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The dysregulation of proliferating and apoptotic processes is a common feature in cancerogenesis. Thus, apoptotic products released into blood circulation are suggested as promising markers for the early cancer detection. However though sensitive assays are available, the lack of organ- and tumor-specificity limits the usefulness of most apoptotic parameters for screening purposes. However, they seem to be valuable for the prognosis and the prediction of response to systemic chemo- or radiotherapy in cancer disease. Here, the relevance of diverse circulating apoptotic markers is reviewed for the clinical management of patients with lung cancer. Among those promising markers are ligands and receptors of the FAS-system, members of the intracellular caspase cascade, cleaved apoptosis substrates such as cytokeratines, nucleosomal DNA, and apoptosis modulators like survivin. The review discusses their role for diagnosis, prognosis and therapy monitoring of lung cancer disease. [ABSTRACT FROM AUTHOR]

Summary: The increasing panel of systemic therapies enables the individual management of cancer patients, even in advanced stages. However, diagnostic tools indicating early the efficacy of therapy are still needed. In prospectively collected sera of 161 patients with recurrent non-small cell lung cancer (NSCLC) receiving second-line chemotherapy, the courses of nucleosomes, cytokeratin-19 fragments (CYFRA 21-1), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and progastrin-releasing peptide (ProGRP) were investigated and correlated with therapy response. At high specificity for detection of progressive disease, most sensitive biomarkers were identified and included in a combination model. High levels and insufficient decreases of nucleosomes and CYFRA 21-1 during the first cycle of therapy indicated poor outcome. Combination of nucleosome concentrations at day 8 and CYFRA 21-1 before start of the second cycle enabled the early detection of progressive disease with a sensitivity of 34.4% at 95% specificity (AUC 0.79) prior to imaging techniques. When cutoffs were fixed at the 90th percentile of responding patients, the combination model achieved sensitivities of 19% at 100% specificity and of 52% at 88% specificity. Thus, nucleosomes and CYFRA 21-1 showed to be valuable for the individual management of patients with recurrent NSCLC. [Copyright &y& Elsevier]

Nucleosomes, complexes of DNA and histone proteins, are released from dying and stressed cells into the blood circulation. Concentrations of circulating nucleosomes in plasma and serum are frequently found to be elevated in various cancers, and also in such acute conditions as stroke, trauma, and sepsis as well as in autoimmune diseases. The first part of this review focuses on the structural and functional properties of nucleosomes, the potential sources of nucleosome release into the circulation, the metabolism of circulating nucleosomes, and their pathophysiological role in disease. It goes on to describe the relevance of circulating nucleosomes in the diagnosis and prognosis of non-malignant conditions such as sepsis, stroke, and autoimmune disease. Finally, it describes the clinical value of nucleosomes in the diagnosis, staging, prognosis, and monitoring of therapy in cancer; in particular, their potential as a new diagnostic tool for the early estimation of response to cytotoxic cancer therapy is emphasized. [ABSTRACT FROM AUTHOR]

Nucleosomes, complexes of DNA and histone proteins, are released during cell death into the blood circulation. Elevated serum and plasma levels have been found in various forms of cancer, but also in autoimmune diseases and acute situations such as stroke, trauma, and during sepsis. Here, the clinical relevance of circulating nucleosomes for diagnosis, staging, prognosis, and therapeutic monitoring of cancer is reviewed. Several studies have shown that levels of nucleosomes are significantly higher in serum and plasma of cancer patients in comparison to healthy controls. However, because of elevations of nucleosome levels in patients with benign diseases relevant for differential diagnosis, they are not suitable for cancer diagnosis. Concerning tumor staging, nucleosome levels correlate with tumor stage and presence of metastases in gastrointestinal cancer, but not in other tumor types. Prognostic value of circulating nucleosomes is found in lung cancer in univariate analyses, but not in multivariate analyses. Circulating nucleosomes are most informative for the monitoring of cytotoxic therapy. Strongly decreasing levels are mainly found in patients with remission of disease, whereas constantly high or increasing values are associated with progressive disease during chemo- and radiotherapy. In addition, therapy outcome is already indicated by the nucleosomal course during the first week of chemo- and radiotherapy in patients with lung, pancreatic, and colorectal cancer as well as in hematologic malignancies. Despite their non-tumor-specificity, kinetics of nucleosomes are valuable markers for the early estimation of therapeutic efficacy and may be helpful to adapting early cancer therapy in the future. [ABSTRACT FROM AUTHOR]

