Detection Of Human Papillomavirus In Single Esophageal And Gastric Cardia Cancer And Concurrent Esophageal And Gastric Cardia Cancers From The Same Patient And Studies On Alternations Of P53, P16, P21^WAF1, MDM2 At High-incidence Area In Henan

1. BACKGROUND AND OBJECTIVEEsophageal cancer (EC) is one of the six most common malignant diseases worldwide. Linzhou of Henan province, Northern China, has been recognized as the highest incidence area for EC in the world, and the predominant histologic type in this area is squamous cell carcinoma (SCC), the incidence rates are around 161/100,000 for men and 103/100,000 for women. However, in distinct areas the incidence rates are remarkably higher, with up to 500 fold variations between low and high risk areas. EC in late stage has a very poor prognosis with a five year survival rate of less than 10%. However, the 5-year survival rate for EC in the early stage could as high as 90%. Up to now, the reasons for these major regional variations in the incidence of this disease are poorly understood. Epidemiological and experimental data suggest that some chemicals, nutritional deficiencies, physical factors, and infectious agents are associated with the development of this malignancy. An aetiological role of certain microorganisms has been implicated (direct carcinogens or promoters).Human papillomavirus (HPV) as one kind of important tumor-related virus has been firmly recognized in cervical cancer. But its oncogenic role in other tumors is still disputed. As to its role in ESCC, it was firstly suggested by Syrjanen 20 years ago, when he reported an involvement of HPV in the development of both benign and malignant SCC of the esophagus, since then, a substantial amount of literatures have accumulated on this subject. But the HPV infection rate in EC varied from 0% to 100 %. There seems to be a common denominator for the low and high detection rates: namely, the geographical distribution of the material.For different detection rates, the possible reasons:①the cases came from different areas where HPV prevalence was different.②many different HPV detection methods have been used. Different methods have different sensitivity and accuracy. Of them, PCR is more sensitive than others, but different primers have different sensitivity.③the samples taken from different parts, of esophagus. HPV only reproduce in those highly differentiated epidermis cells. The number of copies varies widely in different degree of differentiation, which may impact on the results. Linzhou area is one of the highest incidence areas of esophageal cancer in China, even in the world. In recent years, some studies have been performed on the prevalence of HPV in the tissues of esophageal cancer in Linzhou area, but the conclusions were not consistent. Therefore, it is still questionable whether HPV is one of the major causes of esophageal cancer in this high-risk area.In epidemiology, gastric cardia adenocarcinoma (GCA) in China is characterized by its striking geographic distraction, Linzhou in Henan Province has been well documented as the highest incidence area for GCA. The other phenomenon in Linzhou is that the primary SCC and GCA could occur together on the same patient, which has been named as concurrent carcinoma (CC) of the esophagus and gastric cardia by us.In order to explore the prevalence of HPV and alternation of p53, p16, p21WAF1, MDM2 in carcinoma tissues of local residents in Linzhou area and to evaluate their possible roles and interaction in the carcinogenesis, here we present the following experiments. Some useful findings may be provided scientific basis to prevent esophageal cancer.2. MATERIALS AND METHODS2.1 SubjectsEsophageal cancer:A total of 44 surgically resected human EC specimens were recruited from Linzhou City Center Hospital and Yaocun Esophageal Cancer Hospital in Linzhou, Henan, the highest incidence area for both EC and GCA, including 26 males with an average age of 59±7 years old and 18 females with an average age of 56±9 years old.Gastric cardia:All the 18 surgically resected human GCA specimens were collected from the same hospitals as above. Of the 18 cases,13 were males with an average age of 60±8 years old and 5 females with an average age of 