Zhang, Henan

Abstract While combination radiotherapy (RT) and immunotherapy is not novel there is still much to learn about this approach. Some initial pre-clinical and clinical evidence suggests triple therapy with RT and dual immune checkpoint blockade (anti-CTLA-4 + anti-PD-1/PD-L1) is a complimentary technique. We present two cases using the same novel regime of combination RT plus nivolumab and ipilimumab to treat metastatic melanoma. In both cases the burden of disease was significant within a lower extremity. Additionally, in both cases using this treatment plan, we were able establish sustained local disease control with additional systemic benefit which allowed both patients to avoid significant debility from surgical amputation. Given the response and safety we observed, this technique could be used as a potentially safe approach for future patients who are not eligible for traditional RT palliation or clinical trials.

Abstract Immunotherapy targeting immune checkpoints has become a common approach for many types of cancer but currently there are no reliable biomarkers to predict and monitor treatment response. Immunohistochemical staining of PD-L1, which was initially developed as a companion diagnostic, is problematic due to tumor heterogeneity, variations in specimen preparation and multiple scoring schemes. There is, therefore, a critical need for a better biomarker to guide the use of immunotherapy to improve clinical outcomes. We previously reported that higher levels of Bim and CX3CR1/granzyme B in CD8+ T cells were associated with increased response to PD-1 blockade therapy in patients with melanoma; high levels of Bim were also prognostic of poor survival in melanoma patients. In this study, we investigated the roles of Bim and CX3CR1/granzyme B in patients receiving PD-1 blockade therapy (i.e. pembrolizumab) for small bowel adenocarcinoma as part of planned correlative analysis of the ACCRU clinical trial, NCT02949219. Patients with unresectable or metastatic biopsy-proven small bowel adenocarcinoma (excluding ampulla of Vater and appendix) who had at least one prior line of systemic chemotherapy were eligible for the trial. Pembrolizumab (200 mg) was administered over 30 minutes intravenously every 3 weeks until unacceptable toxicity, disease progression, or patient refusal. All patients underwent peripheral blood collection at baseline prior to initiation of treatment and then after 3 cycles of treatment. Levels of Bim and CX3CR1/granzyme B in circulating T cells were quantified by flow cytometry using patients' peripheral blood mononuclear cells. Data was analyzed via Cox proportional hazards model, Wilcoxon Rank-Sum test, and Kaplan Meier curves using SAS 9.4. A total of 35 eligible patients were included in the analysis. Three patients had confirmed response (all partial responses) and 10 had stable disease as their best overall response. Seven patients had disease progression around the second blood draw. The median time from baseline to the second blood draw was 9 weeks. Lower levels of Bim in CD8+CD11ahi T cells at baseline on the percentage scale were associated with treatment response (median 54.5% vs. 79.5%; p=0.0087) and disease control (partial response plus stable disease; median 71.2% vs. 81.3%; p=0.0104) but higher levels led to worse progression-free survival (hazard ratio=1.05, 95% confidence interval 1.01-1.08; p=0.0051). In addition, positive changes in CX3CR1/granzyme B in CD8+CD11ahi T cells from baseline were associated with better overall survival (median 20.2 months vs. 3.7 months, p=0.0086). The levels of PD-1 and Ki-67 in CD8+CD11ahi T cells were not associated with treatment response or survival. In conclusion, Bim and CX3CR1/granzyme B are biomarkers that associate with response and survival benefits respectively of PD-1 blockade therapy. This preliminary data calls for a larger-scale, prospective study to validate their predictive and prognostic utilities. Citation Format: Mojun Zhu, Henan Zhang, Nathan R. Foster, Haidong Dong, Tanios S. Bekaii-Saab, Brandy L. Jaszewski, Patrick M. Boland, Michael J. Overman, Katrina Pedersen, Robert R. McWilliams, Academic and Community Cancer Research United (AACRU). Bim and CX3CR1/granzyme B in circulating CD8+ T cells are predictive biomarkers for PD-1 blockade therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4467.

