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VEGF gene sequence variation defines VEGF gene expression status and angiogenic activity in non-small cell lung cancer
Lung Cancer (2004) 46, 293—298
VEGF gene sequence variation defines VEGF gene expression status and angiogenic activity in non-small cell lung cancer Michael I. Koukourakis a,*, Dimitrios Papazoglou b , Alexandra Giatromanolaki c , George Bougioukas d , Efstratios Maltezos b , Efthimios Siviridis c a
Department of Radiotherapy/Oncology, Democritus University of Thrace, Alexandroupolis, Greece Department of Internal Medicine, Democritus University of Thrace, Alexandroupolis, Greece c Department of Pathology, Democritus University of Thrace, Alexandroupolis, Greece d Department of Thoracic/Cardiac Surgery, Democritus University of Thrace, Alexandroupolis, Greece b
Received 12 February 2004 ; received in revised form 6 April 2004; accepted 15 April 2004
KEYWORDS VEGF; Gene polymorphism; Angiogenesis; Non-small cell lung cancer
Summary Different vascular endothelial growth factor (VEGF) gene polymorphisms have been shown to result in different VEGF gene responsiveness to various stimuli and different capacity for VEGF protein production. In the present study, we examined four VEGF gene polymorphisms in thirty—six individuals with non-small cell lung cancer (NSCLC). Gene polymorphisms were correlated with the VEGF protein expression in cancer cells and the tumor angiogenic activity. The −2578C/C, −634G/G and −1154A/A and G/A alleles in the VEGF gene were linked with low VEGF expression, while the −2578C/A, the −634 G/C and the −1154G/G alleles were linked with high VEGF expression. Tumors with −2578C/C had a significantly lower vascular density (VD) compared to the −2578C C/A. Similarly, cases with the −634G/G VEGF polymorphism had a singinificanltly lower vascular density compared to the combined C/C and G/C groups. In addition, the −1154A/A polymorphism seemed to relate with poor vaccularization but the difference did not reach significance. It is concluded that inherited VEGF sequence variations, which characterize the tumor genome itself, are strong determinants of the molecular VEGF and VEGF-downstream phenotype of NSCLC. The large variation in angiogenicity between tumors of similar histologic morphology emerges as a consequence of the ‘parental’ VEGF gene ability to produce VEGF. © 2004 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author. Present address: Tumor and Angiogenesis Research Group, P.O. Box 12, Alexandroupolis 68100, Greece. Tel.: +30 25510 74622; fax: +30 25510 30349. E-mail address: [email protected] (M.I. Koukourakis).
1. Introduction The importance of vascular endothelial growth factor (VEGF) in the angiogenic process of human malignancies is well established [1]. VEGF is a major angiogenic factor in non-small cell lung cancer (NSCLC), defining poor postoperative outcome [2].
0169-5002/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2004.04.037
294 High VEGF expression and/or high angiogenic activity is noted in about 30—40% of NSCLC [3,4]. The reason for such a divergence of VEGF expression and angiogenic status in tumors of similar histologic type and differentiation remains obscure. To date, several polymorphisms within the VEGF gene have been identified [5—7], and some of them have been correlated with a weak ability for VEGF protein production [6—8]. The present study shows that NSCLC patients with specific VEGF gene polymorphisms develop tumors with low VEGF expression and poor vascularization, suggesting that inherited VEGF sequence variations are strong determinants of the molecular VEGF and VEGF-downstream phenotype of NSCLC.
2. Materials and methods Paraffin embedded tumoral and normal lung tissues, derived from 36 patients with non-small cell lung cancer treated by surgery alone, were retrieved from the archieves of the Department of Pathology, Democritus University of Thrace, Alexandroupolis, Greece.
