Reprogramming of bivalent chromatin states in NRAS mutant melanoma suggests PRC2 inhibition as a therapeutic strategy.

The dynamic evolution of chromatin state patterns during metastasis, their relationship with bona fide genetic drivers, and their therapeutic vulnerabilities are not completely understood. Combinatorial chromatin state profiling of 46 melanoma samples reveals an association of NRAS mutants with bivalent histone H3 lysine 27 trimethylation (H3K27me3) and Polycomb repressive complex 2. Reprogramming of bivalent domains during metastasis occurs on master transcription factors of a mesenchymal phenotype, including ZEB1, TWIST1, and CDH1. Resolution of bivalency using pharmacological inhibition of EZH2 decreases invasive capacity of melanoma cells and markedly reduces tumor burden in vivo, specifically in NRAS mutants. Coincident with bivalent reprogramming, the increased expression of pro-metastatic and melanocyte-specific cell-identity genes is associated with exceptionally wide H3K4me3 domains, suggesting a role for this epigenetic element. Overall, we demonstrate that reprogramming of bivalent and broad domains represents key epigenetic alterations in metastatic melanoma and that EZH2 plus MEK inhibition may provide a promising therapeutic strategy for NRAS mutant melanoma patients

Novel algorithmic approach predicts tumor mutation load and correlates with immunotherapy clinical outcomes using a defined gene mutation set

BACKGROUND: While clinical outcomes following immunotherapy have shown an association with tumor mutation load using whole exome sequencing (WES), its clinical applicability is currently limited by cost and bioinformatics requirements. METHODS: We developed a method to accurately derive the predicted total mutation load (PTML) within individual tumors from a small set of genes that can be used in clinical next generation sequencing (NGS) panels. PTML was derived from the actual total mutation load (ATML) of 575 distinct melanoma and lung cancer samples and validated using independent melanoma (n = 312) and lung cancer (n = 217) cohorts. The correlation of PTML status with clinical outcome, following distinct immunotherapies, was assessed using the Kaplan–Meier method. RESULTS: PTML (derived from 170 genes) was highly correlated with ATML in cutaneous melanoma and lung adenocarcinoma validation cohorts (R(2) = 0.73 and R(2) = 0.82, respectively). PTML was strongly associated with clinical outcome to ipilimumab (anti-CTLA-4, three cohorts) and adoptive T-cell therapy (1 cohort) clinical outcome in melanoma. Clinical benefit from pembrolizumab (anti-PD-1) in lung cancer was also shown to significantly correlate with PTML status (log rank P value < 0.05 in all cohorts). CONCLUSIONS: The approach of using small NGS gene panels, already applied to guide employment of targeted therapies, may have utility in the personalized use of immunotherapy in cancer. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12916-016-0705-4) contains supplementary material, which is available to authorized users

SARS-CoV-2 RT-qPCR testing of pooled saliva samples: A case study of 824 asymptomatic individuals and a questionnaire survey in Japan.

From the beginning of the COVID-19 pandemic, the demand for diagnostic and screening tests has exceeded supply. Although the proportion of vaccinated people has increased in wealthier countries, breakthrough infections have occurred amid the emergence of new variants. Pooled-sample COVID-19 testing using saliva has been proposed as an efficient, inexpensive, and non-invasive method to allow larger-scale testing, especially in a screening setting. In this study, we aimed to evaluate pooled RT-qPCR saliva testing and to compare the results with individual tests. Employees of Philips Japan, Ltd. were recruited to participate in COVID-19 screening from October to December 2020. Asymptomatic individuals (n = 824) submitted self-collected saliva samples. Samples were tested for the presence of SARS-CoV-2 by RT-qPCR in both 10-sample pools and individual tests. We also surveyed participants regarding their thoughts and behaviors after the PCR screening project. Two of the 824 individuals were positive by RT-qPCR. In the pooled testing, one of these two had no measurable Ct value, but showed an amplification trend at the end of the PCR cycle. Both positive individuals developed cold-like symptoms, but neither required hospitalization. Of the 824 participants, 471 responded to our online questionnaire. Overall, while respondents agreed that PCR screening should be performed regularly, the majority were willing to undergo PCR testing only when it was provided for free or at low cost. In conclusion, pooled testing of saliva samples can support frequent large-scale screening that is rapid, efficient, and inexpensive

A questionnaire survey on itching and daily life in patients with atopic dermatitis

No abstract available

CD10-Equipped Melanoma Cells Acquire Highly Potent Tumorigenic Activity: A Plausible Explanation of Their Significance for a Poor Prognosis.

