Abstract 3573: Comprehensive non-invasive tumor sequencing: High fidelity sequencing of tumor-derived circulating cell-free DNA across 300 cancer patients
Authors: Mortimer Stefanie A. , Sebisanovic Dragan , Mei Gangwu , Schiller Benjamin , Siew Lai Mun , Zapanta Aubrey , Eltoukhy Helmy , Talasaz AmirAli 
Published: May 2015

Background: Analysis of circulating tumor nucleic acids by massively parallel DNA sequencing has created an exciting new tool for capturing an accurate, more complete and real-time picture of a patient's tumor profile throughout the body and the course of disease. However, the widespread use of this biomarker has been limited by the high-quality false positives and non-systematic distortion present in current NGS assays, especially where the tumor-derived fraction is low (&lt;1%). These limitations have heretofore impeded routine clinical use of circulating DNA in oncology.

Methods: Our differentiated sequencing assay, Digital Sequencing Technology, has enabled robust, ultra-sensitive and ultra-specific detection of rare genomic abnormalities in cell-free circulating DNA. Digital Sequencing relies on enabling single-molecule sequencing using current next-gen sequencers. Our assay is able to eliminate most of the errors and distortion introduced by these sequencers and produce near-perfect representations of all rare variants.

Results: We have shown that in sequencing 0.1% cancer cell line titrated samples across a broad 80kbp cancer panel, standard Illumina sequencing generates several high-quality false positives with allele frequencies of 0.05-5%, while our novel Digital Sequencing Technology results in high sensitivity and completely error-free detection of rare variants.

Furthermore, we have applied our technology to analyze rare tumor-derived cell-free DNA from more than 300 cancer patient plasma samples collected at multiple sites (various US and international locations) and across different cancer types. We investigated the concordance of tumor mutation profiles derived from circulating cell-free DNA with those derived from matched tumor biopsy samples in colorectal, breast, NSCLC, prostate and melanoma cancers across patients (Stage II-IV). We also studied the presence of de novo mutations (SNV and CNV) in these cancer patient plasma samples. In general, the range of tumor DNA in plasma was determined to be between 0.03%-85.9% in these studies. This comprehensive dataset illustrates the broad power of cell-free DNA analysis as a diagnostic biomarker in cancer patients.

Conclusion: The present work shows the strong potential clinical impact of Digital Sequencing in analysis of circulating cell-free tumor-derived nucleic acids, thereby allowing researchers and clinicians to comprehensively and non-invasively monitor the genetic dimension of cancer throughout the body.

Citation Format: Stefanie A. Mortimer, Dragan Sebisanovic, Gangwu Mei, Benjamin Schiller, Lai Mun Siew, Aubrey Zapanta, Helmy Eltoukhy, AmirAli Talasaz. Comprehensive non-invasive tumor sequencing: High fidelity sequencing of tumor-derived circulating cell-free DNA across 300 cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3573. doi:10.1158/1538-7445.AM2014-3573