Cytology microarrays.

The use of high throughput genetic and expression platforms are generating many candidate diagnostic markers and therapeutic targets for a wide variety of clinical conditions. Tissue microarrays can be used for the evaluation of the utility of many of these markers. However, tissue microarrays can suffer from the limitations associated with sampling and sectioning tissues. We introduce a novel microarray technique based on cell suspensions. Multiple slides can be made, all of which are equally representative of the initial sample. A robotic device was designed that can deposit 60 distinct spots of cytological material on a glass slide. Each spot of cells deposited in this manner may correspond to a unique source. Controlling the number of cells per spot, their distribution within the spot and the size of the spot can be achieved by modifying the viscosity of the cell solution or regulating the amount of fluid deposited. A fully automated analysis of quantitatively stained microarray samples has been performed to quantify the number of cells per spot, the size of spots and the DNA amount per cell in each spot. The reproducibility of these parameters was found to be high.

An analysis of 84244 patients from the British Columbia cytology-colposcopy program.

OBJECTIVE: To determine the diagnostic correlation between referral cytology, initial biopsies and colposcopic impression in patients assessed in a provincial cytology screening program. METHODS: A retrospective review of the computerized cytology screening database for British Columbia (BC), to identify all patients having their first colposcopy between 1986 and 2000 in 24 participating clinics constituted the study population. 84244 patient records were identified for analysis. Colposcopies were performed mainly by 37 general gynecologists as part of a province-wide colposcopy program. Correlation of cytology, colposcopic impression and directed biopsies was performed. RESULTS: The colposcopic impression correlated with the referral cytology within one degree in over 90% of cases. Colposcopists felt cytology underestimated disease in 1.5% and overestimated disease in 8.3%. Cytology-histology correlation within one degree occurred in 82%. Cytology underestimated the result of the biopsies in 2.3% and appeared to overestimate disease in 16.1% of patients. Patients with HSIL cytology had corresponding lesions in 77%, with a further 4.9% having LSIL disease. The predictive accuracy of colposcopy increased with advancing severity of disease expected. As the degree of cytological abnormality worsened, the predictive accuracy of colposcopic diagnosis increased. CONCLUSIONS: Both cytology and colposcopy have high sensitivity but low to moderate specificity. Colposcopy is most accurate in identifying high-grade diseases. Colposcopic impression correlates closely with the cytology diagnosis and combining the two produces optimum results.

Risk of invasive cervical cancer after three consecutive negative Pap smears.

OBJECTIVES: To determine the factors that influence risk of cervical cancer after three consecutive negative Pap smears. METHODS: A cohort study was conducted using data from the British Columbia Cervical Cancer Screening Program and British Columbia Cancer Registry. Analysis was based on a one percent sample of women aged 20-69 years with Pap smears enriched with all invasive cervical cancer cases diagnosed between 1994-99. Screening intervals, after three negative screens, were created with the following variables: age at beginning of interval, interval length, previous cytologic abnormality and previous cervical procedure. The risk of cervical cancer by histologic type was calculated using survival analysis methods. RESULTS: The sample consisted of 10,509 women, who contributed 28,309 intervals, and 371 cervical cancer cases. The incidence rate of invasive squamous cervical cancer increased with time since last screen up to six years. Women with a history of dysplasia remained at elevated risk for squamous cancer, hazard ratio=2.6 (95% confidence interval [CI]=1.9, 3.4) but age or previous procedure were not related to risk. No relationship between time since last screen and non-squamous cancer risk was found although history of a previous procedure was significant. The marginal effectiveness of Pap smears declined with increasing frequency of use. CONCLUSIONS: This study confirmed the preventive effect of Pap smear screening and its dependency on frequency of use. Women with a history of dysplasia, prior to three consecutive negatives, were at increased risk of developing invasive squamous cervical cancer compared with women with no such history.

Cytologic features of villoglandular adenocarcinoma of the uterine cervix: comparison with typical endocervical adenocarcinoma with a villoglandular component and papillary serous carcinoma.

