Analysis of genetic copy number changes in cervical disease progression

Abstract

Background: Cervical dysplasia and tumorigenesis have been linked with numerous chromosomal aberrations. The goal of this study was to evaluate 35 genomic regions associated with cervical disease and to select those which were found to have the highest frequency of aberration for use as probes in fluorescent in-situ hybridization.

Methods: The frequency of gains and losses using fluorescence in-situ hybridization were assessed in these 35 regions on 30 paraffin-embedded cervical biopsy specimens. Based on this assessment, 6 candidate fluorescently labeled probes (8q24, Xp22, 20q13, 3p14, 3q26, CEP15) were selected for additional testing on a set of 106 cervical biopsy specimens diagnosed as Normal, CIN1, CIN2, CIN3, and SCC. The data were analyzed on the basis of signal mean, % change of signal mean between histological categories, and % positivity.

Results: The study revealed that the chromosomal regions with the highest frequency of copy number gains and highest combined sensitivity and specificity in high-grade cervical disease were 8q24 and 3q26. The cytological application of these two probes was then evaluated on 118 ThinPrep samples diagnosed as Normal, ASCUS, LSIL, HSIL and Cancer to determine utility as a tool for less invasive screening. Using gains of either 8q24 or 3q26 as a positivity criterion yielded specificity (Normal +LSIL+ASCUS) of 81.0% and sensitivity (HSIL+Cancer) of 92.3% based on a threshold of 4 positive cells.

Conclusions: The application of a FISH assay comprised of chromosomal probes 8q24 and 3q26 to cervical cytology specimens confirms the positive correlation between increasing dysplasia and copy gains and shows promise as a marker in cervical disease progression.

Figure 1

Mean number signals per specimen for each probe across all histological categories.

Figure 2

Percent change of mean probe copy numbers between histological categories. Values above zero represent positive values (gains) and values below zero represent negative values (losses).

Figure 3

Receiver Operating Characteristic Curve (ROC) for threshold determination using histological samples. The ROC analysis utilized 1 to 10 cells (from right to left on the graph, respectively) for curve determination.

Figure 4

Mean number of abnormal cells for each cytological category using 3q26 or 8q24, 8q24, and 3q26. The 3q26 or 8q24 Abnormal column (black) represents all cells found positive for either 3q26 or 8q24, the 8q24 Abnormal column (diagonal stripes) represents all cells found positive with 8q24, and the 3q26 Abnormal column represents all cells that were found positive by 3q26.