Indiana University-Purdue University Indianapolis (IUPUI) / All platinum (Pt)-based chemotherapeutics exert their efficacy primarily via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of Pt-DNA lesions by nucleotide excision repair and homologous recombination (HR) can substantially reduce the effectiveness of the Pt therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions requires RPA binding to single-stranded DNA (ssDNA). We synthesized structural analogs of our previously reported RPA inhibitor TDRL-505, determined the structure activity relationships and evaluated their efficacy in tissue culture models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro and cellular activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.

Résumé : Il est généralement admis que les cassures double-brin (CDB) de l’ADN sont parmi les lésions les plus toxiques induites par les radiations ionisantes (RI). Les CDBs non ou mal réparées peuvent conduire à une instabilité génomique et à la mort cellulaire. La chimioradiothérapie concomitante est l’une des modalités la plus efficace pour le traitement de certains cancers surtout en stade avancé. Le rendement des CDBs a augmenté quand l’ADN a été irradié en présence de cisplatine avec des électrons de basse énergie (EBEs). Notre étude a pour objectif de réévaluer la contribution des CDBs et d’autres lésions induites par les RI dans la létalité cellulaire. L'effet des RI sur la fonctionnalité de l’ADN plasmidique modifié ou non de façon covalente par le cisplatine a été étudié par mesure de l'efficacité de transformation du plasmide dans E. coli. Les complexes cisplatine-ADN ont été préparés de telle sorte qu’il y avait en moyenne deux adduits de cisplatine par plasmide tel que mesuré par ICP-MS. Nos échantillons ont été irradiés en solution avec des doses croissantes de rayonnements gamma (137Cs). La présence de cisplatine a augmenté la formation des CDBs par un facteur de 2.6 par comparaison avec l'ADN non modifié. Malgré cette augmentation, le rendement des CDBs reste très faible et ne peut pas expliquer la perte de fonctionnalité observée. Alors que, les dommages multiples localisés (LMDS) (non-DSB cluster damage) donnant naissance à des CDBs sous l’action des enzymes de réparation la formamidopyrimidine [fapy]-DNA glycosylase (Fpg) et l’endonuclease III (Nth) où leur rendement a été augmenté d’un facteur de 2.1 lorsque l’ADN a été irradié en présence de cisplatine, ont pu expliquer la perte de fonctionnalité observée. Ces résultats suggèrent que le cisplatine peut agir, non seulement comme un agent chimiothérapeutique, mais aussi comme un radiosensibilisateur efficace par addition d’autres lésions à l’ADN. Aussi, pour la première fois nous avons pu évaluer l’effet des EBEs sur la létalité cellulaire. Des films d'ADN ont été préparés en utilisant la méthode d’adsorption douce sur un substrat de graphite pyrolytique, en présence de 1,3- diaminopropane (Dap[indice supérieur]2+) et ont été irradiées avec des EBEs 10 eV. Nous avons pu conclure, qu’en plus des CSBs, CDBs et des dommages de base, les EBEs sont capables aussi d’induire des LMDS (non-DSB cluster damage) et induire la perte de fonctionnalité de l’ADN. Le rendement des CDBs est très faible d’où ils n’ont pas pu expliquer la perte de fonctionnalité de plasmide observée, après irradiation avec les EBEs. Le rendement très faible des LMDS (non-DSB cluster damage) ne peut pas expliquer la perte de fonctionnalité de l’ADN. Il semble que les EBEs sont capables d’induire des dommages très proches les uns des autres et qui ne peuvent pas être révélés par les enzymes de réparation Fpg et Nth. Plus les dommages sont proches les uns des autres, plus leur réparation est difficile, car une de ces lésions peut inhiber la réparation de l’autre la plus proche. // Abstract : It is generally accepted that DNA double-strand breaks (DSB) are among the most toxic lesions induced by ionizing radiation (IR). Unrepaired or misrepaired DSB can lead to genomic instability and cell death. It is known that concomitant chemoradiation therapy is one of the most preferred methods for the treatment of certain cancers especially in advanced stage. The yield of DSBs was increased when DNA was irradiated with low energy electron (LEEs). The aims of our study was to reassess the contribution of DSBs and other lesions induced by indirect and direct effect of IR in cell lethality. The effect of IR on the DNA functionality of the plasmid modified covalently with cisplatin was studied by measuring the transformation efficiency of the plasmid in E. coli. Cisplatin-DNA complexes were prepared such that there was an average of two cisplatin adducts per plasmid as measured by ICP-MS. Aqueous solutions of the samples were irradiated with 137Cs [gamma]-rays at various doses. Gel electrophoresis analysis shows that cisplatin enhances, by a factor