Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

Consensus statement of the European guidelines on clinical management of HIV-1 tropism testing

Introduction: Testing for HIV tropism is recommended before prescribing a chemokine receptor blocker. To date, in most European countries HIV tropism is determined using a phenotypic test. Recently, new data have emerged supporting the use of a genotypic HIV V3-loop sequence analysis as the basis for tropism determination. The European guidelines group on clinical management of HIV-1 tropism testing was established to make recommendations to clinicians and virologists. Methods: We searched online databases for articles from Jan 2006 until March 2010 with the terms: tropism or CCR5-antagonist or CCR5 antagonist or maraviroc or vicriviroc. Additional articles and/or conference abstracts were identified by hand searching. This strategy identified 712 potential articles and 1240 abstracts. All were reviewed and finally 57 papers and 42 abstracts were included and used by the panel to reach a consensus statement. Results: The panel recommends HIV-tropism testing for the following indications: i) drug-naïve patients in whom toxicity or limited therapeutic options are foreseen; ii) patients experiencing therapy failure whenever a treatment change is considered. Both the phenotypic Enhanced Trofile assay (ESTA) and genotypic population sequencing of the V3-loop are recommended for use in clinical practice. Although the panel does not recommend one methodology over another it is anticipated that genotypic testing will be used more frequently because of its greater accessibility, lower cost and shorter turnaround time. The panel also provides guidance on technical aspects and interpretation issues. If using genotypic methods, triplicate PCR amplification and sequencing testing is advised using the G2P interpretation tool (clonal model) with an FPR of 10%. If the viral load is below the level of reliable amplification, proviral DNA can be used, and the panel recommends performing triplicate testing and use of an FPR of 10%. If genotypic DNA testing is not performed in triplicate the FPR should be increased to 20%. Conclusions: The European guidelines on clinical management of HIV-1 tropism testing provide an overview of current literature, evidence-based recommendations for the clinical use of tropism testing and expert guidance on unresolved issues and current developments. Current data support both the use of genotypic population sequencing and ESTA for co-receptor tropism determination. For practical reasons genotypic population sequencing is the preferred method in Europe.

In vitro activity of fosfomycin against extended-spectrum-beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae: comparison of susceptibility testing procedures

The agar dilution, broth microdilution, and disk diffusion methods were compared to determine the in vitro susceptibility of 428 extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae to fosfomycin. Fosfomycin showed very high activity against all ESBL-producing strains. Excellent agreement between the three susceptibility methods was found for E. coli, whereas marked discrepancies were observed for K. pneumoniae.

Postmortem circulation: a new model for testing endovascular devices and training clinicians in their use.

The development of new medical devices, such as aortic valves, requires numerous preliminary studies on animals and training of personnel on cadavers before the devices can be used in patients. Postmortem circulation, a technique used for postmortem angiography, allows the vascular system to be reperfused in a way similar to that in living persons. This technique is used for postmortem investigations to visualize the human vascular system and to make vascular diagnoses. Specific material for reperfusing a human body was developed recently. Our aim was to investigate whether postmortem circulation that imitates in vivo conditions allows for the testing of medical materials on cadavers. We did this by delivering an aortic valve using minimally invasive methods. Postmortem circulation was established in eight corpses to recreate an environment as close as possible to in vivo conditions. Mobile fluoroscopy and a percutaneous catheterization technique were used to deliver the material to the correct place. Once the valve was implanted, the heart and primary vessels were extracted to confirm its position. Postmortem circulation proved to be essential in several of the cadavers because it helped the clinicians to deliver the material and improve their implantation techniques. Due to the intravascular circulation, sites with substantial arteriosclerotic stenosis could be bypassed, which would have been impossible without perfusion. Although originally developed for postmortem investigations, this reperfusion technique could be useful for testing new medical devices intended for living patients.

Testing of Diagnostic Methods for Detection of Influenza Virusfor Optimal Performance in the Context of an Influenza Surveillance Network

Influenza surveillance networks must detect early the viruses that will cause the forthcoming annual epidemics and isolate the strains for further characterization. We obtained the highest sensitivity (95.4%) with a diagnostic tool that combined a shell-vial assay and reverse transcription-PCR on cell culture supernatants at 48 h, and indeed, recovered the strain

Compositional hypotheses of subcompositional stability and specific perturbation change and their testing

In standard multivariate statistical analysis common hypotheses of interest concern changes in mean vectors and subvectors. In compositional data analysis it is now well established that compositional change is most readily described in terms of the simplicial operation of perturbation and that subcompositions replace the marginal concept of subvectors. To motivate the statistical developments of this paper we present two challenging compositional problems from food production processes.Against this background the relevance of perturbations and subcompositions can beclearly seen. Moreover we can identify a number of hypotheses of interest involvingthe specification of particular perturbations or differences between perturbations and also hypotheses of subcompositional stability. We identify the two problems as being the counterpart of the analysis of paired comparison or split plot experiments and of separate sample comparative experiments in the jargon of standard multivariate analysis. We then develop appropriate estimation and testing procedures for a complete lattice of relevant compositional hypotheses

Methods for testing association between uncertain genotypes and quantitative traits.

