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Major clinical response to nivolumab in relapsed/refractory Hodgkin lymphoma after allogeneic stem cell transplantation

A 25-year-old female with a history of Ewing sarcoma presented with leukopenia (920/μL), neutropenia (180/μL), thrombocytopenia (147,000/μL) and macrocytic anemia (hemoglobin 12.2 g/dL, mean corpuscular volume (MCV) 90.3 fL). Metastatic Ewing sarcoma of the right gluteal muscle with pulmonary metastases had been diagnosed 7 years previously. Prior treatment had included chemotherapy, radiation therapy, and allogeneic hematopoietic stem cell transplantation (alloHSCT).

Donor lymphocyte infusion (DLI), a standard relapse treatment after allogeneic stem cell transplantation (AlloSCT), has limited efficacy and often triggers GVHD. We hypothesized that after AlloSCT tumor-infiltrating donor lymphocytes could be costimulated ex vivo to preferentially activate/expand antitumor effectors. We tested the feasibility and safety of costimulated, tumor-derived donor lymphocyte (TDL) infusion in a phase 1 trial. Tumor was resected from 8 patients with B-cell malignancy progression post-AlloSCT; tumor cell suspensions were costimulated with anti-CD3/anti-CD28 Ab-coated magnetic beads and cultured to generate TDL products for each patient. Costimulation yielded increased proportions of T-bet+FoxP3− type 1 effector donor T cells. A median of 2.04 × 107 TDL/kg was infused; TDLs were well tolerated, notably without GVHD. Two transient positron emission tomography (PET) responses and 2 mixed responses were observed in these refractory tumors. TDL are a feasible, tolerable, and novel donor cell therapy alternative for relapse after AlloSCT. This trial is registered at clinicaltrials.gov as no. NCT00445666.

Background: Chimeric antigen receptor T-cell (CAR-T) therapy is an FDA-approved therapy for relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). A common side effect of CAR-T therapy is cytokine release syndrome (CRS), and its severity ranges from mild to severe, and occasionally resulting in death. Patients at particularly high risk for severe CRS may benefit from earlier supportive care and rescue therapies, such as tocilizumab. Although the median onset of CRS has been reported as two days, no existing prognostic tools adequately assist the bedside clinician with triaging which patients will decompensate and warrant escalation of care. For example, biomarkers such as CRP and ferritin are ineffective in predicting CRS severity. Evaluation of the sublingual microcirculation of patients receiving CAR-T therapy may serve as a valuable surveillance tool. The sublingual microcirculation (defined as blood vessels Methods: Video images are collected in real time. Prior to interpretation, all video clips were assessed for quality based on the current microcirculation consensus document. Each acceptable clip was then analyzed by using two point-of-care (POC) scoring systems: 1) the microvascular flow index (MFI) and 2) the point-of-care microcirculation (POEM) score. The MFI is determined by dividing the video monitor into four equal quadrants and grading the overall flow of each quadrant with a score from 0-3 (0 = no flow; 1 = intermittent flow; 2 = sluggish flow; 3 = normal flow). The POEM score utilizes an ordinal 1-5 scale (1 = critically impaired; 2 = impaired; 3 = normal with marked heterogeneity; 4 = normal with mild heterogeneity; 5 = normal) that is a composite of four measurements assessing flow impairment and heterogeneity. For each enrolled patient, a baseline measurement was made immediately prior to CAR-T cell infusion with follow up measurements occurring every six hours beginning at hour 18 after cell infusion until hour 72. After hour 72, measurements were made daily until they were deemed no longer at risk for CRS. Results: At this time there is mature data on 7 patients. All patients received Axicabtagene Ciloleucel- a CD19 directed CAR-T cell therapy (Yescarta, Kite Pharma). All patients had normal baseline microcirculation (MFI > 2.6, POEM=5) and normal or near-normal microcirculation at the end of the study period. No patients developed severe CRS (grade 3 and above). Three patients developed grade 2 CRS that required tocilizumab. Patients #1 and #2 both had significant microcirculatory impairments ≥12 hours prior to developing symptoms severe enough to warrant tocilizumab. Patient #3 had normal microcirculation through the first four days of therapy and developed hypotension on day six. We captured a subtle change from a normal MFI and POEM score to mild impairment with both scoring algorithms on day five, one day prior to clinical manifestations of decompensation. For logistical reasons, subsequent data were unable to be obtained. MFI and POEM scores for all patients are listed below in Table 1. The remaining four patients developed grade 1 CRS with associated mild microcirculatory changes. Conclusions: In this pilot study, POC microcirculatory assessments were successfully used to monitor patients undergoing CAR-T therapy. Patients with more severe CRS manifested lower MFI and POEM scores and maintained their nadir longer than those with milder CRS. Our data suggest that CAR-T patients developing CRS manifest early signs of sublingual microcirculatory dysfunction. Moreover, these microcirculatory defects present prior to the development of standard clinical abnormalities, such as macrocirculatory derangements. While further investigation is ongoing, this tool could be used for earlier identification of patients at risk for CRS in order to deliver earlier appropriate therapies, and ultimately to improve patient outcomes. Disclosures No relevant conflicts of interest to declare.

Background: A graft-versus-lymphoma effect against diffuse large B-cell lymphoma (DLBCL) is inferred by sustained relapse-free survival after allogeneic stem-cell transplantation; however, there are limited data on a direct graft-versus-lymphoma effect against DLBCL following immunotherapeutic intervention by either withdrawal of immunosuppression or donor lymphocyte infusion (DLI). Materials and methods: An analysis was carried out to determine whether a direct graft-versus-lymphoma effect exists against DLBCL. The analysis was restricted to patients with DLBCL, who were either not in complete remission at day +100 after allogeneic stem-cell transplantation or subsequently relapsed beyond this time point. Results: Fifteen patients were identified as either not in complete remission (n = 13) at their day +100 evaluation or subsequently relapsed (n = 2) and were assessed for subsequent responses after withdrawal of immunosuppression or DLI. Eleven patients were treated with either withdrawal of immunosuppression (n = 10) or a DLI (n = 1) alone; four patients received chemotherapy with DLI to reduce tumor bulk. Nine (60%) patients subsequently responded (complete = 8, partial = 1). Six responses occurred after withdrawal of immunosuppression alone. Six patients are alive (range 42–83+ months) in complete remission without further treatment. Conclusion: The demonstration of sustained complete remission following immunotherapeutic intervention provides direct evidence of a graft-versus-lymphoma effect against DLBCL.

Monoclonal B-cell lymphocytosis (MBL) is a recently described laboratory finding in otherwise healthy individuals. In MBL, a light chain-restricted, clonal B-cell population, often with a chronic lymphocytic leukaemia (CLL) phenotype, is identified by flow cytometry. Although the prognostic significance remains unclear, there is an increased incidence in ageing populations and those with a family history of CLL. During the past decade of MBL study, three families have come to our attention in which prospective sibling haematopoietic stem cell donors were found to have an MBL. These families raise complex bioethical issues with regard to disclosure of research data, eligibility for clinical trials and potential donor transfer of MBL. These issues are explored in this report. Identification of MBL among prospective sibling transplant donors will become a common occurrence in transplant practice as transplantation is increasingly offered to older individuals and those with CLL.

Purpose: We hypothesized that lymphoid-selective host conditioning and subsequent adoptive transfer of sirolimus-resistant allogeneic T cells (T-Rapa), when combined with high-dose sirolimus drug therapy in vivo, would safely achieve antitumor effects while avoiding GVHD. Experimental Design: Patients (n = 10) with metastatic renal cell carcinoma (RCC) were accrued because this disease is relatively refractory to high-dose conditioning yet may respond to high-dose sirolimus. A 21-day outpatient regimen of weekly pentostatin (P; 4 mg/m2/dose) combined with daily, dose-adjusted cyclophosphamide (C; ≤200 mg/d) was designed to deplete and suppress host T cells. After PC conditioning, patients received matched sibling, T-cell–replete peripheral blood stem cell allografts, and high-dose sirolimus (serum trough target, 20–30 ng/mL). To augment graft-versus-tumor (GVT) effects, multiple T-Rapa donor lymphocyte infusions (DLI) were administered (days 0, 14, and 45 posttransplant), and sirolimus was discontinued early (day 60 posttransplant). Results: PC conditioning depleted host T cells without neutropenia or infection and facilitated donor engraftment (10 of 10 cases). High-dose sirolimus therapy inhibited multiple T-Rapa DLI, as evidenced by stable mixed donor/host chimerism. No antitumor responses were detected by RECIST criteria and no significant classical acute GVHD was observed. Conclusions: Immune-selective PC conditioning represents a new approach to safely achieve alloengraftment without neutropenia. However, allogeneic T cells generated ex vivo in sirolimus are not resistant to the tolerance-inducing effects of in vivo sirolimus drug therapy, thereby cautioning against use of this intervention in patients with refractory cancer. Clin Cancer Res; 21(19); 4312–20. ©2015 AACR.

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Herein we report results of a phase I/II trial to evaluate the safety and activity of autologous T-cells engineered to express an affinity-enhanced T-cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4×109 engineered T cells two days after autologous stem cell transplant (ASCT). Infusions were well-tolerated without clinically apparent cytokine release syndrome, despite high IL-6 levels. Engineered T-cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, consistent with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression free survival of 19.1 months. NY-ESO-1/LAGE-1 TCR-engineered T-cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.

