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Asia-Pacific Journal of Clinical Oncology 2014
doi: 10.1111/ajco.12268
ORIGINAL ARTICLE
Soluble CD44 and CD44v6 and prognosis in children with B-cell acute lymphoblastic leukemia Zahra AMIRGHOFRAN,1 Elham ASIAEE2 and Fatemeh M KAMAZANI1 1 Department of Immunology, Autoimmune Disease Research Center and Medicinal and Natural Products Chemistry Research Center and 2Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran
Abstract Aim: CD44v6 is an isoform of CD44 that can be present in soluble form (sCD44v6). The aim of this study is to evaluate the presence of soluble CD44 (sCD44) and sCD44v6 in serum of children with B-cell precursor acute lymphoblastic leukemia (B-ALL) and their relationship with prognosis. Methods: sCD44v6 and sCD44 levels were measured in the sera of patients and healthy children by enzyme-linked immunosorbent assay. The level of the molecules was analyzed in relation to laboratory and clinical characteristics of the patients at presentation and response to therapy. Result: sCD44v6 was significantly lower in patients (103.4 ± 44 ng/mL) than in controls (173.5 ± 73.6 ng/ mL) whereas the serum level of sCD44 showed no significant difference between the groups. In patients, sCD44v6 quantity was inversely correlated with sCD44 level (r = −0.57, P < 0.01). The mean serum level of sCD44 in patients with >20% positivity for CD44 surface expression was greater than that in patients with ≤20% positivity (1345 ± 409 ng/mL vs 1111 ± 390 ng/mL, P = 0.05). sCD44v6 showed no significant association with response to therapy and prognostic factors except the TEL/AML1 positivity, as it was higher in TEL/AML1 positive patients (157.3 ± 55.6 ng/mL) than negative ones (92 ± 43.6 ng/mL, P = 0.036). Conversely, sCD44 was lower in TEL/AML1 positive patients and showed a significant association with white blood cell number, blast percentage and extramedullary involvement. Conclusion: The lower level of sCD44v6 in patients than in controls suggests the possible diagnostic value of this molecule for B-ALL. The presence of an association with established prognostic factors despite of no relationship with disease outcome suggested these molecules for more studies in larger patient cohorts. Key words: acute lymphoblastic leukemia, prognosis, sCD44, sCD44v6.
INTRODUCTION Acute leukemia is a blood progenitor cell disease in bone marrow which occurs due to proliferation of immature lymphoid or myeloid cells. Acute leukemia is divided into acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML); the first one originates from
Correspondence: Professor Zahra Amirghofran PhD, Immunology Department, Medical School, Shiraz University of Medical Sciences, 71345-1798 Shiraz, Iran. Email: [email protected] Conflict of interest: The authors declare no conflicts of interest Accepted for publication 13 July 2014.
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B- and T-cell precursors while the second one originates from granulocyte or monocyte precursors.1 ALL is the most common childhood cancer with a peak onset of 2–5 years of age.2 There are various clinical and paraclinical parameters that are considered as important prognostic factors in ALL patients, for example, white blood cell (WBC) and platelet numbers, cytogenetics, percentage of blast in the blood and bone marrow, and extramedullary involvement (EMI) at the time of diagnosis.3 Age is also an important risk factor as patients with the age of 1–10 and WBC number less than 5 × 109/L have shown a better prognosis.1 CD44 is a surface glycoprotein that expresses on the most hematopoietic and non-hematopoietic cells including malignant cells.4 CD44 is a multifunctional molecule
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involved in adhesion of lymphocytes to endothelium and several extracellular matrix proteins like fibronectin and collagen.5 It takes part in various biological functions of lymphoid cells including lymphopoiesis, migration, homing and signal transduction.6 CD44 standard (CD44s) gene is located on human chromosome 11 and composed of 50–60 kb genomic DNA that contains 20 exons at least. CD44 plays an important role in tumor metastasis7,8 and has many variant isoforms (CD44v) generated by alternative splicing of exons 2v to 10v.9 One of the CD44 variant molecules is CD44v6 which its surface expression on the leukemic cells in ALL patients has been shown.10 CD44 and its variant, CD44v6, can be shed from the surface of the cells into the extracellular space and their soluble forms (sCD44, sCD44v6) are measureable in body fluids.11 In several studies, the presence of sCD44 in the peripheral blood of leukemia patients and its prognostic value has been investigated. In a study of 14 patients with ALL performed by Takeuchi et al., increased sCD44 level in children’s serum was a sign of disease activity.11 Tacyildiz and colleagues’ study on 54 children with ALL showed association of high concentration of sCD44 with poor prognosis.12 Ping and colleagues’ study is among the limited studies on CD44 variants that suggest sCD44v5 and sCD44v6 as well as sCD44 are markers of the severity, prognosis and response to treatment in adult patients with ALL.13 Given the lack of studies on soluble form of CD44v6 in pediatric ALL, in this study we decided to measure the level of sCD44v6 in serum of children with ALL to explore the role of this molecule in this disease and find out its prognostic significance. In order to compare, the level of sCD44 molecule was also measured.
METHODS Patients Forty-eight untreated patients (25 male and 23 female) with newly diagnosed B-cell precursor ALL (B-ALL) entered the study. Their disease was diagnosed on the base of histopathological, immunological and clinical findings. The mean age of the patients was 4.6 ± 3.2 (range 1–16 years). The laboratory and clinical characteristics such as WBC and platelet count, hemoglobin (Hb) concentration, blast percentage in peripheral blood and bone marrow, subtypes according to French– American–British (FAB) classification, EMI such as splenomegaly, hepatomegaly, lymphadenopathy and mediastinal mass, and central nervous system involve-
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Z Amirghofran et al.
ment and cytogenetics were collected from the patient’s medical records. Induction chemotherapy included vincristine, prednisone, L-asparaginase and doxorubicin. For intrathecal chemotherapy, methotrexate was administered once a week for 6 weeks. In 2.5 years of followup, days to achieve complete remission (time to CR), event-free survival (EFS) and overall survival (OS) were recorded. Control group included 33 healthy and age- and sexmatched children. Informed consent was obtained from all patients and controls or their parents. The study was approved by the Shiraz University of Medical Sciences Ethics Committee.
Immunophenotyping and CD44 surface expression Routine immunophenotyping was done with a panel of monoclonal antibodies purchased from Dako (Glostrup, Denmark). Fluorescence isothiocyanate (FITC) or phycoerythrin (PE)-conjugated monoclonal antibodies were used for immunophenotyping of the samples by twocolor flow cytometry. For each sample, 100 μL of whole blood, and then 10 μL conjugated monoclonal antibody (a combination of FITC- and PE-conjugated antibodies) were added. As the controls, a tube without antibody (unstained) and a tube with FITC/PE-conjugated IgG1 (isotype control) were used. After staining, cells were washed and resuspended in tubes by adding 0.5 mL Cellfix solution (Dako). A minimum of 10 000 cells were analyzed for each sample tube. Side scattering versus CD45 expression was used for gating of blasts and then on the other tubes analysis was performed. Patients with ALL were investigated for the surface expression of CD44 molecule using a FITC-conjugated specific monoclonal antibody. The samples were analyzed in a Partec PAS-II flow cytometer (Partec Instruments, Muenster, Germany) with Partec FloMax software. More than 20% reactivity with monoclonal antibodies was considered positive. After determination of patients with B-ALL, they were categorized according to the expression of B-cell lineage markers including CD19, CD10 and CD20 into Pro-B (CD19-only positive), early pre-B (CD19 and CD10 positive) or pre-BALL (all three markers were positive).
