Cancer Liquid Biopsy Research Articles

P37.12 Concordance of Next-Generation Sequencing Between Tissue and Liquid Biopsies in Non-Small Cell Lung Cancer

P37.12 Concordance of Next-Generation Sequencing Between Tissue and Liquid Biopsies in Non-Small Cell Lung Cancer

P89.03 Demonstrating VALUE of Liquid Biopsy for Lung Cancer in a Public Healthcare System

Liquid biopsy (LB) is an important alternative for advanced lung cancer patients in diagnosing resistance or where tissue genotyping has failed. The VALUE study examines clinical outcomes and utility of LB for molecular diagnosis in treatment naive stage IV lung adenocarcinoma patients. [Preliminary data previously presented at ASCO2020. Final study results will be available for WCLC2021] This study is being conducted at 6 Canadian centres (NCT03576937) using Guardant360TM (G360), a validated cell-free DNA next-generation sequencing assay that identifies variants in 74 cancer-associated genes, including fusions and copy number gain. Patients with treatment-naïve advanced non-squamous lung carcinoma, ≤10 pack-year smoking history, and measurable disease are eligible (N=150). Patients receive standard of care tumour tissue (TT) molecular profiling (EGFR, ALK +/- ROS1) and LB. The primary endpoint is response rate to first-line therapy (RECIST 1.1); secondary endpoints include incremental targetable alterations identified through G360 (EGFR, ALK, BRAF, ERBB2, KRAS (G12C), NTRK, MET (amplification, exon 14 skipping), RET, ROS1), turnaround time (TAT) and successful molecular profiling rates. To date, 150 eligible patients have been enrolled. Demographic data are available for the first 100 patients, treatment data for 89 and 60 have response data. Median age is 63 (range 22-91), 63% are female, 85% never smokers, 35% East Asian, 24% ECOG PS>1, and 94% have adenocarcinoma. Actionable targets have been identified in 55% of patients using G360 (EGFR/ALK in 38%), and 38% using standard TT profiling. Concordance between TT and LB was high, 84%, with 8 cases each identified by TT or LB but not the other. TT profiling for EGFR/ALK was unsuccessful in 6% of patients (insufficient tissue, failed biopsy, single biomarker tested only). Fifteen patients (15%) had no ctDNA alterations detected by G360 (low disease burden vs. non-shedding). Of 89 patients receiving first-line treatment, 60% received targeted therapy, 26% chemo-immunotherapy combinations, 10% checkpoint inhibitors alone and 4% were observed. Treatment decisions were informed by G360 alone in 40% and by G360+TT results in 28% (by physician report). Among 51 evaluable patients, ORR was 55% (28/51). Using G360, ORR was 75% (18/24) in those with actionable alterations and 37% (10/27) in those without. Using TT, ORR was 64% (16/25) in those with actionable alterations and 46% (12/26) in those without. Mean TAT was 7.7 days (SD+/-1.6) for LB vs 20.8 days (SD+/- 9.8) for TT. LB using G360 identifies actionable targets beyond tissue profiling alone in newly diagnosed lung cancer patients, has faster TAT and yields similar outcomes with targeted and non-targeted therapy.

Colorectal Cancer-Derived CAT1-Positive Extracellular Vesicles Alter Nitric Oxide Metabolism in Endothelial Cells and Promote Angiogenesis.

