Detection of EGFR mutations in patients with non-small cell lung cancer by high resolution melting. Comparison with other methods

Martínez-Carretero, Carlos; Pascual, Fernando Iguaz; Rus, Antonio; Bernardo, Iván

doi:10.1515/cclm-2016-0353

Cited by 11 publications

(7 citation statements)

References 28 publications

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“…It is established that PCR-HRMA has a lower cost and is an accurate tool for mutation scanning, allowing the identification of variations through comparison of HRM curves; consequently, it is used as screening method or for the detection of known hotspot mutations. Its ability in discriminating known mutations is due to the use of reference material, or if this material is not available or novel variations are present in the samples analyzed, the technique is not able to identify the nucleotide change, but only to detect the presence (54–56). The HRMA curve shape depends on the number and type of nucleotide changes, with different nucleotide changes yielding different curves.…”

Section: Discussionmentioning

confidence: 99%

Pilot investigation of the mutation profile of PIK3CA/PTEN genes (PI3K pathway) in grade 3 endometrial cancer

et al. 2018

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Endometrial cancer (EC) comprises a biological and clinical heterogeneous group of tumors. Several genetic alterations are involved in the development and progression of EC, and may be used for targeted therapy, particularly in patients with advanced-stage EC. In the present study, a combined procedure was developed based on polymerase chain reaction (PCR)-high resolution melting analysis (HRMA) and Sanger sequencing for the evaluation of somatic mutations in selected phosphoinositide 3-kinase (PI3K) catalytic subunit α (PIK3CA; exons 1, 9 and 21) and phosphatase and tensin homolog (PTEN; exons 5, 6, 7 and 8) exons. This combined procedure has the specificity and sensitivity of the two techniques, and overcomes their limitations. A pilot study was performed on 18 selected homogenous EC samples, of grade 3 endometrioid subtype (G3 EEC). First, the feasibility of the combined procedure was investigated to properly identify the presence of somatic mutations on PIK3CA and PTEN, the variations identified were analyzed using Catalogue of Somatic Mutations in Cancer, PolyPhen-2 and Mutation Taster software, and the frequency of mutations/variations was determined in the selected samples. The evaluation of mutational load revealed that the majority of the G3 EEC samples exhibited PIK3CA mutations (39%) and PTEN mutations (67%), and the majority of the samples (83%) had mutations in at least one of the two genes, and 33% had mutations in the two genes. The results of the present pilot study suggested that the cost-effective combined PCR-HRMA and Sanger sequencing procedure may be suitable for identification of PTEN and PIK3CA mutations in G3 EEC and that their frequency was consistent in G3 EEC, indicating that the PI3K pathway serves a pivotal function that may have potential for defining targeted therapy for the treatment of G3 EEC.

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Section: Discussionmentioning

confidence: 99%

Pilot investigation of the mutation profile of PIK3CA/PTEN genes (PI3K pathway) in grade 3 endometrial cancer

et al. 2018

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“…12 EGFR is reported to be overexpressed in 40-80% of NSCLC cases and its mutations in NSCLC are commonly found in people with East Asian ethnicity, adenocarcinoma histological subtype, females and nonsmokers. 11,17 Studies 18,19 have reported that most EGFR mutations occur in exons 18-22 of the tyrosine kinase domains, with the most common mutation being exon 19 deletions and a point mutation in exon 21 (L858R). Furthermore, there are other rare EGFR mutations including substitutions such as glycine 719 with serine, cysteine or alanine in exon 18, which confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs), or mutations associated with resistance to first-generation TKIs such as the T790M mutation in exon 20 or insertions in exon 20.…”

Section: Epidermal Growth Factor Receptor (Egfr)mentioning

confidence: 99%

A review of predictive, prognostic and diagnostic biomarkers for non-small-cell lung cancer: towards personalised and targeted cancer therapy

