ALBERT

Description: Key Points Donor T-Rapa cells were composed of Th1 and Th2 effectors with a reproducible gene expression profile. Preemptive T-Rapa donor lymphocyte infusion was safe and associated with donor engraftment without excessive GVHD.

Description: 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 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.

Description: 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

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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: Post-induction circulating CD8+ T cell counts in TCD recipients with delayed donor T cell engraftment. 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. Patient and Transplant Characteristics and Outcomes 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) Figure Figure

Description: 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.