Aggressive Subgroup Research Articles

Abstract 1494: MicroRNAs encoded at the 14q32 cluster are associated with poor outcome in lung adenocarcinoma

Abstract The aim of this study was to determine whether different histologic subtypes of lung adenocarcinoma have distinct microRNA (miR) expression profiles and to identify miRs associated with aggressive subgroups of surgically-resected lung adenocarcinoma (AC). We profiled the miR expression of 91 surgically-resected lung adenocarcinomas and 10 matched nonmalignant lung tissues using TaqMan OpenArray Human microRNA panel. The top 3 prognostic miRs (miR-411, miR-370 and miR-376a) were validated by quantitative PCR in an independent cohort of 60 lung AC. To determine potential underlying biological processes associated with selected miRs, we used Affymetrix U133A gene expression microarray data from 51 miR profiled lung AC tumors. Two lung AC cells (SK-LU-1 and NCI-H2228) overexpressing miR-411 were transfected with miRCURY LNA microRNA power inhibitors for miR-411 or non-target using Lipofectamine RNAimax and plated into Boyden chambers for assessing cell migration. No significant differences in clinical characteristics were observed among the training (n=91) and the test set (n=60). Patients' characteristics: 45% male; 86% smokers; 52% stage I, 25% stage II, 19% stage III and 3% stage IV; 26% adjuvant treatment. Unsupervised hierarchical clustering of 356 expressed miRs identified 3 major clusters of lung AC that correlated with stage (P = 0.023), tumor differentiation (P < 0.003) and histological type of lung AC based on IASLC-based classification (P < 0.005). Cluster 1 was enriched in lepidic and mucinous invasive AC while cluster 3 included the most solid AC. Patients classified in cluster 3 had a shorter median overall survival (16.8 months) as compared to the other clusters (60 months for cluster 1 and 2, Log-rank P = 0.002). We identified 22 miRs significantly overexpressed in cluster 3 and associated with poor survival. Strikingly, 11 out of 22 were structurally associated by their genomic location on the long arm of chromosome 14 (14q32). The top prognostic miRs encoded at 14q32.2 (miR-370) and 14q32.31 (miR-411 and miR-376a) were validated by qPCR in an independent cohort of 60 lung AC. In the multivariate analysis, higher levels of these 3 miRs had significantly worse overall survival (HR = 3.12, 95% CI 1.32 - 7.34, P = 0.009) after adjusting by age, gender and stage. Gene Ontology enrichment analysis based on genes significantly correlated with miR-411, miR-370 and miR-376a expression revealed an association with cell migration and cell adhesion. MiR-411 knockdown significantly reduced cell migration (P < 0.05) in 2 lung AC cells. This study demonstrated that different histological types of lung AC have distinct miRNA expression profiles. We identified and validated 3 prognostic miRNAs (miR-411, miR-370 and miR-376a) which are encoded at 14q32. These miRNAs associated with aggressive subtypes of lung adenocarcinoma may have potential to be actionable targets in the future. Citation Format: Ernest Nadal, Jules Lin, Rishindra M. Reddy, Nithya Ramnath, Mark B. Orringer, Andrew C. Chang, David G. Beer, Guoan Chen. MicroRNAs encoded at the 14q32 cluster are associated with poor outcome in lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1494. doi:10.1158/1538-7445.AM2014-1494

O6.10 * GLIOMA CELL VEGFR-2 EXPRESSION IMPAIRS CHEMOTHERAPEUTIC AND ANTIANGIOGENIC TREATMENTS IN PTEN-DEFICIENT GLIOBLASTOMA

