STAT5A promoter hypermethylation as a biomarker of immune checkpoint inhibitor response in squamous cell carcinoma in cfDNA liquid biopsies

Background: STAT5A is a downstream transcription factor in the JAK-STAT pathway that regulates immune- and tumor-related gene expression. Hypermethylation of the STAT5A promoter is linked to immune cell depletion and reduced antitumor immune activity in head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma (LSCC) (Liang et al. 2023). We hypothesized that STAT5A promoter hypermethylation (PM) detectable in cfDNA is a predictive biomarker of immune checkpoint inhibitor (ICPI) response in squamous cell carcinomas (SCC). Methods: Guardant360 Liquid (Guardant Health, Palo Alto, CA) was used to detect hypermethylated STAT5A promoter region, defined initially as 5kb upstream from the transcription start sites (TSS). Correlation between methylation and expression for PM regions was determined using The Cancer Genome Atlas (TCGA) data to identify loci where PM was associated with transcriptional silencing. Candidate loci were evaluated in a large cohort of cancer-free samples (n=32k) to ensure analytical specificity. PM status is distinguished relative to constitutively hypermethylated control regions, measuring methylation level relative to total cfDNA. Clinical outcomes analyses used the GuardantINFORM real-world clinico-genomics database. Results: STAT5A PM prevalence as measured by Guardant360 Liquid was concordant with published datasets across tumor types. The assay demonstrated high analytical specificity (negative percent agreement > 98%). STAT5A PM was associated with a shorter time to next treatment (TTNT) among ICPI-treated patients with SCC (n=815, 91% LSCC, 9% HNSCC, unadjusted log-rank p=0.01). Median TTNT for STAT5A PM+ patients (n=16) was 8.6 mo (95% CI, 4.6-10.4) versus 30.7 mo (95% CI, 25.6-38.1) for PM- patients (n=799). In a multivariable Cox proportional hazards model, adjusting for clinical covariates (age, gender, and line of therapy), PM+ remained independently associated with shorter TTNT (HR=2.10; p=0.03). When analyzed by cancer type, the association between STAT5A PM and TTNT was primarily driven by LSCC (HR=2.33; p=0.01). The limited number of PM+ HNSCC cases (n=3) precluded statistical significance in that subgroup. Conclusion: Non-invasive detection of STAT5A PM using Guardant360 Liquid is inversely correlated with real-world ICPI benefit in patients with LSCC, adding to a growing list of biomarkers demonstrated to have predictive significance in ICPI and broadening the role of methylation-based profiling in precision oncology.