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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). 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. 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. 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.

Aim: The need for pharmacogenomic education is becoming more and more urgent. Our aim was to evaluate the progress in pharmacogenomics education since then, and to put forward further recommendations. Methods: A survey was sent to 248 schools of medicine, pharmacy, nursing and health professions around the world. Results: The majority of the study programs (87%) include pharmacogenomics education, which is generally taught as part of the pharmacology curriculum. On average, educators and teachers have selected appropriate and highly relevant pharmacogenomics biomarkers to include in their teaching programs. Conclusions: Based on the results, we can conclude that the state of pharmacogenomics education at the surveyed universities has improved substantially since 2005. ispartof: PHARMACOGENOMICS vol:20 issue:9 pages:643-657 ispartof: location:England status: published

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.

In the EU, the “portrait” of healthcare has undergone many changes down the years, with many adaptations as the EU has evolved. The role of patients has become much more significant as they have gained greater knowledge; there have been giant leaps in innovation, while societal changes and issues (such as the ageing population) have led to different priorities. Today's portrait of healthcare features many perspectives, schools of thought and approaches coming from different stakeholders, different Member States and even different regions within those Member States. One thing that has become very clear is that a one-size-fits-all approach to treatment is outmoded, wasteful and often counterproductive to the health of patients. This includes, in these days of increasing co-morbidities, treating one disease separately, rather than looking at the patient's health issues as a whole. Meanwhile, citizens are being bombarded with often contradictory messages regarding what is “good” or “bad” for them, often in a patronising manner, while the realities of extremely effective preventative measures are often obscured, with a lack of emphasis on screening and early diagnosis. The authors argue that, among other matters, better communication and education are key to improving healthcare in Europe.

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., unstable temperature). We report the results of a pilot study implemented within the EC funded SPIDIA project, aimed to investigate the role of transport and storage of blood samples containing and not containing an RNA stabilizer.Blood was collected from a single donor both in EDTA and in PAXgene Blood RNA tubes. Half of the samples were sent to a second laboratory both at room temperature and at 4°C, whereas the remaining samples were stored at room temperature and at 4°C. Gene expression of selected genes (c-FOS, IL-1β, IL-8, and GAPDH) known to be induced or repressed by ex vivo blood handling and of blood-mRNA quality biomarkers identified and validated within the SPIDIA project, which allow for monitoring changes in unstabilized blood samples after collection and during transport and storage, were analyzed by RT-qPCR.If the shipment of blood in tubes not containing RNA stabilizer is not performed under a stable condition, gene profile studies can be affected by the effects of transport. Moreover, also controlled temperature shipment (4°C) can influence the expression of specific genes if blood is collected in tubes not containing a stabilizer.The use of dedicated biomarkers or time course experiments should be performed in order to verify potential bias on gene expression analysis due to sample shipment and storage conditions. Alternatively, the use of RNA stabilizer containing tubes can represent a reliable option to avoid ex vivo RNA changes.

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]. DNA quality parameters were used to evaluate the laboratory performance within an External Quality Assessment (EQA) [Second SPIDIA-DNA External Quality Assessment (EQA): Influence of pre-analytical phase of blood samples on genomic DNA quality – [4]. These parameters included DNA purity and yield by UV spectrophotometric measurements, the presence of PCR interferences by Kineret software and genomic DNA integrity analysis by Pulsed Field Gel Electrophoresis.Here we present the specific laboratory report of the 2nd SPIDIA-DNA EQA as an example of data and performances evaluation.

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. Evaluated genomic DNA (gDNA) quality parameters were: purity and yield by UV spectrophotometric analysis, PCR interferences by Kineret software and integrity by a dedicated algorithm. Results/conclusions 188 applications have been collected from 26 European countries. A high variability of gDNA integrity was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A dedicated analysis on pre-analytical variables and the evaluated gDNA quality parameters showed that blood storage and DNA extraction procedures influence gDNA integrity. The performances of the participants were improved in comparison with the 1st SPIDIA-DNA EQA, probably due to adopted more stringent pre-analytical conditions.

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.

To confirm the efficacy and safety of bevacizumab/XELOX combination for the treatment of locally advanced or metastatic colorectal cancer (CRC) in Italy.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.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.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.

