Analysis Of Cell-free Nucleic Acids Research Articles

Pre-analytical issues in liquid biopsy – where do we stand?

Abstract It is well documented that in the chain from sample to the result in a clinical laboratory, the pre-analytical phase is the weakest and most vulnerable link. This also holds for the use and analysis of extracellular nucleic acids. In this short review, we will summarize and critically evaluate the most important steps of the pre-analytical phase, i.e. the choice of the best control population for the patients to be analyzed, the actual blood draw, the choice of tubes for blood drawing, the impact of delayed processing of blood samples, the best method for getting rid of cells and debris, the choice of matrix, i.e. plasma vs. serum vs. other body fluids, and the impact of long-term storage of cell-free liquids on the outcome. Even if the analysis of cell-free nucleic acids has already become a routine application in the area of non-invasive prenatal screening (NIPS) and in the care of cancer patients (search for resistance mutations in the EGFR gene), there are still many unresolved issues of the pre-analytical phase which need to be urgently tackled.

Multiparametric liquid biopsy analysis in metastatic prostate cancer.

Molecular profiling of prostate cancer with liquid biopsies, such as circulating tumor cells (CTCs) and cell-free nucleic acid analysis, yields informative yet distinct data sets. Additional insights may be gained by simultaneously interrogating multiple liquid biopsy components to construct a more comprehensive molecular disease profile. We conducted an initial proof-of-principle study aimed at piloting this multiparametric approach. Peripheral blood samples from men with metastatic castrate-resistant prostate cancer were analyzed simultaneously for CTC enumeration, single-cell copy number variations, CTC DNA and matched cell-free DNA mutations, and plasma cell-free RNA levels of androgen receptor (AR) and AR splice variant (ARV7). In addition, liquid biopsies were compared with matched tumor profiles when available, and a second liquid biopsy was drawn and analyzed at disease progression in a subset of patients. In this manner, multiparametric liquid biopsy profiles were successfully generated for each patient and time point, demonstrating the feasibility of this approach and highlighting shared as well as unique cancer-relevant alterations. With further refinement and validation in large cohorts, multiparametric liquid biopsies can optimally integrate disparate but clinically informative data sets and maximize their utility for molecularly directed, real-time patient management.

Liquid Biopsy in Tumor Genetic Diagnosis.

Liquid biopsy involves the analysis of cell-free nucleic acids, mainly circulating free DNA (cfDNA), in bodily fluids such as blood. The obtaining of specimens is easier for patients and less invasive than tissue biopsy, but the method has certain limitations. This review is based on pertinent publications retrieved by a selective literature search. Because the concentration of cfDNA in plasma is less than 0.001%, special amplifying techniques must be used to enable a search for specific mutations. Liquid biopsy can be used in patients with non-small cell lung cancer (NSCLC) if no tissue is available for biopsy; when performed for this indication, it has 67% sensitivity and 94% specificity. If liquid biopsy does not reveal a mutation, this may be due either to the absence of the mutation in the tumor or to the inadequate sensitivity of the measuring technique. This uncertainty associated with negative findings can be reduced by the simultaneous demonstration of reference mutations derived from a primary tumor tissue analysis. In comparison to tissue studies, the search for tumor-specific mutations by liquid biopsy is 70% sensitive and 69% specific; this corresponds to a positive predictive value of 86% and a negative predictive value of 46%. Liquid biopsy and tumor tissue analysis are complementary, rather than alternative, techniques for therapeutically relevant genetic investigation of tumors. Comparative studies are needed so that further indications can be determined for liquid biopsy in the diagnostic evaluation of cancer.

Multi-parametric liquid biopsy analysis of circulating tumor cells (CTCs), cell-free DNA (cfDNA), and cell-free RNA (cfRNA) in metastatic castrate resistant prostate cancer (mCRPC).