of 2.6, the formation of DSB by [gamma]-rays relative to those in unmodified DNA. Despite this increase, the yield of DSBs is very low and cannot explain the loss of functionality observed after transformation with plasmids modified with cisplatin. While locally multiple damaged sites (LMDS) revealed by repair enzymes Fpg (Formamidopyrimidine [fapy]-DNA glycosylase) and Nth (Endonuclease III) as DSB (nonDSB cluster damage), where their yield was increased by a factor of 2.1 when DNA was irradiated in the presence of cisplatin were able to explain the observed loss of DNA functionality. These results suggest that cisplatin may act not only as a chemotherapeutic agent, but also as an effective radiosensitizer by addition of other DNA lesions. For the first time, we could also evaluate the effect of low energy electrons (LEEs) on DNA functionality. Highly ordered DNA films were prepared on pyrolytic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap[superscript]2+) to bind together the plasmids and irradiated with LEE (10 eV). We concluded that in addition to CSBs, DSBs and base damage, LEEs induced the formation of non-DSB cluster damage and also induced the loss of DNA functionality under LEE irradiation. The yields of DSBs and of non-DSB cluster damage are too low and so one unable to explain the loss of DNA functionality. It seems that LEEs are able to induce a high complex damage that cannot be revealed by repair enzymes Fpg and Nth. The high complex damage is difficult to repair possibly because the repair of one lesion, may inhibit the repair of another.

Le glioblastome constitue la tumeur primitive maligne la plus fréquente et la plus agressive du système nerveux central, caractérisée par un pronostic sombre. L’infusion locale dans le parenchyme cérébral du cisplatine a présenté des résultats encourageants sur des modèles précliniques. La nanovectorisation permet de concentrer l’efficacité thérapeutique d’agents anticancéreux à leur cible. Leur fonctionnalisation par des unités d’imagerie offre la possibilité de suivre de façon non invasive par imagerie par résonance magnétique (IRM) leur biodistribution au sein du tissu lésé. Dans cette optique, un copolymère tribloc amphiphile biocompatible a été synthétisé par polymérisations par ouverture de cycle successives à partir d’un oxyde de polyéthylène (PEO). Après micellisation dans l’eau, des complexes de gadolinium ont été greffés sur la couronne de PEO et les fonctions carboxylates situées en périphérie du coeur micellaire se sont réticulées sur le cisplatine, conduisant à la formation d’une prodrogue macromoléculaire de taille nanométrique stable dans le temps. Le potentiel de ces nanoparticules bifonctionnelles comme agents de contraste IRM a été exploré à haut champ magnétique. Une telle vectorisation du cisplatine a en outre permis d’augmenter de façon significative l’accumulation du platine dans deux lignées humaines de glioblastome ainsi que la formation d’adduits à l’ADN par rapport à la drogue libre. L’implication de mécanismes biologiques sous-jacents à cette étude pose la question de l’existence d’autres cibles alternatives critiques des dérivés du platine, remettant en cause le paradigme établi depuis un demi-siècle définissant l’ADN comme la cible ultime du cisplatine. / Glioblastoma is the most frequent and aggressive primary malignant tumor of the central nervous system with a gloomy prognosis. Local infusion of cisplatin within the brain parenchyma exhibited promising results in preclinical models. Nanovectorization of anticancer agents even promotes the concentration of their therapeutic efficiency on their target. Anchorage ofimaging moieties on such smart drug delivery systems further enables the non-invasive monitoring of their biodistribution within the damaged tissue by magnetic resonance imaging (MRI). From this perspective, a biocompatible amphiphilic triblock copolymer was synthesized by successive ring-opening polymerizations from a polyethylene oxide (PEO). After micellization in water, gadolinium complexes were grafted to the PEO corona and the carboxylate functions located at the surface of the micelle’s core were able to cross-link with cisplatin. A stable nano-sized macromolecular prodrug was therefore recovered. Relaxomety measurements at a high magnetic field confirmed the intrinsic potential of these hybrid nanoparticles as alternative MRI contrast agents. Besides, cisplatin vectorization allowed for substantially increasing the accumulation of platinum compounds in two human glioblastoma cell lines as well as the subsequent formation of DNA adducts in comparison with the free drug. Biological mechanisms below this study raise the question whether critical alternative targets of platinum derivatives might exist, thus undermining the old-established paradigm that defines DNA as the ultimate target of cisplatin.

