HPV Quantitative Real-Time PCR
Cervical cancer is one of the major malignancies of the reproductive system. Every eighteen minutes a woman in Europe loses her life due to cervical cancer, while in Cyprus annually 30 deaths are reported from this cause.
The main causative agent of this type of cancer is the Human Papilloma Virus (HPV), mainly transmitted through sexual contact. This virus has a circular structure and consists of a proteins hell (capsule) through which it attaches to the host cells so that its genetic material (DNA) can enter the host. HPV DNA is integrated into the cervical cell genome and induces the activation of oncogenes while supressing the immune response against the virus by cytotoxic T-lymphocytes of the host. Simultaneously, HPV proteins inhibit the repair enzymes during DNA replication in the dividing cells, resulting in genome mutations and uncontrollable cell proliferation.
Such an infection, if not detected early, immediately leads to carcinogenesis. To date, approximately 198 different types of HPV strains have been identified. The percentage of viral genome detection from biopsies is as high as 99.5%. Approximately 40 strains of the virus have been identified in epithelial or skin of the rectum in men and women. The remaining strains usually invade the skin in other areas of the body, the epithelial of the oral cavity, the upper respiratory tract and rarely in other tissues.
Depending on the ability of the strain to induce carcinogenesis in the epithelium, the strains can be categorised into 2 types: high- and low-risk. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, 82 belong to the high-risk types of inducing oncogenesis and are responsible for the development of precancerous and cancerous lesions that lead to the development of cancer in 5-15 years and in rarer cases in 1-2 years. More particularly, type 16 HPV is detected in 50% of cervical cancer cases in Europe. The strains 6, 11, 36, 42, 43, 44, 46, 47, 50 belong in the low-risk types of inducing oncogenesis. These strains can cause genital warts that belong to the benign lesions.
Molecular testing for HPV consists of a comprehensive picture of the risk in developing cervical cancer, assisting in proper prevention, and acting as a reference test for possible carcinogenesis. Recent guidelines suggest that HPV molecular test take place in women over 30 years old or younger women with high-grade intraepithelial lesions. This examination does not replace, in any way, the PAP test unless in the PAP test "atypical epithelial cells of undetermined importance" or "low grade intraepithelial damage" are detected, then the molecular test is considered necessary.
The examination can take place with a cervical smear, a urethral smear, and a semen sample. The Real-Time PCR method is used for the detection of the viral genetic material, while quantifying the viral load present in the cells. The procedure starts with the extraction of nucleic acids from the sample, which is carried out automatically by four state-of-the-art extractors available in our laboratory, minimising the factor human error while maximising the sensitivity of the method. Subsequently, polymerase chain reaction takes place by highly trained molecular biologists of our laboratory, using multipliers manufactured by leading companies in molecular applications, such as QIAGEN and DNA technology.
This method is based on measuring the fluorescence in each propagation cycle. The method uses Taqmanprobe which when hybridized to a target sequence is hydrolysed by Taq-polymerase, resulting in the separation of fluorescence detector and quencher and the fluorescence signal increasing as the HPV target sequence amplifies. FAM, ROX and Cy5 fluorophores are used to detect specific sequences of HPV strains, while HEX fluorophores are used to detect the internal control. The Internal Process Control (IPC) which is added during DNA extraction, is detected in the same reaction as a labelled probe and allows the detection of RT-PCR inhibition by various agents. It also acts as a control by ensuring that viral DNA is isolated from the biological sample, thus preventing false negative results due to insufficient amount of genetic material or improper transfer of the sample. The advantage of this type of PCR is that by using different fluorophores it simultaneously detects in a single reaction the low and high oncogenic risk strains of HPV. The analysis of the results is done by the specialized and experienced staff, thus increasing the reliability of the results given to the candidates for examination.
Due its high sensitivity and simultaneous separation of high and low oncogenic risk strains, this method reduces the number of pathological cases that escape detection by performing the cytological method (Pap test) and increases the percentage of true negative results (i.e., those who do not show malignancy). With the use of advanced technology and the highly trained molecular biologists at A. EVANGELOU MEDICAL LABORATORIES, we are able to deliver the population of Cyprus with this exclusive, innovative and reliable test, contributing significantly to the fight against cervical cancer, controlling all 21 subtypes of HPV, both those at high risk of oncogenicity and those with low risk of oncogenicity.