bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1652: Optogenetic immunoengineering to develop improved anti-cancer immunotherapy
Authors: He Lian , Nguyen Nhung , Han Gang , Huang Yun , Zhou Yubin
Published: July 2017

The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed “Opto-CRAC”) that selectively and remotely controls Ca2+ oscillations and Ca2+-responsive gene expression to regulate the function of model cellular systems, as well as cells of the immune system. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless and remote photoactivation of Ca2+-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded “photoactivatable adjuvant” to improve antigen-specific immune responses to specifically destruct tumor cells. In addition, we have extended similar engineering approaches to photomanipulate membrane contact sites that play critical roles in controlling lipid metabolism and calcium signaling in cancer cells. Our study represents a solid step forward towards the goal of improving spatial and temporal control of anti-cancer therapy through optogenetic immunoengineering.

Note: This abstract was not presented at the meeting.

Citation Format: Lian He, Nhung Nguyen, Gang Han, Yun Huang, Yubin Zhou. Optogenetic immunoengineering to develop improved anti-cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1652. doi:10.1158/1538-7445.AM2017-1652
Abstract 1655: Organotypic 3D models to characterize the molecular requirements for NK and T-cell infiltration
Authors: Sommergruber Wolfgang , Osswald Annika , Hedrich Viola , Majewska Marta , Dolznig Helmut
Published: July 2017

Multiple studies have demonstrated that the presence of tumor infiltrating lymphocytes is strongly correlated with increased survival. However, the level of infiltration within a specific tumor entity and between various tumor types varies widely. Increasing the degree of lymphocyte infiltration in a tumor represents an innovative drug concept. Organotypic 3D models consisting of tumor spheroids, stromal fibroblasts and Natural Killer (NK) or T-cells could represent valuable models to appropriately model infiltration and establish novel therapeutic concepts in the area of immune-oncology. As a first step in the development of a model for lymphocyte infiltration, the cytotoxicity and infiltration of human NK cells in 3D tumor spheroids were analyzed. Different NK cell lines and primary NK cells had a toxic effect on 3D tumor spheroids upon co-culture. Cryosections of co-cultured tumor spheroids showed infiltrated NK cells inside tumor spheroids. Infiltrated NK cells recovered from tumor spheroids were characterized by flow cytometry. Results showed that a higher infiltration correlates with cytotoxicity on tumor spheroids and transcriptomes of infiltrated vs. non infiltrated NK cells differ substantially. IL-12 stimulation increased the killing effect of NK cells on tumor spheroids, inducing higher expression of Perforin, IFNγ and GranzymeB levels. 3D tumor spheroids were embedded in a Matrigel/Collagen mixture with fibroblasts and the effect of compounds on NK cell infiltration was monitored. It was demonstrated that not only IL-12 increased NK cell infiltration, but also several compounds. Moreover, encapsulated 3D models with different cell types (tumor cells, fibroblasts, NK or T-cells) were established at a bioreactor scale to mimic the in vivo situation of complex tumor tissues. We could demonstrate NK and T-cell infiltration into these encapsulated 3D tumor spheroids. These encapsulated model systems permit long-term cultures to study the effect of compounds and/or NK or T-cells on later stages of tumorigenesis. In conclusion, this study demonstrates that organotypic 3D models are a valuable tool for the analysis of the molecular mechanisms that regulate lymphocyte infiltration into tumors.

Citation Format: Wolfgang Sommergruber, Annika Osswald, Viola Hedrich, Marta Majewska, Helmut Dolznig. Organotypic 3D models to characterize the molecular requirements for NK and T-cell infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1655. doi:10.1158/1538-7445.AM2017-1655
Abstract 1660: Humanization of patient derived xenograft (PDX) cancer model mice with peripheral blood mononuclear cells (PBMCs)
Authors: Norum Jens Henrik , Josefsen Dag , Kvalheim Gunnar , Engebråten Olav , Sørlie Therese , Mælandsmo Gunhild M.
Published: July 2017

Immunocompetent transgenic mouse models have for decades served as valuable tools to address the effect of oncogenes and tumor suppressors. Immunodeficient mice have been used to establish patient derived xenograft (PDX) models harboring human tumors. These types of models have been used to study cancer initiation and progression as well as preclinical evaluation of anticancer drugs. One major limiting factor for both these model systems is the immune system; the PDX models lack an immune system and the immune system in immunocompetent mouse models display differences compared to the human immune system. Thus, in cancer research there is a need for preclinical models to study the influence of human immune cells on tumor progression and response to cancer therapies. We have established and optimized protocols for intravenous (IV) or intraperitoneal (IP) injections of isolated human PBMCs, to generate humanized mice harboring human immune cells. Our protocols do not require irradiation or busulfan pretreatment of the animals. Flow cytometry analyses showed that mainly T helper cells, CD4+, and cytotoxic T cells, CD8+, were present in the humanized mice. Immunohistochemistry (IHC) analyses of humanized PDX cancer model mice showed that human lymphocytes were present in the tumor periphery and some very few cells within the tumor, in addition to the human leukocytes in the liver and spleen. The growth rates of the PDX tumors were not affected by the humanization. Our protocols require minimal preparations of the animals and generate humanized PDX mice harboring human lymphocytes locating in close proximity of, as well as inside the PDX tumors. The model systems are suitable for preclinical studies of human, adaptive immune responses in tumor progression and cancer therapies.

