Key considerations for comprehensive validation of an RNA fusion NGS panel
Objectives: Validation of RNA-based NGS assays for the detection of therapeutically targetable gene fusions is challenging. Here, we report systematic validation and quality control monitoring of our targeted fusion panel for the detection of 17 clinically relevant fusion transcripts across several...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-08-01
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| Series: | Practical Laboratory Medicine |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2352551719301313 |
| _version_ | 1811223419126022144 |
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| author | Subit Barua Gary Wang Mahesh Mansukhani Susan Hsiao Helen Fernandes |
| author_facet | Subit Barua Gary Wang Mahesh Mansukhani Susan Hsiao Helen Fernandes |
| author_sort | Subit Barua |
| collection | DOAJ |
| description | Objectives: Validation of RNA-based NGS assays for the detection of therapeutically targetable gene fusions is challenging. Here, we report systematic validation and quality control monitoring of our targeted fusion panel for the detection of 17 clinically relevant fusion transcripts across several tumor types. We implemented this RNA Fusion Panel as a reflex test for tumors lacking DNA driver mutations. Design: Forty-four formalin-fixed, paraffin-embedded (FFPE) or fresh-frozen lung, brain, soft tissue and skin tumors were used to determine the accuracy of the assay. Additional fusion-positive specimens and a calibrated reference standard were used to establish the precision, reproducibility and sensitivity of the assay. All aspects of the validation, including quality control metrics, were performed according to New York State guidelines. Results: For the RNA fusion panel, accuracy, reproducibility and precision studies were above 99%. Reproducibility and sensitivity studies with the reference standard were helpful in identifying inconsistencies. The limit of detection for most RNA fusion transcripts was 50 copies. Application of the RNA fusion assay as a reflex test to 450 tumor samples lacking DNA driver mutations resulted in a 10% increase in diagnostic yield with minimal additional processing time. Conclusions: The validated RNA fusion panel provides clinical utility in therapy selection for patients with solid tumors. By using a sequential testing approach, the RNA fusion assay complements the DNA hotspot assay in identifying clinically relevant variants across many tumor types with minimal additional increase in processing time. |
| first_indexed | 2024-04-12T08:32:37Z |
| format | Article |
| id | doaj.art-173cadb93734432c9543f8cdc618c8d0 |
| institution | Directory Open Access Journal |
| issn | 2352-5517 |
| language | English |
| last_indexed | 2024-04-12T08:32:37Z |
| publishDate | 2020-08-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Practical Laboratory Medicine |
| spelling | doaj.art-173cadb93734432c9543f8cdc618c8d02022-12-22T03:40:08ZengElsevierPractical Laboratory Medicine2352-55172020-08-0121e00173Key considerations for comprehensive validation of an RNA fusion NGS panelSubit Barua0Gary Wang1Mahesh Mansukhani2Susan Hsiao3Helen Fernandes4Department of Pathology and Cell Biology, Columbia University Medical Center, New York, USADepartment of Pathology and Cell Biology, Columbia University Medical Center, New York, USADepartment of Pathology and Cell Biology, Columbia University Medical Center, New York, USADepartment of Pathology and Cell Biology, Columbia University Medical Center, New York, USACorresponding author.; Department of Pathology and Cell Biology, Columbia University Medical Center, New York, USAObjectives: Validation of RNA-based NGS assays for the detection of therapeutically targetable gene fusions is challenging. Here, we report systematic validation and quality control monitoring of our targeted fusion panel for the detection of 17 clinically relevant fusion transcripts across several tumor types. We implemented this RNA Fusion Panel as a reflex test for tumors lacking DNA driver mutations. Design: Forty-four formalin-fixed, paraffin-embedded (FFPE) or fresh-frozen lung, brain, soft tissue and skin tumors were used to determine the accuracy of the assay. Additional fusion-positive specimens and a calibrated reference standard were used to establish the precision, reproducibility and sensitivity of the assay. All aspects of the validation, including quality control metrics, were performed according to New York State guidelines. Results: For the RNA fusion panel, accuracy, reproducibility and precision studies were above 99%. Reproducibility and sensitivity studies with the reference standard were helpful in identifying inconsistencies. The limit of detection for most RNA fusion transcripts was 50 copies. Application of the RNA fusion assay as a reflex test to 450 tumor samples lacking DNA driver mutations resulted in a 10% increase in diagnostic yield with minimal additional processing time. Conclusions: The validated RNA fusion panel provides clinical utility in therapy selection for patients with solid tumors. By using a sequential testing approach, the RNA fusion assay complements the DNA hotspot assay in identifying clinically relevant variants across many tumor types with minimal additional increase in processing time.http://www.sciencedirect.com/science/article/pii/S2352551719301313Next generation sequencingPrecision medicineMolecular oncologyAssayValidationRNA Fusions |
| spellingShingle | Subit Barua Gary Wang Mahesh Mansukhani Susan Hsiao Helen Fernandes Key considerations for comprehensive validation of an RNA fusion NGS panel Practical Laboratory Medicine Next generation sequencing Precision medicine Molecular oncology Assay Validation RNA Fusions |
| title | Key considerations for comprehensive validation of an RNA fusion NGS panel |
| title_full | Key considerations for comprehensive validation of an RNA fusion NGS panel |
| title_fullStr | Key considerations for comprehensive validation of an RNA fusion NGS panel |
| title_full_unstemmed | Key considerations for comprehensive validation of an RNA fusion NGS panel |
| title_short | Key considerations for comprehensive validation of an RNA fusion NGS panel |
| title_sort | key considerations for comprehensive validation of an rna fusion ngs panel |
| topic | Next generation sequencing Precision medicine Molecular oncology Assay Validation RNA Fusions |
| url | http://www.sciencedirect.com/science/article/pii/S2352551719301313 |
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