Effect of an anti-CD54 (ICAM-1) monoclonal antibody (UV3) on the growth of human uveal melanoma cells transplanted heterotopically and orthotopically in SCID mice

We have shown that administration of a novel anti-CD54 monoclonal antibody (UV3) results in long-term survival of SCID mice bearing human myeloma xenografts. Previous studies have demonstrated a link between the expression of CD54 and the progression of uveal melanoma. Our study assessed the expression of CD54 on 7 human uveal melanoma cell lines and 3 cell lines established from uveal melanoma metastases. In vivo studies examined the efficacy of systemic and local administration of UV3 antibody on the progression of uveal melanoma cells transplanted either heterotopically or orthotopically into SCID mice. Five of the 7 primary uveal melanoma cell lines and all 3 of the metastases cell lines expressed CD54. Intraperitoneal injection of either IgG or F(ab')2 fragments of UV3 significantly inhibited the growth of subcutaneous and intraocular melanomas. Subconjunctival injection of either IgG or F(ab')2 fragments of UV3 produced a significant reduction in the growth of intraocular melanomas, even if the antibody was administered after the appearance of intraocular tumors. The results indicate that both primary and metastatic human uveal melanoma cells express CD54. The marked inhibition of intraocular and subcutaneous uveal melanoma progression suggests that UV3 antibody is a promising therapeutic agent for further evaluation in patients with uveal melanoma. This is especially noteworthy, as no existing therapeutic modality prevents metastasis of uveal melanoma or prolongs the survival of patients with uveal melanoma.     

PUMA expression is significantly reduced in human cutaneous melanomas

Cutaneous malignant melanoma is an aggressive form of skin cancer, characterized by strong chemoresistance and poor patient prognosis. The molecular mechanisms underlying its resistance to chemotherapy remain unclear but are speculated to involve the dysregulation of apoptotic pathways. In this study, we sought to determine whether PUMA (p53 upregulated modulator of apoptosis) contributes to human melanoma formation, tumor progression, and survival. We used tissue microarray and immunohistochemistry to examine PUMA expression in 107 primary melanomas, 51 metastatic melanomas, and 64 dysplastic nevi. Here we report that PUMA expression is significantly weaker in primary melanomas compared to dysplastic nevi (P<0.0001), and is further reduced in metastatic melanomas compared to primary tumors (P=0.001). We show that weak PUMA expression in melanoma correlates with poorer overall and disease-specific 5-year survival (P<0.005 and P<0.001, respectively) of melanoma patients and that PUMA expression in tumor tissue is an independent predictor of both overall and disease-specific 5-year survival (P=0.05). Additionally, we show that exogenous PUMA expression in human melanoma cell lines (both wild type and mutant p53) results in significant apoptotic cell death. Our results suggest that PUMA expression may be an important prognostic marker for human melanoma and that adenoviral delivery of PUMA sensitizes melanoma cells to apoptosis.     

First‐in‐Human Proof‐of‐Concept Study: Intralesional Administration of BQ788, an Endothelin Receptor B Antagonist,to Melanoma Skin Metastases

Lessons Learned

• Local tolerance of intralesional treatment of melanoma skin metastases with BQ788 is excellent.
• Hints of efficacy are observed, consisting of both direct effects (decreased expression of endothelin receptor B [EDNRB] and of survival factors, reduced proliferation) and indirect effects (enhanced immune cell infiltration and angiogenesis).
• Patients in future trials should be screened for EDNRB expression level prior to treatment because only patients with high EDNRB-expressing melanomas (four of five) responded to BQ788.
• Future studies should be performed for at least 2 weeks because reduced lesion growth was observed in the only patient that was treated for longer than 1 week.

Background.

This first-in-human proof-of-concept study aimed to check whether safety and preclinical results obtained by intratumoral administration of BQ788, an endothelin receptor B (EDNRB) antagonist, can be repeated in human melanoma patients.

Methods.

Three patients received a single intralesional BQ788 application of 3 mg. After 3–7 days, the lesions were measured and removed for analysis. The administered dose was increased to a cumulative dosage of 8 mg in patient 4 (4 × 2.0 mg, days 0–3; lesion removed on day 4) and to 10 mg in patient 5 (3 × 3.3 mg, days 0, 3, and 10; lesion removed after 14 days). Control lesions were simultaneously treated with phosphate-buffered saline (PBS). All samples were processed and analyzed without knowledge of the clinical findings.

Results.

