Morphometry of nuclei of the normal and malignant prostate in relation to DNA ploidy.

Fine needle biopsies from 70 patients with prostate carcinoma and 10 patients with benign hyperplasia were used to study area, variation in size and form factors of the nuclei by image analysis. The results were related to DNA ploidy of the cell populations as measured by flow cytometry, cytologic grade and patients' survival. Nuclear area differed significantly between benign lesions and tumors. It increased in diploid low-grade tumors from a normal value of 54.2 +/- 3.1 microns2 to 75.6 +/- 5.3 microns2. In aneuploid tumors with an increase in the chromosome number, the nuclear size further increased to about twice that of benign nuclei. Variation in size also differed between benign and malignant epithelium, with a further increase between diploid and gross aneuploid tumors. While nuclear size and variation in nuclear size made it possible to discriminate malignant from benign lesions, form factor did not differ between benign and malignant lesions. In follow-up, however, none of these factors reached significance for predicting survival.     

Differentiating benign nevi from malignant melanoma using DNA microdensitometry and karyometry and maturation: a zonal comparison,correlation and multivariate analysis

OBJECTIVE: To evaluate the diagnostic effectiveness of cytometric features of DNA microdensitometry, karyometry (nuclear morphometry) and maturation and their combinations in separating benign nevi from malignant melanomas. STUDY DESIGN: Tumor cells were measured from each of the superficial, middle and deep zones of 81 melanocytic lesions using video image analysis for nuclear DNA content, chromatin compactness, and nuclear size and shape variables. There were 27 banal compound melanocytic nevi, 20 dysplastic compound nevi, 10 Spitz nevi and 24 malignant melanomas (MM). Maturation of cells with depth into the dermis was also studied by comparing cells from superficial to deep zones. RESULTS: MM showed distinct characteristics of DNA microdensitometry, karyometry and maturation as compared to all groups of benign nevi. There were overall close correlations between nuclear DNA content variables and nuclear size parameters in the total group of 81 lesions. However, there were fewer significant correlations between the various indices in the group of melanomas alone. Using multivariate discriminant analysis, up to 97% of the lesions could be correctly separated as benign or malignant by a combination of five key microdensitometric, karyometric and maturation parameters. CONCLUSION: DNA microdensitometry, karyometry and maturation parameters have independent abilities in identifying individual malignant melanomas. Coevaluation of various cytometric features and maturation profiles offers better diagnostic ability in separating benign nevi from MM.     

Comparative studies of nuclear DNA content in benign and malignant thyroid lesions

Currently available data suggest that DNA aneuploidy is associated with aggressive behavior of and unfavorable prognosis in several malignant human tumors as compared with diploid malignancies. However, the diagnostic and prognostic importance of flow cytometric DNA measurements in the case of thyroid neoplasms remains controversial. Therefore, the aim of our study was to evaluate utility of DNA index (DI) and proliferative index (PI) in distinguishing benign from malignant thyroid lesions taking into account the possible influence of intra-tumor heterogeneity and tissue preparation mode on DNA flow-cytometry measurements. A retrospective study was performed on 71 paraffin-embedded specimens from 57 patients with benign and malignant thyroid pathologies: 13 colloid goitres, 12 parenchymatous goitres, 19 adenomas and 13 carcinomas. In 14 of 57 cases two separate specimens taken from different areas of the same lesion were analysed and DNA parameters were compared. Additionally, flow cytometry DNA analysis was parallelly performed on 3 adjacent but differently processed tissue sections (fresh, formalin-fixed and paraffin-embedded) taken from each of 26 surgically excised thyroid lesions. DNA content was also analysed in both fresh and formalin-fixed twin specimens of normal pig thyroid glands (N = 6). We demonstrated that all tumors diagnosed as thyroid carcinomas were associated with abnormal nuclear DNA content although aneuploidy was not found specific to malignant thyroid tumors. Aneuploid samples of benign thyroid lesions exhibited higher proliferative activity, expressed as mean PI values, than diploid ones. In carcinomas the mean PI values were significantly higher than in benign lesions, independently whether they concerned aneuploid or diploid tissues. Considering intra-tumor heterogeneity, the flow cytometric DNA parameters can be assumed as reproducible despite differences in the mode of tissue fixation and preparation for analysis.     

