No description available.

Introduction/Background: Renal sarcoma constitute about 0.8-2.7% of all primary malignant renal tumor [1]. Of all the different types, Renal Leiomyosarcoma is the most common type (50-60%) which originates from the smooth muscle fibers of the renal pelvis, renal capsule or renal vessel [2]. Case Report: 65-year-old smoker male was seen in the clinic with for progressive right upper quadrant and right flank abdominal from the last 4-5 months, worse with eating (Spicy food) and associated with 30 pounds unintentional weight loss in the last 6 months. Patient denied nausea, vomiting, changes in bowel habits, melena, hematochezia and hematuria. There was an ill-defined firmness in the right upper quadrant on examination and laboratory work up was negative. Patient had an abdominal ultrasound as initial work up to rule out gallbladder pathology that showed right lower quadrant mass attached to the right kidney 13.5x11.9x8.9 cm, later confirmed on Computed Tomography (CT) of abdomen that revealed 14.7 x 10.5 cm fungating multilobular mass attached to the inferior pole of right kidney with extracapsular extension and retroperitoneal lymphadenopathy. Further, metastatic work include CT scan of the chest showed multiple small bilateral pulmonary nodules suggestive of metastatic disease. There was an increase in size of renal mass with SUV 10.8 and no bone metastasis was seen during follow up on positron emission tomography scan. CT guided needle core biopsy of renal mass showed spindle cells arranged in fascicles, increased nuclear polymorphisms and mitotic rates which were positive for desmin, smooth muscles myosin/actin immunostains and negative for CD 34, CD117 and HMB-45. Patient had an elective right radical nephrectomy and resection of leiomyosarcoma. Discussion: Primary renal leiomyosarcoma is very rare, more common in females age 50-60 and involves right kidney [3]. The clinical presentation is similar to renal cell carcinoma include abdominal pain or mass, and hematuria. The most common metastatic sites include liver, lungs, bone, and soft tissue. CT scan can be helpful in diagnosis but cannot differentiate from renal cell carcinoma. Biopsy with histopathology and immunohistochemistry is required to confirm the diagnosis. Management includes surgical approach, radiation and chemotherapy. Radical Nephrectomy is the treatment of choice. Most favorable prognostic factors includes tumor less than 4 cm, low grade, absence of nodal metastasis, and radical surgical treatment. Conclusion: Leiomyosarcoma is a rare aggressive tumor of kidney with higher chances of recurrence and metastasis. Only few case reports have been published so far. Reference: 1: Vogelzang NJ, Fremgen AM, Guinan PD, et al. Primary renal sarcoma in adults: A natural history and management study by the American Cancer Society, Illinois division. Cancer 1993; 71:804-10. 10.1002/1097-0142(19930201)71:33.0.CO; 2-A 2: Kavantzas N, Pavlopoulos PM, Karaitianos I, et al. Renal leiomyosarcoma: Report of three cases and review of the literature. Arch Ital Urol Androl 1999; 71:307-11 3: J. S. Miller, M. Zhou, F. Brimo, C. C. Guo, and J. I. Epstein, “Primary leiomyosarcoma of the kidney: a clinicopathologic study of 27 cases,” The American Journal of Surgical Pathology , vol. 34, no. 2, pp. 238–242, 2010

Les léiomyosarcomes sont des tumeurs malignes mésenchymateuses composées de cellules à différentiation musculaire lisse. Elles présentent des altérations génétiques complexes avec pertes et gains de chromosomes variables selon les tumeurs, sans anomalies moléculaires récurrentes permettant de définir une entité nosologique contrairement aux sarcomes associés à des translocations (sarcome d’Ewing), ou à des mutations (GIST). Les évènements moléculaires pilotant la tumeur restent inconnus et donc aucune thérapie ciblée n’est encore identifiée. L’objectif de ce travail était d’identifier par