantation), except a higher median age of U2AF1 mutated compared to wildtype patients (P=.02). There were no differences regarding transplant-related characteristics between patients with mutated or wildtype U2AF1, SRSF2, and SF3B1 (reduced intensity vs standard conditioning, GvHD prophylaxis, donor age, donor sex, CMV and HLA compatibility between recipient and donor).U2AF1 mutations were associated with a significantly shorter overall survival (OS, median 0.58 vs 3.6 years in mutated vs wildtype patients, respectively, HR 2.54; 95%CI 1.41–4.58; P=.002). The cumulative incidence of relapse (CIR) was higher in U2AF1 mutated compared to wildtype patients (3-year CIR 50% vs 22%, P=.002), while non-relapse mortality (NRM) was similar between mutated and wildtype patients (3-year NRM 43% vs 29%, P=.11). Mutations in SRSF2 and SF3B1 were not associated with OS (P=.98 and P=.44, respectively), CIR (P=.19 and P=.19, respectively), and NRM (P=.49 and P=.44, respectively). In multivariate analysis, when considering variables with P<.15 in univariate analysis (age above or below 55 years, karyotype, stage [MDS vs sAML], CMV serostatus of patient, donor type [related vs unrelated], donor sex), U2AF1 mutations independently predicted shorter OS (HR 2.6; 95%CI 1.39–4.85; P=.011) besides karyotype, stage, CMV serostatus, and donor sex. Mutations in U2AF1 independently predicted higher CIR in multivariate analysis (HR 3.02, 95% CI 1.36–6.7, P=.007). The rates of acute and chronic GvHD were similar in U2AF1 mutated and wildtype patients. Summary:U2AF1 mutations independently predicted worse patient outcome after allogeneic HSCT in MDS and sAML patients in our study due to a higher incidence of relapse. The U2AF1 mutation status may become useful to identify patients at high-risk for relapse after transplantation independent of established prognostic factors. Disclosures:Platzbecker:GlaxoSmithKline: Honoraria, Research Funding.

Characterizing Immunophenotypic and Functional Pseudo-Exhaustion in T Cells From CLL Patients: The Impact of Lenalidomide

AbstractAbstract 564The ability to evade immune destruction is increasingly being recognised as a crucial feature of cancer cells. Chronic lymphocytic leukemia (CLL) is associated with profound defects in T-cell function, resulting in failure of anti-tumor immunity and increased susceptibility to infections. T cells from CLL patients exhibit functional defects and alterations in gene expression, that show similarities to exhausted T cells in chronic viral infections. However, it is unclear whether CLL T cells are truly exhausted, or whether these defects are restricted to expanded populations of CMV specific T cells. We investigated the phenotype and function of CD8+ T cells from CLL patients and controls matched for age and CMV-serostatus. We demonstrate an increased proportion of CCR7- effector T cells in both CLL patients and CMV-seropositive individuals (p<0.05). CD8+ and CD4+ T cells from CLL patients had increased expression of exhaustion markers CD160 and CD244 irrespective of CMV-serostatus (p<0.01), whereas increased PD1 expression on CD8+ T cells was limited to CMV-seronegative patients (p=0.002). CLL CD8+ T cells also showed functional defects in proliferation and cytotoxicity irrespective of CMV-serostatus, with the cytolytic defect caused by a combination of impaired granzyme B packaging into secretory vesicles and non-polarized degranulation. In contrast to virally-induced exhaustion, CLL T cells showed increased production of interferon-γ with increased T-BET expression (p<0.01), normal IL-2 production, and no downregulation of IL-7R. Therefore, while CLL CD8+ T cells exhibit some features of T-cell exhaustion, they show important differences (Table 1). These findings also exclude CMV as the sole cause of T cell defects in CLL.Lenalidomide has recently been demonstrated to have significant clinical activity in CLL. Its mechanism of action in this disease is not well understood, but it thought to act primarily by a combination of CLL cell and immune cell activation. We therefore examined the ability of lenalidomide to repair the observed T cell defects by investigating the impact of this agent on the gene expression profiles and function of CLL T cells. Treatment of CLL CD8+ T cells with lenalidomide increased the expression of 137 genes, while 34 genes were downregulated. The most prominent changes in expression were of genes involved in cytoskeletal signaling including WASF1 (Wiskott-Aldrich syndrome protein, family member 1), and TPM2 (tropomyosin 2). There was also upregulation of genes involved in lymphocyte activation, including TNFSF4 (Tumor necrosis factor ligand superfamily, member 4: OX40L), LAG3 (Lymphocyte-activation gene 3), and TNF, and genes involved in cell proliferation such as IKZF1 (Ikaros) and GRN (Granulin). Although lenalidomide treatment or anti-CD3 stimulation alone had no impact on T-bet expression, co-treatment with both anti-CD3 stimulation and lenalidomide resulted in significantly enhanced T-bet expression and increased production of interferon-γ. In contrast, lenalidomide treatment alone was able to improve T cell cytotoxic function, associated with repair of trafficking of granzyme B into the immunological synapse.In conclusion, T cells from CLL patients exhibit features of T-cell pseudo-exhaustion that are present irrespective of CMV serostatus. Treatment of CLL T cells with lenalidomide results in upregulation of genes involved in proliferation, activation, and cytoskeletal pathways, resulting in repair of the functional T cell defects.Table 1Comparison of the phenotypic and functional defects of T cells from CLL patients with T-cell “exhaustion” in chronic viral infectionsExhausted T cells in chronic viral infectionsT cells from CLL patientsIncreased expression of inhibitory receptorsYesYesAbnormal transcription factor profileYesYesReduced proliferative potentialYesYesDecreased expression of IL-7R (CD127)YesNoDecreased cytokine production ↓IL-2, ↓IFN-γYesNoImpaired cytotoxicityYesYes Disclosures:Riches:Celgene: Research Funding. Gribben:Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

