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  Table of Contents  
ORIGINAL ARTICLE
Year : 2013  |  Volume : 50  |  Issue : 4  |  Page : 333-336
 

Prevalence of ZAP-70 and CD 38 in Indian chronic lymphocytic leukemia patients


1 Department of Medical Oncology, Dr. B. R. A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India
2 Lab Oncology, Dr. B. R. A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India

Date of Web Publication24-Dec-2013

Correspondence Address:
A Sharma
Department of Medical Oncology, Dr. B. R. A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.123620

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 » Abstract 

Aim of Study: Chronic lymphocytic leukemia (CLL) is the most common chronic lympho-proliferative disorder. This study was undertaken to know the prevalence of ZAP-70 and CD 38 in the treatment naive patients of CLL seen at a tertiary care centre of north India. Materials and Methods: ZAP-70 and CD 38 were tested by flow cytometry on peripheral blood samples. ZAP-70 positive and CD 38 positivity was defined as positive expression on 20% and 30% of CLL cells, respectively. Clinico-hematological profile and its correlation with ZAP-70 and CD 38 were assessed in consecutive 80 CLL patients. Results: There were 64 males and median age of the group was 58 years. Sixteen patients (20%) were asymptomatic and diagnosed incidentally. Median total lymphocyte count (TLC) at presentation was 62 × 10 9 /L. Rai stage distribution was: Stage 0-6, stage I-20, stage II-36, stage III-5, and stage IV-13. ZAP-70 and CD 38 positivity were detected in 20 patients (25%) and 29 patients (36%), respectively. Eleven patients were positive and 34 were negative for both ZAP-70 and CD 38 yielding a concordance rate of 56%. There was no statistically significant difference between ZAP-70 and CD 38 positivity and negativity with regard to age, sex, Lymphocyte count, lymphadenopathy, organomegaly, and Rai staging. Conclusion: ZAP-70 and CD 38 positivity were detected 25% and 36%, respectively, with concordance rate of 56%, which is higher than Western literature. There was no correlation of ZAP-70 and CD 38 positivity with age, sex, lymphadenopathy, organomegaly, and Rai staging.


Keywords: CD38, chronic lymphocytic leukemia, ZAP-70


How to cite this article:
Gogia A, Sharma A, Raina V, Kumar L, Gupta R, Kumar R. Prevalence of ZAP-70 and CD 38 in Indian chronic lymphocytic leukemia patients. Indian J Cancer 2013;50:333-6

How to cite this URL:
Gogia A, Sharma A, Raina V, Kumar L, Gupta R, Kumar R. Prevalence of ZAP-70 and CD 38 in Indian chronic lymphocytic leukemia patients. Indian J Cancer [serial online] 2013 [cited 2019 Sep 15];50:333-6. Available from: http://www.indianjcancer.com/text.asp?2013/50/4/333/123620



