Common misdiagnoses in lymphomas and avoidance strategies

Review

Common misdiagnoses in lymphomas and avoidance strategies John K C Chan, Yok-Lam Kwong

Lymphoma diagnosis integrates clinical, morphological, immunophenotypical, and molecular genetic features, as shown in WHO classifications of lymphoid malignancies. Diagnosis of lymphoma is challenging. Reactive lesions such as Kikuchi lymphadenitis, infectious mononucleosis, autoimmune lymphoproliferative syndrome, and immunoglobulin G4-related sclerosing disease can be misdiagnosed as lymphomas. Anaplastic large-cell lymphoma variants that are positive for anaplastic lymphoma kinase, classical Hodgkin’s lymphoma variants, and infarcted lymphomas might be misdiagnosed as reactive disorders. Difficulties with classification of lymphomas are also encountered, such as the distinction of classical Hodgkin’s lymphoma from anaplastic large-cell lymphoma that is negative for anaplastic lymphoma kinase. Interpretation of immunophenotyping results is complicated in some cases by aberrant or crosslineage expression of lymphoid antigens on lymphomas, and the occasional lymphoid antigen expression on nonlymphoid malignancies. Molecular analysis can help to define clonality and lineage, but can be affected by the sensitivity and specificity of tests and cross-lineage gene rearrangement and pseudoclonality. To resolve these issues, a close collaboration between the clinician, histopathologist, and molecular biologist is needed. The aim of this review is to provide pathologists and clinicians with a concise account of these pitfalls and avoidance strategies.

Introduction Diagnosis of lymphoma is an evolving science. Initially based on morphology alone, classification of lymphomas has progressed to include lymphoid lineages, cell types, and clinical features. In WHO classifications, lymphomas are categorised on the basis of morphological, immunophenotypical, genetic, and clinical characteristics. An accurate diagnosis, apart from being pathologically important, provides useful guidance to clinicians for effective treatment. Lymphoma diagnosis, however, is one of the most complicated tasks in histopathology. Importantly, reactive lymphoid disorders need to be distinguished from truly neoplastic lesions, because the exact classification greatly affects patient treatment. Lymphoma classification is not always concordant between histopathologists. For common B-cell lymphomas, the concordance rate between community and specialised lymphoma pathologists is about 95%.1 However, for more difficult subtypes of lymphomas, such as Burkitt’s lymphoma and peripheral T-cell lymphoma, concordance based on morphology could be lower than 50%, although with immunophenotyping this rate might increase to 60–80%.2 Thus, 5% of patients with common B-cell lymphomas and up to 20–40% of patients with less common lymphoma types might be inaccurately diagnosed. Lymphoma diagnosis is based on morphological examination complemented by immunophenotyping and molecular genetic investigations. However, even pathologists with much experience can encounter difficulties in each of these steps.

Reactive lesions often misdiagnosed as lymphoma Kikuchi lymphadenitis Some reactive lymphoid proliferations exhibit a striking increase in large lymphoid cells, and when accompanied by distortion of tissue architecture, can strongly mimic www.thelancet.com/oncology Vol 11 June 2010

lymphoma (panel 1). Kikuchi lymphadenitis is a selflimiting disorder, often affecting young women. It is of unknown cause and more prevalent in Asians than in white people.3 Patients usually present with enlarged lymph nodes in the neck, but are otherwise well. Typical laboratory findings are an increased ESR, lymphocytosis, and neutropenia. Kikuchi lymphadenitis is not infrequently misdiagnosed as lymphoma, because of the presence of many histiocytes and activated T cells in lymph nodes. Morphological features favouring this diagnosis over that of lymphoma are the patchy distribution and non-expansile quality of the lesional areas, abundance of karyorrhectic debris, and presence of admixed medium-sized cells with round nuclei (plasmacytoid dendritic cells).3,4 In doubtful cases, immunohistochemical studies are helpful. In addition to the presence of large T cells (CD3-positive) in the lesion, there are many CD68-positive histiocytes coexpressing myeloperoxidase, and also many CD68-positive CD123positive plasmacytoid dendritic cells.4,5

Lancet Oncol 2010; 11: 579–88 Published Online March 15, 2010 DOI:10.1016/S14702045(09)70351-1 Department of Pathology, Queen Elizabeth Hospital, Hong Kong, China (J K C Chan FRCPath); and Department of Medicine, Queen Mary Hospital, Hong Kong, China (Y-L Kwong FRCPath) Correspondence to: Dr Yok-Lam Kwong, Department of Medicine, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong, Special Administrative Region, China [email protected]

Infectious mononucleosis and other causes of immunoblastic proliferation Reactive immunoblastic proliferation in lymph nodes and other lymphoid tissues can be misdiagnosed as malignant lymphoma because of the presence of a number of large activated lymphoid cells (figure 1).4 The prototype is infectious mononucleosis, a self-limiting lymphoid proliferation related to primary infection with the EpsteinBarr virus. Other common causes are viral infections (such as herpes), reaction to vaccines, and hypersensitivity (including drug reactions). Features suggesting a reactive process include: young patient age (for infectious mononucleosis); presence of some residual normal lymphoid architecture; absence of substantial atypia in large cells; and polymorphous appearance of large cells, with apparent maturation to 579

