Study On Detection Of Tumor Markers In Saliva From Oral Squamous Cell Carcinoma Patients And Its Mechanism

Oral squamous cell carcinoma (OSCC) is the commonest malignant tumor in oral and maxillofacial region. Though great progress had been made on treatment techniques and means, the survival rate of OSCC patients had not been markedly improved with the 5-year survival rate yet about 50% to 70%. Up to now, early detection, early diagnosis and early treatment are still the key points to improve the survival rate and quality of life for OSCC patients. Besides the routine clinical examination, some necessary clinical assist examinations are thought to be useful for clinical diagnosis. Accompanying with the unceasing progress on the techniques of molecular biology and immunology, the research on tumor markers has been gradually deepened, especially on the tumor markers correlated with OSCC, and the tumor markers correlated with OSCC were suggested with potential clinical value on the assistant diagnosis and prognostic monitoring for OSCC patients.As a body fluid existing in oral cavity, saliva contacts directly with the oral mucosa, and it always contains some shedding cells from oral mucosa as well as theirmetabolites and secretions. So, for OSCC patients, if the saliva contains the shedding tumor cells originating from OSCC tissues, the detection of tumor markers in saliva would be potential clinical value on early detection, early diagnosis and prognostic monitoring. As a body fluid, saliva could be used as a detective media with many advantages: the collection method is safe, painless and non-traumatic;the collection technique is easy to manipulate;the collection equipment is simple;and the saliva samples could also be took repeatedly. Several studies had reported the changes of tumor markers in saliva correlated with OSCC such as p53, pl6, microsatellite and so on. Unfortunately, there were few researches reporting the mechanism on the changes of tumor markers in saliva.Cyfra 21-1 is the soluble fragment of cytokeratin 19 (CK19), which is an acid-type cytokeratin with the worst molecular weight of 40 kD among the all the cytokeratins constituting the intermediate filament cytoskeleton. In normal condition, the concentration of Cyfra 21-1 in body fluid is very low, but, the malignant epithelial cells could release a lot of Cyfra 21-1 from the cells into the body fluid. CEA is an embryonic carcinogenic antigen existing mostly in the fetus epithelial tissues of digestive tract, pancreas and lives, and little in adult digestive tract. However, CEA is over expressed in about 95% colorectal carcinoma, gastric carcinoma, pancreatic carcinoma, most non-small cell lung cancer, head and neck squamous cell carcinoma and 50% mammary cancer tissues. In normal condition, CEA is secreted mainly into the digestive tract;but the cancer cells could infiltrate into the surround tissues and secrete the CEA protein from the cancer cells into the body fluid. Telomerase is a ribonucleoprotein aiding to elongate the repeat sequence at the end of the chromosomes. With the cooperation of other factors, telomerase can add the TTAGGG DNA repeats to the 3' end of telomere using a complementary sequence of its intrinsic RNA component as a template. Telomerase activity is also shown expressed in most permanent cell lines and tumors. Escaping from the proliferative limitations of cellularsenescence, telomerase reactivation might be a prerequisite for the development of malignant tumor cells from somatic cells. And the telomerase activity was detected in about 75% of the head and neck squamous cell carcinoma tissues as well as OSCC tissues.In order to investigate the possibility of detection of tumor markers in saliva from OSCC patients as well as the potential clinical application value, we detected the tumor markers correlated with OSCC such as Cyfra 21-1, CEA, and telomerase activity in saliva from OSCC patients before treatment, and the same tumor markers in serum were also detected and compared with those in saliva, with the follow-up data. At last, the CK19 and CEA were detected on protein and mRNA level with immunohistochemistry and quantitive real-time RT-PCR to investigate the relationship between the tumor markers in tissue and saliva, and the mechanism on the changes of tumor markers in saliva was also discussed.Section one: Expression of CK19 soluble fragment CYFRA 21-1, CEA and telomerase activity in saliva from oral squamous cell carcinoma patients Objective: To investigate the expression of preoperative CK19 soluble fragment Cyfra 21-1, CEA and telomerase activity in saliva from OSCC patients as well as their clinical significance.Materials and methods: 42 OSCC patients with intact clinical data and 30 healthy persons as control were included in this section. Among the 42 OSCC patients, Cyfra 21-1 and CEA concentrations in saliva from 30 patients were detected by ELISA method, telomerase activity in saliva from 34 patients were detected by TRAP-PCR-ELISA method. Saliva was collected between 9:00 am and 