Liquid Biopsy Tool Research Articles

MRNAs in urinary nano-extracellular vesicles as potential biomarkers for non-invasive kidney biopsy.

Urinary nano-extracellular vesicles (NVs), including exosomes and microvesicles, are considered potential biomarkers for kidney diseases using liquid biopsies. However, clinical application of urinary NVs has not yet been validated. In the present study, the levels of mRNAs in urinary NVs in animal models of kidney disease were assessed. Urine samples were collected from the animal models and urinary NVs were isolated by ultracentrifugation. Gene expression levels of kidney injury markers in urinary NVs and renal tissue were quantified by reverse transcription-quantitative PCR. The mRNA levels of desmin, a podocyte injury marker, in urinary NVs was markedly increased in the puromycin aminonucleoside (PAN) nephritis model, in parallel with enhanced desmin expression in kidney tissues. The expression of regulator of calcineurin 1 and the podocin to nephrin ratio (PNR) were also increased in the PAN nephritis model. Treatment with prednisolone mitigated these changes in gene expression as well as proteinuria. PNR, which is considered a predictive marker of glomerular dysfunction, in urinary NVs was highly correlated with urinary protein excretion (P<0.01). Furthermore, PNR in urinary NVs of Zucker diabetic fatty rats, a diabetic kidney disease model, was correlated with urinary albumin excretion (P<0.01). These results suggest that changes in mRNA levels of urinary NVs reflect the disease status of kidney tissues and their functional alterations. Collectively, mRNA analysis of urinary NVs may be used as a liquid biopsy tool for improved classification and performance of risk prediction to determine the severity of kidney diseases.

Hypomethylation of the DAZ3 promoter in idiopathic asthenospermia: a screening tool for liquid biopsy

Given the role of the deleted in azoospermia gene in male infertility, whether the somatic deleted in azoospermia methylation status is associated with idiopathic asthenospermia should be determined. To investigate the methylation levels of the deleted in azoospermia promoter in peripheral white blood cells from idiopathic asthenospermia patients relative to those in normozoospermia controls, 61 ethylene diamine tetraacetic acid anticoagulant blood samples were drawn from all participants for DNA isolation. The deleted in azoospermia promoter methylation ratio was detected by MassARRAY-based methylation quantification and confirmed by quantitative methylation-specific polymerase chain reaction. A MassARRAY-based methylation analysis showed that the deleted in azoospermia 3 promoter (0 to − 2 kbp) was significantly hypomethylated in peripheral white blood cells from idiopathic asthenospermia males, specifically one CpG site (− 246 to − 247). Quantitative methylation-specific polymerase chain reaction data further confirmed that the methylation level of the deleted in azoospermia 3 promoter region in idiopathic asthenospermia patients was significantly lower than that in normozoospermia males. The area under the receiver operating characteristic curve determined by quantitative methylation-specific polymerase chain reaction was 0.737 (95% confidence interval: 0.552 to 0.924), with a sensitivity of 53.9% and a specificity of 88.2% at a cut-off level of 74.7%. Therefore, our results suggested that methylation ratio detection of the deleted in azoospermia 3 promoter region by real-time polymerase chain reaction assay is a promising and feasible tool for liquid biopsy in the clinical laboratories. The methylation status of other reported infertility-related genes should also be investigated in peripheral white blood cells.

Liquid biopsy using extracellular vesicle-derived DNA in lung adenocarcinoma.

