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5 pages, 221 KiB

Open AccessEditorial

Liquid Biopsy, an Everchanging Balance between Clinical Utility and Emerging Technologies

by Linda Cucciniello, Lorenzo Gerratana and Fabio Puglisi

Cancers 2022, 14(17), 4277; https://doi.org/10.3390/cancers14174277 - 01 Sep 2022

Cited by 2 | Viewed by 1058

Abstract

To date, tissue biopsy still represents the mainstay for tumor diagnosis and molecular characterization [...] Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

Research

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17 pages, 1829 KiB

Open AccessArticle

Effect of Osimertinib on CTCs and ctDNA in EGFR Mutant Non-Small Cell Lung Cancer Patients: The Prognostic Relevance of Liquid Biopsy

by Galatea Kallergi, Emmanouil Kontopodis, Aliki Ntzifa, Núria Jordana-Ariza, Niki Karachaliou, Evangelia Pantazaka, Haris A. Charalambous, Amanda Psyrri, Emily Tsaroucha, Ioannis Boukovinas, Anna Koumarianou, Dora Hatzidaki, Evi Lianidou, Vassilis Georgoulias, Rafael Rosell and Athanasios Kotsakis

Cancers 2022, 14(6), 1574; https://doi.org/10.3390/cancers14061574 - 19 Mar 2022

Cited by 8 | Viewed by 2770

Abstract

Introduction: Liquid biopsy is a useful tool for monitoring treatment outcome in solid tumors, including lung cancer. The relevance of monitoring CTCs and plasma ctDNA as predictors of clinical outcome was assessed in EGFR-mutant NSCLC patients treated with osimertinib. Methods: Forty-seven EGFR-mutant NSCLC [...] Read more.

Introduction: Liquid biopsy is a useful tool for monitoring treatment outcome in solid tumors, including lung cancer. The relevance of monitoring CTCs and plasma ctDNA as predictors of clinical outcome was assessed in EGFR-mutant NSCLC patients treated with osimertinib. Methods: Forty-seven EGFR-mutant NSCLC patients who had progressed on prior first- or second-generation EGFR inhibitors were enrolled in the study and treated with osimertinib, irrespective of the presence of the T790M mutation in the primary tumor or the plasma. Peripheral blood was collected at baseline (n = 47), post-Cycle 1 (n = 47), and at the end of treatment (EOT; n = 39). CTCs were evaluated in 32 patients at the same time points (n = 32, n = 27, and n = 21, respectively) and phenotypic characterization was performed using triple immunofluorescence staining (CK/VIM/CD45). Results: Osimertinib resulted in an ORR of 34% (2 CR) and a DCR of 76.6%. The median PFS and OS values were 7.5 (range, 0.8–52.8) and 15.1 (range, 2.1–52.8) months, respectively. ctDNA was detected in 61.7%, 27.7%, and 61.5% of patients at baseline, post-Cycle 1, and EOT, respectively. CTCs (CK+/CD45-) were detected in 68.8%, 48.1%, and 61.9% of patients at the three time points, respectively. CTCs expressing both epithelial and mesenchymal markers (CK+/VIM+/CD45-) were detected in 56.3% and 29.6% of patients at baseline and post-Cycle 1, respectively. The detection of ctDNA at baseline and post-Cycle 1 was associated with shorter PFS and OS, whereas the ctDNA clearance post-Cycle 1 resulted in a significantly longer PFS and OS. Multivariate analysis revealed that male sex and the detection of ctDNA at baseline were independent predictors of shorter PFS (HR: 2.6, 95% C.I.: 1.2–5.5, p = 0.015 and HR: 3.0, 95% C.I.: 1.3–6.9; p = 0.009, respectively). Conclusions: The decrease in both CTCs and ctDNA occurring early during osimertinib treatment is predictive of better outcome, implying that liquid biopsy monitoring may be a valuable tool for the assessment of treatment efficacy. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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25 pages, 7521 KiB

Open AccessArticle

From Sampling to Sequencing: A Liquid Biopsy Pre-Analytic Workflow to Maximize Multi-Layer Genomic Information from a Single Tube

by Kendra K. Maass, Paulina S. Schad, Agnes M. E. Finster, Pitithat Puranachot, Fabian Rosing, Tatjana Wedig, Nathalie Schwarz, Natalie Stumpf, Stefan M. Pfister and Kristian W. Pajtler

Cancers 2021, 13(12), 3002; https://doi.org/10.3390/cancers13123002 - 15 Jun 2021

Cited by 16 | Viewed by 6606

Abstract

Liquid biopsies hold great promise for the management of cancer. Reliable liquid biopsy data depend on stable and reproducible pre-analytical protocols that comply with quality measures, irrespective of the sampling and processing site. We established a workflow for plasma preservation, followed by processing, [...] Read more.

