Tomoko Asano

MiR-1290 and its potential targets are associated with characteristics of estrogen receptor α-positive breast cancer

Recent analyses have identified heterogeneity in estrogen receptor α (ERα)-positive breast cancer. Subtypes called luminal A and luminal B have been identified, and the tumor characteristics, such as response to endocrine therapy and prognosis, are different in these subtypes. However, little is known about how the biological characteristics of ER-positive breast cancer are determined. In this study, expression profiles of microRNAs (miRNAs) and mRNAs in ER-positive breast cancer tissue were compared between ER(high) Ki67(low) tumors and ER(low) Ki67(high) tumors by miRNA and mRNA microarrays. Unsupervised hierarchical clustering analyses revealed distinct expression patterns of miRNAs and mRNAs in these groups. We identified a downregulation of miR-1290 in ER(high) Ki67(low) tumors. Among 11 miRNAs that were upregulated in ER(high) Ki67(low) tumors, quantitative RT-PCR detection analysis using 64 samples of frozen breast cancer tissue identified six miRNAs (let-7a, miR-15a, miR-26a, miR-34a, miR-193b, and miR-342-3p). We picked up 11 genes that were potential target genes of the selected miRNAs and that were differentially expressed in ER(high) Ki67(low) tumors and ER(low) Ki67(high) tumors. Protein expression patterns of the selected target genes were analyzed in 256 ER-positive breast cancer samples by immunohistochemistry: miR-1290 and its putative targets, BCL2, FOXA1, MAPT, and NAT1, were identified. Transfection experiments revealed that introduction of miR-1290 into ER-positive breast cancer cells decreased expression of NAT1 and FOXA1. Our results suggest that miR-1290 and its potential targets might be associated with characteristics of ER-positive breast cancer.

Immunohistochemical determination of the miR-1290 target arylamine N-acetyltransferase 1 (NAT1) as a prognostic biomarker in breast cancer

BackgroundThere are many molecular differences between estrogen receptor α (ERα)-positive and ER-negative breast cancers. Recent analyses have shown that the former can be divided into two subtypes, luminal A and luminal B. These differ in response to endocrine therapy and chemotherapy, and in prognosis. In a previous study, we found that microRNA (miR)-1290 that was significantly down-regulated in luminal A tumors and its potential target arylamine N-acetyltransferase 1 (NAT1). The aim of the present study was to determine whether NAT1 is a bona fide target of miR-1290, and to investigate the impact of NAT1 on breast cancer prognosis.MethodsLuciferase reporter assays were employed to validate NAT1 as a putative miR-1290 target gene. Expression of NAT1, ERα, progesterone receptor (PgR) and HER2 was analyzed in 394 breast cancer samples by immunohistochemistry.ResultsNAT1 was confirmed to be a direct target of miR-1290. Levels of expression of NAT1 were positively correlated with those of ERα (P < 0.0001) and PgR (P < 0.0001), but negatively correlated with both tumor grade and size (P < 0.0001). Kaplan-Meier analysis showed that the presence of NAT1 was significantly associated with increased overall survival (OS) (P = 0.0416) in these patients. Similarly, significant associations of NAT1 with disease-free survival (DFS) (P = 0.0048) and OS (P = 0.0055) in those patients who received adjuvant endocrine therapy with tamoxifen (n = 176) were found. Moreover, NAT1 was also significantly associated with increased DFS (P = 0.0025) and OS (P = 0.0007) in the subset of lymph node-positive patients (n = 147). Univariate and multivariate analyses showed significant associations between levels of NAT1 and DFS (P = 0.0005 and 0.019, respectively).ConclusionsWe report that miR-1290 directly targets the NAT1 3′-UTR and that NAT1 protein expression is correlated with improved OS of breast cancer patients. NAT1 is a possible prognostic biomarker for lymph node-positive breast cancer. Thus, miR-1290 and its target NAT1 are associated with important characteristics of breast cancer.

FOXA1 expression after neoadjuvant chemotherapy is a prognostic marker in estrogen receptor-positive breast cancer

