Association between the polymorphisms in exon 12 of hypoxia-inducible factor-1
a
and the clinicopathological features of oral squamous cell carcinoma
Tzong-Ming Shieh
a, Kuo-Wei Chang
b,c, Hsi-Feng Tu
d, Yin-Hua Shih
a, Shun-Yao Ko
e, Yuan-Chien Chen
f,g,
Chung-Ji Liu
b,h,*a
Department of Dental Hygiene, School of Health Care, China Medical University, Taichung, Taiwan
bInstitute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan c
Department of Dentistry, Taipei Veterans General Hospital, Taipei, Taiwan
d
Department of Dentistry, National Yang-Ming University Hospital, Yilan, Taiwan
e
Graduate Institute of Medical Science, Chang Jung Christian University, Tainan, Taiwan
f
Oral and Maxillofacial Surgery, China Medical University Hospital, Taichung, Taiwan
g
Department of Dentistry, College of Medicine, China Medical University, Taichung, Taiwan
hOral and Maxillofacial Surgery, Taipei Mackay Memorial Hospital, Taipei, Taiwan
a r t i c l e
i n f o
Article history: Received 9 January 2010
Received in revised form 6 April 2010 Accepted 19 April 2010
Available online 24 July 2010 Keywords:
Areca chewing
Hypoxia-inducible factor-1a(HIF-1a) Oral squamous cell carcinoma (OSCC) Single nucleotide polymorphism Tumor size
s u m m a r y
Oral squamous cell carcinoma (OSCC) is a common malignancy. The incidence of OSCC is particularly high in some Asian countries because of the popularity of the habit of chewing areca. Hypoxia-inducible fac-tor-1a(HIF-1a) is up-regulated in the hypoxic microenvironment to enhance tumor survival. Five poly-morphisms have been identified in exon 12 of HIF-1aincluding the C1772T polymorphism causing P582S, and the G1790A polymorphism causing A588T of the HIF-1a protein. This study investigated the relationship between these functional polymorphisms and the risk of progression of OSCC. PCR and direct sequencing were utilized to compare the genotypic polymorphism and allelic frequency of HIF-1ain 96 normal controls and 305 OSCC patients. No statistically significant difference in C1772T and G1790A genotypes and allelic frequency between control and OSCC patients was found. However, multivariate analysis indicated that the A carrier of HIF-1aG1790A in OSCC patients was significantly higher in larger tumors than in the contrasting group with an adjusted odds ratio of 2.92. The T carrier of HIF-1aC1772T in buccal cancer patients was significantly higher in the non-areca-chewing group with an adjusted odds ratio of 0.111. The buccal cancer patients with C1772T or G1790A had lower recurrence frequency with an odds ratio of 0.266. These findings may suggest a correlation between the HIF-1a
C1772T and G1790A polymorphisms and the growth of OSCC, and the decrease of OSCC recurrence frequency.
Ó 2010 Elsevier Ltd. All rights reserved.
Introduction
Oral squamous cell carcinoma (OSCC) is a worldwide disease, especially in southern Asian countries. Its high incidence is closely linked to the popularity of the habit of areca (betel) nut chewing.1
Areca nut chewing, alcohol drinking, and cigarette smoking are the greatest risk factors for OSCC in Taiwan.2OSCC is the fourth most
frequent and has the fifth highest mortality of the male cancers in Taiwan. Recently, it has been shown that the age of OSCC pa-tients is younger than before.3 Carcinogenesis of OSCC involves
alterations in cellular proliferation, anti-apoptosis, angiogenesis, migration, and invasion. These tumor progression phenotypes are intimately linked to the abnormalities in molecular regulation machinery.
Hypoxia triggers a cascade of molecular events including angi-ogenesis and cell-cycle control proteins during the development of human tumors. When tumors’ diameters are longer than 2– 3 mm, the centre of the tumor’s microenvironment will become hypoxic.4Hypoxia-inducible factor-1 (HIF-1) is a key regulator of
a broad range of cellular responses to hypoxia and acts in all mam-malian cells. HIF-1 is associated with many physiological responses of cells, including proliferation, differentiation, and viability, and with the genesis and dynamic regulation of blood vessels, glucose, and energy metabolism.5HIF-1
a
is a member of the HIF family and its expression is regulated by the oxygen level.6HIF-1a
is inhibitedby Von-Hippel–Lindau (VHL) protein, which acts as an E3 ubiquitin
1368-8375/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2010.04.009
* Corresponding author at: Oral and Maxillofacial Surgery, Taipei Mackay Memorial Hospital, No. 92, Sec. 2, Zhongshan N. Rd., Zhongshan Dist., Taipei City 10449, Taiwan. Fax: +886 2 2543 3642.
