ccfDNA Concentration Research Articles

P10.24.B PREDICTION OF PCFDNA LEVELS, PROGRESSION-FREE SURVIVAL, AND OVERALL SURVIVAL THROUGH MACHINE-LEARNING MODELS BASED ON MRI-DERIVED RADIOMIC FEATURES IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA

Abstract BACKGROUND Circulating cell-free DNA (ccfDNA) is a promising tool for monitoring patients with high-grade gliomas (HGGs) in addition to follow-up with conventional (cMRI) and advanced MRI (aMRI), which harbors an enormous amount of quantitative subvisual data that can be used to build models predicting disease behavior. Examining the relationship between ccfDNA levels and quantitative features extracted from cMRI and aMRI gives further insight into this novel biomarker, aiming to combine the biological value of ccfDNA and the spatial resolution of image biomarkers to improve HGGs monitoring. MATERIAL AND METHODS Features were extracted from each T1 post-contrast and FLAIR images by using the FLAIR hyperintensity and enhancement automatic segmentation tool (Pyradiomics software, v2.2.0). The radiomic dataset underwent Z-score normalization, feature reduction, and feature selection. Highly correlated features were excluded using a threshold of 0.9 person-correlation-coefficient. The ‘F_regression’ function was used to select the most important features concerning the ccfDNA concentration, Overall survival (OS), and progression-free survival (PFS). This dataset was used as input for four machine learning models: Linear Regression (LR), Support Vector Regression (SVR), Random Forest (RF), and least absolute shrinkage and selection operator (LASSO). Hyperparameter tuning was performed at the first training iteration of each model using a 4-fold cross-validation GridSearchCV and model-specific parameters. The train-test strategy involved randomizing datasets into 80% training and 20% testing subsets across 100 iterations, ensuring robustness and reducing biases. RMSE values of each run were averaged based on sample test frequencies. Root means square error (RMSE) was used to assess the model’s performance. RESULTS 405 features were successfully extracted from T1 post-contrast and FLAIR images from both tumoral masks. A total of 287 were found to be highly correlated and, therefore, removed from the dataset. The ‘F_regression’ with a 90-percentile threshold separates 12 features divided into 3 shapes features relative to FLAIR segmentation, 6 features relative to the T1 post-contrast image, and 3 features for the FLAIR image. The SVR achieves the best RMSE performances when compared to other models when predicting the ccfDNA concentration, reaching an RMSE of 8.58. An analogous strategy was used for the models built to predict PFS and OS reaching an RMSE of 5.89 and 6.77, respectively. CONCLUSION Machine-learning models based on radiomic features combined with clinical variables are valuable tools for predicting OS and PFS in HGGs’ patients. The radiomic signature provides additional information compared to the tumoral volume to forecast ccfDNA levels. Combining both techniques could provide more precise assessment of disease status.

Prognostic Value of Circulating Cell-Free DNA Concentration and Neutrophil-to-Lymphocyte Ratio in Patients with Pancreatic Ductal Adenocarcinoma: A Prospective Cohort Study.

Circulating cell-free DNA (ccfDNA) quantity correlates with the clinical characteristics and prognosis of various cancer types. We investigated whether ccfDNA levels and the neutrophil-to-lymphocyte ratio (NLR) have prognostic value in patients with pancreatic ductal adenocarcinoma (PDAC). Peripheral blood was collected from 82 patients with PDAC prior to any diagnostic procedure or the administration of chemotherapy. Plasma DNA was isolated, and ccfDNA concentration and NLR were determined. We found that ccfDNA levels were correlated with age and tumor burden. Moreover, higher values of NLR (≥3.31) were linked with worse overall survival (OS) (4 vs. 10 months; log rank p = 0.011), and an elevated ccfDNA concentration (≥25.79 ng/mL) was strongly associated with shorter OS (4 vs. 8 months; log rank p = 0.009). According to the results of the multivariable Cox regression analysis, the baseline concentration of ccfDNA was an independent prognostic factor for OS (HR 0.45, 95% CI 0.21-0.97, p = 0.041). Furthermore, the combination of ccfDNA levels with NLR greatly enhanced the prognostic accuracy of PDAC patients. Our study demonstrates that ccfDNA concentration and NLR are independent predictors of survival in PDAC. Subsequent studies should validate this combination as a prognostic indicator in PDAC patients and assess its utility for guiding therapeutic decisions.