Flow cytometric determination of DNA content in malignant and benign bone tumours

The nuclear DNA content of 38 malignant and 25 benign bone tumours was measured by flow cytometry. The specimens were taken either from biopsies or from surgical specimens. Seventeen of 26 primary malignant bone tumours were aneuploid, 15 had a single aneuploid DNA content, and 2 had a biclonal abnormality. Thirteen of 15 osteosarcomas were aneuploid, but only 2 of 6 chondrosarcomas showed an aneuploid DNA content. Six of 12 metastatic malignant bone tumours were also aneuploid. All 25 benign tumours had a diploid DNA content. Cell cycle analysis showed that the proportion of cells in S- and G2M-phases was higher in the malignant compared to benign tumours, indicating a higher proliferative activity. The increase was statistically significant (p less than 0.05) both in diploid and in aneuploid tumours. Among five tumours studied after chemotherapy, four displayed a marked hyperdiploid abnormality. Aneuploidy and high proliferative activity both were highly associated with malignant bone tumours, suggesting that DNA flow cytometry may be an adjunct in the assessment of malignancy of bone tumours.     

Prognostic significance of DNA image cytophotometry for osteosarcoma

OBJECTIVE: To investigate the prognostic significance of DNA image cytophotometric data. STUDY DESIGN: Twenty-six osteosarcomas in patients without lung metastases were investigated for several cytophotometric data. In 24 cases, these data were correlated with the clinical course of the patients to assess the prognostic value of nuclear DNA content in osteosarcomas. RESULTS: Of all osteosarcomas, 96% showed aneuploid DNA content. Patients with tumors having a 2c deviation index (2cDI) of 12.00, DNA malignancy grade (DNA-MG) of 2.0, a mean DNA content (MDC) of 4.95 c, DNA index (DI) of 1.75 or mean nuclear area (MNA) of 130 microns 2 had a significantly lower overall survival rate as compared to those with lower values (P < .05). CONCLUSION: Image cytophotometric features, such as 2cDI, DNA-MG, MDC, DI and MNA, are of prognostic value in patients with osteosarcoma and free of lung metastases.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Nuclear protein content and Ki-67 immunoreactivity in nonneoplastic and neoplastic thyroid cells.

The nuclear DNA content in thyroid tumor cells has been shown to be closely related to the malignant potential of the neoplasm. Besides DNA, nuclear protein (NP) constitutes the major mass of the nucleus. The NP content may vary significantly in relation to the proliferative stage in growing as compared to growth-arrested cells. The increase in NP content associated with the transition from G0 to G1 occurs before the onset of DNA synthesis and may be used to assess growth activity. The nuclear DNA and NP contents were analyzed in 90 nonneoplastic lesions and 75 benign and 62 malignant thyroid tumors. All nonneoplastic specimens were euploid, and 1 of 90 was growth activated. In the group of benign tumors, 59 were euploid, and 16 were aneuploid. Among these there were 5 (9%) of 59 and 6 (38%) of 16 growth-activated specimens, respectively. In the group of malignant tumors 57 of 62 were classified as euploid, and in this group 12 (21%) showed growth activity according to the NP content. Five of 62 were aneuploid, and 3 (60%) of these 5 tumors were growth activated. Evaluation of the growth activity by means of monoclonal antibody Ki-67 was performed on a subgroup of 32 thyroid specimens, both nonneoplastic and neoplastic lesions. Ki-67 immunoreactivity was observed in 0-1.1% cells of 6 nonneoplastic lesions, in 0-3.1% cells of 14 benign cells and in 0.2-3.9% cells of 12 malignant thyroid tumors. Growth activity, as reflected by the NP/DNA ratio and Ki-67 immunoreactivity, appears to be low both in nonneoplastic thyroid lesions and thyroid tumors.     

