Sebastian Mayer

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.

Cancer testis antigens and NY-BR-1 expression in primary breast cancer: prognostic and therapeutic implications

BackgroundCancer–testis antigens (CTA) comprise a family of proteins, which are physiologically expressed in adult human tissues solely in testicular germ cells and occasionally placenta. However, CTA expression has been reported in various malignancies. CTAs have been identified by their ability to elicit autologous cellular and or serological immune responses, and are considered potential targets for cancer immunotherapy. The breast differentiation antigen NY-BR-1, expressed specifically in normal and malignant breast tissue, has also immunogenic properties. Here we evaluated the expression patterns of CTAs and NY-BR-1 in breast cancer in correlation to clinico-pathological parameters in order to determine their possible impact as prognostic factors.MethodsThe reactivity pattern of various mAbs (6C1, MA454, M3H67, 57B, E978, GAGE #26 and NY-BR-1 #5) were assessed by immunohistochemistry in a tissue micro array series of 210 randomly selected primary invasive breast cancers in order to study the diversity of different CTAs (e.g. MAGE-A, NY-ESO-1, GAGE) and NY-BR-1. These expression data were correlated to clinico-pathological parameters and outcome data including disease-free and overall survival.ResultsExpression of at least one CTA was detectable in the cytoplasm of tumor cells in 37.2% of the cases. NY-BR-1 expression was found in 46.6% of tumors, respectively. Overall, CTA expression seemed to be linked to adverse prognosis and M3H67 immunoreactivity specifically was significantly correlated to shorter overall and disease-free survival (p=0.000 and 0.024, respectively).ConclusionsOur findings suggest that M3H67 immunoreactivity could serve as potential prognostic marker in primary breast cancer patients. The exclusive expression of CTAs in tumor tissues as well as the frequent expression of NY-BR-1 could define new targets for specific breast cancer therapies.

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.

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.

Clinical relevance of Cyr61 expression in patients with hormone-dependent breast cancer

Tumor resistance to endocrine therapy triggers estrogen-independent cancer progression, which is a major obstacle to the successful treatment of hormone receptor positive breast cancer (BC). The underlying molecular mechanisms of endocrine resistance are not fully understood yet. The matricellular protein cysteine-rich angiogenic inducer 61 (Cyr61) is associated with tumor invasiveness and the induction of tumorigenesis in various malignancies in vivo and the induction of estrogen-independence and endocrine therapy resistance in BC. The present study evaluated the potential effects and clinical relevance of Cyr61 expression levels in 67 patients with primary non-metastatic BC. Immunohistochemical analysis of formalin-fixed paraffin-embedded tissue sections was performed, and the association between Cyr61 protein expression and clinicopathological factors and survival was analyzed. Cyr61 overexpression was revealed to be significantly associated with a positive estrogen receptor (ER)/progesterone receptor (PR) status (P=0.016) and to the molecular subtype of BC (P=0.039). Compared with patients without Cyr61 overexpression, patients with Cyr61 overexpression exhibited an increased recurrence rate (30.6 vs. 22.6%) and decreased long-term survival (10-year overall survival, 62.9 vs. 69.7%); however, these associations did not reach statistically significant levels in Cox regression model analysis. Similar results were identified in the subgroup analysis of patients with ER/PR positive BC. These results indicate that Cyr61 serves a role in the development of endocrine therapy resistance in BC and is thus a potential therapeutic target to overcome endocrine therapy resistance. However, additional long-term survival analyses with large patient populations are required.

Hyperthermia-driven aberrations of secreted microRNAs in breast cancer in vitro

Abstract Purpose: Expression profile alterations of nine breast cancer (BC)-associated secreted microRNAs (miRs) were determined under microenvironmental alterations occurring in tumour progression, metastasis or specific oncological treatment modalities. Thereto, the potential influence of the exogenic stimuli hypoxia, acidosis and hyperthermia was investigated in vitro. Material and methods: Four established BC cell lines were applied as in vitro BC model systems. Quantitative analyses of secreted microRNA specimens were performed by RNA isolation from cell culture supernatant and subsequent real-time PCR in cells under physiological versus hypoxic, acidic or hyperthermia conditions. Results: The in vitro application of exogenic stimuli hypoxia, extracellular acidosis and hyperthermia caused heterogeneous expression alterations for the investigated secreted miRNA phenotypes. The majority of relevant exogenic stimuli-dependent microRNA expression alterations were restricted to single events displaying distinct cell type and stimulus dependent correlations only. Most remarkably, hyperthermia triggered a uniform significant down-regulatory effect on the expression levels of the three secreted microRNAs miR-10b, miR-15b and miR-139, respectively. The marked decrease in miR-10b and miR-15b levels was detectable in all four, while miR-139 was found significantly reduced in three out of four BC cell lines. Conclusion: Hyperthermia-dependent down-regulatory influence on three distinct BC-related microRNAs in vitro generates translational aspects for clinical BC treatment, since the identified microRNAs miR-10b, miR-15b and miR-139 are known to have oncogenic as well as tumour suppressor functions in BC. However, an evaluation regarding the potential impact of microRNA-related hyperthermia-dependent alterations for innovative BC treatment approaches demands further analysis including in vivo data.