Metastatic castration-resistant prostate cancer (mCRPC) and its treatment significantly affect health-related quality of life (HRQOL). Our objectives were to evaluate and compare patient-reported outcome (PRO) claims granted by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for 5 recently approved mCRPC treatments and to examine key characteristics, development, and measurement properties of the PRO measures supporting these claims against current regulatory standards.Five products approved for treatment of mCRPC by the FDA and the EMA (2010–2013) were examined: enzalutamide, abiraterone, sipuleucel-T, cabazitaxel, and radium Ra 223 dichloride. United States (US) drug approval packages and European Public Assessment Reports were reviewed. PRO claims in the US labels and European Summaries of Product Characteristics and supporting measures were identified. For PRO measures supporting claims, a targeted literature review was conducted to identify information on key characteristics and measurement properties; this information was compared against FDA PRO guidance criteria.Nine PRO “claims” were granted across 4 of 5 products reviewed. The EMA granted more claims  than the FDA . The Brief Pain Inventory–Short Form (BPI-SF) worst pain item supported most pain claims and was the only measure supporting US claims. EMA pain claims were supported by BPI-SF worst pain (n = 2) and average pain (n = 1) items and the McGill Pain Questionnaire Present Pain Intensity component (n = 1). EMA HRQOL claims were supported by the Functional Assessment of Cancer Therapy–Prostate Module (n = 2) and the EuroQol 5 Dimensions with visual analogue scale (n = 1). Pain and prostate cancer–specific HRQOL measures supporting claims met US regulatory standards for construct validity, reliability, and responsiveness; these properties were strongest for the BPI-SF worst pain item. Only the BPI-SF worst pain item has documented content validity in mCRPC.PRO label claims were commonly granted across the mCRPC products reviewed. Among the measures reviewed, only the BPI-SF worst pain item supported US label claims. The BPI-SF worst pain item is recommended for pain assessment for the evaluation of new mCRPC treatments. Prostate cancer (PC) is the most commonly diagnosed solid organ malignancy in the United States (US) and the second-leading cause of cancer deaths among American men . These More than 90% of patients with mCRPC develop bone metastases . Bone iIn 2004, two pivotal trials, TAX-327 and SWOG 9916, supported the approval of docetaxel as the first mCRPC treatment, in combination with prednisone, found to prolong median overall survival by approximately 3 months when compared with the combination of mitoxantrone and prednisone . ConseqThe objectives of this research were to evaluate and compare PRO label claims granted by the FDA and the EMA for these five recently approved mCRPC treatments and to examine the key characteristics, development, and measurement properties of the PRO instruments for which label claims were achieved against current regulatory standards for PROs . The fiThe website Drugs@FDA was searched to identify the latest product label and drug approval package (DAP) for abiraterone, cabazitaxel, enzalutamide, radium Ra 223 dichloride, and sipuleucel-T . RelevaThe labels and SmPCs were reviewed to identify PRO label claims and the measures supporting them. DAPs and EPARs were reviewed to determine any PRO measures that resulted in PRO claims but were not specifically named in the label or SmPC. Any indication of PRO-related comments by the reviewing division or the Study Endpoints and Label Development (SEALD) group were also noted when reviewing US DAPs and labels.For analysis purposes, a PRO claim was defined as any mention of a PRO measure or patient-reported endpoint  anywhere in the product label or SmPC. A product with multiple analyses and results described in the label or SmPC based on the same PRO measure counted as a single claim. Each product may have had more than one PRO claim. PRO label claims were classified based on the following types: symptom (pain), PC-specific HRQOL, and generic HRQOL. Symptom-specific claims based on PRO measures focusing solely on assessment of the concept of pain were classified as “symptom (pain).” HRQOL is a multidimensional concept that represents the patient’s general perception of the effect of illness and treatment on physical, psychological, and social aspects of life . “GenerFrequencies and cross-tabulation of label claim types were calculated using Microsoft Excel 2010 (Excel). PRO label claims and the measures supporting them were also summarized descriptively in tabular format.For select PRO measures achieving label claims, targeted searches of PubMed/Medline (back to 2003) were conducted to identify relevant articles for review based on abstracts in English. Additional desktop searches of instrument websites, the public-access version of PROQOLID, and an internal PRO instrument repository at RTI Health Solutions (RTI-HS) were also conducted to identify relevant information and references, especially those published prior to 2003 .The primary goal of these searches was to identify relevant information sources or publications for individual PRO measures, as available, describing instrument development and psychometric properties  in patients with mCRPC and information on how to determine a clinically meaningful change. We also sought to identify key characteristics of each PRO measure supporting label claims including constructs/subscales, the number of items, response scales, and recall period. All relevant information identified was recorded in tabular format in Excel.The following information concerning the development process was identified for each measure, as available, to inform the assessment of content validity: whether patients with mCRPC were included during the development process, whether input from clinicians or health care professionals (HCPs) treating patients with mCRPC was included during measure development, and whether a literature review was conducted to identify items of importance to patients with mCRPC during instrument development.When extracting relevant information from the source documents for each measure of interest, content validity, internal consistency, test-retest reliability, construct and known-groups validity, and ability to detect change  were defined as per the FDA PRO guidance .Responsiveness is best assessed in the context of a therapeutic intervention that has been shown to be effective. Therefore, to confirm PRO measure responsiveness in patients with mCRPC, targeted searches were completed to identify any phase 2 or 3 randomized controlled trials evaluating treatments for mCRPC that included each relevant PRO measure, especially studies supporting an approved PRO label claim for an mCRPC product of interest. Clinical trials that included the PRO measures but did not evaluate one of the five mCRPC treatments of interest were excluded. Full-text articles were preferred, but abstracts with relevant information were also included. Two recent review articles were also searched to identify studies cited demonstrating relevant PRO measure responsiveness .Finally, published information was sought for each PRO measure of interest to identify recommendations for the amount of change required to justify a clinically meaningful change for an individual with mCRPC.The available data identified regarding instrument characteristics, development, validation, responsiveness, and interpretation for each relevant PRO measure were reviewed against FDA PRO guidance criteria, the highest level of evidence currently required by a regulatory agency to support medical product labeling claims . Key coThe EMA has not released a formal guidance document related to PROs; instead, it has issued a reflections paper that provides broad recommendations regarding HRQOL evaluation, a specific type of PRO in the context of clinical trials to support claims . AlthouFour of the five mCRPC products reviewed received one or more PRO label claims Table . Only siTable As shown in Table Table EU claims related to pain progression and pain response were common to both abiraterone and cabazitaxel SmPCs. The abiraterone SmPC had multiple results described based on positive data from both the BPI-SF worst pain item  and the BPI-SF average pain item (reduction in risk [%] of average pain intensity progression). In contrast, the cabazitaxel SmPC indicated that “there was no statistical difference between both treatment arms in pain progression and pain response” based on the PPI scale from the MPQ. The abiraterone and radium Ra 223 dichloride SmPCs both included HRQOL claims based on the FACT-P. Abiraterone’s claims were based on both the risk (%) of and median time (months) to FACT-P total score degradation, whereas radium Ra 223 dichloride’s claim was based on changes from baseline in FACT-P total score. The radium Ra 223 dichloride SmPC also included claim language based on changes from baseline in the EQ-5D and VAS.Two targeted searches were conducted in PubMed/Medline: one search to identify literature describing the key instrument characteristics, development, and validation information for the BPI-SF worst pain item, BPI-SF average pain item, MPQ PPI, and FACT-P; and the other search to identify literature describing the responsiveness of these measures in clinical registration trials for the five mCRPC treatments of interest.The two PubMed/Medline searches identified a total of 71 articles for review; of these, 65 were excluded because they did not contain relevant information, and 6 were included. Additionally, two papers cited in the review articles examined, two papers obtained from the RTI-HS PRO instrument repository, and one paper identified on an instrument developer’s website were included. Eleven papers, in total, were reviewed and are summarized. One published abstract was identified, and the poster with more extensive information, which was provided by the authors upon request, is summarized. The EuroQoL website also provided relevant information for inclusion in this paper. Finally, key measurement characteristics  for the four measures were pulled from PROQOLID to supplement information found in the papers reviewed. The information identified for the four PRO measures of interest then was evaluated against FDA guidance criteria  for PROTable The BPI-SF is a pain-specific measure developed by Charles Cleeland to assess patient-reported severity (or intensity) of pain (4 items) and impact of pain on daily functioning (11 items) in patients with cancer pain . Two ofThe PPI is a single item developed by Ronald Melzack to measure patient-reported pain intensity with present recall. The PPI is one component and an individually scored subscale of the 20-item MPQ . The PPThe FACT-P is a 39-item questionnaire developed by Dr. David Cella and colleagues at Functional Assessment of Chronic Illness Therapy to assess patient-reported HRQOL and PC-specific symptoms . The FAGiven the emphasis of the FDA PRO guidance on content validity, detailed information on the development of each PRO measure of interest was sought to inform the assessment of each measure’s content validity in patients with mCRPC . AlthouNo evidence was identified that input from clinicians or HCPs treating patients with mCRPC was included in the development of any of the four measures reviewed, nor were items of importance to patients with mCRPC identified from the literature review during instrument development.In summary, only the BPI-SF worst pain item has documented content validity in patients with mCRPC, consistent with FDA criteria.Table Internal consistency was not applicable for assessment for the BPI-SF worst pain and average pain items or the PPI because these are single-item measures. Acceptable internal consistency  was reported for the FACT-P, with Cronbach’s alpha coefficients between 0.78 and 0.83 in patients with mCRPC ].].23]].Test-retest reliability was reported as acceptable  for the BPI-SF worst pain item and FACT-P in two trials evaluating treatment for patients with mCRPC ]. R]. R24]].r) between each of these instruments to assess similar constructs using data from two trials evaluating treatment for patients with mCRPC  in the exact format it is administered in your protocol with instructions on how the instrument will be administered, directions