Figures
Abstract
Vascular endothelial growth factor (VEGF)-mediated angiogenesis contributes to the pathogenesis of B-cell chronic lymphocytic leukaemia (CLL). We investigated the impact of VEGFA gene diversity on the clinical outcome of patients with this disease. A VEGFA haplotype conformed by positions rs699947 (–1540C>A), rs833061 (–460T>C) and rs2010963 (405C>G) and two additional single-nucleotide polymorphisms (SNPs), rs3025039 (936C>T) and rs25648 (1032C>T), were analysed in 239 patients at the time of their CLL diagnosis. Here, we showed that homozygosity for rs699947/rs833061/rs2010963 ACG haplotype (ACG+/+ genotype) correlated with a reduced survival in CLL patients (ACG+/+ vs other genotypes: HR = 2.3, p = 0.002; recessive model). In multivariate analysis, the ACG+/+ genotype was identified as a novel independent prognostic factor (HR = 2.1, p = 0.005). Moreover, ACG homozygosity subdivided patients with CLL with otherwise indolent parameters into prognostic subgroups with different outcomes. Specifically, patients carrying the ACG+/+ genotype with mutated IgVH, very low and low-risk cytogenetics, initial clinical stage, CD38 negative status or early age at diagnosis showed a shorter survival (ACG+/+ vs other genotypes: HR = 3.5, p = 0.035; HR = 3.4, p = 0.001; HR = 2.2, p = 0.035; HR = 3.4, p = 0.0001 and HR = 3.1, p = 0.009, respectively). In conclusion, VEGFA ACG+/+ genotype confers an adverse effect in overall survival in CLL patients with an indolent course of the disease. These observations support the biological and prognostic implications of VEGFA genetics in CLL.
Citation: Lozano-Santos C, Martinez-Velasquez J, Fernandez-Cuevas B, Polo N, Navarro B, Millan I, et al. (2014) Vascular Endothelial Growth Factor A (VEGFA) Gene Polymorphisms Have an Impact on Survival in a Subgroup of Indolent Patients with Chronic Lymphocytic Leukemia. PLoS ONE 9(6): e101063. https://doi.org/10.1371/journal.pone.0101063
Editor: Masaru Katoh, National Cancer Center, Japan
Received: February 8, 2014; Accepted: June 3, 2014; Published: June 27, 2014
Copyright: © 2014 Lozano-Santos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Fundación para la Investigación Biomédica and Fondo de Investigación Sanitaria (FIS CP09-182), Instituto de Salud Carlos III. The authors were funded by the following institutions: NGL by a grant from programa Miguel Servet, Fondo de Investigación Sanitaria, Instituto de Salud Carlos III; CLS by a research grant from Roche Pharma and JMV by Fundación para la Investigación Biomédica Puerta de Hierro. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: CLS received funding from Roche Farma for this work and has no other relationships, conditions or circumstances that present potential competing interests related with this work. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. The rest of the authors declare no competing interests related with this work.
Introduction
A significant variability in the clinical course of B-cell chronic lymphocytic leukemia (CLL) exists as a result of multiple different pathogenic mechanisms. B cell receptor repertoire skewing and stereotypy and differences in the mutational status of the IgVH gene demonstrate an antigen-driven process. Multiple genetic lesions associated with CLL (del13q14, trisomy 12, TP53 deletion, ATM deletion, NOTCH1 and SF3B1 mutations and others) contribute to the initiation and progression of this leukemia. Recently, there has been a growing interest in determining the impact of microenvironmental interactions, such as angiogenesis, in the pathogenesis and progression of CLL.
Vascular endothelial growth factor (VEGF) is a pro-angiogenic factor with multiple roles in tumour formation that is involved in the pathophysiology of many hematologic disorders, including CLL. Several reports have shown an enhanced microvessel density in the bone marrow and lymph nodes in patients with CLL, as a result of a VEGF-dependent angiogenesis associated with an advanced stage of disease [1]–[3]. Furthermore, it has also been reported that the resistance to apoptosis of leukemic cells in CLL is mediated by VEGF-dependent autocrine and paracrine mechanisms of cell survival [4]–[7]. In addition to the angiogenic and antiapoptotic effects on CLL cells, VEGF regulates CLL cell motility [8], [9] and the microenvironment-tumor interactions [10], [11].
Considerable variation in VEGF expression exists among individuals. However, elevated VEGF levels in the serum or plasma of CLL patients positively correlate with disease progression [12] and such patients are more likely to progress rapidly to a more advanced stage of disease [13]. In addition, high levels of expression of one of its receptors, VEGFR2, correlate with shortened survival [14].
