Nodal B-cell lymphoma and Hodgkin lymphoma: current WHO classification and differential diagnostic dilemmas

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

Nodal B-cell lymphoma and Hodgkin lymphoma: current WHO classification and differential diagnostic dilemmas

In order to understand the WHO classification it is necessary to understand the process of normal B-cell development and at what stage key molecular and genetic changes occur that determine phenotype and cell function (Figure 1), although this does not entirely hold for some subtypes, such as chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) and mantle cell lymphoma (MCL) (see subsequently). Consequently the WHO classification, whilst referring where possible to a putative normal counterpart, does not use this as the basis for classification but rather attempts to define homogeneous entities based on a combination of clinical, morphological, immunophenotypical and genetic features. Figure 1 illustrates our present understanding of the evolution of B lymphoid cells from their origin in the bone marrow to plasma cells in the periphery. We now recognize a number of key markers that enable us to identify the different stages of lymphoid development. B cells arise from CD34þ lymphoid progenitor cells in bone marrow. Early pro-B cells express CD34, whilst all pro-B cells express CD10 and TdT. TdT acts to modify heavy chain joining sites, thereby increasing antigen receptor diversity. Pro- and preB cells undergo immunoglobulin (both heavy and light chain) gene rearrangement, selecting a V, D and J segment specific for that cell and all subsequent progeny and deleting the intervening DNA sequences. Recognizing this unique VDJ combination is the basis for identifying clonality in lymphomas. At this stage, the protein product (immunoglobulin/antibody) of the rearranged heavy and light chains is expressed as the antibody classes IgM and IgD. Any of these immature B cells capable of binding selfantigen are deleted by apoptosis. Both CD10 and TdT expression are lost with maturation to a pre-B cell. PAX5 encodes the B-cell lineage specific activator protein, a transcription factor critical for B-cell lineage differentiation that is expressed from the early pre-B-cell stage to the na€ıve mature B-cell stage. Na€ıve mature B cells with rearranged unmutated immunoglobulin genes enter the peripheral circulation and, from there, the lymph node, spleen and MALT tissues. In the spleen and lymph node they are predominantly located in primary lymphoid follicles and the mantle zone. Whilst PAX5 defines commitment to the B-cell lineage, CD19 (in flow cytometry) and CD20 are more commonly used as B-cell lineage markers and are expressed from the early and late pre-Bcell stage, respectively. CD79a is expressed from early pro-B cells to the early plasma cell stage. It is expressed more strongly by mantle than by GC cells. CD5 is typically expressed on T cells. It is thought to associate with, and modulate signalling by, the antigen receptor complex. However, it is also expressed on a minority of circulating and mantle zone B cells, where it is thought to have the same properties in relation to the B-cell receptor (immunoglobulin and associated proteins). It is expressed in both CLL/SLL and mantle cell lymphoma, which are thought to arise from CD5þ subpopulations of na€ıve mature B cells. In the presence of antigen, lymphocytes are attracted to GCs where they are stimulated to proliferate. GC lymphoid cells re-express CD10 and express bcl-6, which is necessary for GC formation, acting as a transcriptional repressor regulating GC B-cell differentiation. These markers are both switched off as the cells mature to plasma cells. The GC has four roles, to produce

Nick Rooney Lucy Foster Richard Byers

Abstract Normal B cells undergo an apparently complex series of morphological phenotypic and molecular changes as part of their transformation from lymphoblasts to plasma cells. Understanding this pathway is the key to understanding the classification of lymphoma. The molecular changes underlying classical and nodular lymphocyte predominant Hodgkin lymphoma help us orientate these diseases with respect to normal B-cell development. This knowledge does not equate with a cellular origin for each tumour but helps us understand the relationship between morphology, phenotype, genotype and the clinical course of the disease. After the introduction of B-cell development, this article describes the nodal B-cell lymphomas, details the associated phenotype and genotype and documents the most important differential diagnoses.

Keywords genotype; Hodgkin; lymphoma; phenotype

Introduction The WHO classification of tumours of the haematopoietic and lymphoid system was updated in 2009 to take account of the latest molecular and phenotypic data.1 In the Best Practice document produced by the Royal College of Pathologists and the British Society of Haematology,2 the differential diagnoses of the lymphomas are discussed and pitfalls outlined. This article is a short summary of these documents concentrating on the nodal appearances while recognizing that the diagnosis may sometimes be made on blood samples and extranodal tissues. In addition to morphology, diagnosis is aided by immunophenotypical and cytogenetic markers, now regarded as essential for accurate classification.

Nick Rooney MD FRCPath is a Consultant Pathologist in the Pathology Sciences North Bristol NHS Trust, Bristol, UK. Lucy Foster

MB ChB

is Pathology Trainee in the North West Deanery UK.

Richard Byers MD FRCPath is a Consultant Pathologist at the Department of Pathology, Manchester Royal Infirmary, Manchester, UK.

DIAGNOSTIC HISTOPATHOLOGY 16:2

54

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

Figure 1 Pathway of lymphoid cells from lymphoblasts in the bone marrow (left), through circulating unmutated lymphocytes and germinal centre B cells to either plasma cell precursors or memory B lymphocytes with mutated immunoglobulin (Ig), which may re-enter the germinal centre (GC) to undergo progressive cycles of mutation and differentiation. Each cell’s immunophenotype can be determined by looking at the horizontal and vertical yellow bars with which it aligns. The vertical bars include the pan-B-cell markers CD20 CD79aþb and PAX5, which have slightly different distributions, and the plasma cell associated proteins MUM1 (IRF4) and CD138. The horizontal bars indicate markers switched on and off during transit through the bone marrow and GC. The blue boxes indicate our current understanding of the relationship between the phenotype and genotype of the tumours and their relationship to normal lymphoid development. Some tumours such as CLL are morphologically homogeneous, which includes two molecular forms, a pre-GC variant (CLLu) with germline Ig genes and a post-GC form (CLLm) with mutated Ig genes. DLBCL and cHL tend to comprise a single cell type morphologically with a common fixed Ig mutation. MZL and FL are characterized by a mixed cell population and progressive Ig gene mutations, indicating continued exposure to the GC environment. Black lines illustrate the putative pathways. NLPHD also falls into this group, each nodule containing related cells that show additional Ig mutations. CLLu e chronic lymphocytic leukaemia, immunoglobulin unmutated, CLLm e chronic lymphocytic leukaemia, immunoglobulin mutated, MCL e mantle cell lymphoma, FL e follicular lymphoma, MZL e marginal zone lymphoma, DLBCL e diffuse large B-cell lymphoma, BL e Burkitt lymphoma, NLPHL e nodular lymphocyte predominant Hodgkin lymphoma, cHL e classical Hodgkin lymphoma, PBL e plasmablastic lymphoma.

a large number of cells, to increase the specificity of the antibody produced, to switch the class of antibody from (IgD and M to G, A and E) and to produce memory B cells. In the dark zone of GCs, there are large numbers of mitoses and a blastic morphology typical of proliferating cells. Increasing specificity is accomplished by somatic hypermutation (random insertion of nucleotides into the DeJ area of the immunoglobulin (Ig) genes) followed by selection. The DeJ sequence is the principal coding area for the antigen binding site. A random mutation might result in more or less avid or even self-reactive antibody, or sometimes a stop codon and no antibody. The cells with the most avid antibody are selected for survival by interaction with follicular dendritic cells (FDCs) bearing the antigen and T cells, and undergo final differentiation into CD138þ and MUM1þ plasma cells and memory B cells. Cells that bind antigen weakly undergo apoptosis and phagocytosis by the tingible body macrophages. Activated GC B cells also undergo Ig heavy chain class switching leading to production, by daughter cells, of a different class of antibody with no change in antigen specificity. Amplification and sequencing of the Ig genes allows us to (i) identify clonality, (ii) identify the presence of somatic hypermutation, indicating whether the cells have been through a GC, and (iii) follow a cell’s progeny as they migrate around the body. Using these techniques, we can identify acute lymphoblastic leukaemia (ALL), some diffuse large B-cell lymphomas (DLBCL),

DIAGNOSTIC HISTOPATHOLOGY 16:2

and a proportion of CLL and MCL as lymphomas composed of pre-GC cells. Follicular lymphoma (FL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) comprise cells with mutated Ig genes with evidence of progressive mutation between tumour sites, as the cells undergo further rounds of GC activity and somatic mutation. The majority of DLBCL, all Burkitt’s lymphoma (BL) and classical Hodgkin lymphoma (cHL) are mutated, indicating their origin from GC cells. Nodal and extranodal marginal zone lymphomas (MZL) and lymphoplasmacytic lymphoma (LPL) are derived from post-GC cells and this is not surprising given that GCs are an essential part of these tumours. Approximately half of CLL is derived from somatically mutated cells (CLLm) possibly representing post-GC memory B cells.

