Liquid biopsy approach to monitor the efficacy and response to CAR-T cell therapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T cells are approved for use in the treatment of hematological malignancies. Axicabtagene ciloleucel (YESCARTA) and brexucabtagene autoleucel (TECARTUS) genetically modified autologous T cells expressing an anti-CD19 scFv based on the FMC63 clone have shown impressive response rates for the treatment of CD19+B cell malignancies, but there remain challenges in monitoring long-term persistence as well as the functional characterization of low-level persisting CAR-T cells in patients. Furthermore, due to CD19-negative driven relapse, having the capability to monitor patients with simultaneous detection of the B cell malignancy and persisting CAR-T cells in patient peripheral blood is important for ensuring timely treatment optionality and understanding relapse. METHODS: This study demonstrates the development and technical validation of a comprehensive liquid biopsy, high-definition single cell assay (HDSCA)-HemeCAR for (1) KTE-X19 CAR-T cell identification and analysis and (2) simultaneously monitoring the CD19-epitope landscape on neoplastic B cells in cryopreserved or fresh peripheral blood. Proprietary anti-CD19 CAR reagents, healthy donor transduced CAR-T cells, and patient samples consisting of malignant B cell fractions from manufacturing were used for assay development. RESULTS: The CAR-T assay showed an approximate limit of detection at 1 cell in 3 million with a sensitivity of 91%. Genomic analysis was additionally used to confirm the presence of the CAR transgene. This study additionally reports the successful completion of two B cell assays with multiple CD19 variants (FMC63 and LE-CD19) and a unique fourth channel biomarker (CD20 or CD22). In patient samples, we observed that CD19 isoforms were highly heterogeneous both intrapatient and interpatient. CONCLUSIONS: With the simultaneous detection of the CAR-T cells and the B cell malignancy in patient peripheral blood, the HDSCA-HemeCAR workflow may be considered for risk monitoring and patient management.

Radiofrequency ablation induces tumor cell dissemination in a mouse model of hepatocellular carcinoma.

BACKGROUND: We tested the hypothesis that radiofrequency ablation (RFA) for hepatocellular carcinoma (HCC) promotes tumor cell release and explored a method for reducing these effects. METHODS: A green fluorescent protein-transfected orthotopic HCC model was established in 99 nude mice. In vivo flow cytometry was used to monitor circulating tumor cell (CTC) dynamics. Pulmonary fluorescence imaging and pathology were performed to investigate lung metastases. First, the kinetics of CTCs during the periablation period and the survival rate of CTCs released during RFA were investigated. Next, mice were allocated to controls, sham ablation, or RFA with/without hepatic vessel blocking (ligation of the portal triads) for evaluating the postablation CTC level, lung metastases, and survival over time. Moreover, the kinetics of CTCs, lung metastases, and mice survival were evaluated for RFA with/without ethanol injection. Pathological changes in tumors and surrounding parenchyma after ethanol injection were noted. Statistical analysis included t-test, ANOVA, and Kaplan-Meier survival curves. RESULTS: CTC counts were 12.3-fold increased during RFA, and 73.7% of RFA-induced CTCs were viable. Pre-RFA hepatic vessel blocking prevented the increase of peripheral CTCs, reduced the number of lung metastases, and prolonged survival (all p ≤ 0.05). Similarly, pre-RFA ethanol injection remarkably decreased CTC release during RFA and further decreased lung metastases with extended survival (all p ≤ 0.05). Histopathology revealed thrombus formation in blood vessels after ethanol injection, which may clog tumor cell dissemination during RFA. CONCLUSION: RFA induces viable tumor cell dissemination, and pre-RFA ethanol injection may provide a prophylactic strategy to reduce this underestimated effect. RELEVANCE STATEMENT: RFA for HCC promotes viable tumor cell release during ablation, while ethanol injection can prevent RFA induced tumor cell release. KEY POINTS: • RFA induced the release of viable tumor cells during the ablation procedure in an animal model. • Hepatic vessel blocking can suppress tumor cells dissemination during RFA. • Ethanol injection can prevent RFA-induced tumor cell release, presumably because of the formation of thrombosis.

Cell-free DNA approaches for cancer early detection and interception.

