Molecule Diagnosis On Micro-metastasis Of Esophageal Carcinoma In Peripheral Blood And Bone Marrow

Esophageal carcinoma, one of six common malignant tumors in the world, is characterized as distinct distributing diversity in epidemiology, forming the diverse incidence in different regions around the world. A high incidence of esophageal carcinoma can be 500 times more than the low incidence region. The death rate and incidence in China is the most in the world, and China takes up half of the new 300,000 patients every year. Although researchers have made rapid progress in the prevention and cure of esophageal carcinoma, this disease causation is unclear and the effect of surgery is dissatisfied, and prognosis is poor. The 5-year-survival after surgery in medium-term and terminal patients is only 10%, although early-stage patients can reach 80%-90%. How to improve the survival rate in the medium-term and terminal patients is the important objective to many researchers. Whether surgery-chemotherapy-radiation therapy, chemotherapy-surgery-radiation therapy or radiation therapy-surgery-chemotherapy is appropriate for the medium-term and terminal patients is searched after by many researchers. In this research the esophageal carcinoma patients are investigated during their treatment to explore if the micrometastasis has happened in bone marrow and peripheral blood, to provide evidence for proper clinical treatment.Many researchers have pay attention to the tumor micrometastasis since the cancer cell was found in the peripheral blood in 1869. Micrometastasis means the cancer cells spreading around and living in the lymphatic system, marrow, liver, lung and other organs during the carcinoma expanding, and there is no clinical symptom. The routine detection methods such as CT, MRI, autoradiography, and pathologic examination are difficult to detect the focus. Micrometastasis is the early-stage of metastasis. Because of the function of patient immunity, the cancer cells are in dormancy. With the development of disease, the cancer cells will suppress the immunoreactions and be in up-growth. As the proportion of cancer cells in the early focus is more than the latter stage, treatment in this stage will be more effective and of great importance for reducing the chance of recrudescence and metastasis. There are three results for the micrometastasis focus: 1, dormancy; 2, elimination; 3, metastasis.Because of the technique restriction, research on Micrometastasis develops slowly. Nowadays the development of immunology and molecular biology make it impossible to detect the micrometastasis in tumors, and make this reahn the focus of research.Many researchers have made comprehensive studies in the micrometastasis on marrow and peripheral blood with various methods, especially on the breast cancer, pancreas cancer, colorectal cancer, and melanoma. Many researches show that there are broad micrometastasis occurring in those tumors without metastasis, and maybe it is an absolute factor affecting long-date survival. Because of the distinct distributing diversity of esophageal carcinoma, researches on the micrometastasis under diversified conditions are few. Researchers in Japan pay more attention to the lymph. Tajima Y and Kodama indicate that there are 50% local lymph micrometastasis in the early-stage esophageal carcinoma. Research on the micrometastasis in marrow and peripheral blood are few reported. O Sullivan GC once uses the Flow cytometry and Immunohistochemistry to study 50 patients and detect 88%-90% occurring micrometastasis in rib marrow, while 15% in ilium marrow, and succeed in inoculating cancer cells from the rib marrow under the rat skin.Cytokeratin(CK) is the component in the epithelia framework. It is expressed inthe normal epidermis, epidermic tumor and transferred tumor cells, and non-expression in blood, marrow and lymph. Transferring tumor cells are in existing if it is expressed in blood, marrow and lymph. CK have more than 20 kinds elements, and CK18, CK19 are tumor marker suggesting micrometastasis at present. And CEA is high-expressed in esophageal carcinoma patient marrow and blood, especially in patients on metastasis and recrudescence. In this research three markers are investigated as the tumor micro transferring.RT-PCR is employed to detect the tumor cells, and foreign researchers testify with the limited-dilution-technology one tumor cell can be detected from 106-107 normal cells. RT-PCR is more sensitive compared to the cell morphologic method and immunochemistry method, especially to the microanalysis for tumor cell. So RT-PCR is the common method to detect the micrometastasis.Nest RT-PCR is employed and lymphocyte in peripheral blood and marrow are used to detect CK18-mRNA, CK19-mRNA and CEA-mRNA genes for the research on whether micrometastasis is occurring in ilium, sternum, rib and peripheral blood on