New ELISA for quantitation of human urokinase receptor (CD87) in cancer.

The serine protease urokinase-type plasminogen activator (uPA), its inhibitor (PAI-1), and its receptor (uPAR; CD87) facilitate cancer cell invasion and metastasis. Whereas uPA and PAI-1 antigen levels determined in tumor tissue extracts of breast cancer patients correlate with disease recurrence and overall survival, the prognostic relevance of uPAR is still a matter of debate. We established two new sandwich-type enzyme-linked immunosorbent assay (ELISA) formats (HU/IIIF10-ELISA and HU/HD13-ELISA) using the epitope-defined monoclonal antibody (mAb) IIIF10 or the conformation-dependent mAb HD13.1, a polyclonal chicken antibody (HU277), and recombinant soluble uPAR (CHO-suPAR) as the standard. The lower detection limit of the assays was at 0.16 ng/ml, with a linear dose-response up to 5 ng/ml of uPAR antigen. Both ELISA formats showed good reproducibility and recovery. The intra-assay and the inter-assay variation coefficients were respectively 4.3% and 11.7% (HU/IIIF10-ELISA) and 4.0% and 10.7% (HU/HD13-ELISA). The recovery rate of uPAR in cell lysates spiked with CHO-suPAR was above 82% and 88%, respectively. With these new ELISA formats, uPAR antigen content in breast cancer tissue extracts and tumor cell lysates was determined and compared to a commercially available ELISA (ADI-ELISA). By all of the three uPAR ELISA formats CHO-suPAR and uPAR present in lysates of non-malignant epithelial cells and stimulated monocytes were quantified with similar sensitivity. Interestingly, in breast cancer cell lines of epitheloid origin a higher uPAR antigen content was determined by the HU/IIIF10-ELISA than the HU/HD13- or ADI-ELISA formats. In lysates of fibroblastic breast cancer cell lines similar uPAR values were obtained with the HU/IIIF10- and ADI-ELISA formats, whereas with the HU/HD13-ELISA significantly lower uPAR concentrations were determined. The prognostic relevance of tumor uPAR antigen was evaluated in 199 primary breast cancer patients with a median follow-up of 24 months. uPAR antigen values above the cut-off of 3.33 ng/mg protein as determined by the HU/IIIF10-ELISA were significantly correlated with short disease-free survival (p=0.025). Results obtained by the other two ELISA formats (HU/HD13-ELISA and ADI-ELISA) were not associated with prognosis. Our findings stress the need of well-characterized antibodies, which detect both uPAR of non-malignant and tumor cells, in setting up a uPAR-ELISA useful for assessing breast cancer patient prognosis.

Expression of p53 and bcl-2 in correlation to clinicopathological parameters, hormone receptor status and DNA ploidy in breast cancers.

The expression of p53 and bcl-2 was immunohistochemically investigated in 61 formalin-fixed, paraffin-embedded invasive breast carcinomas. The study was aimed to elucidate the relationship between both markers and the correlation of p53 and bcl-2, respectively, to clinicopathological variables, to hormone receptor status and to DNA-ploidy. Twenty tumors showed a positive reaction with the monoclonal antibody DO-1 against p53 protein. Its immunohistochemical demonstration was significantly correlated with a tumor size larger than 2 cm, a low estrogen receptor status and DNA-aneuploidy. Bcl-2 was demonstrated in 51 breast cancers. Bcl-2 was preferably seen in low grade and hormone receptor positive tumors. We found a negative correlation between the immunoreactive scores of p53 and bcl-2, but in 17 carcinomas a coexpression of both proteins was seen. Cases with this coexpression did not differ significantly from the other tumors in clinicopathological parameters. In eight of these cases more than 10% of the cells were found to be positive for both markers. In four cases we could show many cells to exhibit both markers as it was assessed by an immunofluorescence double labeling technique.

Demonstration of mouse mammary tumour virus (MuMTV) antigenicity in human milk by means of immunodiffusion technique.

Using the micro-Ouchterlony immunodiffusion technique, we were able to demonstrate mouse mammary tumour virus (MuMTV) antigenicity in the 1.26-1.28 g/ml density fraction prepared from pooled human milk samples after treatment with detergents and ether. The cross-reacting antigen(s) was precipitated by rabbit antisera to MuMTV-B particles prepared from murine milk and intracytoplasmic A particles (iAp) isolated from mouse mammary tumour tissue. By confluence of precipitin lines, the human milk "core" antigen(s) was shown to be identical with the main antigen(s) of iAp which are known to share antigenicity with the MuMTV-B particle cores. Electron microscopy of the human "core" fraction revealed abundant particulate structures of 40-70 nm in diameter. However, the structural entities bearing the cross-reactive antigen(s) remain to be identified.

