The role of dynamic monitoring of plasma cell-free DNA methylation in predicting pathological response in resectable stage IIB-IIIB non-small cell lung cancer: biomarker analyses from a prospective phase II trial.

BACKGROUND: Neoadjuvant chemoimmunotherapy does not benefit all non-small cell lung cancer (NSCLC) patients, and reliable biomarkers are urgently needed. We conducted this prospective phase II trial of neoadjuvant chemoimmunotherapy to explore the role of cell-free DNA (cfDNA) features in pathological response assessment. METHODS: Totally, 100 patients with stage IIB-IIIB NSCLC were enrolled and treated with neoadjuvant toripalimab plus chemotherapy for at least 2 cycles. Targeted methylation panel sequencing and whole methylome sequencing were conducted on 195 cfDNA samples collected from 60 patients before each treatment cycle (C0, C1) and before surgery (BS), with subsequent calculations of methylation fragment ratio (MFR) and chromosome aneuploid of featured fragment (CAFF) scores, respectively. The correlations between MFR or CAFF and pathological response were evaluated. RESULTS: Finally, 83 patients underwent surgery, and 54 (65.1%) patients achieved major pathological response (MPR), including 38 (45.8%) with complete pathological response (pCR). The median MFR and CAFF scores in both the MPR and non-MPR groups significantly decreased after the first cycle, and the MPR group maintained low levels before surgery (P < 0.001). According to pre-defined cut-off values, the MFR and CAFF scores were recategorized as low or high status. Patients with low MFR status at BS (74.5% vs. 11.1%, P < 0.001) or low CAFF status at C1 (73.9% vs. 36.4%, P = 0.031) and BS (76.2% vs. 38.9%, P = 0.008) were more likely to achieve MPR than those with high status. Three dynamic patterns were identified: C0 low, C0 high/C1 low, and C0 high/C1 high. These patterns were further divided by BS low or high status, which indicated distinctive MPR rate (C0 low: BS low vs. high 78.9% vs 0%; C0 high/C1 low: BS low vs. high 73.9% vs. 25%; C0 high/C1 high: BS low vs. high 83.3% vs. 0%). An integrative model was constructed by incorporating immune parameters (PD-L1 and CD8 + CD28- T lymphocytes) and cfDNA features (MFR and CAFF) at C1 and BS, achieving an AUC of 0.86 (95% CI 0.80-0.92). CONCLUSIONS: Dynamic monitoring of cfDNA methylation has potential to predict pathological response of neoadjuvant chemoimmunotherapy in NSCLC. TRIAL REGISTRATION: RENAISSANCE study, NCT04606303, initiated on October 27, 2020.