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Current data using gene expression and DNA modification profiles yield limited information for evaluating the potential resistance to therapies. Recent studies show that the chromatin landscape is dynamic and defines cell identity and disease status. Thus, changes in open chromatin sites could provide important information on disease progression and identify potential therapeutic targets. The majority of breast cancers express estrogen receptor (ER) and initially respond to endocrine therapies blocking ER activity. However, endocrine therapy resistance (ETR) occurs de novo or follows an initial response. Recent studies have identified new drivers of ETR, such as mTOR and CDK4/6. Therapy with these inhibitors in combination with endocrine therapy have improved patient survival, but the molecular mechanism of ETR is not fully understood and additional therapeutic options for ETR are needed. To investigate the molecular mechanisms of ETR, we established in vitro Long-Term Estrogen Deprivation (LTED) model using multiple human ER-positive breast cancer cell lines. These cells showed differential time-dependent patterns of ESR1 expression upon LTED with ESR1 upregulation in some cell lines and downregulation in others. LTEDs with ESR1 upregulation were sensitive to fulvestrant, while the others were resistant. We analyzed time-course of chromatin landscape transitions during acquisition of ETR in ESR1 up- and downregulated LTEDs cells by Assay for Transposase Accessible Chromatin (ATAC-seq) combined with RNA-seq. Because transcription factors play a primary key role in gene expression and in cell-fate changes including response to therapies, we performed bioinformatic analysis to uncover differential open chromatin sites, select enhancer signatures and identify digital footprints of transcription factors. Unlike ChIP-seq, which identifies binding sites for a known transcription factor, ATAC-seq permits analysis of all sites potentially accessible to the transcriptional machinery and predict occupancy by ~700 different transcription factors at once. With these algorithms, we found that Grainyhead-like 2 (GRHL2) is associated with development of both ESR1 overexpressed and downregulated types of LTEDs. Furthermore, RNA-seq analysis showed that GRHL2 target genes are upregulated in LTEDs. Furthermore, in the METABRIC database, high GRHL2 expression is significantly associated with poor outcome in ER+ breast cancer patients treated with endocrine therapy. Taken together, these data suggest that GRHL2 may be a novel important transcription factor in endocrine therapy resistant breast cancer regardless of fulvestrant sensitivity. Our studies provide evidence that chromatin landscape analysis, coupled with the transcription factor network algorithm, is capable of identifying novel biomarkers or therapeutic targets, This approach will expand the range of translational research as it is applicable to many cancers and diseases. Citation Format: Saori Fujiwara, Songjoon Baek, Kaustubh Wagh, Diana Stavreva, Lyuba Varticovski, Gordon Hager. Chromatin landscape analysis based on ATAC-seq and RNA- seq reveals that GRHL2 is a novel key transcription factor for endocrine therapy resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-06.

This is a review article based on the international symposium report of the "US-Japan Conference on Advances in Oncology: Cancer and Infectious Diseases" held online on June 25, 2021, which provided an update on the association between oncology and infectious disease research from cutting-edge basic science to high-impact clinical trials.

The cohesin complex is central to chromatin looping, but mechanisms by which these long-range chromatin interactions are formed and persist remain unclear. We demonstrate that interactions between a transcription factor (TF) and the cohesin loader NIPBL regulate enhancer-dependent gene activity. Using mass spectrometry, genome mapping, and single-molecule tracking methods, we demonstrate that the glucocorticoid (GC) receptor (GR) interacts with NIPBL and the cohesin complex at the chromatin level, promoting loop extrusion and long-range gene regulation. Real-time single-molecule experiments show that loss of cohesin markedly diminishes the concentration of TF molecules at specific nuclear confinement sites, increasing TF local concentration and promoting gene regulation. Last, patient-derived acute myeloid leukemia cells harboring cohesin mutations exhibit a reduced response to GCs, suggesting that the GR-NIPBL-cohesin interaction is defective in these patients, resulting in poor response to GC treatment.

INTRODUCTION AND OBJECTIVE:Alterations of androgen receptor (AR) binding across the human genome and subsequent reprogramming of active regulatory sites are associated with prostate tumorigenesis a...

In the nucleus, genomic DNA is wrapped around histone octamers to form nucleosomes. In principle, nucleosomes are substantial barriers to transcriptional activities. Nuclear non-coding RNAs (ncRNAs) are proposed to function in chromatin conformation modulation and transcriptional regulation. However, it remains unclear how ncRNAs affect the nucleosome structure. Eleanors are clusters of ncRNAs that accumulate around the estrogen receptor-α (ESR1) gene locus in long-term estrogen deprivation (LTED) breast cancer cells, and markedly enhance the transcription of the ESR1 gene. Here we detected nucleosome depletion around the transcription site of Eleanor2, the most highly expressed Eleanor in the LTED cells. We found that the purified Eleanor2 RNA fragment drastically destabilized the nucleosome in vitro. This activity was also exerted by other ncRNAs, but not by poly(U) RNA or DNA. The RNA-mediated nucleosome destabilization may be a common feature among natural nuclear RNAs, and may function in transcription regulation in chromatin., The Eleanor cluster of non-coding RNAs is localised upstream of estrogen receptor-α (ESR1) gene locus in estrogen-deprived breast cancer cells. Fujita et al find that RNA fragments of Eleanor2 and of other non-coding RNAs are able to destabilise nucleosomes in vitro, suggesting a role in transcriptional regulation.