Rapid advancements in the area of early cancer detection have brought us closer to achieving the goals of finding cancer early enough to treat or cure it, while avoiding harms of overdiagnosis. We evaluate progress in the development of early cancer detection tests in the context of the current principles for cancer screening. We review cell-free DNA (cfDNA)-based approaches using mutations, methylation, or fragmentomes for early cancer detection. Lastly, we discuss the challenges in demonstrating clinical utility of these tests before integration into routine clinical care.

[Current Status and Prospects of Liquid Biopsy for Hepatocellular Carcinoma].

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths globally. Early detection and treatment response monitoring of HCC are vital for improved outcomes. Traditional diagnostic methods rely on imaging, serum tumor markers, and invasive liver tissue biopsy. The advent of liquid biopsy has revolutionized cancer diagnostics and management. Liquid biopsy involves the detection and analysis of tumor-derived components, such as circulating tumor DNA, circulating tumor cells, and extracellular vesicles, in various body fluids. These components reflect tumor characteristics, genetic alterations, and functional changes, providing valuable information for HCC diagnosis and treatment response monitoring. Liquid biopsy offers several advantages, including its non-invasive nature, potential for repetitive sampling, and real-time monitoring of disease progression and treatment response. However, challenges remain, including the sensitivity of detection methods, and standardization. In this review, we discuss the methods, current status, and prospects of liquid biopsy for HCC, highlighting its potential as a valuable tool in HCC management.

Heterogeneous circulating tumor cells correlate with responses to neoadjuvant chemotherapy and prognosis in patients with locally advanced breast cancer.

Neoadjuvant chemotherapy (NCT) is the standard treatment for patients with locally advanced breast cancer (LABC). The predictive value of heterogeneous circulating tumor cells (CTCs) in NCT response has not been determined. All patients were staged as LABC, and blood samples were collected at the time of biopsy, and after the first and eighth NCT courses. Patients were divided into High responders (High-R) and Low responders (Low-R) according to Miller-Payne system and changes in Ki-67 levels after NCT treatment. A novel SE-i·FISH strategy was applied to detect CTCs. Heterogeneities were successfully analyzed in patients undergoing NCT. Total CTCs increased continuously and were higher in Low-R group, while in High-R group, CTCs increased slightly during NCT before returning to baseline levels. Triploid and tetraploid chromosome 8 increased in Low-R but not High-R group. The number of small CTCs in Low-R group increased significantly until the last sample, however, remained constant in High-R group. The patients with more CTCs had shorter PFS and OS than those with less CTCs after the eighth course of NCT. Total CTCs following NCT could predict patients' responses. More detailed characterizations of CTC blood profiles may improve predictive capacity and treatments of LABC.

Changes in circulating tumor DNA and outcomes in solid tumors treated with immune checkpoint inhibitors: a systematic review.

BACKGROUND: Quantification of circulating tumor DNA (ctDNA) levels is a reliable prognostic tool in several malignancies. Dynamic changes in ctDNA levels in response to treatment may also provide prognostic information. Here, we explore the value of changes in ctDNA levels in response to immune checkpoint inhibitors (ICIs). METHODS: We searched MEDLINE (host: PubMed) for trials of ICIs in advanced solid tumors in which outcomes were reported based on change in ctDNA levels. ctDNA reduction was defined as reported in individual trials. Typically, this was either >50% reduction or a reduction to undetectable levels. We extracted HRs and related 95% CIs and/or p values comparing ctDNA reduction versus no reduction for progression-free survival (PFS) and/or overall survival (OS). Data were then pooled in a meta-analysis. Variation in effect size was examined using subgroup analyses. RESULTS: Eighteen trials were included in the meta-analysis. ctDNA levels were detectable in all participants in all studies prior to initiation of ICIs. A reduction in ctDNA measured 6-16 weeks after starting treatment was associated with significantly better PFS (HR 0.20; 95% CI, 0.14 to 0.28; p<0.001). Similarly, OS was superior in patients with reduced ctDNA levels (HR 0.18; 95% CI, 0.12 to 0.26; p<0.001). The results were consistent across all disease sites, lines of treatment, magnitude of change (to undetectable vs >50% reduction) and whether treatment exposure comprised single or combination ICIs. CONCLUSIONS: In advanced solid tumors, a reduction in ctDNA levels in response to ICIs is associated with substantial improvements in outcome. ctDNA change is an early response biomarker which may allow for de-escalation of cross-sectional imaging in patients receiving ICIs or support treatment de-escalation strategies.

