Faculty Bibliography

Background Mesothelin (MSLN) is expressed on a variety of solid tumors, including mesothelioma and ovarian, uterine, gastric, pancreatic, and lung cancers.1 However, efforts to target MSLN using cellular therapies have been hampered by severe on-target, off-tumor toxicities associated with damage to normal tissues expressing MSLN.2 To avoid these toxicities, we have developed a logic-gated engineered cell therapy, TmodTM, which is composed of two chimeric antigen receptors (CARs): an activator that targets a tumor-associated antigen and an inhibitory receptor (blocker) gated by an antigen expressed on normal tissue but lost in tumor cells due to loss of heterozygosity (LOH). A2B694 is an MSLN-specific Tmod construct combining a third-generation MSLN CAR with an LIR-1-based inhibitory receptor specific for human leukocyte antigen A*02 (HLA-A*02). Methods Lentivirus encoding i) the CAR, ii) the blocker, and iii) an shRNA targeting b2M was used to transduce T cells from HLA-A*02 donors and generate MSLN Tmod cells. In vitro cytotoxicity measurements were performed using fluorescence-based imaging and luciferase readouts. In vivo assessments were performed in NSG mice subcutaneously implanted with normal cells (MSLN[+]A*02[+]), or tumor cells (MSLN[+]A*02[-]), in the left and right flanks, respectively. Following engraftment, mice were randomized and treated intravenously with MSLN Tmod cells or controls. Grafts were measured via caliper. Results MSLN Tmod cells preferentially killed tumor cells (MSLN[+]A*02[-]) over normal cells (MSLN[+]A*02[+]) in vitro, unlike clinically active comparator M5 CAR T cells, which indiscriminately killed both target cell types (figure 1A). Soluble MSLN, tested across a 0-2 mg/mL range, did not impact MSLN Tmod function. Additionally, in mixed cell cultures where T cells and tumor and normal cells were simultaneously cultured (1:1:1 ratio), MSLN Tmod cells selectively killed tumor targets while sparing normal cells. Further, MSLN Tmod cells cycled between activated and blocked states in vitro when repeatedly challenged with tumor or normal target cells. Finally, while MSLN CAR T cells killed both normal and tumor grafts in vivo, MSLN Tmod cells selectively killed tumor grafts while sparing normal grafts (figure 1B, C). Conclusions A2B694 is an autologous MSLN Tmod cell product that leverages LOH at the HLA locus in cancer cells, providing a mechanism to discriminate between normal and tumor cells. BASECAMP-1 (NCT04981119), an observational study that will identify patients with somatic HLA LOH, is currently recruiting. Eligible patients with metastatic colorectal, pancreatic, or non-small cell lung cancer will be apheresed for a future A2B694 interventional study (EVEREST-2)

Background Chimeric antigen receptor (CAR) T-cell therapy has shown clinical efficacy in hematologic cancers, but success is limited in solid tumors due to a lack of tumor-specific targets that distinguish cancer from normal cells and an immunosuppressive tumor microenvironment.1 Integrating synthetic biology and comprehensive molecular profiling of tumors may provide active and tolerable approaches to CAR T-cell therapy in patients with solid tumors. Human leukocyte antigen (HLA) loss of heterozygosity (LOH) in tumors offers a definitive tumor vs normal discriminator target for CAR T-cell therapy.2 The Tmod platform3,4 is a modular logic-gated CAR T system comprising different versions including a carcinoembryonic antigen (CEA)- or mesothelin (MSLN)-targeting CAR activator and a separate blocker receptor targeting HLA-A*02 or other HLA alleles to protect normal cells. Compared with existing immunohistochemistry (IHC) tests, Tempus xT-Onco is a standard-of-care next-generation sequencing (NGS) assay5 that detects somatic alterations including HLA LOH and generates whole transcriptome RNA data (eg, CEA or MSLN expression) and a tumor immune infiltration profile, which can effectively identify patients appropriate for Tmod CAR T-cell therapy. Methods HLA LOH in solid tumors was assessed with paired germline and somatic DNA sequencing. Common driver mutations, microsatellite instability status, and tumor mutational burden were examined in HLA-A LOH or HLA-A intact cohorts. Tumor expression of CEA and MSLN was evaluated via RNA sequencing and compared with immunohistochemistry (IHC) results. Results A total of 21,053 tumor samples in the Tempus database were compared with their matched-normal samples. HLA-A LOH was detected in 16% of 10,867 advanced solid tumors (table 1) and similar LOH frequencies were observed among common HLA-A alleles. Clinical factors and molecular biomarkers were similar between HLA-A LOH and HLA-A intact cohorts. High CEA expression was seen in IHC-positive patients. Conclusions The frequency of HLA-A LOH in solid tumors in the Tempus database is similar to that reported in the Cancer Genome Atlas.6 Tempus xT-Onco reliably detects HLA LOH and quantifies CEA and MSLN expression. Based on these data, patients with solid tumors are now being prospectively screened for HLA LOH using xT-Onco in an ongoing tissue banking study (BASECAMP-1, NCT04981119), preparing for future interventional protocols