Accumulating scientific evidences strongly support the importance of cancer-derived extracellular vesicles (EV) in organization of tumor microenvironment and metastatic niches, which are also considered as ideal tools for cancer liquid biopsy. To uncover the full scope of proteomic information packaged within EVs secreted directly from human colorectal cancer, we cultured surgically resected viable tissues and obtained tissue-exudative EVs (Te-EV). Our quantitative profiling of 6,307 Te-EV proteins and 8,565 tissue proteins from primary colorectal cancer and adjacent normal mucosa (n = 17) allowed identification of a specific cargo in colorectal cancer-derived Te-EVs, high-affinity cationic amino acid transporter 1 (CAT1, P = 5.0 × 10-3, fold change = 6.2), in addition to discovery of a new class of EV markers, VPS family proteins. The EV sandwich ELISA confirmed escalation of the EV-CAT1 level in plasma from patients with colorectal cancer compared with healthy donors (n = 119, P = 3.8 × 10-7). Further metabolomic analysis revealed that CAT1-overexpressed EVs drastically enhanced vascular endothelial cell growth and tubule formation via upregulation of arginine transport and downstream NO metabolic pathway. These findings demonstrate the potency of CAT1 as an EV-based biomarker for colorectal cancer and its functional significance on tumor angiogenesis. IMPLICATIONS: This study provides a proteome-wide compositional dataset for viable colorectal cancer tissue-derived EVs and especially emphasizes importance of EV-CAT1 as a key regulator of angiogenesis.

Epigenetic Landscape of Liquid Biopsy in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common malignancies and is a major cause of cancer-related deaths worldwide. Thus, there is a clinical need to improve early detection of CRC and personalize therapy for patients with this disease. In the era of precision oncology, liquid biopsy has emerged as a major approach to characterize the circulating tumor elements present in body fluids, including cell-free DNA and RNA, circulating tumor cells, and extracellular vesicles. This non-invasive tool has allowed the identification of relevant molecular alterations in CRC patients, including some indicating the disruption of epigenetic mechanisms. Epigenetic alterations found in solid and liquid biopsies have shown great utility as biomarkers for early detection, prognosis, monitoring, and evaluation of therapeutic response in CRC patients. Here, we summarize current knowledge of the most relevant epigenetic mechanisms associated with cancer development and progression, and the implications of their deregulation in cancer cells and liquid biopsy of CRC patients. In particular, we describe the methodologies used to analyze these epigenetic alterations in circulating tumor material, and we focus on the clinical utility of epigenetic marks in liquid biopsy as tumor biomarkers for CRC patients. We also discuss the great challenges and emerging opportunities of this field for the diagnosis and personalized management of CRC patients.

A potential panel of five mRNAs in urinary extracellular vesicles for the detection of bladder cancer.

BackgroundExtracellular vesicles (EVs) have showed promising potential in liquid biopsy of cancer. In present study, we evaluate the feasibility to diagnose bladder cancer using EVs RNA markers identified from public tissue RNA sequencing data.MethodsWe used urine samples from a cohort of population with suspected bladder cancer. Disease status (i.e., primary or recurrent bladder cancer) was diagnosed by cystoscopy. A prediction model including the expression of multiple RNAs in urinary EVs were developed in training cohort (n=368, 126 bladder cancer and 242 negative controls). The performance of optimal model (ExoPanel) consists of five mRNAs (MYBL2, TK1, UBE2C, KRT7, S100A2) was further assessed by a validation cohort (n=155, 56 bladder cancer and 99 negative controls).ResultsThe performance of ExoPanel in training cohort was AUC 0.7759 (95% CI: 0.7259–0.8260), NPV 90.34% (95% CI: 84.04–94.42%), SN 88.89% (95% CI: 81.75–93.57%), and SP 54.13% (95% CI: 47.63–60.50%) respectively. In the validation cohort, the performance of this model was AUC 0.8402 (95% CI: 0.7690–0.9114), NPV 90.91% (95% CI: 79.29–96.60%), SN 91.07% (95% CI: 79.63–96.67%), and SP 50.51% (95% CI: 40.34–60.63%). Using this model, it is possible to rule out a significant number of non cancer patients, thus reduce the unnecessary operation of cystoscopy.ConclusionsWe discovered a panel of five mRNAs, and evaluated its potential to facilitate bladder cancer diagnosis by analyzing their expression in urinary EVs.

Clinical Perspectives on Liquid Biopsy in Metastatic Colorectal Cancer.