E-mail address:cjliu@ms2.mmh.org.tw(C.-J. Liu).
Contents lists available atScienceDirect
Oral Oncology
ligase. VHL can target the N-terminal transactivation domain (N-TAD) within the oxygen-dependent degradation domain (ODDD) of HIF-1
a
. VHL and HIF-1a
binding are dependent on hydroxylation of two conserved proline residues (Pro-402 and Pro-564), which are oxygen-sensitive.7–10 Up-regulation ofHIF-1
a
has been found in head and neck carcinoma (HNSCC) and carci-nomas of the esophagus, lung, breast, stomach, pancreas, prostate, and kidney.11–13Immunohistochemistry studies demonstrated anassociation between HIF-1
a
expression and OSCC prognosis.14Inaddition, nuclear HIF-1
a
predicts the progression and survival of OSCC.15Several single nucleotide polymorphism (SNP) sites have been found in HIF-1
a
. However, the density of SNP appears particularly high in exon 12, which includes three synonymous SNP (C1720T, G1768A, and A1828T) and two non-synonymous SNP (C1772T and G1790A). These non-synonymous SNP result, respectively, in amino acid changes from proline 582 to serine (P582S) and those from alanine 588 to threonine (A588T). Exon 12 translates a por-tion of HIF-1a
encompassing the N-TAD domain and the partial ODDD region, and has been reported to increase HIF-1a
expres-sion, even under normoxia.16,17 The association between HIF-1a
C1772T or G1790A and the risk or progression of carcinoma of the cervix, endometrium, and the colorectum, and of the esopha-gus, bladder, and kidney has been reported.18–20 Both HIF-1
a
C1772T or G1790A have been demonstrated to increase HIF tran-scription activator function and the numbers of microvascular in HNSCC.16,21The frequency of HIF-1
a
C1772T or G1790A has beenfound to be higher in OSCC relative to controls.22However, only
G1790A has been found to be associated with the risk and progno-sis in a cohort of early-stage OSCC.23 The present study further
stratified that HIF-1
a
C1772T was associated with the size of the OSCC.Materials and methods Subjects
Nine OSCC cell lines were used in the study. SAS, OECM-1, OC3, and SCC25 were kind gifts from Dr. K.-W. Chang (National Yang-Ming University), and HSC3, TW206, SCC4, Cal27, and Ca922 were
kindly gifted by Dr. M.-C. Kao (China Medical University). These OSCC cell lines were cultured using previously used proto-cols.24–26 Three hundred and five OSCC cases without previous treatment and 96 healthy controls were randomly selected from patients who presented for physical examination in the Depart-ment of Oral and Maxillofacial Surgery at Mackay Memorial Hos-pital without prior neoplastic operation, immune disorder or oral lesion. The tumors were staged according to the TNM classifica-tion of malignant tumors defined by the AJCC (2002). People with or without oral cancer risk factors including areca chewing, ciga-rette smoking, and alcohol consumption were defined according Ko et al.2 The study was approved by an institutional review
board. Genotyping
Peripheral blood cells or cell lines underwent DNA isolation using the Blood Mini Kit (Qiagen, Valencia, CA, USA). The ampli-cons containing both HIF-1
a
C1772T and G1790A were obtained by PCR reaction with the following primers: Forward: 50-GAC-ACAGATTTAGACTTGGAG-30, Reverse: 50
-TGGGTAGGAGATGGA-GATGC-30. The PCR reaction condition was 95 °C for 5 min
followed by 35 cycles of 95 °C for 1 min, 59 °C for 1 min, and 72 °C for 1 min, with a final step at 72 °C for 10 min to allow com-plete extension of the PCR fragments. After confirming the integ-rity of the amplicons, genotyping was performed using direct sequencing. After purification of amplicons using a gel extraction kit (Qiaex II Gel Extraction Kit; Qiagen), sequencing was per-formed using a 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA) as instructed by the manufacturer.27The
sequenc-ing primer is the same as the reverse primer used for the PCR reaction. ChromasPro software (Technelysium Pty Ltd, Tewantin, QLD, Australia) was used for the reading of sequences on chromatograms.
