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Nuclear morphometry as a predictor of response to therapy in Wilms tumor: a preliminary report.

Present therapy in Wilms tumor is based upon prognosis. Current trends suggest less intensive chemotherapy and/or radiation therapy for children with favorable histological disease who are considered at low risk for recurrence and increasing the amount of therapy for those with unfavorable histological tumors who are at high risk for recurrence. Currently, pathological stage and histological appearance are used to predict prognosis. We tested a new approach. Histological sections of surgical specimens were obtained from 9 children treated for unilateral Wilms tumor. All patients had favorable histological disease, and received postoperative chemotherapy and/or radiotherapy. These specimens were evaluated by nuclear morphometry with the Hopkins Morphometry System to determine if nuclear shape descriptors could distinguish children who responded to therapy from those whose tumor recurred. Nuclear roundness, ellipticity, convexity and bending energy accurately separated the groups. Nuclear morphometry may provide useful information that will aid in predicting which children with Wilms tumor will respond to therapy and warrants further investigations.

Cell Nucleus

Light and electron microscopical morphometry of pituitary adenomas in hyperprolactinemia.

Two highly differentiated acidophil prolactin-cell adenomas with hyperprolactinemia (group I), 8 large cell chromophobe adenomas with hyperprolactinemia (group II), and 2 small cell chromophobe adenomas (group III), one of which was combined with hyperprolactinemia, were studied immunohistologically. Morphometry was performed on the light- and electron microscopical level. The 11 active adenomas were immunohistologically positive for prolactin, the 12th adenoma with normal prolactin plasma level was negative for prolactin. Light microscopical morphometry displayed significantly more cells of smaller size in the "small cell chromophobe" adenomas, whereas the large cell chromophobe adenomas and the highly differentiated prolactin cell adenomas were not different. Ultrastructural morphometry demonstrated significant differences between highly differentiated prolactin cell adenomas (group I), and large cell chromophobe adenomas (group II). The latter contain smaller "relative volumes" of nucleoli and of secretory granules, whereas the rough endoplasmic reticulum, the Golgi fields and the nuclei were not different. Comparison of large cell chromophobe adenomas (group II), and small cell chromophobe adenomas (group III) revealed significantly larger relative volumes of nuclei and of mitochondria but smaller volumes of rough endoplasmic reticulum and of Golgi fields in the small cell chromophobe adenomas. Significant differences between the active and the inactive adenoma of small cell chromophobe type in the group III were not found. In spite of the low quantity of small cell chromophobe adenomas and of acidophil prolactin cell adenomas, our data demonstrate that there exist distinct and significant light microscopical and ultrastructural differences between the three adenoma types.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoma, Acidophil

Morphometry for the prognosis of acute lymphoblastic leukemia in childhood.

The morphology of acute lymphoblastic leukemia is of prognostic significance, but due to the qualitative subjective nature of the assessment, disagreement between pathologists may be considerable. In order to investigate the adjuvant potential of morphometry, the present study was undertaken. Morphometry was applied in 21 children between 2 and 10 years of age with acute lymphoblastic leukemia (ALL) with a follow-up of at least 5 years. Of these, 9 patients died, 12 (57.1%) survived for at least five years (maximally 12 years). Morphometry appeared to have an important prognostic value, independent of the FAB classification currently in use. Combination of the nuclear-cell area ratio and the cell area gave a rather good discrimination, although these quantitative data from a morphological continuum. The positive results of this pilot study point to the necessity of evaluating the data on a larger material.

Cell Nucleus

Intestinal mucosal morphometry and ileal epithelial renewal in conventional and germ-free rats fed an amylomaize starch diet.

