Detection Of YY1mRNA By RT-PCR In Peripheral Blood From The Patients With Esophageal Squamous Cell Carcinoma And Its Clinical Significances

BackgroundEsophageal squamous carcinoma (SCC) is one of the six most common malignant diseases in the world. Linzhou city(formerly Linxian)and nearby counties in Henan province has been well recognized as the highest incidence and mortality areas for SCC in the world.80% of the SCC patient at the first clinical diagnosis are at advanced stage and have a very poor prognosis, five-year survival rate is only about 10%. The recurrence and death incurred by SCC are ascribed to infiltration, invasion and metastasis of SCC. At present, the clinical stage based on TNM classification and histopathological grade are still used to evaluate the prognosis of SCC patients. However, it has been well recognized that there is discordance between the aforementioned classification and the actual prognosis. For example, the SCC patients with well differentiation may have a worse prognosis than those with poor differentiation, indicating the limitation of these markers in evaluating SCC prognosis.Recent studies by us and other laboratories have showed that the invasion and tumor recurrence are based on the micrometastasis of tumor cells in the peripheral blood, lymph node, bone marrow and abdominal cavity. The significance of detection for micrometastasis indicates not only the recurrence and prognosis but also designing individual therapy. In terms of theory, clinical application and gene change correlated with SCC micrometastasis, it is of crucial importance that identification of sensitive and specific molecular biomarkers for early diagnosis and prognosis of SCC. Recurrence and metastasis are the most mortal factors of this cancer. However, sensitive and specific molecular biomarkers to detect early metastasis have not been available yet. Recent investigations indicate that combined analysis with SELDI-TOF-MS and CGH may provide important clue for key protein and genes involved in esophageal carcinogenesis, and found that YY1 maybe have a close relationship with the development of SCC. Protein YY1 is a zinc finger transcription factor with the structure of GLI-Kruppel protein family, the damage of protein YY1 binding site may be an important way for virus to evade the host cell monitoring system, or even cause normal cells into cancer cells. Recently, few of the report on the expression of YY1mRNA in SCC is found in China and abroad. It has been reported that malignant cells mixed with 1 to10 million normal cells could be identified by highly sensitive reverse transcription polymerase chain reaction (RT-PCR) amplification of tumor-specific mRNA. RT-PCR assay may be the most effective method to detect malignant tumor micrometastasis at present. The present study was undertaken to evaluate the feasibility of detecting YY1 mRNA by RT-PCR in peripheral blood of patients with esophageal cancer and to explore its clinical significance.Materials and Methods1. Esophageal cancer patients54 patients with SCC including 35 males and 19 females undergone esophagectomy at the People's Hospital of Linzhou City were enrolled in this study from 2007 to 2008. The mean age was 54±4.4 years and the age range was 19 to 77, respectively. All patients had not received radioactive therapy or chemotherapy and were followed up after operation until January 2008.9 patients who survived less than 73 weeks were all died of recurrence or metastasis.2. Healthy subjectsThe peripheral blood from 25 healthy subjects including 13 males and 12 females was selected from outpatient examination for early SCC in Linzhou as control group. The mean age was 26±3.2 years and the age range was 21 to 49 years, respectively.3. Blood sample collecting and tissue processingThe peripheral blood samples from 54 SCC patients were collected on the day for operation.5 ml blood samples were collected from peripheral venous of every SCC patient and healthy subject. Mononuclear cells in peripheral blood were isolated by gradient centrifugation according to the manufacturer's protocol and then were frozen at-80℃before RNA extraction.At the same time, all the surgically resected tumor specimens from SCC patients were obtained. The esophageal specimens were cut into two-halves. Half