OBJETIVOS: Caracterizar o perfil de imunoexpressão de MLH1, MSH2, MSH6 e PMS2 em adenocarcinomas gástricos, explorando seu desempenho na identificação de características clínicas e patológicas, bem como sua influência prognóstica isolada e em relação aos demais parâmetros. MÉTODOS: Cento e trinta e três casos de adenocarcinomas gástricos esporádicos localmente avançados (pT2a ou mais) operados no Hospital das Clínicas/FMUSP foram incluídos pela ausência de metástases a distância ao diagnóstico (M0) e caracterizados clínica (idade, sexo e sobrevida) e patologicamente (tamanho, local, tipo de Borrmann, tipo histológico, infiltração vascular, perineural e variáveis de estadiamento locorregional). Amostras de 1,0 mm foram dispostas em micromatrizes teciduais (TMA) para pesquisa imuno-histoquímica das enzimas MLH1, MSH2, MSH6 e PMS2, com detecção por sistema de polímeros curtos conjugados a peroxidase. Casos com resultados negativos ou incertos nas amostras de TMA tiveram repetidas as reações em secções convencionais. A associação entre o estado de expressão dos marcadores com variáveis clínicopatológicas foi avaliada através do teste do qui-quadrado ou exato de Fisher. O impacto dos parâmetros clínico-patológicos e estado de expressão das enzimas na sobrevida foi explorado em modelos uni e multivariados de Cox, com construção de curvas de Kaplan-Meyer. Todas as análises estatísticas foram consideradas significativas ao nível de p<0,05. RESULTADOS: Quarenta e cinco casos (33,6%) exibiram perda de expressão de ao menos uma enzima, sendo frequente a perda de duas (9/45: 20%), três (14/45:31,2%) ou quatro enzimas (7/45:15,5%). Anormalidade mais frequente ocorreu com o MLH1 (26,7%), seguida de MSH6 (23%), PMS2 (21%) e MSH2 (20,8%). Quando avaliadas em conjunto, houve correlação entre o estado de expressão de todos os possíveis pares, com destaque para MLH1/PMS2 (rho=0,467, p<0,001) e MSH2/MSH6 (rho=0,666, p<0,001). Perda de MLH1 associou-se a tipos I/II de Borrmann (p<0,001), fenótipo intestinal de Lauren (p=0,005), tubular/túbulo-papilífero (p=0,009), expansivo de Ming (p=0,027) e infiltração em muscular própria (p=0,011). Com relação a perda de MSH2, tipos I/II de Borrmann (p<0,001), padrões tubular/túbulo-papilífero (p=0,008), intestinal (p=0,001), expansivo (p=0,001), infiltração da muscular própria (p=0,025), reação desmoplásica ausente a discreta (p=0,021) e ausência de infiltração perineural (p=0,016) foram mais frequentes. Perda de MSH6 associou-se aos tipos macroscópicos de Borrmann e histológicos de Lauren, OMS e Ming (p<0,001) e ausência de infiltração perineural (p=0,036). Idade mais avançada (p=0,046), tipos I/II de Borrmann (p=0,002), padrão intestinal de Lauren (p=0,021) e menos frequente infiltração perineural (p=0,035) foram identificados nos casos com perda de PMS2. A co-negatividade para os pares MLH1/PMS2 e MSH2/MSH6, além de reproduzir as associações mencionadas, identificou infiltrado linfoplasmocitário intra/peritumoral acentuado (p=0,011 e p=0,013), reação estromal desmoplásica ausente a leve para MSH2/MSH6 (p=0,037) e tamanho maior do tumor primário para MLH1/PMS2 (p=0,021). Pior sobrevida associou-se ao sexo masculino (LogRank: 5,11, p=0,024), tamanho do tumor (3,98, p=0,046), tipos III/IV de Borrmann (4,75, p=0,029), histologia mucinosa/anel-de-sinete da OMS (8,61, p=0,003) e difuso (11,62, p=0,003), infiltração perineural (12,62, p<0,001), metástase linfonodal (23,25, p<0,001) e estadio TNM (35,60, p<0,001) em análises univariadas. Melhor sobrevida associou-se a perda de MLH1, MSH6 e PMS2 isoladamente (5,46, p=0,019; 6,08, p=0,014; 7,46, p=0,006) e dos pares MLH1/PMS2 (7,89, p=0,005) e MSH2/MSH6 (5,29, p=0,021). Em modelos multivariados compostos pelos parâmetros clínicopatológicos, apenas o sexo masculino (HR=2,42, p=0,047), tipo histológico difuso (4,94, p=0,037) e estadios II, IIIA e IV (2,23, p=0,088; 3,12, p=0,022; 33,24, p=0,005), constituíram variáveis independentes de determinação prognóstica. Nas análises multivariadas incluindo o estado de expressão das enzimas, evidenciou-se que as perdas de PMS2 e do par MLH1/PMS2 associaram-se significativamente a maior sobrevida (3,84, p=0,029 e 9,82, p=0,028).CONCLUSÕES: O presente estudo demonstra o valor da imunohistoquímica na identificação de alterações na expressão de enzimas MMR, sendo a mais frequentemente negativa a MLH1. A frequente co-negatividade aponta para a importância da dimerização na funcionalidade do sistema de reparo. A perda isolada destes marcadores, e especialmente do par MLH1/PMS2, define perfil clínico-patológico característico, permitindo avanços no conhecimento previamente atribuído a fenótipo microssatélite instável conforme determinado em métodos moleculares. Em análises multivariadas, o estado de expressão de PMS2 isoladamente ou do par MLH1/PMS2 constitui fator independente de determinação prognóstica / OBJECTIVES: To characterize immunoexpression profile of MLH1, MSH2, MSH6 e PMS2 in gastric adenocarcinomas, exploring its performance to identify distinctive clinico-pathological features, as well as their prognostic implications in univariate and multivariate analyses. METHODS: A hundred and thirty three cases of locally advanced (pT2a or higher) sporadic gastric adenocarcinomas operated on Hospital das Clínicas/FMUSP were included due to absence of distant metastases at diagnosis (M0). Clinical (age, gender and survival) and pathological features (size, local, Borrmann´s type, histological type, vascular and perineural infiltration and locorregional staging parameters) were characterized. One millimeter samples were placed on tissue microarray blocks (TMA) and immunohistochemical detection of MLH1, MSH2, MSH6 and PMS2 performed on obtained sections, with revelation developed by peroxidase conjugated short-polymer based reagents. Negative or equivocal results obtained with TMA samples were repeated on conventional sections. Association between expression status for these markers and clinico-pathological features were evaluated by chisquare or Fisher´s exact test when appropriate. Impact of clinico-pathological features and expression status on disease specific survival were explored by Cox uni and multivariate models, with Kaplan-Meyer curves being fitted to illustrate these. All statistical results were considered significant at p<0,05. RESULTS: Forty five cases (33.6%) showed