Analytical performance of a PCR assay for the detection of KRAS mutations (codons 12/13 and 61) in formalin-fixed paraffin-embedded tissue samples of colorectal carcinoma

KRAS mutation testing is mandatory before prescribing anti-epidermal growth factor monoclonal antibodies in the treatment of advanced colorectal cancer. We describe the performance of a TaqMelt polymerase chain reaction (PCR) assay—the cobas® KRAS Mutation Test—designed to detect 19 mutations in codons 12, 13, and 61. The limit of detection was determined using DNA blends from cell lines, plasmids, and formalin-fixed paraffin-embedded tissue specimens. Assay performance was compared to Sanger sequencing using a panel of 188 specimens. Discordant specimens were subjected to next generation pyrosequencing (454). Assay repeatability was assessed using a panel of six specimens. A >95% correct mutation call rate was obtained in all specimen types with ~5% mutant alleles at DNA inputs of 0.8–6.3 ng per PCR reaction; 100% detection rate was observed at the recommended DNA input of 50 ng. The positive percent agreement with Sanger was 97.5% (79/81) for codons 12/13 and 85.7% (6/7) for codon 61. Negative percent agreement was 94.4% (101/107) for codon 12/13 and 99.4% (180/181) for codon 61. Nine of 10 discordant specimens yielded 454 results consistent with the cobas® results. With repeated testing, the assay showed a correct call rate of 100% (192/192) for all operators, instruments, reagent lots, and days tested. The cobas® test detects KRAS mutations in codons 12, 13, and 61 at a limit of detection of <5%. The PCR assay was more sensitive and specific than Sanger sequencing, and performance was highly reproducible. Test performance was not influenced by various endogenous interfering substances or common gut microbes

Phospho-ERK and AKT status, but not KRAS mutation status, are associated with outcomes in rectal cancer treated with chemoradiotherapy

<p>Abstract</p> <p>Background</p> <p><it>KRAS </it>mutations may predict poor response to radiotherapy. Downstream events from <it>KRAS</it>, such as activation of <it>BRAF</it>, AKT and ERK, may also confer prognostic information but have not been tested in rectal cancer (RC). Our objective was to explore the relationships of <it>KRAS </it>and <it>BRAF </it>mutation status with p-AKT and p-ERK and outcomes in RC.</p> <p>Methods</p> <p>Pre-radiotherapy RC tumor biopsies were evaluated. <it>KRAS </it>and <it>BRAF </it>mutations were assessed by pyrosequencing; p-AKT and p-ERK expression by immunohistochemistry.</p> <p>Results</p> <p>Of 70 patients, mean age was 58; 36% stage II, 56% stage III, and 9% stage IV. Responses to neoadjuvant chemoradiotherapy: 64% limited, 19% major, and 17% pathologic complete response. 64% were <it>KRAS </it>WT, 95% were <it>BRAF </it>WT. High p-ERK levels were associated with improved OS but not for p-AKT. High levels of p-AKT and p-ERK expression were associated with better responses. <it>KRAS </it>WT correlated with lower p-AKT expression but not p-ERK expression. No differences in OS, residual disease, or tumor downstaging were detected by <it>KRAS </it>status.</p> <p>Conclusions</p> <p><it>KRAS </it>mutation was not associated with lesser response to chemoradiotherapy or worse OS. High p-ERK expression was associated with better OS and response. Higher p-AKT expression was correlated with better response but not OS.</p

Epigenetics Offer New Horizons for Colorectal Cancer Prevention

In recent years, colorectal cancer (CRC) incidence has been increasing to become a major cause of morbidity and mortality worldwide from cancers, with high rates in westernized societies and increasing rates in developing countries. Epigenetic modifications including changes in DNA methylation, histone modifications, and non-coding RNAs play a critical role in carcinogenesis. Epidemiological data suggest that, in comparison to other cancers, these alterations are particularly common within the gastrointestinal tract. To explain these observations, environmental factors and especially diet were suggested to both prevent and induce CRC. Epigenetic alterations are, in contrast to genetic modifications, potentially reversible, making the use of dietary agents a promising approach in CRC for the development of chemopreventive strategies targeting epigenetic mechanisms. This review focuses on CRC-related epigenetic alterations as a rationale for various levels of prevention strategies and their potential modulation by natural dietary compounds

