Abstract
Autocrine and paracrine loop involving vascular endothelial growth factor (VEGF) and its receptor have been described in haematological malignancies. However, scarce literature is present on angiogenesis in paediatric acute lymphoblastic leukemia (ALL) with studies showing controversial results. The aim was to study serum levels of VEGF and its receptors in paediatric ALL at the time of diagnosis and at the end of induction phase and to compare these levels with clinico-haematological parameters in these patients. Serum VEGF, VEGFR-1 and VEGFR-2 levels were measured by enzyme-linked immunoabsorbant assay at diagnosis (day 0) and at the end of induction phase (day 35) in 30 newly diagnosed paediatric ALL patients and in 10 healthy controls. Median s-VEGF was significantly lower at day 0 as compared to day 35 (196.15 vs. 606.75 pg/ml: p < 0.001). s-VEGFR-1 levels were detectable only in 7 patients at day 0 and were below detection level at day 35 in all patients. Median s-VEGFR-2 at day 0 was significantly lower as compared to day 35 (17,577.5 vs. 20,507.5 pg/ml; p = 0.005). Median VEGF-R1 showed an inverse relationship with VEGF-R2 but was statistically insignificant. All patients were in remission at the end of induction. Thus, leukemic infiltration of bone marrow affects angiogenesis and reduces pro-angiogenic markers VEGF and VEGFR-2 in serum possibly due to increased local consumption by blasts. A successful induction leads to clearing of blasts causing restoration of normal hematopoiesis with normalization of VEGF and VEGFR-2 levels.
Keywords: Angiogenesis, VEGF, Paediatric ALL, VEGFR2
Introduction
Role of angiogenesis in tumor development was established long ago in 1971 by Folkman et al. [1]. It has been recognized as an independent prognostic factor in tumors of the lung, breast, esophagus and prostate [2, 3]. Cancer cell promotes angiogenesis early in tumorigenesis due to oncogene driven tumor expression of proangiogenic genes. Vascular endothelial growth factor (VEGF), along with its receptors (VEGFR-1, VEGFR-2 and VEGFR-3) is an important mediator of angiogenesis.
In contrast to solid tumors, in which VEGF and its receptors play a key role in tumor neovascularisation, very few studies have focussed on the importance of this angiogenic growth factor and its receptors in pathogenesis of hematologic malignancies. Autocrine and paracrine loop involving VEGF and its receptors have been described in haematological malignancies [4]. However, literature on VEGF and its correlation with other prognostic factors of paediatric acute lymphoblastic leukemia (ALL) is scarce and shows conflicting results.
ALL is the most common malignancy in children below 15 years of age [5]. Although there has been significant improvement in treatment of ALL, relapse rate is 25% [6] with many showing partial or no response. Risk stratification based on current parameters is incapable of accurately predicting progression of disease. There is therefore, an urgent need to identify additional biomarkers at the time of diagnosis for risk stratification which may have a significant impact on the management protocols in the future.
In the present study, serum levels of VEGF and its receptors (VEGFR-1 and VEGFR-2) were studied in 30 newly diagnosed cases of paediatric ALL at the time of diagnosis (day 0) and at the end of induction phase (day 35) in to order to assess whether serum levels of VEGF and its receptors correlates with treatment response, risk stratification and also as an independent prognostic factor.
Material and Methods
Inclusion and Exclusion Criteria
This prospective longitudinal study was conducted on all newly diagnosed cases of paediatric ALL below18 years of age presenting to the KSCH OPD, casualty and adolescent clinic over a period of 18 months at Department of Paediatrics and Department of Pathology in Lady Hardinge Medical College and Kalawati Saran Children’s Hospital (KSCH), New Delhi. Patients with septicaemia/Disseminated Intravascular Coagulation and who have received corticosteroids for > 7 days prior to hospital admission were excluded from the study. Total of 30 cases along with 10 healthy age and sex matched controls were enrolled. The diagnosis of ALL was made on bone marrow morphology and flow cytometry.