Liquid biopsies hold great promise for the management of cancer. Reliable liquid biopsy data depend on stable and reproducible pre-analytical protocols that comply with quality measures, irrespective of the sampling and processing site. We established a workflow for plasma preservation, followed by processing, cell-free nucleic acid isolation, quantification, and enrichment of potentially tumor-derived cell-free DNA and RNA. Employing the same input material for a direct comparison of different kits and protocols allowed us to formulate unbiased recommendations for sample collection, storage, and processing. The presented workflow integrates the stabilization in Norgen, PAX, or Streck tubes and subsequent parallel isolation of cell-free DNA and RNA with NucleoSnap and NucleoSpin. Qubit, Bioanalyzer, and TapeStation quantification and quality control steps were optimized for minimal sample use and high sensitivity and reproducibility. We show the efficiency of the proposed workflow by successful droplet digital PCR amplification of both cell-free DNA and RNA and by detection of tumor-specific alterations in low-coverage whole-genome sequencing and DNA methylation profiling of plasma-derived cell-free DNA. For the first time, we demonstrated successful parallel extraction of cell-free DNA and RNA from plasma samples. This workflow paves the road towards multi-layer genomic analysis from one single liquid biopsy sample. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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20 pages, 2343 KiB

Open AccessArticle

Circulating Tumor Cell Clusters Are Frequently Detected in Women with Early-Stage Breast Cancer

by Carolina Reduzzi, Serena Di Cosimo, Lorenzo Gerratana, Rosita Motta, Antonia Martinetti, Andrea Vingiani, Paolo D’Amico, Youbin Zhang, Marta Vismara, Catherine Depretto, Gianfranco Scaperrotta, Secondo Folli, Giancarlo Pruneri, Massimo Cristofanilli, Maria Grazia Daidone and Vera Cappelletti

Cancers 2021, 13(10), 2356; https://doi.org/10.3390/cancers13102356 - 13 May 2021

Cited by 23 | Viewed by 4097

Abstract

The clinical relevance of circulating tumor cell clusters (CTC-clusters) in breast cancer (BC) has been mostly studied using the CellSearch^®, a marker-dependent method detecting only epithelial-enriched clusters. However, due to epithelial-to-mesenchymal transition, resorting to marker-independent approaches can improve CTC-cluster detection. Blood [...] Read more.

The clinical relevance of circulating tumor cell clusters (CTC-clusters) in breast cancer (BC) has been mostly studied using the CellSearch^®, a marker-dependent method detecting only epithelial-enriched clusters. However, due to epithelial-to-mesenchymal transition, resorting to marker-independent approaches can improve CTC-cluster detection. Blood samples collected from healthy donors and spiked-in with tumor mammospheres, or from BC patients, were processed for CTC-cluster detection with 3 technologies: CellSearch^®, CellSieve™ filters, and ScreenCell^® filters. In spiked-in samples, the 3 technologies showed similar recovery capability, whereas, in 19 clinical samples processed in parallel with CellSearch^® and CellSieve™ filters, filtration allowed us to detect more CTC-clusters than CellSearch^® (median number = 7 versus 1, p = 0.0038). Next, samples from 37 early BC (EBC) and 23 metastatic BC (MBC) patients were processed using ScreenCell^® filters for attaining both unbiased enrichment and marker-independent identification (based on cytomorphological criteria). At baseline, CTC-clusters were detected in 70% of EBC cases and in 20% of MBC patients (median number = 2, range 0–20, versus 0, range 0–15, p = 0.0015). Marker-independent approaches for CTC-cluster assessment improve detection and show that CTC-clusters are more frequent in EBC than in MBC patients, a novel finding suggesting that dissemination of CTC-clusters is an early event in BC natural history. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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16 pages, 3022 KiB

Open AccessArticle

Longitudinal Evaluation of PD-L1 Expression on Circulating Tumor Cells in Non-Small Cell Lung Cancer Patients Treated with Nivolumab

by Mio Ikeda, Yasuhiro Koh, Shunsuke Teraoka, Koichi Sato, Jun Oyanagi, Atsushi Hayata, Nahomi Tokudome, Hiroaki Akamatsu, Yuichi Ozawa, Katsuya Endo, Masayuki Higuchi, Masanori Nakanishi, Hiroki Ueda and Nobuyuki Yamamoto

Cancers 2021, 13(10), 2290; https://doi.org/10.3390/cancers13102290 - 11 May 2021

Cited by 15 | Viewed by 2144

Abstract

Although programmed death-ligand 1 (PD-L1) expression on tumor tissue is a validated predictive biomarker for a PD-1 pathway blockade in non-small cell lung cancer (NSCLC), longitudinal changes in its expression during treatment remains elusive. Circulating tumor cells (CTCs) are assumed to reflect the [...] Read more.

Although programmed death-ligand 1 (PD-L1) expression on tumor tissue is a validated predictive biomarker for a PD-1 pathway blockade in non-small cell lung cancer (NSCLC), longitudinal changes in its expression during treatment remains elusive. Circulating tumor cells (CTCs) are assumed to reflect the transition of characteristics of the primary tumor undergoing anticancer treatment. Here, we sequentially evaluated the PD-L1 expression on CTCs in NSCLC patients treated with nivolumab. Forty-five patients were enrolled, and CTCs were enriched from 3 mL of peripheral blood using a microcavity array system at baseline and weeks 4, 8, 12, and 24 or until progressive disease. The effective responses to therapy were compared between patients without progressive disease (PD) at week 8 (i.e., non-PD patients) and in those with PD between weeks 4 and 8 (PD patients) in terms of increased vs. decreased or equal CTC status at week 8 (for non-PD patients) or at the point of PD (for PD patients) compared to the baseline. Significantly more non-PD patients were classified as decreased or equal in number and proportion to PD-L1-positive CTCs among the detected CTCs (PD-L1 positivity rates) (p < 0.05). Moreover, progression-free survival was significantly longer in patients with ≥7.7% PD-L1 positivity rates (n = 8) than in those with <7.7% rates (n = 8; p < 0.01) at week 8. These results suggest the predictive significance of the early evaluation of PD-L1 expression on CTCs for maintaining the benefits from nivolumab treatment. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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19 pages, 3432 KiB

