Interplay between LncRNAs and microRNAs in Breast Cancer.

(1) Although long noncoding RNAs (lncRNAs) are known to be precursors of microRNAs (miRNAs), they frequently act as competing endogoneous RNAs (ceRNAs), yet still their interplay with miRNA is not well known. However, their interaction with miRNAs may result in the modulation of miRNA action. (2) To determine the contribution of these RNA molecules in tumor resistance to chemotherapeutic drugs, it is essential to consider not only the oncogenic and tumor suppressive function of miRNAs but also the impact of lncRNAs on miRNAs. Therefore, we performed an extensive search in different databases including PubMed. (3) The present study concerns the interplay between lncRNAs and miRNAs in the regulatory post-transcriptional network and their impact on drugs used in the treatment of breast cancer. (4) Consideration of this interplay may improve the search for new drugs to circumvent chemoresistance.

Characterization of circulating molecules and activities in plasma of patients after allogeneic and autologous intraoral bone grafting procedures: a prospective randomized controlled clinical trial in humans.

BACKGROUND: The objective was to assess whether intraoral bone augmentation procedures have an impact on the patient's plasma levels of circulating nucleic acids, exosomes, miRNA levels and caspase activities. The null hypothesis was tested, that no significant differences between the two groups will be found. METHODS: In this prospective randomized controlled clinical trial 35 systemically healthy non-smoking participants were randomly allocated using sealed envelopes by a blinded clinician not involved in the clinical setting. Plasma samples were collected preoperatively and 3 times postoperatively (immediately, 5 weeks and 4 months postoperatively). The test group consisted of twenty-five patients who received allogeneic bone grafting material and the control group of ten patients who received autologous bone grafts. Levels of cell-free DNA (cfDNA) and microRNAs (miR-21, miR-27a, miR-218) were quantified by real-time PCR, caspase activities and exosome concentrations were determined by ELISA. RESULTS: Statistical evaluation reveled a significantly higher exosome level before surgery (p = 0.013) and the first postsurgical sample (p = 0.017) in the control group compared to the test group. The levels of miR-27a and miR-218 significantly differed between the plasma samples before surgery and after surgery in both groups. The levels of miR-21 only significantly differed between the pre- and postsurgical plasma samples in the test group, but not in the control group. All patients completed the study, no adverse events were recorded. CONCLUSIONS: Our data show the diagnostic potential of the plasma levels of miR-27a, miR-218 and miR-21 in detecting changes in bone metabolism after alveolar bone augmentation. Our very promising results indicate that there might be a high diagnostic potential in evaluating the plasma levels of the before mentioned miRNAs in order to detect bone resorption activities before they become clinically relevant. Trial registration Ethical commission of the Ärztekammer Hamburg, Germany (PV5211) on 11/03/2016 as well as by the German Registry of Clinical Studies (DRKS 00,013,010) on 30/07/2018 ( http://apps.who.int/trialsearch/ ).

Characterization of immunologically detectable T-cell sensitization, Immunohistochemical detection of pro-inflammatory cytokines, and clinical parameters of patients after allogeneic intraoral bone grafting procedures: a prospective randomized controlled clinical trial in humans.

BACKGROUND: The null hypotheses were tested that intraoral bone augmentation using two different allogeneic materials has no impact on the patient's blood levels of material-specific lymphocytes and on the immunohistochemical detection of pro-inflammatory cytokines IL-1α, IL1ß and TNF-α and T-cell markers CD4, CD8 in biopsies of the test groups. METHODS: In this prospective RCT, 60 systemically healthy participants were randomly assigned to two allogeneic test groups (1: Maxgraft®, freeze-dried, multiple donors, and 2: Puros®, solvent-dehydrated, single donor) and an autologous control group (10 patients). Plasma samples were collected pre-(T1) and postoperatively (2 weeks (T2) and 4 months (T3)). The Lymphocyte Transformation Test (LTT) was used for analyzing levels of transformed lymphocytes for type IV immune reactions by 3H-thymidine activity. Bone biopsies were harvested at T3 and immunohistochemically analyzed for IL-1α, IL1ß, TNF-α, CD4, CD8 and correlated with the immunological and clinical findings. RESULTS: A statistically significant difference between the tested materials was observed for LTT measurements at T3 (p = 0.033). Furthermore, three groups were identified: Group A (LTT negative T1-T3, n = 48), group B (LTT positive T1-T3, n = 7), group C (developing positive LTT at T2, n = 5). A highly significant elevation of IL-1α, IL1ß, TNF-α in patients of group C (p = 0.0001) and a significant elevation of CD4+ cells in patients of group B (p = 0.005) was shown. CONCLUSION: Our data show that following allogeneic bone grafting, local and systemic immunological reactions can be detected in some patients. These findings were statistically significant for the timepoint T3 between the tested materials as well as for the groups B and C correlated with group A for both tested materials. Therefore, the null hypotheses were rejected. A preoperative compatibility test for allogeneic materials in order to improve patient safety and the predictability of these materials would be desirable. TRIAL REGISTRATION: Ethical commission of the Ärztekammer Hamburg, Germany (PV5211) as well as by the German Registry of Clinical Studies (DRKS00013010) on 30/07/2018 ( http://apps.who.int/trialsearch/ ).