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Panel 1: Clinical clues suggestive of an incorrect diagnosis of lymphoma • A diagnosis of peripheral T-cell lymphoma not otherwise specified in an otherwise healthy young woman with localised lymphadenopathy is probably incorrect. Seriously consider Kikuchi lymphadenitis, especially if the ESR is raised, neutropenia is present, and lactate dehydrogenase concentrations are not increased. • A diagnosis of peripheral T-cell lymphoma not otherwise specified in a child or teenager with extensive lymphadenopathy is most likely to be wrong, because this lymphoma type is very uncommon in this age-group. Autoimmune lymphoproliferative syndrome should be seriously considered. • A lymph node from a child or young patient showing combined peripheral T-cell lymphoma and Rosai-Dorfman disease almost certainly represents autoimmune lymphoproliferative syndrome. • A diagnosis of diffuse large B-cell lymphoma made on the basis of a tonsillar specimen from a young patient is often incorrect. A probable diagnosis is infectious mononucleosis—review of the histological materials and serological studies can assist with diagnosis. • A diagnosis of peripheral T-cell lymphoma not otherwise specified or angioimmunoblastic T-cell lymphoma is probably incorrect in a patient with multiple sites of lymphadenopathy, but no fever, normal or slightly raised serum lactate dehydrogenase concentrations, and presence of lacrimal or salivary gland enlargement. A more probable diagnosis is IgG4-related lymphadenopathy.

plasmablasts and plasma cells.4,6 With immunostaining, if the large cells are mostly B cells, a polytypic pattern of immunoglobulin staining is reassuring for a reactive process. Presence of a mixture of large B and T cells also lends support to a reactive process. When the large cells are almost all T cells, molecular analysis might be needed to establish clonality.

Autoimmune lymphoproliferative syndrome This syndrome is an immune disease resulting from non-functional or dysfunctional apoptosis of lymphocytes. Most cases are due to inherited germline mutations in the FAS, FASL, or CASPASE genes, with the rare sporadic form resulting from somatic heterozygous mutation in the FAS gene.7–9 Because of impaired apoptosis, lymphoid cells that have proliferated in response to antigens or infections persist after the challenge is over, causing persistent enlargement of the reticuloendothelial system, manifesting as generalised lymphadenopathy and hepatosplenomegaly. Autoimmune cytopenias are common. Susceptibility to infection is not increased, but risk for lymphoma is raised. Typically, the blood has increased circulating CD4-negative-CD8negative (double negative) αβ T cells. Lymph-node pathology is especially alarming, superficially resembling a malignant tumour. Pronounced paracortical expansion by numerous immunoblasts and medium-sized clear cells strongly mimics peripheral T-cell lymphoma not otherwise specified (figure 2).10 A substantial proportion of lymphoid cells are CD4-negative CD8-negative αβ T cells expressing CD57. A presumptive diagnosis can be made by identification of a substantial population of these cells in the tissues or peripheral blood, 580

and a definitive diagnosis by identification of mutation in the relevant apoptosis genes.7 A correct diagnosis needs a high index of suspicion: children with lymph-node biopsy samples showing features suggestive of peripheral T-cell lymphoma not otherwise specified should be regarded as having autoimmune lymphoproliferative syndrome until proven otherwise. Diagnostic likelihood is further heightened if focal areas of the lymph node have features of Rosai-Dorfman disease.11

IgG4-related sclerosing disease IgG4-related sclerosing disease is a recognised clinicopathological syndrome with a favourable response to steroid therapy.12 Developing in middle-aged and elderly patients and with a predominance in men, it is characterised by mass lesions usually affecting exocrine glands and extranodal sites. Common laboratory findings include raised serum IgG4 concentrations (IgG4 being the least common IgG subclass, normally constituting 3–6% of the IgG fraction), and circulating autoantibodies. Pathologically, lesions show lymphoplasmacytic infiltration, sclerosis, phlebitis, and increased IgG4-positive plasma cells.12,13 Although autoimmune pancreatitis often develops, other anatomical sites can be affected, such as the hepatobiliary tree, lacrimal glands, salivary glands, lymph nodes, lung, kidneys, retroperitoneum, aorta, mediastinum, breast, and the CNS. Often, two or more sites are affected.12–15 IgG4-related sclerosing disease in extranodal sites usually seems to be inflammatory. Rare cases can consist of dense infiltrates of small lymphoid and plasma cells, mimicking extranodal marginal-zone lymphoma.13 IgG4related lymphadenopathy is common and often affects many sites, especially mediastinal, intra-abdominal, and axillary regions.16,17 Most cases have reactive follicular hyperplasia and plasmacytosis, mimicking multicentric Castleman disease. However, some cases show pronounced paracortical expansion by a mixture of lymphoid and plasma cells, mimicking angioimmunoblastic T-cell lymphoma or peripheral T-cell lymphoma not otherwise specified.16,17 Disease awareness, appropriate clinical features, and identification of large numbers of IgG4-positive cells will help to establish the diagnosis.

