scholarly journals miRNA Profiling: How to Bypass the Current Difficulties in the Diagnosis and Treatment of Sarcomas

Sarcoma ◽  
2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Angélique Gougelet ◽  
Jennifer Perez ◽  
Daniel Pissaloux ◽  
Anthony Besse ◽  
Adeline Duc ◽  
...  
Keyword(s):  
Chromosomal Aberrations ◽  
Therapeutic Agent ◽  
Mtor Inhibitor ◽  
Molecular Diagnostic ◽  
Low Density ◽  
Complex Karyotype ◽  
Mirna Signature ◽  
Cgh Array ◽  
Array Analysis ◽  
Mirna Profiling

Sarcomas are divided into a group with specific alterations and a second presenting a complex karyotype, sometimes difficult to diagnose or with few therapeutic options available. We assessed if miRNA profiling by TaqMan low density arrays could predict the response of undifferentiated rhabdomyosarcoma (RMS) and osteosarcoma to treatment. We showed that miRNA signatures in response to a therapeutic agent (chemotherapy or the mTOR inhibitor RAD-001) were cell and drug specific on cell lines and a rat osteosarcoma model. This miRNA signature was related to cell or tumour sensitivity to this treatment and might be not due to chromosomal aberrations, as revealed by a CGH array analysis of rat tumours. Strikingly, miRNA profiling gave promising results for patient rhabdomyosarcoma, discriminating all types of RMS: (Pax+) or undifferentiated alveolar RMS as well as embryonal RMS. As highlighted by these results, miRNA profiling emerges as a potent molecular diagnostic tool for complex karyotype sarcomas.

CGH array analysis of pituitary tumors revealed a highly-disrupted genome

2018 ◽  
Author(s):  
Helene Lasolle ◽  
Mad-Helenie Elsensohn ◽  
Eudeline Alix ◽  
Clement Bonnefille ◽  
Jessica Michel ◽  
...  
Keyword(s):  
Pituitary Tumors ◽  
Cgh Array ◽  
Array Analysis

Acute Erythroid Leukemia (AEL) Can Be Separated Into Distinct Prognostic Subsets Based On Cytogenetic and Molecular Genetic Characteristics

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1394-1394
Author(s):  
Vera Grossmann ◽  
Claudia Haferlach ◽  
Susanne Schnittger ◽  
Ulrike Bacher ◽  
Franziska Poetzinger ◽  
...  
Keyword(s):  
Chromosome Banding ◽  
Cox Regression ◽  
Mutation Screening ◽  
Intermediate Risk ◽  
Complex Karyotype ◽  
Cgh Array ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership ◽  
N 93

