Species cross-reacting epithelial and urothelial specific antigens in human fetal, adult, and neoplastic bladder epithelium.

Antiserum from a New Zealand White rabbit was raised against the saline-soluble fraction of homogenates of calf urinary bladder epithelium. After absorption, with human red blood cells, human, calf, and mouse sera, and nonepithelial organs, this antiserum was specific for all human epithelia tested. Further absorptions with nonurinary tract epithelia rendered the antiserum specific for urothelium. Species cross-reactivity was seen between the superficial cell layers of calf, human fetal, and adult bladder epithelia. The antigen appeared to be cytoplasmic and was also present (although to a lesser extent) in human bladder tumor biopsy specimens and human bladder tumor cell lines.

Assessment of axillary lymph node involvement in breast cancer patients with positron emission tomography using radiolabeled 2-(fluorine-18)-fluoro-2-deoxy-D-glucose.

BACKGROUND: The presence of metastatic tumor cells in the axillary lymph nodes is an important factor when deciding whether or not to treat breast cancer patients with adjuvant therapy. Positron emission tomography (PET) imaging with the radiolabeled glucose analogue 2-(fluorine-18)-fluoro-2-deoxy-D-glucose (F-18 FDG) has been used to visualize primary breast tumors as well as bone and soft-tissue metastases. PURPOSE: This study was undertaken to evaluate before surgery the diagnostic accuracy of PET for detection of axillary lymph node metastases in patients suspected of having breast cancer. METHODS: Women who were scheduled to undergo surgery for newly discovered, suspected breast cancers were referred for PET imaging of the axilla region. The women were first clinically examined to determine the status of their axillary lymph nodes (i.e., presence or absence of metastases). Fifty-one women were intravenously administered F-18 FDG and were studied by PET imaging. Attenuation-corrected transaxial and coronal images were visually evaluated by two nuclear medicine physicians (blinded to the patient's medical history) for foci of increased F-18 FDG uptake in the axilla region. All patients underwent surgery for their suspected breast cancers. Axillary lymph node dissection was also performed on all patients with breast cancer, with the exception of four patients who received primary chemotherapy for locally advanced breast cancer. A single pathologist analyzed breast tumor and lymph node tissue specimens. RESULTS: The overall sensitivity (i.e., the ability of the test to detect disease in patients who actually have disease) and specificity (i.e., the ability of the test to rule out disease in patients who do not have disease) of this method for detection of axillary lymph node metastases in these patients were 79% and 96%, respectively. When only patients with primary breast tumors larger than 2 cm in diameter (more advanced than stage pT1; n = 23) were considered, the sensitivity of axillary PET imaging increased to 94%, and the corresponding specificity was 100%. Lymph node metastases could not be identified in four of six patients with small primary breast cancers (stage pT1), resulting in a sensitivity of only 33%. Axillary PET imaging provided additional diagnostic information in 12 (29%) of 41 breast cancer patients with regard to the extension of disease to other sites (i.e., other lymph nodes, skin, bone, and lung). CONCLUSIONS: PET imaging with F-18 FDG allowed accurate and noninvasive detection of axillary lymph node metastases, primarily in patients with breast cancer more advanced than stage pT1. Detection of micrometastases and small tumor-infiltrated lymph nodes is limited by the currently achievable spatial resolution of PET imaging. IMPLICATIONS: In clinical practice, PET imaging cannot substitute for histopathologic analysis in detecting axillary lymph node metastases in breast cancer patients. PET imaging, however, improves the preoperative staging of the disease in breast cancer patients and, therefore, might alter therapeutic regimen options.

Immunohistochemical assessment of individual tumor cells in lymph nodes of patients with non-small-cell lung cancer.

