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Nuclear morphometry as a predictor of disease outcome in gastric cancer.

Nuclear morphometric features including nuclear area (NA), nuclear perimeter (PE), their variation (SDNA, SDPE), and mean largest and smallest nuclear diameters (Dmax, Dmin) were examined in 116 patients with stage I-II gastric adenocarcinoma. Morphometric measurements were compared with tumour differentiation, depth of invasion (pT), lymph node status (pN), and Lauren classification. All morphonuclear features were significantly larger in the intestinal type than in the diffuse type of cancer. None of the morphometric variables was related to TNM status or tumour size. Nuclear area and perimeter and their variation were closely related to survival in univariate analysis, patients with small and regular nuclei surviving longer. In the multivariate analysis, pT, pN, perineural invasion, and the standard deviation of nuclear perimeter (SDPE) were independent predictors of survival. Nuclear morphometry is a quantitative, objective, and highly reproducible method of revealing malignant features in several neoplasms. The results of the present study suggest that nuclear morphometric data may help in defining prognosis in gastric cancer.

Adenocarcinoma↗

An image analysis study on nuclear morphology in metastasized and non-metastasized squamous cell carcinomas of the tongue.

In a retrospective case-control study on 46 metastasized and 34 non-metastasized primary tongue carcinomas, the nuclear morphology and chromatin pattern were assessed in 3 microns thick, formalin-fixed, paraffin-embedded, and Feulgen-stained tissue sections of surgical resection specimens, by means of high-resolution computer-assisted image analysis. The aim of this study was to disclose differences in karyometric features, such as nuclear size-, shape-, and chromatin-pattern features, between these groups, with a view to developing a discriminant function that can predict the occurrence of metastasis for the individual patient. In addition, the lymph node metastases of 31 patients and the normal tongue epithelium of 21 patients were also assessed, to study the possible differences between these two groups and primary tumours. In the metastasized tumours, the chromatin was significantly more condensed (P = 0.01) and exhibited significantly less variation in chromatin condensation (P < 0.001) than in the group of non-metastasized carcinomas. Comparison of lymph node metastases with their primary tumours disclosed only minor differences in chromatin pattern. These findings suggest that only minor genetic differences exist between primary tongue carcinomas and their metastases. Tumour cells of tongue carcinomas showed highly significant differences from cells of normal tongue mucosa for most karyometric features. Logistic regression analysis resulted in a classifier, based on the circularity of the nucleus (CIRC) and the standard deviation of the chromatin condensation (SD COND), to predict the occurrence of lymph node metastases. After cross-validation, the percentages of correct classifications in the group of metastasized and non-metastasized tumours were 72 and 62 per cent, respectively. These results are comparable to the classification results obtained from a classifier based on the clinical T-stage, but our karyometric classification results show a much more equal distribution between the sensitivity and specificity. Karyometric features appeared to be more appropriate to predict metastases than biomarkers such as p53, bcl-2, and Ki-67.

Carcinoma, Squamous Cell↗

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.

Adenocarcinoma↗

Deletion mapping of the tumor suppressor locus involved in colorectal cancer on chromosome band 8p21.

Several somatic genetic alterations have been described in colorectal carcinoma (CRC). Recurrent chromosomal deletions have suggested the presence of tumor suppressor genes (TSG) specifically involved in colorectal carcinogenesis. For one of them, two non-overlapping regions have been proposed on the short arm of chromosome 8, encompassing the LPL and NEFL genes. The short arm of chromosome 8 has been extensively studied in colorectal cancer and in other cancer types. Both regions have been reported as candidate loci for a TSG. In order to delineate a reliable region of deletional overlap on chromosome arm 8p in CRC, a series of 365 CRC samples was selected for the absence of microsatellite instability (RER, replication error); tumor and normal matched DNAs were studied for 54 microsatellite polymorphisms distributed on 8p using multiplex-PCR amplification. After purification of tumor nuclei by flow cytometry based on either the abnormal DNA index or the presence of a high expression of cytokeratin, complete allelic losses on 8p were observed in 48% of cases. Measurement of the DNA index showed that 88% of RER tumors were hyperploid. Complete allelic losses of only part of the short arm were observed on 26 occasions. These data allowed us to define a 1 cM interval of common deletion, flanked by the loci D8S1771 and NEFL, where a putative TSG may be localized.

Animals↗

Nuclear profiles of cancer cells reveal the metastatic potential of gastric cancer.

