Atypical Teratoid Rhabdoid Tumor: two Case Reports and an Analysis of Adult Cases With Implications for Pathophysiology and Treatment

We present the first quantitative analysis of atypical teratoid rhabdoid tumors (ATRT) in adults, including two patients from our own institutions. These are of interest as one occurred during pregnancy and one is a long-term survivor. Our review of pathological findings of 50 reported cases of adult ATRT leads us to propose a solely ectodermal origin for the tumor and that epithelial-mesenchymal transition (EMT) is a defining feature. Thus, the term ATRT may be misleading. Our review of clinical findings shows that ATRT tends to originate in mid-line structures adjacent to the CSF, leading to a high rate of leptomeningeal dissemination. Thus, we hypothesize that residual undifferentiated ectoderm in the circumventricular organs, particularly the pituitary and pineal glands, is the most common origin for these tumors. We note that if growth is not arrested soon after diagnosis, or after the first relapse/progression, death is almost universal. While typically rapidly fatal (as in our first case), long-term remission is possible (as in our second). Significant predictors of prognosis were the extent of resection and the use of chemotherapy. Glial differentiation (GFAP staining) was strongly associated with leptomeningeal metastases (chi-squared p = 0.02) and both predicted markedly worse outcomes. Clinical trials including adults are rare. ATRT is primarily a disease of infancy and radiotherapy is generally avoided in those aged less than 3 years old. Treatment options in adults differ from infants in that cranio-spinal irradiation is a viable adjunct to systemic chemotherapy in the adult population. Given the grave prognosis, this combined approach appears reasonable. As effective chemotherapy is likely to cause myelosuppression, we recommend that stem-cell rescue be available locally

Comprehensive and Integrated Genomic Characterization of Adult Soft Tissue Sarcomas

Sarcomas are a broad family of mesenchymal malignancies exhibiting remarkable histologic diversity. We describe the multi-platform molecular landscape of 206 adult soft tissue sarcomas representing 6 major types. Along with novel insights into the biology of individual sarcoma types, we report three overarching findings: (1) unlike most epithelial malignancies, these sarcomas (excepting synovial sarcoma) are characterized predominantly by copy-number changes, with low mutational loads and only a few genes (, , ) highly recurrently mutated across sarcoma types; (2) within sarcoma types, genomic and regulomic diversity of driver pathways defines molecular subtypes associated with patient outcome; and (3) the immune microenvironment, inferred from DNA methylation and mRNA profiles, associates with outcome and may inform clinical trials of immune checkpoint inhibitors. Overall, this large-scale analysis reveals previously unappreciated sarcoma-type-specific changes in copy number, methylation, RNA, and protein, providing insights into refining sarcoma therapy and relationships to other cancer types

Probing glioblastoma and its microenvironment using single-nucleus and single-cell sequencing

Single-cell (scSeq) and single-nucleus sequencing (snSeq) are powerful tools to investigate cancer genomics at single cell resolution. Multiple studies have recently illuminated intratumoral heterogeneity in glioblastoma, however, the majority focused on molecular complexity of tumor cells, without considering unexplored host cell types that contribute to the microenvironment around tumor. To address the glioblastoma microenvironment composition and potential tumor-host interactions, we performed deep coverage sequencing of freshly resected primary GBM patient tissue without implementing any tumor enrichment strategies. The sequencing resulted in 902 cells and 1186 nuclei, respectively, passing quality control and with low mitochondrial gene percentage. We customized reference transcriptome by listing gene transcript loci as exons to take into account immature RNA, which greatly improved the alignment rate for single-nucleus data. We applied Cell Ranger pipelines (Version 3.0.2) and Seurat package (Version 2.3.1) and discovered 10 clusters in both scSeq and snSeq. Pathway analysis of each cluster signature in scSeq data along with known GBM microenvironment cell signatures revealed glioma tumor population along with surrounding microglia/macrophages, astrocytes, pericytes, oligodendrocytes, T cells and endothelial cells. The analysis of snSeq was able to capture the majority of cell types from patient tissues (tumor and microenvironment cells), but interestingly presented different cell type composition in microenvironment cell types such as microglia/macrophages. Integrating single-cell and single-nucleus transcriptomic data using canonical correlation analysis facilitated a comparison of snSeq and scSeq, contrasting depiction for certain cell types (e.g. NKX6-2 gene in Oligodendrocytes). Differential analysis of pathways between tumor and microenvironment cells unveiled potentially rewired pathways such as double strand break repair pathway. Our results demonstrate the cellular diversity of brain tumor microenvironment and lay a foundation to further investigate the individual tumor and host cell transcriptomes that are influenced not only by their cell identity but also by their interaction with surrounding microenvironment

