Comparative Outcomes for Mature T and NK/T-cell Lymphomas in People with and without HIV and to AIDS-Defining Lymphomas

There are no studies comparing the prognosis for mature T-cell lymphoma (TCL) in people with human immunodeficiency virus (PWH) to people without HIV (PWoH) and to AIDS-defining B-cell lymphomas (A-BCL) in the modern antiretroviral therapy (ART) era. NA-ACCORD and COMPLETE are cohorts that enroll patients diagnosed with HIV and TCL, respectively. In our study 52, 64, 101, 500 and 246 PWH with histological confirmation of TCL, primary CNS, Burkitt's, diffuse large B-cell lymphoma (DLBCL) and Hodgkin's lymphoma (HL) respectively and 450 TCL without HIV were eligible for analysis. At the time of TCL diagnosis, Anaplastic large-cell lymphoma (ALCL) was the most common TCL subtype within PWH. While PWH with TCL diagnosed between 1996-2009, experienced a low 5-year survival probability at 0.23 (95% CI: 0.13, 0.41), we observed a marked improvement in their survival when diagnosed between 2010-2016 (0.69; 95% CI: 0.48, 1; p=0.04) in contrast to TCL among PWoH (0.45; 95% CI: 0.41, 0.51; p=0.53). Similarly, PWH with ALCL diagnosed between 1996-2009 were associated with a conspicuously inferior 5-year survival probability (0.17; 95% CI: 0.07, 0.42) and consistently lagged behind A-BCL subtypes such as Burkitt's (0.43; 95% CI:0.33, 0.57; p=0.09) and DLBCL (0.17; 95% CI: 0.06, 0.46; p=0.11) and behind HL (0.57; 95% CI: 0.50, 0.65; p <0.0001). Despite a small number, those diagnosed between 2010-2016, experienced a remarkable improvement in survival (0.67; 95% CI: 0.3, 1) in comparison to PWoH (0.76; 95% CI: 0.66, 0.87; p=0.58). Thus, our analysis confirms improved overall survival for aggressive B and T-cell malignancies among PWH in the last decade.

Epigenomic translocation of H3K4me3 broad domains over oncogenes following hijacking of super-enhancers

Chromosomal translocations are important drivers of hematological malignancies whereby proto-oncogenes are activated by juxtaposition with super-enhancers, often called enhancer hijacking. We analysed the epigenomic consequences of rearrangements between the super-enhancers of the immunoglobulin heavy locus (IGH) and proto-oncogene CCND1 that are common in B cell malignancies. By integrating BLUEPRINT epigenomic data with DNA breakpoint detection, we characterised the normal chromatin landscape of the human IGH locus and its dynamics after pathological genomic rearrangement. We detected an H3K4me3 broad domain (BD) within the IGH locus of healthy B cells that was absent in samples with IGH-CCND1 translocations. The appearance of H3K4me3-BD over CCND1 in the latter was associated with overexpression and extensive chromatin accessibility of its gene body. We observed similar cancer-specific H3K4me3-BDs associated with super-enhancer hijacking of other common oncogenes in B cell (MAF, MYC and FGFR3/NSD2) and in T-cell malignancies (LMO2, TLX3 and TAL1). Our analysis suggests that H3K4me3-BDs can be created by super-enhancers and supports the new concept of epigenomic translocation, where the relocation of H3K4me3-BDs from cell identity genes to oncogenes accompanies the translocation of super-enhancers.

The Role of NKL Homeobox Genes in T-Cell Malignancies

Homeobox genes encode transcription factors controlling basic developmental processes. The homeodomain is encoded by the homeobox and mediates sequence-specific DNA binding and interaction with cofactors, thus operating as a basic regulatory platform. Similarities in their homeobox sequences serve to arrange these genes in classes and subclasses, including NKL homeobox genes. In accordance with their normal functions, deregulated homeobox genes contribute to carcinogenesis along with hematopoietic malignancies. We have recently described the physiological expression of eleven NKL homeobox genes in the course of hematopoiesis and termed this gene expression pattern NKL-code. Due to the developmental impact of NKL homeobox genes these data suggest a key role for their activity in the normal regulation of hematopoietic cell differentiation including T-cells. On the other hand, aberrant overexpression of NKL-code members or ectopical activation of non-code members has been frequently reported in lymphoid and myeloid leukemia/lymphoma, demonstrating their oncogenic impact in the hematopoietic compartment. Here, we provide an overview of the NKL-code in normal hematopoiesis and discuss the oncogenic role of deregulated NKL homeobox genes in T-cell malignancies.

