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In preclinical work, the inflammatory cytokine IL-1β was shown to be upregulated in pancreatic cancer tumors and to contribute to activation of pancreatic stellate cells and immunosuppression (Das et al 2020). We conducted an open-label multicenter Phase Ib study evaluating gemcitabine, nab-paclitaxel, canakinumab (ACZ885), a high-affinity human anti-interleukin-1β (IL-1β) mAb, and spartalizumab (PDR001), a PD-1 mAb. Eligible subjects had previously untreated metastatic PDA and RECIST measurable disease. The primary objective was to confirm recommended phase II dose by evaluating the incidence of dose limiting toxicities (DLTs) in the first 56 days of dosing in at least 6 evaluable subjects utilizing a Bayesian logistic regression model. Subjects underwent baseline and on-study tissue and blood collection for correlative translational research. Results: 10 subjects were enrolled between Nov 2020 and Mar 2021. At the primary data cut off of May 23, 2021, 6 subjects were evaluable for DLT. There were no DLTs, the recommended Phase II dose was established as: gemcitabine (1000mg/m2 IV) day 1,8,15; nab-paclitaxel (125mg/m2 IV) day 1,8,15; canakinumab (250mg SC) day 1, spartalizumab (400mg IV) day 1; of each 28 day cycle. At the time of an updated database extraction on June 1, 2022, 2 subjects remain on study. Adverse events were consistent with those typically seen with chemotherapy. The most common Grade 3/4 AEs were neutropenia (60%) and anemia (50%), with no fatal AEs. One patient discontinued spartalizumab due to grade 3 pneumonitis. In preliminary efficacy analysis (n=10), there are 3 confirmed PRs, 5 subjects with stable disease, 2 subjects with progression as best response. Individual site data estimates that the 12 month OS rate is 60%; updated RR, PFS and OS data will be reported. Activation of CD8 T cells in peripheral blood and increased serum levels of IFN-induced chemokines CXCL9/10 were observed in both responder and non-responder patients. Using an in vitro suppression assay, we showed that baseline serum from responders could induce myeloid derived suppressor cells, an effect that was abrogated with treatment. Single cell transcriptional profiling, multiplex immunofluorescence and spatial transcriptomics also revealed treatment-dependent shifts in T cell activation state and myeloid cells in the tumors of patients experiencing clinical response. Conclusions: PanCAN-SR1 established the Phase II dose of canakinumab and spartalizumab with chemotherapy in first line metastatic PDA, based upon favorable benefit-risk assessment. Based on our comprehensive analysis of 10 patients treated with combination therapy including IL-1b blockade, we hypothesize that the definitive role of anti-IL-1b in human patients with pancreatic cancer is to reduce systemic immune suppression and to reduce immunosuppressive myeloid cell activation in the tumor. The clinical utility of targeting IL-1β in pancreatic cancer is being evaluated in a randomized Phase II/III study through the Precision Promise clinical trial network. NCT04581343. Citation Format: Paul E. Oberstein, Osama Rahma, Nina Beri, Amy Stoll-D'Astice, Emily A. Kawaler, Igor Dolgalev, Gregor Werba, Victoire Cardo-Ruffino, Naïma Böllenrücher, Andressa Dias Costa, Saloney Nazeer, Matthew Squires, Jonathan Nowak, Dafna Bar-Sagi, Brian Wolpin, Diane M Simeone, Stephanie K Dougan. Primary results of PanCAN-SR1, a phase 1b study evaluating Gemcitabine, nab-Paclitaxel, Canakinumab, and Spartalizumab to target IL-1β and PD-1 in metastatic pancreatic cancer with correlative tissue and blood biomarker analysis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR005.

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression. Improving our understanding of the PDAC TME and its role in response to therapy via in-depth single cell characterization will have broad clinical implications for biomarker development and therapeutic design. In this study, we performed single-cell RNA sequencing on freshly collected human PDAC samples of primary (n=16) or metastatic (n=11) origin, either before (n=20) or after (n=7) chemotherapy. We found a heterogeneous mixture of basal and classical Moffitt cancer cell subtypes in all samples, along with distinct cancer-associated fibroblast (CAF) and tumor-associated macrophage (TAM) subpopulations. We identified the major CAF subpopulations as inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs); within these subpopulations were a very few cells expressing immunogenic features which have previously been associated with antigen presenting CAFs (apCAFs). Tumor-associated macrophages (TAMs) could be categorized into two major subpopulations, C1QC+ TAMs or SPP1+ TAMs, each with distinct functional characteristics. For example, phagocytosis-associated gene sets were enriched in C1QC+ TAMs, while angiogenesis-associated gene sets were enriched in SPP1+ TAMs. Comparison of naïve and chemotherapy treated primary PDAC samples revealed that classical and basal-like cancer cells exhibited similar transcriptional responses to chemotherapy; this contrasts with some previous reports which posited a shift towards a basal-like transcriptional program among treated samples. We further noted that treated samples evinced fewer ligand-receptor interactions, particularly between TIGIT on CD8+ T cells and its ligand on cancer cells. We also identified TIGIT, not PD1, as the major inhibitory checkpoint molecule of CD8+ T cells in the PDAC TME. Altogether, our results suggest that chemotherapy impacts the PDAC TME and may further reduce response to immunotherapy. Citation Format: Daniel Weissinger, Emily A. Kawaler, Gregor Werba, Ende Zhao, Despoina Kalfakakou, Surajit Dhara, Grace Oh, Xiaohong Jing, Nina Beri, Lauren Khanna, Tamas Gonda, Paul E. Oberstein, Cristina Hajdu, Cynthia Loomis, Adriana Heguy, Mara H. Sherman, Amanda W. Lund, Theodore H. Welling, Igor Dolgalev, Aristotelis Tsirigos, Diane M. Simeone. Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR010.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that harbors mutations in homologous recombination (HR) repair genes in up to 25% of cases. Defects in HR impart to tumor cells a specific vulnerability to poly-ADP ribose polymerase inhibitors and platinum-based chemotherapy. However, not all patients respond, and some who initially respond develop resistance and progress. Thus, new therapies that are effective against HR-deficient PDAC are needed. Here, we show that inactivation of the HR pathway in PDAC is associated with overexpression of polymerase theta (Polθ, or POLQ), a key enzyme that regulates the alternative non-homologous end-joining (alt-NHEJ) pathway of double-strand break (DSB) DNA repair. Using both human and murine HR-deficient PDAC models, we find that POLQ knockdown is synthetically lethal with mutations in HR genes (BRCA1, BRCA2, and PALB2) as well as the DNA damage repair gene ATM. Further, we present the first findings that POLQ knockdown enhances cytosolic micronucleus formation and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling. In an in vivo murine model, POLQ inhibition also leads to STING-dependent recruitment of immune cells and enhanced CD8+ T cell infiltration in the BRCA2-deficient tumor microenvironment (TME). Through its role in the alt-NHEJ pathway, we thus find that POLQ is critical for DSB repair in HR-deficient PDAC, and its inhibition presents a novel synthetic lethal approach to suppress tumor growth while simultaneously stimulating an immune response in the PDAC TME. Citation Format: Grace Oh, Annie Wang, Lidong Wang, Jiufeng Li, Gregor Werba, Daniel Weissinger, Ende Zhao, Surajit Dhara, Rosmel Hernandez, Amanda Ackermann, Despoina Kalfakakou, Emily A. Kawaler, Talia Golan, Theodore H. Welling, Agnel Sfeir, Diane M. Simeone. Polymerase theta inhibition activates the cGAS-STING signaling pathway and elicits an immune response in homologous recombination-deficient pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B065.

Thromboembolism (TE) is a common complication in cancer patients and the second leading cause of cancer-related deaths. The incidence of TE varies in different cancer types, with the highest risk in pancreatic ductal adenocarcinoma (PDAC) and in advanced-stage and metastatic cancers. Despite the benefits associated with anti-coagulant therapy for symptomatic TE, the prevention of TE still remains an unmet clinical need due to lack of biomarkers predictive of TE risk and the bleeding risk associated with the routine use of anti-coagulants. Small extracellular vesicles (sEV) mediate cell-to-cell communication. Cancer cells and the tumor microenvironment release large numbers of sEV into the blood circulation and sEVs have displayed a therapeutic and predictive value in systemic diseases. However, the role of sEVs in cancer-associated TE remains to be investigated. Here we show that sEVs from (pre)metastatic lungs of mice with melanoma, breast, lung, and PDAC induce TE in mice and express high levels of integrin beta 2 (ITGB2), while sEVs from tumor cell lines, primary tumors, or other metastasis-bearing organs did not show any pro-thrombotic properties. A specific subtype of interstitial macrophages infiltrating (pre-)metastatic lungs were the main source of ITGB2+ pro-thrombotic sEVs. Blockade of ITGB2 on lung-derived sEVs, or systemically in mice, prevented EV-induced platelet aggregation and TE, and reduced metastasis. Examination of the mechanisms of ITGB2-induced TE showed that EV-associated ITGB2 interacts directly or through fibrin with different binding partners on platelets, and induce their activation and aggregation. Importantly, we found that levels of ITGB2 on sEVs are elevated in the plasma of PDAC patients prior ( Citation Format: Serena Lucotti, Yusuke Ogitani, Candia M. Kenific, Linda Bojmar, Michele Cioffi, Pernille Lauritzen, Henrik Molina, Soeren Heissel, Henry B. Lengel, Xiaohong Jing, Haiying Zhang, Irina Matei, Eileen M. O'Reilly, William R. Jarnagin, David Jones, James B. Bussel, David Kelsen, Jacqueline F. Bromberg, Diane M. Simeone, David Lyden. The lung pro-thrombotic niche drives cancer-associated thromboembolism and metastasis via extracellular vesicle ITGB2 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C028.

Introduction: We have previously shown using the KPC (KrasLSL G12D/+; p53 r172H/+; Pdx1-Cre) mouse model of PDAC that sequential treatment with the DNA hypomethylating agents (HMA) followed by anti-PD-1 led to increased tumor necrosis, slowed tumor growth, increased tumor-infiltrating CD8+ cells, and significantly increased mean survival. However, acquired treatment resistance occurred, with emergence of a specific subtype of M2-polarized putatively immunosuppressive Chi3l3+ macrophages. In this study, we characterize the mechanism of polarization of these cells, define their function, and identify a potential therapeutic strategy to combine with epigentic therapy to prevent unfavourable macrophage polarization and improve the efficacy of epigenetic priming of immunotherapy against PDAC. Methods: Studies were conducted with primary macrophages (PM)e isolated from bone marrow (BMDM) or the peritoneum of 6-8 weeks mice and RAW264.7 cells were also used and exposed to conditioned media from primary KPC cell lines with and without previous treatment with the HMAs decitabine or azacytidine. The effect was compared with polarization by direct HMA, IFN-γ and IL-4. Gene set enrichment analysis was done on RNASeq data from myeloid cells and validated by RT-PCR. We used cytokine arrays and Western blot validation to identify secreted cytokines from KPC cells. Results: We profiled expression of RAW264.7 cells, BMDM and PMs following exposure to IL-4 or IFN-γ and identified a signature associated with each treatment (CD206, Fizz1, Tlr8, IGF1, Mgl2 associated with IL-4 and TNFα, CD86, CD64, CD40, NOS2 associated with IFN-γ). Direct exposure of myeloid cells to hypomethylating agents did not result in a significant polarization. We next used conditioned media of KPC cells to identify a hypomethylation specific effect and found a significant IL-4 like enrichment (Chi3l3, Arg1, Il4i1, Raet1a, Lgals4) and a HMA-specific myeloid signature (CD137, IL1a, Ccl2, Ccl7, Spp1) with treatment. To assess which cytokines might trigger this polarization, we employed cytokine arrays and identified a small number of candidate cytokines that are specific to hypomethylation induced polarization of myeloid cells, including CXCL1/2, CCL2, GM-CSF, FGF-21, IGFBP-6 and ICAM-1. Conclusions: Our results show that paracrine secretion of cytokines from cancer cells treated with HMA drugs, rather than direct effect of hypomethylating therapy on macrophages, is responsible for polarizing macrophages into an immunosuppressive subtype. We further identified several candidate cytokines secreted by cancer cells following hypomethylating therapy that may be relevant for this immunosuppressive polarization of macrophages and might be specific therapeutic targets in combination with hypomethylating therapy. Citation Format: Arturo Orlacchio, Daniel Weissinger, Catherine Do, Benjamin Tycko, Diane M. Simeone, Tamas Gonda. Hypomethylating therapy induces a potential immuno-suppressive myeloid phenotype by altering cancer cell cytokine secretion in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C041.

