Lymphoproliferative disorders in the immunosuppressed

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE III

Lymphoproliferative disorders in the immunosuppressed

IALD associated with primary immune disorders There are more than 60 primary immune disorders, though the prevalence of primary immune disorders is extremely low.6 Those that are more commonly associated with IALD are shown in Table 1. Most IALDs occur in the paediatric age group and are more common in males. Epstein Barr virus (EBV) is pathogenetically involved in most cases. A lack of EBV surveillance by T-cells is central to the pathogenesis.6 From a diagnostic point of view, the lymphoid proliferations resemble classical descriptions of lymphomas and reactive lymphoid lesions seen in the immune competent. Some cases are associated with polymorphous proliferations similar to a subset of PTLDs (discussed later). The precursor lesions to full-blown lymphomas that are seen in some primary immune disorders have characteristic histology and immunophenotype and can still cause diagnostic problems. These will be discussed here. Patients with common variable immune deficiency (CVID) may present with lymphadenopathy or extranodal lesions. Lymph nodes (LN) reveal follicular and paracortical expansion with the presence of large atypical EBV-positive Reed Sternberglike cells in some cases. The hyperplastic process is florid and obliterates the LN architecture. Furthermore, as some patients may have massive lymphadenopathy, these patients are often considered to have lymphomas clinically. The majority of the proliferations are benign, as demonstrated by clinical follow-up and gene rearrangement studies. Similar lymphoid hyperplasias may be seen in the gastrointestinal tract.6e8

Kikkeri N Naresh

Abstract Lymphoproliferative disorders occur at a higher frequency in immunosuppressed patients than in the general population and these are a group of diseases that range from benign lymphoid proliferations to full-blown lymphomas. These proliferations develop as a consequence of immunosuppression. Their histological appearances and immunophenotypic features are varied. While some resemble lymphoid lesions seen in immune competent patients, others have unique characteristics, with some posing diagnostic problems. Furthermore, within some clinical contexts like the post-transplant setting, the distinction between benign and malignant proliferations is blurred. Identification of specific entities requires a clear understanding of clinical context and morphology, together with a wide immunohistochemical panel, investigations for viral association and clonality studies. Precise identification of the disease entity has an impact on patient management and follow-up.

Keywords AIDS; HIV; immune suppression; lymphomas; lymphoproliferative disorders

Introduction

Lymphoproliferative disorders in primary immune disorders

Lymphoproliferative disorders (LPD) in the immunosuppressed are a group of diseases that range from benign polyclonal to malignant monoclonal lymphoid proliferations. They develop as a consequence of immunosuppression. Immunodeficiency may be of congenital, infectious or therapeutic origin. Most immunosuppression associated lymphoproliferative disorders (IALDs) are B-cell-derived. They originate more commonly in extranodal sites, but rarely affect skin, and behave aggressively.1,2 While most are ‘high-grade’ B-cell non-Hodgkin lymphomas (NHL), a few are classical Hodgkin lymphomas. Rare cases have also been shown to be either of T-cell or NK-cell origin.3 With regards to post-transplant lymphoproliferative disorders (PTLDs), the abnormal B-cells in solid organ transplant recipients usually originate from the recipient, while in recipients of bone marrow transplant they are of donor origin.4,5 IALD can be broadly categorized into e 1) those associated with primary immune disorders; 2) human immunodeficiency virus (HIV) associated lymphoproliferative disorders; 3) posttransplant lymphoproliferative disorders (PTLDs); and 4) iatrogenic IALD.

Primary immune disorder Severe combined immune deficiency Hyper-IgM syndrome

Type of lymphoproliferative disorder EBV-associated lesions (DLBCL & cHL); fatal IM EBV-associated lesions (DLBCL & cHL); T-LGL Combined variable immune EBV-associated lesions deficiency (DLBCL & cHL); MZL, LPL, SLL & PTCL X-linked LPD EBV-associated lesions (DLBCL & BL) ALPS NLP-HL, cHL, DLBCL, BL, PTCL Ataxia Telangiectasia Non-leukaemic clonal T-cell proliferations; BL, T-PLL, T-ALL, cHL & NLP-HL WiskotteAldrich syndrome EBV-associated lesions (DLBCL, cHL & LyG) Nijimegen breakage syndrome DLBCL, PTCL, T-ALL, cHL & NLP-HL EBV: EpsteineBarr virus; DLBCL: diffuse large B-cell lymphoma; cHL: classical Hodgkin lymphoma; IM: infectious mononucleosis; LGL: T-cell large granular lymphocyte leukaemia; LyG: lymphomatoid granulomatosis; MZL: marginal zone lymphoma; LPL: lymphoplasmacytic lymphoma; SLL: small lymphocytic lymphoma; PTCL: peripheral T-cell lymphoma; BL: Burkitt lymphoma; NLP-HL: nodular lymphocyte predominant Hodgkin lymphoma; T-PLL: T-cell prolymphocytic leukaemia; T-ALL: T-cell acute lymphoblastic leukaemia/lymphoma.

