Comprehensive cervical cancer screening by multiplexed HPV and biomarker mRNA detection using the Quantigene 2.0 platform

Speakers

Abstract:

Background and aim:
Multiplexing of biomarker panels by microsphere-based technologies are robust and cost-effective methods. The QuantiGene™ Plex Assay (ThermoFisher Scientific) platform quantitates mRNA expression from crude lysates discriminating up to 80 target sequences. We aim at using expression analyses fo Human Papillomavirus (HPV) oncogenes and cellular biomarkers corresponding to cervical dysplasia progression for screening and diagnostic molecular assays. Currently used DNA-based HPV tests are highly sensitive for infection but have low specificity for true disease. This is especially problematic in LMIC like in Africa where HPV prevalence is high, no screening approaches exist, and healthcare systems are overburdened.

Methods:
A 40-plex QuantiGene™ assay was designed to quantify mRNA expression of the E7 oncogene of 18 high-risk HPV genotypes, cellular housekeeping genes for normalization, and 18 well established cellular biomarkers like proliferation markers, tumor suppressors, cancer stem cell transcription factors, and invasive tumor markers. Initially, the lysis procedure, sensitivity and specificity of the target sequences were tested in a pilot assay for feasibility with established cervical cancer cell lines. The suitability of the target panel and the assay procedure was tested on 1400 consecutive prospectively collected clinical samples. By logistic regression and ROC analyses marker sub-panels and cut offs were determined identifying and discriminating the different dysplastic stages and cervical cancer. Costs and workload were compared to a qRT-PCR-based method. The assay was used in two cervical cancer screening studies and compared to standard screening methods.

Results:
Feasibility of the assay was investigated by using established cervical cancer cell lines showing that HPV E7 and cellular biomarker expression was sufficiently sensitive detecting less than 40 cells. Most informative
biomarkers were identified by logistic regression. Characteristic marker panels for the dysplastic stages of cervical intraepithelial neoplasia (CIN)2+, CIN3+, and invasive cervical carcinoma were identified. High-grade dysplasia was detected in clinical smear samples with an accuracy of 90%. The comparison to qRT-PCR showed an acceptable correlation of results with remarkable lower costs and efforts for QuantiGene procedures. Using this assay in screening studies in Ethiopia and Germany resulted in much higher specificity for the detection of true dysplasia than the current standard of care. Importantly, work up of samples is simple (no nucleic acid purification and amplification), robust and especially adequate for
low-tech labs as found in LMIC.

Conclusion:
mRNA quantification by multiplexed bead-based technology is feasible in a cost effective way, simple and robust. Additionally, it presents an attractive means for IVD development for assay systems in cervical cancer screening and research.


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