Cytotoxic T‐lymphocyte‐associated protein 4 expressed by melanoma cells does not affect melanoma‐specific cytotoxic T lymphocytes in the effector phase

Cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) is one of the important molecules that regulate the anti‐melanoma T‐cell response. Currently, there are some reports showing that CTLA‐4 is expressed not only by T cells but also by various kinds of tumor cells, including melanoma cells. However, there is no report that shows the role of CTLA‐4 expressed by melanoma cells in melanoma‐specific cytotoxic T‐lymphocyte (CTL) response. In this report, we confirmed substantial CTLA‐4 expression and the localization of CTLA‐4 in melanoma cell lines and tissues. Also, we examined its impact on melanoma‐specific CTL in vitro, and found that CTLA‐4 expressed by melanoma cells does not affect melanoma‐specific CTL in the effector phase. Our findings suggest the importance of elucidating the role of CTLA‐4 expressed by melanoma cells, particularly in anti‐CTLA‐4 antibody therapy.     

肿瘤浸润淋巴细胞在人黑色素瘤组织中的形态学观察

目的探讨人黑色素瘤组织中肿瘤浸润淋巴细胞（tumor infiltrating lymphocyte，TIL）的形态学特征。方法采用光镜、透射电镜和免疫组化方法观察9例人皮肤黑色素瘤组织中TIL的分布及形态变化。结果肿瘤浸润淋巴细胞主要分布在癌周区，病变早期，TIL数量多、集中分布；病变晚期，TIL数量少、散在分布。病变早期TIL数量明显高于病变晚期（P〈0．01）。电镜下，可见肿瘤浸润淋巴细胞核大，胞质少，核较圆。TIL与肿瘤细胞有接触。结论本试验提示黑色素瘤组织中TIL数量多少与肿瘤的进展有关。TIL与肿瘤细胞关系密切。     

Prevention of anergy induction in cloned T cells by interleukin 12

Abstract A variety of tumors are potentially immunogenic but do not stimulate an effective antitumor immune response in vivo. Tumors may be capable of delivering antigen-specific signals to T cells, but may not deliver the costimulatory signals necessary for full activation of T cells. In this regard, we recently reported that a human melanoma cell line (sMC) expressing MHC class II, was able to induce clonal anergy in a specific, MHC-restricted CD-4+ T cell clone (sTC3). We used this system to investigate the influence of interleukin (IL)-12 on induction of this T cell unresponsiveness. The presence of 10 to 100 U IL-12 during the induction phase of anergy leads to a primary proliferative response of sTC3, which was significantly higher than that induced by IL-12 alone; however, in the absence of IL-12 no proliferation was seen during the induction of anergy. Subsequent optimal stimulation of IL-12 treated cells, but not of those cultured without IL-12, led to substantial IL-2 production and cell proliferation. This indicates that induction of the unresponsive state could be inhibited by IL-12. In addition, we have recently demonstrated that anergic T cell clones can produce high amounts of 1L-10 and that this event was correlated with their impaired ability to produce IL-2. This marked induction of IL-10 can be suppressed if IL-12 is present during initiation of unresponsiveness. However, IL-12 was not able to prime the T cell clone, sTC3, to become resistant against the anergizing stimulus, as this cytokine was only effective when present at the time of anergy induction. These findings indicate that IL-12 is very effective in preventing anergy when present at the same time as the anergizing stimulus, but is unable to prime T cells to resist anergy induction in this system.     

