A BCL2 promoter polymorphism rs2279115 is not associated with BCL2 protein expression or patient survival in breast cancer patients.pdf
RESEARCH Open Access A BCL2 promoter polymorphism rs2279115 is not associated with BCL2 protein expression or patient survival in breast cancer patients Claire J Searle1,2, Ian W Brock1, Simon S Cross3, Sabapathy P Balasubramanian4, Malcolm WR Reed4 and Angela Cox1* Abstract The B-cell CLL/lymphoma 2 (BCL2) gene family encodes pro- and anti-apoptotic proteins that are critical regulators of programmed cell death. Higher levels of BCL2 expression in breast tumours have been shown to be an independent prognostic factor for improved survival from breast cancer. The promoter single nucleotide polymorphism (SNP) rs2279115 has been associated with both BCL2 expression and patient survival. The aim of this study was to attempt to replicate these observations in a cohort of 1015 UK women with breast cancer, and to compare genotype frequencies in cases and controls. In this study, 1015 breast cancer cases and 1034 control subjects were genotyped for the rs2279115 SNP by 5’ nuclease PCR. Paraffin embedded tumour tissue for 342 case subjects was assembled into tissue microarrays, and the level of expression of BCL2 was established by immunohistochemistry. Kaplan Meier survival curves and Cox Proportional Hazards models were used to examine the effect of genotype on patient survival. The effect of SNP genotype on tumour BCL2 protein levels and breast cancer susceptibility was assessed by logistic regression. In this study higher BCL2 expression was significantly associated with improved survival from breast cancer (p = 0.015), in keeping with previous reports. The SNP rs2279115 was not found to be associated with tumour expression of BCL2, (p = 0.77), and neither was it associated with case/control status (p = 0.25). There was no significant association between the SNP and overall survival (p = 0.75). In conclusion, we found that higher tumour BCL2 expression is associated with improved survival from breast cancer, in keeping with previous studies. However, in contrast to a previous report, the promoter SNP rs2279115 was not associated with BCL2 expression or overall survival from breast cancer. Keywords: Breast cancer, BCL2, rs2279115, Survival, SNP Background The balance between cell proliferation and levels of apoptosis is frequently disrupted in tumours, with tumorigenesis being promoted by both the loss of pro- apoptotic signals and the gain of anti-apoptotic mechan- isms (Hanahan & Weinberg 2000; Hanahan & Weinberg 2011). The BCL2 family of proteins plays a crucial role in these processes, by integrating the complex pathways incorporating pro- and anti-apoptotic signals at the mitochondrial membrane (Tsujimoto 2002). The BCL-2 family can be categorised into anti-apoptotic and two pro-apoptotic subgroups. The anti-apoptotic members include BCL2 and Bcl-xL. The pro-apoptotic members can be divided into a “multi-BH domain” group includ- ing Bax and Bak and a BH3-only subgroup (Adams & Cory 2002). However, BCL2 itself seems to act as both an oncogene and a tumour suppressor gene in different tumour types. For example, higher levels of tumour BCL2 expression are associated with poor patient sur- vival from chronic lymphocytic leukaemia (CLL), but with improved survival from breast and colon cancer (Faderl et al. 2002) (Buglioni et al. 1999) (Callagy et al. 2008). The BCL2 gene consists of three exons and two pro- moters; it is located on chromosome 18q21.3. The SNP (rs2279115) is located in the inhibitory P2 promotor of * Correspondence: a.cox@sheffield.ac.uk 1Department of Oncology, CR-UK/YCR Sheffield Cancer Research Centre, University of Sheffield Medical School, Beech Hill Road, Sheffield, UK Full list of author information is available at the end of the article a SpringerOpen Journal © 2012 Searle et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Searle et al. SpringerPlus 2012, 1:38 http://www.springerplus.com/content/1/1/38 the BCL2 gene (Park et al. 2004). The C allele in com- parison to the A allele displayed significantly increased