The value of DNA single cell cytometry for the detection of aneuploidy was assessed in 100 specimens of actinic keratoses and 39 specimens of Bowen's disease. Ten seborrhoeic keratoses and 10 samples of normal epidermis served as negative control groups. Monolayer smears, prepared from formalin-fixed, paraffin-embedded tissues, were Feulgen-stained and used for interactive DNA-cytometry. In each specimen, the DNA content of 150 randomly chosen squamous epithelial cells was measured, using a TV-image analysis system (TAS-plus, Leica, Germany). Aneuploidy was diagnosed if at least three nuclei with a DNA content above 5c (5cEE > or = 3) were found. The aneuploidy rate in actinic keratosis was 69% (69 of 100) and in Bowen's disease was 95% (37 of 39). Another 20 specimens of actinic keratoses and the remaining two specimens of Bowen's disease were diagnosed as suspicious for aneuploidy (0 < 5cEE < 3). The 20 specimens of seborrhoeic keratoses and normal epidermis did not show any nuclei above the 5c level, and were classified as non-aneuploid. This indicates a sensitivity of 76% (106 of 139) and a specificity of 100% (20 of 20). The frequent occurrence of aneuploidy in actinic keratoses and Bowen's disease underlines the character of the lesions as epidermal carcinomas in situ, but does not explain the long-term low incidence of invasive growth.

Grading of tumor malignancy in breast cancer should contribute essential information both for the prospective outcome of the individual patient as well as for TNM staging. In a series of 104 breast cancer patients we tested the prognostic validity and reproducibility of mitotic figure counting compared with TNM staging, Bloom and Richardson grading, DNA single cell cytometry, and morphometry. Four-micrometer thick hematoxylin-eosin-stained routine slides were investigated. Mitotic figures were counted in representative areas of the tumor in 10 159-μm2—sized high power fields (HPFs) at a 400× magnification; the median value was seven and the threshold for the 25th percentile was three. This value should replace the common but prognostically invalid threshold of 10. Univariate survival analysis showed that mitotic figure counting allows the identification of three groups of patients (⩽3, 4 to 20, >20 mitoses per HPF) with significantly different survival probabilities (P < .0001; P = .0178). Depending on the number of mitotic figures, length of survival was significantly different within the group of TIN0 tumors (P = .0082) and the group of T1N1 or T2N0 tumors (P = .0251). In a Cox stepwise regression model mitotic frequency counting added prognostic information to tumor size and was of higher prognostic significance than lymph node status, DNA ploidy, or mean nuclear area. The 95% confidence limit for interobserver reproducibility, tested in 20 cases, was plus/minus 8 mitoses. After quartilization an agreement of 75% was observed.

62 intracranial meningiomas in 60 patients were studied with 18FDG-PET prior to neurosurgery in order to evaluate the relationship between 18FDG uptake and biological aggressiveness of the tumors. Histopathological grading, cellular density, Ki-67 proliferation index and evidence of chromosomal aberrations were used to assess tumor aggressiveness. Significantly elevated 18FDG uptake was found in grade 2- and 3- compared to grade 1-meningiomas, in tumors of high cellularity compared with those of low cellularity, and in meningiomas with an elevated Ki-67 proliferation index (above 2%). The two meningiomas with the most pronounced chromosomal aberrations revealed the highest 18FDG uptake of all cytogenetically studied meningiomas. We conclude that 18FDG-PET is useful for estimating the biological aggressiveness of intracranial meningiomas.

AIM--To investigate the prevalence and amount of polyploidy in fine needle aspiration specimens of the liver, urinary cytospin preparations, and cytospin preparations from pleural and peritoneal fluid. METHODS--Cells from 44 liver smears, 48 urine specimens, and 46 pleural and peritoneal aspirations were examined. After Feulgen restaining the DNA content of 100 randomly selected nuclei was determined using a TAS-plus image analysis system, combined with an automated microscope. RESULTS--Polyploidy was observed up to 16c in the liver, and up to 8c in urothelium and mesothelium. Sixty eight per cent of the liver aspirates contained polyploid nuclei. The rate in urothelium was 20.8% and in mesothelium 6.5%. CONCLUSIONS--Polyploidy in the liver may be interpreted as being associated with tissue differentiation, but the findings in urothelium and mesothelium remain of unknown importance.

In this study we evaluated indirect fluorescence in situ hybridization of the neu oncogene in combination with laser scanning microscopy for the detection and quantification of fluorescence signals in single cells. Cell lines from human tumor tissue with neu oncogene amplification and one nonneoplastic human epithelial cell line from a lactating breast with a neu single copy as the reference were used. After quantitative fluorescence analysis of three mixed cell populations (neoplastic cells and reference cells), we evaluated the cell morphology and location of the fluorescence signal by laser scanning microscopy. Finally, the number of oncogene copies in the tumor cell population could be estimated from the ratio of its mean background-corrected integrated fluorescence intensities to those of the reference cells. In all mixed cultures the ratio of fluorescence intensities was within the limits of oncogene copy numbers known from the literature and evaluated by southern blot analysis. The lowest oncogene copy number possible to quantify was four to eight genomic copies.

