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Transarterial chemoembolization (TACE) is used to treat patients with unresectable hepatocellular carcinoma (HCC). We evaluated the clinical impact of a-fetoprotein (AFP) and circulating cell-free and tumor DNA (cfDNA and ctDNA) changes around the TACE procedure. Our prospective monocentric study enrolled consecutive patients treated with TACE, with samples collected at baseline (D - 1), Day 2 (D + 2) and 1 month (M + 1) after TACE. cfDNA was quantified by the fluorometric method, and ctDNA was quantified by digital polymerase chain reaction designed for two hotspot TERT mutations. Computerized tomography scans or magnetic resonance imaging were performed at M + 1 every 3 months following TACE and independently reviewed. The objective was to identify thresholds of cfDNA, ctDNA and AFP changes associated with progressive disease (PD) using receiver operating characteristic curves. Thirty-eight patients were included from March 2018 to March 2019. All markers significantly increased from D - 1 to D + 2 (P .005), and cfDNA and ctDNA significantly decreased from D + 2 to M + 1 (P .0001). The analysis of changes from D - 1 to M + 1 identified thresholds at +31.4% for cfDNA and 0% for ctDNA that were significantly associated with PD at M + 1 (44.4% [+31.4%] vs 3.8% [≤+31.4%] and 50.0% [0%] vs 5.0% [≤0%], respectively). No significant threshold was identified for AFP. Using a score combining cfDNA and ctDNA, the patients were classified into high- or low-risk PD groups at M + 1, with PD rates of 80.0% vs 4.3% (P = .001) and median progression-free survival times of 1.3 vs 10.3 months (P = .002). Our study suggests that cfDNA and ctDNA increases around the TACE procedure and are associated with therapeutic failure.

BACKGROUND: We previously reported that CEA kinetics are a marker of progressive disease (PD) in metastatic colorectal cancer (mCRC). This study was specifically designed to confirm CEA kinetics for predicting PD and to evaluate CA19-9, cell-free DNA (cfDNA), circulating tumour DNA (ctDNA) and circulating tumour cell (CTC) kinetics. METHODS: Patients starting a chemotherapy (CT) with pre-treatment CEA > 5 ng/mL and/or CA19.9 > 30 UI/mL were prospectively included. Samples were collected from baseline to cycle 4 for CEA and CA19-9 and at baseline and the sixth week for other markers. CEA kinetics were calculated from the first to the third or fourth CT cycle. RESULTS: A total of 192 mCRC patients were included. CEA kinetics based on the previously identified >0.05 threshold was significantly associated with PD (p

Background: The majority of locally advanced cervical cancers (LaCC) are causally related to HPV. We sought to investigate the utility of an ultra-sensitive HPV-DNA next generation sequencing (NGS) assay—panHPV-detect—in LaCC treated with chemoradiotherapy, as a marker of treatment response and persistent disease. Method: Serial blood samples were collected from 22 patients with LaCC before, during and after chemoradiation. The presence of circulating HPV-DNA was correlated with clinical and radiological outcomes. Results: The panHPV-detect test demonstrated a sensitivity and specificity of 88% (95% CI-70–99%) and 100% (95% CI-30–100%), respectively, and correctly identified the HPV-subtype (16, 18, 45, 58). After a median follow up of 16 months, and three relapses all had detectable cHPV-DNA at 3 months post-CRT despite complete response on imaging. Another four patients with radiological partial or equivocal response and undetectable cHPV-DNA at the 3-month time point did not go on to develop relapse. All patients with radiological CR and undetectable cHPV-DNA at 3-months remained disease free. Conclusions: These results demonstrate that the panHPV-detect test shows high sensitivity and specificity for detecting cHPV-DNA in plasma. The test has potential applications in assessment of the response to CRT and in monitoring for relapse, and these initial findings warrant validation in a larger cohort.

The effective management of biliary tract cancers (BTCs) has been hampered by limited options for systemic therapy. In recent years, the focus on precision medicine has made technologies such as next-generation sequencing (NGS) accessible to clinicians to identify targetable mutations in BTCs in tumor tissue (primarily) as well as blood, and to treat them with targeted therapies when possible. It has also expanded our understanding of functional pathways associated with genetic alterations and opened doors for identifying novel targets for treatment. Recent advances in the precision medicine approach allowed us to identify new molecular markers in BTCs, such as epigenetic changes (methylation and histone modification) and non-DNA markers such as messenger RNA, microRNA, and long non-coding RNA. It also made detecting these markers from non-traditional sources such as blood, urine, bile, and cytology (from fine-needle aspiration and biliary brushings) possible. As these tests become more accessible, we can see the integration of different molecular markers from all available sources to aid physicians in diagnosing, assessing prognosis, predicting tumor response, and screening BTCs. Currently, there are a handful of approved targeted therapies and only one class of immunotherapy agents (immune checkpoint inhibitors or ICIs) to treat BTCs. Early success with new targets, vascular endothelial growth factor receptor (VEGFR), HER2, protein kinase receptor, and Dickkopf-1 (DKK1); new drugs for known targets, fibroblast growth factor receptors (FGFRs) such as futabatinib, derazantinib, and erdafitinib; and ICIs such as durvalumab and tremelimumab is encouraging. Novel immunotherapy agents such as bispecific antibodies (bintrafusp alfa), arginase inhibitors, vaccines, and cellular therapy (chimeric antigen receptor—T cell or CAR-T, natural killer cells, tumor-infiltrating lymphocytes) have the potential to improve outcomes of BTCs in the coming years.

While liquid biopsy is still relatively a new concept, the advent of next-generation sequencing (NGS) technologies has recently generated a revolution in the field and will be the focus of this review. Circulating tumor DNA (ctDNA) derives from tumor cells and provides information about the genetic alterations of tumors. However, ctDNA concentration in plasma can be below the level of detection by conventional methods; therefore, screening for actionable genetic information is challenging. Clinical trials exploring targeted and untargeted sequencing to improve the outcomes of ctDNA detection are showing promising results, having reached a limit of detection as low as 0.001% of ctDNA in a background of normal circulating DNA. Most of the challenges related to the sensitivity of detection of ctDNA have been defeated by dint of NGS-based approaches. Despite all the efforts, these methods are still expensive, time-consuming, and require advanced skills for appropriate interpretation. Nevertheless, the technology is rapidly improving, and the expectations for the implementation of liquid biopsy into the clinical practice in the near future are high.

Cancer is one of the leading causes of death worldwide. This life-threatening disease requires novel strategies for the early detection and therapy response prediction. Circulating DNA was first described 70 years ago. However, only the recent evolution in the PCR-based sequencing techniques allow the minimally invasive molecular profiling of circulating mutant DNA from small-volume “liquid biopsies” such as blood, urine, or saliva. In this article, we aim to summarize the fast-growing evidence for cfDNA and exosomal DNA as minimally invasive diagnostic markers in solid tumors and to highlight their opposing diagnostic advantages and disadvantages.

