Fibroblast growth factor receptor-1 protein expression is associated with prognosis in estrogen receptor-positive/human epidermal growth factor receptor-2-negative primary breast cancer.

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.

Prognostic role of PIK3CA mutations of cell-free DNA in early-stage triple negative breast cancer.

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.

Clinical significance of CYLD downregulation in breast cancer.

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-ß signaling, Wnt/ß-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.

Comparison of prognostic values between combined immunohistochemical score of estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, Ki-67 and the corresponding gene expression score in breast cancer.

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<0.0001), but not in Ku-FFPE4 score. The high-risk group according to Ku-FFPE4 score showed contradictory low recurrence rates (Ku-IHC4 score vs Ku-FFPE4 score, 53.1 vs 24.8%), which might be caused by risk-dependently extended error ranges. We show that the Ku-IHC4 score, consisted with semiquantitative measures of immunohistochemistry, provides better prognostic information than the corresponding quantitative RNA measurements. Prognostication tools such as the Ku-IHC4 score may be potentially useful in screening which patients had better be assessed by further testing using other genes rather than ER, PgR, HER2 and Ki-67 to determine critical aspects of therapeutic decision making.

A comprehensive analysis of Aurora A; transcript levels are the most reliable in association with proliferation and prognosis in breast cancer.

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.

Locally Administered Photodynamic Therapy for Cancer Using Nano-Adhesive Photosensitizer.

Photodynamic therapy (PDT) is a great potential anti-tumor therapy owing to its non-invasiveness and high spatiotemporal selectivity. However, systemically administered photosensitizers diffuse in the skin and the eyes for a long duration, which cause phototoxicity to bright light and sunlight. Therefore, following PDT, patients must avoid exposure of to light and sunlight to avoid this phototoxicity. In this study, we have developed a locally administered PDT using nano-adhesive porphyrin with polycations consisting of quaternary ammonium salt groups (aHP) as a photosensitizer. The aHP, approximately 3.0 nm in diameter, adhered the negatively charged cell membrane via electrostatic interaction. The aHP localized to the endosome via cell adhesion and induced apoptosis upon 635 nm light irradiation. On being administered subcutaneously on the tumor, 30% of the injected aHP remained in the administered sites. However, low-molecular-weight hematoporphyrin dihydrochloride (HP) disappeared due to rapid diffusion. PDT with locally administered aHP showed a higher anti-tumor effect after light irradiation at 635 nm for three days compared to low-molecular-weight HP. Intraperitoneal administration of HP caused severe phototoxicity upon irradiation with ultraviolet A at 10 J cm-2, whereas aHP did not cause phototoxicity because its diffusion into the skin could be suppressed, probably due to the high-molecular weight of aHP. Therefore, locally administered PDT with aHP is a potential PDT having high therapeutic efficacy without phototoxicity.

HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC.

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14+ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14+ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

Evaluation of tumor stiffness by elastography is predictive for pathologic complete response to neoadjuvant chemotherapy in patients with breast cancer.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The glucocorticoid receptor associates with the cohesin loader NIPBL to promote long-range gene regulation.

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.

Advances in Oncology in US and Japan: Focusing on Cancer and Infectious Diseases.

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.

Distant Metastasis after Surgery for Encapsulated Papillary Carcinoma of the Breast: A Case Report.

In the absence of clear interstitial invasion, encapsulated papillary carcinoma (EPC) of the breast may be attributed to an extremely good prognosis if handled similarly to ductal carcinoma in situ (DCIS) with suitable local treatment. Here, we report our experience with a case of EPC of the breast that presented with carcinomatous pleuritis and lymphangitis carcinomatosa postoperatively, which rapidly resulted in a poor outcome. A 67-year-old woman was diagnosed with DCIS of the left breast and underwent left partial mastectomy and sentinel lymph node biopsy. EPC was diagnosed because the pathological examination showed no sign of interstitial infiltration. Postoperative radiation therapy was performed. Five years and 9 months postoperatively, the patient began experiencing cough and shortness of breath on exertion. Imaging showed right pleural effusion and consolidation of the lung field, but nothing suggesting local recurrence in the preserved left breast, local lymph nodes, or opposite breast was observed. Postoperative recurrence of breast cancer, carcinomatous pleuritis, and lymphangitis carcinomatosa were diagnosed based on the results of pleural fluid cytology. One month later, multiple brain metastases were found, and the patient died of the primary disease 5 months after recurrence. After surgery for EPC without clear interstitial infiltration, there was a small possibility of a poor outcome from distant metastasis. Therefore, although distant metastasis is uncommon, regular examination and testing should be performed.

Nucleosome destabilization by nuclear non-coding RNAs.

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.

High Quality ATAC-Seq Data Recovered from Cryopreserved Breast Cell Lines and Tissue.

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.

Cancer Navigation Strategy for Endocrine Therapy-Resistant Breast Tumors.

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.

Clinical features of lenvatinib treatment in elderly patients with advanced thyroid cancer.

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) and elderly group (≥75 years old). The median maximum systolic blood pressure (sBP) was significantly different between the two groups (158 mmHg in the younger group vs. 173 mmHg in the elderly group; P=0.042). There were no significant differences in median maximum diastolic blood pressure (94 vs. 95 mmHg; P=1.00), median degree of sBP elevation (43 vs. 55 mmHg; P=0.199) or median days until hypertension diagnosis (2.11 vs. 2.33 days; P=0.436). There were also no significant differences in other toxicities (HFS, proteinuria or diarrhea). In conclusion, lenvatinib should be introduced carefully to elderly patients with DTC, as they tend to present with hypertension during treatment. However, there were no differences in other toxicities between the younger and elderly groups; lenvatinib was fully tolerated in patients with DTC >75 years old.

Roles of long noncoding RNAs in chromosome domains.

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.

Clinical significance of monitoring ESR1 mutations in circulating cell-free DNA in estrogen receptor positive breast cancer patients.

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.

Droplet digital polymerase chain reaction assay for screening of ESR1 mutations in 325 breast cancer specimens.

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.

C6ORF97-ESR1 breast cancer susceptibility locus: influence on progression and survival in breast cancer patients.

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<0.0001), highly proliferative characteristics (P=0.0005 for Ki67, P<0.0001 for nuclear grade) and worse RFS in the ER+/HER2- cohort (P=0.013), whereas the other two C6ORFs showed the inverse associations. Furthermore, C6ORF97 showed significant worse prognostic values especially in luminal B subtype in the publically available data sets. rs2046210 and the upstream gene C6ORF97 might have substantial roles not only in carcinogenesis but also in progression toward a more aggressive phenotype in breast cancer patients, which suggests that functional studies of this locus are imperative.

A cluster of noncoding RNAs activates the ESR1 locus during breast cancer adaptation.

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.