explaining how scores will be derived, and how the statistical analyses will be applied; open-label data are only appropriate for labeling if results are convincing and conclusive; pain intensity should be assessed at screening, and then continued eligibility by pain score should be verified at baseline (i.e. before randomization/dosing); pain intensity should then be recorded daily, over the duration of the trial. There should also be evidence of efficacy over the entire duration of treatment; assessment of the ‘worst pain’ will provide more reliable results than ‘average pain’ over 24 hours.” However, it should be noted that the EOP2 meeting for cabazitaxel took place in 2006 and does not appear to represent the most current thinking by the FDA regarding pain assessment. As discussed earlier by Basch and colleagues, daily collection of pain and analgesic assessment for 7 consecutive days prior to an office visit is recommended over continuous daily collection of pain . This rThe largest area of discord identified between the EMA and the FDA was for HRQOL claims. HRQOL claims were granted by the EMA for both abiraterone  and radium Ra 223 dichloride . Similar HRQOL claims for these products were not granted by the FDA.Comments by FDA reviewers found within the radium Ra 223 dichloride DAP may provide some insight into why a HRQOL claim was not granted by the FDA for this product. Specifically, the reviewer comments on the PRO results from the key pivotal trial for radium Ra 223 dichloride stated that “FACT-P and EQ-5D total scores showed a slight improvement for patients receiving Ra-223 when compared to placebo. When evaluated by visit, the statistical significance in the difference between groups decreased over time. Whether this was due to the loss of anti-cancer effect of Ra-223 with time or was due to decreased data completion rates is not known, although both are likely contributors. The FDA SEALD cites limitations specific to the current Ra-223 application including small number of assessments (maximum of 4 for FACT-P), low rate of completion at week 24+ as well as a small observed magnitude of treatment effect. Despite the limitations in choice of instrument, frequency of assessments and completeness of data, the quality-of-life results are supportive of the overall application in that, on average, the available data appear to trend toward an improvement in the Ra-223 arm and do not show a detriment to quality of life measures or pain in patients treated with Ra-223 when compared to placebo.” Similar comments from FDA reviewers were not available within the abiraterone DAP to further explain why HRQOL claims were not granted for this product.As demonstrated by the SmPC language for both cabazitaxel and radium Ra 223 dichloride, the EMA provided more comprehensive coverage of both the positive and negative PRO outcomes from the registration studies for these products, whereas negative PRO outcomes were discussed within the DAP but did not translate into label language in the US.The results of our research on PRO label claims for mCRPC products recently approved in the US and the EU further support the conclusions from several earlier studies with similar objectives -[37-[3735]-]. SimilaOf the measures evaluated, the BPI-SF worst pain item has the strongest measurement properties in patients with mCRPC. Most importantly, content validity has been established in this population, a key criterion necessary for FDA acceptance in supporting labeling claims. Furthermore, although evidence of known-groups validity was not identified for this measure, test-retest reliability, construct validity, and responsiveness have all been established in patients with mCRPC ]. O]. O34]].The identified evidence in support of the BPI-SF average pain item is weaker when compared with FDA PRO guidance criteria. Without documented evidence of content validity in patients with mCRPC, the FDA is unlikely to grant PRO claims based on this single item. However, the BPI-SF average pain item is recommended for publications supporting future FDA-approved products for mCRPC and may achieve EMA-approved SmPC claims if the data are convincing.Of the three pain measures evaluated, the PPI component of the MPQ has the least evidence to support its future use in clinical trials evaluating mCRPC treatments. When considering evaluation of pain in patients with mCRPC and if the desire is to achieve a product label claim in the US or the EU, the PPI is not recommended.The FACT-P was the only HRQOL measure evaluated against FDA PRO guidance criteria. Key strengths for the FACT-P are established reliability , construct validity, known-groups validity, responsiveness, and established definition for clinically meaningful changes to assist with interpretation in clinical trials evaluating patients with mCRPC . In parMore recently, the National Comprehensive Cancer Network FACT Prostate Symptom Index-17 (NCCN-FACT FPSI-17), a new prostate symptom index, was developed based on qualitative input from patients with advanced (stage 3 and 4) castration-resistant PC, which can be used to examine the effectiveness of noncurative treatments in advanced PC . AlthouNotably, the concept of fatigue, a prevalent disease- and treatment-related symptom of patients with mCRPC, was measured using a fatigue-specific PRO measure (Brief Fatigue Inventory) in registration trials for both abiraterone and enzalutamide based on information provided in each product’s DAP and EPAR . HoweveThere are some notable limitations to our research. First, although this study included the majority of recently approved products for mCRPC, not all mCRPC products were included . SecondOur research findings can be used to inform the development of a PRO strategy for phase 2 and 3 clinical trials designed to support a product label claim for a new mCRPC treatment. The BPI-SF worst pain item is recommended for use in combination with analgesic use assessment to evaluate pain progression and pain palliation. Based on the demonstrated content validity in patients with mCRPC and documented measurement properties in this population, as well as the recent claims achieved for enzalutamide and abiraterone, the BPI-SF worst pain item alone may be supportive of future PRO-related claims in both the US and the EU.Assessment of PC-specific HRQOL using the FACT-P to achieve time to FACT-P total score degradation claims may continue to be accepted by the EMA and incorporated into EU SmPCs, whereas FACT-P data are recommended only for publication purposes in the US. An mCRPC symptom index meeting all FDA PRO guidance criteria, including content validity, likely has a greater chance of supporting both US label and EMA SmPC claims. The EQ-5D is recommended for PRO strategy development for any new mCRPC treatment to identify health utility values for comparison across diseases. EQ-5D with VAS data may also be used to support SmPC claims in the EU; however, in the US, these data are recommended for publication rather than label claims. Finally, fatigue is another important disease- and symptom-related concept in patients with mCRPC that should be considered for assessment in future clinical trials using a validated assessment. Further work is needed in this area to increase the likelihood of successful fatigue-specific product label claims for mCRPC products in the US and the EU.AS: Analgesic scoreBPI: Brief pain inventoryBPI-SF: Brief pain inventory–short formDAP: Drug approval packageEQ-5D: EuroQol 5 dimensionsEMA: European medicines agencyEOP2: End-of-phase-2 meetingEPAR: European public assessment reportEQ-5D: EuroQoL 5 dimensionsEU: European UnionFACT-G: Functional assessment of cancer therapy–generalFACT-P: Functional assessment of cancer therapy–prostate moduleFDA: Food and drug administrationHCP: Health care professionalHRQOL: Health-related quality of lifeICC: Intraclass correlation coefficientmCRPC: Metastatic castration-resistant prostate cancerMPQ: McGill pain questionnaireNCCN-FACT FPSI-17: National comprehensive cancer network FACT prostate symptom index-17NRS: Numeric rating scalePC: Prostate cancerPPI: Present pain intensityPRO: Patient-reported outcomePROQOLID: Patient-reported outcome quality of life instrument databaseRTI-HS: RTI health solutionsSEALD: Study endpoints and label developmentSmPC: Summary of product characteristicsUS: United StatesVAS: Visual analogue scaleVRS: Verbal rating scaleFunding for this study was provided by Boehringer Ingelheim GmbH.At the time of this study, Marci Clark, Nimanee Harris, and Catherine Copley-Merriman were employed by RTI Health Solutions, which provides research and consulting services to help biopharmaceutical companies successfully develop and gain market approval for their products. As salaried employees, Dr. Clark, Ms. Harris, and Ms. Copley-Merriman work with various companies but do not receive direct payment or honoraria from them for services rendered. Dagmar Kaschinski and Ingolf Griebsch are employees of Boehringer Ingelheim GmbH.IG and DK had the idea for the study. MC oversaw its design and contributed to the data collection in collaboration with NH. MC did all the analyses and drafted the paper. IG, DK, MC, NH, and CCM contributed to the design of the manuscript, interpretation of results, and discussion of the findings. All authors read and approved the final manuscript.


A major limitation in the concept of oral administration of paclitaxel and docetaxel is their low oral availability [In the past years, the development of oral formulations of the taxane anticancer drugs paclitaxel  lability . Importalability , co-admiEnhanced brain penetration of anticancer drugs due to P-gp inhibition at the BBB can be a double-edged sword: on the one hand, for specifically targeted anticancer drugs, including many tyrosine kinase inhibitors (TKIs), with a low chance of causing problems in the central nervous system (CNS), it may comparatively safely enhance efficacy of the drugs against brain tumor parts or brain (micro)metastases that are positioned behind a functionally intact BBB. On the other hand, for potentially neurotoxic cytotoxins such as the taxanes, there is a risk of disrupting brain functions upon increased CNS penetration, which may thus be undesirable. Our recent study was set up in part to assess the risk of enhancing taxane brain penetration when applying oral taxane administration regimens.We first examined whether we could substantially increase the oral availability of taxanes (at 10 mg/kg) in mice by simultaneous inhibition of P-gp and CYP3A4 using oral co-administration of elacridar 25 mg/kg) and ritonavir (12.5 mg/kg), and to what extent this would affect P-gp transport at the blood-brain barrier . Compari5 mg/kg ams than P-gp. At low plasma concentrations of paclitaxel, these transporter(s) will contribute substantially to limiting brain accumulation of paclitaxel. Partial inhibition of P-gp by elacridar will then have much less of a noticeable impact. At higher plasma concentrations of paclitaxel, the transport capacity of the other transporter(s) will be saturated and partial inhibition of P-gp will thus have more of an impact. Although at this moment in time we can only speculate on the main underlying mechanism, it is likely that the initially far higher plasma concentrations after intravenous compared to oral administration of paclitaxel caused the difference in brain penetration of orally and intravenously administered paclitaxel when co-administered with the P-gp inhibitor elacridar.These results seemed to contrast with previously reported data on increased relative brain penetration after intravenous administration of paclitaxel (10 mg/kg) with orally administered elacridar (25 mg/kg) to mice . InteresIn conclusion, we believe that co-administration of orally applied taxanes and P-gp inhibitors at low dose may provide a relatively safe strategy to boost plasma exposure of taxanes in patients, as relative brain exposure is unlikely to be higher than that in the currently used intravenous schedules. Furthermore, our data suggest that one should be cautious in predicting drug-drug interactions at the BBB after oral administration of a substrate drug based on data obtained after intravenous administration; especially potentially prominent effects of differences in plasma concentrations of the drugs between the different administration routes should be taken into account.


