Combined DNA Analysis from Stool and Blood Samples Improves Tumor Tracking and Assessment of Clonal Heterogeneity in Localized Rectal Cancer Patients.

Objectives: In this study, stool samples were evaluated for tumor mutation analysis via a targeted next generation sequencing (NGS) approach in a small patient cohort suffering from localized rectal cancer. Introduction: Colorectal cancer (CRC) causes the second highest cancer-related death rate worldwide. Thus, improvements in disease assessment and monitoring that may facilitate treatment allocation and allow organ-sparing "watch-and-wait" treatment strategies are highly relevant for a significant number of CRC patients. Methods: Stool-based results were compared with mutation profiles derived from liquid biopsies and the gold standard procedure of tumor biopsy from the same patients. A workflow was established that enables the detection of de-novo tumor mutations in stool samples of CRC patients via ultra-sensitive cell-free tumor DNA target enrichment. Results: Notably, only a 19% overall concordance was found in mutational profiles across the compared sample specimens of stool, tumor, and liquid biopsies. Conclusion: Based on these results, the analysis of stool and liquid biopsy samples can provide important additional information on tumor heterogeneity and potentially on the assessment of minimal residual disease and clonal tumor evolution.

Assessment of Untargeted Metabolomics by Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry to Define Breast Cancer Liquid Biopsy-Based Biomarkers in Plasma Samples.

An early diagnosis of cancer is fundamental not only in regard to reducing its mortality rate but also in terms of counteracting the progression of the tumor in the initial stages. Breast cancer (BC) is the most common tumor pathology in women and the second deathliest cancer worldwide, although its survival rate is increasing thanks to improvements in screening programs. However, the most common techniques to detect a breast tumor tend to be time-consuming, unspecific or invasive. Herein, the use of untargeted hydrophilic interaction liquid chromatography-mass spectrometry analysis appears as an analytical technique with potential use for the early detection of biomarkers in liquid biopsies from BC patients. In this research, plasma samples from 134 BC patients were compared with 136 from healthy controls (HC), and multivariate statistical analyses showed a clear separation between four BC phenotypes (LA, LB, HER2, and TN) and the HC group. As a result, we identified two candidate biomarkers that discriminated between the groups under study with a VIP > 1 and an AUC of 0.958. Thus, targeting the specific aberrant metabolic pathways in future studies may allow for better molecular stratification or early detection of the disease.

Détection de la mutation T790M de l'exon 20 du gène EGFR dans le cancer du poumon résistant aux inhibiteurs de l'EGFR sur ADN tumoral circulant (biopsie liquide) / Circulating tumour DNA (liquid biopsy)-based EGFR exon 20 T790M mutation detection in advanced non-small cell lung cancer after previous EGFR-directed therapy