// Takashi Takeshita 1 , Yutaka Yamamoto 1 , Mutsuko Yamamoto-Ibusuki 2 , Toko Inao 1 , Aiko Sueta 1 , Saori Fujiwara 1 , Yoko Omoto 1, 3 , Hirotaka Iwase 1 1 Department of Breast and Endocrine Surgery, Graduate School of Medical Science, Kumamoto University, Chuo-ku, Kumamoto, 860–8556, Japan 2 Department of Molecular-Targeting Therapy for Breast Cancer, Kumamoto University Hospital, Chuo-ku, Kumamoto, 860–8556, Japan 3 Department of Endocrine and Breast Surgery, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Hirokoji Agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602–0841, Japan Correspondence to: Hirotaka Iwase, email: hiwase@kumamoto-u.ac.jp Keywords: estrogen receptor-positive metastatic breast cancer, acquired endocrine therapy resistance, cell-free DNA ESR1 mutations in plasma, droplet digital PCR Received: January 11, 2016 Accepted: March 14, 2016 Published: April 19, 2016 ABSTRACT Background: The measurement of circulating cell-free DNA (cfDNA) may transform the management of breast cancer patients. We aimed to investigate the clinical significance of sequential measurements of ESR1 mutations in primary breast cancer (PBC) and metastatic breast cancer (MBC) patients. Results: ESR1 mutations ratio in the PBC groups was used as the minimum cutoff for determining increases in cfDNA ESR1 mutation ratio. An increase in cfDNA ESR1 mutations was found in 13 samples of cfDNA from 12 (28.6%) out of 42 MBC patients. A total of 10 (83.3%) out of 12 MBC patients with increase cfDNA ESR1 mutations showed a poor response to treatment. In survival analysis, increase cfDNA ESR1 mutations may predict a shorter duration of post-endocrine-therapy effectiveness ( P = 0.0033). Methods: A total of 119 patients (253 plasma samples) with breast carcinoma were enrolled in this study. Cases were selected if archival plasma samples were available from PBC before and after treatment and from MBC gathered more than twice at the time of progression. cfDNA was isolated from the 77 PBC patients (154 plasma samples) and from the 42 MBC patients (99 plasma samples). To investigate any changes in each cfDNA ESR1 mutation before and after treatment, we analyzed the difference with cfDNA ESR1 mutations ratio in the first blood sample using droplet digital polymerase chain reaction (ddPCR). Conclusions: We demonstrate that ddPCR monitoring of the recurrent ESR1 mutation in cfDNA of MBC patients is a feasible and useful method of providing relevant predictive information.

Until recently, there had not been an effective systemic chemotherapy for advanced differentiated thyroid carcinoma (DTC); lenvatinib, a multi-tyrosine kinase inhibitor, has been proven effective for DTC, but has also been revealed to have adverse side effects including hypertension, hand-foot syndrome (HFS) and diarrhea. There have been few clinical studies focused on the characteristics, safety concerns or precautions for lenvatinib treatment in elderly patients. The present study administered lenvatinib to 18 patients with DTC in Kumamoto University Hospital (Kumamoto, Japan), with 9 patients in both the younger group (75 years old.

DNA accessibility to transcription regulators varies between cells and modulates gene expression patterns. Several “open” chromatin profiling methods that provide valuable insight into the activity of these regulatory regions have been developed. However, their application to clinical samples has been limited despite the discovery that the Analysis of Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) method can be performed using fewer cells than other techniques. Obtaining fresh rather than stored samples and a lack of adequate optimization and quality controls are major barriers to ATAC’s clinical implementation. Here, we describe an optimized ATAC protocol in which we varied nuclear preparation conditions and transposase concentrations and applied rigorous quality control measures before testing fresh, flash frozen, and cryopreserved breast cells and tissue. We obtained high quality data from small cell number. Furthermore, the genomic distribution of sequencing reads, their enrichment at transcription start sites, and transcription factor footprint analyses were similar between cryopreserved and fresh samples. This updated method is applicable to clinical samples, including cells from fine needle aspiration and tissues obtained via core needle biopsy or surgery. Chromatin accessibility analysis using patient samples will greatly expand the range of translational research and personalized medicine by identification of clinically-relevant epigenetic features.

Recently, research into the development of new targeted therapies has focused on specific genetic alterations to create advanced, more personalized treatment. One of the target genes, fibroblast growth factor receptor‐1 (FGFR1), has been reported to be amplified in estrogen receptor (ER)‐positive subtype breast cancer, and is considered one possible mechanism of endocrine resistance through cross‐talk between ER and growth factor receptor signaling. We performed a comprehensive analysis of FGFR1 at the levels of gene copy number, transcript and protein expression, and examined the relationships between FGFR1 status and clinicopathological parameters, including prognosis in 307 ER‐positive/HER2‐negative primary breast cancer patients treated with standard care at our institute. Most notably, a high level of FGFR1 protein expression was observed in 85 patients (27.7%), and was positively associated with invasive tumor size (P = 0.039). Furthermore, univariate analysis revealed that high FGFR1 protein expression was significantly correlated with poor relapse‐free survival rate (P = 0.0019, HR: 2.63, 95% confidence interval: 1.17–5.98), and showed a tendency towards an increase in recurrent events if the observation period extended beyond the 5 years of the standard endocrine treatment term. FGFR1 gain/amplification was found in 43 (14.0%) patients, which was only associated with higher nuclear grade (P = 0.010). No correlation was found between FGFR1 mRNA expression levels and any clinicopathological factors. Overall, the level of FGFR1 protein expression may be a biomarker of ER‐positive/HER2‐negative primary breast cancer with possible resistance to standard treatment, and may be a useful tool to identify more specific patients who would benefit from FGFR‐1 targeted therapy.