2.1. Immunohistochemistry Tumor sections were immunohistochemically assessed for VEGF expression, using the VG1 monoclonal Ab (recognizing the 121, 165 and 189 isoforms of VEGF) and a modified streptavidin technique,
M.I. Koukourakis et al. as previously reported [2,3]. Briefly, sections were dewaxed and incubated in 0.5% H2 O2 in methanol for 30 min. After microwaving and washing in PBS, sections were incubated with the primary antibody (1:4) for 75 min. After washing in PBS for 5 min, sections were incubated with goat anti-mouse immunoglobulins (1:200) for 30 min (Dako, UK), washed again with PBS for 5 min and incubated with rabbit anti-goat immunoglobulins (1:100) for 30 min. The peroxidaze reaction was developed using diaminobenzidine (Sigma Fast tablets) as chromogen and sections were counterstained with haematoxylin. Normal rabbit immunoglobulin-G was substituted for primary antibody as the negative control (same concentration as the test antibody). The number of cancer cells with strong VEGF cytoplasmic expression was assessed in all optical fields and the median value was used to characterize each case. This value was used in continuous variable analysis. Carcinomas with strong VEGF expression in more than 50% of cancer cells were considered as being of high VEGF reactivity (Fig. 1). Tumor vasculature was highlighted using the anti-CD31 MoAb and the APAAP technique [9]. Briefly, ections were dewaxed, rehydrated and pre-digested with protease type XXIV for 20 min at 37 ◦ C. JC70 (1:20) was applied at room temperature for 30 min and washed in TBS. Rabbit anti-mouse antibody 1 in 50 was applied for 30 min, followed by application of mouse APAAP complex 1 in 1 for 30 min. After washing in TBS, the last two steps were repeated for 10 min each. The color was de-
Fig. 1 PCR for VEGF-634 G/C gene polymorphism (Fig. 1a). Lane 1: 50 bp ladder, Lane 2: PCR product (uncut), Lane 3: negative control, Lanes 4—6: representative RFLPs from G/G, G/C and C/C individuals, respectively. Immunohistocehmical expression of VEGF in non-small cell lung cancer from the patients corresponding to the G/G (low VEGF expression and low angiogenicity, Fig. 1b and c) and CC (strong VEGF expression and high angiogenicity; Fig. 1d and e) lane, respectively.
VEGF polymorphisms and angiogenesis in NSCLC veloped by 20 min incubation with New Fuchsin solution. Three areas of high vascular density (VD) were chosen at 100×, and microvessel counting was performed on 200× fields. The vascular density was assessed in three areas of highest vascularization. The highest value was the ‘maximum vascular density’ (VDmax ) and the median value of these three counts was the ‘median vascular density’ (VDmed ) for each case. Assessment was performed by two independent pathologists (AG, ES) who were blinded to the patient’s and VEGF polymorphism data. Discrepancies were resolved on the conference microscope.
2.2. Assessment of VEGF gene polymorphism Multiple sections (10 m) of each formalin-fixed, paraffin-embedded tissue block were collected. Paraffin wax was removed with xylene, and then the samples were washed with 100% ethanol (the two steps repeated twice). DNA was isolated from the resuspended tissue using a commercially available kit according to the manufacturer’s instructions (QIAamp DNA Mini Kit, QIAGEN Inc., CA, US). Amplification of the four regions of the VEGF gene, containing the polymorphisms −2578C/A, −1154G/A, −634G/C and 936C/T, was carried out in a Mastercycler gradient (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany) thermal cycler in 20 l reaction volumes containing 20 mM Tris—HCl (pH 8.4), MgCl2 , 50 mM KCl, 0.2 mM of each nucleotide, 20 pmol of each of the forward and reverse primers, 1 Unit Platinum Taq polymerase (Gibco, BRL, US) and 500 ng of DNA. MgCl2 concentrations were optimised for each SNP (2.75 mM for −2578C/A, 3.5 mM for −1154G/A, 4 mM for −634G/C and 4.5 mM for 936C/T). Following an initial denaturation step (5 min at 94 ◦ C), samples were subjected to 35—40 rounds of PCR consisting of 94 ◦ C for 40 s, 62 ◦ C (−2578C/A), 60◦ (−1154G/A), 58 ◦ C (−634C/G) or 64 ◦ C (936C/T) for 1 min; and 72 ◦ C for 40 s with a final extension time of 5 min at 72 ◦ C. For the −2578C/A polymorphism the following specific/common primers were used, giving a PCR product of 77 bps: 5 -TAG GCC AGA CCC TGG CAC-3 or 5 -TAG GCC AGA CCC TGG CAA-3 with 5 -TGC CCC AGG GAA CAA AGT-3 . For the −1154G/A polymorphism the following specific/common primers were used, giving a PCR product of 130 bps: 5 -GCC CGA GCC GCG TGT GGA G-3 or 5 -GCC CGA GCC GCG TGT GGA A-3 with 5 -CCC CGC TAC CAGCCG ACT T-3 . For the −634G/C polymorphism the following primers gave a product of 304 bps: forward 5 -ATT TAT TTT TGC TTG CCA TT-3 , reverse 5 -GTC TGT CTG TCT GTC CGT CA-3 while for the
295 936C/T polymorphism the following primers gave a product of 208 bps: forward 5 -AAG GAA GAG GAG ACT CTG CGC AGA GC-3 , reverse 5 -TAA ATG TAT GTA TGT GGG TGG GTG TGT CTA CAG G-3 . The VEGF −634G/C polymorphism was analyzed by digestion of the PCR product with restriction endonuclease BsmFI (New England Biolabs). The −634G allele was cut into two fragments of 193 and 111 bps while the −634C allele remained uncut (304 bps). The VEGF 936C/T polymorphism was analyzed by digestion of the PCR product with restriction endonuclease NlaIII (New England Biolabs). The 936C allele remained uncut (208 bps), while the 936T was cut into two fragments of 122 and 86 bps. PCR products and restriction fragments were loaded directly onto 2% agarose gels (containing 0.5% ethidium bromide), electrophoresed and visualized by photography under UV transillumination.
