Neural-net-based cell deconvolution from DNA methylation reveals tumor microenvironment associated with cancer prognosis.

DNA methylation is a pivotal epigenetic modification that defines cellular identity. While cell deconvolution utilizing this information is considered useful for clinical practice, current methods for deconvolution are limited in their accuracy and resolution. In this study, we collected DNA methylation data from 945 human samples derived from various tissues and tumor-infiltrating immune cells and trained a neural network model with them. The model, termed MEnet, predicted abundance of cell population together with the detailed immune cell status from bulk DNA methylation data, and showed consistency to those of flow cytometry and histochemistry. MEnet was superior to the existing methods in the accuracy, speed, and detectable cell diversity, and could be applicable for peripheral blood, tumors, cell-free DNA, and formalin-fixed paraffin-embedded sections. Furthermore, by applying MEnet to 72 intrahepatic cholangiocarcinoma samples, we identified immune cell profiles associated with cancer prognosis. We believe that cell deconvolution by MEnet has the potential for use in clinical settings.

Distinct Transcriptional Profiles in the Different Phenotypes of Neurofibroma from the Same Subject with Neurofibromatosis 1.

Neurofibromatosis 1 is a prevalent hereditary neurocutaneous disorder. Among the clinical phenotypes of neurofibromatosis 1, cutaneous neurofibroma (cNF) and plexiform neurofibroma (pNF) have distinct clinical manifestations, and pNF should be closely monitored owing to its malignant potential. However, the detailed distinct features of neurofibromatosis 1 phenotypes remain unknown. To determine whether the transcriptional features and microenvironment of cNF and pNF differ, single-cell RNA sequencing was performed on isolated cNF and pNF cells from the same patient. Six cNF and five pNF specimens from different subjects were also immunohistochemically analyzed. Our findings revealed that cNF and pNF had distinct transcriptional profiles even within the same subject. pNF is enriched in Schwann cells with characteristics similar to those of their malignant counterpart, fibroblasts, with a cancer-associated fibroblast-like phenotype, angiogenic endothelial cells, and M2-like macrophages, whereas cNF is enriched in CD8 T cells with tissue residency markers. The results of immunohistochemical analyses performed on different subjects agreed with those of single-cell RNA sequencing. This study found that cNF and pNF, the different neurofibromatosis phenotypes in neurofibromatosis 1, from the same subject are transcriptionally distinct in terms of the cell types involved, including T cells.

Activated Akt expression is associated with the recurrence of primary melanomas and further refines the prognostic and predictive values for relapse in acral melanomas.

A PTEN deficiency leads to the activation of phospho-Akt at serine 473 (p-Akt) and promotes the tumorigenesis of melanomas by coupling with NUAK2 amplification. We tested the prognostic impact of p-Akt and/or NUAK2 expression on the relapse-free survival (RFS) and overall survival (OS) of melanoma patients. Primary tumors from patients with acral melanomas (112), Low-cumulative sun damage (CSD) melanomas (38), and High-CSD melanomas (18) were examined using immunohistochemistry and their prognostic significance was analyzed statistically. The expression of p-Akt was found in 32.1%, 68.4%, and 55.6% of acral, Low-CSD, and High-CSD melanomas, while NUAK2 expression was found in 46.4%, 76.3%, and 50.0%, respectively. Either p-Akt or NUAK2 expression was inversely correlated with the RFS of primary melanoma patients and acral melanoma patients (p-Akt: p < .0001, p < .0001; NUAK2; p = .0005, p < .0001, respectively). Strikingly, multivariate analyses revealed that p-Akt had a significant impact on RFS (Hazard ratio = 4.454; p < .0001), while NUAK2 did not. Further subset analyses revealed that p-Akt expression had an inferior RFS of patients with acral melanomas (Hazard ratio = 4.036; p = .0005). We conclude that the expression of p-Akt has a significant impact on RFS of patients with primary melanomas and can predict the relapse of patients with acral melanomas.

