Development of a quality control material for the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an in vivo marker of oxidative stress, and comparison of results from different laboratories

The measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine is an increasingly popular marker of in vivo oxidative damage to DNA. A random-sequence 21-mer oligonucleotide 5'-TCA GXC GTA CGT GAT CTC AGT-3' in which X was 8-oxo-guanine (8-oxo-G) was purified and accurate determination of the oxidised base was confirmed by a 32P-end labelling strategy. The lyophilised material was analysed for its absolute content of 8-oxo-dG by several major laboratories in Europe and one in Japan. Most laboratories using HPLC-ECD underestimated, while GC-MS-SIM overestimated the level of the lesion. HPLC-ECD measured the target value with greatest accuracy. The results also suggest that none of the procedures can accurately quantitate levels of 1 in 10(6) 8-oxo-(d)G in DNA.

An investigation into the effects of the feedback of knowledge of results on operator performance

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Principal Component Analysis of Results Obtained from Finite-Difference Time-Domain Algorithms

Finite-Differences Time-Domain (FDTD) algorithms are well established tools of computational electromagnetism. Because of their practical implementation as computer codes, they are affected by many numerical artefact and noise. In order to obtain better results we propose using Principal Component Analysis (PCA) based on multivariate statistical techniques. The PCA has been successfully used for the analysis of noise and spatial temporal structure in a sequence of images. It allows a straightforward discrimination between the numerical noise and the actual electromagnetic variables, and the quantitative estimation of their respective contributions. Besides, The GDTD results can be filtered to clean the effect of the noise. In this contribution we will show how the method can be applied to several FDTD simulations: the propagation of a pulse in vacuum, the analysis of two-dimensional photonic crystals. In this last case, PCA has revealed hidden electromagnetic structures related to actual modes of the photonic crystal.

High-speed label-free functional photoacoustic microscopy of mouse brain in action.

We present fast functional photoacoustic microscopy (PAM) for three-dimensional high-resolution, high-speed imaging of the mouse brain, complementary to other imaging modalities. We implemented a single-wavelength pulse-width-based method with a one-dimensional imaging rate of 100 kHz to image blood oxygenation with capillary-level resolution. We applied PAM to image the vascular morphology, blood oxygenation, blood flow and oxygen metabolism in both resting and stimulated states in the mouse brain.

Focused molecular analysis of small cell lung cancer: feasibility in routine clinical practice.

Background: There is an urgent need to identify molecular signatures in small cell lung cancer (SCLC) that may select patients who are likely to respond to molecularly targeted therapies. In this study, we investigate the feasibility of undertaking focused molecular analyses on routine diagnostic biopsies in patients with SCLC.

Methods: A series of histopathologically confirmed formalin-fixed, paraffin-embedded SCLC specimens were analysed for epidermal growth factor receptors (EGFR), KRAS, NRAS and BRAF mutations, ALK gene rearrangements and MET amplification. EGFR and KRAS mutation testing was evaluated using real time polymerase chain reaction (RT-PCR cobas®), BRAF and NRAS mutations using multiplex PCR and capillary electrophoresis-single strand conformation analysis, and ALK and MET aberrations with fluorescent in situ hybridization. All genetic aberrations detected were validated independently.

Results: A total of 105 patients diagnosed with SCLC between July 1990 and September 2006 were included. 60 (57 %) patients had suitable tumour tissue for molecular testing. 25 patients were successfully evaluated for all six pre-defined molecular aberrations. Eleven patients failed all molecular analysis. No mutations in EGFR, KRAS and NRAS were detected, and no ALK gene rearrangements or MET gene amplifications were identified. A V600E substitution in BRAF was detected in a Caucasian male smoker diagnosed with SCLC with squamoid and glandular features.

Conclusion: The paucity of patients with sufficient tumour tissue, quality of DNA extracted and low frequency of aberrations detected indicate that alternative molecular characterisation approaches are necessary, such as the use of circulating plasma DNA in patients with SCLC.

Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.

