the usefulness of immunohistochemistry in the diagnosis of follicular lymphoma in bone marrow...

Hematopathology / IMMUNOHISTOCHEMICAL DIAGNOSIS OF FOLLICULAR LYMPHOMA IN BONE MARROW BIOPSY SPECIMENS

636 Am J Clin Pathol 2002;117:636-643 © American Society for Clinical Pathology

The Usefulness of Immunohistochemistry in the Diagnosisof Follicular Lymphoma in Bone Marrow BiopsySpecimens

Robert B. West, MD, PhD, Roger A. Warnke, MD, and Yasodha Natkunam, MD, PhD

Key Words: Follicular lymphoma; Bone marrow; Immunohistochemistry; bcl-2; bcl-6; CD10; CD23

A b s t r a c t

We used a panel of paraffin antibodies to determinewhether neoplastic and nonneoplastic lymphoidaggregates in the bone marrow can be distinguishedreliably. Formalin-fixed, paraffin-embedded bonemarrow core biopsy specimens with lymphoidaggregates were stained using primary antibodiesdirected against bcl-2, bcl-6, CD5, CD10, CD20, andCD23. We studied 61 cases (26 follicular lymphomaand 35 benign or atypical aggregates). We found thatno single stain is sufficient for identification ofneoplastic lymphoid aggregates. However, thisdistinction was made possible by using a panel ofantibodies. Under the conditions we tested, the mostuseful antibodies were CD10, bcl-2, CD5, and CD20.Most benign or atypical aggregates do not expressCD10 and CD23. In addition, nonneoplastic aggregateshad a large population of T cells. bcl-2 was useful in anarchitectural context for distinguishing neoplasticaggregates. bcl-6 often was expressed in bothneoplastic and nonneoplastic aggregates and, thus,poorly discriminated between these processes. Westudied the expression of CD10 and bcl-6 in selectedlymph nodes in some cases.

Staging for follicular lymphoma (FL) involves assessmentof the bone marrow. The status of the bone marrow has a majoreffect on determining the stage of the disease and may affectsubsequent clinical management. The bone marrow is the mostcommon site of extranodal involvement of FL leading to thedesignation of stage IV disease. Patients with low-stage diseasemay be treated with radiotherapy alone. In contrast, patientswith stage IV disease may require chemotherapy. As an alter-native, the conditions of patients may be monitored withoutproviding treatment for stage IV disease.1

The determination of the presence or absence of low-grade FL in the bone marrow can be diagnostically chal-lenging. In particular, it can be difficult to determine whethersmall lymphoid aggregates found in staging bone marrowbiopsy specimens are benign or indicate involvement by FL.FL typically involves the bone marrow as paratrabecularaggregates composed of cleaved cells, while benignlymphoid aggregates are typically smaller, contain a mixedpopulation of cells, and are nonparatrabecular.2 Staging bonemarrow biopsies may reveal aggregates with a combinationof benign and neoplastic histologic features, making morpho-logic assessment difficult and necessitating immunohisto-logic evaluation for confirmation of the diagnosis.

We addressed the question of whether benign andneoplastic lymphoid aggregates in the bone marrow can bedistinguished reliably by using a panel of immunohistologicmarkers amenable to use in paraffin-embedded tissue. Themarkers we chose for study included antibodies with specifici-ties for CD20, CD5, CD23, bcl-2, CD10, and bcl-6. CD20 andCD5 were chosen to determine the lineage of the cells (B and Tcells, respectively) constituting the lymphoid aggregates. Specif-ically, the CD5 marker was chosen because of its usefulness for

Hematopathology / ORIGINAL ARTICLE

Am J Clin Pathol 2002;117:636-643 637© American Society for Clinical Pathology

detecting small lymphocytic lymphoma/chronic lymphocyticleukemia and mantle cell lymphoma. CD23 was selectedbecause it detects dendritic cells and aids in localizing adendritic cell network associated with lymphoid follicles. bcl-2,CD10, and bcl-6 were selected for their putative roles in thepathogenesis of FL.

