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International audience; Ventricular (VT) and atrial (AT) tachycardias are some of the most common clinical cardiac arrhythmias. For ablation of tachycardia substrates, two clinical diagnosis methods are used: invasive electroanatomical mapping for an accurate diagnosis using electrograms (EGMs) acquired with intracardiac catheters, and localized on the surface mesh of the studied cavities; and noninvasive electrocardiographic imaging (ECGi) for a global view of the arrhythmia, with EGMs mathematically reconstructed from body surface electrocardiograms using 3-D cardio-thoracic surface meshes obtained from CT-scans. In clinics, VT and AT are diagnosed by studying activation time maps that depict the propagation of the activation wavefront on the cardiac mesh. Nevertheless, slow conduction areas-a well-known proarrhythmic feature for tachycardias-and tachycardia specific propagation patterns are not easily identifiable with these maps. Therefore, local characterization of the activation wavefront propagation can be helpful for improving VT and AT diagnoses. The purpose of this study is to develop a method to locally characterize the activation wavefront propagation for clinical data. For this, a conduction velocity vector field is estimated and analyzed using divergence and curl mathematical operators. The workflow was first validated on a simulated database from computer models, and then applied to a clinical database obtained from ECGi to improve AT diagnosis. The results show the relevancy and the efficacy of the proposed method to guide ablation of tachyarrhythmias.

Supplemental Digital Content is available in the text., Background: Sudden cardiac death because of ventricular fibrillation (VF) is commonly unexplained in younger victims. Detailed electrophysiological mapping in such patients has not been reported. Methods: We evaluated 24 patients (29±13 years) who survived idiopathic VF. First, we used multielectrode body surface recordings to identify the drivers maintaining VF. Then, we analyzed electrograms in the driver regions using endocardial and epicardial catheter mapping during sinus rhythm. Established electrogram criteria were used to identify the presence of structural alterations. Results: VF occurred spontaneously in 3 patients and was induced in 16, whereas VF was noninducible in 5. VF mapping demonstrated reentrant and focal activities (87% versus 13%, respectively) in all. The activities were dominant in one ventricle in 9 patients, whereas they had biventricular distribution in others. During sinus rhythm areas of abnormal electrograms were identified in 15/24 patients (62.5%) revealing localized structural alterations: in the right ventricle in 11, the left ventricle in 1, and both in 3. They covered a limited surface (13±6 cm2) representing 5±3% of the total surface and were recorded predominantly on the epicardium. Seventy-six percent of these areas were colocated with VF drivers (P

International audience; Introduction Ventricular fibrillation (VF) is the main cause of sudden cardiac death. Despite its clinical significance, there is still a lack of quantitative tools to evaluate the degree of myocardial electrical organization during VF. This study puts forward novels markers of VF complexity and stability on body surface potential maps (BSPMs), uantifying the ability of signal features determined through principal component analysis (PCA) to be retrieved along the recording.Methods Biventricular potential mapping was performed through a 252-electrode vest in 27 VF patients (25 male, 49 ± 22 years). BSPMs were divided in 0:5-s segments, each projected on a 3D subspace determined through PCA in the preceding segment. At each time instant, the multilead error E between the input signal and its PCA approximation was expressed in terms of normalized amplitude norm |E| and angle cos(E). Additionally, the nondipolar component index (NDI) was determined as the fraction of energy retained by the first 3 PCA eigenvalues in sliding windows of the same duration. In 24 VF recordings (duration 19.8±6.5 s), average values of the aforementioned indices were computed in 4-second windows at the beginning and at the end of the outputparameter series. Temporal changes in VF complexity were verified through an unpaired Student’s t-test. The same analysis was performed in 5 control recordings in sinus rhythm (SR). Results A significant increase in spatiotemporal complexity was observed during VF, quantified by higher NDI and |E| and lower cos(E) values at the end of the episode (p < 0:0001). By contrast, no significant changes between the beginning and the end of the recording were observed during SR (p > 0:05).Conclusions This study demonstrates the ability of PCA to capture and quantify changes in signal complexity during VF in a noninvasive framework.

