Circulating tumor cells isolation: The “post-EpCAM era”

Circulating tumor cells (CTCs) represent a submicroscopic fraction detached from a primary tumor and in transit to a secondary site. The prognostic significance of CTCs in metastatic cancer patients was demonstrated for the first time more than ten years ago. To date, it seems clear enough that CTCs are highly heterogeneous and dynamically change their shape. Thus, the inadequacy of epithelial cell adhesion molecule (EpCAM) as universal marker for CTCs detection seems unquestionable and alternative methods able to recognize a broader spectrum of phenotypes are definitely needed. In this review the pleiotropic functions of EpCAM are discussed in detail and the role of the molecule in the biology of CTCs is critically dissected

EGFR inhibitor as second-line therapy in a patient with mutant RAS metastatic colorectal cancer: circulating tumor DNA to personalize treatment

A 47-year-old male patient presented in March 2016 to our unit with a palpable painless left supraclavicular mass. A whole-body contrastenhanced computed tomography (CT) scan revealed a left supraclavicular lymphadenopathy, transverse colon thickening (3 cm), multiple chest and abdominal lymphadenopathies, and peritoneal carcinomatosis. Colonoscopy revealed a bleeding area at 15 cm from the anal verge; biopsy was performed, and the result was negative for a primary cancer

Complexos macrocíclicos tetraiminodifenólicos de vanádio e ferro : síntese e caracterização estrutural, espectroscópica e eletroquímica