Right-sided aortic arch is a rare congenital defect usually diagnosed incidentally in adults; it is often asymptomatic unless aneurismal disease develops. In half the cases, an aberrant left subclavian artery arises from a Kommerell’s diverticulum; in these cases, congenital heart anomaly is very rarely present.We report a case of incidentally-detected right-sided aortic arch with multiple vascular anomalies including left subclavian artery originating from a Kommerell’s diverticulum, supra-sinus origin of coronary arteries and coronary arteriovenous fistula.Through comprehensive 3-dimensional reconstruction of the aortic arch and surrounding structures we defined anatomical relationships, which is useful for follow-up and treatment. In 1936, Burckhard Friedrich Kommerell first described an aberrant right subclavian artery originating from the descending thoracic aorta of a left-sided arch and associated with persistence of a remnant of the right dorsal aorta. This now called Kommerell’s diverticulum consists of both an aneurysm of the thoracic aorta and an aneurismal orifice of the aberrant subclavian artery ; it is uA 20-year old man was referred to the Department of Cardiology for assessment of a vascular abnormality incidentally detected in a CT scan obtained by the Otorhinolaryngology Department during pre-operative assessment for chronic otitis media. Other than otalgia, the patient reported no symptoms, including dysphagia or dyspnea. Physical examination revealed regular heart sounds without cardiac murmur. CT angiography with a 128-slice multi-detector scanner was performed for definitive diagnosis and comprehensive definition of thoracic anatomy. Multi-directional analysis of reconstructed 3D images of the thoracic aorta and its branches and of the trachea and esophagus (Figure A right-sided aortic arch results from persistence of the right fourth aortic arch with suppression of the corresponding left vessels . The claAs previously mentioned, Kommerell’s diverticulum may cause symptoms of tracheal or esophageal compression, such as dysphagia, dyspnea, stridor, wheezing, cough, choking spells, or chest pain . HoweverA right-sided aortic arch can present with a number of vascular anomalies of the aortic arch system . In thisMost symptoms requiring surgery are secondary to compression of the trachea or esophagus by the aberrant vascular structures . TherefoComprehensive imaging with 3D reconstructions could be the gold standard for detecting multiple vascular malformations and understanding relationships with adjacent structures in these patients.Written informed consent was obtained from the patient for publication of this case report and the accompanying images. A copy of the written consent form is available for review by the Editor of this journal.CT: Computed tomography; 3D: Three-dimensional.The authors declare that they have no competing interests.PJS conducted the clinical diagnosis, conceived the report and drafted the manuscript. SJW gathered patient data and helped to draft the manuscript. LCH wrote the case report and assisted in image acquisition and interpretation. Finally, PJS provided supervision and critically reviewed the manuscript. All authors gave their final acceptance to the submission of this report. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/14/104/prepub


There is a short period during rapid growth when a scoliosis brace is most effective. In standard practice, the in-brace correction is not measured during brace casting. The first assessment of the in-brace correction in the follow-up clinic may trigger brace adjustment for improved spinal alignment resulting in delay of effective treatment and increased number of radiographs. The objective was to investigate if real-time ultrasound (US) can aid orthotists in determining the pad pressure level and location resulting in optimal in-brace correction of the spine.Twenty six subjects participated with 9 candidates  in the intervention group and 17  in the control group. All participants were diagnosed with AIS and prescribed either a full time TLSO or a night time Providence brace. A medical ultrasound (US) system, a custom pressure adjustment system and custom software were used.For the intervention group, each subject first underwent a baseline standing US scan. The orthotist then used a custom standing Providence brace design system to apply pressures against the patient's torso. The applied pad pressures were recorded using our pressure system. The first US assessment scan was acquired. A real-time US spinal image with the coronal curvature measurement was displayed. A comparison between the baseline and the assessment scan was performed. The orthotist then decided if an adjustment was needed in terms of altering the pad locations and magnitudes. Another US scan was taken if a second configuration was required. The orthotist used the best simulated in-brace correction configuration to cast the brace.The orthotist tried a second configuration in 7 out of 9 cases (78%). Among these 7 cases, 5 showed better stimulated in-brace corrections which was used to cast the brace. Ultrasound resulted in immediate improved in-brace correction for 5 out of 9 cases (56%). In the control group, 8 of 17 (47%) subjects required a total of 16 additional brace adjustments requiring a total of 33 in-brace radiographs. Some subjects required multiple adjustments. Three intervention subjects have returned to the follow-up in-brace clinics and the other 6 are scheduled in the next 2 months. One subject required a minor adjustment. The total number of in-brace x-rays was 4.The use of the ultrasound system provided a radiation-free method to determine the optimum pressure level and location to obtain the best stimulated in-brace correction during brace casting.


Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types  in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70–80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available. Formalin-fixed, paraffin-embedded (FFPE) tissue samples stored in diagnostic pathology archives represent an invaluable biobank for retrospective clinical research. In addition, FFPE specimens may be useful in prospective studies omitting the collection of fresh-frozen (FF) specimens. Unfortunately, nucleic acids are more difficult to extract from FFPE tissue because of the need to remove the paraffin and to counteract covalent protein-DNA interactions that result from the fixation process. In addition, fixation delay (i.e. perioperative ischemic time), the fixation process, tissue preparation, paraffin embedding, and archival storage contribute to fragmentation, cross-linking and chemical modification of FFPE tissue-derived nucleic acids. These changes interfere with many classical molecular analyses requiring high quality nucleic acids. Advances in next-generation sequencing (NGS) technologies allow the investigation of genomes, epigenomes and transcriptomes using limited sample material. Moreover, this analysis can be made at a relatively modest cost, considering the massive increase in the amount of information that may be obtained. The power of NGS to analyse in depth large numbers of short sequences potentially makes this an ideal technology to apply to the usually fragmented nucleic acids that may be extracted from FFPE specimens. The development of reliable NGS-based methods for use with low-quality FFPE tissue-derived nucleic acids would open the diagnostic pathology archives to high-throughput profiling, facilitating extensive retrospective clinical studies. Similarly, the ability to use FFPE samples for molecular analysis in prospective studies would be of great benefit, by potentially reducing or even eliminating the need for the tedious collection and storage of cryopreserved clinical samples.et al. who reported NGS-based analysis of DNA (DNA-Seq) isolated from FFPE samples et al. combined pooling of samples and reduction in the amount of input DNA to study genome-wide copy number alterations in FFPE samples et al. applied targeted DNA resequencing to screen FFPE non-small cell lung carcinomas for clinically important EGFR, KRAS and BRAF mutations and found a significant correlation with the results of real-time PCR based analyses performed in parallel et al. applied targeted resequencing of 27 cancer related genes to 16 FF/FFPE paired lung adenocarcinomas and found, that in spite of demonstrable effects of the fixation process, identical single-nucleotide variants could be reliably detected et al. have reported a high-throughput sequencing method for epigenetic profiling based on ChIP-seq, FFPE samples from a mouse leukemia model and from a limited range of human cancers (one seminoma and six breast carcinomas), that allow analysis of histone modifications and transcription factor binding on a genome-wide scale et al.While nucleic acids isolated from FFPE tissues have previously mainly been studied using PCR- and microarray-based methods, there are now reports on the application of NGS to FFPE material. One of the first came from Schweiger et al. were one of the first to describe NGS-based analysis of RNA (RNA-Seq) isolated from FFPE samples et al. reported the successful application of ligation-based miRNA sequencing to cancer samples stored up to 9 years et al. who found that RNA-Seq data derived from FFPE primary breast cancers was of sufficient quality to enable biomarker discovery et al. reported a comparative analysis of five methods for RNA-Seq applied to low-quality RNA (such as that isolated from FFPE tissue) and low-quantity RNA et al. applied RNA-Seq to nine sets of FF and FFPE breast tumour samples stored for up to four years and found good correlation between the paired FF and FFPE expression profiles Weng We expand on these studies in terms of number and diversity of human specimens investigated and reports a systematic analysis of the potential use of NGS-based profiling of human FFPE samples in retrospective and prospective clinical studies. Our study includes (1) the evaluation of different isolation strategies of nucleic acids from human FFPE specimens and matching FF and FFPE specimens; (2) the preparation of targeted genome (DNA Exome-Seq) and whole transcriptome (RNA-Seq) sequencing libraries; and (3) thorough analysis of the NGS data obtained. The primary objectives were to identify and characterize systematic effects of the fixation process on the results obtained as well as critical parameters in the workflow from surgery to analysed NGS data. Available extraction kits were evaluated with