The reciprocal translocation t(14;18), involving the bcl-2oncogene and the immunoglobulin heavy chain joining regionis considered the cytogenetic hallmark of FL.3-8 This translo-cation and the deregulated expression of the bcl-2 protein arefound in 80% to 90% of FLs.9-18 Overexpression of the 26-kdinner mitochondrial membrane bcl-2 protein results in theprevention of apoptosis and prolonged cell survival.18-20

Immunohistologic detection of the bcl-2 protein withinneoplastic follicles, but not in reactive germinal centers, is animportant tool for making the diagnosis of FL.2 CD10 is amembrane-associated neutral endopeptidase widely expressedin human tissues including germinal center (GC) B cells.21

Lymphomas derived from the GC, including FL and a subsetof diffuse large B-cell lymphomas, and acute lymphoblasticleukemias express this antigen.22,23 The differential expressionof CD10 in reactive vs neoplastic lymphocytes has led toincreased interest in this antigen as a potentially important toolin the diagnosis of lymphoma.24,25 The proto-oncogene bcl-6was first noted in translocations involving 3q27 in diffuse largeB-cell lymphoma and FL.26-34 This oncogene encodes aprotein that functions as a transcriptional repressor expressedselectively in GC B cells, CD4 T cells, and cortical thymo-cytes.26,35-39 In transgenic mice deficient in bcl-6, there is anabrogated T cell–dependent antibody response and the lack ofmaturation of GCs.38,40 Apart from translocations involvingbcl-6, somatic mutations in the 5' noncoding region of the bcl-6 gene also have been documented in normal GC B cells andin lymphomas derived from GC B cells, including FL.25,27

Materials and Methods

Case Selection

We included 61 bone marrow biopsy specimens selectedfor the presence of lymphoid aggregates in the study. All casesare from Stanford University Medical Center, Stanford, CA.The majority of the biopsies were performed as part of astaging evaluation for recent diagnoses of FL based on lymphnode biopsies. The minimum length of the biopsy specimenwas 0.7 cm. Both unilateral and bilateral biopsy specimenswere evaluated. In the latter case, only 1 biopsy specimen wasused in the study if both specimens contained lymphoid aggre-gates. Of the 61 cases, 26 contained involvement by FL. Thesecases had diagnoses based on morphologic examination andancillary studies, including immunohistochemical analysis. The

remainder of cases were categorized as atypical or benignlymphoid aggregates. These diagnoses were based on morpho-logic examination and, when needed, immunohistochemicalanalysis. Atypical lymphoid aggregates were those that hadsome but not all features of a neoplastic aggregate, includingmonotonous lymphoid population, medium to large size, orparatrabecular location.41,42 H&E-stained sections andimmunohistochemical markers, if any, from the time of initialdiagnoses were reviewed to confirm the findings. All caseswith benign lymphoid aggregates were obtained from patientswith no history of lymphoma. In 12 of 15 cases with atypicallymphoid aggregates, there was no history of lymphoma, and 3cases had an earlier diagnosis of FL or small B-cell lymphoma.

Immunohistochemical Analysis

Primary antibodies were directed against CD20 (DAKO,Carpinteria, CA), CD5 (Novocastra, Newcastle upon Tyne,England), CD23 (The Binding Site, Birmingham, England), bcl-2 (DAKO), CD10 (Novocastra), and bcl-6 (courtesy of Brunan-gelo Falini, MD, Department of Hematology, Perugia University,Perugia, Italy). Serial 4-µm sections were cut and deparaffinizedin xylene and hydrated in a graded series of alcohol. Antigenretrieval by microwave pretreatment was performed in citric acidbuffer (10-mmol/L concentration, pH 6.0, for 10 minutes) forCD20, in tris(hydroxymethyl)amino-methane buffer (5-mmol/Lconcentration, pH 10.0, for 20 minutes) for CD5 and bcl-2, andin EDTA buffer (1-mmol/L concentration, pH 8.0, for 15minutes) for CD10 and bcl-6. Endogenous peroxidase wasblocked by preincubation with 1% hydrogen peroxide in phos-phate-buffered saline. Detection for CD20, CD5, bcl-2, CD10,and bcl-6 was performed using a modified biotin-streptavidinmethod.43 Only detection for CD23 was performed on an auto-mated staining machine (Ventana Medical Systems, Tucson,AZ). The other stains were processed by hand.

The scoring systems for each antibody varied accordingto the stain. CD20, CD5, and bcl-2 were scored positive if50% or more of the cells within an aggregate showedstaining for these markers. CD23 was scored positive ifmembrane staining of dendritic cells was present. Lymphoidaggregates were scored positive for CD10 if more than 15%of the cells within the aggregate had membrane staining forCD10. bcl-6 was scored positive if lesional cells withinlymphoid aggregates showed nuclear staining for bcl-6.