VEGFA is a gene comprised of eight coding exons and several alternative spliced forms that maps to chromosome region 6p1.2. Genetic polymorphisms have been identified outside of the coding region in the 5′ and 3′ flanking regions, and these polymorphisms seem to have an influence on gene expression. SNPs rs699947 (–1540C>A) and rs833061 (–460T>C) reside in the promoter region, rs2010963 (405C>G) and rs25648 (1032C>T) in the 5′UTR and rs3025029 (1689C>T) in the 3′UTR. These polymorphisms have been associated with a variation in the levels of VEGF protein [15]–[18] and predisposition to cancer development and progression [19]–[23].
Given the reported association of VEGF levels with certain clinical conditions in this leukemia, the present study evaluated whether an association exists between VEGFA genetic variability and its predictive value in determining the prognosis of CLL.
Materials and Methods
Study population
Two hundred and thirty-nine consecutive patients with newly diagnosed CLL from four Hospitals belonging to Grupo GLIMCE in Spain (Hospital Puerta de Hierro Majadahonda, Hospital de Getafe, Hospital Severo Ochoa [Madrid, Spain] and Hospital General [Valencia, Spain]) were enrolled in this retrospective study. Also, 183 age and gender-matched control individuals from the Blood Bank Department of Hospital Puerta de Hierro were analysed in order to evaluate the characteristic distribution of VEGFA single nucleotide polymorphisms (SNPs) in Spanish population from the same area. The diagnosis of CLL was based upon standard morphologic and immunophenotypic criteria. Written informed consent was given by participants for their clinical records to be used in this study. This project was approved by the Ethics Committee of Hospital Puerta de Hierro (Comité Ético de Investigación Clínica Hospital Puerta de Hierro Majadahonda). Progression of disease was defined according to NCI-Guidelines criteria [24].
The patient characteristics are summarised in Table 1. Genetic abnormalities were detected by conventional cytogenetics and FISH analysis and stratified as follows: very low-risk (deletion 13q), low-risk (trisomy 12and normal karyotype) and intermediate-risk (deletion 11q) and high-risk (TP53 deletion) [25]. The mutational status of the IgVH gene was analysed and classified according to ERIC recommendations [26]. CD38 and ZAP-70 expression was determined by flow cytometry.
Genotyping of VEGFA polymorphisms
DNA was extracted from peripheral blood cells using either DNAzol (MRC, Cincinnati, OH) or a Maxwell 16 Blood DNA purification kit (Promega Corp. Madison, CA). VEGFA rs699947 (–1540C>A), rs833061 (–460T>C), rs2010963 (405C>G), rs25648 (1032C>T) and rs3025039 (1689C>T) genotyping was performed using TaqMan MGB probes (Applied Biosystems, Foster City, CA). All reactions were performed in a 10 µL PCR reaction with 20 ng of genomic DNA. Allelic discrimination was determined using 7500 Real Time PCR (Applied Biosystems). To validate the genotyping method and haplotype estimation, selected informative samples were analysed by DNA sequencing in a 3130xl Genetic Analyzer Sequencer (Applied Biosystems). Polymorphic positions were named relative to the reference sequence [Genbank: NM_001171630,1].
Statistical Analysis
Individual haplotypes and their frequencies were estimated based upon a Bayesian algorithm using the Phase program (available at http://www.stat.washington.edu/stephens/phase.html[27]). Linkage disequilibrium (LD) analysis was performed using Haploview software. Pearson’s χ2 test was used to evaluate differences in the distribution of VEGFA SNPs alleles, haplotypes or genotypes between subgroups of patients based upon clinical or laboratory parameters.
Odds Ratio (OR) values (95% confidence interval) for the relative risks were calculated for alleles, genotypes and haplotypes. Additive, dominant (major homozygous plus heterozygous vs minor homozygous) and recessive (major homozygous vs heterozygous plus minor homozygous) models were applied to detect associations between genotypes and the clinical and molecular variables.
