Serum vascular endothelial growth factor levels in patients with medullary thyroid carcinoma

doi:10.1530/EJE-08-0197

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Serum vascular endothelial growth factor levels in patients with medullary thyroid carcinoma

Maria João Bugalho¹^,4, Deolinda Madureira², Carla Espadinha³, Ana Paula Font² and Luís G Sobrinho¹^,4

^¹ Serviço de Endocrinologia^² , Laboratório de Endocrinologia^³ Centro de Investigação de Patobiologia Molecular, Instituto Português de Oncologia de Lisboa Francisco Gentil E.P.E., Rua Professor Lima Basto, 1099-023 Lisboa, Portugal^⁴ Faculdade de Ciências Médicas da Universidade Nova Lisboa, 1169-056 Lisboa, Portugal

(Correspondence should be addressed to M J Bugalho; Email: mjbugalho{at}ipolisboa.min-saude.pt)

Abstract

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Abstract
Introduction
Materials and methods
Results
Discussion
References

Background: High levels of vascular endothelial growth factor (VEGF) havebeen reported in patients with cancers of different origins.There are no data comparing serum VEGF levels of medullary thyroidcarcinoma (MTC) patients with that of the healthy subjects.

Objective: We tried to assess whether serum VEGF concentration in MTC patientsis correlated with tumour extension and whether this markermight be used to further refine the selection of candidatesfor future therapies with receptor tyrosine kinase inhibitors.

Methods: Sera from 57 individuals divided into five groups: group I,healthy individuals (n=14); group II, MTC patients in remission(n=10); group III, MTC patients with residual disease (n=12);group IV, MTC patients with loco-regional disease (n=11) andgroup V, MTC patients with distant metastases (n=10) were analysedfor serum VEGF and calcitonin (CT) levels.

Results: Analysis of serum VEGF did not disclose significant differencesamong the five groups. Mean serum VEGF level of patients withdistant metastases was not significantly different from thatobserved in healthy individuals (319.4±49.78 vs 313.7±43.13ng/l). Serum VEGF levels correlated positively with serum CT(r=0.4891; P=0.0394) for CT values below 2500 ng/l whereas therewas no correlation for CT values above this threshold.

Conclusions: Serum VEGF levels in MTC patients are not significantly differentfrom those found in healthy individuals and did not correlatewith the extension of disease.

Introduction

Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Medullary thyroid carcinoma (MTC) stems from calcitonin (CT)-producingparafollicular cells and accounts for less than 10% of thyroidcarcinomas (1). MTC metastasizes early via lymphatic and haematogenousroutes. Currently, there is no effective systemic therapy.

The genetic hallmarks of MTC are gain-of-function mutationsin the RET proto-oncogene (2). This gene encodes a transmembranetyrosine kinase that is a member of the receptor tyrosine kinases(RTKs) family. Thus, RET became a target for genetic-orientatedtherapy of MTC patients with unresectable disease. Most of thepromising inhibitors of growth factor receptor tyrosine kinasesalso inhibit the vascular endothelial growth factor receptor(VEGFR).

VEGF is a glycoprotein that promotes endothelial regeneration,stimulates formation of collateral blood vessels and increasesvascular permeability. The VEGF family is composed of severalisoforms (VEGF-A, VEGF-B, VEGF-C and VEGF-D) arising from tissue-specificalternative splicing of transcripts from a single VEGF gene.VEGF-A regulates vasculogenesis via two receptor tyrosine kinases:VEGFR1 and VEGFR2. VEGF-C is primarily involved in lymphangiogenesisand acts through VEGFR3 (3).

Increased tissue expression and high serum VEGF levels havebeen reported in patients with cancers of different origins(4, 5, 6, 7, 8, 9, 10) including patients with differentiatedthyroid carcinoma (DTC) (11, 12, 13). Several reports suggestthat the expression of VEGF by thyroid cancer cells is associatedwith a more aggressive phenotype in both animal models and clinicalstudies (14, 15, 16).

The expression of VEGF mRNA by MTC cells was first reportedby Soh et al. (17) and confirmed by others (18, 19). There areno data comparing the serum VEGF levels of MTC patients withthat of the healthy subjects.

Herein, we tried to assess whether serum VEGF concentrationin MTC patients is correlated with tumour extension and whetherthis marker might be used, in the future, to further refineselection of candidates for therapy with RTK inhibitors.