loss of at least one mismatchrepair enzyme, being frequent loss of two (9/45: 20%), three (14/45:31.2%) or even the four evaluated enzymes (7/45:15.5%). The most frequent abnormality addressed MLH1 (26.7%), followed by MSH6 (23%), PMS2 (21%) and MSH2 (20.8%). When analyses were performed in conjunction, correlation was identified for the expression status of all possible pairs, mainly the functional heterodimers MLH1/PMS2 (rho=0.467, p<0.001) and MSH2/MSH6 (rho=0.666, p<0.001). MLH1 loss was associated to Borrmann´s types I/I (p<0.001), Lauren´s intestinal phenotype (p=0.005), tubular/tubulo-papillary architecture (p=0.009), Ming´s expansile type (p=0.027) and infiltration limited by muscular propria (p=0.011). Among cases showing MSH2 loss, Borrmann´s I/I (p<0.001), tubular/tubulo-papillary (p=0.008), intestinal (p=0.001), expansile (p=0.001), muscular propria infiltration (p=0.025), absent to mild stromal desmoplasia (p=0.021) and absent perineural infiltration (p=0.016) were more frequent. MSH6 loss was associated to Borrmann´s gross type and Lauren, WHO and Ming´s histological types (p<0.001), as well as absent perineural infiltration (p=0.036). More advanced age (p=0.046), Borrmann´s types I/I (p=0.002), Lauren´s intestinal morphology (p=0.021) and less frequent perineural infiltration (p=0.035) were identified as associated to PMS2 loss. Conegativity for MLH1/PMS2 and MSH2/MSH6 pairs resumed all the above mentioned associations and additionally identified heavy lymphoplasmacytic infiltrate (p=0.011 e p=0.013), absent to mild stromal desmoplasia for MSH2/MSH6 (p=0.037) and increased primary tumor size for MLH1/PMS2 (p=0.021). In univariate analyses, decreased disease-specific survival was associated to male gender (LogRank: 5.11, p=0.024), tumor size (3.98, p=0.046), Borrmann´s types III/IV (4.75, p=0.029), mucinous/signet-ring cell morphology according to WHO (8.61, p=0.003) and Lauren´s diffuse morphology (11.62, p=0.003), perineural infiltration (12.62, p<0.001), lymph node metastases (23.25, p<0.001) and TNM staging (35.60, p<0.001). Better survival was seen in cases showing loss of MLH1, MSH6 and PMS2 when individually analyzed (5.46, p=0.019; 6.08, p=0.014; 7.46, p=0.006), as well as MLH1/PMS2 (7.89, p=0.005) and MSH2/MSH6 heterodimeric pairs (5.29, p=0.021). In multivariate models addressing clinico-pathological features, only male gender (HR=2.42, p=0.047), diffuse histological type (4.94, p=0.037) and stages II, IIIA and IV (2.23, p=0.088; 3.12, p=0.022; 33.24, p=0.005, respectively) were independent prognostic features. Multivariate analyses including status of MMR enzymes and the most significant clinicopathological features disclosed that PMS2 and MLH1/PMS2 losses were independent predictors of increased disease-specific survival (3.84, p=0.029 e 9.82, p=0.028). CONCLUSIONS: The present study demonstrates immunohistochemical detection of mismatch-repair enzymes as a tool to identify losses of these markers, being the most frequently negative MLH1. The frequently observed co-negativity points toward the importance of heterodimerization of these proteins in functional activity of mismatch-repair system. Losses of these markers, mainly MLH1/PMS2 pair, define a distinctive clinico-pathological profile and add knowledge to the previously reported associations with microsatellite instability as defined by molecular approach. In multivariate analyses, expression status of PMS2, as well as MLH1/PMS2 pair, revealed independent prognostic impact on diseasespecific survival

A instabilidade genômica tem sido amplamente usada para caracterizar células cancerosas. Alterações genéticas em queratose actínica (QA) e carcinoma espinocelular (CEC) foram investigadas pelo método de random amplified polymorphic DNA (RAPD) e análise de microssatélites com o objetivo de encontrar marcadores moleculares para auxiliar o prognóstico e o diagnóstico médico. O DNA foi obtido de pacientes brasileiros cirurgiados e tratados no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, totalizando oito QAs, 24 CECs, e tecidos normais e/ou leucócitos correspondentes. Os microssatélites estudados foram D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280 e D9S287, tendo em vista a detecção de instabilidade genômica representada por perda de heterozigosidade (LOH) e instabilidade de microssatélites (MSI). Os \"primers\" usados para comparar os padrões de RAPD foram OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17 e OPB-19. Foi obtida correlação significativa na progressão de QA (1/8) para CEC (5/22) referente ao microssatélite D6S251. As diferenças nos padrões de DNA obtidos pelo método RAPD comparados aos controles foram maiores em lesões com maior grau de severidade segundo critério histológico. O mesmo padrão RAPD foi observado no controle e no tumor em 27% QA, 24% CEC I, 9% CEC II e 0% CEC III. Estes resultados mostram que o microssatélite D6S251 e o método de RAPD são informativos, podendo ser potenciais candidatos para auxílio no diagnóstico e prognóstico de QA e CEC. / Genomic instability has been widely used to characterize cancer cells. Genetic alterations in human actinic keratosis (AK) and squamous cell carcinomas (SCC) were investigated by the random amplified polymorphic DNA (RAPD) method, and microsatellite analysis. DNA was obtained from Brazilian patients diagnosed and treated in the School of Medicine of University of Sao Paulo out Clinics Hospital. Eight AKs, 24 SCCs, and 4 BCCs, matched to normal skin tissue and/or leukocytes were studied. Microsatellite patterns were obtained with primers specific to amplify D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280, and D9S287, in search of detection Loss of heterozygosity (LOH) and Microsatellite instability (MSI). The RAPD primers were: OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17, and OPB-19. A significant correlation was obtained regarding the progress of AK (1/8) to SCC (5/22) detected with the D6S251 microsatellite. DNA fingerprint obtained with RAPD primers were altered in increasing number of samples, according to their histological degree of differentiation. Similar RAPD patterns were observed in tumor and control in 27% AK, 24% SCC I, 9% SCC II, and zero SCC III. These results suggest microsatellite D6S251 and RAPD method to be potential tools in diagnosis and prognosis of AK and SCC.

