Dong, Haidong

AbstractObjectivesPrevious studies noted discordance of programmed death‐1 (PD‐1) and one of its ligands (PD‐L1) across patient‐matched primary and metastatic clear cell renal cell carcinoma (ccRCC). There are inconsistencies if the primary or metastatic tumor has higher expression, and whether metastatic tumor expression is associated with patient outcome. Thus, we examined PD‐1 and PD‐L1 in patient‐matched tumors using a large number of ccRCC patients with long follow‐up.Materials and MethodsWe analyzed PD‐1 and PD‐L1 using immunohistochemistry in patient‐matched primary and metastatic tumors from 110 ccRCC patients. Concordance was assessed among longitudinal metastatic tumors, as well as across patient‐matched primary and metastatic tumors. Cox proportional hazards regression was used to evaluate the associations of metastatic tumor expression with cancer‐specific survival.ResultsWe observed inter‐metastatic tumor heterogeneity of PD‐1 in 25 (69%) of the 36 patients and of PD‐L1 in seven (19%) patients. Concordance between patient‐matched primary and metastatic tumors was 73% (Kappa = 0.16, 95% CI: −0.003‐0.32). Similarly, concordance of PD‐L1 between metastatic and patient‐matched primary tumors was 78% (Kappa = 0.27, 95% CI: 0.09‐0.46). Both markers demonstrated higher expression in primary vs metastatic tumors. Metastatic tumor expression of PD‐1 was significantly associated with metastatic location (P < .0001) and ccRCC‐specific survival (HR = 2.15, 95% CI: 1.06‐4.36, P = .035).ConclusionsThe expression of PD‐1 and PD‐L1 is discordant across patient‐matched ccRCC tumors, with higher expression in primary tumors. Higher PD‐1 expression was associated with metastatic location and lower cancer‐specific survival. If validated, these results highlight the importance of evaluating these biomarkers in metastatic tissue specifically.

65 Background: The addition of chemotherapy to immune checkpoint inhibitors has been explored clinically in solid tumors in order to overcome one of the challenges of immunotherapy: clinical outcomes are variable, with only a subset of patients achieving durable responses. Further understanding of the immunoregulatory mechanisms of chemotherapy in the setting of immunotherapy is crucial for the development of optimal combinatorial chemo-immunotherapeutic strategies to improve clinical outcomes in patients with advanced cancer. Methods: Metastatic melanoma (MM) patients who failed anti-PD-1 single therapy were subsequently treated with paclitaxel and carboplatin in addition to PD-1 blockade. Using peripheral blood (PB) from MM patients and healthy donor, the phenotypic and functional changes in T cells induced by chemotherapy, in the setting of PD-1 blockade, were examined. Results: Paclitaxel and carboplatin, in combination with PD-1 blockade, were able to induce significant clinical responses in a subset of MM patients who were resistant to anti-PD-1 therapy alone. In patients who responded to this combination, a subset of tumor-reactive effector T cells (CD8+CD11a+PD1+GranzymeB+) survived the treatment, with increased frequency after the addition of chemotherapy. This T cell population preserved its effector phenotype, although ongoing PD-1 blockade was needed to improve its anti-tumor activity. In vitro experiments, using CD8+CD11a+PD1+GranzymeB+isolated from healthy donor PB, also demonstrated its ability to survive chemotherapy treatment. In addition, after cultured with chemotherapy, this T cell subset demonstrated higher cytotoxic T cell functions as well as increased chemotherapy efflux. Conclusions: Chemotherapy regulates a subset of tumor-reactive effector T cells (CD8+CD11a+PD1+GranzymeB+) and increases clinical efficacy of PD-1 blockade. This novel effector T cell population underlies the key cellular and molecular immunoregulatory mechanisms of chemotherapy. It serves as a meaningful marker to measure these collaborative effects and to develop the optimal chemo-immunotherapy strategy to improve clinical responses to current immune checkpoint blocking agents.

