Project description:B-cell chronic lymphocytic leukemia (B-CLL) shows an incredibly high heterogeneity in the clinical course, spanning from rapidly aggressive to completely indolent behaviour. In order to identify the correspondent gene expression variability, we investigated 29 cases of untreated B-CLL using microarray. Firstly, two robust CLL clusters were identified applying multiple unsupervised clustering algorithms. Their separation was mainly determined by the differential expression of several genes included into two gene groups designated OxPhos and Lyn clusters. OxPhos gene cluster, previously identified in a subset of diffuse large B-cell lymphomas, comprises genes coding for some respiratory chain enzymes, ribosomal proteins and translation factors. Moreover, increased levels of genes involved in the regulation of apoptosis and in the proteasome-ubiquitin complex and the down-regulation of LYN gene, member of B-cell receptor pathway, characterized the OxPhos CLL subset. These B-CLL biological clusters did not reveal any preferential distribution of Ig mutated or Ig unmutated CLL prognostic groups. Furthermore, we applied another unsupervised algorithm (Subtractive Unsupervised Analysis) after the exclusion of genes characterizing the previously identified CLL subsets. At this point, we could identify two new CLL clusters, showing a clear association to the Ig mutational status. In addition to the ZAP-70 and LPL genes, the patients with unmutated Ig expressed higher level of some interesting genes involved in B cell activation, cell cycle regulation, apoptosis resistance and angiogenesis. In conclusion, we showed that B-CLL is characterized by an intrinsic heterogeneity in gene expression pattern, which overcomes the influence of the immunoglobulin mutational status on B-cell chronic lymphocytic leukemia profiles. SUBMITTER_CITATION: abstract in Blood, Volume 108, Issue 11 [2780] Angiopoietin-2 Expression in B-Cell Chronic Lymphocytic Leukemia: Association with Clinical Outcome and Immunoglobulin Heavy-Chain Mutational Status. "Rossana Maffei, Silvia Martinelli, Ilaria Castelli, Rita Santachiara, Elena Morandi, Patrizia Zucchini, Marcella Fontana, Anna Maria Colacci, Roberto Serra, Roberto Marasca, Giuseppe Torelli - Oncology and Hematology, University of Modena and Reggio Emilia, Modena, Italy; Excellence Environmental Carcinogenesis, ARPA, Bologna, Italy; Social, Cognitive and Quantitative Sciences, University of Modena and Reggio Emilia, Reggio Emilia, Ita Experiment Overall Design: Twenty-nine B-CLL patients, who have attended the haematology clinic of Modena Hospital, were enrolled in this study. Total RNA from mononuclear cells was isolated using RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturerâ??s instructions For all 29 specimens, fluorescently-labelled cRNA was generated by in vitro transcription using Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo alto, CA, USA) according to the manufacturerâ??s instructions. Amplified cRNA of each patient was labelled with cyanine 5-CTP (Perkin-Elmer, NEN Life Science, Boston, MA, USA) in each experiment. Mix of equal amount of total RNAs from our cohort was used as reference control in all microarray gene-profiling experiments. Amplified reference cRNA was labelled with cyanine 3-CTP (Perkin-Elmer, NEN Life Science, Boston, MA, USA) in each experiment. Moreover, cRNA products were purified using RNeasy columns (Qiagen). 1 ï?g of Cy5-labelled cRNA was mixed with the same amount of Cy3-labelled reference cRNA and then mixed cRNAs were fragmented to an average size of ï?¾50-100 nt by incubation at 60°C for 30 min using in situ Hybridization kit-plus (Agilent). Samples were hybridized on Agilent Human 1A Oligo Microarray, ink-jet printed microarray, comprising 20,173 (60-mer) experimentally validated oligonucleotide probes (features).33 After hybridization for 17 h at 60°C, slides were washed according to Agilent SSC protocol instructions and then scanned using a confocal laser scanner (Agilent Technologies). Experiment Overall Design: Fluorescence data were analysed with Feature Extraction Software v.7.5 (Agilent Technologies). Log10 ratio of the dye-normalized Cy3 and Cy5 channel signals were calculated, then a p-value and a final error were obtained to establish the significance of each feature.
2008-06-12 | E-GEOD-4207 | biostudies-arrayexpress
