Shanxi Province Cervical Cancer Screening Study: a cross-sectional comparative trial of multiple techniques to detect cervical neoplasia.

OBJECTIVE: The aim of this study was to design a cervical cancer screening algorithm for the developing world that is highly sensitive for cervical intraepithelial neoplasia (CIN) II, III, and cancer and highly specific for CIN II and III, making it possible to ablate the transformation zone without histologic confirmation. METHODS: In rural Shanxi Province, China, we examined 1997 women ages 35-45. Each subject underwent a self-test for intermediate and high-risk HPV (by HC-II assay), fluorescence spectroscopy, a liquid-based Pap (read manually and by computer and used as a direct test for HPV), a visual inspection (VIA) diagnosis, and colposcopy with multiple cervical biopsies. RESULTS: Mean age was 39.1 +/- 3.16 years, mean number of births was 2.6 +/- 0.93. Based on tests administered, 4.3% subjects had > or =CIN II. All subjects with > or =CIN II had either a ThinPrep Pap (> or =ASCUS) or a positive HPV direct test. The sensitivity and specificity for the detection of > or =CIN II were, respectively, 83 and 86% for the HPV self-test, 95 and 85% for the HPV direct test, 94 and 78% for the ThinPrep Pap (> or =ASCUS), 77 and 98% for the ThinPrep Pap (> or =HGSIL), 94 and 9% for fluorescence spectroscopy, 71 and 74% for VIA, and 81 and 77% for colposcopy. CONCLUSION: Based on these data and the existing healthcare infrastructure in China, we believe that further refinement of primary HPV screening using centralized labs is indicated. Self-testing in the local villages may be effective with improvements in the devices and techniques.

Utility of liquid-based cytology for cervical carcinoma screening: results of a population-based study conducted in a region of Costa Rica with a high incidence of cervical carcinoma.

BACKGROUND: In a study using a split-sample design, liquid-based cytology (ThinPrep Processor, Cytyc Corporation, Boxborough, MA) was compared with the conventional Papanicolaou (Pap) smear in Guanacaste, Costa Rica. The study provides the first population-based comparison of the ThinPrep screening technology and includes "gold standard" measures of diagnostic accuracy. METHODS: The population-based study was performed among over 8000 women residing in a Costa Rican province with a high incidence of cervical carcinoma. Conventional smears were prepared and diagnosed in Costa Rica, while the residual material on the sampling device was collected into a liquid preservative and shipped to the U.S., where ThinPrep cytologic slides were prepared and diagnosed. Cytologic diagnoses based on the two techniques, categorized according to the Bethesda System, were compared with a "gold standard" final case diagnosis for each patient, also based on Bethesda terminology, that reflected an integrated interpretation of all available data, including cytology, histology, and cervicography. Results were also compared with the results of HPV DNA detection (Hybrid Capture, Digene Corporation, Silver Spring, MD). RESULTS: ASCUS was the threshold for colposcopy referral. There were significantly more women referred according to this threshold with the ThinPrep slide (12.7%) than with the conventional smear (6.7%, P<0.001). Compared with the final case diagnosis, referral by ThinPrep slides detected 92.9% of cases with high grade squamous intraepithelial lesions (HSIL) and 100% of carcinoma cases. Smears detected 77.8% of HSIL and 90.9% of carcinomas. Thus, ThinPrep cytology was significantly more sensitive in the detection of HSIL and cancer (McNemar test, P<0.001). Adjudication of cases in which the ThinPrep and smear diagnoses disagreed, using the final case diagnoses and the HPV DNA test results as reference standards, suggested that the ThinPrep method was detecting additional true SIL as opposed to false-positives. CONCLUSIONS: In a population-based study of high risk women, ThinPrep cytology demonstrated significantly increased sensitivity for detecting HSIL and carcinoma, with a concurrent significant increase in colposcopy referrals.

Prevalence of cervical cancer and feasibility of screening in rural China: a pilot study for the Shanxi Province Cervical Cancer Screening Study.

