Ursula Dürmüller

Immunohistochemical characterization of an anti-epithelial monoclonal antibody (mAB lu-5)

A mouse monoclonal antibody (mAB lu-5) was prepared using a lung cancer cell line as an antigen. The selected clone produces an IgG with a gamma-1 heavy chain and a kappa-light-chain. Immunohistochemical testing of mAB lu-5 on 117 normal tissue biopsies and 474 tumours revealed reactivity with an intracytoplasmic, formaldehyderesistant antigen present in most epithelial and mesothelial cells, but absent in mesenchymal cells. The antibody can therefore be used as a first order, pan-epithelial marker. It proved also useful for fast tumour diagnosis on frozen sections.

Influence of slide aging on results of translational research studies using immunohistochemistry.

Several reports have shown that a long delay between cutting sections and immunohistochemical (IHC) staining can decrease the IHC reaction intensity. However, systematic large-scale studies to investigate to what extent this problem may influence the outcome of translational research studies are lacking. In this study, we used a tissue microarray (TMA) approach to investigate the influence of slide age on comparisons between the results of IHC analyses for estrogen receptor (ER), progesterone receptor (PR), cyclin D1, HER2 (HercepTest), and E-cadherin and clinical outcome in a series of 522 breast cancer patients. Old TMA sections stored for 6 months at 4°C and freshly cut sections were analyzed under exactly identical experimental conditions. As compared to results obtained on freshly cut sections, the frequency of positivity on old sections decreased from 65 to 46% for ER (P<0.0001), from 33 to 18.5% for PR (P<0.0001), from 16.3 to 9.6% for HER2 (P=0.0047), from 45.1 to 37.7% for cyclin D1 (P=0.10), and from 58.9 to 32.9% for E-cadherin (P<0.0001). Despite the lower fraction of positive cases, most associations between IHC data and tumor phenotype that were observed in fresh section analysis were also found when old section data were analyzed. The results confirm that slide aging has a great influence on the intensity of IHC staining in individual cases, but they also suggest that many clinicopathological associations can be detected if suboptimally processed sections are used for IHC.

NY-ESO-1 tumour associated antigen is a cytoplasmic protein detectable by specific monoclonal antibodies in cell lines and clinical specimens

NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients.

Comparative Analysis of the Expression of Tenascin and Established Prognostic Factors in Human Breast Cancer

Tenascin is an extracellular matrix glycoprotein expressed during morphogenesis in embryonal life. It reappears in the stroma of benign and malignant tumors. The distribution of tenascin in variants of fibrocystic disease and infiltrating breast carcinoma was assessed in cryostat sections by immunofluorescence using a polyclonal antibody. The tenascin immunoreactivity was compared with various prognostic factors. In fibrocystic disease (n = 10), tenascin appeared as periductal and periacinar bands. In infiltrating carcinomas (n = 32) the tenascin expression was markedly increased. Tenascin immunoreactivity was noted around the ducts (78%), extended into the distal stroma (56%), or was distributed in smaller (reticular) septa around and within tumor-cell nests (34%). Nineteen percent of infiltrating carcinomas did not express tenascin. None of the patterns correlated with prognostic factors such as nodal metastasis, tumor necrosis, invasion of blood vessels, or with flow cytometry results, such as ploidy and S-phase fraction. However, a significantly higher reticular and periepithelial tenascin expression was noted in cases with increased stromal inflammatory reaction. These findings indicate that the appearance of tenascin is neither an indicator of malignancy nor predictive of invasiveness or metastasis but that it is related to local inflammatory response.

The tumour-associated antigen MAGE-1 is detectable in formalin-fixed paraffin sections of malignant melanoma

The MAGE-1 gene encodes a protein encompassing a HLA-A1-restricted target epitope for cytolytic T lymphocytes. Monoclonal antibodies directed against the MAGE-1 protein were tested for usage in immunohistology of routine pathology material. Seven formalinfixed, paraffin-embedded malignant melanomas were studied by the Avidin-Biotin complex (ABC) method with or without different antigen retrieval methods. Native, frozen tissues from the same tumours were used to validate the results by immunohistochemistry on frozen sections, by PCR for mRNA and by protein demonstration in tissue extracts using western blotting. Of 4 monoclonal antibodies tested, mAB 34B and mAB 77B were highly efficient in detecting MAGE-1 protein in deparaffinised sections with the regular ABC method after microwave pretreatment. In a series of an additional 28 patients 75% expressed MAGE-1, 50% in a substantial proportion. Follow-up studies in 6 patients indicate that the expression pattern remains stable but may change substantially within a short range. Immunohistology is thus a rapid and well-established method that might be used to select and monitor HLA-A1 positive patients with malignant melanoma and other candidate tumours for MAGE-1-directed immuno-therapy.

