J Huazhong Univ Sci Technol[Med Sci] DOI 10.1007/s11596-012-0017-7 32(1):97-102,2012 J Huazhong Univ Sci Technol[Med Sci] 32(1):2012
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Correlation between Physical Status of Human Papilloma Virus and Cervical Carcinogenesis* Kezhen LI ( 李 科 珍 ), Xin JIN ( 金 鑫 ), Yong FANG ( 方 勇 ), Changyu WANG ( 王 常 玉 ), Mei GONG ( 龚 妹 ), Pingbo CHEN (陈平波), Jia LIU (刘 佳), Dongrui DENG (邓东锐), Jihui AI (艾继辉)# Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012
Summary: The prevalence of human papilloma virus (HPV)-16 in patients with cervical cancer, the physical status of HPV-16 in patients with cervical lesions, and the role of HPV-16 integration in cervical carcinogenesis were investigated. HPV genotyping was performed by using PCR approach with the primer GP5+/GP6+ and type-specific primer on biopsy specimens taken operatively from 198 women. Multiple PCR was done to detect physical status of HPV-16 in a series of cervical liquid-based cytology samples and biopsy specimens obtained from different cervical lesions with HPV-16 infection, including 112 specimens with cervical cancer, 151 specimens with CINⅠ, 246 specimens with CINⅡ and 120 specimens with CINⅢ. The results showed that there were 112 cervical cancer samples (56.57% of total cervical cancer patients) with HPV-16 infection. The frequency of HPV-16 pure integration was 65.18% (73/112), 56.57% (47/120), 23.58% (58/246) and 7.95% (12/151) in cervical cancer, CINⅢ, CINⅡand CINⅠ patients respectively. In situ hybridization was performed on some paraffin-embedded sections of CINⅡ, CINⅢ and cervical cancer to verify the physical status of HPV-16 infection. Significant difference was observed between cervical cancer and CINⅠ, CINⅡ, CINⅢ in the frequency of HPV-16 integration (P<0.01). It is suggested that HPV-16 is the most prevalent type and is associated with cervical cancer. In the case of HPV-16 infection there are close associations between the severity of cervical lesions and the frequency of HPV-16 integration. The application of testing HPV genotyping and physical status based on detection of HC-Ⅱ HPV DNA would be in favor of predicting the prognosis of cervical precancerosis and enhancing the screening accuracy of cervical cancer. Key words: human papilloma virus; cervical cancer; genotyping; physical status; integration
As one of the most common gynecological cancers, cervical cancer is a threatening disease to women’s health and survival[1]. In China, approximately 135 000 women suffer from cervical cancer and 53 000 die of it each year[2]. It has been revealed in Nobel prize of 2008 that infection with high-risk human papilloma viruses (HPVs) is a prerequisite for the development of cervical cancer[3]. Oncoprotein E6/E7 can induce the immortalization of host cells through interfering with cell cycle and the unstability of chromosome and result in cervical cancer gradually. HPV DNA can be detected in almost every (99.7%) cervical cancer sample analyzed with HPV-16 being the most common type of HPV found in cervical cancer samples[4]. Although infection of high-risk HPV is recognized as an essential initiating event in cervical tumorigenesis, minority of women in-
fected with HPV will develop an HPV-associated precursors or cervical cancer[5]. With the advancement of molecular biology and cell genetics, integration of HPV DNA into the host genome is observed in most invasive cancers but rarely in cervical intraepithelial neoplasm (CIN)[6, 7] and is thus likely to constitute a key step in oncogenesis. The expression of E6/E7 oncogene and the alteration of host genome after HPV integrates into the host DNA may be the key point during the process of cervical cancer[8]. We used the multiple polymerase chain reaction (PCR) that allows determining the presence or absence of integrated viral genome copies in clinical lesions. This study aimed to investigate physical status of HPV in patients with precancerous lesions or cervical cancer and expected to offer necessary evidence of the role of HPV-16 integration during the onset and progress of the cervical cancer.