Determination of sCD44v6 and sCD44 serum levels From all patients (before chemotherapy) and controls, 3 mL peripheral blood was collected. Sera were isolated by centrifugation (800 g, 15 min at 4°C). Serum samples were stored at −20°C until analysis. sCD44v6 and
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Soluble CD44 and CD44v6 in pediatric ALL
sCD44 in the sera were measured by enzyme-linked immunosorbent assay kits from Bender MedSystems (Vienna, Austria). The limit of detection of sCD44 and sCD44v6 was 0.02 and 0.05 ng/mL, respectively. Briefly, an antihuman sCD44 (or sCD44v6) antibody was coated onto the microwells. About 20 μL of each sample and 50 μL enzyme-conjugated antihuman sCD44 (or sCD44v6) antibody were added. Following 3 h of incubation at room temperature, unbound antibodies were removed by washing and the substrate solution was then added. The reaction was terminated by addition of acid and absorbance was measured at 450 nm. The concentration of the sCD44 (or sCD44v6) was determined using a standard curve prepared by known concentrations of the analytes.
Statistical analysis The data were coded and univariate analysis was performed using the Statistical Package for Social Science (SPSS Inc, Chicago, IL, USA). Quantitative variables were reported as the mean ± standard deviation and categorized by frequency and percentage. The relationship of serum levels of CD44v6 and CD44 to the quantitative parameters of age, Hb, WBC, platelet, and blast percentage, and also the relationship with sex, EMI and CD44 positivity were analyzed with t-test for independent samples or the nonparametric equivalent Mann– Whitney. Correlations between soluble molecules with CD44 surface expression were analyzed by using the Pearson’s correlation test. The EFS was defined as the time from CR achievement to relapse/death. OS was determined from the date of entry into the study. Mean ± standard error was used for durations. A predictive value below 0.05 was considered statistically significant. Analysis of prognostic factors was calculated using the general linear model. In this analysis, a P-value less than 0.2 was used to determine the variables (including sex, age, bone marrow blasts and cytogenetic for sCD44v6 and WBC, platelet, peripheral blood blasts, EMI and cytogenetic for sCD44) that would be entered to the analysis. P-values of 0.05 and 0.1 were considered for adding and removing a variable, respectively. For response to therapy, a univariate analysis of prognostic factors was calculated by Cox regression model. In this analysis, the dependent variables were time to event OS or EFS or time to CR, and the covariates were sCD44, sCD44v6 and other parameters. Multivariate Cox regression analysis was performed for the variables with P-value less than 0.2.
Asia-Pac J Clin Oncol 2014
RESULTS Laboratory and clinical characteristics of the patients are summarized in Table 1. According to the age, patients were divided into two groups of less than 10 years and equal or more than that; the majority of them (89.6%) were less than 10 years old. Most of the patients (81.2%) showed a WBC count ≤ 50 × 109/L. There were no high-risk patients (age ≤ 1 or ≥10 years with WBC > 50 × 109/L). The mean percentages of blast in peripheral blood and bone marrow were 52.1 ± 26.2 and 73.9 ± 22.2, respectively. Cytogenetic analysis for hyperdiploidy or chromosomal translocations was done for 40 patients and it was positive in 15 patients (40% Tel/AML1 and 60% non-Tel/AML1). With respect to B-ALL subtypes, half of the patients had early pre-B, almost 40% had pre-B-ALL and the rest had pro-B-ALL. With regard to clinical findings, 28 patients (58.3%) presented with EMI, among which splenomegaly was the most common (in 25 patients). After chemotherapy, all patients were evaluated for response to treatment. Of the patients, 96.7% entered CR and two patients (4.3%) did not reach a CR (the condition of one patient was unknown). Patients entered remission in 19.1 ± 7.6 days. During 2.5 years of follow-up seven patients (14.6%) died. The EFS and OS were 621 ± 40 days (median, 706 days) and 687 ± 30 days (median, 738 days), respectively (Table 1).
sCD44v6 and sCD44 serum levels in patients and controls and surface expression of CD44 Determination of the serum levels of sCD44v6 showed a significant difference between patients and controls (P < 0.001), as sCD44v6 was lower in patients (103.4 ± 44 ng/mL) than in controls (173.5 ± 73.6 ng/mL) (Fig. 1). With respect to sCD44, its serum level showed no significant difference between patients (1218.9 ± 412.4 ng/mL) and controls (1264.5 ± 383.3 ng/mL, P = 0.61) (Fig. 1). There was a negative correlation between sCD44 and sCD44v6 serum levels in the patients (r = −0.57, P < 0.01) (Fig. 2). The percentage expression of CD44 on leukemia cells of the patients was 23.4 ± 17.3 (range, 1.1–71.6%). Twenty-six patients showed more than 20% positivity for CD44. In these patients, the mean serum level of sCD44 was more than that in CD44 negative ones (1345 ± 409 ng/mL vs 1111 ± 390 ng/mL, P = 0.05).
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Table 1
Z Amirghofran et al.
Characteristics of ALL patients
Variables Patients Age (year) Range 50 Hb (g/dL) Range ≤10 >10 Plt × 109/L Range ≤100 >100 %Blast (PB) Range ≤50 >50 %Blast (BM) Range ≤50 >50 FAB: L1 L2 L 1L 2 Pro-B Early Pre-B Pre-B Pt with EMI Pt without EMI Cytogenetic Positive Negative Tel/AML1 Non-Tel/AML1 CR rate Time to CR (days) EFS (days) OS (days) Death rate
Total 48 4.6 ± 3.2 1–16 43 (89.6) 5 (10.4) 23 (47.9) 25 (52.1) 25.4 ± 3.4 1–200 39 (81.2) 9 (18.8) 7.3 ± 2.6 3.8–13.4 40 (83.3) 8 (16.7) 89.6 ± 114.1 11–520 37 (77.1) 11 (22.9) 52.1 ± 26.2 1–90 14 (50) 14 (50) 73.9 ± 22.2 18–100 9 (18.8) 38 (79.1) 28 (58.3) 15 (31.3) 5 (10.4) 5 (10.4) 24 (50) 19 (39.6) 28 (58.3) 20 (41.7) 15 (31.2) 25 (52.1) 6 (40) 9 (60) (96.7) 19.1 ± 7.6 621 ± 40 687 ± 30 (14.6)
Data represent mean ± standard deviation according to number (%) and mean ± standard error for durations. BM, bone marrow; CR, complete remission; EFS, event-free survival; EMI, extramedullary involvement; FAB, French–American–British classification of ALL; Hb, hemoglobin; OS, overall survival; PB, peripheral blood; Plt, platelet; Pt, patient; WBC, white blood cell.
