The prospect and challenge of liquid biopsy in the diagnosis and treatment of chest malignancy / 中华预防医学杂志

In recent years, the incidence of chest malignant tumors in China has increased year by year, which has seriously threatened the health problems of people. Among them, early screening and intervention of patients with chest malignancies is the key to cancer prevention. Early detection, early diagnosis, and early treatment as the "three early prevention" of clinical practice are conducive to improve the survival rate of tumor patients. As a non-invasive and real-time reflection of tumor status, liquid biopsy has gradually received attention in clinical diagnosis and treatment. Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and exosomes as liquid biopsy "Three carriages" are not only widely used in the diagnosis, monitoring and prognostic evaluation of chest malignancies, but also face many unknown challenges. In this article, the application of liquid biopsy in chest malignancies in recent years is elaborated in detail, which provides a reference for the formulation of clinical tumor prevention and diagnosis and treatment strategies.

Células tumorales circulantes en una paciente con sospecha clínica de mieloma múltiple / Circulating tumor cells in a patient with clinical suspicious of multiple myeloma

Introducción: La circulación de células tumorales en la sangre periférica, conocido como carcinocitemia, es un fenómeno raro y muy poco comunicado en la literatura científica y su diagnóstico diferencial puede constituir un desafío en la práctica clínica. Objetivos: Describir las causas más frecuentes de carcinocitemia, los retos diagnósticos que representa y contribuir a elevar el índice de sospecha de esta entidad. Presentación del caso: Paciente femenina de 71 años de edad que acude por dolores óseos y palidez cutánea. En el examen de sangre periférica se observa células de gran tamaño que recordaron células plasmáticas. El inmunofenotipo por citometría de flujo fue sugestivo de mieloma múltiple isotipo IgM. El ultrasonido de mamas y la tomografía de tórax mostraron lesión tumoral en la mama izquierda. El estudio inmunohistoquímico de la biopsia de médula ósea fue compatible con adenocarcinoma de mamas. La paciente falleció sin haber comenzado tratamiento específico. Conclusiones: Se presenta paciente con células circulantes tumorales secundaria a adenocarcinoma de la mama donde la inmunohistoquímica de la biopsia de médula ósea descartó el diagnóstico de mieloma múltiple sospechado clínica, radiológicamente, por la morfología celular y el inmunofenotipo(AU)

Introduction: The circulating tumor cells in peripheral blood, known as carcinocythemia is a rare and poorly documented phenomenon, that can be a challenge diagnosis. Objectives: To describe the most frequents causes of carcinocythemia, the diagnosis challenges that it represents and to contribute raising awareness of this entity. Case presentation: Female patient, 71-year-old who complained bone pain and skin pale. The peripheral blood smear showed big size cells mimicking plasma cells. The immunophenotype by flow cytometry suggested IgM isotype multiple myeloma. Breast ultrasound and thorax tomography showed a tumor in the left breast. The bone marrow biopsy immunohistochemical was compatible with adenocarcinoma of breast. The patient died short after before receive specific treatment. Conclusions: We present a patient with circulating tumor cells secondary to breast adenocarcinoma where the bone marrow biopsy immunohistochemical ruled out multiple myeloma diagnosis suspected by clinical, image studies, cell morphology and immunophenotype(AU)

Análise de marcadores sanguíneos em pacientes com osteossarcoma, sarcomas de partes moles e tumor desmóide / Analysis of blood markers in patients with soft tissue sarcoma and desmoid tumor

Introdução: O tumor desmoide (TD) é uma neoplasia rara com altas taxas de recorrência local, composto por células fibroblásticas que se caracterizam pela expressão de moléculas-chave, incluindo o filamento intermediário vimentina, ciclooxigenase-2 (COX-2) e ß-catenina nuclear. Células tumorais circulantes (CTCs) isoladas do sangue periférico de pacientes com sarcomas e outras neoplasias podem ser utilizadas como biomarcadores precoces de invasão e disseminação tumoral. A família dos Receptores do Fator de Crescimento Epidérmico (Epidermal Growth Factor Receptor, EGFR) também podem influenciar no processo de invasão das CTCs, na formação de metástases e na recolonização de seus tumores de origem por meio de um processo de "auto-semeadura do tumor". Objetivo: Nosso objetivo foi identificar CTCs no sangue periférico de pacientes com TD ou sarcomas e avaliar a expressão das proteínas ß-catenina, TGF-ßRI (do Inglês, Transforming Growth Factor-ß Receptor I), COX-2 (Cyclooxygenase2), vimentina, GLUT-1 (Glucose Transporter 1), LGR5 (G-Protein Coupled Receptor 5) e EGFR, e sua correlação com sobrevidas global (SG) e livre de progressão (SLP). Materiais e Métodos: Foi realizado um estudo prospectivo de pacientes com diagnóstico inicial ou TD recidivado com doença mensurável. Para sarcomas, utilizamos amostras coletadas de forma prospectiva e retrospectiva. As amostras de sangue de cada paciente foram processadas e filtradas pelo ISET® (Rarecells, França) para isolamento e quantificação de CTCs. A expressão das proteínas foi analisada por imunocitoquímica (ICC). Para análise molecular das CTCs provenientes de pacientes com TD foi padronizado o método de PCR digital. Resultados: Foram incluídos 18 pacientes com TD, todos com CTCs detectáveis, com níveis que variaram entre 0,5­13 CTCs/mL. Encontramos uma concordância da expressão de ß-catenina em CTCs e tumores primários de 42,8% (6/14) dos casos usando ICC e imunohistoquímica, respectivamente. Nos nossos testes prévios de PCR digital, encontramos cópias mutadas de S45Pro em 4 pacientes (40%) e de S45Phe em apenas um paciente (10%). Em contraste, não foram encontradas mutações Th41Ala. Nas amostras de sarcomas, analisamos 30 amostras e encontramos CTCs em 93% dos pacientes e os níveis variaram de 0-11,25 CTCs/mL. Observamos também que a SG dos pacientes positivos para EGFR (p=0,027) eram inferiores às sobrevidas dos pacientes negativos para as mesmas proteínas. Conclusões: Nosso estudo identificou alta prevalência de CTCs em pacientes com TD e sarcomas. A concordânciada expressão de ß-catenina entre tumor primário e CTCs traz novas perspectivas para avaliar a dinâmica das CTCs no compartimento sanguíneo, abrindo novos caminhos para o estudo da biologia e comportamento do TD. Este é o primeiro estudo a demonstrar a expressão da proteína LGR5 em CTCs de pacientes com diferentes tipos de sarcomas, o que pode abrir novas oportunidades para futuras investigações. O próximo passo é caracterizar CTCs em uma coorte maior de pacientes para entender melhor o papel do LGR5 e das demais proteínas no processo de metástases tumorais em sarcomas. Além disso, esses resultados abrem a possibilidade de usar CTCs para prever a dinâmica do TD no momento da progressão da doença e tratamento. Mais estudos com tamanhos de amostra maiores são necessários para validar nossos achados tanto em TD como em sarcomas

