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Plasma circulating cell‐free nucleic acids (ccfNAs) provide an exceptional source of information about an individual's health, yet their biology in healthy individuals during aging remains poorly understood. Here, we present the first integrative multiparametric analysis of the major types of plasma ccfNAs, including nuclear (ccfnDNA) and mitochondrial (ccfmtDNA) DNA, as well as ribosomal (ccfrRNA), messenger (ccfmRNA) and micro‐RNA (ccfmiRNA) in 139 healthy donors aged 19–66 years. We focused on quantity, integrity, and DNA methylation using an optimized experimental workflow that combines highly sensitive analytical methods with the detection of highly repetitive DNA and highly abundant RNA sequences, thereby reducing the required amount of ccfNAs per analysis. We showed a highly significant increase in ccfnDNA levels during aging (p < 0.001), associated with a decrease in its integrity (p < 0.05), while no significant changes were detected in ccfmtDNA levels and ccfDNA methylation. Moreover, a significant increase in ccfmRNA and ccfrRNA (p < 0.05), as well as miR‐483‐5p (p < 0.001) levels was detected during aging, but without any changes in ccfRNA integrity. Finally, we also showed that ccfDNA and ccfRNA levels were correlated (p < 0.001), and a similar pattern was observed for ccfmtDNA and ccfRNA levels, suggesting a possible common release, maintenance, and/or clearance mechanism. Therefore, our study provides an optimized workflow for the global analysis of ccfNAs, enhances the understanding of their biology during aging, and identifies several potential ccfNA‐based biomarkers of aging. We performed the first integrative multiparametric analysis of plasma circulating cell‐free nucleic acids, including nuclear and mitochondrial DNA, as well as ribosomal, messenger, and micro‐RNAs, in a cohort of 139 healthy individuals. We focused on quantity, integrity, and DNA methylation using assays targeting highly repetitive DNA and highly abundant RNA.

Background Advanced gastric cancers are usually associated with incurable conditions for which systemic treatments are indicated. Recent studies suggest that circulating cell-free plasma DNA of tumour origin (tDNA) is a promising non-invasive biomarker that can be used to predict the prognosis and monitor the efficacy of systemic treatments in patients with certain types of cancer. We conducted a pilot study to analyse the potential role of tDNA as a biomarker in patients with advanced gastric cancer. Methods We included 30 patients with locally advanced unresectable or metastatic gastric cancer. We obtained samples (10 mL of total blood) from each patient every 3 months and performed concomitant CT until disease progression or death. Total cell-free circulating DNA (cfDNA) samples were measured using GeneQuant RNA/DNA Calculator-Amersham Pharmacia Biotech (Biochrom) Ltd. The cfDNA was used to evaluate the ALU DNA sequences 247 and 115. The level of tDNA was calculated from the ratio of the expression of ALU DNA sequences and the concentration of total cell-free DNA. We utilized the RECIST criteria 1.1 to evaluate the tumour response. Results Patients with advanced gastric cancer had significantly higher concentrations of cfDNA compared with normal controls ( p  = 0.00015), which allowed us to conclude that the cfDNA in the patients originated from the tumour. We did not find any significant correlation between the level of tDNA and OS or tumour response. However, after the first cycles of chemotherapy (at 3 months), we observed that patients with lower tDNA levels had significantly longer DFS compared with those with higher levels (Cox Regression p  = 0.0228). Conclusions At 3 months after the beginning of chemotherapy, the tDNA levels are correlated with DFS in patients with advanced gastric cancer who receive systemic chemotherapy. tDNA may be a specific, non-invasive and cost effective new biomarker for these patients.

Reliable biomarkers are required to evaluate and manage pancreatic ductal adenocarcinoma. Circulating tumor cells and circulating tumor DNA are shed into blood and can be relatively easily obtained from minimally invasive liquid biopsies for serial assays and characterization, thereby providing a unique potential for early diagnosis, forecasting disease prognosis, and monitoring of therapeutic response. In this review, we provide an overview of current technologies used to detect circulating tumor cells and circulating tumor DNA and describe recent advances regarding the multiple clinical applications of liquid biopsy in pancreatic ductal adenocarcinoma. This review provides an overview of current technologies used to detect circulating tumor cells and circulating tumor DNA and recent advances regarding the multiple clinical applications of liquid biopsy in pancreatic ductal adenocarcinoma. Recent studies focusing on the diagnostic, prognostic, and predictive aspects of circulating tumor cells and circulating tumor DNA in pancreatic ductal adenocarcinoma are also discussed.