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.

// Francesca Salvianti 1, * , Letizia Canu 1, * , Giada Poli 1 , Roberta Armignacco 1 , Cristian Scatena 2 , Giulia Cantini 1 , Alessandra Di Franco 1 , Stefania Gelmini 1 , Tonino Ercolino 1 , Mario Pazzagli 1 , Gabriella Nesi 3 , Massimo Mannelli 1 , Pamela Pinzani 1, * and Michaela Luconi 1, * 1 Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy 2 Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy 3 Department of Surgery and Translational Medicine, University of Florence, Florence, Italy * These authors have equally contributed to the work Correspondence to: Michaela Luconi, email: michaela.luconi@unifi.it Keywords: adrenal carcinoma, miRNA, liquid biopsy, rare tumor, quantitative real time RT-PCR Received: February 27, 2017 Accepted: April 19, 2017 Published: July 10, 2017 ABSTRACT 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

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. Results show that the classification obtained by this ad hoc procedure allows a more accurate evaluation of gDNA integrity with respect to a single approach.

Cell-free nucleic acids circulating in plasma are considered a promising noninvasive tool for cancer monitoring. BRAF(V600E) mutation in cell-free DNA (cfDNA) could represent an appropriate marker for papillary thyroid carcinoma (PTC).Our aim is to investigate the role of BRAF(V600E)-mutated allele in cfDNA as a marker for the diagnosis and follow-up of PTC.BRAF(V600E) allele was detected and quantified by an allele-specific real-time quantitative PCR assay in plasma from 103 patients affected by nodular goiter. As control populations, we enrolled 49 healthy subjects and 16 patients with non-nodular thyroid diseases.The percentage of circulating BRAF(V600E) was significantly different between patients and controls and throughout different cytological categories of ultrasound-assisted fine-needle aspiration. Patients with a histopathological diagnosis of PTC showed a higher percentage of circulating BRAF(V600E) (P = .035) compared to those with benign histology. In 19 patients, a second blood draw, taken 3-6 months after surgery, showed a lower percentage of BRAF(V600E) in cfDNA than the presurgical sample (P.001). The diagnostic performance of circulating BRAF(V600E) was assessed by receiver operating characteristic curve analysis resulting in an area under the curve of 0.797. A cutoff value was chosen corresponding to maximum specificity (65%) and sensitivity (80%). On this basis, we evaluated the predictive value of BRAF(V600E) in Thy 3 patients with a resulting positive predictive value of 33% and a negative predictive value of 80%.The results of the present study provide encouraging data supporting the possibility to take advantage of circulating BRAF(V600E) in the management of PTC.

Participation in proficiency testing (PT) or external quality assessment (EQA) programs allows the assessment and comparison of test performance among different clinical laboratories and technologies. In addition to the approximately 2300 tests for individual genetic disorders, recent advances in technology have enabled the development of clinical tests that quickly and economically analyze the entire human genome. New PT/EQA approaches are needed to ensure the continued quality of these complex tests.To review the availability and scope of PT/EQA for molecular genetic testing for inherited conditions in Europe, Australasia, and the United States; to evaluate the successes and demonstrated value of available PT/EQA programs; and to examine the challenges to the provision of comprehensive PT/EQA posed by new laboratory practices and methodologies.The available literature on this topic was reviewed and supplemented with personal experiences of several PT/EQA providers.Proficiency testing/EQA schemes are available for common genetic disorders tested in many clinical laboratories but are not available for most genetic tests offered by only one or a few laboratories. Provision of broad, method-based PT schemes, such as DNA sequencing, would allow assessment of many tests for which formal PT is not currently available. Participation in PT/EQA improves the quality of testing by identifying inaccuracies that laboratories can trace to errors in their testing processes. Areas of research and development to ensure that PT/EQA programs can meet the needs of new and evolving genetic tests and technologies are identified and discussed.