protéomique de nouvelles cibles potentielles dans les LMS. Nous avons identifié des kinases activées en analysant le profil d’expression et de phosphorylation des protéines de signalisation, sur une série de 13 tumeurs congelées. Parmi celles-ci, une surexpression de Tyro-3, PKCq, et MSH2 et une perte de phosphorylation de FAK Y397 ont été détectées dans les tumeurs comparées au tissu sain. Une classification hiérarchique non supervisée a montré deux groupes de LMS ayant des profils d’expression protéique distincts. Nous nous sommes intéressés à Tyro-3 dont la co-expression avec son ligand Gas6 a été exclusivement associée à la phosphorylation d’Akt dans 8 des 13 LMS. Ces corrélations ont été retrouvées dans les deux lignées SK-LMS-1 et CNIO AA. La déphosphorylation de FAK Y397 a été observée uniquement dans CNIO AA qui exprime fortement Gas6. L’ajout de Gas6 exogène à SK-LMS-1 induit la phospho-Akt, et la déphosphorylation de FAK Y397. La double extinction de l’expression des gènes de Tyro-3 et Axl dans CNIO AA réduit la viabilité des cellules, suggérant un rôle crucial de cette voie dans les LMS / Soft tissue and visceral leiomyosarcoma account for 15% of all sarcomas in adults. Molecular alterations in LMS are not well characterized, with often complex gains and losses of chromosome segments. We investigated the expression or phosphorylation of kinases and downstream signalling molecules in a series of fresh frozen LMS and cell lines with the aim to identify potential targets for targeted therapy. Four proteins were found differentially expressed including Tyro3, a receptor tyrosine kinase. The functional activity of Tyro3 was investigated in 2 LMS cell lines, SK-LMS-1 and CNIO AA. Four proteins and phosphoproteins were found differentially expressed in LMS samples as compared to NSM: an hypophosphorylation of FAK Y397 was observed in all samples while Tyro3, MSH2, and PKC theta were found overexpressed in LMS samples. Gas6, the ligand of Tyro3 was found expressed in 8 of the 13 samples, and the coexpression of Gas6 and Tyro3 was found exclusively associated with Akt phosphorylation. Both SK-LMS-1 and CNIO AA LMS cell lines were found to express Tyro3, while Gas6 expression was only observed in CNIO AA cells. P-Akt was expressed spontaneously in CNIO AA, but not in SKLMS-1, while the opposite figure was observed for phosphorylated FAK Y397. Exposure of SKLMS-1 to exogenous Gas6 induced P-Akt, and resulted in a reduction of FAK Y397 phosphorylation. Transfection of CNIO AA with siRNA directed against, Tyro3 and Axl genes induced a reduction of the expression of the specific proteins, and when combined, significantly reduced CNIO AA cell viability. Gas 6 a ligand of Tyro3 is expressed in a subset of LMS tumors and cell lines, and may exert autocrine activities in a subset of LMS

O objetivo deste estudo foi comparar leiomiomas e leiomiossarcomas de boca por meio de análise histológica e imunoistoquímica, utilizando os marcadores Ki67, p53 e PTEN. A actina de músculo liso foi utilizada para confirmar o diagnóstico. Foram examinados 13 tumores de músculo liso disponíveis nos arquivos do Departamento de Estomatologia da Faculdade de Odontologia da USP, sendo 7 leiomiossarcomas, 4 angioleiomiomas e 2 leiomiomas sólidos. As lâminas foram avaliadas de acordo com o grau de atipia, índice mitótico e presença de necrose. Foi realizada a análise imunoistoquímica para o ki67, p53 e PTEN, por meio da contagem de células positivas em 500 células nas áreas mais representativas. A maioria dos angioleiomiomas não apresentou atipia, mitose ou necrose; atipia discreta foi encontrada nos dois leiomiomas sólidos, um deles com 1 mitose por 10 CGA. Atipia foi observada em todos os leiomiossarcomas, o índice mitótico variou de 0 a 6 mitoses por 10 CGA, e apenas um caso apresentou necrose. Houve positividade para o ki67 em apenas um angioleiomioma e em 5 leiomiossarcomas, o restante dos casos