Metastatic Melanoma Lesions Research Articles

Clinical relevance of serum levels of matrix metallopeptidase‐2, and tissue inhibitor of metalloproteinase‐1 and ‐2 in patients with malignant melanoma

The interaction and/or balance between matrix metallopeptidase (MMP)-2 and tissue inhibitor of metalloproteinase (TIMP)-2 in vivo may play important roles in the process of tumor growth, invasion and metastasis of malignant melanoma. In this study, we investigated the serum levels and immunohistochemical expression of MMP-2, TIMP-1 and TIMP-2 in patients with melanoma and analyzed the correlation with clinicopathological parameters. The level of serum MMP-2 in patients was significantly higher than that of the control. Moreover, the level of MMP-2 was significantly higher than that of the control in patients who were: (i) female; (ii) pT1 and pT4; (iii) with and without lymph node (LN) metastasis; (iv) in stage I and stage IV; (v) with and without recurrence; and (v) alive and dead. The level of serum TIMP-1 in patients with melanoma was significantly higher than that of the control. Among melanoma patients, the level of TIMP-1 with pT4 was significantly higher for patients who were: (i) pT1 and pT3; (ii) with LN metastasis (vs those without); (iii) in stage IV (vs those in stages I, II and III); and (iv) dead (vs those alive). The level of serum TIMP-2 in patients with melanoma was not different from the control. However, the level of TIMP-2 in patients with pT4 was significantly higher than for patients who were: (i) pT1, pT3 and control; (ii) with LN metastasis (vs those without metastasis and control); (iii) with stage IV (vs those in stages I and II and control); (iv) in recurrence (vs control); and (v) dead (vs those alive and control). These results suggest that increased serum levels of TIMP-1 and TIMP-2 reflected the extent of metastatic melanoma lesions, and that serum levels of TIMP-1 may be a new useful marker for melanoma progression.

Bcl-2 and Glutathione Depletion Sensitizes B16 Melanoma to Combination Therapy and Eliminates Metastatic Disease

Advanced melanoma resists all current therapies, and metastases in the liver are particularly problematic. Prevalent resistance factors include elevated glutathione (GSH) and increased expression of bcl-2 in melanoma cells. GSH has pleiotropic effects promoting cell growth and broad resistance to therapy, whereas Bcl-2 inhibits the activation of apoptosis and contributes to elevation of GSH. This study determined the in vivo efficacy of combination therapies administered while GSH and Bcl-2 were individually and simultaneously decreased in metastatic melanoma lesions. Highly metastatic murine B16 melanoma (B16M-F10) cells have elevated levels of both GSH and Bcl-2. B16M-F10 cells were injected i.v. to establish metastatic lesions in vivo. GSH was decreased using an L-glutamine--enriched diet and administration of verapamil and acivicin, whereas Bcl-2 was reduced using oligodeoxynucleotide G3139. Paclitaxel, X-rays, tumor necrosis factor-alpha, and IFN-gamma were administered as a combination therapy. Metastatic cells were isolated from liver to confirm the depletion of GSH and Bcl-2 in vivo. Reduction of Bcl-2 and GSH, combined with partial therapies, decreased the number and volume of invasive B16M-F10 foci in liver by up to 99% (P<0.01). The full combination of paclitaxel, X-rays, and cytokines eliminated B16M-F10 cells from liver and all other systemic disease, leading to long-term survival (>120 days) without recurrence in 90% of mice receiving the full therapy. Toxicity was manageable; the mice recovered quickly, and hematology and clinical chemistry data were representative of accepted clinical toxicities. Our results suggest a new strategy to induce regression of late-stage metastatic melanoma.

Targeting Nodal in malignant melanoma cells

Metastatic melanoma continues to be a significantly deadly cancer with a cure rate of < 20% and a median survival of 6 – 9 months. The aggressiveness associated with metastatic melanoma is largely attributable to its inherent plasticity, a property that is mediated by the secretion of Nodal, a stem-cell associated protein belonging to the transforming growth factor-β superfamily. This is supported by the observations that Nodal expression is limited to invasive vertical growth phase and metastatic melanoma lesions, and that inhibition of Nodal signaling promotes the reversion of metastatic melanoma cells toward a more differentiated, less invasive non-tumorigenic phenotype. Hence, due to its restricted expression pattern and function as a melanoma-tumor-promoter, Nodal (and its signaling partners) present unique targets for both immunologic and pharmacologic therapies.

