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PubMed1998

Altered expression of c-erbB-2, DF3, b72.3, p53 and Ki-67 with progression and differentiation to two distinct histologic types of invasive carcinoma in the MCF10AT human xenograft model of proliferative breast disease.

Human breast epithelial MCF10AT cells form simple ducts in nude/beige mice which eventually become hyperplastic and sporadically progress to carcinomas. Altered immunohistochemical detection of c-erbB-2, DF3, B72.3, p53 and Ki-67 was observed with progression and differentiation to two distinct histologic types of invasive carcinoma. c-erbB-2 and DF3 were detected in 50% and 18% of lesions at the stage of atypical hyperplasia and expression increased to 78% and 54% in invasive adenocarcinomas. In contrast, a group of six unusual undifferentiated tumors with squamoid features did not express c-erbB-2 or DF3, but both B72.3 (4/6) and p53 (6/6) were detected.

Adenocarcinoma↗
PubMed1998

Enhancement of all-trans-retinoic acid efficiency in granulocytic differentiation of HL-60 cells by incorporation into low density lipoprotein.

All-trans-retinoic acid (ATRA) has been proven to lead to complete remission of acute promyelocytic leukemia by inducing differentiation into granulocytes except when an acquired resistance occurred. High levels of low density lipoprotein (LDL) receptor in cancer cells suggested the use of ATRA incorporated into LDL. 50% of HL-60 cell differentiation were obtained with 5 nmoles/l of ATRA-LDL compared to 150 moles/l of ATRA. Maximal differentiation (80%) was reached at 25 nmoles/l and 1,000 nmoles/l respectively. This higher efficiency suggests the involvement of LDL receptor in ATRA-LDL internalization and/or the protection of the drug, from eventual catabolism, by LDL particles.

Binding, Competitive↗
PubMed2004

Analyses of the in vivo trafficking of stoichiometric doses of an anti-complement receptor 1/2 monoclonal antibody infused intravenously in mice.

Complement plays a critical role in the immune response by opsonizing immune complexes (IC) and thymus-independent type 2 Ags with C3 breakdown product C3dg, a CR2-specific ligand. We used a C3dg-opsonized IC model, anti-CR1/2 mAb 7G6, to investigate how such substrates are processed. We used RIA, whole body imaging, flow cytometry, and fluorescence immunohistochemistry to examine the disposition of 0.1- to 2-microg quantities of mAb 7G6 infused i.v. into BALB/c mice. The mAb is rapidly taken up by the spleen and binds preferentially to marginal zone (MZ) B cells; within 24 h, the MZ B cells relocate and transfer mAb 7G6 to follicular dendritic cells (FDC). Transfer occurs coincident with loss of the extracellular portion of MZ B cell CR2, suggesting that the process may be mediated by proteolysis of CR2. Intravenous infusion of an FDC-specific mAb does not induce comparable splenic localization or cellular reorganization, emphasizing the importance of MZ B cells in intrasplenic trafficking of bound substrates. We propose the following mechanism: binding of C3dg-opsonized IC to noncognate MZ B cells promotes migration of these cells to the white pulp, followed by CR2 proteolysis, which allows transfer of the opsonized IC to FDC, thus facilitating presentation of intact Ags to cognate B cells.

Animals↗
PubMed1996

[Image cytometric DNA analysis in bladder tumors].

UNLABELLED: We studied image cytometric DNA analysis of bladder tumors to evaluate malignant potentials of bladder tumors. METHODS: Thirty nine samples were obtained by TUR from 37 patients. Nuclear DNA content of all samples were measured by image cytometer and were determined ploidy pattern by DNA histogram. RESULTS: Of 39 TCC non-diploid pattern was recognized in 50% of grade 1 cases, 73% of grade 2 cases and 100% of grade 3 cases. DNA ploidy was strictly correlated with histological grading in TCC. DNA non-diploid pattern was present in 67% of papillary tumors, 87.5% of non-papillary tumors and 100% in CIS. In diploid pattern 2 of 7 cases with grade 1 and 2 of 4 cases with grade 2 recurred. In non-diploid pattern 1 of 4 cases with grade 1, 4 of 10 cases with grade 2 and 4 of 6 cases with grade 3 recurred. There was no significant correlation between diploid and non-diploid pattern in grade 1, 2, 3. CONCLUSION: Image cytometric DNA analysis may be useful in addition to the classic and prognostic parameters of stage and grade, especially in TCC. The differences between image analysis system and flow cytometric analysis for DNA measurement were discussed.

Adult↗
PubMed1999

Development and validation of a computerized cytomorphometric method to assess the maturation of vaginal epithelial cells.

