Evolving Significance of Tumor-Normal Sequencing in Cancer Care

Abstract

Molecular tests assist at various stages of cancer patient management, including providing diagnosis, predicting prognosis, identifying therapeutic targets, and determining hereditary cancer risk. The current testing paradigm involves germline testing in a subset of patients determined to be at high risk for having a hereditary cancer syndrome, and tumor-only sequencing for treatment decisions in advanced cancer patients. A major limitation of tumor-only sequencing is its inability to distinguish germline versus somatic mutations. Tumor-normal sequencing has emerged as a comprehensive analysis for both hereditary cancer predisposition and somatic profiling. Here, we review recent studies involving tumor-normal sequencing, discuss its benefits in clinical care, challenges for its implementation, and novel insights it has provided regarding tumor biology and germline contribution to cancer.

Keywords: hereditary cancer predisposition; tumor sequencing; tumor-normal sequencing.

Figure 1.. Mutations Reported in Blood-only, Tumor-only, and Paired Tumor-Normal Sequencing.

In blood-only sequencing, only germline variants detected in the blood (see limitations about mosaic and clonal hematopoiesis-related variants) are reported, informing about hereditary cancer predisposition. Tumor-only sequencing detects and reports both germline and somatic mutations, and is unable to distinguish between the two sources. Paired tumor-normal sequencing allows one to accurately both report germline variants related to hereditary cancer predisposition and somatic mutations in the tumor. Mutations called in the tumor that are not present in the paired normal sample are annotated as somatic and are reported in the somatic report.

Figure 2:. Paired Tumor-Normal Analysis Distinguishes Variants of Various Origins.

(A) Germline heterozygous variants are typically detected at ~50% allele fraction in both normal and tumor sample (in the absence of additional copy number-altering or copy-neutral LOH events). (B) Somatic variants are absent from the normal tissue and can be detected at various fractions in the tumor, depending on their time of occurrence. (C) Variants with ~50% allele fraction in the normal sample and at much higher fraction in the tumor are likely germline variants with LOH at that region in the tumor. (D) Variants that are detected in the blood but are absent from the tumor, without the presence of LOH at that locus, likely represent clonal hematopoiesis. (E) Variants detected with low allele fraction in the blood and are present in the tumor may represent mosaic alterations, that can be confirmed by testing of additional non-malignant tissue samples.

Figure 3:

Comparison of blood-only, tumor-only, and tumor-normal sequencing