Section 3 -

Nodular Sclerosis Hodgkin's Lymphoma and Mediastinal Large B-cell Lymphoma: Two Related Diseases? Philippe Gaulard Department of Pathology Hôpital Henri Mondor Créteil , France

Until recently, the cell of origin of the neoplastic cells of Hodgkin lymphoma (HL) has been controversial. This was mainly due to the scarcity of the neoplastic cells, which hampered their analysis and to the ambiguous phenotype of Hodgkin and Reed-Sternberg cells. However, it is now agreed that classical Hodgkin Lymphoma (cHL) is derived from a B-lymphocyte which has lost its B-cell identity. Recent molecular findings, including the gene expression signature, indicate striking similarities between cHL,- at least of nodular sclerosis subtype - and primary mediastinal (thymic) B-cell lymphoma (PMBL), a subset of diffuse large B-cell lymphoma (DLBCL). Nodular Slerosis Hodgkin Lymphoma (NS-HL): Since its first description in 1832 based on clinical pictures, considerable progress has been made regarding the histopathology and the molecular pathogenesis of Hodgkin lymphoma. HL is subdivided into a classical variant (cHL) and a nodular lymphocyte predominant variant which are now considered as 2 distinct disease entities according to the WHO classification [1]. cHL is characterized by very peculiar morphological aspects due to the presence of usually scattered large very atypical neoplastic cells –designated Hodgkin and Reed-Sternberg (H-RS) cells - in an abundant cellular background composed of a variable proportion of reactive lymphocytes, eosinophils, plasma cells, histiocytes and/or epithelioid cells. They have a characteristic phenotype: CD30+, CD15+ (60-70% of cases), CD45-, CD3- with absence of expression of B-cell markers (CD20-, CD79a-, Ig-) and of transcription factors BOB.1, OCT-2 and PU.1 necessary for immunoglobulin (Ig) transcription . However, sensitive immunohistochemical methods now allow the detection of heterogeneous CD20 and/or CD79a staining of H-RS cells in 20-40% of cases (reviewed in 2). EBV infection of H-RS cells is found in around 40% of cases in western countries, more frequently in elderly and in mixed cellularity type. The disease shows a predilection for adults and most frequently arise in lymph nodes and/or anterior mediastinum. In around 40% of patients, systemic symptoms (fever, weight loss, night sweats) are present. According to the WHO classification [1], four histological subtypes are distinguished among cHL, the most frequent being nodular sclerosis Hodgkin lymphoma (NS-HL), which accounts for around 60-70% of cHL. NS-HL shows a nodular growth with collagen bands that surrounds nodules of cHL. H-RS cells frequently have a lacunar aspect due to retraction of the cellular membrane in formalin-fixed tissues. A cellular phase with nodules without overt collagen bands and H-RS cells at the periphery of the nodules or around reactive follicles is recognized whereas the syncytial aspect with sheets of neoplastic cells may be responsible for diagnostic difficulties with non-Hodgkin lymphoma with anaplastic features. The prognostic value of histological grading according to the British national lymphoma investigation group (BNLI) is controversial. This subtype is characterized by its occurrence in children or young adults with a mediastinal mass often associated with supraclavicular and/or cervical lymphadenopathies, usually without systemic dissemination to the spleen, liver and bone marrow, at presentation. EBV is rarely found. Most recent molecular data have been obtained from primary tumors or cell lines originating from NS-HL. Recent findings obtained from microdissected H-RS cells have shown that the malignant cell in cHL is a B cell in most –if not all – cases, with rearrangements of Ig variable region genes with somatic mutations indicating a germinal center transit (review in 3). It is therefore not surprising that clinical and histological overlap can occur with B-cell lymphomas, specially PMBL. Table 1 summarizes the similarities in term of clinical, pathological and molecular features between NS-HL and PMBL. Primary Mediastinal (Thymic) B-cell Lymphoma Among DLBCL, PMBL is a distinct lymphoma subtype arising in the mediastinum, defined as a diffuse