Pre-germinal centre lymphoma Pre-GC lymphoma comprises the unmutated form of CLL/SLL and MCL, though for simplicity this section will also refer to the post-GC somatically hypermutated form of CLL/SLL. Chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) Clinical: CLL/SLL is the most common leukaemia of adults in the Western world with an incidence of 2e6 per 100,000 and accounting for 6.7% of all NHL biopsies. CLL and SLL are

55

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

Cytogenetics & molecular genetics: Ig genes are rearranged,4 whilst approximately 50% of cases are unmutated and 50% have somatic hypermutation, indicating antigen exposure, the latter having a better prognosis. FISH for the t(11;14) characteristic of MCL is useful in differentiating it from CLL/SLL, though immunohistochemistry for cyclin D1 is usually able to resolve this.

phenotypically indistinguishable, SLL being used for lymphomatous presentation without lymphocytosis, and CLL for cases presenting predominantly in the peripheral blood. The clinical course is variable but is typically indolent. Most predominantly nodal cases present with symptomatic generalized lymphadenopathy, though the majority of these patients will eventually develop a leukaemic phase. The exact benign counterpart of the malignant cell in SLL and CLL is unknown.

Prognostic markers: a diffuse pattern of marrow involvement and >30% cells positive for CD38 are poor prognostic features.5 Positivity for ZAP70 by immunohistochemistry is strongly but incompletely correlated with a lack of somatic hypermutation (defined as >98% germline Ig gene sequence), both of which are poor prognostic features.6 About 80% of cases of CLL/SLL have FISH detectable cytogenetic abnormalities, and, of these, 13q14 deletion suggests a favourable prognosis, while trisomy 12, 11q22e23 deletion (targeting the ATM gene) and 17p13 deletion (targeting the p53 gene), predict a poor outcome.7,8

Morphology: nodal involvement, by CLL/SLL is typified by diffuse effacement of lymph node architecture by small slightly irregular B lymphocytes (Figure 2), together with a variable number of socalled proliferation centres (also called pseudofollicles), in which there are prolymphocytes and paraimmunoblasts. The size of such areas does not correlate with prognosis, though, when fused to form large sheets, transformation to DLBCL is likely. Transformation to a high grade B-cell lymphoma, often DLBCL, or socalled Richter’s transformation, occurs in a minority (3.5%) of patients, whilst about 0.5% go on to develop Hodgkin lymphoma, which is often EBV positive.

Morphological differential diagnoses

1 Reactive lymphoid hyperplasia lacks the monomorphous diffuse effacement of nodal architecture and proliferation centres of CLL/SLL. CD5 expression by B cells is restricted to mantle zones in hyperplastic nodes. 2 Mantle cell lymphoma is typified by a monotonous population of CD23 and cyclin D1þ cells, without paraimmunoblasts or proliferation centres. 3 Follicular lymphoma can be mimicked by CLL/SLL in cases where proliferation centres are prominent, but the follicles in FL are more clearly defined than in CLL/SLL, and are

Immunophenotype: CLL/SLL is positive for PAX5, CD22, CD19, CD20 (weak), CD5, CD23 and CD79a, weakly positive for CD79b and sIg but negative for FMC7 and importantly, cyclin D1 (Figure 2). Some cases have an aberrant phenotype, with negativity for either CD5 or CD23. Additionally, CD38 and ZAP70 are of prognostic value,3 though their expression is not diagnostic (Figure 2).

Both CLLu and CLLm comprise small lymphocytes that are CD20þ, CD23þ and CD5þ. Figure 2

DIAGNOSTIC HISTOPATHOLOGY 16:2

56

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

proliferation centres with transformed cells such as centroblasts or paraimmunoblasts are absent (Figure 3a). The cells have dispersed nuclear chromatin and there are small inconspicuous nucleoli. The infiltrate can have a variety of growth patterns. The mantle zone pattern is characterized by broad mantle zones composed of neoplastic cells surrounding reactive GCs. The nodular pattern contains nodules of neoplastic cells, whilst the diffuse pattern shows diffuse effacement by neoplastic cells often with hyalinized small vessels and scattered epithelioid cells. In occasional cases involvement is restricted to the inner mantle zones, so-called ‘‘in situ’’ MCL. Histological transformation to another, high grade, lymphoma, does not occur though aggressive variants of MCL exist, namely blastoid (cells resembling lymphoblasts with dispersed chromatin and high mitotic rate (at least 20e30/10 hpf)) and pleomorphic (large cells with oval or irregular nuclei, pale cytoplasm and prominent nucleoli).

composed of centrocytes and centroblasts, rather than of prolymphocytes and paraimmunoblasts. Additionally, FL is CD10þ and CD5. 4 Marginal zone lymphoma may be confused with an interfollicular pattern of CLL/SLL, in which there are pseudofollicles adjacent to reactive follicles, though CD23 positivity in CLL/SLL clearly distinguishes it from MZL. 5 Lymphoplasmacytic lymphoma can be confused with the few cases (5%) of CLL/SLL with a high degree of plasmacytic differentiation. In these cases, positivity for CD5 and the presence of pseudofollicles distinguish CLL/SLL.

Mantle cell lymphoma (MCL) Clinical MCL accounts for 3e10% of NHL and occurs predominantly in middle-aged to older patients, with a male predominance. It presents in lymph nodes, with or without splenic, marrow or peripheral blood involvement. It can cause cytopaenia due to marrow infiltration, and is commonly associated with peripheral blood lymphocytosis. Extranodal sites, such as the gastrointestinal tract and Waldeyer’s ring, are commonly involved. However, despite its cytologically bland nature and morphological similarity to CLL/SLL, it tends to have a poor prognosis except in cases with limited disease.

Immunophenotype B-cell markers (PAX5, CD19, CD20 and CD79a) are positive. Like CLL/SLL, MCL is CD5 positive, though, unlike CLL/SLL, CD23 is negative and cyclin D1 positive (Figure 3bed). In keeping with pre-GC origin, CD10 is negative. bcl-2 is positive, whilst bcl-6 is negative, though these are not useful in MCL diagnosis. Rare cases are CD5, CD10þ and bcl-6þ. Surface IgM is positive; IgD may or may not be positive (Figure 3).

