INTRODUÇÃO: O papilomavírus humano (HPV) encontra-se amplamente distribuído na população mundial. Apesar da grande maioria das infecções serem transientes, assintomáticas e passíveis de regressão espontânea, a infecção persistente por tipos de alto risco de HPV é necessária para o desenvolvimento de neoplasias intraepiteliais cervicais. Uma vez que apenas uma pequena parcela das infecções progride à lesões malignas após um longo período desde o diagnóstico inicial de lesões precursoras, tem-se iniciado a busca por fatores que possam influenciar na progressão ou na eliminação destas manifestações iniciais. A variabilidade genética viral tem sido apontada como um dos fatores que interagem neste processo. Embora virtualmente todos os tumores da cérvice uterina apresentem o DNA viral, neoplasias em outros sítios anatômicos têm sido apenas em parte correlacionadas com a presença viral, sendo o HPV proposto como um dos agentes causadores de tumores em sítios de cabeça e pecoço. MÉTODOS: Espécimens clínicos de tumores de cabeça e pescoço, fixados em formalina e contidos em parafina (FFPE), provenientes do Instituto do Câncer do Estado de São Paulo (n=79) e da Santa Casa de Misericórida de São Paulo (n=94), tiveram seu DNA extraído, seguido de diagnóstico e genotipagem de HPV pela metodologia de Inno-LiPA. Análises de linhagens moleculares foram realizadas nas amostras HPV-16 positivas. Análise imunohistoquímica de P16INK4a foi realizada em todas as amostras. RESULTADOS: A presença do DNA viral foi encontrada em 24,1% (19/79) dentre a série de tumores provenientes do ICESP, sendo a cavidade oral o sítio em que foi observada a maior proporção de DNA viral (27,1%), enquanto que 13,8% (13/94) dentre os espécimens provenientes da Santa Casa apresentaram-se positivos para HPV, sendo a cavidade oral o sítio em que foi observada a maior proporção do DNA viral (18,1%). O HPV-16 foi o tipo mais prevalente, detectado em 73,4% das amostras HPV positivas provenientes do ICESP e 61,5% das amostras provenientes da Santa Casa. Independente da Instituição, as amostras foram alocadas no clado das linhagens Asiático-Americana e Europeia em 50%, cada uma, entre os 18 tumores HPV-16 positivos em que as análises de linhagem foram possíveis. Não foi observada, nestas séries, correlação entre a superexpressão de P16INK4a e a presença do DNA viral. CONCLUSÃO: Nas amostras analisadas, o DNA de HPV foi detectado em 18,5% dos 173 espécimens. O HPV-16 foi o tipo mais prevalente. Isolados da linhagem Europeia e da linhagem Asiatico-Americano foram detectados em 50% dos casos, cada uma, dentre as amostras HPV-16 analisadas por este estudo / INTRODUCTION: Human papillomaviruses (HPV) are widely distributed worldwide. Although the majority of infections are usually transient, asymptomatic and frequently regress spontaneously, persistent infections by high-risk HPVs are necessary for the development of cervical intraepithelial neoplasia. Once only a small proportion of infections progress to malignant lesions after a long period of time since the initial diagnosis of precursor lesions, the search for factors that might influence the progression or clearence of these early manifestations are currently under way. Viral genetic variability has been proposed as one of the factors interacting in this process. Although virtually all cervix tumors present the viral DNA, neoplasias from other anatomical sites have been only in part correlated with viral presence, and HPV has been proposed as one causative agent in tumors from head and neck sites. METHODS: Clinical specimens of formalin-fixed paraffin embedded head and neck tumors, provided by the Cancer Institute of São Paulo (n=79) and also by the Santa Casa de Misericórdia de São Paulo (n=94), were submitted to DNA extraction and further HPV diagnostic and genotyping by the Inno-LiPA methodology. Molecular lineages analyses were performed in all HPV-16 positive samples. P16INK4a immunohistochemical analyses were conducted in all samples. RESULTS: HPV DNA was detected among 24.1% (19/79) of samples provided by ICESP, tumors from oral cavity presented the highest viral positivity (27.1%), whereas 13,8% (13/94) of the samples from Santa Casa presented HPV DNA, tumors from the oral cavity also presented the highest HPV positivity with 18.1% of viral DNA presence. HPV-16 was the most prevalent type detected in 73.4% and 61.5% of HPV positive ICESP and Santa Casa samples, respectively. Irrespective of the Institution, samples submitted to lineage analyzes were allocated in the Asiatic-American and European phylogenetic branches in 50%, each one, among the 18 tumors HPV-16 positive for which lineage analysis was possible. No correlation between P16INK4a overexpression and HPV DNA presence was observed. CONCLUSION: In this study, HPV DNA was detected in 18.5% among 173 head and neck tumor specimens. HPV-16 was the most prevalent type. The European and the Asiatic-American lineage were detected in 50% of the cases, each one, among the cases HPV-16 positive analyzed

