Lau, Denise

PURPOSE Biliary tract cancers (BTCs) are aggressive cancers that carry a poor prognosis. An enhanced understanding of the immune landscape of anatomically and molecularly defined subsets of BTC may improve patient selection for immunotherapy and inform immune-based combination treatment strategies. METHODS We analyzed deidentified clinical, genomic, and transcriptomic data from the Tempus database to determine the mutational frequency and mutational clustering across the three major BTC subtypes (intrahepatic cholangiocarcinoma [IHC], extrahepatic cholangiocarcinoma, and gallbladder cancer). We subsequently determined the relationship between specific molecular alterations and anatomical subsets and features of the BTC immune microenvironment. RESULTS We analyzed 454 samples of BTC, of which the most commonly detected alterations were TP53 (42.5%), CDKN2A (23.4%), ARID1A (19.6%), BAP1 (15.5%), KRAS (15%), CDKN2B (14.2%), PBRM1 (11.7%), IDH1 (11.7%), TERT (8.4%), KMT2C (10.4%) and LRP1B (8.4%), and FGFR2 fusions (8.7%). Potentially actionable molecular alterations were identified in 30.5% of BTCs including 39.1% of IHC. Integrative cluster analysis revealed four distinct molecular clusters, with cluster 4 predominately associated with FGFR2 rearrangements and BAP1 mutations in IHC. Immune-related biomarkers indicative of an inflamed tumor-immune microenvironment were elevated in gallbladder cancers and in cluster 1, which was enriched for TP53, KRAS, and ATM mutations. Multiple common driver genes, including TP53, FGFR2, IDH1, TERT, BRAF, and BAP1, were individually associated with unique BTC immune microenvironments. CONCLUSION BTC subtypes exhibit diverse DNA alterations, RNA inflammatory signatures, and immune biomarkers. The association between specific BTC anatomical subsets, molecular alterations, and immunophenotypes highlights new opportunities for therapeutic development.

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]

4023 Background: Biliary tract cancers (BTC) are increasingly subtyped by molecular alterations, but little is known about the relationship between gain-of-function mutations and the RNA transcript expression of immune-related pathways. Methods: A sample of retrospective, clinicogenomic and transcriptomic data from de-identified records of patients with BTC in the Tempus database was selected. We then investigated the relationship between the mutational landscape and immune-related RNA signatures of different anatomic and genomic BTC subtypes. Results: The cohort included 455 samples of intrahepatic bile duct (IH) (n=267), gallbladder (GB) (n=153), and extrahepatic bile duct (EH) (n=35) cancer subtypes. Across all subtypes, we detected alterations in TP53 (43.8%), ARID1A (19.8%), KMT2C (18.2%), BAP1 (14.6%), KRAS (12.7%), TERT (12.0%), IDH1 (11.4%), KMT2D (11.0%), LRP1B (11.0%), and PBRM1 (10.7%), along with FGFR2 fusions (2.6%). Potentially actionable biomarkers ( FGFR2 and NTRK1-3 fusions, IDH1 and BRAFV600E mutations, tumor mutational burden [TMB]>10, HER2 expression, and/or microsatellite instability) were identified in 21.1% of all BTC and 28.6% of IH samples. Mutually exclusive alterations observed between subtypes were TP53 & BAP1, KRAS & BAP1, TP53 & IDH1, KRAS & IDH1, and SMAD4 & BAP1 ( P < 0.001 for all). GB was more inflamed based on RNA signatures and classical immune biomarkers, including PD-L1 and TMB. RNA signature analyses revealed a higher expression of immune-related pathways in GB than IH ( P = 0.001) with no differences in comparison with EH. PD-L1 expression and continuous TMB were elevated in GB versus the other anatomical subtypes. Significant associations were noted between particular genetic mutations and immune profiling features (table). Conclusions: BTC subtypes are diverse in DNA alterations, RNA inflammatory signatures, and immune markers. Notably, potentially actionable biomarkers were identified in a sizable portion of the cohort and varied significantly between subtypes. These results provide guidance for targeted therapy development and support the use of multimodal immune profiling for BTC. For example, GB-specific clinical trials may be considered due to the relative increase in immune-related biomarkers observed in GB and the historically limited success of BTC trials.[Table: see text]