Digital Sorting of Pure Cell Populations Enables Unambiguous Genetic Analysis of Heterogeneous Formalin-Fixed Paraffin-Embedded Tumors by Next Generation Sequencing.

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.

Development of a tetraplex PCR assay for CYP2D6 genotyping in degraded DNA samples.

CYP2D6 polymorphism analysis is gaining increasing interest in forensic pharmacogenetics. Nevertheless, DNA recovered from forensic samples could be of poor quality and not suitable for long polymerase chain reaction required to type CYP2D6 gene prior to SNaPshot minisequencing analysis performed to define alleles with different enzymatic activity. We developed and validated following the guidelines of the Scientific Working Group on DNA Analysis Methods a tetraplex PCR yielding four amplicons of 597, 803, 1142, and 1659 bp encompassing the entire CYP2D6 gene to analyze eleven SNP positions by SNaPshot minisequencing. Concordance, sensitivity, and specificity were assessed. The method, applied to thirty-two forensic samples failed to amplify with long PCR, allowed the amplification of CYP2D6 gene in 62.5% of degraded samples. The new tetraplex PCR appears a suitable method for CYP2D6 analysis in forensic pharmacogenetics.

CYP2D6 genotyping in natives and immigrants from the Emilia-Romagna Region (Italy).

Pharmacogenetic testing of drug metabolizing enzyme polymorphisms provides an important tool to improve prescribing decisions, avoiding therapeutic failure and adverse drug reactions. Cytochrome P450 2D6 isoform plays an important role in the metabolism of about 20%-25% of widely used clinical drugs. Interethnic differences in allele frequency distribution of the CYP2D6 gene are well established, but interethnic admixture, introducing variations in population ancestry and resulting in distinct levels of population structure, should be acknowledged in pharmacogenomic studies to avoid inappropriate extrapolation of CYP2D6 data. The aim of the present research was to characterize CYP2D6 polymorphism in a random sample of 122 natives and 175 immigrants from Africa, Asia, and South America living in the Emilia-Romagna region (Italy), considering the present scenario of immigration and back migration events, which is a source of admixture. The results are today consistent with the known interethnic genetic variation, but the observed significant divergence between natives and Africans or South-East Asians predicts that admixture will reshape the population structure and the native metabolic ratio curve requiring, for drug prescription and pharmacogenetics studies, an interdisciplinary approach applied in an appropriate biogeographical and anthropological frame.