NRAS and BRAF mutation frequency in primary oral mucosal melanoma

Oral mucosal melanoma (OMM) is a fatal sarcoma of unknown etiology. Histological morphology and genetic events are distinct from those of its cutaneous counterpart. Mutation and up-regulation of c-kit has been identified in OMM which may activate downstream molecules such as RAS and RAF. These molecules are involved in the mitogen-activated protein kinase (MAPK) pathway leading to tremendous cell proliferation and survival. NRAS and BRAF mutation and protein expression have been studied in other melanoma subtypes. The purpose of this study was to determine RAS protein expression and NRAS and BRAF mutation in 18 primary OMM cases using immunohistochemistry and mutation analysis. Results showed that RAS is intensely expressed in both in situ and invasive OMMs. However, NRAS mutation was only observed in 2/15 polymerase chain reaction (PCR) amplified cases both of which were silent mutations. On the other hand, BRAF missense mutations were observed only in 1/15 cases with PCR amplification. NRAS and BRAF mutations were independent from previously reported c-kit mutations. The classical V600E BRAF mutation was not found; instead a novel V600L was observed suggesting that the oncogenic event in OMM is different from that in skin melanoma. The low frequency of NRAS and BRAF mutations indicate that these genes are not common, but probable events in OMM pathogenesis, most likely independent of c-kit mutation. This record was migrated from the OpenDepot repository service in June, 2017 before shutting down

Localization of oxytalan fiber, type III collagen and BMP family in conventional and desmoplastic ameloblastoma

The histologic hallmark distinguishing desmoplastic ameloblastoma (DA) from conventional ameloblastoma (CA) is its pronounced stromal desmoplasia, and this formed the basis of this investigation. To elucidate the stromal characteristics, localization patterns of oxytalan fibers, type III collagen and BMP family in DA (n=8) was compared with CA (n=24), and periodontal ligament (PL) (n=8). Oxytalan fibers formed apico-occlusal bundles in PL, thick radial bundles around tumor nests in DA, and as scanty fibers in CA. Type III collagen was identified in PL, strongly expressed in DA stroma, but weakly in CA. BMP-2, -3, -4 and -7 expression patterns in tumor epithelium and stroma were more pronounced in DA (including sites of bone formation), than CA. No immunoreactivity for BMP-5 and -6 were detected. Current findings suggest that the stroma in DA is neoplastic and derived from odontogenic ectomesenchyme, and recommends its reclassification as an odontogenic epithelial-ectomesenchymal neoplasm

The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination

Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination

NRAS and BRAF mutation frequency in primary oral mucosal melanoma

Oral mucosal melanoma (OMM) is a fatal sarcoma of unknown etiology. Histological morphology and genetic events are distinct from those of its cutaneous counterpart. Mutation and up-regulation of c-kit has been identified in OMM which may activate downstream molecules such as RAS and RAF. These molecules are involved in the mitogen-activated protein kinase (MAPK) pathway leading to tremendous cell proliferation and survival. NRAS and BRAF mutation and protein expression have been studied in other melanoma subtypes. The purpose of this study was to determine RAS protein expression and NRAS and BRAF mutation in 18 primary OMM cases using immunohistochemistry and mutation analysis. Results showed that RAS is intensely expressed in both in situ and invasive OMMs. However, NRAS mutation was only observed in 2/15 polymerase chain reaction (PCR) amplified cases both of which were silent mutations. On the other hand, BRAF missense mutations were observed only in 1/15 cases with PCR amplification. NRAS and BRAF mutations were independent from previously reported c-kit mutations. The classical V600E BRAF mutation was not found; instead a novel V600L was observed suggesting that the oncogenic event in OMM is different from that in skin melanoma. The low frequency of NRAS and BRAF mutations indicate that these genes are not common, but probable events in OMM pathogenesis, most likely independent of c-kit mutation