Introduction: Several studies have found a relationship between familial breast cancer (BC) and pancreatic cancer (PC). Known genes associated with both BC and PC include BRCA1, BRCA2, PALB2, ATM, STK11, and possibly others. For carriers of pathogenic germline variants (PGVs) in these genes with family history of PC (FHPC), pancreas surveillance is recommended and can lead to an earlier detection of disease. Current genetics practice typically involves use of multi-gene panel (MGP) testing covering all of these genes linked to both cancers, but not all patients have had complete testing leading to likely under appreciation of PC risk in BC cohorts. With improved treatment options and survival for BC and ovarian cancer in PGV carriers, we may begin to observe an increased prevalence of PC in this group. The purpose of our study was to evaluate the clinicopathologic characteristics including genetic testing uptake and outcomes in BC patients with and without a family history of pancreatic cancer (FHPC). Methods: We queried the Institutional Breast Cancer Database, which includes patients diagnosed with BC between January 2010 and December 2018. Variables analyzed included FHPC in a first or second-degree relative, and other clinical and tumor characteristics. Statistical analyses included Pearson’s Chi Square and logistic regression. Results: A total of 232 BC patients (7%) had a positive FHPC, including 115 (50%) with a first-degree relative and 117 (50%) with other relatives affected. When comparing BC patients with and without any FHPC, those with FHPC were 1.93 times more likely to be of White race (p Conclusions: Within a contemporary cohort of newly diagnosed breast cancer patients, 7% had a positive FHPC. Although these patients were more likely than those without FHPC to have genetic testing, the majority, 77%, had incomplete or no genetic testing suggesting likely under-diagnosis of PC risk. Our study underscores the importance of querying and documenting FHPC in patients with BC and the consideration of germline testing and evaluation for pancreas surveillance in these patients and their family members. Citation Format: Annie Wang, Jessica Everett, Jennifer Chun, Elianna Kaplowitz, Diane Simeone, Freya Schnabel. Family history of pancreatic cancer and the prevalence of pathogenic germline variants in a contemporary cohort of newly diagnosed breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-24.

Thromboembolism (TE) is a common complication in cancer patients and the second leading cause of cancer-related deaths. The incidence of TE varies in different cancer types, with the highest risk in lung cancer and pancreatic ductal adenocarcinoma (PDAC), and in advanced-stage and metastatic cancers. Despite the benefits associated with thromboprophylaxis for symptomatic TE, the prevention of TE still remains an unmet clinical need due to lack of biomarkers predictive of TE risk and the bleeding risk associated with the routine use of anti-coagulants. Exosomes are small circulating extracellular vesicles that mediate cell-to-cell communication. Cancer cells and the tumor microenvironment release large numbers of exosomes into the blood circulation and have displayed a therapeutic and predictive value in systemic diseases. Integrins expressed on the surface of exosomes drive their selective organotropism and prepare distant sites for metastatic seeding by establishing favorable pre-metastatic niches. Here we show that exosomes from metastasis-bearing lungs or pre-metastatic lungs of mice with melanoma, breast, lung and pancreatic cancer induce TE in mice and express high levels of integrin beta 2 (ITGB2). Instead, exosomes from tumor cell lines, primary tumors or other metastasis-bearing organs did not show any pro-thrombotic properties. Myeloid cells including monocytes/macrophages and neutrophils infiltrating pre- and post-metastatic lungs were the main source of ITGB2+ pro-thrombotic exosomes. Blockade of ITGB2 on lung-derived exosomes, or systemically in mice, prevented exosome-induced platelet aggregation and TE, and reduced metastasis. Examination of the mechanisms of ITGB2-induced TE showed that exosomal ITGB2 interact directly or through fibrin with different binding partners on platelets, and induce their activation and aggregation. Importantly, we found that exosomal ITGB2 levels are elevated in the plasma of PDAC patients prior to TE events in comparison to PDAC patients with no history of TE, and thus might serve as prognostic biomarker of TE. Together, our results provide the first evidence of the establishment of a pro-thrombotic lung niche in different cancer types. Moreover, we identify exosomal ITGB2 as a new target for the prevention and/or treatment of TE, as well as a potential “liquid biopsy” analyte for the early stratification of patients at high risk of TE. Citation Format: Serena Lucotti, Yusuke Ogitani, Candia M. Kenific, Linda Bojmar, Michele Cioffi, Pernille Lauritzen, Henrik Molina, Soren Heissel, Harry B. Lengel, Xiaohong Jing, Haiying Zhang, Irina Matei, Eileen M. O'Reilly, William R. Jarnagin, David R. Jones, James B. Bussel, David Kelsen, Jacqueline F. Bromberg, Diane M. Simeone, David Lyden. The lung pro-thrombotic niche drives cancer-associated thromboembolism via exosomal ITGB2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3138.

Extracellular vesicles (EVs) are comprised of lipid bound vesicles secreted by cells into the extracellular environment with various roles in cell-to-cell communication. Recent studies have implicated EVs in cell proliferation, epithelial-mesenchymal transition (EMT), metastasis, angiogenesis, and mediating the interaction of tumor cells and tumor microenvironment. To systematically characterize the EVs associated with pancreatic cancer, we performed proteomic and functional analyses on the protein contents of EVs released from pancreatic cancer lines, CAF cell lines and a normal pancreatic epithelial cell line. More than 1400 non-redundant proteins were identified in each EV proteome derived from these cell lines. The common EV proteins identified in these cell lines were enriched in biological processes such as vesicle-mediated transport and exocytosis. Protein networks relevant to pancreatic tumorigenesis, such as of EMT, complement and coagulation components, were significantly enriched in the EVs from cancer cells or CAFs in comparison to normal pancreatic epithelial cells. These findings support the roles of EVs as a potential mediator in transmitting EMT signals and complement response in the tumor microenvironment and possibly contributing to coagulation defects related to cancer development. Citation Format: Sharon Pan, Lisa A. Lai, Diane M. Simeone, David W. Dawson, Yuanqing Yan, Tatjana Crnogorac-Jurcevic, Ru Chen, Teresa A. Brentnall. Proteomics analysis of extracellular vesicles from pancreatic cancer cells and cancer associated fibroblasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5077.

Background: Drug development in PDAC has been disappointing with an extremely low trial success rate despite considerable effort. PrP is a transformative, adaptive clinical trial platform that attempts to correct this by continuously and rapidly evaluating novel therapeutic options while maximizing the probability of patient (pt) randomization to an experimental treatment and nurturing enhanced cooperation among groups representing pt advocacy, pharmaceuticals, translational/clinical academia, and the FDA. This patient-centric study represents a fundamental shift in drug development for PDAC in the United States and aims to become the largest Phase 2/3 registrational study ever launched in this disease. Methods: PrP (NCT04229004) is a clinical trial platform sponsored by the Pancreatic Cancer Action Network (PanCAN) and funded solely through non-government sources. The protocol was finalized based on the FDA 2020 guidance document regarding "complex innovative designs" in registration trials https://www.fda.gov/media/130897/download. It utilizes adaptive randomization along with several trial design and Bayesian statistical innovations provided by Berry Consultants LLC. All pts undergo pre-and on-treatment biopsies with state-of-the-art genomic, transcriptomic, and immune analysis, along with collection of blood samples for research purposes throughout the study. Pts are managed using novel standardized supportive care techniques, and PrP contains 3 sub-protocols involving quality of life, sarcopenia and actigraphy. PrP was launched in 2020, and currently includes 20 US sites. Focused on both 1st and 2nd line treatment of metastatic PDAC, PrP uses an adaptive platform design with 30% of pts randomized between one of the 2 standard of care control arms (gemcitabine + nab-paclitaxel and mFOLFIRINOX) and 70% to experimental arms, currently either SM-88, a cancer metabolism-based agent (Tyme Inc); or Pamrevlumab, an antibody inhibiting the activity of the connective tissue growth factor (Fibrogen Inc.) The study is ongoing with >100 pts enrolled to date. The Data and Safety Monitoring Committee regularly reviews the data and continues to recommend that the trial proceeds as planned. New study arms will be added after review by an Arm Selection Committee that assesses the validity of the treatment target and the adequacy of the preexisting pre-clinical and clinical data. An additional experimental arm is anticipated in 2021. Conclusion: Compared to traditional trial designs, PrP offers several advantages: multiple investigational treatments can be evaluated in parallel over time; only ~ 175 pts per experimental arm required to initiate a regulatory registration; and increased learning from every patient during the trial, altogether resulting in both time saving and a 30-50% cost saving. In effect, PrP has created an entirely new “learning community” and can substantially accelerate drug development for PDAC. Citation Format: Vincent J. Picozzi, Anne-Marie Duliege, Anirban Maitra, Manuel Hidalgo, Andrew Eugene Hendifar, Gregory L. Beatty, Sudheer Doss Doss, Regina Deck, Lynn M. Matrisian, Julie Fleshman, Diane M. Simeone. Precision Promise (PrP): An adaptive, multi-arm registration trial in metastatic pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-050.

Background: A wide range of cancers, including pancreatic cancer, display altered expression of amino acid transporter LAT-1. LAT-1 overexpression has been shown to be an important prognostic factor and predicts chemo resistance in pancreatic cancer (Kaira K, 2012; Altan B, 2018). Radiolabeled amino acids for PET imaging, including 3-18F-l-α-methyl-tyrosine ([18F] FAMT), have successfully leveraged cancer’s aberrant LAT1 expression and amino acid uptake for decades. SM-88 is a denatured D/L racemic form of α-methyl-tyrosine designed for selective uptake and disruption of protein synthesis in cancer cells, as well as disruption of catecholamine production via inhibition of tyrosine hydroxylase. Catecholamines have been reported to potentially have a role in driving pancreatic cancer progression and changes in the tumor immune status (Renz B, Cancer Cell 2018; Calvani M, Oncotarget 2014). Initial clinical and preclinical results with SM-88 indicate that this agent could pose a novel approach in the treatment of pancreatic cancer. Methods and Results: Initial xenograft models of human gastrointestinal cancers using monotherapy SM-88 showed a >50% decrease in tumor growth at three weeks of treatment (p Conclusion: Altered amino acid (LAT1) metabolism has been shown to be a negative prognosticator in pancreatic cancer outcomes and progression. Targeting LAT1 and amino acid pathways has been identified as a therapeutic approach in multiple cancers (Fuchs BC, Semin Ca Biol 2005). Initial and ongoing studies of SM-88 may clarify the potential utility of this approach in the treatment of pancreatic cancer. Citation Format: Martin Fernandez-Zapico, Dae Won Kim, Philip Philip, Alexander Vandell, Jonathan Eckard, Ron Korn, Giuseppe Del Priore, Diane Simeone. Therapeutic potential of targeting amino acid metabolism in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B15.

Recent genomic characterization of PDA reveals that between 20-25 % of PDA harbor recurrent mutations in genes, including BRCA1/2, PALB2, and ATM, which are critical for homologous recombination (HR), an important form of DNA repair. In many patients, these may be germline mutations. This subgroup of PDAs, termed HR-deficient PDA, has emerged as a defined biological entity associated with increased chemoresistance and a more aggressive disease course. The defects in HR observed in these tumors impart cells with a specific vulnerability to PARP inhibitors and platinum-containing therapy. Still, as observed in the case of many other targeted therapies, only a fraction of HR-defective patient tumors respond to PARP inhibition. More so, many patients that initially respond eventually often develop resistance and progress. Therefore, novel therapies which can be effective against HR-defective PDA, alone or in combination with PARP inhibitors or other combinatorial regimens, are urgently needed. We have recently determined that inactivation of the HR pathway is associated with overexpression of polymerase theta (PolO–, also known as POLQ) in PDA. POLQ is a key enzyme that regulates an alternative pathway of DNA repair, known as the alternative non-homologous end-joining (Alt-NHEJ) pathway. In the setting of defective HR, Alt-NHEJ becomes a critical pathway responsible for the repair of DNA breaks and POLQ inhibition in HR-defective tumor cells demonstrates a synthetic lethality phenotype, not observed in cells with intact HR. Furthermore, POLQ knockdown significantly upregulated the cGAS-STING pathway in HR-deficient PDAs linking the DNA damage response to the immune response. Overall, targeting POLQ may represent a novel and in a valuable therapeutic strategy in HR-defective pancreatic cancer, as POLQ inhibitors are currently in development for clinical use. Citation Format: Annie Wang, Andrea Zamperone, Mohammad Sohail, Lidong Wang, Fiyinfolu Balogun, Jiufeng Li, Ende Zhao, Daniel Diolaiti, Agnel Sfeir, Diane M. Simeone. Polymerase theta synthetic lethal interaction in homologous recombination-deficient pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I14.

Objective: Although circulating tumor cells' (CTCs) isolation from pancreatic adenocarcinoma (PaCa) patients is feasible, investigation of their clinical utility has proven less successful than other cancer types, due to poor technology sensitivity and EpCAM-only based CTC assays. Design: We present an integrated technology and biology-based approach using a microfluidic “Carpet chip” to study the biologic relevance of rare CTCs. We isolate both circulating epithelial CTCs (EpCs) and EMT-like CTCs (EMTCs) simultaneously from whole blood of PaCa patients (n=35), targeting two different surface markers: anti-EpCAM and anti-CD133. Gene expression profiling of CTCs was performed with 96 genes (n=17). Results: Recovery of cancer cell lines spiked into whole blood is ≥97% with >76% purity. Of the 35 PaCa patients analyzed, 34 had ≥5 EpCs/mL and 35 had ≥15 EMTCs/mL. Higher numbers of EMTCs were observed in patients with lymph node involvement (N1) compared to patients without. The ratio of EpCs to total number of CTCs marginally decreased with advanced stage, while the ratio of EMTCs increased. Gene expression profiling showed CXCR1 was significantly upregulated in EpCs whereas POU5F1/Oct-4 and MYC were upregulated in EMTCs, demonstrating inherent phenotypic differences. Early-stage patients with significantly better overall survival and/or progression-free survival showed significant higher expression of genes including ERCC1 and TTF1. Conclusions: We demonstrated successful isolation of heterogeneous population of CTCs (EpCs and EMTCs). We recovered significantly higher number of EMTCs in PaCa patients compared to EpCs, reflecting aggressive nature of PaCa. Profiling of CTCs revealed genetic signatures relevant to predicting patients' outcomes. Citation Format: Mina Zeinali, Vasudha Murlidhar, Shamileh Fouladdel, Mathias Hafner, Shimeng Shao, Lili Zhao, Heather Cameron, Armand Iii Bankhead, Jiaqi Shi, Kyle C. Cuneo, Vaibhav Sahai, Ebrahim Azizi, Max S. Wicha, Diane M. Simeone, Sunitha Nagrath. Increased number of EMT-like CTCs relative to epithelial CTCs reveals signatures of poor outcomes in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5579.