Intestinal mucosal morphometry and ileal epithelial renewal were studied in conventional (CV) and germ-free (GF) rats fed either poorly digestible amylomaize or normal maize starch diets. Intestinal morphometry and position of labelled enterocytes were studied at various times after tritiated thymidine injection. With amylomaize starch diet, no difference was observed in the size of crypts (C), villi (V) and C + V between duodenum and jejunum both in CV and GF rats. In the ileum, however, values were significantly lower than those in the duodenum and jejunum. Furthermore, the presence of the microbial flora led to higher values when compared with GF values. Despite the morphological modifications in the ileum, no significant difference was detected in the labelled cell positions and epithelial renewal time between CV and GF values. This suggests that the resistant part of amylomaize starch was responsible for the modification in mucosal morphometry and the longer ileal epithelium renewal time in CV rats which then becomes similar to that in GF rats.

Amylopectin

The relative prognostic significance of nucleolar morphometry in invasive ductal breast cancer.

In a group of 65 patients with invasive ductal breast cancer with adequate follow-up for at least 5 years, the prognostic significance of nucleolar area morphometry was investigated. In general, non-survivors have larger and more pleomorphic nucleoli. A value of the standard deviation of the nucleolar area above 2.49 micron2 was almost exclusively found in non-survivors. This is the more interesting because several of these patients had a low mitotic activity index and small tumours, some of these having negative lymph nodes. Without nucleolar morphometry these patients would have been included in a favourable prognosis group. Although the sensitivity of nucleolar morphometry is low, the method can be used as an additional prognostic indicator. This is more important as nucleoli can in principle be automatically measured with digital image processing computers.

Breast Neoplasms

Morphometry of orthopantomographic mandibular bone changes during radiation therapy.

It is generally considered that mineral loss in the bone must amount to 30 per cent or more before any changes become visible to the naked eye in conventional radiography of the skeleton. Early changes in the mandibular bone during radiation therapy are therefore difficult to detect. Abnormalities of the bone caused by irradiation are vascular and cellular in nature. Morphometry detects the changes earlier than conventional radiography. Nineteen patients with a malignant process in the oral cavity or the naso- or hypopharynx but with no evidence of metastases in the mandibular periosteum or bone marrow were given radiation therapy. They were investigated using morphometry together with orthopantomography. The investigation demonstrated a decrease in the number of trabeculae and an increase in the amount of marrow space and compact bone during irradiation. Thus it is possible to detect early radiographic changes by morphometry, but this method is more laborious than scintigraphy. The abnormalities of the mandibular bone were reversible, which signifies the recovery of bone after irradiation with therapeutic doses.

Adult

Respiratory muscle fiber morphometry. Correlation with pulmonary function and nutrition.

To examine the relationship between nutrition, pulmonary function, respiratory muscle strength, and respiratory muscle morphometry, we compared physiologic data and muscle morphometry obtained from internal intercostal, external intercostal, and latissimus dorsi muscle biopsies in 68 patients who were having a thoracotomy. We stained the biopsies for myosin ATPase and measured the proportions and diameters of the type 1 and type 2 fibers. There were more qualitative changes in the external intercostal muscles than in the other two, and some of these changes related to the incidence of malignancy. There were more type 1 fibers in the external intercostal (64 +/- 10 percent) and internal intercostal muscles (59 +/- 12 percent) than in the latissimus dorsi (44 +/- 13 percent) (p less than 0.005). The mean diameter of the type 2 fibers in the external intercostal muscles was less (44 mu +/- 7 mu) than the diameter in the latissimus dorsi (51 mu +/- 9 mu) and the internal intercostal muscles (52 mu +/- 8 mu) (p less than 0.01). The diameters of both fiber types were greater in men than in women. There was no significant relationship between measures of pulmonary function or respiratory muscle strength and muscle fiber proportions and diameters. There were significant correlations between the percentage of ideal body weight and type 1 and type 2 fiber diameters. We conclude that sex and nutrition influence respiratory muscle morphometry.

Biopsy

Multiparametric discrimination of serous ovarian tumors by analytical morphometry.

