Phase II trial of imatinib mesylate in patients with metastatic melanoma

Metastatic melanoma cells express a number of protein tyrosine kinases (PTKs) that are considered to be targets for imatinib. We conducted a phase II trial of imatinib in patients with metastatic melanoma expressing at least one of these PTKs. Twenty-one patients whose tumours expressed at least one PTK (c-kit, platelet-derived growth factor receptors, c-abl, or abl-related gene) were treated with 400 mg of imatinib twice daily. One patient with metastatic acral lentiginous melanoma, containing the highest c-kit expression among all patients, had dramatic improvement on positron emission tomographic scan at 6 weeks and had a partial response lasting 12.8 months. The responder had a substantial increase in tumour and endothelial cell apoptosis at 2 weeks of treatment. Imatinib was fairly well tolerated: no patient required treatment discontinuation because of toxicity. Fatigue and oedema were the only grade 3 or 4 toxicities that occurred in more than 10% of the patients. Imatinib at the studied dose had minimal clinical efficacy as a single-agent therapy for metastatic melanoma. However, based on the characteristics of the responding tumour in our study, clinical activity of imatinib, specifically in patients with melanoma with certain c-kit aberrations, should be examined.     

Synergistic effects of imatinib and carboplatin on VEGF, PDGF and PDGF-Rα/ß expression in squamous cell carcinoma of the head and neck in vitro

Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy. The development of new treatment modalities in order to improve long-term survival of patients with HNSCC is imperative. Numerous studies have demonstrated that carcinogenesis and tumor cell dissemination is influenced by the tumor microenvironment. The protein-kinase-receptors (PTKs) are essential elements of the intracellular signal transduction pathway and regulate cell growth, development and apoptosis. Cell proliferation, migration, induction of tumor vascularization and carcinogenesis, invasion is regulated by a variety of angiogenic factors, such as PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor) and their respective tyrosine kinase receptors (PDGF-R and VEGF-R). They present promising targets for anti-cancer therapy through abrogation of impaired signaling pathways. Indeed, imatinib, a small molecule drug targeting these protein kinases, has antiproliferative effects in several cancer types. The purpose of this study was to investigate the potential synergism of imatinib and carboplatin on the expression of PDGF, PDGF-R α/? and VEGF in different HNSCC cell lines. Several tumor cell lines were subjected to increasing concentrations of carboplatin (3 and 7.5 μmol/l) and imatinib (18 and 30 μmol/l) and ELISA, immunohistochemical methods and RQ-PRC after 48, 72, 120 and 240 h were used to assess their expression levels. While PDGF-Rα/? expression was unimpaired at lower imatinib concentrations (18 μmol/l), PDGF-Rα/? expression was suppressed at 30 μmol/l, and suppression was enhanced by the presence of carboplatin. By RQ-PCR, a significant reduction of PDGF-Rα/? expression was detected (p<0.5). We observed explicit significant reduction in VEGF levels with increasing concentrations of imatinib and with the combination of the two chemotherapeutic drugs (p<0.5). We report for the first time evidence of synergism of imatinib and carboplatin in suppressing VEGF, PDGF and PDGF-Rα/? expression in HNSCC.     

Expression of the c-kit receptor in choroidal melanomas

The KIT gene encodes c-kit, a transmembrane receptor that has tyrosine kinase activity and plays a role in haematopoiesis, gametogenesis and melanogenesis. The c-kit protein is found in normal cutaneous and choroidal melanocytes, and there is evidence that expression is lost in melanoma. Expression of c-kit was analysed in 57 paraffin-embedded sections of choroidal melanoma specimens and three choroidal melanoma cell lines using immunochemistry and Western blotting. Of the tumour specimens, 75% stained positively for c-kit with a membrane pattern of reactivity. Of the six patients who underwent proton beam therapy before enucleation, five tumours exhibited no c-kit immunoreactivity and the other tumour demonstrated weak staining. Of the three melanoma cell lines used, c-kit expression was observed in only one. No correlations between c-kit positivity and parameters such as cell type, largest macroscopic tumour dimension, scleral invasion or pigmentation were observed. In contrast, a significant positive association was found between c-kit staining and mitotic activity (P = 0.02). However, c-kit expression did not significantly influence survival when evaluated by univariate analysis. In conclusion, c-kit is expressed in most choroidal melanoma tumours. Further analysis should provide new insights into the mechanisms underlying the molecular and cellular changes in choroidal melanomas.     