Background Colorectal cancer (CRC) is the second most common malignancy in developed countries. The mortality is high, with nearly half of patients dying from the disease. The primary treatment of CRC is surgery, and decisions about additional treatment with chemotherapy are based mainly on tumor stage. Novel prognostic markers that identify patients at high risk of recurrence and cancer-related death are needed. The development of CRC has been described in terms of two different pathways; the microsatellite instability (MSI) and chromosomal instability (microsatellite stable, MSS) pathway. More recently, the CpG island methylator phenotype (CIMP), characterized by frequent DNA hypermethylation, has been described as an alternative pathway of tumorigenesis. The event of DNA methylation is dependent on one-carbon metabolism, in which folate and vitamin B12 have essential functions. The purpose of this thesis was to study CIMP in CRC. The specific aims were to investigate the potential role of components of one-carbon metabolism as risk factors for this subgroup of tumors, and the prognostic importance of CIMP status, taking into consideration important confounding factors, such as MSI and tumor-infiltrating T cells. Methods CRC cases and referents included in the Northern Sweden Health and Disease Study (NSHDS, 226 cases and 437 referents) and CRC cases in the Colorectal Cancer in Umeå Study (CRUMS, n=490) were studied. Prediagnostic plasma concentrations of folate and vitamin B12 were analyzed in NSHDS. In both study groups, CIMP status was determined in archival tumor tissue by real-time quantitative PCR using an eight-gene panel (CDKN2A, MLH1, CACNA1G, NEUROG1, RUNX3, SOCS1, IGF2 and CRABP1). MSI screening status and the density of tumor-infiltrating T cells were determined by immunohistochemistry.  Results An inverse association was found between plasma concentrations of vitamin B12 and rectal, but not colon, cancer risk. We also found a reduced risk of CIMP-high and CIMP-low CRC in study subjects with the lowest levels of plasma folate. We found that patients with CIMP-low tumors in both NSHDS and CRUMS had a poorer prognosis compared with CIMP-negative, regardless of MSI screening status. We also found that MSS CIMP-high patients had a poorer prognosis compared with MSS CIMP-negative. The density of tumor-infiltrating T cells and CIMP status were both found to be independent predictors of CRC patient prognosis. A particularly poor prognosis was found in patients with CIMP-low tumors poorly infiltrated by T cells. In addition, the density of T cells appeared to be more important than MSI screening status for predicting CRC patient prognosis. Conclusion Rather than being one disease, CRC is a heterogeneous set of diseases with respect to clinico-pathological and molecular characteristics. We found that the association between risk and plasma concentration of vitamin B12 and folate depends on tumor site and CIMP status, respectively. Patient prognosis was found to be different depending on CIMP and MSI screening status, and the density of tumor-infiltrating T cells.

L’instabilité des séquences répétées microsatellites du génome (courtes répétitions en tandem d’un à cinq nucléotides) est une conséquence de l’inactivation du système MMR (MisMatch Repair), en charge de la réparation des erreurs produites au cours de la réplication de l’ADN. Cette instabilité est associée à un processus de transformation cellulaire original, observé chez l’homme dans des pathologies tumorales fréquentes, nommées MSI (pour Microsatellite Instability). Les localisations primaires les plus fréquentes de ces tumeurs sont le côlon, l’endomètre et l’estomac. Elles peuvent avoir une origine héréditaire (prédisposition familiale ; syndrome de Lynch et apparentés), mais sont dans la majorité des cas de survenue sporadique. La transformation des cellules MMR-déficientes s’observe dans le contexte de l’accumulation de nombreuses mutations somatiques dans l’ADN tumoral. Certaines ont un caractère oncogénique en favorisant la troncature et la perte de fonction de gènes suppresseurs de tumeur ou apparentés, impliqués dans des voies de signalisations diverses et qui contiennent des microsatellites codants (mutations indels d’une à deux paires de base, décalant le cadre de lecture, fréquemment rapportées dans ces tumeurs). Les travaux présentés dans le cadre de mon doctorat visent à mieux comprendre le rôle de l’instabilité microsatellitaire dans la tumorigenèse MSI. Ils s’inscrivent dans le contexte du décryptage et de l’analyse des données de séquençage d’exome de 47 cancers colorectaux primitifs MSI. Dans le contexte d’un niveau élevé d’instabilité génomique caractérisant ces tumeurs, la mise au point par mon laboratoire d’accueil de modèles probabilistes a permis de dresser une liste restreinte de gènes, remarquables par le fait qu’ils sont affectés par des mutations somatiques dont les fréquences sont exceptionnellement élevées ou basses dans l’ADN tumoral. Sous l’hypothèse que de tels évènements somatiques affectent des gènes clés de la tumorigenèse MSI colique, j’ai focalisé mes recherches sur les gènes dont les altérations sont peu fréquentes. Brièvement, j’ai pu démontrer le caractère délétère d’un petit nombre d’altérations microsatellitaires codantes dont la survenue semble soumise à une pression de sélection négative (N=13). Mes résultats indiquent que ces mutations semblent fragiliser le phénotype tumoral des cellules dans lesquelles elles surviennent, la perte de fonction des gènes qu’elles affectent conduisant à diverses conséquences délétères en fonction du gène candidat (e.g. sensibilisation à la mort cellulaire, perte des capacités proliférative et migratoire, ralentissement de la croissance tumorale). Ces résultats rapportent pour la première fois et à grande échelle, la sélection négative de mutations dans