Use of Universal and Type-specific Primers in the Polymerase Chain Reaction for the Detection and Typing of Genital Human Papillomaviruses

By using polymerase chain reaction (PCR), we have developed a system for type-specific as well as universal detection of genital human papillomaviruses (HPVs). Primers and probes for specific detection of HPV-16, -18 and -33 were synthesized from the E7 open reading frame (ORF). They were capable of detecting corresponding HPV types with high specificity and sensitivity. Primers for detection of a broad spectrum of HPV (universal primers) were synthesized from the LI ORF. The universal primers were shown to be capable of amplifying HPV-6b, -11, -16, -18, -33, -52b and -58. The system was applied to various cervical tissue specimens from Japanese patients. They consisted of 26 normal specimens, 18 from cervical dysplasias and 29 from cervical carcinomas. HPV was detected in none of the normal specimens. On the other hand, many of the specimens from cervical dysplasias and carcinomas were found to be positive for HPV, especially HPV-16. Except for one, all the specimens which were positive with the type-specific PCRs were also positive with the universal PCR. Furthermore, substantial numbers of specimens were found to be positive only with the universal PCR. Cloning and sequencing of DNA segments amplified by the universal primers were undertaken to characterize some of the unknown HPVs. Our PCR system may thus be useful for the specific detection of the three major types of oncogenic HPVs and also for the detection of a broad spectrum of HPVs including possibly novel HPV types.     

Use of universal and type-specific primers in the polymerase chain reaction for the detection and typing of genital human papillomaviruses.

By using polymerase chain reaction (PCR), we have developed a system for type-specific as well as universal detection of genital human papillomaviruses (HPVs). Primers and probes for specific detection of HPV-16, -18 and -33 were synthesized from the E7 open reading frame (ORF). They were capable of detecting corresponding HPV types with high specificity and sensitivity. Primers for detection of a broad spectrum of HPV (universal primers) were synthesized from the L1 ORF. The universal primers were shown to be capable of amplifying HPV-6b, -11, -16, -18, -33, -52b and -58. The system was applied to various cervical tissue specimens from Japanese patients. They consisted of 26 normal specimens, 18 from cervical dysplasias and 29 from cervical carcinomas. HPV was detected in none of the normal specimens. On the other hand, many of the specimens from cervical dysplasias and carcinomas were found to be positive for HPV, especially HPV-16. Except for one, all the specimens which were positive with the type-specific PCRs were also positive with the universal PCR. Furthermore, substantial numbers of specimens were found to be positive only with the universal PCR. Cloning and sequencing of DNA segments amplified by the universal primers were undertaken to characterize some of the unknown HPVs. Our PCR system may thus be useful for the specific detection of the three major types of oncogenic HPVs and also for the detection of a broad spectrum of HPVs including possibly novel HPV types.     

High risk HPV types in southern Iranian patients with cervical cancer

This study was undertaken to assess the rate of HPV infection in cervical carcinoma among southern Iranian patients. 101 archival cervical carcinoma tissue samples of a 10 year period were studied for the presence of HPV DNA in southern Iran by a polymerase chain reaction method. In addition, the presence of high risk HPV-16 and HPV-18 genotypes was investigated. In total, 88 (87.1%) of the samples were HPV DNA positive, of which 83 were squamous cell carcinomas and 5 were adenocarcinomas. HPV-16 genotype was detected in 26.7% of HPV positive cervical carcinomas; however, none of the samples were positive for the existence of HPV-18 genotype. Collectively, these results suggest that HPV-16 and HPV-18 are not the frequent high risk HPV types in our patients and circulating HPV types in southern Iranian population are different from many other populations.     

HPV infection in cervical-cancer cases in Russia

The presence of human papillomavirus (HPV) sequences in 21 biopsies from cervical carcinomas, II specimens of tissues adjacent to tumours, 2 specimens of cervical tissues with radiation fibrosis from patients after radiation therapy of cervical cancer and 7 normal epithelial tissues from the patients with other genital tumours were examined by polymerase chain reaction (PCR) and Southern-blot analysis. All tumours were HPV-positive by type-specific PCR and 86% by Southern-blot analysis. In normal epithelial and adjacent tissues, HPV sequences were detected in 20% of samples by Southern-blot analysis and in 70% of samples by PCR, including 2 cases of tissues after radiation therapy. HPV 16 was the most prevalent type in tumours (18/21) as well as in normal epithelial tissues (5/7). One HPV-positive tumour contained HPV18 DNA and 2 were doubly infected with HPVs 16 and 18 (2/21). The persistence of exclusively episomal HPV16 DNA was observed in 5 out of 11 tumours examined: 3 cases of squamous-cell carcinomas on the early stage of tumour progression and 2 advanced tumours (squamous-cell carcinoma and adenocarcinoma). The integration of HPV16 genome was detected in 6 out of 11 tumours, but most of them contained episomal forms of viral DNA simultaneously (5 out of 6). The integrative HPV18 genome was found in 2 tumours examined, and the persistence of episomal forms was also observed in one of them. Our data demonstrate that cervical tumours are associated invariably with high-risk types of HPV in Russia. © 1995 Wiley-Liss, Inc.     

