Study On Clinical Value By Detection Of FAM172A Expressed In Circulating Tumor Cells (CTCs) Of Colorectal Cancer Patients

Objective:The purpose of this study is to assess the clinical value of detecting CTCs/FAM172A in the diagnosis and treatment of colorectal cancer,by classified different types of circulating tumor cells(CTCs)from colorectal cancer patients and measured FAM172A expression in CTCs,via an multiplex RNA in situ hybridization(RAN-ISH)method,and analyzed the correlations between CTCs/FAM172A and typical clinical/pathological variables.Methods:1.Patients were recruited by Zhujiang Hospital from Jun.2015 to Dec.2015,including 45 colorectal cancer patients and 10 colorectal benign tumor.Blood samples(5ml)were collected before surgery or adjuvant chemotherapy from patients with early stage and before palliative chemotherapy from those with advanced disease.2.The identification includes the following steps:erythrocytes were removed using a red blood cell lysis buffer containing ammonium chloride,and then were transferred to the filtration tube and filtered with the help of a pump valve.CTCs were isolated using a calibrated membrane with 8-?m diameter pores.Via an multiplex RNA in situ hybridization(RAN-ISH)method,four-color fluorescent imaging were used to identify and discriminate subgroups of CTCs.Afterwards,FAM172A expression in individual CTCs was measured.3.The Correlations of CTCs with clinical variables were done by contingency table analysis using the Chi-square test.Continuous data were compared using nonparametric tests(Mann-Whitney test for comparison between two groups and Kruskal-Wallis test for comparison among three or more groups).All analyses were conducted by SPSS 20.0.For all the analyses,P<0.05 was considered statistically significant.Results:1.CTCs in peripheral blood of colorectal cancer patients could be classified into three subgroups according to the expressed markers,including epithelial CTCs,biophenotypic epithelial/mCTCs,and mCTCs.Overall,>3 CTCs/5ml was detected in 28 of 45 colorectal cancer patients(62.2%),which was defined as CTCs positive.mCTCs were found in 26 enrolled patients(57.8%);?1 mCTCs/5 ml was defined as mCTCs positive.But,all ten colorectal benign tumor patients were negative for CTCs detection.2.There was no correlation between positive CTCs and most of the clinico-pathologic features(P>0.05).However,We observed a significant association between mCTCs positivity and development of distant metastases in CRC patients.mCTCs were detected in all the patients with distant metastasis,which were significant higher than that in patients who didn't develop distant metastasis(100%vs.51.3%,P<0.05).In addition,mCTCs were also closely related to vascular invasion.Our study showed that mCTCs were more common in patients with vascular invasion(84.6%vs.46.9%,P<0.05).3.Twenty-eight patients with CTCs positive(?3 CTCs/mL)were enrolled for evaluating biomarker expression.FAM172A+ CTCs were detected in 20/28 patients(71.4%).Our study found that the overall expression rate of FAM172A in CTCs was 60.7%,with 56.3%in epithelial CTCs,58.6%in biophenotypic CTCs and 68.8%in mCTCs.4.In the study,we observed a significant association between FAM172A expression and depth of invasion in CRC patients(68.1%in T1-T3 vs.51.3%in T4,P<0.05).Besides,higher nuclcar-associated antigen Ki-67(Ki-67)value was associated with higher FAM172A expression rate(71.3%in Ki-67?60 vs.48.8%in Ki-67>60,P<0.05).In addition,the FAM172A expression rate in mCTCs was closely correlated with metastasis-associated clinico-pathologic features,such as vascular invasion(78.9%vs.37.5%,P P<0.05)and depth of invasion(77.3%in T1-T3 vs.33.3%in T4,P P<0.05)in CRC patients.In addition,we found mCTCs positive rate(66.7%vs.37.5%,P=0.199)and FAM172A expression positive rate(54.5%vs.22.2%,P=0.142)were both higher in high risk groups than that in low risk groups,although they did not reach statistical significances.More samples are need to verify this link.Conclusion:Via an multiplex RNA in situ hybridization(RAN-ISH)method,The CTCs could be classified into three subgroups according to the expressed EMT markers,including epithelial CTCs,biophenotypic epithelial/mCTCs,and mCTCs.FAM172A expression in individual CTCs could be measured and was significant higher in mCTCs than that in epithelial CTCs.Mesenchymal CTCs may closely related to hematogenous metastasis,prompts us to hypothesize whether mCTCs can be a surrogate marker of tumor aggressiveness.And FAM172A gene expression in CTCs may play an important role in the aggressivity and metastasis of colorectal cancer.There results meant that combine CTCs subgroups with FAM172A gene expression may enhance clinical prediction of CRC metastasis and prognosis.