ZAP 70 EXPRESSION IN BONE MARROW BIOPSY OF CHRONIC LYMPHOCYTIC LEUKEMIA AMONG IRAQI PEOPLE

Abstract

Background:Chronic lymphocytic leukemia (CLL) is a chronic B-lineage lymphoproliferativedisorder characterized byspecific morphological and immunophinotyping features.Ithas a variable clinical course and several biological markers which were used to assess the disease activity and prognosis. Objective:This was designed to determine the frequency of Immunohistochemical expression of CD5 and ZAP 70 (zeta associated protein of 70 kDa)in bone marrow biopsy of CLL patients and detect the significance of ZAP 70 immunohistochemical expression in bone marrow biopsy as prognostic factor. Patients and methods: This retrospective cross-sectional studyis conducted on fortyformalin fixed paraffin embedded blocks of chronic lymphocytic leukemia cases collected from teaching laboratories of medical city in Baghdad along with ten control cases of normal bone marrow during October 2010through April 2011.The sections of bone marrow biopsies were processed routinely with immunohistochemical stainfor CD 5 and ZAP 70 and examined by light microscope. Results: Main clinical findings of CLL cases were lymphadenopathy, splenomegaly, hepatomegaly, weight loss and fever respectively while asymptomatic cases constitute only 10 % of the cases. Main laboratory findings were leukocytosis , absolute lymphocytosis,anemia and thrombocytopenia. Most of these cases were within the high risk group by use modified Rai staging system .CD 5 expressions was found in all cases. So, there is no statistical significant association with disease activity or prognosis, while ZAP 70 expressed in 48 % of study cases and our study revealed that there was a statistical significant association between ZAP 70 expression and the clinical stage of the disease using Modified Rai system .Moreover, there was a statistical significant inverse association between ZAP 70 expression with PCV % and platelet count. Whereas no statistical significantassociation between ZAP 70 expression and white blood cell count , lymphocyte % in the Bone Marrow and peripheral blood or bone marrow involvement by lymphocytes. Conclusions: This study revealed that CD 5 is expressed nearly in all cases of CLL and gives information that this disease is mostly a B cell linage neoplasm and carry's no significance with disease progression, ZAP 70 is considered an important prognostic marker in CLL and closely related to clinical stage of the disease. Therefore it can predict a clinical course in CLL, ZAP 70 positive score is in significant association with advanced stage of CLL and usually with worse outcome and there is no significant association between CD 5 and ZAP 70 expression in CLL cases was noted.

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ISSN 2320-5407 International Journal of Advanced Research (2016), Volume 4, Issue 3, 849-854
849
Journal homepage:http://www.journalijar.com INTERNATIONAL JOURNAL
OF ADVANCED RESEARCH
RESEARCH ARTICLE
ZAP 70 EXPRESSION IN BONE MARROW BIOPSY OF CHRONIC LYMPHOCYTIC LEUKEMIA
AMONG IRAQI PEOPLE.
Mohammed S. AL-MosawyMBChB1,KaswerAl-TurahiMBChB2, RaheemMahdyMBChB3.
1. MScHematopathology
2. FICMSpathology.
3. FICMSHematopathology
Manuscript Info Abstract
Manuscript History:
Received: 14 January 2016
Final Accepted: 18 February 2016
Published Online: March 2016
Key words:
CLL,CD5, ZAP 70 expression.
*Corresponding Author
Mohammed S. AL-
MosawyMBChB.
Background:Chronic lymphocytic leukemia (CLL) is a chronic B-lineage
lymphoproliferativedisorder characterized byspecific morphological and
immunophinotyping features.Ithas a variable clinical course and several
biological markers which were used to assess the disease activity and
prognosis.
Objective:This was designed to determine the frequency of
Immunohistochemical expression of CD5 and ZAP 70 (zeta associated
protein of 70 kDa)in bone marrow biopsy of CLL patients and detect the
significance of ZAP 70 immunohistochemical expression in bone marrow
biopsy as prognostic factor .