Liquid biopsy, which generally refers to the analysis of biological components such as circulating nuclear acids and circulating tumor cells in body fluids, particularly in peripheral blood, has shown good capacity to overcome several limitations faced by conventional tissue biopsies. Emerging evidence in recent decades has confirmed the promising role of liquid biopsy in the clinical management of various cancers, including colorectal cancer, which is one of the most prevalent cancers and the second leading cause of cancer-related deaths worldwide. Despite the challenges and poor clinical outcomes, patients with metastatic colorectal cancer can expect potential clinical benefits with liquid biopsy. Therefore, in this review, we focus on the clinical prospects of liquid biopsy in metastatic colorectal cancer, specifically with regard to the recently discovered various biomarkers identified on liquid biopsy. These biomarkers have been shown to be potentially useful in multiple aspects of metastatic colorectal cancer, such as auxiliary diagnosis of metastasis, prognosis prediction, and monitoring of therapy response.

Microfluidic-Based Exosome Analysis for Liquid Biopsy.

Liquid biopsy offers non-invasive and real-time molecular profiling of individual patients, and is thus considered a revolutionary technology in precision medicine. Exosomes have been acknowledged as significant biomarkers in liquid biopsy, as they play a central role in cell-cell communication and are closely related to the pathogenesis of most human malignancies. Nevertheless, in biofluids exosomes always co-exist with other particles, and the cargo components of exosomes are highly heterogeneous. Thus, the isolation and molecular characterization of exosomes are still technically challenging. Microfluidics technology effectively addresses this challenge by virtue of its inherent advantages, such as precise manipulation of fluids, low consumption of samples and reagents, and a high level of integration. Recent advances in microfluidics allow in situ exosome capture and molecular detection with unprecedented selectivity and sensitivity. In this review, the state-of-the-art developments in microfluidics-based exosome research, including exosome isolation approaches and molecular detection strategies, with highlights of the characterization of exosomal biomarkers in cancer liquid biopsy is summarized. The major challenges are also discussed and some perspectives for the future directions of exosome-based liquid biopsy in microfluidic systems are presented.

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

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.

Current Status and Future Perspectives of Liquid Biopsy in Small Cell Lung Cancer.

Approximately 19% of all cancer-related deaths are due to lung cancer, which is the leading cause of mortality worldwide. Small cell lung cancer (SCLC) affects approximately 15% of patients diagnosed with lung cancer. SCLC is characterized by aggressiveness; the majority of SCLC patients present with metastatic disease, and less than 5% of patients are alive at 5 years. The gold standard of SCLC treatment is platinum and etoposide-based chemotherapy; however, its effects are short. In recent years, treatment for SCLC has changed; new drugs have been approved, and new biomarkers are needed for treatment selection. Liquid biopsy is a non-invasive, rapid, repeated and alternative tool to the traditional tumor biopsy that could allow the most personalized medicine into the management of SCLC patients. Circulating tumor cells (CTCs) and cell-free DNA (cfDNA) are the most commonly used liquid biopsy biomarkers. Some studies have reported the prognostic factors of CTCs and cfDNA in SCLC patients, independent of the stage. In this review, we summarize the recent SCLC studies of CTCs, cfDNA and other liquid biopsy biomarkers, and we discuss the future utility of liquid biopsy in the clinical management of SCLC.

Extracellular vesicles as a promising biomarker resource in liquid biopsy for cancer

Extracellular vesicles as a promising biomarker resource in liquid biopsy for cancer

Facile PEG-based isolation and classification of cancer extracellular vesicles and particles with label-free surface-enhanced Raman scattering and pattern recognition algorithm.