Prevalence of human papillomavirus (HPV) infection in Basque Country women using slot-blot hybridization: a survey of women at low risk of developing cervical cancer.

Cervical smears from 1,178 women with cytologically normal cervices and 67 women with cervical intra-epithelial neoplasia (CIN) or cervical carcinoma were analyzed for the presence of HPV 6, 11, 16 and 18 DNA by slot-blot hybridization. HPV DNA was detected in 17% (95% CI: 14%-19%) of the women with a normal smear; 11% of infected women harboured HPV 16 and 18 (95% CI: 9%-13%, each). HPV DNA was detected in 54% (95% CI: 41%-66%) of patients with abnormal smear; the most prevalent virus type in this group was HPV 16 (45%; 95% CI: 32%-38%). In order to verify the slot-blot results, a proportion of the samples was also investigated by PCR. There was 88% correlation between the 2 tests. The high prevalence of HPV 16 and 18 infection demonstrated in our low-cervical-cancer-risk area further support the role of as yet unidentified co-factors as determinants of the different geographic rates of cervical-cancer incidence.     

宫颈癌细胞DNA含量和HPV16的对比研究

作者对３３例宫颈癌患者的活检组织进行了ＤＮＡ含量和ＨＰＶ１６检测。结果显示：宫颈癌细胞ＤＮＡ异倍体率为６６．７％，ＨＰＶ１６阳性率３３．３％。异倍体患者ＨＰＶ１６阳性率显著高于二倍体患者（Ｐ＜０．０５）；在异倍体病例中，不同倍体类型者ＨＰＶ１６阳性率明显不同，其顺序为多异倍体＞非整倍体＞四倍体＞近二倍体。ＨＰＶ１６阳性者的ＤＩ、ＳＰＦ、＞２Ｃ％均显著高于ＨＰＶ１６阴性者（Ｐ＜０．０１）。该结果提示，ＨＰＶ１６不仅与宫颈癌的发生关系密切，而且和癌细胞ＤＮＡ含量、增植特性、肿瘤恶性度也有一定关系。     

宫颈癌组织中人乳头瘤病毒16E6基因突变分析

背景与目的：高危型人乳头瘤病毒（humanpa pillomavirus,HPV）以16型最为常见,HPV16E6是宫颈癌主要的致癌基因之一,特定的E6突变是宫颈癌发生的主要因素之一。本研究主要观察兰州地区宫颈癌组织中是否存在E6突变,并探讨了突变与宫颈癌发生的关系。方法：以23例宫颈癌手术切除标本及5例正常宫颈组织的DNA作为模板,PCR扩增HPV-16E6基因201-523位,PCR产物直接测序．分析其HPV16E6基因的突变规律。结果：PCR扩增结果表明5例正常宫颈组织中HPV16E6阳性率为0％（0／5）,宫颈癌组织中HPV16E6阳性率为82．61％（19／23）,对18例样本PCR产物的测序和序列分析结果表明,6例（33．33％）E6基因与原型相同,12例（66．67％）E6基因发生突变,其中11例（61．11％）发生了350G突变。同时,在1例样本（5．56％）发现了249G突变。结论：兰州地区宫颈癌组织中存在着非常高的HPV感染率,且多数HP116E6发生了突变。     