foram negativos. Em relação ao p53, ocorreu leve positividade para um caso de leiomioma sólido e moderada para o outro; a maioria dos angioleiomiomas foram levemente positivos e apenas um foi negativo; todos os leiomiossarcomas foram positivos, 2 com marcação leve, 4 moderada e um intensa. A expressão do PTEN foi negativa em um leiomioma sólido e intensamente positiva em outro; todos os casos de angioleiomioma foram positivos, sendo que 2 demonstraram positividade leve e 2 moderada; 3 leiomiossarcomas apresentaram positividade moderada e 3 intensa, apenas um foi negativo. Nossos resultados sugerem que a marcação para ki67 e p53 são úteis na diferenciação entre leiomiomas e leiomiossarcomas. O mesmo não foi observado para o anticorpo PTEN. / This study aimed to compare oral leiomyomas and leiomyosarcomas by means of histological and immunohistochemical analysis. Cases from the Stomatology Department, School of Dentistry at University of São Paulo were retrieved. For immunohistochemistry Ki67, p53 and PTEN markers were used. Smooth muscle actin was used to confirm the diagnosis. There were 13 smooth muscle tumors: 7 leiomyosarcomas, 4 angioleiomyomas and 2 solid leiomyomas. For morphological analysis, sections were evaluated for atypia, mitotic index and presence of necrosis. Immunohistochemical analysis of Ki67, p53 and PTEN expression was performed by counting 500 positive cells in the most representative areas. Most angioleiomyomas did not present atypia, mitosis or necrosis; mild atypia was found in two solid leiomyomas, one with 1 mitosis per 10 HPF. Atypia was found in all leiomyosarcomas, the mitotic index varied from 0 to 6 mitosis per 10 HPF, and necrosis was found in only one case. Ki67 expression was positive in one angioleiomyoma and 5 leiomyosarcomas, the remaining cases were negative. For p53, one solid leiomyoma was mildly positive and the other showed moderate positivity; most of angioleiomyomas were mildly positive and only one was negative; all leiomyosarcomas were positive, 2 with mild expression, 4 moderate and one intense. PTEN expression was negative in one solid leiomyoma and intensely positive in the other; all angioleiomyomas were positive, with 2 mildly positive and 2 moderately positive; 3 leiomyosarcomas presented moderate positivity and 3 intense, only one was negative. Our results suggest that ki67 and p53 are useful in the differentiation between leiomyoma and leiomyosarcoma. The same was not found for PTEN.

O objetivo deste estudo foi comparar leiomiomas e leiomiossarcomas de boca por meio de análise histológica e imunoistoquímica, utilizando os marcadores Ki67, p53 e PTEN. A actina de músculo liso foi utilizada para confirmar o diagnóstico. Foram examinados 13 tumores de músculo liso disponíveis nos arquivos do Departamento de Estomatologia da Faculdade de Odontologia da USP, sendo 7 leiomiossarcomas, 4 angioleiomiomas e 2 leiomiomas sólidos. As lâminas foram avaliadas de acordo com o grau de atipia, índice mitótico e presença de necrose. Foi realizada a análise imunoistoquímica para o ki67, p53 e PTEN, por meio da contagem de células positivas em 500 células nas áreas mais representativas. A maioria dos angioleiomiomas não apresentou atipia, mitose ou necrose; atipia discreta foi encontrada nos dois leiomiomas sólidos, um deles com 1 mitose por 10 CGA. Atipia foi observada em todos os leiomiossarcomas, o índice mitótico variou de 0 a 6 mitoses por 10 CGA, e apenas um caso apresentou necrose. Houve positividade para o ki67 em apenas um angioleiomioma e em 5 leiomiossarcomas, o restante dos casos foram negativos. Em relação ao p53, ocorreu leve positividade para um caso de leiomioma sólido e moderada para o outro; a maioria dos angioleiomiomas foram levemente positivos e apenas um foi negativo; todos os leiomiossarcomas foram positivos, 2 com marcação leve, 4 moderada e um intensa. A expressão do PTEN foi negativa em um leiomioma sólido e