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Oncology research is increasingly incorporating molecular detection of circulating tumor DNA (ctDNA) as a tool for cancer surveillance and early detection. However, non-invasive monitoring of conditions with low tumor burden remains challenging, as the diagnostic sensitivity of most ctDNA assays is inversely correlated with total DNA concentration and ctDNA abundance. Here we present the Multiplex Enrichment using Droplet Pre-Amplification (MED-Amp) method, which com-bines single-molecule emulsification and short-round PCR preamplification with digital droplet PCR (ddPCR) detection of mutant DNA template. The MED-Amp assay increased mutant signal by over 50-fold with minimal distortion in allelic frequency. We demonstrate detection of as few as 3 mutant copies in wild-type DNA concentrations ranging from 5 to 50ng. The MED-Amp assay successfully detected KRAS mutant ctDNA in 86% plasma samples obtained from patients with metastatic pancreatic ductal adenocarcinoma. This assay for high-sensitivity rare variant detection is appropriate for liquid biopsy samples, or other limited clinical biospecimens

Basal subtype cancers are deadly malignancies but the molecular events driving tumor lethality are not completely understood. Ataxia-telangiectasia group D complementing gene (ATDC, also known as TRIM29), is highly expressed and drives tumor formation and invasion in human bladder cancers but the factor(s) regulating its expression in bladder cancer are unknown. Molecular subtyping of bladder cancer has identified an aggressive basal subtype, which shares molecular features of basal/squamous tumors arising in other organs and is defined by activation of a TP63-driven gene program. Here, we demonstrate that ATDC is linked with expression of TP63 and highly expressed in basal bladder cancers. We find that TP63 binds to transcriptional regulatory regions of ATDC and KRT14 directly, increasing their expression, and that ATDC and KRT14 execute a TP63-driven invasive program. In vivo, ATDC is required for TP63-induced bladder tumor invasion and metastasis. These results link TP63 and the basal gene expression program to ATDC and to aggressive tumor behavior. Defining ATDC as a molecular determinant of aggressive, basal cancers may lead to improved biomarkers and therapeutic approaches.

Pancreatic ductal adenocarcinoma (PDAC) is a clinically challenging cancer, due to both its late stage at diagnosis and its resistance to chemotherapy. However, recent advances in our understanding of the biology of PDAC have revealed new opportunities for early detection and targeted therapy of PDAC. In this review, we discuss the pathogenesis of PDAC, including molecular alterations in tumor cells, cellular alterations in the tumor microenvironment, and population-level risk factors. We review the current status of surveillance and early detection of PDAC, including populations at high risk and screening approaches. We outline the diagnostic approach to PDAC and highlight key treatment considerations, including how therapeutic approaches change with disease stage and targetable subtypes of PDAC. Recent years have seen significant improvements in our approaches to detect and treat PDAC, but large-scale, coordinated efforts will be needed to maximize the clinical impact for patients and improve overall survival.

OBJECTIVES: Current standard of care imaging, cytology, or cystic fluid analysis cannot reliably differentiate malignant from benign pancreatic cystic neoplasms. This study sought to determine if the metabolic profile of cystic fluid could distinguish benign and malignant lesions, as well as mucinous and non-mucinous lesions. Methods: Metabolic profiling by untargeted mass spectrometry and quantitative nuclear magnetic resonance was performed in 24 pancreatic cyst fluid from surgically resected samples with pathological diagnoses and clinicopathological correlation. Results: (Iso)-butyrylcarnitine distinguished malignant from benign pancreatic cysts, with a diagnostic accuracy of 89%. (Iso)-butyrylcarnitine was 28-fold more abundant in malignant cyst fluid compared with benign cyst fluid (P=.048). Furthermore, 5-oxoproline (P=.01) differentiated mucinous from non-mucinous cysts with a diagnostic accuracy of 90%, better than glucose (82% accuracy), a previously described metabolite that distinguishes mucinous from non-mucinous cysts. Combined analysis of glucose and 5-oxoproline did not improve the diagnostic accuracy. In comparison, standard of care cyst fluid carcinoembryonic antigen (CEA) and cytology had a diagnostic accuracy of 40% and 60% respectively for mucinous cysts. (Iso)-butyrylcarnitine and 5-oxoproline correlated with cyst fluid CEA levels (P 3 cm, ≥ 1 high-risk features, cyst fluid CEA, and cytology are 38%, 75%, 80%, and 75%, respectively. Conclusions: (Iso)-butyrylcarnitine has potential clinical application for accurately distinguishing malignant from benign pancreatic cysts, and 5-oxoproline for distinguishing mucinous from non-mucinous cysts.

© Author(s) (or their employer(s)) 2020., [Objective]: ATM serine/threonine kinase (ATM) is the most frequently mutated DNA damage response gene, involved in homologous recombination (HR), in pancreatic ductal adenocarcinoma (PDAC)., [Design]: Combinational synergy screening was performed to endeavour a genotype-tailored targeted therapy., [Results]: Synergy was found on inhibition of PARP, ATR and DNA-PKcs (PAD) leading to synthetic lethality in ATM-deficient murine and human PDAC. Mechanistically, PAD-induced PARP trapping, replication fork stalling and mitosis defects leading to P53-mediated apoptosis. Most importantly, chemical inhibition of ATM sensitises human PDAC cells toward PAD with long-term tumour control in vivo. Finally, we anticipated and elucidated PARP inhibitor resistance within the ATM-null background via whole exome sequencing. Arising cells were aneuploid, underwent epithelial-mesenchymal-transition and acquired multidrug resistance (MDR) due to upregulation of drug transporters and a bypass within the DNA repair machinery. These functional observations were mirrored in copy number variations affecting a region on chromosome 5 comprising several of the upregulated MDR genes. Using these findings, we ultimately propose alternative strategies to overcome the resistance., [Conclusion]: Analysis of the molecular susceptibilities triggered by ATM deficiency in PDAC allow elaboration of an efficient mutation-specific combinational therapeutic approach that can be also implemented in a genotype-independent manner by ATM inhibition., Main funding is provided by the German Cancer Aid grant to AK (111879). Additional funding came from the Deutsche Forschungsgemeinschaft (DFG) ’Sachbeihilfe’ (KL 2544/1–1, 1–2, 5–1, 7-1) and ’Heisenberg-Programm’ (KL 2544/6–1), the Baden-Württemberg-Foundation ExPoChip and the INDIMEDVerbund PancChip. AK, FA, MI, SB, LW and TS are either Principal Investigators or students of HEIST RTG funded by the DFG GRK 2254/1. AK is an Else-KrönerFresenius Excellence fellow. LP received funds by the Bausteinprogramm of Ulm University. PCH is supported by a Max Eder Fellowship of the German Cancer Aid (111746), a German Cancer Aid Priority Program ’Translational Oncology’ 70112505 and by a Collaborative Research Centre grant (316249678 – SFB 1279) of the German Research Foundation. EH received funding from the German Cancer Aid (PiPAC, 70112505) and the Volkswagenstiftung/Ministry for Science and Culture in Lower Saxony (ZN3222). This work was also supported by the Deutsche Forschungsgemeinschaft (AZ.96/1–3) to NA, by the Deutsche Krebshilfe (111264) to AL and by the German Cancer Aid Priority Program Translational Oncology (70112504) to LW.

