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ORIGINAL ARTICLE
Year : 2021 | Volume
: 46
| Issue : 2 | Page : 99-104
CXCR4 is a valuable poor prognostic marker for chronic lymphocytic leukemia: its higher level of expression is associated with inferior response to therapy and lower disease-free survival
Inas Asfour1, Walaa A Elsalakawy1, Mohamed T.H Sallam2, Mostafa K.El Razzaz MD 1
1 Hematology and Bone Marrow Transplantation Unit, Department of Internal Medicine, Ain Shams University, Cairo, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
Correspondence Address:
Mostafa K.El Razzaz
Department of Internal Medicine and Hematology, Faculty of Medicine, Ain Shams University, Abbassyia, Cairo 11566
Egypt
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ejh.ejh_3_21
Background Chronic lymphocytic leukemia (CLL) is one of the common chronic lymphoproliferative disorders (lymphoid neoplasms). It is characterized by a progressive accumulation of functionally incompetent lymphocytes, which are usually monoclonal in origin. CLL is a heterogeneous disease; thus, in some cases, the disease progresses so slowly that treatment is not required, but in others, a more aggressive form of the disease develops. CXCR4 (CD184) is a chemokine and chemokine receptor pair playing critical roles in tumor genesis. It is overexpressed in many hematological malignancies including acute myeloid leukemia and non-Hodgkin’s lymphoma and generally correlates with a poor prognosis.
Aim To evaluate the clinical utility of CXCR4 expression in patients with CLL as a possible predictor of disease outcome.
Patients and methods This is a prospective study conducted on 33 adult patients with newly diagnosed CLL. Expression of CXCR4 was determined by flow cytometry on either peripheral blood or bone marrow samples. Correlation with the course of the disease and the known CLL prognostic parameters was done initially and after 6 months of follow-up.
Results CXCR4 expression was positively correlated with absolute lymphocytic count, Rai score, β2 microglobulin, and lactate dehydrogenase levels. It was negatively correlated with hemoglobin and platelet counts, overall response rate, and 6-month disease-free survival.
Conclusion We conclude that CXCR4 is a valuable poor prognostic marker for CLL. Its higher level of expression is associated with inferior response to therapy and lower disease-free survival.
Keywords: chronic lymphocytic leukemia, CXCR4, survival
Chronic lymphocytic leukemia (CLL) is one of the chronic lymphoproliferative disorders (lymphoid neoplasms). It is characterized by a progressive accumulation of functionally incompetent lymphocytes, which are usually monoclonal in origin. CLL/small lymphocytic lymphoma is the most prevalent leukemia in older adults in the Western hemisphere, with an estimated 19 000 new cases diagnosed per year in the United States [1]. The disease typically affects older people and is rare in those aged less than 50 years [2]. CLL is characterized by the clonal proliferation and accumulation of mature, typically CD5-positive B cells within the blood, bone marrow (BM), lymph nodes, and spleen [3]. These B cells present a weak proliferation index compared with normal B cells and a defect in apoptosis [4]. However, these cells rapidly undergo spontaneous apoptosis when they are cultured in vitro, suggesting that in vivo factors contribute to their prolonged survival and reinforcing the importance of the microenvironment in this context [5]. There are two systems used for CLL clinical staging. These are Binet staging system and the Rai staging systems [6],[7].
CXCR4 (CD184) is a chemokine and chemokine receptor pair, playing critical roles in tumorigenesis. It is overexpressed in many hematological malignancies, including acute myeloid leukemia and non-Hodgkin’s lymphoma, and generally correlates with a poor prognosis [6].
CXCR4 has been shown to be crucial for homing, development, and function of B cells. The receptor is highly expressed by B cells throughout their development, and mice deficient in CXCR4 (or CXCL12) lack B lymphopoiesis [8],[9].
Patients and methodsPatient selection
This study included 33 newly diagnosed adult patients with CLL. The diagnosis of CLL was based on the WHO classification of tumors of lymphoid tissues.