OS (overall survival) was defined as the interval between the date of diagnosis and the time of death due to CLL. The OS times of patients alive at last follow-up or lost to follow-up were censored. The association between the cumulative probability of OS among genotypes or haplotypes was calculated according to the Kaplan-Meier method, while significant differences between survival curves were evaluated with Mantel’s log-rank test. Ten variables were screened by univariate analysis. Because some chromosomal aberrations resemble other molecular prognostic factors included as covariates (deletion 11q and deletion 17p corresponds to ΔATM and ΔTP53, respectively), these were not included in the multivariate Cox model. CD38 and ZAP-70 were also excluded due to their elevated rate of missing values. All other variables were included in the adjusted analysis to verify their independent prognostic value. Statistical data analyses were performed using the Statistical Package for the Social Sciences (SPSS) software (SPSS 15, Chicago, IL, USA).
Results
Characteristics of VEGFA polymorphisms in the patient cohort
We analysed the genotypes of five SNPs of the VEGFA gene, rs699947 (–1540C>A), rs833061 (–460T>C) rs2010963 (405C>G), rs3025039 (936C>T) and rs25648 (1032C>T), on the basis of their previous description as genetic markers in other cancers and/or their potential effect on gene expression.
The cohort was comprised of 239 CLL patients with a mean age of 68±12 years and a male:female ratio of 1.32∶1. Minor allele frequencies of VEGFA polymorphisms in our CLL cohort were >5% and are summarized in Table 2. We investigated the linkage disequilibrium (LD) of the VEGFA polymorphisms in our study population. As the high LD of the four neighboring loci, rs699947, rs833061, rs2010963 and rs25648, was an evidence for shared segregation, we performed a haplotype analysis (data not shown). To perform an in-depth analysis, haplotypes were estimated from the genotypes defined by loci rs699947, rs833061 and rs2010963. The frequencies of the three main groups of haplotypes in the CLL cohort were ACG (0.39), CTC (0.38) and CTG (0.21), respectively.
All haplotypic and allelic frequencies were highly similar to those presented by a cohort of healthy blood donors from the same area (n = 183, data not shown). Moreover, because the majority of the participants of this study were of Spanish Caucasian origin, all frequencies fall in the same range of series reported in previous studies of other populations of white ethnicity [20], [28], [29].
VEGFA genotypes and CLL survival
We investigated the prognostic value of VEGFA variants in relation to patients’ overall survival (OS). For this, we monitored the entire cohort of 239 patients diagnosed from January 1997 to December 2010, with a median follow-up of 63.2 months (1.4–179.4) and a five-year survival of 77.4%. In this cohort, we studied the prognostic value of the VEGFA rs699947/rs833061/rs2010963 and rs3025039 and rs25648 polymorphisms (Fig. 1).
Kaplan-Maier curves according to: (A) a recessive comparison of rs699947/rs833061/rs2010963 ACG+/+ genotype and, (B) number of copies of ACG haplotype.
Comparison of their genotypes in a recessive genetic model showed that patients with the homozygous rs699947/rs833061/rs2010963 ACG genotype (ACG+/+) presented an increased mortality rate compared with patients with other genotypes (Fig. 1A). The median OS of patients with the ACG+/+ genotype was 84.3 months compared with 173.1 months (p = 0.001) for patients with other genotypes (ACG×/− = 1 copy ACG [ACG+/−] + no copies ACG [ACG−/−]). The adverse effect observed for the ACG haplotype appears to be recessive because patients with 1 copy of the risk haplotype (ACG+/− genotypes) and in the absence of the risk genotype (ACG−/− genotypes) behaved similarly, and no significant difference in median OS was observed between both groups (not reached vs 173.1 months, p = 0.92, Fig. 1B).
In addition to VEGFA genetics, other previously reported variables were also significant epidemiological, clinical and molecular risk factors in OS prediction in our cohort: clinical stage (Binet), IgVH mutational status, ATM deletions, TP53 disruptions, cytogenetic abnormalities, CD38 status and age at diagnosis (data not shown).
In the multivariate Cox regression model for OS, the conventional variables that showed an independent prognostic value were IgVH mutational status, ATM deletions, TP53 deletions and age of diagnosis. Additionally, the VEGFA ACG+/+ genotype was identified as a novel prognostic parameter in OS, independent of other known factors (adjusted HR = 2.1, p = 0.005, Table 3).
Influence of VEGF genetic polymorphisms on clinical and molecular low-risk CLL subgroups
To identify protective or risk interactions, a review of the distribution of VEGFA variability in different subgroups of CLL was undertaken and revealed an asymmetric frequency of the ACG+/+ genotypes in patients with different mutational status of the IgVH genes (n = 222). The frequency of haplotype ACG defined by loci rs699947/rs833061/rs2010963 was 0.49 in the group of patients with poor prognosis (unmutated IgVH genes; UM-CLL) and 0.38 in patients with favourable prognosis (mutated IgVH genes; M-CLL). No asymmetries in the distribution of the haplotype ACG was found between groups defined by other epidemiological, clinical or molecular parameters.