Risk Stratification
Patients were stratified according to National Cancer Institute (NCI) criteria [6] into standard risk, intermediate risk and high risk (Table1).
Table 1.
NCI criteria for risk stratification
| Standard risk | Intermediate risk | High risk all/nhl |
|---|---|---|
| Age 1–9 years | Good risk features but age ≥ 10 years | Any of the above but a prednisone poor responder** |
| Non T-cell | Good risk features but WBC ≥ 50,000/mm3 | Any of the above with high risk cytogenetics |
| Prednisone good responder** | Good risk features but bulky lymph nodes (≥ 5 cm in peripheral region and in chest > 5 cm on CT scan or occupying ≥ 1/3rd diameter on chest X-ray) and/or bulky liver/spleen reaching beyond midway to umbilicus | CNS disease (diagnostic Lumbar Puncture done on D8) |
| No high risk cytogenetics | Good risk T-cell phenotype (WBC < 300,000/mm3 and non Bulky disease, Non-ETP* immunophenotype) | High risk T-cell phenotype (WBC ≥ 100,000/mm3 or Bulky disease or ETP* immunophenotype) |
| WBC < 50,000/mm3 | Testicular disease | T-Cell NHL |
| (Good risk: ETV6-RUNX1 fusion or hyperdipoidy with trisomies of chromosomes 4 and 10) |
Good: No blasts on peripheral smear after 1 week of treatment
Poor: Presence of ≥ 1000 blasts/µl on peripheral blasts after 1 week of treatment
*ETP- Early T-cell Precursor
**Prednisone response:
Minimal Residual Disease
Patients were treated according to the ICICLE (Indian Childhood Collaborative Leukaemia Group) protocol 2013 [7], currently in use in our hospital. Complete remission was defined by the presence of < 5% blasts morphologically in the bone marrow aspirate; and/or absence of blasts in the CSF and/or testes of normal size and density (confirmed on ultrasound) at the end of induction phase. In addition, MRD analysis was done in all cases at the end of induction phase (Day 35 bone marrow aspirate samples) by Beckman Flow cytometer FC500 and Kaluza™ software for analysis using following fluorochrome (FITC-fluorescein isothiocyanate, PE-phycoerythrin, PC5-phycoerythrin cyanin, PC7-phycoerythrin cyanin 7 and ECD- energy-coupled-dye) antibodies: For B Cell phenotype: CD58-FITC, CD10-PE, CD45-ECD, CD38PC5, CD19-PC7, CD34-FITC, CD123-ECD, CD45-FITC, CD20-PC7 and any aberrant marker at day 0. For T cell phenotype: antigen at the time of diagnosis (day 0). MRD positivity was defined as ≥ 0.01% of ALL cells.
Laboratory Tests
The cases at the time of diagnosis (day 0) were subjected to complete hemogram with peripheral smear, liver function test (LFT), kidney function test (KFT), Bone Marrow Aspiration, Cytochemistry and Immunophenotyping.
Serum levels of VEGF, VEGFR-1 and VEGFR-2 were done at the time of diagnosis (day 0) and at the end of induction phase (day 35) in all cases and once in controls.
Blood sample was obtained on day 0 and day 35 in all cases and once in controls. Following clotting, the plain vacutainer was centrifuged at 1000 g (relative centrifugal force) for 10 min and the serum was separated. The serum was removed rapidly and stored at − 70 °C in different aliquots (250–500 μl) till further processing. Multiple freeze thaw cycles of frozen specimens were avoided. Human VEGF ELISA; DRG Kit, Human VEGF-R1 ELISA; Diaclone Kit and Human VEGFR-2/KDR ELISA; BioVendor kit were used to assess serum concentration of VEGF, VEGFR-1 and VEGFR-2 respectively. All ELISA based tests were done using bio-rad microplate ELISA reader and platform washer.
