The usefulness of a novel fully automated PCR-based Idylla test for detection of the BRAF V600E mutation in thyroid tissue: comparison with PNA-clamping PCR, real-time PCR and pyrosequencing

Yeo, Min‐Kyung; Jung, Min-Kyu; Lee, Su Yel; Lee, Young Moo; Hur, Gang Min; Kim, Jin Man

doi:10.1136/jclinpath-2016-204025

Cited by 22 publications

(25 citation statements)

References 22 publications

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“…So far, the use of the combined BRAF and NRAS Idylla cartridge has only been described in colorectal cancer (CRC) and in melanoma . Conversely, in thyroid cancer, the single BRAF cartridge has only been used to genotype formalin‐fixed and paraffin embedded (FFPE) sections obtained from surgical samples . Instead, there are no data available on the use of the Idylla technology on thyroid cytological samples.…”

Section: Methodsmentioning

confidence: 99%

“…In any single case, the tissue block representative of the neoplasm was selected for the Idylla assay. The tissue area with the highest percentage of neoplastic cells (Table ) was marked on a hematoxylin and eosin (H&E) stained slide and microdissected on a corresponding FFPE section (10 μm thickness); then, the microdissected area was inserted into the Idylla cartridge and processed as previously described . The BRAF and NRAS genotyping results were compared with those obtained by processing the simulated FNA (Table ).…”

Section: Methodsmentioning

confidence: 99%

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Rapid On‐site Molecular Evaluation in thyroid cytopathology: A same‐day cytological and molecular diagnosis

Luca

Sgariglia

Nacchio

et al. 2020

Diagnostic Cytopathology

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Background Thyroid fine‐needle aspirates (FNAs) with undetermined morphology can be outsourced to centralized laboratories for comprehensive molecular profiling. When a local, rapid screening rules out easily detectable BRAF and NRAS mutations outsourcing is minimized, leading to cost savings. The fully automated Idylla technology, that does not require trained staff, is an emerging option. However, Idylla platform has only been validated to process formalin fixed paraffin embedded (FFPE) sections. Here we investigate whether also the FNA needle rinse could be genotyped by the same cytopathologist who performs the FNA, a procedure that can be termed rapid on site molecular evaluation (ROME). Methods To validate this approach, the Idylla BRAF and NRAS Test was performed on the rinses from 25 simulated (bench‐top) FNAs, in a first part of the study. Genotyping data were compared with those obtained on matched histological FFPE blocks. The second part of the study was carried out on 25 prospectively collected routine FNAs to assess the performance of the Idylla BRAF and NRAS assay against a gold standard real time polymerase chain reaction method. Results Idylla NRAS‐BRAF Mutation Test was performed on needle rinse as well as histological FFPE blocks. A sensitivity of 88.9%, a specificity of 100.0% were obtained comparing the Idylla NRAS‐BRAF Mutation Test on needle rinse to the reference method. Conclusions The FNA needle rinse can be directly genotyped. This obviates the need of cell block preparation, making possible a rapid combined morphological and molecular evaluation. Since DNA extraction is no longer necessary, the cytopathologist can perform ROME him/herself.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Rapid On‐site Molecular Evaluation in thyroid cytopathology: A same‐day cytological and molecular diagnosis

Luca

Sgariglia

Nacchio

et al. 2020

Diagnostic Cytopathology

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“…Pyrosequencing has the smallest number of defects and the greatest number of advantages, compared to other methods of measuring the percentage of heteroplasmy of mitochondrial genome [40][41][42][43][44] . It provides a unique opportunity to analyze a very short DNA fragment containing the region of the investigated mutation.…”

Section: Discussionmentioning

confidence: 99%

New markers of atherosclerosis: a threshold level of heteroplasmy in mtDNA mutations

