PD-L1 Checkpoint Inhibitors Research Articles

Investigation of the PD-1/PD-L1 checkpoint inhibitor pathway in early-onset pre-eclampsia and in healthy pregnancy

Investigation of the PD-1/PD-L1 checkpoint inhibitor pathway in early-onset pre-eclampsia and in healthy pregnancy

Merkel Cell Carcinoma: Updates on Pathogenesis, Diagnosis, and Management

To describe updates on the pathogenesis, diagnosis, and management of Merkel cell carcinoma (MCC). Sequencing studies revealed that MCCs have either a low mutational burden and integrated Merkel cell polyomavirus (MCPyV), or they have a high number of ultraviolet-associated somatic mutations and no MCPyV. Clinically, prognosis was better for stage III MCC of unknown primary than known primary. Similarly, lack of immunosuppression conferred better prognosis. The immunogenicity of MCC was reflected in high response rates to PD-1/PD-L1 checkpoint inhibitors. MCC is a rare but aggressive neuroendocrine skin cancer associated with advanced age and immunosuppression. Approximately 80% of MCCs are MCPyV driven, whereas MCPyV-negative tumors have mutations in genes such as p53 and RB1. MCC is highly immunogenic, and recently, the anti-PD-L1 antibody avelumab was approved to treat metastatic MCC. Here, we summarized features of the pathogenesis, diagnosis, and management of MCC.

Conventional and PD-L1-expressing Regulatory T Cells are Enriched During BCG Therapy and may Limit its Efficacy

The regulation of immune responses occurring during Bacillus Calmette-Guerin (BCG) therapy need to be better scrutinized in order to identify new targetable pathways for non-muscle invasive bladder cancer treatment. Immunoregulatory mechanisms have emerged as key players in various cancers. While T lymphocytes are crucial for the control of tumor growth, they often include regulatory subsets known to restrain their anti-tumor activity. In this prospective study, we assessed conventional regulatory T cells (cTregs) and PD-L1-expressing CD4 T cells (PD-L1+ Tregs) levels in blood and urine of urothelial cancer (UCa) patients undergoing BCG treatment. Local cTregs were found at higher frequencies than their counterpart in the periphery and induced by bladder tumor cells in vitro. Interestingly, while circulating PD-L1+ Tregs were hardly detectable in the blood of healthy donors and UCa patients, substantial levels were found in patients’ urine. In vitro experiments suggested that BCG infection of urothelial cells could induce PD-L1+ Tregs, partially via an interferon-β-mediated mechanism. Of note, high level of Tregs in urine was associated with rapid recurrence following BCG therapy. Our findings demonstrate that T lymphocytes recruited during BCG therapy encompass a significant fraction of regulatory cells including a non-classical source of PD-L1 and reinforce treatment strategies combining BCG with PD-1/PD-L1 checkpoint inhibitors as promising approaches for non-muscle invasive bladder cancer. Patient summaryWe investigated the presence of particular immune cell types in the urine of bladder cancer patients undergoing Bacillus Calmette-Guerin (BCG) therapy. We identified a cell type that is strongly enriched in the urine after BCG instillation and that may favor tumor recurrence. This immune subpopulation might be targeted for bladder cancer treatment.

Abstract LB-155: Rapid patient derived xenograft avatars that consider tumor heterogeneity for prediction of cancer immunotherapy responses in metastatic renal cell carcinoma patients

Abstract INTRODUCTION AND OBJECTIVES: Patient responses to cancer immunotherapy such as checkpoint inhibitor drugs vary greatly between patients and there remains no biomarker to predict sensitivity to these drugs. We describe a patient derived xenograft (PDX) avatar system that predicts cancer immunotherapy responses in the context of tumor heterogeneity within 8-10 days. This avatar model preserves the original tumor microenvironment and tumor infiltrating lymphocytes (TILs) at a high tumor take rate for evaluation of various immunotherapy drugs. This prediction model also involves tumor infiltrating lymphocyte isolation and primary cell generation for confirming the immunotherapy response in vitro. This PDX avatar platform generates rapid actionable information and readily identifies patients that would maximally benefit from checkpoint inhibitor therapies in a phenotype-based manner. METHODS: Tissue from patients that underwent a nephrectomy for RCC or metastasectomy were engrafted into the chorioallantoic membrane of avian embryos with a portion used for TIL isolation and primary cell generation. PD-1 and PD-L1 checkpoint inhibitors were intravenously injected and high frequency ultrasound imaging and immunophenotyping was used to quantitate changes in tumor volume, vascularity, and TIL expansion in all treatment groups. Isolated TILs were expanded and co-cultured with respective primary cells to confirm and examine immunotherapy response at a molecular level. RESULTS: Patient PDXs demonstrated heterogeneous responses to immunotherapy that relates to presence and expansion of TILs after checkpoint inhibitor injection. Patient #1 demonstrated the greatest response with a >50% reduction in tumor volume and 4X expansion of T-cells over 8 days of treatment. Patient #2 demonstrated intratumoral heterogeneities in terms of tumor volume change after checkpoint inhibitors treatment despite TIL expansion in all treatment groups. Patient #3 showed no response that correlated to lack of TILs in the PDX. In vitro expansion of TILs and co-culture with primary cell line demonstrated a specific immune response via INF-gamma release and changes in immunomodulatory molecules. CONCLUSIONS: This PDX system preserves TILs and permits rapid large scale prediction of tumor specimens to various systemic therapy agents such as checkpoint inhibitors without the need for humanized models. This avatar system is a useful tool to rapidly predict and examine response to checkpoint inhibitors on a per-patient basis to improve the assignment of cancer immunotherapies to specific RCC patients. Citation Format: Mario Cepeda, Matthew Lowerison, Cindy Liu, Yarolslav Fedyshyn, Vidhu Joshi, Paras Shah, John Cheville, R. Houston Thompson, Boorjian Stephen, Bradley Leibovich, Haidong Dong, Hon S. Leong. Rapid patient derived xenograft avatars that consider tumor heterogeneity for prediction of cancer immunotherapy responses in metastatic renal cell carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-155.