Racial Differences in Prognostic Factors and Outcomes in Patients Undergoing Allogeneic Hematopoietic Cell Transplant

AbstractAbstract 2020Racial differences have been identified in a number of cancers, with African Americans (AA) having poorer survival than Caucasians (C). Disparate outcomes are attributed to racial differences in cancer biology, pharmacogenetics, environmental exposures, and/or socioeconomic and cultural factors. Disparities in outcome in patients with acute myeloid leukemia (AML) have been observed, and one study of post remission therapy in AA compared to C with AML revealed a significant delay in post-remission therapy with AA, but no differences in survival (Brady et al, Leuk Res 2011; Mar;35:346). There are limited data on the effects of race on survival after allogeneic hematopoietic cell transplantation (HCT).From January 1997 until December 2011 we identified 789 patients, 58 AA and 731 white, that underwent allogeneic HCT for AML (n=304), myelodysplastic syndrome (n=107), acute lymphoblastic leukemia (n=107), chronic myeloid leukemia (n=87), non-Hodgkin lymphoma (n=105), myeloproliferative neoplasms, (n=26), bone marrow failure syndromes (n=23), Hodgkin lymphoma (n=15), plasma cell neoplasms (n=13), and biphenotypic leukemias (n=2). There were no significant differences between AA and C patients in gender, performance status, co-morbidity score, diagnosis, intensity of transplant, or donor type. However, AA were younger than C (median 40 (range 18–62) vs 47 (range 18–71) years, p=0.003); were more likely to: be in remission at HCT, (74.1% vs 57%, p=0.011); have an HLA mismatched donor, (35.7% vs 13.7%, p<0.001); have positive donor or recipient CMV serostatus, (89.7% vs 69.4%, p<0.001), use TBI as part of their preparative regimen (53.4% vs 39.1%, p=0.032), have a cord blood transplant (20.7% vs 5.7%, p<0.001) and have lower CD34+ cell dose (median 1.49 vs 2.49×106/kg, p=0.023). Time from diagnosis to transplant was longer in AA (median 10.7 (range 1.6–136.9) vs 7.9 (range 0.2–246.4) months, with a trend toward significance, p=0.06). Outcomes were compared using the log-rank or Pepe-Mori test and included infection, incidence and degree of graft versus host disease (GVHD), relapse, non-relapse mortality, relapse free-survival (RFS) and overall survival (OS). In univariable analysis, AA had significantly worse OS (HR 1.41, p=0.026) and RFS (HR 1.36, p=0.044) compared to C. There were no significant differences in incidence or degree of acute or chronic GVHD, infection, or relapse. In multivariable analyses, adjusted for number of prior chemotherapy regimens, prior radiation, co-morbidity score, diagnosis, disease status, donor type and source, HLA mismatch, and CMV status, the OS difference between AA and C became non-significant (HR 1.27, p=0.18). Propensity analyses was used to identify a well-matched cohort of C patients with AA patients, n=47 per group. Among these well-matched groups, CMV infection was higher in AA compared to C, p=0.029, but OS (p=0.92) and RFS (p=0.43) were not significantly different.In conclusion, our study demonstrates important differences between racial populations undergoing allogeneic HCT. We found that AA patients coming into transplant present at a younger age, are more likely to have positive donor or recipient CMV serostatus, and are less likely to have an HLA matched donor. Despite these differences, race in and of itself did not translate into worse survival in controlled analyses. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