Kezhen LI, E-mail:
[email protected] # Corresponding author, Email:
[email protected] * This project was supported by grants from National Natural Science Foundation of China (No. 81001151, No. 30973205) and Natural Science Foundation of Hubei Province (No. 2010CDB09503).
1 MATERIAL AND METHODS 1.1 Clinical Samples Clinical samples were collected from patients treated in Department of Gynecologic Oncology, Tongji Hospital (China). Biopsy specimens collected were bi-
98 sected: one portion was submitted for standard histopathologic diagnosis, while the other portion was stored at –80°C for subsequent molecular analysis. All specimens were histologically verified. The stage of the disease was classified according to International Federation of Gynecology and Obstetrics (FIGO). Patients were 24 to 72 years old (mean 43). Cervical liquid-based cytology samples were taken from women with cytological abnormalities determined by Pap smears for the purpose of HPV detection and genotyping, and all of the samples with HPV-16 infection were selected to undertake the DNA extraction. Examinees were 19 to 67 years old (mean age 38.6 years). The cytological diagnosis of cervical specimens was determined according to the “2001 Bethesda” system for reporting Pap test results and histopathologic diagnoses were verified. In the study group there were 151 (29.21%) cases of CINⅠ (also nominated as low-grade squamous intraepithelial lesions, i.e. LSIL), 246 (47.58%) cases of CINⅡ (classified as high-grade squamous intraepithelial lesions, i.e. HSIL), 120 (23.21%) cases of CINⅢ (also classified as HSIL). Total DNA was extracted from cervical swabs and stored at –80°C. 1.2 Cell Culture SiHa cells were obtained from the American Type Culture Collection (ATCC, USA). Cells were grown on Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cell culture was performed as previously described in 37°C, 5% CO2, and saturated humidity. 1.3 DNA Isolation and Identification DNA from samples was isolated using a DNA isolation kit (Qiagen, China) as recommended by the supplier. Pelleted DNA was resuspended in 200 μL of tridistillated sterile water and stored at −80°C. In order to determine the quality and quantity of isolated DNA, each DNA was analyzed by electrophoresis on 1% agarose gels stained with ethidium bromide and spectrophotometrically. For PCR approach, the primer sequences were as follows: sense primer, 5′-ACACAACTGTGTTCACTAGC-3′; antisense primer, 5′-CAACTTCATCCACGTTCACC-3′. PCR was performed with 20 ng DNA in a final volume of 20 µL using the 2×EasyTaq PCR SuperMix (Beijing TransGen Biotech Co., Ltd, China). Thermocycler conditions were 3 min at 94°C and 35 cycles (30 s at 94°C, 30 s at 50°C, and 2 min at 72°C) and 10 min at 72°C. The products were visualized by 1.2% agarose gel electrophoresis. 1.4 Typing-PCR For HPV typing, we used PCR approach with the primer GP5+/GP6+ as described by Jacobs et al[9], and nucleic acid sequence-based amplification as previously described[10, 11]. To control the accuracy of classification, HPV-16 specific primers were designed to confirm the results. Type-specific PCR was performed with 20 ng DNA in a final volume of 20 µL using the 2×EasyTaq PCR SuperMix (Beijing TransGen Biotech Co., Ltd, China). Specific primer sequences were as follows: sense primer, 5′-CGACCCAGAAAGTTACCAC-3′; antisense primer, 5′-GGTCTTCCAAAGTACGAATG-3′. Thermocycler conditions were 3 min at 94°C and 35 cycles (30 s at 94°C, 30 s at 55°C, and 30 s at 72°C) and 10 min
