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Aberrant expression of CA125/MUC16 is associated with pancreatic ductal adenocarcinoma (PDAC) progression and metastasis. However, knowledge of the contribution of MUC16 to pancreatic tumorigenesis is limited. Here, we show that MUC16 expression is associated with disease progression, basal-like and squamous tumor subtypes, increased tumor metastasis, and short-term survival of PDAC patients. MUC16 enhanced tumor malignancy through the activation of AKT and GSK3β oncogenic signaling pathways. Activation of these oncogenic signaling pathways resulted in part from increased interactions between MUC16 and epidermal growth factor (EGF)-type receptors, which were enhanced for aberrant glycoforms of MUC16. Treatment of PDAC cells with monoclonal antibody (mAb) AR9.6 significantly reduced MUC16-induced oncogenic signaling. mAb AR9.6 binds to a unique conformational epitope on MUC16, which is influenced by O-glycosylation. Additionally, treatment of PDAC tumor-bearing mice with either mAb AR9.6 alone or in combination with gemcitabine significantly reduced tumor growth and metastasis. We conclude that the aberrant expression of MUC16 enhances PDAC progression to an aggressive phenotype by modulating oncogenic signaling through ErbB receptors. Anti-MUC16 mAb AR9.6 blocks oncogenic activities and tumor growth and could be a novel immunotherapeutic agent against MUC16-mediated PDAC tumor malignancy.

Elevated levels of Mucin-16 (MUC16) in conjunction with a high expression of truncated O-glycans is implicated in playing crucial roles in the malignancy of pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms by which such aberrant glycoforms present on MUC16 itself promote an increased disease burden in PDAC are yet to be elucidated. This study demonstrates that the CRISPR/Cas9-mediated genetic deletion of MUC16 in PDAC cells decreases tumor cell migration. We found that MUC16 enhances tumor malignancy by activating the integrin-linked kinase and focal adhesion kinase (ILK/FAK)-signaling axis. These findings are especially noteworthy in truncated O-glycan (Tn and STn antigen)-expressing PDAC cells. Activation of these oncogenic-signaling pathways resulted in part from interactions between MUC16 and integrin complexes (α4β1), which showed a stronger association with aberrant glycoforms of MUC16. Using a monoclonal antibody to functionally hinder MUC16 significantly reduced the migratory cascades in our model. Together, these findings suggest that truncated O-glycan containing MUC16 exacerbates malignancy in PDAC by activating FAK signaling through specific interactions with α4 and β1 integrin complexes on cancer cell membranes. Targeting these aberrant glycoforms of MUC16 can aid in the development of a novel platform to study and treat metastatic pancreatic cancer.

Numerous aspects of cellular signaling are regulated by the kinome – the network of over 500 protein kinases that guides and modulates information transfer throughout the cell. The key role played by both individual kinases and assemblies of kinases organized into functional subnetworks leads to kinome dysregulation being a key driver of many diseases, particularly cancer. In the case of pancreatic ductal adenocarcinoma (PDAC), a variety of kinases and associated signaling pathways have been identified for their key role in the establishment of disease as well as its progression. However, the identification of additional relevant therapeutic targets has been slow and is further confounded by interactions between the tumor and the surrounding tumor microenvironment. Fundamentally, it is an open question as to the degree to which knowledge of the state of the kinome at the protein level is able to provide insight into the downstream phenotype of the cell.In this work, we attempt to link the state of the kinome, or kinotype, with cell viability in representative PDAC tumor and stroma cell lines. Through the application of both regression and classification models to independent kinome perturbation and kinase inhibitor cell screen data, we find that the inferred kinotype of a cell has a significant and predictive relationship with cell viability. While regression models perform poorly, we find that classification approaches are able to predict drug viability effects. We further find that models are able to identify a set of kinases whose behavior in response to perturbation drive the majority of viability responses in these cell lines. Using the models to predict new compounds with cell viability effects and not in the initial data set, we conducted a validation screen that confirmed the accuracy of the models. These results suggest that characterizing the state of the protein kinome provides significant opportunity for better understanding signaling behavior and downstream cell phenotypes, as well as providing insight into the broader design of potential therapy design for PDAC.

To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA-sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.

Desmoplasia describes the deposition of extensive extracellular matrix and defines primary pancreatic ductal adenocarcinoma (PDA). The acellular component of this stroma has been implicated in PDA pathogenesis and is being targeted therapeutically in clinical trials. By analyzing the stromal content of PDA samples from numerous annotated PDA data sets and correlating stromal content with both anatomic site and clinical outcome, we found PDA metastases in the liver, the primary cause of mortality to have less stroma, have higher tumor cellularity than primary tumors. Experimentally manipulating stromal matrix with an anti–lysyl oxidase like-2 (anti-LOXL2) antibody in syngeneic orthotopic PDA mouse models significantly decreased matrix content, led to lower tissue stiffness, lower contrast retention on computed tomography, and accelerated tumor growth, resulting in diminished overall survival. These studies suggest an important protective role of stroma in PDA and urge caution in clinically deploying stromal depletion strategies.

Phosphatidylserine (PS) is often externalized in viable pancreatic cancer cells and is therapeutically targetable using PS-selective drugs. One of the first-line treatments for advanced pancreatic cancer disease, gemcitabine (GEM), provides only marginal benefit to patients. We therefore investigated the therapeutic benefits of combining GEM and the PS-targeting drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), for treating pancreatic ductal adenocarcinoma (PDAC). Using cell-cycle analyses and a cell surface PS-based sorting method in vitro, we observed an increase in surface PS as cells progress through the cell cycle from G1 to G2/M. We also observed that GEM treatment preferentially targets G1 phase cells that have low surface PS, resulting in an increased median surface PS level of PDAC cells. Inversely, SapC-DOPS preferentially targets high surface PS cells that are predominantly in the G2/M phase. Finally, combination therapy in subcutaneous and orthotopic PDAC tumors in vivo with SapC-DOPS and GEM or Abraxane (Abr)/GEM (one of the current standards of care) significantly inhibits tumor growth and increases survival compared with individual treatments. Our studies confirm a surface PS and cell cycle-based enhancement of cancer cytotoxicity following SapC-DOPS treatment in combination with GEM or Abr/GEM. Thus, PDAC patients treated with Abr/GEM may benefit from concurrent administration of SapC-DOPS.

Over 55,000 people in the United States are diagnosed with pancreatic ductal adenocarcinoma (PDAC) yearly, and fewer than 20% of these patients survive a year beyond diagnosis. Chemotherapies are considered or used in nearly every PDAC case, but there is limited understanding of the complex signaling responses underlying resistance to these common treatments. Here, we take an unbiased approach to study protein kinase network changes following chemotherapies in patient-derived xenograft (PDX) models of PDAC to facilitate design of rational drug combinations. Proteomics profiling following chemotherapy regimens reveals that activation of JNK-JUN signaling occurs after 5-fluorouracil plus leucovorin (5-FU + LEU) and FOLFOX (5-FU + LEU plus oxaliplatin [OX]), but not after OX alone or gemcitabine. Cell and tumor growth assays with the irreversible inhibitor JNK-IN-8 and genetic manipulations demonstrate that JNK and JUN each contribute to chemoresistance and cancer cell survival after FOLFOX. Active JNK1 and JUN are specifically implicated in these effects, and synergy with JNK-IN-8 is linked to FOLFOX-mediated JUN activation, cell cycle dysregulation, and DNA damage response. This study highlights the potential for JNK-IN-8 as a biological tool and potential combination therapy with FOLFOX in PDAC and reinforces the need to tailor treatment to functional characteristics of individual tumors.

