P30™
™
Performance Evaluation of LymphoTrack® Clonality Assays on Ion PGM and Ion S5 Platforms
Ying Huang1, Jeff Panganiban1, M. Adam Bouzaffour1, Kasey Hutt1, Martin Blankfard1, and Jeffrey E. Miller1,2,31Invivoscribe, Inc. and 2LabPMM, LLC, San Diego, CA; 3LabPMM, GmbH, Martinsried, Germany

Abstract

Results: Chef/S5 and OT2/S5 Comparison

Introduction: Detection of clonal rearrangements within the immunoglobulin (Ig) and T-cell receptor (TCR) genes in clinical specimens
is used to assist in diagnosis of lymphoproliferative disease (LPD). Next-generation sequencing (NGS)-based clonality assays represent
a major advance compared to the traditional capillary electrophoresis (CE)-based assays as the NGS assays can identify specific clonalsequences that can be tracked in follow-up testing.
The LymphoTrack® Dx Assays can be utilized for routine Ig/TCR clonality detection in LPD diagnosis, where multiple patient samples
and all LymphoTrack Dx Assays can be multiplexed into a single sequencing run, reducing per sample testing costs without loss ofsensitivity. Here we compare the performance of LymphoTrack® Dx (IGH (FR1/2/3), IGK, and TRG) CE-IVD assays (Ion PGM™) to results
when running these tests on the Ion S5™.

IGH FR1
Chef/S5: R2=0.9985
OT2/S5: R2=0.9984

IGH FR2
Chef/S5: R2=0.9695
OT2/S5: R2=0.9685

Methods: LymphoTrack® Dx Assays targeting the IGH, IGK, and TRG loci have been developed for the Ion systems. Consensus primers
targeting V and J gene segments include specific adapter sequences and individual barcodes, thus the PCR products from multiple
independent samples and multiple clonality assays can be combined and sequenced together in a single NGS run. Individual mastermixes were manufactured with 12 indices. Single step PCR amplification of 50 ng DNA input was followed by pooling equimolar
amounts of purified amplicons. Template preparations were performed with either the Ion OneTouch™ 2 system for Ion PGM™ or theIon Chef™ system for Ion S5™. Fastq data from both Ion PGM™ and Ion S5™ were analyzed using LymphoTrack® Dx Software, togenerate frequency distributions, determine V-J usage, identify specific sequences for top sequencing reads, and determine thesomatic hypermutation rate of IGH FR1 amplicons. Limit of detection (LoD), limit of blank (LoB) and linearity were evaluated by testing
serial dilutions of contrived samples with V-J rearrangements in tonsil. Clinical samples were used to assess the clinical performance.

IGH FR3
Chef/S5: R2=0.9534
OT2/S5: R2=0.9568

TRG
Chef/S5: R2=0.9929
OT2/S5: R2=0.9921

IGK
Chef/S5: R2=0.9818
OT2/S5: R2=0.9592

Results: All LymphoTrack® Dx assays have demonstrated LoD to detect 5.0% clonality and excellent linearity for both Ion PGM™andIon
S5™ systems. Great concordance between Ion PGM™ and Ion S5™ systems was demonstrated when testing clonality for all targets
from subject samples.

Conclusions: This study has demonstrated that the performance of CE-IVD LymphoTrack® Dx Assays on Ion PGM™ and Ion S5™
systems is comparable. The LymphoTrack Dx Assays are undergoing full validation for use with the Ion S5™ system and will beavailable soon.

Materials and Methods

Results: Single and 5 Targets on Chef/S5
IGH FR1
Single: R2=0.9994
5 targets: R2=0.9985

IGH FR2
Single: R2=0.9667
5 targets: R2=0.9695

IGH FR3
Single: R2=0.9567
5 targets: R2=0.9534• IGH loci

• IGK loci

IGK
Single: R2=0.9760
5 targets: R2=0.9818

TRG
Single: R2=0.9901
5 targets: R2=0.9929• TRG loci

• LymphoTrack® Dx Assays for the Ion PGM™ were manufactured under cGMP standards per an ISO 13485 certified QMS. Five assaysare available to target IGH FR1/2/3, IGK, and TRG loci, respectively. Each target consists of a one step PCR Master Mixes with 12
different indices to allow multiple samples and targets to be run on the same chip.
• Complimentary bioinformatics software package, LymphoTrack® Dx Software, was developed and validated under ISO13485 designcontrol.
• Limit of detection (LoD), Limit of blank (LoB) and linearity were validated using clonal control DNA diluted in wild-type polyclonal(tonsil) DNA.
• DNA from a variety of samples (peripheral blood, bone marrow aspirates, and formalin-fixed paraffin-embedded (FFPE)) wereextracted using common extraction methods and tested by the LymphoTrack® Dx Assays.
• The Ion PGM™ Hi-Q™ View OT2 Kit and Sequencing Kit were used for PGM runs.
• The Ion 520™ & Ion 530™ Kit-OT2 were used for OT2/S5 runs.
• The Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef were used for Chef/S5 runs.
50 ng DNA• Workflow:

