Diagnostic and prognostic value of DNA image cytometry in myelodysplasia.

The DNA content of erythropoietic cells from 10 patients with refractory anaemia (RA) with megaloblastic changes, who subsequently developed acute non-lymphoblastic leukaemia (ANL), and from seven patients with megaloblastic marrow aspirates due to pernicious anaemia were compared by DNA image cytometry. The DNA distribution, the rate of aneuploid cells exceeding 5c (5cER), and the square deviation index of DNA values from the normal 2c-peak (2cDI) were recorded. Both variables were of diagnostic and prognostic importance for epithelial tumours, malignant lymphomas, and dysplastic lesions. A rate of 5cER greater than 0 was found in eight of 10 myelodysplastic, but in none of seven control cases. Hypodiploidy was equally pronounced in both groups of patients. The 5cE had the highest discriminative value of all variables calculated. The 2cDI was not significantly different in either group. In pernicious anaemia the 2cDI depended mainly on the percentage of S cells, reflecting the defect of DNA synthesis. In RA with megaloblastosis the 2cDI correlated with the percentage of G2 cells, reflecting G2 arrest. In the myelodysplastic group the 2cDI correlated positively with the length of time until ANL developed, indicating the prognostic relevance of 2cDI. Our findings show that in megaloblastic anaemia DNA image cytometry can distinguish myelodysplasia from pernicious anaemia and that it also provides prognostic information.     

Diagnostic value of DNA analysis in effusions by flow cytometry and image analysis. A prospective study on 102 patients as compared with cytologic examination

One hundred twenty-six effusion samples from 102 patients were examined by cytology and flow cytometry (FCM). Overall, there was an 84% correlation between cytologic and FCM results. Of the 36 malignant cases determined by cytologic examination, FCM revealed an aneuploid peak in 20 (56%). Image analysis (IA) performed on the malignant cytologic cases with a diploid flow pattern detected two additional aneuploid peaks. In addition, FCM indicated three aneuploid cases in which cytologic characteristics were initially interpreted as benign (false negative). Aneuploidy was therefore detected in 64% of the malignant effusion specimens by FCM and IA. Twenty-three of the total of 24 aneuploid cases detected by FCM were associated with malignancy (predictive value = 96%). The one nonmalignant case was that of hemorrhagic pancreatitis with infected pseudocyst. FCM is an excellent tool when moderate to large numbers of tumor cells are present, whereas use of IA is advantageous for specimens containing smaller numbers of malignant cells because these can be directly analyzed. When an aneuploid peak is present, a diagnosis of malignancy must be suspected, and, if the initial cytologic screen is negative, a critical review of the cytology slides is justified. In those cases with an equivocal atypical cytology report and an abnormal cytometric histogram, additional investigation is warranted. In some malignancies the tumor cells will be diploid (in this study 36%) and neither FCM nor IA will add to tumor detection, leaving cytologic examination as the definitive technique.     

Interobserver variation in cell selection for DNA image cytometry.

AIMS--To describe a systematic investigation of interobserver differences in interpretation of nuclear morphology in preparations of small cell lung cancer (SCLC). METHODS--The screening/reviewing facility on the highly optimised microscope environment was used to individually tag 127 nuclei, chosen to reflect the spectrum of morphological appearances in nuclear preparations from three biopsy specimens of SCLC. Each nucleus was reviewed and labelled as control (lymphocyte), malignant or unsatisfactory by each of four observers. DNA histograms were plotted for each specimen using the nuclei identified as malignant by each participant. The histograms were compared in terms of identification of DNA stemlines and by calculation of a 5c exceeding rate (5cER). RESULTS--Interobserver variation in assessment of morphology was seen in 55.1% of nuclei. Disagreement occurred most frequently in the malignant/unsatisfactory category. Differences in morphological classification had little influence on histogram assessment by means of visual inspection but did show an effect on 5cER. CONCLUSIONS--There are significant interobserver differences in subjective assessment of nuclear morphology in cytometric preparations. This effect may seriously influence cytometric measurements.     

