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OBJECTIVES: Ductal adenocarcinoma of the pancreas is the fourth most common cancer-associated cause of death in the Western world. The presence of circulating tumor cells (CTCs) can be considered a potential prognostic factor, as these cells represent tumor progression, allowing monitoring of therapeutic efficacy. The objectives of this study were to explore the morphological, molecular, and phenotypic characteristics of CTCs from the blood of patients with pancreatic carcinoma and to correlate the findings with response to treatment, progression-free survival, overall survival (OS), and deep vein thrombosis (DVT). METHODS: Peripheral blood (10 mL) was analyzed before the beginning of treatment after 60 and 120 days. CTCs were detected by using ISET® and characterized by immunocytochemistry. For microRNAs (miRNAs) analysis, peripheral leukocytes from the same patients and healthy individuals (controls) were collected in parallel at baseline. The expression of miRNAs was evaluated (in pool) using TaqMan® Array Human MicroRNA Cards v2.0. RESULTS: Only nine patients were included. The proteins, namely, matrix metalloproteinase-2 (MMP2) and TGFß-RI, were highly expressed (77.7%) in CTCs at baseline; at the first follow-up, MMP2 was predominant (80%) and, at the second follow-up, MMP2 and vimentin were predominant (50%). Circulating tumor microemboli (CTMs) were found in two patients and both presented DVT. The miR-203a-3p was highly expressed in CTCs. The miR-203a-3p is involved in the stimulation of epithelial-to-mesenchymal transition (EMT) and is related to worse OS in pancreatic cancer (TCGA data). CONCLUSION: Due to the low number of patients and short follow-up, we did not observe a correlation between CTCs and response to treatment. However, there was a correlation between CTM and DVT and also miR-203a-3p was highly expressed in CTCs, corroborating the findings of EMT proteins. This study opens the perspectives concerning the dynamic change in the pattern of proteins expressed along with treatment and the use of miRNAs as new targets in pancreatic carcinoma.

OBJETIVOS: O adenocarcinoma ductal do pâncreas é a quarta causa de morte associada ao câncer mais comum no mundo ocidental. A presença de células tumorais circulantes (CTCs) pode ser considerada um potencial fator prognóstico, visto que essas células representam a progressão tumoral, permitindo o monitoramento da eficácia terapêutica. explorar as características morfológicas, moleculares e fenotípicas das células tumorais circulantes (CTCs) do sangue de pacientes com carcinoma pancreático e correlacionar os achados com a resposta ao tratamento, sobrevida livre de progressão, sobrevida global (SG) e trombose venosa profunda (TVP). MÉTODOS: o sangue periférico (10mL) foi analisado antes do início do tratamento e após 60 e 120 dias. As CTCs foram detectadas pelo ISET® e caracterizadas por imunocitoquímica. Para análise de miRNAs, leucócitos periféricos dos mesmos pacientes e indivíduos saudáveis foram coletados em paralelo no início do estudo. A expressão de miRNAs foi avaliada usando TaqMan T Array Human MicroRNA Cards v2.0. RESULTADOS: foram incluídos 9 pacientes. As proteínas MMP2 e TGFß-RI foram altamente expressas (77,7%) nas CTCs no início do estudo. No primeiro acompanhamento, MMP2 era predominante (80%) e no segundo acompanhamento, MMP2 e vimentina eram predominantes (50%). Microêmbolos tumorais circulantes (MTC) foram encontrados em dois pacientes e ambos apresentavam TVP. O miR-203a-3p foi altamente expresso em CTCs. miR-203a-3p está envolvido na estimulação da transição epitelio-mesenquima (TEM) e relacionado a pior SG no câncer pancreático (dados TCGA). CONCLUSÃO: Devido ao baixo número de pacientes e curto seguimento, não observamos correlação entre CTCs e resposta ao tratamento. No entanto, houve uma correlação entre MTC e TVP. Além disso, miR-203a-3p foi altamente expresso em CTCs, corroborando os achados de proteínas EMT. Este estudo abre perspectivas sobre a mudança dinâmica no padrão de proteínas expressas ao longo do tratamento e a utilização de miRNAs como novos alvos no carcinoma pancreático.

