Liquid Biopsy Tool Research Articles

Exploiting Exosomes in Cancer Liquid Biopsies and Drug Delivery.

Exosomes are cell-derived nanovesicles that transfer molecular cargo from donor to recipient cells and mediate intercellular communication. Advancement in elucidating the biological capabilities and functionalities of exosomes has revealed the striking roles of exosomes as conveyors of bioactive molecules across the biological barriers. Tumor-derived exosomes hold great promise to serve as a liquid biopsy tool for cancer diagnosis and prognosis, as large quantities of exosomes are excreted by tumor cells continuously into the circulation, carrying the molecular cargo (DNA, RNA, proteins) reflective of the genetic and signaling alterations in tumor cells. Two inherent characteristics of exosomes offer important opportunities for drug delivery: their superb transcellular permeability and biocompatibility. Exosomes are uniquely capable of encapsulating a variety of payloads and deliver them to the target tissues. This review discusses the potential of tumor-derived exosomes in cancer liquid biopsies as well as the underlying mechanisms. Furthermore, the recent progress of developing exosomes as highly versatile and efficient drug carriers is also summarized.

Enumerating Circulating Tumor Cells with a Self-Assembled Cell Array (SACA) Chip: A Feasibility Study in Patients with Colorectal Cancer.

Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide. Detecting and enumerating circulating tumor cells (CTCs) in patients with colorectal cancer emerged as an important prognostic tool which provides a direct estimate of metastatic potential. Improving the turnaround time and decreasing sample volume is critical for incorporating this liquid biopsy tool into routine practice. The objective of the current study was to validate the clinical feasibility of a self-assembled cell array (SACA) chip, a CTC counting platform with less than 4 h turnaround time, in patients with newly diagnosed colorectal cancers. In total, 179 patients with newly diagnosed colorectal cancers from a single institute were enrolled. Epithelial cell adhesion molecule positive (EpCAM(+)), cluster of differentiation 45 negative (CD45(−)) cells were isolated and enumerated from 2 mL of peripheral vein blood (PB) and inferior mesenteric vein blood (IMV) samples obtained during surgery. We found that the CTC count in PB but not IMV correlates with disease stages. Neoadjuvant chemotherapy did not lead to decreased CTC count in both types of blood samples. With cutoffs of four CTCs per 2 mL of blood, and serum carcinoembryonic antigen (CEA) level of 5 ng/mL, patients with non-metastatic disease were more likely to experience recurrence if they had high PB CTC count and high serum CEA concentration (odds ratio, 8.9). Our study demonstrates the feasibility of enumerating CTCs with a SACA chip in patients with colorectal cancer.

Urine-based liquid biopsy: non-invasive and sensitive AR-V7 detection in urinary EVs from patients with prostate cancer.

Androgen-receptor splice variant 7 (AR-V7) is associated with castration-resistant prostate cancer (CRPC) and resistance to anti-androgen therapy. Despite its clinical importance, the lack of efficient methods for AR-V7 analysis remains a challenge for broader use of this biomarker in routine clinical practice. Herein, we suggest a practical and non-invasive liquid biopsy method for analysis of AR-V7 in the RNA of urine-derived extracellular vesicles (EVs) without the need for blood withdrawal. Urine-derived EVs were isolated by a lab-on-a-disc integrated with six independent nanofiltration units (Exo-Hexa) allowing simultaneous processing of six individual samples. Rapid enrichment of EVs (<30 min) from each 4 mL urine sample was followed by mRNA extraction, and AR-V7 and androgen receptor full-length (AR-FL) mRNA levels in the urinary EVs were quantified by droplet digital polymerase chain reaction (ddPCR) as absolute concentrations (copies per mL). Higher AR-V7 and lower AR-FL expressions were detected in urine-derived EVs from 14 patients with CRPC than in those from 22 patients with hormone-sensitive prostate cancer. Additionally, we found that AR-V7 transcript levels and the AR-V7/AR-FL ratio in urinary EVs were higher in patients with advanced prostate cancer. This study is the first to report that RNA of urine-derived EVs is a reliable source for AR-V7 expression analysis. The proposed method for quantifying AR-V7 in urinary EVs prepared by a lab-on-a-disc is therefore a simple and promising approach to liquid biopsy with great potential for therapeutic impact on prostate cancer.