BACKGROUND: After menopause, declining levels of estrogens may cause vaginal discomfort, or so-called "vaginal atrophy." Evaluation of therapies for vaginal atrophy may be performed using the so-called "maturation index." The maturation index is expressed as the percentage of (para)-basal, intermediate, and superficial epithelial cells in a vaginal smear. Manual assessment of the maturation index is subject to inter- and intraobserver variations. In this study, assessment of the maturation of cells in vaginal smears using automated image analysis was investigated. MATERIALS AND METHODS: Automated assessment, using a commercially available image analysis system, was performed on hematoxylin-eosin-stained cytospin specimens. A training set was constructed by an experienced cytotechnologist, based upon visual classification of stored grey value images. From this, two discriminant functions (DFs) were calculated capable of classifying cells in one of the three types. These cell classifiers were capable of classifying 97% of the cells correctly. Data from automated assessment were compared with those of classical manual counting. Specimens of 13 mature and 6 atrophic vaginal specimens were assessed in duplicate, both manually and by image analysis, using the DFs. RESULTS: No significant interobserver effect was found for image analysis, whereas a significant effect was found for manual counting. Both methods were able to distinguish between matured and atrophic specimens. CONCLUSIONS: It was concluded that for assessment of vaginal maturation, the use of automated image analysis systems is recommended. Besides increased reproducibility, image analysis systems yield additional data describing the size and shape of the cytoplasm and nucleus of cells, which might increase discriminating power.

Adult↗
PubMed1999

Photothermal studies of modulating effect of photoactivated chlorin on interaction of blood cells with bacteria.

BACKGROUND: Bactericidal application of photosensitizers (PS) is a new and promising area. Up to now the action of PS against bacteria was studied without regard to immunocompetent cells, doses of drugs and radiation being used usually are not safe for such cells and therefore there is still no efficient model for PS application. METHODS: Action of chlorin, a plant derived photosensitizer, on a system of interacting cells and microbes was studied. Cell monitoring was done with a photothermal microscopy method. The two bacteria used were gram-positive (S. aureus) and gram-negative (E. coli) in a mixture of basic blood cells and neutrophils. The latter were used to model phagocytosis as fast cell response reaction. RESULTS: The strongest bactericidal effect was found when the cells and photoactivated chlorin act together. Selective bactericidal regime of PS application which does not affect cell viability was demonstrated. CONCLUSIONS: The results obtained suggest that this photosensitizer may enhance cell natural defense capabilities.

Algorithms↗
PubMed1999

Semiquantitative assessment of c-erbB-2 (HER-2) status in cytology specimens and tissue sections from breast carcinoma.

OBJECTIVE: To determine whether various methods of fixation of surgical pathology specimens from breast carcinomas would influence the outcome of evaluation of the expression levels of c-erbB-2 (HER-2). For this, comparisons were made between (1) alcohol-fixed (95%) and air-dried smears from fresh surgical pathology specimens of breast carcinomas, and (2) formalin-fixed, paraffin-embedded tissue sections of the same specimens. STUDY DESIGN: Alcohol-fixed and air-dried smears or touch preparations were made from 30 fresh mastectomy/lumpectomy surgical pathology specimens from breast carcinomas. Immunohistochemistry was performed using the c-erbB-2 primary antibody against the extracellular domain of the c-erbB-2 gene product. Staining was simultaneously performed on formalin-fixed, paraffin-embedded tissue sections of the same specimens. A semiquantitative approach was used for evaluation of immunostaining by three independent investigators, and a consensus was reached. RESULTS: A total of 30 cases were reviewed. Tissue positivity was determined for c-erbB-2 in: 73% of alcohol-fixed specimens (n = 13 [3+] and n = 9 [2+]), 67% of air-dried smears (n = 9 [+3] and n = 11 [+2]) and 47% (n = 8 [+3]) and n = 6 [+2]) of formalin-fixed, paraffin-embedded tissue specimens. All formalin-fixed tissue specimens that were determined to positively express c-erbB-2 were also found to be positive on the alcohol-fixed smears. CONCLUSION: The incidence of c-erbB-2 expression in fresh cytologic material is significantly higher (P < .05) than in formalin-fixed, paraffin-embedded tissue. Alcohol-fixed smears demonstrate a slightly higher percentage of cell staining and stronger intensity of c-erbB-2 expression than the matched, air-dried smears. This is a sensitive and simple processing method that can be routinely applied in surgical pathology or fine needle aspiration biopsy specimens for the detection of c-erbB-2 (HER-2), with clinical implications.

Adenocarcinoma↗
PubMed1999

Nuclear volume in type I gastric intestinal metaplasia.

