Identification and characterization of new biomarkers in aggressive subtypes of breast cancer

En 2015, la récidive tumorale et les métastases du cancer du sein demeurent une cause importante de décès à travers le monde. Toutefois, ces cancers sont souvent hétérogènes car en dépit d’un phénotype similaire, l’évolution clinique et la réponse au traitement peuvent varier considérablement. Il y a donc un intérêt évident à identifier et à caractériser de nouveaux biomarqueurs pour permettre classer les tumeurs mammaires dans des sous-groupes plus homogènes. Notre hypothèse est que chaque cancer mammaire possède des caractéristiques distinctes au plan des altérations du génome et des profils d’expression géniques et que ces changements se traduisent cliniquement par une prédisposition à former des métastases ou à répondre ou non à la chimiothérapie et aux thérapies ciblées. Dans le cadre de nos travaux, nous nous sommes intéressés aux sous-types agressifs de tumeurs mammaires et notamment les cancers de type triple négatif. Nous avons aussi tenté d’identifier des marqueurs capables de distinguer l’une de l’autre les tumeurs de type luminal A et luminal B. Pour ce faire, nous avons d’abord utilisé une stratégie in silico à partir de données publiques (micro-puces d’ADN et séquençage de l’ARN). Nous avons ensuite construit sept micro-matrices tissulaires (TMA) provenant de tissus mammaires normaux et tumoraux fixés à la formaline et enrobés en paraffine. Ces outils nous ont permis d’évaluer par immunohistochimie les niveaux d’expression différentielle des marqueurs suivants : ANXA1, MMP-9, DP103 et MCM2. Ceux-ci ont été comparés aux marqueurs usuels du cancer du sein (ER, PR, HER2, CK5/6 et FOXA1) et corrélés aux données cliniques (survie globale et métastase). Nos résultats indiquent que ces nouveaux marqueurs jouent un rôle important dans l’évolution clinique défavorable des tumeurs de haut grade. Dans un premier article nous avons montré que l’expression d’ANXA1 est dérégulée dans les cancers de type triple-négatif et aussi, dans une certaine mesure, dans les tumeurs HER2+. Nous croyons qu’ANXA1 permet de mieux comprendre le processus d’hétérogénéité tumorale et facilite l’identification des tumeurs de haut grade. Nous proposons également qu’ d’ANXA1 stimule la transition épithélio-mésenchymateuse (EMT) et la formation des métastases. Dans un second temps, nous avons montré que les niveaux d’expression de MMP-9 reflètent la différenciation cellulaire et corrèlent avec les sous-types de cancers mammaires ayant un mauvais pronostic. Nous estimons que MMP-9 permet de mieux comprendre et d’identifier les tumeurs mammaires à haut risque. De fait, la surexpression de MMP-9 est associée à une augmentation des métastases, une récidive précoce et une diminution de la survie globale. Dans le cadre d’un troisième article, nous avons montré que la surexpression du marqueur de prolifération MCM2 s’observe dans les cancers triple-négatifs, HER2+ et Luminal B par comparaison aux cancers luminal A (p< 0.0001). Nos résultats suggèrent qu’en utilisant un seuil de 40% de noyaux marqués, nous pourrions distinguer l’une de l’autre les tumeurs de type luminal A et luminal B. Cela dit, avant de pouvoir envisager l’utilisation de ce marqueur en clinique, une étude de validation sur une nouvelle cohorte de patientes s’impose. En somme, les résultats de nos travaux suggèrent qu’ANXA1, MMP-9 et MCM2 sont des marqueurs intéressants pour mieux comprendre les mécanismes physiopathologiques impliqués dans la progression tumorale et le développement des métastases. À terme, ces nouveaux marqueurs pourraient être utilisés seuls ou en combinaison avec d’autres gènes candidats pour permettre le développement de trousses « multigènes » ou d’essais protéomiques multiplex pour prédire l’évolution clinique des cancers mammaires.

