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14 pages, 2829 KiB

Open AccessArticle

Single Step In Situ Detection of Surface Protein and MicroRNA in Clustered Extracellular Vesicles Using Flow Cytometry

by Hee Cheol Yang and Won Jong Rhee

J. Clin. Med. 2021, 10(2), 319; https://doi.org/10.3390/jcm10020319 - 17 Jan 2021

Cited by 11 | Viewed by 3315

Abstract

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are [...] Read more.

Because cancers are heterogeneous, it is evident that multiplexed detection is required to achieve disease diagnosis with high accuracy and specificity. Extracellular vesicles (EVs) have been a subject of great interest as sources of novel biomarkers for cancer liquid biopsy. However, EVs are nano-sized particles that are difficult to handle; thus, it is necessary to develop a method that enables efficient and straightforward EV biomarker detection. In the present study, we developed a method for single step in situ detection of EV surface proteins and inner miRNAs simultaneously using a flow cytometer. CD63 antibody and molecular beacon-21 were investigated for multiplexed biomarker detection in normal and cancer EVs. A phospholipid-polymer-phospholipid conjugate was introduced to induce clustering of the EVs analyzed using nanoparticle tracking analysis, which enhanced the detection signals. As a result, the method could detect and distinguish cancer cell-derived EVs using a flow cytometer. Thus, single step in situ detection of multiple EV biomarkers using a flow cytometer can be applied as a simple, labor- and time-saving, non-invasive liquid biopsy for the diagnosis of various diseases, including cancer. Full article

(This article belongs to the Special Issue Circulating Biomarkers as a Liquid Biopsy for Cancer)

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14 pages, 1023 KiB

Open AccessArticle

The Clinical Significance of RAS, PIK3CA, and PTEN Mutations in Non-Small Cell Lung Cancer Using Cell-Free DNA

by Won Jin Chang, Jae Sook Sung, Sung Yong Lee, Eun Joo Kang, Nak-Jung Kwon, Hae Mi Kim, Sang Won Shin, Jung Yoon Choi, Yoon Ji Choi, Ju Won Kim, Kyong Hwa Park and Yeul Hong Kim

J. Clin. Med. 2020, 9(8), 2642; https://doi.org/10.3390/jcm9082642 - 14 Aug 2020

Cited by 3 | Viewed by 2810

Abstract

Mutations in the EGFR gene downstream signaling pathways may cause receptor-independent pathway activation, making tumors unresponsive to EGFR inhibitors. However, the clinical significance of RAS, PIK3CA or PTEN mutations in NSCLC is unclear. In this study, patients who were initially diagnosed with NSCLC [...] Read more.

Mutations in the EGFR gene downstream signaling pathways may cause receptor-independent pathway activation, making tumors unresponsive to EGFR inhibitors. However, the clinical significance of RAS, PIK3CA or PTEN mutations in NSCLC is unclear. In this study, patients who were initially diagnosed with NSCLC or experienced recurrence after surgical resection were enrolled, and blood samples was collected. Ultra-deep sequencing analysis of cfDNA using Ion AmpliSeq Cancer Hotspot Panel v2 with Proton platforms was conducted. RAS/PIK3CA/PTEN mutations were frequently detected in cfDNA in stage IV NSCLC (58.1%), and a high proportion of the patients (47.8%) with mutations had bone metastases at diagnosis. The frequency of RAS/PIK3CA/PTEN mutations in patients with activating EGFR mutation was 61.7%. The median PFS for EGFR-TKIs was 15.1 months in patients without RAS/PIK3CA/PTEN mutations, and 19.9 months in patients with mutations (p = 0.549). For patients with activating EGFR mutations, the overall survival was longer in patients without RAS/PIK3CA/PTEN mutations (53.8 months vs. 27.4 months). For the multivariate analysis, RAS/PIK3CA/PTEN mutations were independent predictors of poor prognosis in patients with activating EGFR mutations. In conclusion, RAS, PIK3CA and PTEN mutations do not hamper EGFR-TKI treatment outcome; however, they predict a poor OS when activating EGFR mutations coexist. Full article