Melanoma therapy: Check the checkpoints

Recent mutational and translational studies have revealed that the Ras/Raf/mitogen‐activated protein kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK) pathway plays a key role in melanomagenesis. Mutations in NRAS and BRAF are found in the majority of melanomas resulting in the formation of constitutively active NRAS and BRAF molecules, which leads to the proliferation and survival of melanoma cells through the activation of MEK/ERK signals. Inhibitors of BRAF or MEK significantly extend the progression‐free survival and overall survival of melanoma patients compared with conventional chemotherapies. Combining BRAF and MEK inhibitors further enhances the clinical effectiveness. Cytotoxic T‐lymphocyte‐associated antigen 4 (CTLA‐4) is an immune checkpoint molecule that downregulates T‐cell activation by binding to B7 (CD80/CD86) molecules on antigen‐presenting cells. Programmed death receptor ligand 1 on melanoma cells negatively regulates T‐cell function by binding to the programmed death‐1 (PD‐1) receptor on T cells. Antibodies against CTLA‐4 and PD‐1 also enhance the survival of melanoma patients. In this review, we summarize the clinical effectiveness and adverse events of the BRAF inhibitors, MEK inhibitors and anti‐immune checkpoint antibodies in melanoma treatment.     

A novel surgical margin (1 cm) might be from benefit for patients with dysplastic nevi,thin melanomas,and melanoma in situ: Analysis based on clinical cases

Malignant melanoma is an oncologic disease, whose current management among others includes surgical and immunological therapy. According to the current recommendations of the American Joint Committee on Cancer, the surgical excision of the primary tumor should be performed in two operative sessions, which has several consequences. The following paper will present and discuss six cases of pigmented lesions and the advantages of the one‐step melanoma surgery in their management.     

Sentinel lymph node biopsy for 102 patients with primary cutaneous melanoma at a single Japanese institute

Sentinel lymph node biopsy (SLNB) is a widely accepted standard procedure for patients with clinically localized melanoma. Melanoma prevalence and Clark's subtype differ between Asians and Caucasians. Here, we evaluated our experience on SLNB for cutaneous melanoma in a Japanese population. SLNB was performed for patients with melanoma between July 2000 and June 2014. We retrospectively analyzed 102 patients regarding association of clinicopathological features with sentinel lymph node (SLN) status, melanoma‐specific survival (MSS) and disease‐free survival (DFS). A positive SLN was significantly associated with primary Breslow thickness. Compared with 43 patients with negative SLN, 59 patients with positive SLN had significantly shorter MSS (5‐year survival rate, 94.3% vs 63.2%; P = 0.0002) and DFS (5‐year survival rate, 92.7% vs 63.4%; P = 0.0004). According to our subgroup analyses, nine patients with positive non‐SLN had significantly shorter MSS compared with 32 patients with negative non‐SLN (5‐year survival rate, 32.4% vs 68.5%; P = 0.0273). The survival of 51 Japanese patients with acral lentiginous melanoma (ALM) was not inferior to the survival of patients with other Clark's subtype. Breslow thickness is an important factor for both MSS and DFS, and the status of SLN is the most predictive prognostic factor in Japanese patients with clinically localized melanomas, as in case of Caucasians. Features of ALM may be different between Asians and Caucasians.     

Prognostic value of galectin‐3 in primary cutaneous melanoma

Background Galectin‐3, one of the β‐galactoside‐binding lectins, has been suggested as a marker of disease progression in melanoma patients because of its overexpression observed in recent studies. However, prognostic value of galectin‐3 in primary cutaneous melanoma (PCM) has not been clearly defined. Objectives The aim of the study was to analyse whether the intensity of galectin‐3 expression can predict survival in patients with PMC. Methods Galectin‐3 expression was evaluated using immunohistochemistry in 104 PCM samples, including 71 (68.2%) superficial spreading (SSM) and 33 (31.8%) nodular melanomas (NM). Results Significant difference of galectin‐3 expression between SSM and NM was determined (P < 0.001). Increased galectin‐3 expression was positively correlated with tumour thickness (P < 0.001), Clark (P < 0.001) and Breslow (P < 0.001) stage, mitotic rate (P < 0.001), presence of tumour ulceration (P < 0.001), lymphatic invasion (P = 0.018), positive sentinel lymph node (P < 0.022) and distant metastases (P < 0.001). Kaplan–Meier analysis showed an association between increased galectin‐3 expression and reduced recurrence‐free survival (RFS) (P = 0.001) and reduced disease‐specific survival (DSS) (P = 0.015). In Cox proportional hazards regression analysis, significant predictors of reduced RFS were positive sentinel lymph node (P = 0.025) and lymphovascular invasion (P = 0.021), whereas predictors of DSS were tumour thickness (P = 0.012), lymphovascular invasion (P = 0.047), Clark stage (P = 0.029) and location of tumour on upper extremities (P = 0.024). Conclusions Our results support the potential role of galectin‐3 in PCM development, progression and metastasis. Moreover, galectin‐3 could serve as an additional prognostic marker that might help in further stratifying the risk of disease progression and metastasis in patients with PMC.     