des tumeurs à forte instabilité génomique MSI. Ils ouvrent de nouvelles voies pour la compréhension de ce mode particulier de transformation cellulaire, et sont potentiellement d’intérêt pour la mise au point de thérapies personnalisées pour les patients. / Since the discovery of a link between mismatch repair (MMR) deficiency and cancer, microsatellite instability (MSI) is thought as a process underlying cell transformation and tumour progression and invasion. MSI tumours are a subset of frequent human neoplasms, both inherited and sporadic, associated with several primary locations (colon, stomach, endometrium…). In MMR-deficient cells, MSI generates hundreds of frameshift mutations in genes (MSI Target Genes, MSI-TGs) containing coding microsatellite sequences (e.g. -1/+1 bp, insertions/deletions, i.e. indels). Some of these mutations affect genes with a role in human carcinogenesis and are thus expected to promote the MSI-driven tumorigenic process. During my PhD, I aimed to decipher the role of MSI in colon tumorigenesis. I exploited exome-sequencing data available in my lab that were generated from the analysis of a series of 47 human MSI primary colorectal cancer (CRC). Through biostatistics analysis and mathematical models that we designed to interpret mutation rates in the context of the high background for instability characterizing MSI in CRC, we identified a few microsatellites containing genes coding mutations that were negatively selected in MSI colon tumours (N=13). Under the hypothesis that these events may have a negative impact in colon tumorigenesis, I demonstrated that the silencing of these MSI target genes (siRNA/shRNA) was deleterious for MSI cancer cells using in vitro and in vivo models (impairment of proliferation and/or migration and/or response to chemotherapy and/or tumour growth) (Jonchère*, Marisa*, Greene* et al., submitted).

Introdução: O câncer gástrico (CG) foi recentemente categorizado em subtipos moleculares, os quais incluem os tumores Epstein-Barr (EBV)-positivo e com instabilidade de microssatélites (MSI). Esta distinção pode nos fornecer informações prognósticas e identificar alvos terapêuticos, tais como o PD-L1. Objetivo: O objetivo desse estudo foi avaliar a presença de EBV, MSI e expressão de PD-L1 no CG e suas associações com características clinicopatológicas e prognósticas. Métodos: Foram avaliados retrospectivamente 287 pacientes com CG submetidos à gastrectomia D2 com intenção curativa entre 2009 e 2016, através da técnica de tissue microarray. As proteínas de reparo do DNA (MLH1, MSH2, MSH6, PMS2) e o PD-L1 foram avaliados por imuno-histoquímica. O EBV foi avaliado por hibridação in situ. Resultados: Identificou-se a presença de EBV e MSI em 10,5% e 27% dos CGs, respectivamente. A maioria dos CGs com MSI apresentou perda simultânea da expressão de MLH1 e PMS2 (60%). O CG EBV-positivo associou-se ao sexo masculino (p=0,032), localização proximal (p < 0,001), tipo indeterminado de Lauren (p < 0,001), histologia pouco diferenciada (p=0,043) e infiltrado inflamatório acentuado (p < 0,001). Os tumores com MSI foram associados à idade mais avançada (p=0,002), gastrectomia subtotal (p=0,004), pN0 (p=0,024) e ao estágio pTNM menos avançado (p=0,020). Observou-se a imunoexpressão de PD-L1 em 8,8% dos casos, com expressão predominante no CG EBV-positivo (p < 0,001). O CG com MSI apresentou melhor sobrevida livre de doença (p=0,006) e sobrevida global (p=0,049) comparado ao EBV-negativo/microssatélite estável (MSS). Na análise multivariada, o status MSI/MSS permaneceu como fator de risco independente associado à recidiva da doença. Conclusão: O CG EBV-positivo apresentou aumento da expressão de PD-L1, enquanto o CG com MSI relacionou-se à melhor sobrevida. Ambos os subgrupos representam entidades distintas de CG e sua identificação é viável por técnicas diagnósticas convencionais. A caracterização destes subtipos de CG possibilita a aplicação de terapias direcionadas e permite ampliar o poder prognóstico dos atuais sistemas de classificação e estadiamento / Introduction: Gastric cancer (GC) has recently been categorized in molecular subtypes, which include Epstein-Barr (EBV)-positive and microsatellite instable (MSI) tumors. This distinction provides prognostic information and identifies therapeutic targets, such as PD-L1. Objective: The aim of this study was to evaluate the presence of EBV, MSI and PD-L1 expression in GC and their associations with clinicopathological characteristics and prognosis. Methods: We retrospectively evaluated 287 patients with GC who underwent D2-gastrectomy with curative intent from 2009 to 2016 through tissue microarray technique. DNA mismatch repair proteins (MLH1, MSH2, MSH6, PMS2) and PD-L1 were assessed by immunohistochemistry. EBV was detected by in situ hybridization. Results: The presence of EBV and MSI was identified in 10.5% and 27% of GCs, respectively. Most MSI GCs showed simultaneous loss of MLH1 and PMS2 expression (60%). EBV positivity was related to male (p=0.032), proximal location (p < 0.001), undetermined Lauren type (p < 0.001), poorly differentiated histology (p=0.043) and intense inflammatory infiltrate (p < 0.001). MSI-tumors were associated with older age (p=0.002), subtotal gastrectomy (p=0.004), pN0 (p=0.024) and more initial pTNM stage (p=0.020). PD-L1 immunoexpression was observed in 8.8% of cases, with predominant expression in EBV-positive GC (p < 0.001). MSI correlated with better disease-free survival (p=0.006) and overall survival (p=0.049) compared to the EBV-negative/microsatellite stable (MSS). In the multivariate analysis, the MSI/MSS status remained as independent risk factor associated with disease recurrence. Conclusion: EBV-positive GCs had increased PD-L1 expression, while MSI GC had better survival outcome. Both subgroups are distinct GC entities and their recognition is feasible by conventional diagnostic techniques. The characterization of these GC subtypes enables the application of targeted therapies and allows to extend the prognostic power of current classification and staging systems