Introduction: Desmoid tumor (DT) is a rare neoplasm with high rates of local recurrence, composed of fibroblast cells that are characterized by the expression of key molecules, including the intermediate filament vimentin, cyclooxygenase-2 (COX-2) and ß-catenin. Circulating tumor cells (CTCs) isolated from the peripheral blood of patients with sarcomas and other neoplasms can be used as early biomarkers of tumor invasion and dissemination. The Epidermal Growth Factor Receptor (EGFR) family can also influence the process of CTC invasion, metastasis formation and recolonization of their tumors of origin through a process of "tumor selfseeding". Objective: Our objective was to identify CTCs in the peripheral blood of patients with TD or sarcomas and to evaluate the expression of ßcatenin proteins, transforming growth factor receptor beta I (TGF-ßRI), COX-2 (cyclooxygenase-2), vimentin, GLUT-1 (Glucose transporter 1), LGR5 (Gprotein coupled receptor 5) and EGFR and their relation with progression free (PFS) and overall suvival (OS). Methods: We performed a prospective study of patients with initial diagnosis or relapsed TD with measurable disease. For sarcomas, we used samples collected prospectively and retrospectively. Blood samples from each patient were processed and filtered by ISET® (Rarecells, France) for isolation and quantification of CTCs. Protein expression was analyzed by immunocytochemistry (ICC). For the molecular analysis of CTCs from patients with TD, the digital PCR method was standardized. Results: Eighteen TD patients were included, all with detectable CTCs, with levels ranging from 0.5­13 CTCs/mL. We found a concordance ofß-catenin expression in CTCs and primary tumors of 42.8% (6/14) of cases using ICC and immunohistochemistry, respectively. In our previous digital PCR tests, we found mutated copies of S45Pro in 4 patients (40%) and of S45Phe in only one patient (10%). In contrast, no Th41Ala mutations were found. In the sarcoma samples, we analyzed 30 samples and found CTCs in 93% of the patients and the levels ranged from 0-11.25 CTCs/mL. We also observed that the OS of EGFR positive patients (p=0.027) were lower than the survival of negative patients for the same proteins. Conclusions: Our study identified a high prevalence of CTCs in patients with TD and sarcomas. The agreement of ß-catenin expression between primary tumor and CTCs brings new perspectives to evaluate the dynamics of CTCs in the blood compartment, opening newavenues for the study of the biology and behavior of TD. This is the first study to demonstrate the expression of LGR5 protein in CTCs from patients with different types of sarcomas, which may open new opportunities for future investigations. The next step is to characterize CTCs in a larger cohort of patients to better understand the role of LGR5 and other proteins in the process of tumor metastases in sarcomas. Furthermore, these results open up the possibility of using CTCs to predict the dynamics of TD at the time of disease progression and treatment. More studies with larger sample sizes areneeded to validate our findings in both TD and sarcomas

Proteínas mir-203a-3p e mmp-2 são altamente expressas em células tumorais circulantes de pacientes com carcinoma pancreático / Mir-203a-3p and mmp-2 proteins are highly expressed in circulating tumor cells from patients with pancreatic carcinoma

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.

Circulating tumor cells in the diagnosis and treatment of early and advanced prostate cancer / 中华男科学杂志

Circulating tumor cells (CTC) are tumor cells that escape from the primary or metastatic tumor into the circulatory system, and closely related to cancer metastasis. Since the samples can be obtained through simple and minimally invasive blood sampling operations, CTCs have a great clinical potential. PCa is one of the most common malignant tumors in men. In recent years, many scholars have conducted studies as to whether CTC technology can be used for the diagnosis and treatment of PCa, as well as for more accurate prediction of the risk of progression. This article summarizes the advances in researches relating CTC technology and the diagnosis and treatment of PCa. CTC detection has been developed from simple counting to phenotypic classification, and even to its combination with the determination of the expressions of specific genes (such as AR, AR-V7, etc.) and single-cell sequencing. Some reports showed that CTC technology has a certain significance in the early diagnosis of PCa, but its main value is demonstrated in drug sensitivity and prognosis evaluation in the late stage of the malignancy. The standardized detection methods and reference values of CTCs in PCa will be important research orientations in the near future.

Detection of peripheral blood circulating tumor cells in oral squamous cell carcinoma and its clinical significance / 华西口腔医学杂志

OBJECTIVES@#This study aims to investigate the diagnostic value of peripheral blood circulating tumor cells (CTCs) in oral squamous cell carcinoma (OSCC) and its correlation with the clinicopathological features of OSCC.@*METHODS@#Ninety-three patients diagnosed as OSCC in the First Affiliated Hospital of Zhengzhou University from May 2019 to May 2020 were selected as the experimental group, and 20 healthy volunteers were employed as the control group. The CTCs value of peripheral blood of the patients were measured by CTCs detection technology, and its clinical significance was analyzed.@*RESULTS@#The CTCs values in the experimental group were higher than those in the control group, and the difference was statistically significant (@*CONCLUSIONS@#Peripheral blood CTCs has important clinical value for early screening, auxiliary diagnosis, evaluation of metastasis, and determination of malignant degree, progression, and pathological grade of OSCC and a relatively reliable tumor detection indicator.

Significance of circulating tumor cell monitoring in targeted therapy for gastrointestinal stromal tumors / 中华胃肠外科杂志