Kikkeri N Naresh MBBS CCP MD FRCPath is a Consultant Haematopathologist at the Department of Histopathology, Hammersmith Hospital and Imperial College, London, UK. Competing/conflicts of interest: none.

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Table 1

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Patients with hyper-IgM syndrome can present with extranodal lesions in the gastrointestinal tract, gall bladder or liver. In the LNs, germinal centres are absent. Histologically, these lesions demonstrate accumulations of IgM producing plasma cells at all the involved sites. The peripheral blood B-cells express IgM and IgD, and cells expressing other heavy chains are not seen. These patients harbour mutations in the genes encoding CD40, CD40 ligand, activation-induced cytidine deaminase (AID), uracil DNA glycosylase (UNG) or NF-kB essential modulator (NEMO), which impair B-celleT-cell interaction affecting class-switching in B-cells.6,9 Autoimmune lymphoproliferative syndrome (ALPS) is an uncommon disease that can cause diagnostic problems. Young patients usually present with generalized lymphadenopathy, hepatosplenomegaly, hypergammaglobulinemia, B-cell lymphocytosis and autoimmune manifestations like haemolytic anaemia, idiopathic thrombocytopaenic purpura, urticarial rash, glomerulonephritis, or GuillaineBarre´ syndrome. The median age at initial presentation is around 24 months, but may be up to 15 years. Most patients have lymphadenopathy, while some patients may present with splenomegaly without evidence of peripheral lymphadenopathy. The LNs show relative preservation of their architecture with florid reactive follicular hyperplasia and marked paracortical expansion with the presence of immunoblasts and plasma cells. The majority of the cells in the expanded paracortex are CD3-positive T-cells which are characteristically negative for both CD4 and CD8 (double negative cells) (Figure 1). A good proportion of the paracortical cells are positive for CD57 and cytotoxic molecules (TIA-1 and perforin), but CD56 or CD16 positive cells (NK-cells) are not seen. These paracortical lymphoid cells often have high Ki-67 expression and show frequent mitoses. In contrast to other reactive conditions, apoptotic cells and histiocytes containing apoptotic bodies are conspicuously absent in the paracortex. The paracortical expansion is often extensive and a differential diagnosis of immunoblastic lymphoma, a subtype of diffuse large B-cell lymphoma, may be considered. The lymphoid cell expansion is non-clonal by gene rearrangement studies. Apart from florid follicular hyperplasia, changes in follicles akin to Castleman’s

disease, follicular involution and progressive transformation of germinal centres may be seen. The spleen reveals prominent white pulp and expanded red pulp. The red pulp shows an infiltrate similar to those of the LN paracortex. The lymphoid proliferation in ALPS is related to an impairment of apoptosis as a consequence of inherited heterozygous mutations in the Fas, Fas ligand, caspase 10 or caspase 8 genes. Some of these patients later develop nodular lymphocyte predominant Hodgkin lymphoma or a T-cell rich B-cell lymphoma.2,6,10,11

HIV-associated lymphoproliferative disorders HIV infects CD4-positive T-cells, monocytes and dendritic cells, and has profound immunological effects. The effects on the immune system include cellular and tissue manifestations throughout the ‘lymphoreticular’ system e LN, spleen, extranodal lymphoid tissues, bone marrow and peripheral blood. These ‘lymphoproliferative’ disorders encompass pathologies of both neoplastic and non-neoplastic nature (infective or