No statistical evaluation was possible because of the number of patients (n = 5) and the variability in the mode of administration. No adverse events were observed, regardless of administered dose. All observations were in accordance with results obtained in preclinical studies. Accordingly, no difference in degree of tumor necrosis was detected between BQ788- and PBS-treated samples. In addition, both EDNRB and Ki67 showed decreased expression in patients 2 and 5 and, to a lesser extent, in patient 1. Similarly, decreased expression of EDNRB mRNA in patients 2 and 5 and of BCL2A1 and/or PARP3 in patients 2, 3, and 5 was found. Importantly, semiquantitatively scored immunohistochemistry for CD31 and CD3 revealed more blood vessels and lymphocytes, respectively, in BQ788-treated tumors of patients 2 and 4. Also, in all patients, we observed inverse correlation in expression levels between EDNRB and HIF1A. Finally, in patient 5 (the only patient treated for longer than 1 week), we observed inhibition in lesion growth, as shown by size measurement.

Conclusion.

The intralesional applications of BQ788 were well tolerated and showed signs of directly and indirectly reducing the viability of melanoma cells.     

Ferritin contributes to melanoma progression by modulating cell growth and sensitivity to oxidative stress.

PURPOSE: Employing an in vitro model system of human melanoma progression, we previously reported ferritin light chain (L-ferritin) gene overexpression in the metastatic phenotype. Here, we attempted to characterize the role of ferritin in the biology of human melanoma and in the progression of this disease. EXPERIMENTAL DESIGN: Starting from the LM human metastatic melanoma cell line, we engineered cell clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. These cells were then assayed for their growth capabilities, chemoinvasive properties, and sensitivity to oxidative stress. Additionally, ferritin protein content in primary and metastatic human melanomas was determined by immunohistochemistry. RESULTS: Artificial L-ferritin down-regulation in the LM cells strongly inhibited proliferation and chemoinvasion in vitro and cell growth in vivo. In addition, L-ferritin down-regulated cells displayed enhanced sensitivity to oxidative stress and to apoptosis. Concurrently, immunohistochemical analysis of a human melanoma tissue array revealed that ferritin expression level in metastatic lesions was significantly higher (P < 0.0001) than in primary melanomas. Furthermore, ferritin expression was constantly up-regulated in autologous lymph node melanoma metastases when compared with the respective primary tumors in a cohort of 11 patients. CONCLUSIONS: These data suggest that high ferritin expression can enhance cell growth and improve resistance to oxidative stress in metastatic melanoma cells by interfering with their cellular antioxidant system. The potential significance of these findings deserves to be validated in a clinical setting.     

The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells

Differential gene expression of cell lines derived from a malignant melanoma or its autologous lymph node metastasis using cDNA arrays indicated down-regulation of PRSS11, a gene encoding the serine protease HtrA1, a homolog of the Escherichia coli protease HtrA, in the metastatic line. Stable PRSS11 overexpression in the metastatic cell line strongly inhibited proliferation, chemoinvasion and Nm23-H1 protein expression in vitro, as well as cell growth in vivo in nu/nu mice. A polyclonal anti-HtrA1 serum demonstrated a significantly higher expression in primary melanomas when compared to unrelated metastatic lesions in a human melanoma tissue array, and down-modulation of HtrA1 expression in autologous lymph node melanoma metastases in seven out of 11 cases examined. These results suggest that down-regulation of PRSS11 and HtrA1 expression may represent an indicator of melanoma progression.     

Loss of EphB6 expression in metastatic melanoma

Overexpression of various members of the Eph receptor tyrosine kinases and their ephrin ligands has been frequently reported in cancer. In contrast, a loss of EphB6 gene expression has been correlated with a poor prognosis in human neuroblastoma, suggesting a distinct role for this receptor compared to other family members. More recently, an important role of EphB6 signalling in T-cells has been described, suggesting possibly deleterious immunologic effects of a loss of EphB6 in cancer progression. We investigated the expression of EphB6 in melanocytic tumors. EphB6 mRNA of 22 microdissected tissues (7 benign nevi, 7 melanomas, 8 metastases) and 10 different cell lines (normal melanocytes, non-metastatic/metastatic melanoma cell lines) were measured by quantitative real-time RT-PCR. For visualization of EphB6 protein expression, immunohistochemistry of 32 melanocytic lesions were performed. On the mRNA level, the benign nevi revealed the highest EphB6 expression (mean = 1.43), while melanomas (mean = 0.63) and metastases (mean = 0.08; p=0.001) displayed a progressive and significant reduction of EphB6 expression. Accordingly, established melanoma cell lines with metastatic potential showed low EphB6 expression in comparison to normal melanocytes and to most of the melanoma cell lines. Immunohistochemistry revealed homogeneous staining in common nevi, whereas in malignant melanomas and metastases a heterogeneously positive to completely negative EphB6 staining was observed. Remarkably, Spitz nevi stained similarly to ordinary melanocytic nevi. Taken together, we show that melanoma progression to metastatic disease is associated with a significant reduction of EphB6 gene expression which may have considerable consequences for the prognosis of malignant melanoma patients and possible gene-therapeutic approaches.     