Morphology It is composed of monomorphic, small to medium sized lymphoid cells, with irregular nuclear contours, whilst

Cytogenetics and molecular genetics The genetic hallmark of MCL is presence of the t(11;14)(q13;q32) CCND1/IGH translocation, with resultant over-expression of

Mantle cell lymphoma is positive for CD20, CD5 and cyclin D1. Figure 3

DIAGNOSTIC HISTOPATHOLOGY 16:2

57

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

cyclin D1. This is detected by FISH using a CCND1 break-apart probe rather than specific CCND1/IGH probes, as rare variants (translocations of CCND1 to genes other than IgH) occur. MCL cells, particularly blastoid variants, also carry a high number of secondary chromosomal translocations and molecular alterations affecting regulation of cell cycle, senescence, and cellular response to DNA damage.9 Rare cases are cyclin D1 negative, though these cases usually show increased expression of cyclin D2 or cyclin D3.10 Immunoglobulin genes are rearranged in keeping with postulated origin from a mature B cell and in the majority of case are unmutated, though, like CLL/SLL, MCL is a genetically heterogeneous disease and 15e40% of cases show somatic hypermutation, of lower load than in CLLm. The postulated normal counterpart cell of unmutated MCL is a mature, na€ıve, pre-GC, CD5þ B cell of the inner follicular mantle zone. Cases with somatic hypermutation may have undergone exposure to a GC environment or somatic hypermutation in a non-GC context, as part of the neoplastic process. Though not extensively investigated, a recent study failed to identify clinical or pathological differences between mutated and non-mutated forms of MCL, which may support the occurrence of somatic hypermutation in a non-GC context, as part of the neoplastic process.11

5 B- and T-cell precursor lymphoblastic leukaemia/lymphoma (acute lymphoblastic leukaemia, ALL) can be mimicked by the blastoid variant of MCL, though B- and T-cell ALL are cyclin D1 and TdTþ. 6 Diffuse large B-cell lymphoma (DLBCL), especially if CD5þ, can be mimicked by the pleomorphic and blastoid variants of MCL, though positivity for cyclin D1 (usually) and presence of t(11;14) in MCL excludes DLBCL. 7 B-cell prolymphocytic leukaemia (B-PLL) has an aggressive clinical course and high peripheral blood lymphocytosis. Some patients with MCL can present in a similar manner, with large numbers of atypical peripheral blood lymphocytes that can resemble prolymphocytes. However, in such cases, smaller lymphoid cells more typical of MCL are also present, whilst B-PLL lacks cyclin D1. 8 Hodgkin lymphoma of both nodular lymphocyte predominant and lymphocyte-rich classical types can superficially resemble MCL, though both will have large Hodgkin cells interspersed in a lymphocytic infiltrate, which will be cyclin D1 negative (see subsequently).

Germinal centre lymphoma GC lymphoma comprises follicular lymphoma (FL) diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL) and both classical (cHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL).

Prognostic markers MCL is incurable with a 3e4-year median survival. A high mitotic rate (greater than 10e37/15 hpf) or a high proliferation index (Mi-67/Mib-1 >40%) are adverse prognostic features, in keeping with recent data on gene expression.12 Peripheral blood involvement is a poor prognostic indicator in patients with nodal disease, though some studies report it as a favourable feature in patients without adenopathy. Blastoid or pleomorphic morphology, additional cytogenetic abnormalities, including trisomy 12 and TP53 mutation, and lack of somatic hypermutation have been associated with shorter survival.13

Follicular lymphoma (FL) Clinical: FL is the most common lymphoma in Western populations accounting for 20% of lymphoma diagnoses. The disease is frequently multinodal and presents in stage IIIeIV in middle-aged to elderly patients with a male:female ratio of 1:1.7. Though FL does occur <35 years, such a diagnosis should raise the question of either follicular hyperplasia or NLPHL. Morphology: its postulated normal counterpart cell is the GC B cell and this is reflected in its characteristic follicular growth pattern composed of centroblasts and centrocytes with reduced or absent non-B-cell GC cells, namely tingible body macrophages, and a random distribution of cells within the follicle. If there is a significant blastic component showing a diffuse pattern of growth, without centrocytes, an additional diagnosis of DLBCL is also made.14 The continued maturation from the proliferative blasts through to the shrunken and relatively inactive centrocytes reflects FL’s indolent behaviour and ongoing somatic hypermutation. The degree of maturation, reflected by the number of centroblasts (CB), confers some prognostic significance, so separation into grades 1 (<6 CB/hpf); 2 (6e15 CB/hpf); 3a (>15 CB/hpf with centrocytes) or 3b (>15 CB/hpf without centrocytes) is advised. The distinction between low grade (1 & 2) and high grade3 is of most clinical relevance as this influences management.

Morphological differential diagnoses A number of conditions can resemble MCL morphologically: 1 Reactive follicular hyperplasia (RFH) with broad mantle zones can be confused with the mantle zone pattern of MCL, though usually the mantle zones in RFH are thinner and composed of small round cells. Immunohistochemistry for cyclin D1 is helpful, being negative in RFH. Interfollicular areas in MCL are obscured by a ‘‘cloud’’ of small neoplastic cells. 2 CLL/SLL is morphologically similar to MCL (see above). Critically CLL/SLL is cyclin D1 negative and CD23 positive. 3 Follicular lymphoma (FL) is typically composed of sharply circumscribed follicles, but some cases of MCL have a vaguely nodular pattern, which, when combined with occasional reactive GCs or scattered centroblasts, can mimic FL. Immunostaining is helpful, since FL is CD10þ, but CD5 and cyclin D1. 4 Marginal zone lymphoma (nodal or extranodal ) can be mimicked by rare cases of MCL that have prominent areas of pale monocytoid like cells. The marginal zone pattern of MZL should not be mistaken as mantle zone pattern MCL. Immunostaining for cyclin D1 and FISH for t(11;14) help resolve such problems.

DIAGNOSTIC HISTOPATHOLOGY 16:2

Immunophenotype: the phenotype parallels that of GCs. CD20, CD79a and PAX5 are expressed in FL and bcl-2, bcl-6 and CD10 positivity are the hallmark of the disease. Occasionally CD10 expression may be lost, most notably in grade 3b cases. Bcl-2 expression helps to differentiate FL from follicular hyperplasia as non-neoplastic GC cells are bcl-2 negative. If a follicular growth

58

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

growth pattern of FL with diffuse areas of centroblasts and centrocytes, reversed follicles, floral variants, marginal zone differentiation, etc. that confer some prognostic advantage or disadvantage. However, of most significance is the presence of a grade 3b growth pattern that signifies the presence of DLBCL.16 DLBCL in this context is frequently genotypically related to the original FL and is usually of GC type. Management of grade 1 and 2 involves either observation or oral chemotherapy with rituximab. If the patient with grade 3b is fit enough, management as for DLBCL with R-CHOP is the treatment of choice. The management of grade 3a is the most variable.

pattern is difficult to identify on H&E, immunohistochemistry for CD21 and CD23 may be required to identify FDCs. A very small proportion of tumours may express CD5 or CD23 and this should not discourage a diagnosis of FL if other features are supportive (Figures 4 and 5). Cytogenetics and molecular genetics: the characteristic translocation of the BCL2 gene from chromosome 18 to the vicinity of the immunoglobulin heavy chain gene on chromosome 14 leads to inappropriate GC bcl-2 expression and failure of elimination by apoptosis. t(14;18)(q32;q21) is readily detectable by FISH on cell suspensions, imprint cytology or paraffin sections. A t(14;18) BCL2 rearrangement is less common in grade 3 FLs associated with DLBCL and, in this situation, the use of BCL2 break-apart FISH probes to detect variant translocations should be considered. Ongoing IgH gene somatic hypermutation occurs and different tumour sites may harbour additional somatic hypermutations, indicating continued episodes of progression through the GC.