Blood liquid biopsy has emerged as a way of overcoming the clinical limitations of repeat biopsy by testing for the presence of acquired resistance mutations to therapeutic agents. Despite its merits of repeatability and non-invasiveness, this method is currently only used as a supplemental test due to a relatively low sensitivity rate of 50%–60%, and cannot replace tissue biopsy. The circulating tumor DNAs used in blood liquid biopsies are passive products of fragmented DNA with a short half-life released following tumor cell death; the low sensitivity seen with liquid blood biopsy results from this instability, which makes increasing the sensitivity of this test fundamentally difficult. Extracellular vesicles (EVs) are ideal carriers of cancer biomarkers, as cancer cells secret an abundance of EVs, and the contents of tumor cell-originated EVs reflect the molecular and genetic composition of parental cells. In addition, EV-derived DNAs (EV DNAs) consist of large-sized genomic DNAs and tumor-specific oncogenic mutant DNAs. For these reasons, liquid biopsy using EV DNA has the potential to overcome issues arising from tissue shortages associated with small biopsies, which are often seen in lung cancer patients, and the biopsy product can be used in other diagnostic methods, such as epidermal growth factor receptor (EGFR) mutation testing and next-generation sequencing (NGS). A higher sensitivity can be achieved when EV DNAs obtained from bronchoalveolar lavage fluid (BALF) are used rather than those from blood. BALF, when obtained close to the tumor site, is a promising liquid biopsy tool, as it enables the gathering of both cellular and non-cellular fractions of the tumor microenvironment, and provides increased diagnostic sensitivity when compared to blood.

Reviews on Current Liquid Biopsy for Detection and Management of Pancreatic Cancers

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Pancreatic cancer presents dismal clinical outcomes in patients, and the incidence of pancreatic cancer has continuously increased to likely become the second most common cause of cancer-related deaths by as early as 2030. One of main reasons for the high mortality rate of pancreatic cancer is the lack of tools for early-stage detection. Current practice in detecting and monitoring therapeutic response in pancreatic cancer relies on imaging analysis and invasive endoscopic examination. Liquid biopsy-based analysis of genetic alterations in biofluids has become a fundamental component in the diagnosis and management of cancers. There is an urgent need for scientific and technological advancement to detect pancreatic cancer early and to develop effective therapies. The development of a highly sensitive and specific liquid biopsy tool will require extensive understanding on the characteristics of circulating tumor DNA in biofluids. Here, we have reviewed the current status of liquid biopsy in detecting and monitoring pancreatic cancers and our understanding of circulating tumor DNA that should be considered for the development of a liquid biopsy tool, which will greatly aid in the diagnosis and healthcare of people at risk.

Proteomic Approach for Searching for Universal, Tissue-Specific, and Line-Specific Markers of Extracellular Vesicles in Lung and Colorectal Adenocarcinoma Cell Lines.

Tumor-derived extracellular vesicles (EVs), including exosomes, contain proteins that mirror the molecular landscape of producer cells. Being potentially detectible in biological fluids, EVs are of great interest for the screening of cancer biomarkers. To reveal universal, tissue-specific, and line-specific markers, we performed label-free mass spectrometric profiling of EVs originating from the human colon cancer cell lines Caco-2, HT29, and HCT-116, as well as from the lung cancer cell lines NCI-H23 and A549. A total of 651 proteins was identified in the EV samples using at least two peptides. These proteins were highly enriched in exosome markers. We found 11 universal, eight tissue-specific, and 29 line-specific markers, the levels of which were increased in EVs compared to the whole lysates. The EV proteins were involved in the EGFR, Rap1, integrin, and microRNA signaling associated with metastasis and cancer progression. An EV protein-based assay could be developed as a liquid biopsy tool.

Abstract 6446: Liquid biopsy in colon cancer utilizing exosomal miRNA biomarkers: An initial analysis