Characterization of WU-CART-007, an Allogeneic CD7-Targeted CAR-T Cell Therapy for T-Cell Malignancies

Background T-cell Acute Lymphoblastic Leukemia (T-ALL) / Lymphoblastic Lymphoma (LBL) represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults. Development of CAR-T cell therapies for T-cell cancers has been complicated by induction of fratricide and the high risk of malignant cell contamination of the drug product in the autologous setting. Previously, Cooper et. al., demonstrated that CRISPR/Cas9 gene-editing to delete CD7 prevented self-killing, and deletion of the T-cell receptor alpha constant (TRAC) enabled the use of healthy donor-derived T-cells to manufacture CD7-targeted CAR-T cells without risk of malignancy and mitigating the risk of GvHD. Here we present preclinical data supporting the safety and efficacy of WU-CART-007, an IND ready, off-the-shelf, and fratricide resistant CD7-targeted CAR-T cell for the treatment of CD7+ T-cell malignancies. Methods WU-CART-007 was manufactured using T cells isolated from healthy donors by deletion of CD7 and TRAC, followed by CAR transduction, cell expansion, depletion of residual TCRa/b+ cells and cryopreservation. Donors were confirmed negative for a panel of adventitious viruses. CD7/TRAC deletion and CAR transduction were confirmed by flow cytometry. Off-target editing profile was assessed by GUIDE-Seq. The binding kinetics to human CD7 were conducted by bio-layer interferometry and CD7 selectivity was confirmed by cell microarray with a library of HEK-293 cells expressing approximately 6000 human proteins. The in vitro activity of WU-CART-007 was interrogated by co-culture with human CD7+ CCRF-CEM T-ALL cells and the potential on-target, off-tumor activity was assessed by co-culture with a panel of immune and non-immune primary human cells. In vivo anti-tumor functionality was confirmed in immunocompromised NSG mice after the establishment of low or high tumor burden CCRF-CEM xenografts engineered to express green fluorescent protein (GFP) and click beetle red (CBR) luciferase. The impact of WU-CART-007 on normal hematopoiesis was assessed using CD34+ humanized NCG mice. Results Several successful full-scale manufacturing runs were completed with consistently high dual CD7/TRAC deletion, transduction efficiency, and cell viability. Drug product was primarily composed of a T cell memory phenotype. Off- target nuclease analysis by GUIDE-seq and targeted NGS confirmed no evidence of off-target editing events. The WU-CART-007 scFv exhibited high affinity and exquisite specificity for human CD7. In vitro co-incubation experiments confirmed strong cytotoxicity against CD7-expressing cells including CCRF-CEM T-ALL cells, primary T and NK cells, but not CD7- cells such as myeloid cells, B cells, hepatocytes, astrocytes, cardiomyocytes, epithelial cells, and endothelial cells. Importantly, no cytotoxicity was observed against hematopoietic progenitor cells in human bone marrow or cord blood following co-incubation with WU-CART-007. Similarly, WU-CART-007 treatment of a non-tumor bearing humanized mouse model resulted in transient reductions in CD7+ cells (T-cells and NK cells) but not CD7- cells (myeloid and B cells), and the impacted cells recovered after circulating WU-CART-007 cells were no longer detectable. Assessment of in vivo anti-tumor efficacy revealed that WU-CART-007 effectively inhibited tumor progression (>99% TGI) in both low and high burden CCRF-CEM tumor models and improved survival in a dose-dependent manner, while CAR- cells were inactive, confirming CD7-dependent activity. Conclusions These preclinical studies support the use of WU-CART-007 in clinical trials and highlight the potential of WU-CART-007 to be a well-tolerated and active therapy for patients with CD7+ T-cell malignancies. A first in human Phase 1/2 trial in patients with R/R T-ALL/LBL is currently open for enrollment (NCT# 04984356). Disclosures No relevant conflicts of interest to declare.