Orientador : Fábio Souza NunesDissertação (mestrado) - Universidade Federal do ParanáResumo: O interesse em sistemas químicos capazes de reproduzir a função biológica das enzimas capazes de fixar o nitrogênio do ar, as nitrogenases, tem estimulado estudos de modelos funcionais e/ou estruturais dos sítios ativos destas enzimas. Sabe-se que centros poli(hetero)metálicos de baixo estado de oxidação (molibdênio e ferro; vanádio e ferro) estão presentes nestes sítios ativos. A escolha de ligantes macrocíclicos capazes de acomodar dois centros metálicos unidos por pontes fenolatos, como o ligante tetraiminodifenólico (tidf), mostrou ser uma opção interessante para a obtenção de complexos macrocíclicos heterobinucleares. O início do trabalho envolveu sínteses do ligante tidf através de reações "template" envolvendo sais de chumbo(ll) ou magnésio(ll), resultando nos materiais de partida [Pb(tidf)](N03)2-3H20 (1) e [Mg2(tidf)](N03)2'4H20 (2). A redução de (1) com NaBH4, seguida de desmetalação, originou o ligante tetraaminodifenólico (tadf) (3), entretanto, as várias etapas de síntese e os baixos rendimentos de tadf nos levaram a concentrar nossos estudos com o ligante tidf. As sínteses e operações envolvendo os produtos contendo Fe" e/ou V" foram conduzidas sob atmosfera de N2 (pureza 99,999%) ou sob vácuo de 10~4 Torr, utilizando técnicas de Schlenk ou em "glove-box" ([02] " 0,1 ppm). Os complexos [Fe(tidf)(CH30H)2](N03)2 (7) e [Fe(tidf)(H20)2](CI04)2'H20 (8) foram obtidos através de reações de transmetalação dos complexos (1) e (2) com FeCI2 e [Fe(CH3CN)6](CI04)2 (4), respectivamente. O produto (7) foi obtido como cristais adequados à análise por difratometria de raios-X. Espectroscopia Mõssbauer e magnetoquímica revelaram que os centros de ferro nestes complexos correspondem a Fe" spin alto, com NEF = 5,72 NB (S = 3/2). A voltametria cíclica apresentou um par de ondas com Epa = 0,28 V vs Ag/AgN03 com AEp = 100 mV, atribuídas a um processo monoeletrônico Fe" -> Fe'" quasi-reversível. Os complexos [V0(tidf)(H20)](BF4)2 (9) e [V0(tidf)](CI04)2 (10) foram obtidos por reações de transmetalação do produto (2) com [V(H20)6](BF4)2 (5) e [VO(acac)2], respectivamente. Nas sínteses do produto (9), todas as tentativas de obtenção de um complexo contendo vanádio(ll) levaram à formação de complexos contendo vanadila (Vl v02+), num processo em que se postula uma redução das iminas ligadas ao vanádio a aminas. Os compostos (9) e (10) apresentam uma pequena diferença entre seus valores de potenciais de pico anódicos: 0,82 V (9) e 0,88 V (10) vs Ag/AgN03, atribuídos à oxidação monoeletrônica reversível Vl v02+ Vv02 +. Os valores de momento magnético efetivo obtidos foram de 1,78 JJ.b (9) e 1,72 JJ,B (10) (S = 1/2). Os compostos (7) e (9) também foram caracterizados por espectroscopia de absorção no UV-vísivel e no infravermelho, ressonância paramagnérica eletrônica, espectrometria de massa e espectroeletroquímica. Os complexos binucleares [Fe2(tidf)(CH30H)2](CI04)2 (11) e [VOFe(tidf)(CH3CN)2](BF4)2 (12) foram obtidos, sob a forma de cristais, através de reações de metalação dos mononucleares (8) e (9) com [Fe(CH3CN)6](CI04)2. Entretanto, somente a estrutura de (11) foi resolvida a tempo de ser incluída neste trabalho. Ainda aguardamos a resolução da estrutura do produto (12).Abstract: Interest in the chemistry of low valent complexes containing molybdenum, vanadium and iron has been increased due the occurrence of those metals in the poli(hetero)nuclear active site of nitrogenases, enzymes that catalyze the reduction of dinitrogen. The challenge of preparing model complexes capable of mimetizing the function of nitrogenases encouraged us to choose macrocyclic ligands that can bind two metal ions, bridged by phenolates, as does the tetraiminediphenolic ligand (tidf). We prepared tidf through template reactions using lead(ll) and magnesium(ll) salts, producing the starting materials [Pb(tidf)](N03)2*3H20 (1) and [Mg2(tidf)](N03)2'4H20 (2). The chemical reduction of (1) with NaBH4, followed by demetallation, resulted the tetraaminediphenolic ligand (tadf). However, the high number of synthetic steps and low yields guided us to focus on the tidf ligand. All operations involving materials containing Fe" and/or V" were carried out under N2 (99,999%) atmosphere or 10"4 Torr vacuum, employing Schlenk apparatus or glove-box ([02] ~ 0,1 ppm). The complexes Fe(tidf)(CH30H)2](N03)2 (7) and [Fe(tidf)(H20)2](CI04)2'H20 (8) were prepared through transmetallation reactions of (1) and (2) with FeCI2 and [Fe(CH3CN)6](CI04)2 (4), respectively . The product (7) appeared as suitable crystals for X-ray diffratometry analysis. Mossbauer spectroscopy and magnetochemistry revealed a high spin Fe", with jaef = 5,72 (^b (S = 3/2). Cyclic voltametry presented one wave pair with Epa = 0,28 V vs Ag/AgN03 and AEp = 100 mV, attributed to the quasi-reversible monoelectronic process Fe" ^ Fe'". The complexes [V0(tidf)(H20)](BF4)2 (9) and [V0(tidf)](CI04)2 (10) were obtained by transmetallation of (2) with [V(H20)6](BF4)2 (5) and [VO(acac)2], respectively. All our attempts to prepare a vanadium(ll) complex with tidf led, instead, to the oxovanadium(IV) complex through a sequence of reductions of two imine functions. The compounds (9) and (10) show a small difference between their Epa values, 0,82 V (9) e 0,88 V (10) vs Ag/AgN03, assigned to the monoelectronic reversible oxidation Vl v02+ Vv02. Effective magnetic moment found for these products were 1,78 jib (9) and 1,72 jxB (10) (S = 1/2). Also (7) and (9) were characterized by UV-visible, infrared and epr spectrocopies as well as by mass spectrommetry (FAB) and spectroelectrochemistry. The binuclear complexes [Fe2(tidf)(CH30H)2](CI04)2 (11) and [VOFe(tidf)(CH3CN)2](BF4)2 (12) were obtained as monocrystals by the metallation of the mononuclear complexes (7) and (8) with [Fe(CH3CN)6](CI04)2. The x-ray crystal structure of (11) was presented in this work and we are currently solving the structure of (12)

