Abstract 3372: Cell-free DNA (cfDNA) fragment length patterns of tumor- and blood-derived variants in participants with and without cancer
Authors: Hubbell Earl , Maddala Tara , Venn Oliver , Scott Eric , Tang Susan , Shenoy Archana , Aravanis Alex 
Published: August 2019

Previous studies on transplanted tissue or single cancers indicated that cfDNA variant fragment lengths reflect their respective source. The Circulating Cell-free Genome Atlas (NCT02889978) study provides an opportunity to examine cfDNA variant fragment lengths across tumor types and describe the nature of cfDNA variants derived from different sources.

Blood samples (N=1406) were evaluated from participants with (n=845) and without (n=561) cancer; cancer samples included 339 breast, 118 lung, 69 prostate, 45 colorectal, 27 uterine, 26 pancreas, 26 renal, 24 esophageal, 22 lymphoma, 19 head/neck, and 17 ovarian (113 remaining samples represented cancers with ≤15 samples each). cfDNA and genomic DNA from white blood cells (WBC) were subjected to a high-intensity targeted panel (507 genes, 60000X) with error-corrected sequencing; 533 samples also had matched tumor biopsy tissue subjected to whole-genome sequencing (30X).

Somatic single-nucleotide variants (SNVs; that passed noise filters) were identified and classified using the sequencing results into one of four categories: tumor biopsy-matched (TBM; present in cfDNA and biopsy), WBC-matched (WM; present in cfDNA and WBC), non-matched (NM; low probability [P&lt;0.01] of being WBC-derived), or ambiguous (AMB; unidentifiable source). Fragment lengths of reference and SNV alleles were recorded. A statistical model based on fragment lengths was built to predict the likelihood that an SNV belonged to a WBC-like source without using the WBC sequencing results.

A total of 21604 SNVs were identified. The proportion of SNVs from each category were: 4% TBM, 68% WM, 19% NM, and 8% AMB. The number of samples (non-mutually exclusive) that had each SNV category were 152 TBM, 1338 WM, 499 NM, and 761 AMB.

Across categories, the median (SD) length of fragments containing the reference allele was 167 (16.3). Median (SD) fragment lengths of TBM, WM, NM, and AMB were 156 (22.2), 169 (14.8), 158 (20.8), and 165 (17.8), respectively. AMB and WM median SNV fragment lengths were similar to that of the reference allele, suggesting that fragment length shifts are minimal in SNVs derived from clonal hematopoiesis (CH). Fragment lengths of TBM and NM SNVs were similar; further, most NM SNVs came from cfDNA samples in the cancer cohort, suggesting that NM SNVs may be tumor-derived. As expected in a population with a median (SD) age of 61 (12.2), most SNVs occurred in the WM category.

The prediction model distinguished TBM from WM SNVs with an AUC of 0.87. However, at a specificity of 98% (to match filtering based on WBC sequencing), false-negative rates were 35% (TBM) and 52% (NM).

Together, these data suggest that source prediction based on fragment length alone is less robust than source assignment using individual-matched WBC sequencing, highlighting the importance of accounting for CH-derived SNVs when using targeted cfDNA-based approaches for cancer detection.

Citation Format: Earl Hubbell, Tara Maddala, Oliver Venn, Eric Scott, Susan Tang, Archana Shenoy, Alex Aravanis. Cell-free DNA (cfDNA) fragment length patterns of tumor- and blood-derived variants in participants with and without cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3372.