Gap junction-mediated contraction of myoepithelial cells induces the peristaltic transport of sweat in human eccrine glands.

Eccrine sweat glands play an essential role in regulating body temperature. Sweat is produced in the coiled secretory portion of the gland, which is surrounded by obliquely aligned myoepithelial cells; the sweat is then peristaltically transported to the skin surface. Myoepithelial cells are contractile and have been implicated in sweat transport, but how myoepithelial cells contract and transport sweat remains unexplored. Here, we perform ex vivo live imaging of an isolated human eccrine gland and demonstrate that cholinergic stimulation induces dynamic contractile motion of the coiled secretory duct that is driven by gap junction-mediated contraction of myoepithelial cells. The contraction of the secretory duct occurs segmentally, and it is most prominent in the region surrounded by nerve fibers, followed by distension-contraction sequences of the excretory duct. Overall, our ex vivo live imaging approach provides evidence of the contractile function of myoepithelial cells in peristaltic sweat secretion from human eccrine glands.

GREB1 isoform 4 is specifically transcribed by MITF and required for melanoma proliferation.

Growth regulation by estrogen in breast cancer 1 (GREB1) is involved in hormone-dependent and -independent tumor development (e.g., hepatoblastoma). In this study, we found that a GREB1 splicing variant, isoform 4 (Is4), which encodes C-terminal half of full-length GREB1, is specifically expressed via microphthalmia-associated transcription factor (MITF) in melanocytic melanoma, and that two MITF-binding E-box CANNTG motifs at the 5'-upstream region of GREB1 exon 19 are necessary for GREB1 Is4 transcription. MITF and GREB1 Is4 were strongly co-expressed in approximately 20% of the melanoma specimens evaluated (17/89 cases) and their expression was associated with tumor thickness. GREB1 Is4 silencing reduced melanoma cell proliferation in association with altered expression of cell proliferation-related genes in vitro. In addition, GREB1 Is4 targeting by antisense oligonucleotide (ASO) decreased melanoma xenograft tumor formation and GREB1 Is4 expression in a BRAFV600E; PTENflox melanoma mouse model promoted melanoma formation, demonstrating the crucial role of GREB1 Is4 for melanoma proliferation in vivo. GREB1 Is4 bound to CAD, the rate-limiting enzyme of pyrimidine metabolism, and metabolic flux analysis revealed that GREBI Is4 is necessary for pyrimidine synthesis. These results suggest that MITF-dependent GREB1 Is4 expression leads to melanoma proliferation and GREB1 Is4 represents a new molecular target in melanoma.

Increased anti-oxidative action compensates for collagen tissue degeneration in vitiligo dermis.

Vitiligo is a common depigmentation disorder characterized by the selective loss of melanocytes. In our daily clinic experience, we noticed that the skin tightness of hypopigmented lesions would be more evident in comparison to that of uninvolved perilesional skin in vitiligo patients. Therefore, we hypothesized that collagen homeostasis might be maintained in vitiligo lesions, irrespective of the substantial excessive oxidative stress that occurs in association with the disease. We found that the expression levels of collagen-related genes and anti-oxidative enzymes were upregulated in vitiligo-derived fibroblasts. Abundant collagenous fibers were observed in the papillary dermis of vitiligo lesions in comparison to uninvolved perilesional skin by electron microscopy. The production of matrix metalloproteinases that degraded collagen fibers was suppressed. The deposition of acrolein adduct protein, which is a product of oxidative stress, was significantly reduced in vitiligo dermis and fibroblasts. As part of the mechanism, we found upregulation of the NRF2 signaling pathway activity, which is an important defense system against oxidative stress. Taken together, we demonstrated that the anti-oxidative action and collagen production were upregulated and that the collagen degeneration was attenuated in vitiligo dermis. These new findings may provide important clues for the maintenance of antioxidant ability in vitiligo lesions.

Basal cell carcinoma of the umbilicus associated with sweat gland structures: Two case reports.