4 Background: Membrane expression of PD-L1 on tumor cells is associated with poor survival in patients with advanced malignancies including metastatic melanoma (MM). In addition, tumor-associated PD-L1 has been proposed as a predictor of response to anti-PD-1 therapy, although responses are observed in some cases of PD-L1 negative MM. We hypothesize that tumor-derived PD-L1 has additional systemic effects through the release of biologically active soluble forms of PD-L1 (sPD-L1) into the circulation, which further impedes the anti-tumor immune response and contributes to poor clinical outcomes in MM. Methods: We developed a sPD-L1 ELISA and biochemically confirmed the identity of the detected protein. We measured the levels of sPD-L1 in 276 patients with MM enrolled on 3 clinical trials who had stored pre-treatment plasma samples and 37 healthy volunteers (HV) undergoing blood donation at Mayo Clinic. We also measured sPD-L1 in baseline samples from 38 MM patients treated with anti-PD1 (pembrolizumab) 2 mg/kg every 3 weeks. ROC analysis was used to compute sPD-L1 concentration cut-off value and Wald test was used to asses the difference of overall survival (OS) in patients with low versus high sPD-L1 concentration. Results: We found that sPD-L1 levels were significantly elevated in MM patients compared with HV (p=0.0011). Mean sPD-L1 level in MM was 1.73 ng/mL (range: 0.13-18.29) compared with 0.77 ng/mL (range: 0.11–6.02) for HV. Patients with higher levels (>0.293 ng/mL) had a median OS of 11.3 months compared to 14.8 months for patients with sPD-L1 level ≤ 0.293 ng/mL (p=0.040). Similarly to tumor-related PD-L1 findings, patients who had clinical benefit (OR/PR/SD) after 4 cycles of anti-PD1 had higher sPD-L1 levels at baseline (2.1 vs. 1.1 ng/mL). Conclusions: To our knowledge, our study is the first to report a correlation of sPD-L1 with MM patient outcome, although a larger study is needed to establish the utility of sPD-L1 as an independent prognostic and possibly predictive biomarker.

Introduction Tumor surface matrix metalloproteases ADAM10 and ADAM17 are associated with poor outcomes in multiple malignancies. We previously showed that these proteases shed PD-L1 from lymphoma cell line Karpas-299 to produce soluble PD-L1 (sPD-L1) that induces CD8+ T cell apoptosis (1). While higher levels of soluble PD-L1 are associated with worse prognosis in patients with non-Hodgkin lymphoma treated with standard chemoimmunotherapy (cutoff of 1.52 ng/ml, 3-year OS of 76% versus 89%) (2), it is unknown whether the loss of PD-L1 on the surface of B cell lymphoma cells affects clinical outcomes. We hypothesized that tumors expressing low PD-L1 levels despite high PD-L1 mRNA levels would (1) produce higher ADAM10 or ADAM17 transcript levels and (2) predict poorer prognosis in diffuse large B cell lymphomas. Methods We queried the cancer genome atlas (TCGA) public database for cases of diffuse large B cell lymphoma (DLBCL) for which PD-L1 (CD274) protein levels are reported by reverse phase protein array (RPPA). We then queried TCGA for RNA-Seq data for CD274, ADAM10, and ADAM17 expressed as transcripts per million (FPKM). We re-normalized RPPA data and calculated a PD-L1 protein-to-mRNA ratio for each tumor sample. We then compared groups of high and low PD-L1 protein-to-mRNA ratios for ADAM10 and ADAM17 mRNA expression. We further compared groups of high and low PD-L1 protein-to-mRNA ratios for survival (adjusted for age at diagnosis). All statistical analyses were performed using R Statistical Software (R Foundation). Unpaired student's t-test assessed statistical differences in experimental groups except where otherwise indicated. Figures comprise box plots showing quartile values and individual data points. P<0.05 was considered statistically significant. In figures, p values are denoted <0.05 with *, <0.01 with **, and <0.001 with ***. Results Data were available for a total of 33 diffuse large B cell (DLBC) lymphomas. 