Materials and methods

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Abstract
Introduction
Materials and methods
Results
Discussion
References

Samples

We analysed samples from 57 individuals divided into five groups:group I, healthy subjects/blood donors (n=14); group II, MTCpatients in remission/undetectable serum CT levels and negativeimaging exams (n=10); group III, residual disease/measurableserum CT levels and negative imaging exams (n=12); group IV,loco-regional disease (n=11) and group V, metastatic disease/distantmetastases (n=10).

MTC patients were recruited from the outpatient clinic of theEndocrine Department of the Portuguese Cancer Centre in Lisbon.None had co-morbidities such as diabetes mellitus, hypertensionor heart failure and all were under treatment with laevothyroxinepresenting normal thyrotrophin levels. The MTC diagnosis wasestablished based on histological criteria following total thyroidectomy.

Serum VEGF levels were measured in all individuals. Serum CTlevels were measured in MTC patients. The study was approvedby the local ethics committee.

Serum VEGF measurements

Serum VEGF measurements were performed in batches using theQuantikine Human VEGF sandwich enzyme immunoassay (R&D Systems,Minneapolis, MN, USA). The intra-assay precision, expressedas coefficients of variation, was 4.5–6.7%; the inter-assayprecision was 6.2–8.8% and the sensitivity was <9 ng/l.All assays were performed in duplicate.

Serum CT measurements

Serum CT measurement was performed using an IMA chemiluminescenceassay (IMMULITE 2000, Diagnostic Products Corp., Los Angeles,CA, USA) calibrated against the second international RP 89/620.CT values of <2 ng/l were regarded as undetectable.

Statistical analysis

Statistical analysis was carried out using GraphPad Prism statisticalsoftware (San Diego, CA, USA). Values were expressed as mean±S.E.M.Mean values were compared using the one-way ANOVA test plusTukey's multiple comparison test. CT values were transformedto their logarithms to equalize the variances. Relationshipamong variables was sought using Spearman's rank order correlationcoefficient. Statistical significance was accepted at P<0.05.

Results

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Abstract
Introduction
Materials and methods
Results
Discussion
References

As expected, serum CT levels were significantly different betweenMTC patients with detectable CT concentrations: group III versusIV, P<0.001; group III versus V, P<0.001 and group IVversus V, P<0.05 (Table 1).

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Table 1 Serum calcitonin and vascular endothelial growth factor (VEGF) levels (ng/l) in the five study groups.

Analysis of serum VEGF levels did not disclose significant differencesamong the five groups (Table 1). Likewise, there was no significantcorrelation between serum CT and serum VEGF levels based onthe analysis of groups III–V (r=0.2623; P=0.1402). However,if we subdivide patients according to CT values, below and above2500 ng/l, in the former subgroup serum VEGF levels were positivelycorrelated with serum CT levels (n=18, r=0.4891; P=0.0394),whereas there was no correlation in the latter subgroup (n=15,r=–0.1395; P=0.6199).

Patients with distant metastases (including three with onlyliver metastases, two with only lung metastases, two with liver+lung+bonemetastases, two with liver+lung metastases and one with liverand bone metastases) did not differ significantly from the controlgroup in terms of serum VEGF levels (319.4±49.78 vs 313.7±43.13ng/l).

The lowest serum VEGF levels were observed in groups II andIII although they were not significantly different from thecontrol group.

Discussion

Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

VEGF promotes endothelial regeneration, formation of collateralblood vessels and vascular permeability. High serum VEGF levelshave been reported in patients with cancers of different origins(4, 5, 6, 7, 8, 9, 10) including patients with thyroid carcinomasderiving from follicular cells (11, 12, 13). This study wasdesigned to assess serum VEGF levels in MTC patients. Moreover,we wondered if this marker might be useful to refine selectionof patients for future therapies with tyrosine kinase inhibitors,mainly those without RET mutations, since these molecules sharea moderate affinity for RET, with equivalent or better affinityfor VEGFR, in the low nanomolar range.

We concluded that serum VEGF levels in MTC patients are notsignificantly different from those found in healthy individualsand did not correlate with the extension of disease. Thus, theserum VEGF levels in MTC patients do not appear useful to selectpotential candidates for therapies with tyrosine kinase inhibitors.

Failure to demonstrate high levels of serum VEGF in MTC patientswith distant metastases, in contrast to what happens in patientswith DTC (13), suggests the involvement of other proangiogenicfactors. Recently, Chigurupati et al. (20) have shown that prostatetumour-derived CT, acting via CT receptor, may play an importantrole in prostate tumour growth by regulating intra-tumoral vascularization.We, therefore, wonder whether similar mechanisms might be implicatedin MTC.