Clinical Utility of CD23 and FMC7 Antigen CoexistentExpression in B-Cell Lymphoproliferative

Disorder SubclassificationEjaz Ahmad, Diana Garcia, and Bruce H. Davis*

Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan

Background: CD23 and FMC7 are normal B-cell antigens utilized during diagnostic immunophenotyping ofsuspected lymphoproliferative disorders. However, the diagnostic utility of coexistent antigenic expressionpatterns with simultaneous two-color staining and flow cytometric analysis has not been studied extensively.Methods: Using multiparameter flow cytometry, we evaluated the expression pattern of FMC7 and CD23 in218 cases of B-cell lymphoma from blood and bone marrow specimens. Results: The CD23(�)/FMC7(-)pattern was the most common pattern in patients with chronic lymphocytic leukemia and related variants.The widest variation of patterns was found in patients with follicular cell lymphoma, large cell lymphoma,and Waldenstrom’s macroglobulinemia, a lymphoplasmacytoid disorder, although most cases expressed theCD23-/FMC7(�) pattern. The CD23 and FMC7 antigen, along with the CD5 coexpression pattern, providescritical adjunctive data. These data allow accurate classification of the majority of cases, thereby providinga key aspect of a reliable diagnostic algorithm. The CD23 and FMC7 antigen expression pattern, along withselected other antigens, was predictive of subtypes in >95% of lymphoproliferative cases and narrowed thedifferential diagnosis in the remaining cases. Conclusion: The flow cytometric CD23/FMC7 expressionpattern achieved by multicolor immunophenotyping facilitates accurate and reproducible classification ofB-cell lymphomas and has diagnostic utility. Cytometry (Clin. Cytometry) 50:1–7, 2002.© 2002 Wiley-Liss, Inc.

Key terms: flow cytometry; leukemia; lymphoma; monoclonal gammopathy; immunophenotype; phenotype;diagnostic immunology; disease classification

The accurate diagnosis of lymphoproliferative disorder(LPD) is one of the most challenging in surgical pathology.Several ancillary techniques, such as flow cytometry, cy-togenetics, and molecular DNA-based studies, play an im-portant role in diagnostic accuracy. None of these is de-signed to replace histopathology, but enhances theaccuracy and precision of lymphoma diagnosis and sub-classification. The immunophenotypic approach to lym-phoma classification utilizes a variety of monoclonal anti-bodies, which can be used in different combinations andemployed in routine clinical diagnostic practice as eithertissue immunohistochemistry or multiparameter flow cy-tometry. There is little consensus as to the best approachto flow cytometric combinations of monoclonal antibod-ies. Some attempts to reach consensus agreement on theoptimal and minimal acceptable diagnostic approaches inleukemia and lymphoma diagnosis and classification havebeen published (1–3). However, clinical practice still islargely individualistic and based more on regional appren-tice style teaching rather than on published data.

The use of flow cytometry is being recognized increas-ingly as a necessary diagnostic evaluation for the diagnosis

of B-cell LPDs. Many studies have been performed todetermine the expression patterns of different antigens(e.g., CD5, CD10, CD20, and CD23) on different types ofB-cell LPDs and their utilization for subclassification of thedisorders. The antigens CD23 and FMC7 are expressed onnormal B cells and are utilized commonly in the immuno-phenotypic subclassification of suspected LPD (4–9).However, the diagnostic importance of studying the co-existent expression pattern of these two antigens (CD23and FMC7) has not been studied extensively. Our recentstudy (10) of CD23 and FMC7 antigen coexistent expres-sion patterns in tissue biopsies from patients with LPDindicated diagnostic usefulness in the classification of lym-phoma. For this reason, we designed this study to evaluatecoexistent expression patterns of CD23 and FMC7 incases of B-cell LPD with firmly established diagnosis ofchronic lymphocytic leukemia (CLL), prolymphocytoid

*Correspondence to: Bruce H. Davis, Maine Medical Center ResearchInstitute, 81 Research Drive, Scarborough, ME 04074.