respect to purification of RNA and DNA from selected FFPE human specimens . This was followed by evaluation of the extraction of RNA and DNA from FFPE human specimens  with different routine archival storage times (up to 244 months). Finally, DNA and RNA were extracted from matching FF and FFPE human specimens . Extracted DNA and RNA were used for the preparation of targeted genome (DNA Exome-Seq) and whole transcriptome (RNA-Seq) sequencing libraries and NGS data obtained was analysed, focusing on the discovery of systematic effects of the fixation process.All experiments in this study were conducted on specimens of human origin selected from the frozen tissue biobanks at Aarhus University Hospital, Denmark and from the diagnostic FFPE block archive of the Institute of Pathology, Aarhus University Hospital, Denmark. The tissues were removed in the course of routine surgical procedures and were surplus to diagnostic requirements. Samples were anonymized before use in this study. The study was approved by the Committee on Health Research Ethics of the Central Denmark Region and the Danish Data Protection Agency.the test set). To study the impact of tissue type and storage time after fixation, FFPE blocks from four tissues  were selected, each with four storage times . This experiment is denoted the storage time FFPE set. The largest experiment, the paired FF/FFPE colon set, included 19 paired FF/FFPE colorectal tumour samples  and 13 FF samples of matching normal colon tissue that had been collected 2 – 13 years previously. To test the detection of specific single nucleotide variants, a set of 11 colon samples were selected with known variants in KRAS and BRAF (the colon KRAS/BRAF variants detection set). Furthermore, seven paired FF/FFPE prostate carcinoma samples collected 2 – 11 years previously (the paired FF/FFPE prostate set), eight paired FF/FFPE bladder carcinoma samples collected 5 – 9 years previously (the paired FF/FFPE bladder set) and 10 FF bladder carcinoma samples (the FF bladder signature conservation set) were selected for study. DNA and RNA were isolated from each sample and used to generate libraries for exome sequencing and total-RNA sequencing. The primary objective of the DNA Exome-seq experiments was to compare the detection of genomic variation in matched FF and FFPE samples, including both specific variants of clinical relevance as well as global variations. The RNA-Seq experiments were conducted to allow comparisons to be made of the transcriptional information obtained from matched FF and FFPE samples, including (1) global information on transcriptional activity; (2) specific expression levels in relation to biological status ; and (3) description of an expression signature for bladder tumour progression (in the FF bladder signature conservation set and the paired FF/FFPE bladder set).Study workflows from surgery to final analysis of the NGS data are illustrated in test set , liver , bladder (carcinoma) and colon (carcinoma). Each tissue was represented with four FFPE blocks stored for 2–3, 13–15, 60–61 and 241–244 months, respectively . Only ththe colon KRAS/BRAF variants detection set, The storage time study was followed by purification of DNA and RNA from paired FF/FFPE samples of colon (19 samples), bladder (8 samples) and prostate (7 samples) carcinomas . In addiGenomic DNA libraries were prepared from FF- and FFPE-derived DNA using TruSeq DNA library preparation kits  followed by exome enrichment using TruSeq exome kits (Illumina). gDNA library preparation was done according to the manufacturer's manual with the following modifications. (1) In order to avoid buffer-effects, 1.2 µg of dsDNA was processed using a QIAGEN UF filter plate, washed twice in Buffer EB  and resuspended in 50 µL Buffer EB before being used as input for the TruSeq gDNA library preparation. (2) The number of PCR cycles used for amplification of FFPE-derived libraries was increased from 10 to 11. (3) After the final bead-purification step of the amplified gDNA libraries, 10 µL Buffer EB was added to the libraries, bringing the final volume up to 40 µL. The size distribution of the gDNA libraries was estimated by on-chip electrophoresis (DNA 1000 DNA chip) of a 1 µL sample on an Agilent 2100 Bioanalyzer (Agilent Technologies). The DNA concentration of the libraries was estimated using the KAPA Library Quantification Kit (Kapa Biosystems). Briefly, quantification was done using 10 µL reaction volumes containing 6 µL KAPA reagent/primer mix and 4 µL 1.000.000-fold diluted sample following the recommended procedure and the supplied standards. Quantification was carried out on three independent dilutions of each sample. Exome targeting was performed on pools of up to six gDNA libraries using 500 ng of each gDNA library according to the manufacturer's recommendations. gDNA libraries prepared from FF and FFPE samples were not pooled. After the final bead-purification step of the exome captured and amplified gDNA libraries, 30 µL Buffer EB was added to the libraries, bringing the final volume up to 60 µL. The size distributions of the captured libraries were estimated by on-chip electrophoresis (High sensitivity DNA chip) of a 1 µL sample on an Agilent 2100 Bioanalyzer (Agilent Technologies). The DNA concentration of the libraries was estimated using the KAPA Library Quantification Kit as described above. The exome captured gDNA libraries were combined into 2 nM pooled stocks, denatured and diluted to 10 pM with pre-chilled hybridization buffer and loaded into TruSeq PE v3 flowcells on an Illumina cBot followed by indexed paired-end sequencing (101+7+101 bp) on a Illumina HiSeq 2000 using TruSeq SBS Kit v3 chemistry (Illumina).KRAS and BRAF mutation analysis of the 11 samples in the colon KRAS/BRAF variants detection set were performed as described KRAS codon 12, 13 and 61 and BRAF V600E and codon 442–474 were examined using the Lightscanner (Idaho Technology). Samples with diverging melting curves were further validated by Sanger sequencing on the 3130x Genetic Analyzer (Applied Biosystems)To enable a thorough analysis of the RNA-Seq data, we required the method used for preparation of RNA-Seq libraries to provide strand-specific  and paired-end information as well as facilitating multiplex sequencing. Because of the expected degradation of the RNA from the FFPE tissues, the library preparation method should not be based on selection of poly(A)+ as this would impair library preparation or at least result in a 3′ bias. Obtaining total-RNA-Seq data would furthermore facilitate a more complete view of the transcriptome. When the study was initiated in 2011, the only commercialised kit fulfilling all requirements was the Ribo-Zero technology  for depletion of rRNA followed by library preparation using the ScriptSeq technology . Cytoplasmic and mitochondrial rRNA were removed from total RNA using the Ribo-Zero Magnetic Gold Kit  following the manufacturer's instructions. Briefly, rRNA Removal Solution was added to 500 ng of total RNA and incubated for 10 min at 68°C, then 15 min at room temperature. Probes with hybridized rRNA were removed by adding the RNA-probe mixture to washed Magnetic Beads followed by incubation for 5 min at room temperature, mixing by vortexing, incubation for 5 min at 50°C, magnetization and transfer of the supernatant to a RNase-free tube. The rRNA depleted RNA was purified using Agencourt RNAClean XP Kit . Briefly, a 1.8x volume of AMPure RNAClean XP Beads was added to the rRNA depleted RNA followed by incubation for 15 min at room temperature, two 80% ethanol washes and elution into 30 µL RNase-Free water. The quality of the rRNA depleted RNA was estimated by on-chip electrophoresis (Picochip) of a 1 µL sample on an Agilent 2100 Bioanalyzer (Agilent Technologies). Savant Speed-vac (Thermo Fischer Scientific) was used to reduce the remaining sample volume to 9.5 µL followed by synthesis of directional, paired-end and indexed RNA-Seq libraries using the ScriptSeq v2 kit  following the recommended procedure. Briefly, rRNA depleted RNA was chemically fragmented (unless severely degraded RNA from FFPE tissue was used in which case further fragmentation was omitted), following the procedure described in the appendix in the ScriptSeq manual and cDNA was synthesized from a tagged random hexamer. The cDNA was terminal tagged using a 3′-end blocked and tagged oligo followed by MiniElute (QIAGEN) purification. The di-tagged cDNA was then used as template for PCR  using the FailSafe PCR Enzyme , a forward primer and an indexed/barcoded reverse primer. The amplified libraries were purified using Agencourt XP Kit (Beckman Coulter). Briefly, a 1.0x volume of AMPure XP Beads (Beckman Coulter) was added to each PCR reaction followed by incubation for 15 min at room temperature, two 80% ethanol washes and elution into 20 µL RNase-Free water. The qualities of the RNA-Seq libraries were estimated by on-chip electrophoresis (High Sensitivity DNA chip) of a 1 µL sample on an Agilent 2100 Bioanalyzer (Agilent Technologies). The DNA concentration of the libraries was estimated using the KAPA Library Quantification Kit (Kapa Biosystems) following the recommended procedure and the supplied standards. The RNA-Seq libraries were combined into 2 nM pooled stocks, denatured and diluted to 10 pM with pre-chilled hybridization buffer and loaded into TruSeq PE v3 flowcells on an Illumina cBot followed by indexed paired-end sequencing (101+7+101 bp) on a Illumina HiSeq 2000 using TruSeq SBS Kit v3 chemistry (Illumina).The analytical workflow is described in this section and graphically presented in remove duplicate mapped reads tool was used to remove paired reads that have the same start and end coordinates and are, thus, probably PCR duplicates. Variants were detected in the exome data with the CLC Bio Probabilistic Variant Caller using the following parameters: Minimum coverage  =  10; Variant probability  =  90.0; Maximum expected variants  =  4; Requirement of reads in both directions supporting the call.Paired de-multiplexed fastq files from DNA-exome libraries were imported into the CLC Genomics Workbench  running on CLC Genomics Server . Adaptor sequences and bases with low quality were trimmed and reads were mapped to HG19. The Use reference with annotations option. With this option, reads are mapped against sequences corresponding to the annotated gene and transcript sequences. In the first run, only matches to the forward strand of the genes were accepted (using the strand-specific-forward option); in the second run, the reads that were not mapped