P118®
®
LymphoTrack Low Positive Control and LymphoQuant Internal®
™
Control for MiSeq and Ion S5/PGM LymphoTrack Assays
Jeff Panganiban, Presha Shah, Brandon Givens, Edgar Vigil, Maggie Kaminsky, Ying Huang, Kasey Hutt, and Jeffrey E. Miller
Invivoscribe, Inc., San Diego, CA

Introduction

Results: Calculated vs. Expected Cell Equiv. for Contrived Samples (10-2 to 10-5)
LymphoQuant- IGHV Leader Assay

LymphoQuant- IGH FR1 Assay

Calculated Cell Equivalence vs Expected Cell Equivalence

Calculated Cell Equivalence vs Expected Cell Equivalence

2500

3500

R² = 0.9998

R² = 0.9999

3000
Calculated Cell

2000
Calculated Cell

LymphoTrack® Assays with the associated LymphoTrack software have been developed forMiSeq and Ion S5TM platforms. They are able to detect immunoglobulin (Ig) and T-cell receptor(TCR) clonal rearrangements in suspected lymphoproliferative disease (LPD) clinical specimens.
The detected sample specific clonal V-(D)-J sequence in baseline specimens can be tracked in
the follow-up samples to identify and monitor disease status. Typically, the subject-specificclonal rearrangements in follow-up specimens are present at very low levels (10-4 or below).
When testing these specimens, it is important to include a low positive control (LPC) in the runand to report the estimated clonal cells in a sample. We report the design and development ofthese controls for LymphoTrack Assays: the LPCs run quality controls, and the LymphoQuant™
internal controls (LQIC) used for estimating clonal cells within a specimen.

1500
1000
500

2500
2000
1500
1000
500
00050001,000 1,500 2,000 2,500 3,000 3,500

500

1,000 1,500 2,000 2,500 3,000 3,500
Expected Cell

Expected Cell

Materials and Methods

LymphoQuant- IGH FR3 Assay

LymphoQuant- IGH FR2 Assay

Calculated Cell Equivalence vs Expected Cell Equivalence

Calculated Cell Equivalence vs Expected Cell Equivalence

1400

3000

R² = 0.9815

1200
1000
800
600
400

2000
1500
1000

200

500

000500R² = 0.9998

2500
Calculated Cell

Calculated Cell

Two types of LPC and LQIC were developed, one for Ig (IGHV Leader, IGH FR1, IGH FR2, IGH FR3,and IGK), and one for TCR (TRG and TRB). Each LPC consists of clonal positive cell line DNA
diluted in clonal negative DNA at ~10-4 level. Each LQIC is diluted clonal positive cell line DNA ata concentration of about 50 cell equivalent per µL. All LPCs and LQICs can be detected by theLymphoTrack Assays on either the Illumina MiSeq  or Thermo Fisher Ion S5/PGMTM platforms.
Multiple lots of LPCs and LQICs were made and lot-to-lot variations were evaluated using
contrived samples that were prepared by 10-fold serial dilution of clonal positive cell line DNA
into clonal negative DNA at levels ranging from 10-2 to 10-5.

01,000 1,500 2,000 2,500 3,000 3,500

500

1,000 1,500 2,000 2,500 3,000 3,500
Expected Cell

Expected Cell

Principal of LymphoQuant Internal Control (LQIC)

LymphoQuant- IGK Assay

Calculated Cell Equivalence vs Expected Cell Equivalence

1200

Contrived
SampleExpected # ofClonal Cells

600

10-23077400

10-3308

200

10-4

31

010-5

3

R² = 0.9997

1000
Calculated Cell

Sample without the presence of LQIC
Tested by LymphoTrack Assays
Sample output: % Total reads

800

0500

1,000 1,500 2,000 2,500 3,000 3,500
Expected Cell

Sample in the presence of LQIC
Tested by LymphoTrack Assays

Sample output: Clonal cell equivalents

Results: Comparison LQIC Results between MiSeq and Ion S5TM

Non Clonal cells
Clonal cells

LQIC Frequency Values Tested by LymphoTrack Assays on MiSeq and S5 Platforms

LymphoQuant

MiSeq

Assay

Results: Intra and Inter Lot Variability for LQIC
Intra-Lot Mean and CV% of Frequency for LQIC Tested by Each LymphoTrack Assay
IGHV Leader

IGH FR1

IGH FR2

IGH FR3

N1126.16E-03 35.07 2.15E-03 28.12 7.18E-03 18.84 3.78E-03 55.54 4.40E-03 22.15

2126.82E-03

CV%

8.56

Mean

CV%

Mean

CV%

Mean

Mean (n=12)

CV%

Mean (n=3)

CV%

IGH FR1

2.15E-03

28.12

6.47E-03

14.4

IGH FR2

7.18E-03

18.84

1.06E-02

11.4

IGH FR3

3.78E-03

55.54

4.64E-03

45.8

IGKLot

Mean

CV%

Mean

CV%

Results: Calculated Clonal Cell Equivalence in Follow-Up Sample
Calculated Clonal Cell Equiv. in Follow-up Sample in thePresence of LQIC Tested by IGH FR3 Assay

2.10E-03 28.69 5.68E-03 14.54 3.56E-03 63.63 5.34E-03 23.79Inter-Lot Mean and CV% of Frequency for LQIC Tested by Each LymphoTrack Assay24

IGH FR1

IGH FR2

IGH FR3

10000

IGKMeanCV%

Mean

CV%

Mean

CV%

Mean

CV%

Mean

CV%

6.49E-03

24.4

2.12E-03

27.8

6.43E-03

20.8

3.67E-03

58.3

4.85E-03

24.7

Results: Intra and Inter Lot Variability for LPC

Clonal cells in sample

N

IGHV Leader

S5

1000
100
10101234567Timepoint

Intra-Lot Mean and CV% of Frequency for LPC Tested by Each LymphoTrack Assay
LotN12

IGHV Leader

IGH FR1

IGH FR2

IGH FR3IGK

Mean

CV%

Mean

CV%

Mean

CV%

Mean

CV%

Mean

CV%

12

1.99E-04

41.4

5.93E-05

66.8

2.61E-04

63.6

1.74E-04

65.5

8.53E-04

14.3

122.12E-04

44.1

5.73E-05

62.4

2.73E-04

35.7

1.33E-04

40.2

7.55E-04

20.3

Inter-Lot Mean and CV% of Frequency for LPC Tested by Each LymphoTrack Assay
N24

IGHV Leader

IGH FR1

IGH FR2

IGK

Mean

CV%

Mean

CV%

Mean

CV%

Mean

CV%

Mean

CV%

2.06E-04

42.0

5.83E-05

63.5

2.67E-04

49.8

1.54E-04

58.1

8.06E-04

17.7

AMP Global May 16-18 2019 Hong Kong.

IGH FR3MiSeq® is a registered trademark of Illumina, Inc.
Ion S5TM and Ion PGMTM are trademarks of ThermoFisher Scientific, Inc.

Conclusions• We have developed two types of controls (LPC and LQIC) for LymphoTrack assays
run on the MiSeq and S5 sequencing platforms.
• Both inter- and intra-lot variability were assessed.
• The LQIC allows investigators to estimate clonal cells present in the specimen
suggesting potential applications in detect and monitor minimal residual disease.