(This article belongs to the Special Issue Circulating Biomarkers as a Liquid Biopsy for Cancer)

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10 pages, 2864 KiB

Open AccessArticle

Detection of Circulating Tumor Cells in Renal Cell Carcinoma: Disease Stage Correlation and Molecular Characterization

by Petr Klezl, Eliska Pospisilova, Katarina Kolostova, Jindrich Sonsky, Ondrej Maly, Robert Grill, Ireneusz Pawlak and Vladimir Bobek

J. Clin. Med. 2020, 9(5), 1372; https://doi.org/10.3390/jcm9051372 - 7 May 2020

Cited by 13 | Viewed by 2691

Abstract

The presence of circulating tumor cells (CTCs) in patients with solid tumors is associated with poor prognosis. However, there are limited data concerning the detection of CTCs in renal cell cancer (RCC). The aim of this study is to evaluate the presence of [...] Read more.

The presence of circulating tumor cells (CTCs) in patients with solid tumors is associated with poor prognosis. However, there are limited data concerning the detection of CTCs in renal cell cancer (RCC). The aim of this study is to evaluate the presence of CTCs in peripheral blood of patients with RCC undergoing surgery (n = 186). CTCs were tested before and after surgery as well as during the follow-up period afterwards. In total 495 CTC testing in duplicates were provided. To enrich CTCs, a size-based separation protocol and tube MetaCell® was used. CTCs presence was evaluated by single cell cytomorphology based on vital fluorescence microscopy. Additionally, to standardly applied fluorescence stains, CTCs viability was controlled by mitochondrial activity. CTCs were detected independently on the sampling order in up to 86.7% of the tested blood samples in patients undergoing RCC surgery. There is higher probability of CTC detection with growing tumor size, especially in clear cell renal cell cancer (ccRCC) cases. Similarly, the tumor size corresponds with metastasis presence and lymph node positivity and CTC detection. This paper describes for the first-time successful analysis of viable CTCs and their mitochondria as a part of the functional characterization of CTCs in RCC. Full article

(This article belongs to the Special Issue Circulating Biomarkers as a Liquid Biopsy for Cancer)

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15 pages, 2623 KiB

Open AccessArticle

Rapid and Efficient Isolation of Exosomes by Clustering and Scattering

by Jinhyun Kim, Hoyoon Lee, KyongHwa Park and Sehyun Shin

J. Clin. Med. 2020, 9(3), 650; https://doi.org/10.3390/jcm9030650 - 28 Feb 2020

Cited by 24 | Viewed by 8622

Abstract

Tumor-derived extracellular vesicles (EVs) have become important biomarkers of liquid biopsies for precision medicine. However, the clinical application of EVs has been limited due to the lack of EV isolation practical technology applicable to clinical environments. Here, we report an innovative EV isolation [...] Read more.

Tumor-derived extracellular vesicles (EVs) have become important biomarkers of liquid biopsies for precision medicine. However, the clinical application of EVs has been limited due to the lack of EV isolation practical technology applicable to clinical environments. Here, we report an innovative EV isolation method, which is quick and simple, and facilitates high-yield and high-purity EV isolation from blood. Introducing a cationic polymer in plasma resulted in rapid clustering of anionic EVs and a chaotropic agent can separate EVs from these clusters. Isolated EVs were characterized in terms of size distribution, morphology, surface protein markers, and exosomal RNA. Through performance comparison with various methods, including ultracentrifugation (UC), the present method delivered the highest recovery rate (~20 folds that of UC) and purity ratio (3.5 folds that of UC) of EVs in a short period of time (<20 min). The proposed method is expected to be used in basic and applied research on EV isolation and in clinical applications. Full article

(This article belongs to the Special Issue Circulating Biomarkers as a Liquid Biopsy for Cancer)

