Microinvasive cervical squamous cell carcinoma in Slovenia during the period 2001-2007.

BACKGROUND: Microinvasive squamous cell carcinoma (MISCC) comprises a significant portion of all cervical cancers in Slovenia. Criteria of carcinomatous invasion are well described in the literature, however histopathological assessment of MISCC is difficult, because morphological characteristics can overlap with cervical intraepithelial neoplasia grade 3 (CIN 3) and other pathological changes. The aim of our study was to evaluate the reliability of the histopathological diagnosis of MISCC in Slovenia during the period from 2001 to 2007. MATERIALS AND METHODS: Data on patients with a histopathological diagnosis of cervical MISCC (FIGO stage IA) in the period of 2001 to 2007 were obtained from the Cancer Registry of Slovenia. Histological slides were obtained from the majority of pathology laboratories in Slovenia. We received 250 cases (69% of all MISCC) for the review; 30 control cases with CIN 3 and invasive squamous cell carcinoma FIGO stage IB were intermixed. The slides were coded and reviewed. RESULTS: Among 250 cases originally diagnosed as MISCC, there was an agreement with MISCC diagnosis in 184 (73.6%) cases (of these 179/184 (97.3%) cases were FIGO stage IA1 and 5/184 (2.7%) cases were FIGO stage IA2). Among 179 FIGO stage IA1 cases 117 (65.4%) showed only early stromal invasion. CONCLUSIONS: The retrospective review of cases diagnosed as MISCC during the period 2001-2007 in Slovenia showed a considerable number of overdiagnosed cases. Amongst cases with MISCC confirmed on review, there was a significant proportion with early stromal invasion (depth of invasion less than 1 mm).

Aberrant expression of long noncoding RNAs in cervical intraepithelial neoplasia.

OBJECTIVE: Long noncoding RNAs (lncRNAs) are a unique class of messenger RNA-like transcripts of at least 200 nucleotides in length with no significant protein-coding capacity. Aberrant lncRNA expression is emerging as a major component of the cancer transcriptome. Here, we sought to determine if differential lncRNA expression is a feature of the human cervical intraepithelial neoplasia (CIN) transcriptome. METHODS: Sequence data were derived from 16 long serial analyses of gene expression (L-SAGE) libraries constructed from cervical specimens representing mild (CIN1), moderate (CIN2), and severe (CIN3) histopathologic grades of CIN. A novel lncRNA discovery pipeline was developed to query the expression of lncRNAs within the SAGE data sets. RESULTS: A total of 2,230,370 sequence tags were delineated from the 16 SAGE libraries, representing the expression of 367,482 unique tags at varying abundance. Using a novel stepwise filtering strategy, we analyzed the cervical SAGE libraries and identified the expression profiles of 1056 lncRNAs in the human cervix. We present the first lncRNA expression profile derived from nonneoplastic cervical tissue and establish that changes in lncRNA expression do occur in cervical intraepithelial lesions. Our analysis also shows statistically significant aberrant expression of lncRNAs in the 3 CIN grades, suggesting that these unique noncoding RNA transcripts may contribute to the development and progression of precursor lesions. CONCLUSIONS: Through the analysis of L-SAGE libraries constructed from cervical specimens, we provide the first lncRNA expression profile of the cervix and demonstrate aberrant expression in early-stage neoplasia.

The use of quantitative cytology in identifying high-risk oral lesions in community practice.

OBJECTIVE: This study evaluated whether quantitative cytology (QC) can disclose abnormal DNA content (aneuploidy) and abnormal nuclear morphology of high-risk potentially malignant disorders (PMDs) of the oral mucosa found in the community in reference to clinicohistopathologic features. STUDY DESIGN: A total of 171 patients at community-based clinic with suspicious oral lesions were evaluated with concurrent but independent histopathologic and QC assessments. RESULTS: QC-positive results were associated with oral lesions with higher clinical risk factors: large size, nonhomogeneous surface texture, and located at high-risk anatomic sites. Only 3% of benign/reactive and 5% of low-risk PMDs were QC positive, while 92% of high-risk PMDs and 88% of squamous cell carcinomas (SCCs) were QC positive. The sensitivity and specificity of QC for detection of high-grade dysplasia/SCC were 89% and 97%. CONCLUSIONS: QC could serve as an adjunctive tool for the detection of high-risk PMD/SCC requiring immediate clinical care.

