Mark Bittinger

Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens.Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Deng et al., p. 216This article is highlighted in the In This Issue feature, p. 127.

Cytotoxic T Cells in PD-L1–Positive Malignant Pleural Mesotheliomas Are Counterbalanced by Distinct Immunosuppressive Factors

In malignant pleural mesothelioma, immunohistochemical expression of PD-L1 does not accurately predict whether patients respond to treatment with PD-1 pathway inhibitors. Comprehensive immunoprofiling by flow cytometry uncovered immunophenotypes that improve our understanding of response and resistance to checkpoint blockade. PD-L1 immunohistochemical staining does not always predict whether a cancer will respond to treatment with PD-1 inhibitors. We sought to characterize immune cell infiltrates and the expression of T-cell inhibitor markers in PD-L1–positive and PD-L1–negative malignant pleural mesothelioma samples. We developed a method for immune cell phenotyping using flow cytometry on solid tumors that have been dissociated into single-cell suspensions and applied this technique to analyze 43 resected malignant pleural mesothelioma specimens. Compared with PD-L1–negative tumors, PD-L1–positive tumors had significantly more infiltrating CD45+ immune cells, a significantly higher proportion of infiltrating CD3+ T cells, and a significantly higher percentage of CD3+ cells displaying the activated HLA-DR+/CD38+ phenotype. PD-L1–positive tumors also had a significantly higher proportion of proliferating CD8+ T cells, a higher fraction of FOXP3+/CD4+ Tregs, and increased expression of PD-1 and TIM-3 on CD4+ and CD8+ T cells. Double-positive PD-1+/TIM-3+ CD8+ T cells were more commonly found on PD-L1–positive tumors. Compared with epithelioid tumors, sarcomatoid and biphasic mesothelioma samples were significantly more likely to be PD-L1 positive and showed more infiltration with CD3+ T cells and PD-1+/TIM-3+ CD8+ T cells. Immunologic phenotypes in mesothelioma differ based on PD-L1 status and histologic subtype. Successful incorporation of comprehensive immune profiling by flow cytometry into prospective clinical trials could refine our ability to predict which patients will respond to specific immune checkpoint blockade strategies. Cancer Immunol Res; 4(12); 1038–48. ©2016 AACR.

Fine needle aspirate flow cytometric phenotyping characterizes immunosuppressive nature of the mesothelioma microenvironment

With the emergence of checkpoint blockade and other immunotherapeutic drugs, and the growing adoption of smaller, more flexible adaptive clinical trial designs, there is an unmet need to develop diagnostics that can rapidly immunophenotype patient tumors. The ability to longitudinally profile the tumor immune infiltrate in response to immunotherapy also presents a window of opportunity to illuminate mechanisms of resistance. We have developed a fine needle aspirate biopsy (FNA) platform to perform immune profiling on thoracic malignancies. Matching peripheral blood, bulk resected tumor, and FNA were analyzed from 13 mesothelioma patients. FNA samples yielded greater numbers of viable cells when compared to core needle biopsies. Cell numbers were adequate to perform flow cytometric analyses on T cell lineage, T cell activation and inhibitory receptor expression, and myeloid immunosuppressive checkpoint markers. FNA samples were representative of the tumor as a whole as assessed by head-to-head comparison to single cell suspensions of dissociated whole tumor. Parallel analysis of matched patient blood enabled us to establish quality assurance criteria to determine the accuracy of FNA procedures to sample tumor tissue. FNA biopsies provide a diagnostic to rapidly phenotype the tumor immune microenvironment that may be of great relevance to clinical trials.

Defining an inflamed tumor immunophenotype in recurrent, metastatic squamous cell carcinoma of the head and neck

OBJECTIVES Immune checkpoint inhibitors have demonstrated clinical benefit in recurrent, metastatic (R/M) squamous cell carcinoma of the head and neck (SSCHN), but lacking are biomarkers that predict response. We sought to define an inflamed tumor immunophenotype in this R/M SCCHN population and correlate immune metrics with clinical parameters and survival. METHODS Tumor samples were prospectively acquired from 34 patients to perform multiparametric flow cytometry and multidimensional clustering analysis integrated with next-generation sequencing data, clinical parameters and outcomes. RESULTS We identified an inflamed subgroup of tumors with prominent CD8+ T cell infiltrates and high PD-1/TIM3 co-expression independent of clinical variables, with improved survival compared with a non-inflamed subgroup (median overall survival 84.0 vs. 13.0months, p=0.004). The non-inflamed subgroup demonstrated low CD8+ T cells, low PD-1/TIM3 co-expression, and higher Tregs. Overall non-synonymous mutational burden did not correlate with response to PD-1 blockade in a subset of patients. CONCLUSION R/M SCCHN patients with an inflamed tumor immunophenotype demonstrate improved survival. Further prospective studies are needed to validate these findings and explore the use of immunophenotype to guide patient selection for immunotherapeutic approaches.