Circulating miRNA panels for specific and early detection in bladder cancer.

Bladder cancer is the 9th leading cause of cancer death worldwide. The major problem in bladder cancer is primarily the high recurrence rate after drug treatment and resection. Although conventional screening methods, such as cystoscopy, urinary cytology and ultrasound sonography, have become widely used in clinical settings, the diagnostic performance of these modalities is unsatisfactory due to low accuracy or high invasiveness. Because circulating micro RNA (miRNA) profiles have recently been reported as an attractive tool for liquid biopsy in cancer screening, here, we performed global miRNA profiling of 392 serum samples of bladder cancer patients with 100 non‐cancer samples and 480 samples of other types of cancer as controls. We randomly classified the bladder cancer and control samples into 2 cohorts, a training set (N = 486) and a validation set (N = 486). By comparing both controls, we identified specific miRNA, such as miR‐6087, for diagnosing bladder cancer in the training and validation sets. Furthermore, we found that a combination of 7 miRNA (7‐miRNA panel: miR‐6087, miR‐6724‐5p, miR‐3960, miR‐1343‐5p, miR‐1185‐1‐3p, miR‐6831‐5p and miR‐4695‐5p) could discriminate bladder cancer from non‐cancer and other types of tumors with the highest accuracy (AUC: .97; sensitivity: 95%; specificity: 87%). The diagnostic accuracy was high, regardless of the stage and grade of bladder cancer. Our data demonstrated that the 7‐miRNA panel could be a biomarker for the specific and early detection of bladder cancer.

Potential Diagnosis of Vitreoretinal Lymphoma by Detection of MYD88 Mutation in Aqueous Humor With Ultrasensitive Droplet Digital Polymerase Chain Reaction

The diagnostic workup of patients suspected of having vitreoretinal lymphoma (VRL) is primarily based on vitreous fluid analysis, including the recently emerging myeloid differentiation primary response gene 88 (MYD88) mutation analysis. Aqueous humor paracentesis is a relatively less invasive and safer procedure than taking vitreous fluid specimens, and aqueous humor-based MYD88 mutation analysis would provide an additional liquid biopsy tool to diagnose and monitor patients with VRL. To investigate whether the detection of MYD88 L265P by highly sensitive droplet digital polymerase chain reaction (ddPCR) is feasible in the vitreous fluid and aqueous humor of patients with VRL. This cohort study includes aqueous humor and vitreous fluid samples from patients with VRL who were treated at the University Medical Center Utrecht, in Utrecht, the Netherlands, from August 2005 to August 2017. Ocular fluids were randomized and masked before MYD88 L265P analysis, which was performed using an in-house validated ddPCR platform. Patients with uveitis were included as a comparison group. The presence of MYD88 L265P mutation detected by ddPCR in AH and VF. The study included 96 samples from 63 individuals, including 23 patients with VRL (of whom 10 were female and 13 male, with a mean [SD] age of 72 [7.3] years) and 40 individuals with uveitis (of whom 23 were female and 17 male, with a mean [SD] age of 58 [20.9] years). In 17 of 23 patients with VRL (74%), MYD88 L265P was detected; it was not detected in any of the patients with uveitis. It was detectable in both vitreous fluid and aqueous humor samples. In the paired samples, the mutation was detected in 8 of 9 aqueous humor samples (89%) of the MYD88 L265P-positive vitreous fluid samples. In vitreous fluid, the MYD88 ddPCR test showed a sensitivity of 75% (95% CI, 50%-92%) and a positive predictive value of 100%; in aqueous humor, sensitivity was 67% (95% CI, 42%-92%), and positive predictive value was 100%. Specificity was 100% in both fluids. After treatment, the mutation was no longer detectable in any ocular fluids. The high concordance between aqueous humor and vitreous fluid samples suggests that use of the easily accessible aqueous humor is nearly as informative as vitreous fluid in the identification of key somatic mutations in patients with VRL. This approach may provide an additional minimally invasive tool for accurate diagnosis, detection of recurrence, and monitoring of treatment.