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Figure 1 Determination of sCD44 and sCD44v6 serum levels in patients with acute lymphoblastic leukemia and healthy individuals as controls. A significant difference between sCD44v6 level between patients and controls is observed (***P < 0.001). ( ) patients; ( ) controls.
Figure 2 The relationship between sCD44 and sCD44v6 serum levels in patients with acute lymphoblastic leukemia. A negative correlation between the two molecules is observed (r = −0.57). Correlation is significant at the 0.01 level.
sCD44v6 and sCD44 serum levels in relation to patients’ characteristics Study of the serum levels of sCD44v6 and sCD44 in patients showed no significant difference in men and women. The serum levels of these molecules in patients under 10 years and more than that also showed no significant difference. sCD44v6 and sCD44 had no association with laboratory features of the patients such as Hb concentration, FAB subgroups and platelet numbers. However, sCD44 was significantly higher in patients with WBC > 50 × 109/L (1464.5 ± 459.4 ng/mL) than in patients with WBC ≤ 50 × 109/L (1162.2 ± 385.1
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Soluble CD44 and CD44v6 in pediatric ALL
Figure 3 The relationship between sCD44 serum level and several prognostic factors in patients with acute lymphoblastic leukemia. Data represented the mean ± SD of sCD44 serum level in different groups. WBC, white blood cell (×103/μL); EMI, extramedullary involvement. sCD44 serum level was significantly higher in patients with WBC > 50 × 103/μL and in those with EMI as well as Tel/AML1 negative patients (*P < 0.05, **P < 0.01). ( ) WBC ≤ 50 × 109/L; ( ) WBC > 50 × 109/L; ( ) with EMI; ( ) without EMI; ( ) Tel/ AML1; ( ) non-Tel/AML1.
ng/mL, P = 0.038) (Fig. 3), and also in patients with peripheral blood blast percentage > 50 (1435.4 ± 393.6 ng/mL) than in patients with blast percentage ≤ 50 (1143 ± 351 ng/mL, P = 0.048), indicating the prognostic value of this molecule. With respect to immunophenotype, there were no significant differences in the level of these molecules between different B-ALL subtypes. A significant association between sCD44 level and the presence of EMI was detected (Fig. 3). The level of this molecule was higher in patients with EMI than those without EMI (1337.1 ± 391.7 ng/mL vs 1053 ± 391.3 ng/mL, P = 0.017) and in patients with splenomegaly than those without splenomegaly (1344.3 ± 410.9 vs 1082.5 ± 376.5, P = 0.026). There was no significant relationship between sCD44 and sCD44v6 serum levels and the presence or absence of cytogenetic abnormalities, but in relation to TEL/AML1 positivity sCD44v6 showed a higher level in positive patients than negative ones (157.3 ± 55.6 ng/mL vs 92 ± 43.6 ng/mL, P = 0.036) (Fig. 4), whereas sCD44 showed a different result; it was higher in non-TEL/ AML1 patients (1516.7 ± 367.8 ng/mL) than TEL/ AML1 positive ones (743.9 ± 294.1 ng/mL, P = 0.003) (Fig. 3). Analysis for prognostic factors using the general linear model showed no significant results.
Asia-Pac J Clin Oncol 2014
Figure 4 The relationship between sCD44v6 serum level and several prognostic factors in patients with acute lymphoblastic leukemia. Data represented the mean ± SD of sCD44v6 serum level in different groups. WBC, white blood cell (×103/μL); EMI, extramedullary involvement. sCD44v6 serum level was significantly higher in patients positive for Tel/AML1 (*P < 0.05). ( ) WBC ≤ 50 × 109/L; ( ) WBC > 50 × 109/L; ( ) with EMI; ( ) without EMI; ( ) Tel/AML1; ( ) non-Tel/ AML1.
The mean serum levels of sCD44 in patients who remained alive and those who died at the end of follow-up showed no significant difference (1227 ± 415 and 1166 ± 158 ng/mL, respectively). The same result was obtained for sCD44v6 (101 ± 43 and 116 ± 47 ng/ mL, respectively). No significant difference in the level of these molecules in patients who entered CR and those who did not achieve CR was detected. In univariate Cox regression analysis, sCD44 and sCD44v6 showed no significant influence on OS, EFS or time to CR. Because P-values for sCD44 and sCD44v6 were more than 0.2, no multivariate analysis was performed.
DISCUSSION The primary focus of our study was to measure the difference in sCD44 and sCD44v6 levels between ALL children and their healthy counterparts. The second aim was to find the prognostic significance of these molecules. In recent years, several studies have investigated the presence of the soluble form of CD44 molecule in serum of children with hematologic malignancies.11–16 In a number of these studies, the level of sCD44 in the patients at the time of presentation has been significantly higher than its level in healthy children. In this regard,
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Tacyildiz and colleagues have shown a higher serum CD44 level in 16 Hodgkin’s disease, 13 non-Hodgkin’s lymphoma, 8 Burkitt’s lymphoma and 8 ALL patients than those in 12 healthy children.12 Ahmed and Hassab also found an increased sCD44 level in 30 patients with ALL.15 In contrast to these studies, we did not find a significant difference in sCD44 levels between patient and control groups. Similarly, El-Sharkawy and colleagues showed no significant difference in sCD44 level in 35 ALL patients compared with the 15 controls.14 This inconsistency between the results may be due to differences in genetic characteristics or the number of patients studied. To the best of our knowledge, no previous studies have investigated sCD44v6 in children with ALL and its relation to prognostic factors. However, Ping et al. evaluated changes of sCD44 and sCD446 in 20 adult patients with ALL. Their study showed a higher expression of sCD44 in patients than controls but pretreatment serum sCD44v5 and sCD44v6 concentrations were lower. After treatment, serum sCD44 levels declined, while serums sCD44v5 and sCD44v6 returned to normal.13 In the current study, we also found a significant difference in sCD44v6 level between our patients and controls; the level of this molecule was lower in patients (P < 0.001) which may provide an evidence for the possible protective and diagnostic importance of this molecule in pediatric ALL. CD44 is known to be expressed on various hematopoietic cells including leukemia cells, though its level of expression and release from the surface varies according to the state of cell proliferation or activation.14 El-Sharkawy and colleagues investigated the surface expression of CD44 and several of its variant isoforms including CD44v6, CD44v7/8 and CD44v4 in ALL patients, but contrary to wide expression of CD44 in the patients they found no expression of the variant isoforms except CD44v4 (14.3%) on any of their cases.14 No correlation between CD44 expression and soluble form of CD44 was detected in their study. In contrary, Tacyildiz et al. found a correlation between CD44 expression and sCD44 level in ALL patients.12 In our patients, we also found a relationship between CD44 expression and sCD44 levels that showed at least a part of serum sCD44 levels in childhood leukemia may originate from leukemic cells. With respect to CD44v6, we did not measure the surface expression of this molecule on the leukemic cells in the patients; however, in previous studies on other types of malignancies, dissimilar results have been found; for example, in a study, CD44v6 expression has been positively correlated with
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Z Amirghofran et al.
