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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.

Purpose of review Liquid biopsies have emerged as a noninvasive alternative to tissue biopsy with potential applications during all stages of pediatric oncology care. The purpose of this review is to provide a survey of pediatric cell-free DNA (cfDNA) studies, illustrate their potential applications in pediatric oncology, and to discuss technological challenges and approaches to overcome these hurdles. Recent findings Recent literature has demonstrated liquid biopsies' ability to inform treatment selection at diagnosis, monitor clonal evolution during treatment, sensitively detect minimum residual disease following local control, and provide sensitive posttherapy surveillance. Advantages include reduced procedural anesthesia, molecular profiling unbiased by tissue heterogeneity, and ability to track clonal evolution. Challenges to wider implementation in pediatric oncology, however, include blood volume restrictions and relatively low mutational burden in childhood cancers. Multiomic approaches address challenges presented by low-mutational burden, and novel bioinformatic analyses allow a single assay to yield increasing amounts of information, reducing blood volume requirements. Summary Liquid biopsies hold tremendous promise in pediatric oncology, enabling noninvasive serial surveillance with adaptive care. Already integrated into adult care, recent advances in technologies and bioinformatics have improved applicability to the pediatric cancer landscape.

Background After radiation therapy (RT), circulating plasma cell-free DNA (cfDNA) released in response to RT damage to tissue can be measured within hours. We examined for a correlation between cfDNA measured during the first week of therapy and early and late gastrointestinal (GI) and genitourinary (GU) toxicity. Material and Methods Patients were eligible for enrollment if they planned to receive proton or photon RT for nonmetastatic prostate cancer in the setting of an intact prostate or after prostatectomy. Blood was collected before treatment and on sequential treatment days for the first full week of therapy. Toxicity assessments were performed at baseline, weekly during RT, and 6 months and 12 months after RT. Data were analyzed to examine correlations among patient-reported GI and GU toxicities. Results Fifty-four patients were evaluable for this study. Four (7%) and 3 (6%) patients experienced acute and late grade 2 GI toxicity, respectively. Twenty-two (41%) and 18 (35%) patients experienced acute and late grade 2 GU toxicity, respectively. No patients developed grade 3 or higher toxicity. Grade 2 acute GI toxicity, but not grade 2 acute GU toxicity, was significantly correlated with pre-RT cfDNA levels and on all days 1, 2, 3, 4, and 5 of RT (P < .005). Grade 2 late GI toxicity, but not GU toxicity, was significantly correlated with pre-RT cfDNA levels (P = .021). Conclusions Based on this preliminary study, cfDNA levels can potentially predict the subset of patients destined to develop GI toxicity during prostate cancer treatment. Given that the toxicity profiles of the various fractionations and modalities are highly similar, the data support the expectation that cfDNA could provide a biological estimate to complement the dose-volume histogram. A test of this hypothesis is under evaluation in a National Cancer Institute–funded multi-institutional study.

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

We studied the levels of extracellular nuclear and mitochondrial DNA of blood serum and DNA damage in leukocytes of healthy donors of different sex and age groups. The baseline levels of DNA damage in leukocytes and serum DNA levels were shown to vary greatly among different donors. The baseline level of DNA damage in leukocytes was not associated with the presence of chronic deceases or an occupational health risk for elderly donors. It was found that extracellular DNA concentrations were generally higher in men than in women. There is a tendency towards an increase in the relative mitochondrial DNA copy number determined by ΔCt in women but not in men: the relative mtDNA copy number in elderly individuals varies significantly in both sexes, possibly due to age-related physiological changes. It is necessary to consider the gender and age of patients when using an indicator such as the level of extracellular DNA of blood serum for diagnosis and monitoring.