A novel assay for exosomal and cell-free miRNA isolation and quantification.

The use of disease-specific signatures of microRNAs (miRNAs) in exosomes has become promising for clinical applications, either as biomarkers or direct therapeutic targets. However, a new approach for exosome enrichment and quantification of miRNAs is urgently needed for its clinical application, since the commercial techniques have shortcomings in quantity and quality. To overcome these deficiencies, we developed a new method for purification of exosomes with subsequent miRNA extraction, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and compared our assays with commercial techniques. For the establishment of these methods, numerous reagents, parameters, and combinations thereof were examined. Our new technique for exosome extraction is based on a mannuronate-guluronate polymer (MGP) which avoids co-precipitating plasma proteins. Quality, concentration and biological activity of the isolated exosomes were examined by Western blot, Nanoparticle Tracking Analysis (NTA), and confocal microscopy. A combination of chaotropic and non-chaotropic salts was used to extract miRNAs from plasma, serum, and exosomes, allowing the exclusion of hazardous components, such as phenol/chloroform. The performance of the miRNAs extraction was verified by RT-qPCR. The chemistry and TaqMan probe were also optimized for RT-qPCR. Sensitivity, efficiency, and linearity of RT-qPCR were tested on serial dilutions of synthetic miR-16 and miR-142. Our established procedure covers all steps of miRNA analyses, and measures the levels of either cell-free and exosomal miRNAs in plasma, serum and other body fluids with high performance.

MicroRNA expression studies: challenge of selecting reliable reference controls for data normalization.

Accurate determination of microRNA expression levels is a prerequisite in using these small non-coding RNA molecules as novel biomarkers in disease diagnosis and prognosis. Quantitative PCR is the method of choice for measuring the expression levels of microRNAs. However, a major obstacle that affects the reliability of results is the lack of validated reference controls for data normalization. Various non-coding RNAs have previously been used as reference controls, but their use may lead to variations and lack of comparability of microRNA data among the studies. Despite the growing number of studies investigating microRNA profiles to discriminate between healthy and disease stages, robust reference controls for data normalization have so far not been established. In the present article, we provide an overview of different reference controls used in various diseases, and highlight the urgent need for the identification of suitable reference controls to produce reliable data. Our analysis shows, among others, that RNU6 is not an ideal normalizer in studies using patient material from different diseases. Finally, our article tries to disclose the challenges to find a reference control which is uniformly and stably expressed across all body tissues, fluids, and diseases.

The current role and future directions of circulating tumor cells and circulating tumor DNA in urothelial carcinoma of the bladder.

PURPOSE: Urothelial carcinoma of the bladder (UCB) is clinically and genetically a highly heterogeneous disease. Treatment decisions are usually based on histopathological workup and molecular diagnostics on tissue biopsies of the primary tumor or the metastatic site. Next to completely different molecular genotypes of phenotypically similar tumors, standard biopsies do not unconditionally allow real-time insight during the natural course of disease progression. Indeed, in UCB there is an imperative need of biomarkers for improving clinical staging, detecting minimal residual disease, predicting therapy response and prognosis and finally enabling patient stratification for multimodal, individualized treatment and therapy monitoring.Liquid biopsies of blood-based circulating biomarkers have evolved from bench to bedside in some cancer entities. METHODS: In a narrative review we are summerizing the latest evidence on CTC and ctDNA in muscle-invasive and metastatic UCB. RESULTS: In this review, we summarize the current status, limitations and future needs of circulating tumor cells (CTC) and cell-free circulating tumor DNA (ctDNA) in UCB. Moreover, we discuss the potential clinical application of CTC and ctDNA as prognostic markers at different UCB stages and their value for target therapy guidance. CONCLUSIONS: CTC and ctDNA are promising circulating biomarkers in UCB, but none of both has progressed from bench to bedside yet. These markers may support outcome prognostication, patient counseling follow-up monitoring, and potentially decision-making regarding chemotherapy. Further prospective clinical or randomized studies are urgently warranted.

Characterization of circulating DNA in plasma of patients after allogeneic bone grafting.

OBJECTIVES: Cell-free DNA (cfDNA) harboring mutations has been found in patients with diseases. Experimental studies have shown that cfDNA can be transmitted, leading to transformations in the host. In the present study, we evaluated whether bone allograft material contains cfDNA and whether this foreign cfDNA can be released into the patient's blood circulation. MATERIALS AND METHODS: Plasma samples were collected preoperatively and postoperatively on the same day, at 5 weeks, and 4 months from 25 women who received bone allograft material (test group) from male donors and from 10 women who were treated with autologous graft (control group, only pre- and postoperative samples were collected). DNA was quantified and characterized in bone material and plasma samples by quantitative PCR with primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Y chromosome and gel electrophoresis. DNA in bone material was digested by different concentrations of DNase I. RESULTS: We detected between 1 and 1.8 µg cfDNA fragments at a length around 601 base pairs (bp) and smaller in each 100 mg allograft. Treatment of the allograft with DNase I completely degraded the longer but not the shorter DNA 90-bp fragments. Y-DNA was not detected in the patients' bloodstream at any time during the treatment and follow-up, but elevated levels of circulating cfDNA could be measured immediately postoperatively. CONCLUSIONS: Our results suggest that a transmission of DNA from allografts used for alveolar ridge reconstruction in humans is unlikely. The observed increase in circulating cfDNA in allograft and autograft patients immediately postoperatively may be elicited by the surgical procedure. CLINICAL RELEVANCE: The results support the safety of allograft materials. The results suggest that human allograft materials seem not to release DNA into the blood since we did not measure Y-DNA with our technique.

Specific microRNA signatures in exosomes of triple-negative and HER2-positive breast cancer patients undergoing neoadjuvant therapy within the GeparSixto trial.

BACKGROUND: The focus of this study is to identify particular microRNA (miRNA) signatures in exosomes derived from plasma of 435 human epidermal growth factor receptor 2 (HER2)-positive and triple-negative (TN) subtypes of breast cancer (BC). METHODS: First, miRNA expression profiles were determined in exosomes derived from the plasma of 15 TNBC patients before neoadjuvant therapy using a quantitative TaqMan real-time PCR-based microRNA array card containing 384 different miRNAs. Forty-five miRNAs associated with different clinical parameters were then selected and mounted on microRNA array cards that served for the quantification of exosomal miRNAs in 435 BC patients before therapy and 20 healthy women. Confocal microscopy, Western blot, and ELISA were used for exosome characterization. RESULTS: Quantification of 45 exosomal miRNAs showed that compared with healthy women, 10 miRNAs in the entire cohort of BC patients, 13 in the subgroup of 211 HER2-positive BC, and 17 in the subgroup of 224 TNBC were significantly deregulated. Plasma levels of 18 exosomal miRNAs differed between HER2-positive and TNBC subtypes, and 9 miRNAs of them also differed from healthy women. Exosomal miRNAs were significantly associated with the clinicopathological and risk factors. In uni- and multivariate models, miR-155 (p = 0.002, p = 0.003, respectively) and miR-301 (p = 0.002, p = 0.001, respectively) best predicted pathological complete response (pCR). CONCLUSION: Our findings show a network of deregulated exosomal miRNAs with specific expression patterns in exosomes of HER2-positive and TNBC patients that are also associated with clinicopathological parameters and pCR within each BC subtype.

Plasma microRNA signature is associated with risk stratification in prostate cancer patients.