 » Introduction Top


Chronic lymphocytic leukemia (CLL) is common leukemic disorder in the West, with an estimated incidence in the United States of 5.17 per 100,000, representing 20% of all mature B-cell neoplasm. [1] CLL patients with advanced stage disease and those who progress from early stage disease are treated with chemotherapy. [2] Overall about 70% of all patients will require therapy during the course of the disease. Although Rai staging system predicts overall survival but it does not predict which patients in the early stages (0-I) will progress and require therapy. [3] The traditional prognostic parameters based on routine and well-established tests involving the blood or bone marrow (clinical stage, pattern of bone marrow infiltration, lymphocyte doubling time, beta-2 microglobulin levels, and lactate dehydrogenase level) are useful but they may not accurately predict progression for a given patient. [4],[5] From past few years focus of research in prognostic factors in CLL has changed from clinical to biological factors. The IgVH mutational status, CD 38 and ZAP-70 expression are few of such markers. Till date the strongest independent prognostic factor for survival in CLL is the presence of somatic mutations of the variable region of the immunoglobulin heavy chain gene. [6] Patients with unmutated IgVH gene have aggressive disease, requiring early treatment and often show poor response to chemotherapy. However, IgVH mutation studies are cost and labor intensive and are not widely available at most centers. Therefore, there is a need for simpler, reliable, and easily standardized surrogate marker, which can substitute IgVH mutation testing. The best studied of these new prognostic parameters have been proteins residing on the cell surface (CD-38) or in the cytoplasm (ZAP-70), recurring genetic defects in the CLL cells (detected by fluorescence in-situ hybridisation (FISH)), and the mutation status of Ig VH gene. Zeta-associated protein 70 (ZAP-70) is a tyrosine kinase protein normally expressed in T cells and natural killer cells. ZAP-70 has been reported to be expressed preferentially in CLL unmutated Ig VH. [7] The ligation of B-cell receptor on CLL cells that express ZAP-70 is associated with excessive tyrosine phosphorylation. [8] There are several methods used to study the expression of ZAP-70 in patients with CLL, flow cytometric detection of ZAP-70 is relatively reliable and could be placed into more routine use. [9] CD 38 is a 45-kDa, nonlineage-restricted, type II transmembrane glycoprotein that has many protein functions. It can serve as an ectoenzyme that catalyzes the synthesis and hydrolysis of cyclic ADP-ribose, a Ca2+ mobilizing agent that acts independently of inositol triphosphate. [10] CD 38 also functions as receptor that induces proliferation and increases survival of CLL cells. [11] CD 38 positivity (defined as at least 30% positive cells) is an independent prognostic marker for an unfavorable clinical course in CLL. [12] CD 38 positive patients may progress faster to advanced stage. These patients not only have more aggressive disease but also do not respond to chemotherapy as others do. [13] However, it is not a surrogate marker for Ig VH mutational status. It has been found that there is significant correlation between CD 38 positivity and intermediate/high modified Rai stages, multiple, bulky lymphadenopathy, and splenomegaly. [14],[15]

There are no published data available from India about prevalence of ZAP-70 and CD 38 positivity in Indian CLL patients, so this study was undertaken to know the prevalence of CD 38 and ZAP-70 in CLL patients in a developing country and to correlate this with baseline parameters. With longer follow up we will be able to know the prognostic value of ZAP-70 and CD 38, which will be reported later on.


 » Materials and Methods Top


Between January 2009 and December 2010, consecutive 80 cases of treatment naive patient of CLL were selected for this analysis. All cases fulfilled the National Cancer Institute Working Group Criteria for diagnosis of CLL. [16] This protocol was approved by the ethical committee of the Institute.

Two milliliters of peripheral venous blood was collected in ethylenediaminetetraacetic acid from each patient after taking informed consent as per guidelines of the ethics committee. A standard whole blood lysis method was used for sample preparation. Briefly, 1 Χ 10 6 cells were incubated with preconjugated monoclonal antibodies: CD 38 conjugated to Alexa Fluor 488, CD 19 to phycoerythrin cyanine 5.5 (PE-Cy 5.5), and CD 5 to allophycocyanin (APC), (BD Pharmingen, San Diego, CA, USA) at room temperature (RT) in dark for 20 minutes. Two milliliters of fluorescence activated cell sorter (FACS) Lyse (BD Pharmingen, San Diego, CA, USA) was added and incubated for 2 hours in dark at RT followed by incubation for ZAP-70 phycoerythrin (PE) for 30 minutes (BD Biosciences, San Jose, CA, USA). The cells were then washed and resuspended in phosphate buffer saline and kept at 4΀C till acquisition. A total of 10 5 events were acquired on a flow cytometer (FACS Canto, BD Biosciences, San Jose, CA, USA) equipped with facility for at least 4-color immunophenotyping and analysis was done using FCS express software version 3.0 (Denovo software, Los Angeles, CA, USA). CLL cells were identified as CD 5 + CD 19 + events and expression of CD 38 and ZAP-70 was evaluated on these gated CLL cells. A cut-off value of 30% to define expression of CD 38 and 20% to define expression of ZAP-70 was used [Figure 1].
Figure 1: Lymphocytes were gated based on forward and side scatter (Gate 1; a), followed by gating based on CD 5 + CD 19 + expression (Gate 2; b). Expression of CD 38 and ZAP-70 was observed on CD 5 + CD 19 + CLL cells (c)

