Comparison of Bone Marrow and PeripheralBlood ZAP-70 Status Examined by Flow

Cytometric Immunophenotyping in Patientswith Chronic Lymphocytic Leukemia

Rachel Sheridan,1 Taofic Mounajjed,1 Diane E. Ehrmann,2

Paul E. Hurtubise,1 and Jeffrey A. Schrager1*1Department of Pathology and Laboratory Medicine, Division of Hematopathology,

University of Cincinnati Academic Health Center, Cincinnati, Ohio2Flow Cytometry Section, Quest Diagnostics, Inc., Cincinnati, Ohio

Background: The mutational status of the immunoglobulin heavy chain variable gene in patients withchronic lymphocytic leukemia correlates with prognosis. Patients with mutated IgVH genes fare betterthan those with unmutated genes. Gene expression profiling studies identified the tyrosine kinase ZAP-70to be expressed in unmutated CLL samples. Flow cytometric examination of ZAP-70 expression in tumorcells has been proposed to be a convenient surrogate marker for IgVH mutational status. However, a fewstudies have shown a small number of discordant results between ZAP-70 positivity, IgVH mutational sta-tus, and clinical outcome. There have been no reported studies comparing bone marrow samples with pe-ripheral blood for ZAP-70 expression in CLL patients.Methods: We searched our flow cytometry files from October 2004 through April 2006 and identified

CLL in 311 bone marrow and peripheral blood specimens from 256 patients. We defined ZAP-70 positiv-ity as greater than 30% of the CD19+ B-cells above the isotype control value that coexpress ZAP-70. Sta-tistical analyses were performed using the Fisher exact test and student t-test.Results: A significantly greater number of bone marrow specimens were positive for ZAP-70 when

compared with the number of peripheral blood specimens. Of all the ZAP-70 negative specimens, CLLcells from bone marrow had a greater mean percentage of ZAP-70 positive cells when compared with theCLL cells from peripheral blood. Finally, six patients were identified who were ZAP-70 positive in thebone marrow but ZAP-70 negative in the peripheral blood.Conclusions: These results may be due to either an increase in the false positive rate in bone marrow

specimens or to an intrinsic feature of CLL cells in the compartment that is biologically distinct fromperipheral tumor cells. As prognosis and treatment decisions may be based on ZAP-70 results from eitherspecimen type, it is prudent to further examine this observation. q 2006 International Society for Analytical

Cytology

Key terms: CLL; ZAP-70; flow cytometry; bone marrow; peripheral blood

The mutational status of the immunoglobulin variableheavy (IgVH) chain gene has been shown to have prog-nostic significance in patients with chronic lymphocyticleukemia. Patients with unmutated (germ-line) IgVH

genes tend to have a worse prognosis than those withmutated genes. Most clinical laboratories do not havethe ability to perform sequencing studies to directlyassess the mutational status; therefore, surrogate markersbased on gene expression profiling data have been iden-tified (1). One such marker is ZAP-70, which is a proteintyrosine kinase that is constitutively expressed in thecytoplasm of T-cells. It is part of the signal transductioncascade in T-cell receptor signaling. Many studies have

shown that CLL cells that express ZAP-70 correlate withthe unmutated form of IgVH (1) and thus portend aworse prognosis. Using cell permeabilization techniques,cytoplasmic ZAP-70 expression can be readily assessedby flow cytometric immunophenotyping (FCM) (2,3).Multicolor testing permits gating strategies to distinguish

*Correspondence to: Jeffrey A. Schrager, E-mail: jeffrey.schrager@uc.eduReceived 8 May 2006; Accepted 14 May 2006Published online in Wiley InterScience (www.interscience.wiley.

com).DOI: 10.1002/cyto.b.20128

Cytometry Part B (Clinical Cytometry) 70B:320–321 (2006)

q 2006 International Society for Analytical Cytology

CLL cells from T-cells. To date, a consensus FCM ZAP-70assay has not been widely advanced; however, numerousabstracts and studies have compared various methodolo-gies. Most of these studies have been on peripheralblood samples. Our flow cytometry facility supports alarge regional hematology practice, and we receive ahigh volume of both peripheral blood and bone marrowspecimens in the work-up of patients with a clinical sus-picion or documented case of CLL. Since October 2004,we have been performing ZAP-70 FCM assays on sam-ples containing a monoclonal B-cell population with atypical CLL immunophenotype (CD19+, CD20+, CD5+,CD23+, CD10�, FMC-7�, and surface light chain re-stricted). To our knowledge, a comparison of peripheralblood and bone marrow ZAP-70 status by FCM has notbeen previously published. Here we report our observa-tions in these two types of specimens commonly submit-ted in this clinical setting.