Abstract 4517: Characterization of a standardized immunohistochemical assay for detecting PD-L1 expression in non-small cell lung cancer using anti-PD-L1 clone 73-10

Abstract Background: Recent data have demonstrated the clinical efficacy of anti-PD-1/PD-L1 checkpoint inhibitors in non-small cell lung cancer (NSCLC), and approvals by regulatory authorities in first-line and second-line settings have followed. In selected treatment scenarios, expression of PD-L1 by immunohistochemistry (IHC) is considered predictive of patient outcome. A recent comparative study using the rabbit anti-PD-L1 monoclonal antibody clone 73-10 and other commercialized IHC assays demonstrated strong intra-assay reliability in NSCLC tumor cell scoring and inter-assay differences in the number of tumor cells detected by each assay (Blueprint 2 data presented at IASLC 2017). Here we report precision around a ≥ 1% tumor cell cutoff and PD-L1 expression in 428 procured NSCLC tissues using an immunohistochemical assay developed with this antibody clone. Methods: PD-L1 expression was assessed using a standardized PD-L1 IHC assay. Scoring of tumor specimens was performed on a 0 - 3+ intensity scale, with membrane staining in ≥ 1% of tumor cells at ≥ 1+ intensity defining PD-L1 positivity. Assay precision around this cutoff was assessed in 28 specimens. PD-L1 expression was evaluated in 428 Stage IIIA, IIIB, and IV procured NSCLC tissues. Assay specificity was examined in 30 normal tissues. Results: The PD-L1 IHC 73-10 assay showed > 95% negative, positive, and overall agreement in inter-day, inter-operator, intra-run, inter-instrument, and inter-lot conditions around the ≥ 1% tumor cell cutoff. The lower bound of the 95% confidence interval was > 90% for all agreement types and precision conditions. PD-L1 protein localization in normal tissues was consistent with known patterns. The 428 procured NSCLC samples had primarily squamous cell (39.5%), adenocarcinoma (43.7%) and a mixture of other less common (16.8%) histologic subtypes; 397 (92.8%) of samples were from primary and 31 (7.2%) were from metastatic tumors. Crisp membrane staining was observed in tumor cells. Other labeled cell types included macrophages, lymphocytes, endothelial cells, and fibroblasts that exhibited membrane and/or cytoplasmic staining. Of the 428 procured specimens, 270 (63.0%) were PD-L1-positive based on the ≥ 1% cutoff. Tissues of each NSCLC stage expressed PD-L1 in the full dynamic range from 0% - 100% positive. Conclusions: These data provide insight into the prevalence of PD-L1 expression in procured NSCLC specimens when stained with PD-L1 IHC 73-10, and demonstrate that this assay is precise around a ≥ 1% tumor cell cutoff while exhibiting expected antigen specificity. PD-L1 positivity in the tumor stages examined using this assay is greater than in the literature, where prevalence using different antibodies has been reported at less than 50% in various stages. Citation Format: Darlene Krohn, Mai Nguyen, Marko Srdanov, Christina Samathanam, Peng Duan, Monika Vilardo, Alexander Prenta, Sharika Vasudevan, Landry Nicholson-Legg, Debra Hanks, Aaron R. Ellison. Characterization of a standardized immunohistochemical assay for detecting PD-L1 expression in non-small cell lung cancer using anti-PD-L1 clone 73-10 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4517.