Circadian rhythm and circulating cell-free DNA release on healthy subjects

In the last decade, clinical studies have investigated the clinical relevance of circulating cell-free-DNA (ccfDNA) as a diagnostic and prognosis tool in various diseases including cancers. However, limited knowledge on ccfDNA biology restrains its full development in the clinical practice. To improve our understanding, we evaluated the impact of the circadian rhythm on ccfDNA release in healthy subjects over a 24-h period. 10 healthy female subjects underwent blood sampling at 8am and 20 healthy male subjects underwent serial blood sampling (8:00 AM, 9:00 AM, 12:00 PM, 4:00 PM, 8:00 PM, 12:00 AM, 4 AM (+ 1 Day) and 8 AM (+ 1 Day)). We performed digital droplet-based PCR (ddPCR) assays to target 2 DNA fragments (69 & 243 bp) located in the KRAS gene to determine the ccfDNA concentration and fragmentation profile. As control, half of the samples were re-analyzed by capillary miniaturized electrophoresis (BIAbooster system). Overall, we did not detect any influence of the circadian rhythm on ccfDNA release. Instead, we observed a decrease in the ccfDNA concentration after meal ingestion, suggesting either a post-prandial effect or a technical detection bias due to a higher plasma load in lipids and triglycerides. We also noticed a potential effect of gender, weight and creatinine levels on ccfDNA concentration.

Circulating cell-free-DNA concentration is a good biomarker for diagnosis of colorectal cancer in Mexican patients.

Colorectal cancer (CRC) is the third most common cancer in the world. Overall survival is related to clinical stage: more advanced stages show lower survival rates; therefore, they need to be monitored regularly with new, less invasive and more specific biomarkers. The concentration and integrity index of circulating cell-free DNA (ccfDNA) have been proposed as potential diagnostic and prognostic biomarkers for CRC, however, inconsistent results are still observed in different reports. Here we analyze these potential CRC biomarkers in a Mexican population. In this study, 124 patients with sporadic CRC and 37 healthy individuals were examined as a reference group. The ccfDNA was isolated from plasma samples of all included subjects. The ccfDNA concentration was determined by fluorometry and the integrity index (ALU247/ALU115 ratio) by quantitative PCR amplification (qPCR) of ALU sequences. The results show that ccfDNA concentration was higher in CRC patients than in the reference group (P=0.001). The integrity index showed no significant differences between these groups (P=0.258), except for histological type (P=0.012). A higher ccfDNA concentration was also associated with patients younger than 50 years (P=0.030). The ccfDNA concentration showed significant discriminatory power (AUC: 0.854, C.I.: 0.78-0.92, P=0.001) between patients and the reference group and between tumor-node-metastasis (TNM) stages. In conclusion, ccfDNA concentration proves to be a good diagnostic biomarker for CRC patients, whereas the integrity index did not show diagnostic utility.