Abstract Traditional clinicopathologic variables do not predict recurrence and disease progression in colorectal cancer (CRC) accurately. Development of a liquid biopsy as a means to obtain real-time, clinically meaningful biomarkers to assess progression and response to treatment is critical. Current liquid biopsy techniques utilizing circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have significant limitations. Exosomes are emerging as a promising tool for liquid biopsy due to their stability and role in cancer cell to cell communication. To that end, the purpose of this study is to describe patterns of highly abundant microRNA (miRNA) found in serum exosomes of patients with metastatic colon cancer as a step toward developing an exosomal miRNA signature for liquid biopsy. Next generation sequencing was performed on exosomes isolated from serum of stage IV colon cancer patients (n=9) to determine the miRNA read counts. miRNA data from primary tumors (n=53) of stage IV colon cancer patients and available paired peritumoral tissue (n=8) were obtained from The Cancer Genome Atlas (TCGA) database for qualitative comparison. There were 515 total miRNA shared in common between the serum exosomes and primary tumors and 277 miRNA uniquely present in serum exosomes. In our initial analysis, we identified five miRNA, two in the common pool (mir-486-1 and mir-486-2) and three in the unique group (miR-486-5p, miR-92a-3p, and miR-146a-5p) that were most abundant in serum exosomes and may serve as biomarkers. mir-486-1 and mir-486-2 were the most highly abundant miRNAs in exosomes, but were only expressed at low levels in the tissue. This inverse relationship has been demonstrated by other investigators, with decreased levels of tissue mir-486 associated with more advanced stages of disease and poorer survival. Exosomal miR-92a-3p has been shown to correlate with tumor grade and stage in CRC patients. Delivery of miR-146a-5p by exosomes has been shown to promote tumorigenesis and generate immunosuppressive microenvironment in CRC. In general, the top 10 most abundant exosomal miRNA were not among the most abundant tissue level miRNAs. Our group is currently analyzing the differential expression of serum exosomal miRNA from 52 additional patients with non-metastatic colon cancer (n=14), liver metastases (n=16) and PC (n=12) in comparison to healthy volunteer serum (n=10) to better characterize miRNA patterns at different stages of disease. In summary, highly abundant miRNA found in serum exosomes of patients with metastatic colon cancer demonstrate a different pattern of expression than miRNA found in the primary colon cancer itself. Further analysis of these miRNA patterns from serum exosomes of colon cancer patients with non-metastatic disease, visceral metastases and PC compared to normal volunteers will aid in the development of a serum exosomal miRNA signature. Citation Format: Audrey H. Choi, Paul A. Vallejos, Mei Li M. Kwong, Janviere Kabagwira, Matthew J. Selleck, Nathan R. Wall, Maheswari Senthil. Liquid biopsy in colon cancer utilizing exosomal miRNA biomarkers: An initial analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6446.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n= 151) and plasma-derived (n= 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Recapitulating atherogenic flow disturbances and vascular inflammation in a perfusable 3D stenosis model

Blood vessel narrowing and arterial occlusion are pathological hallmarks of atherosclerosis, which involves a complex interplay of perturbed hemodynamics, endothelial dysfunction and inflammatory cascade. Herein, we report a novel circular microfluidic stenosis model that recapitulates atherogenic flow-mediated endothelial dysfunction and blood-endothelial cell (EC) interactions in vitro. 2D and 3D stenosis microchannels with different constriction geometries were fabricated using 3D printing to study flow disturbances under varying severity of occlusion and wall shear stresses (100 to 2000 dynecm−2). Experimental and fluid simulation results confirmed the presence of pathological shear stresses in the stenosis region, and recirculation flow post stenosis. The resultant pathological flow profile induced pro-inflammatory and pro-thrombotic EC state as demonstrated by orthogonal EC alignment, enhanced platelet adhesion at the stenosis, and aberrant leukocyte-EC interactions post stenosis. Clinical utility of the vascular model was further investigated by testing anti-thrombotic and immunomodulatory efficacy of aspirin and metformin, respectively. Overall, the platform enables multi-factorial analysis of critical atherogenic events including endothelial dysfunction, platelets and leukocyte adhesion, and can be further developed into a liquid biopsy tool for cardiovascular risk stratification.

Synergistic effects of extracellular vesicle phenotyping and AFP in hepatobiliary cancer differentiation.