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The heterogeneity of response to neoadjuvant chemoradiotherapy (NCRT) is still a challenge in locally advanced rectal cancer (LARC). The evaluation of thymidylate synthase (TYMS) and RAD23 homolog B (RAD23B) expression in circulating tumor cells (CTCs) provides complementary clinical information. CTCs were prospectively evaluated in 166 blood samples (63 patients) with LARC undergoing NCRT. The primary objective was to verify if the absence of RAD23B/TYMS in CTCs would correlate with pathological complete response (pCR). Secondary objectives were to correlate CTC kinetics before (C1)/after NCRT (C2), in addition to the expression of transforming growth factor-ß receptor I (TGF-ßRI) with survival rates. CTCs were isolated by ISET and evaluated by immunocytochemistry (protein expression). At C1, RAD23B was detected in 54.1% of patients with no pCR and its absence in 91.7% of patients with pCR (p = 0.014); TYMS- was observed in 90% of patients with pCR and TYMS+ in 51.7% without pCR (p = 0.057). Patients with CTC2 > CTC1 had worse disease-free survival (DFS) (p = 0.00025) and overall survival (OS) (p = 0.0036) compared with those with CTC2 ≤ CTC1. TGF-ßRI expression in any time correlated with worse DFS (p = 0.059). To conclude, RAD23B/TYMS and CTC kinetics may facilitate the personalized treatment of LARC.

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RESUMO -RACIONAL: O adenocarcinoma ductal do pâncreas é a quarta causa de morte associada ao câncer mais comum no mundo ocidental. A presença de células tumorais circulantes (CTCs) pode ser considerada um potencial fator prognóstico, visto que essas células representam a progressão tumoral, permitindo o monitoramento da eficácia terapêutica. OBJETIVOS: explorar as características morfológicas, moleculares e fenotípicas das células tumorais circulantes (CTCs) do sangue de pacientes com carcinoma pancreático e correlacionar os achados com a resposta ao tratamento, sobrevida livre de progressão, sobrevida global (SG) e trombose venosa profunda (TVP). MÉTODOS: o sangue periférico (10mL) foi analisado antes do início do tratamento e após 60 e 120 dias. As CTCs foram detectadas pelo ISET® e caracterizadas por imunocitoquímica. Para análise de miRNAs, leucócitos periféricos dos mesmos pacientes e indivíduos saudáveis foram coletados em paralelo no início do estudo. A expressão de miRNAs foi avaliada usando TaqMan T Array Human MicroRNA Cards v2.0. RESULTADOS: foram incluídos 9 pacientes. As proteínas MMP2 e TGFß-RI foram altamente expressas (77,7%) nas CTCs no início do estudo. No primeiro acompanhamento, MMP2 era predominante (80%) e no segundo acompanhamento, MMP2 e vimentina eram predominantes (50%). Microêmbolos tumorais circulantes (MTC) foram encontrados em dois pacientes e ambos apresentavam TVP. O miR-203a-3p foi altamente expresso em CTCs. miR-203a-3p está envolvido na estimulação da transição epitelio-mesenquima (TEM) e relacionado a pior SG no câncer pancreático (dados TCGA). CONCLUSÃO: Devido ao baixo número de pacientes e curto seguimento, não observamos correlação entre CTCs e resposta ao tratamento. No entanto, houve uma correlação entre MTC e TVP. Além disso, miR-203a-3p foi altamente expresso em CTCs, corroborando os achados de proteínas EMT. Este estudo abre perspectivas sobre a mudança dinâmica no padrão de proteínas expressas ao longo do tratamento e a utilização de miRNAs como novos alvos no carcinoma pancreático.