During recent years, significant advances have been made in the field of molecular therapy in urologic oncology, mainly for advanced renal cell carcinoma. In this hitherto largely treatment-refractory disease, several agents have been developed targeting the von Hippel-Lindau metabolic pathway which is involved in carcinogenesis and progression of the majority of renal cell carcinomas. Although cure may not be expected, new drugs, such as the multikinase inhibitors sorafenib and sunitinib and the mammalian target of rapamycine inhibitor temsirolimus, frequently stabilize the disease course and may improve survival. Fewer data are available supporting molecular therapies in prostate, bladder, and testicular cancers. Preliminary data suggest a potential role of high-dose calcitriol and thalidomide in hormone-refractory prostate cancer, whereas targeted therapies in bladder and testicular cancers are still more or less limited to single-case experiences. The great theoretical potential and the multitude of possible targets and drug combinations, however, support further research into this exciting field of medical treatment of urologic malignancies. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Avaliou-se a expressÃo das proteÃnas bax, bcl-2 e P27 no carcinoma espinocelular invasivo da cavidade bucal atravÃs da tÃcnica da imunohistoquÃmica para conhecer-se o perfil apoptÃtico destes tumores. Buscou-se detectar os fatores epidemiolÃgicos e o comportamento clÃnico dos pacientes acometidos por esta neoplasia. Procurou-se empregar parÃmetros estatÃsticos diferentes do odds ratio para comparaÃÃes posteriores. Analisando-se quarenta e oito pacientes da Santa Casa da MisericÃrdia de Fortaleza, Cearà foi possÃvel conhecer os fatores sexo, escolaridade, renda familiar, tabagismo (forma e tempo de consumo,tempo de abandono, quantidade consumida), etilismo(tipo de bebida, tempo de consumo, quantidade consumida, freqÃÃncia). Com a anÃlise do laudo histopatolÃgico da peÃa cirÃrgica soube-se das informaÃÃes referentes ao tumor em si (localizaÃÃo, gradaÃÃo histolÃgica, tamanho, linfonodos envolvidos). Obteve-se fragmentos da peÃas cirÃrgicas e foram confeccionadas lÃminas para a verificaÃÃo da invasividade atravÃs da coloraÃÃo em hematoxilina e eosina. Em seguida foi realizada a reaÃÃo de imunohistoquÃmica foi pelo mÃtodo da estrepto-avidina-biotina-peroxidase objetivando-se avaliar a expressÃo das proteÃnas supracitadas. As lÃminas com marcaÃÃo positiva foram submetidas a contagem de no mÃnimo mil cÃlulas e este resultado foi empregado nos mÃtodos LI (Labelling Index) e HS(HScore). A avaliaÃÃo dos resultados foi atravÃs de mÃtodos descritivos, sendo os testes realizados o qui-quadrado e o teste exato de Fisher com nÃvel de significÃncia a dez por cento.Os resultados apontaram uma mÃdia de idade de cinqÃenta e sete anos e o maior nÃmero de indivÃduos do sexo masculino, analfabetos, com renda familiar de um salÃrio-mÃnimo na sua maioria. A forma de consumo de tabaco, em maior nÃmero encontrada, foi o cigarro industrializado com vinte anos de consumo. A ingestÃo de bebidas destiladas superou a de fermentadas com mÃdia de vinte anos. O soalho bucal foi a localizaÃÃo com maior nÃmero de casos, com uma mÃdia de tamanho de trÃs e meio centÃmetros, o estadiamento patolÃgico predominante foi o pT2, com gradaÃÃo histolÃgica moderadamente diferenciada em maior quantidade. Dentre os cruzamentos realizados, houve correlaÃÃo estatÃstica entre o tamanho de tumores com as faixas etÃrias(P=0,084 para α=10%); tamanho dos tumores com envolvimento de linfonodos(P=0,085 para α=10%); sexo dos pacientes com envolvimento dos linfonodos (P=0,03 para α=10%). Os resultados das reaÃÃes de imunohistoquÃmica mostraram um maior percentual de casos positivos para bax (77,1%) seguido de P27(45,9%) e bcl-2 (16,6%). As mÃdias dos LI encontradas apontaram bax com 67, 766; seguida de bcl-2 com 10,804; e P27 com 7,989. Cruzando-se os H-scores dos marcadores entre si encontrou-se correlaÃÃo positiva entre bax e P27 (0,245 na correlaÃÃo de Pearson). Os parÃmetros estatÃsticos avaliados foram: mÃdia e seu erro padrÃo; mediana; moda; desvio padrÃo; curtose e seu erro padrÃo; mÃnimo; mÃximo e quartis. Os resultados mostram uma tendÃncia a apoptose nos carcinomas espinocelulares bucais / The evaluation of the expression of bax, bcl-2 and p27 proteins at invasive squamous cell carcinoma of oral cavity was done using the immunohistochemistry technique to know about apoptotic profile of these neoplasms. The epidemiological factors and the clinical behavior of the patients were detected, too. Statistical parameters different