PurposeThe clinical utility of circulating tumor DNA (ctDNA) has been shown in advanced pancreatic ductal adenocarcinoma (PDA). However, diagnostic sensitivity of many ctDNA assays is low in resectable and locally advanced disease, where tumor burden is substantially lower. We have previously described Multiplex Enrichment using Droplet Pre-Amplification (MED-Amp), a multiplexed panel for the detection of the most common oncogenic KRAS mutations in PDA. In this study, we aimed to assess the diagnostic sensitivity of MED-Amp for detection of rare mutant alleles present in the plasma of patients with localized PDA.Experimental DesignWe retrospectively analyzed ninety-eight plasma samples from 51 patients with various stages of localized disease. For comparison, we measured ctDNA levels in 20 additional patients with metastatic PDA. The MED-Amp assay was used to measure the abundance of the four most common KRAS codon 12 mutations (G12C/D/R/V). We correlated the presence and quantity of ctDNA with overall survival (OS) as well as progression-free survival (PFS). Using serial plasma draws, we also assessed the relationship between changes in ctDNA allelic frequency and progression.ResultsKRAS-positive ctDNA was detected in 52.9% of localized PDA and 75% of metastatic samples tested using DNA inputs as low as 2 ng. As previously reported, the presence of KRAS mutant ctDNA was correlated with worse OS for all disease stages (p = 0.02). In patients with localized PDA high ctDNA levels also correlated with significantly worse median OS (533 days vs 1090 days) and PFS (192 days vs 787 days). We also studied a small cohort of serial plasma draws to observe the relationship between ctDNA fold change and PFS. We found 83% of patients with increased fold change in mutant KRAS experienced disease progression (n=6). In contrast, 75% (n=4) of patients with decreased fold change remained disease-free (p=0.03).ConclusionsMED-Amp is a flexible and cost-effective approach for measurement of ctDNA in patients with localized cancer. Though this study focused on KRAS mutation detection, this assay could be adapted for a number of common oncogenic alterations.Statement of translational relevanceOnly 25% of pancreatic ductal adenocarcinoma (PDA) patients with localized disease survive five years post-resection. It is hypothesized PDA undergoes dissemination at the earliest stages of tumor formation, driving formation of occult metastases which go undetected using conventional screening methods. Development of high specificity, high sensitivity biomarkers is critical to improving patient mortality. Circulating tumor DNA (ctDNA) has gained increasing acceptance as a non-invasive prognostic in metastatic disease. However, the sensitivity of most targeted ctDNA assays precludes reliable detection of localized and resected disease. Here, we present a digital droplet PCR assay for multiplexed enrichment and detection of KRAS mutations, the most commonly mutated oncogene in PDA. This assay preserves ctDNA allelic frequency in the original sample, while increasing the molecular signal over 50-fold. This study shows the ctDNA has potential diagnostic value in early-stage PDA, and that digital preenrichment of cell-free DNA increases overall assay sensitivity without sacrificing specificity.

Background and Aims Pancreatic ductal adenocarcinoma is an aggressive disease most often diagnosed after local progression or metastatic dissemination, precluding resection and resulting in a high mortality rate. For individuals with elevated personal risk of the development of pancreatic cancer, EUS is a frequently used advanced imaging and diagnostic modality. However, there is variability in the expertise and definition of EUS findings among gastroenterologists, as well as lack of standardized reporting of relevant findings at the time of examination. Adoption of standardized EUS reporting, using a universally accepted and agreed upon terminology, is needed. Methods A consensus statement designed to create a standardized reporting template was authored by a multidisciplinary group of experts in pancreatic diseases that includes gastroenterologists, radiologists, surgeons, oncologists, and geneticists. This statement was developed using a modified Delphi process as part of the Pancreatic Cancer Early Detection Consortium (PRECEDE) and >75% agreement was required to reach consensus. Results We identified reporting elements and present standardized reporting templates for EUS indications, procedural data, EUS image capture, and descriptors of findings, tissue sampling, and for postprocedural assessment of adequacy. Conclusions Adoption of this standardized EUS reporting template should improve consistency in clinical decision making for individuals with elevated risk of pancreatic cancer by providing complete and accurate reporting of pancreatic abnormalities. Standardization will also help to facilitate research and clinical trial design by using clearly defined and consistent imaging descriptions, thus allowing for comparison of results across different centers.

Background Early detection of pancreatic ductal adenocarcinoma (PDAC) is an important goal for improving survival. Individuals who meet published guidelines for surveillance may be underidentified, and family communication about risk represents a pathway to increasing participation in surveillance. We investigated the uptake of and barriers to surveillance in at-risk relatives of clinic patients. Methods We conducted a retrospective record review of patients with personal or family history of PDAC evaluated over 12 months. The first relative presenting to clinic (proband) reported surveillance status and reasons for nonparticipation for at-risk relatives. Descriptive analyses and Fisher's exact tests were conducted to evaluate differences in surveillance participation. Results Among 193 at-risk relatives, 21% were in surveillance. The primary reasons for nonparticipation were lack of awareness (36%) and lack of interest (24%). Neither the sex nor the cancer status of probands impacted surveillance. At-risk relatives with familial pancreatic cancer (FPC) who also carried relevant pathogenic germline variants (PGVs) were more likely to undergo surveillance than those with FPC or PGVs alone (P = .003). Among families with PGVs, 59% of relatives potentially eligible for surveillance had not completed genetic testing. Conclusion PDAC surveillance is underutilized in high-risk families. Communication interventions to address informational needs and decisional support could improve outcomes.

Background & Aims Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. Methods We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient–derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. Results Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. Conclusions Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy., Graphical abstract