Background and aim Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with lack of effective early detection strategies. There is an incomplete understanding of who is at risk for PDAC development and the contribution of heritability to that risk. Further, efforts at biomarker development for detection of early stage disease have been hampered by small sample sizes, lack of coordination, and inadequate access to high quality clinical data and biospecimens in relevant clinical populations. The PRECEDE Consortium was established to serve as a collaborative international network of PDAC clinical and research centers to accelerate early detection advances by standardizing collection of clinical data and biospecimens from patients at increased risk for PDAC. The consortium goal is to increase the overall survival rate for PDAC to 50% in 10 years by enabling transformative biomarker-driven discoveries in early detection of high-risk premalignant lesions and early stage cancers. Method The PRECEDE Consortium (NCT04970056; precedestudy. org) launched in 2019 and began enrollment in May, 2020. Data and biospecimen sharing are required for centers to join the consortium, which is facilitated through use of standardized data and biospecimen collection, and a centralized database (PRECEDELink) managed by a data coordinating center (Arbor Research). Imaging and clinical sequencing data will be stored and analyzed via a PRECEDE solution in the Amazon Web Services cloud. Participants age 18-90 are enrolled into one of seven cohorts based on personal and/or family history of PDAC and carrier status of pathogenic germline variants (PGV) in cancer predisposition genes (CPG). Three-generation pedigrees are collected at enrolment from participants, and standardized clinical germline testing is offered. Blood sample collection for DNA, plasma, and serum is completed at enrollment, and repeated annually for individuals meeting guidelines for annual surveillance. Results To date, 26 clinical sites have enrolled 2370 participants, with a target of 10,000 participants enrolled from 100 sites over the next 5 years. Among enrolled patients, 55% meet criteria for annual surveillance by MRI or endoscopic ultrasound. Demographics of the cohort to date: 56% female; 73% white; 35% CPG PGV carriers; 32% meet criteria for familial pancreatic cancer. Conclusions The PRECEDE Consortium study is a large international, longitudinal, prospective cohort study designed to accelerate the pace and scale of early diagnosis. Planned projects will address modifiers of risk, penetrance of disease, creating comprehensive risk models for clinical decision-making, and development and validation of biomarker assays. The PRECEDE Consortium provides a unique, innovative platform to bring together key stakeholders (academia, patients, public and private sector) to effect progress