The current study investigated adrenomedullin as a potential autocrine regulator of pancreatic cancer cell function. Adrenomedullin was localized in the neoplastic epithelium of 90% (43 of 48) of human pancreatic adenocarcinomas analyzed by immunohistochemistry and was expressed by 100% (8 of 8) of pancreatic cancer cell lines analyzed by reverse transcription-PCR. Pancreatic cancer cell lines also secreted adrenomedullin into the culture medium as determined by ELISA (5 of 5). Exogenous adrenomedullin treatment of Panc-1, BxPC3, and MPanc96 cells in vitro stimulated cell proliferation, invasion, and nuclear factor κB activity, indicating the ability of the cells to respond to adrenomedullin. Treatment of the cell cultures with an adrenomedullin antagonist inhibited basal levels of proliferation and nuclear factor κB activity, supporting the autocrine function of this molecule. Furthermore, increasing adrenomedullin levels by gene transfer to Panc-1 cells increased, whereas adrenomedullin small hairpin RNA silencing in MPanc96 cells inhibited tumor growth and metastasis in vivo. Adrenomedullin is able to act through at least two different receptors, adrenomedullin receptor (ADMR) and calcitonin receptor–like receptor (CRLR). Reverse transcription-PCR and Western blotting indicated that pancreatic cancer cells expressed only ADMR but not CRLR. In contrast, cells found in the tumor microenvironment, primary human pancreatic stellate and endothelial (HUVEC) cells, expressed both ADMR and CRLR. Small hairpin RNA silencing of ADMR in pancreatic cancer cells blocked adrenomedullin-induced growth and invasion, indicating that this receptor is involved in the autocrine actions of adrenomedullin. These data indicate that adrenomedullin acting via ADMR increases the aggressiveness of pancreatic cancer cells and suggests that these molecules may be useful therapeutic targets. [Cancer Res 2007;67(6):2666–75]

Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor, termed cancer stem cells. Although data have been provided to support this theory in human blood, brain, and breast cancers, the identity of pancreatic cancer stem cells has not been determined. Using a xenograft model in which primary human pancreatic adenocarcinomas were grown in immunocompromised mice, we identified a highly tumorigenic subpopulation of pancreatic cancer cells expressing the cell surface markers CD44, CD24, and epithelial-specific antigen (ESA). Pancreatic cancer cells with the CD44+CD24+ESA+ phenotype (0.2–0.8% of pancreatic cancer cells) had a 100-fold increased tumorigenic potential compared with nontumorigenic cancer cells, with 50% of animals injected with as few as 100 CD44+CD24+ESA+ cells forming tumors that were histologically indistinguishable from the human tumors from which they originated. The enhanced ability of CD44+CD24+ESA+ pancreatic cancer cells to form tumors was confirmed in an orthotopic pancreatic tail injection model. The CD44+CD24+ESA+ pancreatic cancer cells showed the stem cell properties of self-renewal, the ability to produce differentiated progeny, and increased expression of the developmental signaling molecule sonic hedgehog. Identification of pancreatic cancer stem cells and further elucidation of the signaling pathways that regulate their growth and survival may provide novel therapeutic approaches to treat pancreatic cancer, which is notoriously resistant to standard chemotherapy and radiation. [Cancer Res 2007;67(3):1030–7]

Bladder cancer is a common and deadly malignancy but its treatment has advanced little due to poor understanding of the factors and pathways that promote disease. ATDC/TRIM29 is a highly expressed gene in several lethal tumor types, including bladder tumors, but its role as a pathogenic driver has not been established. Here we show that overexpression of ATDC in vivo is sufficient to drive both noninvasive and invasive bladder carcinoma development in transgenic mice. ATDC-driven bladder tumors were indistinguishable from human bladder cancers, which displayed similar gene expression signatures. Clinically, ATDC was highly expressed in bladder tumors in a manner associated with invasive growth behaviors. Mechanistically, ATDC exerted its oncogenic effects by suppressing miR-29 and subsequent upregulation of DNMT3A, leading to DNA methylation and silencing of the tumor suppressor PTEN. Taken together, our findings established a role for ATDC as a robust pathogenic driver of bladder cancer development, identified downstream effector pathways, and implicated ATDC as a candidate biomarker and therapeutic target. Cancer Res; 75(23); 5155–66. ©2015 AACR.

Human chromosome 1p35-p36 has long been suspected to harbor a tumor suppressor gene in pancreatic cancer and other tumors. We found that expression of rap1GAP, a gene located in this chromosomal region, is significantly down-regulated in pancreatic cancer. Only a small percentage of preneoplastic pancreatic intraductal neoplasia lesions lost rap1GAP expression, whereas loss of rap1GAP expression occurred in 60% of invasive pancreatic cancers, suggesting that rap1GAP contributes to pancreatic cancer progression. In vitro and in vivo studies showed that loss of rap1GAP promotes pancreatic cancer growth, survival, and invasion, and may function through modulation of integrin activity. Furthermore, we showed a high frequency of loss of heterozygosity of rap1GAP in pancreatic cancer. Collectively, our data identify rap1GAP as a putative tumor suppressor gene in pancreatic cancer. (Cancer Res 2006; 66(2): 898-906)

The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis as well as leads for therapy. We have used a proteomic approach to identify proteins that commonly induce a humoral response in pancreatic cancer. Aliquots of solubilized proteins from a pancreatic cancer cell line (Panc-1) were subjected to two-dimensional PAGE, followed by Western blot analysis in which sera of individual patients were tested for primary antibodies. Sera from 36 newly diagnosed patients with pancreatic cancer, 18 patients with chronic pancreatitis, 33 patients with other cancers, and 15 healthy subjects were analyzed. Autoantibodies were detected against either one or two calreticulin isoforms identified by mass spectrometry in sera from 21 of 36 patients with pancreatic cancer. One of 18 chronic pancreatitis patients and 1 of 15 healthy controls demonstrated autoantibodies to calreticulin isoform 1; none demonstrated autoantibodies to isoform 2. None of the sera from patients with colon cancer exhibited reactivity against either of these two proteins. One of 14 sera from lung adenocarcinoma patients demonstrated autoantibodies to calreticulin isoform 1; 2 of 14 demonstrated autoantibodies to isoform 2. Immunohistochemical analysis of calreticulin in pancreatic/ampullary tumor tissue arrays using an isoform nonspecific antibody revealed diffuse and consistent cytoplasmic staining in the neoplastic epithelial cells of the pancreatic and ampullary adenocarcinomas. The detection of autoantibodies to calreticulin isoforms may have utility for the early diagnosis of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by therapeutic resistance for which the basis is poorly understood. Here, we report that the DNA and p53-binding protein ATDC/TRIM29, which is highly expressed in PDAC, plays a critical role in DNA damage signaling and radioresistance in pancreatic cancer cells. Ataxia-telangiectasia group D-associated gene (ATDC) mediated resistance to ionizing radiation in vitro and in vivo in mouse xenograft assays. ATDC was phosphorylated directly by MAPKAP kinase 2 (MK2) at Ser550 in an ATM-dependent manner. Phosphorylation at Ser-550 by MK2 was required for the radioprotective function of ATDC. Our results identify a DNA repair pathway leading from MK2 and ATM to ATDC, suggesting its candidacy as a therapeutic target to radiosensitize PDAC and improve the efficacy of DNA-damaging treatment. Cancer Res; 74(6); 1778–88. ©2014 AACR.

Introduction: Approximately 5-10% of pancreatic cancer diagnoses are associated with inherited cancer syndromes and family cancer history assessment is a critical component of identifying these high risk families. We developed and validated a comprehensive web-based family history survey, which collects cancer type and age of diagnosis for first and second degree relatives directly from patients, generating a summary and cancer risk model scores for medical care teams in real-time. We tested feasibility and performance of this tool in an outpatient pancreatic cancer clinic setting. Methods: 161 patients age 18 years and older, with appointments at the University of Michigan Multidisciplinary Pancreatic Tumor Clinic between September 2015 and February 2016 were approached during their clinic visit to complete the web-based survey on an iPad device. Responses were compared to the clinic’s current family history collection method with medical assistants entering data from paper forms into the electronic medical record (EMR) during the intake process. Data completeness, and proportion of patients identified as meeting criteria for genetic referral were compared between the two survey modalities. Results: 92.5% of patients approached agreed to take the iPad survey. Time to complete the survey ranged from 2-50 minutes (mean 12 minutes). Patient ages ranged from 24 to 87 years, with a median age of 64. Patients who were unfamiliar with the iPad technology and/or had difficulty recalling ages of diagnoses of relatives accounted for a greater amount of time taken to complete the survey. Only 6.0% of patients were unable to complete the survey prior to the end of their visit. Of the 128 patients that completed the iPad survey, 82 (64.1%) had a personal history of pancreatic cancer; the remaining 46 (35.9%) presented with a personal or family history suggestive of increased pancreatic cancer risk. Based on clinical criteria for genetic referral for known inherited syndromes related to increased pancreatic cancer risk, we determined that 53.7% of patients with a pancreatic cancer diagnosis met criteria for genetic counseling by iPad survey vs 41.4% by the paper form. Of patients without a pancreatic cancer diagnosis, 23.9% met genetic referral criteria using the iPad tool vs 21.7% using the paper form. Among patients with pancreatic cancer, the most common reasons for meeting high risk criteria were having another family member with pancreatic cancer (12.2%) or having two or more relatives on the same side of the family with cancers associated with increased pancreatic cancer risk (e.g., breast, ovarian, melanoma; 17.1%). Among those without a personal history of pancreatic cancer, the most common reasons for meeting high risk criteria were family history of ovarian cancer (6.5%), and having three or more related family members with the aforementioned cancers (13.0%). Conclusions: Assessment of family history using a web-based tool is feasible, as demonstrated by high participation and completion rates among all age groups. Both iPad and paper surveys identified a significant percentage of patients for genetic referral. Improvements in obtainment of cancer relevant family history and better inclusion of such data in the EMR may play an important role in better screening of high risk individuals for pancreatic and other types of hereditable cancers. Citation Format: Kara Anne Schradle, Heather Gail Cameron, Riya Kumar, Erika Koeppe, Elana Leflein, Cristina Moisa, Matthew Demerath, Elena Martinez Stoffel, Diane M. Simeone.{Authors}. Assessment of family cancer history in a pancreatic cancer clinic: A pilot study of a web-based survey. [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 A11.

Introduction and Objective: Pancreatic cancer is a devastating disease with a 5-year survival rate of less than 6%. By the time of diagnosis, less than 15% of patients have surgically resectable tumors, which is one reason why it has the highest mortality rate among all cancer types. Emerging evidence has pointed the importance of circulating tumor cells (CTCs) in the spread of cancers and metastasis. CTCs are believed to be the most promising cancer biomarkers available in blood. Recent reports also show that these cells could be a good surrogate biomarker for not only prognosis, but also for cancer detection and development of personalized treatment. We have developed an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs. Methods: To investigate the isolation efficiency of our technology before running cancer patients’ samples, PANC-1 cells labeled with cell tracker dye were spiked into 1 mL of whole blood. Using this optimized platform we isolate CTCs from pancreatic cancer patients, followed by downstream analysis . CTCs are characterized using immunocytochemistry for heterogeneity studies among CTC population. Results: Our microfluidic device consists of a size based separation platform in which CTCs are separated from other blood components. This high throughput label free separation device was designed and developed by our lab. After more than 20 generations of device revision, an optimized flow rate of 2.5 ml/min for PANC-1 pancreatic cancer cell line has yielded a 95% recovery of these cells from whole blood, with cancer cells traveling to the second outlet and 80% of the White Blood Cells (WBC) to the first outlet. We have successfully isolated CTCs from over 100 pancreatic cancer patients. Among several patients we were able to characterize different subpopulations using the epithelial marker CK-19 and EpCAM, and the EMT-like marker ZEB1. Conclusion: The implementation of our engineering technology to the study of pancreatic cancer can present novel ways to confront major hurdles in cancer research, such as early detection and the lack of effective therapeutics. The isolation and expansion of CTCs, the heterogeneous population of cells that promote metastasis, can provide meaningful information to elucidate the process of pancreatic tumorigenesis to preempt its fatal result. Citation Format: Lianette Rivera-Baez, Eric Lin, Meghna Waghray, Shamileh Fouladdel, Ebrahim Azizi, Max Wicha, Vaibhav Sahai, Diane Simeone, Sunitha Nagrath.{Authors}. Biomarker independant microfluidic Labyrinth for the high throughput enrichment and characterization of circulating tumor cells in pancreatic cancer. [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 A10.