O câncer de mama é a neoplasia com maior mortalidade entre as mulheres. O emprego de agentes alquilantes no tratamento desta neoplasia pode ocasionar o surgimento de instabilidades genômicas. Tais instabilidades podem estar associadas ao desenvolvimento de neoplasias secundárias como, por exemplo, leucemias. A presente tese avaliou a instabilidade de microssatélites em amostras de sangue, sedimento urinário e plasma de pacientes portadoras de carcinoma mamário ao diagnóstico, 3 e 6 meses após o início do tratamento quimioterápico. Também foi avaliada a concentração do DNA plasmático livre como possível marcador tumoral junto aos marcadores séricos CEA e CA15.3, empregados no acompanhamento do câncer de mama. Foram avaliadas as regiões de microssatélites: Tp53-ALU, Tp53.PCR15.1, BAT 40, BAT26, FMR2 e APC. Entre as 40 pacientes incluídas no presente estudo 88,57% apresentaram instabilidade de microssatélites na fração mononuclear do sangue periférico, 85,8% nas amostras de sedimento urinário e 62,5% no DNA plasmático livre. Não houve concordância significativa entre as instabilidades encontradas nos três tipos de amostra. A concentração de DNA plasmático livre das pacientes quando comparada às doadoras sadias apresentou correlação estatisticamente significativa (p>0,0001), e em paciente em regimes neoadjuvantes que responderam objetivamente à quimioterapia (p=0,02) e não houve correlação com os marcadores séricos CEA e CA15.3. / Breast cancer has the major mortality in women among all kind of cancer. The use of alkylating agents at the treatment of this disease is associated with genomic instability. This instability could be associated with the development of secondary cancer, for example, leukemia. The present thesis evaluated microsatellite instability in blood, pellet cells urinary and plasma in patients with breast cancer at diagnosis, 3 and 6 months after the beginning of chemotherapy. There were evaluated also Free Plasmatic DNA concentration as a possible tumoral marker with the serum markers CEA and CA15.3 used in breast cancer follow up. The microsatellites regions assayed were: Tp53-ALU,Tp53.PCR15.1, BAT 40, BAT26, FMR2 e APC. Among the 40 patients included at the present study 88,57% showed microsallite instability in peripheral mononuclear blood cells, 85,8% in urinary pellet cells samples and 62,5% in Free Plasmatic DNA. There werent statistical significant relationship for the instability found at the three kind of samples assayed. The Free Plasmatic DNA concentration of the patients when compared with healthy donors, showed a statistical significant relationship (p<0,0001). And among patients in neoadjuvant chemotherapy regime who reacted positively by treatment (p=0,02). And there werent statistical significant relationship between Free Plasmatic DNA and serum markers CEA and CA15.3.

A instabilidade genômica tem sido amplamente usada para caracterizar células cancerosas. Alterações genéticas em queratose actínica (QA) e carcinoma espinocelular (CEC) foram investigadas pelo método de random amplified polymorphic DNA (RAPD) e análise de microssatélites com o objetivo de encontrar marcadores moleculares para auxiliar o prognóstico e o diagnóstico médico. O DNA foi obtido de pacientes brasileiros cirurgiados e tratados no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, totalizando oito QAs, 24 CECs, e tecidos normais e/ou leucócitos correspondentes. Os microssatélites estudados foram D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280 e D9S287, tendo em vista a detecção de instabilidade genômica representada por perda de heterozigosidade (LOH) e instabilidade de microssatélites (MSI). Os \"primers\" usados para comparar os padrões de RAPD foram OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17 e OPB-19. Foi obtida correlação significativa na progressão de QA (1/8) para CEC (5/22) referente ao microssatélite D6S251. As diferenças nos padrões de DNA obtidos pelo método RAPD comparados aos controles foram maiores em lesões com maior grau de severidade segundo critério histológico. O mesmo padrão RAPD foi observado no controle e no tumor em 27% QA, 24% CEC I, 9% CEC II e 0% CEC III. Estes resultados mostram que o microssatélite D6S251 e o método de RAPD são informativos, podendo ser potenciais candidatos para auxílio no diagnóstico e prognóstico de QA e CEC. / Genomic instability has been widely used to characterize cancer cells. Genetic alterations in human actinic keratosis (AK) and squamous cell carcinomas (SCC) were investigated by the random amplified polymorphic DNA (RAPD) method, and microsatellite analysis. DNA was obtained from Brazilian patients diagnosed and treated in the School of Medicine of University of Sao Paulo out Clinics Hospital. Eight AKs, 24 SCCs, and 4 BCCs, matched to normal skin tissue and/or leukocytes were studied. Microsatellite patterns were obtained with primers specific to amplify D6S251, D6S252, D9S15, D9S50, D9S52, D9S180, D9S196, D9S280, and D9S287, in search of detection Loss of heterozygosity (LOH) and Microsatellite instability (MSI). The RAPD primers were: OPA-2, OPA-7, OPA-13, OPA-17, OPB-8, OPB-13, OPB-17, and OPB-19. A significant correlation was obtained regarding the progress of AK (1/8) to SCC (5/22) detected with the D6S251 microsatellite. DNA fingerprint obtained with RAPD primers were altered in increasing number of samples, according to their histological degree of differentiation. Similar RAPD patterns were observed in tumor and control in 27% AK, 24% SCC I, 9% SCC II, and zero SCC III. These results suggest microsatellite D6S251 and RAPD method to be potential tools in diagnosis and prognosis of AK and SCC.