SummaryMixed chimaerism and graft rejection are higher after reduced-intensityallogeneic stem cell transplantation (RIST) with T-cell depleted (TCD)allografts. As host immune status before RIST affects engraftment, wehypothesized that targeted depletion of host lymphocytes prior to RISTwould abrogate graft rejection and promote donor chimaerism. Lymphocyte-depleting chemotherapy was administered at conventional doses to subjectsprior to RIST with the intent of decreasing CD4 + counts to

TPS3096 Background: Non-small cell lung cancer (NSCLC) accounts for 84% of lung cancer. Survival has recently been impacted by molecularly targeted therapies and checkpoint inhibitors (CPI), and the promising CPI results implicate a role for the immune system in NSCLC. 10-40% of NSCLC express NY-ESO-1 or MAGE-A10 cancer/testis antigens. These studies will evaluate the safety and antitumor activity of genetically engineered affinity enhanced TCRs (NY-ESO-1c259T or MAGE-A10c796T) directed towards a NYESO-1 or MAGE-A10 derived peptides complexed with HLA-A*02. In addition, correlative studies to evaluate persistence, phenotype, functionality of engineered T cells, mechanisms of resistance and antigen spreading will be performed. Methods: Patients (pt) are screened (NCT02636855) to identify those who have the relevant HLA-A*02 alleles and NY-ESO-1 or MAGE-A10 tumor expression. For entry into either treatment protocol, pt must have Stage IIIb or IV NSCLC, have failed at least one platinum-containing regimen (may have received CPIs), have measurable disease, ECOG 0-1, adequate organ function, and be without brain metastases, history of severe autoimmune disease or current uncontrolled illness. Following apheresis, T cells are isolated and expanded with CD3/CD28 beads, transduced with a lentiviral vector containing the NY-ESO-1c259T or MAGE-A10c795 TCR, and infused into the pt following lymphodepleting chemotherapy with fludarabine and cyclophosphamide. The NY-ESO-1c259T study is a 10 pt study utilizing a dose of 1-6 x 109 transduced T cells. The MAGE-A10c796T first-in-human study is a modified 3+3 design in up to 28 pt with escalating doses of 0.1, 1.0 and 1-6 x 109 transduced T cells, with staggered treatments to allow for safety review; dose escalation will be guided by the DLT observed and by safety review committee guidance. Response to treatment will be assessed by RECIST v1.1 at weeks 4, 8, 16, 24, every 3 months (for 2 yr) and every 6 months until disease progression. Clinical trial information: NCT02588612/NCT02592577.

Rationale Despite the responsiveness of metastatic breast cancer (MBC) to available therapies, the prognosis for patients with MBC that fails to respond or progresses after first-line therapy for metastatic disease is poor and points to the need for new treatments in this clinical setting. High-dose chemotherapy with autologous stem cell rescue has achieved disappointing results when compared with conventional therapy. In contrast, immunotherapy using allogeneic hematopoietic stem cell transplantation (alloHSCT), which has achieved some success in the treatment of other solid tumors (eg, renal cell carcinoma), might offer a new therapeutic approach for advanced-stage MBC. Indeed, initial reports of efficacy using a full, myeloablative transplantation were encouraging.1,2 Pitfalls to the tolerability of alloHSCT include the intensity of high-dose preparative regimens, which are particularly challenging for patients who have received extensive chemotherapy for metastatic disease, and the development of graft-versus-host disease (GVHD). The development of less toxic preparative regimens, based on the observation that much of the therapeutic effect of allogeneic transplantation is mediated by the donor immune system (graft-versus-tumor [GVT] effects) rather than the cytotoxic chemotherapy that was traditionally given, has resulted in a more favorable therapeutic index.3

In the National Cancer Institute's Second Workshop on the Biology, Prevention, and Treatment of Relapse after Hematopoietic Stem Cell Transplantation, the Scientific/Educational Session on the Biology of Relapse discussed recent advances in understanding some of the host-, disease-, and transplantation-related contributions to relapse, emphasizing concepts with potential therapeutic implications. Relapse after hematopoietic stem cell transplantation (HSCT) represents tumor escape, from the cytotoxic effects of the conditioning regimen and from immunologic control mediated by reconstituted lymphocyte populations. Factors influencing the biology of the therapeutic graft-versus-malignancy (GVM) effect—and relapse—include conditioning regimen effects on lymphocyte populations and homeostasis, immunologic niches, and the tumor microenvironment; reconstitution of lymphocyte populations and establishment of functional immune competence; and genetic heterogeneity within the malignancy defining potential for clonal escape. Recent developments in T cell and natural killer cell homeostasis and reconstitution are reviewed, with implications for prevention and treatment of relapse, as is the application of modern genome sequencing to defining the biologic basis of GVM, clonal escape, and relapse after HSCT.

Immune escape from graft-versus-tumor (GVT) contributes to relapse after allogeneic stem cell transplantation (SCT). High-dose radiation (XRT) mobilizes a tumor-specific immune response following direct damage to the tumor and microenvironment. Radiation's abscopal effect is also immune mediated. We hypothesized that local radiation of relapsed tumor would rekindle a GVT effect and yield abscopal tumor responses. We tested the safety and systemic effects of single-fraction XRT followed by donor lymphocyte infusion (DLI) in patients with lymphoid malignancy and refractory relapse after SCT. Eligible subjects (Table) had tumor progression following donor engraftment, withdrawal of immune suppression and/or DLI, and XRT-amenable tumor. Subjects received 8 Gy XRT to 1 or more tumors; all but 1 (with chronic GVHD) received DLI 24 hours later. Clinical monitoring included marrow function, GVHD, XRT toxicity and tumor response. Correlative monitoring for systemic immunologic and GVT effects included FDG-PET imaging, serial peripheral blood sampling, and biopsy of radiated and nonirradiated tumor. Immune effects were characterized by flow cytometry and gene expression analysis. Local XRT toxicity of Grade 3 or less presented as expected in all subjects. Grade 4 neutropenia (2) was transient and GCSF-responsive; GVHD flare was not observed. Unexpected toxicity was observed in 1 subject who developed Grade 4 diffuse alveolar hemorrhage and Grade 3 cardiomyopathy that responded to therapy. Sustained response in radiated sites was observed in 6 of 8 evaluable subjects (nonevaluable: 1 MM bony lesion; 1 subject received further Rx). RECIST responses outside the radiation field included 2 PR and 5 SD. 2 subjects with SD had regression after initial tumor flare. 3 of 4 regressions were transient, with progression by 6 months; 3 subjects demonstrated progression. A single subject who received radiation alone demonstrated systemic tumor response as well as improvement in chronic GVHD (sclerotic skin). By FDG-PET, all subjects' radiated tumors showed substantially decreased uptake at D28; SUV changes were variable at D7. Changes in non-XRT tumors demonstrated far greater variability, even within single subjects. Consistent with systemic immune activation resulting from radiation-DLI therapy, within 1 week, peripheral blood T cell proliferation (%Ki-67+) increased significantly in CD4 and CD8 T cells, as well as FoxP3+ Tregs (Wilcoxon paired nonparametric analyses; p =.004, .004 and .01, respectively). A trend (p=.078) toward increased frequency of Th1 effector CD4 cells (T-bet+CD28-) was also observed. Comparison of gene expression in nonirradiated (abscopal) tumor in 4 patients collected pre and 1 month post XRT was consistent with an IFN-mediated response to cell damage. Genes were upregulated for receptors for nuclear materials released by damaged cells (TLR2, TLR4, CLEC4E), high- and low- affinity FcgR (FCGR1A and FCGR2A) used for phagocytosis, and an annexin receptor for phagocytic cells (FPR2). The inflammasome component genes NLRP3 and CASP1 were upregulated, as was IFIT1, a Type 1 IFN inducible gene. After SCT, XRT results in local tumor regression; abscopal responses were also observed, with systemic immune responses suggested by T cell proliferation in the peripheral blood and upregulation of tissue damage receptors and IFN-inducible genes in nonirradiated tumor. Systemic tumor responses were delayed, with peak regression occurring 3-4 months after XRT, consistent with an immune-mediated effect. GVHD was not observed; significant, reversible pulmonary and marrow toxicities were infrequent. Transient abscopal responses in these DLI-refractory patients suggest plausible synergy between radiation and allogeneic GVT effects. Further investigation into posttransplant immunomodulatory radiation is warranted. Funding: Intramural NCI/CCR and NCI Contract No. HHSN261200800001E Table 1. DX Age/Sex Years Post-Allo XRT Site Donor / DLI CD3 Dose (10e6) XRT Response NonXRT Response NonXRT Response Duration CLL 65/M 2.3 B Neck, L Groin URD/1 -90% -69% 2M HL 36/M 2.8 B Axillae MRD/10 -6% +51% CLL 64/M 1.4 R Axilla URD/1 -82% -19% 3M HL 37/M 7.0 R Axilla MRD/10 -69% +2% 4M MM 60/F 6.9 R Ileum MRD/10 NE PD - HL 25/F 1.2 Lumbar 3-5 URD/1 7% -69% 3M HL 37/F 5.8 R Neck MRD/10 -100% -30% 3M HL 27/M 2.7 L Pelvis MRD/10 NE NE - MCL 57/M 3.1 L Groin URD/1 -100% +28% HL 28/M 1.8 L Neck MRD/None -73% -9% 3M Disclosures No relevant conflicts of interest to declare.