Complexos macrocíclicos Homo- e heterobimetálicos de ferro, manganes, rutenio, vanádio e molibdenio : síntese, propriedades espectroscópicas, eletroquímicas e catálise redox

Orientador : Fábio Souza NunesCo-orientadora : Sueli M. DrechselTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Química. Defesa: Curitiba, 15/05/2008Inclui bibliografiaÁrea de concentraçao : Química inorgânic

The rationale for liquid biopsy in colorectal cancer: focus on circulating tumor cells

Capturing circulating tumor cells (CTCs) and/or circulating tumor DNA from blood, which represents a precious source of biological material derived from both primary and metastatic tumors, has been named a 'liquid biopsy'. While the circulating tumor DNA might be more representative of the bulk of the metastatic tumor, CTCs are thought to reflect more of the metastases-initiating cells. Consequently, a liquid biopsy made of tumor cells and tumor DNA that is able to track cancer evolution, as a fingerprint of the patient's individual tumor, and is easy to perform at every stage of the disease course, sounds attractive. This article mainly focuses on the applications of CTCs to track tumor dynamics in real time using colorectal cancer as a model system. The analysis of viable CTCs at DNA, RNA and protein levels, as well as their expansion in vitro, may allow deep investigation of the features of metastases-initiating cells

Impact of chronic exposure to bevacizumab on EpCAM-based detection of circulating tumor cells

BACKGROUND: Circulating tumor cells (CTCs) are often undetected through the immunomagnetic epithelial cell adhesion molecule (EpCAM)-based CellSearch(®) System in breast and colorectal cancer (CRC) patients treated with bevacizumab (BEV), where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro. METHODS: The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch(®), untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch(®). RESULTS: Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins (CK), while no evidence of epithelial to mesenchymal transition (EMT) in treated cells was observed. The recovery rate of cells through CellSearch(®) was gradually reduced in course of treatment with BEV, being 84%, 70% and 40% at 1, 2 and 3 months, respectively. CONCLUSIONS: We hypothesize that BEV may prevent CellSearch(®) from capturing CTCs through altering EpCAM isoforms

Cultura de segurança do paciente na ótica de trabalhadores e equipes da atenção primária

