ALBERT

Description: Introduction: T cell depletion of the stem cell allotransplant (SCT) has the advantage of reducing incidence and severity of GVHD but can be complicated by relapse and infection. An optimum residual T cell dose in T depleted SCT is not known. To optimize T cell depletion we delivered a series of fixed T cell transplant doses with scheduled T cell add back post-SCT in a series of protocols to determine the T cell doses that secured immune reconstitution, and minimized relapse and infection. Here we retrospectively examined large series of patients (pts) transplanted in a single institution where T cell dose at SCT and add back were quantitated. Unique to these protocols was the precise measurement of CD3 dose/kg at transplant for every patient which enabled us to relate transplant T cell doses to outcome. Patients and methods: 205 pts (106 males, 99 females) underwent HLA identical sibling allogeneic T cell depleted SCT between 1994 and 2014. Diagnosis at SCT included AML (48%), MDS (17%), ALL (27%), CLL (5%), MM (3%). Disease risk at SCT was classified as high (57%) or standard (43%). The median age at SCT was 37 (range: 10-75) yrs. TBI based myeloablative conditioning was used. The graft source was marrow in 20 pts and peripheral blood in 195 pts. GVHD prophylaxis was low dose cyclosporine. Different T cell depleted methods were used consecutively: elutriation, Isolex ®, Cellpro ® CD34, and Miltenyl © CD34 selection. A defined T cell dose was allocated at SCT by protocol ranging from 2 - 50 × 104 CD3+ cells/kg. Various schedules were used to add back T lymphocytes between day 30 to 90 with doses ranging 5 - 60 ×106 CD3+ cells/kg by protocol and no T cell add-back was given in 28 pts in recent protocols. Overall survival (OS) was estimated by the Kaplan-Meier method, and cumulative incidence of relapse and nonrelapse mortality (NRM) was estimated by Gray's method to account for competing risks. Cox proportional hazard regression models were used to assess the association of factors at baseline, day 100 and GVHD with the post-SCT outcomes. Results: At a median follow-up of 8.6 yrs (range: 0.7- 19.8) for surviving pts, 112 pts died (52 from NRM) and 68 pts relapsed. OS was 47%, 43% and 41%, NRM was 24%, 27% and 27%, relapse was 32%, 34% and 38% at 5 yr, 10 yr and 15 yr post SCT, respectively. Grade II-IV and III-IV acute GVHD were 41% and 13%, and chronic GVHD was 42% (25% limited, 17% extensive). In the multivariate models of baseline risk factors that adjusted for age at SCT and disease risk, T-cell doses at SCT did not affect OS, NRM or relapse. A higher dose of CD34+ cells at SCT was significantly associated with better OS and lower NRM. Disease risk was an independent predictor, with high-risk pts having more relapse and worse OS, compared to pts with standard risk. A landmark analysis of 156 pts surviving and relapse-free beyond day 100 was carried out to examine the effects of add back T cell schedules by day 100 and aGVHD. The total T-cell dose at add back by day 100 and different add-back T-cell schedules from day 30-90 had no impact on any outcome, controlling for T-cell dose at SCT. In the models controlling for age, risk, CD34+ and T-cell dose at SCT, pts with grade III-IV aGVHD by day 100 had an increased risk for overall mortality and NRM beyond day 100 (HR= 3 and 3.6, both P

Description: Background. Aplastic anemia (AA), the prototypical bone marrow (BM) failure syndrome, is caused by immune-mediated destruction of hematopoietic stem/progenitor cells (HSPCs). CD8+ cytotoxic T cells with restricted TCR diversity (oligoclonal T cells) are expanded in AA, leading to production of proinflammatory cytokines, such as IFN-γ, which induce apoptosis of HSPCs. Recent studies have identified a new subset of memory T cells with stem cell-like properties, TSCM, which are the least differentiated cells of all distinct memory populations. Functionally, TSCM possess an enhanced capacity for self-renewal and can generate