H. Kosmehl

Molecular variants of fibronectin and laminin: structure, physiological occurrence and histopathological aspects

This review deals with biological and pathological aspects of various isoforms of the matrix molecules fibronectin and laminin. They are generated by different molecular mechanisms: ED-A+ and ED-B+ fibronectin by alternative splicing of pre mRNA, de novo-glycosylated fibronectin by alternative post-translational O-linked glycosylation of the IIICS region, and the laminin isoforms by exchange of single chains of the heterotrimeric molecule. In contrast to the “common” fibronectin, the distribution of ED-B+ and de novo-glycosylated fibronectin is restricted to embryonic tissues; they subsequently reappear in granulation tissue, in fibrosing processes and in tumour stroma. The expression of these so-called oncofetal fibronectins is stimulated by growth factors (TGFβ). The association of the ED-B+ fibronectin with proliferative activity and newly formed vessels identifies this fibronectin variant as a marker of cellular activity in the process of fibrosis and as a suitable agent for the evaluation of tumour angioneogenesis. Initial results suggest a correlation between the amount of ED-B+ and de novo-glycosylated fibronectin in tumour stroma and the behaviour of carcinomas with regard to their invasiveness and propensity for metastatic dissemination. The current nomenclature of the laminin molecule family is presented. The laminin chain constitution of basement membranes switches from embryonic or proliferatively active to adult terminally differentiated tissues [disappearance of the laminin β2 (s) chain] and depends on the tissue type. The discrepancy between the loss of basement membranes (multiple basement membrane defects) in carcinomas and the recently reported increased laminin chain synthesis in these tumours may be explained by abundant laminin chain deposition outside the basement membrane in the carcinoma invasion front, possibly associated with enhanced adhesion of budding tumour cells.

TGFβ and bFGF synthesis and localization in Dupuytren's disease (nodular palmar fibromatosis) relative to cellular activity, myofibroblast phenotype and oncofetal variants of fibronectin

SummaryNodular palmar fibromatosis is a self-limited proliferation of fibro-/myofibroblasts associated with growth factor synthesis and abundant fibronectin extracellular matrix deposition. bFGF and TGFβ are potent modulators of fibro-/myofibroblast proliferation and differentiation. Moreover, in vitro investigations evidenced a TGFβ1-dependent regulation of alternative splicing of fibronectin mRNA. To investigate a possible implication of these growth factors in the tissue formation process of palmar fibromatosis, TGFβ1/2 and bFGF synthesis, as well as TGFβ1/3 and bFGF tissue distribution, is demonstrated by RNA in situ hybridization and/or immunohistochemistry in relation to myofibroblast phenotype development (α-smooth muscle actin, desmin immunohistochemistry), expression of different fibronectin isoforms (ED-A+, ED-B+ and oncofetal glycosylated fibronectin immunohistochemistry, fibronectin RNA in situ hybridization) and cellular activity (cyclin RNA in situ hybridization, Ki-67 immunolabelling). The myofibroblast phenotype (α-smooth muscle actin, desmin), the growth factor synthesis (TGFβ1 and 2, bFGF), fibronectin matrix synthesis (RNA in situ hybridization with cDNA) and ED-A+, ED-B+ and oncofetal glycosylated fibronectin immunostaining are exclusively localized in the active proliferative nodules (Ki-67 immunolabelling and cyclin mRNA demonstration). Whereas the growth factor synthesis is restricted to the proliferative areas of the fibromatosis only, TGFβ1, TGFβ3 and bFGF proteins can also be detected immunohistochemically with a lower intensity in the surrounding aponeurotic tissue. The spatial correlation of myofibroblast phenotype, TGFβ and bFGF synthesis and the occurrence of the oncofetal molecular fibronectin variants (ED-B+ and oncofetal glycosylated fibronectin) in the active proliferative fibromatosis nodules suggests a pathogentic role of these growth factors and matrix components in the tumorous tissue formation process. The presence of the bFGF and TGFβ1/3 proteins in fibroblasts neighbouring the proliferative nodules may point to a recruitment of quiescent aponeurotic fibroblasts in the fibromatous tissue formation process.