In order to enhance the discrimination power in the field of serous ovarian tumors, we applied the software system SAM (Shape Analytical Morphometry) to the analytic studies of biological forms. Besides the usual dimensional evaluations (perimeter, area, maximum diameter and shape index), this procedure permits the description of the nuclear form using analytical parameters: 1) extraction of nucleus fundamental curve; that is a functional curve giving the "smoothing" of the original contour by two parametric equations (separately for x and y values as independent variables); 2) evaluation of nuclei contour irregularities by Fourier analysis; 3) evaluation of shape asymmetry by SAE (Shape Asymmetry Evaluator); that is the ratio between the length of a segment of a parabola interpolating the original curve points, and a straight line joining its extremities for a 180 degrees barycentric rotation according 10 degrees steps. All parameters resulted to be independent and were submitted to multivariate discriminant analysis. We studied 180 nuclei from 18 cases of serous ovarian tumors, (6 benign, 6 borderline and 6 malignant tumors). With respect to the dimensional parameters, the application of analytical morphometry permitted us to reduce the minimum percentage error in the discrimination of the different classes. In fact, in the distinctions of benign and malignant nuclei, the minimum percentage error was 13.30%, against the 18.3% error when using dimensional morphometry. Furthermore, in the comparison of malignant and borderline nuclei there was a reduction of error from 23.3% to 22.5%, and in the comparison of benign and borderline nuclei, the error was reduced from 37.5% to 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Nucleus

A longitudinal study of metacarpal bone morphometry in anorexia nervosa.

Osteoporosis is a known complication of anorexia nervosa. Although calorie and mineral malnutrition may contribute to changes in bone mass and morphometry, hypoestrogenism is thought to be the most important etiologic factor. In a seven-year longitudinal study of six women aged 19 to 35 years with adolescent-onset anorexia nervosa, the objective was to correlate menstruation and bone morphometry. At the onset of the study, five women were amenorrheic and had abnormal metacarpal bone morphometry. After seven years, three women remained amenorrheic and below 85% of ideal body weight. Anteroposterior roentgenographs of the nondominant second metacarpal taken at the beginning and end of the study revealed an increase in medullary canal diameter (p less than 0.03) and medullary area (p less than 0.04) and a decrease in combined cortical thickness (p less than 0.04) and percent cortical area (p less than 0.02). These findings suggest progressive endosteal resorption in the absence of compensatory periosteal apposition. Such bone remodeling characteristics are distinctly abnormal in this age group. The three women who regained menses showed up to one third less endosteal resorption and less cortical thinning than did the three women who remained amenorrheic. Resumption of menses may be an important milestone in preventing further cortical bone loss in anorexia nervosa.

Adult

Some fundamental aspects of morphometry in clinical pathology, demonstrated on a simple, multipurpose analysis system.

The aim of this study was (1) to investigate the value of morphometry, (2) to fix a set of parameters suitable for analyzing diagnostic problems, and (3) to create a general strategy for data storage and for user-friendly data management. The intrinsic value of morphometry lies in the fact that in contrast to other morphologic methods, it permits the presentation of findings in the form of numbers. The following set of morphometric parameters, in the broad sense of the term morphometry, is standard in our laboratory: planimetric parameters (shape descriptors), parameters of the gray value histogram (descriptors of the general gray value distribution), texture parameters (descriptors of the correlation between various image segments), invariant moments (descriptors of the size and localization of textural image segments) and densitometric parameters. The introduction of morphometric procedures into the daily routine is facilitated if data registration and evaluation are performed separately. Original data generated by direct measurement are primary or raw data, which are stored as such. In a separate, second step these raw data are used to compare more or less complex morphometric parameters, which are called "secondary data". A system designed for separate data registration and evaluation can easily be adapted to new methodologic developments. For instance, primary data on objects (gray values, coordinates of the contour) measured one time in the past can be reused at any other time for computing new features from these data. This procedure is comparable to the possibilities in immunohistochemical staining: new immunohistochemical stains can be applied to newly prepared sections of old tissue blocks.

Computer Communication Networks

Comparison of conventional morphometry and image analysis for the solution of histomorphometric problems.

The main differences between conventional morphometry and image analysis for their application in histopathology are pointed out. Conventional morphometry allows the use of contextural informations and a priori knowledge in a convenient manner. This is the most complicated thing in image analysis. The solution of this problem lies on the field of artificial intelligence. On the other hand, image analysis is able to measure densitometrical and textural features, to determine arbitrary quantiles and moments of feature distributions, to match scenes. Therefore the descriptive power of image analysis is much higher than the power of conventional morphometry.

