Clinical Application of ISO and CEN/TS Standards for Liquid Biopsies-Information Everybody Wants but Nobody Wants to Pay For.

BACKGROUND: Liquid biopsies are emerging as valuable clinical biomarkers for cancer monitoring. Although International Organization for Standards (ISO) and Technical Specifications from the European Committee for Standardization (CEN/TS) standardized workflows exist, their implementation in clinical practice is underdeveloped. We aimed to assess the applicability of ISO and CEN/TS standards in a real-world clinical setting, with a particular focus on evaluating the impact of preanalytical parameters and hemolysis on liquid biopsy analysis. METHODS: We evaluated 659 peripheral blood samples from advanced prostate cancer patients against ISO and CEN/TS standards and documented all essential criteria, including tube draw order, filling level, temperature, and time tracking from blood draw to storage. We assessed hemolysis and its effect on circulating tumor DNA (ctDNA) and circulating tumor cell (CTC) analysis. RESULTS: Our results demonstrated a high compliance rate, with 96.2% (634/659) of samples meeting essential ISO and CEN/TS criteria. We did not observe a significant impact on ctDNA or CTC detection rates between hemolytic and nonhemolytic samples. Hemolysis was identified in 12.9% (40/311) of plasma samples from our advanced prostate cancer cohort, and within the draw order of 5 blood collection tubes, hemolysis did not significantly increase from tube 1 to 5. In total, 83.8% (552/659) of blood collection tubes had high fill levels above 80% of nominal filling level. CONCLUSIONS: Our study demonstrates the feasibility and benefits of adhering to ISO and CEN/TS standards in a clinical liquid biopsy study. The standards revealed that hemolysis occurred frequently but did not impair downstream ctDNA and CTC analysis in our cohort of advanced prostate cancer patients.

Residual Humidity in Paraffin-Embedded Tissue Reduces Nucleic Acid Stability.

Molecular diagnostics in healthcare relies increasingly on genomic and transcriptomic methodologies and requires appropriate tissue specimens from which nucleic acids (NA) of sufficiently high quality can be obtained. Besides the duration of ischemia and fixation type, NA quality depends on a variety of other pre-analytical parameters, such as storage conditions and duration. It has been discussed that the improper dehydration of tissue during processing influences the quality of NAs and the shelf life of fixed tissue. Here, we report on establishing a method for determining the amount of residual water in fixed, paraffin-embedded tissue (fixed by neutral buffered formalin or a non-crosslinking fixative) and its correlation to the performance of NAs in quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The amount of residual water depended primarily on the fixative type and the dehydration protocol and, to a lesser extent, on storage conditions and time. Moreover, we found that these parameters were associated with the qRT-PCR performance of extracted NAs. Besides the cross-linking of NAs and the modification of nucleobases by formalin, the hydrolysis of NAs by residual water was found to contribute to reduced qRT-PCR performance. The negative effects of residual water on NA stability are not only important for the design and interpretation of research but must also be taken into account in clinical diagnostics where the reanalysis of archived tissue from a primary tumor may be required (e.g., after disease recurrence). We conclude that improving the shelf life of fixed tissue requires meticulous dehydration and dry storage to minimize the degradative influence of residual water on NAs.

Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling.

Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter- and intra-assay as well as intra- and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity highlighting the importance of extensive validation of the test performance before offering these tests in clinical routine practice.

Human fetoplacental arterial and venous endothelial cells are differentially programmed by gestational diabetes mellitus, resulting in cell-specific barrier function changes.

AIMS/HYPOTHESIS: An adverse intrauterine environment can result in permanent changes in the physiology of the offspring and predispose to diseases in adulthood. One such exposure, gestational diabetes mellitus (GDM), has been linked to development of metabolic disorders and cardiovascular disease in offspring. Epigenetic variation, including DNA methylation, is recognised as a leading mechanism underpinning fetal programming and we hypothesised that this plays a key role in fetoplacental endothelial dysfunction following exposure to GDM. Thus, we conducted a pilot epigenetic study to analyse concordant DNA methylation and gene expression changes in GDM-exposed fetoplacental endothelial cells. METHODS: Genome-wide methylation analysis of primary fetoplacental arterial endothelial cells (AEC) and venous endothelial cells (VEC) from healthy pregnancies and GDM-complicated pregnancies in parallel with transcriptome analysis identified methylation and expression changes. Most-affected pathways and functions were identified by Ingenuity Pathway Analysis and validated using functional assays. RESULTS: Transcriptome and methylation analyses identified variation in gene expression linked to GDM-associated DNA methylation in 408 genes in AEC and 159 genes in VEC, implying a direct functional link. Pathway analysis found that genes altered by exposure to GDM clustered to functions associated with 'cell morphology' and 'cellular movement' in healthy AEC and VEC. Further functional analysis demonstrated that GDM-exposed cells had altered actin organisation and barrier function. CONCLUSIONS/INTERPRETATION: Our data indicate that exposure to GDM programs atypical morphology and barrier function in fetoplacental endothelial cells by DNA methylation and gene expression change. The effects differ between AEC and VEC, indicating a stringent cell-specific sensitivity to adverse exposures associated with developmental programming in utero. DATA AVAILABILITY: DNA methylation and gene expression datasets generated and analysed during the current study are available at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database ( http://www.ncbi.nlm.nih.gov/geo ) under accession numbers GSE106099 and GSE103552, respectively.