ZAP-70 expression and Soluble CD14 level in patients with Chronic Lymphocytic Leukemia

Introduction

CLL is a heterogeneous group of clonal neoplastic pathologies, the proliferation of mature B or T lymphoid cells characterizes CLL⁽¹⁾. The disease is defined by the abnormal proliferation of immune-deficient B cells, which develop in lymph nodes, bone marrow and peripheral blood ⁽²⁾.

The clinical behavior of CLL subgroups differs according to the stage of normal B cell development that originate from; these subgroups are classified as follows:

1- B cells that do not differentiate in germinal centers—lymph node locations where B cells undergo somatic hypermutation and selection during an immune response are the progenitors of CLL cells, which express an unmutated (UM) immunoglobulin heavy-chain variable region gene (IgHV)⁽³⁾.

2- CLL cells express a mutated (M) immune-globulin heavy-chain variable region gene⁽⁴⁾.

Patients with a germline identity of 98% are classified as IgHVUM and have a poor prognosis compared to those with a mutation level of more than 2%, classified as IgHV-M and can survive for decades without treatment ⁽⁵⁾. Somatic mutation level within the variable region of the immunoglobulin heavy chain is the gold standard for CLL prognosis.

Approximately ten years is the median survival time for persons diagnosed with CLL. On the other hand, the prognosis for each patient varies greatly. In some cases, the disease has a slow course and does not affect life expectancy; however, in others, the disease advances quickly, the patient exhibits violent behavior, and their survival rate is less than two- or three-years following diagnosis. The lack of a curative treatment for chronic lymphocytic leukemia (CLL) makes prognosis an essential factor in patient management planning ⁽⁶⁾.

Binet and Rai staging of CLL patients and ZAP-70 could be used as prognostic markers that contribute to this CLL prognostic index ⁽⁷⁾.

 It was shown that ZAP-70 was the most differentiating gene between the two CLL groups, with higher expression in the IgHV-UM CLL group. In addition, research has demonstrated that ZAP-70 outperforms IgHV status as a predictor of therapy necessity⁽⁸⁾. When deciding how to best treat CLL patients, ZAP-70 expression is often used as a prognostic indicator. Different studies aimed to determine whether there was a connection between ZAP-70 expression and the percentage of smudge cells (SCs) on peripheral blood films (PBFs) taken from individuals who had just been diagnosed with CLL⁽⁹⁾.

Almost all of the innate immune system-related CD14 protein is produced by human macrophages. When pathogen-associated molecular pattern (PAMP) lipopolysaccharide (LPS) binds, it helps the body recognize bacteria⁽⁷⁾.  There are two forms of CD14. One is soluble (sCD14) and the other is membrane bound (mCD14), which is immobilized on the membrane by the glycosyl-phosphatidyl-inositol tail⁽¹⁰⁾. Circulating sCD14 is considered a protein of the acute phase because IL-6 promotes hepatic sCD14 expression in response to inflammation and infection. Hence, it will worsen in inflam-matory and infectious diseases, both short- and long-term⁽¹¹⁾. It is widely recognized that inflammation plays a role in several B-cell chronic lymphoid malignancies development, including B-CLL.

The quantity of smudge cells in peripheral blood films of CLL patients may be associated with prognosis, according to researches. Because these cells are fragile, they smear when blood films are made. Cases of acute and chronic leukemia show varying frequencies of these abnormalities on peripheral blood films, but CLL patients often have a higher incidence ⁽¹²⁾.

Aim of the work

To evaluate ZAP-70 expression & sCD14 in patients with CLL and their correlation with hematological parameters, clinical staging and smudge cell percentage.

Subjects and methods

Subjects:

From April 2019 to May 2020, the study conducted at the Clinical Pathology Department at Minia University's Faculty of Medicine and the South Egypt Cancer Institute worked together to compile the data for this study .It was conducted upon sixty subjects, divided into two groups; Group I (patients’ group): Included thirty newly diagnosed CLL Patients, they were classified according to Binet and RAI staging system, while Group II (control group): It included thirty seemed to have a good health . This study did not include patients who had any kind of infection, whether it was acute or chronic, inflammatory disease, or renal disease. The individuals were asked to fill out a detailed medical history that included their age, fever, susceptibility to fatigue, tendency to bleed easily, and any history of hemolytic attacks. They were then examined clinically for signs of illness, such as pale skin, enlarged liver and spleen, and enlarged lymph nodes. Laboratory investi-gation was evaluated for all studied subjects in the form of: routine investigations (Complete blood count (CBC), Erythrocyte sedimentation rate (ESR), Renal function test and liver function test, Lactate dehydrogenase enzyme (LDH) in addition to CD5 and CD19. moreover, special investigations in the form of: Expression of ZAP-70 via Flow Cytometry and sCD 14 (soluble form) by ELISA kits were done.