BackgroundRecent studies have indicated that the response to chemotherapy and the prognostic impact of a pathological complete response (pCR) after neoadjuvant chemotherapy differ among breast cancer subtypes. Predictors of response to chemotherapy and prognostic factors for survival might be different in estrogen receptor (ER)-positive breast cancer.MethodsWomen with Stage II to III ER-positive HER2-negative breast cancer treated with anthracycline and taxane-containing neoadjuvant chemotherapy between 2003 and 2011 were retrospectively analyzed. Expression of forkhead box A1 (FOXA1), B cell lymphoma 2 (BCL2) and microtubule-associated protein tau (MAPT) as well as ER, progesterone receptor, HER2 and Ki67 was examined by immunohistochemistry in pre- and post-treatment specimens. Factors predictive of response to neoadjuvant chemotherapy and distant disease-free survival were analyzed.ResultsTumor grade was positively correlated with Ki67 expression. Expression levels of ER were positively correlated with expression levels of HER2, BCL2, FOXA1 and MAPT in pre-treatment tumors. The Ki67 labeling index was the only factor that was significantly associated with clinical response measured by the reduction of tumor volume and pCR. Lymph node status, expression of ER before neoadjuvant chemotherapy and expression of FOXA1 after neoadjuvant chemotherapy were significantly associated with distant disease-free survival, both by univariate and multivariate analyses.ConclusionsPatients with ER-positive HER2-negative breast cancer should be selected for neoadjuvant chemotherapy. FOXA1 expression could be a prognostic marker in ER-positive breast cancer.

Myoepithelial Carcinoma of the Breast Treated with Surgery and Chemotherapy

Myoepithelial carcinoma (malignant myoepithelioma) of the breast is a rare tumor, for which only a limited number of reports have been published. Most of the reports emphasized diagnosis and pathology but not biological behavior and treatment. We report a 61-year-old patient with breast myoepithelial carcinoma who developed locoregional and distant metastases and received many chemotherapy regimens. She presented with an elastic hard mass of the left breast. Breast conserving surgery was performed as part of both diagnosis and treatment. From the results of histological and immunohistochemical examinations, this case was considered to be a myoepithelial carcinoma. Fifteen months after the completion of adjuvant radiotherapy, distant metastasis of the left parasternal lymph node metastasis developed. She was treated by further excision and received a total of four regimens of chemotherapy including a combination of doxorubicin and cyclophosphamide. She received chemotherapy for 20 months after the diagnosis of metastasis.

HER2 Mutation Status in Japanese HER2-negative Breast Cancer Patients

OBJECTIVE Human epidermal growth factor receptor 2 (HER2) gene amplification is a major therapeutic target in breast cancer, and has been introduced as a predictive biomarker to identify patients who may benefit from therapy with anti-human epidermal growth factor receptor 2 agents. Human epidermal growth factor receptor 2 somatic mutations have been reported in patients without human epidermal growth factor receptor 2 gene amplification. Since these are activating mutations, these patients may also benefit from human epidermal growth factor receptor 2-targeted drugs. METHODS In this study, we searched for human epidermal growth factor receptor 2 mutations in a group of 286 Japanese breast cancer patients with human epidermal growth factor receptor 2-negative tumors. The activating mutations of human epidermal growth factor receptor 2 identified were analyzed by direct Sanger sequencing of two major areas: the extracellular domain at 309-310 and the kinase domain between 755 and 781. RESULTS Two tumors were found to have a human epidermal growth factor receptor 2 somatic mutation; one with I767M mutation and another with D769Y. No mutation was observed in the extracellular domain. One of these patients with human epidermal growth factor receptor 2 mutation recurred early with liver metastasis. CONCLUSIONS Better knowledge of human epidermal growth factor receptor 2 mutation status will help us to choose personalized molecular targeted therapy for use in human epidermal growth factor receptor 2-negative Japanese breast cancer patients.

HER2 mutation status in Japanese HER2-positive breast cancer patients

AbstractBackground Human epidermal growth factor receptor 2 (HER2) gene amplification/overexpression is a major therapeutic target in breast cancer, and has been introduced as a predictive biomarker to identify patients who may benefit from therapy with anti-HER2 agents. HER2 somatic mutations have been reported, and these may influence the effect of HER2-targeted drugs.MethodsHere, we sought HER2 mutations in a group of 135 Japanese breast cancer patients with HER2-positive tumors. We analyzed HER2 mutations by direct Sanger sequencing of two major areas, the extracellular domain at position 309–310 and the kinase domain between 755 and 781.ResultsTwo patients with the HER2 somatic mutation S310F in the extracellular domain were found in this series. One patient with the S310F mutation had a node-negative invasive ductal carcinoma classified as HER2 2+ by the HercepTest and fluorescence in situ hybridization (FISH) positive, and which was estrogen receptor (ER)-negative and progesterone receptor (PgR)-negative. Another patient with the S310F mutation had an apocrine carcinoma with seven lymph nodes positive for metastasis, classified as HER2 3+ by the HercepTest, but which was FISH-negative, as well as ER-negative and PgR-negative. Both patients had received adjuvant single-agent trastuzumab therapy, and had no local recurrence or distant metastasis for five and three years after surgery, respectively.ConclusionsOur data show that HER2 mutations are rare in HER2-positive Japanese breast cancer patients. The two mutations found in this study were identical, S310F. We suggest that in vitro experiments to determine whether the S310F mutation could be involved in resistance to anti-HER2 drugs are worthwhile in future.