Purpose The RadTox assay measures circulating cell-free DNA released in response to radiotherapy (RT)-induced tissue damage. The primary objectives for this clinical trial were to determine whether cell-free DNA numbers measured by the RadTox assay are (1) correlated with body integral dose, (2) lower with proton RT compared with photon RT, and (3) higher with larger prostate cancer RT fields. Patients and Methods Patients planned to receive proton or photon RT for nonmetastatic prostate cancer in the setting of an intact prostate or postprostatectomy were eligible for the trial. Plasma was collected pre-RT and at 5 additional daily collection points beginning 24 hours after the initiation of RT. Data from 54 evaluable patients were analyzed to examine any correlations among RadTox scores with body-integral dose, RT modality (photon versus proton), and RT field size (prostate or prostate bed versus whole pelvis). Results Body integral dose was significantly associated with the peak post-RT RadTox score (P = .04). Patients who received photon RT had a significant increase in peak post-RT RadTox score (P = .04), average post-RT RadTox score (P = .04), and day-2 RadTox score (all minus the pre-RT values for each patient) as compared with patients who received proton RT. Field size was not significantly associated with RadTox score. Conclusion RadTox is correlated with body integral dose and correctly predicts which patients receive proton versus photon RT. Data collection remains ongoing for patient-reported RT toxicity outcomes to determine whether RadTox scores are correlated with toxicity.

Background Correct detection of human cardiomyocyte death is essential for definitive diagnosis and appropriate management of cardiovascular diseases. Although current strategies have proven utility in clinical cardiology, they have some limitations. Our aim was to develop a new approach to monitor myocardial death using methylation patterns of circulating cell-free DNA (cf-DNA). Methods We first examined the methylation status of FAM101A in heart tissue and blood of individual donors using quantitative methylation-sensitive PCR (qMS-PCR). The concentrations and kinetics of cardiac cf-DNA in plasma from five congenital heart disease (CHD) children before and after they underwent cardiac surgery at serial time points were then investigated. Results We identified demethylated FAM101A specifically present in heart tissue. Importantly, our time course experiments demonstrated that the plasma cardiac cf-DNA level increased quickly during the early post-cardiac surgery phase, peaking at 4–6 h, decreased progressively (24 h) and returned to baseline (72 h). Moreover, cardiac cf-DNA concentrations pre- and post-operation were closely correlated with plasma troponin levels. Conclusions We proposed a novel strategy for the correct detection of cardiomyocyte death, based on analysis of plasma cf-DNA carrying the cardiac-specific methylation signature. Our pilot study may lead to new tests for human cardiac pathologies.

The application of precision medicine concept in clinical work needs a period of practice and experience accumulation. The present article introduced an example of functioning approach named “multidisciplinary therapy strategy of precision medicine” (MDTS‐PM), clinical practice and process, decision‐making, and therapies. The MDTS‐PM integrates multidisciplinary experts and develops real‐time therapeutic strategy based on clinical phenomes and gene sequencing of tissue DNA and circulating DNA. The strength of MDTS‐PM is the combination of dynamical clinical phenomes, genetic information, diagnosis, and treatment to make the therapy more targeted and specific. MDTS‐PM provides comprehensive, whole‐process, and personalized diagnosis and treatment services for patients with complex cancer or complex drug resistance progression; provides guidance for further adjustment of drug use; and establishes a multidisciplinary cooperative team, improves the quality of clinical diagnosis and treatment, and optimizes the process of medical services.

Powered by rapid technology developments, biomarkers become increasingly diverse, including those detected at genomic, transcriptomic, proteomic, metabolomic and cellular levels. While diverse sets of biomarkers have been utilized in breast cancer predisposition, diagnosis, prognosis, treatment and management, recent additions derived from lincRNA, circular RNA, circulating DNA together with its methylated and hydroxymethylated forms and immune signatures are likely to further transform clinical practice. Here, we take breast cancer as an example of heterogeneous diseases that require many informed decisions from treatment to care to review the huge variety of biomarkers. By assessing the advantages and limitations of modern biomarkers in diverse use scenarios, this article outlines the prospects and challenges of releasing complimentary advantages by augmentation of multiscale molecular biomarkers.

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.