Malignant lymphoma often misdiagnosed as reactive disorders Reasons that some lymphomas can mimic reactive lesions include: subtle cytological atypia and absence of cellular monotony, as in a small-cell variant of anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL) and extranodal marginal zone lymphoma; paucity of neoplastic cells or abundance of inflammatory cells, as in lymphohistiocytic and paucicellular variants of ALK-positive ALCL, lymphomatoid granulomatosis, certain variants of classical Hodgkin’s lymphoma, and infarcted lymphoma; and similarities in architecture, as in nodular sclerosis Hodgkin’s lymphoma with suppurative necrosis, which can be www.thelancet.com/oncology Vol 11 June 2010

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mistaken for cat-scratch disease and follicular lymphoma, which in turn can be mistaken for reactive follicular hyperplasia (panel 2).

ALK-positive anaplastic large-cell lymphoma variants This disorder is a distinctive form of T-cell lymphoma, characterised by CD30 and ALK expression.18,19 It arises predominantly in children and young adults, with a favourable outcome. In most cases, diagnosis is straightforward because of the abundance of atypical large cells. However, several variants of this disorder cause diagnostic difficulties because of the small size of the neoplastic cells (small-cell variant), abundance of admixed histiocytes and absence of overt atypia in the large neoplastic cells (lymphohistiocytic variant), and paucity of neoplastic cells (hypocellular variant) (figure 3).18–20 Morphological clues to the correct diagnosis are perivascular cuffs of large lymphoid cells, and myxoidoedematous granulation tissue-like stroma in the lymph node.20,21 Judicious application of antibodies against CD30 and ALK in all unusual or unexplained lymphoid proliferations in young patients will also keep to a minimum the possibility of missing ALCL (figure 3).

Figure 1: Infectious mononucleosis affecting tonsil of a teenaged patient Histologically, there is a striking increase of large lymphoid cells. Occasional cells are binucleated, mimicking Reed-Sternberg cells (arrow; original magnification 500×).

A

Classical Hodgkin’s lymphoma variants Several variants of classical Hodgkin’s lymphoma have deceptive histological features that make diagnosis difficult. Reed-Sternberg cells can be confined to monocytoid B-cell clusters, and thus can be missed.22 The fibrohistiocytic variant can be mistaken for reactive fibrohistiocytic proliferation.4 Cases accompanied by sheets of foamy cells can be mistaken for xanthogranulomatous inflammation,23 and those showing prominent suppurative necrosis for cat scratch disease.4 Awareness of these variants combined with clinical features (such as large-mass lesions) will reduce these diagnostic errors.

B

Lymph-node infarction Characterised by ghost shadows of cells without intermingled apoptotic bodies and histiocytes, lymph-node infarction is sometimes mistaken for necrotising lymphadenitis. An important cause is lymphoma, which can undergo infarction spontaneously or after fine-needle aspiration.24 Thus, scrutiny of the peripheral portion of the node for viable lymphoma cells is important. Immunohistochemistry is also helpful, because lymphoid antigens might still be preserved in the necrotic neoplastic cells, and a diagnosis of lymphoma can be strongly suspected if sheets of CD20-positive cells are present.25

Difficulties and errors in classification of lymphoma Complexities of classification Errors in classification of lymphomas are inevitable in view of the complexities of WHO classifications (webappendix). In some instances, misclassifications might be inconsequential in relation to treatment. www.thelancet.com/oncology Vol 11 June 2010

Figure 2: Autoimmune lymphoproliferative syndrome affecting lymph node of a child (A) Lymph node shows paracortical expansion. Lymphoid follicles in the cortex are shown by arrows, and the lymphoid compartment in between (paracortical zone) is substantially broadened (original magnification 50x). (B) In the expanded paracortex, infiltrate of atypical medium-sized and large cells, some with clear cytoplasm is notable. This picture is practically indistinguishable from that of peripheral T-cell lymphoma (original magnification 450×).

See Online for webappendix

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Panel 2: Clinical clues suggestive of an incorrect diagnosis of reactive lymphoid hyperplasia • A diagnosis of cat-scratch disease made on the basis of a mediastinal or supraclavicular lymph node can be incorrect—nodular sclerosis Hodgkin’s lymphoma is more likely. • A diagnosis of reactive lymphoid hyperplasia in a child or young adult with prominent lymphadenopathy, fever, and raised serum lactate dehydrogenase concentrations is most likely to be wrong. Consider anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma.