BACKGROUND: To the authors' knowledge, the cytologic features of villoglandular adenocarcinoma (VGC) have been described in very few publications. The malignant cells are difficult to separate from reactive glandular cells and the majority of VGCs are missed on screening cytology. METHODS: The cytologic findings of a retrospective study of four cases of pure VGC are described and are contrasted with those of papillary serous adenocarcinoma and typical mucinous endocervical adenocarcinoma with a focal component of VGC. RESULTS: Although atypical glandular cells of endocervical origin were reported when the smears from the VGC cases were examined in the screening program, none of the cases was recognized as malignant prior to histologic diagnosis. The smears showed many groups of endocervical glandular cells. Important architectural features included large cohesive groups and sheets of cells showing nuclear crowding and loss of the normal honeycomb pattern. True papillary structures comprising stromal cores covered by well polarized columnar cells with a smooth surface were characteristic. It is important to note that a "feathered edge" appearance of the cell groups was absent. The neoplastic cells were mildly atypical, showing a slight increase in the nuclear-cytoplasmic ratio but minimal hyperchromatism. The cytology smears of four cases of typical adenocarcinoma of endocervical type that had a focal VGC pattern showed cell groups with irregular borders and "feathered" edges comprised of distinctly atypical columnar cells with elongated and irregular hyperchromatic nuclei. Free-lying atypical cells and ball-like clusters of atypical cells also were present in the latter cases but not in pure VGCs. The primary high grade papillary serous adenocarcinomas of the cervix exhibited extreme cytologic atypia that was interpreted readily as malignant. CONCLUSIONS: The diagnosis of VGC on cytology smears often is missed. Papillary fragments, nuclear crowding, and subtle atypia may suggest the diagnosis.

DNA-cytometry of progressive and regressive cervical intraepithelial neoplasia.

A retrospective analysis was performed on archival cervical smears from a group of 56 women with cervical intraepithelial neoplasia (CIN), who had received follow-up by cytology only. Automated image cytometry of Feulgen-stained DNA was used to determine the differences between progressive and regressive lesions. The first group of 30 smears was from women who had developed cancer after initial smears with dysplastic changes (progressive group). The second group of 26 smears with dysplastic changes had shown regression to normal (regressive group). The goal of the study was to determine if differences in cytometric features existed between the progressive and regressive groups. CIN categories I, II and III were represented in both groups, and measurements were pooled across diagnostic categories. Images of up to 700 intermediate cells were obtained from each slide, and cells were scanned exhaustively for the detection of diagnostic cells. Discriminant function analysis was performed for both intermediate and diagnostic cells. The most significant differences between the groups were found for diagnostic cells, with a cell classification accuracy of 82%. Intermediate cells could be classified with 60% accuracy. Cytometric features which afforded the best discrimination were characteristic of the chromatin organization in diagnostic cells (nuclear texture). Slide classification was performed by thresholding the number of cells which exhibited progression associated changes (PAC) in chromatin configuration, with an accuracy of 93 and 73% for diagnostic and intermediate cells, respectively. These results indicate that regardless of the extent of nuclear atypia as reflected in the CIN category, features of chromatin organization can potentially be used to predict the malignant or progressive potential of CIN lesions.

The use of an automated image cytometer for screening and quantitative assessment of cervical lesions in the British Columbia Cervical Smear Screening Programme.

The development of an automated device to screen cervical cytology slides for the detection of pre-invasive lesions of the cervix has been the goal of many individuals for over 30 years. The increasing sophistication of the technology of automation and increasingly powerful computer technology have enabled a number of these systems to reach the stage at which they have become a practical reality. The Department of Cancer Imaging at the British Columbia Cancer Agency has developed such a device over the past few years. This study reports the preliminary results of a trial to determine the reliability of the device for the screening and quantitative assessment of cervical cells. A training set of over 1000 cervical slides was used to train the image cytometer. A test set of 1030 slides was screened by the image cytometer and in the Cytology Screening Laboratory. At the 50% sample split the sensitivity of the image cytometer was 95% for severe dysplasia and 90% for moderate dysplasia, compared with a sensitivity of 90% for both of these lesions using conventional screening. A combination of nuclear texture features was found which can be used for the quantitative assessment of both abnormal cells and apparently normal intermediate cells.

Cytologic features of papillary serous adenocarcinoma of the uterine cervix.

