Circulating tumor cells as a predictor and prognostic tool for metastatic clear cell renal carcinoma: An immunocytochemistry and genomic analysis.

BACKGROUND: Treatment of metastatic clear cell renal carcinoma (mccRCC) has changed dramatically over the past 20 years, without improvement in the development of biomarkers. Recently, circulating tumor cells (CTCs) have been validated as a prognostic and predictive tool for many solid tumors. OBJECTIVE: We evaluated CTCs in blood samples obtained from patients diagnosed with mccRCC. Comparisons of CTC counts, protein expression profiling, and DNA mutants were made in relation to overall survival and progression-free survival. METHODS: CTCs were isolated from 10 mL blood samples using the ISET® system (Isolation by SizE of Tumor Cells; Rarecells, France) and counted. Protein expression was evaluated in immunocytochemistry assays. DNA mutations were identified with next generation sequencing (NGS). RESULTS: Blood samples (10 mL) were collected from 12 patients with mccRCC before the start of first-line systemic therapy, and again 30 and 60 days after the start of treatment. All 12 patients had CTCs detected at baseline (median, 1.5 CTCs/mL; range: 0.25-7.75). Patients with CTC counts greater than the median had two or more metastatic sites and exhibited worse progression-free survival (19.7 months) compared to those with CTC counts less than the median (31.1 months). Disease progression was observed in 7/12 patients during the study. Five of these patients had baseline CTC counts greater than the median, one had higher CTC levels at the second blood collection, and one patient had CTCs present at 1 CTC/mL which positively stained for PD-L1, N-cadherin, VEGF, and SETD2. CTC DNA from six patients with worse outcomes was subjected to NGS. However, no conclusions could be made due to the low variant allele frequencies. CONCLUSION: Detection of CTCs in patients with mccRCC receiving first-line treatment is a feasible tool with prognostic potential since increased numbers of CTCs were found to be associated with metastasis and disease progression.

CD47 Expression in Circulating Tumor Cells and Circulating Tumor Microemboli from Non-Small Cell Lung Cancer Patients Is a Poor Prognosis Factor.

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-ß/transforming growth factor-ß receptor type 1 (TGF-ß/TGFßRI) 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 TGFßRI/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 TGFßRI/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). TGFßRI 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 TGFßRI/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.

Brazilian Society of Surgical Oncology: Guidelines for the management of anal canal cancer.

BACKGROUND: Anal canal squamous cell carcinoma (SCC) is a relatively uncommon neoplasia, and it is mostly a local-regional cancer, of low metastatic potential (only 15%), resulting in cure in most cases treated with definitive chemoradiation. On the other hand, its incidence has been steadily increasing over the last decades, which makes it an important public health problem. In an effort to provide surgeons and oncologists who treat patients with anal cancer with the most updated information based on the best scientific evidence, the Brazilian Society of Surgical Oncology (SBCO) has produced the present guideline for the management of anal canal SCC, focused on the main topics related to daily clinical practice. OBJECTIVES: The SBCO developed the present guidelines to provide recommendations on the main topics related to the management of anal canal squamous cell carcinoma (SCC) based on current scientific evidence. METHODS: Between October 2022 and January 2023, 14 experts met to develop the guidelines for the management of anal canal cancer. A total of 30 relevant topics were distributed among the participants. The methodological quality of a final list with 121 sources was evaluated, all the evidence was examined and revised, and the management guidelines were formulated by the 14-expert committee. To reach a final consensus, all the topics were reviewed in a meeting that was attended by all the experts. RESULTS: The proposed guidelines contained 30 topics considered to be highly relevant in the management of anal canal cancer, covering subjects related to screening recommendations, preventive measures, tests required for diagnosing and staging, treatment strategies, response assessment after chemoradiotherapy, surgical technique-related aspects, and follow-up recommendations. In addition, screening and response assessment algorithms, and a checklist were proposed to summarize the important information and offer an updated tool to assist surgeons and oncologists who treat anal canal cancer and in providing the best care to their patients. CONCLUSION: These guidelines summarize recommendations based on the most current scientific evidence on relevant aspects of anal canal cancer management and are a practical guide to help surgeons and oncologists who treat anal canal cancer make the best therapeutic decisions.

HPV virus and biomarkers of resistance to chemoradiation in circulating tumor cells from patients with squamous cell carcinoma of the anus.