Pancreatic cancer is characterized by the accumulation of a fibro-inflammatory stroma. Accumulation of the stroma is already evident surrounding Pancreatic Intraepithelial Neoplasias (PanINs), common precursor lesions to pancreatic cancer (Hezel et al., 2006). The stroma is abundantly infiltrated by immune cells, and myeloid cells are a predominant population (Clark et al., 2007). Different myeloid subsets have been correlated with tumor promotion and unmasking of anti-tumor immunity (Liou et al., 2015; Long et al., 2016; Mitchem et al., 2013; Stromnes et al., 2014). Both PanINs and pancreatic cancer and commonly associated with oncogenic mutations in the Kras gene (Biankin et al., 2012; Jones et al., 2010; Kanda et al., 2012). Expression of oncogenic Kras in the pancreas of genetically engineered mice recapitulates the PanIN to pancreatic cancer progression, including the accumulation of fibrotic stroma (Hingorani et al., 2003). We have described a mouse model that allows inducible and reversible expression of oncogenic Kras in the pancreas, the iKras* mouse. Inactivation of oncogenic Kras during the PanIN stage or in cancer leads to regression of the epithelial lesions as well as to remodeling of the stroma, indicating that the accumulation of the stroma is regulated by signals derived from oncogenic Kras-expressing epithelial cells (Collins et al., 2012a). In the current study, we have investigated the interaction between epithelial cells and myeloid cells that infiltrate the pancreas. For this purpose, we have used a combination of genetically engineered mice (iKras*p53* mice (Collins et al., 2012b)) and transplantation approaches into CD11b-DTR mice (Duffield et al., 2005), that allow depletion of myeloid cells upon administration of Diphtheria Toxin. Our results show that the infiltration and polarization of macrophages in the pancreas depends on signals derived from oncogenic Kras-expressing epithelial cells, either directly or through activation of a pro-inflammatory subset of stromal fibroblasts. Conversely, myeloid cells infiltration is required for the progression of PanINs and pancreatic cancer. Depletion of myeloid cells prevented KrasG12D driven pancreatic cancer initiation. In pre-established tumors, myeloid cell depletion resulted in arrest of growth or tumor regression. We observed that tumor progression was dependent on myeloid cell-mediated blockade of CD8+ T cell anti-tumor activity. Furthermore, myeloid cells regulate the expression of the Programmed death-ligand 1 (PD-L1) in tumor cells in an EGFR/MAPK dependent manner. Our results show that myeloid cells regulate a complex network of signals that ensure immune suppression within the pancreatic cancer microenvironment. Moreover, we show that depletion of the myeloid cell population restores anti-tumor immunity mediated by CD8+ T cells, a finding with implications for the design of immune therapies for pancreatic cancer. References: Biankin, A. V., Waddell, N., Kassahn, K. S., Gingras, M. C., Muthuswamy, L. B., Johns, A. L., Miller, D. K., Wilson, P. J., Patch, A. M., Wu, J., et al. (2012). Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature 491, 399-405. Clark, C. E., Hingorani, S. R., Mick, R., Combs, C., Tuveson, D. A., and Vonderheide, R. H. (2007). Dynamics of the immune reaction to pancreatic cancer from inception to invasion. Cancer Res 67, 9518-9527. Collins, M. A., Bednar, F., Zhang, Y., Brisset, J. C., Galban, S., Galban, C. J., Rakshit, S., Flannagan, K. S., Adsay, N. V., and Pasca di Magliano, M. (2012a). Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice. J Clin Invest 122, 639-653. Collins, M. A., Brisset, J. C., Zhang, Y., Bednar, F., Pierre, J., Heist, K. A., Galban, C. J., Galban, S., and di Magliano, M. P. (2012b). Metastatic pancreatic cancer is dependent on oncogenic Kras in mice. PLoS One 7, e49707. Duffield, J. S., Forbes, S. J., Constandinou, C. M., Clay, S., Partolina, M., Vuthoori, S., Wu, S., Lang, R., and Iredale, J. P. (2005). Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Invest 115, 56-65. Hezel, A. F., Kimmelman, A. C., Stanger, B. Z., Bardeesy, N., and Depinho, R. A. (2006). Genetics and biology of pancreatic ductal adenocarcinoma. Genes Dev 20, 1218-1249. Hingorani, S. R., Petricoin, E. F., Maitra, A., Rajapakse, V., King, C., Jacobetz, M. A., Ross, S., Conrads, T. P., Veenstra, T. D., Hitt, B. A., et al. (2003). Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4, 437-450. Jones, S., Wang, T. L., Shih Ie, M., Mao, T. L., Nakayama, K., Roden, R., Glas, R., Slamon, D., Diaz, L. A., Jr., Vogelstein, B., et al. (2010). Frequent mutations of chromatin remodeling gene ARID1A in ovarian clear cell carcinoma. Science 330, 228-231. Kanda, M., Matthaei, H., Wu, J., Hong, S. M., Yu, J., Borges, M., Hruban, R. H., Maitra, A., Kinzler, K., Vogelstein, B., and Goggins, M. (2012). Presence of somatic mutations in most early-stage pancreatic intraepithelial neoplasia. Gastroenterology 142, 730-733 e739. Liou, G. Y., Doppler, H., Necela, B., Edenfield, B., Zhang, L., Dawson, D. W., and Storz, P. (2015). Mutant KRAS-induced expression of ICAM-1 in pancreatic acinar cells causes attraction of macrophages to expedite the formation of precancerous lesions. Cancer Discov 5, 52-63. Long, K. B., Gladney, W. L., Tooker, G. M., Graham, K., Fraietta, J. A., and Beatty, G. L. (2016). IFNgamma and CCL2 Cooperate to Redirect Tumor-Infiltrating Monocytes to Degrade Fibrosis and Enhance Chemotherapy Efficacy in Pancreatic Carcinoma. Cancer Discov. Mitchem, J. B., Brennan, D. J., Knolhoff, B. L., Belt, B. A., Zhu, Y., Sanford, D. E., Belaygorod, L., Carpenter, D., Collins, L., Piwnica-Worms, D., et al. (2013). Targeting tumor-infiltrating macrophages decreases tumor-initiating cells, relieves immunosuppression, and improves chemotherapeutic responses. Cancer Res 73, 1128-1141. Stromnes, I. M., Brockenbrough, J. S., Izeradjene, K., Carlson, M. A., Cuevas, C., Simmons, R. M., Greenberg, P. D., and Hingorani, S. R. (2014). Targeted depletion of an MDSC subset unmasks pancreatic ductal adenocarcinoma to adaptive immunity. Gut. Citation Format: Yaqing Zhang, Esha Mathew, Ashley Velez-Delgado, Kristen B. Long, Dongjun Li, Flor M. Mendez, Kevin Flannagan, Andrew D. Rhim, Diane M. Simeone, Gregory L. Beatty, Marina Pasca di Magliano.{Authors}. Myeloid cells are required for PD-1/PD-L1 checkpoint activation and the establishment of an immune-suppressive environment in pancreatic cancer [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 IA21.

We have recently demonstrated that ATDC, a novel oncogenic protein, serves as an invasive switch in pancreatic cancer (PDA) by activation of beta–catenin signaling and upregulation of CD44, resulting in EMT and an invasive phenotype during PanIN progression. To further explore the tumorigenic function of ATDC, we generated a floxed ATDC mouse (A F/F) to evaluate the impact of conditional knockout of ATDC on oncogenic Kras-induced PDA initiation and progression. Pancreas-specific ATDC knockout did not cause any histologic abnormalities in pancreas, up to 1 year of age (n=8). Through a series of crosses of LSL-KrasG12D (K), p53F/+ (P), RosaYFP (Y), Pdx1-Cre (C) and AF/F mice, KrasG12D; CY (KCY); KrasG12D; p53+/-; CY (KPCY), KCYA-/- KPCYA-/- mice were generated. Knockout of ATDC in KPCY mice completely prevented the development of ADM and PanIN lesions in 3 month old mice (n= 8), and resulted in the formation of very rare ADM and PanIN1 lesions (2 out of 8) in KPCYA-/- mice at 12 months of age (n=8). In contrast, all KPCY mice developed extensive PanIN (low and high grade) at 3 months of age (n= 8), with the subsequent development of invasive and metastatic cancer at frequencies similar to that reported in the literature. To determine the possible mechanisms by which ATDC inhibited KrasG12D-induced acinar-ductal metaplasia (ADM), we isolated acini from 1.5 month old KCY and KCYA-/- pancreata and performed in vitro 3D cultures and ADM assays. ADM lesions readily formed in 3-D cultures of acini from KCY mice at 5 days, and this was significantly inhibited in acini isolated from KCYA-/- mice (duct-like structures: 95.1±3.5% to 28.0±2.2%*, KCY vs KCYA-/-, n=3, *p Citation Format: Lidong Wang, Huibin Yang, Ethan V. Abel, Phillip L. Palmbos, Christophe Halbrook, Kenneth Takeuchi, Jiaqi Shi, Yaqing Zhang, Sumithra Urs, Meghna Waghray, Marina Pasca di Magliano, Andrew D. Rhim, Howard C. Crawford, Diane M. Simeone.{Authors}. ATDC is required for KRAS-induced pancreatic tumorigenesis. [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 A62.

Background: Gemcitabine based chemoradiation for locally advanced pancreatic cancer (LAPC) is safe and effective, although most patients remain unresectable and freedom from local progression is not ensured. Targeting cell cycle checkpoints may enhance the efficacy of chemoradiation. Tumor cells commonly have an abnormal G1 checkpoint due to mutations in p53 or its pathway making them reliant on the G2 checkpoint to arrest and repair DNA damage after radiation. In our preclinical studies, WEE1 inhibition with AZD1775 abrogates the G2 checkpoint and sensitizes pancreatic cancer cell lines and xenografts to gemcitabine and radiation. Additionally, AZD1775 attenuates homologous recombination and promotes replication stress in cancer cells. Given these preclinical findings, we designed a phase I dose escalation study of the WEE1 inhibitor AZD1775 in combination with gemcitabine and radiation in patients with LAPC (NCT02037230). Methods: The primary objective of this phase I study is to determine the maximum tolerated dose (MTD) of AZD1775 combined with gemcitabine and radiation in patients with LAPC. Secondary objectives include estimation of the efficacy of this regimen at the target dose and to determine if WEE1 is inhibited by AZD1775 at or below its target dose in surrogate tissues. Protocol therapy consists of the administration of AZD1775 and gemcitabine at an assigned dose level in accordance with a Time-to-Event Continual Reassessment Method (TITE-CRM). Study treatment consists of four 21 day cycles. Gemcitabine is given on day 1 and day 8, and AZD1775 on days 1, 2 and 8, 9 of each cycle. Cycles 2 and 3 are administered with concurrent radiation, 52.5 Gy in 25 daily fractions to gross disease using volumetric modulated arc therapy to spare normal tissues. Following cycle 3, subjects have a 3 week break prior to cycle 4. The MTD will be determined by the development of dose limiting toxicities within the first 105 days of therapy with a target dose limiting toxicity rate of 25%. Tumor assessment using CT or MRI is performed at baseline, 1 month after radiation, and approximately every 3 months afterwards, with response characterized per RECIST. Blood samples obtained at baseline and after cycles 1, 2, and 4 will be used for correlative studies on circulating tumor cells and tumor derived exosomes. Target accrual is 36 patients; to date, 16 patients have been enrolled. Dose Escalation SchemaRadiation (Gy)Gemcitabine(mg/m⁁2)AZD1775 (mg)Level -252.5800100Level -152.51000100Level 052.51000125Level 152.51000150Level 252.51000175 Citation Format: Kyle C. Cuneo, Meredith Morgan, Matthew J. Schipper, Jonathan Maybaum, Mary U. Feng, Mahmoud Al-Hawary, Diane M. Simeone, Leah D. Olson, Vaibhav Sahai, Mark Zalupski, Theodore S. Lawrence. A dose escalation trial of the WEE1 inhibitor AZD1775, gemcitabine, and concurrent radiation in locally advanced pancreatic 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 CT035.

Introduction and Objective: The metastasis of cancer is preceded by the dissemination of cancer cells from the primary tumor site to remote sites via the blood circulation. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival. Immune-affinity based capture, although being the most commonly used method for the isolation of CTCs, offers low throughput (∼1mL/hr) and have considerably cell loss caused by the heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing the physical properties of CTCs have been developed to overcome the limitations, such as micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs yielding the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. The isolated CTC populations were further analyzed for single cell multiplex gene expression analysis. Methods: The PDMS-made inertial microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation of CTCs is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. Device is optimized with MCF-7 and Panc-1 cell line within PBS buffer solution and diluted blood, and is tested in patients with breast cancer on an average of 5 mL of whole blood processed through double devices in series. CTCs isolated were analyzed for tumor specific protein markers and genomic characterization is done using singe cell analysis techniques. Results: Samples are processed through the inertial microfluidic device and CTCs are enriched in second outlet based on size difference between CTCs and blood cells. Device is optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (greater than 90%) and high white blood cells (WBCs) removal (5 log orders). In patient samples, we identified CTCs in 38 of 40 (95%) breast patients with metastatic disease (5.4±4.6 CTC/mL) with low WBC contamination (663±647 WBC/mL). Based on the gene expression, both inter and intra patient heterogeneity of CTCs at the single cell level were discovered among the tested patient samples. Conclusion: The study of CTCs could have a direct impact upon society by presenting novel ways to address one of the major hurdles in cancer research - early detection - and will foster the advancement of science and engineering via the exploration of new druggable targets approaches and the further understanding of the pharmacodynamics. Citation Format: Eric Lin, Lianette Rivera, Shamileh Fouladdel, Hyeun Joong Yoon, Stephanie Guthrie, Jacob Weiner, Yadwinder S. Deol, Evan Keller, Vaibhav Sahai, Diane M. Simeone, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Sunitha Nagrath. Label-free high throughput microfluidic device for the isolation and single cell multiplex gene expression analysis of circulating tumor cells from breast cancer patients. [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 3307.