Effects of imatinib vary with the types of KIT-mutation in gastrointestinal stromal tumor cell lines

Mutations of proto-oncogene c-kit in gastrointestinal stromal tumors (GISTs) are considered to cause a constitutive activation of KIT responsible for their oncogenesis. Imatinib has therapeutic potential for GISTs because of its inhibitory effect on KIT kinase activity. However, no study has been published concerning the effects of imatinib on GIST cells with various types of KIT mutation. To investigate the effects of imatinib on various c-kit mutations found in GISTs, cell proliferation and apoptosis assays were performed in two GIST cell lines with different KIT mutations. One of the cell lines, GIST-T1, revealed a heterozygous deletion of exon 11 in the c-kit, while the other cell line, GIST882, possessed a homozygous missense mutation of exon 13 in the c-kit gene. Imatinib inhibited proliferation and induced apoptosis in both cell lines. Imatinib potently suppressed proliferation of the GIST882 cell line at the concentration of 1.0 microM, whereas it inhibited the GIST-T1 at 0.1 microM. In two types of activating mutant KIT, imatinib could inhibit the constitutive activation of both types of KIT mutant, although the antiproliferative effect on GIST882 was weaker than on GIST-T1. Western blot analysis revealed that apoptosis related proteins were activated or suppressed by imatinib in both cell lines in the respective manner. Our results suggest that the apoptotic signal trans-duction caused by imatinib in GISTs is susceptible to various types of KIT mutation.     

Imatinib mesylate inhibits Leydig cell tumor growth: evidence for in vitro and in vivo activity

Leydig cell tumors are usually benign tumors of the male gonad. However, if the tumor is malignant, no effective treatments are currently available. Leydig cell tumors express platelet-derived growth factor (PDGF), kit ligand and their respective receptors, PDGFR and c-kit. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the c-kit and PDGFR tyrosine kinases, on the growth of rodent Leydig tumor cell lines in vivo and in vitro, and examined, in human Leydig cell tumor samples, the expression of activated PDGFR and c-kit and the mutations in exons of the c-kit gene commonly associated with solid tumors. Imatinib caused concentration-dependent decreases in the viability of Leydig tumor cell lines, which coincided with apoptosis and inhibition of proliferation and ligand-stimulated phosphorylation of c-kit and PDGFRs. Mice bearing s.c. allografts of a Leydig tumor cell line treated with imatinib p.o., had an almost complete inhibition of tumor growth, less tumor cell proliferation, increased apoptosis, and a lesser amount of tumor-associated mean vessel density compared with controls. No drug-resistant tumors appeared during imatinib treatment but tumors regrew after drug withdrawal. Human Leydig cell tumors showed an intense expression of the phosphorylated form of c-kit and a less intense expression of phosphorylated PDGFRs. No activating mutations in common regions of mutation of the c-kit gene were found. Our studies suggest that Leydig cell tumors might be a potential target for imatinib therapy.     

c-kit Expression and mutations in peripheral T cell lymphomas, except for extra-nodal NK/T cell lymphomas