18 of those samples were categorized as having low PD-L1 protein-to-mRNA ratios (cutoff 6.38e-6) with the remaining categorized as high ratio. DLBCL tumors demonstrating low PD-L1 protein-to-mRNA ratios expressed significantly higher ADAM10 (p=0.0237) and ADAM17 (p<0.0001) transcripts than tumors expressing low PD-L1 protein-to-mRNA ratios (Figure 1). Furthermore, patients with tumors demonstrating low PD-L1 protein-to-mRNA ratios experienced significantly poorer survival according to likelihood ratio (6.6, p=0.01) and log rank (4.67, p=0.03) tests (Figure 2). Conclusions Our findings may explain a crucial phenomenon seen in diffuse large B cell lymphoma, namely that many purportedly PD-L1-positive tumors appear to express PD-L1 but do not respond to PD-(L)1 inhibitor therapy (3,4). This may be caused by the activity of ADAM10 and ADAM17 to cleave PD-L1, which we previously showed in lymphoma cell line Karpas-299 (1). This posits pre-treatment ADAM10/ADAM17 inhibition to "boost" PD-(L)1 inhibitor therapy in lymphoma. Our results contrast with previous findings that PD-L1-positive DLBCL progression free survival is inferior to PD-L1-negative DLBCL (5). This may be due to different methods of protein detection (immunohistochemistry versus RPPA) and the lack of localization of PD-L1 expression in tumor cells versus the microenvironment. Notably, this study is only correlative with a limited case number. Larger prospective studies will be needed to further elucidate this relationship. Acknowledgments The results shown here are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga. References 1. Orme et al. AACR Adv Malig Lymphoma 2018. Pres 423 2. Rossille et al. Leukemia 2014. 28:2367-75. 3. Moskowitz et al. Blood 2014 124:290. 4. Armand et al. J Clin Oncol 2013. 31:4199-206. 5. Kiyasu et al. 2015.126:2193-201. Figure Disclosures No relevant conflicts of interest to declare.

Abstract Glioblastoma (GBM) is the most common and lethal primary brain tumor, and novel therapeutic strategies that make a substantial impact on outcomes are sorely needed. Immunotherapies have shown great promise in the treatment of a number of cancers in recent year, and a concerted effort is being made to apply these treatment paradigms to GBM. However, many GBM patients exhibit profound immunosuppression, likely limiting the efficacy of such approaches. The mechanisms of this immunosuppression are poorly understood, but tumor-derived extracellular vesicles (EVs), may play a role. We demonstrate that GBM-derived EVs induce the development of myeloid-derived suppressor cells (MDSCs) and non-classical monocytes (NCMs). We further demonstrate that these EV-induced monocytic cells are immunosuppressive, resulting in impaired T cell proliferation upon co-culture. We found that the immunosuppressive effects of tumor-derived EVs appear to be driven by the induction of these immunosuppressive monocytes, as EV treatment of T cells did not significantly impact T cell proliferation. Further, we sought to characterize the important of programmed death ligand 1 (PD-L1) in the induction of these immunosuppressive monocyte populations. We found PD-L1 to be expressed in the EVs from GBM cell lines, and that modulation in PD-L1 expression via either constitutive overexpression or shRNA-mediated knockdown resulted in concordant changes in expression in tumor-derived EVs. We demonstrate that PD-L1 is important for the induction of NCM but not for MDSCs. Taken together, these findings point to a significant role for tumor-derived EVs in the induction of immunosuppressive monocytes in GBM, and that these cells may be a driving force of systemic immunosuppression. PD-L1 is one factor expressed in EVs that has immunomodulatory properties, but additional EV cargo likely plays a major role in the induction of immunosuppressive cells.

