E846

USING NEXT-GENERATION SEQUENCING TO DETECT CLONAL TRG AND TRB GENE REARRANGEMENTS
Presha Shah1, Ying Huang1, Kasey Hutt1, Jeff Panganiban1, Mark D. Ewalt2, James L. Zehnder3, Tim Stenzel1, and Jeffrey E. Miller1
1Invivoscribe Technologies, Inc., San Diego, CA United States,2University of Colorado, Denver, CO United States, 3Department of Pathology, Stanford University Medical Center, Stanford, CA
United States

BackgroundResults: TRG + TRB Combo Assay

T-cell malignancies arise from transformation and clonal expansion of a single cell. During T-cell development,
the T cell receptor gamma (TRG) locus rearranges prior to the T cell receptor beta (TRB) locus. Combined, TRG
and TRB can identify the vast majority of T-cell rearrangements. Historically, these clonal rearrangements areoften identified by capillary electrophoresis (CE) methods which provide size distribution information, but not
the sequence needed for tracking residual disease during the course of treatment. Recently, next-generation
sequencing (NGS)-based approaches for immune receptor genes have been developed to improve the sensitivityof clonal detection and identify the specific V-(D-)J DNA sequences required to track clones in follow-up testing.
We have developed and validated LymphoTrack® TRG & TRB clonality assays for the Illumina® MiSeq® platform.

MiSeq TRB
Single R2=0.98585
Combo R2=0.98662

Results: Clinical StudyTRB Identiclone

TRB MiSeq

MiSeq TRG
Single R2=0.99242
Combo R2=0.99315

ClonalClonal (%)

Non-Clonal (%)

Material and Methods• Schematic Illustration of the TRB gene

Non-Clonal

15/49 (31%)

34/49 (69%)

ClonalTRBMiSeq

8

Non-Clonal

0111

TRB MiSeq vs. Identiclone
Concordance (%)

95

Sensitivity (%)

89

Specificity (%)

100

PPV (%)

100

NPV (%)92

CE

TRB MiSeq

FFPE + 0037

FFPE + 0037

255 bpResults: Top 5 Sequences % for Normal PB Samples•The workflow for the LymphoTrack® TRG & TRB Assays – MiSeq®

SampleRank

V-geneJ-gene% total reads
none
TRB_J2-7
TRB_J2-6
TRB_J2-5
TRB_J2-4
TRB_J2-3
TRB_J2-2P
TRB_J2-2
TRB_J2-1
TRBJ1-6
TRBJ1-5
TRBJ1-4
TRBJ1-3
TRBJ1-2
TRBJ1-1

45

Amplicons

PoolLibraryAmplicons

RunMiSeq

Analyze
Data• The LymphoTrack® TRG and TRB assays for the MiSeq® were manufactured under cGMP standards andQC tested under a QSR-compliant regulatory system prior to use.

PB 17.7

PB 34.5• Limit of detection (LoD), linearity, precision and reproducibility (P/R) were tested using clonal control DNA
diluted in wild-type polyclonal (tonsil) DNA.
PB 43.3• 12 cell line DNA samples were tested with the TRB assay.
• DNA from 49 FFPE samples were extracted using common extraction methods by collaborators. All samples
were tested with the TRG and TRB assays.
• Libraries were prepared with amplicons generated by PCR using proprietary multiplex master mixes with theconsensus primers targeting all TRG and TRB V, (D) and J exon families, synthesized with MiSeq specificadapters and 24 index sequences optimized for NGS.

PB 45.4

PB 48.2• Libraries were either sequenced for TRB and TRG individually or for TRB + TRG combined.
• LymphoTrack® Software – MiSeq® analyzed FASTQ data from the MiSeq.

PB 9.4• All statistical analyses were performed in JMP.

Results: LOD, LOB and Linearity
ClonalControl
Dilutions(%)

NSize(bp)

Mean %
Reads

10%

5198

5%

5198

MiSeq TRB
LoD R2=0.9835

TRB MiSeq

SampleRank

V-geneJ-geneTotal Reads %111212121211212
12
121

TRBV3-1
TRBV19
TRBV18
TRBV6-3
TRBV7-3
TRBV5-5
TRBV20-1
TRBV5-6
TRBV20-1
TRBV10-3
TRBV4-2
TRBV20-1
TRBV7-9
TRBV15
TRBV19
TRBV10-1
TRBD2
TRBV12-4
TRBD1
TRBV5-1

TRB_J2-3
TRB_J2-5TRBJ1-2
TRB_J2-7
TRB_J2-5
TRB_J2-5
TRB_J2-2
TRB_J2-1
TRB_J2-1
TRB_J2-5
TRB_J2-3
TRB_J2-1
TRB_J2-7
TRBJ1-5
TRB_J2-7
TRBJ1-1
TRB_J2-3
TRBJ1-2
TRBJ1-3
TRBJ1-1

95.29
75.86
69.85
15.79
52.75
24.42
78.50
7.65
40.04
40.71
91.04
48.08
18.85
83.68
8.20
37.33
24.96
76.17
17.33
92.37

CV%

Lower
95%

CCR-CEM
CML-T1

19.12

8.5

17.67

20.57

DND-41

8.78

6.3

8.29

9.27

5198

4.5

12.5

4.00

5.00

1%

5198

1.7

8.2

1.57

1.83

Tonsil

3197

0.1

30.6

0.07

0.13

HPB-ALL
LOUCY
MOLT-3/4
PEER

Results: Precision and Reproducibility
MiSeq TRB
Run 1
Run 20.35
0.26
0.23
0.21
0.21
0.25
0.18
0.18
0.15
0.11
1.08
0.36
0.29
0.27
0.26
0.23
0.19
0.14
0.13
0.13
0.86
0.52
0.51
0.32
0.25
0.85
0.49
0.45
0.35
0.31