Nuclear PTEN expression and clinicopathologic features in a population-based series of primary cutaneous melanoma

Germline mutations of the PTEN tumor-suppressor gene, on 10q23, cause Cowden syndrome, an inherited hamartoma syndrome with a high risk of breast, thyroid and endometrial carcinomas and, some suggest, melanoma. To date, most studies which strongly implicate PTEN in the etiology of sporadic melanomas have depended on cell lines, short-term tumor cultures and noncultured metastatic melanomas. The only study which reports PTEN protein expression in melanoma focuses on cytoplasmic expression, mainly in metastatic samples. To determine how PTEN contributes to the etiology or the progression of primary cutaneous melanoma, we examined cytoplasmic and nuclear PTEN expression against clinical and pathologic features in a population-based sample of 150 individuals with incident primary cutaneous melanoma. Among 92 evaluable samples, 30 had no or decreased cytoplasmic PTEN protein expression and the remaining 62 had normal PTEN expression. In contrast, 84 tumors had no or decreased nuclear expression and 8 had normal nuclear PTEN expression. None of the clinical features studied, such as Clark's level and Breslow thickness or sun exposure, were associated with cytoplasmic PTEN expressional levels. An association with loss of nuclear PTEN expression was indicated for anatomical site (p = 0.06) and mitotic index (p = 0.02). There was also an association for melanomas to either not express nuclear PTEN or to express p53 alone, rather than both simultaneously (p = 0.02). In contrast with metastatic melanoma, where we have shown previously that almost two-thirds of tumors have some PTEN inactivation, only one-third of primary melanomas had PTEN silencing. This suggests that PTEN inactivation is a late event likely related to melanoma progression rather than initiation. Taken together with our previous observations in thyroid and islet cell tumors, our data suggest that nuclear-cytoplasmic partitioning of PTEN might also play a role in melanoma progression.     

Identification of the human PHLDA1/TDAG51 gene: down-regulation in metastatic melanoma contributes to apoptosis resistance and growth deregulation

To identify molecules involved in the progression of human melanoma to metastatic disease, autologous primary and metastatic melanoma cells were compared by differential mRNA display. One cDNA, expressed in primary but not in autologous metastatic cells in three different patients, was cloned and characterized, and shown to be the human homologue of the inducible, immediate early TDAG51/PHLDA1 (pleckstrin-homology-like domain family A, member1) gene. Monoclonal antibodies produced against the PHLDA1 protein revealed homogeneous strong expression by benign melanocytic nevi, and progressively reduced expression in primary and metastatic melanomas in vivo. Analysis of stable cDNA transfectants in two different cell lines revealed that constitutive PHLDA1 expression is associated with reduced cell growth, cloning efficiency, and colony formation but not with alterations in cell cycle parameters. However, PHLDA1 expression was associated with increased basal apoptosis as assessed by live cell annexin V binding, terminal deoxynucleotidyltransferase-dependent nucleotide incorporation, and with increased cleavage of poly(ADP-ribose) polymerase and caspase-9. Constitutive PHLDA1 expression greatly enhances the sensitivity of human melanoma cells to the chemotherapeutic agents doxorubicin and camptothecin. These results suggest that PHLDA1 is constitutively expressed by melanocytic nevi where it may contribute to their benign phenotype. The progressive loss of PHLDA1 expression in melanomas may play a role in deregulated cell growth and apoptosis resistance in these tumors.     

Inducible nitric oxide synthase and nitrotyrosine in human metastatic melanoma tumors correlate with poor survival.

