KRAS Gene Mutation in Patients Undergoing Liver Resections for Colorectal Cancer. Is There an Advantage to Anatomical Resections?

Introduction: Several factors have been described to make a prognostic assessment of patients with liver metastases due to colorectal cancer and to define the benefit of the surgical management of metastatic involvement; one of these factors is the status of the KRAS gene since its mutation is associated with worse outcomes. This study aims to describe the outcomes for a retrospective series of patients after liver resections for metastatic colorectal cancer concerning KRAS gene status. Materials and methods: The study involves a retrospective cohort of patients undergoing liver metastasectomy for colorectal cancer with KRAS mutation study from 2009-2013 at the National Institute of Cancerology in Colombia. Five-year survival analyses (overall and disease-free) were performed according to KRAS mutation status and the type of liver resection performed using the Kaplan-Meier estimate. Results: 35 patients undergoing liver metastasectomy were analyzed, of which 42.8% had KRAS gene mutation. Median overall survival was 34.2 months for patients with KRAS- mutant and 46.5 for non-mutant. The median survival for KRAS-mutant patients with anatomic resections was 43.5 months versus 23.5 months for nonanatomic resections. Conclusions: Performing anatomic resections during liver metastasectomy in patients with KRAS mutants could be associated with an improvement in overall survival. It is necessary to continue building the evidence for adequate decision-making in patients with KRAS mutants who will undergo liver resections.

Introducción: se han descrito varios factores para realizar una evaluación pronóstica de los pacientes con metástasis hepáticas por cáncer colorrectal y definir el beneficio en el manejo quirúrgico del compromiso metastásico; uno de estos factores es el estado del gen KRAS, debido a que su mutación está relacionada con peores desenlaces. El objetivo de este estudio es describir los desenlaces para una serie retrospectiva de pacientes después de resecciones hepáticas por metástasis de cáncer colorrectal en relación con el estado del gen KRAS. Metodología: cohorte retrospectiva de pacientes llevados a metastasectomía hepática por cáncer colorrectal con estudio de mutación KRAS durante el período 2009-2013, en el Instituto Nacional de Cancerología en Colombia. Se realizaron análisis de supervivencia a 5 años (global y libre de enfermedad) según el estado de mutación KRAS y según el tipo de resección hepática realizada mediante el método de Kaplan-Meier. Resultados: se analizaron a 35 pacientes llevados a metastasectomía hepática, de los cuales el 42,8% presentaba mutación del gen KRAS. La supervivencia global media fue de 34,2 meses para los pacientes con KRAS mutado y de 46,5 para los no mutados. La supervivencia media para los pacientes con KRAS mutado con resecciones anatómicas fue de 43,5 meses frente a 23,5 meses en los que se realizaron resecciones no anatómicas. Conclusiones: realizar resecciones anatómicas durante la metastasectomía hepática en los pacientes con KRAS mutado podría estar asociado con una mejoría en la supervivencia global. Se requiere continuar en la construcción de la evidencia que permita una adecuada toma de decisiones de los pacientes con KRAS mutado que serán llevados a resecciones hepáticas.

Kristen Rat Sarcoma Viral Oncogene Mutations in Colorectal Carcinomas at the University Teaching Hospital in Lusaka, Zambia

ackground: The second leading cause of cancerrelated deaths worldwide iscolorectal cancer. With an incidence rate of 4.8 per 100,000, this is Zambia's sixth most prevalent cancer; Methods: This laboratory-based, cross-sectional study examined the frequency of Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation and its association with prognostic factors in colorectal carcinoma cases from the University Teaching Hospital-Adult Hospital (UTHs), Lusaka, Zambia; Results: Thirty (30) formalin-fixed paraffinembedded (FFPE) samples collected between June 2017 and June 2018 were sent to the Lancet laboratories and analyzed for KRAS mutations (codons 12 and 13). One FFPE block did not meet the inclusion criteria and was excluded. The demographic and clinicopathological data were analyzed using STATA 12. Males outnumber females by 20 to nine. The average age of the patient was 45 ± 16 years. The rectum was the location of 44.8% of the tumors, with the majority being conventional adenocarcinoma (CAC) (65.6 %). All cases (100%) had advanced-stage (stages 3 and 4) disease;however, only 27.6% of patient tumors exhibited lymphovascular invasion. KRAS mutation was detected in 11 (37.9%) cases and mainly in left-sided tumors (62.5%). KRAS mutations were only detected in CAC and serrated adenocarcinoma subtypes. No significant associations were observed between the KRAS mutation status and tumor or patient's clinical and sociodemographic factors; Conclusion: We advocate for incorporating KRAS mutation testing into the standard of care for treating colorectal cancer.

