Specific Gene Expression Signature Research Articles

The IRX1/HOXA connection: insights into a novel t(4;11)- specific cancer mechanism.

One hallmark of MLL-r leukemia is the highly specific gene expression signature indicative for commonly deregulated target genes. An usual read-out for this transcriptional deregulation is the HOXA gene cluster, where upregulated HOXA genes are detected in MLL-r AML and ALL patients. In case of t(4;11) leukemia, this simple picture becomes challenged, because these patients separate into HOXAhi- and HOXAlo-patients. HOXAlo-patients showed a reduced HOXA gene transcription, but instead overexpressed the homeobox gene IRX1. This transcriptional pattern was associated with a higher relapse rate and worse outcome. Here, we demonstrate that IRX1 binds to the MLL-AF4 complex at target gene promotors and counteract its promotor activating function. In addition, IRX1 induces transcription of HOXB4 and EGR family members. HOXB4 is usually a downstream target of c-KIT, WNT and TPO signaling pathways and necessary for maintaining and expanding in hematopoietic stem cells. EGR proteins control a p21-dependent quiescence program for hematopoietic stem cells. Both IRX1-dependend actions may help t(4;11) leukemia cells to establish a stem cell compartment. We also demonstrate that HDACi administration is functionally interfering with IRX1 and MLL-AF4, a finding which could help to improve new treatment options for t(4;11) patients.

DENV-specific CD4 T-cells dominantly recognize capsid-derived epitopes and display a cytotoxic phenotype

Abstract While DENV-specific CD8+ T cell responses have been extensively studied, the breadth and specificity of CD4+ T cell responses remains to be defined. Here we map CD4 T cell responses in individuals previously exposed to dengue virus. To identify HLA class II candidates, we utilized a panel of algorithms for sixteen common HLA DR molecules representative of the main DR super types. These efforts led to the identification of 365 epitopes derived from all 10 DENV proteins. In contrast to CD8 T cell targets, the highest number of epitopes was associated with the structural capsid protein (C), followed by nonstructural NS3, NS2A, NS5 and envelope proteins (E). Phenotyping of the responding CD4+ T cell subset revealed a DENV specific T cell subset, specifically expanded in donors carrying an allele associated with protection from severe disease and which is absent in DENV negative donors. This subset of DENV specific CD4 T cells is associated with markers of cytotoxic T cells such as Granzyme B, Perforin, CD107a, T bet, Eomes and co-expression of CD8α. We are currently establishing specific gene expression signatures associated with these characteristic phenotypes, as a function of different HLA restricting alleles. Detailed knowledge of phenotype and function of DENV-specific T cells will aid in the establishment of correlates of protection against severe disease.

RNA-Sequencing of Cytogenetically Normal Acute Myeloid Leukemia to Extend Routine Molecular Diagnostics