2.3. Statistics Statistical analysis and graphs were performed using the GraphPad Prism® 2.01 and the Instat® 3.0 packages (San Diego California USA, www.graphpad. com). The chi-square t-test or the unpaired two-tailed t-test was used for testing relationships between categorical tumor variables, as appropriate. All P-values are two sided and P-values <0.05 were used for significance.
3. Results VEGF protein was strongly expressed in the cytoplasm of 12/36 (33%) lung carcinoma samples examined. Overall, the median % of cancer cells with strong VEGF expression in the 12 cases exhibiting VEGF reactivity was 70% (range 20—90). Using a 50% cut-off point (strong staining in more than 50% of cancer cells), tumors were divided in two groups: (a) negative/low (24 cases) and (b) high (12 cases) VEGF reactivity. The ‘maximum vascular density’ ranged from 14 to 95 (median value 40, mean 45.5, standard deviation/S.D. 21). The ‘median vascular density’ ranged from 11 to 79 (median value 35, mean 35.3, S.D. 17). The −2578CA, the −634GC and the −1154GG alleles were linked with high VEGF expression (Fig. 1). A low VEGF expression in cancer cells was significantly associated with the presence of the −2578CC, −634GG and −1154AA and GA alleles, in the VEGF gene (Table 1). No association of the VEGF 946CT polymorphism with VEGF expression was noted, but as 946C/C was noted only in 1/35
296
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Table 1 VEGF gene polymorphism vs. tumor VEGF expression status VEGF status
% VEGF + cells Negative/low
High
P-value
Median (min—max)
P-value
−2578 C/A AA CA CC
4 10 10
2 10 0
0.02
10 (0—70) 25 (0—90) 0 (0—0)
AA vs. CA: 0.46 AA vs. CC: 0.10 CA vs. CC: 0.0005
−634 G/C CC GC GG
10 2 12
5 5 2
0.03
0 (0—80) 50 (0—80) 0 (0—90)
CC vs. GC: 0.12 CC vs. GG: 0.46 GC vs. GG: 0.04
−1154 G/A AA GA GG
10 11 3
1 4 7
0.009
0 (0—50) 0 (0—80) 65 (0—90)
AA vs. GA: 0.13 AA vs. GG: 0.003 GA vs. GG: 0.03
946 C/T CC CT TT
1 6 17
0 3 9
0.77
0 0 (0—80) 0 (0—90)
CT vs. TT: 0.96
patients it was impossible to assess the role of this gene polymorphism. Analysis of the VD in the groups of patients according to the VEGF gene polymorphism revealed that tumors with −2578CC had a significantly lower vascular density compared to the CA, while AA cases had an intermediate vas-
cularization (Table 2). Similarly, cases with the −634GG VEGF polymorphism had a singinificanltly lower VDmax compared to the combined C/C and G/C groups (P = 0.05). The −1154AA polymorphism seemed also to relate with poor vaccularization but the difference did not reach significance.
Table 2 VEGF polymorphisms and vascular density (VD) Polymorphism
VDmax
VDmed
Mean ± S.D.
P-value
Mean ± S.D.
