Overexpression of the met/HGF receptor in renal cell carcinomas.

The c-met oncogene encodes the receptor for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional cytokine able to mediate morphogenesis as well as mitogenesis, motogenesis and invasiveness of epithelial cells. HGF/SF has been implicated in branching tubulogenesis of the developing kidney and in regeneration after renal injury and nephrectomy. We have examined the expression of the met/HGF receptor in normal human kidney and tissues of the genito-urinary tract, and in 50 kidney neoplasms of different histotypes, using monoclonal antibodies (MAbs) against the met/HGF receptor and immunohistochemistry. In normal kidneys, weak staining restricted to the distal tubules was observed. Transitional cell carcinomas were consistently negative, whereas increased expression at various levels was found in 87% of renal cell carcinomas with different cytological features and histological patterns. Western blot analysis of samples showed that the met/HGF receptor found in the malignant cells exhibits features of the normal receptor. The met/HGF receptor is also overexpressed in a renal cell carcinoma cell line, whose motility is triggered by HGF/SF. Our data suggest that expression of the met/HGF receptor may be involved in the onset and progression of renal cell carcinomas.

Expression of alpha 3 beta 1 integrin receptor and its ligands in human lung tumors.

Increasing experimental evidence demonstrates that malignant transformation is associated with changes in the repertoire of expression of the integrin family of molecules, which mediate cell-matrix and cell-cell interactions. We have analyzed immunohistochemically and immunochemically the expression of VLA-3 integrin and its known ligands, namely, laminin (LM), fibronectin (FN), collagen type IV (Coll IV), nicein (NIC), and entactin/nidogen (ENT), in lung tumors of various histological types. alpha 3 beta 1 was detectable in normal bronchial epithelium and along basement membranes of alveolar walls. In non-small cell lung carcinomas (NSCLC) the integrin was expressed in 82% of the cases, independently of histological type and degree of differentiation of the tumors. On the other hand, only 13% of the small cell lung carcinomas (SCLC) displayed a weak and heterogeneous distribution of the alpha 3 beta 1 complex. Our findings were confirmed immunochemically using long-term tumor cell lines. While the expression of both alpha 3 beta 1 and ligands LM, FN, Coll IV, and Ent correlated in NSCLC with the presence of basement membranes, FN was the only ligand detectable in the stroma of SCLCs. A selective loss of nicein in basement membranes was demonstrated in NSCLC indicating an impairment of expression of this glycoprotein following malignant transformation.

Expression of fibronectin, fibronectin isoforms and integrin receptors in melanocytic lesions.

In vitro studies have demonstrated that fibronectin (FN) can deliver a mitogenic signal to quiescent human melanoma cells and that the alpha 5/beta 1-integrin receptor mediates this stimulus. In view of this finding we have analysed the in vivo expression of FN, and of ED-A and ED-B FN isoforms, in benign and malignant lesions of melanocyte origin. In the same specimens the expression of fibronectin integrin receptors was evaluated. The results demonstrate that, while detection of FN does not correlate with transformation and tumour progression, the expression of the two isoforms is associated with transformation and that only the ED-A variant is found in metastases. Integrin phenotyping disclosed that alpha 3/beta 1 expression is associated with tumour progression, alpha v/beta 3 is a marker of transformation, alpha 4 is rarely expressed and alpha 5 is expressed by about 50% and 30% of the primary and metastatic lesions respectively. Taken together, the results of this study demonstrate that transformation and tumour progression of the melanocyte lineage are associated with modulation of expression of FN isoforms and FN integrin receptors. Furthermore, the expression of alpha 5-integrin in a considerable percentage of primary and metastatic lesions indicates that FN may deliver a proliferative stimulus to melanoma cells in vivo.

Transformation of thyroid epithelium is associated with loss of c-kit receptor.

The receptor for the stem cell factor encoded by the c-kit proto-oncogene is expressed by a number of epithelial cells including thyrocytes. Since malignant transformation may be associated with loss of this receptor (melanoma and breast cancer), we have analyzed its expression in benign (38 cases) and malignant (31 cases) thyroid lesions. While low levels of c-kit are expressed in normal thyroids and in 60% of benign lesions, the receptor is undetectable in 60 and 90% of the follicular and papillary carcinomas, respectively. Northern blot analysis from surgical specimens of carcinomas and from carcinoma cell lines has demonstrated a lack of specific c-kit transcripts. These findings indicate that the c-kit receptor may be involved in the growth control of thyroid epithelium and that this function may be lost following malignant transformation.

