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Human Papillomavirus on Oral Tissue, Saliva and Serum (CDHPOTSS)

The safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Listing a study does not mean it has been evaluated by the U.S. Federal Government. Read our disclaimer for details. Identifier: NCT01043328
Recruitment Status : Completed
First Posted : January 6, 2010
Last Update Posted : March 26, 2013
Information provided by (Responsible Party):
Adriana Demathé, UPECLIN HC FM Botucatu Unesp

Brief 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.

Condition or disease
Squamous Cell Carcinoma

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.

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Study Type : Observational
Actual Enrollment : 80 participants
Observational Model: Case Control
Time Perspective: Retrospective
Official Title: Comparison of Detection of Human Papillomavirus on Tissue, Saliva and Serum
Study Start Date : May 2009
Actual Primary Completion Date : January 2010
Actual Study Completion Date : December 2010

Resource links provided by the National Library of Medicine


The study group is composed by patients with a condition that requires a procedure/surgery for oral squamous cell carcinoma treatment.

INTERVENTIONS: Collect blood, saliva and oral tissue.


Group without oral squamous cell carcinoma but with a condition that requires prosthetic procedure/surgery.

INTERVENTIONS: Collect blood, saliva and oral tissue.

Primary Outcome Measures :
  1. DNA HPV status (positive/negative) [ Time Frame: six months ]

Secondary Outcome Measures :
  1. clinical and pathological characteristics [ Time Frame: six months ]

Biospecimen Retention:   Samples With DNA
This biospecimens will be retained: whole blood, exfoliated cells, saliva and formalin-fixed paraffin-embedded tissues.

Information from the National Library of Medicine

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Ages Eligible for Study:   30 Years to 85 Years   (Adult, Older Adult)
Sexes Eligible for Study:   All
Accepts Healthy Volunteers:   Yes
Sampling Method:   Non-Probability Sample
Study Population
university dental care clinic patients with or without a condition

Inclusion Criteria:

  • Pathological diagnosis of squamous cell carcinoma, presented lesions with primary site in the mouth or oropharynx;
  • Matched controls without a condition.

Exclusion Criteria:

  • Patients that received radiotherapy.

Information from the National Library of Medicine

To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.

Please refer to this study by its identifier (NCT number): NCT01043328

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Glauco Issamu Miyahara
Aracatuba, Sao Paulo, Brazil, 16015050
Sponsors and Collaborators
UPECLIN HC FM Botucatu Unesp
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Principal Investigator: Adriana Demathe, PhD UNESP Dental School
Study Director: Glauco I Miyahara, PhD UNESP Dental School

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Responsible Party: Adriana Demathé, PhD Adriana Demathe, UPECLIN HC FM Botucatu Unesp Identifier: NCT01043328    
Other Study ID Numbers: upeclin/FOA-Unesp-05
First Posted: January 6, 2010    Key Record Dates
Last Update Posted: March 26, 2013
Last Verified: March 2013
Keywords provided by Adriana Demathé, UPECLIN HC FM Botucatu Unesp:
human papillomavirus
nested PCR
oral tissues
Additional relevant MeSH terms:
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Carcinoma, Squamous Cell
Neoplasms, Glandular and Epithelial
Neoplasms by Histologic Type
Neoplasms, Squamous Cell