Gene expression profiling of circulating tumor cells captured by MicroCavity Array is superior to enumeration in demonstrating therapy response in patients with newly diagnosed advanced and locally advanced non-small cell lung cancer.

Background: Circulating tumor cells (CTCs) are a promising non-invasive tool for monitoring therapy response. The only Food and Drug Administration (FDA)-approved test is limited to enumeration of epithelial CTC without further characterization and is not approved for the management of non-small cell lung cancer (NSCLC). Here we use a MicroCavity Array (MCA) system to capture CTC agnostic of epithelial markers for further molecular testing in NSCLC. Methods: CTCs were enumerated by fluorescent microscopy as longitudinal sampling throughout disease management from 213 NSCLC patients. CTC-enriched samples from a subset of 127 patients were interrogated for gene expression by reverse transcription polymerase chain reaction (RT-PCR) using a customized pre-selected panel of 20 genes. Results: At least 1 CTC was detected by enumeration in 53.8% of samples. Most patients had fewer than 5 CTCs (91%) and the highest observed count was 35 CTCs. Enumeration of single CTCs was not prognostic, although detection of CTC clusters at any time point was associated with increased risk of progression [hazard ratio (HR) 3.00, 95% confidence interval (CI): 1.1-8.2, P=0.0318]. In contrast, 124 (97.6%) patients with samples interrogated for gene expression had at least 1 gene detectable in at least 1 sample, and 101 (79.5%) had at least one elevated epithelial gene in at least one timepoint. High expression of BCL2, CD274 [programmed death-ligand 1 (PD-L1)], CDH1, EPCAM, FGFR1, FN1, KRT18, MET and MUC1 were associated with poor prognosis. Patients with CTCs positive for at least 3 epithelial genes at baseline all progressed within 10 months (HR 8.2, P<0.001, 95% CI: 3.2-21.1). BCL2, CD274 (PD-L1), EPCAM and MUC1 remained significant independent prognostic factors in multivariate, time-dependent analyses of progression and death. Conclusions: The selective profile of CTC genes and identification of CTC clusters better correlated with prognosis than enumeration of enriched CTC in NSCLC patients in this study.

Folate receptor-positive circulating tumor cells in the preoperative diagnosis of indeterminate pulmonary nodules.

BACKGROUND: The use of FR + CTC to distinguish lung cancer from benign lung disease has been well studied. However, the effective method to differentiate precursor glandular lesions from benign/malignant pulmonary diseases is rare. METHODS: 380 patients with indeterminate pulmonary nodules were prospectively recruited. Peripheral blood samples were collected from all participants before surgery for analyzing FR + CTC levels. The performance of FR + CTC to identify lung precursor lesions were analyzed by receiver operating characteristic (ROC) curve. RESULTS: FR + CTC can effectively differentiate precursor from benign pulmonary diseases in all included patients (cutoff: 9.22 FU/3 ml, AUC = 0.807, (p < 0.0001, sensitivity: 69.17%, specificity: 82.46%) and patients with single pulmonary lesion (cutoff: 9.03 FU/3 ml, AUC = 0.842, p = 0.0001, sensitivity: 75.20%, specificity: 83.00%). However, FR + CTC cannot differentiate precursor from benign pulmonary diseases in multiple lesions patients (p = 0.110). FR + CTC neither differentiate precursor from malignant pulmonary lesions in all included patients (p = 0.715), single nor multiple lesions patients (p = 0.867, p = 0.692, respectively). Total number of pulmonary nodules, MTD, location (lower vs upper) were independent risk factors for malignancy (AOR, 95% CI: 3.104 (1.525, 6.316), 3.148 (1.722, 5.754), 2.098 (1.132, 3.888), respectively. CONCLUSION: Preoperative FR + CTC can be identified in precursor glandular lesions and utilized to differentiate from benign pulmonary diseases. Total number of pulmonary nodules, MTD, location (lower vs upper) were independent risk factors for malignancy.

