The role of deoxyribonucleic acid image cytometric and interphase cytogenetic analyses in the differential diagnosis, prognosis, and clinical follow-up of hydatidiform moles. A report from the Central Molar Registration in The Netherlands.

OBJECTIVES: To assess the value of deoxyribonucleic acid ploidy in the differential diagnosis and clinical follow-up of hydatidiform moles, the histopathologic features, deoxyribonucleic acid ploidy, and clinical follow-up were compared in 347 cases: 143 complete moles, 52 partial moles, and 152 abortions, of which 56 cases were hydropic abortions with histologic features of triploidy but lacked trophoblastic hyperplasia. STUDY DESIGN: In all cases deoxyribonucleic acid image cytometry was performed, and in 85 of these cases interphase cytogenetics was also performed. RESULTS: With use of deoxyribonucleic acid image cytometry and interphase cytogenetics, a bimodal polyploid deoxyribonucleic acid pattern was present in 97% of complete moles, 27% of partial moles, and 4% of abortions. All these cases of partial mole were reclassified to complete mole on the basis of this deoxyribonucleic acid pattern and the histopathologic features in spite of the presence of fetal blood cells, amnion, or yolk sac. Deoxyribonucleic acid triploidy was found in 95% of the remaining partial moles, in 77% of hydropic abortions with histologic features of triploidy, and in 14% of the remaining abortions. Reliable differentiation between deoxyribonucleic acid triploid partial moles and hydropic abortions with histologic features of triploidy was not possible on basis of the histopathologic features (trophoblastic hyperplasia) or 3.5c exceeding rates. Deoxyribonucleic acid diploidy was found in 1% of complete moles, 23% of hydropic abortions with features of triploidy, and 78% of the remaining abortions. Deoxyribonucleic acid tetraploidy was rarely found (1% of complete moles, 2% of partial moles, 1% of abortions). Persistent gestational trophoblastic disease developed in 33% of the bimodal deoxyribonucleic acid polyploid cases (all complete moles), in 1% of the diploid cases (concerning one of the two diploid complete moles), and in 1% of the triploid cases (partial moles). CONCLUSION: Deoxyribonucleic acid analysis is essential in the diagnosis of hydatidiform moles to decide on clinical follow-up.

Persistent gestational trophoblastic disease: DNA image cytometry and interphase cytogenetics have limited predictive value.

DNA flow cytometry has shown a wider spectrum of DNA content in the complete hydatidiform mole (CM) than the originally reported diploidy. Conflicting results have been published about the relationship of DNA content and the occurrence of persistent gestational trophoblastic disease (PGTD). In the present study, 71 cases of CM and 4 cases of partial mole accompanied by PGTD and 100 cases of CM without PGTD were evaluated with DNA image cytometry for differences in DNA-ploidy pattern, expressed as the 2.5c and 5c exceeding rates. A pilot study of 20 cases of each group was performed using interphase cytogenetics to detect differences in the frequency of numerical chromosomal aberrations and in sex chromosome composition. For this purpose, DNA probes specific for the pericentromeric regions of chromosomes 1 and X and for the long arm of chromosome Y were incubated on 6-micron paraffin tissue sections. The results showed no differences between CMs with or without PGTD; DNA polyploidy occurred in 99% and 98% of cases, respectively; the 2.5c exceeding rate and 5c exceeding rate were 62.6 and 62.4, and 6.5 and 6.0, respectively. The frequency of numerical chromosomal aberrations as detected by interphase cytogenetics was 23.4 and 22.8%. An XY pattern was found in 3 of 20 cases of CM with PGTD and in 4 of 20 cases of CM without PGTD. The four cases of partial mole showed a DNA-ploidy pattern identical to that of a CM. For this reason, they would be better reclassified as CMs, despite the presence of nucleated red blood cells or amnion. Although nuclear atypia and corresponding increased DNA content is pronounced but variable in CMs, the occurrence of PGTD is not related to variations in quantitative DNA content nor to gross heterology or homology in sex chromosomes.

The dynamics of keratin expression in malignant transformation of cervical epithelium: a review.

