Stereological estimates of nuclear volume in normal mucosa and carcinoma in situ of the human urinary bladder

The nuclear volume of the epithelial cells in the human urinary bladder mucosa has been estimated using point sampled intercepts in vertical sections (local vertical windows). The study included 27 specimens: ten from normal bladder mucosa, five from inflamed mucosa, seven from mucosa with flat grade II lesions and five from mucosa with flat grade III lesions. After standard fixation, embedding, sectioning and haematoxylin-eosin staining an unbiased estimate of the mean volume of nuclei sampled with a chance proportion to the volume = vV = pi/3 x (l0)3 was calculated using a frame for orientating the linear test probe in vertical sections. Here l0 is the length of the intercept through a test point hitting a nucleus measured in a random direction through the test point. The weighted mean nuclear volume of bladder mucosa with grade II and grade III lesions (537 microns 3 and 494 microns 3 respectively) was significantly larger than the weighted mean nuclear volume of normal (133 microns 3) and inflamed bladder mucosa (182 microns 3). This simple and fast estimation of nuclear volume seems to provide objective data useful in discriminating between neoplastic and non-neoplastic lesions of the bladder mucosa.     

Prognostic significance of mean nuclear volume in pancreatic adenocarcinoma

OBJECTIVE: To evaluate the correlation of stereologically estimated mean nuclear volume of tumor cells with other clinicopathologic prognostic features and survival in pancreatic ductal adenocarcinoma. STUDY DESIGN: The study included 27 patients with primary pancreatic ductal adenocarcinoma. A stereologic method proposed by Gundersen et al was used for the estimation of mean nuclear volume in hematoxylin and eosin-stained histologic sections of each case. Mean nuclear volume values were compared statistically with histopathologic prognostic feature groups and survival. RESULTS: The mean nuclear volume values of tumor cells ranged from 296.83 to 982.79 microns 3 (mean, 633.906 +/- 212.310). Higher values of mean nuclear volume were significantly related to advanced tumor stage and the presence of distant metastasis (Kruskal-Wallis, P = .036; Mann-Whitney U, P = .020). In contrast, nodal stage, tumor grade, perineural invasion, lymphatic and blood vessel invasion, and size of tumor showed no statistical relation to mean nuclear volume of tumor cells. Mean nuclear volume was found to be a significant predictor of survival in univariate analysis (P = .016). CONCLUSION: Estimation of mean nuclear volume may help in predicting the extent of disease and clinical behavior in pancreatic adenocarcinoma.     

Implications of nuclear diameter in small cell lung carcinoma

The nuclear diameter of 5,117 malignant cells from 42 small cell lung carcinoma (SCLC) patients was assessed either on pretreatment tissue sections (35 cases) or cytologic smears (7 cases) by ocular micrometry. The SCLCs were subtyped as 30 oat cell carcinomas and 12 intermediate cell carcinomas according to the World Health Organization classification, based on the predominant histology of the tumor. The median number of nuclei measured from each patient was 110. All patients were treated identically by sequential hemibody and local irradiation combined with chemotherapy and had a median follow-up time of 310 days. The mean nuclear diameter (+/- standard error) obtained from tissue sections was 8.2 +/- 0.03 microns (median = 8.0), including 7.3 +/- 0.03 microns (median = 7.0) for oat cell cases and 9.5 +/- 0.06 microns (median = 9.0) for intermediate cell cases (P less than .001). In 28.6% of these patients, the nuclear diameter overlapped in the range of 8 microns to 9 microns between both subtypes. Comparisons between the nuclear diameter of primary and metastatic SCLC cells revealed no statistically significant differences. The nuclear diameter of malignant cells correlated with the mitotic index and stage of disease, but did not correlate with the other nuclear morphologic variables or with survival. The only identified prognostic factor was the stage of disease; these results indicate that the nuclear diameter of malignant cells should not be considered a prognosticator or a guide for therapy in SCLC patients.     