Hyperactivation of KRAS and inactivation of CDKN2A [p16] play a prominent role in tumor initiation and progression in a broad spectrum of human cancers. In pancreatic adenocarcinoma, KRAS mutation occurs in 90-95% of cases and is often coupled with inactivation of CDKN2A (>90% incidence), typically by homozygous deletion. Based on the high incidence of these genetic events, the present study addresses the hypothesis that dual targeting of MEK and CDK4/6 represents a viable therapeutic strategy for the treatment of pancreatic adenocarcinoma. A panel of primary and high passage xenograft models was screened for in vitro synergy to the antiproliferative effects of the MEK inhibitor trametinib and the CDK4/6 inhibitor palbociclib. Two models that emerged as highly sensitive to this combination treatment strategy, L3.6pl and UM59, both exhibited G1 cell cycle arrest that was significantly more pronounced in response to the combination compared to either single agent. In vivo studies were subsequently carried out to evaluate the efficacy of this combination in tumor-bearing mice. Mice implanted subcutaneously with L3.6pl or UM59 cells were treated daily by oral gavage for ten days with trametinib (3 mg/kg), palbociclib (100 mg/kg), or the combination of these two agents at these doses. Consistent with in vitro synergy observations, both models proved to be exceptional responders in vivo to this combination treatment strategy, showing a robust response to the combination not seen with either single agent. These results suggest that the combined inhibition of MEK and CDK4/6 may offer a valuable therapeutic strategy for the treatment of pancreatic adenocarcinoma. Studies are ongoing to identify molecular determinants of response in pancreatic tumors that are highly sensitive to this combination approach to guide future patient enrichment strategies. Citation Format: Joel D. Maust, Diane Simeone, Judith Sebolt-Leopold. Cotargeting MEK and CDK4/6 to treat pancreatic adenocarcinoma. [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 3018.

The median survival for patients with locally advanced pancreatic cancer treated with gemcitabine and radiation is approximately 1 year. To develop improved treatment, we have combined a Chk1/2-targeted agent, AZD7762, currently in phase I clinical trials, with gemcitabine and ionizing radiation in preclinical pancreatic tumor models. We found that in vitro AZD7762 alone or in combination with gemcitabine significantly sensitized MiaPaCa-2 cells to radiation. AZD7762 inhibited Chk1 autophosphorylation (S296 Chk1), stabilized Cdc25A, and increased ATR/ATM–mediated Chk1 phosphorylation (S345 Chk1). Radiosensitization by AZD7762 was associated with abrogation of the G2 checkpoint as well as with inhibition of Rad51 focus formation, inhibition of homologous recombination repair, and persistent γ-H2AX expression. AZD7762 was also a radiation sensitizer in multiple tumor xenograft models. In both MiaPaCa-2- and patient-derived xenografts, AZD7762 significantly prolonged the median time required for tumor volume doubling in response to gemcitabine and radiation. Together, our findings suggest that G2 checkpoint abrogation and homologous recombination repair inhibition both contribute to sensitization by Chk1 inhibition. Furthermore, they support the clinical use of AZD7762 in combination with gemcitabine and radiation for patients with locally advanced pancreatic cancer. Cancer Res; 70(12); 4972–81. ©2010 AACR.

Activated T cells may express FOXP3. It is thought that FOXP3 is not a specific marker to determine regulatory T cells (Treg) in humans. Here, we examined the functional phenotype and cytokine profile of the in vitro induced FOXP3+ T cells, primary FOXP3+ and FOXP3- T cells in patients with ulcerative colitis and tumors including colon carcinoma, melanoma, hepatic carcinoma, ovarian carcinoma, pancreatic cancer, and renal cell carcinoma. We observed similar levels of suppressive capacity of primary FOXP3+ T cells in blood, tumors, and colitic tissues. Compared with primary FOXP3- T cells in the same microenvironment, these primary FOXP3+ T cells expressed minimal levels of effector cytokines, negligible amount of cytotoxic molecule granzyme B, and levels of suppressive molecules interleukin-10 and PD-1. Although the in vitro activated T cells expressed FOXP3, these induced FOXP3+ T cells expressed high levels of multiple effector cytokines and were not functionally suppressive. The data reinforce the fact that FOXP3 remains an accurate marker to define primary Tregs in patients with cancer and autoimmune disease. We suggest that the combination of FOXP3 and cytokine profile is useful for further functionally distinguishing primary Tregs from activated conventional T cells. [Cancer Res 2009;69(9):3995–4000]

Pancreatic ductal adenocarcinoma (PDA) is a hierarchal cancer consisting of tumor-initiating cancer stem cells (CSCs) and transiently proliferating bulk tumor cells. Despite this understanding, the biological properties of CSCs remain incompletely defined, including the role of transcriptional programs like epithelial-mesenchymal transition (EMT). Using flow cytometry in conjunction with epithelial-cell surface marker ESA (EpCAM) and mesenchymal-cell surface marker CD44, we identified three subpopulations of PDA cells derived from primary patient samples: CD44highESAlow, CD44lowESAhigh, and CD44highESAhigh cells, each with different biological characteristics. CD44highESAlow cells exhibited a more mesenchymal phenotype characterized by high expression of vimentin and low expression of E-cadherin. CD44lowESAhigh cells, by contrast, highly epithelial in appearance, expressed high levels of E-cadherin and low levels of vimentin. Finally, CD44highESAhigh cells showed a phenotype intermediate between CD44highESAlow and CD44lowESAhigh cells. In isolation, all subpopulations were able to self-renew, however, only CD44highESAhigh cells could give rise to all three cell subpopulations. Additionally, CD44highESAhigh cells exhibited the highest ability to form tumorspheres, an indicator of CSC-functionality, in vitro, and had a greater ability to form tumors in vivo, suggesting that this subpopulation enriched for CSC functional activity. Using microarray analysis, we identified a set 50 genes, including the CSC marker CD24 and the transcription factor HNF1A, which showed significant up-regulation in CD44highESAhigh cells CSC population compared to either CD44highESAlow or CD44lowESAhigh cells. We hypothesized that these genes might be critical to CSC biology in PDA. Using bioinformatics analysis to identify possible transcriptional regulators of these 50 genes, we identified HNF1A as the factor with most highly overrepresented binding sites in the promoter regions of (17/50 genes), suggesting a possible role in regulating CD44highESAhigh-specific genes and serving as a key biological regulator of the CSC state. Supporting this hypothesis, knockdown of HNF1A decreased expression of a number of the candidate genes, including CD24, in the CD44highESAhigh population. By contrast, ectopic expression of HNF1A resulted in the up-regulation of a similar set of candidate genes, including CD24, and promoted the formation of tumorspheres. These findings shine light on the complexity of PDA tumor cell heterogeneity, suggesting the CSCs exist in a state between EMT and mesenchymal-epithelial transition, and implicates HNF1A as a key regulator of CSC-associated genes and CSC functionality. Citation Format: Ethan V. Abel, Masashi Goto, Nikita Ramanathan, Chandan Kumar, Lesa Begley, Michele L. Dziubinski, Lidong Wang, Meghna Waghray, Sumithra Urs, Diane M. Simeone. Pancreatic cancer stem cell function is regulated by HNF1A. [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 2335. doi:10.1158/1538-7445.AM2015-2335

Pancreatic ductal adenocarcinoma (PDAC) arises from two precursor lesions, pancreatic intraepithelial neoplasias and pancreatic cysts, such as intraductal papillary mucinous neoplasias (IPMNs). Since cyst lesions are readily detected on cross sectional imaging, early diagnosis of IPMN-associated PDAC and intervention may be feasible. However, clinical guidelines dictating which IPMNs are high-risk and require surgical resection are suboptimal. Recently, the genomic signature of IPMN-associated carcinomas has been described opening the possibility for targeted genomic analysis. Previous groups have sought to achieve this using conventional methods. However, since IPMN tissue and cyst fluid are limited in quantity and contain inhibitors of PCR, we developed a novel microfluidics-based approach to achieve sensitive and specific targeted amplification and Illumina library construction. We have optimized a novel method to enrich targeted genomic regions for next generation sequencing (NGS) platforms featuring microfluidic partitioning of the sample into uniform picoliter volume droplets containing single molecules of target DNA. All droplets contain all of the primers and PCR reagents, ensuring that every target molecule from the sample is amplified, and after endpoint PCR results in a highly uniform yield that facilitates efficient use of the NGS platform. In addition, the primers contain ‘Illumina tails’ that enable easy sample indexing and loading directly onto a MiSeq without additional library preparation. Here we detail the successful customization and use of the ThunderBolts Cancer Panel, which targets 230 commonly mutated regions in 50 cancer associated genes, with additional primers for commonly mutated genes in PDAC. Addition of the 37 PDAC-relevant primer pairs to the commercially available core panel was very straightforward and resulted in sequencing metrics similar to those of the core panel alone (100% coverage at 100x depth, mean read depth of 2500). Cyst fluid from 30 patients with IPMN resected under Sendai criteria were analyzed. We describe the mutational signature of cyst fluid and relate the presence and quantity of mutations in KRAS, GNAS and PIK3CA to the presence of invasive carcinoma and high grade dysplasia on pathology. Finally, unbiased and high resolution sequencing was obtained using this protocol with as little as 8ng of input DNA. Using picodroplet PCR technology we were able to achieve unprecedented sequencing performance on ultra-low sample inputs. Given the ease of use and customization of the panel, the same platform may be readily adapted to other applications in which samples are limited or are difficult to amplify. Future studies will utilize this platform for an expanded analysis of pancreatic cyst fluid with additional primers. Citation Format: Angelina I. Londoño-Joshi, Erica Pratt, Robert W. Cowan, Michael L. Samuels, Steve Kotsopoulos, Jeff Olson, Francis Long, Michelle A. Anderson, Diane Simeone, Andrew D. Rhim. Sensitive and robust targeted sequencing of pancreatic precancer and tumors using microfluidic single-molecule enrichment. [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 1572. doi:10.1158/1538-7445.AM2015-1572

Establishing the role of circulating tumor cells (CTCs) in tumor progression and metastasis depends both on enumeration and on obtaining sufficient numbers of cells for downstream assays. The numbers of CTCs are few in early stages of cancer, limiting detailed molecular characterization. One approach to overcome this limitation is to expand CTCs, i.e. increase their numbers as a result of cell division. Herein, we have developed a novel in-situ capture and culture methodology for ex-vivo expansion of CTCs using a three dimensional co-culture model, simulating a tumor microenvironment to support tumor development. We have successfully identified CTCs isolated from 14 of 19 early stage lung cancer patients. Expanded lung CTCs expressed tumor specific markers and formed spheroids in culture. CTCs derived xenograft demonstrated tumorigenic capability of these cells. Furthermore, CTCs carried mutations of the TP53 gene identical to those observed in the matched primary tumors. Next-generation sequencing further revealed additional matched mutations between primary tumor and CTCs of cancer-related genes. This strategy sets the stage to further characterize the biology of CTCs derived from patients with early lung cancers, thereby leading to a better understanding of these putative drivers of metastasis. Citation Format: Zhuo Zhang, Hiroe Shiratsuchi, Jules Lin, Guoan Chen, Rishindra M. Reddy, Ebrahim Azizi, Shamileh Fouladdel, Andrew C. Chang, Lin Lin, Hui Jiang, Meghna Waghray, Diane M. Simeone, Max S. Wicha, David G. Beer, Gary Luker, Nithya Ramnath, Sunitha Nagrath. Expansion of CTCs from early stage lung cancer patients using a microfluidic co-culture model. [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 370. doi:10.1158/1538-7445.AM2015-370

Introduction and Objective: Pancreatic cancer is a devastating disease with a 5-year survival rate of less than 6%. By the time of diagnosis, less than 15% of patients have surgically resectable tumors, which is one reason why it has the highest mortality rate among all cancer types. Emerging evidence has pointed the importance of circulating tumor cells (CTCs) in the spread of cancers and metastasis. CTCs are believed to be the most promising cancer biomarkers available in blood. Recent reports also show that these cells could be a good surrogate biomarker for not only prognosis, but also for cancer detection and development of personalized treatment. We have developed an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs. Methods: To investigate the isolation efficiency of our technology before running cancer patients’ samples, PANC-1 cells labeled with cell tracker dye were spiked into 1 mL of whole blood. Using this optimized platform we isolate CTCs from pancreatic cancer patients, followed by downstream analysis. CTCs are characterized using immunocytochemistry for heterogeneity studies among CTC population. Results: Our microfluidic device consists of a size based separation platform in which CTCs are separated from other blood components. This high throughput label free separation device was designed and developed by our lab. After more than 20 generations of device revision, an optimized flow rate of 2.5 ml/min for PANC-1 pancreatic cancer cell line has yielded a 95% recovery of these cells from whole blood, with cancer cells traveling to the second outlet and 80% of the White Blood Cells (WBC) to the first outlet. We have successfully isolated CTCs from over 100 pancreatic cancer patients. Among several patients we were able to characterize different subpopulations using the epithelial marker CK-19 and EpCAM, and the EMT-like marker ZEB1. Conclusion: The implementation of our engineering technology to the study of pancreatic cancer can present novel ways to confront major hurdles in cancer research, such as early detection and the lack of effective therapeutics. The isolation and expansion of CTCs, the heterogeneous population of cells that promote metastasis, can provide meaningful information to elucidate the process of pancreatic tumorigenesis to preempt its fatal result. Citation Format: Lianette Rivera-Baez, Eric Lin, Hyeun Joong Yoon, Meghna Waghray, Shamileh Fouladdel, Ebrahim Azizi, Max Wicha, Diane Simeone, Sunitha Nagrath. Application of label-free microfluidic technologies for the enrichment, expansion and characterization of circulating tumor cells 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 1594. doi:10.1158/1538-7445.AM2015-1594