L’adénocarcinome canalaire du pancréas (ACP) est un problème majeur de santé publique. L’ACP se développe principalement à partir de deux lésions précurseurs : les néoplasies intra-épithéliales pancréatiques et les tumeurs intracanalaires papillaires et mucineuses du pancréas (TIPMP). Les mécanismes moléculaires sous-tendant l’oncogenèse pancréatique sont nombreux. Nous avons étudié le mécanisme de cancérogenèse MSI (MicroSatellite Instability) où il existe une déficience dans le système de réparation des erreurs de réplication de l’ADN ou système MMR (Mismatch Repair). Ce mécanisme de cancérogenèse original est caractérisé par une instabilité génétique de l’ADN affectant les séquences répétées microsatellites du génome. Le phénotype MSI a été décrit dans le syndrome de Lynch (SL), dans lequel il existe une mutation germinale d’un des gènes du système MMR (MLH1, MSH2, MSH6 et PMS2). L’intérêt de l’étude des cancers MSI s’est accru de façon considérable avec le développement des immunothérapies dirigées contre les checkpoints immunitaires (ICK), en particulier PD-1/PD-L1. Nous avons confirmé que la fréquence du phénotype MSI se situe entre 1-2%. Nous avons montré que l’immunohistochimie est la méthode de screening plus adaptée pour l’identification de l’ACP MSI en comparaison avec les techniques de biologie moléculaire. Le phénotype MSI a été plus fréquemment observé dans un contexte de TIPMP. Les cas MSI identifiés présentaient des caractéristiques biologiques évocatrices du SL. Egalement, nos résultats confirment la présence d’un processus de carcinogenèse MSI immunogénique, mais suggèrent des évènements somatiques spécifiques à l’organe d’origine du cancer. Par ailleurs, les ACP MSI étaient caractérisés par un infiltrat inflammatoire riche en lymphocytes cytotoxiques T CD8+ et surexprimaient l’ICK PD-L1 permettant de supposer une probable réponse clinique de l’ACP MSI à l’immunothérapie anti-PD1/PD-L1. / Pancreatic ductal adenocarcinoma (PDAC) is a major health problem in France and around the world. PDAC is developed mainly from two precursor lesions: pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasm (IPMN). There are several molecular mechanisms underlying pancreatic oncogenesis. Particularly, we were interested in the MSI (MicroSatellite Instability) which is due to a defective DNA Mismatch Repair (MMR) system, which normally functions to recognize and repair erroneous insertions, deletions, and mis-incorporation of bases that can arise during DNA replication and recombination. The MSI phenotype was first described in the familial cancer condition known as Lynch syndrome (LS), where the MMR genes MLH1, MSH2, MSH6 or PMS2 harbor germline mutations. Interest in MSI tumors has recently increased after studies have highlighted the concomitant expression of multiple active immune checkpoint (ICK) markers including PD-1 and PD-L1 and the role of the MSI status to predict clinical benefit from immune checkpoint blockade. A Our results indicate that the MSI phenotype occurs in PDAC with a frequency of 1-2%. Our data showed that IHC using antibodies against the four MMR proteins was more sensitive for the assessment of MSI status than PCR-based methods. In addition, we demonstrate for the first time a statistically significant positive association between MSI and IPMNs in PDAC. MSI PDAC, including IPMN, are unlikely to be sporadic since they display molecular features that are usually observed in LS-related neoplasms. Also, our results highlight that an MSI-driven immunogenic pathway to cancer is active in MSI PDACs but suggest that MSI-driven somatic events may be tissue-specific. We observed a stronger lymphocytic tumor infiltration by activated TCD8 cells in MSI PDAC compared to MSS PDAC and found a positive association between PD-L1 expression and MSI status, suggesting that MSI PDAC could be responsive to ICK blockade therapy.

Les cancers MSI (Microsatellite Instable) sont caractérisés par un niveau élevé d'instabilité des séquences répétées de l'ADN, les microsatellites, suite à l'inactivation du système MMR (Mismatch Repair). L'étude de la carcinogenèse MSI a révélé des mutations somatiques (délétion/insertion) de nombreux gènes sur des microsatellites codants, qui décalent le cadre de lecture et engendrent la production de protéines tronquées dont la fonction est le plus souvent perdue. Ces altérations affectent des gènes suppresseurs de tumeurs ou apparentés agissant au niveau de voies de signalisation en rapport avec l'oncogenèse, et sont sélectionnées lorsqu'elles promeuvent le développement tumoral. Ces travaux font état de la découverte de la première mutation d'une chaperonne dans une pathologie tumorale, affectant l'oncogène codant pour HSP110 (Heat Shock Protein) sur un microsatellite non-codant intronique situé dans un site accepteur d'épissage. Cette mutation, conduisant à un saut d'exon, est inéluctable et bi-allélique dans l'ADN tumoral, et entraîne la perte des activités oncogéniques d'HSP110 dans les cellules cancéreuses (effet pro-apoptotique, chimio-sensibilisant, antiprolifératif et de ralentissement de la croissance tumorale). Ces résultats remettent en question les mécanismes de l'oncogenèse MSI et le caractère oncogénique de l'instabilité microsatellitaire. Au niveau physiopathologique et clinique, ils pointent HSP110 comme cible pronostique (facteur prédictif de réponse à la chimiothérapie) et thérapeutique. Je propose une approche de traitement pour les patients avec une tumeur MSI, visant à exacerber le caractère délétère de cette mutation dans les cellules tumorales. / MSI cancers (MicroSatellite Instability) are characterized by widespread instability of DNA repeated sequences, known as microsatellites, due to MMR system (Mismatch Repair) deficiency. Since the detection of this tumor phenotype, most of the oncogenic events reported in these tumors are somatic mutations (1-2 bp insertions or deletions) that affect coding DNA repeats, resulting in frameshifts and inactivation of the corresponding proteins. They accumulate in tumor cells due to positive selection during the MSI-driven tumorigenic process when they promote tumor development by inactivating genes with tumor suppressor-related functions. This work reports the first somatic mutation of a chaperone protein in a cancer so far, i.e. HSP110 (Heat Shock Protein) in MSI colorectal cancer. This mutational event consists in the somatic deletion of the intronic microsatellite, located in the splice acceptor site of HSP110. We demonstrate that it is almost systematic and bi-allelic in these cancers, leading to inactivation of the oncogenic functions of the HSP110 chaperone (pro-apoptotic and anti-proliferative impact leading to chemosensitization of tumor cells and tumor growth decrease). Our findings support an unexpected and paradoxical anticancer impact of the microsatellite instability-driven pathway in mismatch repair-deficient colon cancer. From a pathophysiological and clinical point of view, they highlight HSP110 as a putative relevant prognostic marker (improvement of patients’ response to chemotherapy) and therapeutic target. According to these findings, I propose a therapeutic strategy targeting HSP110 and its mutant for personalized medicine of MSI colon cancer patients.

