11% of APLs in our series were CD56(+) by flow cytometry, which is comparable to data

reported in the literature (11-15%).

CD56 expression correlated with microgranular morphology and high WBC count, as

shown by other authors.

In contrast to a recent study, there was no association of CD56 expression with CD2,

CD7, CD15, CD34, CD117, or HLA-DR positivity. In addition, we did not find an association

with increased risk of relapse in CD56(+) APLs.

H. Olteanu, MD, PhD; A.M. Harrington, MD; S.H. Kroft, MD; *N.J. Karandikar, MD, PhD, and *F.F. Fuda, DO.

Departments of Pathology, Medical College of Wisconsin, Milwaukee, WI and *UT Southwestern Medical Center, Dallas, TX

The Incidence of CD56 Expression by Flow Cytometry in Acute Promyelocytic Leukemia

in Patients Treated with All-Trans Retinoic Acid and Anthracycline Drug Combinations

Conclusions

Introduction

Background: Recent cooperative studies have

linked CD56 positivity in acute promyelocytic

leukemia (APL) with an increased risk of relapse, and

with the presence of immaturity-associated and T-cell

antigens on leukemic promyelocytes. The drawbacks

of these multicenter studies are twofold: lack of

centralized immunophenotypic analysis prevented a

systematic standardization of flow cytometric results,

and a possible selection bias resulting from not all

centers assessing for CD56 expression. Because of

these confounding factors, we studied the expression

of CD56 by flow cytometry in APL with a rigorously

standardized flow cytometric protocol, and correlated

it with clinicopathologic parameters.

Design: 64 consecutive diagnostic APL bone

marrows or peripheral bloods were evaluated by 4-

color flow cytometry and cluster analysis, with

antibodies against CD2, CD3, CD4, CD5, CD7, CD8,

CD10, CD11b, CD13, CD15, CD16, CD19, CD20,

CD33, CD34, CD38, CD45, CD56, CD79a, CD117,

HLA-DR, MPO, and Tdt. An antigen was considered

positive in promyelocytes if >20% cells exceeded a

2% isotype control threshold. CD56 expression

status was correlated with clinical and pathologic

parameters.

Results: 7/64 APLs (11%) were CD56(+).

Comparative clinicopathologic parameters for all APL

cases, based on CD56 expression status, included

age, gender, morphology (microgranular vs.

granular), CBC data, cytogenetic and molecular

findings. A higher proportion of patients with CD56(+)

APL had microgranular morphology (p=0.027), and

they presented with a higher WBC count (p=0.003),

as compared to those with CD56(-) APL. There were

no other immunophenotypic differences between the

two groups.

Conclusion: 11% of APLs in our series are CD56(+)

by flow cytometry, which is comparable to data

reported in the literature (11-15%). CD56 expression

correlated with microgranular morphology and high

WBC count, as shown by other authors. In contrast

to a recent study, there was no association of CD56

expression with CD2, CD7, CD15, CD34, CD117, or

HLA-DR positivity.

Abstract

Materials and Methods

Acute promyelocytic leukemia (APL) is a specific subtype of acute myeloid leukemia

(AML) characterized by distinct morphologic, immunophenotypic, cytogenetic, and

clinical features. The unique chromosomal abnormality, t(15;17), results in the

formation of the PML/RARalpha fusion protein and a subsequent block in

granulocytic differentiation. Clinically, this blockade can be overcome by

pharmacologic doses of all-trans retinoic acid (ATRA), which may also ameliorate the

severe bleeding diathesis that is frequently associated with APL. The combination of

ATRA and chemotherapy as frontline therapy has dramatically improved the outcome

of the disease, so that most patients treated with this regimen are expected to

become long-term survivors. However, as a result of early hemorrhagic death and

early disease relapse, treatment failure is still recorded in 5-30% of patients with APL.

It is therefore useful to identify prognostic markers that are associated with these

adverse events. Of the prognostic factors that may predict for an inferior outcome in

APL, older age, high initial leukocyte count, and low platelet count have been

associated with increased relapse risk. More recent reports have demonstrated that

the expression of CD56 was associated with an unfavorable clinical outcome.