274 Background: Molecular profiling of prostate cancer using liquid biopsies such as CTC capture and cell-free nucleic acid analysis yield informative yet distinct datasets. Additional insights may be gained by simultaneously interrogating multiple liquid biopsy components to construct a more comprehensive molecular disease profile. We have conducted an initial proof of principle study aimed at piloting this multi-parametric approach. Methods: Blood was drawn under an IRB-approved protocol from 20 mCRPC patients. Samples were analyzed simultaneously for the following: CTC enumeration and single CTC and matched white blood cell capture for whole genome amplification and low-pass copy number variation; CTC DNA and matched cfDNA for somatic single nucleotide variant analysis; plasma cfRNA extraction and qRT-PCR for AR, AR-V7, and PCA3. When available, liquid biopsies were compared with matched tumor profiles. Results: Fifteen of 20 patients (75%) had detectable CTCs by CellSearch (range: 1-692/7.5mL, median: 16.5/7.5mL). Thirteen of 20 patients (65%) had detectable SSNVs in CTC DNA and/or matched cfDNA, including mutations in TP53, PIK3CA, HRAS, and EGFR. Matched CTC DNA and cfDNA demonstrated both shared and distinct SSNVs. Copy number analysis of single CTCs was performed in 2 patients, and both had CNVs in multiple cancer relevant genes. A majority of CNVs were present in matched solid tumor profiles, but some were exclusive to the liquid biopsies. Plasma PCA3 and AR transcripts were detected in 18/20 (90%) and AR-V7 in 4/20 (20%) cfRNA samples. Unique SSNVs were detected at second time points at disease progression (more are being collected). Conclusions: In this pilot cohort, simultaneous multi-parametric profiling was feasible for CTC DNA mutations and CNVs, and matched plasma cfDNA mutations and cfRNA gene expression. These disease-specific molecular profiles were often concordant with tumor tissues but also contained new, potentially actionable alterations unique to CTC DNA or cfDNA. Expanded studies will build upon this approach to optimally leverage liquid biopsies for molecularly directed patient management.

Circulating cell-free nucleic acids as potential biomarkers for sarcopenia: a step toward personalized medicine

Sarcopenia is an age-related loss of muscle mass and function, leading to disability, morbidity and increased mortality in older people. Given the relatively high prevalence and related- outcome of the disease, correct diagnosis, screening, monitoring and treatment of sarcopenia are needed in clinical practice. Recent researches have focused on cell-free nucleic acids, which are released into the circulation following cell death, as a potential biomarker of aging and systematic inflammation. It seems that the diagnosis and treatment of sarcopenia can be possible by the help of the analysis of cell-free nucleic acids as noninvasive method.

Prenatal Diagnosis Innovation: Genome Sequencing of Maternal Plasma.

Noninvasive prenatal testing (NIPT) is accomplished by analysis of circulating cell-free fetal nucleic acids in maternal plasma. The advent of massively parallel sequencing (MPS) has enabled NIPT of chromosomal aneuploidies with unprecedented robustness, and these tests are now widely available for clinical use. Moreover, MPS-based NIPT of subchromosomal deletions/duplications and single-gene disorders has also been achieved, and the number of applications is growing. In addition to specific fetal genetic disorders, the whole fetal genome, transcriptome, and methylome have been revealed by deep sequencing of maternal plasma. The analysis of the fetal transcriptome and methylome may yield valuable information on fetal and maternal health. With continued improvement in sequencing technology and reduction in sequencing costs, the analysis of cell-free nucleic acids would play an increasingly important role in prenatal screening, diagnosis, monitoring, and risk stratification of fetal as well as maternal conditions.

An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum.

The analysis of cell-free nucleic acids (cfNAs), which are present at significant levels in the blood of cancer patients, can reveal the mutational spectrum of a tumour without the need for invasive sampling of the tissue. However, this requires differentiation between the nucleic acids that originate from healthy cells and the mutated sequences shed by tumour cells. Here we report an electrochemical clamp assay that directly detects mutated sequences in patient serum. This is the first successful detection of cfNAs without the need for enzymatic amplification, a step that normally requires extensive sample processing and is prone to interference. The new chip-based assay reads out the presence of mutations within 15 minutes using a collection of oligonucleotides that sequester closely related sequences in solution, and thus allow only the mutated sequence to bind to a chip-based sensor. We demonstrate excellent levels of sensitivity and specificity and show that the clamp assay accurately detects mutated sequences in a collection of samples taken from lung cancer and melanoma patients.

Cell-free nucleic acids as biomarkers in dialyzed patients

In this review, we discuss the origin, possible biological meaning, quantitative and qualitative changes in the concentrations of cell-free nucleic acids in human circulation with regard to renal failure and the process of dialysis. We focus on the inflammatory response and apoptosis known to be in close relationship not only with hemodialysis but also with different comorbidities frequently detected in hemodialyzed patients. Hemodialysis itself is able to promote the changes in the quantity and quality of circulating nucleic acid pool, but large spectrum of comorbidities in hemodialyzed subjects can further complicate the interpretations of results of cell-free nucleic acid analysis. Such analysis can provide additional information about the patient prognosis and monitor some aspects of comorbidity development. Recently, it has been shown that the analysis of cell-free nucleic acids may also be worthy in patients on peritoneal dialysis because the cell-free nucleic acids may also be detected in overnight effluents and their examination can be informative with regard to the functional state of the patient's peritoneum. We summarize what is recently known about the use of cell-free nucleic acids as biomarkers in patients with renal failure not only in hemodialysis but also in peritoneal dialysis and we describe the future perspectives in this field.