D. Öfner

In situ assessment of cell proliferation at the invasive front of oral squamous cell carcinomas

In oral squamous cell carcinoma (OSCC) the histopathological malignancy grading of the invasive front has been found to offer the most reliable prognostic parameter. In the present study we compared such tumour front grading of 100 OSCCs with the in situ growth fraction demonstrated by MIB1 immunostaining following wet autoclave antigen retrieval. MIB1 labelling indices (LIs) were estimated both at the invasive front and in the central parts of OSCCs using two different evaluation methods (overall and random counting) to investigate whether MIB1 LIs represent a possible biological background for the tumour front grading. Statistically highly significantly increased MIB1 LIs were found at the invasive tumour fronts with both counting methods compared with the centres of the same tumours. For LI estimation the classic overall counting procedure proved to be superior. However, in contrast to tumour front grading, MIB1 LIs revealed no correlation with the clinical outcome of the patients concerned. Our results demonstrate that the invasive tumour front of an OSCC is composed of (a) tumour subpopulation(s) with higher proliferative activity. However, determination of the proliferative activity by MIB1 of this tumour area offers no prognostic information.

Wet autoclave pretreatment for immunohistochemical demonstration of oestrogen receptors in routinely processed breast carcinoma tissue

SummaryThe immunohistochemical demonstration of oestrogen receptor (OR) was performed on 32 randomly selected and routinely processed breast carcinomas after wet autoclave pretreatment of sections. The autoclave method was compared to the OR status found on frozen sections as well as to alternative pretreatment methods such as enzymatic predigestion and microwave irradiation. Using four different monoclonal antibody clones (H222, LH1, CC4-5, ID5.26), the OR status was evaluated for each of the various pretreatment methods applied. All cases with a high OR content on frozen sections (n = 11) also showed a high OR status on wet autoclave-pretreated paraffin tissues using antibody clones 1D5.26 and CC4-5; in cases with low OR content on frozen sections, no false-negative cases were recorded using only the antibody 1D5.26 neither after wet autoclave nor microwave pretreatment. In addition, with this antibody, OR was detectable after autoclave pretreatment in two cases which were considered to be OR-negative even on frozen sections. When the primary antibody was omitted, no false-positive cases were observed after wet autoclave pretreatment. Thus, in our hands, wet autoclave pretreatment, in combination with the antibody 1D5.26, offers a highly sensitive method for the immunohistochemical demonstration of OR in routinely formalin-fixed, paraffin-embedded sections of breast carcinomas.

Feuchtes Autoklavieren Der einfachere Weg zur Antigendemaskierung

ZusammenfassungMit der Technik des feuchten Autoklavierens wird eine einfache, verläßliche und zeitsparende Methode zur Antigen-Demaskierung an Formalin-fixiertem und Paraffin-eingebettetem Gewebe vorgestellt. Anhand einer Reihe von Antikörpern (Östrogen- und Progesteronrezeptoren, Zytoskelettproteine, verschiedene p53-Antikörper, mdm-2, bcl-2, MIB-1 u. a.) verwendeter Antikörper werden die Vorteile dieser Methode beschrieben. Das feuchte Autoklavieren ermöglicht bei einigen sonst nur am Gefrierschnitt einsetzbaren Antikörpern auch deren Anwendung am Paraffinschnitt. Für den Routinepathologen ist die leichte Handhabung sowie die hohe Reproduzierbarkeit von Vorteil.SummaryWet autoclaving is a simple, reliable and time-effective method for antigen retrieval in routinely processed archival material. Both routine diagnostic (e. g., oestrogen and progesterone receptors, cytoskeletal proteins) and research antibodies (e. g. various p53 antibodies, mdm-2, bcl-2, MIB-1) are reported to demonstrate its application. Wet autoclaving may allow successful application of antibodies in paraffin-embedded tissues designed for use on frozen sections. The technique has the poten-tial to reliably handle up to 200 sections at a time, without evidence of any significant damage to the sections or nuclear morphology.

Demonstration of silver-stained nucleolar organizer region associated proteins (AgNORs) after wet autoclave pretreatment in breast carcinoma: correlation to tumor stage and long-term survival.

Argyrophilic nucleolar organizer region associated proteins (AgNORs) are known to reflect cellular and nucleolar activity. Due to a novel staining procedure, which substantially improves visualisation of AgNORs on formalin-fixed and paraffin-embedded material, AgNORs can be reliably demonstrated as true substructures of the nucleoli. The aim of the present study was to apply a standardized morphometric AgNOR quantification on a large series of breast carcinomas with regard to its prognostic relevance. AgNOR quantity was evaluated on archival tumor tissues of 115 adenocarcinomas of the breast treated with the wet autoclave method prior to standardized silver-staining and morphometric analysis. AgNOR parameters were correlated to prognostic features (steroid hormonal receptor status, tumor type, tumor size, histological grading, pTNM, and UICC stage) carrying out both univariate and multivariate survival analyses. AgNOR number and area were proven to be statistically significantly related (Pearson correlation coefficient: 0.67, Bonferroni adjusted P = 0.0001). Almost all AgNOR parameters, in particular CV (coefficient of variation) of corrected area (δ-area) and CV of number, were statistically significantly correlated to estrogen and progesterone receptor status as well as histological grading of tumors. Increased AgNOR parameters were statistically significantly associated with early tumor relapse and cancer related death. Univariate and multivariate analysis by means of Cox regression revealed independent prognostic significance for CV of δ-area and number of AgNORs. Various AgNOR parameters (CV of number, CV of δ-area, CV of area, mean δ-area, and mean area of AgNORs per nucleus) determined on wet autoclave pre-treated formalin-fixed and paraffin-embedded breast cancer tissues are statistically highly significantly associated with the prognostic outcome, independently predicting tumor-free and overall survival.