Devimistat in Combination with Bendamustine Potently Inhibits Tumor Growth and Promotes CD8+ T Cell Tumor Infiltration in a Syngeneic Mouse Model of CD30+ T-Cell Lymphoma

Introduction: Chimeric antigen receptor-based T cell therapy (CAR-T) has shown great promise in B cell malignancies. CD30-targeted therapies like brentuximab vedotin have activity in T cell malignancies expressing CD30. There is interest in developing CD30-targeted CAR-T therapies for the treatment of relapsed T cell malignancies. As the process of generating autologous T cells takes several weeks, there is a need for active bridging therapeutic regimens that can reduce tumor burden to facilitate subsequent treatment with CAR-T cell therapy. We propose the use of devimistat, a TCA cycle inhibitor, in combination with bendamustine as bridging therapy for the treatment of CD30+ T cell lymphoma (TCL) prior to the use of CAR-T cells. We hypothesize that TCA cycle inhibition will increase the sensitivity of TCL cells to bendamustine, negatively impact the function of immunosuppressive cell populations that depend on mitochondrial metabolism, and create a conducive environment for enhanced CAR-T cell efficacy. To test our hypothesis, we developed a model of CD30+ TCL, evaluated the efficacy of the combination, and characterized its effects by transcriptome profiling of immune cell populations in tumors. Methods: Mouse TCL cell line EL4-LUC2 was engineered to express human CD30 by retroviral transduction. Single and combination in vitro efficacy was evaluated by viability assays. Combination indices (CI) were calculated using Compusyn. CD30+ EL4-LUC2 cells (EL4.CD30) were inoculated subcutaneously into the flanks of C57Bl6 mice, tumor volumes were calculated, and CD30 expression in tumors was evaluated by immunohistochemical staining and flow cytometry. Transcriptome profiling was performed on a representative subset of tumors by single cell RNA sequencing (scRNA-seq). Results: EL4.CD30 cells displayed comparable CD30 expression to the human TCL cell line Karpas 299. EC 50 values for Devimistat and bendamustine in EL4.CD30 cells were 64.9 mM (95% CI: 62.4-67.4) and 101.2 mM (96.2-106.4), respectively; in Karpas 299, EC 50 values for Devimistat and bendamustine were substantially higher: 117.2 mM (114.7-119.6) and 188.2 mM (180.0 - 196.8), respectively. When dosed in combination, devimistat and bendamustine (D/B) showed synergy (CI = 0.5-0.8) at effect levels > 0.9 in 8 of the 16 dose combinations tested. D/B synergy at an effect level > 0.9 was less overt in Karpas 299 with only 2 of 16 tested combination levels displaying CI values of 0.5-0.9. Efficacy studies with EL4.CD30 tumor-bearing mice revealed potent D/B anti-tumor activity relative to vehicle and single-agent treatment groups. Six complete regressions and 4 partial regressions were observed in the D/B group (Fig. 1A). Moreover, tumor growth rates and median survival values were significantly different (p<0.0001) in D/B treated mice (Fig 1B-C). Transcriptome profiling of harvested tumors by scRNA-seq revealed substantial infiltration of cytotoxic T lymphocytes (CTL) exclusively in a combination-treated mouse that responded to therapy (Fig. 2A-C). Direct interrogation of Treg-associated transcripts revealed very low Treg numbers and no clear association to any treatment. Conclusions: Our data demonstrate that D/B has potent pharmacological activity in vitro and in vivo and promotes CTL influx into tumors. This suggests that D/B creates an immunopermissive environment fit for CAR-T cell activity. Subsequent studies will investigate the efficacy of CD30.CAR-T cells following D/B treatment. A Phase II pilot study evaluating the feasibility, safety, and tolerability of D/B in patients with relapsed/refractory T-cell Non-Hodgkin Lymphoma is currently ongoing (NCT04217317). Figure 1 Figure 1. Disclosures Pardee: Rafael Pharmaceuticals: Consultancy, Research Funding; Karyopharm Pharmaceuticals: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genetech: Membership on an entity's Board of Directors or advisory committees; BMS: Speakers Bureau; Pharmacyclics: Speakers Bureau.