Extracellular vesicles and particles (EVPs), which contain the same surface proteins as their mother cells, are promising biomarkers for cancer liquid biopsy. However, most of the isolation methods of EVPs are time-consuming and complicated, and hence, sensitive detection and classification methods are required for EVPs. Here, we report a facile polyethylene glycol (PEG)-based method for isolating and classifying EVPs with label-free surface-enhanced Raman scattering (SERS) and pattern recognition algorithm. There are only three steps in the PEG-based isolation method, and it does not require ultracentrifugation, which makes it a low-cost and easy-to-use method. Three types of common male cancer cell lines, namely leukemia (THP-1), prostate cancer (DU-145), and colorectal cancer (COLO-205), and one healthy male blood sample, were utilized to isolate EVPs. To collect the SERS spectra of EVPs, a novel planar nanomaterial, namely amino molybdenum oxide (AMO) nanoflakes, was applied, with the enhancement factor being obtained as 3.2 × 102. Based on the principal component analysis and support vector machine (PCA-SVM) algorithm, cancer and normal EVPs were classified with 97.4% accuracy. However, among the cancer EVPs, the accuracy, precision, and sensitivity were found to be 90.0%, 90.9%, and 83.3% for THP-1; 86.7%, 80.0%, and 92.3% for DU-145; 96.7%, 83.3%, and 100% for COLO-205, respectively. Thus, this work will improve the isolation, detection, and classification of EVPs and promote the development of cancer liquid biopsies.

Comparing GOZILA and COLOMATE: Ongoing Umbrella/Basket Trials Examining Genetic Testing in Gastrointestinal Malignancies.

Circulating tumor DNA (ctDNA)-based next-generation sequencing (NGS) assays have advantages over classic tissue-based analyses because of their low invasiveness and availability of repeated sampling. Because of the low incidence of target gene alterations such as HER2 or BRAF V600E in gastrointestinal cancers, very large screening platforms are needed to develop genome-based clinical trials. For those reasons, ctDNA-based screening studies are being actively conducted; among them are the GOZILA (Guardant Originates in Zipangu Liquid biopsy Arrival) study in Japan and the COLOMATE (COlorectal Cancer Liquid BiOpsy Screening Protocol for Molecularly Assigned ThErapy) study in the United States. Although only patients with metastatic colorectal cancer (mCRC) who had previously received standard chemotherapies are eligible for the COLOMATE study, patients with various types of solid tumors at any line of treatment are eligible for GOZILA. This broad coverage of the eligible population allows a target of 5000 patients. By contrast, effective screening of selected candidate patients for companion trials using rapid turnaround time by ctDNA-based NGS assay is the key for COLOMATE. The companion trials of targeted therapies in mCRC are similar between GOZILA and COLOMATE. Both studies have identified patients eligible for studies by examining ERBB2 (HER2), BRAF V600E, BRAF non-V600E, and FGFR alterations, as well as MET amplification and rechallenge with anti-EGFR antibodies. The existence of various companion trials for common alterations that can be potential therapy targets on the 2 platforms can lead to future international collaboration.

Single-Cell Analysis of Highly Metastatic Circulating Tumor Cells by Combining a Self-Folding Induced Release Reaction with a Cell Capture Microchip.

Nondestructive analysis of the single-cell molecular phenotype of circulating tumor cells (CTCs) is of great significance to the precise diagnosis and treatment of cancer but is also a huge challenge. To address this issue, here, we develop a facile analysis system that integrates CTCs' capture and molecular phenotype analysis. An isothermal nucleic acid amplification technique named self-folding induced release reaction (sFiR), which has high-efficiency signal amplification capabilities and can run under physiological conditions, is first developed to meet the high requirements for sensitivity and nondestructivity. By combining the sFiR with immune recognition and a single cell capture microchip, the molecular phenotype analysis of a single CTC is realized. As a model, nondestructive analysis of junction plakoglobin (JUP), an overexpressed membrane protein that is closely related to the metastasis of CTCs, is successfully achieved. Results reveal that this sFiR-based analysis system can clearly distinguish the expression of JUP in different cancer cell lines and can present quantitative information on the expression of JUP. Furthermore, the captured and analyzed CTCs maintain their basic physiological activity and can be used for drug sensitivity testing. Considering the excellent performance and ease of operation of the system, it can provide technical support for CTC-based cancer liquid biopsy and drug development.