Optical technologies and molecular imaging for cervical neoplasia: a program project update.

There is an urgent global need for effective and affordable approaches to cervical cancer screening and diagnosis. In developing nations, cervical malignancies remain the leading cause of cancer-related deaths in women. This reality may be difficult to accept given that these deaths are largely preventable; where cervical screening programs have been implemented, cervical cancer-related deaths have decreased dramatically. In developed countries, the challenges of cervical disease stem from high costs and overtreatment. The National Cancer Institute-funded Program Project is evaluating the applicability of optical technologies in cervical cancer. The mandate of the project is to create tools for disease detection and diagnosis that are inexpensive, require minimal expertise, are more accurate than existing modalities, and can be feasibly implemented in a variety of clinical settings. This article presents the status and long-term goals of the project.

Accuracy of optical spectroscopy for the detection of cervical intraepithelial neoplasia: Testing a device as an adjunct to colposcopy.

Testing emerging technologies involves the evaluation of biologic plausibility, technical efficacy, clinical effectiveness, patient satisfaction, and cost-effectiveness. The objective of this study was to select an effective classification algorithm for optical spectroscopy as an adjunct to colposcopy and obtain preliminary estimates of its accuracy for the detection of CIN 2 or worse. We recruited 1,000 patients from screening and prevention clinics and 850 patients from colposcopy clinics at two comprehensive cancer centers and a community hospital. Optical spectroscopy was performed, and 4,864 biopsies were obtained from the sites measured, including abnormal and normal colposcopic areas. The gold standard was the histologic report of biopsies, read 2 to 3 times by histopathologists blinded to the cytologic, histopathologic, and spectroscopic results. We calculated sensitivities, specificities, receiver operating characteristic (ROC) curves, and areas under the ROC curves. We identified a cutpoint for an algorithm based on optical spectroscopy that yielded an estimated sensitivity of 1.00 [95% confidence interval (CI) = 0.92-1.00] and an estimated specificity of 0.71 [95% CI = 0.62-0.79] in a combined screening and diagnostic population. The positive and negative predictive values were 0.58 and 1.00, respectively. The area under the ROC curve was 0.85 (95% CI = 0.81-0.89). The per-patient and per-site performance were similar in the diagnostic and poorer in the screening settings. Like colposcopy, the device performs best in a diagnostic population. Alternative statistical approaches demonstrate that the analysis is robust and that spectroscopy works as well as or slightly better than colposcopy for the detection of CIN 2 to cancer.

Prevalence and type distribution of human papillomavirus in 5,000 British Columbia women--implications for vaccination.

BACKGROUND: Human papilloma virus (HPV) prevalence studies performed in different regions and population groups across Canada would inform public health decisions regarding implementation of anti-HPV vaccines. METHODS: A total of 8,700 liquid-based specimens from 8,660 women aged 13-86 from throughout British Columbia were collected. DNA was isolated from 4,980 of these samples and assessed for HPV prevalence and type distribution. HPV was detected by PCR analysis using tagged GP5+/6+ consensus primers to amplify the L1 region of HPV; typing was done by bi-directional sequencing of PCR products. RESULTS: Overall HPV prevalence was 16.8% (age adjusted 15.5%). Prevalence of high-risk HPV was 13.9, and 10.7% of samples contained HPV16. HPV prevalence was highest in the youngest group of women (<20 years). One-third of HPV positive samples contained more than one HPV type. Percentages of low-grade (LGIL) and high-grade intraepithelial lesions (HGIL) containing high-risk HPV are 52.3 and 79.4%, respectively. CONCLUSIONS: Overall HPV prevalence in this study is within the range of estimates from other studies. The prevalence of HPV16 is higher than what is found in other Canadian and international studies. HPV16 and HPV18 compose a majority of the high-risk virus in this study. Use of current HPV vaccines could considerably reduce HPV-related conditions including cervical cancer and procedures such as colposcopy.

Up regulation in gene expression of chromatin remodelling factors in cervical intraepithelial neoplasia.

