Slug-based epithelial-mesenchymal transition gene signature is associated with prolonged time to recurrence in glioblastoma

Background
We previously identified a precise stage-associated gene expression signature of coordinately expressed genes, including the transcription factor Slug (SNAI2) and other epithelial mesenchymal transition (EMT) markers, present in samples from publicly available gene expression datasets in multiple cancer types. The expression levels of the co-expressed genes vary in a continuous and coordinate manner across the samples, ranging from absence of expression to strong co-expression of all genes. These data suggest that tumor cells may pass through an EMT like process of mesenchymal transition to varying degrees. 

Findings
Here we show that this signature in glioblastoma multiforme (GBM) is associated with time to recurrence following initial treatment. By analyzing data from The Cancer Genome Atlas (TCGA), we found that GBM patients who responded to therapy and had long time to recurrence had low levels of the signature in their tumor samples (P = 3x10^-7^). We also found that the signature is strongly correlated in gliomas with the putative stem cell marker CD44, and is highly enriched among the differentially expressed genes in glioblastomas vs. lower grade gliomas. 

Conclusions 
Our results suggest that long delay before tumor recurrence is associated with absence of the mesenchymal transition signature, raising the possibility that inhibiting this transition might improve the durability of therapy in glioma patients

Genomic Profiling Maps Loss of Heterozygosity and Defines the Timing and Stage Dependence of Epigenetic and Genetic Events in Wilms’ Tumors

To understand genetic and epigenetic pathways in Wilms’ tumors, we carried out a genome scan for loss of heterozygosity (LOH) using Affymetrix 10K single nucleotide polymorphism (SNP) chips and supplemented the data with karyotype information. To score loss of imprinting (LOI) of the IGF2 gene, we assessed DNA methylation of the H19 5V differentially methylated region (DMR). Few chromosomal regions other than band 11p13 (WT1) were lost in Wilms’ tumors from Denys-Drash and Wilms’ tumor-aniridia syndromes, whereas sporadic Wilms’ tumors showed LOH of several regions, most frequently 11p15 but also 1p, 4q, 7p, 11q, 14q, 16q, and 17p. LOI was common in the sporadic Wilms’ tumors but absent in the syndromic cases. The SNP chips identified novel centers of LOH in the sporadic tumors, including a 2.4-Mb minimal region on chromosome 4q24-q25. Losses of chromosomes 1p, 14q, 16q, and 17p were more common in tumors presenting at an advanced stage; 11p15 LOH was seen at all stages, whereas LOI was associated with early-stage presentation. Wilms’ tumors with LOI often completely lacked LOH in the genome-wide analysis, and in some tumors with concomitant 16q LOH and LOI, the loss of chromosome 16q was mosaic, whereas the H19 DMR methylation was complete. These findings confirm molecular differences between sporadic and syndromic Wilms’ tumors, define regions of recurrent LOH, and indicate that gain of methylation at the H19 DMR is an early event in Wilms’ tumorigenesis that is independent of chromosomal losses. The data further suggest a biological difference between sporadic Wilms’ tumors with and without LOI

Correlation of tumor-associated macrophages and clinicopathological factors in Wilms tumor

Background/purpose: Despite high long-term survival rates in patients with Wilms tumor, there is a need to develop better prognostic biomarkers in order to maximize cure while avoiding treatment-associated morbidities. Tumor-associated macrophages have been recently associated with poorer prognosis and increased disease progression in a number of adult cancers. We investigated the relationship between macrophages and clinicopathological fators in this pediatric solid tumor. Methods: Tissue microarray sections of 124 Wilms tumor cases obtained from the Cooperative Human Tissue Network were stained with CD68, a macrophage marker using standard immunohistochemical techniques and quantified using digital image processing techniques. Macrophage densities were correlated by tumor stage, and survival analyses were conducted with available clinical data. Immunohistochemistry was performed on 25 additional Wilms tumor cases obtained from the tumor bank at Columbia University Medical Center and correlated with presence of tumor microvascular invasion. Results: Mean macrophage count densities in stage IV tumors were significantly greater than densities in stage I and III tumors (p=.021, .036). Although the overall and disease-free survival did not differ between high and low macrophage presence groups across all stages, increased macrophage presence was associated with decreased disease-free survival in patients with stage II tumors (p=0.035). Increased macrophage presence may have also correlated with decreased disease-free survival in stage IV patients, but the sample size was too small to allow detection of this difference with significance (p=0.575). Increased macrophage presence was associated with tumor microvascular invasion (p=0.0004). Conclusion: Our results suggest that macrophage recruitment may be associated with disease progression in Wilms tumor. Quantitation of macrophage presence may therefore be a useful adjunct in refining staging algorithms for patients with stage II Wilms tumor. Such data might be useful in the effort to reduce the risk of adverse effects associated with under- or overtreatment of this neoplasm

Aberrant Lymphatic Endothelial Progenitors in Lymphatic Malformation Development

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133− cells isolated from LM fluids. CD133− LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133− LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs.</p

Aberrant Lymphatic Endothelial Progenitors in Lymphatic Malformation Development

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133− cells isolated from LM fluids. CD133− LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133− LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs

Inhibition of cyclo-oxygenase 2 reduces tumor metastasis and inflammatory signaling during blockade of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) blockade is an effective therapy for human cancer, yet virtually all neoplasms resume primary tumor growth or metastasize during therapy. Mechanisms of progression have been proposed to include genes that control vascular remodeling and are elicited by hypoperfusion, such as the inducible enzyme cyclooxygenase-2 (COX-2). We have previously shown that COX-2 inhibition by the celecoxib analog SC236 attenuates perivascular stromal cell recruitment and tumor growth. We therefore examined the effect of combined SC236 and VEGF blockade, using the metastasizing orthotopic SKNEP1 model of pediatric cancer. Combined treatment perturbed tumor vessel remodeling and macrophage recruitment, but did not further limit primary tumor growth as compared to VEGF blockade alone. However, combining SC236 and VEGF inhibition significantly reduced the incidence of lung metastasis, suggesting a distinct effect on prometastatic mechanisms. We found that SC236 limited tumor cell viability and migration in vitro, with effects enhanced by hypoxia, but did not change tumor proliferation or matrix metalloproteinase expression in vivo. Gene set expression analysis (GSEA) indicated that the addition of SC236 to VEGF inhibition significantly reduced expression of gene sets linked to macrophage mobilization. Perivascular recruitment of macrophages induced by VEGF blockade was disrupted in tumors treated with combined VEGF- and COX-2-inhibition. Collectively, these findings suggest that during VEGF blockade COX-2 may restrict metastasis by limiting both prometastatic behaviors in individual tumor cells and mobilization of macrophages to the tumor vasculature

Human cancer cells express Slug-based epithelial-mesenchymal transition gene expression signature obtained in vivo

Background: The biological mechanisms underlying cancer cell motility and invasiveness remain unclear, although it has been hypothesized that they involve some type of epithelial-mesenchymal transition (EMT). Methods: We used xenograft models of human cancer cells in immunocompromised mice, profiling the harvested tumors separately with species-specific probes and computationally analyzing the results. Results: Here we show that human cancer cells express in vivo a precise multi-cancer invasion-associated gene expression signature that prominently includes many EMT markers, among them the transcription factor Slug, fibronectin, and α-SMA. We found that human, but not mouse, cells express the signature and Slug is the only upregulated EMT-inducing transcription factor. The signature is also present in samples from many publicly available cancer gene expression datasets, suggesting that it is produced by the cancer cells themselves in multiple cancer types, including nonepithelial cancers such as neuroblastoma. Furthermore, we found that the presence of the signature in human xenografted cells was associated with a downregulation of adipocyte markers in the mouse tissue adjacent to the invasive tumor, suggesting that the signature is triggered by contextual microenvironmental interactions when the cancer cells encounter adipocytes, as previously reported. Conclusions: The known, precise and consistent gene composition of this cancer mesenchymal transition signature, particularly when combined with simultaneous analysis of the adjacent microenvironment, provides unique opportunities for shedding light on the underlying mechanisms of cancer invasiveness as well as identifying potential diagnostic markers and targets for metastasis-inhibiting therapeutics

Surgical protocol violations in children with renal tumors provides an opportunity to improve pediatric cancer care: a report from the Children’s Oncology Group

BackgroundThe purpose of this study was to evaluate the frequency and characteristics of surgical protocol violations (SPVs) among children undergoing surgery for renal tumors who were enrolled on the Children’s Oncology Group (COG) renal tumor biology and classification study AREN03B2.MethodsAREN03B2 was opened in February 2006, and as on March 31, 2013, there were 3,664 eligible patients. The surgical review forms for 3,536 patients with unilateral disease were centrally reviewed for SPVs. The frequency, type, number of violations, institutional prevalence, and quartiles for SPVs were assessed.ResultsOf the 3,536 patients, there were a total of 505 with at least one SPV (564 total SPVs reported), for an overall incidence of 14.28%. The types of SPVs included a lack of lymph node sampling in 365 (64.7%), avoidable spill in 61 (10.8%), biopsy immediately before nephrectomy in 89 (15.8%), an incorrect abdominal incision in 32 (5.7%), and unnecessary resection of organs in 17 (3.0%). The SPVs occurred in 163 of 215 participating institutions (75.8%). For centers with at least one SPV, the mean number of SPVs reported was 3.10 ± 2.39 (mean ± standard deviation). The incidence of protocol violation per institution ranged from 0 to 67%. Centers with an average of ≤1 case/year had an incidence of SPVs of 12.2 ± 3.8%, those with an average of >1 to 0.05).ConclusionsSPVs that potentially result in additional exposure to chemotherapy and radiation therapy are not uncommon in children undergoing resection of renal malignancies.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134088/1/pbc26083.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134088/2/pbc26083_am.pd

Notch and VEGF pathways play distinct but complementary roles in tumor angiogenesis

Background: Anti-angiogenesis is a validated strategy to treat cancer, with efficacy in controlling both primary tumor growth and metastasis. The role of the Notch family of proteins in tumor angiogenesis is still emerging, but recent data suggest that Notch signaling may function in the physiologic response to loss of VEGF signaling, and thus participate in tumor adaptation to VEGF inhibitors. Methods: We asked whether combining Notch and VEGF blockade would enhance suppression of tumor angiogenesis and growth, using the NGP neuroblastoma model. NGP tumors were engineered to express a Notch1 decoy construct, which restricts Notch signaling, and then treated with either the anti-VEGF antibody bevacizumab or vehicle. Results: Combining Notch and VEGF blockade led to blood vessel regression, increasing endothelial cell apoptosis and disrupting pericyte coverage of endothelial cells. Combined Notch and VEGF blockade did not affect tumor weight, but did additively reduce tumor viability. Conclusions: Our results indicate that Notch and VEGF pathways play distinct but complementary roles in tumor angiogenesis, and show that concurrent blockade disrupts primary tumor vasculature and viability further than inhibition of either pathway alone