CD56 is an isoform of the neural cell adhesion molecule (NCAM) involved in cell-to-

cell interaction; it also may function in cell-mediated cytotoxicity. This antigen is

normally expressed in NK cells and a subset of cytotoxic T cells, and has been also

detected in various hematopoietic neoplasms. Recent studies have linked CD56

positivity in APL with an increased risk of relapse, and with the presence of

immaturity-associated and T-cell antigens on leukemic promyelocytes. The

drawbacks of these multicenter studies are twofold: lack of centralized

immunophenotypic analysis prevented a systematic standardization of flow

cytometric results, and a possible selection bias resulting from not all centers

assessing for CD56 expression. Because of these confounding factors, we studied

the expression of CD56 by flow cytometry in APL with a rigorously standardized flow

cytometric protocol, and correlated it with clinicopathologic parameters.

64 patients with APL from the Medical College of Wisconsin and UT Southwestern

Medical Center, that had flow cytometry (FC) data available for review were included

in the study.

Four-color FC was performed with antibodies against CD2, CD3, CD4, CD5, CD7,

CD8, CD10, CD11b, CD13, CD15, CD16, CD19, CD20, CD33, CD34, CD38, CD45,

CD56, CD79a, CD117, HLA-DR, MPO, and Tdt. Positivity for an antigen was defined

as at least 20% of events beyond a 2% isotype control threshold.

Clinical and laboratory data was available from chart review.

Results

Figure 1. Immunophenotypic findings in a patient with CD56(+) acute promyelocytic

leukemia. Neoplastic promyelocytes are in red, normal T cells are in green, B cells are in

blue, plasma cells are in yellow, and NK cells are in violet.

7/64 APLs (11%) were CD56(+).

Comparative clinicopathologic parameters for all APL cases, based on CD56

expression status, are presented in Table 1. A higher proportion of patients with

CD56(+) APL had microgranular morphology (p=0.027), and presented with a higher

WBC count (p=0.003), as compared to those with CD56(-) APL .

There were no other differences, including immunophenotypic features, between

the two groups.

Table 1. Comparison of clinical and laboratory findings in patients

with acute promyelocytic leukemia (APL), separated based on

CD56 expression status.

Parameter CD56(-) CD56(+) p

n (%), APL 57 (89%) 7 (11%)

Age, median, (range) 42 (20-93) 46 (25-88) 0.941

Age, ≥ 65 years 14.0% 14.3% 1.000

M:F 24:33 2:5 0.695

Microgranular 15.8% 57.1% 0.027

WBC, x10e3/uL,

median, (range)

2.9

(0.43 – 126.8)

54.9

(2.9 – 107.0) 0.003

Hgb, g/dL, median,

(range)

9.7

(5.8 – 14.6)

9.6

(4.8 – 12.0) 0.926

Platelets, x10e3/uL,

median,(range)

27

(3 – 245)

73

(17 – 280) 0.135

DIC 56.1% 42.9% 0.687

Complex cytogenetics 33.6% 14.3% 0.409

Relapse rate 9.6% 14.3% 0.548

Immunophenotype

CD2(+) 41.8% 42.9% 1.000

CD4(+) 14.0% 28.6% 0.299

CD7(+) 7.0% 0% 1.000

CD10(+) 0% 0% 1.000

CD13(+) 98.2% 100% 1.000

CD14(+) 0% 0% 1.000

CD15(+) 87.7% 71.4% 0.253

CD33(+) 98.2% 100% 1.000

CD34(+) 33.3% 57.1% 0.239

CD38(+) 94.7% 100% 1.000

CD45(+) 100% 100% 1.000

CD64(+) 57.9% 71.4% 0.691

CD117(+) 97.8% 100% 1.000

HLA-DR(+) 10.5% 14.3% 0.574

MPO(+) 100% 100% 1.000

Tdt(+) 4.9% 14.3% 0.335