Publications by authors named "Marc Hirschfeld"

38 Publications

The cutoff for estrogen and progesterone receptor expression in endometrial cancer revisited: a European Network for Individualized Treatment of Endometrial Cancer collaboration study.

Hum Pathol 2020 Dec 15;109:80-91. Epub 2020 Dec 15.

Department of Oncology, KU Leuven, 3000, Leuven, Belgium; Center for Gynaecologic Oncology, Netherlands Cancer Institute and Amsterdam University Medical Center, 1066, CX, Amsterdam, the Netherlands.

There is no consensus on the cutoff for positivity of estrogen receptor (ER) and progesterone receptor (PR) in endometrial cancer (EC). Therefore, we determined the cutoff value for ER and PR expression with the strongest prognostic impact on the outcome. Immunohistochemical expression of ER and PR was scored as a percentage of positive EC cell nuclei. Cutoff values were related to disease-specific survival (DSS) and disease-free survival (DFS) using sensitivity, specificity, and multivariable regression analysis. The results were validated in an independent cohort. The study cohort (n = 527) included 82% of grade 1-2 and 18% of grade 3 EC. Specificity for DSS and DFS was highest for the cutoff values of 1-30%. Sensitivity was highest for the cutoff values of 80-90%. ER and PR expression were independent markers for DSS at cutoff values of 10% and 80%. Consequently, three subgroups with distinct clinical outcomes were identified: 0-10% of ER/PR expression with, unfavorable outcome (5-year DSS = 75.9-83.3%); 20-80% of ER/PR expression with, intermediate outcome (5-year DSS = 93.0-93.9%); and 90-100% of ER/PR expression with, favorable outcome (5-year DSS = 97.8-100%). The association between ER/PR subgroups and outcomes was confirmed in the validation cohort (n = 265). We propose classification of ER and PR expression based on a high-risk (0-10%), intermediate-risk (20-80%), and low-risk (90-100%) group.
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http://dx.doi.org/10.1016/j.humpath.2020.12.003DOI Listing
December 2020

Frameshift Variant in Novel Adenosine-A1-Receptor Homolog Associated With Bovine Spastic Syndrome/Late-Onset Bovine Spastic Paresis in Holstein Sires.

Front Genet 2020 30;11:591794. Epub 2020 Nov 30.

Department of Animal Sciences, Faculty of Agricultural Sciences, Institute of Veterinary Medicine, University of Göttingen, Göttingen, Germany.

Since their first description almost 100 years ago, bovine spastic paresis (BSP) and bovine spastic syndrome (BSS) are assumed to be inherited neuronal-progressive diseases in cattle. Affected animals are characterized by (frequent) spasms primarily located in the hind limbs, accompanied by severe pain symptoms and reduced vigor, thus initiating premature slaughter or euthanasia. Due to the late onset of BSP and BSS and the massively decreased lifespan of modern cattle, the importance of these diseases is underestimated. In the present study, BSP/BSS-affected German Holstein breeding sires from artificial insemination centers were collected and pedigree analysis, genome-wide association studies, whole genome resequencing, protein-protein interaction network analysis, and protein-homology modeling were performed to elucidate the genetic background. The analysis of 46 affected and 213 control cattle revealed four significantly associated positions on chromosome 15 (BTA15), i.e., AC_000172.1:g.83465449A>G (-logP = 19.17), AC_000172.1:g.81871849C>T (-logP = 8.31), AC_000172.1:g.81872621A>T (-logP = 6.81), and AC_000172.1:g.81872661G>C (-logP = 6.42). Two additional loci were significantly associated located on BTA8 and BTA19, i.e., AC_000165.1:g.71177788T>C and AC_000176.1:g.30140977T>G, respectively. Whole genome resequencing of five affected individuals and six unaffected relatives (two fathers, two mothers, a half sibling, and a full sibling) belonging to three different not directly related families was performed. After filtering, a homozygous loss of function variant was identified in the affected cattle, causing a frameshift in the so far unknown gene locus LOC100848076 encoding an adenosine-A1-receptor homolog. An allele frequency of the variant of 0.74 was determined in 3,093 samples of the 1000 Bull Genomes Project.
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http://dx.doi.org/10.3389/fgene.2020.591794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734149PMC
November 2020

Endoxifen and fulvestrant regulate estrogen-receptor α and related DEADbox proteins.

Endocr Connect 2020 Dec;9(12):1156-1167

Department of Obstetrics and Gynecology, Medical Center - University of Freiburg, Freiburg, Germany.

Breast cancer (BC) represents the most common type of cancer in females worldwide. Endocrine therapy evolved as one of the main concepts in treatment of hormone-receptor positive BC. Current research focuses on the elucidation of tumour resistance mechanisms against endocrine therapy. In a translational in vitro approach, potential regulatory effects of clinically implemented BC anti-oestrogens on ERα, its coactivators DDX5, DDX17 and other DEADbox proteins as well as on the proliferation markers cyclin D1 and Ki67 were investigated on both the RNA and protein level. BC in vitro models for hormone-receptor positive (MCF-7, T-47D) and hormone-receptor negative cells (BT-20) were subjected to endocrine therapy. Anti-oestrogen-dependent expression regulation of target genes on the transcriptional and translational level was quantified and statistically assessed. Endocrine therapy decreases the expression levels of Ki67, cyclin D1 and ERα in hormone-receptor positive cells. In the hormone-receptor negative cells, the three parameters remained stable after endocrine therapy. Endoxifen triggers a downregulation of DDX5 and DDX23 in MCF-7 cells. Fulvestrant treatment downregulates the expression levels of all investigated DEADbox proteins in MCF-7 cells. In T-47D cells, endoxifen and fulvestrant lead to a decrease of all target gene expression levels. Interestingly, endocrine therapy affects DEADbox RNA expression levels in BT-20 cells, too. However, this result could only be confirmed for DDX1, immunocytologically. The investigated DEADbox proteins appear to correlate with the oestrogen-dependent tumourigenesis in hormone-receptor positive BC and show expression alterations after endocrine treatment.
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http://dx.doi.org/10.1530/EC-20-0281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7774761PMC
December 2020

Mutually distinguishing microRNA signatures of breast, ovarian and endometrial cancers in vitro.

