Heat shock protein 105 is overexpressed in squamous cell carcinoma and extramammary Paget disease but not in basal cell carcinoma.

BACKGROUND: Heat shock protein (HSP) 105 is a 105-kDa protein, recently discovered by serological analysis of recombinant cDNA expression libraries prepared from tumour cells (SEREX), and is still undergoing intensive research. SEREX can define strongly immunogenic tumour antigens that elicit both cellular and humoral immunity. Previous studies have shown that HSP105 is a cancer testis antigen and is overexpressed in various internal malignancies. The expression of HSP105 has not been studied in skin cancers. OBJECTIVES: To assess the expression of HSP105 in skin cancers including extramammary Paget disease (EMPD), cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). METHODS: Samples of EMPD (n = 25), SCC (n = 23, of which three were metastatic lesions) and BCC (n = 23) were collected from patients treated in our department between January 2002 and December 2004. Western blot and immunohistochemical staining methods were used to investigate the expression of HSP105. RESULTS: Results of Western blot analysis showed overexpression of HSP105 in EMPD and SCC, and minimal expression in BCC. Immunohistochemistry results showed that 56% of EMPD, 60% of primary and 100% of metastatic SCC highly expressed HSP105 while only 13% of BCC lesions showed increased staining. CONCLUSIONS: EMPD and SCC overexpress HSP105 while BCC does not. Tumours overexpressing HSP105 present ideal candidates for vaccination by HSP105-derived peptides or DNA.

Regulation of high molecular weight-melanoma associated antigen (HMW-MAA) gene expression by promoter DNA methylation in human melanoma cells.

The human high molecular weight-melanoma associated antigen (HMW-MAA) is a membrane-bound chondroitin sulfate proteoglycan that is variably expressed in a high percentage of melanoma cell lines and tumors. Since the mechanism(s) regulating HMW-MAA expression has(ve) not been defined, in this study, we have examined whether promoter DNA methylation regulates the level of HMW-MAA expression. In melanoma cell lines, the level of HMW-MAA mRNA and protein expression is coordinately regulated, implicating a transcriptional control mechanism. Consistent with a role for regulation by DNA methylation, we have found that a dense CpG island flanks the human HMW-MAA gene transcriptional start site. Methylation-specific PCR and sodium bisulfite DNA sequencing analyses indicate that the HMW-MAA promoter is heavily methylated in melanoma cell lines, melanoma lesions and normal lymphocytes that do not express HMW-MAA; in contrast, the HMW-MAA promoter is not methylated in melanoma cell lines and tumors that express this antigen. In addition, HMW-MAA expression is markedly induced in HMW-MAA-negative melanoma cell lines by incubation with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. In summary, our results establish DNA methylation as a key regulator of HMW-MAA expression by human melanoma cells. This information represents a useful background to optimize immunotherapeutic strategies targeting HMW-MAA.

Comparison of dermoscopic and histopathological findings in a mucous melanoma of the lip.

Mucous melanomas on the lip are very rare. None of the few available reports has provided a comparison of dermoscopic and histopathological findings. We describe a 33-year-old Japanese man with a mucous melanoma on the lower lip and present a comparison of our dermoscopic and histopathological findings.

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.

Specificity and functional characteristics of anti-HLA-A mAbs LGIII-147.4.1 and LGIII-220.6.2.

Only three anti-HLA-A monoclonal antibodies (mAbs) have been described in the literature. Two of them recognize determinants shared by only a few HLA-A allospecificities. The third one, mAb 3G11, recognizes a determinant shared by most HLA-A allospecificities. Being an IgM, the latter mAb is not likely to be a useful probe in immunohistochemical reactions and in functional assays. Therefore, in the present study we have characterized the specificity of the mAbs LGIII-147.4.1 and LGIII-220.6.2. The two mAbs that do not share idiotypic determinants recognize distinct but spatially close antigenic determinants expressed on most of the gene products of the HLA-A locus. Specifically, the determinant recognized by mAb LGIII-220.6.2 is expressed on HLA-A1, -A2, -A3, -A26, -A28, -A29, -A30, -A33, -A36, -A74 and -A80 allospecificities. The determinant recognized by mAb LGIII-147.4.1, which appears to be located on the amino-acid residues 79-83 of the heavy chain, is expressed on all HLA-A allospecificities but HLA-A23, -A24, -A25 and -A32. Because of its broad reactivity, the mAb LGIII-147.4.1 was characterized in a number of assays. It was found to be a useful probe to measure the HLA-A antigen level in serum, to assess the HLA-A restriction of cytotoxic T lymphocytes (CTL) and to monitor HLA-A antigen expression in normal and malignant lesions.