The aim of this study was to establish a unique expression profile of circulating cell-free microRNAs (miRNAs) capable of differentiating between prostate cancer (PCa) patients with high-risk and intermediate-risk Gleason scores. MiRNA expression profiles were determined in plasma samples from 79 treatment-naïve PCa patients, 1-2 follow-up samples after radical prostatectomy (RP) from 51 out of the 79 PCa patients, and 33 healthy men, using a quantitative real-time PCR-based array containing 48 selected miRNAs. We identified 27 up- and 2 downregulated plasma miRNAs in PCa patients compared with healthy men. Most of the upregulated miRNA levels were also associated with increasing PSA levels and Gleason scores. Particularly, the levels of miR-16 (p = 0.002), miR-148a (p = 0.006) and miR-195 (p = 0.006) significantly correlated with high-risk Gleason scores, whereby miR-148a (p = 0.003) was also significantly associated with increasing PSA values. The high miRNA levels before RP remained increased in the postsurgical plasma samples. Our findings show a network of deregulated plasma miRNAs. In particular, miR-16, miR-148a and miR-195 are involved in the regulation of the PI3K/Akt signaling pathway. These miRNAs may be promising therapeutic targets for high-risk PCa stratification.

Circulating Cell-Free miR-373, miR-200a, miR-200b and miR-200c in Patients with Epithelial Ovarian Cancer.

In the present study, we investigated whether circulating cell-free microRNAs serve as potential biomarkers in epithelial ovarian cancer (EOC) patients. Circulating miR-373, miR-200a, miR-200b and miR-200c were quantified in a cohort of 60 EOC patients, 20 patients with benign ovarian diseases and 32 healthy women using quantitative TaqMan MicroRNA assays. The serum concentrations of cell-free miR-373, miR-200a, miR-200b and miR-200c were significantly higher in EOC patients than in healthy women (p = 0.0001). With a sensitivity of 83 % and a specificity of 100 %, the combination of miR-200a, miR-200b and miR-200c could differ between malignant and benign ovarian tumors (p = 0.0001). Elevated levels of these cell-free microRNAs could be detected in FIGO I-II and FIGO III-IV stages, grading G1-2 and G3 and lymph node-negative and -positive EOC. In conclusion, the increased serum levels of this microRNA panel have diagnostic value for distinguishing healthy controls and benign tumors from EOC.

Novel Technology for Enrichment of Biomolecules from Cell-Free Body Fluids and Subsequent DNA Sizing.

Circulating cell-free DNA (ccfDNA) is a promising diagnostic tool and its size fractionation is of interest. However, kits for isolation of ccfDNA available on the market are designed for small volumes hence processing large sample volumes is laborious. We have tested a new method that enables enrichment of ccfDNA from large volumes of plasma and subsequently allows size-fractionation of isolated ccfDNA into two fractions with individually established cut-off levels of ccfDNA length. This method allows isolation of low-abundant DNA as well as separation of long and short DNA molecules. This procedure may be important e.g., in prenatal diagnostics and cancer research that have been already confirmed by our primary experiments. Here, we report the results of selective separation of 200- and 500-bp long synthetic DNA fragments spiked in plasma samples. Furthermore, we size-fractionated ccfDNA from the plasma of pregnant women and verified the prevalence of fetal ccfDNA in all fractions.

Circulating DNA as biomarker in breast cancer.

As the release of tumor-associated DNA into blood circulation is a common event in patients with cancer, screening of plasma or serum DNA may provide information on genetic and epigenetic profiles associated with breast cancer development, progression, and response to therapy. Quantitative testing of circulating DNA can reflect tumor burden, and molecular characterization of circulating DNA can reveal important tumor characteristics relevant to the choice of targeted therapies in individual patients. Contrary to circulating DNA from blood that presents molecular changes in tumor DNA in real time, tissue biopsies can deliver only a spatially and temporally limited snapshot of the heterogeneous tumor. Analyses of circulating DNA might provide prognostic and predictive information and therefore advance personalized medicine. However, standardization of different technical platforms as well as the control of pre-analytical and analytical factors is mandatory before its introduction into clinical practice. In the present review, we discussed technical aspects and clinical relevance of the analyses of circulating plasma/serum DNA in patients with breast cancer.

Diagnostic and prognostic potential of serum miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429 in ovarian cancer patients.

