Quantitative methylation-specific PCR for the detection of aberrant DNA methylation in liquid-based Pap tests

BACKGROUND: Aberrant promoter methylation of selective tumor suppressor genes has been detected in squamous intraepithelial lesions (SIL) and invasive cervical cancer. Identification of methylation profiles of genes that can distinguish high-grade SIL (HSIL) from low-grade SIL (LSIL), and cytologically negative for intraepithelial lesion or malignancy (NILM) residual liquid-based Papanicolaou (Pap) tests may be potentially useful as an ancillary test for cervical cancer screening. METHODS: Using real-time quantitative methylation-specific polymerase chain reaction (PCR) (QMSP), the authors analyzed the frequency and relative level of promoter methylation for DAPK1, IGSF4, SPARC, and TFPI2 in biopsy-confirmed HSIL and LSIL, and NILM residual liquid-based Pap tests. The percentage of methylation (%M) for each gene was calculated using the reference gene, ACTB. The cumulative methylation score for each sample, defined as the sum of %M of all 4 genes, was used to analyze the genes in combination. RESULTS: For each gene analyzed the frequency and relative level of methylation were increased in HSIL compared with combined NILM/LSIL samples. The cumulative methylation scores were significantly higher in HSIL samples (P < .0001). Area under the receiver operating characteristic (ROC) curve (AUC) demonstrated that methylation of each gene could distinguish HSIL from NILM/LSIL samples (AUC range, 0.6-0.67; P < or = .0028). The combination of 4 genes showed improved test performance (AUC = 0.76; P < .0001). There was no significant difference in cumulative methylation in HSIL cases with histologic outcomes of cervical intraepithelial neoplasia grade 2 (CIN2) versus CIN3. There was no association between the methylation of any gene and the presence of human papillomavirus. CONCLUSIONS: The methylation profile of multiple genes in combination can better distinguish HSIL from combined NILM/LSIL samples. Although aberrant DNA methylation has the potential to function as a molecular biomarker of HSIL in liquid-based Pap tests, additional genes that are selectively methylated in HSIL are needed to improve the clinical performance.     

Adjunctive high‐risk human papillomavirus DNA testing is a useful option for disease risk assessment in patients with negative Papanicolaou tests without an endocervical/transformation zone sample

BACKGROUND.

Current guidelines recommend that women with negative Papanicolaou (Pap) test results and no endocervical/transformation zone (EC/TZ) sample return for screening within 12 months. For some women, this represents earlier follow‐up than advocated in several routine screening guidelines. Controversy remains with regard to the correlation between sampling of the EC/TZ, Pap test quality, and disease risk assessment.

METHODS.

A retrospective study was conducted reviewing the results from 143,438 liquid‐based cervical Pap tests performed at a large academic women's hospital between July 2005 and December 2006. Vaginal Pap tests were excluded from the study. Women with any Pap result, women with low–grade squamous intraepithelial lesions (LSILs), and patients with high–grade squamous intraepithelial lesion (HSIL) Pap test results were stratified by 10‐year age groups and according to the presence or absence of an EC/TZ sample (EC/TZS). Women with LSIL and HSIL Pap test results with and without an EC/TZS were also compared for rates of high–risk human papillomavirus (hrHPV) DNA detection.

RESULTS.

Of the total of 143,438 cervical Pap tests performed, 27,359 (19.1%) were reported to be lacking an EC/TZS. The absence of an EC/TZS was found to be highest in adolescents and in mature women aged ≥50 years. The overall detection rate of LSIL was 4.29% and that of HSIL was 0.64%. Both the LSIL and HSIL rates were found to be significantly higher in Pap tests with an EC/TZS compared with Pap tests without an EC/TZS (LSIL: 4.51% vs 3.37% and HSIL: 0.72% vs 0.29%). However, when women with LSILs and HSILs were divided into a group in which EC/TZS was present and a group in which EC/TZS was absent, no significant differences were found to be present with regard to hrHPV DNA rates between the 2 groups.

CONCLUSIONS.

Adjunctive hrHPV DNA testing is effective in stratifying risk for the presence of SIL in women with and without an EC/TZS. This finding is consistent with recently reported data from >9000 patients with negative Pap results, which found that hrHPV DNA–positive test rates are independent of the presence or absence of an EC/TZS. hrHPV DNA results provide a useful new optional adjunctive tool for the objective stratification of disease risk in women with negative Pap tests and no EC/TZS. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.     

Clinical significance of the diagnosis of low‐grade squamous intraepithelial lesion,cannot exclude high‐grade squamous intraepithelial lesion

BACKGROUND:

The diagnosis of low‐grade squamous intraepithelial lesion (LSIL), cannot exclude high‐grade squamous intraepithelial lesion (LSIL‐H) was not included in the 2001 Bethesda System. It is used in some institutions to diagnose cases that fulfill criteria for both the diagnosis of LSIL and atypical squamous cells, cannot exclude high‐grade squamous intraepithelial lesion (ASC‐H). In this study, the authors reviewed their experience with cases reported as LSIL‐H during a 4‐year interval.

