Plasma DNA Analysis Research Articles

Liquid biopsy evaluation of circulating tumor DNA, miRNAs, and cytokines in meningioma patients.

Liquid biopsy is a non-invasive method used to detect cancer and monitor treatment responses by analyzing blood or other bodily fluids for cancer biomarkers. Meningiomas are the most common primary central nervous system tumors, and biomarkers play a crucial role in their diagnosis, prognosis, and treatment monitoring. The World Health Organization (WHO) classifies meningiomas based on tumor grades and molecular alterations in genes such as in NF2, AKT1, TRAF7, SMO, PIK3CA, KLF4, SMARCE1, BAP1, H3K27me3, TERT promoter, and CDKN2A/B. Liquid biopsy, specifically cell-free DNA (cfDNA) analysis, has shown potential for monitoring meningiomas as it can detect ctDNA release in the blood, unaffected by the blood-brain barrier. MicroRNAs (miRNAs) have also been found to be deregulated in various cancers, including meningiomas, presenting potential as diagnostic biomarkers. Additionally, studying cytokines in the tumor microenvironment may aid in establishing prognostic or diagnostic panels for meningiomas. In the present study we analyzed the DNA coming from both the plasma and tumor samples, in addition to analyze miRNA-21 and cytokines in the plasma of 28 meningioma patients. Our findings indicate that the detection of ctDNA in the plasma of meningioma patients is feasible. However, it's important to note that certain challenges persist when comparing plasma DNA analysis to that of tumor tissues. In our study, we observed a paired identification of mutations in only one patient, highlighting the complexities involved. Furthermore, we successfully identified miR-21 and cytokines in the plasma samples. Notably, our analysis of Interleukin 6 (IL-6) unveiled higher expression in the clear cell subtype compared to the other types. Despite the ongoing research, the clinical implementation of liquid biopsy in meningiomas remains somewhat limited. Nevertheless, our promising results underscore the need for further investigation.

Release of Cell-Free Tumor DNA in the Plasma of Uveal Melanoma Patients Under Radiotherapy.

Uveal melanoma (UM) is a tumor of the eye that metastasizes in approximately half of cases. Prognostic testing requires accessibility to tumor tissue, which is usually not available with eye-preserving therapies. Noninvasive approaches to prognostic testing that provide valuable information for patient care are therefore needed. The aim of this study was to evaluate the use of circulating cell-free plasma DNA analysis in UM patients undergoing brachytherapy. The study recruited 26 uveal melanoma patients referred to the department between February and October 2020. Blood samples were collected at various time points before, during, and after treatment, and deep amplicon sequencing was used to identify oncogenic variant alleles of the GNAQ and GNA11 genes, which serve as indicators for the presence of circulating tumor DNA (ctDNA). The results showed that all patients were ctDNA negative before brachytherapy. In 31% of patients, ctDNA was detected during therapy. The variant allele fraction of GNAQ or GNA11 alleles in ctDNA positive samples ranged from 0.24% to 2% and correlates with the largest basal diameter and thickness of the tumor. The findings suggest that brachytherapy increases the presence of tumor DNA in the plasma of UM patients. Thus ctDNA analysis may offer a noninvasive approach for prognostic testing. However, efforts are still required to lower the limit of detection for tumor-specific genetic alterations.

Quantitative analysis of cell-free plasma DNA as a prognostic biomarker in acute ischemic stroke patients

BackgroundStroke has become a leading cause of death and disability worldwide, and despite the introduction of new screening programs, therapies, and monitoring technologies, there is still a need to develop more useful biochemical tests to monitor treatment response and inform clinical decision-making. Cell-free DNA released from damaged neurons in stroke patients may be useful in assessing stroke prognosis. The purpose of this study was to evaluate the role of cell-free DNA and a highly sensitive blood biomarker in acute ischemic stroke. 188 patients with acute ischemic stroke were recruited for the study. The level of cell-free DNA in plasma was estimated using a real-time PCR assay for the β-globin gene (Qiagen-Rotor-Gene Q MDX, Germany). Clinical assessment was performed with the National Institutes of Health Stroke Scale (NIHSS) at the time of admission. After a period of three months from the onset of stroke, (mRS) scores were estimated using the modified Rankin scale.ResultsElevated levels of cell-free DNA were found in patients with higher NIHSS admission scores and mRS 3-month scores (p < 0.05). The regression analysis revealed that the markers are associated with an unfavorable outcome in comparison to other stroke risk factors (R2 = 0.224). Stroke outcome was relatively better in patients with a cell-free DNA level < 10,000 kilogenome equivalents/L (p < 0.05).ConclusionsMeasurement of total cell-free DNA levels is a simpler and less-expensive biomarker suggesting potential clinical application of blood-based test. Cell-free DNA can contribute to the clinical evaluation and optimal management of ischemic stroke patients. This biomarker seems to have the potential to predict the long-term prognosis of acute ischemic stroke.