The current study sought to explore the significance of copy number variations (CNVs) of MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived [avian]) and ALK (anaplastic lymphoma kinase) genes individually as well as their combined impact on clinical outcome and overall survival of patients with neuroblastoma (NB).A total 71 individuals including healthy controls (n = 11), circulating DNA (n = 11), and primary tumors (n = 49) were evaluated to detect CNVs of MYCN and ALK genes using droplet digital polymerase chain reaction. Data were correlated with univariate and multivariate survival analysis.CNVs of MYCN and ALK were detected in 27% and 18.2% from circulating DNA samples. A statistically significant difference in CNVs was noted between healthy controls and circulating DNA samples for MYCN (P = 0.001) and ALK (P = 0.004) genes. Further, we noted70% concordance in CNVs of MYCN (P = 0.030) and ALK (P = 0.040) from primary tumors and concordant plasma samples of patients with NB. Multivariate survival analysis for disease-free survival (P = 0.031) and overall survival (P = 0.011) showed that CNVs of both genes emerged at step 1 and thus remained as significant markers for predicting early recurrence and shorter survival, respectively, for patients with NB.Our study showed that the analysis of circulating DNA by droplet digital polymerase chain reaction is a helpful technique to identify high-risk patients for aggressive therapy at an early stage of disease. We also concluded that codetection of MYCN and ALK is a more powerful tool for identifying high-risk patients with NB. Thus, this study showed a novel coordinately significant prognostic role of MYCN and ALK CNVs.

Background: Cell-free circulating DNA (cfDNA), which can be extracted by liquid biopsy, has been studied as a noninvasive biomarker for various diseases. The potential of cfDNA fragment size and level as a marker for low back pain (LBP) has never been studied. We investigated whether cfDNA is a biomarker of LBP severity in patients with a lumbar degenerative disease (LDD). Methods: Blood samples were obtained from patients with LDD (n = 21) before and immediately after spinal surgery. Plasma DNA was isolated and examined for cfDNA fragment size and concentration. A cohort of healthy volunteers (n = 5) constituted the control group.Results: The cfDNA fragment size tended to be shorter in patients than in healthy controls, but this difference was not significant (P = .224). cfDNA level was significantly higher in LDD patients (mean 0.642±0.199 ng/mL, range 0.302–1.150 ng/mL) than in healthy controls (mean 0.429±0.064 ng/mL, range 0.366–0.506 ng/mL) (P = .029). cfDNA level correlated positively with present pain (r = .421, P = .036), maximum pain (r = .419, P = .037), average pain (r = .566, P = .003), low back pain (r = .403, P = .041), leg pain (r = .480, P = .013), and leg numbness (r = .455, P = .020). cfDNA fragment size did not differ from before to after surgery, but cfDNA level increased postoperatively in patients with LDD. Conclusions: This was the first study investigating whether cfDNA fragment size and level are associated with pain, including LBP, in patients with LDD. Our findings suggest that cfDNA level may be an objective indicator of pain and surgical invasiveness in patients with LDD.

The European Society of Gynaecological Oncology (ESGO), the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG), the International Ovarian Tumour Analysis (IOTA) group and the European Society for Gynaecological Endoscopy (ESGE) jointly developed clinically relevant and evidence-based statements on the preoperative diagnosis of ovarian tumors, including imaging techniques, biomarkers and prediction models. ESGO/ISUOG/IOTA/ESGE nominated a multidisciplinary international group, including expert practising clinicians and researchers who have demonstrated leadership and expertise in the preoperative diagnosis of ovarian tumors and management of patients with ovarian cancer (19 experts across Europe). A patient representative was also included in the group. To ensure that the statements were evidence-based, the current literature was reviewed and critically appraised. Preliminary statements were drafted based on the review of the relevant literature. During a conference call, the whole group discussed each preliminary statement and a first round of voting was carried out. Statements were removed when consensus among group members was not obtained. The voters had the opportunity to provide comments/suggestions with their votes. The statements were then revised accordingly. Another round of voting was carried out according to the same rules to allow the whole group to evaluate the revised version of the statements. The group achieved consensus on 18 statements. This Consensus Statement presents these ESGO/ISUOG/IOTA/ESGE statements on the preoperative diagnosis of ovarian tumors and the assessment of carcinomatosis, together with a summary of the evidence supporting each statement.

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.