Management of PD-1 blockade resistance in metastatic melanoma (MM) remains challenging. Immunotherapy or chemotherapy alone provides limited benefit in this setting. Chemo-immunotherapy (CIT) has demonstrated favorable efficacy and safety profiles in lung cancer. Our pre-clinical study showed that in MM patients who have failed PD-1 blockade, the addition of chemotherapy increases CX3CR1+ therapy-responsive CD8+ T-cells with enhanced anti-tumor activity, resulting in improved clinical response. Here, we examined the clinical outcomes of CIT in MM patients after PD-1 blockade failure and the treatment-related changes in CX3CR1+ therapy-responsive CD8+ T-cells. We reviewed MM patients seen between January 2012 and June 2018 who failed anti-PD-1-based therapy and received subsequent CIT, immune checkpoint inhibitors (ICI) or chemotherapy alone. Overall survival (OS), objective response rate (ORR), event-free survival (EFS), and toxicities were assessed. Among 60 patients, 33 received CIT upon disease progression on PD-1 blockade. At a median follow-up of 3.9 years, the CIT group had a median OS of 3.5 years [95% confidence interval (CI) 1.7–NR] vs. 1.8 years (95% CI 0.9–2; P = 0.002) for those who received subsequent ICI (n = 9) or chemotherapy alone (n = 18), with ORR of 59% vs. 15% (P = 0.0003), respectively. The median EFS was 7.6 months (95% CI 6–10) following CIT vs. 3.4 months (95% CI 2.8–4.1; P = 0.0005) following ICI or chemotherapy alone. Therapy-responsive CX3CR1+CD8+ T-cells showed dynamic increase with successful CIT. CIT showed favorable clinical outcomes and acceptable safety profile in PD-1 blockade-resistant patients. CX3CR1+CD8+ therapy-responsive T-cells can be potentially used for monitoring disease response to CIT.

725 Background: The impact of surgical resection of renal tumors on peripheral immune related cells is not well characterized, and has potential implications as biomarkers for systemic immune therapy are being developed. We sought to assess the effect of surgical resection on circulating cytotoxic T-cells (CTLs) for patients with renal tumors. Methods: We prospectively enrolled 40 patients undergoing partial, radical, or cytoreductive nephrectomy (PN, RN, CN) for unilateral primary renal tumors between 2016 and 2018. Blood draws were performed pre-op, 1 day post-op, and 3 months post-op. Flow cytometry was performed on peripheral blood mononuclear cells (PBMCs). The % of PBMCs expressing CD11a/CD8 (CTLs) were assessed. The % of CTLs expressing PD-1, Bim (a downstream PD-1 pathway pro-apoptotic mediator), CX3CR1/GZMB (an effector memory T-cell phenotype), and Ki67 (a proliferation marker) were assessed. Median changes in % of CTLs were evaluated with the Wilcoxon signed rank test. Comparisons between aggressive (pT3-4, N1, M1, or aggressive histology (high-grade, coagulative necrosis, sarcomatoid dedifferentiation, or specific RCC-variant histologies)) versus indolent tumors were assessed using the Wilcoxon rank sum test. Results: Twenty, 12, and 8 patients underwent RN, PN, and CN, respectively. Thirty, 7, and 3 patients had clear-cell RCC, non-clear cell RCC, and oncocytoma, respectively. Twenty-three and 17 patients had aggressive and indolent tumors, respectively. While there was no significant change at 1 day, by 3 months there were significantly increased CTLs among PBMCs (+1.3%; p=0.004). There was significant decrease in CX3CR1+GZMB+ CTLs (-2.9%; p<0.001) and significant increase in Bim+ CTLs (+3.0%; p=0.03) at 1 day but not 3 months. Interestingly, Ki67+ CTLs increased at day 1 (+0.6%; p<0.001) but decreased by 3 months (-0.6%; p=0.001). There were no significant changes in PD-1+ CTLs at either time point. There were no significant changes in any CTL characteristics at 1 day or 3 months between aggressive and indolent tumors. Conclusions: These findings characterize changing CTL profiles over time with surgical resection of renal tumors which may help guide biomarker development.