Nuclear profiles have been reported as useful prognostic predictors in various cancers. Data from computerized morphometry are objective and can be quickly derived using conventional microscopic analysis, but image analysis of nuclear features has only rarely been applied to investigations of gastric cancer. The aim of this study was to evaluate the correlation between one of these morphological nuclear features and the clinicopathological parameters in patients with gastric cancer. The morphometric nuclear feature (nuclear area) was analysed in 400 patients with gastric cancer. In each case, 300 cancer nuclei on routine haematoxylin and eosin-stained slides were analysed through the use of a computer-assisted image analysis system which traced the nuclear profiles (magnificationx400) on a computer monitor. The morphometric data were compared with the patients' clinicopathological status and survival rate. The mean nuclear area (NA) of cancer cells from 400 cases of gastric cancer was 47.2 microm(2). The NAs of cancer cells from tumours with microvessel invasion (lymphatic or venous invasion), lymph node metastasis or hepatic metastasis at the time of operation were significantly larger than those of cancer cells from tumours without such invasion or metastases. Cytokeratin (CK) immunostaining was performed on 2577 lymph nodes from 91 patients with advanced gastric cancer (pT3, pN0, pM0, stage II) to detect micrometastases. CK-positive lymph nodes were detected in 350 of 2577 lymph nodes (13. 6%) and in 62 of 91 patients (68.1%). The mean NA of cancer cells from 62 tumours with micrometastases (44 microm(2)) was larger than that of cancer cells from 29 tumours without micrometastases (38.8 microm(2), p=0.043), and a significant positive correlation was detected between the NAs of cancer cells from 91 tumours and the number of micrometastatic lymph nodes of 91 patients (rho=0.278, p=0. 008). Cancer cells with large NA correlated strongly with haematogenous and lymph node recurrence or relapse after gastrectomy and the NA of cancer cells was identified as an independent prognostic factor in gastric cancer. Nuclear morphometry is an objective, reproducible, and technically uncomplicated procedure. The NA of cancer cells correlates closely with the metastatic potential of gastric cancer. Nuclear morphometry may therefore be useful for the selection of patients who are at risk of haematogenous or lymph node metastatic recurrence after surgery.

Adenocarcinoma↗

Nuclear cytometric changes in breast carcinogenesis.

Breast cancer is thought to originate through progressively aberrant precursor lesions, paralleled by increasing morphological changes. The aim of this study was to quantify nuclear features by image cytometry in invasive breast cancer and its early (hyperplasia) and late (ductal carcinoma in situ) precursor lesions, in order to objectively describe nuclear changes in the spectrum of proliferative intraductal and invasive breast lesions. Image cytometry was performed on tissue sections of 20 samples of normal breast tissue, 71 of usual ductal hyperplasia (UDH), nine of atypical ductal hyperplasia (ADH), and 11 of well-differentiated and 13 of poorly differentiated ductal carcinoma in situ (DCIS) lesions. The invasive breast carcinomas consisted of 19 well-differentiated and 24 poorly differentiated lesions. Through the spectrum from normal breast tissue to invasive carcinoma, progressive changes in many nuclear features were measured. Significant differences were found between nuclei of florid ductal hyperplasia compared with mild and moderate ductal hyperplastic lesions, suggesting that florid ductal hyperplasia may be a more advanced lesion than assumed and may contain cancer precursor cells. No differences were found between ADH and well-differentiated DCIS, suggesting that these lesions are closely related. Feature values of well-differentiated DCIS were comparable to values found in well-differentiated invasive carcinoma and the same applied to poorly differentiated DCIS and invasive lesions. These results support the hypothesis that breast cancer develops through different routes of progression, one leading to well-differentiated invasive cancer through well-differentiated DCIS, and one leading to poorly differentiated invasive cancer through poorly differentiated DCIS. In conclusion, image cytometry reveals progressive changes in nuclear morphological and subvisual chromatin distribution features in the spectrum from intraductal proliferations to invasive breast cancer. This provides evidence for a progression from usual to atypical ductal hyperplasia and then to invasive cancer, through different routes for well-differentiated and poorly differentiated lesions.

Breast↗

Prognostic value of karyometric and clinical characteristics in renal cell carcinoma. Quantitative assessment of tumor heterogeneity.