Objective: To explore the significance of circulating tumor cell (CTC) monitoring in evaluating the efficacy of targeted therapy for gastrointestinal stromal tumor (GIST). Methods: A prospective cohort study was performed. The data of patients with locally advanced GIST or liver metastasis who were admitted to The Affiliated Hospital of Nantong University from August 2013 to December 2018 were collected. Inclusion criteria: (1) patients aged older than 18 years; (2) patients who were diagnosed with GIST based on pathology; (3) patients without surgery, whose preoperative imaging evaluation of GIST found the violations of the surrounding organs or partial transfer of an estimated difficulty to achieve R0 resection, or the maximum diameter of the tumor > 10 cm, or the liver metastasis, or the expectation of higher risk of surgical complications; (4) patients who were treated with the imatinib 400 mg/d for the first time; (5) Eastern Cooperative Oncology Group (ECOG) score of 0-2. Exclusion criteria: (1) genetic testing revealed a D842V mutation in exon 18 of the PDGFRA gene; (2) alanine aminotransferase and/or aspartate aminotransferase > 2.5 times the normal upper limit; (3) serum total bilirubin >1.5 times of normal upper limit; (4) neutrophil count < 1.5×10(9)/L, or platelet count < 75×10(9)/L, or hemoglobin < 60 g/L; (5) creatinine > normal upper limit; (6) patients had serious cardiovascular and cerebrovascular diseases within 12 months before enrollment; (7) female patients were pregnant or lactating; (8) patients suffered from other serious acute and chronic physical or mental problems, and were not suitable for participating in this study judged by researchers. The patients who could not tolerate treatment regimen, or developed serious adverse reactions and did not follow the medication scheme after enrollment were excluded. Before imatinib treatment and 1-month and 2-month after treatment, quantitative PCR was used to detect the DOG-1 expression of monocytes in peripheral blood, and the ratio of DOG-1/β-actin > 3×10(-5) was used as the CTC positive threshold of GIST. The positive rate of CTC, the efficacy of imatinib treatment (complete response, partial response, stable disease, progressive disease, and occurrence of adverse reactions), and the relationship between CTC positive rate and clinicopathological characteristics of patients were analyzed. Furthermore, the ratio of DOG-1 decrease/baseline DOG-1 after 1-month of treatment was used as an indicator to evaluate whether targeted therapy was effective. The receiver operating characteristic (ROC) curve was rendered, and the area under the curve (AUC) was calculated. Results: A total of 68 GIST patients were enrolled in this study, including 39 cases of locally advanced GIST and 29 cases with liver metastases, 32 males and 36 females with the mean age of (51.2±11.8) (range 31 to 74) years. After 2-month of imatinib treatment, 43 cases were evaluated as partial response, 11 cases as stable disease, and 14 cases as progressive disease, with an effective rate of 79.4% (54/68). During the treatment of imatinib, the incidence of grade 3 or higher adverse reactions was 22.1% (15/68), including 12 cases of grade 3 neutropenia and 3 of grade 4 drug eruption, which were all relieved after conservative treatment. The positive rates of CTC in 68 patients before treatment, 1-month and 2-month after treatment were 66.2% (45/68), 41.2% (28/68) and 23.5% (16/68), respectively. The positive rate of CTC was associated with tumor size, liver metastasis, mitotic count and risk level (all P<0.05). By analyzing the effective group and the ineffective group of targeted therapy, it was found that the positive rate of CTC in the effective group showed a decreasing trend, while the positive rate of CTC in the ineffective group showed an increasing trend. The AUC of predicting the efficacy of targeted therapy for GIST was 0.823 by detecting the change trend of CTC 1-month after treatment (P<0.001). When the DOG-1 content decreased by more than 57.5% 1-month after treatment, it can be used as an indicator to judge the effectiveness of the treatment, whose sensitivity was 72.2% and specificity was 100%. Conclusion: The detection of peripheral blood CTC can evaluate the efficacy of targeted therapy in GIST patients and can provide decision-making basis for further clinical treatment.

Comparação entre expressão de receptores hormonais e de HER-2 entre células tumorais circulantes e metástases de câncer de mama / Comparison of hormonal receptor expression and HER2 status in circulating tumor cells and breast cancer metastases

Introdução: O câncer de mama é a neoplasia mais comum em mulheres. A maioria deles é diagnosticada em estágios iniciais, quando o tratamento visa a cura. Mas apesar dos avanços no tratamento, metástases à distância podem ocorrer. A biópsia das lesões metastáticas é recomendada para confirmar o status do receptor de estrogênio (RE), receptor de progesterona (RP) e receptor do fator de crescimento epidérmico humano 2 (HER2), por ocorrerem discrepâncias nesses padrões entre tumores primários e metástases em até 40% dos casos. As células tumorais circulantes (CTCs) estão relacionadas às evoluções clínicas do câncer de mama e podem potencialmente desempenhar um papel substituto aos procedimentos invasivos de rebiópsia de metástase. A tecnologia ISET® (Isolation by SizE of Tumor Cells, Rarecells-Diagnostics, Paris, França) não é usualmente empregada para detectar CTCs em pacientes com câncer de mama, embora seja reconhecida como uma ferramenta útil em alguns outros tumores. Existem dados emergentes de que a caracterização da expressão proteica das CTC pode refinar seu valor prognóstico. Sabe-se que o fator de transformação de crescimento (TGF-ß) desempenha um papel na progressão e invasividade do câncer de mama. Objetivos: Comparar a expressão de RE, RP e HER2 em tumores primários, CTCs, metástases e avaliar a expressão do receptor TGF-ß tipo 1 (TGF-ß RI) em CTCs como fator prognóstico para sobrevida global. Metodologia: Estudo realizado no A.C.Camargo Cancer Center, Brasil. As amostras de sangue foram coletadas antes da biópsia guiada por tomografia computadorizada de lesões metastáticas suspeitas e processadas pela metodologia ISET®. Os níveis de expressão proteica das CTCs foram comparados aos de tumores primários e metástases e correlacionados aos resultados clínicos. Todos os dados clínicopatológicos foram obtidos dos prontuários médicos. Resultados: Dos 39 pacientes inicialmente incluídos, 27 tiveram tanto a biópsia de metástases quanto a coleta de sangue e foram considerados para análise. As taxas de concordância para a expressão de RE, RP e HER2 entre tumores primários e metástases foram altas. Não foi observada nenhuma perda de expressão de HER2 nas metástases e os tumores triplo negativos mantiveram o mesmo padrão em todas as metástases (p <0,0001). Quando as metástases e CTCs foram classificadas como triplo negativo (TN) ou não ­ TN, as CTCs determinaram alta especificidade (93%), acurácia (84,2%) e valor preditivo negativo (88%). A sobrevida global mediana de pacientes sem expressão de TGF-ß RI em CTCs foi de 42,6 x 20,8 meses para os positivos, clinicamente relevante, porém sem significância estatística (p> 0,05). Conclusões: No câncer de mama, o papel das CTCs detectadas pelo ISET® ainda não está estabelecido. Com este estudo, sugerimos que esta metodologia possa ser útil para avaliar metástases em casos de tumores não TN, assim como a expressão de TGF-ß RI em CTCs, o que pode impactar a sobrevida. Devido à limitação da amostra, estudos futuros devem se concentrar em subtipos específicos de câncer de mama, ampliando a coorte.

Introduction: Breast cancer (BC) is the most common neoplasm in women. Most of BC are diagnosed in early stages, when treatment aims cure. Despite advances in BC treatment, distant metastases may develop. Biopsy of metastatic lesions is recommended to confirm estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status, due to discrepancies in these patterns between primary tumors/metastasis in up to 40% of cases. Circulating Tumor Cells (CTCs) are related to breast cancer outcomes and could potentially play a role surrogating invasive procedures of metastasis rebiopsy. ISET® (Isolation by SizE of Tumor Cells, Rarecells-Diagnostics, Paris, France) technology is not currently employed to detect CTCs in breast cancer patients, although recognized as a useful tool in some other tumors. There are emerging data that characterization of CTC protein expression can refine its prognostic value. Transforming growth factor (TGF)-ß play a role in progression/invasiveness of BC. Objectives: To compare ER, PR and HER2 expression in primary tumors, CTCs, metastases and to evaluate TGF-ß type 1 receptor (TGF- ß RI) expression in CTCs as prognostic factor for overall survival. Methods: Study conducted at the A.C.Camargo Cancer Center, Brazil. Blood samples were processed in ISET® before computed tomography­guided biopsy of suspected metastatic lesions. Protein expression levels in CTCs were compared to those in primary tumors/metastases and correlated to clinical outcomes. All clinicopathological data were obtained from medical records. Results: From the 39 patients initially included, 27 had both biopsy of metastases and blood collection and were considered for analysis. Concordance rates for ER, PR and HER2 expression between primary tumors/metastases were high. No loss of HER2 expression at any metastasis site and retention of the same pattern in all triplenegative (TN) tumors (p <0.0001) were observed. When metastases/CTCs were classified as TN/non­TN, CTCs showed high specificity (93%), accuracy (84.2%) and negative predictive value (88%). The median overall survival of patients with no TGF-ß RI expression in CTCs was 42.6 x 20.8 months for positive ones, clinically relevant but not statistically significant (p>0.05). Conclusions: In BC, the role of CTCs detected by ISET® is not yet established. Here, we could suggest that this methodology may be useful to evaluate metastasis in non-TN cases as also TGF-ß RI expression in CTCs, which may impact survival. Due to sample limitation, future studies must focus on specific subtypes of BC, expanding the cohort.

