ABSTRACT
BACKGROUND: Genomic changes that occur in breast cancer during the course of disease have been informed by sequencing of primary and metastatic tumor tissue. For patients with relapsed and metastatic disease, evolution of the breast cancer genome highlights the importance of using a recent sample for genomic profiling to guide clinical decision-making. Obtaining a metastatic tissue biopsy can be challenging, and analysis of circulating tumor DNA (ctDNA) from blood may provide a minimally invasive alternative. PATIENTS AND METHODS: Hybrid capture-based genomic profiling was carried out on ctDNA from 254 female patients with estrogen receptor-positive breast cancer. Peripheral blood samples were submitted by clinicians in the course of routine clinical care between May 2016 and March 2017. Sequencing of 62 genes was carried out to a median unique coverage depth of 7503×. Genomic alterations (GAs) in ctDNA were evaluated and compared with matched tissue samples and genomic datasets of tissue from breast cancer. RESULTS: At least 1 GA was reported in 78% of samples. Frequently altered genes were TP53 (38%), ESR1 (31%) and PIK3CA (31%). Temporally matched ctDNA and tissue samples were available for 14 patients; 89% of mutations detected in tissue were also detected in ctDNA. Diverse ESR1 GAs including mutation, rearrangement and amplification, were observed. Multiple concurrent ESR1 GAs were observed in 40% of ESR1-altered cases, suggesting polyclonal origin; ESR1 compound mutations were also observed in two cases. ESR1-altered cases harbored co-occurring GAs in PIK3CA (35%), FGFR1 (16%), ERBB2 (8%), BRCA1/2 (5%), and AKT1 (4%). CONCLUSIONS: GAs relevant to relapsed/metastatic breast cancer management were identified, including diverse ESR1 GAs. Genomic profiling of ctDNA demonstrated sensitive detection of mutations found in tissue. Detection of amplifications was associated with ctDNA fraction. Genomic profiling of ctDNA may provide a complementary and possibly alternative approach to tissue-based genomic testing for patients with estrogen receptor-positive metastatic breast cancer.
Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Circulating Tumor DNA/genetics , Clinical Decision-Making , High-Throughput Nucleotide Sequencing/methods , Mutation , Receptors, Estrogen/metabolism , Adult , Aged , Aged, 80 and over , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Follow-Up Studies , Genomics/methods , Humans , Middle Aged , Neoplasm Metastasis , Prognosis , Receptor, ErbB-2/metabolism , Receptors, Estrogen/geneticsABSTRACT
The helicase from hepatitis C virus (HCV NS3h) residing on the C-terminal domain of nonstructural protein 3 was considered to be monomeric by several researchers. Here we demonstrate, based on biochemical kinetic data, that the HCV helicase acts as an oligomer. The increase in the ATPase k(cat) of the NS3h protein with increasing protein concentration provided evidence for oligomerization. A sharp decrease in the unwinding rate was observed when the wild type NS3h was mixed with the ATPase deficient mutants of NS3h protein. This provided strong support for both mixed oligomer formation and subunit interactions for the HCV helicase. Chemical cross-linking of NS3h protein was an inefficient process, but yielded cross-linked protein oligomers of various sizes. The information currently available for HCV helicase is consistent with the hypothesis that oligomers of NS3h are not stable and the helicase subunits exchange during unwinding. Nevertheless, oligomerization of HCV helicase stimulates the ATPase activity, and it is required for the helicase activity.