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. •We present the first visual determination of ccfDNA short fragments from plasma samples by AFM.•AFM analysis confirms that ccfDNA is highly fragmented.•This report describes the first integrated ccfDNA multi-marker Q-PCR analytical test.•ccfDNA concentration, detection of mutated allele, and integrity index are analyzed in this test.•This test provides a fast, cost effective, sensitive, and simple analysis of ccfDNA.

In this study, we evaluated the clinical utility of detecting KRAS mutations in circulating cell‐free (ccf)DNA of metastatic colorectal cancer patients. We prospectively recruited 94 metastatic colorectal cancer patients. Circulating cell‐free DNA was extracted from plasma samples and analyzed for the presence of seven KRAS point mutations. Using the Invader Plus assay with peptide nucleic acid clamping method and digital PCR, KRAS mutations were detected in the ccfDNA in 35 of 39 patients previously determined to have primary tumors containing KRAS mutations using the Luminex method, and in 5 of 55 patients with tumors containing wild‐type KRAS. Curative resection was undertaken in 7 of 34 patients with primary and ccfDNA KRAS mutations, resulting in the disappearance of the mutation from the cell‐free DNA in five of seven patients. Three of these patients had tumor recurrence and KRAS mutations in their ccfDNA reappeared. Epidermal growth factor receptor blockade was administered to 24 of the KRAS tumor wild‐type patients. Of the 24 patients with wild‐type KRAS in their primary tumors, three patients had KRAS mutations in their ccfDNA and did not respond to treatment with epidermal growth factor receptor (EGFR) blockade. We also detected a new KRAS mutation in five patients during chemotherapy with EGFR blockade, before disease progression was detectable with imaging. The detection of KRAS mutations in ccfDNA is an attractive approach for predicting both treatment response and acquired resistance to EGFR blockade, and for detecting disease recurrence. We showed the usefulness of detecting KRAS mutations in cell‐free DNA in patients with KRAS wild‐type and mutated colorectal cancer using our new high‐sensitivity method and digital PCR. It is as an attractive method for predicting patient prognosis, monitoring tumor progression, and predicting response to epidermal growth factor receptor (EGFR) blockade, allowing for more personalized patient care. Moreover, we showed the utility of detection of KRAS mutations in cell‐free DNA as a biomarker for early detection of recurrence in patients with KRAS mutated tumors.

The mitochondrial free radical theory of aging suggests that accumulating oxidative damage to mitochondria and mitochondrial DNA (mtDNA) plays a central role in aging. Circulating cell‐free mtDNA (ccf‐mtDNA) isolated from blood may be a biomarker of disease. Extracellular vesicles (EVs) are small (30–400 nm), lipid‐bound vesicles capable of shuttling proteins, nucleic acids, and lipids as part of intercellular communication systems. Here, we report that a portion of ccf‐mtDNA in plasma is encapsulated in EVs. To address whether EV mtDNA levels change with human age, we analyzed mtDNA in EVs from individuals aged 30–64 years cross‐sectionally and longitudinally. EV mtDNA levels decreased with age. Furthermore, the maximal mitochondrial respiration of cultured cells was differentially affected by EVs from old and young donors. Our results suggest that plasma mtDNA is present in EVs, that the level of EV‐derived mtDNA is associated with age, and that EVs affect mitochondrial energetics in an EV age‐dependent manner. Mitochondrial dysfunction plays a central role in aging. A portion of circulating cell‐free mitochondrial DNA (mtDNA) isolated from blood is present in extracellular vesicles (EVs), which are small, lipid‐bound vesicles that shuttle macromolecules as part of intercellular communication systems. The level of EV‐derived mtDNA declines with age, and these EVs affect mitochondrial energetics in an EV age‐dependent manner.