Mol Med Rep 2020 Nov 27;22(5):4048-4060. Epub 2020 Aug 27.

Department of Gynecology and Obstetrics, Faculty of Medicine and Medical Center, University of Freiburg, D‑79106 Freiburg, Germany.

Early diagnosis and therapy in the first stages of a malignant disease is the most crucial factor for successful cancer treatment and recovery. Currently, there is a high demand for novel diagnostic tools that indicate neoplasms in the first or pre‑malignant stages. MicroRNAs (miRNA or miR) are small non‑coding RNAs that may act as oncogenes and downregulate tumor‑suppressor genes. The detection and mutual discrimination of the three common female malignant neoplasia types breast (BC), ovarian (OC) and endometrial cancer (EC) could be enabled by identification of tumor entity‑specific miRNA expression differences. In the present study, the relative expression levels of 25 BC, EC and OC‑related miRNAs were assessed by reverse transcription‑quantitative PCR and determined using the 2‑ΔΔCq method for normalization against the mean of four housekeeping genes. Expression levels of all miRNAs were analyzed by regression against cell line as a factor. An expression level‑based discrimination between BC and OC cell types was obtained for a subgroup of ten different miRNA types. miR‑30 family genes, as well as three other miRNAs, were found to be uniformly upregulated in OC cells compared with BC cells. BC and EC cells could be distinguished by the expression profiles of six specific miRNAs. In addition, four miRNAs were differentially expressed between EC and OC cells. In conclusion, miRNAs were identified as a potential novel tool to detect and mutually discriminate between BC, OC and EC. Based on a subset of 25 clinically relevant human miRNA types, the present study could significantly discriminate between these three female cancer types by means of their expression levels. For further verification and validation of miRNA‑based biomarker expression signatures that enable valuable tumor detection and characterization in routine screening or potential therapy monitoring, additional and extended in vitro analyses, followed by translational studies utilizing patients' tissue and liquid biopsy materials, are required.
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http://dx.doi.org/10.3892/mmr.2020.11466DOI Listing
November 2020

Skin exhibits of Dark Ronald XX are homozygous wild type at the Warmblood fragile foal syndrome causative missense variant position in lysyl hydroxylase gene PLOD1.

Anim Genet 2020 Oct 17;51(5):838-840. Epub 2020 Jun 17.

Institute of Veterinary Medicine, University of Goettingen, Göttingen, Germany.

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http://dx.doi.org/10.1111/age.12972DOI Listing
October 2020

Preoperative risk stratification in endometrial cancer (ENDORISK) by a Bayesian network model: A development and validation study.

PLoS Med 2020 05 15;17(5):e1003111. Epub 2020 May 15.

Department of Obstetrics and Gynaecology, Radboud University Medical Center, Nijmegen, The Netherlands.

Background: Bayesian networks (BNs) are machine-learning-based computational models that visualize causal relationships and provide insight into the processes underlying disease progression, closely resembling clinical decision-making. Preoperative identification of patients at risk for lymph node metastasis (LNM) is challenging in endometrial cancer, and although several biomarkers are related to LNM, none of them are incorporated in clinical practice. The aim of this study was to develop and externally validate a preoperative BN to predict LNM and outcome in endometrial cancer patients.

Methods And Findings: Within the European Network for Individualized Treatment of Endometrial Cancer (ENITEC), we performed a retrospective multicenter cohort study including 763 patients, median age 65 years (interquartile range [IQR] 58-71), surgically treated for endometrial cancer between February 1995 and August 2013 at one of the 10 participating European hospitals. A BN was developed using score-based machine learning in addition to expert knowledge. Our main outcome measures were LNM and 5-year disease-specific survival (DSS). Preoperative clinical, histopathological, and molecular biomarkers were included in the network. External validation was performed using 2 prospective study cohorts: the Molecular Markers in Treatment in Endometrial Cancer (MoMaTEC) study cohort, including 446 Norwegian patients, median age 64 years (IQR 59-74), treated between May 2001 and 2010; and the PIpelle Prospective ENDOmetrial carcinoma (PIPENDO) study cohort, including 384 Dutch patients, median age 66 years (IQR 60-73), treated between September 2011 and December 2013. A BN called ENDORISK (preoperative risk stratification in endometrial cancer) was developed including the following predictors: preoperative tumor grade; immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), p53, and L1 cell adhesion molecule (L1CAM); cancer antigen 125 serum level; thrombocyte count; imaging results on lymphadenopathy; and cervical cytology. In the MoMaTEC cohort, the area under the curve (AUC) was 0.82 (95% confidence interval [CI] 0.76-0.88) for LNM and 0.82 (95% CI 0.77-0.87) for 5-year DSS. In the PIPENDO cohort, the AUC for 5-year DSS was 0.84 (95% CI 0.78-0.90). The network was well-calibrated. In the MoMaTEC cohort, 249 patients (55.8%) were classified with <5% risk of LNM, with a false-negative rate of 1.6%. A limitation of the study is the use of imputation to correct for missing predictor variables in the development cohort and the retrospective study design.

Conclusions: In this study, we illustrated how BNs can be used for individualizing clinical decision-making in oncology by incorporating easily accessible and multimodal biomarkers. The network shows the complex interactions underlying the carcinogenetic process of endometrial cancer by its graphical representation. A prospective feasibility study will be needed prior to implementation in the clinic.
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http://dx.doi.org/10.1371/journal.pmed.1003111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228042PMC
May 2020

Urinary Exosomal MicroRNAs as Potential Non-invasive Biomarkers in Breast Cancer Detection.

