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Deregulation of the RAS GTPase cycle due to mutations in the three RAS genes is commonly associated with cancer development. Protein tyrosine phosphatase SHP2 promotes RAF-to-MAPK signaling pathway and is an essential factor in RAS-driven oncogenesis. Despite the emergence of SHP2 inhibitors for the treatment of cancers harbouring mutant KRAS, the mechanism underlying SHP2 activation of KRAS signaling remains unclear. Here we report tyrosyl-phosphorylation of endogenous RAS and demonstrate that KRAS phosphorylation via Src on Tyr32 and Tyr64 alters the conformation of switch I and II regions, which stalls multiple steps of the GTPase cycle and impairs binding to effectors. In contrast, SHP2 dephosphorylates KRAS, a process that is required to maintain dynamic canonical KRAS GTPase cycle. Notably, Src- and SHP2-mediated regulation of KRAS activity extends to oncogenic KRAS and the inhibition of SHP2 disrupts the phosphorylation cycle, shifting the equilibrium of the GTPase cycle towards the stalled ‘dark state’., Deregulation of the RAS GTPase cycle due to mutations in RAS genes is commonly associated with cancer development. Here authors use NMR and mass spectrometry to shows that KRAS phosphorylation via Src alters the conformation of switch I and II regions and thereby impacts the GTPase cycle.

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.

Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance.

We describe a comprehensive genomic characterization of adrenocortical carcinoma (ACC). Using this dataset, we expand the catalogue of known ACC driver genes to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1. Genome wide DNA copy-number analysis revealed frequent occurrence of massive DNA loss followed by whole-genome doubling (WGD), which was associated with aggressive clinical course, suggesting WGD is a hallmark of disease progression. Corroborating this hypothesis were increased TERT expression, decreased telomere length, and activation of cell-cycle programs. Integrated subtype analysis identified three ACC subtypes with distinct clinical outcome and molecular alterations which could be captured by a 68-CpG probe DNA-methylation signature, proposing a strategy for clinical stratification of patients based on molecular markers.

e16754 Background: Myosteatosis (adipose deposits in muscle) can be detected on cross-sectional imaging through variations in Skeletal Muscle Density (SMD). Patients with myosteatosis tend to have lower overall survival, increased chemotherapy toxicity, and shorter progression-free intervals across cancer types. We investigated whether changes in myosteatosis during neoadjuvant chemotherapy can predict postoperative morbidity risk in patients with pancreatic ductal adenocarcinoma (PDAC). Methods: This is a retrospective cohort study from 2014-2019 of patients with biopsy-proven PDAC who completed neoadjuvant chemotherapy and R0/1 resection (R1: margin < 1mm or microscopically positive). We obtained preoperative patient (age at diagnosis, baseline body mass index (BMI), sex, race, comorbidities) and treatment data (neoadjuvant chemotherapy regimen and duration, time from completion of systemic therapy to surgery, type of operation). Primary outcomes were postoperative complications and 90-day readmission. Average SMD was measured using imaging analysis software at the L3 level on axial abdominal CT scans at the time of diagnosis and at completion of neoadjuvant therapy (SliceOmatic TomoVision QC, Can). We defined SMDΔ as the decrease in SMD during neoadjuvant chemotherapy. Descriptive statistics and Student’s t-test were performed with STATA. Results: We identified 44 patients who received neoadjuvant chemotherapy, achieved a R0/1 resection, and had available CT scans for body composition evaluation. The postoperative complication rate was 43% (n = 19) and 90-day readmission rate was 30% (n = 13). Lower SMD at diagnosis was associated with increased postoperative delirium (p < 0.01) and 90-day readmission (p = 0.02). Greater SMDΔ was associated with increased ICU utilization (p < 0.01) and tube feeding upon discharge (p = 0.03). There was no significant association between preoperative BMI or albumin and our primary outcomes. Conclusions: Preoperative SMD and SMDΔ, rather than albumin or BMI, can predict postoperative morbidity in PDAC patients who received neoadjuvant chemotherapy. This study provides the framework for future studies to develop and validate a tool to predict postoperative morbidity risk in these patients.

