Defining the signatures of peripheral T-cell lymphoma, with a targeted 20-markers gene expression profiling assay (RT-MLPA)

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 EBV-related transcript, and variants of RHOA (G17V) and IDH2 (R172K/T). By unsupervised hierarchical clustering, our assay accurately identified 21/21 ALK-positive anaplastic large cell lymphomas, 16/16 extranodal NK/T-cell lymphomas, 6/6 hepatosplenic T-cell lymphomas, and 13/13 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 in 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 27/77 not specified T-cell lymphomas: 17 TFH, 5 cytotoxic ALK-negative anaplastic, and 5 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 3 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.