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.

Amelanotic malignant melanoma where primary lesion was discovered 5 years after metastasis in the lymph node.

A 53-year-old Japanese man presented with a subcutaneous nodule in his left inguinal region in 2002. He was diagnosed as having a malignant tumor of the soft tissues at a local hospital; however, an exact diagnosis was not obtained. CYVADIC (cyclophosphamide, vincristine, doxorubicin, and dacarbazine) therapy was done for adjuvant therapy. In 2004, he noticed a nodule on the left heel. Positron emission computed tomography showed abnormal up-take and he consulted our department. He was diagnosed as having amelanotic malignant melanoma and the lesion was resected. There were no metastases in the groin or popliteal lymph nodes. After the evaluation, the tumor was staged at pT3b N1b M0 stage IIIC (Breslow's tumor thickness was 7 mm). In our hospital, we have experienced 16 cases of amelanotic malignant melanoma. Generally, it is reported that the patients with amelanotic malignant melanoma have a poor prognosis, but we have observed no difference in the outcome between the patients with amelanotic malignant melanoma and those with malignant melanoma.

The expression of human high molecular weight melanoma-associated antigen in acral lentiginous melanoma.

The high molecular weight melanoma-associated antigen (HMW-MAA) is a membrane-bound chondroitin sulphate proteoglycan that is highly expressed on the surface of melanoma cells. It represents an attractive target for immunotherapy of malignant melanoma. Previously, it was reported that HMW-MAA was detected in about 20-30% of primary acral lentiginous melanoma (ALM) lesions by immunohistochemical staining (IHC) of frozen sections with monoclonal antibodies (mAbs). In the present study, we examined the expression of HMW-MAA in 95 paraffin-embedded, primary ALM lesions and 13 primary superficial spreading melanoma (SSM) lesions. A total of 51 primary ALM lesions (53.6%) were positive for HMW-MAA. Almost all of these positive cases showed a weak staining intensity. On the other hand, all 13 primary SSM lesions were strongly positive for HMW-MAA expression. Our data showed that the staining intensity of HMW-MAA ALM lesions was weaker than that of SSM. Furthermore, the percentage of HMW-MAA positive staining in ALM lesions was higher than previously reported.

12-O-tetradecanoylphorbol-13-acetate inhibits melanoma growth by inactivation of STAT3 through protein kinase C-activated tyrosine phosphatase(s).

The growth of most melanoma cells in vitro is inhibited by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study, the involvement of the signal transducer and activator of transcription 3 (STAT3) in the TPA-induced growth inhibition of melanoma cells was examined. The in vitro growth and DNA synthesis of five melanoma cell lines, whose STAT3 was activated (phosphorylated), was inhibited by TPA, whereas that of WM35 and WM39 cells, whose STAT3 activity was at negligible levels, was considerably slow and not affected by TPA. Blockade of STAT3 activity by small interfering RNAs suppressed the growth of WM1205Lu cells containing constitutively activated STAT3. Treatment of WM1205Lu cells with TPA decreased both the phosphorylated STAT3 and the DNA-binding activity of STAT3. Pretreatment of WM1205Lu cells with either a protein-tyrosine phosphatase inhibitor or a protein kinase C (PKC) inhibitor prevented the inhibitory effects of TPA on the level of phosphorylated STAT3. The five melanoma cell lines containing phosphorylated STAT3 commonly expressed PKCalpha, PKCdelta, and PKCepsilon. Introduction of the dominant negative mutant of one of these PKC isoforms into WM1205Lu cells inhibited the TPA-induced dephosphorylation of STAT3. A Src inhibitor attenuated the STAT3 phosphorylation in WM1205Lu cells. These results indicate that constitutively activated STAT3 is positively regulated by c-Src and negatively regulated by a PKC-activated tyrosine phosphatase(s) in melanoma cells. Because TPA did not affect c-Src activity, we conclude that the growth inhibitory effect of TPA on melanoma cells is mediated through inactivation of STAT3 by a PKC-activated tyrosine phosphatase(s).

The prevalence of melanocytic nevi on the soles in the Japanese population.