Microfluidic chip for graduated magnetic separation of circulating tumor cells by their epithelial cell adhesion molecule expression and magnetic nanoparticle binding

The enumeration of circulating tumor cells (CTCs) in the peripheral bloodstream of metastatic cancer patients has contributed to improvements in prognosis and therapeutics. There have been numerous approaches to capture and counting of CTCs. However, CTCs have potential information beyond simple enumeration and hold promise as a liquid biopsy for cancer and a pathway for personalized cancer therapy by detecting the subset of CTCs having the highest metastatic potential. There is evidence that epithelial cell adhesion molecule (EpCAM) expression level distinguishes these highly metastatic CTCs. The few previous approaches to selective CTC capture according to EpCAM expression level are reviewed. A new two-stage microfluidic device for separation, enrichment and release of CTCs into subpopulations sorted by EpCAM expression level is presented here. It relies upon immunospecific magnetic nanoparticle labeling of CTCs followed by their field- and flow-based separation in the first stage and capture as discrete subpopulations in the second stage. To fine tune the separation, the magnetic field profile across the first stage microfluidic channel may be modified by bonding small Vanadium Permendur strips to its outer walls. Mathematical modeling of magnetic fields and fluid flows supports the soundness of the design.

Detection of ESR1 Mutations Based on Liquid Biopsy in Estrogen Receptor-Positive Metastatic Breast Cancer: Clinical Impacts and Prospects.

Endocrine therapy is the main treatment option for estrogen receptor-positive (ER+) breast cancer (BC). Compared with other clinical subtypes, ER+ BC patients usually have a more favorable prognosis. However, almost all ER+ BCpatients develop endocrine resistance and disease progression eventually. A large number of studies based on liquid biopsy suggest that ESR1 mutations may play a key role in this process. For patients with ER+ metastatic BC (MBC), ESR1 is an important prognostic factor and may associate with the resistance to endocrine therapy, like aromatase inhibitors. The advances of sequencing technologies allow us to conduct longitudinal monitoring of disease and unveil the clinical implications of each ESR1 sub-clone in ER+ MBC. Moreover, since the ESR1-related endocrine resistance has not been fully addressed by existing agents, more potent cornerstone drugs should be developed as soon as possible. Herein, we reviewed the recent progress of detecting ESR1 mutations based on liquid biopsy and different sequencing technologies in ER+ MBC and discussed its clinical impacts and prospects.

Current Status of Circulating Tumor Cells, Circulating Tumor DNA, and Exosomes in Breast Cancer Liquid Biopsies.

Breast cancer is the most common cancer among women worldwide. Although the five-, ten- and fifteen-year survival rates are good for breast cancer patients diagnosed with early-stage disease, some cancers recur many years after completion of primary therapy. Tumor heterogeneity and clonal evolution may lead to distant metastasis and therapy resistance, which are the main causes of breast cancer-associated deaths. In the clinic today, imaging techniques like mammography and tissue biopsies are used to diagnose breast cancer. Even though these methods are important in primary diagnosis, they have limitations when it comes to longitudinal monitoring of residual disease after treatment, disease progression, therapy responses, and disease recurrence. Over the last few years, there has been an increasing interest in the diagnostic, prognostic, and predictive potential of circulating cancer-derived material acquired through liquid biopsies in breast cancer. Thanks to the development of sensitive devices and platforms, a variety of tumor-derived material, including circulating cancer cells (CTCs), circulating DNA (ctDNA), and biomolecules encapsulated in extracellular vesicles, can now be extracted and analyzed from body fluids. Here we will review the most recent studies on breast cancer, demonstrating the clinical potential and utility of CTCs and ctDNA. We will also review literature illustrating the potential of circulating exosomal RNA and proteins as future biomarkers in breast cancer. Finally, we will discuss some of the advantages and limitations of liquid biopsies and the future perspectives of this field in breast cancer management.

Cancer Extracellular Vesicles: Next-Generation Diagnostic and Drug Delivery Nanotools.