Abstract B27: Improved survival with erdafitinib (JNJ-42756493) and PD-1 blockade mediated by enhancement of anti-tumor immunity in an FGFR2-driven genetically engineered mouse model of lung cancer

Targeted therapies against activated oncogenes, such as receptor tyrosine kinases, have significantly prolonged non-small cell lung cancer (NSCLC) patient survival, but the development of resistance limits the durability of clinical response. Genetic alterations which constitutively activate Fibroblast Growth Factor Receptors (FGFR) have been observed in patients with NSCLC. Erdafitinib (JNJ-42756493), an orally bioavailable pan-FGFR inhibitor discovered as part of a collaboration between Janssen and Astex Pharmaceuticals, has been shown to inhibit FGFR signaling pathways resulting in cell death and tumor growth inhibition in both in vitro and in vivo models of FGFR pathway aberration. Further, erdafitinib has been shown to have favorable pharmaceutical properties with manageable side effects in humans and several clinical trials are currently underway. One potential strategy to enhance the durability of response to targeted therapies, such as FGFR inhibitors, is to couple them with immunotherapy. In this setting, T cell responses primed and activated by increased antigen release resulting from the tumor cell targeted therapy could be enhanced and maintained by T-cell directed checkpoint blockade. To test this hypothesis, we evaluated erdafitinib in combination with an anti-programmed death-1 (PD-1) blocking antibody in an autochthonous FGFR2K660N/p53 genetically engineered mouse model (GEMM) of lung cancer, in which tumors develop within the context of an intact immune microenvironment. Cohorts of tumor bearing FGFR2K660N/p53 mutant mice treated with erdafitinib with or without anti-PD-1 showed significant tumor regressions compared to control and anti-PD-1 alone groups. Despite lack of differences in acute tumor responses between erdafitinib monotherapy and combination therapy, we observed significant survival benefit in the combination group erdafitinib alone (median survival 19.7 weeks vs 13.4 weeks, p Citation Format: Sangeetha Palakurthi, Mari Kuraguchi, Sima Zacharek, Jeff Liu, Dennis Bonal, Wei Huang, Kristin Depeaux, Abha Dhaneshwar, Sam Regan, Dyane Bailey, Martha Gowaski, Mei Zheng, Roderick Bronson, Catherine Ferrante, Enrique Zudaire, Sylvie Laquerre, Mark Bittinger, Kirschmeier Paul, Kathryn Packman, Raluca I. Verona, Kwok-Kin Wong, Matthew V. Lorenzi. Improved survival with erdafitinib (JNJ-42756493) and PD-1 blockade mediated by enhancement of anti-tumor immunity in an FGFR2-driven genetically engineered mouse model of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B27.

Abstract A132: Multi-parametric profiling of non-small cell lung cancers reveals distinct immunophenotypes

Immune checkpoint blockade improves survival in a subset of patients with non-small cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited. To develop a more comprehensive knowledge of the NSCLC tumor immune landscape, we performed comprehensive flow-cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC). Cytometric profiling identified two main tumor immunophenotypes: an immunologically “hot” cluster with high abundance of CD8+ T cells expressing high levels of the inhibitory receptors PD-1 and TIM-3, and an immunologically “cold” cluster with lower relative abundance of CD8+ T cells and expression of inhibitory markers. The “hot” cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function by mRNA analysis, and high PD-L1 expression by IHC. Within the “hot” cluster there was a small subgroup with high granulocytic infiltrates and lower relative T cell abundance, but with high expression of inhibitory receptors by T cells. There was no correlation between immunophenotype and KRAS or EGFR mutation status, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the “hot” cluster. Additionally, ∼20% of cases had high B cell infiltrates and we demonstrate that a subset of these cells display characteristics of immunosuppressive IL-10-producing cells. Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer. Citation Format: Patrick Lizotte, Elena Ivanova, Mark Bittinger, Kwok-Kin Wong. Multi-parametric profiling of non-small cell lung cancers reveals distinct immunophenotypes [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A132.