Background: The Cancer Genome Atlas (TCGA) showed that copy number gain/amplification of FGFR1 was around 10% in primary breast cancer. FGFR1 gene amplification in breast cancer has been reported in some studies, more likely seen in ER-positive subtype. Several preclinical and clinical studies demonstrated that FGFR1 was one of novel targets of therapy for metastatic breast cancer. Previous studies suggested that aberrant FGFR1 expression was associated with poor prognosis, while there was no report that compared copy number aberration, mRNA and protein expression. The aim of this study is to analyze FGFR1 gene copy number, expression levels of FGFR1 mRNA and FGFR1 protein in ER-positive/HER2-negative primary breast cancer, and to examine the relationship between FGFR1 status and clinicopathological parameters including prognosis. Methods: The cohort of this study included 307 ER-positive/HER2-negative primary invasive breast cancer patients treated with standard care at Kumamoto University Hospital between June 2000 and January 2011. We performed a comprehensive analysis of FGFR1 at the levels of gene copy number, mRNA and FGFR1 protein expression analyzed by qPCR, qRT-PCR and immunohistochemistry, respectively. Results: FGFR1 gain/amplification was identified in 43 (14.0%) out of 307 patients. FGFR1 gain/amplification had significantly associated with higher nuclear grade (p=0.010). No correlations between FGFR1 mRNA expression levels and any clinicopathological factors were found. Expression levels of FGFR1 protein was positively associated with invasive tumor size (p=0.039). Modest positive correlations between these three (FGFR1 gene gain/amplification, expression levels of FGFR1 mRNA and FGFR1 protein) were found. The univariate analysis revealed that high FGFR1 protein expression was significantly related to poor prognosis (p=0.0019, HR: 2.63, 95%CI: 1.17-5.98) in terms of relapse-free survival (RFS) but not breast cancer-specific survival. The univariate analysis did not show that any factors except FGFR1 protein expression were significantly associated with RFS in this cohort. Conclusion: Expression levels of FGFR1 protein may be an independent prognostic factor in terms of RFS for ER-positive/HER2-negative breast cancer patients receiving standard care. Citation Format: Tomiguchi M, Yamamoto Y, Yamamoto-Ibusuki M, Yamaguchi R, Fujiki Y, Fujiwara S, Sueta A, Takeshita T, Inao T, Iwase H. FGFR1 protein expression is associated with prognosis in primary breast cancer: A comprehensive analysis of gene copy number, mRNA and protein expression. [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 P4-09-01.

Estrogen receptor (ER) α-positive breast cancers frequently acquire resistance to endocrine therapy. However, recent studies found that a fraction of these tumors overexpress ER, and that estrogen treatment induces apoptosis. We propose a 'cancer navigation' strategy to systematically lead resistant cells to growth arrest and apoptosis.

Endocrine therapy resistance is one of the major challenges in breast cancer therapy today. As the causes are not well understood, therapeutic options are limited. To investigate the molecular mechanisms of endocrine therapy resistance, we established in vitro Long-Term Estrogen Deprivation (LTED) models using multiple human ER-positive breast cancer cell lines. These cells showed differential time-dependent patterns of ESR1 expression with upregulation in some cell lines and downregulation in others. Although all cells continued to grow, none acquired mutations in the ESR1 ligand binding domain, reported to be responsible for endocrine therapy resistance. Current data using gene expression and DNA modification profiles yield limited information for molecular mechanisms of resistance and potential for developing novel therapies. The chromatin landscape is a potential identifier of disease state, and can provide specific information on transcriptional regulation by enhancers. Thus, changes in chromatin open/closed sites could provide important information to predict disease progression and can be used to identify potential therapeutic targets. We analyzed the time-course of chromatin landscape transitions during acquisition of endocrine resistance by the Assay for Transposase Accessible Chromatin (ATAC-seq), and combined these studies with RNA-seq and genomic DNA sequence analysis. ATAC-seq permits the analysis of all sites potentially accessible to the transcriptional machinery. Furthermore, digital footprinting of protected transcription factor motifs within the accessible regions can evaluate the occupancy of hundreds of different transcription factors at one time. We performed bioinformatic analysis of open and closed chromatin sites and selected relevant enhancer signatures. Because transcription factors play a primary role in gene expression and in cell-fate changes, including response to therapies, we derived digital footprints from transcription factor occupancy to correlate this information with phenotypes of LTED cells. Comparing changes in the chromatin landscape with RNA-seq, we created a high throughput algorithm that integrates the information from open/closed chromatin sites, transcription factors occupancy, and gene expression. We found key transcription factors for acquisition of LTED cells; Myc-Max is associated with ESR1 overexpressed LTEDs, whereas Oct1 and Hox genes are associated with LTEDs with absence of ESR1 expression. These methods establish novel mechanisms of endocrine therapy resistance in breast cancer that can also be applied to other tumor types. Our studies provide a rationale for using chromatin landscape and transcription factor networks to expand the range of translational research in the search for novel therapies for breast cancer. These studies will potentially have high impact on development of important novel therapeutic approaches in cancer and many other diseases. Citation Format: Saori Fujiwara. Transcription factor dynamics in endocrine therapy resistance models using a novel chromatin landscape analysis algorithm based on ATAC-Seq [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-24.

Human epidermal growth factor receptors (HERs) are known to play a pivotal role in breast cancer, both as prognostic markers and as therapeutic targets. The importance of Her4 expression is, however, still controversially discussed; there are few reports on the clinical significance of HER4, its splice variants, and cleaved HER4 intracellular domains (4ICD) which function differently depending on their localization in breast cancer. In 238 primary invasive breast cancer patients, we analyzed the expression levels of HER4 extracellular (JM-a and JM-b) and intracellular (CYT-1 and CYT-2) domains as well as 4ICD localization, and tested the relationship with clinicopathological characteristics and prognosis. The predominantly-expressed extracellular domain was JM-a, and lower CYT-2 dominance was a factor related to better relapse-free survival. CYT-2-dominance with higher nuclear 4ICD expression was a favorable prognostic marker especially in patients with the ER+ HER2- subtype treated with endocrine therapy. The absence of cytoplasmic 4ICD staining was related to better prognosis in CYT-1-dominant patients. In conclusion, analysis of splicing variants and 4ICD localization should be considered when targeting HER4 as a novel ER+/HER2- breast cancer treatment.

