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Laminin is a basement membrane glycoprotein whose expression has been widely related to cancer progression. Laminin production by primary breast carcinomas was investigated using immunohistochemistry on archival specimens from a retrospective series with long term follow-up. Laminin production was found to be independent of the clinical and pathological variables analyzed, whereas a statistically significant direct association with the expression of the laminin receptor and a negative association with the differentiation-related antigen Ca-MBr8 were observed. Survival analysis indicated that laminin positivity by itself has no prognostic significance. However, when analyzed together with the laminin receptor expression, laminin was associated with a good prognosis in receptor-negative tumors and with the worst prognosis in receptor-positive tumors.

BACKGROUND The 67-kd laminin receptor is a cell-surface protein that binds laminin with high affinity. In vitro studies suggest that this protein is involved in the progression of human tumors to invasive cancers (metastasis), but there have been few in vivo studies. Identification of such proteins would allow development of therapies aimed at interfering with their mechanisms of action. PURPOSE This large retrospective study was designed to investigate the association of expression of this laminin receptor molecule with established prognostic factors and overall survival in breast carcinoma patients. METHODS We immunohistochemically stained archival paraffin-embedded sections of 1160 primary breast carcinomas, using an immunoperoxidase technique and the MLuC5 monoclonal antibody, which is specific for the 67-kd laminin receptor. Specimens were obtained from consecutive surgeries performed from January 1968 through December 1971. Patients with negative lymph nodes or involved regional nodes had been treated with surgery alone; those with positive axillary nodes had received surgery and radiotherapy. No chemotherapy had been administered until disease recurrence. The statistical analysis was carried out using the logrank method for the survival curves and the actuarial life table to calculate survival rates according to the different prognostic variables. RESULTS We found statistically significant associations between laminin receptor expression and young age (P < .001), premenopausal status (P = .001), positive axillary lymph nodes (P = .01), peritumoral lymphatic invasion (P = .02), and the diameter of the tumor (P = .05). Moreover, the association of expression of the receptor protein with poor prognosis, as indicated by survival curves, was statistically significant (P < .01). For patients with receptor-negative tumors, the survival rate was 50% at 20 years; for those with receptor-positive tumors, the survival rate was 50% at 13 years. Multivariate analysis showed the laminin receptor to be an independent prognostic factor (P = .005), indicating its predictive value in relation to overall survival. CONCLUSIONS Our data suggest that the 67-kd laminin receptor is associated with the metastatic process. IMPLICATIONS These preliminary findings also suggest that hormones may have a regulatory role in the in vivo expression of the 67-kd laminin receptor, which supports the hypothesis that hormone therapy might inhibit expression of the receptor. Studies of expression of this receptor in tumors of patients with extremely different sex hormone levels (e.g., men and pregnant women) are in progress.

Three MAbs, MLuC2, MLuC8 and MLuC9, directed against a molecule that is produced and secreted by carcinoma cells were studied with the aim of developing a double-determinant immunoradiometric assay (DDIRMA). We demonstrated by means of immunoblotting, immunodepletion and DDIRMA techniques, that MLuC9 reacted against the CEA molecule, whereas MLuC2 and MLuC8 reacted against a 90 Kd molecule related to CEA. The DDIRMA performed with the anti-CEA as a catcher MAb and the anti-90 Kd as a tracer MAb was found to be positive with the HT29 soluble extract, which suggests the existence of CEA/90 Kd dimeric molecules. The same reactivity was found when sera from patients with lung carcinomas were tested, which excludes that this molecule could be an artefact due to the cell solubilization procedures. The association between CEA and the 90 Kd molecule was further confirmed by immunodepletion experiments in which the immunoprecipitation with one MAb not only removed the recognized molecule, but also partially immunodepleted the material from the other.

