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) N eoadjuvant 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)(2)(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. (4) 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 amp...

We attempted to develop a highly sensitive and specific method for the detection of circulating tumor DNA (ctDNA) using a digital PCR (dPCR) assay for PIK3CA mutations (i.e., H1047R, E545K, and E542K) in primary breast cancer patients. The sensitivity of the dPCR assay for the mutant alleles was examined using cell lines with PIK3CA mutations and proved to be 0.01 %. Serum samples were collected pre-operatively from 313 stage I-III breast cancer patients, of whom 110 were found to have PIK3CA mutant tumors. The serum samples from these patients with PIK3CA mutant tumors were subjected to the dPCR assay, and 25 (22.7 %) were found to be positive. No PIK3CA mutant ctDNA was detected in the serum samples of 50 healthy women and 30 breast cancer patients with PIK3CA non-mutant tumors. The patients with PIK3CA mutant ctDNA were dichotomized into mutant ctDNA-high (ctDNA(high)) and ctDNA-low (ctDNA(low)) groups based on the median. The ctDNA(high) patients exhibited significantly shorter recurrence-free survival (RFS; P = 0.0002) and overall survival rates (OS; P = 0.0048) compared to those exhibited by the combined ctDNA(low) patient and ctDNA-free patient group. Multivariate analysis revealed that ctDNA(high) status significantly predicted poor RFS and OS and did so independently of conventional histological parameters. These results suggest that dPCR is a highly sensitive and specific method for the detection of PIK3CA mutant ctDNA and that ctDNA(high) but not ctDNA(low) status is a significant and independent prognostic factor for primary breast cancer patients.

Anti-tumor immunity is thought to play a significant role in chemotherapeutic response of breast tumors. In the present study, we investigated whether tumor infiltrating FOXP3+ regulatory T cells, CD8+ cytotoxic T cells, and IL17F+ helper T cells were associated with a pathological complete response (pCR) to neoadjuvant chemotherapy (NAC). Breast cancer patients (stages II and III, n = 180) who were treated with NAC consisting of sequential weekly paclitaxel followed by 5-FU/epirubicin/cyclophosphamide were included for this study. Core needle tumor specimens obtained before NAC were immunohistochemically examined for FOXP3, CD8, and IL17F. Intratumoral infiltration of FOXP3+, CD8+, and IL17F+ T cells was observed in 62.2, 80.0, and 62.2 % of tumors, respectively. FOXP3 and CD8 infiltrates, but not IL17F infiltrate, were significantly (P < 0.001 and P = 0.007, respectively) associated with a high-pCR rate (31.3 and 25.7 %, respectively), and breast tumors with both FOXP3 and CD8 infiltrates showed the highest pCR rate (33.0 %). Multivariate analysis indicated that only FOXP3 infiltrates (P = 0.014) and the conventional predictive factor Ki67 (P = 0.031) were statistically significant and independent predictors of pCR. Breast tumors with FOXP3 and CD8 infiltrates were more likely to achieve pCR. FOXP3 infiltrate, in combination with Ki67, could thus be used as a clinically useful predictor of response to NAC. The possible indirect mechanism through which chemotherapy exerts its anti-tumor activity, i.e., enhancing anti-tumor immunity by inhibiting FOXP3, was also suggested.

The aim of this prospective study was to evaluate the feasibility of periareolar injection of the contrast agent Sonazoid (SNZ) followed by ultrasonography (US) for the identification of sentinel lymph node (SLN) in breast cancer patients with clinically negative node. Patients (n = 100) with T1‐2N0M0 breast cancer received a periareolar injection of SNZ followed by US to identify contrast‐enhanced SLN. Each contrast‐enhanced SLN underwent fine needle aspiration cytology (FNAC) followed by SLN biopsy with a conventional method using blue dye and/or radiocolloid (B/R). In almost all cases, contrast‐enhanced lymphatic vessels were clearly visualized by US soon after the periareolar injection of SNZ and the SLNs were easily identified with an identification rate of 98% (98/100) for SNZ and 100% (100/100) for B/R. The number of SLNs identified by SNZ (SNZ‐SLN) (mean per patient, 1.52) was significantly lower than that identified by B/R (B/R‐SLN) (2.19) (P < 0.0001). Twenty‐five patients with positive SLNs had at least one positive SNZ‐SLN. On a node‐by‐node basis, sensitivity, specificity, and accuracy of FNAC for SNZ‐SLNs (n = 149) were 33.3%, 99.2%, and 85.9%, respectively. Identification of SLN by periareolar injection of SNZ is a technically simple method with an identification rate as high as 98%. SNZ‐SLN thus seems to be a good target for FNAC, but sensitivity of FNAC for SNZ‐SLNs needs to be improved.

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