its level in the serum of colorectal carcinoma patients.17 The serum level of sCD44v6 has been higher in patients with CD44v6-positive gastric carcinoma cells than in those with CD44v6-negative tumor cells,18 and in another study on the same disease no correlation has been found.19 Given the observed correlation between CD44 expression and sCD44 level in this study, and the important role of CD44 as an adhesion molecule involved in tumor metastasis,20 one may ask about the value of sCD44 and sCD44v6 in prognosis and response to therapy in ALL patients. In various studies, the level of sCD44 has been suggested as a valuable marker for monitoring response to therapy and prognosis. Takeuchi et al. have reported a higher sCD44 serum level in the patients at presentation time and relapse compared with its level during CR.11 Increased serum level of sCD44 during relapse has also been reported by Yokota et al., which implies the sCD44 relation to disease status and treatment in the patients.21 In the present study, the level of sCD44 and sCD44v6 molecules in relation to treatment response showed no significant difference between patients who remained alive and those who died at the end of followup, nor in patients who entered CR and those who did not, showing the lack of association of sCD44 and sCD44v6 molecules with response to therapy. These data are in agreement with previous studies that reported no significant association between sCD44 levels with survival and achievement of CR.12,13 We looked for possible correlation between sCD44 and sCD44v6 levels with known prognostic factors in the patients. Our data showed that neither sCD44 nor sCD44v6 level was correlated with sex, age, Hb concentration, nor platelet numbers in the patients. However, with respect to sCD44 a significant correlation with WBC numbers and the percentage of blasts in peripheral blood was observed. Patients with higher WBC numbers and peripheral blood blasts had higher sCD44 level. Further statistical analysis of sCD44 level in our patients revealed a significant increased sCD44 level in patients who had EMI. These data are in line with previous studies in which a relationship between the expression of surface CD44 with EMI has been reported.20 Here, our data indicated that CD44 molecule in soluble form can also be used as a valuable prognostic factor in childhood ALL. Furthermore, sCD44 showed a negative association with TEL/AML1 positivity and was higher in patients without this abnormality. As TEL/AML1 positivity has been introduced as a favorable indicator of prognosis in ALL,22 this finding provides a further evidence for the important role of sCD44 as a poor
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Soluble CD44 and CD44v6 in pediatric ALL
prognostic factor in childhood ALL. In contrast, sCD44v6 was significantly higher in TEL/AML1 positive patients than negative ones which may suggest this molecule as a good prognostic factor. This difference between sCD44v6 and sCD44 results in relation to prognosis seems to be in line with another finding of this study that showed a negative correlation between sCD44 and sCD44v6 levels in the patients. As mentioned before, the generation of CD44 variant isoforms is mainly due to alternative splicing which is under the control of distinct factors from various signaling cascades that are able to recognize specifically pre-mRNA elements of different genes.23 Alterations in these factors in various pathological conditions may alter the production of various CD44 variant isoforms.23 In conclusion, as results of this study showed, sCD44v6 serum level was different between patients and controls which suggest the potential diagnostic and protective values of this molecule for ALL. In the patients, increased sCD44 level was associated with decreased sCD44v6 level. Both sCD44 and sCD44v6 indicated a relationship with prognostic factors. In this regard, sCD44 seems to be more important as it indicated association with more prognostic factors. There are several limitations of this study, for example, inadequate case numbers, inadequate follow-up time, and no sequential measurement of sCD44 and sCD44v6 levels. Further research with the larger number of patients and longer follow-up is recommended to find the precise impact of these molecules in response to therapy and disease outcome.
ACKNOWLEDGMENT The authors would like to thank Dr Mahdi Salehipour and Sare Roosta at the Center for Development of Clinical Studies of Nemazee Hospital for statistical assistance, and also Mr Saeed Malekhosseini for providing valuable assistance. This work is originally described in the MD thesis of one of the authors, E. Asiaee.
REFERENCES 1 Vrooman LM, Silverman LB. Childhood acute lymphoblastic leukemia: update on prognostic factors. Curr Opin Pediatr 2009; 21: 1–8. 2 De Angelis C, Pacheco C, Lucchini G et al. The experience in Nicaragua: childhood leukemia in low income countriesThe main cause of late diagnosis may be “medical delay”. Int J of Pediatr 2012; 2012: 129707.
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3 Fathi M, Amirghofran Z, Shahriari M. Soluble Fas and Fas ligand and prognosis in children with acute lymphoblastic leukemia. Med Oncol 2012; 29: 2046–52. 4 Williams K, Motiani K, Giridhar PV, Kasper S. CD44 integrates signaling in normal stem cell, cancer stem cell and (pre)metastatic niches. Exp Biol Med (Maywood) 2013; 238: 324–38. 5 Salmi M, Karikoski M, Elima K, Rantakari P, Jalkanen S. CD44 binds to macrophage mannose receptor on lymphatic endothelium and supports lymphocyte migration via afferent lymphatics. Circ Res 2013; 7: 1577–82. 6 Bjorklund CC, Baladandayuthapani V, Lin HY et al. Evidence of a role for CD44 and cell adhesion in mediating resistance to lenalidomide in multiple myeloma: therapeutic implications. Leukemia 2014; 28: 373–83. 7 Negi LM, Talegaonkar S, Jaggi M, Ahmad FJ, Iqbal Z, Khar RK. Role of CD44 in tumor progression and strategies for targeting. J Drug Target 2012; 20: 561–73. 8 Wu G, Zhou Y, Li T, Guo J, Zhou Z. Immunohistochemical levels of matrix metalloproteinase-2 and CD44 variant 6 protein in the diagnosis and lateral cervical lymph node metastasis of papillary thyroid carcinoma. J Int Med Res 2013; 41: 816–24. 9 Heider KH, Kuthan H, Stehle G, Munzert G. CD44v6: a target for antibody-based cancer therapy. Cancer Immunol Immunother 2004; 53: 567–79. 10 Khan NI, Cisterne A, Devidas M et al. Expression of CD44, but not CD44v6, predicts relapse in children with B cell progenitor acute lymphoblastic leukemia lacking adverse or favorable genetics. Leuk Lymphoma 2008; 49: 710–8. 11 Takeuchi M, Tanizawa A, Fukumoto Y, Kikawa Y, Mayumi M. Serum soluble CD44 in pediatric patients with acute leukemia. J Pediatr Hematol Oncol 1999; 21: 384–8. 12 Tacyildiz N, Cavdar AO, Yavuz G et al. Serum levels and differential expression of CD44 in childhood leukemia and malignant lymphoma- correlation with prognostic criteria and survival. Pediatr Int 2001; 43: 354–60. 13 Ping C, Jin-xiang F, Lan-yun S, Jun L, Zuo Y. Changes of soluble CD44 peripheral blood of adult patients with acute lymphoblastic leukemia and its implication to clinical outcome. Chinese J Hemorheol 2002; 2: 107–9. 14 El-Sharkawy NM, Hamdy N, Attia E et al. CD44 expression and soluble CD44 as a potential marker of tumor load in pediatric acute leukemia. J Egypt Nat Cancer Inst 2003; 15: 129–35. 15 Ahmed MI, Hassab HM. Study of soluble CD44 and its expression by mononuclear cells in children with acute lymphoblastic leukemia its relation to prognostic factors. Egypt J Immunol 2008; 15: 101–11. 16 Nasu H, Hibi N, Ohyashiki JH et al. Serum soluble CD44 levels for monitoring disease states in acute leukemia and myelodysplastic syndromes. Int J Oncol 1998; 13: 525–30. 17 Amirghofran Z, Jalali SA, Hosseini SV, Vasei M, Sabayan B, Ghaderi A. Evaluation of CD44 and CD44v6 in colorectal carcinoma patients: soluble forms in relation to tumor