proliferation of large neoplastic lymphoid cells with primary involvement of the mediastinum and has a putative thymic B-cell origin. It accounts for 5-10% of adult diffuse large cell lymphoma [1, 4]. PMBL mainly occurs in young adult patients with a female predominance, as a localized disease related to a rapidly growing large anterior mediastinal mass which may result in superior vena cava syndrome. Dissemination at distance is very rare at diagnosis. PMBL discloses peculiar morphologic and phenotypic features. Neoplastic cells frequently show abundant pale cytoplasm and are associated with fibrosis, which tends to compartmentalize the tumor and can mimic a carcinoma or thymoma. They express B-cell lineage antigens (CD19, CD20, CD79a,..), but without surface or cytoplasmic immunoglobulin (Ig) [1, 4, 5, 6], a feature which appears unique among DLBCL. PMBL frequently expresses CD30 – usually weak – and CD23 [7]. Finally, expression of MAL, an integral membrane protein and lipid raft component, has been identified in about 70% of PMBL and is regarded as a characteristic marker of this entity, since it is not found in DLBCL arising in other sites [8, 9]. Therefore, MAL expression could be of value to distinguish PMBL from other DLBCL which can involve mediastinum. It is postulated that PMBL derives from the small subset of thymic B cells with asteroid shape located around the Hassal's corpuscles in the medullary thymus which share with PMBL a CD10-, CD21-, CD23+/-, CD35+/- phenotype [10]. The clinical presentation within the anterior mediastinum andthe recent identification of normal thymic medullary B-cells that express MAL reinforces this hypothesis [9]. PMBLs do not show alterations observed in other DLBCL such as rearrangements of the BCL2, BCL6 and C-MYC genes. They disclose recurrent chromosomal gains in 9p23 in about 60-70% of cases, but also in chromosomes X, 12q31 and 2p [11]. Amplification of JAK2, a gene located at 9p23, which is involved in cytokine-dependent signal transduction, is found in a proportion of cases At chromosome 2p, amplifications of the transcription factor REL gene are frequently observed. Two recent studies have shown thatPMBL presents a unique transcriptional signature, distinct from that of non mediastinal-DLBCL - ie germinal-center (GC) B-cells or activated B (ABC) DLBCL [12, 13]. This signature is characterized by high expression of genes involved in IL4/IL13 (including JAK2, FIG1, CD23), interferon and TNF superfamily signaling pathways (CD30, Fas/CD95, TRAF1, …). The signature also comprises genes coding for adhesion molecules, extra cellular matrix proteins (fibronectin, metalloproteinases,…), and proteins involved in the regulation of cell-stroma interactions, as well as MAL and FIG1/IL4I1 [14]. Interestingly, this transcriptional signature defines a distinct clinical entity that affects young patients, harboring a mediastinal tumor, with preferential loco-regional extension, and a relatively good outcome and allows to distinguish these tumors from DLBCL associated with mediastinal invasion [13].In addition, in agreement with the results of transcriptional analysis, PMBL presents a constitutive activation of Signal Transducer and Activator of Transcription 6 (STAT6), the phosphorylated form of which can be detected in the nuclei of PMBL [15]. This activation of STAT6 seems to be related to mutations of the negative regulator of cytokines signaling, SOCS1 [16] rather than to the kinase JAK2 mRNA overexpression observed in these tumors. Similarities Between PMBL and Classical Hodgkin Lymphoma Interestingly, the signature gene expression of PMBL discloses strong similarities with that of HL-derived cell lines [13] and strongly supports a relationship between PMBL and cHL, especially of the nodular sclerosis subtype. Over one third of the genes that were more highly expressed in PMBL than in other DLBCLs were also characteristically expressed in HL cells [13]. These included a number of cytokines (IL-15, CSF-1, TRAIL) and chemokines (specially TARC, RANTES) and many genes involved in cytokine signaling (IL13R alpha1, JAK2, FIG1, TARC, STAT1) as well as members of the TNF/TNF receptor family (CD30, TRAF, TNFRSF6). These data suggest that PMBL cells