Comparison in a recessive genetic model of the VEGFA rs699947/rs833061/rs2010963 genotypes (ACG+/+ vs ACG×/−) showed that homozygosity for the ACG haplotype was associated with forms of the disease with a worse prognosis. The ACG+/+ genotype was 2.5 times more common among individuals with unfavourable prognosis (UM-CLL) relative to other genotypes (OR = 2.5, two-sided p = 0.012). These recessive genetic associations do not denote an allele-dose effect as frequencies of heterozygous genotypes are not increased in the UM-CLL group (see Table 4). No statistical associations were found between the ACG+/+ genotype and other clinical or molecular variables analysed. Allele or genotype frequencies of polymorphisms rs3025039 and rs25648 also failed to associate with any of the analysed groups (data not shown).
As the aforementioned ACG+/+ genotype was asymmetrically distributed in UM-CLL and M-CLL patients, we analysed the OS independently in the two groups. We did not observe any influence of the ACG+/+ genotype on the OS of the UM-CLL group, but conversely, in the group of patients with a favourable outcome (M-CLL), this genotype significantly correlated with a shorter survival (median OS: ACG+/+ = 105.5 months vs ACG×/− = not reached; p = 0.025; Fig. 2). To further investigate a possible biological influence of this VEGFA genotype on the outcome of low-risk CLL, we performed the analysis within groups of patients defined by other markers of indolent course. We categorized our group of CLL patients by age at diagnosis, cytogenetic abnormalities, CD38 status or Binet stage (Fig. 2). Similarly to the subgroup with favourableIgVHmutational status, in patients with very low or low-risk genetics (deletion 13q, trisomy 12, normal karyotype) a decreased median OS from 173.1 to 101.7 months (p = 1.3×10−5) is associated with the presence of the ACG+/+ genotype. Again, the ACG+/+ genotype correlated with a reduction in the median OS from 173.1 to 105.5 months (p = 0.031) from 173.1 months to 82.3 months (p = 3.8×10−5) and from 173.1 to 84.3 (p = 0.006) in patients with an initial clinical stage (Binet A), negative expression of CD38 and age at diagnosis <65 years, respectively. In contrast, VEGFA genetics in the defined groups of poor outcome does not have any appreciable influence on the clinical course with the exception of the group of patients older than 65 years, in which the OS tended to be worse for those patients with the homozygous ACG+/+ genotype.
Kaplan-Maier curves according to VEGFA ACG+/+ genotype (solid line) and other genotypes (dotted line) in subgroups of patients divided by (A) age at diagnosis, (B) IgVH mutational status, (C) Binet stage, (D) CD38 status and, (E) genetic abnormalities. aVery low (del13q), low (normal karyotype), intermediate (ΔATM) and high-risk (ΔTP53).
Discussion
It has been extensively reported that angiogenesis mediated by the irregular expression of proangiogenic factors such VEGF by leukemic cells plays an important role in the pathogenesis of CLL. In this study, we analysed the contribution of VEGFA genetics in the clinical outcome of the leukaemia.
An analysis of the impact on disease progression of VEGFA SNPs in our population-based CLL cohort demonstrated that thers699947/rs833061/rs2010963 ACG genotype in homozygosity (ACG+/+) is a strong independent predictor of OS. The reduced survival observed in these patients could be related to increased angiogenesis as a result of an enhanced gene expression described for VEGFA variants rs699947, rs833061 and rs2010963. This would constitute a potential biological rationalization to the genetic association described in this work. Nonetheless, the justification to our findings could be due to other VEGF-mediated mechanisms of the pathogenesis of CLL. High-expressor VEGFA genotypes could increase the resistance to apoptosis, the cell motility or influence the microenvironment to generate more aggressive tumoral cells and consequently contribute to the progression of the leukaemia.
