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- Materials and methods
- Disclosure Statement
- Supporting Information
The purpose of the present study was to investigate the association of glutathione S-transferase P1 (GSTP1) expression with resistance to neoadjuvant paclitaxel followed by 5-fluorouracil/epirubicin/cyclophosphamide (P-FEC) in human breast cancers. The relationship of GSTP1 expression and GSTP1 promoter hypermethylation with intrinsic subtypes was also investigated. In this study, primary breast cancer patients (n = 123, stage II–III) treated with neoadjuvant P-FEC were analyzed. Tumor samples were obtained by vacuum-assisted core biopsy before P-FEC. GSTP1 expression was determined using immunohistochemistry, GSTP1 promoter methylation index (MI) using bisulfite methylation assay and intrinsic subtypes using DNA microarray. The pathological complete response (pCR) rate was significantly higher in GSTP1-negative tumors (80.0%) than GSTP1-positive tumors (30.6%) (P = 0.009) among estrogen receptor (ER)-negative tumors but not among ER-positive tumors (P = 0.267). Multivariate analysis showed that GSTP1 was the only predictive factor for pCR (P = 0.013) among ER-negative tumors. Luminal A, luminal B and HER2-enriched tumors showed a significantly lower GSTP1 positivity than basal-like tumors (P = 0.002, P < 0.001 and P = 0.009, respectively), while luminal A, luminal B and HER2-enriched tumors showed a higher GSTP1 MI than basal-like tumors (P = 0.076, P < 0.001 and P < 0.001, respectively). In conclusion, these results suggest the possibility that GSTP1 expression can predict pathological response to P-FEC in ER-negative tumors but not in ER-positive tumors. Additionally, GSTP1 promoter hypermethylation might be implicated more importantly in the pathogenesis of luminal A, luminal B and HER2-enriched tumors than basal-like tumors. (Cancer Sci 2012; 103: 913–920)
Neoadjuvant chemotherapy (NAC) for primary breast cancer patients is known to enhance the operability of patients with advanced tumors previously considered inoperable, as well as making breast-conserving surgery more feasible for patients for whom such surgery was previously not feasible due to large tumor size. In addition, it is well established that patients who show a pathological complete response (pCR) to NAC can have a better prognosis than those who do not,[1-3] so the response to NAC can provide valuable information regarding patient prognosis. These advantages of NAC have led to its widespread use including recently for a growing number of breast cancer patients. However, pCR rates for NAC of only 20–30% of patients are still rather low. Because adverse effects of various degrees of severity are seen in virtually all patients, it seems to be very important to develop predictive factors for the response to NAC to avoid the unnecessary use of NAC for patients who are unlikely to derive benefits from such therapy.
Among predictive factors, estrogen receptor (ER), progesterone receptor (PR), HER2, histological grade (HG) and Ki-67 have been most extensively studied and significant associations of ER negativity, PR negativity, HER2 amplification, high histological grade or high Ki-67 labeling index (LI) with high pCR rates have been reported.[5, 6] In addition, intrinsic subtypes of breast tumors classified originally by molecular profiling and later with a simpler method (immunohistochemistry using various markers) have recently been shown to be associated with pCR.[7, 8] Moreover, identification of a few multi-gene classifiers for prediction of pCR based on DNA microarray analysis has been reported.[9, 10] However, the accuracy of these predictive factors and multi-gene classifiers is still not satisfactory so that more accurate and clinically useful predictive factors need to be developed.
Glutathione S-transferase P1 (GSTP1), which belongs to phase two metabolic enzymes, is instrumental in the detoxification of toxic substances and anticancer drugs by conjugating them with glutathione. Moreover, GSTP1 reportedly inhibits the chemotherapy-induced apoptosis by its direct interaction with the C-terminal of JNK.[12, 13] GSTP1 expression in tumor cells can thus be expected to be associated with resistance to chemotherapy. In fact, several in vitro studies using various human cancer cell lines have indicated that GSTP1 expression is associated with resistance to chemotherapy.[14, 15] As for human breast cancers, GSTP1 expression was found to be associated with resistance to 5-fluorouracil (5-FU), doxorubicin and mitomycin C, as well as to paclitaxel and docetaxel. However, conflicting results have also been reported,[18, 19] so that the relationship between GSTP1 expression and response to chemotherapy remains to be ascertained. Furthermore, even though the standard neoadjuvant chemotherapeutic regimen currently consists of a sequential taxane and anthracycline-based regimen, the predictive value of GSTP1 expression for this standard regimen is yet to be studied.
In the present study, we investigated the association of GSTP1 expression with response to neoadjuvant sequential paclitaxel and anthracycline-based chemotherapy. In addition, we also investigated the association of GSTP1 promoter hypermethylation with GSTP1 protein expression according to intrinsic subtypes since the former is reportedly associated with GSTP1 protein expression and intrinsic subtypes.
