P032

Validation of an NGS based assay for monitoring FLT3 ITD and TKD
variants in AML subjects
Lisa M. Chamberlain, Zhiyi Xie, Andrew R. Carson, Ogeen Kiya, Wenli Huang, Selena Zheng, Christopher Chander, and Jeffrey E. Miller
Invivoscribe Technologies, Inc, San Diego, CA

Introduction

Results: Precision and Reproducibility

Internal tandem duplication (ITD) and tyrosine kinase domain (TKD) variants are clinically
relevant variants in the FMS-like tyrosine kinase 3 (FLT3) gene that can independently promote
constitutive activation of the FLT3 kinase. FLT3 mutations occur in approximately 30% of all
Acute myeloid leukemia (AML) patients and are indicative of poor prognosis. AML patients who
test positive for FLT3 mutations have prolonged overall and event-free survival when tyrosine
kinase inhibitors, such as midostaurin and gliteritinib fumarate are utilized during treatment.
Invivoscribe currently has an FDA approved (USA), MHLW approved (Japan), CE-marked
Leukostrat® CDx FLT3 Mutation Assay, which is used to identify ITD and/or TKD variants.
Leukostrat® CDx is a capillary electrophoresis assay that uses DNA extracted from peripheral
blood or bone marrow mononuclear cells to detect FLT3 mutations with a clinical cutoff of 0.05
signal ratio (SR; equivalent to 4.76% variant allele frequency, VAF). Here we describe thedevelopment and validation of a simple next-generation sequencing FLT3 ITD TKD MiSeq Assaywith high concordance to Leukostrat® CDx assay that is able to detect FLT3 ITD and TKD variants
quantitatively down to at least 0.5% VAF.

Precision and Reproducibility were performed using DNA from samples with known ITD and TKD
mutations diluted into background DNA from a cell line with neither of the representative
mutation types. Low negatives (LN, 1% VAF), high negatives (HN, 3.5 – 4.5%), low positives (LP, 5 –7%), and moderate positives (MP, 10%) samples were run. For the FLT3 ITD assay, one clinicalsample and two cell lines were used with ITD lengths ranging from 21 to 126 bp. For the FLT3 TKD
assay, two clinical samples and one cell line were used. %CV values for samples containing
mutations range from 1.98% to 9.86%, while those for negative samples were N/A for the ITD
assay (all negative replicates had 0 mutant reads), and 10.86% for the TKD assay.
Variance Components analysis was used to estimate variability of operator, run day (testing withFLT3 ITD TKD MiSeq Assay), instrument (MiSeq), and random error, expressed in standard
deviations, and is summarized in Table 3. In Summary: The greatest sources of variability are run day and random effects. The main exceptions to thisrule are the Low Negatives for the 126 bp ITD cell line and Clinical Sample A.
 Variation due to instrument was close to zero for all cell lines and dilution levels except for 30
bp ITD cell line MP level (5.8%) and 126 bp ITD cell line HN, LP, and MP levels (2.0%, 10.1%,and 6.8% respectively).

Materials and Methods
Cell line and clinical sample DNA were previously assayed for FLT3 mutations using theLeukostrat® CDx assay. A panel of contrived samples was generated by diluting genomic DNA
with known ITD and/or TKD variants from both clinical and cell line sources into backgroundgenomic cell line DNA with no variants. Contrived and clinical samples were used to generate
FLT3 ITD and TKD assay libraries via PCR amplification. Libraries were then pooled andsequenced. Sequencing data was analyzed using proprietary Invivoscribe software.
Limit of blank (LoB), limit of detection (LoD), and linearity were demonstrated using 52
replicates of negative samples and 48 replicates of samples with varying mutant input from celllines for ITD and TKD.
Precision and Reproducibility were assessed using replicates of both cell line and clinicalsamples with ITDs ranging from 21 to 126 base pairs and with 3 different TKD mutations.
38 clinical samples were used for clinical concordance. All 38 were used for FLT3 TKD NGS andCDx assays, while only 29 were used for FLT3 ITD NGS and CDx assays.

