Поврзаност на експресијата на генот за теломераза со цервикалниот карцином и неговите прекурзорни лезии при инфекција со хуман папилома вирус

Abstract

Background: Cervical cancer is a preventable disease; however, despite its avoidable nature it remains one of the leading causes of cancer mortality globally, with the highest incidence and mortality rates in low-resource countries. Human papillomavirus infection is certainly an underlying cause of cervical cancer and its precursor lesions with different prevalence of the genotypes depending on the patient’s age, ethnicity, race, severity of the precursor cervical lesions and geographical region. Early events associated with disease progression and cervical cancer include hTERT up-regulation at the transcriptional level mediated by highrisk HPV E6 oncoproteins via indefinite mechanisms with concomitant immortal phenotype of the cell in vitro and increased replicative potential of cells in precancerous cervical lesions and cervical cancer. Aim: The aim of this study was to evaluate the frequency of HPV infection and genotypes among women with normal and abnormal cytological results and its correlation according to the severity of the lesions as well as to investigate the correlation between the TERT gene expression levels and the cytological sub-group and to evaluate the correlation between the HPV infection and TERT gene expression levels as a diagnostic and prognostic marker in cervical precursor lesions and cervical cancer. Materials and methods: This study is a prospective observational case-control study conducted in Obstetrics and Gynaecology Clinic in Pristina and Molecular and Biology- Genetics Department in Skopje. Cervical samples from 214 women (median age 45.28 years; range 20-65) from the Outpatient Clinic in University Clinical Center in Kosovo were tested for HPV-DNA and quantitative TERT gene expression after performing conventional Pap smear. From them, 100 women had normal cytology (NC), 45 cases were ASC-US, 37 LSIL, 7 cases with ASC-H, 15 cases with HSIL and 10 cases with squamous cervical cancer. HPV detection and genotyping was performed by PCR using MY 09/MY11 primers with subsequent genotyping by restriction-fragment length polymorphism (RFLP) analyses using combination of 3 endonucleases. RNA isolates were used for complementary DNA (cDNA) synthesis by reverse transcription. Amplification and relative quantitative real-time polymerase chain reaction (qRT-PCR) was performed using TERT-specific primer pair and gene specific fluorescent TaqMan probes. Quantitative TERT gene expression was calculated relative to the reference G3PDH gene and normalized to the normal cervical cell sample using ΔΔCt method. Two-tailed Mann-Whitney U-test was used for statistical analysis. Results: HPV infection was detected in 109 cases (50.93%). One hundred (46.73%) out of 214 samples were found to have normal cytology (NC) and 114 cases showed some type of cytological abnormalities: 45 (21.03%) ASC-US; 37 (17.29%) LSIL; 7 (3.27%) ASC-H; 15 (7.01%) HSIL, and 10 (4.67%) cervical cancer. The frequency of HPV positivity was dependent on the severity of the cervical lesions (62.22% for ASC-US; 85.71% for ASC-H; 86.49% for LSIL; 93.33% for HSIL and 100% for cervical cancer). Multiple HPV infection did not correlate directly with the severity of the cervical lesions because in 44%, 72%, 71% and 80% of samples with ASC-US, LSIL, ASC-H, and HSIL/CC respectively, only single HPV type was found. Multiple HPV infection with more than two genotypes was found in 18/109 patient samples or 8.41%. The most prevalent genotype found was HPV 53 (26.77%). HPV 16 was the second most prevalent type (22.83%) followed by the third most common type 31 (14.96%). The hTERT mRNA expression significantly correlated with the increased grade of the cervical lesion. A statistically high difference was found between the hTERT normal expression levels and hTERT over-expression in all cytological groups with abnormalities compared to normal control group (p˂0.01). hTERT gene overexpression was associated with 6.31 and 9.20 fold higher risk for developing ASC-US and LSIL compared to patients with normal expression and normal cytology, respectively. In addition, there was a strong correlation between the hTERT gene expression levels and HPV infection, notably in the high-grade lesions and cervical cancer. hTERT gene relative expression values showed 98% specificity and 100% sensitivity as an indicator of cervical lesions particularly for the ACS-H, HSIL and cervical cancer. Conclusions: This study highlights the high HPV prevalence corresponding to the high incidence rate of cervical cancer in Kosovo with the most common types HPV 53 and HPV 16, followed by type 31. Based on epidemiological profile of HPV genotypes in this region, these results might prove auxiliary to establish a platform for cervical cancer screening and prevention strategies in the future. Data from this study also suggest that hTERT expression occurs early in progressive cervical disease and correlates with disease severity and HPV infection. Therefore, it is useful as a diagnostic and prognostic biomarker of the disease progression in HPV- infected subjects.