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.eu) is to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for molecular testing which requires intracellular RNA analytes. To this end, a survey and a pan-European external quality assessment (EQA) were implemented. This report is the summary of the results of that trial. With the European Federation of Laboratory Medicine (EFLM) support, 124 applications for participation in the trial were received from 27 different European countries, and 102 laboratories actually participated in the trial. Each participating laboratory described their respective laboratory policies and practices as well as blood collection tubes typically used in performing this type of testing. The participating laboratories received two identical blood specimens: in an EDTA tubes (unstabilized blood; n=67) or in tubes designed specifically for the stabilization of intracellular RNA in blood (PAXgene® Blood RNA tubes; n=35). Laboratories were requested to perform RNA extraction according to the laboratory's own procedure as soon as possible upon receipt of the tubes for one tube and 24h after the first extraction for the second tube. Participants (n=93) returned the two extracted RNAs to SPIDIA facility for analysis, and provided details about the reagents and protocols they used for the extraction. At the SPIDIA facility responsible for coordinating the study, the survey data were classified, and the extracted RNA samples were evaluated for purity, yield, integrity, stability, and the presence of interfering substances affecting RT-qPCR assays. All participants received a report comparing the performance of the RNA they submitted to that of the other participants. All the results obtained by participants for each RNA quality parameter were classified as "in control", "warning", "out of control" and "missing" by consensus mean analysis. From the survey data, the most variable parameters were the volume of blood collected and the time and storage temperature between blood collection and RNA extraction. Analyzing the results of quality testing of submitted RNA samples we observed a data distribution of purity, yield, and presence of assay interference in agreement with expected values. The RNA Integrity Number (RIN) values distribution was, on the other hand, much wider than the optimal expected value, which led to an "in control" classification, even for partly degraded RNA samples. On the other hand, RIN values below 5 significantly correlated with a reduction of GAPDH expression levels. Furthermore, the distribution of the values of the four transcripts investigated (c-fos, IL-1β, IL-8, and GAPDH) was wide and the RNA instability between samples separated by 24h were similar. Assuming the presence of at least two quality parameters "out of control" as an indication of a critical performance of the laboratory, 33% of the laboratories were included in this group. The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the pre-analytical phase of RNA analysis of blood samples.

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.

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.

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.

The development of new tools able to select specific plant tissue is crucial for gene expression studies. During the last years, the use of laser microdissection, mainly tested on herbaceous plant tissue, has been found to be a useful technique for these purposes. This method is poorly tested on woody species, and so far no studies of gene expression have been applied on forest trees. For this reason the present work proposes the optimization of a functional protocol using laser microdissection pressure catapulting (LMPC) and real-time reverse transcription–polymerase chain reaction (RT-PCR) in bark stem tissue of Norway spruce (Picea abies). Bark tissue fragments were collected from Norway spruce trees and sliced with a cryostat. RNA was extracted from both whole cross-sections and microdissected bark cells. The feasibility of the method was confirmed by the amplification of the α-tubulin, an endogenous gene of P. abies, with efficiency comparable to that obtained from non-microdissected tissue....

article i nfo Background: Although important improvements of downstream molecular in vitro diagnostics assays based on RNA from blood were made, the pre-analytical workflow is still poorly defined. Methods: We performed a multicenter study within the EU-granted SPIDIA project to investigate blood col- lection and shipping influence on the following RNA quality parameters: yield, purity, integrity, RT-qPCR in- terference and IL1B, IL8, FOS and GAPDH gene expression. Two models were designed: Exp A. Ten laboratories collected blood from an own donor into two different tubes (with or without stabilizer) and extracted RNA at two different times; Exp B. Blood was drawn from a single donor and shipped to ten labo- ratories in two different tubes (with or without stabilizer) for RNA extraction. Results: In both models and collection tubes, reliable results were obtained for purity, yield, GAPDH expres- sion, and interferences. A substantial variation in RIN (Exp A) and in transcription levels of IL1B, IL8 and FOS (Exp B) was observed for blood collected in tube without stabilizer tubes. Overall the variability was higher among data obtained from unstabilized blood samples. Conclusions: We defined the experimental setup for a larger ring trial throughout Europe. The chosen down- stream analyses verified their potential, serving as adequate markers to test the quality of blood RNA.

