Interlaboratory evaluation of quality control methods for circulating cell-free DNA extraction.

Analysis of circulating cell-free DNA (ccfDNA) isolated from liquid biopsies is rapidly being implemented into clinical practice. However, diagnostic accuracy is significantly impacted by sample quality and standardised approaches for assessing the quality of ccfDNA are not yet established. In this study we evaluated the application of nucleic acid "spike-in" control materials to aid quality control (QC) and standardisation of cfDNA isolation for use in in vitro diagnostic assays.

Stimulated Expression of CXCL12 in Adrenocortical Carcinoma by the PPARgamma Ligand Rosiglitazone Impairs Cancer Progression.

Adrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis when metastatic and scarce treatment options in the advanced stages. In solid tumors, the chemokine CXCL12/CXCR4 axis is involved in the metastatic process. We demonstrated that the human adrenocortex expressed CXCL12 and its cognate receptors CXCR4 and CXCR7, not only in physiological conditions, but also in ACC, where the receptors' expression was higher and the CXCL12 expression was lower than in the physiological conditions.

Clinical characterization of a novel RAB39B nonstop mutation in a family with ASD and severe ID causing RAB39B downregulation and study of a Rab39b knock down mouse model.

Autism spectrum disorder (ASD) and intellectual disability (ID) often exist together in patients. The RAB39B gene has been reported to be mutated in ID patients with additional clinical features ranging from ASD, macrocephaly, seizures and/or early-onset parkinsonism. Here, we describe a novel RAB39B nonstop mutation [Xq28; c.

Automatic lung segmentation in COVID-19 patients: Impact on quantitative computed tomography analysis.

Purpose: To assess the impact of lung segmentation accuracy in an automatic pipeline for quantitative analysis of CT images.

Methods: Four different platforms for automatic lung segmentation based on convolutional neural network (CNN), region-growing technique and atlas-based algorithm were considered. The platforms were tested using CT images of 55 COVID-19 patients with severe lung impairment.

RAB39B-mediated trafficking of the GluA2-AMPAR subunit controls dendritic spine maturation and intellectual disability-related behaviour.

Mutations in the RAB39B gene cause X-linked intellectual disability (XLID), comorbid with autism spectrum disorders or early Parkinson's disease. One of the functions of the neuronal small GTPase RAB39B is to drive GluA2/GluA3 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) maturation and trafficking, determining AMPAR subunit composition at glutamatergic postsynaptic neuronal terminals. Taking advantage of the Rab39b knockout murine model, we show that a lack of RAB39B affects neuronal dendritic spine refinement, prompting a more Ca-permeable and excitable synaptic network, which correlates with an immature spine arrangement and behavioural and cognitive alterations in adult mice.

Circulating tumour cells and cell-free DNA as a prognostic factor in metastatic colorectal cancer: the OMITERC prospective study.

Background: Within the OMITERC prospective study (OMIcs application from solid to liquid biopsy for a personalised ThERapy of Cancer), we explored the prognostic role of liquid biopsy encompassing cell-free DNA (cfDNA) and circulating tumour cells (CTCs) in KRAS mutated metastatic colorectal cancer (mCRC).

Methods: We defined a workflow including pre-analytical and analytical procedures collecting blood before therapy and every 3 months until disease progression (PD). CTCs were counted by CellSearch® and isolated by DEPArray™.

Organisational challenges, volumes of oncological activity and patients' perception during the severe acute respiratory syndrome coronavirus 2 epidemic.

Background: On February 23rd, the 1st case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was diagnosed at the University Hospital Trust of Verona, Italy. On March 13th, the Oncology Section was converted into a 22-inpatient bed coronavirus disease (COVID) Unit, and we reshaped our organisation to face the SARS-CoV-2 epidemic, while maintaining oncological activities.

Methods: We tracked down (i) volumes of oncological activities (January 1st - March 31st, 2020 versus the same period of 2019), (ii) patients' and caregivers' perception and (iii) SARS-CoV-2 infection rate in oncology health professionals and SARS-CoV-2 infection-related hospital admissions of "active"' oncological patients.

The pre-analytical phase of the liquid biopsy.

The term 'liquid biopsy', introduced in 2013 in reference to the analysis of circulating tumour cells (CTCs) in cancer patients, was extended to cell-free nucleic acids (cfNAs) circulating in blood and other body fluids. CTCs and cfNAs are now considered diagnostic and prognostic markers, used as surrogate materials for the molecular characterisation of solid tumours, in particular for research on tumour-specific or actionable somatic mutations. Molecular characterisation of cfNAs and CTCs (especially at the single cell level) is technically challenging, requiring highly sensitive and specific methods and/or multi-step processes.

Pre-analytical processes in medical diagnostics: New regulatory requirements and standards.