E-mail: davisb@mmc.orgReceived 21 May 2001; Accepted 25 October 2001

Cytometry (Clinical Cytometry) 50:1–7 (2002)DOI 10.1002/cyto.10045

© 2002 Wiley-Liss, Inc.

transformation of chronic lymphocytic leukemia (CLL/PL), prolymphocytic leukemia (PLL), hairy cell leukemia(HCL), mantle cell lymphoma (MCL), marginal zone lym-phoma (MZL), Waldenstrom’s macroglobulinemia (WM),large cell lymphoma (LCL), follicular cell lymphoma(FCL), and Burkitt’s lymphoma (BL) in blood and bonemarrow specimens. We also evaluated other antigens(CD5, CD10, CD11c, CD19, CD20, CD25, and CD103) inall above subclassifications of B-cell LPD in an effort todefine a robust diagnostic algorithm integrating our obser-vations on CD23 and FMC7 coexistent expression.

MATERIALS AND METHODSPatient Samples

From January 1997 to August 1999, samples of periph-eral blood and bone marrow were analyzed retrospec-tively in the Analytical Cytometry Lab at William Beau-mont Hospital (Royal Oak, MI). The samples were takenfrom 218 patients with a firmly established diagnosis ofB-cell LPD. The study was carried out in accordance withthe institutional guidelines for human investigation. Allphenotypic studies were correlated with morphologicfindings and other laboratory studies (molecular diagnos-tic gene rearrangements, cytogenetics, and immunohisto-chemistry) to establish a definitive diagnosis of LPD bytrained hematopathologists at William Beaumont Hospi-tal.. The cases were re-reviewed to confirm diagnosticuniformity for the purposes of this study by the authorsusing the diagnostic criteria of the revised European Amer-ican lymphoma (REAL) classification.

Flow Cytometric Analysis

Peripheral blood (5 ml of EDTA anticoagulated specimen)and bone marrow (1–2 ml of EDTA or heparinized bonemarrow aspirate) were diluted to 15 ml with 0.1% of bovineserum albumin/phosphate-buffered saline (BSA/PBS). Thispreparation was washed with centrifugation at 1,500 rpmtwice more with 0.1% BSA/PBS in an automated cell washer(DAC II, Baxter Scientific, Chicago, IL). Preconjugated mono-clonal antibodies used as a three-color panel for lymphomaon 10 aliquots of the sample included CD2, CD3, CD4, CD5,CD7, CD8, CD10, CD16, CD19, CD20, CD22, CD23, CD38,FMC7, CD45, and kappa and lambda light chains. The com-bination of CD23-PE (Becton-Dickinson ImmunocytometrySystem [BDIS], San Jose, CA) and FMC7-FITC (Immunotech,Marseilles, France, a Beckman Coulter company) was em-ployed on all samples. Immunophenotyping staining proce-dures were standard “whole blood lysis” techniques usingFACSLyse (BDIS). Specimens were analyzed on a FACScanflow cytometer (Becton Dickinson, San Jose, CA) equippedwith Cellquest software or on a Cytoron Absolute flow cy-tometer (Ortho Diagnostic Systems, Johnson & Johnson Co.,Raritan, NJ) equipped with Ortho Research software. Theflow cytometers acquired a minimum of 10,000 cellularevents as list mode files containing forward angle scatter, logside scatter, and three log fluorescence signals. List modefiles were analyzed using WinList software (Verity SoftwareHouse, Topsham, ME) and CD45/side scatter gating methods(11). Expression of the antigen was interpreted and scored

for this study as follows: positive (�), negative (-), and partial(�30%) or minor subset expression (�). This arbitrary dis-tinction at the 30% level for partial expression was selectedusing isotype controls to establish the cursor placement forantigen positivity. Additionally, it refers to 30% of the abnor-mal B-cell population and not to the entire lymphocyte pop-ulation, which reflects some degree of subjectivity or judge-ment.

RESULTSExamples of the various CD23 and FMC7 antigen ex-

pression patterns identified among the 218 cases areshown in Figure 1. As T cells included in any lymphoidpopulation visualized using CD45 side scatter gating anal-ysis of list mode files represent a subpopulation of cellslacking both CD23 and FMC7 expression, interpretationof the clonal B-cell population CD23 and FMC7 expressionmust take this fact into account. The complete results ofCD23 and FMC7 coexpression patterns and LPD diagnos-tic subtypes are summarized in Table 1.