Synergistic effects of interferon‐beta and nivolumab in oral mucosal melanoma

Mucosal melanoma is a rare aggressive cancer with a very poor prognosis. Clinical and pathological characteristics of mucosal melanoma differ from those of cutaneous melanoma and there are no established management guidelines for mucosal melanoma. Complete surgical excision is one of the most effective treatments for localized lesions, while targeted therapies and immunotherapies, such as monoclonal antibodies that target cytotoxic T‐lymphocyte‐associated molecule‐4, and the programmed death (PD)‐1/PD‐ligand 1 pathway inhibitors, are treatment options for unresectable or metastatic lesions. Here, we describe the case of a patient with oral mucosal melanoma with multiple metastases. In our case, local injection of interferon (IFN)‐β with dacarbazine–nimustine–vincristine therapy provided antitumor effects on an invasive tumor on the upper gingiva. Nivolumab therapy produced complete remission of lymph node and bone metastases. In contrast, the remaining in situ portion of oral mucosal melanoma on the hard palate was refractory to IFN‐β monotherapy and nivolumab therapy. However, after administration of nivolumab, peritumoral injection of IFN‐β showed rapid therapeutic effects. Our case suggested that nivolumab upregulated the antitumor effects of IFN‐β, which induced the recruitment of CD8⁺ T cells into the tumor microenvironment contributing to the deletion of tumor cells. Combination therapy of IFN‐β and nivolumab may be a potential treatment option for patients with oral mucosal melanoma.     

Circulating melanoma cells as a potential biomarker to detect metastasis and evaluate prognosis

TNM staging is mainly used to evaluate the prognosis of melanoma patients. Serum biomarkers such as 5‐S‐cysteinyldopa (5‐S‐CD) have occasionally been used but most do not respond until the tumour burden becomes high. Recently, circulating melanoma cells (CMC) have been reported as a possible new biomarker to detect metastasis, monitor treatment response and predict prognosis. The object of this exploratory study was to evaluate the efficacy of CMC to detect metastasis and predict prognosis by cross‐sectional and prospective observational analyses, respectively. Altogether 15 patients with stages II–IV melanoma were enrolled and CMC were enumerated by CellSearch system with cut‐off values of two cells/7.5 mL. Serum 5‐S‐CD and lactate dehydrogenase (LDH) were also measured. The sensitivity of CMC and 5‐S‐CD for the detection of metastasis was 33 and 50%, respectively. The combination of CMC and 5‐S‐CD showed a sensitivity of 67%, the best performance among CMC, 5‐S‐CD, LDH and any combination of two of the markers. Additionally, a 30‐month prospective observation showed that CMC could segregate patients with poorer prognosis. The median survival time for the patients with <2 CMC and those with ≥2 CMC was 19.5 and 4.5 months, respectively. The limitation of this study is the small sample size. These preliminary results indicate CMC may complement the efficacy of 5‐S‐CD to detect metastasis and can be a prognostic marker. Although there is still room for improvement to maximise the sensitivity, the CellSearch system is reproducible, standardised and suitable for multi‐centre studies.     

Is Mcl‐1L the new anti‐apoptotic effector of B‐RAFV600E in melanoma?