Transformation and tumor progression are frequently associated with expression of the alpha 3/beta 1 heterodimer in solid tumors.

The ability of tumor cells to interact with the extracellular matrix (ECM) is functionally mediated by a variety of receptor molecules among which the integrins are a well-characterized family of mediators. In this study we have investigated immunohistochemically the in vivo expression of the alpha 3/beta 1 promiscuous receptor for ECM constituents in a variety of human solid malignancies. Although the receptor appears to undergo changes in distribution patterns, its expression is maintained in a high percentage of primary (76%) and metastatic (82%) tumors. Furthermore, the comparative immunohistochemical evaluation of alpha 3/beta 1 and its ligands, in a selected number of tumors of different histotypes, demonstrated that the expression of this integrin correlates with the presence of at least one ligand, either around nests of neoplastic cells or at the epithelial-stromal interface. The highly conserved expression of alpha 3/beta 1 shown in this study suggests that this receptor may play a role in tumor growth at the primary as well as at the metastatic site.

Noninvasive monitoring of ovarian cancer: improved results using CT with intraperitoneal contrast combined with immunocytology.

The purpose of this study was to evaluate whether CT with intraperitoneal contrast (ipc/CT) in combination with immunocytochemical (ICC) tests could improve the diagnostic accuracy over conventional methods in the follow-up of ovarian carcinoma. Forty-five clinically disease-free ovarian cancer patients eligible for a second-look laparotomy were given an intraperitoneal infusion of positive contrast material followed by pelvic and abdominal CT. After the radiographic examination, the contrast fluid was collected by paracentesis and processed for morphological as well as for immunocytochemical analysis employing a panel of monoclonal antibodies identifying distinct ovarian tumor-associated antigens. While ipc/CT correctly detected the presence of peritoneal recurrences in 22 of 45 (49%) patients, the immunocytochemical tests demonstrated the presence of otherwise undiagnosed microscopic disease in an additional 8 patients (18%). Our results demonstrate that the combination of radiological and immunological methods may represent a more accurate, noninvasive means of monitoring ovarian cancer, thus reducing the need for a second-look laparotomy.

Immunocytochemical diagnosis of amelanotic metastatic melanoma using monoclonal antibodies HMB-45 and Ep1-3.

The analysis of fine-needle aspirates and effusions has proved to be a valuable tool for the cytological identification of metastatic melanoma. Nevertheless, while malignant pigmented cells can be easily detected on cytological specimens, their identification may be very difficult in patients bearing amelanotic lesions. In the present study we have evaluated whether this limitation can be overcome using two monoclonal antibodies (mAbs) HMB45 and Ep1-3, which are highly specific for the melanocyte lineage. These reagents were assayed in immunocytochemical tests performed on cytological material obtained from 50 patients with a past history of melanoma and 463 bearing metastases from a cryptic primary tumour. These mAbs, employed in combination with a panel of monoclonal reagents with well-defined tumour specificity, may improve the accuracy of the conventional cytopathological diagnosis of melanoma metastases in the two groups of patients.

Expression of gp185HER-2 in human cutaneous melanoma: implications for experimental immunotherapeutics.

Over-expression of the HER-2 oncogene correlates with poor prognosis in breast and ovarian carcinomas. Using a sensitive immunohistochemical assay, we have detected low levels of gp185HER-2 in intradermal nevi (78%) and in primary (75%) and metastatic melanomas (58%). The HER-2 gene product expressed by cultured melanoma cells had the expected molecular weight, but no levels of tyrosine phosphorylation could be detected. Consistently, we were unable to inhibit in vitro growth of melanoma cells with an anti-gp 185HER-2 MAb, in conditions in which the growth of SKBr-3 breast-carcinoma cells was severely impaired. However, immunotoxins to gp 185HER-2 were able to kill gp185HER-2-positive melanoma cells. These data indicate that low levels of gp185HER-2 are expressed by the melanocyte lineage, with no correlation with transformation or tumor progression. Nevertheless, gp185HER-2 appears a suitable target for immunotherapy of cutaneous melanoma.

