Genetic alterations in mature B- and T-cell lymphomas – a practical guide to WHO-HAEM5

Abstract The identification of recurrent genomic alterations in tumour cells has a significant role in the classification of mature B- and T-cell lymphomas. Following the development of new technologies, such as next generation sequencing and the improvement of classical technologies such as conventional and molecular cytogenetics, a huge catalogue of genomic alterations in lymphoid neoplasms has been established. These alterations are relevant to refine the taxonomy of the classification of lymphomas, to scrutinize the differential diagnosis within different lymphoma entities and to help assessing the prognosis and clinical management of the patients. Consequently, here we describe the key genetic alterations relevant in mature B- and T-cell lymphomas.


A. B.
Figure 1: Mature B-cell neoplasms, cell of origin and main genetic aberrations.A. B-cell maturations process from the naïve B-cell to memory and plasma B-cell, and their derived B-cell lymphomas.The hallmark translocation and most frequently altered gene or genomic region are included in the box belonging to each entity.Of note, r, denotes rearrangement, genes in brackets are the candidate gene altered by the copy number change; and region/gene indicates that the gene is altered via copy number and/or somatic variants.U, unmutated; M, mutated, CLL, chronic lymphocytic leukaemia; cMCL, conventional mantle cell lymphoma; BL, Burkitt lymphoma; DLBCL-GCB, diffuse large B-cell lymphoma germinal centre subtype; FL, follicular lymphoma; DLBCL-ABC, diffuse large B-cell lymphoma activated subtype; PCM, plasma cell neoplasm; cHL, classical Hodgkin lymphoma; PMBCL, primary mediastinal large B-cell lymphoma; SMZL, splenic marginal zone lymphoma; neg, negative; pos, positive B. The genetic subtypes of DLBCL and the hallmark genetic aberrations of each subtype.Created with BioRender.comSPEN, NOTCH1 and DTX1 [3].On the other hand, in splenic diffuse red pulp small B-cell lymphoma, somatic mutations in BCOR, CCND3, MAP2K1, and genes of the NOTCH pathway are frequently identified, but the prevalence of 7q deletion is lower as compared to SMZL [4].Splenic B-cell lymphoma/leukaemia with prominent nucleoli (SBLPN) of WHO-HAEM5 comprises the former HCL variant (HCLv) of WHO-HAEM4R, cases of CD5 negative B-prolymphocytic leukaemia (B-PLL), and a subset of SMZL.SBLPN lacks the BRAF V600E, but some cases harbour MAP2K1 mutations [5].

Transformations of indolent B-cell lymphomas
Although the genomic profile of transformed indolent B-cell lymphomas is partially defined by the specific genomic alterations of the preceding indolent lymphoma, there are key genes that are commonly altered and associated with the transformation.These genes are mainly affecting the proliferation and survival pathways (MYC rearrangement or amplification, CDKN2A/B deletions, TP53 deletions/mutations), and NF-κB (TNFAIP3 deletions/mutations).
Diffuse large B-cell lymphoma/high grade B-cell lymphoma with MYC and BCL2 rearrangements (DLBCL/HG-BL-MYC/BCL2).Concomitant MYC and BCL2 translocations are the genetic hallmark of this entity.An IG locus is the MYC partner in 50% of the cases, the most common being t(8;14)(q24;q32)/IGH::MYC. The mutational profile of these lymphomas is characterized by somatic variants in genes commonly altered both in FL and transformed lymphomas or Burkitt lymphoma (BL) such as CREBBP, KMT2D, EZH2, SI, TNFRSF14, BCL2, MYC, CCND3 and TP53 [14,15].
High-grade B-cell lymphoma with 11q aberrations is characterized by a gain of 11q23.2-q23.3 and a loss (or copy number neutral loss of heterozygosity in occasional cases) of 11q24.1-qter(Figure 2A).The cases display morphological and immunophenotypic features similar to BL, but the genetic profile is distinct.MYC translocations are by definition absent and no rearrangements of BCL2 nor BCL6 are identified.Somatic variants in BTG2, DDX3X, ETS1, EP300, NFRKB and GNA13 have been detected [17,18].
Primary large B-cell lymphoma of immune-privileged sites represents a new umbrella entity in WHO-HAEM5.It includes primary DLBCL of the central nervous system (CNS), the vitreoretinal compartment, and the testes of immunocompetent patients.Tumours in this umbrella category are characterized by a similar high prevalence of somatic variants in PIM1, MYD88 L265P , and CD79B.Furthermore, genomic alterations in genes involved in immune evasion, such as rearrangements in the PD-L1/PD-L2 loci (9p24), and loss of HLA loci (6p21), and DNA damage, CD-KN2A/B(9p21) are key players [19].The genomic landscape of this entity corresponds to molecular subgroups MCD/C5 [12].Notwithstanding this, various mutational processes contribute to the pathogenesis of primary DLBCL of the CNS [20,21].
Primary mediastinal large B-cell lymphoma (PMBCL) is characterized by activation of JAK-STAT and NF-κB signalling pathways.JAK-STAT is activated via activation of IL13, loss of function mutations in SOCS1 and PTPN1, and gain of function mutations in STAT6 and IL4R.The NF-κB pathway is altered via gains in 2p16.1/REL and inactivating mutations of negative regulators such as TNFAIP3 or NFKBIE [22].In addition, genetic alterations involve genes related to immune evasion such as PD-L1/PD-L2, CD58, B2M, CD274 and CIITA.The genetic profile is in proximity to classic Hodgkin lymphoma.