Biliary cancer, comprising cholangio- and gallbladder carcinomas, is associated with high mortality due to asymptomatic disease onset and resulting late diagnosis. Currently, no robust diagnostic biomarker is clinically available. Therefore, we explored the feasibility of extracellular vesicles (EVs) as a liquid biopsy tool for biliary cancer screening and hepatobiliary cancer differentiation. Serum EVs of biliary cancer, hepatocellular carcinoma, colorectal cancer and non-small cell lung cancer patients, as well as from healthy individuals, were isolated by sequential two-step centrifugation and presence of indicated EVs was evaluated by fluorescence activated cell sorting (FACS) analysis. Two directly tumour-related antigen combinations (AnnV+ CD44v6+ and AnnV+ CD44v6+ CD133+ ) and two combinations related to progenitor cells from the tumour microenvironment (AnnV+ CD133+ gp38+ and AnnV+ EpCAM+ CD133+ gp38+ ) were associated with good diagnostic performances that could potentially be used for clinical assessment of biliary cancer and differentiation from other cancer entities. With 91% sensitivity and 69% specificity AnnV+ CD44v6+ EVs showed the most promising results for differentiating biliary cancers from HCC. Moreover using a combined approach of EV levels of the four populations with serum AFP values, we obtained a perfect separation of biliary cancer and HCC with sensitivity, specificity, positive and negative predictive value all reaching 100% respectively. EV phenotyping, especially if combined with serum AFP, represents a minimally invasive, accurate liquid biopsy tool that could improve cancer screening and differential diagnosis of hepatobiliary malignancies.

Molecular and functional extracellular vesicle analysis using nanopatterned microchips monitors tumor progression and metastasis.

Longitudinal cancer monitoring is crucial to clinical implementation of precision medicine. There is growing evidence indicating important functions of extracellular vesicles (EVs) in tumor progression and metastasis, including matrix remodeling via transporting matrix metalloproteases (MMPs). However, the clinical relevance of EVs remains largely undetermined, partially owing to challenges in EV analysis. Distinct from existing technologies mostly focused on characterizing molecular constituents of EVs, here we report a nanoengineered lab-on-a-chip system that enables integrative functional and molecular phenotyping of tumor-associated EVs. A generalized, high-resolution colloidal inkjet printing method was developed to allow robust and scalable manufacturing of three-dimensional (3D) nanopatterned devices. With this nanochip platform, we demonstrated integrative analysis of the expression and proteolytic activity of MMP14 on EVs to detect in vitro cell invasiveness and monitor in vivo tumor metastasis, using cancer cell lines and mouse models. Analysis of clinical plasma specimen showed that our technology could be used for cancer detection including accurate classification of age-matched controls and patients with ductal carcinoma in situ, invasive ductal carcinoma, or locally metastatic breast cancer in a training cohort (n = 30, 96.7% accuracy) and an independent validation cohort (n = 70, 92.9% accuracy). With clinical validation, our technology could provide a useful liquid biopsy tool to improve cancer diagnostics and real-time surveillance of tumor evolution in patients to inform personalized therapy.

Selective capture of plasma cell-free tumor DNA on magnetic beads: a sensitive and versatile tool for liquid biopsy.

Recently, 'solid tumor biopsies' have been challenged by the emergence of 'liquid biopsies', which are aimed at the isolation and detection of circulating cell-free tumor DNA (ctDNA) in body fluids. Here, we developed and optimized a method for selective capture of ctDNA on magnetic beads (SCC-MAG) for mutation detection in plasma of patients with colorectal cancer (CRC). Blood and tissue samples from 28 CRC patients were included for the detection of KRAS mutations. For the tissue samples, mutation analysis was conducted by high resolution melting (HRM) analysis and sequencing. For the SCC-MAG method, ctDNA was isolated from 200 µl plasma from patients with a mutant KRAS gene. For comparison, ctDNA extraction was carried out using a silica membrane-based method, after which mutations were detected using Intplex allele-specific PCR. The mean ctDNA integrity index in plasma samples of cancer patients was 1.03, comparable with that of silica membrane-derived ctDNA (1.011). Notably, the limit of detection for the SCC-MAG approach was lower than that of the silica membrane method and measured 2.25 pg/ml ctDNA in plasma. Our analyses showed that while the silica membrane-based approach was capable of collecting ctDNA from two out of six CRC patient samples (average Cq 34.23), the SCC-MAG captured ctDNA from all samples with an average Cq of 29.76. We present a robust, reproducible, and highly sensitive method for the analysis of mutation statuses in liquid biopsies. The SCC-MAG method can readily be applied to any nucleic acid target for diagnostic purposes upon careful design of the specific capture probes, and can be multiplexed by several probes to identify multiple targets.