Abstract 5581: Clinical validation of a highly sensitive assay to detect mutations in plasma from advanced lung adenocarcinoma patients

Abstract The use of different liquid biopsy tools including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have shown great promise for the use in personalized cancer diagnostics, treatment monitoring, and research, especially among metastatic patients. The power of ctDNA analyses in early detection of acquired resistance against e.g. EGFR-targeted therapies in non-small cell lung cancer (NSCLC) have been demonstrated in several studies. However, NSCLC patients display lower ctDNA and CTC concentrations than expected and in particular brain metastases pose a challenge, probably due to the blood-brain barrier. In this study, we used a novel highly sensitive assay to detect clinically relevant mutations in ctDNA and detected CTCs in blood from metastatic NSCLC patients. 56 metastatic NSCLC cases were enrolled in this study. 37 patients presented with brain metastases and 20 of them had brain as the only site of metastasis (oligo-metastatic disease). Cell free (cf)DNA was isolated from 1.5-2ml of plasma using the Circulating Nucleic Acid kit (Qiagen). The ctDNA quantity was measured by Qubit Fluorometer (Thermo Fisher) and the quality was assessed by Tapestation (Agilent technologies). The UltraSEEK™ Lung Panel on the MassARRAY® System (Agena Bioscience) was used for the detection of mutations in five genes EGFR, KRAS, BRAF, ERBB2 and PIK3CA covering 67 different hot spot mutation loci. CTCs were detected by the CellSearch system. The total cfDNA concentration yields were low in most cases (median; 21ng/30µl, mean; 39ng/30µl, ± SD 2.6 ng/µl isolated from 1.5-2ml of plasma). Mutations could be detected in 26 samples (46.4%), with the variant allele frequency ranging between 0.1% and 5%. EGFR mutations were detected in 23.2% (13/56), KRAS in 21.4%, ERBB2 in 7.1%, PIK3CA in 3.6% and BRAF in 1.8% of the samples. Mutations were detected in 57.9% of patients with peripheral (non-brain) metastases, whereas 40.5% of brain metastatic patients had mutations detected in blood. Among oligo-metastatic patients, 30.5% had detectable mutations in plasma. In 42 samples, the EGFR mutation status in the primary tumor tissue was also determined. In 90.5% of samples, concordant results between plasma and tissue were found. In two patients, an EGFR mutation was only detected in the plasma, whereas in two other samples the mutation was only detected in the tumor tissue. In 46 patients, CTCs were measured. In 11 patients (23.9%), ≥ 1 CTC was detected. In 64.7% of patients, an informative result was obtained when results from both liquid biopsy approaches were combined. The UltraSEEK technology was able to detect mutations in cfDNA in plasma from NSCLC patients with limited, oligo-metastatic tumor burden, low amounts of total cfDNA, and very low fractions of ctDNA. Parallel assessment of CTCs in patients without detectable ctDNA enlarged the population of patients amenable to successful liquid biopsy analysis. Citation Format: Harriet Wikman, Malte Mohme, Melanie Janning, Alexander Sartori, Katrin Lamszus, Sabine Riethdorf, Darryl Irwin, Manfred Westphal, Sonja Loges, Klaus Pantel. Clinical validation of a highly sensitive assay to detect mutations in plasma from advanced lung adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5581.

