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We report a sensitive PCR-based assay called Repetitive Element AneupLoidy Sequencing System (RealSeqS) that can detect aneuploidy in samples containing as little as 3 pg of DNA. Using a single primer pair, we amplified ∼350,000 amplicons distributed throughout the genome. Aneuploidy was detected in 49% of liquid biopsies from a total of 883 nonmetastatic, clinically detected cancers of the colorectum, esophagus, liver, lung, ovary, pancreas, breast, or stomach. Combining aneuploidy with somatic mutation detection and eight standard protein biomarkers yielded a median sensitivity of 80% in these eight cancer types, while only 1% of 812 healthy controls scored positive., Competing Interests: Competing interest statement: K.W.K., N.P., and B.V. are founders of, hold equity in, and are consultants to Thrive and Personal Genome Diagnostics. K.W.K. and N.P. are on the Board of Directors of Thrive. K.W.K. and B.V. are consultants to Sysmex, Eisai, and CAGE Pharma. B.V. is also a consultant to Nexus, and K.W.K., N.P., and B.V. are consultants to Neophore. C.B. is a consultant to Depuy-Synthes. C.T. is a paid consultant to Thrive and Bayer. C.D. is a consultant to Thrive. The companies named above as well as other companies have licensed previously described technologies related to the work described in this paper from Johns Hopkins University. C.D., J.D.C., R.K., C.B., K.W.K., N.P., and B.V. are inventors on some of these technologies. Licenses to these technologies are or will be associated with equity or royalty payments to the inventors as well as to Johns Hopkins University. Additional patent applications on the work described in this paper may be filed by Johns Hopkins University. The terms of all of these arrangements are being managed by Johns Hopkins University in accordance with its conflict of interest policies. N.R. is a cofounder and Chief Scientific Officer of Inivata.

Malignant peripheral nerve sheath tumors (MPNST) are the deadliest cancer that arises in individuals diagnosed with neurofibromatosis and account for nearly 5% of the 15,000 soft tissue sarcomas diagnosed in the United States each year. Comprised of neoplastic Schwann cells, primary risk factors for developing MPNST include existing plexiform neurofibromas (PN), prior radiotherapy treatment, and expansive germline mutations involving the entire NF1 gene and surrounding genes. PN develop in nearly 30-50% of patients with neurofibromatosis type 1 (NF1) and most often grow rapidly in the first decade of life. One of the most important aspects of clinical care for NF1 patients is monitoring PN for signs of malignant transformation to MPNST that occurs in 10-15% of patients. We perform aneuploidy analysis on ctDNA from 883 ostensibly healthy individuals and 28 patients with neurofibromas, including 7 patients with benign neurofibroma, 9 patients with PN and 12 patients with MPNST. Overall sensitivity for detecting MPNST using genome wide aneuploidy scoring was 33%, and analysis of sub-chromosomal copy number alterations (CNAs) improved sensitivity to 50% while retaining a high specificity of 97%. In addition, we performed mutation analysis on plasma cfDNA for a subset of patients and identified mutations in NF1, NF2, RB1, TP53BP2, and GOLGA2. Given the high throughput and relatively low sequencing coverage required by our assay, liquid biopsy represents a promising technology to identify incipient MPNST. [ABSTRACT FROM AUTHOR]