Abstract Peripheral immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) is well-studied and likely relates to the incidence of serious recurrent infections and second malignancies that plague CLL patients. However, the current paradigms of known immune abnormalities are not able to consistently explain these complications and it is not easy to correct CLL patient immune status. Here, we expand on our preliminary reports that demonstrate bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. We found reduced short-term functional capacity of hematopoietic progenitors in BM using colony forming unit assays (Figure 1A-C) and flow cytometry revealed significant reductions in frequencies of hematopoietic stem and progenitor cell (HSPC) populations (exemplified by Lin-CD34+ HSPCs, Figure 1D). We further report that protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 are overexpressed in distinct HSPC subsets from CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Interestingly, sustained myelopoiesis, evaluated by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays maintained for five weeks, revealed no difference between healthy controls and CLL patients. These new data indicate that when HSPCs are removed from the leukemic microenvironment for ample in vitro culture time, they recover the ability to sustain myelopoiesis. To further assess the impact of the CLL microenvironment on HSPC biology, isolated HSPCs (CD34+ BM cells) from healthy controls were exposed in vitro to known leukemic microenvironment constituents. Exposure to TNFα, a cytokine constitutively produced by CLL B cells, resulted in rapid increases in PU.1 and GATA-2 proteins (Figure 2A-D). Similarly, addition of TNFα to the LTC-IC assay resulted in a striking ablation of myelopoiesis, even at the highest input cell concentration. Further, overexpression of PU.1 and GATA-2 were observed in HSPCs following co-culture with CLL B cells, a result that was not recapitulated when cells were exposed to IL-10, another cytokine constitutively produced by CLL B cells. These findings indicate specific components of the leukemic microenvironment are involved in HSPC modulation. Together, these findings expand on our previous observations of BM hematopoietic dysfunction in untreated CLL patients and offer new molecular insights into the contribution of the leukemic microenvironment on immunodeficiency in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Background:Recurrent mutations in ATM, JAK1/3 and STAT5B genes have been identified in T-cell prolymphocytic leukemia (T-PLL), a mature T cell leukemia with poor survival. However, much remains to be understood regarding the dysregulated pathways involved in the pathogenesis of this aggressive disease. Here, we integrated the analyses of whole exome sequencing (WES) and transcriptomes of a cohort of 12 T-PLL samples to gain further insights into molecular mechanisms of this malignancy. Methods: Leukemic cells were isolated from peripheral blood or bone marrow samples of T-PLL patients. Tumor DNA and RNA, and germline DNA were extracted from purified leukemic T cells and bone marrow derived stromal cells, respectively. WES (n=12) and transcriptome analysis (n=10) were performed and data analyzed with published standard methods (Robinson et al. 2010 and DePristo et al. 2011) Results: 7 paired tumor and germline DNA and 5 tumor-only DNA samples were used for WES analysis. Mutations in ATM and TET2 were detected in 4 (33%) and 1 (8%) patients, respectively. JAK3 (p.M511I, p.Q501H and p.Q503H) or STAT5B (2 p.T628S and 1 p.N642H) mutations each occurred in 3 (25%) cases and were mutually exclusive, consistent with prior reports. Importantly, we found several novel mutations in genes with regulatory roles in the NOTCH signaling pathway. Specifically, 2 somatic mutations p.A3V and p.A3S in NOTCH2 and 2 other mutations (p.S244L and p.A1414V) in NOTCH4 were identified in 4 (33%) patients. One mutation was in the EGF- like and another one in the NODP domain critical for NOTCH-mediated