However, if aggressive lymphomas are misclassified as low-grade lymphomas, patient outcomes are jeopardised because of undertreatment. In poorly fixed tissues, thickly-cut sections, or core needle-biopsy materials, neoplastic cells of high-grade lymphomas, such as lymphoblastic lymphoma, Burkitt’s lymphoma, and diffuse large B-cell lymphoma, in some cases assume a shrunken appearance, mimicking small-cell lymphomas. In such materials, immunostaining for Ki67, a proliferation marker, is very helpful because a high proliferative fraction (>50%) is incompatible with a diagnosis of small cell (low-grade) lymphoma.

Classical Hodgkin’s lymphoma versus ALK-negative anaplastic large-cell lymphoma To distinguish between tumour-cell-rich classical Hodgkin’s lymphoma and ALK-negative ALCL can be difficult, because both entities are characterised by abundant large neoplastic cells with CD30 expression, but do not have expression of conventional B-cell (CD20 or CD79a) and T-cell (CD3) lineage markers, or ALK. Classical Hodgkin’s lymphoma tends to have more prominent coagulative necrosis and eosinophilic infiltration than does ALK-negative ALCL, and diagnosis can be confirmed by positive staining for the B-cell transcription factor Pax-5 in the large cells.26 ALK-negative anaplastic large-cell lymphoma is favoured if Pax-5 is negative, especially when other T-lineage markers (such as CD2, CD4, CD5, and CD7) are positive.

Primary mediastinal large B-cell lymphoma versus classical Hodgkin’s lymphoma Primary mediastinal large B-cell lymphoma, a distinctive form of B-cell neoplasm thought to arise from thymic medullary B cells, affects mainly young adults, with a female predominance, commonly presenting with localised bulky tumours.27 It is characterised histologically by sheets of medium-to-large lymphoma cells, some of which might resemble Reed-Sternberg cells, and frequently sclerosis. Immunophenotypical characteristics are a B-cell phenotype without Ig expression, and frequent expression of CD30 (usually moderate to weak and heterogeneous), CD23, and MAL. Although mediastinal large B-cell lymphoma and Hodgkin’s lymphoma are distinct entities, they can develop in a composite form, or sequentially. This finding is not surprising, in view of the fact that both 582

lymphoma types have more similarities in gene expression profile than they do with conventional diffuse large B-cell lymphoma.28,29 Some cases have overlapping features of the two, which are termed “B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin’s lymphoma”30 in the latest WHO classification. These grey zone lymphomas are aggressive, with a worse outcome than either lymphoma type.31 Morphologically, confluent sheets of pleomorphic cells are usually present, many of which resemble lacunar cells or Reed-Sternberg cells. Necrosis is common. The immunophenotype is intermediate between diffuse large B-cell lymphoma and classical Hodgkin’s lymphoma, such as uniform strong expression of CD20 together with CD30 and CD15, or a discrepancy between morphology and immunophenotype, such as diffuse large B-cell lymphomalike features but with a CD20-negative CD15-positive Epstein-Barr virus positive phenotype, or Hodgkin’s lymphoma-like characteristics but with a CD20-positive CD15-negative phenotype.

Lymphoplasmacytic lymphoma versus marginal zone lymphoma Lymphoplasmacytic lymphoma is a neoplasm consisting of small lymphocytes, plasmacytoid lymphocytes, and plasma cells, usually but not necessarily associated with IgM paraproteinaemia and Waldenstrom macroglobulinaemia.32 Diagnosis needs exclusion of other lymphoma types showing plasmacytic differentiation, especially marginal zone lymphoma. Not unexpectedly, this lymphoma type shows a low reproducibility in diagnosis (56%) in the Non-Hodgkin Lymphoma Classification Project.2 Because no specific molecular markers are known, clinical and laboratory findings should be taken into consideration in formulation of the final diagnosis. However, in some cases, a clear-cut distinction between lymphoplasmacytic lymphoma and marginal zone lymphoma cannot be made.

Plasmablastic lymphoma versus anaplastic plasmacytoma Plasmablastic lymphoma is a large B-cell lymphoma in which the neoplastic cells exhibit plasmablastic differentiation.33 It is typically CD20-negative and thus unlikely to benefit from rituximab therapy, and is immunoreactive for various plasma cell-associated markers such as MUM1 and CD138. Although originally described as a distinctive form of lymphoma affecting the oral cavity in patients with acquired immunodeficiency syndrome,34 plasmablastic lymphoma is now recognised to also affect non-immunocompromised patients in both nodal and extranodal sites. ALK-positive large B-cell lymphoma and primary effusion lymphoma also exhibit plasmablastic differentiation, but are not classified as plasmablastic lymphoma. Much morphological and immunophenotypical overlap arises between plasmablastic lymphoma and anaplastic www.thelancet.com/oncology Vol 11 June 2010

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plasmacytoma, rendering distinction between them very difficult or impossible. Nevertheless, this difference is clinically significant, because plasmablastic lymphoma has an unfavourable outcome despite aggressive therapy, whereas anaplastic plasmacytoma responds well to radiotherapy in some cases, especially in localised extramedullary disease.35 Presence of immunodeficiency, high proliferative fraction, and positivity for Epstein-Barr virus are features favouring a diagnosis of plasmablastic lymphoma, although none are specific. However, plasma cell myeloma, if identified on further work-up, would support a diagnosis of anaplastic plasmacytoma.