Introduction and Objective: A necessary step in distant metastasis is the hematogenous dissemination of cancer cells from the primary tumor site to remote sites. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival in many solid malignancies. Immune-affinity based capture is the most commonly used method for the isolation of CTCs which utilizes antibodies to capture tumor cells expressing specific proteins. However, immune-affinity based approaches offer low throughput (∼1mL/hr) and considerable cell loss (∼20-40%) resulting from heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing physical properties of CTCs have been developed to overcome the limitations of immune-affinity based isolation techniques, including micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs that yields the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. Methods: The PDMS-made microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. The device was optimized with MCF-7 and Panc-1 cell lines spiked into PBS buffer and also diluted blood. It was then tested on 10 mL blood samples from patients with metastatic breast cancer. The separated cells were cytospun and stained to identify CTCs as cytokeratin positive, DAPI positive, CD45 negative cells. Results: Samples were processed through the inertial microfluidic device that enriches CTCs in the second outlet based on size difference between CTCs and blood cells. The device was optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (92% for both spiked samples of MCF-7 and PANC-1 cell lines) and high white blood cells (WBCs) removal (91%). To determine the efficiency of capture of rare cell populations, healthy donor blood samples were spiked with MCF-7/GFP at 100 cells and 95% recovery was obtained. In patient samples, we identified CTCs in 24 of 27 (89%) breast patients with metastatic disease (4.3±4.8 CTCs/mL) with low WBCs contamination (465±473 WBCs/mL). Conclusion: The study of CTCs could have a direct impact upon patient care by presenting a novel CTC isolation method. This technology may be applicable to early detection, and also for monitoring response to treatment. Our approach is superior to current strategies because it is independent of cell surface markers, which may be varied in tumor cells, and may exclude cancer stem cells. Citation Format: Eric Lin, Lianette Rivera, Hyeun Joong Yoon, Shamileh Fouladdel, Jacob Wieger, Stephanie Guthrie, Yadwinder S. Deol, Shawn G. Clouthier, Tahra K. Luther, Diane M. Simeone, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Sunitha Nagrath. Label-free high throughput microfluidic device for the isolation of circulating tumor cells from breast cancer patients. [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 1593. doi:10.1158/1538-7445.AM2015-1593

Usp9x regulates tumor cell survival and responsiveness to targeted-, chemo- and radio-therapy in a broad number of tumors through effects on multiple proteins. The role of Usp9x in human pancreatic cancer is largely unknown, although a recent study demonstrated that Usp9x co-operated in promoting KrasG12D tumorigenesis in mice. To examine Usp9x tumorigenicity and therapeutic potential of Usp9x inhibition in human pancreatic cancer vs. mouse pancreatic tumor models we developed 3D cultures of cell lines established from constitutive (8041) and doxycycline-inducible (4668) KrasG12D/Tp53R172H mouse pancreatic tumors, established human cell lines (BxPC3, PANC1 and MIAPACA2) and spontaneously immortalized human pancreatic patient tumor derived cell lines (UM2, UM6, UM16 and UM76). The effect of Usp9x knockdown (KD), overexpression (OE) or Usp9x activity inhibition by our small molecule deubiquitinase inhibitor EOAI3402143 (G9) were assessed by growth in 2D and 3D culture and anti-tumor efficacy studies in mouse (8041) and human (MIAPACA2) xenograft models. Usp9x KD in constitutive KrasG12D/Tp53R172H 8041 cells led to a 3-fold increase in the number of colonies formed in 3D and Usp9x KD sustained 3D colony growth even after withdrawal of mutant Kras support in KrasG12D/Tp53R172H inducible 4668 cells. Usp9x-OE in 8041 cells decreased 3D colony growth by 2.5-fold. These results contrast with outcomes in human pancreatic tumors where Usp9x KD induced either rapid apoptosis (in MIAPACA2) or reduced 3D colony formation by >50% (in PANC1). Usp9x-OE in PANC1 cells enhanced 3D colony growth (2-fold) and increased invasion activity in Boyden chambers (3.5-fold). Mechanistically, we found differential effects of Usp9x on mutant Kras protein expression levels in mouse vs human cells. In vivo G9 treatment (15 mg/kg, every other day) of mouse 8041 xenografts showed a statistically insignificant trend towards tumor growth inhibition but effectively inhibited MIAPACA2 tumor growth in mice. Usp9x KD in human pancreatic patient tumor cell lines inhibited 3D colony growth by >75% and this activity was phenocopied by G9 with nM efficacy. We conclude that Usp9x acts as a tumor promoter in established human pancreatic cancer and a tumor suppressor in murine cells from genetically engineered pancreatic tumors, possibly through differential regulation of mutant Kras. Overall, these results suggest that Usp9x may be a novel therapeutic target in pancreatic cancer. Note: This abstract was not presented at the meeting. Citation Format: Anupama Pal, Marina Pasca Di Magliano, Diane Simeone, Luke Peterson, Harish Potu, Moshe Talpaz, Nicholas Donato. Usp9x as a novel therapeutic target in human 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 1748. doi:10.1158/1538-7445.AM2015-1748

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Many characteristic mutations in PDAC are known, but this information has so far failed to produce the development of effective treatments, highlighting the need for deeper understanding of the processes that drive tumorigenesis. B-cell specific Moloney murine leukemia virus insertion site 1 (Bmi1), a Polycomb repressive group protein, is upregulated in PDAC and associated with poor prognosis. Our lab has shown that despite an oncogenic K-ras mutation, mice with pancreas specific loss of Bmi1 do not develop precancerous lesions, termed PanINs (Pancreatic Intraepithelial Neoplasia). This lack of PanIN development was also seen in the presence of pancreatitis, which is usually known to synergize with oncogenic K-ras to speed PanIN development. In other cancer types, Bmi1's effect on tumorigenesis was mechanistically linked to its regulation of the Ink4a/ARF genetic locus. However, we found that in PDAC, PanIN initiation was independent of Bmi1 control this locus. Further, impairment in the regulation of ROS generation was seen in vitro in pancreatic cancer cell lines lacking Bmi1. Regulating ROS generation is a vital step in the neoplastic process and has been shown in other systems to be controlled by Bmi1. Overall, in this work we have shown that expression of the Polycomb group protein Bmi1 is necessary for the initiation of pancreatic precancerous lesions, and that the mechanism of Bmi1 requirement is independent of its repression of the Ink4a/ARF genetic locus. Given the recent pre-clinical development of a Bmi1 inhibitor, this work could provide rationale for future treatment of pancreatic cancer, a truly devastating disease. Citation Format: Heather Schofield, Filip Bednar, Meredith Collins, Wei Yan, Yaqing Zhang, Nikhil Shyam, Jaime Eberle, Kenneth Olive, Nabeel Bardeesy, Daisuke Nakada, Diane Simeone, Sean Morrison, Marina Pasca di Magliano. Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism. [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 LB-061. doi:10.1158/1538-7445.AM2015-LB-061

Background & Aims: A critical factor in the poor outcomes in pancreatic cancer is due to it's silent nature until late in the disease process, so early detection has remained a major issue in this type of cancer. Detection, quantification, and molecular characterization of Epithelial circulating tumor cells (CTCs) as well as Epithelial-to-Mesenchymal Transition (EMT)-like CTCs in peripheral blood have been recognized as a means of “liquid biopsy” technique in the diagnosis of cancer and metastasis. Methods: A modified version of CTC chip, CTC Carpet chip was used to investigate the inextricable relationship between circulating epithelial and EMT-like CTCs in 40 pancreatic cancer patients at both protein and RNA levels. Results: The recovery from whole blood spiked with different cancer cell line was validated, achieving a mean efficiency of ∼100% for both HT-29 and Panc-1 cell lines compared to Conclusion: Although the isolation of CTCs from pancretic cancer is possible nowadays, investigation of their clinical utility has proven less successful than the other cancer types, due to poor sensitivity of many CTC assays for patients with PDAC. Herein, we present an integrated microfluidic technology- and biology-based translational approach using bioengineering tools to identify and study the biological relevance of rare CTCs including both circulating epithelial and EMT-like tumor cells simultaneously from the peripheral blood of pancreatic cancer patients by using multiple markers of interest. Citation Format: Mina Zeinali, Vasudha Murlidhar, Shamileh Fouladdel, Mathius Hafner, Shimeng Shao, Ebrahim Azizi, Max S. Wicha, Kyle Cuneo, Diane M. Simeone, Sunitha Nagrath. Analysis of circulating epithelial and EMT-like CTCs in pancreatic cancer using a sensitive microfluidic CTC capture device. [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 1595. doi:10.1158/1538-7445.AM2015-1595

Background: Pancreatic cancer is characterized by the accumulation of a fibro-inflammatory stroma. Myeloid cells are a predominant population within the stroma. Different myeloid cell subsets have been correlated with tumor promotion and unmasking of anti-tumor immunity. Objective: The goal of this study was to determine the effect of myeloid cell depletion on the onset and progression of pancreatic cancer, and to understand the relationship between myeloid cells and T cell infiltration and activity within the pancreatic cancer microenvironment. Methods: Primary mouse pancreatic cancer cells were transplanted in CD11b-DTR mice. CD11b+ cells (most myeloid cell populations) were depleted by Diphtheria Toxin treatment; either at the time of tumor implantation or after tumors had formed. Results Depletion of myeloid cells delayed tumor initiation. In pre-established tumors, myeloid cell depletion resulted in arrest of growth or tumor regression. We observed that tumor regression was dependent myeloid cell-mediated blockade of CD8+ T cell anti-tumor activity. We investigated the mechanism of this inhibition. We found that myeloid cells regulate expression of the immune checkpoint ligand Programmed death-ligand 1 (PD-L1) in the tumor cells in an EGFR/MAPK dependent manner. Conclusions: Our results show that myeloid cells regulate a complex network of signals that ensure immune suppression within the pancreatic cancer microenvironment. Moreover, we show that depletion of the myeloid cell population is sufficient to restore anti-tumor immunity mediated by CD8+ T cells, a finding with implications for the design of immune therapies for pancreatic cancer. Citation Format: YAQING ZHANG, ESHA MATHEW, FLOR MENDEZ, KEVIN FLANNAGAN, DIANE SIMEONE, MARINA PASCA DI MAGLIANO. Myeloid cells are required to establish an immune-suppressive regulatory network in pancreatic cancer by activating the PD-1/PD-L1 checkpoint. [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 LB-205. doi:10.1158/1538-7445.AM2015-LB-205

Pancreatic ductal adenocarcinoma (PDA) is a hierarchal cancer consisting of tumor-initiating cancer stem cells (CSCs) and transiently proliferating bulk tumor cells. Despite this understanding, the biological properties of CSCs remain incompletely defined, including the role of transcriptional programs like epithelial-mesenchymal transition (EMT). Using flow cytometry in conjunction with epithelial-cell surface marker ESA (EpCAM) and mesenchymal-cell surface marker CD44, we identified three subpopulations of PDA cells derived from primary patient samples: CD44highESAlow, CD44lowESAhigh, and CD44highESAhigh cells, each with different biological characteristics. CD44highESAlow cells exhibited a more mesenchymal phenotype characterized by high expression of vimentin and low expression of E-cadherin. CD44lowESAhigh cells, by contrast, highly epithelial in appearance, expressed high levels of E-cadherin and low levels of vimentin. Finally, CD44highESAhigh cells showed a phenotype intermediate between CD44highESAlow and CD44lowESAhigh cells. In isolation, all subpopulations were able to self-renew, however, only CD44highESAhigh cells could give rise to all three cell subpopulations. Additionally, CD44highESAhigh cells exhibited the highest ability to form tumorspheres, an indicator of CSC-functionality, in vitro, and had a greater ability to form tumors in vivo, suggesting that this subpopulation enriched for CSC functional activity. Using microarray analysis, we identified a set 50 genes, including the CSC marker CD24 and the transcription factor HNF1A, which showed significant up-regulation in CD44highESAhigh cells CSC population compared to either CD44highESAlow or CD44lowESAhigh cells. We hypothesized that these genes might be critical to CSC biology in PDA. Using bioinformatics analysis to identify possible transcriptional regulators of these 50 genes, we identified HNF1A as the factor with most highly overrepresented binding sites in the promoter regions of (17/50 genes), suggesting a possible role in regulating CD44highESAhigh-specific genes and possibly a key biological regulator of the CSC state. Supporting this hypothesis, knockdown of HNF1A resulted in decreased expression of a number of the candidate genes, including CD24, in the CD44highESAhigh population. By contrast, ectopic expression of HNF1A resulted in the up-regulation of a similar set of candidate genes, including CD24, and promoted the formation of tumorspheres. These findings shine light on the complexity of PDA tumor cell heterogeneity, suggesting the CSCs exist in a state between EMT and mesenchymal-epithelial transition, and implicates HNF1A as a key regulator of CSC-associated genes and CSC functionality. Citation Format: Ethan V. Abel, Masashi Goto, Michele L. Dziubinski, Chandan Kumar, Nikita Ramanathan, Diane M. Simeone. Pancreatic cancer stem cell function is regulated by HNF1A. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A65.

