H58

Multiple Highly Concordant Assays Facilitate Analyses of Clinical Samples at Different Scales and Sensitivities
Lisa Chamberlain1, Valerie McClain1, Andrew R. Carson1, Angelo Arias1, Ogeen Kiya1, Wenli Huang1, Christopher Chander1, Selena Zheng1, Daniela Hubbard2,Dariel Caguioa2, Zhiyi Xie1, Jordan Thornes2, Bradley Patay1, Timothy Stenzel1, and Jeffrey E. Miller1,2 - 1Invivoscribe, San Diego, CA USA, 2LabPMM, LLC, San Diego, CA USA

Introduction

Results: Large Insertion Detection

Results: Single Nucleotide Variant Detection

The uniform detection of clinically relevant mutations is critical to drug and diagnostics
development. Technologies such as next-generation sequencing (NGS) have improved sensitivityof detection, but to date there is no “catch-all” method that addresses both the broad discovery
phase of development and the more focused approach of target validation. We have developed
several methods of variant detection to address this need for varied sensitivity and scale, utilizing
technologies ranging from capillary electrophoresis (CE) to next-generation sequencing. For
example, Invivoscribe’s proprietary NGS large panel-based assays (such as MyHeme® andMyAML®) and NGS medium panel-based assays (MyMRD®) can be useful in early investigational
stages of drugs and treatments, while highly targeted amplicon-based assays (FLT3-ITD MRD) canmore specifically hone in on clinically relevant biomarkers in later stages of development. Here
we demonstrate the ability to reliably detect clinically actionable NPM1, FLT3-ITD, and FLT3-TKD
variants at varying frequencies in a set of clinical samples across multiple Invivoscribe-developed
assays including CE single gene analysis, NGS-amplicon assays, and NGS small- to medium-scale
targeted multi-variant panels.

We compared performance of CE, amplicon-based, and panel-based NGS methodologies toidentify internal tandem duplications (ITDs) in FLT3. Of the eight clinical samples examined, fivewere positive for the FLT3-ITD; all three assays detected FLT3-ITD at similar frequencies (Table 2).
Following dilution of the clinical samples, low frequency mutations are no longer observable inthe less sensitive CE assays. However, using the more sensitive NGS assays, variants are stilldetected at frequencies of 10-2 and 10-3.

We compared performance across the three assays of a single nucleotide variant in the tyrosine
kinase domain of FLT3 (FLT3-TKD). Two clinical samples were positive for the mutation (asdetermined by CE) and were detected by all three assays (Table 3). As with the other variants,NGS-panel assays detected lower frequency variants that were not detected by the CE assay atfrequencies of 10-2 and 10-3.

SampleDilutionNameAML-03

1Results: Small Insertion Detection
We compared performance of CE, amplicon-based, and panel-based NGS assays to identify afour base pair insertion in NPM1. Of the eight AML samples, five were positive for thisinsertion. The CE- based assay detected mutations in 2 out of 4 samples tested (Table 1), whilethe NGS assay identified NPM1 insertions in five samples.

NGS -Amplicon

CallSizeSR

Var (%)Size

VRF (%)

-N/A

N/A

N/A

N/A

-

N/A

192.520.24

19N/A
150.050.02
0.4-+1925124192

192

51

192

52

192

424
24
72
72
72
24

30.42
N/A
91
50.4
18

24
24
72
72
72
24

4
1
N/A
95
13
121

192
192
24
24
72
72
72
24
24
24
21
21
21-6
1
30.5
N/A
94
13
1
21
20.2
96
14
1
N/A

AML-04

Materials & Methods
Eight clinical AML samples from peripheral blood and bone marrow were tested at various
dilutions using multiple Invivoscribe-developed CE, NGS-amplicon, and NGS-panel assays.
Several dilutions of these samples were tested to demonstrate sensitivity of the NGS assays.
An additional 30+ AML samples were run on the CE and NGS-amplicon assays for FLT3 variants.
In this study, small-scale NGS panels are defined as a few genes, and medium-scaled panels
are 20+ genes. The DNA input for the assays ranged from 50-700 ng of high-quality genomic
DNA. In NGS-Amplicon assays, regions containing variants were amplified by PCR. Up to 24
samples were purified, pooled, and sequenced. Target capture
Table Legend
libraries were generated for NGS panel assays and sequenced. CE Capillary Electrophoresis
The Illumina MiSeq® platform was used for all NGS assays in this SR Signal Ratio
study. Sequencing data was analyzed using proprietary VRF Variant Read Frequency
Invivoscribe MyInformatics® software, which produced variant + Positive detection- Not detected
read frequencies (VRF) defined as variant reads divided by total
Sample not tested
reads.