PURPOSE: This prospective study was designed to evaluate the prognostic relevance and biologic characteristics of a minimal lymphatic tumor load in non-small-cell lung cancer (NSCLC). METHODS: Frozen-tissue sections from 391 regional lymph nodes of 72 patients with completely resected NSCLCs, who were staged as free of metastases (pT1-3, pN0,M0,R0) by clinical tumor staging procedures and histopathologic examinations, were studied. For tumor-cell detection, we applied the alkaline phosphatase-antialkaline phosphatase (APAAP) immunostaining technique with monoclonal antibody Ber-Ep4 against two glycoproteins of 34 and 49 kd present of the surface and cytoplasm of epithelial cells. RESULTS: Individual Ber-Ep4-positive cells were detected in 11 of 72 (15.2%) cancer patients, while positive staining was consistently absent in all sections from control nodes of 24 noncarcinoma patients. No correlation between a positive lymph node finding and either the size or differentiation grade of the primary tumor or the presence of micrometastatic tumor cells in bone marrow assessed by immunocytochemistry with antikeratin monoclonal antibody CK2 was observed. Following a median observation time of 26.0 months (range, 15 to 39), patients with lymph node micrometastases showed a significantly shorter disease-free survival duration than node-negative patients (log-rank test, P = .005). The independence of this prognostic significance was demonstrated by a multivariate analysis (Cox regression model, P = .005). CONCLUSION: Our results provide evidence that the presence of single lung carcinoma cells in lymph nodes is an independent indicator of the disseminatory capacity of an individual primary tumor. Immunohistochemical assessment of micrometastases in lymph nodes is recommended for current tumor staging in NSCLC, as it might lead to better stratification of patients for adjuvant therapy.

Metabolic characterization of breast tumors with positron emission tomography using F-18 fluorodeoxyglucose.

PURPOSE: To evaluate the diagnostic value of position emission tomographic (PET) imaging with F-18 fluorodeoxyglucose (FDG) in differentiating between benign and malignant breast tumors. PATIENTS AND METHODS: Fifty-one patients, with suspicious breast lesions newly discovered either by physical examination or by mammography, underwent PET imaging before exploratory surgery. FDG-PET images of the breast were analyzed visually and quantitatively for objective assessment of regional tracer uptake. RESULTS: Primary breast cancer was identified visually with a sensitivity of 68% to 94% and a specificity of 84% to 97% depending on criteria used for image interpretation. Quantitative analysis of FDG uptake in tumors using standardized uptake values (SUV) showed a significant difference between benign (1.4 +/- 0.5) and malignant (3.3 +/- 1.8) breast tumors (P < .01). Receiver operating characteristic (ROC) curve analysis exhibited a sensitivity of 75% and a specificity of 100% at a threshold SUV value of 2.5. Sensitivity increased to 92% with a corresponding specificity of 97% when partial volume correction of FDG uptake was performed based on independent anatomic information. CONCLUSION: PET imaging allowed accurate differentiation between benign and malignant breast tumors providing a high specificity. Sensitivity for detection of small breast cancer ( < 1 cm) was limited due to partial volume effects. Quantitative image analysis combined with partial volume correction may be necessary to exploit fully the diagnostic accuracy. PET imaging may be helpful as a complimentary method in a subgroup of patients with indeterminate results of conventional breast imaging.

Localization of organ-specific antigens in mouse lung by light and electron microscopy.

Rabbit antisera were raised against whole homogenate and a saline soluble fraction of C57BL mouse lung. After absorption with other mouse organs, both antisera gave specific staining of bronchiolar epithelial and single alveolar cells, using the indirect immunofluorescence and indirect immunoperoxidase techniques on both acetic-alcohol-fixed paraffin-embedded and unfixed frozen 5 micrometer sections. Improved resolution by light microscopy was achieved with 1 micrometer sections of formaldehyde-fixed Araldite-embedded material stained by an indirect immunoperoxidase method after the Araldite had been treated with saturated alcoholic NaOH for 5 min. Clara cells and type II pneumocytes were identifiable as sites of the lung-specific immune reaction. At the electron microscopic level the reaction was localized over the granules of bronchiolar Clara cells. The lamellar bodies of type II pneumocytes were extracted in the ultrathin sections and no immune reaction was observed.

Distribution of tissue polypeptide antigen (TPA) in normal human tissues: Immunohistochemical study on unfixed, methanol-, ethanol-, and formalin-fixed tissues.

