Gyöngyi Munkácsy

Meta-analysis of gene expression profiles associated with histological classification and survival in 829 ovarian cancer samples.

Transcriptomic analysis of global gene expression in ovarian carcinoma can identify dysregulated genes capable to serve as molecular markers for histology subtypes and survival. The aim of our study was to validate previous candidate signatures in an independent setting and to identify single genes capable to serve as biomarkers for ovarian cancer progression. As several datasets are available in the GEO today, we were able to perform a true meta‐analysis. First, 829 samples (11 datasets) were downloaded, and the predictive power of 16 previously published gene sets was assessed. Of these, eight were capable to discriminate histology subtypes, and none was capable to predict survival. To overcome the differences in previous studies, we used the 829 samples to identify new predictors. Then, we collected 64 ovarian cancer samples (median relapse‐free survival 24.5 months) and performed TaqMan Real Time Polimerase Chain Reaction (RT‐PCR) analysis for the best 40 genes associated with histology subtypes and survival. Over 90% of subtype‐associated genes were confirmed. Overall survival was effectively predicted by hormone receptors (PGR and ESR2) and by TSPAN8. Relapse‐free survival was predicted by MAPT and SNCG. In summary, we successfully validated several gene sets in a meta‐analysis in large datasets of ovarian samples. Additionally, several individual genes identified were validated in a clinical cohort.

Aberrant DNA methylation impacts gene expression and prognosis in breast cancer subtypes

DNA methylation has a substantial impact on gene expression, affecting the prognosis of breast cancer (BC) patients dependent on molecular subtypes. In this study, we investigated the prognostic relevance of the expression of genes reported as aberrantly methylated, and the link between gene expression and DNA methylation in BC subtypes. The prognostic value of the expression of 144 aberrantly methylated genes was evaluated in ER+/HER2−, HER2+, and ER−/HER2− molecular BC subtypes, in a meta‐analysis of two large transcriptomic cohorts of BC patients (n = 1,938 and n = 1,640). The correlation between gene expression and DNA methylation in distinct gene regions was also investigated in an independent dataset of 104 BCs. Survival and Pearson correlation analyses were computed for each gene separately. The expression of 48 genes was significantly associated with BC prognosis (p < 0.05), and 32 of these prognostic genes exhibited a direct expression–methylation correlation. The expression of several immune‐related genes, including CD3D and HLA‐A, was associated with both relapse‐free survival (HR = 0.42, p = 3.5E‐06; HR = 0.35, p = 1.7E‐08) and overall survival (HR = 0.50, p = 5.5E‐04; HR = 0.68, p = 4.5E‐02) in ER‐/HER2‐ BCs. On the overall, the distribution of both positive and negative expression–methylation correlation in distinct gene regions have different effects on gene expression and prognosis in BC subtypes. This large‐scale meta‐analysis allowed the identification of several genes consistently associated with prognosis, whose DNA methylation could represent a promising biomarker for prognostication and clinical stratification of patients with distinct BC subtypes.

TP53 mutation hits energy metabolism and increases glycolysis in breast cancer.

Promising new hallmarks of cancer is alteration of energy metabolism that involves molecular mechanisms shifting cancer cells to aerobe glycolysis. Our goal was to evaluate the correlation between mutation in the commonly mutated tumor suppressor gene TP53 and metabolism. We established a database comprising mutation and RNA-seq expression data of the TCGA repository and performed receiver operating characteristics (ROC) analysis to compare expression of each gene between TP53 mutated and wild type samples. All together 762 breast cancer samples were evaluated of which 215 had TP53 mutation. Top up-regulated metabolic genes include glycolytic enzymes (e.g. HK3, GPI, GAPDH, PGK1, ENO1), glycolysis regulator (PDK1) and pentose phosphate pathway enzymes (PGD, TKT, RPIA). Gluconeogenesis enzymes (G6PC3, FBP1) were down-regulated. Oxygen consumption and extracellular acidification rates were measured in TP53 wild type and mutant breast cell lines with a microfluorimetric analyzer. Applying metabolic inhibitors in the presence and absence of D-glucose and L-glutamine in cell culture experiments resulted in higher glycolytic and mitochondrial activity in TP53 mutant breast cancer cell lines. In summary, TP53 mutation influences energy metabolism at multiple levels. Our results provide evidence for the synergistic activation of multiple hallmarks linking to these the mutation status of a key driver gene.