Results

Histologic Features

All lymphoid aggregates studied ranged from thoseoccupying a single high-power field to those that replaced themarrow space. The aggregates were composed predominantly

West et al / IMMUNOHISTOCHEMICAL DIAGNOSIS OF FOLLICULAR LYMPHOMA IN BONE MARROW BIOPSY SPECIMENS


of small lymphocytes. These lymphocytes had cleaved orrounded nuclear morphologic features. In some aggregates,larger cells were admixed, although large cells did not exceed50% of all lymphoid cells constituting the aggregates. Occa-sional GCs were present with rare tingible body macrophages.While some aggregates showed tight clusters of lymphocytes,others were more diffuse, ill-defined groups of atypicallymphoid cells that were interspersed with other bone marrowelements. The lymphoid cells were present in the marrow asparatrabecular or nonparatrabecular aggregates.

Immunohistologic Features

The results of immunohistologic stains are summarized in❚ Table 1❚ . The lymphoid aggregates differed in B- and T-cellcomposition as assessed by CD20 and CD5 staining. Of 26cases of lymphoma aggregates, 23 (88%) had a majority of cellsstaining for CD20. In contrast, both atypical (10/15 [67%]) andbenign (11/20 [55%]) aggregates had more cells staining forCD5. Some aggregates had approximately equal proportions ofB and T cells. A small number of FL aggregates (3/26 [12%])exhibited a broad outer zone rich in CD5+ cells surrounding acentral core of CD20+ cells. Staining for CD23 was higher inFL aggregates (6/26 [23%]) than in atypical (1/15 [7%]) orbenign (1/20 [5%]) lymphoid aggregates. When present, CD23staining highlighted a follicular formation ❚ Image 1❚ .

bcl-2 staining alone did not discriminate among the threetypes of aggregates. While all 26 cases with FL aggregates(100%) stained for bcl-2, 14 (93%) of 15 atypical aggregates and15 (75%) of 20 benign aggregates also stained for bcl-2. bcl-2staining in the benign aggregates was not due to the presence ofT cells, as confirmed by the stain for CD5. In some bone marrowbiopsy specimens with large benign aggregates, bcl-2 was usefulfor outlining the physiologic mantle zone and defining the GC.In these cases, the follicular nature of these aggregates was notreadily apparent on H&E-stained sections ❚ Image 2❚ .

Staining for CD10 was the most striking of all themarkers tested in highlighting the difference among FL,atypical, and benign aggregates. Because other hematopoi-etic elements may also stain for CD10, only aggregates that

showed staining in more than 15% of the cells comprisingthe aggregate were designated positive. By this criterion, 12(46%) of 26 FL aggregates stained for CD10. The stainingpattern was predominantly diffuse; however, in a number oflarger FL aggregates, follicular architecture was present. Thestaining was strongest in the center of the aggregates. Inaddition, the CD10 stain highlighted follicles withinneoplastic aggregates even when a follicular pattern was notevident on H&E-stain sections (Image 2). In contrast, CD10staining was not prominent in atypical (1/15 [7%]) or benign(1/20 [5%]) lymphoid aggregates.

The staining pattern for bcl-6 did not differ substantiallybetween aggregate types. Staining for bcl-6 was present in12 (46%) of 26 FLs, 3 (20%) of 15 atypical aggregates, and5 (25%) of 20 benign aggregates. bcl-6 staining was mostprominent in well-formed neoplastic follicle centers;however, scattered diffuse staining also was seen in atypicaland benign aggregates (Image 2).

A small panel of antibodies was chosen to evaluatewhether FLs could be distinguished immunohistochemicallyfrom nonneoplastic lymphoid aggregates. By using a combi-nation of CD5, CD20, and CD10, 88% (23/26) of FLs couldbe distinguished from nonneoplastic aggregates when thescoring criteria in Table 1 were used.