Lymphoproliferative disorders in HIV patients ‘Reactive’ lymph nodal alterations 1) Florid follicular hyperplasia and acute viral lymphadenitis-like changes 2) Follicular involution 3) Depleted lymph node 4) Castleman-like changes T-cell expansions 1) Diffuse infiltrative lymphocytosis syndrome & HIV-associated CD8þ lymphocytosis syndrome Plasma cell infiltrates 1) Florid plasma cell expansion in lymph nodes, tissues and bone marrow with polyclonal hypergammaglobulinemia HHV-8-associated pathologies 1) Multicentric Castleman disease 2) Multicentric Castleman disease with so-called microlymphoma 3) Multicentric Castleman disease with lymphoma 4) Multicentric Castleman disease with Kaposi sarcoma 5) Multicentric Castleman disease with HHV-8-associated haemophagocytic syndrome Lymphomas specifically occurring in HIV-positive patients 1) Plasmablastic lymphoma 2) Primary effusion lymphoma e classical and ‘solid’ forms 3) Lymphomas arising in multicentric Castleman disease Lymphomas occurring in other immunodeficient states 1) Lesions similar to polymorphic post-transplant lymphoproliferative disorder Lymphomas also occurring in immune competent patients 1) Classical Hodgkin lymphoma 2) Burkitt lymphoma 3) Diffuse large B-cell lymphoma 4) Primary diffuse large B-cell lymphoma of the CNS 5) Marginal zone lymphoma (of mucosa-associated lymphoid tissue (MALT) type) 6) Peripheral T-cell and NK-cell lymphoma

Figure 1 LN in a child with documented autoimmune lymphoproliferative syndrome (ALPS). The LN shows reactive follicles with expanded interfollicular areas. The interfollicular area shows a prominent infiltrate of T-cells positive for CD3 and negative for CD4 and CD8.

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Table 2

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otherwise). Non-neoplastic lesions may precede or coexist with neoplasms.12 There are however, morphological overlaps between neoplasms, infective pathologies and non-neoplastic/ non-infective pathologies. LN histologies and IALDs seen in HIV patients are summarized in Table 2.

cells. During all the stages of HIV infection, and especially during the early phase of follicular hyperplasia, demonstration of HIV infection of FDCs is possible by immunohistochemical staining for the p24-gag protein of HIV.13e16 While some may question ethical aspects of doing so, others would argue that this constitutes part of the diagnostic procedure, consent for which would have been given prior to the biopsy.16a

Reactive lymph node lesions The microscopic appearance of LNs in HIV infection varies with the duration of the disease, from florid follicular hyperplasia in the early phases, followed by follicular involution and, in the late stages to complete effacement of nodal architecture with disappearance of germinal centres (lymphoid depletion). Early phases of HIV infection often present with generalized lymphadenopathy and an LN biopsy at this time often shows florid follicular hyperplasia with giant geographic follicles (of unusual map-like shapes) predominantly composed of centroblasts with a starrysky pattern. These may be accompanied by haemorrhage and the presence of multinucleated cells (polykaryocytes). These follicles are usually deficient in mantle zones sometimes making identification of the follicles difficult (Figure 2). An appreciation of the presence of markedly expanded follicles (explosive follicles) by using an immunostain for follicular dendritic cells (FDC), especially CD21 can be a great help. In the later phases of HIV infection, the LNs show follicular involution and lymphoid depletion. In follicular involution, the FDC meshworks crumble (highlighted on CD21 immunostaining) resulting in fragmentation of follicles, serrated borders of the germinal centres and irregular mantle zones. Furthermore, there is a paucity of follicle centre cells along with encroachment upon follicles by T-cells, mantle or marginal zone cells. As the follicles atrophy, there is complete collapse of the follicular structures with replacement by periodic acid-Schiff (PAS)-positive amorphous material. During lymphoid depletion, the LN is generally paucicellular and there is relative or pseudo-paracortical expansion (in comparison with follicles or cortical areas) and increased vascularity with appearances similar to vascular transformation of the sinuses. The paucicellular paracortical areas in LNs in lymphoid depletion show prominent histiocytes and most of the lymphoid cells present in the paracortical areas are CD8-postive small lymphoid

