Single-cell RNA sequencing of neurofibromas reveals a tumor microenvironment favorable for neural regeneration and immune suppression in a neurofibromatosis type 1 porcine model

Neurofibromatosis Type 1 (NF1) is one of the most common genetically inherited disorders that affects 1 in 3000 children annually. Clinical manifestations vary widely but nearly always include the development of cutaneous, plexiform and diffuse neurofibromas that are managed over many years. Recent single-cell transcriptomics profiling efforts of neurofibromas have begun to reveal cell signaling processes. However, the cell signaling networks in mature, non-cutaneous neurofibromas remain unexplored. Here, we present insights into the cellular composition and signaling within mature neurofibromas, contrasting with normal adjacent tissue, in a porcine model of NF1 using single-cell RNA sequencing (scRNA-seq) analysis and histopathological characterization. These neurofibromas exhibited classic diffuse-type histologic morphology and expected patterns of S100, SOX10, GFAP, and CD34 immunohistochemistry. The porcine mature neurofibromas closely resemble human neurofibromas histologically and contain all known cellular components of their human counterparts. The scRNA-seq confirmed the presence of all expected cell types within these neurofibromas and identified novel populations of fibroblasts and immune cells, which may contribute to the tumor microenvironment by suppressing inflammation, promoting M2 macrophage polarization, increasing fibrosis, and driving the proliferation of Schwann cells. Notably, we identified tumor-associated IDO1+/CD274+ (PD-L1)+ dendritic cells, which represent the first such observation in any NF1 animal model and suggest the role of the upregulation of immune checkpoints in mature neurofibromas. Finally, we observed that cell types in the tumor microenvironment are poised to promote immune evasion, extracellular matrix reconstruction, and nerve regeneration

MmuPV1-Induced Cutaneous Squamous Cell Carcinoma Arises Preferentially from Lgr5+ Epithelial Progenitor Cells

Murine papillomavirus, MmuPV1, causes natural infections in laboratory mice that can progress to squamous cell carcinoma (SCC) making it a useful preclinical model to study the role of papillomaviruses in cancer. Papillomavirus can infect cells within hair follicles, which contain multiple epithelial progenitor cell populations, including Lgr5+ progenitors, and transgenic mice expressing human papillomavirus oncogenes develop tumors derived from Lgr5 progenitors. We therefore tested the hypothesis that Lgr5+ progenitors contribute to neoplastic lesions arising in skins infected with MmuPV1 by performing lineage tracing experiments. Ears of 6–8-week-old Lgr5-eGFP-IRES-CreERT2/Rosa26LSLtdTomato mice were treated topically with 4-OH Tamoxifen to label Lgr5+ progenitor cells and their progeny with tdTomato and, 72 h later, infected with MmuPV1. Four months post-infection, tissue at the infection site was harvested for histopathological analysis and immunofluorescence to determine the percentage of tdTomato+ cells within the epithelial lesions caused by MmuPV1. Squamous cell dysplasia showed a low percentage of tdTomato+ cells (7%), indicating that it arises primarily from non-Lgr5 progenitor cells. In contrast, cutaneous SCC (cSCC) was substantially more positive for tdTomato+ cells (42%), indicating that cSCCs preferentially arise from Lgr5+ progenitors. Biomarker analyses of dysplasia vs. cSCC revealed further differences consistent with cSCC arising from LGR5+ progenitor cells

