Trial Information

Comparison of Detection of Human Papillomavirus on Tissue, Saliva and Serum

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.