BIOM-18. METHYLATION ANALYSIS TO REVEAL THE CELLULAR ORIGIN OF PROGNOSTIC CIRCULATING CELL-FREE DNA IN GLIOBLASTOMA

Abstract We have previously demonstrated an independent association between high levels of plasma circulating cell-free DNA (ccfDNA) concentration and poor survival outcomes in patients with newly diagnosed glioblastoma. Given that somatic mutations are rarely detected in glioblastoma patient plasma, we reasoned that DNA shed by tumor (circulating tumor DNA, ctDNA) was not likely to be a driver of prognostic increases in ccfDNA. To investigate the tissue of origin for this prognostic ccfDNA, we analyzed the plasma ccfDNA methylation signatures of 23 patients with newly diagnosed glioblastoma using the Illumina Infinium EPIC array, a genome-wide DNA methylation technique covering over 850,000 CpG sites. Deconvolution of the ccfDNA methylation signatures based on published reference methylomes revealed an increased proportion of ccfDNA derived from granulocytes in glioblastoma specimens compared to healthy controls (p < 0.0001). Further, this granulocyte proportion was increased in patients with high ccfDNA levels (above median value) compared to patients with low ccfDNA (p = 0.0001). Granulocyte proportion correlated with ccfDNA concentration (Spearman r = 0.64, p = 0.001). The top two gene sets identified by differential methylation analysis followed by gene set enrichment analysis (methylGSA) between high- and low-ccfDNA specimens were “neutrophil activation involved in immune response” (GO:0002283, p = 3.4E-23) and “neutrophil degranulation” (GO:0043312, p = 3.4E-23). Additional analysis of ccfDNA fragment size identified larger fragments ( > 700 bp) as the major source of the prognostic signal (log-rank p = 0.002 for progression-free survival) compared to traditionally sized ccfDNA fragments (50-700 bp, log-rank p = 0.12). Taken together, these data suggest that granulocytes are the primary contributing source of prognostic ccfDNA in glioblastoma. Future studies are needed to determine the mechanism through which granulocyte-derived ccfDNA is associated with clinical outcomes in glioblastoma and to explore related therapeutic opportunities.

Mass Spectrometry as a Highly Sensitive Method for Specific Circulating Tumor DNA Analysis in NSCLC: A Comparison Study.

Simple SummaryWe compared the UltraSEEK™ Lung Panel on the MassARRAY® System (Agena Bioscience) with the FDA-approved Cobas® EGFR Mutation Test v2 for the detection of EGFR mutations in liquid biopsies of NSCLC patients, accompanied with preanalytical sample assessment using the novel Liquid IQ® Panel. For the detection of relevant predictive mutations using the UltraSEEK™ Lung Panel, an input of over 10 ng showed 100% concordance with Cobas® EGFR Mutation Test v2 and detection of all tissue confirmed mutations. In case of lower ccfDNA input, the risk of missing clinically relevant mutations should be considered. The use of a preanalytical ccfDNA quality control assay such as the Liquid IQ® Panel is recommended to confidently interpret results, avoiding bias induced by non-specific genomic DNA and low input of specific tumoral ccfDNA fragments.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® EGFR Mutation Test v2 with the UltraSEEK™ Lung Panel on the MassARRAY® System on detection of EGFR mutations, accompanied with preanalytical sample assessment using the novel Liquid IQ® Panel. 137 cancer patient-derived cell-free plasma samples were analyzed with the Cobas® and UltraSEEK™ tests. Liquid IQ® analysis was initially validated (n = 84) and used to determine ccfDNA input for all samples. Subsequently, Liquid IQ® results were applied to harmonize ccfDNA input for the Cobas® and UltraSEEK™ tests for 63 NSCLC patients. The overall concordance between the Cobas® and UltraSEEK™ tests was 86%. The Cobas® test detected more EGFR exon19 deletions and L858R mutations, while the UltraSEEK™ test detected more T790M mutations. A 100% concordance in both the clinical (n = 137) and harmonized (n = 63) cohorts was observed when >10 ng of ccfDNA was used as determined by the Liquid IQ® Panel. The Cobas® and UltraSEEK™ 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.

Investigating the relationship between ccfDNA concentration, its integrity, and some individual factors in an Iranian population.

