Peripheral T-cell lymphoma with t(6;14)(p25;q11.2) translocation presenting with massive splenomegaly

Recurrent chromosomal translocations associated to peripheral T-cell lymphomas (PTCL) are rare. Here, we report a case of PTCL, not otherwise specified (NOS) with the karyotype 46,Y,add(X)(p22),t(6;14)(p25;q11) and FISH-proved breakpoints in the IRF4 and TCRAD loci, leading to juxtaposition of both genes. A 64-year-old male patient presented with mild cytopenias and massive splenomegaly. Splenectomy showed diffuse red pulp involvement by a pleomorphic medium- to large-cell T-cell lymphoma with a CD2+ CD3+ CD5− CD7− CD4+ CD8+/− CD30− TCRbeta-F1+ immunophenotype, an activated cytotoxic profile, and strong MUM1 expression. The clinical course was marked by disease progression in the bone marrow under treatment and death at 4months. In contrast with two t(6;14)(p25;q11.2)-positive lymphomas previously reported to be cytotoxic PTCL, NOS with bone marrow and skin involvement, this case was manifested by massive splenomegaly, expanding the clinical spectrum of PTCLs harboring t(6;14)(p25;q11.2) and supporting consideration of this translocation as a marker of biological aggressiveness

B-cell lymphomas with discordance between pathological features and clinical behavior

B-cell lymphomas encompass a large number of disease entities clinically ranging from indolent to aggressive. The defining pathological features usually predict clinical course, with small and large B-cell lymphomas correlating to low-grade vs high-grade features, but discordant situations may be encountered. Two sessions of the workshop of the XVIII meeting of the European Association for Haematopathology (EAHP) held in Basel in 2016 addressed this topic. One session illustrated various facets of "aggressiveness" in indolent lymphomas, either peculiar clinical manifestations, cytological variants, or unusual genetic features, as well as several examples of progression or transformation to a more aggressive disease. Another session exemplified large B-cell lymphomas with unexpected indolent behavior including cases arising in well-defined body compartments or in sanctuary sites. This paper describes the features of the cases presented in both groups, highlights the most salient points of discussion raised by the submitters and the panel, and summarizes current knowledge and recommendations relevant to diagnostic pathology practice

FLUORESCENCE IN SITU HYBRIDIZATION (FISH) ENHANCEMENT USING MICROFLUIDIC FLOW FOR AN ACCURATE, FAST AND ECONOMICAL ASSESSMENT OF HER2 STATUS IN BREAST CANCER

The fluorescence in situ hybridization (FISH) is the gold standard in human epidermal growth factor receptor 2 (HER2) status assessment in breast cancer. The dissemination of the technique is impeded by the cost of reagents and long experimental time. For overcoming these limitations, we have developed a new method for implementing FISH for HER2 assessment for tissue analysis based on microfluidic technology

Extra short incubation microfluidic assisted – fluorescence in situ hybridization (ESIMA-FISH)

Fluorescence in situ hybridization (FISH) is a powerful technique for evaluating the HER2 gene status in breast cancer specimen (1). However, most of FISH assays currently used in clinical laboratories are expensive and require long experimental time, up to two days with an overnight incubation (2). Indeed, despite the development of faster FISH probes (HER2 IQFISH pharmDxTM from DAKO, Denmark) cutting the assay time to one day (3), the cost of these new probes is still high (more than 200$/test) and impedes the dissemination of this technique. In this study, we present the extra short incubation microfluidic assisted- fluorescence in situ hybridization (ESIMA-FISH) technique that uses microfluidics to improve FISH for HER2 assessment in breast cancer samples. ESIMA-FISH requires a very short incubation time (35 minutes) and uses 4-fold less probe solution per test. The system is based on a microfluidic chip, developed in our laboratory (4), that is clamped against a microscope slide containing a breast cancer tissue specimen (figure 1). A fluorescent DNA probe solution, specific to the target DNA, is then applied to the tissue section within a thin chamber using the microfluidic system. The probe solution used is obtained by diluting 4 times the standard HER2 IQFISH pharmDxTM probe solution (DAKO, Denmark). Oscillating flows can then be implemented using syringe pumps to improve the delivery of the probe to the tissue. Thanks to this hydrodynamic enhancement of mass transport, the probe-target hybridization efficiency is increased, resulting in overall reductions in the cost and duration of the assay. To validate the ESIMA-FISH technique, several tissue specimens were blindly tested with ESIMA-FISH and standard IQFISH. The results from these two techniques were comparable (figure 2, 3), supporting the possibility of a future clinical use of ESIMA-FISH

Activating mutations in genes related to TCR signaling in angioimmunoblastic and other follicular helper T-cell-derived lymphomas.