BACKGROUND: The highest rates of cervical cancer are found in developing countries. Frontline monitoring has reduced these rates in developed countries and present day screening programs primarily identify precancerous lesions termed cervical intraepithelial neoplasias (CIN). CIN lesions described as mild dysplasia (CIN I) are likely to spontaneously regress while CIN III lesions (severe dysplasia) are likely to progress if untreated. Thoughtful consideration of gene expression changes paralleling the progressive pre invasive neoplastic development will yield insight into the key casual events involved in cervical cancer development. RESULTS: In this study, we have identified gene expression changes across 16 cervical cases (CIN I, CIN II, CIN III and normal cervical epithelium) using the unbiased long serial analysis of gene expression (L-SAGE) method. The 16 L-SAGE libraries were sequenced to the level of 2,481,387 tags, creating the largest SAGE data collection for cervical tissue worldwide. We have identified 222 genes differentially expressed between normal cervical tissue and CIN III. Many of these genes influence biological functions characteristic of cancer, such as cell death, cell growth/proliferation and cellular movement. Evaluation of these genes through network interactions identified multiple candidates that influence regulation of cellular transcription through chromatin remodelling (SMARCC1, NCOR1, MRFAP1 and MORF4L2). Further, these expression events are focused at the critical junction in disease development of moderate dysplasia (CIN II) indicating a role for chromatin remodelling as part of cervical cancer development. CONCLUSION: We have created a valuable publically available resource for the study of gene expression in precancerous cervical lesions. Our results indicate deregulation of the chromatin remodelling complex components and its influencing factors occur in the development of CIN lesions. The increase in SWI/SNF stabilizing molecule SMARCC1 and other novel genes has not been previously illustrated as events in the early stages of dysplasia development and thus not only provides novel candidate markers for screening but a biological function for targeting treatment.

p16 and MIB1 improve the sensitivity and specificity of the diagnosis of high grade squamous intraepithelial lesions: methodological issues in a report of 447 biopsies with consensus diagnosis and HPV HCII testing.

OBJECTIVE: Many investigators are studying the additional value of biomarkers to improve histopathologic agreement, but few are using the same methodologies. Our objectives in this analysis to differentiate High-grade Squamous Intraepithelial lesions (HGSIL) from Low Grade Squamous Intraepithelial Lesions (LGSIL), atypia, and normal were: (1) to examine the rate of Human Papilloma Virus High-Risk positivity (HPV HR+), (2) to compare and grade the basal, parabasal, intermediate, and superficial layer staining of each marker, (3) to determine the optimal qualitative threshold for markers, (4) to compare p16 and MIB1 agreement, and (5) to examine the sensitivities and specificities using each markers alone and together. METHODS: A sample of biopsies from 208 patients were chosen from a total of 1850 patients and 3735 biopsies obtained during the course of ongoing optical trials. At least two independent blinded reviews were performed for each biopsy. A third review was performed if there was a disagreement between the two reviews. Both endocervical and ectocervical samples were stained for p16 and MIB1. A grading system that is delineated in the text ranged from 0 to 3 for both markers and each biopsy was scored by each cell layer. Frequencies, sensitivities, and specificities were calculated using Statistica. An ANOVA was used to compare p16 and MIB1 staining in the epithelial layers. Finally the sensitivity and specificity of each marker alone and together were examined. RESULTS: 453 specimens from 208 patients whose final diagnoses were normal (n=244), low-grade (LG) (n=59), and high-grade (HG) (n=144) were selected for analysis. 447 of 453 specimens were available for staining. Most LG and HG lesions were HPV HR positive. Endocervical samples stained positive less often than ectocervix and often results were discordant from ectocervical results. The analysis by layers showed pronounced increases in staining of both p16 and MIB1 as lesions progressed from normal to LG to HG. The cutoff or threshold for p16 was 0 versus 1-3 while for MIB1 it was 0-1 versus 2-3. Using the intermediate epithelial layer measurement of both p16 and MIB1 in HPV High-Risk Positive separated the normal tissue from LGSIL, normal from HGSIL, and LGSIL from HGSIL by a statistically significant margin (p<0.05). Each marker had sensitivities and specificities for the diagnosis of HGSIL versus LGSIL and normal of approximately 85-90% and this improved by 5% for both sensitivity and specificity when used together (p16 sensitivity 90%, specificity 85%; MIB1 sensitivity 89%, specificity 87%; together sensitivity 94%, specificity 90%). CONCLUSION: Several important methodological issues have been studied. Overall, p16 and MIB1 are promising markers to help pathologists differentiate HG lesions from all else. The staining of the endocervix and the ectocervix do not always agree, and the ectocervix more often stains positive with the presence of HGSIL. Each marker is helpful and both are helpful together. In conclusion, both markers are useful for the confirmation of HG lesions.