of the tissue samples were frozen in liquid nitrogen and then preserved in-80℃freezer. The other part was fixed with 85% alcohol and embedded with paraffin. The pathological diagnosis and TNM Classification (1987) were evaluated after the fixed tissues were processed with routine histological method. The frozen tissues were cut into frozen section with cryostat followed by HE staining for RNA extraction.4. The detection of YY1 mRNA expression in esophageal cancer tissueThe total RNA was extracted from esophageal tumor tissues with the HE frozen slide as the template. RT-PCR was used to detect the expression of YY1 mRNA in frozen esophageal cancer tissue.5. The detection of YY1 mRNA expression in peripheral blood from SCC patients and healthy controlsThe total RNA of mononuclear cells was extracted by TRIzol Reagent from peripheral blood of SCC patients and healthy controls. The RT-PCR assay was applied to detect the expression of YY1 mRNA.6. Follow-up studiesThe cumulative survival rate was calculated by the Kaplan-Meier method and statistical significance was analyzed by the log-rank test. To confirm the statistical significance as a prognosis factors, multivariate analysis was performed with Cox proportional hazard regression model. Chi-squared test was performed to evaluate association between metastasis and YY1 mRNA expression. The significant difference was considered when p value was less than 0.05.Results1. Histopathologic diagnosis54 SCC samples were identified with 27(50.00%) poor differentiation, 20(37.04%) moderate differentiation and 7(12.96%) well differentiation according to histopathologic evaluation. TNM staging indicated 10(18.52%) in stageⅠ,6(11.11%) in stageⅡa,17(31.48%) in stageⅡb,18(33.33%) in stageⅢ,3(5.56%) in stageⅣ. 39 patients (72.22%) had lymph nodes metastases.2. The detection of YY1 mRNA in SCC tissueThe YY1 mRNA expression in tumor tissue was 46.30%(25/54). The YY1 mRNA expression in normal tissue was 9.26%(5/54) and the difference was significant between tumor and normal tissue (P<0.05).3. The detection of YY1 mRNA expression in peripheral blood between SCC patients and healthy subjectsNone of YY1 mRNA expression was detected in blood samples from healthy controls (0/25,0%).42.59% of the SCC patients was positive for YY1 mRNA in blood, and the difference was significant (P<0.05).4. The detection of YY1 mRNA in peripheral blood of SCCThe positive rates of YY1 mRNA in peripheral blood were 42.59%(23/54). Univariate analysis showed that the positive rates of YY1 mRNA in peripheral blood were closely in correlation with both TNM-stage and cancer cell differentiation(P<0.05). Stepwise multivariate analysis showed that "differentiation" and "YY1 mRNA" were the most important factors affecting the prognosis of SCC (P<0.05).5. Comparison on the expression of YY1 mRNA in tumor tissue and in peripheral bloodThe SCC patients with high expression of YY1 mRNA displayed high expression rates of YY1 mRNA in peripheral blood as well (P<0.05). The correlation of YY1 mRNA expression between tumor tissue and peripheral blood was significant in the SCC patients (P＜0.05).6. YYl mRNA expression in peripheral blood and SCC prognosisThere was survival difference between patients with YY1 mRNA positive and negative measured (P<0.05). Thus, the status of YY1 mRNA could predict the prognosis of patients and the patients with negative YY1 mRNA survived longer than those with positive, with a mean survival time of 54 weeks (50,58) and 73 weeks (72,74) and survival rate of 66% and 95%, respectively.Conclusions1.42.59% of the SCC patients are positive for YY1 mRNA in blood and the blood level of YY1 mRNA is well correlated with TNM staging and differentiation, indicating that YY1 may be a promising biomarker for SCC prognosis and for designing treatment protocol and monitoring therapy response.2. None of YY1 mRNA expression is detected in blood samples from healthy controls and in contrast high frequency of YY1 mRNA detection is observed in peripheral blood from SCC, which is consistent with the expression of YY1 mRNA in corresponding tumor tissues. These results suggest that YY1 may be a specific molecular event involved in esophageal carcinogenesis and RT-PCR is a sensitive and specific tool to detect the micrometastasis in peripheral blood.