Abstract
OBJECTIVE To investigate the detection rate of human papilloma virus (HPV) DNA in the Kazakh esophageal carcinoma (EC) patients of Xinjiang.
METHODS We detected the prevalence of a HPV gene in tumor tissues from 318 esophageal squamous cell carcinoma (ESCC). Tumor tissues were kept in formalin and embedded in paraffin. One hundred seventeen samples used crude cell suspension, while the other 201 used the method of DNA extraction with phenol-Tris/chloroform. We analyzed the relevance to EC of Kazakh’s in Xinjiang.
RESULTS In the ESCC samples of Kazakh’s in Xinjiang, total detection rate for HPV DNA was 64.5% (205/318). The positive rate of HPV in group of crude cell suspensions was 82.9% (97/117) compared with the rate of 53.7% (108/201) in the group of DNA extraction. The results in the two groups showed significant diffference (χ2 = 5.711, P < 0.05).
CONCLUSION HPV DNA infection may be one of the most important factors related to EC of Kazakh’s in Xinjiang.
keywords
Introduction
HPV is closely related to certain female genital tract tumors, especially uterine cervix cancer. In recent years, there have been numerous reports on HPV in EC, but results appear to differ. We utilized PCR to detect and analyze HPV infections in 318 EC samples from Kazakh patients in Xinjiang. Primary goals were to examine the relationship between HPV and EC in these patients, and to offer some clue on possible prevention and treatment.
Materials and Methods
Materials
All of the 318 samples obtained in the surgery and embedded in paraffin from the Kazakh patients in Xinjiang were pathologically diagnosed as esophageal squamous carcinoma. The specimens were taken from January 1980 to March 2006, and kept on file in the First Hospital of Medical College of Shihezi University, Xinjiang Yili County Friendship Hospital, Xinhua Hospital, the Fourth Agricultural Reclaimation Division Hospital, Kuitun Yili People Hospital and Xinjiang Autonomous Regional People’s Hospital. Among the total, 117 samples used phenol-CHCl3-isoamyl alcohol. All the samples had been fixed in 10% formalin, preserved in paraffin, subsequently sectioned into 5 μm slices and mounted on APES-treated glass slides, using common H&E staining. The other remaining tissues were used DNA extraction to detect HPV by PCR.
Methods
DNA extraction
Ten 5-μm pieces paraffin-embedded tissues were put into a 2.0-ml sterilized Eppendorf (EP) tube. After paraffin removal and hydration, the lysate (0.02 mmol/L Tris Hcl, 0.02 mmol/LEDTA, 2% SDS) and proteinase K (final concentration: 0.3 mg/ml) were added, with the water bath shaking at 55°C for staying overnight. When tissue was homogenized, isovolumic Tris and saturated phenol (pH 8.0) were added in order. The ratio between Phenol and CHCl3 was 1:1, and that between CHCl3 and isoamyl alcohol 24:1. Each sample was extracted for once, with precipitation of DNA by dehydrated alcohol, and dessication. Fifty to 80 μl of TE (pH 8.0) dissolved the DNA sediment for 12 h~24 h, and DNA extracted was stored in the freezer at -20°C.
Preparation of DNA sample in crude cell suspension[1]
Every 10 pieces of 5 μm paraffin-embedded tissue were put into a 2.0-ml disinfected EP tube. After deparaffinage and hydration, an appropriate quantity of the cell lysate was added based on the amount of the sample. In general, about 500 μl of the cell lysate was added in the pathologic samples, and then a homologous proteinase K (20 mg/ml) was added up to a final concentration of 100 μg/ml for overnight digestion with an aqueous bath at 55°C, until achieving a limpid alimentary juice. Proteinase K was inactivated at 95°C for 10 min, centrifugalized at 10,000 rpm at room temperature for 15 min, and kept at -20°C.