Comprehensive serial analysis of gene expression of the cervical transcriptome.

BACKGROUND: More than half of the approximately 500,000 women diagnosed with cervical cancer worldwide each year will die from this disease. Investigation of genes expressed in precancer lesions compared to those expressed in normal cervical epithelium will yield insight into the early stages of disease. As such, establishing a baseline from which to compare to, is critical in elucidating the abnormal biology of disease. In this study we examine the normal cervical tissue transcriptome and investigate the similarities and differences in relation to CIN III by Long-SAGE (L-SAGE). RESULTS: We have sequenced 691,390 tags from four L-SAGE libraries increasing the existing gene expression data on cervical tissue by 20 fold. One-hundred and eighteen unique tags were highly expressed in normal cervical tissue and 107 of them mapped to unique genes, most belong to the ribosomal, calcium-binding and keratinizing gene families. We assessed these genes for aberrant expression in CIN III and five genes showed altered expression. In addition, we have identified twelve unique HPV 16 SAGE tags in the CIN III libraries absent in the normal libraries. CONCLUSION: Establishing a baseline of gene expression in normal cervical tissue is key for identifying changes in cancer. We demonstrate the utility of this baseline data by identifying genes with aberrant expression in CIN III when compared to normal tissue.

Using LongSAGE to Detect Biomarkers of Cervical Cancer Potentially Amenable to Optical Contrast Agent Labelling.

Sixteen longSAGE libraries from four different clinical stages of cervical intraepithelial neoplasia have enabled us to identify novel cell-surface biomarkers indicative of CIN stage. By comparing gene expression profiles of cervical tissue at early and advanced stages of CIN, several genes are identified to be novel genetic markers. We present fifty-six cell-surface gene products differentially expressed during progression of CIN. These cell surface proteins are being examined to establish their capacity for optical contrast agent binding. Contrast agent visualization will allow real-time assessment of the physiological state of the disease process bringing vast benefit to cancer care. The data discussed in this publication have been submitted to NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE6252.

Results of a pilot study of multispectral digital colposcopy for the in vivo detection of cervical intraepithelial neoplasia.

OBJECTIVE: Fluorescence spectroscopy is a promising technology for the detection of cervical squamous intraepithelial precancers and cancers. To date, many investigators have focused on point spectroscopy as an adjunct to diagnostic colposcopy. A device that visualizes the whole field of the cervix is needed for screening. To that end, we have developed a multispectral digital colposcope that works through the colposcope to image with white light, UV excitation at 345 nm, and blue light at 440 nm excitation. Here, we report the pilot study that precedes a Phase I trial. METHODS: The MDC system is composed of a light source, a colposcope, and a video rate color CCD camera with a frame grabber and takes approximately less than 1 min to make images of the cervix. Patients were measured at baseline and after acetic acid placement with white light, 345 nm excitation, and 440 nm excitation from the xenon arc lamp. The white light is in the visible spectrum, 345 nm excitation is in the UV spectrum and is not visible, and 440 nm excitation is blue light in the visible spectrum. White light generates a pink image of the cervix. 345 nm excitation, the UV light, excites fluorophores to emit a blue image. 440 nm excitation, the blue light, excites fluorophores to emit a green image. The patients underwent a loop excision procedure and the histopathology was inked and cut into 12 sections by the study pathologists. The histopathologic slides were scanned and the images were then reconstructed into maps. A diagnostic algorithm was calculated. The data were preprocessed, transformed, and analyzed by the K-means clustering method. Disease maps were generated using the algorithm and classifier and compared to white light colposcopy and the blue and green images obtained at 345 and 440 nm. RESULTS: Forty-six patients were measured at four clinical sites. Images were made of the cervix with white light, 345 nm excitation, and 440 nm excitation and are presented in the figures. As the study went on, images improved with improvements in the instrument. The white light and fluorescence images are presented with crudely constructed histopathologic maps and algorithmic maps. At 345 nm excitation, the UV light, histologically confirmed CIN appears darker blue; while at 440 nm excitation, the blue light, histologically confirmed CIN appears lighter green. CONCLUSIONS: This pilot study shows that MDC images can be matched to both histopathologic and algorithmic maps. The device and the algorithm are evolving but show promise. A Phase I trial is planned.

