Markers for malignancy in the nuclear texture of histologically normal tissue from patients with thyroid tumors

The chromatin of nuclei from histologically normal-appearing glands in tissue sections from patients with follicular adenoma, papillary carcinoma and follicular carcinoma of the thyroid exhibited changes that were statistically significant when compared to the nuclear chromatin in tissue sections of thyroid from normal individuals. Certain chromatin texture features assumed values approximating those found in tumor cell nuclei. Staining was affected only slightly, and morphometric features, such as nuclear area, roundness and ellipticity, were statistically not different from those in normal controls. In patients with Hürthle-cell tumors, such marker features could not be found. Results from measurements on 30 patients (approximately 1,000 nuclei) are reported.     

Morphometry of nucleoli as an indicator for grade of malignancy of bladder tumors

To establish a new indicator for the classification of human urinary bladder cancers, the nucleoli of normal epithelial and neoplastic cells were analyzed, using morphometric techniques. By electron microscopy, the nucleolar profiles of cells from grade 2 and 3 transitional cell carcinomas were often small and irregular. Morphometry showed that the nucleolar volumes, nucleolar/nuclear volume ratios, volume densities of various nucleolar components, and the numbers of fibrillar centers (FCs) altered significantly with an increase in tumor grade. In particular, an increase in FC numbers in the nuclei of higher grade tumors was associated with a decrease in individual volume. The number of FCs in intact urothelial cells obtained from patients with bladder tumors is significantly larger than in the normal urothelial cells. This may be related to the multicentric origin of bladder cancers. These results suggest that morphometric analysis of nucleoli is useful in evaluating the degree of differentiation and invasive capacity of human bladder tumor cells. In particular, the number and individual volume of FCs may be an indicator of tumor malignancy.     

Morphometry of nucleoli as an indicator for grade of malignancy of bladder tumors

To establish a new indicator for the classification of human urinary bladder cancers, the nucleoli of normal epithelial and neoplastic cells were analyzed, using morphometric techniques. By electron microscopy, the nucleolar profiles of cells from grade 2 and 3 transitional cell carcinomas were often small and irregular. Morphometry showed that the nucleolar volumes, nucleolar/nuclear volume ratios, volume densities of various nucleolar components, and the numbers of fibrillar centers (FCs) altered significantly with an increase in tumor grade. In particular, an increase in FC numbers in the nuclei of higher grade tumors was associated with a decrease in individual volume. The number of FCs in intact urothelial cells obtained from patients with bladder tumors is significantly larger than in the normal urothelial cells. This may be related to the multicentric origin of bladder cancers. These results suggest that morphometric analysis of nucleoli is useful in evaluating the degree of differentiation and invasive capacity of human bladder tumor cells. In particular, the number and individual volume of FCs may be an indicator of tumor malignancy.     

Application of morphometry in tumor pathology

The application of morphometry in tumor pathology is discussed, e.g., its use in studying the biology of tumors, in creating tumor classification(s), in creating methods for the identification of a tumor in the diagnostic context, and in characterizing diagnostic histopathology in absolute terms. In traditional subjective diagnostic histopathology, reproducibility can be defined satisfactorily, but the definition of accuracy is ambiguous; in morphometric histopathology, a satisfactory definition is found for both concepts but it may be difficult to separate them in practice. Morphometric histopathology can study parameters measured from sections or parameters derived from the primary measurements through calculations. In the histopathology of tumors, the following parameters have turned out to be specially valuable: densitometric measurements of nuclei, nuclear area, perimeter and form factors, nucleolar parameters, the number of mitotic cells per area, the cellularity, the volume fraction of the epithelium, and parameters associated with the fraction of tumor tissue in the sample. The standard deviation or other moments of the distribution of these measurements can be more relevant than the mean values of the results. This indicates that more attention should be given to sampling rules, which are important in defining the efficiency of the methods. For rational application of morphometric methods, it is very important to make a distinction between group morphometry and diagnostic morphometry. The latter engenders numerous sources of variation (variation in section thickness, variation in tissue processing, variation in the techniques of measurement, interobserver variation, interlaboratory variation, variation due to subjective interpretation, etc.), which are usually better controlled in group morphometry. The influence on morphometric parameters of variation in section thickness and tissue shrinkage during processing are discussed.     