in the first run were mapped allowing only matches to the reverse strand of the genes (using the strand-specific-backward option). To estimate contamination with rRNA, reads that did not map to the human transcriptome as defined by RefSeq, were mapped strand specific forward towards human rRNA  and un-mapped reads were then mapped strand specific backward towards human rRNA. Mapping Reports and Detailed Mapping Reports were generated from each RNA-Seq analysis for each sample, exported from the CLC Genomics Workbench in Microsoft Excel format, saved as individual tab-delimited text files, and specific data were subsequently extracted and studied. For each experiment, gene-wise mapping matrices of both forward and backward RNA-Seq analyses against the human transcriptome were exported from the CLC Genomics Workbench as tab-delimited text files for exploration and statistical analysis in the R computing environment . This resulted in three different fastq files: paired-end data, single-end data after joining of paired reads, and single-end data after elimination of one of the paired reads due to e.g. low quality. All three types of fastq files were then imported batchwise into the CLC Genomics Workbench  running on a CLC Genomics Server  using CLC Server Command Line Tools . The imported high quality reads were mapped against gene regions and transcripts annotated by Human NCBI REFSEQ October 30, 2012 in two runs of the RNA-Seq Analysis tool of CLC Genomics Workbench. The RNA-seq tool was run with the All data underlying the findings described in the manuscript are fully available without restriction. Access to the sequence data, containing person identifying information, needs signature on a controlled access form, and can be accessed at The European Genome-phenome Archive We studied the potential use of FFPE samples in NGS-based retrospective and prospective clinical studies, focused on detection of systematic effects of the fixation process on variant calls in DNA-Seq and on the expression profiles in RNA-Seq. The study included investigation of methods for isolation and characterization of nucleic acids purified from matching FF and FFPE specimens, preparation and quality control of exome and RNA-Seq NGS libraries, as well as analysis and interpretation of the derived NGS data.FFPE samples were routine diagnostic specimens, removed up to 244 months before the study start. Therefore, detailed information on the handling of samples from surgery through fixation, preparation and storage (e.g. perioperative ischemic times and fixation times) were not available. This is the typical situation for paraffin blocks from a routine pathology archive. Although pathology laboratories use standard protocols, these will vary according to the type and size of specimen, the clinical situation, the time of sampling (i.e. routine vs. out-of-hours specimens), and according to local circumstances and logistics. As a result, it will rarely be possible to estimate the expected quality of extracted nucleic acids based on the available information for a given FFPE sample. Results of the initial DNA and RNA purifications from FFPE tissues, performed in order to identify optimal extraction methods, are summarized in the storage time FFPE set. Only the ExpressArt FFPE RNAready kit was used for RNA purifications from the three paired FF/FFPE sets. Sufficient yields  for preparation of RNA-Seq libraries were obtained from all samples. Storage time of the FFPE blocks was found to have a negative impact on RNA quality, but not on the yield (miRNeasy FFPE (QIAGEN) and ExpressArt FFPE RNAready (Amp Tec) kits resulted in higher yields and less severely degraded RNA as compared with Nucleospin FFPE RNA (Machery-Nagel) . Therefohe yield . HoweverDNA isolated from FFPE tissue was found to be relatively intact, although there was a tendency for degradation to increase with storage time. As dsDNA, in contrast to ssDNA, can be ligated to adapters during the preparation of gDNA-Seq libraries for NGS, it is important to measure the concentration of dsDNA using a suitable method and not to rely on A260 nm measurements, which does not distinguish between dsDNA and ssDNA. The QIAamp DNA FFPE Tissue (QIAGEN) gave higher dsDNA yields than the Nucleospin FFPE DNA kit (Machery-Nagel) .Parallel purification of RNA and DNA from the same FFPE sections was tested using the Nucleospin FFPE RNA/DNA kit (Machery-Nagel) and RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Ambion). However, this resulted in low yields of both RNA and DNA and these purification methods were not further included.the paired FF/FFPE colon set and for eight of the 11 samples of the colon KRAS/BRAF variants detection set, but failed for the remaining 43 FFPE samples (29.5% (18/61) success rate). The Illumina TruSeq Exome-sequencing libraries were obtained by preparation of whole genomic sequencing (gDNA-Seq) libraries followed by capture of the exome target on biotinylated probes, complementary to the exome and UTR regions. A total of 500 ng of gDNA-Seq library was required for the subsequent capture reaction and we found that the major challenge in preparing exome-sequencing libraries from FFPE-derived DNA was to obtain a sufficient amount of the gDNA-Seq libraries. We observed that degraded DNA invariably produced very low levels of gDNA-Seq library but it was not caused by the shorter DNA fragments per se. Thus, the degree of degradation must correlate with another FFPE-related DNA modification that prevents efficient gDNA-Seq library production.Preparation of DNA Exome-Seq libraries was successful for ten of the 19 samples in In agreement with the notion that DNA is degraded over time during FFPE-storage, we found that gDNA-Seq library yields were inversely correlated with the storage time of the FFPE blocks, i.e. the longer an FFPE block had been stored, the lower the gDNA-Seq library yield. Interestingly, a comparison of the use of DNA from FFPE-blocks fixed and then stored for no longer than three months with DNA from control FF samples for library preparation, showed a significant reduction in efficiency, even in these freshly prepared FFPE-blocks. Therefore, in addition to the negative correlation between FFPE block storage time (degradation) and gDNA-Seq library preparation efficiency; there is a general effect of tissue fixation and paraffin embedment that reduces the ability to produce gDNA-Seq libraries from FFPE-derived DNA.During gDNA-Seq library preparation, DNA fragmentation was followed by three enzymatic steps resulting in the addition of a total of 120 bp of adapter sequence. After adapter ligation, gDNA-Seq libraries were PCR amplified. Comparison of the size distribution of the DNA fragments prior to adapter ligation with the unamplified gDNA-Seq library after adapter ligation showed that the FFPE-derived DNA was performing well in these steps of the gDNA-Seq library preparation. This indicated that it was the final PCR amplification step in the library preparation that was compromised when using FFPE-derived DNA as the starting material. This was probably a result of DNA modifications caused by formalin fixation of the tissue. In order to increase the number of successful gDNA-Seq libraries from FFPE-DNA, the number of PCR cycles was increased from 10 to 11.If less than 1 µg of dsDNA was used for gDNA library prep, the complexity of the resulting gDNA and exome library was reduced. This was observed as an increase in the fraction of PCR duplicates in the data. Since only a fraction of FFPE-derived DNA can be amplified during gDNA library prep, this effectively corresponded to an input of less than 1 µg of dsDNA. In agreement with this, we found that the PCR duplication rate was significantly increased in FFPE samples (by 60% – 85%) compared to the FF control samples (approximately 30%), as measured by CASAVA during library QC. The relatively high level of duplicates in the control FF samples was a result of the increase in the number of PCR cycles. The recommendation for TruSeq exomes was to sequence three samples in each flow cell lane. Importantly, sequencing of the libraries after exome targeting showed that the loss in reads due to PCR duplicates in FFPE samples could largely be overcome by sequencing fewer samples in each lane. We found that the sequencing of three FFPE exomes across two lanes resulted in a number of unique reads comparable to that found for the libraries prepared from matching FF-sample DNA or from high quality control DNA sequenced as three samples per lane. Exome capture and sequencing of gDNA-Seq libraries that produced significantly less than 500 ng of gDNA-Seq library resulted in an increase in PCR duplicates to a level where deeper sequencing may be needed to produce meaningful data. This corresponded to an exclusion of all heavily degraded FFPE-derived DNA from exome sequencing using the protocol described here.Neither increasing the amount of dsDNA used as input for gDNA-Seq library preparation from 500 ng to 2 µg nor parallel preparation of two gDNA-Seq libraries followed by pooling before exome capture reduced PCR duplicate level. Because cytosine deamination to uracil is a common form of damage in FFPE-derived DNA, we tested the use of the KAPA HiFi Uracil+ DNA Polymerase (Kapa Biosystems) for library amplification. However, the resulting libraries had a higher PCR duplication level than the libraries prepared using the DNA polymerase provided in the TruSeq DNA library preparation kits.the paired FF/FFPE colon set were available for further analysis. The number of total reads, number of mapped reads, percentage of reads mapped, percentage of duplicates and number of variants called are listed in Exome data from ten FF/FFPE pairs from KRAS and/or BRAF mutations were selected for exome sequencing. Eight of these were successfully sequenced and all previously identified KRAS/BRAF mutations were also identified by exome sequencing with a frequency between 6 – 46% , the bladder and prostate sets) and in the storage time FFPE set). In the colon set, essentially no differences were observed between FF and FFPE libraries in the fraction of reads mapping to the different target groups. A total of 61% of reads mapped to the human transcriptome in the forward orientation and 8% in the backward orientation, while 3% mapped to human rRNA in the forward orientation and 7% in the backward orientation, leaving 21% un-mapped. In the prostate and bladder sets  or not to be mapped (in the prostate set). In the storage time FFPE set  using the RNA-Seq analysis tool of the CLC