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tissue expression and metastasis. J Gastrointest Cancer 2008; 39: 73–8. 18 Zhou DH, Ma ZM, Chen Y. Expression and clinical significance of CD44v6 and sCD44v6 in gastric carcinoma. Zhonghua Zhong Liu Za Zhi 2007; 29: 833–7. 19 Saito H, Tsujitani S, Katano K, Ikeguchi M, Maeta M, Kaibara N. Serum concentration of CD44 variant 6 and its relation to prognosis in patients with gastric carcinoma. Cancer 1998; 83: 1094–101. 20 Kamazani FM, Bahoush GR, Aghaeipour M, Vaeli S, Amirghofran Z. CD44 and CD27 expression pattern in B cell precursor acute lymphoblastic leukemia and its clinical significance. Med Oncol 2013; 30: 359.
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21 Yokota A, Ishii G, Sugaya Y, Nishimura M, Saito Y, Harigaya K. Potential use of serum CD44 as an indicator of tumor progression in acute leukemia. Hematol Oncol 1999; 17: 161–8. 22 Mazloumi SH, Madhumathi DS, Appaji L, Prasannakumari. Combined study of cytogenetics and fluorescence in situ hybridization (FISH) analysis in childhood acute lymphoblastic leukemia (ALL) in a tertiary cancer centre in South India. Asian Pac J Cancer Prev 2012; 13: 3825–7. 23 Cheng C, Sharp PA. Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion. Mol Cell Biol 2006; 26: 362–70.
Asia-Pac J Clin Oncol 2014
Asia-Pacific Journal of Clinical Oncology 2014
doi: 10.1111/ajco.12268
ORIGINAL ARTICLE
Soluble CD44 and CD44v6 and prognosis in children with B-cell acute lymphoblastic leukemia Zahra AMIRGHOFRAN,1 Elham ASIAEE2 and Fatemeh M KAMAZANI1 1 Department of Immunology, Autoimmune Disease Research Center and Medicinal and Natural Products Chemistry Research Center and 2Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran
Abstract Aim: CD44v6 is an isoform of CD44 that can be present in soluble form (sCD44v6). The aim of this study is to evaluate the presence of soluble CD44 (sCD44) and sCD44v6 in serum of children with B-cell precursor acute lymphoblastic leukemia (B-ALL) and their relationship with prognosis. Methods: sCD44v6 and sCD44 levels were measured in the sera of patients and healthy children by enzyme-linked immunosorbent assay. The level of the molecules was analyzed in relation to laboratory and clinical characteristics of the patients at presentation and response to therapy. Result: sCD44v6 was significantly lower in patients (103.4 ± 44 ng/mL) than in controls (173.5 ± 73.6 ng/ mL) whereas the serum level of sCD44 showed no significant difference between the groups. In patients, sCD44v6 quantity was inversely correlated with sCD44 level (r = −0.57, P < 0.01). The mean serum level of sCD44 in patients with >20% positivity for CD44 surface expression was greater than that in patients with ≤20% positivity (1345 ± 409 ng/mL vs 1111 ± 390 ng/mL, P = 0.05). sCD44v6 showed no significant association with response to therapy and prognostic factors except the TEL/AML1 positivity, as it was higher in TEL/AML1 positive patients (157.3 ± 55.6 ng/mL) than negative ones (92 ± 43.6 ng/mL, P = 0.036). Conversely, sCD44 was lower in TEL/AML1 positive patients and showed a significant association with white blood cell number, blast percentage and extramedullary involvement. Conclusion: The lower level of sCD44v6 in patients than in controls suggests the possible diagnostic value of this molecule for B-ALL. The presence of an association with established prognostic factors despite of no relationship with disease outcome suggested these molecules for more studies in larger patient cohorts. Key words: acute lymphoblastic leukemia, prognosis, sCD44, sCD44v6.
INTRODUCTION Acute leukemia is a blood progenitor cell disease in bone marrow which occurs due to proliferation of immature lymphoid or myeloid cells. Acute leukemia is divided into acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML); the first one originates from
Correspondence: Professor Zahra Amirghofran PhD, Immunology Department, Medical School, Shiraz University of Medical Sciences, 71345-1798 Shiraz, Iran. Email: [email protected] Conflict of interest: The authors declare no conflicts of interest Accepted for publication 13 July 2014.
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B- and T-cell precursors while the second one originates from granulocyte or monocyte precursors.1 ALL is the most common childhood cancer with a peak onset of 2–5 years of age.2 There are various clinical and paraclinical parameters that are considered as important prognostic factors in ALL patients, for example, white blood cell (WBC) and platelet numbers, cytogenetics, percentage of blast in the blood and bone marrow, and extramedullary involvement (EMI) at the time of diagnosis.3 Age is also an important risk factor as patients with the age of 1–10 and WBC number less than 5 × 109/L have shown a better prognosis.1 CD44 is a surface glycoprotein that expresses on the most hematopoietic and non-hematopoietic cells including malignant cells.4 CD44 is a multifunctional molecule
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involved in adhesion of lymphocytes to endothelium and several extracellular matrix proteins like fibronectin and collagen.5 It takes part in various biological functions of lymphoid cells including lymphopoiesis, migration, homing and signal transduction.6 CD44 standard (CD44s) gene is located on human chromosome 11 and composed of 50–60 kb genomic DNA that contains 20 exons at least. CD44 plays an important role in tumor metastasis7,8 and has many variant isoforms (CD44v) generated by alternative splicing of exons 2v to 10v.9 One of the CD44 variant molecules is CD44v6 which its surface expression on the leukemic cells in ALL patients has been shown.10 CD44 and its variant, CD44v6, can be shed from the surface of the cells into the extracellular space and their soluble forms (sCD44, sCD44v6) are measureable in body fluids.11 In several studies, the presence of sCD44 in the peripheral blood of leukemia patients and its prognostic value has been investigated. In a study of 14 patients with ALL performed by Takeuchi et al., increased sCD44 level in children’s serum was a sign of disease activity.11 Tacyildiz and colleagues’ study on 54 children with ALL showed association of high concentration of sCD44 with poor prognosis.12 Ping and colleagues’ study is among the limited studies on CD44 variants that suggest sCD44v5 and sCD44v6 as well as sCD44 are markers of the severity, prognosis and response to treatment in adult patients with ALL.13 Given the lack of studies on soluble form of CD44v6 in pediatric ALL, in this study we decided to measure the level of sCD44v6 in serum of children with ALL to explore the role of this molecule in this disease and find out its prognostic significance. In order to compare, the level of sCD44 molecule was also measured.