P-value
−2578 C/A AA CA CC
39.6 ± 17 50.0 ± 25 33.0 ± 11
AA vs. CA: 0.27 AA vs. CC: 0.48 CA vs. CC: 0.01
32.8 ± 14 40.9 ± 19 25.6 ± 11
AA vs. CA: 0.29 AA vs. CC: 0.33 CA vs. CC: 0.01
−634 G/C CC GC GG
46.0 ± 23 55.5 ± 23 34.8 ± 15
CC vs. GC: 0.39 CC vs. GG: 0.13 GC vs. GG: 0.07
37.4 ± 18 44.0 ± 18 28.9 ± 18
CC vs. GC: 0.44 CC vs. GG: 0.19 GC vs. GG: 0.08
−1154 A/G AA GA GG
37.0 ± 9 44.2 ± 26 49.7 ± 23
AA vs. GA: 0.33 AA vs. GG: 0.14 GA vs. GG: 0.59
30.0 ± 8 35.3 ± 21 41.4 ± 19
AA vs. GA: 0.38 AA vs. GG: 0.11 GA vs. GG: 0.47
−946 C/T CC CT TT
46 44.2 ± 28 43.2 ± 19
CT vs. TT: 0.92
41 34.7 ± 23 35.5 ± 16
CT vs. TT: 0.94
VEGF polymorphisms and angiogenesis in NSCLC
4. Discussion The role of VEGF gene sequence variations for tumor development and progression remains unclear. In a study by Stevens et al., carriage of the −460/+405 VEGF polymorphism significantly altered the VEGF promoter activity in response to phorbol esters [10]. Howell et al. reported that VEGF −1154AA genotype define melanomas with less aggressive behavior (thinner vertical growth and less thick tumors) [11]. McCarron et al. observed that the VEGF −1154AA genotype is associated with a reduced risk for prostate cancer development, presumably due to its influence on angiogenesis [12]. In the present study, the −1154AA polymorphism of VEGF was significantly associated with lower VEGF expression compared to the GG, while GA seemed to be related with an intermediate VEGF expression status in non-small cell lung cancer. Vascular density analysis revealed that −1154AA polymorphism was linked with poorer vascularisation compared to G/G but the difference did not reach significance, presumably because of the low number of cases examined. These findings are in accordance with the above mentioned studies by Howell et al. and McCarron et al. [11,12], suggesting an inverse association of the −1154AA polymorphism with tumor aggressiveness. Analysis of additional VEGF polymorphisms revealed that the −2578CC was linked with low VEGF expression and poor vascular density compared to the −2578CA, and so was the −634GG compared to the −634GC. Further studies are required to investigate whether patients with −2578CC, −634GG or −1154AA VEGF polymorphisms produce tumors with an eventual benign clinical course compared to other individuals. The herein presented data are the first to suggest that inherited variations in VEGF sequence, that also characterize the tumor genome, are determinants of the molecular VEGF phenotype in NSCLC and, consequently, of the intratumoral vascular density. Hypoxia is an early event in tumor development resulting in stabilization of the hypoxia inducible factor HIF-1␣ and 2␣ binding of DNA and activation of a variety of genes involved in glycolysis and angiogenesis, including VEGF [13,14]. High expression of VEGF, however, is not observed in a subgroup of NSCLC tumors with activated HIF␣ pathways [15]. It seems that the ability of cancer cells to produce VEGF and exhibit an angiogenic phenotype is a consequence of the ‘parental’ VEGF gene ability to produce VEGF in response to external stimuli, such as hypoxia. Whether the clinical aggressiveness of tumors could be predicted by
297 investigating simply the human genome remains open to question.
Acknowledgements The study was financially supported in part by the Tumour and Angiogenesis Research Group.
References [1] Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev 1997;18:4—25. [2] Giatromanolaki A, Koukourakis MI, Sivridis E, O’Byrne K, Cox G, Thorpe PE, et al. Coexpression of MUC1 glycoprotein with multiple angiogenic factors in non-small cell lung cancer suggests coactivation of angiogenic and migration pathways. Clin Cancer Res 2000;6:1917—21. [3] O’Byrne KJ, Koukourakis MI, Giatromanolaki A, Cox G, Turley H, Steward WP, et al. Vascular endothelial growth factor, platelet-derived endothelial cell growth factor and angiogenesis in non-small-cell lung cancer. Br J Cancer 2000;82:1427—32. [4] Giatromanolaki A, Koukourakis MI, Sivridis E, O’Byrne K, Gatter KC, Harris AL. Invading edge vs. inner (edvin) patterns of vascularisation: an interplay between angiogenic and vascular survival factors defines clinical behaviour of non small cell lung cancer. J Pathol 2000;192:140—9. [5] Brogan IJ, Khan N, Isaac K, Hutchinson JA, Pravica V, Hutchinson IV. Novel polymorphisms in the promoter and 5 UTR regions of the human vascular endothelial growth factor gene. Hum Immunol 1999;60:1245—9. [6] Watson CJ, Webb NJ, Bottomley MJ, Brenchley PE. Identification of polymorphisms within the vascular endothelial growth factor (VEGF) gene: correlation with variation in VEGF protein production. Cytokine 2000;12:1232—5. [7] Renner W, Kotschan S, Hoffmann C, Obermayer-Pietsch B, Pilger E. 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 2000;37:443—8. [8] Awata T, Inoue K, Kurihara S, Ohkubo T, Watanabe M, Inukai K, et al. A common polymorphism in the 5 -untranslated region of the VEGF gene is associated with diabetic retinopathy in type 2 diabetes. Diabetes 2002;51:1635—9. [9] Giatromanolaki A, Koukourakis M, Theodossiou D, Barbatis C, Harris AL, Gatter KC. Comparative evaluation of angiogenesis assessment with anti-Factor VIII and anti-CD31 immunostaining in non small cell lung cancer. Clin Cancer Res 1997;3:2493—500. [10] Stevens A, Soden J, Brenchley PE, Ralph S, Ray DW. Haplotype analysis of the polymorphic human vascular endothelial growth factor gene promoter. Cancer Res 2003;63: 812—6. [11] Howell WM, Bateman AC, Turner SJ, Collins A, Theaker JM. Influence of vascular endothelial growth factor single nucleotide polymorphisms on tumour development in cutaneous malignant melanoma. Genes Immunol 2002;3: 229—32. [12] McCarron SL, Edwards S, Evans PR, Gibbs R, Dearnaley DP, Dowe A, et al. Influence of cytokine gene polymorphisms on the development of prostate cancer. Cancer Res 2002;62:3369—72.