multiple memory T cell populations, and they likely have an important role in controlling immunity. In autoimmune diseases, there is abnormal CD4+ and CD8+ T cell activation. We evaluated TSCM frequency in AA and its association with severity, treatment response, relapse, and changes after immunosuppressive therapy (IST). Further, to evaluate the TSCM in other autoimmune diseases, we examined CD4+ and CD8+ TSCM frequencies in uveitis, systemic lupus erythematosus (SLE), and sickle cell disease (SCD), as compared with healthy controls. Method. We retrospectively analyzed CD4+ and CD8+ TSCM populations by flow cytometry. PB specimens were collected from 55 AA samples and 41 age-matched healthy donor samples. Among 55 AA samples, 21 samples were analyzed at diagnosis and 34 after IST. For comparison, blood samples were obtained from 34 uveitis patients (27 inactive or 7 active cases), 43 SLE patients who met the American College of rheumatology (ACR) criteria for the disease [19 inactive SLE (SLE disease activity index-2K (SLEDAI-2K) score 〈 3; and 24 active SLE (SLEDAI-2K score 〉 3)], and 5 SCD patients who were receiving frequent transfusions. TSCM was defined as CD3+ CD4 (CD8)+ CD45RO- CD45RA+ CCR7+ CD27+ CD95+ population. Results and Discussion. In healthy controls, TSCM represented a relatively small percentage of circulating CD4+ or CD8+ T cells (median 2.4% CD4+ TSCM and 2.1% CD8+ TSCM, Fig. 1A). A significantly higher CD8+ TSCM frequency was detected in AA patients (4.2% vs. 2.1%, p 〈 0.05) while there was no difference in the CD4+ TSCM frequency (p 〉 0.05), compared to controls (Fig. 1B-C). In AA, high CD8+ TSCM frequency at diagnosis correlated with complete (CR) or partial response (PR) to IST [5.0 % in CR and PR vs 2.8 % in non-responders (NR), p 〈 0.05). In AA patients prior to IST (n=21), CD8+ TSCM frequency was not correlated with age, sex, absolute neutrophil count, platelet count, time from diagnosis to therapy, and serum ferritin levels. CD8+ TSCM were significantly increased in the two AA cohorts (with or without IST), relative to controls (p 〈 0.05, respectively). Higher CD8+ TSCM frequency after IST associated with treatment-failure (3.5 % in responders vs 5.5 % in NR or relapse, p 〈 0.05). Stimulation with anti-CD3/CD28 beads successfully induced cytokine production in CD4+ and CD8+ T cells from AA and healthy controls. Elevated IFN- γ and IL-2 levels were seen in CD4+ and CD8+ TSCM in AA compared to healthy controls. We next compared CD4+ or CD8+ TSCM frequency between each patient group (AA, uveitis, SLE, or SCD) and a healthy control group. Among the four patient groups, the uveitis group alone displayed a reduction in CD4+ TSCM frequency (1.8%) relative to the healthy controls (2.4 %; p 〈 0.05). An elevated CD8+ TSCM frequency was observed in AA (4.2 %), uveitis (3.6 %), and SCD (4.3 %), but not in SLE, compared to controls (2.1%; p 〈 0.05) (Fig. 2A). Positive correlation between CD4+ and CD8+ TSCM frequencies was found in AA, autoimmune uveitis, and SLE (Fig. 2B). Evaluation of PD-1 expression revealed that TSCM were the least exhausted T cell compartment, as compared to other types of memory T cells. Immune therapies appeared to have negative effects on the TSCM population both in uveitis and SLE patient cohorts, as well as in AA. Conclusion. We provide evidence for increased circulating CD8+ TSCM in AA, underscoring the importance of this novel subset in regulation of immune responses and pathogenesis of autoimmunity. Our work described previously unknown potential roles of TSCM in AA, such as cytokine secretion correlated with effector functions. Understanding the CD8+ TSCM population may offer new therapeutic strategies and novel mechanistic insight into the various autoimmune diseases. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Townsley: Novartis: Research Funding; GSK: Research Funding. Dumitriu:Novartis: Research Funding; GSK: Research Funding. Young:Novartis: Research Funding; GSK: Research Funding.