Statistical analysis
The Hardy–Weinberg equilibrium was assessed with a
v
2testmodule. Unpaired t-test, Fisher’s exact test,
v
2analysis, binarylo-gistic regression analysis, odds ratio and 95% confidence intervals
Figure 1 Protein domains and five SNP in exon 12 of HIF-1a. The domains of the HIF-1apolypeptide from the N to the C terminal: basic helix–loop–helix (bHLH), PER-ARNT-SIM (PAS) domains, which are involved in HRE binding and dimerization with ARNT (aryl hydrocarbon nuclear receptor translocator); oxygen-dependent degradation domain (ODDD) contains two proline residues (P402 and P564), two transacting domains (TAD), and a nuclear localization signal (NLS). Five SNP in the 12th exon of HIF-1a: C1720T, G1768A, C1772T (P582S), G1790A (A588T), and A1828T and each variation ID, amino acid sequences, and translation codes. This figure was modified according to Tanimoto et al.16
were performed using SPSS 12.0 statistical software (SPSS Inc., Chi-cago, IL, USA) or Prism 5.0 (GraphPad software, San Diego, CA,
USA). Differences between the variants were considered significant when p < 0.05.
Figure 2 Amplification and genotyping of HIF-1ain OSCC cells. (A) PCR reaction. The HIF-1aamplicons were from five OSCC cell lines underwent electrophoresis on 2% agarose gel to evaluate the specificity and integrity. It showed a clear band with motility of 278-bp. (B) Direct sequencing of amplicons. The letters marked with gray boxes were SNP sites. Upper panels, three homozygous wild-type synonymous SNP: C1720T (C/C), G1768A (G/G), and A1828T (A/A). Lower panels, non-synonymous homozygous wild-type SNP C1772T (C/C) and G1790A (G/G) as well as heterozygous SNP C1772T (C/T) and G1790A (G/A).
Results
The HIF-1
a
amplicons were designed containing these five SNP sites in the study. The variation of each HIF-1a
SNP is shown in Fig. 1. C1720T, G1768A, and C1772T were in N-TAD, while only C1772T and G1790A made translation codes change from CCA to TCA, and from GCA to ACA respectively. These two codes caused the amino acid number 582th proline (P) and 588th alanine (A) to change to serine (S) and threonine (T) respectively. The ampli-cons were first found by gel electrophoresis to be 278-bp in size (Fig. 2A), and were subjected to direct sequencing to reveal the sta-tus of all five SNP sites at the same time (Fig. 2B). Although the sequencing revealed clear distinctions among various genotypic patterns in screened OSCC cell lines, only HSC3 and TW206 cells were found to have heterozygous G/A genotype in nucleotide 1790. The other SNP alleles were all of homozygous wild-type. The genotypic analysis was limited to C1772T and G1790A in subjects.Four hundred and one subjects were included in the study for HIF-1
a
genotyping. The demographic data of 305 OSCC patients and 96 controls are shown inTable 1. The ages (mean ± SD) of pa-tients and controls were 52.8 ± 11.1 and 47.2 ± 9.4 years, respec-tively. There was a statistically significant difference between patients and controls. Nearly all patients were male (94.09%), which was significantly different from the control group (84.38%). No difference in oral habits including areca chewing, alcohol consumption and tobacco smoking was found between pa-tients and controls. The most common site of OSCC was on the buc-cal mucosa, followed by the tongue and the gingiva. The clinicopathological parameters were carefully recorded and are shown inTable 1.Analysis of the C1772T polymorphism indicated that in the pa-tients 282 cases were type C/C (92.46%), and 23 cases were type C/ T (7.54%); in the controls 89 cases were type C/C (92.71%), and 7 cases were type C/T (7.29%). No significant differences in genotypic and allelotypic frequencies for the C1772T polymorphism were ob-served between patients and controls. As for the G1790A polymor-phism, in the patients 281 cases were type C/C (92.13%), and 24 cases were type C/T (7.87%); in the controls 7 cases were type G/ G (92.71%) and 89 cases were type G/A (7.29%). No significant dif-ferences in genotypic and allelotypic frequencies for the G1790A polymorphism were observed between patients and controls. Compared with individuals with at least one of the two
polymor-Table 1
Subjects and clinical characteristics.
