Nuclear Morphometric Parameters Research Articles

Nuclear morphometry predicts disease-free interval for clinically localized adenocarcinoma of the prostate treated with definitive radiation therapy.

Men treated for prostate cancer often have unexpected outcomes despite predictive models based on stage, grade and prostate-specific antigen (PSA). Previous results have indicated that nuclear morphometry can predict patient outcome in urologic malignancies. Application of this analytical method in prostate cancer treated with radiation therapy is limited. We have evaluated the predictive ability of nuclear morphometry in such patients. Histologic sections from 23 men with clinically localized adenocarcinoma of the prostate treated with radiation therapy were studied. Nuclear morphometric parameters were assessed using a previously described and validated system. Univariate and multivariate logistic regression analyses and a Cox proportional hazards model were used to assess the ability of nuclear morphometric parameters to predict recurrence and disease-free interval. Ten patients had no recurrence with median follow-up of 47. 5 months, while 13 had recurrence. Gleason grade was not predictive of treatment outcome. Pre-treatment PSA data, available for only 11 patients, were predictive of treatment outcome. Several nuclear morphometric parameters predicted recurrence, including upper quartile of suboptimal circle fit and upper quartile of feret-diameter ratio. A prognostic factor score incorporating these 2 parameters was derived, which predicted disease-free interval (p = 0.0014). Int. J. Cancer (Pred. Oncol.) 84:594-597, 1999.

Nuclear morphometry of the myocardial cells as a diagnostic tool in cases of sudden death due to coronary thrombosis

Sudden cardiac death due to underlying coronary artery thrombosis is one of the leading causes of death. However, in a significant percentage of individuals who died suddenly, no indication of myocardial infarction is found during post-mortem examination, especially when the time interval between appearance of symptoms and death is short. In the present study, we have evaluated certain nuclear morphometric parameters, such as, minimum, maximum, mean and standard deviation of perimeter and area in 20 individuals who died of coronary artery thrombosis, within 1 h from symptoms onset. Furthermore, the above parameters were compared with those of a control population of 20 individuals whose sudden death was caused by traffic accidents. Statistical elaboration of the results by means of t-test, Mann-Whitney ( U-test) and analysis of covariance (adjusting for age), showed a statistically significant difference for all variables except for the minimum area. With stepwise discriminant analysis method, the mean perimeter was selected as the best predictor of cardiac death. Mean perimeter achieved a correct reclassification percentage (based on Fisher's linear discriminant function) of 92.5% (85% and 100% for cases and controls, respectively). Moreover, by applying the cut-off of 172 μm, we could identify the individuals who died suddenly because of coronary artery thrombosis with a specificity of 100% (sensitivity 85%, P<0.001). Our results show that nuclear morphometry of the myocardial cells is a reliable diagnostic tool for the diagnosis of coronary thrombosis based lesion in cases of sudden death, even when methods trying to verify the presence of infarction fail to do so.

Nuclear and nucleolar image analysis of human breast epithelial cells transformed by benzo[a]pyrene and transfected with the c-Ha-ras oncogene.

Changes in nuclear and nucleolar morphometric parameters were investigated by image analysis procedures in human breast MCF-10F epithelial cells expressing different stages of the tumourigenic progression after benzo[a]pyrene (BP) transformation (BP1, BP1-E, and BP1-E1 cell lines), and additionally transfected with the c-Ha-ras oncogene (BP1-Tras cell line). Nuclear pleomorphism was evident in all the transformed cells. The analysis of different morphometric parameters did not show a clear relationship between specific nuclear and nucleolar changes and the expression of the different stages of the tumourigenesis, with the exception of the nucleolar size, which could be associated to the expression of the tumourigenic phenotype, and a nucleolar area/nuclear area ratio, which discriminated the immortalized, the transformed, and the tumourigenic phenotypes from one another. The nuclear morphometric data established for the BP-transformed cells and for the cells additionally transfected with the c-Ha-ras oncogene were suggestive of complex and distinct morphofunctional mechanisms involving the in vitro transformation of the MCF-10F cells. The nuclear changes found in the BP1-Tras cell line were assumed to be related to the additional effects and/or enhanced genomic instability induced by transfection with the ras oncogene.

Prognostic value of nuclear morphometry in feline mammary carcinomas

This study of 30 cats with mammary carcinoma was designed to investigate the relationship between (1) six nuclear morphometric parameters (mean nuclear profile area [MNA], standard deviation of MNA [SDa], coefficient of variation of MNA [CVa], nuclear form factor (p2/4 pi area) [FF], standard deviation of FF [SDf], and coefficient of variation of FF [CVf]) assessed by image analysis, and (2) survival for > 1 year or < 1 year after surgical removal of the tumour. Only the SDf and the CVf appeared to be related to survival. Cats that died within 1 year had an SDf or CVf (or both) higher than the corresponding mean values (SDft and CVft) for all 30 cats; but only four of 16 cases (25%) with a SDf lower than SDft and five of 17 cases (29.4%) with a CVf lower than CVft died within 1 year. The authors conclude that SDf and CVf represent reliable prognostic parameters in feline mammary carcinomas.