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Statistical analysis

Quantitative variables were summarized as median and qualitative variables as proportions. Baseline categorical variables were analyzed using Chi-Square test/Fisher ' s Exact t-test. Multivariate logistic regression for identifying independent predictors for ZAP-70 and CD 38 positivity was performed using STATA software version 11.1(StataCorp, Texas, USA) and P < 0.05 was defined as significant.


 » Results Top


The median age was 57 years (range 28-90 years). There were 64 males and 16 female patients. Eighteen patients were asymptomatic. Out of 80 patients, 6 (7.5%) were in Rai stage 0, 20 (25%) were in Rai stage I, 36 (45%) were in Rai stage II, 5(6.25%) were in Rai stage III, and 13 (16.25%) were in Rai stage IV. The median hemoglobin was 11 g/dL with range of 4.7-16 g/dL. The median total leucocytes count was 62 Χ 10 9 /L and absolute lymphocyte count was 51 Χ 10 9 /L. The median platelet count was 150 Χ 10 9 /L. Out of 80 patients, 26 (32.5%) were in Rai stage 0 and I, 36 (45%) were in Rai stage II, 18 (22.5%) were in Rai stage III and IV. CD 38 was positive in 29 (36.25%) of patients. ZAP-70 was positive in 20 (25%) patients. Stage wise distribution of ZAP-70 and CD 38 positive patients is given in [Table 1]. Out of 80 patients in whom both ZAP-70 and CD 38 were tested, 11 were concordant ZAP-70 + CD 38 + and 34 were ZAP-CD 38-, yielding a concordant rate of 56%. After analyzing ZAP-70 and CD 38 as continuous variables no definite correlation was found with age, sex, lymphadenopathy, organomegaly, and Rai staging [Table 2]. With a median follow up of 17 months, 11 patients of early stage disease progressed; 3 were ZAP-70 positive, 4 were CD 38 positive, and 4 patients were negative for both. There was no difference between progression free period of ZAP-70 and CD 38 positive group.
Table 1: Stage distribution as per ZAP 70 and CD 38


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Table 2: Co-relation of ZAP 70 and CD 38 with age and base line lab parameters


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 » Discussion Top


Rai clinical stage is the most robust and established prognostic factor in CLL. The limitation with this staging system is that a significant percentage of patients with early-stage disease will rapidly escalate to advanced disease that requires therapy. Unfortunately, the Rai system is unable to prospectively differentiate the rapidly evolving patient from more stable patients who may not progress for decades. In a study it was found that patients positive for ZAP-70 in early stage disease had a shorter time to therapy, with a median time from diagnosis to initial therapy of 2.9 years, compared with 9.2 years for ZAP-70-negative patients for the same stages. [7] Compared with three of ZAP-70 positive patient and two of ZAP-70 negative patient progressed to advanced stage in this report. ZAP-70 holds significant promise as a prognostic marker as it has been found that ZAP-70 is highly predictive of time to treatment in a large cohort of early-stage (Rai 0-1) and untreated CLL. [7],[8] Various studies have reported ZAP-70 positivity in CLL ranging from 36% to 57% as given in [Table 3]. The optimal cutoff for defining ZAP-70 positivity was reported as 20% by various authors. [9],[14],[15],[17] Del Poeta et al., and Hus et al. Found significant correlation between high ZAP-70 levels and advanced Rai stage and splenomegaly. [12],[14] In this study among the 80 patients who were tested 25% (20 patients) were ZAP-70 positive. There was no correlation of ZAP-70 with age, sex, hemoglobin, lymphocyte count, organomegaly, and clinical Rai stage. Various studies have reported CD 38 positivity in CLL ranging from 29% to 60%, as given in [Table 3]. In this study 80 patients were tested and 29% were CD 38 positive. Out of 80 patients, 8 (10%) were in Rai stage 0 and I, 15 (18.7%) were in Rai stage II, 6 (7.5%) were in Rai stage III and IV. There is no correlation of CD 38 with age, sex, hemoglobin, lymphocyte count, organomegaly, and clinical Rai stage. Because of short follow-up this data is not mature to do survival analysis.
Table 3: Comparison of ZAP 70 and CD 38 with literature