Open AccessArticle

Detection of Genomically Aberrant Cells within Circulating Tumor Microemboli (CTMs) Isolated from Early-Stage Breast Cancer Patients

by Marco Silvestri, Carolina Reduzzi, Giancarlo Feliciello, Marta Vismara, Thomas Schamberger, Cäcilia Köstler, Rosita Motta, Stefano Calza, Cristina Ferraris, Andrea Vingiani, Giancarlo Pruneri, Maria Grazia Daidone, Christoph A. Klein, Bernhard Polzer and Vera Cappelletti

Cancers 2021, 13(6), 1409; https://doi.org/10.3390/cancers13061409 - 19 Mar 2021

Cited by 10 | Viewed by 3590

Abstract

Circulating tumor microemboli (CTMs) are clusters of cancer cells detached from solid tumors, whose study can reveal mechanisms underlying metastatization. As they frequently comprise unknown fractions of leukocytes, the analysis of copy number alterations (CNAs) is challenging. To address this, we titrated known [...] Read more.

Circulating tumor microemboli (CTMs) are clusters of cancer cells detached from solid tumors, whose study can reveal mechanisms underlying metastatization. As they frequently comprise unknown fractions of leukocytes, the analysis of copy number alterations (CNAs) is challenging. To address this, we titrated known numbers of leukocytes into cancer cells (MDA-MB-453 and MDA-MB-36, displaying high and low DNA content, respectively) generating tumor fractions from 0–100%. After low-pass sequencing, ichorCNA was identified as the best algorithm to build a linear mixed regression model for tumor fraction (TF) prediction. We then isolated 53 CTMs from blood samples of six early-stage breast cancer patients and predicted the TF of all clusters. We found that all clusters harbor cancer cells between 8 and 48%. Furthermore, by comparing the identified CNAs of CTMs with their matched primary tumors, we noted that only 31–71% of aberrations were shared. Surprisingly, CTM-private alterations were abundant (30–63%), whereas primary tumor-private alterations were rare (4–12%). This either indicates that CTMs are disseminated from further progressed regions of the primary tumor or stem from cancer cells already colonizing distant sites. In both cases, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be explored in follow-up and mechanistic studies. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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16 pages, 1202 KiB

Open AccessArticle

Phenotypic and Proteomic Analysis Identifies Hallmarks of Blood Circulating Extracellular Vesicles in NSCLC Responders to Immune Checkpoint Inhibitors

by Davide Brocco, Paola Lanuti, Damiana Pieragostino, Maria Concetta Cufaro, Pasquale Simeone, Giuseppina Bologna, Pietro Di Marino, Michele De Tursi, Antonino Grassadonia, Luciana Irtelli, Laura De Lellis, Serena Veschi, Rosalba Florio, Luca Federici, Marco Marchisio, Sebastiano Miscia, Alessandro Cama, Nicola Tinari and Piero Del Boccio

Cancers 2021, 13(4), 585; https://doi.org/10.3390/cancers13040585 - 03 Feb 2021

Cited by 26 | Viewed by 3285

Abstract

Immune checkpoint inhibitors (ICIs) induce durable clinical responses only in a subset of advanced non-small cell lung cancer (NSCLC) patients. There is a need to identify mechanisms of ICI resistance and immunotherapy biomarkers to improve clinical benefit. In this study, we evaluated the [...] Read more.

Immune checkpoint inhibitors (ICIs) induce durable clinical responses only in a subset of advanced non-small cell lung cancer (NSCLC) patients. There is a need to identify mechanisms of ICI resistance and immunotherapy biomarkers to improve clinical benefit. In this study, we evaluated the prognostic and predictive value of circulating endothelial and leukocyte-derived extracellular vesicles (EV) in patients with advanced NSCLC treated with anti-PD-1/PD-L1 agents. In addition, the relationship between total blood circulating EV proteome and response to ICIs was investigated. An optimized flow cytometry method was employed for the identification and subtyping of blood circulating EVs in 59 patients with advanced NSCLC. Blood samples were collected from patients receiving anti-PD-1/PD-L1 inhibitors (n = 31) or chemotherapy (n = 28). An exploratory proteomic analysis of sorted blood EVs was conducted in a subset of patients. Our results show that a low blood concentration of circulating endothelial-derived EVs before treatment was strongly associated to longer overall survival (p = 0.0004) and higher disease control rate (p = 0.045) in patients treated with ICIs. Interestingly, shotgun proteomics revealed that EVs of responders to anti-PD-1 therapy had a specific protein cargo before treatment. In addition, EV protein cargo was specifically modulated during immunotherapy. We identified a previously unknown association between circulating endothelial-derived extracellular vesicle concentration and immunotherapy-related clinical outcomes. We also observed differences in circulating extracellular vesicle proteome according to anti-PD-1-based treatment response in NSCLC patients. Overall, these results may contribute to the identification of novel circulating biomarkers for rational immunotherapy approaches in patients affected by NSCLC. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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11 pages, 990 KiB

Open AccessArticle

The Origin of Tumor DNA in Urine of Urogenital Cancer Patients: Local Shedding and Transrenal Excretion

by Anouk E. Hentschel, Rianne van den Helder, Nienke E. van Trommel, Annina P. van Splunter, Robert A. A. van Boerdonk, Mignon D. J. M. van Gent, Jakko A. Nieuwenhuijzen and Renske D. M. Steenbergen

Cancers 2021, 13(3), 535; https://doi.org/10.3390/cancers13030535 - 31 Jan 2021

Cited by 8 | Viewed by 2090

Abstract

In urogenital cancers, urine as a liquid biopsy for non-invasive cancer detection holds great promise for future clinical application. Their anatomical position allows for the local shedding of tumor DNA, but recent data indicate that tumor DNA in urine might also result from [...] Read more.