BACKGROUND: Given the recent failure of bevacizumab (BEV) in extending overall survival of patients with newly diagnosed glioblastoma, tissue biomarkers predicting response to antiangiogenic treatments are urgently needed. Here, we hypothesized that loss of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a prerequisite for glioma cell expression of vascular endothelial growth factor receptor (VEGFR)-2 in glioblastoma defining a more aggressive glioblastoma subgroup prone to develop evasive resistance towards antiangiogenic treatments including BEV. METHODS AND RESULTS: Besides its ubiquitous expression on endothelial cells, immunohistochemical analysis of 59 glioblastoma tissues showed glioma cell-bound VEGFR-2 expression was restricted to PTEN-deficient tumor specimens and detectable in 20% of all specimens examined, most pronounced in infiltration zones. In glioma cells, pharmacological and RNAi-mediated inhibition of VEGFR-2 signaling revealed proproliferative, antiinvasive and chemoprotective functions for VEGFR-2 in several experimental paradigms comprising flow cytometry-based cell cycle analyses, in vitro proliferation and invasion assays, organotypic brain slice culture assays, and an MRI-monitored orthotopic mouse model. VEGFR-2-dependent cellular effects were concomitant with activation of three potent resistance factors: 'kappa-light-chain-enhancer' of activated B-cells (NF-κB), protein kinase B (AKT/PKB), and N-myc downstream regulated gene (NDRG)1. Two-photon in vivo microscopy demonstrated that glioma cell expression of VEGFR-2 hampers antiangiogenesis and favors a proinvasive response to treatment with BEV. CONCLUSIONS: Glioma cell expression of VEGFR-2, next to its expression on vessels, indicates a particularly aggressive glioblastoma subgroup prone to develop early resistance to chemotherapy with temozolomide or antiangiogenic treatment with BEV. Loss of PTEN can serve as a readily available surrogate marker to identify those tumors upfront by routine neuropathological methods.

Endometrial Carcinoma Recurrence Score (ECARS) validates to identify aggressive disease and associates with markers of epithelial–mesenchymal transition and PI3K alterations

PurposeOur previously reported 29-gene expression signature identified an aggressive subgroup of endometrial cancer patients with PI3K activation. We here wanted to validate these findings by independent patient series. Patients and MethodsThe 29-gene expression signature was assessed in fresh frozen tumor tissue from 280 primary endometrial carcinomas (three independent cohorts), 19 metastatic lesions and in 333 primary endometrial carcinomas using TCGA data, and expression was related to clinico-pathologic features and survival. The 29-gene signature was assessed by real-time quantitative PCR, DNA oligonucleotide microarrays, or RNA sequencing. PI3K alterations were assessed by immunohistochemistry, DNA microarrays, DNA sequencing, SNP arrays or fluorescence in situ hybridization. A panel of markers of epithelial–mesenchymal transition (EMT) was also correlated to the 29-gene signature score. ResultsHigh 29-gene Endometrial Carcinoma Recurrence Score (ECARS) values consistently validated to identify patients with aggressive clinico-pathologic phenotype and reduced survival. Within the presumed favorable subgroups of low grade, endometrioid tumors confined to the uterus, high ECARS still predicted a poor prognosis. The score was higher in metastatic compared to primary lesions (P<0.001) and was significantly associated with potential measures of PI3K activation, markers of EMT and vascular invasion as an indicator of metastatic spread (all P<0.001). ConclusionsECARS validates to identify aggressive endometrial carcinomas in multiple, independent patients cohorts. The higher signature score in metastatic compared to primary lesions, and the potential link to PI3K activation and EMT, support further studies of ECARS in relation to response to PI3K and EMT inhibitors in clinical trials of metastatic endometrial carcinoma.

A MicroRNA Cluster at 14q32 Drives Aggressive Lung Adenocarcinoma

To determine whether different subtypes of lung adenocarcinoma (AC) have distinct microRNA (miRNA) expression profiles, and to identify miRNAs associated with aggressive subgroups of resected lung AC. miRNA expression profile analysis was performed in 91 resected lung AC and 10 matched nonmalignant lung tissues using a PCR-based array. An independent cohort of 60 lung ACs was used for validating by quantitative PCR the top 3 prognostic miRNAs. Gene-expression data from 51 miRNA profiled tumors was used for determining transcript-specific miRNA correlations and gene-enrichment pathway analysis. Unsupervised hierarchical clustering of 356 miRNAs identified 3 major clusters of lung AC correlated with stage (P = 0.023), tumor differentiation (P < 0.003), and IASLC histologic subtype of lung AC (P < 0.005). Patients classified in cluster 3 had worse survival as compared with the other clusters. Eleven of 22 miRNAs associated with poor survival were encoded in a large miRNA cluster at 14q32. The top 3 prognostic 14q32 miRNAs (miR-411, miR-370, and miR-376a) were validated in an independent cohort of 60 lung AC. A significant association with cell migration and cell adhesion was found by integrating gene-expression data with miR-411, miR-370, and miR-376a expression. miR-411 knockdown significantly reduced cell migration in lung AC cell lines and this miRNA was overexpressed in tumors from patients who relapsed systemically. Different morphologic subtypes of lung AC have distinct miRNA expression profiles, and 3 miRNAs encoded at 14q32 (miR-411, miR-370, and miR-376a) were associated with poor survival after lung AC resection.

SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity.

Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL.

Meta-analysis of the global gene expression profile of triple-negative breast cancer identifies genes for the prognostication and treatment of aggressive breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype lacking expression of estrogen and progesterone receptors (ER/PR) and HER2, thus limiting therapy options. We hypothesized that meta-analysis of TNBC gene expression profiles would illuminate mechanisms underlying the aggressive nature of this disease and identify therapeutic targets. Meta-analysis in the Oncomine database identified 206 genes that were recurrently deregulated in TNBC compared with non-TNBC and in tumors that metastasized or led to death within 5 years. This ‘aggressiveness gene list' was enriched for two core functions/metagenes: chromosomal instability (CIN) and ER signaling metagenes. We calculated an ‘aggressiveness score' as the ratio of the CIN metagene to the ER metagene, which identified aggressive tumors in breast cancer data sets regardless of subtype or other clinico-pathological indicators. A score calculated from six genes from the CIN metagene and two genes from the ER metagene recapitulated the aggressiveness score. By multivariate survival analysis, we show that our aggressiveness scores (from 206 genes or the 8 representative genes) outperformed several published prognostic signatures. Small interfering RNA screen revealed that the CIN metagene holds therapeutic targets against TNBC. Particularly, the inhibition of TTK significantly reduced the survival of TNBC cells and synergized with docetaxel in vitro. Importantly, mitosis-independent expression of TTK protein was associated with aggressive subgroups, poor survival and further stratified outcome within grade 3, lymph node-positive, HER2-positive and TNBC patients. In conclusion, we identified the core components of CIN and ER metagenes that identify aggressive breast tumors and have therapeutic potential in TNBC and aggressive breast tumors. Prognostication from these metagenes at the mRNA level was limited to ER-positive tumors. However, we provide evidence that mitosis-independent expression of TTK protein was prognostic in TNBC and other aggressive breast cancer subgroups, suggesting that protection of CIN/aneuploidy drives aggressiveness and treatment resistance.

Diffuse large B-cell lymphoma with combined TP53 mutation and MIR34A methylation: Another "double hit" lymphoma with very poor outcome?

MiR34A, B and C have been implicated in lymphomagenesis, but information on their role in normal CD19+ B-cells (PBL-B) and de novo diffuse large B-cell lymphoma (DLBCL) is limited. We show that in normal and activated B-cells miR34A-5p plays a dominant role compared to other miR34 family members. Only miR34A-5p is expressed in PBL-B, and significantly induced in activated B-cells and reactive lymph nodes. In PBL-B, the MIR34A and MIR34B/C promoters are unmethylated, but the latter shows enrichment for the H3K4me3/H3K27me3 silencing mark. Nine de novo DLBCL cases (n=150) carry both TP53 mutation and MIR34A methylation ("double hit") and these patients have an exceedingly poor prognosis with a median survival of 9.4 months (P<0.0001), while neither TP53 mutation, MIR34A or MIR34B/C promoter methylation alone ("single hit") influence on survival. The TP53/MIR34A "double-hit" is an independent negative prognostic factor for survival (P=0.0002). In 2 DLBCL-cell lines with both TP53 mutation and promoter methylation of MIR34A, miR34A-5p is upregulated by 5-aza-2'deoxycytidine. Thus, the TP53/MIR34A "double hit" characterizes a very aggressive subgroup of DLBCL, which may be treatable with epigenetic therapy prior to or in combination with conventional immunochemotherapy.