Cell-free DNA (cfDNA) harboring mutations has been found in patients with diseases. Experimental studies have shown that cfDNA can be transmitted, leading to transformations in the host. In the present study, we evaluated whether bone allograft material contains cfDNA and whether this foreign cfDNA can be released into the patient’s blood circulation. Plasma samples were collected preoperatively and postoperatively on the same day, at 5 weeks, and 4 months from 25 women who received bone allograft material (test group) from male donors and from 10 women who were treated with autologous graft (control group, only pre- and postoperative samples were collected). DNA was quantified and characterized in bone material and plasma samples by quantitative PCR with primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Y chromosome and gel electrophoresis. DNA in bone material was digested by different concentrations of DNase I. We detected between 1 and 1.8 μg cfDNA fragments at a length around 601 base pairs (bp) and smaller in each 100 mg allograft. Treatment of the allograft with DNase I completely degraded the longer but not the shorter DNA 90-bp fragments. Y-DNA was not detected in the patients’ bloodstream at any time during the treatment and follow-up, but elevated levels of circulating cfDNA could be measured immediately postoperatively. Our results suggest that a transmission of DNA from allografts used for alveolar ridge reconstruction in humans is unlikely. The observed increase in circulating cfDNA in allograft and autograft patients immediately postoperatively may be elicited by the surgical procedure. The results support the safety of allograft materials. The results suggest that human allograft materials seem not to release DNA into the blood since we did not measure Y-DNA with our technique.

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.

To evaluate three non-invasive assays for the diagnosis of schistosomiasis mansoni in an Egyptian village.Urine was collected for the detection of circulating cathodic antigen (CCA) and cell-free parasite DNA (cfpd) by Point-of-contact (POC)-cassette assay and PCR, respectively. These tests were compared to Kato-Katz (KK) faecal thick smear for detection of Schistosoma mansoni eggs.Disease prevalence by POC-CCA assay was 86%; by PCR it was 39% vs. 27% by KK. Compared to KK, the sensitivity of POC-CCA reached 100%, but its specificity was only 19.2% with 41% accuracy. Sensitivity of the PCR assay for cfpd was 55.56%, and specificity was 67.12% with 64% accuracy. A new end point was calculated for combined analysis of KK, POC-CCA assay and PCR. Sensitivity for the three tests was 52.94%, 90.2% and 76.47%; specificity was 100% for KK and PCR and 18.37% for POC-CCA. The accuracy calculated for the three tests at the end point was 76% for KK, 55% for POC-CCA assay and 88% for PCR.Conventional PCR assay for detection of cfpd provides a potential screening tool for intestinal schistosomiasis with reliable specificity, reasonable accuracy and affordable financial and technical cost.Evaluer trois tests non invasifs pour le diagnostic de la schistosomiase mansoni dans un village égyptien. MÉTHODES: L'urine a été collectée pour la détection de l'antigène cathodique circulant (ACC) et de l’ADN du parasite libéré des cellules (cfpd) par le test en cassette POC (point-of-contact) et par PCR, respectivement. Ces tests ont été comparés au test de Kato Katz (KK) sur frottis fécal épais pour la détection des œufs de Schistosoma mansoni. RÉSULTATS: La prévalence de la maladie par dosage POC-ACC était de 86%; elle était de 39% par PCR contre 27% par KK. Par rapport à KK, la sensibilité du POC-ACC atteignait 100%, mais sa spécificité n’était que de 19,2% avec une précision de 41%. La sensibilité du PCR pour la cfpd était de 55,56% et sa spécificité de 67,12% avec une précision de 64%. Un nouveau seuil a été calculé pour l'analyse combinée des tests KK, POC-ACC et PCR. La sensibilité pour les trois tests était de 52,94%, 90,2% et 76,47%; la spécificité était de 100% pour KK et PCR et de 18,37% pour POC-ACC. La précision calculée pour les trois tests au point seuil était de 76% pour KK, 55% pour le POC-ACC et 88% pour la PCR.Le test PCR conventionnel pour la détection de la cfpd constitue un outil de dépistage potentiel de la schistosomiase intestinale avec une spécificité fiable, une précision raisonnable et un coût financier et technique abordable.

Circulating DNA, also named cell-free DNA (cfDNA), is a series of highly fragmented DNA that is present in the blood circulation and other body fluids of human and is free of extracellular. In certain situations, such as cancer patients, pregnant women, patients undergoing organ transplants, etc., a small number of circulating DNA from “heterologous” cells can be used as markers for genetic testing [1]. Large quantity of autologous cell-free DNA reflects abnormal cell death. Autologous cell-free DNA quantification can be used for the diagnosis and monitoring of related diseases. The “liquid biopsy” technology based on circulating DNA detection has received great attention in many aspects such as prenatal diagnosis, tumor screening, early diagnosis, and treatment monitoring, and prognosis evaluation. Because of its extremely low content, a very large number of studies have been done to develop various methods for quantitative detection [1, 2]. At the moment circulating DNA quantitative detection still faces challenges such as the lack of specification, precision, and standardization in detection processes.

