Recent treatment and prognosis in 643 patients with extramammary Paget's disease.

Information about extramammary Paget's (EMPD) treatment is limited because of the rarity of the disease. The prognosis differs between in situ EMPD and invasive EMPD; therefore, therapy should be planned according to the disease stage. We collected data on 643 EMPD cases treated between 2015 and 2019 in Japan and assessed recent trends in EMPD treatment and prognosis based on the EMPD-oriented TNM staging. Among the 643 patients, 317 had stage 0 (49.3%), 185 had stage I (28.8%), 51 had stage II (7.9%), 18 had stage IIIA (2.8%), 48 had stage IIIB (7.5%) and 24 had stage IV (3.7%) disease. Each stage showed a distinct survival curve, with the exception of stages II and IIIA. Curative surgery was most common in patients with stage 0-III disease. Chemotherapy was the first-line therapy, mainly in patients with stage IIIB and IV disease, most commonly with docetaxel (DTX), followed by DTX + tegafur gimeracil oteracil potassium (TS-1) and TS-1. Patients with local disease exhibited a 4.4% recurrence rate. Univariate analysis revealed no prognostic differences according to age, sex or primary tumour site. SLNB was not related to disease-specific survival. In multivariate analysis, female sex significantly predicted local relapse in stage 0-I (HR 3.09; 95% CI, 1.13-8.43), and initial treatment with curative surgery was significantly protective in terms of disease-specific survival in stage II-IIIA (HR, 0.17; 95% CI, 0.04-0.71) and stage IIIB-IV (HR 0.16; 95% CI, 0.05-0.51). Further clinical studies are needed to improve the prognosis of patients with stage II-IV EMPD.

Future prospects for cancer immunotherapy using induced pluripotent stem cell-derived dendritic cells or macrophages.

Cancer immunotherapy is now the first-line treatment for many unresectable cancers. However, it remains far from a complete cure for all patients. Therefore, it is necessary to develop innovative methods for cancer immunotherapy, and immune cell therapy could be an option. Currently, several institutions are attempting to generate immune cells from induced pluripotent stem cells (iPSCs) for use in cancer immunotherapy. A method for generating dendritic cells (DCs) and macrophages (MPs) from iPSC has been established. iPSC-derived DCs (iPS-DCs) can activate T cells via antigen presentation, and iPSC-derived macrophages (iPS-MPs) attack cancer. Since iPSCs are used as the source, genetic modification is easy, and various immune functions, such as the production of anti-tumour cytokines, can be added. Furthermore, when iPS-DCs and iPS-MPs are immortalized, cost reduction through mass production is theoretically possible. In this review, the achievements of cancer research using iPS-DCs and iPS-MPs are summarized, and the prospects for the future are discussed.

Immunotherapy with 4-1BBL-Expressing iPS Cell-Derived Myeloid Lines Amplifies Antigen-Specific T Cell Infiltration in Advanced Melanoma.

We have established an immune cell therapy with immortalized induced pluripotent stem-cell-derived myeloid lines (iPS-ML). The benefits of using iPS-ML are the infinite proliferative capacity and ease of genetic modification. In this study, we introduced 4-1BBL gene to iPS-ML (iPS-ML-41BBL). The analysis of the cell-surface molecules showed that the expression of CD86 was upregulated in iPS-ML-41BBL more than that in control iPS-ML. Cytokine array analysis was performed using supernatants of the spleen cells that were cocultured with iPS-ML or iPS-ML-41BBL. Multiple cytokines that are beneficial to cancer immunotherapy were upregulated. Peritoneal injections of iPS-ML-41BBL inhibited tumor growth of peritoneally disseminated mouse melanoma and prolonged survival of mice compared to that of iPS-ML. Furthermore, the numbers of antigen-specific CD8+ T cells were significantly increased in the spleen and tumor tissues treated with epitope peptide-pulsed iPS-ML-41BBL compared to those treated with control iPS-ML. The number of CXCR6-positive T cells were increased in the tumor tissues after treatment with iPS-ML-41BBL compared to that with control iPS-ML. These results suggest that iPS-ML-41BBL could activate antigen-specific T cells and promote their infiltration into the tumor tissues. Thus, iPS-ML-41BBL may be a candidate for future immune cell therapy aiming to change immunological "cold tumor" to "hot tumor".

Myasthenic crisis and polymyositis induced by one dose of nivolumab.