PDGFRβ and FGFR2 mediate endothelial cell differentiation capability of triple negative breast carcinoma cells

Triple negative breast cancer (TNBC) is a very aggressive subgroup of breast carcinoma, still lacking specific markers for an effective targeted therapy and with a poorer prognosis compared to other breast cancer subtypes.In this study we investigated the possibility that TNBC cells contribute to the establishment of tumor vascular network by the process known as vasculogenic mimicry, through endothelial cell differentiation.Vascular-like functional properties of breast cancer cell lines were investigated in vitro by tube formation assay and in vivo by confocal microscopy, immunofluorescence or immunohistochemistry on frozen tumor sections. TNBCs express endothelial markers and acquire the ability to form vascular-like channels in vitro and in vivo, both in xenograft models and in human specimens, generating blood lacunae surrounded by tumor cells. Notably this feature is significantly associated with reduced disease free survival. The impairment of the main pathways involved in vessel formation, by treatment with inhibitors (i.e. Sunitinib and Bevacizumab) or by siRNA-mediating silencing, allowed the identification of PDGFRβ and FGFR2 as relevant players in this phenomenon. Inhibition of these tyrosine kinase receptors negatively affects vascular lacunae formation and significantly inhibits TNBC growth in vivo.In summary, we demonstrated that TNBCs have the ability to form vascular-like channels in vitro and to generate blood lacunae lined by tumor cells in vivo. Moreover, this feature is associated with poor outcome, probably contributing to the aggressiveness of this breast cancer subgroup. Finally, PDGFRβ and FGFR2-mediated pathways, identified as relevant in mediating this characteristic, potentially represent valid targets for a specific therapy of this breast cancer subgroup.

Concurrent Activation of Acetylation and Tri-Methylation of H3K27 in a Subset of Hepatocellular Carcinoma with Aggressive Behavior

Analysis of acetylation and tri-methylation of the same residue of histone molecules might identify a subset of hepatocellular carcinoma (HCC) with aggressive behavior. In the present study, we examined acetylation and tri-methylation of lysine 27 on histone H3 (H3K27ac and H3K27me3, respectively) because these two modifications are known to exhibit opposite effects (enhancing and silencing) on gene expression. Neoplastic and non-neoplastic tissues from 198 HCC cases were immunostained with specific monoclonal antibodies against H3K27ac and H3K27me3. The stained tissues were evaluated by an image analyzing program to generate histological scores (H-scores, range 0–300), which were determined by multiplying the percentage of positive-stained cells with the classified immunohistochemical marker intensity (0–3). HCC tissues showed significantly higher H3K27ac (156.7±86.8) and H3K27me3 H-scores (151.8±78.1) compared with the background liver (40.3±33.0 and 64.7±45.6, respectively) (both P<0.001). The cases with H-scores of high-H3K27ac/high-H3K27me3 (n = 54) showed significant correlation with poor differentiation of morphology (P<0.01) and p53-positive staining (P<0.05), and poor prognosis (P<0.01). Confocal microscopy revealed segregated intranuclear localization of both modifications in the individual cancer cells: H3K27ac localization in central euchromatin regions and H3K27me3 in peripheral heterochromatin regions. Concurrent acetylation and methylation at H3K27 occurs in HCC cells in association with p53 abnormalities. These findings demonstrate that image analyzer-assisted H-scores of H3K27ac and H3K27me3 identified an aggressive subgroup of HCC, and could serve as a prognostic marker for HCC.

Selected morphologic features influencing the prognosis of conventional renal cell carcinomas co-expressing P53 and MDM2.

Renal cell carcinoma is the most deadly of common urologic malignancies. The classical prognostic factors, including tumor type, grade and stage, as well as performance status of the patient, offer important information, but there is a need for new biomarkers which could improve the quality of prognostication. It has been proposed that tumors co-expressing P53 and MDM2 could represent a specific, more aggressive subgroup. The aim of the study was to explore this hypothesis using tissue microarrays, using two different anti-P53 antibodies. The material analyzed consisted of 470 cases of renal clear cell carcinoma. Reaction for P53 was positive in 15.1 or 13.2% of cases, depending on the antibody used. Reaction for MDM2 was positive in 37.9% of cases; 6.5 or 5.3% of cases coexpressed P53 and MDM2. Both P53-positive and double P53/MDM2-positive cases were higher grade and more likely to contain a sarcomatoid component, but their stage was similar to negative cases. PAb1081 P53-positive MDM2-positive cases were larger than the rest of the tumors (7.6 cm vs. 6.1 cm, p < 0.001). Our data support the hypothesis of prognostic significance of P53, and double P53/MDM2 positivity, yet further studies are needed to clarify the issue.