Abstract 5536: Simultaneous characterization and quantification of immune cell subpopulations and PD-L1 expressing CTCs in peripheral blood of cancer patients

Abstract Background: Expression of PD-L1 on tumor and immune markers in tumor tissue is associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. However, each alone has limited predictive utility. Multimodal characterization of both the tumor and host immune system is an unmet medical need for the improved prediction of response to immunotherapy. Metastatic lesions are likely to be undersampled and require a liquid biopsy, given tumor heterogeneity and evolution and temporal changes in the host immune system. We sought to examine expression of PD-L1 on circulating tumor cells (CTCs) as well as characterize rare immune cell populations with a noninvasive liquid biopsy. Examining dynamic biomarker changes in longitudinal samples could enable the development of novel diagnostic tools for response prediction and pharmacodynamics studies related to immunotherapy. Methods: Blood samples from lung cancer patients were collected and shipped to Epic Sciences. Contrived samples were also developed, using cancer cell lines spiked into healthy donor (HD) blood. Red blood cells were lysed and nucleated cells were plated onto glass slides. Slides were stained with DAPI and immune cocktails, then imaged. Targets included pan-CK, CD45, PD-L1, CD4, CD8, Ki-67, and TIM-3. Approximately 3 million nucleated cells per slide were examined through advanced digital pathology pipelines to detect and quantify changes in T-cell populations and to assess circulating tumor burden. Results: Epic Sciences' rare cell detection platform has an analytically validated limit of detection of 2 cells/mL of blood. Immuno-panels were developed to profile leukocyte subpopulations and CTCs from a single blood sample and feasibility was demonstrated in cell lines, HD and lung disease patient blood. Quantitation experiments showed that immunomagnetically purified CD8+ and CD4+ cells spiked in 3:1, 1:1, and 1:3 ratios were detected as 3.1:1, 1:1.1, and 1:3.1. Purified CD4 cells were also spiked into healthy donor WBCs at target ratios of 1-10%. Percentages of detected spike-in CD4 cells were linearly correlated with the target ratios with correlation coefficient (r) = 0.96. 22.8% (18/79) lung cancer patients had PD-L1+ CTCs detected, and the majority (91%) of patients had PD-L1+ leukocyte populations. Conclusions: The low limit of detection of the Epic Sciences CTC platform coupled with the ability to archive patient blood samples allowed for retrospective, precise quantification of leukocyte subpopulations and PD-L1 expression on CTCs. Development of a liquid biopsy-based platform that can simultaneously measure immune biomarkers in CTCs as well as on leukocytes will allow for real-time assessment and monitoring of response to immune checkpoint inhibitors. Citation Format: Rachel Krupa, David Lu, Adam Jendrisak, Angel Rodriguez, Nadia Ebrahim, Robin Richardson, Sean Nisperos, Ryon Graf, Jiyun Byun, Yipeng Wang, Mark Landers, Ryan Dittamore. Simultaneous characterization and quantification of immune cell subpopulations and PD-L1 expressing CTCs in peripheral blood of cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5536.

Abstract 5621: FAP-4-1BBL: A novel versatile tumor-stroma targeted 4-1BB agonist for combination immunotherapy with checkpoint inhibitors, T-cell bispecific antibodies, and ADCC-mediating antibodies

Abstract For tumor specific co-stimulation via 4-1BB (CD137) a novel tumor-stroma targeted FAP-4-1BB ligand (FAP-4-1BBL) was designed composed of a trimeric split 4-1BBL, a tumor-stroma targeted 4B9 Fab moiety recognizing fibroblast activation protein (FAP) and a heterodimeric Fc-region devoid of FcgR binding but maintained FcRn binding. In presence of a TCR signal 1 either from a T cell receptor MHCI/peptide interaction or through TCR engagement by a T-cell bispecific antibody, FAP-4-1BBL provides co-stimulation to T cells strictly dependent on cross-linking by FAP-expressing fibroblasts. Similarly, FAP-4-1BBL can provide co-stimulation to NK cells with activated FcgRIIIa signaling. FAP-4-1BBL was tested as monotherapy and combined with CEA-TCB, an anti-CEA T-cell bispecific antibody, in the s.c. gastric MKN45 xenograft model co-grafted with NIH-3T3 fibroblasts in human stem cell engrafted (HSC) NSG mice in comparison to DP47-4-1BBL, an analogous untargeted 4-1BBL fusion protein. For use in syngeneic mouse models in immunocompetent mice muFAP-4-1BB, a murine surrogate made of a mu4-1BB agonistic surrogate antibody fused to the variable region of the FAP antibody 4B9, was used. muFAP-4-1BB was tested as monotherapy and combined with a murine specific CEA-TCB surrogate (muCEA-TCB) and the muPD-L1 specific surrogate antibody 6E11 in the s.c. colorectal MC38-CEA model in CEA transgenic (Tg) C57BL/6 mice. For combination with ADCC-mediating antibodies FAP-mu4-1BBL, a hybrid FAP-mu4-1BBL fusion protein, was generated. FAP-mu4-1BBL was tested as monotherapy and combined with the ADCC-mediating anti-HER2 antibody trastuzumab in the s.c. gastric N87 xenograft model in human CD16 Tg Scid mice. In the s.c. gastric MKN45 xenograft model co-grafted with NIH-3T3 fibroblasts in HSC-NSG mice FAP-4-1BBL resulted in combined anti-tumoral efficacy in combination with CEA-TCB, whereas the respective monotherapies as well as the combination with the untargeted DP47-4-1BBL did not improve anti-tumor efficacy. In the syngeneic s.c. MC38-CEA model in CEA Tg C56BL/6 mice, a model with natural FAP expression due to fibroblast infiltration, the muFAP-4-1BB surrogate antibody resulted in combined anti-tumor efficacy, both combined with muCEA-TCB and muPD-L1 antibodies including the induction of tumor remission. Finally, the hybrid FAP-mu4-1BBL surrogate resulted in improved anti-tumor efficacy combined with the ADCC-mediating antibody trastuzumab in the s.c. gastric N87 xenograft model in human CD16 Tg Scid mice. These data show that FAP-4-1BBL is a versatile combination partner for cancer immunotherapy mediating FAP-dependent co-stimulation to T and NK cells in combination with PD-L1 checkpoint inhibition, TCBs and ADCC-mediating antibodies. Clinical evaluation of this novel therapeutic approach is planned early 2018. Citation Format: Johannes Sam, Christina Claus, Claudia Ferrara, Sabine Lang, Valeria Nicolini, Sara Colombetti, Volker Teichgräber, Stefan Evers, Marina Bacac, Pablo Umana, Christian Klein. FAP-4-1BBL: A novel versatile tumor-stroma targeted 4-1BB agonist for combination immunotherapy with checkpoint inhibitors, T-cell bispecific antibodies, and ADCC-mediating antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5621.

