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Biomarkers in cervical screening: quantitative reverse transcriptase PCR analysis of P16INK4a expression
Gaëlle A V Boulet1, Caroline A J Horvath, Christophe E Depuydt
1Applied Molecular Biology Research (AMBIOR) group, Laboratory for Cell Biology and Histology, University of Antwerp, Groenenborgerlaan, Antwerp, Belgium. gaelle.boulet@ua.ac.be
Summary
Quantitative reverse transcriptase PCR (RT-PCR) is not suitable for cervical cancer screening. Overexpressed biomarker transcripts, like p16(INK4a), are diluted in liquid-based cytology samples, hindering detection of transforming human papillomavirus (HPV) infections.
Area of Science:
- Oncology
- Molecular Biology
- Gynecologic Pathology
Background:
- Human papillomavirus (HPV) infection is a primary cause of cervical carcinogenesis.
- Biomarkers like p16(INK4a) show promise for cervical disease detection, but immunostaining has limitations.
- Quantitative reverse transcriptase PCR (RT-PCR) was explored as a more objective method for biomarker analysis.
Purpose of the Study:
- To evaluate RT-PCR for quantifying p16(INK4a) overexpression in liquid-based cervical cytology (LBC).
- To assess RT-PCR's utility as a biomarker for transforming HPV infections.
- To determine the analytical sensitivity of RT-PCR for detecting biomarker overexpression in LBC.
Main Methods:
- Sixty LBC samples were analyzed for HPV type and p16 expression using immunocytochemistry and RT-PCR.
- RT-PCR analytical sensitivity was assessed by spiking experiments with HeLa and HaCaT cells.
- P16(INK4a) expression by RT-PCR was correlated with cytological diagnosis and HPV status.
Main Results:
- RT-PCR measurement of p16(INK4a) expression did not correlate with cytological diagnosis or HPV status.
- Spiking experiments indicated that approximately 1.0% dysplastic cells are needed for RT-PCR detection of biomarker overexpression.
- RNA from normal cells significantly dilutes biomarker transcripts in typical LBC samples.
Conclusions:
- RT-PCR analysis of biomarker expression is not appropriate for cervical screening.
- The dilution of biomarker transcripts in LBC samples prevents reliable detection of overexpression by RT-PCR.
- Current RT-PCR methods are insufficient for detecting transforming HPV infections via biomarker analysis in cervical cytology.