61±10 years old.Biospy tissue:All the 18 endoscopic biopsy esophageal epithelium as normal control were collected from high-incidence regions. Of the 18 cases,9 were males with an average age of 48±10 years old and 9 females with an average age of 50±11 years old.Paraffin-embedded tissue:All the 30 surgically resected human EC specimens were collected from the same hospitals as above. Of the 30 cases,18 were males with an average age of 57±8 years old and 12 females with an average age of 57±10 years old.CC patients:All the 17 cases patients with CC in this study were from the same hospitals as above. Of the patients,12 were males and 5 were females, with a mean age of 59±7 years in male and 58±9 years in female. All the CC patients were conformed to primary SCC and GCA from the same patient by histopathology.None of the patients had received chemotherapy and/or radiotherapy before operation. All the cases were confirmed as GCA or SCC respectively by histopathology. TNM staging was undertaken based on the criteria by AJCC (2002, AJCC).2.2 Tissue collection and processingWithin 2 hours after surgical excision, the specimens were cut into two-halves longitudinally. Half of the specimen was fixed with 85% ethanol, paraffin-embedded, and sectioned at 5μm for histopathological evaluation and immunohistochemical analysis. The other part of the specimens was stored in the liquid nitrogen first and then transferred in a-80℃freezer for DNA extraction.2.3 Methods2.3.1 DNA extraction for frozen tissueDNA extraction from the SCC and GCA pathology sample was performed with QIAamp DNA mini kit, QIAGEN company DNA mini kitTM (51306).2.3.2 DNA extraction for paraffin-embedded tissueBriefly,10-15 slides were deparaffinized in xylene and graded alcohol, then the lysis buffer (300 mmol/1 NaCl; 50 mmol/1 Tris.hCl pH 8.0; 0.2% SDS) was added into the tube with proteinase K (0.5 mg/ml), and the solution was incubated at 55℃overnight until it became clear. Then DNA was extracted using phenol/chloroform, precipitated with cold alcohol, and dissolved in ion-free water and stored at-20℃2.3.3 Immunohistochemical analysis Avidin-biotin-peroxidase complex (ABC) method was used to analyze the expression of p53, p16, p21WAF1 and MDM2. The classification of immunoreactivity was based on the immunostaining intensity (score as 0,1,2,3) and cell proportion of positive immunostaining (score as 0,1,2,3,4).2.3.4 Identification for the purity and integrity of DNA samplesThe DNA concentration was determined from its optical density. Quality of the extracted DNA was tested by PCR with beta-actin primer:5'-TCA CCC ACA CTG TGC CCA TC-3'and 5'-GAA CCG CTC ATT GCC AAT GG-3'.2.3.5 HPV16 E6 amplificationThe usable DNA went through PCR amplification using primer:5'-TCA AAA GCC ACT GTG TCC TG-3'and 5'-CGT GTT CTT GAT GAT CTG CA-3'targeting HPV-16 E6 gene under conditions at 95℃denaturing for 5 min; at 95℃denaturing for 1 min,55℃annealing for 1 min, and at 72℃prolonging for 1 min with 5 cycles; then at 95℃denaturing for 20s,55℃annealing for 30s, and at 72℃prolonging for 30s with 40 cycles; at 72℃prolonging for 5 min. The PCR product was about 120 bp. The plasmid containing full of length of HPV-16 genome as template was the positive control, and the water as template was the negative control.2.3.6 StatisticsChi-squared test and Kappa test were performed to compare the difference between groups.3. RESULTSHPV16 E6 amplification for 44 EC frozen tissuesAll the forty-four extracted DNA samples showed good quality of DNA after PCR with beta-actin primer. After PCR amplification using HPV-16 E6 specific primer, HPV infection was found in tumor patients for 84.1% (37/44) in Linzhou. There was no association between HPV16 infection and gender, age, differentiation, lymph node metastasis and tumor staging.HPV16 E6 amplification for 18 GCA frozen tissuesAll the eighteen extracted frozen gastric cardia cancer samples showed good quality of DNA after PCR with beta-actin primer. PCR amplification using HPV-16 E6 specific primer, HPV infection was found in tumor patients for 44.4%(8/18) in Linzhou, risk incidence area of esophageal cancer. No association between HPV 16 infection and gender, age, differentiation, lymph node metastasis and tumor