Standard Of Care Chemotherapy Research Articles

Abstract A05: Proteomic analysis of exosome content changes induced by gemcitabine in pancreatic cancer stromal cells

Abstract Despite recent advances in multimodality therapy, pancreatic ductal adenocarcinoma (PDAC) remains the 3rd leading cause of cancer-related deaths in the United States with a 5-year survival rate of 8%. One hallmark of this disease is desmoplasia that consist largely of stromal cells and extracellular matrix (ECM) proteins. The abundant stromal cells provide a unique tumor microenvironment that aid tumorigenesis. Unfortunately, there is little data on the mechanism underlying the stromal cells’ ability to impact chemoresistance in neighboring cancer cells in response to drug treatment. Exosomes are small vesicles secreted into the extracellular environment from cells and evidence is accumulating that exosomes play a key role in intercellular signaling. Researchers have focused on exosomes secreted from cancer cells, but the function of stromal exosomes has not been investigated, especially in pancreatic cancer. As stromal cells comprise the majority of the pancreatic tumor bulk, we hypothesize that stromal cell-derived exosomes help pancreatic cancer cells resist chemotherapy. Thus, it is critical to characterize the complex composition of stromal exosomes. In this study, we analyzed the proteomic content of exosomes collected from stromal cell lines created from PDAC patients. Furthermore, we also determined the change in exosomal proteomic composition after the patient-derived stromal cells were treated with gemcitabine, the current standard of care chemotherapy for patients with PDAC. Our studies have identified possible diagnostic and prognostic biomarkers and suggest novel molecular mechanism responsible for the tumor microenvironment’s ability to regulate chemoresistance in neighboring pancreatic cancer cells. Citation Format: Wei Huang, Peter Feist, Amanda Hummon, Reginald Hill.{Authors}. Proteomic analysis of exosome content changes induced by gemcitabine in pancreatic cancer stromal cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A05.

Abstract A114: Effect of different standard of care chemotherapeutics on anti-PD-L1 responses in syngeneic mouse tumor models

Abstract Immunotherapy with checkpoint inhibitors has proven to be highly effective, with durable responses in a subset of patients. In an effort to broaden the number of responding individuals, overcome resistance to single-agent therapy and extend the duration of responses, combination of immunotherapy with other treatments such as chemotherapy is currently being tested in multiple clinical trials. Importantly, combination of immunotherapy with standard of care chemotherapy could bring the benefits of immunotherapy into earlier lines of treatment. Here, we have evaluated the effects of different classes of chemotherapeutic agents alone or in combination with anti-PD-L1 treatment in syngeneic murine tumor models. We have found that combination of anti-PD-L1 with various alkylating agents, taxanes and platins caused differential changes on the frequency and number of intratumoral immune cell subsets, but did not antagonize the functional changes mediated by anti-PD-L1 treatment. Importantly, by comparing the effects of these chemotherapies in different tumor models, we have observed that their effects are model-specific. These differences may be due to tumor-specific immune composition and tumor intrinsic responses to different chemotherapies. Our results demonstrate that in the models we have tested, chemotherapeutic agents do not appear to directly antagonize the activity of anti-PD-L1 as measured by pharmacodynamic changes in immune cell activation although some differences can be observed in the frequency and number of certain intratumoral immune cell subsets. Additionally, our findings also reveal that tumor models might respond differently to the same chemotherapeutic treatment adding complexity to how we evaluate the effect of combination treatments with chemotherapy. Nonetheless, our results suggest a rationale for combining immunotherapy with chemotherapy in the clinic, although consideration should be taken when generalizing results from mouse tumor models. Citation Format: Rafael Cubas, Marina Moskalenko, Jeanne Cheung, Shiuh-Ming Luoh, Erin McNamara, Marcia Belvin, Jeong Kim, Stephen Gould. Effect of different standard of care chemotherapeutics on anti-PD-L1 responses in syngeneic mouse tumor models [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 A114.

Abstract IA02: Selection of cancer antigens and conditions for success of immunotherapy of cancer involving vaccination

Abstract Cancer antigens of choice include mutant DNA sequences encoding neo-antigens, selected cancer testis antigens, and differentiation antigens, in addition to viral antigens. Neo-antigens and viral antigens are the most attractive, because the T cell repertoire against these antigens has not been blunted by central thymic tolerance. Optimal vaccine platforms include DNA and RNA vaccines as well as synthetic long peptides and selected prime-boost protocols involving recombinant viruses encoding cancer antigens. The most effective cancer vaccines deliver concentrated antigen to both HLA class I and II molecules of DCs, promoting both CD4 and CD8 T cell responses. Suboptimal antigen selection, suboptimal vaccine design, and an immunosuppressive cancer microenvironment are the root causes of the lack of clinical efficacy of cancer vaccines in many clinical trials. Drugs or physical treatments can mitigate the immunosuppressive cancer microenvironment and include chemotherapeutics, radiation, inhibitors of T cell checkpoints, indoleamine 2,3-dioxygenase (IDO) inhibitors, agonists of selected TNF receptor family members, and inhibitors of undesirable cytokines. The specificity of therapeutic vaccination combined with such immunomodulation offers an attractive avenue for the development of future cancer therapies. Clinical benefit of certain therapeutic cancer vaccines has been established, usually by noting prolonged progression free or overall survival. Reviews of the properties and prospects of cancer vaccines have been listed below (1-3). The addition of one or more immunomodulation strategies may further enhance the potential of therapeutic cancer vaccines, including of those that have been only marginally active due to the immunosuppressive cancer microenvironment. We have established clinical benefit of a cancer vaccine as monotherapy in patients with premalignant disease induced by high risk human papilloma virus. Regression of lesions was associated with a better vaccine-induced immune response against the target antigens of the vaccine. However in patients with cervical cancer the vaccine monotherapy failed to install clinically effective immunity. Recently we showed in a pilot trial that vaccine-induced immunity could be markedly enhanced by combination with standard of care chemotherapy for late stage recurrent or metastatic cervical cancer. Increased levels of myeloid-derived suppressor cells were shown to be depleted by the carboplatin and paclitaxel chemotherapy to the low levels of healthy donors, while T cell numbers in PBMC remained intact. Timed vaccination with a single dose of vaccine at the MDSC nadir during chemotherapy led to a robust vaccine-induced T cell response (4). In a subsequent chemo-immunotherapy trial with three vaccine doses, spaced by three weeks, and in cohorts of patients at four different vaccine doses, the initiation and maintenance of robust tumor-specific T cell responses was confirmed and a broad dose response relationship of tumor-specific T cell responses was noted. Preliminary data on clinical outcomes relative to HPV-specific immune responses will be discussed. In summary, therapeutic benefit of cancer vaccines can be maximized in patients with established cancers by improving vaccine design and by using vaccines in combination with standard chemotherapies. In addition cancer vaccines can establish and/or maintain tumor-specific T cells in combination with checkpoint blockade or other immunomodulatory therapies. Finally, cancer vaccines can also help to sustain and expand adoptively transferred tumor-specific T cells.