The present study evaluated the expression and mutations of c-kit in peripheral T-cell lymphomas (PTCLs), except for extra-nodal NK/T cell lymphomas, as a potential target for treatment with imatinib mesylate. Fifty-two patients diagnosed with PTCLs (peripheral T-cell lymhoma, unspecified, 38 cases; angioimmunoblastic T-cell, 7 cases; anaplastic large cell, 7 cases) were enrolled. The immunohistochemistry was performed using standard procedures with anti-c-kit monoclonal IgG, while the c-kit mutations were analysed on paraffin-embedded specimens using PCR-single-stranded conformational polymorphism followed by direct DNA sequencing. The median age of the patients was 52 years (19 to approximately 75 years) with a male-to-female ratio of 69%:31%. Weak expression of c-kit was found in 16 (30.8%) patients, while only 3 (5.8%) patients exhibited mutations in exon 11 or exon 13. The c-kit mutations in exon 11 occurred at codon 558 (AAG --> TAG; Lys --> Stop) and at codon 571 (CTA --> ATA; Leu --> Ile), respectively, while the mutation in exon 13 occurred at codon 634 (CGG --> CGA; Arg --> Arg). The current study only found c-kit mutations in a few patients with PTCLs, except for extra-nodal NK/T cell lymphomas. Therefore, c-kit would not seem to be a good target for a new therapeutic approach to PTCLs.     

An immunohistochemical evaluation of c-kit (CD-117) expression in malignant melanoma, and results of imatinib mesylate (Gleevec) therapy in three patients

In order to determine whether imatinib mesylate (Gleevec), a tyrosine kinase inhibitor that binds the CD-117 (c-kit) receptor, may be of value in the treatment of malignant melanoma, an immunohistochemical analysis of 40 cases of primary and metastatic melanoma was undertaken. Thirty-five of the 40 cases showed 1+ or stronger labelling for CD-117 (up to a maximum of 4+). Three patients with neoplasms showing 4+ staining were selected for imatinib therapy. None responded. c-kit (CD-117) expression in melanoma appears to be common; however, the value of imatinib therapy remains to be proven.     

Expression of activated platelet-derived growth factor receptor in stromal cells of human colon carcinomas is associated with metastatic potential

Platelet-derived growth factor receptor (PDGF-R) expression has been reported in a variety of cancers, including colorectal, breast, lung, ovarian and pancreatic cancers, but the role of PDGF-R expression in the development and progression of colon carcinoma has not yet been elucidated. The purpose of this study was to examine the expression of PDGF and PDGF-R in human colon carcinomas. The expression of PDGF, PDGF-R and phosphorylated PDGF-R (p-PDGF-R) was examined by immunofluorescence in 12 surgical specimens of colon carcinoma and in human colon carcinoma cells growing in the subcutis (ectopic site) and the cecal wall (orthotopic site) of nude mice. In most surgical specimens, tumor cells expressed PDGF-A and -B subunits, without corresponding levels of PDGF-Ralpha and PDGF-Rbeta. PDGF-Rbeta was predominantly expressed by tumor-associated stromal cells and pericytes of tumor vasculature. The expression of PDGF-Rbeta in the stroma was associated with advanced stage disease. Under culture conditions, human colon carcinoma cell lines expressed PDGF-A and -B, but not PDGF-R. In orthotopic tumors, the KM12 cells (Duke's stage B) expressed PDGF-A and -B, but PDGF-Rbeta was expressed only by stromal cells and pericytes in the tumor vasculature. This expression of PDGF-Rbeta by stromal cells and pericytes was higher in tumors growing at the orthotopic site than in those at the ectopic site. The expression of PDGF-Rbeta in the stroma was higher in highly metastatic KM12SM tumors than in low metastatic KM12C tumors. In conclusion, the expression of PDGF-Rbeta in stromal cells is influenced by the organ-specific microenvironment and is associated with metastatic potential.     