Alkylating agents represent the oldest class of anticancer agents with the approval of mechloretamine by the FDA in 1949. Even though their clinical use is far beyond the use of new targeted therapies, they still occupy a major place in the treatment of specific malignancies, sometimes representing the unique option for the treatment of refractory tumors. Here, we are reviewing the major classes of alkylating agents, with a particular focus on the latest generations of compounds that specifically target the minor groove of the DNA. These naturally occurring derivatives have a unique mechanism of action that explains the recent regain of interest in developing new classes of alkylating agents that could be used in combination with other anticancer drugs to enhance tumor response in the clinic.

Prostate cancer is one of the leading causes of cancer-related deaths among men. Several prognostic factors allow differentiation of low-grade tumors from high-grade tumors with high metastatic potential. High-grade tumors are currently treated with hormone therapy, to which taxanes are added when the tumors become resistant to castration. Clinical trials with other anticancer agents did not take into account the genetic backgrounds of the tumors, and most trials demonstrated low response rates. Here we used an in silico approach to screen for drug candidates that might be used as alternatives to taxanes, on the basis of a published expression signature involving 86 genes that could distinguish high-grade and low-grade tumors (Proc Natl Acad Sci USA 103:10991-10996, 2006). We explored the National Cancer Institute databases, which include data on the gene expression profiles of 60 human tumor cell lines and the in vitro sensitivities of the cell lines to anticancer drugs, and we identified several genes in the signature for which expression levels were correlated with chemosensitivity. As an example of the validation of this in silico approach, we identified a set of six genes for which expression levels could predict cell sensitivity to oxaliplatin but not cisplatin. This signature was validated in vitro through silencing of the genes in DU145, LNCaP, and C4-2B prostate cancer cells, which was accompanied by changes in oxaliplatin but not cisplatin cytotoxicity. These results demonstrate the relevance of our approach for the identification of both alternative treatments for high-grade prostate cancers and new biomarkers to predict clinical tumor responses.

Polyphenolic ellagitannins are natural compounds that are often associated with the therapeutic activity of plant extracts used in traditional medicine. They display cancer-preventing activity in animal models by a mechanism that remains unclear. Potential targets have been proposed, including DNA topoisomerases II (Top2). Top2α and Top2β, the two isoforms of the human Top2, play a crucial role in the regulation of replication, transcription, and chromosome segregation. They are the target of anticancer agents used in the clinic such as anthracyclines (e.g., doxorubicin) or the epipodophyllotoxin etoposide. It was recently shown that the antitumor activity of etoposide was due primarily to the inhibition of Top2α, whereas inhibition of Top2β was responsible for the development of secondary malignancies, pointing to the need for more selective Top2α inhibitors. Here, we show that the polyphenolic ellagitannin vescalagin preferentially inhibits the decatenation activity of Top2α in vitro, by a redox-independent mechanism. In CEM cells, we also show that transient small interfering RNA-mediated down-regulation of Top2α but not of Top2β conferred a resistance to vescalagin, indicating that the α isoform is a preferential target. We further confirmed that Top2α inhibition was due to a catalytic inhibition of the enzyme because it did not induce DNA double-strand breaks in CEM-treated cells but prevented the formation of Top2α- rather than Top2β-DNA covalent complexes induced by etoposide. To our knowledge, vescalagin is the first example of a catalytic inhibitor for which cytotoxicity is due, at least in part, to the preferential inhibition of Top2α.