Citation Format: Jens Henrik Norum, Dag Josefsen, Gunnar Kvalheim, Olav Engebråten, Therese Sørlie, Gunhild M. Mælandsmo. Humanization of patient derived xenograft (PDX) cancer model mice with peripheral blood mononuclear cells (PBMCs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1660. doi:10.1158/1538-7445.AM2017-1660
Abstract 1665: MuScreenTM : A well-characterized syngeneic model platform for rapid in vivo screening
Authors: Zhang Lan , Mao Binchen , Shi Qian
Published: July 2017

Background: Syngeneic tumor models, many shelved for decades, have been revived as effective models for immunotherapy, accompanying the clinical success of the immuno-checkpoint inhibitors (e.g. anti-CTLA4, anti-PD1, anti-PD-L1 antibodies). In vitro cell-based screens that are frequently used in oncology to quickly identify responsive cells and assess PD effects, often fail in immuno-oncology, due to immunotherapeutics targeting the complex host immune system. Alternatively, an in vivo screen with a panel of models addresses many of these questions, e.g. PD and efficacy, but may be cost prohibitive.

Material and methods: Leveraging in-house detailed profiling data on our syngeneic models, including efficacy benchmarking with anti PD-1, PD-L1, CTLA-4, OX-40, GITR , LAG3 and TIM3 antibodies, RNAseq data on tumor samples, and FACS analysis on both baseline and post-treatment tumor samples, we created a new in vivo screening tool for immune-oncology: MuScreen. MuScreen includes up to 20 well-characterized syngeneic models in a 3 month screening run. Both PD and efficacy may be determined in the run, allowing researchers to make decisions based on results observed from a large dataset. To address the cost issue, test agents from multiple clients are pooled together for each run (sharing vehicle and other common groups) providing a significant reduction in the number of animals used and the associated costs.

Results: CrownBio has established the largest collection of syngeneic models with well-characterized immunotherapy data. With the three MuScreen runs, we have generated new data on common IO agents (e.g. aPD-1 antibody) and combinations treatments, with FACS analysis, IHC, and efficacy data. Based on these data, a bioinformatics

analyses were further explored to identify the makers/pathways that could be correlated with the efficacy or PD effect of the tested IO treatment.

Conclusions: MuScreen, the first in vivo screening tool for cancer immunotherapeutics, provides detailed response data on a panel of syngeneic models, and may help on biomarker discovery in a cost and time efficient manner.

Citation Format: Lan Zhang, Binchen Mao, Qian Shi. MuScreenTM : A well-characterized syngeneic model platform for rapid in vivo screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1665. doi:10.1158/1538-7445.AM2017-1665
Abstract 1666: Immune consequences of penfluridol treatment associated with inhibition of glioblastoma tumor growth
Authors: Ranjan Alok , Gupta Nehal , Ramachandran Sharavan , Wright Stephen , Srivastava Sanjay
Published: July 2017

Glioblastoma is the most common and lethal brain tumor associated with only 12% median survival rate of patients. Despite the development of advanced surgical, radiation or use of combinations of anti-cancer drugs, treatment for glioblastoma patients is still a challenge. The major contributing factor in glioblastoma progression and resistive nature is its ability to evade the immune surveillance. Hence, modulating the immune system in glioblastoma tumors could be an important strategy for anticancer therapeutics. Penfluridol, an antipsychotic drug has been shown to have anti-cancer properties in our recently published studies. The present study evaluates the immune response of penfluridol in glioblastoma tumors. Our results demonstrated that penfluridol treatment significantly suppressed glioblastoma tumor growth. Our current results demonstrated about 72% suppression of myeloid derived suppressor cells (MDSCs) with penfluridol treatment in mouse bearing U87MG glioblastoma tumors. MDSCs are known to increase regulatory T cells (Treg), which are immunosuppressive in nature and suppresses M1 macrophages that are tumor suppressive in nature. Our results also showed suppression of regulatory T cells as well as elevation of M1 macrophages with penfluridol treatment by 58% and 57% respectively. Decrease in CCL4 as well as IFNγ with penfluridol treatment was also observed indicating decrease in overall tumor inflammation. This is the first report demonstrating immune modulations by penfluridol treatment associated with glioblastoma tumor growth suppression prompting further investigation to establish penfluridol as a treatment option for glioblastoma patients. [Studies supported in part by R01 grant CA129038, awarded by National Cancer Institute, NIH]

Citation Format: Alok Ranjan, Nehal Gupta, Sharavan Ramachandran, Stephen Wright, Sanjay Srivastava. Immune consequences of penfluridol treatment associated with inhibition of glioblastoma tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1666. doi:10.1158/1538-7445.AM2017-1666
Abstract 1667: Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach
Authors: Mann Jacqueline E. , Ludwig Megan L. , Hoesli Rebecca C. , Kulkarni Aditi , Patel Simmy , Kafelghazal Judy , Nesvizhskii Alexey , Brenner J Chad
Published: July 2017

