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Evaluation of next‐generation sequencing‐based clonality analysis of T‐cell receptor gamma gene rearrangements based on a new interpretation algorithm
Author(s) -
Nollet Friedel,
Vanhouteghem Karlien,
Vermeire Stefanie,
Maelbrancke Ellen,
Emmerechts Jan,
Devos Helena,
Cauwelier Barbara
Publication year - 2019
Publication title -
international journal of laboratory hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.705
H-Index - 55
eISSN - 1751-553X
pISSN - 1751-5521
DOI - 10.1111/ijlh.12954
Subject(s) - amplicon , polymerase chain reaction , dna sequencing , amplicon sequencing , reproducibility , gene rearrangement , microbiology and biotechnology , biology , gene , medicine , chemistry , genetics , chromatography , 16s ribosomal rna
T‐cell receptor gene (TRG) rearrangement profiling is an essential component of the workup at diagnosis of T‐cell malignancies. TRG amplification by polymerase chain reaction (PCR) and analysis by capillary electrophoresis (PCR‐CE) is mostly widely used but is hampered by a subjective interpretation of its results and possible false‐positive interpretation of clonality. Several studies evaluated the advantage of TRG rearrangement analysis by Next Generation Sequencing (TRG‐NGS), however few have proposed an adequate data interpretation algorithm. Methods Eighty five fresh and 36 formalin‐fixed paraffin embedded (FFPE) diagnostic samples suspected for a lymphoproliferative disorder were analyzed by PCR‐CE and TRG NGS. Final clinical diagnosis was available for all fresh samples. Reproducibility, analytical specificity and sensitivity of the TRG NGS analysis was evaluated. Results We propose a new interpretation algorithm for TRG NGS data analysis. PCR‐CE and TRG NGS showed identical results in 66/85 (78%) of fresh samples. Sensitivities to detect T‐cell malignancies were comparable (96% versus 92%, respectively). The analysis of FFPE material was significantly more successful by TRG NGS (34/36 cases) in respect to PCR‐CE (16/36 cases), most likely due to the small size of the amplicons. Conclusion Assessment of T‐cell clonality by TRG NGS has a significant added value in the diagnosis of T‐cell disorders as an adjunct to PCR‐CE, particularly in difficult to interpret cases or when analyzing FFPE samples.