Mediastinal gray zone lymphoma (MGZL) displays characteristics intermediate between PMBCL and classic
Hodgkin lymphoma (CHL).MGZL is characterized by somatic variants in SOCS1, B2M, TNFAIP3, GNA13, LRRN3 and NFKBIA [23].This mutational profile is specific of the thymic niche and different from morphologically similar tumours arising in extra-mediastinal regions.A BCL2 rearrangement is present in only 6% of the cases and BCL6 translocations are virtually absent.
High-grade B-cell lymphoma, NOS (HGBL, NOS) is a biologically heterogenous entity.MYC translocations are identified in 50% of the cases, whereas BCL2 (without MYC translocation by definition) and BCL6 rearrangements are less prevalent.No specific mutational profile has been identified in these patients, although somatic variants in genes altered in BL such as ID3, CCND3 and MYC or genes altered in DLBCL, NOS including CREBBP and BCL2 have been reported [24].

Burkitt lymphoma
A MYC rearrangement is the hallmark aberration in BL leading to MYC overexpression.The translocation t(8;14) (q24;q32)/IGH::MYC is present in approximately 80% of the patients (Figure 2A), and, less frequently, MYC is rearranged with the IG light chain loci [25].Deregulation of the ID3-TCF3 axis is highly specific of BL.ID3 and TCF3 alterations are detected in 10-20% and 50-60% of the patients, respectively.Additional somatic variants detected in BL affect MYC, CCND3, TP53, SMARCA4, and GNA13 genes.Overall, four pathways are altered in BL, BCR signalling, proliferation and survival, sphingosine-1-phosphate signalling, and SWI-SNF chromatin remodelling [25].Recent molecular studies support the idea to subclassify BL based on EBV status rather than the traditional epidemiological variants.EBV-BL is characterized by a high frequency of ID3, TCF3 and CCND3 somatic alterations whereas EBV+ BL is defined by a higher number of somatic variants and increased PI3K activity [26].BL may occur in paediatric and adult patients and a recent study reported that genomic alterations such as ID3, DDX3X, ARID1A, and SMARCA4 were more frequent in paediatric patients whereas BCL2 and YY1AP1 have been reported to be particularly altered in adult patients [27].
Plasma cell neoplasms and other diseases with paraprotein are described in detail in another manuscript of this special issue.

T-cell and NK-cell lymphomas
T-cell and NK-cell lymphoproliferations and lymphomas are organized in 8 families in WHO-HAEM5.Figure 3A provides an overview on genetic alterations in different entities of T-cell lymphomas in WHO-HAEM5.

Mature T-cell and NK-cell leukaemias
Chromosomal rearrangements involving 14q11 in T-Prolymphocytic leukaemia (T-PLL) deregulate TCL1A due to a TRA::TCL1A rearrangement (Figure 2B).In a small subsets of T-PLL TCL1A is substituted by MTCP1 on chromosome X; in addition, T-PLL is often characterized by mutations and/or deletions of ATM, EZH2, FBXW10 and CHEK2 genes [29].T-large granular-lymphocytic leukaemia (T-LGLL) is characterized by recurrent STAT5B mutations and other recurrently altered genes such as TET2, TNFAIP3, BCL11B, FLT3 und PTPN23 [30].NK-large granular lymphocytic leukaemia, in contrast, shows activating mutations of STAT3, TET2 and CCL22.Adult T-cell leukaemia/ lymphoma (ATLL), next to its classical association with HTLV-1, shows a plethora of activating mutations such as PLCG1, PRKCB and CARD11 among others and, notably, also structural variations of PD-L1 [31].Sezary syndrome characteristically displays a high mutational burden inferred by UV-induced damage and mutations affecting TCR-signalling, chromatin-modifier, and DNA-damage-response pathways.Finally, aggressive NK-cell leukaemia is characterized by mutations in genes affecting cell signalling, histone modifying molecules and immune checkpoint molecules.