Karyopherin Alpha Proteins Regulate Oligodendrocyte Differentiation

<div><p>Proper regulation of the coordinated transcriptional program that drives oligodendrocyte (OL) differentiation is essential for central nervous system myelin formation and repair. Nuclear import, mediated in part by a group of karyopherin alpha (Kpna) proteins, regulates transcription factor access to the genome. Understanding how canonical nuclear import functions to control genomic access in OL differentiation may aid in the creation of novel therapeutics to stimulate myelination and remyelination. Here, we show that members of the Kpna family regulate OL differentiation, and may play distinct roles downstream of different pro-myelinating stimuli. Multiple family members are expressed in OLs, and their pharmacologic inactivation dose-dependently decreases the rate of differentiation. Additionally, upon differentiation, the three major Kpna subtypes (P/α2, Q/α3, S/α1) display differential responses to the pro-myelinating cues T3 and CNTF. Most notably, the Q/α3 karyopherin <i>Kpna4</i> is strongly upregulated by CNTF treatment both compared with T3 treatment and other Kpna responses. <i>Kpna4</i> inactivation results in inhibition of CNTF-induced OL differentiation, in the absence of changes in proliferation or viability. Collectively, these findings suggest that canonical nuclear import is an integral component of OL differentiation, and that specific Kpnas may serve vital and distinct functions downstream of different pro-myelinating cues.</p></div

Expression of lymphocyte immunoregulatory biomarkers in bone and soft-tissue sarcomas

Despite advances in our understanding of the underlying molecular drivers of sarcomas, few treatments are available with proven benefit for advanced metastatic sarcomas. Immunotherapy has value in this setting for some types of cancers, but sarcomas, with their multiplicity of rare types, have not been characterized in detail for their expression of targetable immune biomarkers. This study provides the most systematic evaluation to date of tumor-infiltrating lymphocytes and immune checkpoint biomarker expression in sarcomas. We examined by morphology and immunohistochemistry 1072 sarcoma specimens representing 22 types, in addition to 236 benign bone and soft-tissue tumors. Genomically-complex sarcoma types-those driven by mutations and/or copy-number alterations-had much higher numbers of tumor-infiltrating lymphocytes than translocation-associated sarcomas. Prior exposure to radiotherapy was associated with increased immune infiltrates. Higher lymphocytic infiltration was associated with better overall survival among the non-translocation-associated sarcomas. Expression of PD-1 and CD56 were associated with worse overall survival. LAG-3 and TIM-3, two emerging immune checkpoints, were frequently expressed in most sarcoma types. Indeed, most cases positive for PD-(L)1 coexpressed one or both of these novel biomarkers, providing a potential rationale in support for trials targeting LAG-3 and/or TIM-3 in conjunction with PD-1 inhibition

Karyopherin alphas are widely expression in OLP and important regulators of OL differentiation.

<p><b>(A)</b><i>Kpna</i> gene expression in OLP exposed only to proliferative factors (PDGFAA, 10 ng/ml and bFGF, 20 ng/ml) for 24h. Expression was quantified from isolated total RNA with NanoString nCounter Gene Expression Assay. A panel of probes was designed for target genes and normalized to housekeeping genes <i>Alas1</i>, <i>Ppia</i>, <i>Gapdh</i>, <i>Actb</i>, and <i>Rps11</i>. Following assay completion, raw data was normalized and analyzed using nSolverTM software. <i>Kpna2</i> is the highest expressing Impα in OLP. <i>Kpna7</i> is not expressed in oligodendrocytes. <b>(B-D)</b> Results from primary OLP pre-treated 1h with increasing concentrations the karyopherin inhibitor importazole (IPZ) or DMSO, then differentiated with T3 (40ng/ml) in the presence of IPZ/DMSO for 9h and harvested for qPCR <b>(B)</b> or immunocytochemistry <b>(C)</b>. <b>(B)</b> Failure of karyopherin functioning in IPZ-treated cultures results in dose-dependent inhibition of differentiation as measured by a decrease in the expression of the OL maturation markers <i>CNP</i> or <i>MBP</i> in qPCR. <b>(C,D)</b> While, at lower doses of IPZ, inhibition of differentiation is seen in the absence of any impact on viability, higher concentrations of IPZ eventually produce apoptosis, quantified as the percentage of DAPI cells that are cleaved-caspse 3 (CC3) positive. <b>(E-I)</b> Extension of differentiation out to 72h with T3 in the presence of 2μM IPZ confirms this reduction in maturation. Results are from primary OLP pre-treated 1h with 2μM IPZ or DMSO, then differentiated with T3 (40ng/ml) in the presence of IPZ/DMSO for up to 72h and harvested for qPCR <b>(E,F)</b> or immunocytochemistry <b>(G-I)</b>. <b>(E,F)</b> Failure of karyopherin functioning in 2μM IPZ-treated cultures results in inhibition of differentiation as measured by a decrease in the expression of the OL maturation markers <i>CNP</i> or <i>MBP</i> in qPCR. <b>(G)</b> Representative image shows maturation markers for immature/mature OL (O4), and mature OL (MBP) in the OL lineage in IPZ-treated cultures and DMSO controls treated with T3 for 72h. Maturation was assessed by quantifying %O4/DAPI <b>(H)</b> and %MBP/DAPI <b>(I)</b> expressing cells. Data presented are mean ± S.E.M. and representative of 3 <b>(A,B,C,E,F)</b> or 5 <b>(H,I)</b> independent cultures. Statistics, <b>(B)</b> One-way ANOVA plus Bonferroni post-test, <b>(D,E,F)</b> Two-way ANOVA plus Bonferroni post-test, <b>(H,I)</b> Student’s t-test, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001. Scale bars, <b>(C,G)</b> 20μm. Individual data values are in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170477#pone.0170477.s001" target="_blank">S1 Data</a></b>.</p

