POL Scientific / JBM / Volume 11 / Issue 2 / DOI: 10.14440/jbm.2024.0002
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RESEARCH ARTICLE

Conventional PCR-based versus next-generation sequencing-based approach for T-cell receptor γ gene clonality assessment in mature T-cell lymphomas: A phase 3 diagnostic accuracy study

Riccardo Donelli1,2 ,  Anna Gazzola3 ,  Claudia Mannu3 ,  Maryam Etebari4 ,  Mohsen Navari5,6 ,  Pier Paolo Piccaluga1,2*
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1 Biobank of Research, IRCCS Azienda Opedaliera-Universitaria di Bologna, Bologna, Italy
2 Department of Medical and Surgical Sciences, Bologna University School of Medicine, Bologna, Italy
3 Hematopathology Unit, IRCCS Azienda Opedaliera-Universitaria di Bologna, Bologna, Italy
4 Health Sciences Research Center, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran
5 Department of Medical Biotechnology, School of Paramedical Sciences, Torbat Heydariyeh University of Medical Sciences, Torbat Heydariyeh, Iran
6 Research Center of Advanced Technologies in Medicine, Torbat Heydariyeh University of Medical Scienc-es, Torbat Heydariyeh, Iran
JBM 2024 , 11(2), e99010013; https://doi.org/10.14440/jbm.2024.0002
Submitted: 8 May 2024 | Revised: 13 June 2024 | Accepted: 20 June 2024 | Published: 10 July 2024
© 2024 by the Journal of Biological Methods published by POL Scientific. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

Background: Clonality assessment is currently the major molecular analysis utilized to support the diagnosis of suspicious lymphoid malignancies. Clonal rearrangements of the V-J segments of T-cell receptor G chain locus (TCRγ or TRG) have been observed in almost all types of T neoplasms, such as T-cell-related non-Hodgkin lymphomas and leukemias. At present, the gold standard for clonality evaluation is multiplex polymerase chain reaction (PCR), plus subsequent capillary electrophoresis/heteroduplex analyses, and/or Sanger sequencing. This approach overcomes the problem with the conventional Southern blot hybridization and is more efficient, simple, fast, and reproducible. In the recent years, the new next-generation sequencing (NGS) technologies provided alternative techniques for the analysis of antigen receptors genes, which presented several advantages, such as increased efficiency, specificity (SP), sensitivity (ST), resolution, and objectivity of the results, leading to a better classification, stratification, and monitoring of lymphoid malignancies. Nonetheless, these technologies are still far from being the new gold standard since further studies are warranted to prove their utility. The present study aimed to assess the diagnostic accuracy of these two methods by comparing a commercial NGS-based assay for the evaluation of TRG locus with the gold standard PCR-based one, to fulfill the requirements of a phase 3 diagnostic accuracy study. Methods: We assessed the TRG gene rearrangements in 72 cases using the conventional and highly-validated PCR-based assay proposed by EuroClonality consortium, an alternative commercial PCR-based assay, namely, IdentiClone® TCR Gamma Gene Rearrangement Assay 2.0, and a commercial NGS-based assay, that is, Invivoscribe LymphoTrack® Dx MiSeq® (both by Invivoscribe Technologies Inc., San Diego, CA, USA), to determine the diagnostic accuracy of the latter, and compare them with reference diagnoses made based on observation of clinical manifestations, cytohistological, and immunohistochemical analyses. Statistical values were calculated using the Oxford CATmaker software package. Results: Using standardized criteria of interpretation, the obtained results showed a diagnostic accuracy of 90.3% (correspondence in 65 out of 72 cases) of the test under investigation, with a ST of 86%, a SP of 95%, a positive predicting value of 94%, and a negative predicting value of 88%, demonstrating that Invivoscribe LymphoTrack® Dx MiSeq® assay had high efficiency and reliability in detecting clonal TRG gene rearrangements in T-cell non-Hodgkin lymphomas. Conclusions: This diagnostic accuracy study yielded comparable results using a validated PCR-based approach and a new NGS-based one. Subsequent studies and cost-effectiveness evaluation are needed to put the NGS-based clonality assessment into routine diagnostic practice.

Keywords
Clonality
T-cell receptor gamma
BIOMED2
PTCL
Lymphoma
Leukemia
Evidence-based medicine
Diagnostic accuracy
Polymerase chain reaction
Next-generation sequencing
LymphoTrack® TRG Assay
Funding
The work reported in this publication was funded by the Italian Ministry of Health, RC-2023- 2778976 (Prof. Piccaluga) and FIRB Futura 2011 RBFR12D1CB.
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Conflict of interest
The funding agency had no role in the design of the study, the collection, analyses or interpretation of data, the writing of the manuscript, or the decision to publish the results. The authors have no conflicts of interest to disclose.
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Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific
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