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame BRAF deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC. Significance: Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. Cancer Discov; 8(9); 1096–111. ©2018 AACR. See related commentary by Collisson, p. 1062. This article is highlighted in the In This Issue feature, p. 1047

Malignancy is accompanied by changes in the metabolism of both cells and the organism1,2. Pancreatic ductal adenocarcinoma (PDAC) is associated with wasting of peripheral tissues, a metabolic syndrome that lowers quality of life and has been proposed to decrease survival of patients with cancer3,4. Tissue wasting is a multifactorial disease and targeting specific circulating factors to reverse this syndrome has been mostly ineffective in the clinic5,6. Here we show that loss of both adipose and muscle tissue occurs early in the development of pancreatic cancer. Using mouse models of PDAC, we show that tumour growth in the pancreas but not in other sites leads to adipose tissue wasting, suggesting that tumour growth within the pancreatic environment contributes to this wasting phenotype. We find that decreased exocrine pancreatic function is a driver of adipose tissue loss and that replacement of pancreatic enzymes attenuates PDAC-associated wasting of peripheral tissues. Paradoxically, reversal of adipose tissue loss impairs survival in mice with PDAC. When analysing patients with PDAC, we find that depletion of adipose and skeletal muscle tissues at the time of diagnosis is common, but is not associated with worse survival. Taken together, these results provide an explanation for wasting of adipose tissue in early PDAC and suggest that early loss of peripheral tissue associated with pancreatic cancer may not impair survival.

PURPOSE: Effective treatment of patients with locally advanced pancreatic cancer is a significant unmet clinical need. One major hurdle that exists is inadequate drug delivery due to the desmoplastic stroma and poor vascularization that is characteristic of pancreatic cancer. The local iontophoretic delivery of chemotherapies provides a novel way of improving treatment. With the growing practice of highly toxic combination therapies in the treatment of pancreatic cancer, the use of iontophoresis for local delivery can potentiate the anti-cancer effects of these therapies while sparing unwanted toxicity. The objective of this study was to investigate the impact of formulation on the electrotransport of the FOLFIRINOX regimen for the development of a new treatment for pancreatic cancer. METHODS: Three formulations of the FOLFIRINOX regimen (5-fluorouracil, leucovorin, irinotecan, and oxaliplatin) were generated at a fixed pH of 6.0 and were referred to as formulation A (single drug solution with all four drugs combined), formulation B (two drug solutions with two drugs per solution), and formulation C (four individual drug solutions). Anodic iontophoresis of the three different formulations was evaluated in orthotopic patient-derived xenografts of pancreatic cancer. RESULTS: Iontophoretic transport of the FOLFIRINOX drugs was characterized according to organ exposure after a single device treatment in vivo. We report that the co-iontophoresis of two drug solutions, leucovorin + oxaliplatin and 5-fluorouracil + irinotecan, resulted in the highest levels of cytotoxic drugs in the tumor compared to drugs delivered individually or combined into one solution. There was no significant difference in plasma, pancreas, kidney, and liver exposure to the cytotoxic drugs delivered by the three different formulations. In addition, we found that reducing the duration of iontophoretic treatment from 10 to 5 minutes per solution resulted in a significant decrease in drug concentrations. CONCLUSIONS: Underlying the difference in drug transport of the formulations were electrolyte concentrations, which includes both active and inactive components. Electrolyte concentrations can hinder or improve drug electrotransport. Overall, balancing electrolyte concentration is needed for optimal electro-transport.

Pancreatic adenocarcinoma (PDAC) remains an extremely lethal disease with few effective therapeutic options. Targeted therapy development has remained largely unsuccessful due to complex tumor heterogeneity and better molecular indicators of response are urgently needed. We have previously identified two distinct molecular tumor subtypes in PDAC, basal-like and classical, which are prognostic and dictate response to chemotherapy. We have also developed a patient derived xenograft (PDX) mouse model that maintains the integrity of patient tumor biology and recapitulates primary tumor subtypes in vivo. Here, we aim to utilize multiplexed kinase inhibitor beads and quantitative mass spectrometry (MIB-MS) to determine baseline kinase expression and adaptive kinome responses to therapy in PDAC PDX tumors. A total of 381 kinases were identified across all samples and tumor specific kinases were isolated by alignment to human peptides. Distinct kinase expression profiles were observed for basal-like and classical tumors. Basal-like tumors showed high expression of kinases involved in receptor tyrosine kinase activity, MAP kinase activity, and clathrin-dependent endocytosis. Classical subtype tumors showed increased expression of kinases involved in metabolism and cytoskeletal regulatory kinases. Differential protein expression was further validated by differential gene expression using RNAseq. MIB profiling of kinome response to EGFR inhibition by erlotinib indicated kinome reprogramming with upregulation of microtubule associated kinases and MAP kinase family members. Overall, these results suggest that basal-like and classical subtype tumors exhibit distinct kinome profiles that present possible subtype specific vulnerabilities to kinase inhibition. Stratifying kinase inhibitor therapies based on tumor subtype could improve response to targeted therapies in PDAC. Citation Format: Yi Xu, Michael East, Ashley Morrison, Gabriela Herrera, Laura Peng, Gary Johnson, Jen Jen Yeh. Proteomic profiling reveals subtype specific kinase expression in pancreatic cancer [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-079.

Background: Preoperative (neoadjuvant) therapy has become the preferred treatment sequencing strategy for patients with localized pancreatic cancer. During neoadjuvant therapy, approximately 30% of patients will experience metastatic disease progression while on treatment. Therefore, tools to aid clinicians to select efficacious first-line chemotherapeutic regimens is a critical unmet need. The most common neoadjuvant chemotherapy regimens used are 5-fluorouracil/irinotecan/oxaliplatin (mFOLFIRINOX) and gemcitabine/nab-paclitaxel (GnP). There is growing data to suggest an association of pancreatic cancer subtype (classical versus basal-like) with treatment response to therapy. Recently, the translation of tumor subtyping to the clinic has been successfully achieved using the Purity Independent Subtyping of Tumors (PurIST) single sample classifier. We aim to assess the clinical response to pancreatic cancer subtype-directed therapy in patients with localized pancreatic cancer. Methods: This is a phase II, multicenter, single-arm clinical trial for previously untreated patients with localized (resectable or borderline resectable) pancreatic cancer. Patients will undergo endoscopic ultrasound guided biopsy of the primary tumor and PurIST classifier to determine classical versus basal-like subtype. Patients with classical subtype will be assigned to mFOLFIRINOX and patients with basal-like tumors will be assigned to GnP. Following two months of therapy, patients will be restaged with a computed tomography scan, carbohydrate antigen (CA19-9) levels, performance status assessment, and a repeat endoscopic ultrasound guided biopsy for research. The primary endpoint is composite clinical response as measured by radiographic response, CA19-9 decline, and performance status following two months of treatment. Correlative endpoints include blood-based biomarkers for association with clinical response and stroma-specific response to therapy. The study has enrolled 4 of the anticipated 41 patients at the time of submission. Clinical Trial information: NCT 04683315. Citation Format: Susan Tsai, Erkut Borazanci, Margaret Gulley, Naim Rashid, Jason Merker, Abdul H. Khan, Phillip Chisholm, Bryan Hunt, Tamara Giorgadze, William Hall, Mandana Kamgar, Douglas B. Evans, Jen Jen Yeh. Phase II clinical trial of subtype directed neoadjuvant therapy in patients with localized pancreatic cancer [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-055.

Circulating tumor cells consist of phenotypically distinct subpopulations that originate from the tumor microenvironment. We report a circulating tumor cell dual selection assay that uses discrete microfluidics to select circulating tumor cell subpopulations from a single blood sample; circulating tumor cells expressing the established marker epithelial cell adhesion molecule and a new marker, fibroblast activation protein alpha, were evaluated. Both circulating tumor cell subpopulations were detected in metastatic ovarian, colorectal, prostate, breast, and pancreatic cancer patients and 90% of the isolated circulating tumor cells did not co-express both antigens. Clinical sensitivities of 100% showed substantial improvement compared to epithelial cell adhesion molecule selection alone. Owing to high purity (>80%) of the selected circulating tumor cells, molecular analysis of both circulating tumor cell subpopulations was carried out in bulk, including next generation sequencing, mutation analysis, and gene expression. Results suggested fibroblast activation protein alpha and epithelial cell adhesion molecule circulating tumor cells are distinct subpopulations and the use of these in concert can provide information needed to navigate through cancer disease management challenges., Diagnostics: Dual selection improves circulating tumor cell assay Using two selection markers, instead of one, can improve the sensitivity of a blood test for circulating tumor cells (CTCs). Steven A. Soper from the University of Kansas in Lawrence, USA, and colleagues analyzed blood samples from patients with cancers of the pancreas, colon, breast, ovaries and prostate, as well blood from healthy donors and those with benign disease. They ran each patient’s blood through two microfluidic devices, one chip targeting the established marker epithelial cell adhesion molecule (EpCAM) and another targeting a new marker, fibroblast activation protein alpha (FAPα). Doing so detected CTCs in nearly all the cancer patients—a substantial improvement over testing for EpCAM CTCs alone. Molecular and genetic analyses of the CTCs showed that those expressing EpCAM and FAPα are distinct subpopulations, and testing for both could prove valuable for clinical management.