Results: Cell Lines

Upper
95%

2.5%

TRBJ1-1
TRBJ1-6
TRB_J2-7
TRBJ1-1
TRB_J2-7
TRBJ1-1
TRB_J2-1
TRBJ1-1
TRB_J2-7
TRB_J2-2
TRBJ1-1
TRB_J2-7
TRB_J2-1
TRBJ1-2
TRBJ1-5
TRBJ1-2
TRB_J2-2
TRB_J2-7
TRB_J2-2
TRB_J2-7
TRB_J2-5
TRB_J2-7
TRBJ1-4
TRB_J2-7
TRB_J2-7
TRB_J2-5
TRBJ1-5
TRBJ1-1
TRBJ1-1
TRB_J2-5

PF-382
RPMI-8402
MOLT-13JURKATHSB-2

4035302520151050

TRBV2
TRBV3-2
TRBV4-2
TRBV5-1
TRBV5-4
TRBV5-6
TRBV5-8
TRBV6-2
TRBV6-4
TRBV6-6
TRBV6-8
TRBV7-1
TRBV7-3
TRBV7-6
TRBV7-8
TRBV9
TRBV10-3
TRBV11-2
TRBV12-3
TRBV12-5
TRBV14TRBV16
TRBV18
TRBV20-1
TRBV23-1
TRBV25-1
TRBV28
TRBV30TRBD2PCRQuantify

TRBD1
TRBD1
TRBD2
TRBV12-4
TRBV4-1
TRBV29-1
TRBD2
TRBD1
TRBD2
TRBV14
TRBV11-2
TRBD2
TRBD2
TRBD1
TRBV2
TRBD2
TRBV4-1
TRBV9
TRBV4-2
TRBV29-1
TRBV2
TRBV20-1
TRBV6-4
TRBV20-1
TRBV12-4
TRBD2
TRBD1
TRBV14
TRBV23-1
TRBD2

FFPE - 0020

FFPE - 0020

none
TRB_J2-7
TRB_J2-6
TRB_J2-5
TRB_J2-4
TRB_J2-3
TRB_J2-2P
TRB_J2-2
TRB_J2-1
TRBJ1-6
TRBJ1-5
TRBJ1-4
TRBJ1-3
TRBJ1-2
TRBJ1-1454035302520151050

TRBV2
TRBV3-2
TRBV4-2
TRBV5-1
TRBV5-4
TRBV5-6
TRBV5-8
TRBV6-2
TRBV6-4
TRBV6-6
TRBV6-8
TRBV7-1
TRBV7-3
TRBV7-6
TRBV7-8
TRBV9
TRBV10-3
TRBV11-2
TRBV12-3
TRBV12-5
TRBV14
TRBV16
TRBV18
TRBV20-1
TRBV23-1
TRBV25-1
TRBV28
TRBV30
TRBD2Purify

123451234512345123451234512345Sample

V-geneJ-gene

TRB
Total Reads %TRB MiSeq

TRG MiSeq

TRB Identiclone (CE)

Sample_0020
Sample_0021
Sample_0022
Sample_0024
Sample_0025
Sample_0026
Sample_0027
Sample_0028
Sample_0029
Sample_0030
Sample_0031
Sample_0032
Sample_0033
Sample_0034
Sample_0035
Sample_0036
Sample_0037
Sample_0038
Sample_0039
Sample_0040

TRBD2
TRBV11-2
TRBV14
TRBV12-4
TRBD1
TRBD2
TRBD2
TRBV14
TRBV12-4
TRBV29-1
TRBD2
TRBD1
TRBV28
TRBV18
TRBV12-4
TRBD1
TRBV6-4
TRBV14
TRBD1
TRBV4-3

TRB_J2-2
TRBJ1-2
TRBJ1-2
TRB_J2-5
TRBJ1-5
TRB_J2-2
TRB_J2-2
TRBJ1-1
TRBJ1-2
TRBJ1-2
TRB_J2-2
TRB_J2-7
TRB_J2-7
TRBJ1-6
TRBJ1-2
TRBJ1-4
TRB_J2-2
TRBJ1-1
TRBJ1-1
TRBJ1-2

0.69
0.50
0.43
39.96
22.22
0.53
2.46
0.84
0.89
1.18
0.68
37.96
33.65
5.10
0.92
22.96
37.47
4.85
28.61
15.69

Non-clonal
Non-clonal
Non-clonal
clonalclonalNon-clonal
Non-clonal
Non-clonal
Non-clonal
Non-clonal
Non-clonal
clonalclonalNon-clonal
Non-clonal
clonalclonalNon-clonal
clonalclonalNon-clonal
Non-clonal
Non-clonal
clonalclonalNon-clonal
Non-clonal
Non-clonal
Non-clonal
Non-clonal
Non-clonal
clonalclonalNon-clonal
Non-clonal
clonalclonalNon-clonal
clonalclonalNon-clonal
Non-clonal
Non-clonal
clonalclonalNon-clonal
Non-clonal
Non-clonal
Non-clonal
Non-clonal
Non-clonal
clonalclonalclonalNon-clonal
clonalclonalNon-clonal
clonalclonalConclusions• The LymphoTrack® TRB Assay – MiSeq® was able to consistently detect all known
TRB clonal rearrangements from cell line DNA.
• Excellent linearity (R2>0.90), sensitivity of clonality (2.5%), and reproducibility(<20% CV) were demonstrated with serial dilutions of contrived cell line DNA.
• Concordance between the LymphoTrack® TRB and CE assays was 95% and between
the LymphoTrack® TRG and TRB assays was 94%.