S-1: changing the facets of adjuvant chemotherapy in pancreatic cancer?

From an oncologists view the treatment progress in curatively resected patients for pancreatic cancer (PDAC) has been frustrating so far. Although, the implementations of new standard of care chemotherapies in metastatic PDAC such as FOLFIRINOX (1) or Gemcitabine/Nab-Paclictaxel were a great advancement (2), adjuvant treatment efforts remained static. The use of adjuvant therapy is recommended by several medical societies (3-5) and is based on numerous trials: the CONKO-001 (6), ESPAC-1 (7) and ESPAC-3 (8) trials defined to start gemcitabine or infusional 5-FU monotherapy as the standard of care within 12 weeks post-operative, only differing in their respective toxicity profiles. These regimens have been a breakthrough at the time. However, novel and more tailored treatment algorithms are warranted to eventually reach a high response rate in only a subset of PDAC patients as “ one pill fits all ” seems to be an updated goal in PDAC.

Abstract B16: Lymphatics in triple-negative breast cancer: Contribution to chemotherapeutic efficacy in vitro and in vivo

Abstract Background: Triple-negative breast cancer (TNBC) accounts for approximately 15% of breast cancers and is associated with a poorer prognosis and higher rate of distant recurrence compared to receptor positive breast cancer. Due to an absence of effective targeted therapies, cytotoxic chemotherapy remains the standard of care for TNBC patients. However, the aggressive nature of these tumors makes preventing progression to metastatic disease a priority in therapeutic strategy. Inhibition of lymphatic activation represents an attractive approach to reduce peritumoral lymphatic vessel recruitment and attenuate cancer cell invasion to lymph nodes and subsequent metastasis to distant sites. However, little is currently known regarding the role of lymphatics in TNBC and how common therapeutic strategies can influence lymphatic reactivity. We hypothesized that inhibiting lymphatic activation in combination with standard of care chemotherapy will enhance therapeutic response in TNBC. Methods: Our lab has developed a novel 3D tissue-engineered tri-culture system that recapitulates the tumor-lymphatic interface within the breast tumor microenvironment. It consists of a porous tissue culture insert on which we seed human lymphatic endothelial cells (LECs) to form a monolayer on the underside. Atop the insert, we place Collagen I gel with human mammary fibroblasts and human breast cancer cells labeled with fluorescent Cell Tracker dye. After gelation, flow is applied via a pressure head and the system equilibrates overnight (16-18h). Drugs are applied via flow and then flushed from the system with basal media. After 48h from first application, gels are removed, dissociated, and analyzed via flow cytometry for death [Live/Dead and apoptosis (Caspase 3/7)], while the inserts are analyzed via microscopy for invaded cancer cells. Animal experiments were performed according to guidelines set forth by the UVA Animal Care and Use Committee. Twenty female Balb/c mice were implanted with 250,000 4T1 cells and treated with either anti-VEGFR3 antibody or IgG control and docetaxel (n=5 per group) or saline control once tumors were palpable. Tumors were dissected and dissociated once they reached 150 mm3, and analyzed via flow cytometry for stromal cell populations. Lungs were analyzed for metastases by cryosectioning and H&E staining. Results: We used our in vitro system to screen the common chemotherapeutics docetaxel, doxorubicin, 5-FU, and carboplatin across three common TNBC cell lines MDAMB231, HCC1806, and HCC38. We found that the presence of LECs significantly increases TNBC cell survival and invasiveness, shifting EC50s for cell death 10-1000 fold (EC50 shift nonlinear regression, p<0.05 for +/- LECs) and doubling to tripling invasion of cancer cells (analysis by two-way ANOVA, p<0.05). This poor response to therapy can be attenuated by treatment with the VEGFR3 inhibitor MAZ51. In vivo, we have seen that adjuvant anti- VEGFR3 therapy can reduce lymphatic endothelial cell populations in the tumor stroma with and without docetaxel chemotherapy when analyzed by flow cytometry (n=4 per group, p<0.05). Additionally, combination-treated mice showed reduced metastasis to the lung. Conclusion: Together, these data could indicate the effectiveness of anti-VEGFR3 therapy as an addition to chemotherapy to reduce metastatic progression of TNBC by limiting tumor-induced and therapy-induced lymphangiogenesis. Studies are ongoing to optimize and understand the ability of this adjuvant therapeutic strategy to increase therapeutic efficacy in TNBC. Citation Format: Alexandra H. Harris, Jennifer M. Munson. Lymphatics in triple-negative breast cancer: Contribution to chemotherapeutic efficacy in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B16.

