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In Sweden, approximately 7000 women are diagnosed with breast cancer annually and 1500 die of their disease. Up to 25% of breast cancer patients considered to be at low risk for recurrence, that is, stage-I or II without lymph node involvement, recur within 5 years (Malmström et al, 2001, 2003). Approximately 15% of all breast cancer patients in Sweden die from their disease within 5 years and 30% die within 10 years of diagnosis. Adjuvant therapies such as radiation, tamoxifen, aromatase inhibitors (AIs), and chemotherapy improve the prognosis, but also confer a risk of adverse side effects (Early breast cancer trialists' collaborative group, 2005; Forbes et al, 2008). Moreover, many patients receive adjuvant therapy without any impact on survival, as most are already cured by surgery alone or the adjuvant therapy chosen does not work as intended. Markers, which would help to better tailor adjuvant therapy to each patient, are urgently needed.

Several genetic polymorphisms in genes such as cytochrome-P450 (CYP)2C8 and CYP2C9, may influence survival after cancer diagnosis due to their role in the metabolism of various breast cancer drugs, including tamoxifen and chemotherapy (** et al, 2005). CYP2C8 and CYP2C9 are polymorphic enzymes. CYP2C8*3 and CYP2C9*2 are the major variant alleles in Caucasian populations (Yasar et al, 2002). Approximately 96% of subjects with the CYP2C8*3 allele also carried a CYP2C9*2 and 85% of subjects who had the CYP2C9*2 variant also carried a CYP2C8*3. CYP2C8*3 is defective in the metabolism of two important CYP2C8 substrates: the anticancer drug paclitaxel (Bahadur et al, 2002) and the physiologically important compound arachidonic acid (AA) (Dai et al, 2001). In addition, variants CYP2C8*4 and CYP2C9*2 and CYP2C9*3 also have a lower metabolic activity than the wild-type variants (Bahadur et al, 2002; Griskevicius et al, 2003; King et al, 2004; Sandberg et al, 2004).

Arachidonic acid is metabolised via three major pathways: the cyclooxygenase pathway, which produces prostaglandins; the lipoxygenase pathway, and finally the CYP epoxygenase pathway (Belton and Fitzgerald, 2003).

CYP2C8 and 2C9 are CYP epoxygenases, which metabolise AA to epoxyeicosatrienoic acids (EETs) (Zeldin et al, 1995; Michaelis et al, 2005), with the most abundant product being 14,15-EET, which promotes angiogenesis in vivo (Medhora et al, 2003). In vitro studies have shown that overexpression of CYP2C9 elicits angiogenesis via activation of the epidermal growth factor receptor (EGFR) (Michaelis et al, 2003). Yasar et al (1999) from a Swedish population of 430 unrelated healthy people.

This is the first study to report an association between decreased odds for lymph node involvement and CYP2C8*4 and an over twofold increased odds for lymph node involvement among CYP2C8/9 *3/*1/*2/*1 carriers with invasive tumour sizes over 20 mm. CYP2C8*3 and CYP2C8*4 have both been reported to have lower metabolic activity than the wild type (Bahadur et al, 2002), but the associations between these polymorphisms and axillary nodal involvement went in opposite directions in the current study. Since CYP2C8*3 was in strong LD with CYP2C9*2, it is likely that the association between CYP2C8*3 and nodal status is actually reflecting the association between the CYP2C9*2 polymorphism and nodal status.

We found that an increasing number of CYP2C8/9 *3/*1/*2/*1 haplotype alleles and especially CYP2C8*3 alleles was associated with increased hazard of early breast cancer-related events in tamoxifen-treated patients. Tamoxifen is a moderate CYP2C8 inhibitor (Walsky et al, 2005), a CYP2C9 substrate (** et al, 2005), and significantly inhibits CYP2C9 activity in breast cancer patients (Boruban et al, 2006). CYP2C9 is involved in tamoxifen activation, although neither CYP2C9*2 nor CYP2C9*3, which have lower activity than the wild type, were significantly associated with the levels of the potent tamoxifen metabolite endoxifen in one study (** et al, 2005). The effect of CYP2C8 polymorphisms on endoxifen levels has, to our knowledge, not been investigated. Since the LD between CYP2C8*3 and CYP2C9*2 was incomplete, we also examined the effect of each SNP separately in the model, and while CYP2C8*3 was associated with significant increased hazard of early breast cancer-related events, CYP2C9*2 was associated with non-significant decreased hazard. If our finding is replicated, the CYP2C8*3 may be used to identify patients who may recur early when treated with tamoxifen and who should be offered additional or different treatment.