Zusammenfassung62 intrakranielle Meningeome von 60 Patienten wurden präoperativ durch 18FDG-PET untersucht, um den Zusammenhang zwischen 18FDG-Aufnahme und biologischem Tumorverhalten zu überprüfen. Als Parameter der Tumoraggressivität dienten histopathologisches Grading, Zelldichte, Ki-67-Proliferationsindex und Nachweis chromosomaler Aberrationen. Wir fanden einen signifikant erhöhten 18FDG-Uptake bei Grad 2- und 3- im Vergleich zu Grad 1-Meningeomen, bei zellreichen gegenüber mäßig zellreichen Meningeomen, und bei Tumoren mit einem erhöhten Ki-67-Proliferationsindex (über 2%). Die beiden Meningeome, bei denen die ausgeprägtesten chromosomalen Aberrationen beobachtet wurden, wiesen den höchsten 18FDG-Uptake unter den zytogenetisch untersuchten Meningeomen auf. Hieraus folgern wir, daß die 18FDG-PET zur Abschätzung der biologischen Aggressivität von intrakraniellen Meningeomen geeignet ist.

Cytometry means quantification of various cellular parameter using different instruments like computer based image analysers, flow cytometers or laser-scan microscopes. Using the more time consuming computer based image analysis, different parameters of possible prognostic validity for tumors of the urothelium or prostate may be quantified, like cellular structures, DNA-ploidy, chromosomal aberrations (interphase cytogenetics) AgNORs and a variety of functional antigens like proliferation markers, hormone receptors or oncogene products. The advantage of this method is that measurements can be performed on selected, morphologically classified cells. Using flow cytometers a representative number of tumor cells can be measured in a short time. With laser-scan microscopes oncogene amplification and chromosomal aberrations may be measured using fluorescence DNA in situ-hybridization. Of all cytometric parameters obtainable at present, only DNA ploidy is of clinical relevance in transitional cell carcinomas as it can predict the probability of tumor recurrence, -progression and of survival time with high accuracy. Similarly, at this time, only DNA measurements are of clinical relevance in prostatic cancer, as they can predict tumor progression, death from cancer, survival probability and sensitivity to hormonal treatment. No other grading parameter is of comparable prognostic significance in prostate cancer.

The diagnostic accuracy of sputum cytology for the diagnosis of bronchial carcinoma using paraffin-embedded, serially sectioned and hematoxylin and eosin-stained specimens was tested in 4,297 sputum samples from 1,889 patients, 219 of whom had bronchial carcinoma. The diagnostic sensitivity depended mainly on the number of investigated samples and was 85.4% with three sufficient sputa. The sensitivity was not influenced by the histologic types, location or TNM stage of the tumor. The specificity of the method was 99.5%. In three cases localization of sputum cytologically diagnosed bronchial carcinomas was not possible immediately (occult carcinomas, pTx); in two of these cases the bronchial carcinomas were located during follow-up. The third patient died without verification of the cytologic diagnosis. According to our results, sputum cytology on serial sections is a valuable instrument for mass screening of high-risk groups for the early detection of bronchial carcinoma. Lower sensitivities of sputum cytology in mass screening programs for the early diagnosis of lung cancer are discussed critically.

DNA cytometry is a powerful tool for grading the malignant potential of prostatic carcinomas, superior to histological or cytological evaluation. Independently of the clinical stage, the probability of tumor progression and death from cancer-specific causes can be assessed for individual patients. The mean nonprogression rate of 'DNA near-diploid' prostatic carcinomas seems to be 85-90% for 5 years. Near-diploid tumors are likely to respond to hormonal therapy. Changes in the DNA pattern towards the normal state under conservative therapy may serve as an early indicator of regression, whereas increasing DNA aneuploidy indicates tumor progression. The diagnostically relevant parameter is the degree of DNA aneuploidy. The parameters for prognostic interpretation of DNA data are not yet sufficiently standardized. DNA cytometry may help the urologist to predict the fate of an individual patient with prostatic cancer and to decide on a hormonal or adjuvant therapy. DNA cytometry is so far not useful for the detection of prostatic neoplasia or its precursors. DNA measurements may be performed by flow or static cytometry, the former method being more rapid, the latter more sensitive excluding the measurement of normal cells. Fine-needle aspirates are most suitable for performing DNA measurements with both methods.

Cellular growth on intraocular lenses can be observed by using a specular microscope. We examined in vivo the cellular pattern on the surface of implanted lenses during the early postoperative period in 63 patients. In every case inflammatory cells were noted on the lens surface at some point during the observation period. These often showed a spindle-like appearance resembling fibroblasts. The density of the cell population increased during the following days but usually did not exceed 100 cells/mm2. From the end of the first week, multinucleated giant cells may appear. In one case, a patient with a severe postoperative iritis and hypopyon demonstrated an extremely pronounced and long-lasting cellular proliferation.