CD8-positive cytotoxic T-cell expansion Polyclonal or oligoclonal expansion of cytotoxic T-cells is seen in HIV-positive patients usually early in the disease process. Diffuse infiltrative lymphocytosis syndrome (DILS) typically involves viscera (salivary glands and lungs being the more common sites), but can also involve peripheral blood, LN and mucosal sites. Clinically, the presentation can resemble Sjo¨gren’s syndrome, although associated autoantibodies such as antinuclear antibodies (ANA), anti-Ro, and anti-La are absent. Histologically, the expansion of the lymphoid population can appear infiltrative and have an angiocentric pattern mimicking NK/T-cell lymphoma. However, the cells are negative for EBV and CD56. CD8þ lymphocytosis syndrome without visceral infiltration is seen as increased numbers of CD8þ lymphocytes with features of large granular lymphocytes in peripheral blood. The lymphoid cells also express CD57, and T-cell receptor (TCR) gene rearrangement studies may at times suggest clonal expansion. However, these clonal expansions do not constitute a lymphoma.17,18 Plasma cell expansion HIV patients may present with prominent plasma cell expansions in bone marrow, mucosal lesions and LN biopsies.19,20 The plasma cells and their nuclei are variable in size and some may show nucleoli. Mitoses may also be seen and Ki-67 expression can be high. Light chain immunohistochemistry may show some degree of deviation from the normal range of kappa:lambda light chain ratio. These patients demonstrate polyclonal hypergammaglobulinaemia and a paraprotein is not seen. Multicentric Castleman disease (MCD) and related disorders MCD presents with constitutional symptoms, generalized lymphadenopathy, hepatosplenomegaly, polyclonal hypergammaglobulinemia and elevated levels of serum interleukin-6 (IL-6) a homologue of which is produced by the human herpes virus type 8 (HHV-8).21,22 Morphological features in MCD are highly variable. Follicles can either be hyperplastic, atrophic or have features similar to those of hyaline vascular subtype of Castleman disease (with lollypop follicles). Interfollicular areas also have a variable morphology with varying degrees of vascular proliferation and plasma cell infiltration. The characteristic feature however, is the presence of larger nucleolated cells with basophilic cytoplasm resembling immunoblasts in the mantle zones (so-called plasmablasts), which infiltrate the follicles in some cases. The numbers of these cells are highly variable, but in most cases they are seen as singly scattered cells without forming sheets of cells. These cells can be easily demonstrated by immunostaining for HHV-8 latent nuclear antigen 1 (LANA-1) (Figure 3). These cells express IgM and show lambda light chain restriction. Despite being monotypic for lambda light chain, the cells are polyclonal. Hence, the

Figure 2 LN from an HIV-positive patient presenting with generalized lymphadenopathy. The LN shows large explosive follicles with a geographic pattern. The germinal centres do not show obvious polarity and the mantle zones are extremely thin and nearly absent in some areas.

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LN from an HIV-positive patient presenting with generalized lymphadenopathy. a An LN with atrophic follicles surrounded by prominent blood vessels. b Large nucleolated lymphoid cells amidst the mantle cells. These have been called ‘plasmablasts’ in the past. c HHV-8-LANA-1 positivity among these ‘plasmablasts’. Figure 3

monotypic nature (with respect to light chain expression) of the HHV-8-infected large lymphoid cells does not reflect a clonal expansion or a lymphoma.23,24 As the morphological features may be subtle and variable, HHV-8-LANA-1 immunostain serves as a sensitive marker. Whether these large HHV-8positive cells express CD20 has caused some controversy. Double immunostaining with HHV-8-LANA-1 and CD20 has shown that CD20 expression of variable intensity can be documented in at least a proportion of HHV-8-positive cells in about one quarter of cases of MCD. The low level of expression of CD20 (often below the level of immunohistochemical detection) is adequate for eliciting a therapeutic response to anti-CD20 (rituximab) treatment.25 In a proportion of MCD, the HHV-8-infected large nucleolated cells (‘plasmablasts’) expand and form sheets either surrounding or involving the follicles (referred to as microlymphomas). It is important to investigate these microlymphomas by Ig rearrangement studies to demonstrate the presence or absence of clonal B-cell expansion to guide further treatment. A small minority of MCD develop full-blown HHV-8-associated large cell lymphomas. HHV-8-associated large B-cell lymphoma most commonly involves LNs and spleen. Rarely other sites like liver, gastrointestinal tract lung or peripheral blood may be involved.

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Sheets of large cells replace the LN or spleen and efface the architecture completely and cells are positive for HHV-8LANA-1.23,24,26 More than one-half of cases of MCD harbour microscopic foci of Kaposi sarcoma (KS) usually involving the capsule or the trabeculae and rarely the hilum of the LN.27 It is crucial to evaluate the LNs involved by MCD carefully for the presence of microscopic KS. When patients with MCD and KS are treated with rituximab, there is often an exacerbation of KS, and it is important to forewarn the treating oncologist of this possibility by documenting the presence of microscopic KS.28 Plasmablastic lymphoma Plasmablastic lymphoma commonly occurs in the oral cavity and has a male predominance (male:female ratio of w7:1). Other mucosal sites such as sinonasal areas, orbit, skin, bone, soft tissues, GIT and lymph nodes may also be presenting sites. Patients present on an average 5 years after the diagnosis of HIV. Most patients are HIV-positive, similar cases are recorded post-transplant and in autoimmune disease and age-related settings. Patients often present at advanced stages with a high International Prognostic Index (IPI). Histologically, the lesion is characterized by a diffuse immunoblastic proliferation with

