Circulating tumor cells (CTC) released into blood from primary cancers and metastases reflect the current status of tumor genotypes, which are prone to changes. Here, we conducted the first comprehensive genomic profiling of CTCs using array-comparative genomic hybridization (CGH) and next-generation sequencing. We used the U.S. Food and Drug Administration-cleared CellSearch system, which detected CTCs in 21 of 37 patients (range, 1-202/7.5 mL sample) with stage IV colorectal carcinoma. In total, we were able to isolate 37 intact CTCs from six patients and identified in those multiple colorectal cancer-associated copy number changes, many of which were also present in the respective primary tumor. We then used massive parallel sequencing of a panel of 68 colorectal cancer-associated genes to compare the mutation spectrum in the primary tumors, metastases, and the corresponding CTCs from two of these patients. Mutations in known driver genes [e.g., adenomatous polyposis coli (APC), KRAS, or PIK3CA] found in the primary tumor and metastasis were also detected in corresponding CTCs. However, we also observed mutations exclusively in CTCs. To address whether these mutations were derived from a small subclone in the primary tumor or represented new variants of metastatic cells, we conducted additional deep sequencing of the primary tumor and metastasis and applied a customized statistical algorithm for analysis. We found that most mutations initially found only in CTCs were also present at subclonal level in the primary tumors and metastases from the same patient. This study paves the way to use CTCs as a liquid biopsy in patients with cancer, providing more effective options to monitor tumor genomes that are prone to change during progression, treatment, and relapse. Cancer Res; 73(10); 2965-75. Ó2013 AACR.

Glioblastoma multiforme (GBM) is the most frequent and aggressive brain tumor in adults. The dogma that GBM spread is restricted to the brain was challenged by reports on extracranial metastases after organ transplantation from GBM donors. We identified circulating tumor cells (CTCs) in peripheral blood (PB) from 29 of 141 (20.6%) GBM patients by immunostaining of enriched mononuclear cells with antibodies directed against glial fibrillary acidic protein (GFAP). Tumor cell spread was not significantly enhanced by surgical intervention. The tumor nature of GFAP-positive cells was supported by the absence of those cells in healthy volunteers and the presence of tumor-specific aberrations such as EGFR gene amplification and gains and losses in genomic regions of chromosomes 7 and 10. Release of CTCs was associated with EGFR gene amplification, suggesting a growth potential of these cells. We demonstrate that hematogenous GBM spread is an intrinsic feature of GBM biology.