METHODS:

Clinical information and histologic follow‐up data were retrieved for Papanicolaou (Pap) tests (PTs) that were diagnosed as LSIL‐H, LSIL, ASC‐H and high‐grade squamous intraepithelial lesion (HSIL) from January 1, 2004 to December 31, 2007.

RESULTS:

Of 235,645 PTs (97% SurePath) that were processed during the study period, the laboratory diagnosed 0.52% as ASC‐H, 2% as LSIL, 0.30% as LSIL‐H, and 0.39% as HSIL. Biopsy follow‐up was available for 47%, 49%, 56.7% and 74% of these cases, respectively. Cervical intraepithelial neoplasia 2 (CIN‐2) and CIN‐3 or more severe lesions (CIN‐3+) were identified on follow‐up cervical biopsy more often in women who had diagnoses of LSIL‐H and ASC‐H (33.14% and 26.33%, respectively) than in women who had a diagnosis of LSIL (16.11%).

CONCLUSIONS:

The similarity of histologic follow‐up results between LSIL‐H and ASC‐H suggested that the management of women who have a diagnosis of LSIL‐H should be similar to the management of women who have a diagnosis of ASC‐H. Cancer (Cancer Cytopathol) 2009. © 2009 American Cancer Society.     

Implementation of the Thinprep imaging system in a tertiary military medical center

BACKGROUND:

The ThinPrep Imaging System (TIS) was implemented at Brooke Army Medical Center (BAMC) in February 2006 and has been a crucial part of the ability of the Department of Pathology and Laboratory Services ability to improve efficiency and turnaround times for Papanicolaou (Pap) test reporting. The increased detection rate of squamous abnormalities, specifically high‐grade squamous intraepithelial lesions (HSIL), has been well documented by many studies. In addition, the TIS has increased productivity for many laboratories. The objective of this study was to evaluate the effects of implementing the TIS at BAMC, a tertiary military medical center. Specifically, the following were assessed: 1) whether the detection of squamous abnormalities was increased with the TIS, 2) how the rate of high‐risk human papillomavirus (HR‐HPV) detection in atypical squamous cells (ASC) of undetermined significance (ASC‐US) cases changed (or did not change) before and after implementation of the TIS, and 3) how the TIS influenced productivity.

METHODS:

All gynecologic cytology at BAMC has been collected and processed using the ThinPrep system since 2002. Before February 2006 and before implementation of the TIS, Pap tests were screened manually by the cytotechnologists. Detection rates of squamous abnormalities were compared between the period from February 2005 to December 2005 (manual screening) and the period from February 2006 to December 2006 (image‐assisted screening). Squamous abnormalities included ASC‐US; ASC, cannot rule out HSIL (ASC‐H); low‐grade squamous intraepithelial lesion (LSIL); HSIL; glandular abnormalities; and malignancies (squamous or glandular). In addition, the rates of HR‐HPV‐positive, HR‐HPV‐negative, and HR‐HPV‐quantity not sufficient were compared for the same periods. During both periods, testing for HR‐HPV was performed only on ASC‐US Pap tests. HR‐HPV was tested with Digene Hybrid Capture 2 methodology. Productivity was calculated as the change in average slides screened per hour before and after imager implementation.

RESULTS:

In total, 107,647 Pap tests were analyzed in the 2005 (54,438 Pap tests) and 2006 (53,209 Pap tests) timeframes. Increases in the detection of ASC‐H, atypical glandular cells (AGC), LSIL, and HSIL were statistically significant. The proportion of negative for intraepithelial lesion or malignancy (NILM) and unsatisfactory cases decreased significantly with implementation of the TIS. The ASC to squamous intraepithelial lesion (ASC:SIL) ratio decreased from 1.5 to 1.0 after TIS implementation. Decreases in the ASC‐US HR‐HPV‐positive proportion and increases in the ASC‐US HR‐HPV‐negative proportion after implementation of the TIS were statistically significant. In our laboratory, a 60% increase in productivity was noted with use of the TIS.

CONCLUSIONS:

Implementation of the TIS at BAMC significantly increased the detection of ASC‐H, AGC, LSIL, and HSIL but had no significant impact on the ASC‐US detection rate. Although the ASC‐US rate did not change, both the HR‐HPV‐positive rate and the ASC:SIL ratio decreased. The data from the current study suggested that, at least initially, the use of imager‐directed screening may increase the number of clinically insignificant ASC‐US Pap tests. Cancer (Cancer Cytopathol) 2009. Published 2009 by the American Cancer Society.     

Comparison of ProEx C with p16INK4a and Ki‐67 immunohistochemical staining of cell blocks prepared from residual liquid‐based cervicovaginal material

BACKGROUND.