Differential induction, isolation, physicochemical and molecular characterization of temperate phages of environmental Vibrio cholerae as evidence of phage mediated Horizontal Gene Transfer

The resurgence of the enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries like India. The southern Indian state of Kerala is endemic to cholera. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. Marine aquaculture settings and mangrove environments of Kerala serve as reservoirs for V. cholerae. The non-O1/non-O139 environmental isolates of V. cholerae with incomplete ‘virulence casette’ are to be dealt with caution as they constitute a major reservoir of diverse virulence genes in the marine environment and play a crucial role in pathogenicity and horizontal gene transfer. The genes coding cholera toxin are borne on, and can be infectiously transmitted by CTXΦ, a filamentous lysogenic vibriophages. Temperate phages can provide crucial virulence and fitness factors affecting cell metabolism, bacterial adhesion, colonization, immunity, antibiotic resistance and serum resistance. The present study was an attempt to screen the marine environments like aquafarms and mangroves of coastal areas of Alappuzha and Cochin, Kerala for the presence of lysogenic V. cholerae, to study their pathogenicity and also gene transfer potential. Phenotypic and molecular methods were used for identification of isolates as V. cholerae. The thirty one isolates which were Gram negative, oxidase positive, fermentative, with or without gas production on MOF media and which showed yellow coloured colonies on TCBS (Thiosulfate Citrate Bile salt Sucrose) agar were segregated as vibrios. Twenty two environmental V. cholerae strains of both O1 and non- O1/non-O139 serogroups on induction with mitomycin C showed the presence of lysogenic phages. They produced characteristic turbid plaques in double agar overlay assay using the indicator strain V. cholerae El Tor MAK 757. PCR based molecular typing with primers targeting specific conserved sequences in the bacterial genome, demonstrated genetic diversity among these lysogen containing non-O1 V. cholerae . Polymerase chain reaction was also employed as a rapid screening method to verify the presence of 9 virulence genes namely, ctxA, ctxB, ace, hlyA, toxR, zot,tcpA, ninT and nanH, using gene specific primers. The presence of tcpA gene in ALPVC3 was alarming, as it indicates the possibility of an epidemic by accepting the cholera. Differential induction studies used ΦALPVC3, ΦALPVC11, ΦALPVC12 and ΦEKM14, underlining the possibility of prophage induction in natural ecosystems, due to abiotic factors like antibiotics, pollutants, temperature and UV. The efficiency of induction of prophages varied considerably in response to the different induction agents. The growth curve of lysogenic V. cholerae used in the study drastically varied in the presence of strong prophage inducers like antibiotics and UV. Bacterial cell lysis was directly proportional to increase in phage number due to induction. Morphological characterization of vibriophages by Transmission Electron Microscopy revealed hexagonal heads for all the four phages. Vibriophage ΦALPVC3 exhibited isometric and contractile tails characteristic of family Myoviridae, while phages ΦALPVC11 and ΦALPVC12 demonstrated the typical hexagonal head and non-contractile tail of family Siphoviridae. ΦEKM14, the podophage was distinguished by short non-contractile tail and icosahedral head. This work demonstrated that environmental parameters can influence the viability and cell adsorption rates of V. cholerae phages. Adsorption studies showed 100% adsorption of ΦALPVC3 ΦALPVC11, ΦALPVC12 and ΦEKM14 after 25, 30, 40 and 35 minutes respectively. Exposure to high temperatures ranging from 50ºC to 100ºC drastically reduced phage viability. The optimum concentration of NaCl required for survival of vibriophages except ΦEKM14 was 0.5 M and that for ΦEKM14 was 1M NaCl. Survival of phage particles was maximum at pH 7-8. V. cholerae is assumed to have existed long before their human host and so the pathogenic clones may have evolved from aquatic forms which later colonized the human intestine by progressive acquisition of genes. This is supported by the fact that the vast majority of V. cholerae strains are still part of the natural aquatic environment. CTXΦ has played a critical role in the evolution of the pathogenicity of V. cholerae as it can transmit the ctxAB gene. The unusual transformation of V. cholerae strains associated with epidemics and the emergence of V. cholera O139 demonstrates the evolutionary success of the organism in attaining greater fitness. Genetic changes in pathogenic V. cholerae constitute a natural process for developing immunity within an endemically infected population. The alternative hosts and lysogenic environmental V. cholerae strains may potentially act as cofactors in promoting cholera phage ‘‘blooms’’ within aquatic environments, thereby influencing transmission of phage sensitive, pathogenic V. cholerae strains by aquatic vehicles. Differential induction of the phages is a clear indication of the impact of environmental pollution and global changes on phage induction. The development of molecular biology techniques offered an accessible gateway for investigating the molecular events leading to genetic diversity in the marine environment. Using nucleic acids as targets, the methods of fingerprinting like ERIC PCR and BOX PCR, revealed that the marine environment harbours potentially pathogenic group of bacteria with genetic diversity. The distribution of virulence associated genes in the environmental isolates of V. cholerae provides tangible material for further investigation. Nucleotide and protein sequence analysis alongwith protein structure prediction aids in better understanding of the variation inalleles of same gene in different ecological niche and its impact on the protein structure for attaining greater fitness of pathogens. The evidences of the co-evolution of virulence genes in toxigenic V. cholerae O1 from different lineages of environmental non-O1 strains is alarming. Transduction studies would indicate that the phenomenon of acquisition of these virulence genes by lateral gene transfer, although rare, is not quite uncommon amongst non-O1/non-O139 V. cholerae and it has a key role in diversification. All these considerations justify the need for an integrated approach towards the development of an effective surveillance system to monitor evolution of V. cholerae strains with epidemic potential. Results presented in this study, if considered together with the mechanism proposed as above, would strongly suggest that the bacteriophage also intervenes as a variable in shaping the cholera bacterium, which cannot be ignored and hinting at imminent future epidemics.