Abstract P4-11-22: High expression of LMTK3 is an independent factor indicating a poor prognosis in Japanese estrogen receptor α-positive breast cancer patients

Over 70% of breast cancers are estrogen receptor α (ERα)-positive, and endocrine therapy targeting estrogen action decreases mortality from breast cancer. However, their efficacy of endocrine therapy is limited by intrinsic and acquired resistance. Recently, a novel protein kinase that regulates ERα activity, lemur tyrosine kinase-3 (LMTK3) has been identified. In this study, we investigated whether LMTK3 mRNA expression and its polymorphisms are associated with prognosis in Japanese breast cancer patients during long-term follow-up. First, we investigated the relationship between mRNA expression of LMTK3 and patient outcome in 242 breast cancers (median follow-up, 6.8 years). The effects of several variables on survival were tested by Cox proportional hazards regression analysis. Next, we performed to analyze LMTK3 rs9989661 and rs8108419 genotyping in 641 breast cancer tissues (median follow-up, 9.2 years) to clarify the prognostic role of these polymorphisms. We showed that high expression levels of LMTK3 mRNA were significantly associated with a shorter overall survival (OS) in all patients. We then, analyzed the impact of LMTK3 mRNA expression on the prognosis of breast cancer according to ERα status. Both disease-free survival and OS were significantly shorter in ERα-positive patients with high LMTK3 mRNA expression receiving adjuvant endocrine therapy than in those patients with low LMTK3 mRNA expression. Notably, the level of LMTK3 mRNA expression was not associated with prognosis in ERα-negative breast cancer patients. Univariate and multivariate Cox regression analysis of factors associated with OS revealed that high LMTK3 mRNA expression was an independent poor prognostic factor in ERα-positive breast cancer patients. We did not find any correlation between LMTK3 genotypes and prognosis of breast cancer patients in our series. Our results suggest that higher LMTK3 expression might be one possible mechanism for endocrine resistance in ERα-breast cancer patients, to be confirmed by further research. Citation Format: Tatsuya Toyama, Tomoko Asano, Shinya Sato, Nobuyasu Yoshimoto, Yumi Endo, Yukari Hato, Dong Yu, Satoru Takahashi, Yoshitaka Fujii. High expression of LMTK3 is an independent factor indicating a poor prognosis in Japanese estrogen receptor α-positive breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-22.

Abstract P4-07-04: Immunohistochemical determination of arylamine N-acetyltransferase 1 (NAT1) as the target of miR-1290 and prognostic biomarker of breast cancer

Introduction: There are large-scale molecular differences between estrogen receptor α (ERα)-positive breast cancer and ER-negative breast cancers. In ERα-positive breast cancer, recent analyses have shown that ERα-positives can be divided into two subtypes such as luminal A and luminal B. These subtypes differ in characteristics such as response to endocrine therapy and chemotherapy, and prognosis. In a previous study, we identified a microRNA, miR-1290, that was significantly down-regulated in luminal A tumors and its potential target arylamine N-acetyltransferase 1 (NAT1) . The aim of this study was to clarify whether NAT1 is a bona fide target of miR-1290, and to investigate the impact of NAT1 on breast cancer prognosis. Methods: Luciferase reporter assay was employed to validate NAT1 as a putative miR-1290 target gene. Protein expressions of NAT1, ERα, progesterone receptor (PgR) and HER2 were analyzed in 394 breast cancer samples by immunohistochemistry. Results: NAT1 was shown to be a direct target of miR-1290 by luciferase reporter assay. Expression levels of NAT1 were positively correlated with expression levels of ERα ( P P P P = 0.0416) in breast cancer patients (n=394). Similarly, significant associations of NAT1 presence were shown with disease-free survival (DFS) ( P = 0.0048) and OS ( P = 0.0055) in patients who received adjuvant endocrine therapy with tamoxifen (n = 176). Moreover, NAT1 presence was also significantly associated with increased DFS ( P = 0.0025) and OS ( P = 0.0007) in lymph node-positive breast cancer patients (n=147). Univariate and multivariate analyses showed significant associations between expression levels of NAT1 and DFS ( P = 0.0005 and 0.019, respectively). Conclusion: We reported that miR-1290 directly targets NAT1 3’-UTR and NAT1 protein expression is correlated with improved OS of breast cancer. Moreover, NAT1 is a possible prognostic biomarker for lymph node-positive breast cancer. Thus, miR-1290 and its potential target NAT1 are associated with characteristics of breast cancer. Citation Format: Yumi Endo, Hiroko Yamashita, Satoru Takahashi, Shinya Sato, Nobuyasu Yoshimoto, Tomoko Asano, Yukari Hato, Mina Yamaguchi, Yu Dong, Tatsuya Toyama. Immunohistochemical determination of arylamine N-acetyltransferase 1 (NAT1) as the target of miR-1290 and prognostic biomarker of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-07-04.

