Hisashi Kanemaru

The circulating microRNA-221 level in patients with malignant melanoma as a new tumor marker

BACKGROUND MicroRNA-221 (miR-221) is known to be abnormally expressed in malignant melanoma (MM) cells, and it favors the induction of the malignant phenotype through down-modulation of p27Kip1/CDKN1B and the c-KIT receptor. This suggests that the serum level of miR-221 might increase in patients with MM and thus could be used as a new tumor marker. OBJECTIVE To evaluate the possibility that the serum miR-221 level can be a marker of MM. METHODS Serum samples were obtained from 94 MM patients and 20 healthy controls. MicroRNAs were purified from serum, and miR-221 levels were measured by quantitative real-time polymerase chain reaction. RESULTS Circulating miR-221 was detectable and could be quantified in serum samples. MM patients had significantly higher miR-221 levels than healthy controls. Among the MM patients, the miR-221 levels were significantly increased in patients with stage I-IV MM compared to those with MM in situ, and the levels were correlated with tumor thickness. Moreover, a longitudinal study revealed a tendency for the miR-221 levels to decrease after surgical removal of the primary tumor, and to increase again at recurrence. CONCLUSIONS Serum levels of miR-221 were significantly increased in MM patients and may be useful not only for the diagnosis of MM, but also for the differentiating MM in situ from stage I-IV MM, and for evaluating tumor progression and monitoring patients during the follow-up period. In addition, considering that the serum levels of miR-221 were correlated with tumor thickness, miR-221 might also be useful as a prognostic marker for patients with MM.

Investigation of FOXM1 as a Potential New Target for Melanoma.

Recent studies have shown that immunotherapies and molecular targeted therapies are effective for advanced melanoma. Non-antigen-specific immunotherapies such as immunocheckpoint blockades have been shown to be effective in the treatment of advanced melanoma. However, the response rates remain low. To improve their efficacy, they should be combined with antigen-specific immunotherapy. Elevated expression of the transcription factor, Forkhead box M1 (FOXM1), has been reported in various human cancers, and it has been shown to have potential as a target for immunotherapy. The purpose of this study was to investigate the FOXM1 expression in human melanoma samples and cell lines, to evaluate the relationship between the FOXM1 expression and the clinical features of melanoma patients and to investigate the association between the FOXM1 and MAPK and PI3K/AKT pathways in melanoma cell lines. We conducted the quantitative reverse transcription PCR (qRT-PCR) and Western blotting analyses of melanoma cell lines, and investigated melanoma and nevus tissue samples by qRT-PCR and immunohistochemistry. We performed MEK siRNA and PI3K/AKT inhibitor studies and FOXM1 siRNA studies in melanoma cell lines. We found that FOXM1 was expressed in all of the melanoma cell lines, and was expressed in 49% of primary melanomas, 67% of metastatic melanomas and 10% of nevi by performing immunohistochemical staining. Metastatic melanoma samples exhibited significantly higher mRNA levels of FOXM1 (p = 0.004). Primary melanomas thicker than 2 mm were also more likely to express FOXM1. Patients whose primary melanoma expressed FOXM1 had a significantly poorer overall survival compared to patients without FOXM1 expression (p = 0.024). Downregulation of FOXM1 by siRNA significantly inhibited the proliferation of melanoma cells, and blockade of the MAPK and PI3K/AKT pathways decreased the FOXM1 expression in melanoma cell lines. In conclusion, FOXM1 is considered to be a new therapeutic target for melanoma.

Case of neuro-Behçet's disease successfully maintained a remission using infliximab for 2 years

Behçets disease is a systemic inflammatory disorder with chronic remissions and relapses. Involvement of the central nervous system, called neuro‐Behçets disease (NBD), is a serious complication. Although corticosteroids and immunosuppressive agents are used, there is no definite standard of treatment with long term‐effects for NBD. Here, we report a female case with NBD successfully treated by infliximab and corticosteroids for 2 years. Our report suggests treatment with infliximab may be effective for long‐term remission of NBD.

