Recent advances upper gastrointestinal lymphomas: molecular updates and diagnostic implications

Approximately one‐third of extranodal non‐Hodgkin lymphomas involve the gastrointestinal (GI) tract, with the vast majority being diagnosed in the stomach, duodenum, or proximal small intestine. A few entities, especially diffuse large B‐cell lymphoma and extranodal marginal zone lymphoma of mucosa‐associated lymphoid tissue, represent the majority of cases. In addition, there are diseases specific to or characteristic of the GI tract, and any type of systemic lymphoma can present in or disseminate to these organs. The recent advances in the genetic and molecular characterisation of lymphoid neoplasms have translated into notable changes in the classification of primary GI T‐cell neoplasms and the recommended diagnostic approach to aggressive B‐cell tumours. In many instances, diagnoses rely on morphology and immunophenotype, but there is an increasing need to incorporate molecular genetic markers. Moreover, it is also important to take into consideration the endoscopic and clinical presentations. This review gives an update on the most recent developments in the pathology and molecular pathology of upper GI lymphoproliferative diseases.


Introduction
Lymphomas involving the gastrointestinal (GI) tract account for approximately 30-40% of extranodal lymphomas. 1,2 The notion of primary GI lymphomas, in principle, refers to tumours arising in the GI tract with the bulk of the tumour localised to a specific GI organ, with or without regional involvement. By extension, the term also encompasses situations in which the clinical presentation is related to lymphoma involvement of a GI site, and staging reveals disseminated disease. In addition, the GI tract can be affected by contiguous involvement or dissemination of a systemic disease. Primary oesophageal lymphomas are extremely rare, accounting for <1% of GI lymphomas, [2][3][4] with most cases probably representing extension from posterior mediastinal masses or gastric primaries. The stomach alone accounts for the majority of GI lymphomas (60% of the cases),~30% arise in the duodenum or small intestine and ileocaecal region, and~10% affect the colon. [1][2][3][4][5][6] Most GI lymphoma cases correspond to entities that are not specific to the GI tract, with specific tumour entities primarily arising in the GI tract representing a smaller proportion, including duodenal-type follicular lymphoma (FL) and intestinal T-cell lymphomas. The prevalence of the different lymphoma entities varies according to the site of involvement, i.e. stomach versus duodenum and small intestine versus colon (Table 1).
In this article, we present an update on the pathological and molecular features of the lymphoid neoplasms that most commonly involve the upper GI tract, with an emphasis on the new guidelines derived from the latest updates of the World Health Organization (WHO) classification of tumours of haematopoietic and lymphoid tissues 7 and the digestive system, 8 and a focus on more recent developments.

Small B-cell lymphomas
Although essentially any small B-cell lymphoma may infiltrate the upper GI tract, primary GI presentation is dominated by extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) in the stomach and the small intestine in general, and duodenal-type FL in that specific site. A diagnostic approach to lympho(plasma)cytic infiltrates in the upper GI mucosa is shown in Figure 1, and Table 2 summarises the antibodies commonly used to assist in the differential diagnosis in routine practice.

M A L T L Y M P H O M A
MALT lymphoma, an indolent B-cell lymphoma recapitulating the morphology and architectural features of mucosa-associated lymphoid tissue (MALT), accounts for 40-50% of primary gastric lymphomas and a much smaller proportion (5-15%) of lymphomas arising in the small intestine. [1][2][3]9,10 Patients are usually adults and present with non-specific symptoms such as dyspepsia, nausea, epigastric pain, or bleeding, or may be asymptomatic. Immunoproliferative small intestinal disease (IPSID) is a rare variant of intestinal MALT lymphoma associated with the secretion of defective a heavy chains, and is largely restricted to the eastern Mediterranean area, the Middle and Far East, and Africa. This disease, which usually develops in the duodenum and jejunum, affects young adults of lower socioeconomic status and living in poor hygienic conditions, who present with malabsorption, diarrhoea, abdominal pain, and weight loss. 11,12 Association with infectious agents MALT lymphomas usually occur in tissues naturally devoid of MALT, and develop in a context of chronic inflammation induced by infections or autoimmune disorders. 13,14 Epidemiological, biological and therapeutic studies have shown a strong link between Helicobacter pylori infection and gastric MALT lymphoma. 15,16 Following an initial study by Wotherspoon et al., several groups have confirmed that eradication of H. pylori following antibiotic therapy induces regression of MALT lymphoma in~75% of early-stage tumours. [17][18][19][20] In earlier reports, H. pylori infection was demonstrated in >90% of cases. [21][22][23] However, recent analyses have shown a significant reduction in the prevalence of H. pylori infection in gastric MALT lymphomas in Europe as compared with the early 1990s. 24 In a retrospective study of >400 cases diagnosed in France between 2002 and 2010, only 57% of the cases were associated with H. pylori. 25 Another point is that the density and detectability of H. pylori have been reported to decrease in association with lymphoma progression. 26,27 In intestinal MALT lymphomas, an association with Campylobacter jejuni infection has been reported, but evidence for a causal link is lacking. 28