Currently, no genotype data are used prior to selection of tamoxifen or aromatase inhibitors for breast cancer patients with ER-positive tumours warranting adjuvant endocrine treatment. CYP2D6 has been shown to significantly affect the levels of endoxifen and clinical outcome after tamoxifen treatment (** et al, 2005; Goetz et al, 2008). To our knowledge there is no association between CYP2D6 and CYP2C8/9, and these genes are located on different chromosomes. It is, therefore, unlikely that the increased risk for early recurrences observed in CYP2C8*3 carriers would be explained by CYP2D6 polymorphisms. Schroth et al. studied CYP2D6, CYP2C19, CYP3A5, CYP2B6, and CYP2C9 in tamoxifen-treated women. They reported that the CYP2C19*17 allele partly compensates for non-functioning CYP2D6 alleles and showed that carriers of one CYP2D6-null allele could be further stratified according to their CYP2C19*17 genotype with respect to tamoxifen response (Schroth et al, 2007). The genotypes of CYP3A5, CYP2B6, and CYP2C9 were not significantly associated with tamoxifen response, which is in line with our finding with respect to CYP2C9*2 and CYP2C9*3, but they did not examine CYP2C8.

The St Gallen guidelines recommend that extensive peritumoral vascular invasion be used as a prognostic factor (Goldhirsch et al, 2007). The degree of angiogenesis is not yet routinely evaluated in the clinical setting in Sweden, and we could, therefore, not evaluate whether there was any association between peritumoral vascular invasion and CYP2C8/9 haplotypes. It is plausible that we could have found an association, since CYP2C9-derived EETs stimulate angiogenesis (Michaelis et al, 2003). CYP2C9-derived EETs do so by a mechanism involving activation of the EGFR (Michaelis et al, 2003). One study reported high expression of EGFR to be inversely correlated with nodal metastases and shorter, distant disease-free survival in a group of breast cancer patients who had received 2 years of adjuvant tamoxifen treatment (Dihge et al, 2008). This is not fully compatible with our finding, as we found both a higher frequency of nodal involvement with the CYP2C8/9 *3/*1/*2/*1 genotype (in tumours larger than 20 mm) and a higher risk of any type of breast cancer recurrence, especially in women treated with tamoxifen. In small tumours, angiogenesis is also a predictor of nodal status (Arisio et al, 2000), and it is thus possible that women with the CYP2C8/9 *3/*1/*2/*1 genotype have tumours that are more likely to metastasise through increased angiogenesis.

The enzyme activity of CYP2C8 and CYP2C9 is not only determined by polymorphic variants, but also by use of CYP2C8- and CYP2C9-inhibiting compounds, including several drugs, as reviewed by Ingelman-Sundberg et al (2007). In the present study we enquired only about the use of all concomitant medications during the past week and lack information on long-term use of any of the medications. However, this information is not relevant in relation to tumour characteristics as the tumours were formed many years prior to diagnosis.

HER-2/neu status was not analysed routinely before November 2005, and we have therefore not been able to evaluate the correlation between HER-2 overamplification and CYP2C8/9 haplotypes. Histological grade was assessed according to the procedure of Elston and Ellis (1991). In the current study histological grade was not associated with early recurrences in either group (data not shown), but the median follow-up time is still short.

Our material consisted of a series of primary breast cancer patients, where the only exclusion criteria were any previous breast cancer diagnosis and other cancer diagnosed within the past 10 years. Approximately 60% of the patients operated on in Lund and 46% of the patients operated on in Helsingborg during the study's enrolment period were included. Our sample from Lund was similar to all patients from Lund with respect to age and ER and PR status. The patients from Helsingborg were somewhat older and had fewer PR-positive tumours as compared with the region as a whole, while the subset of patients who were included in this study was comparable to those included from Lund and the whole South Swedish region with respect to age and hormone-receptor status. There are several potential explanations for why receptor status may be different in Helsingborg patients. The patients in Helsingborg are somewhat older and different sets of antibodies are used at different Departments of pathology. ER and PR status was independently re-assessed by 22 pathologists from nine hospitals and the kappa values were 0.78 for ER and 0.72 for PR in 2003 (Chebil et al, 2003). Provided that these markers are used for selection of breast cancer treatment, quality assurance is ongoing. The frequency of HRT use may also differ between the two cities and HRT treatment is associated with a higher frequency of ER-positive tumours (Glass et al, 2007). As patients who were included did not differ with respect to ER, PR, or prior HRT use, we were unable to explain why patients from Helsingborg in general would have fewer PR-positive tumours.

In conclusion, we found that the frequencies of CYP2C8*3 and CYP2C8*4, and CYP2C9*2 and CYP2C9*3 were comparable to those of healthy European populations. Each copy of the CYP2C8/9 *1/*4/*1/*1 allele was associated with significantly lower risk for nodal involvement, while each copy of the CYP2C8/9 *3/*1/*2/*1 allele was associated with increased risk for nodal involvement in tumours larger than 20 mm. Moreover, the CYP2C8*3 allele was associated with early breast cancer-related events in women treated with tamoxifen. Since this is the first study reporting an association between CYP2C8 and tamoxifen response, and the median follow-up time is still short, the finding warrants confirmation. If confirmed, it is possible that CYP2C8*3 can be used as a genetic marker for prediction of treatment response to tamoxifen.