A fresh look at an old story: revisiting HLA class II antigen expression by melanoma cells

The appearance of human leukocyte antigen (HLA) class II antigens may be associated with malignant transformation of melanocytes. However, the information available regarding the clinical relevance of HLA class II antigen expression, which has been found in approximately 50% of primary and metastatic cutaneous melanoma lesions, is scarce and conflicting. The debate regarding the clinical relevance of HLA class II antigen expression by malignant melanocytes, in conjunction with our increased understanding of HLA class II antigen processing and presentation, their role in the interactions between melanoma cells and components of the adaptive immune system and their potential involvement in melanoma cell migration have provided the impetus to revisit the topic of HLA class II antigen expression by malignant melanocytes. This review will discuss the current knowledge regarding HLA class II antigens, including the presentation pathway; frequency of expression in cell lines and lesions of melanocytic origin; po...

Rab33A: Characterization, Expression, and Suppression by Epigenetic Modification

Rab33A, a member of the small GTPase superfamily, is an X-linked gene that is expressed in brain, lymphocytes, and normal melanocytes, but is downregulated in melanoma cells. We demonstrate that in normal melanocytes Rab33A colocalizes with melanosomal proteins and that a constitutively active GTPase mutant suppresses their transport to the melanosomes. In the brain, Rab33A is present throughout the cortex, as well as in the hippocampal CA fields. A survey of melanocytic lesions demonstrated that aberrant downregulation of Rab33A is an early event that is already prevalent in melanocytes of giant congenital nevi. Analyses of bisulfite-modified DNA revealed that Rab33A is regulated by DNA methylation of a specific promoter region proximal to the transcription initiation site, and that suppression of Rab33A in melanoma cells recapitulates normal processes that control silencing of X-linked genes, but not tissue specific gene expression. This information is important for understanding carcinogenesis as well as other aberrant processes because Rab33A may have an important role in disorders involving X-chromosome-linked genes associated with vesicular transport.

Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes

Through the adoptive transfer of lymphocytes after host immunodepletion, it is possible to mediate objective cancer regression in human patients with metastatic melanoma. However, the generation of tumor-specific T cells in this mode of immunotherapy is often limiting. Here we report the ability to specifically confer tumor recognition by autologous lymphocytes from peripheral blood by using a retrovirus that encodes a T cell receptor. Adoptive transfer of these transduced cells in 15 patients resulted in durable engraftment at levels exceeding 10% of peripheral blood lymphocytes for at least 2 months after the infusion. We observed high sustained levels of circulating, engineered cells at 1 year after infusion in two patients who both demonstrated objective regression of metastatic melanoma lesions. This study suggests the therapeutic potential of genetically engineered cells for the biologic therapy of cancer.

CDNA‐array profiling of melanomas and paired melanocyte cultures

Three paired (from the same donor) sets of melanoma cells and normal melanocytes, established as early-passage cultures from metastatic lesions and the uninvolved skin of three patients, were comparatively cDNA profiled by macroarrays (approximately 1,200 genes) and reverse transcription (RT)-PCR. While 145 gene products were significantly (at least twofold) upregulated or downregulated in at least 1 pair, and 23 were in at least 2 pairs, only 3 (the signal transducer and activator of transcription STAT2, collagen type VI, and CD9) were concordantly modulated (downregulation) in all 3 pairs. Array results were validated by RT-PCR on a small panel of surgically removed nevocellular nevi and metastatic melanoma lesions, and by immunohistochemistry on a large panel of benign and malignant lesions of the nevomelanocytic lineage. The three gene products were downregulated at different stages of melanoma progression. STAT2 was detectable in nevi (5/5) and most primary melanomas (11/12), but was lost in 10/15 metastatic lesions. Collagen type VI was expressed in nevi (5/5) and primary melanomas below a Breslow thickness of 1 mm (3/3), but was lost in 24/24 primary melanomas above this threshold, and in metastatic melanomas (10/10). The tetraspanin CD9 molecule was expressed in 18/18 nevi, but was lost in 20/28 primary melanomas (including thin lesions), and in 24/52 metastatic lesions. These data provide the proof of principle that cDNA profiling of paired melanocyte/melanoma cultures sorts out novel, early signatures of melanocyte transformation that could contribute to the clinical management of patients at high risk of metastatic disease.