An 80-year-old man, who developed multiple lymph node and skin metastasis of malignant melanoma, received nivolumab monotherapy. Two weeks after the first dose, he experienced anorexia and fatigue, and suffered from progressive, severe dyspnea and muscle weakness. We diagnosed him with myocarditis, myositis, and myasthenic crisis induced by nivolumab. We commenced steroid therapy, immune absorption therapy, plasma exchange therapy, and i.v. immunoglobulin therapy, and succeeded in saving his life. Because his serum level of anti-acetylcholine receptor antibodies in a sample collected before nivolumab treatment were positive and were elevated significantly after nivolumab, we suspected that nivolumab triggered a severe autoimmune response, which progressed subclinical myasthenia gravis to myasthenic crisis. We carried out T cell receptor repertoire analysis using next-generation sequencing technologies and identified infiltration of clonally expanded T cell populations in the skeletal muscle after nivolumab treatment, implying a very strong T cell immune response against muscular cells. To avoid severe immune-related adverse events, the exclusion of patients with subclinical autoimmune disease is very important for treatment with immune checkpoint inhibitors.

The proportion of lymphocytic inflammation with CD123-positive cells in lupus erythematous profundus predict a clinical response to treatment.

Lupus erythematosus profundus is a rare inflammatory disorder of subcutaneous fat in patients with lupus ery-thematosus. Previous reports suggested that plasmacytoid dendritic cells, which expressed CD123 and CD303 antigens, play a central proinflammatory role in the patho-genesis of lupus erythematosus. To find the factors that determine the response to treatment, we analysed 23 skin specimens from the patients with lupus erythematosus profundus. The patients with considerable lymphocytic inflammation with high percentages of CD123+ cells in dermis and subcutaneous fat significantly responded to the systemic corticosteroid therapies. On the other hand, the patients with minor lymphocytic inflammation with low percentages of CD123+ cells showed poor response to treatments. The mean percentage of CD123+ cells in patients who showed good response to therapy was significantly higher than those that showed poor response (p = 0.027). These results suggest that the clinical response to treatment of lupus erythematosus profundus could be predicted from the histological features.

Metastasis to hypopharynx from epidermotropic metastatic malignant melanoma.

Previous reports proposed the concept and criteria of epidermotropic metastatic malignant melanoma (EMMM): (a) dermal involvement equal to or broader than the epidermal involvement, (b) atypical melanocytes within the dermis, (c) thinning of the epidermis, (d) widening of the papillary dermis with an epithelial collarette, and (e) vascular invasion of atypical melanocytes. However, it remains unclear whether EMMM also involves the mucosal epithelium. In this case, the patient was diagnosed with EMMM based on the histopathological findings of the patient's multiple skin lesions and clinical course. The patient also developed metastasis to the hypopharynx. Although histopathological findings of the lesion suggested the possibility of melanoma in situ, as the lesion included atypical melanocytes in the mucosal epithelium, the clinical course supported the diagnosis of hypopharyngeal metastasis from EMMM. This case suggests that EMMM may have epitheliotropic features not only in the skin but also in the mucosa.

Deciphering the role of adjuvant therapy in melanoma and its actual benefits.

Numerous clinical trials have demonstrated a significant improvement in recurrence-free survival among melanoma patients receiving high-dose interferon-α, immune checkpoint inhibitors (pembrolizumab, nivolumab), and BRAF/MEK inhibitors (dabrafenib-trametinib). This study aimed to investigate whether these findings hold true in real-world conditions for patients with stage III and IV melanoma. In particular, the study explores the efficacy and side effects of adjuvant therapies, focusing on anti-PD-1 antibodies and BRAF/MEK inhibitors. While clinical trials have shown comparable efficacy, differences in side-effect profiles, especially the persistence of immune-related adverse events with anti-PD-1 antibodies, highlight the need for careful consideration in adjuvant settings. In the absence of established biomarkers for guiding adjuvant therapy decisions, it becomes imperative to transparently communicate the advantages and disadvantages of drug administration to patients. The study also delved into the impact of melanoma subtype and BRAF mutation status on the effectiveness of adjuvant therapy, emphasizing the need for further investigation.

Serum levels of leptin receptor in patients with malignant melanoma as a new tumor marker.