Changes in nurse cell nuclei during synchronous infection with Trichinella spiralis

The rate of enlargement of nuclei was determined on 4-microns-thick sections of synchronously infected mouse thigh muscle. Normal muscle nuclei had a geometric mean volume of 84 microns and a range of 42-170 microns 3. At days 5, 6, 7, 8, and 10 and 6 mo after infection, mean nuclear volume was 177 (100-315) microns 3, 254 (140-462) microns 3, 278 (172-447) microns 3, 681 (407-1,138) microns 3, 512 (326-804) microns 3, and 509 (298-870) microns 3, respectively. Size of nuclei for any given day followed a log normal distribution. On days 7 and 8 after infection, 31% of enlarged nuclei had 2 nucleoli, whereas only 15% had 2 nucleoli on day 10. One percent of enlarged nuclei in 6-mo-old nurse cells had double nucleoli. The number of enlarged nuclei in 6-mo-old nurse cells was determined from serial sections of infected tongue muscle. Each nurse cell contained an average of 40 enlarged nuclei. Sixty-four percent of nurse cells examined (n = 55) had between 30 and 60 enlarged nuclei. However, there was great variation in the range (7-142). These results are discussed in relation to the development of the nurse cell.     

Mean nuclear volume in ovarian papillary serous cystadenocarcinoma

OBJECTIVE: To measure mean nuclear volume (MNV), estimated by the stereologic intercept method, in ovarian serous carcinomas and to compare that between omentum-positive cases (cases with omental metastasis) and -negative cases. STUDY DESIGN: This retrospective study consisted of 29 cases (stage I and II = 13 cases, and stage IIIb = 16 cases) of ovarian papillary serous cystadenocarcinoma. MNV of all cases (at primary and metastatic sites) was estimated with an image cytometer. The nuclear point intersection method was used to measure nuclear volume. Mean intercepted diameters of at least 50 nuclei were measured. MNV was correlated with histologic grade, International Federation of Gynecology and Obstetrics (FIGO) staging of ovarian malignancy and primary tumor versus metastatic deposits. RESULTS: MNVs estimated in omentum-negative (FIGO stage I and II) and omentum-positive (FIGO stage IIIb) cases were 1,022.48 +/- 608.45 and 2,152 +/- 1,317.51 microns 3 (P = .05, Student t test), respectively. Significant differences in MNV were also observed among the different grades of tumor. However, no significant difference was observed between MNVs of primary and metastatic tumors in omentum-positive cases. CONCLUSION: Estimates of MNV on conventional histopathology sections may provide objective and useful criteria for relatively subjective histopathology grading and staging (FIGO stage I and II vs. IIIb).     

Stereological estimates of nuclear volume correlated with histopathological grading and prognosis of bladder tumour

The aim of this retrospective study is to provide morphometric data which make grading of urinary bladder tumours objective and reproducible by stereological estimation of nuclear volume using the principle of estimating of the volume of particles of arbitrary shape. The study includes 92 specimens: 12 from normal bladder mucosa, and 80 from bladder tumours (15 grade I, 45 grade II, 19 grade III and one grade IV according to Bergkvist et al. 1965). After standard fixation, embedding, sectioning and hematoxylin-eosin staining, an unbiased estimate of the mean volume of nuclei sampled with a change proportional to the volume: (Formula: see text) was calculated. Here l0 is the length of the intercept through a test point hitting a nucleus measured in a random direction through the test point. The weighted means of nuclear volume in bladder tumours are spread over a wide range and show a strong correlation with the Bergkvist grade. Moreover, the relationship between the weighted mean volume of nuclei in bladder tumours and the prognosis is very good. Only one of 35 patients with a mean nuclear volume below 300 micron 3 died of bladder cancer whereas 18 of 19 patients with a mean nuclear volume above 500 micron 3 developed invasives tumours or died of their disease. This simple and fast estimate of nuclear volume seems to provide objective data of high prognostic value.     

