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.

First Aid for Skin Tears by Mini Patch Grafting from the Flap Edge.

BACKGROUND: Traumatic skin tears often occur in patients with dystrophic skin. Closing them with adhesive skin closures is useful for patients with a healthy flap, but occasionally fails to cover the entire defect. We describe a simple technique to perform mini patch grafting on the remaining raw surface without damaging healthy skin. DISCUSSION: The skin flap is spread out and fixed with adhesive skin strips to minimize defects first. Small pieces of skin are obtained by trimming the edges of the skin flap with curved tip scissors. They are placed on the defect with free spaces of 3 to 5 mm between each of the grafts. Then the whole wound is covered with a dressing and gauze pads. Healing of the ulcer could be markedly promoted with little enlargement of the skin defect. CONCLUSIONS: Small pieces of skin trimmed out from the edge of skin flap can be used as a mini patch graft that remarkably enhances healing of remaining open surface.

Increased accumulation of extracellular thrombospondin-2 due to low degradation activity stimulates type I collagen expression in scleroderma fibroblasts.

The aim of the present study was to determine the expression and role of thrombospondin-2 (TSP-2) in systemic sclerosis (SSc). Both TSP-2 mRNA levels and protein synthesis in cell lysates were significantly lower in cultured SSc fibroblasts than in normal fibroblasts; however, the TSP-2 protein that accumulated in the conditioned medium of SSc fibroblasts was up-regulated, compared with that of normal fibroblasts, because of an increase in the half-life of the protein. In vivo serum TSP-2 levels were higher in SSc patients than in healthy control subjects, and SSc patients with elevated serum TSP-2 levels tended to have pitting scars and/or ulcers. TSP-2 knockdown resulted in the down-regulation of type I collagen expression and the up-regulation of miR-7, one of the miRNAs with an inhibitory effect on collagen expression. Expression levels of miR-7 were also up-regulated in SSc dermal fibroblasts both in vivo and in vitro. Taken together, these findings suggest that the increased extracellular TSP-2 deposition in SSc fibroblasts may contribute to tissue fibrosis by inducing collagen expression. Down-regulation of intracellular TSP-2 synthesis and the subsequent miR-7 up-regulation in SSc fibroblasts may be due to a negative feedback mechanism that prevents increased extracellular TSP-2 deposition and/or tissue fibrosis. Thus, TSP-2 may play an important role in the maintenance of fibrosis and angiopathy in patients with SSc.

Hair miR-29a levels are decreased in patients with scleroderma.

In the present study, we evaluated the possibility that we can utilize hair shaft miR-29a levels as disease marker of scleroderma. Hair samples were obtained from 20 scleroderma patients, five dermatomyositis patients and 13 controls. microRNAs were purified from hairs as well as skins or sera, and miR-29a levels were measured with quantitative real-time polymerase chain reaction. Mean hair miR-29a levels in scleroderma patients were significantly lower than those in control subjects or dermatomyositis, while expression levels of hair shaft marker keratin 34 were similar among them. There was no strong correlation among the miR-29a levels in the hair, skin and serum of each patient, suggesting that hair microRNAs can be independent biomarkers. We found scleroderma patients with decreased miR-29a levels had contracture of the phalanges at a significantly higher prevalence than those without. To confirm the clinical usefulness of hair microRNAs, large-scale researches are needed in the future.

Existence of Staphylococcus aureus correlates with the progression of extramammary Paget's disease: potential involvement of interleukin-17 and M2-like macrophage polarization.

BACKGROUND: The microbiome plays an important role in the tumour microenvironment (TME). OBJECTIVES: In this study, we investigated the clinical significance of the microbiota in extramammary Paget's disease (EMPD). MATERIALS & METHODS: Patients with EMPD, treated between March 2007 and September 2019 at Kumamoto University Hospital, were investigated retrospectively. Inclusion criteria included: histological diagnosis of EMPD, inspection of the bacterial culture of the cancer lesion using swab sampling, and availability of sufficient tissue in paraffin blocks for immunohistochemistry. For the latter, primary antibodies against IL-17, CD163 and ionized calcium-binding adapter molecule 1 (Iba1) were used. RESULTS: Bacterial cultures of the cancer lesion revealed that Staphylococcus aureus (S. aureus) was highly prevalent in EMPD patients, with dermal invasion or lymph node metastasis, compared to patients without these findings. Furthermore, the number of IL-17-positive cells and CD163-positive M2-like macrophages (pro-tumour macrophages) were increased in EMPD tissues with S. aureus. Moreover, the number of IL-17-producing cells in EMPD tissues positively correlated with the accumulation of CD163-positive M2-like macrophages. In addition, the percentage of CD163-positive cells within Iba-1-positive macrophages (total macrophages) was also significantly elevated in EMPD tissues with S. aureus. CONCLUSION: Based on these findings, S. aureus may exacerbate the pathological condition of EMPD via the accumulation of IL-17 and M2-like macrophages.