AIMS: Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. METHODS: 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. RESULTS: Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p

A comparison of three methods for detecting KRAS mutations in formalin-fixed colorectal cancer specimens.

BACKGROUND: KRAS mutation testing is required to select patients with metastatic colorectal cancer (CRC) to receive anti-epidermal growth factor receptor antibodies, but the optimal KRAS mutation test method is uncertain. METHODS: We conducted a two-site comparison of two commercial KRAS mutation kits - the cobas KRAS Mutation Test and the Qiagen therascreen KRAS Kit - and Sanger sequencing. A panel of 120 CRC specimens was tested with all three methods. The agreement between the cobas test and each of the other methods was assessed. Specimens with discordant results were subjected to quantitative massively parallel pyrosequencing (MPP). DNA blends were tested to determine detection rates at 5% mutant alleles. RESULTS: Reproducibility of the cobas test between sites was 98%. Six mutations were detected by cobas that were not detected by Sanger, and five were confirmed by MPP. The cobas test detected eight mutations which were not detected by the therascreen test, and seven were confirmed by MPP. Detection rates with 5% mutant DNA blends were 100% for the cobas and therascreen tests and 19% for Sanger. CONCLUSION: The cobas test was reproducible between sites, and detected several mutations that were not detected by the therascreen test or Sanger. Sanger sequencing had poor sensitivity for low levels of mutation.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Gene scanning of VDJH-amplified segments is a clinically relevant technique to detect contaminating tumor cells in the apheresis products of multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation.

Contaminating tumour cells in apheresis products have proved to influence the outcome of patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (APBSCT). The gene scanning of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) is a reproducible and easy to perform technique that can be optimised for clinical laboratories. We used it to analyse the aphereses of 27 MM patients undergoing APBSCT with clonally detectable VDJH segments, and 14 of them yielded monoclonal peaks in at least one apheresis product. The presence of positive results was not related to any pre-transplant characteristics, except the age at diagnosis (lower in patients with negative products, P = 0.04). Moreover, a better pre-transplant response trended to associate with a negative result (P = 0.069). Patients with clonally free products were more likely to obtain a better response to transplant (complete remission, 54% vs 28%; >90% reduction in the M-component, 93% vs 43% P = 0.028). In addition, patients transplanted with polyclonal products had longer progression-free survival, (39 vs 19 months, P = 0.037) and overall survival (81% vs 28% at 5 years, P = 0.045) than those transplanted with monoclonal apheresis. In summary, the gene scanning of apheresis products is a useful and clinically relevant technique in MM transplanted patients.

Measuring attachment to life in old age: the Portuguese version of the Positive Valuation of Life Scale (Positive VOL)

Purpose This study aims to present the psychometric properties of the Portuguese version of the Positive Valuation of Life Scale (Lawton et al. in J Aging Ment Healt 13:3–31, 2001). Method Sample included 207 community-dwelling elders (129 women; MAge = 77.2 years, SD = 7.5). The data collection included the translated and adapted Portuguese version of Positive Valuation of Life Scale, Life Satisfac- tion Index Z, Meaning in Life Questionnaire and Geriatric Depression Scale. Results From exploratory factor analysis, two factors emerged, existential beliefs and perceived control, ex- plaining 49 % of the total variance. Both factors were positively related with meaning in life and life satisfaction and negatively related with depression (p\0.05). The values obtained for internal consistency for the total scale and for each subscale were good (a [ 0.75). Conclusion The Portuguese version of Positive VOL Scale represents a reliable and valid measure to capture the subjective experience of attachment to one’s life. The two-factor structure is an update to Lawton’s previous work and in line with findings obtained in the USA (Dennis et al. in What is valuation of life for frail community-dwelling older adults: factor structure and criterion validity of the VOL, Thomas Jefferson University, Center for Applied Research on Aging and Health Research, 2005) and Japan (Nakagawa et al. in Shinrigaku Kenkyu 84:37–46, 2013). Future research is required to investigate VOL predictors and the potential changes toward the end of the life span.