Pathological features
Endoscopically, MALT lymphoma may present as subtle gastritis-like lesions, erosions, and ulcerations, or as diffuse infiltrates and thickened folds, which may form polypoid or exophytic mass lesions. Endoscopic ultrasonography is the most accurate staging method for assessment of the depth of invasion and the status of regional lymph nodes. 29,30 The Lugano and Paris staging systems are most commonly used, with the second describing more accurately the depth of gastric wall invasion, a parameter that may predict the response to anti-H. pylori treatment. 30 Histologically, the cardinal features of MALT lymphoma are: (i) a diffuse and/or nodular infiltrate of neoplastic lymphoid B cells cytologically resembling marginal zone B cells; (ii) lymphoepithelial lesions; and (iii) reactive lymphoid follicles with germinal centres. 31,32 The neoplastic population comprises small to medium-sized lymphoid cells, with a centrocyte-like or monocytoid appearance, which infiltrate diffusely into the mucosa and frequently colonise reactive germinal centres ( Figure 2). There can be cytological variation: some tumours contain a relatively monotonous population, whereas others feature a mixture of cell types. The neoplastic infiltrate also usually contains scattered, large, transformed blastic cells resembling centroblasts or immunoblasts. Plasma cell differentiation is seen in~30% of cases ( Figure 3). The neoplastic plasma cells may appear as normal mature plasma cells, have crystalline inclusions in the cytoplasm, or possess nuclei with Dutcher bodies. Lymphoepithelial lesions, defined as the invasion of individual gastric pits and/or glands by aggregates (three or more) of neoplastic cells, often resulting in oxyphilic changes in the epithelium, constitute a characteristic feature of gastric MALT lymphoma, but are found infrequently in MALT lymphomas of the small intestine. The destruction and disintegration of the epithelial structures induce degenerative epithelial changes manifested by eosinophilic or oncocytic metaplasia. Notably, although lymphoepithelial lesions in the stomach are highly suggestive of MALT lymphoma, occasional lymphoepithelial lesions may be seen in florid gastritis, so they are not specific for a diagnosis of malignancy. Moreover, other lymphomas may show epitheliotropism and produce alterations resembling lymphoepithelial lesions. In particular, epitheliotropism is a defining property of monomorphic epitheliotropic intestinal T-cell lymphoma, which, by morphology alone, may mimic MALT lymphoma in the stomach. Epitheliotropism is commonly seen in extranodal natural killer (NK)/Tcell lymphoma, which rarely involves the stomach, and is occasionally encountered in other B-cell lymphoma types such as diffuse large B-cell lymphoma (DLBCL) or, less commonly, mantle cell lymphoma (MCL).
The lymphoma cells are positive for pan-B-cell antigens (CD19, CD20, CD79a, and PAX5), and usually negative for CD5, with aberrant coexpression of CD43 in approximately one-third of the cases. 33 The rare cases of CD5+ MALT lymphoma must be distinguished from small lymphocytic lymphoma (SLL) and MCL. MALT lymphoma cells are negative for the germinal centre markers CD10 and B-cell

Molecular genetics/pathogenesis
Adding to the growth signals provided by infiltrating H. pylori-specific T cells, 37,38 activation of the B-cell receptor (BCR) and nuclear factor-jB (NF-jB) signalling pathways plays a major role in gastric MALT lymphoma development. Sequence analysis of immunoglobulin (IG) gene rearrangements shows evidence of somatic hypermutations, with an active ongoing somatic hypermutation machinery resulting in intraclonal variation and a biased usage of certain immunoglobulin heavy chain (IGH) genes, such as IGHV3-7 and IGHV1-69, indicating a selective pressure towards antigen-driven clonal expansion. [39][40][41][42][43] Several recurrent chromosomal translocations are associated with MALT lymphomas. They are mutually exclusive, and their relative frequencies vary with the primary site of involvement. The most frequent in the GI tract is the t(11;18)(q21;q21)/BIRC3-MALT1 translocation, found in approximately 25% of gastric and 10-50% of intestinal MALT lymphomas. Its oncogenic activity is attributed to overexpression of MALT1, which is an important mediator of activation of the canonical and non-canonical NF-jB pathways. [44][45][46][47][48] Other recurrent translocations, more commonly observed at extra-GI sites, have been reported in <5% of GI MALT lymphomas in most published series. They include the t(14;18)(q32;q21)/IGH-MALT1, t(1;14)(p22;q32)/IGH-BCL10, and t(3;14) (p14.1;q32)/IGH-FOXP1 translocations. 49,50 In gastric MALT lymphomas, the t(11;18)(q21;q21) translocation is more prevalent in H. pylori-negative cases, correlates with higher-stage disease, and is a factor in the poor response to alkylating agents. 51,52 Conversely, t(11;18)-positive MALT lymphomas are less prone to transformation to DLBCL. 53 When it is present in H. pylori-positive cases, the t(11;18)(q21; q21) translocation is predictive of non-response to or relapse after antibiotic therapy. 21,54 Therefore, fluorescence in-situ hybridisation (FISH) testing for t (11;18) is recommended at initial diagnosis or for the cases that fail to respond to anti-H. pylori therapy. 30,55 The clinical significance of the infrequent IGH-BCL10 rearrangement is not clear, although a small study suggested that cases with t(1;14) or strong nuclear expression of B-cell lymphoma 10 (bcl-10) most likely do not respond to H. pylori eradication. 56 Other aberrations variably reported in gastric MALT lymphoma are deletions of TNFAIP3/A20 (6q23), which encodes an inhibitor of NF-jB, and trisomies/gains of chromosomes 3, 8, and 18. [57][58][59][60] In recent years, a few studies have examined the mutational landscape of MALT lymphomas by means of whole exome sequencing or targeted next-generation sequencing (NGS) technologies. 57,61-63 Distinct genetic profiles were observed in MALT lymphomas from different sites. 63 Overall, only a small number of gastric MALT lymphomas have been analysed, with variations in the methodology in the different studies. Recurrent mutations in genes involved in activation of NF-jB signalling pathways, such as TRAF3 (33%) and TNFAIP3 (25%), were identified, and in general were mutually exclusive with the BIRC3-MALT1 translocation. Interestingly, MALT lymphoma essentially lacks mutations in MYD88, CD79A, or CARD11, which are frequently found in other B-cell lymphomas characterised by constitutive NF-jB activation. Mutations are also found in genes related to B-cell differentiation, notably NOTCH1 (11-25%), and epigenetic modifications (CIITA, TBL1XR1, and TET2), and in tumour suppressor genes. 61 Mutations in GPR34 and PIK3CD, which are frequently found in MALT lymphomas of the salivary glands and thyroid, have not been detected in gastric MALT lymphomas. 63