Competence on demand in DNA image cytometry

Quantitation methods in clinical pathology have to be normalized and standardized both from the instrumental and the methodological point of view to guarantee a defined level of precision and accuracy independent of the site where they are applied. The comparability of results obtained in different laboratories is the basis for the application of standardized diagnostic classification systems and therapeutic schemes. Remote quantitation based on standardized evaluation tools could be a way to reach the goals mentioned above. Diagnostic DNA image cytometry, increasingly used as a routine method in clinical pathology, will serve as an example for demonstrating the feasibility and usefulness of a concept of remote quantitation. We report a system for a remote DNA ploidy analysis, based on client server technology, and accessible via Internet or ISDN connections (Quantitation Server EUROQUANT). This system (i) allows the cytometric measurement of the DNA content of cells for diagnostic purposes, (ii) provides the user with comprehensive quality control of such measurements, (iii) helps in trouble-shooting, and (iv) gives assistance in diagnostic interpretation. The system uses the principles of telepathology and Internet technology. To date, more than 40 laboratories from Europe, USA, and Asia have successfully performed analyses on about 3,000 ploidy data sets.     

Role of DNA flow cytometry and image cytometry on effusion fluid

The objective of the study was to assess the value of DNA flow cytometry (FCM) and image cytometry (ICM) as an adjunct to routine diagnostic cytology. In this prospective study, 100 consecutive effusion fluids were studied for routine cytology, DNA FCM, and in selected cases, ICM. One half of the centrifuged fluid sample was used for routine cytology and the remaining portion was used for DNA FCM. Nuclear area, nuclear diameter, nuclear perimeter, nuclear convex perimeter, nuclear roundess, and nuclear convex area were measured on at least 100 cells by ICM in cytologically malignant or DNA aneuploid cases along with control cases. Clinical follow-up was done in all cases. There were 22 cytologically malignant cases and 78 cytologically benign cases. Among the 22 cytologically malignant cases, there were 11 aneuploid and diploid cases each by DNA FCM. Out of 78 cytologically benign cases, six (7.7%) were aneuploid by DNA FCM. Smears of these cases showed predominantly reactive mesothelial cells, but the DNA histograms showed hypodiploid (one), hyperdiploid (three), tetraploid (one), and hypertetraploid (one) aneuploidy. Follow-up of these cases showed clinical or histologic features of malignancy except in one case of tetraploid aneuploidy, which did not show any features of malignancy and responded well to antitubercular therapy. Therefore, out of 27 malignant effusions, DNA FCM picked up 16 cases and routine cytology detected 22 cases. Sensitivity and specificity of DNA FCM were thus 59.25% and 98.63%, respectively. There was a statistically significant difference (Student's unpaired t-test, P < 0.05) between cytologically malignant cases and control benign cases in all the nuclear morphometric parameters except for nuclear roundness. There was, however, no statistically significant difference of nuclear morphometric parameters between cytologically benign vs. DNA aneuploid cases and control benign cases. DNA FCM is a useful adjunct for routine diagnostic cytology. Visual diagnostic cytology and morphometric digital microscopy miss some cases of malignancy which can be detected by DNA flow cytometry. Diagn. Cytopathol. 2000;22:81-85.     

Role of DNA flow cytometry and immunocytochemical analysis in diagnosis of malignant effusions

Body cavity fluid examination sometimes presents a diagnostic challenge in cytology practice. This study was undertaken to evaluate efficacy of cytomorphology, epithelial membrane antigen immunocytochemistry (EMA-ICC) and DNA flow cytometry (FCM) in detection of malignant cells in effusions. One hundred effusions (55 pleural, 44 ascitic, and 1 pericardial fluid) were studied by cytology, EMA, and FCM. There were 29 malignant and 71 benign cases. On cytology, 28 of 29 malignant cases were diagnosed. With no false positives, the sensitivity and specificity was 96.55% and 100% respectively. FCM detected aneuploidy in 85.71% of cytologically malignant and 4.17% of cytologically benign effusions. EMA was positive in 75% of cytologically malignant and 4.17% of cytologically benign cases. EMA had lower sensitivity than cytology; 75.86% versus 96.55%. Sensitivity and specificity of FCM was 86.21%, and 97.18% respectively. FCM had lower sensitivity than cytology; 86.21% versus 96.55%. Sensitivity increased to 100% (P < 0.05) when the combinations of cytology plus EMA or cytology plus ploidy were applied compared to cytology alone (96.55%). Also, the combination of cytology plus EMA had higher sensitivity than EMA alone (100% versus 75.86%, P < 0.05) and combined cytology plus ploidy had higher sensitivity than ploidy alone (100% versus 86.21%, P < 0.05). The study demonstrates the usefulness of EMA-ICC and DNA FCM as adjuncts to cytology to diagnose malignancy in effusions. FCM in combination with ICC can be further developed to reduce number of false-negative cases on cytology and add objectivity to cytologically doubtful or equivocal cases.     