Human sperm morphometry and smoking, caffeine, and alcohol consumption

The purpose of this study is to determine whether sperm nuclear size, shape, and chromatin texture parameters are associated with lifestyle exposures including smoking, caffeine, and alcohol consumption. Eightysix healthy male volunteers (ages 18–35), recruited through newspaper advertisements, provided a semen, blood, and urine sample and completed a questionnaire concerning demographic and lifestyle exposures. Sperm nuclear size, shape, and chromatin texture parameters were measured using computerized image analysis. Results indicated no associations between the sperm nuclear morphometric parameters and age, smoking, or alcohol consumption. There was weak evidence for an association with caffeine intake. In conclusion, the lifestyle factors smoking, caffeine intake, and alcohol consumption do not appear to significantly affect sperm nuclear size, shape, or chromatin texture in this study population.

Immunohistochemical Expression of p53 Protein, Mitotic Index and Nuclear Morphometry in Primary Malignant Melanoma of the Skin

The immunohistochemical expression of the p53 tumor suppressor protein and the nuclear morphometric parameters were studied in 80 primary skin melanomas. The mitotic index was counted in 64 tumors. In 95% of the tumors p53 positive nuclei were detected, but in only 31% of the cases was the proportion of positive nuclei 3% or higher. The mitotic indices and the mean nuclear areas were not significantly different in melanomas with different p53 expression levels. The p53 positive nuclei had a larger mean nuclear area than the p53 negative nuclei. p53 expression did not increase with melanoma thickness. In Cox's stepwise proportional hazards model, the ratio of mean nuclear long and short axis and melanoma thickness had independent prognostic value, while the level of p53 expression, the mitotic index, and the mean nuclear area were not significantly associated with survival. We conclude that the nuclei expressing p53 protein are larger than p53 negative nuclei. The level of immunohistochemical expression of p53 is low in primary skin melanoma, and it is not valuable as a general prognostic marker for this tumor. p53 expression is not associated with melanoma thickness, indicating that high p53 expression is not a late phenomenon in the progression of this tumor.

P53 quantitative immunocytochemical analysis in breast carcinomas

A series of 200 breast carcinomas was investigated on frozen sections using PAb 1801 p53 monoclonal antibody and streptavidin biotin peroxidase complex. Densitometric analysis of the immunoprecipitates was assessed by processing digitized microscopic images. p53 was observed in the nucleus of 48% of the tumors. Some tumors (14 of 91) tested in parallel on paraffin sections were negative, although positive on frozen sections. Image analysis showed that the surfaces positive with anti-p53 and the staining intensity were decreased ( P < .01) on paraffin sections. The p53 tumor expression was independent of patient age, tumor size, axillary lymph node status, HER-2/ neu and cathepsin D expression, and nuclear morphometric parameters. However, p53 correlated with high histological grade ( P < .01), lack of estrogen receptor (ER) ( P = .0015) and progesterone (PR) ( P = .0065) antigenic sites, pS2 detection ( P = .03), high Ki-67 immunoreactivity ( P = .018), large silver-stained nucleolar organizer region (AgNOR) nuclear surface ratio ( P < .02), and degree of hyperploidy ( P < .03), and was more often observed in the comedocarcinomas. The results suggest that p53 expression in breast carcinomas is not a totally independent prognostic indicator and that the clinical relevance and prognostic significance of p53 expression in breast carcinomas can be reliably assessed provided that the procedures are standardized, particularly with regard to the use of frozen sections and image analysis processing of the immunodetection.

MORPHOMETRIC STUDY OF ADENOMATOID HYPERPLASIA OF THE LIVER BY IMAGE-ANALYSIS TECHNOLOGY

Seven nuclear morphometric parameters in adenomatoid hyperplasia of the liver (AHL), compared with normal liver cells (NLC) and hepatocellular carcinoma (HCC), were analyzed quantitatively by measuring the area (AREA), perimeter (PERIM), maximum diameter (DMAX), minimum diameter (DMIN), the equivalent circle diameter (DCIRCLE), circularity shape factor (FCIRCLE) and shape factor (FSHAPE) using image analysis technology (IAT). HCC could be subclassified into two groups according to the variation degree of FCIRCLE. All of above seven parameters in AHL and HCC were statistically different from NLC; AREA, PERIM, DCIRCLE and FCIRCLE in AHL were different from HCC. The degree of nuclear atypism of AHL was intermediate between NLC and HCC. The results indicate that AHL is not part of normal hepatocytic population morphometrically, but is a group of abnormal liver cells with somewhat nuclear atypia. As AHL has almost the same Value of parameters related to both the nuclear geometry and shapes to those of HCC, especially group 1 of HCC, as well as its frequently malignant transformation, AHL should be considered as an important precancerous lesion of human HCC.