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16 pages, 3453 KiB

Open AccessArticle

Genomic Profiling of Uterine Aspirates and cfDNA as an Integrative Liquid Biopsy Strategy in Endometrial Cancer

by Carlos Casas-Arozamena, Eva Díaz, Cristian Pablo Moiola, Lorena Alonso-Alconada, Alba Ferreiros, Alicia Abalo, Carlos López Gil, Sara S. Oltra, Javier de Santiago, Silvia Cabrera, Victoria Sampayo, Marta Bouso, Efigenia Arias, Juan Cueva, Eva Colas, Ana Vilar, Antonio Gil-Moreno, Miguel Abal, Gema Moreno-Bueno and Laura Muinelo-Romay

J. Clin. Med. 2020, 9(2), 585; https://doi.org/10.3390/jcm9020585 - 21 Feb 2020

Cited by 26 | Viewed by 4081

Abstract

The incidence and mortality of endometrial cancer (EC) have risen in recent years, hence more precise management is needed. Therefore, we combined different types of liquid biopsies to better characterize the genetic landscape of EC in a non-invasive and dynamic manner. Uterine aspirates [...] Read more.

The incidence and mortality of endometrial cancer (EC) have risen in recent years, hence more precise management is needed. Therefore, we combined different types of liquid biopsies to better characterize the genetic landscape of EC in a non-invasive and dynamic manner. Uterine aspirates (UAs) from 60 patients with EC were obtained during surgery and analyzed by next-generation sequencing (NGS). Blood samples, collected at surgery, were used for cell-free DNA (cfDNA) and circulating tumor cell (CTC) analyses. Finally, personalized therapies were tested in patient-derived xenografts (PDXs) generated from the UAs. NGS analyses revealed the presence of genetic alterations in 93% of the tumors. Circulating tumor DNA (ctDNA) was present in 41.2% of cases, mainly in patients with high-risk tumors, thus indicating a clear association with a more aggressive disease. Accordingly, the results obtained during the post-surgery follow-up indicated the presence of ctDNA in three patients with progressive disease. Moreover, 38.9% of patients were positive for CTCs at surgery. Finally, the efficacy of targeted therapies based on the UA-specific mutational landscape was demonstrated in PDX models. Our study indicates the potential clinical applicability of a personalized strategy based on a combination of different liquid biopsies to characterize and monitor tumor evolution, and to identify targeted therapies. Full article

(This article belongs to the Special Issue Circulating Biomarkers as a Liquid Biopsy for Cancer)

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13 pages, 958 KiB

Open AccessArticle

Plasma Lysyl-tRNA Synthetase 1 (KARS1) as a Novel Diagnostic and Monitoring Biomarker for Colorectal Cancer

by Ji Hun Suh, Min Chul Park, Peter C. Goughnour, Byung Soh Min, Sang Bum Kim, Woo Yong Lee, Yong Beom Cho, Jae Hee Cheon, Kang Young Lee, Do-Hyun Nam and Sunghoon Kim

J. Clin. Med. 2020, 9(2), 533; https://doi.org/10.3390/jcm9020533 - 15 Feb 2020

Cited by 8 | Viewed by 3632

Abstract

Colorectal cancer (CRC) is one of the leading causes of world cancer deaths. To improve the survival rate of CRC, diagnosis and post-operative monitoring is necessary. Currently, biomarkers are used for CRC diagnosis and prognosis. However, these biomarkers have limitations of specificity and [...] Read more.

