Abstract
Tumor genomic instability and selective treatment pressures result in clonal disease evolution; molecular stratification for molecularly targeted drug administration requires repeated access to tumor DNA. We hypothesized that circulating plasma DNA (cpDNA) in advanced cancer patients is largely derived from tumor, has prognostic utility, and can be utilized for multiplex tumor mutation sequencing when repeat biopsy is not feasible. We utilized the Sequenom MassArray System and OncoCarta panel for somatic mutation profiling. Matched samples, acquired from the same patient but at different time points were evaluated; these comprised formalin-fixed paraffin-embedded (FFPE) archival tumor tissue (primary and/or metastatic) and cpDNA. The feasibility, sensitivity, and specificity of this high-throughput, multiplex mutation detection approach was tested utilizing specimens acquired from 105 patients with solid tumors referred for participation in Phase I trials of molecularly targeted drugs. The median cpDNA concentration was 17 ng/ml (range: 0.5–1600); this was 3-fold higher than in healthy volunteers. Moreover, higher cpDNA concentrations associated with worse overall survival; there was an overall survival (OS) hazard ratio of 2.4 (95% CI 1.4, 4.2) for each 10-fold increase in cpDNA concentration and in multivariate analyses, cpDNA concentration, albumin, and performance status remained independent predictors of OS. These data suggest that plasma DNA in these cancer patients is largely derived from tumor. We also observed high detection concordance for critical ‘hot-spot’ mutations (KRAS, BRAF, PIK3CA) in matched cpDNA and archival tumor tissue, and important differences between archival tumor and cpDNA. This multiplex sequencing assay can be utilized to detect somatic mutations from plasma in advanced cancer patients, when safe repeat tumor biopsy is not feasible and genomic analysis of archival tumor is deemed insufficient. Overall, circulating nucleic acid biomarker studies have clinically important multi-purpose utility in advanced cancer patients and further studies to pursue their incorporation into the standard of care are warranted.
Highlights
The development of cancer is primarily due to genetic aberrations that drive oncogenesis and determine the clinical manifestations of tumors; these may impact response to treatment [1]
*One patient was subsequently found to be ineligible for this study as he had not exhausted all lines of available antitumor treatments. **Includes non-small cell lung cancer (NSCLC), mesothelioma, sarcoma, glioblastoma, adenocarcinoma of unknown primary (ACUP), cholangiocarcinoma, and cervical, endometrial, duodenal, esophageal, pancreatic and renal cancers. ***circulating plasma DNA (cpDNA) was collected from 101 (97%) patients; it was not possible to draw blood from 1 patient for technical reasons and blood was not collected from 2 patients due to logistical errors. doi:10.1371/journal.pone.0047020.t001
This study has demonstrated, for the first time, the feasibility of multiplex detection of tumor DNA mutations utilizing the multiplex OncoCarta panel from both DNA extracted from formalin-fixed paraffin-embedded (FFPE) archival tumor tissue and cpDNA
Summary
The development of cancer is primarily due to genetic aberrations that drive oncogenesis and determine the clinical manifestations of tumors; these may impact response to treatment [1]. Notable examples include KRAS mutations in colorectal tumors predicting resistance to antiepidermal growth factor receptor (EGFR) targeting monoclonal antibodies (cetuximab [ImClone and Bristol-Myers Squibb]; and panitumumab [Amgen]) [4,5], and KIT mutations predicting antitumor responses to imatinib (Novartis) in gastrointestinal stromal tumors [6] Molecular analysis of these genomic aberrations is usually conducted on archival tumor tissue due to ethical and safety challenges associated with repeated biopsies. In view of the potential for genomic instability, concerns remain, about the validity of this approach of analyzing archival tumor tissue, rather than rebiopsying tumor for molecular analyses at each therapeutic decision point It is unclear if the analysis of archival tumor biopsies taken many years and frequently multiple therapies previously, sufficiently reflects disease biology at time of treatment. Improved strategies to pursue patient molecular stratification are urgently needed
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