Abstract 2774: The triple combination of the FAP-IL2v immunocytokine with PD-L1 checkpoint inhibitory and CD40 agonistic antibodies results in long-term tumor control in the orthotopic PancO2 model

Abstract Introduction: We have previously described the FAP-targeted immunocytokine FAP-IL2v and its combination with PD-L1 checkpoint inhibition. Here we show that the anti-tumoral efficacy of FAP-IL2v can be strongly enhanced by combination with a CD40 agonistic antibody as well as the triple combination of FAP-IL2v with a CD40 agonistic and a PD-L1 checkpoint inhibitory antibody in the syngeneic PancO2 model. Methods: Anti-tumoral efficacy was assessed in the orthotopic intra-pancreatic PancO2 model transfected with Luciferase in C57BL/6 mice. Mice were injected intra-pancreatically on study day 0 with 1x10E5 Panc02-Fluc cells, randomized and weighed. One week after tumor cell injection mice were injected i.p. with the murine specific surrogate immunocytokine muFAP-muIL2v (muIgG1 DAPG) (2 mg/kg, q1wx3), anti-muPD-L1 surrogate antibody (muIgG1 DAPG) (10 mg/kg, q1wx3) and/or the anti-muCD40 surrogate antibody FGK4.5 (muIgG1) (10mg/kg, q1wx1). Bioluminescence imaging (BLI) was performed after ip injection of 150 mg/kg D-Luciferin 10 min before acquisition using IVIS® SPECTRUM. An analogous experiment was performed in C57BL/6 B cell knockout mice. Results: In the orthotopic PancO2-Fluc model the triple combination of FAP-IL2v plus anti-PD-L1 plus anti-CD40 showed superior outcome compared to vehicle and the respective monotherapies in terms of median survival and overall survival in the majority of animals followed by the combination of FAP-IL2v with anti-CD40 and the combinations of anti-PD-L1 with anti-CD40 and FAP-IL2v with anti-PD-L1, respectively. Bioluminescence imaging confirmed the survival outcome of the respective groups by decrease in/absence of bioluminescence signals in the tumor area. Tumor re-challenge experiments showed that the animals with long-term survival were protected both in the C57BL/6 and the C57BL/6 B cell knockout mice. Conclusions: These data show that the anti-tumoral efficacy of FAP-IL2v in the syngeneic PancO2 model can be strongly enhanced by combining it with a CD40 agonistic antibody on top of a PD-L1 checkpoint inhibitory antibody. Most notably, the triple combination of FAP-IL2v with CD40 agonism and PD-L1 checkpoint inhibition resulted in long term survival of the majority of animals. These data provide the preclinical rationale for further clinical investigation of the combination of FAP-IL2v (RG7461) with the PD-L1 antibody atezolizumab and the CD40 agonistic antibody selicrelumab (RG7876). Citation Format: Valeria Nicolini, Inja Waldhauer, Anne Freimoser, Emily Corse, Jahad Charo, Pablo Umana, Christian Klein. The triple combination of the FAP-IL2v immunocytokine with PD-L1 checkpoint inhibitory and CD40 agonistic antibodies results in long-term tumor control in the orthotopic PancO2 model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2774.

Abstract 4346: Integrated whole genome profiling of the immune tumour interaction identifies predictive biomarkers of checkpoint inhibitor response in metastatic cancer