Circulating cell‐free DNA (cfDNA) fragmentomics, which encompasses the measurement of cfDNA length and short nucleotide motifs at the ends of cfDNA molecules, is an emerging field for cancer diagnosis. The utilization of cfDNA fragmentomics for the diagnosis of patients with hepatocellular carcinoma (HCC) caused by hepatitis B virus (HBV) is currently limited. In this study, we utilized whole‐genome sequencing data of cfDNA in samples from patients with HCC (n = 197) and HBV (n = 187) to analyze the association of fragment size selection (< 150 bp) with tumor fraction (TF), copy number variation (CNV) alterations and the change in the proportion of 4‐mer end motifs in HCC and HBV samples. Our analyses identified five typical CNV markers (i.e. loss in chr1p, chr4q and chr8p, and gain in chr1q and chr8q) in cfDNA with a cumulatively positive rate of ˜ 95% in HCC samples. Size selection (< 150 bp) significantly enhanced TF and CNV signals in HCC samples. Additionally, three 4‐mer end motifs (CCCA, CCTG and CCAG) were identified as preferred end motifs in HCC samples. We identified 139 end motifs significantly associated with fragment size that showed similar patterns of associations between patients with HCC and HBV, suggesting that end motifs might be inherently coupled with fragment size by a ubiquitous mechanism. Here we conclude that CNV markers, fragment size selection and end‐motif pattern in cfDNA have potential for effective detection of patients with HCC. Circulating cell‐free DNA (cfDNA) fragmentomics, encompassing the measurement of cfDNA length and short nucleotide motifs at cfDNA ends, is an emerging field in cancer diagnostics. In this study, we utilized whole‐genome sequencing data of cfDNA in patients with hepatocellular carcinoma (HCC). We observed that representative abnormal copy number variation (CNV) alterations and shorter fragment size (< 150 bp) selection enhanced the accuracy of CNV detection and improved the clinical utility of ctDNA in HCC. We also discovered a strong correlation of end motif type to fragment size and revealed similar fragment size characteristics of 4‐mer end motifs between patients with HCC and hepatitis B virus.

The clinical relevance of the colorectal cancer serrated pathway is evident, but the screening of serrated lesions remains challenging. We aimed to characterize the serum methylome of the serrated pathway and to evaluate circulating cell‐free DNA (cfDNA) methylomes as a potential source of biomarkers for the non‐invasive detection of serrated lesions. We collected serum samples from individuals with serrated adenocarcinoma (SAC), traditional serrated adenomas, sessile serrated lesions, hyperplastic polyps and individuals with no colorectal findings. First, we quantified cfDNA methylation with the MethylationEPIC array. Then, we compared the methylation profiles with tissue and serum datasets. Finally, we evaluated the utility of serum cfDNA methylation biomarkers. We identified a differential methylation profile able to distinguish high‐risk serrated lesions from no serrated neoplasia, showing concordance with tissue methylation from SAC and sessile serrated lesions. Serum methylation profiles are pathway‐specific, clearly separating serrated lesions from conventional adenomas. The combination of ninjurin 2 (NINJ2) and glutamate‐rich 1 (ERICH1) methylation discriminated high‐risk serrated lesions and SAC with 91.4% sensitivity (64.4% specificity), while zinc finger protein 718 (ZNF718) methylation reported 100% sensitivity for the detection of SAC (96% specificity). This is the first study exploring the serum methylome of serrated lesions. Differential methylation of cfDNA can be used for the non‐invasive detection of colorectal serrated lesions. We analyzed the serum methylome of precursory lesions of the colorectal cancer serrated pathway and found a differential methylation profile between high‐risk serrated lesions and no serrated neoplasia. Since the methylation profiles in serum cfDNA are pathway‐specific at early stages, they may serve as a source of non‐invasive biomarkers for the detection of serrated lesions in screening programs.