BACKGROUND: The variation in tumor cell differentiation within one renal cell carcinoma, also termed tumor heterogeneity, renders visual tumor grading of these carcinomas difficult. Karyometric analysis enables description of nuclear characteristics of multiple tumor areas. Hence, karyometric analysis can be used to quantify tumor heterogeneity and thus may aid in a more objective grading of renal cell carcinoma. METHODS: In 121 patients with renal cell carcinoma (tumors in International Union Against Cancer [UICC] stages I [5 cases], II [23 cases], III [33 cases], and IV [60 cases]), clinical and karyometric features were studied to obtain routinely applicable prognostic factors. Several parts of the tumor were analyzed to obtain a measure of tumor heterogeneity. Univariate and multivariate Cox regression analyses were used to determine the predictive value of karyometric features independent of tumor stage and other clinical characteristics. RESULTS: The Cox univariate regression analysis showed correlation of several clinical and karyometric characteristics with survival. Of the clinical characteristics, TNM stage, tumor size, weight reduction, and performance status were significantly associated with survival. The karyometric features, especially those measurements associated with tumor heterogeneity (e.g. differences in nuclear size or chromatin texture between tumor subpopulations) were of value in predicting prognosis. In the Cox multivariate regression analysis, the Robson and UICC stages proved to be the most powerful predictors of survival (P < 0.0001). Of the clinical features, weight reduction and performance score were the only characteristics offering additional information regarding tumor stage (P < 0.0001). From the karyometric analysis quantification of anisokaryosis in the tumor at time of diagnosis offered additional prognostic information. Moreover, the differences of karyometric features within the tumor presumably associated with tumor heterogeneity correlated with survival. Using the features from the multivariate analysis, prognostic groups could be defined. CONCLUSION: We conclude that karyometric analysis offers a useful means for quantifying tumor heterogeneity. Multivariate Cox analysis revealed additional value of a grading system based on karyometric analysis to tumor stage. Karyometric analysis can be a useful tool for stratification of patient populations.

Adult↗

Objective malignancy grading of squamous cell carcinoma of the lung. Stereologic estimates of mean nuclear size are of prognostic value, independent of clinical stage of disease.

BACKGROUND: The prognostic value of quantitative histopathologic parameters was evaluated in 55 consecutively treated patients with operable lung carcinoma of squamous (N = 39) and mixed, adenosquamous (N = 16) cell type. Patients alive were followed for at least 12 years. METHODS: Using a projection microscope and a simple test system in fields of vision systematically selected from the whole tumor area of one routine section, five quantitative histopathologic variables were estimated: the mean nuclear volume, the mean nuclear profile area, the density of nuclear profiles, the volume fraction of nuclei to tissue, and the number of mitotic profiles per 10(3) nuclear profiles. For each patient, information was recorded regarding sex, age at diagnosis, and clinical stage of disease. RESULTS: Single-factor analyses showed that a favorable prognosis was associated with early clinical stages (Stages I and II) and young age (P < or = 0.03), and that females tended to do better than males (P = 0.09). Estimates of the mean nuclear volume were of prognostic significance (P = 0.02), small nuclei being associated with the worst prognosis. In a multivariate Cox analysis, clinical stage, age, and mean nuclear volume were found to be parameters of significant, independent prognostic value. CONCLUSIONS: The present feasibility study indicates that estimates of the mean nuclear volume are of prognostic value, independent of the clinical stage of disease. This quantitative histopathologic variable is highly reproducible and easily obtained using an unbiased stereologic method. Thus, the mean nuclear volume may be a parameter of future importance in the clinical management of patients with carcinoma of the lung.

Aged↗

Thyroid carcinoma diagnosis based on a set of karyometric parameters of follicular cells.

BACKGROUND: In the works of various authors, performed with the help of morphometry, an assessment of cell changes was carried out, as a rule, by one sign of their atypia (e.g. area of cell nuclei). Moreover, the selective analysis of morphometric data for certain nosologic forms was performed. Such an approach allowed researchers to develop only additional diagnostic criteria of thyroid carcinoma. At the same time, the use of a set of quantitative signs of cell atypia as a criterion for tumor malignancy made it possible to improve oncologic diagnosis. In this study, which is a first step in the development of a quantitative approach to thyroid carcinoma diagnosis, a system of karyometric parameters was developed with the help of morphometry of cytologic material of patients with major nosologic forms of thyroid pathology (papillary and follicular carcinoma, follicular adenoma, autoimmune thyroiditis, and nodular colloid and diffuse toxic goiter). The significance of each parameter in thyroid carcinoma diagnosis was evaluated. METHODS: Thyroid cells of autopsy, operative, and aspiration biopsy material were the object of the study. One hundred fifty-two preparations with histologically verified diagnosis were studied. Light morphometry was performed with the help of a semiautomatic computer analyzer of images. One hundred fifty thyroid nuclei were assessed in each preparation. RESULTS: Among many karyometric parameters, six were selected: mean area of thyrocyte nuclei, ratio of frequencies, and number of classes on histograms of distribution of their area, coefficient and a free term of the regression equation to a scattered diagram of perimeter and nuclear area, and the discriminant index. Boundary values of these parameters between the malignant and benign pathology were united into an expert system for thyroid carcinoma diagnosis. Clinical trials of the system were performed. On the basis of the obtained results, the informativity (accuracy, sensitivity, specificity) of its karyometric parameters was assessed. The formula for calculation of the weighting coefficient for each parameter was derived, the diagnostic index being determined by the sum of their values. CONCLUSIONS: An expert system was created based on a set of karyometric parameters reflecting the regularities of pathologic changes in thyrocyte nuclei with malignant and benign pathology. The informativity and weighting coefficients included into the system of parameters have been assessed, which made it possible to perform the probabilistic diagnosis of thyroid carcinoma.