Sampling:

Each individual had approximately seven milliliters of venous blood withdrawn under strict aseptic circumstances. This sample was divided as follows; For CBC and flow cytometric analysis, two milliliters of blood were evacuated in a tube containing ethylenediaminetetraacetic acid (EDTA). In order to measure ESR , 1.6 ml of blood was emptied into a tube that already contained 0.4 ml of 3.8% Tri-Sodium Citrate . The rest of the blood was transferred to a plain tube. After the blood had clotted, it was centrifuged. The results of the renal and liver function tests, as well as LDH, were determined using the expressed serum. the remaining serum was stored at -20° for further assessment of sCD14.

• In addition to bone marrow aspiration.

Methodology:

Complete blood count (CBC): by using of CELLTAC G, automated hematology analyzer manufactured by Japan's NIHON KOHDEN CORPORATION, Lieshman stained blood film including differential leucocytic count and smudge cells were counted as percent and confirmed by microscopic examination

ESR: was determined by Westergren Method.

Renal and liver function tests: were analyzed by SELECTRA PRO XL, ELITech group, clinical chemistry automation systems, Netherlands, commercially available kits were used in accordance with the instructions given by the manufacturers.

Flow cytometry evaluation of ZAP-70, CD5, and CD19 expression:

Method for staining: The recommended dilution of antibody for cell labeling and flow cytometry analysis is 1:50 for up to 10⁶ cells/100 μL. The following volumes are for nucleated cell counts up to 10⁶ nucleated cells.  If working with higher cell numbers, make sure to scale up all reagent quantities and total volumes proportionately One hundred microliters of blood sample was added to two tubes that were designated as 1&2 (Control and Test, respectively).. Ten μL of Anti CD5 FITC conjugated antibody, 10 μL of anti CD19 PerCP-Cy5.5 conjugated antibody was added (only to the test tube) and vortex. The cells were left to incubate at room temperature for fifteen minutes at the dark. Phosphate Buffered Saline (PBS) was used twice to wash the cells and remove any unbound antibodies. Use 2 milliliters of lysing solution to lyse the red blood cells.

Since ZAP-70 is an intracytoplasmic antigen, permeabilization is an additional step in its detection. Cells fixation and permeabilization followed centrifugation at 200 g: After addition and mixing of 250 μL of fixation buffer, the mixture was incubated for 20 minutes at room temperature in the dark. Careful aspiration of the supernatant followed centrifugation for 5 minutes. After washing the cells with one milliliter of PBS, they were centrifuged at 300×g for 5 minutes. Carefully aspirate the supernatant. centrifuging the mixture at 300×g for 5 minutes after adding 1 mL of permeabilization buffer then careful aspiration of the supernatant. The test tube only received 20 μL of the AntiZAP-70 PE conjugated antibody, which was then vortexed. Cells were left to incubate for 15 minutes at room temperature in the dark before addition of one milliliter of permeabilization buffer. After that, they were centrifuged at 300×g for 5 minutes, and the supernatant was carefully aspirated. Then, cells were washed twice with PBS before being re-suspended in 300 μL of PBS for the final flow cytometric analysis They were then prepared to acquire data using a BD FACS canto II, U.SA. Gating on B cells (CD5+/CD19+) allowed for the determination of percentages of CLL cells that were positive for ZAP-70. To process the data, the Diva software was utilized.

Assessment of sCD14 by ELISA:

Sinogeneclon Co., Ltd, China (Cat. #SG-10117).

Bone marrow aspirate:

Bone marrow (BM) aspirations were done for patients only by marrow puncture needles (Klima type) either from anterior superior or posterior superior iliac spines and examination of leishman-stained smears.

Ethical statements

The study had local Research Ethics approval of Minia University, Faculty of Medicine, Institutional Review Board 'MUFMIRB' (Approval number: 240:6/2019, Date of approval: 16 June 2019).  Participants were asked to provide written informed consent.  All of the study's procedures adhered to the

ethical standards outlined in the Declaration of Helsinki on Human Research, which is a document put out by the World Medical Association.