Os transportadores da família ABC são proteínas transmembrânicas envolvidas com o tráfego de substâncias endógenas e exógenas do meio intracelular para o extracelular, sendo alvos de estudo na resistência celular a agentes quimioterápicos. O ABCC2 é um transportador transmembrânico que exporta ativamente fármacos aniônicos conjugados e facilita o transporte de agentes anticâncer. O ABCG2 é outro transportador transmembrânico que tem influência na farmacocinética e farmacodinâmica de certos xenobióticos e substratos endógenos; além disso, acredita-se que este gene contribui para a resistência a várias drogas. Por esses motivos identificamos por sequenciamento ou PCR-RFLP os polimorfismos dos genes ABCC2 (Val417Ile, Ser789Phe e Ala1450Thr) e ABCG2 (Val12Met, Gly126stop códon, Gly141Lys) em 90 pacientes portadores de carcinoma epidermóide de cabeça e pescoço (HNSCC) e tentamos correlacionar a presença do polimorfismo com resposta a tratamento que incluiu cisplatina em todos os pacientes. Não encontramos nenhuma correlação entre a presença de polimorfismo para Val12Met, Gly141Lys e Val417Ile, determinados em 68 pacientes tratados exclusivamente com cisplatina e radioterapia, e a resposta ao tratamento. As curvas de sobrevida, determinadas por Kaplan-Meier, considerando polimórfico os pacientes que continham pelo menos um dos alelos alterados e selvagem os que não tivessem nenhum deles, mostraram que os pacientes selvagens para o polimorfismo Val12Met tiveram tendência a uma pior sobrevida (sobrevida mediana de 18,7 meses) em relação aos pacientes polimórficos (sobrevida mediana não atingida; P=0,089 Teste de log-rank), já os pacientes selvagens para o polimorfismo Gly141Lys tiveram tendência a uma pior sobrevida (sobrevida mediana de 15,8 meses) em relação aos pacientes polimórficos (sobrevida mediana de 25,6 meses; P = 0,16 Teste de logrank). Não observamos correlação entre os outros polimorfismos e sobrevida. Quanto ao GLY126stop, somente um paciente foi identificado como polimórfico. Conclusões: No nosso estudo, a freqüência do polimorfismo Val12Met de 10% está próxima dos 18% descrito na população normal (Kobayashi 2004). Nosso trabalho foi o primeiro a correlacionar estes polimorfismos com resposta ao tratamento em pacientes com carcinoma epidermóide de cabeça e pescoço e indica que Val12Met e Gly141Lys e o gene ABCG2 como um todo são candidatos a um estudo maior / ATP binding cassette (ABC) transporters form one of the largest transmembrane protein families. These proteins use cellular ATP to drive the transport of various substrates across cell membranes including many exogenous and endogenous compounds, which includes drugs used in cancer treatment. ABCC2 is an ATP binding cassette transporter which accepts a diverse range of substrates, including glutathione, glucuronide, and sulfate conjugates of many metabolites and xenobiotics. ABCG2 is a member of the ATP binding cassette (ABC) transporters whose function is to pump out of the cell a wide variety of endogenous and exogenous compounds. Widely expressed in stem cells, ABCG2 is also recognized as a universal marker of stem cells. For these reasons we had identified the following polymorphisms of ABCC2 gene: -Val417Ile, Ser789Phe and Ala1450Thr- and of ABCG2 gene as well: -Val12Met, Gly126stop códon, Gly141Lys in 88 patients with head and neck squamous cell carcinoma (HNSCC). Methodology included PCR - RFLP and direct sequencing. Survival analysis was done using Kaplan-Meier curves and response measured by RECIST criteria. Comparisons were done between polymorphic patients in which at least one polymorphism was present as opposed to the patients without the polymorphism. Correlation with response to treatment was studied for Val12Met, Gly141Lys e Val417Il in 68 patients exclusively treated with concomitant cisplatin and radiotherapy and no correlation was found between these markers and treatment response. Patients without the Val12Met presented a trend towards shorter survival (median survival 18.7 months) as compared to polymorphic