Elevated activity and expression of Src-family kinases in human breast carcinoma tissue versus matched non-tumor tissue

Abstract Src-family kinase expression was measured in 52 human mammary tumor (T) specimens compared with non-tumor (NT) tissue from the same patient by enzymatic assays employing a Src-kinase family-specific peptide substrate and by immunoblotting with an antibody recognizing the Src-family kinases Src, Fyn, and Yes. In the T specimens, the mean enzymatic activity was moderately elevated (T: 160u2009fmol ATP min−1u2009mg−1; NT: 115u2009fmolu2009ATPu2009min−1u2009mg−1) with 25 tumor samples having higher activity than the corresponding NT tissue, 17 having lower activity, and no activity detectable in tenu2009T/NT pairs. Immunoblotting revealed clearly elevated expression in 25 tumor tissues and no differences or expression below the detection limit in the remaining T/NT pairs. The data are in agreement with a possible role of Src-family kinases for the biology of mammary carcinoma.

Synthesis and protein distribution of the unspliced large tenascin‐C isoform in oral squamous cell carcinoma

The inclusion or omission of the alternatively spliced region in the tenascin‐C (Tn‐C) mRNA gives rise to the large (Tn‐CL) or small (Tn‐CS) variant, respectively. Tn‐CL is thought to be a typical component of provisional extracellular matrices (ECMs) and is expressed during tumour stroma remodelling. Tn‐CL synthesis has been studied using RNA/RNA in situ hybridization, and Tn‐CL protein distribution, using immunohistochemistry (clone BC‐2), in 18 oral squamous cell carcinomas (OSCCs) of different grades of malignancy. While the Tn‐CL protein was demonstrated within the whole stromal compartment regardless of grade of malignancy, the majority of the Tn‐CL mRNA signal‐bearing cells were carcinoma cells. Only a few stromal myofibroblasts were able to synthesize Tn‐CL, as revealed by α‐smooth muscle actin double staining. In well‐differentiated carcinomas (G1), the Tn‐CL synthesizing carcinoma cells were localized as a single positive cell layer in the tumour stroma interface, particularly in invasive areas. A higher grade of malignancy (G2/G3) is associated with a significantly increased number of Tn‐CL synthesizing carcinoma cells randomly distributed within the invading tumour areas. Double‐staining experiments (Tn‐CL mRNA ISH/BC‐2 immunohistochemistry) indicate that these cells are capable of organizing and depositing a three‐dimensional Tn‐CL matrix. Even though an instructive and/or inductive role of the carcinoma cells in tumour stroma formation cannot be excluded, these results demonstrate that carcinoma cells can directly produce the ECM components of tumour stroma. Copyright

Real Indications for Adrenalectomy in Renal Cell Carcinoma

Objectives: Adrenalectomy is a part of radical nephrectomy because of the surgical oncology principle of a ‘wide margin beyond the malignancy’ and due to concern over possible metastases to the ipsilateral adrenal gland, especially in upper pole tumors. But, neither the frequency, predisposing factors of the renal cell carcinoma nor mechanisms of involvement of the adrenal gland are well defined. We assessed the ipsilateral adrenal involvement in renal cell carcinoma to determine whether ipsilateral adrenalectomy during radical nephrectomy is essential. Material and Method: In a series of 15,347 autopsies in Jena from 1985 through 1996, 272 renal cell carcinoma with 24 adrenal metastases were found. In the same period 9 adrenal metastases were found in 639 radical nephrectomies. Contralateral and bilateral metastases were seen in 15 cases of the autopsy series and in 2 cases of the operative series. Results: The risk of adrenal metastases correlated with multifocal tumors, pleomorphic cell type, anaplastic growth pattern and tumors that were larger than 2.5 cm. Of the 24 renal cell carcinomas with adrenal metastases in the autopsy series, 23 had evidence of widespread disease and 22 had lymph node metastases. A preoperative abdominal computerized tomography was performed in all 9 patients of the operative series with renal cell carcinoma and adrenal involvement. The adrenal gland was considered abnormal in 8 of the 9 cases (88.9%). Only in 1 patient was the computerized tomography incorrectly interpreted as negative. Conclusion: We think adrenalectomy should only be performed if there is radiographic evidence of metastases in the adrenal gland or adrenal infiltration by a large upper-pole tumor is possible. Macroscopically normal adrenal glands should not be removed during tumor nephrectomy because the need and benefit of routine adrenalectomy are extremely limited.

