title: Diffuse large B-cell lymphoma: sub-classification by massive parallel quantitative RT-PCR.
abstract: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity with remarkably variable clinical outcome. Gene expression profiling (GEP) classifies DLBCL into activated B-cell like (ABC), germinal center B-cell like (GCB), and Type-III subtypes, with ABC-DLBCL characterized by a poor prognosis and constitutive NF-κB activation. A major challenge for the application of this cell of origin (COO) classification in routine clinical practice is to establish a robust clinical assay amenable to routine formalin-fixed paraffin-embedded (FFPE) diagnostic biopsies. In this study, we investigated the possibility of COO-classification using FFPE tissue RNA samples by massive parallel quantitative reverse transcription PCR (qRT-PCR). We established a protocol for parallel qRT-PCR using FFPE RNA samples with the Fluidigm BioMark HD system, and quantified the expression of the COO classifier genes and the NF-κB targeted-genes that characterize ABC-DLBCL in 143 cases of DLBCL. We also trained and validated a series of basic machine-learning classifiers and their derived meta classifiers, and identified SimpleLogistic as the top classifier that gave excellent performance across various GEP data sets derived from fresh-frozen or FFPE tissues by different microarray platforms. Finally, we applied SimpleLogistic to our data set generated by qRT-PCR, and the ABC and GCB-DLBCL assigned showed the respective characteristics in their clinical outcome and NF-κB target gene expression. The methodology established in this study provides a robust approach for DLBCL sub-classification using routine FFPE diagnostic biopsies in a routine clinical setting.
authors.affiliation: 1] Department of Pathology, Health Science Center, Peking University, Beijing, China [2] Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, UK.
authors.forename: Xuemin
authors.initials: X
authors.lastname: Xue
authors.affiliation: Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, UK.
authors.forename: Naiyan
authors.initials: N
authors.lastname: Zeng
authors.affiliation: Department of Pathology, Health Science Center, Peking University, Beijing, China.
authors.forename: Zifen
authors.initials: Z
authors.lastname: Gao
authors.affiliation: Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, UK.
authors.forename: Ming-Qing
authors.initials: MQ
authors.lastname: Du
mesh_terms: D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse / Q000145:classification* / Q000235:genetics / Q000473:pathology; D016612:Paraffin Embedding; D020133:Reverse Transcriptase Polymerase Chain Reaction