ABSTRACT - BACKGROUND: Ductal adenocarcinoma of the pancreas is the fourth most common cancer-associated cause of death in the Western world. The presence of circulating tumor cells (CTCs) can be considered a potential prognostic factor, as these cells represent tumor progression, allowing monitoring of therapeutic efficacy. OBJECTIVES: The objectives of this study were to explore the morphological, molecular, and phenotypic characteristics of CTCs from the blood of patients with pancreatic carcinoma and to correlate the findings with response to treatment, progression-free survival, overall survival (OS), and deep vein thrombosis (DVT). METHODS: Peripheral blood (10 mL) was analyzed before the beginning of treatment after 60 and 120 days. CTCs were detected by using ISET® and characterized by immunocytochemistry. For microRNAs (miRNAs) analysis, peripheral leukocytes from the same patients and healthy individuals (controls) were collected in parallel at baseline. The expression of miRNAs was evaluated (in pool) using TaqMan® Array Human MicroRNA Cards v2.0. RESULTS: Only nine patients were included. The proteins, namely, matrix metalloproteinase-2 (MMP2) and TGFβ-RI, were highly expressed (77.7%) in CTCs at baseline; at the first follow-up, MMP2 was predominant (80%) and, at the second follow-up, MMP2 and vimentin were predominant (50%). Circulating tumor microemboli (CTMs) were found in two patients and both presented DVT. The miR-203a-3p was highly expressed in CTCs. The miR-203a-3p is involved in the stimulation of epithelial-to-mesenchymal transition (EMT) and is related to worse OS in pancreatic cancer (TCGA data). CONCLUSION: Due to the low number of patients and short follow-up, we did not observe a correlation between CTCs and response to treatment. However, there was a correlation between CTM and DVT and also miR-203a-3p was highly expressed in CTCs, corroborating the findings of EMT proteins. This study opens the perspectives concerning the dynamic change in the pattern of proteins expressed along with treatment and the use of miRNAs as new targets in pancreatic carcinoma.

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Câncer colorretal (CCR) é altamente incidente; estimam-se, no Brasil, em 2016 16.660 casos novos de CCR em homens e 17.620 em mulheres, de acordo com o Ministério da Saúde. Estudos têm demonstrado que as células tumorais circulantes (CTCs) podem estar envolvidas no processo de metastatização. Desta forma, seu isolamento constitui uma estratégia potencial para o acompanhamento clínico, como método não invasivo. O presente estudo teve como objetivos: 1) Isolar e quantificar CTCs do sangue periférico de pacientes com câncer de reto localmente avançado (CRLA), estádios II e III, para correlacionar seus níveis com exames de imagem e sobrevida livre de doença; 2) Analisar nas CTCs marcadores de resistência ao tratamento (TYMS e RAD23) e correlacionar com resposta à terapia e sobrevida livre de doença. Foram analisadas amostras de 30 pacientes. As amostras foram coletadas antes da quimioterapia neoadjuvante (baseline) e após a mesma (follow-up). As CTCs foram caracterizadas nos dois momentos por imunocitoquímica com anticorpos específicos para os marcadores acima citados e por CISH, avaliando a expressão do mRNA do gene do TYMS. As contagens de CTCs diminuíram entre a primeira e a segunda coletas em pacientes exibindo resposta patológica completa (RPC; p = 0,02) ou resposta parcial (RP; p = 0,01). Em relação à expressão protéica, TYMS estava ausente em 100% das CTCs dos pacientes com RPC (p = 0,001), mas foi expresso em 83% dos não respondedores na segunda coleta (p <0,001). Em paralelo, RAD23b foi expresso em CTCs de 75% dos não respondedores na coleta baseline (p = 0,01) e em 100% dos não respondedores do follow-up (p = 0,001). Surpreendentemente, 100% dos não respondedores expressaram mRNA de TYMS nos dois momentos (p = 0,001). Além disso, TYMS / RAD23b não foram detectadas nas CTCs de pacientes que exibiam RPC (p = 0,001). Encontramos 83,3% de sensibilidade para o mRNA de TYMS no baseline (p = 0,001) e 100% para a expressão da proteína TYMS (p = 0,064) e RAD23B (p = 0,01) no follow- up. Assim, a expressão do RNAm TYMS e / ou TYMS / RAD23b nas CTCs, bem como a cinética das CTCs, têm o potencial de prever não resposta ao tratamento neoadjuvante e evitar cirurgias radicais desnecessárias em pacientes com CRLA com RPC. Com a realização deste trabalho obtivemos uma melhor compreensão a respeito dos mecanismos que envolvem a resistência ao tratamento nos pacientes com CRLA, por meio da análise de CTCs. Auxiliados por estas análises, identificamos novos biomarcadores sanguíneos prognósticos, que poderão ser usados como instrumentos de direcionamento clínico na escolha da melhor terapêutica