Achieved good response of S-1 and docetaxel combination chemotherapy in two patients with metastatic extramammary Paget's disease

Dear Editor, The prognosis of metastatic extramammary Paget’s disease (EMPD) is poor because there are few chemotherapy regimens for it. In previous reports, there have been several effective chemotherapeutic regimens including docetaxel (DOC) monotherapy and paclitaxel/trastuzumab combination therapy. Recently, S-1/DOC combination chemotherapy has been reported to be effective for metastatic EMPD. Herein, we treated two metastatic EMPD patients with S-1/DOC chemotherapy and its therapeutic effects were very effective. Case 1 was a 67-year-old man who had been diagnosed as having EMPD, and received tumor excision and right inguinal/ external iliac lymphadenectomy at another hospital. Within 4 months after the operation, he was admitted our hospital because of back pain. Positron emission tomography/computed tomography (PET/CT) revealed multiple metastases of the spine and pelvic lymph nodes (Fig. 1a). Thus, we administrated p.o. S-1 (80 mg/m, day 1–14) and i.v. DOC (40 mg/m, day 1) therapy every 4 weeks in reference to a previous report. Although he developed grade 3 neutropenia, PET/CT revealed complete response (CR) after three cycles (Fig. 1b). We discontinued S-1/DOC chemotherapy after 10 cycles because maintenance of CR and his own request. However, at 2 months after the discontinuation of S-1/DOC therapy, he had a relapse in the lower abdominal region. Reinstitution of monthly S-1/DOC therapy and additional triweekly paclitaxel/ trastuzumab chemotherapy did not improve the exacerbation. He died 26 months after the initiation of S-1/DOC chemotherapy. Case 2 was a 75-year-old man diagnosed as having EMPD with sentinel lymph node metastasis without distant metastasis. We performed tumor excision and right inguinal lymphadenectomy. After 1 year, PET/CT showed abnormal metastases in abdominal para-aortic lymph nodes without local skin recurrence (Fig. 1c). We administrated the monthly S-1/ DOC chemotherapy. After two cycles, partial response (PR) was shown (Fig. 1d) and he developed grade 1 anorexia, malaise and alopecia. After 10 cycles of S-1/DOC chemotherapy, PET/CT showed metastases in the liver and the lumbar vertebrae. Thus, we reinstituted monthly S-1/DOC therapy and additional triweekly paclitaxel/trastuzumab chemotherapy in reference to a past report. After four cycles, he developed

Platelet-rich plasma therapy is effective for the treatment of refractory skin ulcers in patients with systemic sclerosis

administered. MRI showed no osteomyelitis. We tried PRP therapy to accelerate the wound-healing using a PRP kit (KYOCERA Medical Corporation, Osaka, Japan). The medical procedure was performed with reference to the manufacturer ’ s protocol. We collected 20 mL of the patient ’ s blood using a syringe containing sodium citrate. We then performed centrifugal separation of the blood at 600 g for 10 min. Next, we collected the platelets and leukocyte supernatant from the blood sampling syringe to a separation syringe. We then performed centrifugal separation at 2000 g for 5 min, removed the platelet-poor plasma supernatant from the separation syringe, and used the remaining 2 mL present in the separation syringe as the PRP. Just before using it, we added calcium to activate the platelets. Finally, we put the activated PRP on the surface of the ulcer. This patch of PRP was applied on the place of ulcer for a week. After the fi rst PRP treatment, epithelialization was seen. Therefore, we completed the PRP treatment on the fi rst attempt. The skin defect was almost diminished after 1 month (Figure 1b). The second case was a 75-year-old female with a 14-year history of limited cutaneous SSc, positive for anti-centromere antibodies. Neither endothelin receptor antagonists nor PDE5 inhibitors have been administered before. She developed skin ulcers and gangrene on the right big, second, and little toes. MRI revealed osteomyelitis of the right big and little toes. Because of a lack of responsiveness to conservative therapies, we performed amputation below the right knee. However, cutaneous ulcers appeared on the surgical scar after 2 weeks (Figure 2a). We tried PRP therapy every week as described in Case 1. This resulted in good granulation tissue formation after a week. After 1 month (after the third PRP treatment), her ulcer showed good improvement (Figure 2b). After the fourth PRP treatment, the wound was treated with a full-thickness skin graft (FTSG) harvested from the right inguinal region. One week after the FTSG treatment, the ulcer lesion almost completely healed (Figure 2c). Since the skin involvements in SSc, including ulcers and gangrene, tend to be unresponsive to standard therapies, their Mod Rheumatol, 2015; 25(4): 660–661