Leptin is known to be abnormally expressed in a variety of cancers, and leptin receptors have been reported to be expressed on human melanoma cells. In this study, we evaluated the possibility that the serum levels of leptin receptor could be a tumor marker of malignant melanoma (MM). Serum samples were obtained from 71 patients with MM, and the serum levels of leptin receptor were measured by double-determinant ELISA. Interestingly, serum levels of leptin receptor decreased gradually with the stages of MM, being highest at in situ and lowest at stage IV. There was also a trend of reverse correlation between tumor thickness and serum levels of leptin receptor. To our knowledge, this is the first report investigating the serum levels of leptin receptor in MM, and serum leptin receptor levels may be used as a useful tumor marker of MM.

Intertumor and intratumor heterogeneity of PIK3CA mutations in extramammary Paget's disease.

Although the prognosis of patients with extramammary Paget's disease (EMPD) treated with radical resection is good, the prognosis of EMPD with distant metastasis is very poor. PIK3CA mutations predict a good response to PIK3CA inhibitors. The aim of this study was to investigate the occurrence rate of PIK3CA mutations (including multiple mutations [MM]) related to the intertumor and intratumor heterogeneity in EMPD and to evaluate the correlation between these mutations and clinical parameters of EMPD. We performed droplet digital polymerase chain reaction to detect PIK3CA mutations (E542K, E545K, H1047R, and MM) in 68 patients with EMPD. In addition, we investigated the presence of PIK3CA mutations at multiple sites in 16 patients with PIK3CA mutations to assess the intratumor heterogeneity of PIK3CA mutations in EMPD. The frequency of one or more PIK3CA mutations in patients with EMPD was 30.8% (21/68). The frequency of E542K, E545K, H1047R, and MM were 10.2% (7/68), 13.2% (9/68), 11.7% (8/68), and 4.4% (3/68), respectively. No significant correlation was found between PIK3CA mutation patterns and clinical parameters. Of the 21 patients with PIK3CA mutations, 16 with tissue samples that could be analyzed at multiple sites were examined. The proportion of patients with the same PIK3CA mutations at all sites was 12.5% (2/16). The proportion of patients with the same PIK3CA mutations at least two or more sites, but not at all sites, was 31.2% (5/16). The proportion of patients with no PIK3CA mutations at other sites was 37.5% (6/16). The proportion of patients with other PIK3CA mutations at other sites was 18.7% (3/16). There is intertumor and intratumor heterogeneity of PIK3CA mutations. PIK3CA mutations in EMPD may be progressor mutations in EMPD.

An Autopsy Case of Metastatic Melanoma Originating from the Nasal Cavity That Was Treated With Nivolumab and Ipilimumab.

Background: Recent developments in antibodies targeting checkpoint molecules have improved the overall survival of patients with melanoma. Case Report: A case of metastatic melanoma was treated with antibodies to cytotoxic T-lymphocyte-associated protein 4 and programmed cell death protein 1. Stable disease was achieved but the patient died from systemic metastasis 23 months after the diagnosis of melanoma. An autopsy was performed, and immunohistochemical analysis was carried out using primary melanoma (pre-treatment) and autopsy (post-treatment) samples. The down-regulation of human leukocyte antigen class I and II, melanin, and melanoma antigens was seen in the post-treatment tumor cells. Tumor-infiltrating lymphocyte numbers were significantly reduced in the post-treatment tumor microenvironment. Although programmed death ligand 1 expression was seen in the pre-treatment tumor tissues, it was not seen in the post-treatment tumor tissues. Conclusion: A phenotypical change in the tumor cells was suggested to be associated with the resistance to immune checkpoint inhibitor therapy.

Nucleosome assembly protein 1-like 4, a new therapeutic target for proliferation and invasion of melanoma cells.

BACKGROUND: Melanoma is one of the deadliest skin cancers. The treatment of advanced melanoma has been dramatically improved by immune checkpoint inhibitors and targeted therapies. However, many patients still do not respond to these therapies. OBJECTIVE: To investigate whether NAP1L4 can be a new therapeutic target for melanoma. METHODS: Immunohistochemical analysis of human nevus and melanoma tissues was performed. Real-time RT-PCR and immunoblotting were performed using human samples and melanoma cell lines. Next, we examined the effect of NAP1L4 knockdown in melanoma cell lines using cell migration and invasion assays. To investigate the molecular mechanism related to these results, immunoblotting of p21 and Slug was examined. MMP-2 and MMP-9 activity assays were also performed. Further, pathway analysis between NAP1L4 and MMP-2 was performed. Finally, the effects of NAP1L4 knockdown on cell proliferation, apoptosis, and cell cycle were analyzed. RESULTS: NAP1L4 was overexpressed in melanoma tissues compared to the nevus tissue. NAP1L4 knockdown reduced melanoma cell migration and invasion. NAP1L4 knockdown upregulated p21 and downregulated Slug expression in melanoma cells. NAP1L4 knockdown decreased the active levels of MMP-2 in the supernatant from melanoma cells. NAP1L4 knockdown inhibited apoptosis in camptothecin-induced DNA damage, induced cell cycle arrest at the G1/S phase, and inhibited cell proliferation. CONCLUSIONS: NAP1L4 may play a role in cell migration and invasion in melanoma cells through the regulation of Slug. We propose that NAP1L4 can be a new therapeutic target for proliferation and invasion of melanoma cells.