Background Central nervous system lymphomas (CNSL) is a devastating disease. Currently, a confirmatory biopsy is required prior to treatment. Objective Our investigation aims to prove the feasibility of a minimally-invasive diagnostic approach for the molecular characterization of CNSL. Methods Tissue biopsies from 6 patients with suspected CNSL were analyzed using a 649gene next-generation sequencing (NGS) tumor panel (tumor vs. reference tissue (EDTA-blood)). The individual somatic mutation pattern was used as a basis for the digital PCR analyzing circulating tumor DNA (ctDNA) from plasma and cerebrospinal fluid (CSF) samples, identifying one selected tumor mutation during this first step of the feasibility investigation. Results NGS-analysis of biopsy tissue revealed a specific somatic mutation pattern in all confirmed lymphoma samples (n = 5, NGS-sensitivity 100%) and none in the sample identified as normal brain tissue (NGS-specificity 100%). cfDNA-extraction was dependent on the extraction-kit used and feasible in 3 samples, in all of which somatic mutations were detectable (100%). Analysis of CSF-derived cfDNA was superior to plasma-derived cfDNA and routine microscopic analysis (lymphoma cells: n = 2, 40%). One patient showed a divergent molecular pattern, typical of Burkitt-Lymphoma (HIV+, serologic evidence of EBV-infection). Lumbar puncture was tolerated without complications, whereas biopsy caused 3 hemorrhages. Conclusions Our investigation provides evidence that analysis of cfDNA in central nervous system tumors is feasible using the described protocol. Molecular characterization of CNSL could be achieved by analysis of CSF-derived cfDNA. Knowledge of a tumor’s specific mutation pattern may allow initiation of targeted therapies, treatment surveillance and could lead to minimally-invasive diagnostics in the future. Electronic supplementary material The online version of this article (10.1186/s12885-019-5394-x) contains supplementary material, which is available to authorized users.

Circulating cell‐free DNA (cfDNA) contains circulating tumor DNA (ctDNA), which can be obtained from serial liquid biopsies to enable tumor genome analysis throughout the course of treatment. We investigated cfDNA and mutant ctDNA as potential biomarkers to predict the best outcomes of regorafenib‐treated metastatic colorectal cancer (mCRC) patients. We analyzed longitudinally collected plasma cfDNA of 43 mCRC patients prospectively enrolled in the phase II TEXCAN trial by IntPlex qPCR. Qualitative (KRAS, NRAS, BRAFV600E mutations) and quantitative (total cfDNA concentration, mutant ctDNA concentration, mutant ctDNA fraction) parameters were correlated with overall survival (OS) and progression‐free survival (PFS). When examined as classes or continuous variables, the concentrations of total cfDNA, mutant ctDNA, and, partly, mutant ctDNA fraction prior to regorafenib treatment correlated with OS. Patients with baseline cfDNA > 26 ng·mL−1 had shorter OS than those with cfDNA value below this threshold (4.0 vs 6.9 months; log‐rank P = 0.0366). Patients with baseline mutant ctDNA > 2 ng·mL−1 had shorter OS than those with mutant ctDNA below this threshold (log‐rank P = 0.0154). We show that pretreatment cfDNA and mutant ctDNA levels may identify mCRC patients that may benefit from regorafenib treatment., Circulating cell‐free DNA (cfdNA) was extracted from longitudinal blood collection of 43 mCRC patients treated by regorafenib. Qualitative (RAS, BRAFV600E mutations) and quantitative (cfDNA concentration, mutant circulating tumor DNA (ctDNA) concentration, mutant ctDNA fraction) parameters were correlated with survival. We show that pretreatment cfDNA and mutant ctDNA levels may identify mCRC patients that may benefit from regorafenib treatment.

Cholangiocarcinoma (CCA) are a heterogeneous group of tumors in terms of aetiology, natural history, morphological subtypes, molecular alterations and management, but all sharing complex diagnosis, management, and poor prognosis. Several mutated genes and epigenetic changes have been detected in CCA, with the potential to identify diagnostic and prognostic biomarkers and therapeutic targets. Accessing tumoral components and genetic material is therefore crucial for the diagnosis, management and selection of targeted therapies; but sampling tumor tissue, when possible, is often risky and difficult to be repeated at different time points. Liquid biopsy (LB) represents a way to overcome these issues and comprises a diverse group of methodologies centering around detection of tumor biomarkers from fluid samples. Compared to the traditional tissue sampling methods LB is less invasive and can be serially repeated, allowing a real-time monitoring of the tumor genetic profile or the response to therapy. In this review, we analysis the current evidence on the possible roles of LB (circulating DNA, circulating RNA, exosomes, cytokines) in the diagnosis and management of patients affected by CCA.

Simple Summary We recently came across an intriguing protein while screening for tumour-specific apoptosis inducers. It is known as the transmembrane protein with an EGF-like and two Follistatin-like domains 2 (TMEFF2). The gene was identified and characterized by five different groups almost simultaneously around 2000. Physiological function of TMEFF2 is elusive; however, the protein is reported to be involved in wide-ranging physiological and pathological functions including neuroprotection in Alzheimer’s diseases, interferon induction and one-carbon metabolism. Moreover, the TMEFF2 promoter and 5′-upstream regions harbour a CpG island which is progressively methylated upon progression in a wide variety of cancers. Numerous primary publications suggest the methylation of TMEFF2 as a prognostic and even diagnostic marker in different cancers. The primary literature regarding TMEFF2 is distributed far and wide, and despite having more than 150 primary publications mentioning TMEFF2 (or its aliases) in the title or abstract on PubMed, a comprehensive literature review is not available. We believe the reason behind this is firstly the sheer diversity of subjects of these publications and secondly the numerous primary publications reporting contradictory information about TMEFF2, especially when it comes to its oncogenic versus the onco-suppressive roles. The interest in TMEFF2 is growing again; PubMed returning at least 60 publications mentioning TMEFF2 (or its aliases) within the last year. We have made a laborious effort and written a comprehensive review article on TMEFF2 where we have not only compiled and contextualized the information regarding it but also critically analysed the information in the major primary publications. In addition, we have proposed some answers to the apparent TMEFF2 disagreements on its function. This information could serve as a valuable tool for readers not only about TMEFF2 but also on the dual role of type-I transmembrane proteoglycans (harbouring Follistatin-like domains) in oncogenesis and onco-suppression. Abstract Transmembrane protein with an EGF-like and two Follistatin-like domains 2 (TMEFF2) is a 374-residue long type-I transmembrane proteoglycan which is proteolytically shed from the cell surface. The protein is involved in a range of functions including metabolism, neuroprotection, apoptosis, embryonic development, onco-suppression and endocrine function. TMEFF2 is methylated in numerous cancers, and an inverse correlation with the stage, response to therapy and survival outcome has been observed. Moreover, TMEFF2 methylation increases with breast, colon and gastric cancer progression. TMEFF2 is methylated early during oncogenesis in breast and colorectal cancer, and the detection of methylated free-circulating TMEFF2 DNA has been suggested as a potential diagnostic tool. The TMEFF2 downregulation signature equals and sometimes outperforms the Gleason and pathological scores in prostate cancer. TMEFF2 is downregulated in glioma and cotricotropinomas, and it impairs the production of adrenocorticotropic hormone in glioma cells. Interestingly, through binding the amyloid β protein, its precursor and derivatives, TMEFF2 provides neuroprotection in Alzheimer’s disease. Despite undergoing extensive investigation over the last two decades, the primary literature regarding TMEFF2 is incoherent and offers conflicting information, in particular, the oncogenic vs. onco-suppressive role of TMEFF2 in prostate cancer. For the first time, we have compiled, contextualised and critically analysed the vast body of TMEFF2-related literature and answered the apparent discrepancies regarding its function, tissue expression, intracellular localization and oncogenic vs. onco-suppressive role.

Plasma-based tumor mutational profiling is arising as a reliable approach to detect primary and therapy-induced resistance mutations required for accurate treatment decision making. Here, we compared the FDA-approved Cobas&reg, EGFR Mutation Test v2 with the UltraSEEK&trade, Lung Panel on the MassARRAY&reg, System on detection of EGFR mutations, accompanied with preanalytical sample assessment using the novel Liquid IQ&reg, Panel. 137 cancer patient-derived cell-free plasma samples were analyzed with the Cobas&reg, and UltraSEEK&trade, tests. Liquid IQ&reg, analysis was initially validated (n = 84) and used to determine ccfDNA input for all samples. Subsequently, Liquid IQ&reg, results were applied to harmonize ccfDNA input for the Cobas&reg, tests for 63 NSCLC patients. The overall concordance between the Cobas&reg, tests was 86%. The Cobas&reg, test detected more EGFR exon19 deletions and L858R mutations, while the UltraSEEK&trade, test detected more T790M mutations. A 100% concordance in both the clinical (n = 137) and harmonized (n = 63) cohorts was observed when &gt, 10 ng of ccfDNA was used as determined by the Liquid IQ&reg, Panel. The Cobas&reg, tests showed similar sensitivity in EGFR mutation detection, particularly when ccfDNA input was sufficient. It is recommended to preanalytically determine the ccfDNA concentration accurately to ensure sufficient input for reliable interpretation and treatment decision making.

