Matthew Ren Silver

Analysis of Receptor Tyrosine Kinase ROS1-Positive Tumors in Non–Small Cell Lung Cancer: Identification of a FIG-ROS1 Fusion

Purpose: To deepen our understanding of mutant ROS1 expression, localization, and frequency in non–small cell lung cancer (NSCLC), we developed a highly specific and sensitive immunohistochemistry (IHC)-based assay that is useful for the detection of wild-type and mutant ROS1. Experimental Design: We analyzed 556 tumors with the ROS1 D4D6 rabbit monoclonal antibody IHC assay to assess ROS1 expression levels and localization. A subset of tumors was analyzed by FISH to determine the percentage of these tumors harboring ROS1 translocations. Using specific and sensitive IHC assays, we analyzed the expression of anaplastic lymphoma kinase (ALK), EGFR L858R, and EGFR E746-A750del mutations in a subset of lung tumors, including those expressing ROS1. Results: In our NSCLC cohort of Chinese patients, we identified 9 (1.6%) tumors expressing ROS1 and 22 (4.0%) tumors expressing ALK. FISH identified tumors with ALK or ROS1 rearrangements, and IHC alone was capable of detecting all cases with ALK and ROS1 rearrangements. ROS1 fusion partners were determined by reverse transcriptase PCR identifying CD74-ROS1, SLC34A2-ROS1, and FIG-ROS1 fusions. Some of the ALK and ROS1 rearranged tumors may also harbor coexisting EGFR mutations. Conclusions: NSCLC tumors with ROS1 rearrangements are uncommon in the Chinese population and represent a distinct entity of carcinomas. The ROS1 IHC assay described here is a valuable tool for identifying patients expressing mutant ROS1 and could be routinely applied in clinical practice to detect lung cancers that may be responsive to targeted therapies. Clin Cancer Res; 18(16); 4449–57. ©2012 AACR.

PD-L1 Expression Correlates with Tumor-Infiltrating Lymphocytes and Response to Neoadjuvant Chemotherapy in Breast Cancer.

Wimberly and colleagues analyzed pretreatment biopsies and outcomes from 105 breast cancer patients; they report the association of PD-L1 expression with hormone receptor–negative and triple-negative status and pathologic complete responseand suggest that PD-L1 expression is a biomarker in this treatment cohort. Programmed death 1 ligand 1 (PD-L1) is an immune regulatory molecule that limits antitumor immune activity. Targeting of PD-L1 and other immune checkpoint proteins has shown therapeutic activity in various tumor types. The expression of PD-L1 and its correlation with response to neoadjuvant chemotherapy in breast cancer has not been studied extensively. Our goal was to assess PD-L1 expression in a cohort of breast cancer patients treated with neoadjuvant chemotherapy. Pretreatment biopsies from 105 patients with breast cancer from Yale New Haven Hospital that subsequently received neoadjuvant chemotherapy were assessed for PD-L1 protein expression by automated quantitative analysis with a rabbit monoclonal antibody (E1L3N) to the cytoplasmic domain of PD-L1. In addition, tumor-infiltrating lymphocytes (TIL) were assessed on hematoxylin and eosin slides. PD-L1 expression was observed in 30% of patients, and it was positively associated with hormone-receptor–negative and triple-negative status and high levels of TILs. Both TILs and PD-L1 measured in the epithelium or stroma predicted pathologic complete response (pCR) to neoadjuvant chemotherapy in univariate and multivariate analyses. However, because they are strongly associated, TILs and PD-L1 cannot both be included in a significant multivariate model. PD-L1 expression is prevalent in breast cancer, particularly hormone-receptor–negative and triple-negative patients, indicating a subset of patients that may benefit from immune therapy. Furthermore, PD-L1 and TILs correlate with pCR, and high PD-L1 predicts pCR in multivariate analysis. Cancer Immunol Res; 3(4); 326–32. ©2014 AACR.