The distribution of tissue polypeptide antigen (TPA) was studied in unfixed, methanol-, 95% ethanol-1% acetic acid (EA)-, and formalin-fixed paraffin-embedded sections of all adult human tissues using an indirect immunoperoxidase method. The specific staining patterns were virtually identical in unfixed and alcohol-fixed tissues, but in formalin-fixed tissues this similarity was found only after fixation for up to 24 hr and pretreatment with protease for 15 min. Although prolongation of formalin fixation beyond 48 hr increasingly diminished the TPA reactivity, TPA could still be demonstrated in tissues fixed in formalin for up to 6 months. TPA was found to be a cytoplasmic constituent of almost all adult human duct and cavity lining, simple, and stratified epithelia. TPA was not demonstrated in epidermis, renal proximal convoluted and testicular tubules, basket-like myoepithelial cells, nor in most glandular acini, including hepatocytes and pancreatic acinar cells. The TPA staining was also negative in all non-epithelial tissues, including lymph nodes and bone marrow. The well-defined epithelial distribution and the comparable demonstrability in differently preserved tissues make TPA a useful tool for the identification of cells of epithelial character.

Diversity in immunoreactivity of tumor-derived cytokeratin monoclonal antibodies.

The cytokeratins 8, 18, and 19, expressed in many normal and malignant epithelial cells, were purified from human gastrointestinal tumors and used as immunogen for hybridoma generation. The reactivity pattern of five of the generated ten monoclonal antibodies (MAb) was characterized biochemically and immunohistochemically. All of the generated MAb were reactive with the central rod portion of the cytokeratins, as determined after partial enzymatic degradation, and displayed characteristic reactivity patterns. MAb TS 4 exhibits pan-epithelial immunohistochemical reactivity staining of all epithelial structures, including all layers of epidermis and non-keratinizing squamous epithelium and myoepithelial cells. The determinant involved is present on several different cytokeratins, i.e., nos. 1, 5, 7, 8, and 15, as determined by immunoblotting experiments from different tissues and cell lines. MAb TS 1, TS 3, and TS 7 reveal pluri-epithelial reactivity pattern immunohistochemically, similar to TS 4, but they are unreactive with whole epidermis and with superficial cell layers of non-keratinizing squamous cells. MAb TS 1 was found to be highly specific and reactive only with cytokeratin 8. Furthermore, the TS 1 MAb alone can precipitate the antigen, indicating reactivity with repetitive epitopes on cytokeratin 8. MAb TS 3 and 7 bind to cytokeratins 7 and 8. Finally, MAb TS 8 was found to be immunohistologically the most restricted, in general lacking reactivity to hepatocytes, pancreatic and salivary gland acinar cells, proximal renal tubules, and luminal cells of the epididymis. TS 8 was mainly reactive with cytokeratin 19 and showed weak binding to cytokeratin 8 and 14.

Glucose metabolism of breast cancer assessed by 18F-FDG PET: histologic and immunohistochemical tissue analysis.

UNLABELLED: Breast cancer is characterized by elevated glucose consumption resulting in increased uptake of 18F-FDG. However, tracer uptake varies considerably among tumors imaged with PET. This study compared histologic and immunohistochemical tissue analysis of breast carcinomas with preoperative FDG uptake assessed by PET to identify tumor characteristics that define the degree of tracer accumulation. METHODS: FDG uptake in breast tumors was quantified by calculating standardized uptake values (SUVs) corrected for partial-volume effect and normalized to blood glucose level at the time of tracer injection. The histologic sections of 50 invasive and 6 noninvasive breast carcinomas were analyzed for histologic type, microscopic tumor growth pattern, percentage of tumor cells, presence of inflammatory cells, density of blood vessels, histopathologic grading, tumor cell proliferation (mitotic rate and antibody binding of MIB-1), expression of estrogen and progesterone receptors, and expression of the glucose transporter protein Glut-1. RESULTS: A positive correlation was found between FDG uptake and histologic tumor type (ductal vs. lobular; P = 0.003), microscopic tumor growth pattern (nodular vs. diffuse; P = 0.007), and tumor cell proliferation (MIB-1; P = 0.009). Tumors with diffuse growth patterns had significantly lower SUVs compared with clearly defined tumors. A weak relationship was found between FDG uptake and the percentage of tumor cells (P = 0.06). Lower densities of blood vessels corresponded to higher FDG uptakes (P = 0.08). However, even significant correlations showed poor correlation coefficients. No relationship was found between FDG uptake and the following: tumor size; axillary lymph node status; percentage of necrotic, fibrotic, and cystic compounds; presence of inflammatory cells; steroid receptor status; and expression of Glut-1. CONCLUSION: Histologic and immunohistochemical tissue analysis was unable to sufficiently explain the variation of FDG uptake in breast cancer. The degree of metabolic changes after malignant transformation is most likely explained by a complex interaction between cellular energy demand and tumoral microenvironment. Therefore, FDG PET imaging may not be used to estimate tumor biologic behavior of breast cancer such as differentiation, histopathologic grading, cell proliferation, or axillary lymph node status.