DNA integrity in blood is an emerging biomarker in cancer. Here we report a real time PCR approach for the absolute quantification of four amplicons of 67, 180, 306 and 476 bp in cutaneous melanoma. Three different integrity indexes (180/67, 306/67 and 476/67 ratios) were tested for their ability to reflect differences in plasma cell-free DNA (cfDNA) fragmentation in 79 patients affected by cutaneous melanoma and 34 healthy subjects. All the three integrity indexes showed higher values in melanoma patients in comparison with healthy subjects. According to ROC curve analysis, the ratio 180/67 is the most suitable index to be used in cancer patient selection, even if the combination of the 3 indexes gives the best performance in terms of clinical sensitivity. The most represented fragments in plasma of melanoma patients are those comprised between 181 and 307 bp, while in healthy subjects there is a prevalence of shorter fragments (67–180 bp). In conclusion, DNA integrity indexes can be considered suitable parameters for monitoring cfDNA fragmentation in melanoma patients.

Purpose Individual variability in radiosensitivity is large in cancer patients. Single nucleotide polymorphisms (SNPs) in genes involved in DNA repair and in protection against reactive oxygen species (ROS) could be responsible for such cases of radiosensitivity. We investigated the association between the occurrence of acute reactions in 101 patients with squamous cell carcinoma of the head and neck (SCCHN) after radiotherapy (RT) and five genetic polymorphisms: XRCC1 c.1196A > G, XRCC3 c.722C > T, RAD51 (c.-3429G > C, c.-3392G > T), and GSTP1 c.313A > G. Materials and methods Genetic polymorphisms were detected by high resolution melting analysis (HRMA). The development of acute reactions (oral mucositis, skin erythema and dysphagia) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for biologically effective dose (BED). Results Development of grade ⩾2 mucositis was increased in all patients (chemo-radiotherapy and radiotherapy alone) with XRCC1-399Gln allele (HR = 1.72). The likelihood of developing grade ⩾2 dysphagia was higher in carriers of RAD51 c.-3429 CC/GC genotypes (HR = 4.00). The presence of at least one SNP or the co-presence of both SNPs in XRCC1 p.Gln399Arg /RAD51 c.-3429 G > C status were associated to higher likelihood of occurrence of acute toxicities (HR = 2.03). Conclusions Our findings showed an association between genetic polymorphisms, XRCC1 c.1196A > G and RAD51 c.-3429 G > C, and the development of radiation-induced toxicities in SCCHN patients.

Background BRAF is the most frequently mutated oncogene in melanoma with BRAF V600E mutation accounting for 92% of all BRAF variants. As this event occurs early in melanoma progression, the quantification of BRAF-mutated alleles in plasma may represent a useful biomarker for noninvasive diagnosis and prediction of response to therapy. Methods We propose an assay based on the use of a locked nucleic acid probe and an allele specific primer to measure plasma-circulating BRAF V600E concentration in patients affected by cutaneous melanoma ( n = 55) and non-melanoma skin cancers ( n = 13) as well as 18 healthy subjects. The assay is highly sensitive and accurate in detecting down to 0.3% of mutated allele in plasma. Results A significant difference between the control group and invasive melanomas ( p BRAF V600E concentration, either as relative percentage or absolute values. ROC curve indicated that BRAF V600E absolute concentration has the maximal diagnostic relevance with 97% sensitivity and 83% specificity. Comparison of the results obtained in plasma with those found in the corresponding tissues indicated an 80% concordance. Conclusions The allele specific Taqman-based real-time PCR assay allows the sensitive, accurate and reliable measurement of BRAF V600E mutated DNA in plasma.

Background: The completion of the Human Genome Project has increased the pace of discovery of genetic markers for disease. Despite tremendous efforts in fundamental research, clinical applications still lag behind expectations, partly due to the lack of effective tools to systematically search for and summarize published data relative to the clinical assessment of new diagnostic molecular tests. Methods: Through a collaborative process using published tools and an expert panel, we developed a detailed checklist of the evidence that needs to be collected or produced to evaluate the potential usefulness of a new molecular diagnostic test. This tool is called GETT, for Genetic testing Evidence Tracking Tool. Results: GETT allows 1) researchers to summarize the current evidence and to identify knowledge gaps for further research and; 2) stakeholders to collect data related to a given molecular test and improve their decision-making process. GETT comprises 72 clearly defined items/questions, grouped into 10 categories and 26 sub-themes, including an overview of disease epidemiology and genetics, the available diagnostic tools, and their analytical and clinical performances, availability of quality control programs, laboratory and clinical best practice guidelines, clinical utility, and impact on health care and psycho-social, ethical and legal implications. It also includes a summary of the evidence available and attempts to prioritise knowledge gaps related to the testing. We also compare GETT to other existing frameworks. Conclusions: This systematic evidence-based tracking tool, which is more detailed than existing frameworks and provides clear definition for each item, will help streamline collection of the available evidence to appraise the potential for clinical application of new molecular diagnostic tests and prioritize research to produce the evidence-base relative to the clinical implementation of molecular diagnostic tests. Clin Chem Lab Med 2010;48:1397–407.