AA

Burkitt’s lymphoma versus diffuse large B-cell lymphoma Burkitt’s lymphoma is a very rapidly growing B-cell neoplasm, occurring often in extranodal sites in children and young adults. In the latest WHO classification, the variant of atypical Burkitt’s lymphoma has been eliminated and most cases are merged back into the main diagnostic category of Burkitt’s lymphoma.36 With present methods of morphology, immunophenotyping, and molecular studies, even experts misdiagnose some cases of Burkitt’s lymphoma as diffuse large B-cell lymphoma, and vice versa.37,38 Gene-expression profiling might better define a homogeneous group of Burkitt’s lymphomas than might other techniques, but it is not yet available for diagnostic purposes.37,38 Although Burkitt’s lymphoma usually exhibits a distinctive immunophenotype that is CD20-positive, CD10-positive, BCL6-positive, BCL2-negative, and has a Ki67 index higher than 90%, occasional cases of diffuse large B-cell lymphoma might express the same immunophenotype.36 Conversely, occasional cases of Burkitt’s lymphoma can deviate from this classical immunophenotype, with BCL2-positivity or a lower Ki67 index. Thus, molecular studies should be undertaken if the morphology or immunophenotype is not classical. Fluorescence in-situ hybridisation (FISH) to identify presence of MYC translocation (preferably confirmed to be IG-MYC), together with absence of BCL2 and BCL6 rearrangements, will support a diagnosis of Burkitt’s lymphoma, while excluding transformed follicular lymphoma or the so-called double-hit lymphoma (with simultaneous MYC and BCL2 rearrangements), which has a very poor prognosis.36,39,40 FISH is better than is PCR for detection of MYC and BCL2 gene rearrangements, because it has a much lower false-negative rate. Cases showing borderline features can be accommodated in the category “B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt’s lymphoma”30 in the WHO classification, such as cases with morphological features consistent with Burkitt’s lymphoma but immunophenotypical and molecular features that are not consistent with Burkitt’s lymphoma, or cases showing hybrid morphological features of Burkitt lymphoma and diffuse large B-cell lymphoma.41 MYC translocation is www.thelancet.com/oncology Vol 11 June 2010

BB

Figure 3: Hypocellular variant of ALK-positive anaplastic large-cell lymphoma (A) The low cellularity, granulation tissue-like appearance with myxoid to oedematous stroma, and absence of frankly atypical cells in this lymph node biopsy sample could lead to misdiagnosis of a reactive change (original magnification 400x). (B) Immunostaining for anaplastic lymphoma kinase (ALK) highlights the neoplastic cells, some of which are spindly. In this case, the staining is cytoplasmic but not nuclear, suggesting that the ALK gene is translocated with a partner gene other than the NPM gene (original magnification 200×).

common, but the partner gene is often not the IG gene, and the karyotype is complex. Most cases of double-hit lymphoma would fall into this category.42

Angioimmunoblastic T-cell lymphoma versus peripheral T-cell lymphoma not otherwise specified and atypical lymphoid hyperplasia Angioimmunoblastic T-cell lymphoma is a neoplastic proliferation of germinal centre T-helper cells.43 In some cases, it overlaps morphologically and immunophenotypically with peripheral T-cell lymphoma not otherwise specified, rendering classification difficult. Distinctive clinical features of angioimmunoblastic T-cell lymphoma are systemic disease, polyclonal B-cell activation (manifesting as raised ESR and hypergammaglobulinaemia), and subtle immunosuppression predisposing to infections, such as those from opportunistic microorganisms. This disorder can be complicated by a diffuse large B-cell lymphoma that is positive for Epstein-Barr virus.44 Morphological features favouring diagnosis of angioimmunoblastic T-cell lymphoma over peripheral T-cell 583

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lymphoma not otherwise specified include prominent arborising high endothelial venules, neoplastic cells with round rather than irregularly folded nuclei, frequent presence of clear cells, and an admixture of plasma cells and eosinophils.45 On immunohistochemical study, this disorder typically has extrafollicular proliferation of follicular dendritic cells (CD21-positive).4 Newly available markers for angioimmunoblastic T-cell lymphoma are CD10, PD-1, and CXCL-13, although they are not always positive.43 However, early angioimmunoblastic T-cell lymphoma, such as focal nodal involvement or presence of hyperplastic lymphoid follicles, can be very difficult to distinguish from reactive lymphoid hyperplasia.46,47 Expanded meshworks of follicular dendritic cells are often not present to aid in diagnosis. Identification of a substantial population of medium-sized T cells, especially if shown to be CD10-positive or CXCL-13-positive, would favour a diagnosis of angioimmunoblastic T-cell lymphoma. However, if findings are inconclusive, a designation such as atypical lymphoid hyperplasia might be appropriate, with advice for follow-up and rebiopsy.

