Kirtane, Kedar

209 Background: Metastatic colorectal (CRC), pancreatic (PANC), and gastroesophageal cancers are the leading causes of GI cancer–related mortality (5-y survival: 15%, 3%, and 5%-6%, respectively) (ACS 2022). HLA LOH is a recurrent mechanism of immune escape observed in 15%-20% of GI cancers (Hecht R., ASCO GI 2022). The Tmod platform is a logic-gated chimeric antigen receptor (CAR) T-cell modular system, comprising a carcinoembryonic antigen (CEA)- or mesothelin (MSLN)-targeting CAR activator and a separate HLA-A*02-targeting blocker receptor. Both in vitro/in vivo, Tmod CAR T therapy kills cells with HLA-A*02 LOH (tumor) without harming cells with retained HLA-A*02 expression (normal). However, HLA-A*02 LOH can only be therapeutically exploited if patients are identifiable through a feasible and timely clinical workflow. Methods: We established a biobanking protocol (BASECAMP-1, NCT04981119) to determine whether HLA-A*02 LOH patients can be prospectively identified. Patients with CRC, PANC, or non-small cell lung cancer (NSCLC), and a high risk for incurable relapse, were screened first using a standard HLA assay. Heterozygous HLA-A*02 positive tumor samples were then assessed for LOH using a bioinformatic algorithm applied via the Tempus xT platform. Results: As of Sep 1, 2022, 83 patients were consented at 4 institutions. HLA status was obtained from 70 patients and 28 were identified as HLA-A*02:01 heterozygous (40%; expected frequency based on USA NMDP data, 27.6%). LOH results were available for 16 patients; 4 LOH-positive patients were identified (25%, 2 PANC, 2 NSCLC). The LOH assay sensitivity declines below a tumor purity of 40% (Hecht R., ASCO GI 2022). Six patients had a tumor purity of 20% (all with PANC, a tumor known for high stromal content), limiting possible LOH detection. The impact of tumor purity on LOH sensitivity was highlighted in a patient with a low initial sample tumor purity (30%) that resulted in a 41% probability of HLA-A*02:01 LOH (below positive threshold). A second sample with a higher tumor purity (70%), obtained from formalin-fixed, paraffin-embedded sections, resulted in a 92% probability of HLA-A*02:01 LOH (positive). Conclusions: BASECAMP-1 prospective identification of HLA-A*02 LOH is feasible in the real-world setting. The frequencies of the HLA-A*02 allele and of HLA-A*02 LOH in this cohort mirrored expected population frequencies. LOH results can be obtained within a clinically feasible workflow and timeframe, although samples with a < 40% tumor purity have a reduced sensitivity for LOH detection, an issue recurrently observed in patients with PANC. The BASECAMP-1 strategy enables prospective identification of appropriate patients for future therapeutic clinical trials using Tmod CEA and MSLN logic-gated CAR T cells. Clinical trial information: NCT04981119 .

Head and neck squamous cell cancers (HNSCCs) represent a diverse group of tumors emerging within different mucosal surfaces of the oral cavity, nasopharynx, oropharynx, larynx, and hypopharynx. HNSCCs share common clinical risk factors and genomic features, including smoking, alcohol, age, male sex, aneuploidy, and TP53 mutations. Viral initiating and contributing events are increasingly recognized in HNSCCs. While both Epstein–Barr Virus (EBV) and human papilloma virus (HPV) are observed, EBV is more frequently associated with nasopharyngeal cancers whereas HPV is associated with oropharyngeal cancers. HNSCCs are associated with high tumor mutational burden and loss of tumor suppressor gene function, especially in TP53 and X-linked genes. Multiple lines of evidence suggest that HNSCCs are subject to immunologic surveillance and immune-induced evolutionary pressure that correlate with negative clinical outcomes. This review will discuss genomic mechanisms related to immune-mediated pressures and propose prognostic and therapeutic implications of detectable immune escape mechanisms that drive tumorigenesis and disease progression.

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.

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]