from odds ratio was employed for future comparisons. Forty-eight patients from Santa Casa da MisericÃrdia de Fortaleza, CearÃ, Brazil was analyzed and with this analysis was possible to know the prognostic factors: sex of patients, instruction grade, familiar gains, tobacco consumption (form of consumption, duration of consumption, period of forsaking and quantity consumed), alcohol consumption (nature of drinking, duration of consumption, period of forsaking, quantity consumed, periodicity). In a posterior moment, with the histopathological report from the surgical specimen was possible to know about localization of neoplasm, size of neoplasm, histological grade, nodes involvement and invasiveness. Pieces of the neoplasm were achieved from surgical specimen and glass slides were done to analyze the invasiveness by hematoxilin-eosin coloration. After the immunohistochemistry reaction by streptoavidin biotin technique was done to evaluate the expression of proteins above. The glass slides with positive reaction were submitted under a counting and, at least, one thousand cells were counted. The results from counting were submitted under the LI(Labelling Index) and HS(H Score) methods. The evaluation of the results was made using descriptive methods and the statistical tests were qui-square and Fisherâs exact test with 10% significance. The results showed the mean age was 57 years old, more males than females, analphabets and one minimum wage the mean familiar gain. The main form of tobacco consumption was industrialized cigarette with 20 years of consumption. The swallow of distillers drinking was bigger than fermentation ones with 20 years of drinking at mean. The floor of the mouth was the anatomic site with more number of cases and the mean size of neoplasm was 1.4 inches. The preponderant pathological staging detected was pT2 and the preponderant histological grade was moderately differentiated. Among the cross tabs realized, there were statistical correlation between size of tumors and age (P=0.084; α=10%), between size of tumors and nodes involvement (P=0,085; α=10%), between sex of patients and nodes involvement (P=0.03; α=10%). The results from immunohistochemical reactions were more positive to bax (77.1% of positive cases), P27(45.9% of positive cases) and bcl-2 (16.6% of positive cases). The mean of LI showed bax at first position (67.766); second bcl-2 (10.804) and P27(7.989). The cross tabs among HS showed statistical positive correlation between bax and P27 (0.245 at Pearsonâs correlation). The statistical parameters were: mean and its standard error, median, mode, standard deviation, kurtosis and its standard error, minimum, maximum and percentiles. The conclusions showed apoptosis propensity at oral squamous cell carcinoma

O presente estudo comparou os efeitos da ativação e inibição do EGFR em duas linhagens de câncer de pulmão, cultivadas em monocamada ou esferoides. Os esferoides foram cultivados sem elementos de matriz extracelular. As células A549 e HK2 apresentaram, respectivamente, 3 e 6 cópias do gene ErbB1 por núcleo, embora a expressão de EGFR seja menor nas células HK2. A ativação de EGFR por EGF ou inibição por AG1478 não promoveu mudanças na proliferação celular. Entretanto, as células cultivadas em monocamada, estimuladas com EGF, exibiram alterações na disposição dos microfilamentos de actina e aumento na velocidade de migração celular. UO126 e LY294002 foram adicionados às culturas para inibir, respectivamente, as vias ERK e Akt. A linhagem A549, cultivada em monocamada, não apresentou envolvimento das vias de sinalização de ERK e Akt na migração celular induzida por EGF, mas foi observado o envolvimento dessas vias nos esferoides. Já a linhagem HK2 apresentou o envolvimento de Akt para promover a migração celular após estímulo com EGF nas duas formas de cultivo. / This study compared the effects of activation and inhibition of EGFR in two cell lines of lung cancer, grown in monolayer or spheroids. Spheroids were cultured without extracellular matrix components. HK2 and A549 cells showed, respectively, 3 and 6 ErbB1 gene copies per nucleus, while EGFR expression is lower in the HK2 cells. The activation by EGF or EGFR inhibition by AG1478 did not cause changes in cell proliferation. However, cells cultured in monolayers stimulated with EGF, showed changes in the arrangement of actin microfilaments and increased the speed of cell migration. UO126 and LY294002 were added to the cultures to inhibit, respectively, the ERK and Akt pathways. A549 cells grown in monolayer did not show involvement of ERK and Akt signaling pathways in the cell migration induced by EGF, but was observed involvement of such pathways in the spheroids. HK2 cells showed involvement of Akt to promote cell migration after EGF stimulation in monolayers and in spheroids.

