Akifumi Yamamoto

Updated statistical data for malignant melanoma in Japan.

AbstractBackground. A statistical investigation was conducted of Japanese melanoma patients who had been registered by the Prognosis and Statistical Investigation Committee of the Japanese Skin Cancer Society. The data were obtained from 24 main medical institutions in Japan in the period 1987 to 1996. The patients were classified into two chronological groups; group A, 535 patients registered from 1987 to 1991; and group B, 639 patients registered from 1992 to 1996. As the preliminary data for group A has been reported previously (Gann Monograph on Cancer Research 43), in this report, the survival data of group B were assessed, and compared with the data for group A. Methods. The data analyzed included age, sex, anatomical distribution, clinical features of primary lesions, Clarks subtype, tumor thickness, Clarks level, disease stage, and treatment. The survival rate was assessed by the Kaplan-Meier method, and the significance of differences was determined with the log-rank test. In addition, the results of a nationwide survey of various types of skin malignancies, obtained from 101 medical institutions in Japan between 1987 and 1996, were analyzed. Results. The nationwide survey revealed that the number of patients with malignant melanoma showed a steady increase during the period 1987–1996 in Japan. It is noteworthy that the numbers of actinic keratoses, a type of early squamous cell carcinoma in situ, showed a steep increase in recent years. Results revealed in our study of the melanoma registry for the period 1987–1996 were as follows: (1) the male-to-female ratio was 1 to 1.06, (2) the survival rate of female patients was higher than that of male patients (10-year survival in group B: female, 71.6% vs male, 55.9%), (3) the commonest site of melanoma in both sexes was the sole of the foot, (4) with respect to Clarks subtype, acral lentiginous melanoma was commonest, accounting for about half of all melanomas, (5) nodular melanoma showed the worst prognosis among the subtypes, (6) patients in stage IIIB and stage IV had an unfavorable outcome, with 10-year survivals of less than 50% and less than 10%, respectively, (7) Clarks level of invasion, as well as Breslows tumor thickness of the primary lesions, were confirmed to be important prognostic factors, and (8) prophylactic lymph node dissection in stage II and IIIA and chemotherapy in stage IV seemed to have limited effect on the prognosis. Conclusion. The prognosis of advanced melanoma is poor, as it is highly resistant to chemotherapy. Thus, to improve the prognosis, early detection is mandatory, and this is possible because this neoplasm appears as a distinctive pigmented lesion on the skin.

Malignant Eccrine Spiradenoma: A Case Report and Review of the Literature

BACKGROUND Eccrine spiradenoma is a well-differentiated benign tumor of the sweat glands. Malignant change arising within eccrine spiradenoma is rare. OBJECTIVE We describe a patient with malignant eccrine spiradenoma exhibiting both carcinomatous and sarcomatous differentiation. METHODS Case report and literature review. RESULTS A 37-year-old woman noted enlargement of a left axillary tumor that had been present for 20 years. The tumor was resected and the specimen, measuring 3.0 cm × 1.5 cm, revealed an encapsulated benign eccrine spiradenoma as well as an undifferentiated carcinoma possessing both carcinomatous and sarcomatous components. A transition zone was evident between the benign eccrine spiradenoma and the undifferentiated carcinoma, suggesting that the latter had arisen from the benign tumor. The malignant areas consisted principally of undifferentiated carcinoma (70%), although squamous cell carcinoma (10%), adenocarcinoma (10%), and chondrosarcomatous (10%) components were also present. Numerous mitotic figures were noted within the areas of malignant change, suggesting that the tumor was aggressive in nature. The patient died of systemic metastases 7 months after diagnosis. CONCLUSION Although eccrine spiradenomas are usually benign, they can, on rare occasions, undergo malignant transformation. This case report describes one such occurrence of malignant transformation of a benign eccrine spiradenoma that unfortunately resulted in the patients death from systemic metastases 7 months after diagnosis.

Dendritic cell-based vaccination in metastatic melanoma patients: Phase II clinical trial

Metastatic and chemoresistant melanoma can be a good target of immunotherapy because it is an intractable cancer with a very poor prognosis. Previously, we tested a dendritic cell (DC)-based phase I vaccine, and confirmed that it was safe. In the present study, we performed a phase II trial of a DC vaccine for metastatic melanoma patients with mainly the HLA-A24 genotype, and investigated the efficacy of the vaccine. Twenty-four patients with metastatic melanoma were enrolled into a phase II study of DC-based immunotherapy. The group included 19 HLA-A24-positive (A*2402) patients and 3 HLA-A2-positive (A*0201) patients. The protocol for DC production was similar to that in the phase I trial. Briefly, a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-A2, MAGE-A3 and MART-1 or MAGE-A1) restricted to HLA-A2 or A24 and KLH were used for DC pulsing. Finally, DCs were injected subcutaneously (s.c.) into the inguinal region in the dose range of 1–5×107 per shot. The DC ratio (lin-HLA-DR+) of the vaccine was 38.1±13.3% and the frequency of CD83+ DCs was 25.7±20.8%. Other parameters regarding DC processing were not different from phase I. Immune response-related parameters including the ELISPOT assay, DTH reaction to peptide or KLH, DC injection numbers were shown to be related to a good prognosis. The ELISPOT reaction was positive in 75% of the patients vaccinated. The increase of anti-melanoma antigen antibody titer before vaccination was also shown to be a prognosis factor, but that post-vaccination was not. Based on immunohistochemical analysis, CD8 and IL-17 were not involved in the prognosis. Adverse effects of more than grade III were not seen. Overall survival analysis revealed a significant survival prolongation effect in DC-given melanoma patients. These results suggest that peptide cocktail-treated DC vaccines may be a safe and effective therapy against metastatic melanoma in terms of prolongation of overall survival time.