Kappa statistics to measure interrater and intrarater agreement for 1790 cervical biopsy specimens among twelve pathologists: qualitative histopathologic analysis and methodologic issues.

BACKGROUND: As part of a program project to evaluate emerging optical technologies for cervical neoplasia, we performed fluorescence and reflectance spectroscopic examinations of patients with abnormal Papanicolaou smears. Biopsy specimens were taken from each area and measured optically, and study pathologists performed qualitative histopathologic readings. Several methodologic issues arose in this analysis: (1) the interpathologist and intrapathologist agreement between institutions for the 1790 biopsy specimens; (2) the interinstitutional agreement among the two institutions conducting the trials on 117 randomly chosen biopsy specimens; (3) the interinstitutional agreement among the two institutions and a third expert gynecologic pathologist to ensure the expert readings were comparable to those outside both institutions on 117 randomly chosen biopsy specimens; and (4) an additional three reviews of the 106 difficult biopsy specimens by all three institutions. METHODS: All 1790 specimens from 850 patients were reviewed three times at each institution in blinded fashion; those for which the first and second reviews were identical were not reviewed a third time. A randomly selected sample of 117 specimens was randomly ordered and read by study pathologists at The University of Texas M. D. Anderson Cancer Center, British Columbia Cancer Agency (BCCA), and Brigham and Women's Hospital (BWH). The 106 difficult cases were treated in the same manner as the randomized and random-ordered cases. Generalized, unweighted, and weighted kappas and their 95% confidence intervals were used to assess agreement. Binary comparisons were used to compare diagnostic categories. FINDINGS: The kappas for the three readings of the overall data set using eight-category World Health Organization (WHO) criteria were as follows: 0.66 for the generalized, 0.72 for weighted, and ranged from 0.59 to 0.94 unweighted binary categories; those read using four-category Bethesda criteria: 0.70 for generalized, 0.69 for weighted, and 0.56-0.94 for unweighted binary categories. For the pool versus the study pathologist readings, the eight-category kappa was 0.51 for generalized, 0.72 for weighted, and 0.56-0.82 for unweighted binary categories; for those read using Bethesda criteria: 0.70 for generalized, 0.70 for weighted, and 0.59-0.82 for the unweighted binary categories. The interpathologist and intrapathologist readings were fair by Landis standards at the low end of the diagnostic scale (atypia, human papillomavirus, and CIN1) and substantial to almost perfect at the high end (CIN2, CIN3, and CIS). The randomly selected and randomly ordered sample of 117 specimens read with the WHO system yielded a generalized kappa of 0.45; among the three institutions (M. D. Anderson Cancer Center vs. BCCA, M. D. Anderson vs. BWH, and BCCA vs. BWH), the unweighted kappas were 0.46, 0.41, and 0.49 and the weighted were 0.65, 0.66, and 0.68, respectively; for the Bethesda, a generalized kappa of 0.65, unweighted kappas of 0.66, 0.65, and 0.47, and weighted of 0.74, 0.72, and 0.74. The difficult specimens read with the WHO system yielded a generalized kappa of 0.23; among the three institutions the unweighted kappas were 0.20, 0.30, and 0.37, and the weighted were 0.17, 0.34, and 0.31; for the Bethesda, a generalized kappa of 0.25; among the three institutions, the unweighted kappas were 0.21, 0.32, and 0.37, and the weighted were: 0.07, 0.21, and 0.37, respectively. INTERPRETATION: Kappas in this expert group of pathologists were in the moderate, substantial, and almost perfect ranges for the overall and randomized samples. The randomized sample was representative of the larger sample. The kappa of the specimens for which disagreements arose was, predictably, in the slight range. Our findings will aid both the correlations with optical measurements using fluorescence and reflectance spectroscopy and the quantitative histopathologic analysis of these study specimens.