Down-regulation of MMP-2 expression due to inhibition of receptor tyrosine kinases by imatinib and carboplatin in HNSCC

Squamous cell carcinoma of the head and neck (HNSCC) is the most common neoplasm arising in the upper aerodigestive tract. Unfortunately, the survival for this type of cancer has not improved significantly in the past 25 years. To enhance the survival rate multimodal therapy regimens have been set up. In these regimens chemotherapy plays a pivotal role in the majority of advanced cases. Transmembrane protein- tyrosine kinases (PTK) are fundamental elements of the signal transduction. In consequence, they might be promising targets for cancer therapy. Imatinib (STI 571) was originally designed to inhibit the BCR-ABL tyrosine kinase in chronic myeloid leukemia. But imatinib also has an inhibitory impact on the PTK receptor c-kit and on its PTK activity. Furthermore, growth and invasion of HNSCC are strongly influenced by the extracellular matrix (ECM). The ECM is altered by matrix metalloproteinases (MMP). In this study, we incubated different HNSCC cell lines with rising concentrations of imatinib and/or carboplatin. After an incubation time of up to 10 days, we evaluated c-kit, MMP-2 and MMP-14 by ELISA techniques and immunohistochemical methods. Especially the combination of 7.5 μmol carboplatin with 30 μmol imatinib resulted in a significant decrease in MMP-2 expression in all observed cell lines (p<0.05). We did not demonstrate a significant alteration in c-kit expression by imatinib and carboplatin. We observed an increase in apoptosis in HNSCC cells by the combination of the two observed chemotherapeutic drugs. In all cell lines tested, expression of c-kit and MMP could be demonstrated. Our results indicate that MMP-2 expression was suppressed in the presence of imatinib. Thus, imatinib may exert in part its inhibitory effect on malignant cell growth via the blockage of the signal transduction of PTK receptors. Further studies are warranted, especially one keeping in mind the moderate toxicity of imatinib.     

c-KIT-expressing Ewing tumour cells are insensitive to imatinib mesylate (STI571)

PURPOSE: In order to determine whether Ewing tumour patients may be potential candidates for imatinib mesylate therapy, we analysed the expression of the currently known imatinib mesylate-sensitive tyrosine kinases and tested sensitivity to imatinib mesylate in a panel of eight Ewing tumour cell lines in vitro. METHODS: Expression of the different tyrosine kinases was assessed by flow cytometry and RT-PCR. Sensitivity to imatinib mesylate was analysed using a standard MTT proliferation assay. RESULTS: Flow cytometric and RT-PCR analyses in a panel of eight Ewing tumour cell lines demonstrated expression of several imatinib mesylate-sensitive tyrosine kinases, including c-KIT, platelet-derived growth factor receptor, c-ABL and c-ARG. However, in the MTT proliferation assay, all eight Ewing tumour cell lines were found to be resistant to imatinib mesylate at concentrations ranging from 0.1 to 10 micro M. CONCLUSIONS: Despite the expression of imatinib mesylate-sensitive tyrosine kinases, Ewing tumour cells proved resistant to imatinib mesylate in vitro. This observation has implications for the selection of patients for experimental therapy with imatinib mesylate.     

NRAS and BRAF mutation frequency in primary oral mucosal melanoma

Oral mucosal melanoma (OMM) is a fatal sarcoma of unknown etiology. Histological morphology and genetic events are distinct from those of its cutaneous counterpart. Mutation and up-regulation of c-kit has been identified in OMM which may activate downstream molecules such as RAS and RAF. These molecules are involved in the mitogen-activated protein kinase (MAPK) pathway leading to tremendous cell proliferation and survival. NRAS and BRAF mutation and protein expression have been studied in other melanoma subtypes. The purpose of this study was to determine RAS protein expression and NRAS and BRAF mutation in 18 primary OMM cases using immunohistochemistry and mutation analysis. Results showed that RAS is intensely expressed in both in situ and invasive OMMs. However, NRAS mutation was only observed in 2/15 polymerase chain reaction (PCR) amplified cases both of which were silent mutations. On the other hand, BRAF missense mutations were observed only in 1/15 cases with PCR amplification. NRAS and BRAF mutations were independent from previously reported c-kit mutations. The classical V600E BRAF mutation was not found; instead a novel V600L was observed suggesting that the oncogenic event in OMM is different from that in skin melanoma. The low frequency of NRAS and BRAF mutations indicate that these genes are not common, but probable events in OMM pathogenesis, most likely independent of c-kit mutation.     