Background: P is used in combination with G-CSF to improve the mobilization of peripheral blood hematopoietic stem cells in poor mobilizers. Limited data are available in regard to effects of P on post-transplant outcomes in comparison with non-P chemomobilized patients. Methods: In this retrospective study, we compare the engraftment and the outcomes of 34 patients mobilized with P + G-CSF or just-in-time rescue P in combination with chemotherapy and G-CSF to 143 patients (control group) mobilized with G-CSF with or without chemotherapy in lymphoma and myeloma patients who underwent ASCT between February 2012 and April 2014 at the University of Maryland Greenebaum Cancer Center. Post-transplantation outcomes including infections, hematologic recovery, relapse, progression and survival were recorded. Results: The median number of collected of CD34+ cells/Kg was 5.9 x 10(6) in the P group and 12.3 x 10(6) in the control group (p=0.0002). Median time to neutrophil engraftment (>0.5 × 10(9) /L) was comparable between the 2 groups: 12 days for the P group and 11 for the control group (p=0.28). There was a trend toward a shorter time to platelet engraftment (>20 × 10(9) /L) in the control (12 days) compared to the P group (14 days) (p=0.056). Progression-free survival at 1 year after (ASCT) was 88.2% in the P group and 81.8% in the control group. There was no difference in the overall survival of both groups (p=0.62) Conclusions: Short and long-term engraftment and outcomes after ASCT seem to be comparable in lymphoma and myeloma patients receiving plerixafor compared to chemomobilized patients without plerixafor. This observation support the use of plerixafor + G-CSF or just-in-time rescue P in patients who mobilize poorly without P. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Several recent studies have linked the interactions of programmed death 1 (PD-1) receptor and its ligand (PD-L1) to immunologic control of MM. Expression of PD-L1 on myeloma cells and the abundance of PD-1 on various bone marrow microenvironment components contribute to tumor-mediated immune suppression. We hypothesized that pembrolizumab, a PD-1-blocking antibody, will activate myeloma specific cytotoxic T cells that can be enhanced by pomalidomide in RRMM patients to induce clinical responses. METHODS: In this ongoing single arm, phase II study, 24 patients with RRMM received 28-day cycles of pembrolizumab (at a dose of 200 mg IV) every 2 weeks (in a run off phase, first 6 patients received 200 mg IV every 4 weeks) plus pomalidomide (4 mg daily x 21 days) and dexamethasone 40 mg weekly. Study objectives were measurements of safety and efficacy and assessment of the PD-1 and PD-L1 protein expression in bone marrow samples. RESULTS: The median age was 65 years (range: 41-75); 35% were African American and 71% were men. Of the 24 patients, 75% had prior autologous transplantation and 96% were refractory to last therapy. All patients had received both IMids and Proteosome inhibitors; 75% were double refractory to both IMids and Proteosome inhibitors and additional 21% were refractory to lenalidomide alone. Patients had received a median of 3 lines of prior therapy (range: 1-6). The median time from MM diagnosis to study entry was 4 years (range: 1.2-15). All patients had abnormal cytogenetics: most common were 1q+ (72%) and high-risk FISH (40%) [del 17p, t(4:14) and/or t(14:16)]. There were no infusion-related reactions. Hematologic toxicities (≥ grade 3) were neutropenia (29%), lymphopenia (17%) and thrombocytopenia (8%). Non-hematologic adverse events included (Grade ≤2; ≥3): fatigue (n=12; 1), constipation (n=10; 0), dyspnea (n=9; 2), itching (n=6; 0), muscle spasms (n=6; 0), infection (n=4; 3), hyperglycemia (n=5; 0), edema (n= 4; 0), fever (n=3; 0), palpitation (n=2; 1), rash (n=3; 1) and hypotension (n=3; 0). Events of clinical significance, autoimmune mediated, included hypothyroidism (n=2), transaminitis (n=2), and pneumonitis (n=1). Four patients had pomalidomide dose reductions due to rash, neutropenia, palpitations and fatigue. Two patients died; one after cycle 1 (progressive disease) and one during cycle 2 (sepsis). Objective responses (modified IMWG criteria) were observed in 11 of 22 evaluable patients (50%) including: near complete response (n=3), very good partial response (n=2), partial response (n=6); additionally, 3 patients had minimal response, 6 had stable disease and 2 progressed. At a median follow up of 16 weeks; 17 of 22 patients continued on the study. Reasons for discontinuation included disease progression (n= 4) and protocol violation (n=1). Analysis of pretreatment and post-treatment tumor specimens for PD-1 and PD-L1 is in progress. CONCLUSIONS: Pembrolizumab in combination with pomalidomide and dexamethasone has promising therapeutic activity and an acceptable safety profile in heavily treated RRMM patients. ClinicalTrials.gov number, NCT02289222. Disclosures Off Label Use: Pembrolizumab.

Background: The incidence of MM is 2 to 3 fold higher in blacks than in whites; they present at a younger age and have better overall survival. The biological bases for these disparities remain unclear. Outcome of MM is linked to cytogenetic and molecular changes, both primary (hyperdiploidy and heavy chain (IgH) translocations) and secondary (rearrangements of MYC, activating mutations of NRAS, KRAS or BRAF, and deletions of 17p). Methods: Cytogenomic alterations in consecutive MM patients were assessed using integration of metaphase chromosome analysis by GTG-banding and interphase fluorescence in situ hybridization (iFISH) in CD138-positive cells isolated from fresh BM samples using a protocol of magnetic-activated cell sorting. Changes evaluated included monosomy 13/del(13q), monosomy 17/del(17p), gain of 1q21, and rearrangements of the IGH gene including t(4;14), t(11;14) and t(14;16). Results: Samples from 218 consecutive MM patients were analyzed (Table 1). 108 were from black and 110 were from white patients. Median age for blacks was 59 years (range: 36 - 82) and for whites, 63 years (range: 39 - 83) (p=0.008). Fewer black men than whites were observed (46.3% versus 64.6%, p=0.007). Overall, blacks had fewer abnormal karyotypes compared to whites (18.1% versus 31.8%; p=0.02). Black patients had a lower frequency of non-hyperdiploid karyotypes (8.5% versus 20.6%; p=0.01) and had a trend toward lower frequencies of rearrangements of IGH (30.8% versus 43.5%; p=0.055) than white patients. Most notably, they had significantly lower frequencies of monosomy 17/del(17p) (5.6% versus 18.5%; p=0.003) and monosomy 13/del(13q) (28.9% versus 46.3%; p=0.008). After stratification by age (Figure 1), younger patients showed significantly higher frequencies of the monosomy 17/del(17p) abnormality (p=0.001) and the t(4;14) (p=0.04) than older patients, with the difference more significant in white patients. The associations among molecular cytogenetic abnormalities (Figure 2) showed a different association pattern for black and white patients. White patients with t(11;14) were more likely to have monosomy 13/del(13q) (p=0.003) and gain of 1q21 (p=0.02), while this association was not observed in black patients. Conclusion: Black MM patients had significantly different cytogenetic profiles detected by iFISH on CD-138 selected malignant cells, compared to whites. Black MM patients had a more favorable profile, including lower frequencies of non-hyperdiploid karyotype and of IGH rearrangements. This study supports a biological basis for previously described outcome disparities between black and white patients with MM. Further studies will focus on identifying specific molecular targets and their impact on therapy and on overall outcome. Table 1. Demographics and cytogenetic abnormalities of the MM patients Demographics Black White P-value# Total, n 108 110 Gender, n (%) =0.007* Male 50 (46.30%) 71 (64.55%) Female 58 (53.70%) 39 (35.45%) Age (median) 59 63 =0.008* Chromosome (karyotype) =0.022* Normal 86 (81.90%) 73 (68.22%) Abnormal 19 (18.10%) 34 (31.78%) Hyperdiploidy 8 (7.6%) 8 (7.4%) Non-hyperdiploidy 9 (8.5%) 22 (20.6%) =0.013* 11;14 translocation 2 (1.9%) 4 (3.7%) FISH abnormality -13/del(13q) 31 (28.97%) 50 (46.30%) =0.008* Gain of 1q21 35 (32.71%) 47 (43.52%) =0.103 -17/del(17p) 6 (5.61%) 20 (18.52%) =0.003* IGH rearrangements 33 (30.84%) 47 (43.52%) =0.055^ t(4;14) 7 (6.54%) 13 (12.38%) =0.146 t(11;14) 15 (20.55%) 15 (19.48%) =0.870 t(14;16) 2 (3.85%) 6 (10.71%) =0.175 others 16 (14.95%) 15 (13.89%) =0.824 *means statistical significant (p-value < 0.05), where ^ means marginal significant (0.05 < p-value < 0.10). #p-values come from the Cochran-Mantel-Haenszel tests for categorical variables, and t tests for continuous variables. Associations among eight molecular cytogenetic abnormalities. Each solid black line indicates one abnormality is statistically significantly associated with another abnormality. Figure 1. Distributions of cytogenetic abnormalities by age and race Figure 1. Distributions of cytogenetic abnormalities by age and race Figure 2. Relationship of various cytogenetic abnormalities in the MM patients Figure 2. Relationship of various cytogenetic abnormalities in the MM patients Disclosures No relevant conflicts of interest to declare.