Liquid biopsy by NGS: differential presence of exons (DPE) in cell-free DNA reveals different patterns in metastatic and nonmetastatic colorectal cancer.

Next‐generation sequencing (NGS) has been proposed as a suitable tool for liquid biopsy in colorectal cancer (CRC), although most studies to date have focused almost exclusively on sequencing of panels of potential clinically actionable genes. We evaluated the clinical value of whole‐exome sequencing (WES) of cell‐free DNA (cfDNA) circulating in plasma, with the goal of identifying differential clinical profiles in patients with CRC. To this end, we applied an original concept, “differential presence of exons” (DPE). We determined differences in levels of 379 exons in plasma cfDNA and used DPE analysis to cluster and classify patients with disseminated and localized disease. The resultant bioinformatics analysis pipeline allowed us to design a predictive DPE algorithm in a small subset of patients that could not be initially classified based on the selection criteria. This DPE suggests that these nucleic acids could be actively released by both tumor and nontumor cells as a means of intercellular communication and might thus play a role in the process of malignant transformation. DPE is a new technique for the study of plasma cfDNA by WES that might have predictive and prognostic value in patients with CRC.

Abstract P2-02-21: The utility and correlation of circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) based on HER2 positivity

Abstract Background: CTCs are well-established prognostic and predictive biomarkers for metastatic breast cancer (MBC) and other solid tumors. ctDNA is emerging as a quantitative blood-based biomarker for monitoring genomic alterations and disease progression. We evaluated the clinical utility and correlation of these liquid biopsy molecular tools in a cohort of MBC patients. Methods: CTC samples were obtained from an ongoing, prospective study of blood based prognostic biomarkers for breast cancer patients. At this time, 71 patients and 98 total samples have been collected. CTC enumeration was performed using the CellSearchTM platform (Menarini, IT). Within this cohort, MBC patients who had ctDNA testing were identified. ctDNA testing was performed using Guardant360TM (Guardant Health, CA), a digital next-generation sequencing technology. Two groups were analyzed: (1) HER2-negative patients with CTC ≥ 5 in 7.5 ml of blood (2) HER2-positive patients who had been treated with HER2 targeted therapy. Results: 22 samples (N=16 patients) were found with CTC ≥ 5 (range 8-904) and concurrent ctDNA testing (median timeframe between collection 0 days, range 0-42 days). There was a significant association between number of CTCs and the total number of genomic alterations detected in ctDNA (paired two sample t-test, p=0.012). In addition, CTC enumeration was significantly correlated with somatic alteration burden of the dominant clone (paired two sample t-test, p=0.023). The most common alterations detected in the blood were TP53 (55% of patients, 18 total mutations), PIK3CA (41% of patients, 15 total mutations), and ESR1 (32% of patients, 14 total mutations). For patients with HER2 positivity receiving HER2-targeted therapies (N=16 samples from 11 patients), only 18.8% of samples had detectable CTCs (all less than 5) as compared to 75.0% of samples with detectable ctDNA alterations. In N=12 samples with detectable ctDNA mutations, mean number of genomic alterations was 4.4 with mean somatic mutation burden of 2.95%. CTCs detectedctDNA detectedCTC ≥ 5Mean number of ctDNA alterations+Mean somatic alteration burden+HER2- (only cases with CTC ≥ 5)100% (22/22)100% (22/22)100%6.716.1%HER2+ (all cases)18.8% (3/16)75.0% (12/16)0%4.42.95%+excludes ctDNA samples without detected genomic alterations Conclusions: In HER2-negative MBC patients, CTC enumeration was significantly correlated with the number of ctDNA genomic alterations and somatic alteration burden, indicating the potential for ctDNA as a prognostic, quantitative biomarker of tumor burden. In patients with HER2 positivity, ctDNA may be a more sensitive liquid biopsy tool given the rarity of detecting CTCs detection in this population using the CellSearchTM system. In HER2-positive patients, consideration of size-dependent selection of CTCs using filtration of cells that have undergone epithelial-mesenchymal transition may improve detection in this subgroup. Citation Format: Davis AA, Zhang Y, Behdad A, Taxter T, Strickland K, Santa-Maria C, Flaum L, Cruz MR, Platanias LC, Gradishar WJ, Cristofanilli M. The utility and correlation of circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) based on HER2 positivity [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-02-21.