TPS784 Background: Patients(pts) with metastatic pancreatic cancer (PC) have a median survival of less than one year even with use of multiagent chemotherapy programs. Pancreatic tumors are composed of multiple cell types and a dense extracellular matrix that may support cancer cell proliferation and impede chemotherapy delivery. Cancer-associated fibroblasts (CAF’s) in the tumor microenvironment secrete pro-inflammatory factors and components of the extracellular matrix. In PC laboratory models, engagement of the vitamin D receptor (VDR) by VDR agonists shifts CAFs toward a more quiescent phenotype with reduced tumor growth and improved chemotherapy penetration (Sherman. Cell, 2014). Paricalcitol is a synthetic VDR agonist used in patients with secondary hyperparathyroidism due to chronic kidney disease. A prior pilot study evaluated IV paricalcitol with gemcitabine (G) and nab-paclitaxel (A) before surgical resection in patients with resectable PC (NCT02030860). Methods: Pts with previously-untreated metastatic PC will be enrolled in a two-stage study consisting of a safety run-in and a randomized phase 2 study (NCT03520790). In the run-in stage, 36 pts will be randomized 1:1:1 to G (1000 mg/m2) and A (125 mg/m2) given 3 weeks on and 1 week off plus: (a) paricalcitol 25mcg IV thrice weekly, (b) paricalcitol 16mcg oral daily, or (c) placebo oral daily. Grade 3/4 hypercalcemia or genitourinary stones will be considered dose limiting toxicities.Pts will undergo paired pre- and on-treatment tumor biopsies to examine pharmacodynamic (PD) markers by bulk and single cell RNA sequencing and multiplex immunofluoresence.Assuming safety and supportive PD assessments, the phase 2 study will randomize an additional 76 pts to two treatment arms with GA plus: (a) paricalcitol or (b) placebo.Paricalcitol formulation (IV or oral) will be determined based on data from the run-in stage.The primary endpoint of the phase 2 study is overall survival, with a total of 100 pts needed to identify a hazard ratio of 0.6 with 80% power and one-sided alpha of 0.10.Secondary endpoints include safety, response rate, and progression free survival.Trial funding provided by SU2C, CRUK,Lustgarten Foundation, and AACR. Clinical trial information: NCT03520790.

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

Despite major advancements in cancer treatments, there are still many limitations to therapy including off-target effects, drug resistance, and control of cancer-related symptoms. There are opportunities for local drug delivery devices to intervene at various stages of cancer to provide curative and palliative benefit. Iontophoretic devices that deliver drugs locally to a region of interest have been adapted for the treatment of cancer. These devices have shown promise in pre-clinical and clinical studies for retinoblastoma, skin, bladder, and pancreatic cancers. Herein, we review iontophoretic devices used in the management of cancer.

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.

The underlying molecular basis of pancreatic ductal adenocarcinoma (PDAC) is complex, with multiple genetic, epigenetic, and transcriptomic changes. Five to 10% of PDAC cases have a hereditary basis, with multiple risk-conferring genetic mutations running in the family. All PDAC tumors develop from noninvasive precursor lesions that reflect the temporal accumulation of mutations. Recent advances in high-throughput sequencing technologies have facilitated a better understanding of the diverse mutational landscape of PDAC, reaffirming the role of genes previously known to be mutated (K-ras, CDKN2A, TP53, SMAD4, SLIT/ROBO, SWI/SNF) and revealing novel insights into the differential gene expression patterns and structural variations involved. K-ras mutation, an early event in disease pathogenesis, plays a pivotal role in the initiation, progression, and maintenance of PDAC, with the mutation type impacting disease pathophysiology and prognosis. The subsequent loss of tumor suppressors (CDKN2A, TP53, SMAD4) promotes genetic instability and uncontrolled cellular proliferation. Molecular subtypes with potential prognostic and therapeutic relevance have been identified. The challenge now is to translate this wealth of knowledge to the clinic. This review contains 3 figures, 3 tables and 50 references Key words: familial pancreatic cancer, heterogeneity, molecular subtypes, pancreatic ductal adenocarcinoma, whole genome/exome sequencing

Tumor mitotic rate (TMR) is a known prognostic variable in thin melanoma patients. Its significance in stage II melanoma patients is yet to be demonstrated. Retrospective analysis of a prospective melanoma database from 9/1997 to 7/2015 was performed. All stage II melanoma, with documented TMR, and six months of follow-up were included. We evaluated the association of clinicopathologic variables, TMR, as a continuous and categorical variable with recurrence-free survival (RFS) and overall survival (OS) using Cox proportional hazards modeling. We used a statistical model, X-tile, to develop optimal categorizations of TMR. A total of 265 patient characteristics are included in this study. Recurrences occurred in 82 (30.9%) patients, including 5 local, 41 regional, and 36 distant patients. In multivariate model, ulceration, Breslow, and continuous TMR were associated with worse RFS\OS. Continuous TMR demonstrated worse RFS (hazards ratio [HR] 1.02 (1.00-1.05)) and OS (HR 1.02 (1.00-1.04)), whereas dichotomized TMR (≥1 vs1) was not significant. TMR10.4 mitoses/mm2 has a 5-year RFS\OS of 27.2 and 44.3 per cent, respectively, compared with 57.4 and 71.4 per cent, respectively, for TMR3.2 mitoses/mm2. Continuous TMR predicts incidence of recurrence in stage II melanoma. We propose a new categorization method developed by statistical modeling for optimal stratification that may guide surveillance for this disparate patient population.