patients (median survival not reached, long rank p= 0.089). Although statistical significance was not reached, patients wild type for Gly141Lys polymorphism (median survival 15.8 months) had shorter survival than polymorphic patients (25.6 months, p=0.16). We did not observe any other correlation between other polymorphisms and survival. With respect to Gly126stop, only one patient was identified as polymorphic and survival analysis was not possible. As far as we know this is the first study to try to correlate these polymorphisms with treatment response and survival in HNSCC patients. Although we were unable to draw any definitive conclusions, our results indicate that Val12Met and Gly141Lys deserve to be further studied in the future.

INTRODUÇÃO: O presente trabalho teve como objetivo elaborar análise custoefetividade do esquema de quimiorradioterapia com cisplatina (estratégia 2) comparado ao tratamento radioterápico (estratégia 1) para pacientes portadores de CECCP localmente avançado não elegíveis para tratamento cirúrgico. MÉTODOS: levantamos dados prospectivos de 33 pacientes na estratégia 2 e dados retrospectivos de 29 pacientes tratados no HC-FMUSP e Hospital A.C. Camargo, (estratégia 1). Consideramos a tabela de reembolso do Sistema Único de Saúde (perspectiva SUS) e custos do HC-FMUSP com honorários profissionais, medicamentos, demais insumos e depreciação de equipamentos (perspectiva Institucional). A medida de efetividade foi 1 ano de vida ganho, livre de progressão da doença (SLPD). Calculamos a Razão Incremental Custo Efetividade (RICE). RESULTADOS: 31% dos pacientes da estratégia 1 e 58% na estratégia 2) tiveram 1 ano de SLPD. Na perspectiva SUS o custo total por paciente na estratégia 1 foi de R$ 2.798,52 e R$ 4.938,11 na estratégia 2. Na perspectiva institucional os custos foram R$ 26.798,52 e R$ 5.040,79, respectivamente. A RICE na perspectiva SUS foi de R$ 7.924,00 reais por ano de vida ganho e R$ 8.912,71 na perspectiva institucional. CONCLUSÃO: nas duas perspectivas a estratégia 2 se mostrou custo-efetiva, sendo o custo incremental considerado aceitável, segundo diretrizes do Banco Mundial. / INTRODUCTION: The present study aims to conduct a cost-effectiveness analysis comparing chemoradiotherapy with cisplatine and radiotherapy alone, to treat inoperative advanced head and neck cancer. METHODS: we collected data from 29 patients in a prospective study on chemoradiotherapy with cisplatin, conducted at Hospital das Clínicas HC-FMUSP,(strategy 2). For strategy 1, we collected retrospective data of 33 patients treated with radiotherapy at HC-FMUSP and Hospital A.C. Camargo. We considered only direct costs (personnel, drugs, material and equipment depreciation). We considered, the National Health Service (SUS) reimbursement parameters as the National Security System perspective, and HC-FMUSP costs as the institutional perspectives. We measured effectiveness as one year of diseasefree life gained. We collected costs and effectiveness data and calculated the cost-effectiveness incremental ratio ICER, which expresses additional costs per life year gained, in strategy 2, compared to strategy 1 RESULTS: 31.0% of the patients treated in strategy 1 lived more than 12 months, without disease progression, compared to 58.0% of the patients in strategy 2. According to SUS perspective, the total cost per patient in strategy 1) is R$ 2.798,52 and R$ 4.938,11 in strategy 2. Considering the institutional perspective, total costs are R$ 2.634,36, and R$ 5.040,79 respectively. In SUS perspective, the ICER ratio of strategy 2 compared to 1 is R$ 7.924,00 per lifes year gained. In the institutional perspective, ICER is R$ 8.912,71. We conducted a one way sensitivity analysis to verify our calculations. CONCLUSION: Chemoradioterapy with cisplatin proved more cost-effective than radiotherapy. Using the World Bank guidelines, wich considers the countries GDP per capita an acceptable cost per additional year of life (R$ 12.491,00 in 2006), the incremental cost of both is acceptable.