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Primary effusion lymphoma (PEL) This was first identified as a distinct lymphoma that involves serous body cavities and presents as a pleural effusion, pericardial effusion or ascites, without forming solid tumour masses.33,34 This is a rare form of lymphoma accounting for w3% of all HIV-lymphomas. While the majority of cases present exclusively with effusions, a variant of the disease with solid tumour masses has subsequently been identified, and is referred to as extracavitary PEL. Sites of extracavitary PEL include gastrointestinal tract, skin, lungs, central nervous system (CNS) and LNs. Most patients are HIV-positive, and rare cases are seen in the post-transplant setting and in the elderly. Morphologically, the PEL tumour cells have large immunoblastic or plasmablastic morphology with prominent anaplasia (Figure 5). The cells have deeply basophilic cytoplasm with vacuolation that is better appreciated on cytology specimens. Cells resembling RS cells are also seen. Tumour cells are positive for CD45, CD30, CD38, CD138, Vs38c, EMA and MUM1, and are usually negative for CD20, CD19, CD79a, CD15, CD10 and BCL6. Characteristically, the tumour cells in PEL are co-infected by EBV and HHV-8. They are positive for HHV-8-LANA-1 and EBER (ISH), and EBV-latent membrane protein-1 (LMP-1) is usually absent. Only a small proportion of cases express Ig, and the Ig positive cases express lambda light chains. They consistently lack translocations involving c-MYC, BCL6, BCL2 and BCL1 genes. Overall, PEL has a dismal prognosis, with a median survival of <6 months. The extracavitary variants have a marginally better prognosis.34e36 Around 5% of HIV-lymphomas have features akin to polymorphous PTLD with a range of cell types including RS-like cells. Most cases are EBV associated (Table 3).

Figure 4 Biopsy of a retroperitoneal LN in an HIV-positive patient who also had a lesion in the caecum and terminal ileum. The tumour cells have features of plasmablasts or immunoblasts with pronounced plasmacytic differentiation. The cells are positive for CD138, MUM1 and EBER (EBV e in-situ hybridization), and negative for CD45 and CD20. Ki-67 expression is >90%. This is a case of plasmablastic lymphoma. Although plasmablastic lymphoma typically involves the oral cavity, it can be seen at other sites.

tumour cells having plasmacytic/plasmablastic features (Figure 4). The tumour cells are positive for CD138, CD38, Vs38c, MUM1, CD79a, CD30, EMA and cytoplasmic Ig (most commonly IgG). They are negative or weakly positive for CD45, CD20 and PAX5. EBV association (EBER expression on in-situ hybridization (ISH)) is reported in w75% cases. LMP-1 is rarely expressed.29e31 The classification of monomorphic large cell proliferations with plasmacytic/plasmablastic morphology can cause confusion among many pathologists. There are four common, somewhat overlapping morphologies: plasmablastic lymphoma, some cases of diffuse large B-cell lymphoma (DLBCL) of nongerminal centre (GC) type/immunoblastic type, plasmablastic lymphoma DLBCLs arising on a background of MCD and plasmacytoma/myeloma. Plasmablastic lymphomas are frequently EBV associated; they are negative or weakly positive for CD20, PAX5 and CD45 and usually negative for CD56; they are positive for CD138 and other plasma cell markers.30e32 DLBCL-non-GC type with immunoblastic features is often associated with EBV in the HIV setting, but is often positive for CD20 and negative for CD138. DLBCLs (plasmablastic lymphoma) arising in the background of MCD are associated with HHV-8 and not with EBV, and this can be easily identified with HHV-8-LANA-1 immunostaining. Plasmacytomas/ myelomas express CD138 and other plasma cell markers, are variably positive for CD56 and cyclin D1, and, in most cases, are negative for CD45 and EBV (EBER-ISH). The presence of skeletal lesions and a significant paraprotein are features more likely to be associated with plasmacytoma. In addition to the above-mentioned entities, ‘solid’ or extracavitary forms of primary effusion lymphoma (PEL) may also have plasmablastic morphology (discussed below). A poorly differentiated carcinoma and an amelanotic melanoma may also enter the differential diagnosis in certain cases, and need to be differentiated with appropriate work-up.30e32