The role of miR-210, E2F3 and ephrin A3 in angiosarcoma cell proliferation

BackgroundAlthough angiosarcoma exhibits aggressive progression and is associated with unfavourable prognosis, its pathogenesis is poorly understood.ObjectivesIn the present study, we investigated the possibility that microRNAs play a role in the pathogenesis of angiosarcoma.Materials & methodsmicroRNA expression was evaluated by array analysis and real-time PCR, and protein expression was determined by immunohistochemistry and immunoblotting.ResultsmiR-210 expression was decreased in angiosarcoma cells both in vivo and in vitro. E2F3 and ephrin A3 are putative targets of miR-210, and their protein expressionwas up-regulated in the tumour cells. Knockdown of E2F3 or ephrin A3 resulted in a significant decrease in the number of angiosarcoma cells.ConclusionFurther investigations into the regulatory mechanisms of oncogenesis associated with miR-210/E2F3/ephrin A3 signalling may lead to a new therapeutic approach against angiosarcoma.

Clinical significance of serum vascular endothelial-cadherin levels in inflammatory skin diseases.

Dear Editor: Inflammatory skin diseases are sometimes accompanied by vascular abnormalities; e.g. Auspitz phenomenon in psoriasis or white dermographism in atopic dermatitis (AD)1,2. However, the detailed mechanism(s) and role of such vasculopathy in the pathogenesis of each disease are still unclear. Vascular endothelial (VE)-cadherin is one of the major components of adherens junctions between endothelial cells. The critical role of VE-cadherin is vascular morphogenesis during embryogenesis. Such function of VE-cadherin is regulated by vascular endothelial cell growth factor (VEGF) signaling through the VEGF receptor leads to the increased detachment of endothelial cells and transendothelial permeability by promoting VE-cadherin internalization3. On the other hand, VE-cadherin limits the proliferation of endothelial cells by preventing the internalization of the VEGF receptor4. However, the role of VE-cadherin in the vascular abnormalities of inflammatory skin diseases has not been investigated. Full-length VE-cadherin is an insoluble transmembrane protein, whereas the extracellular domain of VE-cadherin is secreted as a soluble protein through a metalloproteinase-dependent mechanism5. Soluble VE-cadherin may function as an antagonist of full-length VE-cadherin, which is suggested by its inhibitory effect on tumor angiogenesis and tumor growth in vivo6. To date, although soluble VE-cadherin has been detected in serum in vivo, the clinical significance of serum soluble VE-cadherin levels is still unknown. Therefore, in this study, we attempted to evaluate the possibility that the serum VE-cadherin level can be a useful marker for inflammatory skin diseases. Serum samples were obtained from 13 patients with AD, 33 patients with psoriasis, and 7 patients with alopecia areata. Control serum samples were also collected from 18 healthy volunteers. Sera of 12 patients with anaphylactoid purpura, 11 patients with cutaneous polyarteritis nodosa, and 6 patients with angiosarcoma were also included as the disease controls. This research was approved by the Ethics Review Committee in Kumamoto University (No. 177). Written informed consents were obtained before patients and healthy volunteers were enrolled into this study according to the Declaration of Helsinki. The serum soluble VE-cadherin levels were measured with a specific enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA)7 (Fig. 1A). The mean serum VE-cadherin level tended to be lower in patients with AD (1.903 µg/ml) and those with psoriasis (1.915 µg/ml) in comparison to normal subjects (2.511 µg/ml). We found a statistical significance in these decreases (p=0.01 in patients with AD, and p=0.04 in patients with psoriasis). The serum VE-cadherin levels in patients with alopecia (p=0.81), anaphylactoid purpura (p=0.90), cutaneous polyarteritis nodosa (p=0.96), or angiosarcoma (p=0.13) were similar to those in normal subjects. When the cutoff value was set at 1.880 µg/ml on the basis of the normal range provided by the manufacturer, reduced serum VE-cadherin levels were found in 3 of the 18 healthy volunteers (16.7%), 8 of the 13 patients with AD (61.5%), 16 of the 33 patients with psoriasis (48.5%), 1 of the 7 patients with alopecia (14.3%), 4 of the 12 patients with anaphylactoid purpura (33.3%), 2 of the 11 patients with cutaneous polyarteritis nodosa (11.2%), and 0 of the 6 patients with angiosarcoma. Thus, the