Next-generation sequencing of pancreatic juice can detect and quantify tumor-promoting mutations, supporting imaging and cytology findings to predict the degree of dysplasia in patients at high risk for pancreatic cancer. Future studies are needed to optimize this approach and determine how it best fits into clinical practice. Clin Cancer Res; 24(12); 2713–5. ©2018 AACR. See related article by Suenaga et al., p. 2963

Tumors are mixtures of different compartments. While global gene expression analysis profiles the average expression of all compartments in a sample, identifying the specific contribution of each compartment remains a challenge. With the increasing recognition of the importance of non-neoplastic components, the ability to breakdown the gene expression contribution of each is critical. Here, we develop DECODER, an integrated framework which performs de novo deconvolution and single-sample compartment weight estimation. We use DECODER to deconvolve 33 TCGA tumor RNA-seq data sets and show that it may be applied to other data types including ATAC-seq. We demonstrate that it can be utilized to reproducibly estimate cellular compartment weights in pancreatic cancer that are clinically meaningful. Application of DECODER across cancer types advances the capability of identifying cellular compartments in an unknown sample and may have implications for identifying the tumor of origin for cancers of unknown primary., Separating different cell compartments from bulk gene expression data can be challenging. Here the authors present DECODER, which can perform de novo deconvolutions on non-negative matrices including microarray, RNA-seq and ATAC-seq data sets.

Purpose: Molecular subtyping for pancreatic cancer has made substantial progress in recent years, facilitating the optimization of existing therapeutic approaches to improve clinical outcomes in pancreatic cancer. With advances in treatment combinations and choices, it is becoming increasingly important to determine ways to place patients on the best therapies upfront. Although various molecular subtyping systems for pancreatic cancer have been proposed, consensus regarding proposed subtypes, as well as their relative clinical utility, remains largely unknown and presents a natural barrier to wider clinical adoption. Experimental Design: We assess three major subtype classification schemas in the context of results from two clinical trials and by meta-analysis of publicly available expression data to assess statistical criteria of subtype robustness and overall clinical relevance. We then developed a single-sample classifier (SSC) using penalized logistic regression based on the most robust and replicable schema. Results: We demonstrate that a tumor-intrinsic two-subtype schema is most robust, replicable, and clinically relevant. We developed Purity Independent Subtyping of Tumors (PurIST), a SSC with robust and highly replicable performance on a wide range of platforms and sample types. We show that PurIST subtypes have meaningful associations with patient prognosis and have significant implications for treatment response to FOLIFIRNOX. Conclusions: The flexibility and utility of PurIST on low-input samples such as tumor biopsies allows it to be used at the time of diagnosis to facilitate the choice of effective therapies for patients with pancreatic ductal adenocarcinoma and should be considered in the context of future clinical trials.

Tumors are mixtures of different compartments. While global gene expression analysis profiles the average expression of all compartments in a sample, identifying the specific contribution of each compartment remains a challenge. With the increasing recognition of the importance of non-neoplastic components, the ability to breakdown the gene expression contribution of each is critical. To this end, we developed DECODER, an integrated framework which performs de novo deconvolution, and compartment weight estimation for a single sample. We use DECODER to deconvolve 33 TCGA tumor RNA-seq datasets and show that it may be applied to other data types including ATAC-seq. We demonstrate that it can be utilized to reproducibly estimate cellular compartment weights in pancreatic cancer that are clinically meaningful. Application of DECODER across cancer types advances the capability of identifying cellular compartments in an unknown sample and may have implications for identifying the tumor of origin for cancers of unknown primary.

The cell surface expression of B7-H3 across multiple tumor subtypes and its restricted expression in normal tissues make it an attractive target for cancer immunotherapy. We generated chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR-Ts), and tested them in pancreatic ductal adenocarcinoma (PDAC), ovarian cancer (OC) and neuroblastoma (NB) models in vitro and in vivo. The results showed that B7-H3.CAR-Ts effectively controlled the growth of PDAC, OC and NB tumor cells in vitro and in orthotopic, metastatic and patient-derived xenograft (PDX) mouse models. In addition, we found that 4-1BB costimulation promotes lower PD1 expression in B7-H3.CAR-Ts, and thus have superior antitumor activity when targeting PD-L1 constitutively expressing tumor cells. Finally, taking the advantage of the cross-reactivity of B7-H3.CAR with murine B7-H3 (mB7-H3), we investigated the antitumor efficacy and safety of B7-H3.CAR-Ts in a syngeneic pancreatic orthotopic tumor model in immunocompetent mouse. The mB7-H3.CAR-Ts significantly controlled the tumor growth in immunocompetent mice tumor model without decreasing the hematopoietic or immune cell numbers, and no tissue damage in any analyzed organs was observed, these findings further support the safety and efficacy of B7-H3.CAR-Ts for clinical application.

BACKGROUND Surgical resection provides the only potentially curative treatment of pancreatic cancer. Neoadjuvant chemotherapy and/or radiation (NAT) is used to downstage patients with borderline resectable tumors. The objective of this study was to examine the postoperative morbidity and mortality of NAT after pancreaticoduodenectomy (PD) for pancreatic ductal adenocarcinoma (PDA). METHODS Using the American College of Surgeons-National Surgical Quality Improvement Project Targeted Pancreatectomy data, we identified patients who underwent a PD for PDA from 2014 to 2015. Patients were grouped by receipt of NAT 90 days before PD. Bivariable and multivariable analyses was used to compare postoperative outcomes. RESULTS A total of 3748 patients with PDA underwent PD; 926 (24.7%) received NAT. Those in the NAT group had more major vein resections, and longer operating times (all P

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal disease with a 5-year survival rate of 4%. A key hallmark of PDAC is extensive stromal involvement, which makes capturing precise tumor-specific molecular information difficult. Here we have overcome this problem by applying blind source separation to a diverse collection of PDAC gene expression microarray data, including data from primary tumor, metastatic and normal samples. By digitally separating tumor, stromal and normal gene expression, we have identified and validated two tumor subtypes, including a 'basal-like' subtype that has worse outcome and is molecularly similar to basal tumors in bladder and breast cancers. Furthermore, we define 'normal' and 'activated' stromal subtypes, which are independently prognostic. Our results provide new insights into the molecular composition of PDAC, which may be used to tailor therapies or provide decision support in a clinical setting where the choice and timing of therapies are critical.

An optimized peptide substrate was used to measure protein kinase B (PKB) activity in single cells. The peptide substrate was introduced into single cells, and capillary electrophoresis was used to separate and quantify nonphosphorylated and phosphorylated peptide. The system was validated in three model pancreatic cancer cell lines before being applied to primary cells from human pancreatic adenocarcinomas propagated in nude mice. As measured by phosphorylation of peptide substrate, each tumor cell line exhibited statistically different median levels of PKB activity (65%, 21%, and 4% phosphorylation in PANC-1 (human pancreatic carcinoma), CFPAC-1 (human metastatic ductal pancreatic adenocarcinoma), and HPAF-II cells (human pancreatic adenocarcinoma), respectively) with CFPAC-1 cells demonstrating two populations of cells or bimodal behavior in PKB activation levels. The primary cells exhibited highly variable PKB activity at the single cell level, with some cells displaying little to no activity and others possessing very high levels of activity. This system also enabled simultaneous characterization of peptidase action in single cells by measuring the amount of cleaved peptide substrate in each cell. The tumor cell lines displayed degradation rates statistically similar to one another (0.02, 0.06, and 0.1 zmol pg(-1) s(-1), for PANC-1, CFPAC-1, and HPAF-II cells, respectively) while the degradation rate in primary cells was 10-fold slower. The peptide cleavage sites also varied between tissue-cultured and primary cells, with 5- and 8-residue fragments formed in tumor cell lines and only the 8-residue fragment formed in primary cells. These results demonstrate the ability of chemical cytometry to identify important differences in enzymatic behavior between primary cells and tissue-cultured cell lines.