Adenoma↗

Estimation of cell numbers and volumes of bronchiolar epithelium during rabbit lung maturation.

To estimate the numbers and volumes of bronchiolar epithelial cells during lung maturation, we examined rabbits at three time points, 30 days gestation and 4 and 17 weeks postnatal age. Morphometric measures (mean caliper diameter, surface area, and volume) of nonciliated and ciliated bronchiolar cell nuclei, using computer modeling from serial sections, showed a significant decrease in nuclear size for both cell types and a significant increase in cell volume for the nonciliated bronchiolar cell during lung maturation. A shape coefficient (beta) proved to be the most efficient estimator of the number of cells per unit volume when it was used with estimates of the number of nuclei per unit area and the volumetric density of nuclei. Two-dimensional estimates of bronchiolar epithelial cell abundance (the number of nuclei per unit length or area) significantly underestimated the percentage of nonciliated bronchiolar cells as compared to three-dimensional estimates for rabbits 17 weeks of age. We have shown an inverse relationship between nonciliated and ciliated bronchiolar cell abundance during lung maturation. Nonciliated cells decreased while ciliated cells increased. We have confirmed that cytodifferentiation of the nonciliated bronchiolar cell occurs within the first 4 weeks of postnatal development. The volume of the nonciliated bronchiolar cell increased about twofold during development. Because of the concomitant decrease in nuclear volume, the cytoplasm of the cell showed an even greater increase in volume. Within the cytoplasm of the nonciliated bronchiolar cell, glycogen significantly decreased, and agranular endoplasmic reticulum (AER) and mitochondria significantly increased in volume during development. The biosynthesis of AER closely correlated with pharmacological studies of xenobiotic metabolism during rabbit lung maturation.

Animals↗

Immunocytochemical study using a GABA antiserum for the demonstration of inhibitory neurons in the rat locus ceruleus.

The localization of GABA-like immunoreactivity in the locus ceruleus of rats was studied by the peroxidase-antiperoxidase (PAP) method using a purified antibody raised against GABA applied to paraffin sections, with counterstaining by cresylecht violet, and to floating sections for preembedding immunoelectron microscopy. A few medium-sized and some small neurons showed GABA-like immunoreactivity in both nuclei and perikarya. The preferential localization of these immunopositive neurons in the marginal parts of the locus ceruleus suggests that they are inhibitory local circuit neurons located between this center and the afferent fiber systems. Some of the immunoreactive neurons displayed homogeneous and heterogeneous "paired cells" patterns. Occurrence of the GABA-GABA interaction is indicated. Immunopositive bouton forms are located close to every positive and negative neuron. Electron microscopy confirms GABA-like immunoreactivity in both medium-sized and small neurons of the locus ceruleus and demonstrates that immunoreactive boutons are axosomatic and axosoma spine symmetric synapses on immunopositive and immunonegative cell bodies. These immunocytochemical results support the existence of inhibitory interneurons in the locus ceruleus.

Animals↗

Molecular and cytogenetic characterization of 9p- abnormalities.

We report on 2 girls with terminal deletion of the short arm of chromosome 9 with concurrent duplication unrecognizable by routine chromosome studies. The phenotype of the patients was not specifically suggestive of the 9p-syndrome in the absence of trigonocephaly and long philtrum as cardinal manifestations. In addition to psychomotor retardation, their manifestations were mild and include upward slant of palpebral fissures and dolichomesophalangy which are characteristic of del(9p). Chromosome abnormalities were de novo in both cases. The two rearranged chromosomes 9 exhibit similar G-banding patterns and suggested the possible duplication of distal 7p. Fluorescence in situ hybridization (FISH) with a chromosome-7 specific library probe indeed identified that one derivative chromosome 9 was the result of a translocation between chromosomes 7 and 9 [der(9)t(7;9)(p15.3;p24] but failed to detect a signal on the other derivative 9. In the second case, the concurrent abnormality was an inverted duplication of proximal 9p and deletion of distal 9p [inv dup(9)(p13-->p22::p22-->qter)] confirmed by FISH using a chromosome 9 specific library probe. FISH clearly identified the origin of these 2 abnormal chromosomes 9 and provided crucial information for clinical evaluation. We emphasize the importance of utilizing updated cytogenetic and molecular techniques in the precise delineation of subtle or complex abnormalities where there are no useful phenotypic clues.

Abnormalities, Multiple↗