Survivin expression by metastatic melanoma predicts poor disease outcome in patients receiving adjuvant polyvalent vaccine

Survivin and livin are members of the inhibitor of apoptosis protein (IAP) family. We hypothesized that elevated expression levels of these 2 IAP genes in resected advanced-stage metastatic melanoma lesions would be associated with poor disease outcome in patients receiving a polyvalent therapeutic cancer vaccine (Canvaxintrade mark). A quantitative real-time RT-PCR (qRT) assay for survivin and livin genes was used to assess mRNA expression in 63 metastatic melanomas obtained during cytoreductive surgery of American Joint Committee on Cancer (AJCC) stage IV melanoma. Nineteen of 63 metastatic melanoma patients received Canvaxin pre- and postoperatively, and 37 patients received only postoperative Canvaxin. Expression of survivin and livin protein was assessed by immunohistochemistry (IHC) and then correlated with mRNA. Survivin mRNA was detected in 62 of 63 (98%) melanoma specimens ranging from 0-5.96 x 10(4) mRNA copies of total RNA. Lower mRNA copy levels of survivin significantly correlated with improved overall survival among the 37 patients who received Canvaxin postoperatively but not preoperatively (log-rank test, p = 0.023). Among patients with low survivin mRNA copies, those who received postoperative Canvaxin did significantly better than patients who received pre- and postoperative Canvaxin (p = 0.003). Livin mRNA was detectable in 60 of 63 (95%) metastatic melanoma specimens but had no significant prognostic utility. These studies demonstrate that lower levels of survivin in recurrent metastatic melanomas are associated with significantly improved survival in patients receiving postoperative adjuvant immunotherapy. Overall, the study indicates survivin expression in metastatic melanomas can significantly influence disease outcome and patient responses to immunotherapy.

Selective monomorphic and polymorphic HLA class I antigenic determinant loss in surgically removed melanoma lesions

The analysis of human leukocyte antigen (HLA) class I allospecificity expression in malignant lesions has been hampered by the limited availability of HLA class I allospecificity-specific monoclonal antibodies (mAbs) which stain tissues in immunohistochemical (IHC) reactions. During the 12th International Histocompatibility Workshop, the HLA and cancer component made available a panel of mAbs capable of detecting monomorphic, locus- and allo-specific HLA class I antigenic determinants in surgically removed frozen tissue sections by IHC staining. In the present study, we have utilized this panel of mAbs to analyze the expression of HLA class I allospecificities in 33 primary and in 11 metastatic lesions surgically removed from HLA-typed patients with malignant melanoma, as this information contributes to determine the extent of HLA class I antigen abnormalities in melanoma lesions. HLA class I antigens were downregulated in six (18.2%) of the primary lesions and in six (54.5%) of the metastatic lesions. Selective loss of HLA-A and HLA-B antigens was detected in two (6.1%) and in one (3.0%), respectively, of the primary lesions, but in none of the metastases. HLA-A and HLA-B antigens were downregulated in three (9.1%) and four (36.4%) of the primary and metastatic lesions, respectively. Selective loss of one or more HLA class I allospecificities was found in 10 (33.0%) and two (18.0%) of the 33 primary and 11 metastatic melanoma lesions analyzed, respectively. HLA class I antigen abnormalities were present in 16 (48.5%) of the 33 primary lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, two lesions demonstrating selective abnormal reactivity with HLA-B locus-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A and HLA-B locus-specific mAbs, and seven lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). Furthermore, HLA class I antigen abnormalities were present in nine (81.8%) of the 11 metastatic lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A locus-specific mAb, and two lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). It cannot be ruled out that the frequency of HLA class I allospecificity abnormalities is higher, as the expression of several HLA class I allospecificities could not be investigated because of the lack of appropriate probes. The frequency of HLA class I antigen defects in primary lesions was significantly correlated with primary lesion thickness, an important prognostic marker in melanoma, arguing for a potential clinical significance of HLA class I antigen abnormalities in melanoma. In conclusion, the results of the present study (i) demonstrate that the frequency of HLA class I allospecificity abnormalities in primary melanoma lesions is markedly higher than that of total HLA class I antigen downregulation described in the literature; (ii) corroborate our previous findings that staining of melanoma lesions with mAb to monomorphic determinants of HLA class I antigens does not detect selective HLA class I allospecificity loss; and (iii) demonstrate for the first time selective loss of antigenic determinants expressed on HLA class I molecules in melanoma lesions. The latter finding indicates that at least two mAbs recognizing distinct antigenic determinants on the HLA molecule being investigated should be used for IHC staining of tissue sections in order to prove that lack of immunostaining reflects actual loss of the corresponding HLA molecule and not selective loss of antigenic determinants.