In urogenital cancers, urine as a liquid biopsy for non-invasive cancer detection holds great promise for future clinical application. Their anatomical position allows for the local shedding of tumor DNA, but recent data indicate that tumor DNA in urine might also result from transrenal excretion. This study aims to assess the origin of tumor-associated DNA in the urine of 5 bladder and 25 cervical cancer patients. Besides natural voided urine, paired urine samples were collected in which contact with the local tumor was circumvented to bypass local shedding. The latter concerned nephrostomy urine in bladder cancer patients, and catheter urine in cervical cancer patients. Methylation levels of GHSR, SST, and ZIC1 were determined using paired bladder tumor tissues and cervical scrapes as a reference. Urinary methylation levels were compared to natural voided urine of matched controls. To support methylation results, mutation analysis was performed in urine and tissue samples of bladder cancer patients. Increased methylation levels were not only found in natural voided urine from bladder and cervical cancer patients, but also in the corresponding nephrostomy and catheter urine. DNA mutations detected in bladder tumor tissues were also detectable in all paired natural voided urine as well as in a subset of nephrostomy urine. These results provide the first evidence that the suitability of urine as a liquid biopsy for urogenital cancers relies both on the local shedding of tumor cells and cell fragments, as well as the transrenal excretion of tumor DNA into the urine. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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15 pages, 1283 KiB

Open AccessArticle

Preoperative CTC-Detection by CellSearch^® Is Associated with Early Distant Metastasis and Impaired Survival in Resected Pancreatic Cancer

by Harald Hugenschmidt, Knut Jørgen Labori, Elin Borgen, Cathrine Brunborg, Cecilie Bendigtsen Schirmer, Lars Thomas Seeberg, Bjørn Naume and Gro Wiedswang

Cancers 2021, 13(3), 485; https://doi.org/10.3390/cancers13030485 - 27 Jan 2021

Cited by 19 | Viewed by 2631

Abstract

In patients with presumed pancreatic ductal adenocarcinoma (PDAC), biomarkers that may open for personalised, risk-adapted treatment are lacking. The study analysed the impact of CTCs-presence on the patterns of recurrence and survival in 98 patients resected for PDAC with 5–10 years of follow-up. [...] Read more.

In patients with presumed pancreatic ductal adenocarcinoma (PDAC), biomarkers that may open for personalised, risk-adapted treatment are lacking. The study analysed the impact of CTCs-presence on the patterns of recurrence and survival in 98 patients resected for PDAC with 5–10 years of follow-up. Preoperative samples were analysed by the CellSearch^® system for EpCAM+/DAPI+/CK+/CD45-CTCs. CTCs were detected in 7 of the 98 patients. CTCs predicted a significantly shorter median disease-free survival (DFS) of 3.3 vs. 9.2 months and a median cancer specific survival (CSS)of 6.3 vs. 18.5 months. Relapse status was confirmed by imaging for 87 patients. Of these, 58 patients developed distant metastases (DM) and 29 developed isolated local recurrence (ILR) as the first sign of cancer relapse. All patients with CTCs experienced DM. pN-status and histological grade >2 were other independent risk factors for DM, but only CTCs predicted significantly shorter cancer-specific, disease-free and post-recurrence survival. Preoperative parameters did not affect clinical outcome. We conclude that CTC presence in resected PDAC patients predicted early distant metastasis and impaired survival. Preoperative CTCs alone or in combination with histopathological factors may guide initial treatment decisions in patients with resectable PDAC in the future. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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13 pages, 1486 KiB

Open AccessArticle

Improved Prognostic Stratification Using Circulating Tumor Cell Clusters in Patients with Metastatic Castration-Resistant Prostate Cancer

by Chun Wang, Zhenchao Zhang, Weelic Chong, Rui Luo, Ronald E. Myers, Jian Gu, Jianqing Lin, Qiang Wei, Bingshan Li, Timothy R. Rebbeck, Grace Lu-Yao, William K. Kelly and Hushan Yang

Cancers 2021, 13(2), 268; https://doi.org/10.3390/cancers13020268 - 13 Jan 2021

Cited by 17 | Viewed by 3194

Abstract

Liquid biopsy-based biomarkers have advantages in monitoring the dynamics of metastatic castration-resistant prostate cancer (mCRPC), a bone-predominant metastatic disease. Previous studies have demonstrated associations between circulating tumor cells (CTCs) and clinical outcomes of mCRPC patients, but little is known about the prognostic value [...] Read more.