In order to obtain further information on the biological role of the HER2/neu oncoprotein monoclonal antibodies (MAbs) were produced against the p185 extracellular domain. To immunize the mice and screen the hybridoma supernatants we selected a lung adenocarcinoma cell line (Calu-3), which demonstrated an over-expression of p185HER2 measured as the reactivity with polyclonal rabbit serum to the 14-amino-acid carboxy-terminal-HER2/neu. Two MAbs, designated MGR2 (IgG1) and MGR3 (IgG2), selected for reactivity on Calu-3 and negativity on A43I live cells, the reference target cell for EGF receptor expression, were found to immunoprecipitate a 185-kDa molecule. Immunodepletion experiments with the polyclonal antiserum and cross-competition experiments indicated that the 2 reagents recognized 2 different epitopes located on the p185HER2 molecule. One of the 2 MAbs, MGR3, was found to internalize, induce p185HER2 phosphorylation and inhibit tumor cell growth in vitro. These results indicate that MGR3 is directed against a determinant located in the p185HER2 ligand binding site and may compete with the p185HER2 ligand, but is incapable of inducing a complete mitotic signal.

By immunizing a mouse with human metastatic breast tumor cells from patient effusions and infiltrated lymph nodes, a monoclonal antibody (MLuC2), which identifies a new carcinoma-associated marker, was raised. The reactivity of this reagent was studied by immunohistochemistry on live and fixed cells from tumor cell lines and on frozen sections from surgical specimens. Besides reacting with 73% of breast carcinomas, MLuC2 also reacted with 93% of non-small cell lung carcinoma (NSCLC) and with a few normal tissues. The MLuC2-recognized molecule (CaMLuC2), whose MW was 90 KDa according to immunoblotting experiments, was found to be detectable in the serum and could therefore be of particular interest for serological diagnostic applications. Since the CaMLuC2 epitope was not polyexpressed on the bearing molecule, we produced a new generation of MAbs in order to define epitopes coexpressed with CaMLuC2 on the same 90 KDa molecule, and which are therefore suitable to develop a double-determinant immunoradiometric assay (DDIRMA) for the detection of this marker in the sera of lung carcinoma patients. Different analyses by immunohistochemistry, binding inhibition tests and DDIRMA, proved that the two new reagents developed, MLuC8 and MLuC9, recognize the same or closely related epitopes, which are however different from CaMLuC2, but which are all present on the same molecule. Preliminary immunoradiometric tests performed on sera from lung cancer and control patients showed a good specificity but a low sensitivity. In fact, only 42% of the 28 tested sera samples from NSCLC patients scored positive despite the fact that more than 90% of the NSCLC expressed the relevant antigen

Anti-p185HER2 monoclonal antibodies often show intense reactivity with the basement membrane of tumor cells that overexpress the HER2/neu gene product (p185HER2). To evaluate a possible interaction between p185HER2 and adhesion molecules or their receptors, the polarity of p185HER2 was tested in lung carcinoma cell line Calu-3, which overexpresses this protein, in cultures grown as confluent monolayers or as aggregates. MAb immunostaining patterns indicated that p185HER2 is concentrated on the baso-lateral membrane of cells and that it colocalizes with the integrin alpha 6 beta 4 at the cell-cell junctions where laminin is also found. The same membrane region showed intense reactivity with antiphosphotyrosine antibodies. Furthermore, integrin clustering induced by the specific antibody was accompanied by the clustering of p185HER2, as indicated by immunoelectron microscopy, and by a subsequent increase in p185HER2 tyrosine phosphorylation. Treatment with exogenous laminin also resulted in increased basal levels of p185HER2 phosphorylation. These data suggest a physical interaction between the integrin and the oncoprotein that might be functionally relevant in directly controlling the tyrosine phosphorylation of the catalytic domain of p185HER2.

