Mathie P.G. Leers

Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis

A neo‐epitope in cytokeratin 18 (CK18) that becomes available at an early caspase cleavage event during apoptosis and is not detectable in vital epithelial cells is characterized. The monoclonal antibody M30, specific for this site, can be utilized specifically to recognize apoptotic cells, which show cytoplasmic cytokeratin filaments and aggregates after immunohistochemistry with M30, while viable and necrotic cells are negative. The number of cells recognized by the antibody increases after induction of apoptosis in exponentially growing epithelial cell lines and immunoreactivity is independent of the phosphorylation state of the cytokeratins. The generation of the M30 neo‐epitope occurs early in the apoptotic cascade, before annexin V reactivity or positive DNA nick end labelling. In a flow cytometric assay, the majority of the M30‐positive cells appear in the ‘apoptotic’ subG1 peak. Tests with synthetic peptides define positions 387–396 of CK18, with a liberated C‐terminus at the caspase cleavage site DALD‐S, as the ten‐residue epitope of M30. This epitope starts at the end of coil 2 of the predicted CK18 structure, at a probable hinge region, compatible with the sensitivity to proteolytic cleavage. The definition of a specific caspase cleavage site in CK18 as a neo‐epitope can be used for quantification of apoptotic epithelial cells with immunocytochemical techniques and is applicable to both fresh and formalin‐fixed material. Copyright

The expression of beta-catenin in non-small-cell lung cancer: a clinicopathological study.

AIMS: To investigate the expression of beta-catenin in non-small-cell lung cancer (NSCLC) and its clinical significance. METHODS: 101 patients were surgically treated for NSCLC by lobectomy or pneumectomy with systematic lymph node dissection. Follow up was available in all patients, ranging from 24 to 110 months. Immunostaining of tissue sections from primary tumours and (when present) their lymph node metastases was performed and evaluated using a monoclonal antibody against beta-catenin. Correlations were investigated between beta-catenin immunostaining in primary tumours and E-cadherin immunostaining (data available from a previous study), lymph node stage, and survival. RESULTS: There were significant correlations between scores for beta-catenin immunostaining and E-cadherin immunostaining in primary tumours (p = 0.007), and between the beta-catenin immunostaining score in primary tumours and in their lymph node metastases (p = 0.006). An inverse correlation was found between the beta-catenin immunostaining score in primary tumours and lymph node stage N0, N1, or N2 (p = 0.03). According to the Kaplan-Meier survival estimate, the level of beta-catenin expression in primary tumours was a statistically significant prognostic factor (p = 0.01). CONCLUSIONS: Reduced beta-catenin expression in surgically treated NSCLC is clearly associated with lymph node metastasis and an infavourable prognosis. The existence of a functional relation between E-cadherin and beta-catenin is supported by the results of this clinicopathological study.

Heat Pretreatment Increases Resolution in DNA Flow Cytometry of Paraffin-Embedded Tumor Tissue

BACKGROUND Flow cytometry of single-cell suspensions prepared by enzymatic digestion from formalin-fixed, paraffin-embedded tissue suffers from several major drawbacks. The most important factors that influence the results are the high and unpredictable coefficients of variation (CVs) of the G0/G1 peak in the DNA histogram and reduction of propidium iodide (PI) intercalation with DNA, resulting from protein cross-linking by formalin. METHODS In this study we introduce a heating step (2 h incubation in citrate solution at 80 degrees C) prior to a brief pepsin digestion of tissue sections in the protocol for DNA content analysis of formalin-fixed and paraffin-embedded tissue. This new method is compared with established methods for the preparation of cell suspensions from frozen and paraffin-embedded tissues with respect to cell yield, DNA histogram resolution, DNA dye saturation kinetics, cell cycle parameters, and antigen retrieval in various epithelial and nonepithelial tissues. RESULTS The recovery of single cells from the paraffin sections was doubled by the heat treatment step, while the limited time of proteolysis resulted in decreased cell debris. Furthermore, an increased fraction of cells became cytokeratin-positive, while these immunocytochemically stained cells also exhibited a higher mean fluorescence intensity. The DNA histograms prepared from cell suspensions obtained according to this new protocol showed a significantly improved resolution, leading to a better identification of peridiploid cell populations. Heat pretreatment of paraffin-embedded archival tissue sections showed PI saturation kinetics similar to, or even better than, those of fresh unfixed tissues, independent of duration of fixation. CONCLUSIONS This new method, making use of routinely available antigen retrieval principles, thus allows high-resolution DNA analysis of routinely fixed and paraffin-embedded tissue samples. Using external reference cells, inter- and intralaboratory standardization of DNA histograms can be achieved.

