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

Cell-Free DNA Variant Sequencing Using CTC-Depleted Blood for Comprehensive Liquid Biopsy Testing in Metastatic Breast Cancer

Liquid biopsy analytes such as cell-free DNA (cfDNA) and circulating tumor cells (CTCs) exhibit great potential for personalized treatment. Since cfDNA and CTCs are considered to give additive information and blood specimens are limited, isolation of cfDNA and CTC in an “all from one tube„ format is desired. We investigated whether cfDNA variant sequencing from CTC-depleted blood (CTC-depl. B; obtained after positive immunomagnetic isolation of CTCs (AdnaTest EMT-2/Stem Cell Select, QIAGEN)) impacts the results compared to cfDNA variant sequencing from matched whole blood (WB). Cell-free DNA was isolated using matched WB and CTC-depl. B from 17 hormone receptor positive/human epidermal growth factor receptor 2 negative (HR+/HER2−) metastatic breast cancer patients (QIAamp MinElute ccfDNA Kit, QIAGEN). Cell-free DNA libraries were constructed (customized QIAseq Targeted DNA Panel for Illumina, QIAGEN) with integrated unique molecular indices. Sequencing (on the NextSeq 550 platform, Illumina) and data analysis (Ingenuity Variant Analysis) were performed. RNA expression in CTCs was analyzed by multimarker quantitative PCR. Cell-free DNA concentration and size distribution in the matched plasma samples were not significantly different. Seventy percent of all variants were identical in matched WB and CTC-depl. B, but 115/125 variants were exclusively found in WB/CTC-depl. B. The number of detected variants per patient and the number of exclusively detected variants per patient in only one cfDNA source did not differ between the two matched cfDNA sources. Even the characteristics of the exclusively detected cfDNA variants in either WB or CTC-depl. B were comparable. Thus, cfDNA variants from matched WB and CTC-depl. B exhibited no relevant differences, and parallel isolation of cfDNA and CTCs from only 10 mL of blood in an “all from one tube„ format was feasible. Matched cfDNA mutational and CTC transcriptional analyses might empower a comprehensive liquid biopsy analysis to enhance the identification of actionable targets for individual therapy strategies

Integrative statistical analyses of multiple liquid biopsy analytes in metastatic breast cancer

Background!#!Single liquid biopsy analytes (LBAs) have been utilized for therapy selection in metastatic breast cancer (MBC). We performed integrative statistical analyses to examine the clinical relevance of using multiple LBAs: matched circulating tumor cell (CTC) mRNA, CTC genomic DNA (gDNA), extracellular vesicle (EV) mRNA, and cell-free DNA (cfDNA).!##!Methods!#!Blood was drawn from 26 hormone receptor-positive, HER2-negative MBC patients. CTC mRNA and EV mRNA were analyzed using a multi-marker qPCR. Plasma from CTC-depleted blood was utilized for cfDNA isolation. gDNA from CTCs was isolated from mRNA-depleted CTC lysates. CTC gDNA and cfDNA were analyzed by targeted sequencing. Hierarchical clustering was performed within each analyte, and its results were combined into a score termed Evaluation of multiple Liquid biopsy analytes In Metastatic breast cancer patients All from one blood sample (ELIMA.score), which calculates the contribution of each analyte to the overall survival prediction. Singular value decomposition (SVD), mutual information calculation, k-means clustering, and graph-theoretic analysis were conducted to elucidate the dependence between individual analytes.!##!Results!#!A combination of two/three/four LBAs increased the prevalence of patients with actionable signals. Aggregating the results of hierarchical clustering of individual LBAs into the ELIMA.score resulted in a highly significant correlation with overall survival, thereby bolstering evidence for the additive value of using multiple LBAs. Computation of mutual information indicated that none of the LBAs is independent of the others, but the ability of a single LBA to describe the others is rather limited-only CTC gDNA could partially describe the other three LBAs. SVD revealed that the strongest singular vectors originate from all four LBAs, but a majority originated from CTC gDNA. After k-means clustering of patients based on parameters of all four LBAs, the graph-theoretic analysis revealed CTC ERBB2 variants only in patients belonging to one particular cluster.!##!Conclusions!#!The additional benefits of using all four LBAs were objectively demonstrated in this pilot study, which also indicated a relative dominance of CTC gDNA over the other LBAs. Consequently, a multi-parametric liquid biopsy approach deconvolutes the genomic and transcriptomic complexity and should be considered in clinical practice

Multicenter Evaluation of Independent High-Throughput and RT-qPCR Technologies for the Development of Analytical Workflows for Circulating miRNA Analysis

Background: Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID. Methods: Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR. Results: We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiRNA and EVmiRNA miRNA counts, respectively, in NSCLC patients compared to healthy control samples. However, a systematic comparison of selected miRNAs revealed little agreement between high-throughput platforms, thus some miRNAs are detected with one technology, but not with the other. Adding to this, 35% (9 of 26) of selected miRNAs in the cfmiRNA and 42% (11 of 26) in the EVmiRNA fraction were differentially expressed by at least one qPCR platform; about half of the miRNAs (54%) were concordant for both platforms. Conclusions: Changing of (pre)-analytical methods of miRNA analysis has a significant impact on blood test results and is therefore a major confounding factor. In addition, to confirm miRNA biomarker candidates screening studies should be followed by targeted validation using an independent platform or technology

Size distribution of total RNA from cancer patient plasma isolated by membrane affinity columns and ultracentrifugation.

<p>Bioanalyzer sizing of vesicle-derived RNA purified by two methods. Total EV RNA from 2 mL plasma of a melanoma patient was isolated using membrane affinity columns and compared total EV RNA from ultracentrifugation, the current gold standard of EV isolation. Both methods purify RNA of similar size and yield.</p

Scanning electron microscopy and western blot analysis of intact vesicles isolated by membrane affinity capture and ultracentrifugation.

<p>(<b>A</b>) Scanning electron microscopy (SEM; 20.000 x magnification) of a solubilized pellet from ultracentrifugation of pre-filtered (0.8 μm) plasma compared to a non-lysed eluate from the membrane affinity spin column. Both preparations contain vesicle-shaped structures with an expected size range from 50–200 nm (white arrows; scale bar 200 nm) indicating that intact vesicles are eluted from the spin column membrane. (<b>B</b>) Exosomes were isolated from four milliliters of normal human plasma using either the membrane affinity column (lane 2) or ultracentrifugation (UC) method (lane 3). Exosomes were concentrated, washed, then lysed, and exosome protein lysates were processed as described in Materials and Methods. The signal for TSG101 runs close to the predicted molecular weight of 43 kDa, the specificity of the TSG101 antibody was confirmed by positive control HeLa cell lysates and further verified by the absence of the 46 kDa band when probed with secondary antibody only. The blot shown is a representative of at least three separate experiments, indicating that the exosome-enriched protein TSG101 is present in vesicles eluted from the membrane affinity column.</p

RNA extraction from high volumes of plasma and serum using several available commercial kits.

<p>EV RNA from 4 mL of plasma or serum was isolated using membrane affinity (spin column), ultracentrifugation (Ultra) and three commercially available methods based on filtration (Kit B) or polymer-based precipitation (Kit S, Kit I) according to the manufacturers recommendations. The method marked with a star had no procedure for processing of high volumes available. The plot depicts raw C_T values of 5 different individuals using an RT-qPCR assay against the GAPDH mRNA. Only column-based purification and ultracentrifugation efficiently recover RNA from high sample volumes.</p