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.

Digital inmigrants and digital natives expectation collection in the Andalusian Public Health System Virtual Library: communication with our users

The Andalusian Public Health System Virtual Library (Biblioteca Virtual del Sistema Sanitario Público de Andalucía, BV-SSPA) provides access to health information resources and services to healthcare professionals through its Website. This virtual environment demands higher users’ knowledge in order to satisfy of the need of information of our users, as digital natives as digital immigrants, improving at the same time the communication with all of them. 1. To collect clients' views and expectations according to their nature of digital natives and immigrants. 2. To know our online reputation. A Collecting User Expectation Questionnaire will be built, taking into account the segmentation of the BV-SSPA users’ professional groups of the Andalusian Public Health System. A pilot test will be run to check the survey dimensions and items about practices, attitudes and knowledge of our users. Two Quality Function Deployment (QFD) matrices will enable the BV-SSPA services to be targeted to our digital natives or digital immigrants, according to their nature, finding the best way to satisfy their information needs. We provide feedback on BV-SSPA: users can have the opportunity to post feedback about the site via the 'Contact us' section and comment about their experience. And Web 2.0 is a shop window, providing the opportunity to show the comments; and through time, our online reputation will be built, but the BV-SSPA must manage its own personal branding. Web 2.0 tools are a driver of improvement, because they provide a key source of insight into people's attitudes. Besides, the BV-SSPA digital identity will be analyzed through indicators like major search engine referrals breakdown, top referring sites (non search engines), or top search engine referral phrases, among others. Definition of digital native and digital immigrant profiles of the BV-SSPA, and their difference, will be explained by their expectations. The design of the two QFD matrices will illustrate in just one graph the requirements of both groups for tackling digital abilities and inequalities. The BV-SSPA could deliver information and services through alternative channels. On the other hand, we are developing a strategy to identify, to measure and to manage a digital identity through communication with the user and to find out our online reputation. With the use of different tools from quantitative and qualitative methodology, and the opportunities offered by Web 2.0 tools, the BV-SSPA will know the expectations of their users as a first step to satisfy their necessities. Personalization is pivotal to the success of the Site, delivering tailored content to individuals based on their recorded preferences. The valuable user research can be used during new product development and redesign. Besides positive interaction let us build trust, show authenticity, and foster loyalty: we improve with effort, communication and show.

Characteristics and effectiveness of diabetes self-management educational programs targeted to racial/ethnic minority groups: a systematic review, meta-analysis and meta-regression.

BACKGROUND It is not clear to what extent educational programs aimed at promoting diabetes self-management in ethnic minority groups are effective. The aim of this work was to systematically review the effectiveness of educational programs to promote the self-management of racial/ethnic minority groups with type 2 diabetes, and to identify programs' characteristics associated with greater success. METHODS We undertook a systematic literature review. Specific searches were designed and implemented for Medline, EMBASE, CINAHL, ISI Web of Knowledge, Scirus, Current Contents and nine additional sources (from inception to October 2012). We included experimental and quasi-experimental studies assessing the impact of educational programs targeted to racial/ethnic minority groups with type 2 diabetes. We only included interventions conducted in countries members of the OECD. Two reviewers independently screened citations. Structured forms were used to extract information on intervention characteristics, effectiveness, and cost-effectiveness. When possible, we conducted random-effects meta-analyses using standardized mean differences to obtain aggregate estimates of effect size with 95% confidence intervals. Two reviewers independently extracted all the information and critically appraised the studies. RESULTS We identified thirty-seven studies reporting on thirty-nine educational programs. Most of them were conducted in the US, with African American or Latino participants. Most programs obtained some benefits over standard care in improving diabetes knowledge, self-management behaviors and clinical outcomes. A meta-analysis of 20 randomized controlled trials (3,094 patients) indicated that the programs produced a reduction in glycated hemoglobin of -0.31% (95% CI -0.48% to -0.14%). Diabetes knowledge and self-management measures were too heterogeneous to pool. Meta-regressions showed larger reduction in glycated hemoglobin in individual and face to face delivered interventions, as well as in those involving peer educators, including cognitive reframing techniques, and a lower number of teaching methods. The long-term effects remain unknown and cost-effectiveness was rarely estimated. CONCLUSIONS Diabetes self-management educational programs targeted to racial/ethnic minority groups can produce a positive effect on diabetes knowledge and on self-management behavior, ultimately improving glycemic control. Future programs should take into account the key characteristics identified in this review.