Background and objective: The epidermal growth factor receptor (EGFR) plays a major role in cell proliferation of epithelial tissues, and its alterations frequently contribute to oncogenesis. Several common polymorphisms of the EGFR gene have been described, at the level of both coding and regulatory sequences. Some of these polymorphisms are associated with alterations of EGFR expression and/or activity and may have an impact on the activity of anticancer agents. This study aims to analyze the relationships between specific EGFR functional polymorphisms and anticancer drug activity. Method: We investigated, in the panel of 60 human tumor cell lines established by the National Cancer Institute (NCI-60), whether the EGFR polymorphisms −216G>T, −191C>A, Arg521Lys (R521K), Val592Ala (V592A), and Cys624Phe (C624F), and the intron 1 (CA)n repeat were associated with EGFR gene expression and the in vitro cytotoxicity of anticancer agents using data extracted from the NCI database. We also looked for mutations of exons 18–21, known to enhance the activity of tyrosine kinase inhibitors, and the deletion of exons 2–7, associated to the oncogenesis of glioblastomas. Results: In the NCI-60 cell lines, only two mutations were observed, both in exon 19, in a leukemia and melanoma cell line. These mutations have not been described previously in clinical samples and their functional role is uncertain. The allele frequencies of the −216G>T, −191C>A, and R521K single nucleotide polymorphisms (SNPs) in the NCI-60 panel were 33%, 8.5%, and 27%, respectively; the V592A and C624F SNPs were not found in any NCI-60 cell line. The intron 1 CA repeat was highly variable in the cell lines; 32 cell lines having a total number of repeats below 35, and 27 having a total number of repeats above 35. The heterozygous and variant homozygous cell lines for the -216G>T SNP presented a significantly higher expression of the EGFR gene than the homozygous wild-type lines. In contrast, there was no association between the −191C>A or R521K SNPs and EGFR gene expression. No association could be detected between the number of CA repeats in intron 1 and the expression of EGFR. The cell lines having at least one variant T allele at the −216G>T SNP were more sensitive to erlotinib and less sensitive to geldanamycin, topoisomerase I and II inhibitors, and alkylating agents than those without a variant allele. No relationship was detected between anticancer drug sensitivity and the −191C>A SNP. The R521K SNP was associated to lower sensitivity to alkylating agents. The number of CA repeats was associated with significant differences in anticancer drug activity: a high total number of CA repeats (>35 per diploid genome) was associated to increased sensitivity to alkylating agents and topoisomerase I and II inhibitors. Discussion: We provide evidence in this work that EGFR polymorphisms are associated with significant differences in the in vitro cytotoxicity of several types of DNA-interfering agents. Studies attempting a clinical validation of these clues are warranted.

Prostate cancer (PCa) is one of the leading causes of death from cancer in men. Several prognostic factors allow differentiating low-grade from high-grade PCa that are often refractory to chemical castration but are still treated with hormone therapy to which docetaxel or cabazitaxel are added when they become resistant to the anti-androgen. Despite many clinical trials with other chemotherapeutic agents, response rates remain low, pointing towards the need for new alternative therapies to treat these aggressive tumors. Using a rational in silico approach based on the NCI60-cell line panel, we recently identified a signature of 6 genes, the expression of which could predict at the functional level, sensitivity to oxaliplatin but not to cisplatin in DU145, LNCaP and C42B prostate cancer cell lines (Puyo et al. Mol. Pharmacol, 2012). Among them, we focused on SHMT2, the mitochondrial isoform of serine hydroxymethyl transferase involved in the biosynthesis of purines. Downregulation of SHMT2 or absence of SHMT2 catalytic activity was associated with a resistance to oxaliplatin, whereas it had no effect on cisplatin sensitivity, a selectivity that was attributed to the DACH moiety of platinum derivatives. Here, we investigated the role of DNA methylation in this selective response to platinum compounds since SHMTs can indirectly regulate the methylation status of DNA. Variations in global level of methylation as measured by the methylation status of LINE-1 retrotransposon, was associated with differences in sensitivity of PCa cells to platinum compounds. Using our in silico approach, we found significant correlations between SHMT2 expression and cell sensitivity to both demethylating agents azacytidine and decitabine. We also found that treatment of DU145 cells, with high level of global DNA methylation, sensitized cells to platinum compounds. In order to evaluate whether methylation could impair the formation of Pt-adducts in vitro, we used purified oligonucleotides containing a unique site of platination. We show that methylation at specific CpG in the vicinity of the platination site could reduce the kinetics of DNA adducts formation. This inhibition was more pronounced for DACH platin than for cisplatin. We also assessed the effect of transient repression of SHMT2 in LNCaP cells on the methylation status of ∼450,000 CpG sites using the Infinium Meth450K beadchip from Illumina. Preliminary results show that significant changes in methylation status was only observed in a reduced number of CpGs which were not located in genes that are known to be involved in cell response to Pt adducts formation. Together our results demonstrate that SHMT2-mediated specific response to oxaliplatin can be due to both methylation in the vicinity of the platination site and the modulation of the expression of genes that are not directly linked to the processing of DNA-adducts. Citation Format: Stéphane Puyo, Nadine Houédé, Marina Hamant, Pierre Richaud, Jacques Robert, Philippe Pourquier. SHMT2 modulates DNA methylation and differentially affects prostate cancer cell response to platinum derivatives. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4478. doi:10.1158/1538-7445.AM2013-4478

