Hodgkin and non-Hodgkin lymphoma involving bone marrow

Hodgkin and Non-Hodgkin Lymphoma Involving Bone Marrow David Viswanatha, MD, and Kathryn Foucar, MD ● The role of bone marrow examination in diagnosis and staging of patients with Hodgkin lymphoma, B-non-Hodgkin lymphoma, and T-non-Hodgkin lymphoma is reviewed. Optimal routine and specialized bone marrow examination techniques are discussed. The salient morphologic, immunophenotypic, and genetic features of mature and immature B, T neoplasms and classic Hodgkin lymphoma in bone marrow are delineated, along with recommendations to distinguish these overt neoplasms from non-neoplastic processes. INDEX WORDS: Hodgkin, NHL, bone marrow, blood, immunoperoxidase, immunophenotype, lymphoid aggregate © 2003 Elsevier Inc. All rights reserved.

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ONE MARROW examination (BME) is a standard procedure in the evaluation of patients with Hodgkin (HL) and non-Hodgkin lymphomas (NHL). Bone marrow examination may be used in the staging of a patient with an established diagnosis of malignant lymphoma, both at initial diagnosis and after therapy. The bone marrow may also be the initial site of diagnosis in patients who undergo BME for unexplained cytopenias, fever, or other symptomatology.1,2 Documentation of random bone marrow involvement by malignant lymphoma indicates stage IV disease. The preferred site for BME is the posterior iliac crest; only rarely is it necessary to evaluate bone marrow from another site. Because each type of specimen provides complementary information, bone marrow aspirate smears, core biopsy sections with touch imprint preparations, clot sections of bone marrow aspirate, and blood smears ideally would be reviewed on all patients undergoing BME for possible lymphoma.3 The cytologic features of the neoplastic process are best characterized on air-dried preparations such as bone marrow aspirate smears or imprint slides, while the pattern and extent of bone marrow infiltration by malignant lymphoma are best delineated by evaluation of step sections of the bone marrow core biopsy and clot sections.3,4 In addition, anticoagulated bone marrow aspirate specimens should be obtained for possible flow cytometric immunophenotyping, cytogenetic assessment, or various molecular studies. Fluorescence in situ hybridization (FISH) can be performed on air-dried From the Department of Pathology, University of New Mexico, Albuquerque, New Mexico. Address reprint requests to Kathryn Foucar, MD, Department of Pathology, MSC 08-4640, 1 University of New Mexico, Albuquerque, New Mexico 87131; e-mail: [email protected]. © 2003 Elsevier Inc. All rights reserved. 0740-2570/03/2003-0005$30.00/0 doi:10.1053/S0740-2570(03)00026-1 196

preparations such as aspirate smears, imprint preparations, or cytospin slides. Because probes are available for individual chromosomes as well as many common fusion genes, FISH methodology is an important tool in lymphoma diagnosis. Because bone marrow involvement by malignant lymphoma is a focal process, generous bone marrow core biopsy specimens will increase the yield of positive diagnoses.5 Consequently, bilateral iliac crest bone marrow core biopsies are preferred to insure an optimal specimen for evaluation. These core biopsy specimens should be step sectioned for morphologic review, again to optimize in the detection of small focal lesions or infiltrates accompanied by fibrosis that may not be readily detected in aspirate smear preparations. Morphologic review of well-prepared and wellstained bone marrow aspirate and imprint preparations and good quality sections of the bone marrow core biopsy specimens is clearly the most important modality for documenting bone marrow involvement by malignant lymphoma. Appropriate, cost-effective utilization of flow cytometric immunophenotyping and molecular analysis on bone marrow specimens is somewhat controversial. Routine flow cytometric immunophenotyping on morphologically negative cases does not significantly increase the yield of positivity in bone marrow aspirate specimens.6 Likewise, the positive rate for immunoglobulin heavy chain and T-cell receptor gene rearrangement in bone marrow aspirate specimens is lower than morphologic and immunophenotypic assessment of the same specimen, presumably secondary to sampling issues.7,8 Consequently, these specialized techniques should be used on an ad hoc basis, depending on the specific features and clinical questions in an individual case. In contrast, immunoperoxidase assessment is the next most useful modality after morphology in the documentation of bone marrow involvement by malig-

Seminars in Diagnostic Pathology, VOL 20, NO 3 (AUGUST), 2003: pp 196-210

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nant lymphoma. Numerous antibodies work on decalcified, paraffin-embedded tissue.9,10 These antibodies can be utilized to define the immunophenotypic profile of abnormal bone marrow infiltrates; paraffin immunoperoxidase is especially useful in fibrotic, inaspirable specimens. Likewise, immunoperoxidase assessment is also essential in recognizing the subtle, morphologically occult lesions of anaplastic large cell lymphoma, hepatosplenic T-cell lymphoma, and some cases of bone marrow involvement by marginal zone lymphoma. However, immunoperoxidase staining will not, in general, delineate HL and NHL that was not morphologically apparent in the more common malignant lymphoma subtypes in bone marrow. Finally, since benign lymphoid aggregates are commonly encountered in the bone marrow of older patients and in individuals with infectious or autoimmune disorders, distinction between low-grade B-cell lymphoma and benign lymphoid aggregates may be facilitated by immunoperoxidase assessment for B- and T-cell antigens.11,12 For positive bone marrow specimens, the pattern and extent of bone marrow replacement provides useful information for treatment and disease monitoring purposes (Fig 1). These neoplastic infiltrates can occupy: 1) focal non-paratrabecular (random) foci in the intramedullary space, 2) focal paratrabecular lesions which mold to bony trabeculae, 3) diffuse interstitial infiltrates that are admixed among fat cells and hematopoietic elements, 4) diffuse solid infiltrates which efface the entire marrow between bony trabeculae, or 5) bone marrow infiltration consisting of intrasinusoidal/ intravascular foci of neoplastic cells.3 The extent of bone marrow infiltration is derived by estimating the relative proportion of neoplastic to non-neoplastic elements within the core biopsy and clot sections. Although there is generally excellent concordance between the morphologic and immunophenotypic features of lymphoma between medullary and extramedullary sites, occasional patients with bone marrow involvement by malignant lymphoma will exhibit either morphologic or immunophenotypic features discordant from those of the initial extramedullary specimen.2,13-16 This phenomenon of discordant morphology predominates in patients who present with extramedullary large B-cell lymphoma in whom the bone marrow infiltrates consist of small cleaved follicular center cells. It is important to distinguish bone marrow involvement by large cell lymphoma versus predominantly small cleaved cells, since involvement by overt large cell lymphoma portends a much more aggressive clinical