This study aimed to test the diagnostic utility of the DNA integrity index for detection of cancer. Matched serum and plasma samples of 42 patients with various forms of cancer and 17 patients with corresponding benign diseases were analyzed. DNA was isolated from 1 mL serum and plasma using the MagNA Pure LC system. Cell-free DNA was quantified by real-time PCR using the reference gene of the LightCycler t(14;18) kit. Subsequently, the DNA integrity index was calculated as the ratio in relative abundance of 347-bp versus 137-bp PCR products. We found that DNA concentrations were not different in plasma (median: 4.5 ng/mL) and serum (67.1 ng/mL) of patients with benign diseases when compared with values in plasma (5.1 ng/mL) and serum (65.4 ng/mL) of cancer patients. Similarly, the DNA integrity index in plasma (0.38), and serum of patients with benign diseases (0.29) was comparable to values in plasma (0.33) and serum (0.37) of cancer patients. Diagnostic sensitivity of DNA (AUC 0.53) and DNA integrity (AUC 0.45) was poor in plasma, and was increased only slightly by the combination of both (AUC 0.57). In serum, sensitivity of DNA (AUC 0.52) and DNA integrity (AUC 0.64) was higher and was further improved by the combination of both (AUC 0.72) reaching a sensitivity of 30% at 100% specificity. In conclusion, we could not confirm a high diagnostic utility of the DNA integrity index. However, a combination with other markers such as DNA may enhance sensitivity for cancer detection. [ABSTRACT FROM AUTHOR]

The prognostic relevance of blood markers in cerebral stroke is still a matter of controversial debate. In sera of 63 patients, nucleosomes, neuronspecific enolase (NSE), S100 protein, and C-reactive protein (CRP) were determined daily during the first week after cerebral stroke. Infarction volume was quantified by CT or MRI and the clinical status by Barthel Index (BI) at admission, discharge, and after 12 months (prognosis). All markers were evaluated by univariate and multivariate analysis on their prognostic relevance. During observation time (12 months), three patients died and 33 reached complete recovery. Infarction volume, nucleosomes, NSE, S100, and CRP correlated significantly with clinical status at admission. The same markers except CRP and initial BI correlated with recovery after 12 months. Almost all patients with initial BI ‡ 50 reached complete recovery. In patients with initially severe defects (BI < 50), nucleosomes and S100, both at day 3, were found to be prognostically relevant. At 100%-specificity for non-recovery, only nucleosomes maintained their prognostic power (sensitivity 52.6%; p = 0.014), whereas S100 did not (sensitivity 16.7%; p = 0.25). In multivariate analysis, nucleosomes and BI at admission showed independent prognostic relevance (p = 0.039). Circulating nucleosomes and clinical scores provide independent prognostic information concerning the later outcome in patients with initially severe defects after stroke. [ABSTRACT FROM AUTHOR]

Facing an era of promising new antitumor therapies, predictors of therapy response are needed for the individual management of treatment. In sera collected prospectively from 311 patients with advanced non-small cell lung cancer receiving first-line chemotherapy, changes in nucleosomal DNA fragments, cytokeratin-19 fragments (CYFRA 21–1), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and progastrin-releasing peptide (ProGRP) were investigated and correlated with therapy response. In univariate analysis, high levels, slower and incomplete decline in nucleosomal DNA, CYFRA 21–1, and CEA predicted poor outcome. DNA concentrations at day 8 of the first therapeutic cycle and CYFRA 21–1 before start of the second cycle were identified as best predictive variables. In multivariate analysis, they predicted progression with a specificity of 100% in 29% of the cases earlier than imaging techniques. Thus, nucleosomal DNA and CYFRA 21–1 specifically identify a subgroup of patients with insufficient therapy response at the early treatment phase and showed to be valuable for disease management. [ABSTRACT FROM AUTHOR]

