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Abstract
Background: Chronic lymphocytic leukemia (CLL) is prognosticated using the Rai and the Binet's staging. In the past few years, new parameters have been considered for prognostication. One such marker that has been a subject of speculation and found useful by some western studies is zeta-associated protein 70 (ZAP-70). Aim: To investigate the prevalence of ZAP-70 and find out its association with other prognostic markers like Rai and Binet's stage and CD38 in Indian CLL patients. Materials and Methods: Twenty-nine newly diagnosed cases of CLL were selected over 1 year. Immunophenotyping was done and expression of CD38 and ZAP-70 was evaluated on gated CLL cells. Statistical Analysis: Qualitative data were expressed as frequency and percentage. Differences between groups were evaluated using Student's t-test for quantitative data and Chi-square test/Fisher's exact t-test for qualitative variables. A P value less than 0.05 was considered significant. Results and Conclusion: We found a lower prevalence rate of ZAP-70 (2/29, 6.89%) with no association with any of the conventional poor prognostic factors. A large number of our CLL patients fall into the good prognostic group (22/29, ZAP 70−/CD38−) with a least number in the poor prognostic group (2/29, ZAP-70 + CD38+). Also, no association was found between ZAP-70 and CD38. The findings of the present study suggest that the majority of CLL patients in India have a good prognosis, may not require treatment, and have good overall survival. Geographical variations, genetic makeup, and natural history of the CLL could be the cause of such differences from western literature.
Keywords: CD38, chronic lymphocytic leukemia, India, prognosis, Rai and Binet stage, ZAP-70
How to cite this article: Introduction 
Traditionally, chronic lymphocytic leukemia (CLL) is prognosticated using the Rai and the Binet's staging. Though simple and useful, both the systems do not predict the subsequent evolution of the disease and fail to address its dynamic nature. In the past few years, some new molecular and biological parameters have been considered for prognostication. One such marker that has been successfully incorporated for prognostication purposes in CLL is CD38. CD38, a type II transmembrane glycoprotein, is linked with advanced stage, organomegaly, and cytopenias in CLL.[1],[2],[3] Another marker that has been a subject of speculation and found useful by some western studies is zeta-associated protein 70 (ZAP-70). ZAP-70 is a tyrosine kinase protein normally expressed in T cells and natural killer (NK) cells. Recently, few studies have indicated that ZAP-70 may also be expressed by malignant population in CLL and its expression is associated with an adverse prognosis in these patients.[1],[2],[3] However, the same may not hold true in this part of the world where the biological characteristics of the disease may differ. There are few studies to address the relevance of ZAP-70. Thus, the present study was conducted to investigate the prevalence of ZAP-70 in Indian CLL patients and find out its association with other prognostic markers, for example, Rai and Binet's stage and CD38 in our cohort of patients.
Materials and Methods Twenty-nine newly diagnosed cases of CLL were selected over 1 year. Informed consent was taken from all cases. Approval from Institutional Ethics Committee for Human Research was taken. Patients were diagnosed as CLL according to the International CLL Workshop guidelines.[4] A complete hemogram (Beckman and coulter Pvt Ltd) including peripheral blood (PB) smear examination and the differential count was done.
Immunophenotyping was done from lysed whole PB (lyse and wash protocol) using dual laser 5 color flow cytometer (Beckman and coulter Pvt Ltd). Analysis was done using FCS express software version 3.0.
Four-tube antibody panel was employed tagged to flourochromes fluorescein isothiocyanate (FITC), phycoerythrin (PE), ECD, PE- Texas Red, PE- Cyanine 5 (PE-Cy5), and PE- Cyanine 7 (PE-Cy7), respectively:
Kappa/lambda/CD38/CD19/CD45CD5/CD23/CD19/CD45/CD20FMC 7/CD10/CD19/CD45CD45/CD4/CD8/CD3.A fifth tube was added to analyze ZAP-70 using the ZAP-70 kit provided by the manufacturer containing six conjugated antibodies: FITC conjugated CD5, PE-conjugated intracytoplasmic ZAP-70, PE-Cy5 conjugated CD19, PE-Cy7 conjugated CD3, and CD 56 and ECD conjugated CD45. T cells (CD3 positive) and NK cells (CD56 positive) were used as ZAP-70 positive and high positive controls. Normal B-cells (CD19 positive) were used as ZAP-70 negative controls. CLL cells were identified as CD5+ CD19+ events and expression of CD38 and ZAP-70 was evaluated on these gated CLL cells. A cut-off value of 30% to define the expression of CD38 and 20% to define the expression of ZAP-70 was used.[5]
Statistical analysis was performed using MS Excel and SPSS software version 20 (IBM SPSS statistics). Numerical data were expressed as the mean and standard error of the mean. Qualitative data were expressed as frequency and percentage. Differences between groups were evaluated using Student's t-test for quantitative data and Chi-square test/Fisher's exact t-test for qualitative variables. A P value less than 0.05 was considered significant.
Results Out of the total 29, ZAP-70 was expressed by malignant cells in only two cases (6.89%).
The expression of ZAP-70 was compared to other markers influencing prognosis. [Table 1] shows the comparative analysis of ZAP-70 positive and negative CLL patients. No significant association was seen between ZAP-70 expression and any of the following parameters, i.e., advanced age, gender, low hemoglobin (Hb), high total leukocyte count (TLC), high absolute lymphocyte count (ALC), low platelet (PLT), organomegaly, and clinical Rai/Binet stage [Table 1].
Table 1: Comparative analysis of ZAP-70 positive and negative CLL patients (n=29)The expression of ZAP-70 and CD38 was compared for concordance. Out of 29 patients, 2/29 were positive for both ZAP-70+ and CD38+, 22/29 were negative for ZAP-70 − and CD38 − yielding a concordance rate of 82.75%. Rest 5/29 (17.25%) were discordant (ZAP-70−/CD38+). [Figure 1] shows ZAP-70 positivity in one of the cases of CLL.