Os transportadores da família ABC são proteínas transmembrânicas envolvidas com o tráfego de substâncias endógenas e exógenas do meio intracelular para o extracelular, sendo alvos de estudo na resistência celular a agentes quimioterápicos. O ABCC2 é um transportador transmembrânico que exporta ativamente fármacos aniônicos conjugados e facilita o transporte de agentes anticâncer. O ABCG2 é outro transportador transmembrânico que tem influência na farmacocinética e farmacodinâmica de certos xenobióticos e substratos endógenos; além disso, acredita-se que este gene contribui para a resistência a várias drogas. Por esses motivos identificamos por sequenciamento ou PCR-RFLP os polimorfismos dos genes ABCC2 (Val417Ile, Ser789Phe e Ala1450Thr) e ABCG2 (Val12Met, Gly126stop códon, Gly141Lys) em 90 pacientes portadores de carcinoma epidermóide de cabeça e pescoço (HNSCC) e tentamos correlacionar a presença do polimorfismo com resposta a tratamento que incluiu cisplatina em todos os pacientes. Não encontramos nenhuma correlação entre a presença de polimorfismo para Val12Met, Gly141Lys e Val417Ile, determinados em 68 pacientes tratados exclusivamente com cisplatina e radioterapia, e a resposta ao tratamento. As curvas de sobrevida, determinadas por Kaplan-Meier, considerando polimórfico os pacientes que continham pelo menos um dos alelos alterados e selvagem os que não tivessem nenhum deles, mostraram que os pacientes selvagens para o polimorfismo Val12Met tiveram tendência a uma pior sobrevida (sobrevida mediana de 18,7 meses) em relação aos pacientes polimórficos (sobrevida mediana não atingida; P=0,089 Teste de log-rank), já os pacientes selvagens para o polimorfismo Gly141Lys tiveram tendência a uma pior sobrevida (sobrevida mediana de 15,8 meses) em relação aos pacientes polimórficos (sobrevida mediana de 25,6 meses; P = 0,16 Teste de logrank). Não observamos correlação entre os outros polimorfismos e sobrevida. Quanto ao GLY126stop, somente um paciente foi identificado como polimórfico. Conclusões: No nosso estudo, a freqüência do polimorfismo Val12Met de 10% está próxima dos 18% descrito na população normal (Kobayashi 2004). Nosso trabalho foi o primeiro a correlacionar estes polimorfismos com resposta ao tratamento em pacientes com carcinoma epidermóide de cabeça e pescoço e indica que Val12Met e Gly141Lys e o gene ABCG2 como um todo são candidatos a um estudo maior / ATP binding cassette (ABC) transporters form one of the largest transmembrane protein families. These proteins use cellular ATP to drive the transport of various substrates across cell membranes including many exogenous and endogenous compounds, which includes drugs used in cancer treatment. ABCC2 is an ATP binding cassette transporter which accepts a diverse range of substrates, including glutathione, glucuronide, and sulfate conjugates of many metabolites and xenobiotics. ABCG2 is a member of the ATP binding cassette (ABC) transporters whose function is to pump out of the cell a wide variety of endogenous and exogenous compounds. Widely expressed in stem cells, ABCG2 is also recognized as a universal marker of stem cells. For these reasons we had identified the following polymorphisms of ABCC2 gene: -Val417Ile, Ser789Phe and Ala1450Thr- and of ABCG2 gene as well: -Val12Met, Gly126stop códon, Gly141Lys in 88 patients with head and neck squamous cell carcinoma (HNSCC). Methodology included PCR - RFLP and direct sequencing. Survival analysis was done using Kaplan-Meier curves and response measured by RECIST criteria. Comparisons were done between polymorphic patients in which at least one polymorphism was present as opposed to the patients without the polymorphism. Correlation with response to treatment was studied for Val12Met, Gly141Lys e Val417Il in 68 patients exclusively treated with concomitant cisplatin and radiotherapy and no correlation was found between these markers and treatment response. Patients without the Val12Met presented a trend towards shorter survival (median survival 18.7 months) as compared to polymorphic patients (median survival not reached, long rank p= 0.089). Although statistical significance was not reached, patients wild type for Gly141Lys polymorphism (median survival 15.8 months) had shorter survival than polymorphic patients (25.6 months, p=0.16). We did not observe any other correlation between other polymorphisms and survival. With respect to Gly126stop, only one patient was identified as polymorphic and survival analysis was not possible. As far as we know this is the first study to try to correlate these polymorphisms with treatment response and survival in HNSCC patients. Although we were unable to draw any definitive conclusions, our results indicate that Val12Met and Gly141Lys deserve to be further studied in the future.