DEMANDEUR: Institut universitaire de cardiologie et de pneumologie de Québec Université Laval. OBJECTIF DE L'ANALYSE: L'analyse proposée vise à effectuer la détection de la mutation T790M du gène EGFR (EGFR-T790M) responsable de la résistance aux inhibiteurs de tyrosine kinase (ITK) à partir de l'ADN tumoral circulant d'un patient atteint d'un cancer du poumon non à petites cellules (CPNPC). CONTEXTE DE LA DEMANDE: Approximativement 60 % des patients atteints d'un CPNPC et traités avec un ITK de 1re ou de 2e génération vont développer la mutation de résistance EGFR-T790M et avoir besoin d'un changement thérapeutique. Bien qu'une analyse permettant notamment de détecter cette mutation à partir d'une biopsie tissulaire soit consignée dans le Répertoire québécois et système de mesure des procédures de biologie médicale (ci-après nommé « Répertoire ¼), la présente demande vise à détecter la mutation EGFR-T790M à partir d'échantillons de biopsie liquide, une autre méthode de prélèvement qui permet au patient d'éviter la biopsie tissulaire, les risques qui y sont associés et d'obtenir un changement thérapeutique plus rapidement. NOMBRE D'ANALYSES PRÉVUES: Le demandeur avait originalement prévu que, annuellement, entre 160 et 250 tests seraient nécessaires pour servir la population locale et 2 000 pour l'ensemble du Québec. Or, l'inscription de l'osimertinib en 1re intention de traitement des CPNPC avec mutations activatrices de l'EGFR entraîne une réduction importante du nombre de patients dont l'état nécessitera éventuellement une détection de la mutation EGFRT790M par biopsie liquide selon l'utilisation prévue par le demandeur. Les données de la RAMQ ont révélé que quelques patients continuent de recevoir des ITK de 1re et de 2e génération et sont susceptibles de tirer avantage de cette analyse. MÉTHODOLOGIE: La démarche d'évaluation comprend une revue rapide de la littérature scientifique et grise de même que des consultations menées auprès d'experts et d'autres parties prenantes. Un rapport d'évaluation portant sur la pertinence de recourir à la biopsie liquide dans le même contexte que la présente demande a été publié par Health Quality Ontario (HQO) en mars 2020. Ce rapport comporte une évaluation de la performance diagnostique, de l'utilité clinique, de la sécurité, de l'efficience et de l'impact budgétaire de même qu'une analyse des préférences et des valeurs selon la perspective du patient. Le rapport de HQO a été jugé de bonne qualité méthodologique et constitue la principale source de données de la présente évaluation. L'ensemble des données scientifiques, contextuelles et expérientielles a été interprété et apprécié à l'aide d'une grille-synthèse pour guider le processus de délibération du Comité scientifique des analyses de biologie médicale (CSABM). DONNÉES PUBLIÉES: Performance diagnostique: Selon le rapport d'évaluation de Health Quality Ontario publié en 2020, la concordance des résultats d'une détection de la mutation EGFRT790M entre la biopsie liquide et la biopsie tissulaire varierait de 50 % à 96 %. La sensibilité et la spécificité, la valeur prédictive négative (VPN) et la valeur prédictive positive (VPP) sont respectivement de 68 % [46 % - 88 %], 86 % [62 % - 99 %], 61 % et 89 %. L'utilisation de la droplet digital PCR (ddPCR) augmenterait la fréquence de détection positive de la mutation EGFR-T790M par biopsie liquide. Utilité clinique: HQO n'a repéré aucune donnée sur l'utilisation de la biopsie liquide comme méthode de triage (biopsie liquide + biopsie tissulaire) comparativement à la biopsie tissulaire seule. Toutefois, les études consultées soulignent qu'une fraction des porteurs de la mutation EGFR-T790M (17,8 %) sont identifiés grâce à l'utilisation