Widespread expression of parathyroid hormone-related peptide in melanocytic cells.

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: These results suggest that PTH-rP was widely expressed in melanocytic cells; however, the cells did not secrete PTH-rP.

Evaluation of 5-S-cysteinyldopa as a marker of melanoma progression: 10 years' experience.

5-S-Cysteinyldopa (5-S-CD) has been used as a biochemical marker of melanoma progression. In this study, we measured serum levels of 5-S-CD in 2648 samples taken from 218 patients in order to evaluate the usefulness of this parameter in following melanoma progression and prognosis. 5-S-CD levels were significantly elevated above the upper limit of the normal range (10 nmol/l) in stage IV melanoma patients. The sensitivity of elevated serum 5-S-CD levels in detecting distant metastasis was 73%, while the specificity was 98% and the positive predictive value 94%. The sensitivity was improved to 77% when cases of amelanotic melanoma were excluded. Patients without metastases had elevated 5-S-CD values in 5% of the 1480 serum samples. Changes in serum 5-S-CD levels were followed during disease progression until the end stage in 49 patients. In 33% of the patients, elevation of serum 5-S-CD levels preceded clinical detection of visceral metastases, and in 37% elevation of 5-S-CD levels occurred at the same time as visceral metastasis. Patients with elevated 5-S-CD levels before or after surgical treatment had significantly shorter survival times than those with normal levels. These results show that the level of 5-S-CD in the serum is a sensitive and specific marker in predicting distant metastases. Elevated serum levels of 5-S-CD, before or after surgical treatment, is associated with a poor prognosis.

Clear cell sarcoma arising in the retroperitoneum.

Clear cell sarcoma is a rare soft-tissue neoplasm, arising most commonly in the tendons and aponeuroses of young adults. We report here the first female case of clear cell sarcoma arising in the retroperitoneum with clinical features similar to those of malignant ovarian tumors. Aspects of clinical presentation, histopathologic evaluation, and treatment are described.

Loss of beta-catenin expression associated with disease progression in malignant melanoma.

BACKGROUND: beta-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. OBJECTIVES: To examine beta-catenin expression in lesions of melanocytic origin for associations with clinicopathological markers of disease progression and for its significance as a predictor of disease recurrence and prognosis. METHODS: beta-catenin expression was examined by immunoperoxidase staining in 50 melanocytic naevi and 91 primary and 50 metastatic melanomas. RESULTS: beta-catenin was expressed in 96% of melanocytic naevi, in 94% and 65%, respectively, of radial and vertical growth phase primary melanomas, and in 38% of metastatic melanomas. Benign and malignant melanocytic lesions had distinct patterns of beta-catenin localization. Most lesions expressing beta-catenin exhibited cytoplasmic staining; however, over 40% of benign lesions also displayed nuclear staining, which was present only in 10% of primary and 15% of metastatic melanomas. Absent or weak expression of beta-catenin in primary melanomas was associated with several markers of disease progression, including tumour thickness and presence of lymph node metastases. A similar but not statistically significant trend was observed for the association of beta-catenin expression with disease recurrence and prognosis. CONCLUSIONS: These results suggest that loss or downregulation of beta-catenin expression in melanoma cells plays a significant role in progression of the disease.

Metastatic melanoma to the palatine tonsil with a favourable prognosis.

Metastasis to the oral cavity from cutaneous melanoma is rare: fewer than 30 cases of metastatic melanoma to the palatine tonsil have been reported. Tonsil metastasis is haematogenously disseminated and therefore usually has a poor prognosis. We present a case of metastatic melanoma to the palatine tonsil occurring 6(1/2) years after removal of the primary cutaneous lesion. The patient has remained disease-free for 18 months since the removal of skin and tonsil metastases. Immunohistopathologically, HLA class II and costimulatory factor B7-2 molecules were concomitantly expressed on melanoma cells: we suggest that the patient was therefore able to develop antimelanoma T-cell activation resulting in prevention of further metastasis, and thus a favourable prognosis.

