Transitioning T-Cell Clonality Testing to High-Throughput Sequencing

The laboratory community continues to work on the implementation of high-throughput sequencing (HTS) for use in analyzing T-cell clonality at level receptor γ gene (TRG) rearrangements, and latest installment is validation study reported this issue Journal Molecular Diagnostics.1Ho C.C. Tung J.K. Zehnder J.L. Zhang B.M. Validation a next-generation sequencing-based gamma rearrangement diagnostic assay: transitioning from capillary electrophoresis sequencing.J Mol Diagn. 2021; 23: 805-815Abstract Full Text PDF PubMed Scopus (3) Google Scholar To best our knowledge, first clinical-validation TRG-based by HTS, was published 2014 Schumacher et al.2Schumacher J.A. Duncavage E.J. Mosbruger T.L. Szankasi P.M. Kelley T.W. A comparison deep TCRG rearrangements vs traditional assessment lymphoproliferative disorders.Am J Clin Pathol. 2014; 141: 348-359Crossref (36) They compared an HTS assay with conventional electrophoresis.2Schumacher commentary one authors that time listed issues should be included future validations assays clonality3Greiner T.C. Clinical TRG has arrived.Am 302-304Crossref Scholar: proportional representation primers all clinically important genes, equal amplification efficiency primers, using software pipeline accurately identifies variable joining defining criteria clonal result.3Greiner In 2014, al used primers,4Greiner Rubocki R.J. Effectiveness florescent-labeled primer detecting rearrangements.J 2002; 4: 137-143Abstract (52) added adaptors, performed two instruments, Ion PGM System (Thermo Fisher Scientific, Waltham, MA) Illumina MiSeq instrument (Illumina, San Diego, CA).2Schumacher By contrast, Ho al1Ho proprietary LymphoTrack kit (Invivoscribe, CA) instrument. established coverage relevant genes4Greiner their analysis 48 samples, them (CE) same set.2Schumacher set BIOMED CE did not include JγP primer, which different method contains complete including JγP. This identified 2014,3Greiner stated so comparisons can head head. need set, JγP, avoid false-negative results been previously established.5Lawnicki L.C. Chan W.C. Lytle D.M. Greiner distribution segments [gamma] demonstrates multiple sets.J 2003; 5: 82-87Abstract (51) studied 101 specimens, exceeded minimum 59 specimens recommended guideline bioinformatics Association Pathology.6Jennings L.J. Arcila M.E. Corless C. Kamel-Reid S. Lubin I.M. Pfeifer J. Temple-Smolkin R.L. Voelkerding K.V. Nikiforova M.N. Guidelines oncology panels.J 2017; 19: 341-365Abstract (317) al2Schumacher only clinical specimens. From biology perspective, least 50 required detection rare utilizing gene, frequency rate 3%, Vγ11 4%.5Lawnicki Pathology College American Pathologists recommend laboratories validate HTS.7Roy Coldren Karumamurthy A. Kip N.S. Klee E.W. Lincoln S.E. Leon Pullambhatla M. Wang Carter A.B. Standards guidelines validating pipelines: joint recommendation association molecular pathology Pathologists.J 2018; 20: 4-27Abstract (192) It challenging second independently custom-designed software, like TCRDriver package,2Schumacher or system,1Ho verify accuracy region identification. As shown al,1Ho numerous advantages over CE. These requirement lesser amounts DNA, lower false positives, increased objectivity. also found peaks identical size—defined as number base pairs amplified products, detected CE—do always imply identity sequence level, reported.8Rea B. Haun P. Emerson R. Vignali Farooqi Samimi Elenitsas Kirsch I. Bagg Role diagnosis cutaneous lymphoma.J 71: 814-820Crossref (19) One other concept article some discordant between CE- HTS-based approaches appeared result false-positive findings due lack background competition two-tube system. assay, competitive spread out distributions, whereas one-tube compete against each amplification.9Cushman-Vokoun A.M. Connealy Assay design affects interpretation rearrangements: performance BIOMED-2-based assay.J 2010; 12: 787-796Abstract (25) (partly) corroborated samples tested al.1Ho are more difficult interpret than those assay. precludes evaluation isolated fluorescence signals distribution, case observations reinforce move away multitube systems target Cushman-Vokoun 9Cushman-Vokoun recently discussed Armand al.10Armand Derrieux Beldjord K. Wabeke T. Lenze D. Boone E. Bruggemann Evans P.A.S. Gameiro Hummel Villarese Groenen P.J.T.A. Langerak A.W. Macintyre E.A. Davi F. new simple multiplex PCR clonality: comparative EuroClonality Consortium.Hemasphere. 2019; 3: e255Crossref (6) system preferred described single-tube system, single calculation result. Whether read percentage (minimum 4.5%) four-fold higher polyclonal background2Schumacher 2.5%) five-fold fourth most abundant clonotype appropriate remains determined.1Ho progress adoption slow reasons, relatively long turnaround 3 7 days cost Therefore, continue However, shorter run times enabled costs come down, will become CE, leading continued growth HTS. Additionally, becoming central workflow such somatic variant detection, natural transition Ig/T-cell through combined runs. New have flexible prior incantations, may facilitate transition. For both TRB several companies collaborative groups developed kits methods applicable DNA and/or RNA. Some amplicons short enough sequenced paraffin-embedded tissue, studies.1Ho Scholar,2Schumacher Comparisons these provide useful data community. genetic would benefit IG scientifically economically sound framework. while still quite useful, limitations being gold standard comparison. questions remain resolved. Examples include: i) Is adequate, duplicate, many do now obtain reproducible results?; ii) What percent unique define result?; iii) role resolving ambiguous results? Recognizing need, NGS Utility Assessment T/B-cell Clonality working group, chaired Dr. David Viswanatha, address issues. parallel, analogous CE,11Langerak P.J. Brüggemann Bellan Bonello L. G.I. Catherwood Delfau-Larue M.H. Diss P.A. Garcia Sanz Gonzalez Grand Håkansson Liu H. Lombardia Milner B.J. Montes-Moreno Schuuring Spaargaren Hodges van Dongen J.J. EuroClonality/BIOMED-2 reporting Ig/TCR testing suspected lymphoproliferations.Leukemia. 2012; 26: 2159-2171Crossref (290) EuroClonality-NGS group (Dr. Langerak) developing testing, based multicenter studies, Ig biological (Van den Brand, unpublished data). Diagnostics readership encouraged look upcoming documents. Next-Generation Sequencing–Based T-Cell Receptor Gamma Gene Rearrangement Diagnostic Assay: Transitioning Capillary Electrophoresis SequencingThe DiagnosticsVol. 23Issue 7PreviewAssessment importants consideration workup diseases. Although fragment current laboratories, it does information semi-quantitative. Next-generation (NGS)-based attractive alternative size–based methods, given they generate specific allow accurate quantitation. Full-Text CorrectionThe 9PreviewThere error title Commentary entitled, “Transitioning Testing High-Throughput Sequencing” (Volume 23, pages 781–783 July 2021 Diagnostics; DOI:https://doi.org/10.1016/j.jmoldx.2021.05.005). Due during proof correction process, edit requested mistakenly overlooked. correct is: Sequencing”, word “From” removed.