Circulating tumor DNA and liquid biopsy: opportunities, challenges, and recent advances in detection technologies.

Cell-free DNA (cfDNA) refers to short fragments of acellular nucleic acids detectable in almost all body fluids, including blood, and is involved in various physiological and pathological phenomena such as immunity, coagulation, aging, and cancer. In cancer patients, a fraction of hematogenous cfDNA originates from tumors, termed circulating tumor DNA (ctDNA), and may carry the same mutations and genetic alterations as those of a primary tumor. Thus, ctDNA potentially provides an opportunity for noninvasive assessment of cancer. Recent advances in ctDNA analysis methods will potentially lead to the development of a liquid biopsy tool for the diagnosis, prognosis, therapy response monitoring, and tracking the rise of new mutant sub-clones in cancer patients. Over the past few decades, cancer-specific mutations in ctDNA have been detected using a variety of untargeted methods such as digital karyotyping, personalized analysis of rearranged ends (PARE), whole-genome sequencing of ctDNA, and targeted approaches such as conventional and digital PCR-based methods and deep sequencing-based technologies. More recently, several chip-based electrochemical sensors have been developed for the analysis of ctDNA in patient samples. This paper aims to comprehensively review the diagnostic, prognostic, and predictive potential of ctDNA as a minimally invasive liquid biopsy for cancer patients. We also present an overview of current advances in the analytical sensitivity and accuracy of ctDNA analysis methods as well as biological and technical challenges, which need to be resolved for the integration of ctDNA analysis into routine clinical practice.

Accurate Electrochemistry Analysis of Circulating Methylated DNA from Clinical Plasma Based on Paired-End Tagging and Amplifications.

Circulating methylated DNA has been a new kind of cancer biomarker, yet its small fraction of trace total DNA from clinical samples impairs the accurate analysis. Though fluorescence methods based on quantitative methylation specific PCR (qMSP) have been adopted routinely, yet alternative electrochemistry assay of such DNA from clinical samples remains a great challenge. Herein, we report accurate electrochemistry analysis of circulating methylated DNA from clinical plasma samples based on a paired-end tagging and amplifications strategy. Two DNA primers each labeled with digoxigenin (Dig) and biotin are designed for the recognition and amplification of methylated DNA. Paired-end tagging amplicons and avidin-HRP molecules are successively captured on the electrode modified with Anti-Dig. Then HRP executes catalytic reaction to generate amplified signal. The design of paired-end tagging can readily integrate downstream electrochemical amplified reaction, and two heterogeneous amplifications enable high assay sensitivity. As little as 40 pg of methylated genomic DNA (∼10 genomic equivalents) is well identified, and our strategy can even distinguish as low as 1% methylation level. Tumor-specific methylated DNA is clearly detected in the plasma of 10 of 11 NSCLC patients. The high clinical sensitivity of 91% (10/11) indicates the good consistency with clinical diagnosis. Excellent spatial control of electrochemistry allows simpler detection of more methylation patterns compared to fluorescence methods. The developed electrochemical assay is a promising liquid biopsy tool for the analysis of tumor-specific circulating DNA.

DNA Hydrogel with Aptamer-Toehold-Based Recognition, Cloaking, and Decloaking of Circulating Tumor Cells for Live Cell Analysis.