Traditionally, total thyroidectomy (TT) was an inpatient procedure, but recent trends indicate that patients are often discharged on the day of surgery. This has been proven safe for high-volume surgeons but has not been studied for low (

Clinical decision-making in regard to the Bethesda System for Reporting Thyroid Cytopathology for fine needle aspirations (FNAs) is complicated for indeterminate nodules, which make up to 25% of all samples. In the quest to limit unnecessary diagnostic thyroid lobectomies or conversely to proceed directly to oncologic thyroidectomy, gene mutation testing is being increasingly used to augment indeterminate thyroid nodule management. This chapter will discuss the test characteristics, clinical validity, and applicability of the current available molecular genetic tests for differentiated thyroid cancer and introduce future possibilities in this rapidly progressing field. The Afirma gene expression classifier (GEC), a high sensitivity test, can be used to rule out differentiated thyroid cancer, while the 7-gene mutation panel (7-MP), which evaluates mutations in BRAF, NRAS, HRAS, and KRAS mutations as well as translocations of the RET/PTC1, RET/PTC3, and PAX8/PPARγ genes, is a highly specific test and can be used to rule in differentiated thyroid cancer. However, as neither Afirma GEC nor 7-MP has both high sensitivity and specificity, next-generation sequencing panels are being utilized to further improve diagnostic capabilities. Future directions in diagnostics include studying biomarkers such as galectin-3 (GAL3), hector battifora mesothelial-1 (HBME-1), cytokeratin-19 (CK-19), CD-117, and c-kit to improve the utility of existing tests.

In recent years, knowledge of the genetics of differentiated thyroid carcinomas has been rapidly increasing. Mutations identified in papillary thyroid cancer (PTC) can be subdivided into BRAF- or RAS-like and include BRAF, RET/PTC, RAS, TRK, TERT, and EIF1AX. The leading genetic alterations in follicular thyroid carcinoma (FTC) are RAS, PAX8/PPARG, PTEN, PI3K/AKT pathway, and IDH1. Chapter 2 will provide an overview of common mutations and rearrangements in differentiated thyroid carcinoma in association with their clinical and pathologic characteristics and the current state of the rapidly advancing field.

Background Non-coding RNAs have been drawing increasing attention in recent years as functional data suggest that they play important roles in key cellular processes. N-BLR is a primate-specific long non-coding RNA that modulates the epithelial-to-mesenchymal transition, facilitates cell migration, and increases colorectal cancer invasion. Results We performed multivariate analyses of data from two independent cohorts of colorectal cancer patients and show that the abundance of N-BLR is associated with tumor stage, invasion potential, and overall patient survival. Through in vitro and in vivo experiments we found that N-BLR facilitates migration primarily via crosstalk with E-cadherin and ZEB1. We showed that this crosstalk is mediated by a pyknon, a short ~20 nucleotide-long DNA motif contained in the N-BLR transcript and is targeted by members of the miR-200 family. In light of these findings, we used a microarray to investigate the expression patterns of other pyknon-containing genomic loci. We found multiple such loci that are differentially transcribed between healthy and diseased tissues in colorectal cancer and chronic lymphocytic leukemia. Moreover, we identified several new loci whose expression correlates with the colorectal cancer patients’ overall survival. Conclusions The primate-specific N-BLR is a novel molecular contributor to the complex mechanisms that underlie metastasis in colorectal cancer and a potential novel biomarker for this disease. The presence of a functional pyknon within N-BLR and the related finding that many more pyknon-containing genomic loci in the human genome exhibit tissue-specific and disease-specific expression suggests the possibility of an alternative class of biomarkers and therapeutic targets that are primate-specific. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1224-0) contains supplementary material, which is available to authorized users.

