Toshiro Kageshita

Down-Regulation of HLA Class I Antigen-Processing Molecules in Malignant Melanoma: Association with Disease Progression

Expression of the proteasome subunits LMP2 and LMP7, the MHC-encoded transporter subunits TAP1 and TAP2, and HLA Class I antigens was examined by immunoperoxidase staining in 10 nevi and 98 melanoma lesions (60 primary and 38 metastatic), because these molecules play an important role in the presentation of melanoma-associated peptide antigens to cytotoxic T cells. LMP2 was less frequently expressed than LMP7 in primary and metastatic melanoma lesions. TAP1, TAP2, and HLA Class I antigen expression was more frequently (P < 0.05) down-regulated in metastatic than in primary melanoma lesions and in nevi. A synchronous TAP1, TAP2, and HLA Class I antigen down-regulation was observed in 58% of primary and 52% of metastatic lesions. TAP and HLA Class I antigen down-regulation in primary lesions was significantly associated with lesion thickness, stage of disease, reduced time to disease progression, and reduced survival. These results suggest that TAP down-regulation plays a role in the clinical course of malignant melanoma, probably by providing melanoma cells with a mechanism to escape from cytotoxic T lymphocyte recognition during disease progression.

Loss of β‐catenin expression associated with disease progression in malignant melanoma

Background β‐catenin plays a crucial role in the function of cell adhesion molecules and also participates in growth regulatory signalling pathways that may be involved in malignant transformation.

Distribution and Significance of Occult Intraepidermal Tumor Cells Surrounding Primary Melanoma

Primary melanoma can recur at the excision site if not excised with a safety margin of surrounding uninvolved skin. To characterize the nature of residual melanoma in the skin surrounding primary tumors targeted by safety margins, we used array comparative genomic hybridization and fluorescent in situ hybridization to detect and spatially map aberrations in the skin adjacent to acral melanomas. Melanocytic cells with genetic amplifications in histopathologically normal skin (field cells) were detected exclusively in the epidermis in 84% of 19 cases, with a mean extension of 6.1 mm (in situ melanomas) and 4.5 mm (invasive melanomas) beyond the histopathological margin. Genetic profiling of these field cells indicated that they represent an early phase of disease preceding melanoma in situ. The extent of field cells did not correlate with tumor depth or diameter, indicating that tumor depth is not suited to predict the extent of field cells. These results demonstrate that, on acral sites, melanoma field cells extend significantly into seemingly normal skin. These field cells provide a plausible explanation for the tendency of certain melanoma types to recur locally despite apparently having undergone complete excision.

Highly sensitive detection of melanoma at an early stage based on the increased serum secreted protein acidic and rich in cysteine and glypican-3 levels

Purpose: There are no available tumor markers detecting primary melanoma at an early stage. The identification of such serum markers would be of significant benefit for an early diagnosis of melanoma. We recently identified glypican-3 (GPC3) as a novel tumor marker but could diagnose only 40% of melanomas. Thereby, we focused out attention on secreted protein acidic and rich in cysteine (SPARC) overexpressed in melanoma as another candidate for tumor marker. Experimental Design: Secreted SPARC protein was quantified using ELISA in the sera from 109 melanoma patients, five patients with large congenital melanocytic nevus, 61 age-matched healthy donors, and 13 disease-free patients after undergoing a surgical removal. We also quantified GPC3 and 5-S-cysteinyldopa in the same serum samples and compared these markers for their diagnostic value. Results: The serum SPARC concentrations in melanoma patients were greater than those in healthy donors (P = 0.001). When we fixed a cutoff value at the mean concentration plus 2 SD of the healthy donors, the serum SPARC was found to have increased in the sera of 36 of the 109 (33%) melanoma patients, whereas there were three (4.9%) false-positive cases of 61 healthy donors. Surprisingly, 19 of 36 patients showing increased SPARC levels were in stages 0 to II. The serum SPARC level decreased under the cutoff level in 10 of 13 patients after surgical removal. Using SPARC and GPC3 in combination thus enabled us to diagnose 47 of 75 (66.2%) melanoma patients at an early stage (0-II). Conclusions: SPARC or its combination with GPC3 is thus considered a potentially useful tumor marker, especially for melanoma at an early stage.

Differential expression of MART-1 in primary and metastatic melanoma lesions

Summary Twenty-eight primary and 29 metastatic melanoma lesions and 18 pigmented nevi lesions were analyzed by using the immunoperoxidase reaction with anti-MART-1 and anti-gp100 monoclonal antibodies (mAbs). The MART-1 was expressed in 28, 29, and 18, and gp100 was expressed in 27, 28, and eight of these lesions, respectively. Intensity and percentage of stained cells with anti-MART-1 mAb were stronger and higher than those with anti-gp100 mAb. MART-1 was expressed homogeneously in primary melanoma and pigmented nevi, whereas it was heterogeneously expressed in metastatic melanoma lesions. The level of expression of MART-1 in primary melanoma lesions did not correlate with any clinicopathologic parameters. These results suggest that anti-MART-1 mAb is a useful tool for immunohistochemical analysis of melanocytic lesions and also is useful for patients selection and monitoring of antigen-loss variants in clinical trials with the MART-1-based immunotherapy.

Serum levels of sICAM-1 and 5-S-cysteinyldopa as markers of melanoma progression.