de la biopsie liquide pour trier les patients résistants aux inhibiteurs des ITK de 1re et 2e génération et pour lesquels la biopsie tissulaire est évitée. En cas de détection de la mutation EGFR-T790M, l'impact sur la prise en charge demeurerait le même quelle que soit la méthode employée car, lorsque la biopsie est positive, le traitement est amorcé. Un gain de survie sans progression de 9,7 mois a été rapporté pour ces deux méthodes. PERSPECTIVE DES PATIENTS: Selon le rapport de HQO, les patients percevraient la biopsie liquide comme une approche plus rapide et plus pratique du fait qu'ils n'auraient pas à attendre plusieurs semaines pour obtenir un rendezvous pour subir une biopsie tissulaire qui pourrait nécessiter un déplacement vers un centre spécialisé plus éloigné. Ils auraient aussi exprimé de la peur et même de la panique relativement au processus de prélèvement de l'échantillon tissulaire qui nécessite une aiguille de gros calibre. POSITIONS ET ORIENTATIONS D'ORGANISMES D'INTÉRÊ: Le National Comprehensive Cancer Network (NCCN) et plusieurs comités d'experts ont recommandé l'utilisation de la biopsie liquide chez un patient qui est médicalement incapable de supporter l'échantillonnage effractif que requiert la biopsie tissulaire. L'ensemble des sociétés savantes recommandent l'utilisation de la biopsie tissulaire lorsque le résultat de la biopsie liquide est négatif. Toutefois, ils soulignent que la biopsie liquide ne devrait pas remplacer la biopsie tissulaire. En revanche, un résultat positif de la biopsie liquide devrait obtenir la même attention qu'un résultat positif de la biopsie tissulaire. ÉVALUATION ÉCONOMIQUE: La biopsie liquide comme méthode de triage est moins coûteuse et plus efficace que la biopsie tissulaire pour détecter la mutation EGFRT790M. Toutefois, lorsqu'on tient compte des avantages cliniques et des coûts liés à l'usage des traitements subséquents, le ratio coûtutilité incrémental est élevé, en raison notamment de l'inefficience de l'osimertinib. Depuis l'inscription aux listes des médicaments de l'osimertinib comme traitement de 1re intention, la population admissible à une biopsie liquide décroît continuellement. Le nombre actuel de personnes qui recevraient des ITK de 1re et 2e génération a été estimé à 32 selon les données de la RAMQ et à 40 en incluant les patients couverts par une assurance privée. Ces analyses estiment les économies de laboratoire à 460 $, mais elles anticipent que 5 patients supplémentaires pourraient recevoir l'osimertinib pour un impact budgétaire net d'environ 537 000 $ au cours des trois prochaines années. POSITION DES EXPERTS CONSULTÉS: Les experts consultés sont favorables à l'utilisation de la biopsie liquide dans le contexte décrit par le demandeur. Ils n'ont pas soulevé d'enjeu en particulier en dehors de la pertinence restreinte de cette analyse étant donné l'inscription récente du traitement osimertinib en 1re intention. L'émergence rapide de nombreuses indications de la biopsie liquide serait imminente et constituerait une révolution technologique et oncologique. ENJEUX PARTICULIERS: Au Québec, l'analyse est principalement employée en contexte de recherche. Des enjeux éthiques et cliniques liés à la biopsie liquide tels que la présence de faux négatifs en raison d'une sensibilité plus faible et la nécessité d'évaluer la survie globale des patients atteints de CPNPC en appliquant cette technique ont également été rapportés dans la littérature. RECOMMANDATION: Suivant une délibération par les membres du CSABM sur l'ensemble de la preuve, y compris la perspective des experts consultés, l'INESSS recommande d'introduire la détection de la mutation EGFR-T790M sur ADN tumoral circulant au Répertoire.