Localized anal cancer is mostly represented by squamous cell carcinoma of the anus (SCCA) and is cured in ≥80 % of cases by chemoradiation (CRT). Development of techniques for detection/evaluating circulating tumor cells (CTCs) for diagnosis/ prognosis/response to therapy can change the manner we treat/follow SCCA patients. OBJECTIVE: to detect CTCs from patients with SCCA and evaluate the presence of HPV virus, p16 expression and markers related to resistance to CRT (RAD23B/ ERCC1/ TYMS) in CTCs at baseline and after CRT. METHODS: CTCs were isolated/quantified by ISET®, protein expressions were analyzed by immunocytochemistry and HPV DNA was detected by chromogenic in situ hybridization. RESULTS: We enrolled 15 patients: median age was 61 (43-73) years, the majority was women (10/15). CTCs were detected in all patients at baseline (median= 0.4 (0.4-3.33) CTCs/mL) and in 8/9 patients, after CRT (median= 2.33 (0-7.0) CTCs/mL). DNA from HPV was found in CTCs in 14/15 patients (93.33 %) at baseline and in 7/9 (77.7 %) after treatment. At a median follow-up of 22.20 (1.45-38.55) months, three patients expressed ERCC1 in CTCs after treatment, with one of them having disease recurrence. CONCLUSION: We showed that detection of HPV in CTCs from patients with non-metastatic SCCA is feasible and appears to be a sensitive diagnostic method. These results may be clinically useful for better monitoring these patients. However, future larger cohorts may demonstrate whether there is any correlation between the presence of HPV and the expression of screening markers for CRT in SCCA.

Heterogeneity of HER2 Expression in Circulating Tumor Cells of Patients with Breast Cancer Brain Metastases and Impact on Brain Disease Control.

HER2 expression switching in circulating tumor cells (CTC) in breast cancer is dynamic and may have prognostic and predictive clinical implications. In this study, we evaluated the association between the expression of HER2 in the CTC of patients with breast cancer brain metastases (BCBM) and brain disease control. An exploratory analysis of a prospective assessment of CTC before (CTC1) and after (CTC2) stereotactic radiotherapy/radiosurgery (SRT) for BCBM in 39 women was performed. Distant brain failure-free survival (DBFFS), the primary endpoint, and overall survival (OS) were estimated. After a median follow-up of 16.6 months, there were 15 patients with distant brain failure and 16 deaths. The median DBFFS and OS were 15.3 and 19.5 months, respectively. The median DBFFS was 10 months in patients without HER2 expressed in CTC and was not reached in patients with HER2 in CTC (p = 0.012). The median OS was 17 months in patients without HER2 in CTC and was not reached in patients with HER2 in CTC (p = 0.104). On the multivariate analysis, DBFFS was superior in patients who were primary immunophenotype (PIP) HER2-positive (HR 0.128, 95% CI 0.025-0.534; p = 0.013). The expression of HER2 in CTC was associated with a longer DBFFS, and the switching of HER2 expression between the PIP and CTC may have an impact on prognosis and treatment selection for BCBM.

Brazilian Society of Surgical Oncology guidelines for malignant bowel obstruction management.

BACKGROUND: Malignant bowel obstruction (MBO) is a frequent complication in advanced cancer patients and especially those with abdominal tumors. The clinical management of MBO requires a specific and individualized approach based on the disease prognosis. Surgery is recommended. Less invasive approaches such as endoscopic treatments should be considered when surgery is contraindicated. The priority of care for inoperable and consolidated MBO is to control the symptoms and promote the maximum level of comfort. OBJECTIVES: This study aimed to develop recommendations for the effective management of MBO. METHODS: A questionnaire was administered to all members of the Brazilian Society of Surgical Oncology, of whom 41 surgeons participated in the survey. A literature review of studies retrieved from the National Library of Medicine database was conducted on particular topics chosen by the participants. These topics addressed questions regarding the MBO management, to define the level of evidence and strength of each recommendation, and an adapted version of the Infectious Diseases Society of America Health Service rating system was used. RESULTS: Most aspects of the medical approach and management strategies reviewed were strongly recommended by the participants. CONCLUSIONS: Guidelines outlining the strategies for management MBO were developed based on the strongest evidence available in the literature.

MIR-203A-3P AND MMP-2 PROTEINS ARE HIGHLY EXPRESSED IN CIRCULATING TUMOR CELLS FROM PATIENTS WITH PANCREATIC CARCINOMA. / PROTEÍNAS MIR-203A-3P E MMP-2 SÃO ALTAMENTE EXPRESSAS EM CÉLULAS TUMORAIS CIRCULANTES DE PACIENTES COM CARCINOMA PANCREÁTICO.