Purpose: Metastatic breast cancer (MBC) response to allogeneic lymphocytes requires donor T-cell engraftment and is limited by graft-versus-host disease (GVHD). In mice, type-II–polarized T cells promote engraftment and modulate GVHD, whereas type-I–polarized T cells mediate more potent graft-versus-tumor (GVT) effects. This phase I translational study evaluated adoptive transfer of ex vivo costimulated type-I/type-II (T1/T2) donor T cells with T-cell–depleted (TCD) allogeneic stem cell transplantation (AlloSCT) for MBC. Experimental Design: Patients had received anthracycline, taxane, and antibody therapies, and been treated for metastatic disease and a human leukocyte antigen (HLA)-identical–sibling donor. Donor lymphocytes were costimulated ex vivo with anti-CD3/anti-CD28 antibody–coated magnetic beads in interleukin (IL)-2/IL-4–supplemented media. Patients received reduced intensity conditioning, donor stem cells and T1/T2 cells, and monitoring for toxicity, engraftment, GVHD, and tumor response; results were compared with historical controls, identically treated except for T1/T2 product infusions. Results: Mixed type-I/type-II CD4+ T cells predominated in T1/T2 products. Nine patients received T1/T2 cells at dose level 1 (5 × 106 cells/kg). T-cell donor chimerism reached 100% by a median of 28 days. Seven (78%) developed acute GVHD. At day +28, five patients had partial responses (56%) and none had MBC progression; thereafter, two patients had continued responses. Donor T-cell engraftment and tumor responses appeared faster than in historical controls, but GVHD rates were similar and responders progressed early, often following treatment of acute GVHD. Conclusion: Allogeneic T1/T2 cells were safely infused with TCD-AlloSCT, appeared to promote donor engraftment, and may have contributed to transient early tumor responses. Clin Cancer Res; 17(21); 6878–87. ©2011 AACR.

Inflammatory breast cancer (IBC) is a rare clinicopathologic entity with a poor prognosis, lagging far behind any other form of nonmetastatic breast cancer. Since the advent of systemic chemotherapy over 35 years ago, only minimal progress has been made in long-term outcome. Although multiple randomized trials of high-dose chemotherapy and autologous progenitor cell transplantation (ASCT) for the treatment of breast cancer have yielded disappointing results, these data are not necessarily relevant to IBC, a distinct clinical and pathologic entity. Therefore, the optimal multimodality therapy for IBC is not well established, and remains unsatisfactory. We treated 21 women with nonmetastatic IBC with a multimodality strategy including high-dose melphalan (Mel)/etoposide and ASCT. The treatment was overall tolerated with acceptable morbidity, and no post-ASCT 100-day mortality. With a median potential follow-up of approximately 8 years, the estimated progression-free survival (PFS), event-free survival (EFS), and overall survival (OS) at 6 years from on-study date are: 67%, 55%, and 69%, respectively. These results from a small phase II study are among the most promising of mature outcome data for IBC. They strongly suggest, along with results of several already published phase II trials, that ASCT could play a significant role in the first line treatment of IBC.

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Adoptive immunotherapy for cancer has been limited by a lack of antigen specificity, low levels of target expression, and failure to break self-tolerance. We hypothesized that infusion of genetically modified tumor-specific T cells following autologous stem cell transplant (ASCT) may overcome these barriers for multiple myeloma (MM). To test this, we conducted a phase I/II clinical trial ([NCT01352286][1]) in which T cells engineered with an HLA-A*0201 restricted, affinity-enhanced TCR recognizing NY-ESO-1 / LAGE-1 peptides (NY-ESOc259-T), were infused in the setting of profound lymphodepletion that accompanies high-dose chemotherapy given with ASCT. HLA-A*0201 MM patients eligible for ASCT, with antigen positive tumor were enrolled. NY-ESOc259-T was manufactured in a 10 day process using anti-CD3/CD28 microbeads and lentiviral vector, and was administered two days following ASCT. IMWG criteria were used to assess response at day 100 with the addition of a near complete response category (nCR) due to the common occurrence of oligoclonal banding observed following rapid post-ASCT immune reconstitution. Blood and marrow samples were taken at multiple timepoints for serum cytokine analysis, NY-ESOc259-T persistence and trafficking, multiparameter flow analysis to examine the phenotype and function of NY-ESOc259-T, and tumor biomarker analysis. 25 of 29 enrolled patients were infused. A mean of 2.8 × 109 engineered cells were administered (range 8.3 × 108-4.2 × 109), and the average transduction efficiency was 33% (range 30%-45%). Patients tended to have advanced disease (64% chromosomal abnormalities, and 24% prior ASCT). At 3 months, 67% (16/24) and 58% (14/24) of patients were in VGPR and nCR or better, respectively. Infusions were well-tolerated and no cytokine release syndrome was reported. NY-ESOc259-T persisted at 6 months in all but one patient, and in a subset of patients at 2 years; marrow infiltration was consistently observed from day 7 through day 180. NY-ESOc259-T initially displayed a dominant activated effector phenotype which converted towards a dominant effector memory phenotype by 1 year post infusion, in a pattern that mirrored clinical responses. Persisting cells demonstrated a polyfunctional response (IFN-γ and TNF-α) with a cytotoxic (CD107a and granzyme B) signature without overexpression of exhaustion markers (PD-1, LAG-3, and TIM-3). Tumor biomarker analysis is ongoing. MM relapse occurred in 13/25 patients. This data show that NY-ESOc259-T cells exhibit robust trafficking and expansion, durable persistence without exhaustion, and follow a natural immune expansion and contraction pattern consistent with an antigen-driven mechanism of action. Relapse correlated with a loss of persistence or tumor antigen escape, suggesting that targeting multiple antigens and maintenance infusions may increase durable remissions. Citation Format: Aaron Rapoport, Edward Stadtmauer, Luca Melchiori, Ryan Wong, Eduardo Davila, Gwendolyn Binder-Scholl, Tom Holdich, Dan Vogl, Brendan Weiss, Jeffrey Finkelstein, Simon Lacey, Sarah Bond, Marylene Fortin, Yoav Peretz, Joanna Brewer, Alan Bennett, Andrew Gerry, Nick Pumphrey, Helen Tayton-Martin, Lilliam Ribeiro, Ashraf Badros, Saul Yanovich, Nancy Hardy, Jean Yared, Naseem Kerr, Sunita Philip, Sandra Wesphal, Bruce L. Levine, Carl June, Michael Kalos, Bent Jakobsen. NY-ESO T cells administered post ASCT for MM exhibit extended functionality without exhaustion in a natural pattern of effector and memory programming. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4701. doi:10.1158/1538-7445.AM2015-4701 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01352286&atom=%2Fcanres%2F75%2F15_Supplement%2F4701.atom

The curative potential of allogeneic hematopoietic stem cell transplantation in treating cancer is derived in large part from the donor immune system reacting against host or tumor cell antigens to generate a graft-vs-tumor (GVT) effect. Whereas early animal models suggested this allogeneic immune response, evidence for its role in human transplantation was not realized until the late 1970s and early 1980s, when the association between graft-vs-host disease and tumor response became clear. In this chapter, the animal and human data that established the GVT effect as the major therapeutic component of allogeneic hematopoietic stem cell transplantation are reviewed, and ongoing efforts to understand and build on GVT as a therapeutic modality for other tumor types are described. The current thinking on the biology of the effectors and modulators of GVT are discussed. The progress that has been made in clinical application of immunotherapy in the autologous setting and of solid tumor therapy in the allogeneic setting is reviewed. Finally, the potential for continued advances in the efficacy and safety of immunotherapy for cancer is explored, as reflected in recent and ongoing efforts to apply the growing understanding of alloreactivity toward fulfillment of the potential for targeted antitumor therapies.

Mixed chimaerism and graft rejection are higher after reduced-intensity allogeneic stem cell transplantation (RIST) with T-cell depleted (TCD) allografts. As host immune status before RIST affects engraftment, we hypothesized that targeted depletion of host lymphocytes prior to RIST would abrogate graft rejection and promote donor chimaerism. Lymphocyte-depleting chemotherapy was administered at conventional doses to subjects prior to RIST with the intent of decreasing CD4(+) counts to0.05 x 10(9)cells/l. Subjects (n = 18) then received reduced-intensity conditioning followed by ex vivo TCD human leucocyte antigen-matched sibling allografts. All evaluable patients (n = 17) were engrafted; there were no late graft failures. At day +28 post-RIST, 12 patients showed complete donor chimaerism. Mixed chimaerism in the remaining five patients was associated with higher numbers of circulating host CD3(+) cells (P = 0.0032) after lymphocyte-depleting chemotherapy and was preferentially observed in T lymphoid rather than myeloid cells. Full donor chimaerism was achieved in all patients after planned donor lymphocyte infusions. These data reflect the importance of host immune status prior to RIST and suggest that targeted host lymphocyte depletion facilitates the engraftment of TCD allografts. Targeted lymphocyte depletion may permit an individualized approach to conditioning based on host immune status prior to RIST.