Mol Diagn Ther 2020 04;24(2):215-232

Department of Obstetrics and Gynecology, Medical Center, University of Freiburg, Freiburg, Germany.

Introduction: Breast cancer (BC) is the most frequent malignant disease in women worldwide and is therefore challenging for the healthcare system. Early BC detection remains a leading factor that improves overall outcome and disease management. Aside from established screening procedures, there is a constant demand for additional BC detection methods. Routine BC screening via non-invasive liquid biopsy biomarkers is one auspicious approach to either complete or even replace the current state-of-the-art diagnostics. The study explores the diagnostic potential of urinary exosomal microRNAs with specific BC biomarker characteristics to initiate the potential prospective application of non-invasive BC screening as routine practice.

Methods: Based on a case-control study (69 BC vs. 40 healthy controls), expression level quantification and subsequent biostatistical computation of 13 urine-derived microRNAs were performed to evaluate their diagnostic relevance in BC.

Results: Multilateral statistical assessment determined and repeatedly confirmed a specific panel of four urinary microRNA types (miR-424, miR-423, miR-660, and let7-i) as a highly specific combinatory biomarker tool discriminating BC patients from healthy controls, with 98.6% sensitivity and 100% specificity.

Discussion: Urine-based BC diagnosis may be achieved through the analysis of distinct microRNA panels with proven biomarker abilities. Subject to further validation, the implementation of urinary BC detection in routine screening offers a promising non-invasive alternative in women's healthcare.
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http://dx.doi.org/10.1007/s40291-020-00453-yDOI Listing
April 2020

Discovery of potential serum and urine-based microRNA as minimally-invasive biomarkers for breast and gynecological cancer.

Cancer Biomark 2020 ;27(2):225-242

Department of Obstetrics and Gynecology, Medical Center, University of Freiburg, Freiburg, Germany.

Background: Deregulated microRNAs (miRNAs) in breast and gynecological cancer might contribute to improve early detection of female malignancies.

Objective: Specification of miRNA types in serum and urine as minimally-invasive biomarkers for breast (BC), endometrial (EC) and ovarian cancer (OC).

Methods: In a discovery phase, serum and urine samples from 17 BC, five EC and five OC patients vs. ten healthy controls (CTRL) were analyzed with Agilent human miRNA microarray chip. Selected miRNA types were further investigated by RT-qPCR in serum (31 BC, 13 EC, 15 OC patients, 32 CTRL) and urine (25 BC, 10 EC, 10 OC patients, 30 CTRL) applying two-sample t-tests.

Results: Several miRNA biomarker candidates exhibited diagnostic features due to distinctive expression levels (serum: 26; urine: 22). Among these, miR-518b, -4719 and -6757-3p were found specifically deregulated in BC serum. Four, non-entity-specific, novel biomarker candidates with unknown functional roles were identified in urine (miR-3973; -4426; -5089-5p and -6841). RT-qPCR identified miR-484/-23a (all p⩽ 0.001) in serum as potential diagnostic markers for EC and OC while miR-23a may also serve as an endogenous control in BC diagnosis.

Conclusions: Promising miRNAs as liquid biopsy-based tools in the detection of BC, EC and OC qualified for external validation in larger cohorts.
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http://dx.doi.org/10.3233/CBM-190575DOI Listing
July 2020

Osteogenesis imperfecta in a male holstein calf associated with a possible oligogenic origin.

Vet Q 2020 Dec;40(1):58-67

Institute of Veterinary Medicine, University of Goettingen, Göttingen, Germany.

Neuromusculoskeletal anomalies generally in combination with severe clinical symptoms, comprise a heterogeneous group of fairly common and mostly fatal disorders in man and animals. Osteogenesis imperfecta (OI), also known as brittle bone disease, causes bone fragility and deformity. Prominent extra-skeletal accessory manifestations of OI comprise blue/gray sclerae, hearing impairment, lung abnormalities and hypercalciuria. Cases of OI in cattle have been reported. However, no causative mutations have been identified in cattle so far. To report a possible oligogenic origin identified in a calf from clinically healthy parents suffering from OI. A neonatal embryo transfer male Holstein calf developing multiple fractures with bone tissue showing marked osteopenia was used for whole genome re-sequencing as well as its parents. In addition, 2,612 randomly chosen healthy Holstein cattle were genotyped as well as controls. Sixteen candidate genes with potential protein-altering variants were selected revealing non-synonymous variants only within and genes. However, in-depth gene analysis did not result in the identification of a single causative mutation in the OI calf. The analysis of the OI case revealed a possible oligogenic origin of the disease attributable to additive effects of three candidate genes, i.e., , , and . Most OI cases in humans and domestic animals reported so far are caused by distinct dominant or recessive monogenic mutations, therefore a potential oligogenic additive genetic effect is a novel finding. Furthermore, the case presented here demonstrates that cross-species genetic analyses might not always be straightforward.
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http://dx.doi.org/10.1080/01652176.2020.1721611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034473PMC
December 2020

Association of α/β-Hydrolase D16B with Bovine Conception Rate and Sperm Plasma Membrane Lipid Composition.

Int J Mol Sci 2020 Jan 17;21(2). Epub 2020 Jan 17.

Institute of Veterinary Medicine, University of Goettingen, 37077 Goettingen, Germany.

We have identified a Holstein sire named Tarantino who had been approved for artificial insemination that is based on normal semen characteristics (i.e., morphology, thermoresistance, motility, sperm concentration), but had no progeny after 412 first inseminations, resulting in a non-return rate (NR) of -29. Using whole genome association analysis and next generation sequencing, an associated nonsense variant in the α/β-hydrolase domain-containing 16B gene () on bovine chromosome 13 was identified. The frequency of the mutant allele in the German Holstein population was determined to be 0.0018 in 222,645 investigated cattle specimens. The mutant allele was traced back to Whirlhill Kingpin (bornFeb. 13th, 1959) as potential founder. The expression of was detected by Western blotting and immunohistochemistry in testis and epididymis of control bulls. A lipidome comparison of the plasma membrane of fresh semen from carriers and controls showed significant differences in the concentration of phosphatidylcholine (PC), diacylglycerol (DAG), ceramide (Cer), sphingomyelin (SM), and phosphatidylcholine (-ether) (PC O-), indicating that ABHD16B plays a role in lipid biosynthesis. The altered lipid contents may explain the reduced fertilization ability of mutated sperms.
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http://dx.doi.org/10.3390/ijms21020627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014312PMC
January 2020

Circulating non‑coding RNA‑biomarker potential in neoadjuvant chemotherapy of triple negative breast cancer?