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

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

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

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)-ß 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-ß type 1 receptor (TGF-ß 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-ß RI expression in CTCs was 42.6 versus 20.8 months for TGF-ß 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-ß 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.

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.

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

Baseline and Kinetic Circulating Tumor Cell Counts Are Prognostic Factors in a Prospective Study of Metastatic Colorectal Cancer.

The discovery of predictive biomarkers in metastatic colorectal cancer (mCRC) is essential to improve clinical outcomes. Recent data suggest a potential role of circulating tumor cells (CTCs) as prognostic indicators. We conducted a follow-on analysis from a prospective study of consecutive patients with mCRC. CTC analysis was conducted at two timepoints: baseline (CTC1; before starting chemotherapy), and two months after starting treatment (CTC2). CTC isolation/quantification were completed by ISET® (Rarecells, France). CTC expressions of drug resistance-associated proteins were evaluated. Progression-free survival (PFS) and overall survival (OS) were estimated by the Kaplan-Meier method. Seventy-five patients were enrolled from May 2012 to May 2014. A CTC1 cut-off of >1.5 CTCs/mL was associated with an inferior median OS compared to lower values. A difference of CTC2-CTC1 > 5.5 CTCs/mL was associated with a reduced median PFS. By multivariate analysis, CTC1 > 1.5 CTCs/mL was an independent prognostic factor for worse OS. Multi-drug resistance protein-1 (MRP-1) expression was associated with poor median OS. CTC baseline counts, kinetics, and MRP-1 expression were predictive of clinical outcomes. Larger studies are warranted to explore the potential clinical benefit of treating mCRC patients with targeted therapeutic regimens guided by CTC findings.

Prospective Assessment of the Association Between Circulating Tumor Cells and Control of Brain Disease After Focal Radiation Therapy of Breast Cancer Brain Metastases.

PURPOSE: Predicting the risk of early distant brain failure (DBF) is in demand for management decisions in patients who are candidates for local treatment of brain metastases. This study aimed to analyze the association between circulating tumor cells (CTCs) and brain disease control after stereotactic radiation therapy/radiosurgery (SRT) for breast cancer brain metastasis (BCBM). METHODS AND MATERIALS: We prospectively assessed CTCs before (CTC1) and 4 to 5 weeks after (CTC2) SRT and their relationship with the number of new lesions (NL) suggestive of BCBM before SRT. CTC were quantified and analyzed by immunocytochemistry to evaluate the expression of the proteins COX2, EGFR, ST6GALNAC5, NOTCH1, and HER2. Distant brain failure-free survival (DBFFS), the primary endpoint, diffuse DBFFS (D-DBFFS), and overall survival were estimated. Analysis for DBF within 6 months, with death as competing risk, was performed. RESULTS: Patients were included between 2016 and 2018. CTCs were detected in all 39 patients before and in 34 of 35 patients after SRT. After median follow-up of 16.6 months, median DBFFS, D-DBFFS, and overall survival were 15.3, 14.1, and 19.5 months, respectively. DBF at 6 months was 40% with CTC1 ≤0.5 and 8.82% with CTC1 >0.5 CTC/mL (P = .007), and D-DBF at 6 months was 40% with CTC1 ≤0.5 and 0 with CTC1 >0.5 CTC/mL (P = .005) and 25% with NL/CTC1 >6.8 and 2.65% with NL/CTC1 ≤6.8 (P = .063). On multivariate analysis, DBFFS was inferior with CTC1 ≤0.5 (hazard ratio, 8.27; 95% confidence interval, 2.12-32.3; P = .002), and D-DBFFS was inferior with CTC1 ≤0.5 (hazard ratio, 10.22; 95% confidence interval, 1.99-52.41; P = .005). Protein expression was not associated with outcomes. CONCLUSIONS: These data suggest that CTC1 and NL/CTC1 may have a role as a biomarker of early diffuse DBF and as a subsequent guide between focal or whole-brain radiation therapy in patients with BCBM.

A higher platelet-to-lymphocyte ratio is prevalent in the presence of circulating tumor microemboli and is a potential prognostic factor for non-metastatic colon cancer.