For cervical cancer screening to be feasible in developing countries, it must be accurate, inexpensive, and easy to administer. We conducted a pilot study in rural Shanxi Province, People's Republic of China, to determine disease prevalence and study feasibility in preparation for a large-scale comparative trial of 6 screening tests. One-hundred and thirty-six nonpregnant women with no history of hysterectomy, pelvic radiation, or Papanicolaou tests were screened in a rural clinic. Ten percent of the women enrolled reported abnormal vaginal bleeding and 45% reported abnormal vaginal discharge. The tests were the Papanicolaou test (both conventional and ThinPrep), a self-administered swab test by Hybrid Capture II for high-risk human papillomavirus (HPV), a test for high-risk HPV from residual PreservCyt medium, fluorescence spectroscopy, and visual inspection of the cervix by a clinician. All women also underwent colposcopy and biopsies as the reference standard. Biopsies showed 12 of 136 women had >/= high-grade squamous intraepithelial lesions (HGSIL). Screening was completed in 5 half-day sessions, the procedures went smoothly, and local cooperation was enthusiastic. Disease prevalence in Xiangyuan and Yangcheng Counties, Shanxi Province, can be estimated at 8.8% (95% CI, 4.5% to 15.0%). Screening 1000-2000 patients would be sufficient to detect a 10% difference in accuracy between diagnostic tests. The proposed large-scale trial is feasible.

Performance of liquid-based, thin-layer cervical cytology: correlation with reference diagnoses and human papillomavirus testing.

The performance of thin-layer cervical cytology with the use of ThinPrep (Cytyc Corporation, Boxborough, MA) was assessed by comparing the original independent diagnosis of ThinPrep slides and conventional smears prepared from 1780 split samples with the most abnormal diagnosis per patient on the basis of an independent pathologist's masked review and with the detection of cancer-associated types of human papillomavirus (HPV) DNA. Cases were selected on the basis of the original diagnoses to include all discordant pairs (those diagnosed as atypical squamous cells of undetermined significance or higher grade, n = 1017), all concordant abnormal pairs (n = 444), and a random 5% of concordant normal pairs (n = 319). In screening centers, thin-layer cytology detected 135 (70.3%) of 192 women diagnosed as having squamous epithelial lesions or a higher grade in the independent review, whereas locally read smears detected 91 (47.4%) of these patients (P < .001). In hospital-based cytology laboratories, thin-layer cytology detected 308 (86.3%) of 357 women diagnosed with SILs or a higher grade in the independent review, compared with 283 (79.3%) diagnosed with smears (P = .011). Cancer-associated types of HPV DNA were detected in a slightly higher proportion of women with smears diagnosed as SILs than in women with thin-layer cytology diagnosed as SILs, whereas the overall number of HPV-associated SILs diagnosed was higher with thin-layer cytology. These data suggest that the ThinPrep method detects a higher percentage of SILs as defined in a masked, independent review than do concurrently prepared smears and that diagnoses of SILs rendered with ThinPrep correlate with the detection of cancer-associated types of HPV.

ThinPrep Papanicolaou testing to reduce false-negative cervical cytology.

The false-negative Papanicolaou (Pap) smear is the major quality issue currently facing the practitioners of diagnostic cytology. Failure to detect cervical disease in women can be due either to sampling error or to screening error. Sampling errors account for the majority of false-negative Pap tests; the remainder are due to the cervical abnormality either not being detected or not being properly classified during microscopic screening. The ThinPrep Pap test was recently cleared by the United States Food and Drug Administration as significantly more effective than the conventional Pap smear and as a replacement for the conventional method of Pap smear preparation. The ThinPrep Pap test increases detection of squamous intraepithelial lesions and significantly improves specimen quality. These features of the ThinPrep Pap test also have a heretofore unreported direct effect on the false-negative rate of the Pap examination. In two recently conducted clinical trials, the ThinPrep test resulted in a significant reduction in the sampling false-negative rate in women undergoing routine Pap screening. In addition, screening false negatives, as expressed by the false-negative proportion, were reduced from 5.6% with the conventional Pap smear, to 2.2% with the ThinPrep Pap test. More high-grade squamous intraepithelial lesions were detected in the screening populations with the ThinPrep test. The ThinPrep Pap test improves the quality of cervical cancer screening through a reduction in false-negative Pap examinations. This improvement has important implications for women undergoing Pap screening and for those who practice gynecologic cytopathology.