Interactions between tumor cells and laminin or other components of the extracellular matrix are thought to play a role in tumor invasion and metastasis. To analyze these interactions, we examined the expression of 5 types of laminin receptors on 11 cell lines derived from the highly malignant and metastatic tumor small-cell lung cancer (SCLC). The integrins VLA-1, VLA-3, VLA-6 and the 67 KDa monomeric receptor were expressed at various levels, whereas the VLA-2 receptor was absent on the cell lines. Only one cell line expressed none of these laminin receptors. All cell lines co-expressed alpha 6 beta 1 (VLA-6) and the 67-kDa molecule, the only receptors specific for laminin. Analysis of the ability of SCLC cells to bind radiolabeled laminin and to adhere to laminin substrata revealed a correlation between these 2 parameters and the expression of VLA-6 and the 67-kDa monomeric receptor. Cell adhesion was mediated by alpha 6 beta 1, as indicated by inhibition of adhesion using an anti-VLA-6 monoclonal antibody (MAb). Both VLA-6 and the monomeric receptor were up-regulated in vitro by laminin.

A cell line, GBM, was established from a human malignant glioblastoma and was characterized with particular reference to its response to conventional drugs. The GBM cell line exhibited a 73 +/- 7 h doubling time in monolayer cultures. Expression of glial fibrillary acidic and S-100 proteins was observed. Karyotype analysis of GBM cells at early passages revealed the presence of two near-triploid clones (A and B) with multiple chromosome rearrangements; a 100% frequency for clone B was observed in the established cell line. GBM cells had tumorigenic properties, since the s.c. injection of cultured cells into nude mice gave rise to slowly growing tumors. The morphology of GBM cells was retained during in vitro and in vivo passages, as judged by light microscopy. GBM cells were relatively resistant to most conventional drugs; among the tested drugs, only taxol exhibited a marked cytotoxic effect comparable to that found in cells of a different tumor type. GBM cells were found positive for the epidermal growth factor receptor, HER2-neu and P-glycoprotein by flow cytometry of cells labelled with monoclonal antibodies. In spite of the expression of relatively high gamma-glutamyltransferase activity, the intracellular glutathione level was comparable to that of other chemosensitive tumor cells. This glioblastoma cell line is a suitable model for the identification and preclinical studies of new agents and provides an additional system to explore the molecular basis of the intrinsic drug resistance of glioblastoma.

The expression of the epithelial antigen recognised by the MBr1 monoclonal antibody (CaMBr1) was studied on 161 small cell lung carcinoma (SCLC) biopsies. A correlation between the marker expression and the overall survival of the patients was found. To investigate the possible role of CaMBr1 in tumour aggressiveness, the in vivo and in vitro growth capabilities of different SCLC cell lines, in relation to the antigen expression, were analysed. The CaMBr1-positive cell lines displayed a higher growth potential in comparison to CaMBr1-negative cells. The biochemical nature of CaMBr1 was analysed in terms of enzyme sensitivity, molecular weight and comparison with other glycoproteins expressed by SCLC cells. The results indicated the trypsin sensitivity of the molecule, and sialic acid hiding of the CaMBr1 epitope. The increase of MBr1 reactivity after neuraminidase treatment suggests that the CaMBr1 epitope expressed in the SCLC cell line is carried by a sialoglycoprotein.

Two monoclonal antibodies (mAbs), designated MLuC5 and MLuC6, were produced against a human small cell lung carcinoma cell line. They were found to exhibit a superimposable reactivity on different cell lines and on platelets. Moreover, they both immunoprecipitated a 67 kDa molecule from the membrane of the reference target cells. Immunodepletion and cross-inhibition tests indicated that the two mAbs recognize two epitopes closely localized on the same molecule. The MLuC5 mAb was further characterized for its reactivity on platelets. Immunoprecipitation and ELISA assays demonstrate that this mAb recognizes the 67 kDa high affinity laminin receptor. MLuC5 reactivity was evaluated by immunohistochemistry on a variety of normal and tumor tissues, in particular breast specimens including normal epithelium, dysplastic lesions, in situ carcinomas, invasive primary carcinomas and distant metastases. The laminin receptor was found to be strongly expressed in 50% of the infiltrating carcinomas, whereas in situ carcinomas and benign lesions, as well as the normal mammary epithelium, were only weakly and focally positive. In metastatic lesions MLuC5 reactivity was only found in 11% of the samples tested, independently of the site of origin of the lesion.