BACKGROUND: The cytologic features of papillary serous carcinoma of the cervix (PSCC) have not been described in detail previously. In this study the cytologic features of primary, pure PSCC are described and correlated with the histology. Comparison is made with papillary serous ovarian carcinoma metastatic to the cervix. METHODS: Seven cases of primary pure PSCC and five cases of ovarian PSCC metastatic to the cervix were retrieved from the pathology files. The cytology records and slides, and clinical charts were traced and examined retrospectively. RESULTS: Five of the 7 patients with PSCC were younger than 40 years. The smears of PSCC contained many groups of atypical glandular cells. Monolayered sheets of mildly atypical glandular cells with papillary branches were observed only in the cases of primary PSCC. All cases contained multilayered sheets of mildly to moderately pleomorphic glandular cells, pseudopapillary fragments, and tight balls of cells resembling endometrial glandular cells. Squamoid cells with abundant densely staining cytoplasm were also encountered. Free-lying dissociated atypical cells were also present, some of which were markedly atypical. A marked tumor diathesis of inflammatory cells, cell debris, and blood was encountered with primary PSCC but was scanty in cases of papillary serous ovarian carcinoma metastatic to the cervix. CONCLUSIONS: PSCC has a relatively characteristic cytologic appearance which usually differs from other endocervical adenocarcinomas and from metastatic papillary serous carcinoma. However, some features may lead to underdiagnosis or confusion with other entities such as squamous carcinoma or endometrial carcinoma.

Cytological aspects of uterine cervical adenocarcinoma, adenosquamous carcinoma and combined adenocarcinoma-squamous carcinoma: appraisal of diagnostic criteria for in situ versus invasive lesions.

This paper reports the cytological findings based on air-dried smears in a retrospective series of 143 cases of endocervical adenocarcinoma, combined adenocarcinoma-squamous carcinoma and adenosquamous carcinoma drawn from the files of the BC Cancer Registry. Cervical cytology smears were available before biopsy in 131 patients, but in 18 cases the cytology showed no abnormality. Malignant changes or high-grade atypia of glandular and/or squamous cells (defined as moderate or severe dyskaryosis) were detected in 103 cases. In 46 cases, only a high-grade squamous abnormality was detected. Low-grade glandular and/or squamous lesions were detected in nine cases and one showed atypical endometrial-type glands. The cervical smears of 64 cases were reviewed in detail to determine the important cytomorphological criteria of in situ and invasive adenocarcinoma in air-dried smears, the technique used for preparing PAP smears in British Columbia. Endocervical cells were absent in four cases. Numerous (> 10) groups of glandular cells were present in 51 cases. Important clues to the diagnosis of adenocarcinoma included crowding of nuclei, stratification of nuclei, loss of polarity, syncytial balls and papillary groups of glandular cells, nuclear enlargement, nuclear pleomorphism, and the presence of free-lying atypical glandular cells. Nuclear hyperchromatism, chromatin pattern, nuclear borders, nuclear membranes, and numbers and morphology of nucleoli were not helpful criteria in our material. Criteria enabling reliable distinction between in situ and invasive adenocarcinoma and/or mixed adenocarcinoma-squamous carcinoma could not be established.

Apocrine carcinoma of the lip: a case report including immunohistochemical and ultrastructural study, discussion of differential diagnosis, and review of the literature.

The light microscopic, immunohistochemical, and ultrastructural features of a case of apocrine carcinoma of the upper lip of a 54-year-old white man are described. The neoplasm had a cribriform intraductal component resembling apocrine carcinoma of the breast. The tumor had irregular borders and infiltrated skeletal muscle. The neoplastic cells had abundant eosinophilic granular cytoplasm and showed apical decapitation secretion characteristic of apocrine differentiation. The differential diagnosis is discussed with particular reference to distinction of the tumor from oncocytic carcinoma and ductal carcinoma of minor salivary gland.

Exfoliative cytology and electron microscopy in the diagnosis of hairy leukoplakia.

The diagnosis of lesions clinically suggestive of hairy leukoplakia was assessed by light and electron microscopic examination of cytologic smears. We found exfoliative cytology to be a simple and noninvasive technique that can confirm the clinical diagnosis of hairy leukoplakia. Our results suggest that electron microscopy may be more sensitive and reliable than light microscopy in confirming the clinical diagnosis of hairy leukoplakia.

Epidermal thickness measurements in vaginal intraepithelial neoplasia. A basis for optimal CO2 laser vaporization.