OBJECTIVE: To review basic and clinical aspects of cytoskeletal keratin expression in normal cervical epithelia as well as in preneoplastic and malignant epithelia of the uterine cervix. DATA SOURCES: The results of extensive studies from our group and other groups on keratin phenotyping in normal premalignant and malignant cervical epithelia were summarized. METHODS OF STUDY SELECTION: All studies involving keratin expression in the cervix were reviewed, as were general studies on keratin expression in which the cervix was mentioned and studies relevant to understanding cervical cancer etiology (36 studies). DATA EXTRACTION AND SYNTHESIS: From these studies, keratin phenotypes of the various epithelia were derived. The phenotypes were correlated to existing theories on the development of cervical carcinoma. CONCLUSIONS: It is possible to distinguish the various epithelial types in the normal cervix based on their keratin expression patterns. Reserve cells display a bidirectional keratin pattern, comprising keratins typical of both squamous and simple types of differentiation, reflecting the bipotential nature of these cells. Cervical intraepithelial neoplasia can be divided into two subpopulations, one characterized by the reserve cell keratin phenotype and the other by a keratin phenotype typical of nonkeratinizing squamous epithelia. The first population also contains the simple keratins, the relative percentage of which increases with increasing degree of dysplasia. We therefore suggest that these lesions are progressive in nature. Carcinomas show a differentiation-related keratin expression pattern in addition to the basic reserve cell keratin phenotype. Adenocarcinomas also have been shown to express most of the reserve cell keratins. The latter observation indicates a common progenitor for both carcinoma types.

Interphase cytogenetics in paraffin sections of routinely processed hydatidiform moles and hydropic abortions.

The differential diagnosis of complete (CM) and partial (PM) hydatidiform moles and hydropic abortions (HA) can be difficult when based on histology alone. Therefore, a more objective approach of chromosome ploidy analysis as detected by in situ hybridization (ISH) was performed on 6 microns paraffin sections of seven cases, originally classified as three CM, two PM, and two HA with a histologic pattern suggestive of triploidy. Probes for repetitive DNA targets in the (peri)centromeric region of chromosomes 1 and X and in the q arm of chromosome Y were used to determine chromosome ploidy and sex chromosome composition. The findings in the three CM were consistent with diploidy: two copies of chromosomes 1 and X and none of chromosome Y. In the two HA with a histologic pattern suggestive of triploidy, three copies of chromosomes 1 and X and none of chromosome Y confirmed triploidy. Two cases originally classified as PM both appeared to have two copies of chromosome 1 with an XX pattern in one case and an XY pattern in the other case, which is consistent with diploidy instead of triploidy. After reviewing, both cases most likely represented CM. We conclude that interphase cytogenetics by ISH on paraffin sections of hydatidiform moles and hydropic abortions enables chromosome ploidy analysis with preservation of histological context.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytology of urinary sediments as a method of differentiating acute rejection from other causes of declining renal graft function.

We have previously reported that during acute rejection of renal allografts T lymphocytosis and increased HLA-DR expression on tubular epithelial cells can be demonstrated in urinary sediments by incubating cytospin preparations with monoclonal antibodies against T cells and HLA-DR antigen in an indirect alkaline phosphatase technique. We now tested whether immunocytological analysis of urinary sediments can be used to differentiate acute rejection from other causes of declining graft function. For this we retrospectively selected, from a series of urinary samples that were taken either at random or as part of a longitudinal study in unselected graft recipients, those specimens that were taken at the time of increasing creatinine levels, and compared the original immunocytological diagnosis, made without knowledge of clinical data, with the final clinical one. In 44 of 74 evaluable cases an immunocytological diagnosis of rejection was made, which in 37 patients was consistent with the eventual clinical diagnosis. In 28 of 30 cases the diagnosis no rejection proved to be correct. This indicates a sensitivity of 95% and a specificity of 80% for the immunocytological diagnosis of rejection. Of 38 patients who underwent a renal core biopsy, the immunocytological diagnosis was consistent with the histological diagnosis in 36 cases (31 rejections, 5 no rejections). In this subgroup the sensitivity of the immunocytology was 97% and the specificity 83%. We conclude that immunocytological examination of urinary sediments in renal allograft recipients can be a valuable new tool in discriminating acute interstitial rejection from other causes of deteriorating graft function.