A deficiência de vitamina D (dVD) aumenta o risco de morte em pacientes hospitalizados. A injúria de isquemia/reperfusão renal (Isq) ativa vias de necrose e/ou apoptose e proliferação celular. A injúria renal aguda (IRA) induz a ativação de inibidores do ciclo celular, incluindo a p21, uma inibidora de kinase dependente de ciclina, a qual possui efeito protetor na IRA. A p21 é um alvo genômico da 25-hidroxivitamina D [25 (OH) D], a qual, atuando através de receptores de vitamina D (VDRs), possui efeitos imunomoduladores potentes e antiproliferativos, sugerindo a participação deste hormônio na fisiopatologia da doença renal. Desta forma, o objetivo deste estudo foi verificar a participação da deficiência de vitamina D no modelo de isquemia/reperfusão renal em ratos. Foram utilizados ratos Wistar que foram divididos em quatro grupos: controle (C), animais que receberam dieta padrão por 30 dias; dVD, animais que receberam dieta livre de vitamina D por 30 dias; Isq, animais que receberam dieta padrão por 30 dias e no 28º dia foram submetidos ao insulto de isquemia/reperfusão em ambos os rins por 45 minutos; e dVD+Isq, animais que receberam dieta livre de vitamina D por 30 dias e no 28º dia foram submetidos ao insulto de isquemia/reperfusão em ambos os rins por 45 minutos. Ao final dos 30 dias e após 48 horas da realização da isquemia/reperfusão, os animais foram submetidos à eutanásia e amostras de sangue, urina e tecido renal foram coletados para o estudo dos mecanismos de lesão renal. A injúria renal aguda associada à deficiência de vitamina D levou a uma queda da filtração glomerular e aumento da proteinúria; aumento da relação peso renal/peso corporal, sugerindo maior proliferação e hipertrofia; induziu uma diminuição na ativação dos receptores de vitamina D e da expressão da proteína p21 e aumento da expressão de caspase-3; observou-se déficit de concentração urinária com diminuição da expressão da AQP2; e um maior dano morfológico caracterizado pela análise da área intersticial e presença de necrose tubular. Nossos dados mostraram que alterando os níveis da p21 na IRA isquêmica, a vitamina D, via VDRs, controla a inflamação renal, a proliferação e a lesão celular / Vitamin D deficiency (VDD) increases the risk of death in hospitalized patients. Ischemia/reperfusion injury activates pathways of necrosis and/or apoptosis and cell proliferation. Acute kidney injury (AKI) induces the activation of cell cycle inhibitors, including p21, an inhibitor of cyclin-dependent kinase, which has a protective effect on the IRA. The p21 is a genomic target of 25-hydroxyvitamin D [ 25 (OH) D], which, acting through vitamin D receptors (VDRs), has potent antiproliferative and immunomodulatory effects, suggesting the involvement of this hormone in the pathophysiology of renal disease. Thus, the aim of this study was to assess the role of vitamin D deficiency in rats submitted to renal ischemia/reperfusion. Wistar rats were divided into four groups: control (C), animals that received a standard diet for 30 days; VDD, animals that received vitamin D-free diet for 30 days; IRI, animals that received standard diet for 30 days and on day 28 were subjected to the ischemia/reperfusion insult (IRI) in both kidneys for 45 minutes; and VDD+IRI, animals that received vitamin D-free diet for 30 days and on day 28 were subjected to the IRI in both kidneys for 45 minutes. At the end of 30 days and 48 hours after the IRI insult, the animals were euthanized and samples of blood, urine and kidney tissue were collected to study the mechanisms of renal injury. Acute kidney injury associated with vitamin D deficiency led to a decrease in glomerular filtration rate and increased proteinuria; increased relative kidney weight/body weight, suggesting greater proliferation and hypertrophy; induced a decrease in the activation of vitamin D receptors and the p21 protein expression and, increased caspase-3 expression; renal concentration impairment with decreased AQP2 expression, as well as greater morphological damage characterized by interstitial area analysis and presence of tubular necrosis. Our data showed that altering the levels of p21 in ischemic-AKI, vitamin D via VDRs, controls kidney inflammation, proliferation and cell injury

A maior parte dos casos de doença renal policística autossômica dominante (DRPAD) é causada por mutações no gene PKD1 (Polycystic Kidney Disease 1). O insulto por isquemia/reperfusão (IR) constitui-se em uma causa freqüente de lesão renal aguda, incluindo a população de pacientes com DRPAD, mas a relação entre policistina-1 e IR é essencialmente desconhecida. Uma vez que a policistina-1 modula proliferação, diferenciação celular e apoptose em sistemas de cultura de células, sua menor atividade biológica na DRPAD poderia favorecer um maior grau de lesão renal. Utilizamos uma linhagem endogâmica de camundongos 129Sv com uma mutação nula em Pkd1 para testar esta hipótese. Camundongos Pkd1+/- não apresentam cistos renais até 12 semanas de vida, constituindo-se em um modelo puro de haploinsuficiência para este gene. Um insulto IR bilateral de 32 min foi induzido em camundongos machos de 10-12 semanas de idade, heterozigotos e selvagens, por meio do clampeamento reversível de ambos os pedículos renais. Os animais foram analisados 48 h, 7 dias (d) e 14 d após o insulto. Camundongos Pkd1+/- apresentaram FENa, FEK e SCr mais elevadas que animais Pkd1+/+ 48 h após IR. O dano cortical