Thalia Erbes

Feasibility of urinary microRNA detection in breast cancer patients and its potential as an innovative non-invasive biomarker

BackgroundSince recent studies revealed the feasibility to detect blood-based microRNAs (miRNAs, miRs) in breast cancer (BC) patients a new field has been opened for circulating miRNAs as potential biomarkers in BC. In this pilot study, we evaluated to our knowledge for the first time whether distinct pattern of urinary miRNAs might be also applicable as innovative biomarkers for BC detection.MethodsUrinary miRNA expression levels of nine BC-related miRNAs (miR-21, miR-34a, miR-125b, miR-155, miR-195, miR-200b, miR-200c, miR-375, miR-451) from 24 untreated, primary BC patients and 24 healthy controls were quantified by realtime-PCR. The receiver operating characteristic analyses (ROC) and logistic regression were calculated to assess discriminatory accuracy.ResultsSignificant differences were found in the expression of four BC-associated miRNAs quantified as median miRNA expression levels. Urinary miR-155 levels were significantly higher in BC patients compared to healthy controls (1.49vs.0.25; p < 0.001). In contrast, compared to healthy controls, BC patients exhibited significantly lower urinary expression levels of miR-21 (2.27vs.5.07; p < 0.001), miR-125b (0.71vs.1.62; p < 0.001), and miR-451 (0.02vs.0.59 p = 0.004), respectively. The ROC including all miRNAs as well as the group of the four significant deregulated miRNAs separated BC patients from healthy controls with a very high (area under the receiver operating characteristic curve [AUC] = 0.932) and high accuracy (AUC = 0.887), respectively.ConclusionsWe were able to demonstrate for the first time the feasibility to detect distinct BC-dependent urinary miRNA profiles. The expression levels of four urinary miRNAs were specifically altered in our cohort of BC patients compared to healthy controls. This distinct pattern offers the possibility for a specific discrimination between healthy women and primary BC patients. This sustains the potential role of urinary miRNAs as non-invasive innovative urine-based biomarkers for BC detection.

Metabolic profiling of breast cancer: Differences in central metabolism between subtypes of breast cancer cell lines

Although the concept of aerobic glycolysis in cancer was already reported in the 1930s by Otto Warburg, the understanding of metabolic pathways remains challenging especially due to the heterogeneity of cancer. In consideration of four different time points (1, 2, 4, and 7 days of incubation), GC-MS profiling of metabolites was performed on cell extracts and supernatants of breast cancer cell lines (MDA-MB-231, -453, BT-474) with different sub classification and the breast epithelial cell line MCF-10A. To the exclusion of trypsinization, direct methanolic extraction, cell scraping and cell disruption was executed to obtain central metabolites. Major differences in biochemical pathways have been observed in the breast cancer cell lines compared to the breast epithelial cell line, as well as between the breast cancer cell lines themselves. Characteristics of breast cancer subtypes could be correlated to their individual metabolic profiles. PLS-DA revealed the discrimination of breast cancer cell lines from MCF-10A based on elevated amino acid levels. The observed metabolic signatures have great potential as biomarker for breast cancer as well as an improved understanding of subtype specific phenomenons of breast cancer.

Metabolome analysis via comprehensive two-dimensional liquid chromatography: identification of modified nucleosides from RNA metabolism.

Modified nucleosides derived from the RNA metabolism constitute an important chemical class, which are discussed as potential biomarkers in the detection of mammalian breast cancer. Not only the variability of modifications, but also the complexity of biological matrices such as urinary samples poses challenges in the analysis of modified nucleosides. In the present work, a comprehensive two-dimensional liquid chromatography mass spectrometry (2D-LC-MS) approach for the analysis of modified nucleosides in biological samples was established. For prepurification of urinary samples and cell culture supernatants, we performed a cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. In order to establish a 2D-LC method, we tested numerous column combinations and chromatographic conditions. In order to determine the target compounds, we coupled the 2D-LC setup to a triple quadrupole mass spectrometer performing full scans, neutral loss scans, and multiple reaction monitoring (MRM). The combination of a Zorbax Eclipse Plus C18 column with a Zorbax Bonus-RP column was found to deliver a high degree of orthogonality and adequate separation. By application of 2D-LC-MS approaches, we were able to detect 28 target compounds from RNA metabolism and crosslinked pathways in urinary samples and 26 target compounds in cell culture supernatants, respectively. This is the first demonstration of the applicability and benefit of 2D-LC-MS for the targeted metabolome analysis of modified nucleosides and compounds from crosslinked pathways in different biological matrices.