Colorectal cancer (CRC) is highly incident; In Brazil, in 2016, an estimated 16,660 new cases of CRC in men and 17,620 in women, according to the Ministry of Health. Studies have shown that circulating tumor cells (CTCs) may be involved in the metastasization process. Thus, its isolation is a potential strategy for clinical follow-up as a noninvasive method. The present study aims to: 1) Isolate and quantify peripheral blood CTCs from patients with locally advanced rectal cancer (stages II and III) to correlate their levels with imaging and progression-free survival; 2) Analyze treatment resistance markers on CTCs (TYMS and RAD23), and correlate with therapy response and progression-free survival. Samples from 30 patients were analyzed. Samples were collected before and after neoadjuvant chemotherapy (follow-up). CTCs were characterized at both times by immunocytochemistry with specific antibodies to the markers mentioned above and by CISH, evaluating the expression of the TYMS gene mRNA. CTC counts decreased between the first and second collection in patients exhibiting complete pathological response (CPR; p = 0.02) or partial response (PR; p = 0.01). Regarding protein expression, TYMS was absent in 100% of the CTCs of patients with CPR (p = 0.001), but was expressed in 83% of non-responders in the second collection (p <0.001). Meanwhile, RAD23b was expressed in CTCs of 75% of baseline non-responders (p = 0.01) and 100% of follow-up non-responders (p = 0.001). Surprisingly 100% of non-responders expressed TYMS mRNA at both times (p= 0.001). In addition, TYMS / RAD23b was not detected in CTCs of patients with CPR (p = 0.001). We found 83.3% sensitivity for TYMS mRNA at baseline (p = 0.001) and 100% for TYMS protein expression (p = 0.064) and RAD23B (p = 0.01) at follow-up. Thus, TYMS mRNA and/or TYMS / RAD23b in CTCs, as well as CTC kinetics, have the potential to predict non-response to NCRT and to avoid unnecessary radical surgery in patients with LARC and CPR. With this work we obtained a better understanding about the mechanisms involving resistance to treatment in patients with locally advanced rectal cancer, through the analysis of CTCs. With these analyzes, we identified new prognostic blood biomarkers that could be used as clinical guidance tools in choosing the best therapy

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It is believed that the development of metastatic cancer requires the presence of circulating tumor cells (CTCs) , which are found in a patient's circulation as rare abnormal cells comingled with billions of the normal red and white blood cells. The systems developed for detection of CTCs have brought progress to cancer treatment. The molecular characterization of CTCs can aid in the development of new drugs, and their presence during treatment can help clinicians determine the prognosis of the patient. Studies have been carried out in patients early in the disease course, with only primary tumors, and the role of CTCs in prognosis seems to be as important as it is in patients with metastatic disease. The published studies on CTCs have focused on their prognostic significance, their utility in real-time monitoring of therapies, the identification of therapeutic and resistance targets, and understanding the process of metastasis . The analysis of CTCs during the early stages, as a "liquid biopsy," helps to monitor patients at different points in the disease course, including minimal residual disease, providing valuable information about the very early assessment of treatment effectiveness. Finally, CTCs can be used to screen patients with family histories of cancer or with diseases that can lead to the development of cancer. With standard protocols, this easily obtained and practical tool can be used to prevent the growth and spread of cancer. In this chapter, we review some important aspects of CTCs , surveying the disease aspects where these cells have been investigated.

