Expression profiling of microRNAs and isomiRs in conventional central chondrosarcoma

Conventional central chondrosarcoma (CCC) is a malignant bone tumor that is characterized by the production of chondroid tissue. Since radiation therapy and chemotherapy have limited effects on CCC, treatment of most patients depends on surgical resection. This study aimed to identify the expression profiles of microRNAs (miRNAs) and isomiRs in CCC tissues to highlight their possible participation to the regulation of pathways critical for the formation and growth of this type of tumor. Our study analyzed miRNAs and isomiRs from Grade I (GI), Grade II (GII), and Grade III (GIII) histologically validated CCC tissue samples. While the different histological grades shared a similar expression profile for the top abundant miRNAs, we found several microRNAs and isomiRs showing a strong different modulation in GII + GIII vs GI grade samples and their involvement in tumor biology could be consistently hypothesized. We then in silico validated these differently expressed miRNAs in a larger chondrosarcoma public dataset and confirmed the expression trend for 17 out of 34 miRNAs. Our results clearly suggests that the contribution of miRNA deregulation, and their targeted pathways, to the progression of CCC could be relevant and strongly indicates that when studying miRNA deregulation in tumors, not only the canonical miRNAs, but the whole set of corresponding isomiRs should be taken in account. Improving understanding of the precise roles of miRNAs and isomiRs over the course of central chondrosarcoma progression could help identifying possible targets for precision medicine therapeutic intervention

Transcriptomic profiling of the development of the inflammatory response in human monocytes in vitro.

Monocytes/macrophages are key players in all phases of physiological and pathological inflammation. To understanding the regulation of macrophage functional differentiation during inflammation, we designed an in vitro model that recapitulates the different phases of the reaction (recruitment, initiation, development, and resolution), based on human primary blood monocytes exposed to sequential changes in microenvironmental conditions. All reaction phases were profiled by transcriptomic microarray analysis. Distinct clusters of genes were identified that are differentially regulated through the different phases of inflammation. The gene sets defined by GSEA analysis revealed that the inflammatory phase was enriched in inflammatory pathways, while the resolution phase comprised pathways related to metabolism and gene rearrangement. By comparing gene clusters differentially expressed in monocytes vs. M1 and vs. M2 macrophages extracted from an in-house created meta-database, it was shown that cells in the model resemble M1 during the inflammatory phase and M2 during resolution. The validation of inflammatory and transcriptional factors by qPCR and ELISA confirmed the transcriptomic profiles in the different phases of inflammation. The accurate description of the development of the human inflammatory reaction provided by this in vitro kinetic model can help in identifying regulatory mechanisms in physiological conditions and during pathological derangement

Genomic organization and evolution of double minutes/homogeneously staining regions with MYC amplification in human cancer

The mechanism for generating double minutes chromosomes (dmin) and homogeneously staining regions (hsr) in cancer is still poorly understood. Through an integrated approach combining next-generation sequencing, single nucleotide polymorphism array, fluorescent in situ hybridization and polymerase chain reaction-based techniques, we inferred the fine structure of MYC-containing dmin/hsr amplicons harboring sequences from several different chromosomes in seven tumor cell lines, and characterized an unprecedented number of hsr insertion sites. Local chromosome shattering involving a single-step catastrophic event (chromothripsis) was recently proposed to explain clustered chromosomal rearrangements and genomic amplifications in cancer. Our bioinformatics analyses based on the listed criteria to define chromothripsis led us to exclude it as the driving force underlying amplicon genesis in our samples. Instead, the finding of coexisting heterogeneous amplicons, differing in their complexity and chromosome content, in cell lines derived from the same tumor indicated the occurrence of a multi-step evolutionary process in the genesis of dmin/hsr. Our integrated approach allowed us to gather a complete view of the complex chromosome rearrangements occurring within MYC amplicons, suggesting that more than one model may be invoked to explain the origin of dmin/hsr in cancer. Finally, we identified PVT1 as a target of fusion events, confirming its role as breakpoint hotspot in MYC amplification

Integrated genomics analysis of gene and miRNA expression profiles in clear cell renal carcinoma cell lines

