Validation of an optimized tissue-agnostic genome-wide methylome enrichment assay to predict clinical outcomes in patients treated with pembrolizumab.

Abstract

2545 Background: Recent work from the INSPIRE study (PMID38393391) suggests that kinetics of cell-free DNA (cfDNA) methylation profiles reflect immunotherapy treatment response in solid tumors. Here we provide validation data of a tissue-agnostic, genome-wide methylation enrichment assay based on cell free methylated DNA immunoprecipitation and high throughput sequencing (cfMeDIP-seq) designed for clinical use, to determine response to immunotherapy. Methods: This study utilizes samples and clinical data from the INSPIRE study, a single-institution investigator-initiated phase II study of pembrolizumab in multiple solid tumors given every 3 weeks (NCT02644369). A prior published analysis of cfMeDIP used TCGA to develop a classifier and demonstrated an association of response to immunotherapy. In contrast, in this analysis, a novel quantitative and highly specific measurement of ctDNA was estimated using a generative machine learning model trained on differentially methylated regions identified from a large cfMeDIP methylome atlas from individuals with and without cancer. In a blinded validation analysis, Firth’s logistic regressions were used to test differences in objective response (ORR) and clinical benefit rate (CBR) defined as complete or partial response or stable disease > / = 6 cycles between patients with a decrease in ctDNA from baseline to cycle 3 of treatment, and those with an increase in ctDNA. Sensitivity for no objective response, specificity for objective response, and positive and negative predictive values (PPV and NPV) were summarized. Cox regressions and log-rank tests were used to evaluate differences in progression-free survival (PFS) and overall survival (OS) between the two groups. Results: The analysis included 64 unique patients with a median follow up of 18.43 months (a total of 128 samples), including head & neck (n = 9), triple negative breast (n = 10), ovarian (n = 11), melanoma (n = 7), and other mixed solid tumor types (n = 27). A decrease in ctDNA was associated with significantly better objective response than an increase [odds ratio (OR) 33.89 (4.07, 44426.47), p = 0.0001], 58% sensitivity, 100% specificity, 100% PPV and 35% NPV. Significantly better CBR [OR 10.17 (2.74, 55.74), p = 0.0002] was also observed. A decrease in ctDNA was associated with significantly better PFS [hazard ratios (HR) 0.28 (0.15, 0.49) p < 0.0001] and OS [HR 0.42 (0.24, 0.76) p < 0.003]. Conclusions: A clinical tissue-agnostic, genome-wide methylome enrichment approach using cfMeDIP-seq accurately predicts clinical outcomes in patients treated with pembrolizumab in multiple advanced solid tumors. This test provides relative quantification of methylated ctDNA to predict response to immmunotherapy and does not require tumor tissue. This analysis highlights potential generalizability across tumor types in response monitoring. Clinical trial information: NCT02644369 .