Circulating nucleic acids are present in the blood of humans and other vertebrates. During the last 10 years researchers actively studied cell-free nucleic acids present in plasma or serum with great expectations of their use as potential biomarkers for cancer and other pathologic conditions. In the present manuscript the main findings related to the principal characteristics of circulating nucleic acids, the hypothesis on their origin and some methodological considerations on sample collection and extraction as well as on some innovative assay methods have been summarized. Recent reports on the importance of circulating nucleic acids in the intercellular exchange of genetic information between eukaryotic cells have been reviewed.

The European Register of Specialists in Clinical Chemistry and Laboratory Medicine was established by the European Communities Confederation of Clinical Chemistry and Laboratory Medicine (EC4) in 1997. The main aims of the Register are to ensure a high quality of professional standards and practice in the European Union (EU) countries, and to assist free movement of professionals within the EU, in accordance with European Directives. We report here the latest developments on the structure of the register, a description of the application process and data about the number of registered members, and the relationship between the Register and the European Commission Directive of Professional Qualifications.

Summary Diplodia pinea (syn. Sphaeropsis sapinea), a common pathogenic fungus, causes considerable damage in Italy, particularly to pine stands in which trees are subjected to environmental stress. The occurrence of D. pinea in symptomless Pinus nigra shoots was investigated and related to the amount of radiation received by the trees growing on a site in a year, expressed as the Normalized Insolation index (NIi). Twenty-seven pines were selected from nine locations in Trentino (northern Italy). For each pine the incidence of the fungus in apparently healthy shoots was determined by both culturing on an agar medium and application of real-time PCR. The incidence of D. pinea determined by culturing samples taken from asymptomatic trees was 59% (16 of 27 trees), compared with 85% found using real-time PCR (23 of 27 trees). Detection of the pathogen in healthy pine tissue was positively correlated (p

Centenarians represent a group of population displaying the most rapid expansion. This progressive increase of the presence of centenarians is a multi-factorial phenomenon, which is due to the improvements of the environmental conditions and the life style and also to the progress of the medical science. In order to obtain a more reliable estimate of the longevity per gender and territorial entities, we propose two new indicators. (i) The ratio between the ultranonagenarians and the total population above 65 years old (called longevity index: LI%); (ii) the ratio between the centenarians and the total population above 90 years old (called centenarity index: CI%). An analysis of the data of the Italian National Office of Statistics (ISTAT, www.istat.it/Popolazione/Demo) using these two indicators demonstrates that the subjects above the age of 90 are more frequent in the regions of Central and Northern Italy, which are more developed regarding the economic conditions and technological progress. Nevertheless, the Southern and Insular regions of Italy have a higher occurrence of centenarians among the ultranonagenarian population, and also a higher prevalence of male centenarians, as compared to the northern regions. This demonstrates that achievement of the threshold of 100 years old does not require only particular socio-economic conditions, but also an adequate climate and environment, as well as a favorable genetic composition.

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.

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). 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. 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. 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.

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.

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.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).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.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).