Release of Biologically Functional Interferon-Alpha from a Nanochannel Delivery System

Metastatic melanoma lesions often are unresectable due to their size and/or location near critical structures. These lesions represent a significant challenge for the oncologist, because radiation therapy and chemotherapy are infrequently successful in halting tumor growth. Of primary concern is the fact that these lesions are usually painful and present a cosmetic dilemma. We hypothesized that the development of a silicon-based nano-device capable of delivering antitumor compounds (e.g. immune modulators), locally, at a constant rate, to the tumor microenvironment could avoid the toxicity of systemic administration and the inconvenience of frequent clinic visits for local injections. Because of its diminutive size, such a device could be implanted using a minimally invasive procedure in close proximity to unresectable melanoma lesions. The current report uses interferon alpha-2b (IFN-alpha) as a model antitumor agent, since it is commonly used in the treatment of malignant melanoma and metastatic renal cell carcinoma. In this system, IFN-alpha is delivered directly to the tumor microenvironment by a novel nanochannel delivery system (nDS) that is capable of zero order release of small molecules. We have demonstrated that the IFN-alpha released from the nDS is functionally active on both host immune cells and a human melanoma cell line in vitro. This drug delivery platform could be used to develop alternative strategies for the treatment of unresectable tumors.

Expression of galectin-3 in primary and metastatic melanoma: immunohistochemical studies on human lesions and nude mice xenograft tumors

Galectins are a large family of proteins which bind galactoside-containing glycans. Their role in cancer seems to be important since members of the family may mediate cell adhesion and modulate cell growth. Galectin-3 (Gal-3) is expressed in the nucleus, in the cytoplasm and on the cell surface, and can also be secreted into the extracellular matrix. A series of experimental and clinical data have been reported which indicate that Gal-3 may play a putative role in carcinogenesis, cancer progression and the process of metastasis. To study the possible correlation between Gal-3 expression and malignant potential in primary melanoma lesions, we conducted an immunohistochemical study with monoclonal anti-Gal-3 antibody in a series of primary and metastatic melanoma lesions as well as benign skin pigmented lesions. We also developed a xenograft melanoma model in nude mice with two melanoma cell lines (ATCC G-361 and ATCC HT-144) and assessed staining with the Gal-3 antibody in the xenografts and the metastases. The expression of anti-Gal-3 staining was determined semiquantitatively. The expression of Gal-3 was higher in thin primary melanoma lesions than in benign pigmented skin lesions or metastases and seemed to correlate inversely with the aggressiveness as estimated by the Breslow index which is recognized as the main prognostic factor in melanoma. We propose Gal-3 expression in melanoma as a diagnostic and/or a prognostic parameter and suggest that further studies of such a role for Gal-3 are warranted.