Nuclear volume and total optical density in Down syndrome

OBJECTIVE: To estimate nuclear size and integrated optical density of parenchymal cells from various organs in patients with Down syndrome and a control group. STUDY DESIGN: During the years 1988-2000, 14 cases of Down syndrome were found (8 male and 6 female). Ten infants without congenital anomalies died of respiratory distress syndrome and were used as a control group. Five nuclear variables were estimated: area, equivalent diameter, volume of equivalent sphere, roundness and total optical density (TOD). RESULTS: Mean nuclear volume and TOD of thyroid follicular cells were significantly lower in patients with Down syndrome (43.82 +/- 8.95 and 173.81 +/- 32.85 microns 3, respectively) than in the control group (65.46 +/- 15.31 and 234.58 +/- 32.85 microns 3, respectively) (P < .01). Mean hepatocite nuclear volume and TOD were significantly higher in the control group (165.54 +/- 55.42 and 220.84 +/- 51.75 microns 3, respectively) than in trisomy 21 (110.39 +/- 32.97 and 176.58 +/- 28.53 microns 3, respectively) (P < .05). CONCLUSION: The present results suggest altered gene expression in excessive genetic material, especially in thyroid follicular cells.     

Manual versus image analysis estimation of PCNA in breast carcinoma

OBJECTIVE: To compare manual to image analysis estimation of proliferating cell nuclear antigen (PCNA) expression in paraffin sections of breast carcinomas. STUDY DESIGN: Paraffin sections of 51 breast carcinomas were stained with primary antibody to PCNA. Nuclear PCNA expression in 100 randomly selected tumor cells from marked areas was manually graded from 0 to 3. Antigen expression was also calculated by a cell analysis system (CAS-200, Becton Dickinson, Elmhurst, Illinois, U.S.A.) from marked and random microscopic fields. Obtained proliferative index (PI) from both methods was compared. RESULTS: Manually calculated PI correlated strongly with the CAS-200-calculated PI (P < .01). The highest correlation was seen between the CAS-200 PI value and manually calculated PI value using grade 2 and 3 nuclei. A particularly high correlation was noted between the number of positive nuclei and antigen staining area (P < .01) as estimated by the CAS-200. CONCLUSION: Nuclear expression of PCNA and other nuclear antigens can be accurately evaluated by an image analysis system. The speed and objectivity of such machines allow the evaluation of larger parts of tissues and provide more-representative antigen expression profiles.     

Overlap of nuclear diameters in lung cancer cells

The nuclear diameters (NDs) of randomly selected malignant cells from 35 cases of small-cell lung cancer (SCLC; 4,370 nuclei) and 31 cases of non-SCLC (NSCLC; 1,280 nuclei) were measured on the pretreatment tissue sections by ocular micrometry. The mean ND (+/- standard deviation) of malignant cells for SCLC patients was 8.1 +/- 1.5 microns; these cases included 23 oat-cell carcinomas and 12 intermediate-cell carcinomas. The ND of malignant cells for NSCLC patients was 12.8 +/- 2.2 microns; these cases included 17 squamous-cell carcinomas, 12 adenocarcinomas and 2 large-cell carcinomas. The differences of ND between SCLC and NSCLC and between intermediate-cell cancer and NSCLC were highly significant (P = 0.001). However, the malignant cells of 36 (54.5%) of the 66 lung cancer patients had NDs that overlapped in the range of 8 microns to 13 microns. For the 12 intermediate-cell patients, the NDs of the malignant cells overlapped with those of 8 (66.7%) of the 12 adenocarcinomas and 10 (58.8%) of the 17 squamous carcinomas. In contrast, the NDs of only 5 (21.7%) of the oat-cell patients overlapped with those of 5 (41.7%) of the 12 intermediate-cell cases and showed no overlap with NSCLC cases. Since there is overlapping of the nuclear diameters of malignant cells between SCLC and NSCLC patients, nuclear parameters other than the diameter are necessary to differentiate these two major histologic types of lung cancers.     