Introduction: Pancreatic cancer continues to carry a dismal prognosis due to the high failure rates of conventional first line treatments. There is growing interest in the molecular profiling of tumors to guide early initiation of targeted therapies. Nearly all patients undergo endoscopic ultrasound (EUS) fine needle aspiration or biopsy as the initial diagnostic procedure. Therefore, we sought to assess the yield of EUS biopsies in obtaining samples for molecular profiling of pancreatic tumors and investigated the endoscopic factors associated with successful EUS sampling.
Method(s): We performed a search for all EUS-guided needle biopsies done for the indication of suspected pancreatic mass on imaging between January 2017 and January 2022. We then limited our cases to those diagnosed with pancreatic adenocarcinoma and had EUS samples sent for molecular profiling. Molecular profiling was done with next-generation sequencing with either a targeted panel of 648 genes or 324 genes. Differences in tumor size, number of needle passes, and needle gauge size between the successful sampling and non-successful sampling groups were determined by Mann-Whitney U Test using SPSS Statistics.
Result(s): We identified 309 consecutive cases where the diagnosis of pancreatic adenocarcinoma was established by EUS. Fifty-nine EUS biopsies were sent for molecular profiling and of these, fifty-three were sufficient for molecular testing (89.5% success rate). No procedural factors were significantly associated with successful sampling though we observed larger mean tumor sizes (31.3 vs 28 mm) and greater mean number of needle passes (3.4 vs 2.7 mean passes) in the successful sampling group. In Figure, we show the most commonly identified mutations and identify those that at the time had potential clinical impact on therapies. The yield of actionable mutations was 14% in the 53 patients who were successfully tested. (Figure)
Conclusion(s): Our results support that yield of somatic mutation testing is high from standard of care EUS biopsies and no obvious procedural factors were associated with failure of testing. We found that 14% of patients had actionable mutations. As the number of available targeted therapies improve, we expect the impact of this highly technically successful approach to grow (Table)

Background Previously reported single institution data on family history of pancreatic adenocarcinoma (PDAC) showed that most individuals with a germline pathogenic or likely pathogenic variant (PV/LPV) in a PDAC susceptibility gene who were diagnosed with PDAC would not have met current recommendations for PDAC surveillance established by the National Comprehensive Cancer Network, the American College of Gastroenterology, or International Cancer of the Pancreas Screening Consortium. These recommendations rely on the assumption that PV/LPV carriers with family history of PDAC are at greater risk for developing PDAC as compared to carriers without a family history. This study is a multi-site collaboration to validate the previous findings. Methods Individuals with PDAC who had a germline PV/LPV in ATM, BRCA1, BRCA2, EPCAM, MLH1, MSH2, MSH6, PALB2, or PMS2 were assessed for family history of PDAC in first- (FDR) or second-degree relatives (SDR). A comparison group of individuals with PDAC who had no germline PV/LPV identified through multigene panel testing was also assessed. Chi-square and t-tests were used to determine statistical significance. Results Nine institutions compiled a cohort of 196 individuals with PDAC who had a germline PV/LPV in one of the aforementioned genes. See Table 1 for demographics. Fifty (25.5%) had an FDR and/ or SDR affected by PDAC and 146 (74.5%) had no family history of PDAC. The cohort was significantly more likely to have a PDACaffected FDR or SDR than individuals with PDAC who had no germline PV/LPV (p = 0.004). Significance was also reached for affected FDR alone (p = 0.003), but not for affected SDR alone (p = 0.344). See Table 2. Conclusions This multi-site study confirms that most individuals with PDAC and a PV/LPV in ATM, BRCA1, BRCA2, EPCAM, MLH1, MSH2, MSH6, PALB2, or PMS2 would not meet current pancreatic cancer surveillance recommendations because they do not have family history of PDAC. Family history, particularly an affected FDR, enriches the cohort but alone is insufficient in identifying the majority of high-risk individuals who are at risk for developing PDAC. (Table Presented)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies, with a dismal survival rate. Screening the general population for early detection of PDAC is not recommended, but because early detection improves survival, high-risk individuals, defined as those meeting criteria based on a family history of PDAC and/or the presence of known pathogenic germline variant genes with PDAC risk, are recommended to undergo screening with MRI and/or endoscopic ultrasound at regular intervals. The Pancreatic Cancer Early Detection (PRECEDE) Consortium was formed in 2018 and is composed of gastroenterologists, geneticists, pancreatic surgeons, radiologists, statisticians, and researchers from 40 sites in North America, Europe, and Asia. The overarching goal of the PRECEDE Consortium is to facilitate earlier diagnosis of PDAC for high-risk individuals to increase survival of the disease. A standardized MRI protocol and reporting template are needed to enhance the quality of screening examinations, improve consistency of clinical management, and facilitate multiinstitutional research. We present a consensus statement to standardize MRI screening and reporting for individuals with elevated risk of pancreatic cancer.