FDG PET evaluation of granular cell tumor of the breast.

Granular cell tumor is a rare, usually benign neoplasm of neural origin that may arise in virtually any site and, when situated in the breast, can mimic breast carcinoma. We describe a case of granular cell tumor of the breast in a 57-yr-old woman. Clinical evaluation, mammography, sonography and MRI suggested a carcinoma with infiltration of skin and muscle. However, the tumor did not display increased glucose metabolism on PET. Clinical findings, imaging results, histological characteristics and surgical management are discussed.

Model system for isolation of competent ovarian-carcinoma cells from fresh tumor-tissue by a magnetic separation technique (macs).

Flow cytometric analysis of tumor cells in carcinomas is hampered by the presence of a variety of different cells in the tumor tissue and the surrounding stroma. To obtain single competent tumor cells, we have established a model system which can be applied to separate living cells from fresh ovarian carcinoma tissue. Due to the lack of tumor-cell surface specific antibodies, we isolated tumor cells by a procedure called 'negative tumor cell selection'. For this purpose, fresh ovarian carcinoma tissue, immediately after surgery, was subjected to mechanical disintegration using an automated mincing device to obtain a single-cell suspension (approximately 10(7) cells/g). Collagenase D (0.005%) was added to prevent further aggregation. Cells other than tumor cells were then labeled with a set of monoclonal antibodies directed to cell surface antigens: CD3 (T-cells), CD14 (monocytes), CD15 (granulocytes), CD45R (T-/B-cells) and 5B5 (fibroblasts). Anti-isotype antibodies coupled to ferrit microbeads were then reacted with the cell suspension and those cells reacting with the microbeads retained on a steel wool matrix in a magnetic field (1). Tumor cells not reacting with the microbeads were recovered by a simple wash of the steel wool matrix. All incubation steps were at 4 degrees C. This procedure, which takes about 2 hours, enables fast and simple isolation of single, living competent tumor cells from fresh tumor tissue and also from ascitic or pleuritic effusions. In a model system with cultured ovarian carcinoma cells and human leukocytes, tumor cell purity was about 93% and about 97% when re-subjected to the same procedure (respective recovery rates 75% and 50%). The still unlabeled tumor cells can subsequently be analyzed by flow cytometry or by central laser scanning microscopy for the presence of various surface antigens including receptors for proteases or growth factors. Moreover, after detergent treatment and fixation, flow cytometric multiparameter analysis such as simultaneous labeling of intracellular and surface antigens as well as nuclear DNA staining for ploidy and S-phase determination becomes possible.

Distribution of tissue polypeptide antigen (TPA) in normal mucosa, in colitis ulcerosa, in adenomas and in carcinomas of the human colorectum. An immunohistochemical study.

65 carcinomas with their normal resection margins, 30 adenomas of the colorectum, and also ulcerative colitis biopsies from 10 cases were analysed immunohistochemically for pattern and intensity of expression of Tissue Polypeptide Antigen (TPA). In normal colon, and in well- and moderately-differentiated carcinomas, a cell membrane type staining pattern was predominant. In ulcerative colitis, in carcinoma cell groups within mucus of mucinous carcinomas or in single cells at the invasion front of all grades of carcinomas, a strong cytoplasmic type staining pattern was found. The cytoplasmic pattern was also found in poorly differentiated carcinomas, but with weaker staining intensity. The relationship between staining intensity and pattern and carcinoma grade was significant, whereas a similar relationship with the Dukes stages was not significant.

Cytokeratins, smooth muscle actin and vimentin in human normal salivary gland and pleomorphic adenomas. Immunohistochemical studies with particular reference to myoepithelial and basal cells.