Aims: To develop a quantitative real-time PCR (Rt PCR) assay for the early detection of Biscogniauxia nummularia, a xylariaceous fungus that causes strip-canker and wood decay on European beech (Fagus sylvatica L.). Methods and Results: The molecular assay was based on TaqManTM chemistry using species-specific primers and a fluorogenic probe designed on the ITS1 sequence of rRNA gene clusters. The specificity of the oligonucleotides and the probe were tested using the DNA of B. nummularia isolates from different geographic areas, of phylogenetically related species, and of some fungi commonly colonizing European beech bark and wood. A total of 31 symptomless and symptomatic shoots of European beech were collected from three forest sites in the Apennine Mountains of Italy. The percentage of positive detections of B. nummularia with the TaqManTM assay was 78·6%, compared with only 14·3% of positive isolations on growth media for two sites. Conclusions: In shoots, the quantitative Rt PCR assay detected down to 8·0-fg fungal DNA per microgram of total DNA extracted. Significance and Impact of the Study: The assay developed in quantitative Rt PCR, by using TaqManTM chemistry, revealed a rapid and sensitive method useful for the early detection of B. nummularia in symptomless European beech twigs.

Laser-assisted microdissection (LMD) has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with real-time PCR and RT-PCR techniques, it is now feasible to study genetic alterations, gene expression features and proteins in defined cell populations from complex normal and diseased tissues. The process that brings from sample collection to the final quantitative results is articulated in several steps, each of which requires optimal choices in order to end up with high-quality nucleic acid or protein that allows successful application of the final quantitative assays. This review will describe shortly the development of LMD technologies and the principles they are based on. Trying to highlight the advantages and disadvantages of LMD, the main problems related to specimens collection and processing, section preparation and extraction of bio-molecules from microdissected tissue samples have been analysed.

Background: DNA sequencing is a key technique in molecular diagnostics, but to date no comprehensive methodologic external quality assessment (EQA) programs have been instituted. Between 2003 and 2005, the European Union funded, as specific support actions, the EQUAL initiative to develop methodologic EQA schemes for genotyping (EQUALqual), quantitative PCR (EQUALquant), and sequencing (EQUALseq). Here we report on the results of the EQUALseq program. Methods: The participating laboratories received a 4-sample set comprising 2 DNA plasmids, a PCR product, and a finished sequencing reaction to be analyzed. Data and information from detailed questionnaires were uploaded online and evaluated by use of a scoring system for technical skills and proficiency of data interpretation. Results: Sixty laboratories from 21 European countries registered, and 43 participants (72%) returned data and samples. Capillary electrophoresis was the predominant platform (n = 39; 91%). The median contiguous correct sequence stretch was 527 nucleotides with considerable variation in quality of both primary data and data evaluation. The association between laboratory performance and the number of sequencing assays/year was statistically significant (P Conclusions: Considerable variations exist even in a highly standardized methodology such as DNA sequencing. Methodologic EQAs are appropriate tools to uncover strengths and weaknesses in both technique and proficiency, and our results emphasize the need for mandatory EQAs. The results of EQUALseq should help improve the overall quality of molecular genetics findings obtained by DNA sequencing.

The traditional mediterranean diet is associated with a hope for longer survival. It has also been shown that the red wine possesses a protective effect against the oxidative stress. We studied TAC, the DHEAS and the IGF-1 in a group of 26 healthy centenarians, 17 women and 9 men, of the age range of 100--105 years. Furthermore, we analyzed also serum urate and bilirubin levels between drinkers and abstainers. Most of centenarian subjects have been moderate wine consumers (500 ml/day of red wine). These subjects were subdivided as follows: (i) Group A: those who had maintained the style of their dietary habits as compared to the previous years (n=3 males, 10 females); (ii) Group B: those who actually consumed a diet being deficient compared to that of the previous years, but remained moderate drinkers of red wine (n=3 males, 4 females); and (iii) Group C: those who actually consumed a diet being deficient compared to that of the previous years, and at the same time, were abstainers in wine consumption (n=3 males, 3 females). The results show that in men three of the studied parameters decreased from Group A to C to considerable extents, as follows (mean+/-S.D.). TAC: 302.4+/-32.3; 142.0+/-24.1 and 96.4+/-20.1 micromol/l; DHEAS: 3.35+/-0.81; 2.52+/-0.18 and 1.34+/-0.14 micromol/l; IGF-1: 85.7+/-6.7; 76.6+/-6.7 and 65.6+/-2.6 ng/ml, respectively. For the same parameters, the results in the women were: TAC: 258.4+/-12.2; 182.1+/-14.0 and 107.6+/-10.0 micromol/l; DHEAS: 3.85+/-0.16; 2.34+/-0.19 and 2.05+/-0.04 micromol/l; IGF-1: 89.7+/-6.7; 76.6+/-4.7 and 64.2+/-2.7 ng/ml, respectively. We did not find any significant difference in the other serum parameters between drinkers (n=14) and abstainers (n=3) (urate: 267.6+/-52.9, and 289.5+/-80.1; bilirubin: 9.81+/-4.29 and 7.18+/-2.89 micromol/l, respectively). Our data suggest that the deteriorated diet caused a reduction of TAC, DHEAS and IGF-1 in the centenarians. However, red vine consumption exerted a protective effect against this trend, even if this protection is not reaching statistical significance in some cases (in men), which is due most probably to the lower number of male subjects in the study.