Recent treatment advances in chemotherapy and targeted therapy regimens have provided pancreatic cancer patients and providers with better options. Molecular subtyping for pancreatic cancer has made substantial progress in recent years. With these advances, there is now an opportunity to enable precision medicine approaches for patients with pancreatic cancer. Transcriptomic molecular subtyping is currently an active area of study where several subtyping schemas have been proposed. This talk will present some of these schemas and attempt to reconcile them. Many subtyping approaches rely on the clustering of a new sample with an existing group of samples, the ability to determine the subtype of an individual tumor is limited. In addition, in order to use molecular subtypes in the clinic, subtypes need to be clinically replicable, robust, and have clinical implications. Developing a robust method for subtype classification is needed in order to study the role of molecular subtypes in the context of future clinical trials. Citation Format: Jen Jen Yeh. Molecular subtypes - bench to bedside [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 I08.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by relatively low tumor purity and an abundant tumor microenvironment. To dissect the contribution of the biologic components, we developed DECODER, which performs de novo compartment deconvolution and weight estimation of tumor samples. DECODER is a sophisticated framework that integrates runs of non-negative matrix factorization (NMF) and non-negative least square (NNLS) algorithms and can be applied to any non-negative matrices without the need to know the number of resultant factors or compartments. DECODER was used to deconvolve the TCGA pancreatic adenocarcinoma (PAAD) RNA-seq dataset, which resulted in the identification of 7 major compartments (basal tumor, classical tumor, activated stroma, normal stroma, immune, endocrine, and exocrine), confirming prior manual NMF-based solutions. These results were then used for single-sample based weight estimation in the COMPASS trial and ICGC PACA-AU RNA-seq dataset. We saw a significant positive correlation between DECODER immune weight and leukocyte fraction (r = 0.757, p < 0.001) or ESTIMATE immune score (r = 0.773, p < 0.001). Samples with high immune weights corresponded to immune infiltration by histology. A significant correlation was found between the sum of basal and classical tumor weights, and tumor purity based on both ABSOLUTE (r = 0.699, p < 0.001) and methylation (r = 0.71, p < 0.001). Similarly, the sum of activated and normal stroma weights correlated with ESTIMATE stromal score (r = 0.729, p < 0.001). Interestingly, we found that the ratio between the basal and classical compartment (bcRatio) was significantly associated with survival outcome (p = 0.049 in TCGA and 0.008 in ICGC) in all patients and treatment response in basal-like patients (r = 0.884, p < 0.001 in COMPASS trial), suggesting that bcRatio may help explain the molecular basis for tumor behavior in PDAC. DECODER was also applied for de novo deconvolution for all the cancer types in TCGA RNA-seq dataset and identified the cancer type specific compartments. Results from DECODER can then be used for single-sample weight estimation of new samples for any cancer type. In addition, we applied DECODER on the PanCan ATAC-seq dataset containing 23 cancer types in a combined fashion, and identified compartments associated with cancer types or organ systems. This proves that DECODER is highly feasible to data of multiple platforms. In summary, we present an automated method for de novo deconvolution that may be used across tumor and data types. With deconvolved results as the reference, DECODER enables the single-sample weight estimation for a new sample, which is plausible in the clinical setting. Citation Format: Xianlu L. Peng, Richard A. Moffitt, Robert J. Torphy, Keith E. Volmar, Jen Jen Yeh. Compartment deconvolution in pancreatic cancer with biologic and clinical implications [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 B41.

The high prevalence of KRAS mutations and importance of the RalGEF–Ral pathway downstream of activated K-ras in pancreatic ductal adenocarcinoma (PDAC) emphasize the importance of identifying novel methods by which to therapeutically target these pathways. It was recently demonstrated that phosphorylation of RalA S194 by Aurora A kinase (AAK) is critical for PDAC tumorigenesis. We sought to evaluate the AAK-selective inhibitor MLN8237 as a potential indirect anti-RalA–targeted therapy for PDAC. We used a site-specific phospho-S194 RalA antibody and determined that RalA S194 phosphorylation levels were elevated in a subset of PDAC cell lines and human tumors relative to unmatched normal controls. Effects of MLN8237 on anchorage-independent growth in PDAC cell lines and growth of patient-derived xenografts (PDX) were variable, with a subset of cell lines and PDX showing sensitivity. Surprisingly, RalA S194 phosphorylation levels in PDAC cell lines or PDX tumors did not correlate with MLN8237 responsiveness. However, we identified Ki67 as a possible early predictive biomarker for response to MLN8237 in PDAC. These results indicate that MLN8237 treatment may be effective for a subset of patients with PDAC independent of RalA S194 phosphorylation. Ki67 may be an effective pharmacodynamic biomarker to identify response early in the course of treatment. Mol Cancer Ther; 13(1); 122–33. ©2013 AACR.

Pancreatic ductal adenocarcinoma, which accounts for the majority of pancreatic cancers, is a lethal disease with few therapeutic options. Genomic profiling of pancreatic ductal adenocarcinoma has identified a complex and heterogeneous landscape. Understanding the molecular characteristics of pancreatic ductal adenocarcinoma will facilitate the identification of potential therapeutic strategies. We analyzed the gene expression profiles of primary tumors from patients compared to normal pancreas and identified high co-overexpression of core components of the spindle assembly checkpoint, including the protein kinase TTK (also known as MPS-1). We found overexpression of TTK protein in a subset of pancreatic ductal adenocarcinoma primary tumors and cell lines. siRNA-mediated depletion or catalytic inhibition of TTK resulted in an aberrant cell cycle profile, multi- and micro-nucleation, induction of apoptosis, and decreased cell proliferation and transformed growth. Selective catalytic inhibition of TTK caused override of the spindle assembly checkpoint-induced cell cycle arrest. Interestingly, we identified ubiquitin specific peptidase 16 (Usp16), an ubiquitin hydrolase, as a phosphorylation substrate of TTK. Usp16 regulates chromosomal condensation and G2/M progression by deubiquitinating histone H2A and polo-like kinase 1. Phosphomimetic mutants of Usp16 show enhanced proteosomal degradation and may prolong the G2/M transition allowing for correction of replication errors. Taken together, our results suggest a critical role for TTK in preventing aneuploidy-induced cell death in pancreatic cancer.

Purpose Advanced pancreatic cancer is a highly refractory disease almost always associated with survival of little more than a year. New interventions based on novel targets are needed. We aim to identify new genetic determinants of overall survival (OS) in patients after treatment with gemcitabine using genome-wide screens of germline DNA. We aim also to support these findings with in vitro functional analysis. Patients and methods Genome-wide screens of germline DNA in two independent cohorts of pancreatic cancer patients (from the Cancer and Leukemia Group B (CALGB) 80303 and the Mayo Clinic) were used to select new genes associated with OS. The vitamin D receptor gene (VDR) was selected, and the interactions of genetic variation in VDR with circulating vitamin D levels and gemcitabine treatment were evaluated. Functional effects of common VDR variants were also evaluated in experimental assays in human cell lines. Results The rs2853564 variant in VDR was associated with OS in patients from both the Mayo Clinic (HR 0.81, 95% CI 0.70-0.94, p = 0.0059) and CALGB 80303 (HR 0.74, 0.63-0.87, p = 0.0002). rs2853564 interacted with high pre-treatment levels of 25-hydroxyvitamin D (25(OH)D, a measure of endogenous vitamin D) (p = 0.0079 for interaction) and with gemcitabine treatment (p = 0.024 for interaction) to confer increased OS. rs2853564 increased transcriptional activity in luciferase assays and reduced the binding of the IRF4 transcription factor. Conclusion Our findings propose VDR as a novel determinant of survival in advanced pancreatic cancer patients. Common functional variation in this gene might interact with endogenous vitamin D and gemcitabine treatment to determine improved patient survival. These results support evidence for a modulatory role of the vitamin D pathway for the survival of advanced pancreatic cancer patients.