Naturally Selected Anti-CD7 CAR-T Cells without Additional Genetic Manipulations As a Potentially Superior Therapy for T-Cell Malignancies

Introduction To prevent CAR-T fratricide, anti-CD7 CAR (7CAR) T cells used for treating T-cell malignancies are often modified by CD7 ablation via CRISPR/CAS9 gene editing or by co-expression of a CD7-specific protein expression blocker. Both methods require additional genetic manipulations of CAR-T. Here we transduce 7CAR into bulk T cells without CD7 disruption and thereafter allow CAR-T cells to emerge in vitro after fratricidal "natural selection". The biological characteristics of these naturally selected anti-CD7 CAR (NS7CAR) T cells and their potential advantages in treating patients with T-cell malignancies are described. Methods The percentage of CD3 +CD7 - T cells in peripheral blood from either healthy donors (HDs) or patients (PTs) were determined by flow cytometry. Peripheral bulk T cells were positively selected using CD3 magnetic beads, and peripheral CD7 - T cells were negatively selected using CD7 magnetic beads. To avoid contamination from malignant T cells, patients only with CD3 -CD7 + T cell blasts were included in this study. The 7CAR gene cassette comprising of the cDNA of a CD7-specific antibody sequence fused to the coding sequences for the CD8TM-41BB-CD3z signal domains, and the T2A-linked tEGFR was cloned into a lentiviral vector backbone under the control of an EF1α promoter. 7CAR lentiviral transduction of bulk T cells (NS7CAR) or CD7 - T cells (Neg7CAR) were performed two days after CD3/CD28 dynabeads activation. CD7-ablated 7CAR T cells (KO7CAR) were derived by electroporation of bulk T cells with CD7-targeting Cas9-gRNA RNP 24 hours before 7CAR transduction. CAR-T cells were routinely kept in culture for 12 days. The levels of CD7 mRNA, protein, and surface expression were determined respectively by qualitative/quantitative reverse transcription PCR, Western blotting, and flow cytometry. Iv vitro cytotoxic activity for CD7 + tumor cell lines was tested using a flow-cytometry-based cytotoxicity assay. NSG mice engrafted with CCRF-CEM-luciferase cells were used as an animal model to validate the activities of CD7 CAR. Results Three approaches for generating anti-CD7 CAR-T cells were compared: NS7CAR (fratricidal natural selection from bulk T cells after 7CAR transduction), Neg7CAR (7CAR transduction of purified CD7-negative T cells) and KO7CAR (Cas9 RNP CD7 gene ablation). While CD7 - T cells were detectable in HDs of all ages (9.48±0.96%, n=13), we observed a significant increase of this cell population in T-cell acute lymphoblastic leukemia PTs (15.98±0.57%, n=13) (Fig 1A). We next tested the feasibility of using bulk T cells to generate naturally occurring 7CAR T cells without CD7 gene ablation or protein blockage. Three days after 7CAR lentiviral transduction, purified bulk peripheral T cells had a rapid and dramatic phenotypic transition from CD7 + CAR - to CD7 -CAR +(Fig 1B). Although fratricide led to a much lower expansion and viability of 7CAR T cells compared to T- cells without 7CAR transduction, approximately 80% of the 7CAR T cells were viable, making further studies feasible. After 12 days of culture, 7CAR T cells from PTs displayed stronger expansion potential (Fig 1C) and contained a larger CD8 + subpopulation (Fig 1D) as compared to cells derived from HDs. In comparison to Neg7CAR and KO7CAR, the final NS7CAR product displayed a lower expansion capability, but contained a higher percentage of CAR + cells, a larger CD8 +subset and an increased central memory phenotype (Table 2). Interestingly, although the final cells from all three products had no surface CD7 expression, mRNA and total protein were only detected from NS7CAR, but not from Neg7CAR or KO7CAR. Additionally, NS7CAR showed superior cytotoxicity and cytokine release in an in vitro functional test (Fig 1E). In the animal model, the NS7CAR conferred robust protection against leukemia progression with marked reduction in leukemia cell burden in the first two weeks after CAR T- cells injection (Fig 1F&G). Conclusion Among the three approaches, the NS7CAR T cells was significantly enriched in CAR + cells and contained a higher percentage of CD8 + central memory T cells. Importantly, our data indicate that autologous PBMCs from patients were superior to PBMCs of healthy donors in yielding sufficient NS7CAR T cells for therapeutic needs. An investigator-initiated trial is currently ongoing to test the feasibility, efficacy, and safety of NS7CAR T cells for treating T-cell acute lymphoblastic leukemia. Figure 1 Figure 1. Disclosures Liu: SenlangBio: Current Employment. Ba: SenlangBio: Current Employment. Li: SenlangBio: Current holder of individual stocks in a privately-held company.

