Identification of DLBCL subtypes by RT-MLPA classifier and its potential as a rapid, cost-effective routine diagnostic

A study by Victor Bobée from INSERM U1245, Department of Biological Hematology, University of Normandie, Rouen, France, and colleagues studied the use of the reverse transcriptase multiplex ligation-dependent probe amplification classifier (RT-MLPA) in identifying different molecular subtypes of diffuse large B-cell lymphoma (DLBCL). The study was conducted by CALYM, the Consortium for the Acceleration of Innovation and its Transfer in the LYMphoma Field, which is credited by the Carnot Institute. The study was published on 6th November 2017 in the Journal of Molecular Diagnostics.

The three main subtypes of DLBCL are germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal B-cell lymphoma (PMBCL). The treatments for these subtypes are varied, therefore differentiating these subtypes may improve patient outcomes through providing them with the most appropriate treatment. The current ‘gold standard’ diagnostic tool to identify these subtypes is array-based gene expression profiling (GEP)2, but this is not easily used in daily diagnoses.

Study Information

  • The RT-MLPA assay was trained using 70 paraffin-embedded biopsy samples and was further validated using more than 160 independent biopsy samples
  • The assay aimed to identify; GCB, ABC, PMBCL and was extended to include Epstein-Barr virus-positive (EBV+) DLBCL and the ABC MYD88 L265P mutation
  • Affymetrix U133+2 data was used as a reference comparison

Key Findings

  • 218 biopsy samples were used from various sources:
    • 150 DLBCL = GHEDI (Deciphering the Genetic Heterogeneity of Diffuse large B-cell lymphoma in the rituximab era)
    • 38 PMBCL = LNH07-3B trial and Center Henri Becquerel, Rouen, France
    • 30 DLBCL = Center Henri Becquerel
  • 21 genetic markers were included in the assay
  • 11 genes were used to differentiate ABC (e.g. IRF4, IGHM) from GCB (e.g. NEK6) and PMBCL (e.g. TNFRSF8 encoding CD30)
  • RT-MLPA assay correctly identified subtypes 85.0% on average:
    • ABC = 87.3%
    • GCB = 83.6%
    • m-PMBCL = 80%
    • This was more accurate than the Hans IHC algorithm which correctly identified on average 78.8% of subtypes
  • The RT-MLPA assay was highly efficient in identifying the MYD88 L265P mutation and was efficient in identifying EBV+ DLBCL

The authors highlighted the importance of identifying the DLBCL subtype at diagnosis for more targeted medical treatment. The study showed that the RT-MLPA classifier tool detailed was an effective method of identifying the three main DLBCL subtypes, including MYD88 L265P mutated ABC and EBV infection status. Furthermore, the RT-MLPA classifier was proven to be an efficient, rapid, and cost-effective assay since no specialized platforms are required to implement it in a laboratory. This could result in an increased availability and frequency of these diagnostic tests being used in practice. The authors recommended identifying DLBCL subtypes at diagnosis as it has the potential to greatly improve patient outcomes.


Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. It includes three major subtypes termed germinal center B-cell-like, activated B-cell-like, and primary mediastinal B-cell lymphoma. With the emergence of novel targeted therapies, accurate methods capable of interrogating this cell-of-origin classification should soon become essential in the clinics. To address this issue, we developed a novel gene expression profiling DLBCL classifier based on reverse transcriptase multiplex ligation-dependent probe amplification. This assay simultaneously evaluates the expression of 21 markers, to differentiate primary mediastinal B-cell lymphoma, activated B-cell-like, germinal center B-cell-like, and also Epstein-Barr virus-positive DLBCLs. It was trained using 70 paraffin-embedded biopsies and validated using >160 independent samples. Compared with a reference classification established from Affymetrix U133 + 2 data, reverse transcriptase multiplex ligation-dependent probe amplification classified 85.0% samples into the expected subtype, comparing favorably with current diagnostic methods. This assay also proved to be highly efficient in detecting the MYD88 L265P mutation, even in archival paraffin-embedded tissues. This reliable, rapid, and cost-effective method uses common instruments and reagents and could thus easily be implemented into routine diagnosis workflows, to improve the management of these aggressive tumors.

  1. Bobee V. et al. Determination of molecular subtypes of diffuse large b-cell lymphoma using a reverse transcriptase multiplex ligation-dependent probe amplification classifier: a CALYM study. Journal of Molecular Diagnosis. 2017 Nov;19(6):892-904. DOI: 10.1016/j.jmoldx.2017.07.007. [Epub 2017 Oct 17].
  2. Gutiérrez-García G. et al. Grup per l’Estudi dels Limfomes de Catalunya I Balears (GELCAB): Gene-expression profiling and not immunophenotypic algorithms predicts prognosis in patients with diffuse large B-cell lymphoma treated with immunochemotherapy. Blood. 2011 May 5;117(18):4836-43. DOI: 10.1182/blood-2010-12-322362.