Discrimination between benign and malignant melanocytic skin lesions by multivariate analysis, quantitative S-100 immunohistochemistry, nuclear morphometry and DNA cytometry

OBJECTIVE: To determine whether combined quantitative immunohistochemistry of S-100, nuclear morphometry and DNA image cytometry improves discrimination between benign and malignant melanocytic skin lesions (MSLs). STUDY DESIGN: S-100 protein expression was measured in tissue sections of MSLs using an image cytometry system. Localized areas of high S-100 expression were used to identify regions in sequential, facing sections in which morphometric and cytometric features of nuclei, including DNA ploidy, were also measured. RESULTS: Malignant cases had significantly higher S-100 protein staining intensity, larger nuclei and greater DNA content (P < .05). High staining intensity for S-100 protein weakly correlated with variation in size of the mean nuclear area (P = .04) and DNA content (P = .03). Combining the features of nuclear area and DNA integrated optical density in areas of high-intensity staining for S-100 protein discriminated more accurately between 12 benign and 16 malignant areas than any of the features along (P = .0003). CONCLUSION: Combined multivariate quantitative immunohistochemical, morphometric and DNA cytometric analysis greatly improves discrimination between benign MSLs and malignant melanoma. Larger test sets are required to confirm the promising results of this initial study.     

Cell heterogeneity and subpopulations in solid tumors characterized by simultaneous immunophenotyping and DNA content analysis

BACKGROUND: Heterogeneity in human malignant tumors is a well-described phenomenon and of interest with regard to subpopulations with differences in clonality, metastatic potential, and response to therapy under different treatment regimes. The aim of this study was the simultaneous characterization of surface markers and DNA content of solid tumors to identify tumor cell subpopulations and to study the association between the expression of antigens and DNA content. METHODS: In the present study, six different malignant tumors grown as xenografts in nude mice were characterized by five-parameter flow cytometry. Immunophenotyping was performed using a variety of direct fluorescence-conjugated antibodies. In all cases, simultaneous detection of DNA content was done after staining with 7-aminoactinomycin D. RESULTS: Tumor cells were characterized by light scatter properties, antigen expression, and DNA content. Tumor cell heterogeneity, subpopulations, and DNA content-dependent antigen expression were identified. CONCLUSIONS: This method offers the possibility of characterizing solid tumors according to their immunophenotype and DNA content. The results obtained can be used to identify changes in immunophenotypic and DNA profiles of tumor cell populations before and after therapy and might be useful to define parameters predictive for response to therapy.     

Automated analysis of rat breast tumors. Comparison of four methods

Chemically-induced malignant rat breast tumors pose diagnostic dilemmas since the majority are well-differentiated, noninvasive papillary lesions that are barely distinguishable from benign papillary lesions. This study compared several automated modalities to see which best separated benign from malignant breast tumors. Thirty-three carcinogen-induced rat breast tumors (13 adenomas, 10 papillary carcinomas and 10 invasive carcinomas) were evaluated by static (image) cytometry (ICM) of integrated optical density, by flow cytometry (FCM) and by two automated morphometric protocols, contextual analysis and single-gland analysis. DNA ploidy analysis, by either ICM or FCM, did not discriminate between the benign and malignant tumors. Contextual analysis correctly identified 11 of 13 benign and 17 of 20 malignant lesions (P less than .01). Single-gland analysis correctly identified all 13 benign and 17 of 20 malignant lesions (P less than .01). No method distinguished invasive from noninvasive carcinomas. The data suggest that architectural features are more important than nuclear features in differentiating benign from malignant rat breast tumors.     

