Yuka Inoue

Epithelial Paradox: Clinical Significance of Coexpression of E-cadherin and Vimentin With Regard to Invasion and Metastasis of Breast Cancer

Background E‐cadherin and vimentin are regarded as major conventional canonical markers of the epithelial–mesenchymal transition. It is commonly assumed that E‐cadherin is uniformly lost during the process of epithelial–mesenchymal transition. Breast tumor cells typically invade as a cohesive multicellular unit in a process called collective invasion. The aim of this study was to reveal the clinical importance of the expression pattern of E‐cadherin and vimentin in breast cancer. Methods E‐cadherin and vimentin protein expression was evaluated by immunohistochemistry in 176 invasive breast cancer samples. Among these, E‐cadherin and vimentin expression were evaluated in the set of primary site and metastatic lymph nodes in 65 cases. In addition, E‐cadherin and vimentin expression were analyzed by confocal laser scanning microscopy to see E‐cadherin and vimentin localization in the breast cancer cells. Results Both at the primary site and metastatic lymph nodes, both E‐cadherin– and vimentin‐positive tumors had the worst disease‐free and overall survival among all cases. In addition, E‐cadherin and vimentin protein is colocalized within the same tumor cells in a human breast cancer specimen. Conclusion Our present data suggest the existence of an aggressive subpopulation in the primary tumor nest of breast cancer. Micro‐Abstract E‐cadherin and vimentin are regarded as major conventional canonical markers of epithelial–mesenchymal transition. Both E‐cadherin– and vimentin‐positive tumors had the worst prognosis among all cases. Further, E‐cadherin and vimentin protein is colocalized within the same tumor cells, suggesting the existence of an aggressive subpopulation in the primary tumor nest of breast cancer.

Expression of APOBEC3B mRNA in primary breast cancer of Japanese women

Recent studies have identified the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3B (APOBEC3B) as a source of mutations in various malignancies. APOBEC3B is overexpressed in several human cancer types, including breast cancer. In this study, we analyzed APOBEC3B mRNA expression in 305 primary breast cancers of Japanese women using quantitative reverse transcription-PCR, and investigated the relationships between the APOBEC3B mRNA expression and clinicopathological characteristics, prognosis, and TP53 mutations. The expression of APOBEC3B mRNA was detected in 277 tumors and not detected in 28 tumors. High APOBEC3B mRNA expression was significantly correlated with ER- and PR-negativity, high grade and high Ki67 index. The APOBEC3B mRNA expression was highest in the triple-negative and lowest in the hormone receptor-positive/HER2-negative subtypes. The TP53 gene was more frequently mutated in the tumors with high APOBEC3B mRNA expression. High APOBEC3B mRNA expression was significantly associated with poor recurrence-free survival in all cases and the ER-positive cases. These findings were almost consistent with the previous reports from the Western countries. In conclusion, high APOBEC3B mRNA expression was related to the aggressive phenotypes of breast cancer, high frequency of TP53 mutation and poor prognosis, especially in ER-positive tumors.

Abstract P2-05-12: Epithelial paradox; clinical significance of co-expression of E-cadherin and vimentin in invasive breast cancer

