Allo SCT Research Articles

Durable Remissions in Children with Relapsed/Refractory ALL Treated with T Cells Engineered with a CD19-Targeted Chimeric Antigen Receptor (CTL019)

BACKGROUND CARs combine a targeting antibody (scFv) domain with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR, which have resulted in up to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 53 pts with relapsed/refractory (r/r) ALL treated on our pilot trial in pediatric ALL. METHODS T cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with r/r CD19+ ALL. 48/53 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 10 7 to 10 8 cells/kg with a transduction efficiency of 3.6-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, and not their allo donors. RESULTS We treated 53 children and young adults with CD19+ ALL, median age 11y, (4-24y). To assess disease burden after lymphodepleting chemotherapy, pts had BM aspirations performed 1D prior to 1 st CTL019 infusion: 41/53 pts had detectable ALL while 12 were MRD(-). A median of 4.3x10 6 CTL019 cells/kg (1-17.4x10 6 /kg) were infused over 1-2D (1 pt got cells over 3D). There were no infusional toxicities >gr2, although pts who developed fevers within 24h of infusion did not receive a planned 2 nd infusion of CTL019 cells. 50 pts (94%) achieved a CR, including a patient with CD19+ T ALL, 3 did not respond. MRD measured by clinical flow cytometry was All but 5 (90%) of pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 28% of patients and was reversed in all cases with the IL6-receptor antagonist tocilizumab, together with short courses of corticosteroids in 9 pts. Although T cells collected from the 35 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion. Persistence of CTL019 cells can be detected by flow cytometry and/or QPCR, and results in the pharmacodynamic marker of CTL019 function, B cell aplasia, which continued for 3-39 months after infusion in pts with ongoing responses. B cell aplasia has been treated with IVIg without significant infectious complications. CONCLUSIONS: CTL019 cells can undergo robust in vivo expansion and can persist for 3 years or longer in children and young adults with r/r ALL, allowing for the possibility of long-term disease control without subsequent therapy such as SCT. This approach also has promise as salvage therapy for patients who relapse after allo SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with r/r ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CTL019 resistance. Rapid loss of CTL019 cells (prior to 3 months) is associated with a high risk of CD19+ relapse. CTL019 therapy has received Breakthrough Therapy designation from the FDA in pediatric and adult ALL, and phase 2 multicenter registration trials are well underway. Disclosures Grupp: Novartis: Consultancy, Research Funding. Maude: Novartis: Consultancy, Research Funding. Shaw: Novartis: Research Funding. Aplenc: Sigma Tau: Consultancy. Lacey: Novartis: Research Funding. Levine: Novartis: Patents & Royalties, Research Funding. Melenhorst: Novartis: Research Funding. Rheingold: Novartis: Consultancy; Endo: Other: Husband9s employer, has equity interest. Teachey: Novartis: Research Funding. Wood: Novartis Pharmaceuticals Corporation: Employment. Porter: Novartis: Other: IP interest, Research Funding; Genentech: Other: Spouse employment. June: University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding.

Outcomes in Diffuse Large B-Cell Lymphoma (DLBCL) Patients Relapsing after Autologous Stem Cell Transplantation (ASCT): An Analysis of Patients Included in the Coral Study

Introduction and Methods: Salvage chemotherapy followed by high-dose therapy and autologous stem cell transplantation (ASCT) is standard care for relapsed/refractory DLBCL. In the CORAL study, 477 patients were assigned to one of two salvage regimens (R-DHAP or R-ICE). Only the 240 responding patients underwent per protocol ASCT and were randomly assigned to rituximab or observation. The four-year event-free survival (EFS) post ASCT were 52 and 53% for the rituximab and observation groups, respectively (p=.7). Secondary IPI (sIPI) independently predicted EFS, PFS and OS after ASCT (Gisselbrecht et al, JCO 2010 & 2012). Outcome data are limited in DLBCL patients who relapse after ASCT, with reported overall survival (OS) of 9.9 months in rituximab-pretreated patients (Nagle et al, AJH 2013). To shed more light on outcome and prognostic factor in this population, 75 patients included in the CORAL study who relapsed after scheduled BEAM/ASCT were reviewed.Results:Median time between ASCT scheduled in CORAL and relapse was 7.1 months (range 3.1-61.9) with 32.9% relapsing > 12 months. Median age was 56.1y (range 20.9-67.7), M/F ratio 51/24, sIPI 0-2 in 71.6%, >2 in 28.4%. 49.3% were in the rituximab and 45.3% in the observation arm of the CORAL. All patients had previously received rituximab. Third-line therapy consisted of ICE-type (17.3%), DHAP-type (24%), gemcitabine-containing (28%), CHOP-like (13.3%), and miscellaneous regimens (17.3%). Overall response rate to third-line chemotherapy was 44%, with 32% complete response (CR)/CR unconfirmed (CRu), and 12% PR. Among the 75 patients, 16 (21.6%) could eventually be transplanted 3 ASCT and 13 allogeneic SCT with conditioning regimens including fludarabine. Median OS, calculated from time of relapse until death, was 10.0 months (95% CI 6.6-12.6; min: 0.9-max: 55.2 months; median follow-up: 32.8 months) with an estimated 1-y OS of 39.1%. Median OS was statistically different (p=.0007) according to sIPI at CORAL failure: sIPI 0-2: 12.6 months (1-y OS 51.3%), sIPI > 2: 5.3 months (1-y OS 21.6%, HR 2.805). Median OS in patients achieving CR/CRu, PR, or no response after third-line regimen was 37.7 m (1-y OS 90.5%, p<.0001, HR 0.132), 10.0 m (1-y OS 44.4%, p=.03, HR 0.375), and 6.3 months (1-y OS 13.4%), respectively. Median OS of patients who could eventually be transplanted was 17.4 months (1-y OS 68.2%), as compared to a median OS of 8.0 months in those who were not transplanted (1-y OS 31.2%) with a HR of 0.575 (p=.11). Median OS was particularly dismal among patients who relapsed < 6 months after CORAL-scheduled ASCT (5.7 months, n=28), as compared to those relapsing either > 6 and < 12 months (11.3 months, n=21) or > 12 months after ASCT (12.6 months, p=0.01, fig. 1)). In multivariate Cox analysis (with the following variables entered: age, sex, sIPI, response to third line and time between CORAL ASCT and relapse), sIPI >2 (HR 2.464, p=0.01), achievement of CR (HR 0.1, p<.0001) or PR (HR 0.242, p=0.02), and post-ASCT remission lasting < 6 months (HR 2.270, p=0.05) independently predicted for OS.Conclusions: Overall, the outcome of DLBCL patients relapsing after second-line R-DHAP/R-ICE followed by ASCT is poor. However, prognostic factors predicting better outcome in this group are late (> 6 months) relapse, lower sIPI and achieving at least PR after third-line salvage followed by transplantation. In the transplanted patients (allo SCT or second ASCT) a 2-year OS of 50% was observed. Thus, new salvage regimens can be a bridge to transplantation in patients with late relapse and/or low sIPI,. New drugs improving salvage efficacy are urgently needed, especially for patients relapsing < 6 months following ASCT. [Display omitted] DisclosuresSchmitz:Roche, Takeda, Gillead, Riemser und ctilifesciences: Other: Advisory board, Speakers Bureau. Gill:Sanofi Aventis: Research Funding; Roche: Honoraria; AbbVie: Honoraria; Roche: Research Funding. Milpied:Celgene: Honoraria, Research Funding. Briere:St. Louis Hospital, Paris, France: Employment. Thieblemont:St. Louis Hospital, Paris, France: Employment. Salles:Celgene Corporation; Roche and Gilead Sciences: Research Funding; Celgene Corporation; Roche: Speakers Bureau; Calistoga Pharmaceuticals, Inc.; Celgene Corporation; Genentech, Inc.; Janssen Pharmaceutica Products, L.P.; Roche: Consultancy. Gisselbrecht:roche: Research Funding, Speakers Bureau.