NASA/American Cancer Society High-Resolution Flow Cytometry Project - II. Effect of pH and DAPI concentration on dual parametric analysis of DNA/DAPI fluorescence and electronic nuclear volume

BACKGROUND: In the present paper, we describe the effect of 4', 6-diamidino-2-phenylindole (DAPI) dihydrochloride concentration and pH on the resolution of DNA distribution histograms generated by dual-parametric simultaneous analysis of DNA content and electronic nuclear volume (ENV). METHODS: Nuclei from tissue culture cell lines and frozen human solid tumors were isolated in nuclear isolation media containing different concentrations of DAPI, at various pH levels, and analyzed on a NASA/American Cancer Society (ACS) flow cytometer. Samples stained with propidium iodide/hypotonic citrate and analyzed in a Coulter XL flow cytometer were used for comparison. RESULTS: Nuclei stained with DAPI concentration of 1-3 microg/ml, pH 6.0, gave the best resolution for the detection of the near-diploid and near-tetraploid populations. Simultaneous use of ENV and DAPI/DNA fluorescence under these conditions identified subpopulations that otherwise could not be detected by DNA analysis alone. CONCLUSIONS: Staining at 1-3 microg/ml DAPI, pH 6.0, was optimal for the detection of aneuploid populations, especially the near-diploid and/or near-tetraploid populations in human tumors.     

Genome size and metabolic intensity in tetrapods: a tale of two lines

We show the negative link between genome size and metabolic intensity in tetrapods, using the heart index (relative heart mass) as a unified indicator of metabolic intensity in poikilothermal and homeothermal animals. We found two separate regression lines of heart index on genome size for reptiles-birds and amphibians-mammals (the slope of regression is steeper in reptiles-birds). We also show a negative correlation between GC content and nucleosome formation potential in vertebrate DNA, and, consistent with this relationship, a positive correlation between genome GC content and nuclear size (independent of genome size). It is known that there are two separate regression lines of genome GC content on genome size for reptiles-birds and amphibians-mammals: reptiles-birds have the relatively higher GC content (for their genome sizes) compared to amphibians-mammals. Our results suggest uniting all these data into one concept. The slope of negative regression between GC content and nucleosome formation potential is steeper in exons than in non-coding DNA (where nucleosome formation potential is generally higher), which indicates a special role of non-coding DNA for orderly chromatin organization. The chromatin condensation and nuclear size are supposed to be key parameters that accommodate the effects of both genome size and GC content and connect them with metabolic intensity. Our data suggest that the reptilian-birds clade evolved special relationships among these parameters, whereas mammals preserved the amphibian-like relationships. Surprisingly, mammals, although acquiring a more complex general organization, seem to retain certain genome-related properties that are similar to amphibians. At the same time, the slope of regression between nucleosome formation potential and GC content is steeper in poikilothermal than in homeothermal genomes, which suggests that mammals and birds acquired certain common features of genomic organization.     

Ploidy and morphology in osteosarcoma

In a cytometric DNA study of high-grade osteosarcoma, the relationship between DNA content and morphology was analyzed. The investigation, based on microspectrophotometry of tissue sections and flow cytometry (FCM), included both primary lesions and recurrences. FCM analysis, applied to a consecutive series of 47 primary osteosarcomas, disclosed that 2 were diploid and 45 were nondiploid, 8 of which were tetraploid. Multiple aneuploid peaks were detected in 13 tumors. Among the nondiploid tumors, there was no clear relationship between the peak DNA value(s) and the histologic subtype (osteoblastic, chondroblastic, fibroblastic) or grade (III-IV). The proliferative activity, as reflected by the percentage of S-phase cells, could be determined in 38 of the 47 tumors analyzed by FCM. The percentage was higher for aneuploid than for tetraploid lesions; however, the distribution of S-phase cells was not related to the histologic subtype or the grade of the tumors. To assess the reliability of a single sample for FCM, the DNA content of biopsy and surgical specimens was compared in 20 tumors; there was complete agreement in all cases with respect to the classification of the lesion as diploid, tetraploid or aneuploid. Analysis by FCM or microspectrophotometry of 12 local recurrences and 16 metastases and the corresponding 19 primary tumors showed that an aneuploid characteristic of the primary lesion was retained during progression of the disease. In 12 tumors analyzed by microspectrophotometry in tissue sections, comparison of chondroblastic and osteoblastic/fibroblastic areas within the same lesion consistently disclosed hyperploidy in both areas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Algorithm for a DNA-cytophotometric diagnosis and grading of malignancy