Plasma cell infiltrates in polyomavirus nephropathy

Polyomavirus (PV) associated nephropathy (PVAN) has become an important cause of allograft dysfunction. We studied plasma cells (PCs) – which have not yet been characterized – present in the cellular infiltrate of 20 PVAN cases using immunohistochemistry and morphometry. The results were correlated with morphological, clinical and anti‐BK virus serological findings. PC‐rich cellular infiltrates occurred in 50% of cases (>15% PCs in the cellular infiltrate) and in these IgM producing PCs were commonly seen (70%): IgM PC predominance in 50% of cases and a comparable number of IgM and IgG PCs in 20% of cases. We found a significant correlation not just between the absolute numbers (P < 0.034) and the percentage values of IgM PCs (P < 0.004 in relation to all cells) and the serum IgM‐Ab anti‐BKV activity, but also between the ratio of IgG/IgM PCs and the ratio of serum IgG/IgM‐Ab activities (P < 0.0001). We showed that IgM PC counts in biopsies correlate with titers of circulating anti‐BK virus IgM antibodies. Every case except one was C4d negative in peritubular capillaries (PTC). As IgG PCs characterize PC‐rich rejection cases, we suggest that in the presence of IgM PCs in PC‐rich infiltrate with PTC C4d negativity, a search for possible PVAN infection should be initiated.

Localization of thromboxane synthase in human tissues by monoclonal antibody Tü 300.

Using the monoclonal antibody Tü 300 we localized thromboxane synthase, a secondary enzyme of the arachidonic acid cascade, employing the alkaline phosphatase anti-alkaline phosphatase method and indirect double labelling immunofluorescence in frozen sections of human tissues. Aside from platelets, the source of the antigen, all cells of the mononuclear phagocytic system were positive, including epithelioid cells and associated giant cells, starry sky macrophages, dendritic cells of T-cell areas, Langerhans cells and Kupffer cells. In addition, some epithelial cells such as epithelia of tonsillar crypts, reticular epithelia of the thymic cortex and ductular epithelia in liver, pancreas, female breast and salivary glands showed occasional focal reactivity for thromboxane synthase. We suggest that the mAb Tü 300 is a key marker for the macrophage system and the thromboxane generating system in normal and pathological conditions. It may detect functional activities of as yet unknown significance in some specialized epithelial cells.

BK virus large T and VP-1 expression in infected human renal allografts

Objective. We investigated the expression of early and late phase BK virus (BKV) proteins and their interactions with host cell proteins in renal allografts, with ongoing polyomavirus associated nephropathy (PVAN), and correlated this with the nuclear and cell morphology. Methods. Frozen sections from three patients with renal allografts (two biopsies, one explant) with PVAN were analysed by indirect immunofluorescence using BKV specific anti-polyoma large T-antigen and anti-VP-1 antibodies, as well as anti-p53, anti-Ki67, anti-caspase-3, anti-bcl2 and anti-cytokeratin 22 antibodies. Nuclear morphology and size were estimated by DNA Hoechst staining. Results. In infected tubular cells the early and late phases of infection could be distinguished according to expression of large T-antigen or VP-1. The early phase revealed almost normal nuclear proportions, whereas in later phases nuclear size increased about 2 to 3 fold. Expression of large T-antigen was strongly associated with accumulation of p53 in the nucleus, accompanied by the activation of the cell cycle associated cell protein Ki67. In contrast, expression of BKV VP1 correlated only weakly with p53. Virus dependent cell lysis was due to necrosis, since neither caspase 3 nor nuclear nor cytoskeleton changes indicated apoptosis. Conclusion. In our selected patients with PVAN a clear distinction between early and late phases was possible, according to the protein expression patterns of BKV markers. Striking nuclear enlargement is only present in the late phase of infection. In the inflammatory setting of PVAN, BKV dependent effects appear to be mediated by the inhibition of p53, resulting in the activation of the cell cycle. We assume that in PVAN similar BKV mechanisms are operative as in certain in vitro systems.

Heterogenität des Samenblasenamyloids Immunhistochemischer Nachweis von Lactoferrin und Amyloid vom Präalbumin-Transthyretin-Typ