Identificação e correlação molecular e dinâmica de proteínas relacionadas à resistência ao tratamento neoadjuvante em células tumorais circulantes de pacientes com câncer de reto localmente avançado / Molecular and dynamic evaluation of proteins related to resistance to neoadjuvant treatment with chemoradiotherapy in circulating tumor cells of patients with locally advanced rectal cancer

INTRODUÇÃO: A quimiorradioterapia neoadjuvante (QRTN) consolidou-se como a principal estratégia para o tratamento do câncer de reto localmente avançado (CRLA). No entanto, respostas heterogêneas são observadas com o tratamento neoadjuvante, com apenas 15-20% dos pacientes com resposta patológica completa (RPC). Diante da necessidade de estratificar os pacientes em respondedores e não respondedores à QRTN antes do seu início, com o objetivo de aprimorar a seleção daqueles com maior probabilidade de obter uma RPC, vários estudos avaliam a identificação de possíveis biomarcadores. O objetivo primário deste estudo prospectivo foi analisar se a ausência da expressão do homólogo B de RAD23 (RAD23B) e da timidilato sintase (TYMS) nas células tumorais circulantes (CTCs) se correlacionaria com a RPC para os pacientes submetidos à QRTN e, assim, identificar possíveis respondedores ao tratamento. Os desfechos secundários foram avaliar a cinética das CTCs antes (C1) e após QRTN (C2), além da correlação da expressão de marcadores de resposta imune, como o Tumor Growth Factor ß Receptor I (TGF-ßRI) e Programmed Death ligand-1 (PD-L1) com a sobrevida livre de doença (SLD) e sobrevida global (SG). MÉTODOS: Entre 2016 e 2020, 63 pacientes com CRLA (cT3/T4 e/ou N+) submetidos a QRTN foram incluídos no estudo. As CTCs foram isoladas por ISET e avaliadas por imunocitoquímica. A expressão de RAD23B, TYMS, PD-L1 e TGF-ßRI foi avaliada nesta ordem de prioridade de acordo com o objetivo primário do estudo em cada momento de coleta e a disponibilidade de células (contagem de CTCs > 0) na amostra. RESULTADOS: Em C1, RAD23B foi detectado em 54,1% dos pacientes sem RPC e sua ausência em 91,7% dos pacientes com RPC (p = 0,014); Na segunda coleta, dos 13 pacientes com RPC, 10 não apresentaram expressão de RAD23B nas CTCs. Para os pacientes que não obtiveram RPC com QRTN, 51,7% apresentavam a expressão de RAD23B em CTC em C2 (p = 0,06). Na análise univariada (OR =0,077;IC 95%, 0,009-0,661; p = 0,019) e multivariada (OR= 0,064;CI 95%, 0,006-0,75; p = 0,029) para RPC, observamos que a expressão de RAD23B foi associado com menor chance de resposta em comparação com os pacientes com a ausência da expressão do RAD23B na C1. A ausência da expressão da TYMS foi observado em 90% dos pacientes com RPC e sua expressão em 51,7% sem RPC (p = 0,057). Na avaliação da cinética da CTCs pacientes com CTC2> CTC1 (cinética desfavorável) tiveram pior SLD (p = 0,00025) e SG (p = 0,0036) em comparação com aqueles com CTC2 ≤CTC1 (cinética favorável). A expressão de TGF-ßRI em qualquer momento das coletas correlacionou-se com pior SLD (p = 0,059). CONCLUSÃO: Demonstramos uma possível correlação entre a ausência de expressão de RAD23B e TYMS nas CTCs com a RPC, sendo um resultado importante para identificar os respondedores ao tratamento neoadjuvante, ajudando individualizar a abordagem terapêutica. Além disso, a cinética desfavorável e a expressão de TGF-ßRI nas CTCs se correlacionaram com pior sobrevida

INTRODUCTION: Neoadjuvant chemoradiotherapy (NCRT) has established itself as the main strategy for the treatment of locally advanced rectal cancer (LARC). However, heterogeneous responses are observed with neoadjuvant treatment, with only 15-20% of patients with complete pathological response (pCR). Given the need to stratify patients into responders and non-responders to NCRT prior to its initiation, in order to improve the selection of those most likely to obtain a pCR, several studies have assessed the identification of potential biomarkers capable of stratifying and monitoring the patient's response. The primary objective of this prospective study was to analyze whether the absence of RAD23 homolog B expression (RAD23B) and thymidylate synthase (TYMS) in circulating tumor cells (CTCs) would correlate with pCR for patients undergoing NCRT and thus identify possible responders to treatment. The secondary outcomes were to evaluate the kinetics of CTCs before (C1) and after NCRT (C2), in addition to the correlation of the expression of immune response markers, such as Tumor Growth Factor ß Receptor I (TGF-ßRI) and Programmed Death ligand- 1 (PD-L1) with clinical outcomes such as disease-free survival (DFS) and overall survival (OS). METHODS: Between 2016 and 2020, 63 patients (pts) with LARC (cT3 / T4 or N +) submitted to NCRT were included in the study. CTCs were isolated by ISET and evaluated by immunocytochemistry (protein expression). The expression of RAD23B, TYMS, PD-L1 and TGF-ßRI was evaluated in this order of priority according to the primary objective of the study at each time of collection (C1, C2 and C3) and the availability of cells (CTC count> 0) in the sample. RESULTS: In C1, RAD23B was detected in 54.1% of patients without pCR and its absence in 91.7% of patients with pCR (p = 0.014). In the second collection, of the 13 patients with pCR, 10 did not show RAD23B expression in the CTCs. For patients who did not obtain pCR with NCRT, 51.7% had RAD23B expression in CTC in C2 (p = 0.06). In the univariate (OR = 0.077; 95% CI, 0.009-0.661; p = 0.019) and multivariate (OR = 0.064; 95% CI, 0.006-0.75; p = 0.029) logistic regression models for pCR, we observed that the expression of RAD23B was associated with a lower chance of response compared to patients with the absence of RAD23B expression in C1. The absence of TYMS expression was observed in 90% of patients with pCR and its expression in 51.7% without pCR (p = 0.057). In the evaluation of CTCs kinetics patients with CTC2> CTC1 (unfavorable kinetics) had worse DFS (p = 0.00025) and overall survival (OS) (p = 0.0036) compared with those with CTC2 ≤CTC1 (favorable kinetics). TGF-ßRI expression at any time of the collections was correlated with worse DFS (p = 0.059). CONCLUSION: We demonstrated a possible correlation between the absence of RAD23B and TYMS expression in CTCs with pCR, being an important result to identify respondents to neoadjuvant treatment, helping to individualize the therapeutic approach. In addition, the unfavorable kinetics and expression of TGF-ßRI in CTCs correlated with worse survival.