<p>We treated 16 patients with somatostatin analogs combined with α-interferon and achieved a biochemical and/or radiological response in 56% (median duration 22 months). We consider this treatment a good alternative for patients who fail during chemotherapy or who do not want to/cannot receive cytotoxic drugs.</p><p>Thirty-six patients with neuroendocrine tumors were treated with cisplatin combined with etoposide. Of 14 patients with evaluable EPTs, 50% responded radiologically and/or biochemically (median duration 9 months). We consider this treatment useful as first-line medical treatment in aggressive EPTs or in patients failing prior chemotherapy.</p><p>Twenty-eight tumor tissues from EPTs were examined with immunohistochemistry regarding expression of somatostatin receptors (ssts) 1 to 5 on tumor cells and in intratumoral vessels. We found that sst₂ and sst₄ were highly expressed on tumor cells and in vessels. However, sst₃ and sst₅ were lacking in half of the tumor tissues and in most of the vessels. Because of the variability in sst expression, we recommend analysis of each individual’s receptor expression before starting treatment.</p><p>Endocrine pancreatic tumors (EPTs) are rare with an incidence of 4 per million inhabitants. In the majority of cases they grow slowly, but there are exceptions with very rapidly progressing malignant carcinomas. First-line medical treatment is streptozotocin combined with 5-fluorouracil.</p><p>We examined 38 tumor samples regarding expression of tyrosine kinase receptors platelet-derived growth factor receptors (PDGFRs), c-kit and epidermal growth factor receptor (EGFR). We found that the receptors were expressed in more than half of the tumor tissues. Further studies will reveal if tyrosin kinase antagonists can be part of the future treatment arsenal.</p>

We treated 16 patients with somatostatin analogs combined with α-interferon and achieved a biochemical and/or radiological response in 56% (median duration 22 months). We consider this treatment a good alternative for patients who fail during chemotherapy or who do not want to/cannot receive cytotoxic drugs. Thirty-six patients with neuroendocrine tumors were treated with cisplatin combined with etoposide. Of 14 patients with evaluable EPTs, 50% responded radiologically and/or biochemically (median duration 9 months). We consider this treatment useful as first-line medical treatment in aggressive EPTs or in patients failing prior chemotherapy. Twenty-eight tumor tissues from EPTs were examined with immunohistochemistry regarding expression of somatostatin receptors (ssts) 1 to 5 on tumor cells and in intratumoral vessels. We found that sst2 and sst4 were highly expressed on tumor cells and in vessels. However, sst3 and sst5 were lacking in half of the tumor tissues and in most of the vessels. Because of the variability in sst expression, we recommend analysis of each individual’s receptor expression before starting treatment. Endocrine pancreatic tumors (EPTs) are rare with an incidence of 4 per million inhabitants. In the majority of cases they grow slowly, but there are exceptions with very rapidly progressing malignant carcinomas. First-line medical treatment is streptozotocin combined with 5-fluorouracil. We examined 38 tumor samples regarding expression of tyrosine kinase receptors platelet-derived growth factor receptors (PDGFRs), c-kit and epidermal growth factor receptor (EGFR). We found that the receptors were expressed in more than half of the tumor tissues. Further studies will reveal if tyrosin kinase antagonists can be part of the future treatment arsenal.