intensamente positiva em outro; todos os casos de angioleiomioma foram positivos, sendo que 2 demonstraram positividade leve e 2 moderada; 3 leiomiossarcomas apresentaram positividade moderada e 3 intensa, apenas um foi negativo. Nossos resultados sugerem que a marcação para ki67 e p53 são úteis na diferenciação entre leiomiomas e leiomiossarcomas. O mesmo não foi observado para o anticorpo PTEN. / This study aimed to compare oral leiomyomas and leiomyosarcomas by means of histological and immunohistochemical analysis. Cases from the Stomatology Department, School of Dentistry at University of São Paulo were retrieved. For immunohistochemistry Ki67, p53 and PTEN markers were used. Smooth muscle actin was used to confirm the diagnosis. There were 13 smooth muscle tumors: 7 leiomyosarcomas, 4 angioleiomyomas and 2 solid leiomyomas. For morphological analysis, sections were evaluated for atypia, mitotic index and presence of necrosis. Immunohistochemical analysis of Ki67, p53 and PTEN expression was performed by counting 500 positive cells in the most representative areas. Most angioleiomyomas did not present atypia, mitosis or necrosis; mild atypia was found in two solid leiomyomas, one with 1 mitosis per 10 HPF. Atypia was found in all leiomyosarcomas, the mitotic index varied from 0 to 6 mitosis per 10 HPF, and necrosis was found in only one case. Ki67 expression was positive in one angioleiomyoma and 5 leiomyosarcomas, the remaining cases were negative. For p53, one solid leiomyoma was mildly positive and the other showed moderate positivity; most of angioleiomyomas were mildly positive and only one was negative; all leiomyosarcomas were positive, 2 with mild expression, 4 moderate and one intense. PTEN expression was negative in one solid leiomyoma and intensely positive in the other; all angioleiomyomas were positive, with 2 mildly positive and 2 moderately positive; 3 leiomyosarcomas presented moderate positivity and 3 intense, only one was negative. Our results suggest that ki67 and p53 are useful in the differentiation between leiomyoma and leiomyosarcoma. The same was not found for PTEN.

Yes / Background: Soft tissue sarcomas are heterogeneous and a major complication in their management is that the existing classification scheme is not definitive and is still evolving. Leiomyosarcomas, a major histologic category of soft tissue sarcomas, are malignant tumours displaying smooth muscle differentiation. Although defined as a single group, they exhibit a wide range of clinical behaviour. We aimed to carry out molecular classification to identify new molecular subgroups with clinical relevance. Methods: We used gene expression profiling on 20 extra-uterine leiomyosarcomas and cross-study analyses for molecular classification of leiomyosarcomas. Clinical significance of the subgroupings was investigated. Results: We have identified two distinct molecular subgroups of leiomyosarcomas. One group was characterised by high expression of 26 genes that included many genes from the sub-classification gene cluster proposed by Nielsen et al. These sub-classification genes include genes that have importance structurally, as well as in cell signalling. Notably, we found a statistically significant association of the subgroupings with tumour grade. Further refinement led to a group of 15 genes that could recapitulate the tumour subgroupings in our data set and in a second independent sarcoma set. Remarkably, cross-study analyses suggested that these molecular subgroups could be found in four independent data sets, providing strong support for their existence. Conclusions: Our study strongly supported the existence of distinct leiomyosarcoma molecular subgroups, which have clinical association with tumour grade. Our findings will aid in advancing the classification of leiomyosarcomas and lead to more individualised and better management of the disease. / Alexander Boag Sarcoma Fund.