SCAPA (Spacially Addressable Protein Array) – a Novel Protein Array for the Differential Profiling of Ligand-receptor Induced Signalling

While protein microarray technology has been successful in demonstrating its usefulness for large scale high-throughput proteome profiling, performance of antibody/antigen microarrays has been only moderately productive. Immobilization of either the capture antibodies or the protein samples on solid supports has severe drawbacks. Denaturation of the immobilized proteins as well as inconsistent orientation of antibodies/ligands on the arrays can lead to erroneous results. This has prompted a number of studies to address these challenges by immobilizing proteins on biocompatible surfaces, which has met with limited success. Our strategy relates to a multiplexed, sensitive and high-throughput method for the screening quantification of intracellular signalling proteins from a complex mixture of proteins. Each signalling protein to be monitored has its capture moiety linked to a specific oligo ‘tag’. The array involves the oligonucleotide hybridization-directed localization and identification of different signalling proteins simultaneously, in a rapid and easy manner. Antibodies have been used as the capture moieties for specific identification of each signaling protein. The method involves covalently partnering each antibody/protein molecule with a unique DNA or DNA derivatives oligonucleotide tag that directs the antibody to a unique site on the microarray due to specific hybridization with a complementary tag-probe on the array. Particular surface modifications and optimal conditions allowed high signal to noise ratio which is essential to the success of this approach.

Identification of novel expressed sequences, up-regulated in the leucocytes of chronic fatigue syndrome patients

BACKGROUND: Chronic fatigue syndrome (CFS) is an increasing medical phenomenon of unknown aetiology leading to high levels of chronic morbidity. Of the many hypotheses that purport to explain this disease, immune system activation, as a central feature, has remained prominent but unsubstantiated. Supporting this, a number of important cytokines have previously been shown to be over-expressed in disease subjects. The diagnosis of CFS is highly problematic since no biological markers specific to this disease have been identified. The discovery of genes relating to this condition is an important goal in seeking to correctly categorize and understand this complex syndrome. OBJECTIVE: The aim of this study was to screen for changes in gene expression in the lymphocytes of CFS patients. METHODS: 'Differential Display' is a method for comparing mRNA populations for the induction or suppression of genes. In this technique, mRNA populations from control and test subjects can be 'displayed' by gel electrophoresis and screened for differing banding patterns. These differences are indicative of altered gene expression between samples, and the genes that correspond to these bands can be cloned and identified. Differential display has been used to compare expression levels between four control subjects and seven CFS patients. RESULTS: Twelve short expressed sequence tags have been identified that were over-expressed in lymphocytes from CFS patients. Two of these correspond to cathepsin C and MAIL1 - genes known to be upregulated in activated lymphocytes. The expression level of seven of the differentially displayed sequences have been verified by quantifying relative level of these transcripts using TAQman quantitative PCR. CONCLUSION: Taken as a whole, the identification of novel gene tags up-regulated in CFS patients adds weight to the idea that CFS is a disease characterized by subtle changes in the immune system.

Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats

The recent discovery that vitamin E (VE) regulates gene activity at the transcriptional level indicates that VE may exert part of its biological effects by mechanisms which may be independent of its well-recognised antioxidant function. The objective of this study was the identification of hepatic vitamin E-sensitive genes and examination of the effects of VE on their corresponding biological endpoints. Two groups of male rats were randomly assigned to either a VE-sufficient diet or to a control diet deficient in VE for 290 days. High-density oligonucleotide microarrays comprising over 7000 genes were used to assess the transcriptional response of the liver. Differential gene expression was monitored over a period of 9 months, at four different time-points, and rats were individually profiled. This experimental strategy identified several VE-sensitive genes, which were chronically altered by dietary VE. VE supplementation down-regulated scavenger receptor CD36, coagulation factor IX and 5-alpha-steroid reductase type 1 mRNA levels while hepatic gamma glutamyl-cysteinyl synthetase was significantly up-regulated. Measurement of the corresponding biological endpoints such as activated partial thromboplastin time, plasma dihydrotestosterone and hepatic glutathione substantiated the gene chip data which indicated that dietary VE plays an important role in a range of metabolic processes within the liver. (C) 2004 Elsevier B.V. All rights reserved.

Multiplexed quantitative proteomics using differential metabolic 15N-labeling

There are several advantages of using metabolic labeling in quantitative proteomics. The early pooling of samples compared to post-labeling methods eliminates errors from different sample processing, protein extraction and enzymatic digestion. Metabolic labeling is also highly efficient and relatively inexpensive compared to commercial labeling reagents. However, methods for multiplexed quantitation in the MS-domain (or ‘non-isobaric’ methods), suffer from signal dilution at higher degrees of multiplexing, as the MS/MS signal for peptide identification is lower given the same amount of peptide loaded onto the column or injected into the mass spectrometer. This may partly be overcome by mixing the samples at non-uniform ratios, for instance by increasing the fraction of unlabeled proteins. We have developed an algorithm for arbitrary degrees of nonisobaric multiplexing for relative protein abundance measurements. We have used metabolic labeling with different levels of 15N, but the algorithm is in principle applicable to any isotope or combination of isotopes. Ion trap mass spectrometers are fast and suitable for LC-MS/MS and peptide identification. However, they cannot resolve overlapping isotopic envelopes from different peptides, which makes them less suitable for MS-based quantitation. Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry is less suitable for LC-MS/MS, but provides the resolving power required to resolve overlapping isotopic envelopes. We therefore combined ion trap LC-MS/MS for peptide identification with FTICR LC-MS for quantitation using chromatographic alignment. We applied the method in a heat shock study in a plant model system (A. thaliana) and compared the results with gene expression data from similar experiments in literature.

Single tap identification for fast BCI control

One of the major aims of BCI research is devoted to achieving faster and more efficient control of external devices. The identification of individual tap events in a motor imagery BCI is therefore a desirable goal. EEG is recorded from subjects performing and imagining finger taps with their left and right hands. A Differential Evolution based feature selection wrapper is used in order to identify optimal features in the spatial and frequency domains for tap identification. Channel-frequency band combinations are found which allow differentiation of tap vs. no-tap control conditions for executed and imagined taps. Left vs. right hand taps may also be differentiated with features found in this manner. A sliding time window is then used to accurately identify individual taps in the executed tap and imagined tap conditions. Highly statistically significant classification accuracies are achieved with time windows of 0.5 s and more allowing taps to be identified on a single trial basis.

Dynamic recurrent neural network for system identification and control

A dynamic recurrent neural network (DRNN) that can be viewed as a generalisation of the Hopfield neural network is proposed to identify and control a class of control affine systems. In this approach, the identified network is used in the context of the differential geometric control to synthesise a state feedback that cancels the nonlinear terms of the plant yielding a linear plant which can then be controlled using a standard PID controller.