Abstract P4-07-18: PDGFRbeta-induced miR-9 is up-regulated in triple negative breast cancer

Abstract miR-9 has been described as an oncogenic microRNA associated to a metastatic phenotype and able to induce EMT (epithelial-to-mesenchymal transition) through direct targeting of E-cadherin. However, data available concerning the expression and the role of this microRNA in different subgroups of breast cancer are still not exhaustive. Evaluating miR-9 expression by Real-Time PCR in a series of 92 breast cancer specimens (35 luminal, 36 HER2, 21 triple negative), we found that this microRNA is increasingly higher in HER2 and Triple Negative versus ER positive patients (fold change 3 and 8 respectively). Moreover, preliminary analysis of miR-9 expression in correlation with bio-pathological features and clinical data also indicates a trend in association with disease progression. Triple Negative Breast Cancers represent a very aggressive breast cancer subgroup, still lacking specific markers for an effective targeted therapy; we investigated whether miR-9 might play a role in the biology of this tumor subtype. Preliminary data indicate that miR-9 is activated downstream PDGFRbeta, which represents a crucial player in the aggressive phenotype of Triple Negative Breast Cancer. In summary, here we show that miR-9 is significantly upregulated in triple negative breast cancer in comparison with other breast cancer subgroups and is activated downstream PDGFRbeta. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-18.

Extramedullary Multiple Myeloma Associated With Reduced Overall Survival: A Retrospective Single-Center Study

IntroductionMultiple Myeloma (MM) is an incurable neoplasm, however recent advancements in therapies have led to median overall survival of 7-10 years in standard risk patients. High risk patients still succumb to the disease within 3 to 5 years. Traditionally used clinical prognostic markers such as the International Staging System (ISS), the Durie-Salmon (DS) staging system, and cytogenetics do not adequately predict response to novel agents or overall prognosis. We previously presented data identifying high risk sub-groups that succumb to shorter progression free survival (PFS) and overall survival (OS) after hematopoietic stem cell transplant (HSCT). Extramedullary disease (EMD) was a highly significant risk factor for poor survival. MethodsA retrospective single institution cohort study was performed of 251 patients who underwent HSCT for MM between 1/1/2001 and 12/31/2011. Of these 251 patients, 18 were identified as having EMD, which was defined by the presence of ≥ 2 plasmacytomas in organs other than bone marrow or bone at any stage of disease. Data points collected included patient and disease characteristics, ISS and DS stage, cytogenetics and FISH, induction therapy, type and number of HSCT, treatment type and cycles, as well as maintenance therapy. Outcome measures included overall response rate (ORR), PFS and OS. Descriptive statistical analysis was conducted for all primary and secondary endpoints. Univariate and multivariate analysis were conducted using the Cox proportional hazards regression model. ResultsPatients with EMD represented 6.6% of our total population. Patient and disease characteristics are found in table 1. Seven of 18 patients had died by the time of data collection. With a median follow up time of 29 months, the median PFS and OS for the entire group (n= 251) compared to the EMD group (n= 18) were PFS: 22.3 months (95% CI 19.75 – 30.32) vs. 12.9 and OS: 57.3 months (95% CI: 46.52 - 77.77) vs. 17.2 (Figs 1 and 2). Results of univariate and multivariate analysis are in table 2. [Display omitted] Table 2Uni and Multivariate analysis of PFS and OS of Patients with EMDPFSOSHazard Ratio (95% Confidence Interval)Pr>ChiSqHazard Ratio (95% Confidence Interval)Pr>ChiSqUnivariate analysis1.65 (0.84- 3.25)0.1402.43 (1.11 – 5.29)0.026Multivariate analysis1.55 (0.63- 3.83)0.3523.65 (0.14 – 9.36)0.007 [Display omitted] [Display omitted] ConclusionExtramedullary multiple myeloma represents a small but highly aggressive subgroup of multiple myeloma. It prevails as a poor prognostic indicator despite the use of novel agents, yet it is not included in traditionally used staging systems. Further confirmatory studies are needed, and likely, new therapeutic approaches will be required for MM patients with EMD. Disclosures:Chen:Genzyme: supported database used for this study Other. Scott:Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

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High levels of secreted frizzled-related protein 1 correlate with poor prognosis and promote tumourigenesis in gastric cancer