Diagnosis of invasive cervical carcinoma with fluid-based, thin-layer slide preparation method.

Objectives: Although the main role of the Papanicolaou smear is to detect precursors of cervical cancer, the detection of invasive disease is important to identify women at the earliest stage of their disease. This study assesses the ability of the ThinPrep Papanicolaou test to identify invasive cervical cancer as compared to the Papanicolaou smear.Methods: A meta-analysis of five studies previously reviewed by the Food and Drug Administration was performed. There were 61 cases of cervical carcinoma processed by both conventional Papanicolaou smear and ThinPrep methods. Each case was reviewed by cytotechnologists and cytopathologists without knowledge of prior diagnoses. In a sub-study, the filtrate resulting from the preparation of the ThinPrep was analyzed to determine if diagnostic cells passed through the filter membrane.Results: With the ThinPrep method, a diagnosis of cancer was made in 54 cases (88.5%) as compared to a diagnosis of cancer in 52 of these cases with the conventional Papanicolaou smear method (85.2%). In those cases where diagnosis of invasive cancer was not made with the ThinPrep method, all cases were classified as abnormal, ranging from atypical glandular cells of undetermined significance to high-grade squamous intraepithelial lesions. The ThinPrep slides contained tumor diathesis and other contextual clues. Analysis of the ThinPrep filtrate demonstrated that cancer cells did not pass through the filter.Conclusions: These studies indicate that the ThinPrep method is at least equivalent to the conventional Papanicolaou smear method for the demonstration of squamous cell carcinoma, adenocarcinoma, and other cervical malignancies. Diagnostically important cells are not lost through the filter membrane during the preparation of the ThinPrep slide, because they are larger than the 8 µm pore size of the filter.

Validation of the ThinPrep Papanicolaou Test for Cervical Cancer Diagnosis.

OBJECTIVES: Although the accuracy of the Thin-Prep Papanicolaou (Pap) Test has been demonstrated in the detection of precursor lesions to cervical cancer, its performance in identifying invasive cancer has not been adequately evaluated. These studies were designed to determine the effectiveness of the ThinPrep method in diagnosing invasive cervical cancer. MATERIALS AND METHODS: In four clinical studies, 47 cases of cervical cancer were processed by both conventional and ThinPrep techniques, and the cytological diagnoses and detection rates were compared. Additional studies evaluated the potential of cell loss during the ThinPrep filtration process. RESULTS: A diagnosis of cancer was made in 45 cases (95.7%) by the ThinPrep method and in 44 cases by conventional Pap smear (93.6%). In the two cases not diagnosed by the ThinPrep method, one was diagnosed as atypical squamous cells of undetermined significance and one as atypical glandular cells of undetermined significance. In the conventional smears, one of the three cases not identified as cancer was atypical squamous cells of undetermined significance, and the other two cases were unsatisfactory. In addition, the ability of the ThinPrep method to demonstrate tumor diathesis and to retain diagnostic cells by the filtration process was confirmed. CONCLUSIONS: These studies indicate that the ThinPrep method is at least equivalent to the conventional Pap smear method for the demonstration of squarnous cell carcinoma, adenocarcinoma, and other cervical malignancies.

Cervical specimens collected in liquid buffer are suitable for both cytologic screening and ancillary human papillomavirus testing.