residual foi mais severo em heterozigotos que em selvagens em todos os tempos avaliados. A marcação para PCNA também foi mais alta em camundongos Pkd1+/- que Pkd1+/+ 48 h e 7 d pós-IR, enquanto a taxa de apoptose e a infiltração inflamatória intersticial foram maiores em heterozigotos que em selvagens nos seguimentos de 48 h, 7 d e 14 d pós-IR. A expressão renal de p21 foi menor nos camundongos Pkd1+/- que Pkd1+/+ no tempo de 48 h pós-insulto, tanto no nível transcricional como traducional. Análises adicionais realizadas 6 semanas após o insulto IR revelaram dilatação tubular e formação de microcistos nos camundongos haploinsuficientes para Pkd1, assim como fibrose renal aumentada nesses animais, comparados aos camundongos selvagens. Por fim, um insulto de 35 min de isquemia/reperfusão acompanhou-se de uma mortalidade precoce substancialmente maior nos animais Pkd1+/-. Esses achados sugerem que isquemia/reperfusão induza uma lesão mais severa em rins de camundongos haploinsuficientes para Pkd1, um processo aparentemente dependente de uma deficiência relativa da atividade de p21, assim como dilatação tubular e formação de microcistos. Em conjunto, nossos resultados sugerem que a heterozigose para mutação nula em Pkd1 em camundongo (e talvez em humanos) esteja associada a um risco aumentado para lesão renal por isquemia/reperfusão e a um pior impacto desse insulto sobre a progressão da doença renal. / The majority of autosomal dominant polycystic kidney disease (ADPKD) cases are caused by mutations in the PKD1 gene. Ischemia/reperfusion is a frequent cause of acute kidney injury, including the ADPKD patient population, but the relationship between polycystin-1 and ischemia/reperfusion is essentially unknown. Since polycystin-1 modulates cell proliferation, cell differentiation and apoptosis in cell culture systems, its lower biological activity in ADPKD might amplify the degree of renal injury. Using an inbred 129Sv mouse line with a Pkd1-null mutation, 32-min renal ischemia/reperfusion was induced in 10-12 week-old male non-cystic mice, heterozygotes and wild types. The animals were analyzed at 48h, 7 days (d) and 14d after the insult. Pkd1+/- mice showed higher FENa, FEK and SCr than Pkd1+/+ animals at 48h of follow-up. The residual cortical damage was more severe in heterozygotes than wild types at all evaluated time points. The PCNA staining was also higher in Pkd1+/- than Pkd1+/+ mice at 48h and 7d, while cell apoptotic rates and the interstitial inflammatory infiltration were higher in heterozygotes than wild types at 48h, 7d and 14d postischemia/ reperfusion. The expression of p21 was lower in Pkd1+/- than Pkd1+/+ kidneys at 48h, both at the transcriptional and translational levels. Additional analyses performed 6 weeks after the insult showed tubular dilatation and microcyst formation in the haploinsufficient mice, and increased renal fibrosis in these animals compared to wild types. Thirty-fivemin ischemia/reperfusion, at last, was accompanied by a substantially higher early mortality of Pkd1+/- animals. These findings suggest that ischemia/reperfusion induces a more severe injury in kidneys of Pkd1- haploinsufficient mice, a process that is apparently dependent on a relative deficiency of p21 activity, as well as tubular dilatation and microcyst formation. Altogether, our results suggest that mouse Pkd1-null heterozygosity (and maybe human) is associated with a higher risk for renal ischemia/reperfusion injury and with a worse impact of this insult upon renal disease progression.

INTRODUÇÃO: O papilomavírus humano (HPV) encontra-se amplamente distribuído na população mundial. Apesar da grande maioria das infecções serem transientes, assintomáticas e passíveis de regressão espontânea, a infecção persistente por tipos de alto risco de HPV é necessária para o desenvolvimento de neoplasias intraepiteliais cervicais. Uma vez que apenas uma pequena parcela das infecções progride à lesões malignas após um longo período desde o diagnóstico inicial de lesões precursoras, tem-se iniciado a busca por fatores que possam influenciar na progressão ou na eliminação destas manifestações iniciais. A variabilidade genética viral tem sido apontada como um dos fatores que interagem neste processo. Embora virtualmente todos os tumores da cérvice uterina apresentem o DNA viral, neoplasias em outros sítios anatômicos têm sido apenas em parte correlacionadas com a presença viral, sendo o HPV proposto como um dos agentes causadores de tumores em sítios de cabeça e pecoço. MÉTODOS: Espécimens clínicos de tumores de cabeça e pescoço, fixados em formalina e contidos em parafina (FFPE), provenientes do Instituto do Câncer do Estado de São Paulo (n=79) e da Santa Casa de Misericórida de São Paulo (n=94), tiveram seu DNA extraído, seguido de diagnóstico e genotipagem de HPV pela metodologia de Inno-LiPA. Análises de linhagens moleculares foram realizadas nas amostras HPV-16 positivas. Análise imunohistoquímica de P16INK4a foi realizada em todas as amostras. RESULTADOS: A presença do DNA viral foi encontrada em 24,1% (19/79) dentre a série de tumores provenientes do ICESP, sendo a cavidade oral o sítio em que foi observada a maior proporção de DNA viral (27,1%), enquanto que 13,8% (13/94) dentre os espécimens provenientes da Santa Casa apresentaram-se positivos para HPV, sendo a cavidade oral o sítio em que foi observada a maior proporção do DNA viral (18,1%). O HPV-16 foi o tipo mais