Page 133 - Haematologica - Vol. 105 n. 6 - June 2020

PTCL classification using RT-MLPA assay
vival of DUSP22-rearranged ALCL.11,26 In the absence of TP63 rearrangement (data not shown), further investigations are needed to explore the genetic abnormalities in the DUSP22-non rearranged cases.
Within the umbrella category of nodal PTCL of TFH deri- vation, comprising AITL and a number of nodal PTCL pre- viously classified under PTCL-NOS but expressing two or more TFH markers, our study interrogates the biological and clinical significance of two subgroups.1 Indeed, whereas the majority (79%) were attributed to the TFH/AITL class by the molecular classifier, a minority disclosed, in addition to TFH markers, enrichment in Th2 genes. We confirmed a “mixed” TFH/Th2 immunophenotype for nine TFH-PTCL showing a Th2 signature by RT-MLPA. It has been report- ed that Th1 and Th2 cells can express TFH markers, and conversely that TFH cells have the capacity to express Th1 or Th2 cytokines.23,24 It has also been suggested that a sub- set of TFH cells may originate from Th2 lymphocytes in the presence of B cells and that TFH cells can acquire GATA3 expression.25,26 Overall, these data raise the question of Th cell plasticity and the specificity of the criteria required to assess TFH-derived PTCL. Indeed, the current definition of a TFH phenotype based on two or more TFH markers may have some limitations in certain cases,1 and the integration of genetic markers, made possible by the RT-MLPA assay, may be helpful. In this respect, when comparing the “TFH/AITL” class to the other SVM classes, it appeared that, among the four TFH genes in our RT-MLPA panel (BCL6, CXCL13, CXCR5, ICOS), ICOS and then BCL6 appeared less specific than CXCL13 and CXCR5 (data not shown). Further studies, however, are needed to determine whether cases with a “mixed” TFH-Th2 RNA profile or immunophenotype should be considered to be TFH-PTCL or Th2 PTCL-NOS with associated TFH markers. The almost complete absence of RHOA and TET2 mutation in the cases with a Th2-like profile may support the latter hypothesis.
Our targeted RT-MLPA panel confirmed two subgroups among PTCL-NOS, based on the expression of TBX21 and cytotoxic markers or GATA3 and CCR4 in 39% and 31% of cases, respectively, expanding the findings of previous studies.13,14 The prognostic relevance of these two groups is controversial.27 Within the limits of our retrospective study, we failed to demonstrate any significant correlation with outcome between the Th1 and Th2 molecular or pheno- typic subgroups. In addition, all TBX21 cases in our series had a cytotoxic profile by RT-MLPA and immunohisto- chemistry. Among PTCL-NOS with a cytotoxic pheno- type, the RT-MLPA assay highlighted a small group of cases with an EBV signature, now referred to as “nodal T/NK lymphoma EBV-positive” according to the revised WHO classification.1 Whether these cases, confirmed to be EBV-positive in almost all neoplastic cells by EBER ISH, are related to extranodal NK/T lymphoma nasal-type warrants further investigation. Of note, the Th2 signature based on the expression of GATA3 and CCR4 in our panel was char- acteristic, although not specific, of ATLL. The expression of GATA3 and CCR4 together with variable expression of FOXP3 in ATLL is in accordance with a recent study show- ing that the HBZ transcript induces CCR4 expression in CD4 T cells by enhancing GATA3 expression, whereas FOXP3 expression was inconsistent in ATLL. However, the distinction between PTCL-NOS with a Th2-like signature and ATLL requires an investigation into HTLV1 serology and/or viral integration.28,29
Finally, our SVM model proposed a class for most cases, with few discrepancies. It may be a useful tool in combi- nation with pathological evaluation, especially when immunohistochemistry is not conclusive or not available. In this series, 23% of cases diagnosed by default as PTCL- NOS due to incomplete or failure in immunohistochem- istry were classified as TFH/AITL by our assay. Unclassified or misclassified cases by RT-MLPA were limited to TFH- PTCL or AITL rich in reactive CD8-positive cytotoxic cells known to be abundant in a proportion of AITL,30 or cyto- toxic PTCL with various reactive TFH cells. These cases illustrate the contribution of the microenvironment to the molecular signature, especially when tumor cell content is low or heterogeneous, a common problem encountered in all gene-expression methods. Therefore, RT-MLPA results should be interpreted in the light of clinical context, as well as biological and histopathological findings. It is worthy of note that our assay does not provide a final solution for PTCL classification, but rather a step forward that requires extensive reworking.
Overall, this study demonstrates the applicability of a robust and dedicated RT-MLPA assay which is easily trans- posable to the diagnostic workflow. It is simple to use, can be applied to FFPE and frozen samples, integrates genetic features, and is cost-effective; these all make it an attractive alternative to high-throughput technologies in routine practice. Implementation of RT-MLPA in large studies in the future, especially in the setting of clinical trials, may confirm how this adjunct tool can help better classify PTCL and therefore improve the management of these patients in the era of personalized medicine.
Acknowledgments
The authors would like to thank the LYSA-Pathology and the Plateforme de Ressources Biologiques from Henri Mondor Hospital for its technical assistance and the participants of the Tenomic consortium (see Online Supplementary Appendix). This work was supported in part by institutional grants from the Institut National du Cancer (INCA), the Fondation pour la Recherche Médicale (FRM, Equipe Labellisée DEQ20160334875), the Leukemia Lymphoma Society (LLS), the Fondation Force Hémato and the Institut Carnot (CALYM), MEDIC Foundation.
Appendix
Participants of the Tenomic consortium
A. Martin, Hôpital Avicenne, Bobigny, France; I. Soubeyran, P. Soubeyran, Institut Bergonié, Bordeaux, France; A. Pilon, O.Tournilhac, CHU Estaing, Clermont-Ferrand, France; P. Gaulard, C. Copie-Bergman, M.H. Delfau, J. Moroch, E. Poullot, F. Lemonnier, F. Le Bras, J. Dupuis, C. Haioun, Hôpital Henri Mondor, Créteil, France; T. Petrella, L. Martin, JN. Bastié, O. Casasnovas CHU,Dijon, France; B. Fabre, R. Gressin, CHU, Grenoble, France; L. de Leval,B. Bisig, E. Missiaglia, MP Dobay, A. Cairoli, CHUV, Lausanne, Suisse; C. Bonnet, J. Somja, CHU Sart-Tilman,Liège, Belgique; M.C. Copin, B. Bouchindhomme, F. Morschhauser, CHU, Lille, France; B.Petit, M. Delage, A. Jaccard, Hôpital Dupuytren, Limoges, France; F. Berger, B. Coiffier, A. Traverse-Glehen, L. Genestier, E.Bachy, CHU Sud, Lyon, France; T. Rousset, G. Cartron, V. Szablewski, Hôpital Gui de Chauliac-St Eloi, Montpellier, France; S. Thiebault, B. Drenou, Hôpital E. Muller, Mulhouse, France; K. Montagne, C. Bastien, S.Bologna, CHU de Brabois, Nancy, France; C. Bossard, S. Le Gouill, Hôtel-Dieu, Nantes, France; J. Brière, V. Meignin, C. Gisselbrecht, J. Soulier, Hôpital St Louis, Paris, France; B.
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