BACKGROUND: Several new techniques have been developed to improve the sensitivity of cervical carcinoma screening and reduce equivocal cytologic diagnoses referred to as atypical squamous cells of undetermined significance (ASCUS). This study evaluates the effectiveness of combining two newly introduced diagnostic techniques: preparation of thin-layer cytologic slides from ThinPrep liquid buffer and selected Hybrid Capture testing for human papillomavirus (HPV) DNA. Because HPV DNA detection has been strongly associated with the presence of a cervical carcinoma precursor ("squamous intraepithelial lesion," or SIL), HPV testing might be useful for identifying women with ASCUS who have an underlying SIL. METHODS: Two hundred specimens demonstrating diverse cervical abnormalities were selected from a prospective population-based study of 9174 women conducted in Costa Rica. The entire cohort had been screened with conventional cervical smears; ThinPrep slides made from liquid buffer, PAPNET, a computerized slide reading system; and Cervicography. Patients with any abnormal screening test were referred for colposcopy, punch biopsy, and loop excision of cases with high grade cytologic abnormalities not explained by punch biopsy. For this investigation, the results of ThinPrep cytology and HPV testing alone and in combination were compared with the final diagnoses, with an emphasis on the detection of carcinoma and high grade SIL. RESULTS: The 200 subjects studied included 7 women with a final diagnosis of carcinoma, 44 with high grade SIL, 34 with low grade SIL, 51 with a variety of equivocal diagnoses, and 64 with normal diagnoses. A ThinPrep cytologic diagnosis of SIL or carcinoma was made in 39 (76%) of the 51 women with final diagnoses of high grade SIL or carcinoma. Hybrid Capture testing detected carcinoma-associated types of HPV DNA in 100% of women with carcinoma, 75% with high grade SIL, 62% with low grade SIL, 20% with equivocal final diagnoses, and 12% of normal women. If colposcopy referral had been limited to women with a ThinPrep diagnosis of SIL or a diagnosis of ASCUS associated with the detection of carcinoma-associated HPV DNA from the same vial, 100% of women with carcinoma and 80% with high grade SIL would have been examined. To achieve this high sensitivity in the entire population of 9174 women would have required the referral of about 7% of the population. The combined screening strategy would have performed marginally better than optimized conventional screening with referral of any abnormal cytology (ASCUS and above). CONCLUSIONS: A cervical carcinoma screening technique which uses a single sample for cytopathology and HPV testing to triage equivocal diagnoses may be promising if it proves to be cost-effective.

The ThinPrep Pap test. A review of clinical studies.

OBJECTIVE: To review the literature describing the use of the ThinPrep system in gynecologic cytology. STUDY DESIGN: Clinical trials of the ThinPrep Beta and the ThinPrep 2000 performed in the United States were reviewed. In each a single sample was used to first prepare a conventional slide and the remainder to perform a ThinPrep cervical cytologic test. Slides were examined in a blind fashion by cytotechnologists and classified according to Bethesda System terminology. RESULTS: The ThinPrep test provided significantly more effective detection of low grade intraepithelial neoplasia or more severe diagnoses without loss of diagnostic specificity. The ability of the ThinPrep system to detect infection and reactive cellular changes was equivalent to or better than that of the conventional cytologic smear. Specimen adequacy was significantly enhanced with the ThinPrep test by reducing the number of cases classified as "Satisfactory but limited by...". CONCLUSION: The ThinPrep test offers increased detection of cervical disease and a clear improvement in specimen adequacy. In addition to potentially lowering the false negative rate of cervical smears, collection of cells in liquid medium allows additional testing, such as human papillomavirus typing and computer imaging, to provide a more comprehensive diagnosis than that obtainable with the cervical cytologic test.

Automated slide preparation system for the clinical laboratory.

An automated device has been developed to prepare a wide variety of cytology preparations. Using the device, cells collected in suspension are mildly dispersed and transferred to a glass slide using filter-transfer technology. The cell density on the slide is controlled by the instrument. A companion product, the solution used in specimen transport and preparation, provides long-term preservation of diagnostic cells while lysing red blood cells. The overall process yields clean, uniform samples with better visualization of the diagnostic cells. The use of a disposable filter prevents sample-to-sample contamination. Collection of the sample in solution makes it possible to produce multiple slides from one sample.

