Multiplex nested PCR (MNP) assay for the detection of 15 high risk genotypes of human papillomavirus

. According to IARC 5 lip cancer incidence in Brazil for 2007 ranging of 0.8/100,000 (Brasília) to 1.4/100,000 (São Paulo) for man and ranging 0.1/100,000 (Cuiabá) to 0.5/100,000 (Goiânia) for woman.The highest prevalence of lip SCC occurs between the sixth and seventh decades of life. The lower lip is the oral site more affected by SCC and usually diagnosed in stage I 7 . South of Australia is among the regions with the highest incidence of lip SCC, with 13.49 cases/100,000 inhabitants/year in men and 3.21 cases/100,000 inhabitants/year in women. In Europe, specifically in Spain, lip SCC represents a major impact on men (12.70 cases/100,000 inhabitants/year), and in Switzerland presents the highest incidence in women (0.83 cases/100,000 inhabitants/ year) 8 . In France, the lip, mouth and pharynx cancer incidence reaches 38.5/100,000 9 . In Brazil, according to the estimate of National Institute of Cancer for 2008, the incidence of oral cancer was 14.45/100,000 in men and 4.83/100,000 in women non-smokers and non-drinkers suggesting that other factors, including certain viruses, may have implications on the oral carcinogenesis 11,12 . Among the viruses that appear to be related to oral cancer etiology is the human papillomavirus (HPV) [11][12][13] . HPV role in the etiology of cervix carcinoma is clear, however its participation on carcinogenesis in oral cavity and other anatomical sites such skin, esophagus, paranasal conjunctiva, bronchus, larynx, oropharynx only is suggested 13 . Syrjänen and cols. described in 1983 14 a possible link between HPV and oral cancer due to the observation of cytopathic alterations in oral lesions similar to those typically induced by HPV previously found in cervix cancer. Regarding the significance of HPV DNA presence in prognosis of patients with oral SCC, literature data are controversial [15][16][17][18] . However, its has been suggested differences between patients infected and no infected with HPV in relation to sex, age, tumor regions, histological grade, regional metastasis, rate of recurrence and survival 18. Due to the great controversy in the literature, further studies are needed to help clarify the remaining questions that still persist. The aim of this study was to investigate HPV DNA presence in biopsies of lip SCC and correlate with clinical aspects and risk factors. Materials and MethodsThis study was reviewed and approved by Research Ethics Committee of São Paulo State University -Dentistry School of Araçatuba. Thirty-three caucasian patients presenting lip SCC confirmed by histological diagnosis participated in the study.A questionnaire was completed at the initial patient trial asking about age, sex, occupation, tobacco and alcohol consumption and sun exposure. If the patient had oral cancer and none of this risk factors above the risk factor is described as "other risk factors". Smokers were considered those that smoked regularly during any time of life, even those who stopped. Non-smokers were those who never used tobacco. The same criteria were used f...

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“…PCR studies with paraffin-embedded tissues have showed worst results when compared with nPCR [28][29][30] , fact ratified in this study.…”

Section: Discussionsupporting

confidence: 64%

Human papillomavirus (HPV) detection in lip squamous cell carcinoma: correlation with clinical aspects and risk factors

Demathé

Garcia

Mattar³

et al. 2011

Rev. bras. epidemiol.

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“…This assay simultaneously detects and identifies 21 HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 70, 73, 82, 53, 6, 11 and 26). The HPV 16 assay (Supplementary Figure 1) has a sensitivity of 400 viral genome copies per ml at the 95% confidence level (CI) and compares well with our nested PCR method, which was validated against previously published PCR methods (Brestovac et al , 2005). DNA was extracted using the RNA QIAmp RNA viral mini kit (Qiagen, Hilden, Germany).…”

Section: Methodsmentioning

confidence: 68%

Human papillomavirus predicts outcome in oropharyngeal cancer in patients treated primarily with surgery or radiation therapy

et al. 2010

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Objective:This study examines the prognostic significance of human papillomavirus (HPV) in patients with locally advanced oropharyngeal squamous cell carcinoma (SCC) treated primarily with surgery or definitive radiotherapy.Methods:One hundred and ninety-eight patients with Stage 3/4 SCC were followed up for recurrence in any form or death from any cause for between 1 and 235 months after diagnosis. HPV status was determined using HPV E6-targeted multiplex real-time PCR/p16 immunohistochemistry. Determinants of recurrence and mortality hazards were modelled using Cox's regression with censoring at follow-up dates.Results:Forty-two per cent of cancers were HPV-positive (87% type 16). HPV predicted loco-regional control, event-free survival and overall survival in multivariable analysis. Within the surgery with adjuvant radiotherapy (n=110), definitive radiotherapy-alone (n=24) and definitive radiotherapy with chemotherapy (n=47) groups, patients with HPV-positive cancers were one-third or less as likely to have loco-regional recurrence, an event or to die of any cause as those with HPV-negative cancers after adjusting for age, gender, tumour grade, AJCC stage and primary site. The 14 patients treated with surgery alone were considered too few for multivariable analysis.Conclusion:HPV status predicts better outcome in oropharyngeal cancer treated with surgery plus adjuvant radiotherapy as well as with definitive radiation therapy±chemotherapy.