ZusammenfassungOrganlimitierte Amyloidablagerungen in den Samenblasen können im höheren Lebensalter auftreten. Die Samenblasenamyloidose konnte bislang keinem bekannten Amyloidtyp zugeordnet werden. In der vorliegenden Studie wurden bei 50 konsekutiven Autopsiefällen von Männern über 50 Jahren die Samenblasen histologisch untersucht, um die Inzidenz und den Phänotyp der Samenblasenamyloidose besser zu charakterisieren. Bei 7 Patienten (14%) wurde eine Samenblasenamyloidose gefunden. Das Amyloid wurde bei diesen Patienten sowie von einem zusätzlichen Fall histochemisch, immunhistologisch und elektronenmikroskopisch charakterisiert. Die Samenblasenamyloidosen waren bei 6 Patienten (75%) mit geringen Amyloidablagerungen im Herzen kombiniert. Die Samenblasenamyloidosen von 6 Patienten (75%) zeigten eine positive Reaktion mit einem Antikörper gegen Lactoferrin, einem in den Samenblasen produzierten Glykoprotein. Bei 4 dieser Patienten bestanden gleichzeitig Amyloidablagerungen im Herzen. Bei diesen Ablagerungen handelte es sich jedoch um einen anderen Amyloidtyp (Immunglobulin Leichtkettenamyloid AL-lambda-Typ). In 2 Fällen (25%) wurde in Herz und Samenblasen der gleiche Amyloidtyp (Präalbumin-Transthyretin-Typ) identifiziert, so daß bei diesen Patienten die Samenblasenamyloidose im Rahmen einer generalisierten Amyloidose aufgetreten ist. Unsere Untersuchungen zeigen, daß die meisten Amyloidablagerungen der Samenblasen organlimitierte Formen sind, die durch Ablagerung von Lactoferrin verursacht werden. Diese Samenblasenamyloidosen sind von einem Begleitbefall im Rahmen einer systemischen senilen Amyloidose abzugrenzen.SummaryLocalized depositions of amyloid in the seminal vesicles may occur in elderly men. Ear-lier immunohistochemical studies have failed to identify immunoreactivity of known amyloid material. In this autopsy study, all seminal vesicles of males older than 50 years were histologically examined to determine incidence and phenotype of seminal vesicle amyloidosis. Seven out of 50 patients (14%) showed depositions of amyloid in the seminal vesicles. These amyloid depositions as well as one additional case were characterized histochemically, immunohistochemically and electronmicroscopically. All but two of these patients (75%) showed simultaneously amyloid depositions in the heart. Lactoferrin immunoreactivity was found in 6 patients (75%). Lactoferrin is an ironbinding, bacteriostatic glycoprotein, which is produced in the seminal vesicles. Four patients with lactoferrin positive amyloid in seminal vesicle showed different amyloid depositions in the heart (immunoglobulin light chain amyloid AL-lambda). Two cases (25%) showed the same amyloid type in heart and seminal vesicles (prealbumin-transthyretin type amyloid). Our study shows that most amyloidoses of the seminal vesicles are organ-limited depositions of lactoferrin. These forms of localized amyloidosis have to be separated from senile systemic amyloidosis with seminal vesicle involvement.

Hepatitis C Virus Plays No Role in the Pathogenesis of Immunoglobulin A Nephropathy in Liver Cirrhosis

Dr. E.H. Ström, Institute of Pathology, Schönbeinstrasse 40, CH-4003 Basel (Switzerland) Dear Sir, Several studies have demonstrated the presence of IgA nephropathy (IgA-N) in up to 50% of patients with liver cirrhosis [1]. Alcohol-induced cirrhosis has been the most common liver disease associated with IgA-N. However, renal IgA deposits are frequently found in posthepatitic and biliary cirrhosis. Several pathogenetic mechanisms for the renal deposition of IgA have been suggested; impaired clearance of circulating IgA immune complexes by defective Kupffer cells, functional portocaval shunts that allow immune complexes to bypass hepatic degradation, defective biliary transport of IgA and hypocomplementemia. Alcohol abuse, even in the absence of liver disease, has also been suggested. Viral diseases, such as hepatitis B and cytomegalovirus, have been associated with liverrelated and idiopathic IgA-N. The frequent association of hepatitis C virus (HCV) infection with mixed cryoglobulinemia and glomerulonephritis has recently been demonstrated, especially in Italian patients [2]. HCV infection with membranous and membranoproliferative glomerulonephritis are reported in case studies [3, 4], Moreover, Francisco et al. [5] reported a black American patient with chronic active hepatitis related to HCV in whom IgA-N developed in a renal transplant. Recently, antibodies to HCV were found to be present in 32-55% of cirrhotic patients from Spain and France, especially in alcoholic and autoimmune cirrhosis [6, 7]. In a Japanese study of patients with alcoholic cirrhosis, 60% had HCV markers, in most of them, both HCV ribonucleic acid and HCV antibodies were present [8]. We, therefore, investigated the relationship between HCV and cirrhotic IgA-N in an autopsy study of 38 cases of liver cirrhosis (24 alcoholic, 6 posthepatitic, 1 biliary and 7 of uncertain etiology). The presence of HCV antibodies was investigated on frozen stored plasma by the quantitative 2nd-generation enzyme immunoassay Abbott HCV EIA. Positive results were confirmed by the 5-antigen 2nd-generation immunoblot assay Chiron RIB A HCV test system. In 27 cases, fresh-frozen renal tissue was investigated for glomerular IgA by immunofluorescence. Thirteen cases (48.1%) were positive for IgA (8 alcoholic, 2 posthepatitic, 3 of uncertain etiology). Only 2 cases, both posthepatitic cirrhosis without glomerular IgA deposits, were anti-HCV positive.