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the United States and affects 600,000 people each year worldwide. With a five-year survival rate of only 50% and a recent rise in HPV-associated HNSCCs, improved treatment protocols are urgently needed. Evidence of immunosuppression is often reported in HNSCC, making immunotherapy an attractive strategy for the management of this disease. The immune checkpoint inhibitor Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, but only 18% of initial participants in a trial of Pembrolizumab responded, and it remains difficult to predict patients likely to experience benefit. To increase the number of patients that respond to immune checkpoint inhibition, we sought to identify targetable genetic factors modulating PD-L1, a molecule that serves to dampen the anti-tumor immune response. We show that several HNSCC models exhibit mild (2-4 fold) induction of PD-L1 expression following treatment with interferon-γ (adaptive expression), and recent reports suggest that common oncogenic pathways, including the EGFR and PI3K pathways, may provide a novel strategy to regulate PD-L1 expression. To this end, we developed a high throughput screen to identify targetable pathways that may be used to regulate PD-L1 expression in patients with specific genetic lesions. We have employed a Genome-scale CRISPR Knock-Out (GeCKO) screening technique in HNSCC cell lines and selected for genetic knockouts exhibiting altered PD-L1 expression. Stable knockout pools with representation of approximately 300 gRNAs per target gene (>20,000 target genes in the library) were expanded and serially sorted to create stable sub-populations with enhanced PD-L1 expression. These sub-populations were sequenced to identify gRNAs whose knockout causes a change in PD-L1 expression (e.g. genes that repress or enhance PD-L1 expression). We expect that these large-scale screens, when performed over multiple HNSCC cell lines with diverse genetic lesions, will identify patterns of targetable regulators that may ultimately be manipulated in combination with PD-1/PD-L1 inhibitors.

Citation Format: Jacqueline E. Mann, Megan L. Ludwig, Rebecca C. Hoesli, Aditi Kulkarni, Simmy Patel, Judy Kafelghazal, Alexey Nesvizhskii, J Chad Brenner. Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1667. doi:10.1158/1538-7445.AM2017-1667
Abstract 1669: Multiplex immunohistochemical staining of PD-L1, PD-1, CD3, CD4, CD8, CD68, FoxP3, and Ki-67 and image analysis of tumor and invasive margin in human FFPE NSCLC tissue
Authors: Dauffenbach Lisa M. , Sia Gela C. , Zheng Jianping , Jun Natalia , Olsen Eric P. , Kerfoot Christopher A.
Published: July 2017

Evaluating the density of immune cell subtypes and their relative spatial positioning in cancers has become an important tool in understanding response to immune checkpoint inhibitors. The purpose of this study was to investigate protein expression of PD-L1, PD-1, CD3, CD4, CD8, CD68, FoxP3, and Ki-67 with four multiplex immunohistochemical assays (Ki-67+CD8, CD3+CD4+Ki-67, FoxP3+PD-1+CD8, and CD68+PD-L1+CD3) using DAB, red, and green chromogens within formalin-fixed, paraffin embedded non-small cell lung (NSCLC) tissues. The Ki-67+CD8 multiplex was developed to evaluate the density of cytotoxic T cells and the percentage that are proliferating. The CD3+CD4+Ki-67 multiplex characterizes the frequency of CD4+ T cells and evaluate proliferation in this subset. The FoxP3+PD-1+CD8 multiplex evaluates the regulatory T cell subset and evaluates PD-1 expression. The CD68+PD-L1+CD3 multiplex characterizes PD-L1 expression on macrophages and T cells. Cell densities were evaluated in the center of tumor and invasive margin regions, and image analysis was performed to quantitate each stain as a single agent and co-expression within each multiplex assay.

Citation Format: Lisa M. Dauffenbach, Gela C. Sia, Jianping Zheng, Natalia Jun, Eric P. Olsen, Christopher A. Kerfoot. Multiplex immunohistochemical staining of PD-L1, PD-1, CD3, CD4, CD8, CD68, FoxP3, and Ki-67 and image analysis of tumor and invasive margin in human FFPE NSCLC tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1669. doi:10.1158/1538-7445.AM2017-1669
Abstract 1676: EZH2 inhibitors in immunotherapy of melanoma
Authors: Hersey Peter , Gallagher Stuart , Tiffen Jessamy , Shklovskaya Elena , Wilson Stephen , Filipp Fabian
Published: July 2017

EZH2 contains the catalytic subunit of the PRC2 complex, which plays a role in maintenance of stem cell- like state of cancer cells and in the progression of melanoma. Our previous studies have shown that hyperactivation of EZH2 by somatic mutations or copy number gain occurs in 20% of melanoma patients and impacts overall survival. Elevated activity of EZH2 in cancer can be targeted by small molecule drugs (e.g. GSK126) and is associated with sensitivity to apoptotic cell death. Transcriptomic profiling showed that several well-known tumor suppressors such as BOK, NDRG1 and RASSF5 are down-regulated in cells with abnormalities in EZH2, which can be reversed by inhibitor treatment. By using our in vitro EZH2 drug targeting assay we validated genes associated with immune recognition and MHC class II antigen presentation such as HLA A and B,DR and CD74 (HLA DR invariant chain). In addition, ChIP-Seq studies identified adhesion molecules, chemokine ligands, and receptors involved in traffic of lymphocytes to tumors as well as the function of NK cells. Treatment of melanoma with EZH2 inhibitors showed increased killing by NK cells suggesting that an immunosuppressive effect of EZH2 involving both innate and adaptive immunity. In a mouse model of subcutaneous B16 melanoma, a course of daily treatment with an EZH2 inhibitor did not change tumour growth or immune responses in immunosufficient mice but caused a transient 9-fold increase in circulating Ly6G+ [granulocytic] myeloid-derived suppressor cells [MDSCs] suggesting the effect of EZH2 modifiers on the myeloid compartment. In summary, patients with somatic activation of EZH2 may benefit from treatment combinations that include inhibitors of EZH2. Supported by NHMRC program grant 633004, Sydney medical school foundation and Cancer Institute NSW. F.V.F. is grateful for the support of grants CA154887 from the National Institutes of Health, National Cancer Institute and CRCC CRN-17-427258.