A further analysis of VEGFA genetics in CLL subgroups defined by different clinical and molecular parameters assessed an association between the VEGFA ACG+/+ genotype with a molecular marker of aggressive leukaemia: the unmutated status of the immunoglobulin variable gene segments (IgVH). IgVH mutation status parameter discriminates between leukaemia originating from B cells with an immunoglobulin gene submitted (mutated IgVH) or not (unmutated IgVH) to somatic hypermutation in the heavy chain variable region genes during ontogeny. Patients with a high number of mutations tend to have a better prognosis (M-CLL) than those with a low number (UM-CLL). Although the ACG+/+ genotype is less frequent among M-CLL patients, it correlated with a shorter overall survival within this category. A similar adverse effect of the ACG+/+ genotype in disease progression was found when we stratified patients with good prognostic features such as genetic abnormalities of favourable prognosis, Binet stage A, negative expression of CD38 or early age at diagnosis. These results suggest a potential clinical utility of VEGFA genetics as predictors of reduced survival in patients with indolent leukaemia and would allow to further refine the classification of this group of CLL patients. Although our study did not find strong differences in survival associated to the ACG+/+ genotype in patients with poor prognostic features, this observation might be perhaps more apparent than real. The large effect of other molecular factors in subgroups of poor outcome may be masking the differences in survival associated with VEGFA genotypes. Although we observed a large effect of VEGFA genotypes in the subgroup analyses, we were not able to analyse an independent cohort of CLL patients to confirm these differences in survival. In this manner, further studies in other CLL cohorts should be performed to validate these results and would be of relevant interest the analysis of VEGFA genetics and CLL in other ethnic groups. To date, exist few reports studying VEGFA polymorphisms as prognostic markers of CLL. A recent publication of a case-control study in a Polish CLL cohort [30], described an increased frequency of the rs3025039_allele T in patients with advanced stage (Rai III/IV) compared to healthy individuals.
A previous work by Pepper et al [31], found an anti-apoptotic over-expression of VEGF in CLL cells with high levels of CD38, which was associated to the reduced prognosis of the CD38-positive patients. However, we found a similar distribution of the VEGFA polymorphisms in patients with different CD38 expression levels indicating that the elevated VEGF expression of this subgroup is not a result of their VEGFA genetic background.
The VEGFA gene 5′-flanking region is highly variable and contains strongly linked polymorphisms. This increased diversity complicates the characterization of VEGFA haplotypes in the promoter region, but some of these linkages are consistently preserved and associated with disease and high VEGFA expression [18], [23]. Alleles A of rs699947 and C of rs833061 are associated with an upstream 18-nucleotide insertion that could modify the binding of factors in the VEGFA promoter. It has therefore been hypothesized that it could be responsible for the enhanced expression of this gene [20]. It would be interesting to perform further studies analysing the VEGFA levels in serum, the vascularity in the bone marrow and/or the resistance to apoptosis of CLL cells of patients with the VEGFA ACG+/+ genotype in order to explore the implication of this genotype in the physiopathology of the leukemia. A previous report [1], described an increased vascularity in the UM-CLL subset establishing subsequently a relation of a progressive phenotype of the leukaemia with an enhanced or aggressive angiogenic environment. This observation could be the result of the association of the UM-CLL subgroup with the high-expressor VEGFA ACG+/+ genotype that we have reported in this work.
The implication of the presence of increased vascularity in the bone marrow has been described in the outcome of various haematological diseases [32], [33]. This led to the study of the clinical significance of VEGFA genetics in other hematologic malignancies. In acute myeloid leukaemia, the rs699947/rs833061/rs2010963 CTG haplotype and rs3025029 C/C genotype influence the leukaemia-free survival (LFS), event-free survival (EFS) and overall survival (OS) of patients [34]. In chronic myeloid leukaemia, the rs699947 A/A and rs833061 C/C genotypes correlate with progression to advanced disease [35]. VEGFA polymorphisms also associate with clinical parameters in non-Hodgkin’s lymphoma (NHL). In these patients, rs699947 CA and CA + AA genotypes and rs3025039 TT genotypes, as well as, rs699947 A and rs3025039 T alleles are associated with decreased risk for invasion [36]. Furthermore, rs3025039 T allele was found more frequently in NHLs with worse prognosis (patients with International Prognostic Index (IPI)-3 and/or 4) [37].
Elevated serum levels of its receptor, VEGF-R2 were also associated with lymphocytosis, severe anaemia and shortened survival in CLL [14]. These results also support the role of the VEGF system in this leukaemia and suggest a potential benefit in evaluating polymorphisms in VEGF-R2 that might have an impact on CLL outcome.
It has been shown that disruption of proangiogenic factors can inhibit tumour vessel density and growth, and the combination of anti-angiogenic agents with other drugs has certain synergetic effects in improving the OR rate in solid tumours. Although, results of the first clinical trials evaluating anti-VEGF therapy for patients with CLL indicate a lack of efficacy in patients with relapsed/refractory CLL [38], combining VEGF blocking with conventional treatments may be a potential therapeutic approach for patients with CLL. In addition to the hypothetical use of VEGFA SNPs to predict the clinical outcome for CLL, these polymorphisms could be used as markers of clinical efficacy for chemoimmunotherapy with novel agents targeting VEGF or its receptor (VEGFR) to prevent leukaemia progression [39].