Identification of a novel subgroup of melanomas with KIT/cyclin-dependent kinase-4 overexpression

Although many melanomas harbor either activating mutations in BRAF or NRAS, there remains a substantial, yet little known, group of tumors without either mutation. Here, we used a genomic strategy to define a novel group of melanoma cell lines with co-overexpression of cyclin-dependent kinase 4 (CDK4) and KIT. Although this subgroup lacked any known KIT mutations, they had high phospho-KIT receptor expression, indicating receptor activity. Quantitative PCR confirmed the existence of a similar KIT/CDK4 subgroup in human melanoma samples. Pharmacologic studies showed the KIT/CDK4-overexpressing subgroup to be resistant to BRAF inhibitors but sensitive to imatinib in both in vitro and in vivo melanoma models. Mechanistically, imatinib treatment led to increased apoptosis and G(1) phase cell cycle arrest associated with the inhibition of phospho-ERK and increased expression of p27(KIP). Other melanoma cell lines, which retained some KIT expression but lacked phospho-KIT, were not sensitive to imatinib, suggesting that KIT expression alone is not predictive of response. We suggest that co-overexpression of KIT/CDK4 is a potential mechanism of oncogenic transformation in some BRAF/NRAS wild-type melanomas. This group of melanomas may be a subpopulation for which imatinib or other KIT inhibitors may constitute optimal therapy.     

Immunohistochemical Analysis of Platelet-derived Growth Factor Receptor-α, -β, c-kit, c-abl, and Arg Proteins in Glioblastoma: Possible Implications for Patient Selection for Imatinib Mesylate Therapy

Summary Inhibition of tyrosine kinase (TK) receptors by synthetic small molecules has become a promising new therapy option in oncology. The TK inhibitor imatinib mesylate selectively targets PDGFR-α, -β, c-kit, c-abl and arg and has proven successful in the treatment of chronic myeloid leukaemia. In recurrent glioblastoma, phase II therapy trials using imatinib mesylate have been initiated. As only a fraction of patients seems to benefit from imatinib mesylate therapy and due to potential side effects and high costs of imatinib mesylate therapy, selection of the right patients is important. The goal of our study was to assess systematically immunohistochemical expression of the major TKs targeted by imatinib mesylate in glioblastoma, as expression of these factors could be used to select patients for imatinib mesylate therapy. In a cohort of 101 glioblastoma patients, anti-PDGFR-α, -β, c-kit, c-abl and arg protein immunohistochemistry was performed. Expression of these proteins was assessed semi-quantitatively and correlated with patient survival. PDGFR-α and arg expression in tumor cells was widespread in 1/101 cases, respectively. Focal PDGFR-α, -β, c-kit, c-abl and arg immunolabeling was detected in 25/101, 19/101, 4/101, 7/101 and 31/101 cases, respectively. Statistical analysis did not reveal any correlation between expression of the TKs and patient survival. We show here for the first time in a large series of glioblastomas that PDGFR-α, -β, c-kit, c-abl and arg expression is immunohistochemically detectable in a fraction of cases. The value of anti-tyrosine kinase immunolabeling as predictive factor for patient selection remains to be clarified by comparative analysis of tumor tissue of therapy-responders versus non-responders.     