Eighty-four skin lesions clinically suspected of being basal-cell carcinomas were investigated by exfoliative cytology. Specific criteria were found for the cytologic diagnosis of basal-cell carcinoma. All 37 cases of basal-cell carcinoma were correctly classified cytologically and could be differentiated from the 8 cases of squamous-cell carcinoma. There were no false-positive or false-negative diagnoses. Insufficient cellular material was obtained in 17% of the cases. The technique for collecting exfoliated epidermal cells with a new swab is described. DNA image cytometry was used as a diagnostic aid in doubtful cases. DNA image cytometry showed that 83% of the basal-cell carcinomas had an aneuploid nuclear DNA content.

Proposed in this article is a new system for the cytological grading of therapy-induced regression of prostatic carcinoma. Reversible and irreversible criteria in cytoplasm, nuclei, and nucleoli are distinguished and rated independently of each other on a scale of 0 to 2; the cellular regression grades r1-r3 are the sum of rating numbers. Criteria that are invalid for grading regression are pointed out. Results of the clinical testing of this grading system are presented.

In nine cases with the acquired immune deficiency syndrome (AIDS), including four stage I cases, three stage II cases and two stage III cases, DNA image cytometry was performed on Feulgen-stained lymph node imprint smears. Diploidy was found in three cases, tetraploidy in three cases and octoploidy in two cases. Aneuploid DNA distribution patterns were not seen. The lymphoid cells showed an enormously increased proliferation rate. Two cases in stage I revealed characteristic intranuclear DNA inclusions in lymphoid cells. These results indicate that DNA image cytometry may be useful as an adjunct to surgical pathology in certain cases to assist in the differential diagnosis between AIDS and benign conditions of the lymphoid system as well as between AIDS and malignant lymphomas, which usually have aneuploid DNA patterns.

Interactive DNA cytometry was used for the diagnosis of prospective malignancy in 48 smears with borderline lesions (mild and moderate dysplasias) of the uterine cervix. In addition, 183 smears with benign squamous epithelia, 38 with carcinoma in situ and 7 with invasive squamous carcinoma were also measured. Nuclear Feulgen-DNA measurements were performed using various methods, and the resulting data were analyzed by an algorithm for a DNA-cytophotometric diagnosis of malignancy. The results were compared with the data on follow-up and subsequent histologic studies in these cases. There was no false-positive diagnosis in the 183 benign smears and only 1 false-negative diagnosis in the 76 histologically proven squamous-cell carcinomas, which yields a specificity of 100% and a sensitivity of 98.6%. The sensitivity for the detection of subsequent histologically proven malignancy in cases with cytologically mild or moderate dysplasia amounted to 97%. In 13 borderline cases, there was a mean interval of 21 months between the taking of the cytologic smear on which the DNA diagnosis of malignancy was made and the date on which the histologic confirmation of malignancy was made. In 17% of the cytologically dysplastic cases, the DNA diagnosis of malignancy was not verified by subsequent histologic investigation. These results indicate that interactive DNA cytometry is able to detect prospective malignancy in smears from borderline lesions of the uterine cervix with a high sensitivity.

Hematoxylin-and-eosin-stained cytologic smears of sputum from 28 patients with dysplastic and suspicious cell findings were subjected to DNA image cytometry after Feulgen restaining. The nuclear DNA contents were measured with a TV-based image-analysis system, the Leitz TAS plus, combined with an automatic microscope. Computation of DNA data was performed according to an algorithm for the diagnosis and grading of malignancy. Of the 19 cases that were proven to be malignant in the follow-up, either by histologic examination, sputum cytology, fine needle aspiration biopsy or autopsy, the algorithm identified 17 as malignant in a stage (dysplasia) in which cytology was not yet able to present a definite diagnosis of malignancy. Only two cases of bronchial carcinoma were not detected in the state of dysplasia by this procedure. The periods between the DNA diagnosis of malignancy in dysplasia and the morphologic evidence of cancer varied from three days up to six months. Of the 11 cases that had been classified as benign by the algorithm, 9 were confirmed as benign during the clinical follow-up. Rapid DNA image cytometry appears able to separate squamous dysplasias of the lung into precancerous and nonprecancerous lesions.

A TV-based image-analysis system (Leitz TAS plus) combined with a Leitz automatic microscope was used for rapid DNA cytometry for diagnostic purposes. Malignant or suspicious cells found by the cytopathologists in conventionally stained smears were automatically relocated and measured. A program for automatic detection and measurement of nuclear DNA content was developed. DNA data are processed by an algorithm for the diagnosis and grading of malignancy. This diagnostic procedure is performed in real time and yields highly reproducible results. With this equipment, diagnostic DNA cytometry may be introduced in the routine cytology laboratory. Our mode of application does not replace the cytopathologist, but assists him or her in cases of doubtful diagnosis and in the grading of malignant tumors.