Differential diagnosis
A common diagnostic challenge is differentiating gastric MALT lymphoma from H. pylori-associated gastritis. An expansile, destructive infiltrate, the cytological characteristics of the infiltrating cells, Dutcher bodies, infiltration of the muscularis mucosae and prominent lymphoepithelial lesions favour lymphoma, but many biopsies may lack some of these features. Monotypic immunoglobulin expression and/or coexpression of CD43 by the B cells establish malignancy. Morphological assessment with the Wotherspoon scoring system helps in identifying the cases that may benefit from clonality testing. 19,64 Differentiating MALT lymphoma from other small B-cell lymphomas, notably MCL, which shows tropism for the GI mucosa, but also SLL/chronic lymphocytic leukaemia (CLL) and FL, can also be challenging. Immunohistochemistry is key in establishing the correct diagnosis (see below). Because of the morphological overlap between MCL and MALT lymphoma, cyclin D1 immunohistochemistry should be performed in all cases of GI small B-cell lymphoma. 30 MALT lymphomas with extensive plasmacytic differentiation should be distinguished from plasma cell neoplasia, either extramedullary plasmacytoma or dissemination of a multiple myeloma, which occur rarely in the GI tract ( Figure 4). 65 Although large cells are typically present and admixed with the small B cells in MALT lymphoma, sometimes representing up to 20% of the total cells, transformation to DLBCL should be considered when sheets or confluent clusters of large, transformed cells are found outside follicles. Clusters of at least 20 cells or a proportion of >30% large cells are needed to consider a diagnosis of large-cell transformation. Conversely, diffusely scattered large cells, representing 5-10% or even up to 20% of the total population, are not associated with a worse prognosis, as long as they do not form clusters. [66][67][68] If a component of MALT lymphoma is noted in a gastric or intestinal DLBCL, it should be mentioned in the report. Reactive germinal centres in MALT lymphomas, which can be identified by their germinal centre immunophenotype, should not be mistaken as foci of large-cell transformation.

Follow-up biopsies in gastric MALT lymphomas
Irrespective of stage, H. pylori eradication is the initial treatment of choice, and it is recommended that follow-up biopsies are taken 2-3 months after treatment and subsequently twice yearly for 2 years. 30 The evaluation of histological response is based on the Groupe d'Etude des Lymphomes de l'Adulte (GELA) scoring system. 69,70 In this scheme, patients with complete response or probable minimal residual disease, in which small residual lymphoid aggregates are recognised, should be managed with observation and regular follow-up. Although molecular assays have shown that such lymphoid aggregates may harbour the same clonal IG gene rearrangements as the initial lymphoma, molecular testing is not recommended, as no firm correlation has been established between a positive molecular test result and a higher risk of relapse. [71][72][73] Patients with responding residual disease or no-change/stable disease can also be managed with observation for several months, as long as they are asymptomatic, because responses may occur as late as >18 months after the completion of antibiotic therapy. 72,74 In patients not responsive to antibiotic therapy or with progressive disease, there are several therapeutic options, including radiation therapy, chemotherapy, and/or immunotherapy. 30 As higher rates of epithelial preneoplastic lesions (intestinal metaplasia, atrophic gastritis, and dysplasia) and a six-fold increased risk for gastric adenocarcinoma have been reported in patients with gastric MALT lymphoma, [75][76][77][78] it is important, in follow-up biopsies, not only to assess the lymphoid infiltrates but also to screen the biopsies for (pre)neoplastic epithelial lesions.

D U O D E N A L -T Y P E A N D P R I M A R Y I N T E S T I N A L F L
Primary intestinal FL accounts for 5-10% of primary intestinal lymphomas overall, and affects middle-aged to older adults. Approximately half of the cases occur in the second portion of the duodenum, 1,2,79,80 and because these show distinct clinicopathological features-duodenal presentation, low histological grade, indolent behaviour, and excellent outcome-they are now recognised as a distinct disease entity, namely duodenal-type FL. 7 FL cases developing elsewhere along the GI tract, usually in the small intestine or colon, are referred to as primary intestinal FL (Table 3). Most duodenal-type FL cases are discovered incidentally during upper GI endoscopy for surveillance of unrelated pathologies, such as gastritis, gastro-oesophageal reflux disease, or other malignancies, and only a minority of patients present with mass effect symptoms such as obstructive jaundice. 79,[81][82][83] Endoscopically, the lesions appear as one or more nodules or small polyps in the second portion of the duodenum, but multifocal involvement of more distal regions of the intestine, mainly the jejunum, is not uncommon. 81,82 Duodenal-type FL is an indolent disease for which a watchful waiting approach is generally recommended. 82,84 Nodal involvement (stage II) is a risk factor associated with progression. 85 Only a few case reports have documented transformation to DLBCL. [86][87][88] Non-duodenal-type FLs tend to produce larger symptomatic tumour masses that may be obstructive or cause bleeding, and staging is essential to differentiate primary intestinal disease from secondary GI involvement by a systemic (nodal) FL.

Pathological features
In duodenal-type FL ( Figure 5A-D), the neoplastic follicles expand the duodenal mucosa/submucosa and show features of grade 1-2 FL, i.e. non-polarised neoplastic follicles composed primarily of centrocytes with few centroblasts, and lacking a starry-sky appearance, an attenuated mantle zone, and infiltration of centrocytes in the lamina propria outside the follicles. The B cells are CD10+, bcl-6+, and bcl-2+, with a low Ki67 proliferation index. One characteristic feature of duodenal-type FL is the frequent distribution of CD21+ CD23+ follicular dendritic cells at the periphery of the neoplastic germinal centres, referred to as a 'hollowed-out' appearance. Furthermore, duodenal-type FL neoplastic cells preferentially express IgA over IgM, 89,90 and the mucosal homing receptors a4b7 integrin and CCR9 chemokine receptor, which direct lymphocyte trafficking to the intestinal mucosa. 91,92 Non-duodenal-type FL ( Figure 5E-G) tends to be transmural, encompasses cases ranging from grade 1-2 to grade 3, and may be accompanied by sclerosis. It is histologically and phenotypically indistinguishable from systemic FL.