Background and Rationale: Pancreatic ductal adenocarcinoma (PDA) is one of the most stroma-rich cancers that exists. It is hypothesized that stroma contributes to the aggressive nature of PDA. There is strong evidence that mesenchymal stem cells (MSC) are recruited as part of the stroma to the tumor microenvironment. Studies in solid cancers of various organs including lung, stomach and ovaries have reported that MSCs are present in stroma within the cancer tissue and impact tumor biology. However, no published reports on role of MSC in PDA exist, therefore an opportunity exists to identify and functionally characterize MSC in pancreatic cancer and understand their contribution to pancreatic cancer biology. Experimental Design: Human pancreatic cancer associated fibroblasts (CAFs) from patient tumors (n=18) were isolated and grown in low passage cultures. The mesenchymal lineage of the CAF cultures was confirmed by KRAS mutational analysis of both the CAF cells and matching neoplastic epithelial cells. The purity of CAF cultures was also confirmed by immunostaining for immune (CD45) and epithelial (CK19) cell markers. An orthotopic model was established in which 105 GFP labeled tumor cells were mixed with either 105 DsRed labeled CAF or CAF-MSC cells and implanted into the pancreata of NOD-SCID mice (n= 6 mice per group). GFP labeled tumor cells and DsRed CAF/CAF-MSC cells injected alone served as the experimental controls. Growth kinetics and tumor metastasis between different groups were studied using bioluminescence animal imaging and weekly tumor monitoring. Once tumors formed, mice were sacrificed and tumor burden quantified. Blood was collected for analysis of circulating GFP+ tumor cells and RFP+ CAF or CAF-MSC cells. Protein cytokine arrays were performed to identify differentially expressed genes by CAF-MSCs. Results: Primary human CAF cultures were heterogeneous and each contained a unique population of MSCs, as defined by expression of MSC markers (CD90, CD49a, CD73 and CD44), the functional capacity to differentiate into bone, cartilage and adipose cells, and the ability to form colonies when plated at low densities. We observed that CAF-MSCs promoted tumor growth and metastasis to a significantly greater degree than bulk CAF cells, and only CAF-MSCs cells were capable of entering into the circulation. Using human protein cytokine arrays to explore the paracrine factors selectively secreted by CAF-MSC, we found that the cytokine GM-CSF was secreted selectively by CAF-MSCs and not CAFs (n=4). GM-CSF knockdown in CAF-MSCs using targeted shRNA compared to control shRNA resulted in a significant decrease in the development of metastasis in co-injection experiments with primary pancreatic cancer cells using an orthotopic model. Conclusions: Here we provide the first characterization of MSCs within the human PDA microenvironment. We find PDAs have a unique population of MSCs which confer specific biologic properties to PDA. These studies will help us to define important target molecules within the tumor microenvironment involved in key tumor-stromal interactions that may impact therapeutic approaches for patients. This abstract is also presented as Poster B2. Citation Format: Meghna Waghray, Guan Grace, Malica Yalamanchili, Lidong Wang, Michele Dziubinski, Mina Zeinali, Sunitha Nagrath, Diane Simeone. Mesenchymal stem cells in pancreatic cancer possess unique properties in promoting tumor growth and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR03.

Hypoxic conditions exist within pancreatic adenocarcinoma, yet pancreatic cancer cells survive and replicate within this environment. To understand the mechanisms involved in pancreatic cancer adaptation to hypoxia, we analyzed expression of a regulator of hypoxia-induced cell death, Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). We found that BNIP3 was down-regulated in nine of nine pancreatic adenocarcinomas compared with normal pancreas despite the up-regulation of other hypoxia-inducible genes, including glucose transporter-1 and insulin-like growth factor-binding protein 3. Also, BNIP3 expression was undetectable even after hypoxia treatment in six of seven pancreatic cancer cell lines. The BNIP3 promoter, which was remarkably activated by hypoxia, is located within a CpG island. The methylation status of CpG dinucleotides within the BNIP3 promoter was analyzed after bisulfite treatment by sequencing and methylation-specific PCR. Hypermethylation of the BNIP3 promoter was observed in all BNIP3-negative pancreatic cancer cell lines and eight of 10 pancreatic adenocarcinoma samples. Treatment of BNIP3-negative pancreatic cancer cell lines with a DNA methylation inhibitor, 5-aza-2′ deoxycytidine, restored hypoxia-induced BNIP3 expression. BNIP3 expression was also restored by introduction of a construct consisting of a full-length BNIP3 cDNA regulated by a cloned BNIP3 promoter. Restoration of BNIP3 expression rendered the pancreatic cancer cells notably more sensitive to hypoxia-induced cell death. In conclusion, down-regulation of BNIP3 by CpG methylation likely contributes to resistance to hypoxia-induced cell death in pancreatic cancer.

The molecular basis of pancreatic cancer is not understood. Previous attempts to determine the specific genes expressed in pancreatic cancer have been hampered by similarities between adenocarcinoma and chronic pancreatitis. In the current study, microarrays (Affymetrix) were used to profile gene expression in pancreatic adenocarcinoma (10), pancreatic cancer cell lines (7), chronic pancreatitis (5), and normal pancreas (5). Molecular profiling indicated a large number of genes differentially expressed between pancreatic cancer and normal pancreas but many fewer differences between pancreatic cancer and chronic pancreatitis, likely because of the shared stromal influences in the two diseases. To specifically identify genes expressed in neoplastic epithelium, we selected genes more highly expressed (2-fold, p0.01) in adenocarcinoma compared with both normal pancreas and chronic pancreatitis and which were also highly expressed in pancreatic cancer cell lines. This strategy yielded 158 genes, of which 124 were not previously associated with pancreatic cancer. Quantitative-reverse transcription-PCR for two molecules, S100P and 14-3-3sigma, validated the microarray data. Support for the success of the neoplastic cell gene expression identification strategy was obtained by immunocytochemical localization of four representative genes, 14-3-3sigma, S100P, S100A6, and beta4 integrin, to neoplastic cells in pancreatic tumors. Thus, comparisons between pancreatic adenocarcinoma, pancreatic cancer cell lines, normal pancreas, and chronic pancreatitis have identified genes that are selectively expressed in the neoplastic epithelium of pancreatic adenocarcinoma. These data provide new insights into the molecular pathology of pancreatic cancer that may be useful for detection, diagnosis, and treatment.

Deubiquitinase Usp9x has been shown to promote tumor cell survival and resistance to chemo- and radiotherapy through effects on the Mcl-1 pro-survival protein. It is predicted that a small molecule Usp9x inhibitor could have a major impact in cancer therapy. However, recent data has raised caution about therapeutic targeting of Usp9X in cancer. In a study to identify genes that reduce the latency of pancreatic tumor formation in a mutant Kras model, Pérez-Mancera et al. 2012 reported that the Usp9x locus was frequently inactivated in pancreatic cancer cells, resulting in protection from anoikis and enhanced transformation. In order to clarify the role of Usp9X in pancreatic cancer, we used a newly created cell line 4668 established from mouse pancreatic tumors with doxycycline (DOX) inducible expression of KrasG12D and Tp53R172H. Stable Usp9x knockdown (KD) in 4668 cells had no effect on their proliferation or phenotype in 2D cultures. However, withdrawal of DOX from DOX-treated Usp9x KD 4668 cells resulted in 4-fold increase in colony formation, unlike DOX depleted control KD 4668 cells which undergo apoptosis resulting in colony disintegration. DOX treated control KD and Usp9x KD 4668 cells did not show any difference in basal 3D colony formation. These results suggest that Usp9x loss in the absence of continuous mutant Kras/p53 expression is sufficient to sustain 3D cancer cell growth, confirming a tumor suppressor role for Usp9x in a Kras mouse model of pancreatic cancer. To determine if Usp9x also plays a tumor suppressor role in human pancreatic tumors, we did Usp9x KD in three human pancreatic cancer cell lines with varied Kras mutational status; BxPC3 (wild type), PANC1 (heterozygous) and MIA-PACA2 (homozygous) which expressed high basal levels of Usp9x. MIA-PACA2-Usp9x KD cells undergo apoptosis and caspase activation. In contrast, BxPC3-Usp9x KD cells showed no effect on growth in 2D or in 3D culture. However, PANC1-Usp9x KD cells showed more rapid (38% p=0.004) 2D growth, but 3D colony formation was consistently reduced by >50%, (p=0.03). These results suggest that the effect of Usp9x loss in established human pancreatic tumor cells is distinct from that detected in a murine pancreatic tumor model. Distinctions may be partially dependent on their Kras mutational status or Usp9x substrate utilization in human vs. murine cells. To further assess the clinical relevance of our observations, Usp9x KD was evaluated in four recently established, low passage number human pancreatic patient tumor cell lines (UM2, UM6, UM16, UM76). Usp9x KD did not affect 2D growth but fully suppressed 3D colony growth. This activity was phenocopied by our small molecule Usp9x inhibitor, EOAI3402143 (G9), with nM efficacy. Together, these results suggest that possible species distinctions exist in the tumorigenic functions of Usp9x and necessitate further assessment of its role in pancreatic tumor development and treatment. Citation Format: Anupama Pal, Marina Pasca di Magliano, Moshe Talpaz, Michelle Dziubinski, Diane Simeone, Nicholas J. Donato. Role of Deubiquitinase Usp9x in pancreatic cancers: Tumor promoter or tumor suppressor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4434. doi:10.1158/1538-7445.AM2014-4434

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process accounting for the majority of cancer-related deaths. They offer a non-invasive biopsy technique to study tumors and are shown to be useful prognostic indicators. Because of their rarity in the bloodstream, microfluidic isolation techniques are complex and time-consuming, and provide yields of CTCs insufficient or non-viable for studies other than enumeration. Additionally, due to the low processing speeds, the volumes of blood processed remain limited and can be a hindrance to obtaining higher yields of CTCs and their potential use as biomarkers of early diagnosis. Here we report a novel high throughput microfluidic technology, the OncoBean Chip, employing radial flow that introduces a varying shear profile across the device and enables efficient cell capture by affinity at high flow rates. The cell recovery from whole blood was validated with cancer cell lines H1650 and MCF7, and the OncoBean Chip achieved an efficiency >80% at a throughput of 10 mL/hr, a flow rate yet achieved only in physical size based separation techniques. A cell viability of 92.91% shows that the cells recovered at high throughput could still be used for downstream analysis. Clinical competence was demonstrated in blood specimens from breast, pancreatic and lung cancer patients. The OncoBean Chip thus holds potential applications in the diagnosis of early stage cancers, where the low numbers of CTCs could be enriched using this novel device. Citation Format: Vasudha Murlidhar, Rishindra M. Reddy, Mina Zeinali, Svetlana Grabauskiene, Mostafa Ghannad-Rezaie, Max S. Wicha, Diane M. Simeone, Nithya Ramnath, Sunitha Nagrath. Radial flow microfluidic device for high-throughput affinity-based isolation of circulating tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3069. doi:10.1158/1538-7445.AM2014-3069

The prognosis of pancreatic ductal adenocarcinoma (PDAC) is dismal. Very rarely, patients will survive for more than10-years after pancreatic surgery. A few clinicopathological characteristics have been demonstrated to have significant effect on long-term survival. However, the molecular characteristics of the pancreatic adenocarcinomas from these very long-term survivors (VLTS) remain to be uncovered. Our study investigated the tissue proteome of pancreatic tumor and sought to characterize protein signatures associated with pancreatic cancer survival. A comparative label-free proteomics approach was applied to investigate the formalin-fixed paraffin-embedded tumor tissues from five VLTS patients (survival >10 years) and five short-term survival patients (STS, survival Citation Format: Ru Chen, Sheng Pan, Anirban Maitra, Diane Simeone, David Dawson, Teresa Brentnall. Proteins associated with pancreatic cancer survival in patients with resectable pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2866. doi:10.1158/1538-7445.AM2014-2866

Circulating tumor cells (CTCs) have gathered much momentum in recent times with emerging micro and nanotechnologies enabling better recoveries of these rare cells. Future research directions in CTCs significantly depend on obtaining meaningful number of CTCs that are available for downstream assays. This can be achieved by expanding CTCs. However, in early stage cancer patients, the numbers of cells that circulate in the peripheral blood are rare. Therefore until now, expanding CTCs remains to be a serious challenge limiting clinical utility. Microfluidic technologies have demonstrated that these rare cells can be recovered with better efficiency, purity and high viability. Herein, we developed a strategy using microfluidic technology to isolate and expand CTCs. We have successfully expanded CTCs isolated from 16 out of 21 early stage lung cancer patients. Expanded CTCs expressed tumor specific markers and formed spheroids in culture. Furthermore, these expanded CTCs carried the identical cancer related mutations (e.g. TP53 mutation) as their primary tumors. We expect that our model opens up the unprecedented avenues to understand biology of these lethal drivers of metastasis offering opportunities for personalized medicine through target drug evaluations. Citation Format: Jennifer Zhuo Zhang, Hiroe Shiratsuchi, Jules Lin, Guoan Chen, Rishindra M. Reddy, Ebrahim Azizi, Shamileh Fouladdel, Andrew C. Chang, Lin Lin, Diane M. Simeone, Max S. Wicha, David G. Beer, Nithya Ramnath, Sunitha Nagrath. Ex-vivo expansion of circulating tumor cells in early lung cancer using a microfluidic model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3075. doi:10.1158/1538-7445.AM2014-3075