Data analysis using statistical methods:

Data was coded, tabulated, and statistically analyzed using SPSS (Statistical Package for the Social Sciences) software version 25. Descriptive statistics were performed using the mean, standard deviation, and minimum and maximum values for parametric quantitative data. Similar to how numerical and percentage-based approaches were used for nonparametric quantitative data, this one uses

the median. When comparing the two sets of quantitative data that are parametric, we used the independent samples T test; when the data was non-parametric we used Mann Whitney test. A chi-square test is used to analyze qualitative data. The Pearson's correlation coefficient was used for correlation. The area

under the curve (AUC), sensitivity, specificity, PPV, NPV, and accuracy for the cases group prediction were all calculated using ROC curve analysis. The level of significance was considered to be at (P value < 0.05).

Results

This study showed that age range of CLL patients was 45-65 years with mean ± SD (54.8±6.5), they were 18 males (60%) and 12 females (40%) while the age range of controls was 47-61 years with mean ± SD (53.5± 4.6), they were 20 males (66.7%) and 10 females (33.3%) with no significant difference between them regards the age and sex.

It was noted a statistically significant decrease in hemoglobin (HB) level and platelet count in CLL patients when compared to controls (p = 0.0001 for each), while there was a statistically significant increase white blood cell (WBCs) count, absolute lymphocytic count, ESR and Smudge cells in the former comparing to the later (p = 0.0001 for each) (table1).

Discussion

Soluble form of CD14 (sCD14), is one of many indicators being studied for its prognostic value in individuals with B-CLL. Acute phase reactant soluble CD14 functions as a new monocyte-derived survival factor for B-CLL and as Toll-like receptor-4 (TLR-4), a co-receptor for bacterial lipopolysaccharide (LPS) detection ⁽¹³⁾.

Survival, disease progression, and the status of IGHV mutations are all correlated with ZAP-70 expression on CLL ⁽¹⁴⁾.

The predictive usefulness of the smudge cell percentage concerning CLL has been demonstrated in a few studies^{(15, 16)}.

Possible explanations for the markedly reduced HB in the CLL group compared to the control group include replacement of bone marrow by tumor cells or due to development of autoimmune hemolytic anemia which is common in CLL. this finding was supported by several studies ^{(17, 18)}.

Županić-Krmek et al., (2011)⁽¹⁹⁾ showed that the highest incidence of anemia found in CLL (40%) before chemotherapy, more common in women than men (63% vs. 43%), but more severe   in men at the start and at the end of treatment. This anemia was anemia of chronic

disease (53.8%), followed by anemia due to many causes (mostly Anemia due to iron deficiency, hemolytic anemia, marrow suppression due to bone marrow infiltration).

When comparing CLL patients to controls, platelet counts were significantly lower in the former group. Rashid et al., (2018)⁽²⁰⁾ approved that and showed that cytopenia in CLL patients can be caused by various things, including chemotherapy, hypersplenism, auto-immunity, sepsis, and bone marrow failure.

Moreover, Dmitrieva  et al., (2020)⁽²¹⁾  found that CLL patient not only have decreased platelet counts; but also have defect in  aggregation with adenosine diphosphate (ADP), and upon stimulation, decreased binding of CD62P, PAC1, and annexin V occurred . 

Concerning ESR, this study confirmed the findings of Ghaderi et al., (2016)⁽²²⁾. that found that CLL patients had a significantly higher ESR than controls and a higher level is associated with a worse prognosis.

The current study found that compared to the control group, CLL patients had considerably higher white blood cell counts, lymphocyte

percentages, and absolute lymphocytic counts. Hallek et al., (2018)⁽²³⁾ found that at least    5×10⁹/L of lymphocytes in the peripheral blood is necessary for a diagnosis of chronic lymphocytic leukemia^.

Shanafelt et al., (2009)⁽²⁴⁾ Found higher lymphocyte count in CLL patient. furthermore, he reported that independent prognostic value of lymphocyte count after prevelance of biologic/molecular prognostic markers.

The percentage of CD19 & CD5 was also considerably higher in the CLL group com-pared to the healthy participants, according to this study. Numerous studies corroborated these findings ^(25,26,27).