The Prevalence of Human Papillomavirus in Cervical Cancer in Iran

Background: The human papiloma virus (HPV), which is sexually transmitted, and most commonly causesgenital warts, has been linked to cervical intraepithelial neoplasia and invasive carcinoma. Of ninety plus types ofHPV, HPV-16 is the most prevalent in cervical cancer, followed by HPV-18, and HPV-33. As HPV’s implication hasnot been assessed in the Middle East the main focus of this retrospective study was to determine the prevalence ofHPV –16,18, and 33 in cases of cervical cancer from Iran.Material and Methods: This retrospective study covered 100 patients with uterine cervical carcinomas who werereferred to two referral centers for cancer in Tehran-Iran. Pathological blocks were collected for these cases andinitial review of the blocks showed poor specimens in 18 cases, which left 82 cases for the study. These samples werehistologically examined to verify the presence and the type of carcinoma. The next step was in situ hybridzation forthe detection of HPV common DNA. In Situ hybridization was preformed on all samples. Finally, Polymerase ChainReaction (PCR) was preformed for the HPV types 16, 18, and 33. PCR amplification of exon 5 of the p53 gene wasused as an internal control for the integrity of DNA. Takara PCR Human papilloma Detection method was usedwhich includes primer for HPV 16, 18, and 33. Three primers were used alone, or in combination, in order toincrease the sensitivity of the detection.Results: The majority of tumors were squamous cell carcinomas (87%). The rest were adenosquamous carcinomaand adenocarcinomas. None of the 82 different cervical carcinoma tissue samples were found to be positive by in situhybridization. In the PCR samples, amplification of DNA was observed for 69 tumor specimens. In the remainning13cases, the DNA in fixed tissue was degraded, as verified by the absence of an internal control band (p53). Out of thetotal 69 tumors (85.5%) with adequate DNA contained HPV band on PCR. The majority (73.9%) of HPV positivetumors contained HPV-16; the rest (11.6%) demonstrated type 18 and 33. There was no correlation between thehistology of carcinoma and presence of types of HPV.Conclusion: The prevalence of HPV in carcinomas of uterine cervix in Iran is similar to those reported in otherregions of the world. Similarly, it appears that HPV-16 is the most common type associated with cervical cancer inIran. Further studies on larger samples of patients, particularly in those with pre-invasive forms of the disease, areneeded to elucidate the carcinogenic role of HPV types in cervical cancer in Iranian women.     

An association between human papillomavirus 16/18 deoxyribonucleic acid in peripheral blood with p16 protein expression in neoplastic cervical lesions

BACKGROUND: Human papillomavirus (HPV) infection plays a crucial role in cervical carcinogenesis. Apart from the detection of p16 protein in cervical tissues, the feasibility of the presence of HPV DNA in peripheral blood being an auxiliary marker of cervical lesions was examined. METHODS: Peripheral blood samples and cervical tissues, from 36 cervical tissues from high-grade squamous intraepithelial lesions (HSIL) and 31 early invasive cervical cancers (EICC), were analyzed for HPV 16/18 DNA and HPV 16/18 E7 mRNA expression, as well as the in situ expressions of p16 and pRb to investigate the in-between associations. RESULTS: The prevalence of HPV 16/18 DNA in patients with EICC was relatively higher than those of HSIL, in both of cervical tissues and peripheral blood. The presence of HPV 16/18 DNA in peripheral blood was positively correlated with that in cervical tissue, as well as with p16 overexpression in cervical tissues together with a significant correlation between E7 mRNA and pRb and p16 protein expressions. DISCUSSION: A positive correlation between the presence of HPV 16 or 18 DNA in peripheral blood and p16 overexpression in tissues of patients with cervical lesions was confirmed. Together with p16 immunostaining in cervical tissues, the detection of high-risk HPV 16 or 18 DNA in peripheral blood may act as an auxiliary biomarker for HPV-associated neoplastic cervical lesions.     

The physical state of human papillomavirus type 16 DNA in cervical carcinomas of Hong Kong Chinese.

The presence of human papillomavirus (HPV) in 15 cervical carcinoma specimens obtained from Hong Kong Chinese patients was analyzed by Southern blot hybridization studies. In nine (60%) of them, HPV 16 genomes were detected, while two others (13.3%) were found to harbor HPV DNA of unknown type closely related to HPV 16. All of them were classified as squamous cell carcinomas according to WHO guidelines. In addition, the presence of HPV 18 was shown in another two (13.3%) squamous cell carcinoma samples. Among the nine tumors harboring HPV 16, four specimens (44.4%) have HPV in integrated forms, while four others (44.4%) have HPV in episomal forms. The simultaneous presence of both episomal and integrated forms was demonstrated in the remaining tissue sample (11.2%). The result obtained here indicates a strong association between HPV infection and cervical carcinogenesis in Hong Kong Chinese, with HPV 16 prevalent in squamous cell carcinoma. Moreover, the persistence of HPV 16 episomes in some of the tumor specimens suggests that extrachromosomal HPV DNA, possibly acting synergistically with other oncogenic factors, is also capable of inducing cervical cancer.     

Detection and quantitation of human papillomavirus DNA in the plasma of patients with cervical carcinoma.