Romidepsin in Conditioning and Maintenance Mitigates Relapse Risk and Enhances NK-Cell Cytotoxicity in Patients Receiving Allogeneic Stem Cell Transplant for Aggressive T-Cell Malignancies: Results of a Phase I/II Clinical Trial

Background: Allo-SCT is the only curative option for patients with high risk and relapsed/refractory T-cell malignancies. Even among allo-SCT recipients, survival is less than 50% and relapse rates are 55-60%. We developed a clinical trial to decrease relapse after allo-SCT for these patients using romidepsin (rom), a histone deacetylase inhibitor approved for the treatment of relapsed T-cell lymphomas. Based on pre-clinical data demonstrating enhanced and synergistic cell killing with the addition of rom to busulfan (Bu) and fludarabine (Flu) in malignant T-cells, we created a novel transplant regimen (BuFluRom). We hypothesized this regimen, coupled with maintenance rom (m-rom), would enhance malignant T-cell killing, eradicate MRD at transplant, decrease relapse, and stimulate the GVL effect by stimulating NK-cells. Here we present results of this clinical trial, with correlative data evaluating NK-cytotoxicity. This is the first trial designed specifically to treat T-cell malignancies with allo-SCT. (NCT02512497) Methods: This is a phase I/II clinical trial. Eligible patients had: a diagnosis of T-cell leukemia (including T-acute lymphoblastic leukemia) or T-cell lymphoma (cutaneous or peripheral) in at least a partial remission requiring an allo-SCT, <70 years of age, with a matched sibling/unrelated donor. The primary objective was to determine the recommended phase 2 dose (RP2D) of rom from 3 dose levels (1, 2, 3 mg/m2) when combined with BuFlu (AUC 20000 or 16000, Figure). Patients received standard tacrolimus/methotrexate GVHD prophylaxis with ATG for MUDs. Once RP2D was determined, an expansion cohort of up to 30 patients (total) was included. M-rom was initiated between day +28 and +100 for 1 year (2 years max). The effect of rom on NK-cell cytotoxicity was assessed on samples taken pre-transplant, and 1, 3, 6, 12 months post allo-SCT. NK cytotoxicity was assessed by isolating mononuclear cells from patient samples and targeting them against K562 and T-cell lymphoma targets using the calcein-AM assay. Fine-Gray models were used to estimate PFS, OS, and cumulative incidence, and compare survival curves across groups. Results: 21 patients have been enrolled (Table). One DLT was observed (VOD), at dose level 2, and the RP2D of rom in conditioning was determined to be 2 mg/m2. With a median follow-up time of 10.1 months, the median OS has not been reached (3.3-NR months), with a 1 and 3-year OS probability of 62.8% & 55.8%. The median PFS is 28.2 months (3.8-28.1), with 1 and 3 year PFS of 57% & 30.4%. Cumulative incidence (CI) of NRM at day 100 and 1 year were 14.8% and 20%. CI of grade II-IV aGHVD and extensive cGVHD were 47.6% and 18.5%. The CI of relapse (CIR) was 22.8% at 1 year (95% CI 6.6-44.9%). There was no difference between PFS among patients with MRD versus those without MRD prior to transplant (p=0.96), and no difference in 1-year CIR (p=0.9). PFS and CIR at 1 year was substantially better in the lymphoma than leukemia patients (85.7% vs 44%, p=0.049), and (0% vs 32.1%, p=0.05). No patients with PTCL relapsed, and 3/5 patients with T-PLL are alive, disease free. 13/21 (62%) of patients received m-rom with a median number of 10 cycles (range 1-41). (Table) 7 patients experienced grade 3/4 adverse events (AE), though no patients discontinued m-rom due to toxicity. NK-cytotoxicity was higher at each time point in patients who received m-rom compared to those who did not, though there were insufficient patients to reach statistical significance. When NK-cytotoxicity was assessed between the two groups after starting maintenance, NK-cytotoxicity in the m-rom group was significantly higher than in those without m-rom (p=0.05) (Figure). Conclusions: BuFluRom with m-rom is effective at decreasing relapse in patients with T-cell malignancies, with 1-year CI relapse below expected relapse rates for this set of diseases. Toxicities were similar to standard BuFlu alone and the RP2D of rom in conditioning was established at 2 m g/m2. Intriguingly, BuFluRom mitigated the poor outcomes of patients with MRD prior to transplant. Further, early data suggests m-rom enhances NK-cell cytotoxicity post allo-SCT, potentially augmenting the GVL effect and accounting for decreased relapse rates. Long-term follow-up is needed to evaluate these results, but these results suggest the BuFluRom regimen with m-rom could become a new option for patients receiving allo-SCT for T-cell malignancies to mitigate relapse. Figure 1 Figure 1. Disclosures Hosing: Nkarta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Popat: Bayer: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding. Vasu: Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: travel support; Kiadis, Inc.: Research Funding; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. de Lima: Miltenyi Biotec: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. William: Dova Pharmaceuticals: Research Funding; Incyte: Research Funding; Kyowa Kirin: Consultancy; Merck: Research Funding; Guidepoint Global: Consultancy. Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company. Brammer: Kymera Therapeutics: Consultancy; Celgene: Research Funding; Seattle Genetics: Speakers Bureau.