[Immunohistochemical studies of chordomas and choroid tumors]. / Immunhistochemische Untersuchungen an Chordomen und chondroiden Tumoren.

Chordomas are rare slow-growing but locally invasive tumors. They are thought to develop from residues of the notochord. Three chordomas and ten chondroid tumors were investigated by an indirect immunoperoxidase method for expression of the epithelial markers: MAM-6, keratin, tissue polypeptide antigen (TPA) and for carcinoembryonic antigen (CEA). In all the chordomas investigated the antigens MAM-6, keratin and TPA were detected using monoclonal antibodies and conventional antisera, respectively. These data provide further evidence of the epithelial nature and the ectodermal origin of chordomas. Moreover, the findings suggest that immunohistochemical methods may be useful for the differential diagnosis between chordomas and morphologically similar chondroid tumors.

[Heart involvement in rheumatoid arthritis]. / Herzbeteiligung bei Rheumatoid-Arthritis.

It is reported on the malignant course of a case of rheumatoid arthritis, in which the participation of the heart became the cause of death. Here it is stated that we certainly enlarge our knowledge on the participation of the heart by modern cardiological diagnostics, but therapeutically are not yet able to engage decisively in the fateful course of the systemic rheumatoid basic disease. Therefore, efforts about an early cardiologic diagnosis are at present not yet of therapeutic consequences.

[Immunohistochemical demonstration of tissue markers in histopathologic diagnosis]. / Immunhistochemischer Nachweis von Gewebsmarkern in der histopathologischen Diagnostik.

Our experience with the demonstration of tissue markers in histopathology supports their value in differential diagnosis of routine material. Microscopic diagnostic methods are genuinely enriched by the use of such monoclonal antibodies and conventional antisera whose specificity has been adequately demonstrated using indirect peroxidase techniques in cut sections and on cytologic preparations. On the basis of several epithelial markers (carcinoembryonic antigen, keratin, tissue polypeptide antigen, MAM-antigens) and other antigens (protein S-100, chromogranin) examples are given for their application to routine differential diagnosis problems. The detection of these markers has been adapted to standardized indirect immunoperoxidase techniques. More sensitive tissue markers (i.e. antigens which do not withstand formalin fixation and paraffin embedding) should best be dealt with by specialized working groups.

Mammary tumour virus (MTV)-specific immune complex deposites in renal glomeruli of MTV-infected tumour-free mice.

Using direct and indirect immunofluorescence tests MTV-specific immune complex deposits were found in tumour-free female mice of the strains C3H/Bln, CBA/Bln, XVII/BlnfCBA/Bln, and CBA/BlnfXVII/Bln. These deposits consist of immunoglobulin, complement, and MTV antigen(s). The immune complex deposition increases with age. Antibodies eluted from renal tissue homogenate react with both MTV-A and -B particles in immunofluorescence tests performed on mouse mammary tumour slices. By these results the earlier finding of age-dependent spontaneously occurring anti MTV antibodies in naturally MTV-infected mice is confirmed and completed.

Late local and pulmonary recurrence of renal cell carcinoma.

Locally recurrent renal cell carcinoma and multiple pulmonary metastases were successfully resected in a patient 20 years after nephrectomy.

[Immunocytochemical studies of epithelial markers in pleural and peritoneal effusions as an adjunct to cytologic diagnosis]. / Immunzytologischer Epithelmarkernachweis in Pleura- und Aszitespunktaten als Ergänzung zur Zytodiagnostik.

Monoclonal antibodies were used in an indirect immunoperoxidase assay to investigate 83 smears of pleural or peritoneal effusions for expression of the epithelial markers MAM-6, MAM-3, and carcinoembryonic antigen (CEA). All but one smears containing tumor cells according to the evaluation of H&E preparations were positive for one, two or all three markers, the exception being a malignant melanoma. Three of 5 cases, suspicious by routine cytology, exhibited marker expression in a different number of cells and thus confirmed the cytological diagnosis. Five of 63 cytologically negative smears exhibited single cells or small cell clusters positive for up to three markers. Four of these patients were found to suffer from metastatic cancer, as established by clinical follow-up and subsequent biopsy or autopsy, respectively. Felty's syndrome and concomitant serositis were diagnosed and confirmed by autopsy in one of the MAM-6 positive cases with negative cytology. The comparatively strong MAM-6 expression in some mesothelial cells of this patient might have been induced by abnormal stimulation due to the rheumatic disease. The results of this study encourage wider use of immunocytochemistry as an adjunct to cytological diagnosis in effusions.

Monoclonal antibodies to tumor-associated antigens.

