Cell-free DNA (cfDNA) mutations from clonal hematopoiesis: Implications for interpretation of liquid biopsy tests.

Abstract

11526 Background: A large fraction of cfDNA fragments are derived from hematopoietic sources. Somatic alterations in cfDNA can be tumor-derived but also could represent somatic changes associated with clonal hematopoiesis. We performed deep sequencing of both plasma cfDNA and matched white blood cell (WBC) genomic DNA (gDNA) to determine the contribution of clonal hematopoiesis to the variants observed in cfDNA. Four cohorts were investigated: metastatic breast (BC), non-small cell lung (NSCLC), castration-resistant prostate cancer (CRPC), and non-cancer participants (pts). Methods: Metastatic cancer pts with de novo or progressive disease were prospectively enrolled. Non-cancer pts were blood bank donors. Plasma cfDNA and matched WBC gDNA were sequenced using a targeted 508-gene panel (2 Mb) to > 60,000X raw depth. Variant calling used a novel pipeline that employed molecular barcoding for error suppression followed by de novo assembly and graph-based variant calling. Results: Of 151 metastatic cancer pts (48 BC, 49 NSCLC, 54 CRPC), median age was 64 (30-87) with 53% female and 33% treatment naive. Of 47 non-cancer pts, median age was 61 (20-78) with 51% female. Analysis of cfDNA identified 1072 variants (AF > 0.1%, > 2 mutant reads, passing bioinformatic quality filters) which were also detected in WBC gDNA as non-germline ( < 35% allele frequency [AF]) non-synonymous variants. For these cfDNA variants, AF ranged from 0.1-14.4% and correlated with AF in WBC gDNA (r2 = 0.47, p < 0.001). Mutated genes were consistent with clonal hematopoiesis, with the most frequently mutated genes being DNMT3A, TET2, PPM1D, and TP53 (215, 77, 45, and 36 variants, respectively). For both cancer and non-cancer pts (age > 45), median number of overlapping variants was 5 per pt (range 0-22). The number of WBC gDNA and cfDNA variants per individual was positively associated with age (p < 0.001) in both cancer and non-cancer pts (interaction p = 0.08). Conclusions: Somatic cfDNA variants are frequently derived from clonal hematopoiesis and increase with age. Accurate assessment of somatic alterations in cfDNA should account for this phenomenon to distinguish between tumor-derived and WBC-derived variants.