Os tumores de musculatura liso do útero se desenvolvem a partir do miométrio e podem apresentar carcterísticas clínicas malignas e benignas. Dentre eles, o leiomiossarcoma (LMS) é o tumor maligno mais comum, com altas taxas de metástase e recidiva, mesmo sendo diagnosticado em estágios iniciais. Já os leiomiomas (LM) são os tumores benignos mais frequentes em mulheres em idade reprodutiva. Ambos possuem mesma diferenciação celular, porém com comportamentos clínico e biológico bastante distintos, e até o momento não se dispõe de tratamento específico ou curativo. Nesse contexto, a busca por novos alvos moleculares pode contribuir não só para um melhor entendimento dessas neoplasias, como também para a descoberta de novas terapias. Em estudo prévio foi observada a expressão aumentada de FOXO3a nos sarcomas uterinos, em comparação aos LMs e ao miométrio adjacente (MM). Além disso, sua expressão foi crescente de acordo com o potencial de malignidade do tumor. Assim, o objetivo do presente estudo foi avaliar in vitro o efeito de terapia alvo específica para FOXO3a em células de LM e LMS. Para isto, linhagens celulares de MM (ATCC PCS-460-011), LM (THESCs - CRL-4003) e LMS (SK-UT-1 - HTB-114) foram caracterizadas quanto à expressão basal de FOXO3a (gene e proteína) e submetidas a tratamento com Genisteína e Metformina ou inativação do gene por siRNA. Os efeitos dos tratamentos foram avaliados por PCR em tempo real, Western Blot, imunocitoquímica, ensaios de proliferação, migração e apoptose. Nossos resultados mostraram que todos os tratamentos realizados interferiram na capacidade de proliferação e migração das células, com maior inibição após as 48 horas nos LMS e 72h nos LM. O efeito obtido na transfecção com siRNA apresentou maior eficiência após 48 h da transfecção nos LMS e 72h nos LM. Os efeitos da inibição de FOXO3a foram maiores na proliferação e migração dos LM, porém os resultados não foram estatisticamente significativos. Dentre as substâncias testadas, a Metformina apresentou maior efeito sobre a proliferação, migração e viabilidade das linhagens celulares. A Genisteína também apresentou efeito inibitório nas células, porém o controle com veículo também apresentou o mesmo efeito citotóxico. De modo geral, os efeitos obtidos com os fármacos, foram tempo e concentração dependentes. Em conjunto, nossos resultados sugerem um relevante do FOXO3a nos tumores de musculatura lisa uterinos, além de apresentá-lo como potencial alvo para terapia específica / Smooth muscle tumors of the uterus develop from the myometrium and may present benign and malignant clinical features. Among them, leiomyosarcoma (LMS) is the most frequent malignant tumor, with high rates of metastasis and relapse, even when diagnosed in early stages. On the other hand, leiomyomas (LM) are the most frequent benign tumors in women of reproductive age. Both have the same cellular differentiation, but with very different clinical and biological behaviors, and so far no specific or curative treatment is available. In this context, the search for new molecular targets can contribute not only for a better understanding of these neoplasms, but also for the discovery of new therapies. In a previous study, increased expression of FOXO3a in uterine sarcomas was observed, compared to LMs and adjacent myometrium (MM). In addition, its expression was increasing according to the malignancy potential of the tumor. Thus, the aim of the present study was to evaluate in vitro, the effect of specific targeted therapy for FOXO3a on LM and LMS cells. For this, MM (ATCC PCS-460-011), LM (THESCs-CRL-4003) and LMS (SK-UT-1-HTB-114) cell lines were characterized for basal expression of FOXO3a (gene and protein) and subsequently submitted to treatment with metformin and genistein, or silencing of FOXO3a by siRNA. The effects of the treatments were evaluated by real-time PCR, Western Blot, immunocytochemistry, proliferation, migration and apoptosis assays. Our results showed that all treatments interfered in the proliferation and migration capacity of the cells, with greater inhibition after 48 hours for LMS and 72 hours LM. The effect obtained in the transfection with siRNA showed higher efficiency