BackgroundSecreted frizzled-related protein 1 (sFRP1), Wnt signalling regulator, can positively or negatively regulate tumourigenesis and progression. We sought to determine the clinical relevance and the role of sFRP1 in gastric cancer development and progression. MethodsWe investigated the sFRP1 protein expression levels and its clinicopathological correlations using 85 cases of human gastric samples with survival information (JWCI cohort). mRNA levels of sFRP1 and coexpressed genes were analysed using 131-sample cDNA microarray data (Ruijin cohort). The effects of sFRP1 alteration were investigated using cell proliferation, colony formation, migration, and invasion and xenograft models. ResultsWe show that sFRP1 is overexpressed in some human cancers and is significantly associated with lymph node metastasis and decreased overall survival in gastric cancer patients. Using gastric cancer cell models, we demonstrate that sFRP1 overexpression is correlated with the activation of TGFβ (transforming growth factor-beta) signalling pathway and thereby induces cell proliferation, epithelial–mesenchymal transition (EMT), and invasion. Conversely, sFRP1 knockdown shows the opposite effects. Furthermore, sFRP1 overexpression promotes tumourigenesis and metastasis in a xenograft model. ConclusionOur studies demonstrate that sFRP1 is a biomarker for aggressive subgroups of human gastric cancer and a prognostic biomarker for patients with poor survival. Our data provide insight into a crosstalk between Wnt and TGFβ pathways which underlies gastric cancer development and progression.

Hepatosplenic γδ T-cell Lymphoma: An Overview

Peripheral T-cell lymphomas are a heterogeneous group of lymphoid malignancies. Among these, hepatosplenic γδ T-cell lymphoma (HTCL) represents an aggressive and treatment-resistant subgroup for which new avenues of treatment are critically needed. HTCL is characterized by primary extranodal distribution of the malignant cells with typical intrasinusoidal infiltration of the liver, spleen, and bone marrow, which results in hepatosplenomegaly and peripheral blood cytopenias. Another characteristic feature is the expression of γδ T-cell receptors. HTCL exhibits a rapid progressive course and an extremely poor response to currently known therapeutic strategies, with a 5-year overall survival rate of only 7%. In this review, we discuss the clinical, pathologic, and molecular characteristics of this disease, along with the challenges that are associated with its diagnosis and treatment.

Identification of plant extracts sensitizing breast cancer cells to TRAIL

Triple-negative breast cancer (TNBC) is an aggressive heterogeneous cancer subgroup with a higher rate of distant recurrence and a poorer prognosis compared to other subgroups. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive molecule that induces cell death in various tumor cells without causing cytotoxicity to normal cells; however, primary or acquired resistance to TRAIL often limits its efficacy in cancer patients. To develop combination therapies to improve TRAIL efficacy and/or to overcome the resistant mechanism, we screened 138 medicinal plant extracts against TRAIL-sensitive and -insensitive TNBC cell lines, MDA-MB-231 and MDA-MB-468. Among them, 5 plant extracts, Uvaria dac, Artemisia vulgaris, Cortia depressa, Dichasia bengalensis and Cinnamomum obtusifolium did not cause apparent cytotoxicity (<20%) as a single regimen, but showed significant synergistic effects in combination with TRAIL against both cell lines. Moreover, Uvaria dac, Artemisia vulgaris and Cinnamomum obtusifolium were found to suppress the phosphorylation of p65 that is involved in TRAIL-resistant mechanisms. These observations suggest that the identified plant extracts in combination with TRAIL could lead to potential therapeutic benefits for cancer patients in the clinical setting.

Predictors of early distant metastasis in women with breast cancer

Breast cancer is a clinically heterogeneous disease. Approximately 10-15 % of breast cancer patients develop distant metastases within 2 years of diagnosis with a poor 5-year survival rate of 21 %. Little data have been gathered about how some breast cancer metastasizes earlier than expected. The study aimed to identify predictors of distant metastases among breast cancer patients in relation to their clinical and tumour characteristics. The results of the study may have important implications in our understanding of the disease process allowing more aggressive treatment and monitoring of certain subgroups of patients. Retrospective review of 215 patients (54 % early stage and 46 % locally advanced stage) who fulfilled the specified criteria was performed. Twelve variables were considered. Univariate and multivariate logistic regression analyses were performed to identify independent predictors of developing distant metastasis within 24 months after surgery and adjuvant therapy. Of the 215 patients, 27.9 and 17.7 % developed bone and visceral metastasis, respectively. Bone metastasis was significantly dependent on stage, tumour size, lymph node involvement, lymphovascular invasion, estrogen receptor, progesterone receptor and Her/2-neu pattern (p < 0.05). Visceral metastasis was significantly dependent on age, menopausal status, stage, tumour size, lymph node involvement, estrogen receptor, progesterone receptor and Her-2/neu pattern (p < 0.05). Among patients with bone and visceral metastases, 67 and 43 % have triple negative pattern, respectively. Logistic regression provided an accurate model for predicting bone but not visceral metastasis. A significant fraction of breast cancer patients experienced early metastasis. Our data suggest that tumour stage, size and lymph node involvement are major predictors of metastasis. Her-2/neu over-expression alone is not a strong predictor of early metastasis but triple negative breast cancers belong to an aggressive subgroup with early metastatic capacity. Young, premenopausal patients may benefit from aggressive surveillance and treatment since they tend to present with early visceral spread.