The stem-cell-like behavior of cancer cells plays a central role in tumor heterogeneity and invasion and correlates closely with drug resistance and unfavorable clinical outcomes. However, the molecular underpinnings of cancer cell stemness remain incompletely defined. Here, we show that SNHG1, a long non-coding RNA that is over-expressed in ~95% of human muscle-invasive bladder cancers (MIBCs), induces stem-cell-like sphere formation and the invasion of cultured bladder cancer cells by upregulating Rho GTPase, Rac1. We further show that SNHG1 binds to DNA methylation transferase 3A protein (DNMT3A), and tethers DNMT3A to the promoter of miR-129-2, thus hyper-methylating and repressing miR-129-2-5p transcription. The reduced binding of miR-129-2 to the 3'-UTR of Rac1 mRNA leads to the stabilization of Rac1 mRNA and increased levels of Rac1 protein, which then stimulates MIBC cell sphere formation and invasion. Analysis of the Human Protein Atlas shows that a high expression of Rac1 is strongly associated with poor survival in patients with MIBC. Our data strongly suggest that the SNHG1/DNMT3A/miR-129-2-5p/Rac1 effector pathway drives stem-cell-like and invasive behaviors in MIBC, a deadly form of bladder cancer. Targeting this pathway, alone or in combination with platinum-based therapy, may reduce chemoresistance and improve longer-term outcomes in MIBC patients.

Missense mutations at the three hotspots in the guanosine triphosphatase (GTPase) RAS-Gly¹², Gly¹³, and Gln⁶¹ (commonly known as G12, G13, and Q61, respectively)-occur differentially among the three RAS isoforms. Q61 mutations in KRAS are infrequent and differ markedly in occurrence. Q61H is the predominant mutant (at 57%), followed by Q61R/L/K (collectively 40%), and Q61P and Q61E are the rarest (2 and 1%, respectively). Probability analysis suggested that mutational susceptibility to different DNA base changes cannot account for this distribution. Therefore, we investigated whether these frequencies might be explained by differences in the biochemical, structural, and biological properties of KRAS^Q61 mutants. Expression of KRAS^Q61 mutants in NIH 3T3 fibroblasts and RIE-1 epithelial cells caused various alterations in morphology, growth transformation, effector signaling, and metabolism. The relatively rare KRAS^Q61E mutant stimulated actin stress fiber formation, a phenotype distinct from that of KRAS^Q61H/R/L/P, which disrupted actin cytoskeletal organization. The crystal structure of KRAS^Q61E was unexpectedly similar to that of wild-type KRAS, a potential basis for its weak oncogenicity. KRAS^Q61H/L/R-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines exhibited KRAS-dependent growth and, as observed with KRAS^G12-mutant PDAC, were susceptible to concurrent inhibition of ERK-MAPK signaling and of autophagy. Our results uncover phenotypic heterogeneity among KRAS^Q61 mutants and support the potential utility of therapeutic strategies that target KRAS^Q61 mutant-specific signaling and cellular output.

OBJECTIVES/OBJECTIVE:Extracellular vesicles (EVs) are lipid bound vesicles secreted by cells into the extracellular environment. Studies have implicated EVs in cell proliferation, epithelial-mesenchymal transition, metastasis, angiogenesis, and mediating the interaction of tumor cells and microenvironment. A systematic characterization of EVs from pancreatic cancer cells and cancer-associated fibroblasts (CAFs) would be valuable for studying the roles of EV proteins in pancreatic tumorigenesis. METHODS:Proteomic and functional analyses were applied to characterize the proteomes of EVs released from 5 pancreatic cancer lines, 2 CAF cell lines, and a normal pancreatic epithelial cell line (HPDE). RESULTS:More than 1400 nonredundant proteins were identified in each EV derived from the cell lines. The majority of the proteins identified in the EVs from the cancer cells, CAFs, and HPDE were detected in all 3 groups, highly enriched in the biological processes of vesicle-mediated transport and exocytosis. Protein networks relevant to pancreatic tumorigenesis, including epithelial-mesenchymal transition, complement, and coagulation components, were significantly enriched in the EVs from cancer cells or CAFs. CONCLUSIONS:These findings support the roles of EVs as a potential mediator in transmitting epithelial-mesenchymal transition signals and complement response in the tumor microenvironment and possibly contributing to coagulation defects related to cancer development.

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.