Patients and methods: This retrospective cross-sectional studyis conducted
on fortyformalin fixed paraffin embedded blocks of chronic lymphocytic
leukemia cases collected from teaching laboratories of medical city in
Baghdad along with ten control cases of normal bone marrow during October
2010through April 2011.The sections of bone marrow biopsies were
processed routinely with immunohistochemical stainfor CD 5 and ZAP 70
and examined by light microscope.
Results: Main clinical findings of CLL cases were lymphadenopathy,
splenomegaly, hepatomegaly, weight loss and fever respectively while
asymptomatic cases constitute only 10 % of the cases. Main laboratory
findings were leukocytosis , absolute lymphocytosis,anemia and
thrombocytopenia. Most of these cases were within the high risk group by
use modified Rai staging system .CD 5 expressions was found in all cases.
So, there is no statistical significant association with disease activity or
prognosis, while ZAP 70 expressed in 48 % of study cases and our study
revealed that there was a statistical significant association between ZAP 70
expression and the clinical stage of the disease using Modified Rai system
.Moreover, there was a statistical significant inverse association between
ZAP 70 expression with PCV % and platelet count. Whereas no statistical
significantassociation between ZAP 70 expression and white blood cell
count , lymphocyte % in the Bone Marrow and peripheral blood or bone
marrow involvement by lymphocytes .
Conclusions: This study revealed that CD 5 is expressed nearly in all cases
of CLL and gives information that this disease is mostly a B cell linage
neoplasm and carry’s no significance with disease progression, ZAP 70 is
considered an important prognostic marker in CLL and closely related to
clinical stage of the disease. Therefore it can predict a clinical course in CLL,
ZAP 70 positive score is in significant association with advanced stage of
CLL and usually with worse outcome and there is no significant association
between CD 5 and ZAP 70 expression in CLL cases was noted.

ISSN 2320-5407 International Journal of Advanced Research (2016), Volume 4, Issue 3, 849-854
850
Copy Right, IJAR, 2016,. All rights reserved.
Introduction:-
Chronic lymphocytic leukemia (CLL) is a monoclonal disorder characterized by a progressive accumulation of
functionally incompetent lymphocytes in peripheral blood, bone marrow and lymphoid organs (1). It has been known
that the clinical course of CLL is variable, with some patients experiencing indolent disease not requiring any
therapy, whereas others demonstrate a more aggressive course, unresponsive to therapy, with substantially shorter
survival(3). In B-cell chronic lymphocytic leukemia, lymphocytes typically show B-cell surface antigens as
demonstrated by CD19, CD20, CD21, and CD23 monoclonal antibodies. In addition, they express CD5 which is
more typically found on normal T and subset of normal B cells (4, 5).
CD5 is present in nearly all cases of CLL, SLL and the great majority of MCLs. It is generally absent in other B-cell
lymphomas (6).
ZAP-70 gene codes for a 70 KDatyrosine kinase that is normally expressed in T-cells and NK cells. In the June
2003, Wiestner and colleagues report that increased expression of the ZAP-70 gene (as determined by quantitative
analysis of ZAP-70 mRNA) correlates well with unmutatedIgVH status in patients with CLL. Furthermore, they
found that the expression of the ZAP-70 gene protein (assessed by immunohistochemistry) was also found to have a
good correlation with IgVH mutation status (7). Our trial was designed to estimate the rate of immunohistochemical
expression of ZAP 70 in our country among CLL patient and to study its role in CLL prognosis.
Materials and methods:-
Study group:-
Included forty cases (31 males and 9 females) of CLL with age range from 34 to 75 years. Relevant information
were noted on prepared sheet including age, gender, clinical findings (lymphadenopathy , splenomegaly ,
hepatomegaly , pallor , bleeding , infection , weight loss and other ) and laboratory findings ( CBP , peripheral
blood , bone marrow aspirate and biopsy ) .
Control and comparative group:-
Ten cases (7 male and 3 female) of normal bone marrow biopsy were selected their age range from 35 to 70 years.