One obstacle in the translation of advances in cancer research into the clinic is a deficiency of adequate preclinical models that recapitulate human disease. Patient-derived xenograft (PDX) models are established by engrafting patient tumor tissue into mice and are advantageous because they capture tumor heterogeneity. One concern with these models is that selective pressure could lead to mutational drift and thus be an inaccurate reflection of patient tumors. Therefore, we evaluated if mutational frequency in PDX models is reflective of patient populations and if crucial mutations are stable across passages. We examined KRAS and PIK3CA gene mutations from pancreatic ductal adenocarcinoma (PDAC) (n = 30) and colorectal cancer (CRC) (n = 37) PDXs for as many as eight passages. DNA was isolated from tumors and target sequences were amplified by polymerase chain reaction. KRAS codons 12/13 and PIK3CA codons 542/545/1047 were examined using pyrosequencing. Twenty-three of 30 (77%) PDAC PDXs had KRAS mutations and one of 30 (3%) had PIK3CA mutations. Fifteen of 37 (41%) CRC PDXs had KRAS mutations and three of 37 (8%) had PIK3CA mutations. Mutations were 100 per cent preserved across passages. We found that the frequency of KRAS (77%) and PIK3CA (3%) mutations in PDAC PDX was similar to frequencies in patient tumors (71 to 100% KRAS, 0 to 11% PIK3CA). Similarly, KRAS (41%) and PIK3CA (8%) mutations in CRC PDX closely paralleled patient tumors (35 to 51% KRAS, 12 to 21% PIK3CA). The accurate mirroring and stability of genetic changes in PDX models compared with patient tumors suggest that these models are good preclinical surrogates for patient tumors.

Primary patient samples are the gold standard for molecular investigations of tumor biology yet are difficult to acquire, heterogeneous in nature and variable in size. Patient-derived xenografts (PDXs) comprised of primary tumor tissue cultured in host organisms such as nude mice permit the propagation of human tumor samples in an in vivo environment and closely mimic the phenotype and gene expression profile of the primary tumor. Although PDX models reduce the cost and complexity of acquiring sample tissue and permit repeated sampling of the primary tumor, these samples are typically contaminated by immune, blood, and vascular tissues from the host organism while also being limited in size. For very small tissue samples (on the order of 10(3) cells) purification by fluorescence-activated cell sorting (FACS) is not feasible while magnetic activated cell sorting (MACS) of small samples results in very low purity, low yield, and poor viability. We developed a platform for imaging cytometry integrated with micropallet array technology to perform automated cell sorting on very small samples obtained from PDX models of pancreatic and colorectal cancer using antibody staining of EpCAM (CD326) as a selection criteria. These data demonstrate the ability to automate and efficiently separate samples with very low number of cells.

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.

The need to activate thermoplastic surfaces using robust and efficient methods has been driven by the fact that replication techniques can be used to produce microfluidic devices in a high production mode and at low cost, making polymer microfluidics invaluable for in vitro diagnostics, such as circulating tumor cell (CTC) analysis, where device disposability is critical to mitigate artifacts associated with sample carryover. Modifying the surface chemistry of thermoplastic devices through activation techniques can be used to increase the wettability of the surface or to produce functional scaffolds to allow for the covalent attachment of biologics, such as antibodies for CTC recognition. Extensive surface characterization tools were used to investigate UV activation of various surfaces to produce uniform and high surface coverage of functional groups, such as carboxylic acids in microchannels of different aspect ratios. We found that the efficiency of the UV activation process is highly dependent on the microchannel aspect ratio and the identity of the thermoplastic substrate. Colorimetric assays and fluorescence imaging of UV-activated microchannels following EDC/NHS coupling of Cy3-labeled oligonucleotides indicated that UV-activation of a PMMA microchannel with an aspect ratio of ∼3 was significantly less efficient toward the bottom of the channel compared to the upper sections. This effect was a consequence of the bulk polymer's damping of the modifying UV radiation due to absorption artifacts. In contrast, this effect was less pronounced for COC. Moreover, we observed that after thermal fusion bonding of the device's cover plate to the substrate, many of the generated functional groups buried into the bulk rendering them inaccessible. The propensity of this surface reorganization was found to be higher for PMMA compared to COC. As an example of the effects of material and microchannel aspect ratios on device functionality, thermoplastic devices for the selection of CTCs from whole blood were evaluated, which required the immobilization of monoclonal antibodies to channel walls. From our results, we concluded the CTC yield and purity of isolated CTCs were dependent on the substrate material with COC producing the highest clinical yields for CTCs as well as better purities compared to PMMA.

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.

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.