Number of subjects OSCC (n = 305) Normal (n = 96) Age (years) Mean ± SD 52.8 ± 11.1 47.2 ± 9.4 p < 0.0001 Sex Male 287 81 p = 0.003 Female 18 15 Areca chewing No 23 7 p = 0.936 Yes 282 89 Cigarette smoking No 19 7 p = 0.712 Yes 286 89 Alcohol consumption No 28 9 p = 0.954 Yes 277 87 Site Buccal cancer 117 Tongue 62 Gingiva cancer 57 Others 51 Tumor size Small (T1, T2) 126 Large (T3, T4) 179 N stage N = 0 199 N = 1 106 Stage
Early (Stages I and II) 92 Late (Stages III and IV) 213 Differentiation
Poor and moderate 161
Well 144 Perineural invasion No 273 Yes 32 Perivascular invasion No 274 Yes 31 Survival Live 227 Dead 78 Recurrence No 227 Yes 78
Unpaired t-test or Fisher’s exact test.
Table 2
HIF-1apolymorphism in OSCC patients and controls.
Nucleotide Amino acid Genotype or allelotype OSCC cell lines Patients (%) Controls (%)
C1772T P582S C/C 100.00% (9/9) 92.46% (282/305) 92.71% (89/96) C/T 0.00% (0/9) 7.54% (23/305) 7.29% (7/96) T/T 0.00% (0/9) 0.00% (0/305) 0.00% (0/96) Genotype (C/C): (C/T+T/T) P = 1 OR = 0.9643 95% CI = 0.4003–2.323 C 100.00% (18/18) 96.23% (587/610) 96.35% (185/192) T 0.00% (0/18) 3.77% (23/610) 3.65% (7/192) Allelotype C:T P = 1 OR = 0.9657 95% CI = 0.4077–2.287 G1790A A588T G/G 77.78% (7/9) 92.13% (281/305) 92.71% (89/96) G/A 22.22% (2/9) 7.87% (24/305) 7.29% (7/96) A/A 0.00% (0/9) 0.00% (0/305) 0.00% (0/96) Genotype (G/G): (G/A+A/A) P = 1 OR = 0.9209 95% CI = 0.3838–2.210 G 88.89% (16/18) 96.07% (586/610) 96.35% (185/192) A 11.11% (2/18) 3.93% (24/610) 3.65% (7/192) Allelotype G: A P = 1 OR = 0.9239 95% CI = 0.3916–2.179 Fisher’s exact test.
phisms, C1772T or G1790A, of the HIF-1
a
gene, there were no sig-nificant differences between patients and controls (data not shown). The genotypic distribution of C1772T and G1790A in both patients and controls exhibited no deviations from the Hardy– Weinberg equilibrium (Table 2).The association between HIF-1
a
polymorphisms and the clini-copathological features was analyzed using binary logistic regres-sion analysis adjusting for age. Genotypic distribution of C1772T was not associated with any clinicopathological parameters (Table 3). The frequency of the heterozygous G1790A (G/A) geno-type was significantly higher in larger tumors (T3 and T4 tumors; adjusted OR = 2.920, 95% CI = 1.044–8.173; Table 3). The cross analysis of C1772T and G1790A polymorphisms showed a signifi-cant increase in double heterozygous genotypes (C/T or G/A) in lar-ger tumors (adjusted OR = 2.161, 95% CI = 1.071–4.361). The late-stage tumors had a greater increase in heterozygous G/A genotypes than early-stage tumors. However, the difference was not statisti-cally significant (Table 3).Areca chewing is a major risk factor for OSCC and is the most frequent cause of buccal cancer in Taiwan. We identified 117 buc-cal cancer patients in this study and analyzed using binary logistic regression analysis by adjusting age and sex. No significant differ-ences in genotypic and allelotypic frequency for the G1790A poly-morphism were observed between patients and controls (data not shown). However, OSCC patients carrying C1772T polymorphisms showed a significant increase in heterozygous genotypes (C/T) in non-areca-chewing patients (adjusted OR = 0.111, 95% CI = 0.016– 0.789). The frequency of double heterozygous genotypes (C/T or G/A) in buccal cancer is lower recurrence (adjusted OR = 0.266, 95% CI = 0.071–1.002;Table 4).