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.

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.

Background: Cell free circulating DNA (cfcDNA) is a promising diagnostic tool for prostate cancer (PCa). This study aimed to measure the cfcDNA concentration and integrity in PCa patients using quantitative polymerase chain reaction (qPCR) analysis. This study also assessed the correlation between these molecular biomarkers with total prostate-specific antigen (PSA), Gleason score, prostate volume, and age. Methods: Eleven PCa patients and 9 persons with benign prostatic hyperplasia (BPH) were recruited. Blood samples were collected before prostate biopsy and plasma quantified by qPCR amplification of the ALU 115 DNA sequence, with the ratio of ALU 247 to ALU 115 reflecting cfcDNA integrity. Results: There were no significant differences in median, interquartile range (IQR) cfcDNA concentration or cfcDNA integrity between the patients with PCa (47.9 (214.93) ng/mL; 0.61 (0.49)) and persons with BPH (41.5 (55.13) ng/mL, p = 0.382; 0.67 (0.45), p = 0.342). A weakly positive correlation exists between cfcDNA concentration and total PSA (r = 0.200, p = 0.555) but not with age or Gleason score in PCa patients. Conclusion: cfcDNA concentration was relatively nonsignificantly higher in PCa patients in comparison to persons with BPH, whereas cfcDNA integrity was similar in both groups. Though limited in sample size, this study shows that cfcDNA concentration may be a potentially valuable noninvasive biomarker for the diagnosis of PCa.

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.

If the current rate of infection are to be better managed, and future waves of infection kept at bay, it is absolutely necessary that the conditions and mechanisms of exposure to Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) be better understood, as well as the downstream severe or lethal clinical complications. While the identification of notable comorbidities has now helped to define broad risk groups, the idiosyncratic responses of individual patients can generate unexpected clinical deterioration that is difficult to predict from initial clinical features. Thus, physicians caring for patients with COVID-19 face clinical dilemmas on a daily basis. The ability to decipher individual predispositions to SARS-CoV-2 infection or severe illness, in light of variations in host immunological and inflammatory responses, in particular as a result of genetic variations, would be of great benefit in infection management. To this end, this work associates the description of COVID-19 clinical complications, comorbidities, sequelae, and environmental and genetic factors. We also give examples of underlying genomic susceptibility to COVID-19, especially with regard to the newly reported link between the disease and the unbalanced formation of neutrophil extracellular traps. As a consequence, we propose that the host/genetic factors associated with COVID-19 call for precision medicine in its treatment. This is to our knowledge the first article describing elements towards precision medicine for patients with COVID-19.

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.

We demonstrate that the general clinical conditions, risk factors and numerous pathological and biological features of COVID-19 are analogous with various disorders caused by the uncontrolled formation of neutrophil extracellular traps and their by-products. Given the rapid evolution of this disease’s symptoms and its lethality, we hypothesize that SARS-CoV2 evades innate immune response causing COVID-19 progresses under just such an amplifier loop, leading to a massive, uncontrolled inflammation process. This work allows us to propose new strategies for treating the pandemic.

Introduction Cunninghamella bertholletiae although rarely causing mucormycosis, is responsible for the highest mortality among mucormycetes. The diagnosis of mucormycosis is challenged by the absence of specific biomarkers. Herein, we report a fatal case of C. bertholletiae infection and detection of its DNA in the serum by polymerase chain reaction (PCR). Presentation of case A 23-year-old male with refractory osteosarcoma was admitted with multiple lung metastases. He was on oral voriconazole prophylaxis after pulmonary aspergillosis. He suffered from fever during temporary neutropenia following chemotherapy and showed several neurological and respiratory symptoms. Despite liposomal-amphotericin B administration, the symptoms rapidly progressed, and he died five days after the onset of neurological symptoms. We retrospectively evaluated the filamentous fungus isolated after his death from gastric juices. Based on the sequence of the internal transcribed spacer (ITS) region we identified the fungal isolate as C. bertholletiae. A 146-bp portion of the D1/D2 region was quantified by quantitative-PCR using DNA extracted from the serum. C. bertholletiae DNA load in the serum was 18.0 copies/μL on the day of onset of neurological symptoms, with the highest (101.0 copies/μL) on the day of his death. Discussion Detection of circulating DNA of mucormycetes in the blood would greatly enhance the diagnosis of mucormycosis. Rapid diagnosis might alleviate mortality due to mucormycosis. Conclusion The present case-report suggests that the quantification of C. bertholletiae DNA in the serum could be useful for the diagnosis and evaluation of mucormycosis pathogenesis in patients.