Figure 1: A case of CLL showing ZAP-70 positivity in CD19 positive cells (red). Normal CD3 positive T cells (green) and CD56 positive (blue) NK cells were used as positive and high positive internal controls for ZAP-70 Discussion CLL is a heterogenous disease with variable disease courses. Many patients do not require active treatment and just close monitoring while others require treatment with disease progression. So, it becomes important to identify patients who have the propensity to develop severe disease and will require treatment. One of the newest biological markers identified to help in this regard is ZAP-70. In some studies, mostly from the West, ZAP-70 was found to be of paramount importance in identifying CLL patients with a propensity to disease progression and poor overall survival and hence those who will benefit from treatment.[6] The present study was performed to evaluate the same in Indian CLL patients.
In the present study, the prevalence of ZAP-70 in newly diagnosed CLL patients was found as 6.89%. This was much lower than what is reported in the West. Different studies from the West have reported ZAP-70 positivity in CLL in the range of 36% to 57%.[1],[2],[3],[6],[7] Comparatively, the expression of ZAP-70 reported from studies from south Asia including India was lower ranging from 0 to 37%. [Table 2] shows the prevalence rate of ZAP-70 in South Asia including Indian studies.[5],[8],[9],[10] The prevalence in the present study was very low as compared to western studies but similar to those from South Asian studies. The differences may be attributed to different genetic makeup, ethnic origin, and geographical distribution between different populations.[10] An Indian study by Patkar et al.[11] stated that ZAP-70 expression by flow cytometry is weak and not a robust assay as it is represented by small shifts above the baseline threshold set by the autofluorescence control. So, the difference in methods used for analyzing ZAP-70, an intracellular kinase, can also be a contributing factor requiring standardization of flow cytometry methods as also mentioned by Kinawy et al.[3] in their study. Moreover, due to subjectivity in the assay, it is important to observe controls along with the population of interest and the same should be specified along with the data analyzed. We found that the same has not been specified in many studies. In this study, the T cells and the NK cells were used as positive and high positive controls.
Table 2: Comparison of ZAP-70 and CD38 with literature from South Asia including India[5],[8],[9],[10]No significant association was seen between ZAP-70 expression and any of the prognostic parameters including Rai and Binet's stage. Our findings are in contrast to previous Western studies.[1],[2],[3],[7] Del Poeta et al. and Hus et al. found a significant correlation between high ZAP-70 levels and advanced Rai stage and splenomegaly.[1],[12] Kinawy et al.[3] found a significant correlation between ZAP-70 and advanced Rai stage, low Hb, high TLC count, high ALC, and low PLT levels (p < 0.05). However, our findings are similar to that of a large study conducted by Gogia et al.[5] on 80 Indian CLL patients who did not find any correlation between ZAP-70 and the conventional poor prognostic markers. The authors attributed these differences to the biology of CLL disease in the Indian population.
As stated by Kinawy et al.,[3] ZAP-70 and CD38 analyses can stratify CLL patients into three prognostic subgroups viz ZAP-70−/CD38 − with a good prognosis; discordant groups (CD38+/ZAP-70- and CD38-/ZAP-70+) with intermediate prognosis; and ZAP-70+/CD38+ group with the worst prognosis. The concordance rate between ZAP-70 and CD38 was 82.75% with a percentage of ZAP-70+/CD 38+ being 6.89% in the present study. Kinawy et al.[3] found a similar concordance of 90% but a higher percentage of ZAP-70+ CD38 + patients (40%). A majority (75.86%) of the patients in the present study were in the good prognostic group (ZAP-70-/CD38-) with the least number (6.89%) in the poor prognostic group (ZAP-70+ CD38+). Gogia et al.[5] and Saxena et al.[8] found 13.75% and 26% of CLL patients, respectively, in poor prognostic group (ZAP-70+ CD38+) in Indian setting [Table 2].
Also, no association was seen between ZAP-70 and CD38 [Table 1]. Similar findings were reported by Gogia et al.[5] who studied 80 Indian CLL patients. However, our findings contrast the findings of Kinawy et al.[3] who found a significant correlation between ZAP-70 and CD38. These differences could be attributed to a relatively lower prevalence rate of ZAP-70 and a smaller sample size in our study. Also, these findings further suggest that in CLL patients, CD38 may signal independent of ZAP-70 in contrast to the belief that a functional link exists between CD38 and ZAP-70 in neoplastic B-cells and they form part of the same signaling pathway and they intersect in some steps.[7]
To conclude, ZAP-70 was found to be an adverse prognostic marker in CLL by some authors. In this study, we found a lower prevalence rate of ZAP-70 with no association with any of the conventional poor prognostic factors. A large number of our CLL patients fall into the good prognostic group (ZAP 70-/CD38-) with the least number in the poor prognostic group (ZAP-70+ CD38+). Also, no association was found between ZAP-70 and CD38. The findings of the present study suggest that the majority of CLL patients in India have a good prognosis, may not require treatment, and have good overall survival. Geographical variations, genetic makeup, and natural history of the CLL could be the cause of such differences from western literature. The limitation of the present study was the absence of molecular signature of the disease in these patients including immunoglobulin heavy chain gene mutation status, presence of trisomy 12 and del 13q, and mutations for p53 and NOTCH1. This was largely due to economic constraints and a lack of institutional setup for these markers. Larger multi-institutional studies incorporating molecular and cytogenetic work up in addition to flow cytometry will be useful for future analysis.
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References 
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CheckDOI: 10.4103/IJPM.IJPM_1200_20
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