Largest evaluation of acquired resistance to sotorasib in KRAS p.G12C-mutated non–small cell lung cancer (NSCLC) and colorectal cancer (CRC): Plasma biomarker analysis of CodeBreaK100.

102 Background: Sotorasib, a specific, irreversible KRASG12C inhibitor, has been approved in multiple countries for adults with KRAS p.G12C-mutated locally advanced or metastatic NSCLC who received prior systemic therapy based on the global phase 1/2 CodeBreaK100 trial. Here we describe putative mechanisms of acquired resistance to sotorasib from the largest single dataset evaluated to-date. Methods: Patients with advanced KRAS p.G12C-mutated NSCLC or CRC from the CodeBreaK100 Ph1/2 trial who received sotorasib monotherapy at 960 mg once daily were analyzed for efficacy. Primary endpoint was objective response rate (ORR) assessed by central review. To investigate biomarkers of resistance to sotorasib, an exploratory endpoint was defined to examine acquired genomic alterations at disease progression. Plasma samples collected at baseline and progression were analyzed for genomic alterations with the 23-gene Resolution Bioscience ctDx Lung test for NSCLC and the 74-gene Guardant 360 ctDNA test for CRC. Acquired genomic alterations were defined by their absence at baseline and presence at progression. Results: In 174 pts with NSCLC and 91 pts with CRC-treated with sotorasib, the ORR were 41% and 12% respectively. Median progression-free survival and median overall survival were 6.3 months (mos) and 12.5 mos for NSCLC pts and 4.2 mos and 13.4 mos for CRC pts (median follow-up: 22.5 mos NSCLC; 12.5 mos CRC). A total of 67 NSCLC pts and 45 CRC pts had a plasma sample sequenced both at baseline and at progression. At least one new acquired genomic alteration at progression was detected in 19 (28%) NSCLC pts and in 33 (73%) CRC pts (Table). The acquired genomic alterations were heterogeneous in both NSCLC and CRC, with variants detected across multiple genes and pathways. The most prevalent putative pathway of resistance in both NSCLC and CRC was the receptor tyrosine kinase (RTK) pathway. Secondary RAS alterations occurred more frequently in CRC versus NSCLC pts (16% vs. 3%). Conclusions: Based on the largest descriptive dataset to-date, diverse mechanisms of acquired resistance occur in KRAS p.G12C-mutated NSCLC and CRC pts treated with sotorasib. New RTK pathway alterations frequently emerged at progression, highlighting the potential role for combining sotorasib with upstream inhibitors of RTK, such as SHP2 or EGFR inhibitors. Serial plasma DNA analysis revealed acquired resistance patterns that support the development of KRASG12C inhibitor combination therapies. Clinical trial information: NCT03600883. [Table: see text]

Quantitative analysis of plasma DNA in anal cancer patients.

IntroductionThe availability and non-invasiveness of circulating cell-free DNA (cfDNA) opens up new possibilities for real-time serial testing. The relationship between cfDNA concentration, clinical factors and suitability for monitoring was analyzed in patients with newly diagnosed anal squamous cell carcinoma (ASCC).Material and methodsBlood samples were collected at several points during and after treatment. Patients were homogeneously treated with chemoradiotherapy.ResultsThe concentration of cfDNA strongly correlated with the tumor volume (r = 0.9, p = 0.00006) and number of neutrophils (r = 0.706, p = 0.0069). Monitoring of cfDNA levels during treatment showed an increase after initiation of therapy, a peak at the end of treatment with significantly higher values for advanced than in T1/T2 tumors, and a decrease (T3/T4) during follow-up. However, neither the concentration of cfDNA before treatment nor its changes correlated with the response to chemoradiotherapy. There was no association between baseline cfDNA levels and T, N, age and gender.ConclusionsSubstantial changes in plasma cfDNA content can be observed after chemoradiotherapy for ASCC. However, the small number of cases studied makes it difficult to assess the usefulness of the cfDNA test.