Morphological differential diagnoses

1 Reactive follicular hyperplasia is usually distinguished from FL by assessment of the architecture and cellular components of the GC (Figures 4 and 5). Features suggesting RFH are: (i) Zonation into light and dark zones, best seen on CD23 that stains light zones but not dark zones (unlike CD21 that stains both) and Ki67 that stains the dark proliferative zone more strongly. (ii) Apoptosis and tingible body macrophages. (iii) Bcl-2 staining of the neoplastic B cells, but beware of reactive follicles rich in bcl-2þ T cells. (iv) CD10 staining of interfollicular lymphoid cells (not granulocytes). 2 Castleman’s disease can be mistaken for a grade 1 follicular lymphoma if the characteristic architecture is not recognized.

Prognostic markers: current clinical practice uses the Follicular Lymphoma International Prognosis Index (FLIPI) score (a product of age, stage, nodal areas involved, haemoglobin, and serum lactate dehydrogenase (LDH)) to stratify patients, but recent evidence from molecular profiling has suggested that the tumour infiltrating cells may have the largest effect on the prognosis of FL, infiltrating T cells being favourable and infiltrating macrophages an unfavourable feature.15 There are a number of variations in the

Reactive hyperplasia: zonation of the GC light and dark zones is emphasized on CD23 immunostaining. CD20 demonstrates B-cell follicles the centres of which are CD10þ and bcl-2, with a background meshwork of CD21þ, CD23þ follicular dendritic cells. Figure 4

DIAGNOSTIC HISTOPATHOLOGY 16:2

59

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

Follicular lymphoma typically comprises a mixed population of centroblasts and centrocytes. There is no zonation on CD23 immunostaining. Bcl-2 and CD10 immunostain the GC as well as the interfollicular zone. Yellow signal indicates t(14;18) with red and green fusion FISH probes (cf. Figure 4). Figure 5

3 4 5

6

The very tight FDC networks in CD are unlike those seen in FL. Mantle cell lymphoma (see above). CLL/SLL (see above). Marginal zone lymphoma (MZL) with follicular colonization may resemble FL, though, while MZL cells will be bcl-2þ, the colonized follicle centres will be bcl-2; FL will usually show the t(14;18) translocation which is absent in MZL. Hodgkin lymphoma of nodular lymphocyte predominant type (NLPHL) is closely related to FL, sharing many immunophenotypical features. ReedeSternberg-like cells may also be seen in FL so it is important to recognize the dual population of lymphocyte predominant (LP; formerly known as ‘‘L&H’’) cells and small lymphocytes in NLPHL, though the large cells will express CD10 and bcl-6 as in FL. In NLPHL the small lymphocytes are derived from the mantle zone and therefore will express CD23, IgD and bcl-2, but, unlike the centrocytes in FL, they do not express CD10 or bcl-6. FISH for t(14;18) can be helpful in difficult cases, since it will be positive in most cases of FL but negative in NLPHL.

driver is EBV, HHV8 or both (further discussed in Dr Naresh’s review in the April 2010 issue of this journal). The Ki67 proliferation index is typically >40% but may exceed 90%. Bcl-2 expression is variable but when expressed it is associated with a poor prognosis17 (Figure 6). DLBCL is mostly derived from blasts with mutated Ig genes indicating a GC or post-GC development. Gene expression and immunophenotype are used to separate DLBCL into its two groups with regards to presumptive cell of origin: GC type (CD10þ, bcl-6þ or CD10, bcl-6þ, MUM1) or activated B-cell (ABC) type (CD10, bcl-6þ/, MUM1þ).18 Like FL, the GC type have ongoing Ig mutations but the Ig genes in ABC type are fixed and do not undergo further mutation.19 Morphological differential diagnoses of DLBCL include nonhaematological malignancies (anaplastic undifferentiated carcinoma and amelanotic melanoma), acute myeloid leukaemia/ granulocytic sarcoma, lymphoblastic lymphoma, Burkitt lymphoma, peripheral T-cell lymphoma/anaplastic large cell lymphoma, plasmacytoma/myeloma, Hodgkin lymphoma and EpsteineBarr virus (EBV) e induced proliferation/infectious mononucleosis.

Diffuse large B-cell lymphoma This is the second most common form of lymphoma in western countries and the most common high grade tumour accounting for 25e30% of NHL in the West and a higher proportion in developing countries and is reviewed in this issue by Kevin Gatter (see pages 00e00). DLBCL is particularly common in immunodeficiency states of HIV or post-transplant where the

DIAGNOSTIC HISTOPATHOLOGY 16:2

Burkitt lymphoma (BL) Clinical: Endemic BL was originally described in malarial areas of Africa where it is associated in virtually 100% of cases with EBV. A disease of similar morphology and phenotype, sporadic Burkitt lymphoma is seen in Western populations predominantly affecting children and young adults. A third variant;

60

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

Diffuse large B-cell lymphoma of GC type is typically a monotonous population of blasts with CD10 and bcl-2 expression. The high Ki67 index reflects MYC activation, in this case due to MYC translocation (large arrow indicates co-localization of red and green signals (normal chromosome); arrowheads indicate split signal from the other allele with an MYC break-apart probe). In this case, there is also bcl-6 activation by translocation (large arrow indicates co-localization of red and green signals (normal chromosome); arrowheads indicate split signal from the other allele with a bcl-6 break-apart probe). Figure 6

intensive chemotherapy than to the usual CHOP based chemotherapy of other aggressive GC tumours.26

immunodeficiency-associated BL is seen in the immunosuppressed, in particular in individuals with HIV.20,21 BL has a very short doubling time and characteristically involves extranodal sites, often presenting with a rapidly enlarging abdominal mass. Other sites include ovary, kidney and breast. The characteristic tumours of the jaw seen in endemic BL are rarely seen in the sporadic variant. Nodal involvement is more commonly seen in adults than in children and is more characteristic of the immunodeficiency-associated variant. Bone marrow involvement occurs in approximately one third of patients and central nervous system (CNS) involvement occurs in up to 40% of cases.22e24 Because BL may be the first manifestation of immunodeficiency, testing for HIV is recommended.

Immunophenotype: the postulated normal cell of origin is the GC or post-GC B cell. BL is positive for pan-B-cell markers PAX5, CD20 and CD79a by immunohistochemistry or CD19, CD20, and CD79b and sIg by flow cytometry. CD10 and bcl-6 are also positive reflecting the GC phenotype. The Ki67 proliferation index approaches 100%. P53 immunostaining is positive, bcl-2 is usually negative or, in 20% of cases, only weakly positive in neoplastic cells. CD5 and TdT are negative. Cytogenetics and molecular genetics: BL is a GC tumour harbouring somatic hypermutation of the Ig genes and is characterized by the presence of an isolated translocation involving MYC on chromosome 8q24, to the Ig heavy chain region 14q32 or, less commonly, to the lambda or kappa light chain loci. This translocation must be demonstrated by FISH or, in the 10% cases negative by FISH, by other techniques to make a diagnosis of BL.

Morphology: BL is recognized as a diffuse infiltrate of monotonous, medium sized cells with vesicular chromatin and multiple small nucleoli. There is a very high mitotic and apoptotic rate inducing the presence of numerous tingible body macrophages giving the starry-sky appearance. Very few T cells are present. Morphologic appearances are variable and are considered an unreliable guide to diagnosis and, except in classical cases, usually children, with the characteristic phenotype, confirmation of the isolated t(MYC) by FISH or cytogenetics is necessary for diagnosis. Some tumours overlap with DLBCL but share the same molecular signature (‘‘grey zone lymphomas’’) (see also Kevin Gatter’s review on pages 00e00 of this issue).25 The recognition of BL is important because it responds more favourably to

DIAGNOSTIC HISTOPATHOLOGY 16:2

Prognostic markers: BL is an aggressive tumour but complete remission is achievable in endemic and sporadic variants and 2-year survival greater than 70% has been reported.27 Poor prognostic markers include bone marrow and CNS involvement, elevated serum LDH, reflecting increased tumour burden and the presence of unresected tumour >10 cm in diameter. Outcome is better in children than adults.