In vitro anti-neuroblastoma activity of saquinavir and its association with imatinib

Neuroblastoma (NB) has a poor prognosis when in advanced stages, highlighting the need for new therapeutic options. The human immunodeficiency virus (HIV) protease inhibitor saquinavir is active in?vitro against chronic myeloid leukaemia cells, in synergy with the tyrosine kinase inhibitor imatinib. Here, we evaluated the effects of saquinavir, alone or in association with imatinib, on cell proliferation (count of viable cells after trypan blue exclusion), apoptosis (Annexin?V binding) and invasion (through a transwell membrane coated with Matrigel) in SJ-N-KP, IMR5, AF-8, SK-N-SH and SK-N-BE NB lines, all expressing c-kit and PDGF-R (determined by flow cytometry). Saquinavir showed a dose-dependent anti-proliferative and anti-invasive activity on NB lines, increased by the association with imatinib when the two drugs were utilized at clinically attainable concentrations. The same low saquinavir concentrations inhibited in NB cells the nuclear activation of NF-κB (Western immuno-blotting for nuclear NF-κB p50 and p65). Saquinavir at high concentrations also exerted a pro-apoptotic activity on NB lines, significantly increased by the association with imatinib. In conclusion, saquinavir and imatinib are both drugs utilized for long-term therapies, with good oral bioavailability and a well-known toxicity profile. The anti-NB activity of saquinavir and of its association with imatinib suggests a potential usefulness in the treatment of NB, particularly for remission maintenance.     

Differential effects of imatinib mesylate against uveal melanoma in vitro and in vivo

Uveal melanoma is refractory to chemotherapy. The receptor tyrosine kinase inhibitor, imatinib mesylate, has demonstrated antiproliferative effects against uveal melanoma cells in vitro. The effects of imatinib mesylate, alone and combined with the alklyating agent, temozolomide, were examined in vivo as well as in vitro. Proliferation and angiogenic factor production of human uveal melanoma cell lines in response to imatinib mesylate and temozolomide were examined in vitro. Tumor growth, angiogenic factor production, tumor interstitial fluid pressure, and stroma constituents in response to imatinib mesylate and temozolomide were examined in vivo in mice bearing human uveal melanoma xenografts. Imatinib mesylate in vitro antagonized the antiproliferative effects of temozolomide and increased the production of angiogenic factors. In contrast, pretreatment with imatinib mesylate in vivo could improve the antitumor activity of temozolomide. Imatinib mesylate in vivo decreased the production of angiogenic factors in the tumor stroma and tumor interstitial fluid pressure. These effects were transient. Increases in angiogenic factors, interstitial fluid pressure, and tumor infiltrating macrophages were observed with continued imatinib mesylate treatment in vivo. The antitumor effects of imatinib mesylate can vary in vivo when compared with in vitro. Imatinib mesylate can both positively and negatively modify host-tumor interactions in uveal melanoma.     

Imatinib enhances human melanoma cell susceptibility to TRAIL-induced cell death: Relationship to Bcl-2 family and caspase activation

In order to define genetic determinants of primary and metastatic melanoma cell susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we have applied oligonucleotide microarrays to TRAIL-sensitive primary T1 cells and TRAIL-resistant metastatic G1 cells treated or not with TRAIL. T1 and G1 cells are isogenic melanoma cell subclones. We examined 22 000 spots, 4.2% of which displayed differential expression in G1 and T1 cells. Cell susceptibility to TRAIL-mediated apoptosis was found to be correlated with gene expression signatures in this model. Some of the differentially expressed genes were identified as involved in ATP-binding and signaling pathways, based on previously published data. Further analysis provided evidences that c-kit was overexpressed in G1 cells while it was absent in T1 cells. The c-kit inhibitor, imatinib, did not restore TRAIL sensitivity, excluding a role for c-kit in TRAIL resistance in G1 cells. Surprisingly, imatinib inhibited cell proliferation and TRAIL-mediated apoptosis in melanoma cells. We investigated the possible involvement of several molecules, including c-ABL, platelet-derived growth factor receptor (PDGFR), cellular FADD-like interleukin-1 alpha-converting enzyme-like inhibitory protein (c-FLIP)(L/S), Fas-associated DD kinase, p53, p21(WAF1), proteins of B-cell leukemia/lymphoma 2 (Bcl-2) family and cytochrome c. Imatinib did not modulate the expression or activation of its own targets, such as c-ABL, PDGFRalpha and PDGFRbeta, but it did affect the expression of c-FLIP(L), BCL2-associated X protein (Bax) and Bcl-2. Moreover, c-FLIP(L) knockdown sensitized T1 cells to TRAIL-mediated apoptosis, with a sensitivity similar to that of cells previously treated with imatinib. More notably, we found that the resistance to TRAIL in G1 cells was correlated with constitutive c-FLIP(L) recruitment to the DISC and the inhibition of caspase 8, 3 and 9 processing. Moreover, c-FLIP(L) knockdown partly restored TRAIL sensitivity in G1 cells, indicating that the expression level of c-FLIP(L) and its interaction with TRAIL receptor2 play a crucial role in determining TRAIL resistance in metastatic melanoma cells. Our results also show that imatinib enhances TRAIL-induced cell death independently of BH3-interacting domain death agonist translocation, in a process involving the Bax:Bcl-X(L) ratio, Bax:Bcl-X(L)/Bcl-2 translocation, cytochrome c release and caspase activation. Our data indicate that imatinib sensitizes T1 cells by directly downregulating c-FLIP(L), with the use of an alternative pathway for antitumor activity, because PDGFRalpha is not activated in T1 cells and these cells do not express c-kit, c-ABL or PDGFRbeta. Caspase cascade activation and mitochondria also play a key role in the imatinib-mediated sensitization of melanoma cells to the proapoptotic action of TRAIL.     