Statistical Analysis
The Kruskal–Wallis test was used to compare initial levels of VEGF, VEGFR-1, VEGFR-2 in patients and controls, and between various patient groups. Wilcoxon signed rank test was used to compare serum levels of VEGF, VEGFR-1 and VEGFR-2 on day 0 and day 35. Correlation of s-VEGF, VEGFR-1 and VEGFR-2 levels with other variables was done by Spearman’s correlation coefficient. For all statistical tests, a p value less than 0.05 was taken to indicate a significant difference.
Results
The age group of patients ranged from 1.5 to 14 years with median age of 6 years. There was male preponderance with male to female ratio of 3.2:1. The control group (5 males and 5 females) had median age of 6 years (range 2–14 years) (Table 2).
Table 2.
Characteristics of 30 ALL patients at the time of diagnosis
| Characteristic | Value |
|---|---|
| Age, median (range) | 6 years(1.5–14) |
| < 5 years | 14 |
| 5–10 years | 10 |
| > 10 years | 6 |
| Male sex | 23 (76.7%) |
| Hepatomegaly | 28 (93.3%) |
| Splenomegaly | 26(86.7%) |
| Lymphadenopathy | 20(67%) |
| CNS involvement | 3 (10%) |
| WBC(× 103/μl) | 9.9 (0.86–584.77) |
| < 10 | 15 (50.0%) |
| 10–50 | 6 (20%) |
| 50–100 | 3 (10%) |
| > 100 | 6 (20%) |
| BM Blast% | |
| 25–50 | 3 (10%) |
| > 50 | 27 (90%) |
| Immunophenotyping | |
| B-ALL | 2 (6.7%) |
| B-precursor ALL | 6 (20%) |
| CALLA | 18 (60%) |
| T-cell ALL | 4 (13.3%) |
| Cytogenetics* | |
| BCR-ABL+ | 5 (16.7%) |
| TEL-AML+ | 5 (16.7%) |
| t(1:19) | 2 (6.6%) |
| Risk group | |
| High risk | 11 (36.7%) |
| Intermediate risk | 13 (43.3%) |
| Standard risk | 6 (20%) |
| Post induction MRD status | |
| MRD negative | 30 (100%) |
| MRD positive | 0 |
*No data available for remaining cases
The median serum levels of VEGF and VEGFR-2 at the time of diagnosis (day 0) were significantly lower as compared to day 35 levels (p < 0.001 and p = 0.005) in all cases. VEGFR-1 levels were detected only in 7 patients (median value of 0.239 pg/ml, range 0.046–1.162 pg/ml) at the time of diagnosis (day 0). At the end of induction phase (day 35), levels were below detection values in all patients (Table 3).
Table 3.
Comparison of median level of VEGF, VEGF-R1 and VEGF-R2 at day 0 and day 35 in ALL cases
| At the time of diagnosis (Day 0) | At the end of induction phase (Day 35) | P Value | |||
|---|---|---|---|---|---|
| Median | Range | Median | Range | ||
| VEGF (pg/ml) | 196.15 | 104.1–1925.2 | 606.75 | 222–2159 | < 0.001* |
| VEGFR-1 (pg/ml) | 0.239 | 0.046–1.162 | – | – | – |
| VEGFR-2 (pg/ml) | 17,577.5 | 3516–245,500 | 20,507.5 | 9375–270,500 | 0.005* |
Bold front is used to highlight that p value which is statistically significant
MRD status of all patients was assessed at the end of induction phase (Day35) and all 30 cases were in remission with significant increase in median levels of VEGF and VEGFR2 (p < 0.001, p < 0.005 resp).
At the time of diagnosis, the median serum level of VEGF was significantly lower in patients as compared to controls (P1 < 0.001) There was statistically significant increment in the median level at the end of induction phase (P2 < 0.041). The median serum level of VEGFR-2 in cases showed similar trend of increment when compared to controls but these values were statistically insignificant. VEGFR-1 was below detection level in all the controls and in cases at day 35 (Table 4 and 5). However, statistical significance could not be calculated due to lesser number of cases (n = 07).
Table 4.
