Genetic intratumour heterogeneity and clonal evolution in extramammary Paget's disease.

All tumour cells in a patient have shared and non-shared genetic alterations, and the diversity of mutations is described as intratumoural heterogeneity (ITH). Multiregion sequencing is a genome sequencing analytical technique used for multiple, spatially-separated biopsy tissues that may further our understanding of ITH and tumour evolution. Although genetic mutations in extramammary Paget's disease (EMPD) have recently been detected by next-generation sequencing analysis, there have been no reports of ITH based on multiregion sequencing in EMPD. Thus, we clarified the landscape of ITH and tumour evolution in EMPD. We performed whole-exome sequencing on 35 tissues (30 tumour tissues and five normal skin samples as a paired control), collected from five patients with EMPD. The rate of private mutations was significantly higher than that of ubiquitous and shared mutations. Ubiquitous mutations were not present in driver genes, and most driver genes exhibited private and shared mutations. The most frequent base substitution was C>T in almost all lesions, and most mutational signatures corresponded to signature 1, 2, 3, and 8. The types of proposed aetiology in most lesions were based on age and AID/APOBEC family and BRCA1/BRCA2 mutations. Evolutionary trees were characterized by short trunks and long branches due to the extremely high ratio of private mutations. In contrast, pathogenic factors, such as base substitutions, mutational signatures, and proposed aetiology, were shared. Tumour evolution in EMPD appears to be characterized by a high level of genetic ITH with shared background factors.

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.

ANGPTL2-mediated epigenetic repression of MHC-I in tumor cells accelerates tumor immune evasion.

Loss or downregulation of major histocompatibility complex class I (MHC-I) contributes to tumor immune evasion. We previously demonstrated that angiopoietin-like protein 2 (ANGPTL2) promotes tumor progression using a Xp11.2 translocation renal cell carcinoma (tRCC) mouse model. However, molecular mechanisms underlying ANGPTL2 tumor-promoting activity in the tRCC model remained unclear. Here, we report that ANGPTL2 deficiency in renal tubular epithelial cells slows tumor progression in the tRCC mouse model and promotes activated CD8+ T-cell infiltration of kidney tissues. We also found that Angptl2-deficient tumor cells show enhanced interferon γ-induced expression of MHC-I and increased susceptibility to CD8+ T-cell-mediated anti-tumor immune responses. Moreover, we provide evidence that the ANGPTL2-α5ß1 integrin pathway accelerates polycomb repressive complex 2-mediated repression of MHC-I expression in tumor cells. These findings suggest that ANGPTL2 signaling in tumor cells contributes to tumor immune evasion and that suppressing that signaling in tumor cells could serve as a potential strategy to facilitate tumor elimination by T-cell-mediated anti-tumor immunity.

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.

A Japanese case of melanoma of unknown origin with a rare BRAFV600R mutation was successfully treated with BRAF/MEK inhibitors.

Combination therapy with BRAF and MEK inhibitors (BRAF/MEKi) has shown significantly prolonged progression-free survival (PFS) and overall survival (OS) for BRAF mutated melanoma. Over 90% of the activating mutations are BRAFV600E or BRAFV600K changes. There are no reports of BRAFV600R in Japanese patients with melanoma. The third most common BRAF mutation is BRAFV600R. In this case, we detected the BRAFV600R mutation with FoundationOne CDx in a Japanese patient with melanoma.. The patient was treated with BRAF/MEKi and maintained stable disease status for 1 year.

Promising Blood-Based Biomarkers for Melanoma: Recent Progress of Liquid Biopsy and Its Future Perspectives.