Abstract Abstract 1394 Background: Acute erythroid leukemia (AEL) is characterized by a predominant erythroid population and is comprising <5% of adult AML cases. Because of the relative rarity of AEL, few large studies have examined underlying clinical and genetic features. Aims: Molecular and cytogenetic characterization of AEL and identification of genes with prognostic impact. Patients and Methods: We studied an unselected cohort of 94 AEL patients including 32 female and 62 male cases; median age was 69.0 yrs (range: 21.3–88.3 yrs). Survival data was available in 73 cases; median survival was 15.9 months. First, chromosome banding analysis (n=94) was performed. In addition, all cases with normal karyotype (NK) were investigated by CGH arrays (n=32) (Human CGH 12×270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). Further, mutation screening for ASXL1 (n=87), CEBPA (n=94), DNMT3A (n=94), FLT3 (both internal tandem duplication (ITD) (n=93), and tyrosine-kinase domain (TKD) mutations (n=85)), IDH1 (n=93), IDH2 (n=65), NRAS (n=91), KRAS (n=93), MLL-PTD (n=79), NPM1 (n=94), RUNX1 (n=94), TP53 (n=94), and WT1 (n=90) was performed by 454 amplicon deep-sequencing (Roche, Branford, CT), Sanger sequencing or melting curve analyses. CGH array data analysis was performed using Nexus Copy Number 6.0 (BioDiscovery Inc, El Segundo, CA). Results: Cytogenetic data was available for all cases: 48 cases (51.1%) presented an intermediate-risk and 46 (48.9%) cases an unfavorable cytogenetic category according to the MRC Classification. By CGH array analysis 30/32 cases retained a NK, whereas in two cases small aberrations were detected: case 1: deletion of the CEBPA gene, case 2: duplication 11q13.3 to 11q25 including the ATM and MLL gene. Molecular mutations were detected in 85/94 patients (90.4%). 63.5% (54/85) of mutated patients carried one, whereas 36.5% (31/85) of cases harbored two (n=22) or more (n=9) mutations. In detail, TP53 was the most frequently mutated gene (41 cases, 43.6%). Other alterations were detected in NPM1 (15/94; 16.0%); DNMT3A (12/94; 12.8%); ASXL1 (8/87; 9.2%); MLL-PTD (7/79; 8.9%); RUNX1 (8/94; 8.5%); IDH1 (6/93; 6.5%); WT1 (5/90; 5.6%); IDH2 (3/65; 4.6%); NRAS (3/91; 3.3%); KRAS (3/93; 3.2%); FLT3-ITD (3/93, 3.2%), FLT3-TKD (3/85, 3.5%), and CEBPA (1/94). First, we were interested in any correlation with the respective karyotype and observed that NPM1, RUNX1, and WT1 mutations correlated with an intermediate-risk karyotype (NPM1: 15/48 vs 0/46, P<0.001; RUNX1: 8/48 vs 0/46, P=0.006; WT1: 5/46 vs 0/44, P=0.056), whereas TP53mut correlated with the unfavorable karyotype (38/46 vs 3/48, P<0.001). Within the cytogenetically adverse subset TP53mut were associated with complex karyotype (36/38 vs 2/8, P<0.001). In addition, NPM1mut correlated with lower age (56±15 vs 67±13 yrs, P=0.002), whereas mutations in ASXL1, DNMT3A, and TP53 correlated with higher age (73±4 vs 64±15, P=0.001; 71±6 vs 65±14, P=0.015; 71±8 vs 61±15, P<0.001). NPM1mut were associated with longer, and RUNX1mut and TP53mut with shorter OS (OS after 2 yrs: NPM1mut vs wt: 85.1% vs 28.3%, P=0.001; RUNX1mut vs wt: 0% vs 45.2%, P=0.007; TP53mut vs wt: 9.4% vs 61.6%, P=0.001). In the univariable Cox regression analyses mutations in NPM1 (HR 0.12; P=0.004), RUNX1 (HR 3.99; P=0.013), TP53 (HR 3.19; P=0.001), age (HR 4.24, P=0.001) and adverse cytogenetics (HR 2.98, P=0.002) were significantly associated with OS. Independent prognostic factors in multivariable Cox regression analysis were: age (HR 2.6, P=0.047) and RUNX1mut (HR 6.3, P=0.006). Of note, when separating MRC intermediate from MRC adverse cases, we confirmed the longer OS of NPM1 and shorter OS of RUNX1 mutated cases in comparison to NPM1, RUNX1 wt cases (OS after 2 yrs: NPM1mut vs wt: 85.1% vs 46.3%, P=0.027; RUNX1mut vs wt: 0% vs 69.0%, P<0.001). Conclusions: (1) The frequency of cases with complex or other adverse karyotypes within the AEL cohort is very high (48.9%), (2) 93.7% of cases with NK also showed a NK using high-resolution CGH arrays. (3) Overall, a remarkably high mutation frequency of 90.4% was found. (4) NPM1 and RUNX1mut were exclusively detected in the cytogenetically intermediate-risk MRC, TP53 mut predominantly in the MRC adverse group and mainly in cases with complex karyotype. (5) In addition to chromosome banding analysis mutation screening of RUNX1 and NPM1 in cases with intermediate-risk karyotype should be considered for better prognostication. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Bacher:MLL Munich Leukemia Laboratory: Employment. Poetzinger:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Survival Analysis of Myelodysplastic Syndrome (MDS) Patients with Abnormal Karyotype - A Single Group Experience

