Bruno Gomes

A Data Mining Approach for the Detection of High-Risk Breast Cancer Groups

It is widely agreed that complex diseases are typically caused by the joint effects of multiple instead of a single genetic variation. These genetic variations may show very little effect individually but strong effect if they occur jointly, a phenomenon known as epistasis or multilocus interaction. In this work, we explore the applicability of decision trees to this problem. A case-control study was performed, composed of 164 controls and 94 cases with 32 SNPs available from the BRCA1, BRCA2 and TP53 genes. There was also information about tobacco and alcohol consumption. We used a Decision Tree to find a group with high-susceptibility of suffering from breast cancer. Our goal was to find one or more leaves with a high percentage of cases and small percentage of controls. To statistically validate the association found, permutation tests were used. We found a high-risk breast cancer group composed of 13 cases and only 1 control, with a Fisher Exact Test value of 9.7×10− 6. After running 10000 permutation tests we obtained a p-value of 0.017. These results show that it is possible to find statistically significant associations with breast cancer by deriving a decision tree and selecting the best leaf.

Induction of sister chromatid exchange by acrylamide and glycidamide in human lymphocytes: role of polymorphisms in detoxification and DNA-repair genes in the genotoxicity of glycidamide.

Acrylamide (AA) is a probable human carcinogen generated in carbohydrate-rich foodstuffs upon heating. Glycidamide (GA), formed via epoxidation, presumably mediated by cytochrome P450 2E1, is considered to be the active metabolite that plays a central role in the genotoxicity of AA. The aim of this work was to evaluate the cytogenetic damage induced by AA and GA in cultured human lymphocytes by use of the sister chromatid exchange (SCE) assay. Furthermore, this report addresses the role of individual genetic polymorphisms in key genes involved in detoxification and DNA-repair pathways (BER, NER, HRR and NHEJ) on the induction of SCE by GA. While AA induced the number of SCE/metaphase only slightly, especially for the highest concentration tested (2000μM), GA markedly induced SCEs in a concentration-dependent manner up to concentrations of 750μM, leading to an increase in SCEs of up to about 10-fold compared with controls. By combining DNA damage in GA-treated lymphocytes and data on polymorphisms, associations between the induction of SCEs with GSTP1 (Ile105Val) and GSTA2 (Glu210Ala) genotypes are suggested.

DNA Repair and Resistance to Cancer Therapy

Humans are constantly exposed to diverse chemical and physical agents that have the potential to damage DNA, such as reactive oxygen species (ROS), ionizing radiation (IR), UV light, and various environmental, dietary or pollutant chemical agents. The integrity and survival of a cell is critically dependent on genome stability, and cells possess multi‐ ple pathways to repair these DNA lesions. These pathways are diverse and target differ‐ ent types of lesions.

Methods for Studying MicroRNA Expression and Their Targets in Formalin-Fixed, Paraffin-Embedded (FFPE) Breast Cancer Tissues.

Drug resistance remains a burden in cancer treatment. In the past few years molecular genetics brought a new hope with personalized therapy. This individual approach allows the identification of genetic profiles that will respond better to a given treatment and consequently get a better outcome. Recently, physicians received an extra aid with the approval of molecular tools based on gene expression signatures. With these tools, physicians have the capacity to identify the probability of disease recurrence in the first 5 years following diagnosis, a fact that is essential for a more effective adjuvant therapy administration. However, some patients still relapse and acquire drug resistance and aggressive tumors. For that reason, a comprehensive understanding of the molecular players in drug resistance is of extreme importance. MicroRNAs have been described as regulators of various cellular pathways and as predictive and prognostic factors. As broad regulators, microRNAs also interfere with drug metabolism and drug targets. Thus it is of paramount importance to understand which microRNAs are deregulated in breast cancer and try to relate this misexpression with resistance to therapeutics, poor outcomes, and survival. Here, we describe a possible approach to study microRNA expression and respective targets from formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues. FFPE tissues are regularly archived for long periods in pathology departments, and microRNAs are well conserved in these tissues.