Circulating tumor cells (CTCs) contain molecular information on the primary tumor and can be used for predictive cancer diagnostics. Capturing rare live CTCs and their quantification in whole blood remain technically challenging. Here we report an aptamer-trigger clamped hybridization chain reaction (atcHCR) method for in situ identification and subsequent cloaking/decloaking of CTCs by porous DNA hydrogels. These decloaked CTCs were then used for live cell analysis. In our design, a DNA staple strand with aptamer-toehold biblocks specifically recognizes epithelial cell adhesion molecule (EpCAM) on the CTC surface that triggers subsequent atcHCR via toehold-initiated branch migration. Porous DNA hydrogel based-cloaking of single/cluster of CTCs allows capturing of living CTCs directly with minimal cell damage. The ability to identify a low number of CTCs in whole blood by DNA hydrogel cloaking would allow high sensitivity and specificity for diagnosis in clinically relevant settings. More significantly, decloaking of CTCs using controlled and defined chemical stimuli can release living CTCs without damages for subsequent culture and live cell analysis. We expect this liquid biopsy tool to open new powerful and effective routes for cancer diagnostics and therapeutics.

Extracellular Vesicles in Hematological Malignancies: From Biology to Therapy.

Extracellular vesicles (EVs) are a heterogeneous group of particles, between 15 nanometers and 10 microns in diameter, released by almost all cell types in physiological and pathological conditions, including tumors. EVs have recently emerged as particularly interesting informative vehicles, so that they could be considered a true “cell biopsy”. Indeed, EV cargo, including proteins, lipids, and nucleic acids, generally reflects the nature and status of the origin cells. In some cases, EVs are enriched of peculiar molecular cargo, thus suggesting at least a degree of specific cellular packaging. EVs are identified as important and critical players in intercellular communications in short and long distance interplays. Here, we examine the physiological role of EVs and their activity in cross-talk between bone marrow microenvironment and neoplastic cells in hematological malignancies (HMs). In these diseases, HM EVs can modify tumor and bone marrow microenvironment, making the latter “stronger” in supporting malignancy, inducing drug resistance, and suppressing the immune system. Moreover, EVs are abundant in biologic fluids and protect their molecular cargo against degradation. For these and other “natural” characteristics, EVs could be potential biomarkers in a context of HM liquid biopsy and therapeutic tools. These aspects will be also analyzed in this review.

Cancer gene panel analysis of cultured circulating tumor cells and primary tumor tissue from patients with breast cancer.

Although numerous effective therapies have improved the survival rate of patients with breast cancer, a number of patients present with tumor recurrence and metastasis. A liquid biopsy of circulating tumor cells (CTC) is a non-invasive method to obtain tumor cells and may be used as substitute for a tumor tissue biopsy. The present study focuses on determining whether CTC culture is an optimal method of obtaining sufficient amounts of CTCs for molecular analysis. The current study demonstrates a method of isolating and culturing CTCs from patients with breast cancer and the construction of a molecular profile of cultured cells using the Ion AmpliSeq Cancer Gene Panel V2. Gene mutations that were observed in cultured CTCs were compared with those observed in primary tumor tissues. CTCs were isolated and cultured from the blood of six patients with breast cancer. Mutations from the Catalogue Of Somatic Mutation In Cancer (COSMIC) were detected in Platelet-Derived Growth Factor Receptor Alpha, MET (also known as Hepatocyte Growth Factor Receptor), Phosphatase and Tensin Homolog, Harvey Rat Sarcoma Viral Oncogene Homolog, SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin Subfamily B Member 1, Cyclin Dependent Kinase Inhibitor 2A and MutL Homolog 1 genes in 5/6 samples. A comparison between mutations detected in cultured CTCs and mutations detected in primary tumor tissues demonstrated that a large number of mutations that were identified in CTCs were also detected in primary tumor tissues. The results from the current study describe a novel cell culture approach that may be used to obtain an optimal number of CTCs for molecular analysis. This novel approach is able to be used as a tool for liquid biopsy during breast cancer treatment.

Establishment and Application of Prostate Cancer Circulating Tumor Cells in the Era of Precision Medicine.

