Faculty Bibliography

BACKGROUND:Germline BRCA1 and BRCA2 mutations (gBRCAm) can inform pancreatic cancer (PC) risk and treatment but most of the available information is derived from white patients. The ethnic and geographic variability of gBRCAm prevalence and of germline BRCA (gBRCA) testing uptake in PC globally is largely unknown. MATERIALS AND METHODS/METHODS:We carried out a systematic review and prevalence meta-analysis of gBRCA testing and gBRCAm prevalence in PC patients stratified by ethnicity. The main outcome was the distribution of gBRCA testing uptake across diverse populations worldwide. Secondary outcomes included: geographic distribution of gBRCA testing uptake, temporal analysis of gBRCA testing uptake in ethnic groups, and pooled proportion of gBRCAm stratified by ethnicity. The study is listed under PROSPERO registration number #CRD42022311769. RESULTS:A total of 51 studies with 16 621 patients were included. Twelve of the studies (23.5%) enrolled white patients only, 10 Asians only (19.6%), and 29 (56.9%) included mixed populations. The pooled prevalence of white, Asian, African American, and Hispanic patients tested per study was 88.7%, 34.8%, 3.6%, and 5.2%, respectively. The majority of included studies were from high-income countries (HICs) (64; 91.2%). Temporal analysis showed a significant increase only in white and Asians patients tested from 2000 to present (P < 0.001). The pooled prevalence of gBRCAm was: 3.3% in white, 1.7% in Asian, and negligible (<0.3%) in African American and Hispanic patients. CONCLUSIONS:Data on gBRCA testing and gBRCAm in PC derive mostly from white patients and from HICs. This limits the interpretation of gBRCAm for treating PC across diverse populations and implies substantial global and racial disparities in access to BRCA testing in PC.

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression; however, in-depth single cell characterization of the PDAC TME and its role in response to therapy is lacking. Here, we perform single-cell RNA sequencing on freshly collected human PDAC samples either before or after chemotherapy. Overall, we find a heterogeneous mixture of basal and classical cancer cell subtypes, along with distinct cancer-associated fibroblast and macrophage subpopulations. Strikingly, classical and basal-like cancer cells exhibit similar transcriptional responses to chemotherapy and do not demonstrate a shift towards a basal-like transcriptional program among treated samples. We observe decreased ligand-receptor interactions in treated samples, particularly between TIGIT on CD8 + T cells and its receptor on cancer cells, and identify TIGIT as the major inhibitory checkpoint molecule of CD8 + T cells. Our results suggest that chemotherapy profoundly impacts the PDAC TME and may promote resistance to immunotherapy.

Hepato-pancreatico-biliary (HPB) malignancies are difficult-to-treat and continue to to have a high mortality and significant therapeutic resistance to standard therapies. Immune oncology (IO) therapies have demonstrated efficacy in several solid malignancies when combined with chemotherapy, whereas response rates in pancreatic ductal adenocarcinoma (PDA) are poor. While promising in hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), there remains an unmet need to fully leverage IO therapies to treat HPB tumors. We therefore defined T cell subsets in the tumor microenvironment of HPB patients utilizing a novel, multiparameter flow cytometry and bioinformatics analysis. Our findings quantify the T cell phenotypic states in relation to checkpoint receptor expression. We demonstrate the presence of CD103⁺ tissue resident memory T cells (T_RM), CCR7⁺ central memory T cells, and CD57⁺ terminally differentiated effector cells across all HPB cancers, while the anti-tumor function was dampened by expression of multiple co-inhibitory checkpoint receptors. Terminally exhausted T cells lacking co-stimulatory receptors were more prevalent in PDA, whereas partially exhausted T cells expressing both co-inhibitory and co-stimulatory receptors were most prevalent in HCC, especially in early stage. HCC patients had significantly higher T_RM with a phenotype that could confer restored activation in response to immune checkpoint therapies. Further, we found a lack of robust alteration in T cell activation state or checkpoint expression in response to chemotherapy in PDA patients. These results support that HCC patients might benefit most from combined checkpoint therapies, whereas efforts other than cytotoxic chemotherapy will likely be necessary to increase overall T cell activation in CCA and PDA for future clinical development.

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 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

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 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)

Background Solid tumors comprise >90% of cancers. Nonsmall cell lung cancer (NSCLC), metastatic colorectal cancer (CRC), and pancreatic cancer are the leading causes of cancerrelated mortality (5-year overall survival: 26%, 15%, and 11%, respectively).1 Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical efficacy in hematologic malignancies.2,3 However, 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.4,5 To create a therapeutic safety window, Tmod CAR T-cell therapy utilizes dual-signaling receptors to create a robust logic gate capable of killing tumor cells, while leaving healthy cells intact.6,7 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 (figure 1).8,9 Human leukocyte antigen (HLA) loss of heterozygosity (LOH) offers a definitive tumor versus normal discriminator target for CAR T-cell therapy.10 The frequency of HLA LOH among advanced NSCLC, CRC, and pancreatic cancers in the Tempus real-world dataset is 16.3% with a range of 15.6%- 23.1%.11 LOH can be reliably detected using the Tempus xTOnco next-generation sequencing (NGS) assay.12,13 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: 1) To identify patients with somatic HLA LOH eligible for Tmod CAR T-cell therapy, and 2) Subsequent apheresis and manufacturing feasibility for the future EVEREST CEA or MSLN Tmod CAR T-cell studies. Methods BASECAMP-1 (NCT04981119) patient eligibility has 2 parts (figure 2): 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 xTOnco NGS testing; 2) If the tumor demonstrates HLAA* 02:01 LOH and the patient is eligible after screening, the patient will undergo apheresis. Banked T cells will be available for the autologous EVEREST Tmod CAR T-cell therapy interventional study to reduce waiting time at relapse. (Figure Presented)

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)