Although liquid‐based cervicovaginal cytology has high sensitivity for detecting dysplastic/malignant lesions, many pitfalls exist. Cell blocks can be prepared from residual liquid‐based cervicovaginal material and used for immunohistochemistry. The aim of this study was to evaluate a new marker, ProEx C, on cell blocks and its ability to distinguish dysplastic/malignant lesions from morphologically abnormal but benign cells. The results of this study were compared with previously reported results for p16 and Ki‐67 on the same material.

METHODS.

ProEx C is a cocktail of monoclonal antibodies against proteins associated with aberrant S phase cell cycle induction (topoisomerase IIA, minichromosome maintenance protein 2). ThinPrep (CytycCorp., Boxborough, Mass) cervicovaginal specimens from 79 patients were selected. Four cases had no residual abnormal cells in the cell block. On the basis of the cell block diagnosis, 29 cases were negative for intraepithelial lesion or malignancy (NILM), 27 had low‐grade squamous intraepithelial lesions (LSIL), 16 had high‐grade squamous intraepithelial lesions (HSIL), and 3 had squamous cell carcinomas (SCC). Cell block sections were immunostained with ProEx C.

RESULTS.

Thirteen of 16 (81%) cases of HSIL stained positively with ProEx C. Two of 27 (7%) LSIL stained positively, and 2 (7%) cases of NILM stained positively. All 3 cases of SCC were strongly positive (100%). Staining for ProEx C showed a higher positive predictive value compared with p16.

CONCLUSIONS.

ProEx C can be used on cell blocks prepared from residual liquid‐based cervicovaginal cytologic specimens. Being a nuclear only stain, it is cleaner and easier to interpret even in scant specimens. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.     

Pathology correlates of a Papanicolaou diagnosis of low‐grade squamous intraepithelial lesion,cannot exclude high‐grade squamous intraepithelial lesion

BACKGROUND.

The objective of this study was to compare findings after a cytologic report of low‐grade squamous intraepithelial lesion, cannot exclude high‐grade squamous intraepithelial lesion (LSIL‐H) with findings after a report of low‐grade squamous intraepithelial lesion (LSIL).

METHODS.

A review of patient records revealed that 312 women had cytologic findings of LSIL‐H, and 324 consecutive women in a comparison group had cytologic findings of LSIL during 2005. Findings over 6 months after diagnosis were retrieved and analyzed using chi‐square tests, Fisher exact tests, and independent group t tests.

RESULTS.

Histology was available for 194 of 312 women (64%) with LSIL‐H and for 184 of 324 women (57%) with LSIL. Of these, 47 of 194 women (24%) with LSIL‐H had grade 2 cervical intraepithelial neoplasia or greater (CIN2+) versus 13 of 184 women (7%) with LSIL (P < .0001). No cancers were identified. High‐grade SIL cytology was reported in 2 of 105 women who had LSIL (2%) and in 4 of 93 women who had LSIL‐H (4%). Women with LSIL‐H who were positive for CIN2+ were younger than those without CIN2+ (25 years vs 30 years; P = .0067)

CONCLUSIONS.

Clinicians whose laboratories report LSIL‐H should manage women who have LSIL‐H with colposcopy, whereas only serial cytologic surveillance is required after a report of LSIL. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.     

Quantitative detection of molecular markers ProEx C (minichromosome maintenance protein 2 and topoisomerase IIa) and MIB‐1 in liquid‐based cervical squamous cell cytology

BACKGROUND.

In this study, the authors conducted a comparative quantitative evaluation of the proliferation markers ProEx C (an aberrant S‐phase induction marker, human papillomavirus E6‐E7 correlated) and MIB‐1 in squamous intraepithelial lesions (SIL) to identify a biomolecular profile informative for the diagnosis of high‐grade SIL/cervical intraepithelial neoplasia 3 or greater that was complementary to the morphologic Papanicolaou (Pap) test (“biomolecular Pap test”).

METHODS.

After the cytologic diagnosis, reflex immunocytochemistry was carried out on 76 unstained SurePath cell samples (20 routine samples that were negative for intraepithelial lesion or malignancy and 56 positive samples that were selected with matching histology). Both a morphometric analysis with a software imaging analysis system and a quantitative analysis of atypical squamous clusters were performed.

RESULTS.

The quantitative evaluation revealed an excellent, direct correlation between the 2 markers, although ProEx C was more selective and more informative for the progression of low‐ and moderate‐grade lesions, because it only revealed cells in aberrant S‐phase cell cycle. The quantitative morphometric analysis revealed the increased presence of atypical, positive clusters and the percentage of positive cells within, both paralleling the severity of the lesions. The threshold of a 3% ProEx C‐positive nuclear area was useful for splitting lesions into groups with a low risk or high risk of progression.

CONCLUSIONS.

Both ProEx C and MIB‐1 were valid proliferation markers in cytologic preparations, and nuclear positivity was quantified successfully by using computer‐assisted analysis. The analysis of atypical clusters may be a valuable tool in the diagnosis of SIL. The presence of atypical clusters and their positivity for proliferation markers are good first‐glance indicators of lesion grade. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.     

Should LSIL‐H be a distinct cytology category?