cellular differentiation. Two (16.7%) novel missense mutations (p.G174D and p.V836I) were found in MAML2. Three missense mutations (2 p.S583N and 1 p.R190H) occurred in MAML1. Both MAML2 and MAML1 are essential cofactors of the NOTCH signaling pathway. In addition, a somatic mutation in the NOTCH signal regulator DTX2 (p.G268D) was detected. Altogether 7 (58.3%) patients harbored mutations in genes with a regulatory role in the NOTCH pathway. Among the epigenetic modifiers, we found deletions and point mutations in KDM6B and KDM6A in 4 (30%) patients including 2 codon deletions in KDM6B (p.X751Val) and 1 missense mutation each in KDM6B (p.R1016Q) and KDM6A (p.V510G). KDM6A and KDM6B are histone H3 lysine 27 demethylases known to be involved in regulation of DNA repair and play an important role in embryonic differentiation. Transcriptome analyses of 10 T-PLL cases versus normal CD3+ T cells from 5 healthy donors revealed 706 up-regulated and 655 down-regulated genes. Consistent with prior data, TCL1A and TCL1B were significantly overexpressed (23.7 and 13.1 fold, respectively). Using Ingenuity Pathway Analysis, we found PTEN Signaling, glutamate receptor, and axonal guidance signaling to be among the top 3 upregulated pathways. The top upstream regulators implicated for the up-regulated signals were FGF, Wnt/β-catenin and TNF. The top 3 down-regulated pathways were granulocyte adhesion and diapedesis, IL-17A in psoriasis and IL-10 signaling. The top regulators for the down-regulated pathways were LPS, TNF and IFNg. Wnt/β-catenin and Human embryonic stem cell pluripotency signaling were among the top 20 up-regulated pathways. Many genes involved in the regulation of Wnt signal pathway were included in the above pathways, including Wnt ligands (Wnt9A, Wnt4), transmembrane receptors (CDH1, LRP5, FZD6, FZD7, SMO), cytoplasmic factor (AXIN2), and nuclear factors (MYC, SOX12, SOX8, SOX18, CCND1). Summary: Our study identified recurrent mutations in the NOTCH signaling pathway as well as dysregulated embryonic developmental pathway, e.g. Wnt/NOTCH as newly implicated molecular mechanisms in T-PLL pathogenesis for further functional studies. Additionally, novel (KDM6A and KDM6B) and known mutations (ATM, TET2) in chromatin regulatory genes underscore the importance of epigenetic dysregulation in the tumorigenesis of T-PLL. Disclosures Parikh: Pharmacyclics: Honoraria, Research Funding. Mustjoki:Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Ding:Merck: Research Funding.

Abstract Background: CLL patients (pts) treated with novel targeted therapies continue to experience disease progression. Approximately 7-15% of relapsed CLL treated with ibrutinib or venetoclax developed Richter's syndrome (RS), an aggressive high grade lymphoma transformation. Standard chemotherapy has limited efficacy in RS. We hypothesized that checkpoint blockade would re-establish anti-tumor immune response in progressive CLL/RS and here we aim to update the clinical data and correlative analysis of MC1485: a phase 2 study of PD-1 blocking antibody pembrolizumab in relapsed/refractory CLL and RS. Methods: Relapsed/refractory CLL including RS enrolled in the CLL arm of MC1485 trial was reported here. The primary end point of this study is overall response rate (ORR). Pts with prior allogeneic stem cell transplant were excluded. Pembrolizumab 200 mg was administered intravenously every 3 weeks. NCI CTCAE v4.0 and IWCLL 2008 criteria were used for non-hematological adverse events (AE) and for CLL grade hematological AE. Tumor expression of PD-1 and PD-L1 were assessed with standard immunohistochemical (IHC) staining. Percentage expressions of individual antigens were analyzed with whole-slide scanning followed by image analysis with Image-Pro software (Media Cybernetics). Fluorescence in situ hybridization (FISH) was performed on available CLL/RS tissue sections to assess copy number of chromosome 9p region that contains PD-L1 and PD-L2. Results: 25 pts including 16 CLL and 9 RS (biopsy-proven large cell lymphoma) were enrolled in CLL arm. The median age was 69 years (46-81). Twelve (48%) pts had del(17p) or monosomy 17 or TP53 mutation. The median number of prior therapies was 4 (1-10). 