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Table 1.

Incidence of Bone Marrow Involvement in Hodgkin Lymphoma Type

Incidence (%)

NLPHL Classic HL: NSHL MCHL LDHL HIV-1-associated Overall HL

⬍1 ⬍10 20 ⬎50 ⬎60 3-15

Abbreviations: NLPHL, nodular lymphocyte predominant Hodgkin lymphoma; HL, Hodgkin lymphoma; NSHL, nodular sclerosis Hodgkin lymphoma; MCHL, mixed cellularity Hodgkin lymphoma; LDHL, lymphocyte-depleted Hodgkin lymphoma; HIV, human immunodeficiency virus. Data from refs 3, 4, 27-29, 32, 133, 134.

course requiring multi-agent therapy.17,18 Recent publications also note discordance between immunophenotype in comparison studies of extramedullary and medullary sites of disease.19 In general, this site-related immunopehnotypic discordance does not contribute to misdiagnosis, although exceptions do occur.19 HODGKIN LYMPHOMA Hodgkin lymphoma (HL) is a primary lymph nodebased neoplasm that occurs in patients of all ages but primarily in young adults, with a second incidence peak in the elderly.20,21 Despite decades of controversy, strong genetic evidence indicates that the neoplastic cell in HL is a B cell, although rare cases of T-cell receptor gene rearrangement positive HL have been reported.22 The etiology of HL is largely unknown and likely involves both genetic and environmental factors such as Epstein-Barr virus (EBV) infection. Infiltrates of HL are characterized by generally low numbers of Reed-Sternberg (RS) cells or RS variants in a cellular environment that consists of non-neoplastic inflammatory and accessory cells such as lymphocytes, plasma cells, eosinophils, neutrophils, dendritic cells, and histiocytes. These non-neoplastic cells comprise the typical cellular milieu of HL. Overexpression and abnormal patterns of cytokine/chemokine expression are linked to pronounced non-neoplastic cell infiltration that generally accompanies RS cells in all sites.23 In addition, fibrosis is characteristic of cases of classic HL. Recent evidence suggests a role of cytokine autocrine pathways in the generation of fibrosis tissue in cases of HL.24 The recently proposed World Health Organization

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Fig 1. This schematic highlights the patterns of bone marrow infiltration in non-Hodgkin lymphoma. There are 5 patterns of infiltration that can occur individually or in combination. Hodgkin lymphoma in bone marrow characteristically produces discrete, focal, nonparatrabecular aggregates. (Reprinted with permission.12)

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Fig 2. This low power photomicrograph of a bone marrow core biopsy highlights a large discrete lesion in Hodgkin lymphoma as well as several smaller focal discrete infiltrates (H&E).

Fig 3. (A) A Reed Sternberg cell is evident admixed with lymphocytes, plasma cells, eosinophils creating the characteristic Hodgkin milieu; (B) CD30 immunohistochemical stain highlights the membrane and Golgi positivity characteristic of Hodgkin lymphoma.

Fig 4. (A) Heterogeneous small to intermediately-sized mature lymphocytes characterize this case of mantle cell lymphoma: note cyclin D1 positivity in inset; (B) extensive bone marrow infiltration by blastic mantle cell lymphoma is evident on this bone marrow core biopsy: note resemblance to acute lymphoblastic leukemia.

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(WHO) histological classification of HL divides this disorder into 2 primary groups: nodular lymphocyte predominant HL (NLPHL) and classic HL, which is further subdivided into nodular sclerosis, mixed cellularity, lymphocyte rich, and lymphocyte depleted.25 Nodular lymphocyte predominant HL accounts for only 5% of cases of HL overall, and only rarely involves bone marrow. Consequently, discussion will be restricted to classic Hodgkin lymphoma in bone marrow. The identification of bone marrow involvement in patients with HL is linked to B-symptomatology, advanced clinical and radiologic stage, classic HL morphology (especially mixed cellularity), and blood cytopenias.26-29 The incidence of bone marrow involvement in patients with HL overall ranges from 5%-15%; incidence by morphologic and clinical subtype is listed on Table 1. Because of the range of morphologic features in bone marrow in patients with HL, criteria for establishing definitive diagnosis, probable diagnosis, and “suspicious for HL” diagnosis have been delineated: 1. A primary diagnosis of HL can be established in a bone marrow specimen when diagnostic ReedSternberg cells are found in an appropriate cellular environment characteristic of HL. However, caution should be exercised, because NHL may overlap HL in bone marrow. Biopsy of an extramedullary site may be required. Patients with HIV-associated HL may manifest isolated bone marrow involvement by HL.30 2. In patients with an established diagnosis of HL in an extramedullary site, bone marrow involvement can be established even in the absence of diagnostic Reed-Sternberg cells, if mononuclear Reed-Sternberg variant cells with prominent nucleoli are identified in a cellular environment characteristic of HL. 3. A diagnosis of “suspicious for HL” can be rendered in bone marrow specimens in which atypical cells (not true Reed-Sternberg cells or variants) are noted in a cellular environment characteristic of HL. 4. Foci of fibrosis without either typical Reed-Sternberg cells or Reed-Sternberg variants can be designated as “suspicious, but not diagnostic” in bone marrow from patients with a previously established diagnosis of HL.31 Within bone marrow specimens, infiltrates of classic HL may be either focal or diffuse, solid (Fig 2).32 The extent of involvement ranges from small fibrotic foci to extensive diffuse effacement of virtually the entire