Liquid biopsy-based biomarkers have advantages in monitoring the dynamics of metastatic castration-resistant prostate cancer (mCRPC), a bone-predominant metastatic disease. Previous studies have demonstrated associations between circulating tumor cells (CTCs) and clinical outcomes of mCRPC patients, but little is known about the prognostic value of CTC-clusters. In 227 longitudinally collected blood samples from 64 mCRPC patients, CTCs and CTC-clusters were enumerated using the CellSearch platform. The associations of CTC and CTC-cluster counts with progression-free survival (PFS) and overall survival (OS), individually and jointly, were evaluated by Cox models. CTCs and CTC-clusters were detected in 24 (37.5%) and 8 (12.5%) of 64 baseline samples, and in 119 (52.4%) and 27 (11.9%) of 227 longitudinal samples, respectively. CTC counts were associated with both PFS and OS, but CTC-clusters were only independently associated with an increased risk of death. Among patients with unfavorable CTCs (≥5), the presence of CTC-clusters signified a worse survival (log-rank p = 0.0185). mCRPC patients with both unfavorable CTCs and CTC-clusters had the highest risk for death (adjusted hazard ratio 19.84, p = 0.0072), as compared to those with <5 CTCs. Analyses using longitudinal data yielded similar results. In conclusion, CTC-clusters provided additional prognostic information for further stratifying death risk among patients with unfavorable CTCs. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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17 pages, 10326 KiB

Open AccessArticle

Ex Vivo Expansion and Drug Sensitivity Profiling of Circulating Tumor Cells from Patients with Small Cell Lung Cancer

by Hsin-Lun Lee, Jeng-Fong Chiou, Peng-Yuan Wang, Long-Sheng Lu, Chia-Ning Shen, Han-Lin Hsu, Thierry Burnouf, Lai-Lei Ting, Pai-Chien Chou, Chi-Li Chung, Kai-Ling Lee, Her-Shyong Shiah, Yen-Lin Liu and Yin-Ju Chen

Cancers 2020, 12(11), 3394; https://doi.org/10.3390/cancers12113394 - 16 Nov 2020

Cited by 28 | Viewed by 3906

Abstract

Small cell lung cancer (SCLC) represents one of the most aggressive malignancies among cancer types. Not only tumor sample availability is limited, but also the ability for tumor cells to rapidly acquire drug resistance are the rate-limiting bottlenecks for overall survival in current [...] Read more.

Small cell lung cancer (SCLC) represents one of the most aggressive malignancies among cancer types. Not only tumor sample availability is limited, but also the ability for tumor cells to rapidly acquire drug resistance are the rate-limiting bottlenecks for overall survival in current clinical settings. A liquid biopsy capable of capturing and enriching circulating tumor cells (CTCs), together with the possibility of drug screening, is a promising solution. Here, we illustrate the development of a highly efficient ex vivo CTC expansion system based on binary colloidal crystals substrate. Clinical samples were enrolled from 22 patients with SCLC in the study. The CTCs were enriched and expanded from the collected peripheral blood samples. Expanded cells were analyzed for protein expression and observed for drug sensitivity with the use of immunofluorescence and ATP titer evaluation, respectively. Successful CTC spheroid proliferation was established after 4 weeks within 82% of all the collected peripheral blood samples from enrolled patients. Upon immunofluorescence analysis, the enriched cells showed positive markers for EpCAM, TTF-1, synaptophysin and negative for CD45. Additionally, the expanded CTCs demonstrated marked heterogeneity in the expression of E-cadherin and N-cadherin. In a preliminary case series, the drug sensitivity of patient-derived CTC to cisplatin and etoposide was studied to see the correlation with the corresponding therapeutic outcome. In conclusion, our study demonstrates that it is possible to efficiently expand CTCs from SCLC within a clinically relevant time frame; the biomarker information generated from enriched CTCs can assist the selection of effective drugs and improve disease outcome. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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16 pages, 2550 KiB

Open AccessArticle

Circulating miR-16-5p, miR-92a-3p, and miR-451a in Plasma from Lung Cancer Patients: Potential Application in Early Detection and a Regulatory Role in Tumorigenesis Pathways

by Patricia P. Reis, Sandra A. Drigo, Robson F. Carvalho, Rainer Marco Lopez Lapa, Tainara F. Felix, Devalben Patel, Dangxiao Cheng, Melania Pintilie, Geoffrey Liu and Ming-Sound Tsao

Cancers 2020, 12(8), 2071; https://doi.org/10.3390/cancers12082071 - 27 Jul 2020

Cited by 35 | Viewed by 4112

Abstract

Background: Micro(mi)RNAs, potent gene expression regulators associated with tumorigenesis, are stable, abundant circulating molecules, and detectable in plasma. Thus, miRNAs could potentially be useful in early lung cancer detection. We aimed to identify circulating miRNA signatures in plasma from patients with lung adenocarcinoma [...] Read more.

Background: Micro(mi)RNAs, potent gene expression regulators associated with tumorigenesis, are stable, abundant circulating molecules, and detectable in plasma. Thus, miRNAs could potentially be useful in early lung cancer detection. We aimed to identify circulating miRNA signatures in plasma from patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), and to verify whether miRNAs regulate lung oncogenesis pathways. Methods: RNA isolated from 139 plasma samples (40 LUAD, 38 LUSC; 61 healthy/non-diseased individuals) were divided into discovery (38 patients; 21 controls for expression quantification using an 800-miRNA panel; Nanostring nCounter^®) and validation (40 patients; 40 controls; TaqMan^® RT-qPCR) cohorts. Elastic net, Maximizing-R-Square Analysis (MARSA), and C-Statistics were applied for miRNA signature identification. Results: When compared to healthy individuals, 580 of 606 deregulated miRNAs in LUAD and 221 of 226 deregulated miRNAs in LUSC had significantly increased levels. Among the 10 most significantly overexpressed miRNAs, 6 were common to patients with LUAD and LUSC. Further analysis identified three signatures composed of 12 miRNAs. Signatures included miRNAs commonly overexpressed in patient plasma. Enriched pathways included target genes modulated by three miRNAs in the C-Statistics signature: miR-16-5p, miR-92a-3p, and miR-451a. Conclusions: The 3-miRNA signature (miR-16-5p, miR-92a-3p, miR-451a) had high specificity (100%) and sensitivity (84%) to predict cancer (LUAD and LUSC). These miRNAs are predicted to modulate genes and pathways with known roles in lung tumorigenesis, including EGFR, K-RAS, and PI3K/AKT signaling, suggesting that the 3-miRNA signature is biologically relevant in adenocarcinoma and squamous cell carcinoma of the lung. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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15 pages, 2039 KiB

