Abstract
BackgroundHuman papillomavirus (HPV) DNA testing plays a main role in the management of cervical cancer, however to improve the specificity in cervical screening, there is a need to develop and validate different approaches that can identify women at risk for progressive disease.Nowadays, mRNA expression of viral E6 and E7 HPV oncogenes stands up as a potential biomarker to improve cervical screening. We aimed to validate a method for RNA extraction, detect HPV mRNA expression and, assess the relationship between E6/E7 mRNA expression and pathology of patients’ lesions and progression.MethodsThis study included 50 specimens that had been previously genotyped as HPV16, 18, 31, 33 and/or 45. Cervical swabs were extracted with three different RNA extraction methods -Nuclisens manual extraction kit (bioMérieux), High Pure Viral RNA Kit (Roche) and RNeasy Plus Mini kit (Qiagen)-, and mRNA was detected with NucliSens EasyQ HPV version 1 test (bioMérieux) afterwards. Association of oncogene expression with pathology and lesion progression was analyzed for each extraction method.ResultsE6/E7 mRNA positivity rate was higher in samples analyzed with bioMérieux (62%), followed by Roche (24%) and Qiagen (6%). Women with lesions and lesion progression showed a higher prevalence of viral RNA expression than women that had not lesions or with lesion persistence. While bioMérieux revealed a higher sensitivity (77.27%), Roche presented a higher PPV (75%) and an increased specificity (89.28%).ConclusionsExtraction methods based on magnetic beads provided better RNA yield than those based in columns. Both Nuclisens manual extraction kit (bioMérieux) and High Pure Viral RNA Kit (Roche) seemed to be adequate for E6/E7 mRNA detection. However, none of them revealed both high sensitivity and specificity values. Further studies are needed to obtain and validate a standard gold method for RNA expression detection, to be included as part of the routine cervical screening program.
Highlights
Human papillomavirus (HPV) DNA testing plays a main role in the management of cervical cancer, to improve the specificity in cervical screening, there is a need to develop and validate different approaches that can identify women at risk for progressive disease
As some authors have reported the importance of RNA input prior to any HPV RNA assay [17,18,19], we aimed to compare three different RNA extraction methods in order to evaluate the impact of extraction in RNA expression detection
According to Bethesda system, 8% of samples were classified as High-grade squamous intraepithelial lesion (HSIL) (4/50), 36% (18/50) as Atypical squamous cells of undetermined significance (ASCUS) or Low-grade squamous intraepithelial lesion (LSIL) and 56% (28/50) as negative
Summary
Human papillomavirus (HPV) DNA testing plays a main role in the management of cervical cancer, to improve the specificity in cervical screening, there is a need to develop and validate different approaches that can identify women at risk for progressive disease. MRNA expression of viral E6 and E7 HPV oncogenes stands up as a potential biomarker to improve cervical screening. Cancer incidence and HPV genome has two oncogenes, E6 and E7, whose expression increased when these genes are deregulated [4]. The integration of HPV DNA into the host genome is an important event in cancer development and in malignant transformation of cervical lesions [7]. HPV viral oncoprotein expression differs among infection state (active, latent, or persistent) [9,10,11]
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