Most pre-clinical therapy studies use the change in tumor volume as a measure for disease response. However, tumor size measurements alone may not reflect early changes in tumor physiology that occur as a response to treatment. Ultrasonic molecular imaging (USMI) and Dynamic Contrast Enhanced-Perfusion Imaging (DCE-PI) with ultrasound are two attractive alternatives to tumor volume measurements. Since these techniques can provide information prior to the appearance of gross phenotypic changes, it has been proposed that USMI and DCE-PI could be used to characterize response to treatment earlier than traditional methods. This study evaluated the ability of tumor volume measurements, DCE-PI, and USMI to characterize response to therapy in two different types of patient-derived xenografts (known responders and known non-responders). For both responders and non-responders, 7 animals received a dose of 30 mg/kg of MLN8237, an investigational aurora-A kinase inhibitor, for 14 days or a vehicle control. Volumetric USMI (target integrin: αvβ3) and DCE-PI were performed on day 0, day 2, day 7, and day 14 in the same animals. For USMI, day 2 was the earliest point at which there was a statistical difference between the untreated and treated populations in the responder cohort (Untreated: 1.20 ± 0.53 vs. Treated: 0.49 ± 0.40; p < 0.05). In contrast, statistically significant differences between the untreated and treated populations as detected using DCE-PI were not observed until day 14 (Untreated: 0.94 ± 0.23 vs. Treated: 1.31 ± 0.22; p < 0.05). Volume measurements alone suggested no statistical differences between treated and untreated populations at any read-point. Monitoring volumetric changes is the “gold standard” for evaluating treatment in preclinical studies, however, our data suggests that volumetric USMI and DCE-PI may be used to earlier classify and robustly characterize tumor response.

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.

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.

Alterations in the DNA sequences of genes, or mutations, have traditionally been viewed as the primary factors driving tumor progression, however, epigenetic evidence would suggest that some heritable traits are mediated by changes in DNA expression that are not dependent upon alterations in the primary DNA sequence. Advances in the genetic understanding of cancer have, in some instances, allowed for more precise administration of anti-neoplastic therapy. Targeted therapies, the aim of which are to target specific cellular proteins or processes used by the cancer cells, have been advocated to avoid the adverse side effects attributable to a lack of cell specificity associated with traditional chemotherapy. Here we aim to describe the current state of understanding regarding the genetic related causes of cancers, the targeted therapies aimed at killing them and the inter-relationship between these two.

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.

Alterations in the DNA sequences of genes, or mutations, have traditionally been viewed as the primary factors driving tumor progression, however, epigenetic evidence would suggest that some heritable traits are mediated by changes in DNA expression that are not dependent upon alterations in the primary DNA sequence. Advances in the genetic understanding of cancer have, in some instances, allowed for more precise administration of anti-neoplastic therapy. Targeted therapies, the aim of which are to target specific cellular proteins or processes used by the cancer cells, have been advocated to avoid the adverse side effects attributable to a lack of cell specificity associated with traditional chemotherapy. Here we aim to describe the current state of understanding regarding the genetic related causes of cancers, the targeted therapies aimed at killing them and the inter-relationship between these two.

Ect2 is a member of the human Dbl family of guanine nucleotide exchange factors (RhoGEFs) that serve as activators of Rho family small GTPases. Although Ect2 is one of at least 25 RhoGEFs that can activate the RhoA small GTPase, cell culture studies using established cell lines determined that Ect2 is essential for mammalian cell cytokinesis and proliferation. To address the function of Ect2 in normal mammalian development, we performed gene targeting to generate Ect2 knockout mice. The heterozygous Ect2(+/-) mice showed normal development and life span, indicating that Ect2 haplodeficiency was not deleterious for development or growth. In contrast, Ect2(-/-) embryos were not found at birth or postimplantation stages. Ect2(-/-) blastocysts were recovered at embryonic day 3.5 but did not give rise to viable outgrowths in culture, indicating that Ect2 is required for peri-implantation development. To further assess the importance of Ect2 in normal cell physiology, we isolated primary fibroblasts from Ect2(fl/fl) embryos (MEFs) and ablated Ect2 using adenoviral delivery of Cre recombinase. We observed a significant increase in multinucleated cells and accumulation of cells in G2/M phase, consistent with a role for Ect2 in cytokinesis. Ect2 deficiency also caused enlargement of the cytoplasm and impaired cell migration. Finally, although Ect2-dependent activation of RhoA has been implicated in cytokinesis, Ect2 can also activate Rac1 and Cdc42 to cause growth transformation. Surprisingly, ectopic expression of constitutively activated RhoA, Rac1, or Cdc42, known substrates of Ect2, failed to phenocopy Ect2 and did not rescue the defect in cytokinesis caused by loss of Ect2. In summary, our results establish the unique role of Ect2 in development and normal cell proliferation.