Simple Summary Prostate cancer is a major cause of cancer-related death in men worldwide. There is an urgent clinical need for improved prognostic biomarkers to better predict the likely outcome and course of the disease and thus inform the clinical management of these patients. Currently, clinically recognised prognostic markers lack sensitivity and specificity in distinguishing aggressive from indolent disease, particularly in patients with localised, intermediate grade prostate cancer. Thus, there is major interest in identifying new molecular biomarkers to complement existing standard clinicopathological markers. DNA methylation is a frequent alteration in the cancer genome and offers potential as a reliable and robust biomarker. In this review, we provide a comprehensive overview of the current state of DNA methylation biomarker studies in prostate cancer prognosis. We highlight advances in this field that have enabled the discovery of novel prognostic genes and discuss the potential of methylation biomarkers for noninvasive liquid-biopsy testing. Abstract There is a major clinical need for accurate biomarkers for prostate cancer prognosis, to better inform treatment strategies and disease monitoring. Current clinically recognised prognostic factors, including prostate-specific antigen (PSA) levels, lack sensitivity and specificity in distinguishing aggressive from indolent disease, particularly in patients with localised intermediate grade prostate cancer. There has therefore been a major focus on identifying molecular biomarkers that can add prognostic value to existing markers, including investigation of DNA methylation, which has a known role in tumorigenesis. In this review, we will provide a comprehensive overview of the current state of DNA methylation biomarker studies in prostate cancer prognosis, and highlight the advances that have been made in this field. We cover the numerous studies into well-established candidate genes, and explore the technological transition that has enabled hypothesis-free genome-wide studies and the subsequent discovery of novel prognostic genes.

International audience; Introduction: Breast cancer is the most common cancer in women worldwide. Despite the development of targeted therapies that have significantly improved survival, effective breast cancer evaluation is still challenging due to the complexity of this disease. Liquid biopsy might allow the noninvasive real-time monitoring of the tumor course and response to therapy.Areas covered: This review summarizes the latest advances on the various circulating analytes used as liquid biopsy (circulating tumor cells and tumor DNA, and more recently extracellular vesicles) for breast cancer work-up. Several studies have shown that in breast cancer, liquid biopsy can be used to predict disease progression or relapse and to monitor the treatment response. Moreover, circulating analytes are more easily accessible than tissue biopsies and might represent a powerful source of specific knowledge.Expert opinion: The new evidence coming from the increasing number of clinical trials, and the continuous improvements in the technologies to isolate these tumor-derived analytes should strengthen the role of liquid biopsy in the clinic for personalized medicine of breast cancer.

The identification of the biological determinants that shape the response of tumors to medical therapies offers perspectives for better patient stratification and therapeutic targeting. Here, we discuss how genomics could help to improve the surgical treatment of head and neck squamous cell carcinoma (HNSCC). We examine the potential use of genomic analyses for: i) refining and standardizing the indications for surgery, ii) the choice of surgical procedure, and iii) the follow-up of patients with resected tumors. We highlight the studies that used genomics to explore the contribution of tumor biology to the outcome of surgery. We discuss the important developments that are challenging current surgical practice in HNSCC, such as neoadjuvant immunotherapy and the analysis of circulating DNA. Genomic analyses provide practical tools that could help improve the pathological diagnosis and staging of HNSCC, and increase the appreciation of the importance of tumor biology in the outcome of surgery. Identification of biomarkers will likely contribute to a move toward precision surgery of HNSCC, i.e. the personalization of surgical practice based on tumor biology.

Background Endocrine therapy is recommended as a first-line treatment for hormone receptor-positive metastatic breast cancer (HR+MBC) patients. No biomarker has been validated to predict tumor progression in that setting. We aimed to prospectively compare the risk of early progression according to circulating ESR1 mutations, CA-15.3, and circulating cell-free DNA in MBC patients treated with a first-line aromatase inhibitor (AI). Methods Patients with MBC treated with a first-line AI were prospectively included. Circulating biomarker assessment was performed every 3 months. The primary objective was to determine the risk of progression or death at the next follow-up visit (after 3 months) in case of circulating ESR1 mutation detection among patients treated with a first-line AI for HR+MBC. Results Overall, 103 patients were included, and 70 (68%) had progressive disease (PD). Circulating ESR1 mutations were detected in 22/70 patients with PD and in 0/33 patients without progression (p ESR1-mutated patients, 18/22 had a detectable mutation prior to progression, with a median delay of 110 days from first detection to PD. The detection of circulating ESR1 mutations was associated with a 4.9-fold (95% CI 3.0–8.0) increase in the risk of PD at 3 months. Using a threshold value of 25% or 100%, a CA-15.3 increase was also correlated with progression (p p = 0.003, respectively). In contrast to ESR1, the CA-15.3 increase occurred concomitantly with PD in most cases, in 27/47 (57%) with a 25% threshold and in 21/25 (84%) with a 100% threshold. Using a threshold value of either 25% or 100%, cfDNA increase was not correlated with progression. Conclusion The emergence of circulating ESR1 mutations is associated with a 4.9-fold increase in the risk of early PD during AI treatment in HR+MBC. Our results also highlighted that tracking circulating ESR1 mutations is more relevant than tracking CA-15.3 or cfDNA increase to predict progression in this setting. Trial registration ClinicalTrials.gov, NCT02473120. Registered 16 June 2015—retrospectively registered after one inclusion (first inclusion 1 June 2015)

Precision medicine in the clinical management of cancer may be achieved through the diagnostic platform called “liquid biopsy”. This method utilizes the detection of biomarkers in blood for prognostic and predictive purposes. One of the latest blood born markers under investigation in the field of liquid biopsy in cancer patients is circulating tumor DNA (ctDNA). ctDNA is released by tumor cells through different mechanisms and can therefore provide information about the genomic make-up of the tumor currently present in the patient. Through longitudinal ctDNA-based liquid biopsies, tumor dynamics may be monitored to predict and assess drug response and/or resistance. However, because ctDNA is highly fragmented and because its concentration can be extremely low in a high background of normal circulating DNA, screening for clinical relevant mutations is challenging. Although significant progress has been made in advancing the detection and analysis of ctDNA in the last few years, the current challenges include standardization and increasing current techniques to single molecule sensitivity in combination with perfect specificity. This review focuses on the potential role of ctDNA in the clinical management of cancer patients, the current technologies that are being employed, and the hurdles that still need to be taken to achieve ctDNA-based liquid biopsy towards precision medicine. Keywords: Liquid biopsy, Circulating tumor DNA (ctDNA), Cell-free DNA (cfDNA)

Background Patients with BC have a higher amount of cell-free circulating DNA (CFCDNA) in the blood and urine than healthy people. We aimed to verify if the Z-Scan method could analyze the concentrations of uDNA (urinary) and pDNA (plasma) in relation to the time of collection during treatment for patients with bladder cancer. Methods Peripheral blood and urine samples were obtained from 30 patients with BC at the time of diagnosis, 45, 90 and 180 days after initiating treatment; 5 μL of k-DNA (k = u or p) was added in 250 μL a solution of 1:1000 Ethidium Bromide dye (EtBr) in water. Continum laser Nd:YVO4, wavelength λ = 532 nm was used. Samples of uDNA and pDNA in water were submitted to the laser with an incident power of 84.5 mW and an exposure time of 30 ms. Results There was a different concentration of pDNA and uDNA during the treatment of patients using both optical techniques. However, the reaction rate of pDNA and uDNA was similar with spectrophotometry, whereas the z-scan technique presented different values. Conclusion Z-scan technique has potential for use in the differentiation of pDNA and uDNA concentrations, which are distinct in patients with BC and healthy people.