Background and Hypothesis: Pancreatic cancer (PC) has the poorest prognosis of all major cancers, with a 5-year survival of less than 5%. Cell-free DNA (cfDNA) has been well validated as a biomarker that correlates with tumor burden. More recently, a role for cfDNA in tumor progression has also been identified. The latter occurs through activation of Toll-like receptors (TLRs) in both tumor cells and the host environment, which can upregulate prometastatic signaling pathways. In addition, tumor-derived extracellular vesicles (EVs) such as microparticles and exosomes have also been implicated in promoting metastasis by activating proinvasive pathways in tumor cells and preconditioning secondary sites for metastatic establishment. Our laboratory has previously shown that cationic polymers can neutralize cfDNA and abrogate inflammation in disease models of autoimmunity and infection. We investigated the ability of the cationic polymer, PAMAM-G3, to bind and inhibit cfDNA and EVs and thereby inhibit tumor invasion in vitro and metastasis in vivo. Methods and Results: Transwell-Matrigel invasion assays were performed using multiple PC cell lines. Cells were plated in the upper chamber with no serum +/- cfDNA, EVs, or PAMAM-G3. The bottom chamber was plated with complete media as a chemo-attractant. Cells treated with cfDNA, EVs, or PAMAM-G3 were also analyzed for activation of pro-inflammatory pathways (NFkB, MAPK, etc.). The in vivo model was performed in C57B6 mice that were injected with bioluminescent murine PC cells into their spleens and followed for liver metastases with bioluminescent imaging. Experimental mice were treated biweekly with intraperitoneal PAMAM-G3 (20 mg/kg) or saline starting 48 hours after pancreatic tumor cell implantation. PAMAM-G3 significantly inhibited in vitro invasion of pancreatic cancer cell lines in response to cancer patient serum-derived cfDNA and extracellular vesicles. Moreover, PAMAM-G3 (20 mg/kg) treatment led to a significant reduction in liver metastasis without affecting primary tumor growth in vivo. Serum derived from saline-treated mice induced higher levels of invasion than serum from polymer-treated mice and exogenous polymer blocked this effect (**** = p Discussion: Cationic polymers such as PAMAM-G3 may represent a novel class of therapeutics to combat pancreatic cancer metastasis. These polymers can bind and neutralize cfDNA and tumor-derived EVs, thereby blocking activation of pro-inflammatory pathways within tumor cells, and reducing tumor invasion and metastasis. Cationic polymers will be tested in other preclinical models of cancer and in dosing studies for potential progression to clinical translation. Citation Format: Ibtehaj A. Naqvi, Ruwan Gunaratne, Jessica McDade, Douglas Rouse, Jen Jen Yeh, David Pisetsky, Jaewoo Lee, Rebekah White, Bruce Sullenger. Cationic polymer inhibits pancreatic cancer invasion and metastasis [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 4189.

Phase II clinical trials of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors are ongoing and ERK1/2 activation is frequently used as a biomarker. In light of the mutational activation of BRAF and KRAS in colorectal cancer, inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase are anticipated to be promising. Previous studies in pancreatic cancer have found little correlation between BRAF/KRAS mutation status and ERK1/2 activation, suggesting that identifying biomarkers of MEK inhibitor response may be more challenging than previously thought. The purpose of this study was to evaluate the effectiveness of MEK inhibitor therapy for colorectal cancer and BRAF/KRAS mutation status and ERK1/2 activation as biomarkers for MEK inhibitor therapy. First, we found that MEK inhibitor treatment impaired the anchorage-independent growth of nearly all KRAS/BRAF mutant, but not wild-type, colorectal cancer cells. There was a correlation between BRAF, but not KRAS, mutation status and ERK1/2 activation. Second, neither elevated ERK1/2 activation nor reduction of ERK1/2 activity correlated with MEK inhibition of anchorage-independent growth. Finally, we validated our cell line observations and found that ERK1/2 activation correlated with BRAF, but not KRAS, mutation status in 190 patient colorectal cancer tissues. Surprisingly, we also found that ERK activation was elevated in normal colonic epithelium, suggesting that normal cell toxicity may be a complication for colorectal cancer treatment. Our results suggest that although MEK inhibitors show promise in colorectal cancer, KRAS/BRAF mutation status, but not ERK activation as previously thought, may be useful biomarkers for MEK inhibitor sensitivity. [Mol Cancer Ther 2009;8(4):834–43]

Pancreatic cancer is a deadly disease with an annual incidence rate nearly equal to its mortality rate. Incremental improvement in outcome has been seen in the last 25 years, illustrating the critical need for novel approaches and intensive research investment. Expression profiling of pancreatic cancers has led to an explosion of informative gene-expression changes and the identification of new diagnostic and prognostic markers. However, the search for genes that are of functional significance in these large datasets continues to be much more challenging. One approach to focusing on genes or pathways that are likely to be more biologically relevant is to study those that are of prognostic significance. This review will therefore focus on the advantages of a prognostic gene signature for pancreatic cancer, the advances that have been made thus far, the approaches used and the challenges that remain.

Poor delivery and systemic toxicity of many cytotoxic agents, such as the recent promising combination chemotherapy regimen of folinic acid (leucovorin), fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX), restrict their full utility in the treatment of pancreatic cancer. Local delivery of chemotherapies has become possible using iontophoretic devices that are implanted directly onto pancreatic tumors. We have fabricated implantable iontophoretic devices and tested the local iontophoretic delivery of FOLFIRINOX for the treatment of pancreatic cancer in an orthotopic patient-derived xenograft model. Iontophoretic delivery of FOLFIRINOX was found to increase tumor exposure by almost an order of magnitude compared with i.v. delivery with substantially lower plasma concentrations. Mice treated for 7 wk with device FOLFIRINOX experienced significantly greater tumor growth inhibition compared with i.v. FOLFIRINOX. A marker of cell proliferation, Ki-67, was stained, showing a significant reduction in tumor cell proliferation. These data capitalize on the unique ability of an implantable iontophoretic device to deliver much higher concentrations of drug to the tumor compared with i.v. delivery. Local iontophoretic delivery of cytotoxic agents should be considered for the treatment of patients with unresectable nonmetastatic disease and for patients with the need for palliation of local symptoms, and may be considered as a neoadjuvant approach to improve resection rates and outcome in patients with localized and locally advanced pancreatic cancer.

There is currently tremendous interest in developing anti-cancer therapeutics targeting cell signaling pathways important for both cancer cell metabolism and growth. Several epidemiological studies have shown that diabetic patients taking metformin have a decreased incidence of pancreatic cancer. This has prompted efforts to evaluate metformin, a drug with negligible toxicity, as a therapeutic modality in pancreatic cancer. Preclinical studies in cell line xenografts and one study in patient-derived xenograft (PDX) models were promising, while recently published clinical trials showed no benefit to adding metformin to combination therapy regimens for locally advanced and metastatic pancreatic cancer. PDX models in which patient tumors are directly engrafted into immunocompromised mice have been shown to be excellent preclinical models for biomarker discovery and therapeutic development. We evaluated the response of four PDX tumor lines to metformin treatment and found that all four of our PDX lines were resistant to metformin. We found that the mechanisms of resistance may occur through lack of sustained activation of adenosine monophosphate-activated protein kinase (AMPK) or downstream reactivation of the mammalian target of rapamycin (mTOR). Moreover, combined treatment with metformin and mTOR inhibitors failed to improve responses in cell lines, which further indicates that metformin alone or in combination with mTOR inhibitors will be ineffective in patients, and that resistance to metformin may occur through multiple pathways. Further studies are required to better understand these mechanisms of resistance and inform potential combination therapies with metformin and existing or novel therapeutics.

