Association of CYP 2 C 19 Polymorphisms with Survival of Breast Cancer Patients Using Tamoxifen : Results of a Meta-analysis

Breast cancer is a well-known important disease which is health-related, and its incidence is still rising (Darakhshan et al., 2013; Donnelly et al., 2013). Over 14% of female death is due to breast cancer, which is the leading cause of death in women (Parkin et al., 2005; Ibrahim et al., 2012). However, approximately 30% – 50% of estrogen receptor (ER)-positive breast cancer patients do not respond to tamoxifen treatment (Osborne et al., 1998). Tamoxifen is the most commonly prescribed and widely used treatment for the prevention of ER-positive breast cancer (Fisher et al., 2005; Colleoni et al., 2006). It is metabolized by the pathway: the pro-drug, tamoxifen, is converted into N-desmethyl tamoxifen, 4-hydroxy-tamoxifen (4-HT) and the 4-hydroxy-Ndesmethyltamoxifen (endoxifen), which is the result of the oxidation of the N-desmethyl tamoxifen, 4-hydroxytamoxifen (4-HT) (Desta et al., 2004). The evidence suggests that the metabolites 4-hydroxy-tamoxifen (4-HT) and 4-hydroxy-N-desmethyltamoxifen (endoxifen) are the active metabolites. They have at least 100-fold higher


Introduction
Breast cancer is a well-known important disease which is health-related, and its incidence is still rising (Darakhshan et al., 2013;Donnelly et al., 2013).Over 14% of female death is due to breast cancer, which is the leading cause of death in women (Parkin et al., 2005;Ibrahim et al., 2012).However, approximately 30% -50% of estrogen receptor (ER)-positive breast cancer patients do not respond to tamoxifen treatment (Osborne et al., 1998).

Association of CYP2C19 Polymorphisms with Survival of Breast Cancer Patients Using Tamoxifen: Results of a Metaanalysis
Lan Bai 1& , Juan He 1& , Gong-Hao He 1 , Jian-Chang He 1 , Fan Xu 2 , Gui-Li Xu 1 * potency, in terms of binding to ER and suppression of breast cancer cell proliferation (Stearns et al., 2003;Desta et al., 2004;Johnson et al., 2004).
To date, we explored potential effect outcomes of the association between CYP2C19*2, CYP2C19*17, tamoxifen and survival of breast cancer.However, the results were often conflicted and based on small sample size of the cohorts.Therefore, we conducted this metaanalysis to explore a possible estimation of the associations.

Search strategy
Eligible articles were identified by a search of PubMed and Embase database using the following keywords: "breast cancer", "tamoxifen", "CYP2C19" and the last search updated on February 18, 2013.There was no limitation to the language restrictions.Furthermore, to find the additional studies, we also used the referenced lists of all the relevant studies.

Selection criteria
Studies were included if they met the following criteria for further meta-analysis: (1) patients who received tamoxifen with breast cancer; or (2) evaluated the function of CYP2C19*2 or CYP2C19*17; and (3) assessed diseasefree (DFS), recurrence-free (RFS) or OS.

Data extraction
Two investigators (Lan Bai and Juan He) independently performed data according to the included studies.The following information was extracted from each study: first author's surname, year of publication, study location, participants, genotypes compared, and the data of diseasefree survival (DFS) and overall survival (OS).

Assessment of study quality
Study quality was assessed by the Newcastle-Ottawa Scale (Stang et al., 2010).

Statistical analysis
Summary hazard ratios (HRs) with their 95% confidence interval (CI) were used to assess the association between the CYP2C19 genotype and the survival in breast cancer patients.It was the dichotomous data.The I 2 statistic and the p value were used to assess between-study statistical heterogeneity (Higgins et al., 2003).The data of OR were computed using the random effects model.DFS and OS were defined as the primary outcomes and OR as secondary outcome.For evaluating the influence of the results, one-way was performed to evaluate the sensitivity analyses with the influence of individual study on pooled estimates.We performed subgroup analysis by genotypes.The potential publication bias was tested using Egger's funnel plot with pseudo 95% confidence limits.
All meta-analyses were conducted with STATA version 12.0 (StataCorp LP, College Station, TX, USA).
Of all the above, six studies were published as full papers (Werner et al., 2007;Masatsugu et al., 2008;Rikje et al., 2010;Ann et al., 2011;Ron et al., 2011;Beelen et al., 2013).Sufficient data were available according to the studies.Therefore, there was no need to contact specific authors.
There was significant between-study heterogeneity (I 2 =68.3%, p=0.013, Figure 2) for the analysis of five studies in DFS.Furthermore, there was moderate heterogeneity of OS (I 2 =29.7%, p=0.233, Figure 3).Overall, we did not reveal the evidence of obvious asymmetry from the shapes of the funnel plots.