Genomics Workbench. The reads were sequentially mapped strand specific against the forward and then backward human gene regions and transcripts. The reads that did not map to the human transcriptome were mapped sequentially strand specific forward and then backward towards Human rRNA. The proportions of reads mapping to the different targets are presented in der sets , we obseFFPE set , we obsethe storage time set there was a tendency to see an increasing fraction of non-perfect matches as a function of increasing storage time . The complexities of the libraries were estimated by the post mapping fraction of duplicated sequences and are presented in ic reads .To summarize the general tendencies in mapping of FF and FFPE NGS data (using paired-end 2x 101 bp sequencing) from DNA and RNA, we found: (1) A higher fraction of non-specifically mapped reads was observed in data from DNA compared with RNA. (2) There tended to be a higher fraction of non-specifically mapped reads in data from FFPE-derived DNA, while no differences were found between FF and FFPE data in RNA . (3) Thethe prostate set. The paired study design was evident for some pairs which clustered closely together in MDS, while other pairs were more distantly located. It should be recalled that the FF and FFPE samples are indeed sampled from two different tissue locations. As a result, tissue heterogeneity  can be expected to result in variations in the profiling results comparing paired FF and FFPE samples. Paired statistical tests for significantly (FDR<0.05) affected genes in FF and FFPE sample profiles revealed 7,078 genes to be affected in the colon set , 2,984 affected genes in the bladder set  and 6,333 genes affected in the prostate set . Ratio-intensity (MA) plots from the three tests with the significantly affected genes highlighted are presented in To investigate the effects of the FFPE process  on expression profiles, expression values from the paired FF/FFPE samples (RPKM of exonic reads) were plotted and the Pearson Correlations Coefficient was calculated for each set. We found the expression profiles obtained from FF and FFPE samples to be highly correlated, both in the recently collected samples and, importantly, even in samples collected 14 years previously . Analysithe colon set consisted of 12 paired FF/FFPE colon tumour samples, of which six had an additional sample of adjacent normal colon tissue. These six trio-sets of tumour and normal tissue were used to study the concordance between the expression profiles of affected genes in tumour versus normal tissue (both FF) and the profile deduced from the FFPE tumour samples. MDS analysis of the expression profiles of the trio-sets  will generate a background of variation, against which changes in NGS data derived from the paired FF and FFPE samples has to be analysed. In spite of these challenges, we were able to identify clear and close correlations between the expression profiles in RNA-Seq data, and detect similar sequence variants in DNA Exome-Seq data for the FF/FFPE pairs. Thus, in spite of the potential problems of both sampling variation and tumour heterogeneity, our study shows that NGS-based analysis of FFPE samples may provide valid, robust data.et al. who reported that the RNeasy FFPE Isolation Kit (QIAGEN) performed well and was superior to the NucleoSpin FFPE RNA Isolation Kit (Macherey-Nagel) It will rarely be possible to estimate the expected quality of extracted nucleic acids based on the available information for a given FFPE block. Instead, standard methods of purification from FFPE tissue should be adopted and the extracted nucleic acids should be validated to decide if the material is of sufficient quality and quantity to be used for NGS based studies. The different FFPE specimen extraction methods tested varied in both quality and yield of nucleic acids. However, we were able to identify a purification method which, in our hands, was the best among those tested in terms of yield, degradation and (for DNA) fraction of dsDNA. For RNA extraction, our results are in agreement with Boeckx the paired FF/FFPE colon set and for eight samples of the colon KRAS/BRAF variants detection set but failed for the remaining 43 specimens (29.5% (18/61) success rate). The main reason for the failure was non-efficient amplification of the libraries following adaptor ligation, most probably as a result of DNA modifications caused by the fixation and subsequent storage. This underlines the need for improved extraction strategies that are more efficient in reversing these modifications. Since cytosine deamination to uracil is one common type of damage in FFPE-derived DNA, we tested an alternative uracil tolerant DNA polymerase. However, this showed no advantage over standard DNA polymerase. Nonetheless, we believe this may be a fruitful area to explore, and additional DNA polymerases should be tested in the future for their ability to facilitate more efficient amplification of libraries. An alternative approach may be pre-treatment of FFPE-derived DNA with uracil-DNA glycosylase as reported by Do et al.Preparation of DNA Exome-Seq libraries was successful for ten samples from et al. reported that storage time of FFPE blocks had only a minor influence on sequencing quality et al.We and others et al.In contrast to the challenges we encountered when applying NGS to FFPE-derived DNA, we were surprised to be able to prepare functional RNA-Seq libraries from all 56 FFPE samples we tested, including even samples that had been routinely collected and fixed two decades previously, and then stored at room temperature in the routine pathology archive. Our results add to the previously reported studies, which show successful application of NGS for molecular profiling of a range of samples, differing widely in terms of tissue types and storage times. Using sequentially mapping of strand specific RNA-Seq data we found no major differences between FF and FFPE libraries in the fractions of reads mapping to the different targets. However, we did observe some variation between the different tissue sets, mainly caused by an increased fraction of rRNA reads. We found the Ribo-Zero technology for depletion of rRNA to perform well on degraded total RNA in combination with the ScriptSeq method for preparation of strand-specific RNA-Seq libraries. Furthermore, we observed some variation in rRNA contamination as found by Adiconis the paired FF/FFPE bladder set we observed a high correlation for a previously published vs intronic regions in RNA-Seq data from FFPE compared with FF tissues. This has also been observed in previous studies Paired statistical tests for significantly differentially expressed genes comparing benign and malignant colon tumours, revealed 471 and 1,207 genes to be affected in the FF and FFPE sets, respectively, of which 158 genes were common. In In general, we found a higher fraction of non-specifically mapped reads looking at NGS data from DNA compared with RNA analyses. Furthermore, there was a tendency for there to be a higher fraction of non-specifically mapped reads from FFPE compared with FF samples in DNA, but not RNA derived data. In general, the fraction of non-perfectly mapped reads was higher for RNA compared with DNA analyses; furthermore, this fraction was higher in analyses of FFPE compared with FF samples for both DNA and RNA. Increasing storage time tended to increase the fraction of both non-specifically and non-perfectly mapped reads in FFPE derived DNA, but had no apparent effect in FFPE derived RNA or in FF samples.The data from the relatively few specimens we tested should naturally be viewed as preliminary. Moreover, in individual research projects, a decision must be made as to how much data deviation can be tolerated when comparing NGS profiling of FFPE tissues with the gold standard of FF analysis. With these provisos, our results are promising and suggest that NGS can be used to study DNA and RNA from FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available. This has important implications for opening the diagnostic pathology archives to high-throughput molecular profiling for both research and clinical purposes.Figure S1The workflow in the project with specific focus areas highlighted.(TIF)Click here for additional data file.Figure S2The analytical workflow in CLC Genomics Workbench.(TIF)Click here for additional data file.Figure S3Insert sizes in DNA-Exome-Seq libraries from the paired FF/FFPE colon set.(TIF)Click here for additional data file.Figure S4Post-mapping results from the DNA-Exome-Seq libraries from the paired FF/FFPE colon set.(TIF)Click here for additional data file.Figure S5SNVs detected in DNA-Exome-Seq data from the paired FF/FFPE samples.(TIF)Click here for additional data file.Figure S6DNase vs DNase+ExoI treatment.(TIF)Click here for additional data file.Figure S7Pre mapping results from RNA-Seq of the paired FF/FFPE bladder and prostate sets.(TIF)Click here for additional data file.Figure S8Pre mapping results from RNA-Seq of the storage time FFPE set.(TIF)Click here for additional data file.Figure S9Post mapping results from RNA-Seq of the paired FF/FFPE bladder and prostate sets.(TIF)Click here for additional data file.Figure S10Post mapping results from RNA-Seq of the storage time FFPE set.(TIF)Click here for additional data file.Figure S11Fractions of duplicated reads in the RNA-Seq data sets.(TIF)Click here for additional data file.Figure S12FF vs. FFPE in RNA-Seq data from the paired FF/FFPE colon, bladder and prostate sets.(TIF)Click here for additional data file.Figure S13FF normal vs FF carcinoma in RNA-Seq data from the paired FF/FFPE colon set.(TIF)Click here for additional data file.Figure S14Benign vs malignant (FF or FFPE) in the RNA-Seq data from the paired FF/FFPE colon set.(TIF)Click here for additional data file.Table S1Detailed information on the sample sets.(XLSX)Click here for additional data file.Table S2Summarised information on isolated RNA and DNA from the different sample sets.(XLSX)Click here for additional data file.Table S3Summary of the DNA-Exome-Seq data from ten FF/FFPE pairs from the paired FF/FFPE colon set.(XLSX)Click here for additional data file.Table S4Gene ID and test statistics of the 1,494 genes found commonly affected by FFPE. Gene ID and log2 fold-change (logFC.XXX), average log2-counts per million of all libraries in the set (logCPM.XXX), likelihood ratio statistics (LR.XXX), P values  and false discovery rate (FDR.XXX) for the colon (COL), prostate (PRO) and bladder (BLA) sets.(XLSX)Click here for additional data file.