96%, 72% or 48% of pts had received alkylator and anti-CD20 antibody, purine analog or anthracycline. Fifteen (60%) pts had prior ibrutinib and 12 (48%) progressed clinically while receiving ibrutinib, including 6 who progressed to RS and 6 who developed progressive CLL. The median number of pembrolizumab doses that pts received was 3 (1 to 21), administered over a median treatment duration of 11 weeks (1 to 56). Drug-related AE occurred in 21 pts (88%) with the most common ones being neutropenia in 9 (37%), cough in 7 (29%) and dyspnea in 6 (25%) pts. Drug-related grade 3 or above AE occurred in 9 (38%) pts with most common ones being thrombocytopenia (5, 21%), dyspnea (2, 8%), and fatigue (2, 8%). The most common immune-related AE was liver enzyme elevation (12%, 8% G3) and was reversible with therapy interruption or steroid therapy. Based on investigator assessment using the Revised Response Criteria for Lymphomas and IWCLL 2008 criteria, one RS pt had complete response (CR, 4%), 3 RS had partial responses (PR, 12%). Among 9 RS pts, 1 CR (11%), 3 PR (33%), 3 SD (33%) and 2 PD (22%) were observed. Thus the ORR in RS was 44%. No CLL pts achieved a CR/PR, 3 had SD and 9 had progressive disease (PD). The ORR of all pts was 16%. Of the 4 RS who had CR/PR, responses occurred early after 2 cycles of therapy and median duration of therapy for CR/PR pts was 8.3 months with data cut-off by June 30th 2016 (Figure 1A). The causes for therapy discontinuation in RS were PD (3, 33%), alternate therapy (2, 22%), and thrombocytopenia due to increased marrow CLL (1, 11%). Increased marrow CLL was observed in 2 RS pts with PR to RS phase of disease assessed by PET, thus the protocol was amended to allow addition of idelalisib/ibrutinib to control the underlying marrow CLL. For RS pts who had a CR/PR or SD, variable nodal responses were observed (Figure 1B). In particular, 6 RS who had prior ibrutinib experienced CR or PR or SD with nodal responses. After a median follow-up of 10.2 (1.9 to 16.1) months, the median overall survival (OS) for all pts was 10.7 months (95% confidence interval [CI], 5.4 to not reached). The 6 month OS rate for RS and CLL pts were 73% and 59%, respectively. Biomarker assessment using IHC analysis on 10 pts with available tumor/nodal tissues (6 RS and 4 CLL) showed an increased expression of PD-L1 (p = 0.02) and a trend for an increased expression of PD-1 (p = 0.1) in the group of pts with CR/ PR versus pts in the group with no clinical response. One out of 10 tested showed polysomic for chromosome 9p. Conclusion: Pembrolizumab had an acceptable safety profile in CLL and RS patients. We confirmed that pembrolizumab has substantial therapeutic activity in RS. Single-agent pembrolizumab does not appear to have clear activity in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding. Shanafelt:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

Abstract Introduction: CLL is the most prevalent leukemia in the United States (≥120,000 cases) with approximately 5000 deaths per year. The dominant cause of mortality in CLL patients is serious infection. Indeed, about 80% of CLL patients will sustain infectious complications at some point during their disease course and infection risk increases with length of the disease. Steady-state hematopoiesis in BM is required to sustain a peripheral repertoire of blood cells and maintain immune competence. There have been few studies evaluating the hematopoietic capacity of CLL BM and how it may relate to immune competence. Thus, the goal of this study was to determine the status of BM hematopoiesis in untreated and treated CLL patients. Methods and findings: The hematopoietic stem (HSC) and progenitor cell pool (HSPC) in human BM is defined by expression of CD34 and lack of surface markers (Lin-) expressed on their mature progeny. Frequencies of Lin- CD34+ progenitors in BM