Human papillomaviruses (HPVs) play a central role in the development of cervical carcinoma. Plasma DNA from 232 patients taken at diagnosis or after treatment for invasive cervical cancer (n = 175) or carcinoma in situ (n = 57) and 60 normal controls were examined for HPV-16 or HPV-18 E7 DNA by conventional and real-time quantitative PCR assays. We found HPV-16 or HPV-18 E7 DNA in 6.9% (11 of 175) of invasive cervical cancer cases (18.1% of cases positive for HPV-16 or HPV-18 at the genital tract), 1.8% (1 of 57) of carcinoma in situ, and 1.7% (1 of 60) of normal controls by conventional PCR. Quantitative PCR identified the highest concentrations of HPV DNA (copy number of HPV/ml of plasma) in patients with invasive cervical cancer (mean, 11,163; median, 183.5), followed by a level of 8 in the single carcinoma in situ case and 0 copies in the normal control initially positive by conventional PCR. HPV DNA can be detected in the plasma of some patients with HPV-positive cervical tumors. It remains to be demonstrated whether quantitative PCR analysis of HPV DNA in plasma may have utility in patients at high risk of recurrent disease.     

宫颈癌高发区新疆维吾尔族妇女宫颈癌组织中HPV DNA的测定

目的 研究我国宫颈癌高发区新疆维吾尔自治区维族妇女宫颈癌发病与人类乳头状瘤病毒（HPV）的关系。方法 对75例新疆维吾尔族宫颈癌患者癌组织及20例正常宫颈组织,采用PCR（聚合酶链反应）技术检测HPV－C（总的HPV）、HPV16、HPV18及HPV6／11。结果 HPV－C、HPV16、HPV18及HPV6／11在宫颈癌患者及正常宫颈组织中的检出率分别为86．7％、72．0％、12．0％、0及20．0％、10．0％、0、10．0％。HPV16在HPV阳性患者中所占的比例为83．1％,宫颈癌患者中HPV－C、HPV16检出率明显高于正常对照组,鳞癌HPV16阳性率明显高于腺癌,而腺癌HPV18检出率明显高于鳞癌（P＜0．05）。HPV总感染率及HPV16阳性率,在不同临床分期及不同病理分级宫颈癌组织间无显著性差异。结论 HPV感染与我国宫颈癌高发区新疆维吾尔族妇女宫颈癌发病有密切相关,其中HPV16感染在其发病中起主要作用,但HPV感染对宫颈癌的进展影响不大。     

Detection of human papillomavirus (HPV) DNA in carcinoma of uterine cervix and genital tract diseases in Jiangsu province, China]

Pst-1 cleaved DNA restriction fragment length polymorphism (RFLP) of biopsy samples from 41 patients with squamous cell carcinoma of uterine cervix collected in Jiangsu province, China were examined for HPVs by Southern blot hybridization using HPV 16 DNA as a probe. 20 of the 41 samples were positive for HPVs when hybridized under non-stringent condition. HPV was not detectable in samples collected in the same time period from patients with cervical adenocarcinoma (N = 2), vaginal carcinoma (N = 3), vulval carcinoma (N = 1) and benign cervicitis (N = 8). Of the 20 positive samples, 7 (17.1%) had CHPV X1, a new type of NPV previously discovered is China, 6 (14.6%) had HPV 16, 4 (9.9%) had HPV 31, and in 3 (7.2%) the HPV type is as yet undetermined. Our data indicate that HPV 16 and CHPV X1 may be more closely related to cancer of the uterine cervix in Jiangsu province.     

Detection of human papillomavirus in primary hepatocellular carcinoma.

Human papillomaviruses (HPV) have been linked causally to some human cancers such as cervical carcinoma. To determine whether any additional type of human malignancy contained HPV DNA, we examined 16 hepatocellular carcinoma (HCC) specimens by Southern blot technique and by polymerase chain reaction (PCR). One HCC contained HPV 16 DNA as demonstrated by both Southern blot and PCR. Two other HCC samples contained HPV 18-related nucleotide sequences by PCR but were negative by Southern blot of genomic DNA. HPV could have been carried via blood to the liver, thus indirectly supporting the presence of an HPV viremia. Our findings suggest that oncogenic HPV might constitute a cofactor acting synergistically with hepatitis B virus (HBV) in the development of the HCC in these patients. Alternatively, the presence of HPV in the tumor tissue might be the result of an opportunistic infection.     