Subcutaneous panniculitis-like T-cell lymphoma versus primary cutaneous αβ T-cell lymphoma, extranodal natural killer or T-cell lymphoma, and panniculitis Subcutaneous panniculitis-like T-cell lymphoma is a special lymphoma type usually presenting as multiple subcutaneous nodules, most frequently in the arms of legs or trunk, in the absence of other sites of disease. Neoplastic cells typically exhibit a CD8-positive cytotoxic cell phenotype. The definition of this entity has undergone changes in WHO’s classification, in that cases expressing the γδ T-cell receptor (TCR) are excluded and reclassified as primary cutaneous γδ T-cell lymphoma.48 A distinction between them is clinically significant, because the prognosis of this disorder is much better than that of primary cutaneous γδ T-cell lymphoma (5-year survival 82% vs 11%).49 Inadvertent inclusion of cutaneous γδ T-cell lymphoma in genuine cases of subcutaneous panniculitislike T-cell lymphoma led to the erroneous notion that subcutaneous panniculitis-like T-cell lymphoma was an aggressive lymphoma. Features suggestive of primary cutaneous γδ T-cell lymphoma include dermal involvement, subcutaneous involvement and CD56-positivity, and a diagnosis that can be confirmed by positive staining for TCRγδ. Extranodal natural killer (NK) or T-cell lymphoma can show prominent subcutaneous involvement, morphologically mimicking subcutaneous panniculitis-like T-cell lymphoma. Features favouring this diagnosis include presence of dermal involvement, substantial angioinvasion, a CD8-negative-CD56-positive immunophenotype, and positivity for Epstein-Barr virus. However, the distinction between subcutaneous panniculitis-like T-cell lymphoma and panniculitis (including lupus panniculitis) is not always straight584

forward. Overt cytological atypia, such as medium-sized nuclei and irregular nuclear foldings, is suggestive of lymphoma. Paucity of plasma cells, rimming of lymphoid cells around fat spaces, paucity of admixed B-cells, and a CD8-positive cytotoxic immunophenotype also favour a diagnosis of subcutaneous panniculitis-like T-cell lymphoma rather than panniculitis. However, rare cases of subcutaneous panniculitis-like T-cell lymphoma showing minimal cellular atypia are difficult if not impossible to distinguish from panniculitis with certainty, and only follow-up with repeat biopsy sampling will clarify the diagnosis, because cellular atypia often becomes more evident with evolution of the disease.

Immunophenotyping issues Difficulties in lymphoma diagnosis Immunophenotyping is important in lymphoma diagnosis, and is crucial for making treatment decisions. Although lineage assignment is often possible with flow cytometry, or immunohistochemistry with an extensive panel of antibodies; in the routine laboratory, usually a panel of fewer antibodies that identify T cells, B cells, and NK-cell lineages are applied. However, cross-lineage expression of antigens can occasionally arise, creating diagnostic challenges. This outcome has been attributed either to transformation of an early lymphoid progenitor or a normal counterpart lymphoid cell that co-expresses antigens of different lineages, or aberrant antigen expression related to the neoplastic process.

CD20 expression in non-B-cell lymphomas The B-cell antigen CD20 has important roles in B-cell activation, proliferation, and differentiation.50 Expression of CD20 in lymphomas identifies the B-cell lineage and affects treatment, because of availability of the highly effective anti-CD20 antibody, rituximab. However, aberrant expression of CD20 in non-B-cell lymphomas is occasionally encountered. More than 30 cases of CD20-positive T-cell and NK-cell lymphomas have been reported.51–54 The most frequent histological subtype is peripheral T-cell lymphoma not otherwise specified,52 followed by cutaneous peripheral T-cell lymphoma, and mycosis fungoides. Others are angioimmunoblastic T-cell lymphoma, anaplastic large-cell lymphoma, enteropathy associated T-cell lymphoma, and extranodal NK-cell or T-cell lymphoma.51,52,54

Absence of CD20 expression in B-cell lymphomas Treatment of B-cell lymphomas with rituximab has occasionally been reported55 to select CD20-negative lymphoma-cell populations in relapse, possibly occurring in up to 25% of cases. The proposed mechanisms are CD20 gene mutations that affect the antibody-binding domain, downregulation of the CD20 messenger RNA or protein by pretranscriptional and post-transcriptional mechanisms, and epigenetic suppression of CD20 gene expression.55 Furthermore, plasmablastic lymphoma, a B-cell neoplasm, is typically CD20-negative.33 www.thelancet.com/oncology Vol 11 June 2010