Description: INTRODUCTION: Ex-vivo T cell depletion strategies have been widely used to reduce the incidence of graft versus host disease (GVHD) in allogeneic stem cell transplantation (allo-SCT). Although several options of ex-vivo graft manipulation strategy are available, direct comparison between strategies along with relevant biomarkers has been lacking. Here we evaluated cellular and plasma biomarkers in two separate graft manipulation strategies, CD3-CD19 depletion versus CD34+ selection using the Miltenyi CliniMACS and their association with clinical outcomes. METHODS: Forty two subjects with hematological malignancies underwent HLA matched sibling allo-SCT at a single center between 2012 and 2015 and received either an ex-vivo CD3-CD19 depleted, CD34+ negatively selected graft (CD3/19D, n=20) or an ex-vivo CD34+ cell positively selected graft (CD34S, n=22). Both cohorts were treated with the same conditioning regimen of cyclophosphamide, fludarabine, and total body irradiation (600-1200 cGy) and GVHD prophylaxis of low dose cyclosporine. Peripheral blood mononuclear cells and plasma samples were collected at days 14 or 30, 60, 100 post-transplant. Post-transplant cellular immune reconstitution was evaluated by multi-color flow cytometry immunophenotyping, characterizing the subsets of memory T cells, regulatory T cells (Tregs), natural killer (NK) cells, and B cells with various functional markers. The plasma levels of ST2, Reg3α, and sTNFR1 were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The median age at transplant was 48 years (range 17-70) in CD3/19D and 45 years (11-73) in CD34S. At a median follow up of 37 months in CD3/19D and 22 months in CD34S, the major clinical outcomes were similar between two groups; the overall survival (70% and 86%), non-relapse mortality (5% and 4.5%), and cumulative incidence of relapse (35% and 39%) at 2 years, respectively. Two subjects in CD3/19D developed late engraftment failure before day 100 but all other subjects achieved primary neutrophil and platelet recovery. Unexpectedly, the cumulative incidence of grade II-IV acute GVHD was higher in CD3/19D (61%) in comparison to the incidence in CD34S (32%, P=0.07, Figure). The cumulative incidence of extensive chronic GVHD was 33% in CD3/19S and 24% in CD34S. The fraction of Helios negative Tregs post-transplant was significantly lower in CD3/19D (median [interquartile range]: 10.4% [7.1-16.4] at day 30; 4.9% [3.0-8.3] at day 60) compared to CD34S (23.8% [10.7-35.8], P=0.03 at day 30; 8.8% [6.8-18.4], P=0.01 at day 60, Figure). Plasma ST2 levels were significantly higher in CD3/19D (45ng/mL [27-67] at day 14; 33ng/mL [27-62] at day 28) in comparison to CD34S (29ng/mL [19-40], P=0.03 at day 14; 25ng/mL [14-33], P=0.03 at day 28, Figure). In addition, significantly higher CD4 naive T cells, lower effector memory and PD-1 bright CD4 T cells were observed in CD3/19D in comparison to CD34S. NK and B cell profiles were not significantly different between the two groups. CONCLUSION: Both methods of ex vivo TCD were associated with extremely low NRM rates (~5%).We observed a higher cumulative incidence of acute GVHD in the recipients of CD3/19 depleted grafts, accompanied with the distinct biomarker profiles of poor Treg reconstitution and high level of ST2. CD3/19 depletion may have disproportionately depleted Tregs in the graft, leading to uncontrolled tissue damage and GVHD evidenced by higher ST2 levels. Further validation is required to confirm the utility of monitoring Treg reconstitution and ST2 level as biomarkers to predict the outcomes of T cell depleted allo-SCT. Figure 1. Figure 1. Disclosures Battiwalla: NIH/NHLBI: Employment.

Description: Introduction: Multiple myeloma (MM) is an incurable malignancy of B-cell lineage. Introduction of immunomodulatory drugs such as lenalidomide has significantly improved overall survival of patients with MM. Lenalidomide maintenance is currently the standard of care for maintaining remission in MM. However, second primary malignancies are known to arise in patients on lenalidomide though their pathogenesis is not known. We report a patient who developed B-lymphoblastic leukemia (B-ALL) while on lenalidomide, which went into spontaneous remission after stopping lenalidomide. Whole exome sequencing (WES) was performed to examine the mutational landscape and clonal evolution of the different malignant clones. We also aimed to postulate a mechanism for