patients during treatment. Comparing the positive rate in ilium, rib and peripheral blood, and analysising the relationship between and positive rate and TNM stage as well as the pathology stage, study whether the positive rate can be used to speculate on the micrometastasis and exploring the tumor gene.As the reports on the micrometastasis in marrow and peripheral blood are few and the theory and method for detection need improving, the relationship between micrometastasis and survival rate is under more research. And research on the realm will improve understanding on the transferring and recrudescence. If the micrometastasis is common in the patient, the notion of transferring only in the terminal stage patient will be changed. Based on the research the treatment scheme for the patient will be great improved and increasing the long-date survival in medium-term and terminal patients.Part one: Micrometastasis in peripheral blood of patient without metastasis Objective: (1) Detection the expression of CK18 mRNA, CK19 mRNA and CEA mRNA in peripheral blood of patient without metastasis;(2) Comparing the positive rate of expression for three genes;(3) Study the relationship between the positive rate and pathologicstage and pathologic differentiation.Methods: Nested-RT-PCR is employed to detect the expression of CK18 mRNA, CK19 mRNA and CEA mRNA in peripheral blood of 50 patients, and analysis the pathologic factor.Results: (1) The positive rate of CK18 mRNA, CK19 mRNA and CEA mRNA in 50 patients is 38.0%(19/50), 44.0%(22/50), 30.2% (16/50);(2) TNM IIa, lib, III three genes positive rates have a trend of increasing, although difference is not distinct(P>0.05), positive rate has no relationship with the pathologic differentiation (P>0.05);(3) Three genes of 2 patients in Ostage have no expression, and the positive expression of 1 patient in I stage;Conclusion: (1) Nested-RT-PCR is the sensitive and effective method to detect the micrometastasis in peripheral blood;(2) Detection the micrometastasis in peripheral blood is helpful to theestimation the tumor progressing;(3) early-stage and medium-term esophageal carcinoma may havemicrometastasis;Part two: Micrometastasis in marrow of patient without metastasis Objective: (1) Detection the expression of CK18 mRNA, CK19 mRNA and CEA mRNA genes in marrow of patient without metastasis;(2) Comparing the positive rate of expression in ilium, sternum and rib;(3) Study the relationship between the positive rate and pathologic stage and pathologic differentiation.Methods: Nested-RT-PCR is employed to detect the expression of CK18 mRNA, CK19 mRNA and CEA mRNA genes in marrow of 50 patients, and analysis the pathologic factor.Results: (1) The positive rate of CK18 mRNA, CK19 mRNA and CEA mRNA in 50 patients rib is 42.0%(21/50), 52.0%(26/50), 36.0%(18/50); Positive rate in sternum is 36.0%(9/25), 40.0%(10/25), 32.0% (8/25); Positive rate in ilium(one side) is 28.0%(7/25), 36.0%(9/25), 28.0%(7/25);(2) TNM IIa, lib, III three genes positive rates have a trend ofincreasing, although difference is not distinct(P>0.05), positive rate has no relationship with the pathologic differentiation (P>0.05);(3) Three genes of 2 patients in Ostage have no expression, and thepositive expression of CK18 mRNA, CK19 mRNA, CEA mRNA in 1 patient in I stage;Conclusion: (1) Nested-RT-PCR is the sensitive and effective method to detect the micrometastasis in marrow, the positive rate is the highest in rib and lowest in sternum;(2) Detection the micrometastasis in marrow is helpful to the estimation the tumor progressing;(3) early-stage and medium-term esophageal carcinoma may havemicrometastasisPart three: Micrometastasis in marrow and peripheral blood of patient in stage IV;Objective: (1) Detection the expression of CK18 mRNA, CK19 mRNA and CEAmRNA in marrow and peripheral blood of patient in stage IV andbenign;(2) Study the positive rate of three genes in peripheral blood of normalsamples;Methods: Nested-RT-PCR is employed to detect the expression of CK18 mRNA, CK19 mRNA and CEA mRNA in peripheral blood and marrow of 10 patients of stage IV, 10 benign patients and 10 normal samples; Results: (1) The positive rate of CK18 mRNA, CK19 mRNA and CEA mRNA in 50 patients in stage IV is 90.0%(9/10), 90.0%(9/10), 80.0%(8/10); Positive rate in ilium is 100.0%(10/10), 100.0%( 10/10), 90.0%(9/10);(2) The expression of three genes in peripheral blood and peripheral of10 patients in benign stage is negative;(3) The expression of three genes in peripheral blood and peripheral of10 normal samples is negative; Conclusion: (1) Nested-RT-PCR is the sensitive and effective method to detectthe micrometastasis in marrow, the positive rate is the highest in rib and lowest in sternum;(2) Detection the micrometastasis in marrow is helpful to the estimation the tumor progressing;(3) The detection rate in peripheral blood and marrow is the same;...