Correlating Factors And Prognostic Value Of CK20 MRNA Expression In Peripheral Blood Of Colorectal Cancer Patients

BACKGROUNDColorectal cancer is one of the world's most common malignancies. In Europe, itis the second most common cancer both in terms of incidence and mortality. ManyAsian countries, including China, have experienced an increase of two to four times inthe incidence of colorectal cancer during the past few decades. Despite improvedtherapeutic modalities, the survival rate of the patients with colorectal cancer is notimproved profoundly. Circular metastasis in distal organs such as liver and lung is oneof the most common and serious problems influencing prognosis. Detecting circularmetastasis early and identifying the patients at risk of recurrence are essential tomanaging colorectal cancer. The presence of circulating tumor cells (CTCs) in theblood is the basis of circular metastasis. Advances in molecular biological technology,especially using reverse transcription-polymerase chain reaction (RT-PCR) method,have made it possible to detect even a few tumor cells in the blood. Cytokeratin 20(CK20) expression is restricted primarily to epithelial tissue. There is no CK20expression in normal blood other than epithelial tissue. As a result, if the expression ofCK20 mRNA is detected in peripheral blood of patients with colorectal cancer, atopicpresence of CTCs in peripheral blood can be inferred. However, the biologicalsignificance of the expression of CK20 mRNA in peripheral blood of patients withcolorectal cancer, both in the preoperative period and at the time of surgery, remainsunclear. Its correlating factors and the relationship between expression of CK20mRNA in peripheral blood and prognosis are not clear. Ki-67 is a human nuclearantigen associated with cell proliferation. Its prognostic value for malignancy hasbeen known. OBJECTIVETo investigate the relationship between expression of CK20 mRNA inperioperative peripheral blood of patients with colorectal cancer andclinicopathological features and prognosis. And to study the relationship betweenexpression of CK20 mRNA in peripheral blood and expression of CK20, Ki-67 in thecorresponding tumor tissue. Therefore to identify the clinical significance of CK20mRNA expression in peripheral blood of patients with colorectal cancer. And initiallyexamining the usefulness of CK20 mRNA as a predictor for circular metastasis ofcolorectal cancer.METHODSEstablishing CK20 mRNA nested RT-PCR method and its detecting sensitivitywas than estimated by using healthy volunteer's peripheral blood samples andHCT-116 colorectal cancer cells.Fifty-seven patients with sporadic colorectal cancer undergoing operation in ourhospital between April 2000 and March 2001 were selected as experimental group.Each 10 ml peripheral blood of patients was collected both in the preoperative periodand at the time of surgery after resection of tumor. The tumor tissue samples were alsoobtained postoperatively. Their clinicopathological features were registered in detail.Patients received long-term follow-up till death or 5 years after operation.Using nested RT-PCR method to detect expression of CK20 mRNA inpreoperative as well as intraoperative peripheral blood of patients in experimentalgroup. And expression of CK20 and Ki-67 in the corresponding tumor tissue wasexamined using the EnVison^TM immunohistochemical method. The experimental datawas correlated with clinicopathological features and long-term follow-up data.Fifteen patients with colorectal benign diseases admitted in our hospital duringthe same period, as well as 10 healthy volunteers were also selected as control groupin this study. Each 10 ml peripheral blood was collected from them to detect theexpression of CK20 mRNA. RESULTSThe expression of CK20 mRNA could be successfully detected by nestedRT-PCR method when 1 ml healthy volunteer's peripheral blood mixed with morethan 5 HCT-116 colorectal cancer cells.Of 57 patients with sporadic colorectal cancer in experimental group, 35patients were male, 22 patients were female, with an average age of 61.0±14.3 years.There were colon cancer in 24 (proximal colon cancer in 11, distal colon cancer in 13),and rectal cancer in 33.The overall detection rate of CK20 mRNA in perioperative peripheral blood ofpatient in experimental group was 59.6ï¼…(34/57). CK20 mRNA was detected in 24 of57 (42.1ï¼…) preoperative peripheral blood samples, while in 32 of 57 (56.1ï¼…)intraoperative peripheral blood samples (McNemar test, P=0.039). None of peripheralblood samples from control group (patients with