Colorectal cancer (CRC) is one of the leading causes of world cancer deaths. To improve the survival rate of CRC, diagnosis and post-operative monitoring is necessary. Currently, biomarkers are used for CRC diagnosis and prognosis. However, these biomarkers have limitations of specificity and sensitivity. Levels of plasma lysyl-tRNA synthetase (KARS1), which was reported to be secreted from colon cancer cells by stimuli, along with other secreted aminoacyl-tRNA synthetases (ARSs), were analyzed in CRC and compared with the currently used biomarkers. The KARS1 levels of CRC patients (n = 164) plasma were shown to be higher than those of healthy volunteers (n = 32). The diagnostic values of plasma KARS1 were also evaluated by receiving operating characteristic (ROC) curve. Compared with other biomarkers and ARSs, KARS1 showed the best diagnostic value for CRC. The cancer specificity and burden correlation of plasma KARS1 level were validated using azoxymethane (AOM)/dextran sodium sulfate (DSS) model, and paired pre- and post-surgery CRC patient plasma. In the AOM/DSS model, the plasma level of KARS1 showed high correlation with number of polyps, but not for inflammation. Using paired pre- and post-surgery CRC plasma samples (n = 60), the plasma level of KARS1 was significantly decreased in post-surgery samples. Based on these evidence, KARS1, a surrogate biomarker reflecting CRC burden, can be used as a novel diagnostic and post-operative monitoring biomarker for CRC. Full article

(This article belongs to the Special Issue Circulating Biomarkers as a Liquid Biopsy for Cancer)

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20 pages, 2983 KiB

Open AccessArticle

Looking for a Better Characterization of Triple-Negative Breast Cancer by Means of Circulating Tumor Cells

by Manuel Abreu, Pablo Cabezas-Sainz, Thais Pereira-Veiga, Catalina Falo, Alicia Abalo, Idoia Morilla, Teresa Curiel, Juan Cueva, Carmela Rodríguez, Vanesa Varela-Pose, Ramón Lago-Lestón, Patricia Mondelo, Patricia Palacios, Gema Moreno-Bueno, Amparo Cano, Tomás García-Caballero, Miquel Ángel Pujana, Laura Sánchez-Piñón, Clotilde Costa, Rafael López and Laura Muinelo-Romay Show full author list Hide full author list

J. Clin. Med. 2020, 9(2), 353; https://doi.org/10.3390/jcm9020353 - 27 Jan 2020

Cited by 16 | Viewed by 4069

Abstract

Traditionally, studies to address the characterization of mechanisms promoting tumor aggressiveness and progression have been focused only on primary tumor analyses, which could provide relevant information but have limitations to really characterize the more aggressive tumor population. To overcome these limitations, circulating tumor [...] Read more.

Traditionally, studies to address the characterization of mechanisms promoting tumor aggressiveness and progression have been focused only on primary tumor analyses, which could provide relevant information but have limitations to really characterize the more aggressive tumor population. To overcome these limitations, circulating tumor cells (CTCs) represent a noninvasive and valuable tool for real-time profiling of disseminated tumor cells. Therefore, the aim of the present study was to explore the value of CTC enumeration and characterization to identify markers associated with the outcome and the aggressiveness of triple-negative breast cancer (TNBC). For that aim, the CTC population from 32 patients diagnosed with TNBC was isolated and characterized. This population showed important cell plasticity in terms of expression of epithelia/mesenchymal and stemness markers, suggesting the relevance of epithelial to mesenchymal transition (EMT) intermediate phenotypes for efficient tumor dissemination. Importantly, the CTC signature demonstrated prognostic value to predict the patients’ outcome and pointed to a relevant role of tissue inhibitor of metalloproteinases 1 (TIMP1) and androgen receptor (AR) for TNBC biology. Furthermore, we also analyzed the usefulness of the AR and TIMP1 blockade to target TNBC proliferation and dissemination using in vitro and in vivo zebra fish and mouse models. Overall, the molecular characterization of CTCs from advanced TNBC patients identifies highly specific biomarkers with potential applicability as noninvasive prognostic markers and reinforced the value of TIMP1 and AR as potential therapeutic targets to tackle the most aggressive breast cancer. Full article

(This article belongs to the Special Issue Circulating Biomarkers as a Liquid Biopsy for Cancer)

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13 pages, 2713 KiB

Open AccessArticle

Affinity-Enhanced CTC-Capturing Hydrogel Microparticles Fabricated by Degassed Mold Lithography

by Nak Jun Lee, Sejung Maeng, Hyeon Ung Kim, Yoon Ho Roh, Changhyun Hwang, Jongjin Kim, Ki-Tae Hwang and Ki Wan Bong

J. Clin. Med. 2020, 9(2), 301; https://doi.org/10.3390/jcm9020301 - 21 Jan 2020

Cited by 6 | Viewed by 3655

Abstract

Technologies for the detection and isolation of circulating tumor cells (CTCs) are essential in liquid biopsy, a minimally invasive technique for early diagnosis and medical intervention in cancer patients. A promising method for CTC capture, using an affinity-based approach, is the use of [...] Read more.

