A Study Of Combined Detection Of Genetic Biomarkers MRNA In Fecal Samples And Diagnosis Of Patients With Colorectal Cancer.

Colorectal cancer (CRC) is one of the most frequent cancers and the leading to death of important disease of tumors. Colorectal cancer is the second common cause of cancer death in the western world, and is the most severe fatal disease among nonsmokers. In the United States, CRC accounts for 11% of all cancers, with an estimated 130200 new cases and 48100 death in 2001, and in Japan, there are 85000 annual cancer registrations and 35600 death caused by CRC. Because most of patients can be treated successfully if metastasis does not occur, it is important to make an early diagnosis and early treatment. In order to reduce the mortality rate of CRC, the development of the screening test by which the cancer can be diagnosed at an early stage is necessary.To date, the fecal occult blood test(FOBT) has been used widely as the screening test for CRC as a noninvasive way. However, three recent large-scale studies showed that the sensitivity of FOBT was low using total colonoscopy as a reference standard in all subjects. However, the sensitivity of FOBT is quiet low, (15%-35%), which implies that a substantial proportion of colorectal neoplasms may be missed. In addition, the peroxidase activity present in dietary components may lead to falsepositive results, which mandates the need for special dietary restrictions prior to stool collection. With a better understanding of molecular changes associated with CRC development, tumor derived DNA alternations in stool as a noninvasive molecular screening test for colorectal neoplasmas has becoming a "hot spot" of searching. Because genetic alterations are associated directly with the devolopment of neoplasia, they have clear advantages over indirect markers such as FOBT, the disadvantage of DNA-based stool assays is the lack of sensitivity caused by clonal heterogeneity in CRC.RNA-based stool assays have been reported in several preliminary studies, one of which showed that CD44 variant expression in human feces could be detected in 68% of CRC patients by a combination of reverse-transcription polynerase chain reaction (RT-PCR) and southern hybridization. A group of Japanese investigators has demonstrated the potential of detecting cyclooxygenase-2(COX-2)mRNA, which is frequently overexpressed in the fecal samples patients with colorectal cancer, however, whether CK20mRNA is also detected in the stool samples of patients with colorectal cancers remains unknown, whether COX-2mRNA, CK20mRNA, and CD44v6mRNA expression in the stools samples and primary lesions of CRC have hemogenous expression remains unknown. No investigations have been conducted regarding combined detection of genetic biomarkers RNA-based stool assays could improve positive-diagnosis rate of CRC and diagnose cancer-related intestinal obstruction.Purpose:The objective of our study is to determine expression of genetic biomarkers RNA-based stool samples originate from the continuously exfoliate tumor cells shed from the primary lesions of colorectal cancers or neoplasmas, by determining expression of COX-2, CK20 and CD44v6mRNA of the primary lesions and fecal samples of CRC, providing suitable combined RNA-based stool assays for noninvasive screening test of colorectal neoplasmas or cancers. The determination of genetic biomarkers RNA-based stool assays and assessed its usefulness as a noninvasive diagnostic tool for determining cancer-related intestinal obstruction.Method and result:We divide the experiment into several parts:The part 1:Expression of COX-2, CK20 and CD44v6mRNA in primary colorectal carcinoma lesions and exfoliate tumor cells of fecal samples. We carry out the expression of COX-2, CK20 and CD44v6mRNA by using RT-PCR in 38 cases of colorectal primary lesions and exfoliate tumor cells of fecal samples, we found that the agreed positive expression rate of COX-2mRNA was 44.7%(17/38) in two groups samples, the agreed negative expression rate of COX-2mRNA was 39.5%(15/38) in them, their homogenous expression rate was 84.2%(32/38); the agreed positive expression rate of CK20mRNA was 44.7%(17/38) in the primary colorectal lesions and stool samples, the agreed negative expression rate of CK20 mRNA was 36.8%(14/38), their homogenous expression rate was 81.5%(31/38); the agreed positive and negative expression rate of CD44v6mRNA in the primary lesions and fecal samples were 50%(19/38),44.7%(17/38), respectively, the homogenous expression were statistically significantly different from hemogenous expression, p<0.001, respectively, it indicated that the expression of genetic biomarkers RNA-based samples originate from the continously exfoliated tumor cells shed from the primary lesions of colorectal cancers.Fecal samples from 38 patients with colorectal carcinoma before radical resection and from 22 cases of normal healthes severed as controls, we determined the expression of COX-2,CK20 and CD44v6mRNA of fecal exfoliated cells by using RT-PCR assay. We found that the positive expression rate of COX-2mRNA in the group of colorectal cancer and healthy control group were 47.4%(18/38),13.6%(3/22), respectively, the rate was statistically significantly different in two groups, p=0.0083; the postive expression rate of CK20mRNA in the colorectal carcinoma group and healthy control group were 55.3%(21/38),18.2%(4/22), respectively, the rate was statistically significantly different in them, p=0.0050; the postive expression rate of CD44v6mRNA in two groups were 50.0%(19/38),9%(2/22), respectively, the rate was statistially significantly different, p=0.0014, it indicated that the expression of CK20, COX-2 and CD44v6mRNA in fecal samples was closely related to the progresssion of colorectal cancers. The further study found that the combined detection of three genetic biomarkers in fecal samples can improve significantly the positive rate of colorectal cancer, the positive rate was 86.8%(33/38), obviously superior to the positive rate of single genetic biomarkers. p<0.001, the Se, Sp, PPV, NPV and Ac of combined detection of three biomarkers were 81.6%,81.8%,88.6%,72% and 81.7%, respectively.The part 2:Combined detection of genetic biomarkers COX-2, CK20 and CD44v6mRNA and diagnose to cancer-related intestinal obstruction.27 patients diagnosed by X-ray or abdominal CT, while 20 patients diagnosed as cancer-related intestinal obstruction by operation. The expression of COX-2, CK20, CD44v6mRNA of fecal samples were assessed by RT-PCR assay. We found that the diagnosis rate of cancer-related intestinal obstruction patients was 90%(18/20), the Se, Sp, PPV, NPV and Ac of combined detection of three genetic biomarkers were 94.7%,85.7%,90%,85.7%and 88.9%, respectively.Conclusion:From the above date and discussion, we can draw the following conclusion:1.There is a high homogenous expression rate of COX-2, CK20 and CD44v6mRNA in the primary colorectal cancer lesions and fecal samples, it indicated the expression of genetic biomarkers RNA-based samples originate from the continuously exfoliated tumor cells shed from the primary lesions of colorectal cancer.2.Combined detection of expression of COX-2, CK20 and CD44v6 mRNA appears to be useful, noninvasive biological markers for diagnosis of colorectal cancer.3.Combined detection of tumor-derived RNA-based stool assay can be as a noninvasive molecular diagnosis assay for cancer-related intestinal instead of endoscopy.