colorectal benign disease or healthyvolunteers) showed detectable CK20 mRNA.In patients with tumor stagingâ… ,â…¡,â…¢andâ…£, the positive rate of CK20mRNA expression in perioperative peripheral blood was 33.3ï¼…(2/6), 47.4ï¼…(9/19),63.6ï¼…(14/22) and 90.0ï¼…(9/10), respectively. This result demonstrated thatexpression of CK20 mRNA in peripheral blood was significantly associated withadvanced tumor stage (Spearman correlation, P=0.010). Moreover, the rate of CK20mRNA expression in peripheral blood of patients with tumor stagingâ…¢,â…£wassignificantly higher than that of patients with tumor stagingâ… ,â…¡(x² test, P=0.033).There was no significant relationship between expression of CK20 mRNA inperipheral blood and primary tumor location, tumor size, histological pattern, degreeof differentiation (P=0.411, 0.620, 0.449, 0.388, respectively). There was also nosignificant relationship between expression of CK20 mRNA in peripheral blood andserum levels of tumor markers including CEA, CA50, CA19-9, CA242 (P＞0.05).The positive rate of CK20 and Ki-67 expression in tumor tissue of colorectalcancer was 86.0ï¼…(49/57) and 80.7ï¼…(46/57). The positive reaction of CK20 in tumortissue of colorectal cancer was mainly located in cytoplasm and cell membrane, and a small number of nuclei were also stained. The positive reaction of Ki-67 was mainlylocated in the tumor nuclei. With the increase of expressive intensity of CK20 andKi-67 in tumor tissue, the positive rate of CK20 mRNA expression in peripheral bloodwas increased accordingly (P=0.042, P=0.001).The follow-up rate of patients in experimental group was 96.5ï¼…(55/57).Fifty-three patients were suited for survival analysis. The 5-year survival rate ofpatients with positive and negative expression of CK20 mRNA in peripheral bloodwas 43.8ï¼…(14/32) and 90.5ï¼…(19/21) (P=0.001). There was significant relationshipbetween prognosis of patients with colorectal cancer and expression of CK20 mRNAin peripheral blood (Kaplan-Meier survival curves, Log-rank univariate test). Theoverall survival (OS) and progression-free survival (PFS) of patients with CK20mRNA positive peripheral blood were significantly shorter than that of markergene-negative patients(P=0.001, P=0.009). Similarly, there was significantrelationship between prognosis of patients with colorectal cancer and clinical stage(TNM stage). Advanced tumor stage was significantly associated with shorter OS andPFS (P＜0.001, P=0.020). Moreover, the OS and PFS of patients with tumor stagingâ…¢,â…£were significantly shorter than that of patients with tumor stagingâ… ,â…¡(P＜0.001, P=0.007). There was no significant interaction to influence prognosis ofpatients between TNM stage and expression of CK20 mRNA in peripheral blood(multivariate mean square analysis, P=0.090). More expressive intensity of Ki-67 intumor tissue of colorectal cancer was significantly associated with shorter OS ofpatients (P=0.010), while there was no significant relationship between PFS ofpatients and expression of Ki-67 in tumor tissue (P=0.598). The COX regressionmodel analysis showed TNM stage was an independent predictor to the prognosis ofpatients with colorectal cancer (P=0.018), while expression of CK20 mRNA inperipheral blood and expression of Ki-67 in tumor tissue failed(P=0.284, P=0.425).CONCLUSIONS1. Detection of CK20 mRNA by nested RT-PCR to determine the presence ofcirculating tumor cells (CTCs) in peripheral blood of patients with colorectal cancer has high sensitivity and specificity.2. The detection rate of CK20 mRNA in intraoperative peripheral blood issignificantly higher than that of CK20 mRNA in preoperative peripheral blood. Itseems that surgical manipulation may increase the incidence of hematogenousspreading of colorectal cancer cells.3. A direct correlation is demonstrated between expression of CK20 mRNA inperipheral blood and TNM stage of colorectal cancer. The expression of CK20 mRNAin peripheral blood is correlated with advanced stage.4. With the increase of expressive intensity of CK20 and Ki-67 in tumor tissueof colorectal cancer, the positive rate of CK20 mRNA expression in peripheral bloodwas increased accordingly. It seems that expression of CK20 mRNA in peripheralblood is closely related to the differentiation and proliferation of primary colorectalcancer cells.5. The patients whose peripheral blood is CK20 mRNA positive show asignificantly poorer prognosis than patients who are marker gene negative. Largescale and long-term clinical studies are needed to confirm the prognostic value ofgenetically detecting CTCs in the peripheral blood.