Humans

Morphometry and immunocytochemistry.

The use of morphometry (and stereology), especially in conjunction with immunocytochemistry, in surgical and experimental pathology is reviewed. The combined use of morphometry and immunocytochemistry permits the study of secretory products and the distinction between cells that produce them, e.g., as in the pancreas. Several examples to illustrate the application of immunocytochemical techniques in morphometry are presented. For example, using this approach on the pancreas, it was shown that not only B cells, but also A and D cells, seem to undergo pathologic changes in a prediabetic organism. Since morphologic alterations may be distributional as well as quantitative, a model for the assessment and statistical evaluation of the distribution of objects within an area is discussed. Similar procedures were used to analyze the distribution of cytochrome P-450 molecules along intracellular membranes; however, special labeling techniques were necessary for this quantification in the electron microscope. Immunoenzyme cytochemically stained secretions can also be studied morphometrically, and microdensitometry and microfluorometry can also be used to quantify immunocytochemical reactions, as is shown in the analysis of the intralobular distribution of NADPH-cytochrome P-450 reductase in the rat liver. Finally, the BIVAS semiautomatic system for morphometric measurements, developed at the Department of Pathology of the University of Basel, is briefly described.

Animals

Electron microscopic morphometry.

The fundamental concepts of morphometry, the principal correction factors for systematic errors and the basic principles of efficient sampling are outlined for quantitative morphology at the electron microscopic level of resolution. The important usefulness of correlating electron microscopic morphometry with complementary light microscopic morphometry is emphasized.

Animals

The adjuvant prognostic value of nuclear morphometry in stage I malignant melanoma of the skin. A multivariate analysis.

In order to evaluate the adjuvant prognostic value to tumor thickness of other clinical, qualitative histologic and morphometric features, a multivariate analysis was performed in 68 patients with stage I cutaneous malignant melanoma and at least five years of follow-up. Stepwise linear regression analysis confirmed that tumor thickness is the most important prognostic factor; the only additional significant feature was the nuclear correlation coefficient (NCC), which is a measure for the roundness versus ellipsoidicity, regularity and homogeneity of the shape of the nuclei in tissue sections. In general, tumors with ellipsoid (spindle-shaped) nuclei were associated with a better prognosis, even in thick tumors, and tumors with predominantly round nuclei were associated with a poorer prognosis. If the NCC was below 0.94, the prognosis was good irrespective of the tumor thickness: all patients with such a low NCC have survived. Further analysis of this feature showed it to be strongly correlated with the nuclear axes ratio (longest axis divided by the shortest axis). The subtlety of the differences in nuclear shape makes it practically impossible to subjectively detect the significant prognostic level of ellipsoidity, especially in borderline cases. The advantage of the application of morphometry is that it allows the objective description of subjective impressions, and minor shape differences can be detected. The significant adjuvant prognostic value of nuclear morphometry to tumor thickness makes it worthwhile to consider these features together in future prognostic studies concerning malignant melanoma. Furthermore, inclusion of nuclear shape morphometry in clinical pathology reports seems advisable.

Analysis of Variance

[In vitro semi-automated morphometry of preserved human corneal endothelium].

Cellular morphometry allows us to quantify the structural alterations of the corneal endothelium. Our study was a semi-automatized in vitro analysis of fresh and preserved corneal endothelium (MK medium cryopreservation). The equipment used was the Quantimeter 720 connected to a Digital PDP1123 computer. Nineteen corneas were studied and the following cellular parameters were established: perimeter, surface area, form factor and polymorphism coefficient. The computer program used was the SPSS/PC (Statistical Package for the Social Sciences) and the principal statistical tests were the Bartlett test and variance analysis. The statistical computerized study allowed us to compare the four cellular parameters and to establish the incidence rate of the conservation process on endothelial morphometry. The statistical and morphometry methods have proved to be reproducible and reliable and carry a supplementary quantitative element to analyse the influence of the conservation process.