Single-molecule sequencing reveals a large population of long cell-free DNA molecules in maternal plasma

In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5' fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5' 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia.

Combining liquid biopsy and functional imaging analysis in metastatic castration-resistant prostate cancer helps predict treatment outcome.

Plasma tumour DNA (ptDNA) is a potential early noninvasive biomarker of treatment outcome in metastatic castration‐resistant prostate cancer (mCRPC). Herein, we investigated whether pretreatment ptDNA levels reflect metabolic tumour burden in mCRPC and better predict treatment outcome in combination with functional imaging. Targeted next‐generation sequencing was performed to estimate the ptDNA fraction from 102 mCRPC patients receiving abiraterone or enzalutamide. The maximum standardized uptake value (SUVmax), total lesion activity (TLA) and metabolic tumour volume (MTV) were evaluated on 18F‐fluorocholine positron emission tomography/computed tomography. We assessed a Weibull multiple regression model to determine the combined impact of clinical, molecular and imaging characteristics on overall survival (OS) and progression‐free survival (PFS), and to obtain prognostic scores. A significant association was seen between ptDNA and SUVmax, MTV and TLA. For survival analysis, patients were randomly allocated into a training (n = 68) and a validation (n = 34) set. In the training set, multivariable analyses showed that ptDNA, MTV and serum lactate dehydrogenase together with visceral metastasis were independent predictors of both OS and PFS. Prognostic scores were generated, with the identification of three groups of patients with significantly different median OS (29.2, 15.9 and 8.7 months) and PFS (13.3, 7.7 and 3.2 months) probabilities. The differences in median survival between risk groups were confirmed in the validation cohort for both OS and PFS. In our study, we showed that integrating plasma DNA analysis with functional imaging may improve prognostic risk stratification and treatment selection in mCRPC.

Fragmentation Analysis of Plasma DNA Reveals Its Prognostic Value in Gastric Cancer.

Gastric cancer (GC) is a common cause of cancer-related deaths. The poor clinical outcome in GC patients is partially associated with a lack of appropriate diagnostic and prognostic biomarkers. In the present study, we evaluated the diagnostic and prognostic values of cell-free DNA (cfDNA) integrity and the concentration of circulating nucleosomes (cNUCs). In the study, 40 GC patients and 55 GC-free individuals were enrolled. Cell-free DNA integrity was calculated as the ratio of concentration of the longer ACTB (beta-actin) gene fragment to that of the shorter ACTB fragment, measured using quantitative PCR. Circulating nucleosomes were measured by an ELISA-based approach. We found that cfDNA integrity is higher in GC patients than in the control subjects (relative median values 0.51 vs. 0.38, respectively, P = .56) indicating prominent abundance of longer fragments in the patients. The patients with larger tumors (T3-4) had significantly higher cfDNA integrity than those with T1-T2 tumors. We also found GC patients to have higher concentrations of cNUCs in their plasma (relative median values 3.64 vs. 3.1). Importantly, the patients with high cfDNA integrity (i.e., lower fragmentation) had longer overall survival rates at 3 years than those with lower cfDNA integrity (76.5% vs. 38.9%, P = .02). Cell-free DNA fragmentation has a prognostic value. However, it has no diagnostic value in GC.

583P Baseline plasma tumour DNA (ptDNA) correlates with PSA kinetics in metastatic castration-resistant prostate cancer (mCRPC) treated with abiraterone or enzalutamide

Baseline high ptDNA fraction and androgen receptor (AR) copy number (CN) gain identify mCRPC patients (pts) with worse outcomes on abiraterone (abi) or enzalutamide (enza). This study aimed to assess if plasma DNA analysis associates with PSA kinetics.