Comparison of hormonal receptor expression and HER2 status between circulating tumor cells and breast cancer metastases

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.

Tromboembolismo tumoral como causa directa de muerte / Tumor thromboembolism as direct cause of death

Introducción: el embolismo tumoral pulmonar es la presencia de acúmulos de células tumorales en las arterias pulmonares arteriolas y capilares pulmonares. Su incidencia en autopsias ha sido descrita entre 3 y 26 por ciento de los pacientes con tumores sólidos, más frecuentemente de mama, estómago, hígado y pulmón. Objetivo: describir los argumentos clínicos que sugieren la sospecha diagnóstica de tromboembolismo tumoral en una paciente atendida en el Hospital Dr. Carlos J Finlay de La Habana, Cuba. Presentación del caso: paciente femenina de 80 años de edad con antecedentes patológicos personales de cirrosis hepática por virus de hepatitis C. Dos meses antes del ingreso comienza con síntomas generales asociados a edemas en miembros inferiores y diarreas. Ingresa en un servicio de Medicina Interna con el diagnóstico de cirrosis hepática descompensada y la sospecha de hepatocarcinoma. Al quinto día de su ingreso comienza de forma súbita con disnea y fallece. Conclusiones: los hallazgos anatomopatológicos informan como causa directa de muerte tromboembolismo tumoral. La embolia pulmonar tumoral debe ser considerada como diagnóstico diferencial en todo paciente con evidencia de neoplasia que presente disnea(AU)

Introduction: Pulmonary tumor thromboembolism is the presence of accumulations of tumor cells in the pulmonary arterioles and pulmonary capillaries. Its incidence in autopsies has been described between 3 percent and 26 percent of patients with solid tumors, most frequently in breast, stomach, liver and lung tumors. Objective: To describe clinical arguments suggesting the suspected diagnosis of tumor thromboembolism in a patient treated at the "Dr. Carlos J. Finlay'' Hospital in Havana, Cuba. Case presentation: Eighty-year-old female patient with personal pathological antecedents of liver cirrhosis due to hepatitis C virus. Two months before admission she started presenting general symptoms associated with edemaof the lower limbs and diarrhea. She was admitted to an Internal Medicine service with the diagnosis of decompensated hepatic cirrhosis and the suspicion of hepatocarcinoma. On the fifth day after admission, he suddenly began with dyspnea and died. Conclusions: Anatomopathological findings report tumor thromboembolism as a direct cause of death. Pulmonary tumor thromboembolism should be considered in the differential diagnosis of all patients with evidence of neoplasia who are presenting dyspnea(AU)

Avaliação prospectiva da correlação entre células tumorais circulantes e controle da doença encefálica após radioterapia focal de metástases encefálicas de câncer da mama / Prospective assessment of the correlation between circulating tumor cells and control of brain disease after focal radiotherapy of breast cancer brain metastases

Introdução: Predizer o risco de progressão encefálica distante (PED) precoce é um recurso útil e premente para a decisão terapêutica em pacientes candidatas ao tratamento local de metástases encefálicas. Este estudo tem como objetivo analisar a correlação entre células tumorais circulantes (CTCs) e controle da doença encefálica após radioterapia estereotática/radiocirurgia (RTE) de metástases encefálicas de câncer da mama (ME). Métodos: Avaliação prospectiva de CTC antes (CTC1) e 4­5 semanas após (CTC2) a RTE de ME e suas relações com o número de lesões sugestivas de ME (NL). As CTCs foram isoladas e quantificadas pelo método ISET (Rarecells, França) e analisadas por imunocitoquímica para avaliar a expressão das proteínas COX2, EGFR, ST6GALNAC5, NOTCH1 e HER2. Sobrevida livre de progressão encefálica distante (SLPED), o objetivo primário, sobrevida livre de progressão encefálica com envolvimento difuso (SLPED-ED), definida como progressão com mais de 4 novas ME ou carcinomatose meníngea, e sobrevida global (SG) foram estimadas pelo estimador de Kaplan-Meier. Testes de log-rank foram aplicados a fim de comparar as curvas de sobrevida. Para análise multivariada dos fatores prognósticos que afetaram a SPED e SG, foi ajustado o modelo proporcional de Cox. Análise de risco competitivo para SLPED na presença do óbito foi realizada. Resultados: Foram incluídas 39 pacientes entre novembro de 2016 e fevereiro de 2018. A idade mediana no momento da RTE foi 54 (34-70) anos e a avaliação prognóstica graduada doença-específica (DS-GPA) foi 1,5­2 em 17,5% e 2,5­4 em 82,5% das pacientes. CTCs foram detectadas em todas as 39 pacientes antes da RTE e a CTC1 mediana foi 2 CTC/mL. Após a RTE, CTCs foram detectadas em 34 das 35 pacientes (4 mortes entre CTC1 e CTC2) e a CTC2 mediana foi 2,33 CTC/mL. Após seguimento mediano de 16,6 (IC95%: 14,8­18,4) meses, 15 pacientes evoluíram com PED, sendo 6 com progressão encefálica distante com envolvimento difuso (PED-ED), e 16 pacientes faleceram. A SLPED, SLPED-ED e SG mediana foram 15,3, 14,1 e 19,5 meses, respectivamente. A incidência cumulativa, com a morte como risco competitivo, de PED em 6 meses foi 40% nas pacientes com CTC1 ≤ 0,5 e 8,82% nas pacientes com CTC1 > 0,5 CTC/mL (p = 0,007) e a de PED-ED em 6 meses foi 40% nas pacientes com CTC1 ≤ 0.5 e 0 nas pacientes com CTC1 > 0,5 CTC/mL (p = 0,005) e 25% nas pacientes com NL/CTC1 > 6,8 e 2,65% com NL/CTC1 ≤ 6,8 (p = 0,063). Na análise mutivariada, a SLPED foi inferior nas pacientes com CTC1 ≤ 0,5 CTC/mL (HR 8,27, IC95%:2,12­32,3; p = 0,002) e superior nas pacientes com imunofenótipo HER2-positivo (HR 0,128, IC95%:0,025­0,534; p = 0,013), a SLPED-ED foi inferior nas pacientes com CTC1 ≤ 0,5 CTC/mL (HR 10,22, IC95%:1,99­52,41; p = 0,005) e a SG foi superior nas pacientes com imunofenótipo HER2-positivo (HR 0,073, IC95%:0,018-0,288; p < 0,0001) e luminal B (HR 0,224, IC95%:0,062­0,816; p = 0,023) e nas pacientes com NL/CTC1 ≤ 2,2 (HR 0,159, 95% CI 0,05­0,505; p = 0,002). Não houve associação entre a expressão das proteínas nas CTCs e PED e SG. Conclusões: CTC1 foi um fator prognóstico independente de SLPED e SLPED-ED e NL/CTC1 foi um fator prognóstico independente de SG e um potencial fator prognóstico de PED-ED em 6 meses. Estes dados sugerem que CTC1 e NL/CTC1 podem ter um papel como biomarcador da PED-ED precoce, auxiliando a definir o momento e o tipo da radioterapia de resgate a fim de otimizar o controle das ME