Table 3: VRF Components of Variance per Mutation Type and Dilution Level
Sample

FLT3
MutClinical
ITD
Sample A 21bp

30 bp ITD ITD
cell line 30bp

126 bpITD
ITD cell
126bpline

Results: LoD, LoB, and Linearity
LoB
A total of 52 replicates of samples with no FLT3 mutations were run using both the ITD and TKD
assays to calculate LoB. The mean variant read frequencies (VRF) for these samples were 0% for
ITD mutations and 0.1% for TKD mutations (Table 1 and Table 2).
LoD
Replicates of contrived samples were run on the ITD and TKD assays from 100% expected VAF
down to 0.5% expected VAF for ITD and from 50% expected VAF down to 0.5% expected VAF for
TKD. All replicates were detected, thus the LoD is 0.5% for both the ITD and TKD variants (Table1 and Table 2).
Table 1: VRF of contrived and negative
samples determined by FLT3 ITD Assay
FLT3 ITD
Expected VAF (%) N
0.00
52
0.50
61.00
94.00
97.00
910.00950.003100.00
3Mean(%)
0.00
0.50
0.90
3.80
6.00
8.60
45.80
99.99

Min(%)
0.00
0.40
0.79
3.57
5.65
8.37
44.97
99.99

Table 2: VRF of contrived and negative
samples determined by FLT3 TKD Assay

FLT3 TKD
Max(%) CV(%) Expected VAF (%) N-0.00
52
0.00
0.50
60.49
8.39
1.00
90.95
6.49
4.00
94.03
3.74
7.00
96.27
3.16
10.00
98.89
1.9520.00
346.481.70
50.00
399.990

Mean(%)
0.10
0.60
1.10
3.70
6.40
9.70
19.30
49.80

Min(%)
0.09
0.52
0.94
3.51
6.34
9.51
19.04
49.38

Max(%) CV(%)
0.13
8.75
0.65
7.57
1.19
7.29
3.87
3.66
6.55
1.06
10.09
2.14
19.59
1.43
50.19
0.81

Linearity
The contrived samples used to generate data for LoD were then assessed for linearity. A log/log
graph of linearity is displayed in Figure 1. The ITD and TKD results are highly linear withequations very close to y=x, and R2 values of 1.00.
Observed VRF vs. Expected VAF

D835Y
cell line

TKD
D835

LevelN

Average

Operator

Random
Error
SD(%)
SD(%)
SD(%)
2.3e-81.1e-7 (63.6%) 0.000 (0.0%)(12.9%)
0.144 (76.6%) 0.024 (2.0%) 0.076 (21.3%)
0.408 (78.3%) 0.000 (0.0%) 0.215 (21.7%)
0.103 (17.1%) 0.000 (0.0%) 0.227 (82.9%)
1.9e-72.5e-8 (11.8%) 0.000 (0.0%)(88.2%)
0.073 (51.6%) 0.000 (0.0%) 0.071 (48.4%)
0.072 (22.3%) 0.000 (0.0%) 0.136 (77.7%)
0.162 (71.7%) 0.046 (5.8%) 0.091 (22.5%)
1.8e-82.1e-8 (26.8%) 0.000 (0.0%)(26.8%)
0.227
0.000 (0.0%) 0.000 (0.0%)(100.0%)0.295 (30.9%) 0.168 (10.1%) 0.407 (59.0%)
1.302 (74.8%) 0.393 (6.8%) 0.647 (18.4%)
1.8e-7
4.5e-7 (71.6%) 0.000 (0.0%)(28.4%)
0.115 (64.7%) 0.000 (0.0%) 0.085 (35.3%)
0.257 (69.2%) 0.000 (0.0%) 0.171 (30.8%)
0.272 (60.3%) 0.000 (0.0%) 0.221 (39.7%)
1.7e-070.000 (0.0%) 0.000 (0.0%)(100%)
0.078 (17.0%) 0.000 (0.0%) 0.172 (83.0%)
0.089 (23.7%) 0.000 (0.0%) 0.159 (76.3%)
0.020 (0.4%) 0.000 (0.0%) 0.301 (99.6%)
DaySD(%)
LN