11:00 am before primary treatment, and 2~3ml saliva without any stimulation was collected for detection. All the data was analyzed by the statistical software SPSS 10.0. Results: The preoperative Cyfra 21-1 and CEA concentrations in saliva from OSCCpatients were 85.95±78.00ug/L and 102.35±188.13ug/L, respectively;which were significantly higher than those from healthy persons with 42.27± 40.84ug/L (p=0.009) and 8.31 ±4.11ug/L (p=0.001). There was no statistical significance of Cyfra 21-1 and CEA concentrations in saliva from OSCC patients between different clinical stagings and pathological differentiation degrees (p>0.05). For the detection of preoperative CEA concentration in saliva from OSCC patients, it was found no statistical value on prognosis evaluation on OSCC patients;while for Cyfra 21-1, the preoperative Cyfra 21-1 concentration in saliva from the patients with tumor recurrence was significantly higher than that from the patients without tumor recurrence (p=0.023), but no statistical value of Cyfra 21-1 concentration on survival analysis was found. The positive rate of telomerase activity in saliva from OSCC patients was 73.53% (25/34), which was significantly higher than that from healthy persons with 6.67% (2/30), p< 0.001. No statistical significance of telomerase activity positive rate in saliva from OSCC patients was found between different clinical stagings and pathological differentiation degrees (p>0.05). The difference of positive rate was great between the patients with and without tumor recurrence (p=0.051), but no statistical value of telomerase activity positive rate on survival analysis was found. Conclusion: The preoperative Cyfra 21-1 and CEA concentrations, and positive rate of telomerase activity in saliva from OSCC patients increase significantly. There is some prognostic value on detection of preoperative saliva Cyfra 21-1 concentration and telomerase activity for their increase in the OSCC patients with tumor recurrence. Saliva could be used as a detective media for OSCC with potential clinical application value.Section two: Expression of CK19 soluble fragment CYFRA 21-1 and CEA inserum from oral squamous cell carcinoma patients Objective: To investigate the expression of preoperative CK19 soluble fragmentCyfra 21-1 and CEA in serum from OSCC patients as well as their clinical significance, and the correlation of the tumor markers between saliva and serum. Materials and methods: 107 OSCC patients with intact clinical data and 56 healthy persons as control were included in this section. Among the 107 patients, 23 patients also had saliva samples. Both of the preoperative Cyfra 21-1 and CEA concentrations in serum from OSCC patients and healthy persons were detected by ELISA method. 5 ml venous blood was collected before treatment, after centrifugation, the supernatant was used for detection. All the data was analyzed by the statistical software SPSS10.0. Results: The preoperative Cyfra 21-1 concentrations in serum from OSCC patients were 1.14+ 1.19ug/L which was significantly higher than those from healthy persons with 0.40±0.16ug/L (p=0.001). While, the preoperative CEA concentrations in serum from OSCC patients and healthy persons were 2.24±1.35ug/L and 1.94±0.88ug/L, respectively, the difference was not significant (p=0.088). There was no statistical significance of Cyfra 21-1 and CEA concentrations in serum from OSCC patients between different clinical stagings and pathological differentiation degrees (p>0.05). For the detection of preoperative CEA concentration in serum from OSCC patients, it was found no statistical value on prognostic evaluation on OSCC patients, while for Cyfra 21-1, the Cyfra 21-1 concentration in serum from the patients with tumor recurrence and the patients had died was significantly higher than that from the patients without tumor recurrence (p=0.026) and the patients still alive (p=0.001), the higher the serum Cyfra 21-1 was, the shorter the patients lived. By the paired comparison of the tumor markers concentration between serum and saliva, the concentration of Cyfra 21-1 and CEA in saliva was significantly higher than that in serum with p value of 0.001 and 0.014, but no relationship of tumor markers concentration was found between serum and saliva from the same patients. Conclusion: The preoperative Cyfra 21-1 concentrations in serum from OSCC patients increase significantly. There is some prognostic value on the detection ofpreoperative serum Cyfra 21-1 concentration for OSCC, and there is no clinical value of detection of serum CEA for OSCC. The concentration of Cyfra 21-1 and CEA in saliva was significantly higher than that in serum from the same patients.Section three: Expression of CK19 and CEA in cancerous and para-canceroustissues from oral squamous cell carcinoma patientsObjective: To investigate the expression of CK19 and CEA in cancerous and para-cancerous tissues from OSCC patients as well as their relationships with Cyfra 21-1 and CEA concentration in saliva and serum, and the role of expression of adhesive molecule