Validation of RNAi Silencing Efficiency Using Gene Array Data shows 18.5% Failure Rate across 429 Independent Experiments.

No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA) for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal–Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC) of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E−06). Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR) or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E−04). There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency.No independent cross-validation of success rate for studies utilizing small interfering RNA (siRNA) for gene silencing has been completed before. To assess the influence of experimental parameters like cell line, transfection technique, validation method, and type of control, we have to validate these in a large set of studies. We utilized gene chip data published for siRNA experiments to assess success rate and to compare methods used in these experiments. We searched NCBI GEO for samples with whole transcriptome analysis before and after gene silencing and evaluated the efficiency for the target and off-target genes using the array-based expression data. Wilcoxon signed-rank test was used to assess silencing efficacy and Kruskal-Wallis tests and Spearman rank correlation were used to evaluate study parameters. All together 1,643 samples representing 429 experiments published in 207 studies were evaluated. The fold change (FC) of down-regulation of the target gene was above 0.7 in 18.5% and was above 0.5 in 38.7% of experiments. Silencing efficiency was lowest in MCF7 and highest in SW480 cells (FC = 0.59 and FC = 0.30, respectively, P = 9.3E-06). Studies utilizing Western blot for validation performed better than those with quantitative polymerase chain reaction (qPCR) or microarray (FC = 0.43, FC = 0.47, and FC = 0.55, respectively, P = 2.8E-04). There was no correlation between type of control, transfection method, publication year, and silencing efficiency. Although gene silencing is a robust feature successfully cross-validated in the majority of experiments, efficiency remained insufficient in a significant proportion of studies. Selection of cell line model and validation method had the highest influence on silencing proficiency.

Gene expression-based prognostic and predictive tools in breast cancer.

Genomic assays measuring the expression of multiple genes have made their way into clinical practice and their utilization is now recommended by major international guidelines. A basic property of these tests is their capability to sub-divide patients into high- and low-risk cohorts thereby providing prognostic, and in certain settings, predictive decision support. Here, we summarize commercially available assays for breast cancer including RT-PCR and gene chip-based tests. Given the relative uncertainty in cancer treatment, multigene tests have the potential for a significant cost reduction as they can pinpoint those patients for whom chemotherapy proves to be unnecessary. However, concordance of risk assessment for an individual patient is still far from optimal. Additionally, emerging multigene approaches focus on predicting therapy response, which is a black spot of current tests. Promising techniques include the homologous recombination deficiency score, utilization of massive parallel sequencing to identify driver genes, employment of internet-based meta-analysis tools and investigation of miRNA expression signatures. Combination of multiple simultaneous analyses at diagnosis, including classical histopathological diagnostics, monogenic markers, genomic signatures and clinical parameters will most likely bring maximal benefit for patients. As the main driving force behind such genomic tests is the power to achieve cost reduction due to avoiding unnecessary systemic treatment, the future is most likely to hold a further proliferation of such assays.

DUSP4 is associated with increased resistance against anti-HER2 therapy in breast cancer

The majority of patients develop resistance against suppression of HER2-signaling mediated by trastuzumab in HER2 positive breast cancer (BC). HER2 overexpression activates multiple signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade. MAPK phosphatases (MKPs) are essential regulators of MAPKs and participate in many facets of cellular regulation, including proliferation and apoptosis. We aimed to identify whether differential MKPs are associated with resistance to targeted therapy in patients previously treated with trastuzumab. Using gene chip data of 88 HER2-positive, trastuzumab treated BC patients, candidate MKPs were identified by Receiver Operator Characteristics analysis performed in R. Genes were ranked using their achieved area under the curve (AUC) values and were further restricted to markers significantly associated with worse survival. Functional significance of the two strongest predictive markers was evaluated in vitro by gene silencing in HER2 overexpressing, trastuzumab resistant BC cell lines SKTR and JIMT-1. The strongest predictive MKPs were DUSP4/MKP-2 (AUC=0.75, p=0.0096) and DUSP6/MKP-3 (AUC=0.77, p=5.29E-05). Higher expression for these correlated to worse survival (DUSP4: HR=2.05, p=0.009 and DUSP6: HR=2, p=0.0015). Silencing of DUSP4 had significant sensitization effects – viability of DUSP4 siRNA transfected, trastuzumab treated cells decreased significantly compared to scramble-siRNA transfected controls (SKTR: p=0.016; JIMT-1: p=0.016). In contrast, simultaneous treatment with DUSP6 siRNA and trastuzumab did not alter cell proliferation. Our findings suggest that DUSP4 may represent a new potential target to overcome trastuzumab resistance.