Endocrine therapies that blocks estrogen production are effective for estrogen receptor (ER)-positive breast cancer. However, endocrine therapy treated patients eventually experience relapse after a long period of estrogen deprivation. The mechanism underlying acquisition of estrogen independent growth by ER positive breast cancer cells remains unclear. To understand such molecular mechanism, we used a cell model LTED (long term estrogen deprivation) which MCF7 cells were cultured under estrogen deprivation for 4-10 months. In LTED cells, we found that ER encoded gene ESR1 was up-regulated and ER overproduction was essential for estrogen-independent cell growth. We also revealed that RNA transcriptions of the ESR1 and several neighbor genes were co-induced from both coding and non-coding regions in LTED cells, using RNA-sequence. These highly transcribed regions were corresponded to active histone modifications and transcription factor bindings according to publically available genome-wide analyses data. Fluorescence in situ hybridization (FISH) analyses indicated that RNA from the chromatin domain region nearby ESR1 were co-localized and made foci in nucleus. We found non-coding regions that are particularly highly transcribed. FISH analyses indicated that RNAs from these regions might interact with the parental ESR1 gene locus. Recent studies have shown that non-coding RNAs are involved in transcriptional regulation and chromatin regulation. To understand the role of the non-coding RNA, we have generated MCF7 cells lines that lack the non-coding site, using CRISPR/CAS9 system. We found that mRNA transcription of multiple genes including ESR1 were impaired by the deletion. These findings suggested that these non-coding RNAs may be involved in chromatin regulation of the chromatin domain nearby ESR1. In this study, we found non-coding RNAs that control transcription of chromatin domain genes in ER positive breast cancer cells. Such non-coding RNA mediated transcriptional regulation might be critical for endocrine therapy resistance adaptation. Citation Format: Fujiwara S, Saitoh N, Tomita S, Abdalla MO, Iwase H, Nakao M. Non-coding RNAs derived from near the ESR1 gene acts as a transcriptional regulator during estrogen deprivation adaptation of ER positive breast cancer cells. [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 P3-05-10.

Cylindromatosis (CYLD) is a tumor suppressor gene that is mutated in familial cylindromatosis, a rare autosomal dominant disorder associated with numerous benign skin adnexal tumors. CYLD is now known to regulate various signaling pathways, including transforming growth factor-β signaling, Wnt/β-catenin signaling, and NF-κB signaling by deubiquitinating upstream regulatory factors. Downregulation of CYLD has been reported in several malignancies; however, the clinical significance of CYLD expression in many malignancies, including breast cancer, remains to be elucidated. This study investigated the clinical significance of CYLD in breast cancer and its roles in tumor progression. We evaluated CYLD expression in matched normal breast tissue samples and tumor breast tissue samples from 26 patients with breast cancer and in a series of breast cancer cell lines. In addition, by means of immunohistochemistry, we investigated CYLD protein expression and its clinical significance in 244 breast cancer cases. We also analyzed the effects of CYLD repression or overexpression on breast cancer cell viability, cell migration, and NF-κB activity with or without receptor activator of NF-κB ligand (RANKL) stimulation. Breast cancer tissues demonstrated significantly reduced CYLD mRNA expression compared with normal breast tissues. Downregulation of CYLD promoted cell survival and migratory activities through NF-κB activation, whereas CYLD overexpression inhibited those activities in MDA-MB-231 cells. As an important finding, CYLD overexpression also inhibited RANKL-induced NF-κB activation. Our immunohistochemical analysis revealed that reduced CYLD protein expression was significantly correlated with estrogen receptor negativity, high Ki-67 index, high nuclear grade, decreased disease-free survival, and reduced breast cancer-specific survival in primary breast cancer. Moreover, reduced CYLD expression was an independent factor for poor prognosis in breast cancer. CYLD downregulation may promote breast cancer metastasis via NF-κB activation, including RANKL signaling.

Breast elastography (EG), which can objectively evaluate tumor stiffness, has been useful for differentiation of benign and malignant breast lesions. However, the value of EG for prediction of response to systemic therapy is poorly understood. The baseline evaluations of EG in 55 patients who received neoadjuvant chemotherapy were reviewed. We investigated the correlation between tumor stiffness and response to neoadjuvant chemotherapy. Tumor stiffness was evaluated by the Tsukuba elasticity scoring system. The mean EG scores were significant lower for the clinical and pathologic complete response (pCR) groups than for the others. When we categorized patients into two groups according to tumor stiffness, 26 patients were assigned to the low EG group (soft, scores from 1 to 3) and 29 patients were assigned to the high EG group (hard, score 4 and 5). The low EG group had significantly higher clinical complete response and pCR rates than the high EG group (clinical complete response, low EG group 38 % vs. high EG group 10 %, P = 0.024; pCR, low EG group 50 % vs. high EG group 14 %, P = 0.003, respectively). Furthermore, multivariate analysis indicated that estrogen receptor, human epidermal growth factor receptor 2, and low EG (odds ratio 13.04, 95 % confidence interval 1.19–458.28, P = 0.035) were independent predictive factors of pCR. Tumor stiffness evaluated by EG bears predictive potential for response to neoadjuvant chemotherapy. Stiffness evaluated by EG may be recognized as a clinically significant tumor characteristic, comparable to other data obtained by functional imaging techniques.