In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 x 10(4) receptors/cell of 10 microns diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.

In order to study the possible relationship between antigenic phenotype and tumor progression, 63 small cell lung carcinomas (SCLC) biopsies derived from primary or metastatic tumors were tested by immunofluorescence and immunoperoxidase techniques with an anti-carcinoma monoclonal antibody designated MBr1. Primary tumors were found to be less reactive with MBr1 than the local and distant metastatic lesions (57 % versus 75 % and 89 % positivity respectively). A life table analysis on the tested cases indicated an inverse association between the expression of the marker recognized by the MBr1 MAb (CaMBr1) and overall survival (p < 0.01): patients with MBr1-positive tumors showed a shorter survival time in comparison to patients whose tumors did not express the marker. The same correlation between survival and CaMBr1 expression was found even when only the 31 cases of early stage disease patients were considered (p < 0.05). Different tumor aggressiveness or resistance to therapy of MBr1-positive tumors could be responsible for the shorter survival time of the patients.

In order to increase the availability of SCLC cells derived from biopsies, in vivo and in vitro growth methods were investigated. The cells grown in both conditions were periodically monitored for reactivity with 2 monoclonal antibodies (MAbs): MLuC1 directed against SCLC cells and IM1 which recognizes the class II antigen on activated lymphocytes and macrophages. About 50 % of the 28 analyzed SCLC specimens were found to proliferate in one or both systems. The in vitro-grown cells exhibited the same heterogeneity found in the original cell suspensions and moreover, in some cases only normal cells were recovered after several in vitro passages. From the subcutaneous transplanted tumors a large number of MLuC1-positive tumor cells could easily be recovered, thus indicating the validity of the in vivo methodology. The MBr1 MAb, directed against an epithelial antigen, was found to react with about 50 % of the 26 tested tumors, mainly those which demonstrated in vivo and/or in vitro growth capacity. These data suggest that only some tumors, presumably with peculiar biological characteristics, can efficiently grow in these artificial systems.

In order to obtain further information on the biological role of the HER2/neu oncoprotein, 7 new monoclonal antibodies (MAbs) were produced against the p185HER2 extracellular domain. These MAbs, together with two others previously produced, were used to investigate the p185HER2 expression in breast carcinomas and compare the recognized antigenic determinants. The 7 reagents (MGR4,5,6,7,8,9 and 10), were shown to define five distinct epitopes. Three of these MAbs (MGR5,7,10), as well as one previously produced (MGR2), recognize the same epitope (Epitope-1) which seems, therefore, to be highly immunogenic for the murine immune system. Epitope-2 recognized by the MGR4 MAb, appears to be closely related to epitope-1 due to a cross inhibition between MGR4 and MGR10, but not MGR2. Epitope-2 is the only one of the 5 also present on the product of the neu oncogene, the rat analogue of the human HER2/neu gene. None of the reagents against epitope-1 and epitope-2 were found to mediate receptor internalization, whereas MGR6 as well as a previously produced MAb (MGR3), both of which define epitope-3 and MGR8 which defines epitope-4, were found to do so. Epitope-4 like the neu-specific peptide recognized by the reference c-neu Ab3 MAb, was detectable on all p185HER2 positive breast cancer, independently from the quantitative content of the oncoprotein, at variance with the other 4 epitopes whose availability on p185HER2 for the relevant MAbs varied with the degree of overexpression. Epitope-5, recognized by the MGR9 MAb, on the contrary to the other epitopes, was prevalently localized at the basal membrane level of the tumor nodule.