Wg dasl assay

A total of 234 cases of de novo DLBCL were investigated in this study. 168 cases were retrieved from the Haematological Malignancy Diagnostic Service (HMDS) at St James's University Hospital, Leeds (n = 145) and Addenbrooke's hospital, Cambridge (n = 23), based on the availability of lymphoma tissue specimens. 153 of these cases have been classified by cell of origin (COO) using the Illumina WG‐DASL assay used in previous studies 7, 26. The remaining 66 cases were positive for MYC translocation by interphase fluorescence in situ hybridisation (FISH) and identified from five participating centres where the assessment of MYC translocation status is a part of the routine diagnostic workup of DLBCL 27. The diagnosis in each case was established by two expert haematopathologists, and those defined as B‐cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma, in the 2008 WHO classification were included in this study 28. Burkitt lymphoma, DLBCL potentially transformed from a low grade lymphoma, cases with HIV or primary CNS lymphoma were excluded from this study. All the laboratory investigations described below were based on the initial diagnostic lymphoma tissue specimens. Ethical guidelines were followed for the use of archival tissues for research with the approval of the ethics committees of the involved institutions.

Whole genome gene expression analysis was performed using the Illumina (San Diego, CA, USA) WG-DASL assay according to manufacturer's protocol. Briefly, 100 ng of FFPE RNA was converted to cDNA by the WG-DASL assay using biotinylated-tagged random nonamer and oligo (dT) primers. The biontinylated cDNA was then mounted onto a streptavidin-coated support and further extended and ligated by gene-specific oligonucleotides (DAP). Subsequently, PCR amplification was performed. The resulting PCR products were eluted and hybridized to the Illumina Human-Ref v3.0 Beadchip and scanned with the Illumina iScan Reader. The image intensity values from the microarray images generated were then analysed by the GenomeStudio Gene Expression Module (Illumina, San Diego, CA, USA) software. The processed methylation values were subsequently used for further analysis in this study.

To examine the prognostic significance of mRNA expression, microarray data were analyzed as described previously [29 (link)]. In brief, RNA was extracted from formalin-fixed, paraffin-embedded tissue sections with removal of nontumor elements. RNA was extracted using the High Pure RNA Paraffin kit (Roche Diagnostic, Mannheim, Germany) and subsequently, the whole genome cDNA mediated annealing selection and ligation (WG-DASL) assay was performed following the manufacturer’s instructions (Illumina, San Diego, CA). With the exclusion of inadequate samples, a total of 300 patient samples were evaluable and the expression data was deposited in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE44001).