BACKGROUND:

The 2001 Bethesda System for gynecologic cervical cytology reporting classifies squamous intraepithelial lesions into low‐grade (LSIL) and high‐grade (HSIL) lesions. An intermediate term, “low‐grade squamous intraepithelial lesion, cannot exclude high‐grade squamous intraepithelial lesion (LSIL‐H),” has been used in a small percentage of LSIL cases. To the authors' knowledge, little is known regarding the human papillomavirus (HPV) status in patients with LSIL‐H.

METHODS:

A total of 808 SurePath specimens obtained between December 2009 and April 2011 were tested for 40 HPV genotypes using DNA microarray, followed by a confirmatory DNA sequencing assay.

RESULTS:

The infection rate for high‐risk HPV in women with LSIL‐H (92%) was strikingly close to that for women with HSIL (91%), which was higher than that for those with LSIL (74%); atypical squamous cells, cannot rule out high‐grade lesion (ASC‐H) (78%); or LSIL and ASC‐H combined (74%). HPV type 16, the most common carcinogenic HPV genotype, was detected in 36% of women with LSIL‐H, which was significantly higher than that in women with LSIL and ASC‐H combined (13.8%), but less than that in women with HSIL (44.6%). Patients with LSIL‐H and HSIL had similar infection rates for low‐risk/intermediate‐risk HPV genotypes, which were lower than those in LSIL or LSIL and ASC‐H combined.

CONCLUSIONS:

Women found to have LSIL‐H on a Papanicolaou test appear to have a unique HPV distribution pattern that clearly differs from LSIL and is comparable to that for HSIL, suggesting an increased risk of high‐grade lesions over that of women with LSIL. Recognizing LSIL‐H as an independent diagnostic category may help in the early identification of the high‐risk subgroup that may require a management algorithm comparable to that for patients with HSIL. Cancer (Cancer Cytopathol) 2012. © 2012 American Cancer Society.     

The role of human papillomavirus type 16/18 genotyping in predicting high‐grade cervical/vaginal intraepithelial neoplasm in women with mildly abnormal Papanicolaou results

BACKGROUND:

The authors compared the predictive value of type 16 and/or 18 human papillomavirus (HPV) versus non‐16/18 HPV types for high‐grade (grade ≥2) cervical neoplasm/vaginal intraepithelial neoplasm and carcinoma (CIN/VAIN2+) in women with mildly abnormal Papanicolaou (Pap) results (ie, atypical squamous cells of undetermined significance [ASCUS] or low‐grade squamous epithelial lesion [LSIL]).

METHODS:

The authors retrospectively selected Pap specimens with HPV testing results obtained from 243 women (155 with ASCUS and 88 with LSIL Pap results) in their Department of Pathology. HPV genotyping was performed using the EasyChip HPV blot assay. The Pap specimens with HPV16/18 and non‐16/18 HPV types were compared with follow‐up biopsy results. Follow‐up duration ranged from 1 month to 58 months (mean, 26 months).

RESULTS:

In total, 58 of 155 specimens (37%) that had ASCUS and 29 of 88 specimens (33%) that had LSIL were positive for HPV16/18. CIN/VAIN2+ biopsies were identified in 43 of 155 women (28%) with ASCUS and in 28 of 88 women (32%) with LSIL. Women with ASCUS and HPV16/18 had a significantly higher rate (43%) of CIN/VAIN2+ than women with ASCUS and non‐16/18 HPV types (19%; P = .003; odds ratio, 3.10; 95% confidence interval, 1.48‐6.53). There was no statistically significant difference in the rate of CIN/VAIN2+ between women who had LSIL and HPV16/18 (45%) and those who had LSIL and non‐16/18 HPV types (29%; P = .16; odds ratio, 1.96; 95% confidence interval, 0.77‐4.97).

CONCLUSIONS:

HPV genotyping for HPV16/18 improved risk assessment for women with ASCUS Pap results and may be used to predict the risk of CIN/VAIN2+ to better guide follow‐up management. Cancer (Cancer Cytopathol) 2013. © 2012 American Cancer Society.     

Candida and dysbacteriosis: A cytologic,population‐based study of 100,605 asymptomatic women concerning cervical carcinogenesis

BACKGROUND.

The objective of this study was to investigate whether the presence of vaginal Candida or dysbacteriosis predisposes women to an increased susceptibility for (pre)neoplasia over time.

METHODS.

A retrospective, longitudinal, cohort study was performed and was conducted in a population of 100,605 women, each of whom had 2 smears taken over a period of 12 years as part of the Dutch Cervical Screening Program. From these women, a cohort of 1439 women with Candida and a cohort of 5302 women with dysbacteriosis were selected as 2 separate study groups. The control cohort consisted of women who had completely normal cervical smears (n = 87,903 women). These groups were followed retrospectively over time. The odds ratios (OR) for squamous abnormalities in the follow‐up smear for the women in these 3 cohorts were established.

RESULTS.