Pancreatic cancer is a lethal disease with a 5-year survival rate of 4%. The only opportunity for improved survival continues to be complete surgical resection for those with localized disease. Although chemotherapeutic options are limited for the few patients with resectable disease, this problem is even more magnified in the majority (85%) of patients with unresectable or metastastic disease. Therefore, there is an urgent need for improved therapeutic options. The recent success of inhibitors of signal transduction for the treatment of other cancers supports the need to identify and validate aberrant signaling pathways important for pancreatic tumor growth. This review focuses on the validation of specific signaling networks and the present status of inhibitors of these pathways as therapeutic approaches for pancreatic cancer treatment.

Parenteral and oral routes have been the traditional methods of administering cytotoxic agents to cancer patients. Unfortunately, the maximum potential effect of these cytotoxic agents has been limited because of systemic toxicity and poor tumor perfusion. In an attempt to improve the efficacy of cytotoxic agents while mitigating their side effects, we have developed modalities for the localized iontophoretic delivery of cytotoxic agents. These iontophoretic devices were designed to be implanted proximal to the tumor with external control of power and drug flow. Three distinct orthotopic mouse models of cancer and a canine model were evaluated for device efficacy and toxicity. Orthotopic patient-derived pancreatic cancer xenografts treated biweekly with gemcitabine via the device for 7 weeks experienced a mean log2 fold change in tumor volume of –0.8 compared to a mean log2 fold change in tumor volume of 1.1 for intravenous (IV) gemcitabine, 3.0 for IV saline, and 2.6 for device saline groups. The weekly coadministration of systemic cisplatin therapy and transdermal device cisplatin therapy significantly increased tumor growth inhibition and doubled the survival in two aggressive orthotopic models of breast cancer. The addition of radiotherapy to this treatment further extended survival. Device delivery of gemcitabine in dogs resulted in more than 7-fold difference in local drug concentrations and 25-fold lower systemic drug levels than the IV treatment. Overall, these devices have potential paradigm shifting implications for the treatment of pancreatic, breast, and other solid tumors.

Summary We performed integrated genomic, transcriptomic, and proteomic profiling of 150 pancreatic ductal adenocarcinoma (PDAC) specimens, including samples with characteristic low neoplastic cellularity. Deep whole-exome sequencing revealed recurrent somatic mutations in KRAS , TP5 3, CDKN2A , SMAD4 , RNF43 , ARID1A , TGFβR2 , GNAS , RREB1 , and PBRM1 . KRAS wild-type tumors harbored alterations in other oncogenic drivers, including GNAS , BRAF , CTNNB1 , and additional RAS pathway genes. A subset of tumors harbored multiple KRAS mutations, with some showing evidence of biallelic mutations. Protein profiling identified a favorable prognosis subset with low epithelial-mesenchymal transition and high MTOR pathway scores. Associations of non-coding RNAs with tumor-specific mRNA subtypes were also identified. Our integrated multi-platform analysis reveals a complex molecular landscape of PDAC and provides a roadmap for precision medicine.

Pancreatic cancer has the poorest prognosis of all major cancers with a 5-year survival of less than 5%. Cell-free DNA (cfDNA) has been well-validated as a biomarker that correlates with tumor burden. More recently, a role for cfDNA in facilitating tumor progression has also been identified. The latter occurs through activation of nucleic acid sensing receptors such as toll-like receptors (TLRs) in both tumor cells and the host environment, which can upregulate pro-metastatic signaling pathways. In addition, tumor-derived extracellular vesicles such as microparticles and exosomes have also been implicated in promoting metastasis by activating pro-invasive pathways in tumor cells and pre-conditioning secondary sites for metastatic establishment. Our laboratory has previously shown that cationic polymers can scavenge negatively charged nucleic acids like cfDNA and abrogate aberrant inflammation in disease models of autoimmunity and infection. Herein, we investigated the ability of the cationic polymer, PAMAM-G3, to bind and inhibit cfDNA as well as anionic tumor-derived extracellular vesicles using in vitro models of pancreatic cancer cell line invasion. We also evaluated the effect of PAMAM-G3 treatment in a bioluminescent syngeneic murine model of pancreatic cancer metastasis. PAMAM-G3 significantly inhibited in vitro invasion of pancreatic cancer cell lines in response to both cfDNA and tumor-derived extracellular vesicles. Moreover, biweekly intraperitoneal treatment with PAMAM-G3 (20 mg/kg) starting 48 hours after pancreatic tumor cell implantation led to a significant reduction in liver metastasis without affecting primary tumor growth. Thus, cationic polymers such as PAMAM-G3 may represent a novel class of therapeutics to combat pancreatic cancer metastasis. Citation Format: Ibtehaj A. Naqvi, Ruwan Gunaratne, Jessica McDade, Douglas Rouse, Jen Jen Yeh, David Pisetsky, Jaewoo Lee, Rebekah White, Bruce Sullenger. Cationic polymer inhibits pancreatic cancer invasion in vitro and metastasis in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4903. doi:10.1158/1538-7445.AM2017-4903

Circulating tumor cells (CTCs) are cells shed from solid tumors into circulation and have been shown to be prognostic in the setting of metastatic disease. These cells are obtained through a routine blood draw and may serve as an easily accessible marker for monitoring treatment effectiveness. Because of the rapid progression of pancreatic ductal adenocarcinoma (PDAC), early insight into treatment effectiveness may allow for necessary and timely changes in treatment regimens. The objective of this study was to evaluate CTC burden as a biomarker of response to treatment with a oral phosphatidylinositol-3-kinase inhibitor, BKM120, in patient-derived xenograft (PDX) mouse models of PDAC. PDX mice were randomized to receive vehicle or BKM120 treatment for 28 days and CTCs were enumerated from whole blood before and after treatment using a microfluidic chip that selected for EpCAM (epithelial cell adhesion molecule) positive cells. This microfluidic device allowed for the release of captured CTCs and enumeration of these cells via their electrical impedance signatures. Median CTC counts significantly decreased in the BKM120 group from pre- to post-treatment (26.61 to 2.21 CTCs/250 µL, p = 0.0207) while no significant change was observed in the vehicle group (23.26 to 11.89 CTCs/250 µL, p = 0.8081). This reduction in CTC burden in the treatment group correlated with tumor growth inhibition indicating CTC burden is a promising biomarker of response to treatment in preclinical models. Mutant enriched sequencing of isolated CTCs confirmed that they harbored KRAS G12V mutations, identical to the matched tumors. In the long-term, PDX mice are a useful preclinical model for furthering our understanding of CTCs. Clinically, mutational analysis of CTCs and serial monitoring of CTC burden may be used as a minimally invasive approach to predict and monitor treatment response to guide therapeutic regimens.

In this manuscript, we discuss the development and clinical use of a thermoplastic modular microsystem for the high-throughput analysis of CTCs directly from whole blood. The modular system offers some innovative features that address challenges currently associated with many CTC platforms; it can exhaustively process 7.5 ml of blood in less than 45 min with recoveries >90%. In addition, the system automates the post-selection CTC processing steps and thus, significantly reduces assay turnaround time (from selection to enumeration 8 h for many reported CTC platforms). The system is comprised of 3 functional modules including; (i) a thermoplastic CTC selection module composed of high aspect ratio (30 μm × 150 μm) channels containing anti-EpCAM antibodies that is scalable in terms of throughput by employing channel numbers ranging from 50 to 320 – the channel number is user selected to accommodate the volume of blood that must be processed; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for the placement of released cells into a 2D array within a common imaging plane for phenotypic identification. To demonstrate the utility of this system, blood samples from patients with local resectable and metastatic pancreatic ductal adenocarcinoma (PDAC) were analyzed. We demonstrate the ability to select EpCAM positive CTCs from PDAC patients in high purity (>86%) and with excellent yields (mean = 53 CTCs per ml for metastatic PDAC patients) using our modular system. In addition, we demonstrate the ability to detect CTCs in PDAC patients with local resectable disease (mean = 11 CTCs per ml).

Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets. Specific capture of nucleated cells or cells expressing epithelial cell adhesion molecules (EpCAM) was achieved by functionalizing micropallet surfaces with either fibronectin, Matrigel or anti-EpCAM antibody. Surface grafting of poly(acrylic acid) followed by covalent binding of protein A/G enabled efficient capture of EpCAM antibody on the micropallet surface. MCF-7 cells, a human breast adenocarcinoma, were retained on the array surface with 90±8% efficiency when using an anti-EpCAM-coated array. To demonstrate the efficiency of tumor cell retention on micropallet arrays in the presence of blood, MCF-7 cells were mixed into whole blood and added to small arrays (71 mm(2)) coated with fibronectin, Matrigel or anti-EpCAM. These approaches achieved MCF-7 cell capture from ≤10 µL of whole blood with efficiencies greater than 85%. Furthermore, MCF-7 cells intermixed with 1 mL blood and loaded onto large arrays (7171 mm(2)) were captured with high efficiencies (≥97%), could be isolated from the array by a laser-based approach and were demonstrated to yield a high rate of colony formation (≥85%) after removal from the array. Clinical utility of this technology was shown through the capture, isolation and successful culture of CTCs from the blood of mice engrafted with primary human pancreatic tumors. Direct capture and isolation of living tumor cells from blood followed by analysis or culture will be a valuable tool for cancer cell characterization.

Mutations in KRAS drive the oncogenic phenotype in a variety of tumors of epithelial origin. The NF-κB transcription factor pathway is important for oncogenic RAS to transform cells and to drive tumorigenesis in animal models. Recently, TGF-β–activated kinase 1 (TAK1), an upstream regulator of IκB kinase (IKK), which controls canonical NF-κB signaling, was shown to be important for chemoresistance in pancreatic cancer and for regulating KRAS-mutant colorectal cancer cell growth and survival. Here, we show that mutant KRAS upregulates glycogen synthase kinase 3α (GSK-3α), leading to its interaction with TAK1 to stabilize the TAK1–TAB complex to promote IKK activity. In addition, GSK-3α is required for promoting critical noncanonical NF-κB signaling in pancreatic cancer cells. Pharmacologic inhibition of GSK-3 suppresses growth of human pancreatic tumor explants, consistent with the loss of expression of oncogenic genes such as c-myc and TERT. These data identify GSK-3α as a key downstream effector of oncogenic KRAS via its ability to coordinately regulate distinct NF-κB signaling pathways. Significance: GSK-3α functions to promote IKK/NF-κB activity downstream of oncogenic KRAS via stabilization and activation of the TAK1/TAB complex and to promote noncanonical NF-κB activity via control of nuclear levels of NF-κB2. Inhibition of GSK-3 strongly suppresses growth of human pancreatic tumor explants with downregulation of certain oncogenic NF-κB target genes such as c-myc and TERT. Cancer Discov; 3(6); 690–703. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 591

Background and Aims: Mutations in RAS and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers, including up to 25% of lung cancers and over 90% of pancreatic cancers. Ras function as ‘molecular switches’ in a number of signaling pathways that regulate vital cellular functions. Over the past few decades, it has become clear that the activity or the oncogenic potential of Ras is dependent on the non-receptor tyrosine kinase Src to regulate essential cellular pathways for proliferation, differentiation and survival of eukaryotic cells. Recently, we showed that Src binds to and phosphorylates Ras on a conserved tyrosine residue at position 32 within the switch I region to promote Ras GTPase activity. We then identified Shp2 as the critical tyrosine phosphatase that is responsible for the dephosphorylation and activation of Ras. However, it is unknown whether pharmacologic manipulation of Shp2 activity would suppress the growth of Ras-driven pancreatic cancer (PDACs), a highly aggressive disease currently without effective treatment options or cure. Methods: We generated the available Ras, Src, and SHP2 reagents (wild-type, oncogenic, truncation, dominant-negative, catalytically active or inactive, mutants) to determine the structure/function relationship between Ras, Src, and SHP2. The interaction was assessed by immunoprecipitation and western blotting. We utilized several PDAC cell lines and patient-derived xenografts cell lines (PDX-C). The cell growth was evaluated by Alarmar Blue assay. Results: We identified SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signaling. In comparison to K-Ras WT pancreatic cancer cells, the level of phospho-Shp2 was observed to be higher in K-Ras mutated cell lines in both absence and presence of growth factors. Pharmacologic inhibition of Shp2 using a specific cell-permeable Shp2 inhibitor decreased the level of phosphorylation of Shp2 as well as downstream signaling such as pAKT and pERK especially in the presence of growth factor. Notably, we show that treatment with this inhibitor suppressed the cell proliferation in both K-Ras WT and mutated pancreatic cancer cells as well as PDX-C in a caspase-dependent manner. Conclusion: Our results identify SHP2 as a direct activator of Ras and potential therapeutic target for pancreatic cancer driven by a previously ‘undruggable’ oncogenic or hyperactive Ras. Citation Format: Yoshihito Kano, Severa Bunda, Jen Jen Yeh, Zhong-Yin Zhang, Michael Ohh.{Authors}. Novel treatment strategy for pancreatic cancer by targeting the ‘undruggable’ Ras oncoprotein. [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 B13.

Despite the poor long-term survival of patients with pancreatic ductal adenocarcinoma (PDA), there are significant survival probabilities in subgroups of patients that cannot be predicted by surgical, pathological, and clinical features, including stage, surgical margins and perineural invasion. Only in the last year, more than a handful papers reported advanced genomic analyses on multiple components of tumors to dissect molecular heterogeneities that may provide a better understanding of gene expression signatures to predict survival probabilities. Although gene-mutation and expression “omic” signatures hold promise, they are difficult to translate into clinical practice. Therefore, we initiated a two phase unbiased and systematic study to first identify and validate a prognostic biomarker by screening multiple independent RNASeq databases and performing survival analyses, on a total of 493 cases. Second, we performed immunohistochemical analyses on 117 cases to validate the in silico analysis results using a pathology based scoring system. We identified in the discovery dataset that keratin 17 (K17) expression discriminated between rapid and slower progressing PDA among patients with localized disease. K17-positive cases had four times the likelihood of dying from this disease compared to stage-matched K17-negative cases (P= 0.0094; median survival 1.64 years compared to >5 years, respectively). In addition, we found that positive-K17 patients have almost twice the probability of recurrence after chemotherapy, compared to matched stage, negative-K17 counterparts. These critical observations were validated in four independent PDA patient cohorts, indicating the negative prognostic value of K17 in this devastating disease, independent of the patient’s age, sex, and tumor stage and grade. Furthermore, we found that pathology-based quantification of K17 expression by immunohistochemistry in malignant cells was associated with poor-patient outcome (P= 0.0026). Based on the recent molecular “omics” subtyping PDA identified by Bailey et al., 2016 and Moffitt et al., 2015, we found that K17 expression can be used to distinguish the “squamous” and “basal” subtypes presented in these reports, respectively. Equally important, in addition to PDA, we found that high expression of K17 is strongly associated with decreased patient survival in a number of carcinomas, including but not limited to cancers of the lung, breast, liver, stomach. Results consistently indicated that K17 expression is associated with shortened interval to death (Hazard Ratio >2). Collectively, these results suggest that activation of K17 expression across multiple primary tumors is a conserved molecular event in tumors with shared clinical behavior and not anatomic location. Based on mechanistic approaches, we know that K17 is a regulator of cell-cycle progression and directly acts to enhance pancreatic tumor growth, providing insight into the mechanisms that drive PDA tumor biology as a major cause of patient mortality. In conclusion, K17 was identified as a robust and independent clinically relevant prognostic biomarker that may be used to better stratify clinical outcome at the time of initial diagnosis. Citation Format: Luisa Escobar-Hoyos, Elizabeth A. Vanner, Shula Schechter, Lucia Roa-Pena, Jinyu Li, Jinelle Wint, Scott Powers, Ali Akalin, Jela Bandovic, Peter Bailey, David K. Chang, Richard A. Moffit, Jen Jen Yeh, Andrew Biankin, Kenneth R. Shroyer.{Authors}. Translating keratin 17 status to stratify clinically relevant pancreatic cancer heterogeneity and survival. [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 B76.

Tumor stroma (TS) is a cardinal component of pancreatic ductal adenocarcinoma (PDAC); it comprises 60 to 90% of tumor volume and is an emerging treatment target. There are conflicting reports of the role that TS plays in disease progression and its prognostic significance. The objective of this study was to develop a high-throughput, operator independent method for objectively quantifying TS density (TSD) and to evaluate the prognostic significance of TSD in resected tumors. Tissue sections of primary tumor from 126 patients undergoing pancreaticoduodenectomy for PDAC from 1979 to 2008 and followed by observation only were stained with hematoxylin and eosin (H&E). Sections were digitally uploaded and location of tumor was annotated using ImageScope. Annotated images were imported to Definiens Architect. By training on representative areas, an algorithm was created to segment the tissue into different regions-of-interest (ROI): tumor epithelium, TS, and inflammatory infiltrate. Next, the algorithm was used to quantify the above described ROIs in each tissue section. TSD was calculated as tumor stroma area over total tumor area. A cox proportional hazards model was used to compare overall survival (OS) and recurrence free survival (RFS) vs TSD controlling for age, sex, tumor grade, T, N, and overall stage, smoking status, and American Society of Anesthesia (ASA) physical classification score. Median TSD was 0.637. High TSD was significantly associated with RFS (p=0.043) and OS (p=0.037). In the low TSD group, median RFS and OS was 8.9 months and 11.5 months, respectively. In the high TSD group, median RFS and OS was 13.9 and 18.3 months, respectively. In this cohort, 18 patients were identified with an OS greater than 5 years. This cohort had a higher median TSD of 0.71. After excluding those with a survival greater than 5 years, there was still a trend toward high TSD and worse OS (p=0.052). Our novel automated algorithm for quantification of TSD allows for high throughput, reproducible, and objective analysis of H&E stained PDAC slides. The median TSD in our study obtained using our novel algorithm was 0.637, in agreement with prior manual descriptions of PDAC tumors having a TSD of greater than 60%. We also found high TSD to be a positive independent prognostic variable in resected tumors in patients not receiving adjuvant therapy. Further understanding of the balance of TS in restraining tumor progression vs impeding chemotherapeutic delivery is needed to successfully manipulate stroma in the treatment of PDAC. Citation Format: Robert J. Torphy, Rashid Naim, Bentley Midkiff, Keith Volmar, Eric Collisson, Jen Jen Yeh.{Authors}. Stroma in pancreatic cancer: Automated quantification and its prognostic significance. [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 B80.

The stromal compartment surrounding epithelial-derived pancreatic tumors is thought to have a key role in the aggressive phenotype of this malignancy. Emerging evidence suggests that cancer-associated fibroblasts (CAFs), the most abundant cells in the stroma of pancreatic tumors, contribute to the tumor’s invasion, metastasis and resistance to therapy, but the precise molecular mechanisms that regulate CAFs behavior are poorly understood. In this study, we utilized immortalized human pancreatic CAFs to investigate molecular pathways that control the matrix-remodeling and invasion-promoting activity of CAFs. We showed previously that palladin, an actin-associated protein, is expressed at high levels in CAFs of pancreatic tumors and other solid tumors, and also in an immortalized line of human CAFs. In this study, we found that short-term exposure of CAFs to phorbol esters reduced the number of stress fibers and triggered the appearance of individual invadopodia and invadopodial rosettes in CAFs. Molecular analysis of invadopodia revealed that their composition resembled that of similar structures (that is, invadopodia and podosomes) described in other cell types. Pharmacological inhibition and small interfering RNA knockdown experiments demonstrated that protein kinase C, the small GTPase Cdc42 and palladin were necessary for the efficient assembly of invadopodia by CAFs. In addition, GTPase activity assays showed that palladin contributes to the activation of Cdc42. In mouse xenograft experiments using a mixture of CAFs and tumor cells, palladin expression in CAFs promoted the rapid growth and metastasis of human pancreatic tumor cells. Overall, these results indicate that high levels of palladin expression in CAFs enhance their ability to remodel the extracellular matrix by regulating the activity of Cdc42, which in turn promotes the assembly of matrix-degrading invadopodia in CAFs and tumor cell invasion. Together, these results identify a novel molecular signaling pathway that may provide new molecular targets for the inhibition of pancreatic cancer metastasis.

Background Pancreatic cancer is the fourth leading cause of cancer related deaths in the United States with a five-year survival rate of 6%. It is characterized by extremely aggressive tumor growth rate and high incidence of metastasis. One of the most common and profound biochemical phenotypes of animal and human cancer cells is their ability to metabolize glucose at high rates, even under aerobic conditions. However, the contribution of metabolic interrelationships between tumor cells and cells of the surrounding microenvironment to the progression of cancer is not well understood. We evaluated differential expression of metabolic genes and, hence, metabolic pathways in primary tumor and metastases of patients with pancreatic adenocarcinoma. Methods and Findings We analyzed the metabolic gene (those involved in glycolysis, tri-carboxylic acid pathway, pentose-phosphate pathway and fatty acid metabolism) expression profiles of primary and metastatic lesions from pancreatic cancer patients by gene expression arrays. We observed two principal results: genes that were upregulated in primary and most of the metastatic lesions; and genes that were upregulated only in specific metastatic lesions in a site-specific manner. Immunohistochemical (IHC) analyses of several metabolic gene products confirmed the gene expression patterns at the protein level. The IHC analyses also revealed differential tumor and stromal expression patterns of metabolic enzymes that were correlated with the metastasis sites. Conclusions Here, we present the first comprehensive studies that establish differential metabolic status of tumor and stromal components and elevation of aerobic glycolysis gene expression in pancreatic cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a truly devastating disease, primarily due to late diagnosis when curative resection is no longer possible and the lack of accurate early detection biomarkers. Therefore, increasing PDAC survival depends heavily on improving the accuracy and effectiveness of early PDAC detection. Methods:Key PDAC biomarkers were identified through an optimized meta-analysis strategy of raw data in 4 PDAC transcriptome datasets (training sets). Application of identical normalization and statistical testing procedures for each dataset generated a list of differentially expressed genes by empirical Bayes moderated t-statistic. The top ranking PDAC-specific genes from this list that were consistently differentially expressed in these training sets were used for developing a classifier for PDAC. The biomarker panel with the highest sensitivity and specificity in the training sets was validated for diagnostic performance in nine independent validation datasets. A subset of genes in the panel were evaluated by immunohistochemistry (IHC) in human PDAC tissue sections. Cell based assays assessed the effect of two genes on PDAC migration and invasion. Results and Conclusions: A 5-gene classifier panel was identified that disntinguished between PDAC and normal pancreas with high sensitivity, specificity and accuracy in the four training sets. Validation of this 5-gene predictor in 9 independent test sets confirmed the diagnostic accuracy to not only discriminate accurately between PDAC and healthy controls, but also between pancreatitis and PDAC. IHC analysis of two of the proteins encoded by the panel confirmed enhanced expression in PDAC cells. Cell-based experiments demonstrated that both proteins enhance migration and invasion of PDAC cells as well as soft agar growth, suggesting a causal link to PDAC development and progression. The immediate clinical utility upon further prospective validation of this PDAC biomarker panel will be accurate PDAC diagnosis in patients with pancreatic abnormalities on imaging, risk stratification and evaluation of the risk of malignancy in pancreatic cysts. This abstract is also presented as Poster B26. Citation Format: Manoj Bhasin, Octavian Bucur, Ken Ndebele, Jessica Plati, Andrea Bullock, Xuesong Gu, Eduardo Castan, Robert Najarian, Jen Jen Yeh, Channing Der, Jon Cody Haines, Karl Ruping, Rebecca Miksad, Roya Khosravi-Far, Towia Aron Libermann. Transcriptome meta-analysis identifies new 5-gene classifier for early detection of pancreatic cancer. [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 PR07.