It has recently being emphasized that gene-expression profiles are important clinical decision-making tools, and as such must be standardized across hospital laboratories in the same way as pathological investigations. In this study our objective was to independently establish a standardized gene-expression assay system using routinely processed, formalin-fixed, paraffin-embedded (FFPE) tissues. To verify gene expression by quantitative real-time polymerase chain reaction, the most stably expressed reference genes were explored using 30 matched FFPE and fresh frozen (FF) tissues. FFPE specimens from 290 female breast cancer patients were used for further RNA extraction; ESR1 and PGR were measured using 203 matched FFPE and FF specimens and normalized to these reference genes. RNA extracted from FFPE specimens was highly degraded, but almost the same selection of genes was identified—TAF, PUM1, and ACTB, and, for FFPE specimens only, FKBP15. Eventually 88.6% of all the FFPE samples were identified as quantitatively and qualitatively adequate for downstream analysis. The results revealed good correlation and excellent concordance with ERα and PgR protein expression evaluated by immunohistochemistry. Moreover, the distribution of ESR1 and PGR gene expression values was quite reasonable, reflecting differences between the transcriptional mechanisms of the respective genes. We successfully confirmed that our gene-expression analysis system provides good quality control for larger scale assays; it may therefore be suitable for development, in the near future, of a multiple gene assay as a routine clinical judgment tool.

Tumor necrosis factor α plays an important role in the pathogenesis of Crohn’s disease (CD). The effects of infliximab on gastrointestinal bleeding in CD have not yet been fully evaluated. Herein we describe three CD cases who presented with gastrointestinal bleeding and received infliximab treatment. In case 1, double-balloon endoscopy showed a large ulcer with several irregularly shaped ulcers in the terminal ileum; 8 weeks after infliximab administration, complete healing of all lesions was observed. In case 2, double-balloon endoscopy showed linear ulcers and mucosal edema in the jejunum and ileum; 5 weeks after infliximab administration, all lesions were decreased in size and were healed. In case 3, double-balloon endoscopy revealed ulcerations and stenosis in the terminal ileum; 12 weeks after infliximab administration, ulcer healing and an increased diameter of the ileal stenosis were observed. These three cases have been receiving ongoing infliximab maintenance therapy and are currently symptom-free. Infliximab thus appears to be useful for treatment of gastrointestinal bleeding in CD patients.

The cell nucleus is highly organized and functionally compartmentalized. Double-stranded naked DNA is complexed with core histones and assembled into nucleosomes and chromatin, which are surrounded by nuclear domains composed of RNAs and proteins. Recently, three-dimensional views of chromosome organization beyond the level of the nucleosome have been established and are composed of several layers of chromosome domains. Only a small portion of the human genome encodes proteins; the majority is pervasively transcribed into noncoding RNAs whose functions are under intensive investigation. Importantly, the questions of how nuclear retained noncoding RNAs play roles in orchestrating the chromatin structure that have been addressed. We discuss the novel noncoding RNA clusters, Eleanors, which are derived from a large chromatin domain. They accumulate at the site of their own transcription to form RNA clouds in the nucleus, and they activate gene expression in the chromatin domain. Noncoding RNAs have emerging roles in genome regulation that are integrated into the spatial organization of chromatin and the nucleus. WIREs RNA 2017, 8:e1384. doi: 10.1002/wrna.1384 For further resources related to this article, please visit the WIREs website.

PIK3CA is an oncogene that encodes the p110α component of phosphatidylinositol 3‐kinase (PI3K); it is the second most frequently mutated gene following the TP53 gene. In the clinical setting, PIK3CA mutations may have favorable prognostic value for hormone receptor‐positive breast cancer patients and, during the past few years, PIK3CA mutations of cell‐free DNA (cfDNA) have attracted attention as a potential noninvasive biomarker of cancer. However, there are few reports on the clinical implications of PIK3CA mutations for TNBC patients. We investigated the PIK3CA major mutation status of cfDNA as a noninvasive biomarker of cancer using droplet digital polymerase chain reaction (ddPCR), which has high level sensitivity and specificity for cancer mutation, in early‐stage 49 triple negative breast cancer (TNBC) patients. A total of 12 (24.4%) of 49 patients had PIK3CA mutations of cfDNA. In a median follow up of 54.4 months, the presence of PIK3CA mutations of cfDNA had significant impacts on relapse‐free survival (RFS; P = 0.0072) and breast cancer‐specific survival (BCSS; P = 0.016), according to the log‐lank test. In a Cox proportional hazards model, the presence of PIK3CA mutations of cfDNA had significant prognostic value in the univariate and multivariate analysis. Additionally, the presence of PIK3CA mutations of cfDNA was significantly correlated with positive androgen receptor phosphorylated form depending on PI3K signaling pathway (pAR) which is independent favorable prognostic factors of TNBC. We demonstrated that the presence of PIK3CA major mutations of cfDNA could be a discriminatory predictor of RFS and BCSS in early‐stage TNBC patients and it was associated with PI3K pathway‐dependent AR phosphorylation., We demonstrated the presence of PIK3CA major mutations of cfDNA could be discriminatory predictor of RFS and BCSS in early‐stage TNBC patients which may be associated with PI3K pathway dependent AR phosphorylation.

Droplet digital polymerase chain reaction (ddPCR), which could perform thousands of PCRs on a nanoliter scale simultaneously, would be an attractive method to massive parallel sequencing for identifying and studying the significance of low-frequency rare mutations. Recent evidence has shown that the key potential mechanisms of the failure of aromatase inhibitors-based therapy involve identifying activating mutations affecting the ligand-binding domain of the ESR1 gene. Therefore, the detection of ESR1 mutations may be useful as a biomarker predicting an effect of the treatment. We aimed to develop a ddPCR-based method for the sensitive detection of ESR1 mutations in 325 breast cancer specimens, in which 270 primary and 55 estrogen receptor-positive (ER+) metastatic breast cancer (MBC) specimens. Our ddPCR assay could detect the ESR1 mutant molecules with low concentration of 0.25 copies/μL. According to the selected cutoff, ESR1 mutations occurred in 7 (2.5%) of 270 primary breast cancer specimens and in 11 (20%) of 55 ER+ MBC specimens. Among the 11 MBC specimens, 5 specimens (45.5%) had the most common ESR1 mutation, Y537S, 4 specimens (36.3%) each had D538G, Y537N, and Y537C. Interestingly, 2 patients had 2 ESR1 mutations, Y537N/D538G and Y537S/Y537C, and 2 patients had 3 ESR1 mutations, Y537S/Y537N/D538G. Biopsy was performed in heterochrony in 8 women twice. In 8 women, 4 women had primary breast cancer and MBC specimens and 4 women had 2 specimens when treatment was failure. Four of these 8 women acquired ESR1 mutation, whereas no ESR1 mutation could be identified at first biopsy. ddPCR technique could be a promising tool for the next-generation sequencing-free precise detection of ESR1 mutations in endocrine therapy resistant cases and may assist in determining the treatment strategy.