Current progress of liquid biopsy in early diagnosis of hepatocellular carcinoma / 中华肝胆外科杂志

Early diagnosis of hepatocellular carcinoma (HCC) is of great significance in the management of patients. Liquid biopsy is a promising tool to use for early diagnosis of liver cancer by detecting tumor expressions through analyzing circulating tumor components such as circulating tumor DNA, circulating tumor cells and extracellular vesicles. The advantages of using liquid biopsy include easy collection of specimen samples and its good sensitivity and specificity for HCC detection. In this review, recent research progress on liquid biopsy on HCC is discussed with the aim to provide updated information on early diagnosis of HCC.

Clinical value of circulating tumor cell typing and RAS/RAF gene detection in colorectal cancer / 中国医师杂志

Objective:To investigate the correlation between circulating tumor cell (CTC) detection, RAS/RAF gene mutation and clinicopathological characteristics in patients with colorectal cancer (CRC).Methods:The Amplification Refractory Mutation System (ARMS)-polymerase chain reaction (PCR) were used to detect the gene mutation in the tumor tissues of 138 CRC patients in the Third Affiliated Hospital of Sun Yat-sen University from May 2017 to May 2020. At the same time, the venous blood of 138 patients was collected and enriched for CTC genotyping by mRNA in situ hybridization. The correlation between CTC, RAS/RAF gene mutation and clinicopathological features of CRC patients was analyzed.Results:The mutation rates of KRAS, NRAS and BRAF genes were 48.6%(67/138), 5.1%(7/138) and 1.4%(2/138), respectively; The overall positive rate of CTC was 84.1%(116/138). The positive rates of different CTC types were: 23.1%(32/138) in epithelial type, 71.7%(99/138) in mixed type and 12.3%(17/138) in interstitial type respectively. The positive rate of CTC in CRC patients with clinical stage Ⅲ-Ⅳ, lymph node metastasis (N1-N3) and distant metastasis (M1) was significantly higher than that in CRC patients with stage Ⅰ-Ⅱ, no lymph node metastasis (N0) and no distant metastasis (M0) (all P<0.05). The total number of CTC, mixed CTC and interstitial CTC were positively correlated with clinical stage, lymph node metastasis and distant metastasis (all P<0.05). RAS/RAF gene mutation, gender, age, tumor location and tumor differentiation did not affect the positive rate of CTC (all P>0.05). Conclusions:The results of CTC typing are of great research significance for comprehensive treatment, prognosis assessment and stratified management of CRC, among which the interstitial type of CTC may be a high risk factor for the recurrence and metastasis of CRC.

Clinical value of peripheral blood circulating tumor cells in the diagnosis and treatment of prostate cancer / 中国基层医药

Objective:To investigate the clinical value of peripheral blood circulating tumor cells (CTC) in the diagnosis and treatment of prostate cancer.Methods:Sixty-four patients with prostate cancer who received treatment in Xinjiang Production and Construction Corps Hospital, China between June 2018 and May 2020 were included in the cancer group. An additional 35 patients with benign prostatic lesions who concurrently received treatment in the same hospital were included in the benign disease group. Twenty male patients with non-prostate disease were included in the control group. Cell enrichment, separation, staining and identification together with Gleason score and pathological stage were subjected to one-way analysis of variance.Results:The percentage of patients with CTC count ≥ 3 in the cancer, benign disease and control groups was 73.43% (47/64), 17.14% (6/35) and 10.00% (2/20), respectively. The level of prostate-specific antigen in patients with CTC was significantly higher than that in patients without CTC ( t = 2.89, P < 0.05). There was significant difference in CTC count between different Gleason score groups ( F = 3.25, P < 0.05) and between different pathological stage groups ( F = 3.42, P < 0.05). Conclusion:Peripheral blood CTC measurement can be used as an auxiliary method for the differentiation of benign and malignant prostate diseases. CTC count in patients with prostate cancer is correlated with prostate-specific antigen level, Gleason score, and pathological stage. Therefore, peripheral blood CTC measurement plays an auxiliary role in predicting prognosis in patients with CTC. This study is innovative and scientific.

Increased Melanoma-Associated Antigen C2 Expression Affords Resistance to Apoptotic Deathin Suspension-Cultured Tumor Cells.