Int J Oncol 2020 Jan 25;56(1):47-68. Epub 2019 Nov 25.

Department of Obstetrics and Gynecology, Faculty of Medicine, Medical Center‑University of Freiburg, D‑79106 Freiburg, Germany.

Due to the positive association between neoadjuvant chemotherapy (NACT) and the promising early response rates of patients with triple negative breast cancer (TNBC), including probabilities of pathological complete response, NACT is increasingly used in TNBC management. Liquid biopsy‑based biomarkers with the power to diagnose the early response to NACT may support established monitoring tools, which are to a certain extent imprecise and costly. Simple serum‑ or urine‑based analyses of non‑coding RNA (ncRNA) expression may allow for fast, minimally‑invasive testing and timely adjustment of the therapy regimen. The present study investigated breast cancer‑related ncRNAs [microRNA (miR)‑7, ‑9, ‑15a, ‑17, ‑18a, ‑19b, ‑21, ‑30b, ‑222 and ‑320c, PIWI‑interacting RNA‑36743 and GlyCCC2] in triple positive BT‑474 cells and three TNBC cell lines (BT‑20, HS‑578T and MDA‑MB‑231) treated with various chemotherapeutic agents using reverse transcription‑quantitative PCR. Intracellular and secreted microvesicular ncRNA expression levels were analysed using a multivariable statistical regression analysis. Chemotherapy‑driven effects were investigated by analysing cell cycle determinants at the mRNA and protein levels. Serum and urine specimens from 8 patients with TNBC were compared with 10 healthy females using two‑sample t‑tests. Samples from the patients with TNBC were compared at two time points. Chemotherapeutic treatments induced distinct changes in ncRNA expression in TNBC cell lines and the BT‑474 cell line in intra‑ and extracellular compartments. Serum and urine‑based ncRNA expression analysis was able to discriminate between patients with TNBC and controls. Time point comparisons in the urine samples of patients with TNBC revealed a general rise in the level of ncRNA. Serum data suggested a potential association between piR‑36743, miR‑17, ‑19b and ‑30b expression levels and an NACT‑driven complete clinical response. The present study highlighted the potential of ncRNAs as liquid biopsy‑based biomarkers in TNBC chemotherapy treatment. The ncRNAs tested in the present study have been previously investigated for their involvement in BC or TNBC chemotherapy responses; however, these previous studies were restricted to patient tissue or in vitro models. The data from the present study offer novel insight into ncRNA expression in liquid samples from patients with TNBC, and the study serves as an initial step in the evaluation of ncRNAs as diagnostic biomarkers in the monitoring of TNBC therapy.
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http://dx.doi.org/10.3892/ijo.2019.4920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910196PMC
January 2020

Addition of IMP3 to L1CAM for discrimination between low- and high-grade endometrial carcinomas: a European Network for Individualised Treatment of Endometrial Cancer collaboration study.

Hum Pathol 2019 07 3;89:90-98. Epub 2019 May 3.

Department of Obstetrics and Gynaecology, Radboud University Medical Centre, 6500HB, Nijmegen, the Netherlands.

Discrimination between low- and high-grade endometrial carcinomas (ECs) is clinically relevant but can be challenging for pathologists, with moderate interobserver agreement. Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) is an oncofoetal protein that is associated with nonendometrioid endometrial carcinomas but has been limited studied in endometrioid carcinomas. The aim of this study is to investigate the diagnostic and prognostic value of IMP3 in the discrimination between low- and high-grade ECs and its added value to L1CAM. IMP3 and L1CAM expression was assessed in tumors from 378 patients treated for EC at 1 of 9 participating European Network for Individualised Treatment of Endometrial Cancer centers. IMP3 was expressed in 24.6% of the tumors. In general, IMP3 was more homogeneously expressed than L1CAM. IMP3 expression was significantly associated with advanced stage, nonendometrioid histology, grade 3 tumors, deep myometrial invasion, lymphovascular space invasion, distant recurrences, overall mortality, and disease-related mortality. Simultaneous absence of IMP3 and L1CAM expression showed the highest accuracy for identifying low-grade carcinomas (area under the curve 0.766), whereas simultaneous expression of IMP3 and L1CAM was strongly associated with high-grade carcinomas (odds ratio 19.7; 95% confidence interval 9.2-42.2). Even within endometrioid carcinomas, this combination remained superior to IMP3 and L1CAM alone (odds ratio 8.6; 95% confidence interval 3.4-21.9). In conclusion, IMP3 has good diagnostic value and together with L1CAM represents the optimal combination of diagnostic markers for discrimination between low- and high-grade ECs compared to IMP3 and L1CAM alone. Because of the homogenous expression of IMP3, this marker might be valuable in preoperative biopsies when compared to the more patchy L1CAM expression.
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http://dx.doi.org/10.1016/j.humpath.2019.04.014DOI Listing
July 2019

Poor outcome in hypoxic endometrial carcinoma is related to vascular density.

Br J Cancer 2019 05 23;120(11):1037-1044. Epub 2019 Apr 23.

Department of Obstetrics and Gynaecology, Radboud Institute for Health Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.

Background: Identification of endometrial carcinoma (EC) patients at high risk of recurrence is lacking. In this study, the prognostic role of hypoxia and angiogenesis was investigated in EC patients.

