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™. NGS sequencing of CTCs and cfDNA was performed using a panel of cancer/CRC related genes respectively. RESULTS: KRAS mutational status was mostly concordant between tumour tissues and liquid biopsy. The percentage of cfDNA samples with mutations in CRC driver genes was in line with literature. In longitudinal monitoring circulating biomarkers anticipated or overlapped conventional diagnostic tools in predicting PD. The presence of CTCs at baseline was confirmed a negative prognostic marker. CONCLUSIONS: Cell-free DNA and CTCs are readily available candidates for clinical application in mCRC. While CTCs demonstrated a prognostic significance at baseline, cfDNA was confirmed an easily accessible material for monitoring the mutational status of the tumour over time. Moreover, in the longitudinal study, the two markers emerged as complementary in assessing disease progression.

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. The analysis of the liquid biopsy relies on a plethora of methods whose standardisation cannot be accomplished without disclosing criticisms related to the pre-analytical phase. Thus, pre-analytical factors potentially influencing downstream cellular and molecular analyses must be considered in order to translate the liquid biopsy approach into clinical practice. The present review summarises the most recent reports in this field, discussing the main pre-analytical aspects related to CTCs, cfNAs and exosomes in blood samples for liquid biopsy analysis. A short discussion on non-blood liquid biopsy samples is also included.

Evaluation of the liquid biopsy for the detection of BRAFV600E mutation in metastatic melanoma patients.

BACKGROUND: Circulating tumor cells (CTCs) and circulating cell free DNA (ccfDNA) represent a liquid biopsy of a tumor allowing real time disease monitoring especially in advanced stages of cancer, but their analysis is technically challenging. OBJECTIVE: We aimed to demonstrate the feasibility of two different technical approaches to detect the BRAFV600E mutation in the liquid biopsy of 20 metastatic melanoma patients by using both the enriched CTC fraction and circulating ccfDNA from the same blood sample. METHODS: We detected CTCs by a filtration method in 20 metastatic melanoma patients and detected the BRAFV600E variant on CTCs and ccfDNA by an allele-specific qPCR assay; the mutated samples were confirmed by ICE-COLD PCR followed by Sanger sequencing. RESULTS: We found CTCs in 70% of the samples, and identified the BRAFV600E variant on CTCs. We correlated the results with those obtained on ccfDNA from the same blood draw. We found some discordant results between CTCs and ccfDNA. CONCLUSIONS: Our results underline the importance of investigating both CTCs and ccfDNA in a liquid biopsy approach to melanoma patients.

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. miR483/miR483-5p levels were evaluated in plasma samples of 27 patients with ACC before surgery and at follow-up. Statistically significant differences in miR483-5p and miR483 levels were found between stage 1/2 and stage 3/4 ACCs in pre-surgery and post-surgery samples. ROC curve analysis of miR483-5p levels gave a prediction of the clinical stage (accuracy 0.917±0.084), with the best cut-off value of 0.221 ng/ml, prognosticating overall and recurrence-free survival. In a multivariate Cox analysis (HR 16.2, 95%CI[1.39-188.6, P<0.026]), miR483-5p was the only variable that significantly predicted recurrence, but not overall survival. In addition, miR483 and miR483-5p levels correlated with the number of circulating tumor cells (CTCs) detected in the same blood samples, independently of the timing of sampling. In conclusion, we demonstrated that miR483-5p absolute plasma levels in ACC patients are powerful molecular markers that may help in the follow-up of patients after surgery and chemotherapy, and contribute to more accurately classify and predict tumor progression.

Circulating tumor cells and microemboli can differentiate malignant and benign pulmonary lesions.

The presence of circulating tumor cells (CTC) or microemboli (CTM) in the peripheral blood can theoretically anticipate malignancy of solid lesions in a variety of organs. We aimed to preliminarily assess this capability in patients with pulmonary lesions of suspected malignant nature. We used a cell-size filtration method (ScreenCell) and cytomorphometric criteria to detect CTC/CTM in a 3 mL sample of peripheral blood that was taken just before diagnostic percutaneous CT-guided fine needle aspiration (FNA) or core biopsy of the suspicious lung lesion. At least one CTC/CTM was found in 47 of 67 (70%) patients with final diagnoses of lung malignancy and in none of 8 patients with benign pulmonary nodules. In particular they were detected in 38 (69%) of 55 primary lung cancers and in 9 (75%) of 12 lung metastases from extra-pulmonary cancers. Sensitivity of CTC/CTM presence for malignancy was 70.1% (95%CI: 56.9-83.1%), specificity 100%, positive predictive value 100% and negative predictive value 28.6% (95%CI: 11.9-45.3%). Remarkably, the presence of CTC/CTM anticipated the diagnosis of primary lung cancer in 3 of 5 patients with non-diagnostic or inconclusive results of FNA or core biopsy, whereas CTC/CTM were not observed in 1 patient with sarcoidosis and 1 with amarthocondroma. These results suggest that presently, due to the low sensitivity, the search of CTC/CTM cannot replace CT guided percutaneous FNA or core biopsy in the diagnostic work-up of patients with suspicious malignant lung lesions. However, the high specificity may as yet indicate a role in cases with non-diagnostic or inconclusive FNA or core biopsy results that warrants to be further investigated.