PURPOSE: Melanoma-associated antigen C2 (MAGEC2) is an oncogene associated with various types of cancers. However, the biological function of MAGEC2 in circulating tumor cells remains unclear. In this study, we investigated the role of MAGEC2 using adapted suspension cells (ASCs), which were previously developed to study circulating tumor cells (CTCs). METHODS: Differential gene expression in adherent cells (ADs) and ASCs was examined using RNA-seq analysis. MAGEC2 expression was assessed using reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and ChIP-seq analysis. Depletion of MAGEC2 expression was performed using siRNA. MAGEC2-depleted ADs and ASCs were used to investigate changes in the proliferation rate and cell cycle. Then, the protein levels of signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3, and downstream of STAT3 were measured using control and MAGEC2-depleted ADs and ASCs. In ASCs, the direct effect of active STAT3 inhibition with Stattic, a STAT3 inhibitor, was assessed in terms of proliferation and apoptosis. Finally, an Annexin V/7-AAD assay was performed to determine the percentage of apoptotic cells in the Stattic-treated cells. RESULTS: MAGEC2 was highly expressed in ASCs when compared with ADs. Depletion of MAGEC2 reduced the proliferation rate and viability of ASCs. To elucidate the underlying mechanism, the level of STAT3 was examined owing to its oncogenic properties. Tyrosine-phosphorylated active STAT3 was highly expressed in ASCs and decreased in MAGEC2-depleted ASCs. Furthermore, on treating ASCs with Stattic, an active STAT3 inhibitor, the cells were markedly sensitive to intrinsic pathway-mediated apoptosis. CONCLUSIONS: High MAGEC2 expression may play an important role in the survival of ASCs by maintaining the expression of activated STAT3 to prevent apoptotic cell death.

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.

The combination of mammaglobin A and TWIST-1 increases detection of circulating tumor cells in breast cancer / A combinação de mamaglobina A e TWIST-1 aumenta a detecção de células tumorais circulantes no câncer de mama

ABSTRACT Objectives: Breast cancer cells that are released into the bloodstream are called circulating tumor cells (CTCs). CTCs can express different genes, like TWIST-1 and mammaglobin A (MGA). The aims of this study were to analyze the expression of TWIST-1 and MGA in the blood of breast cancer patients to detect CTCs and to assess the association between the presence of CTCs and prognostic parameters of breast cancer. Methods: Prospective study. Total ribonucleic acid (RNA) from blood mononucleated cells was obtained from breast cancer patients (n = 36; age: 51.5 ± 12.5 years) and healthy donors (n = 14; age: 49.4 ± 9.4 years). Real-time polymerase chain reaction (RT-PCR) was performed to analyze the expression of TWIST-1 and MGA. Results: Patient carcinomas - ductal (86.7%), other types (13.3%). MGA gene expression was not detected in the donors' samples, while it was detected in 14% of the patient samples. Overexpression of TWIST-1 gene was observed in 17% of the patient samples. The combined analysis of both markers allowed the detection of CTCs in 27.8% of the samples, resulting in a significant (p < 0.05) sensitivity increase of detection. No significant associations (p > 0.05) were found between expression of the analyzed genes and the breast cancer prognostic factors. Conclusion: Combined analysis of TWIST-1 and MGA increased the sensitivity of CTCs detection compared to the single analysis of each gene. The detection of CTCs was not associated with known prognostic factors, suggesting that it is able to provide clinical information in addition to routine breast cancer clinicopathological parameters.

RESUMEN Objetivos: Las células de cáncer de mama liberadas al torrente sanguíneo se llaman células tumorales circulantes (CTCs). Las CTCs pueden expresar diferentes genes, como TWIST-1 y mamaglobina A (MGA). Los objetivos de este estudio fueron analizar la expresión de TWIST-1 y MGA en la sangre de pacientes con cáncer de mama (CM) para detectar CTCs y evaluar la asociación entre la presencia de CTCs y los parámetros pronósticos del CM. Métodos: Estudio prospectivo. Se obtuvo el ácido ribonucleico (ARN) de las células mononucleadas en la sangre de pacientes con CM (n = 36, edad: 51,5 ± 12,5 años) y donantes sanas (n = 14; edad: 49,4 ± 9,4 años). Se realizó reacción en cadena de la polimerasa en tiempo real (RT-PCR) para analizar la expresión de TWIST-1 y MGA. Resultados: Carcinoma ductal (86,7%), otros tipos (13,3%). No se detectó la expresión del gen MGA en las muestras de las donantes, pero en el 14% de las muestras de las pacientes. Se observó elevada expresión de TWIST-1 en el 17% de las muestras de pacientes con CM. El análisis combinado de ambos marcadores permitió detección de CTCs en el 27,8% de las muestras, resultando en un aumento significativo (p < 0,05) en la sensibilidad de detección. No se encontraron asociaciones significativas (p > 0,05) entre la expresión de los genes y los factores pronósticos. Conclusión: El análisis combinado de TWIST-1 y MGA aumentó la sensibilidad de detección de CTCs en comparación con el análisis de cada gen. La detección de CTCs no se asoció a factores pronósticos conocidos, sugiriendo que podría ofrecer informaciones clínicas adicionales a los parámetros clínico-patológicos de rutina del CM.

