Biomarkers associated with recurrence after salvage surgery for localized anal cancer (AC).

9 Background: While most patients with localized AC are cured by chemoradiation (cRT), some recur and require salvage surgery (SS). No adjuvant therapies are effective after SS. We profiled cRT-refractory AC tumors from SS to identify biomarkers linked to recurrence. Methods: Using an IRB-approved protocol, patients with localized, cRT-refractory AC were classified as recurrent (R; N=19) or non-recurrent (NR; N=10) according to outcome after SS. Digital spatial profiling whole transcriptome analysis (GeoMx) on FFPE AC evaluated expression of 18000+ genes on 40 regions of interest (ROI) for R vs 25 ROIs for NR AC cores, separated into “tumor” (panCK+) and “tumor microenvironment” (TME; panCK-). log2 transformed RNA counts were compared for expression between ROIs of R and NR AC (limma). For GSEA hallmark pathway analysis, normalized enrichment scores (NES) were calculated using genes with differential gene expression. Expression of immune biomarkers using multiplex immunofluorescence (mIF) were quantified (N/mm2) and compared with a student’s t-test. Results: With a median follow up of 26.6 months, median OS (29.1 months vs NR; p< .001) was shorter for R AC. For NR AC, higher expression of genes associated with immune activation (p-adj< .0001 for all) - B2M (fold change (FC) 33.0), HLA-DPA1 (FC 30.1), and HLA-DPB1 (FC 15.0) – and higher NES for “interferon alpha response” (NES 3.2) and “interferon gamma response” (NES 3.1) (FDR < .05 for both) were observed. R AC had higher expression of genes (p-adj < .0001 for all) regulating TGF-beta signaling – LY6K (FC 10.3) and BCAR3 (FC 3.6), and higher “EMT” (NES 2.1) and “TGF-beta” (NES 1.6) GSEA signatures (FDR< .05 for both). On mIF, greater CD3+CD8+ T cells were observed in tumor (148 vs 42 N/mm2; p=.02) and TME (8.2 vs 2.8 N/mm2; p=.008) for NR AC. Increased Thy1+ cancer-associated fibroblasts were noted in tumor (4.2 vs 1.2 N/mm2; p=.04) and in TME (149 vs 69 N/mm2; p=.04) for R AC. Conclusions: Signatures of immune activation were observed with gene expression and mIF in AC patients cured by SS. R AC had higher immune suppressing profiles. These data support developing novel therapeutics that promote immune activation in trials of patients with cRT-refractory AC at high risk for recurrence after SS.