Experience is described in applying monoclonal antibodies (Mabs) to immunohistology, epitope analysis on antigens, and radioimmunoassay. The antibodies (provided by international laboratories) used in the experiments described were directed to the mouse mammary tumor virus, a cell surface component of melanoma cells, several proteins of the human milk fat globule membrane (as markers for cell differentiation in breast cancers) as well as to Carcinoembryonic Antigen (CEA). At least three antigenic determinants were detected on CEA, which appeared as being expressed in different quantities in those organs producing CEA-like molecules (lung, spleen, normal colon mucosa, liver). By means of an inhibition radioimmunoassay for CEA, evidence was obtained that in the lung more than one cross-reacting antigen might exist, one of them of high molecular weight (about or more than 200,000 daltons). In immunoperoxidase tests, several antibodies to CEA were seen to react with pulmocytes and bronchus glands. Summarizing the experience, it is concluded that thorough characterization of monoclonal antibodies by more than one method is a prerequisite to their use for analytical or diagnostic purpose.

Primary renal non-Hodgkin's lymphoma - a difficult differential diagnosis.

INTRODUCTION: Primary renal lymphoma (PRL) as a clinical entity is not undisputed because the kidneys do not contain lymphatic tissue and the mechanism of development of PRLs is unclear. Most of the few cases reported showed rapid systemic progression and a poor prognosis. Although there are no clearly defined diagnostic criteria for renal lymphomas, abdominal and thoracic computed tomography as well as renal and bone marrow biopsy are recommended. 3 cases of renal lymphoma are reported and their diagnosis and management discussed. CASE REPORTS: Between 1996 and 2001, 3 male patients with renal lymphoma were diagnosed and treated at our institution. In patient No. 1, because of persisting macroscopic hematuria a bilateral PRL was diagnosed by renal biopsy, without any detectable lesions on CT imaging. Patient No. 2 presented with a large renal mass which, on biopsy, was diagnosed as a lymphoma. Patient No. 3 showed lymphoma on renal biopsy and bone marrow involvement. All 3 patients were treated with systemic chemotherapy which resulted in death of disease in 2 patients and a complete remission in 1 patient after adjuvant radiotherapy and nephrectomy. CONCLUSION: PRL represents a rare entity which must nevertheless be considered in cases of unusual renal masses or otherwise unexplained renal symptoms. If diagnosed early, cure is possible, and multimodal treatment should be considered.

Solitary cervical lymph node manifestation of a seminoma without detectable primary.

The rare case of a 65-year-old male with a cervical lymph node manifestation as the only tumour manifestation is reported. The diagnostic and therapeutic management of the case is discussed. This represents the fourth case with such a condition reported in the literature.

Immunohistochemical localization of the epithelial marker MAM-6 in invasive malignancies and highly dysplastic adenomas of the large intestine.

MAM-6 antigen is detectable in a large variety of formalin fixed and paraffin embedded normal and neoplastic tissues throughout the body with some exceptions. One such exception is the large intestine in which normal mucosa has been found negative for MAM-6, while colorectal carcinomas express the antigen in almost all cases. We examined the expression of MAM-6 in 147 benign epithelial tumors (adenomas) of the colorectum and compared this with the detectability of the antigen in 123 colorectal cancers and in 108 non-neoplastic mucosa specimens. The tissue samples were subjected to an indirect immunoperoxidase assay applying the MoAb 115D8 which recognizes the epitope "a" of MAM-6. A majority of the carcinomas (99.2%) and of the adenomas (61.4%) exhibited reactivity for MAM-6 "a", whereas only 8 of the 108 non-neoplastic biopsy specimens (= 7.4%) exhibited faint focal staining. In adenomas, positive reactions were only seen in highly dysplastic tissue areas, in part reflecting carcinomata in situ. The reactivity in four non-neoplastic tissue samples could be explained by shedding of the antigen from carcinomas into glandular structures of the mucosa. Two out of 32 cases exhibiting severe colitis were also focally positive. The detectability of MAM-6 "a" in routinely processed specimens from the large intestine is therefore considered as being closely associated with malignant and premalignant processes. According to a limited study with the antibodies 139H2 and 140C1, this holds true for two additional MAM-6 epitopes. MAM-6 might be considered as a marker of severe (premalignant) dysplasia in adenomas of the large intestine. Further studies are needed to clarify if there is a biological (possibly prognostic) difference between MAM-6 positive and negative high epithelial atypia in these adenomas.

Favorable long-term outcome in adult genitourinary low-grade sarcoma.