Primary cutaneous T-cell lymphomas
The genetic constitution of Cutaneous T-cell neoplasms is highly variable, reflecting their highly variable clinical presentation and morphological features.More recent literature has identified rearrangements of the DUSP22-IRF4 locus in a small subset of lymphomatoid papulosis cases, originally described as a characteristic of nodal ALK-negative anaplastic large cell lymphoma.

Enteropathy-associated T-cell lymphoma (EATL) and monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL)
represent distinct entities, the former being strongly associated with celiac disease.Their genetic features are similar, however not identical, with amplifications of 9q34 and deletions at 16q12 seen in a high proportion of EATL, as are activating mutations in JAK1 and STAT3.Loss-of-function mutations in TNFAIP3 result in NF-kB activation [32].In MEITL, next to JAK-STAT deregulation, mutations of SETD2, a histone-lysin 36 methyltransferase obviously is crucial for disease initiation and propagation.
Indolent T-cell-lymphoma of the gastrointestinal tract (GIT) harbours mutations in epigenetic modifying genes such as TET2 and KMT2D, recurrent STAT3::JAK2 fusions and structural alterations of the IL2 gene; interestingly, these alterations are found in different frequencies in CD4-positive and CD8-positive (or double negative) subsets.The finding of recurrent mutations in JAK3 and other genes has built evidence that Indolent NK-cell lymphoproliferation (NKLPD) of the GIT is a neoplastic disease, however, basing on its generally indolent clinical course, this disease has been named "lymphoproliferative disorder" and not "lymphoma".

Hepatosplenic T-cell lymphoma
This lymphoma shows a prototypic isochromosome 7q which forms the basis of the overexpression of CHN2, ABCB1 and other genes.Several mutations in genes of the JAK-STAT pathway and/or epigenetic modifier genes have been described.

Anaplastic large cell lymphoma
Anaplastic large cell lymphoma (ALCL) denotes a group of mature T-cell lymphomas that are similar in their cytomorphology and immunophenotype but clinically and genetically heterogeneous (Figure 2B).ALCL showing rearrangements in 2p23, the location of the ALK gene, constitute 50 % of tumours (Figure 3B-C).In the translocations, more than 20 partner genes are involved deregulating ALK expression, the most frequent one being NPM1 involved in the t(2;5)(p23;q35) [33].Constitutive activation of the tyrosine kinase ALK activates several signalling pathways including PI3K/AKT, mTOR, JAK-STAT3 and STAT5B.In contrast, ALK-negative ALCL feature rearrangements of the DUSP22 and TP63 genes in around 30% and 5% of cases, respectively.These oncogenic fusions involving tyrosine kinase genes other than ALK also lead to STAT3 activation; transcription factors such as IRF4 and MYC have been shown to be essential for the survival of ALCL cells [33].
Breast implant-associated ALCL (BIA-ALCL) occurs approximately 10 years after implant placement and generally runs an indolent clinical course, especially if the lymphoma cells are confined to the peri-implant space and/or occur as an effusion.Although the mutational landscape of BIA-ALCL is not fully elucidated, activating somatic mutations have been described in STAT3, STAT5B, JAK1 and JAK2 and loss of function mutations in SOCS1 and SOCS3 pointing to the importance of activation of the JAK/STAT pathway also in this disease.

Nodal T-follicular helper (TFH) cell lymphoma
An important new concept is the creation of an umbrella entity of nodal T-follicular helper cell lymphomas (nTFH lymphomas) encompassing nTFH-angioimmunoblastic-type, nTFH-follicular-type and nTFH-NOS that have different morphological features but in common a derivation from nodal helper T-cells of the reactive germinal center and an expression of TFH-associated immunophenotypic markers such as PD-1, ICOS, CXCL13, CD10, BCL6 and others [34].Of importance, the gene expression profiles of these three nTFH-types are remarkably similar, as is their mutational landscape featuring mutations in TET2, DNMT3A, IDH2 and RHOA, albeit at differing frequencies.Of importance, TET2 and DNMT3A mutations have been shown to arise in myeloid precursor cells and are classical components of the concept of clonal haematopoiesis, whereas IDH2 and RHOA formation has been shown to occur in somatic, T-cell -com-mitted -tumour cells, thus linking clonal haematopoiesis with T-cell lymphomagenesis.

Peripheral T-cell lymphomas, NOS
The genetic landscape of peripheral T-cell lymphomas (PTCL), NOS, is heterogeneous.Gene expression profiling studies have identified two molecular subtypes, PT-CL-GATA3 and PTCL-TBX21, and these subtypes do also differ in their genetic landscapes with 9p, 10p and 17p deletions mainly occurring in PTCL-GATA3 and PTCL-TBX21 harbouring frequently mutations in epigenetic regulators such as TET2 and DNMT3A.