An algorithm for processing data on nuclear DNA content obtained cytophotometrically was developed (1) to obtain an objective discrimination between benign and malignant lesions in conventional cytologic smears secondarily stained according to Feulgen and (2) to obtain an objective degree of tumor malignancy on a continuous scale of malignancy grades. Investigations in 258 malignant tumors (95 malignant lymphomas, 52 uterine cervix carcinomas, 28 prostate carcinomas, 18 breast carcinomas, 45 malignant bone tumors and 19 larynx carcinomas) and in 74 benign lesions in these organs yielded a diagnostic accuracy of no false-positive, no false-negative and 21% suspicious diagnoses. The probability that "suspicious" cases were malignant was 81%. The overall diagnostic accuracy for non-negative cases thus amounted to 100%. Results in 95 patients with different malignant lymphomas and in 16 patients with squamous-cell carcinoma of the larynx demonstrated the prognostic validity of the DNA-grading system.     

Comparison between slide and flow cytophotometric DNA measurements in breast tumors

Nuclear DNA analysis was performed in 37 human mammary adenocarcinomas in order to elucidate the difficulties and pitfalls connected with the interpretation of DNA histograms obtained using different methodologic approaches. For each tumor, DNA profiles were obtained by means of slide microspectrophotometry on a fine needle aspirate, slide cytophotometry on a 4-micron histologic section and flow cytometry on a suspension prepared from a cube of fresh tissue. When the DNA histograms were interpreted according to criteria usually applied to discriminate low-grade malignant tumors from high-grade malignant tumors, some tumors classified as euploid by one method were classified as aneuploid by another method. The main reasons for this weak correlation seem to be in specimen preparation and in tumor cell representation within the specimen between the methods. Another reason is that slide and flow techniques exhibit different sensitivities for malignancy-associated nuclear DNA changes: minor alterations of the DNA content of the tumor stemlines seem to be more exactly reported by means of the flow technique whereas structural alterations of the nuclear chromatin seem to be more sensitively recorded by means of the slide technique. It is suggested that thorough control of each step of the various DNA analysis procedures and the use of information obtainable by slide and flow techniques taken together may significantly improve the prognostic value of DNA measurements.     

C-BAND HETEROCHROMATIN AND DNA CONTENT IN ZEA MAYS

The number of mitotic chromosomal C-bands, the percent of the genome comprised of C-band heterochromatin, and genome size (4C DNA content) were determined for 22 North American inbred and open-pollinated lines of Zea mays. The number of C-bands ranged from 0 in Tama Knobless Flint to 18 in Zapolate Grande. The percent C-band heterochromatin ranged from 0% in Tama Knobless Flint to 16.9% in Tx601. Genome size varied over 23%: Gaspe Flint had the lowest DNA content (9.82 pg), and Zapolate Grande had the highest (12.12 pg). Genome size and the amount of heterochromatin were significantly correlated. The corn lines were assigned to five maturity zones encompassing a south-to-north range from Mexico to Canada. Significant negative correlations were detected between the amount of C-band heterochromatin and maturity zones, and between DNA content and maturity zones among the lines. It is speculated that the simultaneous selection by man for earlier maturation and plant size may be related to the lower DNA content of corn varieties adapted to higher latitudes. Such selection for larger plants may have been achieved through selection for more cells, which could result from the shorter mitotic cycle time that correlates with reduced DNA amount.     