298 [13] Semenza GL. Hypoxia-inducible factor 1: master regulator of O2 homeostasis. Curr Opin Genet Dev 1998;8:588— 94. [14] Harris AL. Hypoxia: a key regulatory factor in tumour growth. Nat Rev Cancer 2002;2:38—47.
M.I. Koukourakis et al. [15] Giatromanolaki A, Koukourakis MI, Sivridis E, Turley H, Talks K, Pezzella F, et al. Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival. Br J Cancer 2001;85:881—90.
VEGF gene sequence variation defines VEGF gene expression status and angiogenic activity in non-small cell lung cancer Michael I. Koukourakis a,*, Dimitrios Papazoglou b , Alexandra Giatromanolaki c , George Bougioukas d , Efstratios Maltezos b , Efthimios Siviridis c a
Department of Radiotherapy/Oncology, Democritus University of Thrace, Alexandroupolis, Greece Department of Internal Medicine, Democritus University of Thrace, Alexandroupolis, Greece c Department of Pathology, Democritus University of Thrace, Alexandroupolis, Greece d Department of Thoracic/Cardiac Surgery, Democritus University of Thrace, Alexandroupolis, Greece b
Received 12 February 2004 ; received in revised form 6 April 2004; accepted 15 April 2004
KEYWORDS VEGF; Gene polymorphism; Angiogenesis; Non-small cell lung cancer
Summary Different vascular endothelial growth factor (VEGF) gene polymorphisms have been shown to result in different VEGF gene responsiveness to various stimuli and different capacity for VEGF protein production. In the present study, we examined four VEGF gene polymorphisms in thirty—six individuals with non-small cell lung cancer (NSCLC). Gene polymorphisms were correlated with the VEGF protein expression in cancer cells and the tumor angiogenic activity. The −2578C/C, −634G/G and −1154A/A and G/A alleles in the VEGF gene were linked with low VEGF expression, while the −2578C/A, the −634 G/C and the −1154G/G alleles were linked with high VEGF expression. Tumors with −2578C/C had a significantly lower vascular density (VD) compared to the −2578C C/A. Similarly, cases with the −634G/G VEGF polymorphism had a singinificanltly lower vascular density compared to the combined C/C and G/C groups. In addition, the −1154A/A polymorphism seemed to relate with poor vaccularization but the difference did not reach significance. It is concluded that inherited VEGF sequence variations, which characterize the tumor genome itself, are strong determinants of the molecular VEGF and VEGF-downstream phenotype of NSCLC. The large variation in angiogenicity between tumors of similar histologic morphology emerges as a consequence of the ‘parental’ VEGF gene ability to produce VEGF. © 2004 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author. Present address: Tumor and Angiogenesis Research Group, P.O. Box 12, Alexandroupolis 68100, Greece. Tel.: +30 25510 74622; fax: +30 25510 30349. E-mail address: [email protected] (M.I. Koukourakis).
1. Introduction The importance of vascular endothelial growth factor (VEGF) in the angiogenic process of human malignancies is well established [1]. VEGF is a major angiogenic factor in non-small cell lung cancer (NSCLC), defining poor postoperative outcome [2].