Comparison of levels of VEGF, VEGFR-1 and VEGFR-2 of cases and controls
| Parameters (pg/ml) | Day 0 diagnosis (n = 30) | Day 35 remission (MRD negative) (n = 30) | Controls (n = 10) | P1 | P2 |
|---|---|---|---|---|---|
| VEGF | 196.15 | 606.75 | 465.55 | < 0.001 | 0.041 |
| VEGFR-1* | 0.239 | … | … | … | … |
| VEGFR-2 | 17,577.5 | 20,507.5 | 22,267.50 | 0.742 | 0.552 |
Bold front is used to highlight that p value which is statistically significant
P1-Diagnosis versus controls, P2-Remission versus Controls
*n = 7
Table 5.
Normal range of VEGF, VEGFR-1 and VEGFR-2 in controls
| At the time of diagnosis | ||
|---|---|---|
| Median | Range | |
| VEGF (pg/ml) | 465.55 | 404.3–700.5 |
| VEGFR-1 (pg/ml) | BDL* | |
| VEGFR-2 (pg/ml) | 16,407 | 9375–25,782 |
*BDL-below detection level
VEGF levels were found to be high in cases in age group > 10 years, male gender, WBC count > 100 × 103/μl and presence of genetic aberrations. The levels were low (145.2 pg/ml) in standard risk category compared to high risk (153.2 pg/ml) and intermediate group (209.6 pg/ml) (Table 6). Also blast count > 50% was associated with low values (189.3 pg/ml) as compared to those with blast count < 50% (297 pg/ml). None of these showed any statistical significance.
Table 6.
Comparison of VEGF, VEGFR-1 and VEGF-R2 in different groups
| Risk group | VEGF (D0) (pg/ml) | VEGF (D35) (pg/ml) | p value | VEGF-R2 (D0) (pg/ml) | VEGF-R2 (D35) (pg/ml) | p value | VEGF-R1 (D0) (pg/ml) | |
|---|---|---|---|---|---|---|---|---|
| (n = 11) HR | Median | 153.2 | 502.4 | 0.006* | 18,750 | 23,440 | 0.324 | (n = 5) 0.245 |
| (n = 13) IR | Median | 209.6 | 1014 | < 0.001* | 14,065 | 21,095 | 0.324 | (n = 2) 0.173 |
| (n = 6) SR | Median | 145.25 | 607.4 | 0.004* | 19,335 | 16,405 | 0.377 | |
| p value | 0.483 | 0.276 | 0.564 | 0.435 | 0.245 | |||
Bold front is used to highlight that p value which is statistically significant
Serum levels of VEGFR-1 were detected in only seven patients at the time of diagnosis. Amongst these 7 patients, those who had one or more adverse prognostic factors such as age group > 10 years/male group/CNS involvement/Blast count > 50%/T-cell immunophenotype/BCR-ABL translocation and/or High risk group had higher levels. But due to small sample size, these findings were not statistically significant.
VEGFR-2 levels showed inverse correlation with gender at day 35 with females having higher levels than males (r = − 0.401; p = 0.028) There was a significant positive correlation with immunophenotype of patients at both day0 (r = 0.467; p = 0.009) and day35 (r = 0.442; p = 0.014). T-cell ALL showed higher levels as compared to B- cell. (Table 7).
Table 7.