METHODS Patients Forty-eight untreated patients (25 male and 23 female) with newly diagnosed B-cell precursor ALL (B-ALL) entered the study. Their disease was diagnosed on the base of histopathological, immunological and clinical findings. The mean age of the patients was 4.6 ± 3.2 (range 1–16 years). The laboratory and clinical characteristics such as WBC and platelet count, hemoglobin (Hb) concentration, blast percentage in peripheral blood and bone marrow, subtypes according to French– American–British (FAB) classification, EMI such as splenomegaly, hepatomegaly, lymphadenopathy and mediastinal mass, and central nervous system involve-
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ment and cytogenetics were collected from the patient’s medical records. Induction chemotherapy included vincristine, prednisone, L-asparaginase and doxorubicin. For intrathecal chemotherapy, methotrexate was administered once a week for 6 weeks. In 2.5 years of followup, days to achieve complete remission (time to CR), event-free survival (EFS) and overall survival (OS) were recorded. Control group included 33 healthy and age- and sexmatched children. Informed consent was obtained from all patients and controls or their parents. The study was approved by the Shiraz University of Medical Sciences Ethics Committee.
Immunophenotyping and CD44 surface expression Routine immunophenotyping was done with a panel of monoclonal antibodies purchased from Dako (Glostrup, Denmark). Fluorescence isothiocyanate (FITC) or phycoerythrin (PE)-conjugated monoclonal antibodies were used for immunophenotyping of the samples by twocolor flow cytometry. For each sample, 100 μL of whole blood, and then 10 μL conjugated monoclonal antibody (a combination of FITC- and PE-conjugated antibodies) were added. As the controls, a tube without antibody (unstained) and a tube with FITC/PE-conjugated IgG1 (isotype control) were used. After staining, cells were washed and resuspended in tubes by adding 0.5 mL Cellfix solution (Dako). A minimum of 10 000 cells were analyzed for each sample tube. Side scattering versus CD45 expression was used for gating of blasts and then on the other tubes analysis was performed. Patients with ALL were investigated for the surface expression of CD44 molecule using a FITC-conjugated specific monoclonal antibody. The samples were analyzed in a Partec PAS-II flow cytometer (Partec Instruments, Muenster, Germany) with Partec FloMax software. More than 20% reactivity with monoclonal antibodies was considered positive. After determination of patients with B-ALL, they were categorized according to the expression of B-cell lineage markers including CD19, CD10 and CD20 into Pro-B (CD19-only positive), early pre-B (CD19 and CD10 positive) or pre-BALL (all three markers were positive).
Determination of sCD44v6 and sCD44 serum levels From all patients (before chemotherapy) and controls, 3 mL peripheral blood was collected. Sera were isolated by centrifugation (800 g, 15 min at 4°C). Serum samples were stored at −20°C until analysis. sCD44v6 and
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Soluble CD44 and CD44v6 in pediatric ALL
sCD44 in the sera were measured by enzyme-linked immunosorbent assay kits from Bender MedSystems (Vienna, Austria). The limit of detection of sCD44 and sCD44v6 was 0.02 and 0.05 ng/mL, respectively. Briefly, an antihuman sCD44 (or sCD44v6) antibody was coated onto the microwells. About 20 μL of each sample and 50 μL enzyme-conjugated antihuman sCD44 (or sCD44v6) antibody were added. Following 3 h of incubation at room temperature, unbound antibodies were removed by washing and the substrate solution was then added. The reaction was terminated by addition of acid and absorbance was measured at 450 nm. The concentration of the sCD44 (or sCD44v6) was determined using a standard curve prepared by known concentrations of the analytes.
Statistical analysis The data were coded and univariate analysis was performed using the Statistical Package for Social Science (SPSS Inc, Chicago, IL, USA). Quantitative variables were reported as the mean ± standard deviation and categorized by frequency and percentage. The relationship of serum levels of CD44v6 and CD44 to the quantitative parameters of age, Hb, WBC, platelet, and blast percentage, and also the relationship with sex, EMI and CD44 positivity were analyzed with t-test for independent samples or the nonparametric equivalent Mann– Whitney. Correlations between soluble molecules with CD44 surface expression were analyzed by using the Pearson’s correlation test. The EFS was defined as the time from CR achievement to relapse/death. OS was determined from the date of entry into the study. Mean ± standard error was used for durations. A predictive value below 0.05 was considered statistically significant. Analysis of prognostic factors was calculated using the general linear model. In this analysis, a P-value less than 0.2 was used to determine the variables (including sex, age, bone marrow blasts and cytogenetic for sCD44v6 and WBC, platelet, peripheral blood blasts, EMI and cytogenetic for sCD44) that would be entered to the analysis. P-values of 0.05 and 0.1 were considered for adding and removing a variable, respectively. For response to therapy, a univariate analysis of prognostic factors was calculated by Cox regression model. In this analysis, the dependent variables were time to event OS or EFS or time to CR, and the covariates were sCD44, sCD44v6 and other parameters. Multivariate Cox regression analysis was performed for the variables with P-value less than 0.2.