The current management of vaginal intraepithelial neoplasia (VAIN) often involves the use of laser vaporization. A study was performed to measure the epithelial thickness in both premenopausal and postmenopausal women with various grades of VAIN to determine the optimum depth of tissue destruction if laser vaporization is used for therapy. Hematoxylin and eosin-stained tissue sections were examined with light microscopy and measurements made with a calibrated micrometer. Sixty-three biopsies from 56 patients were studied. Patients' ages ranged from 22 to 84 years, with a mean of 56. Thirty-six had a prior history of cervical neoplasia. Thirty-nine patients (70%) had VAIN III, 10 had VAIN II, and the remaining 7 patients had VAIN I lesions. The involved epithelium varied from 0.10 to 1.4 mm in thickness, with a mean of 0.46. Noninvolved vaginal epithelium varied in thickness from 0.10 to 0.70 mm, with a mean of 0.28. Koilocytosis was noted in only 9 of the 63 biopsy specimens. In comparing the thickness of involved epithelium in a given patient to that of an adjacent area of normal-appearing epithelium, the epithelium containing VAIN tended to be thicker. The recommended depth of epithelial destruction with laser vaporization in the literature varies widely and appears to have largely an empiric basis. Our study attempted to provide a scientific basis for laser destruction of these lesions. The results obtained indicate that epithelial destruction to a depth of 1.5 mm, including the zone of thermal necrosis, should be sufficient to destroy epithelium containing VAIN without damage to surrounding structures.

Invasive cancer of the cervix in British Columbia: a review of the demography and screening histories of 437 cases seen from 1985-1988.

The charts of 437 patients with newly diagnosed invasive cervical cancer seen in British Columbia during 1985-1988 were reviewed to determine the age at diagnosis, stage of disease, and histology of the lesions. The patient's immigration status and country of birth were also examined. Two hundred forty-two patients (55%) were age 50 or over and 149 (34%) had preclinical stage IB disease. One hundred seventy of these patients (39%) had never had a cytologic examination before presenting with clinical symptoms, and 45 additional patients had not had cytology for 5 or more years before presenting with invasive disease; thus, a total of 215 cases (49%) fell into the category of "no cytology or cytology longer than 5 years ago." All negative smears were reviewed and in 39 patients (15%) having previous cytology, abnormal cells had been missed or undercalled. Native Indian women made up 10% of the cases of invasive carcinoma, a disproportionately large number as they represent only 2% of the British Columbian population. Of the 27 Native Indian patients in this series, 16 (59%) had never had a cytologic examination. Eighty-five (30%) of 276 women born in Canada had never had a cytologic examination, compared with 34 (71%) of 48 immigrants resident in the country for fewer than 10 years. Twenty-four patients were over the age of 60 with a history of negative Papanicolaou smears. We conclude that, if further gains are to be made in the reduction of death rates from invasive cervical carcinoma, new means must be found to encourage women to participate in Papanicolaou smear screening programs. In particular, special efforts must be made to attract specific groups, such as the Native Indian population and older recent immigrants.

A comprehensive program for cervical cancer detection and management.

OBJECTIVE: The purpose of this study was to document some of the results of a comprehensive provincial cytology and colposcopy program for the year 1988 and also to review the impact on the incidence and mortality rates for a clinical carcinoma of the cervix. STUDY DESIGN: This study is a retrospective analysis of the cytologic results of all patients examined provincially in 1988 and a review of the clinical records of patients diagnosed with invasive cancer and those who died of disease. RESULTS: In 1988 490,985 women (40% of all women over the age of 15 in the population) were screened, with 9.2% showing abnormal cells. A total of 79% of women screened were less than 50 years old and accounted for 86.3% of all abnormal smears. Women less than 35 years old were more likely than older women to have moderate dyskaryosis or worse. CONCLUSION: Intensive comprehensive cytology and colposcopy programs reduce not only the incidence and mortality of clinical carcinoma of the cervix but also rates of in situ disease and other precursors.

A quality-control program for colposcopic practice.

Quality control is accepted as an integral part of the cytology laboratory's activities. However, provision of similar safeguards for colposcopic practice in assessing patients with abnormal cervical cytology has been an elusive problem. This report describes the quality-control program for colposcopy that is used province-wide in British Columbia for 21 hospital-based clinics staffed by 35 colposcopists. Copies of the results of all colposcopic examinations performed are sent to the Central Cytology Laboratory where the program is coordinated and the data are collated, analyzed, and evaluated. These data are used to set standards against which each colposcopist's performance and activity can be measured. One of the quality-control indicators used is a comparison between the colposcopic impression of the lesion and the corresponding direct biopsy diagnosis. Correlation within one histologic degree occurred in 86% of patients examined; this information was used to set a performance standard of 80% as the quality-control index for this particular indicator. Three colposcopists had scores below this standard. Comparisons were also made between presenting cytology, directed biopsy results, initial colposcopic impression, and final colposcopic evaluation (the clinical working diagnosis). The other major aspect of the quality-control program consists of an analysis of all cases of diagnosed invasive cervical cancer in the province in a given year to determine whether any colposcopically related errors occurred in the investigation and management of these cases. This system helps to identify individuals who may benefit from additional training.