Up-regulated type I collagen expression by the inhibition of Rac1 signaling pathway in human dermal fibroblasts.

Tissue remodeling is known to play important roles in wound healing. Although Rac1 is reported to be one of the key signaling molecules in cutaneous wound healing process, the exact mechanisms of Rac1-mediated tissue remodeling is still unknown. This study investigated the role of Rac1 in the regulation of extracellular matrix in cultured human dermal fibroblasts obtained by skin biopsy from three healthy donors. Protein levels of type I collagen in cultured human fibroblasts were increased by the treatment with Rac1 inhibitor NSC23766 in a dose-dependent manner. However, the mRNA levels of alpha2(I) collagen was not altered by the inhibitor. On the other hand, by the addition of inhibitor, half-lives of type I collagen protein were increased and MMP1 levels were reduced. These data suggest that blockade of Rac1 signaling results in accumulation of type I collagen due to decreased collagenase activity. This study also suggests that controlling Rac1 signaling is a new therapeutic approach to chronic/untreatable ulcer.

Brain abscess in an angiosarcoma patient during a disease-free interval.

This is the first case of an angiosarcoma patient with brain abscess, and it might be responsible for skin defect and cranial bone necrosis by surgical excision and radiation. Our patient was treated with 10 courses of triweekly paclitaxel therapy, radical radiotherapy (70 Gy), and surgical excision (2 cm margin apart from a lesion) for angiosarcoma. At two years after the operation he was diagnosed as brain abscess. Brain abscess was managed with antibiotic drugs and drainage, his clinical symptoms improved by these treatments. He achieves replace free survival without the exacerbation of angiosarcoma and brain abscess for three years.

A potential significance of circ_0024169 down regulation in angiosarcoma tissue.

Circular RNAs (circRNAs) are recently characterized non-coding RNAs that have a closed continuous loop. CircRNAs might play important roles in the oncogenesis of several cancers. However, little is known about association between circRNAs and skin tumors. In this study, we tried to demonstrate the expression change of circ_0024169 in angiosarcoma, and to elucidate correlations between circ_0024169 expression in angiosarcoma tissues and clinical manifestation. RNA expression was evaluated by quantitative real-time PCR with TaqMan systems for circ_0024169 and linear isoform CUL5. Both relative circRNA levels (corrected for EEF1A1 levels) and circRNA levels/linear RNA expression ratio were evaluated. We found that both relative circ_0024169 levels and circ_0024169/CUL5 ratio was decreased in normal human dermal microvascular endothelial cells (HDMEC) and angiosarcoma cell line in vitro, compared to squamous cell carcinoma line. circ_0024169/ CUL5 ratio was significantly reduced in angiosarcoma and pyogenic granuloma than other tumors in vivo, which were more evident than decreased relative circ_0024169 levels. On the other hand, relative circ_0024169 levels showed mild inverse correlation with the follow-up periods (duration between the first hospital visit and the last hospital visit/the date of death) of angiosarcoma patients. Taken together, circ_0024169/CUL5 ratio are likely to be useful as a diagnostic biomarker for vascular tumors, whereas circ_0024169 levels may have more potential as a prognostic marker of angiosarcoma. The future studies of the function of circRNAs may lead to the clarification of detailed mechanism of oncogenesis of angiosarcoma.

Overexpression of cyclin-dependent kinase 4 protein in extramammary Paget's disease.

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.

Submental perforator flap: location and number of submental perforating vessels.