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HIV-lymphomas that are similar to those occurring in immune competent patients These include classical Hodgkin lymphoma (cHL), Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), primary DLBCL of the CNS, marginal zone lymphoma (MALT lymphoma) and peripheral T-cell and NK-cell lymphoma.37 Only distinctive aspects with respect to the HIV setting are mentioned below:

Figure 5 LN from an HIV-positive patient showing sheets of cohesive pleomorphic large tumour cells. The tumour cells are positive for CD45 and CD30 and are co-infected with HHV-8 and EBV (positive for EBER and HHV-8-LANA-1). The cells are negative for CD20 and CD138. Ki-67 expression is >80%. The morphological and immunophenotypic features are of the extracavitary variant of primary effusion lymphoma (PEL).

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HIV-lymphomas is often of immunoblastic histology in the HIV setting. They more frequently occur in the cerebrum as multiple lesions, though they can involve other sites.37,38

HIV-lymphomas e viral associations and genes involved HIV-lymphoma Burkitt DLBCL e centroblastic DLBCL e immunoblastic DLBCL e primary CNS Classical Hodgkin lymphoma Primary effusion lymphoma Plasmablastic lymphoma HHV-8-associated LPD PTLD-like

Virus association EBV (30%) EBV (30%) EBV (90%) EBV (nearly 100%) EBV (nearly 100%)

Genes involved MYC (100%) BCL6 (30%)

Histiocyte rich proliferations Histiocytic/epithelioid cell infiltrates in HIV-positive patients often indicate an infective aetiology. However, some neoplasms like cHL can present with prominent histiocytic/epithelioid cell infiltrates, and neoplastic cells can be scanty. Furthermore, the neoplastic cells present may not have the characteristic features of Reed Sternberg cells and resemble mononuclear Hodgkin cells. In the HIV setting, a proportion (w15e20%) of cHL present at unusual sites such as liver or bone marrow, and in some cases, these may be the only sites of documentable disease.44 Apart from the usual immunostains, ISH for EBER is a sensitive marker for cHL in this setting, as nearly 100% of cHL in the HIV setting are EBV associated. Another disease entity associated with histiocytic proliferation in bone marrow trephine biopsies (BMTB) and splenic tissue is haemophagocytosis. The presence of haemophagocytosis should prompt a work-up for HHV-8 and EBV, as these viral infections and associated lymphomas/lymphoproliferative processes can drive the haemophagocytosis.45

EBV & HHV-8 (nearly 100%) EBV (60e75%) HHV-8 (100%) EBV (most of the cases)

Table 3

1) Histological features of HIV-cHL conform more often to mixed cellularity or lymphocyte depletion subtypes. The tumour cell load is unusually high as compared to cHL in the general population.38,39 2) Fibrohistiocytic stromal cells form a major component of the microenvironment in HIV-cHL in contrast to the predominance of small lymphoid cells in cHL seen in the general population.40 3) Almost all cases of HIV-cHL are EBV associated. The EBV genome present in the neoplastic cells is episomal and clonal. The presence of EBV association in nearly 100% of HIV-cHL cases along with the expression of EBV-LMP-1 indicates that EBV probably plays a major role in the pathogenesis of HIV-cHL.41,42 4) HIV-cHL exhibits aggressive clinical behaviour, and is associated with a poor prognosis.41,42 5) Nearly two-thirds of HIV-BL have plasmacytoid differentiation with eccentrically placed nuclei and abundant cytoplasm that contains immunoglobulin. Within the plasmacytoid subset of BL, EBV association is seen in 50e70% cases. In contrast, among HIV-BL that lack plasmacytoid differentiation, w30% are EBV associated.37,38,43 6) BL in the HIV setting shows greater variability in nuclear size and morphology, and a proportion of cases express MUM1. 7) Peripheral blood involvement is less common in HIV-BL compared with HIV-negative patients with BL.37,38,43 8) HIV-DLBCLs can either be of centroblastic or immunoblastic morphology. Centroblastic DLBCL occurs on a background of mild immunosuppression, and is less likely to be EBV positive (30e40%). The EBV-positive centroblastic DLBCLs lack EBV-LMP-1. They have a germinal centre B-cell phenotype, and frequently show BCL6 rearrangement. In contrast, immunoblastic DLBCL usually occurs in the background of severe immunosuppression, and is more likely to be EBV positive (80e90%). The EBV-positive immunoblastic DLBCLs frequently express EBV-LMP-1 and EBNA-2. They have a non-germinal centre B-cell phenotype, and lack BCL6 rearrangement.37,38 9) Among HIV-DLBCLs, brain is the most common extranodal site. Primary DLBCL of the CNS accounting for 15e30% of