Powerful qPCR assays for the early detection of latent invaders: interdisciplinary approaches in clinical cancer research and plant pathology.

Latent invaders represent the first step of disease before symptoms occur in the host. Based on recent findings, tumors are considered to be ecosystems in which cancer cells act as invasive species that interact with the native host cell species. Analogously, in plants latent fungal pathogens coevolve within symptomless host tissues. For these reasons, similar detection approaches can be used for an early diagnosis of the invasion process in both plants and humans to prevent or reduce the spread of the disease. Molecular tools based on the evaluation of nucleic acids have been developed for the specific, rapid, and early detection of human diseases. During the last decades, these techniques to assess and quantify the proliferation of latent invaders in host cells have been transferred from the medical field to different areas of scientific research, such as plant pathology. An improvement in molecular biology protocols (especially referring to qPCR assays) specifically designed and optimized for detection in host plants is therefore advisable. This work is a cross-disciplinary review discussing the use of a methodological approach that is employed within both medical and plant sciences. It provides an overview of the principal qPCR tools for the detection of latent invaders, focusing on comparisons between clinical cancer research and plant pathology, and recent advances in the early detection of latent invaders to improve prevention and control strategies.

Mutational analysis of single circulating tumor cells by next generation sequencing in metastatic breast cancer.

Circulating Tumor Cells (CTCs) represent a "liquid biopsy" of the tumor potentially allowing real-time monitoring of cancer biology and therapies in individual patients.The purpose of the study was to explore the applicability of a protocol for the molecular characterization of single CTCs by Next Generation Sequencing (NGS) in order to investigate cell heterogeneity and provide a tool for a personalized medicine approach.CTCs were enriched and enumerated by CellSearch in blood from four metastatic breast cancer patients and singularly isolated by DEPArray. Upon whole genome amplification 3-5 single CTCs per patient were analyzed by NGS for 50 cancer-related genes.We found 51 sequence variants in 25 genes. We observed inter- and intra-patient heterogeneity in the mutational status of CTCs.The highest number of somatic deleterious mutations was found in the gene TP53, whose mutation is associated with adverse prognosis in breast cancer.The discordance between the mutational status of the primary tumor and CTCs observed in 3 patients suggests that, in advanced stages of cancer, CTC characteristics are more closely linked to the dynamic modifications of the disease status.In one patient the mutational profiles of CTCs before and during treatment shared only few sequence variants.This study supports the applicability of a non-invasive approach based on the liquid biopsy in metastatic breast cancer patients which, in perspective, should allow investigating the clonal evolution of the tumor for the development of new therapeutic strategies in precision medicine.

Urinary carbonic anhydrase IX splicing messenger RNA variants in urogenital cancers.

BACKGROUND: To identify molecular biomarkers for tumor diagnosis and monitoring of disease progression, several noninvasive tests on liquid biopsy have been proposed for different cancers including those of urogenital origin. Among biomarkers, carbonic anhydrase IX (CAIX) has gained attention as it regulates extracellular pH and induces cytoplasmic alkalization contributing to malignant progression and poor treatment outcome. Works on tissues suggested the potential use of CAIX as a tumor biomarker for urogenital malignancies, but only few studies have been performed on its detection in urine. SCOPE: The aim of the present study is the measurement of CAIX messenger RNA (mRNA) in urine sediments of patients affected by kidney, prostate, and bladder cancers to evaluate the clinical sensitivity and specificity of the test. PROCEDURES: The quantification of the total CAIX mRNA concentration and of its full-length isoform (CAIX FL) have been performed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) on RNA extracted from urine sediments of patients affected by urogenital cancers. RESULTS: Urinary total CAIX mRNA expression resulted to be lower in patients with kidney and prostate cancer in comparison with the control group, but no statistically significant difference could be evidenced for bladder cancer. The evaluation of the relative percentage of FL isoform mRNA (FL%) showed a significant increase of FL% in urine from patients with cancer (median = 70.8%) in comparison with the healthy subjects (median = 2.6%) and this finding was confirmed for each cancer type separately. The comparison among receiver operating characteristic curves for total CAIX mRNA, CAIX FL mRNA, and FL% indicated that FL% shows the best diagnostic performance with 90% sensitivity and 72% specificity. Comparison of the results obtained in urine with those found in the corresponding tissues indicated 80% concordance. CONCLUSIONS: The CAIX mRNA expression in urine sediments can be considered a surrogate marker of CAIX expression in tumor tissues of urogenital origin. In particular, the analysis of FL% possesses the best characteristics to be a suitable noninvasive biomarker for urogenital cancer diagnosis.