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Salivary gland tumors comprise a heterogeneous group of lesions with different histological features and diverse clinical pathophysiology. They account for about 3% of all head and neck tumors. Apoptosis plays an important role during morphogenesis of glandular structures, including that of the salivary gland. Recent studies have demonstrated that several microRNAs (miRNAs) are involved in the control of apoptosis. The aim of the present study was to determine the expression of apoptosis-related miRNAs (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29, and miR-34) and their target mRNAs in 25 pleomorphic adenomas, 23 mucoepidermoid carcinomas, and 10 non-neoplastic salivary gland samples by real-time RT-PCR. We observed upregulation of miR-15a, miR-16, miR-17-5p, miR-21, miR-29, and miR-34a in pleomorphic adenomas. The expression of miR-21 and miR-34a was upregulated in 91 and 74% of mucoepidermoid carcinomas, respectively. Downregulation of miR-20a was observed in 75% of pleomorphic adenomas and in 57% of mucoepidermoid carcinomas. APAF1, BAX, BCL2, BID, CASP2, CASP8, DIABLO , and TP53 transcripts were upregulated in both tumor types. BAD transcripts were upregulated in pleomorphic adenomas. CASP3 and CASP6 transcripts were upregulated in mucoepidermoid carcinomas. BCL2, CASP2, CASP6, and CASP8 proteins were mostly absent in mucoepidermoid carcinomas but expressed in few cells in pleomorphic adenomas. Our study provides evidence of alterations in the expression of apoptosis-regulating miRNAs in salivary gland tumors, suggesting possible involvement of these microRNAs in salivary gland tumorigenesis.

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Os tumores de glândulas salivares compreendem um grupo heterogêneo de lesões, apresentando diferentes características histológicas e comportamento clínico diverso, tornando seu diagnóstico uma atividade desafiadora. Por isso, a identificação de marcadores moleculares de diagnóstico e prognóstico é tão importante, especialmente para este grupo de neoplasias. Os miRNAs são pequenas moléculas e modulam múltiplas vias oncogênicas, incluindo as vias apoptóticas. Diversos trabalhos têm demonstrado alterações na expressão de microRNAs em diversos tumores. O processo de apoptose desempenha um papel importante na formação do lúmen durante a organogênese de estruturas glandulares, incluindo o desenvolvimento das glândulas salivares. Por isso a necessidade de novos estudos na busca por novos marcadores moleculares. O objetivo desse trabalho foi a determinação da expressão de microRNAs relacionados à regulação da apoptose (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29a, miR-34a) e a determinação da expressão de genes relacionados ao processo de apoptose (BAD, BAX, BCL2, BID, APAF1, CASP2, CASP3, CASP6, CASP7, CASP8, DIABLO e TP53) e alvos desses microRNAs em amostras de tumores de glândula salivar através da metodologia de RT-PCR em tempo real. Para tanto, foram selecionadas 25 amostras de adenoma pleomórfico 23 de carcinoma mucoepidermóide e 10 amostras de glândulas salivar não neoplásicas, provenientes do Biobanco de tumores do AC Camargo Cancer Center. Como resultado, obtivemos os miR-15a, miR-16, miR-17-5p, miR -21, miR-29a e miR-34a com aumento de expressão em 76,0%, 60,0%, 47,8%, 80,0%, 84,0% e 96,0% das amostras de adenoma pleomórfico, respectivamente. Por outro lado, na análise da expressão do miR-20a, pode-se observar a diminuição da sua expressão em 75,0% das amostras. Nas amostras de carcinoma mucoepidermóide, os microRNAs miR-15a, miR-17-5p e miR-29a apresentaram expressão similar à observada no tecido não-neoplásico em 47,8%, 52,1% e 73,9%, respectivamente. O miR-20a apresentou expressão diminuída em 56,5% das amostras e os microRNAs miR-21 e miR-34 que apresentaram aumento de expressão em 90,9% e 73,9% das amostras de carcinoma mucoepidermóide, respectivamente. O miR-16 não apresentou um padrão de expressão predominante. Na análise dos resultados da expressão gênica, encontramos, nas amostras de adenoma pleomórfico, os genes APAF-1, BAD, BAX, BCL-2, CASP8, DIABLO e TP53 com a expressão aumentada, enquanto os genes CASP2, CASP3, CASP6 e CASP7 se apresentaram com equivalente à observada no tecido não neoplásico. O gene BID não apresentou um padrão predominante de expressão. Nas amostras de carcinoma mucoepidermóide os genes APAF-1, BAX, BCL-2, BID, CASP2, CASP3, CASP6, CASP8, DIABLO e TP53 apresentaram sua expressão aumentada. Os genes CASP7 e BAD encontraram-se com a expressão similar às amostras não-neoplásicas utilizadas como controle. Nossos resultados demonstraram alteração na expressão dos microRNAs miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29 e miR-34a em amostras de adenoma pleomórfico e carcinoma mucoepidermóide das glândulas salivares. Poucos estudos têm avaliado o papel dos microRNAs em tumores de glândula salivar e não existem estudos avaliando a expressão desses microRNAs em tumores de glândula salivar. Os resultados também demonstram alterações na expressão dos genes APAF1, BAD, BAX, BCL-2, CASP8, DIABLO e TP53 nas amostras de adenoma pleomórfico e alteração dos genes APAF1, BAX, BCL2, BID, CASP2, CASP3, CASP6, CASP8, DIABLO e TP53 nas amostras de carcinoma mucoepidermóide. Alguns genes estudados podem ser regulados por mais de um microRNA avaliado em nosso estudo, bem como pelo fato de que diversos outros microRNAs não avaliados neste estudo, podem regular esses mesmos genes; isso poderia explicar a razão de existirem resultados contraditórios.