Discussion
The stability of HIF-1
a
is dependent on proline residues in ODDD. The hydroxylation of HIF-1a
on proline residues in nor-moxia are liable to be recognized by VHL for degradation.7–10Five Table 3HIF-1agenotype compared with clinical parameters.
C1772T G1790A C1772T and G1790A
Genotype Genotype Genotype
C/C C/T G/G G/A C/C or G/G C/T or G/A Site Buccal cancer 107 9 105 11 97 19 Non-buccal cancer 175 14 176 13 162 27 P 0.943 0.41 0.73 OR (95% CI) 0.969 0.403–2.330 1.42 0.612–3.310 1.12 0.589–2.129 Tumor size Small (T1, T2) 116 8 121 5 113 13 Large (T3, T4) 164 15 160 19 146 33 P 0.838 0.04 0.031 OR (95% CI) 1.1 0.433–2.806 2.92 1.044–8.173 2.161 1.071–4.361 N stage N = 0 183 16 184 15 168 30 N = 1 99 7 97 9 91 15 P 0.660 0.77 0.989 OR (95% CI) 0.812 0.322–2.050 1.14 0.480–2.692 1.005 0.519–1.943 Stage
Early (Stages I and II) 85 7 89 3 82 10
Late (Stages III and IV) 197 16 192 21 177 63
P 0.838 0.062 0.129
OR (95% CI) 1.1 0.433–2.807 3.283 0.994–11.415 1.8 0.843–3.845
Differentiation
Poor and moderate 150 11 147 14 136 25
Well 132 12 134 10 123 21 P 0.569 0.56 0.791 OR (95% CI) 1.283 0.545–3.018 0.78 0.333–1.811 0.903 0.426–1.918 Perineural invasion No 252 21 253 20 233 40 Yes 30 2 28 4 26 6 P 0.715 0.3 0.581 OR (95% CI) 0.755 0.168–3.403 1.83 0.581–5.743 1.308 0.504–3.391 Perivascular invasion No 255 19 252 22 234 40 Yes 27 4 29 2 25 6 P 0.214 0.75 0.463 OR (95% CI) 2.087 0.654–6.658 0.78 0.174–3.497 1.43 0.551–3.716 Survival Alive 208 19 210 17 192 35 Dead 74 4 71 7 67 11 P 0.388 0.69 0.815 OR (95% CI) 0.612 0.201–1.867 1.21 0.479–3.033 0.916 0.440–1.910 Recurrence No 209 18 209 18 192 35 Yes 73 5 72 6 67 11 P 0.558 0.97 0.695 OR (95% CI) 0.734 0.261–2.063 0.98 0.372–2.579 0.863 0.413–1.805
SNP sites have been found in exon 12 within ODDD of the HIF-1
a
protein. We found that two out of nine OSCC had the G1790A G/A genotype of HIF-1
a
. Since no polymorphism was found in the syn-onymous SNP in OSCC cells, the importance of these synsyn-onymous SNP was excluded from the risk assessment of OSCC. Further dis-section in OSCC patients and controls also excluded the association between G1790A and the risk of OSCC. One study reported a signif-icant association between G1790A and OSCC susceptibility.22Mu-noz-Guerra et al.23found an association between G1790A and the
risk of T1/T2 OSCC. Although the discrepancy between our studies and previous studies may represent a difference in race or study cohort, it is also likely that our control cohort is too small to signify a statistical difference for risk assessment.22,23The lack of
correla-tion identified between the HIF-1
a
polymorphism and OSCC risk could be supported by the fact that HIF-1a
expressed in the upper layers of normal squamous epithelium could be associated with squamous differentiation rather than oncogenesis.28Studies have demonstrated that C1772T or G1790A can affect the normal physiological function of HIF-1
a
. Both C1772T or G1790A increase HIF-1a
stability and HIF transcription activi-ties16,17,21and this might underlie the increased microvascularityin HNSCC and OSCC.16,21Our results implicated G1790A
polymor-phism in the growth of OSCC as reflected by the presence of a higher frequency of the G/A genotype and the A allelotype in T3/ T4 tumors in relation to contrasting groups. There was also a bor-derline increase in the A allelotype in T4 tumors relative to the remaining tumors (p = 0.0551; OR = 2.407, 95% CI = 0.9962– 5.813; detailed analysis not shown). Munoz-Guerra et al.23 ad-dressed a correlation between the A allelotype and relapse or the short survival of T1/T2 patients. This study further specified the marked association between the G/A genotype and the A allelotype and the tumor size. Since HIF-1
a
is up-regulated during hypoxia, which benefits tumor progression in at the late stage, it is postu-lated that tumors carrying the G1790A polymorphism might exhi-bit HIF-1a
activity for growth even when the oxygen level is sufficient when the tumor size is small.10This postulation needsto be clarified by a study of expression. Our analysis indicated no increase in the G/A genotype and the A allelotype in N+ subjects relative to N0 subjects; therefore, the increase in the G/A genotype
in late-stage subjects could be due mainly to the impact of tumor size.