The inadequate response of immune cells to circulating apoptotic products, such as nucleosomal DNA fragments, is assumed to be a potent stimulus for the production of autoantibodies during the pathogenesis and progression of systemic lupus erythematosus (SLE). Here, we analyzed the levels of circulating nucleosomes, caspases, and C-reactive protein in sera of 244 individuals with various autoimmune diseases (155 with autoimmune hepatic disorders, 25 with ANCA-associated vasculitis, and 64 with various connective tissue diseases), and 32 healthy controls. Nucleosomes and caspase activities were significantly elevated in sera of patients with hepatic autoimmune diseases, connective tissue diseases, and particularly in ANCA-associated vasculitis when compared with healthy individuals. Nucleosomes showed a correlation with caspases, and caspases with C-reactive protein, but nucleosomes did not correlate with C-reactive protein. Serum levels of the apoptotic products, nucleosomes, and caspases are increased in various autoimmune diseases but may not be solely responsible for antinucleosome antibody production in SLE patients. It remains to be clarified whether qualitative changes in nucleosomes are linked with pathogenesis and disease progression in SLE. [ABSTRACT FROM AUTHOR]

Background: Elevated levels of nucleosomal DNA fragments can be detected in plasma and sera of patients with malignant diseases. Methods: We investigated the course of nucleosomal DNA, thymidine kinase, lactate dehydrogenase and leukocytes in sera of 25 patients with acute myeloid leukemia during the first cycle of induction chemotherapy and tested their power to distinguish between patients with complete remission and those with no remission. Results: Almost all patients showed strongly decreasing levels of nucleosomal DNA during the first week, in some cases after initial peaks. In overall analysis of variance, DNA levels could clearly distinguish between patients with complete remission, who had higher DNA values, and those with insufficient response (p = 0.017). The area under the curve of DNA values of days 2-4 after start of therapy (AUC 2-4) discriminated between both groups with a sensitivity of 56% at a specificity of 100%. Further, pretherapeutic levels and AUC 2-4 of nucleosomal DNA correlated significantly with blast reduction after 16 days. A tendency to higher levels in patients with complete response was also found for thymidine kinase, lactate dehydrogenase and leukocytes, however the difference did not reach the level of significance (p = 0.542, p = 0.260, and p = 0.144, respectively). Conclusion: Our results indicate that nucleosomal DNA fragments are valuable markers for the early prediction of therapeutic efficacy in patients with acute myeloid leukemia. [ABSTRACT FROM AUTHOR]

Notwithstanding some improvement in the earlier detection of patients with lung cancer, most of them still present with a late-stage disease at the time of diagnosis. Next to the most frequently utilized factors affecting the prognosis of lung cancer patients (stage, performance, and age), the recent application of biomarkers obtained by liquid profiling has gained more acceptance. In our study, we aimed to answer these questions: (i) Is the quantification of free-circulating methylated PTGER4 and SHOX2 plasma DNA a useful method for therapy monitoring, and is this also possible for patients treated with different therapy regimens? (ii) Is this approach possible when blood-drawing tubes, which allow for a delayed processing of blood samples, are utilized? Baseline values for mPTGER4 and mSHOX2 do not allow for clear discrimination between different response groups. In contrast, the combination of the methylation values for both genes shows a clear difference between responders vs. non-responders at the time of re-staging. Furthermore, blood drawing into tubes stabilizing the sample allows researchers more flexibility. [ABSTRACT FROM AUTHOR]