inhibition of BCL2 promoter activity and binding of nu- clear proteins (Nuckel et al. 2007). In keeping with these findings BCL2 protein expression in B cells from CLL patients carrying the AA genotype was significantly increased compared with CC genotypes (Nuckel et al. 2007). This relationship was also demonstrated in rela- tion to lymph node negative breast cancer in one previ- ous study (Bachmann et al. 2007). In this study higher expression of BCL2 was associated with the A-allele (p = 0.044) and Kaplan-Meier survival analysis revealed a significant association of the AA genotype with improved survival (p = 0.030). This relationship has also been demonstrated in oropharyngeal squamous cell car- cinoma (Lehnerdt et al. 2009) where rs2279115 was sig- nificantly associated with BCL2 expression (p = 0.008) and with overall survival (p = 0.0247). This trend was also demonstrated in renal cancer (Hirata et al. 2009). Many studies have clearly demonstrated that increased BCL2 expression is associated with improved outcome from breast cancer (Yang et al. 2003) (Callagy et al. 2006) (Callagy et al. 2008). (Dawson et al. 2010) (Ali et al. 2012). A multivariate analysis incorporating five pub- lished studies from 11,212 breast cancer cases strongly supported the independent prognostic significance of BCL2 positivity with improved survival (Hazard Ratio (HR) 0.76, 95% Confidence Interval (CI) 0.54-0.74), p <0.001)(Dawson et al. 2010). In addition, expression of BCL2 has been proven to be an independent indicator of favourable prognosis for all types of early-stage breast cancer (Callagy et al. 2008; Dawson et al. 2010). The aim of this study was to use a cohort of breast cancer cases, from the Sheffield Breast Cancer Study (SBCS) to determine whether there is a relationship be- tween the promoter SNP rs2279115 and tumour protein levels of BCL2, and whether this corresponds to any dif- ferences in patient survival. We also confirmed the known association between high levels of tumour BCL2 and improved survival from breast cancer. Materials and methods Subjects Between November 1998 and January 2005, 1274 women with breast cancer and 1271 control subjects were en- rolled in the SBCS. The design and methodology of this case control study have been previously described (Rafii et al. 2002) (Azmy et al. 2004). Briefly, all subjects were residents of South Yorkshire, UK and were of European descent. The breast cancer cases all had histopathologic- ally confirmed breast cancer. The control subjects were women aged between 50 and 65 attending the Sheffield Mammography Screening Service between September 2000 and January 2004, whose mammograms showed no evidence of breast lesions. The study was approved by the South Sheffield Research Ethics Committee (SSREC/ 98/137), and the DNA samples were collected with informed consent from subjects for their use in genetic studies of cancer. Paraffin-embedded tumour tissue was requested from the relevant NHS Histopathology Arch- ive for 342 of the subjects recruited above. Pathological data (including tumour grade, morphology and lymph node status) were obtained from medical pathology records and validated (SSC). Immunohistochemical data for the oestrogen receptor (ER), progesterone receptor (PR), HER2 and cytokeratins 5/6 were available (Blows et al. 2010). Data on all-cause mortality and survival was obtained through the Trent Cancer Registry. Median follow-up for breast cancer cases in September 2009 was 21.6 years including 220 deaths. Determination of BCL2 rs2279115 genotype Blood DNA samples were available from 1015 breast cancer subjects and 1034 controls. These were geno- typed for the SNP rs2279115 using a Taqman 5’ nuclease PCR assay. The Probe sequence was as follows 5’- CTCCCCAGGAGAGAGACAGGGGAGA[G/T]GGGA CGATGAAGGAGCCGGGGACGG-3’, with the FAM probe containing T and the VIC probe containing G. The amplification reaction was performed in a final volume of 5 μL, with 1.0 μL of genomic DNA (10 ng), 0.125 μL of TaqMan™Genotyping Assay, 2.5 μL of Taqman Genotyping Master Mix, and 2.375 μL of water. The thermo- cycling conditions were as follows: 95°C for 10 min followed by 60 cycles of 92°C for 10 s and 60°C for 1 min. Allelic discrimination was carried out using the ABI 7900HT Sequence Detector (Life Technologies) The overall genotype call rate was 96% (980 cases and 981 controls successfully genotyped), and duplicate concordance based on 133 duplicate samples was 99.25%. The observed control genotype frequencies were consistent with Hardy Weinberg equilibrium (p = 0.76). BCL2 immunohistochemistry Tissue micro arrays were constructed from 342 archived paraffin embedded tumour samples from the cancer co- hort. Appropriate regions of tumour (judged by H.