Molecular genetics
Both duodenal-type and other intestinal FLs harbour the t(14;18)(q32;q21)/IGH-BCL2 translocation in the majority of the cases. In duodenal-type FL, the active somatic hypermutation machinery is related to the expression of BTB and CNC homolog 2 (BACH2) instead of activation-induced cytidine deaminase (AID). 83,93 In an effort to shed light on the molecular differences between duodenal-type FL and nodal FL and explain the indolent clinical behaviour of duodenal-type FL, recent studies analysed the genetic landscape of the neoplastic cells and the expression profile of the immune microenvironment. 94,95 Comparison of the mutational landscapes of duodenal-type FL and nodal FL showed no significant differences in the mutation rates of the most recurrently altered genes, including CREBBP, TNFRSF14/HVEM, and EZH2. In contrast, duodenal-type FL showed fewer biallelic or multiple mutations in the histone methyltransferase KMT2D/MLL2 gene and a high frequency of mutations in HVCN1 and EEF1A1, in 22% and 35% of cases, respectively. 94 Interestingly, HVCN1 mutations were reported to correlate with longer progressionfree survival in patients with systemic FL. 96 Conversely, factors related to the microenvironment appear to play a significant role in duodenal-type FL lymphomagenesis. Selective usage of the IGHV4 gene segment in most cases indicates an antigen-driven mechanism of lymphomagenesis. 93,97 Furthermore, gene expression analyses have shown a distinct immune microenvironment profile in duodenal-type FL, characterised by a chronic inflammation gene signature and overexpression of proinflammatory cytokines and chemokines such as CXCL6, TNFSF15, CCL11, CXCL1, CCL21, and CCL20 with its ligand CCR6, with consequent recruitment of Th17 and activated CD4+ T-helper cells into the lymphoma microenvironment. 94,95 M C L MCL is an aggressive type of B-cell lymphoma, accounting for approximately 3-5% of primary GI lymphomas. 2,98 MCL mainly affects elderly and predominantly male individuals. As a primary GI disease, it may present as multiple mucosal polyps, so-called multiple lymphomatous polyposis, more commonly in the lower GI tract. 99,100 Gastric involvement manifests as large cerebroid folds. Presenting symptoms may include abdominal pain, weight loss, diarrhoea, or bleeding. Staging frequently reveals widespread extraintestinal disease (stage IV). Microscopic GI tract involvement is also extremely frequent (84%) in patients with systemic MCL. 101,102 Pathological features MCL is composed of a monotonous small to mediumsized lymphoid cell population, with clefted nuclei, indistinct nucleoli, and scant cytoplasm, that infiltrates the mucosa and submucosa in a nodular or diffuse fashion ( Figure 6). Occasionally, infiltration of epithelial structures is found, resembling lymphoepithelial lesions. 103 Two aggressive cytological variants are recognised, namely blastoid and pleomorphic. Immunophenotypically, the cells typically express CD20, PAX5, CD5, CD43, IgD, bcl-2, and cyclin D1, and are negative for CD23, CD10, and bcl-6. Rare cases with aberrant phenotypes, namely CD5-, CD10+, or bcl-6+, have been described. SOX11 expression is a useful diagnostic marker in the rare cyclin D1-MCL cases. 104 A Ki67 proliferation index of >30% identifies patients with a poor prognosis, and should be reported. 7

