E847

DETECTION OF CLONALITY IN CLINICAL SPECIMENS FROM SUSPECTED B-CELL MALIGNANCIESUSING COMPREHENSIVE IGH (FR1/2/3) LYMPHOTRACK® MiSEQ® AND PGM® ASSAYS
Ying Huang1, Kasey Hutt1, Jeff Panganiban1, Austin Jacobsen1, Nick Wong1, Roshanak Bob2, Stefano A. Pileri3, Loris Bernard3,Elvira Gerbino3, Tim Stenzel1 and Jeffrey E. Miller1
1Invivoscribe, San Diego, USA, 2 Institute for Pathodiagnostik Berlin, Germany and 3Haematopathology Unit at European Institute of Oncology, Milan, Italy

Results: LoD, Linearity, Precision and ReproducibilityBackground
PCR-based capillary electrophoresis (PCR-CE) methods targeting immunoglobulin
heavy chain (IGH) framework 1, 2, 3 (FR1, FR2, FR3), and joining regions (J) arehistorically the gold standard for clonality testing in suspected B-cell malignancies.
Recently, next-generation sequencing (NGS) based approaches for immune receptor
genes have been developed that improve sensitivity and identify the specific V-(D-)J
DNA sequences required to track clones in follow-up testing. We developed
comprehensive LymphoTrack® IGH (FR1, FR2, & FR3) Assays for both the Illumina®
MiSeq® and ThermoFisher Scientific® Ion PGM™ platforms, which detect the vast
majority of rearrangements in a single NGS run. In this pilot study, we compared theperformance of both LymphoTrack® IGH MiSeq and PGM Assays to the IdentiClone®
IGH PCR-CE assay by testing in over 50 anonymized, blinded clinical samples.

ClonalControl
Dilutions
(%)

N1032

MiSeq® IGH FR1

Size(bp)

Mean% Reads

295

5.08

MiSeq® IGH FR2

CV%Size(bp)Mean% Reads

17.5243

15.74

MiSeq® IGH FR3

CV%Size(bp)Mean% Reads

CV%

9.9

104

12.99

6.2

ClonalControl
Dilutions(%)

Ion PGM™ IGH FR1NSize(bp)Mean% Reads

Ion PGM™ IGH FR2

CV%Size(bp)Mean% Reads

Ion PGM™ IGH FR3

CV%Size(bp)Mean% Reads

CV%

1032

295

10.86

4.7

243

19.81

6.9

104

17.98

19.6

548

295

2.58

19.4

243

7.72

8.2

104

7.04

8.4

2.5

48

295

1.28

14.1

243

3.93

9.2

104

3.50

8.0

532

295

5.20

6.7

243

8.80

13.0

104

10.70

9.3

148

295

0.50

29.32431.52

12.5

104

1.43

16.1

116295

0.97

12.4

243

1.91

14.2

104

2.14

13.1

016

varies0.04

34.8

varies

0.06

26.5

104

0.07

28.6

016varies

0.057

35.1

varies0.05

45.9

varies0.08

37.5

MiSeq FR1
Single: R2=0.9971
3 combo: R2=0.9974

MiSeq FR2
Single: R2=0.9939
3 combo: R2=0.9969

PGM FR1
Single: R2=0.9973
3 combo: R2=0.9980

PGM FR2
Single: R2=0.9816
3 combo: R2=0.9714

Materials and Methods

MiSeq FR3
Single: R2=0.9973
3 combo: R2=0.9979PGM FR3Single: R2=0.9633
3 combo: R2=0.9651•The LymphoTrack® IGH FR1/2/3 Assays for the MiSeq® and Ion PGM™ weremanufactured under cGMP standards and QC tested under a QSR-compliant
regulatory system prior to use.
•Limit of detection (LoD), linearity, precision and reproducibility (P/R) werevalidated using clonal control DNA diluted in wild-type polyclonal (tonsil) DNA.

•DNA from a variety of samples (21 from peripheral blood, 1 from bone marrowaspirates, and 37 from FFPE were extracted using common extraction methodsby collaborators. 59 samples were tested by all assays except that FR2 PGM
tested additional 9 samples for total 68 samples.
•Libraries were prepared with amplicons generated by the LymphoTrack® IGH
FR1/2/3 Assay optimized for each NGS platform.
•Libraries were either sequenced for each FR individually or for all FRs (IGHFR1/2/3) combined.

Results: Clinical Study between IGH MiSeq® and Ion PGM™ Assays
MiSeq® IGH Assays
FR1

FR3

FR1/2/3

FR1FR2

FR3

IdentiClone IGH Assays
FR1/2/3

Tube A/B/C
Tube A (FR1) Tube B (FR2) Tube C (FR3)(FR1/2/3)

Clonal (%)

18/59 (31%) 16/59 (27%) 22/59 (37%) 25/59 (42%) 17/54 (32%) 18/68 (27%) 15/40 (38%) 15/40 (38%) 18/59 (31%) 21/59 (36%) 15/40 (38%) 26/59 (29%)

Non-Clonal (%)

37/59 (63%) 41/59 (70%) 36/59 (61%) 32/59 (54%) 32/54 (59%) 27/68 (40%) 24/40 (60%) 22/40 (55%) 21/59 (36%) 21/59 (36%) 24/40 (60%) 20/59 (34%)•LymphoTrack® Software - MiSeq® and LymphoTrack® Software - PGM™ analyzed
FASTQ data from the MiSeq® and the Ion PGM™, respectively.
•When comparing testing results, only samples that met the specimen and data
acceptance criteria for both methods were evaluated

FR2Ion PGM™ IGH Assays

IdentiClone IGH Tube A/B/C

®

MiSeq
FR1/2/3

Clonal

Non-Clonal

Clonal24

1

Non-Clonal

118

IdentiClone IGH Tube A/B/C®

PGM
FR1/2/3

Clonal

Non-Clonal

Clonal220

Non-Clonal

118MiSeq FR1/2/3®

PGM
FR1/2/3

Clonal

Non-Clonal

Clonal220

Non-Clonal032•All statistical analyses were performed in JMP®.
MiSeq FR1/2/3 vs. IdentiClone Tube A/B/C

PGM FR1/2/3 vs. IdentiClone Tube A/B/C

PGM FR1/2/3 vs. MiSeq FR1/2/3Concordance (%)

95.5

97.6

100

Sensitivity (%)

96.0

95.7

100Specificity (%)

94.7

100

100

PPV (%)

96.0

100

100

NPV (%)

94.7

94.7

100

Conclusions
A comprehensive NGS-based LymphoTrack® IGH FR1/2/3 Assay was developed forboth the Illumina® MiSeq® and Thermo Fisher Scientific® Ion PGM™ platforms.
These assays identify clonal IGH V-J rearrangements and the specific clonal DNA
sequences, critical for determining the SHM rate and tracking residual disease.
Excellent concordance was demonstrated between these assays.
MISEQ® is a registered trademark of Illumina, Inc.
ION PGM™ is a trademark of Thermo Fisher Scientific.

EHA, June 22–26 2017, Madrid, Spain