471 Lymphoma progression remains an obstacle after allogeneic HCT, particularly after non-myeloablative conditioning in patients with high-risk histology (non-indolent; Kahl et al; Blood, 2007) and chemotherapy refractory disease (Bishop et al; Cancer, 2010). In murine models, rapamycin-resistant T cells favorably modulated the balance between GVHD, graft rejection, and GVT effects. To translate this, we conducted a multi-center phase II trial (NCT0074490) of T-Rapa cells infused as a pre-emptive DLI after low-intensity allogeneic HCT; here, we report overall outcome of patients with high-risk lymphoma diagnoses, many of whom were chemotherapy refractory. T-Rapa cells were manufactured by ex vivo culture of donor CD4+ T cells using CD3/CD28 co-stimulation and IL-4, IL-2, and rapamycin for 12-days (T-Rapa12) or 6-days (T-Rapa6), and administered (2.5 × 107 cells/kg) at d14 post-HCT. Both populations of T-Rapa cells expressed a mixed Th2/Th1 phenotype with minimal T-Reg content. Patients (n=42) received outpatient-intensity chemotherapy (typically, EPOCH-FR) until CD4 count was < 200 cells/μl, and then received an HLA-matched sibling mobilized allograft and GVHD prophylaxis of cyclosporine plus short-course sirolimus (to d14 post-HCT); conditioning consisted of fludarabine (120 mg/m2) and cyclophosphamide (1200 mg/m2). Table I details the high-risk diagnoses, pre-treatment history (median of 4 prior regimens), remission status at time of HCT (7/42 [17%] in CR), and presence of chemotherapy refractory disease (stable or progressive disease to prior therapy: 23/42 pts, 55%). T-Rapa cell infusion was relatively safe, with no engraftment syndrome or d100 TRM; incidences of grade II-IV acute, late acute, and classical chronic GVHD were 17%, 34%, and 36%, respectively. Initial mixed donor/host chimerism converted to predominate donor chimerism after T-Rapa cell DLI (Table I). Overall median survival probability at 24 months post-HCT is 85.7% for patients with chemotherapy sensitive disease vs. 39.1% for patients with chemotherapy refractory disease ([Fig. 1][1]; p=0.0008). All deaths were due to progressive disease except for 2 infection-related deaths at days 162 and 359 post-HCT; all surviving patients are in CR. Survival was not statistically significantly influenced by DLI type (T-Rapa12 vs. T-Rapa6) or histology-type (NHL vs. HD). Pre-emptive DLI using donor T-Rapa cells after low-intensity conditioning is safe and very effective in patients with high-risk lymphoma diagnoses and chemotherapy sensitive disease; for such patients, future randomized trials should compare low-intensity T-Rapa cell therapy to other transplantation regimens. For patients with high-risk lymphoma diagnoses and chemotherapy-refractory disease, we are evaluating a modification to the current platform that incorporates high-dose sirolimus therapy. | Patient Characteristics | Chimerism Results[c][2] | CD3 | CD15 | |:----------------------------------:| ----------------------- | ---------------------- | ------------------ | ----------- | | Number of Patients | n = 42 | Day 14 post-HCT | 57 (12-97) | 46 (8-94) | | Age (median, range) | 44 (23-68) | Day 28 post-HCT | 77 (37-100) | 76 (29-98) | | Sex (male/female) | (26/16) | | | | | # of Prior Therapies (mean, range) | 4 (1-6) | Day 100 post-HCT | 89 (51-100) | 93 (35-100) | | Histology[a][3] | n=26 total (62%) | Survival Results[d][4] | 24 Mo. Surv. Prob. | | NHL Category | n=12 | ChemoSens/T-R12 | 78.6% (n=7) | | DLBCL | n=5 | ChemoSens/T-R6 | 85.7% (n=12) | | DLBCL-tr | n=2 | ChemoRefr/T-R12 | 41.7% (n=11) | | DLBCL-EBV | n=3 | ChemoRefr/T-R6 | 36.4% (n=12) | | PlasmacytoidDC | n=3 | All NHL | 57.4% (n=26) | | T Cell | n=16 total (38%) | All HD/Grey Zone | 68.8% (n=16) | | HD Category | n=12 | | | | HD | n=4 | | | | Grey Zone | | | | | Chemo. Response[b][5] | 23/42 (55%) | | | | Refractory (SD/PD) | 19/42 (45%) | | | | Sensitive (PR/CR) | | | | | Complete Remission | 7/42 (17%) | | | | At Time of HCT | | | | * DLBCL, diffuse large B cell lymphoma; DLBCL-tr, transformed; DLBCL-EBV, Epstein Barr Virus related; Plasmacytoid DC, dendritic cell; HD, Hodgkins Disease; SD, stable disease; PD, progressive disease; PR/CR is partial or complete response. * [↵][6]a Grey Zone Lymphoma and Hodgkins Disease were pooled for statistical analyses. * [↵][7]b Chemotherapyresponse, as determined after last regimen prior to study entry. * [↵][8]c Chimerism determined by VNTR-PCR at days 14, 28, and 100 post-HCT (mean and range of values shown); CD3, T cell chimerism; CD15, myeloid cell chimerism. * [↵][9]d Survival by Kaplan-Meier analysis; 24 month median survival probability values shown; T-R12 and T-R6 indicates recipient of T-Rapa cells manufactured for 12 days vs. 6 days. Table I ![Figure][10] Disclosures: Levine: TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. June: Novartis: Research Funding, institution owned patents have been licensed by Novartis, institution owned patents have been licensed by Novartis Patents & Royalties. Mato: Celgene, Milennium, Genentech, Seattle Genetics: Speakers Bureau. [1]: #F1 [2]: #fn-4 [3]: #fn-2 [4]: #fn-5 [5]: #fn-3 [6]: #xref-fn-2-1 [7]: #xref-fn-3-1 [8]: #xref-fn-4-1 [9]: #xref-fn-5-1 [10]: pending:yes

Abstract 683 Donor lymphocyte infusion (DLI) is the primary treatment for relapse after allogeneic hematopoietic stem cell transplantation (allotransplant), but is effective in less than half of patients and is associated with significant graft-vs-host disease (GVHD). Furthermore, DLI is not available for cord-blood or many unrelated-donor recipients. We theorized that tumor from patients relapsing post-allotransplant would contain infiltrating donor T- lymphocytes with tumor-specificity. If such cells had been “primed”, but inadequately costimulated for tumor cell killing, costimulation ex-vivo could generate a donor lymphocyte product with increased graft-vs-tumor (GVT) potency. Preclinical feasibility was demonstrated in tumor biopsies from patients with tumor progression after allotransplant. Ex-vivo CD3/CD28 costimulation of tumor-infiltrating lymphocytes with antibody-coated magnetic beads yielded donor products, predominently consisting of effector T-cells; tumor specimens of greater than 1 cm reliably produced donor T cell expansions that would permit clinically relevant cell dosing. Based on these preclinical results, we conducted a Phase 1 study of tumor-derived donor lymphocyte (TDL) therapy in allotransplant recipients with B-lymphoid tumor progression despite full-donor T cell chimerism and DLI, or for which DLI was not available. The primary objectives of (1) feasibility of ex-vivo costimulation of lymphocytes from surgically harvested tumor to generate a TDL product and (2) safety of TDL infusion (TDLI) in patients with relapse after allotransplant were met within the first 7 of 15 planned subjects. All 7 patients had chemotherapy-refractory relapse after allotransplant (diffuse large-B cell lymphoma (DLBCL) = 2; Hodgkin's Lymphoma (HL) = 4; chronic lymphocytic leukemia (CLL) = 1). Tumor was surgically resected. Lymphocytes were released from tumor tissue through mechanical dispersion and cultured with CD3/CD28 antibody-coated magnetic beads in media supplemented with IL-2 (100 IU/ml) for a median of 12 days (range: 7– 42 days). T cells comprised from 0.5 – 63.4% of tumor cell suspensions (culture inocula) and 82 – 99% of expanded products (22- to 144-fold expansion). PCR-based chimerism assessment of products demonstrated that the TDL were of donor origin (median 99% donor). During TDL expansion, markers of activation and effector function increased (CD4: CD40L; CD8: CD137, perforin, NKG2D). Percentages of FoxP3+ Treg cells declined and TBet+ Th1/Tc1 effectors increased in the costimulated TDL product. Expression of CD27 and CD28 has been associated with long-term persistence of infused effector T cells; although these markers declined in culture, expression was retained in a significant proportion of cells from the 5 products that were cultured for less than 14 days. TDL products meeting release criteria for tumor contamination ( Disclosures: June: N/A: Inventor, Government-held Patent.

