|Year : 2016 | Volume
| Issue : 2 | Page : 81-86
NRP-1/CD304 expression in acute leukemia: a potential marker for minimal residual disease detection in acute leukemia
Magdy M El Bordiny1, Ashraf El Ghandour2, Amal R Mansour1, Abeer S El Hadidi1, Rehab A Mohamed Farag1
1 Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Alexandria University, Alexandria, Egypt
|Date of Submission||03-Jan-2016|
|Date of Acceptance||03-Jan-2016|
|Date of Web Publication||15-Jul-2016|
Rehab A Mohamed Farag
Clinical Pathology Department, Faculty of Medicine, Alexandria University, 22 El-Guish Road, El-Shatby, Alexandria - 21526
Source of Support: None, Conflict of Interest: None
Background Neuropilin-1 (NRP-1, BDCA-4, or CD304) is a nontyrosine kinase coreceptor for semaphorins and vascular endothelial growth factor, involved in neural development and tumor angiogenesis.
Objective The aim of this study was to evaluate NRP-1/CD304 expression in acute leukemia to be used as a potential marker for minimal residual disease (MRD) detection using flow cytometry.
Materials and methods This study was conducted on 30 adults with newly diagnosed acute leukemia admitted to the Hematology Unit, Internal Medicine Department, Main Alexandria University Hospitals and Alexandria Research Institute within the period from February 2014 to December 2014, and 10 patients diagnosed with hypersplenism were also included as a control group. The following investigations were carried out for all patients: complete blood picture, bone marrow examination, and immunophenotyping using flow cytometry.
Results This study showed that NRP-1/CD304 expression was statistically higher in B-lineage acute lymphoblastic leukemia (ALL) than in the acute myeloid leukemia and the control group (P = 0.002). There was a positive correlation between NRP-1/CD304 percentage expression and blast cell percentage in the bone marrow (P = 0.039). Follow-up of the patients on day 28 of induction chemotherapy revealed that NRP-1/CD304 expression was statistically higher in those who did not achieve complete remission than those who did (P = 0.035). Moreover, we observed that leukemic patients with extramedullary infiltration had statistically higher NRP-1/CD304 expression than those who did not have extramedullary infiltration (P = 0.023). All patients who achieved complete remission on day 28 of induction chemotherapy had negative MRD (<0.001).
Conclusion It was observed that NRP-1/CD304 was expressed in almost all B-lineage ALL patients in variable degrees. It might be a useful marker in MRD detection of B-lineage ALL patients.
Keywords: minimal residual disease, Neuropilin-1/CD304, vascular endothelial growth factor
|How to cite this article:|
El Bordiny MM, El Ghandour A, Mansour AR, El Hadidi AS, Mohamed Farag RA. NRP-1/CD304 expression in acute leukemia: a potential marker for minimal residual disease detection in acute leukemia. Egypt J Haematol 2016;41:81-6
|How to cite this URL:|
El Bordiny MM, El Ghandour A, Mansour AR, El Hadidi AS, Mohamed Farag RA. NRP-1/CD304 expression in acute leukemia: a potential marker for minimal residual disease detection in acute leukemia. Egypt J Haematol [serial online] 2016 [cited 2020 Feb 19];41:81-6. Available from: http://www.ehj.eg.net/text.asp?2016/41/2/81/186411
| Introduction|| |
Acute myeloid leukemia (AML) is the predominant type of acute leukemia in the adult age group and it accounts for ~80% , whereas acute lymphoblastic leukemia (ALL) accounts for 20% in this age group . Acute leukemia is more common in men than in women.
According to the 2008-2011 National Cancer Registry Program, the incidence of myeloid leukemia in Egypt in patients aged 18 years or more was 26.7/100 000 men and 33.5/100 000 women, whereas the incidence of lymphoid leukemia in patients aged 18 years or more was 33.7/100 000 men and 20.2/100 000 women .
The outcome of ALL in adults is challenging as compared with that observed in children. The rate of disease relapse is much higher in adults. Although cure rates in children approach 90%, no more than 40% of adult patients remain free of leukemia after 5 years, and this rate is much lower in older patients. The relatively poor outcome for ALL in adults is contributed by the low doses of chemotherapy that had traditionally been used in adult patients. However, the prognosis for patients with AML has been improving steadily, particularly for those below 60 years of age, and approximately one-third of this group can expect to achieve long-term cure. For the elderly, the situation is poor and less than 10% of those over 70 years of age can expect long-term remission .