Results of a phase I clinical study using autologous tumour lysate-pulsed monocyte-derived mature dendritic cell vaccinations for stage IV malignant melanoma patients combined with low dose interleukin-2

We conducted a pilot study to assess the feasibility and efficacy of immunotherapy for stage IV malignant melanoma patients resistant to conventional therapies involving vaccination with mature dendritic cells (mDCs) combined with administration of low dose interleukin-2. Autologous monocytes were harvested from a single apheresis and cultured for 7 days with granulocyte–macrophage colony-stimulating factor and interleukin-4, yielding immature dendritic cells (iDCs), which were then cryopreserved until use. For 4 days prior to vaccination, iDCs were exposed to autologous tumour lysate combined with tumour necrosis factor-α to induce terminal differentiation into mDCs. Patients were then vaccinated weekly with 107 mDCs for 10 weeks and given 350–700 kIU of interleukin-2 three times per week. Of the 10 patients in the study, one showed stable disease, seven showed progressive disease, and two showed mixed responses, including partial tumour regression, and were therefore given 20 additional injections. Only minimal adverse events were noted, including localized skin reactions and mild fever (NIH-CTC grade 0–1). Median survival from the first vaccination was 240 days (range 31–735 days). In vitro, melanoma patient-derived dendritic cells (DCs) showed reduced cell surface expression of CD1a antigen on iDCs and reduced CD86 and HLA-DR expression on mDCs. In addition, antigen uptake, chemotaxis and antigen presentation were all attenuated in DCs from the patients. In summary, although improvement of clinical efficacy will require further research, autologous tumour lysate-pulsed monocyte-derived mDCs could be safely harvested, cryopreserved and administrated to patients without obvious complications.

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.

Silencing of tissue factor pathway inhibitor‐2 gene in malignant melanomas

To identify tumor‐suppressor genes inactivated by aberrant methylation of promoter CpG islands (CGIs) in human malignant melanomas, genes upregulated by treatment of cells with a demethylating agent, 5‐aza‐2′‐deoxycytidine (5‐aza‐dC), were searched for using oligonucleotide microarrays in melanoma cell lines, HMV‐I, MeWo and WM‐115. Seventy‐nine known genes with CGIs were identified as being upregulated (≥16‐fold), and 18 of them had methylation of their putative promoter CGIs in 1 or more of 8 melanoma cell lines. Among the 18 genes, TFPI‐2, which is involved in repression of the invasive potential of malignant melanomas, was further analyzed. Its expression was repressed in a melanoma cell line with its complete methylation, and was restored by 5‐aza‐dC treatment. It was unmethylated in cultured neonatal normal epidermal melanocyte, and was induced by ultraviolet B. In surgical melanoma specimens, TFPI‐2 methylation was detected in 5 of 17 metastatic site specimens (29%), while it was not detected in 20 primary site specimens (0%) (p = 0.009). By immunohistochemistry, the 5 specimens with promoter methylation lacked immunoreactivity for TFPI‐2. The results showed that TFPI‐2 is silenced in human malignant melanomas by methylation of its promoter CGI and suggested that its silencing is involved in melanoma metastasis.

Clinical response in Japanese metastatic melanoma patients treated with peptide cocktail-pulsed dendritic cells