Prevalence and number of circulating tumour cells and microemboli at diagnosis of advanced NSCLC.

PURPOSE: Timing and magnitude of blood release of circulating tumour cells (CTC) and circulating tumour microemboli (CTM) from primary solid cancers are uncertain. We investigated prevalence and number of CTC and CTM at diagnosis of advanced non-small cell lung cancer (NSCLC). METHODS: Twenty-eight consecutive patients with suspected stage III-IV lung cancer gave consent to provide 15 mL of peripheral blood soon before diagnostic CT-guided fine-needle aspiration biopsy (FNAB). CTC and CTM (clusters of ≥3 CTC) were isolated by cell size filtration (ScreenCell), identified and counted by cytopathologists using morphometric criteria and (in 6 cases) immunostained for vimentin. RESULTS: FNAB demonstrated NSCLC in 26 cases. At least one CTC/3 mL blood (mean 6.8 ± 3.7) was detected in 17 (65 %) and one CTM (mean 4.5 ± 3.3) in 15 (58 %) of 26 NSCLC cases. No correlation between number of CTC or CTM and tumour type or stage was observed. Neoplastic cells from both FNA and CTC/CTM were positive for vimentin but heterogeneously. CONCLUSIONS: CTC can be detected in two-thirds and CTM in more than half of patients with advanced NSCLC at diagnosis. Reasons underlying lack of CTC and CTM in some advanced lung cancers deserve further investigations.

Single circulating tumor cell sequencing as an advanced tool in cancer management.

Circulating tumor cells (CTCs) shed by the primary tumor and metastases are considered a real-time 'liquid biopsy', reflecting the disease complexity that evolves during progression, showing in its late stages different genetic, epigenetic and expression features. Consequently, heterogeneity and development of characteristic features upon disease progression are the two main goals that emerging technologies should account for in view of a clinical application. Single-cell analysis, now possible due to technological advances, may help elucidate tumor heterogeneity at the CTC level. This review focuses on the necessary steps for the analysis of CTCs at the single-cell level. A concise overview is given on the alternative methods referring in particular to studies on the mutational status of single CTCs from cancer patients.

Prospective evaluation of RASSF1A cell-free DNA as a biomarker of pre-eclampsia.

INTRODUCTION: This study aims to quantify total and fetal cell-free DNA (cfDNA) in maternal plasma at different gestational ages and to assess whether this could represent a reliable predictive marker of pre-eclampsia (PE) before clinical onset. METHODS: We performed a qPCR assay to compare the cfDNA concentration of hypermethylated and unmethylated RASSF1A promoter gene sequences in maternal plasma among 3 groups of pregnant women. These included 17 women with overt PE, 33 women at risk for the disease subsequently differentiated into 9 who developed PE and 24 who did not, and 73 controls. All women at risk were consecutively sampled throughout the whole gestation. RESULTS: Both total and fetal cfDNA had a good diagnostic performance in distinguishing patients with overt PE from healthy controls. When comparing women at risk who developed PE to women at risk who did not, the predictive capability was satisfactory at a gestational age ranging from 17 to 30 weeks. This allowed establishing within this time interval a cut-off value of 735 GE/ml for total cfDNA (87.5% sensitivity and 70.0% specificity), and a cut-off value of 7.49 GE/ml for fetal cfDNA (100% sensitivity and 50% specificity). cfDNA levels turned positive several weeks before the onset of the disease: from 2 to 18 weeks for total cfDNA and from 8 to 17 weeks for fetal cfDNA. DISCUSSION: The simultaneous use of total and fetal cfDNA would allow an accurate monitoring and prevention of PE development thus suggesting that RASSF1A could represent a potential biomarker of PE.

Bevacizumab plus XELOX as first-line treatment of metastatic colorectal cancer: The OBELIX study.