Molecular genetics
The characteristic genetic signature of MCL is the t (11;14)(q13;q32) translocation resulting in IGH-CCND1 fusion and cyclin D1 overexpression, whereas cases that lack cyclin D1 expression often show alternative CCND2 or CCND3 rearrangements. 105,106 Immunogenetic studies have shown a biased usage of certain IGHV genes (IGHV3-21, IGHV4-34, IGHV1-8, and IGHV3-23), suggesting antigen-driven selection, whereas completely unmutated IGHV genes are detected in~30% of the cases, which have a worse prognosis than cases with IGH somatic hypermutations. 107,108 The genomic landscape of MCL is heterogeneous, and comprises mutations in genes involved in DNA damage response (ATM and TP53) and Notch signalling (NOTCH1 and NOTCH2), and in genes affecting RNA metabolism and splicing (EWSR1, DAZAP1, and HNRNPH1). Mutations in TP53, SMARCA4, CELSR3, CCND1 and KMT2D have been found to negatively affect the response to venetoclax-based therapies. [109][110][111] The median survival is 3-5 years, and the majority of cases remain incurable, with multiple relapses. Besides standard chemotherapy, treatment options currently comprise many new drugs such as ibrutinib, lenalidomide, bortezomib, temsirolimus, and venetoclax. 112 S L L Although rare cases of primary SLL of the GI tract have been described, 113 most of them represent secondary involvement in patients with known CLL. It usually consists of a dense and diffuse infiltrate of small round lymphocytes, which may constitute proliferation centres. The lymphoma cells are CD20+ (often with heterogeneous or weak expression), CD5+, CD23+, CD43+, cyclin D1À, and lymphoid enhancer binding factor 1 (LEF1)+. 114,115 Secondary lymphoid follicles or lymphoepithelial lesions typical of MALT lymphoma are usually not seen.  The current diagnostic approach to aggressive lymphomas of B-cell lineage according to the 2017 WHO guidelines is summarised in Figure 7. The first level of assessment remains cytomorphology, which encompasses large cells (centroblastic, immunoblastic, plasmablastic or anaplastic types), Burkitt-type morphology (monomorphic medium-sized cells with basophilic cytoplasm, coarse chromatin and several medium-sized nucleoli, densely packed and associated with a starry-sky pattern), intermediate cases pattern), and blastoid tumours (comprising mediumsized cells with fine chromatin, indistinct nucleoli, and scant cytoplasm). Cases with blastoid morphology may represent blastoid MCL or lymphoblastic neoplasms, which are best identified with an appropriate panel of immunostains. BL is a rather uniform entity, and in cases with typical morphology the diagnosis is confirmed by the demonstration of the expected immunophenotype (CD20+ CD10+ bcl-2-bcl-6+ MYC+, and a Ki67 proliferation close to 100%), and association with Epstein-Barr virus (EBV) infection in a subset of the cases. In cases with typical morphology and immunophenotype, confirmation of a MYC rearrangement by the use of FISH is optional; in cases showing deviations from the typical features (mild cytological pleomorphism and bcl-2 expression), FISH studies are mandatory to assess the MYC status and exclude a DH lymphoma (Figure 8). The majority of lymphomas composed of large B cells remain under the umbrella term of DLBCL, NOS. However, specific entities that may occur as GI neoplasms, such as EBV+ entities (EBV+ DLBCL and EBV+ mucocutaneous ulcer), plasmablastic lymphoma, and the solid form of primary effusion lymphoma, have to be excluded, and FISH studies are also mandatory to identify the small subset of large B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements, because these are classified as HGBCL-DH/TH. 7 D L B C L , N O S DLBCL, NOS usually presents de novo, but some cases represent transformation of a pre-existing systemic or GI small B-cell lymphoma. 5,6 In particular, transformation of systemic FL may occur in the small intestine, whereas MALT lymphoma represents the most common primary GI small B-cell lymphoma that undergoes transformation to DLBCL. DLBCL usually presents as symptomatic ulcerated tumour masses or mucosal thickening. The majority of cases are localised (stage I or II) at diagnosis. [117][118][119] The tumour is composed of large or medium to large lymphoid cells, with centroblastic and/or immunoblastic and, less commonly, anaplastic morphology. The growth is diffuse and obliterates the crypts or glands without the formation of typical lymphoepithelial lesions. A smallcell component may be present in transformed cases,  and the presence of residual follicular dendritic cell meshworks may denote an underlying MALT lymphoma. The lymphoma cells express pan-B-cell antigens (CD20, CD19, CD22, CD79a, and PAX5) and monotypic surface and/or cytoplasmic immunoglobulin, usually IgM, and rarely coexpress CD5. 118 A subset of gastric DLBCLs are associated with H. pylori infection, and there have been sporadic case reports and one prospective study suggesting that H. pylori eradication might be an effective treatment for limited-stage de-novo H. pylori-positive gastric DLBCLs. [120][121][122] In a recent retrospective study from China, it was found that a positive H. pylori status in gastric DLBCL was associated with a higher proportion of stage I-II disease and was a predictor of less aggressive behaviour and better outcome. 117 It is mandatory, according to WHO recommendations, that DLBCL, NOS be classified into 'germinal centre B-cell-like (GCB)' and 'non-GCB' or 'activated B-celllike (ABC)' subtypes. 7 In diagnostic practice, this is usually achieved by means of immunohistochemical algorithms designed as surrogates of mRNA-based expression signatures. The Hans classifier, based on the assessment of three markers (CD10, bcl-6, and MUM1) is one of the most widely used. 123 Other alternatives incorporating other markers, such as FOXP1 and/or GCET-1, have been proposed. 124 Methods based on mRNA expression profiling that are applicable to routinely processed samples are also available, e.g. the Lymph2Cx assay or multiplex ligation-dependent probe amplification (MLPA) assay, but they are less widely used. 125,126 In the stomach, the non-GCB/activated cases outnumber tumours with a GCB immunophenotype (58-70% versus 30-42%) 68,117,118,127,128 ; conversely, intestinal DLBCLs are more often of the GCB type. [129][130][131] Those transformed from MALT lymphomas are more often CD10-and bcl-2-than are de-novo DLBCLs, but usually express bcl-6. 26,127,132 The diagnostic work-up of DLBCL, NOS should also include the assessment of bcl-2 and MYC expression, with positive cut-offs set at 50% of cells with cytoplasmic bcl-2 expression and 40% with MYC+ nuclei. Many cases are immunohistochemically bcl-2+ or MYC+ in the absence of BCL2 or MYC rearrangements. Importantly, the so-called 'double-expressor' bcl-2+/MYC+ status ( Figure 9) identifies a group of patients who have more aggressive disease and poorer outcomes. 133,134 Although a subset of primary gastric DLBCLs arise by transformation of MALT lymphomas, specific translocations associated with MALT lymphoma, such as the t(11;18)/BIRC3-MALT1 translocation, are rare in DLBCLs, supporting the view that these cases are less prone to transform. 135 Regarding the recurrent translocations associated with DLBCL, BCL2 rearrangements are overall rarely found in primary GI DLBCL, and~10% carry MYC translocations, which is similar to the rate in nodal cases. [136][137][138][139] BCL6 rearrangements, which have been found in association with transformation of MALT lymphoma, are present in 48% of gastric DLBCLs and 30% of intestinal DLBCLs. 132,139 Some studies suggest that BCL6 rearrangements may be associated with a better prognosis. 127,140 Recently, two seminal studies using a multiplatform genomic approach, consisting of gene expression profiling, whole exome sequencing, structural variants, and DNA copy number analysis, led to the identification of several genetic subgroups based on enrichment of driver mutations and signalling pathways. 141,142 Interestingly, both studies identified a genetic subgroup defined by the association of structural alterations of BCL6 and NOTCH2 mutations, encompassing both non-GCB and GCB gene expression signatures, which may be related to marginal zone lymphomas.

E B V -A S S O C I A T E D D I S E A S E S
A variety of large B-cell lymphoma entities are characterised by a more or less frequent association with EBV infection (Table 4). 7 Overall these entities are rare, but may involve the GI tract. They have in common the tendency to occur in individuals with some dysfunction of the systemic or local immune system. Given the spectrum of EBV-associated entities, the relevance of EBV testing for proper diagnosis, and the clinical importance of recognising EBV-associated entities, we advocate performing EBV testing by means of in-situ hybridisation for EBV-encoded small RNAs (EBERs) in a broad range of settings. EBV+ DLBCL, NOS ( Figure 10) and EBV+ mucocutaneous ulcer include a broad range of clinicopathological features, ranging from highly aggressive diseases to self-limited localised conditions, with some overlap between the two entities. 143 The lesions contain EBV+ CD20+ large cells, often including scattered Reed-Sternberg-like cells, and a polymorphic inflammatory background. The immunophenotype is usually of the activated B-cell type, with frequent CD30 expression, but a subset of cases are CD10+. 144 EBV+ DLBCL, NOS is generally reported in patients without any known immune deficiency disorder (after exclusion of 'specific' types of EBV+ DLBCL, such as plasmablastic lymphoma), and can occur at any age, but usually in older adults. In a series from Asia, EBV+ DLBCLs represented approximately 7-10% of gastric primaries, and the presence of EBV was negatively correlated with outcome. 119 EBV positivity was found to correlate with higher expression of programmed death-ligand 1 (PD-L1) in the tumour microenvironment, a finding that might have therapeutic impact in the immunotherapy era. 118 EBV+ mucocutaneous ulcer causes superficial ulcerative lesions in the skin or mucosae in elderly individuals, in the setting of iatrogenic immunosuppression (methotrexate, azathioprine, cyclosporine, mycophenolate) for rheumatoid arthritis or inflammatory bowel disease, in human immunodeficiency virus (HIV)-infected individuals, or post-transplantation. [145][146][147] Whereas EBV+ mucocutaneous ulcer has a predilection for the lower GI tract, a recent study from Japan reported that GI EBV+ DLBCLs occurring in non-immunosuppressed individuals have a predilection for the stomach, which was the main localisation of disease for low-stage lesions and was often involved in cases with multifocal lesions. 144 Plasmablastic lymphoma is composed of large cells, ranging from immunoblasts to plasmablasts with or without a component of mature plasma cells, and is characterised by extinction of the B-cell transcription programme and a plasma cell immunophenotype. 148 Recent data provide evidence that the transcriptome of plasmablastic lymphoma is more closely related to that of multiple myeloma than to that of other B-cell malignancies. 149 The GI tract is the most common site of presentation, after the oral cavity, and~50% of primary GI plasmablastic lymphomas are diagnosed in the stomach or proximal small intestine. [150][151][152][153] Although plasmablastic lymphoma was initially described in HIV+ individuals, the majority of cases nowadays occur in HIV-patients with other causes of immunosuppression or apparently immunocompetent. 151,154,155 Plasmablastic lymphoma is essentially negative for CD45, CD20, and PAX5, negative or variably positive for CD79a, and positive for CD38, CD138, and VS38c, with expression of the transcription factors PRDM1/BLIMP-1, MUM1, and XBP1. Epithelial membrane antigen (EMA) and CD30 are also commonly expressed. Cytoplasmic immunoglobulin is detected in 50-70% of the cases, and CD56 is variably expressed. 150,151,153 Approximately half of the cases are EBV+ (EBER+; Epstein-Barr nuclear antigen 1 (EBNA1)+). 150,151,153 MYC rearrangements are found in 50% of the cases, usually with an IG gene as the translocation partner. [151][152][153]156 Recent NGS-based studies have reported recurrent mutations affecting the JAK-STAT (STAT3, JAK1, SOCS1, JAK2, and PIM1), RAS-mitogen-activated protein kinase (MAPK) (NRAS, KRAS, BRAF, and MAP2K1) and Notch (NOTCH1, SPEN, and NCOR2) signalling pathways, providing the genetic basis for new therapeutic interventions. 149,157

