Proficiency Testing to Assess Technical Performance for CTC-Processing and Detection Methods in CANCER-ID.

BACKGROUND: Multiple technologies are available for detection of circulating tumor cells (CTCs), but standards to evaluate their technical performance are still lacking. This limits the applicability of CTC analysis in clinic routine. Therefore, in the context of the CANCER-ID consortium, we established a platform to assess technical validity of CTC detection methods in a European multi-center setting using non-small cell lung cancer (NSCLC) as a model. METHODS: We characterized multiple NSCLC cell lines to define cellular models distinct in their phenotype and molecular characteristics. Standardized tumor-cell-bearing blood samples were prepared at a central laboratory and sent to multiple European laboratories for processing according to standard operating procedures. The data were submitted via an online tool and centrally evaluated. Five CTC-enrichment technologies were tested. RESULTS: We could identify 2 cytokeratin expressing cell lines with distinct levels of EpCAM expression: NCI-H441 (EpCAMhigh, CKpos) and NCI-H1563 (EpCAMlow, CKpos). Both spiked tumor cell lines were detected by all technologies except for the CellSearch system that failed to enrich EpCAMlow NCI-H1563 cells. Mean recovery rates ranged between 49% and 75% for NCI-H411 and 32% and 76% for NCI-H1563 and significant differences were observed between the tested methods. CONCLUSIONS: This multi-national proficiency testing of CTC-enrichment technologies has importance in the establishment of guidelines for clinically applicable (pre)analytical workflows and the definition of minimal performance qualification requirements prior to clinical validation of technologies. It will remain in operation beyond the funding period of CANCER-ID in the context of the European Liquid Biopsy Society (ELBS).

Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows.

BACKGROUND: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. METHODS: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. RESULTS: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. CONCLUSIONS: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.

Multicenter Evaluation of Circulating Plasma MicroRNA Extraction Technologies for the Development of Clinically Feasible Reverse Transcription Quantitative PCR and Next-Generation Sequencing Analytical Work Flows.

BACKGROUND: In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters. METHODS: By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals. RESULTS: There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias. CONCLUSIONS: The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.

Small Nucleolar RNA Score: An Assay to Detect Formalin-Overfixed Tissue.

Although there are millions of formalin-fixed paraffin-embedded (FFPE) tissue blocks potentially available for scientific research, many are of questionable quality, partly due to unknown fixation conditions. We analyzed FFPE tissue biospecimens as part of the NCI Biospecimen Preanalytical Variables (BPV) program to identify microRNA (miRNA) markers for fixation time. miRNA was extracted from kidney and ovary tumor FFPE blocks (19 patients, cold ischemia ≤2 hours) with 6, 12, 24, and 72 hours fixation times, then analyzed using the WaferGen SmartChip platform (miRNA chip with 1036 miRNA targets). For fixation time, principal component analysis of miRNA chip expression data separated 72 hours fixed samples from 6 to 24 hours fixed samples. A set of small nuclear RNA (snRNA) targets was identified that best determines fixation time and was validated using a second independent cohort of seven different tissue types. A customized assay was then developed, based on a set of 24 miRNA and snRNA targets, and a simple "snoRNA score" defined. This score detects FFPE tissue samples with fixation for 72 hours or more, with 79% sensitivity and 80% specificity. It can therefore be used to assess the fitness-for-purpose of FFPE samples for DNA or RNA-based research or clinical assays, which are known to be of limited robustness to formalin overfixation.

A high rate of telomeric sister chromatid exchange occurs in chronic lymphocytic leukaemia B-cells.