Comparative analysis of nuclear and mitochondrial DNA from tissue and liquid biopsies of colorectal cancer patients

The current standard for molecular profiling of colorectal cancer (CRC) is using resected or biopsied tissue specimens. However, they are limited regarding sampling frequency, representation of tumor heterogeneity, and sampling can expose patients to adverse side effects. The analysis of cell-free DNA (cfDNA) from blood plasma, which is part of a liquid biopsy, is minimally invasive and in principle enables detection of all tumor-specific mutations. Here, we analyzed cfDNA originating from nucleus and mitochondria and investigated their characteristics and mutation status in a cohort of 18 CRC patients and 10 healthy controls using targeted next-generation sequencing (NGS) and digital PCR. Longitudinal analyses of nuclear cfDNA level and size during chemotherapy revealed a decreasing cfDNA content and a shift from short to long fragments, indicating an appropriate therapy response, while shortened cfDNAs and increased cfDNA content corresponded with tumor recurrence. Comparative NGS analysis of nuclear tissue and plasma DNA demonstrated a good patient-level concordance and cfDNA revealed additional variants in three of the cases. Analysis of mitochondrial cfDNA surprisingly revealed a higher plasma copy number in healthy subjects than in CRC patients. These results highlight the potential clinical utility of liquid biopsies in routine diagnostics and surveillance of CRC patients as complementation to tissue biopsies or as an attractive alternative in cases where tissue biopsies are risky or the quantity/quality does not allow testing.

Complementary detection of genomic alterations in metastatic castration-resistant prostate cancer (mCRPC) from CheckMate 9KD through analyses of tumor tissue and plasma DNA.

5038 Background: Accurate analysis of the genomic alteration landscape within tissue- and plasma-derived tumor DNA using next-generation sequencing (NGS) may provide insights into specific patient populations that benefit from different therapies. The interchangeable use of tissue- and plasma-based assessments may benefit patients when tissue availability is limited, a common occurrence in individuals with mCRPC. To understand the potential sources of technical and biological variability in this setting, we performed comprehensive comparative analyses across 3 NGS platforms, using samples from patients enrolled in CheckMate 9KD, a phase 2 study of nivolumab combined with docetaxel, rucaparib, or enzalutamide for patients with confirmed mCRPC (NCT03338790). Methods: We performed retrospective integrated analyses of sequence and structural alterations identified through comprehensive genomic profiling (CGP) of DNA obtained from formalin-fixed, paraffin-embedded tissue specimens and cell-free DNA obtained from plasma. Tissue-based analysis was performed using the FoundationOne assay (F1, 395 genes), while the FoundationACT (FACT, 70 genes) and GuardantOMNI (OMNI, 500 genes) assays were used for plasma-based analysis. Analysis was performed on samples from 103 patients for which datasets from all 3 assays were available. Inter-platform analysis considered variants with ≥ 0.50% variant allele fraction and common to the shared pairwise targeted regions, while excluding synonymous variants. Results: Through broad profiling of DNA obtained from tissue and plasma, we uncovered previously identified recurrent alteration of AR, TP53, PTEN, and TMPRSS2 fusion with ETS genes. Additionally, we found that 42% (F1), 45% (FACT), and 34% (OMNI) of patients harbored a combination of germline and somatic mutations in homologous recombination repair pathway genes. Across all samples, median tumor mutational burden was 3.5 mutations per megabase (mut/Mb) by F1 and 8.6 mut/Mb by OMNI. Inter-platform variant analyses demonstrated concordance of 52% for F1 vs FACT, 40% for F1 vs OMNI, and 75% for FACT vs OMNI. Conclusions: Overall, these data demonstrate the value of integrated tissue and liquid biopsy profiling in mCRPC. Both technical and biological sources of variation, including panel size, mutation detection algorithms, variant annotation and reporting, analytical performance, circulating tumor DNA levels, and tumor heterogeneity, may be captured differently by tissue- and plasma-based techniques, accounting for the discordance in reported results. Clinical trial information: NCT03338790.

Combining molecular and functional imaging in metastatic castration-resistant prostate cancer (mCRPC).

