scholarly journals Not enough can be enough: feasibility of the Idylla EGFR mutation test when reuse of stained tissue slides is the only option available

2021 ◽  
pp. jclinpath-2021-207726
Author(s):  
Cristiana Ercolani ◽  
Anna Di Benedetto ◽  
Claudia Bonomo ◽  
Paolo Visca ◽  
Aldo Palange ◽  
...  
Keyword(s):  
Diagnostic Accuracy ◽  
Ad Hoc ◽  
In Situ Hybridisation ◽  
Molecular Analyses ◽  
Dna Yield ◽  
Mutational Status ◽  
Formalin Fixed Paraffin ◽  
Assay Performance ◽  
Formalin Fixed Paraffin Embedded ◽  
Pathological Material

AimsThe minimally invasive procedures used in the diagnostic workup of patients with advanced non-small cell lung cancer (NSCLC) often provide poor yields of pathological material suitable for molecular analyses. Not infrequently, the DNA yield from small biopsies/cytological samples is insufficient for the assessment of genomic biomarkers that inform personalised therapies. The Idylla EGFR mutation test (IEMT) has been specifically designed to process formalin-fixed paraffin-embedded sections without requiring preliminary DNA extraction.This study aims to evaluate the diagnostic accuracy of IEMT when used to analyse archival histopathology material. More specifically, our objective was to establish whether or not different staining procedures could affect assay performance.MethodsTwenty NSCLC samples were selected accordingly to EGFR mutational status. To mimic archived stained material, sections were subjected to H&E staining, fluorescent in situ hybridisation analyses or immunodetection by immunohistochemistry before being processed for IEMT.ResultsParallel assessment of EGFR mutational status by IEMT on stained sections and next-generation sequencing on DNA yielded a concordant result in 50 out of 60 tests (83.3%). The discoloration of H&E of the archived sample was found to be the optimal procedure to highlight all the actionable alterations of EGFR.ConclusionsIEMT can provide remarkable diagnostic accuracy for the assessment of EGFR mutational status also when the only source of pathological material available for molecular analyses is represented by H&E stained sections. Ad hoc supervision by a qualified molecular biologist is in any case recommended.

Sodium Alginate versus Plasma Thrombin Cell Blocks in Diagnostic Cytopathology: A Comparative Analysis

2021 ◽  
pp. 1-7
Author(s):  
Shruti Gupta ◽  
Upasana Gautam ◽  
Shaily Susheilia ◽  
Baneet Bansal ◽  
Radha Uppal ◽  
...  
Keyword(s):  
Sodium Alginate ◽  
Molecular Analysis ◽  
Low Cost ◽  
Nuclear Marker ◽  
Fine Needle Aspirates ◽  
Dna Yield ◽  
Morphological Evaluation ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

<b><i>Background:</i></b> Cell blocks (CBs) are an essential adjunct in cytopathology practice. The aim of this study was to compare 2 techniques of CB preparation – plasma thrombin (PT) method with sodium alginate (SA) method for overall cellularity, morphological preservation, obscuring artefacts, immunocytochemistry (ICC), suitability for molecular analysis, and cost of preparation. <b><i>Design:</i></b> A total of 80 fine-needle aspirates from various sites and serous effusion samples were included. Of these cases, by random selection, 40 each were prepared by PT method and SA methods, respectively. The haematoxylin-eosin-stained sections from the formalin-fixed, paraffin-embedded CBs from both methods were evaluated in a blinded fashion by 2 cytopathologists and scored for cellularity, artefacts, and morphological preservation and analysed by χ<sup>2</sup> test with Yates correction. We evaluated 6 cases from each method by ICC for a range of membrane, cytoplasmic and nuclear marker expression. DNA was extracted from four cases to evaluate their utility for molecular analysis. <b><i>Results:</i></b> CB sections from PT and SA techniques showed comparable cellularity and excellent cytomorphological preservation. Blue gel-like artefacts were common in the SA technique but did not interfere with morphological evaluation. ICC staining results were also similar. DNA yield and utility for PCR were also comparable. The SA-CB cost half that of PT-CB (USD 0.4 vs. USD 1). <b><i>Conclusion:</i></b> SA technique of CB preparation is an excellent low-cost alternative to PT method for CB preparation.