Citation Format: Peter Hersey, Stuart Gallagher, Jessamy Tiffen, Elena Shklovskaya, Stephen Wilson, Fabian Filipp. EZH2 inhibitors in immunotherapy of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1676. doi:10.1158/1538-7445.AM2017-1676
Abstract 1678: A flow-based immunoprofiling strategy for interrogating system-wide leukocyte composition in response to glioblastoma
Authors: Baker Gregory J. , Thiagarajan P.S. , Palaniappan Sucheendra K. , Davis Stephanie H. , Moore Jodene K. , Sorger Peter K.
Published: July 2017

Glioblastoma (GBM) is a deadly brain cancer that suppresses cellular immunity through the production of anti-inflammatory cytokines/catabolites, expression of immune checkpoint molecules, and induction of tolerogenic immune cell subsets. While preclinical models have shown each of these mechanisms to negatively impact survival outcome, it remains unclear how these distinct processes manifest themselves within the context of a coordinated immune system and whether additional immunosuppressive mechanisms exist. To address these questions, we have developed an extensible in vivo screening platform to holistically determine how GBM affects cellular immune organization. The method allows for temporal assessment of leukocyte population dynamics in response to tumor progression. Leukocytes derived from lymphoid tissues of tumor-bearing mice are isolated and immunolabeled with a 12-color optimized immunofluorescence panel (OMIP) targeting several major leukocyte lineages. Single-cell data is acquired by flow cytometry and is clustered in an unsupervised manner based on cell-surface protein expression. A vector-based classification system is then used to map phenotypically distinct clusters onto known immune cell lineages. The platform allows for the extraction of immune signatures whose divergence from tumor-naïve standards may be quantified and statistically analyzed; such signatures may also be correlated with response/resistance to therapy. The approach has been validated on orthotopic GL261 glioma in syngeneic C57BL6/J mice, yet it is readily amenable to the study of diverse model systems, including de novo tumors driven by clinically-relevant oncogenes. Alternative flow cytometry antibody panels may easily be substituted to probe more specific immune cell subsets of interest. Systems biology approaches applied to the field of tumor-induced immunosuppression hold promise in identifying novel network-level immune signatures, facilitating a better understanding of immunotherapeutic drug action, and driving next generation high-dimensional biomarker discovery.

Citation Format: Gregory J. Baker, P.S. Thiagarajan, Sucheendra K. Palaniappan, Stephanie H. Davis, Jodene K. Moore, Peter K. Sorger. A flow-based immunoprofiling strategy for interrogating system-wide leukocyte composition in response to glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1678. doi:10.1158/1538-7445.AM2017-1678
Abstract 1679: BET inhibitors suppress PD-L1 expression in pancreatic stellate cells
Authors: Ebine Kazumi , DeCant Brian T. , Collier Katharine A. , Pham Thao N. , Kumar Krishan , Munshi Hidayatullah G.
Published: July 2017

Single agent treatment with T-cell checkpoint inhibitors has not been effective in pancreatic ductal adenocarcinoma (PDAC) patients. The PDAC stroma, which can account for as much as 80-90% of the tumor mass, can act as a physical and an immunologic barrier to T-cell-mediated therapies. Transgenic mouse models have shown that ablation of pancreatic stellate cells (PSCs), key regulators of fibrosis in vivo, can sensitize PDAC tumors to immune checkpoint therapies. Recently, inhibitors targeting bromodomains and extra-terminal (BET) proteins, a number of which are currently being evaluated in clinical trials for solid tumors, were shown to induce stellate cells to become quiescent and decrease collagen production. The BET family of proteins binds to acetylation motifs present in histones and enables recruitment of transcription factors and other chromatin regulators during RNA transcription. We have found that PSCs express significantly increased PD-L1 levels compared to pancreatic cancer cell lines. We show that BET inhibitors, and in particular specific knockdown of BRD4 protein, decrease basal and IFN-γ-mediated PD-L1 expression in primary stellate cells. We also show that, in contrast to a recent report using cancer cells, c-MYC does not mediate basal or IFN-γ-mediated PD-L1 expression in stellate cells. Instead, we show that the IFN-γ-mediated PD-L1 expression is regulated by IRF1, suggesting cross talk between BRD4 and IRF1 in the regulation of PD-L1 expression. Ongoing in vivo experiments will evaluate the role of BET inhibitors in the regulation of PD-L1 in mouse models of pancreatic cancer.