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4952-4952
Author(s):  
Muhammad Nadeem ◽  
Naomi Galili ◽  
Muhammad Mumtaz ◽  
Peter Daya ◽  
Jason Cheskis ◽  
...  
Keyword(s):  
Survival Analysis ◽  
High Risk ◽  
Median Survival ◽  
Chromosomal Aberrations ◽  
Risk Groups ◽  
Low Risk ◽  
Complex Karyotype ◽  
Abnormal Karyotype ◽  
High Risk Disease ◽  
Social Security Death Index

Abstract Abstract 4952 Introduction: MDS are a heterogeneous group of hematologic disorders associated with clonal evolution of abnormal erythroid, myeloid and megakaryocytic lineages. Risk of transformation to acute myeloid leukemia (AML) is determined by IPSS score. Common chromosomal aberrations include abnormalities of 5, 7, 8, 20 and Y. The present survival analysis is unique in that only one team of experts was involved in diagnosis and management of these patients over more than two decades. Material and Methods: Data of patients with abnormal karyotype either at first presentation or during the course of MDS was collected retroactively. FAB, IPSS and WHO classification were used. Dates of death were retrieved from social security death index. Statistical analyses were performed by using SPSS version 18. Results: Of 709 patients with abnormal karyotypes, 292(41%) were classified as RA (or refractory cytopenias), 80 (11%) RARS, 253(36%) RAEB, 45(6%) CMMoL and 34(5%) RAEB-t and 5 (1%) were unclassified. There were 271 (37%) females and 438 (63%) males with a median age of 67 yrs and 68 yrs respectively. The most frequent abnormalities affected chromosome 5 (279 or 40%); del5q/-5 with other changes was seen in 233 (83. 5 %) and isolated del5q/-5 in 79 (28. 3%). Chromosome 7 abnormalities were found in 181 (25. 5%) patients with 38 (21%) having isolated del7q/-7. Chromosomal 8 abnormality was seen in 174 (24. 5%) patients and 71 (41%) had isolated trisomy 8. Other frequently involved chromosomes were 20 and Y affecting 158 (22. 3%) and 55 (7. 8%) patients respectively. Complex karyotype with 3 or more chromosomal aberrations was seen in 201 (28. 3%) patients. Data on 700 patients was available for analysis when all chromosomal aberrations were considered according to various IPSS risk categories. The median survival was 73 months for low risk (74 patients), 33. 7 months for int-1 (303 patients), 13 months for int-2 (227 patients) and 11. 5 months for high risk (96 patients) (p=0. 000) groups. By cytogenetic abnormalities, the best median survival of 82 months (39/229) was seen in patients with del5q/-5 and low risk disease. Other risk groups with de5q/-5 showed 32, 11 and 9 months in int-1, int-2 and high risk disease respectively (p=<0. 05). The worst median survival was in patients with high risk disease and del7q/-7 (7. 4 months, 33/127) and in patients with complex karyotype (8 months, 55/197). Conclusion: Deletion 5q patients show the best median survival among low and int-1 risk groups. Our data show considerable improvement in median survival of high risk patients compared to the earlier reported survival (11. 5 versus 4 months) which probably reflects improvement due to the use of hypomethylating agents. This improvement in survival gains more significance when considering the fact that we have used the data of only those patients with chromosomal aberrations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Newly Established Murine Cell Line as a Model for Hepatocellular Cancer in Non-Alcoholic Steatohepatitis