Introduction: Predicting the risk of early distant brain failure (DBF) is a useful and demanding resource for management decisions in patients who are candidates to local treatment of brain metastasis. This study aims to analyze the correlation between circulating tumor cells (CTCs) and brain disease control after stereotactic radiotherapy/radiosurgery (SRT) for breast cancer brain metastasis (BM). Methods: Prospective assessment of CTCs before (CTC1) and 4­5 weeks after (CTC2) SRT for BM and its relations with the number of suggestive lesions of BM (NL). CTCs were isolated and quantified by the ISET method (Rarecells, France) and analyzed by immunocytochemistry to evaluate the expression of the proteins COX2, EGFR, ST6GALNAC5, NOTCH1 e HER2. Distant brain failure-free survival (DBFFS), the primary endpoint, diffuse distant brain failure-free survival (D-DBFFS), defined as progression with more than 4 new BM or meningeal carcinomatosis, and overall survival (OS) were estimated by Kaplan-Meier estimator. Log-rank tests were applied in order to compare the survival curves. For multivariate analysis of prognostic factors that affected DBFFS and OS, the Cox proportional model was adjusted. Competing risk analysis for DBFFS in the presence of death was performed. Results: 39 patients were included between November 2016 and February 2018. The median age at SRT was 54 (34-70) years and the diagnosis-specific graded prognostic assessment (DS-GPA) was 1.5­2 in 17.5% and 2.5­4 in 82.5% of them. CTCs were detected in all 39 patients before SRT and the median CTC1 was 2 CTC/mL. After SRT, CTCs were detected in 34 of 35 patients (4 deaths between CTC1 and CTC2) and the median CTC2 was 2.33 CTC/mL. After a median follow-up of 16.6 (95% CI: 14.8­18.4) months, there were 15 patients with DBF, being 6 with diffuse distant brain failure (D-DBF), and 16 deaths. The median DBFFS, D-DBFFS and OS were 15.3, 14.1 and 19.5 months, respectively. The cumulative incidence, with death as competing risk factor, of DBF at 6 months was 40% in patients with CTC1 ≤ 0.5 and 8.82% in patients with CTC1 > 0.5 CTC/mL (p = 0.007) and of D-DBF at 6 months was 40% in patients with CTC1 ≤ 0.5 and 0 in patients with CTC1 > 0.5 CTC/mL (p = 0.005) and 25% in patients with NL/CTC1 > 6.8 and 2.65% with NL/CTC1 ≤ 6.8 (p = 0.063). On multivariate analysis, DBFFS was inferior in patients with CTC1 ≤ 0.5 CTC/mL (HR 8.27, 95% CI 2.12­32.3; p = 0.002) and superior in patients with immunophenotype HER2-positive (HR 0.128, 95% CI 0.025­0.534; p = 0.013), D-DBFFS was inferior in patients with CTC1 ≤ 0.5 CTC/mL (HR 10.22, 95% CI 1.99­52.41; p = 0.005) and OS was superior in patients with immunophenotype HER2-positive (HR 0.073, 95% CI 0.018-0.288; p < 0.0001) and luminal B (HR 0.224, 95% CI 0.062­0.816; p = 0.023) and in patients with NL/CTC1 ≤ 2.2 (HR 0.159, 95% CI 0.05­0.505; p = 0.002). There was no association between protein expression in CTCs and DBF and OS. Conclusions: CTC1 was an independent prognostic factor of DBFFS and D-DBFFS and NL/CTC1 was an independent prognostic factor of OS and a potential prognostic factor of D-DBF at 6 months. These data suggest that CTC1 and NL/CTC1 may have a role as a biomarker of early D-DBF, helping define the timing and type of salvage radiotherapy in order to optimize the control of BM

Circulating Tumor Cell Number Is Associated with Primary Tumor Volume in Patients with Lung Adenocarcinoma / 결핵

Análise de marcadores sanguíneos relacionados a prognóstico em câncer de reto localmente avançado / Analysis of prognostic-related blood markers in locally advanced rectal cancer

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

Evaluating mmp-2 and tgfß-ri expression in circulating tumor cells of pancreatic cancer patients and their correlation with clinical evolution / Avaliação da expressão de mmp-2 e tgfß-ri em células tumorais circulantes de pacientes com câncer de pâncreas e sua correlação com evolução clínica

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.

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.

Impacto prognóstico de células tumorais circulantes e potencial papel preditor de resposta ao tratamento em carcinoma epidermóide de cabeça e pescoço localmente avançado / Prognostic impact and potential predictive role of circulating tumor cells in locally advanced head and neck squamous cell carcinoma