RESUMO Objetivos: As células cancerígenas da mama liberadas na corrente sanguínea são chamadas de células tumorais circulantes (CTCs). As CTCs podem expressar diferentes genes, como TWIST-1 e mamaglobina A (MGA). Os objetivos deste estudo foram analisar a expressão de TWIST-1 e MGA no sangue de pacientes com câncer da mama (CM) para detectar CTCs e avaliar a associação entre a presença de CTCs e os parâmetros prognósticos do CM. Métodos: Estudo prospectivo. O ácido ribonucleico (RNA) das células mononucleadas no sangue foi obtido de pacientes com CM (n = 36, idade: 51,5 ± 12,5 anos) e doadoras saudáveis (n = 14; idade: 49,4 ± 9,4 anos). Reação da cadeia da polimerase em tempo real (RT-PCR) foi realizada para analisar a expressão de TWIST-1 e MGA. Resultados: Carcinoma ductal (86,7%), outros tipos (13,3%). A expressão do gene MGA não foi detectada nas amostras das doadoras, mas foi observada em 14% das amostras das pacientes. Superexpressão de TWIST-1 foi observada em 17% das amostras dos indivíduos com CM. A análise combinada de ambos os marcadores permitiu a detecção de CTCs em 27,8% das amostras, resultando em um aumento significativo (p < 0,05) na sensibilidade da detecção. Associações significativas (p > 0,05) entre a expressão dos genes e os fatores prognósticos não foram encontradas. Conclusão: A análise combinada de TWIST-1 e MGA aumentou a sensibilidade da detecção de CTCs em comparação com a análise de cada gene. A detecção de CTCs não foi associada a fatores prognósticos conhecidos, sugerindo que ela pode fornecer informações clínicas adicionais aos parâmetros clinicopatológicos de rotina do CM.

Application progress of liquid biopsy in the diagnosis and treatment of biliary tract cancer / 中华肝胆外科杂志

With the development of precision medicine and individualized treatment, tissue biopsy in cancer patients diagnosis and therapy has been broadly used. However, because it’s hard to collect enough samples for biliary tract tumors, liquid biopsy was broadly applied for the diagnosis. In liquid biopsy, circulating tumor cells, circulating tumor DNA, and tumor-derived exosomes carrying tumor-specific information are released from tumor tissue into blood, bile, and other body fluids, which makes tumor biopsy samples easily to be obtained in a non-invasive way. At the same time, through a series of morphological and molecular measurements as well as genetic characterization, liquid biopsy can be used to look for the new early diagnostic markers, and therapeutic targets, monitoring progression and prognosis of diseases. This article outlined the current technology used to detect circulating tumor cells, circulating tumor DNA, and tumor-derived exosomes, and summarizes the latest advances in the clinical application of liquid biopsy in biliary tract cancers.