Adult

Theoretical aspects of diagnostic histopathology and their relation to morphometry.

The theoretical aspects of diagnostic histopathology are discussed and their relationship to morphometry is examined. In diagnostic pathology, one tends to look at certain structural features, overlooking other details regarded as unimportant. In fact, the pathologist is continuously reducing the images under observation into a new simplified reality, based on a theoretical model (concept). The model is composed of features in a structural interrelation that is thought to be specific for the pathologic diagnosis being considered. The pathologist also selects appropriate regions for study in order to enhance the possible successful application of the appropriate criteria for the given diagnosis; the diversity of different tissues within, between and at different levels requires a vast experience in order to select the appropriate criteria in a specific situation. Analytical histopathology and morphometry require a more conscious construction of a model comprising the concept of the pathologic process and the criteria for its diagnosis. The theoretical background subconsciously used in the pathologist's daily practice is thus the essential knowledge for the application of morphometry in diagnostic histopathology, the basis on which elements for measurement or counting and on which compartments for calculation are selected.

Biopsy

The effect of anorexia nervosa on bone morphometry in young women.

Changes in bone morphometry during chronic undernutrition were evaluated in 14 young women with anorexia nervosa (mean age +/- SEM = 25.5 +/- 4.4 yrs). Bone morphometry studies using the second metacarpal of the left hand showed significant depression for percent cortical area (p less than 0.05); cortical area (p less than 0.001) and combined cortical thickness (p less than 0.01) as compared to age, sex and race matched controls. A trend (p less than 0.10) was observed in study subjects for reductions in bone width and total area. Using percent cortical area (PCA) as the standard, subjects had mean cortical bone morphometry equivalent to 60-year-old women. Appendicular bone mass is significantly decreased in adults with anorexia nervosa. Anorexia nervosa should be considered in the differential diagnosis of osteopenia in young women, and serves as a model for studying the effects of chronic calorie and mineral malnutrition on bone remodeling at the time of attainment of peak bone mass.

Adult

The value of morphometry to classic prognosticators in breast cancer.

In 271 breast cancer patients with adequate follow-up for at least 5.5 and maximally 12 years, the value of morphometry to classic prognosticators of breast cancer (tumor size and axillary lymph node status) was assessed. Previous studies had indicated the value of this quantitative microscopic technique. Apart from quantitative microscopic features, subjective qualitative features such as nuclear and histologic grade were assessed as well. Univariate life-table analysis showed the significance (p less than 0.001) of several features such as lymph node status, tumor size, nuclear and histologic grade, and several morphometric variables (mitotic activity index, mean and standard deviation of nuclear area). Cellularity index was also significant (p = 0.02). Survival analysis with Cox's regression model, using a stepwise selection as well as backwards elimination, pointed to three features: mitotic activity index, tumor size, and lymph node status. Mitotic activity was the most important prognostic feature, but the combination of these three features in a multivariate prognostic index had even more prognostic significance. Kaplan-Meier curves showed that the 5-year survival of lymph node-negative patients (n = 146) is 85%, versus 93% in patients with a "good prognosis index" (n = 150). For lymph node-positive patients (n = 125), 5-year survival was 55%, compared with 47% in the "high index" (poor prognosis) patients (n = 121). Logistic discriminant analysis with 5.5-year follow-up as a fixed endpoint (191 survivors and 80 nonsurvivors) essentially gave the same results. Application of two instead of one decision threshold (e.g., numerical classification probability 0.60 and 0.40) decrease the number of false-negative and false-positive outcomes, however, with a number of patients falling in the class "uncertain." Thus, in agreement with other studies, morphometry significantly adds to the prognosis prediction of lymph node status and tumor size. Mitotic activity index is the best single predictor of the prognosis. An additional index advantage is that the multivariate model results in a continuous index variable that can be subdivided in many classes with an increasing risk of recurrence, so that more refined clinical therapeutic decision making is possible in individual patient care. The morphometric techniques are inexpensive and fairly simple and therefore can be applied in most pathology laboratories.

Actuarial Analysis