Enhancer of Zeste Homolog 2 (EZH2) and Global H3K27 Trimethylation Expression During Progression of Thyroid Cancer

none7siBackground: EZH2 is a histone lysine methyltransferase that is a member of the polycomb group protein family. EZH2 plays an important role in the epigenetic maintenance of H3K27 trimethylation (H3K27me3). Abnormal EZH2 expression has been associated with various cancers. We examined the expression of EZH2 and H3K27me3 to determine their roles in thyroid cancer progression. Design: Anaplastic thyroid carcinomas (ATC, n=35), poorly differentiated thyroid carcinomas (PDTC, n=21), papillary thyroid carcinomas (PTC, n=28), follicular variant of papillary thyroid carcinomas (FVPTC, n=29), follicular thyroid carcinomas (FTC, n=28), follicular adenomas (FA, n=31) and normal thyroids (NT, n=10) on a tissue microarray (TMA) were analyzed by immunohistochemistry (IHC) using formalin-fixed paraffin-embedded tissues. Antibodies to EZH2 (Novus, 1:100) and H3K27me3 (Cell Signaling, 1:200) were used. IHC was scored based on the intensity and percentage of nuclear staining. Survival analysis was performed using the statistical computing software R. Results: Both EZH2 and H3K27me3 showed strong nuclear staining. The highest level of EZH2 expression was observed in ATC (97%) followed by PDTC (90%). Levels were lower in FA (71%), FTC (64%), PTC (79%) and FVPTC (79%). EZH2 expression was significantly higher in ATC than in well-differentiated carcinomas (P<0.04). H3K27me3 expression was high in both benign and malignant thyroid tumors as well as in NT. H3K27me3 expression was not significantly different in higher-grade tumors compared to well-differentiated carcinomas and FA. The carcinoma types expressing the highest levels of EZH2 (ATC and PDTC) had worse prognoses than the carcinoma types with lower expression (P<0.001). Conclusions: EZH2 is expressed in thyroid malignancies, and its expression correlates with higher-grade tumors. EZH2 effects may also include H3K27me3-independent mechanisms. Our findings suggest that EZH2 may play an important role in thyroid tumor progression.mixedSundling, Kaitlin; Montemayor-Garcia, Celina; Hardin, Heather; Buehler, Darya; Asioli, Sofia; Righi, Alberto; Lloyd, Ricardo .Sundling, Kaitlin; Montemayor-Garcia, Celina; Hardin, Heather; Buehler, Darya; Asioli, Sofia; Righi, Alberto; Lloyd, Ricardo

The Merkel Cell Polyomavirus T Antigens Function as Tumor Promoters in Murine Skin

Merkel cell polyomavirus (MCPyV) causes the majority of human Merkel cell carcinomas (MCC), a rare but highly aggressive form of skin cancer. We recently reported that constitutive expression of MCC tumor-derived MCPyV tumor (T) antigens in the skin of transgenic mice leads to hyperplasia, increased proliferation, and spontaneous epithelial tumor development. We sought to evaluate how the MCPyV T antigens contribute to tumor formation in vivo using a classical, multi-stage model for squamous cell carcinoma development. In this model, two chemical carcinogens, DMBA and TPA, contribute to two distinct phases of carcinogenesis&mdash;initiation and promotion, respectively&mdash;that are required for tumors to develop. By treating the MCPyV transgenic mice with each chemical carcinogen, we determined how the viral oncogenes contributed to carcinogenesis. We observed that the MCPyV T antigens synergized with the tumor initiator DMBA, but not with the tumor promoter TPA, cause tumors. Therefore, the MCPyV tumor antigens function primarily as tumor promoters, similar to that seen with human papillomavirus (HPV) oncoproteins. These studies provide insight into the role of MCPyV T antigen expression in tumor formation in vivo and contribute to our understanding of how MCPyV may function as a human DNA tumor virus

