Mohamed Guled

CDKN2A, NF2, and JUN Are Dysregulated Among Other Genes by miRNAs in Malignant Mesothelioma—A miRNA Microarray Analysis

Malignant mesothelioma (MM) is an aggressive cancer arising from mesothelial cells, mainly due to former asbestos exposure. Little is known about the microRNA (miRNA) expression of MM. miRNAs are small noncoding RNAs, which play an essential role in the regulation of gene expression. This study was carried out to analyze the miRNA expression profile of 17 MM samples using miRNA microarray. The analysis distinguished the overall miRNA expression profiles of tumor tissue and normal mesothelium. Differentially expressed miRNAs were found in tumor samples compared with normal sample. Twelve of them, let‐7b*, miR‐1228*, miR‐195*, miR‐30b*, miR‐32*, miR‐345, miR‐483‐3p, miR‐584, miR‐595, miR‐615‐3p, and miR‐885‐3p, were highly expressed whereas the remaining nine, let‐7e*, miR‐144*, miR‐203, miR‐340*, miR‐34a*, miR‐423, miR‐582, miR‐7‐1*, and miR‐9, were unexpressed or had severely reduced expression levels. Target genes for these miRNAs include the most frequently affected genes in MM such as CDKN2A, NF2, JUN, HGF, and PDGFA. Many of the miRNAs were located in chromosomal areas known to be deleted or gained in MM such as 8q24, 1p36, and 14q32. Furthermore, we could identify specific miRNAs for each histopathological subtype of MM. Regarding risk factors such as smoking status and asbestos exposure, significantly differentially expressed miRNAs were identified in smokers versus nonsmokers (miR‐379, miR‐301a, miR‐299‐3p, miR‐455‐3p, and miR‐127‐3p), but not in asbestos‐exposed patients versus nonexposed ones. This could be related to the method of assessment of asbestos exposure as asbestos remains to be the main contributor to the development of MM.

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.

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.

Array comparative genomic hybridization analysis of olfactory neuroblastoma

Olfactory neuroblastoma is an unusual neuroectodermal malignancy, which is thought to arise at the olfactory membrane of the sinonasal tract. Due to its rarity, little is understood regarding its molecular and cytogenetic abnormalities. The aim of the current study is to identify specific DNA copy number changes in olfactory neuroblastoma. Thirteen dissected tissue samples were analyzed using array comparative genomic hybridization. Our results show that gene copy number profiles of olfactory neuroblastoma samples are complex. The most frequent changes included gains at 7q11.22–q21.11, 9p13.3, 13q, 20p/q, and Xp/q, and losses at 2q31.1, 2q33.3, 2q37.1, 6q16.3, 6q21.33, 6q22.1, 22q11.23, 22q12.1, and Xp/q. Gains were more frequent than losses, and high-stage tumors showed more alterations than low-stage olfactory neuroblastoma. Frequent changes in high-stage tumors were gains at 13q14.2–q14.3, 13q31.1, and 20q11.21–q11.23, and loss of Xp21.1 (in 66% of cases). Gains at 5q35, 13q, and 20q, and losses at 2q31.1, 2q33.3, and 6q16–q22, were present in 50% of cases. The identified regions of gene copy number change have been implicated in a variety of tumors, especially carcinomas. In addition, our results indicate that gains in 20q and 13q may be important in the progression of this cancer, and that these regions possibly harbor genes with functional relevance in olfactory neuroblastoma.

Array comparative genomic hybridization reveals frequent alterations of G1/S checkpoint genes in undifferentiated pleomorphic sarcoma of bone.

Undifferentiated pleomorphic sarcoma of bone (UPSb) is a rare tumor often difficult to differentiate from fibrosarcoma of bone (FSb), diagnostically. We applied array comparative genomic hybridization (array CGH) to screen for genes with potential importance in the tumor and compared the results with alterations seen in FSb. Twenty‐two fresh frozen tissue specimens from 20 patients (18 primary tumors and 4 local recurrences) with UPSb were studied. DNA was isolated and hybridized onto Agilent 244K CGH oligoarrays. The hybridization data were analyzed using Agilent DNA Analytics Software. The number of changes ranged from 2 to 168 (average = 66). Losses were most frequently seen at 8p, 9p, 10, 13q, and 18q, and gains at 4q, 5p, 6p, 7p, 8q, 12p, 14q, 17q, 19p, 20q, 22q, and X. Homozygous deletions of CDKN2A, RB1, TP53, and ING1 were seen in 8/20, 7/20, 3/20, and 2/20 cases, respectively. Hypermethylation of both p16INK4a and p14ARF was found in two cases with loss at CDKN2A. Inactivation either of CDKN2A, RB1, or TP53 was detected in 18/20 cases. One case showed high level gains of CDK4 and MDM2. Frequent gains were seen at MYC, PDGFRA, KIT, and KDR. Immunohistochemical positivity of KIT, PDGFRA, KDR, and PDGFRB was found in 8/14, 5/14, 4/14, and 4/14 cases, respectively. The regions most significantly discriminating between UPSb and FSb included RB1 and MYC. No homozygous deletions of RB1 were found in FSb. In conclusion, our analysis showed the disruption of G1/S checkpoint regulation to be crucial for the oncogenesis of UPSb.