RNA interference and its clinical applications

RNA interference is a type of posttranscriptional gene silencing, when short RNA molecules suppress the function of RNAs and block gene expression. Double-stranded RNAs or short interfering RNAs injected into cells activate the RNA-induced silencing complex which degrades the target messenger RNA. The short RNAs produced inside the cell are called micro RNAs. These form a hairpin and then have the same function as double-stranded RNAs. RNA interference is an evolutionary important mechanism having a role in the protection against transposon and viral infection and regulate gene expression. While a number of studies demonstrate the in vivo applicability of RNAi, the first potential clinical trials are arising. So far it has been used to treat viral infections, inhibit macula degeneration, decrease the level of cholesterol in blood, treat cancer and neurodegenerative diseases. However, its application is hampered by ineffective bioinformatics algorithms unable to design effective short interfering RNAs, by low delivery efficiency and by the limited use to temporary antagonist gene silencing. The most important advantage of its application is the exceptional specificity resulting minimal side-effects. For this reason therapies based on RNA interference can be expected to spread in the near future.

Abstract P5-08-22: DUSP4 is associated with increased resistance against anti-HER2 therapy in breast cancer

Background. The majority of patients develop resistance against suppression of HER2-mediated signaling by trastuzumab in HER2 positive breast cancer (BC). HER2 overexpression activates multiple signaling pathways, including the mitogen-activated protein kinase (MAPK) cascade. MAPK phosphatases (MKPs) are essential regulators of MAPKs and participate in many facets of cellular regulation, including proliferation and apoptosis. We aimed to identify whether differential MKPs are associated with resistance to targeted therapy in patients previously treated with trastuzumab. Methods. Using Affymetrix HGU133plus2 gene chip data of 88 HER2-positive, trastuzumab treated BC patients, candidate MKPs were identified by Receiver Operator Characteristics analysis performed in R. Genes were ranked using their achieved area under the curve (AUC) values and were further constricted to those markers significantly associated to worse survival. Functional significance of the two strongest predictive biomarkers was evaluated in vitro experiments after gene silencing in the HER2 overexpressing, trastuzumab resistant breast cancer cell lines SKBR-3-TR and JIMT-1. Results. Out of 10 investigated MKPs, the strongest predictive genes were DUSP4/MKP2 (AUC=0.75, p=0.0096) and DUSP6/MKP3 (AUC=0.77, p=5.29E-05). Furthermore, higher expression for these correlated to worse survival in 221 HER2 positive BC patients (DUSP4: HR=1.6, p=0.04 and DUSP6: HR=1.8, p=0.0053). Silencing of DUSP4 had significant sensitization effects - viability of DUSP4 siRNA transfected, trastuzumab treated cells decreased significantly compared to scramble-siRNA transfected, trastuzumab treated controls (SKBR-3-TR: p=0.016; JIMT-1: p=0.016). In contrast, simultaneous treatment with DUSP6 siRNA and trastuzumab did not alter cell proliferation. Conclusions. Our findings suggest that DUSP4 is involved in the development of trastuzumab resistance in HER2 positive BC. Citation Format: Gyorffy B, Munkacsy G, Esteva FJ, Miquel TP, Menyhart O. DUSP4 is associated with increased resistance against anti-HER2 therapy in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-22.

Danubian Biobank Konzorcium: a Duna menti egyetemek „biobanking” tevékenységének összehangolása@@@The Danubian Biobank Initiative: synchronizing the biobanking activities of the Danube universities

