Allison M. Cushman-Vokoun, Solomon Connealy, Timothy C. Greiner

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Assay Design Affects the Interpretation of T-Cell Receptor Gamma Gene Rearrangements Allison M. Cushman-Vokoun, Solomon Connealy, Timothy C. Greiner The Journal of Molecular Diagnostics Volume 12, Issue 6, Pages 787-796 (November 2010) DOI: 10.2353/jmoldx.2010.090183 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic diagram of multiplex reactions used in Part 1. A: Tri-color assay with a complete multiplex reaction in one tube with three different fluorescently labeled Jγ joining region primers (Jγ1/Jγ2-NED, JγP-FAM, and JγP1/JγP2-JOE) mixed with five unlabeled Vγ variable region primers. B: One-color assay with a similar reaction as in (A); however, all three joining region primers have the same fluorochrome tag (NED). C: Partial multiplex assay is performed in which either JγP1/JγP2-NED or JγP-NED is combined with the five variable region primers in separate tubes. The Journal of Molecular Diagnostics 2010 12, 787-796DOI: (10.2353/jmoldx.2010.090183) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 A: Three representative cases of clonal rearrangements from paraffin-embedded T-cell lymphomas, using either Jγ1/Jγ2 (left panel), JγP1/P2 (biallelic, middle panel), or JγP1/P2 plus JγP (biallelic, right panel). Each reaction contained all five forward Vγ variable region primers and all three differentially labeled Jγ joining primers. B: Polyclonal peripheral blood lymphocyte DNA amplified with five forward Vγ variable region primers and labeled Jγ joining primers. Tri-color: a complete multiplex reaction using three separate fluorochrome tags on joining primers (Jγ1/Jγ2-NED-black, JγP-FAM-blue, and JγP1/JγP2-JOE-green) produces three polyclonal populations of one size range. One-color: the same multiplex reaction is performed; however, all three joining primers are labeled with the same NED fluorochrome, demonstrating a combination of all three polyclonal populations into one distribution. JγP1/P2 or JγP: DNA was amplified using five variable primers and a single joining region primer, JγP1/JγP2-NED or JγP-NED, respectively. Note the single peak in the JγP figure, which could be mistaken for a clonal rearrangement if not compared to the larger distribution of rearrangements seen in the tri-color or one-color figure. (x axis scale = bp; y axis = relative fluorescence units). The Journal of Molecular Diagnostics 2010 12, 787-796DOI: (10.2353/jmoldx.2010.090183) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Ten clinical cases amplified by three approaches. Tri-color (left column): the 10 cases are amplified with Jγ1/Jγ2-NED, JγP1/JγP2-JOE, and JγP-FAM labeled primers in a single tube labeled with three separate fluorochrome tags. Note that amplification with JγP1/2 (green) in five cases (A) or JγP (blue) in five cases (B) yields a small population of rearrangements with no peak greater than two times the height of the Jγ1/Jγ2 polyclonal background. One-color (middle column): the three NED-labeled joining primers yield a single polyclonal distribution. Separate JγP1/P2 or JγP (right column): Five variable region primers and a single fluorescent NED-labeled joining primer [JγP1/P2 (A) or JγP (B)]. Each case now shows one or two peaks that are more than two times the JγP1/P2 (A) or JγP (B) polyclonal background. The electropherograms in the right hand column would produce an interpretation of a false-positive clonal TCRγGR as a result of assay design using the same primers. (x axis scale = bp; y axis = relative fluorescence units). The Journal of Molecular Diagnostics 2010 12, 787-796DOI: (10.2353/jmoldx.2010.090183) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Representative comparison of cases analyzed with the tri-color, one-color, and the IVS TCRG Gene Clonality Assay (Tubes A and B). A: PBL, 1 μg DNA. Note pseudoclonal spikes in tubes A and B of the TCRG Gene Clonality Assay. B: Diffuse large B-cell lymphoma, Southern blot result was negative for a TRB@ gene rearrangement. Both the one-tube assays and the BIOMED-2 assay show an oligoclonal result with multiple pseudoclonal spikes present. C: Reactive lymphocytosis, Southern blot result negative for a TRB@ gene rearrangement. Whereas the one-tube assays are negative, two peaks greater than 2× RPB are identified in IVS tube A (arrows) that would be interpreted as clonal using the 2× RPB threshold, but would be interpreted as negative if a 5× RPB threshold was used. D: Peripheral T-cell lymphoma, Southern blot result positive for a TRB@ gene rearrangement. Discordant results with a biallelic Jγ1/Jγ2 and a JγP rearrangement identified in the one-tube, tri-color method, the latter not identified in the IVS System. The peak in tube B is one-fifth the intensity of the clonal peak in A and is not JγP. The Journal of Molecular Diagnostics 2010 12, 787-796DOI: (10.2353/jmoldx.2010.090183) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions