Novel, Ultra Deep Next-Generation Sequencing Of Plasma Cell-Free Dna From Patients With Advanced Cancers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PABackground: Plasma cell-free (cf) DNA from cancer patients offers an easily obtainable and repeatedly applicable source of DNA for mutation analysis, which provides attractive alternative to tumor tissue testing. Novel ultrasensitive multiplex technologies using small amounts of DNA are needed for further implementation of plasma cfDNA testing in personalized therapy.Methods: We have developed an ultra-deep next-generation sequencing method for somatic BRAF mutation detection in 5 ng of cfDNA with high sensitivity and specificity, which was expanded to include 30 (full exon tiling: ERBB2, TP53, FGFR1, PTEN, MET, KRAS, ALK, CTNNB1, KIT, NRAS, BRAF, PIK3CA, EGFR, MAP2K1, PIK3R1, DDR2, NF1, MITF, AKT3, PHLPP1, HGF, HOXD8, MEK2, hotspot containing exons: AKT1, HRAS, GNA11, GNAQ, RAC1; copy number variations: ERBB2, FGFR1, MET, BRAF, EGFR, MITF, HGF, HOXD8; gene fusions: RET1, ROS, and ALK) and then 58 common cancer related genes (previous panel + APC, AR, FGFR2, FGFR3, FGFR-4, SMO, FBXW7, NOTCH1, CDKN2B, IDH1, IDH2, GNAS, KDR, STK11, ESR1, VHL, ATM, CDH1, TRKA, TRKB, TRKC, TSC1, TSC2, Fltr3, FOXL2, CDKN2A, PDGFRA, IGF1R). Each cfDNA fragment was uniquely barcoded and amplified prior to Illumina target enrichment workflow, followed by ultra-deep sequencing (u003e10,000X). Proprietary data processing and analysis tools were developed to enable sensitive detection of rare mutant molecules over high wild-type background (detection of 1 in 1,000 - 10,000 molecules). Results were compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care from a CLIA-certified laboratory.Results: Initially, cfDNA was extracted from plasma samples of 24 patients with advanced cancers (melanoma, n = 9; colorectal, n = 5; non-small cell lung, n = 2; papillary thyroid, n = 2; other cancers, n = 6) and 5ng were used for BRAF mutation analysis. BRAF mutations were detected in 71% (17/24) of plasma samples and in 88% (21/24) of archival tumor samples, resulting in concordance in 87% (20/24) of cases. Subsequently, we extracted plasma samples from additional 13 patients with advanced cancers (colorectal, n = 4; melanoma, n = 3; other cancers, n = 6) and tested 14-30ng of cfDNA for the presence of alterations in 30 to 58 cancer related genes. Both ultra-deep sequencing of plasma cfDNA and standard tissue sequencing detected median of 2 alterations per patient and dominant oncogenic alterations previously detected in the tumor tissue were also found in plasma cfDNA. Additional 80 samples are being analyzed and results will be presented at the meeting.Conclusions: Detecting common oncogenic alterations using ultra-deep sequencing of plasma cfDNA is feasible with an acceptable level of concordance with testing of tumor tissue and should be further investigated for testing in patients with advanced cancer.Citation Format: Filip Janku, Helen J. Huang, Nishma M. Ramzanali, David S. Hong, Daniel D. Karp, Xuyu Cai, Yue Zhao, Neeraj Salathia, Jill Waters, Li Liu, Rick Klausner, Funda Meric-Bernstam, Jian-Bing Fan. Novel, ultra deep next-generation sequencing of plasma cell-free DNA from patients with advanced cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2414. doi:10.1158/1538-7445.AM2015-2414