P212

CLINICAL ASSESSMENT OF CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)
SAMPLES FOR SOMATIC HYPERMUTATION STATUS BY NEXT-GENERATIONSEQUENCING AND SANGER SEQUENCING1• Next-generation sequencing (NGS) based LymphoTrack® Dx SHM assays (MiSeq® IGH FR1, MiSeq Leaderand PGM IGH FR1) have been developed to address these limitations.

• The current method used to determine SHM status requires two steps: a PCR/capillary electrophoresis(CE) step for IGH FR1 to detect clonality, followed by a Sanger sequencing step. This multistep approachis labor intensive and time consuming.

• The clinical performance of LymphoTrack® Dx IGH assays to detect SHM status was assessed with 50
anonymous, blinded CLL samples (17 BM and 33 PB) in comparison with the traditional PCR/Sanger
method.

Diagnostic Molecular Pathology Laboratory, Memorial Sloan-Kettering Cancer Center, New York, United States
2
Invivoscribe Technologies, Inc., San Diego, United States
1• The LymphoTrack Dx IGH FR1 Assay - MiSeq was able to detect 50 out of 50 samples with 31 (62%) samples
exhibiting SHM rate ≥ 2.0 and 19 (38%) samples exhibiting SHM rate < 2.0.

FIGURE 2. WORKFLOW

No. of samples with indicated resultb

CDR2

JH

PurifyAmplicons

PCR

CDR3
J-primers

Quantify
Amplicons

Pool LibraryAnalyze
DataRun MiSeq

PCR

PurifyAmplicons

Quantify
Amplicons

Pool
Library

Genomic DNA (50 ng) was amplified with consensus primers
using a single multiplex PCR. Amplicons were purified using
the Agencourt® AMPure XP PCR Purification system before
pooling of the libraries. Amplicon libraries were quantified
before being loaded onto the MiSeq or PGM. MiSeq data wasPrepareTemplate

Analyze
DataRun PGM

generated using the MiSeq v2 Reagent kit (500 cycles) for FR1or the MiSeq v3 Reagent kit (600 cycles) for the Leader assay.
PGM data was generated using the Ion PGM Template OT2
400 kit and Sequencing 400 kit. IGH Leader was not analyzed
on the PGM due to length limitations of the PGM platform.

V –J SEQUENCE FREQUENCIES : TOP 200 SEQUENCES
Rank

Sequence

LengthRaw
count

V-geneJ-gene% total Cumulative Mutation rate to In-frame(Y/N)
reads%
partial V-gene (%)

1GCCTCTGGATTCA…

290354037

IGHV3-43_01

IGHJ6_02

61.69

61.69

5.24

Y

Y

97.82

2

GCCTCTGGATTCA…

2878147

IGHV3-43_01

IGHJ6_02

1.42

63.10

5.24

YY

97.82

3GCCTCTGGATTCA…

2906798

IGHV3-43_01

IGHJ6_02

1.18

64.29

5.68

Y

Y

97.82

4

GCCTCTGGATTCA…

2906602

IGHV3-43_01

IGHJ6_02

1.15

65.44

5.68

Y

Y

97.82

40

5

GCCTCTGGATTCA…

2904082

IGHV3-43_01

IGHJ6_02

0.71

66.15

5.24

Y

Y

97.82

30

6GCCTCTGGATTCA…

2902364

IGHV3-43_01

IGHJ6_020.4166.56

5.68

YY

97.82

7

GCCTCTGGATTCC…

290

1672

IGHV3-43_01

IGHJ6_02

0.29

66.85

5.68

YY

97.82

8

GCCTCTGGATTCA…

2901494

IGHV3-43_01

IGHJ6_02

0.26

67.11

5.68

YY

97.82

9

GCCTCTGGATTCA…

290

1361

IGHV3-43_01

IGHJ6_02

0.24

67.35

5.24

YY

97.38

10

GCCTCTGGATTCA…

2901314

IGHV3-43_01

IGHJ6_02

0.23

67.58

5.68

YY

97.82

90% TOTAL READS

805020100

A typical MiSeq IGH FR1 result from a BM sample showing evidence forclonality with one sequence at 61.69% of the total reads and mutation
rate at 5.24%. Only the top 10 sequences are presented here. FASTQ
data from either MiSeq or PGM can be analyzed by LymphoTrack® Dx

S-T-

PPV (%)NPV (%)

30 (9 BM and 21PB)

0017 (6 BM and 11PB)

100

100

100

100MiSeq Leader

30 (9 BM and 21 PB)

01c (PB)

16 (6 BM and 10 PB)

100

94

97

100

PGM FR130 (9 BM and 21 PB)

0017 (6 BM and 11 PB)

100

100

100

100

S, standard (CE/Sanger for FR1); T, NGS test; +, positive (SHM ≥ 2.0%); -, negative (SHM < 2.0); PPV, positive predictive value; NPV, negative predictive value.
bThree samples were not evaluable by standard Sanger method.
cAdditional mutations occur upstream of FR1 primer increasing the SHM rate from 1.8% to 2.4%.
aFIGURE 4. SHM CONCORDANCE
MiSeq IGH FR1 versus CE/Sanger/IMGT

Software running on a Windows PC. LymphoTrack® Dx Software
generates frequency distributions, DNA sequences, V-J assignment
and usage, and SHM status. The SHM status is determined two ways: 1)alignment statistics obtained from the BLAST algorithm for the top

No StopV-coveragecodon (Y/N)

clonal read for the calculation of mismatches and gaps to the V-genereference sequence; 2) the mutation rate is calculated as the sum ofmismatches and gaps divided by the V-gene length.