OBJETIVOS: Caracterizar o perfil de imunoexpressão de MLH1, MSH2, MSH6 e PMS2 em adenocarcinomas gástricos, explorando seu desempenho na identificação de características clínicas e patológicas, bem como sua influência prognóstica isolada e em relação aos demais parâmetros. MÉTODOS: Cento e trinta e três casos de adenocarcinomas gástricos esporádicos localmente avançados (pT2a ou mais) operados no Hospital das Clínicas/FMUSP foram incluídos pela ausência de metástases a distância ao diagnóstico (M0) e caracterizados clínica (idade, sexo e sobrevida) e patologicamente (tamanho, local, tipo de Borrmann, tipo histológico, infiltração vascular, perineural e variáveis de estadiamento locorregional). Amostras de 1,0 mm foram dispostas em micromatrizes teciduais (TMA) para pesquisa imuno-histoquímica das enzimas MLH1, MSH2, MSH6 e PMS2, com detecção por sistema de polímeros curtos conjugados a peroxidase. Casos com resultados negativos ou incertos nas amostras de TMA tiveram repetidas as reações em secções convencionais. A associação entre o estado de expressão dos marcadores com variáveis clínicopatológicas foi avaliada através do teste do qui-quadrado ou exato de Fisher. O impacto dos parâmetros clínico-patológicos e estado de expressão das enzimas na sobrevida foi explorado em modelos uni e multivariados de Cox, com construção de curvas de Kaplan-Meyer. Todas as análises estatísticas foram consideradas significativas ao nível de p<0,05. RESULTADOS: Quarenta e cinco casos (33,6%) exibiram perda de expressão de ao menos uma enzima, sendo frequente a perda de duas (9/45: 20%), três (14/45:31,2%) ou quatro enzimas (7/45:15,5%). Anormalidade mais frequente ocorreu com o MLH1 (26,7%), seguida de MSH6 (23%), PMS2 (21%) e MSH2 (20,8%). Quando avaliadas em conjunto, houve correlação entre o estado de expressão de todos os possíveis pares, com destaque para MLH1/PMS2 (rho=0,467, p<0,001) e MSH2/MSH6 (rho=0,666, p<0,001). Perda de MLH1 associou-se a tipos I/II de Borrmann (p<0,001), fenótipo intestinal de Lauren (p=0,005), tubular/túbulo-papilífero (p=0,009), expansivo de Ming (p=0,027) e infiltração em muscular própria (p=0,011). Com relação a perda de MSH2, tipos I/II de Borrmann (p<0,001), padrões tubular/túbulo-papilífero (p=0,008), intestinal (p=0,001), expansivo (p=0,001), infiltração da muscular própria (p=0,025), reação desmoplásica ausente a discreta (p=0,021) e ausência de infiltração perineural (p=0,016) foram mais frequentes. Perda de MSH6 associou-se aos tipos macroscópicos de Borrmann e histológicos de Lauren, OMS e Ming (p<0,001) e ausência de infiltração perineural (p=0,036). Idade mais avançada (p=0,046), tipos I/II de Borrmann (p=0,002), padrão intestinal de Lauren (p=0,021) e menos frequente infiltração perineural (p=0,035) foram identificados nos casos com perda de PMS2. A co-negatividade para os pares MLH1/PMS2 e MSH2/MSH6, além de reproduzir as associações mencionadas, identificou infiltrado linfoplasmocitário intra/peritumoral acentuado (p=0,011 e p=0,013), reação estromal desmoplásica ausente a leve para MSH2/MSH6 (p=0,037) e tamanho maior do tumor primário para MLH1/PMS2 (p=0,021). Pior sobrevida associou-se ao sexo masculino (LogRank: 5,11, p=0,024), tamanho do tumor (3,98, p=0,046), tipos III/IV de Borrmann (4,75, p=0,029), histologia mucinosa/anel-de-sinete da OMS (8,61, p=0,003) e difuso (11,62, p=0,003), infiltração perineural (12,62, p<0,001), metástase linfonodal (23,25, p<0,001) e estadio TNM (35,60, p<0,001) em análises univariadas. Melhor sobrevida associou-se a perda de MLH1, MSH6 e PMS2 isoladamente (5,46, p=0,019; 6,08, p=0,014; 7,46, p=0,006) e dos pares MLH1/PMS2 (7,89, p=0,005) e MSH2/MSH6 (5,29, p=0,021). Em modelos multivariados compostos pelos parâmetros clínicopatológicos, apenas o sexo masculino (HR=2,42, p=0,047), tipo histológico difuso (4,94, p=0,037) e estadios II, IIIA e IV (2,23, p=0,088; 3,12, p=0,022; 33,24, p=0,005), constituíram variáveis independentes de determinação prognóstica. Nas análises multivariadas incluindo o estado de expressão das enzimas, evidenciou-se que as perdas de PMS2 e do par MLH1/PMS2 associaram-se significativamente a maior sobrevida (3,84, p=0,029 e 9,82, p=0,028).CONCLUSÕES: O presente estudo demonstra o valor da imunohistoquímica na identificação de alterações na expressão de enzimas MMR, sendo a mais frequentemente negativa a MLH1. A frequente co-negatividade aponta para a importância da dimerização na funcionalidade do sistema de reparo. A perda isolada destes marcadores, e especialmente do par MLH1/PMS2, define perfil clínico-patológico característico, permitindo avanços no conhecimento previamente atribuído a fenótipo microssatélite instável conforme determinado em métodos moleculares. Em análises multivariadas, o estado de expressão de PMS2 isoladamente ou do par MLH1/PMS2 constitui fator independente de determinação prognóstica / OBJECTIVES: To characterize immunoexpression profile of MLH1, MSH2, MSH6 e PMS2 