Interaction of the calf uterine estrogen receptor with acceptor sites in heterologous chicken target cell nuclei

Estrogen receptor (ER) from chicken liver and calf uterus were used to study the capacity and the characteristics of the receptor binding sites (acceptor sites) in chicken target cell nuclei. Binding studies were performed at a physiological salt concentration of 0.15 M KCl. Binding of liver ER to liver nuclei was temperature-dependent, showing a 9-fold increase between 0 and 28 degrees C. The maximal number of acceptor sites measured in this cell-free system (280 sites/nucleus) was considerably lower than measured in nuclei after in vivo administration of estrogen (820 sites/nucleus). Moreover incubation of nuclei with the liver ER preparation resulted in a substantial breakdown of nuclear DNA, making this ER less suitable for DNA binding studies. The temperature-activated calf uterine receptor bound to liver nuclei at 0 degrees C, at which temperature no DNA degradation was measured. To all chicken cell nuclei tested, the receptor bound with a high affinity (Kd = 0.4-1.0 nM). Nuclear binding displayed tissue specificity: oviduct greater than heart, liver greater than spleen greater than erythrocytes and was salt dependent. Calf uterine ER binding in liver nuclei ranged from 3000-6000 acceptor sites per nucleus when assayed under conditions of a constant protein or a constant DNA concentration. Nuclei isolated from estrogen-treated cockerels bound a 2-fold lower number of calf uterine ER complexes when compared to control nuclei. Incubation of nuclei with a fixed concentration of [3H]ER from liver and increasing concentrations of uterine non-radioactive-ER also resulted in a reduced binding of the liver receptor. Both types of experiments suggest that liver and uterine ER compete for a common nuclear acceptor site. Our data demonstrate that the ER from calf uterus is very useful as a probe to examine the nature of the acceptor sites in heterologous chicken target cell nuclei. The assay system functions at 0 degrees C, a temperature at which no DNA degradation occurs.     

Interaction of calf uterine estrogen receptors with chicken target cell nuclei

The calf uterine estrogen receptor (ER) was used to study the capacity and the characteristics of the acceptor sites in chicken target cell nuclei. The temperature-activated ER is bound at 0 degrees C with a high affinity to all chicken cell nuclei tested (Kd = 0.4-1.0 nM). The nuclear binding displayed tissue specificity: oviduct greater than liver, heart greater than spleen greater than erythrocytes and was salt-dependent. ER binding to liver nuclei measured in 0.15 M KCl varied between 3000 and 6000 acceptor sites per nucleus. Liver nuclei isolated from estrogen-treated cockerels showed a 2-fold lower binding capacity than nuclei from non-treated chickens. When nuclei were incubated with [3H]ER from embryo liver and increasing concentrations of uterine non-radioactive-ER a progressive inhibition of the binding of the liver ER was found. These experiments suggest that liver and uterine ER compete for a common acceptor site. Liver nuclei charged in vitro with calf uterine ER were digested at 0 degree C with DNAase I and micrococcal nuclease. Both enzymes excised the ER in the form of a chromatin-ER complex. A considerable portion was associated with nucleosomal subunits and a minor fraction was associated with a nuclease-sensitive, protein-poor fraction of the chromatin.     

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.     

Quantitative ultrastructural study of nuclei from exfoliated benign and malignant mesothelial cells and metastatic adenocarcinoma cells

Various approaches, including morphometric image analysis, are currently being used to improve the distinction between diffuse mesothelioma and metastatic adenocarcinoma of the serous membranes. Since exfoliated cells of malignant mesotheliomas were thought to have nuclear profile contours with greater irregularity than the similar profiles in metastatic adenocarcinoma cells in pleural effusions, this and other nuclear parameters were measured in ultrastructurally examined preparations from three cases of reactive mesothelial hyperplasia, seven examples of diffuse mesothelioma and three cases of metastatic adenocarcinoma (with primaries in the ovary, esophagus and prostate). Contrary to the subjective impression, the nuclei in metastatic adenocarcinomas actually had a mean nuclear contour index greater than that found in diffuse mesotheliomas; statistically, the difference was not significant. Likewise, such other nuclear parameters as nuclear area, condensed chromatin area and contour index, percentage of condensed chromatin and number of condensed chromatin clumps per nuclear profile did not discriminate between malignant mesotheliomas and adenocarcinomas metastatic to pleural surfaces. These morphometric results quantitate the similarities in nuclear size, nuclear shape and condensed chromatin arrangement in these two types of tumor and explain why the cytopathologist has such great difficulty in distinguishing between exfoliated mesothelioma and adenocarcinoma cells in most cases.     