after 48 hours of transfection in LMS and 72 hours in LM. The effects of inhibition of FOXO3a were greater in the proliferation and migration of the LM, but the results were not statistically significant. Among the substances tested, Metformin had a greater effect on proliferation, migration and viability of the cell lines. Genistein also had an inhibitory effect on the cells, but the control with the vehicle also presented the same cytotoxic effect. In general, the effects obtained with the drugs were time and concentration dependent. Together, our results suggest a relevant role of FOXO3a in uterine smooth muscle tumors, in addition to presenting it as a potential target for specific therapy

Os tumores de musculatura liso do útero se desenvolvem a partir do miométrio e podem apresentar carcterísticas clínicas malignas e benignas. Dentre eles, o leiomiossarcoma (LMS) é o tumor maligno mais comum, com altas taxas de metástase e recidiva, mesmo sendo diagnosticado em estágios iniciais. Já os leiomiomas (LM) são os tumores benignos mais frequentes em mulheres em idade reprodutiva. Ambos possuem mesma diferenciação celular, porém com comportamentos clínico e biológico bastante distintos, e até o momento não se dispõe de tratamento específico ou curativo. Nesse contexto, a busca por novos alvos moleculares pode contribuir não só para um melhor entendimento dessas neoplasias, como também para a descoberta de novas terapias. Em estudo prévio foi observada a expressão aumentada de FOXO3a nos sarcomas uterinos, em comparação aos LMs e ao miométrio adjacente (MM). Além disso, sua expressão foi crescente de acordo com o potencial de malignidade do tumor. Assim, o objetivo do presente estudo foi avaliar in vitro o efeito de terapia alvo específica para FOXO3a em células de LM e LMS. Para isto, linhagens celulares de MM (ATCC PCS-460-011), LM (THESCs - CRL-4003) e LMS (SK-UT-1 - HTB-114) foram caracterizadas quanto à expressão basal de FOXO3a (gene e proteína) e submetidas a tratamento com Genisteína e Metformina ou inativação do gene por siRNA. Os efeitos dos tratamentos foram avaliados por PCR em tempo real, Western Blot, imunocitoquímica, ensaios de proliferação, migração e apoptose. Nossos resultados mostraram que todos os tratamentos realizados interferiram na capacidade de proliferação e migração das células, com maior inibição após as 48 horas nos LMS e 72h nos LM. O efeito obtido na transfecção com siRNA apresentou maior eficiência após 48 h da transfecção nos LMS e 72h nos LM. Os efeitos da inibição de FOXO3a foram maiores na proliferação e migração dos LM, porém os resultados não foram estatisticamente significativos. Dentre as substâncias testadas, a Metformina apresentou maior efeito sobre a proliferação, migração e viabilidade das linhagens celulares. A Genisteína também apresentou efeito inibitório nas células, porém o controle com veículo também apresentou o mesmo efeito citotóxico. De modo geral, os efeitos obtidos com os fármacos, foram tempo e concentração dependentes. Em conjunto, nossos resultados sugerem um relevante do FOXO3a nos tumores de musculatura lisa uterinos, além de apresentá-lo como potencial alvo para terapia específica / Smooth muscle tumors of the uterus develop from the myometrium and may present benign and malignant clinical features. Among them, leiomyosarcoma (LMS) is the most frequent malignant tumor, with high rates of metastasis and relapse, even when diagnosed in early stages. On the other hand, leiomyomas (LM) are the most frequent benign tumors in women of reproductive age. Both have the same cellular differentiation, but with very different clinical and biological behaviors, and so far no specific or curative treatment is available. In this context, the search for new molecular targets can contribute not only for a better understanding of these neoplasms, but also for the discovery of new therapies. In a previous study, increased expression of FOXO3a in uterine sarcomas was observed, compared to LMs and adjacent myometrium (MM). In addition, its expression was increasing according to the malignancy potential of the tumor. Thus, the aim of the present study was to evaluate in vitro, the effect of specific targeted