Background Atypical melanocytic neoplasms present a therapeutic dilemma. Current consensus is to perform a sentinel lymph node (SLN) biopsy as part of management. However, it is unclear whether this is required in all patients. We present our experience with sentinel lymphadenectomy in these patients and examine the clinical and pathologic variables associated with a positive SLN. Study Design A prospectively maintained melanoma database was queried for patients with controversial melanocytic lesions. All patients between January 1997 and January 2009 were included. Demographic and pathologic information was collected and correlated with results of SLN biopsy. Results Thirty-one patients underwent SLN biopsy. Median patient age was 19 years (range 5 to 59 years) and median tumor Breslow depth was 1.35 mm. Five patients (16%) had a positive SLN. Those with a positive SLN were younger (median 11 vs 23.5 years, p=0.02) and had a greater Breslow depth (median 1.90 vs 1.09; p=0.03) than those who were SLN negative. Median follow-up was16 months for patients with at least 6 months of follow-up time and there have been no recurrences identified. Conclusions We report an SLN positive rate of 16% in patients with atypical melanocytic tumors. Younger age and greater Breslow depth are associated with having a positive SLN. These results confirm earlier work demonstrating the importance of SLN biopsy in this disease and highlight the need to measure Breslow depth in these lesions so that they can be appropriately stratified as to the need for SLN biopsy.

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.

211 Background: COMPASS is a prospective study with the primary aim to identify predictive mutational and transcriptional features in advanced PDAC for improved patient stratification and treatment selection. Methods: Patients with advanced PDAC were prospectively recruited prior to first-line combination chemotherapy for whole genome sequencing (WGS) and RNA sequencing (RNASeq). Fresh tumor tissue was acquired by image guided percutaneous core needle biopsy of locally advanced primary or metastatic tumors. Laser capture microdissection was performed for all cases to ensure high-resolution genomic analyses. Primary endpoint was feasibility to report WGS results prior to first disease assessment CT scan at 8 weeks. The main secondary endpoint was discovery of patient subsets with predictive mutational and transcriptional signatures. Results: Of 63 patients who underwent a tumor biopsy between December 2015 and June 2017, WGS and RNASeq were successful in 62 (98%) and 60 (95%), respectively. Genomic results were reported at a median of 35 days (range 19-52 days) from biopsy, meeting the primary feasibility endpoint. Three patients with an ‘unstable’ genomic subtype, including two with a novel ‘duplicator’ phenotype, responded well to m-FOLFIRINOX. Of two cases with the same germline BRCA2 mutation, only the chemotherapy responder had loss of heterozygosity and genomic hallmarks of double stranded break repair deficiency. Approximately 25% of tumors displayed the basal-like RNA expression signature and these were chemotherapy resistant, with tumor shrinkage mainly observed in those with the classical RNA subtype (P = 0.003). Thirty percent of patients had potentially actionable genetic alterations. Conclusions: Prospective comprehensive genomic profiling of advanced PDAC is feasible and our early data indicate that chemotherapy response differs among patients with different genomic/transcriptomic subtypes providing the impetus for further studies. Clinical trial information: NCT02750657.

277 Background: PDAC is a lethal disease with poor survival even when detected at an early stage. Recurrence rates after surgical resection remain high. Recently, two distinct molecular subtypes of PDAC (basal-like and classical) have been identified with basal-like tumors demonstrating inferior outcomes. We hypothesize that differences in tumor immunogenicity may contribute to this aggressive biology and predict response to immune checkpoint inhibitors. Methods: RNA sequencing was performed on formalin-fixed paraffin embedded samples of 60 resected PDAC patients. We evaluated previously published immune gene expression signatures comprised of 1400 genes and used a single sample classifier to determine molecular subtypes. Results: Table 1 summarizes patient characteristics in our cohort. There were 35 classical and 25 basal-like tumors. PFS was significantly shorter in patients with basal-like compared to classical subtypes (9 vs 15 mo, p = 0.006). In a multivariable model with molecular subtype, lymph node and margin status, subtype was the only independent predictor of PFS (p=0.028). Unsupervised clustering identified two distinct immune groups that were associated with molecular subtypes (p=0.038) with higher expression of immune genes in basal-like tumors. Basal-like tumors were significantly associated with an immunosuppressive signature (p