Computer-assisted immunocytochemical determination of breast cancer steroid receptors on cytological smears of excised surgical specimens compared with frozen sections

BACKGROUND: Due to the widespread use of fine needle aspirate biopsy the practice of determining estrogen (ER) and progesterone (PR) receptors in breast carcinoma from cytological smears (CS) is becoming very common. The aim of this study was to determine concordance between ER and PR assessed by immunocytochemical assay (ICA) on CS and FS both evaluated by image analysis since we have found no data in literature on this. METHODS: 104 breast carcinoma cases were selected. For all cases ER and PR determination was performed on CS, obtained by light scraping of the freshly cut surface of the excised surgical tumors at the time of frozen section diagnosis, and FS using the same monoclonal antibodies. Computer-assisted image analysis was performed in all cases using CAS 200. Results were expressed as percent positive area of neoplastic nuclei compared with total nuclear area of the examined neoplastic cells. RESULTS: Good correlation was demonstrated between percent positive nuclear neoplastic area by ER-ICA on CS and FS (r = 0.759; P < 0.0001). Concordance of results was 90.19% (P < 0.001). Good correlation was also demonstrated between percent positive nuclear neoplastic area by PR-ICA in CS and FS (r = 0.889; P < 0. 0001). Concordance of results was 97.02% (P < 0.0001). CONCLUSIONS: Our data suggest that ICA on CS with automated image analysis is efficient in evaluating ER and PR content in human breast cancer, especially when CS is the only method pathologists have to evaluate receptor status e.g. in advanced breast cancer cases when neoadjuvant therapy is necessary before surgery or when surgery is impossible.     

Tissue microarray-based immunohistochemical study can significantly underestimate the expression of HER2 and progesterone receptor in ductal carcinoma in situ of the breast

Abstract

The accuracy of immunohistochemical (IHC) analysis on tissue microarray (TMA)-based studies largely depends on the uniformity of the staining pattern for a given antibody and minimal intratumor heterogeneity of a given tumor. Our study was designed to investigate the concordance of expression in TMA and whole sections of estrogen receptor (ER), progesterone receptor (PR) and HER2 using IHC analysis for ductal carcinoma in situ (DCIS) of the breast. Seventy-five consecutive cases of DCIS were retrieved, reviewed and used to construct the TMA. IHC analysis of the expression of ER, PR, and HER2 were performed on TMA and whole sections of the corresponding cases, and the results were compared. The specificity and sensitivity for TMA-based assays were 87.0, 75.9, 90.6 and 90.4%, and 76.1, 27.3 for ER, PR and HER2, respectively. The concordance and discordance were 89.3, 76.0 and 72.0%, and 6.7, 13.3 and 16.0% for ER, PR, HER2, respectively. The kappa values were 0.83, 0.89 and 0.42 for ER, PR and HER2, respectively. The non-concordance rates were inversely related to core number, with 46.67, 22.67 and 11.56% for one core, two cores, and three cores, respectively, per marker per case (p < 0.001), but not associated with tumor size. Our results showed that the intratumor heterogeneity and the number of cores have a great impact on the results of TMA-based studies. Increasing the number of tissue cores per case may help improve the accuracy and concordance with whole section results. Although TMA remains an effective tool for translational research, we should be cautious in our interpretation of these results.     

Immunocytochemical versus biochemical receptor determination in normal and tumorous tissues of the female reproductive tract and the breast

The development of highly specific and sensitive monoclonal antibodies directed against human estrogen (ER) and progesterone receptors (PR) provides a new approach in precise histochemical receptor location independent of hormone binding. Over the years receptor determination was the domain of the radioligand-binding assay, in which receptors are measured by tritiated ligand and unbound ligand is removed by the dextran-coated charcoal (DCC) procedure. Presented here are the results and experiences obtained by the classic DCC and the immunocytochemical method in the different normal and tumorous tissues of the female reproductive tract and the breast. The results of both methods were compared, and overall concordance of the results was found to vary considerably among the different types of tissue analyzed. Best agreement (86%) was found for PR determination in breast cancer, and the lowest rate of concordance for ER determination in fibrocystic disease of the breast. Special attention was directed toward the heterogeneity of receptor distribution in the specimens examined. In all tissues investigated, ER and PR were located in the nuclei of cells in both paraffin and frozen sections. Staining intensity varied among different cell types and from cell to cell for a single cell type, as well as in tumorous and normal tissues. In breast cancer, randomly scattered single cell receptor positivity was distinguished from focal/clonal positivity. Paraffin-embedded lymph node metastases showed significantly weaker staining as compared with their respective primary tumors. In the normal ovary, the corpus luteum and the stromal layer of the outer cortex were revealed as highly receptive elements for progestins, whereas ER was barely demonstrable in the normal ovary. Benign serous and mucinous ovarian tumors showed opposite ER and PR distribution among the stromal and epithelial components. Of special interest were the highly significant changes in ER and PR content in the stromal and glandular cells of the different layers of the normal endometrium throughout the menstrual cycle.     