Abstract This report describes a new human B7-like gene designatedB7-H2. Cell surface expression of B7-H2 protein is detected in monocyte-derived immature dendritic cells. Soluble B7-H2 and immunoglobulin (Ig) fusion protein, B7-H2Ig, binds activated but not resting T cells and the binding is abrogated by inducible costimulator Ig (ICOSIg), but not CTLA4Ig. In addition, ICOSIg stains Chinese hamster ovary cells transfected with B7-H2 gene. By suboptimal cross-linking of CD3, costimulation of T-cell proliferation by B7-H2Ig is dose-dependent and correlates with secretion of interleukin (IL)-2, whereas optimal CD3 ligation preferentially stimulates IL-10 production. The results indicate that B7-H2 is a putative ligand for the ICOS T-cell molecule.

This report describes a new human B7-like gene designatedB7-H2. Cell surface expression of B7-H2 protein is detected in monocyte-derived immature dendritic cells. Soluble B7-H2 and immunoglobulin (Ig) fusion protein, B7-H2Ig, binds activated but not resting T cells and the binding is abrogated by inducible costimulator Ig (ICOSIg), but not CTLA4Ig. In addition, ICOSIg stains Chinese hamster ovary cells transfected with B7-H2 gene. By suboptimal cross-linking of CD3, costimulation of T-cell proliferation by B7-H2Ig is dose-dependent and correlates with secretion of interleukin (IL)-2, whereas optimal CD3 ligation preferentially stimulates IL-10 production. The results indicate that B7-H2 is a putative ligand for the ICOS T-cell molecule.

Abstract Background: High risk CLL patients treated with novel targeted therapies continue to experience disease progression (PD), and more than 50% of these PD patients evolve into Richter's syndrome (RS), (Maddocks, Jama Oncol 2015). Standard chemotherapy for large cell lymphoma has limited efficacy (median overall survival <12 months) in RS. Novel immunotherapy using a PD-1 antibody to block immune checkpoint signals to activate the cytotoxic T -cell has significant efficacy in relapsed Hodgkin lymphoma (Ansell, NEJM 2015). We conducted a phase 2 trial to test the safety and clinical efficacy of the anti-PD-1 antibody pembrolizumab in patients with relapsed/refractory CLL and relapsed low grade B non-Hodgkin lymphoma (B-NHL) (MC1485 trial). Methods: MC1485includes two arms: patients with relapsed/refractory CLL (including RS) and patients with relapsed B-NHL. The main end points of this study are safety and overall response rate. The first 6 patients enrolled in either arm were included in a designed safety interim analysis. Here we report the initial interim analysis from the CLL arm. Relapsed/refractory CLL including RS who had ECOG performance 0-2 and normal organ function were included. Patients with active autoimmune disease or interstitial lung disease were excluded. Pembrolizumab 200 mg was administered intravenously every 3 weeks until progression, excessive toxicity, or completion of 2 years of therapy. Soluble plasma PD-L1 levels were measured in these 6 patients using an established ELISA assay (Frigola, Clin Cancer Res, 2011). CD8+ T cell subsets were assessed for their functional status using an established analysis (Liu, Oncoimmunology. 2013) at baseline and after 2 cycles of therapies. Results: As of July 27th, 2015, 16 relapsed/refractory CLL patients including 5 RS patients were enrolled. The median age was 71 years (58-81). The median number of prior therapies was 3 (1-6). All CLL patients had received chemoimmunotherapy (FCR/PCR). 4 out of 5 RS patients had received anthracycline-containing chemotherapy. 5 out of 8 patients who had received prior ibrutinib therapy progressed on ibrutinib. 