Molecular profiling of ctDNA in pancreatic cancer: Opportunities and challenges for clinical application

Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related mortality within the next decade, with limited effective treatment options and a dismal long-term prognosis for patients. Genomic profiling has not yet manifested clinical benefits for diagnosis, treatment or prognosis in PDAC, due to the lack of available tissues for sequencing and the confounding effects of low tumour cellularity in many biopsy specimens. Increasing focus is now turning to the use of minimally invasive liquid biopsies to enhance the characterisation of actionable PDAC tumour genomes. Circulating tumour DNA (ctDNA) is the most comprehensively studied liquid biopsy analyte in blood and can provide insight into the molecular profile and biological characteristics of individual PDAC tumours, in real-time and in advance of traditional imaging modalities. This can pave the way for identification of new therapeutic targets, novel risk variants and markers of tumour response, to supplement diagnostic screening and provide enhanced scrutiny in treatment stratification. In the roadmap towards the application of precision medicine for clinical management in PDAC, ctDNA analyses may serve a leading role in streamlining candidate biomarkers for clinical integration. In this review, we highlight recent developments in the use of ctDNA-based liquid biopsies for PDAC and provide new insights into the technical, analytical and biological challenges that must be overcome for this potential to be realised.     

Quantification of cell-free DNA in blood plasma and DNA damage degree in lymphocytes to evaluate dysregulation of apoptosis in schizophrenia patients

Oxidative DNA damage has been proposed as one of the causes of schizophrenia (SZ), and post mortem data indicate a dysregulation of apoptosis in SZ patients. To evaluate apoptosis in vivo we quantified the concentration of plasma cell-free DNA (cfDNA index, determined using fluorescence), the levels of 8-oxodG in cfDNA (immunoassay) and lymphocytes (FL1-8-oxodG index, flow cytometry) of male patients with acute psychotic disorders: paranoid SZ (total N = 58), schizophreniform (N = 11) and alcohol-induced (N = 14) psychotic disorder, and 30 healthy males. CfDNA in SZ (N = 58) does not change compared with controls. In SZ patients. Elevated levels of 8-oxodG were found in cfDNA (N = 58) and lymphocytes (n = 45). The main sources of cfDNA are dying cells with oxidized DNA. Thus, the cfDNA/FL1-8-oxodG ratio shows the level of apoptosis in damaged cells. Two subgroups were identified among the SZ patients (n = 45). For SZ-1 (31%) and SZ-2 (69%) median values of cfDNA/FL1-8-oxodG index are related as 1:6 (p < 0.0000001). For the patients with other psychotic disorders and healthy controls, cfDNA/FL1-8-oxodG values were within the range of the values in SZ-2. Thus, apoptosis is impaired in approximately one-third of SZ patients. This leads to an increase in the number of cells with damaged DNA in the patient's body tissues and may be a contributing cause of acute psychotic disorder.     

Targeted Tissue and Cell-Free Tumor DNA Sequencing of Advanced Lung Squamous-Cell Carcinoma Reveals Clinically Significant Prevalence of Actionable Alterations

Background

Major guidelines do not recommend routine molecular profiling of lung squamous-cell carcinoma (LUSC) because the prevalence of actionable alterations is thought to be low. Increased utilization of next-generation sequencing (NGS), particularly with cell-free circulating tumor DNA, facilitates reevaluation of this premise.

More attention should be paid to pregnant women who fail non-invasive prenatal screening

ObjectiveWe discuss how to handle failure of first-pass non-invasive prenatal screening (NIPS) and investigate the pregnancy outcomes after second-pass failure.MethodsA total of 35,187 pregnant women underwent NIPS in a single center. Those who failed first-pass NIPS were re-tested after a repeat blood draw. Those who failed again were offered genetic counseling. We recorded antenatal data and pregnancy outcomes.ResultsA total of 273 (0.78%) women failed the first test. On re-testing, 220 (80.59%) yielded reliable results and 53 failed the test again. Women with higher total cell-free DNA (cfDNA) levels evidenced a lower NIPS success rate (40%) and a higher incidence of adverse pregnancy outcomes.ConclusionsMost women who failed first-pass NIPS yielded reliable results on repeat testing, especially those with lower fetal fraction. Higher concentrations of cfDNA in maternal plasma were associated with poorer pregnancy outcomes. Such women require special attention, thus early medical intervention, to avoid an adverse prognosis.     

Liquid biopsy is a valuable tool in the diagnosis and management of lung cancer

Liquid biopsy refers to the use of various body fluids to test for circulating biological elements derived from the tumor. Liquid biopsy has taken on an increasingly important role in lung cancer diagnosis, molecular characterization, surveillance, monitoring, and determining mechanisms of resistance. These assays can utilize various sources of cell-free DNA (cfDNA) including blood, pleural fluid, urine, and others to detect tumor associated alterations. With the increasing power of next-generation sequencing technologies and the development of assays such as digital droplet PCR, rare tumor alleles can be detected in cfDNA to determine key characteristics of the tumor. Current assays, while effective, are still challenged by limited sensitivity and capacity to single genes or small panels of genes, though this is rapidly expanding. Nevertheless, testing of cfDNA has been shown to be valuable in detecting resistance to targeted inhibitors, particularly for detection of T790M in EGFR and monitoring response to therapy. With the continued development of more powerful and sensitive assays, these techniques will empower clinicians to better characterize early stage disease and can be used in the screening of high-risk patients, which may eliminate the requirement for tissue diagnosis in some settings. That said, since the majority of these alterations are not specific to lung cancer, there will continue to be a need for tissue in at least the initial diagnosis. Used in conjugation with tissue sampling, these assays will assist the treating clinician and the pathologist to better characterize individual tumors, even in the setting of limited tissue.     