141 Background: Recently, plasma tumour DNA (ptDNA) has been identified as a potential early noninvasive biomarker of treatment response in mCRPC patients ( Conteduca, Br J Cancer 2020). In this study, we sought to determine whether pre-treatment ptDNA could accurately reflect metabolic tumor burden in mCRPC and if it could be in combination with functional imaging could provide better prognostication. Methods: Between October 2011 and June 2016, 102 plasma samples from mCRPC patients treated with abiraterone or enzalutamide were collected. Targeted next-generation sequencing was performed to determine baseline ptDNA fraction. Maximum standardized uptake value (SUVmax), total lesion activity (TLA), and metabolic tumour volume (MTV) were calculated on 18F-fluorocholine positron emission tomography/computed tomography. A Weibull multiple regression model was adopted to evaluate the combined impact of clinical, molecular and imaging features on overall survival (OS) and to obtain a prognostic score. Each variable was allotted a “partial score” that depended on the size of the regression coefficient. Total scores ranged between 0 and 5.85 and assigned patients to 3 different risk groups according to 18-months survival probability: group I, &gt;70%; group II 30%-70%; and group III, &lt;30%. We estimated OS probabilities by the exponential model and by the Kaplan-Meier method. Results: We observed a significant association between ptDNA levels dichotomized as below or above median plasma tumor fraction (low ptDNA≤0.188 versus high ptDNA&gt;0.188) and median SUVmax (p&lt;0.0001), MTV (p=0.0005) and TLA (p&lt;0.0001). Patients were randomly divided into a training set (n=68) and a validation set (n=34). In the training cohort, we performed a multivariable analysis showing that visceral metastasis, serum LDH, MTV and ptDNA were independent predictors of OS [HR=2.64, 95%CI 1.32-5.26, p=0.006; HR=3.69, 95%CI 1.98-6.87, p&lt;0.0001; HR=1.91, 95%CI 1.13-3.21, p=0.015; and HR=2.64, 95%CI 1.32-5.26, p=0.003, respectively]. In the training set, median OS was significantly different among the 3 risk groups (risk group I, 29.2 months [95% CI, 18.3 to 37.0 months]; risk group II, 15.9 months [95% CI, 10.6 to 24.0 months]; and risk group III, 8.7 months [95% CI, 6.3 to 15.4 months]; p&lt;0.0001). Similar results were observed in the validation set groups (risk group I, 23.4 months [95% CI, 8.1 to 38.5 months]; risk group II, 13.3 months [95% CI, 3.7 to 18.0 months]; and risk group III, 7.3 months [95% CI, 2.6 to 11.8 months]; p=0.001). Conclusions: Integrating plasma DNA analysis with functional imaging may improve prognostic risk stratification and treatment selection in mCRPC patients. A larger prospective evaluation is now warranted.

Frequency of S492R mutations in the epidermal growth factor receptor: analysis of plasma DNA from patients with metastatic colorectal cancer treated with panitumumab or cetuximab monotherapy

ABSTRACT Background Antibodies against epidermal growth factor receptor (EGFR), panitumumab, a fully human monoclonal antibody, and cetuximab, a human/mouse chimeric monoclonal antibody, have shown clinical efficacy in metastatic colorectal cancer (mCRC). In the phase 3 noninferiority ASPECCT (ClinicalTrials.gov, NCT01001377) study, panitumumab was demonstrated to be noninferior to cetuximab and provided a similar overall survival benefit for patients with chemotherapy-refractory wild-type KRAS exon 2 mCRC. However, some patients eventually develop resistance to anti-EGFR therapy. EGFR p.S492R mutation was previously identified as conferring resistance to cetuximab, but not to panitumumab. Methods This biomarker study analyzed plasma samples from ASPECCT collected at both baseline and posttreatment. Results No EGFR p.S492R mutations were identified at baseline; however, after treatment the EGFR p.S492R mutation was detected in 1% of patients treated with panitumumab versus 16% of those treated with cetuximab, supporting that, in a large population, this mutation is more likely to be induced by cetuximab than by panitumumab. There were, however, no significant differences in progression-free survival or overall survival between patients who were wild-type compared with those with the S492R mutation within the cetuximab arm or the overall population. Conclusions These results may support targeting treatment to small patient subgroups based on the presence of emerging EGFR mutations and provide a molecular rationale for rechallenging with a different anti-EGFR agent in patients who develop resistance. Prospective studies are needed to evaluate the efficacy of panitumumab in the EGFR p.S492R mutant population.

Identification and characterization of extrachromosomal circular DNA in maternal plasma

We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at ∼202 and ∼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5'-untranslated regions (5'-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications.