N. LUCHI, P. CAPRETTI, P. PINZANI, C. ORLANDO AND M. PAZZAGLI. 2005. Aims: Real-time PCR, based on TaqMan chemistry, was used to detect Biscogniauxia mediterranea, a fungalpathogen that after a long endophytic phase may cause charcoal disease in oak trees.Methods and Results: Specific primers and probe were designed and tested on axenic cultures of B. mediterraneaand other fungi commonly colonizing oaks. Twig samples were collected in Tuscany from apparently healthyoaks (Quercus cerris, Quercus ilex and Quercus pubescens) growing near trees infected with the fungus. Twigs weredivided into two groups: one for isolation in agar plates, and one for real-time PCR after DNA extraction. Thedetection limit of the assay was 0AE01 pg/DNA, whereas the amounts of fungal DNA detected in asymptomatictissue were >0AE5pglg )1 total DNA extracted. In the apparently healthy twigs the frequency of isolation found onagar was 25AE0%, much lower than that with real-time PCR (96AE4%).Conclusions: Real-time PCR is a sensitive and fast technique able to specifically detect and quantify the DNA ofB. mediterranea in oak tissue.Significance and Impact of the Study: This diagnostic method is a precise tool to localize fungi in symptomlessplant tissues and promises to advance our understanding of fungal infection during their latent phase.Keywords: Biscogniauxia mediterranea, fungi, latent infections, Quercus sp., real-time polymerase chain reaction.

Pinus nigra (Austrian pine) is a tree widely planted in central and southern Europe in the reforestation of areas with poor soils. One of the major constrains of P. nigra is a fungal disease caused by Sphaeropsis sapinea which causes shoot tip dieback and tree mortality. This paper describes a real-time quantitative PCR assay based on the partial sequence of the small subunit ribosomal RNA of S. sapinea to detect and quantify DNA of the fungal pathogen in S. sapinea inoculated shoots. The study found that real-time quantitative PCR was a reliable technique to diagnose this disease and suggests that it can also be used to study fungal behaviour in host tissue and particularly to quantify fungal growth in the latent or endophytic phase.

P. PINZANI, L. BONCIANI , M. PAZZAGLI, C. ORLANDO, S. GUERRINI AND L. GRANCHI. 2004. Aims: To develop a real-time polymerase chain reaction (PCR) method for rapid detection and quantification ofOenococcus oeni in wine samples for monitoring malolactic fermentation.Methods and Results: Specific primers and fluorogenic probe targeted to the gene encoding the malolacticenzyme of O. oeni were developed and used in real-time PCR assays in order to quantify genomic DNA either frombacterial pure cultures or wine samples. Conventional CFU countings were also performed. The PCR assayconfirmed to be specific for O. oeni species and significantly correlated to the conventional plating method both inpure cultures and wine samples (r ¼ 0AE902 and 0AE96, respectively).Conclusions: The DNA extraction from wine and the real-time PCR quantification assay, being performed in ca6 h and allowing several samples to be concurrently processed, provide useful tools for the rapid and directdetection of O. oeni in wine without the necessity for sample plating.Significance and Impact of the Study: Rapid quantification of O. oeni by a real-time PCR assay can improve thecontrol of malolactic fermentation in wines allowing prompt corrective measures to regulate the bacterial growth.Keywords: fluorogenic probes, lactic acid bacteria, malolactic enzyme, malolactic fermentation, TaqMan

