Uncovering actionable genetic alterations and immune predictive biomarkers for anal squamous cell carcinomas in the era of immunotherapy: PD-L1 and beyond.

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3518 Background: Squamous cell carcinoma of the anal canal (SCAC) is a rare cancer with limited treatments. Immune checkpoint inhibitors (ICIs) use is routine for recurrent/refractory disease, but predictive biomarkers remain elusive. We analyzed the largest dataset of SCAC to date, focusing on PD-L1 expression. Methods: Next-generation sequencing of DNA (592 genes or WES) and RNA (WTS) was tested at Caris Life Sciences (Phoenix, AZ). PD-L1 was tested by immunohistochemistry (IHC) (SP142) and grouped as high (2+ and ≥5%), low (1-2 and 1-5%), and negative (0) CPS. dMMR/MSI-H was tested by IHC/NGS and tumor mutation burden (TMB)-High was defined as >10 mt/MB. RNA expression data was used to estimate the tumor microenvironment (TME) using QuantiSEQ. RNA signatures predictive of ICI response (interferon gamma [IFNγ]; T-cell inflamed signature [TIS]) were tested. X2/Fisher-Exact was used with significance shown as P-value adjusted for multiple comparisons (Q<0.05). Real-world overall survival (rwOS) was from insurance claims and calculated from tissue collection to last contact; time-on-treatment (TOT) was from start to finish of ICI. Results: Of 1242 SCAC samples, 64.3% (798) expressed PD-L1, 41.7% (518) showed high PD-L1 expression, 1.2% were dMMR/MSI-H and 14.5% were TMB-high. TMB-H trended higher in PD-L1 low vs PD-L1 negative (16.3% vs 10.2%, p=0.036). No trend was seen for dMMR/MSI-H. Mutations in PIK3CA (38.3% vs 21.7%) and CASP8(5.3% vs 0.3%) were significantly higher in PD-L1 high vs negative. Similar trends were seen in NF1 (2.4% vs 0.4%) and ARID1A (2.7% vs 0.6%). The opposite was noted for JAK2 (0% vs 2.6%), BAP1 (1.1% vs 3.6%) mutations, and FGFR3 amplifications (0% vs 2.2%). For PD-L1 high vs negative, infiltration of Tregs, M1 macrophages, neutrophils, CD8+ cells, and cancer-associated fibroblasts decreased with PD-L1 expression (fold change [FC]: 0.67-0.80, Q<0.05). Expression of immune-oncology (IO) markers IDO1, PDCD1, IFNG, CD274, HVACR2, CTLA4, LAG3, CD80, CD86 and PDCD1LG2 decreased with PD-L1 expression (FC:0.35-0.73, Q<0.05). Similarly, TIS and IFNg scores decreased with PD-L1 IHC (Q<0.05). In 316 SCAC patients treated with ICIs, PD-L1 high had longer TOT compared to PD-L1 negative (N=168 vs 80, 5.5m vs 2.9m, HR 0.758, 95% CI 0.579 to 0.992, p=0.044) while PD-L1 low (N=68) showed a TOT in between (3.2m) but not significantly different compared to high or negative. No differences in rwOS were observed. Conclusions: PD-L1 is expressed in over 50% of SCACs, while dMMR/MSI-H is rare. High PD-L1-expressing tumors have higher PIK3CAand CASP8 mutations and overexpress IO markers, are inflamed, and have prolonged treatment times with ICIs. This is the largest study to date reporting SCAC genomic profiles and identifies the utility of PD-L1 as a predictive biomarker of IO efficacy. This discovery needs to be confirmed in prospective trials.