| BackgroudNasopharyngeal carcinoma mostly happened in southern China, especially in Guangdong At present, the exact etiology of NPC has not been studied clearly,a large number of researches show it is closely related to genetic factors, EB virus infection, environmental factors and dietary factors. The incidence of this disease among men was higher than that of women, and mostly happened in the 40-50 years old patients. Radiation therapy is the main treatment for nasopharyngeal carcinoma, with advances in imaging and radiotherapy technology, the 5-year survival in patients at an early stage is more than 70%. But most of the early onset of nasopharyngeal carcinoma have no apparent specificity, but also lack effective early diagnostic markers.Most has been developed to an advanced stage when they see a doctor with cervical lymph node metastasis or distant metastasis. This will undoubtedly greatly reduce the clinical cure rate of nasopharyngeal carcinoma, survival rates and quality of life also greatly reduced. EB virus has been shown to correlate with nasopharyngeal carcinoma. A variety of specific antigens and antibodies against EB virus are the main means for early diagnosis and screening of nasopharyngeal carcinoma, commonly VCA-IgA and EA-IgA. But sensitivity and specificity are not high. This is not only likely to let the early tumors avoid being diagnosed, but also to increase the mental burden of non-cancer patients. To explore and determine early diagnosis of NPC, is an important task for researchers. With the rapid development of molecular biology, the detection of tumor-associated molecular markers opened a new chapter for cancer diagnosis, prognosis and follow-up to assess the efficacy.The free nucleic acid molecule in periphery blood of the cancer patients is the spotlight of medical workers.There are a variety of free nucleic acids in the peripheral blood of normal subjects and cancer patients. The detection of specific nucleic acid related to tumors has an important value in diagnosis and treatment of cancer patients. Telomeres are hot for life science research in recent years. Telomerase activation is closely related to the malignant tumors. Telomerase is a reverse transcriptase which carry their own templates, responsible for lengthening of telomeres in cells.Human telomerase reverse transcriptase (hTERT) is the subunit of telomerase.It can synthesize telomeric DNA using its RNA template and add to the ends of chromosomes to maintain the stability chromosome and prolong the life of cells and even make the cells immortalized. hTERT only expressed in cells, such as stem cells, germ cells and the majority of tumor cells which have telomerase activity. The expression levels of hTERT mRNA was positively correlated to telomerase activity.Numerous studies have confirmed hTERT is highly expressed in nasopharyngeal carcinoma and the vast majority of malignant tumors, but not in normal human cells. Recently people has done a lot of work in hTERT mRNA in the plasma. It is expected to become a new molecular markers for early diagnosis, assess the efficacy and prognosis of cancer.Circulating tumor cells is one of the hot research in recent years.Studies have demonstrated the circulating tumor cells are connected with the diagnosis and tumor recurrence and metastasis. At the same time monitoring the variation of circulating tumor cells during treatment can help evaluate the therapeutic effect,at any time to adjust treatment accordingly in order to select the best individualized treatment plan. Therefore, in-depth understanding of circulating tumor cells for cancer diagnosis, treatment and prognosis are is of great significance.Numerous studies have using real-time quantitative PCR method to detect the amounts of circulating tumor cells in tumor patients, with tumor-specific molecular markers as the carrieres.They have been used in micrometastases of lung cancer,breast cancer, prostate cancer.To sum up:the circulating tumor cells and tumor-specific RNA in plasma are both of great value in cancer research. Combined detection of tumor cells in peripheral blood and free RNA for tumor diagnosis and treatment is also not yet been reported. We will use a forward-looking research methods to detect the circulating tumor cells in peripheral blood and