The Danubian Biobank Initiative: synchronizing the biobanking activities of the Danube universities. Aging disorders pose an increasing challenge for the public health care systems in Europe. An important approach to cope with this task is the identification of relevant novel disease genes and the control of risk factors using new technological capabilities. A key element in this process is the availability of well classified, large enough patient cohorts and the establishment of quality-controlled central banks for DNA, serum, plasma, and cells/tissues/RNA/proteins together with the development of an IT based infrastructure to provide samples and data required for biomedical studies. The Danubian Biobank initiative connects universities, associated teaching hospitals and endpoint-related rehabilitation clinics along the Danube river and in neighbouring regions. The scientific network focuses on diabetes-related endpoints, vascular disease (e.g. myocardial infarction, stroke, arterial thrombosis, kidney failure), metabolic disease (e.g. obesity, diabetes, metabolic syndrome), and neurodegenerative disorders (e.g. dementia syndromes, Parkinsonism). Task forces are set up for the relevant topics of the biobank project including patient recruitment, sample and data management, public health, epidemiology and genetics, enabling technologies, and research strategies. The project aims to select the most relevant and promising scientific targets utilizing the core competences developed in the individual partner institutions. For this purpose a series of dedicated workshops and conferences are organized as well as joint research grant proposals are submitted.Aging disorders pose an increasing challenge for the public health care systems in Europe. An important approach to cope with this task is the identification of relevant novel disease genes and the control of risk factors using new technological capabilities. A key element in this process is the availability of well classified, large enough patient cohorts and the establishment of quality-controlled central banks for DNA, serum, plasma, and cells/tissues/RNA/proteins together with the development of an IT based infrastructure to provide samples and data required for biomedical studies. The Danubian Biobank initiative connects universities, associated teaching hospitals and endpoint-related rehabilitation clinics along the Danube river and in neighbouring regions. The scientific network focuses on diabetes-related endpoints, vascular disease (e.g. myocardial infarction, stroke, arterial thrombosis, kidney failure), metabolic disease (e.g. obesity, diabetes, metabolic syndrome), and neurodegenerative disorders (e.g. dementia syndromes, Parkinsonism). Task forces are set up for the relevant topics of the biobank project including patient recruitment, sample and data management, public health, epidemiology and genetics, enabling technologies, and research strategies. The project aims to select the most relevant and promising scientific targets utilizing the core competences developed in the individual partner institutions. For this purpose a series of dedicated workshops and conferences are organized as well as joint research grant proposals are submitted.

[The Danubian Biobank Initiative: synchronizing the biobanking activities of the Danube universities].

The Danubian Biobank Initiative: synchronizing the biobanking activities of the Danube universities. Aging disorders pose an increasing challenge for the public health care systems in Europe. An important approach to cope with this task is the identification of relevant novel disease genes and the control of risk factors using new technological capabilities. A key element in this process is the availability of well classified, large enough patient cohorts and the establishment of quality-controlled central banks for DNA, serum, plasma, and cells/tissues/RNA/proteins together with the development of an IT based infrastructure to provide samples and data required for biomedical studies. The Danubian Biobank initiative connects universities, associated teaching hospitals and endpoint-related rehabilitation clinics along the Danube river and in neighbouring regions. The scientific network focuses on diabetes-related endpoints, vascular disease (e.g. myocardial infarction, stroke, arterial thrombosis, kidney failure), metabolic disease (e.g. obesity, diabetes, metabolic syndrome), and neurodegenerative disorders (e.g. dementia syndromes, Parkinsonism). Task forces are set up for the relevant topics of the biobank project including patient recruitment, sample and data management, public health, epidemiology and genetics, enabling technologies, and research strategies. The project aims to select the most relevant and promising scientific targets utilizing the core competences developed in the individual partner institutions. For this purpose a series of dedicated workshops and conferences are organized as well as joint research grant proposals are submitted.Aging disorders pose an increasing challenge for the public health care systems in Europe. An important approach to cope with this task is the identification of relevant novel disease genes and the control of risk factors using new technological capabilities. A key element in this process is the availability of well classified, large enough patient cohorts and the establishment of quality-controlled central banks for DNA, serum, plasma, and cells/tissues/RNA/proteins together with the development of an IT based infrastructure to provide samples and data required for biomedical studies. The Danubian Biobank initiative connects universities, associated teaching hospitals and endpoint-related rehabilitation clinics along the Danube river and in neighbouring regions. The scientific network focuses on diabetes-related endpoints, vascular disease (e.g. myocardial infarction, stroke, arterial thrombosis, kidney failure), metabolic disease (e.g. obesity, diabetes, metabolic syndrome), and neurodegenerative disorders (e.g. dementia syndromes, Parkinsonism). Task forces are set up for the relevant topics of the biobank project including patient recruitment, sample and data management, public health, epidemiology and genetics, enabling technologies, and research strategies. The project aims to select the most relevant and promising scientific targets utilizing the core competences developed in the individual partner institutions. For this purpose a series of dedicated workshops and conferences are organized as well as joint research grant proposals are submitted.