CD10 has been widely used in cancer diagnosis. We previously demonstrated that its expression in melanoma increased with tumor progression and predicted poor patient survival. However, the mechanism by which CD10 promotes melanoma progression remains unclear. In order to elucidate the role of CD10 in melanoma, we established CD10-overexpressing A375 melanoma cells and performed DNA microarray and qRT-PCR analyses to identify changes in the gene expression profile. The microarray analysis revealed that up-regulated genes in CD10-A375 were mostly involved in cell proliferation, angiogenesis, and resistance to apoptosis; down-regulated genes mostly belonged to the categories associated with cell adhesion and migration. Accordingly, in functional experiments, CD10-A375 showed significantly greater cell proliferation in vitro and higher tumorigenicity in vivo; CD10 enzymatic inhibitors, thiorphan and phosphoramidon, significantly blocked the tumor growth of CD10-A375 in mice. In migration and invasion assays, CD10-A375 displayed lower migratory and invasive capacity than mock-A375. CD10 augmented melanoma cell resistance to apoptosis mediated by etoposide and gemcitabine. These findings indicate that CD10 may promote tumor progression by regulating the expression profiles of genes related to cell proliferation, angiogenesis, and resistance to apoptosis

CD10 exhibits higher resistance to anticancer drugs.

<p>(A-C) Histogram (upper) and dot plot (lower) showing the proportion of Annexin V- and Annexin V/PI-positive cells 24 hours after treatment with anticancer drugs. CD10-A375 or mock-A375 cells were treated with etoposide (B) or gemcitabine (C) overnight. Cell were harvested and stained with Annexin V and PI, and 10,000 events were counted by flow cytometry. Without any treatment (A), the number of Annexin V-positive apoptotic or Annexin V/PI-positive dead cells was low and there was no difference between CD10-A375 and mock-A375. Under the treatment with the anticancer drugs, however, the number of apoptotic cells increased remarkably. CD10-A375 cells (red line) displayed a lower proportion of Annexin V-positive or Annexin V/PI-positive dead cells induced by anticancer drugs than mock-A375 (blue line), showing stronger resistance. Isotype control is shown with a gray line.</p

Establishment of CD10-A375 melanoma cells and gene expression profiles.

<p>(A) Stable transfection of CD10-A375 cells was confirmed by flow cytometry. After selection with G418, CD10-A375 or mock-A375 cells were examined for CD10 protein expression with PE-conjugated anti-hCD10 antibody. CD10-A375 exhibited marked surface expression of CD10 (gray shaded area), whereas mock-A375 did not (solid gray line). Isotype IgG control is shown with a gray dashed line. (B) Western blot analysis of CD10 and GAPDH in the wild type- and transfected- A375 cells. Successful transfection of CD10 was also confirmed in CD10-A375 cells by Western blot analysis. (C and D) Microarray analysis of gene expression in CD10-A375 vs. mock-A375 melanoma cells. (C) Heat map generated using MeV4.6 from the 1,247 probes of genes significantly differentially expressed between CD10-A375 and mock-A375 (<i>n</i> = 4 per group). A red color represents a higher-than-average standardized expression value, whereas a green color represents a lower-than-average one. Examples of differentially expressed genes are listed and sorted into major known functional categories in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149285#pone.0149285.t002" target="_blank">Table 2</a>. (D) Heat map of the selected genes as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149285#pone.0149285.t002" target="_blank">Table 2</a>. (E) qRT–PCR validation analysis of representative genes that were significantly upregulated in CD10-A375 (<i>n</i> = 3). Each value was normalized for ACTB (β-actin), and the results are expressed as fold change over mock-A375. Primers used in the assay are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149285#pone.0149285.t002" target="_blank">Table 2</a>. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001; unpaired t test.</p

CD10 decreases cell migration and invasion.

<p>Cell migration assay (A-D) and invasion assay (E) of the CD10-A375 or mock-A375 cells were performed in the absence (A, B) or presence (C, D) of mitomycin C (10 μl/ml). (A and C) Representative images showing the wound gap filled by CD10-A375 (right) or mock-A375 cells (left) cultured in the absence of mitomycin C for 87 hours (A) or in its presence at a concentration of 10 μl/ml for 24 hours (C). (B and D) Graphs showing the time course changes in the percentage of wound healing after 87 hours of incubation without mitomycin C (B) or after 24 hours of incubation with mitomycin C (D). CD10-A375 (white dot) displayed lower migratory capacity both in the absence and in the presence of mitomycin C, compared with mock-A375 (black dot). ***<i>P</i> < 0.001 by two-way ANOVA. Results are representative of at least three independent experiments, and values represent averages of three independent measurements along the wound. (E) A graphic result of cell invasion assay of CD10-A375 vs. mock-A375 cells. Cells were plated on polycarbonate membrane inserts coated with uniform layer of basement membrane in 24-well plates (Cell Biolabs) and incubated for 72 hours. The invading cells were stained and quantified as indicated in the graph at 560 nm.</p