Angioimmunoblastic T-cell lymphoma (AITL) and other lymphomas derived from follicular T-helper cells (TFH) represent a large proportion of peripheral T-cell lymphomas (PTCLs) with poorly understood pathogenesis and unfavorable treatment results. We investigated a series of 85 patients with AITL (n = 72) or other TFH-derived PTCL (n = 13) by targeted deep sequencing of a gene panel enriched in T-cell receptor (TCR) signaling elements. RHOA mutations were identified in 51 of 85 cases (60%) consisting of the highly recurrent dominant negative G17V variant in most cases and a novel K18N in 3 cases, the latter showing activating properties in in vitro assays. Moreover, half of the patients carried virtually mutually exclusive mutations in other TCR-related genes, most frequently in PLCG1 (14.1%), CD28 (9.4%, exclusively in AITL), PI3K elements (7%), CTNNB1 (6%), and GTF2I (6%). Using in vitro assays in transfected cells, we demonstrated that 9 of 10 PLCG1 and 3 of 3 CARD11 variants induced MALT1 protease activity and increased transcription from NFAT or NF-κB response element reporters, respectively. Collectively, the vast majority of variants in TCR-related genes could be classified as gain-of-function. Accordingly, the samples with mutations in TCR-related genes other than RHOA had transcriptomic profiles enriched in signatures reflecting higher T-cell activation. Although no correlation with presenting clinical features nor significant impact on survival was observed, the presence of TCR-related mutations correlated with early disease progression. Thus, targeting of TCR-related events may hold promise for the treatment of TFH-derived lymphomas

Approche moléculaire à la pathobiologie des lymphomes T périphériques

Peripheral T-cell lymphomas (PTCLs) constitute a heterogeneous group of clinically aggressive lymphoid neoplasms derived from mature T and NK cells, accounting for approximately 10% of all non-Hodgkin lymphomas. With rare exceptions, the molecular mechanisms underlying their pathogenesis are still unknown, and the demarcations between different entities are often unclear.The first part of our work aimed at identifying oncogenic alterations in angioimmunoblastic T-cell lymphomas (AITLs), one of the most common PTCL entities in western countries. We focused our investigations on MAF and BCL6, encoding transcription factors that are physiologically expressed in follicular helper T cells from which AITLs have been shown to arise. By analogy with B-cell lymphomagenesis and on the grounds of experimental murine models, we postulated that these genes might be targeted by chromosomal translocations and/or aberrant somatic hypermutations in AITLs. In a series of 97 PTCLs including 48 AITLs, we searched for MAF and BCL6 gene rearrangements and copy number alterations by fluorescence in situ hybridization (FISH). No structural or numerical MAF or BCL6 abnormalities were observed in the neoplastic cells of AITLs. In two samples we identified a t(3;14)(q27;q32)/BCL6-IGH translocation in bystander B blasts, as demonstrated by FICTION analysis combining FISH with CD20 immunofluorescence. The 5’ regulatory region of the BCL6 gene was analyzed in 20 AITLs by direct sequencing of PCR amplification products. Although several reported single nucleotide polymorphisms (SNPs) were observed, no acquired somatic mutation could be identified.The second part of our investigations regarded PTCLs characterized by the expression of CD30: ALK+ and ALK- anaplastic large cell lymphomas (ALCLs), and the CD30+ subset of PTCLs, not otherwise specified (NOS). We aimed at characterizing their molecular and immunophenotypic features in order to gain insights into their biology and improve their demarcations. By gene set enrichment analysis (GSEA) based on published gene expression profiling (GEP) datasets, we showed that CD30+ PTCLs, NOS, compared to CD30- tumors, were significantly enriched in ALK- ALCL-related genes. To validate these and previous GEP findings, we studied by immunohistochemistry a series of 80 PTCLs representative of CD30+ and CD30- PTCLs, NOS, ALK+ and ALK- ALCLs. Twenty-one proteins were selected among the molecules most differentially expressed between these subgroups, mainly involved in T-cell receptor (TCR) signaling. CD30+ PTCLs, NOS differed significantly from CD30- samples, most notably by the downregulation of TCR signaling-associated molecules, while displaying important overlaps with other CD30+ PTCLs, particularly ALK- ALCLs. Survival analyses showed a tendency for CD30- PTCLs, NOS to have a worse outcome compared to CD30+ subgroups, although the differences were not significant.In conclusion, our findings advocate against the hypothesis that molecular alterations targeting the MAF and BCL6 genes might contribute to the pathogenesis of AITLs. The characterization of CD30+ PTCLs, on the other hand, suggests that the expression of CD30 might discern two biologically distinct subgroups within the heterogeneous PTCL, NOS category, while questioning the legitimacy of the current distinction between CD30+ PTCLs, NOS and ALK- ALCLs. The putative clinical relevance of these revisited demarcations will need to be confirmed in larger series of patients./Les lymphomes T périphériques (PTCL) constituent un groupe hétérogène de néoplasies cliniquement agressives dérivées des lymphocytes T et NK matures, représentant environ 10% des lymphomes non hodgkiniens. Les anomalies moléculaires qui sous-tendent leur pathogénie sont en grande partie méconnues et les frontières entre les différentes entités souvent mal définies.La première partie de notre travail a visé l’identification d’altérations oncogéniques dans le lymphome T angioimmunoblastique (AITL), l’une des entités les plus fréquentes de PTCL dans les pays occidentaux. Nous avons centré nos recherches sur les gènes MAF et BCL6, qui encodent des facteurs de transcription physiologiquement exprimés par les cellules T helper folliculaires dont dérivent les AITL. Par analogie avec la lymphomagenèse B et sur la base de modèles murins, nous avons postulé que ces gènes pouvaient être la cible de translocations chromosomiques et/ou d’hypermutations somatiques aberrantes dans les AITL. Dans une série de 97 PTCL comprenant 48 AITL, nous avons recherché par hybridation in situ fluorescente (FISH) la présence de réarrangements géniques et de variations du nombre de copies de MAF et BCL6. Aucune anomalie structurelle ou numérique de ces gènes n’a été observée dans les cellules néoplasiques d’AITL. Néanmoins, une translocation t(3;14)(q27;q32)/BCL6-IGH a été mise en évidence dans deux échantillons au sein de blastes B d’accompagnement, comme démontré par une analyse de FICTION combinant la FISH avec une immunofluorescence pour CD20. Par séquençage direct des produits d’amplification PCR, nous avons de plus analysé, dans 20 AITL, la région régulatrice en 5’ du gène BCL6. Bien que plusieurs polymorphismes mononucléotidiques aient été identifiés, aucune mutation somatique acquise n’a pu être mise en évidence.La deuxième partie de nos recherches s’est rapportée aux PTCL exprimant l’antigène CD30: les lymphomes anaplasiques à grandes cellules (ALCL) ALK+ et ALK- et le sous-groupe CD30+ des PTCL sans spécificité (NOS). Nous avons poursuivi leur caractérisation moléculaire et immunophénotypique dans le but de mieux appréhender leur biologie et préciser leurs délimitations. Par gene set enrichment analysis (GSEA), basée sur des données transcriptomiques publiées, nous avons démontré que les PTCL, NOS CD30+ étaient significativement plus riches en gènes associés aux ALCL ALK- que les néoplasies CD30-. Pour valider ces résultats et d’autres données transcriptomiques, nous avons étudié par immunohistochimie une série de 80 PTCL représentatifs de PTCL, NOS CD30+ et CD30-, ALCL ALK+ et ALK-. Nous avons sélectionné 21 protéines différentiellement exprimées entre ces sous-groupes, souvent impliquées dans la signalisation du récepteur des lymphocytes T (TCR). Les PTCL, NOS CD30+ se sont révélés significativement différents par rapport aux néoplasies CD30-, notamment par une sous-expression de molécules liées à la signalisation du TCR, tout en montrant d’importants chevauchements avec les autres PTCL CD30+, plus particulièrement avec les ALCL ALK-. Les analyses de survie ont décelé une tendance à un pronostic plus sombre pour les PTCL, NOS CD30- par rapport aux sous-groupes CD30+, bien que les différences ne se soient pas révélées significatives.En conclusion, nos observations plaident contre l’hypothèse selon laquelle des anomalies moléculaires ciblant les gènes MAF et BCL6 contribueraient à la pathogénie des AITL. D’autre part, la caractérisation des PTCL CD30+ suggère que l’expression de CD30 pourrait discriminer deux sous-groupes biologiquement distincts au sein de la catégorie hétérogène des PTCL, NOS, et remet en cause le bien-fondé de la séparation actuelle entre PTCL, NOS CD30+ et ALCL ALK-. La pertinence clinique de ces frontières revisitées devra être confirmée dans des séries incluant un plus grand nombre de patients