Noninvasive circulating tumor DNA (ctDNA) can be used to predict breast cancer recurrence and prognosis. In this study, we detected 226 and 114 somatic variants in tumor DNA from 70 primary breast cancer (PBC) patients (98.59%) and ctDNA from 48 patients (67.61%), respectively. Gene frequencies of tumor DNA and ctDNA significantly correlated (R2 = 0.9532, P < 0.0001), and tumor‐derived variants were detectable in the blood of 43 patients. ctDNA was more often detected in locally advanced/metastatic and nonluminal patients. Multivariate analysis revealed that individual N stage (P < 0.001) and hormone receptor (HR) status (P = 0.001) could independently predict the detectability of tumor‐derived mutations in blood. The maximal variant allele frequency of ctDNA was significantly higher in patients with stage IV/M1 (P = 0.0136) and stage T3/T4 (P = 0.0085) cancers. Finally, clonal variants in tumor DNA were more easily traced in ctDNA than subclonal variants (84.62% vs 48.75%). In conclusion, ctDNA fragments concordant with tumor DNA can be consistently detected in the majority of tested PBC patients, which may enable noninvasive genomic profiling of PBC, particularly for patients with advanced‐stage tumors and positive HR status. Detection of noninvasive circulating tumor DNA (ctDNA) is a promising method for evaluating tumor burden for breast cancer. However, the detectability of ctDNA is heterogeneous for each patient. In this study, we investigated the clinicopathological and biological factors affecting detectability of ctDNA, which may help in identifying suitable cohorts for ctDNA‐based liquid biopsy.

Glioblastoma (GBM) is the most common type of glioma and is uniformly fatal. Currently, tumour heterogeneity and mutation acquisition are major impedances for tailoring personalized therapy. We collected blood and tumour tissue samples from 25 GBM patients and 25 blood samples from healthy controls. Cell‐free DNA (cfDNA) was extracted from the plasma of GBM patients and from healthy controls. Tumour DNA was extracted from fresh tumour samples. Extracted DNA was sequenced using a whole‐genome sequencing procedure. We also collected 180 tumour DNA datasets from GBM patients publicly available at the TCGA/PANCANCER project. These data were analysed for mutations and gene–gene fusions that could be potential druggable targets. We found that plasma cfDNA concentrations in GBM patients were significantly elevated (22.6 ± 5 ng·mL−1), as compared to healthy controls (1.4 ± 0.4 ng·mL−1) of the same average age. We identified unique mutations in the cfDNA and tumour DNA of each GBM patient, including some of the most frequently mutated genes in GBM according to the COSMIC database (TP53, 18.75%; EGFR, 37.5%; NF1, 12.5%; LRP1B, 25%; IRS4, 25%). Using our gene–gene fusion database, ChiTaRS 5.0, we identified gene–gene fusions in cfDNA and tumour DNA, such as KDR–PDGFRA and NCDN–PDGFRA, which correspond to previously reported alterations of PDGFRA in GBM (44% of all samples). Interestingly, the PDGFRA protein fusions can be targeted by tyrosine kinase inhibitors such as imatinib, sunitinib, and sorafenib. Moreover, we identified BCR–ABL1 (in 8% of patients), COL1A1–PDGFB (8%), NIN–PDGFRB (8%), and FGFR1–BCR (4%) in cfDNA of patients, which can be targeted by analogues of imatinib. ROS1 fusions (CEP85L–ROS1 and GOPC–ROS1), identified in 8% of patient cfDNA, might be targeted by crizotinib, entrectinib, or larotrectinib. Thus, our study suggests that integrated analysis of cfDNA plasma concentration, gene mutations, and gene–gene fusions can serve as a diagnostic modality for distinguishing GBM patients who may benefit from targeted therapy. These results open new avenues for precision medicine in GBM, using noninvasive liquid biopsy diagnostics to assess personalized patient profiles. Moreover, repeated detection of druggable targets over the course of the disease may provide real‐time information on the evolving molecular landscape of the tumour. Personalized therapy of patients with glioblastoma (GBM) is challenging owing to tumour heterogeneity. Here, we extracted and sequenced cell‐free DNA (cfDNA) from the plasma of 25 GBM patients and tumour DNA from fresh tumour samples. We found that cfDNA concentrations in the plasma of GBM patients were significantly elevated, as compared to healthy controls. Moreover, we identified unique mutations and gene–gene fusions in the cfDNA and tumour DNA of GBM patients, some of which could be therapeutically targeted by tyrosine kinase inhibitors.