prevalente, detectado em 73,4% das amostras HPV positivas provenientes do ICESP e 61,5% das amostras provenientes da Santa Casa. Independente da Instituição, as amostras foram alocadas no clado das linhagens Asiático-Americana e Europeia em 50%, cada uma, entre os 18 tumores HPV-16 positivos em que as análises de linhagem foram possíveis. Não foi observada, nestas séries, correlação entre a superexpressão de P16INK4a e a presença do DNA viral. CONCLUSÃO: Nas amostras analisadas, o DNA de HPV foi detectado em 18,5% dos 173 espécimens. O HPV-16 foi o tipo mais prevalente. Isolados da linhagem Europeia e da linhagem Asiatico-Americano foram detectados em 50% dos casos, cada uma, dentre as amostras HPV-16 analisadas por este estudo / INTRODUCTION: Human papillomaviruses (HPV) are widely distributed worldwide. Although the majority of infections are usually transient, asymptomatic and frequently regress spontaneously, persistent infections by high-risk HPVs are necessary for the development of cervical intraepithelial neoplasia. Once only a small proportion of infections progress to malignant lesions after a long period of time since the initial diagnosis of precursor lesions, the search for factors that might influence the progression or clearence of these early manifestations are currently under way. Viral genetic variability has been proposed as one of the factors interacting in this process. Although virtually all cervix tumors present the viral DNA, neoplasias from other anatomical sites have been only in part correlated with viral presence, and HPV has been proposed as one causative agent in tumors from head and neck sites. METHODS: Clinical specimens of formalin-fixed paraffin embedded head and neck tumors, provided by the Cancer Institute of São Paulo (n=79) and also by the Santa Casa de Misericórdia de São Paulo (n=94), were submitted to DNA extraction and further HPV diagnostic and genotyping by the Inno-LiPA methodology. Molecular lineages analyses were performed in all HPV-16 positive samples. P16INK4a immunohistochemical analyses were conducted in all samples. RESULTS: HPV DNA was detected among 24.1% (19/79) of samples provided by ICESP, tumors from oral cavity presented the highest viral positivity (27.1%), whereas 13,8% (13/94) of the samples from Santa Casa presented HPV DNA, tumors from the oral cavity also presented the highest HPV positivity with 18.1% of viral DNA presence. HPV-16 was the most prevalent type detected in 73.4% and 61.5% of HPV positive ICESP and Santa Casa samples, respectively. Irrespective of the Institution, samples submitted to lineage analyzes were allocated in the Asiatic-American and European phylogenetic branches in 50%, each one, among the 18 tumors HPV-16 positive for which lineage analysis was possible. No correlation between P16INK4a overexpression and HPV DNA presence was observed. CONCLUSION: In this study, HPV DNA was detected in 18.5% among 173 head and neck tumor specimens. HPV-16 was the most prevalent type. The European and the Asiatic-American lineage were detected in 50% of the cases, each one, among the cases HPV-16 positive analyzed

Le glioblastome (GBM), tumeur de plus haut grade du système nerveux central (OMS grade 4) a un pronostic très sombre, quelque soit le traitement, lié à une résistance à l’apoptose. L’erlotinib (Tarceva®, OSI 774) est un inhibiteur de la tyrosine kinase du récepteur au facteur de croissance épithélial (EGFR). L’hyper-expression et l’amplification du gène de l’EGFR dans 40 à 60% des GBM, fourni un rationnel pour utiliser l’erlotinib. Nous avons montré sur U87-MG et DBTRG-05MG, deux lignées de GBM, l’absence d’apoptose avec l’erlotinib, liée soit à un déficit en pro-caspase 3, soit à une accumulation d’αB-crystalline bloquant l’activation de la caspase-3. L’absence d’apoptose dévie alors la cellule vers l’autophagie. L’inhibition de l’autophagie par ARN interférents ou par la chloroquine permet d’obtenir une synergie avec l’erlotinib en induisant la mort des cellules tumorales à des doses acceptables.Les GBM ont composition cellulaire hétérogène, avec un petit nombre d’éléments appelés cellules souches cancéreuses (CSC). Douées d’auto-renouvellement, elles participent à la propagation tumorale et à la résistance aux traitements. Nous avons testé l’erlotinib sur trois lignées issues de GBM humains, ayant deux modes de croissance distincts selon les conditions de milieu: en neurosphères (NS) et de type adhérent. Erlotinib a un effet inhibiteur minime sur les trois lignées adhérentes, alors que l’effet est significatif sur les lignées NS, traduisant l’importance de la voie d’EGFR pour les NS. Dans les lignées en NS, l’erlotinib est efficace sur les cellules progénitrices, mais n’a pas d’action ni sur les cellules initiatrices de NS, ni sur les cellules différenciées. L’auto-renouvellement des NS n’est pas non plus altéré. L’association cyclopamine, inhibiteur pharmacologique de la voie de Hedgehog, -erlotinib est synergique en bloquant la croissance et l’initiation des NS, laissant présager une efficacité sur les CSC. Les résultats obtenus sur ces différents modèles permettent d’une part de préciser certains mécanismes de résistance des cellules de GBM, et aussi d’orienter les indications et le choix des traitements susceptibles d’être les plus efficaces. / Glioblastoma (GBM) is the most common primary central nervous system tumor in adults and the prognosis remains dismal, any treatment used. Epidermal Growth Factor Receptor (EGFR) is amplified, overexpressed, and/or mutated in GBM, making it a rational for therapy. Erlotinib, an EGFR kinase inhibitor is strongly associated with clinical response in several cancers. We showed for U87-MG and DBTRG-05MG, two human GBM cell lines, that erlotinib can’t trigger apoptosis, related either to accumulation of αB-crystallin capable to impair caspase 3 cleavage, or to constitutive deficit for procaspase 3 in DBTRG-05MG. Apoptosis deficit switches the cell to autophagic process. Inhibition of autophagy with RNA interference or chloroquine resulted in sensitization of U87 and allowed a synergistic effect with erlotinib at therapeutic doses.Moreover, GBM showed a heterogeneous cell composition with cancer stem cells, progenitors and more differentiated cells. In this study, we test erlotinib in vitro on other GBM models: three cell lines established from surgically resected GBM specimens, grown along two features adherent and neurospheres. On the three differentiated adhering cell lines, erlotinib had only a moderate activity. Conversely, on neurosphere forming cell lines, erlotinib induced a strong inhibition of cell growth related to the EGFR amplification and EGFR expression. A short erlotinib exposure induced cell death primarily in nestin-positive cells; however it was found without effect on neurosphere initiating activity and self renewal. These results suggest that EGFR pathway activation is essential for the proliferation of GBM progenitor cells but dispensable for stem-like cancer cells self–renewal. As Hedgehog pathway is known to be activated in neural stem cells, we assayed the Hedgehog pathway inhibitor cyclopamine in association with erlotinib. While each drug separately was without effect on sphere initiation, their combination led to a 25 fold decrease in the sphere number (p=0.0004).These in vitro models are convenient to investigate resistance mechanisms in GBM. Furthermore, they focus on the necessity to exploit drug combinations for greatest efficiency.

Le domaine de l'oncologie progresse de manière rapide, surtout depuis l'avènement des thérapies ciblées. Parmi elles, les inhibiteurs de tyrosine kinase multicible (ITK) antiangiogéniques ont fait la preuve de leur efficacité dans plusieurs types de cancers métastatiques. Le sorafenib, le sunitinib, le pazopanib, l'axtinib, et le regorafenib sont aujourd'hui utilisés en pratique courante. Ces premières thérapeutiques ont ouvert la voie au développement de nombreuses autres molécules ciblant d'autres récepteurs TK (crizotinib, céritinib). Les ITK ciblant les récepteurs de l'angiogénèse inhibent des récepteurs membranaires tels que les VEGFR, les PDGFR, les FGFR etc. Ces molécules améliorent généralement survie et/ou survie sans progression dans les essais cliniques pivots mais il existe une grande variabilité interindividuelle en termes de bénéfice clinique. Nous avons cherché à mettre en évidence des biomarqueurs moléculaires prédictifs de la réponse, afin de mieux sélectionner les patients susceptibles de bénéficier de ces ITKs. L'objectif final de ce travail est ainsi de mieux sélectionner les patients candidats à ce traitement, mais il est également médico économique. La part la plus importante de ce travail est axée sur le regorafenib, qui a fait preuve de son efficacité dans les cancers colorectaux métastatiques prétraités et les tumeurs stromales gastro-intestinalesen échec d'imatinib et de sunitinib. Cependant, aucun paramètre clinique ou histologique n'a été identifié pour sélectionner les patients potentiels pouvant bénéficier de ce traitement, ou, à l'inverse pour éviter de traiter les patients chez lesquels la balance bénéfice/risque est défavorable. Ce travail a été réalisé dans le cadre de programme ProfiLER (NCT01774409) en partenariat avec les plateformes de génomique tumorale (Centre de Recherche en Cancérologie de Lyon, Centre Léon Bérard). Cette étude avait pour objectif de tester l'hypothèse que l'ensemble des altérations des gènes codant pour les kinases cibles d'un ITK donné pourrait être associé au bénéfice clinique de ce traitement. Dans notre étude, nous avons observé que les cancers des patients présentant un bénéfice clinique accumulent des gains chromosomiques sur les gènes cibles, et à l'inverse, les cancers des patients nonrépondeurs possèdent plutôt un profil inverse. L'index génomique, un paramètre évaluant l'instabilité chromosomique ne permet pas de différencier les patients répondeurs, mais nous avons mis en évidence que l'accumulation de certains gains sur les gènes cibles est associée à une meilleure survie. Nous avons ainsi proposé un nouveau concept : celui de TTC (Tumor Target Charge), la somme des gains sur les gènes cibles ; et à l'inverse, celui de TTL (Tumor Target Loss), la somme des pertes sur les gènes cibles. En nous appuyant sur ces définitions de TTC et TTL, nous avons généré un algorithme nommé SUMSCAN traduisant donc la somme des gains et des pertes sur les gènes cibles. Le score SUMSCAN a été appliqué à une première cohorte composée essentiellement de patients ayant un cancer colorectal métastatique et traités par regorafenib, ainsi qu'à une 2ème cohorte de validation composée des patients ayant différentes pathologies néoplasiques. Chez les patients ayant un cancer colorectal « moléculairement sélectionné», la médiane de survie sans progression était de 9 mois contre 3 mois dans