MiSeq IGH FR1 versus PGM IGH FR1151510

R = 0.98
10105050051015MiSeq IGH FR1 versus MiSeq IGH Leader

15R = 1.00

R = 0.91

MiSeq FR1 Mutation Rate (%)

FIGURE 3. LYMPHOTRACK Dx SHM SOFTWARE60S-T+

Specificity (%)

LYMPHOTRACK Dx PGM IGH FR1 SHM ASSAY

LymphoTrack® Dx SHM assays have been developed for both the MiSeq and PGM
NGS platforms. Both assays and NGS platforms were used in this study. The MiSeq
SHM Assay employs two master mixes. One amplifies genomic DNA between theupstream leader (VHL) region and the downstream joining (J) region of the IGH gene.
The other amplifies from the framework1 (FR1) to J region. Amplicon products fromVHL/J primers span the entire variable (V) region. Amplicon products from FR1/Jprimers encompass portions of the FR1 region to the downstream J region. The PGM
Assay only employs FR1/J primers. The proprietary V and J consensus primers weredesigned and adapted to enable the PCR products to be sequenced on either theMiSeq or PGM platform. The IGH Leader and IGH FR1 assays for MiSeq use 24 indicesallowing analysis of 22 patient samples. IGH FR1 assay for PGM uses 12 indicesallowing analysis of 10 patient samples. Turnaround time for the FR1 analysis of 22patient samples on either platform is 3 days. Analysis for the IGH Leader is 4 days dueto the increased sequencing length of the amplicon.

70

S+T-Sensitivity (%)

MiSeq Leader Mutation Rate (%)

CDR1
FR1-primers

FR3

LYMPHOTRACK Dx MISEQ IGH FR1/LEADER SHM ASSAY

S+T+

PGM FR1 Mutation Rate (%)

Leader-primers

FR2• The LymphoTrack Dx IGH FR1 Assay - PGM was able to detect 50 out of 50 samples with 31 (62%) samples
exhibiting SHM rate ≥ 2.0 and 19 (38%) samples exhibiting SHM rate < 2.0.

MiSeq FR1

CE/Sanger/IMGT Mutation Rate (%)

FR1J• The LymphoTrack Dx IGH Leader Assay MiSeq was able to detect 50 out of 50 samples with 32 (64%)
samples exhibiting SHM rate ≥ 2.0 and 18 (36%) samples exhibiting SHM rate < 2.0.

• The standard CE/Sanger method was able to detect 47 out of 50 samples with 30 (64%) samplesexhibiting SHM rate ≥ 2.0 and 17 (36%) samples exhibiting SHM rate < 2.0. Three samples were notevaluable and were excluded for comparison in Table 1.

Test

D1

TABLE 1. SENSITIVITY, SPECIFICITY AND PREDICTIVE VALUES OF THE THREE LYMPHOTRACK Dx ASSAYS FOR DETERMINING SHM STATUSA

Materials and Methods

V2Results• Somatic hypermutation (SHM), is an important process to increase the affinity of immunoglobulin
molecules. The presence of ≥ 2% SHM is an important prognostic factor for CLL samples.

L2

Maria Arcila , Ying Huang* , Kasey Hutt , Jeff Panganiban , Tessara Baldi ,1222
Khedoudja Nafa , Jordan Thornes , Jeffrey E. Miller and Michael Klass

Introduction

FIGURE 1. PRIMER DESIGN

20510MiSeq FR1 Mutation Rate (%)• A comprehensive NGS assay has been developed for both MiSeq and PGM platforms that identifies
clonal IGH V-J rearrangements, associated specific V-J region DNA sequences and determines theSHM status in CLL specimens as well as likelihood of expression with the determination of thesequence being in frame, the absence of stop codons and the degree of V coverage.

15500510MiSeq FR1 Mutation Rate (%)• These NGS assays have demonstrated excellent clinical concordance for determining SHM status ascompared to the standard PCR/Sanger sequencing method.

Conclusions• A comprehensive NGS assay has been developed for both MiSeq and PGM platforms that identifies clonal
IGH V-J rearrangements, associated specific V-J region DNA sequences and determines the SHM status in
CLL specimens as well as likelihood of expression with the determination of the sequence being in frame,
the absence of stop codons and the degree of V coverage.

15• These NGS assays have demonstrated excellent clinical concordance for determining SHM status ascompared to the standard PCR/Sanger sequencing method.