Subvisual chromatin changes in cervical epithelium measured by texture image analysis and correlated with HPV.

OBJECTIVES: In this study, we are testing the hypothesis that human papillomavirus (HPV) positivity is correlated with chromatin texture in the cell. Interim analyses are important since this study involves 2000 patients and generates 6000 biopsy specimens that will be subjected to quantitative histopathological analysis and correlated to HPV positivity as measured by the Hybrid Capture II test (Digene; Gaithersberg, MD) and both HPV-DNA and mRNA by the polymerase chain reaction (PCR). The studies of optical technologies, from which we derive this sample, use the colposcopically directed and histopathologically classified cervical biopsy as the gold standard. In this report, we describe the results of an interim analysis of quantitative histopathology and chromatin texture as correlates of HPV infection using the cyto-savant system in cytologically and histopathologically negative specimens. METHODS: A group of 1544 patients entered the optical technology trials, generating 3275 biopsies and 1544 Papanicolaou readings. Two hundred forty-eight patients were cytologically and histopathologically negative. Study pathologists reviewed histologic samples 3 times in a blinded fashion. Non-overlapping, quantitatively stained nuclei were selected from the samples by the pathologists. HPV testing was done using the PCR method and the Hybrid Capture II test. Statistical analysis involved the creation of a classification matrix using a linear discriminant analysis. The matrix was trained on HPV-positive cells by PCR. The analysis included the random creation of both a training set and a validation set that were classified based on the discrimination score obtained by correlating nuclear texture with HPV positivity. RESULTS: The sensitivity of the classification was 52-54% and the specificity was 77-78%. Overall, a 68% predicted accuracy was achieved for both the training set and the test set. The agreement of a test and training set shows that the sets created randomly are indeed similar, and that the discrimination score worked equally well in both sets of cells. Once a cell-by-cell algorithm for HPV positivity was derived, HPV positivity was recalculated on the basis of cell-by-cell texture features. HPV positivity was then recalculated on both a per-biopsy basis and a per-patient basis. For HPV 16 and 18, the positivity rate was 70% on a per-biopsy basis and 73% on a per-patient basis. CONCLUSIONS: Although these results are preliminary, they suggest that texture features reflecting chromatin condensation may correlate with HPV positivity. The current sample is histologic, the analysis suggests that in a cytologic sample, HPV positivity could be detected or confirmed by texture features computed as part of an HPV-associated score. Additional biologic markers could be used as needed. While this study was performed on histologic samples, a study of cytologic samples would be more useful. Future studies will examine chromatin texture compared to HPV integration and mRNA HPV expression.

Costs of colposcopy services and their impact on the incidence and mortality rate of cervical cancer in Canada.

OBJECTIVE: Organized cervical cancer screening services consisting of conventional Papanicolaou cervical smears, colposcopy, and related treatment modalities are readily available in all provinces. The purpose of this report was to study the impact of colposcopy usage and costs on cervical cancer incidence and mortality rates in several Canadian provinces. Knowledge of such information is essential before newer technology such as liquid-based cytology and human papillomavirus testing is introduced or replaces the traditional systems used. MATERIALS AND METHODS: The Ministries of Health of five provinces were contacted and asked to furnish information on the number of colposcopic services and fee-for-service costs for these and for cryosurgery, carbon dioxide laser vaporization, loop electrosurgical excisions, and cold-knife conizations for the year 2000. Canadian Cancer Society estimates of incidence and mortality rates for cervical cancer were also obtained. RESULTS: All provinces had similar incidence and mortality rates for cervical cancer; however, the number of colposcopic services on a per-capita basis varied substantially, with Manitoba and Ontario having rates that were approximately two or three times higher. Fee-for-service payments for colposcopy were similar in the Provinces studied but unit costs for surgical treatment services were highest in Ontario and British Columbia. CONCLUSIONS: Although both the incidence and mortality rates for cervical cancer in Canada fell dramatically after the Walton Report in 1976, these rates have plateaued over the past decade despite widespread availability of colposcopy and related ambulatory treatment services. Higher rates of colposcopy usage do not seem to result in lower incidence rates for this disease. Unit costs for colposcopy are similar among the provinces reviewed, but substantial difference exists for certain treatment services. Additional studies are recommended before the widespread introduction or replacement of existing methods with newer, more costly techniques.