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medullary space.3,27,32 The distribution of cell types within the background cellular environment of classic HL is highly variable. In some cases, especially in immunosuppressed HIV-1 positive patients, cytologically bland histiocytes may predominate and mask the rare admixed RS cells. In other patients, the discrete fibrotic foci of HL contain abundant lymphocytes, eosinophils, and plasma cells (Fig 3A). Because HL lesions within bone marrow are generally associated with fibrosis, RS cells are seldom identified on aspirate smears.29 Indeed, the bone marrow core biopsy specimen is essential for the diagnosis of bone marrow involvement by HL. In classic HL, the Reed-Sternberg cells and RS variants are typically CD30 and CD15 positive (Fig 3B). These cells generally, but not always, lack B- and T-cell antigen expression, although variable CD20 positivity occurs in a significant subset of cases. Reed-Sternberg cells also characteristically lack common leukocyte antigen (CD45) in classic HL.25 Although in many cases the bone marrow features of HL may be straightforward, in other cases overlap with various NHL’s presents a diagnostic challenge. In these borderline cases, it is wise to recommend lymph node biopsy for confirmation, since both B- and T-NHL can closely mimic HL in bone marrow specimens.3 NON-HODGKIN LYMPHOMA

Bone Marrow Involvement by B-NHL B-cell non-Hodgkin lymphomas (B-NHL) commonly involve the bone marrow, although the frequency differs by lymphoma subtype (Table 2). Knowledge of the characteristic patterns of bone marrow involvement by B-NHL can both establish a diagnosis and suggest a more specific subclassification in many cases in which the marrow is a primary biopsy site, particularly when combined with phenotypic and molecular or cytogenetic findings. Furthermore, the bone marrow may be used for molecular residual disease monitoring, such as in the evaluation of treatment response in patients undergoing bone marrow transplantation for low-grade NHL. Precursor B-lymphoblastic leukemia/lymphoma is an immature B-cell neoplasm representing a continuum of disease distribution.33 The vast majority of these tumors predominantly involve blood and bone marrow, designated as precursor B-lineage acute lymphoblastic leukemia (B-ALL). However, primary nodal disease can occur as B-lymphoblastic lymphoma (B-LBL), and this diagnosis is arbitrarily established when there are less than 25% accompanying lymphoblasts in the mar-

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KATHRYN FOUCAR Table 2.

Lymphoma Subtype

Incidence of BM Involvement (%)

Bone Marrow Involvement in B-NHL

1° Pattern of BM Core Biopsy Involvement (2° patterns)

Cytologic Features

Immunophenotypic Features

Genotype

Comments Biologic continuum with B-lineage ALL; rule out myeloid leukemia, Burkitt leukemia/ lymphoma, or small round blue cell tumor Atypical presentations may be difficult to distinguish from MCL requiring cyclin D1 immunostaining Mast cells in aspirate particles a helpful feature; associated with IgM paraproteinemia and clinical syndrome Waldenstro¨ ms macroglobulinemia Nuclear features may be blastic or prolymphocytoid; may have marked leukemic presentation May be subtle in core sections requiring additional levels SMZL may represent difficult differential diagnosis with hairy cell leukemia in occasional cases

Precursor Blymphoblastic lymphoma/leukemia

40-60

Patchy, interstitial (diffuse, focal nodules)

Small-medium sized blasts

Immature B-cell, CD19 (CD20 usually negative), CD79a, CD10, CD34, TdT, HLA-DR, no surface Ig

Clonal Ig and TCR gene rearrangements; ALL-type translocations in a minority

Small lymphocytic lymphoma

45-75

Nodular, nonparatrabecular (interstitial or diffuse effacing)

Small lymphocytes, hypercondensed chromatin, occasional prolymphocytes

⫹12, del (13q), del (17p)

Lymphoplasmacytic lymphoma

75-90

Vaguely nodular, nonparatrabecular (interstitial or diffuse effacing)

Spectrum of small lymphocytes, plasmacytoid small lymphocytes and plasma cells; Dutcher bodies

Mature B-cell coexpressing CD5 and CD23; weak surface monotypic Ig; subset CD38⫹ with apparently poor prognosis Mature B-cell, usually CD5 and CD23 negative; monotypic surface and cytoplasmic Ig

Mantle cell lymphoma

50-80

Follicular lymphoma

40-70

Nodular, nonparatrabecular and focal loosely paratrabecular (interstital or diffuse effacing) Tightly paratrabecular (nodular or diffuse effacing)

Small-medium sized lymphocytes with condensed chromatin, irregular nuclei, no significant large transformed cells Small-medium sized cleaved lymphocytes, admixed large transformed cells Small-medium sized “monocytoid” cells, nucleoli may be prominent; plasmacytoid features in some cases, or “bipolar” villous lymphocytes (SMZL) Large atypical lymphocytes, prominent nucleoli, pleomorphic