Evaluation of the Oncomine Comprehensive Assay v3 panel for the detection of 1p/19q codeletion in oligodendroglial tumours

2021 ◽  
pp. jclinpath-2021-207876
Author(s):  
Rola H Ali ◽  
Mona Alateeqi ◽  
Hiba Jama ◽  
Noor Alrumaidhi ◽  
Ali Alqallaf ◽  
...  
Keyword(s):  
Copy Number ◽  
In Situ Hybridisation ◽  
Fluorescence In Situ Hybridisation ◽  
Ion Torrent ◽  
Robust Detection ◽  
Targeted Next Generation Sequencing ◽  
Formalin Fixed Paraffin ◽  
Custom Made ◽  
Formalin Fixed Paraffin Embedded ◽  
Diffuse Gliomas

AimsAccurate assessment of 1p/19q codeletion status in diffuse gliomas is of paramount importance for diagnostic, prognostic and predictive purposes. While targeted next generation sequencing (NGS) has been widely implemented for glioma molecular profiling, its role in detecting structural chromosomal variants is less well established, requiring supplementary informatic tools for robust detection. Herein, we evaluated a commercially available amplicon-based targeted NGS panel (Oncomine Comprehensive Assay v3) for the detection of 1p/19q losses in glioma tissues using an Ion Torrent platform and the standard built-in NGS data analysis pipeline solely.MethodsUsing as little as 20 ng of DNA from formalin-fixed paraffin-embedded tissues, we analysed 25 previously characterised gliomas for multi-locus copy number losses (CNLs) on 1p and 19q, including 11 oligodendrogliomas (ODG) and 14 non-oligodendroglial (non-ODG) controls. Fluorescence in-situ hybridisation (FISH) was used as a reference standard.ResultsThe software confidently detected combined contiguous 1p/19q CNLs in 11/11 ODGs (100% sensitivity), using a copy number cut-off of ≤1.5 and a minimum of 10 amplicons covering the regions. Only partial non-specific losses were identified in non-ODGs (100% specificity). Copy number averages of ODG and non-ODG groups were significantly different (p<0.001). NGS was concordant with FISH and was superior to it in distinguishing partial from contiguous losses indicative of whole-arm chromosomal deletion.ConclusionsThis commercial NGS panel, along with the standard Ion Torrent algorithm, accurately detected 1p/19q losses in ODG samples, obviating the need for specialised custom-made informatic analyses. This can easily be incorporated into routine glioma workflow as an alternative to FISH.

scholarly journals Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens

2006 ◽  
Vol 54 (7) ◽  
pp. 773-780 ◽  
Author(s):  
Kiyohiro Hamatani ◽  
Hidetaka Eguchi ◽  
Keiko Takahashi ◽  
Kazuaki Koyama ◽  
Mayumi Mukai ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Rt Pcr ◽  
Molecular Analyses ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Tissue Specimens ◽  
Formalin Fixed

scholarly journals A protocol for good quality genomic DNA isolation from formalin-fixed paraffin-embedded tissues without using commercial kits

2021 ◽  
Author(s):  
Fazlur Rahman Talukdar ◽  
Irena Abramovic ◽  
Cyrille Cuenin ◽  
Christine Carreira ◽  
Nitin Gangane ◽  
...  
Keyword(s):  
Dna Isolation ◽  
Middle Income ◽  
Dna Yield ◽  
Genomic Dna Isolation ◽  
Novel Approach ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Commercial Kits ◽  
Formalin Fixed

DNA isolation from formalin fixed paraffin embedded (FFPE) tissues for molecular analysis has become a frequent procedure in cancer research. However, the yield or quality of the isolated DNA is often compromised, and commercial kits are used to overcome this to some extent. We developed a new protocol (IARCp) to improve better quality and yield of DNA from FFPE tissues without using any commercial kit. To evaluate the IARCp performance, we compared the quality and yield of DNA with two commercial kits, namely NucleoSpin DNA FFPE XS (MN) and QIAamp DNA Micro (QG) isolation kit. Total DNA yield for QG ranged from 120.0 to 282.0 ng (mean 216.5 ng), for MN: 213.6 to 394.2 ng (mean 319.1 ng), and with IARCp the yield was much higher ranging from 775.5 to 1896.9 ng (mean 1517.8 ng). Moreover, IARCp has also performed well in qualitative assessments. Overall, IARCp represents a novel approach to DNA isolation from FFPE which results in good quality and significant amounts of DNA suitable for many downstream genome-wide and targeted molecular analyses. Our proposed protocol does not require the use of any commercial kits for isolating DNA from FFPE tissues, making it suitable to implement in low-resource settings such as low and middle-income countries (LMICs).