CMV-Replication After Allogeneic Stem Cell Transplantation Is Associated with a Gvhd-Independent Reduced Relapse Risk in Lymphoma

Abstract 1635 Background:We have previously showed that a CMV- reactivation after allogeneic HSCT is associated with a reduced risk for leukemic relapse and improved overall survival in patients with AML (Elmaagacli et al Blood 2011). Further, experimental data in a lymphoma mice model reported from Erlach et al. showed that coinfection with murine CMV revealed a strong anti-lymphoma effect by induction of apoptosis in lymphoma cells and improving rate of overall survival in mice after transplant. Methods:This prompted us to investigate the influence of early replicative CMV infection in 94 (median age [years]: 45, 18 – 70) patients with lymphoma, who received transplants from unrelated (n=67) or related (n=27) donors. Patients were transplanted from HLA-identical (n=74), HLA-mismatched (n=20). 13 patients were transplanted for indolent lymphoma (FL n=11, CLL n=2), 67 patients for aggressive lymphoma (B-lineage n=35, T-lineage n=27, transformed n=5), 11 patients for MCL and 3 patients for HD. The disease stages of patients at HSCT were CR in 20 patients, PR in 40 patients, refractory in 30 patients and untested in 2 patients. 55 patients (59%) received previous autograft and 82 patients (87%) were treated prior to transplant with at least 3 chemotherapy lines. The hematopoietic cell transplantation specific comorbidity index (HCT-CI) were 0–2 in 76 patients (81%) and 3+ in 18 patients (19%). Myeloablative preparative regimen was applied in 60 patients (64%) while 34 patients (36%) received a RIC. Sixty-eight % of patients (n=48) were at risk for CMV reactivation based on either patient or donor pretransplant CMV serostatus. CMV replication as detected by pp65 antigenemia assay occurred in 34 patients (36%). Results:Taking all competing risks into account, the cumulative incidence of progressive free survival (PFS) at 5 years after alloSCT was 38 % (95 % confidence limit [95 % CL]: 31 – 45) in patients without as compared to 20 % (95 % CL: 9 – 31) in patients with early pp65 antigenemia (p<0.018). In multivariate analysis including parameters as grades II-IV acute graft-versus-host disease (GvHD), chronic GvHD, disease stage, chemorefractory, previous chemotherapy lines and pp65 antigenemia, CMV replicative status was confirmed as a strong independent predictor of PFS (hazard ratio [HR]: 0.29, 95 % CL: 0.08 – 1.00, p<0.049) together with chronic GvHD (HR: 0.32, 95 % CL: 0.13 – 0.80, p<0.016), and chemorefractory HR: 3.3, 95 % CL: 1.28 – 8.4, p<0.013). The anti-lymphoma effect was detectable across all lymphoma subsets and was most pronounced in patients with chemotherapy refractory lymphoma or refractory disease of lymphoma. However, Overall survival rate did not differ in both groups (51.9% for patients with CMV-replication versus 50.7% without n.s.) Conclusions:This is the first report which demonstrates a strong and independent effect of early CMV replication on the PFS in patients with lymphoma. This effect deserves further and more comprehensive studies with regard to its clinical relevance and the underlying anti-lymphoma mechanisms.Cumulative incidence of progression-free survival (PFS) stratified by posttransplant HCMV pp65-antigenemia and cell type of aggressive lymphoma. Disclosures:No relevant conflicts of interest to declare.