Prostate cancer (PCa) is one of the leading causes of death from cancer in men. High Gleason grade PCa are characterized by aggressive tumors with poorly differentiated cells and a high metastatic potential. They are often refractory to chemical castration but still treated with hormone therapy to which docetaxel or cabazitaxel are added when they become resistant to the anti-androgen. Despite many clinical trials with other chemotherapeutic agents, response rates remain low pointing towards the need for new alternative therapies. Using an in silico approach based on a 86 genes signature which could distinguish between low-grade and high-grade tumors, we identified 9 genes (PCCB, SHMT2, DPM1, RHOT2, RPL13, CD59, EIF4AI, CDKN2C, JUN) for which expression levels across the NCI-60 cell lines panel was significantly correlated (p< 0.05) to oxaliplatin but not to cisplatin sensitivity. Among them, SHMT2 which is overexpressed in high grade PCa, encodes the mitochondrial isoform of serine hydroxymethyl transferase, which converts glycine to serine, which is then exported to the cytoplasm to generate the one-carbon units that are required for the biosynthesis of purines. SiRNA-mediated donwregulation of SHMT2 in DU145 or LNCaP prostate tumor cell lines rendered cells resistant to oxaliplatin but not to cisplatin. CHO 51-11 cells which express a catalytically inactive SHMT2 were also resistant to oxaliplatin but not to cisplatin as compared to CHO K1 control cells. The selective resistance to oxaliplatin was not due to the oxalate moiety since addition of oxalate to cisplatin could not restore the level of sensitivity of CHO cells to oxaliplatin. Selectivity could however be attributed to the diamino cyclohexane (DACH) moiety of oxaliplatin since CHO 51-11 cells were also resistant to dichloro(1,2-diaminocyclohexane)-platinum(II). Resistance to oxaliplatin could also be achieved by SHMT1 downregulation (the cytoplasmic form of SHMT) or by an alteration of the pool of glycine and serine. SHMTs can indirectly regulate the methylation status of DNA, and we showed that siRNA-mediated downregulation of SHMTs in hypomethylated LNCaP cells increased the global level of DNA methylation, as measured by 4 CpGs methylation status of LINE-1. Interestingly, increased level of global methylation status was associated with increased resistance to oxaliplatin. Altogether, our results identified oxaliplatin as a potential alternative for the treatment of high grade prostate cancers and SHMT2 (and SHMT1) as selective markers of oxaliplatin sensitivity which involves, at least in part, the regulation of global levels of DNA methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2665. doi:1538-7445.AM2012-2665