Detection of numerical chromosome aberrations using in situ hybridization in paraffin sections of routinely processed bladder cancers.

An improved protocol for in situ hybridization (ISH) to routinely processed, paraffin-imbedded tissue sections from transitional bladder carcinoma (TCC) is presented. The protocol to detect numerical chromosome aberrations involved treatment of sections with thiocyanate prior to proteolytic digestion, resulting in reproducible ISH reactions. It was used to explore the influence of nuclear truncation in the detection of numerical chromosome aberrations and the detection of tumor cells among stromal and inflammatory cells, to compare the flow cytometric DNA index with chromosome copy number, and to study chromosome heterogeneity within tumors. For this study, a DNA probe for the chromosome region 1q12 was used. Hybridization of model systems with known chromosome numbers, such as sections of paraffin-embedded lymph nodes, paraffin-embedded human peripheral lymphocytes, T24 and Molt-4 cells with two, three, and four chromosomes 1, respectively, showed in at least 50% of the cells the proper number of chromosome hybridization signals in standard 6-microns-thick sections. Depending on the size of the nucleus, a certain percentage of the cells showed lower copy numbers as a result of truncation. In four cases of normal urothelium in paraffin sections, the percentage of nuclei with more than two chromosome spots did not exceed 5%. Comparison of the number of ISH signals, as detected in ethanol-fixed single cell suspensions of 11 TCCs [five flow cytometric (FCM) diploid, three FCM aneuploid, and three FCM tetraploid], with ISH results obtained in paraffin sections of the same tumors showed that typical numerical chromosome aberrations, such as trisomy and tetrasomy up to nonasomy, could be detected. However, the real chromosome copy number is underestimated, especially in tumors with high copy numbers, as detected in the single cell suspensions of the same tumors. Hybridization of a TCC with extremely large nuclei (DNA index = 3.2) containing six to nine ISH signals as detected in the isolated tumor cells, showed that an indication of these real chromosome copy numbers could be obtained in 6-microns paraffin sections. The accuracy for the detection of the chromosome copy number was even higher in cases where hybridization signals were counted in the mitotic cells. Furthermore, chromosome heterogeneity was detected by ISH using centromeric probes for chromosomes 7, 9, and 18, even though nuclei are truncated in the section. The surplus value of ISH on paraffin sections, as compared with ISH on isolated tumor cells, can be summarized as follows. (a) The focal tumor cell areas with chromosome aberrations can be recognized in the sections and be correlated with the histologic appearance.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a hormone-producing ovarian carcinoma cell line.

An ovarian carcinoma cell line (OTN 11) was produced from the ascitic fluid of a patient with a moderately to well differentiated papilliferous cystadenocarcinoma of the ovary. The cell line was characterized using electron microscopy karyotyping, immunohistochemical techniques with monoclonal antibodies against keratins as epithelial markers, and the monoclonal antibodies OV-TL 3 and OC 125 as ovarian carcinoma markers. These techniques revealed the epithelial and adenocarcinomatous nature of the cell line and the presence of ovarian carcinoma-related surface markers. The adenocarcinomatous nature of the cell line also became apparent after heterotransplantation of cell suspensions into nude mice and nude rats, in which adenomatous tumor structures were formed. These xenografts had the same ultrastructural and immunohistochemical properties as the cell line. Despite the adenocarcinomatous character of the tumor the cultured cells release estradiol into the culture medium. We may conclude that OTN 11 is an ovarian carcinoma cell line which has retained highly differentiated functions, such as the production of an ovarian hormone.

Population screening for cervical cancer in the region of Nijmegen, The Netherlands 1976-1985.