宫颈癌和宫颈上皮内瘤变中表皮生长因子受体表达与人乳头瘤病毒16和(或)18感染及其相互关系

目的探讨宫颈癌发生发展过程中,表皮生长因子受体(EGFR)与人乳头瘤病毒(HPV)的作用及其相互关系。方法宫颈癌组60例,选自1997年至2001年间中山大学肿瘤防治中心住院初治的宫颈癌病例,临床分期Ⅰa～Ⅱb期;宫颈上皮内瘤变(CIN)组40例;正常上皮对照组30例。以免疫组化S-P法检测宫颈组织EGFR的表达,以PCR检测HPV16和(或)HPV18感染。结果正常上皮组、CIN组和宫颈癌组的EGFR中强表达率呈梯度上升,分别为0、42.5%和76.7%,差异有统计学意义(P<0.05)。正常上皮组、CIN组和宫颈癌组的HPV16和(或)HPV18感染率分别为6.7%、67.5%和58.3%,宫颈癌组和CIN组的感染率均显著高于正常上皮组(P=0.000),但宫颈癌组与CIN组之间,差异无统计学意义(P=0.355)。肿瘤侵袭程度超过宫颈1/2间质者,EGFR中强表达率显著高于未达1/2间质者(89.2%:56.5%,P=0.004)。宫颈管侵袭者HPV16和(或)HPV18感染率显著高于无侵袭者(88.2%:46.5%,P=0.003)。EGFR与HPV之间无显著相关性(P>0.05)。EGFR与HPV均未显示与宫颈癌预后有关。结论EGFR和HPV与宫颈癌的发生发展有关;EGFR、HPV16和(或)HPV18与宫颈癌预后无关,EGFR与HPV16和(或)HPV18无显著相关性。     

Detection of human papillomavirus DNA in primary and metastatic lesions of carcinoma of the cervix in women from Okinawa, Japan

Of 351 patients with invasive cervical cancer treated at Ryukyu University Hospital, Okinawa, 293 who were tested for human papilloma virus (HPV) DNA of the primary cervical lesion before the initiation of treatment were considered for the study. The polymerase chain reaction (PCR) was performed using fresh specimens, immediately after sampling. In 250 of 293 patients (85.3%), HPV DNA was detected in cervical tumor by PCR using L1 consensus primer. The positive rate by histology was 89.9% in squamous cell carcinoma, 93.8% in adenosquamous carcinoma, and 51.4% in adenocarcinoma. The former two figures were significantly higher than the latter (p < 0.001 and p = 0.002). Concerning identification of HPV types, HPV 16 was most predominant in squamous cell carcinoma, whereas type 18 was relatively high in adenocarcinoma. However, the type distribution of HPV was different to some extent from those in other countries. During treatment, 489 nodal and other tissue samples were obtained from 113 of 250 HPV DNA-positive patients, and were submitted to an assay of HPV DNA. HPV DNA was amplified in all 55 metastatic samples and also in 12 of 434 nonmetastatic tissues (2.8%). HPV types specified in these samples were always identical with the HPV types determined in their primary tumors. In 154 samples from 29 of 43 HPV DNA-negative patients, HPV DNA was not detected, either in 14 metastatic samples or in 140 histologically benign samples. Cancer-free, but HPV DNA-positive nodal, liver, and pulmonary tissues could be interpreted to be already involved at the time of examination, by observing the clinical course of the disease over time.     

Prevalence of Human Papillomavirus subtypes 16 and 18 among Yemeni Patients with Cervical Cancer

Background: The Human Papillomavirus (HPV) is a DNA tumor virus that causes epithelial proliferation. There are more than 100 HPV subtypes, of which 13 subtypes are regarded as high risk subtypes that can cause cancers of epithelial mucosal surfaces. High risk human papilloma viruses (HR-HPV) subtypes 16 and 18 plays a major role in the etiology of cervical cancer worldwide. Therefore, the aim of this study was to screen for the existence of HPV16 and HPV18 among Yemeni women with cervical lesions. Methodology: Formalin fixed paraffin wax processed tissue blocks were retrieved for 200 patients (150 were previously diagnosed with cervical cancer and the remaining 50 were diagnosed with different benign conditions). Results: Of the 200 cervical cancer tissue specimens, HR-HPV 16 was identified in 74/200 (37%) samples and couldn’t be recognized in 126/200(63%) tissue samples. HR-HPV 18 was identified in 32/200 (16%) specimens and couldn’t be recognized in 168/200(84%) tissue specimens. Conclusion: HR-HPV subtypes were prevalent among Yemeni women with cervical cancer, with significant increase of HR-HPV subtype 16 over the HR-HPV subtype 18.