Introduction: A novel non-invasive blood-based cell-free DNA (cfDNA) analysis incorporating genomic and epigenomic assessment has demonstrated high sensitivity and specificity in patients with newly diagnosed colorectal cancer (CRC) (Kim, et al. CCR, 2019). Using an improved epigenomic analysis, we tested a new cohort of individuals with either a negative colonoscopy for advanced neoplasia (CRC or advanced adenoma) or newly diagnosed early-stage CRC.

Methods: Whole blood samples were collected from 162 patients with a known diagnosis of CRC (pre-operative; 20 Stage I; 98 Stage II; 39 Stage II; 5 Stage IV), 38 self-declared cancer-free donors, and 205 individuals who were screen-negative for advanced neoplasia by colonoscopy. 5-8mL of plasma was isolated and total cfDNA was extracted and partitioned based on methylation level. Sequencing libraries were prepared and enriched using an integrated genomic and epigenomic cfDNA panel (Guardant Health, Redwood City, CA, USA). Sequencing results were used to analyze genomic, methylation, and fragmentomic signals. By using a thermodynamic model to approximate the physical binding process of methylation partitioning, modeling cfDNA fragmentation with increased resolution, and training on colonoscopy screen-negative samples, we improved the performance of the cfDNA epigenomic analysis. A training cohort of 117 colonoscopy screen-negative controls, 38 self-declared cancer-free controls, and 49 CRC patients (10 Stage I; 21 Stage II; 13 Stage III; 5 Stage IV) was used to train a linear model to combine these multimodal signals. The same cohort was used to establish the classification threshold prior to generating results for any of the validation samples.

Results: The assay performance was tested on a blinded held-out cohort of 113 CRC patients (10 Stage I; 77 Stage II; 26 Stage III) and 88 colonoscopy screen-negative controls. Median age was 66 years (39-86) for CRC patients (52% female) and 57 years (20-84) in the colonoscopy screen-negative cohort (73% female). Overall sensitivity for CRC detection was 90.3% (90% Stage I; 88% Stage II; 96% Stage III) and specificity was 96.6%. We define a per sample ‘signal-to-noise-ratio' (SNR) score as the number of standard deviations (SD) from the mean model score observed in the colonoscopy screen-negative cohort. In these terms, our cfDNA detection threshold is 3.8 SD above the mean. The median SNR observed per stage was 6.8 SD (1.2 - 8.6) for Stage I, 6.9 SD (-0.18 - 8.6) for Stage II, and 7.3 SD (2.4 - 8.6) for Stage III.

Conclusion: These results provide further support that an integrated genomic and epigenomic cfDNA assay consistently provides sufficient sensitivity and specificity for clinical detection of early-stage CRC. A prospective CRC screening study in a larger cohort of participants is needed to further validate assay performance.

Citation Format: Oscar Westesson, Haley Axelrod, Jason Dean, Yupeng He, Paul Sample, Elena Zotenko, Ruth McCole, Mohit Goel, Charbel Eid, Jessica Kurata, Yu Kong, Anna Hartwig, Matthew Snyder, Will Greenleaf, Victoria M. Raymond, Darya Chudova, Ariel Jaimovich, AmirAli Talasaz. Integrated genomic and epigenomic cell-free DNA (cfDNA) analysis for the detection of early-stage colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2316.