Clear cell renal cell carcinoma (ccRCC) is the most common and malignant tumor in the adult kidney, representing 75-80% of renal primary malignancies. Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene, by mutation, deletion and promoter methylation, occurs in most sporadic ccRCCs and in all inherited cases. Recent evidences showed that microRNAs (miRNAs) are often dysregulated in many tumors, including ccRCC. We used Caki-1, Caki-2 and A498 cell lines as in vitro model of ccRCC pathology, and HK-2 (normal proximal tubular epithelial cell line) as reference sample. We characterized the VHL status by direct sequencing and the HIF status by western blot. Affymetrix microarray technology was applied to assess miRNA (onto GeneChip\uae miRNA Array) and gene expression profiles (onto GeneChip\uae Human Gene 1.0 ST Array). Analysis of common differentially expressed miRNAs (DEMs) in RCC cell lines outlined specific miRNAs to be involved in ccRCC and in cancer (e.g. miR-145, miR-29a, miR-342-3p, miR-205, miR-183, miR-197, miR-132, miR-146a and miR-34a). Functional enrichment analysis of common differentially expressed genes (DEGs) highlighted some genes involved in leucocyte transendothelial migration, focal adhesion and p53 signalling pathways (e.g. ATM, FER, CDC27 and GRB10). Additionally, we conducted an integrated analysis to combine gene and miRNA expression profiles and to reconstruct miRNA-gene post-transcriptional regulatory networks involved in RCC pathology. We also compared our expression data with RCC datasets publicly available from NCBI GEO microarray repository. We selected potentially interesting miRNAs and target genes for further validation (by qPCR). This integrated analysis approach may help to unravel the molecular complexity characterizing ccRCC biology, and it will facilitate the elucidation of regulatory circuits important for tumorigenesis and the biological processes under relevant post-transcriptional regulation in ccRCC

Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues

<p>Abstract</p> <p>Background</p> <p>Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues.</p> <p>Methods</p> <p>We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation).</p> <p>Results</p> <p>A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed.</p> <p>Conclusions</p> <p>ccRCC primary cultures are a reliable <it>in vitro </it>model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches aimed to study genes or pathways involved in ccRCC etiopathogenesis and to identify novel clinical markers or therapeutic targets. Moreover, SNP array technology proved to be a powerful tool to better define the cell composition and homogeneity of RCC primary cultures.</p

Analisi del numero di copie e sequenza del DNA in campioni sequenziali di melanoma alla diagnosi e recidiva=DNA copy number and whole-exome sequencing analyses in sequential myeloma samples at diagnosis and relapse

Introduction. Multiple myeloma (MM) is a plasma cell (PC) malignancy characterized by a marked genetic heterogeneity at onset, followed by further genomic complexity acquired during disease progression and particularly after treatment. To gain insight into the molecular evolution associated with MM progression, we investigated sequential samples of 7 MMs and 1 primary PC leukemia (pPCL) by genome-wide DNA copy number analysis and whole-exome sequencing (WES). Methods. Highly purified PC samples obtained at diagnosis and relapse after first line therapy (7 symptomatic MMs and 1 pPCL) were subjected to genome-wide DNA profiling (all cases) and WES (1 MM and 1 pPCL samples, with matched negative controls). Copy number data were generated on Affymetrix CytoScan HD Array, using the Chromosome Analysis Suite software. Single sample analysis was performed with default parameters and setting the Reference Model. WES was carried out on Illumina GAIIx platform and variant calling was performed using Mutect algorithm, by separately comparing primary and progression samples to its matched normal control. Results. Concerning regions of prognostic importance, 1p loss was identified as a novel lesion or evolving from a sub-clone in 3 relapsed samples, whereas 1q gain or 17p loss were respectively acquired in two cases. Notably, some alterations, present at diagnosis only in sub-clones, were detected in the majority of tumor cells in at least one of 5 relapsed MMs. Such lesions involved single or combined gains or losses of whole chromosomes (chr) 3, 8, 9, 18, 20, aberrations of short/long arms of chrs 11, 13, 14, 15, 18, 21 or smaller altered regions on chrs 4, 10, 12, 16, 17, 19. Interestingly, deletions involving chr 5q (3/8 relapsed MMs) or 8q (2/8 relapsed MMs) were detected as de novo acquired lesions. Furthermore, we investigated by WES two cases, a MM progressed to secondary PCL (sPCL) and a pPCL patient at diagnosis and relapse. Each patient showed a dynamic mutational pattern, involving both the acquisition and the loss of a large number of point mutations. Specifically, 19 genes were exclusively mutated in MM at diagnosis and 66 only in sPCL phase, whereas 12 genes were mutated in both conditions; in pPCL patient, 138 genes were evidenced at diagnosis and 166 at relapse, while 78 were commonly altered. Genes acquiring mutations in disease course were mostly involved in DNA repair, histone methylation, protein metabolism, regulation of NF-kB cascade, focal adhesion and MAPK signaling pathways. Concerning genes frequently altered in MM, it is worth reporting mutations of TP53 and CYLD at relapse, in sPCL and pPCL, respectively. Conclusions. Our data highlight the importance of using high-throughput approaches to provide insights into the definition of genetic alterations potentially related to mechanisms of drug resistance and MM progression