Circulating cell-free DNA (ccfDNA) increases in some pathologic conditions like cancer. We aimed to investigate the correlation between some individual factors and the ccfDNA level in peripheral blood of Iranian in relation to prostate cancer. 30 patients with prostate cancer (PCa), 40 with benign prostate hyperplasia (BPH), and 30 controls were studied. Personal information, ccfDNA concentration, and the integrity index were assessed for the correlation between the disease and different factors. The results were statistically analyzed using SPSS software. In PCa group, no association was found between total ccfDNA, BMI, BPH background, non-cancerous diseases, medications, PCa length, and job (p-value > 0.05). But, total ccfDNA had statistical associations with weight, family history of cancer, and location (p-value < 0.05). No association was between the integrity of ccfDNA, weight, the background of BPH, and family history of cancer. But, the integrity of ccfDNA was significantly associated with BMI and PCa length (p-value < 0.05).In BPH group, no association between total ccfDNA or the integrity of ccfDNA and the assessed factors was obtained (p-value > 0.05). In the normal group, neither statistical association was found between total ccfDNA, weight, BMI, and job, nor between the integrity of ccfDNA, weight, BMI, non-cancerous disease, drug, job, and location (p-value > 0.05). But, a statistical association was found between the integrity of ccfDNA and family history of cancer in the recent group (Based on 95% CI and P-value less than 0.05). ccfDNA and its integrity as possible prostate cancer biomarkers under the influence of individuals' physiological status are prone to the pathologic changes toward the disease. Further simultaneous study of the target groups could clarify this matter.

The value of the plasma circulating cell-free DNA concentration and integrity index as a clinical tool for prostate cancer diagnosis: a prospective case-control cohort study in an Iranian population.

Introduction: Prostate cancer (PCa) is the most common cancer among men and the second cause of cancer death among men. For early detection and differentiating PCa from benign prostate hyperplasia (BPH) tissue biopsy has been used for decades. However, circulating cell-free DNA (ccfDNA) testing is a noninvasive, fast, easily repeatable, and sensitive liquid biopsy for cancer detection. Hence, we aimed to investigate the value of the ccfDNA concentration and integrity index in peripheral blood of a population of Iranian prostatic patients for early diagnosis of the disease.Materials and methods: 100 subjects including 30 PCa, 40 BPH, and 30 healthy individuals were selected. ccfDNA was extracted from fresh blood plasma, and its total concentration and the integrity index were estimated by amplification of ALU115 and ALU247 repeat elements using quantitative real-time PCR.Results: In the PCa group, the ccfDNA concentration and its integrity were significantly higher than that of the BPH and healthy groups (P-value <0.001 and P-value <0.001). The ccfDNA concentration and its integrity were higher in BPH compared to the healthy group, although it was not statistically significant (P-value =0.836 and P-value =0.053, respectively).Conclusion: A significant relation between ccfDNA concentration, its integrity, and PCa suggests that the liquid biopsy can be used as a noninvasive early diagnostic biomarker. Determination of a cutoff or a diagnostic range value of the measured parameters for healthy, BPH, and PCa subjects in more samples of Iranian population results in timely, correct, and early detection, which results in better treatment outcomes. Moreover, this method may reduce overdiagnosis and overtreatment procedures.

Evaluation of circulating cell free DNA in plasma as a biomarker of different thyroid diseases

IntroductionMany studies have been done on proteomics, genomics, epigenetic, immunogenetics in many body fluids. Among these, circulating cell-free DNA (ccfDNA) entered the literature in 1948, but it has not been studied for many years due to technological deficiencies. Following recent advances, geno-metastasis has been mentioned and new research is needed in this area. ccfDNA is known to be an important biomolecule in this regard. ObjectiveThe presence of cell-free DNA in the circulatory system may offer a tremendous opportunity to provide novel biomarkers for thyroid diseases. This experimental study was conducted to determine the amount of ccfDNA in different thyroid diseases, then to evaluate whether the ccfDNA concentration varied between the disease groups and control group. MethodsIn total, we included 121 individuals in the present study. We collected blood samples and then determined the ccfDNA concentration in plasma of collected blood samples from three groups: thyroiditis (n=33), benign (n=37), and malignant (n=30) and from a control group (n=21). ResultsThe median values of the ccfDNA groups were found as 1610, 1665, 1685 and 576ng/mL for the thyroiditis, benign, malign, and control groups, respectively. Findings showed that the ccfDNA of the three groups was significantly higher than the control (p<0.0001). Each group was compared in terms of ccfDNA and the p-values of benign-thyroiditis, benign-malign, and thyroiditis-malign were 0.09, 0.65, and 0.29, respectively. ConclusionsThe clear differences between thyroid diseases and controls suggest that ccfDNA is worthy of attention as a biomarker for further evaluation of different thyroid diseases. Likewise, it might indicate a clear tendency that ccfDNA can also be used to distinguish different thyroid diseases.