therapy for FOXO3a on LM and LMS cells. For this, MM (ATCC PCS-460-011), LM (THESCs-CRL-4003) and LMS (SK-UT-1-HTB-114) cell lines were characterized for basal expression of FOXO3a (gene and protein) and subsequently submitted to treatment with metformin and genistein, or silencing of FOXO3a by siRNA. The effects of the treatments were evaluated by real-time PCR, Western Blot, immunocytochemistry, proliferation, migration and apoptosis assays. Our results showed that all treatments interfered in the proliferation and migration capacity of the cells, with greater inhibition after 48 hours for LMS and 72 hours LM. The effect obtained in the transfection with siRNA showed higher efficiency after 48 hours of transfection in LMS and 72 hours in LM. The effects of inhibition of FOXO3a were greater in the proliferation and migration of the LM, but the results were not statistically significant. Among the substances tested, Metformin had a greater effect on proliferation, migration and viability of the cell lines. Genistein also had an inhibitory effect on the cells, but the control with the vehicle also presented the same cytotoxic effect. In general, the effects obtained with the drugs were time and concentration dependent. Together, our results suggest a relevant role of FOXO3a in uterine smooth muscle tumors, in addition to presenting it as a potential target for specific therapy

Jusqu'à présent, les modifications de la réplication de l'ADN entre cellules normales et cancéreuses ont été peu étudiées. Dans ce travail, nous avons utilisé le séquençage des fragments d'Okazaki, une technique récemment développée au laboratoire, pour déterminer la directionalité des fourches de réplication dans plusieurs lymphomes de Burkitt (LB), qui surexpriment l'oncoprotéine Myc à la suite de translocations chromosomiques spécifiques, ainsi que dans des lignées lymphoblastoides contrôles (LLC) et dans des léiomyosarcomes (LMS). Les profils de directionalité des fourches de réplication permettent de déduire la localisation et l'efficacité des sites d'initiation et de terminaison de la réplication le long du génome. Nous avons observé de nombreuses (~2000) différences de zones d'initiation entre les lignées Raji (LB) et GM06990 (LLC) ainsi qu'entre les lignées BL 79 et IARC385, une paire LB/LLC provenant d'un même patient. Nous avons détecté un nombre comparable de différences en comparant deux à deux les lignées étudiées. Cependant, les profils de BL79 et de Raji (deux LB) sont un peu plus proches l'un de l'autre que de la lignée contrôle GM06990. Ceci suggère l'existence de changements de la réplication récurrents dans les lignées LB. L'importance des différences observées entre les lignées IARC385 et GM06990 indique de façon surprenante une grande variabilité entre les LLC normales, provenant de différents individus. De façon intéressante, de nombreuses différences observées entre les lignées LB et LLC sont associées à des changements de l'expression des gènes ou de la liaison de l'oncoprotéine Myc. La comparaison des profils des deux LMS avec tous les profils disponibles au laboratoire montre que c'est à celui de fibroblastes normaux (IMR90) qu'ils ressemblent le plus. Ceci suggère que les cellules de tumeurs musculaires lisses auraient subi une transformation fibroblastique au cours de la tumorigénèse. Des données récentes suggèrent que les champs magnétiques peuvent perturber certains processus cellulaires comme l'assemblage du cytosquelette. Nous avons utilisé le séquençage de fragment d'Okazaki pour rechercher d'éventuels effets d'un champ magnétique sur la réplication de l'ADN chez la levure. Aucun effet du champ magnétique sur la directionalité des fourches de réplication n'a été détecté. / Changes in DNA replication profiles between cancer and normal cells have been poorly explored. In this work, sequencing of Okazaki fragments, a novel methodology developed in the laboratory, was used to determine replication fork directionality (RFD) in several Burkitt's lymphomas (BL), which overexpress the Myc oncoprotein due to specific chromosomal translocations, and control normal lymphoblastoid cell lines (LCL), and in leiomyosarcomas (LMC). RFD profiles allow to