la cohorte de patients non sélectionnés (X. JIANG et al, Oncotarget, 2015). Nous avons pu montrer que le principe de ce score pronostique pouvait s'appliquer aux autres antiangiogéniques multi-ITKs.. Nous sommes ainsi en cours de validation de ce score pour la prédiction de la survie sur de larges populations de patients présentant divers types tumoraux : sarcome des tissus mous, carcinomes ovariens de haut grade, carcinome rénal carcinome de la thyroïde, etc.) / Small molecule antiangiogenic tyrosine kinase inhibitors (TKI), such as regorafenib, sorafenib, sunitinib, pazopanib, axitinib, and cabozantinib, are active in a variety of advanced cancers, including renal cell carcinoma (RCC), gastrointestinal stromal tumors (GIST), hepatocellular carcinoma (HCC), colorectal cancer (CRC) and thyroid cancers. Predictive criteria for response to these multiple kinase inhibitors (MTKI) are not as well determined as for tumors harboring key driver alterations, such as BCR-ABL translocations in chronic myeloid leukemia (CML), KIT-mutant GIST, BRAF-mutant melanoma, and ALK-positive non-small cell lung cancer among others. Regorafenib, for instance, has been shown to yield a progression-free survival (PFS) improvement in pretreated metastatic colorectal cancer (mCRC) and in imatinib and sunitinib refractory gastrointestinal stromal tumors (GIST). We report that the antitumor activity of MTKIs in tumors lacking a well-defined oncogenic driver is strongly correlated with copy number alterations of genes encoding the protein kinases targeted by these drugs. A concept of tumor target charge (TTC), defined as the total gains of the genes encoding for targets of MTKIs as well as tumor target loss (TTL) was developed, and correlated to response to regorafenib in 2 cohorts of patients composed of mCRC and STS patients. A predictive model, called SUMSCAN, was conceived as a binary classifier to identify patients as either good or poor candidates for use of MTKIs. Moreover, the PFS and OS of patients with a favorable SUMSCAN score were significantly improved. Importantly, SUMSCAN predicted exclusively response to regorafenib, but not the response to conventional chemotherapy in mCRC

<p>Despite advancements in therapies, such as surgery, irradiation (IR) and chemotherapy, outcome for patients suffering from glioblastoma (GBM) remains fatal; the median survival time is only about 15 months. Even with novel therapeutic targets, networks and signaling pathways being discovered, monotherapy with such agents targeting such pathways has been disappointing in clinical trials. Poor prognosis for GBM can be attributed to several factors, including failure of drugs to cross the blood-brain-barrier (BBB), tumor heterogeneity, invasiveness, and angiogenesis. Development of tumor resistance, particularly to temozolomide (TMZ) and IR, creates a substantial clinical challenge.</p><p> </p><p>The primary focus of the work described herein was to develop a modeling and simulation approach that could be applied to rationally develop novel combination therapies and dose regimens that mitigate resistance development. Specifically, TMZ was combined with small molecule inhibitors that are either currently in clinical trials or are approved drugs for other cancer types, and which target the disease at various resistance signaling pathways that are induced in response to TMZ monotherapy. To accomplish this objective, an integrated PKPD modeling approach was used. A PK model for each drug was first defined. PK models were subsequently linked to a PD model description of tumor growth dynamics in the presence of a single drug or combinations of drugs. A key outcome of these combined PKPD models was tumor static concentration (TSC) curves of TMZ in combination with small molecule inhibitors that identify combination drug exposures predicted to arrest tumor growth. This approach was applied to TMZ in combination with abemaciclib (a dual CDK4/6 small molecule inhibitor) based on data from a published study evaluating abemaciclib (ACB) efficacy in combination with TMZ in a U87 GBM xenograft model. TSC was also constructed for TMZ in combination with RG7388 (MDM2 inhibitor) based on the data from an in-vivo study that evaluated effects on tumor growth suppression of these small molecule inhibitors in combination with TMZ in GBM 10 patient derived xenografts.</p><p>In GBM 43 mouse xenografts, emergence of resistance to TMZ treatment was identified. Thus, a resistance integrated PKPD model was developed to predict tumor growth kinetics after treatment with TMZ in GBM 43 tumors. Population PK models in immune deficient NOD.Cg-<em>Prkdc^scid Il2rg^tm1Wjl</em>/SzJ (NSG) mice for TMZ and small molecule inhibitors (GDC0068/RG7112) were developed based on a combination of data obtained from an in-vivo study and published sources. Subsequently, PK models were linked to tumor volume data obtained from GBM 43 subcutaneous xenografts. Model parameters quantifying tumor volume dynamics were precisely estimated (coefficient of variation < 40%) compared to a base tumor growth inhibition model in GBM 43 that did not incorporate resistance development. Graphical diagnostics of the resistance incorporated PKPD tumor growth inhibition model demonstrated a superior fit compared to the base model, and accurately captured the emergence of resistance to the TMZ monotherapy treatment observed in the GBM 43 patient derived xenograft model.</p>