Combined malignancy associated change and contextual analysis for computerized classification of cervical cell monolayers.

Previous efforts to automate cervical cell analysis have focused on algorithms that use measurements of high resolution images of individual cells for 'rare event' detection of abnormal cells, sometimes in combination with cluster analysis of low resolution images. A new methodology was developed in which intermediate cell markers (indicating malignancy associated change) and contextual analysis of slide architecture were performed on the same digital images at 0.33 mu pixel resolution. A total of one hundred and forty-six cases that were either negative or at least high grade squamous intraepithelial lesions (HGSIL) were prepared as monolayers, and about 20 fields per slide were analyzed. Features most important for intermediate cell marker analysis were those that measured the variation in nuclear texture measurements across the slide as well as densitometric features. The most discriminatory contextual features were those that measured the variation of the arrangement of cells within a cluster across the slide. Linear discriminants were calculated using the most important features from both types of analysis; they provided smear classification accuracies of 71-86%.

Computerized contextual analysis in diagnosis.

Cervical cytology specimens prepared as monolayers were analyzed with a combination of high-resolution single-cell image analysis and lower-resolution contextual image analysis. For the latter, quantitative features of cluster architecture, object distribution and background were used to mimic the pathologists global assessment of a specimen. Contextual analysis correctly classified 81% of the cases for diagnosis, about on a par with the companion single-cell cytometric analysis. Contextual and single cell analysis complemented each other, yielding greater accuracy when the two methods were combined. Additional applications of contextual analysis are discussed.

Homogeneous sampling accounts for the increased diagnostic accuracy using the ThinPrep Processor.

Recent clinical trials indicated that the ThinPrep method of sample preparation has greater diagnostic sensitivity than the conventional direct Papanicolaou smear. The authors hypothesized that nonhomogeneous cell sampling during transfer from the sampling device to the microscope slide was a contributing factor to the reduced accuracy of the conventional direct Pap smears in these trials. To test this hypothesis, four direct smear methods were compared with the newly developed, fluid-based, filter-transfer method. Counts of epithelial cells on conventional smears showed that only a fraction of the available epithelial cells on the sampling devices (medians, 6.5% to 62.5%) was actually deposited on the slides. In all 27 cases studied with the ThinPrep method, equivalent diagnostic material was obtained on each of the replicate slides prepared per specimen. This identifies a new source of error, preparation error, in conventional smears.

Measurement of subvisual changes in cervical squamous metaplastic cells for detecting abnormality.

Quantitative measures of visually normal squamous metaplastic cells exfoliated from the uterine cervix were obtained to test the hypothesis that these cells, like intermediate squamous and endocervical columnar cells, show subvisual evidence of atypia in cases of bonafide squamous intraepithelial neoplasia. The cells identified as squamous metaplastic were obtained from 14 abnormal (dysplastic) and 9 diagnostically negative cases. Although the cell populations so grouped showed no statistically significant differences in overall cell size, nuclear area or nuclear/cytoplasmic ratios, there were significant differences in nuclear and cytoplasmic densitometric features and in nuclear texture features. A combination of three features (nuclear density, texture and cytoplasmic density) permitted 76% of the cells to be categorized correctly as originating in normal versus abnormal slides. It is concluded that selected quantitative features of exfoliated metaplastic cell populations may contribute to improved diagnostic accuracy in automated screening for cervical abnormalities.

High-resolution and contextual analysis for the diagnosis of fine needle aspirates of breast.