CLL

All of the diagnosed cases of CLL (n � 121) expressedCD23 and CD5. Of these 121 cases, 105 cases (88%) wereCD23(�), but did not coexpress FMC7. In the remaining15 cases (12%), only a minor subset of the clonal B lym-phocytes coexpressed FMC7.

CLL/PL

In this diagnostic category, defined as 10–55% of thelymphocytes showing prolymphocytoid morphology, sixof eight cases (75%) were CD23(�). Of these six cases,only one was negative for FMC7, four showed a minorsubpopulation of FMC7 positive cells, and one case fullycoexpressed CD23 and FMC7. The remaining two caseswere FMC7 (�) but did not coexpress CD23.

PLL

The two cases with a PLL diagnosis were CD23(-) andFMC7(�).

MCL

All 15 cases were CD23(-) and expressed FMC7.

WM

Six cases were reviewed under this category, which isconsidered a form of lymphoplasmacytoid leukemia/lym-phoma (LPL). Four cases (67%) were CD23(-) andFMC7(�). One case coexpressed CD23 and FMC7 and theother was CD23(�) and FMC7(-).

MZL

All 20 cases were CD23(-) and FMC7(�). These casescomprised cases of lymph node-based disease with bloodor marrow involvement and cases of classical splenic MZL.

2 AHMAD ET AL.

FCL

Of 24 cases, 17 (71%) were CD23(-) and FMC7(�). Theremaining seven cases (29%) were FMC7(�) with a minorsubpopulation of CD23 (�) cells.

HCL

All 14 cases were CD23(-) and FMC7(�).

Diffuse LCL

The largest variation of patterns was seen in this group.Unlike that found in the CLL group, the pattern spread

was due to variability in the expression of both CD23 andFMC7. These six cases showed three different patterns ofCD23 and FMC7 coexpression. Two cases were CD23(-)and FMC7(�), two cases were CD23(�) with a subpopu-lation of FMC7(�), and the remaining two cases lackeddetectable expression of both CD23 and FMC7 (dual neg-ative).

BL

Both cases were CD23(-) and FMC7(�).

FIG. 1. Coexistent expression patterns of CD23 and FMC7 commonly seen in various B-cell LPDs. The abnormal lymphoid populations are present invarious proportions, but represent a significant population among lymphoid cells gated in blood and bone marrow samples using CD45 and side scatteranalysis methods. Patterns of CD23 and FMC7 coexpression as categorized in the study include CD23(�)/FMC7(�) (histogram A), CD23(�)/FMC7(�)(histograms B,H), CD23(�)/FMC7(�) (histogram C), CD23(�)/FMC7(�) (histogram D), CD23(�)/FMC7(�) (histograms E,F), CD23(�)/FMC7(�)(histogram G), and CD23(�)/FMC7(�) (histogram I). Benign T cells in the CD45/side scatter gated population also constitute a population of CD23(�)and FMC7(�) lymphoid cells.

3CLINICAL UTILITY OF CD23 AND FMC7

Various studies (12–14) have reported a relationshipbetween CD20 expression and the FMC7 antigen epitope.In particular, these studies raise technical issues withregard to simultaneous multicolor immunophenotypicstaining with FMC7 and anti-CD20 and raise questions asto the diagnostic usefulness of the FMC7 antigen. Ourimmunophenotypic panel for the study of our 218 cases ofB-cell LPD utilized an anti-CD20 reagent (clone L27, peri-dinin chlorophyll protein labeled; Becton Dickinson) in aseparate tube (along with anti-immunoglobin light chainantibodies) from that of the FMC7. Thus, we comparedthe CD20 expression with that of FMC7 expression with-out concern for steric hindrance of antibody binding. Weobserved no reliably consistent correlation between CD20and FMC7 expression among our cases (Fig. 2).