IL-33 synergizes with IgE-dependent and IgE-independent agents to promote mast cell and basophil activation

ObjectiveMast cell and basophil activation contributes to inflammation, bronchoconstriction, and airway hyperresponsiveness in asthma. Because IL-33 expression is inflammation inducible, we investigated IL-33-mediated effects in concert with both IgE-mediated and IgE-independent stimulation.MethodsBecause the HMC-1 mast cell line can be activated by GPCR and RTK signaling, we studied the effects of IL-33 on these pathways. The IL-33- and SCF-stimulated HMC-1 cells were co-cultured with human lung fibroblasts and airway smooth muscle cells in a collagen gel contraction assay. IL-33 effects on IgE-mediated activation were studied in primary mast cells and basophils.ResultIL-33 synergized with adenosine, C5a, SCF, and NGF receptor activation. IL-33-stimulated and SCF-stimulated HMC-1 cells demonstrated enhanced collagen gel contraction when cultured with fibroblasts or smooth muscle cells. IL-33 also synergized with IgE receptor activation of primary human mast cells and basophils.ConclusionIL-33 amplifies inflammation in both IgE-independent and IgE-dependent responses.

Abstract 1310: Programmed death ligand-1 (PD-L1) heterogeneity in non-small cell lung cancer (NSCLC)

Early phase I trials with monoclonal antibodies targeting PD-1/PD-L1 have demonstrated durable clinical responses in patients with NSCLC. However, the prognostic/predictive role of tumor PD-L1 expression has not been determined. Current assays for evaluation of PD-L1 protein expression are not standardized. Here, we demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF), and compare results obtained using three PD-L1 antibodies targeting the intracellular (IC) and extracellular (EC) domains. We measured PD-L1 protein expression using two antibodies against the IC domain (E1L3N [Cell Signaling Technology] and SP142 [Spring Biosciences Inc.]) and one antibody binding the EC domain (E1J2J [Cell Signaling Technology]) in 49 NSCLC whole tissue sections and a TMA with the same 49 cases. Mel624 cells stably transfected with PD-L1, as well as Mel624 parental cells and human term placenta were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA® method of QIF. For IHC, PD-L1 positivity was determined using previously reported cut-points for tumor (1%, 5%, and 50%) and stroma (5%). IHC inter-assay concordance was evaluated using kappa coefficient. AQUA scores were measured in an average of 28.6 fields of view per case or by TMA at two-fold redundancy. Linear regression coefficients (R2) were used to compare antibody QIF scores. Tumor-infiltrating lymphocytes were scored in hematoxylin/eosin stained slides using current consensus guidelines. Chromogenic IHC assays showed fair to poor concordance (kappa 0.124 - 0.552) for all cut-points. QIF showed PD-L1 immunopositivity was heterogeneous for all three PD-L1 antibodies. R2 values were lower when E1J2J (EC) was compared to E1L3N and SP142 (IC) (R2 = 0.090 and 0.079), while coefficients were higher when the IC antibodies were compared (R2 = 0.658). E1L3N and SP142 significantly correlated with high TILs (p = 0.007 and p = 0.021) but E1J2J did not (p = 0.281). E1J2J significantly correlated with older age (p = 0.038), but no other clinicopathological feature. SP142 significantly correlated with lymph node positive status (p = 0.030) Objective determination of PD-L1 protein levels in NSCLC reveals significant heterogeneity within tumors. There is significant inter-assay variability, even between assays detecting the same protein domain. This could be due to different antibody affinities, cross reactivity, or distinct target epitopes. Efforts to determine the clinical value of these observations are underway. Citation Format: Joseph McLaughlin, Kurt A. Schalper, Daniel E. Carvajal-Hausdorf, Vasiliki Pelekanou, Vamsidhar Velcheti, Herbert Haack, Matthew R. Silver, Roy Herbst, Patricia LoRusso, David L. Rimm. Programmed death ligand-1 (PD-L1) heterogeneity in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1310. doi:10.1158/1538-7445.AM2015-1310

Abstract A006: Analysis of immune checkpoint control protein co-expression in breast and ovarian cancer using novel rabbit monoclonal antibodies and multiplex IHC

With an increasing number of biomarkers, and often limited availability of biopsy material, there is a growing need for multiplexed assays for both research and clinical purposes. IHC based solutions are particularly attractive in the field of immuno-oncology, as maintaining special context within the tumor microenvironment provides meaningful, and potentially actionable, information. Immuno-assays with high specificity and sensitivity are a powerful tool; however, there are challenges associated with antibody based multiplexing when more than 2-3 markers are required. A multiplex IHC solution that bypasses antibody species/isotype concerns, while providing signal amplification, was used to examine the co-expression of immune checkpoint control proteins, such as PD-L1, B7-H3, B7-H4, VISTA, in breast and ovarian cancer. Citation Format: Matthew R. Silver, Jennifer E. Ziello, Herbert Haack. Analysis of immune checkpoint control protein co-expression in breast and ovarian cancer using novel rabbit monoclonal antibodies and multiplex IHC. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A006.