Abstract 4898: Effects of chemotherapeutic agents on the tumor immune microenvironment

Abstract Immunotherapeutic agents have shown dramatic success in oncology in recent years and several have been approved in melanoma and lung cancer. One strategy for extending the benefit of immunotherapy is to evaluate these agents in combination with standard of care chemotherapy, with the goal of bringing the benefit of immunotherapy into earlier lines of treatment. We have carried out a systematic evaluation of the effects of different classes of chemotherapeutic agents (including alkylating agents, platinum based agents, and taxanes) on the tumor immune microenvironment of syngeneic murine tumor models (CT26 and MC38) in two different strains of mice (BALB/c and C57BL/6). We found that different chemotherapeutic agents caused markedly different changes in the composition and cell phenotype of the tumor immune environment. The majority of chemotherapeutic agents resulted in a mild to moderate increase in CD8+ T cells in the tumor. Some agents also altered the activation state of these CD8+ cells as measured by the level of PD-1 and IFN-gamma. A subset of the chemotherapeutic agents resulted in profound changes in the number of CD4+ cells, including regulatory T cells, as well as changes in myeloid subpopulations including monocytes, neutrophils, and macrophages. In addition to single agents studies, we carried out combination studies of selected chemo agents with simultaneous addition of anti-PD-L1. Several of these combinations caused pronounced increases in efficacy above single agent treatment. We will present the combination pharmacodynamic marker changes and efficacy of these treatments. Our findings demonstrate that chemotherapeutic agents can stimulate the tumor immune environment, at least transiently, through multiple mechanisms. Different subsets of chemotherapeutic agents display unique changes in the tumor immune infiltrate, and may require different immunotherapeutic approaches. These findings provide a rationale for testing immunotherapy-chemotherapy combinations in the clinic. Citation Format: Marcia P. Belvin, Shiuh-Ming Luoh, Jeanne Cheung, Erin McNamara, Rafael Cubas, Jeong Kim. Effects of chemotherapeutic agents on the tumor immune microenvironment. [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 4898.

Abstract 1209: Istiratumab (MM-141), a bispecific antibody targeting IGF-1R and ErbB3, inhibits pro-survival signaling in vitro and potentiates the activity of standard of care chemotherapy in vivo in ovarian cancer models

Abstract Insulin-like growth factor receptor 1 (IGF-1R) signaling has been implicated in the pathogenesis of ovarian cancer. However, clinical trials evaluating monospecific IGF-1R inhibitors have demonstrated limited clinical efficacy. Our data indicate that ErbB3, a member of the ErbB receptor tyrosine kinase family, can activate pro-survival AKT signaling in response to IGF-1R blockade and may represent a potential escape route in the development of resistance to therapy. Istiratumab (MM-141), an IGF-1R and ErbB3 directed bispecific antibody, inhibits ligand activation of these signaling pathways and degrades IGF-1R and ErbB3 receptor-containing complexes, leading to inhibition of downstream pro-survival signaling. Here we tested the activity of istiratumab, alone and in combination with chemotherapy, in in vitro and in vivo models of ovarian cancer. Anti-proliferative activity of istiratumab monotherapy was evaluated in a panel of ovarian cancer cell lines in vitro. The effects of istiratumab and the ligands IGF-1 and heregulin on IGF-1R- and ErbB3-mediated survival signaling were tested by ELISA and immunoblotting. Co-treatment assays with istiratumab and chemotherapy investigated mechanisms of synergy and additivity. Anti-tumor activity of istiratumab, alone and in combination with chemotherapy, was tested in in vivo ovarian xenograft tumor models. Our results indicated that istiratumab monotherapy inhibits ovarian cancer cell line proliferation in vitro. In addition, istiratumab blocked ligand-mediated resistance to chemotherapy. Co-treatment of istiratumab, ligands or chemotherapy indicated a strong correlation between drug activity and IGF-1R expression. Furthermore, co-treatment of chemotherapies and ligands potentiated AKT activation, which was inhibited by istiratumab. In vivo studies showed that istiratumab potentiates the activity of chemotherapy in ovarian xenograft tumor models. Our findings demonstrate that co-inhibition of IGF-1R and ErbB3 signaling with istiratumab can potentiate standard of care chemotherapies in ovarian tumor models and warrant further investigation of istiratumab as a potential therapy for ovarian cancer patients. Citation Format: Michael D. Curley, Gege Tan, Isabel Yannatos, Adam Camblin, Sergio Iadevaia, Chrystal Louis, Alexey Lugovskoy. Istiratumab (MM-141), a bispecific antibody targeting IGF-1R and ErbB3, inhibits pro-survival signaling in vitro and potentiates the activity of standard of care chemotherapy in vivo in ovarian cancer models. [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 1209.

Abstract 774: Proteogenomic characterization of high-grade serous ovarian cancer

Abstract High grade serous ovarian carcinoma (HGSOC) is a highly lethal gynecological malignancy, in large part because most patients develop resistance to the standard of care chemotherapy with platinum and taxanes. We performed deep proteomic and phosphoproteomic characterization on ovarian high-grade serous carcinomas (HGSC) previously characterized genomically by the The Cancer Genome Atlas (TCGA) as part of the Clinical Proteomic Tumor Analysis Consortium (CPTAC; http://proteomics.cancer.gov/programs/cptacnetwork). We constructed an integrated proteogenomic landscape of HGSC and identified functional modules statistically associated with outcomes including platinum resistance and overall survival. Integration of genomic data such as copy number alterations (CNA) with global protein abundance data revealed several chromosomal regions that have significant trans effects on protein expression. These genetically affected proteins were used to construct statistical models capable of predicting survival outcomes. Trans-affected proteins were enriched in proliferation, cell motility and invasion, and immune regulation, hallmarks of cancer. We used statistical methods to identify functional pathways able to discriminate between short surviving and long surviving patients. We found that protein abundance and phosphorylation indicated pathway activity that was able to clearly discriminate between these two groups, but that transcriptional expression and CNAs were not. We integrated proteomic abundance and phosphorylation state to elucidate specific signaling interactions and pathways differentially active in tumors from patients with short overall survival as well as to predict novel kinase substrate relationships important in HGSC. Finally, we identified druggable pathways that are differentially active across proteomic subtypes. A key point arising from our multimodal analysis is that combining different types of molecular data from these tumors greatly increases the ability to identify biologically relevant differences between groups. Using a Lasso-based regression approach to integrate data we show that the most robustly predictive survival models can be obtained using integrated CNA, transcriptome, proteome, and phosphoproteome data than with any of the individual sources of data. Citation Format: Jason E. McDermott, Samuel Payne, Debjit Ray, Vladislav Petyuk, Ronald Moore, Marina Gritsenko, Richard Smith, Karin Rodland. Proteogenomic characterization of high-grade serous ovarian cancer. [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 774.

