H09

Detec3ng B-Cell Clonality in Clinical Samples using a Comprehensive®
NGS LymphoTrack Dx IGH FR1/2/3 Assay
Ying Huang1, Kasey HuA1, Jeﬀ Panganiban1, AusDn Jacobsen1, Nick Wong1, Duy Duong1, Arnold M. Raem2, Jeﬀrey E. Miller1, and Tim Stenzel1
1Invivoscribe Technologies, San Diego, USA, 2Arrows Biomedical Deutschland GmbH, Mȕnster, Germany

AbstractIon PGM™ IGH Results: LoD, Linearity, Precision and ReproducibilityIntroduc)on: PCR-based methods targe3ng immunoglobulin heavy chain (IGH) frameworks 1, 2, 3 (FR1, FR2, FR3), and joining region(JH) are the current gold standard for clonality tes3ng in suspected B-cell prolifera3ons. Recently, next-genera3on sequencing (NGS)
based approaches for immune receptor genes have been suggested to improve sensi3vity and iden3fy the speciﬁc V-(D-)J DNA
sequence required to track clones in follow-up tes3ng. We developed the comprehensive LymphoTrack Dx® IGH FR1/2/3 Assay for boththe Illumina® MiSeq® and Thermo Fisher Scien3ﬁc® Ion PGM™ plaZorms, which detect the vast majority of rearrangements in a single
NGS run. Here, we report a pilot study on detec3ng suspected B-cell clonality using the LymphoTrack Dx® IGH FR1/2/3 Assay.

FR1Single: R2=0.9973
3 combo: R2=0.9980

FR2Single: R2=0.9816
3 combo: R2=0.9714

Methods: The LymphoTrack Dx® IGH FR1/2/3 Assay was been developed for both the MiSeq® and Ion PGM™ plaZorms. The proprietary
V and J consensus primers were designed and adapted to enable the PCR products to be sequenced directly on either the MiSeq® orIon PGM™ plaZorm. Each MiSeq® IGH FR master mix uses 1 of 24 diﬀerent indices, allowing analysis of 22 samples with 2 controls.
Each Ion PGM™ IGH FR master mix uses 1 of 12 diﬀerent indices, allowing analysis of 10 samples with 2 controls. One-step PCR wasfollowed by amplicon puriﬁca3on. Puriﬁed equimolar amounts of amplicons were pooled to form a library, which was loaded onto theMiSeq® or Ion PGM™. The sequencing data was analyzed using Invivoscribe® LymphoTrack Dx® bioinforma3cs sobware, which sorts thesequences by both index and FR, generates frequency distribu3ons, and determines the soma3c hypermuta3on (SHM) rate of FR1amplicons.
FR3
Single: R2=0.9633
3 combo: R2=0.9651

Results: The analy3cal performance of the LymphoTrack Dx® IGH FR1/2/3 Assay on both plaZorms was evaluated using contrived
samples with known V-J rearrangements. Both NGS assays demonstrated excellent linearity (R2>0.96), clonality sensi3vity (2.5%), andreproducibility (<20% CV). The clinical performance of the IGH Assay was evaluated with over 40 clinical samples that have beentested by control methods. Results demonstrated that clonality detec3on missed by one FR was detected by another FR, increasing
the overall detec3on rate. Both the MiSeq® and Ion PGM™ NGS assays demonstrated excellent concordance in detec3ng clonality. In
addi3on, data generated with the MiSeq® and the Ion PGM™ assays were concordant.
Conclusions: A comprehensive LymphoTrack Dx® IGH FR1/2/3 Assay was developed for both MiSeq® and Ion PGM™ plaZorms thatiden3ﬁes clonal IGH V-J rearrangements and DNA sequences. Assay performance demonstrated that combining FR1, FR2, and FR3
helps to decrease the false-nega3ve rate due to failure in ampliﬁca3on of any individual targeted FR region. The inter-assay
concordance in clonality detec3on using assays developed for the MiSeq® and Ion PGM™ plaZorms was excellent.