Assessment of hormone receptors in breast carcinoma by immunocytochemistry and image analysis. II. Estrogen receptors

Frozen sections of 30 invasive breast carcinomas were stained for estrogen receptors (ERs) and the tumor cell proliferative rate by an immunoalkaline phosphatase technique. The stained sections were evaluated for ER by the microTICAS image analysis system. Seventeen tumors were ER positive and 13 were ER negative by image analysis. There was 93% concordance between the ER results obtained by image analysis and those obtained by biochemical methods. One case that was ER negative by image analysis was weakly positive by biochemical assay; a second case was ER positive by image analysis but ER negative by biochemical assay. Twelve of the 17 ER-positive tumors were diffusely positive while 5 displayed considerable intratumoral heterogeneity, with tumor cells exhibiting a broad range of intensity of receptor expression. In most cases, the image analysis ER status coincided with the progesterone receptor (PR) status, but in a large minority of cases (41%) the ER status and the PR status differed. Tumors with a high growth fraction (greater than 30%), as measured by Ki-67 immunostaining, were uniformly ER negative. The results of this investigation suggest that immunohistochemical staining of frozen sections for ER aided by automated image analysis (1) reliably detects the receptor in breast carcinoma, (2) allows for the assessment of heterogeneity within tumors and (3) may be used as part of a panel of antibodies to markers of potential prognostic importance in a single small tissue sample.     

Nuclear morphometry and AgNOR quantification: computerized image analysis on ovarian mucinous tumor imprints

OBJECTIVE: To determine the morphometric characteristics of nuclei and silver-stained nucleolar organizer regions (AgNORs) on cytologic imprints and their value in differential cytodiagnosis of benign, atypical proliferative (borderline) and malignant ovarian mucinous tumors. STUDY DESIGN: Forty-six mucinous ovarian tumor imprints (16 benign, 15 borderline, 15 malignant), were analyzed. Nuclear area, outline, "shape factor" and "form factor" were measured on Papanicolaou-stained smears. AgNOR quantification included 7 variables related to the number and area of single, cluster, total and relative AgNOR content per nucleus and the size distribution of AgNORs. RESULTS: Nuclear area and shape factor allowed distinguishing borderline and malignant tumors. The nuclear area in benign tumors was larger than that in borderline tumors; malignant tumors had the highest values. Single and cluster AgNORs were statistically significantly different in borderline tumors compared with malignant tumors, except for the cluster AgNOR area. The total AgNOR area, number and relative area increased from benign through malignant tumors, with statistically significant differences among all groups. By AgNOR size distribution, small AgNORs discriminate malignant from borderline and benign tumors. CONCLUSION: Combining nuclear morphometry and AgNOR analysis on cytologic imprints could be a diagnostically useful method in the assessment of mucinous ovarian tumors.     

A comparative study in twelve mammalian species of volume densities, volumes, and numerical densities of selected testis components, emphasizing those related to the Sertoli cell

