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Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer deaths worldwide. Recent studies have shown that cancer stem cells (CSCs) are an important cause of tumor recurrence and metastasis. We hypothesized that CSCs marker CD166-positive CRC and colorectal adenoma (CAD) cells consist of more hotspot mutations than CD166-negative CRC and colorectal adenoma cells. To verify this, formalin fixed paraffin embedded tissue specimens from 42 patients each with CRC and CAD were recruited and CD166 immunohistochemical (IHC) staining followed by macrodissection was performed. DNA extracted was used for quantitative polymerase chain reaction detection on a somatic mutation array. Results showed that the immunoreactivity of CD166 protein had significant difference among CRC, CAD, and normal colorectal epithelial tissues (NCET) (P < 0.0001, Kruskal-Wallis test). Moreover, nucleotide changes were found in APC, KRAS, P53, PIK3CA, FBXW7 and SRC genes. Among those genes, KRAS exon 2 mutations were validated in another cohort of 70 CRC and 72 CAD specimens. Results showed that the difference in percentage of KRAS exon 2 mutations between CD166 positive and CD166 negative CRC specimens was significant (P < 0.05, chi-square test). Long term follow-up of the CRC patients showed that CD166-positive KRAS exon 2 mutations was useful in discriminating CRC patients with worse outcome. This study has provided evidence that KRAS exon 2 mutations are concentrated in CD166-positive cancer cells, with prognostic significance in CRC, and those mutations are also detected in CAD.

// Sze Chuen Cesar Wong 1 , Moon Tong Cheung 2 , Lewis Lai Yin Luk 2 , Vivian Ha Man Lee 2 , Pak Tat Chan 2 , Hin Fung Andy Tsang 1 , Evelyn Yin Kwan Wong 1 , Vivian Weiwen Xue 1 , Amanda Kit Ching Chan 3 and John Kwok Cheung Chan 3 1 Department of Health Technology and Informatics, Hong Kong Polytechnic University, Hong Kong, China 2 Department of Surgery, Queen Elizabeth Hospital, Hong Kong, China 3 Department of Pathology, Queen Elizabeth Hospital, Hong Kong, China Correspondence to: Sze Chuen Cesar Wong, email: cesar.wong@polyu.edu.hk Keywords: prognostic significance; cytokeratin 20 lymph node; vascular endothelial growth factor A mRNA; chromodomain helicase DNA binding protein 4 mRNA Received: May 05, 2017 Accepted: November 28, 2017 Published: December 19, 2017 ABSTRACT BACKGROUND: Cytokeratin 20-positive cells in lymph nodes from pN0 colorectal cancer (CRC) patients were detected previously by us. The aims of this study were to investigate which tumor metastasis-related genes were involved and their potential clinical significance. RESULTS: Fourteen of 84 (17%) genes were differentially expressed by at least 2-fold. Among them, 10 genes were up-regulated whereas 4 genes were down-regulated. Those differential expressed genes were validated in the second cohort of specimens. Follow-up analysis for 60 months showed that patients with lymph node vascular endothelial growth factor A (VEGF-A) mRNA and chromodomain helicase DNA binding protein 4 (CHD4) mRNA expression higher than the median copies had significantly shorter time to recurrence than those with lower than the median copies. Multivariate analysis showed that VEGF-A mRNA, CHD4 mRNA and lymphatic vessel involvement were independent prognostic factors for disease recurrence. CONCLUSIONS: VEGF-A mRNA and CHD4 mRNA were up-regulated in CK20-positive pN0 lymph nodes and they may have prognostic significance in pN0 CRC patients. METHODS: Two cohorts of lymph node specimens from pN0 CRC patients of each with and without CK20-positive cells were recruited. In the first cohort, tumor metastasis genes were profiled using gene expression arrays. Differential expressed genes were validated in the second cohort. Moreover, their prognostic significance was examined by following-up the second cohort of patients with CK20-positive cells for 60 months and all histopathological findings were correlated to recurrence.