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“Somatic” and “pathogenic” - is the classification strategy applicable in times of large-scale sequencing?
Ferrata Storti Foundation
Haematologica 2019 Volume 104(8):1515-1520
Constance Baer, Wencke Walter, Stephan Hutter, Sven Twardziok, Manja Meggendorfer, Wolfgang Kern, Torsten Haferlach and Claudia Haferlach
MLL Munich Leukemia Laboratory, Munich, Germany
Introduction
In the early days of sequencing only a small number of bases was evaluated because of the labor-intensive nature of the procedure. Genes were identified to play a role in the pathogenesis of neoplasms in animal models and cell lines. Subsequently, these mutations were analyzed in samples from patients and their impact on prognosis was evaluated. The list of examples is long: e.g. TP53 was found to be universally mutated across cancers,1 and NPM1 is now among the most frequently analyzed genes in acute myeloid leukemia2 and the mutation defines its own acute myeloid leukemia subtype in the current World Health Organization classification.3
High-throughput sequencing has changed the landscape. It is now possible to sequence a huge number of genes up to exomes and even whole genomes in a comparably short time at affordable cost. The challenge is no longer the sequenc- ing, but rather the evaluation of the results and the interpretation of their impact on diagnosis, prognosis or therapeutic decisions. This has led to some major changes in the way we view sequencing data. In 2015, the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) recommended changing the terms mutation and polymorphism to “variant”. Variants are then further subdivided into five categories depending on the likelihood of their association with the disease.4 The definition was designed for hereditary diseases and therefore addresses germline variants.
The vast majority of genetic events in cancer are somatic.5 Acquired variants represent potential drug targets or biomarkers. Testing a sample, e.g. of colon can- cer, for mutations is frequently performed by comparing variants from a biopsy to those in leukocytes as reference to identify only somatic variants (tumor-normal comparison).6,7 The classical tumor-normal workflow is challenging in studies with large or historic cohorts because of additional sequencing costs, or limited avail- ability of reference material. Leukemia represents another challenge, since blood cannot be used as the reference material. In addition, the growing knowledge of genetic complexity and tumor heterogeneity challenges the historic binary variant classifications (mutation, polymorphism) in the somatic field as well (Figure 1).
Today
Ideally, the results of tumor sequencing are compared to those of reference material with an unaltered germline sequence. Clonal hematopoiesis of indetermi- nate potential has made us familiar with the idea that mutations are acquired as part of the aging process.8 Blood cells are strongly affected by the continuous accu- mulation of somatic changes as a consequence of lifelong proliferation,9 but the phenomenon could apply to all types of reference material.10,11 Tissue formed of cells that divide less quickly (e.g. cerebral tissue)12 would be preferred as a refer- ence; however, this is not a practical approach for routine analysis. Easily accessi- ble sources of reference material are hair follicles, nails, urine, T cells, fibroblast cultures, buccal swabs, saliva, and skin biopsies, but poor DNA yield and the pres- ence of leukemic cell contamination (e.g. DNA from blood in nails) are potential challenges to the use of such material.13-15
In the absence of available reference material, the variant allele frequency (VAF) can be used to distinguish germline from somatic variants. A germline variant is present with a 50% (heterozygous) or 100% (homozygous) VAF. An acquired vari- ant is usually present with a lower VAF, because it is not present in all cells. The caveat is that other factors can also contribute to VAF. Firstly, technical issues
Correspondence:
TORSTEN HAFERLACH
torsten.haferlach@mll.com
Received: February 10, 2019. Accepted: May 15, 2019. Pre-published: July 4, 2019.
doi:10.3324/haematol.2019.218917
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/104/8/1515
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