Interpretability and power of genome-wide association studies can be increased by imputing unobserved genotypes, using a reference panel of individuals genotyped at higher marker density. For many markers, genotypes cannot be imputed with complete certainty, and the uncertainty needs to be taken into account when testing for association with a given phenotype. In this paper, we compare currently available methods for testing association between uncertain genotypes and quantitative traits. We show that some previously described methods offer poor control of the false-positive rate (FPR), and that satisfactory performance of these methods is obtained only by using ad hoc filtering rules or by using a harsh transformation of the trait under study. We propose new methods that are based on exact maximum likelihood estimation and use a mixture model to accommodate nonnormal trait distributions when necessary. The new methods adequately control the FPR and also have equal or better power compared to all previously described methods. We provide a fast software implementation of all the methods studied here; our new method requires computation time of less than one computer-day for a typical genome-wide scan, with 2.5 M single nucleotide polymorphisms and 5000 individuals.

Testing the validity of a set of diagnostic criteria for sensory neuronopathies: a francophone collaborative study.

There are no validated criteria for the diagnosis of sensory neuronopathy (SNN) yet. In a preliminary monocenter study a set of criteria relying on clinical and electrophysiological data showed good sensitivity and specificity for a diagnosis of probable SNN. The aim of this study was to test these criteria on a French multicenter study. 210 patients with sensory neuropathies from 15 francophone reference centers for neuromuscular diseases were included in the study with an expert diagnosis of non-SNN, SNN or suspected SNN according to the investigations performed in these centers. Diagnosis was obtained independently from the set of criteria to be tested. The expert diagnosis was taken as the reference against which the proposed SNN criteria were tested. The set relied on clinical and electrophysiological data easily obtainable with routine investigations. 9/61 (16.4 %) of non-SNN patients, 23/36 (63.9 %) of suspected SNN, and 102/113 (90.3 %) of SNN patients according to the expert diagnosis were classified as SNN by the criteria. The SNN criteria tested against the expert diagnosis in the SNN and non-SNN groups had 90.3 % (102/113) sensitivity, 85.2 % (52/61) specificity, 91.9 % (102/111) positive predictive value, and 82.5 % (52/63) negative predictive value. Discordance between the expert diagnosis and the SNN criteria occurred in 20 cases. After analysis of these cases, 11 could be reallocated to a correct diagnosis in accordance with the SNN criteria. The proposed criteria may be useful for the diagnosis of probable SNN in patients with sensory neuropathy. They can be reached with simple clinical and paraclinical investigations.

One person with two DNA profiles: a(nother) case of mosaicism or chimerism.

Nuclear DNA markers, such as short tandem repeats (STR), are widely used for crime investigation and paternity testing. STR were used to determine whether a piece of tissue regurgitated by a dog was part of the penis of a dead, emasculated, man. Unexpectedly, when analyzing the recovered material and a blood sample from the deceased, five out of the 18 loci differed. According to the results, one could have concluded that these samples originated from two different persons. However, taking into account contextual information and data from complementary genetic analyses, the most likely hypothesis was that the deceased was a genetic mosaic or a chimera. Within a forensic genetic context, such genetic peculiarities may prevent associating the perpetrator of an offense with a stain left at a crime scene or lead to false paternity exclusions. Fast recognition of mosaics or chimeras, adapted sampling scheme, as well as careful interpretation of the data should allow avoiding such pitfalls.

Mosaico mediterráneo. El Arte como herramienta de diálogo intercultural

El presente trabajo de investigación doctoral reflexiona sobre el aporte de las prácticas artísticas contemporáneas en los procesos de diálogo entre culturas en el Mediterráneo. En las dinámicas de percepción, acercamiento y (re)conocimiento del otro y de las culturas otras, todos los medios informativos y de comunicación pueden ser, por un lado, válidos y efectivos como, por otro, pueden desviar de la verdad y difundir fácilmente numerosos prejuicios. Frente a la “síntesis” operada por los medios de comunicación tradicionales, donde “el Otro” es a menudo objeto de representaciones estereotipadas, las prácticas artísticas, en su sentido más amplio, contraponen un “análisis” que puede permitir, en diferentes casos, la eliminación de las barreras y un mayor acercamiento a la cultura otra. En la presente investigación se quiere trazar un itinerario teórico que parte de las hipótesis orientalistas, pasa por las filosofías del encuentro con el otro; los estudios sobre la participación; las actuales reflexiones sobre la estética relacional; las definiciones históricas y socio-políticas del Mediterráneo; hasta llegar a reconocer en el arte, en la creación artística, el medio de comunicación más eficaz y penetrante a la hora de refinar el conocimiento y (re)descubrir los diferentes aspectos de las culturas de los países y de los pueblos del Mediterráneo. En el espacio plural del Mediterráneo las prácticas artísticas y creativas actuales actúan como un campo de experimentación y un territorio de debate valioso e importante para el desarrollo del discurso intercultural.