Risk of invasive cervical cancer after Pap smears: the protective effect of multiple negatives.

OBJECTIVE: To determine the relationship between the number of initial negative Pap smears and risk of subsequent cervical cancer. DESIGN: A cohort study was conducted using data from the British Columbia Cervical Cancer Screening Program and British Columbia Cancer Registry. The analysis used a random sample (1%) of women aged 20-69 with Pap smears and all cases of invasive cervical cancer diagnosed between 1994 and 1999. Each negative screen defined the beginning of a screening interval and intervals longer than five years were truncated. The following variables were created for each interval: age at the beginning of the interval, interval length, previous cytological abnormality, previous cervical procedure and number of preceding consecutive negative screens. The relationship between these variables and risk of squamous cervical cancer was determined using survival analysis methods. RESULTS: A total of 388 cases of invasive cervical cancer (252 squamous) were included in the study from a study population of over 3.3 million Pap smears. The risk of invasive squamous cancer increased with time since the last negative screen, history of cytological abnormality and history of cervical therapeutic procedure. Risk was not significantly related to age (P=0.2) but was highest in women aged 30-49. Multiple consecutive negative pap smears were associated with reduced risk in women with a history of moderate atypia (P<0.0001), but not in women without a history (P=0.4). CONCLUSIONS: Multiple consecutive negative cytology was not associated with reduced risk of invasive cervical cancer in women with no history of cytological abnormality.

Quantitative histopathological analysis of cervical intra-epithelial neoplasia sections: methodological issues.

OBJECTIVES: As part a Program Project to evaluate emerging optical technologies for cervical neoplasia, our group is performing quantitative histopathological analysis of biopsies from 1,800 patients. Several methodological issues have arisen with respect to this analysis: (1) Finding the most efficient way to compensate for staining intensity variation with out losing diagnostic information; (2) Assessing the inter- and intra-observer variability of the semi-interactive data collection; and (3) the use of non-overlapping cells from the intermediate layer only. METHODS: Non-overlapping quantitatively stained nuclei were selected from 280 samples with histopathological characteristics of normal (199), koilocytosis (37), CIN 1 (18), CIN 2 (10) and CIN 3 (16). Linear discriminant analysis was used to assess the diagnostic information in three different feature sets to evaluate and compare staining intensity normalization methods. Selected feature values and summary scores were used to evaluate intra- and inter-observer variability. RESULTS: The features normalized by the internal subset of the imaged cells had the same discriminatory power as those normalized by the control cells and by both normalization methods seem to have additional discriminatory power over the set of features which do not require normalization. The use of the internal subset decreased the image acquisition time by approximately 50% at each center, respectively. The intra- and inter-observer variability was of a similar size. Good performance was obtained by measuring the intermediate layer only. CONCLUSION: The use of intensity normalization from a subset of the imaged non-overlapping intermediate layer cells works as well as or better than any of the other methods tested and provides a significant timesaving. Our intra- and inter-observer variability do not seem to affect the diagnostic power of the data. Although this must be tested in a larger data set, the use of intermediate layer cells only may be acceptable when using quantitative histopathology.

Exploratory analysis of quantitative histopathology of cervical intraepithelial neoplasia: objectivity, reproducibility, malignancy-associated changes, and human papillomavirus.

