Persistent infection of high-risk human papillomavirus (HPV) has been recognized as the direct cause of cervical carcinoma. Therefore, detection and genotyping of HPV are important to cervical-cancer screening. In this study, we have evaluated the efficacy of flow-through hybridization and gene chip (HybriMax) on HPV genotyping through comparison of the results with Hybrid Capture II (HC-II) and
The human papillomavirus (HPV) belongs to the
Therefore, it is of considerable clinical value to establish a reliable and convenient method to detect and genotype HPV [
591 out of 7520 women who accepted liquid-based cytology examination in China-Japan Friendship Hospital from August 2004 to May 2005 were randomly selected for detection of the 21 HPV genotypes by HybriMax, and their mean age was 35.4 ± 7.7 (ranging from 20 to 64). Among them, 138 women (mean age was 35.8 ± 7.8), who diagnosed within normal limits with cervical cytology for at least two years without any cervical disease or operation, were described as “normal”. A total of 453 women were diagnosed with abnormal cytology, and the mean age was 34.1 ± 6.9. Cytological diagnosis (according to the 2001 Bethesda System) of those 453 patients were as follows: 161 cases with atypical squamous cells (ASC), 187 cases with low-grade squamous intraepithelial lesion (LSIL), 105 cases with high-grade squamous intraepithelial lesion (HSIL), or squamous cell carcinoma (SCC). Patients were classified into 6 groups according to their histopathology diagnosis from specimens of olcposcopic biopsy, loop electrosurgical excision procedure (LEEP), or cold-knife conization. These 6 groups include 152 cases of chronic cervicitis, 101 cases of cervical intraepithelial neoplasia (CIN, which indicates that dysplasia is seen on a biopsy of the cervix) I (mild dysplasia), 77 cases of CIN II (moderate to marked dysplasia), 76 cases of CIN III (severe dysplasia to carcinoma
With liquid-based cytology, samples were taken with the cervical brush at gynecological examination for HPV DNA testing. The collected specimens were stored at 4°C and processed within two weeks. It is prohibitive to apply vaginal douching three days prior to the collection of samples or to have sexual intercourse within one day. Sampling should not be carried out during menstrual period. All the positive cytological samples were confirmed by biopsies.
HPV genotyping by HybriMax was performed using an HPV GenoArray Test Kit (HybriBio Ltd., Chaozhou, China). This assay can determine 21 HPV types, including 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68), five low-risk HPV types (6, 11, 42, 43, and 44), and two unknown-risk types (53 and CP8304), by the flow-through hybridization technique using HPV DNA amplified by PCR. In brief, 0.5 mL specimen was centrifuged at 13,000 g for 15 min with KUBOTA 6930. Then, the supernatant was removed and the pellet was resuspended in 200
The flow-through hybridization was made on a prewarmed instrument at 45°C, and the HybriMem HPV-21 DNA microarray membrane was placed, which is marked with 21 HPV-genotype probes including HPV 6, 11, 42, 43, 44, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and cp8304. The number of samples tested in a batch could be adjusted from 1 to 15 as required. The PCR products were denatured at 95°C for 5 min just before hybridization and then was chilled on ice for at least 2 min. We mixed the PCR products with hybridization solutions and then added the mixture into sample wells to proceed with flow-through hybridization for about 5–10 min. The membrane was washed with hybridization solution, and the empty space was blocked without reaction. Adding NBT/BCIP solution to display the results, a positive result was indicated by a clearly visible indigo dot. The HPV-genotype result was determined according to the position of the HPV-genotype probes on the microarray chip. Multiple dots indicated multiple infections.
413 samples were detected by the commercially available HC-II assay (Digene Co., Gaitherburg, MD, USA). The probes used were designed to detect 13 types of high-risk HPV, including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. This enzyme-linked immunosorbent assay is based on a sandwich hybridization followed by a nonradioactive alkaline phosphatase reaction with chemiluminescence in the microplate. Samples were classified as positive for HPV DNA if their chemiluminescence was more than 1.0 pg/mL of control.
101 paraffin-embedded specimens were detected by ISH for HPV 16/18. It was performed with REMBRANDT universal DISH and AP Detection Kit (PanPath, The Netherlands). After being dewaxed and hydrated, paraffin sections (5-6
The statistical analysis was performed using the SPSS software (version 10.0) for chi-square test and Kappa index was calculated to evaluate the accordance of the results.