Application of Molecular Diagnostic Techniques for Viral Testing.

Nucleic acid amplification techniques are commonly used currently to diagnose viral diseases and manage patients with this kind of illnesses. These techniques have had a rapid but unconventional route of development during the last 30 years, with the discovery and introduction of several assays in clinical diagnosis. The increase in the number of commercially available methods has facilitated the use of this technology in the majority of laboratories worldwide. This technology has reduced the use of some other techniques such as viral culture based methods and serological assays in the clinical virology laboratory. Moreover, nucleic acid amplification techniques are now the methods of reference and also the most useful assays for the diagnosis in several diseases. The introduction of these techniques and their automation provides new opportunities for the clinical laboratory to affect patient care. The main objectives in performing nucleic acid tests in this field are to provide timely results useful for high-quality patient care at a reasonable cost, because rapid results are associated with improvements in patients care. The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. This review is an up-to-date of the main nucleic acid techniques and their clinical applications, and special challenges and opportunities that these techniques currently provide for the clinical virology laboratory.

Comparative evaluation under routine conditions of the nitrate reduction assay, the proportion assay and the MGIT 960 assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis

The performance of the nitrate reductase assay (NRA) was compared with the proportion method (PM) on Lowenstein-Jensen medium and the BACTEC MGIT960 assay under routine conditions using 160 clinical isolates of Mycobacterium tuberculosis with a high proportion of resistant strains. The mean time to obtain results was 8.8 days and the overall agreements between NRA and PM and NRA and M960 were 95% and 94%, respectively. NRA was easy to perform and represents a useful tool for the rapid screening of drug-resistant M. tuberculosis strains in low-resource countries.

Effects of social support on the clinical course of Crohn's disease.

BACKGROUND: Social support has been found to be protective from adverse health effects of psychological stress. We hypothesized that higher social support would predict a more favorable course of Crohn's disease (CD) directly (main effect hypothesis) and via moderating other prognostic factors (buffer hypothesis). METHODS: Within a multicenter cohort study we observed 597 adults with CD for 18 months. We assessed social support using the ENRICHD Social Support Inventory. Flares, nonresponse to therapy, complications, and extraintestinal manifestations were recorded as a combined endpoint indicating disease deterioration. We controlled for several demographic, psychosocial, and clinical variables of potential prognostic importance. We used multivariate binary logistic regression to estimate the overall effect of social support on the odds of disease deterioration and to explore main and moderator effects of social support by probing interactions with other predictors. RESULTS: The odds of disease deterioration decreased by 1.5 times (95% confidence interval [CI]: 1.2-1.9) for an increase of one standard deviation (SD) of social support. In case of low body mass index (BMI) (i.e., 1 SD below the mean or <19 kg/m(2)), the odds decreased by 1.8 times for an increase of 1 SD of social support. In case of low social support, the odds increased by 2.1 times for a decrease of 1 SD of BMI. Low BMI was not predictive under high social support. CONCLUSIONS: The findings suggest that elevated social support may favorably affect the clinical course of CD, particularly in patients with low BMI. (Inflamm Bowel Dis 2010;).

Primary resistance of HIV to antiretrovirals among individuals recently diagnosed at voluntary counselling and testing centres in the metropolitan region of Recife, Pernambuco

Determining the prevalence and type of antiretroviral (ARV) resistance among ARV-naïve individuals is important to assess the potential responses of these individuals to first-line regimens. The prevalence of primary resistance and the occurrence of recent infections among individuals with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) were identified among recently diagnosed patients at five sexually transmitted disease/AIDS testing and counselling centres in the metropolitan region of Recife (RMR), Pernambuco, Brazil, between 2007-2009. One-hundred and eight samples were analysed using the Calypte® BED assay. Males predominated (56%), as did patients aged 31-50 years. Twenty-three percent presented evidence of a recent HIV infection. The median CD4+ T lymphocyte count was 408 cells/mm³ and the median viral load was 3.683 copies/mL. The prevalence of primary resistance was 4.6% (confidence interval 95% = 1-8.2%) based on criteria that excluded common polymorphisms in accordance with the surveillance drug resistance mutation criteria. The prevalence of resistance to non-nucleoside reverse transcriptase, nucleoside/nucleotide reverse transcriptase and protease inhibitors were 3.8%, 1.5% and 0.8%, respectively. Fifty-seven percent of strains were from clade B, 37.7% were clade F and 3.1% were clade C; there were no statistically significant differences with respect to resistance between clades. Recent infection tended to be more common in men (p = 0.06) and in municipalities in the south of the RMR (Jaboatão dos Guararapes and Cabo de Santo Agostinho) (p = 0.046). The high prevalence of recent infection and the high prevalence of non-B strains in this poor Brazilian region merit further attention.