 In the present study, we found that ZAP-70 percentage evaluated by flow cytometry & the concentration of sCD14 were significantly higher in CLL patients when compared with healthy individuals.

Similarly, Al-Kahiry et al., (2018)⁽¹²⁾ and Khaliq et al., (2019)⁽⁸⁾ reported significant relationship between ZAP-70 percentage and advanced disease stage and poorer response to therapy and reported high serum levels of sCD14 in CLL patients compared with healthy individuals .

Patients with Binet stage A CLL and more than 20% ZAP-70 positive leukemic cells showed worse prognosis and shorter life times compared to patients with less than 20% ZAP-70 positive cells, according to Crespo et al., (2003)⁽²⁸⁾.

Seiffert et al., (2010) ⁽²⁹⁾ reported that increased expression of soluble CD14 by monocytes in CLL patients.

In this study, a statistically significant negative correlation between ZAP-70 percentage & sCD14 with LDH level in CLL patients. However, non-significant correlations between ZAP-70 percentage & sCD14 with HB level, TLC, platelet count, absolute lymphocytic count, CD19 and CD5 percentage. 

The results were consistent with those of Al-Kahiry et al., (2018) ⁽¹²⁾ but they ran counter to those of Mohammed (2021)^(30). who discovered no correlation between LDH levels and ZAP-

70 expression. The present study also found a significant positive correlation between sCD14 levels and ZAP-70 percentages, suggesting that sCD14 could serve as a substitute marker for ZAP-70. This agreed with the findings of Al-Kahiry et al., (2017)^(31), who found a significant positive correlation between sCD14 and the percentage of ZAP-70.

 Additionally, the current study reported that a statistically significant positive correlation between sCD14 level and ZAP-70 percentage, which reflect that sCD14 could be used as an alternative marker for ZAP-70.

This was in agreement with Al-Kahiry et al., (2017)⁽³¹⁾ who reported that sCD14 has a signi-ficant positive correlation to the percentage of ZAP-70. 

This study's findings of a positive correlation between sCD14 level, Binet staging, and RAI staging in group I were supported by previous research showing that sCD14 levels in CLL patients were associated with disease progression Callea et al., (1999) ⁽³²⁾  and Al-Kahiry et al., ( 2017)  ^{( 31)} and  Abdelgader et al., 2020)  ⁽⁹⁾, more over  sCD14 level is much higher in advanced stages of bone marrow failure (Binet C and Rai III-IV) and in patients with a high risk CLL prognostic index compared to those with early stage illness or low or intermediate risk.

The correlation with the percentage of ZAP-70 expression was statistically significant. Conse-quently, it is a crucial indicator for CLL patients' prognosis ⁽³³⁾.

There was a statistically significant negative correlation between sCD14 and ZAP-70 and smudge cell percentage in CLL patients. A higher percentage of smudge cells (SCs) (>30%) was found to be associated with better prognosis parameters in CLL patients. Since flow cytometric evaluation of ZAP-70 is not readily available, as patients with high smudge cells percentage (≥30%) the expression of ZAP-70 ranged from 1.1-2%, concentration of sCD14 ranged from 2.8-3.1%. While patients with low smudge cells percentage (<30%), the expression of ZAP-70 ranged from 4.7-9.7%, concentration of sCD14 ranged from 6.7-11.1%,

We agree with AlKahiry et al., (2018) ⁽¹²⁾ that there is a strong negative association between SC percentage in peripheral blood films of newly diagnosed CLL patients and ZAP-70 expression, suggesting that the former could be utilized instead.

 The results showed that the prognosis for chronic lymphocytic leukemia (CLL) improved with increasing percentages of SCs and decreasing expression of ZAP-70. Lastly, he mentioned that a better prognosis for CLL is associated with lower sCD14 concentration and ZAP-70 expression.

 According to Johansson et al., (2010) ⁽¹⁵⁾, One new prognostic marker in chronic lymphocytic leukemia (CLL) is the percentage of smudge cells, which correlates with ZAP-70 and has been reported to be considerably negative in some patients.

Enumeration of SCs is a marker of chronic lymphocytic leukemia (CLL) prognosis, according to Marionneaux et al. (2021) ⁽³⁴⁾.

 Sall et.al, (2022) ⁽³⁵⁾   found that SCs less than 30% in CLL associated with a poor prognosis. In developing countries, SCs percentage in CLL could be used as a reliable prognostic marker, Easy and accessible.

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