Each step done for the control group was in parallel with the study group.
Positive control slides:-
Parallel positive control sections were processed with each set of immunostaining. Positive controls of tonsil tissue
involved by benign reactive inflammation which is known to express CD 5 and ZAP 70 (8)respectively were used
with each run.
Negative control slides:-
Normal bone marrow biopsies were taken as negative control in each run (8).
Immunohistochemical staining protocol:-
The immunostaining method used in the current study was LabelledStrept - Avidin Biotin (LSAB+) technique
which was applied for both CD5 and ZAP 70 staining(17):
Evaluation of all the study cases including:-
1. Reevaluation of all available slides of peripheral blood smear and bone marrow aspirate and biopsy for
CLL cases and assess with their related notes of diagnosis.
2. From bone marrow biopsy of each case two sections had been taken and were stained
immunohistochemically for CD 5 and ZAP 70. The slides were examined by light microscope and scanned
on low and high power (10 xs, 40 xs and 100 xs).
3. Modified Ray staging system was adopted for staging of patients with CLL. Accordingly, they were
classified into three groups: low, intermediate and high risk groups. (Low-risk patients: With only
lymphocytosis in the PB and BM, Intermediate-risk patients: With lymphocytosis and lymphadenopathy

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and/orhepatosplenomegaly and High-risk patients: With lymphocytosis together with anemia (Hb< 11 g/do)
(stage III) and/or thrombocytopenia (18).
Scoring system:-
The scoring system for CD 5 were scored positive if 50% or more of the cells within an aggregate showed cellular
membrane and/or cytoplasmic staining pattern (5) , while , the scoring system for ZAP 70 were scored positive if
20% and more of the cells within an aggregate showed nuclear and cytoplasmic stain (9,10,11) .
Statistical analysis:-
Was performed with SPSS 13 (statistical package for social sciences) and Excel 2003 programs. Data analysis was
done using t- test, analysis of variance (ANOVA) and chi –square test for tables with frequencies, percentages,
range mean and standard deviation, P value was (0.05).
Results:-
The maximum age group (45%) was within the 7th decade of life and more, while (35%) were within the 6thdecade,
(17.5%) within the 5th decade and only (2%) was within the 4th decade. In our study male constitute 77.5% (31/40)
of patient and female were 22.5 % (9/40) so, male to female ratio was about 3.4:1. The most common clinical
features of CLL cases in this study were lymphadenopathy (72.5%) followed by splenomegaly (45%), hepatomegaly
(22.5%), weight loss (20 %) and fever (17.5 %).
Regarding to clinical stage (52.5%) was in the high risk group, (30 %) were in the intermediate risk group and
(17.5%) were in the low risk group by use modified Rai staging system.
Hematological parameters in study group:-
The mean PCV percent was were less than normal range to age and gender in 43% of cases. The platelet count was
less than normal range in 50% of cases. The WBC count was more than normal range in 95% of cases. The
lymphocyte percent in peripheral blood was (89.4 + 9.3) % of circulating white blood cells and majority with typical
mature looking lymphocytes. The mean lymphocyte percent in bone marrow aspirate was (85.6 +13) % of all
nucleated cells. Bone marrow histological lymphocytes involvement was diffuse pattern in 55% of CLL cases,
interstitial in 30% of cases and mixed in 15% of cases.
There was a statistical significant positive association between Bone Marrow percentage of lymphocytes and
peripheral blood lymphocytosis while, there was a statistical significant inverse association between lymphocyte %
in the Bone Marrow and PCV % and platelet count . As in table 1:
Table 1 the hematological parameters of patients included in the study (N=40)
Range
Mean
SD
PCV %
21%- 46%
34.67 %
6.90
Platelet count(x109\L)
29-320
136.57
75.37
*WBC count
6.8-458
102.34
97.75
**L.C. (%) in peripheral
blood
61% -100%
89.4
9.35
**L.C. (%) in bone marrow
aspirate
40 % - 99 %
85.60
13.04
Immunohistochemical results:- 40 cases (100 %) were positive for CD5 as shown in table 1.
