Abstract
BackgroundHuman papillomaviruses (HPV) are the aetiological agents of certain benign and malignant tumours of skin and mucosae; the most important of which is cervical cancer. Also, the incidence of ano-genital warts, HPV-anal cancer and oropharyngeal cancers are rising. To help ascertain a useful PCR detection protocol for oropharyngeal cancers, we directly compared three commonly used primer sets in detection of HPV from different clinical samples.MethodsWe compared PGMY09/11, MY09/11 and GP5+/6+ primers sets in PCRs of 34 clinically diagnosed samples of genital warts, cervical brushings (with associated histological diagnosis) and vulval biopsies. All negative samples were subsequently tested using the previously reported PGMY/GP PCR method and amplicons directly sequenced for confirmation and typing. An optimised PCR protocol was then compared to a line blot assay for detection of HPV in 15 oropharyngeal cancer samples.ResultsPGMY09/11 primers detected HPV presence in more cervical brushing (100%) and genital wart (92.9%) samples compared to MY09/11 (90% and 64.3%) and GP5+/6+ (80% and 64.3%) primer sets, respectively. From vulval biopsies, HPV detection rates were: MY09/11 (63.6%), GP5+/6+ (54.5%) and PGMY09/11 (54.5%). PGMY/GP nested PCR demonstrated that HPV was present, and direct sequencing confirmed genotypes. This nested PCR protocol showed detection of HPV in 10/15 (66.7%) of oropharyngeal cancer samples.ConclusionsPGMY09/11 primers are the preferred primer set among these three for primary PCR screening with different clinical samples. MY09/11 and GP5+/6+ may be used (particularly for cervical samples) but demonstrate lower detection rates. A nested PCR approach (i.e. a PGMY-GP system) may be required to confirm negativity or to detect low levels of HPV, undetectable using current primary PCR methods, as demonstrated using oropharyngeal cancer samples.
Highlights
Human papillomaviruses (HPV) are the aetiological agents of certain benign and malignant tumours of skin and mucosae; the most important of which is cervical cancer
We evaluated HPV detection in DNA from three common types of genital clinical samples; directly comparing genital warts, Cervical intra-epithelial neoplasia (CIN) brushings and vulval intra-epithelial neoplasia (VIN) lesions using the MY09/11, GP5+/6+ and PGMY09/11 primer sets in primary polymerase chain reaction (PCR) and with subsequent comparison with a nested PCR approach (PGMY-GP) [25]
We suggest that nested PCR should be performed on clinical specimens if the initial PGMY PCR is negative, if only to confirm HPV negativity
Summary
Human papillomaviruses (HPV) are the aetiological agents of certain benign and malignant tumours of skin and mucosae; the most important of which is cervical cancer. Strong epidemiological and molecular evidence has demonstrated human papillomavirus (HPV) to be the aetiological agent of both benign (warts, papillomas) and malignant tumours (subsets of ano-genital and oropharyngeal carcinomas) [1]. Strong evidence linking HPV to the development of approximately 20-50% (depending on the anatomical site) [7,8] of head and neck cancers has been accumulating [9,10,11]. This is an issue of great global importance as head and neck cancer is the 5th most common cancer in the world [12,13]. Mortality has not improved substantially over the last few decades [14], due to late diagnosis (75% of cases) and/or recurrent primary malignancies, and remains at 40-50% at 5 years [15,16]
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