staging.HPV16 E6 amplification for 18 biospy esophageal epitheliumUsing the same method, HPV infection rate was found in endoscopic biopsy esophageal epithelium as normal control was collected from high-incidence regions for33.3%(6/18).HPV16 E6 amplification for 30 paraffin-embeddedUsing the same method, HPV infection rate was found in frozen sample of esophagus for 84.1% and 53.3%(16/30) from paraffin-embedded tissue, respectively. But comparing the positive rate of frozen and paraffin-embedded samples, the difference of HPV infection in DNA level was significant (P< 0.05).HPV 16 E6 amplification for 17 CCAll the extracted DNA samples showed good quality of DNA after PCR with beta-actin primer. After PCR amplification using HPV-16 E6 specific primer, HPV infection was found in tumor patients from both tissue, with an infection rate of 47.1% (8/17) in SCC and 29.4% in GCA. There have no significant differences among them by statistics (P> 0.05).Immunohistochemical staining for 17 CCIn CC patients, the positive immunohistochemical staining for p53, p16, p21WAF1 and MDM2 was observed both in SCC and GCA with different degrees. In SCC, the overexpression rates of p53 was the highest with 64.7%(11/17), followed by MDM2, p16 and p21WAF1 with 58.8%(10/17),41.2%(7/17) and 41.2%(7/17), respectively. In GCA, the overexpression rates of MDM2, p53, p16 and p21WAF1 were similar, with 70.6%(12/17),64.7%(11/17),58.8%(10/17) and 47.1%(8/17), respectively. Frequent immunostaining pattern for MDM2 and p53 was "Diffuse" and "Scatter". In p16, and p21WAF1, "Scatter" immunostaining pattern was predominant. High coincidence rates for MDM2, p53, p16 and p21WAF1 positive staining was observed in SCC and GCA from the same patients, and were 70.6%(12/17),64.7%(11/17),58.8%(10/17) and 47.1%(8/17), respectively.Comparison for HPV infection and p53, p16, p21WAF1, MDM2 immunohistoche-mical staining in 17 CCIn CC patients, correction was analyed both in SCC and GCA with HPV infection and immunohistochemical staining for p53, p16, p21WAF1 and MDM2. In GCA, the correction of HPV infection and p53, p21WAF1, MDM2 were the highest for 1.0, followed by p16 for 0.338, respectively. In SCC, the correction of HPV infection and p53, p16 and p21WAF1, MDM2 were similar, with 0.335,0.335,1.0 and 0.637, respectively.4. CONCLUSIONS4.1 The prevalence of HPV16 in the esophageal cancer patients was higher than that gastric cardia cancer and healthy control subjects, which indicate that high-risk HPV plays an important role in the development of esophageal cancer of risk-incidence areas. Statistical analyses show that the prevalence of HPV is not dependent on gender, age, differentiation, lymph node metastasis and tumor staging.4.2 The incidence of gastric cardia adenocarcinoma is also very high in this area, higher detection rate of HPV in gastric cardia adenocarcinoma indicate that HPV may be an aetiological factor in carcinogenesis of GCA.4.3 HPV DNA was amplified with a high detection rate in endoscopic biopsy healthy subjects in risk incidence area show HPV as a tumor promoters preceding carcinogenesis progression.4.4 30 cases of pair integrity esophageal cancer sample, the fresh(frozen) and paraffin- embedded tissues amplified using the same approach, the two organizations HPV16 E6 in the detection rate was 84.1% and 53.3%, respectively. The matching test, (P<0.01); the limitations of method itself should be excluded, paraffin-embedded tissues was significantly lower than fresh tissue. This may be fixed and all organizations in the process, the DNA breakdown, the formation of some of the smaller fragments.4.5 The CC patients (the same individual), and HPV in the SCC in the detection rate of 47.1%(8/17), the detection rate was 29.4%(5/17) in the GCA. When comparing the two no significant difference (P> 0.05). HPV infection may suggest that the high incidence area of common participation in the esophagus and gastric cardia cancer process, the two have similar carcinogenic risk factors.4.6 For CC patients, HPV infection and p53, p16, p21WAF1, MDM2 protein changes associated, Which p53, p21WAF1, MDM2 protein changes associated with more GCA, Suggest that the evolution of the process of carcinogenesis may have different molecular basis.