Mature B-cell coexpressing CD5 but not CD23; surface Ig relatively bright; FMC-7 positive; Cyclin D1 positive Mature B-cell co-expressing CD10 in most cases; Bcl-2 and Bcl-6 positive Mature B-cell; no characteristic aberrant antigen profile

Mature B-cell; subset co-expressing CD10; Bcl-6 positive; Bcl2 in a subset

t(14;18)/BCL2-IgH in 20-30%; 3q27/BCL6 rearrangement in 30%; complex karyotypes

Milieu of abundant reactive small lymphocytes and/or histiocytes; occasional large atypical lymphocytes Large atypical lymphocytes, prominent nucleoli, pleomorphic Medium sized cells with dispersed, but coarse chromatin, several small nuclei, deep blue cytoplasm with vacuoles; uniform cytology

Mature B-cell; Bcl-6 positive, EBV negative; CD15 and CD30 negative

Not well characterized

Mature B-cell (majority); some cases co-express CD5⫹ Mature B-cell coexpressing CD10 and monotypic surface Ig; Bcl-6 positive but Bcl-2 negative; Ki-67 positive in ⬇100% cells

Not well characterized

Rare cases may be of T-cell lineage

t(8;14)/cMYC-IgH, or variant t(2;8) and t(8;22); EBV positivity variable (100% in endemic vs. ⬍40% in sporadic)

Biologic continuum with Burkitt leukemia; variant cytology includes Burkitt-like and plasmacytic (in AIDS patients)

⬇100 in SMZL; less frequent in EN-MZL and N-MZL

Nodular, nonparatrabecular and intra-sinusoidal

Diffuse large B-cell lymphoma

8-35

Nodular destructive and/or diffuse effacing (interstitial)

T-cell rich or histiocyterich large B-cell lymphoma

0-62

Nodular destructive and/or diffuse effacing

Intravascular large B-cell lymphoma

10-20

Subtle intrasinusoidal aggregates of large atypical cells

Burkitt lymphoma/leukemia

20-35

Interstitial (diffuse effacing)

Marginal zone lymphoma (mainly primary splenic)

⫺8, del (6q) described in marrow LPL; t(9;14)/PAX5-IgH in primary nodal LPL

t(11;14)/BCL1-IgH

t(14;18)/BCL2-IgH in 85-90%

del (7q) in 40-50% of SMZL; ⫹3 in EN-MZL; t(11;18)/API2MALT1 in subsets of EN-MZL

Abbreviations: SMZL, splenic marginal zone lymphoma; EN-MZL, extranodal marginal zone lymphoma; N-MZL: nodal marginal zone lymphoma. Data from references 3, 13, 17, 18, 33, 34, 38, 39, 42-49, 52-59, 62-74, 76-83, 88-92.

May rarely present as a solitary intraosseous mass; “Downgraded” paratrabecular foci of FL type lesions may be seen in staging marrows for DLBCL Numerous small Tcells (CD4⫹/CD57-) and lack of small B-cells is characteristic

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row.33 Morphologic bone marrow involvement is observed in a significant number, but not all cases of primary nodal presentations of B-LBL (Table 2). In such cases, the aspirate preparation reveals a sub-population of lymphoblasts characterized by finely dispersed chromatin, high nuclear to cytoplasmic ratio and minimal cytoplasm. Nucleoli are general inconspicuous however, can occasionally be more prominent and occasional cytoplasmic vacuoles may be evident. The bone marrow biopsy core reveals patchy, interstitial, “leukemic” type infiltrates of small to medium size cells with blastic nuclei (Table 2).3,33 Flow cytometric studies of the marrow will reveal a sub-population of immature B-lymphoid cells expressing weak CD45, CD10, HLA-DR, with variable CD34 and TdT.33 Tumor genetic studies show clonotypic rearrangements of the immunogloblin heavy chain gene, as well as the aberrant T cell ␥ or ␦ gene rearrangements in the majority of cases.34 Cytogenetics studies may reveal clonal karyotype abnormalities, or in a subset of cases in children, characteristic ALL-type translocations.34 Caution should be exercised in interpreting bone marrows in children or adolescents with nodal B-LBL as the marrow aspirates in these individuals may demonstrate increased benign precursor B-cells (hematogones), thus leading to a potential misdiagnosis of marrow involvement by B-LBL. Immunophenotypic and molecular analyses can be helpful in differentiating hematogones from B-LBL cells.35-37 Other types of B-NHL that may involve the bone marrow are mature B-cell disorders. Chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) are synonymous entities, with the specific nomenclature applied depending upon the primary site(s) of tissue involvement. CLL is a common mature B-lymphoproliferative disorder, which always involves the peripheral blood and bone marrow by definition. Patients with predominantly nodal based disease, namely SLL, have a relatively high incidence of bone marrow involvement, varying from focal to extensive in degree (Table 2). Bone marrow aspirate smears show a population of small monotonous round lymphocytes with minimal cytoplasm, “hypercondensed” chromatin and inconspicuous nucleoli.38,39 The bone marrow biopsy in CLL/SLL can display several patterns, but generally patchy nonparatrabecular aggregates and/or interstitial collections are most commonly encountered.3,38,39 In some cases, diffuse, enfacing infiltrates replace the bone marrow space; this pattern has been associated with a poor clinical outcome.40,41 Immunophenotyping and genetic studies can be very useful in