REQUESTER: Institut universitaire de cardiologie et de pneumologie de QuébecUniversité Laval. PURPOSE OF TEST: The test is designed to detect the EGFR gene T790M mutation (EGFRT790M), which is responsible for resistance to tyrosine kinase inhibitors (TKIs), in circulating tumour DNA in non-small cell lung cancer (NSCLC) patients. BACKGROUND TO THE REQUEST: Approximately 60% of NSCLC patients treated with a 1st- or 2ndgeneration TKI will develop the EGFR-T790M resistance mutation and require a change in therapy. Although a test used to detect this mutation from a tissue biopsy is listed in the Répertoire québécois et système de mesure des procédures de biologie médicale (hereinafter the "Répertoire"), the present request concerns the detection of the EGFRT790M mutation in samples obtained by liquid biopsy, an alternative specimen collection method that enables the patient to avoid a tissue biopsy and the associated risks, and to obtain a change in therapy more quickly. EXPECTED NUMBER OF TESTS: The requester had originally anticipated that between 160 and 250 tests would be required annually to serve the local population and 2000 for all of Québec. However, the listing of osimertinib as a 1st-line therapy for NSCLC with EGFR-activating mutations is resulting in a significant reduction in the number of patients who will possibly require EGFRT790M mutation testing by liquid biopsy, based on the use anticipated by the requester. RAMQ data show that a few patients continue to receive 1st- or 2nd-generation TKIs and are likely to benefit from this test. METHODOLOGY: The assessment process included a rapid review of the scientific and grey literature, and consultations with experts and other stakeholders. An assessment report on the relevance of using liquid biopsy in the same context as this request was published by Health Quality Ontario (HQO) in March 2020. The report includes an assessment of its diagnostic performance, clinical utility, safety, cost-effectiveness and budget impact, and an assessment of patient preferences and values. The HQO report was deemed to be of good methodological quality and is the main source of data for the present assessment. All the scientific, contextual and experiential data were interpreted and assessed using a synthesis grid to guide the Comité scientifique des analyses de biologie médicale (CSABM)'s deliberation process. PUBLISHED DATA: Diagnostic performance: According to the Health Quality Ontario assessment report published in 2020, the concordance rate for EGFR-T790M mutation detection results between liquid biopsy and tissue biopsy ranges from 50% to 96%. The sensitivity and specificity, negative predictive value (NPV) and positive predictive value (PPV) are 68% [46%-88%], 86% [62%-99%], 61% and 89%, respectively. The use of droplet digital PCR (ddPCR) appears to increase the rate of positive detection of the EGFR-T790M mutation by liquid biopsy. Clinical utility: HQO did not find any data on the use of liquid biopsy as a triage test (liquid biopsy + tissue biopsy) versus tissue biopsy alone. However, the studies examined note that a proportion of EGFR-T790M mutation carriers (17.8%) are identified through the use of liquid biopsy to detect 1st- or 2nd-generation TKI-resistant patients, who thus avoid a tissue biopsy. If the EGFR-T790M mutation is detected, the impact on management would remain the same, regardless of the method used, because when the biopsy is positive, treatment is initiated. A 9.7-month gain in progression-free survival was reported for both methods. PATIENT PERSPECTIVE: According to the HQO report, patients perceived liquid biopsy as a faster and more convenient approach because they would not have to wait several weeks to get an appointment for a tissue biopsy, which could require a trip to a specialized centre that might be further away. They also expressed fear and even panic about the tissue sample collection procedure, in which a large-gauge needle is used. POSITIONS AND PERSPECTIVES OF ORGANIZATIONS OF INTEREST: The National Comprehensive Cancer Network (NCCN) and a number of expert panels have recommended the use of liquid biopsy in patients who are medically unable to tolerate the invasive specimen collection involved in a tissue biopsy. All the learned societies recommend the use of tissue biopsy when the liquid biopsy result is negative. However, they stress that liquid biopsy should not replace tissue biopsy. In any event, a positive liquid biopsy result should be given the same attention as a positive tissue biopsy result. ECONOMIC EVALUATION: Liquid biopsy as a triage test is less expensive and more effective than tissue biopsy in detecting the EGFR-T790M mutation. However, when the clinical benefits and costs of the subsequent treatments are factored. in, the incremental cost-utility ratio is high, in part because of osimertinib's non-cost-effectiveness. Since the listing of this drug as a 1st-line therapy in the formularies, the liquid biopsy-eligible population has been steadily decreasing. The current number of people receiving 1st- or 2nd-generation TKIs was estimated to be 32, based on RAMQ data, and 40 if patients with private insurance are included. These analyses estimate the laboratory savings at $460, but they anticipate that 5 additional patients could receive osimertinib, for a net budget impact of approximately $537,000 over the next 3 years. POSITION OF THE EXPERTS CONSULTED: The experts consulted support the use of liquid biopsy in the context defined by the requester. They did not raise any specific issues, apart from the limited relevance of this test, given the recent listing of osimertinib as a 1st-line therapy. The rapid emergence of a large number of indications for liquid biopsy appears to be imminent and would constitute a technological and oncologic revolution. SPECIFIC ISSUES: In Québec, liquid biopsy is used primarily in research settings. Ethical and clinical issues associated with this test, such as false negatives due to lower sensitivity and the need to assess overall survival in NSCLC patients in whom this technique is used, have also been reported in the literature. RECOMMENDATION: Based on the deliberation, by the CSABM's members, of all the evidence, including the perspective of the experts consulted, INESSS recommends that circulating tumour DNA-based EGFR-T790M mutation detection be included in the Répertoire