Comparative assessment of DNA analysis in effusions by lmage analysis and flow cytometry

Cytologic evaluation of body cavity fluids is useful to detect malignancy within the pleural and peritoneal spaces. A definitive diagnosis cannot always be made on cytologic evaluation alone. As malignant processes may show abnormal DNA content, DNA analysis of effusions may be useful. Therefore, we determined the DNA content of 37 effusions by flow cytometry (FC) and image analysis (IA) using the CAS 200. Of the 37 fluids evaluated, 18 were cytologically malignant, 15 benign, and four atypical. Overall, 22 fluids (60%) showed concordance between FC and IA. None of the benign fluids were aneuploid. All showed diploid histograms or diploidy with increased proliferating cells. Three of four atypical fluids had increased proliferating cells by either FC or IA, whereas one was diploid by both methods. Aneuploidy was detected in 13 malignant fluids: five were aneuploid by both methods and eight by only one method. IA identified aneuploidy in five of those eight cases, while three were identified by FC. Three of the cytologically malignant fluids were diploid by both methods, and two showed increased proliferating cells by IA and diploidy by FC. The specificity of both methods was 100%. However, the sensitivity of identifying a malignant fluid by aneuploidy is low, 44% for FC and 55% for IA. IA appears to identify small aneuploid populations more frequently than FC. The detection of aneuploidy in effusions is highly suggestive of malignancy, and the combination of both techniques gives the highest detection rate (72%). However, neither are as sensitive as traditional cytologic evaluation with the occasional use of additional histochemical stains. © 1994 Wiley-Liss, Inc.     

Atypical cells in effusions: Diagnostic value of cell image analysis combined with immunocytochemistry

The reliable identification of tumor cells in populations of atypical cells occurring in body cavity effusions is a well-known diagnostic problem. In order to improve tumor cell detection and to predict disease progression, we developed a cell scoring strategy based on a combination of DNA cytophotometry and immunocytochemistry. For this purpose, morphologically atypical cells obtained from 33 effusion samples were submitted to DNA content analysis and tested for Ber-EP4 immunoreactivity. It turned out that elevated DNA content alone has a low specificity (true negative ratio) and sensitivity (true positive ratio) in predicting disease outcome, whereas Ber-EP4 immunoreactivity alone has a high specificity (100%) but a low sensitivity (56%). In contrast, the use of a scoring system combining the two techniques and relating scores to the previous disease state and the cytomorphology of the atypical cells results in highly specific and sensitive prediction of the disease outcome. We therefore suggest that this approach is a valuable tool for reliably identifying tumor cells in effusions containing populations of cytologically suspect cells. Diagn Cytopathol 1996;15: 263–269. © 1996 Wiley-Liss, Inc.     

Computed cell cycle and DNA histogram analyses in image cytometry in breast cancer.

AIMS--To analyse the cell cycle and DNA histogram components in data from DNA static cytometry and, in particular, to investigate the influence of the length of time the slides are exposed to the light of the cytophotometer in evaluating the G0/G1 peak. METHODS--DNA static cytometry was performed on 18 Feulgen stained imprints and six histological sections taken from six breast carcinomas. The total optical density values obtained were analysed using software commercially available as Multicycle. DNA flow cytometry was performed on the same cases. RESULTS--The proportions of nuclei related to the cell cycle components from DNA static cytometric data, obtained from Feulgen stained cytological smears, were almost identical with those obtained from DNA flow cytometric data. Moreover, additional information was obtained from the DNA static cytometry frequency histogram and the proportions of nuclei below the diploid G0/G1 peak and above the G2 phase. Discrepancies between DNA static cytometry and DNA flow cytometry were seen in the large coefficients of variation of the G0/G1 peaks obtained with the former method of analysis, even though a better correspondence was found when the exposure time of the slides to the light of the cytophometer was conspicuously shortened. The information obtained from histological sections seemed to be similar to that obtained from DNA flow cytometry when a single cell population was present; a single cell population was detected in two out of the three cases in which two distinct populations had been present in DNA flow cytometry. CONCLUSIONS--The computer analysis of DNA static cytometric data obtained from Feulgen stained cytological specimens provides the type of information on the cell cycle which is usually obtainable only from DNA flow cytometry. Correspondence with the DNA data from histological sections, however, was poor.     