Abstract B99: Immune profiling of NSCLC tumors and matching normal lung samples by multicolor flow cytometry

With the recent advancements in cancer immunotherapy, understanding of tumor microenvironment becomes crucial to progressing oncology research. Standard method for tumor immune characterization today is traditional IHC technique with 1-2 color staining which presents certain limitations. In order to simultaneously identify immune infiltrate cellular components and parallel it with the analysis of expression of activation/suppression markers in various subpopulations, we developed a multicolor flow cytometry pipeline for freshly resected tumors. Thirty one individual biomarkers were used in several antibody staining panels with suffficientdesigned redundancy to allow crossover and address intra-experiment variability. Aliquots of the samples were frozen and stained on separate days to test for inter-assay variability. We performed flow cytometry analysis of 23 NSCLC tumors (17 adenocarcinoma, 1 adenosquamous and 5 squamous carcinoma subtypes) and matcheding normal lung tissue. Unsupervised clustering ofby 70 parameters, including percentages of cellular subtypes as well as modulator marker expression, showed distinct segregation of normal and tumor tissue samples. The immune infiltrate in resected tumors exhibited significantly decreased CD66b+ granulocytes and CD56+ NK cells (especially cytotoxic CD56+CD16+ NK cells) and highly increased CD3+ T and CD19+ B cells when compared to flow analysis of normal lung tissue. Adeno and squamous carcinomas were not segregated into separate groups by clustering analysis but rather joined together to form 3 subgroups defined by exhaustion/activation marker expression. Grouping the tumor samples by the expression of the clinically relevant PD-L1 marker in immune cells indicated that “high PD-L1” tumors tend to have highly activated T cells populations. This finding correlates with the latest views on high PD-1/PD-L1 expressing immune cells being “negatively regulated” rather than “exhausted” as the result of their extensive interaction with cancer cells. As we continue to characterize the immune profile of NSCLC tumors and paired normal lung, in conjunction with genetic and clinical information, we aim to further understanding molecular and clinical correlates that influence the tumor microenvironment and to ultimately of the mechanisms of immune response to overcome both innate and acquired resistance to immune therapy agents. Citation Format: Elena Ivanova, Robert E. Jones, Mark M. Awad, Mark A. Bittinger, Meghana M. Kulkarni, Shohei Koyama, Christina G. Almonte, Abigail A. Santos, Jessie M. English, Julianne Barlow, William G. Richards, Peter S. Hammerman, Scott J. Rodig, Raphael Bueno, Kwok-Kin Wong. Immune profiling of NSCLC tumors and matching normal lung samples by multicolor flow cytometry. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B99.

Abstract A69: An epigenetic-focused CRISPR/Cas9 screen to identify regulators of IFNγ-induced PD-L1 expression

