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Expression of the 90K Tumor-Associated Protein in Benign and Malignant Melanocytic Lesions

2002; Elsevier BV; Volume: 119; Issue: 1 Linguagem: Inglês

10.1046/j.1523-1747.2002.17642.x

1523-1747

Anna Maria Cesinaro, Gian Paolo Trentini, Clara Natoli, Antonino Grassadonia, Nicola Tinari, Stefano Iacobelli,

Skin and Cellular Biology Research

To the Editor: Histopathologic differentiation of melanoma from benign melanocytic lesions can be extremely difficult (Barnhill et al., 1999Barnhill R.L. Argenyi Z.B. From L. et al.Atypical Spitz nevi/tumors: lack of consensus for diagnosis, discrimination from melanoma, and prediction of outcome.Hum Pathol. 1999; 30: 513-520Abstract Full Text PDF PubMed Scopus (350) Google Scholar;Reed, 1999Reed R.J. Dimensionalities: borderline and intermediate melanocytic neoplasia.Hum Pathol. 1999; 30: 521-524Abstract Full Text PDF PubMed Scopus (16) Google Scholar), as a subset of melanomas mimic conventional (Suster et al., 1987Suster S. Romero M. Bubis J.J. Verrucous pseudonaevoid melanoma.J Surg Oncol. 1987; 36: 134-137Crossref PubMed Scopus (27) Google Scholar;Wong et al., 1995Wong T.-Y. Suster S. Duncan L.M. Mihm M.C. Nevoid melanoma mimicking spindle and epithelioid cell nevus and verrucous dermal nevus.Hum Pathol. 1995; 26: 171-179Abstract Full Text PDF PubMed Scopus (73) Google Scholar) or Spitz's nevi (Muhlbauer et al., 1983Muhlbauer J.E. Margolis R.J. Mihm Jr, M.C. Reed R.J. Minimal deviation melanoma: a histological variant of cutaneous malignant melanoma in its vertical growth phase.J Invest Dermatol. 1983; 80: 635-655Crossref Google Scholar;Smith et al., 1989Smith K.J. Barrett T.L. Skelton H.G. Lupton G.P. Graham J.H. Spindle cell and epithelioid cell nevi with atypia and metastasis (malignant Spitz nevus).Am J Surg Pathol. 1989; 13: 931-939Crossref PubMed Scopus (190) Google Scholar;Wong et al., 1995Wong T.-Y. Suster S. Duncan L.M. Mihm M.C. Nevoid melanoma mimicking spindle and epithelioid cell nevus and verrucous dermal nevus.Hum Pathol. 1995; 26: 171-179Abstract Full Text PDF PubMed Scopus (73) Google Scholar), but behave aggressively (Reed et al., 1975Reed R.J. Ichinose H. Clarke W.H. Mihm M.C. Common and uncommon melanocytic naevi and borderline melanoma.Semin Oncol. 1975; 2: 119-147PubMed Google Scholar;Reed, 1978Reed R.J. Minimum deviation melanoma.Am J Surg Pathol. 1978; 2: 215-220Crossref PubMed Google Scholar;Muhlbauer et al., 1983Muhlbauer J.E. Margolis R.J. Mihm Jr, M.C. Reed R.J. Minimal deviation melanoma: a histological variant of cutaneous malignant melanoma in its vertical growth phase.J Invest Dermatol. 1983; 80: 635-655Crossref Google Scholar;Phillips et al., 1986Phillips M.E. Margolis R.J. Meerot Y. Sober A.J. Reed R.J. Muhlbauer J.E. Mihm M.C. The spectrum of minimal deviation melanoma. a clinicopathologic study of 21 cases.Hum Pathol. 1986; 17: 796-806Abstract Full Text PDF PubMed Scopus (55) Google Scholar). Therefore, the identification of molecular markers that allow an accurate diagnosis of melanoma is of utmost importance. Studying such markers could also improve the understanding of the biologic events involved in the melanocyte transformation to a highly invasive and metastatic tumor. 90K is a large oligomeric protein composed of a ≈ 90 kDa subunit, originally identified as a tumor-secreted antigen in the culture medium of human breast cancer cells (Iacobelli et al., 1986Iacobelli S. Arno E. D'Orazio A. Coletti G. Detection of antigens recognized by a novel monoclonal antibody in tissue and serum from patients with breast cancer.Cancer Res. 1986; 46: 3005-3010PubMed Google Scholar). After cDNA cloning (Ullrich et al., 1994Ullrich A. Sures I. D'Egidio M. et al.The secreted tumor-associated antigen 90K is a potent immune stimulator.J Biol Chem. 1994; 269: 18401-18407Abstract Full Text PDF PubMed Google Scholar), the sequence of 90K showed that it contained a cystein-rich domain, which is homologous to that found in the SRCR group A family of proteins and is strictly related to the immunoglobulin superfamily (Resnick et al., 1994Resnick D. Pearson A. Krieger M. The SRCR superfamily: a family reminiscent of the Ig superfamily.Trends Biochem Sci. 1994; 19: 5-8Abstract Full Text PDF PubMed Scopus (301) Google Scholar). 