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

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

e16287 Background: Pancreatic ductal adenocarcinoma (PDA) has been refractory to therapeutic targeting of the immune microenvironment. In preclinical work, IL-1β was upregulated in PDA tumors, and in mouse models, IL-1β expression led to activation of pancreatic stellate cells and immunosuppression. We hypothesize that blockade of IL-1β and PD-1 will result in significant alterations in immune and fibroblast subsets within the PDA microenvironment. Methods: We conducted an open-label multicenter Phase Ib study evaluating gemcitabine and nab-paclitaxel with canakinumab (ACZ885), a high-affinity human anti-interleukin-1β (IL-1β) mAb, and spartalizumab (PDR001), a mAb directed against human PD-1. 10 subjects with untreated metastatic PDA and RECIST measurable disease were enrolled. The primary objective was to identify a recommended phase II/III dose by evaluating the incidence of DLTs in the first 56 days. All subjects underwent baseline and on-study tissue and blood collection for extensive exploratory correlative studies. Secondary objectives including safety and tolerability and preliminary assessment of clinical activity. Results: 10 subjects were enrolled between November 2020 and March 2021. 6 out of 10 were evaluable for the dose confirmation. In the dose confirmation analysis there were no dose limiting toxicities (DLTs) and the recommended Phase II/III dose was established as; gemcitabine (1000 mg/m2 IV) and nab-paclitaxel (125 mg/m2 IV) on day 1,8,15; canakinumab (250 mg via subcutaneous injection) and spartalizumab (400 mg IV) on day 1; of each 28 day cycle. Adverse events were consistent with those seen with chemotherapy and were predominately hematologic. A preliminary efficacy analysis confirms 1 PR and 7 pts with SD. Two of the 7 pts have been treated for 11 and 12 cycles, respectively, without progression. Activation of CD8 T cells in peripheral blood and increased serum levels of IFN-induced chemokines CXCL9/10 were observed in patient samples. Using an in vitro suppression assay, we showed that baseline serum, from patients deriving clinical benefit, could induce myeloid derived suppressor cells, but that this effect was abrogated with treatment. Single cell transcriptional profiling also revealed treatment-dependent shifts in T cell activation state and myeloid cells in the tumors of patients experiencing clinical benefit. Conclusions: In this Phase Ib study, we established the Phase II/III dose of canakinumab and spartalizumab with chemotherapy in first line metastatic PDA. Translational studies suggest putative biomarkers that may help identify responding pts. This novel combination is being evaluated in a randomized Phase II/III study through the Precision Promise clinical trial network. Clinical trial information: NCT04581343.

TPS4188 Background: The success rate of drug development in PDAC is disappointingly low.PrP is a transformative, adaptive platform clinical trial designed to continuously evaluate many novel therapeutic options while increasing the probability that patients (pts) are randomized to effective experimental therapies. It cultivates enhanced cooperation among groups representing pts advocacy, pharmaceutical companies, academia, and the FDA. This patient-centric study aims to become the largest Phase 3 registrational study in PDAC and represents a fundamental shift in drug development for PDAC in the United States (US). Methods: PrP (NCT04229004) is a platform clinical trial sponsored by the Pancreatic Cancer Action Network (PanCAN), developed based on the FDA 2020 guidance document regarding "complex innovative designs" in registration trials https://www.fda.gov/media/130897/download . It utilizes adaptive randomization and other Bayesian statistical innovations provided by Berry Consultants LLC, including the “time machine” which uses all previously randomized controls for each arm, suitably adjusted for line of therapy and the time period of the arm. Focused on 1st and 2nd line treatment of mPDAC, PrP uses an adaptive platform design with randomization to one of 2 control arms (gemcitabine + nab-paclitaxel (GA) or mFOLFIRINOX, 30% of pts) or experimental therapy (70% of pts). Candidate experimental arms are reviewed by an Arm Selection Committee based on validity of the treatment target and strength of the pre-clinical and clinical data. The primary endpoint is overall survival (OS). Pts undergo pre- and on-treatment biopsies with state-of-the-art genomic, transcriptomic, and immune analysis, along with a serial collection of blood-based research samples. Pts are managed using novel supportive care techniques; PrP contains 3 sub-protocols evaluating quality of life, sarcopenia, and actigraphy. PrP launched in 2020 and has enrolled > 130 pts; 30 US sites have been selected with 17 currently active. Current experimental arms include: (i) GA + Pamrevlumab, an anti-CTGF Ab, (ii) Racemetyrosine monotherapy, a cancer metabolism-based therapy (for follow-up of patients) and (iii) an immuno-oncology arm in activation. Other arms are in the planning stages. Compared to traditional designs, PrP offers several advantages: multiple investigational treatments evaluated in parallel over time; ̃175 pts per experimental arm required to initiate a regulatory registration; and continuous learning from every patient, resulting in significant savings of time and resources. PrP has created an entirely new learning environment for accelerating drug development in PDAC. Clinical trial information: NCT04229004.

TPS2676 Background: Solid tumors comprise > 90% of cancers. Metastatic colorectal cancer, non-small cell lung cancer, and pancreatic cancer are among the leading causes of cancer-related mortality (5-year overall survival: 14%, 6%, and 3%, respectively) (ACS. 2021). Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical efficacy in hematologic malignancies (Neelapu S. et al. N Engl J Med. 2017). Translating engineered T-cell therapies to solid tumors has proven to be challenging due to a lack of tumor-specific targets that can discriminate cancer cells from normal cells. Previous studies using carcinoembryonic antigen (CEA) T-cell receptors and mesothelin (MSLN) CARs resulted in dose-limiting on-target, off-tumor toxicities (Parkhurst M, et al. Mol Ther. 2011; Tanyi J. Cellicon Valley '21). To create a therapeutic safety window, Tmod CAR T-cell therapy utilizes dual-signaling receptors to create a robust NOT logic gate capable of killing tumor cells, while leaving healthy cells intact (Hamburger A, et al. Mol Immunol. 2020). The 2 receptors in Tmod CAR T-cell therapy comprise an activator that recognizes an antigen on the surface of tumor cells that may also be present on normal cells, such as CEA and MSLN, and a blocker that recognizes a second surface antigen from an allele lost only in tumor cells. The frequency of HLA LOH among advanced GI solid tumor cancers in the Tempus real-world dataset is 16.3% with a range of 15.6%-20.8% between colorectal, pancreatic, and gastroesophageal tumors (Hecht R. et al. ASCO-GI 2022. Abstract #190). As such, HLA LOH offers a definitive tumor versus normal discriminator target for CAR T-cell therapy. Different activator/blocker combinations can be engineered with the Tmod platform technology and may be applied to T cells and natural killer cells in autologous and allogeneic settings. BASECAMP-1 is a currently enrolling observational study with key objectives of 1) To identify patients with somatic HLA LOH eligible for Tmod CAR T-cell therapy, and 2) To obtain leukapheresis and feasibility for the future EVEREST Tmod CAR T-cell trial. Methods: BASECAMP-1 (NCT04981119) patient eligibility has 2 parts: 1) Patients will be initially screened to identify germline HLA-A*02 heterozygosity by central NGS. If HLA-A*02 heterozygosity is confirmed, primary archival tumor tissue will be analyzed for somatic mutations by xT-Onco NGS testing. 2) If the tumor demonstrates HLA-A*02 LOH and the patient is eligible after screening, the patient will undergo leukapheresis. Banked T cells will be available for the autologous EVEREST Tmod CAR T-cell therapy interventional study to reduce waiting time at relapse. Clinical trial information: NCT04981119.

Needle-compatible miniaturized optoelectronic sensor may help pancreatic cancer diagnosis., Pancreatic cancer is one of the deadliest cancers, with a 5-year survival rate of

Summary: Patient-derived tumor organoids (TOs) are emerging as high-fidelity models to study cancer biology and develop novel precision medicine therapeutics. However, utilizing TOs for systems-biology-based approaches has been limited by a lack of scalable and reproducible methods to develop and profile these models. We describe a robust pan-cancer TO platform with chemically defined media optimized on cultures acquired from over 1,000 patients. Crucially, we demonstrate tumor genetic and transcriptomic concordance utilizing this approach and further optimize defined minimal media for organoid initiation and propagation. Additionally, we demonstrate a neural-network-based high-throughput approach for label-free, light-microscopy-based drug assays capable of predicting patient-specific heterogeneity in drug responses with applicability across solid cancers. The pan-cancer platform, molecular data, and neural-network-based drug assay serve as resources to accelerate the broad implementation of organoid models in precision medicine research and personalized therapeutic profiling programs.