Citation Format: Kazumi Ebine, Brian T. DeCant, Katharine A. Collier, Thao N. Pham, Krishan Kumar, Hidayatullah G. Munshi. BET inhibitors suppress PD-L1 expression in pancreatic stellate cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1679. doi:10.1158/1538-7445.AM2017-1679
Abstract 1681A: Overcoming pd1 targeting antibody resistance using combination strategies
Authors: Mirjolet Jean-François , Ramelet Maxime , France Damien
Published: July 2017

PD-1 targeting antibodies (nivolumab, pembrolizumab) are now approved in various tumor types either as second line treatment (locally advanced or metastatic urothelial carcinoma, advanced renal cell carcinoma, recurrent or metastatic head and neck squamous cell carcinoma, classical Hodgkin lymphoma) or even as first line therapy (metastatic melanoma, metastatic non-small cell lung cancer). Despite an increase in response rate as well as survival, there is still issue with resistance to PD-1 targeting therapies. Selection biomarkers as well as response biomarkers are still under intensive research. In addition, combination strategies are needed to increase response rate and overcome resistance.

Combination strategies were therefore tested in one well characterized syngeneic mouse model, i.e. EMT-6 breast carcinoma. The model is known to be highly sensitive to CTLA-4 targeting antibody (optimum tumor to control ratio (T/C) of 3%), a moderate responder to PD-1 targeting treatment (best T/C of 54%) and a well immune infiltrated tumor. Antibodies targeting co-stimulatory receptor such as OX40, GITR and 4-1BB as well as co-inhibitory receptor such as Tim3 were combined to anti-PD-1 antibody. In all cases, combination arm produces better therapeutic activity when compared to single treatment alone. Moreover, both strategies, meaning combination of two antibodies targeting co-inhibitory receptors (PD-1 + Tim3) and combination targeting one co-inhibitory (i.e. PD-1) and one co-stimulatory receptors (OX40, GITR or 4-1BB) worked. Detailed results will be presented including response rate, immune infiltrates and cytokines profiles in order to rationally choose the best therapeutic treatment.

Citation Format: Jean-François Mirjolet, Maxime Ramelet, Damien France. Overcoming pd1 targeting antibody resistance using combination strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1681A. doi:10.1158/1538-7445.AM2017-1681A
Abstract 1699: Impact of whole blood interferon gamma production for patient selection in vaccination with autologous formalin-fixed tumor vaccine
Authors: Sakamoto Naoyuki , Ishikawa Takeshi , Okayama Tetsuya , Yasuda Tomoyo , Doi Toshifumi , Tanigawa Mari , Naito Yuji , Itoh Yoshito , Yoshikawa Toshikazu
Published: July 2017

Introduction:

Immune checkpoint blockade by anti-CTLA-4 antibody and anti-PD-1 antibody led to clinical breakthrough for the treatment of patients with solid tumors. Moreover, recent reports have shown that neoantigens are important functional targets mediating response to immune checkpoint inhibitors. Therefore, sequential combination of personalized cancer vaccines and immune checkpoint blockade has emerged as a potential therapeutic strategy. However, existing drawbacks is that it is difficult to identify robust biomarkers for patient selection in vaccination with cancer vaccine. In this study, we examined whether the development of delayed-type hypersensitivity (DTH) response, measured after vaccination with autologous formalin-fixed tumor vaccine (AFTV), correlates to parameters of immune function such as whole blood cytokine levels and peripheral regulatory T cells (Treg) in patients with solid tumors.

Methods:

Subjects comprised 52 patients with solid malignancy. AFTV treatment consisted of 3 courses of vaccination performed at intervals of 2 weeks. DTH test was performed before and after vaccination. A positive DTH response was defined as a ≥ 10mm diameter erythema/induration. We evaluated whole blood cytokine production after phytohemagglutinin (PHA) stimulation using the bioplex array system. We also assessed the number of peripheral Tregs as well as routine hematological and biochemical parameters.

Results:

DTH positive patients and DTH negative patients did not differ with respect to the number of peripheral Tregs, leukocytes, lymphocytes or granulocyte/lymphocyte ratio. Whole blood IFN-γ production levels after phytohemagglutinin (PHA) stimulation at baseline in DTH positive patients were significantly higher than those in DTH negative patients (p=0.0397).

Conclusion:

These data indicate that whole blood IFN-γ production at baseline is likely to be useful to assess immunologic competence in vaccinated patients with AFTV. Consequently, clinical utility of whole blood IFN-γ production, as immunomonitoring method for patient selection in vaccination with cancer vaccine, is required to investigate in larger clinical studies in the future.

Citation Format: Naoyuki Sakamoto, Takeshi Ishikawa, Tetsuya Okayama, Tomoyo Yasuda, Toshifumi Doi, Mari Tanigawa, Yuji Naito, Yoshito Itoh, Toshikazu Yoshikawa. Impact of whole blood interferon gamma production for patient selection in vaccination with autologous formalin-fixed tumor vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1699. doi:10.1158/1538-7445.AM2017-1699
Abstract 1710: Providing confidence around computational tissue analysis using heterogeneity assessments
Authors: Schnatwinkel Carsten , Rudmann Daniel , Aeffner Famke , Bajwa Jasmeet , Hutnick Natalie , Sharp Michael , Chu Gerry , Alvarez JD
Published: July 2017

Background: Though the techniques to interrogate the appearance of a biomarker in tissue sections have greatly advanced, there are limitations as to how representative an analysis of a tissue section is compared to the entire diseased tissue. Depending on the heterogeneous expression level of a biomarker, tissue sampling can result in different interpretations of the biomarker’s appearance, and hence could potentially lead to a false therapeutic intervention.