Prognostic DNA methylation markers for sporadic colorectal cancer: a systematic review

Background Biomarkers that can predict the prognosis of colorectal cancer (CRC) patients and that can stratify high-risk early stage patients from low-risk early stage patients are urgently needed for better management of CRC. During the last decades, a large variety of prognostic DNA methylation markers has been published in the literature. However, to date, none of these markers are used in clinical practice. Methods To obtain an overview of the number of published prognostic methylation markers for CRC, the number of markers that was validated independently, and the current level of evidence (LoE), we conducted a systematic review of PubMed, EMBASE, and MEDLINE. In addition, we scored studies based on the REMARK guidelines that were established in order to attain more transparency and complete reporting of prognostic biomarker studies. Eighty-three studies reporting on 123 methylation markers fulfilled the study entry criteria and were scored according to REMARK. Results Sixty-three studies investigated single methylation markers, whereas 20 studies reported combinations of methylation markers. We observed substantial variation regarding the reporting of sample sizes and patient characteristics, statistical analyses, and methodology. The median (range) REMARK score for the studies was 10.7 points (4.5 to 17.5) out of a maximum of 20 possible points. The median REMARK score was lower in studies, which reported a p value below 0.05 versus those, which did not (p = 0.005). A borderline statistically significant association was observed between the reported p value of the survival analysis and the size of the study population (p = 0.051). Only 23 out of 123 markers (17%) were investigated in two or more study series. For 12 markers, and two multimarker panels, consistent results were reported in two or more study series. For four markers, the current LoE is level II, for all other markers, the LoE is lower. Conclusion This systematic review reflects that adequate reporting according to REMARK and validation of prognostic methylation markers is absent in the majority of CRC methylation marker studies. However, this systematic review provides a comprehensive overview of published prognostic methylation markers for CRC and highlights the most promising markers that have been published in the last two decades

Postoperative outcomes in oesophagectomy with trainee involvement

BACKGROUND: The complexity of oesophageal surgery and the significant risk of morbidity necessitates that oesophagectomy is predominantly performed by a consultant surgeon, or a senior trainee under their supervision. The aim of this study was to determine the impact of trainee involvement in oesophagectomy on postoperative outcomes in an international multicentre setting. METHODS: Data from the multicentre Oesophago-Gastric Anastomosis Study Group (OGAA) cohort study were analysed, which comprised prospectively collected data from patients undergoing oesophagectomy for oesophageal cancer between April 2018 and December 2018. Procedures were grouped by the level of trainee involvement, and univariable and multivariable analyses were performed to compare patient outcomes across groups. RESULTS: Of 2232 oesophagectomies from 137 centres in 41 countries, trainees were involved in 29.1 per cent of them (n = 650), performing only the abdominal phase in 230, only the chest and/or neck phases in 130, and all phases in 315 procedures. For procedures with a chest anastomosis, those with trainee involvement had similar 90-day mortality, complication and reoperation rates to consultant-performed oesophagectomies (P = 0.451, P = 0.318, and P = 0.382, respectively), while anastomotic leak rates were significantly lower in the trainee groups (P = 0.030). Procedures with a neck anastomosis had equivalent complication, anastomotic leak, and reoperation rates (P = 0.150, P = 0.430, and P = 0.632, respectively) in trainee-involved versus consultant-performed oesophagectomies, with significantly lower 90-day mortality in the trainee groups (P = 0.005). CONCLUSION: Trainee involvement was not found to be associated with significantly inferior postoperative outcomes for selected patients undergoing oesophagectomy. The results support continued supervised trainee involvement in oesophageal cancer surgery