low serum VE-cadherin levels may be more specific to AD or psoriasis than cutaneous vasculitis or vascular tumor. Fig. 1 (A) Serum soluble VE-cadherin levels in patients with various skin diseases. The serum levels of VE-cadherin determined by using ELISA are shown on the ordinate; the horizontal bars show the mean value in each group. The dotted line indicates the cutoff. ... In the receiver operating characteristic curve analysis of patients with AD (Fig. 1B), the area under curve (AUC) was 0.77 (95% confidence interval [95% CI], 0.59~0.96). An AUC of >0.7 indicates that the serum VE-cadherin levels can effectively distinguish patients with AD from normal subjects. On the other hand, the AUC was 0.67 (95% CI, 0.53~0.82) in patients with psoriasis, indicating that serum VE-cadherin is less useful in diagnosing psoriasis. Accordingly, serum VE-cadherin may be more effective for diagnosing AD than psoriasis. Next, we determined the association of serum VE-cadherin levels with the clinical and serological features of patients with AD (Table 1); 5 disease activity markers (SCORAD, percentage of eosinophil counts, serum lactate dehydrogenase levels, serum thymus and activation-regulated chemokine levels, and serum immunoglobulin E level) and the duration of the disease (between symptom onset and the first visit to the hospital) were evaluated. However, we could not find a significant difference in these factors between patients with reduced VE-cadherin levels and those with normal levels. On the other hand, in patients with psoriasis, when 4 activity indicators (psoriasis area and severity index score, body surface area of the involved skin, arthritis, and nail change) and disease duration were evaluated (Table 1), patients with reduced VE-cadherin levels showed a significantly lower prevalence of nail change (p=0.03). Thus, serum VE-cadherin levels are correlated with clinical symptom in patients with psoriasis but not in patients with AD. Taken together, our results suggest that the serum VE-cadherin level in patients with AD can be the diagnostic marker rather than the disease activity marker, whereas that in patients with psoriasis may be more useful as the marker for disease activity. Table 1 Association of serum VE-cadherin levels with clinical and serological features This is the first report measuring serum VE-cadherin levels in patients with various skin diseases. Furthermore, as far as we searched, decreased serum VE-cadherin levels have not been reported in human diseases. Although the role of VE-cadherin in skin diseases is unknown, skin erythema, for example, is one of the common features of AD and psoriasis and is caused by dilated vessels. As described above, soluble VE-cadherin may function as an antagonist of full-length VE-cadherin. Thus, lower serum soluble VE-cadherin levels may activate transmembrane full-length VE-cadherin, which may contribute to the pathogenesis of AD and psoriasis through erythema formation. Clarifying the mechanism by which VE-cadherin-mediated vascular abnormality contributes to the pathogenesis may lead to the understanding of these diseases and to novel therapeutic strategies. However, this study has a limitation; although the VE-cadherin levels were lower in patients with psoriasis than in healthy volunteers, the nail change is less frequent in patients with psoriasis with decreased VE-cadherin levels. This paradoxical finding may be due to the small number of patients. A larger study with an increased number of patients is needed in the future.

Combination chemotherapy with S-1 and docetaxel for cutaneous angiosarcoma resistant to paclitaxel

The prognosis of cutaneous angiosarcoma is very poor compared with that of other skin malignancies. The main reason for this is the limited regimens of chemotherapy available for angiosarcoma, because it is resistant to most common chemotherapeutic agents. Therefore, there is an urgent need to identify new treatment options. Recently, S-1 and docetaxel therapy was reported to be effective for advanced gastric cancer and metastatic extramammary Pagets disease. Therefore, we treated paclitaxel-resistant angiosarcoma patient with S-1/docetaxel chemotherapy. The progression-free survival was 5.0 months although grade 3 adverse events such as diarrhea and neutropenia developed. Our data need to be confirmed in a large number of patients, but S-1/docetaxel chemotherapy as an additional regimen seems to be an effective treatment option for paclitaxel-resistant angiosarcoma.