Human papillomavirus (HPV) DNA copy number is dependent on grade of cervical disease and HPV type

Abstract

The association between human papillomavirus (HPV) DNA copy number and cervical disease was investigated. Viral DNA copy number for the most common high-risk HPV types in cervical cancer (types 16, 18, 31, and 45) was determined in cervical cytobrush specimens from 149 women with high-grade cervical intraepithelial neoplasias (CIN II-CIN III), 176 with low-grade CIN (CIN I), and 270 with normal cytology. Quantitative, PCR-based fluorescent assays for each of the HPV genotypes and for the beta-globin gene were used. The amount of cellular DNA increased significantly with increasing disease; thus, HPV was expressed as copies per microgram of cellular DNA. The assay had a dynamic range of >10(7), allowing documentation for the first time of the wide range of HPV copy numbers seen in clinical specimens. Median HPV DNA copy number varied by more than 10(4) among the viral types. HPV16 was present in the highest copy number; over 55% of HPV16-positive samples contained more than 10(8) copies/microgram. Median copy number for HPV16 showed dramatic increases with increasing epithelial abnormality, an effect not seen with the other HPV types. HPV16 increased from a median of 2.2 x 10(7) in patients with normal cytology, to 4.1 x 10(7) in CIN I patients, to 1.3 x 10(9) copies/microgram in CIN II-III patients. Even when stratified by cervical disease and viral type, the range of viral DNA copies per microgram of cellular DNA was quite large, precluding setting a clinically significant cutoff value for "high" copy numbers predictive of disease. This study suggests that the clinical usefulness of HPV quantitation requires reassessment and is assay dependent.

FIG. 1

Sample cellular DNA content. Shown are sample cellular DNA amounts (micrograms), calculated from β-globin signals, stratified by disease grade.

FIG. 2

High-risk HPV copy number distribution in patient specimens. The copy number ranges were selected to maximize the number of patients in each category. The segments (striped, reverse-stippled, stippled, and checkered boxes) represent numbers of patient specimens with copy numbers of >10⁸, >10⁶, >10⁴, and >0 per μg, respectively, for each of the HPV types.

FIG. 3

High-risk HPV copy number per microgram stratified by HPV type and sample histology. The median HPV copy numbers per microgram were calculated for each of the three disease categories. The stippled, reverse-stippled, and striped bars indicate median (log [copy numbers per microgram of cellular DNA]) in normal, CIN I, and CIN II-III samples, respectively. (A) All HPV-positive samples (including multiple infections). A Kruskal-Wallis test of the significance of the copy number differences among normal, CIN I, and CIN II-III samples for the four HPV types returned the following P values: HPV16, P = 0.03; HPV18, P = 0.45; HPV31, P = 0.97; and HPV45, P = 0.30. (B) Singly infected samples. P values were as follows: HPV16, P = 0.03; HPV18, P = 0.12; HPV31, P = 0.73; and HPV45, P = 0.94.