// Jessica Garcia 1, 2, 3, 4, ** , Eric Dusserre 1, 3, 4, ** , Valerie Cheynet 3, 5 , Pierre Paul Bringuier 6 , Karen Brengle-Pesce 3, 5 , Anne-Sophie Wozny 1, 7 , Claire Rodriguez-Lafrasse 1, 3, 4, 7 , Gilles Freyer 4, 8, 9 , Marie Brevet 4, 6, 9 , Lea Payen 1, 2, 3, 4, 7, * and Sebastien Couraud 4, 9, 10, * 1 Laboratoire de Biochimie et Biologie Moleculaire, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, 69310, Pierre Benite, France 2 Centre de Recherche en Cancerologie de Lyon, INSERM 1052, CNRS 5286, Universite Claude Bernard Lyon 1, Lyon, 69003, France 3 Laboratoire Commun de Recherche Hospices Civils de Lyon – BioMerieux, Centre Hospitalier Lyon Sud, 69310, Pierre Benite, France 4 Institut de Cancerologie des Hospices Civils de Lyon, CIRculating CANcer Program (CIRCAN), 69002 Lyon, France 5 Medical Diagnostic Discovery Department, BioMerieux, 69290 Craponne, France 6 Service d’Anatomie et de Cytologie Pathologiques, Groupement Hospitalier Est, Hospices Civils de Lyon, 69500, Bron, France 7 Faculte de Pharmacie de Lyon (IPSB), Universite de Lyon1, Lyon, 69008, France 8 Service d’Oncologie Medicale, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Lyon, 69003, France 9 EMR 3738 Ciblage Therapeutique en Oncologie, Faculte de Medecine Lyon Sud, Universite Lyon 1, 69600, Oullins, France 10 Service de Pneumologie Aigue Specialisee et Cancerologie Thoracique, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon, Lyon, 69003, France * Authors share co-senior authorship ** Authors share co-first authorshop Correspondence to: Lea Payen, email: lea.payen-gay@chu-lyon.fr Keywords: lung cancer, EGFR mutation, circulating-free DNA, liquid biopsy, digital PCR Received: December 22, 2016 Accepted: June 26, 2017 Published: September 21, 2017 ABSTRACT Non invasive somatic detection assays are suitable for repetitive tumor characterization or for detecting the appearance of somatic resistance during lung cancer. Molecular diagnosis based on circulating free DNA (cfDNA) offers the opportunity to track the genomic evolution of the tumor, and was chosen to assess the molecular profile of several EGFR alterations, including deletions in exon 19 (delEX19), the L858R substitution on exon 21 and the EGFR resistance mutation T790M on exon 20. Our study aimed at determining optimal pre-analytical conditions and EGFR mutation detection assays for analyzing cfDNA using the picoliter-droplet digital polymerase chain reaction (ddPCR) assay. Within the framework of the CIRCAN project set-up at the Lyon University Hospital, plasma samples were collected to establish a pre-analytical and analytical workflow of cfDNA analysis. We evaluated all of the steps from blood sampling to mutation detection output, including shipping conditions (4H versus 24H in EDTA tubes), the reproducibility of cfDNA extraction, the specificity/sensitivity of ddPCR (using external controls), and the comparison of different PCR assays for the detection of the three most important EGFR hotspots, which highlighted the increased sensitivity of our in-house primers/probes. Hence, we have described a new protocol facilitating the molecular detection of somatic mutations in cancer patients from liquid biopsies, improving their diagnosis and introducing a less traumatic monitoring system during tumor progression.

// Jie Cheng 1, 2 , Katarina Cuk 1, 2 , Jorg Heil 3 , Michael Golatta 3 , Sarah Schott 3 , Christof Sohn 3 , Andreas Schneeweiss 3, 4 , Barbara Burwinkel 1, 2, * and Harald Surowy 1, 2, * 1 Division of Molecular Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, Germany 2 Molecular Biology of Breast Cancer, Department of Gynecology and Obstetrics, University of Heidelberg, Heidelberg, Germany 3 Department of Gynecology and Obstetrics, University Women’s Clinic, Heidelberg, Germany 4 National Center for Tumor Diseases, University of Heidelberg, Heidelberg, Germany * These authors have share the last authorship Correspondence to: Barbara Burwinkel, email: B.Burwinkel@dkfz.de Keywords: breast cancer, recurrence, circulating DNA integrity, biomarker Received: October 11, 2016 Accepted: March 26, 2017 Published: April 24, 2017 ABSTRACT Non-invasive blood-based molecule markers are evaluated as promising biomarkers these days. Here we investigated the potential of cell-free circulating DNA Integrity (cfDI) as blood-based marker for the prediction of recurrence during the follow-up of breast cancer patients within a prospective study cohort. cfDI was determined in plasma of 212 individuals, by measuring ALU and LINE1 repetitive DNA elements using quantitative PCR. A significant decrease of cfDI in recurrent breast cancer patients was observed. The group of patients who had impending recurrence during the follow-up had significant lower cfDI compared to the group of non-recurrent patients (P < 0.001 for ALU and LINE1 cfDI). cfDI could differentiate recurrent breast cancer patients from non-recurrent breast cancer subjects (area under the curve, AUC = 0.710 for ALU and 0.704 for LINE1). Univariate and multivariate analysis confirmed a significant association of recurrence and cfDI. Breast cancer patients with a lower cfDI had a much higher risk to develop recurrence than the patients with a higher cfDI (P = 0.020 for ALU cfDI and P = 0.019 for LINE1 cfDI, respectively). Further we show that cfDI is an independent predictor of breast cancer recurrence. In combination with other molecular markers, cfDI might be a useful biomarker for the prediction for breast cancer recurrence in clinic utility. We propose that cfDI might also be useful for the prediction of recurrence during the follow-up of other cancers.

Cetuximab (CTX) has been used to treat metastatic colorectal cancer (mCRC) with wild-type (wt) RAS and BRAF genes. Meanwhile HER2 amplification reportedly denoted CTX-resistant mCRC tumors. We investigated whether monitoring of HER2 amplification in circulating DNA allowed early detection of mCRC progression and CTX resistance. We analyzed HER2 amplification in circulating DNA at 8-week intervals using ddPCR from 36 patients with RASwt/BRAFwt mCRC, who progressed after CTX treatments between July 2015 and January 2018. Of the 36 patients, 5 (13.9%) exhibited dynamic fluctuations of HER2 amplification in plasma in the course of CTX treatment, of whom 2 were positive for HER2 amplification in matched tumor specimens at baseline (per FISH). All 5 primary sites were left side: 3 rectums and 2 descending colon. HER2 ratio fluctuations in circulating DNA not only reflected changes in tumor volume, but their obvious increases presaged CT-documented progress by an average lead time of 2 months. Interestingly, progression-free survival did not significantly differ between these 5 patients and those without HER2 amplification (HR 1.06, 95% CI 0.40–2.77, P = 0.909). Plasma HER2 amplification detected by ddPCR changed over time and predicted resistance to CTX, by an average lead time of 2 months. Further study is needed to validate our findings.