Plasma Circulating Tumor DNA and Clonal Hematopoiesis in Metastatic Renal Cell Carcinoma

BackgroundThere is a lack of molecularly-informed biomarkers for patients with metastatic renal cell carcinoma (RCC). Plasma cell-free DNA (cfDNA) sequencing is a minimally-invasive alternative to tissue for profiling the genome in other cancers but relevance in metastatic RCC remains unclear.Materials and MethodsWhole blood was collected from 55 patients with metastatic RCC. Plasma cfDNA and leukocyte DNA were subjected to targeted sequencing across 981 cancer genes. Matched tumor tissue from 14 patients was analyzed.ResultsThirty-three percent of patients had evidence for RCC-derived circulating tumor DNA (ctDNA), significantly lower than patients with metastatic prostate or bladder cancer analyzed using the same approach. Among ctDNA-positive patients, ctDNA fraction averaged only 3.9% and showed no strong association with clinical variables. In these patients, the most commonly mutated genes were VHL, BAP1, and PBRM1, and matched tissue concordance was 77%. Evidence of somatic expansions unrelated to RCC, such as clonal hematopoiesis of indeterminate potential, were detected in 43% of patients. Pathogenic germline mutations in DNA repair genes were detected in 11% of patients. CtDNA-positive patients had shorter overall survival and progression-free survival on first-line therapy. Patients with evidence of clonal hematopoiesis of indeterminate potential had an intermediate prognosis compared with ctDNA-positive and -negative patients.ConclusionsCfDNA sequencing enables straightforward characterization of the somatic RCC genome in a minority of patients with metastatic RCC. Owing to low ctDNA abundance, and the presence of non-RCC derived somatic clones in circulation, cfDNA sequencing may not be a simple pan-patient alternative to tissue biopsy in metastatic RCC.     

Relationship between plasma cell-free DNA (cfDNA) and prognosis of TACE for primary hepatocellular carcinoma

BackgroundOur study aims to investigate changes in cell-free DNA (cfDNA) concentration and integrity in primary hepatocellular carcinoma (PHC) patients before and after transcatheter arterial chemoembolization (TACE) treatment and their influence on the evaluation of prognosis of the disease.MethodsA total of 84 PHC patients admitted to the Affiliated Hospital of Nanjing University of Chinese Medicine from December 2016 to December 2017 were included as the study group, while 55 healthy people served as the control group. Plasma cfDNA concentration and integrity were determined using qRT-PCR. The correlation between cfDNA concentration/integrity and clinical characteristics of PHC patients were analyzed. A ROC curve was used to investigate the sensitivity and specificity of cfDNA as detection indices. Univariate and multivariate analyses were used to analyze factors affecting recurrence in PHC patients and compare recurrence-free survival (RFS) of PHC patients with high cfDNA expression and low cfDNA expression.ResultsPlasma cfDNA concentration and integrity were significantly higher in PHC patients before TACE treatment than in healthy people and significantly lower after treatment than before (P<0.05). The cfDNA concentration was significantly correlated with tumor size, lymph node metastasis, TNM stage, and BCLC stage, while cfDNA integrity was significantly correlated with tumor size, TNM stage, and BCLC stage (P<0.05). ROC results showed that the area under the curve (AUC) value of cfDNA concentration was the largest, with an optimal cut-off of 10.51 ng/mL. Multivariate regression analysis for COX showed that the TNM stage, cfDNA concentration, and AFP were independent risk factors that affected PHC patients’ survival.ConclusionsPlasma cfDNA concentration in PHC patients is more sensitive and specific than any other tumor marker. It is an independent risk factor for PHC patients treated with TACE. Therefore, it is hypothesized cfDNA is a potential biomarker for prognostic evaluation of PHC patients treated with TACE.     

The value of liquid biopsy in diagnosis and monitoring of diffuse large b-cell lymphoma: recent developments and future potential

Introduction: Diffuse large B-cell lymphomas (DLBCL) represent a heterogeneous subset of non-Hodgkin lymphomas (NHL) that demonstrate many molecular alterations and somatic mutations, all of which are targets for the recent development of biomarkers that use various molecular biological techniques. These non-invasive emerging biomarkers will be used in the next few years to better monitor the response to immunochemotherapeutic treatments with the aim of completely eradicating the disease in order to cure it.

Areas covered: In this review, the authors conducted a literature search to identify and summarize the major advances in liquid biopsy techniques for DLBCL that are useful for diagnosis and monitoring minimal residual disease (MRD). The authors report on the major technological leaps represented by the main MRD tools (sequencing of clone-specific rearrangements of immunoglobulin genes and sequencing of somatic mutations in circulating tumor plasma DNA) and present the expected future developments and the impact of these new tools on clinical practice.

Expert commentary: The monitoring of somatic mutations in tumor plasma cell-free DNA represents a promising tool for liquid biopsy, which will in the future allow non-invasive monitoring that will be used at any time to follow the response to the treatment.     

Prediction model for malignant pulmonary nodules based on cfMeDIP-seq and machine learning

Cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) is a new bisulfite-free technique, which can detect the whole-genome methylation of blood cell-free DNA (cfDNA). Using this technique, we identified differentially methylated regions (DMR) of cfDNA between lung tumors and normal controls. Based on the top 300 DMR, we built a random forest prediction model, which was able to distinguish malignant lung tumors from normal controls with high sensitivity and specificity of 91.0% and 93.3% (AUROC curve of 0.963). In summary, we reported a non–invasive prediction model that had good ability to distinguish malignant pulmonary nodules.