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CD3 expression in non-T cell lymphomas The CD3 molecule consists of four chains: δ, ε, γ,
, and binds either TCRδβ or TCRγδ to form the TCR-CD3 complex. Although CD3 is regarded as the most specific T-lineage marker, it can rarely be expressed in non-T-cell lymphomas. Pyothorax-associated lymphoma expresses CD3 in about 30% of cases, and occasionally other T-lineage antigens, such as CD2 and CD4.56 Expression of T-lineage markers in B-cell lymphomas seems to be especially frequent in the setting of immunodeficiency.57,58 Rare cases of sporadic diffuse large B-cell lymphomas have also been reported to be positive for CD3.59 NK-cell lymphomas express cytoplasmic CD3ε but not surface CD3, whereas T-cell lymphomas express both. Polyclonal antiserum or monoclonal antibodies against CD3ε (such as PS1 or LN10) that are available on formalinfixed specimens stain both T-cell and NK-cell lymphomas (cytoplasmic CD3ε). However, monoclonal anti-CD3 antibodies such as Leu4, OKT3, and UCHT1, when used in flow cytometry or applied to immunohistochemical analysis of frozen sections, detect solely surface CD3, which is present only in T-cell but not NK-cell lymphomas. For this reason, when immunophenotyping is undertaken on paraffin-fixed specimens, lymphomas of NK-cell lineage can in some cases be mistaken for T-cell lymphomas because of the CD3ε expression. This caveat accounts for the early belief that T-cell lymphomas were much more prevalent in Asian populations, constituting up to 20% of all lymphomas.60 When strict WHO diagnostic criteria are applied, peripheral T-cell lymphoma in Asian populations have been identified to constitute no more than 10% of all lymphomas, the rest being NK-cell lymphomas.61

Expression of pan-lymphoid markers on non-lymphoid malignancies The CD45 molecule (leucocyte common antigen) is expressed on all haematolymphoid cells apart from erythroid cells and megakaryocytes, and at especially high levels on lymphoid cells. Immunoreactivity for CD45 is highly indicative of a lymphoid malignancy. However, CD45 is rarely expressed in various carcinomas and sarcomas.62,63 It is essential, therefore, that immunophenotyping be interpreted in the appropriate morphological context.

Avoidance strategy When non-commensurate morphological and immunophenotypical features are encountered, a panel of markers should be used for lineage assignment. PAX-5, a transcription factor that activates B-cell specific genes and represses lineage-inappropriate genes, is a highly reliable B-lineage marker.64 The Ig-associated α protein CD79a is also a useful B-lineage marker, although it can infrequently be positive in peripheral T-cell lymphomas65 and NK-cell or T-cell lymphomas.66 Other T-cell markers (such as CD2, CD7, or LAT), CD56, or cytotoxic markers can help to establish the lineage of T-cell or NK-cell www.thelancet.com/oncology Vol 11 June 2010

lymphoma.67 To distinguish between the latter two, flow cytometry or immunophenotyping on fresh smears or cryostat sections with monoclonal anti-CD3 antibodies is most helpful, because the test is negative in NK-cell but positive in T-cell lymphomas; although such materials are often unavailable for these studies.

Difficulties in clonality assessment with gene rearrangement studies Cases needing clonality analysis During lymphocyte development, antigen receptor genes undergo rearrangement. This stepwise rearrangement of the IG or TCR gene joins V, D, and J gene segments, with nucleotides deleted or randomly inserted at the joining sites, leading to a large diversity of antigen receptors. Reactive lymphoid proliferations have polyclonally rearranged IG or TCR genes, whereas malignant lymphoid lesions show clonal rearrangement. In clinical practice, standard histopathological assessment supplemented by immunophenotyping will often result in a definite diagnosis. However, up to 10% of cases might need clonality analysis, owing to an inconclusive diagnosis, unusual histopathological or immunophenotypical features, or incongruent clinical and histopathological findings.68 PCR is most often used to study IG and TCR rearrangements. The IG heavy chain (IGH), κ (IGK) and λ (IGL) genes; and the TCR β(TCRB), γ (TCRG), and δ (TCRD) genes are valid PCR targets. However, difficulties might arise with use of these molecular markers for lymphoma lineage assignment.

False-negative PCR for IG gene clonal rearrangement in B-cell lymphomas PCR for IGH generally uses consensus VH and JH primers, or family-specific VH and consensus JH primers. However, failed primer annealing due to imprecise primer binding to all potential V and J segments, and somatic hypermutation in the IGH gene in germinal centre and post-germinal centre lymphomas69 could lead to falsenegative PCR. Thus, detection rates of clonal IGH rearrangement range from less than 85% in diffuse large B-cell lymphoma and follicular lymphoma (germinal centre lymphoma) to less than 94% in marginal zone B-cell lymphoma (post-germinal centre lymphoma).70 With combination PCR for IGH, IGK, and IGL genes, almost all mature B-cell neoplasms can be shown to have clonally rearranged IG genes.70