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at 72°C. The products were visualized by 1.2% agarose gel electrophoresis. The PCR amplicons were excised from the agarose gel and extracted using the QIAquick Gel Extraction Kit from Qiagen and sequenced by Invitrogen Co. (USA). HPV sequences were then compared to available HPV genome data (GenBank) by using BLASTN v.2.2 (NIH). 1.5 Multiple PCR All HPV-16 positive cases were further examined for analysis of the physical status of their HPV DNA. Three sets of primers which had crossed fragments of each other for the E2 segments were used for HPV-16 genome amplification[12]. If these three fragments of the sample were not amplified together, it meant this sample was pure integrated. The primer sequences were as follows: fragment A (475 bp): sense primer, 5′-AGGACGAGGACAAGGAAAA-3′, antisense primer, 5′ACTTGACCCTCTACCACAGTTACT-3′; fragment B (477 bp): sense primer, 5′-TTGTGAAGAAGCATCAGTAACT-3′, antisense primer, 5′-TAAAGTATTAGC ATCACCTT-3′; fragment C (176 bp): sense primer, 5′-GTAATAGTAACACTACACCCATA-3′, antisense primer, 5′-GGATGCA GTATCAAGATTTGTT-3′. PCR as performed with 100 ng DNA in a final volume of 20 µL using the 2×EasyTaq PCR SuperMix (Beijing TransGen Biotech Co., Ltd, China). Thermocycler conditions were as follows: 5 min at 94°C and 35 cycles (1 min at 94°C, 30 s at 60°C, and 1 min at 72°C) and 10 min at 72°C. The products were visualized by 1.2% agarose gel electrophoresis. All of the three fragments of the sample were amplified with multiple PCR for analysis of the episomal or mixed infection status of their HPV DNA. The primers were designed to amplify the outer-to-outer of E2 and part of E7. The primer sequences were as follows: E2 (1145 bp): sense primer, 5′-ACGAAAACGGAAATCCAGTG-3′, antisense primer: 5′-AATCCGTCCTTTGTGTGAGC-3′; E7 (282 bp): sense primer, 5′-TGCTCAGAGGAGGAGGATG-3′, antisense primer: 5′-GGTCTTCCAAAGTACGAATG-3′. PCR was performed with 200 ng DNA in a final volume of 50 µL using the 2×EasyTaq PCR SuperMix (Beijing TransGen Biotech Co., Ltd, China). Thermocycler conditions were as follows: 5 min at 94°C and 35 cycles (1 min at 94°C, 30 s at 64°C, and 1.5 min at 72°C) and 10 min at 72°C. Each product was visualized by 1.2% agarose gel electrophoresis. The gray scale per area (Ⅰa) of the electrophoresis strip was measured and analyzed to calculate the ratio of the Ⅰa of the amplicon of E2 and E7. If the ratio got close to 1, indicating HPV was of episome in the sample. 1.6 In situ Hybridization Slides were prehybridized for 1 h at 45°C. Hybridization was carried out overnight at 37°C with 1 μg/mL digoxin acylated oligonucleotide probe complementary to the HPV16 E6 and E7 coding region (5′-ATGTTTCAGGACCCACAGGAGCGACCCAGAAAGTTACCACAGTTA-3′). Negative controls were similarly processed by omitting the probe. An alkaline phosphatase-conjugated anti-digoxin antibody (Boehringer Mannheim, China) was added to bind to the hybridized probe. The slides were then incubated with nitroblue
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tetrazolium/5-bromo-4-chloro-3-indolyl phosphate for 30 min and counterstained once with nuclear fast red. The evaluation of the hybridization signal: the positive hybridization signals showed amethyst, violet or black particles in the nucleus. In each sample, the percentage of cells expressing HPV DNA and the image of hybridization signals was determined. Two observers rated the samples blindly; we used the average of their evaluations as the final result. 1.7 Statistical Analysis The results obtained by different methods were entered into Microsoft Excel on a personal computer and processed manually. The differences between groups were evaluated by Chi-square (χ2) test (SPSS Statistics for Windows), with P value under 0.05 being statistically significant.