Synergistic anti-tumor efficacy of immunogenic adenovirus ONCOS-102 (Ad5/3-D24-GM-CSF) and standard of care chemotherapy in preclinical mesothelioma model.

Malignant mesothelioma (MM) is a rare cancer type caused mainly by asbestos exposure. The median overall survival time of a mesothelioma cancer patient is less than 1-year from diagnosis. Currently there are no curative treatment modalities for malignant mesothelioma, however treatments such as surgery, chemotherapy and radiotherapy can help to improve patient prognosis and increase life expectancy. Pemetrexed-Cisplatin is the only standard of care (SoC) chemotherapy for malignant mesothelioma, but the median PFS/OS (progression-free survival/overall survival) from the initiation of treatment is only up to 12 months. Therefore, new treatment strategies against malignant mesothelioma are in high demand. ONCOS-102 is a dual targeting, chimeric oncolytic adenovirus, coding for human GM-CSF. The safety and immune activating properties of ONCOS-102 have already been assessed in phase 1 study (NCT01598129). In this preclinical study, we evaluated the antineoplastic activity of combination treatment with SoC chemotherapy (Pemetrexed, Cisplatin, Carboplatin) and ONCOS-102 in xenograft BALB/c model of human malignant mesothelioma. We demonstrated that ONCOS-102 is able to induce immunogenic cell death of human mesothelioma cell lines in vitro and showed anti-tumor activity in the treatment of refractory H226 malignant pleural mesothelioma (MPM) xenograft model. While chemotherapy alone showed no anti-tumor activity in the mesothelioma mouse model, ONCOS-102 was able to slow down tumor growth. Interestingly, a synergistic anti-tumor effect was seen when ONCOS-102 was combined with chemotherapy regimens. These findings give a rationale for the clinical testing of ONCOS-102 in combination with first-line chemotherapy in patients suffering from malignant mesothelioma.

Abstract P2-06-07: PRKCQ regulates taxol sensitivity of triple negative breast cancer cells via IL-6/Stat3 signaling

Abstract Background/Rationale. While some patients with triple negative breast cancer achieve long-term remission with chemotherapy, many have cancers that are chemotherapy resistant. The lack of targeted therapies for this subtype also makes some triple negative cancers difficult to treat and control. PRKCQ, a member of the novel protein kinase C family, is preferentially expressed in triple negative breast cancers compared to ER+/Luminal cancers. We previously reported that PRKCQ expression drives growth-factor independent growth, anoikis resistance and migration of breast epithelial cells. In addition, PRKCQ is required for in vivo growth of triple negative breast cancers tumor xenografts. We sought to determine if PRKCQ expression modulates sensitivity of triple negative breast cancer cells to standard of care chemotherapy and whether PRKCQ inhibition could be a strategy to induce death of chemotherapy-resistant triple negative breast cancer cells. Methods. We determined the effects of modulating PRKCQ expression, using PRKCQ cDNA or shRNA vectors, on Doxorubicin or Taxol treatment-induced effects on triple negative breast cancer cells, including those that are relatively chemotherapy resistant at baseline. We determined the mechanisms by which PRKCQ expression regulates sensitivity to Taxol. Results. Increased PRKCQ expression in MCF-10A breast epithelial cells suppresses the apoptosis-inducing effects of Doxorubicin or Taxol treatment. PRKCQ-induced Taxol resistance is dependent on PRKCQ kinase activity. PRKCQ-expressing MCF-10A cells secrete enhanced levels of IL-6, leading to the autocrine activation of Stat3; IL-6/Stat3 activation is necessary for PRKCQ-induced resistance to Taxol. Finally, downregulation of PRKCQ sensitized MDA-231-Luc cells to Taxol treatment and induced apoptosis of these cells which are relatively resistant to Taxol at baseline. Conclusions. PRKCQ regulates sensitivity to standard of care chemotherapies used in the treatment of triple negative breast cancer. IL-6/Stat3 signaling induced by PRKCQ kinase activity is responsible for resistance to the effects of Taxol treatment. Targeting PRKCQ therefore could be an attractive strategy to overcome chemotherapy resistance of a subset of triple negative breast cancers. Citation Format: Irie HY, Byerly J. PRKCQ regulates taxol sensitivity of triple negative breast cancer cells via IL-6/Stat3 signaling. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-06-07.

KEYNOTE-177: First-line, open-label, randomized, phase III study of pembrolizumab (MK-3475) versus investigator-choice chemotherapy for mismatch repair deficient or microsatellite instability-high metastatic colorectal carcinoma.

