A task force of invited experts in the field of diagnostic DNA image cytometry, especially consisting of participants from the PRESS (Prototype Reference Standard Slides) and EUROPATH (European Pathology Assisted by Telematics for Healthcare) projects, but open to any other scientist or physician revealing experience in that new diagnostic procedure (names are given in the Annex A) agreed upon the following updated consensus report during the 5th International Congress of the ESACP 1997 in Oslo. This report is based on the preceeding one [9] and on results of the above mentioned European research projects. It deals with the following items: <list><list-item>&#x2013; Biological background and aims of DNA image cytometry,</list-item><list-item>&#x2013; Principles of the method,</list-item><list-item>&#x2013; Basic performance standards,</list-item><list-item>&#x2013; Diagnostic interpretation of DNA measurements,</list-item><list-item>&#x2013; Recommendations for practical use.</list-item></list>

G. Haroske

F. Giroud

A. Reith

A. B&#xf6;cking

Part I: Basic Considerations and Recommendations for Preparation, Measurement and Interpretation

Cyclin E is a part of the cell cycle machinery and aberrantly expressed in several malignancies including breast cancer. Since cyclin E is cell cycle specifically expressed, we wanted to examine the relation between proliferation and expression of cyclin E with special attention to tumours with overexpression of the protein. Seventy-four breast tumours were analysed for the expression of cyclin E by immunohistochemistry and Western blotting and related to the growth fraction determined by Ki-67. Significant correlations were obtained between the growth fraction, the percentage of cyclin E positive cells, the intensity of cyclin E and total amount of cyclin E determined by Western blotting. The majority of the tumours had less cyclin E than Ki-67 positive cells indicating a conserved cell cycle specific expression of the protein which further was supported by flow cytometric analysis of breast cancer cell lines. The cell cycle specificity of cyclin E was found even in tumours with inactivated retinoblastoma protein (pRB) demonstrating the existence of a pRB independent regulation of cyclin E. A fraction of the tumours had considerably elevated cyclin E levels that were not in relation to the proliferative activity as observed for the other tumours. These tumours were in general highly proliferative and considered to overexpress cyclin E. Patients with tumours of high proliferative activity, high total cyclin E levels or disproportionally elevated cyclin E expressions in relation to proliferation had significantly increased risk of death in breast cancer, whereas the intensity of the immunohistochemical cyclin E staining did not affect the survival.

Niels H. Nielsen

Conny Arnerl&#xf6;v

Stefan Cajander

G&#xf6;ran Landberg

Cyclin E Expression and Proliferation in Breast Cancer

Liver macrophages, which are involved in the different types of hepatitis, may indirectly induce hepatic fibrogenesis, since they have the possibility to activate hepatic stellate cells and fibroblasts by secretion of TGF-&#x03B2; , TNF-&#x03B1; and IL-1. To evaluate variations of the number of liver macrophages and their subpopulations, a quantification was carried out in normal human liver tissue, fatty liver, fatty liver hepatitis and hepatitis B. Identification was performed by the mab PG-M1 (anti-CD68) and, comparatively, four lectins, Griffonia simplicifolia agglutinin I (GSA-I), Erythrina cristagalli agglutinin (ECA), peanut agglutinin (PNA) and soybean agglutinin (SBA). A slight decrease in the frequency of macrophages in pericentral fields was observable in fatty liver and fatty liver hepatitis as compared to normal liver tissue. On the other hand, the number of CD68+ cells was significantly enhanced in hepatitis B with moderate and severe inflammatory activity. The highest incidence of macrophages was found in portal tracts of liver with fatty liver hepatitis and, particularly, hepatitis B. The fraction of cells stained by ECA, PNA or SBA did not increase significantly under pathological conditions. In contrast, the percentage of GSA-I binding macrophages was higher in liver parenchyma of hepatitis B and in portal tract macrophages in fatty liver hepatitis and also hepatitis B. In conclusion, our results indicate that GSA-I may aid in the detection of the subpopulation of activated macrophages which are assumed to play a pivotal role in liver pathology.

Stephan E. Baldus

Thomas K. Zirbes

Ion-Corin Weidner

Uta Flucke

Elke Dittmar

Juergen Thiele

Hans P. Dienes

Comparative Quantitative Analysis of Macrophage Populations Defined by CD68 and Carbohydrate Antigens in Normal and Pathologically Altered Human Liver Tissue