Prostate cancer (PC) is the second most common cancer in men and is the fifth leading cause of cancer-related deaths worldwide. Additionally, there is concern for overdiagnosis and overtreatment of PC. Thus, selection of an appropriate candidate for active surveillance as well as more accurate and less invasive tools for monitoring advanced PC is required. Circulating tumor cells (CTCs) have emerged as a liquid biopsy tool; there have been several reports on its role, technologies, and applications to various cancers, including PC. Liquid biopsy using CTCs has been gaining attention as a minimal invasive tool for investigation of biomarkers and for prognosis and assessment of response to therapies in patients with PC. Because of the lower invasiveness of liquid biopsy using CTCs, it can be performed more frequently; accordingly, personalized disease status can be successively determined at serial time points. CTC analysis enables detection of genomic alterations, which is drug-targetable, and it is a potential tool for monitoring response to therapeutic agents in patients with PC. This review focuses on the characteristics, technologies for analysis, and advantages and disadvantages of CTCs as a liquid biopsy tool and their application in PC. Finally, we propose future directions of CTCs.

Tumour-associated circulating microparticles: a novel liquid biopsy tool for screening and therapy monitoring of colorectal carcinoma and other epithelial neoplasia

Up to date, novel tools for low-cost, minimal invasive cancer surveillance, cancer screening and treatment monitoring are in urgent need. Physicians consider the so-called liquid biopsy as a possible future tool successfully achieving these ultimate goals. Here, we aimed to identify circulating tumour-associated MPs (taMPs) that could aid in diagnosing minimal-invasively the presence and follow up treatment in non-small cell lung carcinoma (NSCLC), colorectal carcinoma (CRC) and pancreas carcinoma (PaCa). Tumour-associated MPs (taMPs) were quantified after isolation by centrifugation followed by flow cytometry analysis from the serum of cancer patients with CRC (n = 52), NSCLC (n = 38) and PaCa (n = 10). Healthy subjects (n = 55) or patients with struma nodosa (thyroid nodules) (n = 43) served as negative controls. In all three types of tumour entities, the presence of tumour was associated with an increase of circulating EpCAM+ and EpCAM+CD147+ taMPs. The presence of CD147+EpCAM+ taMPs were specific to tumour-bearing patients thus allowing the specific distinction of malignancies from patients with thyroid nodules. Increased level of EpCAM single positive MPs were, in turn, also detected in patients with thyroid nodules. Importantly, EpCAM+CD147+ taMPs correlated with the measured tumour-volume in CRC patients. EpCAM+ taMPs decreased at 7 days after curative R0 tumour resection suggesting a close dependence with tumour presence. AUROC values (up to 0.85 and 0.90), sensitivity/specificity scores, and positive/negative predictive values indicated a high diagnostic accuracy of EpCAM+CD147+ taMPs. Taken together, EpCAM+CD147+ double positive taMPs could potentially serve as novel promising clinical parameter for cancer screening, diagnosis, surveillance and therapy monitoring.

Abstract 2249: Detection of EGFR mutations in circulating cell-free DNA of non-small cell lung cancer patients by next-generation sequencing

