Ioana Borze

Integrative analysis of microRNA, mRNA and aCGH data reveals asbestos- and histology-related changes in lung cancer

Lung cancer has the highest mortality rate of all of the cancers in the world and asbestos‐related lung cancer is one of the leading occupational cancers. The identification of asbestos‐related molecular changes has long been a topic of increasing research interest. The aim of this study was to identify novel asbestos‐related molecular correlates by integrating miRNA expression profiling with previously obtained profiling data (aCGH and mRNA expression) from the same patient material. miRNA profiling was performed on 26 tumor and corresponding normal lung tissue samples from highly asbestos‐exposed and non‐exposed patients, and on eight control lung tissue samples. Data analyses on miRNA expression, and integration of miRNA and previously obtained mRNA data were performed using Chipster. A separate analysis was used to integrate miRNA and previously obtained aCGH data. Both known and new lung cancer‐associated miRNAs and target genes with inverse correlation were discovered. Furthermore, DNA copy number alterations (e.g., gain at 12p13.31) were correlated with the deregulated miRNAs. Specifically, thirteen novel asbestos‐related miRNAs (over‐expressed: miR‐148b, miR‐374a, miR‐24‐1*, Let‐7d, Let‐7e, miR‐199b‐5p, miR‐331‐3p, and miR‐96 and under‐expressed: miR‐939, miR‐671‐5p, miR‐605, miR‐1224‐5p and miR‐202) and inversely correlated target genes (e.g., GADD45A, LTBP1, FOSB, NCALD, CACNA2D2, MTSS1, EPB41L3) were identified. In addition, over‐expression of the well known squamous cell carcinoma‐associated miR‐205 was linked to down‐regulation of the DOK4 gene. The miRNAs/genes presented here may represent interesting targets for further investigation and could eventually have potential diagnostic implications.

MicroRNA profiling differentiates colorectal cancer according to KRAS status

Recent studies have shown the important role of microRNAs (miRNAs) in a variety of biological processes, and in its ability to distinguish tumors according to their prognostic and predictive properties. To identify miRNA signatures associated with colorectal carcinoma (CRC) and with KRAS status, we studied, using Agilents miRNA microarrays, miRNA expression in primary tumors from 55 metastatic CRC patients, including 15 with mutant and 40 with wild‐type KRAS. Comparing these with normal colon tissue, we identified 49 miRNAs—including 19 novel miRNAs—significantly deregulated in tumor tissue. The presence of the KRAS mutation was associated with up‐regulation of miR‐127‐3p, miR‐92a, and miR‐486‐3p and down‐regulation of miR‐378. Increased expression of miR‐127‐3p and miR‐92a in KRAS mutant tumors was significantly confirmed by quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) (P < 0.05). We identified some predicted target genes of differentially expressed miRNAs between mutated and wild‐type KRAS, such as RSG3 and TOB1, which are involved in apoptosis and proliferation. Target prediction and pathway analysis suggest a possible role for deregulated miRNAs in nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and G protein‐coupled receptor signaling pathways.

MicroRNA profiling predicts survival in anti-EGFR treated chemorefractory metastatic colorectal cancer patients with wild-type KRAS and BRAF

Anti-EGFR monoclonal antibodies (anti-EGFRmAb) serve in the treatment of metastatic colorectal cancer (mCRC), but patients with a mutation in KRAS/BRAF and nearly one-half of those without the mutation fail to respond. We performed microRNA (miRNA) analysis to find miRNAs predicting anti-EGFRmAb efficacy. Of the 99 mCRC patients, we studied differential miRNA expression by microarrays from primary tumors of 33 patients who had wild-type KRAS/BRAF and third- to sixth-line anti-EGFRmAb treatment, with/without irinotecan. We tested the association of each miRNA with overall survival (OS) by the Cox proportional hazards regression model. Significant miR-31* up-regulation and miR-592 down-regulation appeared in progressive disease versus disease control. miR-31* expression and down-regulation of its target genes SLC26A3 and ATN1 were verified by quantitative reverse transcriptase polymerase chain reaction. Clustering of patients based on miRNA expression revealed a significant difference in OS between patient clusters. Members of the let-7 family showed significant up-regulation in the patient cluster with poor OS. Additionally, miR-140-5p up-regulation and miR-1224-5p down-regulation were significantly associated with poor OS in both cluster analysis and the Cox proportional hazards regression model. In mCRC patients with wild-type KRAS/BRAF, miRNA profiling can efficiently predict the benefits of anti-EGFRmAb treatment. Larger series of patients are necessary for application of these miRNAs as predictive/prognostic markers.