Efficacy of Tie2 Receptor Antagonism in Angiosarcoma12

Angiosarcomas are malignant endothelial cell tumors with few effective systemic treatments. Despite a unique endothelial origin, molecular candidates for targeted therapeutic intervention have been elusive. In this study, we explored the tunica internal endothelial cell kinase 2 (Tie2) receptor as a potential therapeutic target in angiosarcoma. Human angiosarcomas from diverse sites were shown to be universally immunoreactive for Tie2. Tie2 and vascular endothelial growth factor receptor (VEGFR) antagonists inhibited SVR and MS1-VEGF angiosarcoma cell survival in vitro. In the high-grade SVR cell line, Tie2 and VEGF antagonists inhibited cell survival synergistically, whereas effects were largely additive in the low-grade MS1-VEGF cell line. Xenograft modeling using these cell lines closely recapitulated the human disease. In vivo, Tie2 and VEGFR inhibition resulted in significant angiosarcoma growth delay. The combination proved more effective than either agent alone. Tie2 inhibition seemed to elicit tumor growth delay through increased tumor cell apoptosis, whereas VEGFR inhibition reduced tumor growth by lowering tumor cell proliferation. These data identify Tie2 antagonism as a potential novel, targeted therapy for angiosarcomas and provide a foundation for further investigation of Tie2 inhibition, alone and in combinations, in the management of this disease

Long Non-Coding RNA MALAT1 Expression in Thyroid Tissues and Tumors

Background: Long non-coding RNAs (lncRNAs) participate in transcription and in epigenetic or post-transcriptional regulation of gene expression, and may contribute to carcinogenesis. MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1), a lncRNA that participates in the regulation of cell cycle and migration, is known to be deregulated in multiple cancers. Some studies suggest MALAT1 may function as both an oncogene and a tumor suppressor. We analyzed the expression of the MALAT1in thyroid tumors and compared its expression to miR-146b-5p, a microRNA known to be deregulated in papillary thyroid cancer. Design: Tissue microarrays (TMAs) were constructed with formalin-fixed paraffin-embedded (FFPE) tissues of normal thyroid ( NT, n=10), nodular goiters (NG, n=10), follicular adenoma (FA, n=32), follicular carcinoma (FCA, n=28), papillary thyroid carcinoma ( PTC n=28), follicular variant of papillary thyroid carcinoma( FVPTC, n=29), poorly differentiated thyroid carcinomas (PDC, n=21) and anaplastic thyroid carcinoma (ATC, n=35). TMA sections were analyzed by in situ hybridization (ISH) using RNAscope technology with a MALAT1 probe (Advanced Cell Diagnostics). ISH for miR-146b-5p was also performed on the same set of TMAs (Exiqon). qRT-PCR was performed on a subset of the TMA cases (n=16). The results of the MALAT1 TMA ISH were analyzed with Vectra imaging technology, Nuance\uae and inForm \uae software. Results: MALAT1 was highly expressed in NT, NG and in benign and malignant thyroid tumors predominantly in the nucleus, but also in the cytoplasm. The highest levels of MALAT1 wwere observed in PTCs which was significantly higher than in NT (p=0.014) and FVPTC (p=0.016). In contrast NT expressed higher levels of MALAT1 than PDC (p=0.015) or ATC (p<0.001). qRT-PCR analyses supported the ISH findings. Expression of miR-146b-5p was highest in PTC (89%) followed by FVPTC (41%) and was lowest in ATC (8%). Conclusions: MALAT1 is highly expressed in NT tissues and thyroid tumors with increased expression during progression from NT to PTCs. However both MALAT1 and miR-146b-5p are downregulated in ATC compared to PTCs, suggesting that MALAT1 may function both as an oncogene and as a tumor suppressor in different thyroid tumors and that non-coding RNAs may regulate the development of PTCs and ATCs. Category: Endocrine Patholog

Plasmacytoid Dendritic Cells Are Dispensable during Primary Influenza Virus Infection