Neuropilins (NRPs) are transmembrane glycoprotein receptors that play an important role in various biological processes, including axonal guidance, angiogenesis, tumorigenesis, and the immunologic response . Two Neuropilin genes, NRP-1 and NRP-2, have been identified. NRP-1 (also known as CD304, BDCA-4, VEGF165R) was originally identified as a receptor for class III semaphorins (SEMA3s) mediating neuronal guidance and axonal growth , and then was subsequently found to bind to vascular endothelial growth factor (VEGF), which is a critical proangiogenic factor that induces proliferation and migration of endothelial cells to tumor vasculature .
NRP-1/CD304 was consistently absent on peripheral blood lymphocytes, granulocytes, eosinophils, and platelets . Moderate-to-strong expression of NRP-1/CD304 was detected on cells consistent with plasma dendritic cells ,. Moreover, humans express NRP-1/CD304 on red blood cell progenitors and B-lymphoblasts in the bone marrow . It was demonstrated that AML blasts not only express VEGF but also its receptors . It was observed that there was no association of NRP-1/CD304 expression with an AML FAB subtype; however, NRP-1/CD304 was expressed on blasts in AMLs with the inv(16) cytogenetic abnormality . It was found that NRP-1 is frequently expressed on B-ALL blasts, and weakly expressed in normal bone marrow B-cell progenitors, while gradually decreasing during maturation, to be completely lost at later stages of B-cell. The expression of NRP-1 on B-cell progenitors may explain its frequent higher expression in precursor B-ALL than in mature ALL .
The aim of this study was to evaluate NRP-1/CD304 expression in patients with ALL and those with AML with aberrant marker expression (e.g. AML with CD19) using flow cytometry at diagnosis and on day 28 of induction chemotherapy to evaluate the possibility of using this marker for minimal residual disease (MRD) detection and correlates its expression with clinical outcome after 6 months of treatment.
| Materials and methods|| |
The study was carried out on 40 patients divided into two groups: 30 adult patients (≥18 years old) newly diagnosed with acute leukemia (ALL and AML with aberrant expression) admitted to the Hematology Unit, Internal Medicine Department, Main University Hospital of Alexandria and Alexandria Research Institute during the period from February 2014 to December 2014. There were 16 male and 14 female patients with a mean age of 31.77 ± 13.67 years, and 10 patients diagnosed with hypersplenism of matched age and sex were recruited as a control group.
All patients were subjected to full history taking and complete clinical examination, complete blood count (Sysmex KX-21 N; Sysmex, Kobe, Japan), bone marrow aspirate examination, and immunophenotyping by means of flow cytometry using Becton Dickinson Biosciences (Franklin Lakes, USA), FACS Calibur flow cytometry analyzer equipped with Cell Quest Pro software (Franklin Lakes, USA).
Flow cytometry 
Extensive panel of fluorescein isothiocyanate (FITC) and phycoerythrin (PE), CD34 (PerCP), and CD19 (APC) for the diagnosis of acute leukemia was applied:
Primary panel was applied for all cases:
CD7, CD5, CD19, CD14, CD13, CD33, HLA-DR, CD34, CD45, and CD10.
Secondary panels were applied if needed:
CD11b, CD64, Cyt3, Cyt-MPO, and Cyt CD22.
Almost all of the above monoclonal antibodies were purchased from Immunostep (Salamanca, Spain); however, CD10 was obtained from DAKO (Glostrup, Denmark), CD19 was obtained from BD Pharmigen, and PE-conjugated NRP-1/CD304 was obtained from Immunostep.
The results of NRP-1/CD304 were expressed as the percentage of positively stained cells within the gated blast population. The cutoff value was 20% or greater of the gated cells .
Follow-up BMA samples from B-ALL cases on day 28 after induction chemotherapy were analyzed by means of flow cytometry for MRD detection, using the four color laboratory panel for MRD detection that comprised the following Monoclonal antibodies: TDT (FITC), CD10 (PE), CD34 (PerCP), and CD19 (APC). This combination was shown to be quite pertinent to differentiate residual leukemic blasts from hematogones, based on the difference in fluorescence intensity displayed by both . TDT (FITC) was obtained from BD Biosciences (San Jose, California, USA), CD10 (PE) was purchased from DAKO, and CD19 (APC) and CD34 (PerCP) were obtained from BD Pharmigen.