Neoadjuvant therapy for pancreatic cancer: Limitations and advances of response assessment (Review).

Neoadjuvant therapy (NAT) has been widely recommended for managing patients with borderline resectable pancreatic cancer and resectable tumors with high risk factors. Accurate evaluation of the response after NAT is crucial to decide surgery, which then improves the rate of R0 resection and avoids meaningless surgery. The response to NAT is currently evaluated by conventional radiological examination and changes of serum CA19­9 levels. However, these assessments cannot accurately reflect the response to NAT. This article describes the limitations and advances of NAT response evaluation in pancreatic cancer. The values of some traditional imaging techniques, including positron emission tomography, endoscopic ultrasound, and diffusion weighted magnetic resonance imaging, are discussed, as well as novel imaging modalities or biomarkers, such as radiomics, dual energy computed tomography and liquid biopsy.

Salivary metabolomics for the diagnosis of periodontal diseases: a systematic review with methodological quality assessment.

INTRODUCTION: Early diagnosis of periodontitis by means of a rapid, accurate and non-invasive method is highly desirable to reduce the individual and epidemiological burden of this largely prevalent disease. OBJECTIVES: The aims of the present systematic review were to examine potential salivary metabolic biomarkers and pathways associated to periodontitis, and to assess the accuracy of salivary untargeted metabolomics for the diagnosis of periodontal diseases. METHODS: Relevant studies identified from MEDLINE (PubMed), Embase and Scopus databases were systematically examined for analytical protocols, metabolic biomarkers and results from the multivariate analysis (MVA). Pathway analysis was performed using the MetaboAnalyst online software and quality assessment by means of a modified version of the QUADOMICS tool. RESULTS: Twelve studies met the inclusion criteria, with sample sizes ranging from 19 to 130 subjects. Compared to periodontally healthy individuals, valine, phenylalanine, isoleucine, tyrosine and butyrate were found upregulated in periodontitis patients in most studies; while lactate, pyruvate and N-acetyl groups were the most significantly expressed in healthy individuals. Metabolic pathways that resulted dysregulated are mainly implicated in inflammation, oxidative stress, immune activation and bacterial energetic metabolism. The findings from MVA revealed that periodontitis is characterized by a specific metabolic signature in saliva, with coefficients of determination ranging from 0.52 to 0.99. CONCLUSIONS: This systematic review summarizes candidate metabolic biomarkers and pathways related to periodontitis, which may provide opportunities for the validation of diagnostic or predictive models and the discovery of novel targets for monitoring and treating such a disease (PROSPERO CRD42020188482).

Improved Assessment of Response Status in Patients with Pancreatic Cancer Treated with Neoadjuvant Therapy using Somatic Mutations and Liquid Biopsy Analysis.

PURPOSE: To evaluate somatic mutations, circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) in patients with Pancreatic ductal adenocarcinoma (PDAC) with pathologic complete response (pCR) to neoadjuvant therapy (NAT) and find their associations with outcome. EXPERIMENTAL DESIGN: Thirty-six patients with PDAC with pCR were identified from 2009 to 2017. Macrodissection was performed on resected specimens to isolate DNA from 332 regions of interest including fibrosis, normal duct, normal parenchyma, and undefined ductal cells (UDCs). Cell-free DNA and CTCs were also extracted. Next-generation sequencing was used to detect mutations of KRAS, CDKN2A, SMAD4, TP53, GNAS, and BRAF. RESULTS: KRAS mutation was detected in UDCs and fibrosis while SMAD4, TP53, and GNAS were only seen in UDCs. Patients with TP53 mutation showed relatively worse overall survival (HR, 3.596, 95% CI, 0.855-15.130; P = 0.081). Five patients available for CTCs data were all positive for CTCs and seven of 16 patients with pCR were detected with ctDNA at surgery. We proposed a new concept of regression assessment combining genomic analysis of resected specimens and liquid biopsy data for PDAC, namely, molecular complete response (mCR). Three of six patients with mCR recurred as compared with six in 15 non-mCR patients. Seven of 15 non-mCR patients died during follow-up, while there was only one in six patients with mCR. CONCLUSIONS: This study first reports that somatic mutations, CTCs, and ctDNA existed even in patients with PDAC with pCR to NAT, which could possibly predict early recurrence and reduced survival. The current regression evaluation system of PDAC needs to be reassessed at a molecular level.