Prophylaxis of intra- and postoperative nausea and vomiting in patients during cesarean section in spinal anesthesia

Background This paper describes a randomized prospective study conducted in 308 patients undergoing caesarean section in spinal anaesthesia at a single hospital between 2010 and 2012 to find a suitable anti-emetic strategy for these patients. Material/Methods Spinal anesthesia was performed in left prone position, at L3/L4 with hyperbaric 0.5% Bupivacaine according to a cc/cm body height ratio. There were no opioids given peri-operatively. The patients received either no prophylaxis (Group I) or tropisetron and metoclopramide (Group II) or dimenhydrinate and dexamethasone (Group III), or tropisetron as a single medication (Group IV). The primary outcome was nausea and/or vomiting (NV) in the intraoperative, early (0–2 h) or late (2–24 h) postoperative period. Multivariate statistical analysis was conducted with a regression analysis and a backward elimination of factors without significant correlation. Results All prophylactic agents significantly reduced NV incidence intraoperatively. Relative risk reduction for NV by prophylaxis was most effective (59.5%) in Group II (tropisetron and metoclopramide). In Group III (dimenhydrinate and dexamethasone), NV risk was reduced by 29.9% and by 28.7% in Group IV (tropisetron mono-therapy). The incidence of NV in the early (0–2 h) and the late (2–24 h) postoperative period was low all over (7.8%), but the relative risk reduction of NV in the early postoperative period was 54.1% (Group IV), 45.1% (Group III), and 34.8% (Group II), respectively. In the late postoperative period, there was no significant difference between the 4 groups. Conclusions We recommend a prophylactic medication with tropisetron 2 mg and metoclopramide 20 mg for patients during caesarean section. These agents are safe, reasonably priced, and highly efficient in preventing nausea and vomiting.

Hypoxia-Dependent mRNA Expression Pattern of Splicing Factor YT521 and Its Impact on Oncological Important Target Gene Expression

The ubiquitously expressed splicing factor YT521 (YTHDC1) is characterized by alternatively spliced isoforms with regulatory impact on cancer‐associated gene expression. Our recent findings account for the prognostic significance of YT521 in endometrial cancer. In this study, we investigated the hypoxia‐dependency of YT521 expression as well as its differential isoform activities on oncological important target genes. YT521s potential regulatory influence on splicing was investigated by a minigene assay for the specific target gene CD44. Functional splicing analysis was performed by YT521 knock‐down or overexpression, respectively. In addition, YT521 expression was determined under hypoxia. The two protein‐generating YT521 mRNA isoforms 1 and 2 caused a comparable, specific induction of CD44v alternative splicing (P < 0.01). In a number of oncological target genes, YT521 upregulation significantly altered BRCA2 expression pattern, while YT521 knock‐down created a significant regulatory impact on PGR expression, respectively. Hypoxia induced a specific switch towards the processing of two non‐protein‐coding mRNA variants, of which one is described for the first time in this study. The presented study underlines the comparable regulatory potential of both YT521 isoforms 1 and 2, on the investigated target genes in vivo and in vitro. Hypoxia induces a specific switch in YT521 expression pattern towards the two non‐protein coding mRNA variants, the already characterized isoform 3 and the newly discovered exon 8‐skipping isoform. The altered YT521 alternative splicing is functionally coupled with nonsense‐mediated decay and can be interpreted as regulated unproductive splicing and transcription with consecutive impact on the processing of specific cancer‐associated genes, such as BRCA2 and PGR.

Neoadjuvant chemotherapy in breast cancer significantly reduces number of yielded lymph nodes by axillary dissection

BackgroundNeoadjuvant chemotherapy (NC) is an established therapy in breast cancer, able to downstage positive axillary lymph nodes, but might hamper their detectibility. Even if clinical observations suggest lower lymph node yield (LNY) after NC, data are inconclusive and it is unclear whether NC dependent parameters influence detection rates by axillary lymph node dissection (ALND).MethodsWe analyzed retrospectively the LNY in 182 patients with ALND after NC and 351 patients with primary ALND. Impact of surgery or pathological examination and specific histomorphological alterations were evaluated. Outcome analyses regarding recurrence rates, disease free (DFS) and overall survival (OS) were performed.ResultsAxillary LNY was significantly lower in the NC in comparison to the primary surgery group (median 13 vs. 16; p < 0.0001). The likelihood of incomplete axillary staging was four times higher in the NC group (14.8% vs. 3.4%, p < 0.0001). Multivariate analyses excluded any influence by surgeon or pathologist. However, the chemotherapy dependent histological feature lymphoid depletion was an independent predictive factor for a lower LNY. Outcome analyses revealed no significant impact of the LNY on local and regional recurrence rates as well as DFS and OS, respectively.ConclusionNC significantly reduces the LNY by ALND and has profound effects on the histomorphological appearance of lymph nodes. The current recommendations for a minimum removal of 10 lymph nodes by ALND are clearly compromised by the clinically already established concept of NC. The LNY of less than 10 by ALND after NC might not be indicative for an insufficient axillary staging.