90K was identified independently as a ligand of the lactose-specific S-type lectin, galectin-3 (formerly known as Mac-2), and also named Mac-2 binding protein (Koths et al., 1993Koths K. Taylor E. Halenbeck R. Casipit C. Wang A. Cloning and characterization of a human Mac-2-binding protein, a new member of the superfamily defined by the macrophage scavenger receptor cysteine-rich domain.J Biol Chem. 1993; 268: 14245-14249Abstract Full Text PDF PubMed Google Scholar). 90K is observed in many tissues and in biologic fluids (Koths et al., 1993Koths K. Taylor E. Halenbeck R. Casipit C. Wang A. Cloning and characterization of a human Mac-2-binding protein, a new member of the superfamily defined by the macrophage scavenger receptor cysteine-rich domain.J Biol Chem. 1993; 268: 14245-14249Abstract Full Text PDF PubMed Google Scholar;D'Ostilio et al., 1996D'Ostilio N. Sabatino G. Natoli C. Ullrich A. Iacobelli S. 90K (Mac-2BP) in human milk.Clin Exp Immunol. 1996; 104: 543-546Crossref PubMed Scopus (37) Google Scholar) and is overexpressed by the majority of human tumors (Iacobelli et al., 1986Iacobelli S. Arno E. D'Orazio A. Coletti G. Detection of antigens recognized by a novel monoclonal antibody in tissue and serum from patients with breast cancer.Cancer Res. 1986; 46: 3005-3010PubMed Google Scholar), with serum levels frequently elevated, as in patients with melanoma (Natoli et al., 1994Natoli C. Ortona L. Tamburrini E. et al.Elevated serum levels of a 90,000 daltons tumor-associated antigen in cancer and in infection by human immunodeficiency virus (HIV).Anticancer Res. 1994; 14: 1457-1460PubMed Google Scholar). In A375 human melanoma cells, 90K was found to promote homotypic cell–cell adhesion by cross-linking of surface-bound galectin-3 residues (Inohara et al., 1996Inohara H. Akahani S. Koths K. Raz A. Interactions between galectin-3 and Mac-2-binding protein mediate cell-cell adhesion.Cancer Res. 1996; 56: 4530-4534PubMed Google Scholar). Results from binding studies in vitro demonstrated β1 integrin-mediated cell adhesion for 90K, as well as interactions with some collagens and fibronectin (Sasaki et al., 1998Sasaki T. Brakebusch C. Engel J. Timpl R. Mac-2 binding protein is a cell adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin.EMBO J. 1998; 17: 1606-1613Crossref PubMed Scopus (197) Google Scholar). This suggests that 90K may play a part in embolization of disseminating tumor cells in circulation and favors the progression and metastatization. In this study, we verified whether 90K could be a reliable immunohistochemical marker of melanoma. We also evaluated the ability of melanoma cells to adhere to 90K and other extracellular matrix proteins. Pathologic specimens, achieved from the section of Pathologic Anatomy, comprehended the series of benign and malignant melanocytic lesions listed in Table I. An immunohistochemical reaction was performed using the anti-90K monoclonal antibody 1A4.22 (Tinari et al., 1997Tinari N. D'Egidio M. Iacobelli S. et al.Identification of the tumor antigen 90K domains recognized by monoclonal antibodies SP2 and L3 and preparation and characterization of novel anti-90K monoclonal antibodies.Biochem Biophys Res Commun. 1997; 23: 367-372Crossref Scopus (17) Google Scholar), and results were evaluated as positive or negative cytoplasmic staining, scored in three grades (+, ++, +++) and in three patterns of distribution (diffuse, focal, patchy); weak staining was recorded as +/–. Cell adhesion assay was performed on cells of the human melanoma cell line MEL-8863, from metastatic human melanoma (Mecchia et al., 2000Mecchia M. Matarrese P. Malorni W. et al.Type I consensus interferon (CIFN) gene transfer into human melanoma cells up-regulates p53 and enhances cisplatin-induced apoptosis: implications for new therapeutic strategies with IFN-alpha.Gene Ther. 2000; 7: 167-179Crossref PubMed Scopus (24) Google Scholar), using recombinant 90K protein (Ullrich et al., 1994Ullrich A. Sures I. D'Egidio M. et al.The secreted tumor-associated antigen 90K is a potent immune stimulator.J Biol Chem. 1994; 269: 18401-18407Abstract Full Text PDF PubMed Google Scholar), laminin, and fibronectin (Calbiochem, San Diego, CA). The immunohistochemical results (see Table I and Figure 