between age matched healthy subjects (HS) (n=22) and untreated CLL patients (n=10) were compared. We note that the Lin+ cocktail used included an antibody to CD19, eliminating dilution of CD34+ HSPC frequency due to variation in leukemic B cell infiltration. CD34+ HSPCs were significantly reduced in untreated CLL patients (6.33±3.01% vs. 2.04±2.33% in HS vs. CLL, p=0.0004). Next we evaluated total CFU responding cells within the residual CD34+ cells to determine if they were functionally compromised. Numbers of total CFU-responsive CD34+ progenitors (CFU-GM, CFU-E, and CFU-GEMM) were reduced when assayed from purified CLL patient CD34+ cells compared to HS. Thus, while HSPC are present in CLL patient BM, they are functionally compromised even after removal from the tumor microenvironment. To determine if reductions in CFUs were accompanied by decreased myeloid and erythroid lineage differentiated progeny, we evaluated frequencies and absolute numbers of CD14+ monocytes and CD71+ erythroid progenitors by flow cytometry and found both were severely reduced in CLL patient BM. To determine if the reductions in myeloid/erythroid lineage cells was due to deficiencies in their precursor HSPCs, we evaluated frequencies and absolute numbers of HSC, multipotent progenitors (MPP), and common myeloid progenitors (CMP). Proportions, but not absolute numbers, of HSC, MPP, and CMP were significantly reduced in CLL patient BM. Frequencies of CD14+ monocytes and CD71+ erythroid precursors were also significantly reduced in patients with low BM tumor burden (designated as 20-40% BM involvement), suggesting that the hematopoietic dysfunction is not solely due to tumor burden. Comparative analysis of frequencies of CD14+ monocytes in BM to frequencies of monocytes in peripheral blood revealed a strong association, suggesting that for some immune cell types the blood immune repertoire may be reflective of BM dysfunction. In addition to BM evaluation of untreated CLL patients, we also studied a cohort of post-therapy patients (n=23). Not surprisingly, we found considerable heterogeneity in the extent of BM hematopoietic reconstitution. Nevertheless, we observed robust BM hematopoiesis post therapy in a subset of treated patients (n=9 of 23). Summary: Our findings reveal BM hematopoietic dysfunction (HD) in CLL not solely due to the degree of tumor burden. Importantly, the BM HD is reversible by effective therapies. The HD is manifested by reduced numbers and function of HSPCs and production of their mature progeny. Of significance is the severe reduction in monocytic and granulocytic cells found in CLL BM. This is an important finding as these two innate cell types provide critical first responder protection against invading pathogens. Our study also uncovered that levels of blood monocytes can mirror their diminished production in BM. Future studies will focus on determining the mechanism(s) that reduce the ability of CLL BM to sustain immune cell production in addition to determining if restoration of hematopoiesis is a necessary outcome for more effective therapies. Disclosures Ding: Merck: Research Funding. Kay:Pharmacyclics: Research Funding; Tolero Pharmaceuticals: Research Funding; Acerta: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-Sys: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract T-cell prolymphocytic leukemia (T-PLL) is a rare disease with an aggressive clinical course and a median overall survival of less than three years. Although almost 75% of T-PLL patients are reported to harbor translocations causing the activation of the proto-oncogene TCL1A, T-PLL is genetically heterogenous: most T-PLL patients also have mutations or deletions in the ATM gene and the genes involved in the JAK-STAT pathway are mutated in 76% of cases. There is an urgent need for more rational based therapies, but clinical trials are difficult to perform due to the rareness of the disease. Here, we systematically explored the diversity of drug responses in T-PLL patient samples ex vivo using a drug sensitivity