International audiencePlasmacytoid dendritic cells (pDC) are thought to be pivotal in the first line of defense against viral infections. Although previous studies have suggested that pDC regulate the immune response against respiratory syncytial virus, their role in pulmonary infection with influenza virus has remained unclear. Using mice with GFP-tagged pDC, we observed a marked increase in pDC numbers in the lung airways 3 days after intranasal infection with influenza virus A/PR/8/34. To further investigate their potential involvement in the disease, we made use of pDC-deficient IkarosL/L mice. In the absence of pDC, the recruitment of T cells to the bronchoalveolar space was delayed, which could be reversed by the adoptive transfer of pDC before infection. Surprisingly, however, when compared with wild-type animals, IkarosL/L mice revealed a similar course of disease, as determined by weight loss, viral titers, levels of neutralizing Ab, and lung pathology. Moreover, the activation and differentiation of influenza-specific CD8+ effector T cells was unaltered in the absence of pDC, as was the generation of CD8+ memory T cells. Taken together, our study suggests that pDC regulate the accumulation of T cells in the bronchoalveolar space during early influenza virus infection, but are dispensable for the control of this disease

Plasmacytoid dendritic cells are dispensable during primary influenza virus infection

International audiencePlasmacytoid dendritic cells (pDC) are thought to be pivotal in the first line of defense against viral infections. Although previous studies have suggested that pDC regulate the immune response against respiratory syncytial virus, their role in pulmonary infection with influenza virus has remained unclear. Using mice with GFP-tagged pDC, we observed a marked increase in pDC numbers in the lung airways 3 days after intranasal infection with influenza virus A/PR/8/34. To further investigate their potential involvement in the disease, we made use of pDC-deficient IkarosL/L mice. In the absence of pDC, the recruitment of T cells to the bronchoalveolar space was delayed, which could be reversed by the adoptive transfer of pDC before infection. Surprisingly, however, when compared with wild-type animals, IkarosL/L mice revealed a similar course of disease, as determined by weight loss, viral titers, levels of neutralizing Ab, and lung pathology. Moreover, the activation and differentiation of influenza-specific CD8+ effector T cells was unaltered in the absence of pDC, as was the generation of CD8+ memory T cells. Taken together, our study suggests that pDC regulate the accumulation of T cells in the bronchoalveolar space during early influenza virus infection, but are dispensable for the control of this disease

Merkel Cell Carcinoma Analysis of Outcomes: A 30-Year Experience

<div><p>Background</p><p>Merkel cell carcinoma (MCC) is an aggressive cutaneous malignancy with poor prognosis. Limited data exists to guide treatment decisions. Here we report on our institutional experience and outcomes treating patients with MCC.</p><p>Methods</p><p>A database search (1984-2014) of patients treated at the University of Wisconsin Hospital and Clinics was used to identify patients with histologically confirmed MCC. Patient, tumor, and treatment characteristics were examined via review of medical records. Statistical analyses were performed to assess outcomes and associated prognostic factors.</p><p>Results</p><p>A total of 87 patients with MCC were identified with a median follow-up of 17 months (mean: 38, range: 0-210 months). Two and five-year overall survival rates were 53.9% and 32.8%, respectively. Recurrence was documented in 31.0% of patients (85.2% locoregional, 48.1% distant and 33.3% both). Patients with a history of immunosuppression exhibited significantly worse survival (hazard ratio, 2.01; 95% CI, 1.1-3.7) when compared to immune-competent individuals. The head and neck region was the most common location of primary lesion (N=49) followed by the extremities (N=31). Upper extremity primaries predicted significantly better overall survival (hazard ratio, 0.48; 95% CI, 0.23-0.99) while lower extremity primaries did not have significantly better results (hazard ratio, 0.5; 95% CI, 0.21-1.2) in comparison to head and neck site of primary. Nodal involvement (hazard ratio, 2.95; 95% CI, 1.5-5.79) was also a negative prognostic factor associated with poor overall survival when compared with clinically node negative patients. Primary tumor size > 2 cm (hazard ratio, 1.76; 95% CI, 0.91-3.4) was not associated with survival.</p><p>Conclusions</p><p>This study highlights the role of various factors in determining prognosis of Merkel cell carcinoma; history of immunosuppression, nodal involvement, and head/neck primary predicted worse overall survival. These findings suggest that improvements in both distant and locoregionally directed therapies might play an important role in control of MCC and identify areas for future study.</p></div