Re: Polymorphisms of Death Pathway Genes FAS and FASL in ...

Table 1. FAS and FASL genotype and allele frequencies of patients with breast cancer and healthy control subjectsⴱ

CORRESPONDENCE FAS ⫺1377G⬎A, No. (%)

Re: Polymorphisms of Death Pathway Genes FAS and FASL in Esophageal Squamous-Cell Carcinoma We have read with great interest the recent work by Sun et al. (1) about the role of functional FAS and FASL gene polymorphisms and esophageal squamouscell carcinoma risk. In addition to the report of Sun et al., polymorphisms of the FAS/FASL system have also been shown to be associated with acute myeloid leukemia (2), cervical cancer (3), and lung cancer (4). To analyze the role of FAS/FASL polymorphisms for breast cancer, we performed a case– control study that included 500 female breast cancer patients and 500 healthy sex- and age-matched control subjects. The study was approved by the Ethical Committee of the Medical University Graz. Written informed consent was obtained from all participating subjects. All subjects were Caucasian. FAS –1377G⬎A, FAS – 670A⬎G, and FASL – 844C⬎T genotypes were determined by a 5⬘-nuclease assay (TaqMan; Applera Austria Handels, Vienna, Austria). Primer and probe sets were designed and manufactured by the Applied Biosystems Assay-by-Design custom service (Applera Austria Handels). The following sequences of primers and probes were used to detect FAS and FASL polymorphisms: for FAS –1377G⬎A, forward primer (5⬘-GCT CAGAGTGTGTGCACAAG-3⬘), reverse primer (5⬘-ACTGTTAGTGCCATGAG GAAGAC-3⬘), G-probe (5⬘-VIC-CTG GCACGCCCAGG-NFQ-3⬘, where VIC is the fluorescent dye, NFQ is the nonfluorescent quencher, and the underlined base indicates the polymorphic site), and A-probe (5⬘- FAM-CTGGCACAC CCAGG-NFQ-3⬘, where FAM is the fluorescent dye); for FAS – 670A⬎G, forward primer (5⬘-CTATGGCGCAA CATCTGTACTTTT-3⬘), reverse primer (5⬘-TCCATCTTGTGGCTGCAACAT3⬘), G-probe (5⬘-VICCATTCCAG GAACGTC-NFQ-3⬘), and A-probe (5⬘FAM-CATTCCAGAAACGTC-NFQ-

Control subjects

Case patients

P†

GG GA AA

401 (80.7) 92 (18.5) 4 (0.8) 0.101

371 (74.3) 120 (24.0) 8 (1.6) 0.136

.045‡

AA AG GG

135 (27.3) 246 (49.7) 114 (23.0) 0.479

114 (22.8) 264 (52.9) 121 (24.2) 0.507

.27

CC CT TT

204 (41.9) 216 (44.4) 67 (13.8) 0.359

210 (42.9) 229 (46.8) 50 (10.2) 0.336

.23

A allele frequency FAS ⫺670A⬎G, No. (%) G allele frequency FASL ⫺844C⬎T, No. (%) T allele frequency

.017

.23

.31

ⴱFAS ⫺1377G⬎A, FAS ⫺670A⬎G, and FASL ⫺844C⬎T genotype data were available for 497, 495, and 487 control subjects and 499, 499, and 488 case patients, respectively. †Calculated by the chi-square test. ‡P ⫽ .017, if a dominant effect (GA ⫹ AA versus GG) is assumed.

3⬘); and for FASL ⫺844C⬎T, forward primer (5⬘-CCTGGGTGACAGAGT GAGACT-3⬘), reverse primer (5⬘AGGCTGCAAACCAGTGGAA-3⬘), Cprobe (5⬘-VIC-TTTGTATTTCGCAAT GTT-NFQ-3⬘), and T-probe (5⬘-FAM CTTTGTATTTCACAATGTT-NFQ-3⬘). As a quality control, 95 samples were reanalyzed; results were identical for all samples. Characteristics of the study population have been described previously (5,6). At the time of breast cancer diagnoses, case patients were between 28 and 84 years of age, with a mean (⫾ standard deviation) age of 57 (⫾11) years. Control subjects were agematched to the case patients (⫾1 year), and their mean age was 57 (⫾11) years with a range of 28 – 84 years. Genotype distribution did not deviate from the Hardy–Weinberg equilibrium in case patients or control subjects. Genotype frequencies are summarized in Table 1. The FAS –1377A allele was associated with a higher breast cancer risk, whereas genotype and allele frequencies of FAS – 670A⬎G and FASL – 844C⬎T were not statistically significantly different between case patients and control subjects. In a co-dominant model (genotype codes: GG ⫽ 0, GA ⫽ 1, AA ⫽ 2), the odds ratio of the FAS –1377A variant for breast cancer was 1.42 (95% confidence interval ⫽ 1.08 to 1.88; P ⫽ .013). Although both FAS polymorphisms were in tight linkage disequilibrium (P⬍.001), the FAS – 670G⬎A polymorphism appeared to have no statistically significant influence on breast cancer risk. The FAS