Background To detect acute schistosomiasis, low-intensity infections, or to verify the success of treatment with praziquantel, highly sensitive test methods are required. The aim of this study was therefore to demonstrate the performance of Schistosoma mansoni specific DNA detection in serum and urine using real-time polymerase chain reaction (PCR) in an endemic area before and after treatment. Methods The study pursued a 1-week and 20-weeks longitudinal design with a treatment intervention among 36 study participants aged 18 to 70 years in the community of Kayenze, a fishing village in Ilemela district on the southern shore of Lake Victoria in north-western Tanzania between February and June 2018. Blood, urine and stool samples were collected from each participant to diagnose Schistosoma mansoni infection before and two times after treatment with praziquantel using serum- and urine based real-time PCR, point-of-care circulating cathodic antigen (POC-CCA) rapid diagnostic test and the microscopic Kato-Katz (KK) method. Kappa coefficient (κ) was used to estimate the agreement between these diagnostic tests compared to a combined “gold standard” of positive results by serum-based real-time PCR and/or positive egg counts determined by KK. Kendall’s Tau rank correlation was used to examine the relationship between cycle threshold (Ct)-values and egg counts and the Wilcoxon signed-rank test was used to compare the median Ct-values of the different examination time points. Results By using the combined “gold standard” of the parasitological Kato-Katz test and/or serum-based real-time PCR, a S. mansoni prevalence of 77.1% could be determined at baseline. In terms of sensitivity, serum-based real-time PCR (96.3%) and POC-CCA assay (77.8%) showed the highest results. The detection of DNA from urine samples showed the lowest sensitivity (33.3%). Treatment with praziquantel resulted in a significantly reduced prevalence of S. mansoni. No infection could be detected by Kato-Katz, with the POC-CCA test only 33.3%. The analysis of the median Ct values over time (which were determined by the serum-based real-time PCR) showed that the Ct decreases significantly shortly after treatment (from 30.3 to 28) and increases above baseline level (34.9) three months later. Conclusions The data presented here show that the serum-based real-time PCR exhibits excellent diagnostic accuracy, in contrast to the use of urine as sample material for S. mansoni DNA detection. However, as circulating DNA does not necessarily reflect the persistence of living worms in schistosomiasis, this method is less well suited to verify the success of treatment with praziquantel.

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)

Invasive mucormycosis is a fungal infection with high mortality. Early diagnosis and initiation of appropriate treatment is essential to improve survival. However, current diagnostic tools suffer from low sensitivity, leading to delayed or missed diagnoses. Recently, several PCR assays for the detection of Mucorales DNA have been developed. We retrospectively assessed the diagnostic and kinetic properties of a commercial Mucorales PCR assay (MucorGenius, PathoNostics) on serial blood samples from patients with culture-positive invasive mucormycosis and found an overall sensitivity of 75%. Importantly, a positive test preceded a positive culture result by up to 81 days (median eight days, inter-quartile range 1.75&ndash, 16.25). After initiation of appropriate therapy, the average levels of circulating DNA decreased after one week and stabilized after two weeks. In conclusion, detection of circulating Mucorales DNA appears to be a good, fast diagnostic test that often precedes the final diagnosis by several days to weeks. This test could be especially useful in cases in which sampling for culture or histopathology is not feasible.