DNA Repair Perspectives in Thyroid and Breast Cancer: The Role of DNA Repair Polymorphisms

One of the great challenges of modern molecular biology is the integration of new genetic information into procedures that can be implemented in rapid, cost effective and reliable methods to genotype, phenotype, identify gene function, and development treatment for the disease. One of the major impacts of such methods and procedures is the increase of our knowledge and understanding of human biology leading to the recognition of the importance of molecular factors in disease aetiology. The immediate consequence of such knowledge is an increased ability for pathology diagnostic and for the identification of presymptomatic individuals or those susceptible to specific diseases, improving our ability for disease prognosis and to develop more efficient therapeutic strategies. Every organism is exposed to hazardous agents in its environment on a continual basis. As a result, organisms have evolved sophisticated pathways that are considered an environmental response machinery, to minimize the biological consequences of hazardous environmental agents. A large number of human genes, including the ones involved in the environmental response machinery, are subject to genetic variability, which can be associated with the altered efficiency of a biological pathway (Perera & Weinstein, 2000). So, an individual’s risk for developing a disease stemming from an environmental exposure might be dependent on the efficiency of his/her own unique set of environmental response genes. These genes are usually involved in the metabolism of environmental carcinogens, in the repair of DNA lesions induced by exogenous and endogenous carcinogens, and in the control of the cell cycle. Individual polymorphic forms in those genes have been associated with individual susceptibility to different types of cancer namely in breast and thyroid cancer (Conde et al., 2009; Gaspar et al., 2004; Pabalan et al., 2010; Peng et al., 2011; Silva et al., 2005; Silva et al., 2006b; Silva et al., 2006a; Silva et al., 2009; Silva et al., 2010; Silva et al., 2007). Several enzymes have evolved for the detoxification of xenobiotic compounds, and their gene expression is induced in response to the presence of numerous compounds (e.g., polycyclic aromatic hydrocarbons found in tobacco smoke). An inefficient detoxification of reactive endogenous or exogenous compounds ultimately leads to lesions in DNA, which should be repaired by DNA repair mechanisms, ought to reduce cancer risk. As a

Genetic Susceptibility in Acute Pancreatitis: Genotyping of GSTM1, GSTT1, GSTP1, CASP7, CASP8, CASP9, CASP10, LTA, TNFRSF1B, and TP53 Gene Variants.

Objectives Genetic testing could play a critical role in diagnosis and prognosis of acute pancreatitis (AP) and guide effective therapeutic interventions. We hypothesized that genetic polymorphisms in apoptosis and oxidative stress genes could determine incidence or severity in AP. Methods We conducted a hospital-based case-control study in a white Portuguese population (133 AP patients and 232 age- and sex-matched healthy controls) to evaluate the role of 15 gene polymorphisms (2 deletions and 13 single nucleotide polymorphisms [SNPs]) in oxidative stress (GSTM1, GSTT1, GSTP1) and apoptosis genes (CASP7, CASP8, CASP9, CASP10, LTA, TNFRSF1B, TP53) in AP. Criteria for AP were abdominal pain, hyperamylasemia, and contrast-enhanced computed tomography. Results The presence of GSTM1 is associated with increased susceptibility for AP, and the GSTP1 Val105Ile SNP is associated with an increased risk for AP in men. CASP9 Phe136Leu/Phe136Phe SNPs (heterozygotes) increases the risk for mild AP (odds ratio, 3.616; 95% confidence interval, 1.151–11.364; P < 0.05), whereas the homozygotic genotype of CASP9 Ala28Val decreases risk for mild AP (odds ratio, 0.296; 95% confidence interval, 0.091–0.963; P < 0.05). Conclusions Our results suggest that variations in GSTM1, GSTP1, and CASP9 may influence risk for AP.

Abstract 2747: Synergistic antileukemic activity of F14512 in combination with AraC

New antileukemic agents are urgently needed to achieve improvement of the survival of patients suffering from Acute Myelogenous Leukemia (AML). F14512 combines an epipodophyllotoxin core with a spermine moiety that targets cells with a high demand for polyamines such as cancer cells. F14512 exhibits a high level of in vivo anti-tumor activity in a series of experimental murine and human solid tumor models and has entered into clinical phase one. In this study, we investigated the in vivo antileukemic activity of F14512 against cell line or primary human AML models and evaluated its potential in combination with the reference antileukemic agent, AraC. HL60 cells or AML cells collected from different patients were engrafted onto NSG mice. Using flow cytometry and q-PCR analysis, we demonstrated that multiple administrations of F14512 for 3 weeks resulted in an extensive reduction of AML cell number in the bone marrow and blood of treated mice. We also showed in vitro and in vivo synergistic activity of F14512 in combination with AraC in HL-60 and primary AML models when both compounds were used at suboptimal doses. The mechanisms triggering primary leukemic cell death were investigated and our results indicated that senescence could be involved. Collectively, these results demonstrate that F14512 exhibits a marked in vivo antileukemic activity, supporting the Phase I clinical trials of this novel promising drug candidate. 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 2747. doi:1538-7445.AM2012-2747

Abstract 1925: Trifluoromethylated artemisinin dimers demonstrate a potent anti-cancer activity in vitro and in vivo