The dysbacteriotic cohort was significantly more likely to have low‐grade squamous intraepithelial lesions (LSIL) and high‐grade squamous intraepithelial lesions (HSIL+) in their follow‐up smear (OR, 1.85; 95% confidence interval [95% CI], 1.28–2.67 and OR, 2.00; 95% CI, 1.31–3.05, respectively) compared with women in the control group. In contrast, the Candida cohort had no significantly increased or decreased risk of developing SIL. The equivocal diagnosis ‘atypical squamous cells of undetermined significance’ was rendered significantly more often in the follow‐up smear of both study cohorts (Candida cohort: OR, 1.42; 95% CI, 1.03–1.95; dysbacteriotic cohort: OR, 1.44; 95% CI, 1.22–1.71).

CONCLUSIONS.

The results from this study indicated that the presence of Candida vaginalis was not associated with an increased risk for SIL over time. In contrast, women with dysbacteriosis had a significantly increased risk of developing (pre)neoplastic changes. These findings should be taken into account in further research concerning predisposing factors for cervical carcinogenesis. Cancer (Cancer Cytopathol) 2007. © 2007 American Cancer Society.     

Evaluation of quantity and staining pattern of human papillomavirus (HPV)‐infected epithelial cells in thin‐layer cervical specimens using optimized HPV‐CARD assay

BACKGROUND.

Testing for human papillomavirus (HPV) is used in the triage of women with a cervical cytology of atypical squamous cells of undetermined significance (ASCUS). A fluorescent in situ hybridization assay was developed for the detection of HPV using the catalyzed receptor deposition technique (HPV‐CARD). In this study, the utility of this assay was tested for the detection of HPV in liquid‐based cervical cytology specimens.

METHODS.

A total of 195 liquid‐based cytology specimens were analyzed using the HPV‐CARD assay. The results from the assay were compared with HPV polymerase chain reaction (PCR) and typing results. The number of HPV‐infected cells and the staining pattern was correlated with the cytology classification.

RESULTS.

A 91% concordance between HPV‐CARD and PCR was observed for the detection of high‐risk HPV viruses. A 78% concordance was observed for specimens that were negative for HPV. In ASCUS, low‐grade squamous intraepithelial lesion (LSIL), and high‐grade squamous intraepithelial lesion (HSIL) categories, the average number of HPV‐positive cells per slide was 19 cells, 127 cells, and 450 cells, respectively. The number of cells with a punctate staining, suggestive of HPV integration, was 21% in ASCUS, 34% in LSIL, and 46% in HSIL specimens.

CONCLUSIONS.

The results of the current study indicate positive correlations between the severity of the disease and the increased overall quantity of HPV‐positive epithelial cells in cervical cytology specimens and accumulation of cells with punctate staining suggestive of integrated HPV. In summary, the developed HPV‐CARD assay was found to provide novel information regarding the proportion and staining pattern of HPV‐infected epithelial cells in different cytologic categories of cervical specimens. Cancer (Cancer Cytopathol) 2007. © 2007 American Cancer Society.     

A validation study of the FocalPoint GS imaging system for gynecologic cytology screening

BACKGROUND:

Studies of the performance of the automated FocalPoint Guided Screening (FPGS) imaging system in gynecologic cytology screening relative to manual screening have yielded conflicting results. In view of this uncertainty, a validation study of the FPGS was conducted before its potential adoption in 2 large laboratories in Ontario.

METHODS:

After an intense period of laboratory training, a cohort of 10,233 current and seeded abnormal slides were classified initially by FPGS. Manual screening and reclassification blinded to the FPGS results were then performed. Any adequacy and/or cytodiagnostic discrepancy between the 2 screening methods subsequently was resolved through a consensus process (truth). The performance of each method's adequacy and cytodiagnosis vis‐a‐vis the truth was established. The sensitivity and specificity of each method at 4 cytodiagnostic thresholds (atypical squamous cells of undetermined significance or worse [ASC‐US+], low‐grade squamous intraepithelial lesion or worse [LSIL+], high‐grade squamous intraepithelial lesion or worse [HSIL+], and carcinoma) were compared. The false‐negative rate for each cytodiagnosis was determined.

RESULTS:

The performance of FPGS in detecting carcinoma, HSIL+, and LSIL+ was no different from the performance of manual screening, but the false‐negative rates for LSIL and ASC‐US were higher with FPGS than with manual screening.

CONCLUSIONS:

The results from this validation study in the authors' laboratory environment provided no evidence that FPGS has diagnostic performance that differs from manual screening in detecting LSIL+, HSIL+, or carcinoma. Cancer (Cancer Cytopathol) 2013;121:189–196. © 2013 American Cancer Society.     

Impact of the more restrictive definition of atypical squamous cells introduced by the 2001 Bethesda System on the sensitivity and specificity of the Papanicolaou test

BACKGROUND.