Values of serum neuron-specific enolase, circulating tumor cells and lactate dehydrogenase levels in the diagnosis and treatment of small cell lung cancer / 中国基层医药

Objective:To investigate the values of serum neuron specific enolase (NSE), circulating tumor cells (CTC) and lactate dehydrogenase (LDH) levels in the diagnosis and treatment of small cell lung cancer (SCLC).Methods:Ninety patients with SCLC who received treatment in the Second Affiliated Hospital of Zhejiang Chinese Medical University, China between December 2017 and December 2019 were retrospectively included in the observation group. Ninety healthy subjects who concurrently received lung examination in the same hospital were included in the healthy control group. An additional 90 patients with benign lung disease were included in the benign lung disease group. Serum NSE, CTC and LDH levels were determined in each group. The values of serum NSE, CTC and LDH levels in the diagnosis of SCLC were analyzed. Serum NSE, CTC and LDH levels were compared between before and after chemotherapy and their values in the treatment of SCLC were analyzed.Results:There were significant differences in serum NSE, CTC and LDH levels between three groups ( F = 359.789, 188.873 and 768.704, all P < 0.001). Serum NSE, CTC and LDH levels in the benign lung disease group were significantly greater than those in the healthy control group and significantly lower than those in the observation group. The receiver operating characteristic curve (ROC curve) analysis showed that the AUC values of serum NSE, CTC and LDH levels in the diagnosis of SCLC were 0.995, 0.953 and 0.987, respectively. The diagnostic accuracy was very high. The value at the maximum tangent point of Youden's index of serum NSE, CTC and LDH levels at the left-upper corner of the ROC curve was taken as the most appropriate cut-off value. The sensitivity and specificity of the most appropriate cut-off value of serum NSE, CTC and LDH levels in the prediction of SCLC were 100.0%/94.4%/91.1% and 94.4%/88.3%/100.0%, respectively. Therefore, serum NSE, CTC and LDH levels were of high values in the predication of SCLC. After chemotherapy, serum NSE, CTC and LDH levels in patients with SCLC were significantly lower than those before chemotherapy [NSE: (12.26 ± 3.26) μg/L vs. (18.36 ± 4.64) μg/L; CTC: (3.54 ± 1.08) counts/5 mL vs. (7.34 ± 1.30) counts/5 mL; LDH: (24.61 ± 9.66) U/L vs. (50.29 ± 16.29) U/L, t = 10.205, 12.864, 21.330, all P < 0.001). Serum NSE, CTC and LDH levels in SCLC patients in whom treatment was effective were significantly lower than those in SCLC patients in which treatment was not effective ( t = 8.111, 7.347, 10.731, all P < 0.001). Spearman correlation results showed that serum NSE, CTC and LDH levels were significantly negatively correlated with curative effects ( r = -0.562, -0.562, -0.758, all P < 0.05). Conclusion:Serum NSE, CTC and LDH levels are highly expressed in SCLC patients, which can be used as markers for early clinical diagnosis and treatment of SCLC.

Short-term efficacy of pemetrexed combined with cisplatin in the treatment of malignant pleural effusion and its effect on serum carbohydrate antigen 199 level and circulating tumor cells / 中国基层医药

Objective:To investigate the short-term efficacy of pemetrexed combined with cisplatin in the treatment of malignant pleural effusion and its effect on serum carbohydrate antigen 199 level and circulating tumor cells.Methods:Sixty patients with advanced lung cancer complicated by malignant pleural effusion who received treatment in Healthcare Group of Cixi Third People's Hospital, China from January 2017 to January 2020 were included in this study. They were randomly assigned to receive intrapleural injection of cisplatin (cisplatin alone group, n = 30) or intrapleural injection of cisplatin combined with intravenous injection of pemetrexed (cisplatin + pemetrexed group, n = 30) after thoracic drainage. Before and 1 month after treatment, pleural effusion was measured to evaluate clinical efficacy and improvement in quality of life. Serum carcinoembryonic antigen level, serum carbohydrate antigen 199 level and circulating tumor cells were determined. Adverse reactions during the treatment were recorded. Results:Total effective rate and the rate of improvement in quality of life in the cisplatin + pemetrexed group were 66.67% (20/30) and 70.00% (21/30), respectively, which were significantly higher than those in the cisplatin alone group [40.00% (12/30) and 43.33% (13/30), χ2 = 4.286, 4.344, both P < 0.05]. After treatment, serum carcinoembryonic antigen and serum carbohydrate antigen 199 levels in each group were significantly decreased compared with before treatment (both P < 0.05). Serum carcinoembryonic antigen and serum carbohydrate antigen 199 level in the cisplatin + pemetrexed group were (22.26 ± 5.13) ng/mL and (20.12 ± 4.35) U/mL, respectively, which were significantly lower than those in the cisplatin alone group [(31.64 ± 6.46) ng/mL, (28.07 ± 5.61) U/mL, t = 6.228, 3.134, both P < 0.05). In the cisplatin alone group, there was no significant difference in the proportion of circulating tumor cells before and after treatment ( P > 0.05). In the cisplatin + pemetrexed group, the proportion of circulating tumor cells after treatment was significantly lower than that before treatment ( χ2 = 4.286, P < 0.05). After treatment, there was no significant difference in the proportion of circulating tumor cells between the two groups ( P > 0.05). During the treatment, there were no significant differences in the incidences of rash, nausea and vomiting, leukopenia, and anemia between the two groups (all P > 0.05). Conclusion:Pemetrexed combined with cisplatin in the treatment of malignant pleural effusion exhibits better short-term efficacy than cisplatin alone. The combined therapy is more conducive to relieving clinical symptoms and improving the quality of life with higher safety than monotherapy.

