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. This is the first reported quality control scheme targeting ccfDNA for pre-analytical phase studies. Methods: Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR). Results: The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile. Conclusions: The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).

Circulating microRNAs (miRNAs) are promising non‑invasive biomarkers whose expression may be affected by confounding factors, including hemolysis, that should be considered in studies of miRNA discovery. The present study proposes a methodology for evaluating the impact of hemolysis on the expression of miRNAs. An experiment of in vitro controlled hemolysis was designed for assessing if changes in the expression of eight miRNAs observed to be circulating in plasma may be associated with hemolysis, and also to estimate the level of red blood cell (RBC) contamination in plasma samples where the expression of these miRNAs will be measured. It was confirmed that four miRNAs, miR‑16, miR‑92a, miR‑451 and miR‑486, known to be present in blood cells, were influenced by contamination of RBCs. Furthermore, it was demonstrated that miR‑378 and miR‑30c are hemolysis‑independent and that the expression of miR‑320 and miR‑324‑3p was associated with the level of RBC contamination. This procedure is proposed as a tool for the evaluation of the influence of hemolysis on candidate circulating miRNA biomarkers prior to their analysis in plasma samples.

Cell-free DNA in blood (cfDNA) represents a promising biomarker for cancer diagnosis. Total cfDNA concentration showed a scarce discriminatory power between patients and controls. A higher specificity in cancer diagnosis can be achieved by detecting tumor specific alterations in cfDNA, such as DNA integrity, genetic and epigenetic modifications. The aim of the present study was to identify a sequential multi-marker panel in cfDNA able to increase the predictive capability in the diagnosis of cutaneous melanoma in comparison with each single marker alone. To this purpose, we tested total cfDNA concentration, cfDNA integrity, BRAF^V600E mutation and RASSF1A promoter methylation associated to cfDNA in a series of 76 melanoma patients and 63 healthy controls. The chosen biomarkers were assayed in cfDNA samples by qPCR. Comparison of biomarkers distribution in cases and controls was performed by a logistic regression model in both univariate and multivariate analysis. The predictive capability of each logistic model was investigated by means of the area under the ROC curve (AUC). To aid the reader to interpret the value of the AUC, values between 0.6 and 0.7, between 0.71 and 0.8 and greater than 0.8 were considered as indicating a weak predictive, satisfactory and good predictive capacity, respectively. The AUC value for each biomarker (univariate logistic model) was weak/satisfactory ranging between 0.64 (BRAF^V600E) to 0.85 (total cfDNA). A good overall predictive capability for the final logistic model was found with an AUC of 0.95. The highest predictive capability was given by total cfDNA (AUC:0.86) followed by integrity index 180/67 (AUC:0.90) and methylated RASSF1A (AUC:0.89). An approach based on the simultaneous determination of three biomarkers (total cfDNA, integrity index 180/67 and methylated RASSF1A) could improve the diagnostic performance in melanoma.

Currently used CA15–3 and CEA have found their clinical application particularly in the follow-up of patients with advanced disease. Novel biomarkers are urgent, especially for improving early diagnosis as well as for discriminating between benign and malignant disease. In the present study, we used a proteomic approach based on surface-enhanced laser desorption/ionization–time of flight–mass spectrometry screening with the aim of identifying differentially expressed 2–30 kDa proteins in plasma of patients with malignant (65 cases) and benign (88 cases) breast lesions with respect to 121 healthy controls. We found that the most promising SELDI peaks were those corresponding to hepcidin-25 and ferritin light chain. We evaluated the capability of these peaks in predicting malignant and benign breast lesions using the area under the receiver operating characteristic curve (AUC). The results showed a good capacity to predict malignant breast lesions for hepcidin-25 [AUC: 0.82; 95% confidence interval (CI) 0.75–0.90] and ferritin light chain (AUC: 0.86; 95% CI 0.79–0.92). Conversely, a weak and satisfactory capability to predict benign breast lesion was observed for hepcidin-25 (AUC: 0.63; 95% CI 0.41–0.85) and ferritin light chain (AUC: 0.73; 95% CI 0.49–0.97). A significant association between HER2 status and hepcidin-25 was observed and the distribution of transferrin and ferritin were found significantly different in patients with breast cancer when compared with that of controls. This study provides evidence that hepcidin and ferritin light chain level in plasma may be of clinical usefulness to predict malignant and benign disease with respect to healthy controls.

Animal experimentation is a vast ecosystem that tries to make different issues such as legislative, ethical and scientific coexist. Research in animal experimentation has made many strides thanks to the 3Rs principle and the attached legislative decrees, but for this very reason, it needs to be evenly implemented both among the countries that have adhered to the decrees and among the team members who design and execute the experimental practice. In this article, we emphasize the importance of the 3Rs principle’s application, with a particular focus on the concept of Reduction and related key aspects that can best be handled with the contribution of experts from different fields.