The Bethesda 2001 (B2001) classification system raised the threshold for reporting atypical squamous cells. The category of Atypical Squamous Cells of Undetermined Significance (ASCUS)‐favor reactive has been eliminated with a recommendation for cytologists to judiciously downgrade those Papanicolaou (Pap) tests that would formerly have been in this category to Negative for Intraepithelial Lesion or Malignancy (NILM). The effect of this change on sensitivity and specificity of the Pap test is not yet known.

METHODS.

A total of 535 consecutive SurePath Pap tests interpreted as ASCUS during a period from March 1 through December 31, 2001, with follow‐up polymerase chain reaction(PCR)‐based human papilloma virus (HPV) testing, were independently reviewed by 3 pathologists and 1 cytotechnologist who reinterpreted these tests according to Bethesda 2001 criteria. Follow‐up HPV and biopsy results were compiled for a 5‐year period.

RESULTS.

By consensus of the 4 observers, 169 (32%) of the ASCUS cases were downgraded to NILM. These cases showed a lower rate of underlying high‐risk HPV infection (11% vs 30%) and cervical intraepithelial neoplasia of grades 2 to 3 (CIN 2/3) (5% vs 10%) on follow‐up than those tests that were reinterpreted as ASCUS or higher. Nine women with follow‐up CIN 2/3 would have had the Pap test interpreted as NILM instead of ASCUS under Bethesda 2001 (20% of all CIN 2/3 found). Individual reviewers downgraded 29% to 42% Pap tests to NILM including those of 5 to 10 women with follow‐up CIN 2/3.

CONCLUSIONS.

The ASCUS threshold established by B2001 prevents a sizeable subset of women from having follow‐up for ASC. However, as a consequence, a few women with CIN 2/3 are downgraded to NILM. The cost savings thus achieved must be weighed against the lost opportunities to detect CIN 2/3. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.     

Histopathologic follow-up and human papillomavirus DNA test results in 290 patients with high-grade squamous intraepithelial lesion Papanicolaou test results

BACKGROUND:

The study documents histopathologic outcomes and high‐risk (hr) human papillomavirus (HPV) test results in a large cohort of patients with high‐grade squamous intraepithelial lesion (HSIL) liquid‐based cytology (LBC) Pap test results.

METHODS:

A total of 352 patients with HSIL results (338 cervical and 14 vaginal) who had hrHPV testing and 290 patients with biopsy follow‐up were studied. hrHPV detection rates were compared at different ages, with or without an endocervical/transformation zone sample (EC/TZS), and for cervical and vaginal HSIL Pap smears. Histopathologic follow‐up findings were also compared. hrHPV‐negative HSIL slides were re‐evaluated in a blinded manner.

RESULTS:

A total of 325 of 338 (96.2%) cervical HSIL and 12 of 14 (87.5%) vaginal HSIL tested hrHPV‐positive. A total of 271 of 281 (96.4%) EC/TZS‐positive cervical HSIL and 54 of 57 (94.7%) EC/TZS‐negative cervical HSIL tested hrHPV‐positive. The percentage of hrHPV‐positive HSIL declined slightly with increasing age. 197 of 273 (72.3%) hrHPV‐positive cervical HSIL had histopathologic cervical intraepithelial neoplasia (CIN) 2/3+ follow‐up, including 8 squamous carcinomas, compared with 4 of 12 (33.3%) hrHPV‐negative HSIL with CIN2/3 (no carcinomas). 167 of 241 (69.2%) EC/TZS‐positive HSIL had CIN2/3+ follow‐up, compared with 34 of 44 (77.3%) EC/TZS‐negative HSIL. Equivocal HSIL morphology characterized some HPV‐negative HSIL without CIN2/3+ follow‐up.

CONCLUSIONS:

hrHPV was detected in LBC vials from 96.2% of 338 cervical HSIL and 85.7% of 14 vaginal HSIL. CIN2/3+ was significantly more likely with hrHPV‐positive cervical HSIL than with hrHPV‐negative cervical HSIL. Presence or absence of an EC/TZS did not significantly impact HSIL hrHPV or CIN2/3+ rates. Some hrHPV‐negative HSIL cases may represent HSIL cytologic mimics. Cancer (Cancer Cytopathol) 2011;. © 2011 American Cancer Society.     

Papanicolaou smear history in women with low‐grade cytology before cervical cancer diagnosis

BACKGROUND.

The purpose of the current study was to examine the screening histories of women diagnosed with invasive cervical cancer (ICC) in 2000 who had previous Papanicolaou (Pap) smears deemed to be unsatisfactory or with low‐grade findings that did not lead to biopsy.

METHODS.

A total of 252 Pap smears from 47 women taken between 1992 and 2000 were included in the study; 247 smears were reexamined at the laboratory of origin before the study and all 252 were then reexamined independently by 2 experienced cytotechnicians and 2 cytopathologists.

RESULTS.