Establishment of a novel TB-ARMS qPCR method for kras mutation detection / 生物工程学报

A simple, robust and highly sensitive TB-ARMS method based on qPCR technique was developed to detect kras mutations. The technique was evaluated, and its clinical application was investigated. Mutation specific primers for eight common kras mutations and wild type gene targeted blockers were designed and optimized. Moreover, a mutant-enriched condition was used in to improve the sensitivity and specificity of mutation detection. Constructed plasmids carrying mutant kras genes, as well as confirmed wild type genomic DNA, were used as standard samples for evaluation of the methodology. The performance of our new method was validated by comparing the results of our method with that of a commercial kras kit in testing 40 clinical samples. Preoperative plasma samples, as well as paired tissue samples, were tested in parallel for evaluation of its clinical application. We have developed a new TB-ARMS method for kras mutation detection that can detect minor mutant alleles with a frequency as low as 0.01% in a heterogeneous sample. We have successfully demonstrated its 0.01% detection sensitivity with highly specific mutant amplification in conjunction with selective wild type suppression by blocker under a mutant-enriched reaction condition. We also showed that our TB-ARMS method was more accurate than the commercial kras kit, which is widely used presently. Furthermore, we have validated our method as an efficient liquid biopsy method, and the results of the plasma DNA detection with our TB-ARMS method were in consistent with the sequencing results of paired tissue samples. In conclusion, our TB-ARMS qPCR method could be effectively applied in kras mutation test for clinical tissue samples, as well as for liquid biopsy samples such as plasma.

KRAS Mutation Test in Korean Patients with Colorectal Carcinomas: A Methodological Comparison between Sanger Sequencing and a Real-Time PCR-Based Assay

BACKGROUND: Mutations in the KRAS gene have been identified in approximately 50% of colorectal cancers (CRCs). KRAS mutations are well established biomarkers in anti–epidermal growth factor receptor therapy. Therefore, assessment of KRAS mutations is needed in CRC patients to ensure appropriate treatment. METHODS: We compared the analytical performance of the cobas test to Sanger sequencing in 264 CRC cases. In addition, discordant specimens were evaluated by 454 pyrosequencing. RESULTS: KRAS mutations for codons 12/13 were detected in 43.2% of cases (114/264) by Sanger sequencing. Of 257 evaluable specimens for comparison, KRAS mutations were detected in 112 cases (43.6%) by Sanger sequencing and 118 cases (45.9%) by the cobas test. Concordance between the cobas test and Sanger sequencing for each lot was 93.8% positive percent agreement (PPA) and 91.0% negative percent agreement (NPA) for codons 12/13. Results from the cobas test and Sanger sequencing were discordant for 20 cases (7.8%). Twenty discrepant cases were subsequently subjected to 454 pyrosequencing. After comprehensive analysis of the results from combined Sanger sequencing–454 pyrosequencing and the cobas test, PPA was 97.5% and NPA was 100%. CONCLUSIONS: The cobas test is an accurate and sensitive test for detecting KRAS-activating mutations and has analytical power equivalent to Sanger sequencing. Prescreening using the cobas test with subsequent application of Sanger sequencing is the best strategy for routine detection of KRAS mutations in CRC.

Prevalence of KRAS mutations in metastatic colorectal cancer: A retrospective observational study from India.