8 patients had 17p-/TP53 mutation. Herein, we report preliminary results from the interim analysis of CLL arm including 4 RS and 2 CLL as well as the 5th RS who had response evaluation by PET. The median cycles of therapy administered was 4 (3-7). One patient experienced grade 2 cytokine release syndrome with transient febrile neutropenia, grade 3 headache and skin rashes. These symptoms resolved with steroid therapy. One patient experienced grade 3 myalgia and was also treated with steroids. The other patients in this safety cohort tolerated pembrolizumab well. The most common drug-related adverse events were grade 1 dyspnea (33%, 2/6) and grade 1 anemia (33%, 2/6). Based on investigator assessment using the Cheson 2007 International Working Group Criteria, 4 out of 5 RS patients had responded to therapy. One patient had a complete remission after 2 cycles and remains in remission. One patient had almost complete PET response after two cycles and is classified as PR as her bone marrow have not been re-evaluated yet. Two patients had notable responses with nodal and skin lymphoma improvements before they showed potential evidence of progressive diseases. The remaining 1 RS and 2 CLL had stable disease and continued on therapy at the last follow-up. Soluble PD-L1 levels (sPD-L1, n=6) were measured at baseline and after 2 cycles of therapies. 3 RS patients who had responded to therapy had decreased or stable sPD-L1 levels. The 4th RS and two CLL had increased sPD-L1 levels and had not demonstrated clinical response. CD8 T-cell subset analyses performed blindly (n=4) showed increased CD8 T cell function in one of the 4 patients tested. This patient who demonstrated CD8 T cell functional enhancement is the only patient in the 4 tested patients who had notable response to pembrolizumab. Conclusion: Pembrolizumab is tolerated in relapsed CLL and RS patients. Early efficacy observed in heavily pretreated RS patients including patients progressed on both anthracycline-based treatment and ibrutinib indicate PD-1 inhibition is a promising novel approach in patients with RS. Our CD8 T cell subset analysis may be used as a biomarker assay to predict clinical response in the era of novel immunotherapy. This trial and further correlative analysis is ongoing and will be presented at the ASH meeting. Disclosures Ding: Merek: Research Funding. Shanafelt:Cephalon: Research Funding; Pharmactckucs: Research Funding; Janssen: Research Funding; Hospira: Research Funding; Polyphenon E Int'l: Research Funding; Celgene: Research Funding; Glaxo-Smith_Kline: Research Funding; Genentech: Research Funding. Kay:Tolero Pharma: Research Funding; Hospira: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Celldex: 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 Resistance to chemotherapy remains a major obstacle to improving outcomes of cancer treatment. Recent data suggest intrinsic functions of immune checkpoint proteins, including PD-L1, that may be distinct from their immunoregulatory roles. We found that colorectal cancer (CRC) cells with mutant BRAF(V600E) or KRASalleles show increased PD-L1 transcripts and expression of cell surface PD-L1 protein compared to those with wild-type copies. Furthermore, ectopic expression of BRAFV600E or mutant KRAS induced PD-L1 whose upregulation was attenuated by inhibition of RAS-RAF-MEK-ERK signaling. Combined knockdown of MEK/ERK effectors c-JUN and YAP markedly reduced PD-L1. Knockout of PD-L1 in RKO cells resulted in a reduction in chemotherapy-induced DNA double strand breaks (pH2AX) and caspase-3 cleavage compared to parental cells. Results were confirmed in PD-L1 knockout MC38 murine CRC cells where re-expression of PD-L1, but not its deletion mutants, was shown to induced pH2AX and apoptosis. In cells with knockout of PD-L1, reductions in p-AKT and the BH3-only proteins BIM and BIK were observed that could be restored by re-expression of PD-L1. Treatment of cells with an anti-PD-L1 antibody reduced p-AKT, BIM and BIK, and also attenuated chemotherapy-induced apoptosis. Re-expression of BIM in PD-L1 knockout cells restored apoptosis. Murine tumor xenografts generated from cells with knockout of PD-L1 displayed resistance to oxaliplatin-induced tumor regression compared to tumors derived from parental cells. Using TCGA datasets, human CRCs with high vs low PD-L1 mRNA levels were significantly associated with better survival. In summary, we identified a non-immune function of tumor cell-intrinsic PD-L1 that upregulates BH3-only proteins BIM and BIK to promote apoptosis, thereby suggesting its potential as a predictive biomarker. Citation Format: Daofu Feng, Krishnendu Pal, Debabrata Mukhopadhyay, Xin Liu, Haidong Dong, Shengbing Huang, Frank A. Sinicrope. Tumor intrinsic PD-L1 is mediated by BRAFV600E and can regulate drug-induced apoptosis in human colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 705.