BACKGROUND: Acral lentiginous melanoma (ALM) is the most common type of melanoma in Japan. The association between ALM and acral nevus has not been elucidated. OBJECTIVE: To investigate the prevalence and dermatoscopic patterns of plantar melanocytic nevi on the soles in the Japanese and to evaluate the relationship between acral nevi and ALM. METHODS: All outpatients (N = 1697) and melanoma patients (N = 104) were included. We examined the number, size, and dermatoscopic images of nevi. RESULTS: In the control group, the prevalence of plantar nevi was 10.9%, and the mean size was 3.8 +/- 2.4 mm. The prevalence of nevi in patients with ALM and melanoma in situ on the soles was 8.6% and that of patients with melanoma on other sites was 14.5%. The main dermatoscopic pattern was "parallel furrow" in both groups. LIMITATIONS: This was a clinical observational study only. CONCLUSION: The number, size, and dermatoscopic patterns of nevi on the soles of patients with ALM and melanoma in situ on the soles did not differ from those of the control group.

Multiple antigen-targeted immunotherapy with alpha-galactosylceramide-loaded and genetically engineered dendritic cells derived from embryonic stem cells.

Numerous tumor-associated antigens (TAA) have been identified and their use in immunotherapy is considered to be promising. For TAA-based immunotherapy to be broadly applied as standard anticancer medicine, methods for active immunization should be improved. In the present study, we demonstrated the efficacy of multiple TAA-targeted dendritic cell (DC) vaccines and also the additive effects of loading alpha-galactosylceramide to DC using mouse melanoma models. On the basis of previously established methods to generate DC from mouse embryonic stem cells (ES-DC), 4 kinds of genetically modified ES-DC, which expressed the melanoma-associated antigens, glypican-3, secreted protein acidic and rich in cysteine, tyrosinase-related protein-2, or gp100 were generated. Anticancer effects elicited by immunization with the ES-DC were assessed in preventive and also therapeutic settings in the models of peritoneal dissemination and spontaneous metastasis to lymph node and lung. The in vivo transfer of a mixture of 3 kinds of TAA-expressing ES-DC protected the recipient mice from melanoma cells more effectively than the transfer of ES-DC expressing single TAA, thus demonstrating the advantage of multiple as compared with single TAA-targeted immunotherapy. Loading ES-DC with alpha-galactosylceramide further enhanced the anticancer effects, suggesting that excellent synergic effects of TAA-specific cytotoxic T lymphocytes and natural killer T cells against metastatic melanoma can be achieved by using genetically modified ES-DC. With the aid of advancing technologies related to pluripotent stem cells, induced pluripotent stem cells, and ES cells, clinical application of DC highly potent in eliciting anticancer immunity will be realized in the near future.

A pilot study of human interferon beta gene therapy for patients with advanced melanoma by in vivo transduction using cationic liposomes.

BACKGROUND: Cationic liposomes containing the human interferon beta (HuIFNbeta) gene (IAB-1) was used for the clinical trial for glioma patients. HuIFNbeta gene therapy showed much higher anti-tumor activity compared with the administration of HuIFNbeta protein for melanoma. These results suggest that HuIFNbeta gene therapy is an attractive strategy for the treatment of melanoma. METHODS: Stage IV or III melanoma patients with cutaneous or subcutaneous metastatic lesions were enrolled in this pilot study. IAB-1 was dissolved by sterile PBS at a concentration of 30 microg DNA/ml and was injected into cutaneous or subcutaneous metastatic nodules three times a week for 2 weeks and the effect on the injected and non-injected metastatic lesions was evaluated. RESULTS: Clinical responses were as follows (five patients): mixed response (MR) and no change in each one patient, and progressive disease in three patients. In the MR patient, the IAB-1 injected lesion disappeared clinically and histopathologically and one-half of IAB-1 non-injected skin metastases were transiently inflamed and mostly regressed. In the responded non-injected lesions of this patient, histopathologically, infiltration of CD4 positive T cells was observed around the melanoma cells in the dermis, which expressed the HLA-Class II antigen. Adverse events due to this gene therapy were not recognized in any of the patients. CONCLUSIONS: The efficacy of this gene therapy was generally insufficient; however, some immunological responses were recognized in one patient. No adverse events were observed. HuIFNbeta gene therapy could be an attractive strategy for treatment of a variety of malignancies, including melanoma, though some modifications should be required.

Differential expression of heat shock protein 105 in melanoma and melanocytic naevi.