DISCUSSIONUtilization of flow cytometry in the diagnosis and sub-

classification of B-cell LPD is very sensitive and specific(1,15). Immunophenotypic characterization of certainsubgroups of B-cell LPD is well documented and generallywell accepted as the major defining feature of some spe-cific LPD entities (1, 8–10, 15–25). Specific LPD entitiesdefined by immunophenotype with minimal morphologiccorrelation include CLL (CD5�, CD10-, CD23�, FMC7-),CLL/PL (CD5�, CD10-, CD23�, FMC7�), MCL (CD5�,CD10-, CD23-, FMC7�), MZL (CD5-, CD10-, CD23-,FMC7�), and HCL (CD5-, CD10 -/�, CD23-, FMC7�,CD25�, CD11c�, CD103�). Our findings indicate thatthe coexistent expression pattern of CD23 and FMC7 canadd further information toward the reproducible subclas-sification of LPD in blood and bone marrow specimens,similar to that previously reported for diagnostic tissuebiopsies (10).

After showing the monoclonality for kappa or lambdalight chains, the expression of CD5 on B cells is a mostimportant step in the subclassification of B-cell LPD. TheB-cell LPD that expresses CD5 includes CLL and it variantand MCL. The presence of CD5 expression in B-cell CLL iswell documented and is recognized in a recent classifica-tion system (18–22). Our findings closely parallel recentstudies indicating that CD5- CLL, if truly an entity, consti-tutes a very minor population of patients with this form oflow-grade B-cell leukemias (20). Most of these reportedCD5- B-cell CLLs show more aggressive clinical behavior(20,26). In our study, none of 121 cases of B-cell CLL were

truly negative for CD5 expression, all expressed CD23,and only 15 cases show a subpopulation that expressesFMC7. CLL/PL and PLL are considered variants related toCLL, although the true origin of PLL remains controversial.Both of these variants are different from B-cell CLL by theirclinical behavior and immunophenotypical properties(26). When B-cell CLL is being transformed into CLL/PL,the percentage of FMC7� cells increases as does theexpression of surface immunoglobulin. The expression ofCD5 remains on these neoplastic cells during this trans-formation. In our study, we observe the same pattern ofpersistent CD5 expression with FMC7 in cases of CLL/PL(n � 8) and PLL (n � 2). Only one of eight cases of CLL/PLwas negative for FMC7. Both our cases of PLL were posi-tive for FMC7. Our findings indicate the pattern ofCD23(�) and FMC7(�) to be very characteristic of CLL/PL, thus providing a means of indicating patients with apotentially more aggressive clinical course and worthy ofcloser clinical follow-up. A minor subset of CLL cases (15of 121) also had coexistent expression of CD23 and FMC7,which we speculate to be cases in the process of earlytransformation into CLL/PL. As the number of PLL cases islimited in this study, we hesitate to generalize from ourexperience of only two cases, other than to confirm pre-vious observations of PLL being FMC7� (19,22). Anothersubgroup in B-cell LPD that expresses CD5 is mantle cellleukemia/lymphoma (21,23). Recognition of this sub-group is of clinical importance due to the relatively pooroutcome and potentially different clinical management forpatients with MCL (26,27). Expression of CD23 in B-cellCLL and coexpression of CD23 and FMC7 in CLL/PL andPLL distinguish these more indolent LPD subtypes fromMCL (CD23- and FMC7�) as initially described by Stein etal. (28). There are few reported cases of MCL with CD23expression (23). All cases (N � 15) in our study of MCLwere CD23(-) and FMC7(�).

In cases of CD5- B-cell LPD, the diagnostic possibilitiesincludes MZL, FCL, HCL, WM, LCL, and BL. Although MZLis typically CD5-, there are rare cases reported that showCD5 expression (29,30). Most of these cases presentedwith more advanced disease and frequent relapses, whichis unusual for this low-grade B-cell LPD. All of our cases ofMZL were CD5-, CD23-, and FMC7�. Follicular center celllymphoma is clonal B lineage with a common expressionof CD10 and/or CD38, which is typically CD5- (31). In our

Table 1Observed Immunophenotypic Patterns of CD23 and FMC-7 in LPD Subtypes*

CD23/FMC-7pattern

LPD diagnosisCLL CLL/PL PLL WM MCL MZL HCL FCL LCL BL

�/� 106 1 0 1 0 0 0 0 0 0�/� 15 4 0 0 0 0 0 0 0 0�/� 0 1 0 1 0 0 0 0 0 0�/� 0 2 2 4 15 20 14 17 2 2�/� 0 0 0 0 0 0 0 7 2 0�/� 0 0 0 0 0 0 0 0 2 0

*Scoring of antigen expression: �, positive; �, negative; �, dim or partial expression.