Correlation of mean nuclear area with estrogen receptor content in aspiration cytology of breast carcinoma

Thin needle aspirates of 42 consecutive breast carcinomas were obtained at the time of excisional biopsy. Nuclear diameters of 100 cells from each case were measured, and the nuclear areas were calculated. The concomitantly acquired histologic sections were reviewed and assigned a histologic grade according to the National Surgical Adjuvant Breast Project protocol no. 4. Estrogen receptor (ER) content was analyzed by both the DCCA and SDGA techniques. The ER content of each case was then compared to both the mean nuclear area of the cells on the cytologic smears and the histologic grade. All 16 cases with mean nuclear areas of less than 60 sq micrometer contained significant levels of ER (greater than 10 fmol/mg protein), as did 6 of 11 cases with nuclei between 60 and 90 sq micrometer. Only 5 of 15 cases with nuclei larger than 90 sq micrometer contained significant ER levels. Comparison of the sensitivity, specificity and predictive values of both techniques suggests that a quantitative assessment of nuclear area in cytologic thin needle aspirates correlates more closely with ER content than does histologic grading.     

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

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

Growth behavior of human pancreatic carcinoma xenograft correlates with nuclear features.

Two human pancreatic ductal adenocarcinomas with different growth rates were serially transplanted into nude mice. Feulgen-stained 4 microns sections and imprints from the xenografts were studied with a VICOM automated image analysis system. After pooling the results from two passages, with three mice in each passage, it was shown that of 23 nuclear parameters measured the following were correlated with a fast tumor growth rate: in sections, a decrease in heterogeneity of the chromatin and an increase in perimeter and nuclear area; in imprints, an increase in lesser diameter, in mean grey level difference between second neighboring pixels, and in total integrated optical density (DNA content). Several parameters differed significantly between passages, and between animals in the same passage. These findings suggest that the growth speed of pancreatic tumors may be predicted by nuclear parameters.     

Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.

An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.     

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

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

Terminal uridine nick end labelling and mitosis in breast carcinoma. Correlation with tumor grade and p53 overexpression

OBJECTIVE: To study the relationship of cell death and proliferation to histologic grade and p53 expression in invasive carcinoma of the breast. STUDY DESIGN: A total of 31 cases of infiltrating duct carcinoma of the breast were randomly selected. The terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling (TUNEL) reaction and p53 immunostaining were performed on representative paraffin-embedded tissue sections. Mitotic and apoptotic indices (MI and AI) were also measured on hematoxylin-eosin-stained sections. Histologic grade of infiltrating duct carcinoma was performed with the help of the Nottingham modification of the Bloom-Richardson system. Tumor grade and p53 overexpression were correlated with MI, AI and AI detected by TUNEL. RESULTS: There were a total of 31 infiltrating duct carcinomas of the breast, of which 13 cases were grade 1 and nine cases each were grade 2 and 3. Cells with positive TUNEL showed a strong brown nuclear positivity. TUNEL showed positivity from the periphery of the nuclear margin to the central portion. AI detected by TUNEL did not correlate with tumor grade (ANOVA, P > .05). MI was significant only in grade 1 versus grade 3 and 2 versus grade 3 carcinomas (ANOVA, P < .01). The morphologic apoptotic index was significant only in grade 1 versus grade 3 carcinomas. Nine cases showed p53 overexpression, and the rest of the cases were negative for p53 immunostaining. MI, AI and TUNEL were not significantly different in p53-negative and -positive groups. Pearson's correlation coefficient showed that AI and MI were significantly related, but there was no significant relation between AI detected by TUNEL and MI. CONCLUSION: MI is still more useful than AI or AI detected by TUNEL in differentiating various grades of carcinoma of the breast.     