<i>Kpna4</i> is an important regulator of CNTF-induced oligodendrocyte differentiation.

<p><b>(A-C)</b> Confocal imaging of Kpna4 expression <i>in vivo</i> in P1 <b>(A)</b> and P14 <b>(C)</b> thoracolumbar mouse spinal cord sections. Kpna4 is expressed in multiple cells within the CNS including APC⁺Olig2⁺ OLs (representative cells indicated with white arrows), but not by immature Olig2⁺APC^- cells (yellow arrowhead). The region outlined in <b>(A)</b> is magnified in panels to the right. Panel <b>(B)</b> shows a high power image of Olig2⁺ cells in thoracolumbar spinal cord at P1. Cells show both nuclear (<b>B,</b> red arrowheads) and cytoplasmic Kpna4 immunoreactivity (<b>B,</b> white arrowheads). <b>(D)</b> qPCR data from OLP nucleofected with <i>siKpna4</i> or <i>siNT</i> control for 24h. Silencing is efficient and is selective for <i>Kpna4</i>. <b>(E,F)</b> Representative confocal image and associated quantification from primary mouse OLP silenced for <i>Kpna4</i> versus nontargeting control, then allowed to proliferate in the presence of the mitogens PDGFAA and bFGF for 24h. Active proliferation was assessed by immunocytochemistry for Ki67, confocal imaging, and quantitation of %Ki67/DAPI cells. <b>(G-I)</b> To directly compare responses of Kpna4-deficient cells and controls to CNTF vs. T3 in parallel, primary cultures were nucleofected with siRNA for <i>Kpna4</i> or nontargeting (NT) control, then treated with either CNTF or T3. <b>(G)</b> Representative confocal image and associated quantification from primary mouse OLP nucleofected with <i>siKpna4</i> or <i>siNT</i> control and then differentiated with T3 (60 ng/ml) or CNTF (100 ng/ml) for 72h. This image shows maturation markers for OLP (NG2), immature/mature OL (O4), and mature OL (MBP) in the Olig2⁺ OL lineage in <i>Kpna4</i>-deficient cultures and controls treated with T3 or CNTF. Maturation was assessed by quantifying %O4/DAPI <b>(H)</b> and %MBP/DAPI <b>(I)</b> expressing cells. Maturation was reduced in CNTF-treated cultures. In T3-treated cultures, only a slight reduction was observed in the proportion of MBP expressing cells, which did not reach significance, illustrating a differential impact of silencing <i>Kpna4</i> depending on the growth factor added to induce differentiation. <b>(J,K)</b> The reduction in maturation markers coincided with a loss in complexity of branching morphology, a marker of OL maturity, as assessed by fractal analysis. Fractal results are represented by the box-counting fractal dimension (Db). <b>(L,M)</b> Cell death was unchanged in these cultures regardless of treatment with CNTF <b>(L)</b> or T3 <b>(M)</b>, measured by both assessments of apoptosis (%Cleaved-caspase 3 (CC3)/DAPI) and total cell number (DAPI counts per field). Data are mean ± S.E.M. and representative of 3 <b>(D,F,L,M)</b> or 5 <b>(H-K)</b> independent cultures. Statistics, <b>(D,F,L,M,J,K)</b> Student’s t-test, <b>(H,I</b>) Two-way ANOVA plus Bonferroni test, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. Scalebars, <b>(A)</b> 50μm, 25μm inset, <b>(B)</b> 10μM, <b>(C)</b> 25μm, <b>(E)</b> 20μm, <b>(G)</b> 20μm. Individual data values are in <b>S1 data</b>.</p