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Post-transplant lymphoproliferative disorders (PTLDs) The incidence of PTLD is influenced by many factors e the type of immunosuppressive regimen and the intensity of suppression, type of transplant, EBV-sero-negativity at the time of transplant (an important risk factor), age at transplant (children have higher incidence) and use of T-cell depletion in bone marrow transplants (these are at a higher risk (w22%) of PTLD). The incidence of PTLD varies as per the organ transplanted e it is lowest in renal and bone marrow transplant patients (1%), intermediate in hepatic and cardiac transplant patients (1e2%) and highest in heart/lung, lung or intestinal transplant patients (>5%). Patients who receive unrelated umbilical cord blood transplants with a non-myeloablative regimen containing antithymocyte globulin have a PTLD incidence of 17%.46e51 Most of the PTLD are of B-cell lineage; T-cell PTLD is reported to be less than 5e15% of total PTLD cases. Rare cases of NK-cell lymphoma are also encountered. More than two-thirds of B-PTLD are EBV associated and less than one-third of T-PTLD is EBV-related.3,51e53 Generally EBV-negative PTLD occurs later (5 or more years after the transplant) compared with EBV-positive cases.54,55 Rare cases of B-PTLD are reported to be associated with HHV-8 and some of them belong to the PEL category.56 The sites of involvement can be LNs or extranodal. Early lesions commonly affect tonsils and adenoids. Other extranodal tissues that are involved by PTLD include gastrointestinal tract, lungs, liver and the allograft in solid organ transplants. Allograft involvement by PTLD is more common among early onset PTLD.51 The classification of PTLD is detailed in Table 4. Early lesions classified as plasmacytic hyperplasia show architectural preservation of the LN (sinusoidal architecture) or tonsillar crypts. The medulla is expanded in the case of LNs and the interfollicular areas in tonsils, showing numerous plasma cells, small lymphoid cells and bland immunoblasts. Some cases may have floridly reactive follicles. The lymphoid cells and

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may indicate polyclonal, oligoclonal or monoclonal B-cell expansion.51,57 Early lesions tend to occur in children and young adults who have not been exposed to EBV before the transplant. These tend to involve tonsils, LNs and adenoids. Most early lesions spontaneously regress following reduction in immune suppression. Rarely, IM-like lesions can be fatal.51 Lesions classified as polymorphic PTLD (P-PTLD) are destructive lesions with disruption of tissue architecture and the presence of necrosis, often geographic in nature. The cellular infiltrate is polymorphic, with the presence of small and intermediate sized lymphoid cells, plasmacytoid cells, plasma cells, immunoblasts and RS-like cells (Figure 6). There is often prominent mitotic activity. The lesion can be mistaken for Hodgkin lymphoma. There may also be foci of monomorphism. However, plasma cells, immunoblasts and a spectrum of cells with plasmacytic features are what distinguish this lesion from monomorphic PTLD. Typically the lymphoid cells are positive for CD20. The large atypical cells resembling RS cells express CD20 and CD30, and are negative for CD15. The proliferation often harbours one or more clones of cells as demonstrated by light chain restriction studies, immunoglobulin gene rearrangement studies or clonality studies based on EBV terminal repeats. The IgV genes show mutations, but without evidence of ongoing mutations in 75% of the cases, and the rest are unmutated.51,58,59 P-PTLD accounts for 20e80% of all PTLDs, and is more frequent in children. Reduction of immune suppression results in regression in some, while many other would require specific treatment.51 Monomorphic PTLD resembles lymphomas seen in immune competent individuals and includes DLBCL, BL, plasmacytoma and T/NK-cell lymphoma. The DLBCLs more often have immunoblastic morphology with pleomorphism and multinucleation. Though there may be some admixture with smaller cells, thereby raising the differential diagnosis of P-PTLD, large transformed

