High resolution melting analysis of KRAS, BRAF and PIK3CA in KRASexon 2 wild-type metastatic colorectal cancer

Guedes, João Miranda; Veiga, Isabel; Rocha, Patrícia; Pinto, Pedro; Pinto, Carla; Pinheiro, Manuela; Peixoto, Ana; Fragoso, Maria; Raimundo, A.; Ferreira, P.; Machado, Manuela; Sousa, Nuno; Lopes, Paula; Araújo, António; Macedo, Joana Espiga de; Alves, Fábio Abreu; Coutinho, C.; Henrique, Rui; Santos, Lúcio Lara; Teixeira, Manuel R.

doi:10.1186/1471-2407-13-169

Cited by 41 publications

(31 citation statements)

References 50 publications

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“…Thus, according to recent data 12–17% of wild-type KRAS exon 2 (codons 12/13) patients harbour a mutation in KRAS exons 3 and 4 and NRAS exons 2, 3 and 4 3 6 13. In our study this percentage was 15.31% (figure 4).…”

Section: Discussionsupporting

confidence: 65%

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KRAS,NRASandBRAFmutations in Greek and Romanian patients with colorectal cancer: a cohort study

Negru

Papadopoulou²,

Apessos³

et al. 2014

BMJ Open

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ObjectivesTreatment decision-making in colorectal cancer is often guided by tumour tissue molecular analysis. The aim of this study was the development and validation of a high-resolution melting (HRM) method for the detection of KRAS, NRAS and BRAF mutations in Greek and Romanian patients with colorectal cancer and determination of the frequency of these mutations in the respective populations.SettingDiagnostic molecular laboratory located in Athens, Greece.Participants2425 patients with colorectal cancer participated in the study.Primary and secondary outcome measures2071 patients with colorectal cancer (1699 of Greek and 372 of Romanian origin) were analysed for KRAS exon 2 mutations. In addition, 354 tumours from consecutive patients (196 Greek and 161 Romanian) were subjected to full KRAS (exons 2, 3 and 4), NRAS (exons 2, 3 and 4) and BRAF (exon 15) analysis. KRAS, NRAS and BRAF mutation detection was performed by a newly designed HRM analysis protocol, followed by Sanger sequencing.ResultsKRAS exon 2 mutations (codons 12/13) were detected in 702 of the 1699 Greek patients with colorectal carcinoma analysed (41.3%) and in 39.2% (146/372) of the Romanian patients. Among the 354 patients who were subjected to full KRAS, NRAS and BRAF analysis, 40.96% had KRAS exon 2 mutations (codons 12/13). Among the KRAS exon 2 wild-type patients 15.31% harboured additional RAS mutations and 12.44% BRAF mutations. The newly designed HRM method used showed a higher sensitivity compared with the sequencing method.ConclusionsThe HRM method developed was shown to be a reliable method for KRAS, NRAS and BRAF mutation detection. Furthermore, no difference in the mutation frequency of KRAS, NRAS and BRAF was observed between Greek and Romanian patients with colorectal cancer.

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Section: Discussionsupporting

confidence: 65%

“…Two of the aforementioned studies (performed by Vaughn et al ,3 and Douillard et al 6) also analysed NRAS gene in KRAS exon 2 wild-type patients 13. Mutation frequency of NRAS exon 2 was 1.9% and 3.4% (weighted average 2.77%), while exon 3 was mutated with a percentage of 3.1% and 4% (weighted average 3.64%).…”

Section: Discussionmentioning

confidence: 99%

KRAS,NRASandBRAFmutations in Greek and Romanian patients with colorectal cancer: a cohort study

Negru

Papadopoulou²,

Apessos³

et al. 2014

BMJ Open

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“…For e.g., it has been used for detection of mutations in KRAS, BRAF and PIK3CA in metastatic colorectal cancer [26–29]. HRMA has been recently adapted for the detection of mutations at target sites introduced by genome editing reagents in Aedes aegypti and zebrafish [30].…”

Section: Discussionmentioning

confidence: 99%

Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents

et al. 2017

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Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that eliminates a PCR amplifiable target) revealed these cryptic DSBs and facilitated the determination of the true efficacy of genome editing reagents. The results indicated that in HEK293T cells, approximately 40% of the DSBs introduced by genome editing, were available for participation in HDR.

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“…Clinical NGS testing (8) for an actionable CRC gene panel Nelson, Turbyville et al 4 identified a novel NRAS internal tandem duplication (ITD) of 10 amino acids within the switch II domain. The prevalence of such RAS ITDs in CRC is unknown, and at the time of initial diagnosis (2015), we found literature reports of only three prior CRC patients with this type of duplication in KRAS (2,9,10). Subsequently, other reports of RAS ITDs have emerged including one report of a pediatric patient with a hematologic malignancy (11) and a report of RAS-family ITDs in up to 2% of vascular malformation/overgrowth syndrome cases (12).…”

Section: Introductionmentioning

confidence: 97%

RASInternal Tandem Duplication Disrupts GAP-binding to Activate Oncogenic Signaling

Nelson

Turbyville

Dharmaiah

et al. 2019

Preprint

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Molecular testing of oncogenic RAS mutations in colorectal cancer (CRC) have led to increased identification of mutations in patients with CRC. NRAS-mutated CRC has not been well characterized because it is less common (<6%) than KRAS mutations. Here, we report a novel 10 amino acid internal tandem duplication (ITD) in NRAS, which disrupts the switch II domain, in a patient with widely disseminated CRC. Hotspot next generation sequencing of a brain metastasis identified the NRAS ITD and a TP53 missense mutation (p.P275F). Whole exome sequencing of the primary tumor and two metastatic lesions (lung and brain) confirmed that the NRAS ITD and TP53 mutation were conserved between the primary tumor and both metastatic tumors, and identified an additional pathogenic mutation in CSMD1 (a tumor suppressor gene). Structural biology and biochemical analyses demonstrated that the NRAS ITD prevented binding to GAP protein, leading to sustained RAS activation, increased interaction with RAF, and downstream MAPK activation. Additionally, we provide the first crystal structure of the RAS ITD. In conclusion, these studies indicate that the NRAS ITD was the probable primary driver mutation of this aggressive CRC. Identical or biologically similar ITDs in NRAS and KRAS may be rare drivers of CRC and other aggressive malignancies. Nelson, Turbyville et al. 3

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High resolution melting analysis of KRAS, BRAF and PIK3CA in KRASexon 2 wild-type metastatic colorectal cancer

Cited by 41 publications

References 50 publications

KRAS,NRASandBRAFmutations in Greek and Romanian patients with colorectal cancer: a cohort study

KRAS,NRASandBRAFmutations in Greek and Romanian patients with colorectal cancer: a cohort study

Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents

RASInternal Tandem Duplication Disrupts GAP-binding to Activate Oncogenic Signaling

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