1) demonstrated that all 65 malignant lesions, including metastasizing Spitz's nevi and metastases, were positively stained (++/+++); however, only 18 of 44 benign lesions (also including the nine residual nevi in melanomas) gave a positive reaction, which was weak (+/–) in eight cases, and mild or moderate (+/++) in the remaining 10, represented by Spitz's nevi. Altogether these results demonstrated that the specificity and sensitivity of 90K in differentiating melanocytic lesions is 59.09% and 100%, respectively (Table II). Cell adhesion experiments showed adhesion of MEL-8863 to 90K, laminin and fibronectin higher than adhesion to the negative control bovine serum albumin (p < 0.0001) (Figure 2a), fibronectin being the best substrate. Competition experiments were also performed and the results are shown in Figure 2(b).Table I90K staining in benign and malignant melanocytic lesionsCasesTotal cases (no.)Positive cases (no.)Distribution patternScoreLentigo simplex50Junctional nevus52Diffuse+/–Intradermal nevus51Focal+/–Compound nevus105Focal (2)+/–Patchy (3)Spitz's nevus1010Diffuse+/++Metastasizing22Focal (1)+/++Spitz's nevusDiffuse (1)Melanoma in nevusaResidual nevi were negative.99Diffuse++/+++In situ melanoma66Diffuse+++Melanoma ≤ 0.76 mm1616Diffuse (12)++/+++Patchy (4)Melanoma > 0.76 to1414Diffuse (10)++/+++≤ 3.00 mm88Patchy (4)++/+++Melanoma > 3.00 mmDiffuse (4)Patchy (4)Cutaneous metastasis66Diffuse+/++Nodal metastasis33Diffuse+/++Bone metastasis11Focal++a Residual nevi were negative. Open table in a new tab Table II90K immunostaining in detecting malignancy in melanocytic lesionsPositiveNegativeTotalBenign lesionsaIncluding nine residual nevi in melanoma.182644Malignant lesionsbIncluding two metastasizing Spitz's nevi.65065Total8326(95% confidence interval Binomial exact test)109Sensitivity (65/65)100.00%94.48%100.00%Specificity (26/44)59.09%43.25%73.66%Positive predictive value (65/83)78.31%67.91%86.61%Negative predictive value (26/26)100.00%86.77%100.00%a Including nine residual nevi in melanoma.b Including two metastasizing Spitz's nevi. Open table in a new tab Figure 2Cell adhesion to 90K protein. (A) Cells from the human melanoma cell line MEL 8863 were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% (vol/vol) penicillin (100 U per ml), 1% (vol/vol) streptomycin (100 U per ml), and 1% (vol/vol) L-glutamine (all from Gibco-BRL, Grand Island, NY), maintained at 37°C in 5% CO2 and passaged every week. Cells (50,000 cells per well) were seeded in 96-well microtiter plates and coated with 100 µl of 90K, laminin, and fibronectin (10 µg per ml in phosphate-buffered saline) overnight at 4°C. One hour before seeding, plates were saturated with 1% bovine serum albumin in RPMI at 37°C. Cells were trypsinized, washed, and resuspended at 500,000 per ml in RPMI serum free with 0.1% bovine serum albumin. One hundred microliters (50,000 cells) were seeded in the wells and, after 1 h of incubation at 37°C, nonadherent cells were removed by gentle washing in phosphate-buffered saline and adherent cells fixed with 95° ethanol for 10 min and stained with 100 µl of 4% crystal violet for 30 min. The absorbance at 550 nm was measured on a plate reader after solubilization with 50 µl of 0.25% Triton-X100. Data represent the mean ± SD of three different experiments done in quadruplicate. (B) Adhesion assay was performed seeding MEL 8863 cells in 96-well microtiter plates precoated with 100 µl of 90K (10 µg per ml) in presence of the function blocking anti-β1 antibody 4B4 (Coulter, Hialeah, FL), anti-90K SP-2 and 1A4.22 antibodies (Tinari et al., 1997Tinari N. D'Egidio M. Iacobelli S. et al.Identification of the tumor antigen 90K domains recognized by monoclonal antibodies SP2 and L3 and preparation and characterization of novel anti-90K monoclonal antibodies.Biochem Biophys Res Commun. 