The sites and numbers of submental perforator vessels were examined using a Doppler probe in 21 volunteers. The subcutaneous vascular network from each perforator was studied in three cases of dissection of upper-neck lymph nodes among the volunteers. A perforator from the submental vessels was noted in all 21 volunteers: a single perforator in 13 cases, and double perforators in eight. The main perforator, which had some subdermal branches, was located 31.8 (8.3) mm in front of the facial artery that was palpated at the mandible. Five patients who presented with skin defects on the cheek and the chin had the submental perforator flap reconstructed, excluding the platysma muscle. All flaps covered the wounds. The submental perforator flap was useful for reconstructing skin defects on the cheek and the chin, because the site of the submental perforator was stable and raising the flap was easy, and the colour and texture matches were acceptable.

Intratumor dihydropyrimidine dehydrogenase mRNA expression levels are decreased in extramammary Paget's disease.

S-1, a 5-fluorouracil (5-FU)-based anti-cancer agent, is an important drug for treating metastatic extramammary Paget's disease (EMPD). Although intratumor expression levels of 5-FU metabolism enzymes have been studied widely in many solid tumors, no studies have examined on the expression levels of thymidylate synthase (TS), orotate phosphoribosyl-transferase (OPRT) or dihydropyrimidine dehydrogenase (DPD) in skin cancers. The aim of this study was to estimate the intratumoral mRNA expression levels of these genes in EMPD by real time PCR. Intratumoral DPD mRNA levels were decreased in EMPD compared to those in normal skin, but its intratumoral DPD mRNA expression levels were not correlated with clinical manifestations. Intratumoral DPD mRNA levels were positively correlated with OPRT mRNA levels in EMPD. Based on these results, low expression of intratumoral DPD mRNA in EMPD may contribute to the pathogenesis of this disease.

The expression of miR-124 increases in aged skin to cause cell senescence and it decreases in squamous cell carcinoma.

Skin senescence is induced by various factors including intrinsic aging and extrinsic aging. The current study compared the expression of microRNAs in young facial skin and senescent facial skin, and this study identified skin aging-related microRNAs. According to the results from a microRNA PCR Array, miR-124 was the microRNA that increased the most in senescent skin compared to young skin. Real-time PCR with a greater number of samples indicated that the increase in miR-124 levels in senescent facial skin was statistically significant. In situ hybridization was performed, and results indicated that the signal for miR-124 was evident in keratinocytes of senescent skin but not in those of young skin. The morphology of cultured normal human epidermal keratinocytes (NHEKs) transfected with a miR-124 mimic changed to an enlarged and irregular shape. In addition, the number of NHEKs positive for senescence-associated ß-galactosidase (SA-ß-gal) increased significantly as a result of the overexpression of the miR-124 mimic. The expression of miR-124 increased in UVB-irradiated NHEKs compared to controls in a dose-dependent manner. Expression of miR-124 in A431, a human cutaneous squamous cell carcinoma (SCC) cell line, decreased significantly compared to that in NHEKs. Forced overexpression of miR-124 as a result of the transfection of a miR-124 mimic in A431 resulted in the significant suppression of the proportion of cancer cells. The current results indicated that miR-124 increases as a result of cell senescence and that it decreases during tumorigenesis. The effect of supplementation of miR-124 in an SCC cell line suggests that senescence induction therapy with microRNA may be a new therapeutic approach for treatment of SCC.

The role of miR-210, E2F3 and ephrin A3 in angiosarcoma cell proliferation.

BACKGROUND: Although angiosarcoma exhibits aggressive progression and is associated with unfavourable prognosis, its pathogenesis is poorly understood. OBJECTIVES: In the present study, we investigated the possibility that microRNAs play a role in the pathogenesis of angiosarcoma. MATERIALS & METHODS: microRNA expression was evaluated by array analysis and real-time PCR, and protein expression was determined by immunohistochemistry and immunoblotting. RESULTS: miR-210 expression was decreased in angiosarcoma cells both in vivo and in vitro. E2F3 and ephrin A3 are putative targets of miR-210, and their protein expression was up-regulated in the tumour cells. Knockdown of E2F3 or ephrin A3 resulted in a significant decrease in the number of angiosarcoma cells. CONCLUSION: Further investigations into the regulatory mechanisms of oncogenesis associated with miR-210/E2F3/ephrin A3 signalling may lead to a new therapeutic approach against angiosarcoma.

