Assessments of intratumor genetic heterogeneity (ITGH) by whole-exome sequencing (WES) can be rendered unreliable by technical artifacts at typical depths of sequencing coverage, according to a study published in Cell Reports.1 As much as 80% of the discordance in breast tumor somatic genetic variation might be artifactual “noise,” the research team concluded.

“The resolution of standard sequencing technologies is limited by the chemistry employed to label the bases in the DNA, and by the hardware platform used to detect the label,” said study coauthor Christos Hatzis, PhD, associate professor at the Yale School of Medicine and director, bioinformatics, breast medical oncology at Yale Cancer Center, New Haven, Connecticut.

“This detection threshold does not cause any problems when identifying dominant (or clonal) somatic mutations that are present in most cancer cells in the tumor,” Dr Hatzis said. “However, genetic intratumor heterogeneity is driven by genetic subclones, each characterized by different sets of subclonal mutations that are present in only a small number of genetically distinct cancer cells.”

Detecting those subclonal mutations is a significant challenge because their frequency is close to the detection limit of standard coverage for WES, Dr Hatzis told Cancer Therapy Advisor.

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The findings might also apply to assessment of tumor mutational burden (TMB), which has been approved as a therapeutic biomarker, Dr Hatzis noted.

“It is not known how the sequencing depth, bioinformatics pipelines, and preanalytical factors such as tumor cellularity and intratumor heterogeneity may affect estimation of TMB,” he said. “We plan to address next these important questions.”

The researchers used 3 different WES pipelines and then validated results with high-depth amplicon sequencing.

“The cancer-only pipeline was unreliable, with about 69% of the identified somatic variants being false positive,” they reported.1 “Even with matched normal DNA, where 82% of the somatic variants were detected reliably, only 36% to 78% were found consistently in technical replicate pairs.”

Those findings are “both compelling and somewhat disconcerting,” said Adrian Lee, PhD, director of the Institute for Precision Medicine at University of Pittsburgh and the University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania. “Sequencing has evolved and has become more powerful over time — but challenges remain. Not having a reproducible assay is a problem. Sensitivity and specificity are absolutely crucial.”

The study bolsters existing evidence that WES can be noisy, Dr Lee said. “It is important that researchers understand that it is a multifaceted problem, not just the depth of sequencing,” Dr Lee cautioned. “There are several challenges.”

It is not entirely clear what the implications might be for variants of unknown significance (VUS), Dr Lee said. “That’s a key question.”