DNA extraction from Ca Ski cells as a positive control
Ca Ski cells were incubated in a culture solution with 10% FBS DMEM up to the amount of 3 × 106. Five hundred μl of cell lysate and 10 mg/ml of RNase were added, with an aqueous bath at 37°C for 30 min, and then proteinase K (20 mg/ml) was added in to reach a final concentration of 100 μg/ml, with an aqueous digestion for overnight at 55°C, until achieving a limpid digestive juice. The suspension was regularly extracted from phenol, CHCl3 and isoamyl alcohol, at the ratio of 25:24:1 and then precipitated in the iced dehydrated alcohol, after that DNA extracted was dissolved in 500 μl TE.
HPV detection by PCR
Before HPV detection by PCR, all the samples were treated, then β-globin PCR was conducted as an internal control, so as to measure the quality and quantity of DNA. Based on the β-globin PCR result, the negative or weak positive samples were rejected. Further HPV PCR detection was performed on the appropriate samples. By comparing the advantages and disadvantages of various HPV primers reported previously, a HPVL1(GP5+/6+), the general type primer of HPV PCR[2] was chosen, with 5’-TTG GAT CCT TTG TTA CTG TGG TAG ATA CTA C-3’ as the upstream primer and 5’-TTG GAT CCG AAA AAT AAA CTG TAA ATC ATA TTC-3’ as the downstream primer. The primers were synthesized by the Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. The PCR reaction system includes 10 × PCR buffer (2.5 μl), primer (0.2 μmol/L), various dNTPs, 0. 5 U Taq DNA polymerase (200 μmol/L), and 25 μl of distilled water. The reaction requirements are as following: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 45 s, annealing at 53.5°C for 45 s, elongation at 72°C for 45 s, with a total of 40 cycles and a re-elongation at 72°C for 10 min. Using 3% agarose gel electrophoresis (160 V), a 10 μl of PCR amplification product was observed through a UV gel formatter and pictures taken 30 min later. A 150 bp HPV segment could be seen in the biogel imaging system. Positive control is Ca Ski cell DNA (a donation from Professor Ke Yang’s lab, Beijing Institute for Cancer Prevention and Treatment) (HPV-16 positive), and normal esophageal samples that had been confirmed to be HPV negative were chosen for negative control, and water was taken as the template for blank control.
Sequencing
Purification and sequencing of the PCR products were conducted by the Shanghai Gene Core BioTechnologies Co., Ltd. (Fig. 2).
Statistical analysis
Grouping of the esophageal carcinoma cases were conducted according to DNA preparative method. The χ2-test was used for statistical treatment, the value of α = 0.05 was considered to be a significant standard in statistical analysis.
Results
Results of the PCR detection are shown in Table 1 & Fig. 1. Of all 318 EC cases, 205 were HPV and DNA positive, with the total positive rate of 64.5%. In 117 of the total EC cases, whose DNA samples were prepared in crude cell suspensions, 97 were HPV and DNA positive, with a positive rate of 82.9%. And in 201 cases with a regular DNA extraction method (phenol-CHCl3), 108 were HPV and DNA positive, and the positive rate was 53.7%. There were statistically significant difference between the two (χ2 = 5.711, P < 0.05).
Comparison between the DNA detection results of the HPVL1 using 2 preparation methods.
Electrophoretic results of HPV-L1 PCR amplification product, the arrowheads show the specific products. M: DNA marker; 1~7: detected samples; 8: negative control; 9; positive control; 10: blank control.
Sampling and sequencing of the HPV-L1 PCR amplification products.
It was found that, after a pathological analysis of the total HPV infection rate in the DNA of ESCC samples in Kazarkh, that there was no statistical significance in the total HPV infection rate among the differentiation degrees of the ESCC (Table 2).
Analysis of the general HPV infection status in ESCC based on pathologic data.
Discussion
The EC is one of the common malignant tumors that jeopardize the human health. Main pathology types include squamous carcinoma and adenocarcinoma, accounting for over 90% in EC. In China, squamous carcinoma accounts for over 80% of the EC cases[3-5].