Despite recognition of the malignant potential of human melanomas, the mechanisms responsible for the pathobiological characteristics contributing to tumor growth, vascular invasiveness, and distant organ metastasis remain undefined. Recent studies have shown that various human tumors express an inducible form of nitric oxide synthase (iNOS) and nitrotyrosine (NT), which suggests a mechanistic role of tumor-associated nitric oxide (NO) in tumorigenesis. We investigated iNOS and NT expression by immunohistochemistry in 20 human metastatic melanoma tissue specimens specifically with respect to iNOS-expressing cell types in the tumor area, pathological and clinical response to systemic therapy, potential role as a prognostic indicator, and NT formation. Our results showed that melanoma cells from 12 of 20 tumors express iNOS, yet the expression of this molecule in the tumor did not correlate with pathological or clinical response to therapy. More importantly, iNOS and NT expression by the melanoma cells strongly correlated with poor survival in patients with stage 3 disease (P < 0.001 and P = 0.020, respectively), suggesting a pathway whereby iNOS might contribute to enhanced tumor progression. In conclusion, our findings strongly suggest that iNOS expression has potential to be considered as a prognostic factor and NO as a critical mediator of an aggressive tumor phenotype in human metastatic melanomas.     

Role for beta3 integrins in human melanoma growth and survival

The role of alphaIIbbeta3 integrin in regulating platelet function is well appreciated, whereas its role in tumor progression and metastasis is not. The purpose of our study was to determine a functional relevance to expression of alphaIIbbeta3 integrin in cells derived from human solid tumors. A study of human melanoma biopsies (n = 24) showed that alphaIIbbeta3 expression increased with tumor thickness, which is indicative of metastatic propensity. Expression of alphaIIbbeta3 was 8% (+/-1.8), 33% (+/-10.4) and 62% (+/-5) in melanomas ranging in thickness from 0-1.5 mm, 1.5-4.0 mm and >4 mm, respectively; alphavbeta3 was equally high all categories. To determine biological function, we stably transfected alphaIIbbeta3 into human melanoma cells that express alphavbeta3, but not alphaIIbbeta3. Surface expression of alphavbeta3 remained unaltered between alphaIIbbeta3 (+) and mock transfected counterparts. The alphaIIbbeta3 (+) cells possessed increased ability to adhere, spread and migrate on fibrinogen. They had decreased ability to attach, spread and migrate on vitronectin. Immunocytochemistry showed that expression of alphaIIbbeta3 displaced alphavbeta3 from focal contact points. When implanted subcutaneously into SCID mice, the alphaIIbbeta3 (+) cells developed approximately 4-fold larger tumors when compared to their mock counterparts and the level of apoptosis was reduced within the tumors. Results suggest that co-expression of the 2 beta3 integrins, alphavbeta3 and alphaIIbbeta3, in human melanoma cells enhanced cell survival and promoted growth in vivo.     

Cytoplasmic localization of the oncogenic protein Ski in human cutaneous melanomas in vivo: functional implications for transforming growth factor beta signaling.

The oncogenic protein Ski associates with Smad proteins and counteracts their activation of gene expression and growth inhibition in response to transforming growth factor beta (TGF-beta). Here we show that Ski protein levels are increased in all 44 human melanoma tumor tissues analyzed in vivo. In addition, Ski subcellular localization changes from nuclear, in preinvasive melanomas (melanomas in situ), to nuclear and cytoplasmic in primary invasive and metastatic melanomas. Furthermore, Ski/Smad association in the cytoplasm seems to prevent Smad3 nuclear translocation in response to TGF-beta. The biological significance of Ski overexpression in melanomas was established by showing that down-regulation of Ski levels, by antisense Ski vectors, restored TGF-beta-mediated growth inhibition. Such inhibition is apparently mediated by up-regulation of the cyclin-dependent kinase-I p21(Waf-1) and inhibition of cyclin-dependent kinase 2 activity. Our results suggest that high levels of Ski in human melanomas produce a disruption of TGF-beta signaling phenotypically similar to that in cells harboring mutations in TGF-beta receptors or Smad proteins, and this may represent a significant event in the progression of melanomas in vivo.     

