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Next-generation sequencing of disseminated tumor cells.

ABSTRACT: Disseminated tumor cells (DTCs) detected in the bone marrow have been shown as an independent prognostic factor for women with breast cancer. However, the mechanisms behind the tumor cell dissemination are still unclear and more detailed knowledge is needed to fully understand why some cells remain dormant and others metastasize. Sequencing of single cells has opened for the possibility to dissect the genetic content of subclones of a primary tumor, as well as DTCs. Previous studies of genetic changes in DTCs have employed single-cell array comparative genomic hybridization which provides information about larger aberrations. To date, next-generation sequencing provides the possibility to discover new, smaller, and copy neutral genetic changes. In this study, we performed whole-genome amplification and subsequently next-generation sequencing to analyze DTCs from two breast cancer patients. We compared copy-number profiles of the DTCs and the corresponding primary tumor generated from sequencing and SNP-comparative genomic hybridization (CGH) data, respectively. While one tumor revealed mostly whole-arm gains and losses, the other had more complex alterations, as well as subclonal amplification and deletions. Whole-arm gains or losses in the primary tumor were in general also observed in the corresponding DTC. Both primary tumors showed amplification of chromosome 1q and deletion of parts of chromosome 16q, which was recaptured in the corresponding DTCs. Interestingly, clear differences were also observed, indicating that the DTC underwent further evolution at the copy-number level. This study provides a proof-of-principle for sequencing of DTCs and correlation with primary copy-number profiles. The analyses allow insight into tumor cell dissemination and show ongoing copy-number evolution in DTCs compared to the primary tumors.

SUBMITTER: Moller EK

PROVIDER: S-EPMC3876274 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Next-generation sequencing of disseminated tumor cells.

Møller Elen K EK Kumar Parveen P Voet Thierry T Peterson April A Van Loo Peter P Mathiesen Randi R RR Fjelldal Renathe R Grundstad Jason J Borgen Elin E Baumbusch Lars O LO Naume Bjørn B Børresen-Dale Anne-Lise AL White Kevin P KP Nord Silje S Kristensen Vessela N VN

Frontiers in oncology 20131231

Disseminated tumor cells (DTCs) detected in the bone marrow have been shown as an independent prognostic factor for women with breast cancer. However, the mechanisms behind the tumor cell dissemination are still unclear and more detailed knowledge is needed to fully understand why some cells remain dormant and others metastasize. Sequencing of single cells has opened for the possibility to dissect the genetic content of subclones of a primary tumor, as well as DTCs. Previous studies of genetic c ...[more]

PMID: 24427740

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Project description:Treatment of aggressive glioblastoma multiforme (GBM) must be based on very precise histological and molecular diagnostic of GBM type. According to the WHO guidelines, only tissue biopsy is a relevant source of cellular material evaluated in the diagnostic process to specify the tumor features. Nevertheless, obtaining a GBM biopsy is complicated and relies mostly on resection surgery. Evaluating circulating free DNA and/or circulating tumor cells (CTCs) in the clinic, using a liquid biopsy could represent a non-invasive cancer care optimization. In the present study, the peripheral blood of patients undergoing GBM resection (n = 18) was collected and examined for CTCs. The feasibility of GBM molecular diagnostics from a simple non-invasive peripheral blood withdrawal was evaluated. The size-based enriched CTCs were analyzed using cytomorphology and their origin confirmed based on mutational analysis. In addition, shared DNA mutations in CTCs and in primary tumor tissue were searched. For the identification of CTCs, next generation sequencing (NGS) was used. The GeneReader™ sequencing platform enables targeted sequencing of a 12-gene panel and direct evaluation of detected gene variations using QIAGEN Clinical Insight Analyze (QCI-A) software with a special algorithm for liquid biopsy sequencing analysis. Herein, we present a standard operating procedure for CTC enrichment in GBM patients, CTC in vitro culture, CTC cytomorphological evaluation, and NGS analysis of CTCs using the QIAGEN Actionable Insights Tumor (ATP) Panel. CTCs were present in all tested patients (18/18). The NGS data generated for formalin-fixed paraffin-embedded (FFPE) primary tumor tissues and CTCs reached significantly high-quality parameters. The comparisons between different sample types (CTCs vs. primary tumors) and sampling area (different primary tumor regions) showed a significant level of concordance, indicating CTC testing could be used for patient monitoring and recurrence awareness. Notably, more mutations were detected when analyzing CTC samples compared with the paired primary tumors (n = 3). The results confirm the feasibility of using CTCs as a source of tumor DNA in a diagnostic process, especially when evaluating the molecular characteristics of GBMs. A major advantage of the presented NGS approach for detecting CTCs is the simultaneous identification of several markers relevant for GBM diagnostics, allowing molecular diagnostics on cytological specimens and potential administration of innovative targeted therapies.

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Next-generation sequencing of disseminated tumor cells.

Publications

Next-generation sequencing of disseminated tumor cells.

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