Progress of circulating tumor cells in primary bone tumor / 肿瘤研究与临床

Some primary bone tumors are prone to hematogenous metastasis and after that, the therapeutic effect is not that good and prognosis is poor. Circulating tumor cells (CTC) shed from the tumor cells of primary or metastatic focus and then enter into blood circulation. CTC may appear in the early stage of the tumor, which can implant in distant organs to form metastatic sites and self-implant in the primary sites leading to the tumor recurrence; CTC are closely related with the prognosis of patients with tumors. In most primary bone tumors, CTC are heterogeneous compared with primary tumor cells. Studying CTC from various aspects can provide a basis for the early diagnosis and treatment of primary bone tumors. This review summarizes the current researches of CTC in common primary bone tumors, and expects the future of research direction and application practice in clinic.

Efficacy of folate-coupled quantum dots immunomagnetic beads method for detecting circulating tumor cells in epithelial ovarian cancer and the association of circulating tumor cells with clinicopathological features / 肿瘤研究与临床

Objective:To explore the effectiveness of folate-coupled quantum dots (FA-QD) immunomagnetic beads method for detecting circulating tumor cells (CTC) in epithelial ovarian cancer and the association of CTC with clinicopathological features of tumor patients.Methods:A total of 67 ovarian cancer patients in Shanxi Provincial People's Hospital from August 2019 to January 2020 were selected. Ovarian cancer SKOV-3 cells were divided into 5 cell number gradients (0, 100, 150, 200, 300), the detection rates of CTC were compared by using epithelial cell adhesion molecule (EpCAM) immunomagnetic beads (single standard method) and FA-QD immunomagnetic beads method (double standard method). The number of CTC in peripheral blood of ovarian cancer patients was detected by using FA-QD immunomagnetic beads method, and those with positive CTC under fluorescence microscope were treated as CTC positive patients. The association of CTC with clinicopathological factors and tumor markers of tumor patients was analyzed.Results:The average capture efficiency rate of CTC in SKOV-3 cells detected by FA-QD immunomagnetic beads method (83.4%) was higher than that by EpCAM immunomagnetic beads method (70.3%). Among 67 patients of ovarian cancer, the proportion of CTC positive patients was 30.0% (3/10) in stage Ⅰ-Ⅱ, 91.9% (34/37) in stage Ⅲ, 95.0% (19/20) in stage Ⅳ, and the difference was statistically significant ( P < 0.05). The proportion of CTC positive patients with lymph node metastasis was higher than that of patients without lymph node metastasis [97.1% (33/34) vs. 69.7% (23/33)], and the difference was statistically significant ( P < 0.05). The proportion of CTC positive patients with human epididymis protein 4 (HE4)>110 pmol/L was lower than that of patients with HE4 ≤ 110 pmol/L [58.8% (10/17) vs. 92.0% (46/50)], and the difference was statistically significant ( P = 0.005). There were no statistically significant differences in the proportion of CTC positive patients stratified by age, menopause, pathological differentiation, distant metastasis, carbohydrate antigen (CA) 125, CA199, carcino-embryonic antigen (CEA) (all P > 0.05). Conclusions:FA-QD immunomagnetic beads method can effectively detect CTC in peripheral blood of patients with epithelial ovarian cancer. The level of CTC in patients with epithelial ovarian cancer is related to lymph node metastasis, clinical TNM stage and HE4 level.