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RESULTS Laboratory and clinical characteristics of the patients are summarized in Table 1. According to the age, patients were divided into two groups of less than 10 years and equal or more than that; the majority of them (89.6%) were less than 10 years old. Most of the patients (81.2%) showed a WBC count ≤ 50 × 109/L. There were no high-risk patients (age ≤ 1 or ≥10 years with WBC > 50 × 109/L). The mean percentages of blast in peripheral blood and bone marrow were 52.1 ± 26.2 and 73.9 ± 22.2, respectively. Cytogenetic analysis for hyperdiploidy or chromosomal translocations was done for 40 patients and it was positive in 15 patients (40% Tel/AML1 and 60% non-Tel/AML1). With respect to B-ALL subtypes, half of the patients had early pre-B, almost 40% had pre-B-ALL and the rest had pro-B-ALL. With regard to clinical findings, 28 patients (58.3%) presented with EMI, among which splenomegaly was the most common (in 25 patients). After chemotherapy, all patients were evaluated for response to treatment. Of the patients, 96.7% entered CR and two patients (4.3%) did not reach a CR (the condition of one patient was unknown). Patients entered remission in 19.1 ± 7.6 days. During 2.5 years of follow-up seven patients (14.6%) died. The EFS and OS were 621 ± 40 days (median, 706 days) and 687 ± 30 days (median, 738 days), respectively (Table 1).
sCD44v6 and sCD44 serum levels in patients and controls and surface expression of CD44 Determination of the serum levels of sCD44v6 showed a significant difference between patients and controls (P < 0.001), as sCD44v6 was lower in patients (103.4 ± 44 ng/mL) than in controls (173.5 ± 73.6 ng/mL) (Fig. 1). With respect to sCD44, its serum level showed no significant difference between patients (1218.9 ± 412.4 ng/mL) and controls (1264.5 ± 383.3 ng/mL, P = 0.61) (Fig. 1). There was a negative correlation between sCD44 and sCD44v6 serum levels in the patients (r = −0.57, P < 0.01) (Fig. 2). The percentage expression of CD44 on leukemia cells of the patients was 23.4 ± 17.3 (range, 1.1–71.6%). Twenty-six patients showed more than 20% positivity for CD44. In these patients, the mean serum level of sCD44 was more than that in CD44 negative ones (1345 ± 409 ng/mL vs 1111 ± 390 ng/mL, P = 0.05).
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Table 1
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Characteristics of ALL patients
Variables Patients Age (year) Range 50 Hb (g/dL) Range ≤10 >10 Plt × 109/L Range ≤100 >100 %Blast (PB) Range ≤50 >50 %Blast (BM) Range ≤50 >50 FAB: L1 L2 L 1L 2 Pro-B Early Pre-B Pre-B Pt with EMI Pt without EMI Cytogenetic Positive Negative Tel/AML1 Non-Tel/AML1 CR rate Time to CR (days) EFS (days) OS (days) Death rate
Total 48 4.6 ± 3.2 1–16 43 (89.6) 5 (10.4) 23 (47.9) 25 (52.1) 25.4 ± 3.4 1–200 39 (81.2) 9 (18.8) 7.3 ± 2.6 3.8–13.4 40 (83.3) 8 (16.7) 89.6 ± 114.1 11–520 37 (77.1) 11 (22.9) 52.1 ± 26.2 1–90 14 (50) 14 (50) 73.9 ± 22.2 18–100 9 (18.8) 38 (79.1) 28 (58.3) 15 (31.3) 5 (10.4) 5 (10.4) 24 (50) 19 (39.6) 28 (58.3) 20 (41.7) 15 (31.2) 25 (52.1) 6 (40) 9 (60) (96.7) 19.1 ± 7.6 621 ± 40 687 ± 30 (14.6)
Data represent mean ± standard deviation according to number (%) and mean ± standard error for durations. BM, bone marrow; CR, complete remission; EFS, event-free survival; EMI, extramedullary involvement; FAB, French–American–British classification of ALL; Hb, hemoglobin; OS, overall survival; PB, peripheral blood; Plt, platelet; Pt, patient; WBC, white blood cell.
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Figure 1 Determination of sCD44 and sCD44v6 serum levels in patients with acute lymphoblastic leukemia and healthy individuals as controls. A significant difference between sCD44v6 level between patients and controls is observed (***P < 0.001). ( ) patients; ( ) controls.
Figure 2 The relationship between sCD44 and sCD44v6 serum levels in patients with acute lymphoblastic leukemia. A negative correlation between the two molecules is observed (r = −0.57). Correlation is significant at the 0.01 level.
sCD44v6 and sCD44 serum levels in relation to patients’ characteristics Study of the serum levels of sCD44v6 and sCD44 in patients showed no significant difference in men and women. The serum levels of these molecules in patients under 10 years and more than that also showed no significant difference. sCD44v6 and sCD44 had no association with laboratory features of the patients such as Hb concentration, FAB subgroups and platelet numbers. However, sCD44 was significantly higher in patients with WBC > 50 × 109/L (1464.5 ± 459.4 ng/mL) than in patients with WBC ≤ 50 × 109/L (1162.2 ± 385.1
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Soluble CD44 and CD44v6 in pediatric ALL
Figure 3 The relationship between sCD44 serum level and several prognostic factors in patients with acute lymphoblastic leukemia. Data represented the mean ± SD of sCD44 serum level in different groups. WBC, white blood cell (×103/μL); EMI, extramedullary involvement. sCD44 serum level was significantly higher in patients with WBC > 50 × 103/μL and in those with EMI as well as Tel/AML1 negative patients (*P < 0.05, **P < 0.01). ( ) WBC ≤ 50 × 109/L; ( ) WBC > 50 × 109/L; ( ) with EMI; ( ) without EMI; ( ) Tel/ AML1; ( ) non-Tel/AML1.
ng/mL, P = 0.038) (Fig. 3), and also in patients with peripheral blood blast percentage > 50 (1435.4 ± 393.6 ng/mL) than in patients with blast percentage ≤ 50 (1143 ± 351 ng/mL, P = 0.048), indicating the prognostic value of this molecule. With respect to immunophenotype, there were no significant differences in the level of these molecules between different B-ALL subtypes. A significant association between sCD44 level and the presence of EMI was detected (Fig. 3). The level of this molecule was higher in patients with EMI than those without EMI (1337.1 ± 391.7 ng/mL vs 1053 ± 391.3 ng/mL, P = 0.017) and in patients with splenomegaly than those without splenomegaly (1344.3 ± 410.9 vs 1082.5 ± 376.5, P = 0.026). There was no significant relationship between sCD44 and sCD44v6 serum levels and the presence or absence of cytogenetic abnormalities, but in relation to TEL/AML1 positivity sCD44v6 showed a higher level in positive patients than negative ones (157.3 ± 55.6 ng/mL vs 92 ± 43.6 ng/mL, P = 0.036) (Fig. 4), whereas sCD44 showed a different result; it was higher in non-TEL/ AML1 patients (1516.7 ± 367.8 ng/mL) than TEL/ AML1 positive ones (743.9 ± 294.1 ng/mL, P = 0.003) (Fig. 3). Analysis for prognostic factors using the general linear model showed no significant results.
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Figure 4 The relationship between sCD44v6 serum level and several prognostic factors in patients with acute lymphoblastic leukemia. Data represented the mean ± SD of sCD44v6 serum level in different groups. WBC, white blood cell (×103/μL); EMI, extramedullary involvement. sCD44v6 serum level was significantly higher in patients positive for Tel/AML1 (*P < 0.05). ( ) WBC ≤ 50 × 109/L; ( ) WBC > 50 × 109/L; ( ) with EMI; ( ) without EMI; ( ) Tel/AML1; ( ) non-Tel/ AML1.