A study was undertaken to confirm earlier work on a smaller number of patients that had suggested that medium-resolution contextual analysis complements high-resolution individual cell analysis for cytomorphometric classification of fine needle aspirate smears of breast. The objectives of this study were to improve and verify the method. Sixty-one biopsy-confirmed hematoxylin and eosin-stained aspirate smears of breast were restained using the Feulgen technique. Individual nuclei were digitized at a resolution of 0.25 micron. Features describing size, shape, density and texture were extracted from the images. Individual cell analysis correctly classified 84% of cases, contextual analysis correctly classified 70% of cases, and the combined use of both techniques resulted in 87% classification accuracy. However, if fibroadenoma cases are excluded, the combined correct classification rate is 93%. Geometric and densitometric features contributed most to correct classification in individual cell analysis, while the most important contextual feature was the number of clusters per scene. We conclude that the addition of quantitative measures of smear patterns, termed "contextual analysis," improves automated classification schemes.

Uncoupling of phospholipase C from receptor regulation of [Ca2+]i in T84 colonic cells by prolonged exposure to phorbol dibutyrate.

The T84 colonic cell line, a cultured Cl- secretory cell, elevates intracellular free Ca2+ [( Ca2+]i) in a concentration-dependent manner when exposed to carbachol or histamine. As determined with a fluorescence microscope imaging system, exposure of T84 cells to 100 microM carbachol or histamine resulted in an immediate [Ca2+]i rise of approximately 50-80 nM in all cells. Preincubation of monolayers for 1 h or longer with 0.4 microM phorbol 12,13-dibutyrate (PDB) reduced the number of cells which responded to histamine or carbachol and reduced the magnitude of the increase in the responding cells. This effect reached its maximum after 2 h and persisted for at least 24 h of PDB incubation. Binding of quinuclidinyl benzilate, a cholinergic receptor antagonist, indicated that down-regulation of external receptors was not an explanation for this effect. Examination of phospholipase C activity in T84 cell membranes showed increased basal activity in PDB-treated compared with control cells. Measurement of inositol phosphates generated by intact cells using myo-[3H]inositol incorporation or receptor binding assays showed that 2 h of incubation with PDB elevated basal levels of inositol 1,4,5-trisphosphate and prevented any further carbachol-induced generation of inositol trisphosphate. Probably as a consequence, both total cell calcium and Ca2+ ionophore-releasable calcium were decreased after 2 h of PDB incubation. Membrane-associated protein kinase C activity was elevated after a 2 h exposure to PDB but was below the level of detection after 24 h with PDB. Protein kinase C antagonists neither duplicated nor blocked the uncoupling of carbachol receptors induced by long term treatment with PDB. The results suggest that prolonged PDB incubation caused uncoupling and elevation of phospholipase C activity from cholinergic and histaminergic receptor regulation resulting in increased basal levels of inositol 1,4,5-trisphosphate. Protein kinase C apparently is not involved directly in the mechanism that leads to these effects.

An image analysis method for assessment of prognostic risk in prostate cancer: a pilot study.

Fully automated computerized image analysis at medium resolution (1 micron per pixel space) was applied in a study of 17 patients with stage D1 prostate cancer. For this pilot study, patients were selected on the basis of very good or very poor outcome. This selection was made in the hope of identifying morphometric features that are useful in prognostic assessment. Nine patients with good outcome were alive after 7 or more years of follow-up and eight patients with poor prognosis were dead of disease in less than 3 years. All patients were treated with 125I seed implantation to the prostate and pelvic lymph node dissection. Hormone therapy was not administered until the time of distant failure. Routine hematoxylin and eosin tissue sections of lymph nodal tissue bearing metastatic neoplasm were used for this analysis. A minimum of eight scenes per case was analysed. Of 50 measured parameters on each cluster, five (gray level distribution, number of cell clusters per scene, bending energy, average cluster area and cluster polarity) were useful to distinguish patients with good outcome from those with a poor outcome. Thirteen of the 17 patients were correctly classified by image analysis (P = 0.044, Fischer's exact test). By comparison, flow cytometry of the identical tissue samples correctly classified 14 of 17 patients (diploid, good outcome; aneuploid, poor outcome; P = 0.009). Only one patient was incorrectly classified by both image analysis and flow cytometry, implying a complementary prognostic role for the two methods. The encouraging result, successful identification of useful morphometric features, justifies a larger study of unselected patients.