Treatment of Chronic Graft Versus Host Disease with a Combination of B-Cell Depletion and Tyrosine Kinase Inhibition

Chronic Graft Versus Host Disease (cGVHD) affects about 60% of all patients receiving an allogeneic hematopoietic stem cell transplantation (HSCT) and surviving beyond day 100. Incidence of cGVHD is rising because of the widespread use of Peripheral Blood Stem Cells (PBSC) as stemcell source. Chronic GVHD accounts for high morbidity and mortality rates. Affected patients require long term use of immunosuppressive drugs, mainly corticosteroids, which lead to development of severe side effects. Both the manifestations of cGVHD as the side effects from immune suppressive drugs account for a decrease in quality of life when cGVHD develops. Therefor new therapeutic strategies are urgently needed.Therapeutic approaches for cGVHD are limited. Generally recommended first line therapy consists of glucocorticoid therapy combined with a calcineurin inhibitor. Options for second line therapy are numerous but no consensus on the most favourable choice of agents has been reached.However as both monotherapy with rituximab and monotherapy with tyrosine kinase inhibition have shown to be effective in reducing cGVHD symptoms . We aimed to test whether the sequential therapy of the anti CD20 antibody rituximab followed by a 6 month treatment period with the tyrosine kinase inhibitor nilotinib is a good treatment strategy for patients with sclerotic cGVHD (EudraCT nr 2008-004125-42).We treated 26 patients with a combination of 4 weekly infusions of rituximab followed by a 6 month period of treatment with nilotinib (300mg b.i.d.). Patients were evaluated monthly for 13 months and sequential blood samples and skin biopsies were analyzed. All patients gave informed consent before enrollment.3 patients experienced severe side effects from either rituximab or nilotinib treatment and therefor were taken off study. Of the remaining 13 patients who thus far have completed the study protocol, 60% showed a (partial) response (3 patients went from NIH scoring 'severe' to 'moderate', 1 patient went from NIH scoring 'moderate' to 'no cGVHD'). There is also a significant decrease in cGVHD affected body surface area (Figure 1). Moreover, two out of four patients who suffered from severe ulcerations at the start of the study had a complete resolution of ulcers at the end of the treatment period. Patients with a (partial) response also showed a decrease in self attributed severity of cGVHD and their immunosuppressive drugs could be tapered.The sequential therapy of B-cell depletion and tyrosine kinase inhibition provides a new and interesting alternative treatment option for this difficult and heavily pretreated patient category.Table 1Baseline characteristics of participants.Patients26Male sex20Mean age in years(range)47,521-70Primary diagnosisAML7Multiple myeloma5ALL4Lymphoma4MDS2CLL1Myeloproliferative disorder2aplastic anemia1Mean days after allo SCT(range)1368411-3820Ulcerative chronic GvHD at start5NIH grade severity cGVHD at startsevere20moderate6 [Display omitted] DisclosuresOff Label Use: Anti CD20 antibody rituximab is used for sclerotic chronic GVHD. Tyrosine kinase inhibitor nilotinib is used for sclerotic chronic GVHD..

Clinical Significance of Bone Marrow Imaging of Appendicular Skeletons By Low-Dose Multi-Detector Computed Tomography in Patients with Bone Marrow Failure Syndrome

Background: Aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic syndrome (MDS) are the heterogeneous group of bone marrow failure syndrome (BMFs). AS they often show profound hypocellular marrow, the diagnosis is often difficult by bone marrow and laboratory examination alone. Red to yellow marrow conversion occurs with age in the appendicular skeleton (AS), where red marrow is converted to yellow marrow until the age of early 20s. Although abnormal distribution of red marrow in appendicular skeleton were previously reported in small series of patients with MDS, leukemia and lymphoma by MRI, no further study has published so far. Here, we examined distribution of red marrow in AS by low-dose multi-detector CT (MDCT) in BMFs patients, and analyzed the relationship between the abnormal medullary pattern in AS and laboratory variables. The relationship between the MDCT pattern and subsequent development of leukemic transformation on survivals was analyzed in patients with BMFs.Patients: We retrospectively reviewed the medical records of 138 untreated patients (AA n=36, PNH n=5, and MDS n=97) with BMFs diagnosed in the Department of Hematology/Oncology at Kameda Medical Center, Kamogawa, Japan, from July 2008 to June 2014. Follow-up MDCTs were evaluated in 28 MDS patients when they were diagnosed as overt AML (MDS/tAML). Retrospective review of clinical and laboratory features including complete blood count, % of bone marrow blast, chromosomal analysis, and International Prognostic Scoring System (IPSS) at diagnosis was performed. WHO classification of patients with MDS was as follows: RCUD (n=21), RARS (n=2), RCMD (n=26), RAEB (n=43), and, MDS unclassified (MDS-U) (n=5). Leukemia-free survival (LFS) and overall survival (OS) were analyzed in 73 patients with MDS who were ≥65 years of age and ineligible for allogeneic stem cell transplantation (allo SCT) by the Kaplan-Meier.CT image acquisition and Image analysis: Non-enhanced CT examinations were performed from the skull to the knees by MDCT scanner (Aquilion 64, Tohshiba, Tokyo, Japan). Bony canal of humeral and femoral bone were visualized by coronal and sagittal axis image reconstruction. Medullary CT density of humerus and femurs were measured and the results were expressed as Hounsfield unit (HU). As the normal adult bone marrow was composed of rich adipocytes and called yellow marrow, it is represented by low density CT value between -30 to -100 HU. The value above -30 HU observed in long bony canals was considered as high density. Medullary pattern of AS were categorized as follows: (1) fatty pattern; showing a low signal density marrow (2) focal pattern; showing abnormally focal high density lesions (3) diffuse pattern; showing uniformly high density marrow.Results: All 36 patients with AA showed a fatty (n=13, 36.1%) or focal (n=23, 63.9%) pattern in medullary AS on MDCT, and none of them showed diffuse pattern. Five patients with PNH showed as follows: fatty/focal/diffuse, 1/3/1. Ninety-seven patients with MDS showed as follows: fatty/focal/diffuse, 24/46/27. Patients with MDS who showed diffuse pattern had a significantly low hemoglobin concentration compared to those with fatty or focal pattern (p=0.03). Among the patients with MDS, most of the patients with RCUD (n=21), RARS (n=2), RCMD (n=26), MDS-U (n=5) showed the fatty or focal pattern (fatty or focal/diffuse pattern; RCUD (18/3), RARS (2/0), RCMD (21/5), MDS-U (5/0)), but approximately half (46%) of patients with RAEB showed diffuse pattern (fatty/focal/diffuse pattern; 7/17/19). In addition, patients with transformed to MDS/tAML showed either focal (n=10, 35.7%) or diffuse (n=18, 64.3%) pattern and none of them showed fatty pattern. In 73 patients with MDS who were ≥65 years of age and ineligible for allo SCT, the group with focal or diffuse pattern had significantly shorter LFS and OS compared to the group with fatty pattern (p=0.01, p=0.05, respectively). Patients with focal pattern in AS showed longer LFS than those with diffuse pattern (p=0.05), but difference was not statistically significant in OS (p=0.22).Conclusions: This study showed that MDCT imaging of the appendicular skeletons provided important information for the diagnosis and prognosis of patients with BM. In patients with MDS, focal or diffuse pattern on MDCT showed negative prognostic impact on LFS and OS, and these patterns appeared to reflect the status of disease. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quantitative Detection of DNMT3A R882H Mutation in Course of Treatment of Acute Myeloid Leukemia Patients