The mean serum levels of sCD44 in patients who remained alive and those who died at the end of follow-up showed no significant difference (1227 ± 415 and 1166 ± 158 ng/mL, respectively). The same result was obtained for sCD44v6 (101 ± 43 and 116 ± 47 ng/ mL, respectively). No significant difference in the level of these molecules in patients who entered CR and those who did not achieve CR was detected. In univariate Cox regression analysis, sCD44 and sCD44v6 showed no significant influence on OS, EFS or time to CR. Because P-values for sCD44 and sCD44v6 were more than 0.2, no multivariate analysis was performed.
DISCUSSION The primary focus of our study was to measure the difference in sCD44 and sCD44v6 levels between ALL children and their healthy counterparts. The second aim was to find the prognostic significance of these molecules. In recent years, several studies have investigated the presence of the soluble form of CD44 molecule in serum of children with hematologic malignancies.11–16 In a number of these studies, the level of sCD44 in the patients at the time of presentation has been significantly higher than its level in healthy children. In this regard,
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Tacyildiz and colleagues have shown a higher serum CD44 level in 16 Hodgkin’s disease, 13 non-Hodgkin’s lymphoma, 8 Burkitt’s lymphoma and 8 ALL patients than those in 12 healthy children.12 Ahmed and Hassab also found an increased sCD44 level in 30 patients with ALL.15 In contrast to these studies, we did not find a significant difference in sCD44 levels between patient and control groups. Similarly, El-Sharkawy and colleagues showed no significant difference in sCD44 level in 35 ALL patients compared with the 15 controls.14 This inconsistency between the results may be due to differences in genetic characteristics or the number of patients studied. To the best of our knowledge, no previous studies have investigated sCD44v6 in children with ALL and its relation to prognostic factors. However, Ping et al. evaluated changes of sCD44 and sCD446 in 20 adult patients with ALL. Their study showed a higher expression of sCD44 in patients than controls but pretreatment serum sCD44v5 and sCD44v6 concentrations were lower. After treatment, serum sCD44 levels declined, while serums sCD44v5 and sCD44v6 returned to normal.13 In the current study, we also found a significant difference in sCD44v6 level between our patients and controls; the level of this molecule was lower in patients (P < 0.001) which may provide an evidence for the possible protective and diagnostic importance of this molecule in pediatric ALL. CD44 is known to be expressed on various hematopoietic cells including leukemia cells, though its level of expression and release from the surface varies according to the state of cell proliferation or activation.14 El-Sharkawy and colleagues investigated the surface expression of CD44 and several of its variant isoforms including CD44v6, CD44v7/8 and CD44v4 in ALL patients, but contrary to wide expression of CD44 in the patients they found no expression of the variant isoforms except CD44v4 (14.3%) on any of their cases.14 No correlation between CD44 expression and soluble form of CD44 was detected in their study. In contrary, Tacyildiz et al. found a correlation between CD44 expression and sCD44 level in ALL patients.12 In our patients, we also found a relationship between CD44 expression and sCD44 levels that showed at least a part of serum sCD44 levels in childhood leukemia may originate from leukemic cells. With respect to CD44v6, we did not measure the surface expression of this molecule on the leukemic cells in the patients; however, in previous studies on other types of malignancies, dissimilar results have been found; for example, in a study, CD44v6 expression has been positively correlated with
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its level in the serum of colorectal carcinoma patients.17 The serum level of sCD44v6 has been higher in patients with CD44v6-positive gastric carcinoma cells than in those with CD44v6-negative tumor cells,18 and in another study on the same disease no correlation has been found.19 Given the observed correlation between CD44 expression and sCD44 level in this study, and the important role of CD44 as an adhesion molecule involved in tumor metastasis,20 one may ask about the value of sCD44 and sCD44v6 in prognosis and response to therapy in ALL patients. In various studies, the level of sCD44 has been suggested as a valuable marker for monitoring response to therapy and prognosis. Takeuchi et al. have reported a higher sCD44 serum level in the patients at presentation time and relapse compared with its level during CR.11 Increased serum level of sCD44 during relapse has also been reported by Yokota et al., which implies the sCD44 relation to disease status and treatment in the patients.21 In the present study, the level of sCD44 and sCD44v6 molecules in relation to treatment response showed no significant difference between patients who remained alive and those who died at the end of followup, nor in patients who entered CR and those who did not, showing the lack of association of sCD44 and sCD44v6 molecules with response to therapy. These data are in agreement with previous studies that reported no significant association between sCD44 levels with survival and achievement of CR.12,13 We looked for possible correlation between sCD44 and sCD44v6 levels with known prognostic factors in the patients. Our data showed that neither sCD44 nor sCD44v6 level was correlated with sex, age, Hb concentration, nor platelet numbers in the patients. However, with respect to sCD44 a significant correlation with WBC numbers and the percentage of blasts in peripheral blood was observed. Patients with higher WBC numbers and peripheral blood blasts had higher sCD44 level. Further statistical analysis of sCD44 level in our patients revealed a significant increased sCD44 level in patients who had EMI. These data are in line with previous studies in which a relationship between the expression of surface CD44 with EMI has been reported.20 Here, our data indicated that CD44 molecule in soluble form can also be used as a valuable prognostic factor in childhood ALL. Furthermore, sCD44 showed a negative association with TEL/AML1 positivity and was higher in patients without this abnormality. As TEL/AML1 positivity has been introduced as a favorable indicator of prognosis in ALL,22 this finding provides a further evidence for the important role of sCD44 as a poor
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Soluble CD44 and CD44v6 in pediatric ALL
prognostic factor in childhood ALL. In contrast, sCD44v6 was significantly higher in TEL/AML1 positive patients than negative ones which may suggest this molecule as a good prognostic factor. This difference between sCD44v6 and sCD44 results in relation to prognosis seems to be in line with another finding of this study that showed a negative correlation between sCD44 and sCD44v6 levels in the patients. As mentioned before, the generation of CD44 variant isoforms is mainly due to alternative splicing which is under the control of distinct factors from various signaling cascades that are able to recognize specifically pre-mRNA elements of different genes.23 Alterations in these factors in various pathological conditions may alter the production of various CD44 variant isoforms.23 In conclusion, as results of this study showed, sCD44v6 serum level was different between patients and controls which suggest the potential diagnostic and protective values of this molecule for ALL. In the patients, increased sCD44 level was associated with decreased sCD44v6 level. Both sCD44 and sCD44v6 indicated a relationship with prognostic factors. In this regard, sCD44 seems to be more important as it indicated association with more prognostic factors. There are several limitations of this study, for example, inadequate case numbers, inadequate follow-up time, and no sequential measurement of sCD44 and sCD44v6 levels. Further research with the larger number of patients and longer follow-up is recommended to find the precise impact of these molecules in response to therapy and disease outcome.
ACKNOWLEDGMENT The authors would like to thank Dr Mahdi Salehipour and Sare Roosta at the Center for Development of Clinical Studies of Nemazee Hospital for statistical assistance, and also Mr Saeed Malekhosseini for providing valuable assistance. This work is originally described in the MD thesis of one of the authors, E. Asiaee.
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