Comment[ArrayExpressAccession] E-GEOD-48182 MAGE-TAB Version 1.1 Public Release Date 2013-09-25 Investigation Title Molecular classification of mature aggressive B cell lymphoma using digital multiplexed gene expression on formalin-fixed paraffin-embedded biopsy specimens [FFPE] Comment[Submitted Name] Molecular classification of mature aggressive B cell lymphoma using digital multiplexed gene expression on formalin-fixed paraffin-embedded biopsy specimens [FFPE] Experiment Description The most frequent mature aggressive B-cell lymphomas are diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Patients suffering from molecularly defined BL (mBL) but treated with a regimen developed for DLBCL show an unfavorable outcome compared to mBL treated with chemotherapy regimens for BL. Distinguishing BL from DLBCL by conventional histopathology is challenging in lymphomas that have features common to both diseases (aggressive B-cell lymphoma unclassifiable with features of DLBCL and BL [intermediates]). Moreover, DLBCL are a heterogeneous group of lymphomas comprising distinct molecular subtypes: the activated B-cell (ABC)-like, the germinal center B-cell-like (GCB) and the unclassifyable subtype as defined by gene expression profiling (GEP). Attempts to replace GEP with techniques applicable to formalin-fixed paraffin-embedded (FFPE) tissue led to algorithms for immunohistochemical stainings (IHS). Disappointingly, the algorithms yielded conflicting results with respect to their prognostic potential, raising concerns about their validity. Furthermore, IHS algorithms did not provide a fully resolved classification: They did not identify mBL; nor did they separate ABC from unclassified DLBCL. 29 diffuse large B-Cell lymphoma samples were hybridized to HGU133A Affymetrix GeneChips. In addition, this study contains 22 already published samples whereas 11 of them contribute to GSE22470, 6 contribute to GSE10172, 3 to GSE44164 and 2 to GSE4475. No re-normalisation of published samples was performed. We used digital multiplexed gene expression (DMGE) with FFPE derived RNA to classify agressive B-cell lymphomas. Our assay comprised only 30 genes (10 for the detection of mBL and 20 for the detection of ABC and GCB). We chose these genes by reanalysis of the microarray data reported in a previous study. 39 samples from mature aggressive B-cell lymphomas were analyzed using DMGE (nCounter, NanoString Technologies Inc., Seattle, WA, USA) of FFPE- and fresh-frozen derived RNA. All cases were previously characterized by the Molecular Mechanisms of Malignant Lymphoma (MMML) consortium using the Affymetrix GeneChip technology (gold standard of classification). Please note that there are total 40 FFPE-derived and 50 fresh-frozen derived samples, with 39 samples derived from both materials (allowing direct comparison). Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Kohler Masque-Soler Szczepanowski Kohler Spang Klapper Person First Name Christian Neus Monika Christian Rainer Wolfram Person Mid Initials Wilhelm W Person Email christian.kohler@ur.de Person Affiliation University of Regensburg Person Address Institute of Functional Genomics, University of Regensburg, Josef-Engert Str. 9, Regensburg, Bavaria, Germany Person Roles submitter Protocol Name P-GSE48182-1 P-GSE48182-3 P-GSE48182-4 P-GSE48182-2 P-GSE48182-5 Protocol Description Data processing was done using the R-package NanoStringNorm (Waggot et al, Bioinformatics 2012). Final data normalization was performed using the quantile normalization (Bolstadt et al, Bioinformatics 2003) ID_REF = VALUE = Normalized nCounter data on log2 scale NA Straight after a RNA quality measurement and with a known RNA concentration for each sample, 300ng were able to be hybridised with our custom-designed Reporter Probe (each with a specific 5’-end colour code signal) and Capture Probe (marked with biotin at the 3’ end). Formalin-fixed paraffin-embedded material was cut in five 10μm-thick pieces per sample. Extraction of RNA was done according to the manufacturer’s instructions (ExpressArt FFPE Clear RNAready Kit, AmpTec, Hamburg, Germany). Fresh-frozen blocks’ RNA was extracted as described in Hummel et al, NEJM 2006). The RNA quality was analyzed with the Agilent RNA 6000 Nano Chips (Agilent Technologies, Santa Clara, California, USA) following the product’s protocol. Further material quality control is present after RNA processing at the nCounter instrument for digital multiplexed gene expression due to the company’s recommendations. After overnight hybridisation the samples were ready to load to the PrepStation where they were washed with a two-step magnetic bead-purification process and loaded in the 12-well cartridges. Afterwards the cartridges were loaded to the Digital Analyzer (DA, nCounter, NanoString Technologies Inc., Seattle, WA, USA). The DA resolution was set to high, meaning that 280 fields of view (FOV) were counted per sample. A comma-separated-value (.csv) file was obtained after processing. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name DLBCL MOLECULAR SUBTYPE MOLECULAR.DIAGNOSIS Experimental Factor Type dlbcl molecular subtype molecular.diagnosis Publication Title Molecular classification of mature aggressive B-cell lymphoma using digital multiplexed gene expression on formalin-fixed paraffin-embedded biopsy specimens. Publication Author List Masqu�-Soler N, Szczepanowski M, Kohler CW, Spang R, Klapper W PubMed ID 24030260 Publication DOI 10.1182/blood-2013-06-508937 Comment[SecondaryAccession] GSE48182 Comment[GEOReleaseDate] 2013-09-25 Comment[ArrayExpressSubmissionDate] 2013-06-21 Comment[GEOLastUpdateDate] 2013-09-25 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE48182_raw_data_FFPE.txt SDRF File E-GEOD-48182.sdrf.txt