Abstract Immune checkpoint inhibitors are fast becoming a key therapy in the medical oncologist's toolbox across a diverse array of tumour types. The excitement surrounding the utility of these emerging agents is marred by the heterogeneous response observed among patients, highlighting the urgent need for clinically applicable predictive biomarkers. Significant progress has been made in identifying potentially relevant biomarkers including mutation burden and immune infiltration. However, it is still unclear which biomarkers are most predictive across various disease types, and how to integrate various markers of response and resistance for clinical interpretation. We hypothesized that comprehensive genomic profiling, combined with clinical data, would reveal differences in the immuno-oncologic phenotype that could be used as biomarkers for response to checkpoint inhibitor therapy. The Personalized OncoGenomics (POG) study at BC Cancer performs whole genome and transcriptome sequencing of metastatic disease across a diverse array of cancer types to comprehensively characterize cancers and inform clinical therapeutic decision-making. Here we performed in-depth genomic profiling of 64 POG patients who received immune checkpoint inhibitors encompassing multiple disease types including skin, lung, breast, pancreatic and colorectal cancers and sarcomas. Single nucleotide variants, structural variants, copy number alterations, and RNA expression derived from whole genomic and transcriptomic data were used to characterize genomic instability, neoantigen landscape, immune infiltration, and other potential biomarkers of clinical response to checkpoint inhibitors. This collection of putative immune-oncologic biomarkers were integrated into a multivariate model to stratify markers of the tumour immune response. Our analysis shows limited association between PD-L1 expression and checkpoint inhibitor response, consistent with the inadequate effectiveness of this as a universal marker, and shows stronger association between signatures of T cell infiltration based on RNA-Seq data. We also observed patients with low mutation burden that respond to checkpoint inhibitors and show high levels of immune infiltration, and cases with high mutation burden that harbour mechanisms of resistance including reduction in predicted neoantigen diversity. Additional mechanisms of therapeutic resistance observed in post treatment biopsies highlight disruption of antigen presentation and JAK1 mutations in resistant tumours. Our study helps to define which features distinguish patients most likely to respond to checkpoint inhibitors, and uses these features in selecting patients for immunotherapy treatment within the POG clinical trial. Citation Format: Hillary Pearson, Laura Williamson, Erin Pleasance, Scott Brown, Emma Titmuss, Martin Jones, Stuart Zong, Payal Sipahimalani, Yussanne Ma, Steve Jones, Robert Holt, Marco Marra, Janessa Laskin. Integrated whole genome profiling of the immune tumour interaction identifies predictive biomarkers of checkpoint inhibitor response in metastatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4346.

Collateral Damage: Insulin-Dependent Diabetes Induced With Checkpoint Inhibitors.

Insulin-dependent diabetes may occur in patients with cancers who are treated with checkpoint inhibitors (CPIs). We reviewed cases occurring over a 6-year period at two academic institutions and identified 27 patients in whom this developed, or an incidence of 0.9%. The patients had a variety of solid-organ cancers, but all had received either anti–PD-1 or anti–PD-L1 antibodies. Diabetes presented with ketoacidosis in 59%, and 42% had evidence of pancreatitis in the peridiagnosis period. Forty percent had at least one positive autoantibody and 21% had two or more. There was a predominance of HLA-DR4, which was present in 76% of patients. Other immune adverse events were seen in 70%, and endocrine adverse events in 44%. We conclude that autoimmune, insulin-dependent diabetes occurs in close to 1% of patients treated with anti–PD-1 or –PD-L1 CPIs. This syndrome has similarities and differences compared with classic type 1 diabetes. The dominance of HLA-DR4 suggests an opportunity to identify those at highest risk of these complications and to discover insights into the mechanisms of this adverse event.

PD-L1 immunostaining scoring for non-small cell lung cancer based on immunosurveillance parameters.

Non-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer death globally, and new immunotherapies developed and under development targeting PD-1/PD-L1 checkpoint inhibition require accurate patient selection to assure good clinical outcome. PD-L1 immunohistochemistry is the current biomarker assay used for patient selection, but still imprecise in predicting therapy response. Exploring this issue, we performed computational tissue analysis of PD-L1 immunostaining in procured NSCLC tissues (n = 50) using the Merck KGaA anti-PD-L1 clone MKP1A07310. Staining patterns and PD-L1 cut-off points were interrogated using relevant cancer immune-surveillance biomarkers. Groups with high PD-L1 expression levels (above 25/50% staining cut-off points) were enriched for a biomarker profile in the tumor-nest and microenvironment indicating escape from host-immunity, as represented by increased numbers of cells positive for CD8 and Granzyme B (immune-effectors), FOXP3 (immune-suppressive), and CD68 (P < 0.05). Manual analysis of PD-L1 staining patterns identified tumors with an immune-induced reactive pattern relevant for immunotherapy that would ordinarily be excluded by the arbitrary 25% staining threshold (P < 0.05). Conversely, some cases with completely or predominantly immune-independent constitutive PD-L1 staining patterns that indicate insensitivity to immunotherapy may have been incorrectly selected using this staining cut-off point criterion. Therefore, we propose differentiation of reactive vs constitutive PD-L1 staining patterns to improve the accuracy of this biomarker assay in selecting NSCLC patients for PD-1/PD-L1 immunotherapy.