Abstract 521 Ex-vivo culture of murine donor CD4+ T cells using rapamycin, co-stimulation, and IL-4 yielded a defined T cell population (T-rapa cells) that favorably modulated the balance between GVHD, graft rejection, and GVT effects. To translate these findings, we conducted a multi-center clinical trial (NCT0074490) to evaluate T-rapa cell therapy after allogeneic HCT. T-rapa cells were manufactured by ex vivo culture of donor CD4+ T cells using CD3/CD28 co-stimulation in media containing IL-4, IL-2, and rapamycin. T-rapa cells had a mixed Th2/Th1 phenotype with minimal Treg content (intra-cellular flow, n=48 products; median transcription factor expression: 11.5% [GATA-3], 5.1% [T-bet], and 0.1% [FoxP3]). Median T-rapa cell cytokine secretion (pg/ml; re-stimulation at harvest) was 1.3 [IL-4], 20.6 [IL-5], 9.7 [IL-10], 23.7 [IL-13], 34.7 [IFN-g], and 17.1 [IL-2]. Patients received an HLA-matched sibling, T cell-replete, G-CSF mobilized allograft, and GVHD prophylaxis of cyclosporine plus short-course sirolimus (to d14 post-HCT). Two protocol arms evaluated T-rapa cell therapy after induction chemotherapy and outpatient, low-intensity preparative chemotherapy (Table I). First, patients (n=25) were accrued to arm A to evaluate T-rapa infusion at d +14 post-HCT; subsequently, accrual was initiated to arm B (n=25) to evaluate T-rapa infusion on d0 of HCT. Arm A was then expanded to n=40 patients. Patients accrued to arms A and B were similar for recipient age, high-risk malignancy diagnosis, chemotherapy refractoriness, and prior regimen number (Table I). Most recipients were not in remission at the time of HCT. High-risk NHL was the most frequent diagnosis (25/65 patients), followed by non-high-risk NHL (11/65), AML/MDS (8/65), myeloma (7/65), CLL (6/65), Hodgkin's disease (5/65), and CML (3/65). Arm A and B recipients had similar mean donor myeloid cell chimerism at d +14, +28, and +100 (arm A, 43%, 74%, and 89%; arm B, 50%, 62%, and 84%). At d +14, arm A and B recipients also had mixed donor T cell chimerism (mean values, 60% in each arm; Table I). At d +28 and +100, T cell chimerism increased in arm A to 80% and 89%; in arm B, these values increased to only 67% and 69%. Four recipients on arm B had < 10% donor T cell chimerism at d +100; in contrast, the lowest donor T cell chimerism value observed at d +100 on arm A was 36%. T-rapa therapy on arm A was relatively safe as there was: no engraftment syndrome, a 10% rate of acute grade II to IV GVHD, a 67% incidence of chronic GVHD, and no transplant-related mortality (Table I). On arm A, 37.5% (15/40) of recipients are in sustained complete remission, with a median survival probability of 63.6% at 24 months post-HCT. Therefore, pre-emptive donor lymphocyte infusion with ex-vivo manufactured T-rapa cells that express a balanced Th2/Th1 effector phenotype represents a novel approach to safely accelerate alloengraftment and harness allogeneic GVT effects after low-intensity conditioning.Table IArm AArm BLow-Intensity Regimen Induction Chemotherapy1EPOCH-FREPOCH-FR 2Terminal Chemotherapy3Flu (120 mg/m2)EPOCH-FRCy (1200 mg/m2)T-Rapa Cell TimingD +14 post-HCTD 0 of HCTPatient Characteristics & of Patients Accrued4025 Age (median, range)55 (25–67)51 (32–66) & of Prior Regimens3 (1–6)3 (1–8) High-Risk Malignancy65% (26/40)52% (13/25) Chemotherapy Refractory50% (20/40)48% (12/25) CR (at time of HCT)25% (10/40)8% (2/25)% Donor T Cell ChimerismMean Median (Range)Mean Median (Range)Day 14 post-HCT6061(8–97)6060(4–100)Day 28 post-HCT8089(27–100)6773(10–100)Day 100 post-HCT8993(36–100)6982(0–100)Clinical Results Engraftment Syndrome0% (0/40)0% (0/25) Acute GVHD10% (4/40)23% (5/22) Chronic GVHD67% (22/33)75% (15/20) Complete Remission38% (15/40)28% (7/25) Transplant-related Mortality0% (0/40)0% (0/25) Percent Survival65% (26/40)40% (10/25) Median Survival27.5 mo11.2 mo Survival Prob. at 24 mo63.6%44.0%1EPOCH-FR, EPOCH with fludarabine (Flu) and rituximab.2Terminal (preparative) chemotherapy administered one week prior to HCT.3Flu/Cy [cyclophosphamide] doses are total doses, given over 4 days (Cy dose is 75% lower than 4800 mg/m2 “reduced-intensity” Cy dose). Disclosures: No relevant conflicts of interest to declare.

Abstract 3451 Relapse is the major cause of treatment failure and death following reduced-intensity (RI) allogeneic hematopoietic stem cell transplantation (HSCT) for non-Hodgkin's lymphoma (NHL), yet there are no reports that focus specifically on the natural history of relapse in this patient population. We assessed relapse risks and outcomes on 120 consecutive patients (median age = 52 years; range, 28–71 years) with NHL (indolent = 43; aggressive = 77) who all received a T-cell replete allograft from HLA-matched siblings following a reduced-intensity conditioning regimen (fludarabine/cyclophosphamide). All patients were assessed for disease status at 1, 3, 6, 12, 18, 24 months post-transplant, and annually thereafter or as clinically indicated. Median event-free survival (EFS) from transplant date for all 120 patients was 11.3 months (range, 0.5–105.5+ months) with a 5-year EFS = 35%. Median EFS (months) was assessed for the following pre-transplant characteristics: chemo-resistant vs. chemo-sensitive = 3.3 vs. 39.1, p = 0.0001; pre-transplant disease status - CR vs. PR vs. SD vs. PD = not reached (NR) vs. NR vs. 11.2 vs. 1.7, p Disclosures: No relevant conflicts of interest to declare.

Allograft T cell depletion (TCD) reduces acute graft-vs. host disease (aGVHD) after myeloablative hematopoietic stem cell transplantation (HSCT). Lower incidences of aGVHD reported after reduced-intensity stem cell transplantation (RIST) may reflect delayed donor T cell engraftment. We compared the incidence of aGVHD following RIST with TCD (19 patients (pts) vs. T cell replete (TCR) allografts (20 pts)(Table). There was no difference in the incidence aGVHD, occurring in 71% of TCD recipients (median onset Day 47) and 70% of TCR recipients (median onset Day 25). After TCD, 100% of those who engrafted prior to any DLI developed aGVHD compared with 56% of those who engrafted after DLI, including two pts who developed “late-acute” aGVHD after Day 100, upon completion of donor T cell engraftment. T cell engraftment after TCD was uneven: engraftment kinetics were associated with residual host circulating CD8+ T cell counts after immune depletion; and aGVHD did not occur in the setting of mixed T cell chimerism (Figure). These observations demonstrate that aGVHD can occur with very low doses (105/kg) of allograft T cells after RIST. Protection from aGVHD conferred by mixed T cell chimerism may be lost with full donor T cell engraftment. With limited donor T cell numbers, host T cells appear to determine kinetics of engraftment and of aGVHD after RIST. Figure: 1. Post-induction circulating CD8+ T cell counts in TCD recipients with delayed donor T cell engraftment. 2. After TCD, all subjects who developed aGVHD did so at or after the establishment of T cell full donor T cell chimerism, and occasionally prior to any DLI. Shaded triangle represents the theoretical area in which values would fall if subjects developed aGVHD prior to complete donor T cell engraftment. Arrows: DLI. | Protocol | TCD | TCR | |:---------------------------------------------------------------------------------------------------------------------------------------------------------------- | ----------------------- | ---------------------- | | Flu/Cy: Fludarabine and cyclophosphamide; EPOCH-F: Etoposide, doxorubicin, vincristine, cyclophosphamide, prednisone and fludarabine; FDC: Full donor chimerism. | | | Median (range) | Median (range) | | Recipient Age | 43 years (32 – 56) | 44 (19 – 67) | | CMV Risk | 14/19 | 16/20 | | Pretransplant Immune Depletion | Flu/Cy | EPOCH-F | | Conditioning Regimen | Flu/Cy | Flu/Cy | | Pre-conditioning Host Cell Counts: | | | CD3 | 86 (1 – 701) | 140 (21 – 441) | | CD4, p=0.017 | 44 (1 – 156) | 71 (12 – 191) | | CD8 | 34 (0 – 555) | 55 (2 – 309) | | NK | 58 (0 – 376) | 88 (3 – 467) | | Day 0 CD3 Cell Count | 1 (1 – 6) | 5 (0 – 42) | | Allograft CD34+ cells/kg | 7.75 x 106 (5.1 – 12.9) | 7.68 x 106 (4.6–18.4) | | Allograft CD3+ cells/kg | 1.0 x 105 (preset) | 3.63 x 108 (1.5 – 8.3) | | Donor T cell engraftment | Day +70 (14 – 180) | 14 (14 – 100) | Patient and Transplant Characteristics and Outcomes ![Figure][1] Figure [1]: pending:yes