TPS789 Background: Mismatch repair deficient (dMMR) tumors are characterized by high mutational load and lymphocyte infiltration and may be good candidates for immune checkpoint blockade. Pembrolizumab is a monoclonal antibody against PD-1 that is designed to block its interaction with PD-L1 and PD-L2 and thus allow an antitumor immune response. In the KEYNOTE-016 proof-of-concept study, pembrolizumab showed promising antitumor activity against dMMR tumors in patients (pts) with treatment-refractory metastatic colorectal carcinoma (mCRC). KEYNOTE-177 is an international, randomized trial designed to evaluate the efficacy and safety of pembrolizumab compared with standard-of-care (SOC) chemotherapy in the first-line setting for dMMR or microsatellite instability-high (MSI-H) mCRC. Methods: Key eligibility criteria include age ≥ 18 y, confirmed MSI-H or dMMR mCRC, ECOG PS 0-1, no active autoimmune disease or brain metastases, and no prior therapy for metastatic disease. Pts will be randomized 1:1 to receive either pembrolizumab 200 mg Q3W or investigator’s choice of SOC chemotherapy. Chemotherapy must be chosen prior to randomization; options include mFOLFOX6 or FOLFIRI alone or in combination with bevacizumab or cetuximab. Treatment will continue until PD, unacceptable toxicity, pt/investigator decision, or completion of 35 cycles (pembrolizumab only). Response will be evaluated every 9 wk per RECIST v1.1 by central review and per RECIST adapted for immunotherapy response patterns. Eligible pts may continue pembrolizumab beyond initial RECIST-defined progression. Pts in the SOC arm who have PD and meet crossover criteria may be eligible to receive pembrolizumab for up to 17 treatment cycles. AEs will be assessed throughout treatment and for 30 d thereafter (up to 90 d for serious AEs) and graded per NCI CTCAE v4.0. Pts will be followed for survival every 9 wk. PFS per RECIST v1.1 is the primary end point; OS and ORR are key secondary end points. Other end points include duration of response and health-related quality of life. Planned enrollment in KEYNOTE-177 is 270 pts.

Abstract IA21: Pediatric medulloblastoma: Drug screens and preclinical studies

Abstract Medulloblastoma (MB) is the most common malignant brain tumor that develops in the cerebellum. MB occurs mostly in children between the ages of 3-7 years. Human MBs are classified into four subgroups: Wingless (WNT), Sonic Hedgehog (SHH), Group 3 (G3) and G4, each of which with distinct molecular signatures. SHH MBs with MYCN amplification and TP53 mutations and G3 MBs represent the most aggressive subgroups and the least curable with current therapeutic regimen. Whole genome sequencing and gene expression analysis of human G3 MBs demonstrated C-MYC (MYC) overexpression, from gene amplification in ~17% of cases, stem like properties and high levels of EZH2 and histone 3 lysine 27 trimethylation (H3K27me3) marks. G3 MBs have few, recurrent oncogenic point mutations that might be targeted therapeutically but contain large chromosomal copy number changes and an aberrant epigenome. We developed a mouse model of G3 MB using orthotopic transplantation as well as in utero electroporation approaches by enforcing the expression of Myc with loss of p53 function that shares many characteristics of its human counterpart including high levels of Ezh2 and H3K27me3 marks. A high throughput screen of FDA-approved drugs on mouse G3 MB neuropsheres identified Pemetrexed and Gemcitabine that, in combination with standard of care chemotherapy, greatly increased the survival of mice bearing mouse or human G3 MBs. Because genetic alterations found in human G3 MBs occur in epigenetic regulators, we tested several drugs that target the epigenome to re-establish normal gene expression profiles, including those of tumor suppressor genes. Using a preclinical drug development pipeline, we evaluated nine compounds that are believed to modulate the epigenome. Of the nine compounds tested we found that the nucleoside analog 5-fluoro-2′-deoxycytidine (FdCyd) markedly reduced the proliferation of G3 MBs in vitro at nanomolar concentrations. Detailed intracranial PK studies confirmed that systemically administered FdCyd exceeded concentrations in the tECF (tumor extra cellular fluid) necessary to inhibit tumor cell proliferation in vivo. However, despite promising in vitro activity and in vivo PK properties, FdCyd was completely ineffective in treating mouse and human G3 MBs in vivo. Our studies highlight the requirement for a comprehensive and integrated preclinical drug development pipeline with mouse and human medulloblastoma to identify the best candidate drug to be translated into the clinic and to avoid taking a drug forward that looks feasible in in vitro screens but which efficacy does not translate into an in vivo setting. Citation Format: Marie Morfouace, Yogesh Patel, Anang Shelat, Daisuke Kawauchi, Giles W. Robinson, Kip Guy, Richard J. Gilbertson, Clinton Stewart, Amar Gajjar, Martine F. Roussel. Pediatric medulloblastoma: Drug screens and preclinical studies. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr IA21.

A66 - (R)-a-Lipoic acid reduces sensorial peripheral neurotoxicity in breast cancer patients receiving weekly paclitaxel

Background: Sensorial peripheral neuropathy (SPN) is a common adverse event occurred patients (pts) treated with microtubule targeting agent, including paclitaxel (P). P neurotoxicity is determined by microtubule structure destruction, resulting in an assoplasmic transport system alteration with subsequent cellular death. Three-four grade (G) SPN incidence in weekly P ranges approximately from 9-24% in metastatic breast cancer pts to 4-8% in adjuvant setting. Furthermore, SPN recovery can be long and not completely solved. To date, there is no a standard of care for chemotherapy induced SPN. Recently, an oral liquid (R)-&agr;-Lipoic acid (ALA) formulation proved efficacy in SPN, diabetic polyneuropathy and radiculopathy opposing oxygen free radicals damage on peripheral nerve fibers. Material (patients) and methods: Based on clinical data observed in diabetic polyneuropathy, we conducted a pilot study to evaluate the effect of an innovative oral liquid ALA formulation (300 mg vial once a day) on SPN, occurred in breast cancer pts treated with adjuvant/neoadjuvant weekly P. ALA was administered until clinical evidence of SPN deterioration or complete resolution. SPN was evaluated according to NCI CTcriteria, version 4.0. Results: From December 2014 to March 2015, we collected data from 20 breast cancer women (median age 58 years, range 35-74) treated with adjuvant (16 patients) or neoadjuvant (4 patients) epirubicin combined to cyclophosphamide followed by weekly P (80 mg/mq for 12 weeks). All pts had a negative medical history for neuropathy and/or diabetes. During P treatment, 12 (60%) pts developed a G1 SPN, 6 (30%) a G2 PN and 2 (10%) a G3 SPN, respectively. SPN median time onset from the beginning of P was 35 days (range 14-84). Pts started to intake ALA at SPN onset. G1/2/3 PN improved in 5 (42%)/3 (50%)/1 (50%), respectively, stabilized in 5 (42%)/ 2 (33%)/1 (50%) respectively and worsened in 2 (16%)/1 (17%)/0 pts respectively. Overall, 16 (80%) of 20 treated women had a SPN relied. Median time to response was 21 days (range 7-84). Seventeen (85%) pts completed P chemotherapy; 1 needed a P dose reduction and 2 stopped P (at IX and X cycle, respectively), due to SPN worsening. ALA treatment was well tolerated, excepting 2 pts who experienced heartburn, resolved after ALA withdrawal. Conclusions: Although preliminary, ALA appears to have a role in reducing SPN induced by weekly P. Further studies are needed to prospectively evaluate these promising findings.