Atypical Teratoid Rhabdoid Tumor: Two Case Reports and an Analysis of Adult Cases with Implications for Pathophysiology and Treatment

We present the first quantitative analysis of atypical teratoid rhabdoid tumors (ATRT) in adults, including two patients from our own institutions. These are of interest as one occurred during pregnancy and one is a long-term survivor. Our review of pathological findings of 50 reported cases of adult ATRT leads us to propose a solely ectodermal origin for the tumor and that epithelial–mesenchymal transition (EMT) is a defining feature. Thus, the term ATRT may be misleading. Our review of clinical findings shows that ATRT tends to originate in mid-line structures adjacent to the CSF, leading to a high rate of leptomeningeal dissemination. Thus, we hypothesize that residual undifferentiated ectoderm in the circumventricular organs, particularly the pituitary and pineal glands, is the most common origin for these tumors. We note that if growth is not arrested soon after diagnosis, or after the first relapse/progression, death is almost universal. While typically rapidly fatal (as in our first case), long-term remission is possible (as in our second). Significant predictors of prognosis were the extent of resection and the use of chemotherapy. Glial differentiation (GFAP staining) was strongly associated with leptomeningeal metastases (chi-squared p = 0.02) and both predicted markedly worse outcomes. Clinical trials including adults are rare. ATRT is primarily a disease of infancy and radiotherapy is generally avoided in those aged less than 3 years old. Treatment options in adults differ from infants in that cranio-spinal irradiation is a viable adjunct to systemic chemotherapy in the adult population. Given the grave prognosis, this combined approach appears reasonable. As effective chemotherapy is likely to cause myelosuppression, we recommend that stem-cell rescue be available locally

Inhibition of NF-κB function suppresses glioma cell migration in vitro and invasion ex vivo.

<p>Treatment with 50 µM SN50 peptide inhibitor suppresses migration of (A) T98G and SNB19 glioma cells in a radial migration assay when compared with untreated (NT) or scrambled peptide treated (SN50M) cells (*; p<0.0001). (B) SNB19 glioma cells stably expressing GFP were implanted into the bilateral putamen on rat organotypic brain slices. Implanted cells were then treated with SN50 peptide inhibitor, scrambled peptide (SN50M) or left untreated (NT) and observed at 48 hrs. Depth of invasion was then calculated from Z-axis images collected by confocal laser scanning microscopy. The mean value of the depth of invasion was obtained from four independent experiments (*; p<0.0001). Treatment with 20 µM NF-κB functional inhibitor (BAY-11-7082) suppresses migration of (C) T98G and SNB19 glioma cells in a radial migration assay when compared with untreated (NT) or DMSO treated (VC) cells (*; p<0.0001). (D) Similar ex vivo brain slice invasion assay as described in B with NF-κB functional inhibitor (BAY-11-7082) demonstrated reduced invasion of T98G and SNB19 glioma cells. The mean value of the depth of invasion was obtained from four independent experiments (*; p<0.0001).</p

Reciprocal Activation of Transcription Factors Underlies the Dichotomy between Proliferation and Invasion of Glioma Cells

<div><p>Histology of malignant glioma depicts dense proliferative areas rich in angiogenesis as well as dissemination of neoplastic cells into adjacent brain tissue. Although the mechanisms that trigger transition from proliferative to invasive phenotypes are complex, the dichotomy of cell proliferation and migration, the “Go or Grow” hypothesis, argues for specific and coordinated regulation of these phenotypes. We investigated transcriptional elements that accompany the phenotypes of migration and proliferation, and consider the therapeutic significance of the “Go or Grow” hypothesis. Interrogation of matched core and rim regions from human glioblastoma biopsy specimens <i>in situ</i> (n = 44) revealed higher proliferation (Ki67 labeling index) in cells residing at the core compared to the rim. Profiling activated transcription factors in a panel of migration-activated versus migration-restricted GBM cells portrayed strong NF-κB activity in the migratory cell population. In contrast, increased c-Myc activity was found in migration-restricted proliferative cells. Validation of transcriptional activity by NF-κB- or c-Myc-driven GFP or RFP, respectively, showed an increased NF-κB activity in the active migrating cells, whereas the proliferative, migration restricted cells displayed increased c-Myc activity. Immunohistochemistry on clinical specimens validated a robust phosphorylated c-Myc staining in tumor cells at the core, whereas increased phosphorylated NF-κB staining was detected in the invasive tumor cells at the rim. Functional genomics revealed that depletion of c-Myc expression by siRNA oligonucleotides reduced cell proliferation <i>in vitro</i>, but surprisingly, cell migration was enhanced significantly. Conversely, inhibition of NF-κB by pharmacological inhibitors, SN50 or BAY-11, decreased both cell migration <i>in vitro</i> and invasion <i>ex vivo</i>. Notably, inhibition of NF-κB was found to have no effect on the proliferation rate of glioma cells. These findings suggest that the reciprocal and coordinated suppression/activation of transcription factors, such as c-Myc and NF-κB may underlie the shift of glioma cells from a “growing-to-going” phenotype.</p></div

Migrating glioma cells promote activation of the transcription factor NF-κB whereas migration-restricted glioma cells display high c-Myc activation.