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confirming the diagnosis of CLL/SLL. These features are summarized in Table 2 and described in further detail in the section on CLL in this volume. Briefly, the principal findings in CLL/SLL include a population of CD23 positive B-cells with weak monotypic surface immunoglobulin and aberrant co-expression of CD5 antigen. Expression of CD38 antigen is seen in a subset of cases and appears to be associated with more aggressive disease in some studies.42-47 Cytogenetic analysis may reveal one of several recurrent chromosomal abnormalities including trisomy 12, and deletions of (13q).48 In the bone marrow core biopsy or clot preparation, immunohistochemistry for cyclin D1 protein is helpful in distinguishing mantle lymphoma from CLL/ SLL.49 Bone marrow involvement by lymphoplasmacytic lymphoma (LPL) is common and is responsible for the clinical syndrome of Waldenstro¨ m’s macroglobulinemia (WM).50,51 Classical LPL/WM involves the bone marrow and spleen. Patients with LPL develop serum IgM paraproteinemia, often with symptoms of hyperviscosity syndrome.50,51 Key features of LPL are summarized in Table 2. Bone marrow aspirate morphology demonstrates a variably abundant infiltrate of small “CLL-like” lymphocytes with condensed chromatin, small lymphocytes with plasmacytoid cytoplasmic features, and mature plasma cells.3,39,52 This spectrum of lymphoplasmacytic cytology serves to distinguish LPL from CLL/SLL. In addition, the finding of increased mast cells within and around bone marrow aspirate particles is a very helpful, if not absolutely specific feature of LPL.53 Bone marrow core biopsy findings are remarkable for mainly patchy interstitial or loosely nodular infiltrates of small lymphocytes and plasma cells, along with scattered large transformed cells typically accounting for less than 5%-10% of the lymphoid cells. Dutcher bodies (intranuclear pseudoinclusions of immunogloblin) may be seen in plasmacytoid lymphocytes or plasma cells and are better appreciated with a PAS stain. LPL is a B cell neoplasm expressing panB– cell markers, as well as surface and cytoplasmic light chain restriction, but generally lacks CD5 and CD23 antigens.52,54 Although previous genetic studies of node-based LPL revealed the presence of the t(9;14)/ PAX5-IgH gene fusion, more recent studies have shown different karyotypic findings for bone marrow predominant LPL.55-57 Mantle cell lymphoma (MCL), a particularly aggressive subtype of small B-lymphocyte lymphoma, involves the bone marrow in at least 50% of cases.58 MCL may occasionally present with substantial blood

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and bone marrow disease,59 including uncommon blastic cytologic variants resembling acute leukemia.60,61 Morphologically, bone marrow aspirates in patients with classic MCL show a monotonous small to intermediate sized lymphoid population (Fig 4A). Lymphocytes have condensed, round to irregularly shaped nuclei, usually with inconspicuous nucleoli and minimal to moderate cytoplasm. Although most MCL lymphocytes are of smaller size, a degree of cytological heterogeneity is often present and blastic or prolymphocytoid features may also be noted (Fig 4B).62-64 The bone marrow core biopsy reveals infiltrates of small lymphocytes with a conspicuous absence of large transformed large cells. The predominant pattern is that of nonparatrabecular nodular aggregates, although focal loosely paratrabecular collections of cells may be also seen.3,65 The diagnosis of MCL in the marrow is suggested by flow cytometry showing a population of phenotypic B cells with moderately intense surface immunoglobulin, and co-expression of CD5 but not CD23 antigen. The definitive diagnosis of MCL is established by demonstration of cyclin D1 overexpression by immunohistochemistry, or by the presence of the t(11;14)/BCL1-IgH locus genetic rearrangement by cytogenetic or FISH assays.49,66-73 A synopsis of MCL in the bone marrow is presented in Table 2. Follicular lymphoma (FL), the most common of the low grade B-lymphocyte non-Hodgkin lymphomas in North America, involves the bone marrow in approximately 40%-70% of cases (Table 2). Marrow involvement by FL is often less apparent in bone marrow aspirate then biopsy preparations. The bone marrow aspirate in FL will show an atypical population of small to intermediate size lymphocytes with irregular, cleaved nuclei and inconspicuous nucleoli (Fig 5A). Scattered large transformed (noncleaved) cells may be seen. The bone marrow biopsy core reveals focal or multifocal involvement by follicular lymphoma in a highly characteristic paratrabecular pattern, with tumor cells closely apposed to the trabeculae (Fig 5B).3,38 These paratrabecular lesions may be subtle, requiring careful scrutiny or deeper sectioning of the core biopsy. Foci of FL in the bone marrow are frequently accompanied by fibrosis, resulting in poorer representation in aspirate specimens, leading to potential false negative results by flow cytometry. If tumor cells are present in the bone marrow aspirate, these will mark positively for pan-B– cell antigens, with co-expression of the germinal center-associated antigen CD10 in most cases.74 Cytogenetic and molecular analyses can confirm the presence of the t(14;18)/BCL2-IgH abnormality in the