The distribution of immunostaining in normal major salivary gland and in 12 pleomorphic adenomas was studied using monospecific monoclonal antibodies to a number of cytokeratins, including cytokeratin 14, to smooth muscle actin and vimentin. A number of these antibodies enabled a distinction to be made between structural components of the normal gland, and to relate this to the different structures of pleomorphic adenomas. In the normal gland, the luminal duct cells expressed cytokeratins 7, 8, 18 and 19. Three antibodies were of particular value for the characterization of normal myoepithelial and basal cells; while the antibody to smooth muscle actin and the cytokeratin antibody Ks8.12 mutually exclusively stained the myoepithelial (basket) cells and the basal duct (light) cells, respectively, the recently established monospecific antibodies to cytokeratin 14 showed specific immunostaining with both cell types. These three antibodies left luminal cells virtually unstained. Ck 13 was found occasionally in single luminal excretory duct cells. Antibodies to cytokeratins 1/2, 10 and 10/11 did not show any staining in the normal gland. In the pleomorphic adenomas, the staining pattern of the two-layered tubular formation resembled that of the normal gland ducts: tumour luminal cells showed the characteristic, although more irregular, expression of cytokeratins 7, 8, 18 and 19; the outer cells resembled normal ductal basal cells with their anti-cytokeratin 14/Ks8.12-epitope staining and in that they virtually lacked staining for smooth muscle actin. Trabecular formations and cells in myxoid areas were reactive with Ks8.12 and for cytokeratin 14, occasionally also for cytokeratins 7, 18 and 19. Epidermoid cell islets expressed mainly cytokeratin 14 and inconsistently the squamous epithelial cytokeratin 13 and the epidermal cytokeratin 10/11. Vimentin was found in cells of myxoid areas. The results support the postulate that some of the normal duct basal cells act as reserve cells and can give rise to tumour formation with a primitive myxoid or trabecular pattern and a more differentiated tubular or epidermoid configuration.

Amyloid goiter and arthritides after kidney transplantation in a patient with systemic amyloidosis and Muckle-Wells syndrome.

A case of hereditary AA amyloidosis with Muckle-Wells syndrome is described. After a successful kidney transplantation for chronic renal failure due to renal amyloid deposits at age 21, the patient, a white female now 26 years of age, developed a large amyloid goiter as a manifestation of the systemic amyloidosis and recurrent monarthritides. Both observations are novel for this disease. Subtotal thyroidectomy and oral colchicine administration, known to be effective in preventing complications of familial Mediterranean fever, another hereditary type of AA amyloidosis, proved highly effective in the management of this unusual case.

p53 alterations in thymic epithelial tumours.

The prognosis of thymic epithelial tumours depends on malignant behaviour that cannot always be predicted on histological grounds. This study aimed at identifying a molecular marker that would be useful in overcoming the drawbacks of histopathology. Forty-four thymic epithelial tumours were analysed for alterations of the tumour suppressor gene p53 using immunohistochemistry (antibodies D0-1 and CM-1) and PCR-based single-strand conformation polymorphism and DNA sequencing. Histological and clinical evaluation and also p53 analysis revealed three major tumour groups: non-organotypic thymic carcinomas with frequent p53 alterations (7/9) and occurrence of p53 gene mutations (2/9); malignant thymomas with frequent p53 alterations but without p53 gene mutations (11/18); and benign thymomas with rare p53 alterations and without p53 gene mutations (2/17). In non-organotypic thymic carcinomas p53 was detected with both antibodies. In contrast, thymomas lacked immunoreaction with D0-1 suggesting alteration of the antibody-binding site. Overall immunohistochemical results correlated with clinical stages (P < 0.01), pathohistology (P < 0.01), and survival times (P < 0.05). We consider immunohistochemical p53 detection to be a useful new prognostic factor for the evaluation of thymic epithelial tumours.

MRI of the breast--histopathologic correlation.

132 solid breast masses have been examined at our institution by MR and have consequently been histopathologically correlated. T1- and T2-weighted SE and multi-echo sequences have been evaluated visually. It was found that signal intensities of tissues on T2-weighted images correlated with the contents of fibrosis, cells or water. Thus in some lesions (which consisted of different tissue components), a characteristic internal structure was visible on T2-weighted images, reflecting their histopathologic structure. Corresponding to their different composition, differences of signal intensity have also been noted between those fibroadenomas with a high contents of fibrosis and all other well-circumscribed breast lesions (fibroadenomas, carcinomas). However, for the majority of lesions with irregular contours a discrimination based on signal intensities or calculated T1- and T2-values did not seem possible. This overlap can also be explained by the macroscopically similar composition (amount of fibrosis, water or cells) of benign and malignant irregular lesions.