lenalidomide-induced ALL. Case history and methods: A 59-year-old female with history of rheumatoid arthritis was diagnosed with IgG-κ MM and treated with 8 cycles of bortezomib, Lenalidomide and dexamethasone followed by lenalidomide-maintenance (10 mg/day). At 36 months after initiation of treatment for MM, she developed lymphopenia and her bone marrow (BM) biopsy showed 38% leukemic B lymphoblasts. Lenalidomide was discontinued and a follow-up BM biopsy done 2 months later showed spontaneous complete remission of ALL, which was confirmed at 8 months. She remains in complete remission of ALL and MM without any specific treatment at 12 months after the diagnosis of ALL. BM aspirate samples at diagnosis of MM, on lenalidomide-maintenance with MM in remission, at diagnosis of ALL, and after stopping lenalidomide with MM and ALL in remission were used to perform WES with 2x150 bp reads and 100x coverage utilizing the Illumina Hiseq. The raw reads were mapped to reference genome (hg19) and compared with peripheral blood T-lymphocytes as germ line control. Variants were annotated using dbSNP, CLINVAR and COSMIC through Mutect. Clonal evolution was analyzed by SciClone. The study was approved by Institutional Review Board. Results: The burden of somatic mutations was significantly higher at diagnosis of MM when compared to the three other time points. Analysis of clonal architecture revealed the distinct clustering of mutations at specific time points (Figure). Most mutations detected at diagnosis of MM (e.g. NOTCH2, BTG1, BCLAF1) disappeared after treatment for MM. Similarly, most mutations detected only at diagnosis of ALL (e.g.PIK3CD, CDK16) became undetectable at spontaneous remission. Interestingly, clones with mutations of IGSF3 (immunoglobulin superfamily) and CXXC4 (Wnt signaling pathway) were detectable while the patient was on lenalidomide and at diagnosis of ALL but disappeared after stopping lenalidomide, which suggests that these clones gained pro-survival advantage from lenalidomide. Only a few mutations (GSDMC, NBPF20, ANAPC1) persisted in both MM and ALL stages. GSDMC is a gasdermin family member which may modulate function of MYC. NBPF20 has been described in relapsed pediatric ALL. ANAPC1 is a cell cycle gene whose transcription is regulated by Ikaros. Loss of repressor function of Ikaros was recently reported to deregulate ANAPC1 expression and cause mitotic progression of ALL in vitro. As lenalidomide is known to induce degradation of Ikaros, we hypothesize that lenalidomide may create a favorable selection pressure for B-cell clones harboring mutations in Ikaros-dependent genes. Conclusions: Clonal evolution analysis suggests that MM and ALL arose from different B-cell sub-clones, which was consistent with previous observation. However, there are a few shared mutations between MM and pre-B ALL, which may be responsible for leukemogenesis in our case. Lenalidomide may affect intracellular protein interactions to induce selection of rare B-cell clones evolving into secondary ALL. Also, our case demonstrated that simply stopping lenalidomide may lead to spontaneous and durable regression of ALL. Transcriptomic and proteomic analysis including Ikaros expression is required to further understand the mechanism of appearance of ALL and its regression after stopping lenalidomide. Figure Disclosures Shlomchik: BlueSphere Bio: Other: Founder and Equity Interest.

Description: Long term allogeneic stem cell transplantation (allo-SCT) survivors face a 2.3 fold increase risk of premature cardiovascular (CV) related death compared to the general population. A reliable screening strategy to identify allo-SCT survivors at risk for CV-related disease is therefore warranted to minimize future events. Cardiac CT is an emerging non-invasive imaging technology with high sensitivity for detecting coronary artery disease (CAD) and high negative predictive value to exclude the presence of CAD. We conducted the first prospective non-randomized single institution study to evaluate Agatston coronary calcium scoring by CT with concomitant coronary CT angiograms as a tool to identify the survivors at risk for CV disease. Sixteen asymptomatic post allo-SCT survivors (11 males; 5 females) with median age of 45 years (range 22-66) at transplant underwent coronary calcium scoring and contrast enhanced coronary CT angiograms at a median follow up of 5 years post transplant. 