Background: E-cadherin and vimentin are now regarded as major and conventional canonical markers of epithelial-mesenchymal transition (EMT). It is commonly assumed E-cadherin is uniformly lost during the process of EMT. We previously reported that the elevated expression of vimentin contributes to the aggressive phenotype in invasive breast cancer. On the other hand, the role of E-cadherin in breast cancer biology might be unclear and more complex. Although, cell cohesion during breast cancer invasion is often overlooked, accumulating evidences indicate breast tumor cells are typically cohesive and often display membrane-localized E-cadherin in both the primary tumor and distant metastases, termed collective invasion. Multiple mechanisms have emerged to address how epithelial breast tumors invade. Aims: The aim of this study is to reveal the clinical importance of the expression of E-cadherin and vimentin in breast cancer. Methods: The E-cadherin and vimentin protein expression were evaluated by immunohistochemistry (IHC) in 177 invasive breast cancer samples. Among these, E-cadherin and vimentin expression were evaluated in the set of primary breast cancer and metastatic lymph nodes in 65 cases. Results: The positive vimentin expression was highly correlated with poor disease-free survival (DFS) and overall survival (OS) (p=0.019 and p=0.0044), however, the E-cadherin expression alone did not correlate with prognosis. Interestingly, Both E-cadherin and vimentin positive tumor had the worst DFS and OS among all breast cancer (p=0.03 and p=0.0089). Vimentin expression was highly correlated between primary tumors and metastatic lymph nodes. However, E-cadherin expression levels were significantly elevated in metastatic lymph nodes (p=0.0017), Co-expression of E-cadherin and vimentin in the metastatic lymph nodes also showed worst DFS and OS (p=0.12 and p=0.027). Conclusions: Co-expression of E-cadherin and vimentin seems to be associated with the most aggressive phenotype and poorest prognosis in breast cancer, and positive E-cadherin expression may not always play roles for tumor suppression. Citation Format: Yamashita N, Tokunaga E, Inoue Y, Tanaka K, Ueo H, Saeki H, Oki E, Maehara Y. Epithelial paradox; clinical significance of co-expression of E-cadherin and vimentin in invasive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-12.

Clinical Significance of the Wild Type p53-Induced Phosphatase 1 Expression in Invasive Breast Cancer

&NA; The nuclear expression of wild type p53‐induced phosphatase 1 (Wip1) protein was found to be positive in 21 patients (10.4%) out of 201 breast cancer patients in our study. The protein phosphatase magnesium dependent 1 delta DNA copy number was significantly correlated with Wip1 protein expression, which was positively correlated with p21 expression. Tumors with positive Wip1 expression and negative p21 expression showed the poorest prognosis of all tumors examined. Background: Wild type p53‐induced phosphatase 1 (Wip1), encoded by the protein phosphatase magnesium dependent 1 delta (PPM1D), inhibits p53. PPM1D amplification has been reported in breast cancer. Breast cancer can sometimes develop without a tumor protein 53 (TP53) mutation. In these cases, the p53 pathway might be disrupted by alternative mechanisms, and Wip1 is reported to be a key molecule involved. Materials and Methods: Primary invasive ductal carcinoma specimens were obtained from 201 cases, for which archival tissue samples for immunohistochemistry were available. We evaluated Wip1 and p21 protein expression (201 cases), Wip1 mRNA expression (63 cases), PPM1D DNA copy number (71 cases) and TP53 status (36 cases) using available samples among the 201 cases, and analyzed their relationships with clinicopathological factors and prognosis. Results: The nuclear expression of Wip1 protein was positive in 21 cases (10.4%). The PPM1D DNA copy number was significantly correlated with Wip1 protein expression. All cases with PPM1D amplification by single‐nucleotide polymorphism comparative genomic hybridization array showed positive nuclear Wip1 expression. Wip1 protein expression was positively correlated with p21 expression. The tumors with positive Wip1 and negative p21 expression showed the poorest prognosis among all tumor types. Conclusion: The protein expression of Wip1 might be regulated by PPM1D amplification, independent of TP53 status. Positive Wip1 and negative p21 expression was associated with the poorest prognosis and suggests the loss of p53 function.

Abstract 3696: Biological significance of the wild-type p53-induced phosphatase 1(Wip1) expression in invasive breast cancer