Single-step RT-qPCR for detection of extracellular vesicle microRNAs in vivo: a time- and cost-effective method.

Emerging evidence suggests that extracellular vesicle (EV)-associated microRNAs (miRNAs) are a potential diagnostic tool for liquid biopsy in various human diseases. However, the experimental procedure for the detection of EV-associated miRNAs (EV-miRNAs) from body fluids is relatively complex and not cost-effective. Due to the limited amount of EVs and EV-RNAs, a column-based RNA purification, which is an expensive approach, is often used to detect EV-miRNAs via reverse transcription-quantitative real-time PCR (RT-qPCR). Here, we developed and validated a simple and cost-effective method (single-step RT-qPCR) in which we directly detect EV-miRNAs without RNA purification from the EVs. We validated this protocol using the EVs isolated from mouse broncho-alveolar lavage fluid (BALF) and serum. The obtained EVs were first lysed in the EV-lysis buffer, followed by RT-qPCR without isolation and purification of RNAs. We successfully detected the designated miRNAs from lysed EVs; 106 to 107 EVs were optimal to detect the EV-miRNAs using the single-step RT-qPCR. In our previously published work, using the conventional RT-qPCR method, we have reported that miR-142 and -223 are dramatically upregulated in both BALF and serum EVs after lung infection. Hence, we reassessed and confirmed the level of EV-miR-142/223 using the newly developed single-step RT-qPCR. Notably, inhibition of RNase activity in the lysed EVs remains crucial for the detection of EV-miRNAs. Moreover, repeated freeze-thaw cycling significantly interferes the EV-miRNA quantification. Collectively, the single-step RT-qPCR for the detection of EV-miRNAs in vivo will potentially provide a fast, accurate, and convenient way to quantify circulating and/or body fluid-derived EV-miRNAs. This method may potentially be applied to the diagnostic blood testing used in the medical centers or research laboratories.

A Clinical Study on Detection of Dysplastic Cells Through Saliva.

Saliva is an ideal tool for liquid biopsy as it is noninvasive, easily collected, and easily transported. Saliva is used to detect various biomarkers in oral cancer patients, but none used for detecting salivary dysplastic cells in potentially malignant and malignant disorders. To detect dysplastic cells from saliva in oral cancer and potentially malignant patients and compare with wedge biopsy. All potentially malignant and malignant cases of oral cavity were included in this study. In these patients, unstimulated saliva was collected and centrifuged. The concentrated salivary substrate was stained by Haematoxylin and Eosin (H&E) stain, PAP (Papanicolaou) method and May Grunwald Giemsa stain (MGG) and analyzed. Wedge biopsy was performed from the representative area in the oral cavity and compared with salivary cells. In our study, out of 24 patients, 16 cases were malignant, and 8 were potentially malignant. 12 patients were found to be positive for atypical cells, 10 patients were found to be positive for dysplastic cells and atypical cells, and two patients were negative for atypical cells/dysplastic cells. In 10 patients who were positive for dysplastic cells, all were diagnosed histologically as malignancies (P = 0.001). The two patients who were negative for atypical cells/dysplastic cells showed precursor epithelial lesion with hyperplasia in the biopsy. Few neutrophils were found in 15 patients and increased in 9 patients (P = 0.039). Saliva is readily available, and can be collected noninvasively for detecting dysplastic cells.