In this study the results of a 9-year cervical screening program in the region of Nijmegen, The Netherlands are presented. All women aged 35 through 54 were invited every 3 years for a cervical smear. Overall attendance rates were 74%, 67%, and 63% at first, second, and third screening, respectively. The number of histologically confirmed severe epithelial abnormalities discovered in women who were screened for the first time was 3.8 per thousand smears, 1.0 per thousand in women who were screened twice and 0.7 per thousand in women who were screened three times in the program. Three years after the start of the screening program the incidence of invasive squamous cell cancer started to decline in the women aged 35 through 54 years. On the basis of the results of this study we may conclude a screening policy with an interval of 3 years to be a safe procedure. Whether this interval is the most efficient cannot be concluded. We have the impression that an interval of 4 to 6 years may lead to comparable results.

Cervical cancer survival in Nijmegen region, The Netherlands, 1970-1985.

Survival rates were computed for 359 women diagnosed with invasive cervical cancer between 1970 and 1985. The 5-year survival rate for the entire group was 67%. Survival was better in the period 1976-1980. Extension of the tumor and age at diagnosis were important prognostic factors. The effects of clinical stage, age at diagnosis, and year of diagnosis were studied simultaneously with the proportional hazards model. The hazard rate increased with increasing age and increasing clinical stage. Year of diagnosis had effect on survival only in case of IIB tumors. For the other clinical stages there was no significant effect on survival of year of diagnosis.

The effectiveness of cervical screening: a population-based case-control study.

The cervical smear histories of 36 women with invasive cervical cancer were compared to those of 120 age-matched controls, drawn from local registrar's offices. Of the cases 47% were screened at least once, while for the controls this figure was 68%. The relative risk of getting invasive cervical cancer for women screened at least once compared to women who were never screened was 0.32. The most important confounding factor was age at first intercourse. Contrary to other studies however, it was found that women who were younger when having first intercourse were screened more often. After correcting the relative risk of screened vs unscreened for age at first intercourse, the relative risk became 0.22. When the length of the interval since the last smear was considered, the relative risk was 0.18 when the smear was made between 2 and 5 years earlier and 0.30 when this smear was made more than 5 years earlier. These results support the assumption that screening is effective in the prevention of invasive cancer of the uterine cervix. Even a screening interval of more than 5 years provides considerable protection.

Sputum cytology by the Saccomanno method in diagnosing lung malignancy.

From 1,488 patients, satisfactory sputum was available for cytologic diagnosis. Overall diagnoses were correct in 85.4% of patients, false negative in 193 patients (13.0%), and false positive in 24 patients (1.6%). In patients with a malignant lung process, cytologic diagnoses were correct in 228 patients (54.2%) and false negative in 193 patients (45.8%). In patients with primary lung cancer, the proportion of correct positive diagnoses increased from 0.47 to 0.87 with one to five sputum specimens examined. In patients with metastatic disease, the figures were 0.35 with one specimen examined and 0.38 with two and more sputum specimens. Cytologic typing accuracy was 67% for large-cell carcinomas, 73% for adenocarcinomas, 91% for small-cell lung cancers, and 98% for squamous-cell carcinomas. For the clinically most relevant groups of nonsmall-cell lung cancer and small-cell lung cancer, these figures were 99% and 91%, respectively.

Relationship between the diagnosis of epithelial abnormalities and the composition of cervical smears.

The significance of endocervical columnar cells as a high-quality parameter of cervical smears was studied. In a cohort of women with two successive screenings, the consistency of the cellular composition of the cervical smears and the relation between the cellular composition of the smears and the frequency of the diagnosis of abnormal epithelial changes was investigated. At the first screening, a significantly higher number of epithelial abnormalities was found in smears with endocervical columnar cells than in smears without endocervical columnar cells. A significantly higher number of abnormal epithelial changes was found on the second screening in smears from women whose smears from the first screening did not contain endocervical columnar cells than in smears from women whose smears from the first screening did contain endocervical columnar cells. The presence of endocervical cells should be considered a very important indicator of the quality of cervical smears. The chance of missing an abnormal epithelial change is increased in smears without endocervical columnar cells. When endocervical columnar cells are absent, the smear should be considered to be of unreliable quality and a repeat smear should be taken after a short interval, unless the absence of columnar cells can be satisfactorily explained.

Immunochemical demonstration of keratin and vimentin in cytologic aspirates.