Thorough etio-epidemiologic survey and intervention were performed based on various factors, such as living habits, physical geographic environment, hereditary factors and microelement of the people from the high-risk regions, however, the exact pathogeny remains unknown now. In years, more and more attention on microbic infection in the environmental factors was paid. Studies on the correlation between HPV and the pathogeny of esophageal carcinoma were highlighted in the first place of the previous. HPV is a kind of virus with a group of small double-stranded DNA. At present, more than 120 subtypes of HPV have been separated, among which 15 high-risk subtypes of HPVs causing infection have been proved to be the essential factors that cause the uterine cervix cancer (UCC), therefore the high-risk type of HPV infection is the precondition of UCC[6,7].
In 1982, Syrjanen put forward for the first time that the morphologic change of esophageal carcinoma was much similar to the genital tract wart, therefore, it was believed that HPV may relate to the development of esophageal carcinoma. Afterwards there were quite a few reports about the relationship between HPV and esophageal carcinoma. Comparing with the HPV infection in the UCC tissue, the infection rate in the ESCC differs greatly in different countries or regions, and even in the same region. What results in the diversity may be relevant to sample source, pathological sample position, quantity of the sample, environmental and geographical factors, hereditary susceptibility, and different methods of detection. In our study, PCR technique was used to amplify HPVL1 gene for detecting the HPV infection rate, and this method is the first choice of screening the HPV infection[8]. The results of detection showed that HPV infection of Xrate was 64% in the esophageal carcinoma patients in Kazakh of Xinjiang, which was in accord with the previous reports. The results in our study suggest that the HPV infection is likely related to the existence of esophageal carcinoma of Kazakh in Xinjiang.
In our experiment, a quality evaluation on the DNA of all ESCC samples was first conducted using a β-globin primer, therefore, the HPV PCR false-negative problem resulted from the DNA quality was precluded. The quality of the template DNA and selection of the PCR primer were the two key factors that cause the significant difference in the HPV PCR detection rate[9]. There was an inverse ratio between the length of template DNA segments and PCR amplification efficiency[10]. It was found by Walboomers etc.[11] that 2 pairs of primer were most efficient in amplification, and PCR product sizes (length) were respectively 100 bp and 209 bp. The bigger the size of the product segment, the lower the positive rate was. In the experiment, all the ESCC samples came from the surgery as pathological specimens which had been fixed in formalin and embedded in paraffin. The conservation time of the samples was different. And DNA was severely destroyed, with broken pieces of unequal sizes[12,13]. According to the routine requirement of PCR, DNA should be extracted. Since the amount of specimen was small in each case, and a part of the samples should be reserved for other succedent experiments, the crude cell suspension of the samples was used in some cases chosen from our experiment, based on the literature references. The method can both avoid the contamination in the process of DNA extraction and decrease the loss of DNA. According to the references, the efficiency of PCR amplification in the DNA prepared by this method is similar to that in the purified DNA, or even superior to the template preparation in the purified DNA because during the purification, DNA is probably lost owing to the phenol extraction, therefore, the result shows decrease in sensibility[14,15]. To assure the reliability of the experiment result, and to compare with the DNA extracted by the routine method (phenol-CHCl3), our experiment method in DNA preparation can fully meet the template requirement of the routine PCR, with the results in agreement with the reports from the literature.
It has been shown in our research that the HPV infection on DNA is likely to be one of the significant factors in the incidence of esophageal carcinoma in Kazakh of Xinjiang. The significance of our study is subject to a contrast with normal tissues, to analysis for clinicopathologic data and to further epidemiology study.
Footnotes
This work was supported by grants from State Key Development Program of Basic Research of China (No.2005CCA03700/2007CB516804), Science Foundation of Ministry of Education of China (No.206167), and National Natural Science Foundation of China (No.30660161).
- Revision received August 11, 2008.
- Accepted December 22, 2008.
- Copyright © 2009 by Tianjin Medical University Cancer Institute & Hospital and Springer
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