Keratin, luminal epithelial antigen and carcinoembryonic antigen in human urinary bladder carcinomas. An immunohistochemical study.

14 urinary bladder carcinomas of all main types were investigated with antisera to "broad spectrum keratin" (aK), "luminal epithelial antigen" (aLEA) and carcinoembryonic antigen (aCEA), using an indirect immunoperoxidase method on formalin fixed paraffin embedded sections. Keratin and LEA were both present in normal transitional epithelium, papilloma and carcinoma in situ whereas CEA was absent. Transitional cell carcinomas reacted with both aK and aLEA whereas CEA was seen only in a few foci. In squamous metaplasia and squamous carcinoma reaction with aK was particularly strong, while LEA was almost lacking and CEA was present in necrotic centres. In adenocarcinomas aK and aLEA reacted equally while aCEA reacted only on the surface.

Immunohistochemical localisation of keratin and luminal epithelial antigen in myoepithelial and luminal epithelial cells of human mammary and salivary gland tumours.

Rabbit antisera to human 40-63 000 MW epidermal keratin, one batch with restricted distribution of reactivity from an initial (aK1) and one with "broad spectrum" distribution of reactivity from a late bleeding (aK), and to "luminal epithelial antigen" (aLEA) were applied to formalin fixed paraffin embedded sections of human normal and neoplastic mammary and salivary glands using an indirect immunoperoxidase method. aK1 reacted with myoepithelial cells, aLEA with luminal epithelial cells and aK with both cell types in normal mammary and salivary gland. In breast carcinomas the majority of intraluminal and infiltrating carcinoma cells reacted with aLEA but not with aK1 which reacted only with surrounding myoepithelial cells. aK reacted with both myoepithelial cells and with intraluminal and infiltrating tumour cells. In the salivary gland adenomas the majority of cells reacted with aK, and those cells arranged in a tubular fashion reacted with aLEA.

Immunoelectron microscopic localisation of keratin and luminal epithelial antigens in normal and neoplastic urothelium.

Immunoelectron microscope cytochemistry was carried out on 2% paraformaldehyde fixed, 50 mu sections of normal urothelium and bladder carcinoma cells in culture using antisera raised in rabbits to human 40-63 000 MW epidermal "broad spectrum" keratin and calf urothelial "luminal epithelial antigen" (aLEA) Both the unconjugated and indirect immunoperoxidase-DAB techniques were used before routine embedding. The localisation of both keratin and luminal epithelial antigen (LEA) was similar in normal and neoplastic cells and reaction product was associated not only with tonofilaments but also lining membrane vesicles and on fine filaments in the cytoplasmic ground substance.

[Simpson's atherectomy of the peripheral arteries: early results and follow-up]. / Simpson-Atherektomie peripherer Arterien--Frühergebnisse und Nachkontrollen.

Over a seventeen-month period, a percutaneous transluminal removal of stenosing plaque material from leg and pelvic arteries was performed successfully and without complication in 43 patients. A complete atherectomy in the femoropopliteal vessels succeeded in 99% of cases. In the pelvic region, the primary results were much lower (58%). After six months, the angiographically checked restenosis rate was 17% for femoropopliteal vessels and 11% for iliac arteries, and the corresponding Kaplan-Meier cumulative patency rates were 73.8 and 78.7% respectively. Simpson's atherectomy is the only method of its kind that is therapeutically effective and diagnostically significant, since the removed plaque can be used for further tests.

[Simpson's atherectomy in embolizing leg artery stenoses]. / Simpson-Atherektomie embolisierender Beinarterienstenosen.

We report on the treatment and follow-up of six patients with an unilateral "blue toe" syndrome. This is caused by atheromatous micro-embolisation to the digital arteries and requires urgent attention due to the painful cutaneous necroses and impending digital gangrene. In all patients, Simpson's atherectomy of proximally situated femoropopliteal stenoses caused the pre-gangrenous digital changes to heal completely. In a mean observation period of ten months no relapse occurred. The embolising material was presumably parietal fibrino-platelet thrombi which could be observed with angioscopy and were regularly detectable histologically in the excised tissue.