<p>T98G and SNB19 glioma cells were infected with lentivirus expressing the binding element for either the transcription factor NF-kB and a green fluorescent protein (GFP) reporter or the transcription factor c-Myc and a red fluorescent protein (tdTomato) reporter. Cells were plated in a migration stimulating environment and imaged after 48 hrs. (A) & (B) Quantitative analysis of GFP positive T98G and SNB19 glioma cells respectively, at the core and the rim in radial monolayer assay (n = 5). (C) & (D) Quantitative analysis of tdTomato positive T98G and SNB19 glioma cells respectively, at the core and the rim in radial monolayer assay (n = 5). (E) & (F) Fluorescent micrographs (20X) of mCMV control GFP vector infected T98G and SNB19 glioma cells respectively. (G) & (H) Fluorescent micrographs (20X) of control tdTomato vector infected T98G and SNB19 glioma cells respectively. (I) & (J) Fluorescent micrographs (20X) of NF-κB reporter vector infected T98G and SNB19 glioma cells respectively. (K) & (L) Fluorescent micrographs (20X) of tdTomato reporter vector infected T98G and SNB19 glioma cells respectively. Green cells are GFP positive and blue cells are not expressing the GFP protein but are stained with Hoescht stain. Red cells are tdTomato positive and blue cells are not expressing the tdTomato protein but are stained with Hoescht stain. Fluorescent micrographs of core regions are depicted by “C” and corresponding rim regions are depicted by “R”. Error bar represent the standard deviation of n = 5 observations. Asterisk (*) represents p value <0.05.</p

Knockdown of c-Myc using siRNA increases glioma cell migration and reduces glioma cell proliferation.

<p>Western blot analysis (α-tubulin used as a loading control) to confirm reduction of c-Myc in the glioma cell lines (A) SNB19 and (D) T98G transfected with two c-Myc–specific siRNA. Untransfected cells and cells transfected with a control siRNA (luciferase) are shown for comparison. Treatment with two separate c-Myc siRNA increased migration of (B) SNB19 and (E) T98G cells in a radial migration assay when compared with untreated or luciferase-transfected cells (p<0.001, 2-tailed Student’s t-test). Treatment with two c-Myc siRNA suppressed proliferation of (C) SNB19 and (E) T98G as demonstrated by alamar blue assay when compared with untreated or luciferase transfected cells. Cell Death siRNA (Qiagen) was utilized in the proliferation assay as internal experimental control.</p

Transcription Factor Profiling of Migrating Glioma Cells vs.

<p><b>Migration-Restricted Glioma Cells.</b> Tumor cells were seeded in either migration-activated “sparse” condition or in migration-restricted “dense” condition on SF767 glioma-derived ECM. Nuclear lysates were collected and hybridized with biotinylated-DNA probes specific to 19 transcription factors as per Marligen’s transcription kit and assayed using the Luminex 200. Two independent biological replicates were performed with each sample in triplicate. Ratios of the averaged mean fluorescent intensities for each transcription factor for sparse over dense were calculated for each biological set and average values of two biological replicates are plotted in the heat map above (For ratios of the average MFI for each biological replicate is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072134#pone.0072134.s002" target="_blank">Figure S2</a>). The heat map was constructed using a conditionally formatted color range. Green boxes represent the transcription factors activated when cells were in a migration-activated condition (sparse/dense ratios ≥1.5). Red boxes represent transcription factors activated when cells were in a migration-restricted condition (sparse/dense ratios ≤0.6). Yellow boxes indicate no change in transcription activity (sparse/dense ratios between 0.65 and 1.5).</p

Glioma tumor specimens show differential activation of c-Myc and NF-κB in core and invasive rim.

<p>Immunohistochemistry of matched glioma core and rim sample from a glioma invasion specific tissue microarray (n = 45). Phosphorylated c-Myc nuclear protein expression is greater at the glioma tumor core than the rim regions of tumor. Representative 10X images of core (A) and rim (C) of a GBM sample. 20X images of core (B) and rim (D) of the same GBM sample. Phosphorylated NFκB nuclear protein expression is greater at the glioma tumor rim than the core regions of tumor. Representative 10X images of core (E) and rim (G) of a GBM sample. 20X images of core (F) and rim (H) of the same GBM sample. Black arrows represent positively stained nuclear regions of the glioma tumor cells.</p