A Multiplex PCR-Based Assay for Detection of Plasma Cell Free DNA Integrity as a Marker of Bladder Cancer

Background: Cell free DNA (cfDNA) analysis in patient plasma has been suggested for screening of different cancers. Healthy cells may shed more degraded DNA than non-apoptotic tumor cells. Objectives: Our goal was to check whether DNA fragments extracted from patient’s plasma with bladder cancer (BC) had higher integrity than DNA purified from plasma of healthy controls. Methods: cfDNA was extracted from the plasma of a healthy control group and patients with BC. The association between clinical status and length of DNA fragments was examined. Plasma cfDNA integrity was examined using multiplex PCR with specific primers producing progressively long PCR fragments (200, 400 & 800-bp). Length and concentration of PCR amplicons obtained from BC patients and healthy individuals were compared. Results: Using a multiplex PCR assay, the used p53 primers efficiently amplified the increasingly long fragments. High molecular weight DNA fragments (400 & 800-bp) in the plasma cfDNA are associated with the presence of cancer. Moreover, elevation of the 200-bp fragment concentration in the plasma cfDNA is significantly (p < 0.002) associated with the presence of cancer. Conclusions: The detected long DNA fragments in plasma cfDNA is associated with BC indicating that the multiplex PCR assay of plasma cfDNA integrity could be a useful future marker for the detection of BC.

A Multiplex PCR-Based Assay for Detection of Plasma Cell Free DNA Integrity as a Marker of Bladder Cancer

Background: Cell free DNA (cfDNA) analysis in patient plasma has been suggested for screening of different cancers. Healthy cells may shed more degraded DNA than non-apoptotic tumor cells. Objectives: Our goal was to check whether DNA fragments extracted from patient’s plasma with bladder cancer (BC) had higher integrity than DNA purified from plasma of healthy controls. Methods: cfDNA was extracted from the plasma of a healthy control group and patients with BC. The association between clinical status and length of DNA fragments was examined. Plasma cfDNA integrity was examined using multiplex PCR with specific primers producing progressively long PCR fragments (200, 400 & 800-bp). Length and concentration of PCR amplicons obtained from BC patients and healthy individuals were compared. Results: Using a multiplex PCR assay, the used p53 primers efficiently amplified the increasingly long fragments. High molecular weight DNA fragments (400 & 800-bp) in the plasma cfDNA are associated with the presence of cancer. Moreover, elevation of the 200-bp fragment concentration in the plasma cfDNA is significantly (p < 0.002) associated with the presence of cancer. Conclusions: The detected long DNA fragments in plasma cfDNA is associated with BC indicating that the multiplex PCR assay of plasma cfDNA integrity could be a useful future marker for the detection of BC.

Antenatal screening for fetal trisomies using microarray-based cell-free DNA testing: A systematic review and meta-analysis.

To evaluate the test accuracy of non-invasive prenatal testing (NIPT) for fetal trisomy 21, 18, and 13 using cell-free (cf) DNA analysis in maternal plasma with microarray quantitation. Systematic review and meta-analysis. Searches in MEDLINE, Pre-MEDLINE, EMBASE, Web of Science, and the Cochrane Library to 09.07.2018. Five studies analyzing 3074 samples, including 187 trisomy 21, 43 trisomy 18, and 19 trisomy 13 cases, were identified. Risk of bias was high in all studies, introduced particularly by exclusions from analysis and by the role of the sponsor. Sensitivity of microarray-based cfDNA testing was 99.5% (95%CI 96.3%-99.9%) for trisomy 21, 97.7% (95%CI 87.9%-99.6%) for trisomy 18, and 100% (95%CI 83.2%-100%) for trisomy 13. Specificity was 100% (95% CI 99.87%-100%) for trisomy 21, 99.97% (95%CI 99.81%-99.99%) for trisomy 18, and 99.97% (95%CI 99.81%-99.99%) for trisomy 13. Pooled test failure rate was 1.1%. A direct comparison of microarray- and sequencing-based cfDNA found equivalent test accuracy. Included studies suggest that NIPT using microarray-based cfDNA testing has high sensitivity and specificity for detecting fetal trisomy 21, 18, and 13. However, the evidence base is small and at high risk of bias.

Multimodal Approach to Outcome Prediction in Metastatic Castration-Resistant Prostate Cancer by Integrating Functional Imaging and Plasma DNA Analysis.