Epidermal thickness and skin appendage involvement in vulvar intraepithelial neoplasia.

Colposcopically directed biopsies obtained from 165 women with vulvar intraepithelial neoplasia (VIN) were studied to determine both the thickness of the dysplastic epithelium and the depth of involved and noninvolved skin appendages and ultimately to provide a morphometric basis for laser therapy for the condition. Multiple measurements per section were made, and the mean epithelial thickness was calculated for each biopsy. The mean (+/- SD) thickness of the epithelium for all grades of VIN was 0.52 +/- 0.23 mm, with the lesions ranging in thickness from 0.10 to 1.90 mm. The thickness of the involved epithelium varied little, regardless of the location of the lesions. The study indicated that VIN frequently is a multifocal disease most commonly affecting the central vulvar structures, with the posterior half of the vulva the area affected most often. The difference in thickness between the various grades of VIN was not of practical clinical significance from a therapeutic point of view. The measurements suggest that laser vaporization to a depth of 1.0 mm, including the zone of thermal necrosis, should be sufficient to destroy most epidermal lesions without skin appendage involvement. If the initial biopsy showed involvement of adjacent hair follicles or sebaceous glands, deeper tissue destruction would be necessary to theoretically achieve greater than 90% elimination of the disease.

Organisation and results of the cervical cytology screening programme in British Columbia, 1955-85.

A screening programme to detect preinvasive carcinoma of the cervix was started in British Columbia in 1949. Since 1970 the number of women who have been screened at least once has been maintained at about 85% of the population at risk. More than 500,000 cervical smears are being examined each year in the central laboratory. There has been an appreciable increase in the number of cases and rates of carcinoma in situ seen since 1970, particularly in women between 20 and 30 years of age. Since the programme started over 26,000 cases of squamous carcinoma in situ have been detected and treated. The incidence of clinically invasive squamous carcinoma of the cervix has fallen by 78% during the period under review, and mortality from squamous carcinoma of the cervix has fallen by 72%. A colposcopy programme, introduced throughout British Columbia over the past 12 years, has been important in reducing the problems of managing preinvasive lesions, particularly in younger women. It is concluded that the reduction in morbidity and mortality from invasive squamous cancer of the cervix in British Columbia over the past 30 years is directly attributable to the province wide screening programme and that a large potential increase in invasive cervical cancer rates among younger women is being prevented.

A comprehensive internal quality control system for a large cytology laboratory.

The British Columbia Cervical Cytology Program is operated through the Central Laboratory at the Cancer Control Agency in Vancouver and processes all of the gynecologic Papanicolaou smears collected by 3,200 physicians throughout the province of British Columbia. The laboratory receives approximately 2,400 smears per day, and the program currently processes in excess of 500,000 smears annually. This article describes the methods that have been developed for ensuring that adequate quality control is present in the screening and interpretation of half a million smears per year, both at the cytotechnologist and at the cytopathologist level. The results of the quality control program, which was considerably modified in 1985, are also presented. The modified program shows a significant improvement in the number of undercalled and overcalled cases detected in two comparable six-month periods.

Confirmation of genital herpes simplex viral infection by an immunoperoxidase technique.

Over the 12-month period from April 1984 to April 1985, 512,000 gynecologic (Papanicolaou) smears were examined in the Provincial Screening Program in British Columbia. During this time, 307 patients were found to have smears that contained cells consistent with, or suggestive of, a herpes simplex viral (HSV) infection. The Papanicolaou-stained smears from these 307 cases were subsequently restained, without prior destaining, using an immunoperoxidase technique specific for type 2 HSV (HSV-2) and cross reactive with HSV-1. Of the 205 smears containing cells considered to be consistent with a herpes infection, 187 were positive using the immunoperoxidase technique. Of the 102 smears showing reactive cell changes though unlikely to be causes by an HSV infection, only 5 were positive using the immunoperoxidase technique. The results show that the immunoperoxidase technique is a rapid and reliable method of confirming a suspected diagnosis of herpetic infection and that it is particularly useful in those patients in whom the Papanicolaou smear findings are equivocal.