Sentinel lymph nodes study in 30 cases of melanoma.

In 30 cases of melanoma, we attempted to detect sentinel lymph nodes using 1-2% patent blue dye and were able to detect them in 27 cases (90%): 19 cases out of 21 cases in the groin area (90%), 5 out of 5 cases in the axilla area (100%), and 3 out of 4 cases in the neck area (75%). The numbers of sentinel lymph nodes were one in 16 cases, two in 7 cases, three in 2 cases, and four in 2 cases. The cases with three and four nodes were all in the groin area. In 22 cases, tumor metastasis was negative in sentinel lymph nodes. Sentinel lymph nodes were detected in 36 out of 174 samples, and tumor metastasis was negative except in these sentinel lymph nodes (false negative 0%). In the groin area, sentinel lymph nodes were located around the femoral and great saphenous vein junction. In the axilla area, sentinel lymph nodes were located in the central, lateral and subscapular lymph nodes. In the head and neck area, sentinel lymph nodes were found in the submandibular and occipital lymph nodes. The positions of sentinel lymph nodes differed a little with tumor location. By accumulating cases, it should become possible to predict the positions of sentinel lymph nodes before operations. Sentinel lymph node biopsy is easy and requires only a small incision.

Growth inhibition of human malignant melanoma transfected with the human interferon-beta gene by means of cationic liposomes.

Among the various types of human interferons, human interferon-beta (HuIFNbeta) has the strongest anti-proliferative activity against human melanoma cell lines. Therefore, we investigated the growth inhibitory effect of a cationic liposome containing the HuIFNbeta gene on human melanoma cell lines in vitro and in vivo. After transfection with liposomes containing the HuIFN-beta gene, human melanoma cell lines produced HuIFNbeta in the culture medium at levels ranging from 67 to 3.8 IU/ml on day 6, and growth of the cells was inhibited by 71-92%. Moreover, six injections of liposomes containing the HuIFNbeta gene completely eradicated human melanoma nodules transplanted onto the backs of nude mice 40 days after the first injection. Histological analysis of the injected nodules revealed that the HuIFNbeta gene transfection induced apoptosis of the human melanoma cells. These data suggest that transfection of the HuIFNbeta gene using cationic liposomes is a promising candidate for gene therapy of human melanoma.

Tissue factor expression and serum level in patients with melanoma does not correlate with disease progression.

Not only does tissue factor (TF) play a crucial role in hemostasis and thrombosis, but it is also involved in tumor progression and metastatic potency in some malignant tumors. We evaluated the clinical relevance of TF expression in melanocytic tumors and TF serum level in patients with malignant melanoma. TF expression in benign and malignant melanocytic lesions was examined by immunoperoxidase staining in 20 nevi, 41 primary, and 24 metastatic melanoma lesions. TF was detected in 94, 95, and 100% of these lesions, respectively. The staining pattern was membranous and cytoplasmic both in nevi and melanoma cells. This finding was confirmed by western blot analysis using cultured human melanocytes, nevi cells, and melanoma cell lines. TF was also expressed on blood vessels in benign and malignant melanocytic lesions. Expression of TF in primary melanoma lesions was not associated with any clinicopathological variables. In addition, the serum level of TF was elevated in 14% of patients with melanoma; however, it was not correlated with disease progression. These results suggest that TF was ubiquitously expressed in melanocytic cells and its expression was not correlated with disease progression and/or metastatic potency of melanoma cells.

Clinical significance of MART-1 and HLA-A2 expression and CD8+ T cell infiltration in melanocytic lesions in HLA-A2 phenotype patients.

MART-1 is a good candidate antigen for immunotherapy against HLA-A2 patients with melanoma, since it is a highly immunogenic antigen recognized by HLA-A2 and HLA-B45 restricted CD8+ cytotoxic T cells and expressed in the majority of melanoma lesions. In the present study the expression of MART-1 and HLA-A2 on melanocytic cells and CD8+ T cell infiltration was immunohistochemically analyzed. MART-1 was expressed in most melanocytic lesions, while HLA-A2 was down-regulated with melanoma disease progression. Furthermore, concomitant down-regulation of MART-1 and HLA-A2 in melanoma cells was correlated with poor prognosis. These findings suggest both MART-1 and HLA-A2 expression in melanoma lesions should be analyzed for selection of patients eligible for MART-1 based immunotherapy and monitoring for emergence of melanoma cells resistant to T cell therapy.