Background Prostate cancer diagnosis using the PSA test remains controversial because of overdiagnosis and overtreatment of potentially indolent cancers. There remains a need to increase the diagnostic lead time and to target treatment to patients with significant disease. One possible approach to overcome the limitations of PSA is to screen men for the molecular signature of early PCA, monitor the rate of disease progression and target treatment to patients who are likely to benefit from it. Such an approach requires a large panel of markers that define a molecular clock for PCA. We recently developed a panel of 19 markers for the non-invasive detection of PCA from urine DNA. It raised the possibility that additional methylation markers could be successfully analyzed from urine DNA, a prerequisite for increasing the diagnostic lead time and enabling disease monitoring. Methods We developed semi-quantitative polymerase chain reaction assays for 13 additional markers and determined their methylation status in 150 urine DNAs from 94 patients with elevated PSA. Eighty five samples were obtained following DRE and 65 samples were from first void. We combined the data of the 13 new markers with the previously reported 19 markers and calculated the sensitivity, specificity, negative and positive predictive values at every threshold from one to 32 positive markers. Results Using 10of32 positive markers as the threshold to recommend a biopsy yields a sensitivity of 81% (95% CI 0.68–0.93) and 93% (95% CI 0.84–1.02) and a specificity of 76% (95% CI 0.63–0.88) and 77% (95% CI 0.63–0.91) from DRE and FV DNA, respectively. The PPV was 71% and 77% and the NPV was 85% and 93% from DRE and FV, respectively. Conclusions This study shows that large marker panels can be analyzed from urine DNA without loss of sensitivity or specificity. Using 32 markers improved the stratification of patients undergoing screening for PCA particularly for patients below the 10of32 threshold. The results show the utility of larger biomarker panels for PCA diagnosis and suggest that the development of the panels needed to monitor disease progression could be successfully accomplished. Electronic supplementary material The online version of this article (10.1186/s40164-019-0137-x) contains supplementary material, which is available to authorized users.

International audience; Circulating cell-free DNA (cfDNA) is one of the fastest growing and most exciting areas in oncology in recent years. Its potential clinical uses cover now each phase of cancer patient management care (predictive information, detection of the minimal residual disease, early detection of resistance, treatment monitoring, recurrence surveillance, and cancer early detection/screening). This review relates the recent advances in the application of circulating DNA or RNA in oncology building on unpublished or initial findings/work presented at the 10th international symposium on circulating nucleic acids in plasma and serum held in Montpellier from the 20th to the 22nd of September 2017. This year, presenters revealed their latest data and crucial observations notably in relation to (i) the circulating cell-free (cfDNA) structure and implications regarding their optimal detection; (ii) their role in the metastatic or immunological processes; (iii) evaluation of miRNA panels for cancer patient follow up; (iv) the detection of the minimal residual disease; (v) the evaluation of a screening tests for cancer using cfDNA analysis; and (vi) elements of preanalytical guidelines. This work reviews the recent progresses in the field brought to light in the meeting, as well as in the most important reports from the literature, past and present. It proposes a broader picture of the basic research and its potential, and of the implementation and current challenges in the use of circulating nucleic acids in oncology.

The standard diagnostic approach to cancer relies on the analysis of a surgical or biopsy tumor sample from a primary or metastatic site obtained at a given time point. However, due to intratumoral heterogeneity and selection of different subpopulations in metastatic lesions, the analysis of a single tissue specimen will provide only a limited characterization of the molecular makeup of the disease. Monitoring of the molecular tumor characteristics by serial analyses of cell-free, circulating nucleic acids promises to capture the dynamic nature and heterogeneity of the disease and support monitoring and treatment decisions. The analysis of circulating cell-free tumor DNA is continuously evolving with improvements in detection limits and reduction of the operating costs and represents a set of research tools that is poised to enter clinical care. Here we will discuss cell-free circulating DNA, potential clinical applications, and current limitations and challenges.

In recent years, the introduction of new molecular techniques in experimental and clinical settings has allowed researchers and clinicians to propose circulating-tumor DNA (ctDNA) analysis and liquid biopsy as novel promising strategies for the early diagnosis of cancer and for the definition of patients’ prognosis. It was widely demonstrated that through the non-invasive analysis of ctDNA, it is possible to identify and characterize the mutational status of tumors while avoiding invasive diagnostic strategies. Although a number of studies on ctDNA in patients’ samples significantly contributed to the improvement of oncology practice, some investigations generated conflicting data about the diagnostic and prognostic significance of ctDNA. Hence, to highlight the relevant achievements obtained so far in this field, a clearer description of the current methodologies used, as well as the obtained results, are strongly needed. On these bases, this review discusses the most relevant studies on ctDNA analysis in cancer, as well as the future directions and applications of liquid biopsy. In particular, special attention was paid to the early diagnosis of primary cancer, to the diagnosis of tumors with an unknown primary location, and finally to the prognosis of cancer patients. Furthermore, the current limitations of ctDNA-based approaches and possible strategies to overcome these limitations are presented.

The monitoring of therapy during the treatment of cancer patients is currently assessed by the levels of circulating tumor cells or by PET/CT scans. Neither approach has the sensitivity or specificity to be very sure of the efficacy of the treatment. Moreover, PET/CT scans can be both comparatively expensive and produce low levels of radiation for the patient. The advent of the possibility of using circulating DNA released from the tumor permits (1) a possible early marker of the presence of the cancer, (2) an indication of the success of the primary treatment, (3) an indication of the early presence of possible metastasis, (4) a marker of the success of secondary subsequent treatment, (5) determining which patients can benefit from a particular treatment, and (6) offering a prognosis. These aspects will be discussed concerning the application of circulating tumor DNA analysis to the monitoring of cancer patients undergoing therapy.

Liquid biopsy, based on the circulating tumor cells (CTCs) and cell-free nucleic acids has potential applications at multiple points throughout the natural course of cancer, from diagnosis to follow-up. The advantages of doing ctDNA assessment vs. tissue-based genomic profile are the minimal procedural risk, the possibility to serial testing in order to monitor disease-relapse and response to therapy over time and to reduce hospitalization costs during the entire process. However, some critical issues related to ctDNA assays should be taken into consideration. The sensitivity of ctDNA assays depends on the assessment technique and genetic platforms used, on tumor-organ, stage, tumor heterogeneity, tumor clonality. The specificity is usually very high, whereas the concordance with tumor-based biopsy is generally low. In patients with renal cell carcinoma (RCC), qualitative analyses of ctDNA have been performed with interesting results regarding selective pressure from therapy, therapeutic resistance, exceptional treatment response to everolimus and mutations associated with aggressive behavior. Quantitative analyses showed variations of ccfDNA levels at different tumor stage. Compared to CTC assay, ctDNA is more stable than cells and easier to isolate. Splice variants, information at single-cell level and functional assays along with proteomics, transcriptomics and metabolomics studies can be performed only in CTCs.

Circulating tumor DNA (ctDNA) in the plasma or serum of cancer patients provides an opportunity for non-invasive sampling of tumor DNA. This ‘liquid biopsy’ allows for interrogations of DNA such as quantity, chromosomal alterations, sequence mutations and epigenetic changes, and can be used to guide and improve treatment throughout the course of the disease. This tremendous potential for real-time ‘tracking’ in a cancer patient has led to substantial research efforts in the ctDNA field. ctDNA can be distinguished from non-tumor DNA by the presence of tumor-specific mutations and copy number variations, and also by aberrant DNA methylation, with both DNA sequence and methylation changes corresponding to those found in the tumor. Aberrant methylation of specific promoter regions can be a very consistent feature of cancer, in contrast to mutations, which typically occur at a wide range of sites. This consistency makes ctDNA methylation amenable to the design of widely applicable clinical assays. In this review, we examine ctDNA methylation in the context of monitoring disease status, treatment response and determining the prognosis of cancer patients.