Methods: Tumour slides from EC patients were stained by immunofluorescence for carbonic anhydrase IX (CAIX) as hypoxic marker and CD34 for assessment of microvessel density (MVD). CAIX expression was determined in epithelial tumour cells, with a cut-off of 1%. MVD was assessed according to the Weidner method. Correlations with disease-specific survival (DSS), disease-free survival (DFS) and distant disease-free survival (DDFS) were calculated using Kaplan-Meier curves and Cox regression analysis.

Results: Sixty-three (16.4%) of 385 ECs showed positive CAIX expression with high vascular density. These ECs had a reduced DSS compared to tumours with either hypoxia or high vascular density (log-rank p = 0.002). Multivariable analysis showed that hypoxic tumours with high vascular density had a reduced DSS (hazard ratio [HR] 3.71, p = 0.002), DDFS (HR 2.68, p = 0.009) and a trend for reduced DFS (HR 1.87, p = 0.054).

Conclusions: This study has shown that adverse outcome in hypoxic ECs is seen in the presence of high vascular density, suggesting an important role of angiogenesis in the metastatic process of hypoxic EC. Differential adjuvant treatment might be indicated for these patients.
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http://dx.doi.org/10.1038/s41416-019-0461-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738053PMC
May 2019

Christmas disease in a Hovawart family resembling human hemophilia B Leyden is caused by a single nucleotide deletion in a highly conserved transcription factor binding site of the gene promoter.

Haematologica 2019 11 7;104(11):2307-2313. Epub 2019 Mar 7.

Institute of Genetics, University of Bern, Bern, Switzerland.

Hemophilia B is a classical monogenic, X-chromosomal, recessively transmitted bleeding disorder caused by genetic variants within the coagulation factor IX gene (). Although hemophilia B has been described in dogs, it has not yet been reported in the Hovawart breed. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males were reported to suffer from recurrent hemorrhagic episodes. A blood sample from one of these males with only 2% of the normal concentration of plasma factor IX together with samples from seven relatives were provided. Next-generation sequencing of the mother and grandmother revealed a single nucleotide deletion in the promoter. Genotyping of the deletion in 1,298 dog specimens including 720 Hovawarts revealed that the mutant allele was only present in the aforementioned Hovawart family. The deletion is located 73 bp upstream of the start codon in the conserved overlapping DNA binding sites of hepatocyte nuclear factor 4α (HNF-4α) and androgen receptor (AR). The deletion only abolished binding of HNF-4α, while AR binding was unaffected as demonstrated by electrophoretic mobility shift assay using human HNF-4α and AR with double-stranded DNA probes encompassing the mutant promoter region. Luciferase reporter assays using wildtype and mutated promoter fragment constructs transfected into Hep G2 cells showed a significant reduction in expression from the mutant promoter. The data provide evidence that the deletion in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.
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http://dx.doi.org/10.3324/haematol.2018.215426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821609PMC
November 2019

Added Value of Estrogen Receptor, Progesterone Receptor, and L1 Cell Adhesion Molecule Expression to Histology-Based Endometrial Carcinoma Recurrence Prediction Models: An ENITEC Collaboration Study.

Int J Gynecol Cancer 2018 03;28(3):514-523

Objectives: Endometrial carcinoma mortality is mainly caused by recurrent disease, and various immunohistochemical markers to predict recurrences have been studied. Loss of the estrogen receptor (ER) and progesterone receptor (PR) and the presence of the L1 cell adhesion molecule (L1CAM) are promising markers, but their combined value has not been studied.

Materials And Methods: Expression of ER, PR, and L1CAM was immunohistochemically determined in 293 endometrial carcinomas from 11 collaborating European Network for Individualized Treatment of Endometrial Cancer centers. Estrogen receptor, PR, or L1CAM staining was considered positive or negative when expressed by greater than or equal to 10% or less than 10% of the tumor cells, respectively. The association between these markers and clinicopathological markers, and their combined value in predicting survival were calculated, both in the entire cohort and in a selected groups of stage I endometrioid and low-risk stage I endometrioid carcinomas.

Results: Estrogen receptor and PR were negative in 19% and 28% of the cases, respectively, and L1CAM was positive in 18%. All 3 were associated with advanced stage, high-grade, nonendometrioid histology, lymphovascular space invasion (LVSI), and reduced disease-free survival. Only advanced stage, loss of PR, and LVSI were associated with reduced disease-free survival in multivariate analysis. A prognostic model including these 3 markers was superior to 1 including only the 3 immunohistochemical markers, which was superior to the traditional model. In both the stage I endometrioid and the low-risk stage I endometrioid groups, only loss of PR was associated with reduced disease-free survival.

Conclusions: Loss of ER and PR, and the presence of L1CAM are associated with high risk characteristics, and loss of PR is the strongest predictor of recurrent disease. Although a combination of these 3 markers is slightly superior to the traditional histological markers, a prognostic model including stage, PR expression, and LVSI is the most promising model in the identification of high risk carcinomas. In the stage I endometrioid carcinomas, PR immunohistochemistry appears to be of additional value in predicting recurrences.
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http://dx.doi.org/10.1097/IGC.0000000000001187DOI Listing
March 2018

Targeted Proteomics Identifies Proteomic Signatures in Liquid Biopsies of the Endometrium to Diagnose Endometrial Cancer and Assist in the Prediction of the Optimal Surgical Treatment.

Clin Cancer Res 2017 Nov 8;23(21):6458-6467. Epub 2017 Aug 8.

Biomedical Research Group in Gynecology, Vall Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, CIBERONC, Barcelona, Spain.