2371 Validation of a novel PD-L1 assay for bladder cancer circulating tumor cells

OBJECTIVES/SPECIFIC AIMS: Bladder cancer patients being considered for immune checkpoint blockade are often judged on immunohistochemical staining for the checkpoint target protein PD-L1 in the original surgery or biopsy sample. However, sampling error or the clinical evolution of most patients’ cancer can render the original PD-L1 assessment no longer accurate. In contrast, circulating tumor cells (CTCs) allow serial noninvasive sampling of the current tumor status throughout a patient’s clinical course including those with the highest metastatic potential. We therefore sought to develop a method for quantifying PD-L1 expression in CTCs towards addressing inherent limitations of current UC management. METHODS/STUDY POPULATION: This work utilizes both cancer cell lines as well as patient samples. Positive and negative control cancer cell lines were assessed via “industry standard” antibodies for PD-L1 expression via Western blots and immunofluorescence, and a threshold-based method was developed for reliable quantification. PDL-1 expression was additionally verified via interferon-mediated up-regulation. CTCs isolated from bladder cancer patient samples via a density centrifugation method were then assessed for PD-L1 via the same antibodies. RESULTS/ANTICIPATED RESULTS: We will show preliminary preclinical and clinical data that validates the sensitivity and specificity of our assay. A case study will be presented that illustrate the potential useful of the novel approach we describe and which should be complementary to current clinical practices. In a patient with metastatic bladder cancer, this method effectively detected the PD-L1 expression in CTCs taken at a time coincident to when the patient derived an excellent response to the PD-L1 checkpoint inhibitor Pembrolizumab. DISCUSSION/SIGNIFICANCE OF IMPACT: This work highlights the potential utility of CTCs in the management of bladder cancer. It may be the case that this assay in conjunction with current methods of patient selection for immunotherapy may allow for better response prediction than either method alone.

Abstract B42: Targeting cancer cell CCR2 enhances synergistic immune surveillance in breast cancer

Abstract High expression of the C-C chemokine receptor type 2 (CCR2) in tumor samples and serum correlates with poor prognosis in human breast cancer, which has sparked interest in targeting the CCR2 pathway for therapeutic benefit. Importantly, a human CCR2 inhibitor (PF-04136309) has been investigated in clinical trials, and is currently being studied in a phase 1b/2 trial for pancreatic cancer (NCT02732938). Previous studies have focused on the protumor effects of CCR2 signaling in inflammatory monocytes. However, CCR2 is also expressed in breast cancer cells, but its potential function in cancer cells is poorly understood. To determine the specific roles of CCR2 in cancer and host cells, respectively, we established an orthotopic breast cancer mouse model by injecting primary cancer cells from MMTV-PyMT; Ccr2+/+ or MMTV-PyMT; Ccr2-/- mice into the mammary glands of wide-type or Ccr2 null hosts. In this cancer model, deleting Ccr2 in host cells, including monocytes, did not alter tumor growth. However, when tumor cells had lost Ccr2, immune surveillance was much more efficient, with greater infiltration and expansion of cytotoxic CD8+ T cell lymphocytes (CTLs) that efficiently recognized and destroyed the cancer cells, resulting in significant inhibition of tumor growth. Consistently, the delayed tumor growth of Ccr2 null cancer cells was fully reversed when these same cancer cells were transplanted into athymic (immunodeficient) mice. The reduced growth of tumors derived from Ccr2-/- cancer cells was associated with upregulation of IFN-γ response genes and genes involved in MHC class I presentation, as well as decreased expression of checkpoint inhibitor PD-L1 in cancer cells. Finally, greater infiltration of CD103+ dendritic cells (DCs) in Ccr2-/- tumor microenvironment also contributed to increased CTL function by cross presentation. Taken together, our results show that CCR2-expressing breast cancer cells orchestrated global changes in the infiltration of CTLs and DCs, leading to effective immune suppression. This suggests that CCR2 may be an immunotherapeutic target in breast cancer. Citation Format: Miriam R. Fein, Xue-Yan He, Ana S. Almeida, Arnaud Pommier, Emilis Bružas, Anaïs Eberhardt, John Erby Wilkinson, Camila dos Santos, Mikala Egeblad. Targeting cancer cell CCR2 enhances synergistic immune surveillance in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B42.

Immunotherapy in Older Adults With Advanced Cancers: Implications for Clinical Decision-Making and Future Research.

Immunotherapy has expanded the therapeutic landscape for advanced cancers, including solid tumors and lymphomas. For many patients with cancer, these agents have been shown to have substantial efficacy and favorable toxicity compared with cytotoxic agents, particularly in the second-line setting. With the advent of anti-PD-1 and anti-PD-L1 checkpoint inhibitors, combination immunotherapy- and chemoimmunotherapy-based strategies have emerged as promising novel regimens to improve cancer-related outcomes. Older adults age 65 or older represent the growing majority of patients diagnosed with cancer. However, older adults are under-represented in clinical trials in general, as well as in the landmark studies that led to approval of these immunotherapy agents. Because of increasing age and attendant multimorbidity and impaired functional status, many of these patients seen in the community-based oncology practices would not have been considered eligible for such studies. Thus, the results of these studies are difficult to generalize to a broader patient population with these competing risks. Furthermore, robust evaluation of toxicities, effect on quality of life and functional status, and aging-related (i.e., immunosenescence) and immunotherapy-related changes affecting the immune system remain underexplored research areas for older adults. This review examines the role of immunotherapy and its unique issues, specifically in older adults with lung cancer, bladder cancer, and lymphomas.

