In:
Cancer Research, American Association for Cancer Research (AACR), Vol. 77, No. 13_Supplement ( 2017-07-01), p. 2737-2737
Abstract:
Background: Circulating tumor DNA (ctDNA) is rapidly emerging as a viable alternative to tissue molecular diagnostics for the detection of actionable oncogenic mutations. In non-small cell lung cancer, identifying the emergence of EGFR T790M in patients receiving first line EGFR tyrosine-kinase inhibitors (TKIs) is a clinical need. Obtaining a tissue biopsy to determine T790M status is associated with significant cost and patient morbidity. In a retrospective analysis of the TIGER-X trial, patients had similar therapeutic response to a 3rd generation TKI, independent of the sample type positive for T790M; tissue, plasma, or urine. This indicates that non-invasive urine liquid biopsy is a viable diagnostic option (Wakelee et al, ASCO, 2016). We report on the expansion of single mutation assays to a multiplex-panel of clinically actionable mutations having analytical properties with similar ultra-sensitive detection. Methods: We previously developed two next generation sequencing methodologies with high sensitivity and specificity for ctDNA detection: 1) A series of individual ultrasensitive ctDNA hotspot assays capable of detecting ultra-short DNA fragments (31-45 base pairs) and a 0.01% lower limit of detection (LLoD). The short sized amplicons coupled with mutant allele enrichment enabled high clinical sensitivity for plasma and urine ctDNA (Reckamp et al, J Thorac Oncol., 2016) 2) A multiplex panel using sequence-specific synchronous coefficient of drag alteration (SCODA) technology that enriches for mutant ctDNA to obtain a LL0D of 0.001%-0.03%, (Pel et al, PNAS, 2009; Kidess et al, Oncotarget, 2015). By combining a multiplex of short amplicons with SCODA enrichment capabilities, we developed an assay enriched for over 200 clinically actionable driver and resistance mutations within 7 oncogenes. Results: An iterative process was used to design and optimize primer and enrichment probe length and placement to produce a panel where mean coverage across hotspot regions was within 2 to 3-fold (read uniformity) and greater than 1,000-fold enrichment of mutant DNA prior to sequencing. Technical validation was performed utilizing in-house and commercially available model systems for plasma and urine demonstrating mutation detection from inputs ranging from hundreds to fewer than 10 copies and LLoD as low as 0.001%. Clinical performance evaluation of the newly developed multiplex assay is ongoing using urine and plasma ctDNA from patients with advanced cancer. Conclusions: High sensitivity is critical when identifying targetable driver mutations as well as emergence of resistance mutations which can inform therapeutic decision making. By combining short amplicon and mutation enrichment technology, we developed an ultra-sensitive ctDNA assay for use in sample types with highly degraded and fragmented DNA. Citation Format: Karena A. Kosco, Matthew Wiggin, Peter J. Croucher, David Broemeling, Shiloh Guerrero, Laura Mai, Kurt Krummel, Victoria M. Raymond, Mark G. Erlander, Andre Marziali, Karsten Schmidt. Development of a clinically actionable, ultra-sensitive multi-oncogene panel compatible with ctDNA testing in urine and plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2737. doi:10.1158/1538-7445.AM2017-2737
Type of Medium:
Online Resource
ISSN:
0008-5472
,
1538-7445
DOI:
10.1158/1538-7445.AM2017-2737
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2017
detail.hit.zdb_id:
2036785-5
detail.hit.zdb_id:
1432-1
detail.hit.zdb_id:
410466-3
Permalink