BACKGROUND: One of the genetic alterations implicated in tumor progression in colorectal cancers (CRCs) are abnormalities in Kristen Rat Sarcoma (KRAS) gene. Evaluation of KRAS mutation status is an important prognostic factor and has predictive value in deciding first line therapy based on monoclonal antibodies such as Cetuximab and Panitumumab in metastatic CRCs. MATERIALS AND METHODS: In this retrospective study, we analyzed 7 different somatic mutations in Exon 2 of KRAS gene in 299 unselected incidental CRC patients who visited the hospital for clinical management during the period 2009–2013. Most of the tumors were primarily originating from colon and rectum; nevertheless, there were a few from rectosigmoid, sigmoid, ceacum and anal canal in the study group. Genomic DNA extracted from paraffin embedded tumor tissues was screened for 7 point mutations located in Codons 12 and 13 of KRAS gene, using Scorpions amplified refractory mutation system real time polymerase chain reaction technology. Statistical analysis was performed to assess bivariate relationship between different variables that includes: mutation status, mutation type, tumor location, tumor morphology, age and sex. RESULTS: Prevalence of mutation in Codons 12 and 13 was 42.8% in the study group. Well‑differentiated tumors had significantly more mutation positivity than moderately and poorly differentiated tumors (P = 0.001). 92% of the mutations were from Codon 12 and 8% in Codon 13. Glycine to Arginine was relatively more common in rectosigmoid followed by ceacum, while Glycine to Alanine mutation was relatively more prevalent in sigmoid, followed by rectum and rectosigmoid. CONCLUSION: The results suggest a prevalence of KRAS mutation at 42.8% in Indian population indicating that this testing is very crucial for targeted therapy management in metastatic CRC in India. Further analysis on mutation status of other homologues such as NRAS and downstream partner, v‑raf murine sarcoma viral oncogene homolog B1, would add value to understanding the role of anti‑epidermal growth factor receptor therapy in CRC management.

RAS Mutation is a Local Tumour Event and Not a Field Change in Pancreatobiliary Tumours.

Background: KRAS mutation (KRM) is the earliest, most common mutation in pancreatic cancer. Accurate assessment of tumour KRM status in pancreatobiiary tumours is relevant in an era of targeted molecular therapies. Aim: To assess KRM in tumour and non-tumourous margin tissue in patients undergoing a pancreatic resection. Study Design: Original research, retrospective review of prospectively collected specimens. Place and Duration of Study: Patients who had undergone pancreaticoduodenectomy and distal pancreatic resection at the Royal Adelaide Hospital from 2011-2012 were consented for the study. Methods: Patient demographics, background history and tumour details were collated. Tumour tissue and margin areas were macrodissected from FFPE tissue sections following identification by a pathologist. DNA was prepared from the tissue using the QIAamp FFPE Tissue kit (Qiagen GmbH, Hilden Germany). KRM at codons 12 and 13 was assessed using SNaPShot TM (Applied Biosystems, Warrington UK) in tumour tissue and non-tumourous margin tissue. Fourteen patients were included in the study. The median age of the patients in the study was 68 (range 57-86) years. The M : F ratio was 8 : 6. Results: Twelve patients had adenocarcinomas (5 pancreatic; 4 ampullary, 3 biliary) and two had benign mucinous tumours. Six patients with adenocarcinomas had KRM (5@codon 12 and 1@codon 13). Margin tissue was negative for KRM in all the tested patients (p<0.016 Fisher) particularly, in those with tumour KRM. Tumours with KRM were associated with larger tumours 30(22-65) mm vs 20(15-35) mm [median(range)](p = .045 – MW-U). Nodal disease occurred in 6/6 with KRM vs 2/6 without KRM (p = .61 – Fisher). Conclusions: KRM is a local tumour event and not a field change. This suggests that testing for KRM should be reliant on tumour tissue and not surrounding normal margin tissue. KRM was associated with larger malignant tumours and a trend towards nodal disease.