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Intratumor heterogeneity (ITH) enable us to understand the evolution of cancer. ITH and evolution of colorectal cancer (CRC) has not been well studied. In this prospective study, we recruited different stages of 68 CRC patients with primary tumor at right-sided colon, left-sided colon and rectum. We performed high-depth whole exome sequencing of 206 multi-region tumor samples including primary tumors, lymph node metastasis (LN) and extranodal tumor deposits (ENTD). Our result showed extreme ITH with Darwinian pattern of CRC evolution, evolution pattern of left-sided CRC was more complex and divergent than right-sided CRC and both LN and ENTD were of polyclonal in origin. Extensive ITH was found in driver mutations in KRAS and PIK3CA genes, suggesting major limitations of single biopsies in clinical diagnosis for the CRC patients. In conclusion, our study showed the Darwinian pattern of CRC evolution with differences in evolution pattern between right-sided and left-sided CRC patients.

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