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Citation: Blood Cancer Journal (2016) 6, e464; doi:10.1038/bcj.2016.76
www.nature.com/bcj
LETTER TO THE EDITOR
Association of VEGFA-2578 C4A polymorphism with
clinicopathological aspects and outcome in follicular
lymphoma patients
Blood Cancer Journal (2016) 6, e464; doi:10.1038/bcj.2016.76;
published online 26 August 2016
Angiogenesis (AG) based on immunohistochemistry (IHC) for
related proteins is a rather contradictory prognostic marker in
follicular lymphoma (FL).1,2 These inconsistencies may result from
the variety of treatments in different cohorts, and also from the
complex tumor–host interaction in this disease.3
The vascular endothelial growth factor A (VEGFA), produced
both by lymphoma and microenvironment cells, mediates AG4
and seems to interfere in lymphangiogenesis (LG) in lymphomas.5
A single-nucleotide polymorphism (SNP) in the promoter region of
the VEGFA gene, with a C → A substitution at − 2578 nucleotide
position (rs699947), had its wild-type genotype CC associated
with a higher protein expression when compared with the CA
or AA genotypes in immunoassay-based studies.6–8 More
recently, the VEGFA CC genotype was associated with increased
risks of mantle cell lymphoma (MCL)9 and multiple myeloma,10
suggesting an influence of this locus in the development of
mature lymphoplasmacytic neoplasias. The SNP did not influence the survival of MCL11 and diffuse large B-cell lymphoma
(DLBCL) patients,12 but a VEGFA haplotype, including the
rs699947 locus, determined worse prognosis in chronic lymphocytic leukemia (CLL) patients.13 To the best of our knowledge, the roles of VEGFA-2578 C4A SNP in FL susceptibility,
clinicopathological features and outcome are still unknown, and,
therefore, these were the aims of this study. Moreover, we
investigated a possible functional role of rs699947 in FL, by
combining genotyping with the analysis of AG and LG markers
in the patients’ biopsies.
Newly diagnosed FL patients (N = 171; median age: 56 years; 78
males, 93 females; 99 of histological grade 1 or 2, 30 of grade 3A,
42 unclassified; 41 of Ann Arbor stage I or II, 130 of III or IV) and
controls (209 blood donors; median age: 50 years; 103 males, 106
females) were included in the study from January 1999 to
December 2014 (Supplementary Table S1), after Ethics Committees approvals. Patients received CHOP (N = 24), R-CHOP (N = 115)
or other regimens (N = 17) as induction therapy; 15 patients were
maintained in watch and wait regimen.
Genotypes were identified in DNA of peripheral blood by realtime polymerase chain reaction, using a Taqman SNP Genotyping
Assay (catalog #4351379, ThermoFisher Scientific, Foster City, CA,
USA). Replicates were performed in 10% of the reactions,
achieving 100% of concordance. Eighty-two patients' diagnostic
paraffin blocks, arranged as a tissue microarray, served for IHC
analyses using anti-VEGFA, anti-CD34 and anti-D2-40. The slides
were scanned at 20 × magnification in Aperio Scanscope XT
(Aperio Technologies, Vista, CA, USA) and submitted to algorithms
for analysis (Supplementary Table S2), in a blinded fashion.
The Hardy-Weinberg equilibrium (HWE) was verified using χ2
statistics for the goodness-of-fit. Differences between groups were
analyzed by the χ2 or Fisher’s exact test. Two-tailed t-tests were
performed to compare IHC scores between groups of patients.
Logistic regression models assessed associations between genotypes and clinicopathological features. Event-free survival (EFS)
and overall survival (OS) encompassed time from diagnosis until
relapse, progressive disease, death due to disease effects or last
follow-up, and time from diagnosis until death by any cause or last
follow-up, respectively, and were considered only in the 139
patients treated with CHOP or R-CHOP. EFS and OS probabilities
were estimated by the Kaplan–Meier method, and curves were
compared by the log-rank test. The Cox hazards model was used
to identify variables predicting EFS and OS. Variables with P o 0.15
in univariate analyses were included in multivariate analysis.
Significant results of Cox analyses were validated using a
bootstrap resampling study to investigate the stability of risk
estimates (1000 replications). Differences were significant when
P o0.05.
Both patients (χ2 = 0.0002, P = 0.98) and controls (χ2 = 1.73, P = 0.18)
satisfied the HWE at the VEGFA-2578 C4A locus. The mean age of
patients was higher than that of controls (56 vs 50 years, Po0.001).
Similar frequencies of VEGFA CC genotype were seen in patients and
controls (39.2% vs 36.8%, P = 0.34), after adjustment by differences in
age of groups, and individuals with the CC genotype were under
similar risks of FL than the ones carrying CA or AA genotypes (odds
ratio: 1.12, 95% confidence interval (CI): 0.72–1.75). However, patients
harboring the VEGFA CC genotype had 2.11- (95% CI: 1.11–4.02) and
2.34 (95% CI: 1.10–4.97)-fold increased chances of presenting B
symptoms and belonging to high or intermediate Follicular
Lymphoma International Prognostic Index (FLIPI) groups, respectively
(Supplementary Table S1).
