High-Resolution Melting Analysis for Rapid Detection of<i>KRAS, BRAF,</i>and<i>PIK3CA</i>Gene Mutations in Colorectal Cancer

Simi, Lisa; Pratesi, Nicola; Vignoli, Marina; Sestini, Roberta; Cianchi, Fabio; Valanzano, Rosa; Nobili, Stefania; Mini, Enrico; Pazzagli, Mario; Orlando, Claudio

doi:10.1309/lwdy1axhxuulnvhq

Cited by 162 publications

(145 citation statements)

References 40 publications

(51 reference statements)

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“…Using a pre-screening approach in codons 12 -13 with high-resolution melting curve analysis (Simi et al, 2008) followed by sequencing, we found 35% cases with a mutation in codons 12 -13, in agreement with the literature. However, we also report two cases presenting a mutation in codon 19 (c.57G4C, p.Leu19Phe), without mutations in codons 12 -13.…”

Section: Sirsupporting

confidence: 90%

KRAS mutation status in colorectal cancer to predict response to EGFR targeted therapies: the need for a more precise definition

et al. 2008

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Section: Sirsupporting

confidence: 90%

KRAS mutation status in colorectal cancer to predict response to EGFR targeted therapies: the need for a more precise definition

et al. 2008

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“…In a previous study 29 on colorectal cancer, we demonstrated the high sensitivity of HRM analysis, which allowed identifying at least 5% of mutated alleles in a background of wild-type DNA. Even though HRM analysis has been widely applied for the screening of somatic variants in biopsies of solid cancers, only a few previous applications of HRM analysis have been reported in the screening of cytological material, as fixed fresh cells 36 or scraped cells from archival slides 37 obtained from needle aspiration.…”

Section: Discussionmentioning

confidence: 98%

“…Screening the FNAB samples provided a satisfactory resolution of melting for all of the DNA sites of interest and allowed the amplification of all genes simultaneously. To evaluate the theoretical sensitivity of our method, a detection limit was calculated, as described previously, 29 by using serial dilution of positive controls (cell lines CCRF-CEM, SW948, HT1197, SK-MEL28) in wild-type DNA (from MCF-7 cell line) for each gene under study. We were able to detect the presence of mutated DNA up to 5% in a background of wild-type DNA (data not shown).…”

Section: Mutation Scanning By Hrm Analysis and Sequencingmentioning

confidence: 99%

A High-Resolution Melting Protocol for Rapid and Accurate Differential Diagnosis of Thyroid Nodules

Mancini

Pinzani

Pupilli

et al. 2012

The Journal of Molecular Diagnostics

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Thyroid cancer is the most common malignancy of the endocrine system, accounting for approximately 1% of all malignancies in Western countries. 1 The incidence of thyroid cancer has increased 2.6-fold in the last 30 years. This change is attributed not only to an increment in papillary thyroid carcinoma, 2 but also to more wisely conducted medical surveillance and improvements in diagnostic tools. 3 The large majority of thyroid nodules, as discovered with the use of new diagnostic imaging techniques, are asymptomatic and benign. In this context, diagnostic studies are becoming essential to identify the small fraction of thyroid nodules that harbor malignant disease and to predict when surgery is indicated. Fine-needle aspiration biopsy (FNAB) has emerged over the past 30 years as an accurate and cost-effective procedure for the preoperative screening of thyroid nodules, representing the gold standard for differential diagnosis of benign and malignant nodules. 4 Under recent guidelines, 5,6 cytological smears are classified in five categories for the diagnostic report: Thy 1, nondiagnostic; Thy 2, benign or negative for malignant cells; Thy 3, all follicular lesions (including atypia/ follicular lesion of undetermined significance and follicular neoplasm or suspicious for follicular neoplasm); Thy 4, suspicious; and Thy 5, diagnostic for malignancy.Although the overall accuracy of FNAB is considered excellent, approximately 30% of cytological aspirates do not allow definitive diagnosis of malignancy, because of intrinsic and unavoidable characteristics of samples. 7 The major limitations of FNAB procedures are linked to inadequate and indeterminate specimens and, in that sense, are also linked respectively to the nondiagnostic or follicular lesions categories. 8,9 Thus, a clinical need emerges for the characterization of aspirates with suspicious features but with unsatisfactory cellularity, to allow accurate distinction of benign from malignant forms of follicular lesions.Several somatic mutations have been identified in thyroid cancer, stimulating the search for genetic alterations in FNAB that could increase the diagnostic accuracy of traditional cytology. Numerous studies have demonstrated that identification of specific mutations in cytological specimens can assist in the diagnosis by FNAB and in the clinical decision to excise the nodule and to intensify the follow-up. 10 -16 In most cases, genetic alterations are represented by activating mutations of oncogenes that are mutually exclusive and linked to distinct histological subtypes, with a demonstrated pathogenic role in thyroid cell transformation as effectors of the RAS/RAF/ MAPK signaling cascade.The presence of BRAF mutations, a frequent alteration in papillary carcinoma (PTC), evolves into an unregulated activation of the intracellular MAPK pathway that can promote tumorigenesis and tumor progression. The main mutation of BRAF, identified exclusively in PTC, affects