Open AccessArticle

Shallow Whole-Genome Sequencing from Plasma Identifies FGFR1 Amplified Breast Cancers and Predicts Overall Survival

by Chantal Bourrier, Jean-Yves Pierga, Laura Xuereb, Hélène Salaun, Charlotte Proudhon, Michael R. Speicher, Jelena Belic, Ellen Heitzer, Brian Paul Lockhart and Nolwen Guigal-Stephan

Cancers 2020, 12(6), 1481; https://doi.org/10.3390/cancers12061481 - 06 Jun 2020

Cited by 13 | Viewed by 3277

Abstract

Background: Focal amplification of fibroblast growth factor receptor 1 (FGFR1) defines a subgroup of breast cancers with poor prognosis and high risk of recurrence. We sought to demonstrate the potential of circulating cell-free DNA (cfDNA) analysis to evaluate FGFR1 copy numbers [...] Read more.

Background: Focal amplification of fibroblast growth factor receptor 1 (FGFR1) defines a subgroup of breast cancers with poor prognosis and high risk of recurrence. We sought to demonstrate the potential of circulating cell-free DNA (cfDNA) analysis to evaluate FGFR1 copy numbers from a cohort of 100 metastatic breast cancer (mBC) patients. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue samples were screened for FGFR1 amplification by FISH, and positive cases were confirmed with a microarray platform (Oncoscan^TM). Subsequently, cfDNA was evaluated by two approaches, i.e., mFAST-SeqS and shallow whole-genome sequencing (sWGS), to estimate the circulating tumor DNA (ctDNA) allele fraction (AF) and to evaluate the FGFR1 status. Results: Tissue-based analyses identified FGFR1 amplifications in 20/100 tumors. All cases with a ctDNA AF above 3% (n = 12) showed concordance for FGFR1 status between tissue and cfDNA. In one case, we were able to detect a high-level FGFR1 amplification, although the ctDNA AF was below 1%. Furthermore, high levels of ctDNA indicated an association with unfavorable prognosis based on overall survival. Conclusions: Screening for FGFR1 amplification in ctDNA might represent a viable strategy to identify patients eligible for treatment by FGFR inhibition, and mBC ctDNA levels might be used for the evaluation of prognosis in clinical drug trials. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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15 pages, 2390 KiB

Open AccessArticle

Novel Gene Fusions in Glioblastoma Tumor Tissue and Matched Patient Plasma

by Lan Wang, Anudeep Yekula, Koushik Muralidharan, Julia L. Small, Zachary S. Rosh, Keiko M. Kang, Bob S. Carter and Leonora Balaj

Cancers 2020, 12(5), 1219; https://doi.org/10.3390/cancers12051219 - 13 May 2020

Cited by 13 | Viewed by 5035

Abstract

Sequencing studies have provided novel insights into the heterogeneous molecular landscape of glioblastoma (GBM), unveiling a subset of patients with gene fusions. Tissue biopsy is highly invasive, limited by sampling frequency and incompletely representative of intra-tumor heterogeneity. Extracellular vesicle-based liquid biopsy provides a [...] Read more.

Sequencing studies have provided novel insights into the heterogeneous molecular landscape of glioblastoma (GBM), unveiling a subset of patients with gene fusions. Tissue biopsy is highly invasive, limited by sampling frequency and incompletely representative of intra-tumor heterogeneity. Extracellular vesicle-based liquid biopsy provides a minimally invasive alternative to diagnose and monitor tumor-specific molecular aberrations in patient biofluids. Here, we used targeted RNA sequencing to screen GBM tissue and the matched plasma of patients (n = 9) for RNA fusion transcripts. We identified two novel fusion transcripts in GBM tissue and five novel fusions in the matched plasma of GBM patients. The fusion transcripts FGFR3-TACC3 and VTI1A-TCF7L2 were detected in both tissue and matched plasma. A longitudinal follow-up of a GBM patient with a FGFR3-TACC3 positive glioma revealed the potential of monitoring RNA fusions in plasma. In summary, we report a sensitive RNA-seq-based liquid biopsy strategy to detect RNA level fusion status in the plasma of GBM patients. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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18 pages, 2319 KiB

Open AccessArticle

Pre-Analytical and Analytical Variables of Label-Independent Enrichment and Automated Detection of Circulating Tumor Cells in Cancer Patients

by Claudia Koch, Simon A. Joosse, Svenja Schneegans, Okka J. W. Wilken, Melanie Janning, Desiree Loreth, Volkmar Müller, Katharina Prieske, Malgorzata Banys-Paluchowski, Ludwig J. Horst, Sonja Loges, Sven Peine, Harriet Wikman, Tobias M. Gorges and Klaus Pantel

Cancers 2020, 12(2), 442; https://doi.org/10.3390/cancers12020442 - 13 Feb 2020

Cited by 26 | Viewed by 3803

Abstract

Circulating tumor cells (CTCs) are promising tools for risk prediction and the monitoring of response to therapy in cancer patients. Within the EU/IMI CANCER-ID consortium, we validated CTC enrichment systems for future inclusion into clinical trials. Due to the known heterogeneity of markers [...] Read more.