Background Estrogen receptor (ER) positive breast cancer can often be treated by hormone therapy; however a certain population of ER-positive patients become resistant to hormone therapy after long-term hormone treatment. Ethinylestradiol (EE2) is a derivative of estrogen, which has shown promising effects in these patients. Methods We successfully obtained tissue samples from 6 patients undergoing EE2 treatment and examined 13 well-known breast cancer-related factors by immunohistochemistry. Of the 6 patients, 5 responded but one patient did not. Results Before EE2 treatment, staining for both ER and androgen receptor (AR) was strong in the nucleus, and the progesterone receptor (PgR) was almost no staining. EE2 treatment significantly down-regulated ER and up-regulated PgR while nuclear and cytosolic AR were oppositely down- and up-regulated, respectively. Cytosolic staining of BRCA1 was significantly up-regulated by EE2 whereas nuclear staining tended to decrease. Individual comparisons suggested less induction of PgR and decreasing AKT but increasing pAKT in the non-responder following EE2 treatment. Conclusions Our observations revealed that EE2 activated ER downstream genes; however it did not stimulate cell growth. This suggests that hormone resistant cells might receive growth signals from a non-genomic pathway and this may be reflected in their sensitivity to EE2 treatment.

Estrogen receptor-α (ER)-positive breast cancer cells undergo hormone-independent proliferation after deprivation of oestrogen, leading to endocrine therapy resistance. Up-regulation of the ER gene (ESR1) is critical for this process, but the underlying mechanisms remain unclear. Here we show that the combination of transcriptome and fluorescence in situ hybridization analyses revealed that oestrogen deprivation induced a cluster of noncoding RNAs that defined a large chromatin domain containing the ESR1 locus. We termed these RNAs as Eleanors (ESR1 locus enhancing and activating noncoding RNAs). Eleanors were present in ER-positive breast cancer tissues and localized at the transcriptionally active ESR1 locus to form RNA foci. Depletion of one Eleanor, upstream (u)-Eleanor, impaired cell growth and transcription of intragenic Eleanors and ESR1 mRNA, indicating that Eleanors cis-activate the ESR1 gene. Eleanor-mediated gene activation represents a new type of locus control mechanism and plays an essential role in the adaptation of breast cancer cells., Estrogen-receptor-positive breast cancer cells undergo hormone-independent proliferation after long-term oestrogen deprivation and become resistant to endocrine therapies. Here, the authors report a cluster of noncoding RNAs important for this adaptation process.

Genome-wide association studies have identified a single-nucleotide polymorphism (SNP) to be associated with an increased risk of breast cancer. The biology of one of the susceptibility locus C6ORF-ESR1 and whether it also contributes to progression of established disease has not yet been ascertained. We examined the association of rs2046210 and its six linkage disequilibrium SNPs with clinicopathological characteristics, prognosis, and gene expression levels of ESR1 and the C6ORFs (C6ORF97:CCDC170, C6ORF211, C6ORF96:RMND1) in 344 breast cancer tissue samples and 253 corresponding samples of adjacent normal tissue. Tumor genotypes with homozygous risk alleles were more frequent than normal tissues. The tumor genotypes of rs2046210 and rs6929137 with homozygous risk alleles showed worse relapse-free survival (RFS, P=0.038 and P=0.031, respectively), whereas no notable associations were observed with either clinicopathological characteristics or expression of the peripheral genes. Higher C6ORF97 expression correlated with ER negativity (P

The cell nucleus is highly organized and functionally compartmentalized. Double-stranded naked DNA is complexed with core histones and assembled into nucleosomes and chromatin, which are surrounded by nuclear domains composed of RNAs and proteins. Recently, three-dimensional views of chromosome organization beyond the level of the nucleosome have been established and are composed of several layers of chromosome domains. Only a small portion of the human genome encodes proteins; the majority is pervasively transcribed into noncoding RNAs whose functions are under intensive investigation. Importantly, the questions of how nuclear retained noncoding RNAs play roles in orchestrating the chromatin structure that have been addressed. We discuss the novel noncoding RNA clusters, Eleanors, which are derived from a large chromatin domain. They accumulate at the site of their own transcription to form RNA clouds in the nucleus, and they activate gene expression in the chromatin domain. Noncoding RNAs have emerging roles in genome regulation that are integrated into the spatial organization of chromatin and the nucleus. WIREs RNA 2017, 8:e1384. doi: 10.1002/wrna.1384 For further resources related to this article, please visit the WIREs website.