PCR
PurifyAmplicons
Quantify
Amplicons
Pool Library

Results: OT2/PGM and Chef/S5 for Clinical Samples*Total reads

PCR MM
Sample Type
Id

LengthVg

Jg

Top %reads

S5PGM

S5PGM

S5PGM

S5PGM

S5PGM

id01

BM

125,836

65,573

135

135

Vg8

Vg8

Jg1/2

Jg1/2

22.22

22.95

id02

PB

128,593

67,483

145

145

Vg9

Vg9

JgP

JgP

0.97

0.82

id03

BM

121,253

61,727

153

144

Vg10

Vg10

JgP1

Jg1/2

0.42

1.06

id04

PB

158,328

78,668

152

152

Vg9

Vg9

JgP1

JgP1

13.44

12.81

id07

PB

157,917

78,141

142

142

Vg10

Vg10

Jg1/2

Jg1/2

9.83

10.18

id08

BM

145,657

70,777138142Vg8

Vg3

Jg1/2

Jg1/2

0.92

1.03

id09

BM

142,184

71,685

151

151

Vg10

Vg10

JgP1

JgP1

3.16

3.01

id10

BM179,240

86,093

161

161

Vg3

Vg3

JgP2

JgP2

4.35

4.69

id11

PB

149,402

76,159

149

149

Vg3

Vg3

JgP2

JgP2

6.84

6.97

id12

PB

124,543

64,658

135

135

Vg8

Vg8

Jg1/2

Jg1/2

2.58

2.73

id13

Pos control

191,660

92,662

147

147

Vg11

Vg11

Jg1/2

Jg1/2

7.09

7.17

id14

Neg control

181,054

88,382

147

144

Vg10

Vg10

JgP1

Jg1/2

0.42

0.37

*TRG samples as an example.

(Single Target or 5 Targets)

Run Time and Cost Comparison
OT2PGM

OT2

S5ChefS5

Results: OT2/PGM and OT2/S5 Comparison
IGH FR1
OT2/PGM: R2=0.9977
OT2/S5: R2=0.9984

IGH FR2
OT2/PGM: R2=0.9776
OT2/S5: R2=0.9685

IGH FR3
OT2/PGM: R2=0.9586
OT2/S5: R2=0.9568

OT2/PGM

OT2/S5*Chef/S5

Hands on time

2 hr

1.1 hr

0.3 hr

Instrument time per run

16.5 hr

11.5 hr17.5 hrReagents cost per sequencing run$935$1,202$1,189

Reagents cost per sample (12 samples/run)

$78

$100

$99

Reagents cost per sample (12 samples x 5 targets/run)

$16

$20

$20

* Ion Chef can do 2 runs per setup.

Conclusions

IGK
OT2/PGM: R2=0.9698
OT2/S5: R2=0.9592

TRG
OT2/PGM: R2=0.9911
OT2/S5: R2=0.9921• Excellent correlation was observed when comparing the performance of LymphoTrack® Dx Assays on Ion
PGM™ and Ion S5™ systems.
• The per sample testing costs can be significantly reduced and TATs improved by combining multiple samples
from up to 5 LymphoTrack Dx PGM/S5 Assays into a single sequencing run.
• The provided bioinformatics software sorts the complex PGM- or S5-NGS data for easy analysis andvisualization of individual samples.
• The Ion S5™ can produce 2x to 5x more reads with less hands-on time than the Ion PGM™.
• With the LymphoTrack® Dx Assays, the same reagents and workflow can be utilized for subject Ig/TCR clonality
testing and for tracking of clonal populations. The LymphoTrack Dx Assays are undergoing full validation for
use with the Ion S5™ system and will be available soon.

Ion PGM™, Ion Hi-Q™, Ion 510™, Ion 520™, Ion 530™ and Ion S5™ are trademarks of Thermo Fisher Scientific.

AMP Europe 2018, April 30 – May 2, Rotterdam, Netherlands