515 CRS-207, a mesothelin-targeted immunotherapy, in combination with standard of care chemotherapy as treatment for malignant pleural mesothelioma (MPM)

515 CRS-207, a mesothelin-targeted immunotherapy, in combination with standard of care chemotherapy as treatment for malignant pleural mesothelioma (MPM)

Abstract 4236: FAK inhibitor VS-6063 (defactinib) targets mesothelioma cancer stem cells, which are enriched by standard of care chemotherapy

Abstract Malignant pleural mesothelioma (MPM) is an aggressive tumor in the lining of the lung often resulting from prior exposure to asbestos. Median overall survival with standard of care chemotherapy is only 12 months from diagnosis. This poor prognosis may be attributable at least in part to cancer stem cells (CSCs) which are resistant to chemotherapy and can mediate cancer recurrence and progression. Focal adhesion kinase (FAK) has been shown to play an essential role in the survival, self-renewal and tumor-initiating capability of CSCs. Accordingly, the FAK inhibitor VS-6063 is currently being tested in patients with MPM following disease control on standard pemetrexed/platinum chemotherapy (COMMAND, ClinicalTrials.gov NCT01870609). Aldehyde dehydrogenase (ALDH) activity was validated as a CSC marker in MPM by assessment of tumor-initiating capability in mice. As compared to ALDH-negative cells, sorted ALDH-positive MM87 MPM cells showed 35-fold greater tumor initiating capacity when implanted in limiting dilutions into immunodeficient mice. Indeed, 50 ALDH-positive cells were sufficient to generate sizeable tumors in 3 weeks illustrating the aggressive nature of MPM CSCs. Treatment of a human MPM cell line with pemetrexed enriched ALDH-positive CSCs 6-fold, with similar CSC enrichment by cisplatin. In direct contrast, the FAK inhibitor VS-6063 markedly reduced the proportion of CSCs. The enrichment of MPM CSCs was similarly observed in samples from 11 tested patients following first line chemotherapy. Patient specimens post treatment with pemetrexed and cisplatin showed an elevated ALDH immunohistochemistry H-score and an increase in expression of CSC genes such as CD133 as compared to matched MPM biopsies taken from the same patients prior to chemotherapy. To assess drug effects on tumor-initiating capacity, MM87 merlin-low MPM and H28 merlin-high MPM cell lines were treated in vitro with VS-6063, pemetrexed or the combination and subsequently implanted into mice. While control and pemetrexed-treated MPM cells showed robust tumor initiation, cells treated with VS-6063 alone or VS-6063 plus pemetrexed showed little or no tumor initiating capacity. Accordingly, in tumor biopsies from MPM patients treated for 12 days with VS-6063, tumor pFAK (Y397) and expression of CSC genes such as CD133 were reduced. MPM patient-derived xenograft (PDX) tumors were employed to model the clinical scenario. Compared to control, tumor growth was blocked by 2-week treatment with pemetrexed/cisplatin. Tumors then grew rapidly upon cessation of pemetrexed/platinum treatment, whereas tumor growth was substantially delayed by FAK inhibitor treatment after cessation of chemotherapy. These data provide strong rationale for the current clinical testing of VS-6063 following treatment with pemetrexed plus platinum to potentially prolong time to progression in patients with mesothelioma. Citation Format: Jonathan A. Pachter, Vihren N. Kolev, Laurel Schunselaar, Irina M. Shapiro, Raphael Bueno, Paul Baas, Qunli Xu, David T. Weaver. FAK inhibitor VS-6063 (defactinib) targets mesothelioma cancer stem cells, which are enriched by standard of care chemotherapy. [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 4236. doi:10.1158/1538-7445.AM2015-4236

Abstract 2639: Preclinical evaluation and biomarker identification for the anti-LGR5 mAb BNC101 in K-Ras mutant CRC and other solid tumor indications