BACKGROUND: As part of a project to evaluate emerging optical technologies for cervical neoplasia, our group is performing quantitative histopathological analyses of biopsy specimens from 1,190 patients. Objectives in the interim analysis are (a) quantitatively assessing progression of the neoplastic process of cervical intraepithelial neoplasia (CIN)/squamous intraepithelial lesions (SIL), (b) detecting malignancy-associated changes (MACs), and (c) phenotypically measuring human papillomavirus (HPV) detected by DNA testing. METHODS: The diagnostic region of interest (ROI) from immediately adjacent sections were imaged, and the basal lamina and surface of the superficial layer were delimited. Nonoverlapping quantitatively stained nuclei were selected from 1,190 samples with histopathological characteristics of normal (929), koilocytosis (130), CIN 1 (40), CIN 2 (23), and CIN 3/carcinoma in situ (CIS) (68). A fully automatic procedure located and recorded the center of every nucleus in the region of interest (ROI). We used linear discriminant analysis to assess the changes between normal and CIN 3/CIS. RESULTS: Scores computed from the cell-by cell features and the clinical grade of CIN/SIL were highly correlated, as were those of the architectural features and the clinical grade of CIN/SIL. We found even higher correlations between a combination of cell-by-cell and architectural scores, and clinical grade. Using these scores, we found MACs in the normal biopsy specimens from patients with high-grade CIN/SIL. Furthermore, the same scores correlated with the molecular detection of HPV. CONCLUSIONS: Quantitative histopathology can be used in large clinical trials as an objective and reproducible measure of CIN/SIL. Detectable phenotypic changes correlate well with CIN/SIL neoplastic progression. It can also be used to infer the presence of CIN/SIL (MACs) and molecular changes associated with increased risk of cancer development (high-risk HPV).

The quality of community colposcopic practice.

OBJECTIVE: To estimate the quality of community colposcopic practice in British Columbia through an assessment of the degree of correlation between colposcopy, cytology, and histology. METHOD: We reviewed all new-patient colposcopies in British Columbia during 2001 by 37 gynecologists in 24 hospital-based clinics. RESULTS: Colposcopic impression closely mirrored the referral cytology diagnosis in 89.8% of cases. As with cytology-biopsy comparisons, discordant cases were more likely to be overestimates of disease rather than underestimates, 18.8% versus 1.8%. Overestimates were usually biopsy sampling errors rather than false positive cytology. The overall correlation between cytology and biopsy was considered satisfactory in 79.4% of cases. Satisfactory agreement between the colposcopic diagnosis and accompanying biopsies occurred in 86.8% of patients. Five colposcopists had performance scores below this standard. Colposcopy with a sensitivity of 90.3% and a specificity of 57.3% as practiced in this provincial program would appear to be of a satisfactory level. The rate of intraepithelial or invasive disease increased from 40.6% in patients with low-grade squamous intraepithelial changes to 91.9% in patients with suspicious or malignant cytology. The value of the colposcopic impression to identify disease correlated best with the higher the grade of disease predicted (64.6% to 92.6%). CONCLUSION: A measure of the colposcopic proficiency in the community can be estimated by comparing the level of agreement between the presenting cytology, colposcopic impression, and corresponding directed biopsies. The results of this study would indicate that 5 individuals had practice standards that were below average. An integrated cytology-colposcopy program facilitates the assessment and identification of below-average practice standards in a community.

Cervical cleaning improves Pap smear quality.

BACKGROUND: Cervical Papanicolaou (Pap) smear screening is an effective method of detecting cytological changes in the cervix before they lead to cervical cancer. However, the quality of a Pap smear can be compromised by inflammatory exudate, inadequate cellularity or failure to sample the transformation zone. We evaluated the effect of routine cervical cleaning on Pap smear quality. METHODS: In a primary care setting, we compared the quality of Pap smears obtained after cervical cleaning (with a dry, oversized cotton swab) with the quality of historical control slides obtained from the same women without prior cervical cleaning. The results for both groups were then compared with statistical averages for the province of British Columbia. RESULTS: Inflammatory exudate was reported in 1 (0.3%) of the 334 study smears and 72 (11.0%) of the 652 control smears (p < 0.001). Inadequate endocervical or metaplastic squamous cells were reported in 11 (3.3%) of the study smears and 90 (13.8%) of the control smears (p < 0.001). Inadequate cellularity was reported in 13 (3.9%) of the study smears and 9 (1.4%) of the control smears (p = 0.01). There were similar statistical differences between the study group and provincial averages. The results for the control group did not differ significantly from provincial averages (inflammatory exudate, 11.3%; inadequate endocervical cells, 14.7%; and poor cellularity, 2.7%). INTERPRETATION: Prior cervical cleaning with an oversized cotton swab was associated with a lower frequency of smears with inflammatory exudate or inadequate endocervical cells and, to a lesser degree, a higher frequency of smears with inadequate cellularity.