To further assess the expression of CD10 and bcl-6 in theFL cases, corresponding lymph node biopsy specimens withFL from selected patients were evaluated ❚ Table 2❚ and ❚ Image

3❚ . Staining for CD10 and bcl-6 was assessed separately infollicle center and interfollicular compartments and comparedwith staining for these markers in bone marrow lymphoidaggregates and the interstitium. With 1 exception, staining forCD10 in the lymph node follicles and bone marrow aggre-gates was similar. Lymph nodes with a high number of cellsstaining for CD10 (70% or more) in the follicle showed highernumbers of cells staining for CD10 (15% or more) in the bonemarrow aggregates. For bcl-6, the percentage of stained cellswas substantially less in the bone marrow than in lymph nodespecimens. The majority of the bone marrow biopsy speci-mens entirely lacked staining for bcl-6. Thus, designation of

❚ Table 1❚Summary of Immunohistologic Staining of Lymphoid Aggregates in 61 Bone Marrow Core Biopsy Specimens*

Criterion for Scoring†

≥≥50% ≥≥50% >0% >0% >15% ≥≥50%

CD20 CD5 CD23‡ bcl-6 CD10 bcl-2

Follicular lymphoma (n = 26) 23 (86) 4 (15) 6 (23) 12 (46) 12 (46) 26 (100)Atypical aggregates (n = 15) 4 (27) 10 (67) 1 (7) 3 (20) 1 (7) 14 (93)Benign aggregates (n = 20) 11 (55) 11 (55) 1 (5) 5 (25) 1 (5) 15 (75)

* Data are given as number (percentage) of cases scored positive as defined by the scoring criteria described in the “Materials and Methods” section.† Cases were scored positive if more than the percentage of lesional cells indicated at the top of each column showed staining for the particular marker.‡ Staining of the dendritic cell network is indicated.



bcl-6 positivity was set at the lower threshold of 5% forassessment of the bone marrow environment. Similar to CD10staining, there was a correlation between numbers of cellsstaining for bcl-6 in lymph node follicles and bone marrowaggregates. While the staining for bcl-6 in lymph node and inbone marrow biopsy specimens was variable in the same case,the presence or absence of staining for CD10 and bcl-6 wassimilar between the 2 sites.

Discussion

By using a select panel of antibodies directed againstantigens pertinent to the pathogenesis of FLs, we studied

whether the staining patterns of these antibodies reliablyseparate low-grade FL from atypical and benign lymphoidaggregates involving the bone marrow.

An increased number of cells in bone marrow aggre-gates staining for CD20 or a decreased number of cellsstaining for CD5 were found in 80% of bone marrow biopsyspecimens involved by FL aggregates. In contrast, all aggre-gates designated atypical or benign showed more CD5+ cellsconstituting the lymphoid aggregate. In addition, CD23 andbcl-2 were useful for identifying architectural features thatdiscriminate between neoplastic and nonneoplastic aggre-gates. Staining for CD23 was detected in more cases of FLthan cases of atypical or benign lymphoid aggregates;however, case by case, this staining pattern was not sufficiently

A B

C D

❚ Image 1❚ bcl-2 and CD23 staining in the bone marrow. A, Benign aggregate (H&E, original magnification ×150). B,Immunoreactivity with the same aggregate (bcl-2, original magnification ×300). C, Paratrabecular neoplastic lymphoidaggregate (H&E, original magnification ×300). D, Immunoreactivity of the same aggregate (CD23, original magnification ×300).



discriminatory to warrant the use of CD23 to distinguish FLfrom nonneoplastic aggregates.

Staining for bcl-2 was present in all 3 types of lymphoidaggregates tested in the present study. Previous studies havesuggested that bcl-2 alone is useful for discriminating FLfrom benign lymphoid aggregates.44 Although we found thatthe absence of bcl-2 was highly specific for benign lymphoidaggregates, the frequent expression of bcl-2 in lymphoidcells constituting benign and atypical aggregates did notmake bcl-2 a sensitive or specific marker for the detection ofFL in bone marrow biopsy specimens in our study. A similarresult was obtained by Skalova and Fakan.45

CD10 expression is almost entirely contained withinfollicle center cells in physiologic follicles and in FL involving

the lymph node. However, CD10 staining also has beenreported in interfollicular neoplastic B cells.25 In the presentstudy, we found that CD10 stained a greater number of casesof FL involving the bone marrow (46%) than atypical (7%) orbenign (5%) lymphoid aggregates. However, the numbers ofcells that stained for CD10 in each type of aggregate was notdissimilar enough to warrant the use of CD10 as a marker todiscriminate among the 3 types of lymphoid aggregates.