Cells have evolved different pathways in order to tolerate damage produced by different cytotoxic agents. Each agent reacts differently with DNA causing formation of different types of adducts, each eliciting the SOS stress response to induce different cellular repair pathways. One such type of substrate generated by cytotoxic agents is the DNA double strand break (DSB). The main pathway to repair such damage in the cell is through a process of recombination. In this thesis, I specifically examined the anti-cancer therapeutic agent cisplatin, which forms single- and double-strand breaks in DNA, and methylating agents, which are proposed to also be capable of forming such breaks. Neither type of agent can directly form these breaks; however, they leave a signature type of damage lesion which is recognized by different repair processes. The mismatch repair (MMR) status of a mammalian cell or an Escherichia coli dam mutant relates directly to the sensitivity of the cells to the agents mentioned above. As the dam gene product plays an important role in this pathway and in other processes in the cell, when mutated, dam cells are more sensitive to methylating agents and cisplatin than wildtype. A combination of dam and either mutS or mutL restores resistance to the same agents to wild type levels. Therefore, mismatch repair sensitizes dam bacteria to these agents. The rationale for this comes from examining the viability of dam mutants, as dammutants are only viable because they are highly recombinogenic. The presence of MMR-induced nicks or gaps results in the formation of DSBs that require recombination to restore genomic integrity. Mismatch repair proteins inhibit recombination between homeologous DNA. Homeologous recombination (recombination between non-identical, but similar, DNA sequences) is only possible when the MMR proteins, MutS and MutL, are absent. It is postulated that this is because MutS recognizes the homeologous DNA and subsequently slows down or aborts recombination completely. The double mutant, dam mutS/L shows wild type levels of sensitivity to cisplatin because mismatch repair is no longer recognizing the adducts and recombinational repair is allowed to continue. Human cells behave in an analogous fashion to the bacterial dam mutant, showing sensitivity to cisplatin and methylating agents. When an additional mutation in a mismatch repair gene is present, the cells become as resistant as wild type. Therefore, the E. coli dammutant is a useful model system to study this mechanism of drug resistance. DNA containing cisplatin adducts or lesions resulting from methylation are substrates for other types of repair processes such as nucleotide excision repair and base excision repair; however they have also been implicated as substrates for MMR and recombinational repair. The goal of the work in this thesis was two-fold. The first was to identify the gene products and mechanism necessary for repair of cisplatin damage by recombination. The second was to examine the mechanism of cisplatin toxicity, and specifically how MMR proficiency aids in the cytotoxicity of this drug by preventing recombination. Using the duplicated inactive lac operon recombination assay, we were able to determine the requirements for spontaneous and cisplatin-induced recombination, the RecBCD and RecFOR pathways. We were also able to further postulate that the cisplatin- induced signature damage recognized by recombination was the double strand break, likely formed from fork stalling and regression or a subsequent collapse during DNA synthesis, thus requiring these pathways for repair. This observation led to the experiments involving examination of the mechanism of cisplatin toxicity and where MMR could inhibit specific steps of recombination with DNA containing cisplatin lesions. Low levels of cisplatin lesions slowed the rate of RecA-mediated strand transfer in vitro, likely due to its ability to form a large bend in the DNA. MutS bound to cisplatin lesions in the DNA during heteroduplex formation in the RecA strand exchange step of recombination, inhibiting branch migration, and aborting the reaction. In order for MutS to inhibit recombination with cisplatin lesions, the results in the work in Chapter IV, show that binding to the lesion requires the C-terminus of MutS to be present, possibly due to a requirement for tetramerization of the protein, a domain contained in the C-terminus of MutS. This antirecombination function is different than the mutation avoidance function of MutS, as binding of mismatches requires only dimers. This differential sensitivity for cisplatin versus a mismatch was further exemplified in Chapter V, the experiments with dna mutants, where the greatest difference in sensitivity was observed for a dnaE mutant (catalytic subunit of polIII), which was as sensitive to cisplatin as a dam mutant, but fairly resistant to treatment with MNNG. This is indicative of the potency of a cisplatin adduct to block polymerase progression, versus a mismatch which poses little problem to synthesis. Recombination is invoked to repair DSBs caused by the cisplatin lesions through the RecBCD and FOR pathways after fork regression or collapse. A main conclusion from these studies is that a cisplatin lesion is processed differently than a mismatch. The mechanism of how a cisplatin lesion is processed, forming the DSB which invokes recombinational repair is still unclear and continues to be investigated.