Somatostatin receptor scintigraphy with 111In-DOTA-lanreotide and 111In-DOTA-Tyr3-octreotide in patients with stage IV melanoma: in-vitro and in-vivo results

The overexpression of somatostatin receptors (SST-Rs) on various tumour cells provides the molecular basis for the successful use of radiolabelled SST analogues in clinical oncology. The objective of the study was to evaluate the tumour binding of In-1,4,7,10-tetraazacyclo-dodecane-N,N',N',N'-tetraacetic acid-lanreotide (In-DOTA-LAN) and In-DOTA-tyrosine-octreotide (In-DOTA-Tyr-OCT) in patients with stage IV melanoma. In addition, we evaluated the potential antiproliferative effect of SST analogues, together with an assessment of the functionality of SST-Rs, on four melanoma cell lines. Twenty-three patients with advanced metastatic melanoma underwent scintigraphy. Thirty-eight of 61 lesions (62%) were positively imaged with In-DOTA-LAN, whereas 23 (37%) were negative. With In-DOTA-Tyr-OCT, 10 of the 23 documented lesions (43%) were positive and 13 (56%) were negative. In vitro, cell lines showed no growth inhibition in the presence of SST analogues and no influence on cell cycle distribution was found with the addition of SST analogues to cultured cells. In addition, no functional surface SST-Rs could be demonstrated on these cell lines. Taken together, our results demonstrate the visualization of metastatic melanoma in a high percentage of patients, probably due to binding of SST analogues to SST-Rs on tumour vessels or infiltrating immune cells. Judging from our data, however, there is no evidence of functional SST-R expression on melanoma cells.     

NY-ESO-1 tumour associated antigen is a cytoplasmic protein detectable by specific monoclonal antibodies in cell lines and clinical specimens

NY-ESO-1 gene encodes a novel member of the cancer/testis (CT) family of human tumour-associated antigens (TAA). Specific monoclonal antibodies (mAb) have identified the corresponding gene product in lysates of tumour cell lines as a 22 kDa protein but no data are available concerning its intracellular location or distribution within neoplastic tissues. We have generated NY-ESO-1 specific mAbs recognizing the target molecule in cytospin preparations and in sections from clinical tumour specimens. These reagents identify NY-ESO-1 TAA in melanoma cell lines expressing the specific gene as a cytoplasmic protein, sharing the intracellular location of most MAGE TAA. In a series of 12 melanoma specimens, specific staining, limited to neoplastic cells, was detectable in the five cases where NY-ESO-1 gene expression was observed. In two of them over 90% of tumour cells showed evidence of positive staining. Lower percentages of positive neoplastic cells ranging between single cells and 50% were observed in the remaining tumours. These data suggest that active specific immunotherapies targeting NY-ESO-1, alone or in combination with other TAA could be of high clinical relevance in sizeable subgroups of melanoma patients.     

