OBJECTIVES: Breast cancer (BC) is the most common neoplasm in women. Biopsy of metastatic lesions is recommended to confirm estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status as there are discrepancies in these patterns between primary tumors and metastases in up to 40% of the cases. Circulating tumor cells (CTCs) are related to BC outcomes and could potentially be an alternative to the invasive procedures of metastasis rebiopsy. ISET® technology is not currently employed to detect CTCs in patients with BC. Emerging data support that the characterization of CTC protein expression can refine its prognostic value. Transforming growth factor (TGF)-β plays a role in BC progression and invasiveness. Thus, in this study, we aimed to compare ER, PR, and HER2 expression in primary tumors, CTCs, and metastases and evaluate TGF-β type 1 receptor (TGF-β RI) expression in CTCs as prognostic factor for progression free survival (PFS) and overall survival (OS). METHODS: This prospective study was conducted at the A.C. Camargo Cancer Center, Brazil. Blood samples were processed in ISET® (Isolation by SizE of Tumors, Rarecells, France) before computed tomography-guided biopsy of suspected metastatic lesions. Protein expression levels in CTCs were compared to those in primary tumors/metastases (medical records). RESULTS: Of the 39 patients initially included, 27 underwent both biopsies of metastases and blood collection and were considered for analysis. The concordance rates for ER, PR, and HER2 expression between primary tumors and metastases were high. No loss of HER2 expression at any metastasis site and retention of the same pattern of protein expression in all triple-negative (TN) tumors (92.5%, 81.5% and 96.2% respectively) (p<0.0001) was observed. When metastases/CTCs were classified as TN/non-TN, CTCs showed high specificity (93%), accuracy (84.2%), and negative predictive value (88%). The median OS of patients without TGF-β RI expression in CTCs was 42.6 versus 20.8 months for TGF-β RI expression-positive ones (p>0.05). CONCLUSION: The role of CTCs detected by ISET has not yet been established in BC. Here, we suggest that this methodology may be useful to evaluate metastasis in non-TN cases as well as TGF-β RI expression in CTCs, which may impact patient survival. Due to sample limitations, future studies must focus on specific BC subtypes and an expansion of the cohort.

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.

RESUMO Racional: A metástase é comum no diagnóstico de câncer de pâncreas; presença de marcadores de transição epitélio-mesenquimal nas células tumorais circulantes (CTCs) podem sugerir pior prognóstico. Objetivo: Correlacionar o número de CTCs no sangue periférico de pacientes com tumor de pâncreas localmente avançado ou metastático e expressão de proteínas envolvidas na transição epitélio-mesenquimal (TEM) nas CTCs com características clínicas, sobrevida livre de progressão (SLP) e global (SG). Método: Estudo prospectivo realizado por meio de coletas de sangue periférico em três tempos distintos. As CTCs foram quantificadas pelo sistema ISET e analisadas por imunocitoquímica. Proteínas envolvidas na TEM (vimentina, TGFß-RI e MMP2) foram analisadas em todas as CTCs. Resultados: Foram incluídos 21 pacientes. A mediana de CTCs detectadas foi de 22, 20 e 8 CTCs/8 ml de sangue no baseline, primeiro e segundo seguimentos, respectivamente. Na correlação entre número de CTCs e as características clínicas levantadas, SLP, SG não houve correlação estatisticamente significante. Nos marcadores de TEM não houve diferença de SLP e SG entre os grupos que apresentaram e não apresentaram marcação. Conclusão: As CTCs não se mostraram relevantes na comparação dos achados clínicos, SLP e SG em pacientes com câncer de pâncreas. No entretanto, pode ser que para a análise de marcador seja útil, como observado pelas curvas separadas de expressão de MMP-2 e TGFß-RI nas CTCs.

ABSTRACT Background: Metastasis is common in the diagnosis of pancreatic cancer, and the presence of epithelial-mesenchymal transition markers in circulating tumor cells may suggest worse prognosis. Aim: To correlate the number of circulating tumor cells (CTCs) in the peripheral blood of patients with a locally advanced or metastatic pancreatic tumor and the protein expression involved in epithelial-mesenchymal transition (EMT) in CTCs with clinical characteristics, progression-free survival (PFS) and overall survival (OS). Method: This was a prospective study conducted using peripheral blood samples collected at three different times. CTCs were quantified by the ISET test and analyzed by immunocytochemistry. Proteins involved in EMT (vimentin, TGFß-RI and MMP2) were analyzed in all CTCs. Results: Twenty-one patients were included. Median CTCs detected were 22, 20 and 8 CTCs/8 ml blood at baseline, first and second follow-up, respectively. No statistically significant correlation was found in correlating the number of CTCs and the evaluated clinical characteristics, PFS, or OS. There was no difference in PFS and OS among the EMT markers in the groups with and without markers. Conclusion: CTC analysis was not relevant in this sample for comparing clinical findings, PFS and OS in patients with pancreatic cancer. However, marker analysis in CTCs could be useful for the MMP-2 and/or TGFß-RI expression, as observed by the separate PFS curve.

O acometimento leptomeníngeo por metástases tumorais pode ocorrer em tumores sólidos, sendo chamado de carcinomatose meníngea (CM), e também em doenças linfoproliferativas. Tumores de mama, pulmão e melanoma são os principais responsáveis pelos casos, e adenocarcinoma é a histologia mais frequentemente encontrada. A CM é um evento tardio na evolução da doença e caracteriza-se por sinais e sintomas neurológicos multifocais. O diagnóstico se faz pela avaliação conjunta do quadro clínico, neuroimagem e estudo do líquido cefalorraquidiano. O maior controle da doença sistêmica obtido com as novas modalidades terapêuticas e a baixa penetração de drogas no sistema nervoso central, aliados ao desenvolvimento nos métodos de neuroimagem observado nas últimas décadas, são fatores que respondem por um aumento na incidência desta apresentação. A quimioterapia intratecal é o tratamento de escolha, porém, frequentemente paliativo. O prognóstico é reservado, sendo que o melhor performance status pode selecionar um subgrupo de pacientes com melhor evolução.

The involvement of the leptomeninges by metastatic tumors can be observed in solid tumors, in which case it is termed meningeal carcinomatosis (MC), and in lymphoproliferative malignant disease. It is more common in breast and lung cancer, as well as melanoma, with adenocarcinoma being the most frequent histological type. MC is usually a late event, with disseminated and progressive disease already present and, it is characterized by multifocal neurological signs and symptoms. Diagnosis is based on the evaluation of clinical presentation, cerebrospinal fluid and neuroimaging studies. The better systemic disease control is observed with new therapeutic agents, and the development of neuroimaging methods is responsible for the increasing incidence of such metastatic evolution. Intrathecal chemotherapy is generally the treatment of choice, although frequently palliative. Prognosis is guarded, although a higher performance status may indicate a subgroup of patients with a more favorable outcome.