150 Background: Prostate cancer (PCa) is one of the leading causes of death from cancer in men. High Gleason grade prostate cancers are characterized by aggressive tumors with poorly differentiated cells and a high metastatic potential. They are often refractory to chemical castration but still treated with hormone therapy to which docetaxel or cabazitaxel are added when they become resistant to the anti-androgen. Despite many clinical trials with other chemotherapeutic agents, response rates remain low. Moreover, none of these trials took into account the tumor grade. Methods: We used an in silico approach to screen for drug candidates that could be used as an alternative to taxanes, based on a 86 genes signature which could distinguish between low-grade and high-grade tumors. We extracted from the NCI60 panel databases the expression profiles of the 86 genes across 60 human tumor cell lines and the corresponding in vitro cytotoxicity data of 152 drugs and looked for correlation between their expression level and cell sensitivity to each of these drugs. Results: Among the 86 genes, we identified 9 genes (PCCB, SHMT2, DPM1, RHOT2, RPL13, CD59, EIF4AI, CDKN2C, JUN) for which expression levels across the 60 cell lines was significantly correlated (p< 0.05) to oxaliplatin but not to cisplatin sensitivity. This signature was validated at the functional level since repression of each of these genes conferred a significant change in the sensitivity of PCa cell lines to oxaliplatin but not cisplatin. Conclusions: Our results provide a proof of concept that gene expression signature specific from high grade PCa could be used for the identification of alternative therapies to taxanes. They could also be used to select patients for further clinical trials and as predictive markers of response to these drugs, which represents a further step forward towards personalized therapy of PCa.

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Prostate cancer is one of the leading causes of death from cancer in men. The prognosis of patients is defined according to staging, PSA levels and Gleason score, which differentiates low grade (Gleason < 4) and high grade (Gleason ≥ 4) cancers. High-grade tumors are associated with high metastatic potential and poor prognosis. They are treated with hormone therapy, to which microtubule poisons are added when they become resistant to the anti-androgen. Despite many clinical trials with other chemotherapeutic agents, response rates remain low. Moreover, none of these trials took into account the tumor grade. We therefore envisaged a new rational in silico approach to screen for drug candidates that could be used as an alternative to docetaxel, based on an expression signature of 86 genes that could distinguish low-grade and high-grade tumors, with a reliability of 81% (PNAS 2006 103:10991-6). We explored the NCI databases, which allow access to both gene expression profiles of 60 human tumor cell lines and their in vitro sensitivity to thousands of anticancer drugs and extracted the expression profiles of the 86 genes’ signature. We calculated for each gene the Pearson coefficients of correlation (r) between their expression level in the 60 cell lines and cell sensitivity to 152 core anticancer compounds. We found that the expression of 11 genes was associated with sensitivity to oxaliplatin. They include DPM1, PCCB, ATP5G3, and SHMT2 genes involved in metabolism; RHOT2, CD59 and JUN genes involved in signal transduction, the CDKN2C cyclin-dependent kinase inhibitor gene, RPL13 and EIF4A1 genes involved in translation, and the unknown [FLJ35093][1] gene. This signature seems specific to DACH platinum derivatives since no correlation was found with the sensitivity to cisplatin. Functional validation of this signature was performed in vitro using the prostate cancer cell lines DU145 and LNCaP and the benign prostate hyperplasia (BPH) cells as “normal” cells. We measured the effect of siRNA-mediated downregulation of each of the 11 genes on the sensitivity to oxaliplatin or cisplatin by clonogenic or MTT assays. Preliminary data confirmed our in silico results for 7 out of the 11 genes, for which donwregulation induced a significant change in IC50 for oxaliplatin (but not cisplatin) in DU145 and/or LNCaP cells. For EIF4A1 we found a correlation opposite to that obtained from in silico results. Also, inhibition of CDKN2C could affect the sensitivity to cisplatin in LNCaP cells. Conversely, no effect of each siRNA tested could be observed in BPH cells, suggesting that functional validation should be tested in a tumoral background. Our new rational approach allowed the identification of oxaliplatin as an alternative therapy for high grade prostate cancers. It also identified a potential gene expression signature that could be used to predict tumor response to oxaliplatin (and potentially DACH platinum derivatives) in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5064. doi:10.1158/1538-7445.AM2011-5064 [1]: /lookup/external-ref?link_type=GENPEPT&access_num=FLJ35093&atom=%2Fcanres%2F71%2F8_Supplement%2F5064.atom