Cancer cells protect their telomere ends from erosion through reactivation of telomerase or by using the Alternative Lengthening of Telomere (ALT) mechanism that depends on homologous recombination. Chronic lymphocytic leukaemia (CLL) B cells are characterized by almost no telomerase activity, shelterin deregulation and telomere fusions. To characterize telomeric maintenance mechanisms in B-CLL patients, we measured their telomere length, telomerase expression and the main hallmarks of the ALT activity i.e. C-circle concentration, an extra-chromosomal telomere repeat (ECTR), and the level of telomeric sister chromatid exchange (T-SCE) rate. Patients showed relative homogenous telomere length although almost no TERT transcript and nearly no C-circle were evidenced. Nevertheless, compared with normal B cells, B-CLL cells showed an increase in T-SCE rate that was correlated with a strong down-regulation of the topoisomerase III alpha (TOP3A) expression, involved in the dissolution of Holliday Junctions (HJ), together with an increased expression of SLX1A, SLX4, MUS81 and GEN1, involved in the resolution of HJ. Altogether, our results suggest that the telomere maintenance mechanism of B-CLL cells do not preferentially use telomerase or ALT. Rather, the rupture of the dissolvasome/resolvasome balance may increase telomere shuffling that could homogenize telomere length, slowing telomere erosion in this disease.

Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts.

Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts.

Method validation for preparing urine samples for downstream proteomic and metabolomic applications.

BACKGROUND: Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was validated for fitness-for-purpose in terms of key downstream endpoints. METHODS: Urine processing was optimized for centrifugation conditions on the basis of microparticle counts at room temperature (RT) and at 4°C. The optimal protocol was validated for performance (microparticle counts), and for reproducibility and robustness for centrifugation temperature (4°C vs. RT) and brake speed (soft, medium, hard). Acceptance criteria were based on microparticle counts, cystatin C and creatinine concentrations, and the metabolomic profile. RESULTS: The optimal protocol was a 20-min, 12,000 g centrifugation at 4°C, and was validated for urine collection in terms of microparticle counts. All reproducibility acceptance criteria were met. The protocol was robust for centrifugation at 4°C versus RT for all parameters. The protocol was considered robust overall in terms of brake speeds, although a hard brake gave significantly fewer microparticles than a soft brake. CONCLUSIONS: We validated a urine processing method suitable for downstream proteomic and metabolomic applications. Temperature and brake speed can influence analytic results, with 4°C and high brake speed considered optimal. Laboratories and biobanks should ensure these conditions are systematically recorded in the scope of accreditation.

Mouse natural killer (NK) cells express the nerve growth factor receptor TrkA, which is dynamically regulated.

BACKGROUND: Nerve growth factor (NGF) is a neurotrophin crucial for the development and survival of neurons. It also acts on cells of the immune system which express the NGF receptors TrkA and p75(NTR) and can be produced by them. However, mouse NK cells have not yet been studied in this context. METHODOLOGY/PRINCIPAL FINDINGS: We used cell culture, flow cytometry, confocal microscopy and ELISA assays to investigate the expression of NGF receptors by NK cells and their secretion of NGF. We show that resting NK cells express TrkA and that the expression is different on NK cell subpopulations defined by the relative presence of CD27 and CD11b. Expression of TrkA is dramatically increased in IL-2-activated NK cells. The p75(NTR) is expressed only on a very low percentage of NK cells. Functionally, NGF moderately inhibits NK cell degranulation, but does not influence proliferation or cytokine production. NK cells do not produce NGF. CONCLUSIONS/SIGNIFICANCE: We demonstrate for the first time that mouse NK cells express the NGF receptor TrkA and that this expression is dynamically regulated.

Novel method for isolating untouched rat natural killer cells with higher purity compared with positive selection and fluorescence-activated cell sorting.

Natural killer (NK) cells are important effectors of both innate and adaptive immune responses. Although human and mouse NK cells are extensively characterized, much less is known about the rat cells, partly because of the current lack of reliable isolation techniques. We aimed to develop a method for isolating highly pure 'untouched' rat NK cells by negative selection from splenocytes. Thereafter, we characterized them phenotypically and functionally in comparison with those isolated by positive selection targeting the NKR-P1 receptor. Our novel method isolated highly pure untouched NK cells reproducibly with 97 ± 0.7% (n = 7), 96.6 ± 0.8% (n = 3) and 88.3 ± 1.5% (n = 9) in LEWIS, Fischer and athymic nude rats, respectively. The positively selected NK cells were less homogeneous and exhibited undesired method-related activation profiles. Resting negatively selected NK cells were less proliferative and less robust compared with positively selected NK cells. Although resting positively selected NK cells were more cytotoxic, interleukin-2 (IL-2) activation increased the cytotoxicity of negatively selected cells three-fold. The negatively selected NK cells responded to cross-linking of the NKR-P1 receptor by calcium mobilization from intracellular stores. However, combined IL-2 and IL-12 activation resulted in significantly more interferon-γ release from positively selected NK cells. This new NK-cell isolation method will allow a deeper insight into rat NK-cell phenotypes and the roles of their receptors in the biology of these cells.

