Paul H. M. H. Theunissen

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.

Gain of chromosome 7, as detected by in situ hybridization, strongly correlates with shorter survival in astrocytoma grade 2

The clinical course of astrocytoma grade 2 (A2) is highly variable and is not reflected by morphological characteristics. Earlier studies using small series of A2 cases suggest that in situ hybridization (ISH) with chromosome‐specific DNA probes allows for frequent detection of aneusomy 1, trisomy 7, and monosomy 10. The role of trisomy 7 in astrocytoma carcinogenesis is disputed, however, because of its presence in non‐neoplastic brain tissue, as detected by karyotyping. Our objective was to investigate whether there was a correlation between chromosomal aberrations and survival in a series of 47 cases of A2. All cases were evaluated for numerical aberrations of chromosomes 1, 7, and 10 by ISH. Chromosomal aberrations were detected in 68% of cases of A2. Trisomy/polysomy 7 was seen in 31 cases (66%), 22 of which (47%) had a high percentage of this numerical aberration. Only 11 of these 22 cases also showed aneusomy for 1 or 10. No cells or only a few cells with aberrations were detected in non‐neoplastic control samples. Using Kaplan‐Meier analysis, trisomy/polysomy 7 correlated significantly with shorter survival. Hence, as determined by ISH, trisomy/polysomy 7 is absent in non‐neoplastic brain tissue and is frequently detected in A2, correlating with the malignant progression of the disease.

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.

Improved pre-operative mediastinal staging in non-small-cell lung cancer by serial sectioning and immunohistochemical staining of lymph-node biopsies

OBJECTIVE Mediastinal staging of non-small-cell lung carcinoma (NSCLC) by mediastinoscopy suffers from a low sensitivity, leading to a number of patients with unforeseen N2 disease at thoracotomy. This study was undertaken to assess whether pre-operative staging could be improved by serial sectioning and immunohistochemical staining of mediastinoscopy biopsies. METHODS In 183 consecutive patients with NSCLC, a thoracotomy was performed after a thorough mediastinal staging by computed tomography scan and cervical mediastinoscopy. In 158 patients (88%), a mediastinal node dissection was performed, revealing unforeseen N2 disease in 24 cases (15%). The preserved mediastinoscopy biopsies of these patients were retrospectively serially sectioned and stained with MNF 116. RESULTS Metastases could be identified in seven cases (30%), reducing unforeseen N2 disease from 15 to 10%. The number of patients who could theoretically benefit from neo-adjuvant therapy would have been increased by at least 10%. CONCLUSIONS Pre-operative mediastinal staging can be improved considerably by serial sectioning and immunohistochemical staining of mediastinoscopic biopsy specimens.

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

Intranodal and extranodal tumour growth in early metastasised non-small cell lung cancer: problems in histological diagnosis.

AIM--To question the observer reliability or agreement of reports on the intranodal and extranodal tumour growth patterns in early metastasised non-small cell lung cancer (NSCLC). METHODS--In a pilot study original histological sections of mediastinal lymph node metastases from NSCLC obtained by lymph node dissection (n = 82) or by mediastinoscopy (n = 62) were examined and classified independently by three pathologists as extranodal, intranodal, or indefinite. After clear criteria for these growth patterns had been defined sections were re-examined and recategorised one year later. Interobserver agreement was examined for both investigations. RESULTS--In the dissected lymph nodes the kappa value improved significantly from 0.52 (moderate agreement) at the first investigation to 0.72 (good agreement) at the second. In the mediastinoscopic lymph node biopsy specimens an increase in kappa value from 0.50 at the first to 0.67 at the second examination was found, although this improvement was not significant. In mediastinoscopic biopsy specimens a very high proportion of tissue samples showed indefinite tumour extension. CONCLUSION--Good reproducibility of intranodal and extranodal growth patterns in the histological examination of mediastinal lymph node metastases can be achieved, provided that pathologists use strictly defined criteria. In mediastinoscopic biopsy specimens it is often impossible to differentiate between intranodal and extranodal tumour growth.