&.E staining) were selected from the blocks and 0.6 mm tripli- cate tissue cores were punched out from these regions using a custom precision instrument (Beecher Instrument Inc., Sun Prairie, US). These were then transferred into re- cipient paraffin blocks in a specific orientation. 5 μm sec- tions from the array blocks were dried, deparaffinised and rehydrated before blocking endogenous peroxidase with a solution of 2% hydrogen peroxide in methanol. The sec- tions were then subjected to antigen retrieval by micro- wave treatment in 10 Mm tri-sodium citrate. This was Searle et al. SpringerPlus 2012, 1:38 Page 2 of 8 http://www.springerplus.com/content/1/1/38 (Callagy et al. 2008) (Dawson et al. 2010). Anti-apoptotic BCL2 members act as repressors of apoptosis by block- ing the release of cytochrome c, whereas pro-apoptotic members act as promoters (Ghobrial et al. 2005). The contrasting effect on survival of tumour BCL2 expres- sion in breast cancer as opposed to non-Hodgkin lymph- oma may well be due to the importance of the careful equilibrium between tumour BCL2 protein expression and other pro-apoptotic members such as Bax, rather than on BCL2 tumour protein quantity alone (Reed 1997) (Cory et al. 2003). Unfortunately the exact mech- anism that underpins this difference is not fully under- stood. In vitro studies in a variety of different cell types have found that high levels of BCL2 protein expression in tumours can result in striking growth inhibition (Pietenpol et al. 1994). In human breast cancer cell lines there is an inverse correlation between the expression of BCL2 and mutant p53 and that this relationship could lead to down-regulation of BCL2 tumour protein expres- sion (Haldar et al. 1994). Other studies have suggested a function of BCL2 protein in lengthening the cell cycle (O’Reilly et al. 1996) (Knowlton et al. 1998) (Lipponen et al. 1995). The relationship between tumour BCL2 protein ex- pression and oestrogen has also been widely debated. It has been suggested that the intrinsic and extrinsic path- ways which make up the two main routes involved in breast cancer cell apoptosis regulation, are both induced when oestrogen binds to the oestrogen receptor. Both pathways result in the activation of caspase leading fi- nally to apoptosis (Lewis-Wambi & Jordan 2009). Leung and Wang found that a breast cancer cell line treated with the oestrogen 17β-oestradiol resulted in up- regulation of BCL2 mRNA and protein, but down- regulation of Bcl-x(L) mRNA and protein . They did not find this result with other sex hormones. They specu- lated that different members of the BCL2 family proteins may be regulated through different pathways and that these pathways may be modulated by 17β-oestradiol (Leung & Wang 1999). Tumour BCL2 protein expres- sion status has also been previously strongly associated with PR and ER expression (Nadler et al. 2008) (Lee et al. 1997). Our data are consistent with previous obser- vations that BCL2 is a strong independent prognostic marker for breast cancer survival (Dawson et al. 2010). The SNP rs2279115 has been associated with BCL2 expression in CLL and breast cancer from node negative patients (Bachmann et al. 2007). The study by Bachmann et al. found that higher expression of BCL2 was asso- ciated with the A-allele (P = 0.044) in lymph node negative patients only. This also corresponded to an improved survival in this group (HR (95% CI) 3.2 (1.03,9.93) p = 0.044). Lymph node negative patients who were homozygous for the C allele had a