Comparison between image and flow DNA cytometry in non-Hodgkin's lymphomas

To assess the reliability of DNA estimation in cytological material, Feulgen lymph node imprints from 22 cases of malignant non-Hodgkin's lymphomas were examined by image cytometry (ICM) for both ploidy and cell kinetics, and the results obtained were compared with flow cytometry (FCM). The DNA distribution pattern was less accurate with ICM than with FCM; however, a high correlation was found between proliferative indices (r = 0.91) and between aneuploidy rates (agreement in about 86% of the cases) determined by FCM and ICM. Moreover, DNA tetraploid (or near-tetraploid) stem lines were more easily detected by ICM, due to the morphological selection of lymphomatous cells. The proliferation rate and the aneuploidy frequency according to morphological classification were in agreement with larger, previously reported studies with FCM. Therefore, ICM appears to supply additional complementary information to that obtained with FCM, particularly for the study of heterogeneous cell populations, which may be usefully applied to refine the large cell lymphoma subclassification.     

Comparison of DNA histograms by standard flow cytometry and image cytometry on sections in Barrett's adenocarcinoma

Background  

The purpose of this study was to compare DNA histograms obtained by standard flow cytometry (FC) and high fidelity image cytometry on sections (ICS) in normal gastrointestinal mucosa and Barrett's adenocarcinoma (BAC).     

Diagnostic value of DNA flow cytometry combined with fine needle aspiration biopsy in lymphomas

The nuclear DNA content of cells from 45 malignant lymphomas and from 60 benign lymph nodes obtained by fine needle aspiration was analysed to investigate the diagnostic value of DNA flow cytometry combined with routine diagnostic cytology in lymphomas. DNA aneuploidy was found in 43 per cent of lymphomas of high grade malignancy (NCI Working Formulation) but only rarely in lymphomas of intermediate- or low-grade malignancy or in Hodgkin's disease, and never in benign lymph nodes. The median percentage of proliferative cells (S + G2/M) was 22.6 per cent in diploid high-grade lymphomas, 15.3 per cent in intermediate-, and 8.1 per cent in low-grade lymphomas, as compared with 4.9 per cent in benign lymph nodes (P less than 0.0001). If the presence of DNA aneuploidy or more than 12 per cent of proliferative cells is used as a criterion for malignancy, the diagnostic accuracy of DNA flow cytometry in detecting lymphoma is 81 per cent. DNA flow cytometry suggested correct diagnosis in 10 of the 19 false positive, false negative, or indeterminate cytological findings encountered during the study. It is concluded that DNA flow cytometry combined with fine needle aspiration biopsy has diagnostic value in lymphomas, but false negative results are common especially in low-grade lymphomas; the method should therefore be used in conjunction with light microscopy.     

Prognostic value of cells with more than 5c DNA content in node-negative breast cancer as determined by image cytometry from tissue sections

The aim of this investigation was to study the prognostic significance of 5c cells (presence of cancer cells with >5c DNA content; ie, over 18 pg of DNA per nucleus) in axillary node-negative breast cancer. Tissue sections (3 μm) from 134 tumors were stained for DNA using the Feulgen method and screened for the percentage of 5c cells with the CAS 200 image analysis system (Cell Analysis System, Inc, Lombard, IL). Cancer cells with a DNA content exceeding the 5c level were found in 45% (60 of 134) of the cases, accounting for a median of 0.2% (range, 0.05% to 1.05%) of all cells. The presence of 5c cells was associated with a high histologic grade of the tumor (P = .0001), a large number of mitoses (P < .0001), flow cytometric DNA aneuploidy and high S-phase fraction (P = .0002 and P < .0001, respectively), and c-erbB-2 oncoprotein and p53 tumor suppressor gene product overexpression (P = .0002 and P = .0006, respectively). Patients with 5c cell-positive tumors had a significantly worse 8-year survival rate (P = .003) than those with 5c cell-negative tumors. Subgroup analysis showed that the presence of 5c cells had a prognostic impact in low malignancy tumors, ie, in well-differentiated (grade I or II) and slowly proliferating tumors. Our findings suggest that determination of 5c cells may be a useful additional prognostic factor in axillary node-negative breast cancer. It adds prognostic information, especially in cases that are otherwise thought to have a favorable course.     

Evaluation of DNA content in gastric dysplasia and carcinoma by image cytometry

In 30 cases of gastric dysplasia and 10 cases of gastric carcinoma, DNA content was studied by IBAS image analysis system. The mean DNA level increased steadily with the advance of histologic gradation, and the highest DNA content was observed in gastric carcinoma. No case of aneuploidy was found in mild dysplasia. In moderate dysplasia, aneuploid cells were occasionally encountered. Severe dysplasia had a lower percentage (4.48%), and gastric carcinoma was characterized by a high percentage of aneuploid cells (14.54%).     

Clinical importance of DNA content in rectal cancer measured by flow cytometry.