Identification of potential mechanisms of toxicity after di-(2-ethylhexyl)-phthalate (DEHP) adult exposure in the liver using a systems biology approach

Phthalates are industrial additives widely used as plasticizers. In addition to deleterious effects on male genital development, population studies have documented correlations between phthalates exposure and impacts on reproductive tract development and on the metabolic syndrome in male adults. In this work we investigated potential mechanisms underlying the impact of DEHP on adult mouse liver in vivo. A parallel analysis of hepatic transcript and metabolic profiles from adult mice exposed to varying DEHP doses was performed. Hepatic genes modulated by DEHP are predominantly PPARalpha targets. However, the induction of prototypic cytochrome P450 genes strongly supports the activation of additional NR pathways, including Constitutive Androstane Receptor (CAR). Integration of transcriptomic and metabonomic profiles revealed a correlation between the impacts of DEHP on genes and metabolites related to heme synthesis and to the Rev-erbalpha pathway that senses endogenous heme level. We further confirmed the combined impact of DEHP on the hepatic expression of Alas1, a critical enzyme in heme synthesis and on the expression of Rev-erbalpha target genes involved in the cellular clock and in energy metabolism. This work shows that DEHP interferes with hepatic CAR and Rev-erbalpha pathways which are both involved in the control of metabolism. The identification of these new hepatic pathways targeted by DEHP could contribute to metabolic and endocrine disruption associated with phthalate exposure. Gene expression profiles performed on microdissected testis territories displayed a differential responsiveness to DEHP. Altogether, this suggests that impacts of DEHP on adult organs, including testis, could be documented and deserve further investigations.

Genes driving potato tuber initiation and growth: identification based on transcriptional changes using the POCI array

The increasing amount of available expressed gene sequence data makes whole-transcriptome analysis of certain crop species possible. Potato currently has the second largest number of publicly available expressed sequence tag (EST) sequences among the Solanaceae. Most of these ESTs, plus other proprietary sequences, were combined and used to generate a unigene assembly. The set of 246,182 sequences produced 46,345 unigenes, which were used to design a 44K 60-mer oligo array (Potato Oligo Chip Initiative: POCI). In this study, we attempt to identify genes controlling and driving the process of tuber initiation and growth by implementing large-scale transcriptional changes using the newly developed POCI array. Major gene expression profiles could be identified exhibiting differential expression at key developmental stages. These profiles were associated with functional roles in cell division and growth. A subset of genes involved in the regulation of the cell cycle, based on their Gene Ontology classification, exhibit a clear transient upregulation at tuber onset indicating increased cell division during these stages. The POCI array allows the study of potato gene expression on a much broader level than previously possible and will greatly enhance analysis of transcriptional control mechanisms in a wide range of potato research areas. POCI sequence and annotation data are publicly available through the POCI database (http://pgrc.ipk-gatersleben.de/poci).

The identification of QTL controlling ergot sclerotia size in hexaploid wheat implicates a role for the Rht dwarfing alleles

The fungal pathogen Claviceps purpurea infects ovaries of a broad range of temperate grasses and cereals, including hexaploid wheat, causing a disease commonly known as ergot. Sclerotia produced in place of seed carry a cocktail of harmful alkaloid compounds that result in a range of symptoms in humans and animals, causing ergotism. Following a field assessment of C. purpurea infection in winter wheat, two varieties ‘Robigus’ and ‘Solstice’ were selected which consistently produced the largest differential effect on ergot sclerotia weights. They were crossed to produce a doubled haploid mapping population, and a marker map, consisting of 714 genetic loci and a total length of 2895 cM was produced. Four ergot reducing QTL were identified using both sclerotia weight and size as phenotypic parameters; QCp.niab.2A and QCp.niab.4B being detected in the wheat variety ‘Robigus’, and QCp.niab.6A and QCp.niab.4D in the variety ‘Solstice’. The ergot resistance QTL QCp.niab.4B and QCp.niab.4D peaks mapped to the same markers as the known reduced height (Rht) loci on chromosomes 4B and 4D, Rht-B1 and Rht-D1, respectively. In both cases, the reduction in sclerotia weight and size was associated with the semi-dwarfing alleles, Rht-B1b from ‘Robigus’ and Rht-D1b from ‘Solstice’. Two-dimensional, two-QTL scans identified significant additive interactions between QTL QCp.niab.4B and QCp.niab.4D, and between QCp.niab.2A and QCp.niab.4B when looking at sclerotia size, but not between QCp.niab.2A and QCp.niab.4D. The two plant height QTL, QPh.niab.4B and QPh.niab.4D, which mapped to the same locations as QCp.niab.4B and QCp.niab.4D, also displayed significant genetic interactions.