AIM: To confirm the efficacy and safety of bevacizumab/XELOX combination for the treatment of locally advanced or metastatic colorectal cancer (CRC) in Italy. METHODS: This multicentric, prospective, open-label study included patients with CRC previously untreated with chemotherapy. Patients were administered bevacizumab in combination with XELOX. The primary efficacy end-point was progression-free survival (PFS). Secondary end-points included time to overall response (TOR), duration of response (DOR), time to treatment failure (TTF) and overall survival (OS). The incidence and type of adverse events AEs and severe AEs were evaluated. Also, the mutational status of BRAF and KRAS was assessed by high resolution melting and direct sequencing, and quality of life (QoL) was measured by the EuroQoL EQ-5D questionnaire at baseline and at the last visit. RESULTS: The intention-to-treat population included 197 patients (mean age: 62.3 ± 9.9 years, 56.4% males). At baseline, 16/34 evaluable subjects (47.1%) harbored a KRAS and/or a BRAF mutation; the mean QoL index was 80.2 ± 14.3. First-line therapy was given for 223.7 ± 175.9 d, and after a mean follow-up of 387.7 ± 238.8 d all patients discontinued from the study mainly for disease progression (PD, 45.4%) and AEs (25.4%). Median PFS was 9.7 mo (95%CI: 8.4-10.5) and the median values for secondary end-points were: TOR = 3.9 mo (95%CI: 2.6-4.7), DOR = 8.5 mo (95%CI: 7.3-10.3), TTF = 6.7 mo (95%CI: 6.0-7.7) and OS = 23.2 mo (95%CI: 20.1-27.2). Patients carrying at least one lesion had a lower overall response rate (66.7% vs 88.9%) and a lower probability of achieving complete or partial response than those without mutations, but the difference in relative risk was not statistically significant (P = 0.2). Mean EQ-5D-3L raw index score significantly decreased to 74.9 ± 19.1 at the last visit (signed-rank test, P = 0.0076), but in general the evaluation on QoL perceived by patients was good. CONCLUSION: The efficacy of bevacizumab in combination with XELOX in terms of PFS in patients with aCRC or mCRC in Italy was confirmed, with acceptable toxicity.

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. 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).

Heterogeneity of PIK3CA mutational status at the single cell level in circulating tumor cells from metastatic breast cancer patients.

Circulating Tumor Cells (CTCs) represent a "liquid biopsy of the tumor" which might allow real-time monitoring of cancer biology and therapies in individual patients. CTCs are extremely rare in the blood stream and their analysis is technically challenging. The CellSearch(®) system provides the enumeration of CTCs with prognostic significance in patients with metastatic breast cancer (mBC), but it does not allow their molecular characterization, which might be useful to identify therapeutically relevant targets for individualized treatment. Combining the CellSearch(®) and DEPArray™ technologies allows the recovery of single CTCs as a pure sample for molecular analysis. The purpose of the study was to investigate the heterogeneity of PIK3CA mutational status within single CTCs isolated from individual mBC patients. CTCs were enriched and enumerated by CellSearch(®) in blood samples collected from 39 mBC patients. In 20 out of 39 patients enriched samples with ≥5 CTCs were sorted using DEParray™ to isolate single CTCs or pools of CTCs to be submitted to Whole Genome Amplification (WGA) before sequencing analysis. In 18 out of 20 patients, it was possible to perform PIK3CA sequencing on exons 9 and 20. Twelve subjects were wild type (wt) for the PIK3CA gene. PIK3CA status could also be assessed in pools of CTCs in seven of these patients, with consistent wt status found. Six patients (33%) had a PIK3CA mutation identified. In 2 of the six patients, molecular heterogeneity was detected when mutational analysis was performed on more than one single CTC, including one patient with loss of heterozygosity on both single and pooled CTCs, and one patient with three different PIK3CA variants on single CTCs but PIK3CA wt status on pooled CTC samples. In six out of the 18 cases PIK3CA status was also evaluable on a primary tumor sample. In one of the six cases a discordance in PIK3CA status between the primary (wild-type) and the matched CTC (exon 20 mutation) was observed. This study demonstrates the feasibility of a non-invasive approach based on the liquid biopsy in mBC patients. Moreover, our data suggest the importance of characterizing CTCs at the single cell level in order to investigate the molecular heterogeneity within cells from the same patient.

Implementation of a companion diagnostic in the clinical laboratory: the BRAF example in melanoma.

