Abstract
Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.
Highlights
Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification
A total of 23 formalin-fixed paraffin-embedded samples from 23 patients were obtained from the tissue bank of the Pathology Department, Leiden University Medical Center and from European Institute of Oncology, Milan, according to the medical ethical guidelines described in the Code for Proper Secondary Use of Human Tissue established by the Dutch Federation of Medical Science and IEO ethical committee internal procedures, respectively
Small formalin-fixed paraffin-embedded (FFPE) tissue cores taken from representative tumor areas are used routinely in pathology
Summary
Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Background of normal diploid cells dilutes the signal associated with quantitative genomic features like copy number variations (CNVs), loss of heterozygosity (LOH) and simple homozygous/heterozygous status of a variant in a tumor cell subpopulation This strongly impairs their accurate detection by NGS, and limits CNV analysis to samples having relatively high tumor-content and high tumor-gene amplification levels. Sequencing artifacts, induced by DNA damages associated with FFPE preparation protocol[4,5] together with tumor heterogeneity[6] further exacerbate the technical difficulties of identifying the presence of true somatic alterations in a sensitive and specific way These challenges are relevant across a broad range of specialties, www.nature.com/scientificreports/.
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