infection
Fig. 1 Direct gel electrophoretogram of each DNA M: Marker; 1, 2: Samples 1 and 2
2 RESULTS 2.1 Quality and Quantity of Isolated DNA Most ratios of A260/A280 of DNA extracted from cervical lesions, which were detected with ultraviolet spectrophotometer, were over 1.8 and the average of DNA density was 0.32 μg/μL. The distinct objective straps were visible after direct electrophoresis of each DNA or amplifying house-keeping gene (β-Globin) with PCR (fig. 1 and 2). 2.2 Detection of HPV Types The types of HPV infected were detected as described in fig. 3. There were 112 cervical cancer samples (56.57% of total cervical cancer patients) with HPV-16
Fig. 2 Gel electrophoretogram of amplicon of house-keeping gene (β-Globin) of DNA extracted from samples by PCR assays C: Negative control; M: 1 Kb marker; 1-10: Samples 1-10, respectively
Fig. 3 Schematic diagram of HPV typing detection
2.3 Physical Status of HPV-16 Infection There were 73 samples with pure integrated HPV DNA in 112 cervical cancer samples with HPV-16 infection and the positive ratio was 65.18%. The remaining 39 samples of cervical cancer were further examined with multiple PCR. The frequency of HPV-16 pure integra-
CINⅠ CINⅡ CINⅢ CC Total
tion was 56.57% (47/120), 23.58% (58/246) and 7.95% (12/151) in CINⅢ, CINⅡ and CINⅠ patients respectively. Significant difference was observed between cervical cancer and CINⅠ, CINⅡ or CINⅢ in the frequency of HPV-16 integration (P<0.01, table 1).
Table 1 Physical status of HPV-16 in patients with CIN and cervical cancer (CC) Integrated form Episomal form Mixed form Samples (n) Ratio (%) Samples (n) Ratio (%) Samples (n) Ratio (%) 12 7.95 125 87.81 14 9.27 58 23.58 164 66.67 24 9.76 47 39.17 53 44.17 20 16.67 73 65.18 17 15.18 22 19.64 190 30.21 359 57.07 80 12.72
Total (n) 151 246 120 112 629
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Fig. 4 Gel electrophoretogram of HPV integration detected by PCR A, B and C: Three fragments of amplicon; Control: Negative control without template DNA; siha cell: Positive control of integration; Patients 1 and 2: Two samples with distinct break sites; Patient 3:Sample with episome or mixed HPV infection; M: Marker
Based on gray scales per area of electrophoresis strap by multiple PCR, 17 episomal samples (15.18%) and 22 samples with mixed (19.64%) HPV-16 infection were detected in patients with cervical cancer. In situ hybridization was performed on some paraffin-embedded sections of CINⅡ, CINⅢ and cervical cancer to verify the physical status of HPV-16 infection. There were three kinds of positive signals in the host’s cells (fig. 6): (1) dispersed pattern: the positive hybridization signals showed the diffuse distribution of free bodies in the nuclear and perinuclear spaces; (2) granular pattern: the positive hybridization signals showed the massive distribution in the nuclear and perinuclear spaces; (3) mixed type: the positive hybridization signals showed the diffuse distribution and the massive distribution coexisted in the nuclear and perinuclear spaces. These images represented viral physical status of epi-
somal form, integrated form and mixed form respectively, and corresponded with results of PCR.
Fig. 5 Gel electrophoretogram of HPV integration detected by multiple PCR
siha cell: Negative control of pure integration; Patient 1: Sample with episomal HPV infection; Patients 2 and 3: Samples with mixed HPV infection
Fig. 6 Physical status of HPV16 detected by in situ hybridization (×200) A: In CINⅡ, the positive hybridization signals showed the diffuse distribution of free bodies in the nuclear and perinuclear spaces; B: In cervical cancer, the positive hybridization signals showed the massive distribution in the nuclear and perinuclear spaces, suggesting that HPV-DNA was in the integration status; C: In CINⅢ, the positive hybridization signals showed the diffuse distribution and the massive distribution coexisted in the nuclear and perinuclear spaces, suggesting that HPV-DNA was in the mixed form.