A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells.

Turning non-inflamed (cold) tumors into inflamed (hot) tumors is important for maximizing the effect of immune checkpoint inhibitors (ICIs) against malignancies. We showed that lactate, a product of the Warburg effect, inhibited the efficacy of ICIs and suppressed IL-12 p40 expression in dendritic cells (DCs) through reducing NF-κB p65, p50, and c-Rel DNA-binding activity to the IL-12 p40 promoter. Additionally, lactate promoted the expression of early growth response protein 1 (EGR1), whose expression was increased in human invasive melanoma compared with non-invasive melanoma. We also found that EGR1 interacts with serum response factor (SRF) and represses the expression of CD80 in DCs. These findings suggest that lactate and its induced EGR1 are key factors that turn hot tumors into cold tumors and may represent targets in cancer treatment with ICIs.

Liquid biopsy-based analysis by ddPCR and CAPP-Seq in melanoma patients.

BACKGROUND: The development of BRAF/MEK inhibitors in patients with metastatic melanoma harboring BRAF mutations has garnered attention for liquid biopsy to detect BRAF mutations in cell-free DNA (cfDNA) using droplet digital PCR (ddPCR) or next-generation sequencing methods. OBJECTIVE: To investigate gene mutations in tumor DNA and cfDNA collected from 43 melanoma patients and evaluate their potential as biomarkers. METHODS: ddPCR and CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) techniques were performed to detect gene mutations in plasma cfDNA obtained from patients with metastatic melanoma. RESULTS: Gene variants, including BRAF, NRAS, TP53, GNAS, and MET, were detectable in the plasma cfDNA, and the results were partially consistent with the results of those identified in the tissues. Among the variants examined, copy numbers of MET mutations were consistent with the disease status in two melanoma patients. CONCLUSION: Liquid biopsy using CAPP-Seq and ddPCR has the potential to detect tumor presence and mutations, especially when tissue biopsies are unavailable. MET mutations in cfDNA may be a potential biomarker in patients with metastatic melanoma.

Cytomegalovirus Enterocolitis in a Patient with Refractory Immune-Related Colitis.

Although most immune-related adverse events (irAEs) secondary to immune checkpoint inhibitors can be managed with immunosuppressive therapies; they can induce reactivation of infectious diseases, including cytomegalovirus (CMV). Here, we show a case of CMV enterocolitis during steroid therapy for an irAE. A 77-year-old man with unresectable malignant melanoma was treated with ipilimumab. He suffered from immune-related colitis (irColitis) and was treated with methylprednisolone. Although corticosteroids initially improved his symptoms, CMV reactivation occurred and colitis was exacerbated. Antiviral therapy improved his symptoms without augmenting the immunosuppressive therapy. CMV colitis should be considered when a patient with irColitis shows resistance to immunosuppressive therapy.

Methyl-CpG binding domain protein 3: a new diagnostic marker and potential therapeutic target of melanoma.

Methyl-CpG binding domain protein 3 (MBD3) belongs to the methyl-CpG binding protein family. MBD3 facilitates the initiation of neural stem cell reprogramming. Melanoma originates in melanocytes derived from neural crest stem cells; therefore, we investigated the role of MBD3 in melanoma. MBD3 was overexpressed in melanoma compared with pigmented nevi. MBD3 knockdown had no effect on the proliferation of melanoma cells (A375 and A2058 cells). Contrarily, it significantly reduced the migration and invasion of A375 cells, but had no significant effect on A2058 cells. Furthermore, MBD3 knockdown reduced N-cadherin protein levels and matrix metalloproteinase-2 (MMP-2) activity in A375 cells, but had no significant effect on A2058 cells. Based on these results, the MBD3 expression level may be a useful biomarker for the diagnosis of melanoma. Thus, MBD3 has potential as a novel therapeutic target for some melanoma patients.

