Vlasta Korenkova

Functional, Genetic, and Epigenetic Aspects of Base and Nucleotide Excision Repair in Colorectal Carcinomas

Purpose: DNA repair capacity (DRC) is a determinant not only of cancer development but also of individual response to therapy. Previously, altered base and nucleotide excision repair (BER and NER) have been described in lymphocytes of patients with sporadic colorectal cancer. We, for the first time, evaluate both excision repair capacities in human colon biopsies to study their participation in colorectal tumorigenesis. Experimental design: Seventy pairs of tumor and adjacent healthy tissues were analyzed for BER- and NER-specific DRC by a comet repair assay. Tissue pairs were further compared for expression levels of a panel of 25 BER and NER genes complemented by their promoter methylation status. Results: We observed a moderate increase of NER-DRC (P = 0.019), but not of BER-DRC in tumors. There was a strong correlation between both tissues for all investigated parameters (P < 0.001). However, 4 NER (CSB, CCNH, XPA, XPD) and 4 BER (NEIL1, APEX1, OGG1, PARP1) genes showed a 1.08- to 1.28-fold change difference in expression in tumors (P < 0.05). Individual gene expression levels did not correlate with overall DRC, and we did not detect any aberrant methylation of the investigated genes. Conclusions: Our complex analysis showed that tumor cells are not deficient in BER and NER, but rather follow patterns characteristic for each individual and are comparable with adjacent tissue. Alteration of excision repair pathways is not a pronounced event in colorectal carcinogenesis. This study shows the feasibility of DRC evaluation in human solid tissues, representing a complex marker of multigene DNA repair processes. Clin Cancer Res; 18(21); 5878–87. ©2012 AACR.

Biomarkers for Monitoring Pre-Analytical Quality Variation of mRNA in Blood Samples

There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.

Tumor-initiating cells of breast and prostate origin show alterations in the expression of genes related to iron metabolism

The importance of iron in the growth and progression of tumors has been widely documented. In this report, we show that tumor-initiating cells (TICs), represented by spheres derived from the MCF7 cell line, exhibit higher intracellular labile iron pool, mitochondrial iron accumulation and are more susceptible to iron chelation. TICs also show activation of the IRP/IRE system, leading to higher iron uptake and decrease in iron storage, suggesting that level of properly assembled cytosolic iron-sulfur clusters (FeS) is reduced. This finding is confirmed by lower enzymatic activity of aconitase and FeS cluster biogenesis enzymes, as well as lower levels of reduced glutathione, implying reduced FeS clusters synthesis/utilization in TICs. Importantly, we have identified specific gene signature related to iron metabolism consisting of genes regulating iron uptake, mitochondrial FeS cluster biogenesis and hypoxic response (ABCB10, ACO1, CYBRD1, EPAS1, GLRX5, HEPH, HFE, IREB2, QSOX1 and TFRC). Principal component analysis based on this signature is able to distinguish TICs from cancer cells in vitro and also Leukemia-initiating cells (LICs) from non-LICs in the mouse model of acute promyelocytic leukemia (APL). Majority of the described changes were also recapitulated in an alternative model represented by MCF7 cells resistant to tamoxifen (TAMR) that exhibit features of TICs. Our findings point to the critical importance of redox balance and iron metabolism-related genes and proteins in the context of cancer and TICs that could be potentially used for cancer diagnostics or therapy.

Molecular characteristics of mismatch repair genes in sporadic colorectal tumors in Czech patients

BackgroundMismatch repair (MMR) genes are known to be frequently altered in colorectal cancer (CRC). Both genetics and epigenetics modifications seems to be relevant in this phenomenon, however it is still not clear how these two aspects are interconnected. The present study aimed at characterizing of epigenetic and gene expression profiles of MMR genes in sporadic CRC patients from the Czech Republic, a country with one of the highest incidences of this cancer all over Europe.MethodsExpression levels and CpG promoter methylation status of all MMR genes were evaluated in DNA from tumor and adjacent mucosal samples of 53 incident CRC patients.ResultsWe have found significantly increased transcription levels in EXO1 gene in tumor tissues (P = 0.05) and significant over-expression of MSH3 gene in colon tumors when compared to adjacent mucosal tissues (P = 0.02). Interestingly, almost all MMR genes were differently expressed when localization of tumors was compared. In particular, colon tumors showed an up-regulation of EXO1, MSH2, MSH3, MSH6, and PMS2 genes in comparison to rectal tumors (P = 0.02). Expression levels of all MMR genes positively correlated between each other. The promoter methylation of MLH1 gene was observed in 9% of CRC tissues only.ConclusionsIn our study, we have observed different pattern of MMR genes expression according to tumor localization. However, a lack of association between methylation in MMR genes and their corresponding expressions was noticed in this study, the relationship between these two aspects is worthy to be analyzed in larger population studies and in pre-malignant stages.

The focus on sample quality: Influence of colon tissue collection on reliability of qPCR data

Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing.