Background: External quality assurance (EQA) programs for diagnostic tests based on nucleic acid amplification have not been widely implemented in clinical laboratories and remain limited to few tests. Development of specific EQA programs based on application-based proficiency testing for any diagnostic molecular target is challenging. Development of EQA trials based on methodologic proficiency testing and directed to the evaluation of analytical aspects common to the majority of PCR-based tests may be valuable. Methods: We developed an EQA program for evaluation of DNA extraction and amplification and analysis of products after PCR. Participants received a package containing primers and reference materials to evaluate three specific controls for, respectively, DNA extraction (quality and quantity), PCR performance (specificity and efficiency), and interpretation of results after electrophoresis. Each participant was asked to return to the organizers a form with their numerical results and an aliquot of all amplified samples for joint evaluation. Results: Results varied in all phases of the experimental procedure: preamplification, amplification, and post-PCR interpretation. To give a general estimation on the quality of performances for each laboratory, we designed a score scheme in which the results of any specific action were evaluated on the basis of the distribution around the median consensus values. The maximum possible score was 84. On the basis of total score obtained by each laboratory, we created a qualitative ranking list that provided the final interpretation of results as excellent (>63 points; n = 4 laboratories), good (53–63 points; n = 13), sufficient (42–52 points; n = 15), poor (31–41 points; n = 3), and not acceptable ( Conclusions: This survey demonstrates the importance of EQA trials based on methodologic proficiency testing directed to evaluation of analytical aspects common to the majority of PCR-based tests.

Careful normalization is essential when using quantitative reverse transcription polymerase chain reaction assays to compare mRNA levels between biopsies from different individuals or cells undergoing different treatment. Generally this involves the use of internal controls, such as mRNA specified by a housekeeping gene, ribosomal RNA (rRNA), or accurately quantitated total RNA. The aim of this study was to compare these methods and determine which one can provide the most accurate and biologically relevant quantitative results. Our results show significant variation in the expression levels of 10 commonly used housekeeping genes and 18S rRNA, both between individuals and between biopsies taken from the same patient. Furthermore, in 23 breast cancers samples mRNA and protein levels of a regulated gene, vascular endothelial growth factor (VEGF), correlated only when normalized to total RNA, as did microvessel density. Finally, mRNA levels of VEGF and the most popular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were significantly correlated in the colon. Our results suggest that the use of internal standards comprising single housekeeping genes or rRNA is inappropriate for studies involving tissue biopsies.

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. Methods 157 DNA samples deriving from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. 157 DNA samples derived from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), which aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. Results/conclusions Our results demonstrate that blood sample storage and DNA extraction procedures influence gDNA integrity and that the same blood sample which underwent a long range multiplex PCR based analytical test can provide different results if the adopted pre-analytical procedures are not standardized.

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.

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.

Telomerase is a ribonucleoprotein enzyme that compensates for the progressive erosion of chromosomal ends, called telomeres. In most somatic cells telomerase expression is repressed and telomeres progressively shorten after each cell division, causing cell senescence. Conversely telomerase is active in most human cancers, maintaining the integrity of chromosome ends and representing an important step in cell immortalization and carcinogenesis. The large and increasing interest in telomerase was motivated by the demonstration that more than 90% of human cancers are telomerase positive, whereas most normal tissues or benign tumors contained low or undetectable telomerase activity. We addressed the most recent data on telomerase detection in urological malignancy. Approaches to telomerase inhibition as a future anti-cancer therapy are also discussed.We comprehensively reviewed the most recent and significant publications in this field using current issues of specific journals and a MEDLINE search.Telomerase is often expressed in bladder (90%), prostate (80%) and renal (69%) carcinoma. A variable but significant percent of normal tissues from tumor adjacent zones or noncancer samples are positive for telomerase. The clinical role of telomerase is still questionable in renal cancer, while important insights into the diagnostic role of telomerase in bladder and prostate carcinoma are increasing. Telomerase detection in exfoliated cells collected with urine or bladder washings seems a promising tool for the diagnosis and management of bladder cancer.Larger perspective studies of larger groups of patients are required to discover an appropriate role for telomerase when assessing these tumors. The improvement of quantitative methods to evaluate the expression of telomerase is a cornerstone in the complete clarification of the clinical relevance of telomerase.