PDF file - 96KB, Summary of patient characteristics and number of chromosomal aberrations determined by aCGH analyses (S1); Summary of the full genomic analyses of CRC derived CTC and the corresponding tumor tissue (S2); UICC stages of the patients enrolled in the mutational analysis (S3); Characteristics of the patients enrolled in the CTC transcriptional analysis (S4); Distribution of sex and UICC stage in patients enrolled in the CTC transcriptional analysis (S5); Summary of patient characteristics and source of CTC isolation for the CTC transcriptional analysis (S6); Clinical information regarding patients enrolled in the CTC mutational analysis (S7); Overview of the real-time PCR primer sequences used in the CTC transcriptional analysis (S8); Overview of the pre-amplification PCR primer sequences used for the mutational profiling of patient-derived CTC and tumor tissue (S9); Summary of the primer sequences used for sequencing analyses and detection of common CRC mutation (S10); Summary of the primer sequences used for noncoding and coding MSI analyses (S11).
ARTICLE ABSTRACTThe prognosis of colorectal cancer is closely linked to the occurrence of distant metastases. Systemic dissemination is most likely caused by circulating tumor cells (CTC). Despite the fundamental role of CTC within the metastatic cascade, technical obstacles have so far prevented detailed genomic and, in particular, phenotypic analyses of CTC, which may provide molecular targets to delay or prevent distant metastases. We show here a detailed genomic analysis of single colorectal cancer–derived CTC by array comparative genomic hybridization (aCGH), mutational profiling, and microsatellite instability (MSI) analysis. Furthermore, we report the first gene expression analysis of manually selected colorectal cancer–derived CTC by quantitative real-time PCR (qRT-PCR) to investigate transcriptional changes, enabling CTC to survive in circulation and form distant metastases. aCGH confirmed the tumor cell identity of CellSearch-isolated colorectal cancer–derived CTC. Mutational and MSI analyses revealed mutational profiles of CTC to be similar, but not identical to the corresponding tumor tissue. Several CTC exhibited mutations in key genes such as KRAS or TP53 that could not be detected in the tumor. Gene expression analyses revealed both a pronounced upregulation of CD47 as a potential immune-escape mechanism and a significant downregulation of several other pathways, suggesting a dormant state of viable CTC. Our results suggest mutational heterogeneity between tumor tissue and CTC that should be considered in future trials on targeted therapy and monitoring of response. The finding of upregulated immune-escape pathways, which may be responsible for survival of CTC in circulation, could provide a promising target to disrupt the metastatic cascade in colorectal cancer. Cancer Res; 74(6); 1694–704. ©2014 AACR.