Plasma protein-based signature predicts distant metastasis and induction chemotherapy benefit in Nasopharyngeal Carcinoma.

Rationale: Currently, for locoregionally advanced nasopharyngeal carcinoma (LA-NPC), there is no effective blood-based method to predict distant metastasis. We aimed to detect plasma protein profiles to identify biomarkers that could distinguish patients with NPC who are at high risk of posttreatment distant metastasis.Methods: A high-throughput antibody array was initially applied to detect 1000 proteins in pretreatment plasma from 16 matched LA-NPC patients with or without distant metastasis after radical treatment. Differentially expressed proteins were further examined using a low-throughput array to construct a plasma protein-based signature for distant metastasis (PSDM) in a cohort of 226 patients.Results: Fifty circulating proteins were differentially expressed between metastatic and non-metastatic patients and 18 were proven to be strongly correlated with distant metastasis-free survival (DMFS) in NPC. A PSDM signature consisting of five proteins (SLAMF5, ESM-1, MMP-8, INSR, and Serpin A5) was established to assign patients with NPC into a high-risk group and a low-risk group. Patients in the high-risk group had shorter DMFS (P < 0.001), disease-free survival (DFS) (P < 0.001) and overall survival (OS) (P < 0.001). Moreover, the PSDM performed better than N stage and Epstein-Barr virus (EBV) DNA load at effectively identifying patients with NPC at high risk of metastasis. For patients in the high-risk group, induction chemotherapy significantly improved DMFS, DFS, and OS.Conclusions: The PSDM could be a useful liquid biopsy tool to effectively predict distant metastasis and the benefit of induction chemotherapy in patients with LA-NPC.

Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA

Circulating cell-free DNA (cfDNA) comprises small DNA fragments derived from normal and tumor tissue that are released into the bloodstream. Recently, methylation profiling of cfDNA as a liquid biopsy tool has been gaining prominence due to the presence of tissue-specific markers in cfDNA. We have previously reported cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) as a sensitive, low-input, cost-efficient and bisulfite-free approach to profiling DNA methylomes of plasma cfDNA. cfMeDIP-seq is an extension of a previously published MeDIP-seq protocol and is adapted to allow for methylome profiling of samples with low input (ranging from 1 to 10 ng) of DNA, which is enabled by the addition of 'filler DNA' before immunoprecipitation. This protocol is not limited to plasma cfDNA; it can also be applied to other samples that are naturally sheared and at low availability (e.g., urinary cfDNA and cerebrospinal fluid cfDNA), and is potentially applicable to other applications beyond cancer detection, including prenatal diagnostics, cardiology and monitoring of immune response. The protocol presented here should enable any standard molecular laboratory to generate cfMeDIP-seq libraries from plasma cfDNA in ~3-4 d.

Surface Plasmon Resonance for Biomarker Detection: Advances in Non-invasive Cancer Diagnosis.

Biomarker-based cancer analysis has great potential to lead to a better understanding of disease at the molecular level and to improve early diagnosis and monitoring. Unlike conventional tissue biopsy, liquid biopsy allows the detection of a large variety of circulating biomarkers, such as microRNA (miRNA), exosomes, circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and proteins, in an easily accessible and minimally invasive way. In this review, we describe and evaluate the relevance and applicability of surface plasmon resonance (SPR) and localized SPR (LSPR)-based platforms for the detection of different classes of cancer biomarkers in liquid biopsy samples. Firstly, we critically discuss unsolved problems and issues in capturing and analyzing biomarkers. Secondly, we highlight current challenges which need to be resolved in applying SPR biosensors into clinical practice. Then, we mainly focus on applications of SPR-based platforms that process a patient sample aiming to detect and quantify biomarkers as a minimally invasive liquid biopsy tool for cancer patients appearing over the last 5 years. Finally, we describe the analytical performances of selected SPR biosensor assays and their significant advantages in terms of high sensitivity and specificity as well as accuracy and workflow simplicity.