Survival Analysis of Patients with T-Cell Lymphoma or T-Cell Large Granular Leukemia and Concomitant Plasma Cell Dyscrasias

Introduction: Sporadic cases of patients with a combined T-cell malignancy and plasma cell dyscrasia (PCD) have been reported in the literature. While the most commonly observed association is with T-cell large granular lymphocytic leukemia (T-LGLL) and PCD, there are case reports of other T-cell malignancies and PCD, such as peripheral T-cell lymphoma (PTCL) and angioimmunoblastic T-cell lymphoma (AITL) with multiple myeloma (MM). Nearly one-third of MM patients develop clonal T-cell populations that share a similar immunophenotype to T-LGLL, suggesting likely under diagnosis of concomitant T-cell malignancies in patients with PCD. However, with the limited data available regarding the overlap between PCD and T-cell malignancy, the significance, associated pathogenesis, and impact on survival outcomes is unknown. The purpose of this study is to describe the outcomes of patients with overlapping T-cell malignancies and PCD in order to determine the survival outcomes and ultimately make management/diagnostic recommendations. Methods: In this IRB approved study, we retrospectively evaluated patients with concomitant T-cell malignancy and PCD at Ohio State University from 2010-2020. Patients were identified using a database search for T-cell malignancies as well as PCD. All patients that were included met the 2016 World Health Organization diagnostic criteria for their respective T-cell malignancy and PCD. Progression-free survival (PFS) was measured as time from the start of treatment until first progression, death, or last follow-up according to the Kaplan-Meier method with median survival times and 95% confidence intervals reported. Results: A total of 21 patients, with a median follow-up time of 22 months (range 1-153), were included in this analysis. Baseline demographics are in table 1. The most common T-cell malignancy was T-LGLL (11/21; 52%) and the most common PCD were MGUS (8/21; 38%) and MM (8/21; 38%). Ten (48%) patients presented with a T-cell malignancy as their primary malignancy, 9 (43%) presented with a PCD as their primary malignancy, 1 (5%) patient was diagnosed with both at the same time, and for 1 (5%) patient it is unknown. Within the cohort, 62% (13/21) of patients received primary treatment for their T-cell malignancy and 38% (8/21) of patients received primary treatment for their PCD. Of the 7 patients that had their PCD clone 4 were treated concomitantly and 3 were treated for only their T-cell malignancy. Overall, 9/21 (42.9%) of patients had progression of their T-cell malignancy. A summary of outcomes is provided in Table 2. 54.6% of patients with T-LGLL and 60% of patients with AITL/PTCL experienced progressive T-cell disease and no patients with CTCL had progressive T-cell disease. The median overall survival (OS) across all patients was 4.1 years. Median OS was not reached for patients with T-LGLL, 1.7 years for AITL/PTCL, and 12.4 years for CTCL. PFS was 11 months for patients with T-LGLL, 1 year for AITL/PTCL, and 12.37 years for CTCL. Survival probability is shown in Figure 1. The rates of progression, OS, and PFS were consistent with previously published data for patients with these T-cell malignancies. Conclusions: Herein, we characterize a cohort of patients with concomitant T-cell malignancies and PCD with an emphasis on survival outcomes. Our data suggests that there is no PFS or OS difference for patients with T-cell malignancies and concomitant PCD when treated with standard T-cell directed therapy. There is the potential that treating a patient's T-cell malignancy may lead to resolution of their PCD clone, even without therapy directed at the PCD. While our data is limited by small sample size, this report represents the largest data set available in this rarely described patient population. Larger retrospective cohort studies are needed to further characterize this population and validate these findings. For patients with T-cell malignancies as the primary diagnosis with concomitant PCD, treatment with standard T-cell directed therapies is recommended with continued follow-up and monitoring of the concomitant PCD. Figure 1 Figure 1. Disclosures Bumma: Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees. Porcu: Viracta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Daiichi: Honoraria, Research Funding; Kiowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spectrum: Consultancy; DrenBio: Consultancy. Brammer: Seattle Genetics: Speakers Bureau; Celgene: Research Funding; Kymera Therapeutics: Consultancy.