Exometabolom analysis of breast cancer cell lines: Metabolic signature

Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach.

Gastrin-releasing Peptide Receptor Imaging in Breast Cancer Using the Receptor Antagonist 68Ga-RM2 And PET

Introduction: The gastrin-releasing peptide receptor (GRPR) is overexpressed in breast cancer. The present study evaluates GRPR imaging as a novel imaging modality in breast cancer by employing positron emission tomography (PET) and the GRPR antagonist 68Ga-RM2. Methods: Fifteen female patients with biopsy confirmed primary breast carcinoma (3 bilateral tumors; median clinical stage IIB) underwent 68Ga-RM2-PET/CT for pretreatment staging. In vivo tumor uptake of 68Ga-RM2 was correlated with estrogen (ER) and progesterone (PR) receptor expression, HER2/neu status and MIB-1 proliferation index in breast core biopsy specimens. Results: 13/18 tumors demonstrated strongly increased 68Ga-RM2 uptake compared to normal breast tissue (defined as PET-positive). All PET-positive primary tumors were ER- and PR-positive (13/13) in contrast to only 1/5 PET-negative tumors. Mean SUVMAX of ER-positive tumors was 10.6±6.0 compared to 2.3±1.0 in ER-negative tumors (p=0.016). In a multivariate analysis including ER, PR, HER2/neu and MIB-1, only ER expression predicted 68Ga-RM2 uptake (model: r2=0.55, p=0.025). Normal breast tissue showed inter- and intraindividually variable, moderate GRPR binding (SUVMAX 2.3±1.0), while physiological uptake of other organs was considerably less except pancreas. Of note, 68Ga-RM2-PET/CT detected internal mammary lymph nodes with high 68Ga-RM2 uptake (n=8), a contralateral axillary lymph node metastasis (verified by biopsy) and bone metastases (n=1; not detected by bone scan and CT). Conclusion: Our study demonstrates that 68Ga-RM2-PET/CT is a promising imaging method in ER-positive breast cancer. In vivo GRPR binding assessed by 68Ga-RM2-PET/CT correlated with ER expression in primary tumors of untreated patients.

HNRNP G and HTRA2-BETA1 regulate estrogen receptor alpha expression with potential impact on endometrial cancer

BackgroundEstrogen receptor alpha (ERa/ESR1) expression is regulated by alternative splicing. Its most frequently detectable exon7 skipping isoform (ERaD7) is a dominant negative variant. Elevated expression of ERaD7 was already detected in endometrial cancer (EC), while its potential prognostic significance has not been characterized so far. Exon7 contains potential binding sites for the two functional splicing regulatory opponents, HNRNPG and HTRA2-BETA1 known to trigger opposite effects on EC outcome.This study served to elucidate the influence of HNRNPG and HTRA2-BETA1 on ERa exon7 splicing regulation and the impact of ERaD7 concentration on type 1 EC outcome.MethodsFunctional in vitro experiments for HNRNPG and HTRA2-BETA1 in regard to the regulatory impact on endogenous and exogenous ERaD7 splicing were performed. Additionally, real-time PCR determined mRNA levels of ERaD7, HNRNPG and HTRA2-BETA1 in 116 type 1 EC patients.ResultsHNRNPG and HTRA2-BETA1 were found to be specific regulators of ERa exon7 splicing. While HTRA2-BETA1 promoted exon7 inclusion, HNRNPG antagonized this effect by inducing exon7 skipping (p = 0.004). ERaD7 was detected in 71 out of 116 type 1 EC specimens. Statistical analyses revealed an inverse correlation between ERaD7 mRNA levels and tumor grading (p = 0.029), FIGO stage (p = 0.033) as well as lymph node metastases (p = 0.032), respectively. Furthermore, higher ERaD7 expression could be correlated to an improved disease-specific survival (p = 0.034).ConclusionsOur study demonstrates antagonistic regulatory effects of HNRNPG and HTRA2-BETA1 on ERa exon7 splicing with potential impact on type 1 EC clinical outcome due to the consecutively variable expression levels of the ERa isoform D7.

Comprehensive Two-Dimensional Liquid Chromatography in MetabolomeAnalysis

Metabolomics deals with analysis of highly complex biological samples resulting in insufficient separation of relevant metabolites from matrix substances with one-dimensional liquid chromatography. Comprehensive twodimensional liquid chromatography (2D-LC) is a promising technique for the analysis of complex samples because of its increased peak capacity. Comprehensive 2D-LC-MS/MS has been applied for a targeted metabolomics approach regarding metabolites from RNA catabolism. Combination of a Zorbax Eclipse Plus C18 column with a Zorbax Bonus-RP column resulted in increased peak capacity, minimisation of matrix effects and excellent reproducibility.