Page 126 - Haematologica - Vol. 105 n. 6 - June 2020

F. Drieux et al.
Figure 1. Unsupervised hierarchical clustering of peripheral T-cell lymphoma (PTCL) entities other than PTCL-not otherwise specified (NOS) using reverse transcrip- tase-multiplex ligation-dependent probe amplification (RT-MLPA) (n=153). The assay was used to classify angioimmunoblastic T-cell lymphomas (AITL) (n=30), PTCL with T-follicular helper phenotype (TFH) (n=33), anaplastic large cell lymphomas (ALCL) (n=55), adult T-cell lymphomas (ATLL) (n=13), hepatosplenic T-cell lymphoma (HSTL) (n=6), and natural killer (NK)-TCL (n=16). Differential gene expression is depicted according to a red (positive) to blue (negative) color scale, and concordance with histopathological diagnosis (Pathology). Two main branches were observed: the left branch divided in 6 HTSL (C1), 50 TFH-PTCL/AITL (C2), 12 ATLL with 13 TFH- PTCL (C3), and 24 ALK-negative ALCL (C4), and the right branch contained two clusters of 16 NKTCL (C5) and 31 cytotoxic ALCL (C6).
Overall and progression-free survival analysis was performed using the Kaplan-Meier method and the log-rank test. The Mann-Whitney test was used to analyze continuous data and the Fisher exact test to analyze categorical data.
Results
Design and validation of the RT-MLPA assay
The study design is presented in Online Supplementary Figure S1. The gene set and sequences of the RT-MLPA probes are shown in Table 1 and Online Supplementary Table S1, respectively. The panel was designed to include several genes encoding immunohistochemical or genetic markers routinely used for the diagnosis of PTCL and genes of interest selected from previous transcriptomic and genomic studies.9,10,12,13,18 It includes genes related to the major CD4 and CD8 T-cell subsets, genes defining the main subsets of Th cells [TFH (CXCL13, CXCR5, ICOS, BCL6), Th1 (TBX21, IFNγ), Th2 (GATA3, CCR4), and Treg (FOXP3)], as well as genes encoding cytotoxic molecules (PRF, GZMB). CD30 and ALK were chosen to identify ALCL and CD56 and EBER1 (Epstein-Barr virus encoding small RNA) were selected to identify hepatosplenic T-cell lymphoma (HSTL) and NKTCL. We also included the RHOAG17V and IDH2R172K/T variants, as the most prevalent hotspot mutations of TFH-derived PTCL.
We obtained RT-MLPA profiles for all 230 PTCL of the classification cohort. Representative RT-MLPA profiles for each entity are shown in Online Supplementary Figure S2.
Table 1. Gene panel designed for the reverse transcriptase-multiplex ligation-dependent probe amplification assay.
Family genes and other targets
Main T-cell subsets
TFH
Th1
Th2
Treg
NK-associated
Activation Virus Mutations
Other
Genes
CD4 CD8 TCRα
CXCL13 CXCR5 BCL6 ICOS
TBX21 IFN
GATA3
CCR4 FOXP3 CD56
PRF
GZB
CD30
EBER RHOAm G17V
IDH2m R172K/T ALK
Detection method in the routine practice
Immunohistochemistry Immunohistochemistry Not applicable
Immunohistochemistry Not applicable Immunohistochemistry Immunohistochemistry
Immunohistochemistry Not applicable
Immunohistochemistry
Not applicable
Immunohistochemistry
Immunohistochemistry and cytotoxic Immunohistochemistry Immunohistochemistry
Immunohistochemistry In situ hybridization
AS-PCR, other sequencing methods
AS-PCR, other sequencing methods, immunohistochemistry
Immunohistochemistry, FISH
AS-PCR: allele-specific polymerase chain reaction; NK: natural killer; FISH: fluores- cence in situ hybridization.
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