Expression of the c-Met/HGF receptor in human melanocytic neoplasms: demonstration of the relationship to malignant melanoma tumour progression.

The c-MET proto-oncogene encodes the receptor for the Hepatocyte Growth Factor/Scatter Factor, which is known to mediate mitogenic, motogenic and invasive responses of several cell types. We have analysed by immunohistochemistry and biochemically the expression of c-MET in benign and malignant melanocytic lesions. The Met/HGF receptor which in the melanocytic lineage displays the structural features of the authentic receptor was undetectable in tissue melanocytes and in nevocytic nevi. Only four out of 23 primary melanomas scored positive. Expression was increased to a significant level in 17 out of the 44 metastatic lesions examined. The c-MET expression was homogeneous in multiple metastases from the same patients. Comparative analyses showed both lack of correlation with the expression of the tumour progression associated ICAM-1 adhesion molecule and, in 23% of cases, co-expression with the c-KIT encoded receptor. These findings show that the c-MET gene is expressed at late stages of melanoma progression and suggest that the presence of Met/HGF receptor may contribute to the acquisition of an invasive phenotype.

Vla-3 distribution in normal and neoplastic non-lymphoid human tissues.

Using monoclonal antibody (mAb) M-Kid 2 to the alpha 3 beta 1 heterodimer, we have evaluated immunohistochemically the in vivo expression of the Vla-3 integrin in normal and transformed non-lymphoid human tissues. In normal tissues the alpha 3 beta 1 complex displays a polarized distribution at the baso-lateral aspect of most keratinizing and glandular epithelia. In addition the integrin is detected in perineurium, basal lamina of smooth muscular fibers, vascular media, podocytes and Bowman's capsule, myoepithelial cells of the parotid and breast, and in pulmonary alveoli. Neoplastic transformation is associated with qualitative and quantitative changes in expression of this integrin. The loss of polarized distribution often occurs in various malignancies. Furthermore, a significant decrease in expression occurs in 13% of the colon-rectum carcinomas, 75% of the ductal invasive, and 40% of the lobular invasive breast carcinomas. Among the lung malignancies tested, the small cell lung carcinomas (SCLC) were found to be consistently unreactive with mAb M-Kid 2. Analysis of Vla-3 expression in established tumor cell lines demonstrated that the integrin is almost invariably expressed by the plastic adherent cell subpopulations.

Integrin expression in cutaneous malignant melanoma: association of the alpha 3/beta 1 heterodimer with tumor progression.

The cell-surface heterodimers of the integrin family of molecules, which mediate cell-cell and cell-substratum interactions, are likely to be functionally relevant in local and metastatic tumor growth. In the present study we have analyzed whether the alpha 3/beta 1 receptor for collagen, laminin and fibronectin undergoes changes in expression during tumor progression in cutaneous malignant melanoma (CMM). The results of this study have demonstrated that, while low levels of VLA3 expression are detectable in benign lesions, in primary melanomas the heterodimer undergoes progressive increase in expression which correlates with the degree of dermal invasiveness. Metastatic lesions were found VLA3 positive in 82% of cases. Furthermore, the heterodimer is homogeneously expressed in multiple autologous metastases. The presence of VLA3 correlates with detection of at least one of the ligands in 45% of the cases studied. These findings provide additional evidence that tumor progression in CMM is associated with changes in integrin phenotypes which include the alpha 3/beta 1 heterodimer.

Localization of ahe alpha 6 and beta 4 integrin subunits in normal human non-lymphoid tissues.

The alpha 6/beta 4 integrin, of undefined receptor activity, has been shown to be expressed in a variety of murine epithelial cells. To gain information on the role of this heterodimer in tissue architecture as well as in malignant transformation we have performed an extensive immunohistochemical analysis of normal human tissues using monoclonal antibodies to alpha 6 and beta 4 subunits. Because alpha 6 is known to associate also with the beta 1 subunit to form a non-promiscuous receptor for laminin, the expression of beta 1 chain was also evaluated. The results of this study have shown that the alpha 6 chain has a wide distribution in tissues, including small vessels and peripheral nerves. alpha 6 colocalizes with beta 4 and beta 1 in most epithelial cells at the basolateral or basal aspect abutting the basement membrane. In a minority of tissues lacking beta 4, the alpha 6 chain is coexpressed with beta 1. These findings demonstrate that the expression of alpha 6/beta 1 laminin receptor and alpha 6/beta 4 heterodimer is phylogenetically conserved, suggesting that they are likely to play an important role in cellular scaffolding through binding to laminin and to still uncharacterized ligand/s present in basement membranes.