Abstract 4595: Development and validation of a novel EGF receptor-neutralizing monoclonal antibody

The epidermal growth factor receptor (EGFR) is a cell-surface receptor for members of the epidermal growth factor family (EGF-family). EGFR dimerizes upon ligand binding, which leads to autophosphorylation, downstream signaling and ultimately internalization. In cancer, EGFR mutations and amplification have been linked to cell proliferation and survival. Two EGFR neutralizing monoclonal antibodies are currently FDA approved, which includes Cetuximab (Erbitux, BMS; mouse/human chimeric) and Panitumumab (ABX-EGF, Amgen; fully humanized). However, these antibodies are often unavailable to researchers studying this important pathway. Here we describe the development and validation of a new rabbit monoclonal EGFR neutralizing antibody (clone D1D4J). D1D4J was validated using cell-based models and immunofluorescence imaging. D1D4J binds EGFR and neutralizes EGF-induced activation and internalization. Compared to other Research Use Only (RUO) neutralizing antibodies on the market, D1D4J shows superior performance and is a useful research tool in the EGFR signaling space. Citation Format: Krystyna Zuberek Hincman, Christopher A. Manning, Michael R. Nelson, Michael Lewis, Roy Scialdone, Jaime Darce, Stephen R. Lutz, Evans Burford, Herbert Haack, Matthew R. Silver. Development and validation of a novel EGF receptor-neutralizing monoclonal antibody. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4595.

Dissecting the tumor micro-environment in triple negative breast cancer identifies a mutually exclusive expression pattern of the immune co-inhibitory molecules B7-H4 and PD-L1

B7-H4 is a member of the B7 family of co-regulatory receptors. It is believed to negatively regulate T cell function and has been associated with poor prognosis in renal cell and ovarian cancers. We performed an unbiased analysis of TCGA gene expression data and identified triple negative breast cancer (TNBC) as having the greatest absolute B7-H4 mRNA level of all tumors analyzed. Recent clinical studies with anti-PD-1 or PD-L1 therapies have reported promising activity in TNBC leading us to investigate check point molecule expression (PD-1, PD-L1 and B7-H4) as well as expression of immune cell infiltration (CD8, Fox P3) in archival samples from a cohort of 96 TNBC patients collected at Yale University. We developed a specific and sensitive immunohistochemistry (IHC) assay for evaluating B7-H4 protein and used an immunofluorescence-based multiplex IHC for assessing combinations of checkpoint molecules in the TNBC samples. The majority of tumors had detectable B7-H4 expression, whereas PD-L1 expression was restricted to a subset of TNBC patients (~20% having >5% PD-L1 positive cells). Multiplex IHC and flow cytometry studies showed that the majority of B7-H4 expression was restricted to the tumor epithelial cells, while the CD45+ immune cells were negative for B7-H4 expression. Interestingly, a majority of the B7-H4 high tumors were negative or showed scant PD-L1 staining. In addition, cells that are B7-H4 positive are negative for PD-L1 staining, suggesting that B7-H4 and PD-L1 checkpoint proteins may act in a mutually exclusive manner. B7-H4 expression was not associated with overall survival, disease stage, nodal status, or other clinical characteristics. In contrast, PD-L1, PD-1, and CD8 expression all conferred a significant survival advantage in TNBC, thus highlighting the importance of the immune response in this disease. Upon further investigation, and contrary to published literature, we were unable to show a definitive immunosuppressive role of B7-H4. However B7-H4 over-expression in CT-26 syngeneic in vivo model accelerated tumor growth. The unique expression pattern of B7-H4 on TNBC suggests an opportunity for targeted approaches with possible immunomodulatory activity. Additional work is needed to further clarify the immunological mechanisms of B7-H4, but we believe its unique expression pattern makes B7-H4 an attractive target for the treatment of TNBC.