INTRODUÇÃO: O presente trabalho teve como objetivo elaborar análise custoefetividade do esquema de quimiorradioterapia com cisplatina (estratégia 2) comparado ao tratamento radioterápico (estratégia 1) para pacientes portadores de CECCP localmente avançado não elegíveis para tratamento cirúrgico. MÉTODOS: levantamos dados prospectivos de 33 pacientes na estratégia 2 e dados retrospectivos de 29 pacientes tratados no HC-FMUSP e Hospital A.C. Camargo, (estratégia 1). Consideramos a tabela de reembolso do Sistema Único de Saúde (perspectiva SUS) e custos do HC-FMUSP com honorários profissionais, medicamentos, demais insumos e depreciação de equipamentos (perspectiva Institucional). A medida de efetividade foi 1 ano de vida ganho, livre de progressão da doença (SLPD). Calculamos a Razão Incremental Custo Efetividade (RICE). RESULTADOS: 31% dos pacientes da estratégia 1 e 58% na estratégia 2) tiveram 1 ano de SLPD. Na perspectiva SUS o custo total por paciente na estratégia 1 foi de R$ 2.798,52 e R$ 4.938,11 na estratégia 2. Na perspectiva institucional os custos foram R$ 26.798,52 e R$ 5.040,79, respectivamente. A RICE na perspectiva SUS foi de R$ 7.924,00 reais por ano de vida ganho e R$ 8.912,71 na perspectiva institucional. CONCLUSÃO: nas duas perspectivas a estratégia 2 se mostrou custo-efetiva, sendo o custo incremental considerado aceitável, segundo diretrizes do Banco Mundial. / INTRODUCTION: The present study aims to conduct a cost-effectiveness analysis comparing chemoradiotherapy with cisplatine and radiotherapy alone, to treat inoperative advanced head and neck cancer. METHODS: we collected data from 29 patients in a prospective study on chemoradiotherapy with cisplatin, conducted at Hospital das Clínicas HC-FMUSP,(strategy 2). For strategy 1, we collected retrospective data of 33 patients treated with radiotherapy at HC-FMUSP and Hospital A.C. Camargo. We considered only direct costs (personnel, drugs, material and equipment depreciation). We considered, the National Health Service (SUS) reimbursement parameters as the National Security System perspective, and HC-FMUSP costs as the institutional perspectives. We measured effectiveness as one year of diseasefree life gained. We collected costs and effectiveness data and calculated the cost-effectiveness incremental ratio ICER, which expresses additional costs per life year gained, in strategy 2, compared to strategy 1 RESULTS: 31.0% of the patients treated in strategy 1 lived more than 12 months, without disease progression, compared to 58.0% of the patients in strategy 2. According to SUS perspective, the total cost per patient in strategy 1) is R$ 2.798,52 and R$ 4.938,11 in strategy 2. Considering the institutional perspective, total costs are R$ 2.634,36, and R$ 5.040,79 respectively. In SUS perspective, the ICER ratio of strategy 2 compared to 1 is R$ 7.924,00 per lifes year gained. In the institutional perspective, ICER is R$ 8.912,71. We conducted a one way sensitivity analysis to verify our calculations. CONCLUSION: Chemoradioterapy with cisplatin proved more cost-effective than radiotherapy. Using the World Bank guidelines, wich considers the countries GDP per capita an acceptable cost per additional year of life (R$ 12.491,00 in 2006), the incremental cost of both is acceptable.