Of the 47 cases of ICC, 35 were squamous cell carcinoma, 10 were adenocarcinoma, and 2 were other types. On reexamination at the laboratory of origin, 24 cases were upgraded and in the study group 27 cases were upgraded to diagnoses requiring biopsy. On reexamination at the laboratory of origin, it was found that the first high‐grade squamous intraepithelial lesion (HSIL) could have been diagnosed on average 4.2 years earlier than it was originally (95% confidence interval [95% CI], 3.3–5.1 years). On reexamination by the study group the first diagnosis of HSIL was made in smears dating from 5.4 years before the diagnosis of ICC (95% CI, 4.5–6.2 years).

CONCLUSIONS.

The study confirms that unsatisfactory and low‐grade Pap smears imply a risk of developing high‐grade lesions at a later date and shows that in a screening program a subgroup of smears may be diagnosed as unsatisfactory or low grade despite the presence of high‐grade findings that are detectable on reexamination. Cancer (Cancer Cytopathol) 2007. © 2007 American Cancer Society.     

DNA methylation analysis in liquid‐based cytology for cervical cancer screening

Cervical cancer is the second most common type of cancer in women worldwide. Preinvasive disease can be detected by cervical cytology. All currently available cytology technologies rely on the visual analysis of exfoliated cells from the uterine cervix. Improvement of conventional cytological screening has been proposed by the introduction of molecular‐based markers applied to liquid‐based cytology (LBC), the suspension of cells collected from the cervix. DNA methylation changes occur very early in carcinogenesis and identification of appropriate DNA methylation markers in such samples should be able to distinguish high‐grade squamous intraepithelial lesions (HSIL) from nonspecific cytology changes and the normal cervix. To address this potential, we have undertaken a proof‐of‐principle study of methylation status of LBC samples from HSIL cytology cases compared against matched normal controls. Using quantitative methylation‐specific PCR on 28 genes, we found SOX1, HOXA11 and CADM1 to significantly discriminate between the groups analyzed (p < 0.01). Area under the receiver operating characteristic (ROC) curve (AUC) demonstrated that methylation of SOX1, HOXA11 and CADM1 could discriminate between HSIL cases and controls with high sensitivity and specificity (AUC 0.910, 0.844 and 0.760, respectively). The results were further validated in an independent set. This proof‐of‐principle study is the first to validate the results in an independent case/control set and presents HOXA11, a gene that is important for cervical development, as a potentially useful DNA marker in LBC samples. Further assessment of these preliminary estimates will need to be performed in a larger cohort to confirm clinical utility. © 2009 UICC     

The significance of the Papanicolaou smear diagnosis of low-grade squamous intraepithelial lesion cannot exclude high-grade squamous intraepithelial lesion

BACKGROUND: Cervical cytologic specimens that show a low-grade squamous intraepithelial lesion (LSIL) occasionally contain a few cells that are suspicious for, but not diagnostic of, a high-grade squamous intraepithelial lesion (HSIL). In such cases, a diagnosis of LSIL cannot exclude HSIL is rendered. The objective of the current study was to assess the positive predictive value (PPV) for HSIL in follow-up cervical biopsies for these cases. METHODS: One hundred forty-four women with a Papanicolaou (Pap) diagnosis of LSIL cannot exclude HSIL and their follow-up cervical biopsies were reviewed. Results were compared with a control group of 155 women with a Pap diagnosis of LSIL. A subset of biopsies was tested and typed for human papillomavirus (HPV) DNA by polymerase chain reaction amplification using consensus primers followed by restriction fragment length polymorphism analysis. HPVs were scored as low-risk or high-risk types. RESULTS: Women with LSIL cannot exclude HSIL had a higher incidence of HSIL (PPV = 29%) on follow-up cervical biopsy than the control group (PPV = 15%, P < 0.01). In addition, SIL, indeterminate grade was diagnosed in 10% of cervical biopsies in the study group as compared with 4% in controls. Review of Pap smears from the study group showed that there were 3 types of cells suspicious for a high-grade lesion: atypical squamous metaplastic cells (62%), atypical keratinized cells (20%), and dysplastic cells of borderline nuclear-to-cytoplasm ratio (18%). HPV analysis confirmed the presence of high-risk HPV types in the study cases with high-grade cervical biopsies. CONCLUSIONS: Women with a Pap diagnosis of LSIL cannot exclude HSIL appear to be more likely to harbor a high-grade lesion than those diagnosed with LSIL alone. Its use appears warranted. Women with this diagnosis merit appropriate clinical follow-up to exclude HSIL.     