Morphometric analysis of AgNORs in tubular and papillary parts of canine mammary gland tumors

OBJECTIVE: To test the value of the silver staining nucleolar organizer regions (AgNORs) technique on canine mammary gland tumors using image analysis and to estimate differences in AgNOR parameters in structurally different parts of canine mammary gland tumors. STUDY DESIGN: Analysis was performed on 13 complex type and 10 simple type malignant canine mammary gland tumors containing tubular and/or papillary structures. Ten normal mammary glands were used as controls. Morphometric analysis was done by a computer-assisted image analysis system and consisted of evaluation of nuclear area, number and area of AgNORs per nuclear area, ratio of nuclei with five or more AgNORs, nuclear perimeter, area fraction between nuclear area and area of AgNORs, and area, equivalent diameter, volume equivalent sphere, perimeter and circularity of a singular AgNOR. RESULTS: Distinct differences were detected between normal and malignant mammary gland tissue for all measured parameters. There were no significant differences between the tubular and papillary parts of the same tumor or between the tubular and papillary parts of complex and simple type tumors. CONCLUSION: Despite the fact that no significant differences were found for AgNOR parameters between papillary and tubular structures of mammary gland tumors, the results of grouping tumors by the number of AgNORs indicate that this might help with classification of canine mammary gland tumors.     

Flow cytometric DNA analysis on fine needle aspiration biopsies of liver lesions

Flow cytometric DNA analysis on fine needle aspiration biopsies of liver lesions The DNA cell content of 39 fine needle aspiration biopsies (FNAs) from five benign liver lesions, nine hepatocellular carcinomas (HCCs), and 25 metastatic tumours was analysed in a prospective fashion by flow cytometry (FCM). All benign lesions were diploid. Aneuploidy was found in five (55.6%) HCCs and in nine (36%) metastatic tumours. DNA index (DI) differences were not significant. The S-phase fraction (SPF) was higher in the malignant tumours, both combined (P < 0.02) and separated primary and metastatic (P < 0.05). We could not demonstrate an association between diploidy and percentage of benign hepatocytes in the smears of malignant tumours. The serum alpha-fetoprotein (AFP) level did not correlate with ploidy, DI, or SPF in the HCCs. In conclusion, ploidy and DI do not discriminate between benign and malignant liver lesions, but the SPF is higher in malignant tumours. DNA analysis does not help to distinguish primary from metastatic liver tumours. The presence of benign hepatocytes in samples from malignant tumours does not seem to influence the analysis of ploidy by FCM.     

Discriminant analysis of image karyometric variables in benign and malignant melanocytic skin lesions

OBJECTIVE: To perform a quantitative analysis to identify which of 7 nuclear morphometry-related variables are of diagnostic value in distinguishing benign from malignant melanocytic skin lesions. STUDY DESIGN: At the Institute of Pathology, University of Nis, formalin-fixed, paraffin-embedded skin biopsies from 23 cases of benign nevi (18 intradermal and 5 junctional) and 25 cases of primary nodular malignant melanomas were retrieved. Specimens were routinely stained with hematoxylin and eosin and analyzed using a computer-assisted interactive image analysis system. Nuclear area, equivalent diameter, volume of equivalent sphere, perimeter, mean chord, circularity and integrated optical density were estimated after manual editing of binary images. RESULTS: In univariate analysis, 6 features were found to be significantly different between the benign and malignant groups (P < .0001); all measured nuclear variables (except circularity) were higher in malignant melanomas. No significant differences were found among lesions with respect to nuclear shape. Using discriminant function analysis, a correct diagnosis was achieved in 95.8% of benign nevi cases and 84.0% of malignant melanoma cases. The best discriminant variable was nuclear area. CONCLUSION: Image analysis is diagnostically relevant to the evaluation of melanocytic lesions of the skin. The area of the nucleus appeared to have potential for differentiating benign from malignant tumors and can be estimated in the course of routine histology.