BackgroundMetastatic, chemotherapy-resistant melanoma is an intractable cancer with a very poor prognosis. As to immunotherapy targeting metastatic melanoma, HLA-A2+ patients were mainly enrolled in the study in Western countries. However, HLA-A24+ melanoma patients-oriented immunotherapy has not been fully investigated. In the present study, we investigated the effect of dendritic cell (DC)-based immunotherapy on metastatic melanoma patients with HLA-A2 or A24 genotype.MethodsNine cases of metastatic melanoma were enrolled into a phase I study of monocyte-derived dendritic cell (DC)-based immunotherapy. HLA-genotype analysis revealed 4 cases of HLA-A*0201, 1 of A*0206 and 4 of A*2402. Enriched monocytes were obtained using OptiPrep™ from leukapheresis products, and then incubated with GM-CSF and IL-4 in a closed serum-free system. After pulsing with a cocktail of 5 melanoma-associated synthetic peptides (gp100, tyrosinase, MAGE-2, MAGE-3 and MART-1 or MAGE-1) restricted to HLA-A2 or A24 and KLH, cells were cryopreserved until used. Finally, thawed DCs were washed and injected subcutaneously (s.c.) into the inguinal region in a dose-escalation manner.ResultsThe mean percentage of DCs rated as lin-HLA-DR+ in melanoma patients was 46.4 ± 15.6 %. Most of DCs expressed high level of co-stimulatory molecules and type1 phenotype (CD11c+HLA-DR+), while a moderate number of mature DCs with CD83 and CCR7 positive were contained in DC products. DC injections were well tolerated except for transient liver dysfunction (elevation of transaminases, Grade I-II). All 6 evaluable cases except for early PD showed positive immunological responses to more than 2 melanoma peptides in an ELISPOT assay. Two representative responders demonstrated strong HLA-class I protein expression in the tumor and very high scores of ELISPOT that might correlate to the regression of metastatic tumors. Clinical response through DC injections was as follows : 1CR, 1 PR, 1SD and 6 PD. All 59 DC injections in the phase I study were tolerable in terms of safety, however, the maximal tolerable dose of DCs was not determined.ConclusionsThese results suggested that peptide cocktail-treated DC-based immunotherapy had the potential for utilizing as one of therapeutic tools against metastatic melanoma in Japan.

Carbonic Anhydrase II Is a Tumor Vessel Endothelium ^ Associated Antigen Targeted by Dendritic Cell Therapy

Tumor-associated antigens are promising candidates as target molecules for immunotherapy and a wide variety of tumor-associated antigens have been discovered through the presence of serum antibodies in cancer patients. We previously conducted dendritic cell therapy on 10 malignant melanoma patients and shrinkage or disappearance of metastatic tumors with massive necrosis occurred in two patients. In this study, we found a 29-kDa protein against which antibody was elicited by dendritic cell therapy in one of the two patients. Matrix-assisted laser desorption ionization-time of flight/mass spectrometry analysis of the protein isolated by two-dimensional electrophoresis combined with Western blots revealed that the 29-kDa protein was carbonic anhydrase II (CA-II). Immunohistochemistry of the tumors and normal tissues showed that CA-II was expressed in the tumor vessel but not in normal vessel endothelium. CA-II expression in tumor endothelium was observed as well in other cancers including esophageal, renal, and lung cancers. In an in vitro angiogenesis model, CA-II expression of normal human vein endothelial cells was significantly up-regulated when cells were cultured in the acidic and hypoxic conditions indicative of a tumor environment. These findings suggest that CA-II is a tumor vessel endothelium–associated antigen in melanoma and other cancers, and elicitation of serum anti–CA-II antibody by dendritic cell therapy may be associated with good clinical outcome including tumor reduction.

Interferon-β 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-β (HuIFN-β) against malignant melanoma. In vitro study revealed that HuIFN-β 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-β was different among cell lines. In addition, we administered HuIFN-β 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-β, histopathological examination revealed apoptotic changes of melanoma cells along with dense lymphohistiocytic infiltration. The present study confirmed direct and indirect inhibitory effects of HuIFN-β on human melanoma cells and suggests that local higher concentration of HuIFN-β is needed to eradicate melanoma lesions.

Applicability of radiocolloids, blue dyes and fluorescent indocyanine green to sentinel node biopsy in melanoma

Patients with primary cutaneous melanoma underwent sentinel node (SN) mapping and biopsy at 25 facilities in Japan by the combination of radiocolloid with gamma probe and dye. Technetium‐99m (99mTc)‐tin colloid, 99mTc‐phytate, 2% patent blue violet (PBV) and 0.4% indigo carmine were used as tracers. In some hospitals, 0.5% fluorescent indocyanine green, which allows visualization of the SN with an infrared camera, was concomitantly used and examined. A total of 673 patients were enrolled, and 562 cases were eligible. The detection rates of SN were 95.5% (147/154) with the combination of tin colloid and PBV, 98.9% (368/372) with the combination of phytate and PBV, and 97.2% (35/36) with the combination of tin colloid or phytate and indigo carmine. SN was not detected in 12 cases by the combination method, and the primary tumor was in the head and neck in six of those 12 cases. In eight of 526 cases (1.5%), SN was detected by PBV but not by radiocolloid. There were 13 cases (2.5%) in which SN was detected by radiocolloid but not by PBV. In 18 of 36 cases (50%), SN was detected by radiocolloid but not by indigo carmine. Concomitantly used fluorescent indocyanine green detected SN in all of 67 cases. Interference with transcutaneous oximetry by PVB was observed in some cases, although it caused no clinical trouble. Allergic reactions were not reported with any of the tracers. 99mTc‐tin colloid, 99mTc‐phytate, PBV and indocyanine green are useful tracers for SN mapping.