Methylation of viral and host genes and severity of cervical lesions associated with human papillomavirus type 16

Methylation of human papillomavirus (HPV) and host genes may predict cervical cancer risk. We examined the methylation status of selected sites in HPV16 and human genes in DNA extracted from exfoliated cervical cell samples of 244 women harboring HPV16‐positive cancer or cervical intraepithelial neoplasia (CIN) or negative for intraepithelial lesions or malignancy (NILM). We quantified the methylation of CpG sites in the HPV16 L1 gene (CpG 6367 and 6389) and in the human genes EPB41L3 (CpG 438, 427, 425) and LMX1 (CpG 260, 262, 266, 274) following bisulfite treatment and pyrosequencing. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic utility of methylation level for the different sites and for a joint predictor score. Methylation in all sites significantly increased with lesion severity (p < 0.0001). Area under the curve (AUC) was highest among the CIN2/3 vs. cancer ranging from 0.786 to 0.853 among the different sites. Site‐specific methylation levels strongly discriminated CIN2/3 from NILM/CIN1 and cancer from CIN2/3 (range of odds ratios [OR]: 3.69–12.76, range of lower 95% confidence bounds: 1.03–4.01). When methylation levels were mutually adjusted for each other EPB41L3 was the only independent predictor of CIN2/3 vs. NILM/CIN1 contrasts (OR = 9.94, 95%CI: 2.46–40.27). High methylation levels of viral and host genes are common among precancerous and cancer lesions and can serve as independent risk biomarkers. Methylation of host genes LMX1 and EPB41L3 and of the viral HPV16 L1 sites has the potential to distinguish among precancerous lesions and to distinguish the latter from invasive disease.     

Feasibility of clinical detection of cervical dysplasia using angle‐resolved low coherence interferometry measurements of depth‐resolved nuclear morphology

This study sought to establish the feasibility of using in situ depth‐resolved nuclear morphology measurements for detection of cervical dysplasia. Forty enrolled patients received routine cervical colposcopy with angle‐resolved low coherence interferometry (a/LCI) measurements of nuclear morphology. a/LCI scans from 63 tissue sites were compared to histopathological analysis of co‐registered biopsy specimens which were classified as benign, low‐grade squamous intraepithelial lesion (LSIL), or high‐grade squamous intraepithelial lesion (HSIL). Results were dichotomized as dysplastic (LSIL/HSIL) versus non‐dysplastic and HSIL versus LSIL/benign to determine both accuracy and potential clinical utility of a/LCI nuclear morphology measurements. Analysis of a/LCI data was conducted using both traditional Mie theory based processing and a new hybrid algorithm that provides improved processing speed to ascertain the feasibility of real‐time measurements. Analysis of depth‐resolved nuclear morphology data revealed a/LCI was able to detect a significant increase in the nuclear diameter at the depth bin containing the basal layer of the epithelium for dysplastic versus non‐dysplastic and HSIL versus LSIL/Benign biopsy sites (both p < 0.001). Both processing techniques resulted in high sensitivity and specificity (>0.80) in identifying dysplastic biopsies and HSIL. The hybrid algorithm demonstrated a threefold decrease in processing time at a slight cost in classification accuracy. The results demonstrate the feasibility of using a/LCI as an adjunctive clinical tool for detecting cervical dysplasia and guiding the identification of optimal biopsy sites. The faster speed from the hybrid algorithm offers a promising approach for real‐time clinical analysis.     

DNA methylation profiling of cervical squamous intraepithelial lesions using liquid-based cytology specimens: an approach that utilizes receiver-operating characteristic analysis

BACKGROUND: Cervical carcinoma is a common malignancy among women worldwide, and its pathogenesis is related causally to human papillomavirus infection. The progression from precursor squamous intraepithelial lesions to cervical carcinoma requires additional genetic and epigenetic alterations that have not been characterized fully. The authors examined aberrant promoter methylation of multiple tumor suppressor genes in precursor squamous intraepithelial lesions. METHODS: A multiplex, nested, methylation-specific polymerase chain reaction approach was used to examine promoter methylation of 15 tumor suppressor genes in high-grade squamous intraepithelial lesions (HSIL, n = 11), low-grade squamous intraepithelial lesions (LSIL, n = 17), and negative tissues (n = 11) from liquid-based cytology samples. The area under the receiver-operating characteristic (ROC) curve was determined for individual methylated tumor suppressor genes and for gene combinations to evaluate test performance for the ability of methylation profiles to distinguish HSIL cytology samples from combined LSIL/negative cytology samples. RESULTS: Aberrant promoter methylation of DAPK1 and IGSF4 occurred at a high frequency in HSIL samples and was absent in LSIL and negative samples. There was a significant trend toward increased methylation with the increased severity of lesions, and the mean number of methylated genes was significantly higher in HSIL samples compared with LSIL and negative samples. Using the area under the ROC curve as a measure of test performance, the methylation of IGSF4 and DAPK1 had areas that were significantly greater than 0.5; thus, each had the ability to distinguish HSIL samples from combined LSIL/negative samples. The areas under the curve for the best two-gene combination (IGSF4/DAPK1) and the best three-gene combination (IGSF4/DAPK1/HIC1) were not statistically different from the best individual tumor suppressor gene (IGSF4) in distinguishing HSIL samples from combined LSIL/negative samples. CONCLUSIONS: Aberrant promoter methylation of tumor suppressor genes is an epigenetic alteration that occurs during neoplastic progression to cervical carcinoma. The methylation status of multiple tumor suppressor genes can be evaluated using ROC analysis to determine methylation profiles that can distinguish HSIL samples from combined LSIL/negative samples.