93P Circulating cell-free DNA fragmentation profiles during systemic therapy of advanced-stage non-small cell lung cancer patients

Circulating cell-free DNA (cfDNA) may be used for monitoring response to systemic treatment in patients with advanced carcinomas. Tumor-derived cfDNA is characterized by a shift of fragment sizes. The aim of the study was to evaluate longitudinally the changes in cfDNA fragmentation profile and correlate it with disease progression in non-small cell lung cancer (NSCLC) patients receiving anticancer systemic therapy. We recruited 14 patients aged 36 - 72 years at diagnosis with advanced stage IIIb/IV NSCLC and performance status 0-2 at diagnosis. All patients were initially tested on PD-L1 expression as well as on mutations in the EGFR and the ALK genes. Patients with positive PD-L1 expression (n=3) received immunotherapy pembrolizumab as the first-line therapy, those with mutations in the EGFR gene (n=3) received tyrosine kinase inhibitor - gefitinib, one ALK-positive patient received alectinib, while other patients received standard chemotherapy - Carboplatin/Etoposide (n=3) or Carboplatin/Paclitaxel (n=4). Consecutive plasma samples were collected at diagnosis, during systemic treatment and after termination of the therapy. cfDNA was extracted from 0.5 mL of plasma using magnetic-based MagMax cfDNA extraction kit, and quantified using Qubit HS dsDNA assay and ddPCR. Contamination with genomic DNA was determined using a B-cell-specific ddPCR assay. Fragment size distribution of cfDNA was determined by Agilent Bioanalyzer HS dsDNA assay. Concentrations of cfDNA measured by Qubit (∼14.3 ng/mL of plasma) showed good correlation to the absolute quantification determined by ddPCR (∼13.22 ng/mL of plasma). In a 38% of samples there was small contamination by peripheral blood cells gDNA. Besides expected fraction of cfDNA (168 bp mode) observed in all samples, we observed in 48% of samples an ultra-short fraction of cfDNA (50bp mode). Application of magnetic-based cfDNA extraction method allowed us to detect an ultra-short cfDNA fraction, previously unrecognized in lung cancer patients. Studies to correlate it to progression-free survival are ongoing.