The objective of this study is to assess the expression of heat shock protein 105 (HSP105) in melanoma and benign melanocytic lesions. The expression of HSP105 in 62 human melanoma samples--46 primary and 16 metastatic lesions--and 42 melanocytic naevi samples, was assessed by immunohistochemistry. Western blotting was performed on melanoma cell lines, melanoma tissues with matched normal skin and melanocytic naevi. The Mann-Whitney test was used for statistical analysis and significance was considered to be P less than 0.05. Seventy-four per cent of the primary melanoma lesions and 88% of the metastatic lesions overexpressed HSP105 by immunohistochemistry. The majority of melanocytic lesions (95%) were negative (P<0.05). Western blotting detected high expression of HSP105 in melanoma cell lines and tissues. The expression of HSP105 was related to the invasiveness of the lesions. Melanocytic naevi expressed HSP105 at a level that was similar to that of normal skin. Our results show that high expression of HSP105 is associated with malignant melanoma especially advanced and metastatic lesions. The results suggest that HSP105 analysis may be a helpful tool as a poor prognostic indicator and as a diagnostic aid in problematic lesions; in addition, melanoma can be included in the growing list of tumours overexpressing HSP105 to be targeted for potential HSP105-based therapeutic strategies.

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.

Interferon-beta therapy for malignant melanoma: the dose is crucial for inhibition of proliferation and induction of apoptosis of melanoma cells.

We investigated the anti-tumor effect of human interferon-beta (HuIFN-beta) against malignant melanoma. In vitro study revealed that HuIFN-beta not only inhibited proliferation of melanoma cells (seven cell lines: MM-AN, MM-BP, MM-LH, MM-RU, PM-WK, RPM-EP, RPM-MC) but also induced apoptosis in a dose dependent fashion, though the sensitivity to HuIFN-beta was different among cell lines. In addition, we administered HuIFN-beta into cutaneous metastatic lesions of melanoma and evaluated clinical and histopathological effects. Although the size of the metastatic cutaneous lesion did not change by the intralesional injection of HuIFN-beta, histopathological examination revealed apoptotic changes of melanoma cells along with dense lymphohistiocytic infiltration. The present study confirmed direct and indirect inhibitory effects of HuIFN-beta on human melanoma cells and suggests that local higher concentration of HuIFN-beta is needed to eradicate melanoma lesions.

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.

Identification of a novel cancer-testis antigen CRT2 frequently expressed in various cancers using representational differential analysis.

PURPOSE: Cancer-testis antigens are promising targets for cancer immunotherapy. Identification of additional cancer-testis antigens with frequent expression in various cancers was attempted using representational differential analysis (RDA) and immunogenicity evaluation. EXPERIMENTAL DESIGN: cDNAs preferentially expressed in testis were enriched using RDA by subtraction between testis and normal tissues. Thirty clones showing cancer-testis-like expression based on EST database analysis were evaluated by reverse transcription-PCR. A potential antigen, CRT2, was identified and its expression was analyzed with a newly generated anti-CRT2 antibody. The immunogenicity of CRT2 was examined based on reactivity with serum immunoglobulin G (IgG) from cancer patients, using Western blot and ELISA analysis, and on in vitro induction of tumor-reactive CTLs from HLA-A24 transgenic mice and human peripheral blood lymphocytes. RESULTS: CRT2 was expressed in elongated spermatids of testis among normal tissues and in various cancer cell lines and tissues. The recombinant CRT2 protein was recognized by serum IgG from patients with various cancers in Western blot and ELISA analyses. A CRT2-derived peptide was identified as an HLA-A24-restricted T-cell epitope that induced tumor-reactive CTLs. CONCLUSION: CRT2 was identified as a new cancer-testis antigen expressed in elongated spermatids of testis and in cancer tissues (particularly melanoma) that is recognized by serum IgG from cancer patients. An HLA-A24-restricted T-cell epitope capable of inducing tumor-reactive CTLs was identified, suggesting that CRT2 may be useful for cancer diagnosis and immunotherapy.

Giant malignant peripheral nerve sheath tumor of the scalp.

Herein, we describe a rare case of giant malignant peripheral nerve sheath tumor of the head in a 38-year-old Japanese man. The tumor measured 210 mm at its largest diameter and was ulcerated, hemorrhagic, multilocular and non-mobile. It should be noted that the patient stubbornly refused to see a doctor for a long time, resulting in the extreme growth of the tumor. We suspect a psychological basis for this behavior. Dermatohistopathological findings of the biopsy indicated ancient schwannoma and total excision was therefore performed. However, after 4 months, the patient developed multiple metastases and died. Post-mortem skin biopsy revealed features of malignant peripheral nerve sheath tumor. We performed immunohistochemical studies on the primary and recurrent lesions and concluded that there was a difference in the expression of Ki67 and p16. We propose that the expressions of Ki67 and p16 should be checked for all lesions of peripheral nerve sheath tumor for distinguishing benign from malignant forms.

