Objective
The chromosome 3q26 region is a biomarker for cervical cancer. Women with low-grade squamous intraepithelial lesions (LSIL) currently are referred for immediate colposcopy. The objective of this study was to determine the negative predictive value of the 3q26 amplification test for the persistence or regression of LSIL.
Study Design
Archival thin layer cytologic slides of 47 women (14–67 years old) with LSIL were linked to histologic and cytologic end points. To determine 3q status, the slides were hybridized for the chromosome 3q26 region and for the centromere of chromosome 7, as a control, with the use of the standard fluorescent in situ hybridization methods.
Results
The negative predictive value of 3q26 gain for the development of cervical intraepithelial neoplasia grade 2/3 within 1 year was 93% (95% confidence interval, 68– 100); after 21 months, its negative predictive value was 100% (95% confidence interval, 29–100).
Conclusion
The 3q26 gain might help identify women with LSIL who do not need colposcopy.
Cervical cancer screening has been one of the most successful, but one of the most costly, public health screening programs in the past century. Although the burden of cervical cancer death remains high in developing countries without screening programs, the challenge for continued cervical cancer control in developed countries is to refine screening so that those women who truly are at risk for cervical cancer can be targeted. Current screening programs intensively screen the women least likely to experience cervical cancer.
In the current cytologic screening system, human papillomavirus (HPV) testing has proved valuable for triage after an atypical cytologic test to identify which women are likely to benefit from colposcopy. On the other hand HPV testing is not an effective triage test for women with low-grade squamous intraepithelial lesion (LSIL) screening results, because a large percentage of women will be infected with high-risk HPV types but never progress to a precancerous lesion; the lesion often will clear within 2 years of detection. At this time, the clinical recommendation for women with LSIL cytologic condition is to undergo colposcopy and biopsy. This recommendation sends approximately 3% of the US screened population to colposcopy annually; the subsequent prevalence of detected cervical intraepithelial neoplasia (CIN) 2/3 is approximately 15%, which is a very low yield at a high cost. Other HPV-based tests have not produced effective clinical triage for women with LSIL results.
The discordance between the high prevalence of high-risk HPV infections and much lower incidence of cervical cancer indicates that additional factors also play a role in oncogenesis. Specifically, the 3q26 region contains sequences for the RNA component of the human telomerase gene, which serves as a template for telomere addition, which is a potential basis for telomerase-based cell immortalization. A 3q26 gain (3q gain) increases in frequency as the severity of the cytologic specimen approaches cervical cancer. There are relatively infrequent gains in 3q in women with LSIL cytologic findings, but up to 70% of women with high-grade squamous intraepithelial lesions (HSILs) and 100% of women with cervical cancer express 3q gain. Testing for 3q gain has been assessed in small numbers of women with LSIL cytologic findings to predict which will have CIN 2/3 lesions. The true clinical value of this biomarker, though, is its potential power to indicate which women with LSIL cytologic findings are less likely to progress to CIN 2/3 or cancer and hence can be followed more conservatively.
The objective of this study was to determine the negative predictive value of the 3q26 amplification test for persistence or regression of the LSIL abnormality.
Materials and Methods
Cytologic screening
A set of cervical cytologic specimens with an index diagnosis of LSIL was collected from 2 independent cytopathology laboratories (B.D., M.M.). The set was identified from 64,693 Papanicolaou smears that were collected in New England between 2001 and 2003. From the 1050 specimens with a diagnosis of LSIL, a set of 54 specimens that (1) were readily accessible, (2) had follow-up cytologic or histologic findings, and (3) had an index cytologic slide available for analysis was selected at random. Five of the 54 cases were excluded because of discordant interpretation by the study cytopathologist and the initial clinical diagnosis. Two cases were excluded because of low cellularity and/or inadequate fluorescence in situ hybridization (FISH) quality after hybridization of the index cytologic slide. The index cytologic slides were de-identified before forwarding for 3q analysis, and no patient clinical information was provided. Because the samples were anonymous and no patient information was provided, patient informed consent and institutional review board approval was not required. The women ranged from 14–67 years in age at the index cytologic finding. We stratified the patients into 3 groups in the following manner: the first group of women had a histologic biopsy within 1 year of the index cytologic finding. The second group of women had a follow-up examination for a corresponding histologic biopsy >1 year from the index cytologic finding. The third group of women had only follow-up cytologic review >1 year after the index cytologic finding. Inclusion criteria for keeping the archival slides in the study were agreement between the clinical cytologic diagnosis and the reviewing cytopathologist for the index LSIL. Each slide had been Papanicolaou-stained. Microscopic fields with cells that met the diagnostic criteria for LSIL were identified and recorded. The index slides were subjected to secondary review, according to the Bethesda criteria, by a board-certified cytopathologist (R.W.) who was blinded to the follow-up cytologic and histologic specimens. No information was available about treatments received by the women during the follow-up periods.