14

1.70E-07

4.2e-8 (23.5%)

HN
LP
MP

28
28
28

3.665
7.335
10.4780.000 (0.0%)
0.000 (0.0%)
0.000 (0.0%)

LN

28

2.10E-07

0.000 (0.0%)

HN
LP
MP

28
28
28

3.826
6.017
8.814

0.000 (0.0%)
0.000 (0.0%)
0.000 (0.0%)

LN

14

6.60E-08

2.7e-8 (41.7%)

HN

28

2.471

0.000 (0.0%)

LP
MP

28
28

10.161
20.2770.000 (0.0%)
0.000 (0.0%)LN30

6.4 e-70.000 (0.0%)

HNLPMP

30
30
30

3.326
7.261
10.6610.000 (0.0%)
0.000 (0.0%)
0.000 (0.0%)

LN

28

1.70E-07

0.000 (0.0%)

HN
LPMP

42
42
28

3.598
6.448
9.623

0.000 (0.0%)
0.000 (0.0%)
0.000 (0.0%)

R² (ITD): 1.00MiSeq

SD

%CV

0.0004

5.4

0.165
0.461
0.25

4.2
5.9
2.4

0.0005

5.1

0.102
0.154
0.192

2.5
2.5
20.000290.227

9.2

0.53
1.506

4.8
6.1

0.0007

10.1

0.143
0.309
0.354.1
43.1

0.0004

3.9

0.189
0.182
0.301

5.2
2.8
3.1

Clinical concordance was performed on 29 samples for FLT3 ITD and 38 samples for FLT3 TKD. VRF
data is graphed along with ITD insert size data with PPA, NPA, and OPA in Figure 2. There was only
one discordant call. This call was for a FLT3 TKD that was near the cutoff region for the CDx assay(0.05 signal ratio = 4.76%)

Figure 2: Clinicalconcordance between FLT3CDx assay and the FLT3 ITD
TKD reference assay is very
goodMiSeq VRF (%) =-0.002316 +0.8694548*CDx %R2 = 0.988

FLT3 ITD FLT3 ITD
Concordance NGS
NGS
Positive Negative
FLT3 CDx Positive
TKD
CDx Negative
PPA=
94.1%

ITD MiSeq Insert Size =0.3449575 +0.9992808*ITD CDx insert
size
R2 = 0.999

MiSeq VRF (%) =-0.000635 +0.9858896*ITD CDx %MutantR2 = 0.999

ITD

TKD

70.0%Y (TKD) = -8.117e-5 + 0.986*X

Total Variation

Results: Clinical ConcordanceAssay

Y (ITD) = -0.002633 + 0.9832*X
100.0%

Figure 1: Observed VRF
values of contrived andnegative samples for FLT3
ITD and FLT3 TKD graphedagainst the expected VAF
demonstrate highly linear
results down to 0.5% forboth ITD and TKD.

Clinical TKD
Sample B I836

Variation due to1021

NPA=100%

OPA=97.4%

FLT3 ITD FLT3 ITD
Concordance NGS
NGS
Positive Negative
FLT3 CDx Positive
ITD
CDx Negative
PPA=
100%

50.0%R² (TKD): 1.00
40.0%

1650024

NPA=100%

OPA=100%

30.0%
20.0%

Conclusions

10.0%
7.0%
5.0%
4.0%

NGS based methodology allows for lower LoB (0% for ITD, 0.1% for TKD) and LoD (0.5% forboth ITD and TKD) with highly concordant results compared to capillary electrophoretic
detection of FLT3 ITD and TKD mutations. The FLT3 ITD TKD MiSeq Assay provides asensitive method for detecting variants in AML subjects.

3.0%
2.0%

1.0%
0.7%
0.5%
0.4%
0.3%
0.3%

1.0%

2.0%

4.0%

10.0%

Expected VAF

AMP Global, May 16-18 2019, Hong Kong

30.0%

100.0%