Statistical analysis of the data
Data were analyzed using IBM SPSS software package, version 20.0 (Chicago, IL, USA). Qualitative data were described using number and percentage. Quantitative data were described using range (minimum and maximum), mean, SD, and median. Significance of the obtained results was judged at the 5% level.
| Results|| |
Expression of CD304 by means of flow cytometry revealed that there were 11 patients (36.7%) who showed positive expression for NRP-1/CD304 ([Figure 1]a), whereas 19 patients were negative (63.3%) at diagnosis ([Figure 2]a). B-lineage ALL patients had significantly higher NRP-1/CD304 expression levels compared with the AML and the control group (P = 0.002). There was a significant positive correlation between NRP-1 and blast percentage in the bone marrow (rs = 0.379, P = 0.039). Moreover, there was a statistically significant correlation between lymphadenopathy and NRP-1 (P = 0.019). Expression of NRP-1/CD304 was statistically higher in those who did not achieve complete remission (P = 0.035). There were no statistically significant differences between NRP-1/CD304-positive and NRP-1/CD304-negative patients as regards age, sex, hemoglobin level, leukocyte count, platelets, blast cells in peripheral blood and bone marrow, and the presence of hepatomegaly. There were statistically significant differences between them as regards splenomegaly (P = 0.003) and lymphadenopathy (P = 0.001) ([Table 1]). Moreover, we observed that leukemic patients with extramedullary infiltration had statistically higher NRP-1/CD304 expression than those who did not have extramedullary infiltration (P = 0.023). Extramedullary infiltrations included renal, osseous, ovarian, and testicular infiltration.
|Figure 1 (a) Positive expression of CD304 in a B-ALL patient at diagnosis. (b) Negative expression of CD304 in the same patient on day 28 of treatment. ALL, acute lymphoblastic leukemia|
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|Figure 2 (a) Negative expression of CD304 in B-ALL patient at diagnosis. (b) Negative expression of CD304 in the same patient on day 28 of treatment. ALL, acute lymphoblastic leukemia|
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|Table 1 Comparison between positive and negative cases according to demographic data |
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The patients were followed up on day 28 of induction chemotherapy: 17 patients achieved complete remission ([Figure 1]b and [Figure 2]b), eight patients were refractory, and five patients died; the cause of death in almost all of them was due to serious infections such as pneumonia as a result of severe absolute neutropenia. We continued following up the patients for 6 months after completing induction: 11 patients maintained complete remission, four patients relapsed, and 15 patients died. There was no statistically significant correlation between NRP-1/CD304 percentage expression and clinical outcome of the patients at day 28 or after 6 months (P = 0.859 and 0.887, respectively).
Detection of minimal residual disease
MRD was negative in all patients who achieved complete remission (MRD <0.001) on day 28 of induction chemotherapy. After 6 months, 11 patients maintained negative MRD, but one patient relapsed, and the remaining five patients died.
| Discussion|| |
The present study was conducted on 30 newly diagnosed patients with acute leukemia and 10 patients diagnosed with hypersplenism serving as a control group.
We started by studying the differential expression of CD304 on the surface of the blasts and lymphocytes in the control group. We used CD45 mean fluorescence intensity (MFI) to differentiate between the blasts, which are dim for CD45, and the lymphocytes, which are bright for CD45. We found that NRP-1/CD304 percentage expression on blast cells ranged from 1.41 to 11.56% and for lymphocytes it ranged from 0.0 to 0.39%; MFI on blast cells was 14.20-279.07 and for lymphocytes it was 0.0-51.29. There were statistically significant differences between blast cells and lymphocytes in normal bone marrow as regards percentage expression and MFI (P = 0.004 and 0.028, respectively). NRP-1/CD304 expression was then evaluated in leukemic patients; it was expressed in all patients with variable degrees of expression, ranging from 0.11 to 95.84% for B-ALL patients, and, for AML patients, NRP-1/CD304 ranged from 0.08 to 0.93%. Our results revealed that NRP-1/CD304 expression was significantly higher in B-lineage ALL than in the AML and the control group (P = 0.002).