Immune checkpoint blockade (ICB) has demonstrated efficacy in multiple cancers, offering the potential of long-term disease control not achievable with cytotoxic or targeted therapies. However, the field has not yet achieved the crucial next steps - the expansion of the response rate and achievement of clinical efficacy in so-called "cold tumours". Mechanistic studies of tumour-type specific immunosuppressive pathways can reveal underlying biological hurdles to immunotherapy and offer new therapeutic insights. Our finding that tumour-derived IL-1β mediates immunosuppression in pancreatic cancer has precipitated a new clinical trial.

Patients with cancer often confront the decision of whether to continue high-dose chemotherapy at the expense of cumulative toxicities. Reducing the dose of chemotherapy regimens while preserving efficacy is sorely needed to preserve the performance status of these vulnerable patients, yet has not been prioritized. Here, we introduce a dual pronged approach to modulate the microenvironment of desmoplastic pancreatic tumors and enable significant dose deescalation of the FDA-approved chemotherapeutic nanoliposomal irinotecan (nal-IRI) without compromising tumor control. We demonstrate that light-based photodynamic priming (PDP) coupled with vitamin D3 receptor (VDR) activation within fibroblasts increases intratumoral nal-IRI accumulation and suppresses protumorigenic CXCL12/CXCR7 crosstalk. Combined photodynamic and biochemical modulation of the tumor microenvironment enables a 75% dose reduction of nal-IRI while maintaining treatment efficacy, resulting in improved tolerability. Modifying the disease landscape to increase the susceptibility of cancer, via preferentially modulating fibroblasts, represents a promising and relatively underexplored strategy to enable dose deescalation. The approach presented here, using a combination of three clinically available therapies with nonoverlapping toxicities, can be rapidly translated with minimal modification to treatment workflow, and challenges the notion that significant improvements in chemotherapy efficacy can only be achieved at the expense of increased toxicity.

Despite tremendous gains in the molecular understanding of exocrine pancreatic cancer, the prognosis for this disease remains very poor, largely because of delayed disease detection and limited effectiveness of systemic therapies. Both incidence rates and mortality rates for pancreatic cancer have increased during the past decade, in contrast to most other solid tumor types. Recent improvements in multimodality care have substantially improved overall survival, local control, and metastasis-free survival for patients who have localized tumors that are amenable to surgical resection. The widening gap in prognosis between patients with resectable and unresectable or metastatic disease reinforces the importance of detecting pancreatic cancer sooner to improve outcomes. Furthermore, the developing use of therapies that target tumor-specific molecular vulnerabilities may offer improved disease control for patients with advanced disease. Finally, the substantial morbidity associated with pancreatic cancer, including wasting, fatigue, and pain, remains an under-addressed component of this disease, which powerfully affects quality of life and limits tolerance to aggressive therapies. In this article, the authors review the current multidisciplinary standards of care in pancreatic cancer with a focus on emerging concepts in pancreatic cancer detection, precision therapy, and survivorship.

Human chromosome 9p21.3 is susceptible to inactivation in cell immortalization and diseases, such as cancer, coronary artery disease and type-2 diabetes. Although this locus encodes three cyclin-dependent kinase (CDK) inhibitors (p15INK4B, p14ARF and p16INK4A), our understanding of their functions and modes of action is limited to the latter two. Here, we show that in vitro p15INK4B is markedly stronger than p16INK4A in inhibiting pRb1 phosphorylation, E2F activity and cell-cycle progression. In mice, urothelial cells expressing oncogenic HRas and lacking p15INK4B, but not those expressing HRas and lacking p16INK4A, develop early-onset bladder tumors. The potency of CDKN2B/p15INK4B in tumor suppression relies on its strong binding via key N-terminal residues to and inhibition of CDK4/CDK6. p15INK4B also binds and inhibits enolase-1, a glycolytic enzyme upregulated in most cancer types. Our results highlight the dual inhibition of p15INK4B on cell proliferation, and unveil mechanisms whereby p15INK4B aberrations may underpin cancer and non-cancer conditions., The human chromosome locus 9p21.3 is a tumour suppressor hub which encodes three CDK inhibitors, p15INK4B, p14ARF and p16INK4A. Here, the authors show that p15INK4B inhibits the cell cycle and glycolysis in a murine model of HRas + ‐mediated urothelial carcinoma and has a more relevant role as a tumour suppressor than its neighbouring p16INK4A.

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3β) a key regulator of glycolysis. Pharmacological inhibition of GSK3β results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3β inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC.

190 Background: Metastatic colorectal (CRC), pancreatic (PANC), and gastroesophageal (GE) cancers are the leading causes of GI cancer–related mortality (5-yr survival rate, 14%, 3% and ̃5-6%, respectively). T-cell immunotherapy targeting GI-associated tumor antigens has been attempted, but efficacy has been constrained by on-target off-tumor toxicity, limiting the therapeutic window. The Tmod (TM) platform is an AND-NOT logic-gated CAR T modular system, versions of which have a CEA- or MSLN-targeting CAR activator and a separate HLA-A*02-targeting blocker receptor to protect normal cells. Tmod CAR T exploits HLA LOH, common in GI malignancies (10-33% in primary solid tumors [TCGA]) and can kill tumor cells without harming healthy cells in vitro and in vivo. However, the prevalence of HLA LOH across GI tumors is unknown in the real-world setting. We utilized the Tempus xT oncology NGS database of patients with multiple GI tumors. From a standard-of-care NGS assay, GI cancer patients can be readily identified for HLA LOH and future treatment with Tmod CAR T therapy. Methods: The occurrence of HLA LOH in GI tumors of 1439 patients was assessed using paired germline and somatic DNA sequencing using a research assay [6]. CRC, PANC and GE patients with ≥ stage 3 were then extracted, and rates of HLA LOH were identified (ie, whether loss occurred across high-frequency HLA-A alleles). In addition, mutations in KRAS and BRAF, as well as MSI status were stratified to determine any association with HLA-A LOH. Results: HLA-A LOH was detected in 830 (17.3%) of all solid tumor records, and a similar proportion when all GI cancer records were analyzed (17.0%). For GI subtypes, these values ranged from 13.5% to 23.1% (Table). No high-frequency HLA-A allele (A*01, A*02, A*03, A*11) was more likely to be lost. Clinical biomarkers ( KRAS, BRAF and MSI status) were not associated with HLA-LOH. Conclusions: The frequency of HLA LOH among advanced solid tumor cancers in this dataset is 17.3%, with a range of 13.5-23% between CRC, PANC and GE. The HLA LOH frequency observed in these GI tumors is consistent with that in primary tumors from TCGA, which also used germline-matched and tumor samples. Clinical biomarkers were not associated with HLA LOH. Tempus NGS was able to identify HLA LOH, which can be used for Tmod CAR T therapy to an enhanced therapeutic window. Identification of these patients in BASECAMP-1 (NCT04981119) will enable novel Tmod CAR T therapy. [Table: see text]