Backgrounds The wild-type p53-induced phosphatase 1(Wip1) is a member of the serine/threonine protein phosphatases, and plays an important role in the nucleus as one of the key components in the DNA damage response (DDR) network. Wip1 is encoded by the protein phosphatase magnesium dependent 1 delta (PPM1D), sited on locus 17q23. PPM1D amplification has been reported in breast cancer. Aims We evaluated the expression of Wip1 mRNA, Wip1 protein and PPM1D DNA copy number to clarify the relationship between Wip1 expression and the clinicopathological features and prognosis to determine the biological significance of Wip1. Materials and Methods Breast cancer cells (MCF7, T47D, MDA-MB231, HCC1937, HS578T, BT20 and SKBr3) were used for Wip1 expression analysis and copy number analysis. Primary invasive ductal carcinoma specimens were obtained from Japanese patients who underwent surgery without neoadjuvant chemotherapy or endocrine therapy. Wip1 mRNA expression was evaluated in 140 cases by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Wip1 protein expression was evaluated in 192 cases by immunohistochemistry (IHC). The PPM1D DNA copy number was analyzed by genomic PCR in 33 breast cancer cases and by the single-nucleotide polymorphism-Comparative Genomic Hybridization (SNP-CGH) array in 12 cases. The effects on the cell growth of the Wip1 inhibitor (GSK2830371) was analyzed by the viability assay in MCF7. Results Wip1 mRNA expression was significantly higher in MCF7, luminal type cell line. Wip1 mRNA expression was divided into four groups, very high, high, low and very low. The very high Wip1 mRNA expression was significantly associated with positive estrogen receptor (ER) expression (p = 0.02). There was no significant correlation between Wip1 mRNA expression and the prognosis. Wip1 protein nuclear expression was positive in 21 cases (10.9%). There was no significant association between the Wip1 protein expression levels and the clinicopathological factors and the prognosis. PPM1D DNA copy number significantly correlated with Wip1 protein expression (p = 0.0035). Amplification at 17q23 was detected in 6 cases by SNP-CGH array, and all of these six cases showed positive nuclear Wip1 expression. PPM1D amplification was not observed in Wip1 negative cases. In the cell viability assay, the suppression of the MCF7 cell growth was observed by Wip1 inhibitor. Conclusions Wip1 nuclear protein expression may be regulated by PPM1D amplification, and the Wip1 inhibitor may have the therapeutic effects for the breast cancer with PPM1D amplification or high Wip1 protein expression. Citation Format: Yuka Inoue, Nami Yamashita, Eriko Tokunaga, Hiroyuki Kitao, Kimihiro Tanaka, Hiroki Ueo, Hiroshi Saeki, Eiji Oki, Yoshihiko Maehara. Biological significance of the wild-type p53-induced phosphatase 1(Wip1) expression in invasive breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3696.

Abstract P5-13-05: The relationship between the expression of FOXA1 and GATA3 and the efficacy of neoadjuvant endocrine therapy

Background. The estrogen receptor (ER)/ GATA3/ Forkhead box A1 (FOXA1) network is necessary for the ERα functional signature specific to luminal type breast cancers. High expression of FOXA1 indicates a good prognosis in ER-positive breast cancer. However, little is known about the association between the expression of FOXA1 and GATA3, and the efficacy of neoadjuvant endocrine therapy (NAE). This study investigated their predictive potential for NAE and the changes of their expression after NAE. Methods. The expression of ER, progesterone receptor (PgR), Ki67, FOXA1, and GATA3 were analyzed by immunohistochemistry in 66 patients with hormone receptor-positive/ human epidermal growth factor receptor 2 (HER2)-negative breast cancer who had been treated with NAE between March 2003 and December 2012 at Kyushu University Hospital, National Kyushu Cancer Center, and Sagara Hospital. The association between the expression of biological marker and the efficacy of NAE, and their expression changes after NAE were evaluated. Results. The median age of the patients was 60 years (range, 30–84 years). Pre- and post-menopausal patients were 24 (36.4%) and 42 (63.6%). Endocrine agents that were administered are as follows: aromatase inhibitors (AIs) for 42 patients (63.6%), luteinizing hormone-releasing hormone (LHRH) agonist plus AI for 10 patients (15.2%), LHRH agonist plus tamoxifen for 13 patients (19.7%). NAE yielded a partial response (PR) in 21 patients (31.8%) and stable disease (SD) in 45 patients (68.2%). Breast conserving surgery was performed in 56 patients (84.8%) and mastectomy was performed in 10 patients (15.2%). Preoperative Endocrine Prognostic Index (PEPI) score was 0 in 10 patients (15.2%) and 1 or greater (score 1 ≤) in 56 patients (84.8%). Pre-treatment FOXA1 expression was positively correlated with GATA3 (P = 0.0003) and PgR (P = 0.0138). Post-treatment Ki67 expression was significantly lower in tumors, which achieved PR compared with those with SD (P = 0.0007). The expression of PgR, Ki67, and FOXA1 was significantly lower in post-treatment tumors compared with those in pre-treatment samples (p 20%, the expression of Ki67 and FOXA1 were significantly lower in post-treatment tumors (P Conclusions. FOXA1 expression correlated with PgR expression, and was reduced significantly after NAE. These results suggest that blocking the effect of estrogen might reduce FOXA1 expression. Citation Format: Tanaka K, Tokunaga E, Inoue Y, Ueo H, Yamashita N, Sagara Y, Ohi Y, Taguchi K, Ohno S, Okano S, Kitao H, Oki E, Oda Y, Maehara Y. The relationship between the expression of FOXA1 and GATA3 and the efficacy of neoadjuvant endocrine therapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-13-05.