In concordance with these findings, Meyerson et al.  found that NRP-1/CD304 was frequently expressed on B-ALL blasts, whereas its expression was less frequent on AML blasts and consistently absent on peripheral blood lymphocytes. Similarly, Karjalainen et al.  demonstrated its expression above baseline bone marrow levels in all of the B-ALL samples and two-thirds of the AML samples, and they reported that expression in the B-ALL samples was stronger than that in AML. Moreover, Younan et al.  found that NRP-1/CD304 was expressed in 80 and 95% of ALL and AML patients, respectively, with levels higher in patients than in controls and in ALL than in AML patients. Hagag et al.  revealed highly significant differences in NRP-1 expression between B-lineage ALL lymphoblasts and control lymphocytes.
As regards expression of NRP-1/CD304 in AML, NRP-1/CD304 expression was less than that in the control group.
In contrast to this result, a study by Kreuter et al.  found that NRP-1/CD304 expression was significantly increased in AML patients when compared with controls. Sallam et al.  also observed that NRP-1 expression using flow cytometry showed a highly significant increase in de novo and relapsed AML patients, compared with the control group, and on using real-time PCR no statistical significance was found in de novo and relapsed AML patients, compared with the control group.
The present study showed a statistically significant correlation between NRP-1/CD304 and blast cell percentage in the bone marrow. This was also observed by Younan et al. , who found that the expression of NRP-1/CD304 was significantly correlated with high (>90%) blasts percentage in the bone marrow of both AML and ALL patients. Moreover, Lu et al.  demonstrated that a higher level of NRP-1/CD304 mRNA was expressed in leukemias and increased NRP-1/CD304 expression was directly correlated with blast percentage in the bone marrow of patients with AML, suggesting that NRP-1/CD304 expression correlates with disease severity; they also observed that NRP-1/CD304 promoted proliferation and chemotaxis of leukemic cells in response to VEGF, and that the inhibition of NRP-1/CD304 functions may provide a new therapeutic strategy for the treatment of AML. In another study by Lu et al. , they showed that the increased NRP-1 expression was directly correlated with the blast percentage bone marrow of AML patients (r = 0.5, P < 0.05). Hagag et al.  also found that NRP-1/CD304 expression significantly associated with bone marrow blast percentage.
In this work, the patients were followed up on day 28 of induction chemotherapy and it was noticed that there was a statistically significant difference in NRP-1/CD304 percentage level between patients (ALL and AML) who achieved complete remission and those who did not achieve remission (P = 0.035); the level was higher in those cases who did not achieve complete remission. This is in agreement with the findings of Younan et al. , who found that there was a statistically significant difference after 28 days in NRP-1 level between patients (AML and ALL) who achieved complete remission and those who did not, with the level being higher in those cases who did not achieve complete remission.
In the current study, it was revealed that there were no statistically significant differences between CD304 expression at diagnosis and the clinical outcome of the 11 positive cases at day 28 of induction chemotherapy and after 6 months of treatment (P = 0.859 and 0.887, respectively). In contrast to our findings, Younan et al.  suggested that NRP-1/CD304 was significantly associated with acute leukemia and that its level might serve as an indicator for disease progression. Hagag et al.  suggested that NRP-1/CD304 was significantly expressed in children with B-lineage ALL and had a bad prognostic impact on the course of this disease. Coustan Smith et al.  observed that NRP-1/CD304 had been associated with drug resistance and disease progression in AML.
In the present study, MRD was performed for the patients who achieved complete remission on day 28 and it was negative for all patients at day 28 of induction chemotherapy, and it was also observed that NRP-1/CD304 was expressed in variable degrees in almost all cases of B-ALL at diagnosis; it can be considered a promising potential marker that can be used in the detection of MRD. This finding needs to be confirmed after testing CD304 expression in the established MRD panel of antibodies in a large cohort of patients. This was also recommended by Meyerson et al. , who demonstrated the usefulness of NRP-1/CD304 for the detection of MRD. The use of CD304 enhanced discrimination of the blasts from other cells. This is in agreement with a recent study by Solly et al. , who suggested the stability of CD304 expression on the blast population during treatment, which must be further evaluated in a larger cohort of patients. Moreover, Coustan Smith et al.  demonstrated that NRP-1 is one of the most differentially expressed markers between ALL blast cells and normal B-cells precursors.
| Conclusion|| |
Our results provided a useful information about the reliability of this marker, as NRP-1/CD304 expression appeared stable during treatment, especially when combined with other stable markers. Overall, our data highlighted CD304 as a promising MRD marker in B-ALL patients. Thus, the identification of leukemic cell-specific markers will help to improve the sensitivity of flow cytometry-based MRD quantification.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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