Clinically relevant low-frequency Next Generation Sequencing variants in hereditary cancer patients: an operational multi-step algorithm for laboratory managing

AUTORI

ABSTRACT

The improved sensitivity of Next Generation Sequencing (NGS), in comparison to Sanger sequencing, results in increased identification of unusual findings. Typically, heterozygous germline variants are expected to be detected with NGS at a 50% read-frequency. However, NGS may identify clinically relevant variants at significantly different frequencies. Those in the 5.0-30% range are generally filtered out as low-quality data. Nevertheless, low-frequency variants could be truly present in DNA samples from peripheral blood, due to clonal hematopoiesis of indeterminate potential (CHIP) or constitutional mosaicism, where the variant is only present in a fraction of cells, as the result of a post-zygotic mutation during embryonic development. So, hereditary cancer analysis needs to be improved. Clinically relevant variants that result less than 30% in reads frequency, should be evaluated. Initially, sequencing artifacts and chimerism due to bone marrow transplantation should be excluded. Then, analyses on secondary normal tissues should be performed. If clinically relevant variants with low frequency are not confirmed except for blood, these mutations should be considered as CHIP events. Otherwise, if the analyses in non-transformed samples yield positive results, they would suggest the patients’ constitutional mosaicisms. Testing parents and offspring could provide definitive proof that the low-frequency variants are germline heritable mosaicisms. These results would have a profound impact on mosaic patients’ surveillance, prophylactic surgery, treatment options, and their family members risk assessment.

BIBLIOGRAFIA

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