Morphometric studies were performed on 12 mammalian species (degu, dog, guinea pig, hamster, human, monkey, mouse, opossum, rabbit, rat, stallion, and woodchuck) to determine volume density percentage (Vv%), volume (V), and numerical density (Nv) of seminiferous tubule components, especially those related to the Sertoli cell, and to make species comparisons. For most species, measurements were taken both from stages where elongate spermatids were deeply embedded within the Sertoli cell and from stages near sperm release where elongate spermatids were in shallow crypts within the Sertoli cell. Montages, prepared from electron micrographs, were used to determine Vv% of Sertoli cell components in seminiferous tubules. Excluding the tubular lumen, the Sertoli cell occupied from a high of 43.1% (woodchuck) to a low of 14.0% (mouse) of the tubular epithelium. There was a strong negative correlation (r = -0.83; P less than 0.005) of volume occupancy of Sertoli cells with sperm production. Nuclear volume, as determined by serial reconstruction using serial thick sections, ranged from a high of 848.4 microns 3 (opossum) to a low of 273.8 microns 3 (degu). There was no correlation (r = 0.02) of nuclear volume with volume occupancy (Vv%) in the tubule. Sertoli cell volume was determined by point-counting morphometry at the electron-microscope level as the product of the nuclear size and points determined over the entire cell divided by points over the nucleus. Sertoli cell V ranged from 2,035.3 microns 3 (degu) to 7,011.6 microns 3 (opossum) and was highly correlated (r = 0.85; P less than 0.001) with nuclear size. However, there was no significant correlation between the Sertoli cell size (V) and volume occupancy (Vv%; r = 0.13) or sperm production (r = -0.21). Stereological estimates of the numerical density (Nv) of Sertoli cells ranged from a high of 101.9 x 10(6) (monkey) to a low of 24.9 x 10(6) (rabbit) cells per cm3 of testicular tissue. There was no correlation of numerical density of Sertoli cells with sperm production (r = 0.002). A negative correlation was, however, observed between the numerical density of the Sertoli cells and the Sertoli cell size (r = -0.79; P less than 0.002). Data from the present study are compared with those previously published. This is the first study to compare Sertoli cell morphological measurements using unbiased sampling techniques. Morphometric data are provided which will serve as a basis for other morphometric studies.     

Glucocorticoid receptors in mouse mammary tumors: Specific binding to nuclear components.

The specific interaction of glucocorticoids with nuclei of mouse mammary tumor was studied in vitro by incubation of the tissue with [3H]dexamethasone at 25 degrees. It was demonstrated that the mammary tumors contain a limited number of specific nuclear binding sites which were saturated with low hormone concentrations (10-8 M)9 The concentrations of specific binding sites in the nuclei were related to the concentration of cytoplasmic binding sites of unincubated tissues and varied between individual tumors. The binding component in the nuclei appeared to be a protein and was easily solubilized with 0.4 M KCl containing buffers. The ability of various corticoids to block the nuclear localization of the steroid correlated well with their glucocorticoid potency. Estradiol and progesterone at concentrations of 10-6 M were also effective in competing for the glucocorticoid receptor binding sites. However, while the glucocorticoids such as hydrocortisone and corticosterone translocated to nuclear sites also specific for dexamethasone, estradiol and progesterone competed for the cytoplasmic binding sites and did not translocate to the nucleus. The possible significance of the interaction of various steroids with the glucocorticoid receptors in mammary tumors is discussed.     

Comparison of different antibodies for detection of progesterone receptor in breast cancer

Monoclonal antibodies directed against human estrogen receptor (ER) and progesterone receptor (PR) have been used extensively for biochemical and immunohistochemical detection of receptors independent of hormone-binding assays. These antibodies have been valuable both for experimental work and for detection of receptors in clinical breast cancer specimens. The purpose of this study was to characterize the sensitivity and specificity of different antibodies for detection of PR by immunohistochemistry (IHC) of formalin-fixed paraffin breast carcinoma sections. The panel of twelve antibodies included two new ones (PgR636 and PgR1294) produced prospectively to be resistant to formalin fixation and paraffin embedding. Fifty-nine breast carcinomas, having known PR levels by biochemical ligand-binding assay, were used to prepare multitumor paraffin-embedded tissue blocks for characterization of the PR antibodies. Of all the antibodies tested, both PgR636 and PgR1294 stained the highest percentage of breast carcinomas known to be positive by the biochemical assay (95-98%) and they exhibited the highest concordance with the biochemical assay (88-90%). The PgR636 and PgR1294 antibodies, along with one other, PR 88, also gave the highest intensity of nuclear staining, while PgR636 and PgR1294 stained the highest mean percentage of tumor cell nuclei. Antigen retrieval was not necessary for PR immunostaining by PgR636 and PgR1294 in most tumors and other tissues examined, but did slightly increase the staining intensity. The majority of the other antibodies tested were highly dependent on antigen retrieval; only PR 88 and KD 68 antibodies approached the performance of PgR636 and PgR1294 without antigen retrieval. These results indicate that PgR636 and PgR1294 are optimal antibodies for IHC detection of PR in routine paraffin tissue blocks.     