Technologies for the detection and isolation of circulating tumor cells (CTCs) are essential in liquid biopsy, a minimally invasive technique for early diagnosis and medical intervention in cancer patients. A promising method for CTC capture, using an affinity-based approach, is the use of functionalized hydrogel microparticles (MP), which have the advantages of water-like reactivity, biologically compatible materials, and synergy with various analysis platforms. In this paper, we demonstrate the feasibility of CTC capture by hydrogel particles synthesized using a novel method called degassed mold lithography (DML). This technique increases the porosity and functionality of the MPs for effective conjugation with antibodies. Qualitative fluorescence analysis demonstrates that DML produces superior uniformity, integrity, and functionality of the MPs, as compared to conventional stop flow lithography (SFL). Analysis of the fluorescence intensity from porosity-controlled MPs by each reaction step of antibody conjugation elucidates that more antibodies are loaded when the particles are more porous. The feasibility of selective cell capture is demonstrated using breast cancer cell lines. In conclusion, using DML for the synthesis of porous MPs offers a powerful method for improving the cell affinity of the antibody-conjugated MPs. Full article

(This article belongs to the Special Issue Circulating Biomarkers as a Liquid Biopsy for Cancer)

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18 pages, 1360 KiB

Open AccessArticle

Sequential Circulating Tumor Cell Counts in Patients with Locally Advanced or Metastatic Hepatocellular Carcinoma: Monitoring the Treatment Response

by Kun-Ming Rau, Chien-Ting Liu, Yu-Chiao Hsiao, Kai-Yin Hsiao, Tzu-Min Wang, Wei-Shan Hung, Yu-Li Su, Wei-Ching Liu, Cheng-Hsu Wang, Hsueh-Ling Hsu, Po-Heng Chuang, Ju-Chien Cheng and Ching-Ping Tseng

J. Clin. Med. 2020, 9(1), 188; https://doi.org/10.3390/jcm9010188 - 10 Jan 2020

Cited by 19 | Viewed by 3275

Abstract

Hepatocellular carcinoma (HCC) is among the most common causes of cancer death in men. Whether or not a longitudinal follow-up of circulating tumor cells (CTCs) before and at different time points during systemic/targeted therapy is useful for monitoring the treatment response of patients [...] Read more.

Hepatocellular carcinoma (HCC) is among the most common causes of cancer death in men. Whether or not a longitudinal follow-up of circulating tumor cells (CTCs) before and at different time points during systemic/targeted therapy is useful for monitoring the treatment response of patients with locally advanced or metastatic HCC has been evaluated in this study. Blood samples (n = 104) were obtained from patients with locally advanced or metastatic HCC (n = 30) for the enrichment of CTCs by a negative selection method. Analysis of the blood samples from patients with defined disease status (n = 81) revealed that those with progressive disease (PD, n = 37) had significantly higher CTC counts compared to those with a partial response (PR) or stable disease (SD; n = 44 for PR + SD, p = 0.0002). The median CTC count for patients with PD and for patients with PR and SD was 50 (interquartile range 21–139) and 15 (interquartile range 4–41) cells/mL of blood, respectively. A longitudinal analysis of patients (n = 17) after a series of blood collections demonstrated that a change in the CTC count correlated with the patient treatment response in most of the cases and was particularly useful for monitoring patients without elevated serum alpha-fetoprotein (AFP) levels. Sequential CTC enumeration during treatment can supplement standard medical tests and benefit the management of patients with locally advanced or metastatic HCC, in particular for the AFP-low cases. Full article

(This article belongs to the Special Issue Circulating Biomarkers as a Liquid Biopsy for Cancer)