A companion diagnostic test provides information that is essential for the safe and effective use of a corresponding therapeutic product as indicated in the drug instructions. The implementation of a companion diagnostic follows the rules of a molecular test for somatic mutations in a routine clinical laboratory environment and needs guidance on practical aspects, including the choice of the proper analytical method and the procedures for internal and external quality controls. Selection of the appropriate assay for detection of genetic alterations depends on several factors: the type of mutation under study, the sample to be assayed and its preparation procedure. In addition, the results of a molecular assay require a complex interpretation process of the analytical data as the patient's genotype, the translation of the identified variant into a predicted phenotype and knowledge on restrictions of the method used. In relation to these aspects herein we report an opinion paper of the Working Group Personalized Laboratory Medicine jointly constituted by the European Federation of Laboratory Medicine (EFLM) and by the European Society of Pharmacogenomics and Theranostics (ESPT) using, as an example, the BRAF genotype analysis in tumor tissue samples for identification of melanoma patients that can benefit treatment with BRAF inhibitors. The manuscript is focused on the following aspects: i) medical rationale, ii) methodologies of analysis, iii) laboratory performance evaluation and iv) the laboratory specific report for the clinicians. The critical evaluation of these aspects would be useful for the implementation of a companion diagnostic in the clinical laboratory.

Tumor-Related Methylated Cell-Free DNA and Circulating Tumor Cells in Melanoma.

Solid tumor release into the circulation cell-free DNA (cfDNA) and circulating tumor cells (CTCs) which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A) is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma. The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs. RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET) as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC). The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic) than in healthy subjects (Pearson chi-squared test, p < 0.001). The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC) in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive and metastatic melanomas. Our data suggest that cell-free tumor DNA and CTCs represent two complementary aspects of the liquid biopsy which may improve the diagnosis and the clinical management of melanoma patients.

Feasibility of a workflow for the molecular characterization of single cells by next generation sequencing.

The purpose of the study was to explore the feasibility of a protocol for the isolation and molecular characterization of single circulating tumor cells (CTCs) from cancer patients using a single-cell next generation sequencing (NGS) approach. To reach this goal we used as a model an artificial sample obtained by spiking a breast cancer cell line (MDA-MB-231) into the blood of a healthy donor. Tumor cells were enriched and enumerated by CellSearch(®) and subsequently isolated by DEPArray™ to obtain single or pooled pure samples to be submitted to the analysis of the mutational status of multiple genes involved in cancer. Upon whole genome amplification, samples were analysed by NGS on the Ion Torrent PGM™ system (Life Technologies) using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies), designed to investigate genomic "hot spot" regions of 50 oncogenes and tumor suppressor genes. We successfully sequenced five single cells, a pool of 5 cells and DNA from a cellular pellet of the same cell line with a mean depth of the sequencing reaction ranging from 1581 to 3479 reads. We found 27 sequence variants in 18 genes, 15 of which already reported in the COSMIC or dbSNP databases. We confirmed the presence of two somatic mutations, in the BRAF and TP53 gene, which had been already reported for this cells line, but also found new mutations and single nucleotide polymorphisms. Three variants were common to all the analysed samples, while 18 were present only in a single cell suggesting a high heterogeneity within the same cell line. This paper presents an optimized workflow for the molecular characterization of multiple genes in single cells by NGS. The described pipeline can be easily transferred to the study of single CTCs from oncologic patients.

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. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

Circulating cell-free DNA in cancer.

This papers deals with the preanalytical and analytical phase of cell-free DNA analysis, highlighting some criticism on sample collection and extraction. We describe a method to accurately quantify total cfDNA in plasma and our particular approach to the measurement of tumor deriving cfDNA.

Circulating tumor cells detection and counting in uveal melanomas by a filtration-based method.

Uveal melanoma is one of the most deadly diseases in ophthalmology for which markers able to predict the appearance of metastasis are needed. The study investigates the role of circulating tumor cells (CTC) as a prognostic factor in this disease. We report the detection of circulating tumor cells by Isolation by Size of Epithelial Tumor cells (ISET) in a cohort of 31 uveal melanoma patients: we identified single CTCs or clusters of cells in 17 patients, while the control population, subjects with choroidal nevi, showed no CTC in peripheral blood. The presence of CTCs did not correlate with any clinical and pathological parameter, such as tumor larger basal diameter (LBD), tumor height and TNM. By stratifying patients in groups on the basis of the number of CTC (lower or higher than 10 CTC per 10 mL blood) and the presence of CTC clusters we found a significant difference in LBD (p = 0.019), Tumor height (p = 0.048), disease-free and overall survival (p < 0.05). In conclusion, we confirm the role of CTC as a negative prognostic marker in uveal melanoma patients after a long follow-up period. Further characterization of CTC will help understanding uveal melanoma metastasization and improve patient management.