T-cell neoplasms
GI T-cell neoplasms are overall markedly less frequent than B-cell lymphomas, but they encompass several distinct entities. Although the majority of the cases develop in the intestine, a small proportion of the cases may arise in or disseminate to the stomach, duodenum or, uncommonly, oesophagus. The recently revised WHO classification introduced significant changes in the classification of intestinal T-cell lymphomas. The two subtypes of enteropathy-associated T-cell lymphoma (EATL), types I and II, which were previously considered to be variants of the same disease, have been reclassified as two distinct diseases [EATL, formerly type I EATL, and monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), formerly type II EATL], owing to differences in their epidemiology and clinical features, morphology, phenotype, and genetic features, as highlighted in recent studies. 7,158,159 Intestinal T-cell lymphoma, NOS is a novel category created to include the cases of primary intestinal T-cell lymphoma not meeting the criteria for either EATL or MEITL. In addition, indolent T-cell lymphoproliferative disorder of the GI tract (ITLDP) represents a newly recognised group of clonal monomorphic proliferations of small T cells showing a superficial/mucosal distribution along the GI tract, and usually characterised by indolent behaviour and a chronic relapsing course. 160,161 NK-cell enteropathy (NKCE) represents another rare indolent disorder derived from NK cells. Other mature T/NK-cell lymphoma entities, such as extranodal NK/T-cell lymphoma, nasal type (ENKTCL), can also primarily or secondarily involve the GI tract, and their diagnosis may be challenging.
E A T L EATL, the most common form of primary intestinal T-cell lymphoma, is a neoplasm derived from intraepithelial T cells, and occurs in individuals with coeliac disease (CD) (gluten-sensitive enteropathy). The prevalence is highest in Europe, and especially northern Europe, whereas the disease is essentially non-existent in Asia. [162][163][164][165] The median age at presentation is >60 years. Patients typically have a history of CD or refractory CD (RCD), but a substantial proportion of the cases arise de novo, and these may be associated with a better outcome. Patients with EATL present with GI symptoms that often culminate in intestinal perforation and peritonitis, or obstruction. B symptoms are common (30-40%). EATL most commonly affects the small bowel, and rarely the large intestine or stomach (8% of the cases). 163,164 Lesions are frequently multifocal. The prognosis is poor, with a median survival of 7 months. 166 Pathological features EATL ( Figure 11) presents as ulcerative lesions or tumour masses. Histologically, EATL is composed of pleomorphic, medium-sized to large, occasionally anaplastic, lymphoid cells with a high mitotic rate. A mixed inflammatory infiltrate is typically present, usually rich in histiocytes and eosinophils, and may sometimes obscure the lymphoma cells. 8 Angiocentricity and angioinvasion can lead to extensive necrosis. Enteropathic features [villous atrophy, crypt hyperplasia, and increased intraepithelial lymphocytosis (IEL)] may be seen in the adjacent or distant mucosa. The neoplastic cells are CD2+ CD3+ CD4À CD5À CD7+ CD8À/+ CD56À with an activated cytotoxic phenotype and a high Ki67 index. CD30 is usually expressed by a large proportion of cells. 167 The majority of the cases are negative for T-cell receptor (TCR) expression ('TCR-silent'); 25% are positive for TCRab, and TCRcd expression has been described in a few cases. Expression of CD103 (human mucosal lymphocyte antigen 1, integrin aEb7), which is found in normal intraepithelial lymphocytes, is often at least partially preserved. Overexpression of p53 is seen in many cases.
Whole transcriptome sequencing showed differences in the gene expression profiles of EATL and MEITL, further supporting their reclassification as distinct entities. 158 Genes expressed at higher levels in EATL include STAT3, STAT5A, IRF1/4, and TGM2, which encodes transglutaminase 2, a known autoantigen in CD, highlighting the association of EATL with this disorder. Furthermore, EATL was also characterised by overexpression of interferon-c signalling pathway genes, a pattern reported in untreated CD patients. 177