2019 ◽  
Vol 20 (22) ◽  
pp. 5658
Author(s):  
Andreas Kroh ◽  
Jeanette Walter ◽  
Herdit Schüler ◽  
Jochen Nolting ◽  
Roman Eickhoff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Chromosomal Aberrations ◽  
Mtor Inhibitor ◽  
Hepatocellular Cancer ◽  
Stem Cell Markers ◽  
Dependent Manner ◽  
Cancer Stem Cell Markers ◽  
Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) has become a major risk factor for hepatocellular cancer (HCC) due to the worldwide increasing prevalence of obesity. However, the pathophysiology of NASH and its progression to HCC is incompletely understood. Thus, the aim of this study was to generate a model specific NASH-derived HCC cell line. A murine NASH-HCC model was conducted and the obtained cancer cells (N-HCC25) were investigated towards chromosomal aberrations, the expression of cell type-specific markers, dependency on nutrients, and functional importance of mTOR. N-HCC25 exhibited several chromosomal aberrations as compared to healthy hepatocytes. Hepatocytic (HNF4), EMT (Twist, Snail), and cancer stem cell markers (CD44, EpCAM, CK19, Sox9) were simultaneously expressed in these cells. Proliferation highly depended on the supply of glucose and FBS, but not glutamine. Treatment with a second generation mTOR inhibitor (KU-0063794) resulted in a strong decrease of cell growth in a dose-dependent manner. In contrast, a first generation mTOR inhibitor (Everolimus) only slightly reduced cell proliferation. Cell cycle analyses revealed that the observed growth reduction was most likely due to G1/G0 cell cycle arrest. These results indicate that N-HCC25 is a highly proliferative HCC cell line from a NASH background, which might serve as a suitable in vitro model for future investigations of NASH-derived HCC.

miRNA Expression in Multiple Myeloma as Predictive Model of Response to Bortezomib.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4918-4918 ◽  
Author(s):  
Paola Neri ◽  
Kathy Gratton ◽  
Li Ren ◽  
Adnan Mansoor ◽  
Peter Duggan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Mirna Expression ◽  
Response To Therapy ◽  
Differentially Expressed ◽  
Mirna Signature ◽  
Prediction Of Response ◽  
Mirna Profiling

Abstract Abstract 4918 Background Bortezomib therapeutic efficacy is well established in multiple myeloma (MM) however response to this therapy remains difficult to predict with resistant disease observed in nearly 20% of MM patients. Through DNA microarrays, predictive models of response to stem cell transplant and Bortezomib were reported correlating mRNA expression data with disease outcomes and response to therapy. MicroRNAs (miRNAs) are a key class of small, non-coding RNA molecules that modulate post-transcriptional regulation of gene expression and were recently described to be involved in deregulation of gene expression in many cancers including MM. Little evidence however is available concerning the role of miRNA expression in the prediction of response to Bortezomib in MM. We aimed to assess the expression of miRNAs in a panel of Bortezomib highly sensitive and relatively resistant MM cell lines as well as primary MM cells and identify miRNA expression patterns that are associated with response to Bortezomib. Methods We have used miRNA microarrays (Affymetrix miRNA GeneChip) as well as liquid phase Luminex microbead miRNA profiling (Flexmir, Luminex) to profile miRNA expression in MM cell lines (MM1S, KMS11, INA6, U266) and sorted CD138+ bone marrow PCs from MM patients prior to treatment with Bortezomib (n=5; 3 sensitive and 2 resistant) and PCs from a healthy normal donor (n=1). The MM cell lines included in this analysis were classified as sensitive (S) or resistant (R) based on their Bortezomib IC50 at 48 hours (IC50 for MM1S and KMS11 ∼ 5 nM versus INA6 and U266 ∼ 20nM). For the microarray studies the hybridization signal values for the multiple probes for each miRNA were obtained and normalized with the use of miRNA QC tool (Affymetrix) and analyzed using Partek Genomics Suite software. Thereafter, filters were applied to identify the miRNA probes whose normalized signal were at least 2 folds differentially expressed between sensitive (MM1S) and resistant (INA6) cell lines with a P value < 0.05 (ANOVA) and a FDR of 0.05. Bortezomib sensitive (n=3) and resistant (n=2) primary MM samples were subjected to the same miRNA array analysis and filtering. Liquid phase Luminex microbead miRNA profiling (FlexmiR) was used for the confirmation (MM1S and INA6) and validation of the array results in other MM cell lines KMS11 (IC50 5nM) and U266 (IC50 20nM). Results Using Affymetrix miRNA GeneChip we identified 22 differentially expressed miRNA with overexpression of miR-155, miR-342-3p, miR-181a and b, miR-128, miR-20b and downregulation miR-let-7b, miR-let-7i, miR-let-7d, miR-let-7c, miR-222, miR-221, miR-23a, miR-27a and miR-29a in bortezomib relatively resistant (INA6) versus sensitive (MM1S) cell line. These results were confirmed in INA6 and MM1S cells with the use of Luminex microbead miRNA profiling and validated to be similarly differentially expressed between KMS11 (sensitive) and relatively U266 (resistant) cell lines. Furthermore, TargetScan algorithms and Ingenuity Pathway Analysis software were used to identify predicted miRNAs-targeted mRNAs or potentially regulated networks and included genes involved in cell cycle regulation, cell growth, apoptosis and ubiquitin-conjugation pathways. Lastly to further investigate the clinical relevance of miRNAs in MM in terms of prediction of response and outcome to Bortezomib, we correlated miRNA expression profile of sorted CD138+ bone marrow PCs from Bortezomib sensitive (n=3) and resistant (n=2) MM patients with their response to therapy. Unsupervised analysis of the data revealed that the Bortezomib sensitive MM patients clustered with MM1S cell line while resistant patients segregated into the INA6 cluster. Conclusion In summary, we have described a MM miRNA signature, which includes miRNAs that modulate the expression of proteins critical to myeloma pathogenesis and is predictive of response to Bortezomib. Further validation of this miRNA signature in a larger cohort of Bortezomib-treated MM patients is ongoing. Disclosures Stewart: Glaxo-Smith-Kline: Research Funding.