PD-L1 (B7-H1, CD274) is a clinically validated immuno-oncology target, which is often over-expressed on the surface of tumor cells. PD-L1 binds to PD-1 expressed on T cells generating an immunosuppressive signaling response that limits T cell activation and facilitates immune evasion. The tumor microenvironment often recruits immune cells that produce a number of secreted factors, including IFNγ, a potent inducer of PD-L1 expression on tumor cells. Blocking IFNγ-induced PD-L1 expression with small molecules could be a potential alternative to antibody-based PD-L1/PD-1 blockade. Recent studies on human patient samples indicate that the level of PD-L1 expression on tumor cells is inversely related to the level of DNA methylation at the PD-L1 promoter (Gettinger et al, 2015), suggesting that PD-L1 expression is epigenetically silenced in tumor cells with low PD-L1 expression. Therefore, cytokines that induce PD-L1 expression on tumor cells, such as IFNγ, may regulate the activity of epigenetic silencing factors at the PD-L1 promoter, and identification of these epigenetic factors could provide novel therapeutic targets to block PD-L1 expression on tumor cells. CRISPR/Cas9 gene editing has recently emerged as a powerful technology for phenotypic screening. To identify potential epigenetic regulators of IFNγ-induced PD-L1 on tumor cells, several murine tumor cell lines were treated with IFNγ and PD-L1 expression was monitored by flow cytometry. The ovarian cancer cell line ID8 demonstrated high PD-L1 expression following IFNγ stimulation, and stable Cas9 expressing clones were generated. A high expressing Cas9 clone was selected for follow-up transduction with a sgRNA library targeting >350 known epigenetic factors, plus numerous positive and negative controls. Library transduced cells were evaluated in two separate screening streams: i) an enrichment screen to identify genes regulating IFNγ-induced PD-L1 expression, and ii) a depletion screen to identify genes essential for the growth of ID8 tumor cells. For the enrichment screen, sgRNA transduced cells were treated with IFNγ, and FACS was performed to collect cells with low PD-L1 expression i.e. cells refractory to IFNγ-induced PD-L1 expression. Genomic DNA was then isolated from the sorted cells and sgRNA sequences were quantified by next-generation sequencing (NGS). As an important validation of the FACS-based screening format, the most highly enriched sgRNAs in the low PD-L1 population were PD-L1 itself, and the canonical mediators of IFNγ signaling JAK1/2 and STAT1. For the depletion screen, sgRNA transduced cells were cultured for up to 14 days, with cell pellets collected on day 0, 3, 7 and 14 for NGS quantification of sgRNAs. All positive and negative controls scored as expected and several epigenetic factors were strongly depleted indicating an essential role for ID8 cell growth. In summary, CRISPR/Cas9 gene editing is a powerful screening technology for the identification of factors essential for cell growth, and when paired with FACS, is a useful methodology to identify factors regulating expression of immune checkpoint molecules. Gettinger et al, Journal of Clinical Oncology , 2015 ASCO Annual Meeting, Vol 33, No 15 (May 20 supplement), 2015: 3015 Citation Format: Troy A. Luster, Meghana Kulkarni, Erica Fitzpatrick, Lauren Badalucco, Jessie English, Kwok-Kin Wong, Mark Bittinger. An epigenetic-focused CRISPR/Cas9 screen to identify regulators of IFNγ-induced PD-L1 expression. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A69.

Abstract 5436: Targeting oncoproteins via disruption of proteostasis: Identification of oncoprotein destabilizing agents using luciferase tagged oncoproteins

Since many oncoproteins are considered undruggable targets, we have developed and validated a new screening strategy to identify small molecules that inhibit oncoprotein activity by causing their degradation. Recently, the mechanism of action for the class of drugs known as immunomodulators (IMiDs; e.g. thalidomide, lenalidomide and pomalidomide) has uncovered a novel oncogene targeting strategy. IMiDs commandeer an E3 ligase complex that contains the E3 recognition subunit cereblon and divert its activity toward two transcription factors IKZF1 and IKZF3, which play critical roles in hematopoietic cells. This mechanism explains the efficacy of IMiDs in multiple myeloma and may translate into a general approach to target traditionally undruggable oncoproteins. We generated cell lines stably expressing IKZF1as a firefly luciferase fusion protein with coexpression of Renilla luciferase. A pilot HTS campaign was run to uncover novel drugs that can mimic the effects of IMiDs. A cell line expressing the MYC-firefly fusion protein was used for counter-screening. We used pomalidomide as our positive control and it reduced firefly luciferase signal ≈75% with no change in the renilla luciferase signals. This control resulted in a Z prime value typically >0.5. The pilot screen of 3,850 compounds resulted in 39 “hits” that reduced firefly luciferase >35%. More importantly, both thalidomide and lenalidomide were found to be active in the screen. Confirmation testing revealed that 75% of “hits” repeated, but also had activity in the MYC-firefly cell line. Actives were tested in western blots for their ability to decrease fusion protein abundance. Many active compounds did not reduce protein levels in western blots and represent nonspecific hits (e.g. luciferase inhibitor). In conclusion, we present a screening paradigm with the potential to uncover novel proteostasis disruptors by expressing luciferase tagged oncoproteins. Citation Format: Richard E. Middleton, Greg Olsen, Russell McSweeney, Gang Lu, Wenhua Gao, Justin Roberts, Michael R. McKeown, Mark A. Bittinger, Kwok-Kin Wong, James E. Bradner, William G. Kaelin. Targeting oncoproteins via disruption of proteostasis: Identification of oncoprotein destabilizing agents using luciferase tagged oncoproteins. [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 5436. doi:10.1158/1538-7445.AM2015-5436