Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study
Background: Multiplex PCR based on consensus primers followed by capillary electrophoresis and Sanger sequencing are considered as the gold standard method for the evaluation of clonality and somatic hypermutation in lymphoid malignancies. As an alternative, the next-generation sequencing (NGS) of i...
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MDPI AG
2023-09-01
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| Series: | Cancers |
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| Online Access: | https://www.mdpi.com/2072-6694/15/18/4624 |
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| author | Anna Gazzola Mohsen Navari Claudia Mannu Riccardo Donelli Maryam Etebari Pier Paolo Piccaluga |
| author_facet | Anna Gazzola Mohsen Navari Claudia Mannu Riccardo Donelli Maryam Etebari Pier Paolo Piccaluga |
| author_sort | Anna Gazzola |
| collection | DOAJ |
| description | Background: Multiplex PCR based on consensus primers followed by capillary electrophoresis and Sanger sequencing are considered as the gold standard method for the evaluation of clonality and somatic hypermutation in lymphoid malignancies. As an alternative, the next-generation sequencing (NGS) of immune receptor genes has recently been proposed as a solution, due to being highly effective and sensitive. Here, we designed a phase III diagnostic accuracy study intended to compare the current gold standard methods versus the first commercially available NGS approaches for testing immunoglobulin heavy chain gene rearrangements. Methods: We assessed IGH rearrangements in 68 samples by means of both the NGS approach (LymphoTrack<sup>®</sup> IGH assay, and LymphoTrack<sup>®</sup> IGH somatic hypermutation assay, run on Illumina MiSeq) and capillary electrophoresis/Sanger sequencing to assess clonality and somatic hypermutations (SHM). Results: In comparison to the routine capillary-based analysis, the NGS clonality assay had an overall diagnostic accuracy of 96% (63/66 cases). Other studied criteria included sensitivity (95%), specificity (100%), positive predictive value (100%) and negative predictive value (75%). In discrepant cases, the NGS results were confirmed by a different set of primers that provided coverage of the IGH leader sequence. Furthermore, there was excellent agreement of the SHM determination with both the LymphoTrack<sup>®</sup> FR1 and leader assays when compared to the Sanger sequencing analysis (84%), with NGS able to assess the SHM rate even in cases where the conventional approach failed. Conclusion: Overall, conventional Sanger sequencing and next-generation-sequencing-based clonality and somatic hypermutation analyses gave comparable results. For future use in a routine diagnostic workflow, NGS-based approaches should be evaluated prospectively and an analysis of cost-effectiveness should be performed. |
| first_indexed | 2024-03-10T22:57:10Z |
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| language | English |
| last_indexed | 2024-03-10T22:57:10Z |
| publishDate | 2023-09-01 |
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| series | Cancers |
| spelling | doaj.art-461c3d27fb444c88881c62e1324257ef2023-11-19T09:56:24ZengMDPI AGCancers2072-66942023-09-011518462410.3390/cancers15184624Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy StudyAnna Gazzola0Mohsen Navari1Claudia Mannu2Riccardo Donelli3Maryam Etebari4Pier Paolo Piccaluga5Hematopathology Unit, IRCCS Azienda Opedaliera-Universitaria di Bologna S. Orsola-Malpighi, 40138 Bologna, ItalyDepartment of Medical Biotechnology, School of Paramedical Sciences, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh 95196-33787, IranHematopathology Unit, IRCCS Azienda Opedaliera-Universitaria di Bologna S. Orsola-Malpighi, 40138 Bologna, ItalyBiobank of Research, IRCCS Azienda Opedaliera-Universitaria di Bologna, 40138 Bologna, ItalyHealth Sciences Research Center, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh 33787-95196, IranBiobank of Research, IRCCS Azienda Opedaliera-Universitaria di Bologna, 40138 Bologna, ItalyBackground: Multiplex PCR based on consensus primers followed by capillary electrophoresis and Sanger sequencing are considered as the gold standard method for the evaluation of clonality and somatic hypermutation in lymphoid malignancies. As an alternative, the next-generation sequencing (NGS) of immune receptor genes has recently been proposed as a solution, due to being highly effective and sensitive. Here, we designed a phase III diagnostic accuracy study intended to compare the current gold standard methods versus the first commercially available NGS approaches for testing immunoglobulin heavy chain gene rearrangements. Methods: We assessed IGH rearrangements in 68 samples by means of both the NGS approach (LymphoTrack<sup>®</sup> IGH assay, and LymphoTrack<sup>®</sup> IGH somatic hypermutation assay, run on Illumina MiSeq) and capillary electrophoresis/Sanger sequencing to assess clonality and somatic hypermutations (SHM). Results: In comparison to the routine capillary-based analysis, the NGS clonality assay had an overall diagnostic accuracy of 96% (63/66 cases). Other studied criteria included sensitivity (95%), specificity (100%), positive predictive value (100%) and negative predictive value (75%). In discrepant cases, the NGS results were confirmed by a different set of primers that provided coverage of the IGH leader sequence. Furthermore, there was excellent agreement of the SHM determination with both the LymphoTrack<sup>®</sup> FR1 and leader assays when compared to the Sanger sequencing analysis (84%), with NGS able to assess the SHM rate even in cases where the conventional approach failed. Conclusion: Overall, conventional Sanger sequencing and next-generation-sequencing-based clonality and somatic hypermutation analyses gave comparable results. For future use in a routine diagnostic workflow, NGS-based approaches should be evaluated prospectively and an analysis of cost-effectiveness should be performed.https://www.mdpi.com/2072-6694/15/18/4624clonalityimmunoglobulin heavy chainBIOMED2lymphomaleukemiaevidence-based medicine |
| spellingShingle | Anna Gazzola Mohsen Navari Claudia Mannu Riccardo Donelli Maryam Etebari Pier Paolo Piccaluga Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study Cancers clonality immunoglobulin heavy chain BIOMED2 lymphoma leukemia evidence-based medicine |
| title | Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study |
| title_full | Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study |
| title_fullStr | Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study |
| title_full_unstemmed | Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study |
| title_short | Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study |
| title_sort | single step ighv next generation sequencing detects clonality and somatic hypermutation in lymphoid malignancies a phase iii diagnostic accuracy study |
| topic | clonality immunoglobulin heavy chain BIOMED2 lymphoma leukemia evidence-based medicine |
| url | https://www.mdpi.com/2072-6694/15/18/4624 |
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