Laboratory methods
Slides were soaked in xylene (Fisher Scientific, Pittsburgh, PA) overnight to remove the coverslips then further destained in fresh xylene for a minimum of 1 hour, in isopropyl alcohol for 30 minutes, and in 100% ethanol for an additional 30 minutes. Slides were hydrated in 85% and 75% ethanol then treated with 1.5% ammonium hydroxide (Acros Organics, Geel, Belgium) in 70% ethanol for 20 minutes. After being rinsed with tap water, slides were treated with an acid alcohol solution for 30 seconds to complete the destaining process.
In preparation for FISH, slides were placed in a sodium citrate solution (Zymed Laboratories Inc, San Francisco, CA) for 30 minutes at 80°C, rinsed in deionized water for 1 minute, then treated with proteinase K solution for 20 minutes at 37°C, followed by a 5-minute wash in phosphate-buffered saline solution. The proteinase treatment was then repeated, followed by fixation in 10% formaldehyde (Fisher Scientific), 50 mmol/L MgCl 2 (Sigma-Aldrich, St. Louis, MO) in phosphate-buffered saline solution for 10 minutes. Finally, the slides were rinsed in 2X saline sodium citrate at room temperature and dehydrated in an alcohol gradient. Each slide was examined under both visible light and a fluorescence microscope to assess background. Slides with high background were treated for an additional 30 seconds with acid alcohol then dehydrated in an alcohol gradient.
Slides with <1000 cells were considered inadequate. Slides were hybridized with a probe for the chromosome 3q26 region, labeled with Spectrum Gold (Abbott Molecular, Des Plaines, IL) and a probe for the centromeric alpha-repeat sequence of chromosome 7, as a control, labeled with Spectrum Aqua (Abbott Molecular). The probe for the 3q26 region comprised a series of bacterial artificial chromosome clones that spanned a 500-kilobase region that contained the human telomerase gene TERC and the gene for the alpha catalytic subunit of phosphatidylinositol 3-kinase. Probe mix in hybridization buffer was added to each slide and covered with a glass coverslip. Coverslips were sealed with rubber cement, and the samples were cohybridized by denaturing at 73°C, for 6 minutes then hybridized at 37°C for 48 hours with a ThermoBrite (Abbott Molecular). After hybridization, slides were washed for 2 minutes in 2X SSC, 0.3% NP40 (Abbot Molecular) at 73°C, and 2 minutes at room temperature in 2X SSC, 0.1% NP40. The slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), dehydrated, and cover slipped with antifade-containing mounting medium (44 mmol/L 1,4-diazabicyclo[2.2.2]octane [Fisher Scientific]; 89.55% glycerol, 20 mmol/LTris-HCl, pH 8.0) and placed at –20°C for 15 minutes to enhance signals.
Microscopic analysis
The index cytologic slide for each subject was examined by FISH analysis. After hybridization, areas of interest that had been recorded previously were examined with a fluorescent microscope (BX 51; Olympus, Center Valley, PA) equipped with SpectrumGold, SpectrumAqua, and DAPI filters (Abbot Molecular) for nuclei with abnormal DAPI and FISH signals. The number of target nuclei scored and the number of FISH signals for 3q and CEP 7 were enumerated with either a ×60 or ×100 dry objective. Approximately 15–20 fields were examined for each slide.
A case was considered positive for 3q gain by having ≥2 cells with ≥5 3q-FISH signals ( Figure ). Our rationale for this was that a Papanicolaou smear requires a single abnormal cell for a case to be regarded as abnormal; therefore, by requiring 2 abnormal cells, we set a more stringent threshold. We avoided some of the difficulties that have been encountered in previous studies based on the percentage of nuclei with 3 copies of 3q caused, in part, by artifacts overlapping cells and split FISH signals. It also allowed us to exclude likely tetraploid nuclei from the analysis, because the significance of tetraploidies is not yet well understood. This approach provided for a stringent threshold for determining positivity for 3q gain.
