Defining signatures of peripheral T-cell lymphoma with a targeted 20-marker gene expression profiling assay.

Peripheral T-cell lymphoma comprises a heterogeneous group of mature non-Hodgkin lymphomas. Their diagnosis is challenging, with up to 30% of cases remaining unclassifiable and referred to as "not otherwise specified". We developed a reverse transcriptase-multiplex ligation-dependent probe amplification gene expression profiling assay to differentiate the main T-cell lymphoma entities and to study the heterogeneity of the "not specified" category. The test evaluates the expression of 20 genes, including 17 markers relevant to T-cell immunology and lymphoma biopathology, one Epstein-Barr virus-related transcript, and variants of RHOA (G17V) and IDH2 (R172K/T). By unsupervised hierarchical clustering, our assay accurately identified 21 of 21 ALK-positive anaplastic large cell lymphomas, 16 of 16 extranodal natural killer (NK)/T-cell lymphomas, 6 of 6 hepatosplenic T-cell lymphomas, and 13 of 13 adult T-cell leukemia/lymphomas. ALK-negative anaplastic lymphomas (n=34) segregated into one cytotoxic cluster (n=10) and one non-cytotoxic cluster expressing Th2 markers (n=24) and enriched in DUSP22-rearranged cases. The 63 TFH-derived lymphomas divided into two subgroups according to a predominant TFH (n=50) or an enrichment in Th2 (n=13) signatures. We next developed a support vector machine predictor which attributed a molecular class to 27 of 77 not specified T-cell lymphomas: 17 TFH, five cytotoxic ALK-negative anaplastic and five NK/T-cell lymphomas. Among the remaining cases, we identified two cell-of-origin subgroups corresponding to cytotoxic/Th1 (n=19) and Th2 (n=24) signatures. A reproducibility test on 40 cases yielded a 90% concordance between three independent laboratories. This study demonstrates the applicability of a simple gene expression assay for the classification of peripheral T-cell lymphomas. Its applicability to routinely-fixed samples makes it an attractive adjunct in diagnostic practice.

Authors' Reply.

Authors' Reply to the Letter to the Editor by Y. Lynn Wang (MYD88 mutations and sensitivity to ibrutinib therapy).

Determination of Molecular Subtypes of Diffuse Large B-Cell Lymphoma Using a Reverse Transcriptase Multiplex Ligation-Dependent Probe Amplification Classifier: A CALYM Study.

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.