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Targeted next-generation sequencing is becoming increasingly common as a clinical diagnostic and prognostic test for patient- and tumor-specific genetic profiles as well as to optimally select targeted therapies. Here, we describe a custom-developed, next-generation sequencing test for detecting single-nucleotide variants (SNVs) and short insertions and deletions (indels) in 93 genes related to gastrointestinal cancer from routine formalin-fixed, paraffin-embedded clinical specimens. We implemented a validation strategy, based on the College of American Pathologists requirements, using reference DNA mixtures from cell lines with known genetic variants, which model a broad range of allele frequencies. Test sensitivity achieved >99% for both SNVs and indels, with allele frequencies >10%, with high specificity (97.4% for SNVs and 93.6% for indels). We further confirmed test accuracies using primary formalin-fixed, paraffin-embedded colorectal cancer specimens characterized by alternative and conventional clinical diagnostic technologies. Robust performance was observed on the formalin-fixed, paraffin-embedded specimens: sensitivity was 97.2% and specificity was 99.2%. We also observed high intrarun and inter-run reproducibility, as well as a low cross-contamination rate. Overall assessment using cell line samples and formalin-fixed, paraffin-embedded samples showed that our custom next-generation sequencing assay has consistent detection sensitivity down to 10% variant frequency.

Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel–Lindau (VHL) tumor suppressor. Roles for noncoding cis-regulatory aberrations in ccRCC tumorigenesis, however, remain unclear. Analyzing 10 primary tumor/normal pairs and 9 cell lines across 79 chromatin profiles, we observed pervasive enhancer malfunction in ccRCC, with cognate enhancer-target genes associated with tissue-specific aspects of malignancy. Superenhancer profiling identified ZNF395 as a ccRCC-specific and VHL-regulated master regulator whose depletion causes near-complete tumor elimination in vitro and in vivo. VHL loss predominantly drives enhancer/superenhancer deregulation more so than promoters, with acquisition of active enhancer marks (H3K27ac, H3K4me1) near ccRCC hallmark genes. Mechanistically, VHL loss stabilizes HIF2α–HIF1β heterodimer binding at enhancers, subsequently recruiting histone acetyltransferase p300 without overtly affecting preexisting promoter–enhancer interactions. Subtype-specific driver mutations such as VHL may thus propagate unique pathogenic dependencies in ccRCC by modulating epigenomic landscapes and cancer gene expression. Significance: Comprehensive epigenomic profiling of ccRCC establishes a compendium of somatically altered cis-regulatory elements, uncovering new potential targets including ZNF395, a ccRCC master regulator. Loss of VHL, a ccRCC signature event, causes pervasive enhancer malfunction, with binding of enhancer-centric HIF2α and recruitment of histone acetyltransferase p300 at preexisting lineage-specific promoter–enhancer complexes. Cancer Discov; 7(11); 1284–305. ©2017 AACR. See related commentary by Ricketts and Linehan, p. 1221. This article is highlighted in the In This Issue feature, p. 1201

Objective This study aimed to provide an individualized assessment of fetal trisomy 21 and trisomy 18 status for twin pregnancies by maternal plasma DNA sequencing. Method Massively parallel sequencing was performed on the plasma/serum DNA libraries of eight twin pregnancies and 11 singleton pregnancies. The apparent fractional fetal DNA concentrations between genomic regions were assessed to determine the zygosities of the twin pregnancies and to calculate the fetal DNA concentrations of each individual member of dizygotic twin pairs. Z-scores were determined for the detection of trisomy 18 and trisomy 21. Results Circulating DNA sequencing showed elevated chromosome 21 representation in one set of twins and elevated chromosome 18 representation in another pair of twins. Apparent fractional fetal DNA concentration analysis revealed both sets of twins to be dizygotic. The fractional fetal DNA concentrations for each individual fetus of the dizygotic twin pregnancies were determined. Incorporating the information about the fetal DNA fraction, we ascertained that each fetus contributed adequate amounts of DNA into the maternal circulation for the aneuploidy test result to be interpreted with confidence. Conclusion Noninvasive prenatal assessment of fetal chromosomal aneuploidy for twin pregnancies can be achieved with the use of massively parallel sequencing of cell-free DNA in maternal blood. © 2013 John Wiley & Sons, Ltd.

BACKGROUND The current methods for distinguishing the zygosities of twins include ultrasound scanning, which is nondefinitive, and amniocentesis, which is invasive. We explored the use of massively parallel sequencing of maternal plasma DNA for the noninvasive prenatal assessment of the zygosities of twin pregnancies. METHODS Plasma DNA was extracted from blood collected from 8 women pregnant with twins. Target enrichment and massively parallel sequencing were performed for each plasma DNA library. Apparent fractional fetal DNA concentrations were calculated for multiple genomic regions by determining the ratio of minor to major alleles among single-nucleotide polymorphism sites. Variations in the apparent fractional fetal DNA concentrations between genomic regions were used to infer whether individual fetuses in a twin pair were genotypically different and hence dizygotic. RESULTS The extent of the variation in the apparent fractional fetal DNA concentration across chromosomes was 0.82–1.35 SDs for monozygotic twin pregnancies and 2.42–4.80 SDs for dizygotic twin pregnancies. The proportions of apparent fractional fetal DNA concentration values that deviated beyond the range expected for stochastic variation were 0.00%–1.93% for monozygotic twin pregnancies and 36.2%–78.1% for dizygotic twin pregnancies. After identifying a pair of twins as likely dizygotic, the method also allowed determination of the fractional fetal DNA concentrations contributed by the individual fetuses of a dizygotic twin pair. CONCLUSIONS Noninvasive prenatal determination of twin zygosity by maternal plasma DNA sequencing is feasible. It is also possible to determine the relative fractional fetal DNA concentrations for each fetus for dizygotic twin pregnancies.