Tomohiro Miyake

GSTP1 expression predicts poor pathological complete response to neoadjuvant chemotherapy in ER-negative breast cancer

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)

Indication for sentinel lymph node biopsy for breast cancer when core biopsy shows ductal carcinoma in situ

BACKGROUND The use of sentinel lymph node biopsy (SLNB) for ductal carcinoma in situ (DCIS) is controversial. METHODS A total of 103 primary breast cancer patients who were diagnosed with DCIS by needle biopsy preoperatively and underwent initial SLNB were analyzed retrospectively. RESULTS No sentinel nodal metastasis was detected in 66 patients with the final diagnosis of DCIS. However, 2 (5.4%) of 37 patients with invasive ductal carcinoma at final diagnosis had positive sentinel nodes. Multivariate logistic regression analysis identified 2 independent significant predictors of existence of invasive components: presence of a palpable tumor (odds ratio, 4.091; 95% confidential interval, 1.399-11.959; P = .010) and tumor size of 2.0 cm or larger on magnetic resonance imaging (odds ratio, 4.506; 95% confidence interval, 1.322-15.358; P = .016). CONCLUSIONS Initial SLNB should be considered for patients diagnosed with DCIS by needle biopsy when they have a high risk for harboring invasive ductal cancer preoperatively.

BRCA1 promoter methylation of normal breast epithelial cells as a possible precursor for BRCA1-methylated breast cancer

The breast cancer susceptibility gene 1 (BRCA1) and glutathione S‐transferase P1 (GSTP1) promoters are reportedly often methylated in breast cancer tissues. Their methylation status in surrounding normal breast tissues has not been examined thoroughly although this may well be important for a better understanding of breast carcinogenesis. In this study, BRCA1 and GSTP1 promoter methylation was examined by methylation‐specific PCR assay. Patients with BRCA1‐methylated (n = 15) or BRCA1‐unmethylated (n = 15) tumors and those with GSTP1‐methylated (n = 9) or GSTP1‐unmethylated (n = 11) tumors were included in the present study. Methylation status of manually micro‐dissected normal epithelial cells from the formalin‐fixed paraffin‐embedded sections of normal breast tissues adjacent to and distant from the tumors was examined at multiple sites (n = 1–5). Of the 15 patients with BRCA1‐methylated tumors, 9 harbored BRCA1 promoter methylation in at least one site of the normal breast tissues. However, no BRCA1 promoter methylation was observed at any site of the normal tissues of the 15 patients with BRCA1‐unmethylated tumors. No GSTP1 promoter methylation was observed in the normal tissues regardless of the methylation status of the tumors. The presence of BRCA1 promoter methylation in the normal tissues was confirmed in the epithelial cells enriched with the magnetic‐activated cell sorting method. Our findings suggest that a small proportion of normal breast epithelial cells with BRCA1 promoter methylation can be precursor cells from which BRCA1‐methylated breast tumors may originate. This does not apply to GSTP1 promoter methylation.

Pilot study of intraperitoneal administration of paclitaxel and oral S-1 for patients with peritoneal metastasis due to advanced gastric cancer

BackgroundThere is no standard treatment for peritoneal dissemination from gastric cancer. A novel combination chemotherapy has been introduced for patients with advanced gastric cancer with peritoneal metastasis.MethodsThis pilot study was performed on four patients to confirm safety and efficacy. They were diagnosed with unresectable gastric cancer with severe peritoneal dissemination by staging laparoscopy, or with metastasis to the transverse colon. We selected combined chemotherapy with both paclitaxel and S-1. Paclitaxel at 60 mg/m2 or 60 mg/body was administered intraperitoneally on days 1 and 8 and S-1, at 80–120 mg/body, was administered orally for 14 days followed by 7 days’ rest, as one course. After five courses of this therapy, the primary gastric tumors were evaluated by conventional examinations, and second-look laparoscopy was performed to assess the efficacy of the treatment against the peritoneal metastases.ResultsAfter five courses, primary tumor reductions were confirmed, and no cancer cells were detected on pathocytological investigation during second-look laparoscopy in any of the patients. Three patients underwent total gastrectomy with lymph node dissection and one underwent left upper abdominal evisceration. Final histological staging showed two stage 3 and two stage 4 patients. The intraperitoneal administration of paclitaxel and the oral administration of S-1 were well tolerated. Three patients died, at 8, 15, and 29 months, respectively, after the initial treatment, and one has been alive for 54 months without recurrence.ConclusionThis chemotherapy can be used in the treatment of patients with peritoneal metastasis of gastric cancer.

Detection of ESR1 mutations in plasma and tumors from metastatic breast cancer patients using next-generation sequencing

PurposeLiquid biopsy using digital PCR (dPCR) has been widely used for the screening of ESR1 mutations, since they are frequently identified in the hotspot. However, dPCR is limited to the known mutations. Therefore, we aimed to analyze the utility of next-generation sequencing (NGS) to discover novel ESR1 mutations.MethodsWhole exon sequencing of the ESR1 gene using NGS was performed in 16 primary and 47 recurrent tumor samples and 38 plasma samples from hormone receptor-positive metastatic breast cancer patients. Functional analyses were then performed for the novel mutations we detected.ResultsWe identified no mutations in primary tumors and six mutations in five recurrent tumors, including three types of known mutations (Y537C, Y537N, and D538G) and two novel mutations (E279V and G557R). We also identified seven mutations in five plasma samples, including three types of known mutations (S463P, Y537S, and D538G) and one mutation not reported in COSMIC database (L536H). All nine patients with ESR1 mutations were treated with aromatase inhibitors (AIs) prior to sampling, and the mutations were frequently detected in patients who received AI treatments in the metastatic setting. Among the three novel mutations (E279V, L536H, and G557R), L536H, but not E279V and G557R, showed ligand-independent activity. All three mutant proteins showed nuclear localization and had no relation with non-genomic ER pathways.ConclusionsAlthough the molecular mechanisms of the E279V and G557R mutations remain unclear, our data suggest the utility of NGS as a liquid biopsy for metastatic breast cancer patients and the potential to identify novel ESR1 mutations.