Two-color, multiparametric synthesis phase fraction (SPF) analysis of cytokeratin-labeled epithelial cells was flow cytometrically performed on both benign (SPFb) and malignant tissue samples (if available, SPFt) from 132 mastectomy/lumpectomy specimens. These data were then correlated with clinicopathologic features, including (1) tumor differentiation, (2) the proportion of tumor comprised of duct carcinoma-in situ (DCIS), and (3) the histology of accompanying benign breast tissue, classified by predominant microscopic pattern as intact, normal terminal duct lobular units (NTDLU, 34&#x25; of cases), atrophic (AT, 33&#x25; of cases), proliferative fibrocystic (PFC, 26&#x25; of cases), and non-proliferative fibrocystic (NPFC, 7&#x25; of cases). SPFt was inversely correlated with extent of DCIS (DCIS =0 &#x2013; 20&#x25; tumor volume &#x2013; 12.7&#x25; mean SPFt, vs. DCIS &#x3e;20&#x25; tumor volume &#x2013; 6.4&#x25; mean SPFt, p = 0.001). SPFt also correlated with the histology of background benign breast tissue (NTDLU &#x2013; 14.8&#x25; mean SPFt vs. AT &#x2013; 6.9&#x25; mean SPFt vs. PFC &#x2013; 12.7&#x25; mean SPFt, p = 0.05) but it did not correlate with patient age or SPFb (overall mean =0.73&#x25;). SPFb was correlated with patient age (&#x003E;56 yr &#x2013; 0.59&#x25; mean SPFb vs. &#x003C; yr &#x2013; 0.84&#x25; mean SPFb, p = 0.02), with background histology (NTDLU &#x2013; 1.1&#x25; mean SPFb vs. AT &#x2013; 0.43&#x25; mean SPFb vs. PFC &#x2013; 0.70&#x25; mean SPFb, p &#x003C; 0.02) and with the grade of the neoplasm (well/moderate &#x2013; 0.58&#x25; mean vs. poorly differentiated &#x2013; 0.85&#x25; mean, p = 0.04). Patients having a background of PFC were significantly older than patients with a background of NTDLU (45.2 yr vs. 60.2 yr, p = 0.01).We conclude: (1) breast carcinomas arising from a background of more actively cycling pre-involutional or proliferative fibrocystic epithelium have a greater proliferative fraction than tumors arising from atrophic epithelium, implying that the differentiation status of target cells may impact the effect(s) of tumorigenic events; (2) PFC may represent delayed, abnormal or interrupted involution rather than a hyperproliferative state relative to NTDLU, suggesting that it facilitates neoplasia by extending the period of exposure to promoter agents such as endogenous hormones, and (3) lower SPFt in breast neoplasia with more abundant &#x201c;residual&#x201d; DCIS may reflect a lengthier pre-invasive disease interval due to intrinsically less aggressive phenotype.

Daniel W. Visscher

Debra S. Gingrich

Carmen Leon-Armin

Pamela Tabaczka

John D. Crissman

Histopathologic and Flow-Cytometric Analysis of Neoplastic and Benign “background” Tissue in Breast Carcinoma Resections

Cells from 7 patients operated on for thyroid cancer were investigated. Samples of cells from the carcinoma and from the normal thyroid tissue were cultured with and without TSH stimulation. For light microscopy, serial sections of cells were cut and the size of nucleoli was measured and the number of nucleoli per cell counted. At the electron microscopic level the number and the volume of the fibrillar centres (FC) were estimated taking the Swiss cheese effect into account. The areal densities of FC, the fibrillar and granular component in nucleoli were determined by point counting. The results indicate that the malignant transformation has no influence on the size of the FC, but the observed numbers as well as the total area of FC are larger in cancer cells than in the normal thyroid epithelial cells. The nucleolar density of the fibrillar component is larger and that of the granular component is smaller in thyroid carcinoma cells than in non&#x2010;malignant thyroid epithelial cells (p = 0.0001). Thus simple morphometry at the electron microscopic level might be helpful to discriminate between thyroid epithelial cells and thyroid carcinoma cells in culture.

Lars Andersen

Lars Kayser

Niels Keiding

Jens Thomsen

A Morphometric Analysis of Nucleoli in Cultured Carcinoma Cells of the Human Thyroid

Oral papilloma is a benign tumourous lesion. Part of this lesion is associated with human papillomavirus (HPV) infection. We analysed the genetical and histopathological evidence for HPV type 2 infection in three oral papillomas. Southern blot hybridization showed HPV 2a sequence in one lesion. Cells of the positive specimen appeared to contain high copy numbers of the viral DNA in an episomal state. In situ staining demonstrated virus capsid antigen in koilocytotic cells and surrounding cells in the hyperplastic epithelial layer. Two other specimens contained no HPV sequences by labeled probe of full length linear HPVs 2a, 6b, 11, 16, 18, 31 and 33 DNA under low stringency hybridization conditions. These results showed the possibility that HPV 2 plays a role in oral papilloma.

Taihei Yamaguchi

Masanobu Shindoh

Akira Amemiya

Nobuo Inoue

Masaaki Kawamura

Hiroshi Sakaoka

Masakazu Inoue

Kei Fujinaga

Detection of Human Papillomavirus Type 2 Related Sequence in Oral Papilloma

Multi parameter flow cytometrical assays permit simultaneous assessment of proliferation, differentiation, and inflammation parameters. In this study, the validation of TO-PRO-3 iodide (TP3) compared to propidium iodide (PI) and DE-K10 compared to RKSE60 were evaluated in tape stripping induced hyperproliferation. No occlusion, Duoderm (intermediate occlusion) and Blenderm (maximal occlusion) were used as a model to evaluate the effect of occlusion on epidermal regeneration. Proliferation in the keratin 10-negative compartment measured with TP3 proved to be a good approximation of proliferation measured with PI. Other epidermal subpopulations (keratin 10-dim and -bright cells) did not make a relevant contribution to hyperproliferation. DE-K10 is probable more sensitive than RKSE60 to distinguish populations that differ in degree of differentiation. Occlusion of tape stripped skin resulted in decreased proliferation and increased differentiation. This effect was most pronounced with maximal occlusion. This study showed that occlusion is a therapy, which realises normalisation of hyperproliferative skin disorders.