Chronic kidney disease is often accompanied by hyperparathyroidism. Cinacalcet, a recent addition to the medical armamentarium, has proven efficacious. It is unclear whether cinacalcet use has any impact on the postoperative course in patients progressing to surgery. The records of 77 patients operated on for renal hyperparathyroidism were reviewed. Sixty-three were treated before the use of cinacalcet and 14 after. Ten subtotal and 67 total parathyroidectomies were performed. Mean nadir serum calcium was similar (6.6 ± 1.3 vs 6.2 ± 1.4 mg/dL). More patients taking cinacalcet preoperatively required intravenous calcium postoperatively (62%) than those treated before its use (41%), although this did not reach statistical significance ( P = 0.09). In those undergoing total parathyroidectomy, cinacalcet use preoperatively (n = 11) led to a lower postoperative nadir calcium (5.8 ± 1.7 vs 6.6 ± 1.3 mg/dL) as compared with those who did not receive it (n = 56) ( P = 0.05). This translated to a greater need for intravenous calcium infusion postoperatively (72 vs 38%) ( P = 0.03). These data suggest a somewhat more aggressive postoperative course in patients who fail calcimimetic and require surgery. This may be useful to inform physicians and patients of expectations postoperatively, although it is not likely to alter management.

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.

The follow-up of patients with cutaneous melanoma is controversial. Current recommendations suggest routine history and physical examination every 3 to 6 months for the first 3 years and correlate studies including laboratory tests and radiographic imaging. However, the utility of these recommendations are unclear. The purpose of this study was to determine the impact of routine imaging on the method of detection of first recurrence in patients with stage II and sentinel lymph node–positive stage III melanoma. We analyzed a prospective database of all cutaneous melanoma patients treated at our institution from 1997 to 2005 who had at least 2 years of follow-up. The method of detection of initial recurrence was analyzed. One hundred eighteen patients with stage II (n = 83) or III (n = 35) melanoma who were followed for at least 2 years were identified. Forty-three of these patients developed recurrence (median time to recurrence, 14 months). Site of first recurrence was as follows: 4 local, 17 in transit, 7 regional lymph node, and 15 distant. Twenty-nine recurrences (67%) were either patient detected or symptomatic. Eleven (26%) were detected by the physician at routine follow-up. Only three (7%) were identified by imaging (two chest X-ray and one brain magnetic resonance imaging) in an otherwise asymptomatic patient. Two-thirds of all initial recurrences of cutaneous melanoma were either detected by a patient or were symptomatic, with most of the remainder detected during routine physical examination. Routine imaging added little value in the detection of initial recurrence.

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.

Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.

Colorectal cancer (CRC) is the third most common cancer in the world and the second leading cause of cancer deaths in the US and Spain. The molecular mechanisms involved in the etiology of CRC are not yet elucidated due in part to the complexity of the human gut microbiota. In this study, we compared the microbiome composition of 90 tumor and matching adjacent tissue (adjacent) from cohorts from the US and Spain by 16S rRNA amplicon sequencing in order to determine the impact of the geographic origin on the CRC microbiome. Data showed a significantly (P < 0.05) higher Phylogenetic Diversity (PD) for the US (PD Adjacent = 26.3 ± 5.3, PD Tumor = 23.3 ± 6.2) compared to the Spanish cohort (PD Adjacent = 18.9 ± 5.9, PD Tumor = 18.7 ± 6.6) while no significant differences in bacterial diversity were observed between tumor and adjacent tissues for individuals from the same country. Adjacent tissues from the Spanish cohort were enriched in Firmicutes (SP = 43.9% and US = 22.2%, P = 0.0001) and Actinobacteria (SP = 1.6% and US = 0.5%, P = 0.0018) compared to US adjacent tissues, while adjacent tissues from the US had significantly higher abundances of Fusobacteria (US = 8.1% and SP = 1.5%, P = 0.0023) and Sinergistetes (US = 0.3% and SP = 0.1%, P = 0.0097). Comparisons between tumor and adjacent tissues in each cohort identified the genus Eikenella significantly over represented in US tumors (T = 0.024% and A = 0%, P = 0.03), and the genera Fusobacterium (T = 10.4% and A = 1.5%, P =, IA is a Fulbright Scholar. The Microbiome Core Facility is supported in part by the NIH/NIDDK grant P30 DK34987. The Instituto Universitario de Oncología del Principado de Asturias receives financial support from the Fundación Caja de Ahorros de Asturias and Consejería de Educación del Principado de Asturias.