Differential response of primary and metastatic melanomas to neutrophils attracted by IL-8

IL-8 is a strong chemoattractant for neutrophils, and it is constitutively produced by many tumors, including human melanomas. To determine the biologic importance of IL-8 for melanoma cells from primary and metastatic lesions, we transduced selected cell lines constitutively producing low levels of IL-8 with IL-8 cDNA using a replication-deficient adenoviral vector. Nontumorigenic SBcl2 primary melanoma cells formed tumors when transduced with increasing plaque-forming units of IL-8 per cell. However, at high IL-8 transduction levels (100 ng/ml/10(5) cells in 48 hr), tumor growth was impaired due to massive neutrophil infiltration. A similar biphasic response was observed in WM115 primary melanomas, which are tumorigenic but not metastatic. Depletion of neutrophils with an antibody that blocks the accumulation of granulocytes at the site of inflammation enabled transduced primary melanomas secreting high levels of IL-8 to survive and grow. In contrast, highly tumorigenic and metastatic 451Lu cells showed marked increases in tumor growth and number of metastatic foci in the lungs depending on the expression levels of IL-8. Cytotoxicity assays with isolated neutrophils confirmed the preferential killing of primary over metastatic melanoma cells. SBcl2 cells stimulated by IL-8 to form tumors in immunodeficient mice were induced to produce VEGF, suggesting that the angiogenic response is enhanced due to increased growth factor production. Our results demonstrate that nontumorigenic primary melanomas depend on IL-8 stimulation in vivo for growth and that tumor growth depends on the level of neutrophil infiltration. Metastatic melanomas proliferate in vivo independently of infiltrating neutrophils.     

Tumorigenicity and dissemination of primary and metastatic human melanomas implanted into different sites in athymic nude mice

In the present study, the growth features and metastatic capacity of human primary and metastatic melanomas transplanted by different routes in nude mice was examined. Eight different human melanoma early cell cultures derived from 3 primary tumors, 1 local recurrence and 4 metastatic lesions of 6 melanoma patients were characterized for cell surface HLA (class I and II) and melanoma-associated antigens and karyotype. These tumors were then transplanted in CD-1 outbred nude mice by subcutaneous, intraperitoneal and intrasplenic routes. It was found that 7 out of 8 melanomas were tumorigenic after subcutaneous implantation without giving rise to metastases; 5 out of 7 melanomas grew when injected intraperitoneally and 3 of them disseminated to peritoneal organs and infiltrated intraperitoneal lymph nodes, liver and pancreas; of 8 melanomas implanted intrasplenically 4 grew in the spleen or invaded intraperitoneal lymph nodes, liver, pancreas, ovaries or lungs. Primary and metastatic melanomas did not differ in the pattern of dissemination. In fact, 2 out of 4 metastases and 1 of 3 primary tumors and 1 recurrence disseminated after intrasplenic or intraperitoneal inoculation. Heterogeneity in the growth pattern of different metastases of the same melanoma patient was also found. No correlation could be detected between the metastatic ability of melanoma cells studied and clinical stage of patients, tumor cell karyotype abnormalities, modal number or with the antigenic phenotype.     

The low molecular weight cyclin E isoforms augment angiogenesis and metastasis of human melanoma cells in vivo

Immunohistochemical analysis has consistently shown that cyclin E is up-regulated in human malignant melanomas in vivo. Here we analyzed such expression in more detail and show that cyclin E is overexpressed and present in low molecular weight (LMW) isoforms in metastatic melanoma and in a subset of primary invasive melanoma tumor tissues, but not in benign nevi. Human metastatic melanoma cell lines, but not normal melanocytes, also expressed the LMW cyclin E forms. The biological significance of these findings was established by showing that overexpression of two LMW cyclin E forms named cyclin E truncated 1 [cyclinE(T1)] and cyclin E truncated 2 [cyclinE(T2)] in a low tumorigenic and non-metastatic primary cutaneous melanoma cell line generated angiogenic tumors with prominent perineural invasion compared with full-length cyclin E. In addition, cyclin E(T1)- and cyclin E(T2)-expressing melanoma cells displayed a dramatic increase in the incidence and number of metastases in an experimental lung metastasis assay. Together, these results indicate that the LMW cyclin E forms are functional and likely act as regulators of human melanoma tumor progression and invasion.     

Expression of p16, p27, p53, p73 and Nup88 proteins in matched primary and metastatic melanoma cells

Cutaneous melanoma is a tumor with high metastatic potential, but the mechanisms leading to progression are still not fully understood. In this study, we examined whether p16, p27, p53, p73 and Nup88 proteins were involved in the progression from primary to metastatic melanomas by immunocytochemistry and Western blotting in eleven melanoma cell lines from five matched primary and metastatic melanomas. We demonstrated that the primary and metastatic melanomas expressed differently p16, p27, p73 and Nup88 proteins. When expressed in the primary melanoma cells p16 and p27 were lost or reduced in almost all the metastatic melanoma cell lines. In contrast, p73 and Nup88 were expressed in most of the tested melanoma cell lines and were increased in the metastatic melanomas. p53 was expressed at the same level in both the primary and metastatic melanoma cells. These data suggest that a reduced expression of p16 and p27 and an enhanced expression of p73 and Nup88 might play an important role in the progression of melanoma from primary tumor to metastasis.     