Introduction:DNMT3A mutation is one of the most common somatic mutations in acute myeloid leukemia (AML) patients with normal karyotype. The most frequent mutation is located at R882 codon in the methyltransferase domain. Because of prognostic significance and high stability during the disease evolution, DNMT3A mutations might represent highly informative biomarkers for prognosis and outcome of disease.Methods: Using allele-specific PCR with a Blocking reagent (ASB-PCR assay) for the quantitative detection of DNMT3A R882H mutation, we analyzed 350 follow-up samples from 28 AML patients in complete remission (CR) after induction and consolidation treatment and allogeneic stem cell transplantation (alloSCT). Seventeen patients included in the follow-up analysis harbored a NPM1 mutation. Using a well-established marker for the detection of minimal residual disease (MRD) allowed to estimate the stability of DNMT3A mutation in CR and complete molecular remission (molCR). In addition, we analyzed FLT3, IDH1, and IDH2 mutations in diagnostic and follow up samples and donor chimerism after alloSCT.Results: We found the persistence of DNMT3A R882H mutations in all patients in CR after standard therapy. On the contrary, after alloSCT, DNMT3A R882H mutation was not found in patients with CR and complete donor chimerism. In relapse of leukemia, an increasing of both NPM1 and DNMT3A mutated alleles were shown all cases.Conclusion: Persistence of DNMT3A mutation after standard chemotherapy could indicate the origin of mutation in the early pre-leukemic stem cells. The loss of correlation between NPM1 and DNMT3A in CR could be associated with leukemic clone evolution. It is impotant to note, that the removal of mutated leukemic stem cells after allo SCT indicates therapeutic options allo SCT for high risk AML patients. Increased of both DNMT3A and NPM1 mutated alleles in relapse indicates the presence of both mutations, at least partly, in the same leukemic clone.We conclude that quantitative detection of DNMT3A R882H mutations at different time points of AML disease could provide additional information about the role of mutations in development and progression of AML. DisclosuresBlau:BMS: Honoraria; MSD: Honoraria; Celgene: Honoraria, Research Funding; AMGEN: Honoraria; JAZZ Pharma: Honoraria; Shire: Honoraria; Janssen: Honoraria, Research Funding; Baxalta: Honoraria.

Efficacy of Azacitidine (AZA) in Autoimmune and Inflammatory Disorders (AID) Associated with Myelodysplastic Syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML)

Background: AID are seen in 10-30% of MDS and CMML. After initial response to steroids, AID are often poorly controlled and steroid-sparing drugs are difficult to use due to the underlying MDS/CMML. Some case reports suggest a beneficial role of AZA treatment in AID associated to MDS/CMML.Methods: We retrospectively analyzed 22 MDS/CMML patients (pts) with AID who received AZA in French centers between January 2007 and May 2014.Results: Median age of the 22 pts was 70y (range 41-84), including 6F/16M. Diagnosis of MDS/CMML preceded AID (n=8) by a median of 17 months (mo), was concomitant with (n=7) or followed AID (n=7) by a median of 20 mo. 14 pts had lower risk IPSS and 8 higher risk IPSS. AID diagnosis included: Behçet's disease (n=4), polymyalgia rheumatica (n=4), relapsing polychondritis (RP) (n=4), giant cell arteritis (n=2), Sweet's syndrome (n=2), SLE (n=2), Sjögren's (n=1), adult onset Still's disease (n=1), unclassified small vessel vasculitis (n=1) and seronegative polyarthritis (n=1). One pt presented with both RP and Sweet's syndrome. 12 (55%) AID were considered atypical and 10 (45%) fulfilled complete diagnostic criteria. Before AZA, 21 pts (96%) had received steroids and 16 (73%) a median of 2 immunomodulatory drugs (IMD) (range 1-6) in addition to steroids.At AZA onset, AID was still active (n=14), in PR (n=6) and in CR (n=2). 19 pts still reported asthenia (n=17), weight loss (n=8), fever >38°5 (n=8), rheumatologic signs (n=15), skin involvement (n=10), oral aphtosis (n=4). 9 pts were steroid dependent or resistant. 20 pts were treated with steroids (91%, median dose of 23 mg/d). 13 pts were non-responders to IMD and 11 received concomitant IMD, without efficacy in 7 pts.AZA was the first-line treatment of MDS/CMML in 13 pts (59%) and was in 9 pts preceded by: intensive chemotherapy (n=3), LEN (n=2), HY (n=2), allo SCT (n=2) and LDaraC (n=2). Median interval between MDS/CMML diagnosis and AZA onset was 9 mo (range 1-93). At AZA onset, IPSS was low (n=4), int-1 (n=7), int-2 (n=8) and high (n=3). Pts received a median of 6 cycles of AZA (range 3-14); 1 pt received 3 cycles, 5 received 5 cycles; 9 pts received more than 6 cycles. Complete remission (CR) of AID was observed in 16 pts (73%), partial remission (PR) in 3 pts (14%) and no effect or worsening of AID in 3 pts (14%). All responses were observed within 3 cycles, but 8 PR of AID after 3 cycles became CR after 6 mo. Discontinuation of steroids was possible in 3 pts (14%) and dose reduction in 14 pts (64%), to a median of 9 mg/d, (range 0-30), (p=0.001). Discontinuation of IMD was possible in 7 pts (32%). Altogether, dose reduction or discontinuation of steroids and/or IMD after AZA was possible in 16 cases (73%). Good response of AID symptoms (including PR and CR) or discontinuation of IST was noted in 19 patients (86%). Median CRP decreased from 40 mg/L to 15 mg/L (p=0.28). Response of MDS/CMML to AZA (IWG2006 criteria) was seen in 12 pts (55%), including CR in 9 pts (41%), PR in 1, SD+HI-E in 1, mCR in 1, SD in 8 and PD in 2. No uncommon side effects to AZA were seen.MDS/CMML and AID evolution was concordant in 13 pts (59%): both improved (n=11) or worsened (n=2). AID improved while MDS worsened (n=8) and vice versa (n=1). With a median FU from response to AZA of 8 mo, only 3 relapses of AID were seen after 3 (n=1) and after 19 mo (n=2), and 5 pts had a response > 12 mo. Seven pts (32%) progressed to AML and 10 pts (45%) had died, with 8 deaths occurring in IPSS high or int 2 pts. Median survival from AZA onset was 16 months in IPSS high or int-2 MDS and was not reached in IPSS low and int-1 MDS.Conclusions: AZA frequently seems effective in controlling steroid-dependent AID associated with MDS/CMML, but prospective studies are necessary to confirm those findings. DisclosuresFenaux:JANSSEN: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; NOVARTIS: Honoraria, Research Funding.

Cyclophosphamide Followed By Busulphan in Place of Standard BuCy Regimen for Patients Undergoing Allogeneic Stem Cell Transplantation: A Preliminary Experience from a Single Centre in India

Cyclophosphamide Followed By Busulphan in Place of Standard BuCy Regimen for Patients Undergoing Allogeneic Stem Cell Transplantation: A Preliminary Experience from a Single Centre in India

Management of CML blast crisis and the role of SCT

Progression of chronic myeloid leukemia to blast crisis (CML BC) is still a phenomenon that is only incompletely understood. In up to 90% of BC patients, additional chromosomal aberrations (ACA) are reported. In up to 77% mutations are detected by means of deep genome sequencing. In gene expression profiles blast crisis appears as a disease distinct from CML. Treatment continues to be mostly unsuccessful unless allo SCT is offered. Treatment failure indicates point of no return. Additional chromosomal aberrations (ACA) are associated with CML blast crisis, being non-random in chromosomal distribution. Major route ACA are +8 (34%), +Ph (30%), i(17q) (20%), +19 (13%), +21 (7%), +17 (5%),

T Cells Engineered with a Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Have Long Term Persistence and Induce Durable Remissions in Children with Relapsed, Refractory ALL

BACKGROUNDCARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains. We have previously reported on CTL019 cells expressing an anti-CD19 CAR. Infusion of these cells results in 100 to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including sustained CRs in adults and children with ALL (Grupp et al., NEJM 2013, Maude et al., NEJM 2014). We now report on outcomes and longer follow up of the first 30 pts with relapsed, refractory ALL treated on our pilot trial in pediatric ALL.METHODST cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into children with relapsed or refractory CD19+ ALL. 26/30 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency of 11-45%. T cells for manufacturing were collected from the pt regardless of prior SCT status, not allo donors.RESULTS30 children median age 10y (5-22y) with CD19+ ALL were treated. 25/30 pts had detectable disease on the day before CTL019 cell infusion, while 5 were MRD(-). A median of 3.6x106 CTL019 cells/kg (1.1-18x106/kg) were infused over 1-3 days. There were no infusional toxicities >grade 2, although 9 pts developed fevers within 24 hrs of infusion and did not receive a planned 2nd infusion of CTL019 cells. 27 pts (90%) achieved a CR, including a patient with T cell ALL aberrantly expressing CD19+. 3 did not respond. MRD measured by clinical flow cytometry was negative in 23 responding pts and positive at 0.1% (negative at 3 mo), 0.09%, 0.22%, and 1.1% in 4 pts. With median follow up 8 mo (1-26 mo), 16 pts have ongoing CR, with only 3 patients in the cohort receiving subsequent treatment such as donor lymphocyte infusion or SCT, 6-month EFS measured from infusion is 63% (95% CI, 47-84%), and OS is 78% (95% CI, 63-95%). CTL019 cells were detected in the CSF of 17/19 pts and 2 pts with CNS2a disease experienced a CR in CSF. 10 pts with a CR at 1 mo have subsequently relapsed, half with CD19(-) blasts. 2/5 pts who relapsed with CD19(-) disease had previously been refractory to CD19-directed blinatumomab and subsequently went into CR with CTL019. [Display omitted] All responding pts developed grade 1-4 cytokine release syndrome (CRS) at peak T cell expansion. Detailed cytokine analysis showed marked increases of IL6 and IFNγ (both up to 1000x), and IL2R. Treatment for CRS was required for hemodynamic or respiratory instability in 37% of patients and was rapidly reversed in all cases with the IL6-receptor antagonist tocilizumab, together with corticosteroids in 5 pts. Although T cells collected from the 21 pts who had relapsed after allo SCT were median 100% donor origin, no GVHD has been seen. Grade 4 CRS was strongly associated with high disease burden prior to infusion and with elevations in IL-6, ferritin (suggesting macrophage activation syndrome) and C reactive protein after infusion.Persistence of CTL019 cells detected by flow cytometry and/or QPCR, and accompanied by B cell aplasia, continued for 1-26 months after infusion in pts with ongoing responses. QPCR showed very high levels of CTL019 proliferation, with all patients achieving peak levels >5000 copies/ug gDNA and 26 patients with peak levels >15,000 copies/ug gDNA. B cell aplasia has been treated with IVIg without significant infectious complications. Probability of 6-mo CTL019 persistence by flow was68% (95% CI, 50-92%) andrelapse-free B cell aplasia was 73% (95% CI, 57-94%). CONCLUSIONSCTL019 cells can undergo robust in-vivo expansion and can persist for 2 years or longer in pts with relapsed ALL, allowing for the possibility of long-term disease response without subsequent therapy such as SCT. This approach also has promise as a salvage therapy for patients who relapse after allo-SCT with a low risk of GVHD. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL; however, recurrence with cells that have lost CD19 is an important mechanism of CLT019 resistance. CTL019 therapy has received Breakthrough Therapy designation from the FDA in both pediatric and adult ALL, and phase II multicenter trials have been initiated. DisclosuresGrupp:Novartis: Consultancy, Research Funding. Barrett:Novartis: Research Funding. Chew:Novartis: Research Funding. Lacey:Novartis: Research Funding. Levine:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding. Rheingold:Novartis: Consultancy. Shen:Novartis: Employment. Wood:Novartis Pharma: Employment. Porter:Novartis: managed according to U Penn Policy Patents & Royalties, Research Funding. June:Novartis: Research Funding, Royalty income Patents & Royalties.

A Phase II Study of the Combination of FCR-Lite and Lenalidomide Followed By Lenalidomide Maintenance in Front-Line CLL: The FCR2 Regimen

Introduction: Fludarabine, cyclophosphamide, rituximab (FCR) remains the standard of care for the treatment of newly diagnosed, fit CLL pts requiring therapy. However, the FCR side effects profile is non-trivial and CLL pts with poor risk features have inferior outcomes. Strategies aimed at minimizing toxicity without compromising efficacy have been reported including modifications of the optimal dosing and duration of FCR using a dose-reduced approach (Foon et al, “FCR lite”) or reduction in number of FCR cycles based on MRD status (Strati et al). In the era of biological agents, both preclinical and clinical data have shown the efficacy of lenalidomide (Len) in CLL likely through its role as an immunomodulator and interference with interactions between CLL and its microenvironment. In CLL, Len presents a clinical opportunity both in combination with chemotherapy and as maintenance strategy. This was the rationale for our trial using Len in combination with FCR and as maintenance to improve outcomes and shorten therapy.Methods: We conducted a phase 2 cohort study examining the combination of dose-reduced FCR and Len (“FCR2”) followed by Len maintenance for treatment naïve, CLL pts requiring therapy (NCIWG criteria). Eligible pts (age ≥18 yr, ECOG 0-2, CrCl ≥30 ml/min, absence of AIHA) were treated with 4-6 cycles of FCR2 (D1-3 fludarabine 20 mg/m2, D1-3 cyclophosphamide 150 mg/m2, D1&15 rituximab 500 mg/m2 every 28 days). Len was administered on D 8-28 of each cycle (starting dose of 5 mg increasing to 10 mg and 15 mg in cycle ≥ 2 based on toxicity algorithm). Pts who were MRD (-) in PB and BM (multicolor flow cytometry) initiated Len maintenance after cycle 4 FCR2 (otherwise proceeded to 6 cycles). Supportive care included asa 81 mg, pegfilgrastim, and antimicrobial prophylaxis (ciprofloxacin, bactrim, fluconazole, acyclovir). Daily Len maintenance started two months after FCR2 completion in responding pts for a total of 12 months (5-15 mg based on toxicity algorithm). The primary study endpoint was the proportion of CR pts after cycle 4 of FCR2 with ≥ 8/19 (≥ 40%) CR+CRi considered a positive result and worthy of further study (α 0.05, β 90%). Secondary endpoints included MRD status, ORR, OS, PFS and toxicity (CTCAE V. 4.0). We report the primary endpoint and preliminary data on non-survival secondary endpoints.Results: 25 pts met inclusion criteria for enrollment of which 22 pts enrolled and 19 were evaluable (consent was withdrawn on 3 pts after ≤ 4 FCR2 cycles due to pt preference n=1, physician preference n=2, one additional pt withdrew consent after cycle 5 to proceed with Allo SCT.) Baseline characteristics expressed as median (range) were as follows: age 62.5 yr (42-75 yr), ECOG PS 1 (0-2), WBC 115.4 x 109/L (8.7-301.7), Hb 12.2 g/dL (7.8-15.3), Plt 131 x 109/L (60-178), IgG 602 mg/dL (294-1018), β2mg 2.8 μg/mL (1.74-6.69), LDH u/L 471 (132-1022), BM % lymphs 75 (25-100%), baseline SPD 23.4 (2.9-63.5). In addition 30% pts had Rai stage III-IV, 53% unmutated-CLL, 5% del17p and 30% del11q. After 4 cycles of FCR2 (n=19) response rates were as follows CR 47%, CRi 5%, PR 42%, SD 5% (pts were MRD neg in 29% BM and 56% PB samples, 3 pts (16%) were MRD neg in both PB and BM). After 6 cycles of FCR2 (n=16), response rates improved and were as follows: 63% CR, 13% CRi, 19% PR, 5% PD (pts were MRD neg in 50% BM and 72% PB samples). Table 1 shows MRD status following 4 and 6 cycles of FCR2. During induction, 12 pts required a treatment interruption or dose reduction of Len per protocol (median dose of len was 10 mg). The most common grade 3-4 toxicities were as follows (% events): neutropenia (51%), leukopenia (20%), hyperglycemia (5%), NTP fever (5%). Grade 3-4 TLS and tumor flare were not noted. The med follow up for the entire cohort is 13.1 months (7.3-22.5 months). One progression has occurred in a pt who withdrew from study after 2 cycle of FCR2. One death has occurred in a pt with del17p who died on D+255 following allogeneic SCT in CR from the complications of cGVHD. 10 pts have gone on to initiate Len maintenance.Conclusions: FCR-lite in combination with Len is feasible and demonstrates encouraging clinical activity in a high-risk, CLL pt population with an acceptable toxicity profile. A significant proportion of pts are MRD neg in PB and/or BM following FCR2. Addition of Len to FCR may minimize chemotherapy exposure without compromising outcome. Table 1:MRD StatusFollowing Cycle 4 FCR2Following Cycle 6 FCR2Bone Marrow29%50%Peripheral Blood56%72% DisclosuresMato:Celgene : Honoraria, Research Funding, Speakers Bureau.