Hypothesis: Digital image analysis has demonstrated tremendous value in quantifying many features related to biomarker distribution and expression in biological tissues. The information can be collected for various indications and biomarkers and a phenotypic signature can be established that describes a biomarker representation across indications. Moreover, the assessment of new samples can be compared to the established phenotypic signature and a confidence score applied in support to the determined endpoint.

Approach: For a proof of concept, 6 prostate cancer samples were processed and a single section was collected after every 100microns. A total of 7 sections per sample were stained for the lymphocyte marker CD3, and the number of positive target cells were determined in the tumor and tumor microenvironment using tissue Image Analysis (tIA™). To assess how indicative the evaluation of a single tissue section would be for the entire tumor, the heterogeneity level was determined on the section level as well as by random grid analysis on each individual section. Both criteria were utilized to define an indication and biomarker specific confidence interval and heterogeneity score.

Conclusion: The combination of IHC and tIA is a powerful tool to convert complex data into meaningful interpretations. tIA is also a capable tool to catalogue valuable information about the biomarker’s expression pattern across different disease stages and hence could be used to evaluate how representative a single biomarker evaluation is in the grand scheme. Ultimately, we demonstrated a technique that can be applied to any biomarker and would assist in guiding therapeutic decisions.

Citation Format: Carsten Schnatwinkel, Daniel Rudmann, Famke Aeffner, Jasmeet Bajwa, Natalie Hutnick, Michael Sharp, Gerry Chu, JD Alvarez. Providing confidence around computational tissue analysis using heterogeneity assessments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1710. doi:10.1158/1538-7445.AM2017-1710
Abstract 1712: Detection of circulating tumor cells in stage IV non-small cell lung cancer
Authors: Shishido Stephanie N. , Bazhenova Lyudmila , Carlsson Anders , Hicks James , Kuhn Peter
Published: July 2017

Non-Small Cell Lung Cancer (NSCLC) is the most common cause of cancer related deaths in both men and women. Some lung cancer patients have a unique challenge of a tissue access due to tumor location or baseline lung disorders such as emphysema and chronic obstructive pulmonary disease (COPD). Liquid biopsy technologies can overcome tissue assess difficulties. However, as of today there are no commercially approved assays for CTC enumeration in lung cancer. Multiple non-commercial assays exist and some show correlation of CTC enumeration and changes in CTC counts on therapy with prognosis and response to therapy. Outside of absolute number of CTC there is little published data regarding prognostic significance of CTC morphologic heterogeneity in late stage NSCLC and its ability to predict treatment outcomes. Here we investigate CTC counts in 81 patients with stage IV NSCLC using a fluid phase biopsy and high definition (HD) diagnostic pathology imaging of all nucleated cells. HD-CTCs were detected in 51 (63%) patients at initiation of therapy with a median of 2.20 (range 0-509.20) and a mean of 26.21 HD-CTCs/mL (±15.64). There was no correlation between the absolute number of HD-CTCs at the time of initiation of new therapy and patient outcomes. A subset of 25 patients was further analyzed to determine the significance of HD-CTC kinetics, which may follow three distinct patterns: an increase in HD-CTCs with therapy, unchanged HD-CTCs numbers (stable), and a decline in HD-CTCs numbers. Patients that experienced an increasing, stable, or decreasing HD-CTC profile had an overall change of 118.40, 0.54, and 81.40 HD-CTCs/mL respectively during the first 3 months. The overall survival (OS) for increasing profiles was 31.08 months (±13.08, median 32.46, range 15.11-43.50), for stable profiles OS was 8.82 months (±3.57, median 7.23, range 3.91-19.75), and for decreasing profiles it was 15.30 months (±6.39, median 14.06, range 3.81-28.06) months. CTCs are identifiable in patients with stage IV NSCLC, but correlation of absolute HD-CTC counts with disease progression was not statistically significant. However, change in CTC counts were predictive of OS in patients with metastatic lung cancer receiving chemotherapy.

Citation Format: Stephanie N. Shishido, Lyudmila Bazhenova, Anders Carlsson, James Hicks, Peter Kuhn. Detection of circulating tumor cells in stage IV non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1712. doi:10.1158/1538-7445.AM2017-1712
Abstract 1714: Translating CTCs for clinical use: melanoma patient-derived CTCs evolution in xenografts
Authors: Vishnoi Monika , Liu Haowen N. , Boral Debasish , Yin Wei , Sprouse Marc L. , Thiery Jean P. , Glitza Isabella C. , Marchetti Dario
Published: July 2017