Unique microRNA profile in Dupuytren's contracture supports deregulation of β -catenin pathway

Dupuytrens contracture, a proliferative disease of unknown origin, is characterized by an abnormal fibroblast proliferation process. Evidence from numerous microRNA (miRNA) studies shows that miRNAs have a vital function in many biological processes, for instance, in cellular signaling networks, cell growth, tissue differentiation, and cell proliferation. Our aim was to characterize, to our knowledge for the first time, the miRNA-expression profile of Dupuytrens contracture. The miRNAs identified may have a function in the pathogenesis of Dupuytrens contracture by targeting and regulating important pathways. We compared the miRNA-expression profile of 29 Dupuytrens contracture patients with that of control samples (fibroblast cells and palmar fascia). Some of the miRNAs identified in our Dupuytrens contracture samples, including miR-29c, miR-130b, miR-101, miR-30b, and miR-140-3p, were found to regulate important genes related to the β-catenin pathway: WNT5A, ZIC1, and TGFB1. Expression profiles of these genes reanalyzed from published gene-expression data from similar patient material correlated with our miRNA results. Analysis was also performed for groups of patients with recurrent/non-recurrent and patients with hereditary/non-hereditary Dupuytrens contracture, but no significant differences appeared in miRNA-expression profiles of these groups. Identification of unique miRNA expression in Dupuytrens contracture may lead to the development of novel molecular therapy for its treatment.

CanGEM: mining gene copy number changes in cancer

The use of genome-wide and high-throughput screening methods on large sample sizes is a well-grounded approach when studying a process as complex and heterogeneous as tumorigenesis. Gene copy number changes are one of the main mechanisms causing cancerous alterations in gene expression and can be detected using array comparative genomic hybridization (aCGH). Microarrays are well suited for the integrative systems biology approach, but none of the existing microarray databases is focusing on copy number changes. We present here CanGEM (Cancer GEnome Mine), which is a public, web-based database for storing quantitative microarray data and relevant metadata about the measurements and samples. CanGEM supports the MIAME standard and in addition, stores clinical information using standardized controlled vocabularies whenever possible. Microarray probes are re-annotated with their physical coordinates in the human genome and aCGH data is analyzed to yield gene-specific copy numbers. Users can build custom datasets by querying for specific clinical sample characteristics or copy number changes of individual genes. Aberration frequencies can be calculated for these datasets, and the data can be visualized on the human genome map with gene annotations. Furthermore, the original data files are available for more detailed analysis. The CanGEM database can be accessed at http://www.cangem.org/.

Differentiating soft tissue leiomyosarcoma and undifferentiated pleomorphic sarcoma: A miRNA analysis.

The rare and highly aggressive adult soft tissue sarcomas leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS) contain complex genomes characterized by a multitude of rearrangements, amplifications, and deletions. Differential diagnosis remains a challenge. MicroRNA (miRNA) profiling was conducted on a series of LMS and UPS samples to initially investigate the differential expression and to identify specific signatures useful for improving the differential diagnosis. Initially, 10 high‐grade LMS and 10 high‐grade UPS were profiled with a miRNA microarray. Two cultured human mesenchymal stem cell samples were used as controls. 38 and 46 miRNAs classified UPS and LMS samples, respectively, into separate groups compared to control samples. When comparing the two profiles, miR‐199b‐5p, miR‐320a, miR‐199a‐3p, miR‐126, miR‐22 were differentially expressed. These were validated by RT‐PCR on a further series of 27 UPS and 21 LMS for a total of 68 cases. The levels of miR‐199‐5p and miR‐320a, in particular, confirmed the microarray data, the former highly expressed in UPS and the latter in LMS. Immunohistochemistry was performed on all 68 cases to confirm original diagnosis. Recently reported LMS‐ and UPS‐associated genes were correlated with miRNA targets based on target algorithms of three databases. Several genes including IMP3, ROR2, MDM2, CDK4, and UPA, are targets of differentially expressed miRNAs. We identified miRNA expression patterns in LMS and UPS, linking them to chromosomal regions and mRNA targets known to be involved in tumor development/progression of LMS and UPS.