Polyphenolic ellagitanins are natural compounds that are often associated with the therapeutic activity of plant extracts used in traditional medicine. They display cancer-preventing activity in animal models at concentrations found in diets by a mechanism which remains unclear. Potential targets have been proposed, including topoisomerases II (Top2) which are involved in the regulation of replication, transcription and chromosome segregation. Top2α and Top2β, the two human isoforms of Top2, are the target of anticancer agents such as etoposide (VP-16). Antitumoral activity of VP-16 is primarily due the inhibition of top2α, whereas inhibition of top2β is responsible for the development of secondary malignancies, pointing towards the need for more selective top2α inhibitors. In this study, vescalagin, methylvescalagin, acutissimin B (acuB), and epiacutissimin B (epiB) were tested for their selectivity against Top2α and Top2β. Using kinetoplast DNA as a substrate, we showed that Top2α-mediated decatenation was inhibited by ≈ 50% by all 4 compounds at 1 µM, whereas only minor inhibition of top2β could be observed ( 100 fold). We confirmed that vescalagin was a catalytic inhibitor of Top2α, since no double-strand break formation or phosphorylation of H2AX could be observed in treated CEM cells (50 µM, 2h). This was confirmed by the absence of Top2α-DNA cleavage complexes formation as measured by the Immuno-Complex of Enzyme (ICE) assay. Since polyphenolic ellagitanin are known to induce ROS, we investigated the potential contribution of ROS on vescalagin-induced cytotoxicity. Indeed, vescalagin treatment induced single-strand breaks (SSB) in both CEM and CEM VM1 cell lines. We showed that treatment with N-acetylcystein (NAC) prevented the formation of vescalagin-induced SSB in both cell lines but had no significant effect on vescalagin-induced cytotoxicity in CEM or CEM-VM1 cells. This precludes the role of ROS in vescalagin induced cytotoxicity by specific inhibition of Top2α. Together, our data suggest that vescalagin is a new polyphenolic ellagitanin compound which specifically inhibits Top2α in vitro and in cells at pharmacological concentrations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2531. doi:10.1158/1538-7445.AM2011-2531

Prostate cancer is one of the leading causes of death from cancer in men and its incidence is constantly rising. In most cases, the prognosis of patients is defined according to staging, PSA levels and Gleason score, which differentiates low grade (Gleason < 4) and high grade (Gleason ≥ 4) cancers. High-grade tumors are associated with high metastatic potential and poor prognosis. They are currently treated with hormone therapy, to which docetaxel is added when they become resistant to the anti-androgen. However, response rates remain very low. Clinical trials with other chemotherapeutic agents such as mitoxantrone, vinorelbine or oxaliplatin have been performed, yet with limited success. It was reported that an expression signature of 86 genes could distinguish low-grade and high-grade tumors, with a reliability of 81% (True et al. PNAS 2006 103:10991-6). We used an in silico approach to explore the NCI databases, which allow access to both gene expression profiles of 60 human tumor cell lines and their in vitro sensitivity to thousands of anticancer drugs. After extraction of the expression profiles of the 86 genes of the True's signature, we calculated, for each gene, the Pearson coefficients of correlation (r) between their expression level in the 60 cell lines and cell sensitivity to 140 core anticancer compounds, expressed as the antilog of IC50. A positive r-value is associated to drug efficacy, whereas a negative r-value is associated to drug resistance. Among the 86 genes, the expression of 45 genes was correlated with the sensitivity to various compounds with an r-value +0.3 (p 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 2692.