Assessment of prognostic and diagnostic value of some biomarkers in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is an increasing problem worldwide. Determining a prognosis is important for the management of HCC. AIM: We aimed to investigate the impact of interleukin (IL)-29, galectin-3, leptin, fibronectin and protease-activated receptor-1 on the prognosis and diagnosis of patients with HCC. MATERIALS AND METHODS: 60 HCC patients (75% male) and 20 healthy volunteers (70% male) were enrolled in this prospective study. Serum samples were obtained during the first admission before any adjuvant or metastatic treatments were administered. Serum biomarkers were determined using ELISA kits. RESULTS: All patients had cirrhosis, and the Child - Pugh stages were as follows: 61.5% Child - Pugh A, 35.9% Child - Pugh B and 2.6% Child - Pugh C (61.7% hepatitis B virus, 11.7% hepatitis C virus, 6.7% hepatitis B virus + hepatitis C virus, 11.7% alcoholic and 8.3% cryptogenic). Fifty-three percent of the HCC patients died within a median of 7.5 months. The mean serum level of IL-29 in patients with HCC was higher than that in the control group (32.55 pg/ml vs 11.46 pg/ml, p < 0.015). Galectin-3 levels were significantly higher in the HCC group (6.7 ng/ml vs 1.38 ng/ml, p < 0.001). Fibronectin levels were higher in the control group than in the HCC group (260 635 ng/ml vs 257 353 ng/ml). However, the mean protease-activated receptor-1 and leptin levels were similar between the two groups (p > 0.05). The biomarkers were divided into two groups according to their median level. In the log rank analysis, biomarkers had no effect on survival (p > 0.05). CONCLUSIONS: IL-29 and galectin-3 levels were significantly higher in HCC patients. Although IL-29 and galectin-3 can be used as diagnostic markers for HCC, they had no prognostic value in HCC patients.

Assessment of Pre-Analytical Sample Handling Conditions for Comprehensive Liquid Biopsy Analysis.

Liquid biopsies as a minimally invasive approach have the potential to revolutionize molecular diagnostics. Yet, although protocols for sample handling and the isolation of circulating tumor DNA (ctDNA) are numerous, comprehensive guidelines for diagnostics and research considering all aspects of real-life multicenter clinical studies are currently not available. These include limitations in sample volume, transport, and blood collection tubes. We tested the impact of commonly used (EDTA and heparin) and specialized blood collection tubes and storage conditions on the yield and purity of cell-free DNA for the application in down-stream analysis. Moreover, we evaluated the feasibility of a combined workflow for ctDNA and tumor cell genomic testing and parallel flow cytometric analysis of leukocytes. For genomic analyses, EDTA tubes showed good results if stored for a maximum of 4 hours at room temperature or for up to 24 hours when stored at 4°C. Spike-in experiments revealed that EDTA tubes in combination with density gradient centrifugation allowed the parallel isolation of ctDNA, leukocytes, and low amounts of tumor cells (0.1%) and their immunophenotyping by flow cytometry and down-stream genomic analysis by whole genome sequencing. In conclusion, adhering to time and temperature limits allows the use of routine EDTA blood samples for liquid biopsy analyses. We further provide a workflow enabling the parallel analysis of cell-free and cellular features for disease monitoring and for clonal evolution studies.

Urine as a biological modality for colorectal cancer detection.

Introduction: The increasing incidence of colorectal cancer (CRC) in young adults warrants early and preferably noninvasive diagnostic modalities. Although the current stool-based assays have had good performance indicators for CRC detection, the overall poor uptake remains a challenging issue. However, alternative blood and urine markers are emerging.Areas covered: This paper discusses the various urinary biomarkers available for the detection of CRC. The more commonly encountered drawbacks are the small number of studies and the size of the study population. We discuss the role of microRNA and ProstaglandinE2 in CRC detection. The emergence of new, low-cost technologies, specifically in the detection of volatile organic compounds (VOCs), presents a promising future. We postulate possible mechanisms for the origin of these VOCs in urine and their role in carcinogenesis.Expert opinion: Urinary biomarkers provide an alternative option to the stool-based screening tests. MicroRNA and ProstaglandinE2 have shown utility in CRC detection. Evidence so far suggests that VOCs could also be a potential biomarker for the detection of CRC. In addition to its interaction within the colon lumen, this altered 'VOC signature' might also play a role in carcinogenesis. Low-cost technology may enable such diagnostic methods to be utilized at the point of care.

Diagnostic Power of DNA Methylation Classifiers for Early Detection of Cancer.

DNA methylation-based epigenetic signatures have become valuable cancer biomarkers. We highlight the advantages of liquid biopsy based DNA-methylation profiling for noninvasive diagnosis of early stage cancers and discuss the advanced analytical approaches developed by commercial and academic partnerships.