- Top of page
- Materials and methods
- Disclosure Statement
- Supporting Information
The main purpose of the present study was to investigate whether GSTP1 expression was associated with resistance to P-FEC in human breast cancers since GSTP1 is thought to be implicated in chemoresistance through the detoxification of chemotherapeutic agents and inhibition of chemotherapy-induced apoptosis. First, we investigated the relationship of GSTP1 expression with various clinicopathological parameters and found that GSTP1 expression was significantly associated with ER negativity and PR negativity, findings that are consistent with those previously reported.[18, 31] In addition, GSTP1-positive tumors tended to be low histological grade (HG1) in ER-positive tumors but not in ER-negative tumors. Because GSTP1 promoter hypermethylation has been shown to play an important role in silencing GSTP1, we investigated the relationship between this hypermethylation and GSTP1 expression. We were able to show that GSTP1-positive tumors were significantly less likely than GSTP1-negative tumors to possess GSTP1 promoter hypermethylation, while ER-negative and PR-negative tumors were less likely than their opposites to possess GSTP1 promoter hypermethylation, confirming its important role in silencing GSTP1. Interestingly, GSTP1 MI was significantly higher in HG2 tumors than HG1 tumors in ER-positive tumors, while there was no association between GSTP1 MI and HG in ER-negative tumors. These results seem to suggest that GSTP1 hypermethylation plays an important role in the pathogenesis of ER-positive tumors with relatively high histological grade (HG2). This is consistent with the hypothesis that loss of GSTP1 expression caused by promoter hypermethylation results in the diminished detoxification of DNA-damaging estrogen metabolites such as E2-2,3-Q and E2-3,4-Q[32, 33] and in the development of breast tumors with relatively high histological grade.
Next, we investigated the relationship between GSTP1 expression and clinical response (reduction rate) to paclitaxel or P-FEC. We were able to show that GSTP1 expression was associated with a poor response to paclitaxel as well as to P-FEC in both ER-positive and ER-negative tumors, indicating that GSTP1 plays a significant role in the suppression of antitumor activity of P-FEC irrespective of ER status. In contrast, univariate and multivariate analysis of the pathological response to P-FEC showed that only GSTP1 expression was significantly associated with a lower pCR rate in ER-negative tumors but not in ER-positive tumors. It has been well established that the pCR rate of ER-positive tumors in response to NAC is lower than that of ER-negative tumors, and the pCR rate was in fact significantly lower in ER-positive tumors (11.7%) than ER-negative tumors (41.3%) in the present study also (P < 0.001). Thus, a low pCR rate might mask the impact of GSTP1 on the resistance to P-FEC in ER-positive tumors. It is also possible that ER-positive tumors possess other important resistant mechanisms than GSTP1, which together determine the overall pathological response.
We also investigated the relationship between GSTP1 expression or GSTP1 promoter hypermethylation and intrinsic subtypes. We found that luminal A, luminal B and HER2-enriched tumors were more likely to harbor GSTP1 promoter hypermethylation than basal-like tumors, which suggests that GSTP1 promoter hypermethylation might be implicated more importantly in the pathogenesis of luminal A, luminal B and HER2-enriched tumors than basal-like tumors. It is suggested that luminal A, luminal B and HER2-enriched tumors originate from luminal progenitor cells.[34, 35] Thus, it is speculated that loss of GSTP1 expression results in DNA damage caused by estrogen metabolites,[32, 33] and that luminal progenitor cells, which are thought to be ER positive, are more frequently affected since ER-positive cells are considered to have a higher concentration of estrogens.
Another interesting finding of the present study being consistent with a previous report was that GSTP1 promoter hypermethylation was rarely observed in basal-like tumors, while most of the basal-like tumors expressed GSTP1. Recently, Honeth et al. reported that basal-like tumors had a breast cancer stem cell-like phenotype. These results seem to suggest that breast cancer stem cells feature high GSTP1 expression. Indeed, Croker and Allan very recently reported that GSTP1 was strongly expressed in breast cancer stem cells (ALDH1+/CD44+). Because basal-like tumors show a higher Ki-67 LI than HER2-enriched tumors and a high Ki-67 LI is well known to be associated with a good response to chemotherapy, basal-like tumors could be expected to show a higher pCR rate than HER2-enriched tumors, but in fact we found that the reverse was true. It seems that this lower pCR rate of basal-like tumors can be partially explained by their higher GSTP1 expression.
In conclusion, we found that GSTP1 expression was associated with a lower clinical response to P-FEC irrespective of ER status and with a lower pCR rate of ER-negative but not ER-positive tumors. GSTP1 seems to have the potential to be used for clinical identification of, especially, ER-negative breast tumors, which are unlikely to derive benefits from chemotherapy (P-FEC). In addition, our results might suggest that GSTP1 promoter hypermethylation is implicated more importantly in the pathogenesis of luminal A, luminal B and HER2-enriched tumors than basal-like tumors. Our observations presented here are preliminary and need to be confirmed by a future study covering a larger number of patients. Thus, the clinical significance of GSTP1 in the prediction of response to chemotherapy still remains to be established in future.