Materials and Methods• The CE-marked LymphoTrack Dx® IGH FR1/2/3 Assays for the MiSeq® and Ion PGM™ were manufactured under cGMP
standards and QC tested under a QSR-compliant regulatory system prior to use.
• Limit of detec3on (LoD), linearity, precision and reproducibility (P/R) were calculated using clonal control DNA diluted
in wild-type polyclonal (tonsil) DNA.
• DNA from a variety of samples (35 from peripheral blood, 4 from bone marrow aspirates, and 1 from lymph node)
were extracted using common extrac3on methods by collaborators.
• Libraries were prepared with amplicons generated by the LymphoTrack Dx® IGH FR1/2/3 Assay op3mized for each
NGS plaZorm.
• Libraries were either sequenced for each FR individually or for all FRs (IGH FR1/2/3) combined.
• LymphoTrack Dx® Sobware-Ion PGM™ IGH FR1

ClonalControlDilu)ons(%)

N10Size(bp)Mean% Reads

32295

532

1
0CV%Size(bp)Mean% Reads

10.86

4.7

243

295

5.20

6.7

16295

0.97

16varies

0.057

FR1
Clonal (%)

FR1Single: R2=0.9971
3 combo: R2=0.9974

CV%Size(bp)Mean% Reads

CV%

19.81

6.9

104

17.98

19.6

243

8.80

13.0

104

10.70

9.3

12.4

243

1.91

14.2

104

2.14

13.1

35.1

varies

0.0545.9varies

0.08

37.5MiSeq® IGH Assays

the Ion PGM™, respec3vely.

MiSeq® IGH Results: LoD, Linearity, Precision and Reproducibility

Ion PGM™ IGH FR3Results: Clinical Study between IGH MiSeq® and Ion PGM™ Assays

MiSeq® and LymphoTrack Dx® Sobware - PGM™ analyzed FASTQ data from the MiSeq® and• All sta3s3cal analyses were performed in JMP®.

Ion PGM™ IGH FR2

FR2Ion PGM™ IGH Assays

FR3

FR1/2/3

FR1FR214/40 (35%) 13/40 (33%) 12/40 (30%) 15/40 (38%) 13/40 (33%) 13/40 (33%) 15/40 (38%) 15/40 (38%)

Ion PGM™ FR1

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

ClonalNonClonal

Ion PGM™ FR2

Clonal

Non-Clonal

131023MiSeq®
FR2Clonal
NonClonal

Ion PGM™ FR3®

MiSeq
FR3

FR3
Single: R2=0.9973
3 combo: R2=0.9979

ClonalNonClonal

Concordance (%)
Sensi)vity (%)
Speciﬁcity (%)
PPV (%)NPV (%)
FR1
R2=0.7682

MiSeq® IGH FR1
N10Size(bp)Mean% Reads

322955482.5MiSeq® IGH FR2

CV%Size(bp)Mean% Reads

5.0817.5243295

2.58

19.4

48295

1.28

148295016varies

FR1/2/3

Non-Clonal (%) 25/40 (63%) 25/40 (63%) 25/40 (63%) 23/40 (58%) 24/40 (60%) 27/40 (68%) 24/40 (60%) 22/40 (55%)

MiSeq®
FR1

ClonalControlDilu)ons
(%)

FR3

Clonal

Non-Clonal

121

124Ion PGM™ FR1/2/3

Clonal

Non-Clonal

120223Clonal

Non-Clonal

14

1

121

ClonalMiSeq
FR1/2/3 NonClonal®

FR1FR2

FR3

FR1/2/3

971009693100959296929695
86100
100929593959395

FR2
R2=0.8491

FR3R2=0.8827

MiSeq® IGH FR3

CV%Size(bp)Mean% Reads

CV%

15.74

9.9

104

12.99

6.2

243

7.72

8.2

104

7.04

8.4

14.1

243

3.93

9.2

104

3.50

8.0

A comprehensive NGS-based LymphoTrack Dx® IGH FR1/2/3 Assay was developed for both the Illumina® MiSeq® and Thermo Fisher
Scien3ﬁc® Ion PGM™ plaZorms. These assays iden3fy clonal IGH V-J rearrangements and the speciﬁc clonal DNA sequences, cri3cal for
determining the SHM rate and tracking residual disease. Excellent concordance was demonstrated between these assays.

0.50

29.3

243

1.52

12.5

104

1.43

16.1

AMP Global, April 3–5 2017, Berlin, Germany

0.04

34.8

varies

0.06

26.5

104

0.07

28.6

Conclusions