In May 2017, the European In Vitro Diagnostic Regulation (IVDR) entered into force and will apply to in vitro diagnostics from May 26th, 2022. This will have a major impact on the in vitro diagnostics (IVD) industry as all devices falling under the scope of the IVDR will require new or re-certification. It will also affect health institutions developing and using in-house devices.

The paramount role of cytokines and chemokines in papillary thyroid cancer: a review and experimental results.

Our study demonstrates that (C-X-C motif) ligand 9 and 11 (CXCL9, CXCL11) chemokines were absent basally in non-neoplastic thyroid (TFC) and papillary thyroid carcinoma (PTC) cells. Interferon (IFN)γ induced the chemokine secretion in TFC and PTC, while tumor necrosis factor (TNF)α induced it only in PTC. IFNγ+TNFα induced a synergistic chemokines release in PTC, and at a lower level in TFC.

New insights in the clinical and translational relevance of miR483-5p in adrenocortical cancer.

Adrenocortical cancer (ACC) is a rare aggressive malignancy. Recent ACC integrated genomics analysis contributed to redefine the risk groups on molecular basis, including tumor microRNAs (miRs), detectable also in the bloodstream. We developed a quantitative real-time (RT) assay for the measurement of miR483 and miR483-5p absolute levels in plasma samples.

Data and performances evaluation of the SPIDIA-DNA Pan-European External Quality Assessment: 2nd SPIDIA-DNA laboratory report.

Within the EU-SPIDIA project (www.spidia.eu), the quality parameters of blood genomic DNA were defined [SPIDIA-DNA: an External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses - [1]; Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience - [2]; Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: the SPIDIA experience - [3].

Effects of Transport and Storage Conditions on Gene Expression in Blood Samples.

Background: Inappropriate handling of blood samples might induce or repress gene expression and/or lead to RNA degradation affecting downstream analysis. In particular, sample transport is a critical step for biobanking or multicenter studies because of uncontrolled variables (i.e.

Second SPIDIA-DNA External Quality Assessment (EQA): Influence of pre-analytical phase of blood samples on genomic DNA quality.

Background: In order to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for DNA molecular testing, two pan-European External Quality Assessments (EQAs) were implemented within the European Commission funded project SPIDIA. Here we report the results of the 2nd SPIDIA EQA that has been implemented on the basis of the 1st DNA EQA with the inclusion of some stringent conditions related to blood storage temperature and time.

Methods: SPIDIA facility sent to all the participants the same blood sample to be processed by their own procedure following SPIDIA suggestion for time and temperature storage.

Influence of storage conditions and extraction methods on the quantity and quality of circulating cell-free DNA (ccfDNA): the SPIDIA-DNAplas External Quality Assessment experience.

Background: Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the pre-analytical phase on ccfDNA.

Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: The SPIDIA experience.

In this note, we present an ad hoc procedure that combines qualitative (visual evaluation) and quantitative (ImageJ software) evaluations of Pulsed-Field Gel Electrophoresis (PFGE) images to assess the genomic DNA (gDNA) integrity of analyzed samples. This procedure could be suitable for the analysis of a large number of images by taking into consideration both the expertise of researchers and the objectiveness of the software. We applied this procedure on the first SPIDIA DNA External Quality Assessment (EQA) samples.

Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia.

The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells.

Anti-miR21 oligonucleotide enhances chemosensitivity of T98G cell line to doxorubicin by inducing apoptosis.

Various signal transduction pathways seem to be involved in chemoresistance mechanism of glioblastomas (GBMs). miR-21 is an important oncogenic miRNA which modulates drug resistance of tumor cells. We analyzed the expression of 5 miRNAs, previously found to be dysregulated in high grade gliomas, in 9 pediatric (pGBM) and in 5 adult (aGBM) GBMs.

Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience.

Background: DNA integrity is a critical part of the definition of genomic DNA (gDNA) quality and can influence downstream molecular applications. Pre-analytical variables as sample storage and DNA extraction methods can influence the quality and quantity of isolated DNA and affect molecular test performances. The aim of this paper is to investigate the role of blood sample storage and DNA extraction procedures on gDNA integrity and gDNA fragmentation impact on a molecular test.

SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA.

SPIDIA-DNA: an External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses.

Background: The EC-funded project SPIDIA is aimed to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for DNA molecular testing. To this purpose, a survey and a pan-European External Quality Assessment (EQA) were implemented.

Methods: SPIDIA facility sent to all the participants the same blood sample to be processed without time or temperature limitation.

SPIDIA-RNA: first external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

The diagnostic use of in vitro molecular assays can be limited by the lack of guidelines for collection, handling, stabilization and storage of patient specimens. One of the major goals of the EC funded project SPIDIA (www.spidia.