IntroductionDespite the discovery of new genetic alterations the cytogenetically normal acute myeloid leukemia (CN-AML) subset is still insufficiently characterized. As mRNA-sequencing (RNA-seq) is becoming more accessible, sequencing of the transcriptome could reveal not only information regarding deregulated expression patterns in leukemias, but also enable mutational evaluation of expressed alleles. To address this issue we performed RNA-seq of 5 AML patients from diagnostic bone marrow aspirates to assess the validity of the following: 1) remission samples can be used as control, 2) concordance between standard clinical laboratory analysis and RNA-seq data, and 3) implementation of existing microarray data repository to infer expected mutational status of the sequenced samples based on expression patterns.MethodsDiagnostic samples were selected among patients with high leukemic blast fraction (mean of 75%) and paired remission samples with very low, or no detectable, molecular minimal residual disease. A panel of recurrent somatic mutations was assessed by means of quantitative PCR (qPCR) and fragment analysis for sequencing comparisons. Sequencing was performed on single HiSeq lane aimed at minimum 66 million reads per sample (AROS Applied Biotechnology, Aarhus, Denmark). Biomedical Genomics Workbench 2 was employed for alignment, mutation calling and analysis of differential expression (Robinson-Smyth exact test, Bonferroni corrected). R and Mathematica were used for comparison of expression patterns from the individual samples in conjunction with CN-AML (251) and control bone marrow (73) data from microarray repositories (Gene Expression Omnibus, GSE15434 and GSE13159).Results259 genes were differentially expressed as defined by the following thresholds: normalized fold-change > 2, expression range values > 10 RPKM and p<0.05. Of these, 41 genes were upregulated in AML samples (p<0.01 subset heatmap is shown in fig. 1A). Supervised clustering of the samples on the basis of differential expression patterns efficiently divided the data into 2 groups according to diagnosis and remission status (fig. 1A, dendrogram). A median of 61 mutations per sample was observed (35 to 675). Thirty-four mutations occurred twice or more (fig. 1B, size reflects transcript fraction of mutated allele).A close correlation between routine molecular diagnostics and sequencing data was found. As expected, routine minimal residual disease marker WT1 was in agreement and found to be clearly expressed at diagnosis, but not at time of remission.By comparing patient expression patterns with NPM1, FLT3 or CEPBA mutation specific microarray expression signatures we were largely able to deduce expected mutational status for each patient (fig. 1C, showing the individual matching fraction of mutation specific gene expression signature in terms of upregulated or downregulated) in favor of qPCR analysis shown in table 1.Table 1Comparison of mutational status from qPCR/RNA-seqFLT3mutNPM1mutIDH1mutWT1mutCEBPAmutKITmut#1+/++/++/+-/--/--/-#2+/++/+-/--/--/--/-#3+/++/--/--/--/--/-#4-/-+/-+/++/--/--/-#5+/+-/--/--/--/--/-ConclusionsThis approach serves as a proof of the concept that RNA-sequencing can be directly implemented in the routine laboratory. Moreover, transcriptome data such as these can extend the molecular survey in a dynamic manner by aiding in therapy-related decision-making for the application of targeted therapy and for delineating the reasons for treatment. While publicly available repositories of RNA-seq data are being generated for referencing, it is possible to include microarray data to support molecular classification of the individual patients, as is shown here. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

EZH2 dependent epigenetic landscape in adult T cell leukemia and Tax immortalized cells

Epigenetic regulations globally determine gene transcription. Recent studies have revealed that expression changes and genetic mutations of epigenetic factors cause epigenetic imbalance in cancers. We previously reported that aberrant expression of Polycomb repressive complex 2 (PRC2) components causes constitutive NF-κB activation through silencing of miR-31 in ATL cells. However, the underlying mechanisms by which the epigenetic imbalance is induced and maintained remain to be elucidated. Here, we conducted ChIP-on-chip and transcriptome analyses of ATL and normal CD4+ T cells and found that the epigenetic reprogramming closely associates with ATL specific gene expression signature. Leukemic cell-specific silencing of cell cycle regulator, CDNK1A, was correlated with H3K27me3 level. In addition, orchestrated loss of microRNAs and multiple transcription factors appears to be mediated by the epigenetic mechanism. EZH2 upregulation and H3K27me3 accumulation were found in HTLV-1-infected populations derived from ATL patients as well as asymptomatic carriers. Furthermore, EZH2 inhibition blocked Tax-dependent cell growth and its immortalization in vitro. Intriguingly, the Tax-triggered immortalizing cells partially mimicked the methylation pattern observed in ATL cells, suggesting that the epigenetic alterations are closely involved in immortalization of infected cells and disease progression. In parallel, we found that over expression of PRC2 core components was induced by active signaling cascades including NF-κB in ATL cells. Collectively, our results suggest a coherent positive feedback mechanism comprised of PRC2 and NF-κB signaling, which stabilizes the epigenetic rearrangement and phenotypic outcomes in HTLV-1 infected cells. Since pharmacological inhibition of EZH2 selectively killed ATL and HTLV-1 infected cells in ex vivo culture, targeting the epigenetic elements will hold great promise in treatment and prevention of ATL and HTLV-1-related diseases.

The novel atypical retinoid ST5589 down-regulates Aurora Kinase A and has anti-tumour activity in lymphoma pre-clinical models.