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vast majority of follicular lymphomas.74,75 This genetic rearrangement underlies the over-expression of Bcl-2 protein in most FL; however, the presence of Bcl-2 protein is not itself diagnostic of FL, being abnormally expressed (via different molecular mechanisms) in many non-Hodgkin’s lymphomas. A pattern of lymphomatous marrow involvement identical to classic FL occurs in a subset of diffuse large B-cell lymphomas (DLBCL) and has been termed “discordant” or “downgraded” lymphoma in this setting.13,17,18 All three sub-categories of marginal zone lymphomas (MZL) can infiltrate the bone marrow, but splenic MZL (SMZL) is characterized by consistent and sometimes extensive marrow and blood involvement, as compared with nodal (N-MZL) and extranodal (ENMZL types of disease (Table 2).76 Bone marrow aspirate preparations in patients with disseminated MZL show a population of small to medium sized lymphocytes with moderately condensed nuclei and relatively abundant cytoplasm; nucleoli may be conspicuous, particularly in SMZL. In some cases of SMZL, cells may appear quite plasmacytoid, or may show “bipolar” cytoplasmic projections (so-called “villous” lymphocytes).77 Bone marrow core biopsy sections reveal nonparatrabecular nodules, or discrete intrasinusoidal infiltrates of predominantly small lymphocytes with moderately abundant pale cytoplasm typical of monocytoid B-cells (Fig 6).76,77 In some instances, reactive germinal centers with a cuff of MZL cells are apparent.76 Flow cytometry and immunohistochemistry are important in confirming the diagnosis and assessing the extent of lymphomatous involvement, but there is no distinct antigenic profile that is highly characteristic of MZL. Cytogenetic or FISH studies can be helpful, as many cases of SMZL harbor a del (7q), whereas ENMZL tumors may show the presence of trisomy 3, or in some EN-MZL subtypes, the t(11;18)/AP12-MALT1 abnormality.78-83 Diffuse, aggressive large B-cell lymphomas generally present at a lower clinical stage in contrast to the small B-lymphocyte lymphomas. Accordingly, the incidence of bone marrow involvement is lower, in the range of 20%-30% overall.17 However, the nature of involvement and corresponding prognostic significance differs among cases of DLBCL with positive bone marrow status. In contrast to “discordant” (“downgraded” or low grade) marrow involvement resembling FL as described above, “concordant” marrow involvement in DLBCL (defined as the presence of lymphomatous marrow infiltrates with a predominant large cell component) is relatively infrequent in DLBCL, but is

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Fig 5. (A) A small cleaved follicular center cell is evident in this bone marrow aspirate smear; (B) a minute focus of paratrabecular lymphoma is evident in this bone marrow core biopsy from a patient with follicular lymphoma.

Fig 6. CD20 highlights the distinctive sinusoidal pattern of infiltration in this bone marrow core biopsy from a patient with splenic marginal zone lymphoma.

Fig 7. (A) Characteristic cytologic features of bone marrow involvement by large cell lymphoma are evident on this bone marrow aspirate smear; (B) an extensive area of infiltration by B-large cell lymphoma is evident on this bone marrow core biopsy section.

Fig 8. (A) Individual anaplastic large cell lymphoma cells are admixed with hematopoietic elements in this bone marrow biopsy section (note resemblance to megakaryocytes); (B) CD30 immunohistochemical staining highlights these individually dispersed lymphoma cells in anaplastic large cell lymphoma; (C) alk 1 positivity in nucleus and cytoplasm is evident in this bone marrow core biopsy from a child with disseminated anaplastic large cell lymphoma.

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nonetheless associated with a poor prognosis.17,18,84 An important role for the hematopathologist in the staging of DLBCL in bone marrow biopsies is thus to distinguish true large cell disease from discordant lymphoma involvement, as these entities carry such different prognostic implications. The bone marrow aspirate involved by large B-cell lymphoma demonstrates a population of large lymphoid cells with dispersed nuclear chromatin, prominent nucleoli and a moderate amount of basophilic cytoplasm (Fig 7A). These cells may resemble agranular blasts in Wright-Giemsa stain. Plasmacytoid features may rarely be seen and a spectrum of smaller atypical lymphocytes may be present. The core biopsy section reveals a destructive diffuse or nodular cellular infiltrate effacing the marrow space (Fig 7B).17,18 Large cells predominate, although an admixture of small and medium size irregular lymphocytes may also be apparent. DLBCL may also rarely present as a solitary intraosseous lesion. The diagnosis of DLBCL in the marrow is supported by immunophenotyping studies, revealing a monotypic large B-cell population, a subset of which will express CD10 antigen.85 Cytogenetic or molecular assays will demonstrate the t(14;18)/BCL2IgH abnormality in approximately 10%-30% of germinal center-derived DLBCL.85-87 A summary of features, including additional genetic findings in DLBCL is listed in Table 2. Some specific histologic subtypes of DLBCL deserve additional comments with regards to bone marrow involvement (Table 2). T-cell rich or histiocyterich B-cell lymphomas (TCRBCL/HRBCL) are relatively distinct when identified in the marrow. The incidence of marrow involvement by TCRBCL/HRBCL has been reported to be variable.88 The marrow infiltrates in these entities tend to resemble the histology of the primary nodal lesion, namely a discrete population of small mature lymphocytes and/or histiocytes, within which few large atypical lymphocytes (⬍10%) are apparent.85,88 Diagnosis can be enhanced by the use of immunohistochemistry in the core biopsy, demonstrating scattered CD20 positive lymphoma cells in a dense background of small T-lymphocytes or histiocytes. Intravascular large B-cell lymphoma (IVBL), also known as “angiotropic” large cell lymphoma, is rare but highly unique. Although many affected individuals are reportedly discovered post-mortem, others have been diagnosed during life.89,90 IVBL can be seen as aggregates of large atypical cells within sinusoidal blood vessel lumina in the marrow core biopsy.89 Although bone marrow involvement in IVBL is considered relatively uncommon in comparison to brain and