Endometrial cancer (EC) diagnosis relies on the observation of tumor cells in endometrial biopsies obtained by aspiration (i.e., uterine aspirates), but it is associated with 22% undiagnosed patients and up to 50% of incorrectly assigned EC histotype and grade. We aimed to identify biomarker signatures in the fluid fraction of these biopsies to overcome these limitations. The levels of 52 proteins were measured in the fluid fraction of uterine aspirates from 116 patients by LC-PRM, the latest generation of targeted mass-spectrometry acquisition. A logistic regression model was used to assess the power of protein panels to differentiate between EC and non-EC patients and between EC histologic subtypes. The robustness of the panels was assessed by the "leave-one-out" cross-validation procedure performed within the same cohort of patients and an independent cohort of 38 patients. The levels of 28 proteins were significantly higher in patients with EC ( = 69) compared with controls ( = 47). The combination of MMP9 and KPYM exhibited 94% sensitivity and 87% specificity for detecting EC cases. This panel perfectly complemented the standard diagnosis, achieving 100% of correct diagnosis in this dataset. Nine proteins were significantly increased in endometrioid EC ( = 49) compared with serous EC ( = 20). The combination of CTNB1, XPO2, and CAPG achieved 95% sensitivity and 96% specificity for the discrimination of these subtypes. We developed two uterine aspirate-based signatures to diagnose EC and classify tumors in the most prevalent histologic subtypes. This will improve diagnosis and assist in the prediction of the optimal surgical treatment. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-0474DOI Listing
November 2017

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

Oncol Lett 2017 Aug 16;14(2):2334-2340. Epub 2017 Jun 16.

Department of Obstetrics and Gynecology, Medical Center - University of Freiburg, D-79106 Freiburg, Germany.

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 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.
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http://dx.doi.org/10.3892/ol.2017.6406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529991PMC
August 2017

Hypoxia- and acidosis-driven aberrations of secreted microRNAs in endometrial cancer in vitro.

Oncol Rep 2017 Aug 13;38(2):993-1004. Epub 2017 Jun 13.

Department of Obstetrics and Gynecology, Medical Center - University of Freiburg, Freiburg, Germany.

Due to their post-transcriptional regulatory impact on gene expression, microRNAs (miRNA, miRs) influence decisively cellular processes of differentiation, proliferation and apoptosis. In oncogenic pathways various miRNAs exert either oncogenic or tumor suppressor activities in a stage-specific manner. Dysregulation of miRNA expression pattern has been associated with several human cancers including endometrial cancer (EC). In the present study, expression profile alterations of EC associated secreted miRNAs were determined under the microenvironmental stress situations hypoxia and acidosis occurring in tumor progression and metastasis. The potential influence of hypoxia and acidosis vs. control conditions on the expression levels of 24 EC-relevant miRNA types was quantitatively accessed via real-time PCR in three established EC in vitro models. Expression data were analyzed statistically. In vitro application of hypoxia resulted in downregulation of miR-15a, miR-20a, miR-20b and miR-128-1 in Ishikawa cells (type I EC) and upregulation of miR-21 in EFE-184 cells (type I EC). Acidosis triggered upregulation of tumor promoting miR-125b in AN3-CA cell (type II EC), whereas in Ishikawa cells (type I EC) miRNAs with tumor suppressive function were found altered in divergent directions, both up- (let-7a) and down- (miR-22) regulated. Our current findings emphasize the functional importance of secreted miRNAs in the immediate response of EC cells to exogenic stress situations such as the typical tumor epiphenomena hypoxia and acidosis. Focusing on the specific potential of secreted, thus circulating miRNA molecules, alterations in expression levels not only influence intracellular gene expression and signaling cascades, but also transfer the induction of (tumor)biological cellular changes to adjacent cells.
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http://dx.doi.org/10.3892/or.2017.5717DOI Listing
August 2017

Cyr61 Expression Pattern and Association with Clinicopathological Factors in Patients with Cervical Cancer.

Anticancer Res 2017 05;37(5):2451-2456

Department of Obstetrics and Gynecology, Medical Center - University of Freiburg, Freiburg, Germany.

Background/aim: The pro-angiogenic Cyr61 protein has been associated with tumorigenesis and cancer progression in different gynecological carcinomas. In this study, we evaluated the potential impact and clinical relevance of Cyr61 expression in patients with primary non-metastatic cervical cancer (CC).

Patients And Methods: Cyr61 expression was assessed in tissue specimen of 48 patients with primary CC by immunohistochemical analysis. Expression levels were scored and correlated to clinico-pathological factors and outcome data.

Results: High Cyr61 expression levels were present in 54.2% of CC tissues. Associations with histological grade (p=0.030), depth of tumor invasion (p=0.007) and GOG score (p=0.027) were observed. Patients who overexpressed Cyr61 displayed an increased death rate (30.8% vs. 18.2%) and a decreased 5-year-survival (76.9% vs. 86.4%).

Conclusion: Our data indicate a potential functional impact of Cyr61 in development and the progression of CC. The definite tumor-relevant function (suppressive/promoting) of Cyr61 in CC and the prognostic relevance of Cyr61 overexpression has to be evaluated in larger cohorts.
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http://dx.doi.org/10.21873/anticanres.11585DOI Listing
May 2017

Absence of epithelial atypia in B3-lesions of the breast is associated with decreased risk for malignancy.

Breast 2017 Feb 17;31:144-149. Epub 2016 Nov 17.

Department of Obstetrics and Gynecology, Medical Center - University of Freiburg, Germany; Faculty of Medicine, University of Freiburg, Germany.

Introduction: Lesions of uncertain malignant potential (B3) represent a heterogeneous group with an overall risk for malignancy of 9.85-35.1% after total resection. Positive predictive values (PPV) for malignancy vary depending on B3 subtype. The aim of this study was to evaluate the PPV for malignancy in B3 lesions and to determine the clinical significance of atypia-dependent sub-classification (a = without epithelial atypia; b = with epithelial atypia) of B3 into B3a and B3b and papillary lesions (PL) in PLa and PLb.

Methods: 219 patients with histopathologically proven B3 lesions on core needle/vacuum-assisted biopsy who subsequently underwent diagnostic excision biopsy were included in this study. PPVs for malignancy were reported for B3 in general and all B3 sub-categories. Logistic regression analysis identified associations between B3-subgroups and outcome after excision biopsy as well as the impact of clinical and diagnostic findings on excision diagnosis.