Despite the marked improvements in the treatment of lymphomas, there is still a need for new therapeutic agents. Synthetic retinoids represent a class of compounds with anti-cancer activity. Here, we report the preclinical activity of a new member of this class, the ST1926-derivative ST5589, in lymphomas. ST5589 presented a dose-dependent anti-proliferative activity in almost all of the 25 lymphoma cell lines analysed, with a median 50% inhibitory concentration of 433 nM. Apoptosis was observed in 8/11 cell lines. ST5589 induced changes in the gene expression profiles of the cell lines, including the down-regulation of Aurora Kinase A (AURKA). Specific gene expression signatures were associated with a higher sensitivity to the compound and combination of ST5589 with carfilzomib revealed the importance of proteasome activity in mediating the anti-tumour activity of ST5589. In conclusion, we have identified a new mechanism of action of atypical retinoids as anti-cancer compounds, and the encouraging results obtained with the new ST1926-derivative ST5589 provide the basis for further developments of the compound.

No specific gene expression signature in human granulosa and cumulus cells for prediction of oocyte fertilisation and embryo implantation.

In human IVF procedures objective and reliable biomarkers of oocyte and embryo quality are needed in order to increase the use of single embryo transfer (SET) and thus prevent multiple pregnancies. During folliculogenesis there is an intense bi-directional communication between oocyte and follicular cells. For this reason gene expression profile of follicular cells could be an important indicator and biomarker of oocyte and embryo quality. The objective of this study was to identify gene expression signature(s) in human granulosa (GC) and cumulus (CC) cells predictive of successful embryo implantation and oocyte fertilization. Forty-one patients were included in the study and individual GC and CC samples were collected; oocytes were cultivated separately, allowing a correlation with IVF outcome and elective SET was performed. Gene expression analysis was performed using microarrays, followed by a quantitative real-time PCR validation. After statistical analysis of microarray data, there were no significantly differentially expressed genes (FDR<0,05) between non-fertilized and fertilized oocytes and non-implanted and implanted embryos in either of the cell type. Furthermore, the results of quantitative real-time PCR were in consent with microarray data as there were no significant differences in gene expression of genes selected for validation. In conclusion, we did not find biomarkers for prediction of oocyte fertilization and embryo implantation in IVF procedures in the present study.

Microarray analysis of Arabidopsis under gold exposure to identify putative genes involved in the synthesis of gold nanoparticles (AuNPs).

Very little is known about the genes responsible for Au uptake, reduction and detoxification in plants, which indeed essential to understand the complex trait of AuNP biosynthesis. We designed a targeted experiment to elucidate the response of plant at transcriptional level under Au exposure, and a microarray was performed on root tissue treated with AuCl4− in the absence of nutrient media to record specific gene expression signature. Here, we describe the experimental procedures and data analysis in detail to reproduce the results (available at GEO database under GSE55436) published by Shukla et al. (2014) [1] in the Frontiers in Plant Sciences. The data produced from this study provide significant information of genes which may be used to enhance the AuNP biosynthesis.