Circulating tumor cells (CTCs) are promising tools for risk prediction and the monitoring of response to therapy in cancer patients. Within the EU/IMI CANCER-ID consortium, we validated CTC enrichment systems for future inclusion into clinical trials. Due to the known heterogeneity of markers expressed on CTCs, we tested the Parsortix^® system (ANGLE plc) which enables label-independent CTC enrichment from whole blood based on increased size and deformability of these tumor cells compared to leukocytes. We performed extensive comparisons both with spiked-in blood models (i.e., MDA-MB-468 tumor cell line cells spiked at very low concentration into blood from healthy donors) and validated the protocol on actual clinical samples from breast, lung, and gastrointestinal cancer patients to define optimal conditions for CTC enrichment. Multiple parameters including cassette gap, separation pressure, and cell fixatives were compared in parallel. Also, the compatibility of blood collection tubes with whole genome amplification of isolated tumor cells was demonstrated and we furthermore established a workflow for semi-automated CTC detection using a quantitative cell imager. The established workflow will contribute to supporting the use of size-based CTC enrichment platforms in clinical trials testing the clinical validity and utility of CTCs for personalized medicine. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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Review

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17 pages, 1044 KiB

Open AccessReview

Rationale for Early Detection of EWSR1 Translocation-Associated Sarcoma Biomarkers in Liquid Biopsy

by Felix I. L. Clanchy

Cancers 2021, 13(4), 824; https://doi.org/10.3390/cancers13040824 - 16 Feb 2021

Cited by 3 | Viewed by 2402

Abstract

Sarcomas are mesenchymal tumours that often arise and develop as a result of chromosomal translocations, and for several forms of sarcoma the EWSR1 gene is a frequent translocation partner. Sarcomas are a rare form of malignancy, which arguably have a proportionally greater societal [...] Read more.

Sarcomas are mesenchymal tumours that often arise and develop as a result of chromosomal translocations, and for several forms of sarcoma the EWSR1 gene is a frequent translocation partner. Sarcomas are a rare form of malignancy, which arguably have a proportionally greater societal burden that their prevalence would suggest, as they are more common in young people, with survivors prone to lifelong disability. For most forms of sarcoma, histological diagnosis is confirmed by molecular techniques such as FISH or RT-PCR. Surveillance after surgical excision, or ablation by radiation or chemotherapy, has remained relatively unchanged for decades, but recent developments in molecular biology have accelerated the progress towards routine analysis of liquid biopsies of peripheral blood. The potential to detect evidence of residual disease or metastasis in the blood has been demonstrated by several groups but remains unrealized as a routine diagnostic for relapse during remission, for disease monitoring during treatment, and for the detection of occult, residual disease at the end of therapy. An update is provided on research relevant to the improvement of the early detection of relapse in sarcomas with EWSR1-associated translocations, in the contexts of biology, diagnosis, and liquid biopsy. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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13 pages, 314 KiB

Open AccessFeature PaperReview

Emerging Roles of Urine-Derived Components for the Management of Bladder Cancer: One Man’s Trash Is Another Man’s Treasure

by Sarah Minkler, Fabrice Lucien, Michael J. Kimber, Dipak K. Sahoo, Agnes Bourgois-Mochel, Margaret Musser, Chad Johannes, Igor Frank, John Cheville, Karin Allenspach and Jonathan P. Mochel

Cancers 2021, 13(3), 422; https://doi.org/10.3390/cancers13030422 - 23 Jan 2021

Cited by 13 | Viewed by 3205

Abstract

Urinary bladder cancer (UBC) is the most common malignancy of the urinary tract in humans, with an estimated global prevalence of 1.1 million cases over 5 years. Because of its high rates of recurrence and resistance to chemotherapy, UBC is one of the [...] Read more.

Urinary bladder cancer (UBC) is the most common malignancy of the urinary tract in humans, with an estimated global prevalence of 1.1 million cases over 5 years. Because of its high rates of recurrence and resistance to chemotherapy, UBC is one of the most expensive cancers to treat, resulting in significant health care costs. The development of innovative molecular and cellular tools is necessary to refine patient stratification and help predict response to treatment. Urine is an underused resource of biological components shed from bladder tumors, such as exfoliated cells and extracellular vesicles, that could serve as molecular fingerprints and provide valuable biological insights into tumor phenotype and mechanisms of resistance to chemotherapy. Additionally, characterization of urine-derived extracellular vesicles and cells could be used as reliable biomarkers for prediction of response to neoadjuvant therapy. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

23 pages, 9483 KiB

Open AccessReview

The Validity and Predictive Value of Blood-Based Biomarkers in Prediction of Response in the Treatment of Metastatic Non-Small Cell Lung Cancer: A Systematic Review

by Frederik van Delft, Hendrik Koffijberg, Valesca Retèl, Michel van den Heuvel and Maarten IJzerman

Cancers 2020, 12(5), 1120; https://doi.org/10.3390/cancers12051120 - 30 Apr 2020

Cited by 5 | Viewed by 3276

Abstract

With the introduction of targeted therapies and immunotherapy, molecular diagnostics gained a more profound role in the management of non-small cell lung cancer (NSCLC). This study aimed to systematically search for studies reporting on the use of liquid biopsies (LB), the correlation between [...] Read more.