1997; 23: 367-372Crossref Scopus (17) Google Scholar) at a concentration of 10 µg per ml, anti-galectin 3M3/38 antibody, or 50 mM lactose. Anti-galectin-3 and lactose did not affect the binding of MEL 8863 cells to 90K, as well as 1A4.22 antibody, whereas adhesion was competed by the anti-90K antibody SP-2; moreover, β1 function-blocking antibody 4B4 markedly reduced the binding of cells to 90K. Data represent the mean ± SD of three different experiments done in quadruplicate.View Large Image Figure ViewerDownload (PPT) Studies on markers of malignancy in melanoma have focused mainly on oncogene alterations (Newton Bishop, 1997Newton Bishop J.A. Molecular pathology of melanoma.Cancer Metastasis Rev. 1997; 16: 141-154Crossref PubMed Scopus (28) Google Scholar), but little is known about other molecules, such as glycoconjugate ligands, that interact with extracellular matrix and may be related to malignant transformation and tumor progression. In this study, 90K appeared to represent a potential marker of melanocytic cell transformation and a factor possibly involved in tumor invasion and metastasis. Although the expression of 90K is not absolute in distinguishing between benign and malignant lesions, this marker was consistently expressed in all types of melanoma, but not in normal melanocytes at the dermoepidermal junction nor in conventional nevi. Only a minority of these nevi showed a weak and usually focal staining. Also, in melanomas arising in nevus, the benign part of the lesion was negative, whereas the melanoma cells were strongly positive. Metastases were immunohistochemically positive for 90K (confirmed by immunoblotting, not shown), thus providing evidence that 90K is expressed also by secondary lesions. On the other hand, 90K does not appear to be of value in differentiating Spitz's nevus from melanoma. Indeed, Spitz's nevi showed a diffuse, although less intense, positivity when compared with melanoma, and a similar positivity was observed in the two cases of metastasizing Spitz's nevus. Intriguingly, these data also suggest a role for 90K as an early marker of neoplastic transformation of melanocytic lesions, as well as raise some questions about the real nature of Spitz's nevus. This is further substantiated by the finding that 90K expression was stronger and more diffuse in "in situ" melanoma than in infiltrating lesions and metastases. Our findings show that the expression of 90K can help to distinguish between benign and malignant melanocytic lesions, with the exception of Spitz's nevus, which presents roughly the same degree of expression of malignant melanoma. There was no difference in the expression between melanomas of different thickness, with the exception of the stronger positivity characterizing the "in situ" lesions. Therefore, 90K does not seem to represent a prognostic factor that may predict the clinical outcome for patients. Our in vitro experiments show that this protein, absorbed on a plastic substrate, causes the adhesion of melanoma cells through a β1-integrin-mediated process. It has recently been documented that 90K is a cell-adhesive, secreted glycoprotein that accumulates in the extracellular matrix (Sasaki et al., 1998Sasaki T. Brakebusch C. Engel J. Timpl R. Mac-2 binding protein is a cell adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin.EMBO J. 1998; 17: 1606-1613Crossref PubMed Scopus (197) Google Scholar). As β1-integrins are important components of the adhesion receptor repertoire on melanoma cells (Schadendorf et al., 1993Schadendorf D. Gawlik C. Haney U. Ostmeier H. Suter L. Czarnetzki B.M. Tumour progression and metastatic behaviour in vivo correlates with integrin expression on melanocytic tumours.J Pathol. 1993; 170: 429-434Crossref PubMed Scopus (99) Google Scholar;Hieken et al., 1995Hieken T.J. Ronan S.G. Farolan M. Shilkaitis A.L. Kim D.K. Das Gupta T.K. Beta 1 integrin expression in malignant melanoma predicts occult lymph node metastases.Surgery. 1995; 118: 669-673Abstract Full Text PDF PubMed Scopus (22) Google Scholar), the interaction between 90K present in the extracellular matrix and β1-integrins on the cell surface may play a part in the interaction between tumor cells and surrounding microenvironment; however, our immunohistochemical data do not determine whether this favors a more invasive behavior. In conclusion, this study demonstrated that 90K is highly expressed in melanoma as compared with normal melanocytes and benign nevi. Our data suggest that the immunohistochemical evaluation of 90K could potentially provide useful diagnostic information in selected patients with melanoma. This work was supported by a grant from COFIN MIUR and a grant from the Università di Modena e Reggio Emilia.