4 AHMAD ET AL.

study, all our cases were CD5-, CD10�, FMC7�. Theexpression of CD23 was variable, as 17 cases were nega-tive and 7 cases showed a positive subpopulation of ab-normal cells. These two patterns of coexistent expres-sions were similar to our previous observations of FCL intissue biopsies (10). Interestingly, although only 24 casesof FCL were studied in these blood and bone marrowspecimens, the majority of cases lacked significant CD23expression. In our previous study of 62 cases of FCL fromtissue biopsy specimens (10), 45 of 64 cases exhibitedsignificant CD23 expression The significance of this ap-parent anatomical difference is unknown, but one might

speculate this phenotypic difference could relate to eitherclonal evolution in advanced stage disease or a predilec-tion for FCL cells lacking CD23 to enter the peripheralblood circulation and seed the bone marrow.

Previous studies by Hubl et al. (12) suggested that FMC7antigen expression paralleled that of CD20, implying theinformation to be redundant. However, their methodol-ogy paired FMC7 in the same staining antibody cocktailwith CD20, which has been reported to block potentiallythe FMC7 binding epitope (13,14). Recent evidence bySerke et al. (13) indicated that the FMC7 antibody is in factbinding to a CD20-related epitope. However, the level of

FIG. 2. Expression of CD20 does not parallel FMC7 expression in LPDs. Eleven cases of various types of LPD are shown. They demonstrate CD20 andFMC7 antigen density, but no consistent relationship of the expression level or pattern is observed. [Color figure can be viewed in the online issue, whichis available at www.interscience.wiley.com.]

5CLINICAL UTILITY OF CD23 AND FMC7

expression of the FMC7 epitope and CD20 expression, atleast in neoplastic LPD cells, is far from a perfect correla-tion. Our results of CD20 expression measured separatelyfrom the sample studied for FMC7 showed an imperfectrelationship and indicated that FCM7 expression providesdiagnostic information incremental to that of CD20 ex-pression levels. The histograms in Figure 2 were selectedto show the spectrum of interrelationships in the bindingof the CD20 and FMC7 clones used in our study and donot represent an exhaustive study into the relationship ofthese two B-cell epitopes. The difference in our findingsfrom those of Hubl et al. (12) might be due to cloneselection or to the fact that the expression of these twoantigenic sites was measured in separate tubes. Althoughthe purpose of this study was not focused on the relation-ship between CD20 clones and FMC7 epitope expression,our observations seemingly refute the concept that FMC7expression can be predicted simply by other reagentsdirected to CD20.

In summary, we found that simultaneous staining ofB-cell neoplasms for expression of CD23 and FMC7 is auseful tool to assist in the reproducible subclassification ofLPD in blood and bone marrow, particularly in thoseinstances where it is clinically relevant for prognostic andtherapeutic purposes. The CD23/FMC7 pattern of coex-pression, when used in combination with other monoclo-nal antibodies, has a similar diagnostic utility as the CD5/CD20 expression pattern. We believe that CD23, FMC7,and CD5 expression in clonal B-cell populations providesa diagnostic algorithm (Fig. 3) that can be utilized adjunc-tively with other data. In our experience, this resulted inthe accurate classification of �95% of B-cell LPD. Thisstudy on blood and bone marrow specimens yielded sim-ilar patterns in various subtypes of LPD to those observedin our previous study on tissue biopsies (10), thus makingthe proposed diagnostic algorithm useful in all types of

diagnostic specimens. We conclude that the simultaneousCD23/FMC7 coexistent expression pattern, as defined byflow cytometric immunophenotyping in conjunction withother antibodies, is a valuable contribution toward accu-rate and reproducible classification of B-cell LPD. Theprognostic significance of the CD23 and FMC7 expressionpattern in lymphoma requires more extensive study, butmay be worthwhile, especially in low-grade LPD, where aheterogeneity of CD23 and FMC7 antigen coexistent ex-pression patterns occurs.

ACKNOWLEDGMENTSWe are grateful to Katharine A. Steel, Cindy Sounart-

Miscovitch, and Carole A. Ceckowski for their experttechnical assistance with flow cytometric analysis.

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7CLINICAL UTILITY OF CD23 AND FMC7