In conclusion, VEGFA genetic diversity appears to influence the physiopathology of CLL. The genotyping of polymorphisms in this gene can be performed easily using a rapid PCR method and, together with other parameters, these genetic markers might be utilized both in clinical practice and in further studies of CLL to upgrade patients’ prognosis and select target therapies.
Acknowledgments
We are indebted to all individuals who kindly donated blood samples and to the blood bank, Biobank staff and all technicians in the Molecular Cytogenetics laboratory of the Hospital Puerta de Hierro who helped with the sample collection and characterization. We are also grateful to Teresa Hernando and Jose Luis Bueno who helped with the epidemiologic aspects of the work.
Author Contributions
Conceived and designed the experiments: NGL. Performed the experiments: CLS JMV. Analyzed the data: JAGM NGL. Wrote the paper: NGL. Obtained the cytogenetic and molecular data: BFC NP RC JAGM. Provided clinical cases to the study: PSG FC JAGV BN JAGM. Performed the immunophenotyping analyses: JAGV. Reviewed the statistical analysis: IM JMG. Analysed the data: NGL JAGM CLS.
References
- 1. Maffei R, Martinelli S, Castelli I, Santachiara R, Zucchini P, et al. (2010) Increased angiogenesis induced by chronic lymphocytic leukemia B cells is mediated by leukemia-derived Ang2 and VEGF. Leuk Res 34: 312–321 S0145-2126(09)00319-1 [pii];10.1016/j.leukres.2009.06.023 [doi].
- 2. Kini AR, Kay NE, Peterson LC (2000) Increased bone marrow angiogenesis in B cell chronic lymphocytic leukemia. Leukemia 14: 1414–1418.
- 3. Peterson L, Kini AR (2001) Angiogenesis is increased in B-cell chronic lymphocytic leukemia. Blood 97: 2529.
- 4. Farahani M, Treweeke AT, Toh CH, Till KJ, Harris RJ, et al. (2005) Autocrine VEGF mediates the antiapoptotic effect of CD154 on CLL cells. Leukemia 19: 524–530 2403631 [pii];10.1038/sj.leu.2403631 [doi].
- 5.
Bairey O, Boycov O, Kaganovsky E, Zimra Y, Shaklai M, et al. (2004) All three receptors for vascular endothelial growth factor (VEGF) are expressed on B-chronic lymphocytic leukemia (CLL) cells. Leuk Res 28: 243–248. S014521260300256X [pii].
- 6. Lee YK, Bone ND, Strege AK, Shanafelt TD, Jelinek DF, et al. (2004) VEGF receptor phosphorylation status and apoptosis is modulated by a green tea component, epigallocatechin-3-gallate (EGCG), in B-cell chronic lymphocytic leukemia. Blood 104: 788–794 10.1182/blood-2003-08-2763 [doi];2003-08-2763 [pii].
- 7. Gehrke I, Gandhirajan RK, Poll-Wolbeck SJ, Hallek M, Kreuzer KA (2011) Bone marrow stromal cell-derived vascular endothelial growth factor (VEGF) rather than chronic lymphocytic leukemia (CLL) cell-derived VEGF is essential for the apoptotic resistance of cultured CLL cells. Mol Med 17: 619–627 molmed.2010.00210 [pii];10.2119/molmed.2010.00210 [doi].
- 8. Till KJ, Spiller DG, Harris RJ, Chen H, Zuzel M, et al. (2005) CLL, but not normal, B cells are dependent on autocrine VEGF and alpha4beta1 integrin for chemokine-induced motility on and through endothelium. Blood 105: 4813–4819 2004-10-4054 [pii];10.1182/blood-2004-10-4054 [doi].
- 9. Ugarte-Berzal E, Redondo-Munoz J, Eroles P, del Cerro MH, Garcia-Marco JA, et al. (2010) VEGF/VEGFR2 interaction down-regulates matrix metalloproteinase-9 via STAT1 activation and inhibits B chronic lymphocytic leukemia cell migration. Blood 115: 846–849 blood-2009-08-239426 [pii];10.1182/blood-2009-08-239426 [doi].