BACKGROUND: Radiotherapy is a common treatment for brain metastases. However, it is commonly associated with central nervous system (CNS) toxicity, leading to functional disability. There are no biomarkers capable of detecting preclinical neurotoxicity for the purpose of early detection of radiation-induced CNS damage. Here we explore the utility of cell-free circulating DNA (cfDNA), shed from dying cells, for detection of brain cell death in the context of brain metastases. Using comparative methylome analysis we have previously identified 12 genomic loci showing brain-specific DNA methylation patterns, including markers for neurons, oligodendrocytes and astrocytes. These brain-specific methylation markers were identified in the plasma of patients suffering from multiple sclerosis, as well as traumatic and ischemic brain damage. We hypothesize that brain-derived cell-free DNA (bncfDNA) can be identified in patients suffering from brain metastases receiving radiotherapy, and can potentially be used as a biomarker to help guide treatment. MATERIAL AND METHODS: We recruit oncological patients treated by brain radiotherapy for brain metastases. We serially assess each patient before, during and after treatment by neurological examination. MRI studies are performed before and after radiotherapy in 2–3 months intervals. In each study visit a blood sample is collected for bncfDNA. We measure bncfDNA from serial blood samples of each patient, and correlate bncfDNA levels with clinical assessment. RESULTS: Preliminary results on samples from 22 patients show elevation of bncfDNA in patients suffering from brain metastases (average 8501 copies/ml; range 0-112336 copies/ml) compared with an extremely low background in healthy individuals (average 6 copies/ml; range 0–33 copies/ml); p

Classical Hodgkin lymphoma (cHL) accounts for 30% of all lymphomas [1]. Currently, 20–25% of cHL cases relapse or are refractory after first-line standard treatments, leading to the need to better ...

The accurate detection of ultralow allele frequency variants in DNA samples is of interest in both research and medical settings, particularly in liquid biopsies where cancer mutational status is monitored from circulating DNA. Next-generation sequencing (NGS) technologies employing molecular barcoding have shown promise but significant sensitivity and specificity improvements are still needed to detect mutations in a majority of patients before the metastatic stage. To address this we present analytical validation data for ERASE-Seq (Elimination of Recurrent Artifacts and Stochastic Errors), a method for accurate and sensitive detection of ultralow frequency DNA variants in NGS data. ERASE-Seq differs from previous methods by creating a robust statistical framework to utilize technical replicates in conjunction with background error modeling, providing a 10 to 100-fold reduction in false positive rates compared to published molecular barcoding methods. ERASE-Seq was tested using spiked human DNA mixtures with clinically realistic DNA input quantities to detect SNVs and indels between 0.05% and 1% allele frequency, the range commonly found in liquid biopsy samples. Variants were detected with greater than 90% sensitivity and a false positive rate below 0.1 calls per 10,000 possible variants. The approach represents a significant performance improvement compared to molecular barcoding methods and does not require changing molecular reagents.

Background Cell-free DNA (cfDNA) can be detected in the circulation of healthy individuals, but is found in higher concentrations in cancer patients. Furthermore, mutations in tumor cells can be identified in circulating DNA fragments. This has been the subject of significant interest in the field of cancer research, but little has been published in thyroid cancer. Objectives To assess all available evidence on the use of circulating cfDNA in the diagnosis, management and surveillance of patients with differentiated thyroid cancer, and collate it into a systematic review to guide future research. Methods A comprehensive literature search on the measurement of cfDNA in thyroid cancer was undertaken, and results from relevant studies collated into a systematic review. Results Nine studies were identified, with varying methodologies and findings. Key techniques and findings are summarized. Conclusion There is limited but promising evidence that somatic mutations in thyroid cancer can be detected in circulating cfDNA and are associated with more advanced disease. Further research is required to develop a clinically useful tool based on cfDNA to improve the management of thyroid cancers.

Background Early screening for prostate cancer (PCA) remains controversial because of overdiagnosis and overtreatment of clinically insignificant cancers. Even though a number of diagnostic tests have been developed to improve on PSA testing, there remains a need for a more informative non-invasive test for PCA. The objective of this study is to identify a panel of DNA methylation markers suitable for a non-invasive diagnostic test from urine DNA collected following a digital rectal exam (DRE) and/or from first morning void (FV). A secondary objective is to determine if the cumulative methylation is indicative of biopsy findings. Methods DRE and FV urine samples were prospectively collected from 94 patients and analyzed using 24 methylation-specific quantitative PCR assays derived from 19 CpG islands. The methylation of individual markers and various combinations of markers was compared to biopsy results. A methylation threshold for cancer classification was determined using a target specificity of 70%. The average methylation and the number of positive markers were also compared to the result of the biopsy, and the area under the receiver operating characteristic curves (AUCs) were calculated. Results Methylation of all 19 markers was detected in FV and DRE DNAs. Combining the methylation of two or more markers improved on individual marker results. Using 6of19 methylated markers as the threshold for cancer classification yielded a specificity of 71% (95% CI, 0.57–0.86) from both DRE and FV and a sensitivity of 89% (95% CI, 0.79–0.97) from DRE and 94% (95% CI, 0.84–1.0) from FV. The negative predictive value at the 6of19 threshold was ≥ 90 for both DNA types. Conclusions PCA-specific methylation was detected in both FV and DRE DNA. There was no significant difference in diagnostic accuracy at the 6of19 threshold between DRE and FV urine DNA. The results support the development of a non-invasive diagnostic test to reduce unnecessary biopsies in men with elevated PSA. The test can also provide patients with personalized recommendations based on their own methylation profile. Electronic supplementary material The online version of this article (10.1186/s13148-018-0524-x) contains supplementary material, which is available to authorized users.

Management of localized and advanced prostate cancer benefits from several therapeutic options with a surprising improvement in terms of clinical outcome. The selection of patients more likely to benefit from a specific approach still remains a key issue as well as the early identification of patients with aggressive disease which could benefit from a more aggressive treatment strategy. The lack of reliable bio-marker in castration resistant setting able to monitor response to treatment and early inform about tumor progression is an emerging issue. Accordingly, circulating DNA and circulating tumor cells appears a promising and attractive approach despite to date practical applications of these techniques are few and not validated. The aim of this review of the literature is to explore current knowledge on liquid biopsy in prostate cancer focusing on possible future applications.

// Nicolas Macagno 1, 2, * , Frederic Fina 1, 3, * , Nicolas Penel 4 , Corinne Bouvier 1, 2 , Isabelle Nanni 5 , Florence Duffaud 6, 7 , Raquel Rouah 5 , Bruno Lacarelle 8 , L'houcine Ouafik 5 , Sylvie Bonvalot 9 and Sebastien Salas 6, 7 1 Department of Pathology, Assistance Publique Hopitaux de Marseille Timone Hospital, Marseille, France 2 Aix-Marseille University, Medical Faculty, CRO2, UMR 911 (Equipe IV), Marseille, France 3 ID-Solutions, Grabels, France 4 Department of General Oncology, Oscar Lambret Center, Lille, France 5 Department of Molecular Oncology, Assistance Publique Hopitaux de Marseille, Marseille, France 6 Department of Oncology, Assistance Publique Hopitaux de Marseille Timone Hospital, Marseille, France 7 Aix-Marseille University, Medical Faculty, Marseille, France 8 Department of Medical Biology, Assistance Publique Hopitaux de Marseille Timone Hospital, Marseille, France 9 Department of Surgery, Institut Curie, PSL Univeristy, Paris, France * These authors contributed equally to this work Correspondence to: Sebastien Salas, email: sebastien.salas@ap-hm.fr Keywords: ddPCR; cfDNA; CTNNB1; desmoid; prognosis Received: October 27, 2017 Accepted: February 25, 2018 Published: April 06, 2018 ABSTRACT Since desmoid tumors (DT) exhibit an unpredictable clinical course, with stabilization and/or spontaneous regression, an initial “wait-and-see” policy is the new standard of care–thus, the actual challenge is to identify early factors of progression. We present a method of detection of CTNNB1 mutations using a targeted digital droplet PCR (ddPCR) on cell-free DNA (cfDNA) extracted from blood samples of 31 DT patients. Furthermore, we analyzed the correlation between DT evolution and plasmatic concentration of total and mutated cfDNA at the time of diagnosis. Circulating copies of CTNNB1 mutants (ctDNA) were detected in the plasma of 6 patients (33%) but their concentration was not correlated with evolution of the tumor. Concentration of total cfDNA was higher in the plasma of patients with progressive desmoids ( p = 0,0009). Using a threshold 1375, it was possible to predict desmoid evolution for 65% of patients by measuring the quantity of circulating DNA in their plasma as early as the time of diagnosis. Albeit showing that the detection of CTNNB1 mutants is possible in the plasma of patients harboring a desmoid tumor, the results of this preliminary study raise the hypothesis that most of the circulating DNA detected in their plasma is derived from non-neoplastic cells, most likely normal neighboring tissues being actively invaded. Our results open the perspective of using cfDNA as a biomarker to predict prognosis at the time of diagnosis and assess tumor dynamics to optimize the treatment strategy.