MAGE-A3 is a frequent tumor antigen of metastasized melanoma

MAGE-3 or MAGE-A3 is one of the best-characterized tumor antigens. Due to its tumor-restricted expression pattern and its recognition by both cytotoxic and helper T cells it constitutes a promising tumor antigen for anticancer immunotherapy, notably of malignant melanoma. Surprisingly, however, only very limited information is available on the frequency and consistency of its expression in metastatic melanoma lesions. We have now investigated the presence of MAGE-A3 mRNA in 316 tumor samples from 147 melanoma patients by RT-PCR. MAGE-A3 mRNA was detectable in 62% of metastases, and expression did not depend on the site of the metastases (skin, lymph node, and internal organs), age, sex, or duration of disease. Southern blot hybridization of the PCR product enhanced sensitivity of detection, and 26% more samples (13/50 samples tested) scored positive, indicating an even higher MAGE-A3 mRNA frequency than determined by simple ethidium bromide gel analysis. In 62 patients, we were able to investigate MAGE-A3 expression in several metastases from the same patient, and unexpectedly, both MAGE-A3-positive and MAGE-A3-negative metastases were found in 32% of these patients (20 of 62). Immunohistochemistry (using mAb 57B) demonstrated that the expression pattern was usually also heterogeneous with positively and negatively stained tumor cells within one metastasis. However, most (90%) of the metastases (47/52) gave a partially positive signal. Taken together, MAGE-A3 is a common and frequent tumor antigen in metastasized melanoma, but its expression is often heterogeneous.

T cells associated with tumor regression recognize frameshifted products of the CDKN2A tumor suppressor gene locus and a mutated HLA class I gene product.

The dramatic tumor regression observed following adoptive T cell transfer in some patients has led to attempts to identify novel Ags to understand the nature of these responses. Nearly complete regression of multiple metastatic melanoma lesions was observed in patient 1913 following adoptive transfer of autologous tumor-infiltrating lymphocytes. The autologous 1913 melanoma cell line expressed a mutated HLA-A11 class I gene product that was recognized by the bulk tumor-infiltrating lymphocytes as well as a dominant T cell clone derived from this line. A second dominant T cell clone, T1D1, did not recognize the mutated HLA-A11 product, but recognized an allogeneic melanoma cell line that shared expression of HLA-A11 with the parental tumor cell line. Screening of an autologous melanoma cDNA library with clone T1D1 T cells in a cell line expressing the mutated HLA-A11 gene product resulted in the isolation of a p14ARF transcript containing a 2-bp deletion in exon 2. The T cell epitope recognized by T1D1, which was encoded within the frameshifted region of the deleted p14ARF transcript, was also generated from frameshifted p14ARF or p16INK4a transcripts that were isolated from two additional melanoma cell lines. The results of monitoring studies indicated that T cell clones reactive with the mutated HLA-A11 gene product and the mutated p14ARF product were highly represented in the peripheral blood of patient 1913 1 wk following adoptive transfer, indicating that they may have played a role in the nearly complete tumor regression that was observed following this treatment.

The melanoma inhibitor of apoptosis protein: a target for spontaneous cytotoxic T cell responses.

The identification of tumor antigens which expression is essential for the survival of tumor cells is a new avenue to prevent antigen loss variants emerging due to immunoselection, particularly during immune therapy. The melanoma inhibitor of apoptosis protein, ML-IAP (also named livin) counteracts apoptosis induced by death receptors, hypooxgenic conditions, or chemotherapeutic agents. Thus, elevated expression of ML-IAP renders melanoma cells resistant to apoptotic stimuli and thereby potentially contributes to the oncogenic phenotype. Here, we demonstrate that T cells in a large proportion of melanoma patients infiltrating the tumor or circulating in the peripheral blood specifically recognize ML-IAP-derived peptides. Interestingly, the responses against the peptide epitope ML-IAP280-289 were not restricted to melanoma patients but present among peripheral blood T cells in a few healthy controls. In situ peptide/HLA-A2 multimer staining, however, confirmed the infiltration of ML-IAP-reactive cells into the tumor microenvironment. Moreover, ML-IAP-reactive T cells isolated by magnetic beads coated with peptide/HLA-A2 complexes were cytotoxic against HLA-matched melanoma cells. In conclusion, out data strongly indicate ML-IAP as a suitable target for immunologic intervention.