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–1377A/– 670G haplotype was associated with breast cancer (odds ratio ⫽ 1.39, 95% confidence interval ⫽ 1.02 to 1.90; P ⫽ .038), but this association was less pronounced than that of the –1377G⬎A polymorphism alone. In summary, our data suggest that the FAS –1377G⬎A polymorphism is associated with breast cancer, similar to its association with esophageal squamouscell carcinoma (1). Interestingly, two other functional polymorphisms of the FAS/FASL system, FAS – 670A⬎G and FASL – 844C⬎T, were not related to breast cancer risk in our study. This finding could be the result of the different ethnicities of study populations and/or different molecular mechanisms of breast and esophageal tumorigenesis, respectively. Consequently, we urge that the association between polymorphisms of the FAS/FASL system and cancer be investigated in additional studies. PETER KRIPPL UWE LANGSENLEHNER WILFRIED RENNER HERWIG KÖPPEL HELLMUT SAMONIGG

REFERENCES (1) Sun T, Miao X, Zhang X, Tan W, Xiong P, Lin D. Polymorphisms of death pathway genes FAS and FASL in esophageal squamous-cell carcinoma. J Natl Cancer Inst 2004;96:1030 – 6. (2) Sibley K, Rollinson S, Allan JM, Smith AG, Law GR, Roddam PL, et al. Functional FAS promoter polymorphisms are associated with increased risk of acute myeloid leukemia. Cancer Res 2003;63:4327–30. (3) Lai HC, Sytwu HK, Sun CA, Yu MH, Yu CP, Liu HS, et al. Single nucleotide polymorphism

Journal of the National Cancer Institute, Vol. 96, No. 19, October 6, 2004

at Fas promoter is associated with cervical carcinogenesis. Int J Cancer 2003;103:221–5. (4) Wang LE, Cheng L, Spitz MR, Wei Q. Fas A670G polymorphism, apoptotic capacity in lymphocyte cultures, and risk of lung cancer. Lung Cancer 2003;42:1– 8. (5) Krippl P, Langsenlehner U, Renner W, Yazdani-Biuki B, Wolf G, Wascher TC, et al. A common 936 C/T gene polymorphism of vascular endothelial growth factor is associated with decreased breast cancer risk. Int J Cancer 2003;106:468 –71. (6) Krippl P, Langsenlehner U, Renner W, Yazdani-Biuki B, Koppel H, Leithner A, et al. The 5A/6A polymorphism of the matrix metalloproteinase 3 gene promoter and breast cancer. Clin Cancer Res 2004;10:3518 –20.

NOTES Affiliations of authors: Division of Oncology (PK, UL, HS) and Division of Angiology (HK) in the Department of Internal Medicine, and Clinical Institute of Medical and Chemical Laboratory Diagnostics (WR), Medical University Graz, Graz, Austria. Correspondence to: Peter Krippl, MD, Division of Oncology, Medical University Graz, Auenbruggerplatz 15, A-8036 Graz, Austria (e-mail: [email protected]). DOI: 10.1093/jnci/djh289

RESPONSE Recently, we reported a statistically significant association between genetic polymorphisms in the death pathway genes FAS and FASL and risk of esophageal squamous-cell carcinoma among the Chinese (1). Krippl et al. now extend the results to breast cancer, showing a similar association between the FAS –1377G⬎A polymorphism and risk of the cancer among Caucasians. However, they did not observe the same effect for the other two functional polymorphisms, FAS – 670A⬎G and FASL – 844C⬎T. There are several possible explanations for the inconsistent results between their study on breast cancer and our study on esophageal squamous-cell carcinoma. First, as Krippl et al. point out, the inconsistencies may be the result of the different ethnicities of the study populations. We have noted in our article that the allelic and genotype frequencies of the investigated FAS and FASL polymorphisms vary greatly with ethnicity (2–7). In our study, both FAS variant genotypes were associated with increased risk for esophageal squamouscell carcinoma because the two polymorphisms were in almost complete linkage disequilibrium (LD; D⬘ ⫽ .99;