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13 pages, 2198 KiB

Open AccessArticle

A Novel Saliva-Based miRNA Signature for Colorectal Cancer Diagnosis

by Óscar Rapado-González, Blanca Majem, Ana Álvarez-Castro, Roberto Díaz-Peña, Alicia Abalo, Leticia Suárez-Cabrera, Antonio Gil-Moreno, Anna Santamaría, Rafael López-López, Laura Muinelo-Romay and María Mercedes Suarez-Cunqueiro

J. Clin. Med. 2019, 8(12), 2029; https://doi.org/10.3390/jcm8122029 - 20 Nov 2019

Cited by 49 | Viewed by 5117

Abstract

Salivary microRNAs (miRNAs) are of high interest as diagnostic biomarkers for non-oral cancer. However, little is known about their value for colorectal cancer (CRC) detection. Our study aims to characterize salivary miRNAs in order to identify non-invasive markers for CRC diagnosis. The screening [...] Read more.

Salivary microRNAs (miRNAs) are of high interest as diagnostic biomarkers for non-oral cancer. However, little is known about their value for colorectal cancer (CRC) detection. Our study aims to characterize salivary miRNAs in order to identify non-invasive markers for CRC diagnosis. The screening of 754 miRNAs was performed in saliva samples from 14 CRC and 10 healthy controls. The differential expressed miRNAs were validated by RT-qPCR in 51 CRC, 19 adenomas and 37 healthy controls. Receiver operating characteristic (ROC) curves and logistic regression models were performed to analyze the clinical value of these miRNAs. Twenty-two salivary miRNAs were significantly deregulated in CRC patients vs. healthy individuals (p < 0.05) in the discovery phase. From those, five upregulated miRNAs (miR-186-5p, miR-29a-3p, miR-29c-3p, miR-766-3p, and miR-491-5p) were confirmed to be significantly higher in the CRC vs. healthy group (p < 0.05). This five-miRNA signature showed diagnostic value (72% sensitivity, 66.67% specificity, AUC = 0.754) to detect CRC, which was even higher in combination with carcinoembryonic antigen (CEA) levels. Overall, after the first global characterization of salivary miRNAs in CRC, a five-miRNA panel was identified as a promising tool to diagnose this malignancy, representing a novel approach to detect cancer-associated epigenetic alterations using a non-invasive strategy. Full article

(This article belongs to the Special Issue Circulating Biomarkers as a Liquid Biopsy for Cancer)

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Review

Jump to: Research

17 pages, 966 KiB

Open AccessReview

The Potential Role of Liquid Biopsies in Advancing the Understanding of Neuroendocrine Neoplasms

by Dinakshi Shah, Angela Lamarca, Juan W Valle and Mairéad G McNamara

J. Clin. Med. 2021, 10(3), 403; https://doi.org/10.3390/jcm10030403 - 21 Jan 2021

Cited by 6 | Viewed by 2400

Abstract

Tumour tissue as a source for molecular profiling and for in vivo models has limitations (e.g., difficult access, limited availability, single time point, potential heterogeneity between primary and metastatic sites). Conversely, liquid biopsies provide an easily accessible approach, enabling timely and longitudinal interrogation [...] Read more.

Tumour tissue as a source for molecular profiling and for in vivo models has limitations (e.g., difficult access, limited availability, single time point, potential heterogeneity between primary and metastatic sites). Conversely, liquid biopsies provide an easily accessible approach, enabling timely and longitudinal interrogation of the tumour molecular makeup, with increased ability to capture spatial and temporal intra-tumour heterogeneity compared to tumour tissue. Blood-borne biomarker assays (e.g., circulating tumour cells (CTCs), circulating free/tumour DNA (cf/ctDNA)) pose unique opportunities for aiding in the molecular characterisation and phenotypic subtyping of neuroendocrine neoplasms and will be discussed in this article. Full article

(This article belongs to the Special Issue Circulating Biomarkers as a Liquid Biopsy for Cancer)

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