Introdução: O papel prognóstico de células tumorais circulantes (CTCs) em câncer de cabeça e pescoço localmente avançado (CCPLA) ainda não está determinado, devido a resultados conflitantes em estudos prévios, a maioria utilizando técnicas dependentes de citoqueratina para identificação e contagem de CTCs. O objetivo primário deste estudo é determinar a taxa de detecção utilizando o método ISET, o papel prognóstico e potencial papel preditivo de CTCs em CCPLA. Métodos: Prospectivamente amostras de sangue de pacientes com CCPLA não metastático, estágios III/IV, foram analisadas para CTCs antes e após o tratamento, em dois cenários: cirurgia curativa inicial e radioterapia (RT) adjuvante e candidatos a estratégia não cirúrgica (irressecáveis ou preservação de órgão) com RT concomitante a quimioterapia (QT) ou cetuximabe, precedida ou não por QT de indução (QTI). Resultados: Foram incluídos 83 pacientes e a taxa de detecção de CTCs baseline foi de 94% (78/83). A contagem de CTCs se correlacionou significativamente com sobrevida, com um aumento relativo de 18% no risco de óbito (HR=1,18; CI95%: 1,06-1,31; p<0,001), 16% no risco de progressão (HR=1,16; CI95%: 1,04-1,28; p=0,004) e uma redução de 26% na chance de resposta completa ao tratamento (OR=0.74; CI95%: 0.58-0.95; p=0.022) para cada aumento de uma CTC. Pacientes com CTCs < 6,5/mL apresentaram estimativa de sobrevida global (SG) em dois anos de 85,6% x 22,9% para CTCs ≥ 6.5/mL (HR=0,18; CI95%: 0,06-0,49; P<0,0001) e pacientes com CTCs ≤ 3.8/mL uma estimativa de sobrevida livre de progressão (SLP) em dois anos de 71,8% x 37% para CTCs > 3.8/mL (HR=0,32; CI95%:0,15-0,67; p=0,001). Após o tratamento, contagens altas de CTCs (ponto de corte 6,6/mL) se correlacionaram significativamente com pior SG (HR=0,12; CI95%: 0,06-0,40; p<0,001) e SLP (HR=0,19; CI95%: 0,06-0,59; p=0,001). No subgrupo de tratamento não cirúrgico (n=67), presença de microêmbolos (ME) se correlacionou significativamente com pior SG (HR=3,01; CI95%: 1,06-8,52; p=0,029) e SLP (HR=3,84; CI95%: 1,62-9,11; p<0,001). Neste subgrupo, CTCs altas (>3,8/mL) e ME foram identificados como potenciais preditores do benefício de QTI. Expressão de MRP-7 em ME baseline se relacionou a pior SG (HR=3,49; CI95%: 1,01-12,04; p=0,047) e SLP (HR=3,62; CI95%: 1,08-12,13; p=0,037) e expressão de TFGßRI nas CTCs após o tratamento a pior SG (HR=3,60; 1,03-12,59; p=0,032). Expressão de ß-tubulina III nas CTCs se relacionou a pior SG em pacientes recebendo QTI (p=0.012). Pacientes com cinética favorável de CTCs tiveram melhor SG (HR=0,22; IC95%: 0,07-0,67; p=0,004) e SLP (HR=0,33; CI95%: 0,13-0,84; p=0,015). Conclusões: Contagem de CTCs baseline se correlacionaram com sobrevida e resposta ao tratamento e, junto com ME, são potenciais fatores preditivos do benefício de QTI. Contagens de CTCs altas após o tratamento e cinética desfavorável também foram prognósticos. Expressão de biomarcadores em CTCs e ME tem papel prognóstico e preditivo em CCPLA

Introduction: The prognostic role of circulating tumor cells (CTCs) in locally advanced head and neck squamous cell carcinoma (LAHNSCC) is yet to be determined, with conflicting results in previous trials, the majority utilizing cytokeratin dependent techniques for identification and counting of CTCs. The primary objective of this study is to determine the detection rates using the ISET method, the prognostic and potential predictive role of CTCs in LAHNSCC patients (pts) treated with a curative intent. Methods: In this prospective study, peripheral blood samples of pts with non-metastatic LAHNSCC, stages III/IV, were analyzed for CTCs before and after treatment, in two scenarios: curative surgical resection and adjuvant radiotherapy (RT) and candidates for a non-surgical strategy (unresectable or organ preservation) with RT concurrent with chemotherapy (CT) or cetuximab, preceded or not by induction CT (ICT). Results: Eighty-three pts were included, and the detection rate of baseline CTCs was 94% (78/83). The CTCs counts were significantly correlated with survival, with an a relative increase of 18% in the risk of death (HR=1.18; CI95%: 1.06-1.31; p<0.001), 16% in the risk of progression (HR=1.16; CI95%: 1.04-1.28; p=0.004) and a reduction of 26% in the odds of complete response to treatment (OR=0.74; CI95%: 0.58-0.95; p=0.022) for each increase of one CTC. Pts with CTCs < 6,5/mL had an estimated two year overall survival (OS) of 85.6% x 22.9% for CTCs ≥ 6.5/mL (HR=0.18; CI95%: 0.06-0.49; P<0.0001) and pts with CTCs ≤ 3.8/mL had an estimated two year progression free survival (PFS) of 71.8% x 37% for CTCs > 3.8/mL (HR=0.32; CI95%:0.15-0.67; p=0.001). After treatment, high CTCs counts (cut-off 6,6/mL) were significantly correlated with worse OS (HR=0.12; CI95%: 0.06-0.40; p<0.001) and PFS (HR=0.19; CI95%: 0.06-0.59; p=0.001). In the non-surgical subgroup (n=67), the presence of microemboli (ME) was correlated with a significantly worse OS (HR=3.01; CI95%: 1.06-8.52; p=0.029) and PFS (HR=3.84; CI95%: 1.62-9.11; p<0.001). In this subgroup, high CTCs counts (>3,8/mL) and ME were identified as potential predictive factors for efficacy of ICT. Baseline expression of MRP-7 in ME was related to a worse OS (HR=3.49; CI95%: 1.01-12.04; p=0.047) and PFS (HR=3.62; CI95%: 1.08-12.13; p=0.037) and TFGßRI expression in CTCs after treatment to a worse OS (HR=3.60; 1.03-12.59; p=0.032). ß-tubulin III expression in CTCs was related to a worse OS in pts receiving ICT (p=0.012). Patients with favorable kinetics of CTCs had better OS (HR=0.22; IC95%: 0.07-0.67; p=0.004) and PFS (HR=0.33; CI95%: 0.13-0.84; p=0.015). Conclusions: Baseline CTCs counts were correlated with survival and response to treatment and, along with ME, potential predictive factors of ICT benefit. High CTCs counts after treatment and unfavorable kinetics were also prognostic. Biomarker expression in the CTCs and ME have prognostic and predicitive role in LAHNSCC

Correlation between circulating tumor cells and clinicopathological features of early breast cancer / 中南大学学报(医学版)

To investigate the correlation between the number of peripheral blood circulating tumor cells (CTCs) and clinicopathological features of early breast cancer. 
 Methods: The clinical and pathological data from 100 patients with early breast cancer treated by a breast cancer treatment team in the Department of Breast Surgery, Second Xiangya Hospital, Central South University, were collected from January 2017 to December 2018. For these patients, their peripheral blood CTCs were detected, enumerated and typed by CanpatrolTM CTC assay.
 Results: The positive rate of CTCs was 90% in peripheral blood of patients with early breast cancer, and the majority of molecular phenotypes was hybrid CTCs (55.6%). The positive rate of CTCs was only related to the pathological type of tumor (P<0.05), but not to other clinicopathological features. No correlation between clinicopathological features and the total number of CTCs, the number of epithelial CTCs or the number of hybrid CTCs was found. However, the number of mesenchymal CTCs was significantly correlated with the expression of hormone receptors and Ki-67 (r=0.200, P<0.05), and there was a significant correlation between the proportion of mesenchymal CTCs and the expression level of Ki-67 (r=0.213, P<0.05).
 Conclusion: The number of CTCs is not correlated with all clinicopathological features, but patients with negative hormone receptor and high expression of Ki-67 probably have more hybrid CTCs.