Prognostic significance of activated Akt expression in melanoma: a clinicopathologic study of 292 cases.

PURPOSE: Akt is a serine/threonine kinase that leads to stimulation of cell cycle progression, cell proliferation, and inhibition of apoptosis. To investigate the role of Akt in melanoma pathogenesis, we examined the expression of phospho-Akt (p-Akt; Ser-473) in melanocytic lesions at different stages and analyzed the correlations between the p-Akt expression level and clinicopathologic factors and patient survival. PATIENTS AND METHODS: We evaluated the p-Akt expression in 12 cases of normal nevi, 58 cases of dysplastic nevi, 170 cases of primary melanomas, and 52 cases of melanoma metastases using tissue microarray and immunohistochemistry. RESULTS: Strong p-Akt expression was observed in 17%, 43%, 49%, and 77% of the biopsies in normal nevi, dysplastic nevi, primary melanoma, and melanoma metastases, respectively. Significant differences for p-Akt staining pattern were observed between normal nevi and primary melanomas (P < .05), and between primary melanomas and melanoma metastases (P < .001). Furthermore, our Kaplan-Meier survival curves showed that strong p-Akt expression is inversely correlated with both overall and disease-specific 5-year survival of patients with primary melanoma (P < .05 for both). Strikingly, our multivariate Cox regression analysis revealed that p-Akt is an independent prognostic factor in low-risk melanomas (thickness < or = 1.5 mm; relative risk, 6.44; 95% CI, 1.28 to 32.55; P = .018). CONCLUSION: The expression of p-Akt increases dramatically with melanoma invasion and progression and is inversely correlated with patient survival. In addition, p-Akt may serve as an independent prognostic marker and help to identify those patients with low-risk melanomas who are at increased risk of death.     

A new Mr 55,000 surface protein implicated in melanoma progression: association with a metastatic phenotype

Emergence of the invasive phenotype is a key event in the progression of human melanoma from benign proliferative lesions to malignant lesions. Recently we successfully selected in vivo from a poorly metastatic M4Beu. human melanoma cell line two variants (7GP and T1P26) that generate a higher frequency of spontaneous metastases to the lungs into immune-suppressed neonatal rats. Both cell lines showed no significant differences in the integrin profile of the subunits analyzed except for beta3, which was reduced to a background level in metastatic variants. To investigate how these variant sublines of human melanomas manage to sustain growth in the absence of alpha(v)beta3, a subtractive immunization approach was used to elicit host antibody response against cell surface proteins expressed on metastatic variants. In this study, a new monoclonal antibody (MoAb), LY1, that is highly specific for the 7GP and T1P26 variants, was isolated. LY1 identifies a membrane protein of Mr 55,000 on melanoma variants with epitopes that were resistant to sugar-cleaving enzymes. Immunostaining cells from variants by LY1 showed that staining is distributed to the cell periphery with high labeling intensity at the cell-to-cell contact points. This MoAb significantly inhibited invasion of metastatic variants through a reconstituted basement membrane (Matrigel) in vitro. Moreover, tumor growth of melanoma variants was dramatically affected in vivo with this MoAb. In vitro studies indicate that the LY1 MoAb does not inhibit chemotactic migration of the metastatic variants, the adhesion of tumor cells to vitronectin, collagen IV, fibronectin, and laminin, or cell proliferation. Expression of this antigen is high in human striated muscle, heart, spleen, brain, and lung and absent in kidney, liver, and pancreas. Using 59 fixed, paraffin-embedded archival tissues of human melanomas and nevi, LY1-reactive cells were not observed in melanocytes, nevi, or radial growth phase primary melanomas. In sharp contrast, LY1 selectively stained melanocytes derived from the vertical growth phase of many primary melanomas and metastatic melanomas. These results provide evidence that the Mr 55,000 protein expressed by selected variants with increased metastatic properties in vivo plays a functionally important role in determining metastasis. This molecule may represent a new metastatic risk marker in human melanoma and may be of biological importance in the identification of fatal metastatic subpopulations that have acquired competence for metastasis production.