OPINION STATEMENT: Because the recent success of novel therapeutic approaches has dramatically changed the clinical management of melanoma, less invasive and repeatable monitoring tools that can predict the disease status, drug resistance, and the development of side effects are increasingly needed. As liquid biopsy has enabled us to diagnose and monitor disease status less invasively, substantial attention has been directed toward this technique, which is gaining importance as a diagnostic and/or prognostic tool. It is evident that microRNA, cell-free DNA, and circulating tumor cells obtained via liquid biopsy are promising diagnostic and prognostic tools for melanoma, and they also have utility for monitoring the disease status and predicting drug effects. Although current challenges exist for each biomarker, such as poor sensitivity and/or specificity and technical problems, recent technical advances have increasingly improved these aspects. For example, next-generation sequencing technology for detecting microRNAs or cell-free DNA enabled high-throughput analysis and provided significantly higher sensitivity. In particular, cancer personalized profiling by deep sequencing for quantifying cell-free DNA is a promising method for high-throughput analysis that provides real-time comprehensive data for patients at various disease stages. For wide clinical implementation, it is necessary to increase the sensitivity for the markers and standardize the assay procedures to make them reproducible, valid, and inexpensive; however, the broad clinical application of liquid biopsy could occur quickly. This review focuses on the significance of liquid biopsy, particularly related to the use of blood samples from patients with melanoma, and discusses its future perspectives.

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.

Analysis of microsatellite instability using Promega panel in dermatofibrosarcoma protuberans.

Dermatofibrosarcoma protuberans (DFSP) is a rare neoplasm derived from fibroblasts. Although the frequency of microsatellite instability (MSI) in skin cancer is reported to be less than 5%, there is only one report of the status of MMR in DFSP. The only analytical report of microsatellite stability in which Promega panel is not used, showed that the frequency of MSI-high, MSI-low and microsatellite stable (MSS) cases was 13.9% (5/36), 16.7% (6/36) and 69.4% (25/36), respectively. Thus, the aim of this study was to evaluate the status of MMR in 36 patients with DFSP diagnosed at Kumamoto University. MSI analysis using the Promega panel showed that all cases were MSS, which indicated the absence of MSI in DFSP. This result indicates that the status of MMR may not be useful for the potential therapeutic application of pembrolizumab and the pathogenesis of DFSP may not involve MSI.

A protocol for quantifying lymphocyte-mediated cytotoxicity using an impedance-based real-time cell analyzer.

Current standard assays to analyze lymphocyte-mediated antitumor cytotoxicity employ radioisotopic or fluorescent labels. However, such assays are not suitable for real-time analysis. Here we describe a protocol that facilitates the analysis of lymphocyte-mediated toxicity using a label-free, impedance-based real-time cell analyzer. This analyzer measures cellular electrical impedance, expressed as the cell index value, noninvasively and continuously. In contrast with label-dependent assays, this protocol simultaneously generates real-time killing curves useful for quantifying lymphocyte-mediated cytotoxicity in real time. For complete details on the use and execution of this protocol, please refer to Kanemaru et al. (2021).

Questionnaire Survey Regarding Troubles and Concerns Related to Clinical Research Based on the Clinical Trial Act for Clinicians and Academics.

The Clinical Trial Act came into force in 2018 in Japan. A questionnaire survey was conducted with personnel at Kumamoto University Hospital engaged in research and development, to explore their perceptions of troubles and concerns about clinical research related to the Clinical Trial Act. We collected 127 comments about troubles and 149 about concerns. Text mining (co-occurrence network and hierarchical cluster analysis) was used to extract the characteristics or tendencies in these comments. The analysis extracted 18 key terms for troubles and 21 for concerns. Most troubles and concerns had to do with concrete examples of clinical research or protocols and biostatistics information. Our results emphasized the importance of clinical research support organizations, and suggested that appropriate workshops and information covering specific situations are necessary to perform clinical research under the new regime.

Genomic landscape of circulating tumour DNA in metastatic extramammary Paget's disease.

Although cancer personalized profiling by deep sequencing (CAPP-Seq) of cell-free DNA (cfDNA) has gained attention, the clinical utility of circulating tumour DNA (ctDNA) in extramammary Paget's disease (EMPD) has not been investigated. In this study, genomic alterations in the cfDNA and tumour tissue DNA were investigated in seven patients with metastatic EMPD. CAPP-Seq revealed mutations in 18 genes, 11 of which have not yet been reported in EMPD. The variant allele frequency of some of the mutated genes reflected the disease course in patients with EMPD. In one patient, the mutation was detected even though imaging findings revealed no metastasis. In another patient with triple EMPD (genital area and both axilla), cfDNA sequencing detected the mutation in a rib metastatic lesion, which was also detected in both axilla lesions but not the genital region. Investigations of the ctDNA may be useful towards the elucidation of clonal evolution in EMPD.

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.