The positive rate of HybriMax for 13 types of HR-HPV (detected by HC-II) was 73.7% (303/413) and the positive rate of HC-II was 70.7% (292/413) (Table
Correlation between HybriMax and HC-II in detecting HPV DNA.
Groups | Cases | Positive rate % ( |
Kappa index | |
---|---|---|---|---|
HC-II | HybriMax | |||
Normal | 79 | 24.0 (19) | 31.6 (25) | 0.75 |
Chronic cervicities | 110 | 76.3 (84) | 81.8 (90) | 0.781 |
CIN I | 82 | 82.9 (68) | 80.4 (66) | 0.755 |
CIN II | 48 | 87.5 (42) | 87.5 (42) | 0.619 |
CINI III | 56 | 91.1 (51) | 96.4 (54) | 0.548 |
SCC | 18 | 100 (18) | 100 (18) | — |
Condyloma acuminata | 20 | 0.5 (10) | 0.4 (8) | 0.8 |
| ||||
Total | 413 | 70.7 (292) | 73.4 (303) | 0.814 |
If Kappa index ≥ 0.75, the accordance rates were regarded as excellent; if 0.4 ≤ Kappa index ≤ 0.75, the accordance rates were regarded as good. Abbreviations: CIN I: cervical intraepithelial neoplasia grade I; CIN II: cervical intraepithelial neoplasia grade II; CINI III: cervical intraepithelial neoplasia grade III; SCC: squamous cell carcinoma.
In addition, we found that there were 21 cases with HC-II-negative and HybriMax-positive diagnoses, including 8 cases of HPV18 (or multiple infections that include HPV18), 5 cases of HPV 68 (or that include HPV 68), 4 cases of HPV 31 (or that include HPV 31), 2 cases of HPV 33 (or that include HPV 33), and 2 cases of HPV 51 (or that include HPV 51). Another two cases with genotyping results of HPV 66, 2 cases with HPV 6, one case with HPV 6/11, and one case with HPV 44 by HybriMax were HC-II positive, the results of which may be out of the HC-II testing limits.
Table
The accordance of HybriMax and ISH for HPV 16/18 detection.
HybriMax | ISH | |
---|---|---|
Positive cases ( |
63 | 56 |
Positive rate (%) | 62.4 | 55.4 |
Accordance rate (%) | 89.1 | |
Kappa index | 0.776 |
Table
Positive rates of different HPV genotypes in each group detected by HybriMax.
HPV type | Group no. of patients % | Normal | ||||||
---|---|---|---|---|---|---|---|---|
Total | A | B | C | D | E | F | ||
16 | 28.9 | 19.7 | 21.8 | 32.5 | 47.4 | 55.6 | 15.0 | 8.0 |
18 | 16.8 | 14.5 | 14.9 | 15.6 | 21.1 | 25.9 | 20.0 | 6.5 |
31 | 9.7 | 11.2 | 6.9 | 10.4 | 13.2 | 3.7 | 5.0 | 1.4 |
33 | 9.9 | 6.6 | 11.9 | 13.0 | 10.5 | 14.8 | 5.0 | 0.7 |
35 | 0.2 | 0.7 | — | — | — | — | — | — |
39 | 4.2 | 5.3 | 4.0 | 3.9 | 3.9 | — | 5.0 | 1.4 |
45 | 0.6 | 0.7 | 1.0 | 1.3 | — | — | — | — |
51 | 4.0 | 3.3 | 2.0 | 10.4 | 2.6 | 3.7 | — | 0.7 |
52 | 19.0 | 11.8 | 21.8 | 26.0 | 25.0 | 22.2 | 5.0 | 3.6 |
53 | 8.6 | 10.5 | 8.9 | 6.5 | 6.6 | 3.7 | 15.0 | 2.2 |
56 | 4.0 | 4.6 | 7.9 | 2.6 | 1.3 | — | — | 0.7 |
58 | 19.1 | 14.5 | 22.8 | 18.2 | 28.9 | 18.5 | — | 3.6 |
59 | 1.1 | 1.3 | 2.0 | 1.3 | — | — | — | — |
66 | 5.1 | 3.9 | 5.9 | 3.9 | 5.3 | 11.1 | 5.0 | — |
68 | 8.4 | 9.9 | 10.9 | 6.5 | 6.6 | 7.4 | — | 7.2 |
81 | 8.8 | 8.6 | 7.9 | 13.0 | 7.9 | 3.7 | 10.0 | 0.7 |
6 | 2.9 | 0.7 | 5.0 | 1.3 | 1.3 | — | 30.0 | 0.7 |
11 | 4.4 | 2.6 | 3.0 | — | 2.6 | — | 55.0 | 2.9 |
42 | 0.2 | 0.7 | — | — | — | — | — | — |
43 | — | — | — | — | — | — | — | — |
44 | 0.8 | — | 2.0 | 1.3 | — | — | 5.0 | — |
Group A: chronic cervicitis; group B: CIN I; group C: CIN II; group D: CIN III; group E: SCC; group F: condyloma acuminata.
