Poster # I27

Development of LymphoTrack® Bioinformatics Methods:
Clonality Testing, Somatic Hypermutation and Minimal Residual Disease
Kasey Hutt, Jeff Panganiban, Austin Jacobsen, Jordan Thornes, Jeffrey E Miller, and Michael Klass
Invivoscribe® Technologies, Inc., San Diego, CA, United States

Introduction• Minimal Residual Disease (MRD) is a key prognostic indicator for determining efficacy oftreatment.
• IGHV Somatic Hypermutation (SHM) is a naturally occurring process to increase B-cell receptor
diversity, and serves as a positive prognostic factor in Chronic Lymphocytic Leukemia (CLL).

Measured Read Frequency (Log10)

20-1

15LeaderR² = 0.9946

10FR1R² = 0.9871-1-2-3-4-5-6-2Dilution Level (Log10)• Next-Generation Sequencing (NGS) assays for measuring these disease markers are becoming thecutting edge of diagnostic methods, but require the development of new bioinformatics tools
to confidently analyze and report the results in a simple manner. We have developed theLymphoTrack® bioinformatics tools to analyze IGH, IGK and TRG genes on the Illumina® MiSeq® andThermo Fisher Ion PGM® platforms, for the purpose of measuring clonality, IGHV SHM and MRD.

Sanger IMGT Mutation Rate (%)• Clonality assessment is an important diagnostic indicator for the malignant transformationof lymphoid cells.

Results-3
R² = 0.9962-4-5

5-600Materials and Method

FR1
Leader-primers

DFR2

CDR1
FR1-primers

FR3
CDR2

FIGURE 4. SHM CONCORDANCE
LymphoTrack® SHM calculations for IGHV FR1 and Leader closely
match results generated by Sanger sequencing and IMGT
mutation rate calculations.

SHM CalculationJStitch PairedEnd Reads*

JH
CDR3

Filter Noise
Reads

Quality
FilterAlign To
Reference

Generate
Summary

IGH Linearity

J-gene% Total Cumulative Mutation Rate to In-Frame
No Stop
V-Coverage
Reads%
Partial V-Gene (%)(Y/N)
Codon (Y/N)

IGHV3-43_01

IGHJ6_02

61.69

61.69

5.24

YY

97.82

IGHV3-43_01

IGHJ6_02

1.42

63.10

5.24

YY

97.82

IGHV3-43_01

IGHJ6_02

1.18

64.29

5.68

YY

97.826602

IGHV3-43_01

IGHJ6_02

1.15

65.44

5.68

YY

97.82

2904082

IGHV3-43_01

IGHJ6_02

0.71

66.15

5.24

YY

97.82

2902364

IGHV3-43_01

IGHJ6_02

0.41

66.56

5.68

YY

97.82

GCCTCTGGATTCC…

290

1672

IGHV3-43_01

IGHJ6_02

0.29

66.85

5.68

YY

97.82

8

GCCTCTGGATTCA…

290

1494

IGHV3-43_01

IGHJ6_02

0.26

67.11

5.68

Y

Y

97.82

9

GCCTCTGGATTCA…

290

1361

IGHV3-43_01

IGHJ6_02

0.24

67.35

5.24

Y

Y

97.38

10

GCCTCTGGATTCA…

290

1314

IGHV3-43_01

IGHJ6_02

0.23

67.58

5.68

Y

Y

97.82

Rank

Sequence

Length

Raw
Count

1

GCCTCTGGATTCA…

290354037

2

GCCTCTGGATTCA…

2878147

3GCCTCTGGATTCA…

2906798

504

GCCTCTGGATTCA…

290405GCCTCTGGATTCA…

306GCCTCTGGATTCA…

20780% Total Reads

FIGURE 2. BIOINFORMATICS WORKFLOW
LymphoTrack® bioinformatic analysis begins on MiSeq® with stitching of paired reads (PGM® analysis
begins at the next step). The resulting sequence file is then filtered for noisy reads (non-specific orfragments that are too short), and reads that do not pass quality are removed. These reads are thenaligned to the reference sequence database, and numerous statistics are calculated. SHM rate is
calculated from the alignable portion of the V-gene. MRD detection requires the previous
identification of a clonal read, and for the user to input the amount of DNA being assayed. MRD
results are reported for exact matches, and very similar matches to the clone submitted by the user.
A confidence value is assigned to these results that tell the user the reliability of result, which is basedon how much DNA was assayed, as well as the read depth achieved for that sample. The confidence
is based on theoretical samplings using a binomial distribution.

7060

100V-gene

Detected % IVS - 0013

FIGURE 1. PRIMER DESIGN
LymphoTrack® assays have been developed for both the MiSeq® and PGM® NGS platforms,for the IGH, IGK and TRG loci. Each assay employs a separate master mix to amplify therespective upstream V-region (or Leader) with the downstream J-region. Depicted is theIGH locus. The MiSeq® assays use 24 indices, allowing for parallel analysis of 22 samples(plus 2 controls). PGM® assays use 12 indices, allowing for parallel analysis of 10 samples(plus 2 controls).

FIGURE 6. MRD DILUTION CONCORDANCE
Dilution experiment shows strong linear concordance
between dilution level and measured read frequency.

MRD Detection

864

Combo R² = 0.9961

2TRG Linearity

IGK Linearity

1090

FIGURE 5. MRD OUTPUT EXAMPLE
Example output for MRD module. Results include the read count andfrequency, as well as confidence metrics for various levels of detection.
Additional outputs will include sequence files for all reads.

Clonality
Assessment*MiSeq® only stepJ-primers

20Single R² = 0.9951

122016128Combo R² = 0.9905

4SingleR² = 0.9897

02468Contrived % IVS - 0013

10121086Combo R² = 0.9916

4Single2R² = 0.9910

000

Detected % IVS - 0009

V1015NGS Invivoscribe Mutation Rate (%)

Detected % IVS - 0030

L505101520Contrived % IVS - 0030

2502468Contrived % IVS - 0009

1012FIGURE 7. MULTIPLEXING CAPABILITY
LymphoTrack® Software is able to analyze samples mixed with all different target loci (IGH, IGK and TRG) even when sharing barcodes without a significant change in signal.

Conclusions• LymphoTrack® bioinformatics analysis accomodates a wide variety of important diagnostic andprognostic indicators. The straightforward presentation of results ensures an equally uncomplicateddetermination of clonality, SHM, and MRD.

• LymphoTrack® bioinformatics analysis also provides the resources necessary to delve deeper

into the data by providing full sequence information and V-J assignment, allowing for an
unprecedented level of granularity previously unattainable using capillary electrophoresis data.

VJ-Rearrangement

FIGURE 3. LYMPHOTRACK® SOFTWARE OUTPUTS

A typical MiSeq® IGH FR1 result from a bone marrow sample showing evidence for clonality with one sequence at 62% of total reads and mutation rate of 5%. Top 10 unmerged reads are presented inthe table. FASTQ data from either MiSeq® or PGM® can be analyzed by LymphoTrack® Software running on a Windows® PC. The LymphoTrack® Software generates frequency distributions, DNA sequences,
V-J assignment and usage, and SHM status. An add-on module allows for the tracking of clonotypes for MRD purposes.

LymphoTrack® Assays are for research use only (RUO). Not for use in diagnostic procedures.
LymphoTrack® Dx Assays are CE-IVD diagnostic products, not available for sale or use within North America.