OBJECTIVE: To retrospectively compare nuclear size of epithelial cells in type I intestinal metaplasia (IM) from various pathologic lesions of gastric mucosa. STUDY DESIGN: Endoscopic mucosal biopsies with type I IM from intestinal type gastric carcinoma (n = 25), chronic gastritis (n = 40) and benign ulcer (n = 32) cases were analyzed. After standard fixation, embedding, sectioning, routine hematoxylin and eosin and alcian blue--periodic acid--Schiff reaction (pH 1.0 and 2.5) staining were used for identification of IM. The mean point sampled nuclear intercept was estimated by the original test system and 100x objective at a total magnification of 1,200x. To obtain the mean nuclear volume, the cubed nuclear intercept was multiplied by pi/3. In each case 100 epithelial cell nuclei were analyzed. RESULTS: In type I IM in gastric carcinoma cases there was significantly greater nuclear volume (118.34 +/- 10.32 micron 3) than in type I IM in other pathologic states of gastric mucosa (77.72 +/- 8.58 micron 3). CONCLUSION: The karyometric findings of the present study suggest a difference between type I IM found in benign pathologic states and type I IM found in gastric mucosa surrounding carcinoma, despite morphologic and histochemical similarities. Nuclear volume may be used in early detection of precancerous states of gastric mucosa.

Adenocarcinoma↗
PubMed1999

The Ebbeson-Tang formula. Theoretical background of an alternative to the disector in stereologic cell count studies.

OBJECTIVE: To evaluate the formula of Ebbeson and Tang (FET) with respect to the disector (DS) principle. STUDY DESIGN: The DS principle has been proposed for avoiding cell count bias. DS is a slice of tissue, and from it those cells by area are counted in microscopy; the cells are not in contact with one of the surfaces of the slice. The resulting number divided by the thickness of the DS gives an accurate figure for cell number by volume. FET applies two sections of different thickness, usually cut adjacent. Cells seen in the sections are counted by area, the figures are subtracted from each other, and the difference is divided by the difference between the thicknesses of the sections. The result is cell number by volume: NV = (NA1 - NA2)/(t1 - t2). RESULTS: FET and the DS principle superficially appear different. However, from a geometric point of view they are based on the same principle. When the thickness of the thinner section of FET approaches zero, the situation is in all respects equal to the DS principle. The formula for DS can thus be written: NV = (NA1 - NA2)/t1. CONCLUSION: The result proves that in principle DS and FET are equivalent methods of counting cell numbers by volume in tissues. FET may be more easily applied in histopathology practice because visual comparison of the sections is not necessary. Section thickness, however, has to be measured from vertically embedded sections or with scanning laser confocal microscopy. FET shares the stereologically unbiased character of the DS principle and is independent of the size and shape of structures counted.

Cell Count↗
PubMed1999

Total number and size distribution of hepatic metastases of carcinoma.

OBJECTIVE: To quantify the susceptibility of carcinoma to hepatic metastases by studying autopsy livers with carcinoma metastases, the primary sites of which were mostly the digestive organs. STUDY DESIGN: We developed a stereologic method of estimating the total number, N, and the size distribution of metastatic tumors in the liver based on a geometric model of spherical nodules with varying radii, r. This method proved to be sufficiently reliable by disector analysis simultaneously performed in some cases; it gave an approximate result. This method was applied to the 31 autopsy cases. Correlation and regression analyses were performed among N, the mean radius of nodules, rmean, and conventional pathologic features of the primary tumor. RESULTS: The estimates of N ranged from 10 to 3.2 x 10(5). A close negative correlation was confirmed between N and rmean. Neither significant correlation nor regression was observed among N and the other pathologic factors of the primary tumors. CONCLUSION: N turned out to serve as a useful index for evaluating the metastatic potential of a carcinoma. However, investigation has yet to be made to determine biologic factors in the primary tumor closely associated with N.

Carcinoma↗
PubMed1999

Statistical considerations in the methodology of quantifying immunocompetent cells in tumors.

OBJECTIVE: To quantify immunocompetent cell (IC) density for different testicular tumors and antibodies and to verify if the results of the comparisons depend on the antibody used. STUDY DESIGN: T-lymphocytes were studied with CD3 and UCHL1 antibodies and macrophages with MAC387 and CD68 in 43 patients with seminomas, mature teratomas, immature teratomas and embryonal carcinomas of the testis. Counts were expressed as number of IC per square millimeter. RESULTS: Use of different antibodies produced significant differences in the IC cell number; moreover, in the case of macrophages, the tumor type sequence by increasing cell number was different according to the antibody used. CONCLUSION: These results suggest the existence of a statistical interaction between the type of tumor and antibody.