Differential diagnosis
The diagnosis of EATL in the upper GI tract may be challenging, especially that of de-novo cases if enteropathic features are not identified. In those instances, if the pathological findings are consistent, a diagnosis of EATL should be proposed, and tested with serological investigations and human leukocyte antigen genotyping. In cases with no evidence of CD, a diagnosis of intestinal T-cell lymphoma, NOS is more appropriate. Cases with an anaplastic large-cell morphology and strong CD30 expression may histologically resemble anaplastic large-cell lymphoma (ALCL), but primary intestinal ALCL is very rare, and this diagnosis can be considered only after thorough clinical investigation to exclude underlying CD.
A clinical context of RCD may raise the differential diagnosis between EATL and type II RCD. Type II RCD features villous atrophy and intraepithelial expansion of slightly atypical lymphocytes, which are usually monoclonal and immunophenotypically abnormal, similar to that found in EATL, including loss of surface CD3 expression by flow cytometry, and loss of CD4 and CD8 expression. However, type II RCD is an 'in-situ' precursor to EATL, lacking the inflammatory background, predominantly large-cell morphology, infiltration with possible mass formation, necrosis and angioinvasion seen in EATL. CD30 expression is rarely seen in type II RCD, and is considered to be a sign of progression to EATL. 8,178 M E I T L MEITL is a lymphoma derived from intraepithelial lymphocytes. It is unrelated to CD, and is composed of medium-sized cells with round nuclei and pale cytoplasm, featuring epitheliotropism to the adjacent mucosa and lacking the inflammation and necrosis characteristic of EATL. MEITL occurs worldwide, but is the main form of primary intestinal T-cell lymphoma in Asia. 163,179,180 The disease occurs in older adults, with a male predominance.
Patients may have non-specific symptomatology, such as abdominal pain and diarrhoea, or present with perforation, obstruction, abdominal masses, bleeding, or ascites. MEITL usually involves the jejunum, and less commonly the ileum or duodenum, sometimes with concurrent colonic lesion(s). Presentation in the colon or stomach is rare. Dissemination to regional lymph nodes and various distant organs may occur. The bone marrow is rarely involved (5%). [180][181][182][183] The prognosis is universally poor, despite aggressive treatment, and the median overall survival is 7-14.8 months. 179,180 Pathological features MEITL ( Figure 12) usually presents as an ulcerated, sometimes bulky, mass. The tumour comprises monotonous, small to medium-sized lymphoid cells with pale cytoplasm, dispersed chromatin, and inconspicuous nucleoli. Some cases show some pleomorphism but, unlike in EATL, there is no prominent inflammatory infiltrate, and necrosis is limited to the surface. The peritumoral mucosa shows prominent epitheliotropism and lesser involvement of the submucosa and muscularis propria. The distant mucosa may contain patchy foci with increased IEL without villous atrophy. 179,181,183,184 Nevertheless, rare reports of cases with concomitant CD challenge the sporadic nature of the neoplasm. 185 Immunohistochemically, the tumour cells are typically CD2+, CD3+, CD7+, CD4À, and CD5À. 163,176,179,181,183 Most cases are positive for CD8, CD56, and TIA1. Granzyme B is detected in two-thirds of the cases. Aberrant expression of CD20 or other B-cell antigens occurs in 20% of the cases, and CD30 is mostly negative. Either TCRab or TCRcd is usually expressed, whereas a small subset are TCRsilent. [179][180][181]186,187 MATK expression has been reported as a characteristic marker in the majority of the cases. 179,188 A recent study identified spleen tyrosine kinase (SYK) as a distinctive marker for MEITL (95% versus 0% in EATL), pointing to a role of enhanced TCR signalling in MEITL. 186 EBER expression may be seen in scattered B cells, but is consistently absent in the neoplastic T cells.

Molecular and genetic features
The majority of cases show monoclonal rearrangements of the TR genes. 179,180,189 Genomic copy number alterations in common with EATL include gains of chromosomes 9q, 1q, and 7q. 158,172,184 Alterations of chromosome 8, i.e. loss or gain of 8q, which includes MYC, are significantly more frequent in MEITL than in EATL. 172,179 A characteristic feature of MEITL consists of SETD2-disruptive mutations or deletions, which are present in >90% of the cases. 159 SETD2 encodes a lysine methyltransferase responsible for trimethylation of lysine 36 in histone H3. Immunohistochemically, SETD2-mutated cases have undetectable or reduced SETD2 expression, with concomitant low expression of histone H3 trimethylated on lysine 36. 159 Mice with SETD2 loss showed expansion of cd T cells, suggesting novel roles of the gene in T-cell development and an early event in lymphomagenesis. 158 Inactivation of SETD2 has been demonstrated in many different tumour types, and seems to play a role in both disease initiation and disease progression. 190 Other genes mutated in MEITLs and related to epigenetics include the histone acetyltransferase gene CREBBP (26%) and EP300, EZH2, and ARID1, which are frequently mutated in B-cell lymphomas, whereas other genes that are frequently mutated in peripheral T-cell lymphomas (PTCLs), such as TET2, DNMT3A, and IDH1/2, are usually wild-type in MEITLs. 159 Mutations activating the JAK-STAT pathway (especially in STAT5B, JAK3, and SH2B3) are present in a large proportion of the cases. Mutually exclusive alterations affecting the MAPK pathway (BRAF, KRAS, NRAS, and TP53) are observed more frequently than in EATL, collectively in~80% of the cases. 159,176 Mutations of GNAI2, which is a gene encoding a subunit of guanine nucleotide-binding protein, have been reported in 21% of MEITLs. 191 Whole transcriptome sequencing analysis showed that genes overexpressed in MEITL as compared with EATL include FASLG, SYK, TGBR1, and NCAM1 (consistent with CD56 expression). 158 Furthermore, enrichment for an NK-like cytotoxic signature, a known function of intraepithelial lymphocytes, was detected in MEITL. 192

Differential diagnosis
The features of MEITL are usually sufficiently distinctive from those of EATL. Even if an increase in IEL may be seen in the mucosa distant from MEITL, villous atrophy is, in principle, absent. Expression of CD56 (and CD8) may suggest a diagnosis of ENKTCL ( Figure 13), but MEITL is EBV-. In some cases, expression of B-cell markers may be confusing, and lead to consideration of a B-cell lymphoma. ITLPD of the GI tract is a new provisional entity defined as a clonal T-cell lymphoproliferation involving the GI mucosa, usually following a chronic and indolent course. 7 ITLPD can occur at any age, but it usually occurs in middle-aged adults, with a slight male predominance (1.5:1). Symptoms include diarrhoea, vomiting, bloating, and abdominal pain. Some patients have been misdiagnosed as having RCD, and others have a history of inflammatory bowel disease, autoimmunity, or infection. 161,193,194 Mucosal involvement is frequently multifocal, with a predilection for the small bowel and colon rather than the stomach. Mesenteric lymph nodes may be enlarged. Isolated cases of progression to other sites have been reported. 161,[193][194][195][196][197] Most patients are alive with persistent disease after several years of follow-up. Complete remission has been reported in two cases, 161,198 and death from disease progression has been reported at 10-27 years after diagnosis in seven patients. 194,196,[199][200][201] Pathological features