A recent report has unveiled a novel mechanism by which oncogenic BRAF signalling might trigger apoptotic resistance in melanoma by selectively affecting the expression of Bcl‐2 family member Mcl‐1L (Exp Dermatol 2013: 22 : 767). Correlation of Mcl‐1 splice variants and B‐RAF mutational status was determined in a panel of melanoma cell lines. In vivo validation of this mechanism, which is supported by recent literature, might provide novel therapeutic approaches such as the use of targeted Mcl‐1 inhibitors to improve outcome in melanoma.     

Interdisciplinary management recommendations for toxicity associated with interferon‐alfa therapy

Adjuvant interferon‐α (IFN‐α) therapy in patients with melanoma has been established as standard therapy since more than 10 years.During IFN‐α therapy, flu‐like symptoms, gastrointestinal disorders, arthralgias and neuropsychiatric symptoms are the most common side effects. The management and prophylaxis of these side effects have been improved by a more detailed understanding of pathophysiologic mechanisms and increased clinical experience. New insights in the relevance of detection of autoantibodies and development of autoimmunity have influenced the clinical pathway substantially. This review covers the pathomechanisms, incidence and optimized therapy of IFN‐α‐associated side effects.     

Circulating CLA+ T lymphocytes as peripheral cell biomarkers in T‐cell‐mediated skin diseases

T lymphocytes expressing the CLA antigen constitute a subset of effector memory lymphocytes that are functionally involved in T‐cell‐mediated cutaneous diseases. Skin‐seeking lymphocytes recirculate between inflamed skin and blood during cutaneous inflammation. Many studies in different T‐cell‐mediated inflammatory cutaneous diseases have clearly related their pathologic mechanisms to CLA+ T cells. Based on common features of these cells in different cutaneous disorders mediated by T cells, we propose that circulating CLA+T cells could constitute very useful peripheral cellular biomarkers for T‐cell‐mediated skin diseases.     

Efficacy of combined radiotherapy and anti‐programmed death 1 therapy in acral and mucosal melanoma

Some studies showed that clinical response to immune check point inhibitors is lower in acral and mucosal melanoma than in cutaneous melanoma. Although the synergistic effect of radiotherapy (RT) and ipilimumab has been reported in patients with brain metastasis, the efficacy of combined RT and anti‐programmed death 1 (PD‐1) therapy for acral and mucosal melanoma is unclear. The present study aimed to evaluate the efficacy of combined RT and anti‐PD‐1 therapy for acral and mucosal melanoma. We retrospectively analyzed patients with acral or mucosal melanoma who were treated with anti‐PD‐1 and RT at Sapporo Medical University Hospital. In 10 patients (acral, 3; mucosal, 7), the response rate (RR) and the disease control rate (DCR) were 40% and 60%, respectively. As regards mucosal melanoma, four of the seven patients had achieved complete response + partial response, and three had progressive disease (RR = 57.1%). Meanwhile, two of the three patients with acral melanoma had stable disease and one had progressive disease (RR and DCR were 0% and 66.6%, respectively). Except for the patients treated with palliative RT for bone metastasis in the present study, the RR was 50% (4/8 patients), and the DCR was 75% (6/8 patients). Vitiligo developed after RT in five (50%) patients at a median duration of 2 months after RT. The clinical response and the high occurrence of vitiligo suggest that the combination of RT and anti‐PD‐1 therapy could be effective in some patients with mucosal melanoma.     

Prognostic signs in melanoma: state of the art

Prognoses for melanoma patients are currently based on statistically confirmed parameters, above all the Breslow thickness and number of lymph node and/or distant metastases. However, metastases can develop even with "thin" melanomas (< 0.7 mm), while survival has been recorded in patients with tumours classified as "thick" (> 4 mm). This review of the literature examines the most recent advances in prognostic markers for melanoma (serological, immunohistochemical, histological, genetic and surgical). These markers offer interesting possibilities in terms of diagnostic certainty, identification of early growth phases and estimation of the tumour's potential for progression and metastasis. It is reasonable to assume that their combined use can provide useful information for formulating prognoses that are not only statistically valid but also individualized.