61

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

though the LP cells themselves are not on the plasma cell pathway and are MUM1.35 PAX5 is the most useful B-cell marker since the nuclear staining highlights the positive tumour cell in a negative Tcell rosette. The use of CD21 can highlight the nodules within an FDC network (Figure 7).

Morphological differential diagnoses

1 B lymphoblastic leukaemia is composed of lymphoblasts and is easily distinguished from BL by its immunophenotype (TdT positive, Ki67 index <90%) and by a lack of MYC rearrangement on cytogenetics. 2 Blastoid mantle cell lymphoma lacks the MYC translocation and has a proliferation index of <90%. It is cyclin D1þ, bcl2þ and CD10 by immunohistochemistry. 3 DLBCL is usually distinguishable morphologically and phenotypically from classical Burkitt lymphoma, but some DLBCLs may show variable CD10 and bcl-2 expression. In difficult cases, where there is a high proliferation index, the demonstration of an isolated MYC rearrangement is crucial. DLBCL may also have a MYC rearrangement, but in combination with other translocations (Figure 6). 4 HIV related lymphoma including DLBCL should be considered in HIVþ patients. 5 Non-haematological small round blue cell tumours including Ewing sarcoma/PNET, neuroblastoma, embryonal rhabdomyosarcoma, and small cell carcinoma, can be distinguished by appropriate immunohistochemical panels, with or without cytogenetic analysis.

Cytogenetics and molecular genetics: LP cells have clonally rearranged immunoglobulin heavy chain genes but genetic tests are not routinely used in diagnosis of NLPHL. Prognostic markers: prognosis is related to stage and, as most cases present at an early stage, it is typically good, although relapse is common. In approximately 3e5% cases transformation to large B-cell lymphoma like lesions occurs, but the prognosis is good if the disease remains localized. Morphological differential diagnoses

1 Reactive follicular hyperplasia with progressive transformation of germinal centres (PTGC ) is a benign disorder which often co-exists with NLPHL or may precede or follow its diagnosis. The diagnosis can be made on morphological grounds, the presence of hyperplastic follicles with GCs favouring a diagnosis of PTGC. 2 Follicular lymphoma can be differentiated by the presence of small follicles with centrocytes not seen in NLPHL and an absence of CD20 positive LP cells and of CD3 positive sT cell rosettes. NLPHL also lacks the t(14;18) characteristic of FL. 3 Lymphocyte rich variant of cHL (LRCHL) can look very similar to NLPHL on standard sections. The phenotype of the tumour cells in LRCHL is CD30 positive CD79a negative, OCT-2 or BOB-1 negative, CD20 only stains scattered cells. The background lymphocytes are T cells and do not form the typical rosettes of NLPHL. 4 Nodular sclerosis variant of cHL (NScHL) which may not show pronounced sclerosis in its cellular phase may be confused with NLPHL, though the Hodgkin cells in NScHL are morphologically different to those in NLPHL and are CD30 and CD15 positive. 5 T-cell and histiocyte-rich large B-cell lymphoma (THRLBCL) is usually a diffuse tumour while NLPHL has a nodular growth pattern. The background lymphocytes in THRLBCL are TIA1þ and do not form the CD57þ PD1þ rosettes typical of NLPHL. THRLBCL is also a clinically aggressive disease and presents at an advanced stage with B symptoms. However, a diffuse growth pattern in NLPHL may be seen and this can be difficult to separate from THRLBCL. 6 CLL/SLL may contain cells with morphological features of RS cells, so it is always important to check the phenotype of the background lymphocytes.

Hodgkin lymphoma (HL): overview In the WHO classification HL is separated into classical Hodgkin lymphoma (cHL) and nodular lymphocyte predominant (NLPHL) Hodgkin lymphoma. These two distinct entities share one feature, namely the presence of small numbers of GC B-cell derived tumour cells in a sea of non-malignant cells, but phenotype, molecular features and progression are different. However on morphology alone, lymphocyte-rich cHL can resemble NLPHL so careful analysis of the phenotype of the tumour cells is essential.28e30 CHL variants account for 95% of all Hodgkin lymphomas. Nodular lymphocyte predominant Hodgkin lymphoma Clinical: NLPHL is a disease of the cervical, axillary and groin nodes usually presenting in stage I or II with lymphadenopathy, but no constitutional symptoms. Morphology: the characteristic growth pattern is large nodules of small B lymphocytes partially or completely effacing the architecture of the node. A co-existent diffuse infiltrate may be present in some cases. Within the nodules there is a subpopulation of large B cells with folded nuclei and a nucleolus at the connection of the folds, formerly called popcorn cells or L&H cells but currently referred to as LP cells. The postulated normal counterpart of the malignant LP cell is a GC B cell at the centroblastic stage of differentiation. The background comprises small B lymphocytes of GC type. Diffuse areas are composed of small lymphocytes mixed with histiocytes. There may be some overlap with THRLBCL but the detection of one nodule showing classical features of NLPHL on a diffuse background should resolve this.31

Classical Hodgkin lymphoma Clinical: The phenotype of cHL is shared by all histological variants but this obscures very different epidemiology, so it should not be regarded as a single disease.36 These are primarily nodal diseases with frequent involvement of different extranodal sites according to subtype. There is a bimodal distribution for age at presentation with a peak at 15e35 years and a smaller peak in later life. Men are more commonly affected than women with the exception of the nodular sclerosis variant, which has an equal gender distribution.

Immunophenotype: all the tumour cells express B-cell markers CD20, CD79a, PAX5 and the immunoglobulin transcription factors OCT-2 and BOB-1. They lack CD30 and CD15, though CD30 may stain reactive B-cell blasts that are normal components of GCs.32,33 They are surrounded by a rosette of reactive GC T-cells positive with CD3, CD4, CD57 and PD1.34 The T cells also express MUM1,

DIAGNOSTIC HISTOPATHOLOGY 16:2

62

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

Nodular lymphocyte predominant Hodgkin lymphoma. Pax5þ LP cells set within nodules of small Pax5þ CD20þ B lymphocytes, surrounded by rosettes of CD57 (and PD1) positive T cells. CD23 immunostaining demonstrates a nodule with an expanded FDC network adjacent to reactive germinal centres with tight FDC networks. Figure 7

Morphology: all variants share the presence of the classical large binucleate ReedeSternberg cells and mononuclear Hodgkin cells (HRS cells) set in a background of lymphocytes, histiocytes, eosinophils and fibroblasts. Nodular sclerosis (NScHL), the most common form of HL in Western populations is characterized by the presence of large nodules separated by bands of fibrosis. The delicate cytoplasm of HRS cells may shrink away from the nucleus, especially if poorly fixed, giving rise to lacunar cells. This variant may have a syncytial growth of HRS cells, sometimes with central necrosis, which can be confused with other tumours or even granulomas. Mediastinal involvement is common. Mixed cellularity (MCHL) occurs with increased frequency in disadvantaged populations and the immunosuppressed and is more frequently EBV positive. Granulocytes (particularly eosinophils), histiocytes sometimes forming granulomas and plasma cells are present in varying proportions. Abdominal and splenic involvement is seen in this variant. Lymphocyte depleted (LDcHL) is the least common variant of cHL and, before the use of immunohistochemistry, was easily confused with other tumours. It is rarely diagnosed now as a primary disease. Lymphocyte rich (LRcHL) is easily confused with NLPHL on HþE. This variant occurs with increased frequency in the head and neck and post-nasal space. It was originally described as ‘‘Follicular Hodgkin’s Disease’’ until the diffuse growth pattern was also recognized.37 Atrophic follicles identified with CD21 are typically present at the edge of the nodules. The phenotype of the HRS cells is the same as other forms of CHL.