Multi-parameter flow cytometric analysis with detection of the Ki67-Ag in paraffin embedded mammary carcinomas

In the present study we describe a novel multiparameter flow cytometric (FCM) assay to estimate the fraction of cycling cells in epithelial tumors derived from fresh frozen as well as archival material. To this end, MCF-7 cells as well as a series of breast carcinomas (n = 10; fresh frozen as well as formalin fixed and paraffin embedded) were stained using a panel of different antibodies directed against the Ki67-Ag (DAKO/PC, MIB-1, Ki-S5, and poly-Ki67) for a 3-parameter cytokeratin/Ki67-Ag/DNA FCM analysis. Whereas all Ki67-Ag antibodies work equally well in the methanol fixed cell line, MIB-1 and Ki-S5 epitopes are retained in cell suspensions mechanically derived from fresh frozen tissue. Only antibody Ki-S5 shows specific nucleolar staining patterns in cell suspensions prepared by trypsin digestion of formalin fixed, paraffin embedded tissue sections. A good correlation was found between the fractions of Ki67-Ag-positive epithelial cells measured in cell suspensions derived from fresh frozen and the corresponding formalin fixed and paraffin embedded tumor samples. Furthermore, the fraction of Ki67-Ag-positive epithelial cells as determined by 3-parameter FCM correlated very well with the Ki67-Ag-labeling index in paraffin embedded tissue sections.

Trivariate flow cytometric analysis of paraffin-embedded lung cancer specimens: Application of cytokeratin subtype specific antibodies to distinguish between differentiation pathways

The aim of the present study was to investigate whether trivariate FCM analysis, for the simultaneous detection of two different CK subtypes in combination with DNA content, can be applied to paraffin embedded samples of different types of non-small cell lung cancer in order to evaluate the cell cycle of individual sublines. Single cell suspensions were prepared from 50 microm thick paraffin sections of 22 lung carcinomas by pepsin digestion and immunostained with CK-antibodies which were chosen to distinguish glandular differentiation (adenocarcinomas) and squamous differentiation. There was a good correlation between the immunocytochemical results of the different CK antibodies in tissue sections and in the corresponding single cell suspensions. Gating for CK-positivity revealed a higher S-phase fraction as compared to the ungated cell population. The tumor cells in adenocarcinoma cases were specifically recognized by CK7 antibodies, while well-differentiated squamous cell carcinomas were specifically stained for CK14 and/or CK17. In poorly differentiated squamous cell carcinomas simultaneous expression of CK7 and CK17 was detected in a subpopulation of the tumor cells, next to cells positive for CK7 or CK17 alone. The trivariate FCM analysis allowed the separate estimation of ploidy status and cell cycle parameters in the three different cell populations of these, apparently (phenotypically) heterogeneous, malignancies.

Evaluation of the membrane attack complex of complement for the detection of a recent myocardial infarction in man

The diagnosis of an acute myocardial infarction (MI) can be cumbersome for pathologists. Even with a positive nitroblue tetrazolium (NBT) reaction, haematoxylin and eosin (H&E) evaluation of the myocardial tissue can remain inconclusive. Early signs presumed diagnostic for myocardial infarction, such as hypereosinophilia, waviness, and contraction band necrosis, have to be considered non‐specific and are probably reversible signs of ischaemia. Several studies implicate the complement system, and especially complement factor C9, as part of the membrane attack factor (MAC), in cardiomyocyte damage during MI. In a post‐mortem study on well‐documented cardiological autopsies, we evaluated the use of complement factor C9 immunostaining as a marker for the detection of very recent MI. Forty‐three tissue samples from 40 patients were obtained from the left ventricular free wall only, a region that can be specifically attributed to one corresponding coronary artery. As some patients presented with MIs of various stages in that perfusion area, in total 57 observations were possible. C9 immunostaining specifically detected irreversibly damaged (=infarcted) cardiomyocytes, as is implied by the lytic activity of C9/MAC binding to cell membranes. Most interesting was the group of clinically suspected, NBT‐positive MIs resulting from very recent myocardial ischaemia. In this population, where H&E evaluation by (cardio‐) experienced pathologists was not conclusive, C9 immunostaining clearly pointed towards myocardial infarction in 47% of the cases. In conclusion, C9 immunostaining, routinely practicable in the pathology laboratory, has an additional value in discriminating between reversible ischaemia and infarcted cardiomyocytes in very early MIs. Copyright