Abstract P5-05-02: Prognostic impact of single-nucleotide polymorphisms (SNPs) in or near the ZNF423 and CTSO genes in estrogen receptor (ER)-positive breast cancer patients receiving adjuvant endocrine therapy

Background : Selective estrogen receptor modulators (SERMs) can reduce the occurrence of breast cancer in high-risk women by 50%. Recently, a genome-wide association study identified SNPs in or near the ZNF423 (rs8060157) and CTSO (rs10030044) genes that were associated with breast cancer risk during SERM therapy and these SNPs were reported to be involved in estrogen-dependent induction of BRCA1 expression (Ingle JN. et al . Cancer Discovery 2013). Materials and methods : A total of 588 breast carcinomas collected between 1983 and 2003 were available for polymorphism assay. TaqMan pre-designed SNP genotyping assays for ZNF423 rs8060157 and CTSO rs10030044 were used. We investigated whether these SNPs are associated with prognosis in breast cancer patients. The effects of several variables on survival were tested by Cox proportional hazards regression analysis. Results : Estrogen receptor (ER)-positive breast cancer patients receiving adjuvant endocrine therapy with the genotype GG at CTSO rs10030044 showed significantly shorter disease-free survival (DFS) and overall survival (OS) ( P = 0.0024 and P = 0.0003, respectively). On the other hand, this genotype were not associated with prognosis in ER-negative breast cancer patients. Multivariate Cox regression analysis revealed that the GG genotype at CTSO rs10030044 was an independent poor prognostic factor in ER-positive breast cancer patients receiving adjuvant endocrine therapy (OS: RR = 1.86; 95%CI, 1.18 to 2.85). The SNP, ZNF423 rs8060157, was not associated with prognosis in this study. Conclusion: We show that the genotype GG at CTSO rs10030044 is an independent factor indicating poor prognosis in ER-positive breast cancer patients receiving adjuvant endocrine therapy. Citation Format: Yukari Hato, Yumi Endo, Nobuyasu Yoshimoto, Tomoko Asano, Mina Yamaguchi, Satoru Takahashi, Tatsuya Toyama. Prognostic impact of single-nucleotide polymorphisms (SNPs) in or near the ZNF423 and CTSO genes in estrogen receptor (ER)-positive breast cancer patients receiving adjuvant endocrine therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-02.

High Expression of LMTK3 is an Independent Factor Indicating a Poor Prognosis in Estrogen Receptor α-Positive Breast Cancer Patients

OBJECTIVE Over 70% of breast cancers are estrogen receptor alpha-positive, and endocrine therapy targeting estrogen action decreases mortality from breast cancer. Recently, a novel protein kinase that regulates estrogen receptor alpha activity, lemur tyrosine kinase-3, has been identified. In this study, we investigated whether messenger RNA expression and polymorphisms of the gene encoding the kinase, LMTK3, are associated with prognosis in breast cancer patients during long-term follow-up. METHODS First, we investigated the relationship between messenger RNA expression of LMTK3 and patient outcome in 219 breast cancers. The effects of several variables on survival were tested by Cox proportional hazards regression analysis. Next, we performed LMTK3 genotyping in 471 breast cancers to clarify the prognostic role of these polymorphisms. RESULTS Our data showed that LMTK3 expression level was not associated with prognosis in all patients. We then analyzed the impact of LMTK3 mRNA expression on the prognosis of breast cancer according to estrogen receptor alpha status. Both disease-free survival and overall survival were significantly shorter in estrogen receptor alpha-positive patients with high LMTK3 expression receiving adjuvant endocrine therapy than in those patients with low LMTK3 expression. Multivariate Cox regression analysis revealed that high LMTK3 expression was an independent poor prognostic factor in estrogen receptor alpha-positive breast cancer patients. We did not find any correlation between LMTK3 genotypes and prognosis of breast cancer patients in our series. CONCLUSIONS Our results show that high expression of LMTK3 is an independent prognostic factor in estrogen receptor alpha-positive breast cancer patients receiving adjuvant endocrine therapy.