The concept of studying tumor-derived circulating tumor cells (CTCs) from a simple blood-draw of a patient, has acquired significant attention in recent years. This relatively non-invasive approach opens many avenues in understanding and monitoring quite a number of cancers where correlations have since been made with CTCs and patient survival as well as patient response to therapeutics. Additionally, studies geared towards identifying genetic markers as well as gene profiling of CTCs are ongoing in efforts to develop early screening markers as well as gain a more holistic understanding of the various forms of the disease. Moreover, the concept of personalized medicine in treating several types of cancer is deemed indispensable due to the highly heterogeneous nature of the disease. Extensive studies involving genetic material of CTCs are expected to considerably develop effective therapeutics that are tailored to meet each patient's need. However, several challenges need to be overcome in order to harness the wealth of information that can be gained from CTC studies, such as the rarity of such cells among other blood cells, where CTCs occur as rarely as 1 to 1 million among other mononuclear blood cells. In this vein we discuss the development of an integrated, continuous microfluidic device designed to isolate and label CTCs disseminated from cancer tumors. The 3-part device initially exploits the size disparity of the majority of CTCs with other blood cells and couples this with the use of size-based inertial forces to presort CTCs from whole blood at 1.2mL/min. The CTC-rich fluid stream is then mixed passively, on-chip, with EpCAM coated micro-beads which allows the cells and beads to mix at the micron length scale. This is followed by a brief period of incubation which allows cells to be sufficiently labeled, where the majority of cells have experienced at least two-thirds magnetic bead coverage using this mixing-incubation approach. Thereafter on-chip magnetic sorting of CTCs from any remaining blood cells is achieved by application of an external magnet along the magnetic sorter of the integrated device. Cells with as little as one-third bead coverage experience magnetic deflection to the CTC collection stream. To identify CTCs, cells were stained positively for DAPI and CK-19, and CD45 was used to distinguish leukocytes. We demonstrate that the isolated CTCs are not only viable but that the range of WBC decontamination is the lowest yet reported in literature. Based on PANC-1 cell-line experiments with the device, an 80% recovery rate of cancer cells from other blood cells was consistently achieved. Testing of metastatic patient samples with the device has yielded high counts of CTCs, coupled with high purity rates. We believe that the use of this system in characterizing CTCs and studying cancer will contribute to understanding this aggressive disease in a non-invasive, efficient manner. Citation Format: Rhonda M. Jack, Meggie MG Grafton, Robert A. Cieslak, Denise C. Jue, Cathy Griffith, Danika Rodrigues, Sunitha Nagrath, Diane Simeone. Continuous isolation, labeling and collection of viable CTCs using an integrated microfluidic device. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3477. doi:10.1158/1538-7445.AM2014-3477

Membrane bound extracellular vesicles, including microvesicles and exosomes, are secreted by both normal and cancerous cells into the extracellular space and in blood circulation. These circulating extracellular vesicles (cirEVs) and exosomes in particular, are recognized as a potential source of disease biomarkers and also carry signature molecules for early stage cancer detection. However, to exploit the use of circulatory exosomes as a new diagnostic tool, a rapid, high-throughput and reproducible method is required for their isolation and to allow their molecular analysis. We have developed a simple and a low cost microfluidic-based platform to analyze cirEVs enriched in exosomes directly from blood serum in a single step allowing simultaneous capture and quantification of exosomes. To capture exosomes with specificity, we employed a microfluidic device “ExoChip” functionalized with antibodies against exosomes to capture cirEVs. Subsequent staining with a fluorescent dye that specifically labels the exosomes, we quantitated exosomes using a standard plate-reader. Ten independent ExoChip experiments performed using serum obtained from five pancreatic cancer patients and five healthy individuals revealed a statistically significant increase (2.34±0.31 fold, p Citation Format: Shailender S. Kanwar, Christopher J. Dunlay, Diane M. Simeone, Sunitha Nagrath. Simultaneous isolation and quantification of circulating exosomes for cancer biomarker discovery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5591. doi:10.1158/1538-7445.AM2014-5591

Introduction and Objective: Circulating tumor cells (CTCs) have been identified in the art in peripheral blood from cancer patients and are likely the origin of metastatic disease. Isolation and capture of CTCs represent a potential alternative to invasive biopsies during diagnosis of disease. In the recent past, vVarious microfluidic- based CTC isolation technologieschips have been developed to capture, indentify, sort, and enumerate CTCs. but, they need to be more sensitive, reliable, and specific for CTC detection in early stage cancer patients. Here we present a microfluidic CTC-chip with functionalized nanomaterials to capture and culture CTCs. Methods: Micro-sized structuresposts functionalized with anti-epithelial-adhesion-molecule antibodies (Anti-EpCAM) are is the common approachstructures to capture CTCs. Here we present a microfluidic device with functionalized planar structure to capture CTCs. The CTC-chipmicrofluidic device has a PDMS chamber and patterned gold postatterns . functionalized with graphene oxides as nanomaterial and anti-epithelial-cell-adhesion-molecule antibodies (Anti-EpCAM). To investigate the capture efficiency before running cancer patients’ samples, a low number of cells were spiked into 1 mL of whole blood. We stained the captured cells with cytokeratin, CD45, and DAPI to identify CTCs. For clinical studies, we searched for CTCs in 10 pancreatic cancer patients, 6 breast cancer patients, 4 early lung cancer patients, and 5 healthy donors. Results: It is much easier to scan, count and grow captured cells in comparison with previous micro-sized post-based structure. CTCs were isolated by assembling Graphene oxide on the planar structures for enhanced sensitivity. is easy to functionalize the surface and sensitive to detect biomaterial. The functionalized graphene oxide has been used to selectively capture MCF-7 breast cancer cells with a capture rate higher than 8790 %. This new CTC-chip identified CTCs in the peripheral blood of patients with pancreatic, breast, and lung cancer in 20 of 20 samples (100%). Conclusion: We present a sensitive CTC-chip using graphene oxides as nanomaterial to isolate, capture, identify, and characterize extremely rare CTCs. This CTC-chipIt is much easier to scan, count, and grow captured cells in comparison with previous micro-sized post-based structure. We believe this CTC-chip offers great potential for clinical research on cancer. Citation Format: Hyeun Joong Yoon, Tae Hyun Kim, Zhuo Zhang, Nithya Ramnath, Max S. Wicha, Daniel F. Hayes, Diane M. Simeone, Sunitha Nagrath. Integrated micro nanotechnology for the sensitive isolation of circulating tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5109. doi:10.1158/1538-7445.AM2013-5109

Bladder cancer is the 5th most common malignancy and a significant cause of morbidity and mortality, but the molecular events leading to its development are incompletely understood. We have identified an oncogene, Ataxia-Telangiectasia Group D Complementing (ATDC) gene, which drives formation and progression of pancreatic and bladder tumors. To better characterize the oncogenic function of ATDC, we generated transgenic (tg) mice which overexpress ATDC and the predominant phenotype of these mice was the development of non-invasive and muscle-invasive urothelial carcinomas. Tg bladder tumors were histologically identical to human tumors and displayed a similar gene expression signature to human invasive bladder tumors. ATDC expression was assessed in a tissue microarray and was highly up-regulated in 70% of human invasive bladder tumors (173/252). High expression of ATDC correlated with more advanced stage disease and worse survival after chemotherapy (p = 0.002). ATDC knockdown in human bladder cancer cell lines correlated with increased sensitivity to chemotherapy in vitro and decreased proliferation and invasion both in vitro and in vivo in a human bladder cancer xenograft model. To understand the mechanism of ATDC-mediated tumor formation, we modulated ATDC expression in normal and bladder cancer cells lines. ATDC overexpression down-regulated PTEN levels via DNA promoter methylation by DNMT3A in both tg mouse tumors and human cell lines. IHC analysis of ATDC, PTEN and DNMT3A expression in human bladder tumors specimens demonstrated similar findings. ATDC was also found to bind to p53 and inhibit p53 mediated functions. These findings establish ATDC as a novel determinant of bladder cancer initiation, progression and resistance to treatment which mediates tumorigenesis by down-regulation of PTEN expression and inhibition of p53. Citation Format: Phillip Palmbos, Lidong Wang, Huibin Yang, Taylor Detzler, Gina Ney, Justin Hart, Stephanie Daignault-Newton, L. Priya Kunju, Chandan Kumar-Sinha, Monica Liebert, Mats Ljungman, Diane Simeone. ATDC drives bladder cancer formation by promoting methylation of the PTEN promoter and inhibition of p53 function. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 313. doi:10.1158/1538-7445.AM2013-313

Protein kinases represent a most effective class of therapeutic targets in cancer cells, and potent inhibitors of oncogenic kinases including ABL, AKT, ALK, AURKs, BRAF, CDKs, EGFR, ERBB2, FGFRs, KIT, MAPKs, MET, PIK3CA, PLKs, RET, SRC, S6Ks, and VEGFR are in clinical use, trials, or development. Presence of activating mutations, gene fusions or copy number amplification of many of these kinases predict responsiveness to specific inhibitors, for example, imatinib for BCR-ABL-positive chronic myeloid leukemia, trastuzumab and lapatinib for ERBB2-amplified breast cancers, gefitinib for lung cancers with kinase domain mutations in EGFR, and crizotinib for lung cancers with ALK gene fusions. However, tumors with defined kinase aberrations represent a very small proportion of all cancers, and we hypothesize that potentially most, if not all, cancers are dependent on specific kinases, and that “Outlier Expression” of specific kinases in individual samples may be indicative of selection during clonal evolution, and thus may represent a therapeutic avenue. To explore this hypothesis, we analyzed RNA-sequencing data from a compendium of 482 cancer and benign samples belonging to 25 different tissue types to define sample-specific ‘kinome’ expression profiles. Comparing the expression of kinases within a sample and across sample sets, we identified distinct ‘outlier kinases’ in individual samples, defined as genes showing the highest statistically significant levels of absolute and differential expression. Frequently observed outlier kinases included known therapeutic targets like ERBB2 and FGFR4 in breast cancer, distinct from MET, AKT2, and PLK2 in pancreatic cancer. Outlier kinases imparted sample-specific dependencies in various cell lines as assessed by siRNA knockdown or pharmacologic inhibition in vitro and in vivo. Outlier expression of polo-like kinases (PLKs) observed in a subset of KRAS-dependent pancreatic cancer cell lines conferred increased sensitivity to the PLK inhibitor BI 6727. Together, our results suggest that outlier kinases represent effective personalized therapeutic targets that are readily identifiable through RNA-sequencing of tumors. Next, to help translate these observations into treatment options, we are attempting to establish 3D tumoroid cultures from tumor samples that can be used to optimize combinations of therapeutics including outlier kinase inhibitors. Citation Format: Chandan Kumar-Sinha, Vishal Kothari, Iris Wei, Sunita Shankar, Shanker Kalyana-Sundaram, Lidong Wang, Linda W. Ma, Pankaj Vats, Catherine Grasso, Dan Robinson, Yi-Mi Wu, Xuhong Cao, Diane M. Simeone, Arul M. Chinnaiyan. Sample specific outlier kinase expression identified by RNA-Seq provide targets for precision therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3136. doi:10.1158/1538-7445.AM2013-3136

Pancreatic cancer has one of the highest mortality rates of all cancers. The mortality is associated with both resistance to therapeutics and late stage detection. Often by the time symptoms are presented the disease has advanced to an incurable state. Due to the aggressive nature of pancreatic cancer and the difficulty in obtaining biopsies, it has been challenging to examine the behavior and characteristics of pancreatic tumors over time. Recently, circulating tumor cells (CTCs) have been isolated from peripheral blood of cancer patients and correlated with survival and therapeutic response. CTCs can also be analyzed to provide insight into tumor genetics and metastatic capabilities. Since CTCs are present in early cancers, they have the potential to be used as an early screening marker of metastatic disease. Especially for pancreatic cancer, isolating CTCs would perhaps allow for detection prior to advanced disease states and aid in determining appropriate therapeutic treatments. We demonstrate an integrated microfluidic device to detect CTCs in pancreatic cancer patients. The three-part device which integrates an inertial pre-sorter, a passive mixer and a magnetic sorter can process 1ml of blood in less than 20min to achieve nearly 100% pure cancer cells at the outlet. High-throughput pre-sorting of CTCs from the diluted blood is achieved using the inertial sorter and further specificity and isolation of the CTCs is achieved through magnetic labeling and re-sorting. First diluted whole blood is run through the inertial spiral sorter at nearly 2ml/min. The curvature of the spiral focuses and aligns cancer cells to the inner channel wall, thus making it possible to enrich the cancer cells. The outlet of the spiral is designed to collect the enriched CTC stream, which are then passed through a passive mixer with EpCAM-labeled superparamagnetic beads. Only the cancer cells will be labeled with the magnetic beads as leukocytes and erythrocytes do not express the epithelial markers that are targeted during the mixing process. The mixed and labeled sample is sorted by a continuous flow magnetic sorter, where the application of an external magnetic field deflects the magnetically labeled cancer cells to a single collection outlet. Once isolated, the highly pure CTC population can be counted, genetically profiled, immunostained, or cultured for further analysis. Cells were stained using CK7/8, N-cadherin, CD45, and DAPI to assess epithelial and mesenchymal characteristics. Isolating pancreatic CTCs with both high sensitivity and high specificity can enable earlier cancer detection and downstream molecular analysis can identify key targets for pancreatic cancer treatment. Thereupon, the development of effective CTC isolation tools will improve patient prognosis and therapeutic treatment plans. Citation Format: Meggie MG Grafton, Rhonda M. Jack, Robert Cieslak, Danika Rodriguez, Somya Sharma, Diane M. Simeone, Sunitha Nagrath. An integrated microfluidic device for circulating tumor cell detection in pancreatic cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1457. doi:10.1158/1538-7445.AM2013-1457