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

BackgroundSolid tumors comprise >90% of cancers. Metastatic colorectal cancer, non-small cell lung cancer, and pancreatic cancer are among the leading causes of cancer-related mortality (5-year overall survival: 14%, 6%, and 3%, respectively).1Chimeric antigen receptor (CAR) T-cell therapy demonstrated clinical outcomes in hematologic malignancies.2 3 However, translating engineered T-cell therapies to solid tumors proves difficult due to a lack of tumor-specific targets that discriminate cancer cells from normal cells. In previous studies, the use of a carcinoembryonic antigen T-cell receptors and mesothelin CARs both resulted in dose-limiting on-target, off-tumor toxicities.4 5 TmodTM CAR T-cell therapy addresses these challenges by leveraging dual receptors to create a robust AND NOT signal integrator capable of killing tumor cells, while leaving healthy cells intact (figure 1).6 Tmod platform technology is a versatile system that may be applied to T cells and natural killer cells in autologous and allogeneic settings.HLA LOH offers a definitive tumor versus normal discriminator target for CAR T-cell therapy.6 7 The 2 receptors comprise an activator that recognizes an antigen present on the surface of normal and tumor cells and a blocker that recognizes a second surface antigen from an allele lost only in tumor cells. HLA LOH has been observed in ~13% across all solid tumors and up to 33% of pancreatic cancers.8 New technologies have shown higher HLA LOH rates; however, it is unclear whether patients with HLA LOH in their primary tumor tissues are at higher risk for recurrence. BASECAMP-1 is an observational study with key objectives: 1) To determine and identify patients with somatic HLA LOH eligible for Tmod CAR T-cell therapy, and 2) Subsequent leukapheresis and manufacturing feasibility for future Tmod CAR T-cell trials.MethodsBASECAMP-1 (NCT04981119) patient eligibility has 2 parts (figure 2): 1) Patients will be initially screened to identify germline HLA-A*02 heterozygosity by central next-generation sequencing (NGS). If HLA-A*02 heterozygosity is confirmed, primary archival tumor tissue will be analyzed by xT-Onco NGS testing9 to determine if somatic tumor HLA-A*02 LOH is present; 2) If the tumor demonstrates HLA-A*02 LOH and the patient screens eligible, the patient will undergo leukapheresis. Patients enrolled in the study who undergo leukapheresis will be evaluated for safety 7 days post-leukapheresis and followed for relapsed status. Banked T cells will be available for subsequent autologous Tmod CAR T-cell therapy at the time of relapse.Abstract 491 Figure 1Illustration of the Tmod T cell engaging with tumor cells with somatic loss of HLA-A*02 and with normal cellsAbstract 491 Figure 2Study schema. HLA, human leukocyte antigen; LOH, loss of heterozygosity; NGS, next generation sequencingTrial RegistrationNCT04981119ReferencesAmerican Cancer Society. Cancer Facts & Figures 2021. Atlanta: American Cancer Society; 2021.Neelapu S, Locke F, Bartlett N, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377(26):2531–2544.Maude S, Laetsch T, Buechner J, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378(5):439–448.Parkhurst M, Yang J, Langan R, et al. T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 2011;19(3):620–626.Haas AR, Tanyi JL, O’Hara MH, et al. Phase I study of lentiviral-transduced chimeric antigen receptor-modified T cells recognizing mesothelin in advanced solid cancers. Mol Ther. 2019;27(11):1919–1929.Hamburger A, DiAndreth B, Cui J, et al. Engineered T cells directed at tumors with defined allelic loss. Mol Immunol 2020;128:298–310.Hwang M, Mog B, Douglass J, et al. Targeting loss of heterozygosity for cancer-specific immunotherapy. Proc Natl Acad Sci U S A 2021;118(12):e2022410118.The Cancer Genome Atlas (TCGA) Research Network. https://www.cancer.gov/tcga. Accessed June 2021.Perera J, Mapes B, Lau D, et al. Detection of human leukocyte antigen class I loss of heterozygosity in solid tumor types by next-generation DNA sequencing. J Immunother Cancer 2019;7(suppl 1):103.

Definitive chemoradiotherapy for unresectable pancreatic cancer has traditionally involved 5-fluorouracil-based chemotherapy. Our institution has a long history of combining gemcitabine and radiotherapy (RT), and performed a retrospective review of all patients treated in this manner. We reviewed the records of 180 patients treated from 1999 to 2012. Mean RT dose was 40.9 Gy in 2.2-Gy fractions, and targeted only radiographically apparent disease. Ninety-six percent of patients received full-dose gemcitabine-based chemotherapy with RT. Kaplan-Meier was used to analyze time-to-event endpoints, and Cox regression models were used to assess significant prognostic variables. Eighty-nine percent of patients completed RT without a toxicity-related treatment break. Median follow-up was 10.2 months. Twenty-nine percent of patients had a radiographic decrease in primary tumor size following treatment. Median overall survival was 11.8 months, time to distant metastasis (TDM) was 6.7 months, and time to local recurrence (TLR) was 8.3 months. On multivariate analysis, male sex, lower performance status, and higher posttreatment CA 19-9 level predicted for worse overall survival. Posttreatment, CA 19-9 was also associated with TDM and TLR, and radiographic tumor response was associated with better TLR. Definitive chemoradiation using full-dose gemcitabine is well tolerated and achieves survival outcomes comparable to reported trials in the literature.

Purpose: To evaluate safety and preliminary efficacy of metronomic 5-fluorouracil plus nab-paclitaxel, bevacizumab, leucovorin, and oxaliplatin (FABLOx) in patients with newly diagnosed metastatic pancreatic cancer (MPC). Methods: A total of 12 treatment-naive patients (aged 18–65 years, Eastern Cooperative Oncology Group performance status [ECOG PS] ≤1) with MPC received 5-fluorouracil 180 mg/m2 per day (days 1–14 continuous infusion); nab-paclitaxel 75 mg/m2, leucovorin 20 mg/m2, and oxaliplatin 40 mg/m2 (days 1, 8, and 15); and bevacizumab 5 mg/kg (days 1 and 15) administered intravenously in each 28-day cycle. The primary end-point was incidence of dose-limiting toxicities (DLTs) in cycle 1. Safety was further evaluated as a secondary end-point; preliminary efficacy was also examined. Results: Two DLTs (grade 3 anemia requiring transfusion and grade 3 mucositis unresponsive to treatment within 4 days of onset) were observed in one of six patients enrolled in dose cohort 1. Cohort 1 was expanded from 6 to 12 patients to further evaluate safety, per the investigators' recommendation. All patients discontinued treatment. The most common grade ≥3 adverse events were abdominal pain, fatigue, mucositis, and decreased neutrophil count. Objective response rate was 33% (four partial responses). Median progression-free survival (PFS) and overall survival (OS) were 5.6 (95% confidence interval [CI], 1.7–11.3) and 9.9 (95% CI, 4.4–13.2) months, respectively; 1-year PFS and OS rates were 12.2% (95% CI, 0.7–40.8) and 38.9% (95% CI, 12.6–65.0). Conclusion: FABLOx is feasible and tolerable in patients newly diagnosed with MPC. However, preliminary efficacy data are inconclusive for continued investigation in a phase II trial.

Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting the DNA damage response (DDR) and replication stress. We show that patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum and PARP inhibitor therapy in vitro and in vivo. We generated a novel signature of replication stress with potential clinical utility in predicting response to ATR and WEE1 inhibitor treatment. Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy.Abstract FigureSTATEMENT OF SIGNIFICANCEWe define therapeutic strategies that target subgroups of PC using novel signatures of DNA damage response deficiency and replication stress. This potentially offers patients with DNA repair defects therapeutic options outside standard of care platinum chemotherapy and is being tested in clinical trials on the Precision-Panc platform.

Purpose The treatment of cancer is complex, which can overwhelm patients and lead to poor comprehension and recall of the specifics of the cancer stage, prognosis, and treatment plan. We hypothesized that an oncologist can feasibly record and deliver a custom video summary of the consultation that covers the diagnosis, recommended testing, treatment plan, and follow-up in < 5 minutes. The video summary allows the patient to review and share the most important part of a cancer consultation with family and caregivers. Methods At the conclusion of the office visit, oncologists recorded the most important points of the consultation, including the diagnosis and management plan as a short video summary. Patients were then e-mailed a link to a secure Website to view and share the video. Patients and invited guests were asked to respond to an optional survey of 15 multiple-choice and four open-ended questions after viewing the video online. Results Three physicians recorded and sent 58 video visit summaries to patients seen in multidisciplinary GI cancer clinics. Forty-one patients logged into the secure site, and 38 viewed their video. Fourteen patients shared their video and invited a total of 46 visitors, of whom 36 viewed the videos. Twenty-six patients completed the survey, with an average overall video satisfaction score of 9 on a scale of 1 to 10, with 10 being most positive. Conclusion Video visit summaries provide a personalized education tool that patients and caregivers find highly useful while navigating complex cancer care. We are exploring the incorporation of video visit summaries into the electronic medical record to enhance patient and caregiver understanding of their specific disease and treatment.

The identification of molecularly defined subgroups of Pancreatic ductal Adenocarcinoma (PDAC) has the potential to transform clinical practice. There is now a growing consensus that PDAC can be divided into transcriptomic subtypes with 2 broad linages referred to as Classical (Pancreatic) and Squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal linage specification, HNF4A and GATA6, switch metabolic profiles from Classical (Pancreatic) to predominantly Squamous, with GSK3B a key regulator of glycolysis. Pharmacological inhibition of GSK3B results in selective sensitivity in the Squamous subtype, however a subset of these Squamous PDCLs acquired rapid drug tolerance. Using chromatin accessibility maps, we identify subtype specific chromatin landscapes and unique promoter usage between subpopulations of Squamous PDCLs can affect drug tolerance. Our findings demonstrate that a chromatin-based framework can be used to identify subtypes of PDAC that are indistinguishable by gene expression profiles which could refine patient selection for precision medicine.

Sensitive and reproducible platforms have been developed for detection, isolation, and enrichment of circulating tumor cells (CTCs)-rare cells that enter the blood from solid tumors, including those of the breast, prostate gland, lung, pancreas, and colon. These might be used as biomarkers in diagnosis or determination of prognosis. CTCs are no longer simply detected and quantified; they are now used in ex vivo studies of anticancer agents and early detection. We review what we have recently learned about CTCs from pancreatic tumors, describing advances in their isolation and analysis and challenges to their clinical utility. We summarize technologies used to isolate CTCs from blood samples of patients with pancreatic cancer, including immunoaffinity and label-free physical attribute-based capture. We explain methods of CTC analysis and how findings from these studies might be used to detect cancer at earlier stages, monitor disease progression, and determine prognosis. We review studies that have expanded CTCs for testing of anticancer agents and how these approaches might be used to personalize treatment. Advances in the detection, isolation, and analysis of CTCs have increased our understanding of the dissemination and progression of pancreatic cancer. However, standardization of methodologies and prospective studies are needed for this emerging technology to have a significant effect on clinical care.

Pancreatic ductal adenocarcinoma (PDA) is characterized by a dense stroma consisting of a prevalence of activated fibroblasts whose functional contributions to pancreatic tumorigenesis remain incompletely understood. In this study, we provide the first identification and characterization of mesenchymal stem cells (MSC) within the human PDA microenvironment, highlighting the heterogeneity of the fibroblast population. Primary patient PDA samples and low-passage human pancreatic cancer–associated fibroblast cultures were found to contain a unique population of cancer-associated MSCs (CA-MSC). CA-MSCs markedly enhanced the growth, invasion, and metastatic potential of PDA cancer cells. CA-MSCs secreted the cytokine GM-CSF that was required for tumor cell proliferation, invasion, and transendothelial migration. Depletion of GM-CSF in CA-MSCs inhibited the ability of these cells to promote tumor cell growth and metastasis. Together, these data identify a population of MSCs within the tumor microenvironment that possesses a unique ability, through GM-CSF signaling, to promote PDA survival and metastasis. Significance: The role of stroma in pancreatic cancer is controversial. Here, we provide the first characterization of MSCs within the human PDA microenvironment and demonstrate that CA-MSCs promote tumorigenesis through the production of GM-CSF. These data identify a novel cytokine pathway that mediates mesenchymal–epithelial cross-talk and is amenable to therapeutic intervention. Cancer Discov; 6(8); 886–99. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 803

Background Pancreatic cancer is characterised by the accumulation of a fibro-inflammatory stroma. Within this stromal reaction, myeloid cells are a predominant population. Distinct myeloid subsets have been correlated with tumour promotion and unmasking of anti-tumour immunity. Objective The goal of this study was to determine the effect of myeloid cell depletion on the onset and progression of pancreatic cancer and to understand the relationship between myeloid cells and T cell-mediated immunity within the pancreatic cancer microenvironment. Methods Primary mouse pancreatic cancer cells were transplanted into CD11b-diphtheria toxin receptor (DTR) mice. Alternatively, the iKras* mouse model of pancreatic cancer was crossed into CD11b-DTR mice. CD11b + cells (mostly myeloid cell population) were depleted by diphtheria toxin treatment during tumour initiation or in established tumours. Results Depletion of myeloid cells prevented Kras G12D -driven pancreatic cancer initiation. In pre-established tumours, myeloid cell depletion arrested tumour growth and in some cases, induced tumour regressions that were dependent on CD8 + T cells. We found that myeloid cells inhibited CD8 + T-cell anti-tumour activity by inducing the expression of programmed cell death-ligand 1 (PD-L1) in tumour cells in an epidermal growth factor receptor (EGFR)/mitogen-activated protein kinases (MAPK)-dependent manner. Conclusion Our results show that myeloid cells support immune evasion in pancreatic cancer through EGFR/MAPK-dependent regulation of PD-L1 expression on tumour cells. Derailing this crosstalk between myeloid cells and tumour cells is sufficient to restore anti-tumour immunity mediated by CD8 + T cells, a finding with implications for the design of immune therapies for pancreatic cancer.