Matrin-3 plays an important role in cell cycle and apoptosis for survival in malignant melanoma.

BACKGROUND: A previous study revealed that matrin-3 is an essential component in maintaining fibroblast growth factor 2 (FGF2)-mediated undifferentiation of neural stem cells (NSCs) using a proteomics approach. Malignant melanoma (MM) arises from melanocytes that originate from neural crest stem cells during development. Additionally, it has been reported that the expression of FGF2 is positively correlated with the progression of MM. OBJECTIVE: We expected that matrin-3, as a downstream component of FGF2, might be associated with the aggressiveness or differentiation of MM. METHODS: Matrin-3 expression was measured in human melanoma patient tissues and human MM cell lines. We analyzed the effect of matrin-3 siRNA on the proliferation of human MM cell lines and focused on cell cycle progression and apoptosis. We carried out in vivo xenograft tumor experiments by implanting A375 cells transfected with matrin-3 shRNA. RESULTS: Matrin-3 was highly expressed in MM, and matrin-3 knockdown inhibited the proliferation of melanoma cellsin vivo and in vitro. Furthermore, we found that matrin-3 knockdown led to an accumulation of cells in the G1 phase and an increase in apoptotic cell number. CONCLUSION: Our results suggest that matrin-3 could be a new therapeutic target for the treatment of MM.

Circulating tumor necrosis factor-α DNA are elevated in psoriasis.

In psoriasis, tumor necrosis factor (TNF)-α is a key pro-inflammatory cytokine that activates keratinocytes to produce other inflammatory mediators. In addition, increased serum or plasma TNF-α levels are considered to be biomarkers of psoriasis. Circulating cell-free DNA (cfDNA) originates from apoptotic or necrotic cells and reflects the severity of cellular damage. Although cfDNA has recently attracted attention as a marker in the diagnosis and prognosis of various disorders, there are few reports of its clinical implications in the field of dermatology including psoriasis. The aim of this study was to investigate whether the TNF-α gene is present in the cfDNA, and whether its levels can be utilized as a biomarker for patients with psoriasis. cfDNA was isolated from serum samples of 79 patients with psoriasis vulgaris and 29 with psoriatic arthritis. The levels of TNF-α in the cfDNA were assessed by droplet digital polymerase chain reaction. In this study, we made two novel findings. First, circulating TNF-α DNA levels in the cfDNA were significantly higher in patients with psoriasis than in healthy controls. In addition, the area under the curve was 0.91, suggesting that serum TNF-α DNA levels are effective as a diagnostic biomarker. Second, the levels of TNF-α DNA copies in the cfDNA were positively correlated with the Psoriasis Area and Severity Index (PASI) score in the group of patients with a PASI score higher than 10. Generally, a PASI score of more than 10 is defined as severe psoriasis; therefore, the levels of TNF-α DNA copies in the cfDNA could be a biomarker for severity in patients with severe psoriasis. Further studies are needed to establish serum TNF-α DNA levels as a novel biomarker of psoriasis.

Case series of cutaneous T-cell lymphomas treated with bexarotene-based therapy.

Bexarotene is useful for both early and advanced cutaneous T-cell lymphoma (CTCL), and is sometimes applied to ultraviolet-tolerant early CTCL patients as one of the first-line therapies in the real world. However, continuous administration of bexarotene is sometimes difficult because of its adverse events (AE). Development of an appropriate protocol for bexarotene that can induce a consistent response for CTCL without severe AE (SAE) is needed. We retrospectively investigated 29 Japanese cases of CTCL and evaluated the efficacy of treatment and incident ratios of all AE and SAE. Objective response rate (ORR) for the overall cohort was 65.5%. ORR of the 300 mg/m2 cohort (conventional dose) was 76.2%, while that of the 150-300 mg/body (low dose) with narrowband ultraviolet B light (NBUVB) cohort was 37.5%. Mean event-free survival was 10.0 months for all patients, 6.7 months for the bexarotene conventional-dose cohort and 19.1 months for the low-dose with NBUVB cohort. The incident ratio of total SAE for all patients was 20.7%. The incident ratio of total SAE was 23.8% for the conventional-dose cohort and 12.5% for the low-dose with NBUVB cohort. Our present study suggests that low-dose bexarotene plus NBUVB therapy is well-tolerated and could be one of the optimal therapies for advanced CTCL.