An integrated analysis of molecular aberrations in NCI-60 cell lines

<p>Abstract</p> <p>Background</p> <p>Cancer is a complex disease where various types of molecular aberrations drive the development and progression of malignancies. Large-scale screenings of multiple types of molecular aberrations (e.g., mutations, copy number variations, DNA methylations, gene expressions) become increasingly important in the prognosis and study of cancer. Consequently, a computational model integrating multiple types of information is essential for the analysis of the comprehensive data.</p> <p>Results</p> <p>We propose an integrated modeling framework to identify the statistical and putative causal relations of various molecular aberrations and gene expressions in cancer. To reduce spurious associations among the massive number of probed features, we sequentially applied three layers of logistic regression models with increasing complexity and uncertainty regarding the possible mechanisms connecting molecular aberrations and gene expressions. Layer 1 models associate gene expressions with the molecular aberrations on the same loci. Layer 2 models associate expressions with the aberrations on different loci but have known mechanistic links. Layer 3 models associate expressions with nonlocal aberrations which have unknown mechanistic links. We applied the layered models to the integrated datasets of NCI-60 cancer cell lines and validated the results with large-scale statistical analysis. Furthermore, we discovered/reaffirmed the following prominent links: (1)Protein expressions are generally consistent with mRNA expressions. (2)Several gene expressions are modulated by composite local aberrations. For instance, CDKN2A expressions are repressed by either frame-shift mutations or DNA methylations. (3)Amplification of chromosome 6q in leukemia elevates the expression of MYB, and the downstream targets of MYB on other chromosomes are up-regulated accordingly. (4)Amplification of chromosome 3p and hypo-methylation of PAX3 together elevate MITF expression in melanoma, which up-regulates the downstream targets of MITF. (5)Mutations of TP53 are negatively associated with its direct target genes.</p> <p>Conclusions</p> <p>The analysis results on NCI-60 data justify the utility of the layered models for the incoming flow of cancer genomic data. Experimental validations on selected prominent links and application of the layered modeling framework to other integrated datasets will be carried out subsequently.</p

Association of genome-wide DNA copy number data and transcriptional profile in renal carcinoma

The work comprised in this PhD thesis described the development of a novel mathematical and statistical framework to analyse and combine, at genome-wide level, gene expression profile and DNA copy number data obtained by high-throughput oligonucleotide microarray platform. This dual strategy is now considered the most effective to understand the genetic causes underlying neoplastic diseases and identify interesting regions and genes with potential clinical application as novel tumor markers. In this thesis, we applied this combined approach to study the clear cell renal carcinoma (ccRCC) pathology, using firstly a human metastatic cell line as in vitro model and then a collection of clinical tumor tissue samples. Considering the physical position of genes along the genome, high-throughput gene expression data were used to assemble a regional transcriptional activity profile. In the meantime, a genome-wide DNA copy number map was assembled by high-throughput SNP mapping technology, thus identifying recurrent aberrations that might be novel candidate regions characterizing all or subsets of ccRCC samples. To filter the large amount of array-based data and narrow down the hundreds of candidate regions to those whose altered expression level was attributable to underlying chromosomal alterations, regional gene expression data were combined with DNA copy number alteration map at genome-wide level. After confirming a strong association between aneuploidy and regional transcriptional activity profiles, we identified a set of regions showing concomitant DNA alteration and modulated expression level and, within, particularly interesting genes as novel candidate RCC-related markers. Overall, this study demonstrates the efficacy of the combination of DNA and RNA profiles to improve the specificity of analysis and increase the possibility of identifying the genetic causes underlying ccRCC pathology, so highlighting candidate genes that are actively involved in the causation or mainteinance of the malignant phenotype

Integration of whole genome SNP mapping and transcriptional data in clear renal cell carcinoma primary cell cultures

Renal cell carcinomas (RCCs) comprise a heterogeneous group of tumors representing 2%-3% of all adult cancers. Among RCCs, the clear cell histotype (cRCC) is the most common, representing 80% of all primary renal neoplasias. cRCC can be familial or sporadic, with recurrent genomic deletions or translocations, suggesting that genetic mechanisms are involved in early tumorigenesis. In the context of an Italian research oncological project aimed to the identification of new molecular markers for cRCC at different tissues levels using an integrated molecular approach (DNA/RNA/protein), we performed a whole transcriptome/genome integrated analysis of primary cell cultures prepared from tumor, cortical and medullar surgical tissues using the Affymetrix microarray technology. In particular we use either U133 Plus 2.0 and Human Mapping 10K GeneChips\uae that allow the analysis of 54.000 human transcripts and 11.550 Single Nucleotide Polymorphisms (SNP) distributed over all human chromosomes respectively. Results of gene expression profiling of cRCC versus Universal Human Reference RNA (hRefRNA) highlight a total of 829 modulated transcripts (393 down- and 436 up- regulated). Cortex tissues show 961 modulated transcripts compared with hRefRNA (213 down- and 748 up-regulated), whereas medullar tissues evidence 824 modulated transcripts (220 down- and 604 up-regulated). Analysis of genomic instability on triads of RCC primary cultures\u2013tumor biopsies\u2013normal blood samples is in progress. A complex data mining strategy for integrating whole transcriptome/genome results using several bionformatics tools (GCOS, GDAS, Copy Number Tool, dCHIP, GenMAPP, IGB, DAVID, PubMATRIX ) will be shown. This work was supported by MIUR-FIRB grants n\ub0RBNE01HCKF and n\ub0RBNE01TZZ8