A highly sensitive microfluidic system to capture circulating tumor cells from whole blood of cancer patients is presented. The device incorporates graphene oxide into a thermoresponsive polymer film to serve as the first step of an antibody functionalization chemistry. By decreasing the temperature, captured cells may be released for subsequent analysis.

Summary There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

TPS789 Background: Patients with metastatic pancreatic cancer who have progressed on two prior lines of therapy have a poor prognosis with an overall survival in the range of 2-2.5 months. (Manax, et al. J Clin Oncol 37, 2019 suppl 4; abstr 226). There is currently no standard of care for these patients that has demonstrated improved outcomes. SM-88 (D,L-alpha-metyrosine; racemetyrosine [USAN]) is a proprietary dysfunctional tyrosine derivative and is the backbone of SM-88 used with MPS (Methoxsalen 10mg, Phenytoin 50mg and Sirolimus 0.5mg; all administered daily). SM-88 monotherapy was relatively well tolerated, with improvement in survival in select patients with heavily pretreated PDAC who achieved stable disease on therapy (HR 0.08, p = 0.02). Circulating tumor cells (CTC’s) were prognostic and decreased on therapy with SM-88 potentially identifying a subgroup of PDAC that may be most likely to benefit from therapy (Noel et al. Annal Oncol V30, Suppl 4, 2019). Preliminary radiomic analysis of the largest metastases at baseline suggested the same benefits including a correlation with baseline CTCs, changes in CTCs on therapy and OS (Ocean et al, Annal Oncol, V30, Suppl 5, 2019). Here, we describe a randomized, open-label, phase 2/3 trial evaluating the efficacy of SM-88 + MPS vs physician’s choice treatment as third line therapy for patients with metastatic PDAC. Methods: This is a multi-center Phase 3 study of patients ≥18 years with metastatic PDAC that progressed after 2 lines of chemotherapy (gemcitabine [gem] and 5-fluorouracil [5-FU] based) with an ECOG

Purpose An ASCO provisional clinical opinion (PCO) offers timely clinical direction to ASCO’s membership and other health care providers. This PCO addresses identification and management of patients and family members with possible predisposition to pancreatic adenocarcinoma. Methods ASCO convened an Expert Panel and conducted a systematic review of the literature published from January 1998 to June 2018. Results of the databases searched were supplemented with hand searching of the bibliographies of systematic reviews and selected seminal articles and contributions from Expert Panel members’ curated files. Provisional Clinical Opinion All patients diagnosed with pancreatic adenocarcinoma should undergo assessment of risk for hereditary syndromes known to be associated with an increased risk for pancreatic adenocarcinoma. Assessment of risk should include a comprehensive review of family history of cancer. Individuals with a family history of pancreatic cancer affecting two first-degree relatives meet criteria for familial pancreatic cancer (FPC). Individuals (cancer affected or unaffected) with a family history of pancreatic cancer meeting criteria for FPC, those with three or more diagnoses of pancreatic cancer in same side of the family, and individuals meeting criteria for other genetic syndromes associated with increased risk for pancreatic cancer have an increased risk for pancreatic cancer and are candidates for genetic testing. Germline genetic testing for cancer susceptibility may be discussed with individuals diagnosed with pancreatic cancer, even if family history is unremarkable. Benefits and limitations of pancreatic cancer screening should be discussed with individuals whose family history meets criteria for FPC and/or genetic susceptibility to pancreatic cancer. Additional information is available at www.asco.org/gastrointestinal-cancer-guidelines .

The tumor microenvironment plays a vital role in therapeutic resistance in pancreatic adenocarcinoma. We have created a 3D model to study the tumor microenvironment ex vivo that integrates tumor cells with tumor associated macrophages (TAMs) and cancer associated fibroblasts (CAFs). The microtumor model recreates the complex histology of a primary tumor and can be analyzed histologically or with viability assays for therapeutic testing. Microtumors demonstrate increased resistance to gemcitabine when compared to tumor cells alone in a 3D spheroid, highlighting the contribution of TAMs and CAFs when designing models for therapeutic testing.

The tumor microenvironment plays a vital role in therapeutic resistance in pancreatic adenocarcinoma. We have created a 3D model to study the tumor microenvironment ex vivo that integrates tumor cells with tumor associated macrophages (TAMs) and cancer associated fibroblasts (CAFs). The microtumor model recreates the complex histology of a primary tumor and can be analyzed histologically or with viability assays for therapeutic testing. Microtumors demonstrate increased resistance to gemcitabine when compared to tumor cells alone in a 3D spheroid, highlighting the contribution of TAMs and CAFs when designing models for therapeutic testing.

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The biological properties of pancreatic cancer stem cells (PCSCs) remain incompletely defined and the central regulators are unknown. By bioinformatic analysis of a human PCSC-enriched gene signature, we identified the transcription factor HNF1A as a putative central regulator of PCSC function. Levels of HNF1A and its target genes were found to be elevated in PCSCs and tumorspheres, and depletion of HNF1A resulted in growth inhibition, apoptosis, impaired tumorsphere formation, decreased PCSC marker expression, and downregulation of POU5F1/OCT4 expression. Conversely, HNF1A overexpression increased PCSC marker expression and tumorsphere formation in pancreatic cancer cells and drove pancreatic ductal adenocarcinoma (PDA) cell growth. Importantly, depletion of HNF1A in xenografts impaired tumor growth and depleted PCSC marker-positive cells in vivo. Finally, we established an HNF1A-dependent gene signature in PDA cells that significantly correlated with reduced survivability in patients. These findings identify HNF1A as a central transcriptional regulator of PCSC properties and novel oncogene in PDA., eLife digest Pancreatic ductal adenocarcinoma is the most common form of pancreatic cancer. It is also one of the deadliest types of cancer: fewer than one in ten patients live for five years after being diagnosed with the disease. Several reasons can explain this poor outcome including that the cancer is often diagnosed late, when tumor cells have already spread, and that there are not many effective treatments for it. Pancreatic tumors contain different types of cancer cells with different properties. Among these are the so-called pancreatic cancer stem cells. These aggressive cells produce copies of themselves, contributing to tumor growth and spread. They can also help tumors to resist chemotherapy and radiotherapy. New treatments that specifically target cancer stem cells could therefore prove important for treating pancreatic cancer. It is still not clear what makes pancreatic cancer stem cells so aggressive, or how they differ from the rest of the cells in a tumor. Abel et al. therefore looked for proteins that were more abundant in human pancreatic cancer stem cells than in other, less aggressive cancer cells with the idea that these proteins are likely to be important for the behavior of the pancreatic cancer stem cells. Abel et al. found that a protein called HNF1A is enriched in pancreatic cancer stem cells. Experimentally reducing the levels of HNF1A in cells taken from human pancreatic cancers caused the cells to grow less well and form smaller tumors when injected into the pancreases of mice. These tumors contained few cancer stem cells, suggesting that HNF1A is important for maintaining the stem cell state. Further experiments showed that HNF1A increases the amount of many other proteins inside cells, including one that controls the activity of normal stem cells. Given the importance of HNF1A to pancreatic cancer stem cells, finding ways to prevent this protein from working could lead to new treatments for pancreatic cancer. At the moment there are no drugs that target HNF1A. Further research is therefore needed to develop new drugs that work against HNF1A or one of the other proteins that it affects.