Expression of fibronectin splice variants and oncofetal glycosylated fibronectin in the synovial membranes of patients with rheumatoid arthritis and osteoarthritis

ObjectiveThe aim of this study was to define and compare the expression of fibronectin (Fn) isoforms in synovial tissue of patients with rheumatoid arthritis (RA) and osteoarthritis (OA).MethodsUsing monoclonal antibodies specific for total Fn, extra domain (ED)-A Fn, ED-B Fn, and oncofetal glycosylated Fn, we studied the expression of the Fn isoforms in synovium. Furthermore, in situ hybridization for the detection of ED-B Fn mRNA including a double labeling technique for the detection of cell type was applied.ResultsStrong expression of total Fn, ED-A Fn, oncofetal glycosylated Fn and, to a lesser extent, ED-B Fn could be demonstrated in the synovial lining layer in both RA and OA. Stromal and vessel expression of Fn isoforms was more prominent in RA tissue. Pannus tissue showed strong labeling with ED-B Fn.ConclusionThe expression of alternatively spliced isoforms of Fn is associated with tissue remodeling and, as a partial process of this phenomenon, with neovascularization rather than underlying disease, X-ray status, or parameters of acute inflammation. In the lining layer, Fn expression correlates with hyperplasia associated with cell recruitment but not with proliferative status. Most remarkably, the expression of ED-B Fn in pannus tissue seems to be associated with the invasive phenotype described in RA tissue.

Multifocality in Renal Cell Carcinoma: A Bilateral Event?

Objectives: The major disadvantage of nephron-sparing surgery for renal cell carcinoma is the risk of local recurrence. This is most likely a manifestation of undetected small additional tumors in the renal remnant. To define more clearly the incidence and nature of unilateral and bilateral multifocal tumors, an autopsy study was undertaken. Materials and Methods: In a series of 14,793 autopsies from 1985 to 1995, 260 renal cell carcinomas were found. In all cases of renal cell carcinoma a search for small renal lesions was performed in the apparently normal-appearing portion of the kidneys. Every kidney was serially and systematically cut (5 mm) to probe for intraparenchymal lesions. Results: Of the 260 renal cell carcinomas 36 cases (13.85%) had multifocal malignant and/or benign nodules. The number of the additional nodules ranged from 2 to 18. 12% of the malignant multifocal carcinomas were limited to the ipsilateral kidney and 88% were bilateral. The average size of the multifocal renal lesions was 8.7 × 9.0 × 9.5 (range 3–23) mm. Renal cell carcinomas with low stage and good grading have a higher incidence of multifocal nodules. No significant difference was found with regard to metastasized and nonmetastasized renal cell carcinomas. In 38.1% of all chromophilic renal cell carcinomas additional nodules were found. Conclusions: Multifocality in renal cell carcinomas cannot be predicted reliably, although the papillary histological pattern, good grading and low staging seems to be associated with a higher incidence of multifocality. Nearly 90% of the multifocal nodules were bilateral.

Increased Transforming Growth Factor β1 Plasma Level in Patients with Renal Cell Carcinoma:A Tumor-Specific Marker?

