Inference of Germline Mutational Status and Evaluation of Loss of Heterozygosity in High-Depth, Tumor-Only Sequencing Data.

Abstract

Inherited germline defects are implicated in up to 10% of human tumors, with particularly well-known roles in breast and ovarian cancers that harbor <i>BRCA1/2</i>-mutated genes. There is also increasing evidence for the role of germline alterations in other malignancies such as colon and pancreatic cancers. Mutations in familial cancer genes can be detected by high throughput sequencing (HTS), when applied to formalin-fixed paraffin-embedded (FFPE) tumor specimens. However, due to often lack of patient-matched control normal DNA and/or low tumor purity, there is limited ability to determine the genomic status of these alterations (germline versus somatic) and to assess the presence of loss of heterozygosity (LOH). These analyses, especially when applied to genes such as <i>BRCA1/2</i>, can have significant clinical implications for patient care. LOHGIC (LOH-Germline Inference Calculator) is a statistical model selection method to determine somatic-versus-germline status and predict LOH for mutations identified via clinical grade, high-depth, hybrid-capture tumor-only sequencing. LOHGIC incorporates statistical uncertainties inherent to HTS as well as specimen biases in tumor purity estimates, which we use to assess <i>BRCA1/2</i> mutations in 1,636 specimens sequenced at Rutgers Cancer Institute of New Jersey. Evaluation of LOHGIC with available germline sequencing from <i>BRCA1/2</i> testing, demonstrates 93% accuracy, 100% precision, and 96% recall. This analysis highlights a differential tumor spectrum associated with <i>BRCA1/2</i> mutations. LOHGIC can assess LOH status for both germline and somatic mutations. It also can be applied to any gene with candidate, inherited mutations. This approach demonstrates the clinical utility of targeted sequencing in both identifying patients with potential germline alterations in tumor suppressor genes as well as estimating LOH occurrence in cancer cells, which may confer therapeutic relevance.