Two decades after the discovery of the first animal microRNA (miRNA), the number of miRNAs in animal genomes remains a vexing question. Here, we report findings from analyzing 1,323 short RNA sequencing samples (RNA-seq) from 13 different human tissue types. Using stringent thresholding criteria, we identified 3,707 statistically significant novel mature miRNAs at a false discovery rate of ≤0.05 arising from 3,494 novel precursors; 91.5% of these novel miRNAs were identified independently in 10 or more of the processed samples. Analysis of these novel miRNAs revealed tissue-specific dependencies and a commensurate low Jaccard similarity index in intertissue comparisons. Of these novel miRNAs, 1,657 (45%) were identified in 43 datasets that were generated by cross-linking followed by Argonaute immunoprecipitation and sequencing (Ago CLIP-seq) and represented 3 of the 13 tissues, indicating that these miRNAs are active in the RNA interference pathway. Moreover, experimental investigation through stem-loop PCR of a random collection of newly discovered miRNAs in 12 cell lines representing 5 tissues confirmed their presence and tissue dependence. Among the newly identified miRNAs are many novel miRNA clusters, new members of known miRNA clusters, previously unreported products from uncharacterized arms of miRNA precursors, and previously unrecognized paralogues of functionally important miRNA families (e.g., miR-15/107). Examination of the sequence conservation across vertebrate and invertebrate organisms showed 56.7% of the newly discovered miRNAs to be human-specific whereas the majority (94.4%) are primate lineage-specific. Our findings suggest that the repertoire of human miRNAs is far more extensive than currently represented by public repositories and that there is a significant number of lineage- and/or tissue-specific miRNAs that are uncharacterized.

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

Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth-leading cause of cancer-related death in the United States and is projected to become the second leading cause by 2030. Most patients present with advanced disease and die within 12 months of diagnosis. Recent genomic studies of primary pancreatic cancer resection specimens have identified several molecular alterations and genomic subtypes of the disease that may guide precision medicine approaches to clinical management. However, the molecular landscape of metastatic PDAC has been less well characterized. Moreover, biopsy-driven studies in metastatic PDAC have been historically very challenging due to the aggressive course of this disease as well as the low-volume and heterogeneous nature of biopsies that makes deep molecular characterization difficult. Insufficient genomic analysis of a patient’s tumor early in their disease course is a major barrier to enrollment on clinical trials of targeted therapies. To address these limitations, we have implemented a multi-disciplinary clinical and research biopsy protocol to enable real time comprehensive molecular characterization of metastatic PDAC biopsy specimens. We have performed core needle biopsies of metastatic lesions in the liver or peritoneal cavity in 42 patients at the time of initial presentation. A low rate of complications was observed, with only a single patient having a self-limited hemorrhagic complication after liver biopsy. On average, 4-6 separate biopsy specimens were collected from each patient for histopathology and genomic analysis. Whole exome sequencing (WES) was performed in a CLIA-certified laboratory and a comprehensive molecular report of somatic alterations and selected pathogenic germline variants was returned to the referring clinician with a typical turn-around time of 3-5 weeks. We observed a striking incidence of recurrent germline and somatic alterations in DNA-damage repair genes, such as BRCA2, ATM and CHEK2. We also observed alterations in genes with known therapeutic implications, such as BRAF, RNF43, STK11 and ROS, and in select cases, these results guided choice of second or third line therapy. In parallel to WES, we performed RNA sequencing on bulk tumor tissue and readily identified expression signatures defining multiple subtypes of tumor and stroma that may have prognostic or therapeutic implications for tumor- or stroma-directed therapies. Collectively, these results demonstrate the feasibility and value of real-time genomic characterization of metastatic PDAC and provide a path forward for improved stratification and enrollment of PDAC patients on molecularly defined clinical trials. Citation Format: Andrew J. Aguirre, Scott Carter, Nicholas Camarda, Arezou Ghazani, Jonathan Nowak, Annacarolina Da Silva, Lauren Brais, Dorisanne Ragon, Devin McCabe, Lori Marini, Kristin Anderka, Karla Helvie, Nelly Oliver, Ana Babic, Paul Shyn, Douglas Rubinson, Anuj Patel, James Cleary, Nadine McCleary, Matthew Kulke, Thomas Clancy, Leona Doyle, Jason Hornick, Christine Ardito-Abraham, Ruth Yu, Michael Downes, Ronald Evans, Richard A. Moffitt, Jen Jen Yeh, William C. Hahn, Charles Fuchs, Robert Mayer, Nikhil Wagle, David Tuveson, Levi A. Garraway, Brian M. Wolpin. Real-time genomic characterization of metastatic pancreatic cancer to enable precision medicine [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 3036. doi:10.1158/1538-7445.AM2017-3036