Prognostic Analysis of GATA2 Mutations in CEBPA-Mutated Acute Myeloid Leukemia

BackgroundCEBPA mutations are found in approximately 10% of acute myeloid leukemia (AML). Recent studies revealed an association between CEBPA and GATA2 mutations. GATA2 gene encodes a transcription factor involved in hematopoiesis. In two recent studies (Grossmann et al., BJH 2013; Fasan et al., Leukemia 2013), GATA2 mutations appeared to be associated specifically with double CEBPA mutations and improved overall survival (OS). In contrast, another study failed to show any impact on OS (Green et al., BJH 2013). The aim of our study was to investigate the prognostic significance of GATA2 mutations in a large cohort of patients with CEBPA-mutated AML.Patients and methodsWe studied a cohort of 128 patients with CEBPA-mutated AML treated with intensive chemotherapy. The entire coding region of CEBPA, NPM1 exon 12, and GATA2 exons 4 to 6 (that encode the two multifunctional zinc finger domains of the protein) were screened on genomic DNA by PCR and direct Sanger sequencing. FLT3 internal tandem duplications (FLT3-ITD) were screened on genomic DNA by PCR and fragment analysis. Additionally, transcriptome analysis was performed with Affymetrix HG U133 Plus 2.0 array in pre-treatment samples from 72 patients for which RNA was available.ResultsMedian age at AML diagnosis in the whole cohort was 50 years (range, 3-80). Almost all patients belonged to the intermediate cytogenetic risk-group (n=117), of which 90 had a normal karyotype. The remaining patients had favorable (n=1) or adverse cytogenetics (n=4). CEBPA mutations were distributed as follows: 29 single-mutated (sm) cases with N-terminal (N-ter) mutations, 12 sm cases with C-terminal (C-ter) mutations, 80 double-mutated (dm) cases with both N-ter and C-ter mutations, 2 cases with homozygous N-ter and 5 cases with homozygous C-ter mutations. GATA2 mutations were found in 29/128 patients (23%) and were significantly associated with CEBPA dm cases (4/41 sm vs 25/87 dm: Fisher’s exact test p=0.022). NPM1 mutations were detected in 12 patients. They were specifically associated with CEBPA sm cases (12/40 sm vs 0/78 dm, p<0.001) and mutually exclusive with GATA2 mutations (12/93 GATA2 wild-type vs 0/25 GATA2 mutated cases, p<0.001). In contrast with previous studies, FLT3-ITD and GATA2 mutations did co-occur in our cohort, with 3 GATA2 mutants identified in 10 FLT3-ITD positive patients. Transcriptome analysis revealed that GATA2 mutations were not associated with a specific gene expression signature. As previously described, we found that CEBPA dm AML harbored a specific gene expression profile distinct from CEBPA sm cases. Since AML with homozygous CEBPA mutations were found to have a similar gene expression signature as CEBPA dm AML, we decided to pool them together for prognostic analysis. Overall, complete remission was achieved in 113 patients (88%), of whom 36 relapsed (estimated cumulated incidence of relapse [CIR] at 3 and 5 years, 39%). Neither age nor karyotype or gene mutations (including NPM1, FLT3, GATA2 and type of CEBPA mutation) significantly influenced CIR in multivariate analysis. Five-year OS was estimated at 58% in the whole cohort, with longer OS in cases with normal karyotype (p=0.05) and double CEBPAmutations (sm vs dm, p=0.03). In multivariate analysis, younger age (p=0.020) and normal karyotype (p=0.029) remained the only factors significantly associated with a longer OS.ConclusionsThis study confirmed the strong association between GATA2 mutations and CEBPA double mutations, in line with previous studies. However, we did not find any prognostic impact of GATA2 mutations in our cohort of CEBPA-mutated AML. DisclosuresNo relevant conflicts of interest to declare.

Deciphering the Epigenetic Landscape of Relapsed Pediatric Acute Lymphoblastic Leukemia