The miR-183~96~182 cluster promotes tumorigenesis in a mouse model of medulloblastoma

Medulloblastoma is the most common malignant pediatric brain tumor. Some are thought to originate from cerebellar granule neuron progenitors (CGNPs) that fail to undergo normal cell cycle exit and differentiation. The contribution of microRNAs to the initiation and progression of medulloblastoma remains poorly understood. Increased expression of the miR-183∼96∼182 cluster of microRNAs has been noted in several aggressive subgroups. We identified that expression of miR-183∼96∼182 was higher in medulloblastomas with Pten gene loss in the background of the activated sonic hedgehog (Shh) signaling pathway. Ectopic miR-183∼96∼182 expression in CGNPs synergized with exogenous Shh to increase proliferation and its role depended on hedgehog signaling activation. Our findings suggest a new microRNA cluster, the miR-183∼96∼182, functionally collaborates with the Shh signaling pathway in the development of medulloblastomas in mice.

Understanding the Role of Mutations in SF3B1 and Splicing in Chronic Lymphocytic Leukemia

AbstractAbstract SCI-15A major challenge in the treatment of chronic lymphocytic leukemia (CLL) is the tremendous clinical heterogeneity of this disease, ranging from indolent disease kinetics to a rapidly fatal course despite aggressive therapy. While some prognostic markers have been established, the advent of deep-sequencing technologies has enabled identification of most or all genetic alterations underlying malignancy, which is leading to new genetic prognostic markers across cancers. We recently sequenced the exomes of 91 CLLs with matched germline controls and identified nine recurrently mutated genes, six pathways involved in CLL pathogenesis (1), as well as new prognostic factors. From this study, a striking finding was the high frequency of mutations in SF3B1, part of the catalytic core of the spliceosome. We found that 14 independent SF3B1 mutations (15% of 91 CLL samples) localized to a single region of the gene (with unknown function) with 50 percent being identical K700E mutations. This finding has been since verified by other investigators. SF3B1 mutations were significantly associated with del(11q), a cytogenetic abnormality that is associated with aggressive disease, and whose minimally deleted region contains the critical DNA damage response gene ATM. Importantly, a Cox multivariate regression model identified mutated SF3B1 as an independent predictive marker of poor prognosis. Mechanistically, CLL samples harboring SF3B1 mutation demonstrated altered pre-mRNA splicing, with increased intron retention in known spliceosome target genes. Finally, SF3B1-K700E was also identified as a recurring mutation in myelodysplasia. In short, mutations in SF3B1 are positively selected in CLL, alter splicing patterns, and represent a novel oncogenic mechanism in hematologic malignancies. Our genetic and clinical evidence suggests that altered pre-mRNA splicing is a novel cancer pathway of importance to CLL and to blood malignancies in general. An ongoing focus of investigation in our group is trying to understand the CLL-promoting activity of SF3B1 mutation by linking alterations in transcripts with functional changes in B-cell biology. Precise identification of driver transcripts and their functional characterization will enable the development of novel therapeutics that can target this pathway more efficaciously and with minimal toxicity. This is especially important in the treatment of a clinically aggressive subgroup of CLL for which effective therapies are presently unavailable. Spliceosome targeting drugs have been identified—discovered through medicinal screens of natural products with tumoricidal properties, and only later found to target the SF3b complex—and may have promise for this subset of patients. Elucidation of this novel cancer pathway has broad implications for cancer biology, from the vantage of both understanding disease mechanism and the potential for therapeutic targeting. Disclosures:No relevant conflicts of interest to declare.