PCR-Based Cervical Cytology HPV Screening Tests Require High Analytical Sensitivity To Ensure the Inclusion of All Patients with CIN3 among Test Positives
MF Evans, Z Peng, CSC Adamson, TL St. John, G Leiman, K Cooper. University of Vermont, Burlington, VT; Fletcher Allen Health Care, Burlington, VT
Background: The relationship of cervical cytology sample HPV viral load to a patient's subsequent histopathological diagnosis is contentious. Additionally, there is uncertainty about the sensitivity required of PCR-based cytological HPV screening tests to ensure the inclusion of all patients with CIN3 among test positives. It has been suggested that a high 'analytical' sensitivity will result in the detection of patients with clinically irrelevant amounts of HPV and that PCR assays should be optimized in terms of 'clinical' sensitivity. This study has examined HPV16 viral load in relation to lesion grade and has investigated what represents a clinically relevant amount of HPV16.
Design: A novel quantitative (q) PCR assay for the HPV16 L1 gene was developed using Plexor technology (Promega Corp) and optimized for the consistent detection of 10 copies of HPV16 in a high background of human sequences. The assay was applied to DNA extracts (50ng) from 126 (13 ASC-US, 31 ASC-H, 12 LSIL, 64 HSIL, 6 NILM) known HPV16 positive routine cervical scrape samples. Histological follow-up data (39 benign changes, 5 CIN1, 26 CIN2, 49 CIN3, and 1 SCC) were available for all 121 patients with abnormal cytology. For each sample, HPV16 viral load data were normalized as ratios of qPCR data for the GAPDH housekeeping gene.
Results: Normalized viral loads ranged from 5.3 x 10-5 to 3.1 x 103 copies of HPV16 per cell equivalent (mean 70.7, median 1.6, SD 337.5). Viral load did not distinguish different grades of abnormal cytology from each other [p=0.58]; NILM sample HPV16 viral load was significantly lower than in abnormal samples [p=0.0001]. Viral load did not predict biopsy histological grade; there was no significant difference in the amounts of HPV16 preceding a diagnosis of benign changes, CIN1, CIN2, or CIN3 [p=0.75]. Regarding CIN3, preceding viral loads ranged from 4.6 x 10-4 to 3.1 x 103 HPV16 copies per cell (mean 86.4, median 1.7, SD 448.2), or, 16.0 x 100 to 52.5 x 106 HPV16 copies per 50ng DNA sample; CIN3 viral load was significantly higher in patients aged 16-25 years of age (n=21) than in patients aged 26-35 (n=22) [p=0.01].
Conclusions: These data show that HPV viral load testing is a poor predictor of histopathological grade and suggest that PCR-based HPV screening tests (currently under FDA-review) require application at high analytical sensitivity to avoid false negative HPV tests from potential CIN3 patients.
Tuesday, March 10, 2009 1:45 PM
Platform Session: Section F, Tuesday Afternoon