Abstract [Background] Mutational analysis is essential for recent personalized therapeutic approaches of non-small cell lung cancer (NSCLC) patients. Circulating cell-free DNA (cfDNA) from plasma is an emerging topic as a tool for liquid biopsy and possibly enable repetitive genetic diagnosis or monitoring of cancer status. However, detection of cfDNA from the circulation still remains challenging because of its highly fragmented and low concentration nature. In this study, we analyzed cfDNA samples by targeted deep sequencing at high coverage. [Materials and Methods] Twenty matched DNA samples were collected from metastatic NSCLC patients with EGFR mutation, which was detected by a clinical EGFR mutant enriched PCR assay using PNA-LNA PCR clamp method. These samples were extracted from tissue samples of fresh tumor tissue except one formalin fixed paraffin embedded (FFPE) sample (tDNA), and plasma samples (cfDNA). Mutation hotspots were enriched by multiplex PCR using the GeneRead DNAseq Targeted Panels V2 Human Tumor Actionable Mutations panel (QIAGEN, Hilden, Germany), followed by library construction, and sequenced on the MiSeq sequencer. Resulting data was analyzed using CLC Genomics Workbench and Genomics Server and mapped to the full human genome. [Results and discussion] The mean read depth in target region was 59,000 reads. Matched EGFR driver mutations between cfDNA and tDNA were detected with an overall concordance of 25% (5 of 20 samples). The concordance rate of exon21 L858R point mutation, exon19 deletions and exon20 T790M point mutation were 32.5% (3 of 8 samples), 16.7% (2 of 12 samples), and 66.7% (2 of 3 samples), respectively. The mutant allele frequencies in cfDNA ranged from 0.1% to 44.5% (median, 0.3%). While further improvement is mandatory, these results suggest that targeted deep sequencing of cfDNA at high coverage may have a potential utility as a non-invasive mutational analysis. In addition, there seemed to be a tendency of better detection rate for point mutations (45.5%) than deletions (16.7%), while the difference didn't reach statistical significance (p = 0.19), which were corresponding to previous reports. Citation Format: Ken Suzawa, Shuta Tomida, Takahiro Matsubara, Kadoaki Ohashi, Yuho Maki, Hiromasa Yamamoto, Mizuki Morita, Junichi Soh, Hiroaki Asano, Kazunori Tsukuda, Katuyuki Kiura, Shinichiro Miyoshi, Shinichi Toyooka. Detection of EGFR mutations in circulating cell-free DNA of non-small cell lung cancer patients by next-generation sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2249.

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool.

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2(-) (ΔΔct) (13). Control values were used as baseline and results are shown in logarithmic scale.

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-ΔΔct 13. Control values were used as baseline and results are shown in logarithmic scale.

Tumour-associated circulating microparticles: A novel liquid biopsy tool for screening and therapy monitoring of colorectal carcinoma and other epithelial neoplasia

Up to date, novel tools for low-cost, minimal invasive cancer surveillance, cancer screening and treatment monitoring are in urgent need. Physicians consider the so-called liquid biopsy as a possible future tool successfully achieving these ultimate goals. Here, we aimed to identify circulating tumour-associated MPs (taMPs) that could aid in diagnosing minimal-invasively the presence and follow up treatment in non-small cell lung carcinoma (NSCLC), colorectal carcinoma (CRC) and pancreas carcinoma (PaCa). Tumour-associated MPs (taMPs) were quantified after isolation by centrifugation followed by flow cytometry analysis from the serum of cancer patients with CRC (n = 52), NSCLC (n = 40) and PaCa (n = 11). Healthy subjects (n = 55) or patients with struma nodosa (thyroid nodules) (n = 43) served as negative controls. In all three types of tumour entities, the presence of tumour was associated with an increase of circulating EpCAM+ and EpCAM+CD147+ taMPs. The presence of CD147+EpCAM+ taMPs were specific to tumour-bearing patients thus allowing the specific distinction of malignancies from patients with thyroid nodules. Increased level of EpCAM single positive MPs were, in turn, also detected in patients with thyroid nodules. Importantly, EpCAM+CD147+ taMPs correlated with the measured tumour-volume in CRC patients. EpCAM+ taMPs decreased at 7 days after curative R0 tumour resection suggesting a close dependence with tumour presence. AUROC values (up to 0.85 and 0.90), sensitivity/specificity scores, and positive/negative predictive values indicated a high diagnostic accuracy of EpCAM+CD147+ taMPs. Taken together, EpCAM+CD147+ double positive taMPs could potentially serve as novel promising clinical parameter for cancer screening, diagnosis, surveillance and therapy monitoring.