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Comparison Between Single PCR and Nested PCR in Detection of Human Papilloma Viruses in Paraffin-embedded OSCC and Fresh Oral Mucosa
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Oral and Maxillofacial Surgery.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Oral and Maxillofacial Surgery.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Oral and Maxillofacial Surgery.
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2015 (English)In: In Vivo, ISSN 0258-851X, E-ISSN 1791-7549, Vol. 29, no 1, 65-70 p.Article in journal (Refereed) Published
Abstract [en]

Background: Infection with human papilloma virus (HPV) has been implicated as one of the risk factors for the development of oropharyngeal cancer. Many different HPV tests exist, and information regarding their specific technical, analytical, and clinical properties is increasing. Aim: This study aimed to compare the level of detection of HPV using two reliable polymerase chain reaction (PCR) methods, nested PCR (NPCR) and single PCR (SPCR), in archival paraffin-embedded oral squamous cell carcinoma (OSCC) samples and fresh oral mucosa specimens. Materials and Methods: The presence of HPV genome in two groups of tissue samples was analyzed: (i) 57 paraffin-embedded OSCC samples from Sudan and (ii) eight healthy fresh oral mucosal samples from Swedish volunteers. The specimens were tested by SPCR with primer pair MY9/MY11 and NPCR using GP5+/GP6+ primer sets. Results: Eighteen (32%) out of the 57 paraffin-embedded OSCC samples, and five (62%) out of the eight fresh clinically healthy samples were found to be HPV-positive with NPCR. With SPCR, four (7%) out of the paraffin-embedded OSCC samples were HPV-positive. A statistically significant difference between HPV-positive and -negative samples was found when comparing NPCR and SPCR in OSCC and fresh oral mucosa (p<0.0001). The comparative test between SPCR and NPCR showed 100% sensitivity and 69% specificity for OSCC. Conclusion: The use of the GP5+/GP6+ nested PCR increased the positivity rate, efficiency rate and sensitivity of HPV detection in oral samples significantly and should be considered as the method of choice.

Place, publisher, year, edition, pages
2015. Vol. 29, no 1, 65-70 p.
Keyword [en]
HPV, nested PCR, single PCR, paraffin embedded, OSCC, fresh oral mucosa
National Category
Surgery
Identifiers
URN: urn:nbn:se:uu:diva-245198ISI: 000348134300011PubMedID: 25600532OAI: oai:DiVA.org:uu-245198DiVA: diva2:790813
Available from: 2015-02-25 Created: 2015-02-25 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Oral cancer with special reference to virus detection and quantitative gene expression
Open this publication in new window or tab >>Oral cancer with special reference to virus detection and quantitative gene expression
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background. Head and neck cancers (HNC) are among the most common malignancies worldwide, and about 90–92% of oral neoplasias are oral squamous cell carcinomas (OSCC). Alcohol and tobacco consumption have been recognized as the main risk factors for OSCC development. Oncogenic viruses, such as human papillomavirus (HPV) or Epstein-Barr virus (EBV), as well as genetic alterations may also contribute to tumour formation. 

Aims. To study the prevalence of HPV, EBV, Herpes simplex type-1 (HSV-1), and HPV-16 and their integration status as well as the molecular mechanisms that can serve as a basis for the development of OSCC.

Results. In Paper I we reported a statistically significant increase in the prevalence of HPV-16 in oral epithelial dysplasia (OED) and OSCC samples compared to controls. A statistically significant increase was also seen in integrated HPV-16 compared to episomal viral forms when comparing OED and OSCC samples. Paper II reported the detection of HSV-1 in 54% of healthy samples, in 36% of oral leukoplakia samples, and 52% of OSCC samples. However, these differences were not statistically significant. In Paper III we reported a statistically significant increase in the detection of HPV-positive samples when comparing nested polymerase chain reaction (PCR) with single-PCR results in OSCC and fresh oral mucosa. Paper IV reported that the highest prevalence of HPV (65%) was seen in Sudan, while an HSV-1 prevalence of 55% and an EBV prevalence of 80% were seen in the UK. Finally, Paper V reported that the mRNA levels of Bcl-2, keratin 1, keratin 13, and p53 were significantly lower and that the level of survivin was significantly higher in the OSCC samples of the toombak users than in their paired control samples. Significant downregulation in keratin 1 and keratin 13 expression levels was found in the OSCC samples of the non-toombak users relative to their normal control samples.

Conclusion. HPV-16 integration was increased in oral epithelial dysplasia and OSCC compared to normal oral mucosa. Nested PCR is a more accurate method of establishing HPV prevalence in samples containing low copy numbers of HPV DNA. HPV and EBV may be a risk factor in OSCC development. Our findings confirmed the role of survivin in OSCC carcinogenesis and survivin might be interesting as a biomarker to be monitored. The results presented here provide both clinical and biological insights that will bring us closer to the goal of managing this disease and improving treatment and outcomes for future patients.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 72 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1179
Keyword
HPV, EBV, HSV-1, Oral Squamous Cell Carcinoma, Leukoplakia, apopto-sis, cell cycle regulation, intermediate filament proteins.
National Category
Natural Sciences Medical and Health Sciences
Research subject
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-275516 (URN)978-91-554-9476-6 (ISBN)
Public defence
2016-03-18, Skoog-salen, Akademiska sjukhuset, Ingång 79, Uppsala, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2016-02-26 Created: 2016-02-04 Last updated: 2016-03-09

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Jalouli, MirandaJalouli, JamshidHirsch, Jan-MichaelSand, Lars

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