False-negative PCR for TCR-gene clonal rearrangement in T-cell lymphomas PCR for the TCR genes generally targets TCRD, TCRG, and TCRB genes. The complex structure of the TCRα (TCRA) gene precludes its routine detection by PCR. TCR gene rearrangement takes place in a hierarchical mode, with TCRD rearranging first, followed by TCRG (generating TCRγδ), TCRB, and TCRA (generating TCRαβ). However, almost all T cells expressing TCRαβ 585

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Search strategy and selection criteria We searched PubMed from January, 1989, until October, 2009, for reports in English with the keywords “lymphoma”, “T-cell”, “B-cell”, “natural killer cell”, “Hodgkin”, “immunophenotyping”, and “molecular analysis”. We also searched the reference lists of selected reports so that all relevant articles could be reviewed. Some articles before 1989 were included if they were pertinent.

have rearranged TCRG, and most T-cells expressing TCRγδ have rearranged TCRB. In the routine laboratory, PCR for TCRG is most often done, because primer design is easier with the restricted TCRG germline repertoire.71 However, TCRG gene rearrangement is not able to be identified in at least 25% of anaplastic large-cell lymphoma, and 4–8% of other mature T-cell lymphomas.71 With combination PCR for TCRG, TCRD, and TCRB, clonal TCR rearrangement can be shown in almost all mature T-cell lymphomas, although 20% of anaplastic large-cell lymphoma remains negative.71 Whether this outcome is attributable to technical limitations or a genuine absence of TCR rearrangement is unknown.

Cross-lineage gene rearrangement Use of gene rearrangement studies for lymphoma lineage assignment is confounded by the occurence of crosslineage gene rearrangement. In genuine B-cell lymphomas, clonal TCR rearrangement is identified in 13–50% of cases, dependent on the histological subtypes examined.70 This finding might be due to the presence of a small population of T cells with a restricted repertoire, the true presence of a clonal T-cell population, or crosslineage gene rearrangement. The clue to the first two situations is the presence of only weak clonal PCR products, and rearrangement at two or more loci. Crosslineage gene rearrangement is often shown as a strong predominant PCR product with rearrangement at a single locus. Similarly, when PCR for IG genes is undertaken for genuine T-cell lymphomas, apparent clonal patterns are identified in more than 30% of angioimmunoblastic T-cell lymphoma, and 4–9% of other mature T-cell lymphomas.71 The explanation is similar to TCR gene rearrangement in B-cell lymphomas. The high frequency of IG gene rearrangement in angioimmunoblastic T-cell lymphoma could in part be related to the presence of B cells infected with Epstein-Barr virus that might be oligoclonal or truly clonal. These findings suggest that gene rearrangement results cannot be used solely for lymphoma lineage designation.

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a misdiagnosis has occurred, or a minor lymphoma population has been missed. In reactive lymphoid proliferations in response to infections (Epstein-Barr virus, cytomegalovirus, or HIV) or other inflammatory process (sarcoidosis, Hashimoto thyroiditis, or Sjögren’s syndrome), an oligoclonal or seemingly monoclonal pattern might rarely be observed, owing to chronic antigen-driven selection of populations of B and T cells.72 In these disorders, PCR products are usually weak and present in a heavy polyclonal background. An apparent dominant clonal pattern (pseudoclonality) might occasionally be observed in reactive lesions, because of restricted template availability. In small biopsy samples, a pseudoclonal pattern can be observed in some cases in PCR for IGH if the lymphoid infiltrate consists of a small number of B-cells.73 Similarly, because the TCRD gene has a small germline repertoire, a pseudoclonal pattern can result from use of a small tissue sample.72 Some lymphoid tissues can also create difficulties in diagnosis. A normal germinal centre can consist of 1–5 B-cell clones.74 PCR of the IG gene of a single germinal centre might therefore yield an oligoclonal to a monoclonal pattern. This outcome might cause diagnostic difficulties when small biopsy samples, such as from the gastrointestinal tract, are studied.72

Avoidance strategy Interpretation of clonality study results might need collaboration between the clinician, histopathologist and molecular biologist. Use of a comprehensive panel of PCR primers is preferable to reduce rates of false negatives. Clinicians have to be aware of cross-lineage gene rearrangements, and molecular results should always be considered together with histopathological and immunophenotypical findings.

Conclusion With careful assessment of the clinical history and methodical morphological examination complemented by appropriate immunophenotyping, most cases of lymphomas can be correctly diagnosed and classified. Molecular analysis will be helpful if doubts exist, especially when the size or quality of the biopsy limits a comprehensive morphological and immunophenotypical examination. However, some cases will remain challenging, necessitating teamwork from the clinician, pathologist, and molecular biologist. Contributors Both authors developed the idea for this paper, and wrote and approved the review.

Clonal IG or TCR gene rearrangement in non-neoplastic lymphoid lesions

Conflicts of interest The authors declared no conflicts of interest.

Most non-neoplastic lymphoid lesions show polyclonal patterns on IG or TCR gene rearrangement studies. When a clonal pattern is identified in a reactive lesion, pathological studies should be undertaken to establish if

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