Abstract BNC101 is a monoclonal antibody (mAb) targeting LGR5 that has successfully completed IND-enabling studies in preparation for Phase I clinical studies in 2015. LGR5 is a validated cancer stem cell (CSC) receptor overexpressed in colorectal cancer (CRC), pancreatic cancer and most other solid tumors. Loss and gain-of-function studies indicate that LGR5 is a functional cancer receptor involved in tumor growth and survival. Sorted LGR5+ primary CRC are highly tumorigenic compared to LGR5- cells in limiting dilution in vivo xenograft studies. BNC101 was selected as the lead LGR5 candidate for clinical development using Bionomics’ CSC Rx Discovery™ platform. Key lead selection criteria for BNC101 were anti-tumor and anti-CSC activity in vitro and in vivo, in patient-derived xenograft (PDX) tumor models from multiple tumor types. CSC activity was evaluated by functional in vivo limiting dilution assays (LDA) using tumor cells treated with BNC101 +/- standard-of-care chemotherapy (SOC) combinations. In LDA studies with the K-Ras mutant CRC PDX tumors CT1 and CT3, 5/8 (63%) and 6/8 (75%) mice implanted with serially diluted cells from BNC101 treated tumors remained tumor free, compared to 1/8 (13%) and 2/8 (25%) in the control groups. Combination with SOC further improved BNC101 activity against CT3 tumors, where 8/8 mice re-implanted for LDA had 6 months progression-free-survival (vs 3/8 in chemo group). In the pancreatic PDX model JH109, BNC101 combined with SOC flat-lined established tumors in 3/7 mice, compared to 0/7 tumors eradicated with control SOC alone. BNC101 is also active in multiple pancreatic, triple-negative breast (TNBC) and small-cell lung (SCLC) cancer xenograft models. Ongoing translational studies have identified a panel of Wnt genes modulated by BNC101 treatment that could potentially be used to identify BNC101 responsive versus resistant patients in the clinic. Furthermore, BNC101 treatment significantly reduced LGR5+ HLA+ circulating-tumor-cells (CTCs) in the peripheral blood of tumor-bearing mice, providing a pharmacodynamic (PD) biomarker of target engagement to further evaluate in clinical studies. Altogether, the preclinical data package supports our therapeutic hypothesis that BNC101 targeting of LGR5+ CSCs will significantly improve PFS and OS in solid tumor indications. Additional translational biomarker data to support the clinical development of BNC101 will also be presented. Citation Format: Peter Chu, Kristen Smith, Farbod Shojaei, Colin Walsh, John Norton, Jose Iglesias, Christopher Reyes. Preclinical evaluation and biomarker identification for the anti-LGR5 mAb BNC101 in K-Ras mutant CRC and other solid tumor indications. [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 2639. doi:10.1158/1538-7445.AM2015-2639

Abstract 3311: Molecular targeted photoimmunotherapy as a treatment for bladder cancer

Abstract Introduction: Bladder cancer (BCa) is a common cancer and is the most expensive malignancy to treat from diagnosis to death. Although no new therapies have been introduced in the past two decades, data from ‘The Cancer Genome Atlas (TCGA)’ confirms that Epidermal Growth Factor Receptor (EGFR) family and its downstream signaling molecules are altered in almost 72% of muscle invasive bladder cancer (MIBC) cases. However, anti-EGFRs to date have failed to demonstrate any benefit over standard of care chemotherapy. We have applied a technique of employing monoclonal antibodies (mAbs) conjugated with photo-activatable compounds (IRDye 700Dx, a phthalocyanine dye) that are activated by near-infrared light (NIR). When incubated with the conjugate, exposure to NIR destroys only targeted cells. We explored photoimmunotherapy (PIT) in a panel of BCa cell lines. The purpose of this study was to study the efficacy and mechanism of action of PIT using the anti-EGFR panitumumab (Pan)-IR700 immunoconjugate as a selective therapeutic strategy for bladder cancer. Methods: Using flow cytometry, the surface expression of EGFR was profiled in several BCa cell lines. The cytotoxicity of Pan-IR700 was analyzed using LIVE/DEAD and IC50 was measured using the MTS assay. The type of cell death was examined by fluorometric caspase assay, Annexin V-PI staining, and TEM. The mechanism of cell death was elucidated using fluorometric measurement of reactive and singlet oxygen species. Results: Using flow cytometry, the surface expression of EGFR was profiled in several BCa cell lines and cell lines with increasing amounts of surface EGFR (RT4, TCCSUP, 5637 and UMUC-5) were selected for further studies. Pan-IR700 rapidly killed UMUC-5 cells (high EGFR expressions) with an IC50 of 4 nM at 4 J/cm2 NIR. In TCCSUP cells (low surface EGFR), the same IC50 of Pan-IR700 could be achieved at 64 J/cm2 of NIR. No significant cytotoxicity was observed in the presence of IR700 or NIR alone, or in cell lines without any EGFR expression. Absence of any caspases and presence of most cells in the late apoptosis/necrosis quadrant of Annexin V-PI staining suggested that PIT kills cells by necrosis. In TEM, these cells showed classic features of necrotic cell death such as swollen organelles, disintegrated plasma membrane and depleted cytoplasm and nucleoplasm. Cells undergoing PIT showed production of ROS and singlet oxygen species in large amounts immediately after exposure to NIR. However, only the singlet oxygen quencher (NaN3) and not the ROS quencher (Trolox) was able to protect the cells from PIT induced cell death indicating partial involvement of singlet oxygen in PIT-induced necrosis of cells. Conclusions: PIT is a new targeted treatment for bladder cancer. Our data demonstrate that Pan-IR700-induced PIT selectively and efficiently kills EGFR-expressing bladder cancer cells in vitro and therefore warrants further preclinical therapeutic studies in in vivo bladder cancer models. Citation Format: Reema Railkar, Quentin Li, Srinivas Vourganti, Sam J. Brancato, Peter L. Choyke, Hisataka Kobayashi, Piyush K. Agarwal. Molecular targeted photoimmunotherapy as a treatment for bladder cancer. [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 3311. doi:10.1158/1538-7445.AM2015-3311

Abstract 1339: Preclinical combination activity of SGN-CD19A and CVAD in patient-derived B-lineage acute lymphoblastic leukemia models