Cyclophosphamide Followed By Busulphan in Place of Standard BuCy Regimen for Patients Undergoing Allogeneic Stem Cell Transplantation: A Preliminary Experience from a Single Centre in India

▪Conditioning regimens are an important issue determining the outcome of hematopoietic stem cell transplantation (HSCT). Altering the administration order of Busulphan (Bu) and Cyclophosphamide (Cy) during conditioning from conventional method of administering Bu followed by Cy had resulted in an improved toxicity profile in few animal and subsequent human studies. However the data substantiating this approach is limited.We retrospectively analyzed all consecutive patients receiving allogeneic stem cell transplant (Allo SCT) with myeloablative conditioning from 2009 to 2013. A total of 40 patient received Allo SCT of which 18 patient received Bu-Cy and 22 patients received Cy-Bu conditioning regimen. The Bu–Cy conditioning regimen consisted of i.v. Bu 0.8 mg/kg administered every 6 h (16 doses) on days −7 to −4, followed by i.v. Cy 60 mg/kg on days −3 and −2. Patients with the Cy–Bu regimen received i.v. Cy 60 mg/kg on days −7 and −6; followed by i.v. Bu 0.8 mg/kg administered every 6 hr (16 doses) on days −5 to −2. GVHD prophylaxis was given with Cyclosporine A and methotrexate. Common Terminology Criteria for Adverse Events version 3.0 (CTCAE) was used for assessment of toxicity. The diagnosis of sinusoidal obstruction syndrome (SOS) was based on modified Seattle criteria.Pre-transplant characteristics were comparable in the two cohorts (Table 1a and 1b). Time to platelet engraftment was earlier in the Cy-Bu cohort (21 days vs. 16 days; P=0.008) (Table 2).Treatment related side effects were similar in both the groups except hepatotoxicity which was higher in Bu-Cy as compared to Cy-Bu group (10 (55.16%) vs. 3 (13.64%); p=0.03). There was no significant difference in treatment related mortality (TRM) at day 100; however there was trend towards higher TRM at day 30 in Bu-Cy group (3 vs. none; p=0.083).There was no difference in aGVHD incidence, grade or stage of organ involved between the 2 groups.As in previous studies hepatotoxicity in the present analysis was found to be less in patients who received Cy-Bu as the conditioning regimen and there was earlier platelet engraftment in this group. These findings suggest Cy-Bu has better toxicity profile than conventional Bu-Cy regimen. However further prospective studies are required to confirm these findings.Table 1aBaseline CharacteristicsPatient Characteristics Bu-Cy (n=18)Cy-Bu (n=22)P valueSexMale15160.424Age (in yrs)Median (Range)17 (1-50)16 (1-48)≤18913≥1899Underlying DiseaseAML14120.415ALL34CML13MDS02Primary Myelofibrosis01Pre-transplant Remission StatusCR11110.482Performance status (ECOG)0430.3141916HSCT Comorbidity Index014150.341115222310Table1bBaseline CharacteristicsTransplant characteristicsBu-CyCy-BuP valueTime from diagnosis to TransplantMean (days) + SD548.16750.567HLA MatchingHLA identical (6/6)17210.884HLA mismatch (5/6)11DonorMatched Sibling17160.884UCB11Donor AgeMedian(Range)20 (0-47)16 (0-50)0.781Donor SexMale5140.034Female128Sex mismatch12130.458Female Donor in Male patient1170.064Harvest SourcePeripheral Blood (PB)15210.269Bone Marrow (BM)20Cord Blood (UCB)11CD 34 Count(x106 cells/kg)Mean+SD5.12+2.605.66+2.370.499CD 3 Count(x107 cells/kg)Mean25.517.50.200Table 2ResultsOutcomeBu-CyCy-BuPANC recovery14 (11-30)11 (8-32)0.119Platelet recovery21 (17-44)16 (11-27)0.008Platelets transfused6 (1-10)4 (1-15)0.166Days of GCSF18 (12-36)14 (9-37)0.126D100 Complete Donor Chimerism9 (90%)12(85.71%)1.000Transplant Response15/17 (88.24%)19/22 (86.36%)1.000 Days of Antimicrobial use13 (0-34)13 (0-36)0.83 Hepatotoxicity (grade3-4)10/18 (55.56%)3/22 (13.64%)0.030Nephrotoxicity (grade3-4)3/18 (16.67%)1/22 (4.55%)0.204Mucositis (grade3-4)6/18 (33.33%)6/22 (27.27%)0.677Any Grade 3-4 toxicity10/18 (55.56%)9/22 (40.91%)0.356D30 TRM3 (16.67%)00.083D100 TRM4 (22.22%)2 (10%)0.395Follow up28.67 months7.6 monthsAcute GVHD3/18 (16.67%)5/22 (22.73%)0.709 DisclosuresNo relevant conflicts of interest to declare.

T Cells Engineered With a Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Produce Significant In Vivo Proliferation, Complete Responses and Long-Term Persistence Without Gvhd In Children and Adults With Relapsed, Refractory ALL

▪ BackgroundCARs combine a single chain variable fragment (scFv) of an antibody with intracellular signaling domains into a single chimeric protein. We previously reported on CTL019 cells expressing a CAR with intracellular activation plus costimulatory domains. Infusion of these cells results in 100 to 100,000x in vivo proliferation, durable anti-tumor activity, and prolonged persistence in pts with B cell tumors, including 1 sustained CR in a patient with ALL (Grupp, et al. NEJM 2013). We now report on outcomes and longer follow up from our pilot studies treating 20 pts (16 children and 4 adults) with relapsed, refractory ALL. MethodsT cells were lentivirally transduced with a CAR composed of anti-CD19 scFv/4-1BB/CD3ζ, activated/expanded ex-vivo with anti-CD3/anti-CD28 beads, and then infused into pts with relapsed or refractory CD19+ ALL. 17/20 pts received lymphodepleting chemotherapy the week prior to CTL019 infusion. The targeted T cell dose range was 107 to 108 cells/kg with a transduction efficiency (TE) of 11-45%. On the adult protocol, the target dose was 5 x 109 total cells split over 3 days with a TE of 6-31%. 11 pts had relapsed ALL after a prior allogeneic SCT. T cells were collected from the pt, regardless of prior SCT status, and not from allo donors. All pts s/p allo SCT had to be 6 mos s/p SCT with no GVHD or GVHD treatment. Results16 children median age 9.5 y (5-22y) and 4 adults median age 50y (26-60y) with CD19+ ALL were treated. One child had T cell ALL aberrantly expressing CD19. 14/16 pediatric pts had active disease or +MRD after chemotherapy on the day prior to CTL019 cell infusion, while 2 were MRD(-). 3 of 4 adults had active disease prior to lymphodepleting chemotherapy, while 1 was in morphologic CR. Lymphodepleting chemotherapy varied with most receiving a Cytoxan-containing regimen the week prior to CTL019. A median of 3.7x106 CTL019 cells/kg (0.7-18x106/kg) were infused over 1-3 days.There were no infusional toxicities >grade 2, although 5 pts developed fevers within 24 hrs of infusion and did not receive planned subsequent infusions of CTL019 cells. 14 patients (82%) achieved a CR, including the patient with CD19+ T ALL, 3 did not respond, and 3 are pending evaluation. 11/17 evaluable pts have ongoing BM CR with median follow up 2.6 mo (1.2-15 mo). Three patients with a CR at 1 month have subsequently relapsed, 1 with CD19(-) disease. Median follow-up as of August 1, 2013 was 2.6 mo (1-15 mo) for all pts.All responding pts developed some degree of delayed cytokine release syndrome (CRS), concurrent with peak T cell expansion, manifested by fever, with variable degrees of myalgias, nausea, anorexia. Some experienced transient hypotension and hypoxia. Detailed cytokine analysis showed marked increases from baseline values of IL6 and IFNγ (both up to 1000x), and IL2R, with mild or no significant elevation in systemic levels of TNFα or IL2. Treatment for CRS was required for hemodynamic or respiratory instability in 7/20 patients and was rapidly reversed in all cases with the IL6-receptor antagonist tocilizumab (7 pts), together with corticosteroids in 4 pts. Although T cells collected from the 11 pts who had relapsed after allo SCT were generally 100% of donor origin, no GVHD has been seen. Persistence of CTL019 cells detected by flow cytometry and/or QPCR in pts with ongoing responses continued for 1-15 months after infusion, resulting in complete B cell aplasia during the period of CTL019 persistence. Pts have been treated with IVIg without any unusual infectious complications. One child who entered a CR subsequently developed MDS with a new trisomy 8 in ALL remission and has gone to SCT, and 1 child developed a single leukemia cutis lesion at 6 mo, still BM MRD(-). ConclusionsCTL019 cells are T cells genetically engineered to express an anti-CD19 scFv coupled to CD3ζ signaling and 4-1BB costimulatory domains. These cells can undergo robust in-vivo expansion and can persist for 15 mo or longer in pts with relapsed ALL. CTL019 therapy is associated with a significant CRS that responds rapidly to IL-6-targeted anti-cytokine treatment. This approach has promise as a salvage therapy for patients who relapse after allo-SCT, and collection of tolerized cells from the recipient appears to have a low risk of GVHD. CTL019 cells can induce potent and durable responses for patients with relapsed/refractory ALL. Multicenter trials are being developed to test this therapy for ALL in the phase 2 setting. Disclosures:Grupp:Novartis: Research Funding. Chew:Novartis: Patents & Royalties. Levine:Novartis: cell and gene therapy IP, cell and gene therapy IP Patents & Royalties. Litchman:Novartis Phamaceuticals: Employment, Equity Ownership. Rheingold:Novartis: Research Funding. Shen:Novartis Pharmaceuticals: Employment, Equity Ownership. Wood:Novartis Pharmaceuticals: Employment, Equity Ownership. June:Novartis: Patents & Royalties, Research Funding.

The Pan- JAK Inhibitor Ruxolitinib Impairs T-Cell Activation, Cytokine Production and Proliferation In Vivo and In Vitro

Myelofibrosis (MF) is a clonal myeloproliferative neoplasm, in which the JAK2-V617F mutation is frequently observed. The appearance in up to 50% of the cases makes the JAK2 mutation attractive as therapeutical target. In 2012 Ruxolitinib (Ruxo) a pan-JAK inhibitor was approved for the treatment of MF and showed efficacy in disease treatment, irrespectively of the JAK2V617 mutation status. Currently allogeneic stem cell transplantation (allo SCT) remains the only curative treatment option for MF. To further improve transplant outcome in MF reduction of spleen size and constitutional symptoms prior transplantation is a reasonable target. Harnessing graft versus myelofibrosis post transplantation by immune-modulating drugs may help to reduce the risk of relapse. Ruxolitinib may be used as pre- and post-transplantation drug to improve transplant outcome. However the impact of Ruxolitinib on the immune system, especially on T-cells, is poorly understood. Here we investigated the effects of Ruxolitinib on T-cells in vivo and in vitro.T-cells from healthy donors were isolated by magnetic cell sorting to pan CD3+, CD4+ and CD8+ fraction. All three different cell subsets were cultured with different dosages of Ruxolitinib (100, 250, 500, 750nM and 1µM) for additional 48h. Thereafter cells were analysed for cell growth, cell death, RNA expression, immune phenotype.Additionally, immune profiles of 9 patients were analysed for the changes of the T-cell compartment during the treatment with Ruxolitinib over a period of 3 weeksT –cells from healthy donors showed a dosage dependent impairment in the proliferation capacity compared to non-treated control cells (4.1x106 CD3 cells /ml vs. 1.9x106 CD3 cells /ml, p<0.05), additionally KI67 expression was reduced from 48% in control cells to 12% in 100nM treated CD3 cells and 9% in 500nM treated CD3 cells, p<0.05. Strikingly apoptotic cell death increased from 11% in control cells to 43% and 48% in 100nM and 500nM Ruxo treated cells, p<0.03.Analysing the immune phenotype of Ruxo treated CD3, CD4 and CD8 cells we found a significant reduction in the expression of activation marker like CD25 and HLA-DR (38% vs. 6% and 4.5% respectively, p<0.05 and 63% vs. 47% and 40% respectively, p<0.05). Furthermore, we found that the effector cells, marked by CCR7/CD45RA expression, decreased in the CD8 compartment from 22% to 10.5% and 7.8% respectively, p<0.05. When analysing regulatory T-cells we also observed a decrease in a dose dependent manner (4% vs. 1.2% and 0.8%, p=0.05). While control Treg showed a KI67 expression of >60%, Ruxo (100nM) treated T-reg did not expressed KI67. Likewise to CD8 effector cells and Tregs we found a decrease in pro-inflammatory TH1 and TH17 cells in vitro (27% vs. 14% and 12% for TH1 cells and 6% vs. 4% and 4% for TH17 cells).Next, we analysed mRNA expression and found that pro-inflammatory cytokines like IL23, IL18, IL7 were down regulated after Ruxo treatment. To in contrast to pro- inflammatory cytokines, p53 and cell cycle inhibitor of the cip/waf locus showed to be up regulated in CD3 and CD4 cells suggesting that the observed increase in apoptosis in T-cells is mediated by p53.We next investigated the impact of Ruxolitinib on T-cells in patients. Therefore we analysed the blood of patients treated with Ruxolitinib in weekly intervals. Likewise to in vitro CD3 cells showed a decrease which turned to be significant after two and three weeks of treatment (1560/µl vs. 688/µl and 410/µl, p<0.05), this was mainly through the reduction of CD8+ T-cells (630/µl before treatment vs. 250/µl at week 2 and 200/µl at week 3, p <0.05). We also observed a decrease of CD3+/ HLA-DR+ (as activation marker) from 355/µl before to 130/µl and 70/µl however this did not reached statistical significance. The same was found for Tregs in vivo (5.6% vs. 2.3% and 1.9%, respectively).These data argue that treatment of T-cells by Ruxolitinib impairs their proliferation capacity by inducing apoptosis through an up regulation of p53. This increase of cell death applies all analysed T-cell compartments, and thereby may explains why Ruxo treated T-cells were less able to show a pro-inflammatory as well as regulatory phenotype. Disclosures:No relevant conflicts of interest to declare.

Consolidation Chemotherapy for Adults With AML in First Remission: Is There a Best Choice?

Consolidation Chemotherapy for Adults With AML in First Remission: Is There a Best Choice?

Recurrent opportunistic infections in a post-transplant lymphoma patient

Introduction: Infections and graft-versus-host disease (GVHD) are the most notable problems after allogeneic stem cell transplantation (Allo SCT). Case Report: A 50-year-old patient underwent an Allo SCT for transformed follicular lymphoma (t-FL). Transplant course of the patient was complicated by GVHD and recurrent, severe, opportunistic infections including pneumonia caused by two fungal pathogens simultaneously, recurrent bacteremia with no apparent source, septic arthritis caused by an unusual pathogen and eventually developed progressive multifocal leukoencephalopathy (PML). Conclusion: This case illustrates the diversity of opportunistic infections affecting patients undergoing Allo SCT and the complexity involved in diagnosis and treatment of such infections.

A subset of CD8+ T cells acquiring selective suppressive properties may play a role in GvHD management

Difficulty in segregating graft-versus-tumor effect (GvT) from graft-versus-host disease (GvHD) remains a major limitation of allogeneic stem cell transplantation (Allo SCT). Naturally occurring regulatory T cells have been suggested to suppress alloreactive T cells involved in GvHD; however, their non-selective suppressive effect raises concern regarding probable attenuation of the GvT effect. Recent studies suggested inducible CD8 (iCD8) cells to be useful in suppressing autoimmune reactions, although their function in the Allo SCT setting has not been fully explored. The current study assessed in-vitro the properties of iCD8 T cells, generated in response to allogeneic dendritic cells (DCs), imitating the Allo SCT conditions. CD25− peripheral blood mononuclear cells (PBMCs) were stimulated with allogeneic DCs in mixed lymphocyte culture (MLC). The resultant iCD8+CD25+ population was isolated and assessed for phenotypic markers, cytokine expression profile, cell proliferation, inhibitory capacity and anti-viral response. The generated CD8+CD25+FOXP3+ T cells selectively inhibited the primary allogeneic response, without attenuating T cell response against other stimuli, such as mitogens or a cytomegalovirus (CMV) recall antigen. In conclusion, iCD8+CD25+ cells suppress allogeneic stimulation, while maintaining the capacity to respond to infectious pathogens. These cells could be potentially efficient in the Allo SCT setting, where GvHD prevention is required.