Morphometric Analysis of Melanocytic Tumors

Because the differential diagnosis of melanocytic tumors is sometimes difficult, we performed a morphometric analysis of 20 benign nevi, 20 malignant melanomas, and 10 Spitz's nevi. The nuclear pleomorphism and the maturation to the depth of the tumor were quantified by measuring nuclear area and nuclear density at serial levels of the tumor (every 220 microns of thickness). Data obtained were analyzed statistically. Linear regression was used to represent the variation of nuclear area to the depth, and the standard deviation of nuclear areas reflected nuclear pleomorphism. We attempted to separate different tumors from each other using the Jacknife procedure and morphometric data. Using the Jacknife procedure rather than the usual histological procedures, we were able to distinguish benign nevi from Spitz's nevi and malignant melanoma in as many as 92% of cases, but we distinguished Spitz's nevi from malignant melanoma in only up to 60% of cases. In conclusion, we found it difficult to distinguish between Spitz nevi and melanoma using nuclear morphometric parameters. Other studies using new morphometric parameters are needed in order to improve our ability to make this discrimination.

DIGITAL IMAGE-ANALYSIS OF NUCLEAR MORPHOMETRY, DNA-PLOIDY AND AGNORS IN BREAST-CARCINOMA CELL IMPRINTS

A series (n = 322) of breast carcinomas was investigated from 1991 to 1993 using digital image analysis. Nuclear morphometry and DNA content, and AgNORs were evaluated on cell imprints from fresh tissue samples which were further stored frozen (-80-degrees-C). Data were correlated to morphological prognostic factors and immunocytochemical expression of cell markers assessed on frozen sections and evaluated by densitometry after image analysis processing. Nuclear morphometric parameters, DNA nuclear content and AgNORs were independent from the tumor size, histological grades, and the tumor content of immunodetectable pHER-2/neu, Cathepsin D, ER, PR, pS2, and p53. But, DNA index and hyperploidy degree correlated with the mitosis index (p < 0.01) and Ki67 immunostaining (p = 0.003, p < 0.0001) whereas the shape factor and nucleus large diameter correlated with the degree of cell anisocytosis (p < 0.01). Nuclear surface and large diameter were greater in ductal carcinomas (p = 0.028) and in comedocarcinomas (p < 0.001) than in lobular carcinomas. These results suggest that image analysis processing provides accurate data to refine histoprognostic grading and additional parameters to evaluate tumor proliferative activity.

A Study of Hepatocellular Carcinoma Using Morphometric and Densitometric Image Analysis

Hepatocellular carcinoma is often difficult to diagnose in cytologic material and biopsy specimens. To demonstrate the utility of image analysis in discriminating benign and malignant hepatocytes, 42 malignant cell groups were compared with 26 benign cell groups with a wide range of nuclear morphology in hematoxylin and eosin-stained histologic sections from 42 patients with hepatocellular carcinoma. Nuclear measurements were performed with a relatively inexpensive microcomputer-based image analysis system using a highly flexible imaging software package. Twenty-two nuclear morphometric and densitometric parameters were evaluated. The best single discriminator of benign and malignant cells was the nuclear major axis. Classification of the test samples using optimized linear discriminant functions achieved the following positive predictive values (PV+) and negative predictive values (PV-) for hepatocellular carcinoma: 95.0% PV+ and 85.7% PV- for the major axis; 90.5% PV+ and 84.6% PV- for five densitometric parameters; 100% PV+ and 86.7% PV- for three morphometric parameters; and 95.5% PV+ and 100% PV- for nine combined morphometric/densitometric parameters. These results demonstrate that multivariate linear discriminant functions of nuclear features measured by image analysis can be used to classify benign and malignant hepatocytes accurately.