E-cadherin in OSCC tissues, also try to find the cause of increase concentration of preoperative Cyfra 21-1 and CEA in saliva from OSCC patients. Materials and methods: 33 OSCC patients with intact clinical data were included in this section. The samples including the cancerous and para-cancerous tissues were collected during the surgical operation, each tissue sample was divided into two parts, one for protein detection of CK19, CEA and E-cadherin by immunohistochemistry, the other for mRNA detection of CK19 and CEA by quantitive real-time RT-PCR method. All the data was analyzed by the statistical software SPSS 10.0. Results: The positive rate and positive intensity of CK19 protein expression in cancerous tissues from OSCC patients were significantly higher than those in pare-cancerous tissues with p<0.001, and the CK19 mRNA level in cancerous tissues were 2.21 folds higher than that in para-cancerous tissues with p=0.020. Significant correlations were found between the CK19 protein expression and the pathological differentiation degree of cancerous tissues as well as the CK19 mRNA transcript level and the pathological differentiation degree of cancerous tissues, with the p value of 0.002 and 0.025, respectively, the poorer pathological differentiation degree, the higher the CK19 protein and mRNA level. Unfortunately, there was no statistical value on the patients' clinical staging judgment for both CK19 protein expression andmRNA transcript level(p>0.05). When the correlation analysis was performed among the tissue CK19 protein, tissue CK 19 mRNA, serum Cyfra 21-1 and saliva Cyfra 21-1, only the positive correlation was found between the tissue CK19 protein and tissue CK 19 mRNA (p=0.003) as well as the tissue CK19 protein and saliva Cyfra 21-1 (p=0.051). No significant difference of positive rate and positive intensity of CEA protein expression were found between the cancerous and para-cancerous tissues. Although, the CEA mRNA level in cancerous tissues were 2.45 folds higher than that in para-cancerous tissues, the difference was not significant (p=0.732). When analyzing the correlation among the tissue CEA protein, tissue CEA mRNA, serum CEA and saliva CEA, positive correlation was also found between the tissue CEA protein and tissue CEA (P=0.029) as well as the tissue CEA protein and saliva CEA (p=0.051). Moreover, no correlation was found between tissue CK19 and CEA expressions of gene and protein, nor between serum Cyfra 21-1 and serum CEA. However, positive correlation was found between saliva Cyfra 21-1 and saliva CEA with p=0.035. The positive intensity of E-cadherin protein in cancerous tissues were significantly lower than that in para-cancerous tissues with p=0.014, the E-cadherin protein decreased at the invasive front of tumors and superficial surface of oral mucosa.Conclusion: In OSCC tissues, CK19 gene and protein expressions increase, E-cadherin protein expression decreases, while, there is no change of CEA gene and protein expressions. There is significant correlation between the tissue CK19, CEA gene expression and their protein expression, there is also great correlation between the tissue CK19, CEA protein expression and saliva Cyfra 21-1, CEA. The decrease of E-cadherin in cancerous tissues plays an important role in the increase of saliva tumor markers concentration. The CK19 gene and protein expressions are correlated with pathological differentiation degrees with potential clinical application value.Summary:1. Saliva tumor markers such as Cyfra 21-1 concentration, CEA concentration and positive rate of telomerase activity increase significantly in OSCC patients, and the tumor markers of Cyfra 21-1 and CEA concentrations in saliva are higher than those in serum from the same patients. The potential clinical application value is suggested on the detection of tumor marker in saliva for OSCC.2. Serum Cyfra 21-1 concentration increases significantly in OSCC patients and correlate with prognosis in OSCC patients with good clinical application value. There is no use of serum CEA concentration for OSCC patients.3. In OSCC tissues, the CK19 gene and protein expressions increase and correlate with pathological differentiation degrees, E-cadherin protein expression decreases, while, there is no change of CEA gene and protein expressions. There is positive correlation between tissue CK19, CEA gene and protein expressions as well as tissue CK19, CEA protein expression and saliva Cyfra 21-1, CEA.4. CK19 immunohistochemistry and quantitive real-time RT-PCR techniques are clinical useful for pathological differentiation degree judgment in OSCC tissues.5. The increase of tumor markers in saliva from OSCC patients is caused to some extent by the increase of tissue protein expression, which is also caused by the increase of tissue gene transcription.6. The increase of tumor markers in saliva from OSCC patients is also partly caused by the decrease of tissue E-cadherin protein expression, which could lead to the easier epithelial cells shedding off from the oral mucosa into saliva.