Beside their widely recognised efficacy as antimalaric drugs, artemisinin derivatives are also known for their cytotoxic properties since the early nineties. More recently, artemisinin dimers (ADs) were shown to possess an increased potential as anti-cancer agents due to their ability to inhibit cancer cells proliferation in the low nanomolar range. Taking advantage of an original chemistry, we synthesized a new series of trifluoromethylated artemisnin dimers which proved to be particularly potent depending on the size and nature of the linker. Different mode of linkage were performed by either carbon 16 or carbon 10 and were tested in vitro and in vivo. Best combination was found in a hybrid linking position delivering non-symmetrical C10-C16 ADs. One of the lead compound of this series, F98458 demonstrated a moderate (but statiscally significative) anti-tumor activity in mice against a human melanoma xenograft that is, to our knowledge, the first demonstration of in vivo antitumor properties of ADs in preclinical models. Many efforts to understand the molecular origin of the cytotoxic effect of monomeric artemisinin derivatives have been undertaken. One of the aims of this study was to investigate the mechanism of action of ADs in comparison with artemisinin: the antimalaric drug artesunate, a representative monomeric derivative of artemisinin, is known to induce DNA damage and ROS generation while our ADs did not under the same experimental conditions. Moreover ADs did not induce apoptosis (caspase 3/7, annexin V), leading us to investigate cell death related to senescence and autophagy. We have also synthesized partially reduced ADs possessing only one endoperoxide functionality. These compounds did show the same potency than non reduced ADs, underlaying that a tight molecular recognition is involved in its biological response. All these observations suggest that monomeric artemisinin derivatives such as artesunate and ADs possess distinct modes of action. The high in vitro potency associated with the in vivo response led us to synthesize affinity probes in order to identify the molecular target of ADs which could provide fundamental informations for further developpement of these compounds as anti-cancer agents. This work currently underway will also be discussed. 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 1925. doi:1538-7445.AM2012-1925

Abstract B37: F14512, a polyamine-vectorized anticancer drug, triggers senescence cell death mechanism.

Drugs specifically vectorized to cancer cells may offer a reinforced activity, resulting in an improved therapeutic index. In this context, F14512 exploits the Polyamines Transport System (PTS) to accumulate into cells and exhibits an enhanced anti-proliferative activity on a large panel of tumor cell lines as compared to etoposide. At the molecular level, the spermine tail of F14512 contributes to enhance the water solubility of the drug, and reinforces the activity of the drug toward its primary molecular target, topoisomerase II. This observation has encouraged the set up of a phase 1 clinical study with F14512 in patients with relapsed or refractory acute myeloid leukemia (AML). In order to determine how these molecular properties translate at the cellular level, we have compared the kinetic of cell cycle modulation associated with the inhibition of cell proliferation. F14512 proved to be >30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent, nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased -galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 as compared to etoposide, and this difference may account for their distinct pharmacological profiles and the superior activity of F14512 in vivo. The identification of such response markers for senescence are currently pursued on in vivo MX-1 models by following a transcriptomic analysis focused on cell death cascades including apoptosis and senescence. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor response, applicable in clinical trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B37.

Abstract A214: F14512, a novel targeted cytotoxic agent exhibits a marked antileukemic activity, alone and in combination with AraC.

Chemotherapy remains mainly used for the treatment of Acute Myelogenous Leukemia (AML). However, in the past 3 decades limited progress has been achieved in improving the long-term disease-free survival. Therefore the development of more effective drugs for AML represents a high level of priority. F14512 combines an epipodophyllotoxin core targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. In fact the polyamine moiety facilitates F14512 selective uptake by tumor cells via the polyamine transport system, a machinery frequently overactivated in cancer cells. In this study, we report the in vivo antileukemic activity of F14512 against human AML models, established from patient samples. AML cells, collected from 3 different patients, were established onto NSG mice (LAM-2, LAM-7 and LAM-18). These 3 AML samples exhibited a normal karyotype, with FLT3-ITD, NPM1 and DNMTA3 mutations which proved stable over serial transplantations in vivo. After multiple i.v. administrations of F14512, 3 times a week for 3 weeks, an extensive reduction of AML cell number (98–99%) was observed in LAM-2 and LAM-7 - bearing mice. This antileukemic activity was recorded on the basis of flow cytometry, q-PCR and histology assessments. The effects of F14512 on LAM-18 bearing mice were marginal with an inhibition of AML cell growth of 42%. We also show in vitro and in vivo synergistic effects of F14512 in combination with AraC, one of the frontline chemotherapeutic agents for AML. These results were obtained using the HL-60 cell line. The activity of F14512 in combination with AraC will be further investigated in patient AML models. Collectively, these results demonstrate that F14512 exhibits a marked in vivo antileukemic activity, supporting its clinical development. Phase I clinical trials in onco-hematology are on going with this novel promising drug candidate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A214.