BACKGROUND: Owing to late diagnosis in advanced disease stages, prognosis of patients with epithelial ovarian cancer (EOC) is poor. The quantification of deregulated levels of microRNAs could facilitate earlier diagnosis and improve prognosis of EOC. METHODS: Seven microRNAs (miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429) were quantified in the serum of 180 EOC patients and 66 healthy women by TaqMan PCR microRNA assays. Median follow-up time was 21 months. The effects of miR-7 and miR-429 on apoptosis, cell proliferation, migration and invasion were investigated in two (EOC) cell lines. RESULTS: Serum levels of miR-25 (P=0.0001) and miR-93 (P=0.0001) were downregulated, whereas those of miR-7 (P=0.001) and miR-429 (P=0.0001) were upregulated in EOC patients compared with healthy women. The four microRNAs discriminated EOC patients from healthy women with a sensitivity of 93% and a specificity of 92%. The levels of miR-429 positively correlated with CA125 values (P=0.0001) and differed between FIGO I-II and III-IV stages (P=0.001). MiR-429 was an independent predictor of overall survival (P=0.011). Overexpressed miR-429 in SKOV3 cells led to suppression of cell migration (P=0.037) and invasion (P=0.011). Increased levels of miR-7 were associated with lymph node metastases (P=0.0001) and FIGO stages III-IV (P=0.0001). Overexpressed miR-7 in SKOV3 cells resulted in increased cell migration (P=0.001) and invasion (P=0.011). Additionally, the increased levels of miR-376a correlated with FIGO stages III-IV (P=0.02). CONCLUSIONS: Our data indicate the diagnostic potential of miR-7, miR-25, miR-93 and miR-429 in EOC and the prognostic potential of miR-429. This microRNA panel may be promising molecules to be targeted in the treatment of EOC.

Data Normalization Strategies for MicroRNA Quantification.

BACKGROUND: Different technologies, such as quantitative real-time PCR or microarrays, have been developed to measure microRNA (miRNA) expression levels. Quantification of miRNA transcripts implicates data normalization using endogenous and exogenous reference genes for data correction. However, there is no consensus about an optimal normalization strategy. The choice of a reference gene remains problematic and can have a serious impact on the actual available transcript levels and, consequently, on the biological interpretation of data. CONTENT: In this review article we discuss the reliability of the use of small RNAs, commonly reported in the literature as miRNA expression normalizers, and compare different strategies used for data normalization. SUMMARY: A workflow strategy is proposed for normalization of miRNA expression data in an attempt to provide a basis for the establishment of a global standard procedure that will allow comparison across studies.

Circulating tumor cells and circulating tumor DNA.

Solid tumors derived from epithelial tissues (carcinomas) are responsible for 90% of all new cancers in Europe, and the main four tumor entities are breast, prostate, lung, and colon cancer. Present tumor staging is mainly based on local tumor extension, metastatic lymph node involvement, and evidence of overt distant metastasis obtained by imaging technologies. However, these staging procedures are not sensitive enough to detect early tumor cell dissemination as a key event in tumor progression. Many teams have therefore focused on the development of sensitive assays that allow the specific detection of single tumor cells or small amounts of cell-free tumor DNA in the peripheral blood of cancer patients. These methods allow the detection and characterization of early metastatic spread and will provide unique insights into the biology of metastatic progression of human tumors, including the effects of therapeutic interventions.

Changes in serum levels of miR-21, miR-210, and miR-373 in HER2-positive breast cancer patients undergoing neoadjuvant therapy: a translational research project within the Geparquinto trial.

Trastuzumab and lapatinib are established treatments for patients with HER2 (human epidermal growth factor receptor 2)-positive breast cancer with different mechanisms of action. The focus of this study is to investigate, whether altered expression levels of potentially relevant microRNAs (miRs) in serum are associated with response to trastuzumab or lapatinib. Circulating miR-21, miR-210, and miR-373 were quantified with TaqMan MicroRNA assays in serum of 127 HER2-postive breast cancer patients before and after neoadjuvant therapy and in 19 healthy controls. Patients received chemotherapy combined with either trastuzumab or lapatinib within the prospectively randomized Geparquinto trial. The association between miR levels and pathological response (pCR) to therapy and type of therapy was examined. Serum levels of miR-21 (p = 5.04e-08, p = 1.43e-10), miR-210 (p = 0.00151, p = 1.6e-05), and miR-373 (p = 7.87e-06, p = 1.75e-07) were significantly higher in patients before and after chemotherapy than in healthy women. Concentrations of miR-21 (p = 5.73e-08), miR-210 (p = 0.000724), and miR-373 (p = 0.00209) increased further after chemotherapy. A significant association of higher serum levels of miR-373 with advanced clinical tumor stage could be detected (p < 0.002). An association of miR-21 levels before (p = 0.0091) and after (p = 0.037) chemotherapy with overall survival of the patients could be detected, independent of type of anti-HER2 therapy. No association of circulating miRs with pCR was found. Our findings demonstrate a specific influence of neoadjuvant therapy on the serum levels of miR-21, miR-210, and miR-373 in breast cancer patients together with a prognostic value of miR-21.