scholarly journals Development of a Low-Density DNA Microarray for Detecting Tick-Borne Bacterial and Piroplasmid Pathogens in African Cattle

2019 ◽  
Vol 4 (2) ◽  
pp. 64 ◽  
Author(s):  
Babette Abanda ◽  
Archile Paguem ◽  
Mbunkah Daniel Achukwi ◽  
Alfons Renz ◽  
Albert Eisenbarth
Keyword(s):  
Dna Microarray ◽  
Low Cost ◽  
Molecular Diagnostic ◽  
Multiple Infections ◽  
Screening Methods ◽  
Low Density ◽  
African Cattle ◽  
Specificity And Sensitivity ◽  
Northern Cameroon ◽  
Widespread Availability

In Africa, pathogens transmitted by ticks are of major concern in livestock production and human health. Despite noticeable improvements particularly of molecular screening methods, their widespread availability and the detection of multiple infections remain challenging. Hence, we developed a universally accessible and robust tool for the detection of bacterial pathogens and piroplasmid parasites of cattle. A low-cost and low-density chip DNA microarray kit (LCD-Array) was designed and tested towards its specificity and sensitivity for five genera causing tick-borne diseases. The blood samples used for this study were collected from cattle in Northern Cameroon. Altogether, 12 species of the genera Anaplasma, Ehrlichia, Rickettsia and Theileria, and their corresponding genus-wide probes including Babesia were tested on a single LCD-Array. The detection limit of plasmid controls by PCR ranged from 1 to 75 copies per µL depending on the species. All sequenced species hybridized on the LCD-Array. As expected, PCR, agarose gel electrophoresis and Sanger sequencing found significantly less pathogens than the LCD-Array (p < 0.001). Theileria and Rickettsia had lower detection limits than Anaplasma and Ehrlichia. The parallel identification of some of the most detrimental tick-borne pathogens of livestock, and the possible implementation in small molecular-diagnostic laboratories with limited capacities makes the LCD-Array an appealing asset.

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