Standardisierte AgNOR-Färbemethode für formalinfixiertes und paraffineingebettetes Material

ZusammenfassungIn dieser Studie wird eine Methode zum Nachweis von sog. argyrophilen Nukleolus Organisator Regionen assoziierten Proteinen (AgNORs) vorgestellt, die es erstmalig erlaubt, diese als Teilstrukturen des Nukleolus an formalinfixiertem und paraffineingebetteten Gewebe zu erkennen. Mit einer Vorbehandlung durch feuchte Autoklavierung werden am archivierten Material Färbeergebnisse erzielt, wie sie an alkoholfixiertem Frischmaterial erreicht wurden. Sämtliche Färbeartefakte entfallen mit dieser neuen Methode. Weitere Vorteile bieten die einfache Handhabung sowie die Tatsache, daß auch bei unterschiedlicher Fixations- und Einbettungsdauer standardisierte und somit vergleichbare Ergebnisse erzielt werden. Das vorgestellte Verfahren bietet somit die Möglichkeit, die Wertigkeit der AgNOR-Quantifizierung auch an archiviertem Paraffinmaterial in retrospektiven Studien reproduzierbar zu evaluieren.SummaryVisualization of proteins associated with nucleolar organizer regions proteins (AgNORs) on formalin-fixed and paraffin-embedded archival tissues is substantially improved after application of wet autoclave pretreatment. Silver staining results are comparable to those obtained on tissues processed in alcoholbased fixatives, illustrating AgNORs as substructures of the nucleoli without any staining artefacts. A highly reproducible staining quality was achieved irrespective of tissue origin or duration of formalin fixation. As a result of this novel and simple method, the grounds have been prepared for standardized AgNOR quantification on archival material.

Possible relation of p53 and mdm-2 oncoprotein expression in thyroid carcinoma: A molecular-pathological and immunohistochemical study on paraffin-embedded tissue

Routinely processed tissues from a series of benign and malignant thyroid lesions were immunohistochemically investigated with antibodies against p53 and mdm-2. p53 was immunolocalized in <10% of nuclei in 2/80 nodular goiters, 2/60 follicular adenomas, 26/68 follicular carcinomas, 7/40 papillary carcinomas, 3/10 “insular” carcinomas, and 10/31 anaplastic carcinomas. More than 10% positively stained nuclei were found in 2 widely invasive follicular, 2 insular, and 15 anaplastic carcinomas. All p53-positive cases showed a concomitant immunohistochemical mdm-2 expression; an immunohistochemical colocalization on serial section was demonstrated in 12 anaplastic carcinomas. Screening by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of these 12 cases revealed no relevant mutations in the coding regions of exons 2–11 of the p53 gene. Additionally, 1 follicular adenoma, 6 follicular carcinomas (4 minimally and 2 widely invasive), 1 papillary, and 2 poorly differentiated insular carcinomas were mdm-2 positive without immunohistochemically detectable p53 expression. These results provide evidence that wild-type p53 expression in thyroid carcinomas may be associated with mdm-2 induced formation of stable complexes. However, the role of p53 mutations and p53 protein inactivation owing to other factors (e.g., mdm-2) in the progression of thyroid carcinomas is still poorly understood.

Immunohistochemical demonstration of chromogranin A, chromogranin B, and secretoneurin in primary non-small-cell carcinomas of the lung

Fifty-three primary non-small-cell lung carcinomas (NSCLC) were immunohistochemically investigated with antibodies against chromogranin A, chromogranin B, and ecretoneurin. All 3 peptides were focally immunolocalized in 3 of 25 adenocarcinomas and in 2 of 6 large-cell anaplastic carcinomas in more than 20% of tumor cells. Two of 15 squamous-cell carcinomas showed chromogranin B reactivity in more than 20% of tumor cells. Neuroendocrine (NE) differentiation was also demonstrated in lymphnode metastases of large-cell anaplastic carcinomas, in 1 adenocarcinoma, and in 1 squamous-cell carcinoma, with NE differentiation of the respective primary tumors. All tumors with NE differentiation exhibited (large cell) anaplastic tumor areas. We conclude that NE differentiation should be immunohistochemically proven or excluded, particularly in NSCLC with anaplastic components. Chromogranin B and secretoneurin are proposed as useful additional neuroendocrine markers for demonstration of NE differentiation in lung carcinomas.