Abstract 2226: Validation of a liquid biopsy tool to identify point mutations in pediatric brain tumor patients

Abstract The use of liquid biopsies to diagnose and monitor tumors has been widely explored especially in adult cancers. In pediatric cancers, the feasibility of this method as a clinical tool is still to be established. Pediatric low grade gliomas (PLGG) are characterized by mutations in the MAPK pathway, and 20% harbour BRAFV600E mutation, which can also be found in paediatric patients with Langerhans cell histiocytoma (LCH), thyroid carcinoma and melanomas. Specific histone mutations have been described in pediatric high grade gliomas(HGG) and diffuse midline pontine gliomas(DIPG). Midline gliomas are not amenable to a gross total resection; however, a biopsy is needed for pathological and molecular diagnosis. In this scenario, liquid biopsy is of utmost importance and has the potential to spare the risk of morbidity with surgical procedures, determine diagnosis and prognosis, as well as serve as a tool to guide and monitor response to therapy with targeted agents. The objective of this study is to evaluate the use of droplet digital PCR (ddPCR) for identification of point mutations in cerebro-spinal fluid (CSF) or plasma of BRAF V600E or H3K27M positive pediatric patients. CSF was collected from a total of 51 patients, and plasma from 55 patients. ctDNA was extracted from 3 ml of plasma or 2 ml of CSF, and pre-amplified prior to ddPCR, which was conducted on the RainDance system. For BRAFV600E: Forty-five patients had CSF samples available for analysis, 8 had known positive brain tumors, 32 negative brain tumors, 5 normal controls; 5/8 positive cases had BRAFV600E mutation in ctDNA from CSF, one of the patients with negative CSF and positive tumor was on treatment with BRAF inhibitors. Plasma was analysed in 17 patients with known positive brain tumors and additionally 10 patients negative for BRAFV600E, all of the plasma samples were negative for the mutation. For H3K27M: Thirty patients had CSF samples for analysis 1 was a known positive tumor, 27 negative and 2 unknown status (radiological diagnosis of DIPG). Of the 3 DIPG samples 2 were positive, one with unknown status of the primary tumor. Plasma was also analysed for H3K27M mutation in 7 patients with known positive brain tumors, one DIPG with unknown status and 7 negative samples. All plasma samples tested negative for the mutation. Sensitivity and Specificity for this assay was respectively 62% and 97% for BRAF and 66% and 98 % for H3K27M in CSF. The sensitivity in plasma for brain tumors is poor, however seems better for non-CNS lesions. In summary, we show that liquid biopsy with analysis of CSF ctDNA is feasible with high specificity, important in the context of a diagnoses tool. Moving forward larger cohorts need to be validated towards the goal of implementing ddPCR as a clinical tool for diagnosis of specific point mutations in pediatric patients with CNS tumors. Citation Format: Liana Nobre, Michal Zapotocky, Monique Johnson, Jonathan Wasserman, Oussama Abla, Jim Whitlock, Uri Tabori, Cynthia Hawkins. Validation of a liquid biopsy tool to identify point mutations in pediatric brain tumor patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2226.

Exo-miRNAs as a New Tool for Liquid Biopsy in Lung Cancer.

Lung cancer is the predominant cause of cancer-related deaths. The high mortality rates are mainly due to the lack of diagnosis before the cancer is at a late stage. Liquid biopsy is a promising technique that could allow early diagnosis of lung cancer and better treatment selection for patients. Cell-free microRNAs have been detected in biological fluids, such as serum and plasma, and are considered interesting biomarkers for lung cancer screening and detection. Exosomes are nanovesicles of 30–150 nm and can be released by different cell types within the tumor microenvironment. Their exosomal composition reflects that of their parental cells and could be potentially useful as a biomarker for lung cancer diagnosis. This review summarizes the state-of-the-art of circulating microRNAs (miRNAs) in lung cancer, focusing on their potential use in clinical practice. Moreover, we describe the importance of exosomal miRNA cargo in lung cancer detection and their potential role during lung carcinogenesis. Finally, we discuss our experience with the analysis of circulating exosomal miRNAs in the bioMILD screening trial.