infer the location and efficiency of replication initiation and termination sites genome-wide. A larger number (~2000) of differences in replication initiation zones were observed genome-wide between Raji (BL) and GM06990 (LCL), and between BL79 and IAR385, a BL / LCL pair of cell lines established from a single patient. Comparably large numbers of changes were slightly more similar to each other than to GM06990. This suggests the occurrence of some recurrent replication changes in BL cell lines. The large number of changes observed between IARC385 and GM06990 also indicates an unexpectedly large variation between normal LCLs of different individuals. Interestingly, many changes in RFD profiles between BLs and and LCLs are associated with cell-type specific gene expression and differential binding of the Myc oncoprotein. Comparison of the two LMS profiles with all RFD profiles available in the laboratory reveals that they most resemble normal fibroblasts (IMR90). This suggests that the smooth muscle cancer cells might have undergone a fibroblastic transformation during tumorigenesis. Magnetic fields have been reported to perturb cellular processes such as cytoskeleton assembly. Sequencing of Okazaki fragments was used in a preliminary investigation of the possible effects of magnetic fields on DNA replication in yeast cells. No effect of magnetic fields on replication fork directionality were observed.

Les léiomyosarcomes (LMS) sont des tumeurs d’origine mésenchymateuse caractérisées par une différenciation musculaire lisse. La voie de signalisation PI3K/mTOR (qui contrôle la prolifération et la survie cellulaire) joue un rôle majeur dans le développement de ces tumeurs. De nos jours, cette voie est devenue une cible thérapeutique majeure en oncologie. Cette étude est la première qui évalue le bénéfice thérapeutique de l’inhibition de la voie PI3K/mTOR pour des patients atteint de LMS. Nous avons mis en évidence qu’une double inhibition de PI3K et mTOR est associée à une activité antitumorale supérieure à celle observée avec une inhibition de PI3K ou mTOR seule. Nous avons également montré que l’inhibition de la voie PI3K/mTOR est associée à une activation paradoxale de la voie MAPK et qu’un ciblage concomitant de cette voie est associé à une synergie antitumorale in vitro et in vivo. Afin de caractériser les mécanismes de résistance secondaire à l’inhibition de la voie PI3K/mTOR, nous avons développé in vitro et in vivo un modèle de résistance secondaire à l’inhibiteur double cible PI3K/mTOR. Nous avons notamment détecté une sous-population de cellules résistantes à l’inhibiteur et ayant des caractéristiques proches de celles des cellules souches. Nous avons mis en évidence que l’inhibition pharmacologique d’EZH2, une protéine cruciale du complexe Polycomb, permet de restaurer la sensibilité des modèles résistants. Ces résultats apportent de nouvelles perspectives thérapeutiques pour les patients atteints de LMS. / Leiomyosarcomas (LMS) are tumors of mesenchymal origin characterized by a smooth cell differentiation. The PI3K/mTOR pathway has been shown to play a crucial role in the tumorigenesis of LMS. Several agents targeting this pathway are under clinical development for the treatment of solid tumors and hematological malignancies. We report here the first study evaluating its potential therapeutic benefit for patients with LMS. We have demonstrated that dual inhibition of PI3K and mTOR is associated with more effective antitumor activity than agents targeting PI3K or mTOR only. We have also shown that PI3K and mTOR inhibition is associated with a paradoxal activation of the MAPK pathway and that combined treatment with MEK inhibitor resulted in synergistic antitumor activity in vitro and in vivo. Moreover, we developed in vitro and in vivo resistant model to dual PI3K/mTOR inhibitor. Interestingly, we have found that a cancer stem cell-like subpopulation may be involved in treatment resistance. We have shown that pharmacological inhibition of EZH2, a crucial protein of the Polycomb complex, is able to reverse dual PI3K/mTOR inhibitor resistance in vitro and in vivo. These results provide new therapeutic strategies for patients with LMS.