Detection of EGFR and KRAS mutations on trans-thoracic needle aspiration of lung nodules by high resolution melting analysis

Fassina, Ambrogio; Gazziero, Alessia; Zardo, Davide; Corradin, Matteo; Aldighieri, Enrico; Rossi, Gian Paolo

doi:10.1136/jcp.2009.067587

Cited by 62 publications

(44 citation statements)

References 48 publications

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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: 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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Section: Discussionmentioning

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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“…24,41,42 Use of antibodies that are specific to mutated protein present a potentially less expensive and more routine method for mutation detection. However, new technologies and methods may soon neutralize any cost difference between DNA and protein analyses.…”

Section: Discussionmentioning

confidence: 99%

Standardization of Epidermal Growth Factor Receptor (EGFR) Measurement by Quantitative Immunofluorescence and Impact on Antibody-Based Mutation Detection in Non–Small Cell Lung Cancer

Dimou

Agarwal

Anagnostou

et al. 2011

The American Journal of Pathology

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Challenges in measurement of epidermal growth factor receptor (EGFR) protein expression have led to conflicting data on its prognostic value and discontinuation of its use for prediction of response. Herein is described a quantitative standardized assay for EGFR and its use in a series of retrospective cohorts of patients with nonsmall cell lung cancer (NSCLC). The AQUA technology of quantitative immunofluorescence was used in conjunction with Western blot analysis to calculate the absolute concentration of EGFR in two independent NSCLC cohorts (170 from Yale New Haven Hospital and 335 from Sotiria and Patras Hospitals in Greece). EGFR and mutated EGFR were measured using D38B1 antibody and two mutation-specific antibodies. All patients positive or borderline for mutation-specific antibody were genotyped. A threshold for reproducible detection of EGFR was defined as 0.85 ng/ g total protein. EGFR expression demonstrated no prognostic value in either cohort. The mutation rate was 1.79% in the Yale cohort, and 1.52% in the Sotiria/Patras cohort, with no antibody detection-based false-positive cases. No mutations were detected for EGFR concentrations <1.46 ng/ g total protein. In summary, accurate measurement of EGFR still shows no prognostic value in NSCLC. In these two population-based cohorts, the antibody-based EGFR mutation rate was lower than has been frequently reported.

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“…The genes tested have included EGFR KRAS, BRAF and PIK3CA in the lung specimens and BRAF, RET, RAS, TRK and PPRg in the thyroid nodules. 6,7,10,23,[30][31][32][33][34][35][36][37][38] Due to the low analytical sensitivity of direct sequencing, ultrasensitive techniques such as high resolution melting, realtime PCR and pyrosequencing have been utilized to improve detection sensitivity. 7 However, assessment of multiple genes in cytological specimens is sometimes not possible, because traditional platforms require large amounts of DNA.…”

Section: Discussionmentioning

confidence: 99%

Next-generation sequencing-based multi-gene mutation profiling of solid tumors using fine needle aspiration samples: promises and challenges for routine clinical diagnostics

et al. 2014

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Increasing use of fine needle aspiration for oncological diagnosis, while minimally invasive, poses a challenge for molecular testing by traditional sequencing platforms due to high sample requirements. The advent of affordable benchtop next-generation sequencing platforms such as the semiconductor-based Ion Personal Genome Machine (PGM) Sequencer has facilitated multi-gene mutational profiling using only nanograms of DNA. We describe successful next-generation sequencing-based testing of fine needle aspiration cytological specimens in a clinical laboratory setting. We selected 61 tumor specimens, obtained by fine needle aspiration, with known mutational status for clinically relevant genes; of these, 31 specimens yielded sufficient DNA for next-generation sequencing testing. Ten nanograms of DNA from each sample was tested for mutations in the hotspot regions of 46 cancer-related genes using a 318-chip on Ion PGM Sequencer. All tested samples underwent successful targeted sequencing of 46 genes. We showed 100% concordance of results between next-generation sequencing and conventional test platforms for all previously known point mutations that included BRAF, EGFR, KRAS, MET, NRAS, PIK3CA, RET and TP53, deletions of EGFR and wild-type calls. Furthermore, next-generation sequencing detected variants in 19 of the 31 (61%) patient samples that were not detected by traditional platforms, thus increasing the utility of mutation analysis; these variants involved the APC, ATM, CDKN2A, CTNNB1, FGFR2, FLT3, KDR, KIT, KRAS, MLH1, NRAS, PIK3CA, SMAD4, STK11 and TP53 genes. The results of this study show that next-generation sequencing-based mutational profiling can be performed on fine needle aspiration cytological smears and cell blocks. Next-generation sequencing can be performed with only nanograms of DNA and has better sensitivity than traditional sequencing platforms. Use of next-generation sequencing also enhances the power of fine needle aspiration by providing gene mutation results that can direct personalized cancer therapy.

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Detection of EGFR and KRAS mutations on trans-thoracic needle aspiration of lung nodules by high resolution melting analysis

Cited by 62 publications

References 48 publications

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

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

Standardization of Epidermal Growth Factor Receptor (EGFR) Measurement by Quantitative Immunofluorescence and Impact on Antibody-Based Mutation Detection in Non–Small Cell Lung Cancer

Next-generation sequencing-based multi-gene mutation profiling of solid tumors using fine needle aspiration samples: promises and challenges for routine clinical diagnostics

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