Direct Assessment of Single-Cell DNA Using Crudely Purified Live Cells: A Proof of Concept for Noninvasive Prenatal Definitive Diagnosis.

Noninvasive testing techniques are often used for fetal diagnosis of genetic abnormalities but are limited by certain characteristics, including noninformative results. Thus, novel methods of noninvasive definitive diagnosis of fetal genetic abnormalities are needed. The aim of this study was to develop a single-cell DNA analysis method with high sensitivity and specificity that enables direct extraction of genetic information from live fetal cells in a crude mixture for simultaneous evaluation. Genomic DNA from circulating fetal CD45-CD14- cells, an extremely rare cell type, extracted from 10-mL samples of maternal peripheral blood, was extracted using a single-cell-based droplet digital (sc-dd) PCR system with a modified amount of polymerase. A hexachloro-6-carboxyfluorescein-labeled RPP30 probe was used as an internal control and a 6-carboxyfluorescein-labeled SRY probe as a target. The results indicated that no droplets generated with samples from pregnant women carrying female fetuses were positive for both probe signals, whereas droplets prepared with samples from pregnant women carrying male fetuses were positive for both probe signals. The latter was considered a direct assessment of genetic information from single circulating male fetal cells. Thus, the modified sc-ddPCR system allows the detection of genetic information from rare target cells in a crudely purified cell population. This research also serves as a proof of concept for noninvasive prenatal definitive diagnosis.

Preanalytical Variables for the Genomic Assessment of the Cellular and Acellular Fractions of the Liquid Biopsy in a Cohort of Breast Cancer Patients.

Liquid biopsy allows assessment of multiple analytes, providing temporal information with potential for improving understanding of cancer evolution and clinical management of patients. Although liquid biopsies are intensely investigated for prediction and response monitoring, preanalytic variables are of primary concern for clinical implementation, including categories of collection method and sample storage. Herein, an integrated high-density single-cell assay workflow for morphometric and genomic analysis of the liquid biopsy is used to characterize the effects of preanalytical variation and reproducibility of data from a breast cancer cohort. Following prior work quantifying performance of commonly used blood collection tubes, this study completes the analysis of four time points to assay (24, 48, 72, and 96 hours), demonstrating precision up to 48 hours after collection for assay sensitivity, highly reproducible rare cell enumeration, morphometric characterization, and high efficiency and capacity for single-cell genomic analysis. For the cell-free analysis, both freezing and use of fresh plasma produced similar quality and quantity of cell-free DNA for sequencing. The genomic analysis (copy number variation and single-nucleotide variation) described herein is broadly applicable to liquid biopsy platforms capable of isolating cell-free and cell-based DNA. Morphometric parameters and genomic signatures of individual circulating tumor cells were evaluated in relation to patient clinical response, providing preliminary evidence of clinical validity as a potential biomarker aiding clinical diagnostics or monitoring progression.

Liquid Biopsy Applications in the Clinic.

The global liquid biopsy industry is expected to exceed $US5 billion by 2023. One application of liquid biopsy technology is the diagnosis of disease using biomarkers found in blood, urine, stool, saliva, and other biological samples from patients. These biomarkers could be DNA, RNA, protein, or even a cell. More recently, the use of cell-free DNA from plasma is emerging as an important minimally invasive tool for clinical diagnosis. The development of technology has increased the diversity of its application. Here, we discuss how liquid biopsies have been used in the clinic, and how personalized medicine are likely to use liquid biopsies in the near future.

Evolving Significance of Tumor-Normal Sequencing in Cancer Care.

Molecular tests assist at various stages of cancer patient management, including providing diagnosis, predicting prognosis, identifying therapeutic targets, and determining hereditary cancer risk. The current testing paradigm involves germline testing in a subset of patients determined to be at high risk for having a hereditary cancer syndrome, and tumor-only sequencing for treatment decisions in advanced cancer patients. A major limitation of tumor-only sequencing is its inability to distinguish germline versus somatic mutations. Tumor-normal sequencing has emerged as a comprehensive analysis for both hereditary cancer predisposition and somatic profiling. Here, we review recent studies involving tumor-normal sequencing, discuss its benefits in clinical care, challenges for its implementation, and novel insights it has provided regarding tumor biology and germline contribution to cancer.