Regulated expression of vascular cell adhesion molecule-1 in human malignant melanoma.

Expression of the endothelial adhesion molecule VCAM-1 was studied in human malignant melanoma lines by flow cytometry. Clones 2/4 and 2/14 (derived from the same lesion) had appreciable levels of VCAM-1 expression, whereas clone 2/21 and the lines A2058, Mel24, and A375 were negative. Clone 2/14 was selected for further analysis. Exposure to tumor necrosis factor (TNF) markedly augmented VCAM-1 on melanoma cells. Surface VCAM-1 was associated with expression of specific transcripts that were augmented by TNF. Analysis by reverse transcriptase and polymerase chain reaction using appropriate primers revealed that TNF-stimulated melanoma cells expressed both 7 and 6 immunoglobulin domain transcripts with predominance of the longer species. Tumor necrosis factor--stimulated melanoma cells bound more VLA-4-expressing cells (melanoma and monocytes) than resting tumor cells and anti-VCAM-1 monoclonal antibodies significantly inhibited binding, thus suggesting that surface VCAM-1 on melanoma is functional. Analysis of melanoma tissue sections demonstrated that VCAM-1 is not a marker of transformation of melanocytes because it can be detected in benign nevi. Although, unlike ICAM-1, VCAM-1 is not correlated with tumor progression, its expression in a fraction of primary melanomas indicates that it may play a role in regulating host immune response and homotypic interactions in some malignant melanomas.

Expression of c-kit receptor in normal and transformed human nonlymphoid tissues.

The protooncogene c-kit encodes a tyrosine kinase with a molecular weight of 145,000, highly related to the platelet derived growth factor/colony stimulating factor receptors. Mutations of the murine gene result in impairment of hematopoiesis, gametogenesis, and of the melanocyte cell lineage. In order to elucidate c-kit functions in development and oncogenesis we have analyzed immunohistochemically its expression in human normal and transformed nonlymphoid tissues. The receptor has been detected in spermatogonia, melanocytes, and unexpectedly, in astrocytes, renal tubules, parotid cells, thyrocytes, and breast epithelium. While the gene product is expressed in seminoma, lung tumors, and melanoma of low invasiveness, no detectable levels have been detected in thyroid and breast carcinomas, astrocytomas, and invasive melanomas. In breast tumors these findings were confirmed by paired, Northern blot analysis of RNA preparations from normal and transformed tissue. The present results demonstrate that the c-kit receptor plays a role in the development of a larger spectrum of cell lineages. Furthermore, on the basis of the transformation associated changes, we speculate that, while in some cell types, c-kit expression positively regulates mitogenesis and is selected for in neoplastic transformation, in other tissues the c-kit pathway is involved in morphogenesis and differentiation and is, therefore, negatively selected in the course of tumor progression.

Expression of glutathione transferase pi in benign and malignant lesions of the melanocyte lineage.

Intrinsic and acquired resistance to chemotherapeutic agents represents the major clinical obstacle in the control of most tumours. In vitro studies have established that multiple mechanisms, including changes in drug uptake and efflux and in detoxifying enzymes, are responsible for drug resistance. Among the latter, glutathione S transferases (GST) have been recognized to play a relevant role. In the present study we have evaluated GST pi immunohistochemically as well as enzymatically in benign and malignant primary and metastatic lesions of the melanocyte lineage. A parallel analysis of the multiple drug resistance (MDRI) gene product was performed in a representative number of specimens. Results of this study demonstrate that while GST pi is constitutively expressed by the melanocyte lineage, independently from the transformed stage, MDRI p-glycoprotein is detected with a significantly lower frequency. These findings clearly indicate that GST pi represents the major detoxifying metabolic pathway of the melanocyte lineage and may be responsible for the high degree of inherent resistance of malignant melanoma to available cytostatic treatments.

