TT45

Detection of Clonal TRG and TRB Gene 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 MedicalCenter, Stanford, CA USA

BackgroundResults: TRG /TRB LoD, LoB and Linearity

T-cell malignancies arise from transformation and clonal expansion of a single cell. During T-celldevelopment, the T cell receptor gamma (TRG) locus rearranges prior to T cell receptor beta (TRB) locus.
Combined, TRG and TRB assays can identify the vast majority of T-cell rearrangements in T-cell as well assome B-cell malignancies (e.g., B-ALL). Historically, clonal rearrangements have been identified using
capillary electrophoresis (CE) methods, which provide size distribution information, but not the sequenceneeded for tracking minimal residual disease (MRD) throughout the course of treatment. Recently, we havedeveloped next-generation sequencing (NGS) assays for immune receptor genes that improve both thesensitivity of clonality detection and identify the specific V(D)J DNA sequences required to track clones infollow-up MRD testing. Here we present data from the LymphoTrack® TRG and TRB Clonality Assays
developed for the Illumina® MiSeq® platform and compare results with the IdentiClone® CE-based assays.

CE-0041

MiSeq® TRG
Combo R2 =0.9924
Single R2 =0.9923

MiSeq® TRB
Combo R2 =0.9792
Single R2 =0.9789•Pool
Library% Total Reads% Total Reads

2520403020100

Vb2-Jb1
Vb2-Jb2
Vb2-none
Vb3-Jb1
Vb3-Jb2
Vb3-none
Vb4-Jb1
Vb4-Jb2
Vb4-none
Vb5-Jb1
Vb5-Jb2
Vb5-none
Vb6-Jb1
Vb6-Jb2
Vb6-none
Vb7-Jb1
Vb7-Jb2
Vb7-none
Vb9-Jb1
Vb9-Jb2
Vb9-none
Vb10-Jb1
Vb10-Jb2
Vb10-none
Vb11-Jb1
Vb11-Jb2
Vb11-none
Vb12-Jb1
Vb12-Jb2
Vb12-none
Vb13-Jb1
Vb13-Jb2
Vb13-none
Vb14-Jb1
Vb14-Jb2
Vb14-none
Vb15-Jb1Vb15-Jb2
Vb15-none
Vb16-Jb1
Vb16-Jb2
Vb16-none
Vb17-Jb1
Vb17-Jb2
Vb17-none
Vb18-Jb1
Vb18-Jb2
Vb18-none
Vb19-Jb1
Vb19-Jb2
Vb19-none
Vb20-Jb1
Vb20-Jb2
Vb20-none
Vb21-Jb1
Vb21-Jb2
Vb21-none
Vb23-Jb1
Vb23-Jb2
Vb23-none
Vb24-Jb1
Vb24-Jb2
Vb24-none
Vb25-Jb1
Vb25-Jb2
Vb25-none
Vb27-Jb1
Vb27-Jb2
Vb27-none
Vb28-Jb1
Vb28-Jb2
Vb28-none
Vb29-Jb1
Vb29-Jb2
Vb29-none
Vb30-Jb1
Vb30-Jb2
Vb30-none
Db1-Jb1
Db1-Jb2
Db1-none
Db2-Jb1
Db2-Jb2
Db2-nonenone-Jb1
none-Jb2
none-none

Vg9-JgP2
Vg9-none
Vg10-Jg1/2
Vg10-JgP
Vg10-JgP1
Vg10-JgP2
Vg10-none
Vg11-Jg1/2
Vg11-JgP
Vg11-JgP1
Vg11-JgP2
Vg11-none

Vg4-JgP2
Vg4-none
Vg5-Jg1/2
Vg5-JgP
Vg5-JgP1
Vg5-JgP2
Vg5-none
Vg8-Jg1/2
Vg8-JgP
Vg8-JgP1
Vg8-JgP2
Vg8-none
Vg9-Jg1/2
Vg9-JgP
Vg9-JgP10

Vg2-Jg1/2
Vg2-JgP
Vg2-JgP1
Vg2-JgP2
Vg2-none
Vg3-Jg1/2
Vg3-JgP
Vg3-JgP1
Vg3-JgP2
Vg3-none
Vg4-Jg1/2
Vg4-JgP
Vg4-JgP15RunMiSeq®

Analyze
Data

Clonal ControlDilutions(%)

Size(bp)

Mean% Reads

CV%105
2.5
10147
147
147
147
varies

8.66
4.59
2.39
0.94
0.62

10.4
12.0
11.7
13.8
40.3

Size(bp)

Mean% Reads

CV%198198
198
198
varies

17.63
9.35
5.25
2.04
0.17

16.7
18.4
19.6
20.0
46.8

Results: TRG/TRB MRD Detection•Limit of detection (LoD), linearity, precision and reproducibility (P/R) were tested using clonal control
DNA diluted in wild-type polyclonal (tonsil) DNA for TRB assay.

LymphoTrack® TRG Assay-MiSeq®

•

DNA from 60 formalin-fixed paraffin-embedded (FFPE) samples were extracted using common
extraction methods by collaborators. All samples were tested with TRG and TRB Assays .