Salvage Therapy with the Combination of ARA-C and Gemtuzumab Ozogamicin in Post-Allo-SCT High Risk AML Patients Is Feasible and Results in Modest Responses.

AbstractAbstract 2629Acute myeloid leukemia (AML) relapse is often associated with a poor outcome, in particular in the post allogneic stem cell transplantation ( Allo-SCT) setting. Therapeutic options in patients relapsing early after SCT are further limited partially due to the risk of increased toxicity. In patients who did not develop graft versus host disease ( GVHD), reduction or cessation of immunosuppression therapy may result in the achievement of a graft versus leukemia (GvL) effect. However, this is usually only beneficial in patients with a low disease burden. Gemtuzumab Ozogamicin (GO) was introduced into therapy of CD33 positive AML patients as a targeted therapy with a relatively safe extra-medullary toxicity profile. Although withdrawn from the USA market by the FDA, recent studies have shown efficacy of combining GO with chemotherapy in newly diagnosed AML patients. We hypothesized that in patients with AML relapsing early after an Allo-SCT, combination of GO with single agent chemotherapy will result in disease control for a sufficient amount of time to allow a trial of GvL induction. Therapy consisted of high dose ARA-C (1 gr/m2) for 4 days followed by one dose of GO, 9 mg/m2 on day 5. To decrease hematopoietic toxicity, therapy was supported by donor stem cells, when available. Responding patients which did not develop GVHD, where eligible for cell mediasted immunotherapy with a donor lymphocyte infusion(DLI) or a second Allo-SCT. 16 patients (F=10, M=6), median age 52 years (range 26–63) are included in this analysis. The majority (14/16) of patients were previously transplanted for a high risk AML (secondary AML-6, unfavorable cytogenetics-5, FLT3-ITD-3); disease status at SCT were CR1 (n=8), CR2 (n=4), untreated 1st relapse (n=1) and at diagnosis (n=1, MDS with excess of blasts). AML relapse occurred at a median of 104 days (range 15–403) after SCT. ARA-C/GO therapy was supported with donor stem cells in 8/16 patients and was relatively well tolerated. One patient died in aplasia in day +2 due to sepsis resulting in overall 6% induction mortality. Five patients developed veno occlusive disease of the liver ( VOD )(36% of evaluable patients); all were mild and resolved with symptomatic care. Responses were achieved in 8/15 (53%) evaluable patients, all CR/CRp. Four patients in CR/CRp received further therapy with DLI (n=3) or Allo SCT from a different donor (n=1), and 2 non-responding patients received a second Allo-SCT after second line salvage chemotherapy. At long follow up, 1 patient (6%) is alive in and continuous remission (1040 days after therapy). In patients surviving induction, death occurred due to relapse (n=10) or severe GVHD (n=5). Median EFS and OS were 55 and 133 days, respectively in the entire cohort. Median EFS was longer in responding patients (156 vs. 16 days, p<0.0001), while median OS was not significantly different in these 2 patients cohorts (204 vs. 33 days, p=0.2).ARA-C/GO combination is a valid option for salvage therapy in AML patients relapsing after an Allo-SCT and is well tolerated. Although responses are usually of a short duration, they may allow immunogenic manipulation in the absence of an already established significant GVHD, and may result in a long term disease control in a minority of patients. Disclosures:No relevant conflicts of interest to declare.

Durable Remission of Renal Cell Carcinoma After Donor Lymphocyte Infusion Is Unavoidably Linked with Graft Versus Host Disease As Illustrated by the Detection of Allo Reactive T Cells Recognizing a Novel Minor Histocompatibility Antigen Encoded by the FUCA2 Gene

AbstractAbstract 4467Allogeneic stem cell transplantation (allo SCT) followed by donor lymphocyte infusion (DLI) can be applied as immunotherapeutic intervention to treat malignant diseases. In hematological malignancies, remissions can be observed both in the presence and absence of graft versus host disease (GvHD), illustrating that graft versus leukemia and GvHD can be clinically separated. For efficient induction of an allo immune response, professional (hematopoietic) antigen presenting cells (APC) are required, which may result in skewing towards recognition of minor histocompatibility antigens (MiHA) selectively expressed by hematopoietic cells. However, for treatment of solid tumors including renal cell carcinoma (RCC), recognition of MiHA on non hematopoietic cells is essential. Therefore in these malignancies APC have to present broadly expressed MiHA to be targeted on non hematopoietic cells, making it inevitable that the graft versus tumor (GvT) effect is accompanied by GvHD.Here we describe the treatment of a patient who presented with progressive metastatic clear cell RCC with T cell depleted non myeloablative allo SCT. At 7 months after allo SCT the patient received DLI at a single dose of 0.5*107 T cells/kg. Acute skin GvHD occurred within 30 days after DLI and developed soon thereafter into persistent extensive chronic GvHD for several years that eventually resolved after prolonged topical and systemic immune suppression. Whereas the patient was mixed chimeric after allo SCT (84%, 80% and 95% donor after 2, 3 and 5 months, respectively), treatment with DLI resulted in conversion to full donor chimerism, which remained complete during the following years. The strong immune response was accompanied by an approximately 50% reduction in size of the measurable lung metastasis (according to RECIST criteria) and slight regression of the smaller lung lesions. To characterize the specificity of the immune response, we measured known MiHA, and detected a disparity for the previously identified MiHA LRH-1. T cells specific for this MiHA were detectable in peripheral blood at the onset of GvHD (0.14% of CD8+ T cells). Isolated LRH-1 specific T cells strongly recognized patient derived EBV-LCL, but were irresponsive to monocyte derived dendritic cells (monoDC), fibroblasts, keratinocytes, and a panel of LRH-1+ RCC cell lines and are therefore considered irrelevant for the GvT effect. In addition, we directly cloned activated CD8+ T cells and isolated an HLA-B*07 restricted T cell clone with unknown specificity. Whole genome association scanning unraveled a patient specific single nucleotide polymorphism in the FUCA2 gene encoding a novel MiHA which we designated LB-FUCA2-1V. High frequencies of circulating LB-FUCA2-1V specific T cells were detected after DLI (1.9% of CD8+ cells). Moreover, in contrast to LRH-1 specific T cells, LB-FUCA2-1V specific T cells recognized monoDC, and skin derived keratinocytes and fibroblasts after IFN-γ pretreatment. Our data also show recognition of 4 RCC cell lines, and therefore support a dominant role for LB-FUCA2-1V specific T cells in mediating tumor regression. Unfortunately, coinciding with the gradual resolvement of chronic GvHD, the GvT effect declined and eventually the metastases became progressive almost 3 years after allo SCT. In an attempt to re-initiate the GvT effect, escalating DLI doses of 0.2*107, 0.5*107, 1*107 and 5*107 T cells/kg were given. No GvHD developed but also no GvT effect could be achieved and the patient died of progressive disease 8.5 years after allo SCT.In conclusion, we observed a strong donor derived immune response targeting the broadly expressed LB-FUCA2-1V MiHA that durably controlled metastatic clear cell RCC. Initiation of this response likely depended on the presence of patient hematopoiesis, including APC, since all attempts to re-induce the MiHA specific response to treat disease progression with escalating doses of DLI under full donor chimerism were ineffective. Our data suggest that successful treatment of solid tumors may require induction of an effective immune response against broadly expressed MiHA, inevitably inducing damage to normal tissue cells resulting in GvHD. Disclosures:No relevant conflicts of interest to declare.

Allogenic Transplantation in Lymphomas: A Focused Analysis On Aggressive Lymphomas and Factors Predictive of Outcome in a Series of 179 Pts Treated At John Theurer Cancer Center.