0169-5002/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2004.04.037
294 High VEGF expression and/or high angiogenic activity is noted in about 30—40% of NSCLC [3,4]. The reason for such a divergence of VEGF expression and angiogenic status in tumors of similar histologic type and differentiation remains obscure. To date, several polymorphisms within the VEGF gene have been identified [5—7], and some of them have been correlated with a weak ability for VEGF protein production [6—8]. The present study shows that NSCLC patients with specific VEGF gene polymorphisms develop tumors with low VEGF expression and poor vascularization, suggesting that inherited VEGF sequence variations are strong determinants of the molecular VEGF and VEGF-downstream phenotype of NSCLC.
2. Materials and methods Paraffin embedded tumoral and normal lung tissues, derived from 36 patients with non-small cell lung cancer treated by surgery alone, were retrieved from the archieves of the Department of Pathology, Democritus University of Thrace, Alexandroupolis, Greece.
2.1. Immunohistochemistry Tumor sections were immunohistochemically assessed for VEGF expression, using the VG1 monoclonal Ab (recognizing the 121, 165 and 189 isoforms of VEGF) and a modified streptavidin technique,
M.I. Koukourakis et al. as previously reported [2,3]. Briefly, sections were dewaxed and incubated in 0.5% H2 O2 in methanol for 30 min. After microwaving and washing in PBS, sections were incubated with the primary antibody (1:4) for 75 min. After washing in PBS for 5 min, sections were incubated with goat anti-mouse immunoglobulins (1:200) for 30 min (Dako, UK), washed again with PBS for 5 min and incubated with rabbit anti-goat immunoglobulins (1:100) for 30 min. The peroxidaze reaction was developed using diaminobenzidine (Sigma Fast tablets) as chromogen and sections were counterstained with haematoxylin. Normal rabbit immunoglobulin-G was substituted for primary antibody as the negative control (same concentration as the test antibody). The number of cancer cells with strong VEGF cytoplasmic expression was assessed in all optical fields and the median value was used to characterize each case. This value was used in continuous variable analysis. Carcinomas with strong VEGF expression in more than 50% of cancer cells were considered as being of high VEGF reactivity (Fig. 1). Tumor vasculature was highlighted using the anti-CD31 MoAb and the APAAP technique [9]. Briefly, ections were dewaxed, rehydrated and pre-digested with protease type XXIV for 20 min at 37 ◦ C. JC70 (1:20) was applied at room temperature for 30 min and washed in TBS. Rabbit anti-mouse antibody 1 in 50 was applied for 30 min, followed by application of mouse APAAP complex 1 in 1 for 30 min. After washing in TBS, the last two steps were repeated for 10 min each. The color was de-
Fig. 1 PCR for VEGF-634 G/C gene polymorphism (Fig. 1a). Lane 1: 50 bp ladder, Lane 2: PCR product (uncut), Lane 3: negative control, Lanes 4—6: representative RFLPs from G/G, G/C and C/C individuals, respectively. Immunohistocehmical expression of VEGF in non-small cell lung cancer from the patients corresponding to the G/G (low VEGF expression and low angiogenicity, Fig. 1b and c) and CC (strong VEGF expression and high angiogenicity; Fig. 1d and e) lane, respectively.
VEGF polymorphisms and angiogenesis in NSCLC veloped by 20 min incubation with New Fuchsin solution. Three areas of high vascular density (VD) were chosen at 100×, and microvessel counting was performed on 200× fields. The vascular density was assessed in three areas of highest vascularization. The highest value was the ‘maximum vascular density’ (VDmax ) and the median value of these three counts was the ‘median vascular density’ (VDmed ) for each case. Assessment was performed by two independent pathologists (AG, ES) who were blinded to the patient’s and VEGF polymorphism data. Discrepancies were resolved on the conference microscope.