peripheral free hTERT mRNA expression levels in plasma in nasopharyngeal carcinoma patients by real-time quantitative reverse transcription polymerase chain reactin (Q-PCR) to understand its clinical value of diagnosis and treatment in nasopharyngeal carcinoma.ObjectiveTo explore the quantitative measurement of hTERT mRNA in the plasma and circulating tumor cell of the patients with nasopharyngeal carcinoma using real-time quantitative reverse transcription polymerase chain reactin (Q-PCR) techniqueand. To explore the diagnostic and curative efficacy of hTERT mRNA in the plasma and circulating tumor cell of the patients with nasopharyngeal carcinoma,and explore the correlation between the two, to further explore the source of hTERT mRNA in plasma.Methods1.hTERT mRNA in plasma:33 cases were from patients with nasopharyngeal carcinoma, in department of Otolaryngology Head and Neck Surgery, ZhujiangHospital, Southern Medical University.All patients before treatment were examined by chest radiographs, abdominal B ultrasound, head and neck enhanced CT, MRI, whole body bone scan (SPECT) in routine to determine the tumor violations of the scope and clinical stage. Inclusion criteria:had not received previous radiation therapy; no distant metastasis; no more than 5 days of treatment interruption; chemotherapy and radiotherapy in patients signed consent; pathology of poorly differentiated squamous cell nasopharyngeal carcinoma; not associated with a second primary cancer; age<75 years; Karnofsky score>70.Exclusion criteria:recurrence after radiotherapy treatment; unfinished treatment; combined with other serious medical disorders (stroke, diabetes, hypertension, heart disease, etc.); pathology is not of poorly differentiated squamous cell nasopharyngeal carcinoma; distant metastasis; pregnant or lactating women; severe bone marrow dysfunction, and any other conditions for chemotherapy contraindications. Nasopharyngeal 2008 clinical staging criteria was used for staging system including four cases of stage Ⅰ,5 cases of stage Ⅱ,17 cases of stage Ⅲ,7 cases of stage Ⅳ; 11 cases of T1,6 cases of T2,13 cases of T3,3 cases of T4; 4 cases of NO; 5 cases of Nl,17 cases of N2,7 cases of N3; all cases were M0, pathology detection of all undifferentiated squamous cell carcinoma. Span of 33 patients aged 23-70 years old, with a median age of 49 years, including 16 males and 17 females. Radiotherapy Solution:All patients were in 5-15 days after the end of induction chemotherapy radiotherapy. IMRT tumor area (GTV) prescriptions for 68-72Gy, the amount of each division 2.2-2.4Gy, exposure to 5 times a week. Chemotherapy Solution:induction chemotherapy for all patients were with platinum (cisplatin/Springs platinum)+5-FU,a dose of DDP 60-100 mg/m2d1+5-FU 500-1000 mg/m2d2-5.Some patients on the basis of this added the docetaxel/cyclophosphamide, docetaxel,dose of 60-75mg/m2d1. Concurrent chemotherapy using cisplatin three weeks of chemotherapy (cisplatin 50-80mg/m2) once or cisplatin chemotherapy once a week (20-40mg/m2). Patients of I, II stage used radiotherapy directly; patients of III, IV stage used two courses of induction chemotherapy before radiotherapy. Peripheral blood of 2mL were collected before treatment, after induction chemotherapy and after radiotherapy between 6:00 to 9:00,which were anticoagulated EDTA, centrifuged 10 minutes by 2000rpm within 2 hours.Plasma was separated and stored at -80℃ refrigerator for use.24 healthy controls were from department of Otolaryngology Head and Neck Surgery, ZhujiangHospital, Southern Medical University,and specimens were treated with the former. The expressions of hTERT mRNA in plasma of patients with nasopharyngeal carcinoma before and after treatment (chemotherapy or radiotherapy) were detected by real-time PCR technology, with 24 healthy volunteers as controls.24 healthy controls were from Zhujiang Hospital ENT inpatients.They were all chronic sinusitis, chronic otitis media, vocal cord polyp and other common diseases.The patients in this group approved the consent of the Zhujiang Hospital Southern Medical University Ethics Committee and informed consent by the patient. Then its association with clinicopathological factors were also analyzed.2. hTERT mRNA in circulating tumor cell of the patients with nasopharyngeal carcinoma:The 