Post-treatment recovery of suboptimal DNA repair capacity and gene expression levels in colorectal cancer patients

DNA repair in blood cells was observed to be suboptimal in cancer patients at diagnosis, including colorectal cancer (CRC). To explore the causality of this phenomenon, we studied the dynamics of DNA repair from diagnosis to 1 yr follow‐up, and with respect to CRC treatment. Systemic CRC therapy is targeted to DNA damage induction and DNA repair is thus of interest. CRC patients were blood‐sampled three times in 6‐mo intervals, starting at the diagnosis, and compared to healthy controls. DNA repair was characterized by mRNA levels of 40 repair genes, by capacity of nucleotide excision repair (NER), and by levels of DNA strand breaks (SBs). NER and base excision repair genes were significantly under‐expressed (P < 0.016) in patients at diagnosis compared to controls, in accordance with reduced NER function (P = 0.008) and increased SBs (P = 0.015). Six months later, there was an increase of NER capacity, but not of gene expression levels, in treated patients only. A year from diagnosis, gene expression profiles and NER capacity were significantly modified in all patients and were no longer different from those measured in controls. All patients were free of relapse at the last sampling, so we were unable to clarify the impact of DNA repair parameters on treatment response. However, we identified a panel of blood DNA repair‐related markers discerning acute stage of the disease from the remission period. In conclusion, our results support a model in which DNA repair is altered as a result of cancer.

One generalist or several specialist species? Wide host range and diverse manipulations of the hosts’ web-building behaviour in the true spider parasitoid Zatypota kauros (Hymenoptera: Ichneumonidae)

Parasitoid wasps of the Polysphincta genus‐group are highly specialised on their spider hosts, and most of them are known to manipulate their hosts into building a special web in which the parasitoid pupates. Trophic niche and the plasticity of host use were investigated in the koinobiont parasitoid Zatypota kauros Gauld from Queensland, Australia. We found that Z. kauros attacks spider hosts from different families, each differing widely in their web‐building behaviours, which makes it unique in the breadth of its host range. Molecular analyses revealed that the taxon Z. kauros contains three divergent mitochondrial lineages. Lineage A was associated exclusively with spiders of the genus Anelosimus (Theridiidae), which builds tangle webs; lineage B was associated with the genus Cyrtophora (Araneidae), which weaves tent webs; and lineage C was associated with a broad range of hosts, including spiders of both the families Araneidae and Theridiidae. Unique host manipulations could be observed in the web‐building behaviours of the different host groups. Nevertheless, nuclear data from two ribosomal genes and three introns did not add any support to the existence of different evolutionary lineages, nor did they coincide with the different host groups. The partial correspondence of mitochondrial lineage and host use, together with an apparent mito‐nuclear conflict might indicate maternal effects or very recent and/or incomplete speciation in this taxon. Given their wide host range and intriguing interactions with their hosts, the Z. kauros complex represents a promising system for studying parasitoid specialisation and its potential impact on speciation.

Abstract 2946: Gene expression profiling in circulating cells (CTCs) of breast carcinoma patients - a tool for early metastasis detection and therapy individualization

Background: Tumor cell dissemination is an early process in breast cancer (BC) and circulating tumor cells (CTCs) are considered potential surrogate marker for the detection and characterization of minimal residual disease. Here we monitored hematogenous micrometastasis in BC patients by gene expression profiling of CTCs based on CTC-abundance in blood and expression of 35 oncomarkers on the mRNA level by multimarker quantitative PCR (qPCR). Characteristics of the early dissemination process and gene expression profile changes were studied by analyzing tumor tissue and CTCs of primary and metastatic BC patients. Methods: In primary BC five ml blood was collected before and after chemotherapy (n=87 patients). In metastatic BC 5 ml blood of 72 patients was studied either at the time of relapse of BC or at a documented progressive BC before receiving new therapy. All samples were analyzed for CTCs using the AdnaTest BreastCancer (AdnaGen AG, Langenhagen, Germany). CTCs were immunomagnetically enriched using the AdnaTest BreastCancerSelect followed by RNA isolation, subsequent gene expression analysis by reverse transcription and multiplex-PCR for the detection of EpCAM, MUC-1 and HER-2 transcripts. RNA from formalin fixed paraffin embedded (FFPE)-tumor tissue (n=46) was isolated with RecoverAll® (Ambion). After reverse transcription the cDNA was gene-specifically pre-amplified for multimarker qPCR analysis on the Biomark® (Fludigm, USA) microfluidic chip for 48 genes in each of 48 samples (2034 rxn in total). qPCR data were analyzed with GenEx ver. 5.0 (MultiD, SE) and correlated to available clinical data. Results: 286 CTC samples were analyzed in total. Blood sample was considered positive in the AdnaTest if at least one marker was measured above the cut-off level in the sample. CTCs were found in 29/87 (33,3%) of primary BC patients before chemotherapy and in 10/87 (11,5 %) patients after chemotherapeutical treatment. Among metastatic BC patients 48/72 (67%) were CTC positive. Gene expression analysis of the CTCs in metastatic BC patients and FFPE samples revealed 20 genes that were differentially expressed (p Conclusion: Gene expression profiling of CTCs is a potential discriminator of patients with therapy sensitive and therapy resistant tumor cell populations and therefore with good and inferior prognosis. The predictive value of expression profiles in CTCs for therapeutic interventions will be prospectively evaluated. Study has been supported by IGA NS 9776-3 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2946.