Alpha(v)beta3 expression on blood vessels and melanoma cells in primary lesions: differential association with tumor progression and clinical prognosis.

The alpha(v)beta3 integrin has emerged as a key mediator in angiogenesis. Its role in tumor-induced angiogenesis is supported by its up-regulation in vivo in the vasculature of a number of different types of carcinoma. The potential clinical significance of alpha(v)beta3 expression on blood vessels in carcinomas is suggested by its association with tumor progression. Currently no information is available about the clinical significance of alpha(v)beta3 expression on the vasculature of lesions of melanocytic origin. Since we have previously found that alpha(v)beta3 expression on melanoma cells in primary lesions is associated with a poor prognosis, in the present study we have compared alpha(v)beta3 expression on blood vessels and on cells of melanocytic origin in nevi and in malignant melanoma lesions. In addition we have examined the lesions for expression of the alpha(v) subunit to gain information on the regulation of alpha(v)beta3 expression on endothelial cells and on cells of the melanocyte lineage. alpha(v)beta3 expression on endothelial cells and on melanocytic cells was a relatively sensitive and specific marker for malignant lesions. However, alpha(v)beta3 expression on endothelial cells in primary melanoma lesions was not associated with the prognosis of the disease. The alpha(v) subunit and the alpha(v)beta3 complex were differentially expressed on endothelial cells and on melanocytic cells, implying that different regulatory pathways control their expression. This finding may account for the differential clinical significance of alpha(v)beta3 expression on tumor vasculature and on melanoma cells we observed in our patient cohort. Lastly, alpha(v)beta3 may be a useful target for immunotherapeutic approaches in melanoma because of its high expression on the vasculature of all metastatic lesions tested and its restricted distribution in normal tissues.

Myelodysplastic syndrome following treatment of malignant melanoma with vincristine, ACNU, and dacarbazine.

Therapy-related myelodysplastic syndrome is a rare adverse effect in melanoma patients elicited by chemotherapy. We report a case of myelodysplastic syndrome following treatment of malignant melanoma with alkylating agents. Peripheral blood showed a remarkable suppression of three cell lineages, and the bone marrow was slightly hypercellular. However, no morphological abnormalities were detected in the peripheral blood or the bone marrow, and chromosomal analysis was normal.

Differential clinical significance of alpha(v)Beta(3) expression in primary lesions of acral lentiginous melanoma and of other melanoma histotypes.

Despite its potential clinical relevance, alpha(v)beta(3) 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 alpha(v)beta(3) 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 alpha(v)beta(3) 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 alpha(v)beta(3) 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, alpha(v)beta(3) 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.

Hypercalcaemia in melanoma patients associated with increased levels of parathyroid hormone-related protein.

We analysed serum levels of calcium and parathyroid hormone-related protein (PTH-rP) in 59 patients with melanoma by immunoradiometric assay, since only a few studies have been conducted on hypercalcaemia and serum levels of PTH-rP in patients with melanoma. Hypercalcaemia was found in seven of the 59 patients with melanoma. The serum level of PTH-rP was increased in three of the 59 patients and two of the three showed hypercalcaemia. All the patients with hypercalcaemia and/or an increased level of PTH-rP had stage IV melanoma. These findings suggest that hypercalcaemia is much more common than previously reported in patients with advanced stage melanoma and that an increased serum level of PTH-rP is one of the causative factors in hypercalcaemia in melanoma. Analysis of the serum PTH-rP level will contribute to the monitoring of the clinical course of patients with advanced stage melanoma.

Low frequency of neutralizing antibodies against natural interferon-beta during adjuvant therapy for Japanese patients with melanoma.

The incidence of therapy-induced neutralizing interferon-beta (IFN-beta) antibodies was investigated in 41 Japanese patients with melanoma treated with a combination of chemotherapy and subcutaneous injections of natural IFN-beta. Only one patient (2.4%) developed anti-IFN-beta antibodies. This finding differs from those of previous studies with Caucasian patients in which subcutaneous injections of natural IFN-beta resulted in a high incidence of anti-IFN-beta antibodies. This difference may be related to ethnic group of patients, treatment schedule, pharmacological preparation of IFN-beta and/or technical assay system used in each study.