1CE Data

AML-05
AML-08
AML-09AML-12

AML-18AML-58

1/10
1/100
11/10
11
1/10
1/100
11/10
1/100
11/10
1/100
1+

24.08

0.03

3+

N/A
72.18

N/A
10.01N/A
91+

240.2218+-21

N/A

18.03

N/A

95

N/A

212121-Figure 1. Concordance between methods (R2)

AML-03

11
1/10
1/100
11/10
11
1/10
1/100
11/10
1/100
1
1/10
1/100
1

AML-04
AML-05
AML-08
AML-09AML-12

AML-18AML-58

CE Data

NGS-AmpliconMedium-Scale NGS Panel

Call

Variant

VRF(%)

Variant

VRF(%)+

insTGTA
insTGTA

N/A
46.9
3.5

insTCTG

40.6

insTCTG
insTCTG
insTCTG
insTGCA

N/A
46.1
2.6
0.3
31.0

insTCTG

34.6-

N/A

insTGTAinsTGTA
insTGTA
insTCTG
insTCTG
insTCTG
insTCTG
insTCTG
insTGCA
insTCTG
insTCTG
insTCTG
insTCTG
insTCTG
-N/A
37.6
2.4
0.2
28.9
4.3
N/A
34.7
2.6
0.3
31.2
3.9
0.4
38.2
4.3
0.4
N/A

+-

21

7.17
0.76
N/A

-

96

N/A

Figure 2. NGS-Amplicon by CEcomparison for FLT3-ITD
NGS-Amplicon VRF(%)

Dilution

95

Measured allele frequencies are highly similar between the assays, with R2 values of linear fit
ranging from 0.77 to 0.99 (Figure 1). The lowest R2 values are observed when comparing resultsin amplicon-based versus hybridization-based assays due to the difference in amplification bias
between the two methods. A study conducted with an additional 31 AML samples indicatedexcellent linear fit between the CE-based assay and the NGS-amplicon based assay, with an R2
value of 0.999 (Figure 2).

Table 1. Variant: NPM1
Sample NameNGS-Panel
Small-Scale
Medium-Scale
Size VRF (%) Size VRF (%)

Table 3. Variant: FLT3-TKD
SampleName
AML-03
AML-04

Dilution

CE Data111/10
1/100

NGS -Amplicon

Call

Variant

VRF (%)

D835E
D835Y
-

1.1
1.3
N/A
N/A--

N/A
N/A

NGS-Panel
Small-Scale
Medium-Scale
Variant VRF (%) Variant VRF (%)
-N/A
N/A
N/A

1.3
1.3
N/A
N/A
N/A
N/A
N/A

AML-05

1+AML-08

11
1/10
1/100
11/10
1/100---

N/A

D835E
D835Y-

1+D835V
D835Y

28.1
17.3

D835V
D835Y

30.2
16.9-

N/A

AML-09AML-12

AML-18

1/10
1/100

AML-58

1-

D835E
D835Y
D835V
D835Y
D835V
D835Y
D835V
D835Y-N/A
N/A
N/A
N/A
1.1
1.3
N/A
N/A
N/A
N/A
N/A
N/A
N/A
28.1
17.1
4.8
2.6
0.7
N/A
N/A

Additionally, 39 clinical AML samples with varying TKD variant allele frequencies (2%-56%; asdetected by the CE-based CDx assay) were tested using the NGS-amplicon based FLT3-TKD assay.
The NGS assay correlates with excellent linearity when compared with the CE assay (Figure 3).

Figure 3. NGS-Amplicon by CE comparison for FLT3-TKD
NGS-Amplicon VRF (%)

Table 2. Variant: FLT3-ITD

Linear Fit
y = -0.0023 + 0.86x
R2 = 0.9889
Observations = 39

CE Var (%)
Linear Fit
y= 0.34 + 0.99x
R2 = 0.9994
Observations = 31

CE Var (%)

CE Var (%): Percentage of variant
converted from signal ratio

Conclusions
The ever-changing landscape of drug and molecular diagnostics development requires the diligent
application of both cost-effective and scale-appropriate assays. Invivoscribe® assays offer excellent
detection and frequency concordance of clinically actionable variants in this set of AML clinicalsamples, demonstrating reliable variant analysis across assays of different methodologies (CE andNGS) and sensitivities. These assays could be used interchangeably as required for different stagesof drug development or clinical treatments. Coupling flexibility of scale (with the associateddifferences in cost and specimen requirements) with reliability of detection is of criticalimportance for the evaluation and improvement of patient outcomes and personalized medicine.

AMP, November 16 –18 2017, Salt Lake City, USA