Abstract
Circulating cell-free DNA (cfDNA) released from cancerous tissues has been found to harbor tumor-associated alterations and to represent the molecular composition of the tumor. Recent advances in technologies, especially in next-generation sequencing, enable the analysis of low amounts of cfDNA from body fluids. We analyzed the exomes of tumor tissue and matched serum samples to investigate the molecular representation of the tumor exome in cfDNA. To this end, we implemented a workflow for sequencing of cfDNA from low serum volumes (200 μl) and performed whole-exome sequencing (WES) of serum and matched tumor tissue samples from six non-small cell lung cancer (NSCLC) patients and two control sera. Exomes, including untranslated regions (UTRs) of cfDNA were sequenced with an average coverage of 68.5x. Enrichment efficiency, target coverage, and sequencing depth of cfDNA reads were comparable to those from matched tissues. Discovered variants were compared between serum and tissue as well as to the COSMIC database of known mutations. Although not all tissue variants could be confirmed in the matched serum, up to 57% of the tumor variants were reflected in matched cfDNA with mutations in PIK3CA, ALK, and PTEN as well as variants at COSMIC annotated sites in all six patients analyzed. Moreover, cfDNA revealed a mutation in MTOR, which was not detected in the matched tissue, potentially from an untested region of the heterogeneous primary tumor or from a distant metastatic clone. WES of cfDNA may provide additional complementary molecular information about clinically relevant mutations and the clonal heterogeneity of the tumors.
Highlights
Since circulating cell-free DNA was first shown to carry somatic aberrations, its utility for molecular characterization of tumor diseases has been demonstrated in several recent studies [1,2,3,4]
All patients were diagnosed with advanced, lymph node-positive stage III tumors, three squamous cell carcinoma (SCC) and three lung adenocarcinoma (LUAD)
By comparing thes target genes and the COSMIC reference to the whole-exome sequencing (WES) data, we identified a broad range of non-small cell lung cancer (NSCLC)-associated somatic mutations in tissue and matched cell-free DNA (cfDNA) (Table 3)
Summary
Since circulating cell-free DNA (cfDNA) was first shown to carry somatic aberrations, its utility for molecular characterization of tumor diseases has been demonstrated in several recent studies [1,2,3,4]. Tissue biopsies are associated with the risk of invasive procedures and often provide only limited information about the heterogeneous molecular composition of the tumor and its genetic causes. Recent studies demonstrated the analysis of cfDNA as potential minimal-invasive surrogate for cancer diagnostics and prognostics [7,8,9]. Detection of cfDNA in the circulation of cancer patients after surgery could potentially indicate minimal residual disease, which may eventually lead to disease recurrence [13, 14]
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