Background Extramammary Paget's disease (EMPD) is a rare malignant skin cancer. One of the hallmarks of cancers, including EMPD, is an enhancement of aerobic glycolysis, which is also known as the Warburg effect. In the last step of glycolysis, the enzyme lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactic acid, the accumulation of which contributes to the creation of an acidic tumour microenvironment. This in turn results in immunosuppression in various types of cancers. However, the contribution of these pathways has not been well-studied in EMPD. Objective To investigate the significance of the Warburg effect and its contribution to the tumour immune microenvironment in EMPD. Methods The mRNA expression levels of molecules involved in glycolysis and immune-related cytokines were examined by ddPCR. The number of immune cells was assessed by immunohistochemistry (IHC). Results The levels of two glycolytic enzymes, HK2 and LDHA, in tumour tissues were significantly increased compared to those in paired-normal tissues. IHC analyses revealed increased numbers of PD-L1 + , PD-1 + , CD163 + M2 macrophages, Iba1 + macrophages and Foxp3 + Tregs that were associated with high LDHA levels in EMPD. ddPCR demonstrated that multiple cytokines including IL-4, IL-6, IL-10, TGF-b and CCL-2 were upregulated and associated with high LDHA levels in EMPD. Statistical analyses showed that IL-6 mRNA expression correlated with the number of CD163 + , Iba-1 + and Foxp3 + cells. Conclusion The Warburg effect contributes to immunomodulation in the tumour microenvironment and further elucidation may lead to better understanding of the pathogenesis of EMPD.

Background: Angiosarcoma is a rare malignant tumor with a poor prognosis. It is known that hypoxic condition activates tumor progression in several cancers. Additionally, hypoxic tumor microenvironment accelerates immune escape. However, the presence and significance of hypoxia in angiosarcoma has not been adequately investigated. Objective: To study the role of hypoxia in the progression of angiosarcoma. Methods: The protein level of hypoxia inducible factor-1α (HIF-1α) in angiosarcoma was examined using immunohistochemistry and immunoblotting. To study the effect of hypoxia on tumor progression, cell proliferation, migration, invasion, and tube formation assays were performed in angiosarcoma cells. The influence of tumor cell supernatant in hypoxia from angiosarcoma cells on immune escape and angiogenesis was analysed to investigate the modulatory effect of hypoxia on tumor microenvironment of angiosarcoma. The molecular mechanism related to these results was investigated using immunoblotting and real time RT-PCR. Results: HIF-1α protein was over-expressed in angiosarcoma tissues and cell lines under hypoxic conditions, and there was heterogeneity of oxygen supply in angiosarcoma. Hypoxia enhanced the proliferation, migration, and invasion abilities and inhibited tube formation in angiosarcoma cells. Tumor cell supernatant in hypoxia from angiosarcoma cells activated the monocyte invasion ability, facilitated its differentiation into M2-like macrophages, and suppressed cell-adhesion. These in vitro results were compatible to the pathological findings of angiosarcoma patients. Conclusion: Hypoxia plays a major role in progression of angiosarcoma cells by enhancing cell proliferation, migration, and invasion and by modulating the tumor microenvironment.

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.

Summary Background Although carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA) are useful markers for extramammary Paget disease (EMPD), serum CEA and CYFRA levels are not elevated in most patients with EMPD without metastasis. Cell‐free (cf)DNA has attracted attention as an indicator of clinical conditions in several cancers. Objectives To identify further useful biomarkers for the detection of EMPD, including early lesions, and to study the clinical implications of cfDNA in EMPD. Methods cfDNA were isolated from serum of patients with EMPD with and without metastasis, and from healthy volunteers. Serum extracts were amplified using polymerase chain reaction. Results Serum cfDNA levels were significantly elevated in patients with EMPD with or without metastasis compared with those in healthy controls. Serum cfDNA was a better diagnostic marker for the presence of EMPD than serum CYFRA. Moreover, the postoperative serum cfDNA levels were significantly lower than those from the preoperative samples, and the change in serum cfDNA levels reflected the clinical courses of patients with EMPD treated with chemotherapy. Conclusions Taking the evidence together, serum cfDNA levels may be a useful marker for diagnosis and disease progression in EMPD. What's already known about this topic? Serum levels of carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA) are not elevated in most patients with extramammary Paget disease (EMPD) without metastasis. Cell‐free (cf)DNA has attracted attention as an indicator of clinical conditions in several cancers. There are few reports of the clinical implications of cfDNA in dermatology. What does this study add? Serum cfDNA levels were significantly elevated in patients with EMPD with or without metastasis compared with those in healthy controls. Postoperative serum cfDNA levels were significantly lower than those from the preoperative samples. Changes in serum cfDNA levels reflected the clinical courses of patients with EMPD treated with chemotherapy. What is the translational message? Serum cfDNA levels in patients with EMPD are a useful marker for the detection of EMPD, including localized EMPD. Changes in serum cfDNA levels in an individual patient may reflect the clinical course of EMPD.

Cyclin‐dependent kinase 4 and 6 (CDK4/6) plays an important role in cell cycle progression, and the CDK4/6–cyclin D1 complex controls the cell cycle transition from G1 phase to S phase. CDK4 is enhanced in several types of cancers and CDK4/6 inhibitors attenuate the proliferation of several types of cancer in vitro/in vivo. The purpose of our study was to investigate the expression pattern of CDK4 and evaluate its clinical importance in extramammary Paget's disease (EMPD). Almost all EMPD tissues were positive for CDK4, and metastatic lesions had a similar immunostaining intensity to primary lesions. In addition, CDK4 protein levels were positively correlated with those of cyclin D1 protein. Taken together, CDK4 may assume a crucial role in EMPD progression.