Human Papillomavirus on Oral Tissue, Saliva and Serum
Condition: Squamous Cell Carcinoma
MeSH terms: Carcinoma, Squamous Cell
Study type: Observational

Summary: 
      Human papillomavirus (HPV) is one of the most prevalent infections in the world with several
      millions of new cases diagnosed yearly. Oral HPV infection may be associated with different
      diseases of oral cavities including some cases of oropharyngeal cancer.

      The aim of this report is to detect the presence of HPV DNA in samples of biopsies, oral
      swabs, saliva and serum of patient with oral squamous cell carcinoma (OSCC) and controls. We
      hoped to find there is correlation among the presence of HPV DNA in the several biological
      materials and if it is possible to use the saliva as screening to HPV DNA detection. The
      presence of tumor HPV DNA in blood may be of diagnostic and prognostic value.
    

Detailed description: 
      This study will made at the buccal cancer center of UNESP and involved forty patients (n =
      40) and forty controls. Serum samples, oral swabs and saliva will collect at the date of
      diagnosis before therapy and will store at -80 ºC until analysis. Formalin-fixed
      paraffin-embedded oral squamous cell carcinoma tissues and other biologic samples will
      process with phenol/chloroform extraction method.

      Tumors from 40 OSCC patients at the UNESP University will be obtained from biopsy with prior
      consent, along with corresponding venipuncture blood, saliva collection and exfoliated buccal
      cells samples. From controls will be obtained all samples except biopsy tissues. Clinical
      information including tumor location, stage, and nodal status will be recorded.

      Clotted blood specimens will be centrifuged at low speed for 5 min, and the serum was stored
      at - 80 °C before DNA extraction. Serum samples (400 ml) will be used for DNA extraction.
      Whole saliva and exfoliated buccal cells will be digested in proteinase K at 48°C during two
      hours, serum and tumor tissue samples will be digested in proteinase K at 48°C overnight,
      followed by phenol/chloroform extraction and ethanol precipitation of DNA for all samples.
      After resuspension in 50 ml of distilled water, the mean working DNA concentrations will be
      100-150 ng/ml per serum and tissues samples and 30-50 ng/ ml per whole saliva and exfoliated
      buccal cells samples. For beta-globin PCR will be used 150 to 300 ng of purified total
      cellular DNA, to assess the quality of the DNA using the PCR primers GH20 and PC04. After
      confirmation of the presence and integrity of genomic human DNA, the same amount of DNA will
      be testing for HPV DNA by nested polymerase chain reaction (PCR) in all samples. In first PCR
      round degenerate consensus primers MY11 and MY09 will be using to amplify fragments of 450
      bp. HPV DNA will amplified in a second round by GP5+ and GP6+ primer sets. The other reaction
      components will be: 10.9 microlitres of ultra-pure water, 2.5 microlitres PCR buffer 10X, 4mM
      MgCl2, 15 pmol dNTPs and 1 unit of Platinum Taq DNA polymerase. Approximately 150-300 ng of
      genomic DNA from each sample will be add to the mixture. The same amount of Hela cells, with
      up to 4 copies of HPV-18 per cell, will be used as positive control for HPV infection. The
      negative control will be composed by all PCR components except DNA. The mixture underwent
      initial denaturation to 94ºC for 10 min, before 40 PCR cycles (94ºC for 1 min; 55ºC for 1
      min; 72ºC for 40) and 72°C for 4 minutes. For nPCR, two microliters of the product from the
      first reaction will be used directly in a reaction containing: 0,02 mM of each primer GP5+
      and GP6+(Invitrogen Life Technologies®, Brazil), which produce a 150 pb DNA fragment. The
      remaining reaction components and conditions will be as described for the first round of PCR,
      except for the annealing temperature that will be reduced to 43ºC. Ten microliters of the
      nPCR products will be fractionated by electrophoresis in a 8% polyacrylamide gel, for 3 hours
      at 100 volts. Band visualization will be performed by staining with silver nitrate solution.
      Samples will be scored as either HPV DNA-positive or negative based on the inspection of
      silver nitrate stained bands. PCR amplification will be performed in triplicates for each
      sample. Samples will be classified as positive or negative based on gel analysis.

      Differences in proportion will be evaluated by means of Fisher's exact test. A P value of
      less then 0.05 will be considered statistically significant. These statistical calculations
      will be performed using SPSS, version 10.0, for Windows.
    

Observational model: Case-Control
Time perspective: Retrospective
Sampling method: Non-Probability Sample
Study population: 
        university dental care clinic patients with or without a condition
      
Biospecimen description: 
      This biospecimens will be retained: whole blood, exfoliated cells, saliva and formalin-fixed
      paraffin-embedded tissues.
    
Keywords: human papillomavirus; nested PCR; saliva; serum; oral tissues