Abstract P5-06-06: Expression of APOBEC3B in primary breast cancer of Japanese women

Background: Human cancer genomes contain tens of thousands of mutations. APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) family of cytidine deaminases normally function in innate immune responses that protects against retrovirus and retrotransposon propagation. However, these enzymes can also deaminate cytosines in the host genome and generate C to T mutations. APOBEC3B is overexpressed in several human cancer types, and this overexpression correlates with the pres[not]ence of the APOBEC3B mutation signature. Recent studies have demonstrated APOBEC3B as a source of mutations in various malignancies including breast cancers. However, the relationships between the expression of APOBEC3B in breast cancer and the clinicopathological features have not been fully elucidated. Aims: To investigate the expression of APOBEC3B mRNA in primary breast cancers and to evaluate the relationships between the APOBEC3B mRNA expression and the clinicopathological characteristics and prognosis in the primary breast cancer of Japanese women. Methods: Specimens were obtained from 305 patients with primary breast cancers who underwent surgery without neoadjuvant systemic therapy. APOBEC3B mRNA expression was analysed using quantitative reverse transcription-PCR (qRT-PCR). The APOBEC3B expression level was normalized to that of the constitutive housekeeping gene TATA binding protein (TBP). Four breast cancer subtypes were determined by the immunohistochemical analysis of ER, PR and HER2; hormone receptor (HR; ER and/or PR)+/HER2-, HR+/HER2+, HR-/HER2+(HER2) and triple negative (TN). Results: Expression of APOBEC3B mRNA was detected in 277 tumors, while it was not detected in 28 tumors. The APOBEC3B expression was significantly higher in ER-negative, PR-negative, high grade tumors. The APOBEC3B expression was positively correlated with Ki67 index and highest in TN and lowest in HR+/HER2- subtype. There were no correlations between the APOBEC3B expression and age, tumor size, lymph node metastasis and the stage. The APOBEC3B expression was not statistically different between invasive ductal carcinoma and DCIS, suggesting that the APOBEC3B expression is related to the carcinogenesis of breast cancer. High expression of APOBEC3B was significantly associated with the poor recurrence free survival in all cases and ER-positive cases; however, APOBEC3B expression was not related to the prognosis in ER-negative tumors. Conclusions: The high APOBEC3B expression was related to the aggressive phenotype of breast cancer and the poor prognosis. Citation Format: Eriko Tokunaga, Nami Yamashita, Kimihiro Tanaka, Yuka Inoue, Hiroshi Saeki, Eiji Oki, Hiroyuki Kitao, Yoshihiko Maehara. Expression of APOBEC3B in primary breast cancer of Japanese women [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 P5-06-06.