Antibodies, Neoplasm↗
PubMed1999

Fraction of neoplastic cells in breast cancer samples. Correction for the oncogene amplification test.

OBJECTIVE: To develop the necessary correction for the polymerase chain reaction-based oncogene amplification test applied to paraffin-embedded tissue sections. STUDY DESIGN: The theoretical basis for making the correction in test results and for determining the neoplastic cell fraction was clarified, and a method for microscopic sampling was developed. The cell count bias was corrected by applying an equation by Ebbeson and Tang and using two adjacent tissue sections of different thickness, cut from the block from which DNA was isolated. RESULTS: There was no need to determine the absolute thicknesses of the sections. Section thickness, as a relevant feature, was cancelled out in developing the formula for estimating the cell fraction. The correction was applied in practice, and when tested on 10 breast cancer samples, the correlation between amplification estimates from two observers was good (r = .96). CONCLUSION: This method is valuable for interpreting the test results based on paraffin sections and for developing simpler quantitating methods for special test applications. The study also showed that amplifications may be more common than expected on the basis of the crude results of this type of test.

Breast Neoplasms↗
PubMed1999

Morphometric prognostic index in breast cancer.

OBJECTIVE: To assess the ability of the morphometric prognostic index (MPI) in predicting clinical outcome in a group of breast cancer patients with short-term follow-up and to assess the relationship between MPI and other prognosticators. STUDY DESIGN: The study group consisted of 63 cases of breast cancer. Follow-up data were available for 48 patients. MPI values were calculated, and degree of nuclear and tubular differentiation was investigated in each tumor. S-phase fraction (SPF), estrogen and progesterone receptors were also studied. RESULTS: The group of patients with MPI values < 0.60 had percent values of disease-free survival significantly higher than did those with MPI values > or = 0.60. Furthermore, significant direct correlations were found between MPI and degree of nuclear atypia and between MPI and SPF. Significant inverse relationships were found between MPI and tumor progesterone receptor levels and between MPI and degree of histologic tubular differentiation. CONCLUSION: The validity of MPI as a prognosticator in breast cancer was confirmed, even in a limited number of patients observed in short-term follow-up. MPI seems to be a reliable and economical prognosticator in selecting breast cancer patients for adjuvant chemotherapy.

Adenocarcinoma↗
PubMed1999

Basic aspects of and recent developments in telepathology in Europe, with specific emphasis on quality assurance.

Telepathology is the diagnostic work of a pathologist from a distance and includes all specific fields of diagnostic pathology, such as frozen section services, expert consultation, cytometric and histometric measurement, and continuous education. For about 15 years experience has been collected at several universities in the United States and Europe based upon analog telephone lines (9.2 kbaud), digitized lines (ISDN, 64 kbaud), broad band connections (1.5 Mbaud) and the World Wide Web (28 kbaud). Potential use can be expected in the application of telepresentation, remote slide preparation, remote central diagnostics and telediscussion. The transfer of still images is well developed; that of live images is used in only a few institutions for frozen section services. The image quality and spatial resolution as well as the transfer speed are sufficient for expert consultations, morphometric measurements, quality assurance and education. All applications focus on discontinuous work flow. Although the European Community focuses on user needs and standardization aspects of telepathology by sponsoring a widespread telepathology project (Europath), implementation of telepathology into routine application in the continuous work flow has still to be developed. The technical equipment has still to be adjusted to the labor flow charts in routine pathologic diagnostic procedures. Telepathology seems to be the appropriate technique to offer both improvement in diagnostic quality and inclusion of the "control institution" into diagnostic responsibility.

Critical Pathways↗
PubMed1999

Proliferative activity as detected by immunostaining with Ki-67 and proliferating cell nuclear antigen in benign and malignant epithelial lesions of the human parotid gland.

