BackgroundExosomal microRNAs (miRNAs) are promising candidate biomarkers for diagnosis or prognosis for breast cancer. We investigated the prognostic role of exosomal miRNAs in serum samples derived from patients with breast cancer and compared miRNA expression between serum and tumor tissues.MethodsThe miRNA profile derived from exosome between breast cancer patients with recurrence (n = 16) and without recurrence (n = 16) were compared by miRNA PCR array. Further, we examined the expression of miRNAs derived from tissues in the patients with breast cancer with (n = 35) and without recurrence (n = 39) by qRT-PCR.ResultsOf 384 miRNAs, three miRNAs (miR-338-3p, miR-340-5p, and miR-124-3p) were significantly upregulated and eight (miR-29b-3p, miR-20b-5p, miR-17-5p, miR-130a-3p, miR-18a-5p, miR-195-5p, miR-486-5p, and miR-93-5p) were significantly downregulated in the patients with recurrence. We evaluated the expression of the miRNAs in tumor tissues. The patients with recurrence had higher levels of miR-340 at their primary site as well as in the serum. In contrast, miR-195-5p, miR-17-5p, miR-93-5p, and miR-130a-3p, derived from tumor tissues that were downregulated in the serum from patients with recurrence, were higher in the patients with recurrence than in those with no recurrence. In logistic regression analysis, miR-340-5p, miR-17-5p, miR-130a-3p, and miR-93-5p were significantly associated with recurrence.ConclusionsSeveral exosomal miRNAs may be useful biomarkers to predict breast cancer recurrence. We show the different expression patterns of miRNAs between tumor tissues and serum. These findings may suggest selective mechanism of release of exosomal miRNAs by cancer cells to regulate their progression.

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.

BackgroundThe measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients.MethodsThe subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients. We developed multiplex droplet digital PCR assays to verify the clinical significance of ESR1 and PIK3CA mutations both in a snapshot and serially in these patients.ResultscfDNA ESR1 and PIK3CA mutations were found in 28.9% and 24.6 % of MBC patients, respectively. The relation between ESR1 or PIK3CA mutations and clinical features showed that ESR1 mutations occurred mostly in patients previously treated by ET, which was not the case for PIK3CA mutations. The analysis of the clinical impact of those mutations on subsequent lines of treatment for the 69 MBC patients revealed that both ESR1 and PIK3CA mutations detection were related to a shorter duration of ET effectiveness in univariate analysis but only for ESR1 mutations in multivariate analysis. The monitoring of cfDNA in a subset of 52 patients showed that loss of ESR1 mutations was related to a longer duration of response, which was not the case for PIK3CA mutations.ConclusionsWe have demonstrated the clinical significance of on-treatment ESR1 mutations both in a snapshot and serially in comparison with PIK3CA mutations.

The somatic activation of PI3K/AKT pathway mutations, PIK3CA and AKT1, and ESR1 mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive procedure to quickly assess and monitor disease progression or therapeutic effect in breast cancer (BC) patients, but the clinical significance of these mutations in late treatment lines (TLs) remains unclear. The subjects of this study were a total of 251 plasma samples from 128 estrogen receptor-positive (ER+) BC patients. Of these plasma samples, 133 were from 73 primary BC (PBC) patients, and 118 plasma samples were from 68 metastatic BC (MBC) patients. We developed droplet digital PCR (ddPCR) assays to verify the clinical significance of PIK3CA, AKT1, and ESR1 mutations in these patients. cfDNA PIK3CA mutations were observed in 15.1% of the PBC patients, while a cfDNA AKT1 mutation was observed in 1.4% of patients, and cfDNA ESR1 mutations were observed in 2.7% of patients. Patients with detectable cfDNA PIK3CA mutations were not associated with clinical outcomes. According to the TL, the prevalence of the PIK3CA and ESR1 mutations in cfDNA were lower in early TLs compared with late TLs. In the early TL group, patients with cfDNA PIK3CA mutations had a shorter time to treatment failure (TTF) than patients without mutations (P = 0.035). However, there was no statistically significant difference between patients with or without cfDNA ESR1 mutations. However, in the late TL group, patients with cfDNA ESR1 mutations had a shorter TTF than patients without mutations (P = 0.048). However, there was no statistically significant difference between patients with or without cfDNA PIK3CA mutations. Since the prevalence of cfDNA AKT1 mutation is low in both PBC and MBC patients, the impact of AKT1 mutations on the prognosis remains unclear. We have demonstrated the difference in the clinical significance of the hotspot PIK3CA, AKT1, and ESR1 mutations in cfDNA for each TL in ER+ BC patients.Keywords: Estrogen receptor-positive breast cancer, Cell-free DNA, PIK3CA mutations, AKT1 mutation, ESR1 mutations Endocrine therapy (ET) resistance occasionally occurs during the treatment of primary breast cancer (PBC) and inevitably results in metastatic BC (MBC). Recently, the focused mechanisms of ET resistance include hyperactivation of PI3K/AKT pathway. Importantly, somatic activating mutations of PIK3CA and AKT1 affect the magnitude of PI3K/AKT activation [1]. Meta-analysis of the BC literature shows that the PIK3CA mutation is present in 20%-40% of all BCs, making this gene the second most frequently mutated in BC, with most mutations being expressed in 'hotspots' in the helical domain (exon (Ex) 9) or the catalytic domain (Ex20) [2]. A somatic mutation in the plekstrin homology domain of AKT1:p.Glu17Lys is found in approximately 4% of breast tumors [3]. Conversely, recent evidence describing next generation sequencing showed that another key potential mechanism of the failure of ET involves activating

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 (Po0.0001), highly proliferative characteristics (P = 0.0005 for Ki67, Po0.0001 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.