The detection of circulating DNA is considered a promising strategy in cancer patients. Digital PCR has emerged as a sensitive method able to quantify both circulating free and tumour DNA.The aim of this study was to prospectively evaluate the clinical value of a chip-based digital PCR for the detection of circulating DNA.Digital PCR was used in 34 metastatic colorectal cancer patients to detect and quantify circulating free and tumour DNA based on K-ras mutational status. Clinical outcomes were analyzed according to circulating DNA measurements.Digital PCR yielded a detection rate of 69% for circulating tumour DNA. The median concentrations of circulating free and tumour DNA were 20 and 6.8 ng/mL, respectively, with significant correlation between both biomarkers (p0.001). Median overall survival was 4.8 months in patients with high circulating free DNA (75% quartile) versus not reached in patients with a low level (25% quartile) (p=0.029). Moreover, median overall survival was significantly decreased in patients with detectable circulating tumour DNA compared to those without (respectively 11.8 months versus not reached, p=0.04).Chip-based digital PCR is a simple and non-invasive method allowing the efficient detection of circulating DNA. Our results highlight that levels of these circulating markers may have a potential prognostic value.

Liquid biopsy has the potential to provide information about cancers without invasive biopsy, using circulating biomarkers. These include proteins, RNA and DNA. They can be used in detection, diagnosis, monitoring and detection of recurrence. While protein-based tumour markers have been used in routine pathology for many years, the ability to detect mutations in circulating DNA is relatively new, and poised to enter clinical practice. A number of issues remain, and it is important that such markers are fully validated before they enter clinical practice. Evidence of clinical utility and cost effectiveness are major hurdles, but it is likely that the use of liquid biopsy in defined settings could benefit cancer patients substantially.

Introduction There is much interest in the use of noninvasive biomarkers in the management of lung cancer, particularly with respect to early diagnosis and monitoring the response to intervention. Cell-free tumor DNA in patients with cancer has been shown to hold potential as a noninvasive biomarker, in which the response to treatment may be evaluated using a blood test only. Multiple technologies have been suggested as being appropriate to measure cell-free tumor DNA. Microdroplet digital polymerase chain reaction (mdPCR) has a number of attributes that suggest it may be a useful tool for detecting clinically relevant genetic events. It offers precise and accurate quantitation of mutant alleles, including rare variants. Methods We evaluate the performance of mdPCR in the analysis of DNA extracted from reference standards, tumor biopsies, and patient plasma. Results The potential of mdPCR to detect clinically relevant mutations is demonstrated, in both formalin-fixed paraffin-embedded material and plasma. Furthermore, we show that mdPCR can be used to track changes in peripheral blood biomarkers in response to treatment and to detect the emergence of drug-resistant clones. Conclusions MdPCR has potential as a tool to detect and quantify tumor-derived mutational events in cell-free DNA from patients with lung cancer.

Background: Cell-free DNA integrity (cfDI) has been identified as a potential diagnostic biomarker for different types of cancer, suggesting the importance of liquid biopsy. Methods: This systematic review aims to assess the diagnostic performance of cfDI in cancer. After an extensive search of literature published through February 28, 2017, 25 articles that included 40 studies were identified. The descriptions of all the studies were analyzed. The sensitivity, specificity, positive and negative likelihood ratios, diagnostic ORs, weighted symmetric summary receiver operating characteristic curve, and the area under the curve (AUC) of cfDI in these studies were calculated. Results: Aberrant results of cfDI were observed. Some studies observed an increased cfDI in cancer patients, while some studies confirmed a decreased cfDI compared with healthy or benign controls. Conclusions: This review suggests that cfDI is controversial as a blood-based biomarker of cancers, although the sensitivity and AUC were relatively high. Impact: cfDI shows heterogeneity between different studies; more perspective studies are needed to further assess its diagnostic performance, especially with other markers in combination. Cancer Epidemiol Biomarkers Prev; 26(11); 1595–602. ©2017 AACR.

Background: Circulating DNA can be used to measure the total cell-free DNA (cfDNA) and for detection and quantification of tumor-specific genetic alterations in the peripheral blood, and the broad clinical potential of circulating DNA has attracted increasing focus over the past decade. Concentrations of circulating DNA are high in metastatic colorectal cancer (CRC), and the total levels of cfDNA have been reported to hold strong prognostic value. Colorectal tumors are characterized by a high frequency of well known, clinically relevant genetic alteration, which is readily detected in the cfDNA and holds potential for tailoring of palliative therapy and for monitoring during treatment. This review aims to present the current literature which has specifically reported data on the potential utility of cfDNA and on tumor-specific mutations in metastatic colorectal cancer (mCRC). Method: Methodological, biological and clinical aspects are discussed based on the most recent development in this specific setting, and eligible studies were identified by systematic literature searched from Pubmed and EMBASE in addition to conference papers and communications. Results: The literature regarding cfDNA in CRC is broad and heterogeneous concerning aims, nomenclature, methods, cohorts and clinical endpoints and consequently difficult to include in a single systematic search. However, the available data underline a strong clinical value of measuring both total cfDNA levels and tumor-specific mutations in the plasma of patients with mCRC, pre- and during systemic therapy. Conclusion: This paper had gathered the most recent literature on several aspects of cfDNA in mCRC, including methodological, biological and clinical aspects, and discussed the large clinical potential in this specific setting, which needs to be validated in carefully designed prospective studies in statistically relevant cohorts.

International audience

Intermittent hypoxia (IH) a hallmark characteristic of obstructive sleep apnea (OSA), is proposed as a major determinant of processes involving tumor growth, invasion and metastasis. To examine whether circulating DNA (cirDNA) in blood plasma reflects changes in tumor cells under IH conditions, we used a xenografted murine model. Mice engrafted with TC1 epithelial lung cancer cells and controls were exposed to IH or room air (RA) conditions. Plasma cirDNA amounts were significantly increased in mice exposed to IH (p