Association between CYP2C19 genotype and OS
We pooled two studies (Werner et al., 2007;Rikje et al., 2010) for this analysis.The data of HRs ranged from 0.26 to 0.61 in the studies.Pooling of data from two studies did not show toward to increase survival of disease with an OR of 0.46 (95% CI=0.21 -1.01, Figure 3).With a moderate heterogeneity (I 2 =29.7%, p=0.233).

Discussion
The overall results of this meta-analysis focused on the function of CYP2C19 genotype for survival of the breast cancer patients using tamoxifen.No meta-analysis has been performed, and our meta-analysis is the first one on this association.In addition, a sensitivity analysis was performed by removing one study at a time to reflect the influence after excluding the individual study.Estimates changed quite little, suggesting that the result is stable in this meta-analysis.
The exact biological mechanism of the function of CYP2C19 is understood.Previous studies suggest that CYP2C19 is involved in the conversion of tamoxifen to its active metabolites (Crewe et al., 2002;Boocock et al., 2002;Stearns et al., 2003;Desta et al., 2004;Jin et al., 2005).4-HT has estrogen receptor more markedly affinity than tamoxifen (Johnson et al., 2004).Furthermore, prior studies suggest that 4-HT is metabolized by CYP2C19, which is the main metabolized product.These findings (Ruiter et al., 2009;Justenhoven et al., 2009;Lu et al., 2012) about the common associations of CYP2C19 and tamoxifen suggest that CYP2C19 genotypes play an important role in women suffering from breast cancer.It is well documented that the patients who are postmenopausal ER positive would get more benefit from receiving tamoxifen than pre-menopausal ones (Sukasem et al., 2012).
In this meta-analysis, significant between-study heterogeneity was detected (I 2 = 68.3%,p = 0.013) for the analysis of five studies in DFS.Furthermore, there was moderate heterogeneity of OS (I 2 = 29.7%,p = 0.233).The results can be influenced by the ways of the experiment of the studies, the groups of the population, environmental exposures and sample size.As a measure of heterogeneity between these confounding factors or some unknown factors.Due to limited number of studies and sufficient information heterogeneity, our ability was limited to detect heterogeneity by subgroup analysis.Heterogeneity was assessed by I 2 statistic among studies, which is defined for low (< 25%), moderate (~ 50%), and high (> 75%) heterogeneity (Higgins et al., 2003).It is possible that other limitations of these studies may partially contribute to the heterogeneity.For this reason, the random-effect model (DerSimonian Laird method) was conducted when there exists a significant I 2 >50%.In our meta-analysis, Funnel plot and Egger's test were used to test the publication bias.The results suggest that the publication bias affects on the results in this study and the results of our meta-analysis are not relatively stable.
Our meta-analysis suggested that the CYP2C19 genotype was associated with DFS, but not associated with OS.This result indicated that the CYP2C19 genotype is associated with the survival in breast cancer patients, which are consistent with previous studies.Zafra-Ceres et al. (Zafra-Ceres et al., 2013) demonstrated that CYP2C19*2 can serve as a predictive factor for survival in breast cancer patients treated with tamoxifen in the Spanish population (Schaik et al., 2011).Ruiter et al. (Ruiter et al., 2010) also suggests that CYP2C19 genotype may possibly be a predictive factor for survival in breast cancer patients after using tamoxifen.In the current study, we also performed subgroup analyses based on genotype, to further evaluate the value of CYP2C19 genotype for survival in breast cancer patients who received tamoxifen.It has even been suggested that CYP2C19 genotype may possibly be a predictive factor for survival in breast cancer patients who used tamoxifen.

Limitations of the Meta-analysis
Other potential limitations should be taken into account in this meta-analysis.First, the selection bias were not avoided since our study was reported in English and identified in PubMed and Embase.Second, the studies of the meta-analysis were often small-scale, and patient selection bias may be possible.Third, publication bias was not investigated.Although this bias can be estimated using funnel plot in STATA.Thus, the likelihood of bias from published studies should be minimized.
In summary, this meta-analysis explores the association between CYP2C19 genotypes and the survival of breast cancer patients.Our results supporting the association of the CYP2C19*2 and CYP2C19*17 genotypes may possibly be a predictive factor for survival in breast cancer women.Previous Meta-analysis have not found a similar association between them.Because of the between-study heterogeneity in small size of cohorts, further larger scale studies should focus on investigating.There is an evidence suggesting that CYP2C19 genotypes might be useful in predicting tamoxifen efficacy in post-menopausal women.The ER positive postmenopausal patients with CYP2C19*17 variant will benefit from tamoxifen.Our meta-analysis supplies the way of the potential effect outcomes of the association between CYP2C19*2, CYP2C19*17, tamoxifen and survival of breast cancer patients.Hopefully, further studies can verify the results to clinical relevance in a larger population.
Figure 2. Forest Plot of Meta-Analysis of Disease-Free Survival