RATIONALE: Basal cell carcinoma (BCC) arising in the umbilicus is relatively rare, and in particular, there have been few reports mentioning peritumoral sweat gland structures histopathologically. We herein, report 2 cases of umbilical BCC with sweat gland structures within and around the tumor. PATIENT CONCERNS: A 61-year-old woman had a 2-year history of black exudative plaque in her umbilicus, and an 80-year-old woman had a 6-month history of dark brownish plaque in the umbilicus, with exudation 2 months prior to her first visit. DIAGNOSES: Based on the histopathological finding, both cases were confirmed as BCC. The results of immunohistochemical staining showed that the tumor cells were Ber-EP4 positive. In addition, EMA-positive glandular structures were seen within and around the tumor. INTERVENTIONS: Curative resection at the level of the linea alba on the bottom side was performed. OUTCOMES: No relapse has been observed since resection in either patient. LESSONS: We herein report 2 cases of umbilical BCC with sweat glands and ducts. Although whether peri- and/or intra-tumor sweat gland structures are the source of the tumor or arise by transdifferentiation from tumor cells remains unclear, these findings may provide clues to help understand the morphopathogenesis of umbilical BCC in the future.

WT1 Trio Peptide-Based Cancer Vaccine for Rare Cancers Expressing Shared Target WT1.

No standard treatment has been established for most rare cancers. Here, we report a clinical trial of a biweekly WT1 tri-peptide-based vaccine for recurrent or advanced rare cancers. Due to the insufficient number of patients available for a traditional clinical trial, the trial was designed for rare cancers expressing shared target molecule WT1. The recruitment criteria included WT1-expressing tumors as well as HLA-A*24:02 or 02:01. The primary endpoints were immunoglobulin G (IgG) antibody (Ab) production against the WT1-235 cytotoxic T lymphocyte (CTL) epitope and delayed-type hypersensitivity (DTH) skin reactions to targeted WT1 CTL epitopes. The secondary endpoints were safety and clinical efficacy. Forty-five patients received WT1 Trio, and 25 (55.6%) completed the 3-month protocol treatment. WT1-235 IgG Ab was positive in 88.0% of patients treated with WT1 Trio at 3 months, significantly higher than 62.5% of the weekly WT1-235 CTL peptide vaccine. The DTH positivity rate in WT1 Trio was 62.9%, which was not significantly different from 60.7% in the WT1-235 CTL peptide vaccine. The WT1 Trio safety was confirmed without severe treatment-related adverse events, except grade 3 myasthenia gravis-like symptoms observed in a patient with thymic cancer. Fifteen (33.3%) patients achieved stable disease after 3 months of treatment. In conclusion, the biweekly WT1 Trio vaccine containing the WT1-332 helper T lymphocyte peptide induced more robust immune responses targeting WT1 than the weekly WT1-235 CTL peptide vaccine. Therefore, WT1-targeted immunotherapy may be a potential therapeutic strategy for rare cancers.

Nivolumab-induced systemic lymphadenopathy occurring during treatment of malignant melanoma: a case report.

Nivolumab is an anti-programmed cell death protein 1 monoclonal antibody that exhibits significant efficacy in treating melanoma and other malignancies. However, various nivolumab-induced immune-related adverse events (irAEs) have been reported, and differentiating irAEs from tumor progression is sometimes difficult. Here, we report a case of reactive lymphadenopathy occurring after treatment with nivolumab. A 56-year-old man with stage IIIC melanoma received adjuvant therapy with nivolumab after wide local excision. He developed systemic lymphadenopathy and autoimmune hemolytic anemia 1 month after receiving seven cycles of nivolumab. Pathological analysis of a cervical lymph node biopsy specimen revealed no metastatic lesion or any other malignancy, including lymphoma. Thus, the patient was diagnosed with nivolumab-induced reactive lymphadenopathy. Systemic corticosteroids were administered to reduce hemolysis, which led to the resolution of lymphadenopathy. When progressive lymphadenopathy is observed in a patient who received immune checkpoint inhibitor therapy, reactive lymphadenopathy should be carefully distinguished from progression to lymphoid metastasis, and biopsy should be performed if needed.

CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory.

Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8+ Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8- CCR8+ tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8+ effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.

A phase I dose-escalation, safety/tolerability, and preliminary efficacy study of the intratumoral administration of GEN0101 in patients with advanced melanoma.

Despite recent advance in immunotherapy agents, safe new therapies that enhance the effects of immune checkpoint inhibitors are still required to develop. We previously demonstrated that hemagglutinating virus of Japan-envelope (HVJ-E) induced not only direct tumor cell death but also antitumor immunity through the activation of T and natural killer (NK) cells, thereafter, developed a manufacturing process of HVJ-E (GEN0101) for clinical use. We here performed a phase Ia clinical trial of intratumoral GEN0101 administration in six patients with stage IIIC or IV malignant melanoma. The primary aim was to evaluate the safety and tolerability of GEN0101, and the secondary aim was to examine the objective tumor response. Patients were separated into two groups (n = 3 each) and received a low dose of 30,000 and high dose of 60,000 mNAU of GEN0101. All patients completed a two-week follow-up evaluation without severe adverse events. The overall response rate was 33% (2 of 6), with 2 partial responses in the high-dose group and 2 with stable disease, and 2 with progressive disease in the low-dose group. Local complete or partial responses were observed in 11 of 18 (61%) target lesions. One patient demonstrated shrinkage of lung metastases after the treatment. The activity of NK cells and interferon-γ levels were increased in the circulation, indicating augmentation of antitumor immunity by GEN0101. This trial showed not only the safety and tolerability but also the significant antitumor effect of GEN0101, suggesting that GEN0101 might be a promising new drug for patients with advanced melanoma.

Tumor response evaluation in patients with malignant melanoma undergoing immune checkpoint inhibitor therapy and prognosis prediction using 18F-FDG PET/CT: multicenter study for comparison of EORTC, PERCIST, and imPERCIST.

OBJECTIVE: In malignant melanoma patients treated with immune checkpoint inhibitor (ICI) therapy, three different FDG-PET criteria, European Organization for Research and Treatment of Cancer (EORTC), PET Response Criteria in Solid Tumors (PERCIST), immunotherapy-modified PERCIST (imPERCIST), were compared regarding response evaluation and prognosis prediction using standardized uptake value (SUV) harmonization of results obtained with various PET/CT scanners installed at different centers. MATERIALS AND METHODS: Malignant melanoma patients (n = 27) underwent FDG-PET/CT examinations before and again 3 to 9 months after therapy initiation (nivolumab, n = 21; pembrolizumab, n = 6) with different PET scanners at five hospitals. EORTC, PERCIST, and imPERCIST criteria were used to evaluate therapeutic response, then concordance of the results was assessed using Cohen's κ coefficient. Log-rank and Cox methods were employed to determine progression-free (PFS) and overall (OS) survival. RESULTS: Complete metabolic response (CMR)/partial metabolic response (PMR)/stable metabolic disease (SMD)/progressive metabolic disease (PMD) with harmonized EORTC, PERCIST, and imPERCIST was seen in 3/5/4/15, 4/5/3/15, and 4/5/5/13 patients, respectively. Nearly perfect concordance between each pair of criteria was noted (κ = 0.939-0.972). Twenty patients showed progression and 14 died from malignant melanoma after a median 19.2 months. Responders (CMR/PMR) showed significantly longer PFS and OS than non-responders (SMD/PMD) (harmonized EORTC: p < 0.0001 and p = 0.011; harmonized PERCIST: p < 0.0001 and p = 0.0012; harmonized imPERCIST: p < 0.0001 and p = 0.0012, respectively). CONCLUSIONS: All harmonized FDG-PET criteria (EORTC, PERCIST, imPERCIST) showed accuracy for response evaluation of ICI therapy and prediction of malignant melanoma patient prognosis. Additional studies to determine their value in larger study populations will be necessary.