Background Circulating cell-free DNA (cfDNA) has recently been recognized as a resource for biomarkers of cancer progression, treatment response, and drug resistance. However, few have demonstrated the usefulness of cfDNA for early detection of cancer. Although aberrant DNA methylation in cfDNA has been reported for more than a decade, its diagnostic accuracy remains unsatisfactory for cancer screening. Thus, the aim of the present study was to develop a highly sensitive cfDNA-based system for detection of primary breast cancer (BC) using epigenetic biomarkers and digital PCR technology. Methods Array-based genome-wide DNA methylation analysis was performed using 56 microdissected breast tissue specimens, 34 cell lines, and 29 blood samples from healthy volunteers (HVs). Epigenetic markers for BC detection were selected, and a droplet digital methylation-specific PCR (ddMSP) panel with the selected markers was established. The detection model was constructed by support vector machine and evaluated using cfDNA samples. Results The methylation array analysis identified 12 novel epigenetic markers (JAK3, RASGRF1, CPXM1, SHF, DNM3, CAV2, HOXA10, B3GNT5, ST3GAL6, DACH1, P2RX3, and chr8:23572595) for detecting BC. We also selected four internal control markers (CREM, GLYATL3, ELMOD3, and KLF9) that were identified as infrequently altered genes using a public database. A ddMSP panel using these 16 markers was developed and detection models were constructed with a training dataset containing cfDNA samples from 80 HVs and 87 cancer patients. The best detection model adopted four methylation markers (RASGRF1, CPXM1, HOXA10, and DACH1) and two parameters (cfDNA concentration and the mean of 12 methylation markers), and, and was validated in an independent dataset of 53 HVs and 58 BC patients. The area under the receiver operating characteristic curve for cancer-normal discrimination was 0.916 and 0.876 in the training and validation dataset, respectively. The sensitivity and the specificity of the model was 0.862 (stages 0-I 0.846, IIA 0.862, IIB-III 0.818, metastatic BC 0.935) and 0.827, respectively. Conclusion Our epigenetic-marker-based system distinguished BC patients from HVs with high accuracy. As detection of early BC using this system was comparable with that of mammography screening, this system would be beneficial as an optional method of screening for BC. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0788-z) contains supplementary material, which is available to authorized users.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer arising from the bile ducts with a need for earlier diagnosis and a greater range of treatment options. KRAS/NRAS mutations are common in ICC tumours and 6&ndash, 32% of patients also have isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene mutations associated with metabolic changes. This feasibility study investigated sequencing circulating tumour DNA (ctDNA) combined with metabolite profiling of plasma as a method for biomarker discovery in ICC patients. Plasma was collected from four ICC patients receiving radio-embolisation and healthy controls at multiple time points. ctDNA was sequenced using Ampliseq cancer hotspot panel-v2 on Ion Torrent PGM for single nucleotide variants (SNV) detection and with Illumina whole genome sequencing for copy number variants (CNV) and further targeted examination for SNVs. Untargeted analysis of metabolites from patient and control plasma was performed using liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS). Metabolite identification was performed using multi-parameter comparisons with analysis of authentic standards, and univariate statistical analysis was performed to identify differences in metabolite abundance between patient and control samples. Recurrent somatic SNVs and CNVs were identified in ctDNA from three out of four patients that included both NRAS and IDH1 mutations linked to ICC. Plasma metabolite analysis revealed biomarker metabolites associated with ICC and in particular 2-hydroxyglutarate (2-HG) levels were elevated in both samples from the only patient showing a variant allele in IDH1. A reduction in the number of CNVs was observed with treatment. This study demonstrates that ctDNA and metabolite levels can be identified and correlated in ICC patient blood samples and differentiated from healthy controls. We conclude that combining genomic and metabolic analysis of plasma offers an effective approach to biomarker identification with potential for disease stratification and early detection studies.

Background: Anti-CD19 chimeric antigen receptor (CAR19) T-cells have significant activity in patients with relapsed/refractory DLBCL (rrDLBCL). While the majority of rrDLBCL patients receiving axicabtagene ciloleucel (Axi-cel)achieve complete responses, a significant subset of patients experience disease progression (Locke FL, et al. Lancet Oncol. 2019). Circulating tumor DNA (ctDNA) analysis has demonstrated utility for predicting therapeutic benefit in DLBCL, as well as for detecting emergent resistance mechanisms to targeted therapies. Here we apply cell-free DNA (cfDNA) analysis to patients receiving Axi-cel, to characterize molecular responses, resistance mechanisms, and to track CAR19 cells. Methods: We performed Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) on DNA from germline and plasma samples collected prior to CAR T-cell infusion, multiple time-points post infusion, and, where available, at the time of relapse from 30 patients receiving Axi-cel for rrDLBCL at Stanford University. We designed a novel hybrid-capture panel and analysis pipeline designed to detect both tumor variants, as well as Axi-cel specific recombinant retroviral sequences to quantify CAR19 levels in cfDNA. Tumor variants were identified prior to and following Axi-cel therapy to assess for emergent variants, and Axi-cel specific sequences were quantified. Results: The median follow-up for the 30 patients after Axi-cel infusion was 10 months, with 47% (14/30) of patients experiencing disease progression after Axi-cel therapy. We identified an average of 164.3 SNVs per case (range:1-685) before Axi-cel therapy; the most common coding variants identified at baseline were in MLL2 (29.2%), BCL2 (22.5%), and TP53 (19.3%). When treated as a continuous variable, pretreatment ctDNA levels were prognostic of PFS (HR 2.16, 95% CI 1.11-4.21, P=0.02). Using a previously established ctDNA threshold to stratify disease burden (2.5 log10(hGE/mL); Kurtz et al. JCO 2018), we observed significantly superior PFS in patients with low pretreatment ctDNA levels treated with Axi-cel (Fig. 1A). In the majority of Axi-cel treated patients (62.9%), ctDNA was detectable at day 28, and PFS was significantly longer in patients with undetectable ctDNA at this time-point (Fig. 1B). Multiple putative resistance mechanisms were identified at relapse after Axi-cel, including emergent variants in CD19, HVEM, and TP53, as well as copy number gains in PD-L1 (Fig. 1C). For example, in one patient, a CD19 stop-gain mutation, which was not detected prior to treatment or at the time of the first interim PET scan, emerged at the time of relapse (Fig. 1D). Finally, we found cfDNA evidence for Axi-cel DNA in 74% of patients 28 days after therapy, including in patients without evidence of circulating CAR T-cells in PBMCs. Axi-cel levels in cfDNA as measured by CAPP-Seq were significantly correlated with CAR19 flow cytometry (Pearson r=0.55, P=.015; Fig. 1E). Conclusions: Baseline and interim ctDNA measurements have prognostic significance in DLBCL patients being treated with CAR19 T-cells, and potential emergent resistance mutations, including in CD19, can be identified in patients via cfDNA analysis. Quantification of CAR19 T-cells using cfDNA is significantly correlated with flow cytometric quantification, indicating that these cells can be quantified via cfDNA. Taken together, these data indicate that cfDNA analysis is a powerful tool for predicting response to CAR19 therapy, identifying genomic determinants of resistance and quantifying CAR19 cells, which may in turn inform the next therapeutic steps. Figure 1: A) Kaplan Meier analysis of PFS, with patients stratified based on pre-Axi-cel therapy ctDNA level, above and below a previously established threshold (2.5 log10[haploid Genome Equivalents/mL]). B) A Kaplan Meier plot depicting PFS stratification for patients with detectable versus undetectable ctDNA at day 28 after Axi-cel infusion. C) Oncoprint depicting selected emergent and baseline tumor variants in progressors and non-progressors after Axi-cel therapy. D) Change in mean ctDNA variant allele frequency (VAF) and emergence of a CD19 stop-gain mutation (CD19 pTrpX) at the time of relapse in a patient who initially achieved a CR at day 28 after CAR19 infusion. E) Relationship between CAR19 T-cell quantification by cfDNA and flow cytometry. (ND: Not detected) Disclosures Kurtz: Roche: Consultancy. Chabon:Lexent Bio Inc: Consultancy. Khodadoust:Corvus Pharmaceuticals: Research Funding. Majzner:Xyphos Inc.: Consultancy; Lyell Immunopharma: Consultancy. Mackall:Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board; Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Diehn:Roche: Consultancy; Quanticell: Consultancy; Novartis: Consultancy; AstraZeneca: Consultancy; BioNTech: Consultancy. Miklos:Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees. Alizadeh:Genentech: Consultancy; Janssen: Consultancy; Pharmacyclics: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Chugai: Consultancy; Roche: Consultancy; Pfizer: Research Funding.