The Role Of Novel Biomarkers In Diagnosis Of Colorectal Cancer

Colorectal cancer(CRC)is the third most comnon cancer worldwide and remains the Fourth leading cause of cancer death in the United States and Europe.Early detection of colon cancer is key to improving survival rates because more than 90%of those diagnosed with early-stage CRC live 5 or more years after treatment.Several different procedures and tests have been used for CRC screening,including colonoscopy,the fecal occult blood test,and the fecal immunochemical test.However,in the United States,only 65%of those older than 50 years of age have undergone any traditional form of CRC screening.An effective alternative strategy may be to develop reliable plasma biomarkers so as to improve screening possibilities.Proteomic studies have showed that the expression of inter alpha-trypsin inhibitor heavy chain H3(ITIH3)and interalpha-trypsin inhibitor heavy chain H4(ITIH4)in plasma of Apcmin/+ mice model of early colorectal cancer or polyp was significantly different from that of the control group.Polymeric immunoglobulin receptor(PIGR)has been confirmed that it has a distinct differential expression in plasma of patients with gastric cancer and controls.However,the diagnostic value of plasma levels of ITIH3,ITIH4 and PIGR in human colorectal cancer remains unclear.Therefore,this study mainly aimed at detecting the expression of ITIH3,ITIH4 and PIGR in human colorectal cancer patients’ plasma,and compared with that of healthy controls,in order to find promising plasma biomarkers for CRC diagnosis.The main results of this paper are as follows:1.In total,101 patients with colorectal cancer and 156 healthy controls were enrolled in this study.There was no statistical difference between the two groups in gender,age,smoking and drinking,all p values are greater than 0.05.2.The concentrations of ITIH3,ITIH4 and PIGR proteins in plasma samples of participants were assessed using enzyme-linked immunosorbent assay(ELISA).The results showed the plasma ITIH3 level in CRC patients was significantly lower than that in healthy controls(p<0.001),while the plasma ITIH4 concentration in CRC patients was significantly higher than that of healthy controls(p<0.001).There was no signimcant difference in plasma PIGR expression between CRC patients and healthy controls(p＝0.113).3.Receptor operating characteristic(ROC)curve was adopted to estimate the diagnostic power of the two proteins differentially expressed in plasma of CRC patients.The AUC of ITIH4 was 0.801(95%Cl:0.745-0.857,p＝0.000),higher than that of ITIH3(95%Cl:0.571-0.704,p=0.000).The AUC of the ROC for combined ITIH3 and ITIH4 was 0.827(95%Cl:0.776-0.877,p=0.000),which was even higher than that of carcinoembryonic antigen(CEA).The combined ROC curves of TIMP-1,CEA,ITIH3 and ITIH4 are also analyzed,the diagnostic accuracy was the highest(AUC=0.962,95%Cl:0.940-0.985,p<0.000),when the cutoff value was set at 0.705,the corresponding sensitivity and specificity was 0.917 and 0.908.4.The net reclassification improvement(NRI)was adopted to evaluate the incremental predictive ability of ITIH3/ITIH4 when added to TIMP-1/CEA.The results showed that when ITIH3 and/or ITIH4 was added to TIMP-1/CEA,the diagnostic accuracy of CRC was significantly improved,compared with TIMP-1 or CEA alone.5.Further,the mRNA and protein expression of ITIH3 and ITIH4 in human colorectal cancer and normal colorectal tissues were detected by reverse transcription polymerase chain reaction(RT-PCR)and inmunohistochemistry staining(IHC).The results showed that the expression of ITIH3 mRNA and protein in CRC tissues was significantly lower than that in normal colorectal tissues(p<0.01,p＝0.000),while the expression of ITIH4 mRNA and protein in CRC tissues was significantly higher compared to normal colorectal tissues(p<0.01,p=0.000).The differential expression of ITIH3 and ITIH4 in CRC tissues compares to normal colorectal tissues showed the same trend as that in plasma.6.The Kaplan Meier curve was also conducted,and Log-rank single factor test was adopted to compare the difference of overall survival curve distribution between high and low plasma ITIH3/ITIH4 concentrations in CRC patients.The results showed that there was no statistical difference(p>0.05).7.The expression of ITIH3/ITIH4 mRNA and its clinical and pathological characteristics in CRC tissues were analyzed by linkedOmics database.The results showed that the expression of ITIH3 mRNA in CRC tissues with different degrees of lymph node infiltration was statistically significant(p＝0.033).The expression level of ITIH3 mRNA in CRC tissue of MSI-H group was lower than that of MSS group(p=0.036),which showed statistical significance.However,there was no significant correlation between the expression of ITIH3 mRNA and TNM stage,tissue type,metastasis and radiotherapy.The expression of ITIH4 mRNA in CRC tissue of MSI-H group was higher than that of MSS group.However,there was no significant correlation between the expression of ITIH4 mRNA and lymph node infiltration,TNM stage,tissue type,metastasis and radiotherapy.Above all,this study confirmed that plasma ITIH3 and ITIH4 could be used as effective biomarkers to differentiate CRC patients from healthy controls.The sensitivity and specificity was more than 0.900,when combined ITIH3 and ITIH4 with CEA and TIMP-1,which supported the superior clinical value of ITIH3 and ITIH4 as biomarkers of CRC detection and provided a new strategy for clinical screening CRC.Also,the results of prognosis evaluation showed that ITIH3/ITIH4 was not related to the prognosis of CRC patients.The results of RT-PCR and IHC analysis showed that the expression trend of ITIH3 and ITIH4 protein and mRNA in CRC tissues was consistent with that in plasma,which further confirmed the credibility and clinical significance of plasma results and laid a foundation for its clinical application as reliable biomarkers for CRC detection.In the future,the molecular mechanism of ITIH3 and ITIH4 in pathogenesis of CRC needs to be further studied.