Adaptive resistance to MEK inhibitors (MEK-Is) typically occurs via induction of genes for different receptor tyrosine kinases (RTKs) and/or their ligands, even in tumors of the same histotype, making combination strategies challenging. SHP2 (PTPN11) is required for RAS/ERK pathway activation by most RTKs, and might provide a common resistance node. We found that combining the SHP2 inhibitor SHP099 with a MEK-I inhibits proliferation of multiple cancer cells in vitro. PTPN11 knockdown/MEK-I had similar effects, while expressing SHP099-binding mutants conferred resistance, demonstrating that SHP099 was on-target. This combination was efficacious in xenograft and/or genetically engineered models of KRAS-mutant pancreas cancer and ovarian cancer and in wild-type RAS-expressing triple negative breast cancer. Biochemical studies show that SHP099 impedes SOS/RAS/MEK/ERK1/2 reactivation in response to MEK-Is and blocks ERK1/2-dependent transcriptional programs. SHP099 alone also inhibited RAS activation in some, but not all, KRAS-mutant lines. Hence, SHP099/MEK-I combinations could have therapeutic utility in multiple malignancies.

Adaptive resistance to MEK inhibitors (MEKi) typically occurs via induction of genes for different receptor tyrosine kinases (RTK) and/or their ligands, even in tumors of the same histotype, making combination strategies challenging. SHP2 (PTPN11) is required for RAS/ERK pathway activation by most RTKs and might provide a common resistance node. We found that combining the SHP2 inhibitor SHP099 with a MEKi inhibited the proliferation of multiple cancer cell lines in vitro. PTPN11 knockdown/MEKi treatment had similar effects, whereas expressing SHP099 binding–defective PTPN11 mutants conferred resistance, demonstrating that SHP099 is on-target. SHP099/trametinib was highly efficacious in xenograft and/or genetically engineered models of KRAS-mutant pancreas, lung, and ovarian cancers and in wild-type RAS-expressing triple-negative breast cancer. SHP099 inhibited activation of KRAS mutants with residual GTPase activity, impeded SOS/RAS/MEK/ERK1/2 reactivation in response to MEKi, and blocked ERK1/2-dependent transcriptional programs. We conclude that SHP099/MEKi combinations could have therapeutic utility in multiple malignancies. Significance: MEK inhibitors show limited efficacy as single agents, in part because of the rapid development of adaptive resistance. We find that SHP2/MEK inhibitor combinations prevent adaptive resistance in multiple cancer models expressing mutant and wild-type KRAS. Cancer Discov; 8(10); 1237–49. ©2018 AACR. See related commentary by Torres-Ayuso and Brognard, p. 1210. This article is highlighted in the In This Issue feature, p. 1195

To understand tissue architecture, it is necessary to understand both which cell types are present and the physical relationships among them. Single-cell RNA-Seq (scRNA-Seq) has made significant progress towards the unbiased and systematic identification of cell populations within a tissue, however, the characterization of their spatial organization within it has been more elusive. The recently introduced ‘spatial transcriptomics’ method (ST) reveals the spatial pattern of gene expression within a tissue section at a resolution of a thousand 100 µm spots across the tissue, each capturing the transcriptomes of multiple cells. Here, we present an approach for the integration of scRNA-Seq and ST data generated from the same sample, and deploy it on primary pancreatic tumors from two patients. Applying our multimodal intersection analysis (MIA), we annotated the distinct micro-environment of each cell type identified by scRNA-Seq. We further found that subpopulations of ductal cells, macrophages, dendritic cells, and cancer cells have spatially restricted localizations across the tissue, as well as distinct co-enrichments with other cell types. Our mapping approach provides an efficient framework for the integration of the scRNA-Seq-defined subpopulation structure and the ST-defined tissue architecture in any tissue.

The ineffectiveness of chemotherapy in patients with pancreatic cancer highlights a critical unmet need in pancreatic cancer therapy. Two commonly mutated genes in pancreatic cancer, KRAS and CDKN2A, have an incidence exceeding 90%, supporting investigation of dual targeting of MEK and CDK4/6 as a potential therapeutic strategy for this patient population. An in vitro proliferation synergy screen was conducted to evaluate response of a panel of high passage and patient-derived pancreatic cancer models to the combination of trametinib and palbociclib to inhibit MEK and CDK4/6, respectively. Two adenosquamous carcinoma models, L3.6pl and UM59, stood out for their high synergy response. In vivo studies confirmed that this combination treatment approach was highly effective in subcutaneously implanted L3.6pl and UM59 tumor-bearing animals. Both models were refractory to single-agent treatment. Reverse-phase protein array analysis of L3.6pl tumors excised from treated animals revealed strong downregulation of COX-2 expression in response to combination treatment. Expression of COX-2 under a CMV-driven promoter and shRNA knockdown of COX-2 both led to resistance to combination treatment. Our findings suggest that COX-2 may be involved in the improved therapeutic outcome seen in some pancreatic tumors that fail to respond to MEK or CDK4/6 inhibitors alone but respond favorably to their combination.

Induction of DNA damage by ionizing radiation (IR) and/or cytotoxic chemotherapy is an essential component of cancer therapy. The ataxia telangiectasia group D complementing gene (ATDC, also called TRIM29) is highly expressed in many malignancies. It participates in the DNA damage response downstream of ataxia telangiectasia-mutated (ATM) and p38/MK2 and promotes cell survival after IR. To elucidate the downstream mechanisms of ATDC-induced IR protection, we performed a mass spectrometry screen to identify ATDC binding partners. We identified a direct physical interaction between ATDC and the E3 ubiquitin ligase and DNA damage response protein, RNF8, which is required for ATDC-induced radioresistance. This interaction was refined to the C-terminal portion (amino acids 348-588) of ATDC and the RING domain of RNF8 and was disrupted by mutation of ATDC Ser-550 to alanine. Mutations disrupting this interaction abrogated ATDC-induced radioresistance. The interaction between RNF8 and ATDC, which was increased by IR, also promoted downstream DNA damage responses such as IR-induced γ-H2AX ubiquitination, 53BP1 phosphorylation, and subsequent resolution of the DNA damage foci. These studies define a novel function for ATDC in the RNF8-mediated DNA damage response and implicate RNF8 binding as a key determinant of the radioprotective function of ATDC.