Sazonova¹,

Рыжкова²,

Sinyov³

et al. 2017

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How to cite this article: Sazonova MA, Ryzhkova AI, Sinyov VV, Galitsyna EV, Orekhova VA, Melnichenko AA, Orekhov AN, Ravani AL, Sobenin IA. New markers of atherosclerosis: a threshold level of heteroplasmy in mtDNA mutations. Vessel Plus 2017;1:182-91. Aim:The aim of the present article was the detection of threshold heteroplasmy level of mitochondrial DNA mutations, above which a patient is at increased risk of atherosclerotic lesions. Besides, this parameter was detected for mutations, in which after reaching threshold heteroplasmy level, a protective antiatherogenic effect started to appear. Methods: The participants of the study were 700 women and men from the Moscow region. Fragments of DNA, amplified by polymerase chain reaction, were analyzed with pyrosequencing technology. Then on the basis of pyrograms' peaks in the samples, the heteroplasmy level of the investigated mitochondrial genome mutations was detected. Results: The threshold heteroplasmy level of 11 investigated mutations (m.5178C>A, m.15059G>A, m.652delG, m.13513G>A, m.14846G>A, m.652insG, m.12315G>A, m.3336T>C, m.1555A>G, m.14459G>A, m.3256C>T) in individuals with atherosclerotic plaques or thickening of the intima-medial layer of carotid arteries was detected. Conclusion: Using the method developed in our laboratory, the authors managed to determine threshold heteroplasmy levels of 11 mitochondrial genome mutations associated

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“…Due to its high sensitivity, PNA identified 67.3% of BRAF mutated samples, which is, to our knowledge, one of the highest percentages of BRAF mutations reported in the literature [4, 5, 7]. To our knowledge PNAs have been described one time only, very recently, for BRAF detection in melanomas; in addition they have been efficiently applied to BRAF testing for colorectal cancer and papillary thyroid carcinoma (PTC) [31, 37–40]. …”

Section: Discussionmentioning

confidence: 99%

Heterogeneity and frequency of BRAF mutations in primary melanoma: Comparison between molecular methods and immunohistochemistry

et al. 2016

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Finding the best technique to identify BRAF mutations with a high sensitivity and specificity is mandatory for accurate patient selection for target therapy. BRAF mutation frequency ranges from 40 to 60% depending on melanoma clinical characteristics and detection technique used.Intertumoral heterogeneity could lead to misinterpretation of BRAF mutational status; this is especially important if testing is performed on primary specimens, when metastatic lesions are unavailable.Aim of this study was to identify the best combination of methods for detecting BRAF mutations (among peptide nucleic acid – PNA-clamping real-time PCR, immunohistochemistry and capillary sequencing) and investigate BRAF mutation heterogeneity in a series of 100 primary melanomas and a subset of 25 matched metastatic samples.Overall, we obtained a BRAF mutation frequency of 62%, based on the combination of at least two techniques. Concordance between mutation status in primary and metastatic tumor was good but not complete (67%), when agreement of at least two techniques were considered. Next generation sequencing was used to quantify the threshold of detected mutant alleles in discordant samples. Combining different methods excludes that the observed heterogeneity is technique-based. We propose an algorithm for BRAF mutation testing based on agreement between immunohistochemistry and PNA; a third molecular method could be added in case of discordance of the results. Testing the primary tumor when the metastatic sample is unavailable is a good option if at least two methods of detection are used, however the presence of intertumoral heterogeneity or the occurrence of additional primaries should be carefully considered.

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The usefulness of a novel fully automated PCR-based Idylla test for detection of the BRAF V600E mutation in thyroid tissue: comparison with PNA-clamping PCR, real-time PCR and pyrosequencing

Abstract: This study validates that the Idylla test is a sensitive and specific method to detect BRAF V600E in FFPE thyroid tissues. A simple, quick and easy to handle Idylla test is a useful and reliable molecular technique to evaluate BRAF mutations.

Cited by 22 publications

References 22 publications

Rapid On‐site Molecular Evaluation in thyroid cytopathology: A same‐day cytological and molecular diagnosis

Rapid On‐site Molecular Evaluation in thyroid cytopathology: A same‐day cytological and molecular diagnosis

New markers of atherosclerosis: a threshold level of heteroplasmy in mtDNA mutations

Heterogeneity and frequency of BRAF mutations in primary melanoma: Comparison between molecular methods and immunohistochemistry

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