The aim of this study was to assess the in vitro effects of graphene nanoribbons (O-GNR) noncovalently functionalized with PEG-DSPE ]) on some of the key hematological and vascular components of the circulatory system.The systemic administration of graphene nanoribbons for a variety of Transmission electron microscopy was used to characterize the nanoparticles. ELISA-based assays, bright-field microscopy, transmission electron microscopy and colorimetric assays were used to assess toxicological effects.Our findings taken together indicate that low concentrations of O-GNR-PEG-DSPE (<80 µg/ml) are relatively nontoxic to the hematological components, and could be employed for diagnostic and therapeutic applications especially for diseases of the circulatory system. Graphene nanoribbons are a class of carbon-based nanostructures derived from multiwalled carbon nanotubes that have been shown to have unique properties and high potential for drug-delivery applications in recent studies from our group. However, further development of this nanoparticle for biomedical applications will be possible only after its interactions with components of the circulatory system are suitably characterized. Toward that goal, this study is aimed at identifying potential toxicities of graphene nanoribbons in the circulatory system. Results from this study will give us indications about safe dosages and lay the foundation toward further animal studies. Examination of the interactions between nanoparticles and cells and proteins present in tissues or circulating body fluids  is necessary while characterizing biological effects of nanoparticles ,2. Depenin vivo biomedical applications often involves their intravenous, intramuscular and intraperitoneal injection. This can result in interaction of the particles with different components of the circulatory system including blood proteins, clotting factors, blood cells and components of the immune and allergy response system. Thus, hematological toxicity of nanoparticles is a very critical component of its overall toxicological assessment. Hematological toxicity of nanoparticles has been extensively investigated in recent years. Reports suggest that manifestation of nanoparticle-induced hematological toxicity may vary and include increased or decreased cell counts (red and white blood cells), activation or inhibition of the immune response system, hemolysis, endothelial dysfunction and allergic responses. For example, gold nanoparticles ) (PEG-DSPE)) on various cell lines and stem cells demonstrated that they exhibit a significantly different cellular uptake characteristics and cytotoxicity profile compared with other types of graphene nanoparticles including graphene nanoplatelets ) decreases protein binding to the nanoparticles and prevents RBC hemolysis or activation of allergen, immune activation or blood clotting cascade.The study shows that functionalizing graphene nanoribbons with PEG-DSPE -PEG-DSPEs can potentially interact and cause structural changes in a small fraction of exposed RBCs.O-GNR-PEG-DSPEs show a concentration dependent decrease in cell viability when exposed to endothelial cells with approximately 15% decrease at 100 µg/ml and approximately 40% decrease at 600 µg/ml exposure concentrations.The toxicity observed is probably due to high cellular uptake of O-GNR-PEG-DSPEs.O-GNR-PEG-DSPEs are safe for biomedical applications up to a concentration of 80 µg/ml in the blood


Canine intervertebral disc degeneration can lead to intervertebral disc disease. Mild degenerative changes in the structure of the canine intervertebral disc can be identified in magnetic resonance images, whereas these changes are not visible in computed tomographic images. Therefore, one aim of this study was to detect whether colour-coded computed tomography enhances the visibility of mild degenerative changes in the canine disc structure compared to non-contrast computed tomography. Furthermore, the study aimed to detect if intervertebral disc degeneration could be classified with a higher reliability in colour-coded images than in non-contrast images.Computed tomographic image studies of 144 canine intervertebral discs were coloured using three different lookup tables. Canine intervertebral disc degeneration was evaluated by three observers using a 5-grade classification system and compared to the evaluation of non-contrast CT and MRI images. A moderate to almost perfect intraobserver and a moderate to substantial interobserver agreement were found depending on the used colour code. On comparing non-contrast and colour-coded CT significant differences were found by one observer only. Significant differences in evaluation were found in grading intervertebral disc degeneration in MRI and colour-coded CT.Intervertebral disc degeneration could not be classified with a higher reliability on colour-coded images compared to non-contrast images. Furthermore, colour-coded CT did not enhance the visibility of mild degenerative changes in disc structure compared to non-contrast CT.However, the better intraobserver agreement and the subjective impression of the observers highlighted that the usage of colour encoded CT data sets with a wide range of tonal values of few primary and secondary colours may facilitate evaluation. Canine intervertebral disc (IVD) disease is a frequent result of IVD degeneration , 2. DiagTherefore, one aim of the present study was to test whether the colour-encoding enhances intra- and interobserver agreement in the evaluation of canine IVD degeneration. Furthermore, the study aimed to detect whether colour-coded CT ameliorates the visibility of early degenerative changes in the canine IVD structure compared to non-contrast CT.MRI and CT records of client-owned dogs which were presented at the Small Animal Clinic, University of Veterinary Medicine Hannover, Foundation between April 2011 and March 2012 were reviewed in this retrospective study. Cases were included if MRI and CT records of the same intervertebral disc spaceswere available, data sets were obtained on the same day and the dogs were suspicious of having spinal cord compression. Data sets of 43 client-owned dogs met the inclusion criteriaAcquired non-contrast CT DICOM data sets were exported to ImageJThree observers with different degrees of experience in evaluating computed tomographic images ; observer 2, 5 years of experience (VGZ); observer 3, 24 years of experience (IN)) evaluated all images on standard computer screens using ImageJ. A self-developed scoring system was used to evaluate IVD degeneration . Bowker’s test showed no significant differences in evaluation of IVD degeneration. The similar results of evaluation of the different colour-coded images can be seen in Fig. Comparison of Colour16- and Spectrum-coloured images yielded substantial agreement, while the comparison of Union Jack images with Colour16 images and Spectrum images showed moderate to substantial agreement Table . Limits p-value of the weighted kappa test showed a significant correlation in evaluation, being smaller than 0.0001. All three observers achieved no significantly different results in the evaluation of non-contrast and Colour 16 images analysed in Bowker’s test. Furthermore, the comparison of non-contrast CT images with Spectrum and Union Jack images from observers 1 and 3 was not significantly different, while significant differences in evaluation were found from observer 2 between non-contrast and Spectrum and non-contrast and Union Jack-coloured images. These differences in the evaluation of observer 2 can be seen in Fig. Comparison of non-contrast and Colour16 images and Spectrum images yielded a moderate to substantial agreement, while agreement between non-contrast and Union Jack images showed substantial agreement among all three observers Table . The corThe normal IVD can be easily distinguished from the surrounding tissue in Colour 16 images Fig.  are limeIn Spectrum images the IVD shows several yellow, orange and red tonal values. IVD material can be better differentiated from the surrounding tissue in Spectrum images than in non-contrast CT. Hyperattenuating annular regions show more yellow tonal values and can be identified easily. Setting disc mineralisations apart from IVD material is difficult due to a high brightness of the colours. Especially in the periphery of mineralisations lime green tonal values make the contour to the yellow IVD material poorly defined. Therefore, the mineralisations are optically larger in size in Spectrum images than in non-contrast CT.In Union Jack images, dark blue IVD material is hard to distinguish from the surrounding tissue. Slight changes in attenuation in the periphery of the IVD are easy to see as white, cloudy areas of white and light blue pixels within the dark blue disc material. Mineralisations are seen as bright white regions in the dark blue disc with a clear silhouette. Hyperattenuating parts of mineralised IVD material are red and contrast strongly with the white colours.All observers reported that the changes could be seen more easily in Union Jack-coloured images than in Colour 16 and Spectrum images.p-value <0.0001).Comparison of MRI and colour-coded CT images yielded a slight to fair intraobserver agreement with the following weighted kappa coefficients and associated limits of confidence: Colour 16 and MRI 0.34 , 0.15  and 0.19 , respectively; Spectrum and MRI 0.31 , 0.13 , 0.21 , respectively; Union Jack and MRI 0.31 , 0.20 , 0.22, respectively. Statistical analysis using Bowker’s test showed significant differences in evaluation of intervertebral disc degeneration in all colour-coded images compared to evaluation of Pfirrmann Grade in MRI (The aim of the present study was to test whether colour-coded CT images simplify the evaluation of data sets and enhance the visibility of degenerative changes in the structure of the canine IVD. Therefore, the reliability of evaluation of IVD degeneration in colour-coded data was tested and the evaluation of IVD degeneration in non-contrast and colour-coded CT data was compared.Evaluation of canine IVD degeneration in colour-coded CT images using the proposed grading system is possible due to a predominantly good intra- and interobserver reproducibility .There were no statistically significant differences in the evaluation of IVD degeneration in different colour-coded CT images although the observers preferred the Union Jack coloured images. Furthermore, a higher reliability could be found in the evaluation of Union Jack coloured images compared to the other colour-coded methods. On comparing the lookup-table Colour16 with the other two colour-coded methods, fewer tonal values are used in that method. Thus, a small number of tonal values did not facilitate evaluation. On comparing Spectrum with Union Jack more tonal values of several primary and secondary colours are used in Spectrum while the Union Jack is limited to 191 tonal values of the colours red, blue, white and black. Consequently, a wider range of tonal values may facilitate the evaluation of CT images, while the usage of several primary and secondary colours seems not to provide any additional benefits. In Union Jack coloured images the human eye perceives the different colours with the retinal cones as well as the rods. The grey values of non-contrast CT images are only perceived by the retinal rods , 14. TheThe comparison of the evaluation of IVD degeneration in colour-coded and non-contrast CT images shows that the usage of colours does not enhance the reliability in evaluation compared to the evaluation of non-contrast CT images. This may be a result of the familiarisation of all observers with the non-contrast CT images.A further aim of this study was to enhance the visibility of IVD degeneration in CT images. Significant differences in evaluation of IVD degeneration in non-contrast and colour-coded CT images were found by one observer. Kovacs et al. defined a cut-off point of normal and abnormal IVD degeneration between Pfirrmann Grades 2 and 3 for the classification of IVD degeneration in MRI images , 18. OurIn conclusion, the colour-encoding does not lead to a better intra- and inter-observer agreement than the evaluation of non-contrast CT images. Furthermore, colour-coded CT did not ameliorate the identification of early degenerative changes in IVD in comparison to non-contrast CT. The three different lookup-tables used in the present study to colour CT images had no statistically significant effect on evaluation when compared with one another. However, the better intraobserver agreement and the subjective impression of the observers highlighted that the usage of colour encoded CT data sets with a wide range of tonal values of few primary and secondary colours may facilitate evaluation.