Introduction: Given the dismal outcome of relapsed pediatric ALL, there is an urgent need to identify underlying drug resistance mechanisms. We have previously discovered that chemosensitivity can be restored by epigenetic reprogramming (Bhatla et al, Blood 2012). Based on our prior work, we hypothesize that epigenetic changes play a major role in mediating chemoresistance and relapse in pediatric ALL. To develop a comprehensive map of relapse specific epigenetic alterations and to understand the impact of epigenetic alterations on the relapse specific gene expression signature, we have embarked on an unbiased genome-wide approach to map the location of key histone marks by chromatin immunoprecipitation sequencing (ChIP-seq) in diagnosis-relapse patient pairs with B-lymphoblastic leukemia.Methods: To date, we have performed ChIP-seq on 13 matched diagnosis/relapse cryopreserved bone marrow samples from patients enrolled on Children’s Oncology Group protocols. We assessed histone marks associated with promoters (H3K4me3, H3K9ac), enhancers (H3K27Ac) and those which are rather widely distributed in euchromatin and heterochromatin (H3K9me3, H3K27me3). 51-cycle single-end sequencing was performed using the Illumina HiSeq2000 Analyzer. Reads were aligned to the Human reference genome (assembly hg19) using the Burrows-Wheeler Alignment tool (BWA, v0.7.7) and post-preocessed using Samtools (v0.1.18). Enriched binding sites (“peaks”) were determined by the peak-calling algorithm, MACS2 (v2.0.10.20131216) using a q-value of 0.01 to define significance. Histone peak deposition on the promoters and enhancer regions were correlated with gene expression data from microarrays obtained from NCI’s TARGET initiative (Therapeutically Applicable Research to Generate Effective Treatment) on the same patients. Promoter regions were defined as 3 kb upstream and downstream of Transcription Start Site (TSS). Super-enhancers were identified by executing the ROSE algorithm (Hnisz et al, Cell 2013).Results: Promoter and enhancer region analysis was carried out only on activating histone marks (H3K4me3, H3K9ac and H3K27Ac) due to their expected uniform and enriched deposition in these regions. We observed that approximately 50% of the genes exhibited transcriptional activation or repression with respective concordant gain or loss of activating histone marks in the majority of patients, while the regulation of rest of the genes seemed independent of histone modification. Next, we sought to determine the impact of histone modification from diagnosis to relapse on the gene expression signature previously established in a cohort of 49 diagnosis-relapse patient pairs (Hogan et al, Blood 2011). Of 60 genes, 46 genes had one or more activating histone marks differentially deposited in the 6 kb promoter region in one or more of the patient samples analyzed and showed concordant expression. Furthermore, differentially up-regulated relapse specific genes such as FOXM1, FANCD2, PRMT7, CENPM and PTBP1 showed concordant deposition of activating histone marks in approximately 50% of relapse samples. Likewise, 5 down-regulated genes including SMEK2 and FOXP1 had concordant loss of these marks in approximately 50% of relapse samples. In order to identify the compendium of distal regulatory enhancers that may govern transcription, we generated chromatin state maps based on the histone modification H3K27ac, which depicts active enhancers. This analysis suggested that the super-enhancers deposited adjacent to genes having higher expression at diagnosis relative to relapse (eg. JARID2, TLE4, ETS1, EBF1 and CIITA), are implicated in transcriptional regulation. Likewise, genes involved in DNA replication and repair such as PHB and TOP3B and those involved in immune regulation such as CD34, IGLL1 and LMO2 were up-regulated with concordant gain of super-enhancers at the time of relapse.Conclusions: In a pilot ChIP-seq analysis of 13 ALL diagnosis/relapse pairs, we have identified several candidate genes, whose transcription appear to be epigenetically regulated and are markers of aggressive disease. Our study further implicates a potential use for epigenetic therapy for the treatment of relapsed ALL. DisclosuresNo relevant conflicts of interest to declare.

Telomerase Inhibition Effectively Targets Mouse and Human AML Stem Cells and Delays Relapse following Chemotherapy

Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer maintained by rare populations of leukemia stem cells (LSCs). Selective targeting of LSCs is a promising approach for treating AML and preventing relapse following chemotherapy, and developing such therapeutic modalities is a key priority. Here, we show that targeting telomerase activity eradicates AML LSCs. Genetic deletion of the telomerase subunit Terc in a retroviral mouse AML model induces cell-cycle arrest and apoptosis of LSCs, and depletion of telomerase-deficient LSCs is partially rescued by p53 knockdown. Murine Terc(-/-) LSCs express a specific gene expression signature that can be identified in human AML patient cohorts and is positively correlated with patient survival following chemotherapy. In xenografts of primary human AML, genetic or pharmacological inhibition of telomerase targets LSCs, impairs leukemia progression, and delays relapse following chemotherapy. Altogether, these results establish telomerase inhibition as an effective strategy for eliminating AML LSCs.

De-regulation of gene expression and alternative splicing affects distinct cellular pathways in the aging hippocampus.

Aging is accompanied by gradually increasing impairment of cognitive abilities and constitutes the main risk factor of neurodegenerative conditions like Alzheimer's disease (AD). The underlying mechanisms are however not well understood. Here we analyze the hippocampal transcriptome of young adult mice and two groups of mice at advanced age using RNA sequencing. This approach enabled us to test differential expression of coding and non-coding transcripts, as well as differential splicing and RNA editing. We report a specific age-associated gene expression signature that is associated with major genetic risk factors for late-onset AD (LOAD). This signature is dominated by neuroinflammatory processes, specifically activation of the complement system at the level of increased gene expression, while de-regulation of neuronal plasticity appears to be mediated by compromised RNA splicing.

