15. Interpreting TP53 variants identified by NGS in the setting of complex karyotypes: examples of potential cryptic copy number alterations and copy-neutral loss of heterozygosity

      Diagnosis and follow-up of hematologic malignancies often involve both cytogenetic and molecular testing, and while they target different types of genetic aberrations, the two have findings that are complementary. Chromosome analysis provides a low-resolution genome-wide view, whereas, next-generation sequencing (NGS) offers a high resolution view but may overlook the larger picture. One way in which these technologies complement each other is in the identification of copy number aberrations (CNAs). CNAs can be determined from NGS data by quantitatively assessing the number of amplicon reads (coverage) from one case compared to all cases in the batch (log normalized copy number state or LN-CNS), which can then be correlated with concurrent karyotype studies. We reviewed hematologic malignancy cases received in our laboratories over a period of one year to identify samples with possible loss of heterozygosity (LOH) for the TP53 locus based on NGS allele frequency. Of 21 cases with possible LOH, loss of 17p was confirmed in 14 cases by cytogenetic studies, either karyotype or fluorescence in situ hybridization (FISH). Of the seven cases without evidence of a loss by cytogenetics, six revealed a normal LN-CNS by NGS, suggestive of copy-neutral LOH. The remaining case showed abnormal LN-CNS by NGS. Importantly, this case had an apparently balanced translocation involving 17p (confirmed by FISH) that was discrepant with the NGS results. Overall, we conclude that using the read depth from NGS panels may aid in determining the genetic mechanism of disease. To illustrate the usefulness of CNA detection via NGS, we present three interesting hematologic malignancy cases with LOH for the TP53 locus in patients with complex karyotypes but no TP53 loss identified by cytogenetic testing, highlighting how integrating molecular and cytogenetic methods can provide a more complete genetic diagnosis.
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