Platelet Derived Growth Factor (PDGF) autocrine components in human tumor cell lines

Tumor cells may stimulate their own proliferation through an autocrine mechanism by simultaneously producing growth factors and growth factor receptors. We now report that numerous human tumor-derived cell lines simultaneously express the genes for platelet-derived growth factor (PDGF) A and B chains and the PDGF receptor (PDGF-R). Measurement of mRNA transcribed from these genes showed that among 16 malignant glioma cell lines tested, 15 expressed the PDGF A gene, 12 expressed the PDGF B gene, and 13 expressed the PDGF-R gene. Of three osteosarcoma lines, three expressed PDGF A, two expressed PDGF B, and three expressed PDGF-R. For eight malignant melanoma lines, seven expressed PDGF A, five expressed PDGF B, and three expressed PDGF-R genes. Thus, 13 of 16 malignant glioma, 3 of 3 osteosarcomas, and 3 of 8 malignant melanoma cell lines expressed the PDGF receptor gene and either or both PDGF genes. Five cell lines were tested for production of biologically active PDGF and PDGF receptor protein. Media conditioned by each of the five cell lines induced tyrosine phosphorylation of a protein identical in size to the PDGF receptor. These five cell lines also produced PDGF receptor protein as measured by Western blot analysis or metabolic labeling and immunoprecipitation using PDGF-R antibodies. The PDGF receptors of these cell lines were activated by human platelet PDGF or by recombinant AA or BB homodimers. Intracellular interaction of these receptors with the growth factor simultaneously produced may provide continuous stimulation to the proliferation of these cells.     

KIT receptor is expressed in more than 50% of early-stage malignant melanoma: a retrospective study of 261 patients

The c-kit gene encodes a transmembrane receptor (KIT) with tyrosine kinase activity which is a specific target for anti-cancer therapy. We investigated KIT expression in a group of patients with early-stage malignant melanoma. Primary tumour specimens obtained from 261 radically resected patients with stage I and II malignant melanoma were examined for KIT expression. Formalin-fixed, paraffin embedded tissues were stained with the polyclonal rabbit anti-human anti-KIT antibody (Dako Cytomation Inc., Carpenteria, California, USA). Patients were classified into four groups according to the level of expression (0%, <30%, 30-60% and >60%). Univariate and multivariate analyses examining the impact of KIT expression, Breslow thickness, Clark level and microscopic ulceration on disease-free survival were performed. Within the population of 261 patients with early-stage melanoma with 62 recurrences during a follow-up of 64 months, KIT expression was found in 144 cases (55%). KIT was expressed in more than 60% of cells in 20 patients (8%), in 30-60% of cells in 64 patients (24%) and in less than 30% of cells in 60 patients (23%). KIT expression was not found in 117 patients (45%). In univariate analyses, the influence of KIT expression on disease-free survival was not proven (P=0.4956; log-rank test). Increasing Breslow thickness, a higher Clark level, the presence of microscopic ulceration and a higher stage were significantly associated with a shorter disease-free survival (P<0.0001; log-rank test in all cases). In multivariate analysis, Breslow thickness, stage and KIT expression were significant negative prognostic factors for a shorter disease-free survival (P<0.0001, P=0.0028, P=0.0488, respectively; stepwise Cox regression model). It can be concluded that KIT is expressed in more than one-half of early-stage malignant melanoma. KIT may serve as an additive prognostic factor to Breslow thickness and stage within the tested population. The therapeutic impact of KIT expression in malignant melanoma is uncertain. Results of ongoing pilot phase II studies may validate the efficacy of imatinib mesylate in malignant melanoma expressing KIT.