33 cases of,enrolled patients with nasopharyngeal carcinoma were from September 2013 to June 2014, department of Otolaryngology Head and Neck Surgery, ZhujiangHospital, Southern Medical University.Inclusion and exclusion criteria were as the first chapter,33 patients are also the same of the first chapter. Two tubes of blood of experimental groups were collected,the first tube was processed as the first chapter,the second tube was collected as follows with collecting peripheral blood mononuclear cells for use.This study was passed ethical audit by ZhujiangHospital, Southern Medical University; the collection and testing experiments of pathology specimen were confirmed by the patients. The peripheral venous blood in patients with nasopharyngeal were collected before and after treatment between 6:00 to 9:00.The blood of the first tube was abandoned and the second 2mL blood was taken into sterile heparin vacuum tubes. Lymphocyte separation was added and centrifugal 20 minutes by 2500r/min within 2 hours.Interface cells were collected to give the mononuclear cell layer (tumor cells in this layer), and finally washed once with PBS buffer,-80℃ preservation.20 healthy controls were from department of Otolaryngology Head and Neck Surgery, ZhujiangHospital, Southern Medical University,and specimens were treated as the former. The expressions of hTERT mRNA in circulating tumor cell of the patients with nasopharyngeal carcinoma before and after treatment (chemotherapy or radiotherapy)were detected by real-time PCR technology, with 20 healthy volunteers as controls.20 healthy controls were from Zhujiang Hospital ENT inpatients.They were all chronic sinusitis, chronic otitis media, vocal cord polyp and other common diseases.The patients in this group approved the consent of the Zhujiang Hospital Southern Medical University Ethics Committee and informed consent by the patient.Then its association with clinicopathological factors were also analyzed.Results1. The results showed that the relative expression of hTERT mRNA in plasma in patients with nasopharyngeal carcinoma (10.75±4.29) was significantly higher than those in healthy volunteer group (1.17±0.46,P< 0.05). The expression of hTERT mRNA in plasma of patients with nasopharyngeal carcinoma significantly correlated with clinical staging, tumor size, and degree of nodal metastasis (P<0.05).But it did not significantly correlate with gender and age (P>0.05). And the expression of hTERT mRNA in plasma in the early stage(Ⅰ、Ⅱ stage) after radiotherapy (3.43±1.42) obviousely depressed comparing with pre-treatment(5.60±2.33); the late stage(Ⅲ、Ⅳ stage) after chemotherapy(10.68±2.48,P< 0.05) and radiotherapy (3.13±1.69,P< 0.05) significantly depressed comparing with pre-treatment (12.68±3.08).2.The results showed that:1)The relative expression of hTERT mRNA in healthy volunteer group was 1.09±0.40 and 10.65±4.28 (P<0.05),in peripheral blood with nasopharyngeal carcinoma 2)The expression of hTERT mRNA in peripheral blood with nasopharyngeal carcinoma significantly correlated with clinical stage, tumor invasion range, and lymph node metastasis (P<0.05).3) The expression of hTERT mRNA after-treatment (5.59±2.32) depressed comparing with pre-treatment (10.65±4.28).The correlation coefficient between the relative expression of hTERT mRNA in plasma and circulating tumor cells in nasopharyngeal carcinoma is 0.981, highly correlated.ConclusionThe expression of hTERT mRNA in plasma and circulating tumor cells of patients with nasopharyngeal carcinoma increased significantly,and radiotherapy and chemotherapy can effectively inhibit the expression of the gene. Moreover, the expression of this gene in plasma and circulating tumor cells of patients with nasopharyngeal carcinoma are closely related to the clinical pathologic factors such as clinical staging, tumor size, and degree of nodal metastasis. The quantitative measure of hTERT mRNA in plasma and circulating tumor cells can provide useful data for early diagnosing and observing curative effect in nasopharyngeal carcinoma.The relative expression between the of hTERT mRNA in plasma and circulating tumor cells in nasopharyngeal carcinoma areh ighly correlated.It may partly explain the source of hTERT mRNA in plasma. |
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