Simple SummaryExtracellular vesicles (EVs) are secreted continuously from different cell types. The composition of EVs, like proteins, nucleic acids and lipids is linked with the cells of origin and they are involved in cell-cell communication. The presence of EVs in the majority of the body fluids makes them attractive to investigate and define their role in physiological and in pathological processes. This review is focused on EVs with dimensions between 30 and 150 nm like exosomes (EEVs). We described the biogenesis of EEVs, methods for isolation and their role in cancer as innovative diagnostic tools and new drug delivery systems.Nanosized extracellular vesicles (EVs) with dimensions ranging from 100 to 1000 nm are continuously secreted from different cells in their extracellular environment. They are able to encapsulate and transfer various biomolecules, such as nucleic acids, proteins, and lipids, that play an essential role in cell‒cell communication, reflecting a novel method of extracellular cross-talk. Since EVs are present in large amounts in most bodily fluids, challengeable hypotheses are analyzed to unlock their potential roles. Here, we review EVs by discussing their specific characteristics (structure, formation, composition, and isolation methods), focusing on their key role in cell biology. Furthermore, this review will summarize the biomedical applications of EVs, in particular those between 30 and 150 nm (like exosomes), as next-generation diagnostic tools in liquid biopsy for cancer and as novel drug delivery vehicles.

The Diagnostic and Prognostic Value of a Liquid Biopsy for Esophageal Cancer: A Systematic Review and Meta-Analysis.

Simple SummaryThe “liquid biopsy” is a novel concept for detecting circulating biomarkers in the peripheral blood of patients with various cancers, including esophageal cancer. There are two main methods to identify circulating cancer related biomarkers such as morphological techniques or molecular biological techniques. There are some differences in the sensitivity and specificity for detecting circulating tumor cells (CTCs) or circulating markers between each method. Although it is still challenging to determine strong candidates for early diagnosis and predicting prognosis in patients with esophageal cancer, our meta-analysis might be a milestone for the future development of liquid biopsies in use with esophageal cancer.Esophageal cancer is among the most aggressive diseases, and circulating tumor cells (CTCs) have been recognized as novel biomarkers for various cancers over the past two decades, including esophageal cancer. CTCs might provide crucial clinical information for predicting cancer prognosis, monitoring therapeutic responses or recurrences, or elucidating the mechanism of metastasis. The isolation of CTCs is among the applications of a “liquid biopsy”. There are various technologies for liquid biopsies, and they are classified into two main methods: cytometric or non-cytometric techniques. Here, we review a total of 57 eligible articles to summarize various technologies for the use of a liquid biopsy in esophageal cancer and perform a meta-analysis to assess the clinical utility of liquid biopsies as a prognostic and diagnostic biomarker technique. For prognostic evaluation, the pooled hazard ratio in the cytometric assay is relatively higher than that of the non-cytometric assay. On the other hand, a combination of multiple molecules, using a non-cytometric assay, might be a favorable biomarker technique for the early diagnosis of esophageal cancer. Although determining strong evidence for a biomarker by using a liquid biopsy is still challenging, our meta-analysis might be a milestone for the future development of liquid biopsies in use with esophageal cancer.

Liquid biopsy in cancer using the Z-scan technique: a new approach to discover biomarkers in cancer.

Aim:Biomarkers have been broadly studied as a tool for the diagnosis and prognosis for different types of cancer. Z-scan is a kind of measurement technique that generates a nonlinear refractive index (n2). Today, Z-scan has been used in oncology to discriminate between solid tumors and to identify tumor circulating cell-free DNA in liquid samples.Materials & methods:According to the inclusion and exclusion criteria defined in this review, 35 articles were selected.Conclusion:The use of this technique for this kind of measurement will allow for a rapid and precise diagnosis of different types of tumor and may lead to better therapeutic approaches.

Current Status of Circulating Tumor DNA Liquid Biopsy in Pancreatic Cancer.

Pancreatic cancer is a challenging disease with a low 5-year survival rate. There are areas for improvement in the tools used for screening, diagnosis, prognosis, treatment selection, and assessing treatment response. Liquid biopsy, particularly cell free DNA liquid biopsy, has shown promise as an adjunct to our standard care for pancreatic cancer patients, but has not yet been universally adopted into regular use by clinicians. In this publication, we aim to review cfDNA liquid biopsy in pancreatic cancer with an emphasis on current techniques, clinical utility, and areas of active investigation. We feel that researchers and clinicians alike should be familiar with this exciting modality as it gains increasing importance in the care of cancer patients.