Identification of sentinel lymph nodes by contrast-enhanced ultrasonography with Sonazoid in patients with breast cancer: a feasibility study in three hospitals

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.

PAM50 for prediction of response to neoadjuvant chemotherapy for ER-positive breast cancer

PurposeThere is an urgent need for the development of a predictor of response to chemotherapy for ER-positive breast cancer which is less chemosensitive than for ER-negative breast cancer in order to avoid unnecessary chemotherapy. In the present study, intrinsic subtyping by PAM50 was evaluated for its ability to predict a response to chemotherapy.Patients and MethodsFor this study, 124 patients with ER-positive breast cancer treated with neoadjuvant sequential paclitaxel and FEC (NAC) were evaluated. Tumor biopsy specimens obtained before NAC were subjected to intrinsic subtyping (IS) by gene expression (GE) using PAM50 (PAM50-IS) or immunohistochemistry (IHC-IS).ResultsOf the PAM50-ISs (Luminal A, Luminal B, HER2-enriched, and Basal-like), GE-Luminal A showed the lowest pCR rate (1.9%), and multivariate analysis revealed that GE-Luminal A was a significant (P = 0.031) predictor of non-pCR independently of other clinicopathological parameters, including Ki67, and tumor-infiltrating lymphocytes. Of the IHC-ISs, on the other hand, IHC-Luminal A was not significantly associated with pCR. We also found that breast tumors with low ER levels (1–9%), like ER-negative tumors, were mostly GE-HER2-enriched and GE-Basal-like, and more sensitive to NAC than those with high ER levels (≥ 10%).ConclusionsGE-Luminal A intrinsically subtyped by PAM50 was the least sensitive to NAC and very unlikely to attain pCR. IHC-Luminal A identified by IHC, on the other hand, was not significantly predictive of pCR. In addition, PAM50 revealed that tumors with low ER (1–9%) were more like ER-negative tumors than ER-positive tumors, and most such cases should therefore would better be treated with chemotherapy.

Mutational Analysis of AKT1 and PIK3CA in Intraductal Papillomas of the Breast with Special Reference to Cellular Components

The pathologic feature of intraductal papillomas is defined as a papillary structure composed of a fibrovascular stromal core lined by luminal epithelial cells and myoepithelial cells. We used droplet digital PCR for the mutational analysis of AKT1 (E17K) and PIK3CA (H1047R, E542K, and E545K) in 60 papillomas. AKT1 and PIK3CA mutations were detected in 12 (20%) and 17 (28%) of the papillomas, respectively. In five tumors harboring mutations, mutational analysis of AKT1 or PIK3CA was performed separately using luminal epithelial cells and myoepithelial cells sorted using anti-cytokeratin 19 antibody and anti-α smooth muscle actin antibody. The two types of cells from a given papilloma had the identical mutation. Three patients with the PIK3CA mutation-positive papilloma developed breast cancers at the resection site of the papilloma, but none of these subsequent breast cancers had the PIK3CA mutation. These results indicate that a papilloma stems from a bipotent progenitor cell that contains the AKT1 or PIK3CA mutation and proliferates and differentiates to form the papilloma. Papilloma can be a risk factor for developing breast cancer but is unlikely to be its obligate precursor.

Endocrine sensitivity of estrogen receptor-positive breast cancer is negatively correlated with aspartate-β-hydroxylase expression.

Although prognostic markers for early estrogen receptor (ER)‐positive breast cancer have been extensively developed, predictive markers for adjuvant endocrine therapy are still lacking. Focusing on the mechanisms underlying endocrine resistance, we investigated whether the endocrine sensitivity of ER‐positive breast cancer cells was correlated with the expression of aspartate‐β‐hydroxylase (ASPH), which is involved in the development of hepatocellular carcinoma. ASPH expression in ER‐positive and tamoxifen‐resistant breast cancer cells was upregulated by the MAPK and phosphoinositide‐3 kinase (PI3K) pathways, which both play pivotal roles in endocrine resistance. In the clinical setting, ASPH expression was negatively correlated with recurrence‐free survival of luminal B breast cancer patients that received adjuvant endocrine therapy, but not in patients that did not receive adjuvant endocrine therapy. Luminal B breast cancer is one of the intrinsic molecular subtypes identified by the Prediction Analysis of Microarray 50 (PAM50) multiple gene classifier, and because of its poor response to endocrine therapy, chemotherapy in addition to endocrine therapy is generally required after surgical resection. Our results suggest that the endocrine sensitivity of luminal B breast cancer can be assessed by examining ASPH expression, which promotes the consideration of a prospective study on the association between ASPH expression at the mRNA and protein levels in luminal B breast cancer and subsequent response to endocrine therapy.