P⬍.001). However, according to the data presented by Krippl et al., the LD in their study population, if any, seems to be much weaker. Given this difference in LD, the null association with the – 670A⬎G variant in their study suggests that this polymorphism might have no functional effect on cancer; rather, it may be only a genetic tag of the functional –1377G⬎A polymorphism (or others) in our study population but not in theirs. In this regard, it would be interesting for them to analyze the effect of the – 670A⬎G variant in the context of haplotype with the –1377G⬎A variant. Second, the difference in the results for the FASL polymorphism could reflect the different molecular mechanisms in the formation of different cancers. In fact, we also have extended our study to lung cancer and breast cancer, and we have observed a statistically significant association between these polymorphisms in the FAS/ FASL system and risk of lung cancer (Zhang X, Miao X, Sun T, Qu S, Xiong P, Tan W, et al.: unpublished results). However, neither the FAS –1377G⬎A nor the FASL – 844C⬎T variant was associated with breast cancer, which is similar to the observation by Krippl et al., although a statistically significant interaction between these two polymorphisms was observed (odds ratio ⫽ 2.64, 95% confidence interval ⫽ 1.45 to 4.81 [Xiong P, Miao X, Sun T, Tan W, Zhang X, Lin D: unpublished data]). These results imply that FAS and FASL polymorphisms may be less important in breast carcinogenesis. Third, other confounding factors, such as ethnically mixed study populations, should also be considered as a potential source of bias. It has been shown that the genotype frequencies of the FAS – 670A⬎G polymorphism in populations defined as Caucasian but with different origins, such as the United Kingdom, The Netherlands, and the United States, are statistically significantly different (3,4,6). Given these findings, caution should be taken in cancer association studies to avoid genetic heterogeneity of the study population. The general categorization of a human population as Caucasian, Asian, or African might not be sufficient for cancer– genetic association studies, especially

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when the mutations of interest are fairly recent in origin. In conclusion, although our studies suggest an association of polymorphisms in FAS and FASL with the risk of certain cancers, we agree with the proposal that more studies should be done for comparison. TONG SUN XIAOPING MIAO XUEMEI ZHANG DONGXIN LIN

REFERENCES (1) Sun T, Miao X, Zhang X, Tan W, Xiong P, Lin D. Polymorphisms of death pathway genes FAS and FASL in esophageal squamous-cell carcinoma. J Natl Cancer Inst 2004;96:1030 – 6. (2) Huang QR, Manolios N. Investigation of the -1377 polymorphism on the Apo-1/Fas promoter in systemic lupus erythematosus patients using allele-specific amplification. Pathology 2000;32:126 –30. (3) Sibley K, Rollinson S, Allan JM, Smith AG, Law GR, Roddam PL, et al. Functional FAS promoter polymorphisms are associated with increased risk of acute myeloid leukemia. Cancer Res 2003;63:4327–30. (4) Nelson HH, Kelsey KT, Bronson MH, Mott LA, Karagas MR. Fas/APO-1 promoter polymorphism is not associated with nonmelanoma skin cancer. Cancer Epidemiol Biomarkers Prev 2001;10:809 –10. (5) Coakley G, Manolios N, Loughran TP Jr, Panayi GS, Lanchbury JS. A Fas promoter polymorphism at position -670 in the enhancer region does not confer susceptibility to Felty’s and large granular lymphocyte syndromes. Rheumatology (Oxford) 1999;38:883– 6. (6) Wu J, Alizadeh BZ, Veen TV, Meijer JW, Mulder CJ, Pena AS. Association of FAS (TNFRSF6)-670 gene polymorphism with villous atrophy in coeliac disease. World J Gastroenterol 2004;10:717–20. (7) Wu J, Metz C, Xu X, Abe R, Gibson AW, Edberg JC, et al. A novel polymorphic CAAT/ enhancer-binding protein beta element in the FasL gene promoter alters Fas ligand expression: a candidate background gene in African American systemic lupus erythematosus patients. J Immunol 2003;170:132– 8.

NOTES Affiliation of authors: Department of Etiology and Carcinogenesis, Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Correspondence to: Dongxin Lin, MD, Department of Etiology and Carcinogenesis, Cancer Institute, Chinese Academy of Medical Sciences, Beijing 100021, China (e-mail: dlin@public. bta.net.cn). DOI: 10.1093/jnci/djh290

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