Table1. CD5 expression in CLL cases
CD 5 expression
Number of cases
Percent
Positive
40/40
100 %
Negative
Zero/40
Zero %
Total
40
100%

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852
The staining character and localization of CD 5 in CLL and control group in our study are shown in figure1:-
Figure 1 bone marrow tissue of CLL stained with CD 5 most of cells show brown membrane and cytoplasmic stain
(CD5 positive) (x 100).
ZAP 70 expression:- 19 cases (48%) were positive for ZAP 70 and 21 cases (52%) were negative for ZAP 70
among CLL cases while, all control group were negative and the score distribution for CLL cases is shown in table
2:-
Table 2: ZAP 70 score distribution for CLL cases.
ZAP 70 score
Number of cases
Percent
Negative
21/40
52 %
Positive
19/40
48 %
Total
40
100 %

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853
The staining character and localization of ZAP 70 in CLL and control group in our study are shown in figure2:-
Figure 2 bone marrow tissue of CLL stained with ZAP 70more than 20% of cells show brown nuclear and
cytoplasmic stain (Zap 70 positive) (x 100).
There was a statistical significant associationbetween ZAP 70 expression and Modified Rai staging in CLL
cases.Also, There was a statistical significant inverse association between ZAP 70 expression and PCV percent and
platelet count, but there was no statistical significant association between ZAP 70 expression and WBC count ,
lymphocyte percent or morphology in the peripheral blood or bone marrow aspirate as shown in table 3 using t.
test
Table 3: The correlation between ZAP 70 expression and hematological parameters
ZAP 70 score
N
Mean
SD
Minimum
Maximum
p-value
Platelet count
(x109/L)
Negative
21
156.571
63.3
34.00
320.00
0.05
Inverse association
positive
19
119.157
35.6
29.00
320.00
PCV (%)
Negative
21
38.04
6.06
22.00
46.00
0.001
Inverse association
positive
19
31.31
5.49
21.00
40.00
WBC count
(x109/L)
Negative
21
80.061
68.4
12.00
252.00
0.05
No ass0ciation
positive
19
127.163
119.
6.8
458.00
LC % in
peripheral blood
Negative
21
88.04
10.1
61.00
99
0.05
No association
positive
19
90.89
8.44
73.00
100
LC % in BM
aspirate
Negative
21
82.333
15.6
40
96
0.05
No association
positive
19
89.210
8.35
66
99
Discussion:-
CD5 is present on nearly all cases of CLL/SLL, and the great majority of MCLs. It is generally absent in other B-
cell lymphomas (6). In our study, CD 5 was 100% positive in study group while in all of control group it is found
100% negative. Accordingly, it carries no significance in this study behind the confirmation of B-cell CLL
diagnosis.

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ZAP-70 expression correlates with immunoglobulin heavy-chain variable region gene mutational status in
CLL/SLL, and can be detected reliably using immunohistochemical methods (13) . In our study the frequency of ZAP
70 positive score was 48%,whereas the control group was negative for ZAP 70 totally.
Similar results were reported by Cameron Yin et al and MarjanErtault, Maria Elena Noguera, et al. who had use
immunophinotyping study of ZAP 70 expression in bone marrowof CLL cases by flowcytometry technique (14, 15). In
addition, our results were slightly more than Michele; Rachel ET. Al. who found (41%) of CLL cases in their study
were positive for ZAP 70 by immunohistochemistry technique (16). The present study revealed that there was no
significant association between ZAP 70 score with gender,age, clinical presentation or bone marrow histology of
patient. Nevertheless there was a significance association with modified Rai stagingsystemexhibited in the large
percent (73.68 % ) of positive ZAP 70 group with high risk group , (21.05 %) with intermediate risk group and (5.26
%) with low risk group . This significant association reflects the importance of ZAP 70 as a prognostic marker in
CLL.