The DNA content of 369 rectal cancers was measured by flow cytometry. One hundred and four (28%) were diploid, 252 (68%) were aneuploid, and 13 (3.5%) were tetraploid. Diploid cancers were associated with an improved 5 year survival (p less than 0.001) and were more likely to present at an early stage. DNA content, however, did not confer independent prognostic information in a Cox model based on four discrete pathological variables. Patients were classified by a new system of prognostic grouping and those with a very good or a very poor outlook were removed leaving 137 prognostic group III patients. No further substratification of this group by DNA content or by four additional pathological variables could be achieved. As the new prognostic system is not improved by the addition of ploidy, routine adoption of flow cytometry in the assessment of rectal cancer cannot be recommended.     

Clinical application of flow cytometry for DNA analysis of solid tumors.

Recent developments of flow cytometry (FCM) technology which make multiple correlative biological measurements on normal and neoplastic cells is affecting areas of diagnostic pathology as well as research fields, and a general understanding of FCM techniques is essential for pathologists. Today, FCM DNA measurements of tumors also becomes routine in the clinical and/or pathological laboratory for aid in cancer diagnosis and cancer treatment. It can also contribute to diagnosis of tumors as a supplemental method to conventional histopathology, and DNA ploidy and the percentage of S-phase fraction are considered as complementary prognostic parameters independent of the stage of disease. This article reviews clinical applications of flow cytometry focusing on the DNA measurements of solid tumors, and related practical issues, such as the methodology for nuclear DNA measurement, interpretation of DNA histograms and the relationship of DNA ploidy and S-phase fraction to clinical and pathological features of human solid tumors.     

A prospective comparison of DNA quantitation by image and flow cytometry

Advances in computer and video technology suggest that image analysis may be practical method of measuring DNA that also allows visual confirmation of cell type. The purpose of this study was to prospectively compare DNA quantitation from 92 solid tumors in which DNA indices had been measured by image analysis of touch preparations (CAS 100) and flow cytometry of cell suspensions (FACScan). For 81 cases, there was excellent correlation between the two methods. For nine cases, however, an aneuploid population, usually near tetraploid, was identified by image but not by flow cytometry. Three cases had aneuploid peaks by flow cytometry that were not identified by image. Although these methods show good correlation, rare populations may be missed by CAS, presumably because of sampling errors in the touch preparation. Aneuploid populations may also be missed by flow cytometry, either because of cell loss during processing or because visual identification by image can increase sensitivity.     

DNA analysis of cardiac myxomas: flow cytometry and image analysis

Cardiac myxoma is the most common primary tumor of heart, but there is a longstanding controversy over whether it is a true neoplasm or a reactive lesion. We analyzed 24 cardiac myxomas from 22 patients: 22 by DNA flow cytometry and five by image analysis. Two myxomas were aneuploid; one of those analyzed by flow cytometry, and the other by image analysis. Proliferative fractions (S + G2/M) were high in three tumors from patients with multiple myxomas (mean, 15.9%; SD, 4.0%) as compared with 12 solitary uncomplicated myxomas (mean, 7.7%; SD, 6.0%). S-phase and proliferative fractions were low in embolic, recurrent, and solitary myxomas. The presence of aneuploidy in some myxomas supports a neoplastic origin for this tumor.     

DNA quantitation of Wilms'' tumour (nephroblastoma) using flow cytometry and image analysis.

AIMS: To compare flow cytometry (FCM) with image analysis (IA) in the DNA quantitation of Wilms' tumour (WT) and to correlate data so obtained with recognised clinical and pathological prognostic parameters. METHODS: Thirty six patients with histologically proved WT diagnosed between 1980-89 were investigated. Fifteen patients had stage I disease, 10 stage II, six stage III, two stage IV and three stage V. Suspension of nuclei obtained by pepsin digestion of paraffin wax embedded tumour tissue was analysed using a FAC-Scan flow cytometer, and a CAS-100 image analyser. RESULTS: Tumours were concordant in most instances, however, IA identified aneuploidy in two tumour samples which were diploid by FCM. Aneuploidy was detected in 5/33 tumours with favourable histology and 3/3 with unfavourable histology. Three of 28 patients with Stage I, II and V disease and 5/8 patients with stage III and IV had aneuploid tumours. All patients with unfavourable histology died of disease. In the group with favourable histology, 4/5 patients with aneuploid tumours developed recurrent disease compared with 1/27 diploid tumours (p less than 0.0001). CONCLUSIONS: Ploidy may be a useful additional prognostic indicator in Wilms' tumour with favourable histology. Larger scale studies are needed to confirm the relation of ploidy to survival in early stage WT.