The LymphoTrack®

Libraries were prepared with amplicons generated by PCR using proprietary multiplex master mixes withthe consensus primers targeting all TRG and TRB V, D, and J exon fa``milies, synthesized with MiSeq®
specific adapters and 24 index sequences optimized for the NGS platform.

•Libraries were either sequenced for TRB and TRG individually or for TRB + TRG combined.

•LymphoTrack® Software - MiSeq® was used to analyze FASTQ data generated from the MiSeq®.

•

When comparing testing results, only samples that met the specimen and data acceptance criteria forboth methods were evaluated

1.E-02

Non-Clonal

Clonal180

Non-Clonal620

1.E-03Contrived Cell Line

Concordance (%)

86.4

Sensitivity (%)

75.0

Specificity (%)

100.0

PPV (%)

100.0

NPV (%)

76.9

TRGMiSeq ®

TRG MiSeq® vs. TRG IdentiClone® 2.0

TRG Identiclone 2.0

n=51

R² = 0.9993

1.E-04

ClonalResults: Clinical Study Comparing TRG MiSeq® and IdentiClone® Assays

1.E-01

Clonal
Non-Clonal

Clonal

Non-Clonal

141234Control Cell Line

Concordance (%)

94.1

Sensitivity (%)

87.5

Specificity (%)

97.1

PPV (%)

93.3

NPV (%)

94.4

1.E-05
1.E-06
1.E-01Results: Clinical Study for LymphoTrack® TRG + TRB Assays - MiSeq®
1.E-021.E-03

1.E-04

1.E-05

1.E-06

Clonal DNA dilutions

TRB MiSeq®

TRG MiSeq®

TRB + TRG MiSeq®

Clonal19/60 (32%)

16/60 (27%)

20/60 (33%)

Non-Clonal

41/60 (68%)

43/60 (73%)

39/60 (65%)

LymphoTrack® TRB Assay-MiSeq®

• All statistical analyses were performed in JMP®.

LT TRB: Contrived Cell Line Dilutions

Conclusion

1.E-01

1.E-02

Measured %reads• The LymphoTrack® MiSeq® TRB assay demonstrated excellent linearity (R2>0.90), sensitivity (as low as2.5%), and reproducibility (<20%CV) testing serial dilutions of contrived cell line DNA.
• Concordance between the LymphoTrack® TRB - MiSeq® and IdentiClone® TRB assays was 86.4%.
• Concordance between the LymphoTrack® TRG - MiSeq® and IdentiClone® TRG assays was 94.1%• Concordance between the combined TRB+TRG LymphoTrack® MiSeq® and IdentiClone® assays was84.1%• Excellent linearity (R2>0.90) for contrived cell line, reproducibility (<20% CV) for control cell line, andclonal detection for MRD at 10-5 sensitivity was demonstrated for LymphoTrack® MiSeq® TRG and TRB
assays.

TRBMiSeq ®

TRB MiSeq® vs. TRB IdentiClone® (Tube A/B/C)

TRB Identiclone

n=44

LT TRG: Contrived Cell Line Dilutions

Measured %reads

Contrived cell line was used for MRD detection at 10-2, 10-3, 10-4, 10-5 and 10-6 sensitivity with an internal
control spiked in to reactions at approximately 1000 cell equivalence.

TRB Positive (Clonal)

Results: Clinical Study between TRB MiSeq® and IdentiClone® Assays

TRB MiSeq®

TRG and TRB Assays for the MiSeq® were cGMP manufactured, QC tested, andanalyzed using LymphoTrack® bioinformatics software developed under full design control consistent
with our ISO13485-compliant regulatory system.

•30

TRG Positive (Clonal)

Workflow for the LymphoTrack® TRG & TRB Assays - MiSeq®
Quantify
Amplicons

50

10

Results: TRG/TRB Precision and Reproducibility

Schematic Illustration of the TRB gene

PurifyAmplicons

60

4515

•

•

50

Schematic Illustration of the TRG gene

PCR

LymphoTrack TRB Assay - V – J Sequence Frequencies

35

TRG MiSeq®
•CE-0041

LymphoTrack TRG Assay - V – J Sequence Frequencies

40

Material and Methods•

Results: TRG/TRB Positive Clinical Sample

1.E-03R² = 0.9956

Control Cell Line

1.E-05

1.E-06
1.E-01

1.E-02

1.E-03

1.E-04

Clonal DNA dilutions

1.E-05

1.E-06

TRB +TRG IdentiClone®

n=44

Contrived Cell Line
1.E-04

TRB + TRG
MiSeq ®

Clonal

Non-Clonal

Clonal180

Non-Clonal719

TRB + TRG MiSeq® vs. IdentiClone®
Concordance (%)
Sensitivity (%)
Specificity (%)
PPV (%)NPV (%)

84.1
72.0
100.0
100.0
73.1

MiSeq ® is a registered trademark of Illumina, Inc.

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