10-year Framingham cardiovascular risk scores (incorporating age, sex, total cholesterol, HDL cholesterol, systolic BP, HTN, smoking status) were also calculated at time of screening. Two were classified as high risk, 1 intermediate and 13 as low risk. Iodinated IV contrast was administered for coronary artery visualization and IV hydration given to patients with decreased creatinine clearance. Non-obstructive CAD was detected in seven (44%) patients. Additionally, four (25%) of these subjects had aortic root calcification. Lesion distributions by arterial territory were: left main 5.8%, left anterior descending 35.3%, left circumflex 29.4% and right coronary artery 29.4%. Characteristics of coronary plaques were: 47% calcified, 47% mixed calcified / non-calcified, and 6% non-calcified. In those with CAD, the median coronary calcium score was 55 (range: 0-992) (p

Description: Abstract 328 Low CD4+ T cell counts early post-SCT are associated with a greater risk of developing chronic GVHD (cGVHD). However, the factors affecting post graft CD4+ T cell recovery are not known. Furthermore, the CD4 T cell subset correlating best with protection from cGvHD is not defined. We hypothesized that the donor immune repertoire determines CD4+ reconstitution after SCT. We studied 220 donor-recipient pairs undergoing a myeloablative matched-sibling T-cell depleted SCT for a variety of hematological malignancies including AML, ALL, CML, and MDS. Median donor and patient age was 36 years (range, 9 – 70). Median CD34+ cell dose was 5.56 ×106/kg (range, 2.3–14.5), with a fixed T cell dose per protocol of 1–5 ×104/kg. Ninety-four patients (43%) developed chronic GvHD (32 limited, 64 extensive). Prior to SCT a cryopreserved mononuclear fraction (PBMC) was obtained by leukopheresis on all donors. PBMC from 139 donors were stained with fluorescently conjugated antibodies against CD3, CD4, CD8, CD27, CD45RA, CD31, CD25, CD14, CD19, FOXP3, and Helios and acquired on a custom-built LSR Fortessa (BD) flow cytometer. The following T cell subsets were examined: naive, CD27+CD45RA+; central memory, CD27+CD45RA-; effector memory, CD27-CD45RA-; and effector T cells, CD27-CD45RA+. Regulatory T cells (Tregs) and Treg subsets were identified within the CD4+ T cell population using a combination of FOXP3 and Helios The combination of FOXP3 with Helios has recently been shown to identify natural, thymus-derived Tregs (nTregs), whereas CD4+FOXP3+ T cells lacking Helios represent a pool of induced Tregs (Thornton et al., 2010, J Immunol. 184:3433–3441). Combined expression of CD31 and CD45RA in the CD4+ and nTreg populations was used to identify recent thymic emigrants (RTE). Data were analyzed by FlowJo (Treestar, Ashland, OH, version 8.6.6), and both univariate and multivariate analysis analyses using the Cox regression models with right censored time-to-event variables were performed on the readouts (SPSS 15.0, S-Plus 8 and Prism 4 Software). All p-values were computed based on the log-rank test statistics. Absolute lymphocyte counts in 220 donors were 2.11/μl (range 0.89 – 4.12). A low absolute lymphocyte count was significantly correlated with a higher cumulative probability for time to incidence of extensive cGVHD (HR 2.38, p = 0.0008). This relationship between lymphocytes and cGVHD was retained in CD4+ T cell subsets: CD3+CD4+ (HR 2.48, p = 0.005); CD3+CD4+CD25+ 〈 30 cells/μL (HR 2.1, p = 0.02); nTregs 〈 8 cells/μL (HR 1.97, p = 0.043). RTE of total CD4 did not correlate with RTE nTregs, indicating that nTreg production varies among donors with similar levels of thymic activity. Furthermore recipients of donors with low RTE nTregs relative to overall thymic output had a significantly higher likelihood of developing extensive chronic GvHD (HR 2.78, p=0.0014). In multivariate analysis including patient age, acute GvHD (grade II – IV), graft direction (female donor to male recipient vs. other combinations), disease and status at transplant, and administration of post SCT donor lymphocyte infusion, only thymic output of nTregs (HR = 0.8, p = 0.037) and graft direction (HR = 2.1, p = 0.023) remained significant risk factors for extensive cGvHD. These data show that the thymic output of nTreg varies inherently among donors. Donors with low thymic output of nTreg may recreate a less tolerant immune system in the recipient leading to cGVHD of greater severity. (ZAM and JJM contributed equally to this work). Disclosures: No relevant conflicts of interest to declare.