Progression of human cutaneous melanoma is associated with loss of expression of c-kit proto-oncogene receptor.

Mutations at the white spotting (w) locus in mice have deleterious effects on germ cells, melanocytes and hematopoietic stem cells. The w locus encodes the c-kit tyrosine-kinase receptor whose ligand is the product of the SI locus. Using monoclonal antibodies (MAb(s)) to the extracellular domain, we have evaluated the expression of c-kit in normal and transformed melanocytes. This cell lineage synthesizes a receptor with a mw of 145 kDa. The gene product is expressed in epidermal melanocytes and in a fraction of nevocytic and blue nevi. In primary melanomas, loss of the receptor is observed in more invasive lesions. Only 30% of the metastatic lesions express detectable levels of the receptor. These findings demonstrate that the c-kit product is down-regulated in melanocytes following malignant transformation. The functional relevance of this modulation remains to be evaluated.

Changes in expression of alpha 6/beta 4 integrin heterodimer in primary and metastatic breast cancer.

The alpha 6/beta 4 integrin complex has been shown to be expressed in murine tissues at the basolateral aspect of most epithelial cells including the mammary epithelium, thus suggesting that this heterodimer may interact with components of the basement membrane. Because transformation of mammary epithelium frequently results in disappearance of basement membranes and loss of cell polarisation we have analysed in the present study whether expression of the alpha 6/beta 4 complex is altered in human breast tumours. The results of the present study confirm that in human mammary gland alpha 6 and beta 4 subunits colocalise at the basolateral aspect of the epithelium. While in benign breast lesions this distribution pattern remains mostly unchanged, in primary carcinomas the expression of both chains is either redistributed over the cells surface or significantly reduced. This altered pattern of expression is paralleled by the lack of detection of basement membrane laminin and collagen type IV. In metastatic lesions the expression of the heterodimer is maintained in most of the lymphnodal foci, but less frequently detected in metastasis localised in the pleural cavity and in parenchymal tissues. These findings indicate that in breast epithelium expression of the alpha 6/beta 4 heterodimer is modulated by the presence of basement membrane and is possibly influenced by microenvironmental factors as suggested by the different pattern of alpha 6/beta 4 expression in nodal and extranodal metastatic foci.

Immunocytodiagnosis of atypical hyperplasia and endometrial carcinoma in post-menopausal women.

In the present study we have evaluated whether monoclonal antibodies (MAbs) B72.3 and AR-3 which display, on histological preparations, a differential reactivity with normal and transformed endometrium, could be a useful adjunct to endometrial cytology in the identification of pre-neoplastic and neoplastic conditions. Immunocytochemical (ICC) tests, using the 2 reagents, were performed on normal cycling endometrium and on hyperplastic and malignant lesions collected by the endocyte technique both from 86 surgically resected specimens and from 62 postmenopausal symptomatic and asymptomatic outpatients. The results obtained showed that the combination of the 2 MAbs can complement conventional morphology in the identification of pre-malignant atypical lesions and endometrial carcinoma of unclear cytological features, thus allowing a selection of those patients who are candidates for fractional curettage.

The use of a panel of monoclonal antibodies can lower false-negative diagnoses of peritoneal washings in ovarian tumors.

A correct surgical staging of ovarian carcinoma and the identification of persistent microscopic disease at second-look surgery largely rely on the cytologic examination of peritoneal washings (PW). Nevertheless, the morphologic analysis of these fluids frequently provides false-negative findings. As shown in other areas of cytodiagnosis, monoclonal antibodies (MoAb) to tumor-associated antigens may be a useful adjunct to overcome the limitations of conventional cytopathologic examination of PW. To evaluate this question, immunocytochemical tests were done using a panel of four MoAb to ovarian carcinoma-associated antigens (B72.3, MOv18, MOv19, and OC-125) to analyze 117 PW sampled during initial surgical staging and 121 PW harvested at second-look operations. The results of this study showed that immunocytochemical tests using the combination of the four reagents could improve cytodiagnosis more than 15% in both groups of PW. Thus a significant fraction of patients could be correctly staged and treated or become potentially curable by second-line salvage therapy.