scholarly journals Blood-based RAS mutation testing: concordance with tissue-based RAS testing and mutational changes on progression

2020 ◽  
Vol 16 (28) ◽  
pp. 2177-2189 ◽  
Author(s):  
Theodora Germetaki ◽  
Camille Nicholls ◽  
Richard A Adams ◽  
Michael Braun ◽  
Jane Rogan ◽  
...  
Keyword(s):  
Ras Mutation ◽  
Mutation Status ◽  
First Line Therapy ◽  
Mutational Status ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Longitudinal Plasma ◽  
Good Concordance ◽  
Colorectal Cancer Patients ◽  
Formalin Fixed

Aim: To determine the concordance between plasma and tissue RAS mutation status in metastatic colorectal cancer patients to gauge whether blood-based testing is a viable alternative. We also evaluated the change in mutation status on progression. Materials/methods: RAS testing was performed on plasma from patients commencing first-line therapy (OncoBEAM™ RAS CEIVD kit). Results were then compared with formalin-fixed paraffin embedded tumor samples. Results: The overall percentage agreement (concordance) was 86.0% (86/100), which demonstrates that blood-based testing is an alternative to tissue-based testing. Reproducibility was 100% between three laboratories and 20% showed changes in their RAS mutational status on progression. Conclusion: These results show good concordance between tissue and plasma samples and suggest the need for longitudinal plasma testing during treatment to guide management decisions.

scholarly journals Clinical Performance Evaluation Of The Idylla™ Egfr Mutation Test On Formalin-Fixed Paraffin-Embedded Tissue Of Non-Small Cell Lung Cancer

2020 ◽  
Author(s):  
Mercedes Delgado-Garcia ◽  
Birgit Weynand ◽  
Lourdes Gómez Izquierdo ◽  
María José Hernández Barrera ◽  
Ángela María Blanco Lobo ◽  
...  
Keyword(s):  
Egfr Mutation ◽  
Small Cell ◽  
Test Method ◽  
Egfr Mutations ◽  
Small Cell Lung ◽  
Diagnostic Agreement ◽  
Mutational Status ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract Background detection of epidermal growth factor receptor ( EGFR) mutations in exons 18-21 is recommended in all patients with advanced Non-small-cell lung carcinoma due to the demonstrated efficiency of the standard therapy with tyrosine kinase inhibitors in EGFR mutated patients. Therefore, choosing a suitable technique to test EGFR mutational status is crucial to warrant a valid result in a short turnaround time using the lowest possible amount of tissue material. The Idylla™ EGFR Mutation Test is a simple, fast and reliable method designed for the detection of EGFR mutations from formalin-fixed paraffin-embedded samples. The aim of this study was the Clinical Performace Evaluation of the Idylla™ EGFR Mutation Test on the Idylla™ System. Methods EGFR mutational status was determined on 132 archived formalin-fixed paraffin-embedded tissue sections with Idylla™ technology. Results were compared with the results previously obtained by routine method in the reference lab (Therascreen ® EGFR RGQ PCR v2, Qiagen in Molecular Pathology lab, Hospital Universitario Virgen del Rocío de Sevilla). Results the overall agreement between results obtained with the Idylla™ EGFR Mutation Test and the Comparator test method was 95.38% (with 1-sided 95% lower limit of 91.7%) showing Positive Diagnostic Agreement of 93.22% and Negative Diagnostic Agreement of 97.18%, with a Limit Of Detection ≤5%. Conclusions the Idylla™ EGFR Mutation Test passed its clinical validity performance characteristics for accuracy.

scholarly journals Clinical performance evaluation of the Idylla™ EGFR mutation test on formalin-fixed paraffin-embedded tissue of non-small cell lung cancer