Liquid biopsy has the great potential of detecting early diseases before deterioration and is valued for screening abnormalities at early stage. In oncology, circulating DNA derived from shed cancer cells reflects the tissue of origin, so it could be used to locate tissue sites during early screening. However, the heterogenous parameters of different types limit the clinical application, making it inaccessible to encompass all the cancer types. Instead, for reproducible scenario as pregnancy, fetal cell-free DNA has been well utilized for screening aneuploidies. Noninvasive and convenient as is, it would be of great value in the next decades far more than early diagnosis. This review recapitulates the discovery and development of tumor and fetal cell-free DNA. The common factors are also present that could be taken into consideration when collecting, transporting, and preserving samples. Meanwhile, several protocols used for purifying cell-free DNA, either classic ones or through commercial kits, are compared carefully. In addition, the development of technologies for analyzing cell-free DNA have been summarized and discussed in detail, especially some up-to-date approaches. At the end, the potential prospect of circulating DNA is bravely depicted. In summary, although there would be a lot of efforts before it’s prevalent, cell-free DNA remains a promising tool in point-of-care diagnostic medicine.

The advent of targeted therapy in non-small-cell lung cancer (NSCLC) has made the routine molecular diagnosis of EGFR mutations crucial for optimal patient management. Obtaining tumour tissue for biomarker testing, especially in the setting of re-biopsy, can present many challenges. A potential alternative source of tumour dna is circulating cell-free tumour-derived DNA (CTDNA). Although CTDNA is present in low quantities in plasma, the convenience of sample acquisition and the increasing reliability of detection methods make this approach a promising one. The various performance characteristics of both digital and nondigital platforms are still variable, and a standardized approach is needed that will make those platforms reliable clinical tools for the detection of EGFR sensitizing mutations and resistance mutations, including the T790M resistance mutation. Information derived from ctdna can be used to assess tumour burden, to identify genomic-based resistance mechanisms, and to track dynamic changes during therapy.

We present the results of our technical validation process in establishing the analysis of circulating tumor DNA (ctDNA) as a diagnostic tool. Like most cells in our body, tumor cells shed DNA in the blood flow. Analysis of ctDNA mutational content can provide invaluable information on the genetic makeup of a tumor, and assist oncologists in deciding on therapy, or in following residual disease. However, low absolute amounts of circulating DNA and low tumor fraction constitute formidable analytical challenges. A key step is to avoid contamination with genomic DNA from cell lysis. Several brands of specialized blood collection tubes are available to prevent leukocyte lysis. We show that they are not equally efficient, depending on storage temperature and time before plasma preparation. We report our analysis of preanalytical factors pertaining to ctDNA analysis (tubes, transportation time, temperature) and our conclusions in terms of instructions to prescribing physicians. We also stress the importance of proper DNA quality control and compare several methods, including a differential amplicon length PCR technique which allows determination of multiple QC parameters from minimal amounts of DNA. Altogether, these data provide useful practical information to diagnostic laboratories wishing to implement the assay of ctDNA in clinical practice.

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)

// 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.

Resume Introduction L’identification d’une mutation activatrice du gene codant pour le recepteur a l’epidermal growth factor (EGFR) est un facteur predictif d’efficacite des inhibiteurs de tyrosine kinase d’EGFR (ITK). La recherche de ces mutations peut etre difficile a un stade avance de la maladie en raison du caractere parfois invasif des biopsies et de la taille souvent limitee des echantillons tumoraux. Observation Nous rapportons le cas d’une patiente âgee de 79 ans et atteinte d’un adenocarcinome pulmonaire metastatique. La recherche de mutations activatrices du gene EGFR sur biopsie bronchique s’est averee non contributive en raison d’une faible quantite de cellules tumorales. Le mauvais etat general de la patiente et son refus de prelevements invasifs ont empeche la realisation de nouvelles biopsies. L’analyse de l’ADN tumoral circulant (ADNc) a permis de detecter une deletion de l’exon 19 et de lui proposer un ITK avec a l’issue une reponse prolongee. Conclusion L’analyse de l’ADNc peut permettre d’identifier une mutation activatrice d’EGFR dans les adenocarcinomes pulmonaires. Cette recherche peut etre utile chez les patients au terrain fragilise et en cas d’echec ou d’impossibilite de biopsies tumorales. L’initiation d’un ITK de l’EGFR est possible sur la base de ce resultat, comme le precise l’autorisation de mise sur le marche du gefitinib.

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.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

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.