Abstract B027: Combining total cell-free DNA (cfDNA) and circulating tumor (ctDNA) to enhance the clinical sensitivity of ddPCR assays to detect minimal residual disease (MRD) in stage IIIB/C/IV melanoma patients on adjuvant immunotherapy in CheckMat

Abstract Background: In patients (pts) with resected, stage III/IV melanoma receiving adjuvant immunotherapy, pre-treatment (pre-Tx) and on-treatment (on-Tx) ctDNA measurements are associated with relapse free survival; however, clinical adoption has been hindered by suboptimal assay sensitivity. Methods: We used analytically validated, mutation-specific ddPCR assays to measure ctDNA and cfDNA in pre- and on-Tx plasma samples from pts with resected stage IIIB/C/IV melanoma enrolled in CheckMate 238 (NCT02388906) receiving either adjuvant Nivolumab (NIVO) or Ipilimumab (IPI). Assay choice was personalized based on detection of one of the 7 melanoma hot-spot mutations in pt tumors: BRAF V600E/K, NRAS Q61R/L/K, or TERT promotor C228T or C250T. We evaluated associations between ctDNA detection, or high cfDNA based on cutpoint analysis (>6,000 copies/ml of plasma) and recurrence-free survival (RFS), pre-Tx tumor biomarkers including tumor cell PD-L1 expression, IFNg gene signature, CD8 infiltration, and tumor mutational burden. Survival analysis was performed using univariable and multivariable Kaplan-Meier and Cox regression models. Results: Assay mutations were identified in 87% of resected tumors. In the pre-Tx samples, ctDNA alone was detected in 94/753 (12%) pts. The detection rate increased to 20% when assay positivity was defined as either detectable ctDNA or high cfDNA. The sensitivity of ctDNA detection to predict recurrence ≤6 months(m) from pre-Tx samples increased from 25% (ctDNA alone) to 33% when high cfDNA was added to detectable ctDNA; specificity decreased to 84% from 91% (ctDNA alone). The patient subgroup defined by the combined metric of ctDNA+ or high cfDNA in pre- Tx samples was significantly enriched with higher stage of disease, elevated LDH, tumor cell PD-L1 expression < 5%, and tumor IFNg-RNA signature < median. Pts with detectable pre-Tx ctDNA or high cfDNA was associated with shorter RFS in both NIVO (median RFS 20.5 m [95%CI, 2.88, 59.15]) and IPI (median RFS 7.75 m [95%CI, 2.76, 24.08]) arms compared to those with undetectable ctDNA and low cfDNA (NIVO (median RFS 47.95 m [95%CI, 11.09, 59.89]) and IPI (median RFS 24.15 m [95%CI, 5.78, 58.38])). In a multivariable model that included sex, age, treatment, and IFNg gene signature, ctDNA+ or cfDNA high was an independent predictor of shorter RFS [HR 1.624 (95%CI, 1.275, 2.068), p<0.001]. When combining pre-Tx and on-Tx measurements at week 3 (w3) and w7, the assay performance generally increased compared to pre-Tx alone. For example, pre-Tx assay positivity had a 38% positive predictive value (PPV) and 81% negative predictive value (NPV) to predict recurrence at <6 m. In pts who were undetectable at pre-Tx but became positive at w3 and remained positive at w7, PPV increased to 62% and NPV increased to 90%. Conclusions: Incorporating quantitative measurements of cfDNA with ctDNA detection in the evaluation of pre-Tx and on- Tx plasma samples improves the clinical performance of mutation-specific ddPCR assays to detect MRD in patients with resected stage III and stage IV melanoma. Citation Format: Mahrukh M Syeda, Jennifer M Wiggins, Josephine Alegun, Saim Ali, Soutrik Mandal, Tracy (Hao) Tang, Keyur Desai, Sonia Dolfi, Daniel J Tenney, Paolo A Ascierto, James Larkin, Michele Del Vecchio, Jeffrey S Weber, David Polsky. Combining total cell-free DNA (cfDNA) and circulating tumor (ctDNA) to enhance the clinical sensitivity of ddPCR assays to detect minimal residual disease (MRD) in stage IIIB/C/IV melanoma patients on adjuvant immunotherapy in CheckMate 238 [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B027.