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.

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.

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