- 10. Edelmann J, Klein-Hitpass L, Carpinteiro A, Fuhrer A, Sellmann L, et al. (2008) Bone marrow fibroblasts induce expression of PI3K/NF-kappaB pathway genes and a pro-angiogenic phenotype in CLL cells. Leuk Res 32: 1565–1572 S0145-2126(08)00126-4 [pii];10.1016/j.leukres.2008.03.003 [doi].
- 11. Ghosh AK, Secreto CR, Knox TR, Ding W, Mukhopadhyay D, et al. (2010) Circulating microvesicles in B-cell chronic lymphocytic leukemia can stimulate marrow stromal cells: implications for disease progression. Blood 115: 1755–1764 blood-2009-09-242719 [pii];10.1182/blood-2009-09-242719 [doi].
- 12.
Molica S, Vitelli G, Levato D, Gandolfo GM, Liso V (1999) Increased serum levels of vascular endothelial growth factor predict risk of progression in early B-cell chronic lymphocytic leukaemia. Br J Haematol 107: 605–610. bjh1752 [pii].
- 13. Gora-Tybor J, Blonski JZ, Robak T (2005) Circulating vascular endothelial growth factor (VEGF) and its soluble receptors in patients with chronic lymphocytic leukemia. Eur Cytokine Netw 16: 41–46.
- 14. Ferrajoli A, Manshouri T, Estrov Z, Keating MJ, O’Brien S, et al. (2001) High levels of vascular endothelial growth factor receptor-2 correlate with shortened survival in chronic lymphocytic leukemia. Clin Cancer Res 7: 795–799.
- 15. Koukourakis MI, Papazoglou D, Giatromanolaki A, Bougioukas G, Maltezos E, et al. (2004) VEGF gene sequence variation defines VEGF gene expression status and angiogenic activity in non-small cell lung cancer. Lung Cancer 46: 293–298 S0169500204002053 [pii];10.1016/j.lungcan.2004.04.037 [doi].
- 16. Prior SJ, Hagberg JM, Paton CM, Douglass LW, Brown MD, et al. (2006) DNA sequence variation in the promoter region of the VEGF gene impacts VEGF gene expression and maximal oxygen consumption. Am J Physiol Heart Circ Physiol 290: H1848–H1855 01033.2005 [pii];10.1152/ajpheart.01033.2005 [doi].
- 17.
Renner W, Kotschan S, Hoffmann C, Obermayer-Pietsch B, Pilger E (2000) A common 936 C/T mutation in the gene for vascular endothelial growth factor is associated with vascular endothelial growth factor plasma levels. J Vasc Res 37: 443–448. 54076 [pii];54076 [doi].
- 18. Watson CJ, Webb NJ, Bottomley MJ, Brenchley PE (2000) Identification of polymorphisms within the vascular endothelial growth factor (VEGF) gene: correlation with variation in VEGF protein production. Cytokine 12: 1232–1235 10.1006/cyto.2000.0692 [doi];S1043-4666(00)90692-6 [pii].
- 19. Awata T, Inoue K, Kurihara S, Ohkubo T, Watanabe M, et al. (2002) A common polymorphism in the 5′-untranslated region of the VEGF gene is associated with diabetic retinopathy in type 2 diabetes. Diabetes 51: 1635–1639.
- 20.
Brogan IJ, Khan N, Isaac K, Hutchinson JA, Pravica V, et al. (1999) Novel polymorphisms in the promoter and 5′ UTR regions of the human vascular endothelial growth factor gene. Hum Immunol 60: 1245–1249. S0198-8859(99)00132-9 [pii].
- 21. Hansen TF, Spindler KL, Lorentzen KA, Olsen DA, Andersen RF, et al. (2010) The importance of −460 C/T and +405 G/C single nucleotide polymorphisms to the function of vascular endothelial growth factor A in colorectal cancer. J Cancer Res Clin Oncol 136: 751–758 10.1007/s00432-009-0714-1 [doi].
- 22. Jain L, Vargo CA, Danesi R, Sissung TM, Price DK, et al. (2009) The role of vascular endothelial growth factor SNPs as predictive and prognostic markers for major solid tumors. Mol Cancer Ther 8: 2496–2508 1535-7163.MCT-09-0302 [pii];10.1158/1535-7163. MCT-09-0302 [doi].
- 23. Stevens A, Soden J, Brenchley PE, Ralph S, Ray DW (2003) Haplotype analysis of the polymorphic human vascular endothelial growth factor gene promoter. Cancer Res 63: 812–816.
