Accurate classification of GCB/ABC Diffuse Large B cell Lymphoma using a simple and rapid RT-MLPA assay: a CALYM study

Abstract

Diffuse large B-cell lymphoma, the most common non-Hodgkin lymphoma, is subdivided into germinal center B-cell–like and activated B-cell–like subtypes. Unfortunately, these lymphomas are difficult to differentiate in routine diagnosis, impeding the development of treatments. Patients with these lymphomas can benefit from specific therapies. We therefore developed a simple and rapid classifier based on a reverse transcriptase multiplex ligation dependent probe amplification assay and 14 gene signature. When compared to the Affymetrix U133+2 gold standard, all 46 samples 95% CI [92%, 100%]) of a validation cohort classified by both techniques were attributed to the expected subtype. Similarly, 93% of the 55 samples (95% CI [82%, 98%]) of a second independent series characterized using a mid-throughput gene expression profiling method were classified correctly. Unclassifiable sample proportions reached 13.2% and 13.8% in these cohorts, comparable to the frequency originally reported. The developed assay was also sensitive enough to obtain reliable results from preserved samples and flexible enough to include prognostic factors such as MYC/BCL2 co-expression. Finally, in a series of 135 patients, both overall (P =0.01) and progression-free (P =0.004) survival differences between the two subtypes were confirmed. As the multiplex ligation dependent probe amplification methodology is already in use and requires only common instruments and reagents, it could easily be applied to clinical trial patient stratification to help in treatment decisions.