Biomarkers for treatment personalization in metastatic castration-resistant prostate cancer (mCRPC) could help improve patient outcomes. Multiple tests on blood have reported associations with poorer outcome, including serum lactate dehydrogenase (LDH), chromogranin A (CGA), neutrophil:lymphocyte ratio (NLR), and, recently, copy number (CN) of androgen receptor (AR) in plasma DNA. Biologic data suggest an association between choline uptake and AR signaling. We aimed to integrate 18F-fluorocholine (FCH) uptake on positron emission tomography/computed tomography (PET/CT) scanning with plasma AR CN and other routinely obtained circulating biomarkers to evaluate their association with outcome. We determined plasma AR CN by digital droplet polymerase chain reaction from 105 mCRPC samples collected before abiraterone (n = 65) or enzalutamide (n = 40) therapy in the before (n = 26) and after (n = 79) chemotherapy settings. Pretreatment serum LDH, CGA, and NLR were also measured. FCH-PET/CT scan was performed at baseline, and maximum standardized uptake value (SUVmax), total lesion activity (TLA), and metabolic tumor volume (MTV) were calculated. Main end points were the correlation of FCH-PET/CT parameters with circulating biomarkers and their impact on outcome. Plasma AR CN gain was observed in 27 patients (25.7%), and it correlated significantly with higher median SUVmax, TLA, and MTV values (P < .001). Kaplan-Meier curves showed significantly worse progression-free survival and overall survival in patients with plasma AR gain and higher SUVmax, TLA, and MTV values (P < .001 in each prognostic group). Conversely, no association was reported for prostate-specific antigen response. On multivariable analysis of overall survival, we showed as independent factors AR gain (hazard ratio [HR], 1.92; 95% CI, 1.07 to 3.47; P = .029), presence of visceral metastasis (HR, 3.04; 95% CI, 1.66 to 5.58; P = < .001), LDH (HR, 2.95; 95% CI, 1.72 to 5.05; P < .001), NLR (HR, 3.51; 95% CI, 2.14 to 5.74; P < .001), serum CGA (HR, 3.36; 95% CI, 1.99 to 5.67; P < .001), and MTV (HR, 2.09; 95% CI, 1.25 to 3.50; P = .005). Our results indicate the potential usefulness of integrating functional imaging with plasma DNA analysis and other noninvasive biomarkers as a tool to improve treatment selection for CRPC. A larger prospective evaluation is warranted.

Effects of Quantification Methods, Isolation Kits, Plasma Biobanking, and Hemolysis on Cell-Free DNA Analysis in Plasma.

Cell-free DNA (cfDNA) has become a promising noninvasive clinical marker widely studied in early disease detection, monitoring, and therapy selection. However, there is lack of data on a number of cfDNA-associated procedural features such as blood plasma biobanking conditions, isolation, and quantification methods that should be taken into account as they can affect downstream applications. In this study cfDNA from 125 plasma samples from healthy individuals were isolated using three different commercial kits (bead and vacuum based). Yield of cfDNA, distribution of cfDNA fragments and absolute amount of miR-223 were estimated. Moreover, the impact of different plasma biobanking conditions and hemolytic plasma were evaluated. In conclusion, results showed that quantification method (fluorescence or microcapillary electrophoresis based) has a major impact in estimating cfDNA amount. Samples isolated by QIAamp showed a higher amount of larger (around 300 bp) DNA fragments and miRNA yield, suggesting possible applications for multiomics approach. On the other hand, the highest cfDNA yield was obtained in samples isolated by the MagMAX Isolation Kit. This kit also showed lowest coefficient of variation and low miRNA yield. Plasma storage conditions and hemolysis affected performance of isolation kits differently.

Methylation analysis of plasma DNA informs etiologies of Epstein-Barr virus-associated diseases

Epstein-Barr virus (EBV) is associated with a number of diseases, including malignancies. Currently, it is not known whether patients with different EBV-associated diseases have different methylation profiles of circulating EBV DNA. Through whole-genome methylation analysis of plasma samples from patients with nasopharyngeal carcinoma (NPC), EBV-associated lymphoma and infectious mononucleosis, we demonstrate that EBV DNA methylation profiles exhibit a disease-associated pattern. This observation implies a significant potential for the development of methylation analysis of plasma EBV DNA for NPC diagnostics. We further analyse the plasma EBV DNA methylome of NPC and non-NPC subjects from a prospective screening cohort. Plasma EBV DNA fragments demonstrate differential methylation patterns between NPC and non-NPC subjects. Combining such differential methylation patterns with the fractional concentration (count) and size of plasma EBV DNA, population screening of NPC is performed with an improved positive predictive value of 35.1%, compared to a count- and size-based only protocol.