Dose-dependent benefits of quercetin on tumorigenesis in the C3(1)/SV40Tag transgenic mouse model of breast cancer

Breast cancer is the leading cause of cancer related death in women. Quercetin is a flavonol shown to have anti-carcinogenic actions. However, few studies have investigated the dose-dependent effects of quercetin on tumorigenesis and none have used the C3(1)/SV40 Tag breast cancer mouse model. At 4 weeks of age female C3(1)/SV40 Tag mice were randomized to one of four dietary treatments (n = 15–16/group): control (no quercetin), low-dose quercetin (0.02% diet), moderate-dose quercetin (0.2% diet), or high-dose quercetin (2% diet). Tumor number and volume was assessed twice a week and at sacrifice (20 wks). Results showed an inverted ‘U’ dose-dependent effect of dietary quercetin on tumor number and volume; at sacrifice the moderate dose was most efficacious and reduced tumor number 20% and tumor volume 78% compared to control mice (C3-Con: 9.0 ± 0.9; C3-0.2%: 7.3 ± 0.9) and (C3-Con: 2061.8 ± 977.0 mm3; and C3-0.2%: 462.9 ± 75.9 mm3). Tumor volume at sacrifice was also reduced by the moderate dose compared to the high and low doses (C3-2%: 1163.2 ± 305.9 mm3; C3-0.02%: 1401.5 ± 555.6 mm3), as was tumor number (C3-2%: 10.7 ± 1.3 mm3; C3-0.02%: 8.1 ± 1.1 mm3). Gene expression microarray analysis performed on mammary glands from C3-Con and C3-0.2% mice determined that 31 genes were down-regulated and 9 genes were up-regulated more than 2-fold (P < 0.05) by quercetin treatment. We report the novel finding that there is a distinct dose-dependent effect of quercetin on tumor number and volume in a transgenic mouse model of human breast cancer, which is associated with a specific gene expression signature related to quercetin treatment.

The effect of freeze-thaw cycles on gene expression levels in lymphoblastoid cell lines.

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene expression. We report a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene expression levels in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. We found that previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that while insight drawn from gene regulatory studies in mature LCLs may generally not be affected by the artificial nature of the LCL model system, many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures.

Ikaros deletions in BCR-ABL-negative childhood acute lymphoblastic leukemia are associated with a distinct gene expression signature but do not result in intrinsic chemoresistance.

Ikaros, the product of IKZF1, is a regulator of lymphoid development and polymorphisms in the gene have been associated with the acute lymphoblastic leukemia (ALL). Additionally, IKZF1 deletions and mutations identify high-risk biological subsets of childhood ALL [Georgopoulos et al. Cell 1995;83(2):289-299; Mullighan et al. N Engl J Md 2009;360(5):470-480]. To discover the underlying pathways modulated by Ikaros we performed gene expression and gene ontology analysis in IKZF1 deleted primary B-ALL pediatric patient samples. To validate downstream targets we performed qPCR on individual patient samples. We also created IKZF1 knockdown B-ALL cell lines with over 50% reduction of Ikaros, mimicking haplosufficient Ikaros deletions, and again performed qPCR to investigate the downstream targets. Finally, to understand the association of Ikaros deletion with a poor prognosis we challenged our IKZF1 knockdown cell lines with chemotherapy and compared responses to IKZF1 wild-type controls. We report a specific gene expression signature of 735 up-regulated and 473 down-regulated genes in IKZF1 deleted primary B-ALL pediatric patient samples. Gene ontology studies revealed an up-regulation of genes associated with cell adhesion, cytoskeletal regulation, and motility in IKZF deleted patient samples. Validated up-regulated target genes in IKZF1 deleted patient samples included CTNND1 and PVRL2 (P = 0.0003 and P = 0.001), and RAB3IP and SPIB (P = 0.005 and P = 0.032) were down-regulated. In further studies in IKZF1 knockdown cell lines, apoptosis assays showed no significant chemoresistance. IKZF1 knockdown alone does not impart intrinsic chemotherapy resistance suggesting that the association with a poor prognosis may be due to additional lesions, microenvironmental interactions with the bone marrow niche, or other factors.