The problem posed by exam choice on the comparability of results in the Finnish matriculation examination

The article focuses on the upper secondary matriculation examination in Finland as a school leaving and university entrance examination. The presented research addresses the question of whether increased choice of the subject-specific examinations has the potential to undermine the comparability of examination results and to direct students’ choices not only in the examination but already beforehand at school. The authors refer to Finland’s tradition of more than 160 years of a national examination connecting the academic track of upper secondary schools with universities. The authors explain the Finnish system by describing the adoption of a course-based (vs. class- or year-based) curriculum for the three-year upper secondary education and the subsequent reforms in the matriculation examination. This increases students’ choices considerably with regard to the subject-specific exams included in the examination (a minimum of four). As a result, high-achieving students compete against each other in the more demanding subjects while the less able share the same normal distribution of grades in the less demanding subjects. As a consequence, students tend to strategic exam-planning, which in turn affects their study choices at school, often to the detriment of the more demanding subjects and, subsequently, of students’ career opportunities, endangering the traditional national objective of an all-round pre-academic upper secondary education. This contribution provides an overview of Finnish upper secondary education and of the matriculation examination (cf. Klein, 2013) while studying three separate but related issues by using data from several years of Finnish matriculation results: the relation of the matriculation examination and the curriculum; the problems of comparability vis-à-vis university entry due to the increased choice within the examination; the relations between students’ examination choices and their course selection and achievement during upper secondary school. (DIPF/Orig.)

Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR

With an increased emphasis on genotyping of single nucleotide polymorphisms (SNPs) in disease association studies, the genotyping platform of choice is constantly evolving. In addition, the development of more specific SNP assays and appropriate genotype validation applications is becoming increasingly critical to elucidate ambiguous genotypes. In this study, we have used SNP specific Locked Nucleic Acid (LNA) hybridization probes on a real-time PCR platform to genotype an association cohort and propose three criteria to address ambiguous genotypes. Based on the kinetic properties of PCR amplification, the three criteria address PCR amplification efficiency, the net fluorescent difference between maximal and minimal fluorescent signals and the beginning of the exponential growth phase of the reaction. Initially observed SNP allelic discrimination curves were confirmed by DNA sequencing (n = 50) and application of our three genotype criteria corroborated both sequencing and observed real-time PCR results. In addition, the tested Caucasian association cohort was in Hardy-Weinberg equilibrium and observed allele frequencies were very similar to two independently tested Caucasian association cohorts for the same tested SNP. We present here a novel approach to effectively determine ambiguous genotypes generated from a real-time PCR platform. Application of our three novel criteria provides an easy to use semi-automated genotype confirmation protocol.

Response to chemotherapy is predictive in relation to longer overall survival in an individual patient combined-analysis with pleural mesothelioma

Background: There is currently no early predictive marker of survival for patients receiving chemotherapy for malignant pleural mesothelioma (MPM). Tumour response may be predictive for overall survival (OS), though this has not been explored. We have thus undertaken a combined-analysis of OS, from a 42 day landmark, of 526 patients receiving systemic therapy for MPM. We also validate published progression-free survival rates (PFSRs) and a progression-free survival (PFS) prognostic-index model. Methods: Analyses included nine MPM clinical trials incorporating six European Organisation for Research and Treatment of Cancer (EORTC) studies. Analysis of OS from landmark (from day 42 post-treatment) was considered regarding tumour response. PFSR analysis data included six non-EORTC MPM clinical trials. Prognostic index validation was performed on one non-EORTC data-set, with available survival data. Results: Median OS, from landmark, of patients with partial response (PR) was 12·8 months, stable disease (SD), 9·4 months and progressive disease (PD), 3·4 months. Both PR and SD were associated with longer OS from landmark compared with disease progression (both p < 0·0001). PFSRs for platinum-based combination therapies were consistent with published significant clinical activity ranges. Effective separation between PFS and OS curves provided a validation of the EORTC prognostic model, based on histology, stage and performance status. Conclusion: Response to chemotherapy is associated with significantly longer OS from landmark in patients with MPM. © 2012 Elsevier Ltd. All rights reserved.