Abstract Combining targeted therapies with standard of care chemotherapy is a proven method for improving outcomes for cancer patients. SGN-CD19A is an antibody-drug conjugate (ADC) targeting the B-lineage specific marker CD19, and it is currently in phase 1 clinical trials for treatment of B-lineage acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). HyperCVAD (cyclophosphamide, vincristine, doxorubicin, and dexamethasone) is a common frontline chemotherapy regimen used to treat adult ALL. To explore the potential for combining SGN-CD19A with these agents (CVAD) ALL patient-derived xenografts (PDX) were developed, as these models are known to more closely reflect clinical disease. PDX were established by intravenous implant of 1-5×106 unsorted ALL patient bone marrow isolate cells into irradiated NOD scid IL2Rgammanull (NSG) mice. Initially, patient isolates were comprised of a wide-ranging (20-77%) population of CD19+/CD10+/CD45low blast cells and a varied population (5-70%) of CD19+/CD10+/CD45high cells. After implant, cells homed to and expanded in the bone marrow, thereafter progressing into the spleen and peripheral blood. After passage in mice, phenotyping revealed an enrichment of the CD19+/CD10+/CD45low blast cells and a loss of the CD19+/CD10+/CD45high population. These passaged cells were utilized for subsequent activity experiments. To evaluate PDX sensitivity to SGN-CD19A, a single dose at 1, 3 or 10 mg/kg was administered when bone marrow in sentinel animals contained between 5-24% blast cells. Activity was measured by the extent of ALL blast cell reduction in the bone marrow compared to untreated mice. SGN-CD19A elicited a dose-dependent depletion (71-99%) of blast cells in bone marrow by day 14 post dose. Like SGN-CD19A, CVAD also produced a dose-dependent depletion of the ALL blasts. Next, PDX models were used to examine SGN-CD19A in combination with CVAD. SGN-CD19A + CVAD produced a statistically superior depletion of blast cells when compared to SGN-CD19A or CVAD alone. To better understand these combined therapies, NALM-6 and RS4;11 cell line disseminated xenografts were also evaluated. SGN-CD19A + CVAD significantly prolonged survival compared to either single treatment alone in these cell line derived xenografts as well. Remarkably, SGN-CD19A in combination with vincristine (NALM-6, RS4;11) or dexamethasone (RS4;11) resulted in a survival advantage that was similar to that of SGN-CD19A + CVAD. When SGN-CD19A was tested in combination with CVAD and CVAD components in the PDX models, it was observed that SGN-CD19A potentiated the single-agent activity of vincristine, cyclophosphamide and doxorubicin. Together, these data suggest that SGN-CD19A can combine with a frontline ALL chemotherapy regimen and potentially improve outcomes for patients with ALL. Citation Format: Ivan J. Stone, Tina Albertson, Che-Leung Law. Preclinical combination activity of SGN-CD19A and CVAD in patient-derived B-lineage acute lymphoblastic leukemia models. [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 1339. doi:10.1158/1538-7445.AM2015-1339

Abstract 2497: Immune checkpoint inhibitors enhance benefits of modified vaccinia virus Ankara to improve survival in preclinical models of cancer

Abstract TG4010 is a Modified Vaccinia virus Ankara (MVA) expressing human interleukin 2 and the human mucin1 (MUC1) tumor associated antigen. TG4010 has demonstrated clinical benefit for advanced non small cell lung cancer patients in combination with standard of care chemotherapy in two phase 2 randomized and controlled clinical trials (NCT00415818 and NCT1383148). Immunotherapy based on the use of immune checkpoint blockers such as anti PD-1 and anti PD-L1 has also demonstrated efficacy in phase 2 trials. Hence, the combination of both approaches appears to be of great interest considering the high unmet medical need of this pathology. We developed experimental primary tumor and lung metastasis models to evaluate the rationale of combining both approaches at the preclinical level. Using two CT26 cell lines expressing different target antigens (β-galactosidase and MUC1) we evaluated the impact on overall survival of combined MVA-based and immune checkpoint-based immunotherapies. Synergistic increase in overall survival was observed in the therapeutic CT26-CL25 primary tumor and lung metastasis models upon treatment of mice with the combination of MVA-βgal and anti CTLA4 or anti PD-1 in comparison with either treatment alone. We provide evidence that TG4010 synergized with immune checkpoint inhibitors to increase overall survival in the therapeutic CT26-MUC1 tumor models in comparison with either treatment administrated independently. These observations were associated with an increase in the frequency and the quality of antigen-specific tumor infiltrating CD8+ T cells. These studies pave the way for the evaluation of combinatorial therapies including TG4010 and immune checkpoint blockers in the clinic. Citation Format: Karola Rittner, Christelle Remy-Ziller, Julie Hortelano, Isabelle Farine, Micael de Meyer, Virginie Nourtier, Murielle Gantzer, Christine Thioudellet, Philippe Slos, Xavier Preville. Immune checkpoint inhibitors enhance benefits of modified vaccinia virus Ankara to improve survival in preclinical models of cancer. [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 2497. doi:10.1158/1538-7445.AM2015-2497

Abstract 5517: Polymeric nanogel-based treatment regimen for enhanced efficacy and sequential administration of synergistic drug combination in pancreatic cancer

Abstract Pancreatic cancer (PC) is one of the most lethal malignancies, due to aggressive tumorigenicity, early metastasis and development of drug resistance to standard care chemotherapy. Since its approval in 1997, Gemcitabine has been the first-line treatment for advanced disease. However, there is no standard second-line therapy after Gemcitabine failure. The combination of Gemcitabine with Cisplatin (CDDP) has been explored in clinical trials for metastatic disease and our preliminary in vitro studies with the free drugs on T3M4 Simple Cells (COSMC deleted cells) showed that synergy of the combination is schedule-dependent, and Gemcitabine administration followed by CDDP showed the most potent cytotoxic activity. However, this combination proved to be only marginally effective in actual practice due to combined increased toxicity of both the agents. We have shown that encapsulation of CDDP in polymeric nanogels with cross-linked ionic cores enhanced its tumor accumulation and improved its safety profile. Additionally, sustained release profile of CDDP from nanogels allows for the administration of free Gemcitabine and CDDP loaded nanogels in a single injection while still retaining schedule-dependent synergy of the combination. Pancreatic ductal adenocarcinoma cells are known to express truncated O-glycans (Tn and STn antigens) and it is anticipated that decorating the nanogels with an antibody directed against this antigen will further enhance their uptake by tumor cells while reducing off-target accumulation. Citation Format: Kruti S. Soni, Michael A. Hollingsworth, Prakash Radhakrishnan, Tatiana K. Bronich. Polymeric nanogel-based treatment regimen for enhanced efficacy and sequential administration of synergistic drug combination in pancreatic cancer. [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 5517. doi:10.1158/1538-7445.AM2015-5517