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visceral organs, astute morphologic observation along with ancillary immunohistochemical studies for B- and T-cell antigens can establish the diagnosis in this site. Analogous to the situation for precursor B-lymphoblastic leukemia/lymphoma, Burkitt lymphoma/leukemia (BL) is one disease with variable distribution (ie, predominant extramedullary versus bone marrow). Marrow involvement in BL occurs in approximately one-third of cases and reflects high tumor burden.3,91 The marrow aspirate reveals a relatively uniform population of medium sized lymphocytes with a moderate amount of deeply basophilic cytoplasm containing several vacuoles.3,38,91 Nuclei are blastic with coarsely dispersed chromatin and one to several small nucleoli. In marrow core biopsy sections, a discrete interstitial infiltrate is apparent, although limited foci with a diffuse pattern may be encountered.3 The tumor cells demonstrate round, medium sized nuclei with several small nucleoli. The cytoplasm is moderately abundant with distinct, “squared-off” cytoplasmic borders. Mitoses are usually pronounced and apoptotic bodies, along with increased scattered tingible body macrophages are often noted. Cytologic variants may be encountered, including a plasmacytoid type more prevalent among immunodeficient patients, and “Burkittlike” lymphoma.91,92 The latter entity is characterized by a slightly greater degree of nuclear variability, while retaining the other characteristics of BL.92 Phenotyping studies in BL demonstrate a mature B-cell population with monotypic surface light chain expression and the presence of CD10 antigen. Other key phenotypic details are presented in Table 2. The diagnosis of BL is greatly supported by finding the t(8;14)/cMYC-IgH gene fusion or variants by classical cytogenetics or FISH analyses.92,93 Epstein-Barr virus may be detected in subsets of both BL and Burkitt-like lymphomas92,93 (Table 2).

Bone Marrow Involvement in T-NHL Because T-NHL’s are substantially less common than B-NHL in Western countries, the frequency with which T-NHL is encountered in bone marrow specimens is much less than corresponding than mature B lymphomas.94,95 In addition, T-cell neoplasms are associated with systemic cytokine-mediated phenomena such as hemophagocytic syndrome that may be evident in bone marrow specimens in patients even in the absence of actual lymphomatous infiltration. The incidence, primary pattern of bone marrow infiltration, cytologic features, and classic immunophenotype of bone marrow involvement by precursor and

HODGKIN AND NON-HODGKIN LYMPHOMAS IN BONE MARROW Table 3.

Bone Marrow Involvement in T-NK Lymphomas

Incidence BM Involvement (%)

10 Pattern (20 patterns)

Precursor Tlymphoblastic lymphoma Mycosis fungoides/Se´ zary syndrome Peripheral T-cell lymphoma, unspecified

20-60

Patchy, interstitial

Lymphoblastic features with scant cytoplasm

20-25

Patchy, interstitial

30-75

Focal, nonparatrabecular

Nuclear convolutions, hyperchromatic, scant to moderate cytoplasm Variable, mixed or large cell lymphoma

Angioimmunoblastic Tcell lymphoma

50-80

Focal, nonparatrabecular

Polymorphous with immunoblasts

Anaplastic large cell lymphoma

10-20

Hepatosplenic T-cell lymphoma

100

Aggressive NK leukemia

⬎90

Isolated individual Very large multi-lobulated cells cell with moderate cytoplasm; RS-like cells may be present Intravascular/ Medium-sized cells, intrasinusoidal variable nuclear irregularity, scant to moderate cytoplasm Interstitial, diffuse Variable features; usually solid medium to large with blastic to condensed chromatin and distinct cytoplasmic granules

Lymphoma Subtype

205

Cytologic Features

Classic Immunophenotype

Comments

Immature T-cell phenotype: TdT⫹, variable CD34, CD1 Mature T: often CD4⫹, CD7⫺

Biologic continuum with T-ALL

True NK: cytoplasmic CD3␧⫹, EBER⫹, TIA-1⫹, CD56⫹

Usually presents with leukemic features in blood, bone marrow; T-cell receptor gene rearrangement absent

May be subtle, highlighted by CD3, CD4 Mature T cell, no May show morphologic distinctive IP; may show overlap with nonaberrant antigen neoplastic disorders expression profile and HL. Mature T cell, may show May show morphologic aberrant antigen overlap with nonexpression profile, neoplastic disorders CD10⫹ and HL Mature T cell: CD30⫹,. Can be very subtle; EMA⫹, ALK 1⫹, requires I P (CD30, TIA-1⫹, CD45v⫹, EMA, ALK 1 T-cell ag⫹ Typically very subtle, Mature T cell: CD3⫹, requires IP (CD3, TIA-1⫹, ␥␦⫹ TIA-1)

HL, Hodgkin lymphoma; NK, natural killer; T-ALL, T-acute lymphoblastic leukemia; v, variable. Data from references 3, 4, 27, 28, 95, 107, 109, 110, 113, 114, 133, 134.

matue T/NK lymphomas are presented on Table 3. Precursor T-lymphoblastic lymphoma is on a biologic continuum with T-acute lymphoblastic leukemia, and the distinction between these 2 diagnostic designations is generally based on the extent of bone marrow infiltration.3,27 Precursor T-lymphoblastic lymphoma cases show patchy foci of lymphoblasts rather than the extensive, diffuse bone marrow infiltration that typifies T-acute lymphoblastic leukemia; involvement of the bone marrow is relatively common in T-LL patients and may be highlighted by immunoperoxidase staining for CD3, CD34, and TdT.3,27,96 The remaining T-NHL that can be encountered in bone marrow exhibit a mature (peripheral) T-cell phenotype. Although mycosis fungoides/Se´ zary syndrome may exhibit peripheral blood involvement, especially in patients with generalized erythroderma (ie, Se´ zary syndrome), bone marrow involvement is documented in only a minority of cases and may consist of subtle, patchy interstitial foci of cerebriform cells.97,98 Immunophenotyping enhances detection of these inconspicuous infiltrates.99 The incidence of bone marrow involvement in peripheral T-cell lymphoma unspecified (PTCL) is high;