DIAGNOSTIC HISTOPATHOLOGY 16:2

Immunophenotype: all the tumour cells express CD30, the vast majority express PAX5 and approximately 75% express CD15 on immunohistochemistry. It is very difficult to identify cHL on flow cytometry. Since the synthesis of immunoglobulin is crippled, Bcell markers CD20, CD79a and Ig transcription factors OCT-2 and BOB-1 are only expressed on a few cells. These are very important markers for separating cHL from NLPHL and DLBCL. Cytogenetics and molecular genetics: microdissection studies have demonstrated that the neoplastic HRS cells of cHL contain clonal gene rearrangements in more than 98% of cases, whilst, though some have clonal T-cell receptor gene arrangements, such cases are rare. HRS cells carry a high load of somatic hypermutations in their Ig genes, indicating their origin from the GC. Between disease sites the mutations are fixed. This and the presence of MUM1 indicates cHL’s relationship to the plasma cell pathway and to some DLBCL, but distinguishes it from NLPHL. Despite this, HRS cells have lost much of the B-cell specific expression programme and have acquired inappropriate gene products that promote proliferation and abrogate apoptosis. Specifically NFkB is constitutively activated, inducing NFkB target genes.38 Comparative genomic hybridization has demonstrated recurrent gains in sub-regions in 2p, 9p and 12q and amplification in 4p16, 4q23e24 and 9p23e24, though these are not used diagnostically39 (Figure 8). Prognostic markers: in more than 85% of cases cHL is a curable disease with chemo- and radiotherapy and prognosis depends on stage at presentation. The cHL variants show variable prognosis

63

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

Classical Hodgkin lymphoma. ReedeSternberg cells are surrounded by lymphocytes histiocytes and eosinophils, and are CD30þ, CD15þ and MUM1þ, but OCT-2 and BOB-1. Surrounding small B lymphocytes are a useful positive control for OCT-2 and BOB-1. Figure 8

in order from best to worst: lymphocyte rich, nodular sclerosis, mixed cellularity, lymphocyte depleted.

diagnostic subtypes. These have been referred to as ‘‘grey zone lymphomas’’ and the WHO 2008 classification recognizes the difficulty such cases pose by the introduction of two indeterminate subtypes of lymphoma, specifically B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma (discussed further by Dr Gatter in this issue).

Morphological differential diagnoses: cHL has several morphological subtypes, each of which may be mimicked by a range of other diseases. In general ReedeSternberg-like cells may be seen in DLBCL, CLL/SLL, carcinoma and melanoma so it is important to check the phenotype of the tumour cells. Thorough immunohistochemical analysis will resolve the issue in each of these cases. Apart from the above, other specific differential diagnoses that should be considered include: 1 Anaplastic large cell lymphoma contains characteristic hallmark cells (kidney shaped nuclei) of ALCL that may express CD30 and MUM1. It is a T neoplasm that may lack expression of T-cell markers but it should not express PAX5. 2 Peripheral T-cell NHL may contain large atypical cells resembling Hodgkin cells, but in cHL such cells are positive for CD15 and negative for CD45. 3 NLPHL (see above). 4 Infectious mononucleosis (IM) may present with floridly reactive nodes in which there are immunoblasts and Hodgkin like cells. In addition IM contains apoptosis and tingible body macrophages, which are lacking in cHL, whilst the immunoblasts in IM express T-cell markers, and are CD15 and CD30 negative.

Post-germinal centre lymphoma Post-GC lymphoma comprises those lymphomas in the plasma cell pathway, including the ABC variant of DLBCL and some CLL/SLL as described above. The low grade tumours are characterized by plasma cell differentiation and include marginal zone lymphomas (MZL) and lymphoplasmacytic lymphoma (LPL). Marginal zone lymphoma (MZL): overview The postulated normal counterpart of both extranodal or nodal MZL is a post-GC marginal zone (MZ) B cell. The MZ is, strictly speaking, restricted to the spleen. It is poorly developed in most lymph nodes, though monocytoid B cells, representing the nodal equivalent of marginal zone B cells have a similar immunophenotype. The MZ contains a heterogeneous group of cells, most of which have rearranged, mutated immunoglobulin loci, indicating post-GC phenotype. However, a minor proportion are unmutated, suggesting that they have not been exposed to the GC. MZ B cells are slightly larger than mantle zone B cells, and have paler, more-irregular nuclei. They express CD19 and CD20 and

‘‘Grey zone lymphoma’’ Increasing sophistication in immunophenotypic and genetic analysis over the past decade has resulted in the discovery of lymphomas which do not fit neatly into previously defined

DIAGNOSTIC HISTOPATHOLOGY 16:2

64

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

have the phenotype of memory B cells. They are strongly positive for IgM, IgG, or IgA, and a subpopulation exhibits weak IgD staining. They also express bcl-2, CD21, and CD27, and are negative for CD5, CD10, CD23. MZL occurs is seen in three separate diseases, extranodal or mucosa-associated lymphoid tissue (MALT) type (see also Dr Bacon’s review in this issue), splenic type (to be discussed in Dr Wilkin’s review in the March 2010 issue of this journal) and nodal type.40 The appearances and phenotype when of MZL in the lymph node are similar, so the clinical context is important to allow assignment to the correct MZL category.

can resemble FL, though CD10 and bcl-6 staining will be negative in cases of follicular colonization. Furthermore such follicles will be bcl-2 negative and will have a high Ki67 index. 4 Mantle cell lymphoma can have a marginal zone pattern surrounding reactive follicular centres, though the cells in MCL are more centrocytic, lacking monocytoid features and without the accompanying large cells seen in MZL. Finally, cyclin D1 staining is useful to distinguish between them. 5 CLL/SLL is rarely confused with MZL, though approximately 10% of cases of SLL have an interfollicular pattern, surrounding reactive follicles. CD23 and CD5, both positive in CLL/SLL and negative in MZL, are helpful in making this distinction. 6 Lymphoplasmacytic lymphoma. Cases showing extensive plasmacytic differentiation can be difficult to distinguish from LPL. No reliable morphological discriminators have been identified, though clinical features, such as the presence of significant lymphadenopathy and low serum paraprotein levels point towards a diagnosis of MZL. However, as there is little difference between the treatments of the two, this distinction is perhaps not critical.

Nodal marginal zone lymphoma Clinical: Nodal MZL accounts for only 1.5e2% of all lymphomas, with similar age distribution to extranodal MZL, most cases occurring in adults with a median age of 60 years. Morphology: MZL is typified by a heterogeneous mix of small B cells, including marginal zone, centrocyte-like cells, monocytoid cells, small lymphocytes and scattered immunoblasts and centroblast-like cells; plasma cell differentiation is present in (an) approximately 30% of cases. The tumour cells infiltrate lymph nodes in a perisinusoidal and interfollicular pattern, surrounding reactive follicles and expanding into interfollicular areas; they may also show follicular colonization. In some cases there may be a diffuse pattern and immunostaining for FDC may be needed to detect follicular remnants. Some cases have frequent large transformed cells, but a proliferation index <40%.

Lymphoplasmacytic lymphoma (LPL) Clinical LPL is a neoplasm of small B lymphocytes, plasmacytoid lymphocytes and plasma cells, usually involving the bone marrow, often with splenomegaly, and sometimes also involving lymph nodes. It is often associated with an IgM paraproteinaemia. LPL occurs in older adults with a median age in the 60s. It is a diagnosis reached after all other lymphomas with plasmacytic differentiation have been excluded.

