TT45 Detection of Clonal TRG and TRB Rearrangements Using Next-Generation Sequencing Presha Shah1, Ying Huang1, Kasey Hutt1, Jeff Panganiban1, Edgar Vigil1, Roshanak Bob2 ,Mark D. Ewalt3, James L. Zehnder4, Tim Stenzel1, and Jeffrey E. Miller1 1Invivoscribe Technologies, Inc., San Diego, CA USA, 2Institute for Pathodiagnostik Berlin, Germany, 3University of Colorado, Denver, CO USA, 4Department of Pathology, Stanford University Medical Center, Stanford, CA USA

Background Results: TRG /TRB LoD, LoB and Linearity Results: TRG/TRB Positive Clinical Sample

T-cell malignancies arise from transformation and clonal expansion of a single cell. During T-cell CE-0041 CE-0041 MiSeq® TRG MiSeq® TRB LymphoTrack TRG Assay - V – J Sequence Frequencies LymphoTrack TRB Assay - V – J Sequence Frequencies 2 50 development, the gamma (TRG) locus rearranges prior to T cell receptor beta (TRB) locus. Combo R =0.9924 Combo R2=0.9792 60 Single R2=0.9923 Combined, TRG and TRB assays can identify the vast majority of T-cell rearrangements in T-cell as well as Single R2=0.9789 45 50 some B-cell malignancies (e.g., B-ALL). Historically, clonal rearrangements have been identified using 40 35 capillary electrophoresis (CE) methods, which provide size distribution information, but not the sequence 40 30 needed for tracking minimal residual disease (MRD) throughout the course of treatment. Recently, we have 25 30 developed next-generation sequencing (NGS) assays for that improve both the 20 % Total Reads % Total Reads 20 sensitivity of clonality detection and identify the specific V(D)J DNA sequences required to track clones in 15 10 follow-up MRD testing. Here we present data from the LymphoTrack® TRG and TRB Clonality Assays 10 5 developed forthe Illumina®MiSeq® platform and compare results with the IdentiClone® CE-based assays. 0 0 JgP JgP JgP JgP JgP JgP JgP JgP Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 Jb1 Jb2 ------JgP1 JgP2 JgP1 JgP2 JgP1 JgP2 JgP1 JgP2 JgP1 JgP2 JgP1 JgP2 ------JgP1 JgP2 JgP1 JgP2 none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none none ------Jg1/2 Jg1/2 Jg1/2 Jg1/2 Jg1/2 Jg1/2 Jg1/2 Jg1/2 ------Vg2 Vg3 Vg4 Vg5 Vg8 Vg9 Vb2 Vb2 Vb3 Vb3 Vb4 Vb4 Vb5 Vb5 Vb6 Vb6 Vb7 Vb7 Vb9 Vb9 Db1 Db1 Db2 Db2 Vg2 Vg2 Vg3 Vg3 Vg4 Vg4 Vg5 Vg5 Vg8 Vg8 Vg9 Vg9 Vg10 Vg11 none none Vg2 Vg3 Vg4 Vg5 Vg8 Vg9 Vb10 Vb10 Vb11 Vb11 Vb12 Vb12 Vb13 Vb13 Vb14 Vb14 Vb15 Vb15 Vb16 Vb16 Vb17 Vb17 Vb18 Vb18 Vb19 Vb19 Vb20 Vb20 Vb21 Vb21 Vb23 Vb23 Vb24 Vb24 Vb25 Vb25 Vb27 Vb27 Vb28 Vb28 Vb29 Vb29 Vb30 Vb30 Vb2 Vb3 Vb4 Vb5 Vb6 Vb7 Vb9 Db1 Db2 Vg2 Vg3 Vg4 Vg5 Vg8 Vg9 Vg10 Vg10 Vg11 Vg11 Vg10 Vg11 none Vb10 Vb11 Vb12 Vb13 Vb14 Vb15 Vb16 Vb17 Vb18 Vb19 Vb20 Vb21 Vb23 Vb24 Vb25 Vb27 Vb28 Vb29 Vb30 Material and Methods Results: TRG/TRB Precision and Reproducibility Vg10 Vg11 TRG Positive (Clonal) TRB Positive (Clonal) • Schematic Illustration of the TRG gene TRG MiSeq® TRB MiSeq® Results: Clinical Study between TRB MiSeq® and IdentiClone® Assays Clonal Control Size Mean Size Mean Dilutions CV% CV% • Schematic Illustration of the TRB gene (bp) % Reads (bp) % Reads TRB MiSeq® vs. TRB IdentiClone® (Tube A/B/C) (%) TRB Identiclone Concordance (%) 86.4 10 147 8.66 10.4 198 17.63 16.7 n=44 Clonal Non-Clonal Sensitivity (%) 75.0 • Workflow for the LymphoTrack® TRG & TRB Assays - MiSeq® 5 147 4.59 12.0 198 9.35 18.4 Clonal 18 0 2.5 147 2.39 11.7 198 5.25 19.6 Specificity (%) 100.0 Purify Quantify Pool Run Analyze TRB PCR 1 147 0.94 13.8 198 2.04 20.0 MiSeq ® PPV (%) 100.0 Amplicons Amplicons Library MiSeq® Data Non-Clonal 6 20 0 varies 0.62 40.3 varies 0.17 46.8 NPV (%) 76.9 • The LymphoTrack® TRG and TRB Assays for the MiSeq® were cGMP manufactured, QC tested, and analyzed using LymphoTrack® bioinformatics software developed under full design control consistent Results: TRG/TRB MRD Detection Results: Clinical Study Comparing TRG MiSeq® and IdentiClone® Assays with ourISO13485-compliant regulatory system. • Limit of detection (LoD), linearity, precision and reproducibility (P/R) were tested using clonal control LymphoTrack® TRG Assay-MiSeq® DNA diluted in wild-type polyclonal(tonsil)DNA forTRB assay. TRG MiSeq® vs. TRG IdentiClone® 2.0 • DNA from 60 formalin-fixed paraffin-embedded (FFPE) samples were extracted using common LT TRG: Contrived Cell Line Dilutions TRG Identiclone 2.0 1.E-01 n=51 Concordance (%) 94.1 extraction methods bycollaborators. Allsamples were tested with TRG and TRB Assays . Clonal Non-Clonal Sensitivity (%) 87.5 • Contrived cell line was used for MRD detection at 10-2, 10-3, 10-4, 10-5 and 10-6 sensitivity with an internal 1.E-02 R² = 0.9993 Clonal 14 1 TRG Specificity (%) 97.1 control spiked in to reactions at approximately1000 cell equivalence. 1.E-03 MiSeq ® PPV (%) 93.3 • Libraries were prepared with amplicons generated by PCR using proprietary multiplex master mixes with Contrived Cell Line Non-Clonal 2 34 the consensus primers targeting all TRG and TRB V, D, and J exon fa``milies, synthesized with MiSeq® 1.E-04 Control Cell Line NPV (%) 94.4 Measured %reads specific adapters and 24 index sequences optimizedforthe NGS platform. 1.E-05