and resistance testing (DSRT) system including 306 oncology drugs (approved or investigational). We also aimed to determine any associations between the genetic aberrations and drug sensitivities in T-PLL patients. Primary mononuclear cells were gathered from 30 T-PLL patients for drug testing. Cells were plated in 384-well plates and subjected to the 306 substances using a 10,000-fold concentration range. After 72 hours, cell viabilities were measured, the results were depicted as dose-response curves for each compound, and differential drug sensitivity scores (sDSS), representing leukemia-specific responses, were computed by comparing patient samples to healthy donors. Drug response profiles across patients were clustered and visualized by hierarchical clustering. The subgroups resulting from the clustering were statistically compared using a two-sample t-test to understand which drug classes were driving the grouping. To delineate heterogeneous pathway dependencies, drug sensitivities were correlated with somatic genetic variants and recurrent chromosomal aberrations. Genetic characterization was performed by targeted amplicon sequencing of tumor cells to profile known recurrent genetic variants (STAT5b, IL2RG, JAK1, JAK3, ATM). Information on chromosomal aberrations (TCL1A translocations, ATM deletions) was derived from parallel clinicopathologic databases. Amplicon sequencing revealed that 70% of T-PLL patients (21/30) harbored a mutation in genes involved in the JAK-STAT pathway (JAK1, JAK3, STAT5b or IL2RG). The most prevalent mutation led to an M511I amino acid exchange in the JAK3 protein (26% of patients). Interestingly, the STAT5b mutations (5/30) did not coexist with any of the JAK mutations in our cohort. Based on DSRT analysis, all T-PLL samples were sensitive to the CDK-inhibitor SNS-032 and the anti-cancer antibiotic actinomycin D. Next, we clustered patients using sDSS values for all 306 different substances, and this showed that patient samples could be divided into 3 main groups, based on their drug responses (Figure). According to two-sample t-test, the grouping was driven by the selective sensitivities of Groups II and III to HDAC inhibitors (belinostat, panobinostat, quisinostat, CUDC-101 and vorinostat) and the selective sensitivity of Group III to PI3K/AKT/mTOR inhibitors (AZD-8055, MK-2206, apitolisib, dactolisib, PF-04691502, ZSTK474, and omipalisib), HSP90 inhibitors (BIIB021, luminespib, alvespimycin, and tanespimycin) as well as JAK inhibitors (ruxolitinib, momelotinib, tofacitinib, gandotinib). Group I samples were on the other hand relatively resistant to these classes of drugs. Surprisingly, despite the prevalence of the signature event of activation of TCL1 (the established AKT coactivator) in nearly all cases, only a subset of cases (group III) responded to PI3K/AKT/mTOR inhibitors. Strikingly, the grouping of selective responses to HDAC, JAK, PI3K/mTOR/Akt and HSP90 inhibitors did not link to the presence of JAK/STAT mutations, TCL1A translocations, or ATM deletion status. Ex vivo drug screening of primary T-PLL samples revealed heterogenous selective drug responses in specific drug classes (such as HDAC-, JAK-, HSP90- and PI3K/Akt/mTOR-inhibitors). Surprisingly, the drug response patterns did not correlate with known recurrent genetic aberrations suggesting that sequencing for recurrent genetic biomarkers cannot easily be turned into effective therapeutic strategies in T-PLL, and that further elucidation of the biological pathways driving T-PLL is needed. Figure 1. Mutation status and clustering of HDAC-, PI3K/mTOR/Akt-, HSP90-, and JAK-inhibitor responses in PLL patients based on sDSS values Figure 1. Mutation status and clustering of HDAC-, PI3K/mTOR/Akt-, HSP90-, and JAK-inhibitor responses in PLL patients based on sDSS values Disclosures Koschmieder: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences, Research Funding; Novartis Foundation: Research Funding; Baxalta/CTI: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen Cilag: Other: Travel reimbursement for scientific conferences ; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences. Wennerberg:Pfizer: Honoraria, Research Funding. Ding:Merek: Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Abstract Introduction T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-Cell malignancy typically associated with aggressive clinical course. Leukemic T-cells demonstrate post-thymic T-cell phenotypes (Tdt-, CD1a-, CD5+, CD2+ and CD7+) and commonly are CD4+/CD8- T-cells, but co-expression of CD4+/CD8+ or CD8+/CD4- have also been observed. Rearrangement of chromosome 14 involving TCL1 locus is the cornerstone for T-PLL diagnosis which leads to overexpression of oncogene TCL1. As a binding partner for TCL1, AKT expression correlates with worse prognosis. Despite advances in identification of novel mutations, therapeutic option is limited with most patients having very short survival. Herein, we performed a detailed analysis of specific T-cell subsets affected in T-PLL and a comprehensive genomic analysis by whole exome sequencing (WES) and RNA sequencing. We also explored the preclinical efficacy of targeting AKT activation by AKT inhibitor MK2206 on leukemic T-cells isolated from T-PLL patients. Methods T-PLL leukemic cells were isolated from blood or marrow samples of T-PLL patients (n=9) and were tested for involved T-cell subsets by multicolor flow cytometric analysis. Leukemic T-cells were treated with escalating doses of MK2206 (0 to 10 µM) for about 80 hours and were assessed for their apoptosis levels using Annexin V flow cytometric analysis. Whole exome sequencing (WES) and RNA sequencing were conducted using genomic DNA and RNA isolated from purified leukemic T cells from T-PLL patients. Results Phenotypic analysis of T-cell subsets in T-PLL patients showed that leukemic T-cells in 4 untreated T-PLL patients are consistent with naïve T-cell phenotype (CD45RA+, CD45RO-, CCR7+). Leukemic T-cells in 3 of the untreated patients and in 2 of the treated patients have central memory T (CD45RA-, CD45RO+, CCR7+) and effector memory T phenotypes (CD45RA-, CD45RO+,CCR7-) respectively. MK2206 treatment was able to induce dose-dependent apoptosis on the isolated PBMC (containing > 90% leukemic T-cells) of T-PLL patients (n=4) (IC50: 5 µM). FISH analysis found a rearrangement of TCL1 locus on chromosome 14 in all T-PLL cases (n=9). Three cases have also been detected to have del (11q) involving ATM locus and one patient has both del(11q) and del(17p). Subsequent WES (n=7) and RNA sequencing (n=6) analysis revealed recently reported mutations in ATM (frame shift mutation and early stop mutation W579*) in 2 cases, JAK3 (M511I) in 2 cases and STAT5B (T628S and N642H) in 2 cases. Importantly, novel somatic mutations in gene IKZF1 (N159S), NTRK1 (R33W), AP2A2 (P514L), HDAC8 (I115R), RARB (G90W) and TNIP2 (K104Q) were detected by WES. Mutations in EML4 (L548W and F304S) and VAV3 (C282Y and splice site mutation) were also identified in 2 different T-PLL cases. RNA sequencing revealed several fusion transcripts resulting in early stop of several different genes including PTPRT, L3MBTL1, UCKL1 in one T-PLL case. Conclusion T-PLL leukemic T-cells are consistent with either naïve or central memory T-cell subsets in untreated patients. The AKT inhibitor MK-2206 was capable of inducing apoptosis and could be a potential therapeutic agent for T-PLL patients. In addition, we detected known mutations in ATM and JAK-STAT pathways. Novel mutations in genes involving DNA binding and chromatin remodeling (IKZF1, HDAC8) or kinase signal pathway (NTRK1, TNIP2, VAV3, EML4) were uncovered. These results suggest that further therapeutic approaches could be developed to target DNA binding factors or JAK-STAT or AKT-TCL1 signal pathway with an ultimate goal to improve survival of T-PLL patients. Disclosures Off Label Use: Off label use of MK2206: MK-2206 is an AKT inhibitor obtained from Selleck Chemicals and is used to treat leukemic cells in vitro to test the leukemic sensitivity to AKT inhibition. .