Classification of post-transplant lymphoproliferative disorders Early lesions Plasmacytic hyperplasia Infectious mononucleosis-like lesions Polymorphic PTLD Monomorphic PTLD B-cell neoplasms DLBCL Burkitt lymphoma Plasmacytoma Plasmacytoma-like Others T-cell neoplasms (w15%) Peripheral T-cell lymphoma, unspecified Hepatosplenic T-cell lymphoma Others Classical Hodgkin lymphoma-like PTLD Table 4

plasma cells do not show light chain restriction, and immunoglobulin gene rearrangement studies suggest a polyclonal B-cell expansion. The proliferation is often positive for EBV.51 Early lesions classified as infectious mononucleosis-like PTLD show similar architectural preservation. The paracortex is markedly expanded by a large number of immunoblasts along with T-cells and plasma cells. The immunoblasts form a prominent component of the lesion with frequent binucleate forms and cells resembling Hodgkin cells. The cells are EBV positive with expression of both EBER (ISH) and EBV-LMP-1. Results of immunoglobulin gene rearrangement studies are variable, and

A patient within the first 100 days of receiving an allogeneic bone marrow stem cell transplant developed lymphadenopathy. An LN shows a diffuse polymorphous infiltrate of cells with areas of necrosis. a A polymorphic infiltrate of cells of varying sizes. Many of the cells have plasmacytoid features and some cells have features of immunoblasts. There are also cells resembling Hodgkin cells (not seen in the figure provided). The cells are positive for CD45, CD20 and vs38C, and kappa light chain restriction is present. A proportion of cells expresses EBV-latent membrane protein-1 b. This case is an example of polymorphic PTLD. Figure 6

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Biopsy from an ulcerated skin and subcutaneous lesion in a patient who had been on long term methotrexate treatment for an autoimmune disease. The base of the ulcer shows large atypical cells with features of immunoblasts that are admixed with plasma cells, plasmacytoid cells and lymphoid cells a. The large atypical cells are positive for CD20, CD30, MUM1 and EBER (EBV e in-situ hybridization) b. They also show lambda light chain restriction. This case exemplifies an iatrogenic immune deficiency associated lymphoproliferative disorder. The lesion resolved completely on withdrawal of methotrexate. Figure 7

blasts predominate. These show demonstrable heavy chain expression in w50% cases, which is usually IgG or IgA. These DLBCLs are also more often CD30 positive. The EBV-positive DLBCLs tend to have a non-germinal centre (non-GC) phenotype (based on the Hans algorithm60) and the EBV-negative DLBCLs have GC phenotype. In almost all cases of monomorphic B-PTLD, a clonal population can be identified by immunoglobulin gene rearrangement studies. Most cases have mutated IgV genes with only some showing ongoing mutations. BCL6 hypermutation (but not translocations) and RAS or P53 mutation is frequent. MYC rearrangement is only seen in the BL subtype of monomorphic B-PTLD. A proportion of the plasmacytoma-like-PTLD are EBV associated.51,59e62 With regards to cHL-type PTLD, histological features are akin to mixed cellularity or lymphocyte-depleted subtypes. It is crucial to use rigid immunophenotypic criteria for diagnosis, and demonstration of positivity for CD30 and CD15, in the absence of expression of CD45 and OCT-2/BOB.1, is recommended. Care should be taken not to diagnose polymorphic-B-PTLD with Hodgkin-like cells as cHL-type PTLD. cHL-type PTLD is rare and occurs almost exclusively in renal-transplant patients, and is almost always EBV associated.1,51,63 T-NK-cell PTLD is less common. These show features akin to peripheral T-cell lymphoma, unspecified; hepatosplenic T-cell lymphoma; extranodal NK/T-cell lymphoma, nasal type; and other types.51

morphological overlap with PTLD. The classical setting are patients treated with methotrexate or those treated with tumour necrosis factor-alpha (TNF-a) antagonists, like infliximab, adalimumab and etanercept.64 IIALDs arising on a background of methotrexate treatment are better characterized. Histologically, these cases have features of DLBCL (35e60%), cHL (12e25%), polymorphic PTLD (w15%) and follicular lymphoma (5e10%). BL, MALT lymphoma and peripheral T-cell lymphoma, unspecified are less common. Nearly 40% of methotrexate-related IIALD are EBV associated (Figure 7). Among cHL, EBV association is seen in w80% cases. Withdrawal of methotrexate treatment in these patients results in regression of disease in <20e40% of DLBCL and w25% of cHL. This regression is more common among EBV-positive cases. In nearly half of the cases that regress, remission is short, necessitating chemotherapy. In contrast to methotrexate-related IALDs, cases associated with TNF-a-antagonist medication do not regress following discontinuation of the drug.64 A

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