Results: The overall PPV rate was 10.0% (22/219). Excision histology exhibited a higher malignancy rate in PLb (2/7; PPV: 28.6%) than in PLa (6/127; PPV: 4.7%) (p = 0.057) and in B3b (12/50; PPV: 24.0%) compared to B3a category (8/165; PPV: 4.8%) (p < 0.001).

Discussion: These findings support the necessity of B3 lesion sub-classification into B3a and B3b and of PL into PLa and PLb when considering epithelial atypia. The determination of atypia status represents a relevant factor in risk-stratification for clinical management of B3 lesions. Should future studies using the sub-classification of PL confirm these results, observation may be a safe option for the clinical management of patients with asymptomatic PLa lesions.
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http://dx.doi.org/10.1016/j.breast.2016.11.007DOI Listing
February 2017

L1CAM expression in endometrial carcinomas: an ENITEC collaboration study.

Br J Cancer 2016 Sep 9;115(6):716-24. Epub 2016 Aug 9.

Department of Obstetrics and Gynaecology, TweeSteden Hospital, Doctor Deelenlaan 5, Tilburg 5042AD, The Netherlands.

Background: Identification of aggressive endometrioid endometrial carcinomas (EECs) and non-endometrioid carcinomas (NEECs) is essential to improve outcome. L1 cell adhesion molecule (L1CAM) expression is a strong prognostic marker in stage I EECs, but less is known about L1CAM expression in advanced-stage EECs and NEECs. This study analyses L1CAM expression in a clinically representative cohort of endometrial carcinomas.

Methods: The expression of L1CAM was immunohistochemically determined in 1199 endometrial carcinomas, treated at one of the European Network for Individualized Treatment of Endometrial Cancer (ENITEC) centres. Staining was considered positive when >10% of the tumour cells expressed L1CAM. The association between L1CAM expression and several clincopathological characteristics and disease outcome was calculated.

Results: In all, L1CAM was expressed in 10% of the 935 stage I EECs, 18% of the 160 advanced stage EECs, and 75% of the 104 NEECs. The expression of L1CAM was associated with advanced stage, nodal involvement, high tumour grade, non-endometrioid histology, lymphovascular space invasion, and distant recurrences in all cases, and with reduced survival in the EECs, but not in the NEECs.

Conclusions: The expression of L1CAM is a strong predictor of poor outcome in EECs, but not NEECs. It is strongly associated with non-endometrioid histology and distant spread, and could improve the postoperative selection of high-risk endometrial carcinomas. The value of L1CAM expression in the preoperative selection of high-risk endometrial carcinomas should be studied.
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http://dx.doi.org/10.1038/bjc.2016.235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023774PMC
September 2016

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

Int J Hyperthermia 2016 09 5;32(6):630-42. Epub 2016 Jul 5.

a Department of Gynaecology and Obstetrics, Medical Centre , University of Freiburg , Freiburg , Germany ;

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.
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http://dx.doi.org/10.3109/02656736.2016.1161832DOI Listing
September 2016

Alterations of the exo- and endometabolite profiles in breast cancer cell lines: A mass spectrometry-based metabolomics approach.

Anal Chim Acta 2016 Jun 28;925:34-42. Epub 2016 Apr 28.

Center for Biological Systems Analysis ZBSA, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany. Electronic address:

In recent years, knowledge about metabolite changes which are characteristic for the physiologic state of cancer cells has been acquired by liquid chromatography coupled to mass spectrometry. Distinct molecularly characterized breast cancer cell lines provide an unbiased and standardized in vitro tumor model reflecting the heterogeneity of the disease. Tandem mass spectrometry is a widely applied analytical platform and highly sensitive technique for analysis of complex biological samples. Endo- and exometabolite analysis of the breast cancer cell lines MDA-MB-231, -453 and BT-474 as well as the breast epithelial cell line MCF-10A has been performed using two different analytical platforms: UPLC-ESI-Q-TOF based on a scheduled precursor list has been applied for highlighting of significant differences between cell lines and HPLC-ESI-QqQ using multiple reaction monitoring has been utilized for a targeted approach focusing on RNA metabolism and interconnected pathways, respectively. Statistical analysis enabled a clear discrimination of the breast epithelial from the breast cancer cell lines. As an effect of oxidative stress, a decreased GSH/GSSG ratio has been detected in breast cancer cell lines. The triple negative breast cancer cell line MDA-MB-231 showed an elevation in nicotinamide, 1-ribosyl-nicotinamide and NAD+ reflecting the increased energy demand in triple negative breast cancer, which has a more aggressive clinical course than other forms of breast cancer. Obtained distinct metabolite pattern could be correlated with distinct molecular characteristics of breast cancer cells. Results and methodology of this preliminary in vitro study could be transferred to in vivo studies with breast cancer patients.
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http://dx.doi.org/10.1016/j.aca.2016.04.047DOI Listing
June 2016

BMI and Pathologic Complete Response to Neoadjuvant Chemotherapy in Breast Cancer: A Study and Meta-Analysis.

Clin Breast Cancer 2016 08 9;16(4):e119-32. Epub 2016 Mar 9.

Department of Obstetrics and Gynecology, Medical Center - University of Freiburg, Freiburg, Germany.

Introduction: There is only limited data from clinical practice on the relevance of body mass index (BMI) on pathologic complete response (pCR) following neoadjuvant chemotherapy (NAC) in patients with breast cancer (BC).

Patients And Methods: The impact of BMI on pCR and survival outcome was examined in 324 patients with primary non-metastatic BC. An additional meta-analysis was performed on the current data and relevant previously published studies in clinical practice.