• Libraries were either sequenced for TRB and TRG individuallyorforTRB + TRG combined. 1.E-06 Results: Clinical Study for LymphoTrack® TRG + TRB Assays - MiSeq® ® 1.E-01 1.E-02 1.E-03 1.E-04 1.E-05 1.E-06 • LymphoTrack Software - MiSeq® was used to analyze FASTQ data generated from the MiSeq®. Clonal DNA dilutions • When comparing testing results, only samples that met the specimen and data acceptance criteria for TRB MiSeq® TRG MiSeq® TRB + TRG MiSeq® both methods were evaluated LymphoTrack® TRB Assay-MiSeq® • All statistical analyseswere performed in JMP®. LT TRB: Contrived Cell Line Dilutions Clonal 19/60 (32%) 16/60 (27%) 20/60 (33%) Conclusion 1.E-01 Non-Clonal 41/60 (68%) 43/60 (73%) 39/60 (65%) • The LymphoTrack® MiSeq® TRB assay demonstrated excellent linearity (R2>0.90), sensitivity (as low as 1.E-02 2.5%), and reproducibility(<20%CV) testingserial dilutionsofcontrived cell line DNA. R² = 0.9956 1.E-03 • Concordancebetween the LymphoTrack® TRB - MiSeq® and IdentiClone® TRB assays was 86.4%. TRB +TRG IdentiClone® TRB + TRG MiSeq® vs. IdentiClone® Contrived Cell Line n=44 • Concordancebetween the LymphoTrack® TRG - MiSeq® and IdentiClone® TRG assays was 94.1% 1.E-04 Control Cell Line Clonal Non-Clonal Concordance (%) 84.1 • Concordance between the combined TRB+TRG LymphoTrack® MiSeq® and IdentiClone® assays was Measured %reads Sensitivity (%) 72.0 1.E-05 Clonal 18 0 84.1% Specificity (%) 2 TRB + TRG 100.0 • Excellent linearity (R >0.90) for contrived cell line, reproducibility (<20% CV) for control cell line, and 1.E-06 MiSeq ® PPV (%) 100.0 clonal detection for MRD at 10-5 sensitivitywas demonstrated for LymphoTrack® MiSeq® TRG and TRB 1.E-01 1.E-02 1.E-03 1.E-04 1.E-05 1.E-06 Non-Clonal 7 19 Clonal DNA dilutions NPV (%) 73.1 assays.

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AMP, November 16 –18 2017, Salt Lake City, USA