CXCR4 expression was measured by flow cytometry. The level of CXCR4 expression has been correlated with the known CLL prognostic parameters and with the course of the disease and the outcome after 6 months of follow-up.
All procedures performed in our study were in accordance with the ethical standards of the ethical committee of Faculty of Medicine, Ain Shams University, and with the 1975 Helsinki Declaration as revised in 1983. Informed consent was obtained from all individual participants included in the study.
Flow cytometry methodology
Sampling
Blood was collected from each patient aseptically by withdrawing venous blood by a single puncture technique of the antecubital vein. Samples were dispensed gently into ethylene diamine tetra-acetic acid dipotassium salt (K2-EDTA)-containing vacutainer tubes (final concentration of 1.5 mg/ml) to perform CXCR4 by flow cytometry.Approximately 3–4 ml BM aspirate was obtained and divided as follows:Half to 1 ml of BM aspirate was obtained; smears were prepared and stained with Leishman and MPO stains for morphological and cytochemical examination.Three milliliters of BM aspirate was obtained on K2-EDTA for IPT and PCR.Samples were sent to each laboratory respectively on the same day of collection.Flow cytometric immunophenotyping
In the current study, we performed flow cytometric analysis of CXCR4 expression on B lymphocytes (supplied by Beckman Coulter, Hialeah, Florida, USA).
BM or peripheral blood samples were processed on the same day of sample collection. Counting was done using Coulter LH750 cell counter (Beckman Coulter) and the total leucocytic count (TLC) was adjusted to be around 5.0×109/l using phosphate buffered saline 120 Mm NaCl, 2.7 mM KCl, 10 mM phosphate buffer, PH 7.4 (commercially available from Sigma, St Louis, Missouri, USA). Overall, 50 μl of adjusted samples was aliquoted in each of the sample tubes and then 5 μl of each monoclonal antibody was added.
After incubation for 15 min at room temperature protected from light, 1–2 ml of ammonium chloride-based erythrocyte-lysing solution was added to every tube [8.29 g (0.15) NH4Cl, 1 g (10 mM) KHCO3, 0.037 g (0.1 mM) EDTA, and 1 l distilled water, adjusted to PH 7.3]. Tubes were vortexed then analyzed using Coulter Navios flow cytometer.
Definition of response to treatment
Response assessment was defined according to the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) criteria [10].
Statistical methods
Data were coded and entered using the statistical package SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, Illinois, USA), version 22. Data were summarized using mean and SD for quantitative data and using frequency (count) and relative frequency (percentage) for categorical data.
Comparisons between quantitative variables were done using the nonparametric Mann–Whitney tests and for categorical data using the χ2 test. Correlations between quantitative variables were done using Spearman correlation coefficient. P values less than 0.05 were considered as statistically significant.
ResultsThe current study included 33 newly diagnosed adult CLL patients. The mean age of patients was 53.79 years (range, 42–80 years). Twenty-one patients were males (63.6%) and 12 were females (36.4%).
On examination, five (15.2%) patients had lymphadenopathy, six (18.2%) patients had hepatosplenomegaly, and 19 (57.6%) patients had positive B symptoms.
According to the revised Rai score, 22% of our studied patients were low risk, 27% were intermediate risk, and 51% were high risk. The mean TLC was 58.13×109/l (range, 16.5–219×109/l), and mean absolute lymphocyte count was 61.18×109/l (range, 8.25–154.84×109/l). The mean hemoglobin level was 10.65 g/dl (range, 6–15.5 g/dl), and mean platelet count was 177.4×109/l (range, 16–329×109/l).
The mean CXCR4 expression was 58.57% (range=10.1–93%). The cutoff value for CXCR4 positivity was 20%, where 32 patients out of the 33 were positive and one patient only was negative. CXCR4 expression was not influenced by age or sex of the patients. The mean β2 microglobulin was 2.46 mg/l (range, 1–4.1 mg/l), with 54.5% of the patients with levels less than 3.5 mg/l and 45.5% with levels more than or equal to 3.5 mg/l. The mean lactate dehydrogenase (LDH) level was 1043.76 IU/l (range, 346–1890 IU/l).
The initial level of CXCR4 expression was significantly positively correlated with TLC, absolute lymphocytic count, LDH, β2 microglobulin, percentage of BM lymphocytes, and both Rai stage and revised Rai score, and negatively correlated with both hemoglobin and platelet count ([Table 1] and [Figure 1]).
The level of CXCR4 expression was highly correlated with B symptoms ([Table 2] and [Figure 2]).
With respect to response, patients included in the present study experienced progressive disease (PD) in 13 (39.3%) cases, complete response (CR) in two (6.06%) cases, partial response (PR) in 10 (30.03%) cases, and no response in eight (24.24%) cases. The mean CD184 expression was lowest in the CR group, and highest in the PD group, denoting the inverse relation between CXCR4 (CD184) expression and response to treatment ([Table 3]).
Table 3 Correlation between initial CXCR4 level and response to treatmentThe level of CXCR4 expression at 6 months of follow-up was significantly correlated with TLC, absolute lymphocytic count, hemoglobin, platelet, LDH, and β2 microglobulin ([Table 4]).
Table 4 Correlation between CXCR4 level at 6 months and different variablesAs for the cytogenetic studies, CLL panel was done by fluorescence in situ hybridization (FISH) in only 15 patients; this panel included 11q−, +12, 13q−, and 17p−. At diagnosis, 80% of those patients had normal cytogenetic parameters (CLL panel by FISH was normal), whereas only 20% of patients had abnormal cytogenetic in the form of 11q−, +12, 13q−, and 17p− by FISH technique. CD38 was positive in three (37.5%) of eight studied patients. ZAP70 was positive in two (28.6%) of seven studied patients, and the remaining five (71.4%) were negative. Regarding Coombs test, five (29.4%) of 17 patients were positive, and the remaining 12 (70.6%) were negative ([Table 5]).
DiscussionCLL is the leading malignant B cell tumor in the western world. The disease is characterized by its heterogeneous pathological nature, indolent natural history, and variable clinical outcomes [11],[12].
A crucial process in the CLL pathogenesis is CLL cells homing in the BM microenvironment. It is thought that many molecular pathways are recruited in this process [13].
Mesenchymal stromal cells, nurse-like cells, and lymphoma-associated macrophages, together with T cells, natural killer cells, and extracellular matrix components, share in the dialog with the neoplastic B cells. B-cell receptor signaling, activation via TNF family members (i.e., BAFF, APRIL), and tissue homing chemokine receptors and adhesion molecules are important in the interaction between malignant B cells and their microenvironment [14].
CXCR4 is a chemokine and chemokine receptor pair, playing crucial roles in tumor genesis. Overexpression of CXCR4 is characteristic of many hematological malignancies, including AML and NHL, and generally correlates with a poor prognosis [6].
So, the present study aimed to determine clinical utility of CXCR4 in patients with CLL in predicting disease outcome and planning treatment regimen.
Patients included in the present study comprised 21 (63.6%) males and 12 (36.4%) females, with a mean age of 53.79±8.05 years. Similarly, in the study of El-Ghammaz et al. [15], males outnumbered females (38 vs. 33), and the median age of the studied patients was 57 years. In the study of Abousamra et al. [16], males constituted 66.2% of the studied patients with CLL, and the mean±SD patients’ age was 59.9±6.78 years.
On examination, five (15.2%) patients of our series had lymphadenopathy, six (18.2%) patients had hepatosplenomegaly, and 19 (57.6%) patients had positive B symptoms. These findings accord with that reported by Hassan et al. [17], who reported splenomegaly in 66.7% and hepatomegaly in 43.3% of 30 patients with CLL. However, in the study of Kamel et al. [18], among 78 patients with newly diagnosed CLL, 49.3% of patients had hepatomegaly, 64.2% had splenomegaly, and 83.6% had lymphadenopathy. This is simply explained by the heterogeneous patients’ characteristics among various studies according to disease stage and treatment response.