Assessment of Urinary MicroRNAs by Quantitative Polymerase Chain Reaction in Diabetic Nephropathy Patients.

Urinary microRNAs show promise as noninvasive biomarkers in renal disease. Here, we describe a detailed protocol for the column-based extraction and quantification of miRNAs by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) from urine samples.

Liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients.

BACKGROUND: Hepatitis B is a major public health problem in China. Accurate liver injury assessment is essential for clinical evidence-based treatment. Liver biopsy is considered the gold standard method to stage liver disease, but it is not widely used in resource-limited settings. Therefore, non-invasive liquid biopsy tests are needed. AIM: To assess liver injury in hepatitis B patients using quantified cell free DNA combined with other serum biomarker as a liquid biopsy-based method. METHODS: A cohort of 663 subjects including 313 hepatitis B patients and 350 healthy controls were enrolled. Ultrasound-guided liver biopsies followed by histopathological assessments were performed for the 263 chronic hepatitis B patients to determine the degree of liver injury. Cell-free DNA was quantified using a novel duplex real-time polymerase chain reaction assay. RESULTS: Compared with healthy controls, patients with hepatitis B virus (HBV) infection had significantly higher plasma DNA, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and HBV DNA levels (P < 0.01). Serum ALT, AST, bilirubin, and plasma DNA levels of patients with marked-severe inflammation were significantly higher than those with mild-moderate inflammation (P < 0.01). There was a statistically significant correlation between hepatocyte inflammation severity and serum bilirubin (R 2 = 0.673, P < 0.01) or plasma DNA (R 2 = 0.597, P < 0.01) levels. The areas under the curves of serum ALT, bilirubin, plasma DNA, and their combination to distinguish between patients with mild-moderate and marked-severe inflammation were 0.8059, 0.7910, 0.7921, and 0.9564, respectively. CONCLUSION: The combination of plasma DNA, serum ALT, and bilirubin could be a candidate liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients.

Multilaboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements.

We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.

Electric Field-Induced Release and Measurement Liquid Biopsy for Noninvasive Early Lung Cancer Assessment.

Previously, we detected circulating tumor DNA that contained two EGFR mutations (p.L858R and exon19 del) in plasma of patients with late-stage non-small-cell lung carcinoma (NSCLC) using the electric field-induced release and measurement (EFIRM) platform. Our aim was to determine whether EFIRM technology can detect these mutations in patients with early-stage NSCLC. Prospectively, 248 patients with radiographically determined pulmonary nodules were recruited. Plasma was collected before biopsy and histologic examination of the nodule. Inclusion criteria were histologic diagnosis of benign nodule (control) and stage I or II adenocarcinoma harboring either p.L858R or exon19 delEGFR mutations. Plasma samples were available from 44 patients: 23 with biopsy-proven benign pulmonary nodules and 21 with stage I or II adenocarcinoma (12 p.L858R and 9 exon19 delEGFR variants). Samples were analyzed for the EGFR mutations using the EFIRM platform. Assay sensitivity was 92% for p.L858R (11 of 12 samples positive) and 77% for exon19 del (7 of 9 samples positive). Specificity was 91% with two false-positive results in 23 patients with EGFR-positive nodules and 95% for the entire 44-patient series. Concordance was 100% with identical mutations discovered in plasma and nodule biopsy. The EFIRM platform is able to noninvasively detect two EGFR mutations in individuals with early-stage NSCLC.

Simple and cost-effective laboratory methods to evaluate and validate cell-free DNA isolation.

OBJECTIVE: In the present study, we investigated different simple and cost effective methods to evaluate and validate cell free DNA (cfDNA) isolation. The ability of the QIAamp DNA Blood Mini Kit method to extract cfDNA was assessed by several approaches, including purification of endogenous cfDNA and exogenous spike-in control material, prior to plasma extraction, and followed by quantitative-PCR. RESULTS: Using QIAamp DNA Blood Mini kit, nearly 27% (380 bp) to 35% (173 bp) cfDNA was recovered with a higher recovery of smaller size cfDNA (173 bp) in comparison to larger ones (380 bp). These simple laboratory methods can be used to assess the efficiency of any cfDNA isolation method.