METHODS

Our flow cytometry files were searched for peripheralblood and bone marrow cases accessioned from October2004 through April 2006 with an immunophenotypeconsistent with CLL (surface immunoglobulin light chain-restricted B-cell population positive for CD19, CD20,CD5, CD23, and negative for FMC-7). Standard FCM assayconditions were employed. For the detection of ZAP-70,lysed and washed cells were stained for surface markersfollowed by a permeabilization step using Caltag Fix andPerm kit reagents. Anti-ZAP-70 monoclonal antibody(FITC-conjugated clone 1E7.2, Caltag) was used to bind in-tracellular ZAP-70. Samples were acquired on BeckmanCoulter EPIC-C flow cytometry instrument. Quadrantswere set based on isotype controls, and ZAP-70 status wasrecorded as positive if 30% or more of the B-cells ex-pressed more ZAP-70 when compared with the isotypecontrol quadrant marker. T-cells, which express brightZAP-70, served as an internal positive control.

RESULTS AND DISCUSSION

A retrospective search of our files identified 311 pe-ripheral blood and bone marrow samples from 256patients with CLL. The patients ranged in age from 25 to95 with a mean age of 68 years. The male to female ratiowas 1.6:1. 193 samples were from peripheral blood and118 samples were from bone marrow. ZAP-70 determina-tions were performed in 91% (176 of 193) of the periph-eral blood samples and in 75% (89 of 118) of the bonemarrow specimens. We found a significant differencebetween the number of bone marrow and peripheralblood specimens that were ZAP-70 positive. Ninety-onepercent (81/89) of the bone marrow samples were ZAP-

70 positive; whereas, only 69% (121/176) of the periph-eral blood specimens were positive (Fisher’s exact testtwo-sided P-value <0.0001) (Table 1).Furthermore, in the cases that were ZAP-70 negative

the mean percentage of ZAP-70+ B-cells was significantlyhigher (Student t-test two-sided P-value ¼ 0.0076) in thebone marrow (21.6%, SD ¼ 3.7) when compared withthat of the peripheral blood samples (14.0%, SD ¼ 7.6).Thus on statistical grounds, the CLL cells in the bonemarrow specimens, defined by the 30% cut-off to beZAP-70 negative, are different from the CLL cells in theZAP-70 negative peripheral blood specimens. Whetherthis represents a true biologic difference in CLL cells ofthe two compartments is unknown.In addition, six patients were identified with discord-

ant ZAP-70 results. Their bone marrows were ZAP-70positive while their peripheral blood samples were nega-tive. In one of these six, the specimens were collectedtwo days apart. The possibility of a biologic difference inCLL cells in the bone marrow when compared withthose in the peripheral blood may exist. One studyrecently identified ZAP-70 positivity in activated non-neo-plastic B-cells (4). Perhaps activational status may differ-entiate CLL cells in the two compartments. Regardless ofthe cause, however, additional studies are needed com-paring peripheral blood and bone marrow in relation toIgVH mutational status, ZAP-70 expression, and clinicaloutcome.

LITERATURE CITED1. Wiestner A, et al. ZAP-70 expression identifies a chronic lympho-

cytic leukemia subtype with unmutated immunoglobulin genes, in-ferior clinical outcome, and distinct gene expression profile. Blood2003;101:4944–4951.

2. Gibbs G, et al. Comparison of flow cytometric methods for the mea-surement of ZAP-70 expression in a routine diagnostic laboratory.Clin Lab Haematol 2005;27:258–266.

3. Schroers R, et al. Combined analysis of ZAP-70 and CD38 expres-sion as a predictor of disease progression in B-cell chronic lympho-cytic leukemia. Leukemia 2005;19:750–758.

4. Cutrona G, et al. B lymphocytes in humans express ZAP-70 whenactivated in vivo. Eur J Immunol 2006;36:558–569.

Table 1Results of Retrospective Study of Flow Cytometry Files

Bone marrow Peripheral blood

No. of Specimenstested for ZAP-70 89 176

ZAP-70(+) specimens 81 (91%) 121 (69%)ZAP-70(�) specimens 8 (9%) 55 (31%)Mean % of B-cellscoexpressing ZAP-70in ‘‘negative’’ samples 21.6 (3.7)a 14.0 (7.6)a

aValues in parentheses indicate standard deviation.

321ZAP-70 IN BONE MARROW VS. PERIPHERAL BLOOD IN CLL PATIENTS

Cytometry Part B: Clinical Cytometry DOI 10.1002/cyto.b