2019 ◽  
Author(s):  
Mercedes Delgado-Garcia ◽  
Birgit Weynand ◽  
Lourdes Gómez Izquierdo ◽  
María José Hernández Barrera ◽  
Ángela María Blanco Lobo ◽  
...  
Keyword(s):  
Egfr Mutation ◽  
Clinical Performance ◽  
Small Cell ◽  
Egfr Mutations ◽  
Small Cell Lung ◽  
Diagnostic Agreement ◽  
Mutational Status ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract BackgroundDetection of epidermal growth factor receptor ( EGFR) mutations in exons 18-21 is recommended in all patients with advanced Non-small-cell lung carcinoma due to the demonstrated efficiency of the standard therapy tyrosine kinase inhibitors in EGFR -mutated patients. Therefore, choosing a suitable technique for testing EGFR mutational status is crucial to warrant a valid result in a short turnaround time using the lowest possible amount of tissue material. The Idylla™ EGFR Mutation Test is a simple, fast and reliable method designed for the detection of EGFR mutations from formalin-fixed paraffin-embedded sample. The aim of this study was the evaluation of the Idylla™ EGFR Mutation Test on the Idylla™ System conducting a Clinical Performance Evaluation.MethodsEGFR mutational status was determined on 132 archived formalin-fixed paraffin-embedded tissue sections with Idylla™ technology. Results were compared with the results previously obtained by routine method in the reference lab (Therascreen ® EGFR RGQ PCR v2, Qiagen in Molecular Pathology lab, Hospital Universitario Virgen del Rocío de Sevilla)ResultsThe overall agreement between results obtained with the Idylla™ EGFR Mutation Test and the Comparator test method was 95.38% (with 1-sided 95% lower limit of 91.7%) showing Positive Diagnostic Agreement of 93.22% and Negative Diagnostic Agreement of 97.18%, with a Limit Of Detection ≤5%.ConclusionsThe Idylla™ EGFR Mutation Test passed its clinical validity performance characteristics for accuracy.

Rapid EGFR evaluation from used H&E, IHC and FISH diagnostic slides with the Idylla platform

2021 ◽  
pp. jclinpath-2020-207315
Author(s):  
Josè Nunnari ◽  
Paolo Graziano ◽  
Lucia Anna Muscarella ◽  
Antonio Rossi ◽  
Lucia Rosalba Grillo ◽  
...  
Keyword(s):  
Tissue Sample ◽  
Correct Diagnosis ◽  
In Situ Hybridisation ◽  
Growth Factor Receptor ◽  
Diagnostic Process ◽  
Fluorescence In Situ Hybridisation ◽  
Small Tumour ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues

AimsDiagnostic tumour samples are mandatory for morphologic and molecular diagnosis of non-small cell lung cancer (NSCLC) to establish the best therapeutic approach. In the presence of small tumour tissue sample, the pathologist needs to make responsible choices to achieve a correct diagnosis and save material for subsequent molecular evaluations. Nevertheless, in some instances, the diagnostic process can lead to tissue depletion. The automated Idylla epidermal growth factor receptor (EGFR) mutation test has been developed to rapidly process formalin-fixed paraffin-embedded (FFPE) pathologic material, without previous DNA extraction. This study aimed to test whether this platform is suitable for the reuse of H&E, immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) diagnostic slides.MethodsA training set of 19 FFPE tissues with known EGFR status was revaluated on H&E slides. Fourteen of them were also tested using IHC and FISH treated specimens. An additional series of 25 H&E, IHC or FISH slides of NSCLC cases tested for EGFR mutation at an external institution was blindly assessed as a validation cohort.ResultsCombining the two sets, 32 of 32 classical ex19dels and p.L858R were correctly identified. Three uncommon mutations (p.G719X, p.L861Q and ex20ins) were also detected. Four discrepancies were related to rare ex19del/ins not included in the Idylla list of detectable mutations. Two p.T790M variants were missed on one FFPE and two H&E slides but were detected using IHC and FISH sections from the same FFPE blocks.ConclusionsThe Idylla EGFR mutation test is highly reliable using differently treated tumour specimens and should be validated in larger studies.

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