Correlation of VEGF and its receptors with clinico-hematological parameters
| Parameters | VEGF (D0) (pg/ml) | VEGFR-1 (D0) (pg/ml) | VEGFR-2 (D0) (pg/ml) | VEGF (D35) (pg/ml) | VEGFR-2 (D35) (pg/ml) | |
|---|---|---|---|---|---|---|
| Age (years) | r | 0.16 | 0.378 | 0.126 | − 0.29 | 0.015 |
| p value | 0.399 | 0.403 | 0.506 | 0.120 | 0.938 | |
| Gender | r | 0.273 | 0.612 | − 0.292 | 0.25 | − 0.401* |
| p value | 0.144 | 0.144 | 0.117 | 0.182 | 0.028 | |
| WBC (× 103/µl) | r | − 0.043 | 0.286 | − 0.253 | − 0.054 | − 0.088 |
| p value | 0.822 | 0.535 | 0.178 | 0.778 | 0.643 | |
| HB (g/dl) | r | 0.059 | 0.429 | 0.298 | − 0.131 | 0.03 |
| p value | 0.757 | 0.337 | 0.11 | 0.490 | 0.876 | |
| Platelet (× 103/µl) | r | − 0.07 | 0.505 | − 0.18 | − 0.006 | − 0.06 |
| p value | 0.713 | 0.248 | 0.34 | 0.977 | 0.753 | |
| PS blast% | r | 0.24 | − 0.143 | − 0.313 | − 0.086 | − 0.128 |
| p value | 0.238 | 0.76 | 0.12 | 0.674 | 0.533 | |
| CNS involvement | r | 0 | 0 | − 0.019 | 0.032 | 0.257 |
| p value | 1 | 1 | 0.919 | 0.866 | 0.170 | |
| BM BLAST% | r | − 0.273 | − 1.000** | 0.014 | 0.097 | 0.04 |
| p value | 0.244 | 0.952 | 0.684 | 0.867 | ||
| Flow cytometry | r | − 0.165 | − 0.094 | 0.467** | − 0.182 | 0.442* |
| p value | 0.384 | 0.842 | 0.009 | 0.336 | 0.014 | |
| Cytogenetic | r | 0.166 | 0.154 | − 0.234 | 0.111 | − 0.208 |
| p value | 0.382 | 0.741 | 0.212 | 0.559 | 0.269 | |
| Risk group | r | − 0.087 | − 0.474 | 0.025 | 0.198 | − 0.226 |
| p value | 0.648 | 0.282 | 0.897 | 0.293 | 0.230 |
No correlation/relationship was observed among the serum levels of VEGF, VEGFR-1 and VEGFR-2 in respect to each other.
Discussion
VEGF is a homodimeric (40–45 kDa) glycoprotein located on the short arm of chromosome 6. It is expressed in many types of cancer, the increased expression in tumors is caused by several environmental factors including hypoxia inducible factor (HIF-1), low pH, inflammatory cytokines (e.g. interleukin-6), growth factors (e.g. basic fibroblast growth factor), sex hormones (both androgens and estrogens), and chemokines (e.g. stromal-cell–derived factor 1) [8]
Five VEGF isoforms are produced from VEGF gene by alternating splicing [9]. These various VEGF forms bind to three tyrosine-kinase receptors: VEGFR-1, VEGFR-2 and VEGFR-3. Activation of these receptors results in up-regulation of genes involved in mediating the proliferation and migration of endothelial cells and promoting their survival and vascular permeability [10, 11].
Acute lymphoblastic leukemia, like other cancers, is angiogenesis dependent and lymphoblasts are the likely source of VEGF [12].
In this study, serum levels of vascular endothelial growth factor and its receptors (VEGFR-1 and VEGFR-2) were evaluated at the time of diagnosis (day 0) and at the end of induction phase (day 35) in 30 newly diagnosed paediatric ALL cases by ELISA method.
The median VEGF levels in all patients were lower at the time of diagnosis as compared to controls. There was a statistically significant increase in levels at the end of induction phase in all patients. This phenomenon has been explained as a result of increased consumption of angiogenic factors by blast cells. The clearing of blasts from marrow and restoration of normal hematopoiesis leads to normalization of VEGF levels in serum [13–15]
Patients in higher age group (> 10 years), WBC count > 100 × 103/μl, genetic translocations and male gender had higher levels of VEGF at the time of diagnosis as compared to other groups. Our findings are similar to Avramis et al. [16] who found that high levels of VEGF facilitate survival and growth of leukemic blast and disease progression and was correlated with unfavourable prognostic factors. However, the VEGF levels were not statistically significant in our study. This could be due to small sample size. Pule et al. [17] however did not find any correlation of microvessel density (MVD) with outcome and prognostic criteria.