In contrast with CD10 expression, we found that bcl-6staining frequently was present in FL (46%) compared withatypical (20%) or benign (25%) lymphoid aggregates in thebone marrow. The reason for this differential expression ofCD10 and bcl-6 is unclear. There may be elements in the bonemarrow microenvironment that up-regulate the expression of

A B

C D

❚ Image 2❚ A typical example of follicular lymphoma involving the bone marrow. A, Paratrabecular aggregates of predominantlysmall cleaved cells (H&E, original magnification ×60). B-D, Immunohistologic stains highlight the lymphoid aggregates tovariable degrees (B, bcl-2, original magnification ×60; C, CD10, original magnification ×60; D, bcl-6, original magnification ×60).



❚ Table 2❚Comparative Immunohistochemical Results on Bone Marrow Follicular Lymphomas and Their Lymph Node Counterparts*

CD10 bcl-6

Lymph Node Lymph Node

Case No. Follicle Center Interfollicular Area Bone Marrow Follicle Center Interfollicular Area Bone Marrow

1 90 5 90 100 30 52 90 20 70 75 15 53 70 70 70 70 10 54 10 0 5 15 15 05 100 20 5 90 5 06 70 15 30 70 10 207 50 10 5 90 10 58 90 10 50 70 5 30

* Data are given as the percentage of cells positive for CD10 or bcl-6.

A B

C D

❚ Image 3❚ Comparison of staining patterns for bcl-6 and CD10 in follicular lymphoma involving the lymph node and the bone marrow.A and B, Immunohistologic stains highlight the follicle center within a lymph node (A, bcl-6, original magnification ×150; B, CD10,original magnification ×300). C and D, Only scattered cells stain for bcl-6 (C), while CD10 highlights the majority of the cells within aneoplastic aggregate in the bone marrow (D) (C, bcl-6, original magnification ×300; D, CD10, original magnification ×300).



bcl-6 in reactive aggregates. Alternatively, the expression ofCD10 in reactive aggregates may be suppressed in the bonemarrow. Regardless, despite expression of bcl-6 in lymphnodes of FL, bcl-6 reactivity does not distinguish FL fromnonneoplastic lymphoid aggregates in the bone marrow.

Interestingly, staining for both CD10 and bcl-6 wasincreased in bone marrow aggregates involved by FL, as 67%(8/12) of FLs with staining for CD10 in bone marrow aggre-gates also showed staining for bcl-6. This coexpression mayindicate that these lymphoma cells have a follicular centric“program” compared with those that express either CD10 orbcl-6. However, further work on a larger number of cases isneeded to make this subcategorization possible. The increasednumbers of cells staining for bcl-6 in benign lymphoid aggre-gates was a surprising finding. bcl-6 expression, typicallyfound in follicle center cells, has been reported to occuroutside follicle centers.25 It is unclear whether bcl-6 proteinexpression in these benign aggregates represents the differ-ence in the microenvironment between the lymph node andthe bone marrow. Our results also indicate that the stainingpattern of bone marrow FL approximates the staining patternof the follicle center cells rather than the paracortical cellswithin the corresponding lymph nodes involved by FL. Oneexplanation for this finding is that FL is derived from 2 typesof cells, follicular and interfollicular cells, and that it is thefollicular cells in the lymph node that preferentially involvethe bone marrow. The second explanation is that the microen-vironment for follicular and other indolent lymphomas mayhave significant influence on the phenotype of the neoplasticcells. Thus, the microenvironment in the bone marrow mayevoke a response by the lymphoma cells that is similar to thefollicle center environment rather than the paracortical envi-ronment. This latter explanation may be applicable to ourobservations of CD10 and bcl-6 staining in lymph node folli-cles and bone marrow aggregates.

We have shown that no single antibody that we tested inthis study reliably distinguishes FL from nonneoplasticaggregates involving the bone marrow. This includes bcl-2, amarker previously thought to have discriminatory valuewhen used alone. Our results indicate, however, that using apanel of antibodies including B- and T-cell markers, bcl-2and CD10, in addition to morphologic examination, providesuseful information for making this diagnostic distinction.

From the Department of Pathology, Stanford University MedicalCenter, Stanford, CA.

Supported in part by grant CA34233 from the NationalCancer Institute, National Institutes of Health, Bethesda, MD.

Address reprint requests to Dr West: Dept of Pathology,Stanford University Medical Center, 300 Pasteur Dr, Stanford,CA, 94305-5302.

Acknowledgment: We thank Elizabeth Domenay for experttechnical assistance.

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