Nuclear roundness factor and local failure from definitive radiation therapy for prostatic carcinoma

Of 375 patients with prostatic carcinoma treated definitively with radiation therapy at this institution with at least a 5 year follow-up, 23 patients failed locally only, 72 failed with distant metastasis only, 60 had both local and distant failure, while 220 showed no evidence of disease. In search for a possible marker for local failure following radiation therapy, we examined several nuclear morphometric parameters which have been shown to correlate with the biologic aggressiveness of this disease. The 23 locally failed only patients were matched with 23 no evidence of disease patients for stage, grade, treatment modality, prior surgery, age at diagnosis and race. Archival hematoxylin and eosin slides were obtained for 22 of the 23 matched pairs, and morphometric features, including nuclear roundness factor and nuclear area, as well as numbers of nucleoli were assessed using computer-assisted image analysis in both tumor cells and normal prostatic epithelium. Tumor nuclei from the locally failed only patients had significantly higher nuclear roundness factor values ( p = 0.0089) compared with tumor cells from no evidence of disease patients. Analysis of these data by clinical stage demonstrated no significant differences between the locally failed only and no evidence of disease patients. Likewise, there were no significant differences in nuclear roundness factor values of locally failed only and no evidence of disease patients with poorly or moderately well-differentiated tumors. However, there was a highly significant difference ( p = 0.0012) in the nuclear roundness factor values of locally failed only and no evidence of disease patients with well-differentiated tumors. Thus, there appears to be a subset of patients with well-differentiated adenocarcinoma of the prostate who have significantly more irregular tumor nuclei and who fail locally only following definitive radiation therapy.

Correlation of nuclear morphometry and DNA ploidy in rectal cancer

Several investigators have used morphometric measurements to determine differences in the nuclear size and shape of normal and neoplastic colorectal tissue. Changes in nuclear morphometric parameters have also been shown to correlate with prognosis in a variety of noncolorectal cancers. The association of nuclear morphometry with prognostic indicators in rectal cancer has not been well studied. Measurements of the nuclear area, perimeter, longest cord, and circularity factor from 39 primary rectal adenocarcinomas were compared with DNA content, degree of tumor differentiation, Dukes' class, and patient survival. Nuclear circularity was found to correlate with DNA ploidy. Nondiploid tumors with a DNA index greater than 1.3 had significantly more circular nuclei than tumors with diploid or near-diploid DNA content. There was no correlation between nuclear morphometry and Dukes' class or patient survival. Significant increases in DNA content of rectal cancers appear to be reflected by measurable changes in nuclear shape. Nuclear morphometric measurements may provide useful information in the study of the progression of neoplastic changes in colorectal cancer.

Quantitative studies on the DNA content and morphometric features of liver cell dysplasia

The DNA content and morphometric features of hepatocellular carcinoma (HCC) and liver cell dysplasia (LCD), including nuclear area, nuclear perimeter, nuclear maximum diameter and nuclear circle diameter, were quantitatively determined by means of image analysis technology. The results showed that in comparison with normal hepatocytes, LCD had a markedly increased DNA content and nuclear morphometric parameters, but the values were lower than those for HCC. LCD showed a slight increase in nuclear atypia represented by the nuclear irregular index, which was also less than HCC. The findings indicate that LCD may be a precancerous lesion of HCC, to the cells in an abnormal proliferative state.

Nuclear morphometric analysis of non-hodgkin’s lymphoma

Nuclear morphometric data on resin-embedded sections have been utilized to classify 50 cases of non Hodgkin's lymphoma (NHL) and to compare with morphological classification. Nuclear morphometric parameters studied were maximum diameter (Dmax), area, perimeter, form factor (FF) and nuclear contour index (NCI). The first three are determinants of the size, and the latter two indicate the shape of nuclei. Based on Dmax, small (4.5-5.5 microns), intermediate (5.5-7.0 microns) and large (7.0-12.0 microns) cell lymphomas were identified, which correlate with the low, intermediate and high grade lymphomas respectively of the Working Formulation. Determination of form factor further helped to subclassify these cases into lymphomas of B cell other than of follicular centre cell origin (FF = 0.7-1.0), follicular centre cell lymphomas (FF = 0.55-0.75) and T-cell lymphomas (FF less than 0.55). Pleomorphism factor is an objective assessment of the degree of morphological pleomorphism and is generally proportional to the grade of malignancy.

Objective identification of cell cycle phases and subphases by automated image analysis

Frequency distributions of integrated optical density, perimeter, projection, area, form factor, average optical density, and mean dispersion path of nuclear images of Feulgen-stained HeLa S3 cells were obtained by automated image analysis at the base threshold of 0.04 OD. The mean values and standard deviations of these geometric parameters were then computed versus increasing values of threshold (0.08--0.32 OD). There is clear evidence of differential chromatin dispersion and convolution during the cycle of synchronized HeLa S3 cells at different times after selective mitotic detachment. The combination of average OD, form factor, and mean dispersion path at base threshold with the threshold dependence of nuclear morphometric parameters permits objective identification of cell cycle phases and their subphases, by characterizing variations in chromatin geometry within and between phases, regardless of whether DNA content remains constant (early G1, middle G1, late G1), varies only slightly (late G1-early S or late S-G2 transitions), or varies significantly (early S-middle S).