Since all the patients achieved remission by the end of induction phase, and also due to short follow up duration, relationship between MRD positive to therapy and VEGF/VEGFR levels could not be established.
The serum levels of VEGFR-1 was detected in only 7 patients at the time of diagnosis. The levels at day 35 were below detection in all the patients. Although the levels were higher in patients with presence of one or more poor prognostic factors, But due to small number of positive cases statistical significance could not be established. The reason for low detection levels could be due to internalization or secretion blockade of receptors as evidenced by study of El obeid et al. [18].
The median VEGFR-2 levels were low at the time of diagnosis. There was significant increment in median level at the end of induction phase. VEGFR-2 levels showed significant inverse correlation only with female gender at day 35 and a significant positive correlation with T-cell ALL at both day 0 and day 35.
Clinical trials for surrogate biomarkers of antiangiogenesis conducted by Children’s Oncology Group (COG) showed significant increase in plasma levels of VEGF and reduced levels of soluble VEGFR-2 after treatment with tyrosine kinase inhibitors targeting VEGF receptors [19]. Kalra et al. [2] hypothesed stating that low levels of VEGF at the time of diagnosis reflect a higher expression of VEGFR on leukemia cells that bounds to soluble VEGF, thus decreasing unbound form of VEGF proportionate to tumor burden via ligand receptor interaction. However, in our study, patients at the time of diagnosis (day 0) had lower levels of VEGF and VEGFR-2 whereas VEGFR-1 was detectable. At the end of induction phase (day 35), VEGF and VEGFR-2 levels normalized whereas VEGFR-1 became undetectable (similar to controls).
VEGFR-1 negatively regulates the signals of VEGFR-2 and it suppresses the VEGFR-2-mediated proliferation of endothelial cells [9, 20], we give a hypothesis saying that there is inverse relationship of VEGFR-1 levels with VEGFR-2 and expression of VEGFR-1 decreases after end of induction phase.
Conclusion
Our study showed that ALL patients at the time of diagnosis (day 0) have lower levels of VEGF and VEGFR-2 and detectable levels of VEGFR-1. This reflects that angiogenesis is affected in ALL and pro-angiogenic markers VEGF and VEGFR2 are reduced in serum possibly due to increase local consumption by blasts. At day 35, as blasts decreases, there is normalization of angiogenesis with VEGF and VEGFR-2 levels becoming normal and VEGFR-1 becoming undetectable. Although, levels of VEGF-R1 showed an inverse relationship with VEGF-R2 levels, but no statistical significance could be established possibly due to small sample size which needs to be confirmed in a large cohort.
Thus we conclude that serum levels of pro-angiogenic factor-VEGF and VEGFR-2 can be used as additional biomarkers at the time of diagnosis for prediction of progression of disease. However, since all cases achieved remission at the end of induction, thus comparison between MRD positive and negative status with VEGF and its receptors could not be established. Therefore, more studies with larger patient population and longer duration of follow up is desirable to understand the interaction of VEGF and its receptors with each other and their role in angiogenesis in childhood acute lymphoblastic leukemia.
Author Contributions
RM: Review of literature, performed the tests, collection and compilation of data, contributed to conception and design, analysis and interpretation of data, drafting the article and preparation of manuscript and final approval of the version to be published. She agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. AN: contributed to conception and design, analysis and interpretation of data, revising it critically for content, and final approval of the version to be published. She agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. SS: contributed to conception and design, analysis and interpretation of data, revising it critically for content, and final approval of the version to be published. She agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. JC: contributed to conception and design, acquisition of data and interpretation of data, drafting the article or revising it critically for important intellectual content and final approval of the version to be published He agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the manuscript.
Funding
None.
Compliance with Ethical Standards
Conflict of interest
RM, AN, SS and JC declare that they have no conflict of interests.
Ethical Committee Approval
The approval of the Ethic Committee of the Lady Hardinge Medical College and Associated Hospitals was obtained about this study (2014/136).
Informed Consent
Informed written consent was obtained from all individual participants included in the study.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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