Immunophenotype: the cells have the phenotype of marginal zone B cells and are positive for pan-B markers (CD20, CD79a), and bcl-2, with variable expression of CD138 depending on the degree of plasma cell differentiation; light chain restriction is usually present, predominantly of kappa chains. They typically express IgM, and less often IgA or IgG. They are negative for CD5, CD10, CD23 and bcl-2. CD21 highlights disrupted networks of FDCs.41

Morphology It is postulated to arise from a post-follicular B cell early in the plasma cell pathway. In lymph nodes it produces paracortical infiltrates with a sinusoidal pattern, which can be subtle in early cases, though advanced disease often produces diffuse effacement of nodal architecture. Consequent upon origin from a plasma cell precursor, it is characterized by a mixture ranging from small lymphocytes, through plasmacytoid cells, with features intermediate between plasma cells and lymphocytes, to mature plasma cells, though transformed plasmablasts may also be present. Often the plasma cells are aggregated close to blood vessels whilst the lymphocytes and plasmacytoid cells are more dispersed. Dutcher and Russell bodies are common and crystalline Ig deposits may also be seen in macrophages. There is often an accompanying reactive infiltrate including mast cells and macrophages sometimes forming granulomas.

Cytogenetics and molecular genetics: immunoglobulin heavy and light chains are rearranged and show somatic hypermutation, consistent with post-GC derivation. Trisomies 3, 7 and 18 have been reported but the chromosomal translocations associated with extranodal MZL lymphoma of MALT type are not seen. Prognostic markers: morphological, immunohistochemical or genetic prognostic markers are lacking, though the FLIPI index is predictive of survival. Morphological differential diagnoses

1 Reactive lymphoid hyperplasia may contain aggregates of reactive monocytoid cells in sinusoids, which can resemble MZL. MZL usually shows destructive spread outside the sinusoids, together with large sheets of monocytoid cells, irregular monocytoid and large transformed cells. It also shows clonal rearrangement of the Ig genes and the infiltrating cells are bcl-2 positive. 2 Extranodal MZL of MALT or MZL of spleen. Nodal involvement by extranodal MZL of MALT or MZL of spleen needs to be considered in all cases. 3 Follicular lymphoma. Cases in which there is follicular colonization by the neoplastic cells, with infiltration of the GCs,

DIAGNOSTIC HISTOPATHOLOGY 16:2

Immunophenotype The cells are positive for pan-B markers (PAX5, CD19, CD20, CD79a), for bcl-2 and variably for CD38 and CD25. They are SIgþ, usually IgMþ and IgDþ but rarely IgGþ or IgAþ. Therefore Bcl-6, CD5, CD10, CD103, CD23 and CD138 are negative, though CD138 is positive in the plasma cells (Figure 9). Cytogenetics and molecular genetics Ig genes are rearranged, usually with somatic hypermutation, though without ongoing mutations, consistent with a post-GC origin. There are no specific chromosomal, or molecular abnormalities

65

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

Lymphoplasmacytic lymphoma (LPL) is a mixture of small CD20 and CD79a positive B lymphocytes and CD138 positive plasma cells. Figure 9

typical for LPL, though trisomies 3, 4 and 18 have been infrequently reported and some cases have a deletion of 6q.42,43

markers. Advanced age, cytopaenias, especially anaemia, and high beta-2 microglobulin levels are unfavourable features.44,45 Cases with large numbers of transformed cells also have worse survival, though formal grading of this has not been done, whilst cases with deletion of 6q have been associated with other adverse prognostic features.46

Prognostic markers Morphological and molecular prognostic markers are lacking, though there are several clinical and laboratory prognostic

Immunophenotype of tumour cells in nodal B-cell lymphoma Tumour ALL Mantle cell CLL/SLL FL NLPHL DLBCL gc DLBCL abc cHL Plasmablastic MZL

CD20  þ þ þ þ þ þ   þ

CD79a þ þ þ þ þ þ þ   þ

PAX5 þ þ þ þ þ þ þ þ  þ

OCT-2/BOB-1 n/a n/a n/a þ þ þ þ  þ þ

CD10 þ   þ  þ/    

BCL-6 n/a   þ þ þ þ/   

MUM1       þ þ þ þ

CD138       þ  þ þ

CD5  þ þ   /þ /þ   

CD23   þ /þ  

Cyclin D1  þ    

  

  

TdT þ          

þ/ Majority of cases positive (see text). /þ Some cases are positive.

Table 1

DIAGNOSTIC HISTOPATHOLOGY 16:2

66

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

13 Samaha H, Dumontet C, Ketterer N, et al. Mantle cell lymphoma: a retrospective study of 121 cases. Leukemia 1998; 12: 1281e7. 14 Ott G, Katzenberger T, Lohr A, et al. Cytomorphologic, immunohistochemical, and cytogenetic profiles of follicular lymphoma: 2 types of follicular lymphoma grade 3. Blood 2002; 99: 3806e12. 15 Dave SS, Wright G, Tan B, et al. Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells. N Engl J Med 2004; 351: 2159e69. 16 Vitolo U, Ferreri AJ, Montoto S. Follicular lymphomas. Crit Rev Oncol Hematol 2008; 66: 248e61. 17 Tibiletti MG, Martin V, Bernasconi B, et al. BCL2, BCL6, MYC, MALT 1, and BCL10 rearrangements in nodal diffuse large B-cell lymphomas: a multicenter evaluation of a new set of fluorescent in situ hybridization probes and correlation with clinical outcome. Hum Pathol 2009; 40: 645e52. 18 Rossi D, Gaidano G. Molecular heterogeneity of diffuse large B-cell lymphoma: implications for disease management and prognosis. Hematology 2002; 7: 239e52. 19 Lossos IS, Alizadeh AA, Eisen MB, et al. Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas. Proc Natl Acad Sci U S A 2000; 97: 10209e13. 20 Raphael M, Gentilhomme O, Tulliez M, Byron PA, Diebold J. Histopathologic features of high-grade non-Hodgkin’s lymphomas in acquired immunodeficiency syndrome. The French Study Group of Pathology for Human Immunodeficiency Virus-Associated Tumors. Arch Pathol Lab Med 1991; 115: 15e20. 21 Wright DH. Burkitt’s lymphoma: a review of the pathology, immunology, and possible etiologic factors. Pathol Annu 1971; 6: 337e63. 22 Song KW, Barnett MJ, Gascoyne RD, et al. Haematopoietic stem cell transplantation as primary therapy of sporadic adult Burkitt lymphoma. Br J Haematol 2006; 133: 634e7. 23 Mwanda OW. Clinical characteristics of Burkitt’s lymphoma seen in Kenyan patients. East Afr Med J 2004: S78e89. 24 Mwanda OW. Aspects of epidemiological and clinical features of patients with central nervous system Burkitt’s lymphoma in Kenya. East Afr Med J 2004: S97e103. 25 Dave SS, Fu K, Wright GW, et al. Molecular diagnosis of Burkitt’s lymphoma. N Engl J Med 2006; 354: 2431e42. 26 Aldoss IT, Weisenburger DD, Fu K, et al. Adult Burkitt lymphoma: advances in diagnosis and treatment. Oncology (Williston Park) 2008; 22: 1508e17. 27 Choi MK, Jun HJ, Lee SY, et al. Treatment outcome of adult patients with Burkitt lymphoma: results using the LMB protocol in Korea. Ann Hematol 2009. 28 Anagnostopoulos I, Hansmann ML, Franssila K, et al. European Task Force on Lymphoma project on lymphocyte predominance Hodgkin disease: histologic and immunohistologic analysis of submitted cases reveals 2 types of Hodgkin disease with a nodular growth pattern and abundant lymphocytes. Blood 2000; 96: 1889e99. 29 de Jong D, Bosq J, MacLennan KA, et al. Lymphocyte-rich classical Hodgkin lymphoma (LRCHL): clinico-pathological characteristics and outcome of a rare entity. Ann Oncol 2006; 17: 141e5. 30 Nam-Cha SH, Montes-Moreno S, Salcedo MT, Sanjuan J, Garcia JF, Piris MA. Lymphocyte-rich classical Hodgkin’s lymphoma: distinctive tumor and microenvironment markers. Mod Pathol 2009. 31 Boudova L, Torlakovic E, Delabie J, et al. Nodular lymphocytepredominant Hodgkin lymphoma with nodules resembling T-cell/histiocyte-rich B-cell lymphoma: differential diagnosis between