Description: Introduction . T-cell large granular lymphocytosis (T-LGL) is a low grade lymphoproliferative disorder, often clinically manifest as bone marrow failure. Treatment with immunosuppressive therapies is effective, but the dominant clone may persist even in responding patients. The pathogenesis of T-LGL has not been fully elucidated. In this study, we performed single cell RNA sequencing (sc-RNA seq) and V(D)J profiling to discern clonotypes and gene expression patterns of T lymphocytes from T-LGL patients who were sampled before and after treatment. Methods. Blood was obtained from patients participating in a phase 2 protocol of alemtuzumab as second line therapy (NCT00345345; Dumitriu B et al, Lancet Haematol 2016). Leukapheresis was performed in 13 patients (M/F 7/6; median age 51 years, range 26-85) before and after 3-6 months alemtuzumab administration and in 7 age-matched healthy donors. Cryopreserved blood was enriched for T cells with the EasySep Human T cell Isolation Kit (Stem cell). sc-RNA seq was performed on the 10XGenomics Chromium Single Cell V(D)J + 5' Gene Expression platform, and sequencing obtained on the HiSeq3000 Platform. Barcode assignment, alignment, unique molecular index counting and T cell receptor sequence assembly were performed using Cell Ranger 2.1.1. Results. Four hundred fifty thousand cells from 13 patients and 107,000 cells from 7 healthy donors were profiled. We measured productive TCR chains (which fully span the V and J regions, with a recognizable start codon in the V region and lacking a stop codon in the V-J region, thus potentially generating a protein). We detected at least one productive TCR α-chain in 50%, one productive TCR β-chain in 69% and paired productive αβ-chains in 47% of all cells. There was loss of TCR repertoire diversity in patients which was quantified by Simpson's diversity index; most patients showed oligoclonal or, less frequently, monoclonal expansion of the TCR repertoire (Fig. A). Regardless of clinical response, alemtuzumab treatment did not correct the low TCR repertoire diversity. TCR repertoires can be classified as "public", when they express identical TCR sequences across multiple individuals, or "private", when each individual displays distinct TCR clonotypes. No TCRA or TCRB CDR3 homology among patients was observed: most TCR clonotypes appeared to be private. Our data suggests that T-LGL is etiologically heterogenous disease, consistent with T cell expansion in response to a variety antigens, in diverse HLA contexts, or randomly. Despite differences of TCR among patients and healthy donors, and the presence of large clones in patients, distribution of TCR diversity followed the power law distribution in healthy donors and patients (Fig. B, showing the negative linear relationship between logarithmic expression of clone frequency and clone size). The observed distribution is consistent with a somatic evolution model, in which cell fitness depends on cellular receptor response to specific antigens and stimulation of cells by cytokine and other signals from the environment; fitted clones have higher birth-death ratios and thus expand (Desponds J et al, PNAS 2016). CD4 and CD8 T cells can be virtually separated by imputation from their transcriptomes (Fig. C). Comparison of gene expression between patients and healthy donors showed dysregulation of genes involved in pathways related to the immune response and cell apoptosis, consistent with a pathophysiology of T cell clonal expansion. We used diffusion mapping, which localizes datapoints to their eigen components in low-dimesional space, to characterize sources contributing to the gene expression phenotype: the first component was mainly from T cell activation and the second was associated with TCR expression. In LGL the T cell transcriptome appeared to be shaped by both lineage development and TCR rearrangement. Conclusion. We describe at the single cell level T clonal expansion profiles in T-LGL, pre- and post-treatment. Single cell analysis allows accurate recovery of paired α and β chains in the same cell and demonstrates a continuum of cell lineage differentiation. We found a range of differences in transcriptome and TCR repertoires across patients. Transcriptome data, coupled with detailed TCR-based lineage information, provides a rich resource for understanding of the pathology of T-LGL and has implications for prognosis, treatment, and monitoring in the clinic. Figure. Figure. Disclosures Young: GlaxoSmithKline: Research Funding; CRADA with Novartis: Research Funding; National Institute of Health: Research Funding.

