{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T02:16:39Z","timestamp":1784168199919,"version":"3.55.0"},"reference-count":35,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2023,9,19]],"date-time":"2023-09-19T00:00:00Z","timestamp":1695081600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Italian Ministry of Health","award":["RC-2023- 2778976"],"award-info":[{"award-number":["RC-2023- 2778976"]}]},{"name":"Italian Ministry of Health","award":["RBFR12D1CB"],"award-info":[{"award-number":["RBFR12D1CB"]}]},{"name":"FIRB Futura","award":["RC-2023- 2778976"],"award-info":[{"award-number":["RC-2023- 2778976"]}]},{"name":"FIRB Futura","award":["RBFR12D1CB"],"award-info":[{"award-number":["RBFR12D1CB"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>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\u00ae IGH assay, and LymphoTrack\u00ae 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\u00ae 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.<\/jats:p>","DOI":"10.3390\/cancers15184624","type":"journal-article","created":{"date-parts":[[2023,9,19]],"date-time":"2023-09-19T02:47:52Z","timestamp":1695091672000},"page":"4624","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Single-Step IGHV Next-Generation Sequencing Detects Clonality and Somatic Hypermutation in Lymphoid Malignancies: A Phase III Diagnostic Accuracy Study"],"prefix":"10.3390","volume":"15","author":[{"given":"Anna","family":"Gazzola","sequence":"first","affiliation":[{"name":"Hematopathology Unit, IRCCS Azienda Opedaliera-Universitaria di Bologna S. Orsola-Malpighi, 40138 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohsen","family":"Navari","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnology, School of Paramedical Sciences, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh 95196-33787, Iran"},{"name":"Research Center of Advanced Technologies in Medicine, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh 95196-33787, Iran"},{"name":"Bioinformatics Research Center, Mashhad University of Medical Sciences, Mashhad 91779-48564, Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claudia","family":"Mannu","sequence":"additional","affiliation":[{"name":"Hematopathology Unit, IRCCS Azienda Opedaliera-Universitaria di Bologna S. Orsola-Malpighi, 40138 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Riccardo","family":"Donelli","sequence":"additional","affiliation":[{"name":"Biobank of Research, IRCCS Azienda Opedaliera-Universitaria di Bologna,  40138 Bologna, Italy"},{"name":"Department of Medical and Surgical Sciences, Institute of Hematology and Medical Oncology \u201cL&A Ser\u00e0gnoli\u201d, Bologna University School of Medicine, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maryam","family":"Etebari","sequence":"additional","affiliation":[{"name":"Health Sciences Research Center, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh 33787-95196, Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9432-0595","authenticated-orcid":false,"given":"Pier Paolo","family":"Piccaluga","sequence":"additional","affiliation":[{"name":"Biobank of Research, IRCCS Azienda Opedaliera-Universitaria di Bologna,  40138 Bologna, Italy"},{"name":"Department of Medical and Surgical Sciences, Institute of Hematology and Medical Oncology \u201cL&A Ser\u00e0gnoli\u201d, Bologna University School of Medicine, 40126 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,9,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2192","DOI":"10.1038\/sj.leu.2400887","article-title":"Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations","volume":"11","author":"Langerak","year":"1997","journal-title":"Leukemia"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2257","DOI":"10.1038\/sj.leu.2403202","article-title":"Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936","volume":"17","author":"Langerak","year":"2003","journal-title":"Leukemia"},{"key":"ref_3","first-page":"167","article-title":"Use of IGK gene rearrangement analysis for clonality assessment of lymphoid malignancies: A single center experience","volume":"1","author":"Mannu","year":"2011","journal-title":"Am. 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