PD-L1 inhibition with avelumab for metastatic Merkel cell carcinoma

ABSTRACTIntroduction: Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer that lacks durable responses to traditional chemotherapy.Areas covered: After MCC was shown to be an immunogenic tumor, small trials revealed high objective response rates to PD-1/PD-L1 checkpoint inhibitors. The JAVELIN Merkel 200 (NCT02155647) trial tested the use of avelumab, a human IgG1 monoclonal antibody against PD-L1, in metastatic MCC. Avelumab recently became the first approved drug for metastatic MCC.Expert commentary: By conducting broad phase I studies assessing the safety of avelumab and a small phase II study demonstrating efficacy in this rare orphan tumor type, avelumab gained accelerated approval for the treatment of metastatic MCC. Additional studies are needed to determine how the antibody-dependent cellular cytotoxicity (ADCC) competent Fc region of avelumab contributes to disease control.Remaining questions: Longer follow-up will determine the durability of checkpoint blockade in controlling metastatic MCC. Additional studies will assess the utility and safety of adjuvant checkpoint blockade in patients with excised MCC. How to increase response rates by combining PD-1/PD-L1 blockade with other treatment approaches needs to be explored. In addition, treatment options for MCC patients who fail or do not respond to avelumab need to be identified.

Atezolizumab for the treatment of colorectal cancer: the latest evidence and clinical potential

ABSTRACTIntroduction: Atezolizumab is a fully humanized, engineered monoclonal antibody that specifically targets PD-L1, key molecule in the cancer-immunity pathway. Atezolizumab is currently approved for the treatment of metastatic non-small-cell lung cancer and advanced urothelial carcinomas.Areas covered: In this review, we will present the available data supporting the efficacy of atezolizumab for the treatment of metastatic colorectal cancer (mCRC). We will also provide an update on the ongoing/future clinical trials evaluating the role of atezolizumab for the treatment of CRC in different settings (alone or in combination with other checkpoint inhibitors and/or targeted therapies). So far, a small subgroup of mCRC (those with deficiency in mismatch repair - dMMR) appears to benefit significantly from checkpoint inhibitors. As expected, further research is needed to develop biomarkers, effective therapeutic strategies and novel combinations to overcome immune escape resistance and achieve better responses with minimal toxicities.Expert opinion: Interim analyses from ongoing early-phase studies in mCRC have shown encouraging activity of atezolizumab in combination with chemotherapy and/or targeted therapies, especially with MEK inhibitor cobimetinib. Within the next few years, this PD-L1 checkpoint inhibitor will likely be included as one of the treatment options for CRC, at least for patients with dMMR.

Abstract MS1-2: New clinical approaches for TNBC

Abstract Over the last decade, triple-negative breast cancer (TNBC) has been the subject of intense clinical investigation. Importantly, an improved understanding of the critical role of DNA damage repair deficiency in TNBC has led to effective therapeutic strategies to target BRCA1 and BRCA2 mutation-associated TNBCs with platinum chemotherapy agents and PARP inhibitors. A major area of increasing clinical relevance focuses on the treatment of BRCA1 and BRCA2 mutation-associated breast cancers after the development of resistance to platinum and PARP inhibitor therapy. Lurbinectedin, a novel marine derived DNA damaging agent, has shown activity in BRCA1 and BRCA2 mutation-associated cancers, including those with platinum resistance. While therapeutic strategies for TNBC patients with BRCA1 and BRCA2 mutations have increased, most TNBC patients lack an underlying germline mutation in these genes and continue to have high unmet clinical need. Multiple lines of investigation are currently underway. Androgen receptor (AR) blockade has shown preliminary proof-of-concept in AR expressing TNBCs. While monotherapy with PD-1/PD-L1 checkpoint inhibitors has shown modest activity in metastatic TNBC, multiple studies are exploring the role of these agents in combination with chemotherapy, targeted therapies and other immunotherapeutic agents. Given the high prevalence of TP53 mutations in TNBC, strategies to target additional cell cycle checkpoints including ATR, ATM, Wee1 and others may hold promise. Antibody-drug conjugates targeting Trop-2, LIV-1 and GPNMB expressing TNBCs have also shown clinical activity and are advancing in clinical development. Citation Format: Telli M. New clinical approaches for TNBC [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr MS1-2.

Single-cell protein analysis in support of biomarker evaluation for breast cancer immunotherapeutics.