Differential regulation of MAGE-A1 promoter activity by BORIS and Sp1, both interacting with the TATA binding protein.

BACKGROUND: As cancer-testis MAGE-A antigens are targets for tumor immunotherapy, it is important to study the regulation of their expression in cancers. This regulation appears to be rather complex and at the moment controversial. Although it is generally accepted that MAGE-A expression is controlled by epigenetics, the exact mechanisms of that control remain poorly understood. METHODS: We analyzed the interplay of another cancer-testis gene, BORIS, and the transcription factors Ets-1 and Sp1 in the regulation of MAGE-A1 gene expression performing luciferase assays, quantitative real-time PCR, sodium bisulfite sequencing, chromatin immunoprecipitation assays and pull down experiments. RESULTS: We detected that ectopically expressed BORIS could activate and demethylate both endogenous and methylated reporter MAGE-A1 promoter in MCF-7 and micrometastatic BCM1 cancer cell lines. Overexpression of Ets-1 could not further upregulate the promoter activity mediated by BORIS. Surprisingly, in co-transfection experiments we observed that Sp1 partly repressed the BORIS-mediated stimulation, while addition of Ets-1 expression plasmid abrogated the Sp1 mediated repression of MAGE-A1 promoter. Both BORIS and Sp1 interacted with the TATA binding protein (hTBP) suggesting the possibility of a competitive mechanism of action between BORIS and Sp1. CONCLUSIONS: Our findings show that BORIS and Sp1 have opposite effects on the regulation of MAGE-A1 gene expression. This differential regulation may be explained by direct protein-protein interaction of both factors or by interaction of MAGE-A1 promoter with BORIS alternatively spliced isoforms with different sequence specificity. We also show here that ectopic expression of BORIS can activate transcription from its own locus, inducing all its splice variants.

Circulating nucleic acids as biomarkers in breast cancer.

During tumor development, tumor cells release their nucleic acids into the blood circulation. This process occurs by apoptotic and necrotic cell deaths along with active cell secretion, resulting in high levels of circulating DNA, mRNA, and microRNA in the blood of patients with breast cancer. As circulating cell-free tumor nucleic acids may reflect the characteristics of the primary tumor and even of micrometastatic cells, they may be excellent blood biomarkers for screening breast cancer. Assays that allow the repetitive monitoring of patients by using blood samples as liquid biopsy may be efficient in assessing cancer progression in patients whose tumor tissue is not available. This review evaluates the recent data on the potential use of circulating cell-free nucleic acids as biomarkers for breast cancer.

Deregulated serum concentrations of circulating cell-free microRNAs miR-17, miR-34a, miR-155, and miR-373 in human breast cancer development and progression.

BACKGROUND: MicroRNAs (miRs) are small, noncoding RNAs that target genes involved in tumor development and progression. In the current study, we investigated the use of circulating miR concentrations as biomarkers in the serum of breast cancer patients. METHODS: We analyzed serum samples from 120 patients with primary breast cancer after surgery and before chemotherapy (M0, classified into 3 subgroups of 40 patients with progesterone/estrogen-positive, HER2-positive, and triple-negative cancer), 32 patients with overt metastasis (M1), and 40 healthy women. Using quantitative TaqMan MicroRNA PCR, we measured the relative concentrations of 6 circulating microRNAs (miR-10b, -17, -34a, -93, -155, and -373) known to be relevant for tumor development and progression. The data were correlated with clinicopathologic risk factors, with particular reference to HER2 and hormone receptor status of the primary tumor and the presence of metastases. RESULTS: The relative serum concentrations of circulating miR-34a [P = 0.013, area under the curve (AUC) 0.636], miR-93 (P = 0.001, AUC 0.699), and miR-373 (P = 0.0001, AUC 0.879) were significantly different between M0 breast cancer patients and healthy women, whereas miR-17 (P = 0.002, AUC 0.679) and miR-155 (P = 0.0001, AUC 0.781) were differently expressed between M0 and M1 patients. Increased concentrations of miR-373 were associated with negative HER2 status of the primary tumor (P = 0.0001). Deregulated concentrations of miR-17 (P = 0.019) and miR-34a (P = 0.029) were detected in patients with progesterone/estrogen receptor-positive and -negative status, respectively. CONCLUSIONS: Our findings indicate that serum concentrations of deregulated microRNAs may be linked to a particular biology of breast carcinomas favoring progression and metastatic spread.