3 DISCUSSION Cervical cancer ranks the second most common cancer among women worldwide. It is reported by WHO in 2010 that there are about 50 million new cervical cancer cases and more than 250 000 patients died of cervical cancer each year worldwide. Approximately 80% of cervical cancer cases in the world occur in developing countries. In China, there are almost 131 500 new cases of cervical cancer and 20 000 to 30 000 deaths annually. These morbidity and mortality of cervical cancer in China is second only by Chile in the world and is arising year by year[13]. Further more, the age tendency of patients with cervical cancer is making more youthful.
Therefore, the prevention and treatment of cervical cancer in China is difficult and important. Numerous epidemiological, clinical and experimental studies have established the involvement of HPVs, particularly the “high-risk” (HR) HPV types, as indispensable carcinogenic agents for the development of cervical cancer, and almost all cases of cervical cancers show the presence of HR-HPV infection. Accordingly, screening of HR-HPV has been accepted as one part of screening schedule of cervical cancer. How to detect genotypes of HPV quickly and punctually is the investigative objective that many experts devote themselves to do. HC-Ⅱ HPV DNA detecting has been generally applied but it can’t offer an accurate typing of HPV and thus limited the application of this technology[14]. Other methods
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on trial in clinical diagnosis for instance GenoArray test (Hybriobio Ltd., HongKong) have not been certificated by FDA and SDA until now. Therefore, PCR is the acknowledged and commonly used facility in detecting nucleic acid. So HPV genotyping was performed using traditional PCR test and was validated through repeatedly sequencing in this study. It was found the prevalence of HPV-16 was 56.57% in cervical cancer in this study. As we know, HPV-16 is the most prevalent type and is associated with conjunctival papillomas/carcinomas and CIN and cervical cancers[15]. It was reported by WHO that the prevalence of HPV-16 was 56.6% in cervical cancer, 39.17% in HSIL, 23.58% in LSIL and 7.95% in normal cervix in 2009[16]. The WHO’s report is similar with this study and further supports the viewpoint that HPV-16 is the main infected subtype in the patients with cervical cancer. It has been well established that HPV is necessary but insufficient cause of cervical cancer and its precursors. Thus, additional risk factors are needed, which can be environmental, host- or virus-related factors, such as HPV type, integration or viral load for progression to cervical cancer. HPV DNA can be found in cervical materials in three forms: those are in episomal forms, integrated forms or mixed forms that contain both. Currently, an area of very active research interest is HR-HPV DNA integration into the host genome. It was reported the frequencies of integration of HPV-16 were 50% to 83%[17] and mixed form was the frequent one[18]. It has been confirmed that the integration of HPV DNA into the host genome is observed in majority of invasive cancers[19] but rarely in condyloma which is caused by LR-HPV[20]. So integration of HPV DNA into the host genome is thus likely to constitute a key step in oncogenesis. Numerous researches indicate that the frequency of integration is arising in CINⅠ, CINⅡ-Ⅲ and cervical cancer. It means the integration of HPV DNA into the host genome is a significant molecular biology event during the transformation of CIN to cervical cancer. We found the frequency of HPV-16 viral integration was 65.18% in cervical cancer and increased in parallel with the severity of cervical lesions (P<0.001). It means in the case of single HPV-16 infection there were close associations between the severity of cervical lesions and the frequency of HPV-16 integration (P<0.001), which is consistent with most of findings of molecular epidemiology previously reported[21-24]. However, Sathish found that frequencies of HPV-16 integration into the host genome in CIN and cervical cancer were all 68%[25] and thus some scholars presumed that the integration of HPV was not the crucial factor during the development of cervical cancer. So there are contraries about the physical status of HPV-16 in cervical lesions and the role of integration. Along with the acceptance of the causal relation of HR-HPV and cervical cancer, screening of HR-HPV associated with cervical cytopathology has been accepted as parts of screening schedule of cervical cancer. Since the integration of HPV DNA into the host genome is observed in majority of invasive cancers but rarely in condyloma and CIN and there is close correlation between the type of HPV and the frequency of virus integration[26], the application of testing HPV genotyping and
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