Plasma free DNA: Evaluation of temperature-associated storage effects observed for Roche Cell-Free DNA collection tubes.

IntroductionStandardized pre-analytical blood sample procedures for the analysis of circulating cell-free DNA (ccfDNA) are still not available. Therefore, the present study aimed at evaluating the impact of storage conditions related to different times (24 and 48 h) and temperatures (room temperature (RT) and 4 - 8 °C) on the plasma ccfDNA concentration of blood samples drawn into Cell-Free DNA collection tubes (Roche Diagnostics GmbH, Mannheim, Germany).Materials and methodsVenous blood from 30 healthy individuals was collected into five 8.5 mL Cell-Free DNA Collection Tubes (Roche Diagnostics GmbH) each. Plasma samples were processed at time point of blood collection (tube 1), and after storage under the following conditions: 24 h at RT (tube 2) or 4-8 °C (tube 3), and 48 h at RT (tube 4) or 4 - 8 °C (tube 5). Circulating cell-free DNA concentrations were determined by EvaGreen chemistry-based droplet digital PCR (ddPCR).ResultsNo statistically significant differences between median (interquartile range) plasma ccfDNA concentrations (ng/mL) at time point of blood collection (3.17 (2.13 – 3.76)) and after storage for 24 h (RT: 3.02 (2.41 – 3.68); 4-8 °C: 3.21 (2.19 – 3.46)) and 48 h (RT: 3.13 (2.10 – 3.76); 4-8 °C: 3.09 (2.19 – 3.50)) were observed (P values from 0.102 – 0.975).ConclusionsNo unwanted release of genomic DNA from white blood cells could be detected in plasma samples after tube storage for 24 and 48 h regardless of storage temperature.

Assessment of the circulating cell-free DNA marker association with diagnosis and prognostic prediction in patients with lymphoma: a single-center experience

Circulating cell-free DNA (ccfDNA) has been shown to be associated with the clinical characteristics and prognosis of cancer patients. Our objective was to assess whether the concentration and integrity index of ccfDNA in plasma may be useful for diagnosing and monitoring the progression of patients with lymphoma. We included plasma samples from 174 lymphoma patients and 80 healthy individuals. The total concentration of ccfDNA was determined using a fluorometry method, and the DNA integrity index (DII), which is the ratio of longer to shorter DNA fragments, for the APP gene was detected using real-time quantitative PCR. The median levels of the ccfDNA concentration and the DII in patients with lymphoma were significantly higher than those in controls (both P < 0.0001). Increases in the ccfDNA concentration and the DII were associated with advanced stage disease, elevated lactate dehydrogenase levels, and a higher prognosis score. In patients with diffuse large B cell lymphoma (DLBCL), high levels of ccfDNA (both concentration and the DII) showed an inferior 2-year progression-free survival (PFS) (P = 0.001; P < 0.0001, respectively). Our study provides quantitative and qualitative evidence in favor of using ccfDNA analysis in lymphoma patients for diagnostic and prognostic assessments.