Clinical Application of Circulating Tumor Cells in Gastric Cancer

Early detection and accurate monitoring of cancer is important for improving clinical outcomes. Endoscopic biopsy and/or surgical resection specimens are the gold standard for diagnosing gastric cancer and are also useful for selecting therapeutic strategies based on the analysis of genomic/immune parameters. However, these approaches cannot be easily performed because of their invasiveness and because these specimens do not always reflect tumor dynamics and drug sensitivities during therapeutic processes, especially chemotherapy. Accordingly, many researchers have tried to develop noninvasive novel biomarkers that can monitor real-time tumor dynamics for early diagnosis, prognostic evaluation, and prediction of recurrence and therapeutic efficacy. Circulating tumor cells (CTCs) are metastatic cells that are released from the primary tumors into the blood stream and comprise a crucial step in hematogenous metastasis. CTCs, as a liquid biopsy, have received a considerable amount of attention from researchers since they are easily accessible in peripheral blood, avoiding the invasiveness associated with traditional biopsy techniques; they can also be used to derive clinical information for monitoring disease status. In this review, with respect to CTCs, we summarize the metastatic cascade, detection methods, clinical applications, and prospects for patients with gastric cancer.

Circulating Tumor Cells: Liquid Biopsy for Early Detection of Cancer

Cancer is a complex, heterogeneic, and dynamic disease involving multiple gene-environment interactions, and affecting numerous biological pathways. As such, the development of reliable and robust non-invasive platforms constitutes a vital step toward realizing the potential of precision medicine. Distant metastases harbor unique genomic characteristics that are not detectable in the corresponding primary tumor of the same patient, and metastases located at different sites show considerable intra-patient heterogeneity. Thus, the analysis of the resected primary tumor alone or, if possible, re-evaluation of tumor characteristics based on the biopsy of the most accessible metastasis, may not reveal sufficient information for treatment decisions. Here, we propose that this dilemma can be solved by a new diagnostic concept: liquid biopsy, that is, the analysis of therapeutic targets and drug resistance-conferring gene mutations in or on circulating tumor cells (CTCs). Finally, the analysis of the resected primary tumor alone may provide misleading information with regard to the characteristics of metastases, the key target for systemic anticancer therapy. Liquid biopsies are noninvasive tests using blood or fluids that detect CTCs or the products of tumors, such as fragments of nucleotides or proteins that are shed into biological fluids from the primary or metastatic tumors. Such biopsies are expected to be informative or easily accessible tools to provide comprehensive information regarding cancers beyond conventional biopsies. Thus, this review addresses the use of CTCs in cancer detection, diagnosis and monitoring and discusses the direction of its clinical application in cancer patient care.

Identificação de células tumorais circulantes e outros componentes sanguíneos em pacientes com câncer de cólon localizado / Identification of circulating tumor cells and other blood componentes in localized colon cancer

Introdução: A detecção precoce do câncer de cólon proporciona altas taxas de cura, no entanto, há pacientes que apresentam recidiva local e metástase à distância. As células tumorais circulantes (CTCs) e os microêmbolos circulantes (MEs) desempenham um papel importante nestes processos. Objetivo: avaliar o papel de CTCs e MEs em pacientes com câncer de cólon localizado. Material e métodos: foram coletados 10 mL de sangue de pacientes com câncer de cólon pré-cirúrgico, pré-adjuvância e 6 meses após o final do tratamento. As amostras foram processadas no dispositivo ISET® e as CTCs foram fixadas com formaldeído e identificadas por imunocitoquímica. Para detecção de expressão de RNAm, foi realizada a técnica de hibridização in situ cromogênica. O DNA foi extraído das membranas sem formaldeído e analisado por PCR digital em gotas. Resultados: no módulo I, foram incluídos 69 pacientes (18 com estágio I, 15 com estágio II e 36 com estágio III). A taxa de detecção de CTCs na primeira coleta foi de 94,2%, de 94,6% no primeiro seguimento e de 100% no segundo seguimento. Foi observada uma queda global na mediana de CTCs ao longo do tempo. No segundo seguimento, a expressão de ERCC1 e de ß-galactosidase nas CTCs foi mais encontrada em pacientes com estágio III (p= 0,03 e p= 0,04, respectivamente). A expressão de ERCC1 com alto índice de positividade (IP) nas CTCs, no segundo seguimento foi determinante de sobrevida livre de recidiva (SLR) inferior (p= 0,014). Foi encontrada uma correlação positiva entre o nível de CTCs e a porcentagem de células TReg (p= 0,01) e negativa entre o nível de CTCs e a porcentagem de linfócitos CD3+ (p= 0,01). Pacientes com alta Platelet-to-Lymphocyte Ratio (PLR) encontravam-se em sua maioria com estadiamentos patológicos II de alto risco e III (p= 0,014). Alta PLR foi determinante de SLR inferior (p= 0,01). No módulo II (pacientes com tumores de alto risco, submetidos à quimioterapia adjuvante) foi encontrada uma correlação positiva entre os níveis de CTC e CEA nos casos que tiveram recidiva da doença (p= 0,001). Alto IP de ERCC1 no segundo seguimento foi determinante de SLR significantemente inferior (p= 0,013). Conclusões: CTCs foram encontradas em altas taxas nos pacientes com câncer de cólon localmente avançado. A avaliação do sistema imunológico dos pacientes juntamente com as CTCs demonstrou ser uma ferramenta promissora para acompanhamento destes pacientes

Introduction: The early detection of colon cancer provides high cure rates, however, there are patients that present local relapse and distant metastasis. Circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) play a crucial role in these processes. Objectives: to evaluate the role of CTCs and CTM in non-metastatic colon cancer patients. Material and methods: 10 mL of blood were collected from colon cancer patients prior to the surgery, prior to the adjuvant treatment, and 6 months after the treatment end. Samples were processed in the ISET® device and CTCs were formaldehyde-fixed and identified by immunocytochemistry. For mRNA expression in situ hybridization was applied. The DNA was extracted from the non-fixed CTCs and analyzed by droplet digital PCR. Results: there were 69 patients included (18 at stage I, 15 at stage II, and 36 at stage III) at module I. The CTC detection rate at baseline was 94.2%, at first follow-up was 94.6%, and at second follow-up was 100%. It was observed an overall drop in CTC median over time. At second follow-up, ERCC1 and ß-galactosidase expression in CTCs was most commonly found in stage III patients (p= 0.03 and p= 0.04, respectively). High positivity index (PI) of ERCC1 in CTC at second follow-up was determinant of inferior relapse-free survival (RFS) (p= 0.014). It was found a positive correlation between CTC levels and the percentage of TReg cells (p= 0.01) and a negative correlation between CTC levels and the percentage of CD3+ lymphocytes. Patients with high Platelet-to-Lymphocyte Ratio (PLR) were mostly found in high-risk stage II and III patients (p= 0.014). High PLR was determinant of inferior RFS (p= 0.01). At module II (patients with high-risk tumors, treated with adjuvant chemotherapy), it was found a positive correlation between CTC and CEA levels in the cases that shown disease progression (DP) (p= 0.001). High PI of ERCC1 at second follow-up had shown significantly worse RFS (p= 0.013). Conclusions: CTCs were found in high rates in localized colon cancer patients. Additionally, the evaluation of the patient's immune combined with the CTCs showed to be a promising tool to monitoring these patients