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

BACKGROUND: The differentiation of true sentinel lymph nodes from nonsentinel lymph nodes is difficult in cases of multiple radiolabeled or dyed lymph nodes. METHODS: We 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. RESULTS: Sentinel 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. CONCLUSIONS: Dynamic 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).

Acute myocardial infarction induced by cisplatin-based combination chemotherapy for malignant melanoma: a case report.

A 61-year-old woman with stage IV malignant melanoma suffered acute myocardial infarction during the third course of chemotherapy with cisplatin, dacarbazine, nimustine hydrochloride and tamoxifen citrate, despite no serious problem occurring during the first and second courses of chemotherapy. Since this patient, excluding menopause, had no significant risk factor for coronary heart disease before the start of chemotherapy, the infarction was likely to be attributable to the chemotherapy regimen. Chemotherapy should be used cautiously in patients with coronary risk factors before treatment is begun.

Determination of eumelanin in human urine.

Normal and malignant melanocytes produce melanins and melanin-related metabolites, most of which are retained in the cells but some are secreted into the blood and then excreted in the urine. In this study, we developed a method to measure levels of eumelanin in urine samples and evaluated its clinical significance in comparison with the melanin-related metabolites 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) and 5-S-cysteinyldopa (5-S-CD), and with pheomelanin, measured after degradation as 4-amino-3-hydroxyphenylalanine (4-AHP). The method is based on the production of pyrrole-2,3,5-tricarboxylic acid (PTCA) on permanganate oxidation of eumelanin, followed by quantification by liquid chromatography. For 118 urine samples from 10 control subjects, mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD and 4-AHP were 19, 67, 37 and 59 micromol/mol creatinine respectively. In melanoma patients (n = 45), the mean urinary excretions of PTCA, 6H5MI2C, 5-S-CD, and 4-AHP were 91, 926, 4070 and 3530 micromol/mol creatinine respectively. Median level of PTCA in melanoma patients was elevated 2.1-fold compared with control subjects. The degrees of elevation for 6H5MI2C, 5-S-CD, and 4-AHP were 1.8-, 22- and 6.2-fold respectively. Thus, although urinary PTCA is of little clinical value in following the progression of melanoma, urinary 4-AHP appears to be of considerable value in this respect.

Increased expression of germinal center-associated nuclear protein (GANP) is associated with malignant transformation of melanocytes.

BACKGROUND: Germinal center-associated nuclear protein (GANP) is a newly cloned molecule that is up-regulated in the germinal center B cells. Although GANP functions in the regulation of DNA repair during replication and survival of B cells, little is known about its expression in melanocytic cells. OBJECTIVES: To investigate whether GANP and phosphorylated-GANP (P-GANP) are expressed in cultured human melanocytes and melanoma cells and in benign and malignant melanocytic lesions. In addition, we aim to determine whether GANP and P-GANP are associated with malignant transformation of melanocytic lineage. METHODS: GANP and P-GANP expression in cultured melanocytic cells was analyzed by immunostaining and in vitro kinase assay. GANP and P-GANP expression in melanocytic lesions was analyzed by immunohistochemistry. RESULTS: GANP and P-GANP were up-regulated in cultured melanoma cells compared to melanocytes. GANP and P-GANP were restricted to nucleus of melanocytes but co-expressed in cytoplasm of melanoma cells. On the other hand, GANP and P-GANP were widely expressed at various levels in melanocytic nevi and melanoma lesions with nuclear and cytoplasmic staining pattern. Melanoma cells showed a stronger intensity of GANP and P-GANP than melanocytic nevus cells, however the staining intensity in primary melanoma lesions was not associated with any clinicopathological variables. Cytoplasmic GANP and P-GANP expression was associated with MCM3 and Ki67 expression. CONCLUSIONS: These data suggest, for the first time, that GANP and P-GANP are up-regulated in cultured melanoma cells compared to melanocytes and also they are widely expressed in benign and malignant melanocytic tumor cells.