2.2. Assessment of VEGF gene polymorphism Multiple sections (10 m) of each formalin-fixed, paraffin-embedded tissue block were collected. Paraffin wax was removed with xylene, and then the samples were washed with 100% ethanol (the two steps repeated twice). DNA was isolated from the resuspended tissue using a commercially available kit according to the manufacturer’s instructions (QIAamp DNA Mini Kit, QIAGEN Inc., CA, US). Amplification of the four regions of the VEGF gene, containing the polymorphisms −2578C/A, −1154G/A, −634G/C and 936C/T, was carried out in a Mastercycler gradient (Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany) thermal cycler in 20 l reaction volumes containing 20 mM Tris—HCl (pH 8.4), MgCl2 , 50 mM KCl, 0.2 mM of each nucleotide, 20 pmol of each of the forward and reverse primers, 1 Unit Platinum Taq polymerase (Gibco, BRL, US) and 500 ng of DNA. MgCl2 concentrations were optimised for each SNP (2.75 mM for −2578C/A, 3.5 mM for −1154G/A, 4 mM for −634G/C and 4.5 mM for 936C/T). Following an initial denaturation step (5 min at 94 ◦ C), samples were subjected to 35—40 rounds of PCR consisting of 94 ◦ C for 40 s, 62 ◦ C (−2578C/A), 60◦ (−1154G/A), 58 ◦ C (−634C/G) or 64 ◦ C (936C/T) for 1 min; and 72 ◦ C for 40 s with a final extension time of 5 min at 72 ◦ C. For the −2578C/A polymorphism the following specific/common primers were used, giving a PCR product of 77 bps: 5 -TAG GCC AGA CCC TGG CAC-3 or 5 -TAG GCC AGA CCC TGG CAA-3 with 5 -TGC CCC AGG GAA CAA AGT-3 . For the −1154G/A polymorphism the following specific/common primers were used, giving a PCR product of 130 bps: 5 -GCC CGA GCC GCG TGT GGA G-3 or 5 -GCC CGA GCC GCG TGT GGA A-3 with 5 -CCC CGC TAC CAGCCG ACT T-3 . For the −634G/C polymorphism the following primers gave a product of 304 bps: forward 5 -ATT TAT TTT TGC TTG CCA TT-3 , reverse 5 -GTC TGT CTG TCT GTC CGT CA-3 while for the
295 936C/T polymorphism the following primers gave a product of 208 bps: forward 5 -AAG GAA GAG GAG ACT CTG CGC AGA GC-3 , reverse 5 -TAA ATG TAT GTA TGT GGG TGG GTG TGT CTA CAG G-3 . The VEGF −634G/C polymorphism was analyzed by digestion of the PCR product with restriction endonuclease BsmFI (New England Biolabs). The −634G allele was cut into two fragments of 193 and 111 bps while the −634C allele remained uncut (304 bps). The VEGF 936C/T polymorphism was analyzed by digestion of the PCR product with restriction endonuclease NlaIII (New England Biolabs). The 936C allele remained uncut (208 bps), while the 936T was cut into two fragments of 122 and 86 bps. PCR products and restriction fragments were loaded directly onto 2% agarose gels (containing 0.5% ethidium bromide), electrophoresed and visualized by photography under UV transillumination.
2.3. Statistics Statistical analysis and graphs were performed using the GraphPad Prism® 2.01 and the Instat® 3.0 packages (San Diego California USA, www.graphpad. com). The chi-square t-test or the unpaired two-tailed t-test was used for testing relationships between categorical tumor variables, as appropriate. All P-values are two sided and P-values <0.05 were used for significance.
3. Results VEGF protein was strongly expressed in the cytoplasm of 12/36 (33%) lung carcinoma samples examined. Overall, the median % of cancer cells with strong VEGF expression in the 12 cases exhibiting VEGF reactivity was 70% (range 20—90). Using a 50% cut-off point (strong staining in more than 50% of cancer cells), tumors were divided in two groups: (a) negative/low (24 cases) and (b) high (12 cases) VEGF reactivity. The ‘maximum vascular density’ ranged from 14 to 95 (median value 40, mean 45.5, standard deviation/S.D. 21). The ‘median vascular density’ ranged from 11 to 79 (median value 35, mean 35.3, S.D. 17). The −2578CA, the −634GC and the −1154GG alleles were linked with high VEGF expression (Fig. 1). A low VEGF expression in cancer cells was significantly associated with the presence of the −2578CC, −634GG and −1154AA and GA alleles, in the VEGF gene (Table 1). No association of the VEGF 946CT polymorphism with VEGF expression was noted, but as 946C/C was noted only in 1/35
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Table 1 VEGF gene polymorphism vs. tumor VEGF expression status VEGF status
% VEGF + cells Negative/low
High
P-value
Median (min—max)
P-value
−2578 C/A AA CA CC
4 10 10
2 10 0
0.02
10 (0—70) 25 (0—90) 0 (0—0)
AA vs. CA: 0.46 AA vs. CC: 0.10 CA vs. CC: 0.0005
−634 G/C CC GC GG
10 2 12
5 5 2
0.03
0 (0—80) 50 (0—80) 0 (0—90)
CC vs. GC: 0.12 CC vs. GG: 0.46 GC vs. GG: 0.04
−1154 G/A AA GA GG
10 11 3
1 4 7
0.009
0 (0—50) 0 (0—80) 65 (0—90)
AA vs. GA: 0.13 AA vs. GG: 0.003 GA vs. GG: 0.03
946 C/T CC CT TT
1 6 17
0 3 9
0.77
0 0 (0—80) 0 (0—90)
CT vs. TT: 0.96
patients it was impossible to assess the role of this gene polymorphism. Analysis of the VD in the groups of patients according to the VEGF gene polymorphism revealed that tumors with −2578CC had a significantly lower vascular density compared to the CA, while AA cases had an intermediate vas-
cularization (Table 2). Similarly, cases with the −634GG VEGF polymorphism had a singinificanltly lower VDmax compared to the combined C/C and G/C groups (P = 0.05). The −1154AA polymorphism seemed also to relate with poor vaccularization but the difference did not reach significance.
Table 2 VEGF polymorphisms and vascular density (VD) Polymorphism
VDmax
VDmed
Mean ± S.D.
P-value
Mean ± S.D.
P-value
−2578 C/A AA CA CC
39.6 ± 17 50.0 ± 25 33.0 ± 11
AA vs. CA: 0.27 AA vs. CC: 0.48 CA vs. CC: 0.01
32.8 ± 14 40.9 ± 19 25.6 ± 11
AA vs. CA: 0.29 AA vs. CC: 0.33 CA vs. CC: 0.01
−634 G/C CC GC GG
46.0 ± 23 55.5 ± 23 34.8 ± 15
CC vs. GC: 0.39 CC vs. GG: 0.13 GC vs. GG: 0.07
37.4 ± 18 44.0 ± 18 28.9 ± 18
CC vs. GC: 0.44 CC vs. GG: 0.19 GC vs. GG: 0.08
−1154 A/G AA GA GG
37.0 ± 9 44.2 ± 26 49.7 ± 23
AA vs. GA: 0.33 AA vs. GG: 0.14 GA vs. GG: 0.59
30.0 ± 8 35.3 ± 21 41.4 ± 19
AA vs. GA: 0.38 AA vs. GG: 0.11 GA vs. GG: 0.47
−946 C/T CC CT TT
46 44.2 ± 28 43.2 ± 19
CT vs. TT: 0.92
41 34.7 ± 23 35.5 ± 16
CT vs. TT: 0.94
VEGF polymorphisms and angiogenesis in NSCLC
4. Discussion The role of VEGF gene sequence variations for tumor development and progression remains unclear. In a study by Stevens et al., carriage of the −460/+405 VEGF polymorphism significantly altered the VEGF promoter activity in response to phorbol esters [10]. Howell et al. reported that VEGF −1154AA genotype define melanomas with less aggressive behavior (thinner vertical growth and less thick tumors) [11]. McCarron et al. observed that the VEGF −1154AA genotype is associated with a reduced risk for prostate cancer development, presumably due to its influence on angiogenesis [12]. In the present study, the −1154AA polymorphism of VEGF was significantly associated with lower VEGF expression compared to the GG, while GA seemed to be related with an intermediate VEGF expression status in non-small cell lung cancer. Vascular density analysis revealed that −1154AA polymorphism was linked with poorer vascularisation compared to G/G but the difference did not reach significance, presumably because of the low number of cases examined. These findings are in accordance with the above mentioned studies by Howell et al. and McCarron et al. [11,12], suggesting an inverse association of the −1154AA polymorphism with tumor aggressiveness. Analysis of additional VEGF polymorphisms revealed that the −2578CC was linked with low VEGF expression and poor vascular density compared to the −2578CA, and so was the −634GG compared to the −634GC. Further studies are required to investigate whether patients with −2578CC, −634GG or −1154AA VEGF polymorphisms produce tumors with an eventual benign clinical course compared to other individuals. The herein presented data are the first to suggest that inherited variations in VEGF sequence, that also characterize the tumor genome, are determinants of the molecular VEGF phenotype in NSCLC and, consequently, of the intratumoral vascular density. Hypoxia is an early event in tumor development resulting in stabilization of the hypoxia inducible factor HIF-1␣ and 2␣ binding of DNA and activation of a variety of genes involved in glycolysis and angiogenesis, including VEGF [13,14]. High expression of VEGF, however, is not observed in a subgroup of NSCLC tumors with activated HIF␣ pathways [15]. It seems that the ability of cancer cells to produce VEGF and exhibit an angiogenic phenotype is a consequence of the ‘parental’ VEGF gene ability to produce VEGF in response to external stimuli, such as hypoxia. Whether the clinical aggressiveness of tumors could be predicted by
297 investigating simply the human genome remains open to question.
Acknowledgements The study was financially supported in part by the Tumour and Angiogenesis Research Group.
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