and H-RS cells have activated a similar set of signalling pathways. In this regard, both diseases display activation of NF-kB [12, 17] and constitutive activation of STAT6 [15]. However, contrary to PMBL, STAT6 activation in cHL might be due to autocrine IL-4/IL-13 secretion. In addition, PMBL and cHL exhibit common genetic abnormalities including gains in chromosomes 9p24 and 2p, which are associated with amplifications of the JAK2 and REL loci respectively. Interestingly, SOCS1m utations have also been reported in CHL [18]. As shown in Table 1, NS- HL also share many clinical and pathological features with PMBL, with common presentation in young patients, as mediastinal tumours associated with fibrosis and absence of Ig expression. Furthermore, there are rare cases showing borderline morphologic features between PMBL and cHL, "composite lymphomas" and reports of nodular sclerosis HL patients who developed PMBL as a second malignancy within one year following treatment (reviewed in 19). Altogether, these findings provide further evidence that PMBL and NS-HL may represent related tumours. It is tempting to speculate that similarities between both diseases may reflect a common ancestor from a peculiar thymic B-cell subset and/or early common oncogenic events. However, both diseases would acquire different secondary genetic events leading to differences in term of pathological features (ie polymorphous inflammatory infiltrate in cHL), outcome as well as biological differences. The latter include downregulation of mature B-cell genes that is characteristic of HL, but is not observed in PMBL. If the scenario of a common cell of origin is valid, that would also imply that cHL should be regarded as an heterogeneous disease comprising a nodular sclerosis entity with mediastinal presentation related to PMBL and a mixed cellularity entity which discloses a more disseminated presentation in elderly and strong association with EBV. Whatever the biological significance, the similarities between both diseases may explain the occurrence of cases of mediastinal "Gray zone" tumours that combine morphologic and phenotypic features of both NS-HL and PMBL, and provide a diagnostic challenge to pathologists [19]. In the absence of definitive criteria, the diagnosis in such cases should be based on the distinctive phenotype of PMBL, ie usually CD15-, but positive for CD45, CD20, CD79a and transcription factors PU.1, OCT-2 and the coactivator BOB.1. Expression of CD23 and BCL6 could be also used as additional diagnostic criteria for PMBL. Conclusion Recent data further demonstrate that PMBL can be regarded as a clinicopathologic entity among DLBCL, distinct from the GC and ABC DLBCL subgroups. The overlapping molecular findings – such as gains on chromosome 9, MAL overexpression and similarities in gene expression profiling - described both in PMBL and NS-HL strongly support a pathogenic relationship between both diseases. The better understanding of the molecular pathways– including the involvement of the NF-kB pathway and STAT6 activation – may provide rational to more specific treatments which could improve the prognosis of PMBL patients in the future, and might be also used to cure refractory NS-HL patients. Table 1: Comparison of the main clinical, pathological, phenotypic and genetic features between PMBL and nodular sclerosis Hodgkin lymphoma PMBL cHL (nodular slerosis) Mediastinal presentation Young patients (women) Fibrosis Response to radiotherapy Gains in chromosomes 9p, 2p Ig- CD30+/- (weak, heterogeneous) Activation of NF-kB pathway STAT6 activation SOCS1 mutations MAL+ Clear cells Sternberg cells No polymorphous infiltrate CD20+, CD79a+ Oct-2+, BOB.1+, PU.1+ CD15-, EBV- Mediastinal presentation Young patients (male:female ratio=1) Fibrosis Response to radiotherapy Gains in chromosomes 9p, 2p Ig- CD30+ Activation of NF-kB pathway STAT6 activation SOCS1 mutations MAL - (+) Hodgkin, Reed Polymorphous CD20-(+), CD79-(+) Oct-2-, BOB.1-, PU.1- CD15+, EBV+/- Abbreviation : Ig, immunoglobulins Differences are indicated in italics References Jaffe ES, Harris NL, Stein H, Vardiman J (Eds). World Health Organization Classification of Tumours. 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