OBJECTIVES: To report our experience treating sarcomas in 20 consecutive patients. METHODS: Pretreatment and follow-up data were obtained from 20 adult patients consecutively treated between 1992 and 1998 for primary or locally recurrent genitourinary sarcoma. RESULTS: Eight patients (40%) were classified as having high-grade and 12 (60%) low-grade disease. Except for 3 patients, the primary treatment was surgery alone. The median follow-up was 52 months. The actuarial disease-specific 5-year survival rate was 84% in all patients and was 100% for patients with Memorial Sloan Kettering Cancer Center (MSKCC) Stages 1-2 and 54% in MSKCC Stages 3-4. The disease-specific survival was significantly better in low-grade tumors (log-rank test, P = 0.0063) and inguinal-scrotal tumors (P = 0.019), tumors 5 cm or less (P = 0.039), and MSKCC Stages 1-2 tumors (P = 0.0035). CONCLUSIONS: The results of this study with a high proportion of low-grade, low-stage, and inguinal-scrotal sarcomas demonstrate the favorable prognosis of these subgroups.

Epithelial markers for paraffin-embedded human tissues. Immunohistochemistry with monoclonal antibodies against milk fat globule antigens.

About 200 human tumours and corresponding normal tissue samples were investigated by immunoperoxidase tests for the expression of MAM-3, MAM-5 and MAM-6 antigens, which had previously been defined by monoclonal antibodies to human milk fat globule membranes. All tissue specimens had been treated for routine histopathology, i.e. fixed in formalin and embedded in paraffin. One of the antigens, MAM-6, appeared to be an important epithelial marker, present in all normal and neoplastic breast tissue samples, in about 80% of non-mammary normal tissues and in more than 90% of non-mammary epithelial tumours. It could never be detected in normal and neoplastic mesenchymal and neuroectodermal structures. Direct comparison with the distribution of Carcinoembryonic Antigen (CEA), Tissue Polypeptide Antigen (TPA) and keratin provided clear evidence that MAM-6 is different from these well known epithelial markers. MAM-3 proved to be an additional important marker exhibiting a characteristic distribution pattern in those epithelial tissues investigated. In contrast to MAM-6, it could never be detected in renal cell cancers and carcinomas of the prostate gland, thus allowing differential diagnosis on the basis of immunohistochemistry. MAM-5, known to be associated with lactoferrin, was mainly detectable in secretory organs and their tumours. In the group of breast tumours, its expression was mainly seen in lobular cancers. These findings suggest a use for these new markers for routine histopathology.

Monoclonal antibodies to epithelial sialomucins recognize epitopes at different cellular sites in adenolymphomas of the parotid gland.

Fourteen monoclonal antibodies (MAbs) to epithelial sialomucins were studied for their immunohistochemical reactivity on serial sections of 14 formalin-fixed and paraffin-embedded adenolymphomas of the parotid gland. Two types of reactivity were observed, suggesting different cellular distribution of the corresponding epitopes. Most antibodies reacted with the luminal membrane of the columnar tumor cells (type-A reaction). The other reaction (type B) was observed with the membrane of basal epithelial cells. The antibodies could be ranked according to their tendency to show type-A and/or type-B reactions. MAb Cal was the only one with a pure type-A reaction. A strong tendency to type-A reactivity (with traces of type-B reactions) was observed for the antibodies HMFG-2, M8, E29 and NCRC-II. Several antibodies gave good type-B reactions in addition to strong type-A reactivity (MAbs 126E7, 115G2, 115D8, 140C1, F36/22, 139H2). MAb DF3 showed equally strong reactions with both cell types. A clear-cut preference of the reactions with basal cells was seen with the antibodies HMFG-1 and 115F5. This subclassification of the antibodies is in accordance with epitope mapping data, obtained by conventional blocking studies reported in the literature.

Spontaneous regression of hepatocellular carcinoma confirmed by surgical specimen: report of two cases and review of the literature.

Two cases with spontaneous regression of a histologically confirmed hepatocellular carcinoma (HCC) are presented. This rarely seen phenomenon of a spontaneous tumor involution is discussed and compared with the current literature. The clinical symptoms were very similar to that of a liver abscess. A 56-year-old male suffered from a multicentric, highly differentiated, trabecular HCC. First symptoms were epigastric pain, septic fever and arthritis. The tumor marker AFP was constantly normal and no hepatitis could be verified. A resection of the tumor was performed. In patient 2, a 74-year-old male, a multicentric, clear cell HCC was found. The patient had completely recovered from hepatitis type B and within the liver tissue no viruses could be identified. Clinical symptoms were mainly characterized by upper abdominal pain and septic fever. AFP was excessively elevated (3850 ng/ml) but returned to normal preoperatively. In both cases, the specimen showed a subtotal necrotic HCC with insignificant amounts of vital tumor cells. Neither patient had a liver cirrhosis macroscopically, however patient 2 had local periportal fibrosis histologically. After 24 and 41 months of follow-up, respectively, both patients are in good health