RAS/PI3K Crosstalk and Cetuximab Resistance in Head and Neck Squamous Cell Carcinoma

Cetuximab, an antibody directed against the EGF receptor, is an effective clinical therapy for patients with head and neck squamous cell cancer (HNSCC). Despite great clinical promise, intrinsic or acquired cetuximab resistance hinders successful treatment outcomes but little is known about the underlying mechanism. To study the role of oncogenic HRAS in cetuximab resistance in HNSCC, the frequency of oncogenic HRAS mutations was determined in a cohort of 180 genomic DNAs from head and neck cancer specimens. We also used a combination of cetuximab-resistant cell lines and a transgenic mouse model of RAS-driven oral cancer to identify an oncogenic RAS-specific gene expression signature that promotes cetuximab resistance. Here, we show that activation of RAS signaling leads to persistent extracellular signal-regulated kinase 1/2 signaling and consequently to cetuximab resistance. HRAS depletion in cells containing oncogenic HRAS or PIK3CA restored cetuximab sensitivity. In our study, the gene expression signature of c-MYC, BCL-2, BCL-XL, and cyclin D1 upon activation of MAPK signaling was not altered by cetuximab treatment, suggesting that this signature may have a pivotal role in cetuximab resistance of RAS-activated HNSCC. Finally, a subset of patients with head and neck cancer with oncogenic HRAS mutations was found to exhibit de novo resistance to cetuximab-based therapy. Collectively, these findings identify a distinct cetuximab resistance mechanism. Oncogenic HRAS in HNSCC promotes activation of ERK signaling, which in turn mediates cetuximab resistance through a specific gene expression signature. Clin Cancer Res; 20(11); 2933-46. ©2014 AACR.

Multiparametric diagnostics of cardiomyopathies by microRNA signatures

The diagnosis of cardiomyopathies by endomyocardial biopsy analysis is the gold standard for confirmation of causative reasons but is failing if a sample does not contain the area of interest due to focal pathology. Biopsies are revealing an extract of the current situation of the heart muscle only, and the need for global organ-specific or systemic markers is obvious in order to minimize sampling errors. Global markers like specific gene expression signatures in myocardial tissue may therefore reflect the focal situation or condition of the whole myocardium. Besides gene expression profiles, microRNAs (miRNAs) represent a new group of stable biomarkers that are detectable both in tissue and body fluids. Such miRNAs may serve as cardiological biomarkers to characterize inflammatory processes, to confirm viral infections, and to differentiate various forms of infection. The predictive power of single miRNAs for diagnosis of complex diseases may be further increased if several distinctly deregulated candidates are combined to form a specific miRNA signature. Diagnostic systems that generate disease-related miRNA profiles are based on microarrays, bead-based oligo sorbent assays, or on assays based on real-time polymerase chain reactions and placed on microfluidic cards or nanowell plates. Multiparametric diagnostic systems that can measure differentially expressed miRNAs may become the diagnostic tool of the future due to their predictive value with respect to clinical course, therapeutic decisions, and therapy monitoring. We discuss here specific merits, limitations and the potential of currently available analytical platforms for diagnostics of heart muscle diseases based on miRNA profiling. Contains 34 references.

XBP1 promotes triple-negative breast cancer by controlling the HIF1α pathway

Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization1. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE12 and its substrate XBP13. Previous studies report UPR activation in various human tumors4-6, but XBP1's role in cancer progression in mammary epithelial cells is largely unknown. Triple negative breast cancer (TNBC), a form of breast cancer in which tumor cells do not express the genes for estrogen receptor, progesterone receptor, and Her2/neu, is a highly aggressive malignancy with limited treatment options7, 8. Here, we report that XBP1 is activated in TNBC and plays a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumor growth and tumor relapse and reduced the CD44high/CD24low population. Hypoxia-inducing factor (HIF)1α is known to be hyperactivated in TNBCs 9, 10. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and imply that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.