higher risk of death than AA homozygous patients, with heterozygous women being intermediate in risk. In the present data we found no association between rs2279115 and tumour expression of BCL2 in the whole cohort, or when results were subdivided into Table 2 Level of BCL2 protein expression according to lymph node status, tumour grade, morphology, ER, PR, HER2 and CK5/6 status Low BCL2 n (%) High BCL2 n (%) p valuea Node status No nodal Involvement 20 (64.5) 140 (70.0) Nodal Involvement 11 (35.5) 60 (30.0) 0.54 Grade 1 3 (8.8) 51 (24.9) 2 7 (20.6) 114 (55.6) 3 24 (70.6) 40 (19.5) 4x10-9 Morphology Ductal 28 (80.0) 159 (75.7) Lobular 2 (5.7) 22 (10.5) Other 5 (14.3) 29 (13.8) 0.68 ER status Negative 27 (79.4) 31 (15.3) Positive 7 (20.6) 171 (84.7) 1x10-14 PR status Negative 20 (62.5) 53 (26.6) Positive 12 (37.5) 146 (73.4) 5x10-5 HER2 status Negative 29 (82.9) 193 (92.3) Positive 6 (17.1) 16 (7.7) 0.07 CK5/6 status Negative 23 (67.6) 180 (90.5) Positive 11 (32.4) 19 (9.5) 0.0002 BCL2 immunohistochemistry scores were grouped into low (scores 0–1) and high (scores 2–3) a Pearson χ2 test. Table 3 Level of BCL2 protein expression of according to rs2279115 genotype Genotype Low BCL2 n (%) High BCL2 n (%) Odds Ratio 95% CI p value AA 12 (13.3) 78 (86.7) 1.00 AC 19 (15.6) 103 (84.4) 0.83 0.38 1.82 0.65 CC 4 (11.1) 32 (88.9) 1.23 0.37 4.10 0.74 TOTAL 35 (100) 213 (100.0) BCL2 immunohistochemistry scores were grouped into low (scores 0–1) and high (scores 2–3). Table 4 Genotype frequencies for SNP rs2279115 in case and control subjects Genotype controls n (%) cases n (%) Odds Ratio 95% CI p value AA 290 (29.6) 314 (32.0) 1.00 AC 475 (48.4) 477 (48.7) 0.93 0.76 1.14 0.47 CC 216 (22.0) 189 (19.3) 0.81 0.63 1.04 0.098 TOTAL 981 (100) 980 (100.0) Searle et al. SpringerPlus 2012, 1:38 Page 6 of 8 http://www.springerplus.com/content/1/1/38 patient with lymph node positive or lymph node negative disease. We also found no association with survival for the different genotypes. Assuming a base- line survival proportion of 0.84 in lymph node nega- tive cases, our study would have been expected to detect a hazard ratio of 3.2 between homozygous gen- otypes (as was found by Bachmann et al. 2007), hav- ing 80% power to detect hazard ratio of 1.8. However, we are unable to exclude effects smaller than this. It is possible that there may be genotypic effects on sur- vival of similar or smaller magnitude to those of BCL2 expression (Callagy et al. 2008; HR = 1.64); this study is underpowered to detect these. In conclusion we have no evidence to support the SNP rs2279115 as a prognostic biomarker for breast cancer patients. Higher BCL2 expression has been Figure 4 Kaplan-Meier survival functions according to rs2279115 genotype. Overall survival based on 934 case subjects with total time at risk 6765.43 years. Numbers at risk at the end of the 10-year analysis period were 158 (AA), 221 (AC), and 102 (CC). Hazard ratio (95% CI) 1.03 (0.86, 1.24), p = 0.75. Figure 5 Kaplan-Meier survival functions in lymph node negative and positive subjects according to rs2279115 genotype. Overall survival based on 786 subjects with total time at risk 5808.75 years. A shows lymph node negative subjects and B shows lymph node positive subjects. Numbers at risk at the end of the 10-year analysis period were A: 103 (AA), 146 (AC), 79 (CC) and B: 38 (AA), 54 (AC), 18 (CC). Hazard ratios (95% CI) for A were 0.97 (0.73, 1.30), p = 0.85 and for B were 1.20 (0.88, 1.64), p = 0.24. Searle et al. SpringerPlus 2012, 1:38 Page 7 of 8 http://www.springerplus.com/content/1/1/38 conclusively proven to correlate with improved survival and further studies are required to explore its use as a prognostic indicator. Competing interest The authors declare that they have no competing interests. Authors’ contribution The study was designed by AC and MWR. Patient diagnosis and recruitment, and clinical data collection was carried out by MWR and SPB. Pathology data collection and generation of tissue microarrays was carried out by SSC. Genotyping, immunohistochemistry, and scoring of the immunohistochemical data was carried out by CJS and