Hendrikus J. Dubbink, Peggy N. Atmodimedjo, Ronald van Marion, Niels M

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Presentation transcript:

Diagnostic Detection of Allelic Losses and Imbalances by Next-Generation Sequencing Hendrikus J. Dubbink, Peggy N. Atmodimedjo, Ronald van Marion, Niels M.G. Krol, Peter H.J. Riegman, Johan M. Kros, Martin J. van den Bent, Winand N.M. Dinjens The Journal of Molecular Diagnostics Volume 18, Issue 5, Pages 775-786 (September 2016) DOI: 10.1016/j.jmoldx.2016.06.002 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Overview on marker location and single-nucleotide polymorphism (SNP)-based loss of heterozygosity (LOH) analysis by targeted next-generation sequencing. A: Location of SNP and microsatellite markers on chromosomes 1p and 19q (not at scale, size bars shown in Mbp). The relative positions and distribution of the microsatellite markers (green dots above) and SNPs (blue dots below) on chromosomes 1p and 19q used in this study are indicated. The SNP and microsatellite markers and their exact locations are shown in Tables 1 and 2. B: Explanatory figure showing the possible outcomes of SNP-based LOH analysis of chromosome 1p. The x axis shows the chromosomal position in million base pairs (Mbp), and the y axis shows the percentage variant (B-allele frequency). Blue dots reflect normal and red dots reflect aberrant bi-allele frequency. The Journal of Molecular Diagnostics 2016 18, 775-786DOI: (10.1016/j.jmoldx.2016.06.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Distribution of B-allele frequencies of single-nucleotide polymorphisms (SNPs) in next-generation sequencing (NGS) analysis of normal brain tissues. NGS analyses of 10 normal autopsy brain formalin-fixed, paraffin-embedded tissues were performed. The mean B-allele frequencies of chromosome 1p SNPs, which are either homozygous referent, homozygous variant, or heterozygous, are shown. Corresponding standard deviations were determined based on the number of times the SNPs were homozygous referent, homozygous variant, or heterozygous, which may vary per SNP. The x axis shows the chromosomal position in million base pairs (Mbp), and the y axis shows the percentage variant (B-allele frequency). Blue dots reflect normal and red dots reflect aberrant bi-allele frequency. The Journal of Molecular Diagnostics 2016 18, 775-786DOI: (10.1016/j.jmoldx.2016.06.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Representative examples of single-nucleotide polymorphism (SNP)-based loss of heterozygosity (LOH) analysis of glioma tissues. Hematoxylin and eosin stain of the corresponding astrocytoma with an estimated neoplastic cell content of 60% (A), and an oligodendroglioma with 90% neoplastic cell content (B). C: Astrocytoma without obvious genomic alteration of chromosomes 1p (above) and 19q (below). The x axis shows the chromosomal position in million base pairs (Mbp), and the Y axis shows the percentage variant (B-allele frequency). D: Oligodendroglioma with typical 1p/19q codeletion. Note the similar degree of LOH (ie, shifts relative to the location of nonaffected informative SNPs) of both chromosome 1p (above) and 19q (below). The x axis shows the chromosomal position in million base pairs (Mbp), and the y axis shows the percentage variant (B-allele frequency). The dot color helps to identify normal (blue) versus aberrant (red) bi-allele frequency. In case of a high percentage of tumor cells, dots with aberrant B-allele frequencies may become blue because they fall within the arbitrarily set normal range of 0–5% or 95–100%. Original magnification: ×20 (A and B). The Journal of Molecular Diagnostics 2016 18, 775-786DOI: (10.1016/j.jmoldx.2016.06.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Sensitivity of single-nucleotide polymorphism (SNP)-based compared with microsatellite-based loss of heterozygosity (LOH) analysis. A: Schematic view of serial dilution preparation from oligodendroglioma tissue (>90% neoplastic cells) with known 1p/19q deletion and adjacent normal tissue. SNP- and microsatellite-based LOH analyses were performed in parallel with the same amount of DNA from the same DNA preparations. B: Serial dilution analysis to test the sensitivity of SNP-based LOH detection. Only results of the chromosome 1p analysis are shown. Experiments were performed in duplicate. The results are indicated per experiment by blue dots and red crosses, respectively. The estimated percentages of neoplastic cells are indicated in the graphs. Calculated outcomes of SNP-based LOH analysis of indicated different admixtures of normal and neoplastic cells are indicated by the lines. The x axis shows the chromosomal position in million base pairs (Mbp), and the y axis shows the percentage variant (B-allele frequency). C: Serial dilution analysis to test the sensitivity of microsatellite-based LOH detection. Results with one (D1S513) of five other chromosome 1p markers are shown. Experiments were performed in duplicate, and one representative experiment is shown. Similar results were obtained with other markers (data not shown). Estimated percentages of neoplastic cells are indicated between the graphs. The x axis shows the DNA fragment size in base pairs (bp), and the y axis shows the relative fluorescence unit (RFU). The Journal of Molecular Diagnostics 2016 18, 775-786DOI: (10.1016/j.jmoldx.2016.06.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Examples of partial imbalance as observed in single-nucleotide polymorphism (SNP)-based loss of heterozygosity (LOH) analysis of glioma tissues. Unambiguous (partial) allelic imbalance was observed in case of SNP-based LOH analysis, but not with in-parallel microsatellite-based analysis (data not shown). A: Glioma 46 shows a large telomeric imbalance of chromosome 1p; with classic fluorescent in situ hybridization probes this tumor might have been considered 1p/19q co-deleted. Glioma 36 (B) and glioma 37 (C) show both telomeric imbalances of chromosome 19q. See additional information on these gliomas, including percentages of neoplastic cells, in Table 3 and Supplemental Table S1. The x axis shows the chromosomal position in million base pairs, and the y axis shows the percentage variant (B-allele frequency). The color of the dot indicates normal (blue) versus aberrant (red) bi-allele frequency. The Journal of Molecular Diagnostics 2016 18, 775-786DOI: (10.1016/j.jmoldx.2016.06.002) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions