ANTICANCER RESEARCH 27: 4121-4126 (2007)

The Glycoprotein Ib· VNTR Polymorphism is Associated with Risk for Oral Cancer

ELEFTHERIOS VAIRAKTARIS1*, ZOE CHARALAMBOUS SEREFOGLOU1*, CHRISTOS YAPIJAKIS1,2, STAVROS VASSILIOU1, EMEKA NKENKE3, DIMITRIS AVGOUSTIDIS1, ANTONIS VYLLIOTIS1, PANAGIOTIS STATHOPOULOS1, FRIEDRICH W. NEUKAM3 and EFSTRATIOS PATSOURIS4

Departments of 1Oral and Maxillofacial Surgery, 2Neurology and 4Pathology, University of Athens Medical School, Athens, Greece; 3Department of Maxillofacial Surgery, Universität Erlangen, Klinik und Poliklinik für Mund-, Kiefer-, Gesischtschirurgie, Erlangen D-91054, Nürnberg, Germany

Abstract. Background: We have previously found an p=0.027, respectively) compared to the control group. association of platelet glycoprotein Ia polymorphism with Conclusion: The VNTR polymorphism of the GPIb· gene, increased risk for oral cancer. The purpose of this study was to which affects the structure and function of this platelet investigate the possible relation of another platelet glycoprotein, glycoprotein, seems to be associated with risk for oral cancer, Ib· (GPIb·), with oral oncogenesis. Patients and Methods: especially in patients without a family history of cancer. The variable number of tandem repeats (VNTR) polymorphism of the GPIb· gene, which affects the protein's structure and Oral squamous cell carcinoma (OSCC) is a common function, was examined in 162 Greek and German patients malignancy, characterized by a fairly poor prognosis (1). with oral squamous cell carcinoma and 225 healthy controls of Carcinogenesis in the oral region is known to involve equivalent age, gender and ethnicity. Results: The B allele genetic alterations and exposure to other factors, such as frequency detected, representing higher platelet activation, in the tobacco and alcohol (2). Recently, common polymorphisms patient group and in the subgroups of patients without family in genes involved in angiogenesis, inflammation and history either of cancer or thrombophilia were significantly thrombosis have also been associated with increased risk for elevated in comparison with that of the control group (p=0.03, oral cancer (3-9). One such factor, previously correlated p=0.016 and p=0.036, respectively). The D allele frequency with cerebrovascular disease and cancer, is platelet (lower platelet activation) was significantly lower in comparison glycoprotein Ib· (GPIb·) (10-12). with controls only in patients with family history of GPIb· is the largest polypeptide of a major platelet thrombophilia. The frequency of B/B homozygotes was complex called the membrane glycoprotein (GP) Ib-IX-V significantly increased in the total group of patients and the complex (13, 14). GPIb· is disulfide-linked to GPIb‚, and subgroup of patients with a family history of thrombophilia, in non-convalently complexed with GPIX and GPV in the comparison with the control group (p=0.042 and p=0.043, ratio 2:2:2:1 (14). GPIb-IX complex is a platelet receptor respectively), while the frequency of heterozygotes for the C/B for another large multimeric glycoprotein, von Willebrand alleles was significantly lower in the subgroups of patients with factor (vWF), and plays a major role in mediating platelet a family history of cancer and thrombophilia (p=0.036 and adhesion to the subendothelium (15, 16). The GPIb-IX-V complex binding to subendothelial vWF at high shear rates is an initial step in the cascade of events which lead to thrombosis (17, 18). GPIb· is not only pivotal to the process *Both authors contributed equally to this work. of arterial thrombosis but is also highly expressed in epithelial and tumor cells (12, 19). The levels of GPIb· Correspondence to: Dr. Eleftherios Vairaktaris, MD, DDS, Ph.D., expression in tumor cells are associated with motility, Department of Oral and Maxillofacial Surgery, University of invasiveness and cell differentation (12). Athens Medical School, “Attikon” Hospital, Rimini 1, GR-12462, A functional polymorphism has been located in the Greece. Tel: +302106443035, Fax: +302106443803, e-mail: [email protected] polymorphic site to the heavily O-glycosylated region of the GPIb·, affecting its stucture (20). This polymorphism was Key Words: Platelet glycoprotein Ib·, GPIb·, oral cancer, found to result from a variable number of tandem repeats polymorphism, oncogenesis, thrombophilia. (VNTR) of 39 bp of GPIb·: either one repeat (D allele),

0250-7005/2007 $2.00+.40 4121 ANTICANCER RESEARCH 27: 4121-4126 (2007) two (C allele), three (B allele), or four repeats (A allele) (21, 22). At the protein level, each repeat leads to addition of 13 amino acids moving the vWF-binding domain out of the platelet membrane (22, 23). This may result in the exposure of the molecule to greater shear forces and thus lower the thresold for shear-induced interaction with vWF and subsequent platelet activation (23). A previous study did not detect any association between the GPIb· VNTR polymorphism and breast cancer, while another found a significant correlation of GPIb· expression with breast Figure 1. Agarose gel electrophoresis of VNTR genotyping PCR products cancer aggressiveness (24, 25). Some studies of large patient in five studied individuals. The GPIb· gene B allele corresponds to 276 cohorts have detected a strong association between the bp, the C allele to 237 bp and the D allele to 198 bp. 1: Molecular weight GPIb· VNTR polymorphism and venous or arterial marker; 2: B/D heterozygote; 3, 4: C/C homozygotes; 5, 6: B/C thrombosis, while other studies with smaller patient heterozygotes. numbers have not found any such association (26-31). We have previously detected an association of another platelet glycoprotein polymorphism with risk for oral ratios with a 95% confidence interval (CI). A p-value less than 0.05 cancer, namely GPI· C807T (6). In order to determine was considered statistically significant. whether the GPIb· VNTR polymorphism is also associated with oral cancer, we investigated this polymorphism in Results patients with oral squamous cell carcinoma and matched healthy controls. The prevalence of detected GPIb· genotypes, allele and carrier frequencies are shown in Table I. The data for the Patients and Methods two tested populations (Greek and German healthy controls) were analyzed together since there were no A total of 387 individuals of Greek and German origin were significant differences in genotype or allele frequency of the enrolled after informed consent was obtained, consisting of 225 GPIb· polymorphism between the two populations. healthy controls and 162 patients with oral squamous cell carcinoma In comparison with controls, the frequency of the B of similar gender, age and ethnicity. In addition to clinical allele, representing higher platelet activation, was presentation, a biopsy with pathological diagnosis of tumor stages I-IV and family history regarding cancer and thrombophilia were significantly elevated in the patient group and in the available. The age of patients ranged between 40-84 years, with a subgroups of patients without a family history either of mean age of 58.5±10.1 years. The age of the controls varied cancer or thrombophilia (p=0.03, p=0.016, p=0.036, between 38-92 years, while the mean age was 56.9±14.3 years. Sixty respectively; Table I). The D allele frequency (lower platelet of the patients (37%) had one or two first degree relatives with activation) was significantly lower in comparison with cancer and their age range 41-83 years (58.7±10.2) did not differ controls only in patients with family history of significantly from the whole group of patients. Furthermore, 32 thrombophilia (p=0.035). Finally, there were no major patients (19.8%) had one or two first-degree relatives with idiopathic thrombosis and an age range of 44-75 years (58.0±9.9), differences in the frequencies of the three GPIb· alleles due again with no statistical difference compared to the whole group. to categorizations of gender, age, age at onset of oral Sixteen patients (9.9%) had a positive family history for both cancer cancer, or cancer stage. and thrombosis and an age range of 48-74 with a mean age of Compared to the control group, only the homozygotes for 56.3±8.0 years. Nearly all patients (95.0%) were smokers and about the higher activation B allele were significantly increased in a third of them were alcohol abusers (32.5%). Most of the studied the total group of patients (p=0.042), as well as in patients individuals worked in a low-risk environment (with the exception of with a family history of thrombophilia (p=0.043; Table I). one patient and three controls who worked in chemical factories). DNA was isolated using the NucleoSpinì (Macherey-Nagel In the subgroups of patients with a family history of cancer GmbH & Co, Düren, Germany). Molecular detection of the GPIb· and thrombophilia, the frequency of heterozygotes for the polymorphism in the GPIb· gene was performed by restriction C/B alleles was significantly lower (p=0.036 and p=0.027, fragment length polymorphism typing, as described elsewhere (24). respectively). No statistically significant difference in the An example of observed alleles is illustrated in Figure 1. frequency of genotypes or alleles of the controls was ® The statistical analyses were performed using SAS software observed in the subgroups of patients with initial or (version 9.0; SAS Worldwide Headquarters SAS Institute Inc., advanced stages of cancer, or with and without tobacco or Cary, NC, USA). The frequencies of alleles and genotypes of the whole group and subgroups of patients were compared to the alcohol abuse (data not shown). respective frequencies of the controls using Fisher's exact test. The Interestingly, compared to individuals with the referent Mantel-Haenszel method was used for the calculation of all odds C/C genotype, B/B homozygotes have an almost double

4122 Vairaktaris et al: Association of GPIb· with Oral Cancer relative risk for oral squamous cell carcinoma (odds ratio (OR) 1.74, 95% confidence interval (CI) 0.7-4.4; Table I).

Additionally, B/B homozygotes with family history of 1.29 thrombophilia have an even greater risk for developing oral

cancer (OR 3.29, 95% CI 0.9-11.8; Table I). 0.036 -value OR (CI) P p= Discussion

Platelet glycoprotein Ib· is highly expressed in tumor cells and the level of its expression is associated with motility, invasiveness and distant metastasis (12, 19, 24, 25, 31). The 0.31 30 N.S. 0.98 0.28 13.2% N.S. 0.90 GPIb· VNTR polymorphism, which affects the structure 3.29 12 N.S. 1.51 and function of GPIb·, was investigated in patients with oral cancer and matched healthy controls in order to detect 0.027 0.035 0.043 whether it affects risk of cancer in the oral region. -value OR (CI) (%) P p= p= p= Despite the modest number of studied individuals, the findings of this study seem to support the association of

VNTR polymorphism with risk for oral cancer. In s with regard to family history of cancer and thrombophilia). comparison with controls, the B allele (higher platelet activation) frequency was significantly higher in the total

group of patients and their subgroup without positive family 1.47 29.2% N.S. 1.27 29.7% history of cancer or thrombophilia. Furthermore, the D allele frequency (lower platelet activation) was reduced in 0.016 value OR (CI) (%) P- the total group and most subgroups of patients, while this p= trend reached a significant value in the subgroup of patients with a family history of thrombophilia. The present study indicates that the VNTR polymorphism B allele is associated with oral cancer risk, while the D allele plays a rather prophylactic role. The B allele has 3 tandem repeats of 39 bp, while the D allele has 0.77 28 N.S.2 1.25 only one (21, 22). Therefore, the GPIb· protein with the

longer amino acid sequence corresponding to that of the B 0.036 -value OR (CI) (%) P allele may be exposed out of the platelet membrane, p= lowering the threshold for greater shear-induced interaction with vWF, resulting in easier as well as irreversible platelet (%) activation (22, 23). It is well-known that in malignancies, tumor cells undergo complex interactions with vascular endothelium and . In a human osteosarcoma study,

it was shown that the initiation of the platelet aggregating 1.74 8 N.S. 1.55 10 N.S. 1.84 6 activity of tumor cells required their interaction with 0.03 1.29 22.7% N.S. 0.99 33.8% platelet membrane GPIIb/IIa, but for irreversible platelet 0.042 -value OR (CI) Patients with Patients without Patients with Patients without p= P aggregating activity the platelet membrane glycoprotein p= GPIb· was responsible (19). The fact that the B allele (higher platelet activation) frequency was not significantly increased in the subgroup of patients with a family history of either cancer or thrombophilia may suggest that the GPIb· polymorphism (%) (%) (0.9%) (0%) (0.01-3.3) (0%) (0.01-6.4) (0%) (0.01-7.01) (0%) (0.04-18.5)(0.01-4.1) (0%) (1.8%) (1.2%) (0.1-7.01) (3.3%) (0.3-23)(0.13-9.4) (1.5%) (0.01-3.5) (0.0%) (0.0%) (0.02-9.2) (4.4%) (11.1%) (0.7-4.4) (13.3%) (0.51-4.7)(0.54-6.3) (9.8%) (18.8%) (0.9-11.8) (9.2) (0.55-4.2) (23.6%) (19.8%) (0.4-1.3)(16.9%) (6.7%) (13.6%) (0.4-1.5) (0.24-2.4)(0.7-2.25) (27.5%) (13.3%) (0.3-2.1) (6.3%) (13.7%)(0.51-1.8) (15.4%) (0.4-1.9) (0.1-1.04)(0.13-1.5) (23.1%) (6.3%) (0.55-1.7) has a minor effect in oral carcinogenesis. Similar to the case (52.4%) (54.3%)(68.8%) (63.3%) (49%) (50.8%) of the minor contribution of GPI· in risk for oral cancer (6), it seems that many factors regulate cell/cell and cell -value was used for genotype comparisons; odds ratios (OR) are age-adjusted; CI: 95% confidence interval; N.S.: not significant. p substratum adhesion. Therefore, each one of them might Prevalence of GPIb· (VNTR) polymorphism in healthy controls and patients with oral cancer (total group subgroup D allele 14.7% 11.3% N.S. 0.77 13.6% N.S. 0.93 9.9% N.S. 0.67 4.2% frequencyB allele 22.9% 29.6% (0.5-1.3) (0.5-1.8) (0.36-1.25) (0.07-1.1) (0.53-1.5) slightly affect adhesion of platelets to the extracellular Frequency (0.9-1.9) (0.6-1.7) (0.97-2.2) (0.67-2.4) (0.87-1.9) C/DD/BPrevalence of variant alleles 38 2 22 N.S. 0 N.S.0 0.16 0.800.3 N.S. 8 0 N.S. N.S. 0.86 0.32 14N.S. 0 0.81 N.S. 0.90N.S. 2 0 0.19 N.S. 0.44 20 N.S. 0.97 C/CD/DB/B 118 4 88 10 2 18 1 (referent) 38 N.S 0.83 2 N.S. 1 (referent) 2.6 50 0N.S.0 0.18 1 (referent) 0.45 N.S. 22 N.S. 2 1.11 1 (referent) 66 1 (referent) C/B 53 32 N.S 0.78 4 Fisher's tumor cell and contribute to the risk for oral carcinogenesis. Table I. Genotype Controls Patients Family history of cancer Family history of thrombophilia

4123 ANTICANCER RESEARCH 27: 4121-4126 (2007)

In conclusion, the VNTR polymorphism of the GPIb· 12 Grossi IM, Hatfield JS, Fitzgerald LA, Newcombe M, Taylor gene, which affects the structure and function of this platelet JD and Honn KV: Role of tumor cell glycoproteins glycoprotein, seems to be associated with risk for oral cancer, immunologically related to glycoproteins Ib and IIb/IIIa in tumor cell-platelet and tumor cell matrix interactions. especially in patients without a family history of cancer. Federation of American Societies for Experimental Biology 2(8): 2385-2395, 1998. Acknowledgements 13 Massberg S, Brand K, Grüner S, Page S, Müller E, Müller I, Bergmeier W, Richter T, Lorenz M, Konrad I, Nieswandt B This work was co-funded by the European Social Fund and National and Gawaz M: A critical role of platelet adhesion in the Resources (EPEAEK II “Pythagoras” 70/3/7391) grant to E.V. initiation of atherosclerotic lesion formation. J Exp Med 196(7): 887-896, 2002. 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26 Gonzalez-Conejero R, Lozano ML, Rivera J, Corral J, Iniesta 30 Ishida F, Ito T, Takei M, Shimodaira S, Kiyoshi K and JA, Moraleda JM and Vicente V: Polymorphisms of platelet Kivasawa K: Genetic linkage of Kozak sequence polymorphism membrane glycoprotein Ib· associated with arterial thrombotic of the platelet glycoprotein Ib alpha with human platelet disease. Blood 92(8): 2771-2776, 1998. antigen-2 and variable number of tandem repeats 27 Ozelo MC, Origa AF, Aranha FJP, Mansur AP, Annichino- polymorphisms, and its relationship with coronary artery Bizzacchi JM, Costa FF, Pollak ES and Arruda VR: Platelet disease. Br J Haemaot 111: 1247-1249, 2000. glycoprotein Ib· polymorphisms modulate the risk for 31 Baker RI, Elkelboom J, Lofthouse E, Staples N, Afshar- myocardial infarction. Thromb Haemost 92: 384-386, 2004. Kharghan V, López JA, Shen VS, Berndt MC and Hankey G: 28 Mikkelsson J, Perola M, Penttilä A and Karhunen PJ: Platelet Platelet glycoprotein Ib· Kozak polymorphism is associated with glycoprotein Ib· HPA-2 Met/VNTR B haplotype as a genetic an increased risk of ischemic stroke. Blood 98(1): 36-40, 2001. predictor for myocardial infarction and sudden cardiac death. Circulation 104: 876-880, 2001. 29 Kenny D, Muckian C, Fitzgerald DJ, Cannon CP and Shields DC: Platelet glycoprotein Ib· receptor polymorphisms and Received June 14, 2007 recurrent ischaemic events in acute coronary syndrome patients. Revised July 19, 2007 J Thrombosis and 13(1): 13-19, 2002. Accepted September 27, 2007

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