With the introduction of targeted therapies and immunotherapy, molecular diagnostics gained a more profound role in the management of non-small cell lung cancer (NSCLC). This study aimed to systematically search for studies reporting on the use of liquid biopsies (LB), the correlation between LBs and tissue biopsies, and finally the predictive value in the management of NSCLC. A systematic literature search was performed, including results published after 1 January 2014. Articles studying the predictive value or validity of a LB were included. The search (up to 1 September 2019) retrieved 1704 articles, 1323 articles were excluded after title and abstract screening. Remaining articles were assessed for eligibility by full-text review. After full-text review, 64 articles investigating the predictive value and 78 articles describing the validity were included. The majority of studies investigated the predictive value of LBs in relation to therapies targeting the epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) receptor (n = 38). Of studies describing the validity of a biomarker, 55 articles report on one or more EGFR mutations. Although a variety of blood-based biomarkers are currently under investigation, most studies evaluated the validity of LBs to determine EGFR mutation status and the subsequent targeting of EGFR tyrosine kinase inhibitors based on the mutation status found in LBs of NSCLC patients. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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22 pages, 1902 KiB

Open AccessReview

Cell-Free DNA Methylation Profiling Analysis—Technologies and Bioinformatics

by Jinyong Huang and Liang Wang

Cancers 2019, 11(11), 1741; https://doi.org/10.3390/cancers11111741 - 06 Nov 2019

Cited by 39 | Viewed by 8399

Abstract

Analysis of circulating nucleic acids in bodily fluids, referred to as “liquid biopsies”, is rapidly gaining prominence. Studies have shown that cell-free DNA (cfDNA) has great potential in characterizing tumor status and heterogeneity, as well as the response to therapy and tumor recurrence. [...] Read more.

Analysis of circulating nucleic acids in bodily fluids, referred to as “liquid biopsies”, is rapidly gaining prominence. Studies have shown that cell-free DNA (cfDNA) has great potential in characterizing tumor status and heterogeneity, as well as the response to therapy and tumor recurrence. DNA methylation is an epigenetic modification that plays an important role in a broad range of biological processes and diseases. It is well known that aberrant DNA methylation is generalizable across various samples and occurs early during the pathogenesis of cancer. Methylation patterns of cfDNA are also consistent with their originated cells or tissues. Systemic analysis of cfDNA methylation profiles has emerged as a promising approach for cancer detection and origin determination. In this review, we will summarize the technologies for DNA methylation analysis and discuss their feasibility for liquid biopsy applications. We will also provide a brief overview of the bioinformatic approaches for analysis of DNA methylation sequencing data. Overall, this review provides informative guidance for the selection of experimental and computational methods in cfDNA methylation-based studies. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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Other

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8 pages, 331 KiB

Open AccessBrief Report

Circulating Methylated DNA to Monitor the Dynamics of RAS Mutation Clearance in Plasma from Metastatic Colorectal Cancer Patients

by Chiara Nicolazzo, Ludovic Barault, Salvatore Caponnetto, Marco Macagno, Gianluigi De Renzi, Angela Gradilone, Francesca Belardinilli, Enrico Cortesi, Federica Di Nicolantonio and Paola Gazzaniga

Cancers 2020, 12(12), 3633; https://doi.org/10.3390/cancers12123633 - 04 Dec 2020

Cited by 10 | Viewed by 2106

Abstract

The clearance of RAS mutations in plasma circulating tumor DNA (ctDNA) from originally RAS-mutant metastatic colorectal cancer (mCRC) has been recently demonstrated. Clinical trials investigating whether RAS mutant mCRC who “convert” to wild-type in plasma might benefit from EGFR blockade are ongoing. Detection [...] Read more.

The clearance of RAS mutations in plasma circulating tumor DNA (ctDNA) from originally RAS-mutant metastatic colorectal cancer (mCRC) has been recently demonstrated. Clinical trials investigating whether RAS mutant mCRC who “convert” to wild-type in plasma might benefit from EGFR blockade are ongoing. Detection of tumor-specific DNA methylation alterations in ctDNA has been suggested as a specific tool to confirm the tumoral origin of cell-free DNA. We monitored RAS clearance in plasma from patients with RAS-mutant mCRC at baseline (pre-treatment) (T0); after 4 months of first-line therapy (T1); at the time of first (T2) and second (T3) progression. A five-gene methylation panel was used to confirm the presence of ctDNA in samples in which RAS mutation clearance was detected. At T1, ctDNA analysis revealed wild-type RAS status in 83% of samples, all not methylated, suggesting at this time point the lack of ctDNA shedding. At T2, ctDNA analysis revealed wild-type RAS status in 83% of samples, of which 62.5% were found methylated. At T3, 50% of wild-type RAS samples were found methylated. Non-methylated samples were found in patients with lung or brain metastases. This five-gene methylation test might be useful to confirm the presence of ctDNA in RAS wild-type plasma samples. Full article

(This article belongs to the Special Issue Liquid Biopsy in Cancer)

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