Results: Multivariable regression analysis identified lymph vascular invasion (odds ratio [OR], 0.05; 95% confidence interval [CI], 0.01-0.18; P = .0000), grading 3 (OR, 3.12; 95% CI, 1.59-6.12; P = .0009), and HER2/neu status (OR, 4.76; 95% CI, 1.86-12.18; P = .011) as independent factors for pCR after NAC. There was no association between pCR and continuous or categorical BMI. Various additional subgroup analyses of molecular BC subtypes (triple-negative, luminal-like, HER2-luminal, HER2-like) and BMI also showed no association. These findings were confirmed by the meta-analysis. Except for one subgroup analysis in which overweight and obese patients were combined as one group, no association between BMI and pCR as well as survival outcome was found.

Conclusions: BMI was not established as a relevant clinical factor. Only lymph vascular invasion, grading 3, luminal-like, and HER2/like BC subtype showed predictive and prognostic impact in patients with BC receiving NAC.
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http://dx.doi.org/10.1016/j.clbc.2016.02.018DOI Listing
August 2016

Exometabolom analysis of breast cancer cell lines: Metabolic signature.

Sci Rep 2015 Aug 21;5:13374. Epub 2015 Aug 21.

Center for Biological Systems Analysis ZBSA, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany.

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.
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http://dx.doi.org/10.1038/srep13374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4544000PMC
August 2015

Alternative splicing of synuclein gamma in endometrial cancer: identification of a novel isoform.

Oncotarget 2015 Sep;6(26):22553-63

Department of Obstetrics and Gynecology, University Medical Center, Freiburg, Germany.

Synuclein gamma (SNCG) is under consideration as a potential biomarker in cancer biology. Up to date four different SNCG variants are described. Due to growing evidence suggesting correlations between aberrant alternative splicing processes and cancer progression, we investigated the effects of peritumoural conditions on expression pattern of SNCG in endometrial cancer (EC) in vitro. Compared to breast cancer cell lines, mRNA expression levels of all known SNCG isoforms 1-4 are significantly reduced in EC cell lines. We identified a novel alternatively spliced variant of isoform 2 (isoform 2 short) which is found highly expressed in EC cell lines. Hypoxia and acidosis trigger an up-regulation of isoform 2 short. EC cell lines are characterized by low SNCG protein levels under control conditions, but exhibit a significant increase triggered by hypoxia and acidosis. In addition we analysed the potential association between SNCG protein expression and clinico-pathological parameters in human EC samples. Our findings indicate a grade-dependent induction of SNCG protein expression in endometrial cancer. We identified for the first time a novel isoform of SNCG that is found specifically expressed in EC. Our results also strongly indicate the existence of a corresponding protein of isoform 2 short that potentially plays a critical role in EC cancer progression.
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http://dx.doi.org/10.18632/oncotarget.4155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673181PMC
September 2015

RON alternative splicing regulation in primary ovarian cancer.

Oncol Rep 2015 Jul 19;34(1):423-30. Epub 2015 May 19.

Department of Obstetrics and Gynecology, University Medical Center Freiburg, Freiburg, Germany.

The proto-oncogene recepteur d'origine nantais (RON, MST1R) and its alternatively spliced variants are involved in various tumor biological processes, such as cell motility, adhesion, proliferation, apoptosis and epithelial-to-mesenchymal transition (EMT). RON overexpression and the occurrence of specific alternatively spliced RON isoforms have been detected in ovarian cancer. In the present study, we evaluated the role and regulation of cancer-related RON splicing isoforms in primary ovarian cancer. Expression of RON variants (RONΔ165, RONΔ160) was determined in 45 primary ovarian cancer and 4 physiological ovarian tissue specimens by RT-PCR and western blot analysis. The results were correlated to clinicopathological parameters. Additionally, expression of splicing factors with known involvement in RON alternative splicing regulation was examined. Increased RON levels were detected in all tumor samples (p=0.001) without differences between the primary tumors and metastases. Alternative RON variants were present in the majority of tumor samples (39 of 45; 86.67%). Potential RONΔ165 occurred more often (82.22%) than potential RONΔ160 or RONΔ155 (24.40%). Several significant correlations of RON and splicing factor expression [e.g. ASF/SFRS1 (p=0.035)] were detected. Correlations of RON expression to clinicopathological parameters were not observed. Significant splicing factor interactions (e.g. SRp55/SRp75: p<0.001) were observed in tumor samples with alternative RON splicing. Our data demonstrated upregulated RON isoform expression and significant changes in splicing factor expression in primary ovarian cancer. These findings account for an essential regulatory interplay of splicing factor-driven alterations in the RON alternative splicing pattern with subsequent tumor biological consequences in ovarian cancer.
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http://dx.doi.org/10.3892/or.2015.3995DOI Listing
July 2015

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

BMC Cancer 2015 Mar 28;15:193. Epub 2015 Mar 28.

Department of Obstetrics and Gynecology, University Medical Center Freiburg, Hugstetterstr. 55, Freiburg, 79106, Germany.

Background: Since 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.

Methods: Urinary 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.

Results: Significant 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.

Conclusions: We 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.
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http://dx.doi.org/10.1186/s12885-015-1190-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383066PMC
March 2015

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

BMC Cancer 2015 Feb 27;15:86. Epub 2015 Feb 27.

Department of Obstetrics and Gynecology, University Medical Center Freiburg, Hugstetterstr 55, 79106, Freiburg, Germany.

Background: Estrogen 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.

Methods: Functional 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.

Results: HNRNPG 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).

Conclusions: Our 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.
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http://dx.doi.org/10.1186/s12885-015-1088-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355463PMC
February 2015

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

BMC Cancer 2014 Jan 3;14. Epub 2014 Jan 3.

Department of Gynaecology and Obstetrics, University Medical Center Freiburg, Hugstetter Street 55, 79106 Freiburg, Germany.

Background: Neoadjuvant 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).

Methods: We 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.

Results: Axillary 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.

Conclusion: NC 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.
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http://dx.doi.org/10.1186/1471-2407-14-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3884010PMC
January 2014