Purpose: The most worrying problem with renal cell carcinoma (RCC) seems to be the prediction of metastases by means of tumor-specific markers. Therefore, much effort is committed to the development of new markers. Materials and Methods: The level of latent transforming growth factor β1 (TGF-β1) was measured in plasma samples by ELISA. These samples were collected from patients with RCC before they underwent radical nephrectomy, from patients 1 h after extracorporeal lithotripsy, from patients with pyelonephritis, and from healthy controls. Results: In all cases of RCC the levels of latent TGF-β1 in plasma were much higher (n = 20, 41.0 ± 13.9 ng/ml, range 19.3–78.1 ng/ml) than in healthy controls (n = 20, 3.8 ± 2.9 ng/ml, range 0.6–9.9 ng/ml, p < 0.0001). The TGF-β1 levels in plasma after extracorporeal lithotripsy (n = 20, 7.4 ± 4.64 ng/ml, range 2.9–21.7 ng/ml, p < 0.01) and in patients suffering from pyelonephritis (n = 20, 18.93 ± 14.2 ng/ml, range 4.2–46.7 ng/ml, p < 0.001) were also higher than in healthy controls. Conclusion: We conclude that increased levels of latent TGF-β1 are common in the plasma of RCC patients. The TGF-β1 plasma level in RCC was found to be significantly higher than in cases of inflammation. Thus, TGF-β1 is a possible tumor-prognostic marker in RCC.

Differential expression of fibronectin splice variants, oncofetal glycosylated fibronectin and laminin isoforms in nodular palmar fibromatosis

The tissue formation process in nodular palmar fibromatosis (Morbus Dupuytren) was investigated by the demonstration of fibronectin splice variants (ED-A and ED-B fibronectin), de novo glycosylated fibronectin and laminin isoforms (A, M, B1, B2, s chains) in association to the proliferative activity (Ki-67 antigen) and the occurrence of myofibroblast phenotype (alpha-smooth muscle actin, desmin). The proliferative noduli of the fibromatosis were characterized by a diffuse immunostaining for alpha-smooth muscle actin, and single cells positive for desmin and the Ki-67 antigen. In contrast to the surrounding aponeurosis as extracellular matrix, components of the whole proliferative noduli were defined: ED-A, ED-B and de novo glycosylated fibronectin, B1 and B2 laminin chain, tenascin and collagen type IV. The demonstration of the A and M laminin chain was restricted to a few cells of the proliferative noduli. S laminin could be visualized in the majority of palmar aponeurotic fibroblasts. As revealed by mRNA, in situ hybridization a de novo synthesis of fibronectin could only be detected within proliferative noduli. There is a positive correlation between the myofibroblast phenotype formation, cellular proliferation and the occurrence of ED-A and ED-B containing fibronectin, as well as de novo glycosylated fibronectin in Dupuytrens disease. The ultrastructural irregularities of myofibroblastic basal lamina and the heterogeneity of the myofibroblast phenotype are equivalent to the variability of laminin isoform immunostaining.

Quantitative Evaluation of Apoptosis and Proliferation in Renal Cell Carcinoma. Correlation to Tumor Subtype, Cytological Grade According to Thoenes-Classification and the Occurrence of Metastasis

To analyse growth characteristics of human renal cell tumors, 66 renal cell carcinomas and one oncocytoma were investigated concerning the proliferative activity by immunohistochemical demonstration of the Ki-67 antigen (clone MIB1) and the apoptotic rate using the terminal deoxynucleotidyl-transferase mediated dUTP-fluorescin nick end labelling (TUNEL) method. The TUNEL method indicates DNA double strand breaks considered as a hallmark of programmed cell death (apoptosis). Apoptotic cells were observed in 57 of 67 cases. The apoptotic rate (percentage of stained tumor cells) varied from 0% to 54.1%. GI carcinomas possessed a statistically significant higher apoptotic rate than GII/GIII carcinomas. The proliferation index (percentage of Ki-67 labelled cells) ranged from 0.09% to 22.3%. The well differentiated carcinomas (GI) showed statistically lower proliferative activity than moderate and poorly differentiated carcinomas (GII/GII). The clear cell variant of renal cell carcinoma expressed a higher apoptotic rate than the chromophilic variant. A statistical correlation between apoptosis/proliferation and occurrence of metastasis could not be established. In progression from well to less differentiated renal cell carcinoma the decrease of apoptotic rate, as well as the increase of the proliferative activity, contributes to a rapid tumor growth.