Diagnostic Detection of Allelic Losses and Imbalances by Next-Generation Sequencing

Abstract

Cancer cells are genomically unstable and accumulate tumor type-specific molecular aberrations, which may represent hallmarks for predicting prognosis and targets for therapy. Co-deletion of chromosomes 1p and 19q marks gliomas with an oligodendroglioma component and predicts a better prognosis and response to chemotherapy. In the current study, we present a novel method to detect chromosome 1p/19q co-deletion or loss of heterozygosity (LOH) in a diagnostic setting, based on single-nucleotide polymorphism (SNP) analysis and next-generation sequencing (NGS). We selected highly polymorphic SNPs distributed evenly over both chromosome arms. To experimentally determine the sensitivity and specificity of targeted SNP analysis, we used DNAs extracted from 49 routine formalin-fixed, paraffin-embedded glioma tissues and compared the outcome with diagnostic microsatellite-based LOH analysis and calculated estimates. We show that targeted SNP analysis by NGS allows reliable detection of 1p and/or 19q deletion in a background of 70% of normal cells according to calculated outcomes, is more sensitive than microsatellite-based LOH analysis, and requires much less DNA. This specific and sensitive SNP assay is broadly applicable for simultaneous allelic imbalance analysis of multiple genomic regions and can be incorporated easily into NGS mutation analyses. The combined mutation and chromosomal imbalance analysis in a single NGS assay is suited perfectly for routine glioma diagnostics and other diagnostic molecular pathology applications.

Interim results from the CATNON trial (EORTC study 26053-22054) of treatment with concurrent and adjuvant temozolomide for 1p/19q non-co-deleted anaplastic glioma: a phase 3, randomised, open-label intergroup study

“…In Europe and Australia, 1p/19q co-deletion status was assessed locally by microsatellite analysis, whereas in North America fluorescent in-situ hybridisation was used. 6,7 MGMT promoter methylation status was assessed in two central laboratories with quantitative PCR, as previously described. 8 If methylation status was not available before patients were randomly assigned to treatment, patients were classified as having indeterminate status.…”

Combined mutation and copy-number variation detection by targeted next-generation sequencing in uveal melanoma

“…These highly polymorphic regions with a global minor allele frequency of at least 45% were selected based on data found in the NCBI SNP database. 26 For chromosome 3, 21 amplicons were designed, due to the clinical relevance. The DNA input varied between 3 and 10 ng, depending on the amount of DNA available per sample.…”

Molecular clonality analysis of esophageal adenocarcinoma by multiregion sequencing of tumor samples

“…A sample was evaluated as having locus LOH when ≥50% of the informative SNPs in that locus demonstrated LOH (variant in tumor DNA <40 or >60%) [16]. In addition, identified tumor suppressor gene mutations also supplied information about possible LOH (loss of the wild type allele) by the relative frequency of the mutant DNA sequence compared to the normal wild type sequence in the tumor samples.…”