in gastric adenocarcinomas, exploring its performance to identify distinctive clinico-pathological features, as well as their prognostic implications in univariate and multivariate analyses. METHODS: A hundred and thirty three cases of locally advanced (pT2a or higher) sporadic gastric adenocarcinomas operated on Hospital das Clínicas/FMUSP were included due to absence of distant metastases at diagnosis (M0). Clinical (age, gender and survival) and pathological features (size, local, Borrmann´s type, histological type, vascular and perineural infiltration and locorregional staging parameters) were characterized. One millimeter samples were placed on tissue microarray blocks (TMA) and immunohistochemical detection of MLH1, MSH2, MSH6 and PMS2 performed on obtained sections, with revelation developed by peroxidase conjugated short-polymer based reagents. Negative or equivocal results obtained with TMA samples were repeated on conventional sections. Association between expression status for these markers and clinico-pathological features were evaluated by chisquare or Fisher´s exact test when appropriate. Impact of clinico-pathological features and expression status on disease specific survival were explored by Cox uni and multivariate models, with Kaplan-Meyer curves being fitted to illustrate these. All statistical results were considered significant at p<0,05. RESULTS: Forty five cases (33.6%) showed loss of at least one mismatchrepair enzyme, being frequent loss of two (9/45: 20%), three (14/45:31.2%) or even the four evaluated enzymes (7/45:15.5%). The most frequent abnormality addressed MLH1 (26.7%), followed by MSH6 (23%), PMS2 (21%) and MSH2 (20.8%). When analyses were performed in conjunction, correlation was identified for the expression status of all possible pairs, mainly the functional heterodimers MLH1/PMS2 (rho=0.467, p<0.001) and MSH2/MSH6 (rho=0.666, p<0.001). MLH1 loss was associated to Borrmann´s types I/I (p<0.001), Lauren´s intestinal phenotype (p=0.005), tubular/tubulo-papillary architecture (p=0.009), Ming´s expansile type (p=0.027) and infiltration limited by muscular propria (p=0.011). Among cases showing MSH2 loss, Borrmann´s I/I (p<0.001), tubular/tubulo-papillary (p=0.008), intestinal (p=0.001), expansile (p=0.001), muscular propria infiltration (p=0.025), absent to mild stromal desmoplasia (p=0.021) and absent perineural infiltration (p=0.016) were more frequent. MSH6 loss was associated to Borrmann´s gross type and Lauren, WHO and Ming´s histological types (p<0.001), as well as absent perineural infiltration (p=0.036). More advanced age (p=0.046), Borrmann´s types I/I (p=0.002), Lauren´s intestinal morphology (p=0.021) and less frequent perineural infiltration (p=0.035) were identified as associated to PMS2 loss. Conegativity for MLH1/PMS2 and MSH2/MSH6 pairs resumed all the above mentioned associations and additionally identified heavy lymphoplasmacytic infiltrate (p=0.011 e p=0.013), absent to mild stromal desmoplasia for MSH2/MSH6 (p=0.037) and increased primary tumor size for MLH1/PMS2 (p=0.021). In univariate analyses, decreased disease-specific survival was associated to male gender (LogRank: 5.11, p=0.024), tumor size (3.98, p=0.046), Borrmann´s types III/IV (4.75, p=0.029), mucinous/signet-ring cell morphology according to WHO (8.61, p=0.003) and Lauren´s diffuse morphology (11.62, p=0.003), perineural infiltration (12.62, p<0.001), lymph node metastases (23.25, p<0.001) and TNM staging (35.60, p<0.001). Better survival was seen in cases showing loss of MLH1, MSH6 and PMS2 when individually analyzed (5.46, p=0.019; 6.08, p=0.014; 7.46, p=0.006), as well as MLH1/PMS2 (7.89, p=0.005) and MSH2/MSH6 heterodimeric pairs (5.29, p=0.021). In multivariate models addressing clinico-pathological features, only male gender (HR=2.42, p=0.047), diffuse histological type (4.94, p=0.037) and stages II, IIIA and IV (2.23, p=0.088; 3.12, p=0.022; 33.24, p=0.005, respectively) were independent prognostic features. Multivariate analyses including status of MMR enzymes and the most significant clinicopathological features disclosed that PMS2 and MLH1/PMS2 losses were independent predictors of increased disease-specific survival (3.84, p=0.029 e 9.82, p=0.028). CONCLUSIONS: The present study demonstrates immunohistochemical detection of mismatch-repair enzymes as a tool to identify losses of these markers, being the most frequently negative MLH1. The frequently observed co-negativity points toward the importance of heterodimerization of these proteins in functional activity of mismatch-repair system. Losses of these markers, mainly MLH1/PMS2 pair, define a distinctive clinico-pathological profile and add knowledge to the previously reported associations with microsatellite instability as defined by molecular approach. In multivariate analyses, expression status of PMS2, as well as MLH1/PMS2 pair, revealed independent prognostic impact on diseasespecific survival