A genome-wide test of the differential susceptibility hypothesis reveals a genetic predictor of differential response to psychological treatments for child anxiety disorders

Background: The differential susceptibly hypothesis suggests that certain genetic variants moderate the effects of both negative and positive environments on mental health and may therefore be important predictors of response to psychological treatments. Nevertheless, the identification of such variants has so far been limited to preselected candidate genes. In this study we extended the differential susceptibility hypothesis from a candidate gene to a genome-wide approach to test whether a polygenic score of environmental sensitivity predicted response to Cognitive Behavioural Therapy (CBT) in children with anxiety disorders. Methods: We identified variants associated with environmental sensitivity using a novel method in which within-pair variability in emotional problems in 1026 monozygotic (MZ) twin pairs was examined as a function of the pairs’ genotype. We created a polygenic score of environmental sensitivity based on the whole-genome findings and tested the score as a moderator of parenting on emotional problems in 1,406 children and response to individual, group and brief parent-led CBT in 973 children with anxiety disorders. Results: The polygenic score significantly moderated the effects of parenting on emotional problems and the effects of treatment. Individuals with a high score responded significantly better to individual CBT than group CBT or brief parent-led CBT (remission rates: 70.9%, 55.5% and 41.6% respectively). Conclusions: Pending successful replication, our results should be considered exploratory. Nevertheless, if replicated, they suggest that individuals with the greatest environmental sensitivity may be more likely to develop emotional problems in adverse environments, but also benefit more from the most intensive types of treatment.

Differential proteome analysis of mature and germinated embryos of Araucaria angustifolia

Araucaria angustifolia is an endangered Brazilian native conifer tree. The aim of the present work was to identify differentially expressed proteins between mature and germinated embryos of A. angustifolia, using one and two dimensional gel electrophoresis approaches followed by protein identification by tandem mass spectrometry. The identities of 32 differentially expressed protein spots from two dimensional gel maps were successfully determined, including proteins and enzymes involved in storage mobilization such as the vicilin-like storage protein and proteases. A label free approach, based on spectral counts, resulted in detection of 10 and 14 mature and germinated enriched proteins, respectively. Identified proteins were mainly related to energetic metabolism pathways, translational processes. oxidative stress regulation and cellular signaling. The integrated use of both strategies permitted a comprehensive protein expression overview of changes in germinated embryos in relation to matures, providing insights into the this process in a recalcitrant seed species. Applications of the data generated on the monitoring and control of in vitro somatic embryos were discussed. Published by Elsevier Ltd.

Differential expression of new LTA splice variants upon lymphocyte activation

Lymphotoxin alpha (LTA) is a member of the TNF cytokine superfamily, produced principally by lymphocytes. It plays an important role in immune and inflammatory responses. Many TNF superfamily members have functionally important isoforms generated by alternative splicing but alternative splicing of LTA has never been studied. The known LTA protein is encoded by a transcript containing four exons. Here we report seven new LTA splice variants, three of them evolutionary conserved. We demonstrate their presence in cytoplasmic RNA suggesting that they could be translated into new LTA isoforms. We observed that their expression is differentially regulated upon activation of peripheral blood mononuclear cells and lymphocyte subpopulations (CD4+, CD8+, and CD19+). Our data suggest that the new LTA splice variants might play a role in the regulation of the immune response. (C) 2007 Elsevier Ltd. All rights reserved.

Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma

The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue were obtained from six patients. A custom-built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down-regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave-one-out patient cross-validation. Among the down-regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein-coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (P<0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (P<0.05), were confirmed in clear cell RCC by Real-time RT-PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells. (C) 2008 Wiley-Liss, Inc.