Cancer is primarily a disease in which late diagnosis is linked to poor prognosis, and unfortunately, detection and management are still challenging. Circulating tumor cells (CTCs) are a potential resource to address this disease. Cell fusion, an event discovered recently in CTCs expressing carcinoma and leukocyte markers, occurs when ≥2 cells become a single entity (hybrid cell) after the merging of their plasma membranes. Cell fusion is still poorly understood despite continuous evaluations in in vitro/in vivo studies. Blood samples from 14 patients with high-grade serous ovarian cancer (A.C. Camargo Cancer Center, São Paulo, Brazil) were collected with the aim to analyze the CTCs/hybrid cells and their correlation to clinical outcome. The EDTA collected blood (6 mL) from patients was used to isolate/identify CTCs/hybrid cells by ISET. We used markers with possible correlation with the phenomenon of cell fusion, such as MC1-R, EpCAM and CD45, as well as CEN8 expression by CISH analysis. Samples were collected at three timepoints: baseline, after one month (first follow-up) and after three months (second follow-up) of treatment with olaparib (total sample = 38). Fourteen patients were included and in baseline and first follow-up all patients showed at least one CTC. We found expression of MC1-R, EpCAM and CD45 in cells (hybrid) in at least one of the collection moments. Membrane staining with CD45 was found in CTCs from the other cohort, from the other center, evaluated by the CellSearch^® system. The presence of circulating tumor microemboli (CTM) in the first follow-up was associated with a poor recurrence-free survival (RFS) (5.2 vs. 12.2 months; p = 0.005). The MC1-R expression in CTM in the first and second follow-ups was associated with a shorter RFS (p = 0.005). CEN8 expression in CTCs was also related to shorter RFS (p = 0.035). Our study identified a high prevalence of CTCs in ovarian cancer patients, as well as hybrid cells. Both cell subtypes demonstrate utility in prognosis and in the assessment of response to treatment. In addition, the expression of MC1-R and EpCAM in hybrid cells brings new perspectives as a possible marker for this phenomenon in ovarian cancer.

Circulating tumor cells (CTCs) and/or circulating tumor microemboli (CTM) from non-small cell lung cancer (NSCLC) patients may be a non-invasive tool for prognosis, acting as liquid biopsy. CTCs interact with platelets through the transforming growth factor-β/transforming growth factor-β receptor type 1 (TGF-β/TGFβRI) forming clusters. CTCs also may express the Cluster of Differentiation 47 (CD47) protein, responsible for the inhibition of phagocytosis, the “don’t eat me” signal to macrophages. Objectives: To isolate, quantify and analyze CTCs/CTMs from metastatic NSCLC patients, identify TGFβRI/CD47 expression in CTCs/CTMs, and correlate with progression-free survival (PFS). Methods: Blood (10 mL) was collected at two time-points: T1 (before the beginning of any line of treatment; T2 (60 days after initial collection). CTCs were isolated using ISET^®. Immunocytochemistry was conducted to evaluate TGFβRI/CD47 expression. Results: 45 patients were evaluated. CTCs were observed in 82.2% of patients at T1 (median: 1 CTC/mL; range: 0.33–11.33 CTCs/mL) and 94.5% at T2 (median: 1.33 CTC/mL; 0.33–9.67). CTMs were observed in 24.5% of patients and significantly associated with poor PFS (10 months vs. 17 months for those without clusters; p = 0.05) and disease progression (p = 0.017). CTMs CD47+ resulted in poor PFS (p = 0.041). TGFβRI expression in CTCs/CTMs was not associated with PFS. Conclusion: In this study, we observed that CTC/CTM from NSCLC patients express the immune evasion markers TGFβRI/CD47. The presence of CTMs CD47+ is associated with poor PFS. This was the first study to investigate CD47 expression in CTCs/CTM of patients with NSCLC and its association with poor PFS.

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-β receptor I (TGF-βRI) 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-βRI 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.

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.

Introduction: Cancer-associated thrombosis (CAT) is a major cause of morbidity and mortality in oncology patients. There are no accurate risk assessment tools to predict venous thromboembolism (VTE). Circulating tumor cells (CTCs), circulating tumor microemboli (CTM), and high platelet-lymphocyte ratio (PLR) may predispose to VTE. Objective: To evaluate correlations of CTCs, CTM, and PLR with VTE and recurrence-free survival (RFS) in gastric cancer patients. Material and Methods: Patients with gastric cancer (localized and metastatic disease) were recruited (March 2016 to April 2017). CTCs were analysed by ISET at two timepoints: before neoadjuvant treatment (CTC1) and after surgery/before adjuvant therapy (CTC2) for patients with localized disease, and before first-line chemotherapy (CTC1) and after 6 months (CTC2) for patients with metastases. VTE incidence was determined retrospectively. RFS was estimated by Kaplan-Meier analysis. Results: We evaluated 93 patients. According to Khorana scores, 63 (67.7%) patients were at intermediate and 30 (32.3%) were at high risk for VTE. VTE incidence was 20.4% and CTM were found in 39.8%. VTE developed in 7/37 (18.9%) CTM-positive and in 11/50 (22%) CTM-negative patients (p=0.93). When PLR >288, VTE occurred in 7/14 patients (p=0.005). PLR also associated with poor RFS (p<0.0001). CTC2 was associated with poor RFS (p<0.0001). CTC2, PLR and VTE were independent prognostic factors for RFS (p=0.005, 0.043, and <0.0001, respectively). Conclusion: PLR is a prognostic indicator for VTE and RFS in gastric cancer patients. Neither CTC, nor CTM improved risk stratification for VTE in our studied population. PLR, CTC2, and VTE were independent prognostic factors for RFS.