Exosomes as novel prognostic biomarker in potentially resectable colorectal cancer liver metastatic (CCLM) patients.

3558 Background: Target therapies and new surgical strategies deeply modify the history of CCLM patients (pts). Several prognostic scoring systems have been developed but no one is able to identify pts who should be excluded from a potentially useless surgery. Currently research is committed in identifying early biomarkers able to discern pts who could benefit from an aggressive approach. Exosomes are arising as promising biomarkers in cancer. The aim of this pivotal study was to analyze the association among exosome levels during CCLM-pts treatment, clinical outcomes and the KRAS status. Methods: We enrolled 22 pts with CCLM candidate to preoperative chemotherapy (pCT) and subsequent liver surgery. A blood sample was collected before pCT, after surgery, monthly during follow-up and at progression (PD). Exosomes were isolated by ultracentrifugation and characterized by standard methods. Exosomes concentration was assessed by Bradford assay. We adopted ddPCR™ KRAS G12/G13 Screening Kit to evaluate the KRAS status in exosomal DNA (e-DNA). Results: 22 CCLM pts received pCT and underwent liver surgery: 5 major hepatectomies and 17 multiple liver resections. Changes in exosomes plasma levels were found to correlate with each treatment step,resulting reduced after pCT and surgery and increased at PD, respectively (p = 0.0026). Pts with higher baseline exosome levels experimented shorter PFS than those with lower levels (p = 0.0033 HR 0.2). No association was found between exosome levels after liver surgery and disease free interval nor overall survival. KRAS status on e-DNA was evaluated on 10 pts in baseline, in pCT, after surgery, and in PD samples. In 8 out of 10 pts e-DNA displayed the same mutational status than the one detected on tumor DNA. Changes in e-DNA KRAS copies were found statistically significant in pCT vs surgery and pCT vs PD (p = 0.039; p = 0.04). Conclusions: Our study suggests a prognostic role of exosome levels in CCLM pts. Moreover, we showed that KRAS mutational status could be monitored during the post-surgery follow up by analyzing e-DNA. Overall, our data confirm the potential role of exosomes in liquid biopsy tool to monitor molecular changes during the treatment of CCLM pts.

Review: Peering through a keyhole: liquid biopsy in primary and metastatic central nervous system tumours.

Tumour molecular profiling by liquid biopsy is being investigated for a wide range of research and clinical purposes. The possibility of repeatedly interrogating the tumour profile using minimally invasive procedures is helping to understand spatial and temporal tumour heterogeneity, and to shed a light on mechanisms of resistance to targeted therapies. Moreover, this approach has been already implemented in clinical practice to address specific decisions regarding patients’ follow‐up and therapeutic management. For central nervous system (CNS) tumours, molecular profiling is particularly relevant for the proper characterization of primary neoplasms, while CNS metastases can significantly diverge from primary disease or extra‐CNS metastases, thus compelling a dedicated assessment. Based on these considerations, effective liquid biopsy tools for CNS tumours are highly warranted and a significant amount of data have been accrued over the last few years. These results have shown that liquid biopsy can provide clinically meaningful information about both primary and metastatic CNS tumours, but specific considerations must be taken into account, for example, when choosing the source of liquid biopsy. Nevertheless, this approach is especially attractive for CNS tumours, as repeated tumour sampling is not feasible. The aim of our review was to thoroughly report the state‐of‐the‐art regarding the opportunities and challenges posed by liquid biopsy in both primary and secondary CNS tumours.