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 » Conclusion Top


The present study was aimed to study the incidence of two prognostic markers namely ZAP-70 and CD 38 positivity in CLL patients. ZAP-70 and CD 38 positivity were detected in 20 patients (25%) and 29 patients (36%), respectively, with concordance rate of 56%. There was no correlation of ZAP-70 and CD 38 positivity with age, sex, lymphadenopathy, organomegaly, and Rai staging. The low prevalence rate of ZAP-70 and high prevalence of CD 38 was due to biology of disease in the Indian population.

 
 » References Top

1.Dighiero G, Binet JL. Chronic lymphocytic leukemia. Hematol Cell Ther 1996;38:S41-61.  Back to cited text no. 1
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6.Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999;94:1848-54.  Back to cited text no. 6
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7.Rassenti LZ, Huynh L, Toy TL, Chen L, Keating MJ, Gribben JG, et al. ZAP70 compared with Immunoglobulin heavy-chain gene mutation status a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med 2004;3513-901.  Back to cited text no. 7
    
8.Del Principe MI, Del Poeta G, Buccisano F, Maurillo L, Venditti A, Zucchetto A, et al. Clinical significance of ZAP 70 protein expression in B cell Chronic Lymphocytic Leukemia Blood 2006;108:853-61.  Back to cited text no. 8
    
9.Crespo M, Bosch F, Villamor N, Bellosillo B, Colomer D, Rozman M, et al. ZAP 70 expression as a surrogate for immunoglobulin variable region mutations in chronic lymphocytic leukemia. N Engl J Med 2003;348:1764-75.  Back to cited text no. 9
    
10.Ibrahim S, Keating M, Do KA, O'Brien S, Huh YO, Jilani I, et al. CD 38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood 2001;98:181-6.  Back to cited text no. 10
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11.Tinhofer I, Rubenzer G, Holler C, Hofstaetter E, Stoecher M, Egle A, et al. Expression levels of CD 38 in T cells predict course of disease in male patients with B chronic lymphocytic leukemia. Blood 2006;108:2950-6.  Back to cited text no. 11
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12.Del Poeta G, Maurillo L, Venditti A, Buccisano F, Epiceno AM, Capelli G, et al. Clinical significance of CD 38 expression in chronic lymphocytic leukemia. Blood 2001;98:2633-9.  Back to cited text no. 12
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13.Schroers R, Griesinger F, Trumper L, Haase D, Kulle B, Klein-Hitpass L, et al. Combined analysis of ZAP 70 and CD 38 expression as a predictor of disease progression in B cell chronic lymphocytic leukemia. Leukemia 2005;19:750-8.  Back to cited text no. 13
    
14.Hus I, Podhorecka M, Bojarska-Junak A, Rolinski J, Schmitt M, Sieklucka M, et al. The clinical significance of ZAP70 and CD 38 expression in B cell chronic lymphocytic leukemia. Ann Oncol 2006;17:683-90.  Back to cited text no. 14
    
15.D'Arena G, Tarnani M, Rumi C, Vaisitti T, Aydin S, De Filippi R, et al. Prognositc significance of combined analysis of ZAP 70 and CD 38 in chronic lymphocytic leukemia. Am J Haematol 2007;82:787-91.  Back to cited text no. 15
    
16.Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, O'Brien S, et al. National Cancer Institute sponsored Working Group guidelines for chronic lymphocytic leukemia: Revised guidelines for diagnosis and treatment. Blood 1996;87:4990-7.  Back to cited text no. 16
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    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]

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