The serum levels of the soluble form of intercellular adhesion molecule-1 (slCAM-1) and 5-S-cysteinyldopa (5-S-CD) were determined by double determinant immunoassay and high-performance liquid chromatography, respectively. Fifty-three melanoma patients (stage 1, 12 patients; stage II, 11; stage III, 19; and stage IV, 11; total number of samples, 116) and 31 age- and sex-matched healthy control subjects were analysed. Both the slCAM-1 and 5-S-CD levels were significantly higher in stage IV patients than those in stage I, II and III patients (slCAM-1; all P<0.001; 5-S-CD; all P<0.05). The serum levels of 5-S-CD were elevated only in stage IV patients. slCAM-1 levels elevated gradually with disease progression. Testing of sequential bleedings showed that both slCAM-1 and 5-S-CD levels were elevated in most of the patients whose disease had progressed. However 5- S-CD levels were not elevated in those patients whose metastases were amelanotic. There was a statistically significant correlation between slCAM-1 and 5-S-CD levels (R=0.6 55, P< 0.001). Serum levels of 5-S-CD may be a useful parameter for monitoring the clinical course of the disease in patients whose metastases are melanotic. Analysis of both slCAM-1 and 5-S-CD levels in serum will contribute greatly to monitoring the clinical course of melanoma patients.

Management of sentinel lymph nodes in malignant skin tumors using dynamic lymphoscintigraphy and the single-photon-emission computed tomography/computed tomography combined system

BackgroundThe differentiation of true sentinel lymph nodes from nonsentinel lymph nodes is difficult in cases of multiple radiolabeled or dyed lymph nodes.MethodsWe examined the locations of sentinel lymph nodes in melanoma and other malignant skin tumors by using dynamic lymphoscintigraphy and the single-photon-emission computed tomography/computed tomography (SPECT/CT) combined system.ResultsSentinel lymph nodes were detected in 45 of the 53 patients examined using only the ordinary blue dye method (85%), and were detected in all 35 patients examined using the SPECT/CT method (100%). Twenty of the 35 patients mentioned above had one sentinel lymph node. Multiple sentinel lymph nodes were demonstrated in the head and neck areas using the SPECT/CT method. Significant differences (P = 0.0015) in the numbers of sentinel lymph nodes were found between the blue dye method only and the SPECT/CT method in the neck area. Popliteal sentinel lymph nodes were recognized in three patients, and cubital sentinel lymph nodes were recognized in two patients. Two patients had plural regional lymph nodes: one had popliteal and groin sentinel lymph nodes, while the other had cubital and axillary sentinel lymph nodes. The probe counts of the popliteus and cubitus were significantly lower (P = 0.0241) than the counts in the groin, axilla, and neck areas. Micrometastatic sentinel lymph nodes were recognized in four patients, and two patients had metastases in both sentinel and nonsentinel lymph nodes.ConclusionsDynamic lymphoscintigraphy was useful when we were concerned about cubital and popliteal lymph nodes. The SPECT/CT combined system was useful in recognizing the anatomical location of sentinel lymph nodes before biopsy. The detection rate of sentinel lymph nodes using the SPECT/CT method was always better than that with the blue dye method (P = 0.0197).

Biochemical and immunohistochemical analysis of cathepsins B, H, L and D in human melanocytic tumours.

We carried out biochemical and immunohistochemical analyses of cathepsins B, H, L and D in human melanocytic tumours using monospecific antibodies against rat cathepsins. In Western blot analysis, anti-rat cathepsin antibodies reacted with the cathepsins from normal human tissues and human malignant melanoma. However, the molecular profiles of the cathepsins from human melanoma were slightly different from those of the rat cathepsins, suggesting a distinct intracellular processing mechanism for cathepsins in human melanoma. Although cathepsins B, H, L and D were expressed in primary and metastatic melanomas and pigmented naevi immunohistochemically, the intensity of staining in metastatic melanomas was stronger than in primary melanomas and pigmented naevi. These findings suggest that anti-rat cathepsin antibodies may be useful in biochemical and/or immunohistochemical analysis of human melanocytic tumours.

Functional Characterization of an scFv-Fc Antibody that Immunotherapeutically Targets the Common Cancer Cell Surface Proteoglycan CSPG4

Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4(+) melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited in vitro growth and migration of tumor cells and in vivo growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal-regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4.

Differential clinical significance of αvΒ3 expression in primary lesions of acral lentiginous melanoma and of other melanoma histotypes

Despite its potential clinical relevance, αvβ3 expression has been analyzed only in a limited number of melanoma lesions, mostly nodular melanoma (NM) and superficial spreading melanoma (SSM). Therefore, in the present study, we have correlated αvβ3 expression in 33 acral lentiginous melanomas (ALMs), 6 lentigo maligna melanomas, 7 mucosal melanomas, 12 NMs and 9 SSMs with their antigenic profile, with their histo‐pathological characteristics and with the clinical course of the disease. Furthermore, we have compared αvβ3 expression in ALM lesions with that in NM and SSM lesions since this information helps to clarify the relationship of the latter 2 histotypes with ALM. Such a relationship is uncertain since ALM has a clinical course similar to that of NM and SSM despite different antigenic profiles and biological characteristics. The level of αvβ3 expression in primary lesions was not correlated with that of high‐m.w. melanoma‐associated antigen and intercellular adhesion molecule‐1, with lesion thickness and with disease recurrence in ALM but was significantly correlated with these 4 parameters in the other melanoma histotypes analyzed. Therefore, αvβ3 expression appears to have a differential clinical significance in ALM and in the other histotypes of melanoma we have analyzed since it appears to play a significant role in the progression of the disease only in non‐ALM histotypes. Int. J. Cancer (Pred. Oncol.) 89:153–159, 2000.