Circulating tumor cells (CTCs) are known to disseminate from primary/metastatic tumors, survive in the circulation, and develop competency to colonize distant organs. Dissecting CTC heterogeneity is therefore crucial to provide improve prognostic and diagnostic tools (concept of “liquid biopsy”). However, the clinical significance of CTC heterogeneity is still unknown. We hypothesized that melanoma patient blood-derived, lineage-negative cells (CD45-/CD34-/CD73-/CD90-/CD105-) contain melanoma cell subpopulations (Melan A+, S100+, CD146+ and NG2+ CTCs) that evolve in vivo and achieve metastatic potency when reaching an organ-specific niche. We enriched Lin-negative cells from peripheral blood of melanoma patients clinically diagnosed with or without brain metastasis (MBM) by multi-parametric flow cytometry; and analyzed CTC heterogeneity in xenografts by longitudinal capturing and interrogation at the single-cell level (DEPArrayTM platform). First, we evaluated the presence of melanoma CTC subpopulations in Lin-negative cells and developed Lin-negative murine xenograft models. Human-cell derived melanoma metastasis in xenografts confirmed that CTCs require metastasis-potentiating factors present in the Lin-negative population. Second, we performed multi-parametric flow cytometry to isolate human-derived HLA+/Melan A+/- CTCs/DTCs population from murine blood and bone marrow at T0, Tmid and Tend phases of metastasis. We interrogated the differential levels of CTCs/DTCs in HLA+/Melan A+ cell population and established their neoplastic identity by genetic profiling and the expression of melanoma markers at single-cell level. Third, we dissected longitudinal transcriptional signatures of CTCs vs DTCs in association with MBM onset. We assessed the comprehensive EMT scores of CTCs and DTCs transcriptomics and identified the pathways associated with CTC/DTCs dynamic functional states depending upon melanoma progression. Collectively, this study provide important insights to identify novel EMT-related biomarkers of MBM progression. Further elucidation of these CTC biomarkers and properties may provide novel therapeutic strategies that can be clinically useful in melanoma patients which are yet to develop MBM.

Citation Format: Monika Vishnoi, Haowen N. Liu, Debasish Boral, Wei Yin, Marc L. Sprouse, Jean P. Thiery, Isabella C. Glitza, Dario Marchetti. Translating CTCs for clinical use: melanoma patient-derived CTCs evolution in xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1714. doi:10.1158/1538-7445.AM2017-1714
Abstract 1720: Circulating tumor cell (CTC) enrichment and DNA mutation detection by sequencing
Authors: Kou Ruqin , Zhao Eric , Leung Conrad , Gogoi Priya , Chow Will , Wang Yixin
Published: July 2017

Molecular analysis of circulating tumor cells (CTCs) is hindered by low sensitivity and high level of background leukocytes of currently available CTC enrichment technologies. The Celsee PREP100 device captures CTCs with high sensitivity and allows the captured CTCs to be retrieved for molecular analysis. To evaluate the potential of utilizing PREP100 in CTC molecular analysis, we first prepared mock samples using cultured prostate cancer cells PC3 and LnCAP and analyzed captured cells by PCR amplicon sequencing. Using Celsee PREP100, we were able to recover the average of 79% (ranging from 40% to 100%) of 110 - 1,100 PC3 cells and 60 - 1,500 LnCAP cells and detect p.K139fs*3 deletion in PC3 cells and T877A mutation in LnCAP cells. In addition, we also spiked in the two cell lines into normal donor blood samples and analyzed the captured cells by PCR amplicon sequencing. Preliminary experiments showed that 40% of 250 spiked PC3 cells and 74% of 50 spiked LnCAP cells were captured. Further, we were able to detect p.K139fs*3 deletion in the retrieved 50 - 1,000 spiked PC3 cells. Our results illustrate that Celsee PREP100 system represents a promising technology for capturing and molecular characterization of CTCs.

Citation Format: Ruqin Kou, Eric Zhao, Conrad Leung, Priya Gogoi, Will Chow, Yixin Wang. Circulating tumor cell (CTC) enrichment and DNA mutation detection by sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1720. doi:10.1158/1538-7445.AM2017-1720
Abstract 1737: ALK rearrangement analysis in circulating tumor cells of lung cancer patients
Authors: Jeon Min Kyung , Kim Young Hun , Hwang Eunjoo , Lee Hye Seon , Uh Ji-hyun , Kim Myoung Shin , Park JooKyung , Jeon Byung Hee , Lee Se-Hoon
Published: July 2017

The evaluation of ALK rearrangement in non-small-cell lung cancer (NSCLC) is a significant tool when considering chemotherapy. It is not always possible to perform a tumor biopsy in patients. We suggest isolation and culturing of circulating tumor cells (CTCs) as an alternative tool to a tumor biopsy for the diagnosis of ALK rearrangement. From 22 patients with NSCLC harboring ALK rearrangement, blood samples were collected and divided into two parts: one for immunofluorescence staining of CTC marker and the other for culturing of CTCs. Both samples were processed by size-based filtration, and Cultured CTCs were analyzed for EML4-ALK translocation by fluorescence in situ hybridization (FISH) using Vysis ALK break apart FISH probe kit. CTC culturing was successful in 18 of 22 cases (81.8%). Among 18 cases of successful CTC cultures, 13 cases showed ALK rearrangement positivity (72.2%). Therefore, we suggest that the CTCs can be used as an alternative method to tissue biopsy for diagnosing ALK rearrangement. In addition, this method may have clinical applications including serial blood sampling for the development of personalized cancer therapy based on individual genomic information.