Flow cytometric DNA and phenotype analysis in pathology

Abstract. This meeting report summarizes the presentations of three different groups that are active in the field of flow cytometry (FCM) in relation to diagnosing and classification of proliferative disorders. The report starts with the contribution from Regensburg about the developments in DNA FCM, the progression to dual parameter determinations, and combination of immunophenotyping in combination with DNA. In the second part, the use of FCM for the detection of isolated tumor cells in the peripheral blood from patients with prostate or breast cancer is discussed in a contribution from Münster. In the third part, from Heerlen, the use of multi-parameter FCM on formalin-fixed paraffin-embedded tissues from solid tumors is discussed as a new development and application in routine surgical pathology.

Early apoptotic responses in transgenic mouse mammary carcinoma for photodynamic therapy.

BACKGROUND Male transgenic mice expressing the human RAS gene on an FVB strain background develop adenocarcinoma of the breast between 7 and 8 weeks of age. We have utilized this mammary tumour model to investigate apoptotic responses following photodynamic therapy (PDT) with a chlorin-based, water-soluble photosensitizer. METHODS Detection of apoptosis was accomplished by use of the antibody M30 against a neo-epitope of caspase-cleaved cytokeratin 18 that becomes available at an early stage of the apoptotic cascade. Mice bearing multiple tumours were injected with the photosensitizer intraperitoneally, and following a drug-light interval of 96h, 40J/cm(2) of 652nm laser light was applied to one tumour per animal, while the other tumours were protected from light to serve as host controls. The M30 antibody was used for standard immunohistochemistry of tumour sections and flow cytometric detection of epitope expression coupled to cell cycle analysis in tumour cell populations retrieved from paraffin blocks. RESULTS M30 staining was significantly increased within 2h following light treatment and persisted until 96h after treatment. Flow cytometric analysis for the S-phase fraction (SPF) of tumour cells post-PDT showed a substantial decrease in SPF at 2h post PDT, and recovery of SPF within 96h. CONCLUSIONS Cytokeratin 18 cleavage seems to be both an early and ongoing event during the cellular response to PDT. Calculating the M30/SPF ratio at both 2h and 96h suggested distinct cellular dynamics at early and late time points, and we propose the M30/SPF ratio as a tumour dynamic index (TDI) to monitor events post PDT.

9 Clinical flow cytometry of solid tumors

Publisher Summary Immunophenotyping by multiparameter flow cytometric (FCM) can provide a rapid accurate method for both identifying unique cell populations and describing their functional status. This can be achieved because antibodies to cellular proteins can be conjugated or labeled with different-colored fluorochromes and combined in panels for staining heterogeneous cell populations. Traditionally, FCM has never been the core business of surgical pathology. However, immunohistochemistry as an additional tool for the surgical pathologist is not always satisfactory—for example, clonality assessment in B-cell lymphomas is still difficult to perform with immunohistochemistry because of lack of staining contrast between surface immunoglobulins and extracellular immunoglobulins. In addition, solid tumors are heterogeneous of composition, and they consist of a mixture of normal stromal, inflammatory, and malignant cells. This heterogeneous cell composition is one of the limiting factors for reproducible quantification—for example, of steroid hormone receptor expression in breast carcinomas. Immunohistochemistry is sensitive to many external factors interfering with accurate quantification of receptor content. Staining intensity is influenced by the kind and duration of fixation, thickness of the tissue section, incubation conditions of the primary antibodies, choice and concentration of the chromogens, and subjective scoring by the investigators.