10 Background: Triple-negative breast cancer (TNBC) is being tested for PD-1/PD-L1 checkpoint inhibitors combination therapy. The relationship between tumor expression of PD-L1 and patient outcomes has been established using immunohistochemistry (IHC) biomarker assays across various malignancies. PD-L1 is expressed in tumor and immune cells within the tumors in TNBC, but only immune-cell PD-L1 is associated with response to anti-PD-L1 (atezolizumab) so differentiating tumor vs. immune PD-L1 expression is essential. The currently available IHC biomarker assays that relay on tissue biopsies struggle to provide clinically meaningful identification of responders vs. non-responders. Poor sensitivity and specificity is partially attributed to tumor heterogeneity that is not well-represented in these biopsies. In addition, PD-L1 expression may vary over time due to disease progression or treatment. Since tissue biopsy collection is often an invasive procedure, it is restricted. Needle biopsies can mitigate some of these issues as a minimally invasive method to probe multiple regions within several cancer lesions across several time points. MILO, a new single cell Western Blot (WB) technology allows quantitative measurements of multiple protein biomarkers within one cell in small samples. Methods: To evaluate the capability of MILO to differentiate between PD-L1 in immune and tumor cell populations, we interrogated BC cell lines as well as peripheral blood mononuclear cells (PBMCs) mixed in known ratios. We used MDA-MB-231 that shows high PD-L1 and low HER2 expression and BT-474, which shows the inverse expression profile for these biomarkers along with PBMCs as PD-L1+ immune cells. Primary breast tumor tissues are currently interrogated to establish utility in needle biopsies. Results: MILO demonstrated heterogeneity at the single-cell level of HER2 and PD-L1 expression in BC tumor cells and enabled differentiation between PD-L1 expression in immune vs. tumor cells using cell-specific markers and low cell numbers. Conclusions: MILO can improve patient selection and prediction of response to immune checkpoint inhibition by promoting the utility of needle biopsies to complement IHC of core biopsies.

Nivolumab for the treatment of urothelial cancers

ABSTRACTIntroduction: Advanced urothelial cancer patients had limited therapeutic options following failure of platinum-based chemotherapy. The recognition that anti-PD1/PDL1 immune checkpoint inhibitors lead to dramatic and durable responses in a subset of patients has transformed the therapeutic landscape of these cancers. Since May 2016, five agents targeting this pathway have been approved by the US FDA, including the PD-1 inhibitor nivolumab.Areas covered: The purpose of this paper was to review the safety, activity and efficacy of nivolumab, an anti-PD1 checkpoint inhibitor for the treatment of locally-advanced or metastatic urothelial cancers. Future therapeutic perspectives were also discussed.Expert commentary: Nivolumab is one of five anti-PD1/PDL1 checkpoint inhibitors approved for treatment of advanced urothelial cancers. While durable responses can be observed, only 15 – 24% of patients are likely to respond. To date, there are no validated biomarkers, including PDL1 expression, which might accurately identify patients who are likely to respond. Many different biomarkers are currently under active investigation. Future direction for therapeutic development is likely to involve combination therapies with PD1/PDL1 agent as the therapeutic backbone.

Abstract A058: Simultaneous characterization of rare immune cell subpopulations and PD-L1-expressing CTCs in peripheral blood of cancer patients

Abstract Background: Expression of PD-L1 on tumor and immune markers in tumor tissueis associated with improved response to PD-1 and PD-L1 checkpoint inhibitors. However, the PD-L1 biomarker has limited predictive utility. Multimodal characterization of both tumor and host immune system is an unmet medical need for the improved prediction of immuno-oncology therapy. Given tumor heterogeneity and evolution and temporal changes to the host immune system, metastatic lesions are likely to be undersampled and require a liquid biopsy. We sought to examine the expression of PD-L1 on circulating tumor cells (CTCs) as well as characterize rare immune cell populations with a noninvasive liquid biopsy. Examining dynamic biomarker changes in longitudinal samples could enable the development of novel diagnostic tools for response prediction and pharmacodynamics studies related to immunotherapy. Methods: Blood samples from cancer patients were received and shipped to Epic Sciences. Contrived blood samples were also developed utilizing spike-in of cancer cell line controls into healthy blood samples. RBCs were lysed, and nucleated cells plated onto glass slides utilizing the Epic Sciences process. Slides were stained for nucleus DAPI in addition to IF cocktails and scanned, with targets including CK, CD45, PD-L1, CD4, CD8, Ki-67, LAG-3, and TIM-3. Approximately 3 million nucleated cells were examined through advanced Digital Pathology pipelines to detect and quantify the changes in T-cell populations to infer immune activation, exhaustion, suppression, and circulating tumor burden. Results: Epic’s rare cell detection platform has an analytically validated limit of detection of 1 cell/mL of blood. Three immuno-panels were developed to profile leukocytes subpopulations and CTCs simultaneously. 30 out of 33 lung cancer patients had PD-L1 leukocytes detected, and the incidences of PD-L1+ leukocytes widely ranged from 0 to 0.138% (average 0.0213%, median 0.0118%). PD-L1+ CTCs were observed in the presence of both high and low counts of PD-L1+ leukocyte populations. Conclusions: Epic Sciences CTC platform’s low limit of detection coupled with ability to archive patient blood samples allowed precise quantification of leucocyte subpopulations (CD4 and CD8, etc.) and PD-L1 expression on CTCs retrospectively. Development of a liquid biopsy-based platform that is capable of simultaneously measuring immune biomarkers in CTCs as well as leucocytes will allow real-time assessment and monitoring of response to immune checkpoints inhibitors. Citation Format: Adam Jendrisak, Angel Rodriguez, Nadia Ebrahim, Jiyun Byun, Ryon Graf, Yipeng Wang, Mark Landers, Ryan Dittamore. Simultaneous characterization of rare immune cell subpopulations and PD-L1-expressing CTCs in peripheral blood of cancer patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A058.