- 24. Cheson BD, Bennett JM, Grever M, Kay N, Keating MJ, et al. (1996) National Cancer Institute-sponsored Working Group guidelines for chronic lymphocytic leukemia: revised guidelines for diagnosis and treatment. Blood 87: 4990–4997.
- 25. Rossi D, Rasi S, Spina V, Bruscaggin A, Monti S, et al. (2013) Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia. Blood 121: 1403–1412 blood-2012-09-458265 [pii];10.1182/blood-2012-09-458265 [doi].
- 26. Ghia P, Stamatopoulos K, Belessi C, Moreno C, Stilgenbauer S, et al. (2007) ERIC recommendations on IGHV gene mutational status analysis in chronic lymphocytic leukemia. Leukemia 21: 1–3 2404457 [pii];10.1038/sj.leu.2404457 [doi].
- 27. Stephens M, Smith NJ, Donnelly P (2001) A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 68: 978–989 S0002-9297(07)61424-4 [pii];10.1086/319501 [doi].
- 28.
Freathy RM, Weedon MN, Shields B, Hitman GA, Walker M, et al. (2006) Functional variation in VEGF is not associated with type 2 diabetes in a United Kingdom Caucasian population. JOP 7: 295–302. v07i03a05 [pii].
- 29. Girnita DM, Webber SA, Ferrell R, Burckart GJ, Brooks MM, et al. (2006) Disparate distribution of 16 candidate single nucleotide polymorphisms among racial and ethnic groups of pediatric heart transplant patients. Transplantation 82: 1774–1780 10.1097/01.tp.0000250656.33731.08 [doi];00007890–200612270–00046 [pii].
- 30. Wrobel T, Mazur G, Dzietczenia J, Gebura K, Kuliczkowski K, et al. (2013) VEGF and bFGF gene polymorphisms in Polish patients with B-CLL. Med Oncol 30: 456 10.1007/s12032-013-0456-4 [doi].
- 31. Pepper C, Ward R, Lin TT, Brennan P, Starczynski J, et al. (2007) Highly purified CD38+ and. Leukemia 21: 687–696 2404587 [pii];10.1038/sj.leu.2404587 [doi].
- 32. Aguayo A, Kantarjian H, Manshouri T, Gidel C, Estey E, et al. (2000) Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes. Blood 96: 2240–2245.
- 33. Hussong JW, Rodgers GM, Shami PJ (2000) Evidence of increased angiogenesis in patients with acute myeloid leukemia. Blood 95: 309–313.
- 34. Kim DH, Lee NY, Lee MH, Sohn SK, Do YR, et al. (2008) Vascular endothelial growth factor (VEGF) gene (VEGFA) polymorphism can predict the prognosis in acute myeloid leukaemia patients. Br J Haematol 140: 71–79 BJH6887 [pii];10.1111/j.1365–2141.2007.06887. x [doi].
- 35. Kim DH, Xu W, Kamel-Reid S, Liu X, Jung CW, et al. (2010) Clinical relevance of vascular endothelial growth factor (VEGFA) and VEGF receptor (VEGFR2) gene polymorphism on the treatment outcome following imatinib therapy. Ann Oncol 21: 1179–1188 mdp452 [pii];10.1093/annonc/mdp452 [doi].
- 36. Diao LP, Yu XM, Gao YH, Li Y, Liu HS, et al. (2009) Association of VEGF genetic polymorphisms with the clinical characteristics of non-Hodgkin’s lymphoma. J Cancer Res Clin Oncol 135: 1473–1481 10.1007/s00432-009-0650-0 [doi].
- 37. Wrobel T, Mazu G, Dzietczenia J, Gebura K, Kuliczkowski K, et al. (2013) VEGF and bFGF gene polymorphisms in patients with Non-Hodgkin’s lymphoma. BioMed Research International 2013: 159813 10.1155/2013/159813 [doi].
- 38. Shanafelt T, Zent C, Byrd J, Erlichman C, LaPlant B, et al. (2010) Phase II trials of single-agent anti-VEGF therapy for patients with chronic lymphocytic leukemia. Leuk Lymphoma 51: 2222–2229 10.3109/10428194.2010.524327 [doi].
- 39. Shanafelt TD, Byrd JC, LaPlant B, Zent CS, Call T, et al. (2009) Pretreatment angiogenic cytokines predict response to chemoimmunotherapy in patients with chronic lymphocytic leukaemia. Br J Haematol 146: 660–664 BJH7811 [pii];10.1111/j.1365-2141.2009.07811.x [doi].