Regarding laboratory data, patients included in the current study had a mean hemoglobin level of 10.65±3 g/dl, platelets count of 177.4±82×109/l, TLC of 58.1±49.5×109/l, and lymphocyte count of 61±48.6×109/l. These data generally accord with that reported by the study of Payandeh et al. [19], in their 109 patients with CLL research.
Other laboratory data showed elevated β2 microglobulin in 15 (45.5%) patients and elevated LDH in 26 (78%) patients. Both markers are important prognostic factors for CLL as reported by Labib et al. [20]. Prognostic Rai classification in the present study involved low-risk disease in seven (22.0%) patients, intermediate-risk disease in nine (27.0%) patients, and high-risk disease in 17 (51.0%) patients. This is in accordance with the study of Assem et al. [21], who found that 48.0% of their study patients were classified as high-risk patients, whereas the remainder is of low and intermediate risk.
Further cytogenetic and immunological analysis of our study patients revealed abnormal cytogenetics in 20% of 15 cases, positive CD38 in 37.5% out of eight cases, positive ZAP-70 in 28.6% out of seven cases, and positive Coombs test in 29.4% 17 cases. In comparison, abnormal cytogenetics was detected in 52% and CD38 was positive in 60% of patients included in the study of Saad et al. [22], on 25 patients with CLL, whereas in the study of Abousamra et al. [16], ZAP-70 was positive in 47.8% of patients.
With respect to response, patients included in the present study experienced PD in 13 (39.3%) cases, CR in two (6.06%) cases, PR in 10 (30.03%) cases, and no response in eight (24.24%) cases. Correspondingly, El-Kinawy et al. [23] reported unfavorable outcome in most patients (55.0%) in their series evaluating the prognostic factors in 40 patients with CLL.
Correlation analysis of CXCR4 with other clinical and laboratory values declared a statistically significant inverse correlation between CXCR4 levels and hemoglobin concentration and platelet count and a statistically significant correlation between CXCR4 and TLC, lymphocyte count, LDH, β2 microglobulin, and Rai stage at the start of the study and after 6 months.
These data are in agreement with the study of Barretina et al. [24], who studied CXCR4 expression in B-cell CLL and noted a significant correlation between CXCR4 and TLC and lymphocyte count.
Although no studies reported the relation between CXCR4 and LDH in patients with CLL, the study of Guo et al. [25] found a statistically significant correlation between CXCR4 and LDH levels in patients with diffuse large cell lymphoma.
Moreover, the study of Ghobrial et al. [26] noted a significant increase in CXCR4 with increased Rai stage in their study investigating the clinical relevance of chemokine receptor expression on the progression of B-cell CLL in 45 patients with B-cell CLL.
Perusing relation between CXCR4 expression and the clinical data in the present study, we noticed a significantly higher CXCR4 values in patients with positive B symptoms. The study of Kubeczko et al. [27] noted that patients with CLL with positive B symptoms had significantly higher levels of multiple chemokines, including IFNγ, CCL3, and CCL4, but not as in our study.
Our results agree with the study by Kriston et al. [28], who studied 80 patients with CLL and found an inverse correlation between BM stromal cell-induced apoptosis protection and their CXCR4 expression level. Furthermore, CLL cells with low CXCR4 expression were more dependent on the microenvironmental stimuli for survival.
ConclusionCXCR4 expression in patients with CLL showed a statistically significant inverse correlation with hemoglobin concentration and platelet count.
In addition, it showed a statistically significant positive correlation with TLC, lymphocyte count, LDH, β2 microglobulin, and Rai stage at the start of the study and after 6 months.
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Conflicts of interest
There are no conflicts of interest.
References
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