Nuclease sensitivity of estradiol-charged estrogen receptor binding sites in nuclei isolated from normal and neoplastic rat mammary tissues

The interaction of partially purified calf uterine estradiol-charged estrogen receptor ([3H]ER) with rat nuclei was studied in vitro. We previously observed a significantly greater number of [3H]ER binding sites (at saturation) in nuclei of R3230AC mammary tumors from intact vs ovariectomized (ovex) rats with no difference in the affinity of [3H]ER binding for these nuclei. We now report on the nuclease sensitivity of [3H]ER binding sites in nuclei from these tumors and from normal rat tissues. Digestion of tumor nuclei with deoxyribonuclease I (DNase I) prior to incubation with [3H]ER in vitro resulted in a progressive loss of [3H]ER binding capacity, which was not accompanied by alterations in the affinity of [3H]ER for the nuclei (Kd = 1-3 nM). A significantly lower concentration (P less than 0.005) of DNase I eliminated 50% of the [3H]ER binding sites in nuclei of tumors from intact hosts (8 unit.min/ml) compared to tumors from ovex hosts (22 unit.min/ml). These results indicate that DNA regions capable of binding ER are more susceptible to DNase I digestion in tumors from intact rats than those from ovex hosts, suggesting that the endogenous hormonal milieu is responsible, at least in part, for maintenance of nuclease-sensitive DNA conformations in this hormone-responsive mammary tumor. The amount of DNase I required to eliminate 50% of [3H]ER binding to nuclei from lactating mammary gland, liver, and kidney ranged from 14 to 56 unit.min/ml. Therefore, accessibility of [3H]ER binding sites to nuclease digestion in normal rat tissue is generally less than that of R3230AC tumors.     

Development of human cerebellar nuclei. Morphometric study

The morphometric development of the human cerebellar nuclei was examined in 9 fetuses (16-40 weeks of gestation; WG), an infant (2 months old) and 2 adults (16 and 63 years old). With the morphological observation of serial sections of the brain containing the cerebellar nuclei, the authors measured sections to get several morphometric parameters: the volume of nuclear column and number, packing density and cell body area of neurons. Each nucleus (dentate, emboliform, globose and fastigial nucleus) was recognized even at 16 WG. Nerve cells containing Nissl bodies were observed in all nuclei after 23 WG. Degenerative changes were detected in some neurons for every nucleus at 21 and 23 WG. Three stages were observed in the developmental course of nuclear volume and neuronal packing density: the primary or undifferentiated stage at 16 WG, the secondary stage with variability at 21-32 WG and the tertiary stage with monotonous increase (nuclear volume) or gradual decrease (neuronal packing density) after 35 WG. No significant correlation between neuronal number and gestational age was noticed for every nucleus. The analysis of cell body area (neuronal size) demonstrated that the dentate neurons developed after the intermediate or fastigial neurons. It is concluded that there is a critical period between slightly before 20 WG and slightly after 30 WG, matched with the secondary stage in the development of the cerebellar nuclei.     

Histological grade in breast cancer: association with clinical and biological features in a series of 229 patients

In order to study the association of histological grade (HG) with specific clinical and biological parameters which may influence the clinical behavior of infiltrating ductal carcinomas of the breast (IDC), we analyzed in 229 tissue samples the cytosolic concentrations of estrogen receptor (ER), progesterone receptor (PR), pS2, cathepsin D, hyaluronic acid (HA) and tissue-type plasminogen activator (t-PA), as well as those of the erbB2 oncoprotein, epidermal growth factor receptor (EGFR), HA, CD44v5 and CD44v6 in the cell membrane fraction. Likewise, we considered size, ploidy, S-phase fraction and axillary node involvement as variables of the study. The transition from HG1 to HG2 and from HG2 to HG3 was accompanied by a number of common features: global increase in size, greater number of tumors >2.0 cm, decrease in membrane hyaluronic acid concentrations, increased cell proliferation (S-phase >7%) and greater aneuploidy. Other events observed during the transition from HG2 to HG3 were a decrease in ER, PR, t-PA and cytosolic hyaluronic acid. These results led us to consider that HG is associated with certain clinical-biological changes that may help explain its value as a prognostic factor in breast carcinomas.