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“…Although type-specific PCR primer sets allow the identification of individual genotypes of HPV, the need to perform multiple and parallel amplifications for each sample imposes severe constraints on throughputs. Recently, two independent multiplex PCR methods have been reported and allow the detection and genotyping of mucosotropic HPV, including infections with multiple genotypes (6,36). These simple PCRbased methods are capable of detecting multiple HPV genotypes in a single reaction tube on the basis of the product size.…”

Section: Discussionmentioning

confidence: 99%

“…Sixteen HPV genotypes (genotypes 6,11,16,18,30,31,33,35,39,45, 51, 52, 56, 58, 59, and 66) were detected by multiplex PCR in a single tube. PCRs were performed with a multiplex PCR kit (Qiagen Inc., Valencia, CA), according to the manufacturer's instructions, with minor modifications.…”

Section: Methodsmentioning

confidence: 99%

Genotyping of Human Papillomaviruses by a Novel One-Step Typing Method with Multiplex PCR and Clinical Applications

Nishiwaki¹,

Yamamoto²,

Tone³

et al. 2008

J Clin Microbiol

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We describe here a rapid, high-throughput genotyping procedure that allows the simultaneous detection of 16 high-and low-risk genital human papillomavirus (HPV) types by multiplex PCR in a single reaction tube. Multiplex PCR is based on the amplification of HPV DNA by sets of HPV genotype-specific primers, and the genotypes of HPV are visually identified by the sizes of amplicons after they are separated by capillary electrophoresis. The procedure does not include a hybridization step with HPV-specific probes and is rapid and labor-saving. We detected all 16 HPV genotypes (types 16, 58, 52, 51, 56, 31, 18, 39, 66, 59, 6, 33, 30, 35, 45, and 11) with a high sensitivity and a high degree of reproducibility. By using this newly developed method, we conducted a pilot study to examine the correlation between the prevalence and genotype distributions of HPV and the cytological group classifications for 547 cervical samples. Compared with the group of samples considered normal (14.7%), there was a significant increase in the prevalence of HPV in women with atypical squamous cells of unknown significance (61.3%), low-grade intraepithelial lesions (75.8%), and high-grade intraepithelial lesions (HSILs) (82.2%). The prevalence and distribution of type 58 were correlated with cytological malignancies, with the highest prevalence in women with HSILs. In conclusion, the novel multiplex PCR method described appears to be highly suitable not only for the screening of cervical cancer precursor lesions but also for the characterization of genotype distributions in large-scale epidemiological studies and HPV vaccination trials.Accumulating evidence indicates that persistent infection with high-risk human papillomaviruses (HPVs) is indeed a major causative factor in the development of cervical intraepithelial neoplasia and invasive cervical carcinoma (42,10,8,5,40,27,11,30). The HPV family includes over 100 genotypes, 30 to 40 of which are mucosotropic, and at least 15 types of the mucosotropic HPVs have been linked to cervical cancer (5,8,10,42). In addition, some of these types are also related to other cancers of the genital tract (21,22) and to cancers of other organs (14, 28). Light microscopic examination of a Papanicolaou (Pap)-stained smear is of primary importance for the detection of cervical cancer precursor lesions. It has been demonstrated that concomitant testing for DNA of the highrisk HPV types by the Pap test can clearly identify women at high risk for cervical cancer, particularly if persistent infection by high-risk HPV types is diagnosed (5,8,10,11,27,30,40). Furthermore, HPV genotyping is of critical importance for the investigation of the clinical behavior and the epidemiology of HPV infection, for population studies for HPV vaccination trials, and for monitoring of the efficacy of HPV vaccines. Several genotyping methods have been developed in order to identify high-risk HPV in liquid-based cytology (LBC) samples and tissue samples (1,12,34). The molecular techniques that have been applied for HPV DNA detect...

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Multiplex nested PCR (MNP) assay for the detection of 15 high risk genotypes of human papillomavirus

Cited by 17 publications

References 29 publications

Human papillomavirus (HPV) detection in lip squamous cell carcinoma: correlation with clinical aspects and risk factors

Human papillomavirus (HPV) detection in lip squamous cell carcinoma: correlation with clinical aspects and risk factors

Human papillomavirus predicts outcome in oropharyngeal cancer in patients treated primarily with surgery or radiation therapy

Genotyping of Human Papillomaviruses by a Novel One-Step Typing Method with Multiplex PCR and Clinical Applications

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