M. M. van Rossum

J. M. Mommers

P. C. M. van de Kerkhof

G. J. de Jongh

C. A. E. M. van Hooijdonk

P. E. J. van Erp

Multi Parameter Flow Cytometric Assessment of Regenerative Epidermis with Special Reference to the Antiproliferative Effect of Occlusion

The p53 tumour suppressor gene has an important role in the the maintenance of genome stability and its mutational inactivation may be at the origin of aneuploidy in cancer cells. The aim of this study was to determine whether p53 mutations were associated to DNA aneuploidy, as assessed by flow cytometry, in colorectal adenocarcinomas. Analysis of p53 mutations spectrum of the sorted nuclei was done by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing. Overall, we studied 20 adenocarcinomas, the corresponding control mucosa, and 7 lymph node metastases.Five tumours (25&#x25;) were DNA diploid, while 15 tumours (75&#x25;) were composed of DNA aneuploid and diploid subpopulations. DNA diploid control mucosa and adenocarcinomas showed no p53 mutations, while 60&#x25; of the tumours with DNA aneuploidy had p53 mutations. Therefore, p53 mutations occurred significantly more often in DNA aneuploid than in DNA diploid tumours (p &#x003C; 0.04, Fisher&#x2019;s exact test). Incidences of DNA aneuploidy and p53 mutations in lymph node metastases were 60 and 86&#x25;, respectively. In all tumours showing a p53 mutation, the wild-type allele was not or only bearly visible in DNA aneuploid cells suggesting that, in such cells, aneuploidy is accompanied by complete p53 functional inactivation.The present observations suggest that p53 mutations may have a role in the origin of aneuploidy at late stages of colorectal carcinogenesis.

Paola Campomenosi

Paola Assereto

Massimo Bogliolo

Gilberto Fronza

Angelo Abbondandolo

Antonella Capasso

Patrizia F. Bellomo

Roberto Monaco

Anna Rapallo

Andrea Sciutto

Roberto Orecchia

Elio Geido

Walter Giaretti

p53 Mutations and DNA Ploidy in Colorectal Adenocarcinomas

The proliferative capacity (&#x25;S&#x2010;phase fraction), DNA ploidy, apoptosis frequency (DNA fragmentation) and steroid hormone receptor status (estrogen receptor, ER; progesterone receptor, PR) of 110 samples of human breast tissues with ductal invasive carcinoma were measured using biochemical and cytofluorimetric procedures. The DNA fragmentation had a left&#x2010;skewed frequency distribution and an overall median value of 1.64&#x25;, whilst the median &#x25;S&#x2010;phase fraction was 8&#x25;. The median &#x25;DNA fragmentation and &#x25;S&#x2010;phase fraction were 1.96&#x25; and 16&#x25; in hyperdiploid tumours (n=29; DNA index &#x3e;1.1) higher than in hypodiploid tumors (n=10; DNA index 0.96), 0.38&#x25; and 7.5&#x25;. DNA diploid tumours (n=71) had median &#x25;DNA fragmentation and &#x25;S&#x2010;phase values of 1.68&#x25; and 6&#x25;, consistently lower than the median values of DNA hyperdiploid tumours. The ER content of hypodiploid tumours was about one half (median: 5.9 fmol/mg) the median values in hyperdiploid (10.6 fmol/mg) and diploid tumours (14.6 fmol/mg). This may correlate with the lowest frequency of apoptosis in hypodiploid tumours, at least when measured by biochemical methods which only detect cells in the late phases of apoptosis. In contrast, the median PR was lowest in hyperdiploid tumours than in hypo and/or diploid tumours. The &#x25;S&#x2010;phase/&#x25;fragmented DNA ratio for the hypodiploid tumours was 19.7, significantly higher than the ratios for hyperdiploid (8.2) and diploid tumours (3.6). These findings indicated that there is an imbalance between proliferative capacity and cell death or growth arrest in human breast tumours. This imbalance may well be linked to a loss of steroid hormone control.

Gabriele A. Losa

Riccardo Graber

Apoptotic Cell Death and the Proliferative Capacity of Human Breast Cancers

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Analytical Cellular Pathology provides a forum for pathologists and medical practitioners working in the cellular pathology field. Topics covered include cytology, carcinogenesis, cell receptors, biomarkers, diagnostic pathology, and immunopathology.

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Analytical Cellular Pathology provides a forum for pathologists and medical practitioners working in the cellular pathology field. Topics covered include cytology, carcinogenesis, cell receptors, biomarkers, diagnostic pathology, and immunopathology.

Analytical Cellular Pathology