Chromosomal biomarkers for detection of human papillomavirus associated genomic instability in epithelial cells of cervical cytology specimens

Abstract

The goal of this study was to compare how accumulation of chromosomal aberrations in human papillomavirus (HPV)-infected cells correlates with the severity of cervical dysplastic lesions. We assessed the frequency of genomic alterations for 35 different loci in a pilot biopsy study and selected two loci (3q26 and 8q24) with the highest frequency of copy number gains found in high-grade dysplasia and cancer. These probes were labeled with gold and red fluorophores and combined with HPV biotin-labeled probes for subsequent detection using a tyramide signal amplification system with a green fluorophore. Cells that were both HPV positive and chromosomally abnormal were designated as "double-positive cells." Cervical cytology specimens from 235 patients were used for this blinded study. The average number of double-positive cells increased from two cells in patients with a cytological interpretation of atypical squamous cells of undetermined significance to 22 cells in low-grade squamous intraepithelial lesion and 99 cells in high-grade squamous intraepithelial lesion, reflecting an accumulation of chromosomal abnormality with disease progression. Using a cutoff of four or more double-positive cells as the criterion for the presence of a cervical intraepithelial neoplasia 2 or 3 lesion, we demonstrated that low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion cytology specimens with underlying cervical intraepithelial neoplasia 2/3 histology showed positive test results in more than 80% of cases. Correlation of 3q26 and 8q24 aneusomy with concurrent HPV infection may thus serve as a biomarker of genetic instability in HPV-infected cells.

Figure 1

A representative image of HPV staining and chromosome probes 3q26 (TERC) and 8q24 (MYC) signals observed in cervical epithelial cells after FISH assay. Each individual signal for the TERC and MYC probes constitutes one copy of the respective gene. The images consist of DAPI staining of cell nuclei, HPV staining (green) of infected cells, chromosome probe 3q26 (TERC) visualized using a gold filter, and chromosome probe 8q24 (MYC) visualized using a red filter. Column A represents a cell episomally infected with HPV, column B represents a cell with a mixed HPV infection, and column C represents an integrated HPV infection.

Figure 2

Average quantity of HPV-positive cells, chromosomally abnormal cells, and double-positive cells per cytological category.

Figure 3

Average quantity of HPV-positive cells, chromosomally abnormal cells, and double-positive cells per histological category.

Figure 4

Relationships between HPV-infected and chromosomally abnormal cervical epithelial cells depicted in a Venn diagram. The numbers beneath the diagram circles reflect the percentage of HPV-infected cells that demonstrated chromosome copy gains for either one or both chromosome markers (3q26 and 8q24 probes).