Cell-free DNA (cfDNA) has become widely recognized as a promising candidate biomarker for minimally invasive characterization of various genomic disorders and other clinical scenarios. However, among the obstacles that currently challenge the general progression of the research field, there remains an unmet need for unambiguous universal cfDNA nomenclature. To address this shortcoming, we classify in this report the different types of cfDNA molecules that occur in the human body based on its origin, genetic traits, and locality. We proceed by assigning existing terms to each of these cfDNA subtypes, while proposing new terms and abbreviations where clarity is lacking and more precise stratification would be beneficial. We then suggest the proper usage of these terms within different contexts and scenarios, focusing mainly on the nomenclature as it relates to the domains of oncology, prenatal testing, and post-transplant surgery surveillance. We hope that these recommendations will serve as useful considerations towards the establishment of universal cfDNA nomenclature in the future. In addition, it is conceivable that many of these recommendations can be transposed to cell-free RNA nomenclature by simply exchanging "DNA" with "RNA" in each acronym/abbreviation. Similarly, when describing DNA and RNA collectively, the suffix can be replaced with "NAs" to indicate nucleic acids. [ABSTRACT FROM AUTHOR]

• Available cancer biomarkers are associated with low diagnostic performance. • Satellite 2 DNA amount is higher in the blood circulation of patients with cancer. • Satellite 2 DNA may be a universal biomarker for the detection of cancer. Currently, cancer biomarkers are associated with low diagnostic performance, and the notion of using cell-free DNA (cfDNA) as a surrogate cancer biomarker is a subject of ongoing research efforts. Pericentromeric satellite repeats were shown to expand in tumor cells. Here, we hypothesized that the increased release of satellite DNA into the circulation might be a basis for developing a biomarker for the detection of cancer. The study included patients with different cancer types, and controls without cancer. Human satellite 2 repeat (HSATII) from chromosomes 1, 10, or 16 was amplified using extracted DNA from plasma or direct application of diluted plasma in PCR. We first showed that HSATII DNA levels were higher in patients with cancer than in controls relative to LINE1 element, with chr10-HSATII being the most relevant. Absolute quantification in digital PCR showed much higher levels of chr10-HSATII in patients with breast cancer compared with healthy individuals. Subsequently, employing diluted plasma also revealed that HSATII DNA was present in increased levels in patients with cancer including breast, gastric, lung or bile cancers, sarcoma or Hodgkin's lymphoma than in controls with an AUC of 94% in the ROC curve. This proof-of-concept study reveals the high potential of HSATII DNA as a surrogate cancer biomarker. [ABSTRACT FROM AUTHOR]

Through various pathways of cell death, degradation, and regulated extrusion, partial or complete genomes of various origins (e.g., host cells, fetal cells, and infiltrating viruses and microbes) are continuously shed into human body fluids in the form of segmented cell-free DNA (cfDNA) molecules. While the genetic complexity of total cfDNA is vast, the development of progressively efficient extraction, high-throughput sequencing, characterization via bioinformatics procedures, and detection have resulted in increasingly accurate partitioning and profiling of cfDNA subtypes. Not surprisingly, cfDNA analysis is emerging as a powerful clinical tool in many branches of medicine. In addition, the low invasiveness of longitudinal cfDNA sampling provides unprecedented access to study temporal genomic changes in a variety of contexts. However, the genetic diversity of cfDNA is also a great source of ambiguity and poses significant experimental and analytical challenges. For example, the cfDNA population in the bloodstream is heterogeneous and also fluctuates dynamically, differs between individuals, and exhibits numerous overlapping features despite often originating from different sources and processes. Therefore, a deeper understanding of the determining variables that impact the properties of cfDNA is crucial, however, thus far, is largely lacking. In this work we review recent and historical research on active vs. passive release mechanisms and estimate the significance and extent of their contribution to the composition of cfDNA. [ABSTRACT FROM AUTHOR]