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“…High-resolution melting has been used clinically to detect somatic changes in select exons of oncogenes such as EGFR, 53 KRAS, 54 PDGFRA, 55 KIT, 56 BRAF, 57 and TP53. 58 In tumors with mixed populations of tumor and normal cells, the sensitivity of scanning (Ͻ10%) is superior to sequencing (ϳ20%).…”

Section: Mutation Scanning By Meltingmentioning

confidence: 99%

LightCycler Technology in Molecular Diagnostics

Lyon

Wittwer

2009

The Journal of Molecular Diagnostics

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From the first report of the LightCycler as a real-time polymerase chain reaction (PCR) instrument 1 clinical laboratories quickly realized its potential as a diagnostic tool. Quantitative applications important for infectious disease and oncology applications were followed by genotyping assays that took advantage of accurate temperature control to melt DNA amplicons or probe/amplicon duplexes. The first Food and Drug Administration-cleared molecular genetic assays were developed for this platform, specifically genotyping single base variants in F5 and F2. According to a College of American Pathologists survey, 2 ϳ30% of clinical laboratories report using the LightCycler for these two assays. This article will review the history of LightCycler technology, focusing on those applications widely incorporated into molecular diagnostics. In addition we will review recent developments that may impact future clinical testing.Real-time PCR instruments simultaneously amplify and detect, eliminating the need to open tubes containing PCR amplicon and therefore reducing the risk of future contamination. Fluorescent dyes or probes allow continuous monitoring as template DNA is amplified.3 By monitoring fluorescence every cycle, the amount of original target can be calculated. By monitoring fluorescence as the temperature changes, genotyping and heterozygote scanning can be performed by melting analysis, often removing the need for downstream analysis.The first two platforms developed for real-time PCR were the LightCycler (Roche, Indianapolis, IN) and the ABI 7700 (Applied Biosystems, Foster City, CA). The instruments were as different as the groups that created them. At the time, ABI was the undisputed corporate leader of PCR technology and the 7700 was a large 96-well laser-based instrument focused on throughput.

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High-Resolution Melting Analysis for Rapid Detection ofKRAS, BRAF,andPIK3CAGene Mutations in Colorectal Cancer

Cited by 162 publications

References 40 publications

KRAS mutation status in colorectal cancer to predict response to EGFR targeted therapies: the need for a more precise definition

KRAS mutation status in colorectal cancer to predict response to EGFR targeted therapies: the need for a more precise definition

A High-Resolution Melting Protocol for Rapid and Accurate Differential Diagnosis of Thyroid Nodules

LightCycler Technology in Molecular Diagnostics

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