Description: INTRODUCTION: The potent graft versus leukemia (GVL) effect of allogeneic stem cell transplantation (allo-SCT) is considered as a blueprint for cellular immunotherapy. However, failure of GVL leads to relapse of underlying leukemia, the major cause of death after allo-SCT. In solid tumors, higher tumor mutation burdens are associated with better response to check point inhibitors which implies the importance of neoantigen specific T-cell functions in cancer immunity. In contrast, the frequencies of somatic mutations in acute leukemia are generally low, therefore the role of neoantigens in GVL remains undetermined. Here, we developed a platform to screen for potential neoantigens by performing whole exome sequencing (WES) and RNA sequencing (RNAseq) in matched samples: leukemic blasts at relapse after allo-SCT, recipient T cell controls, and donor cells. METHODS: Leukemic blasts from patients in relapse were enriched by flow sorting from bone marrow aspirate or peripheral blood samples. Recipient T cells were isolated from pre-transplant peripheral blood as germline controls, and donor monocytes or CD34-positive cells were used as hematopoietic-lineage cell controls. WES was performed to 100X coverage, paired with RNAseq 40M reads per sample. Somatic mutations were detected with mutect and mpileup, followed by annotation with SnpEff. High confidence somatic mutations were subjected to pVAC-seq for neoantigen predictions. RESULTS: Six patients with relapsed acute leukemia (AML 5, ALL 1) after allo-SCT and their transplant donors (matched sibling 3, haplo-identical 3) had suitable samples available for analysis. On average, somatic mutations were identified in 297 genes (range 108- 609) by comparing leukemic blasts and germline control T cells. Among those mutations, potential candidates of neoantigen were identified in five out of six subjects. Allele frequencies of mutant genes varied. Most of neoantigens were predicted to bind HLA of both class I (median 5, range 0-15) and class II (median 6, range 0-12). One subject had only HLA class II restricted peptides as predicted neoantigens. Of interest majority of antigens were derived from molecules known to play important roles in leukemia or tumor biology which include ETV6, CCNY, IDH2, PTPN11, SF3B1, and TP53. Evolution analysis of neoantigen showed an emergence of new antigens in relapsed leukemia while a few driver gene mutations persisted after allo-SCT (Figure). CONCLUSION: Our in-silico analysis demonstrated the possibility that somatic mutation in acute leukemia could serve as putative neoantigens applicable for novel immunotherapy after allo-SCT. The binding capacity of mutant peptides to class I and II HLA implies the importance of both CD4 and CD8 contributions to anti-neoantigen immunity. Next, we will search for neoantigen specific T cells exerting an anti-leukemia effect to validate the GVL potential of these mutations in allo-SCT. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Allogeneic CD19-CAR VSTs are well tolerated by patients with relapsed B-cell malignancies post-HSCT. At periods of CD19-CAR VST persistence, these cells demonstrate antitumor activity.

Description: Abstract 654 Unrelated cord blood (UCB) transplantation is a useful alternative for patients with hematological malignancies or non-malignant hematological disorders lacking an HLA matched donor. However, outcomes for patients with severe aplastic anemia (SAA) undergoing either a single or dual UCB transplant have been disappointing. A recent EBMT/ Eurocord study reported engraftment and 3 year survival rates of only 51% and 38% respectively (Perrault de Latour, Biol Blood Marrow Transplant 2011). We investigated whether co-infusion of a single UCB unit with CD34+ selected cells from a haploidentical relative following a highly immunosuppressive conditioning regimen could improve transplant outcome for patients with SAA refractory to immunosuppressive therapy that lack an HLA matched donor. Subjects with SAA and life-threatening neutropenia (ANC 500 by day 42, 7 of 8 achieving a UCB-derived ANC 〉500 cells/μl. The median time to neutrophil recovery was 10 days (range 10–18 days). One patient failed to engraft with the cord unit, but has had sustained engraftment from the haploidentical donor, and is transfusion independent with a normal neutrophil count 〉25 months post transplant. Acute GVHD grade II developed in 2 patients and one developed limited chronic GVHD. Early T-cell engraftment was predominantly UCB in 7 cases; on day 21, T-cell chimerism was a median 100% cord in origin (range 0–100%). In contrast, myeloid chimerism at engraftment was predominantly haplo-donor in origin and showed 3 phases of engraftment: 1) early myeloid engraftment from the haplo-CD34+ cell donor 2) delayed myeloid engraftment from the cord unit resulting in dual myeloid chimerism and 3) disappearance of the haplo-donor cells with transition towards full cord donor myeloid chimerism (see figure). Mixed lymphocyte reactivity assays performed on post transplant PBMCs showed increasing alloreactivity of cord blood T-cells against the haploidentical donor during the period when myeloid chimerism transitioned towards cord, indicating that the disappearance of haplo-donor myeloid cells occurred as a consequence of rejection by engrafting cord blood T-cells. At a median follow-up of 9 months (range 75 days to 3 years), 7 patients survive, and all are transfusion–independent. One patient died 14 months after transplantation from complications related to CMV pneumonitis. In conclusion, transplantation of haploidentical CD34+ cells can shorten the time to neutrophil recovery in SAA pts undergoing a single UCB transplant. Furthermore, durable full engraftment from donor haploidentical CD34+ cells can occur in the context of cord graft failure. These data suggest co-infusion of allogeneic cord blood with haploidentical CD34+ cells can improve the outcome of UCB transplantation for SAA. Disclosures: Wilder: NCI: Funded in part by NCI contract No. HHSN261200800001E.