Salivary gland tumors comprise a heterogeneous group of lesions, with different histological features and different clinical behavior, imposing a significant challenge on the diagnosis of these tumors. Therefore, the identification of molecular markers for diagnosis and prognosis is so important, especially for this group of malignancies. miRNAs are small noncoding RNAs that modulate several biological processes, including apoptotic pathways. Several studies have demonstrated changes in microRNA expression in various tumors. The process of apoptosis plays an important role during the organogenesis of glandular structures, including the development of salivary glands. For that reason the need for further studies in the search for new molecular markers. The aim of this study was to determine the expression of microRNAs related to the regulation of apoptosis (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29a, miR-34a) and the expression of genes (BAD, BAX, BCL2, IDB, APAF1, CASP2, CASP3, CASP6, CASP7, CASP8, DIABLO and TP53) related to the apoptotic process and that are target of these microRNAs in salivary gland tumor samples by real time RT-PCR methodology. Twenty-five pleomorphic adenoma samples 23 mucoepidermoid carcinoma samples and 10 nonneoplastic salivary gland samples were selected from the Biobank of the AC Camargo Cancer Center. Our results demonstrated that microRNAs miR15a, miR-16, miR-17-5p, miR-21, and miR-29a miR-34a presented an increased expression in 76.0%, 60.0%, 47.8%, 80.0% 84.0% and 96.0% of the pleomorphic adenoma samples, respectively. Moreover, miR-20a expression was decreased in 75.0% of samples. microRNAs miR-15a, miR17-5p and miR-29a presented expression similar to that observed in non- neoplastic tissue. The miR-20a expression was decreased in 56.5% of samples and microRNAs miR-21 and miR-34 presented increased expression in 90.9% and 73.9% of the mucoepidermoid carcinoma samples, respectively, and miR-16 presented the same number of samples with increased expression and expression similar to that observed in nonneoplastic samples (43.5%). With regard to gene expression, we have observed APAF-1, BAD, BAX, BCL-2, CASP8, DIABLO and TP53 genes with increased expression in pleomorphic adenoma samples, while CASP2, CASP3, CASP6 and CASP7 genes presented expression similar to that observed in non-neoplastic samples. The BID gene did not presented a predominant pattern of expression. In mucoepidermoid carcinoma samples, APAF-1, BAX, BCL-2, BID, CASP2, CASP3, CASP6, CASP8, DIABLO and TP53 genes presented increased expression. The CASP7 and BAD genes presented expression similar to that observed in non-neoplastic samples. Our results demonstrated normal expression of microRNAs miR-15a, miR16, miR-17-5p, miR-20a, miR-21, miR-29 and miR-34a in mucoepidermoid carcinoma and pleomorphic adenoma of the salivary glands. Few studies have evaluated the role of microRNAs in salivary gland tumors and there are no studies evaluating the expression of these microRNAs in salivary gland tumors. The results also demonstrated changes in the expression of APAF1, BAD, BAX, BCL-2, CASP8, DIABLO and TP53 genes in pleomorphic adenoma samples and alteration of APAF1, BAX, BCL2, IDB, CASP2, CASP3, CASP6, CASP8, DIABLO and TP53 genes in mucoepidermoid carcinoma samples. Some studied genes may be regulated by more than one microRNA evaluated in our study as well as the fact that several other microRNAs not evaluated in this study, can regulate these same genes; this could explain why there are contradictory results.

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