The higher nuclear HIF-1
a
labeling indices in tissues signifi-cantly correlate with OSCCs of larger size, lymph node metastasis, and more advanced clinical stage15; and C1772T could provideHIF-1
a
with higher stability.17 Our findings were in agreement withprevious studies in OSCC denoting no association between C1772T and the risk or progression of such tumors.22,23However,
these findings conflict with those in HNSCC and carcinomas in the endometrium, esophagus, colorectum, and prostate.16–20
Although HIF-1
a
expression modulates tumor progression and sur-vival in hypoxic microenvironments, there are additional crucial factors, whose regulation is independent from hypoxia, being in-volved in the OSCC progression.24,29The interaction between thesefactors and HIF-1
a
for OSCC progression requires clarification. Most of the OSCC patients have buccal cancer, which is caused by betel nut chewing in Taiwan. The recurrence rate of buccal can-cer patients with C/T or G/A heterozygous HIF-1a
genotypes is low. The result was different to HIF-1a
expression in breast cancer, which was significantly predictive of metastasis risk and of re-lapse.30 Both C1772T and G1790A HIF-1a
make HIF-1a
proteinmore stable to wild-type (C/C and G/G) to increase the HIF tran-scription activator function.16,17,21 Factors inhibiting the HIF-1
(FIH-1) protein that interact with amino acid 531–826 of HIF-1
a
include the C1772T and G1790A polymorphism sites.31Both poly-morphism sites might decrease the binding affinity between FIH-1 and HIF-1
a
and increase the transcription activator function of HIF-1 protein. The expression of HIF-1a
affects many physiological functions that have complicated regulation mechanisms.32 Themechanism of endometrial carcinoma recurrence with higher HIF-1
a
expression but lower microvessel density is still un-clear.32,33The induction of HIF-1 will increase mRNA expressionof the N-Myc downstream regulated gene-1 (NDRG1, tumor sup-pressor gene).34 Whether NDRG1 was specifically induced by
HIF-1 in buccal cancer is still unknown .These suppositions need more evidence and information before they are proven.
In conclusion, C1772T and G1790A of the HIF-1
a
gene were important factors for enhancing the betel nut effect of the buccal site and increasing the tumor size in oral cancer respectively. CarryTable 4
HIF-1agenotype compared with risk factors and recurrence in buccal cancer.
C1772T G1790A C1772T and G1790A
Genotype Genotype Genotype
C/C C/T G/G G/A C/C or G/G C/T or G/A Areca chewing No 7 2 18 0 24 2 Yes 110 7 99 11 93 17 P 0.028 0.999 0.234 OR (95% CI) 0.111 0.016–0.789 94,321,330 0–0 0.318 0.048–2.099 Cigarette smoking No 11 1 14 0 21 1 Yes 106 8 103 11 96 18 P 0.996 0.999 0.996 OR (95% CI) 0.000 0–0 18,559,156 0–0 0.000 0–0 Alcohol consumption No 16 1 8 0 26 1 Yes 101 8 109 11 91 18 P 0.566 0.999 0.786 OR (95% CI) 0.519 0.055–4.892 128,982,514 0–0 1.357 0.151–12.196 Recurrence No 79 9 82 8 82 16 Yes 38 0 35 3 35 3 P 0.999 0.544 0.050 OR (95% CI) 0.000 0–0 0.647 0.159–2.639 0.266 0.071–1.002
anyone of these polymorphism sites of buccal cancer patients were low recurrence.
Conflicts of interest statement None declared.
Acknowledgments
This study was supported by a Research Grant (CMU97-205) from China Medical University, and NSC97-2314-B-039-041 from the National Science Council, Taiwan.
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