Human epidermal growth factor receptor 2 (HER2) is amplified in human breast cancers in which therapy targeted to HER2 significantly improves patient outcome. We re-visited the use of real-time quantitative polymerase chain reaction (qPCR)-based assays using formalin-fixed paraffin-embedded (FFPE) tissues as alternative methods and investigated their particular clinical relevance.DNA and RNA were isolated from FFPE specimens and HER2 status was assessed by qPCR in 249 consecutive patients with primary breast cancer. Concordance with results forg immunohistochemistry (IHC) and in situ hybridization (ISH), clinical characteristics and survival was assessed.HER2 gene copy number had a stronger correlation with clinicopathological characteristics and excellent concordance with IHC/ISH results (Sensitivity: 96.7 %; concordance: 99.2 %). HER2 gene expression showed inadequate sensitivity, rendering it unsuitable to determine HER2 status (Sensitivity: 46.7 %; concordance: 92.1 %), but lower HER2 gene expression, leading to the classification of many cases as "false negative", contributed to a prediction of better prognosis within the HER2-amplified subpopulation.Quantitative HER2 assessments are suggested to have evolved their accuracy in this decade, which can be a potential alternative for HER2 diagnosis in line with the in situ method, while HER2 gene expression levels could provide additional information regarding prognosis or therapeutic strategy within a HER2-amplified subpopulation.

In the clinical diagnosis of breast cancer, immunohistochemistry panels with estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2) and Ki-67 are routinely used, and they have been proposed for the classification of breast tumors into distinct subtypes. Gene expression analysis with formalin-fixed paraffin-embedded material have also become widely available recently, but the prognostic values of corresponding gene panels compared with these four immunohistochemical panels had never tested. We independently evaluated the 5-year relapse risk-estimation scores using semiquantitative data of four immunohistochemical panels (Ku-IHC4 score) and compared these with the results of four-gene expression profiling of formalin-fixed paraffin-embedded specimens (Ku-FFPE4 score) in a consecutive series of 235 primary invasive breast cancer patients. Ku-IHC4 score was revealed to be an independent predictor of recurrence other than Ku-FFPE4 score in a multivariate model analyzed by classical clinical parameters (Ku-IHC4 score vs Ku-FFPE4 score; χ(2): 14.2 vs 2.5, P: 0.0002 vs 0.11). When patients were trichotomized into high-, intermediate- and low-risk groups using the thresholds determined from the approximately calculated 5-year relapse rate, Kaplan-Meier analyses showed a significant difference among the three groups in Ku-IHC4 score (log-rank, P

Survivin plays a key role in the initiation and progression of breast cancer. However, its prognostic relevance to breast cancer patients has long been a matter of debate. The purpose of this study was to examine the expression of survivin and its role in predicting clinical outcome in a series of human breast cancer cases both at the mRNA and protein level.Formalin-fixed paraffin-embedded tumor tissues from 245 female patients with invasive breast cancer and 13 patients with ductal carcinoma in situ were examined for survivin mRNA by quantitative real-time RT-PCR (RT-qPCR). In addition, 237 of these tumors with invasive breast cancer were available for immunohistochemistry (IHC). The relationship between survivin status and clinicopathological characteristics and prognosis was evaluated.RT-qPCR revealed that high levels of survivin mRNA were strongly associated with high nuclear grade, positive axillary lymph nodes, negative hormone receptor status, positive Her2 amplification, higher Ki67 labeling index, and presence of vascular invasion. In the Cox proportional regression model analysis, survivin mRNA was shown to be a significant univariate parameter for relapse-free survival (RFS), distant relapse-free survival (DRFS), and breast cancer-specific survival (BCSS) as well as a significant multivariate parameter for RFS, DRFS, and BCSS. In hormone receptor (HR)-positive/Her2-negative subtype cases, survivin mRNA expression was also an independent predictor in terms of DRFS. Immunohistochemically, positive staining was seen in the cytoplasm and/or nucleus of cancer cells, although this did not correlate with the mRNA level, and harbored no prognostic value.High mRNA expression of survivin was an independent marker of poor prognosis both in the entire cohort and in the HR-positive/Her2-negative subtype, whereas the protein expression of survivin was not. These findings suggest that RT-qPCR can provide more reliable data than IHC in validating the prognostic significance of survivin for breast cancer patients.

Human epidermal growth factor receptor type 2 (Her2)/ErbB2 plays a key role in the initiation and progression of invasive breast cancer. However, the prognostic relevance to breast cancer patients of the other ErbB family members has long been a matter of debate. In a series of 250 primary invasive breast cancer patients, we performed a comprehensive analysis of ErbB1–4 at the levels of mRNA expression and gene copy number using real-time quantitative PCR. The relationship between the status of ErbB1–4 and the clinicopathological characteristics or prognosis was evaluated. The mRNA expression of ErbB2, but not the other ErbB genes, was significantly correlated to copy number (P = 0.0005). ErbB3 and ErbB4 mRNA expression were positively correlated to each other (P

Ki67 is a protein associated with cell cycle activity and shows a good correlation with the growth fraction, which has been proposed as a prognostic or predictive marker in breast cancer. In this study, we aimed to analyze the expression levels of Ki67 (MKI67) messenger RNA (mRNA) derived from formalin-fixed paraffin-embedded (FFPE) tissues for comparison with the immunohistochemical Ki67 labeling index, and investigate the correlation coefficients with clinical outcomes. We analyzed the data of Ki67 mRNA from FFPE and matched fresh-frozen (FF) tissues based on a real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assay system in 203 cases of primary invasive breast cancer. The correlation between Ki67 mRNA expression of either FFPE or FF specimens and Ki67 labeling index was positive, as was the correlation between the FFPE and FF results (P