Simultaneous expression of keratan sulphate epitope (a sulphated poly-N-acetyllactosamine) and blood group ABH antigens in papillary carcinomas of the human thyroid gland

Summary The monoclonal antibody 5-D-4 recognizes heavily sulphated forms of keratan sulphate epitope. It reacted strongly with the cell surfaces of most thyroid papillary carcinomas from all the individuals examined, independently of the blood group of the patients. Cells of follicular variants of papillary carcinomas were also labelled by 5-D-4. In contrast, no labelling with this antibody was observed in other types of thyroid neoplasms, or in normal tissues. The reactivity of 5-D-4 with papillary carcinomas was markedly reduced or abolished by prior digestion with endo-β-galactosidase keratanase II, or N-glycosidase F. Although keratanase digestion had no effect on 5-D-4 labelling, it revealed the binding sites ofGriffonia simplicifolia agglutinin II (GSA-II), which recognizes terminalN-acetylglucosamine in a limited number of carcinoma cells from some individuals. Blood group ABH antigens, which are simultaneously expressed together with keratan sulphate epitope in cancer cells, were eliminated by digestion with endo-β-galactosidase and N-glycosidase F, but were resistant to keratanase and keratanase II treatment. These results indicate that keratan sulphate oligosaccharides are cancer-associated and are probably oncofoetal antigens, as are the blood group antigens in human thyroid glands. The results suggests that poly-N-acetyllactosamine, which is ubiquitously and consistently produced in papillary carcinomas, is modified in two different ways: sulphation on the 6-position of at least some units of either galactose, orN-acetylglucosamine or both, and decoration of non-reducing termini with the blood group antigens. Along with the endo-β-galactosidase-GSA-II labelling procedure, labelling with 5-D-4 may be a useful diagnostic means for distinguishing papillary carcinoma from other types of thyroid neoplasms.     

Mean nuclear volume in squamous cell carcinoma of the vulva

OBJECTIVE: To compare mean nuclear volume (MNV) estimated by the stereologic intercept method in lymph node-positive and -negative cases of squamous cell carcinoma of the vulva. STUDY DESIGN: This retrospective study consisted of 53 cases (lymph node metastasis, n = 19; cases without lymph node metastasis, n = 34) of squamous cell carcinoma of the vulva. MNV was estimated with the help of an image cytometer. The nuclear point intersection method was used to measure MNV. The mean nuclear volumes of both lymph node-positive and -negative cases were compared. RESULT: MNV in the lymph node-negative and -positive cases was 717.0 +/- 533.1 and 1,961.4 +/- 1,369.6 microns 3, respectively (P < .000, Mann-Whitney U test). There was a significant difference in MNV between the 2 groups of tumors. CONCLUSION: The observations from the present study suggest that estimation of MNV of malignant squamous cells from the vulva on conventional histopathology sections may provide an objective and useful diagnostic tool in predicting lymph node metastasis.     

Stereological analysis of neuron, glial and endothelial cell numbers in the human amygdaloid complex

Cell number alterations in the amygdaloid complex (AC) might coincide with neurological and psychiatric pathologies with anxiety imbalances as well as with changes in brain functionality during aging. This stereological study focused on estimating, in samples from 7 control individuals aged 20 to 75 years old, the number and density of neurons, glia and endothelial cells in the entire AC and in its 5 nuclear groups (including the basolateral (BL), corticomedial and central groups), 5 nuclei and 13 nuclear subdivisions. The volume and total cell number in these territories were determined on Nissl-stained sections with the Cavalieri principle and the optical fractionator. The AC mean volume was 956 mm(3) and mean cell numbers (x10(6)) were: 15.3 neurons, 60 glial cells and 16.8 endothelial cells. The numbers of endothelial cells and neurons were similar in each AC region and were one fourth the number of glial cells. Analysis of the influence of the individuals' age at death on volume, cell number and density in each of these 24 AC regions suggested that aging does not affect regional size or the amount of glial cells, but that neuron and endothelial cell numbers respectively tended to decrease and increase in territories such as AC or BL. These accurate stereological measures of volume and total cell numbers and densities in the AC of control individuals could serve as appropriate reference values to evaluate subtle alterations in this structure in pathological conditions.