Antibodies to intermediate filament proteins were used to characterize tumor cells present in peritoneal and pleural effusions and in thin needle aspirates from palpable lymph nodes. Metastatic adenocarcinoma cells (breast, ovary, endometrium, cervix, colon and stomach) as well as squamous-cell carcinomas and mesotheliomas stained specifically with antibodies to keratin while mesenchymally derived tumor cells (lymphomas, melanoma, fibrosarcoma and neurofibrosarcoma) were positive only for vimentin. Especially in cases of lymph node aspirates, keratin staining in cells was a direct indication of metastatic carcinoma. Antibodies to these different components of the cytoskeleton can thus be used in cytopathologic diagnosis when a definitive diagnosis cannot be made on the basis of conventional cytologic features.

Pressure-fixation method of transferring cells from polycarbonate filters to glass slides.

A technique for the preparation of cytology slides is presented by which cells collected on a polycarbonate membrane filter are transferred to a slide by means of simultaneous pressure and fixation. Using cervical samples as a model, the influence of the filtration rate, filter pore size and duration of pressure application on cell recovery was analyzed. The present version of the preparation procedure uses manual techniques that define the operating characteristics of a fully automated procedure.

The significance of endocervical cells in the diagnosis of cervical epithelial changes.

A study was made of the relationship of endocervical columnar cells to the cytologic identification of abnormality in the cervix. From a population of women participating in a cervical cancer screening program, a group of women was selected whose smears theoretically should have contained endocervical columnar cells. Endocervical cells were present in 93.1% of the smears and absent in 6.9%. A significantly higher number of moderately and severely atypical epithelial changes was found in smears with endocervical cells as compared with smears without endocervical cells.

Quantitative light microscopy of atypical mesothelial cells and malignant cells in ascitic fluid.

Light microscopic cytologic diagnosis of pleural and peritoneal effusions--specifically, the differentiation between atypical or reactive mesothelial cells and malignant cells--remains a problem. During the past several years, various labor-intensive special techniques have been used to obtain a better understanding of the morphology of these cell types. The present study, performed in part to provide a basis for developing more advanced quantitative cytologic techniques, employed visual light microscopic cytometry using an ocular micrometer on 750 malignant and 750 atypical mesothelial cells at a magnification of 1,000X. Our results statistically confirmed the importance of diagnostic features used in routine cytologic evaluation. In addition to the previously reported differences between cellular, nuclear and nucleolar size in malignant cells and atypical mesothelial cells in effusions, the percentage of irregular nucleoli was shown to be an important differential feature, averaging 58.8% in malignant cells as compared with 6.6% in atypical mesothelial cells.

Thionine-Feulgen-Congo red staining of cervical smears for the BioPEPR image-analysis system.

A staining method developed for use with BioPEPR (Biological Precision Encoding and Pattern Recognition), an automated image-analysis system for cervical smears, is described. The stain is a combination of the Feulgen procedure, with thionine-SO2 as the Schiff reagent, and Congo red, which is used as a counterstain. The stain resulted in smears suitable both for microscopic diagnosis and for BioPEPR measurements made on photonegatives at a single wavelength 545 nm. A high level of reproducibility and accuracy of nuclear and cytoplasmic area measurements was obtained. Nuclear integrated optical density could be well measured and was shown to be useful in discriminating between normal and abnormal cells. Using a combination of morphologic features, a high level of cell classification accuracy was reached. The possibility of using the stain for more detailed studies is discussed.

The development of a cervical smear preparation procedure from the BioPEPR image analysis system.

The preparation procedure for the BioPEPR automated image analysis system is described. Cervical cells are collected in a preservative solution and disaggregated by an automated syringing apparatus, giving about 50% single cells. After centrifugation, the preservative solution is discarded, 2% polyethyleneglycol in 50% ethanol (carbowax) is added, and the cell solution is automatically spread onto a glass slide, resulting in a cell density of about 50 cells/sq mm in the central area of the smear. Increasing cell density is shown to lead to a gradual linear decrease in single cells. All preparation steps have been critically evaluated, and the possibility of implementing this preparation procedure in a fully automated system is discussed.