Background: Estrogen receptor (ER) positive breast cancer can be treated by endocrine therapy; however a certain population of ER-positive patients becomes resistant to endocrine therapy after long-term estrogen deprivation treatment. ESR1 gene alterations, mutations, translocation and amplification, have been reported as one of the reasons. Patients and Methods: We successfully obtained metastatic tissue samples from 30 patients with resistance to aromatase inhibitor (AI), and 7 patients with resistance to ethinylestradiol (EE2). ER and PgR expression by immunohistochemistry, and ESR1 gene amplification using FISH and copy number variation (CNV) by PCR, and ESR1 gene mutation including Y537N, Y537C, Y 537S and D538G by droplet digital PCR, were examined, in order to evaluate the endocrine responsiveness. Results: High expression of ER (median; 90%) and low expression of PgR (median 5%) were seen in majority of the patients treated by AI. Signal enhancement of DNA-FISH (> 2.0 fold) was seen in 2 (8%) of 25 patients, and signal gain (1.0< ≤2.0 fold) was seen in 11 (44%) of 25 patients. The increasing of DNA-CNV of ESR1 gene was seen in 3 cases of the 25 examined cases. There was no relationship between DNA-FISH and CNV of ESR1. High frequent ESR1 mutations were seen in 7 (28%) at Y537N, 4 (16%) at Y537C, 11 (44%) at Y 537S, and 2 (8%) at D538G. Double mutation, Y537N/S, was seen in 6 (24%) patients, who tended to unresponsive to subsequent endocrine therapy. After EE2 treatment, ER expression was decreased and PgR expression was increased, but there was no relationship between EE2 treatment and ESR1 gene alteration. Conclusion: In the patients with resistance to AI, high ER / low PgR expression was frequently seen, which was no correlation to ESR1 DNA-amplification. The long non-coding RNA in ESR1 gene locus might be related to this discrepancy (Tomita et al; Nat Commun. 2015). Furthermore, accumulation of ESR1 gene mutations tended to be related to endocrine unresponsiveness. Citation Format: Hirotaka Iwase, Takashi Takeshita, Mutsuko Yamamoto-Ibusuki, Touko Inao, Aiko sueta, Saori Fujiwara. ESR1 gene alterations in the metastatic breast cancer with resistance to aromatase inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A40.

Purpose: We aimed to develop a droplet digital Polymerase Chain Reaction (ddPCR)-based method for the sensitive detection of estrogen receptor (ER) α (ESR1) mutations of tumor tissues and the corresponding cell-free DNA (cfDNA) in recurrent/ metastatic breast cancer. Experimental Design: We investigated a total of 325 tumor specimens (270 primary breast cancer specimens and 55 ER-positive recurrent/metastatic tumor specimens, in which 33 recurrent/ metastatic tumor specimens had the corresponding plasma). We investigated the quantification of rare ESR1 mutations, four representative types, Y537S, Y537N, Y537C, and D538G in 33 extracted genomic DNA (gDNA) and its corresponding cfDNA using ddPCR system that simultaneously performed thousands of PCRs on a nanoliter scale. Results: A total of 33 (60 %) of 55 recurrent/ metastatic breast cancer specimens had the corresponding plasma which were evaluated for ESR1 mutations' analysis for cfDNA. We verified the correlation of each ESR1 mutation between cfDNA and tissue gDNA. Only ESR1 Y537C was correlated between cfDNA and tissue gDNA (r = 0.56, P = 0.0006). ESR1 Y537S in tissue gDNA were tend to be correlated Y537N in cfDNA (r = 0.35, P = 0.043). CEA (Carcinoembryonic antigen), which is one of the representative breast cancer tumor markers, was correlated with ESR1 Y537S in cfDNA (r = 0.74, P < 0.0001) and tend to be correlated with Y537S in tissue gDNA (r = 0.36, P = 0.033) and D538G in cfDNA (r = 0.54, P = 0.0011). We also evaluated the correlation between the presence of ESR1 mutations and best overall response (BOR) to post-endocrine therapy. BOR to post-endocrine therapy was 25 stable disease (SD) and 8 progressive disease (PD) in 33 patients. Using each selected cut-off, a total of 4 (50%) of 8 PD patients had ESR1 cfDNA mutations as follow: each patient had Y537S, Y537N, D538G, and double mutations (Y537S and D538G). The presence of ESR1 cfDNA mutations was much higher in PD group, compared with SD group (P = 0.0073) Conclusions: We demonstrated the sensitive detection and accurate quantification of low frequency ESR1 mutations in 33 tumor tissues and the corresponding cell-free DNA (cfDNA) using ddPCR assay in recurrent/ metastatic breast cancer. Our results highlight that ESR1 mutations may be of clinical relevance for metastatic breast cancer patient. Citation Format: Takashi Takeshita, Yutaka Yamamoto, Mutsuko Yamamoto-Ibusuki, Toko Inao, Aiko Sueta, Saori Fujiwara, Hirotaka Iwase. Clinical significance of ESR1 mutations by cell-free DNA in recurrent/metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A44.

1061 Background: Preclinical study has revealed that tumor stiffness can force tumor progression. Tumor stiffness appears to be caused by increased matrix cross-linking and changing in a number of ...

Background Aurora A kinase, a centrosomal serine/threonine kinase which plays an essential role in chromosome segregation during cell division, is commonly amplified and/or over expressed in human malignancies. Aurora A is suggested to be one of the proliferation parameters which is an independent prognostic factor for early invasive breast cancer patients; however the individual clinical or prognostic relevance of this gene has been a matter of debate. Methods A comprehensive analysis of Aurora A at the levels of gene expression, gene copy number and protein expression was performed for 278 primary invasive breast cancer patients; and the correlation with clinical outcomes were investigated. Results Aurora A gene expression level not only correlated with gene amplification, but was also significantly associated with several clinicopathological parameters and patient prognosis. Patients with higher nuclear grade, negative progesterone receptor status and higher Ki67 expressed higher levels of Aurora A mRNA, which was associated not only with poor relapse-free survival (RFS) but was also found to be a significant multivariate parameter for RFS. Aurora A protein expression was also significantly associated with clinicopathological characteristics; lymph node status, nuclear grade, estrogen receptor status and Ki67, but not with prognosis. By contrast, Aurora A gene amplification correlated with tumor size, nuclear grade and Ki67, and had no prognostic value. Conclusion Our data indicate that Aurora A gene expression is an effective tool, which defines both tumor proliferation potency and patient prognosis.