The 10 most common genotypes and their infection rates with abnormal cytological diagnosis in turn (descending) were HPV 16 (28.9%), 52/58 (19.0%), 18 (16.8%), 33 (9.9%), 31 (9.7%), 81 (8.4%), 53 (8.6%), 68 (8.4%), 66 (5.1%), and 43 (0%). The 10 most common genotypes in normal groups were HPV 16 (8.0%), 68 (7.2%), 18 (6.5%), 52/58 (3.6%), 11 (2.9%), 53 (2.2%), 31/39 (1.4%), and 33 (0.7%), while HPV 35, 45, 59, 66, 42, 43, and 44 were not detected.
The 10 most common genotypes in different groups (in descending order) were as follows: HPV 16, 18/58, 52, 31, 53, 68, 81, 33, and 39 in the group of chronic cervicitis; HPV 58, 16/52, 18, 33, 68, 53, 56/81, and 31 in the group of CIN; HPV 16, 52, 58, 18, 33/81, 31/51, 53, and 68 in CIN; HPV 16, 58, 52, 18, 31, 33, 81, 53/68, and 66 in CIN III; HPV 16, 18, 52, 58, 33, 66, 68, and 31/51/53 in group of SCC. We come to the conclusion that HPV 16, 18, 52, 58, 33, and 31 were the 6 most common HPV types that can infect the patients with cervical lesions. The most common HPV types causing condyloma acuminata were HPV 11 (with a total positive rate of 55.0%) and HPV 6 (with a total positive rate of 30.0%).
The positive rates of the 6 most common genotypes in different groups were shown in Figure
The positive rates of the 6 most common genotypes in different groups. It shows the positive rates of the 6 most common genotypes in different groups. CIN: cervical intraepithelial neoplasia, SCC: squamous cell carcinoma. HPV 16 was the most frequent type in almost all the groups (except for CIN I, less than HPV 58); the positive rate increased with the development of disease. Abbreviations: CIN I: cervical intraepithelial neoplasia grade I; CIN II: cervical intraepithelial neoplasia grade II; CINI III: cervical intraepithelial neoplasia grade III; SCC: squamous cell carcinoma.
Previous studies have documented that HPV plays a central role in the etiology of cervical cancer [
Flow-through hybridization is the most efficient method for molecular hybridization [
Among the 21 cases which HC-II diagnosed negative while HybriMax revealed positive, there were 8 cases of HPV 18 and 5 cases of HPV 68, which probably suggested that HC-II was less sensitive to those HPV types. There were some samples detected HPV 6, 11, 44, and 66 positive by HybriMax, present also positive by HC-II, which should be negative (not included in the range of genotype which can detected by HC-II). It was indicated that there was cross-reaction between the probes of the HC-II HPV types. It had been reported that the probes of HC-II can have a cross-reaction with less than 22 types of HPV DNA other than 13 types of HC-II [
In addition, this study revealed that, in China, the 6 most common genotypes in cervical lesions were HPV 16, 18, 52, 58, 33, and 31 included in cervical cancer. The recent international prevalence surveys by the International Agency of Research on Cancer (IARC) reported that the most common HPV types of invasive cancer were 16 (57.4%), 18 (16.6%), 45 (6.8%), 31 (4.3%), 33 (3.7%), 52 (2.5%), 58 (2.3%), 35 (2.2%), 59 (1.5%), and 56 (1.3%), but the study did not include the Chinese population [
This study also found that the positive rates of HR-HPVs in groups of abnormal cytological diagnoses were prominently higher than that of normal groups. It has been revealed that abnormality of cytology is strongly related to HPV infection. Therefore, it is suggested that HPV detection is especially important for women with abnormal cytological findings.
This study was supported by Zhejiang provincial key medical discipline construction program of China (no. GJSX-010-004).