OBJETIVO: Analisar se a cultura de segurança do paciente entre os profissionais da atenção primária à saúde difere entre as equipes de saúde. MÉTODOS: Estudo transversal, quantitativo, realizado nos meses de abril e maio de 2017, em um município da região Sul do Brasil. Participaram 144 profissionais que responderam ao questionário “Pesquisa sobre Cultura de Segurança do Paciente para Atenção Primária”. Os dados foram analisados no programa Statistical Analysis Software e expressos em porcentagens de respostas positivas. Os preceitos éticos estabelecidos para pesquisas com seres humanos foram cumpridos. RESULTADOS: A cultura de segurança do paciente é positiva entre 50,81% dos profissionais e as dimensões “seu serviço de saúde” (63,39%) e “segurança do paciente e qualidade” (61,22%) obtiveram as maiores médias de respostas positivas. Identificou-se diferenças significativas entre as equipes de saúde da família e de saúde bucal (α = 0,05 e p < 0,05), nas dimensões “segurança do paciente” (p = 0,0274) e “trabalho no serviço de saúde” (p = 0,0058). CONCLUSÕES: Concluiu-se que, apesar de próxima da média, a cultura de segurança do paciente entre os profissionais da Atenção Primária à Saúde é positiva e existem diferenças na cultura de segurança entre as equipes de saúde da família e saúde bucal em relação às equipes de atenção básica.OBJECTIVE: Analyze if the patient safety culture among professionals in the primary health care differs among health care teams. METHODS: Cross-sectional and quantitative study conducted in April and May 2017, in a city in Southern Brazil. A total of 144 professionals who responded to the questionnaire “Survey on Patient Safety Culture in Primary Health Care” participated in the study. Data were analyzed in the Statistical Analysis Software program and expressed in percentage of positive responses. The ethical principles established for research with human beings were applied. RESULTS: Patient safety culture is positive among 50.81% of the professionals, and the dimensions “your health service” (63.39%) and “patient safety and quality” (61.22%) obtained the highest average of positive responses. Significant differences were found between the family health and oral health teams (α = 0.05 and p < 0.05), in the dimensions “patient safety” (p = 0.0274) and “work at the health service” (p = 0.0058). CONCLUSIONS: We concluded that, although close to the average, patient safety culture among professionals in the Primary Health Care is positive and that there are differences in safety culture between family health and oral health teams in comparison with the primary health care teams

Liquid Biopsy in Rare Cancers: Lessons from Hemangiopericytoma

Hemangiopericytoma (HPT) is a rare mesenchymal tumor of fibroblastic type and for its rarity is poorly studied. The most common sites of metastatic disease in patients with intracranial HPT are the bone, liver, and lung, suggestive for an hematogenous dissemination; for this reason, we investigated, for the first time, the presence of circulating tumor cells (CTCs) in hemangiopericytoma patient by CellSearch® and SceenCell® devices. Peripheral blood samples were drawn and processed by CellSearch, an EpCAM-dependent device, and ScreenCell®, a device size based. We found nontypical CTCs by CellSearch system and the immunofluorescence analysis performed on CTCs isolate by ScreenCell demonstrated the presence of single CTCs and CTC clusters. The molecular characterization of single CTCs and CTC clusters, using antibodies directed against EpCAM, CD34, cytokeratins (8, 18, and 19), and CD45, showed a great heterogeneity in CTC clusters. We believe that the present study may open a new scenario in the rare tumors: the introduction of the liquid biopsy and the molecular characterization of circulating tumor cells could lead to personalized targeted treatments and also for rare tumors

Transient disappearance of RAS mutant clones in plasma: A counterintuitive clinical use of EGFR inhibitors in RAS mutant metastatic colorectal cancer

Genomic studies performed through liquid biopsies widely elucidated the evolutionary trajectory of RAS mutant clones under the selective pressure of EGFR inhibitors in patients with wild type RAS primary colorectal tumors. Similarly, the disappearance of RAS mutant clones in plasma has been more recently reported in some patients with primary RAS mutant cancers, supporting for the first time an unexpected negative selection of RAS mutations during the clonal evolution of mCRC. To date, the extent of conversion to RAS wild type disease at the time of progression has not been clarified yet. As a proof of concept, we prospectively enrolled mCRC patients progressing under anti-VEGF based treatments. Idylla™system was used to screen RAS mutations in plasma and the wild type status of RAS was further confirmed through IT-PGM (Ion Torrent Personal Genome Machine) sequencing. RAS was found mutant in 55% of cases, retaining the same plasma mutation as in the primary tumor at diagnosis, while it was found wild-type in 45%. Four patients testing negative for RAS mutations in plasma at the time of progression of disease (PD) were considered eligible for treatment with EGFR inhibitors and treated accordingly, achieving a clinical benefit. We here propose a hypothetical algorithm that accounts for the transient disappearance of RAS mutant clones over time, which might extend the continuum of care of mutant RAS colorectal cancer patients through the delivery of a further line of therapy