Cost-utility analyses undertaken to inform decision making regarding colorectal cancer (CRC) require a set of health state utility values (HSUVs) so that the time CRC patients spend in different health states can be aggregated into quality-adjusted life-years (QALY). This study reviews CRC-related HSUVs that could be used in economic evaluation and assesses their advantages and disadvantages with respect to valuation methods used and CRC clinical pathways. Fifty-seven potentially relevant studies were identified which collectively report 321 CRC-related HSUVs. HSUVs  vary markedly and this adds to the uncertainty regarding estimates of cost-effectiveness. There are relatively few methodologically robust HSUVs that can be directly used in economic evaluations concerned with CRC. There is considerable scope to develop new HSUVs which improve on those currently available either by expanded collection of generic measures or by making greater use of condition-specific data, for example, using mapping algorithms.The online version of this article (doi:10.1186/s13561-016-0115-5) contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide . CRC wasThere are generic preference-based measures (PBM), such as the EQ-5D, SF-6D, 15D and the HUI3, where generic health states are valued using a tariff based on the preferences of the general public elicited using methods such as the time trade-off (TTO) and the standard gamble (SG).An alternative approach is to identify a number of relevant cancer-specific health states  and to value these health states directly, again using methods such as the TTO and the SG. In this case the valuations are potentially made by cancer patients themselves, health care professionals or the general public.A variation on this second approach is to develop a preference-based algorithm with which a full range of cancer-specific health states can be valued. Two such measures, the EORTC-8D and the QLU-C10D, are based on items from the Quality of Life Questionnaire C30 (QLQ-C30).Finally, a mapping algorithm can be used to transform cancer-specific data such as the EORTC QLQ-C30 and the Functional Assessment of Cancer Therapy-General (FACT-G) into generic PBMs.There are four ways by which the required HSUVs can be empirically generated:This paper reviews CRC-related HSUVs that could be used in economic evaluations and assesses their advantages and disadvantages with reference to the valuation methods used and CRC clinical pathways.www.nice.org.uk) to minimise the chance of missing relevant studies. Economic filters were used when searching for evidence on generalist databases, such as MEDLINE. A simplified search was undertaken without using economic filters, for evidence on economics databases such as HEED. A further search was run on non-economic databases, including MEDLINE, to capture studies that are relevant to mapping. Search strategies are reported in The literature was searched to identify CRC-related HSUVs for use in economic evaluation. MEDLINE, MEDLINE In-Process & Other Non-Indexed Citations, Embase (up to 30 October 2015) and Health Economic Evaluations Database  were searched using the keywords colorectal cancer, health-related quality of life, QALY and economic evaluation. The search was restricted to studies in English. The search was broadened to include the National Institute for Health and Care Excellence (NICE) website . All included studies were read and any disagreements were resolved by discussion between the two reviewers. Of the 285 papers identified as potentially relevant 228 were excluded because they did not report CRC-related HSUVs but presented psychometric validation studies without internal validation properties, the values were previously reported in other included studies, they involved unspecified or not clearly specified CRC-related health state utility values, or a primary mapping function was not reported.A total of 57 studies were included in the reviews  and methodological characteristics  were collected for the 57 studies . FindingOf the 57 studies, eleven were set in the US –14, eighThe 57 studies included in this review reported a total of 368 CRC-related HSUVs. All reported HSUVs are summarised in Additional file Thirty-two studies collected health state information from CRC patients using generic PBMs, and have applied health state tariffs based on the preferences of the general public. These studies generally collect data from patients recruited to trials, usually several hundred patients and at multiple time points. The most widely reported generic PBM is the EQ-5D valued using the UK (TTO-derived) value set with some exceptions , 57, 58 Fourteen studies directly valued CRC health states. Preferences have been elicited either using the TTO method with patients or a surrogate group , 48–50 oAnother approach has recently been developed which offers an alternative to using directly valued health states from the literature. The EORTC-8D is a cancer-specific PBM derived from the EORTC QLQ-C30 . It utilThe absence of data on generic PBMs from most cancer trials has generated considerable interest in mapping algorithms, from cancer-specific measures such as the Functional Assessment of Cancer Therapy-General (FACT-G) and the EORTC QLQ-C30 to measures such as the EQ-5D and the SF-6D , 64. WhiFor the purposes of estimating QALYs it is usually necessary to have information on HSUVs at several points along the clinical pathway. Evaluations of screening or diagnosis require valuations at the time of the intervention and subsequently following treatment.Only one study has reported CRC-screening related HSUVs as presented in Table Sixteen colostomy-related HSUVs were reported from 4 studies , 12, 35.Wong and colleagues  reportedHSUVs for hypothetical health states related to therapy for locally recurrent rectal cancer were higher among rectal cancer patients than health care professionals when measured using SG .Two sets of rectal cancer-related HSUVs were reported at different time points and at different levels of surgery using EQ-5D and TTO values assigned by the UK general public , 67. HowBest et al.  elicitedSkin toxicity is a common AE related to epidermal growth factor receptor (EGFR) agents. Improved HSUVs related to an EGFR were demonstrated using HUI3 among mCRC patients when compared with best supportive care. Health-related quality of life (HRQL) was measured in mCRC patients and valued by the public . Skin toHRQoL measured directly from patients is not always possible in mCRC; so around 30 carers were used a number of times as a proxy because terminally ill mCRC patients would have difficulties in understanding SG or TTO techniques , 48–50. Ness et al.  reportedOf five studies reporting HSUVs of different CRC stages HSUVs were clustered ranging from 0.732–0.87 with an exception of one study 0.25–0.74 , 22, 33.There is no shortage of HSUVs available for those wishing to estimate the cost-effectiveness of diagnostic and treatment strategies with respect to CRC. Those assessing cost-effectiveness face a number of challenges: first, justifying their selection of values when there is no set of values that are clearly superior to all others, and second, negotiating trade-offs between the advantages and disadvantages of the available values.This choice can be simplified where there is an agreed hierarchy regarding the appropriateness of different approaches to generating HSUVs. In order to aid resource allocation and decision making within a tax-funded healthcare system, economic evaluation needs population values for specific health states related to CRC. The preferences of the public are generally deemed appropriate when health services are largely paid for by taxpayers . Some agResearchers usually confront a series of trade-offs and must make judgements about the importance of having all HSUVs used in an economic evaluation come from the same source, or at least obtaining all the HSUVs using the same methods. The number of HSUVs required will in part depend on where in the clinical pathway the intervention being assessed is located. The earlier in the pathway, the larger the number of potentially relevant health states and the less likely it is that all the required HSUVs can come from a single study. Even with clear preferences over the type of measure and the source of values the decision over which values to use can be challenging since the ranking of methods or sources might change in particular circumstances. For example, trial data is not always to be preferred to observational data if the latter provide much larger numbers of observations and are more representative of patients in routine clinical practice. Also a directly collected generic PBM might not always be preferred to the same measure obtained through mapping, for example, if the latter allowed the valuation of a wider range of CRC-related health states. It is uncertain if HSUVs related to mCRC valued by CRC survivors are more relevant than those by health care professionals when making decisions. Also whether or not HSUVs for mCRC valued by early CRC patients are more reliable than those valued by patients with different types of metastatic cancer, has received little attention.HSUVs have been measured by a surrogate group such as oncology nurses, pharmacists or other health care professionals , 48–50. Given the absence of generic PBM data from many trials, existing mapping algorithms could be more fully utilised as an additional means of deriving HSUVs for economic evaluation of CRC, and also exploratory studies to derive HSUVs for colorectal cancer health states from EORTC QLQ-C30 data using the EORTC-8D or QLU-C10D are warranted.Although there are a number of algorithms for mapping from cancer-specific scales to generic PBMs this approach has not been frequently reported with respect to CRC. Cancer-specific scales, such as the QLQ-C30, capture a number of clinical and domain-specific effects that might not be captured when using generic PBMs . MappingImportant questions associated with HSUVs for the economic evaluation of CRC remain unanswered. What is the most accurate way of measuring and valuing HRQoL in CRC? Is it better to collect HRQoL data directly from a small number of CRC patients over a follow-up period ? The stuThis review highlights gaps in the evidence and opportunities for informative research. The most appropriate way to measure and value CRC-related health states should be studied. Developing a set of criteria for selecting the most appropriate HSUVs that fits the analyst’s purpose is encouraged. It is not known whether the mCRC-related HRQoL of CRC survivors is more representative than those derived from a small surrogate group. Also, whether mCRC-related HSUVs valued by early CRC patients are more appropriate than those valued by patients with different types of metastatic cancer for economic evaluation has been under-researched.CRC-related HSUVs vary markedly between studies and across methods. Despite the number of HSUVs published, there is not a set of HSUVs that are methodologically robust with a full range of values for health states of interest appropriate for the use in economic evaluation of CRC. There is considerable scope for new HSUVs to be developed which improve on those currently available and consequently to produce better estimates of QALYs and cost-effectiveness in order to better inform resource allocation and healthcare decision making. In addition, the use of existing mapping algorithms to derive CRC-related HSUVs should be further explored.


N = 1000), we injected sub-threshold oscillations that were either asynchronous or synchronous, resulting in time-lagged or zero-lag correlations in spiking activity, respectively. We then examined the ability of neurons to respond reliably and discriminately to stimuli (large depolarizing events) delivered to random subsets of the population. We measured reliability (Cwithin) by examining the correlations between responses to the same stimulus, and discriminability (Cbetween) as the correlation between responses to different stimuli. Asynchronous networks responded with moderate reliability and high discriminability (green circles – Figure . In sum, our work highlights a novel form of trade-off between asynchrony and synchrony. We suggest that living neuronal networks may take advantage of both forms of coding depending on the context and requisites of information processing.Recordings of neuronal networks in cortex show evidence of asynchronous – or out-of-phase – activity. Cells are known to generate asynchronous outputs despite strongly shared synaptic inputs , yet the