Chronic sun exposure-related fusion oncogenes EGFR-PPARGC1A in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) differs from SCC of other organs in its strong association with chronic sun exposure. However, the specific driver mutations in cSCC remain unknown. Fusion genes in established cSCC cell lines (A431 and DJM-1) were predicted by transcriptome sequence, and validated by Sanger sequence, fluorescence in situ hybridization and G-banding. By transcriptome sequencing, we identified fusion gene EGFR-PPARGC1A in A431, which were expressed in 31 of 102 cSCCs. The lesions harboring the fusion gene tended to be located in sun-exposed areas. In vivo cutaneous implantation of EGFR-PPARGC1A-expressing NIH3T3 induced tumors resembling human cSCC, indicating its potent tumorigenicity. NIH3T3 transfected with EGFR-PPARGC1A as well as A431 showed increased cell proliferation activity. With regard to underlying mechanism, EGFR-PPARGC1A protein causes constitutive tyrosine phosphorylation, and induces the phosphorylation of wild-type full-length epidermal growth factor receptor (EGFR) by dimerization. Conversely, the RNAi-mediated attenuation of EGFR or CRISPR/Cas9-mediated knockdown of the fusion gene in A431 led to a decrease in the cell number, and may have therapeutic value. Our findings advance the knowledge concerning genetic causes of cSCC and the function of EGFR, with potential implications for new diagnostic and therapeutic approaches.

Cytokine expression profiles in the sera of cutaneous squamous cell carcinoma patients.

We focused on the interaction of cytokines in squamous cell carcinoma (SCC), and determined the expression profile of multiple cytokines in the serum of each patient with SCC in the present study. Serum samples were obtained from 12 SCC patients and 7 normal subjects. Four cytokines (IFN-γ, IL-6, GM-CSF, and TGF-ß) were selected because they are reported to be involved in keratinocyte proliferation and SCC progression. Serum levels were measured using ELISA. We found a statistically significant increase of serum IFN-γ levels in SCC patients compared to those in normal subjects, and areas under the curve (AUC) of 0.82 for the serum levels of IFN-γ were higher than those for other cytokine levels according to ROC curve analysis. Patients with increased IFN-γ levels had a significantly more severe cancer stage. Furthermore, the combination of IFN-γ levels and TGF-ß levels could improve the AUC to 0.84. We also found there was a significant correlation between IFN-γ levels and GM-CSF levels or between GM-CSF levels and TGF-ß levels only in SCC patients. Our results suggest that the combination of IFN-γ levels and TGF-ß levels is more effective to diagnose SCC, while serum levels of IFN-γ alone are useful to evaluate tumor progression. Furthermore, expression of these cytokines was not independent, but may be regulated by common upstream factors (e.g. cytokines or methylation) in SCC patients, and such factors may play some roles in the pathogenesis of SCC.

Case of Paecilomyces lilacinus infection occurring in necrotizing fasciitis-associated skin ulcers on the face and surrounding a tracheotomy stoma.

A 28-year-old man undergoing treatment for hemophagocytic syndrome developed Paecilomyces lilacinus infection in skin ulcers on the face and in the tracheotomy stoma. While his bone marrow was suppressed by chemotherapy with dexamethasone, cyclosporin and etoposide for hemophagocytic syndrome, dental infection led to subacute necrotizing fasciitis caused by Pseudomonas aeruginosa on the right side of the face, resulting in a large area of soft tissue defects. Etoposide was discontinued, and prophylactic treatment with itraconazole was initiated. The ulcers resulting from necrotizing fasciitis were treated conservatively using trafermin and alprostadil alfadex ointment 0.003 %, and near-complete re-epithelialization occurred, except on the right lower eyelid, right buccal mucosa and perioral area. However, 6 weeks later, pustules/crusts started to form and break down repeatedly, leading to expansion of tissue defects on the face. Direct microscopic examination revealed fungal elements, and fungal culture identified Paecilomyces lilacinus suspicious twice some other day. Based on DNA extraction from the isolated fungus, this fungal strain was identified as Paecilomyces lilacinus. Cyclosporin and itraconazole were discontinued, and treatment with liposomal amphotericin B and a tapering dose of steroids was initiated. Cure was achieved in approximately 2.5 months after treatment initiation, and no relapse has been observed. The most important factor that ultimately contributed to the resolution of fungal infection might have been release of immunosuppression by discontinuing cyclosporin and tapering steroids.