VEGFA and D240 protein expressions in tumor samples were similar
in patients with distinct SNP genotypes (data not shown), but FL
microvessel density was increased in patients with the CC genotype
compared with others (3.3 × 10 − 4 vs 2.4 × 10 − 4 vessels/µm2)
(Figures 1a–c).
At the study end (May 2016), 116 patients were alive and 23
patients had died. The median follow-up time was 41.0 months
(1.7–196.1). The 60-month EFS and OS for all patients were 56.5%
and 80.5%, respectively. At this time, both EFS and OS were
shorter in patients with B symptoms (40.9% vs 67.9%, P o 0.01;
66.4% vs 90.1%, P o 0.01), FLIPI of high risk (33.2% vs 68.3%,
P o0.01; 59.4% vs 91.0%, P o 0.01) and VEGFA CC genotype
(42.4% vs 68.3%, P = 0.02; 65.3% vs 91.1%, P o 0.01), respectively
(Figures 1d and e). Bulky disease predicted only worse OS (65.0%
vs 87.7%, P = 0.03), and bone marrow infiltration was marginally
associated with shorter EFS (37.0% vs 59.6%, P = 0.05). No
differences in EFS or OS were seen in patients treated with CHOP
or R-CHOP (Figures 1f and g) (Kaplan–Meier estimates). In
univariate Cox analysis, B symptoms, FLIPI score and VEGFA-2578
C4A predicted EFS and OS, and bulky disease influenced only OS.
In multivariate analysis, B symptoms predicted EFS, bulky disease
predicted OS and VEGFA-2578 C4A influenced both EFS and OS.
Univariate and multivariate analyses showed that patients with
the VEGFA CC genotype had a 1.84- and 1.82-fold higher risk of
presenting an event and a 3.76- and 3.35-fold increased risk of
progression to death than others, respectively. All associations
Letter to the Editor
2
Figure 1. (a) Represents microvessel density measured by CD34 staining in biopsies from CC vs CA+AA patients (P = 0.02, two-tailed t-test); six
cases out of 82 showed technical unsuitability for microvessel analysis. (b, c) Show extreme images of cases with the respective
abovementioned genotypes with high and low microvascular densities, respectively (CD34 staining, 20 × magnification). (d, e) Represent the
estimated EFS and OS of FL patients treated with CHOP/R-CHOP stratified by the VEGF-2578 C4A genotypes. (f, g) Show the estimated EFS
and OS of FL patients submitted to CHOP or R-CHOP regimens in first-line treatment.
Blood Cancer Journal
Letter to the Editor
3
Table 1.
Association of clinicopathological features and VEGFA-2578 C4A genotypes with survival in follicular lymphoma patients
Characteristics
Univariate Cox analysis
Multivariate Cox analysis
N (%)
EFS HR (95% CI)
P-value
OS HR (95% CI)
P- value
EFS HR (95% CI)
P-value
OS HR (95% CI)
P-value
B symptoms
Present
Absent
55
84
2.76 (1.58–4.80)
Reference
o0.01a
3.85 (1.58–9.38)
Reference
o0.01b
2.18 (1.15–4.11)
Reference
0.01c
2.96 (0.98–8.90)
Reference
0.05
Bulky diseased
Present
Absent
39
94
0.96 (0.51–1.82)
Reference
0.92
2.38 (1.02–5.53)
Reference
0.04e
NA
NA
2.59 (1.01–6.63)
Reference
0.04f
Bone marrow infiltration
Present
Absent
64
75
1.70 (0.98–2.97)
Reference
0.05
1.44 (0.62–3.31)
Reference
0.38
1.38 (0.75–2.54)
Reference
0.28
Not considered
Reference
NA
FLIPI scored
High risk
Low/intermediate risk
41
96
2.24 (1.26–3.95)
Reference
o0.01g
5.01 (2.01–12.44)
Reference
o0.01h
1.55 (0.83–2.89)
Reference
0.16
2.55 (0.93–6.96)
Reference
0.06
Treatment
CHOP
R-CHOP
24
115
1.61 (0.84–3.08)
Reference
0.14
2.21 (0.93–5.24)
Reference
0.07
1.12 (0.55–2.25)
Reference
0.74
1.52 (0.58–4.00)
Reference
0.39
VEGFA-2578 C4A
CC
CA+AA
54
85
1.84 (1.06–3.17)
Reference
0.02i
3.76 (1.54–9.16)
Reference
1.82 (1.02–3.24)
Reference
0.04k
3.35 (1.22–9.12)
Reference
0.01l
o0.01j
Abbreviations: CI, confidence interval; EFS, event-free survival; FLIPI, Follicular Lymphoma International Prognostic Index; HR, hazard ratio; N, number of
patients; NA, not applicable; OS, overall survival; %, percentage. In univariate and multivariate Cox analysis: aPbootstrap o0.01, bPbootstrap o0.01,
c
Pbootstrap = 0.01, dThe number of patients differed from the total treated with CHOP/R-CHOP included in analysis of survival (N = 139) because it was not
possible to obtain the information of interest in some patients. ePbootstrap = 0.03, fPbootstrap = 0.04, gPbootstrap o0.01, hPbootstrapo 0.01,
i
Pbootstrap = 0.02, jPbootstrapo 0.01, kPbootstrap = 0.04, lPbootstrap = 0.02. Significant differences between groups are presented in bold letters.