OBJECTIVE: A retrospective immunohistochemical study of parotid gland lesions was designed to evaluate the diagnostic and prognostic value of the proliferating cell nuclear antigen (PCNA) and Ki-67 with monoclonal antibodies PC 10 and MIB-1, respectively. STUDY DESIGN: Tissue samples comprised normal parotid gland (N, n = 10), chronic sialadenitis (CS, n = 8), Warthin's tumor (W, n = 10), benign pleomorphic adenoma (BPA, n = 8), mucoepidermoid carcinoma (MEC, n = 13), carcinoma in pleomorphic adenoma (CPA, n = 8) and adenoid cystic carcinoma (ACC, n = 12). The morphometric parameters for PCNA and MIB-1 comprised the PI and MI labelling indices (the numerical percentage of positive nuclei), NAP and NAM (the numerical density of positive nuclei), and NPI and NMI (volume corrected index). RESULTS: The values of MIB-1 parameters increased progressively in benign lesions in comparison with the N group and in malignant neoplasms in comparison with nonneoplastic groups and benign lesions. Values for all parameters in BPA were significantly lower than those in malignant groups. Spearman rank correlation analysis showed a highly positive correlation between the morphometric parameters and severity of the lesions. The mean values of MI and NMI were significantly higher in patients who died of the malignant tumors than in those who survived. The same quantitative parameters for PCNA did not differ significantly from those obtained for MIB-1 and showed similar trends. CONCLUSION: PCNA and MIB-1 indices are reliable markers for discriminating between benign and malignant tumors of the parotid gland, and the parameters PI, MI, NPI and NMI may have prognostic applications.

Adenocarcinoma↗
PubMed1999

Inhibition of DNA synthesis in isolated human endometrial cells by a levonorgestrel-releasing intrauterine device.

OBJECTIVE: To estimate the effect of an intrauterine device (IUD) releasing 20 micrograms levonorgestrel (LNG) per 24 hours on DNA synthesis in human endometrial cells before and after 12 months of use. STUDY DESIGN: Endometrial specimens were collected from the anterior or posterior wall of the miduterus from 6 females on cycle day 10-12 before insertion of the IUD and after 12 months of use. RESULTS: Previous results from our group did not reveal any influence on endometrial DNA cell content when a levonorgestrel IUD releasing 2 micrograms/24 h was used for 12 months in a group of fertile females. In this study, the IUD release rate, 20 micrograms LNG/24 h, was statistically significantly different from the results in the previous studies. The effect of the levonorgestrel IUD on endometrial proliferation was dose dependent, and a significant correlation could be found between continuous exposure to LNG and inhibition of DNA synthesis in endometrial cells. CONCLUSION: Inhibition of proliferative activity in endometrial cells seems to be reflected by a decrease in DNA synthesis per cell nucleus and contributes to the clinical performance of the LNG-releasing IUD.

Adult↗
PubMed1999

DNA ploidy and nuclear morphometry for the classification of dysplastic nevi.

OBJECTIVE: To examine the diagnostic value of DNA ploidy and nuclear morphometric features in sporadic dysplastic nevi as compared to those in compound nevi and melanoma. STUDY DESIGN: DNA ploidy profiles plus seven direct and three derived nuclear features were obtained in a series of 120 melanocytic skin neoplasms (30 dysplastic nevi [DN], 30 melanomas [MM], 60 compound nevi [CN]) and the results compared. RESULTS: DNA ploidy separated melanomas from benign melanocytic skin neoplasms with 96.5% accuracy in classifying the grouped cases. The derived nuclear shape factor Form PE and nuclear axis ratio were the most successful discriminants separating DN from MM but allowed only 73.3% correct classification of cases. Separation of DN from CN was best achieved using Form PE and mean nuclear area (74.4% correctly classified). Results from compound nevi in subjects < 25 years of age fell between those for DN and MM. CONCLUSION: Quantitative nuclear cytologic characteristics in sporadic dysplastic nevi span a range seen in common nevi through to those in thin melanomas. Cytologic changes in sporadic dysplastic nevi overlap those seen in other melanocytic skin neoplasms. Therefore, other reproducible morphometric features need to be assessed in order to further refine the histopathologic diagnosis of this entity.

Adult↗
PubMed1999

In vitro effects of elastase on periosteum of long bones: an histochemical, immunohistochemical and morphometric study.

The aim of the study was to determine the in vitro effects of porcine pancreatic elastase on the periosteum of long bones and to what extent the effects are selective for the elastic fibres of the tissue. Twenty-eight new-born chicks' tibiae were incubated for 1 or 3 hours in different experimental conditions (PBS, 30 or 60 units (U)/ml of porcine pancreatic elastase) or immediately formalin fixed. The tibiae were then processed for histo-chemical (Verhoeff and van Gieson stain), immunohistochemical (anti-elastin antibody) and histomorphometric analysis. A decrease of periosteal elastic fibres in all the specimens incubated with elastase in comparison with non incubated specimens was evident. The effect of elastase was easily detectable even at the lower concentration (30 U/ml) and at the shorter time of incubation (1 h). The amount of elastic fibres decreased in accordance with the rise of enzyme levels and incubation time, while periosteal collagen fibre content was not substantially modified by elastase activity. Present data are a prerequisite to evaluate the in vitro and in vivo effects of experimental destruction of periosteal elastic fibres by elastase and to assess the role of these fibres in the growth process of long bones.

Animals↗