There was a significant inverse association between ZAP 70 expression and PCV percent and platelet count, but
there was no significant correlation between ZAP 70 score and WBC count, morphology orlymphocyte % in the
bone marrow or peripheral blood.
References:-
1. Brenner H,GondosA, Pulte D (2008) Trends in long- term survival of patients with CLL from the 1980s to the
early 21st century. Blood 111: 4916 – 21.
2. Elter T, Halleck M, Engert A. Fludarabine in CLL. Expert OpinPharmacother. Aug 2006; 7(12):1641-51.
3. Rodney T.MillerEP, M.D., director of IHC ZAP 70in CLL 2003.
4. Hallek M, Cheson BD, Catovsky D, et al. Guidelines for the diagnosis and treatment of CLL: a report from the
International Workshop on CLLupdating the National Cancer Institute-Working Group 1996
guidelines. Blood. Jun 15 2008; 111(12):5446-56.
5. Rossi S, Laurino L, Furlanetto A, Chinellato S, Orvieto E, Canal F, Facchetti F, Dei Tos AP. Rabbit monoclonal
antibodies: a comparative study between a novel category of immune reagents and the corresponding mouse
monoclonal antibodies. Am J ClinPathol. 2005; 124:1-8.
6. Dighiero G. Unsolved issues in CLL biology and management. Leukemia 2003; 17:2385-2391.
7. Grever M, Andritsos LA, Lozanski G. CLL. In: Abeloff MD, Armitage JO, Niederhuber JE, Kastan MB,
McKenna WG, eds. Abeloff’s Clinical Oncology. 4th ed. Philadelphia, Pa: Elsevier Churchill Livingstone;
2008: chap 108.
8. Carreras J, Villamor N, Colomo L, Moreno C, Cajal S, Crespo M, et al. IHC analysis of ZAP70 expression in
B-cell lymphoid neoplasms. J Pathol 2005;205;507-13.
9. Karen W, Susan M, O’Brien MD .CLL: Diagnosis and Treatment .Mayo Clin Proc. 2006;81(8):1105-1129.
10. Rassenti L, Huynh L, Toy T, et al.ZAP-70 compared with immunoglobulin heavy-chain gene mutation status as
a predictor of disease progression in CLL. N Engl J Med. 2004; 351: 893-901.
11. Del Principe M, Del Poeta G, Buccisano F, et al. Clinical significance of ZAP-70 protein expression in B-cell
CLL. Blood. 2006; 108: 853-861.
12. Chen L, Apgar J, Huynh L, Dicker F, Giago-McGahan T, Rassenti L, et al. ZAP-70 directly enhances IgM
signaling in CLL. Blood 2005;105:2036-41.
13. Sup SJ, Domiati-Saad R, Kelley TW, Steinle R, Zhao X, Hsi ED. ZAP-70 expression in B-cell hematologic
malignancy is not limited to CLL/SLL. Am J ClinPathol 2004;122:582-7.
14. Cyclin E but not bcl-2, bax or mcl-1 is differentially expressed in ZAP 70-positive and ZAP 70-negative B-CLL
cells by Christian Bogner . Michael Sandherr et.al , Ann Hematol (2006) 85: 458–462.
15. ZAP-70 Protein Expression and CD38 Positivity in B-cell CLL by MarjanErtault-Daneshpouy, Maria Elena
Noguera, et. al. , Clinical Advances in Hematology & Oncology Volume 6, Issue 1 January 2008.
16. ZAP70 Expression Assessed by IHC on Peripheral Blood: A Simple Prognostic Assay for Patients With CLL
by Michele; Rachel , et. al. Applied IHC & Molecular Morphology: December 2007 - Volume 15 - Issue 4 - pp
471-476
17. Dennis E. Chenoweth, Marc Key, ed. Immunohistochemical Staining Methods, 4th ed. DAKO, 2006.
18. Ghia P, Ferreri AM, Galigaris-Cappio F. Chronic lymphocytic leukemia. Crit Rev Oncol/Hematol. 2007; 234-
246.