COLD-PCR enhanced melting curve analysis improves diagnostic accuracy for KRAS mutations in colorectal carcinoma

<p>Abstract</p> <p>Background</p> <p><it>KRAS </it>mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (<it>EGFR</it>) in patients with metastatic colorectal cancer. Sensitive methods are required to reliably detect <it>KRAS </it>mutations in tumor samples due to admixture with non-mutated cells. Many laboratories have implemented sensitive tests for <it>KRAS </it>mutations, but the methods often require expensive instrumentation and reagents, parallel reactions, multiple steps, or opening PCR tubes.</p> <p>Methods</p> <p>We developed a highly sensitive, single-reaction, closed-tube strategy to detect all clinically significant mutations in <it>KRAS </it>codons 12 and 13 using the Roche LightCycler^®instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (<it>T_c</it>) of 81°C increased the sensitivity of the assay >10-fold for the majority of <it>KRAS </it>mutations.</p> <p>Results</p> <p>We compared the COLD-PCR enhanced melting curve method to melting curve analysis without COLD-PCR and to traditional Sanger sequencing. In a cohort of 61 formalin-fixed paraffin-embedded colorectal cancer specimens, 29/61 were classified as mutant and 28/61 as wild type across all methods. Importantly, 4/61 (6%) were re-classified from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significant <it>KRAS </it>mutations by COLD-PCR DNA sequencing. Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background.</p> <p>Conclusions</p> <p>We have developed and validated an inexpensive, rapid, and highly sensitive clinical assay for <it>KRAS </it>mutations that is the first report of COLD-PCR combined with probe-based melting curve analysis. This assay significantly improved diagnostic accuracy compared to traditional PCR and direct sequencing.</p

Integration of decision support systems to improve decision support performance

Decision support system (DSS) is a well-established research and development area. Traditional isolated, stand-alone DSS has been recently facing new challenges. In order to improve the performance of DSS to meet the challenges, research has been actively carried out to develop integrated decision support systems (IDSS). This paper reviews the current research efforts with regard to the development of IDSS. The focus of the paper is on the integration aspect for IDSS through multiple perspectives, and the technologies that support this integration. More than 100 papers and software systems are discussed. Current research efforts and the development status of IDSS are explained, compared and classified. In addition, future trends and challenges in integration are outlined. The paper concludes that by addressing integration, better support will be provided to decision makers, with the expectation of both better decisions and improved decision making processes

Morbidity associated with "self-rated health" in epithelial ovarian cancer survivors

<p>Abstract</p> <p>Background</p> <p>Epithelial ovarian cancer survivors (EOCSs) frequently report multiple complaints after their treatment. The objective was to study somatic and mental morbidity in EOCSs associated with their Self- Rated Health (SRH) assessed by a single item.</p> <p>Findings were compared to age-matched controls from the general population.</p> <p>Methods</p> <p>In a cross -sectional follow-up design 189/287 (66%) EOCSs treated at The Norwegian Radiumhospital 1979–2003 responded to a mailed questionnaire on demographic data, and somatic and mental morbidity. SRH last week was rated on item #29 of the European Organization and Treatment of Cancer Quality of Life Questionnaire in 84/189 (97%) of responding EOCSs. For comparisons "good" and "poor" SRH groups were defined by the median score on the SRH item.</p> <p>Results</p> <p>EOCSs with "poor SRH" had higher level of somatic symptoms, anxiety, depression and fatigue than those with "good SRH" (p < .001). In multivariate analyses somatic symptoms, age and fatigue, were significantly associated with the SRH score in EOCSs, but not the cancer-related variables (FIGO stage, recurrence in < 6 months or chemotherapy ever). The model explained 70% of the variance in SRH in linear and 77% in logistic regression analyses. The distribution of the SRH scores in EOCSs did not differ significantly from that of normative controls; however a higher proportion of controls recorded a high SRH score.</p> <p>Conclusion</p> <p>SRH is strongly related to common somatic complaints, impairment and fatigue but not to cancer-related variables. A single question concerning SRH last week might be a quick screening method for collecting important information on symptoms in EOCSs, in addition to cancer – related questions.</p

Frequency-dependent selection in vaccine-associated pneumococcal population dynamics

Many bacterial species are composed of multiple lineages distinguished by extensive variation in gene content. These often cocirculate in the same habitat, but the evolutionary and ecological processes that shape these complex populations are poorly understood. Addressing these questions is particularly important for Streptococcus pneumoniae, a nasopharyngeal commensal and respiratory pathogen, because the changes in population structure associated with the recent introduction of partial-coverage vaccines have substantially reduced pneumococcal disease. Here we show that pneumococcal lineages from multiple populations each have a distinct combination of intermediate-frequency genes. Functional analysis suggested that these loci may be subject to negative frequency-dependent selection (NFDS) through interactions with other bacteria, hosts or mobile elements. Correspondingly, these genes had similar frequencies in four populations with dissimilar lineage compositions. These frequencies were maintained following substantial alterations in lineage prevalences once vaccination programmes began. Fitting a multilocus NFDS model of post-vaccine population dynamics to three genomic datasets using Approximate Bayesian Computation generated reproducible estimates of the influence of NFDS on pneumococcal evolution, the strength of which varied between loci. Simulations replicated the stable frequency of lineages unperturbed by vaccination, patterns of serotype switching and clonal replacement. This framework highlights how bacterial ecology affects the impact of clinical interventions.Accessory loci are shown to have similar frequencies in diverse Streptococcus pneumoniae populations, suggesting negative frequency-dependent selection drives post-vaccination population restructuring

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Effects of alirocumab on types of myocardial infarction: insights from the ODYSSEY OUTCOMES trial

Aims  The third Universal Definition of Myocardial Infarction (MI) Task Force classified MIs into five types: Type 1, spontaneous; Type 2, related to oxygen supply/demand imbalance; Type 3, fatal without ascertainment of cardiac biomarkers; Type 4, related to percutaneous coronary intervention; and Type 5, related to coronary artery bypass surgery. Low-density lipoprotein cholesterol (LDL-C) reduction with statins and proprotein convertase subtilisin–kexin Type 9 (PCSK9) inhibitors reduces risk of MI, but less is known about effects on types of MI. ODYSSEY OUTCOMES compared the PCSK9 inhibitor alirocumab with placebo in 18 924 patients with recent acute coronary syndrome (ACS) and elevated LDL-C (≥1.8 mmol/L) despite intensive statin therapy. In a pre-specified analysis, we assessed the effects of alirocumab on types of MI. Methods and results  Median follow-up was 2.8 years. Myocardial infarction types were prospectively adjudicated and classified. Of 1860 total MIs, 1223 (65.8%) were adjudicated as Type 1, 386 (20.8%) as Type 2, and 244 (13.1%) as Type 4. Few events were Type 3 (n = 2) or Type 5 (n = 5). Alirocumab reduced first MIs [hazard ratio (HR) 0.85, 95% confidence interval (CI) 0.77–0.95; P = 0.003], with reductions in both Type 1 (HR 0.87, 95% CI 0.77–0.99; P = 0.032) and Type 2 (0.77, 0.61–0.97; P = 0.025), but not Type 4 MI. Conclusion  After ACS, alirocumab added to intensive statin therapy favourably impacted on Type 1 and 2 MIs. The data indicate for the first time that a lipid-lowering therapy can attenuate the risk of Type 2 MI. Low-density lipoprotein cholesterol reduction below levels achievable with statins is an effective preventive strategy for both MI types.For complete list of authors see http://dx.doi.org/10.1093/eurheartj/ehz299</p