morphologic assessment can be enhanced by the detection of aberrant patterns of T-cell antigen expression in these cases.100-104 Analogous to the variability of morphologic features in lymph node sections, bone marrow involvement in PTCL may be associated with prominent vascularity, polymorphous infiltrates with large atypical cells, and reticulin fibrosis.3,100 Because of this heterogeneity of cell types, distinction from HL and PTCL may be problematic; immunophenotyping and molecular analyses can facilitate in making these diagnostic distinctions. Lymph node biopsy is recommended for inconclusive cases, and a definitive primary diagnosis of PTCL on bone marrow specimens should be approached with caution.3 Angioimmunoblastic T-cell lymphoma (AITL) is a systemic disease that characteristically involves lymph node, spleen, and bone marrow.105,106 Bone marrow infiltration in AITL mimics lymph node in that vascular proliferation, polymorphous infiltration of small, intermediate and large immunoblast-like cells, and reticulin fibrosis are all characteristic features.3,27 The expression of CD10 on the neoplastic T cells in AITL has been recognized recently as an immunophenotypic feature of this disorder.105

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Anaplastic large cell lymphoma (ALCL) is unique among the mature T-cell lymphomas in that a cytogenetic abnormality [t(2;5)] is the defining genetic feature in patients with systemic disease.107-110 Unlike other mature T-cell neoplasms, ALCL is common in pediatric patients.25,111 Bone marrow involvement in ALCL is uncommon and is notably inconspicuous on standard morphologic review of H&E stained bone marrow core biopsy specimens. In fact, immunoperoxidase staining for CD30, EMA, and ALK 1 substantially increase the yield of positive diagnoses in bone marrow (Figs 8 A, B, and C). The large multi-lobulated neoplastic cells tend to be individually dispersed within normal hematopoietic elements making recognition of this neoplastic infiltrate a challenge. In some cases distinction for megakaryocytes and/or Reed Sternberg cells is problematic (Fig. 8A).112-114 In addition, the small cell variant of ALCL shows an even greater propensity for blood and bone marrow involvement, and the distinction between these neoplastic T cells and other types of lymphoid disorders is notably challenging without comprehensive immunophenotyping including CD30, EMA, ALK 1, and TIA-1.115-117 Cytogenetic FISH assessment for t(2;5) can also facilitate the diagnosis in these cases. Hepatosplenic T-cell lymphoma is uncommon in clinical practice. Bone marrow findings consist of morphologically occult intravascular/intrasinusoidal infiltrate that may occur in conjunction with benign hemophagocytic syndrome.118,119 Consequently, immunophenotypic staining for CD3 and TIA-1 is essential on all staging bone marrow specimens performed in patients with an established diagnosis of hepatosplenic T-cell lymphoma, even those cases which appear negative by morphologic review. These neoplastic cells are often small to intermediate in size and exhibit variable nuclear irregularity; the patchy intravascular/intrasinusoidal localization is highlighted by immunoperoxidase staining. Likewise, aggressive NK leukemia/lymphomas are very rare disorders that are not frequently encountered in clinical practice, although bone marrow involvement is characteristic in this disorder.120,121 Aggressive NKcell leukemia/lymphomas are NK cell-derived neoplasms comprised of cells that retain many morphologic and immunophenotypic similarities to normal circulating large granular lymphocytes.120,122,123 These cells are medium to large and have abundant cytoplasm that contains distinct granules. Although nuclei are generally mature appearing, greater cytologic atypia, immaturity, and larger sized neoplastic cells may be

KATHRYN FOUCAR

evident in some cases.124 Infiltrates in bone marrow may be extensive with individual cell necrosis and apoptosis. In addition, proliferation of cytologically bland histiocytes with prominent hemophagocytosis may be noted.120,124,125 The immunophenotypic profile of aggressive NK leukemia/lymphoma parallels that of the nasal type of NK cell lymphoma with expression of CD56, CD2, CD3␧, TIA-1, and granzyme. Also analogous to their nasal counterpart, aggressive NK cell leukemia/lymphomas are EBVassociated neoplasms.120,126 These rare NK neoplasms must be distinguished from lymphoblastic lymphoma, acute myeloid leukemias, and recently described dendritic leukemia/lymphomas.127-131 CONCLUSION The successful identification of B and T/NK cell lymphomatous infiltrates in bone marrow is essential for optimal medical management. In many patients this is relatively straightforward, based on morphologic assessment of well-prepared aspirate smears and clot/core biopsy sections. In other patients, especially those with T-cell disorders, immunophenotyping is essential in highlighting morphologically occult infiltrates. Specialized studies such as multicolor flow cytometric immunophenotyping and genetic analyses (FISH, karyotyping, molecular) may all be useful diagnostic adjuncts to answer specific differential diagnostic questions. Recognition of therapy-related changes is also of considerable importance in evaluating bone marrow lymphoid infiltrates following treatment, more so in light of the current widespread use of anti-CD20 monoclonal antibody therapy. In the latter case, the presence of T-cell aggregates may morphologically mimic residual small B-lymphocyte lymphoma.132 Reliance on CD20 immunostaining may be thus misleading in the setting of anti-CD20 monoclonal antibody therapy, requiring the use of additional B-cell markers by flow cytometry or immunohistochemistry to delineate persistent disease. Molecular analyses for antigen receptor gene clonality, or translocation-associated markers such as BCL2 gene rearrangements can also be used to effectively assess for the presence and relative level of minimal residual lymphoma in specific circumstances. Finally, lymphomatous infiltrates must be reliably distinguished from non-neoplastic lymphoid infiltrates in bone marrow. REFERENCES 1. Ponzoni M, Li C-Y: Isolated bone marrow non-Hodgkin’s lymphoma: A clinicopathologic study. Mayo Clin Proc 69:37-43, 1994

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