Reduced-intensity conditioning is less potently tumoricidal than myeloablative regimens; thus, RIST relies more upon graft-versus-tumor (GVT) effects for disease eradication. However, variation in baseline host immune status contributes to inconsistent donor engraftment and may impede maximal GVT effects after RIST. We hypothesized that targeted immune depletion (TID) with conventional-dose chemotherapy before RIST might facilitate donor engraftment, thereby potentiating GVT effects. Thus, we evaluated dose-adjusted (DA-) EPOCH-F, a novel regimen of etoposide, prednisone, vincristine, cyclophosphamide (Cy), doxorubicin, and fludarabine (Flu), in a phase II manner in 83 pts with lymphoid malignancies undergoing RIST on 3 sequential, prospective clinical trials between 1999 and 2005. Pts received at least 1 and no more than 3 cycles of DA-EPOCH-F, targeting a peripheral blood CD4+ T cell count < 100 cells/μL. Pts achieving this CD4 count after only 1 or 2 cycles then proceeded to Flu/Cy conditioning and RIST from HLA-matched related donors. Pts otherwise proceeded to RIST after 3 cycles or if disease progression occurred during DA-EPOCH-F, regardless of CD4 count. After 2002, pts with CD20+ malignancies (n=28) also received rituximab (R) on day 1 of each DA-EPOCH-F cycle. Pt characteristics: median age 50 yrs; diagnoses Hodgkin lymphoma (14%), DLBCL (28%), other aggressive NHL (37%), follicular NHL (12%), CLL or other indolent NHL (8%); chemosensitive disease in 48%; median 3 prior regimens, prior autologous stem cell transplant in 28%; poor-risk IPI, FLIPI, IPF score, or Rai stage in 36%. Pts received 1 (n=26), 2 (n=20), or 3 (n=37) cycles of DA-EPOCH-F before RIST. Median CD4 count declined from 284 cells/μL at enrollment to 79 cells/μL after DA-EPOCH-F, which similarly depleted CD8+ T cells and B cells. DA-EPOCH-F toxicities were mainly hematologic and transient. Responses to DA-EPOCH-F were CR/CRu (11%), PR (27%), SD (35%), and PD (23%). Response rates differed by histology (P=0.006); DLBCL or other aggressive NHL were less likely to respond. Baseline CD4 counts were higher in DA-EPOCH-F responders than in non-responders (median 374 vs. 181 cells/μL; P=0.003), despite a similar extent of prior therapy. Full donor chimerism was present by day 14 after RIST in 85% of pts; no graft rejection occurred. The absolute lymphocyte count (ALC) after DA-EPOCH-F was more strongly associated with donor chimerism after RIST (P=0.02) than was CD4+ or other lymphocyte subsets. We retrospectively compared immune depletion in DA-EPOCH-F recipients with that observed in 17 previously untreated pts receiving 6 cycles of DA-EPOCH-R for mantle cell NHL. Although pts receiving DA-EPOCH-F before RIST had lower baseline ALC and all lymphocyte subsets except NKT cells, relative depletion of ALC, T and B cells from baseline levels was surprisingly similar with DA-EPOCH-F and DA-EPOCH-R. We conclude that DA-EPOCH-F effectively provides TID and disease control or stability in most pts with lymphoid malignancies prior to RIST. This strategy is associated with rapid conversion to full donor chimerism and may be useful to enhance potential GVT effects after RIST.

Relapse of hematologic malignancy is a major problem after allogeneic hematopoietic stem cell transplantation (alloHSCT), with limited treatment options of proven benefit other than donor lymphocyte infusions (DLI). Additionally, toxicity from treatment with cytotoxic chemotherapy after alloHSCT may differ from conventional treatments, and could include graft-versus-host disease, allograft failure, or increased risk of infection. We retrospectively reviewed treatment outcomes of 25 patients who received cytotoxic chemotherapy as treatment for relapsed hematologic malignancies after reduced-intensity, matched-sibling donor alloHSCT. Combination therapies included: EPOCH+/− rituximab (n=12 patients); “7&3” AML induction (ida/ara-c) (n=2); FLAG (n=2); asparaginase/6-MP/vincristine (n=1); R-ICE (n=1); MIME (n=1); BVP (n=1); and bortezomib/doxorubicin/dexamethasone (n=1). Single-agent therapies included: bortezomib (n=6); gemcitabine (n=4), vinorelbine (n=3); vincristine (n=1); methotrexate (n=1); and intrathecal methotrexate and/or cytarabine (n=5). A total of 133 cycles or doses were administered, of which 20 were supported with donor stem cell boosts, and another 9 were followed by nonmobilized DLI. There were 52 Grade 3 or higher toxicity episodes recorded, most commonly: neutropenia (10 episodes); infection with neutropenia (5 episodes); thrombocytopenia (6 episodes); and anemia (3 episodes). There were 12 episodes of CMV reactivation in 8 of 15 patients at risk, including 2 cases of pneumonitis and 1 case of colitis. GVHD flares were considered definitely chemotherapy-induced (2), possibly chemotherapy-induced (7), and unlikely chemotherapy-induced (2). Unusual events after treatment included culture-negative sepsis-like illness after bortezomib (n=2), secondary (11q23) AML of donor origin after one cycle of EPOCH (n=1) and diffuse alveolar hemorrhage after EPOCH (n=1). Seven patients achieved CR with therapy for post-transplant relapse, including three durable remissions. Four patients achieved PR and seven achieved disease stabilization. Median survival after starting cytotoxic therapy was 263 days. Timing of relapse appeared to be associated with survival, with median overall survival of 95 days for patients requiring therapy before Day 100 vs. 508 days for patients after Day 100 (p=0.0008). Treatment of relapse with cytotoxic chemotherapy after alloSCT is feasible and can result in durable remissions in a minority of patients. However, administration of cytotoxic therapy after alloHSCT requires monitoring and support for hematologic toxicities, GVHD and CMV reactivation; selected patients may benefit from prophylactic stem cell boosts or immune suppression. Survival after Cytotoxic Therapy for Relapse after alloHSCT Survival after Cytotoxic Therapy for Relapse after alloHSCT

6546 Background: Despite being the most common non-Hodgkin’s lymphoma, there have been no specific reports on the use of reduced-intensity (RI) allogeneic stem cell transplantation (alloSCT) to treat patients (pts) with diffuse large B-cell lymphomas (DBCL). This may be due to a lack of definitive evidence for a therapeutic graft-versus-lymphoma (GVL) effect against DLBC. We undertook a retrospective analysis to assess clinical outcomes and evidence of a GVL effect in DLBC pts undergoing RI alloSCT. Methods: The analysis was limited to 18 pts with primary refractory (n = 6) or relapsed (n = 12) DLBC. The median age was 43 years (range: 31–61); median number of previous treatments was 3 (range: 2–9). Nine (50%) pts had undergone autologous transplantation. Three (16%) pts were determined to have chemo-sensitive disease to last treatment prior to RI alloSCT. All pts received a RI conditioning regimen consisting of fludarabine (30 mg/m2/d × 4d) and cyclophosphamide (1200 mg/m2/d × 4d) followed by a T-cell replete allograft from HLA-matched siblings. Results: Median potential follow-up from transplant is 43 months. Seven (39%) pts developed grade II-IV acute GVHD. Response at day +100 post-transplant was as follows: complete response (CR/CRu) = 5; partial response = 5; progressive disease = 8. Nine of 17 (53%) evaluable pts developed chronic GVHD. Median progression-free survival (PFS) was 4.8 months; however, PFS after 9 months post-transplant was 31% with 5 pts in continuous CR/CRu > 12 months post-transplant. Among 14 pts who were not in CR/CRu (n = 12) or progressed after achieving a CR/CRu (n = 2) at day +100 post-transplant, 8 (57%) subsequently achieved a CR/CRu after removal of immune suppression and/or donor lymphocyte infusion (DLI) ± chemotherapy. Seven of these 8 pts remain in continuous (median = 34 months; range: 6–55+) CR/CRu without further treatment. Median survival for all 18 pts was 19 months with survival probability of 40% plateauing at 25 months post-transplant. Conclusions: The clinical observations of sustained CR/CRu after withdrawal of immune suppression and DLI suggest that a GVL effect exists against DLBC. RI alloSCT should be considered as a treatment option for pts with primary refractory and relapsed DLBC. No significant financial relationships to disclose.

6540 Background: Significant variation in host immune status may influence outcomes after reduced-intensity (RI) allogeneic stem cell transplantation (alloSCT). We have investigated a strategy of targeted immune depletion (TID) with conventional chemotherapy to deplete host T cells and achieve a minimal disease state prior to RI alloSCT. The aim of TID is to rapidly establish complete donor chimerism after RI alloSCT in order to potentiate a graft-versus-tumor (GVT) effect. In a prospective phase II trial (NIH 03-C-0077), we evaluated the effect of TID on donor chimerism, acute graft-versus-host disease (GVHD), and clinical outcome. Methods: Thirty-one patients (pts) with relapsed and refractory hematologic malignancies (NHL = 16; HL = 4; CLL/PLL = 4; MDS/AML = 3; other = 4) were enrolled. Median age was 57 years (range: 31–71). All pts received EPOCH-F (etoposide, prednisone, vincristine, cyclophosphamide, adriamycin, fludarabine) ± rituximab (R) as TID to deplete host CD4+ cells 95%) was observed in 74% and 81% of pts at day +14 and +28 post-transplant, respectively. The incidence of grade II-III acute GVHD was 42% with no cases of grade IV acute GVHD. The median potential follow-up from transplant is 25 months. Actuarial treatment-related mortality at 1 and 2 years was 3% and 8%, respectively. Event-free survival probabilities at 1 and 2 years post-transplant are 65% and 49%, respectively. Ten pts are alive and event-free >24 months post-transplant. The overall survival probabilities at 1 and 2 years are 84% and 64%, respectively. Conclusions: TID prior to RI alloSCT results in rapid, complete donor engraftment and may potentiate GVT effects. This treatment strategy was associated with very low TRM and favorable outcomes in an older patient population with advanced hematologic malignancies. No significant financial relationships to disclose.