Single Capture High Throughput Sequencing Assay for Combined V(D)J Clonality Analysis and Oncogene Mutations in the Diagnosis of T and B Lymphoid Malignancies

Introduction The diagnosis of B and T cell malignancies relies on the demonstration of B-cell (BCR) or T-cell (TCR) antigen receptor clonality. This can be studied through the analysis of V(D)J rearrangements of BCR and TCR genes by PCR (van Dongen Leukemia 2003) or, more recently, by high-throughput sequencing (HTS). Amplification of a clonal population with a "primers approach" could fail in case of hybridization problems due to too fragmented DNA, somatic mutations or polymorphic variations. Here we evaluated the performance of a HTS capture system for the analysis of B and T-cell clonality in clinical samples from mature T or B malignancies. We further combined this technology to concomitant sequencing of oncogenes of interest. Patients and Methods DNA was extracted from 58 tumoral samples from fresh/frozen (FF) cells or tissues or formalin-fixed paraffin-embedded tissue (FFPE) (n=19). These samples comprised various T-cell [i.e. 1T-cell prolymphocytic leukemia, 1 T large granular lymphocytic leukemia, 2 Sézary syndrome, 4 peripheral T-cell lymphoma not otherwise specified, 14 angioimmunoblastic T-cell lymphoma] or B-cell [i.e. 14 chronic lymphocytic leukemia, 1 mantle cell lymphoma, 5 diffuse large B-cell lymphoma, 1 grey-zone lymphoma, 13 Hodgkin lymphoma, 1 Poppema, 2 Waldenström and 1 multiple myeloma] malignancies. The Biomed-2 PCR technique was used as standard for assessing the performance of TRG, IGH and IGK clonality analysis. An extensive panel of capture probes was designed (SureSelect XT HS2 DNA system, Agilent Technologies) that covered the variable (V), + diversity (D) and junction (J) segments of the IGH, IGK, TRG, TRB loci and diagnostic/theranostic genes of interest i.e. B2M, BTK, CARD11, CD28, DNMT3A, IDH2, JAK3, PLCG1, PLCG2, ROHA, SOCS1, STAT3, STAT5B, STAT6, TET2, TNFAIP3, TP53. Paired-End sequencing was performed on a MiSeq system (Illumina) in 300, 500 and 600 cycles. Analysis of clonality profiles was performed using Vidjil software and SeqOne. Results HTS runs resulted in a median total read count of 1,6M (0.7-2.9) per sample. V(D)J rearrangements were identified with a median of 1503 reads (189-6824) per sample. Five samples were excluded because less than 300 rearranged reads were obtained. The number of rearranged reads and of clonotypes identified are influenced by the number of sequencing cycles (300<500 or 600) but not by the quality of DNA (FFPE vs FF). Analyses of tumoral samples with HTS versus PCR were compared. For the IGH locus (n=47), comparable PCR/HTS clonal (n=22) and polyclonal (PCL, n=20) profiles were identified. One discordant case showed a clonal PCR profile and a PCL HTS profile but the IGK was clonal. For the IGK locus (n=23), 10 clonal and 12 PCL cases were similar with both techniques. One case appeared discordant with a PCL PCR profile but a clonal HTS profile. For the TRG locus (n=31), PCR and HTS profiles were similar in 14 clonal, 5 oligoclonal and 9 PCL cases respectively. Three