Introduction: Alterations in cell-free DNA concentration (cfDNA) over time have been studied in diseased or injured patients or analyzed in athletes during exhaustive exercise. However, no fluctuations have been examined over a short time course in healthy humans at rest so far, wherefore the aim of this study was to examine individual variations at different time points within 75 min. Methods: Serial blood drawing was performed in 14 healthy female volunteers at rest within 75 min. Plasma DNA was quantified by real-time qPCR, and absolute levels were analyzed together with relative variations. cfDNA alterations were moreover analyzed in consideration of potential volunteer-related impact factors (e.g., pulse) and were compared to alterations of plasma CK and AST. Results: Absolute cfDNA concentration ranged from 0.6 to 3.4 ng/ml. Regarding alterations over time, positive and negative variations were identified, whereby the interdecile range of fold changes was from 0.5 to 1.4. The maximum fold change was determined at 10 min. No relations were found between cfDNA levels and the analyzed individual factors. Conclusion: We evidenced the variability of cfDNA in healthy humans at rest within a short time course. The determined variations should serve in future studies to distinguish small cfDNA increases after minor trauma from natural fluctuations. Without such reference of intra-individual variation at rest, it would not be feasible to distinguish an injury from a fluctuation with certainty. Thus, a basis was established for the application of cfDNA as biomarker for the detection of mild injuries in forensic biomechanics. [ABSTRACT FROM AUTHOR]

Abstract: Extracellular nucleic acids are present in plasma, serum, and other body fluids and their analysis has gained increasing attention during recent years. Because of the small quantity and highly fragmented nature of cell-free DNA in plasma and serum, a fast, efficient, and reliable isolation method is still a problem and so far there is no agreement on a standardized method. We used spin columns from commercial suppliers (QIAamp DNA Blood Midi Kit from Qiagen; NucleoSpin Kit from Macherey-Nagel; MagNA Pure isolation system from Roche Diagnostics) to isolate DNA from 44 plasma samples in parallel at laboratories in Berlin and Munich. DNA in all samples was quantified by real-time PCR on a LightCycler 480 using three different targets (GAPDH, ß-globin, ERV). The quantities of cell-free DNA for the different isolation methods and genes varied between medians of 1.6ng/mL and 28.1ng/mL. This considerable variation of absolute DNA values was mainly caused by the use of different isolation methods (p<0.0001). Comparable results were achieved by the use of the genes GAPDH and ERV while higher values were obtained by use of ß-globin. The laboratory site had only minor influence on DNA yield when manual extraction methods were used. [Copyright &y& Elsevier]

Abstract: Background: We evaluated the utility of post-treatment plasma levels of the circulating bone-morphogenetic protein-6-specific mRNA (cBMP6 mRNA), cell-free DNA (cf-DNA), apoptotic nucleosomes and Histone H3 lysine 27 trimethylation (H3K27me3), in discriminating metastatic prostate cancer (PCa) from organ confined, locally controlled disease. Methods: Peripheral blood was taken from the patients at the end of therapy, and quantitative PCR was performed to amplify cBMP6 mRNA or cf-DNA from plasma while apoptotic nucleosomes and H3K27me3 were determined by ELISA-based approaches. Following blinded measurements, the markers were compared between the patients with local (n=22), local advanced (n=11) or metastatic disease (n=28). Results: Of the four markers investigated, the cBMP6 mRNA and H3K27me3 levels revealed significant differences between the three subgroups. We found higher levels of cBMP6 mRNA in the patients with metastases than in those with localized (p=0.001) or local advanced disease (p=0.05). When compared to cBMP6, H3K27me3 displayed an inverse distribution and was significantly lower in the patients with metastatic disease than in those with localized (p=0.05) or local advanced disease (p=0.024). There was no correlation between the different markers and total PSA levels or Gleason score at diagnosis. Conclusion: Our study provides evidence that post-treatment analysis of cBMP6 mRNA and H3K27me3 may be used to distinguish metastatic PCa from organ confined, locally controlled disease. [Copyright &y& Elsevier]