AbstractAbstract 3120▪▪This icon denotes a clinically relevant abstract Introduction:Chemo-immunotherapy (i.e. rituximab combinations) has clearly had a significant impact on the outcome of all B-cell NHL both in terms of PFS and OS. However in the relapse/refractory setting a large proportion of pts still do very poorly especially in aggressive subtypes including DLBCL and MCL. Salvage therapy followed by HDT-ASCT in relapsed DLBCL remains the standard, though pts with early failures (<1y) and/or prior exposure to rituximab still show dismal results (CORAL data). In MCL the use of HDT-ASCT in the relapse setting is debated given the frequency of chemo-resistance leading to poor results even in second CR. The use of allogeneic transplantation was developed based on observations c/w with a clear GVL effect in NHL as illustrated by pts going into remission after DLI injections. The development of non-myeloablative approaches has allowed expansion of use of allogeneic BMT in relapsed/refractory NHL. We report here one of the largest series (179 consecutive pts) with relapsed/refractory lymphoma focusing on overall survival and outcome predictors. Methods:Utilizing Kaplan-Meier survival and Cox regression methods, we report on the outcome of 179 consecutive pts with relapsed/refractory lymphoma who underwent allogeneic stem cell transplantation at the John Theurer Cancer Center between 1995–2012. The primary study endpoint was overall survival (OS) assessed by chart and SSDI database review. Secondary study endpoints included examination of the association between overall survival and allogeneic stem cell source, donor source, development of GVHD, pre-transplant chemo-sensitivity and prior failure to HDT-ASCT (second transplant). The proportional hazards assumption was met for this analysis. Results:Survival data on 179 pts (median age 48, range 20–71) were analyzed, representing 86 deaths and 5720 total months at risk (median follow up=12.3 months). Baseline characteristics included: ECOG PS (med 1, range 0–2), diagnosis (25% DLBCL, 21% HD, 20% MCL, 13% FCL, 13% PTCL, 8% other), donor source (50% matched SIB, 31% MUD, 19% mismatched MUD), stem cell source (73% PB, 23% BM, 6% Cord) and prior autologous SCT (38%). The median OS for the entire cohort was 31.2 months. OS KM curves by selected aggressive NHL subtypes are represented in Figure 1. We performed COX regression analyses to address outlined secondary endpoints. In univariate analyses statistically significant inferior outcomes were associated with the use of mismatched unrelated donor (HR 1.4, p=.01, Figure 2), bone marrow donor stem cells vs. PBSCT (HR=1.7 p=.04), pre-transplant stable/refractory disease (HR 1.8, p=.03), absence of cGVHD (HR=4.7, p<.001) and presence of acute GVHD (HR 2.8, p=.001). No difference in OS was detected whether pts had undergone allogeneic SCT as a second transplant (med time between auto/allo=20.9 months) following relapse after auto SCT (HR 1.14, CI .75–1.73, p=.5). Conclusions:This series represents a large cohort of poor risk, relapsed/refractory lymphoma pts treated consecutively with allogeneic stem cell transplantation over a > 10-year period at our institution with the following observations: 1.These data clearly demonstrate that a sizable subset of pts with either HL or aggressive NHL (MCL, DLBCL, PTCL) can enjoy long disease-free survival following allo SCT (beyond the expectation of standard salvage chemotherapy).2.Relapse following autologous SCT should not preclude consideration of allo SCT as second salvage.3.New strategies to improve outcome include our ongoing TH2 study which led to reduced GVH incidence and promising outcome.4.Our ongoing study looking at expanded donor derived T-cells is showing promising results in reducing GVH incidence and improving outcome, including in aggressive NHL, and will be presented on a separate presentation. [Display omitted] [Display omitted] Disclosures:Mato:Celgene: Speakers Bureau; Millennium: Speakers Bureau; Seattle Genetics: Speakers Bureau; Genentech: Speakers Bureau. Goldberg:Eisai: Speakers Bureau. Feldman:Allos: Speakers Bureau; celgene: Speakers Bureau; Seattle Genetics: Speakers Bureau; Merck: Speakers Bureau. Goy:Milennium: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity’s Board of Directors or advisory committees; Seattle Genetics: Membership on an entity’s Board of Directors or advisory committees; Pharmacyclics: Membership on an entity’s Board of Directors or advisory committees; J & J: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Membership on an entity’s Board of Directors or advisory committees.

Clinical Significance of Mixed Phenotype in Adult Patients with Philadelphia Chromosome-Positive Acute Leukemia–no Prognostic Impact in the Imatinib Era.

Abstract 2574 Background:Mixed phenotype acute leukemia (MPAL) has historically been known as biphenotypic acute leukemia (BAL), and novel diagnostic criteria for this disease entity are described in the World Health Organization (WHO) classification 4th edition. As the most common recurrent genetic abnormality observed in MPAL is the bcr-abl fusion gene, Philadelphia chromosome-positive MPAL (Ph+MPAL) has been recognized as one distinctive disease entity. The prognosis of Ph+B-cell acute lymphoblastic leukemia (Ph+B-ALL) has been dramatically improved with the introduction of imatinib, and the goal of this study was to determine whether imatinib results in a survival benefit in the context of Ph+MPAL. Patients and Methods:We retrospectively analyzed 42 consecutive adult patients who were diagnosed with Ph+AL between January 2001 and March 2012 at Gunma University Hospital and Saiseikai Maebashi Hospital in Gunma, Japan. Ph+AL was diagnosed based on detection of the bcr-abl fusion gene with the polymerase chain reaction method and the presence of more than 20% of blasts in the peripheral blood and/or bone marrow. Patients with a previous history of chronic myelogenous leukemia were excluded. The lineage of leukemia cells was defined according to the WHO classification 4th edition. All patients received intensive chemotherapy and concurrent administration of imatinib. The c2-test was used for comparison of binary variables. The Mann-Whitney U test was used for comparison of continuous variables. Overall survival (OS) rate was estimated by the Kaplan-Meier method and were compared using the log-rank test. P < 0.05 was considered as statistically significant. Results:According to the WHO classification 4th edition, 13 (31%) patients were categorized as Ph+MPAL (positive for both myeloid and B-cell lineage), 27 (64%) patients were categorized as B-cell lineage acute lymphoblastic leukemia (Ph+B-ALL), and two (5%) patients were categorized as acute myeloid leukemia (Ph+AML). Patients with Ph+AML were excluded from this study, as the number of patients was relatively small. Of the 40 Ph+AL patients, 23 patients were men, and 17 were women, and the median age was 53 years (range, 16–75 years). Age, sex, white blood cell counts, lactate dehydrogenase levels, and the prevalence of additional cytogenetic abnormalities at diagnosis were not significantly different when comparing the groups, although patients with Ph+MPAL showed significantly higher frequency of major bcr-abl gene than those with Ph+B-ALL (69% and 19%, respectively; p < 0.01). Immunophenotypic analysis revealed that Ph+MPAL patients expressed CD10 and CD34 with significantly lower frequency than Ph+B-ALL patients. Notably, positivity of myeloid antigens (CD13 and 33) was similar between both groups. The complete response (CR) rates after the initial induction therapy were not significantly different when comparing Ph+MPAL and Ph+B-ALL (100% vs. 85%, respectively, p = 0.14). Likewise, the 5-year-OS rate was similar when comparing patients with Ph+MPAL and Ph+B-ALL (55% vs. 53%, respectively, p = 0.87). Of the 13 patients with Ph+MPAL, six patients received AML-type chemotherapy, and seven patients received ALL-type chemotherapy as the initial induction therapy. All patients achieved CR after the initial induction therapy, and there was no significant difference in 5-year OS according to the therapeutic strategy (AML-type vs. ALL-type), (50% vs. 63%, respectively, p = 0.71). Among 12 patients younger than 65 years old who were alive at >3 months after the diagnosis, eight patients underwent allogeneic hematopoietic stem cell transplantation (allo SCT). The 5-year OS was significantly better for patients who underwent allo-SCT than for those who received chemotherapy alone (58% vs. 20%, respectively, p = 0.05). Conclusion:Among adult Ph+AL patients, mixed phenotype was more frequently observed than expected, and Ph+MPAL patients showed unique clinical features, including immunophenotype and the type of bcr-abl fusion gene. Although BAL has been considered as a negative prognostic factor, Ph+MPAL patients showed comparable prognosis to those with Ph+B-ALL who received imatinib-containing intensive chemotherapy. Therefore, the established theory that mixed phenotype is associated with poor outcomes should be revisited among these patients. Disclosures:No relevant conflicts of interest to declare.