Treatment of refractory or recurrent malignancy with donor lymphocyte infusion (DLI) after allogeneic hematopoietic stem cell transplantation (alloHSCT) is often not curative, with graft-vs-tumor (GVT) effects frequently accompanied by graft-versus-host disease (GVHD), requiring immune suppression that compromises efficacy. After alloHSCT, chimeric T lymphocytes infiltrating residual tumor (chimeric TIL) may provide enhanced antigen specificity and maintain tumor-specific homing. Compared with DLI, they may have a better GVT effect with less GVHD. Based on the success of autologous TIL therapy for melanoma, we tested the hypothesis that enhanced GVT with limited GVHD could be achieved through administration of ex-vivo activated chimeric TIL after alloHSCT. Preclinical TIL production carried out on several tumor types from non-transplanted patients demonstrated effective T cell isolation, expansion and activation using anti-CD3/CD28 bead co-stimulation, yielding a 10- to 30-fold expansion of CD3+ cells. Clinical evaluation of chimeric TIL therapy was initiated with a 51 year-old woman for metastatic breast cancer whose disease progressed after a T cell-depleted reduced-intensity alloHSCT with delayed DLI from a 6/6 HLA-matched sibling donor and subsequent conventional therapy plus DLI. Two weeks after administration of unmanipulated DLI, two thoracic metastases were surgically removed. T cells were liberated from 9.4 cm of tumor using enzymatic digestion and mechanical dispersion, lymphocyte-enriched by density gradient separation, and expanded for 14 days through co-stimulation with anti-CD3/CD28-coated magnetic beads (3:1 bead-to-total nucleated cell ratio) and media containing IL-2 (100 or 1000 IU/mL). This process yielded 42.5 x 106 cells, 33% expressing CD3, and generated 14.7 x 109 chimeric TIL, 85% expressing CD3 (a 3.1-log T cell expansion). There was no tumor contamination of the T cell product by immunohistochemistry. Flow cytometry demonstrated that the CD4/CD8 T cell ratio increased from 1.3 to 1.9 after expansion. Three infusions of the chimeric TIL product were given in a dose-escalating manner (5, 25 and 100 x 106 CD3+ cells/kg). A fourth infusion was given in conjunction with low-dose IL-2 (6mu SQ per day x 3D). CT scan performed after each infusion monitored disease response, with progressive disease the indication for the next dose administration. No infusion-related or delayed toxicities were observed, except for rigors following IL-2 administration. The patient shows no evidence of GVHD, even after the highest dose of 108 allogeneic T cells. Evaluation of the remaining thoracic lesion demonstrated progressive disease after the first two chimeric TIL dose-levels, transient disease stability one month after the third dose-level, and stable disease one month after the fourth dose with IL-2. This is the first clinical report of the application of chimeric TIL and represents a novel approach for developing new forms of adoptive immunotherapy in the setting of alloHSCT.

Allogeneic hematopoietic stem cells (HSC) generally engraft rapidly and completely after myeloablative conditioning. However, with reduced-intensity conditioning (RIC), mixed chimerism and graft failure are more common. Host immune status and HSC number are factors known to affect engraftment after reduced-intensity stem cell transplantation (RIST). In addition, donor T cells within the allograft may also influencethe kinetics of donor engraftment after RIST. To evaluate this, we performed a controlled comparison of engraftment outcomes among 3 groups undergoing RIST, varying by ex vivo T cell depletion (TCD) or in vivo depletion of activated T cells with methotrexate (MTX) to prevent graft-versus-host disease (GVHD). Group I (n = 50) received T cell replete (TCR) peripheral blood stem cells (PBSC) with cyclosporine (CSA) alone for GVHD prophylaxis. Group II (n = 17) received ex vivo TCD PBSC (positive/negative selection with T cell add-back to uniform dose of 1 x 105 CD3+ cells/kg) with CSA alone for GVHD prophylaxis. Group III (n = 31) received TCR PBSC with CSA plus MTX (5 mg/m2 IV x 4 doses) for GVHD prophylaxis. The 3 groups were similarly immunosuppressed from prior therapy before RIST (median absolute lymphocyte counts 330/μL, 260/μL, and 307/μL for Groups I, II, and III, respectively), and received an identical RIC regimen (fludarabine/cyclophosphamide) plus comparable numbers of filgrastim-mobilized PBSC from HLA-matched sibling donors (median 7.9 x 106, 7.6 x 106, and 6.8 x 106 CD34+ cells/kg, respectively; median 3.6 x 108, 1.0 x 105, and 3.2 x 108 CD3+ cells/kg, respectively). Hematopoietic recovery was slowest in Group III, consistent with the myelosuppressive effects of MTX (Table). A greater proportion of patients in Group I achieved complete donor chimerism (≥ 95%) by day +28 than in Groups II or III (P < 0.025), and at day +100, mixed donor chimerism persisted more often in Groups II and III than in Group I patients (P < 0.01). Correspondingly, early (< day +42) occurrence of grade 3–4 acute GVHD, before initiation of planned sequential donor lymphocyte infusions (DLI) in Group II, was more frequent in Group I than in either Groups II or III (p=0.08). Table: Hematopoietic Recovery, Engraftment, and GVHD Group Days to ANC > 500, median (range) Days to plt > 100, median (range) Donor chimerism ≥ 95% Early acute GVHD, grades 3–4 Day +28 Day +100 I 9 (7–13) 15.5 (12-42) 37/44 (84%) 36/38 (95%) 9/50 (18%) II 9 (7–10) 17.5 (11–40) 8/17 (47%) 9/14 (65%) 0/17 (0%) III 14 (7–21) 21.5 (12–85) 23/31 (74%) 21/31 (68%) 2/31 (6%) Thus, the deletion of T cells by either ex vivo TCD or in vivo MTX administration measurably alters the kinetics and degree of donor T cell engraftment after RIST. These observations provide evidence that donor T cells are an independent factor affecting engraftment of allogeneic HSC after RIST by compensating for incomplete host immune ablation. These data also support the hypothesis that a graft-versus-host effect plays a significant role in engraftment after RIST. Manipulation of donor T cells through graft engineering techniques may be a useful strategy to enhance engraftment in the setting of RIST.

Relapse is a major cause of treatment failure after allogeneic hematopoietic stern cell transplantation (alloHSCT). Treatment options for relapse have been inadequate, and the majority of patients ultimately die of their disease. There is no standard approach to treating relapse after alloHSCT. Withdrawal of immune suppression and donor lymphocyte infusions are commonly used for all diseases; although these interventions are remarkably effective for relapsed chronic myelogenous leukemia, they have limited efficacy in other hematologic malignancies. Conventional and novel chemotherapy, monoclonal antibody therapy, targeted therapies, and second transplants have been utilized in a variety of relapsed diseases, but reports on these therapies are generally anecdotal and retrospective. As such, there is an immediate need for well-designed, disease-specific trials for treatment of relapse after alloHSCT. This report summarizes current treatment options under investigation for relapse after alloHSCT in a disease-specific manner. In addition, recommendations are provided for specific areas of research necessary in the treatment of relapse after alloHSCT.

In the National Cancer Institute’s Second International Workshop on the Biology, Prevention, and Treatment of Relapse after Hematopoietic Stem Cell Transplantation, the Scientific/Educational Session on Autologous Transplantation addressed the role of novel agents and immunomodulatory strategies in management of relapse after autologous hematopoietic stem cell transplantation (AHSCT). Concepts were illustrated through in-depth discussion of multiple myeloma, with broader discussion of areas relevant for relapse of other malignancies as well as in the setting of allogeneic transplantation. Dr. Hari provided an overview of the epidemiology of relapse after AHSCT in multiple myeloma, addressing clinical patterns, management implications, and treatment options at relapse, highlighting the implications of novel therapeutic agents in initial, maintenance and relapse treatment. Dr. Avigan discussed current concepts in tumor vaccine design, including whole-cell and antigen-specific strategies, use of an AHSCT platform to reverse tumor-associated immunosuppression and tolerance, and combining vaccines with immunomodulatory agents to promote establishment of durable antitumor immunity. Dr. Hsu reviewed the immunogenetics of natural killer (NK) cells and general NK biology, the clinical importance of autologous NK activity (e.g., lymphoma and neuroblastoma), as well as the impact of existing therapies on promotion of NK-cell activity (e.g., immunomodulatory drugs, monoclonal antibodies) and strategies for enhancing autologous and allogeneic NK-cell effects through NK-cell gene profiling.