MicroRNA microarrays on archive bone marrow core biopsies of leukemias—Method validation

Due to availability of bone marrow core biopsies (CB) in many pathology laboratories, we evaluated the quality and the biological information of the miRNA profiling using 9 acute lymphoblastic leukemia (ALL) and 9 chronic myeloid leukemia (CML) matched CB and bone marrow aspirates (BA). Technical replicates showed reproducible results across platforms and clustered together in hierarchical clustering analysis; and matched samples showed similar biological content having common differentially expressed miRNAs against the same control samples. We showed, that CBs, which have underwent decalcification in addition to formalin-fixation, are suitable for miRNA profiling.

miRNA expression profiles in myelodysplastic syndromes reveal Epstein–Barr virus miR-BART13 dysregulation

Recently, the microRNA (miRNA) signature has been used for better characterization and understanding of the pathogenesis of different malignancies, including myelodysplastic syndromes (MDS). MDS are a heterogeneous group of stem cell disorders in which the genetic and molecular defects are not well defined. In the present study, we applied array based miRNA profiling to study 19 bone marrow cell samples of de novo MDS compared with eight healthy individuals. In addition, integration of the miRNA profiling data with our previous array comparative genomic hybridization data, from the same cohort of patients, was performed. We observed up-regulation of hsa-miR-720 and hsa-miR-21, and down-regulation of hsa-miR-671-5p and one human virus miRNA (Epstein–Barr virus miR-BART13) in MDS samples compared with normal samples. In our study, the copy number alteration harboring miRNA was not affecting miRNA expression, but a distinct microRNA expression pattern was observed, not only in MDS compared with controls, but also between MDS entities.

JAK2V617F mutation and spontaneous megakaryocytic or erythroid colony formation in patients with essential thrombocythaemia (ET) or polycythaemia vera (PV)

The in vitro cultures of erythroid (BFU-E) and megakaryocytic (CFU-Meg) progenitors have been useful diagnostic tools in myeloproliferative disorders (MPD). However, after the discovery of the JAK2V617F mutation, their diagnostic role has been uncertain. In this single-centre retrospective study we analyzed JAK2V617F mutation in 58 ET and 42 PV patients diagnosed according to WHO criteria and compared the results with those of colony forming assays with special emphasis on CFU-Meg growth. 91% of PV and 57% of ET patients had JAK2V617F mutation and they all showed spontaneous BFU-E growth. However, endogenous BFU-E formation was also seen in nine JAK2V617F mutation negative patients displaying also a normal JAK2 exon 12 allele. Endogeneous CFU-Meg colony formation was found in 59% of PV and 53% of the ET patients. A subgroup of ET patients (n=7) displayed sole spontaneous CFU-Meg growth without spontaneous BFU-E growth. They all were JAK2 mutation negative, but one of them had MPL mutation. In conclusion, in vitro cultures of haematopoietic progenitors are sensitive diagnostic tools in the present group of 100 MPD patients revealing also JAK2 mutation negative ET and PV patients displaying sole spontaneous CFU-Meg or BFU-E growth.

Oligoarray comparative genomic hybridization in polycythemia vera and essential thrombocythemia

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative disorders (MPD) characterized by expansion of one or more myeloid lineages, which by definition results in erythrocytosis in PV and thrombocytosis in ET. The etiology and pathogenesis are still largely unknown. At