T(6;9)(p22;q34)/DEK-NUP214-rearranged pediatric myeloid leukemia: an international study of 62 patients

Acute myeloid leukemia with t(6;9)(p22;q34) is listed as a distinct entity in the 2008 World Health Organization classification, but little is known about the clinical implications of t(6;9)-positive myeloid leukemia in children. This international multicenter study presents the clinical and genetic characteristics of 62 pediatric patients with t(6;9)/DEK-NUP214-rearranged myeloid leukemia; 54 diagnosed as having acute myeloid leukemia, representing <1% of all childhood acute myeloid leukemia, and eight as having myelodysplastic syndrome. The t(6;9)/DEK-NUP214 was associated with relatively late onset (median age 10.4 years), male predominance (sex ratio 1.7), French-American-British M2 classification (54%), myelodysplasia (100%), and FLT3-ITD (42%). Outcome was substantially better than previously reported with a 5-year event-free survival of 32%, 5-year overall survival of 53%, and a 5-year cumulative incidence of relapse of 57%. Hematopoietic stem cell transplantation in first complete remission improved the 5-year event-free survival compared with chemotherapy alone (68% versus 18%; P<0.01) but not the overall survival (68% versus 54%; P=0.48). The presence of FLT3-ITD had a non-significant negative effect on 5-year overall survival compared with non-mutated cases (22% versus 62%; P=0.13). Gene expression profiling showed a unique signature characterized by significantly higher expression of EYA3, SESN1, PRDM2/RIZ, and HIST2H4 genes. In conclusion, t(6;9)/DEK-NUP214 represents a unique subtype of acute myeloid leukemia with a high risk of relapse, high frequency of FLT3-ITD, and a specific gene expression signature.

Myocarditis—Personalized Medicine by Expanded Endomyocardial Biopsy Diagnostics

Myocarditis and dilated cardiomyopathy (DCM) are acute or chronic disorders of myocardium. The gold standard for final confirmation of causative reasons of these heart muscle diseases is the endomyocardial biopsy (EMB) analysis. Due to focal pathology, diagnostics are failing if the EMB does not contain the area of interest. Personalized medicine comprises the genetic information together with the phenotypic and environmental factors to yield a tailored healthcare for each individual and removes the limitations of the “one-size-fits-all” therapy approach. This provides the opportunity to translate therapies from bench to bedside, to diagnose and predict disease, and to improve patient-tailored treatments based on the unique signatures of a patient’s disease. Furthermore, novel treatment schedules can be identified which have eventually the chance to enhance long-term survivals. Global biomarkers such as specific gene expression signatures or miRNA profiles not only have the potential to reduce this problem but also add valuable information for individualized therapy decisions. In future, multiplex approaches allowing rapid and absolutely reliable identification of inflammatory or virally-induced myocardial diseases will replace singleplex methods such as direct detection of viral genomes in one single biopsy. Gene or miRNA profiles are upcoming diagnostic biomarkers for cardiomyopathies which are not only detectable in tissue samples but in body fluids as well. Consequently, a systemic diagnostic approach by determination of distinct expression pattern in e.g., peripheral blood samples will support the characterization of distinct cardiomyopathies by means of non-invasive methods.

Placental Macrophages Are Impaired in Chorioamnionitis, an Infectious Pathology of the Placenta

Pregnancy is dependent on maternal-fetal tolerance that may be compromised because of infections or inflammation of the placenta. In this study, we examined whether the context of placental immune tolerance affected the functions of resident macrophages and if their functions were altered during chorioamnionitis, an infectious pathology of the placenta. Macrophages from at-term placentas expressed CD14, exhibited macrophage microbicidal functions, but were less inflammatory than monocyte-derived macrophages. Moreover, placental macrophages spontaneously matured into multinucleated giant cells (MGCs), a property not exhibited by monocyte-derived macrophages, and we detected MGCs of myeloid origin in placental tissue. Compared with placental macrophages, MGCs exhibited a specific phenotype and gene expression signature, consisting of increased cytoskeleton-associated gene expression along with depressed expression of inflammatory response genes. Furthermore, placental macrophages from patients with chorioamnionitis were unable to form MGCs, but this defect was partially corrected by incubating these placental macrophages with control trophoblast supernatants. MGCs formation likely serves to regulate their inflammatory and cytocidal activities in a context that imposes semiallograft acceptance and defense against pathogens.