Differential downregulation of endoplasmic reticulum-residing chaperones calnexin and calreticulin in human metastatic melanoma

Characterization of the molecular basis of tumor recognition by T cells has shown that major histocompatibility complex (MHC) class I molecules play a crucial role in presenting antigenic peptide epitopes to cytotoxic T lymphocytes. MHC class Ia downregulation has been repeatedly described on melanoma cells and is thought to be involved in the failure of the immune system to control tumor progression. Proper assembly of MHC class I molecules is dependent on several cofactors, e.g. the chaperones calnexin and calreticulin residing in the endoplasmic reticulum. Alterations in the expression of these chaperones may have important implications for MHC class I assembly, peptide loading, and presentation on the tumor cell surface and thus may contribute to the immune escape phenotype of tumor cells. In the present study, we compared melanoma lesions representing different stages of tumor progression with regard to the expression of calnexin and calreticulin in tumor cells by means of immunohistochemistry. Metastatic melanoma lesions exhibited significant downregulation of calnexin as compared to primary melanoma lesions. In contrast, chaperone calreticulin was expressed in melanoma cells of primary as well as of metastatic lesions. Our data suggest that chaperone-downregulation, particularly calnexin-downregulation, may contribute to the metastatic phenotype of melanoma cells in vivo. Consistently, conserved chaperone expression in metastatic melanoma lesions may be a useful criterion for selection of patients for treatment with T cell-based immunotherapies.

Heparan sulphate epitope-expression is associated with the inflammatory response in metastatic malignant melanoma.

Heparan sulphate (HS) represents a heterogeneous class of molecules on cell membranes and extracellular matrices. These molecules are involved in a variety of biological processes, including immune responses, through their binding and functional modulation of proteins. Recently a panel of HS-epitope-specific, human single chain antibodies have been generated by phage display, facilitating analysis of the structural heterogeneity of HS in relation to pathological conditions. In a pilot study a heterogeneous staining pattern in melanoma metastases was observed with one of the clones (EW4G1). Using a double-staining technique, the expression of this epitope was studied in 12 metastatic melanoma lesions in relation to the presence of a CD3(+) cell infiltrate. Different staining patterns with EW4G1 were observed in the different lesions. The different staining patterns were associated with the presence and pattern of inflammation with CD3(+) cells. A pronounced staining pattern of blood vessels with EW4G1 was associated with a more or less brisk presence of CD3(+) cells, while a pronounced staining of tumour cells or tumour cell matrix or absence of staining with EW4G1 was associated with absence of CD3(+) cells. These results suggest a dualistic role for HS in the recruitment and intratumoural migration of CD3(+) cells, depending on the location of expression of its epitope recognized by EW4G1. Further characterization of the structural diversity of HS and its function in T-cell recruitment and migration is therefore warranted, since detailed understanding of this relation may provide new targets for therapeutic intervention, such that better homing and migration of T cells (in)to tumours might be achieved in immunologically based treatment strategies.

DOTA alpha-melanocyte-stimulating hormone analogues for imaging metastatic melanoma lesions.

Scintigraphic imaging of metastatic melanoma lesions requires highly tumor-specific radiopharmaceuticals. Because both melanotic and amelanotic melanomas overexpress melanocortin-1 receptors (MC1R), radiolabeled analogues of alpha-melanocyte-stimulating hormone (alpha-MSH) are potential candidates for melanoma diagnosis. Here, we report the in vivo performance of a newly designed octapeptide analogue, [betaAla(3), Nle(4), Asp(5), D-Phe(7), Lys(10)]-alpha-MSH(3-10) (MSH(OCT)), which was conjugated through its N-terminal amino group to the metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) to enable incorporation of radiometals (e.g., indium-111) into the peptide. DOTA-MSH(OCT) displayed high in vitro MC1R affinity (IC(50) 9.21 nM). In vivo [(111)In]DOTA-MSH(OCT) exhibited a favorable biodistribution profile after injection in B16-F1 tumorbearing mice. The radiopeptide was rapidly cleared from blood through the kidneys and, most importantly, accumulated preferentially in the melanoma lesions. Lung and liver melanoma metastases could be clearly imaged on tissue section autoradiographs 4 h after injection of [(111)In]DOTA-MSH(OCT). A comparative study of [(111)In]DOTA-MSH(OCT) with [(111)In]DOTA-[Nle(4), D-Phe(7)]-alpha-MSH ([(111)In]-DOTA-NDP-MSH) demonstrated the superiority of the DOTA-MSH(OCT) peptide, particularly for the amount of radioactivity taken up by nonmalignant organs, including bone, the most radiosensitive tissue. These results demonstrate that [(111)In]DOTA-MSH(OCT) is a promising melanoma imaging agent.