EBV-positive NK/T-cell lymphomas
Among the classically EBV-positive T-cell and NK-cell lymphomas, extranodal NK/T-cell lymphoma is frequently arising -as the name implies -in extranodal sites such as the nasal cavity and the paranasal sinuses, but also in the skin, GIT, testis and other locations.It is characteristically associated with deletions in 6q21-25 harbouring several tumour suppressor genes such as PRDM1 and others and features recurrent mutations in JAK-STAT pathway genes, epigenetic regulators, tumour suppressor genes (such as TP53) and RNA helicases.In contrast, EBV-positive nodal T-cell and NK-cell lymphoma is a nodal neoplasm mainly diagnosed in Asian patients and often shows mutations in TET2, PIK3CD, DDX3X and STAT3 [35].

EBV-positive T-and NK-cell lymphoid proliferations and lymphomas of childhood
The family of EBV-positive T-cell-and NK-cell lymphoid proliferations and lymphomas of childhood represents a group of uncommon disorders characterized by EBV-infection of T-and NK-cells and shows variable clinical features ranging from indolent and localized forms to systemic disease characterized by constitutional symptoms and high clinical aggressiveness.The genetic landscape of these dis-eases has been incompletely characterized.Somatic mutations have been identified in DDX3X (e. g. in severe mosquito bite allergy suggesting that this is a neoplastic disease) and in systemic chronic active EBV disease [36].

Conclusions
Genetic data can be helpful or are occasionally even essential in the diagnosis of, and sometimes highly relevant in treatment decisions in haematological malignancies (Table 1).
The identification of specific chromosomal rearrangements using gold standard techniques such as conventional cytogenetics and molecular cytogenetics (FISH) are essential to identify hallmark aberrations specific to some subtypes of mature lymphomas, such as t(8;14)/IGH::MYC translocation in BL.Besides, NGS technologies have been important to identify somatic variants crucial in a subset of entities and defining new target vulnerabilities.Taken together, the identification of genetic alterations in mature B-and T-cell lymphomas have impact in the diagnosis, risk stratification, and ultimately clinical management, of the patients.

Figure 2 :
Figure 2: Chromosomal alterations in mature B-and T-cell neoplasms.A. Chromosomal alterations observed in specific entities of mature B-cell lymphomas.The rearranged genes are labelled according to the colour design of FISH dual colour dual fusion probes for the translocations t(14;18), t(11;14) and t(8;14), FISH break-apart probes for the loci MYC, BCL2, BCL6 and IRF4, and locus specific FISH probes for the 11q aberrations.B. Chromosomal rearrangements detected in entities of T-cell neoplasms.The rearranged genes are labelled based on the colour design of FISH break-apart probes for the loci TCL1A, TRA, ALK, TP63 and DUSP22.For the inv(14), t(14;14) and t(X;14), the genomic rearrangements and the derivative chromosomes generated from the chromosomal rearrangements are represented.Created with BioRender.com.

Figure 3 .
Figure 3. Main genetic aberrations in T/NK-cell malignancies and ALK rearrangements in Anaplastic large cell lymphomas.A. The mature subgroup of T/NK-cell malignancies is divided into leukaemic, nodal and extranodal.The most frequently altered genes or genomic regions are included in the box belonging to each entity.T-PLL, T-Prolymphocytic leukaemia; T-LGLL, T-large granular-lymphocytic leukaemia; NK-LGLL, NK-large granular lymphocytic leukaemia; ATLL, Adult T-cell leukaemia/lymphoma; nTFH, Nodal T-follicular helper; PTCL-NOS; peripheral T-cell lymphomas not otherwise specified; ALCL, Anaplastic large cell lymphoma; CTCL, Cutaneous T-cell lymphoma; BIA-ALCL, Breast implant-associated ALCL; HTCL, Hepatosplenic T-cell lymphoma; EATL, Enteropathy-associated T-cell lymphoma; MEITL, monomorphic epitheliotropic intestinal T-cell lymphoma; ENKT, extranodal NK/T-cell lymphoma.B. Staining of ALK using immunohistochemistry showed a large proportion of ALK positive cells.C. Fluorescence in situ hybridisation using an ALK break-apart probe.A cell without break is shown on the left and an ALK-positive cell on the right, showing one fusion signal (green & red signals) corresponding to the unarranged allele, and one green and one red signal indicating a break in ALK gene.

Enteropathy
Gains or rearrangement PD-L1/PD-L2Useful for treatment with PD1 inhibitor in relapse/ refractory cHL cell leukaemia/lymphoma PLCG1 and CARD11 mutations Useful for diagnosis TP53 and PRKBC mutations Associated with inferior clinical behaviourIntestinal T-cell and NK-cell lymphoid proliferations and lymphomas anaplastic large cell lymphoma (ALK+ ALCL)ALK rearrangements Appropriate for diagnosis and useful for ALK inhibitors treatment

Table 1 :
Clinical relevance of genetic alterations in mature B and T-cell neoplasms