Indolent T/NK-cell entities
Endoscopy may show nodules, mucosal polyps, fissures, and erosions. Some cases have no mucosal anomalies. Histologically, ITLPD features a non-destructive proliferation of small, monomorphic lymphocytes, which may infiltrate the muscularis mucosae or submucosa, and is associated with crypt hyperplasia. Mitoses and apoptoses are both inconspicuous; vascular invasion and necrosis are absent. Villous atrophy is infrequent, but IEL may be encountered. Scattered granulomas, lymphoid follicles or admixed eosinophils may be seen. Transformed cases comprise atypical cells and resemble PTCL. Immunohistochemically, ITLPD is positive for CD3, CD2, and TCRbF1. 161,194,201 Some cases show downregulation or loss of CD5 and/or CD7 expression. Most reported cases are CD4+, and less frequently CD8+, but double-negative (CD4À CD8À) or doublepositive (CD4+ CD8+) immunophenotypes have been described. They are negative for T follicular helpercell markers. A recent study investigating the cell of origin found that the CD4+ and double-positive cases featured Th1 (T-bet+), Th2 (GATA3+) or hybrid Th1/ Th2 (T-bet+ GATA3+) profiles, whereas the majority of CD8+ cases and the double-negative cases showed a Th2-polarised (GATA3+) phenotype. 194 CD8+ cases express TIA1, but are negative for other cytotoxic markers. CD103 may be expressed, CD56 is usually negative, and there is no association with EBV. The Ki67 proliferation index is typically very low (<5%). In one case, aberrant expression of CD20 was observed. Transformed cases may express MUM1/ IRF4, CD30, CD25, perforin, and granzyme B.

Molecular genetics
Genetic data on ITLPD are scarce, but indicate a burden of genetic aberrations lower than that in EATL/MEITL, and suggest a different pathogenesis for CD4+ and CD8+ cases. 161,194,201 A study focused mostly on CD8+ ITLPD did not find phospho-STAT3 expression or STAT3 mutations. 161 A STAT3-JAK2 fusion was reported in four of five cases of CD4+ ITLPD by Sharma et al., 201 but a lower prevalence was found in subsequent studies, and the fusion has not been identified in CD8+, CD4+ CD8+ or CD4 À CD8 À cases. 194 Cases harbouring the STAT3-JAK2 fusion show phosphorylation of STAT5, and might be sensitive to JAK inhibitors. 201,202 STAT3 mutations and SOCS1 deletion are other recurrent alterations found essentially in CD4+ cases. 161,194 Other genetic aberrations include loss-of-function mutations in epigenetic modifier genes (TET2, DNMT3A, and KMT2D) and structural alterations of IL2 in CD8+ cases. 194 Differential diagnosis ITLPD should be distinguished from inflammatory disorders, and from primary intestinal lymphomas. This distinction is particularly important, because the natural history is completely different, and ITLPDs do not either require or respond to chemotherapy. Clues to the correct diagnosis include the superficial nature of the lesions; the absence of mass formation and/or a destructive growth pattern; the bland cytology; and, the low proliferation index. evidence to substantiate NK-cell lymphoma. 203 The disorder was first described in the stomach, and has also been reported as 'lymphomatoid gastropathy'. [204][205][206] NKCE is a rare disease, with <50 cases documented so far (reviewed in Dargent et al.; see references therein). 178,[203][204][205][206][207][208][209] Most cases occurred in middle-aged to older individuals, who were either asymptomatic or presented with mild and rather nonspecific GI symptoms. The majority of the cases occurred in the stomach, duodenum, or several upper GI sites, or involved both the upper and lower GI mucosa. Several gastric cases had concomitant H. pylori infection. The clinical course entails spontaneous resolution or persistence of the lesions for up to several years in some patients, despite various types of therapy. Unlike ITLPD of the GI tract, it has not yet been shown to progress to a more aggressive disease or cause nodal involvement. At endoscopy, the altered mucosa often consists of erythematous, flat or polypoid lesions, superficial erosions, or ulcers. Histologically (Figure 14), NKCE features an atypical diffuse lymphoid infiltrate composed of medium-sized to large cells, with moderate clear or slightly eosinophilic cytoplasm, fine nuclear chromatin, and inconspicuous nucleoli. The infiltrate expands the lamina propria, with occasional epitheliotropism and glandular destruction, but lacks angiocentricity and necrosis. The atypical cells feature a CD2 +/À CD3+ CD4 À CD5 À CD7+ CD8 À/+ CD56+ NKcell immunophenotype, with an activated cytotoxic profile. The Ki67 proliferation index is variable, and is sometimes elevated. 203,[205][206][207][208] The cells in NKCE are EBV-, which is a major feature distinguishing it from ENKTCL.
All cases lack clonal TR gene rearrangements, consistent with an NK-lineage derivation. In a recent study using a large haematology-oriented NGS panel, a somatic JAK3 mutation consisting of a small inframe deletion in exon 12 was identified in three of 10 NKCE cases. 207 Other non-recurrent mutations involving PTPRS, AURKB, AXL, ERBB4, IGF1R, PIK3CB, CUL3, CHEK2, RUNX1T1, CIC, SMARCB1 and SETD5 were found in other cases. 207 These findings support the neoplastic nature of the disorder, and may suggest potential therapeutic targets in some patients. 207 Conclusion A variety of lymphomas occur in the upper GI tract, and they are primarily diagnosed on endoscopic biopsies, which pose specific challenges, owing to limited sampling. The diagnosis of lymphoid infiltrates in these tissues primarily relies on careful morphological assessment complemented by immunophenotypic studies. Clonality assays, FISH studies and additional molecular genetic tests are complementary tools that are becoming increasingly useful or even mandatory to provide complete characterisation and accurate categorisation of the tumours. Importantly, endoscopic and clinical features are essential for providing a correct interpretation of the rare T-cell proliferations encountered in the stomach or small intestine.