seen in Cox analyses were validated by bootstrap studies based on
1000 samples (Table 1).
We initially observed that the rs699947 SNP was unimportant in
FL development, but had a relevant role in determining tumor
burden, aggressiveness and vascularization at diagnosis. In fact,
increased blood vessels may determine non-Hodgkin lymphoma
progression, probably due to increased supplies of oxygen and
nutrients to tumor cells.2,4
Second, we found lower EFS and OS in patients with B
symptoms, bulky disease and FLIPI of high risk, as expected, and
observed that patients with the VEGFA CC genotype presented
poorer outcome than others. No association between rs699947
and patients' outcome was found in a large study in DLBCL and in
a small MCL cohort.11,12 In contrast, a VEGFA haplotype, including
the rs699947 locus, had deleterious effects in CLL patients.13 Our
results and those found in CLL support that AG modulation by
rs699947 may be relevant driving indolent lymphoproliferative
diseases to unfavorable outcomes.
The VEGFA CC genotype was previously associated with higher
protein expression in the peripheral blood of 30 healthy
individuals7 and 52 ovarian cancer patients.8 We found, however,
similar VEGFA expressions in tumors derived from FL patients with
distinct genotypes, suggesting that the SNP does not affect
protein levels. Interestingly, the final AG product, that is,
microvessel density, was increased in biopsies of patients carrying
the CC genotype. It seems, thus, that the functional role of
rs699947 also exists in FL, but is not dependent on local VEGFA
production, as similarly reported in other AG studies for FL.5,14 It is
possible, however, that rs699947 affects systemic VEGFA levels in
FL patients, as described in the abovementioned immunoassay
studies.7,8 Complementarily, the lack of LG modulation by VEGFA2578 C4A in our study suggests an exclusive role of the CC
genotype in AG, which could increase resistance to apoptosis and
tumor cell migration, leading ultimately to disease progression, as
previously modeled.13 Another report, in contrast, observed that
higher VEGFA levels in lymphoma samples were correlated with
increased LG.5 However, only 11 FL cases were analyzed by the
authors, hampering a more direct comparison with our results. In
addition, our findings do not support a tumor VEGFA modulation
dependent on rs699947. Growing evidences, however, point
toward regulation of LG by the VEGFC gene, which might guide
future investigations.15
In summary, we present the first evidence regarding VEGFA2578 C4A SNP role in clinicopathological aspects and as an
independent prognostic factor in FL. However, we are aware that
larger cohorts of various parts of the world and studies of systemic
VEGFA production in patients carrying the different genotypes of
the SNP are needed to give support to the findings.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This study was funded by FAPESP (grant no. 2014/09854-5) and CAPES (grant no.
62/2014).
AUTHOR CONTRIBUTIONS
MGRA carried out the genotyping, immunohistochemical scoring, analyzed the
data and wrote the manuscript. RRM supervised immunohistochemical scoring.
BABC, DMT, NRA, CGWB and SCA provided medical care to the patients, as well
as clinical information. SFA supervised the immunohistochemical studies.
VJ and LCSP designed and supervised the study and the final manuscript.
All authors approved the final manuscript.
Blood Cancer Journal
Letter to the Editor
4
GRA de Mendonça1, ABC Brito1, RM Rocha2, MT Delamain3, R de
Andrade Natal1, FA Soares2, GWB Colleoni4, CA Souza1,3,
J Vassallo2,5 and CSP Lima1
1
Faculty of Medical Sciences, University of Campinas, Campinas,
Brazil;
2
Department of Pathology, AC. Camargo Cancer Center, São Paulo,
Brazil;
3
Hematology and Hemotherapy Center, University of Campinas,
Campinas, Brazil;
4
Department of Clinical and Experimental Oncology, Federal
University of São Paulo, São Paulo, Brazil and
5
Laboratory of Molecular and Investigative Pathology, Faculty of
Medical Sciences, University of Campinas, Campinas, Brazil
E-mail: carmenl@fcm.unicamp.br
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