The role of fine needle aspiration biopsy in the diagnosis of melanoma

The role of fine needle aspiration biopsy (FNAB) is discussed in the follow up of patients with the diagnosis of malignant melanoma. The review is based on literary data and the author's own material. The primary role of FNAB is to confirm metastatic or recurrent melanoma lesions. US or CT guided FNAB is valuable in the diagnosis of visceral metastases. FNAB has limited role in the diagnosis of primary melanomas except in cases with unusual clinical presentation (e.g. oral mucosa). In spite of the well-known cytology the diagnosis can be difficult due to the inherent histological variation of malignant melanomas, especially in cases with unusual localisation and amelanotic tumor presentation when immunocytochemistry is needed. The known clinical history of melanoma is very helpful.

Downregulation of tapasin expression in progressive human malignant melanoma.

Tumor cells with alterations in the MHC class I peptide-loading complex are unable to load peptide antigens onto MHC class I molecules. These alterations result in destabilization of MHC class I expression on the tumor cell surface and thus play a critical role in escape from immunological recognition by the acquired cellular immune system. By forming physical links between class I heavy chains and TAP molecules, a component of the class I peptide-loading complex, tapasin, plays an important role in the assembly of MHC class I molecules with peptides in the endoplasmic reticulum. In the present study, we compared 104 human melanoma lesions representing different stages of tumor progression for their expression of tapasin in tumor cells by immunohistochemistry. Tapasin downregulation was significantly associated with tumor progression. Whereas 100% of melanomata in situ and 96.2% of primary melanomas with a Breslow index of <or=0.75 mm showed strong expression of tapasin, significant downregulation of tapasin was observed in 25% of primary melanomas with a Breslow index >0.75 mm as well as in 21.1% metastatic melanoma lesions. The downregulation of tapasin in advanced stages of human melanoma may reflect the accumulation of alterations in the antigen-presenting/processing machinery associated with neoplastic progression. These alterations may lead to failure of the acquired cellular immune system to control progression and metastatic spread of melanoma cells in vivo, and thus contribute to the immune escape phenotype of human melanoma cells.

Widespread expression of parathyroid hormone-related peptide in melanocytic cells

Parathyroid hormone-related peptide (PTH-rP) was associated with the syndrome of hypercalcaemia of malignancy. An increased serum level of PTH-rP could occur in patients with advanced melanoma. We examined PTH-rP expression in cultured melanocytic cell lines and in lesions of melanocytic origin for associations with clinicopathological variables of disease progression. We measured the supernatant and cell lysate level of PTH-rP in cultured melanoma cells to clarify whether melanoma cells secrete PTH-rP. PTH-rP expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) in cultured melanocytic cell lines and by immunoperoxidase staining in 18 melanocytic naevi, 40 primary melanoma and 19 metastatic melanoma lesions. The supernatant level of PTH-rP was measured with an immunoradiometric assay. RT-PCR products of PTH-rP mRNA were detected in six of eight melanoma cell lines; however, neither naevus cells nor melanocytes showed positive products. On the other hand, immunohistochemical analysis showed that PTH-rP was widely expressed both in benign and malignant melanocytic lesions. In addition, PTH-rP expression was not associated with any clinicopathological variables. Cell lysate but not the supernatant of melanoma cells showed high PTH-rP levels. These results suggest that PTH-rP was widely expressed in melanocytic cells; however, the cells did not secrete PTH-rP.