Adhesion molecules and cytokine expression in fibromyalgia patients: increased L-selectin on monocytes and neutrophils.

Several lines of evidence implicate the immune system in the pathophysiology of fibromyalgia (FM). We investigated the role of cytokines and adhesion molecules involved in immune cell trafficking and the influence of 1.5 mg of dexamethasone (DEX) per os on their expression. L-selectin was elevated on monocytes and neutrophils of FM patients. Differences in group response to DEX were observed for CD11b on NK cells, sICAM-1 and IL-2. This study shows a slight disturbance in the innate immune system of FM patients, and suggests an enhanced adhesion and recruitment of leukocytes to inflammatory sites.

Humoral immune responses to a protective peptide-conjugate against measles after different prime-boost regimens.

The current live-attenuated measles vaccine leaves many children unprotected until they reach the recommended age of vaccination. We have previously shown that the short peptide corresponding to the hemagglutinin noose epitope (HNE) of the measles virus (MV) hemagglutinin protein induced virus-neutralizing antibodies even in the presence of protective levels of anti-whole virus-specific antibodies. Here we investigate the immunogenicity of HNE peptide-conjugates of diphtheria or tetanus toxoid in mice after active and passive priming with antibodies against the peptide, toxoids and conjugates. Both conjugates induced high titers of peptide antibodies which crossreacted with the virus and protected against a lethal intracranial challenge with a rodent-adapted measles virus, even after active priming with homologous or heterologous toxoid or conjugate. Peptide-specific epitopic suppression was stronger after passive priming with carrier or conjugate antibodies, but diphtheria toxoid as a carrier was less susceptible to suppression than tetanus toxoid and suppression was overcome by an additional boost. Furthermore, prior immunization with peptide-conjugate did not interfere with the development of a complete response to a subsequent injection of MV, suggesting that the benefits of a follow-up vaccination with the current live-attenuated vaccine would not be lost. These results underline the potential of these peptide-based conjugates as vaccine candidates for use in early infancy to close the window of susceptibility before the live-attenuated vaccine can be administered.

Functional fine-mapping and molecular modeling of a conserved loop epitope of the measles virus hemagglutinin protein.

Neutralizing and protective monoclonal antibodies (mAbs) were used to fine-map the highly conserved hemagglutinin noose epitope (H379-410, HNE) of the measles virus. Short peptides mimicking this epitope were previously shown to induce virus-neutralizing antibodies [El Kasmi et al. (2000) J. Gen. Virol.81, 729-735]. The epitope contains three cysteine residues, two of which (Cys386 and Cys394) form a disulfide bridge critical for antibody binding. Substitution and truncation analogues revealed four residues critical for binding (Lys387, Gly388, Gln391 and Glu395) and suggested the binding motif X7C[KR]GX[AINQ]QX2CEX5 for three distinct protective mAbs. This motif was found in more than 90% of the wild-type viruses. An independent molecular model of the core epitope predicted an amphiphilic loop displaying a remarkably stable and rigid loop conformation. The three hydrophilic contact residues Lys387, Gln391 and Glu395 pointed on the virus towards the solvent-exposed side of the planar loop and the permissive hydrophobic residues Ile390, Ala392 and Leu393 towards the solvent-hidden side of the loop, precluding antibody binding. The high affinity (Kd = 7.60 nm) of the mAb BH216 for the peptide suggests a high structural resemblance of the peptide with the natural epitope and indicates that most interactions with the protein are also contributed by the peptide. Improved peptides designed on the basis of these findings induced sera that crossreacted with the native measles virus hemagglutinin protein, providing important information about a lead structure for the design of more stable antigens of a synthetic or recombinant subunit vaccine.