Fish and chips.

peer reviewedAcademic hospital laboratories should offer patients the possibility to have the most accurate diagnosis by the development of new analyses, such as molecular biology tests including FISH (Fluorescent In Situ Hybridization) and chips (microarrays,...). The purpose of this article is to describe the principles and the potential applications of these techniques

Molecular classification of T-cell lymphomas.

T-cell neoplasms encompass a heterogeneous group of relatively rare disease entities. This review, focused on lymphoblastic tumors (T-ALL/LBL) and nodal-based peripheral T-cell lymphomas (PTCL), summarizes recent advances in the molecular characterization of these diseases. In T-ALL/LBL, molecular subgroups delineated by gene expression profiling correlate with leukemic arrest at specific stages of normal thymocyte development and different oncogenic pathways, and seem to be of interest for prognosis prediction. Angioimmunoblastic T-cell lymphoma (AITL), one of the most common PTCL entities, comprises neoplastic cells with a molecular signature similar to normal follicular helper T cells, and this cellular derivation might account for several of the peculiar aspects of this disease. Except in ALK-positive anaplastic large cell lymphoma, defined by ALK gene fusions, chromosomal translocations are otherwise rare in PTCLs, but some recurrent rearrangements might be associated with distinct lymphoma subtypes. In PTCL, not otherwise specified (PTCL, NOS), novel molecular biomarkers of potential therapeutic interest have been recently identified