Morphological differential diagnoses

1 CLL/SLL can show plasmacytoid differentiation, though these are CD5 and CD23 positive, whilst LPL is negative for both. 2 Follicular lymphoma can show plasmacytoid differentiation, but such cases are CD10 and bcl-6 positive, whilst FISH may demonstrate presence of a t(14;18) translocation. 3 Plasma cell myeloma. The small cell variant of myeloma may be difficult to distinguish histologically from LPL. The clinical context is important and most cases of plasma cell myeloma are composed of larger cells and secrete IgG or IgA and only rarely IgM. 4 Marginal zone B-cell lymphoma (see above) (Table 1).

Summary The classification of lymphomas is complex. Because the tumour remains with the patient for many years, the correct diagnosis is essential for optimum clinical management. Fortunately, as we understand more about the molecular pathways in lymphoid cells, we can categorize the tumours more accurately. It is essential therefore both to have access to all the diagnostic modalities and to understand their relative contributions to the diagnosis and classification of lymphomas. A

REFERENCES 1 Swerdlow SH, Campo E, Harris NL, et al., eds. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: IARC, 2008. 2 Parker ABB, Devereux S, Gatter G, et al. Best practice in lymphoma diagnosis and reporting. London: British Committee for Standards in Haematology with Royal College of Pathologists, 2008. 3 Morilla A, Gonzalez de Castro D, Del Giudice I, et al. Combinations of ZAP-70, CD38 and IGHV mutational status as predictors of time to first treatment in CLL. Leuk Lymphoma 2008; 49: 2108e15. 4 Ghia P, Caligaris-Cappio F. The origin of B-cell chronic lymphocytic leukemia. Semin Oncol 2006; 33: 150e6. 5 Moreno C, Montserrat E. New prognostic markers in chronic lymphocytic leukemia. Blood Rev 2008; 22: 211e9. 6 Wiestner A, Rosenwald A, Barry TS, et al. ZAP-70 expression identifies a chronic lymphocytic leukemia subtype with unmutated immunoglobulin genes, inferior clinical outcome, and distinct gene expression profile. Blood 2003; 101: 4944e51. 7 Quintero-Rivera F, Nooraie F, Rao PN. Frequency of 50 IGH deletions in B-cell chronic lymphocytic leukemia. Cancer Genet Cytogenet 2009; 190: 33e9. 8 Mehes G. Chromosome abnormalities with prognostic impact in Bcell chronic lymphocytic leukemia. Pathol Oncol Res 2005; 11: 205e10. 9 Bea S, Ribas M, Hernandez JM, et al. Increased number of chromosomal imbalances and high-level DNA amplifications in mantle cell lymphoma are associated with blastoid variants. Blood 1999; 93: 4365e74. 10 Wlodarska I, Dierickx D, Vanhentenrijk V, et al. Translocations targeting CCND2, CCND3, and MYCN do occur in t(11;14)-negative mantle cell lymphomas. Blood 2008; 111: 5683e90. 11 Schraders M, Oeschger S, Kluin PM, et al. Hypermutation in mantle cell lymphoma does not indicate a clinical or biological subentity. Mod Pathol 2009; 22: 416e25. 12 Hartmann EM, Ott G, Rosenwald A. Molecular outcome prediction in mantle cell lymphoma. Future Oncol 2009; 5: 63e73.

DIAGNOSTIC HISTOPATHOLOGY 16:2

67

Ó 2009 Elsevier Ltd. All rights reserved.

MINI-SYMPOSIUM: HAEMATOPATHOLOGY UPDATE I

32

33 34

35

36 37 38

39

40 Mollejo M, Camacho FI, Algara P, Ruiz-Ballesteros E, Garcia JF, Piris MA. Nodal and splenic marginal zone B cell lymphomas. Hematol Oncol 2005; 23: 108e18. 41 Arcaini L, Lucioni M, Boveri E, Paulli M. Nodal marginal zone lymphoma: current knowledge and future directions of an heterogeneous disease. Eur J Haematol 2009. 42 Wong KF, So CC, Chan JC, Kho BC, Chan JK. Gain of chromosome 3/3q in B-cell chronic lymphoproliferative disorder is associated with plasmacytoid differentiation with or without IgM overproduction. Cancer Genet Cytogenet 2002; 136: 82e5. 43 Mansoor A, Medeiros LJ, Weber DM, et al. Cytogenetic findings in lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia. Chromosomal abnormalities are associated with the polymorphous subtype and an aggressive clinical course. Am J Clin Pathol 2001; 116: 543e9. 44 Garcia-Sanz R, Montoto S, Torrequebrada A, et al. Waldenstrom macroglobulinaemia: presenting features and outcome in a series with 217 cases. Br J Haematol 2001; 115: 575e82. 45 Dhodapkar MV, Jacobson JL, Gertz MA, et al. Prognostic factors and response to fludarabine therapy in patients with Waldenstrom macroglobulinemia: results of United States intergroup trial (Southwest Oncology Group S9003). Blood 2001; 98: 41e8. 46 Ocio EM, Schop RF, Gonzalez B, et al. 6q Deletion in Waldenstrom macroglobulinemia is associated with features of adverse prognosis. Br J Haematol 2007; 136: 80e6.

nodular lymphocyte-predominant Hodgkin lymphoma and T-cell/histiocyte-rich B-cell lymphoma. Blood 2003; 102: 3753e8. Uherova P, Valdez R, Ross CW, Schnitzer B, Finn WG. Nodular lymphocyte predominant Hodgkin lymphoma. An immunophenotypic reappraisal based on a single-institution experience. Am J Clin Pathol 2003; 119: 192e8. Lee AI, Lacasce AS. Nodular lymphocyte predominant Hodgkin lymphoma. Oncologist 2009. Nam-Cha SH, Roncador G, Sanchez-Verde L, et al. PD-1, a follicular Tcell marker useful for recognizing nodular lymphocyte-predominant Hodgkin lymphoma. Am J Surg Pathol 2008; 32: 1252e7. Carbone A, Gloghini A, Cabras A, Elia G. Differentiating germinal center-derived lymphomas through their cellular microenvironment. Am J Hematol 2009; 84: 435e8. Nakatsuka S, Aozasa K. Epidemiology and pathologic features of Hodgkin lymphoma. Int J Hematol 2006; 83: 391e7. Ashton-Key M, Thorpe PA, Allen JP, Isaacson PG. Follicular Hodgkin’s disease. Am J Surg Pathol 1995; 19: 1294e9. Schmitz R, Stanelle J, Hansmann ML, Kuppers R. Pathogenesis of classical and lymphocyte-predominant Hodgkin lymphoma. Annu Rev Pathol 2009; 4: 151e74. Hartmann S, Martin-Subero JI, Gesk S, et al. Detection of genomic imbalances in microdissected Hodgkin and ReedeSternberg cells of classical Hodgkin’s lymphoma by array-based comparative genomic hybridization. Haematologica 2008; 93: 1318e26.

DIAGNOSTIC HISTOPATHOLOGY 16:2

68

Ó 2009 Elsevier Ltd. All rights reserved.