Impact of Pneumatic Tube System Transport of Whole Blood Samples on Free DNA Concentration in Human Plasma

To the Editor: During the last decade the interest in using circulating cell-free DNA (ccfDNA)1 as a biomarker for the early diagnosis, prognosis, and monitoring of a variety of clinical conditions has increased rapidly. In daily clinical practice, it has been shown that preanalytical variables such as blood sampling, handling-procedurevariations, and blood storage conditions affect the ccfDNA concentration in human plasma (1). In this respect, optimal handling of blood samples between venipuncture and plasma preparation may be considered essential for downstream processes (1, 2) since time delay and/or agitation could trigger the release of genomic DNA from white blood cells (1). To reduce laboratory turnaround time, sample transport by pneumatic tube systems (PTS) has been established in many hospitals. However, rapid accelerations and decelerations during the PTS transport may cause shear stress and can potentially affect various laboratory measurements for coagulation, clinical chemistry, hematology, or blood gas analyses (3). At present, the literature lacks studies investigating the impact of PTS transport on quantitative ccfDNA measurements. Therefore, we compared ccfDNA plasma concentrations between paired whole …

Circulating DNA as a Strong Multimarker Prognostic Tool for Metastatic Colorectal Cancer Patient Management Care.

Circulating cell-free DNA (ccfDNA) is a valuable source of tumor material obtained from a simple blood sampling that enables noninvasive analysis of the tumor genome. Our goal was to carry out a multiparametric analysis of ccfDNA and evaluate its prognostic value by investigating the overall survival (OS) of 97 metastatic colorectal cancer patients (mCRC). Qualitative parameters (determination of the main KRAS exon2 and BRAF V600E mutations) and quantitative parameters (total ccfDNA concentration, mutant ccfDNA concentration, the proportion of mutant ccfDNA, and ccfDNA integrity index) were determined simultaneously in a single run using a unique Q-PCR multimarker approach (100% success rate). The median follow-up time was 36 months and median OS was 22 months. Patients showing high ccfDNA levels had significantly shorter OS (18.07 months vs. 28.5 months, P = 0.0087). Moreover, multivariate analysis revealed that a high ccfDNA level is an independent prognostic factor (P = 0.034). All ccfDNA parameters were of prognostic interest: patients with higher levels of mutant ccfDNA and higher mutation loads for the detected mutations had shorter OS (P = 0.0089 and P = 0.05, respectively). In addition, the level of ccfDNA fragmentation correlated positively with decreased OS in the exclusive KRAS/BRAF-mutant cohort of patients (P = 0.0052) and appeared as a strong independent prognostic factor (P = 0.0072), whereas it was not significant in the exclusive KRAS/BRAF WT cohort of patients (P = 0.67). Our data provide for the first time qualitative and quantitative evidence in favor of multiparametric ccfDNA analysis in mCRC patients for prognostic assessment. Clin Cancer Res; 22(12); 3067-77. ©2016 AACR.

Multi-marker analysis of circulating cell-free DNA toward personalized medicine for colorectal cancer

Development of a Q-PCR-based assay for the high-performance analysis of circulating cell-free DNA (ccfDNA) requires good knowledge of its structure and size.In this work, we present the first visual determination of ccfDNA by Atomic Force Microscopy (AFM) on plasma samples from colorectal cancer (CRC) patients and healthy donors. In addition to the examination of fragment size distribution profile as performed by Q-PCR, this analysis confirms that ccfDNA is highly fragmented and that more than 80% of ccfDNA fragments in CRC plasma are below 145 bp. We adapted an Allele-Specific Blocker (ASB) Q-PCR to small ccfDNA fragments to determine simultaneously the total ccfDNA concentration, the presence of point mutation, the proportion of mutated allele, and a ccfDNA integrity index. The data validated analytically these four parameters in 124 CRC clinical samples and 71 healthy individuals. The multi-marker method, termed Intplex, enables sensitive and specific non-invasive analysis of tumor ccfDNA, which has great potential in terms of cost, quality control, and easy implementation in every clinical center laboratory.