Citation Format: Min Kyung Jeon, Young Hun Kim, Eunjoo Hwang, Hye Seon Lee, Ji-hyun Uh, Myoung Shin Kim, JooKyung Park, Byung Hee Jeon, Se-Hoon Lee. ALK rearrangement analysis in circulating tumor cells of lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1737. doi:10.1158/1538-7445.AM2017-1737
Abstract 1762: Keratin 17 identifies prognostic subtypes of pancreatic ductal adenocarcinoma
Authors: Escobar-Hoyos Luisa F. , Leiton Cindy , Vanner Elizabeth , Roa-Pena Lucia , Li Jinyu , Powers Scott , Akalin Ali , Bandovic Jela , Bailey Peter , Chang David , Moffitt Richard , Yeh Jen Jen , Biankin Andrew , Shroyer Kenneth
Published: July 2017

Recent RNA sequencing (RNASeq) studies from Bailey et al., 2016, Moffitt et al., 2015 and Collisson et al., 2011 reported that mRNA expression from bulk tumor defines molecular subtypes of pancreatic ductal adenocarcinoma (PDAC) that are highly correlated with patient survival. These studies independently identified Keratin 17 (K17) mRNA overexpression as one of over 20 upregulated transcripts that define the RNA signature of the most lethal PDACs. Studies in our lab determined that neither K17 gene missense mutations nor copy-number alterations explain the upregulation of K17 mRNA expression by malignant cells. In addition, we found that K17 expression, measured at the protein level by immunohistochemistry or at the level of mRNA by RNASeq, is sufficient to stratify patients by short- vs long- term survival at baseline after resection. These retrospective survival analyses were performed in four independent patient cohorts using a uniform threshold to define low- vs high- K17 patients (total n= 558). High-K17 cases were twice as likely to die from this disease compared to stage-matched low-K17 cases (P values < 0.05). Furthermore, we determined that K17 expression is associated with outcome after Gemcitabine treatment using the Bailey et al., 2016 patient cohort (n= 94). This is the first study to show that the expression of a single gene, K17, can accurately subtype PDAC at initial diagnosis. In conclusion, K17 was identified as a robust and independent, clinically relevant, prognostic and predictive biomarker to stratify clinical outcome at the time of initial diagnosis and to potentially inform clinical decisions regarding chemotherapeutic intervention.

Citation Format: Luisa F. Escobar-Hoyos, Cindy Leiton, Elizabeth Vanner, Lucia Roa-Pena, Jinyu Li, Scott Powers, Ali Akalin, Jela Bandovic, Peter Bailey, David Chang, Richard Moffitt, Jen Jen Yeh, Andrew Biankin, Kenneth Shroyer. Keratin 17 identifies prognostic subtypes of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1762. doi:10.1158/1538-7445.AM2017-1762
Abstract 1763: A novel RNA in situ hybridization approach highly sensitive for detection of Merkel cell polyomavirus
Authors: Wang Lisha , Harms Paul W. , Palanisamy Nallasivam , Carskadon Shannon , Cao Xuhong , Siddiqui Javed , Patel Rajiv M. , Zelenka-Wang Sylvia , Johnson Timothy M. , Durham Alison , Su Fengyun , Shukla Sudhanshu , Mehra Rohit , Chinnaiyan Arul M.
Published: July 2017

Background: Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine tumor of the skin. Merkel cell polyomavirus (MCPyV) plays an oncogenic role in the majority of MCC tumors. Detection of MCPyV in MCC tumors has diagnostic utility, and potentially prognostic and therapeutic implications. We investigated whether RNAscope, a novel RNA in situ hybridization (ISH) method for detection of RNA transcripts in tissues at the single-cell/single-molecule level, is useful for MCPyV detection.

Methods: We applied an RNAscope probe targeting MCPyV T antigen transcripts on tissue microarrays (TMAs) and full tissue sections encompassing 91 MCC tumors from 75 patients, 14 carcinomas of other types, and various normal tissues. qPCR and immunohistochemistry (IHC) for MCPyV were performed on 58 and 88 cases, respectively. qPCR was also performed on 18 cases of normal skin to establish background levels of MCPyV in non-tumor tissues.

Results: RNA-ISH demonstrated the presence of MCPyV in 46.2% (42/91) of cases. A total of 58 samples had data from all the three detection modalities (qPCR, RNA-ISH and IHC). RNA-ISH demonstrated 100% concordance with qPCR results, whereas IHC had a slightly lower concordance (96.6%). RNA-ISH demonstrated higher agreement than IHC among TMA cores from the same case, and between TMA cores and matched full tissue sections. Of samples with background inflammatory cells, 5 of 37 (15.6%) showed moderate nonspecific staining of lymphocytes by IHC, whereas RNA-ISH lacked background.

Conclusions: RNA-ISH is comparably sensitive to qPCR for detection of MCPyV, and allows for correlation with tissue morphology. Our findings support RNA-ISH as a highly effective method for MCPyV detection in tumors.

Citation Format: Lisha Wang, Paul W. Harms, Nallasivam Palanisamy, Shannon Carskadon, Xuhong Cao, Javed Siddiqui, Rajiv M. Patel, Sylvia Zelenka-Wang, Timothy M. Johnson, Alison Durham, Fengyun Su, Sudhanshu Shukla, Rohit Mehra, Arul M. Chinnaiyan. A novel RNA in situ hybridization approach highly sensitive for detection of Merkel cell polyomavirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1763. doi:10.1158/1538-7445.AM2017-1763