Studies of Small DNA Virus Encoded Oncogenes in Viral Carcinogenesis Using Laboratory Model Systems

Study subjects:

1. Inclusion criteria:

   • Age: 18 - 65 years old.
   • Women who are positive for HPV diagnosed by routine screening. Women willing to participate in the study and sign an informed consent

2. Exclusion criteria:

   • Age extremes (less than 18 years old or more than 65 years old).
   • Immuno-comprised patients, patients under steroid therapy or chemotherapy, or patients with serious medical illness that could affect their immune system.
   • Unknown medical history.

   Study Groups:

   • Group 1 (Cases): Patients known to have Cancer cervix with any degree of malignancy (diagnosed by routine screening or any other diagnostic tests)
   • Group 2 (Controls): Women sharing the same inclusion and exclusion criteria, but not known to have Cancer cervix

   Aim of The Study:

   1. Studying the role of HPV as an example of small DNA viruses in cell transformation and carcinogenesis.
   2. Determining the most HPV genotypes associated with high risks of cancer cervix occurrence.
   3. Detection of the HPV oncogenes playing role cell transformation and malignancy.

   4. Studying the role of viral DNA integration in cellular transformation and carcinogenesis.

      Study methods:

      Samples collection and storing:

      • Samples will be obtained from the cervix with the brush or swab\ by following the instructions corresponding to the type of collecting device.
      • Collection tubes will be stored at room temperature (15-30 °C).
      • Samples will be sent to the laboratory in less than 14 days following collection
      • The tubes can be preserved for 2-3 weeks at room temperature.

      HPV detection:

      • HPV cannot be propagated in tissue culture, and therefore, in most cases its accurate identification relies on molecular biology techniques, such as polymerase chain reaction (PCR).

      HPV Genotyping:

      • PCR-RFLP shows good discriminatory power by differentiating between HR and LR HPV genotypes, and it is possible to identify single or multiple infections .
      • In this technique, the amplified DNA is digested by restriction enzymes, resulting in DNA fragments of various lengths. Each fragment length is characteristic of a certain HPV genotype.
      • The commonest restriction enzymes are BamHI, Dd6eI, HaeIII, HinfI, PstI and RsaI.

      HPV Oncogenes and oncoproteins:

      • The main techniques used to detect mRNA for E6/E7 oncogenes are two commercial assays: PreTectW Proofer and APTIMAW HPV Assay. These techniques are based on transcription-mediated amplification of full-length E6/E7 transcripts using PCR.

      Statistical analysis:

      • Data will be analyzed by one-way analysis of variance using SPSS software version 24.0. Values will be expressed as mean ± S.D. For comparison between 2 groups, Student's t test will be used to determine whether the cases was significantly different from the control. Differences will be considered statistically significant at P\<0.05, while P\<0.01 will represent more significant change.