cases were discordant with oligoclonal PCR profiles but a clonal HTS profile. Overall in the 38 cases of B-cell malignancies, 27 and 11 cases had a concordant B-cell clonal or PCL profile with PCR and HTS. Among PCL cases, only one was discordant with a clonal HTS profile. This case and 3 other PCL cases were Hodgkin lymphomas which all disclosed another mutation (i.e. TP53, TNFAIP3, SOCS1). Of the 20 cases of T-cell malignancies, 14 displayed a clonal TRG profile with PCR and HTS. Among them, 13 showed oncogene mutations that confirmed the oncogenic nature of the clonal proliferation. Among 6 patients with a non-clonal PCR TRG profile, two cases of AITL and T-LGL had a discordant clonal TRG HTS profile and both also had specific mutations (SOCS1, RHOA and STAT3 respectively). Two other AITL samples showed a T-PCL profile with PCR and HTS but also had a mutation/CNV (RHOA, SOCS1). Conclusion A very good performance of B and T cell clonality assessment was obtained here with capture-HTS compared to Biomed-2 PCR. The combined identification of mutation/CNV allowed to confirm the malignant character in cases of clonal or PCL lymphoproliferations, while concomitantly specifying the type of lymphoproliferative disorder. The combined capture-HTS of B and T repertoires and oncogenes of diagnostic or theranostic interest thus appears as an efficient, accurate and useful approach for the diagnosis of mature B and T lymphoid malignancies in clinical practice. Disclosures No relevant conflicts of interest to declare.

CCR4 as a Therapeutic Target for Cancer Immunotherapy

CCR4 is a chemokine receptor mainly expressed by T cells. It is the receptor for two CC chemokine ligands, CCL17 and CCL22. Originally, the expression of CCR4 was described as highly selective for helper T type 2 (Th2) cells. Later, its expression was extended to other T cell subsets such as regulatory T (Treg) cells and Th17 cells. CCR4 has long been regarded as a potential therapeutic target for allergic diseases such as atopic dermatitis and bronchial asthma. Furthermore, the findings showing that CCR4 is strongly expressed by T cell malignancies such as adult T cell leukemia/lymphoma (ATLL) and cutaneous T cell lymphomas (CTCLs) have led to the development and clinical application of the fully humanized and glyco-engineered monoclonal anti-CCR4 Mogamulizumab in refractory/relapsed ATLL and CTCLs with remarkable successes. However, Mogamulizumab often induces severe adverse events in the skin possibly because of its efficient depletion of Treg cells. In particular, treatment with Mogamulizumab prior to allogenic hematopoietic stem cell transplantation (allo-HSCT), the only curative option of these T cell malignancies, often leads to severe glucocorticoid-refractory graft-versus-host diseases. The efficient depletion of Treg cells by Mogamulizumab has also led to its clinical trials in advanced solid tumors singly or in combination with immune checkpoint inhibitors. The main focus of this review is CCR4; its expression on normal and malignant T cells and its significance as a therapeutic target in cancer immunotherapy.