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.

An integrated analysis of miRNA and gene copy numbers in xenografts of Ewing's sarcoma

BackgroundXenografts have been shown to provide a suitable source of tumor tissue for molecular analysis in the absence of primary tumor material. We utilized ES xenograft series for integrated microarray analyses to identify novel biomarkers.MethodMicroarray technology (array comparative genomic hybridization (aCGH) and micro RNA arrays) was used to screen and identify copy number changes and differentially expressed miRNAs of 34 and 14 passages, respectively. Incubated cells used for xenografting (Passage 0) were considered to represent the primary tumor. Four important differentially expressed miRNAs (miR-31, miR-31*, miR-145, miR-106) were selected for further validation by real time polymerase chain reaction (RT-PCR). Integrated analysis of aCGH and miRNA data was performed on 14 xenograft passages by bioinformatic methods.ResultsThe most frequent losses and gains of DNA copy number were detected at 9p21.3, 16q and at 8, 15, 17q21.32-qter, 1q21.1-qter, respectively. The presence of these alterations was consistent in all tumor passages. aCGH profiles of xenograft passages of each series resembled their corresponding primary tumors (passage 0). MiR-21, miR-31, miR-31*, miR-106b, miR-145, miR-150*, miR-371-5p, miR-557 and miR-598 showed recurrently altered expression. These miRNAS were predicted to regulate many ES-associated genes, such as genes of the IGF1 pathway, EWSR1, FLI1 and their fusion gene (EWS-FLI1). Twenty differentially expressed miRNAs were pinpointed in regions carrying altered copy numbers.ConclusionIn the present study, ES xenografts were successfully applied for integrated microarray analyses. Our findings showed expression changes of miRNAs that were predicted to regulate many ES associated genes, such as IGF1 pathway genes, FLI1, EWSR1, and the EWS-FLI1 fusion genes.

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.

Frequent deletion of CDKN2A and recurrent coamplification of KIT, PDGFRA, and KDR in fibrosarcoma of bone--an array comparative genomic hybridization study.

Very little is known about the genetics of fibrosarcoma (FS) of bone. We applied array comparative genomic hybridization (CGH) to identify genes and genomic regions with potential role in the pathogenesis of this tumor. Seventeen patients with FS of bone were included in the study. Array CGH analysis was carried out in 13 fresh frozen tissue specimens from 11 of these patients (nine primary tumors and four local recurrences). DNA was extracted and hybridizations were performed on Agilent 244K CGH oligoarrays. The data were analyzed using Agilent DNA Analytics Software. The number of changes per patient ranged from 0 to 132 (average = 43). Losses were most commonly detected at 6q, 8p, 9p, 10, 13q, and 20p. CDKN2A was homozygously deleted in 7/11 patients. Hypermethylation of both p16INK4a and p14ARF was found in 1/14 patients. An internal deletion of STARD13 was found in a region with common losses at 13q13.1. The most frequent gains were seen at 1q, 4q, 5p, 8q, 12p, 15q, 16q, 17q, 20q, 22q, and Xp. Single recurrent high level amplification was detected at 4q12, including KIT, PDGFRA, and KDR. No activating mutations were found in any of them. Immunohistochemistry revealed expression of PDGFRA and/or PDGFRB in 12/17 samples. Moreover, small regions of gains pinpointed genes of particular interest, such as IGF1R at 15q26.3 and CHD1L at 1q21.1. In conclusion, our analysis provided novel findings that can be exploited when searching for markers for diagnosis and prognosis, and targets of therapy in this tumor type.

Abstract 5196: Different miRNAs expression in leiomyosarcoma and undifferentiated pleomorphic sarcoma

Rare and highly aggressive adult soft tissue sarcomas, leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS), contain complex genomics characterized by a multitude of rearrangements, amplifications and deletions. Since differential diagnosis between the two sarcoma histotypes continues to remain a challenge, we attempted to identify a specific signature useful for improving the differential diagnosis, and we performed miRNA profiling on series of LMS and UPS samples stored at Rizzoli biobank. First, we investigated the different miRNA expression profile in 20 primary sarcoma samples, 10 high-grade LMS and 10 UPS, MFH-like, using miRNA array technique. The top 5 differentially expressed (p Citation Format: Laura Pazzaglia, Mohamed Guled, Ioana Borze, Maria Serena Benassi, Piero Picci, Sakari Knuutila. Different miRNAs expression in leiomyosarcoma and undifferentiated pleomorphic sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5196. doi:10.1158/1538-7445.AM2014-5196