IWB. CJS and AC performed statistical analyses and CJS and AC drafted the manuscript. All authors read and approved the final manuscript. Ethical standards All experiments completed as part of this study comply with the current laws in the United Kingdom. Acknowledgements This project was supported by Yorkshire Cancer Research awards S295 and S299. We would like to thank all the women who took part in the SBCS, and Helen Cramp and Dan Connley for patient recruitment and data management respectively. CJS was funded by an Academic Clinical Fellowship from the UK National Institute of Health Research. Author details 1Department of Oncology, CR-UK/YCR Sheffield Cancer Research Centre, University of Sheffield Medical School, Beech Hill Road, Sheffield, UK. 2Department of Clinical Genetics, Chapel Allerton Hospital, Chapeltown Road, Leeds, UK. 3Academic Unit of Pathology, Department of Neuroscience, University of Sheffield Medical School, Beech Hill Road, Sheffield, UK. 4Academic Unit of Surgical Oncology, CR-UK/YCR Sheffield Cancer Research Centre, University of Sheffield Medical School, Beech Hill Road, Sheffield, UK. Received: 12 September 2012 Accepted: 17 September 2012 Published: 23 October 2012 References Adams J, Cory S (2002) The BCL2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2:647–656 Ali H et al (2012) A Ki67/BCL2 index based on immunohistochemistry is highly prognostic in ER-positive breast cancer. 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Science 226(4678):1097–1099 Yang Q et al (2003) Prognostic value of Bcl-2 in invasive breast cancer receiving chemotherapy and endocrine therapy. Oncol Rep 10:121–125 doi:10.1186/2193-1801-1-38 Cite this article as: Searle et al.: A BCL2 promoter polymorphism rs2279115 is not associated with BCL2 protein expression or patient survival in breast cancer patients. SpringerPlus 2012 1:38. Searle et al. SpringerPlus 2012, 1:38 Page 8 of 8 http://www.springerplus.com/content/1/1/38
Title: A BCL2 promoter polymorphism rs2279115 is not associated with BCL2 protein expression or patient survival in breast cancer patients ​
Authors: Claire J Searle, Ian W Brock, Simon S Cross, Sabapathy P Balasubramanian, Malcolm WR Reed, Angela Cox ​

Publisher: SpringerPlus
Date: 23 October 2012 ​
Abstract: The B-cell CLL/lymphoma 2 (BCL2) gene family encodes pro-and anti-apoptotic proteins that are critical regulators of programmed cell death. ​ Higher levels of BCL2 expression in breast tumours have been shown to be an independent prognostic factor for improved survival from breast cancer. ​ The promoter single nucleotide polymorphism (SNP) rs2279115 has been associated with both BCL2 expression and patient survival. ​ The aim of this study was to attempt to replicate these observations in a cohort of 1015 UK women with breast cancer, and to compare genotype frequencies in cases and controls. ​ In this study, 1015 breast cancer cases and 1034 control subjects were genotyped for the rs2279115 SNP by 5’ nuclease PCR. ​ Paraffin embedded tumour tissue for 342 case subjects was assembled into tissue microarrays, and the level of expression of BCL2 was established by immunohistochemistry. ​ Kaplan Meier survival curves and Cox Proportional Hazards models were used to examine the effect of genotype on patient survival. ​ The effect of SNP genotype on tumour BCL2 protein levels and breast cancer susceptibility was assessed by logistic regression. ​ In this study higher BCL2 expression was significantly associated with improved survival from breast cancer (p = 0.015), in keeping with previous reports. ​ The SNP rs2279115 was not found to be associated with tumour expression of BCL2, (p = 0.77), and neither was it associated with case/control status (p = 0.25). ​ There was no significant association between the SNP and overall survival (p = 0.75). ​ In conclusion, we found that higher tumour BCL2 expression is associated with improved survival from breast cancer, in keeping with previous studies. ​ However, in contrast to a previous report, the promoter SNP rs2279115 was not associated with BCL2 expression or overall survival from breast cancer. ​