Colorectal cancer is a common and often deadly cancer. Circulating tumor cells (CTCs) have been implicated as a potentially valuable prognosis factor. The detection of circulating tumor microemboli (CTM) and of simple blood component parameters that reflect inflammatory status, such as the platelet-to-lymphocyte ratio (PLR) and neutrophil-to-lymphocyte ratio (NLR), may provide information about tumor progression. The aim of this study was to explore the importance of CTCs, CTM, PLR, and NLR prospectively in non-metastatic colon cancer progression. CTCs were enriched using ISETⓇ (Isolation by SizE of Tumor cells) and identified by immunocytochemical exclusion of leukocytes. We evaluated CTCs and blood cell parameters in a cohort of 69 stage I-III colon cancer patients (52.2% men; median age, 61 years; age range, 19-87 years) at a baseline timepoint prior to resection surgery. The median of CTC levels at baseline was 20 cells/8 mL (0-94) and higher levels were associated with CTM presence (p = 0.02). CTM were found in 18 (26.1%) patients. Of 18 stage I patients, 33.3% had CTM and of 51 stages II or III patients, 13.7% had CTM (p = 0.08). Patients with a high PLR (>124) were mostly (75.6%) diagnosed with high-risk stages II/III cancer (stages I/low-risk II, 24.4%; p = 0.014). All 8 patients that had disease recurrence during follow-up had a high PLR (p = 0.02 vs. low PLR). NLR was not significantly associated with disease stage or recurrence. The present results indicate that CTCs and PLR analyses may be clinically useful for colon cancer management and risk stratification.

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.

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.

EGFR expression in circulating tumor cells from high-grade metastatic soft tissue sarcomas.

INTRODUCTION: Soft tissue Sarcomas (STS) are rare malignances, with high mortality rates. Half of patients develop metastasis. The presence of isolated Circulating Tumor Cells (CTCs) and Circulating Tumor Microemboli (CTM) in the blood may be early markers of tumor invasion. Epidermal Growth Factor (EGF) family receptors can also influence this process. OBJECTIVES: to quantify CTCs and identify CTM as well as the EGF Receptor (EGFR) protein expression in these cells and correlate with clinical outcome in metastatic STS. MATERIALS AND METHODS: Approximately 8mL of blood was prospectively collected from patients with different types of high-grade STS, before the beginning of chemotherapy. The samples were processed and filtered by ISET (Rarecells, France) for the isolation and quantification of CTCs and CTMs. EGFR expression was analyzed by immunocytochemistry (ICC) on CTCs/ CTMs. RESULTS: We analyzed 18 patients with median age of 49 years (18-77 y). The positivity for EGFR protein expression in CTCs was observed in 93.75% of the patients. This result shows that targeting EGFR positive CTCs from STS origen can be translated in clinical benefit for some patients. In addition, if target therapy is chosen, the EGFR expression in CTCs can be used in follow-up to measure treatment effectiveness. CONCLUSIONS: This is the first study to demonstrate the expression of EGFR protein in CTCs from sarcoma patients. It may open an area for future investigations. The next step is to characterize CTCs in a larger cohort of patients to better understand the role of EGFR in sustaining tumor metastasis in sarcomas.

Evaluation of incidence, significance, and prognostic role of circulating tumor microemboli and transforming growth factor-ß receptor I in head and neck cancer.

BACKGROUND: Circulating tumor microemboli (CTM) are clusters of circulating tumor cells (CTCs), involved in metastasis, as also transforming growth factor-ß (TGF-ß). The purpose of this study was to verify their role in progression-free survival (PFS). METHODS: Blood from patients with locally advanced head and neck squamous cell carcinoma (HNSCC; n = 53) was analyzed in 2 moments. TGF-ß receptor I (TGF-ßRI) expression was evaluated by immunocytochemistry. RESULTS: Comparing CTM1 (baseline) with CTM2 (first follow-up), patients with CTM1-positive disease who became CTM2-negative were classified as favorable (PFS 20 months). Patients with unfavorable evolution (CTM1-negative/CTM2-positive), had PFS of 17.5 months. Patients always CTM-negative showed PFS of 22.4 months, those always positive, 4.7 months (P < .001). The TGF-ßRI expression in the first follow-up correlated with poor PFS (12 × 26 months; P = .007), being an independent prognostic factor (hazard ratio [HR] = 6.088; P = .033). CONCLUSION: CTM1/2, TGF-ßRI expression, and unfavorable CTM kinetics may represent poor prognosis in locally advanced HNSCC.

Circulating Tumor Cells as Cancer Biomarkers in the Clinic.

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