Osei

Lumini

Gunasekara³

et al. 2019

J Radiother Pract

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Introduction: Lung cancer has a high mortality rate mainly due to the lack of early detection or outward signs and symptoms, thereby often progressing to advanced stages (e.g., stage IV) before it is diagnosed. However, if lung cancers can be diagnosed at an early stage and also if clinicians can prospectively identify patients likely to respond to specific treatments, then there is a very high potential to increase patients’ survival. In recent years, several investigations have been conducted to identify cancer biomarkers for lung cancer risk assessment, early detection and diagnosis, the likelihood of identifying the group of patients who will benefit from a particular treatment and monitoring patient response to treatment. Materials and Methods: This paper reports on the review of 19 current clinical and emerging biomarkers used in risk assessment, screening for early detection and diagnosis and monitoring the response of treatment of non-small-cell lung cancers. Conclusion: The future holds promise for personalised and targeted medicine from prevention, diagnosis to treatment, which take into account individual patient’s variability, though it depends on the development of effective biomarkers interrogating the key aberrant pathways and potentially targetable with molecular targeted or immunologic therapies. Lung cancer biomarkers have the potential to guide clinical decision-making since they can potentially detect the disease early, measure the risk of developing the disease and the risk of progression, provide accurate information of patient response to a specific treatment and are capable of informing clinicians about the likely outcome of a cancer diagnosis independent of the treatment received. Moreover, lung cancer biomarkers are increasingly linked to specific molecular pathway deregulations and/or cancer pathogenesis and can be used to justify the application of certain therapeutic or interventional strategies.

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“…As a result, accurate and sensitive molecular techniques and methods for EGFR T790M mutation analysis are acquired. The current methods for detecting of T790M mutation in clinical practice include real-time PCR assays ( [4]), allele-specific (AS) PCR ( [5]), amplification refractory mutation system (ARMS) ( [1]), droplet digital PCR (ddPCR) ( [1,5]), highresolution melting (HRM) ( [6]), Sanger sequencing ( [6]) and next-generation sequencing (NGS) ( [7]). Among those PCR-based methods, some do not allow to detect low-abundance EGFR T790M mutation, while others are highly sensitive.…”

Section: Introductionmentioning

confidence: 99%

“…It reduces the limitations in low-abundance mutation detection by use of a critical denaturation temperature (Tc) during PCR amplification. Low-abundance mutation enrichment by use of COLD-PCR has been demonstrated in combination with several downstream approaches such as TaqMan-based real-time PCR ( [16]), HRM ( [17]), Sanger sequencing ( [6]) and pyrosequencing ( [14]). When combined with pyrosequencing, COLD-PCR can improve mutation detection and identification by approximately 5-fold to 35-fold ( [14]).…”

Section: Introductionmentioning

confidence: 99%

The use of COLD-PCR and pyrosequencing for sensitive detection of EGFR T790M mutation

Chen

Zhang

et al. 2021

E3S Web Conf.

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A sensitive and convenient method for the detection of epidermal growth factor receptor (EGFR) T790M mutation in non-small cell lung cancer (NSCLC) patients with acquired resistance to tyrosine kinase inhibitors (TKIs) would be desirable to guide treatment strategy. Consequently, studies have focused on sensitive characterization of EGFR T790M mutation. Herein, two methods of co-amplification at lower denaturation temperature PCR (COLD-PCR) and pyrosequencing were combined (COLDPCR/ pyrosequencing) for detecting EGFR T790M mutation. Evaluation of mutation-containing dilutions revealed that the sensitivities of COLD-PCR/pyrosequencing and conventional PCR/pyrosequencing assays for the detection of the T790M mutation were 0.1 and 5%, respectively, indicating a 50-fold increase in sensitivity. When the T790M mutation in 20 clinical NSCLC samples who had relapsed under firstgeneration EGFR TKI were further determined using COLD-PCR/pyrosequencing and conventional PCR/pyrosequencing, the detection rates were 35% (7/20) and 25% (5/20), respectively. All patients who were positive for the T790M mutation with conventional PCR/pyrosequencing were also found to be positive with COLD-PCR/pyrosequencing. The discordant cases were 2 samples with no T790M mutation detected with conventional PCR/pyrosequencing, but which were positive with COLD-PCR/pyrosequencing. COLD-PCR/pyrosequencing is a sensitive and cost-effective tool for detecting the T790M mutation which will permit an improvement of therapeutic management.

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Detection of EGFR mutations in patients with non-small cell lung cancer by high resolution melting. Comparison with other methods

Abstract: HRM is a good method for mutational screening in EGFR. It is able to detect any change in the sequence of exons 18-21, providing high cost/effectiveness, but samples with low tumor burden may produce false negatives results.

Cited by 11 publications

References 28 publications

Pilot investigation of the mutation profile of PIK3CA/PTEN genes (PI3K pathway) in grade 3 endometrial cancer

Pilot investigation of the mutation profile of PIK3CA/PTEN genes (PI3K pathway) in grade 3 endometrial cancer

A review of predictive, prognostic and diagnostic biomarkers for non-small-cell lung cancer: towards personalised and targeted cancer therapy

The use of COLD-PCR and pyrosequencing for sensitive detection of EGFR T790M mutation

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