EHA-3919

Small Customizable NGS Based Target Capture Panels Detect Variantsin Clinical Specimens at Frequencies as Low as 0.5%

Lisa Chamberlain1, Andrew R. Carson1, Valerie McClain1, Bradley Patay1, Ogeen Kiya1, Wenli Huang1, Daniela Hubbard2, Dariel Caguioa2, Zhiyi Xie1, Jordan Thornes2, Timothy Stenzel1 and Jeffrey E. Miller1,21Invivoscribe Inc., San Diego, CA, USA, 2LabPMM LLC, San Diego, CA, USA

Abstract

Results: LOD, LOB and Linearity

Results: Precision and Reproducibility

Background: The use of large scale hybridization panels in early stages of clinical trials for novel
therapies elicits a plethora of information for targeted biomarkers. However, as therapeutictargets are further characterized large panels generate an overly broad set of data, compromisingsensitivity in the selected biomarker subset. Therefore, once biomarker targets are identified, theuse of smaller hybridization panels can facilitate specific variant detection by analyzing specificgenomic regions of interest with greater sensitivity than larger gene panels and greater breadththan that available via PCR-based assays. Modifications of laboratory methods for small scale
panels allow for the maintenance of high analytic quality with finely targeted panels. Our smallpanels (~10 kb) focus on 1-4 genes, allowing for high-multiplexing of samples on sequencers, andreduced costs/processing times without compromising accuracy.

To assess the LoD and linearity of our small target-capture panel assays, contrived samples were assayedusing a target-capture panel covering 3 genes and 4 expected variants.

Libraries were generated by 2 operators on 2 days and were run on 2 different instruments. In total, 24
replicates each of 5%, 2%, 1%, and 0.5% (DNA:DNA) contrived samples were run.

Linearity data is graphed in Figure 1. Each expected variant is graphed separately; and displays data from
DNA:DNA% with 3 replicates for 25% and 10% and 4 replicates for 5%, 2%, 1%, and 0.5%. Equations for lineof fit and R2 values are listed on each graph. R2 values are significant, ranging from 0.994 to 0.998.

For reproducibility, the acceptance criterion was set so no more than two 1% contrived samples wereundetected. All contrived samples ≥1% DNA:DNA test positive for all 4 expected mutations, demonstrating
excellent reproducibility of this assay.

LoD was established at 0.5% (Table 1). Of particular interest, we note that Expected SNV 4 is reliably at
detected at a lower VAF of 0.25% (due to variable representation in the contrived sample).

Precision analysis observed %CV of variant allele frequency (VAF), with the goal of this metric to be ≤30%.
Data is presented in Table 2, and all expected variants pass this criterion.

No expected mutations were detected in 100% background DNA samples (N=2), LoB was established foreach expected variant as the background variant rate + 5*stdev.

Day-to-day, operator-to-operator, and instrument-to-instrument variation analysis is presented in Figure 2.
Overall there is very little variance, most of which is from variations in DNA:DNA% and random (residual)
sources.

Aims: To assess the sensitivity, linearity, and agreement of small NGS target capture panels withother assays.
Methods: Two separate next generation sequencing-target capture assays were developed withbioinformatics software. One panel contained 3 genes, including fms related tyrosine kinase 3(FLT3); the second covers only CD274 (PD-L1). Libraries were made, hybridized with baits, andsequenced. Testing was carried out by spiking in fixed amounts of mutant DNA into wild type DNA
to determine the linearity and sensitivity of the assays. Sequencing libraries were generated bycapturing with baits from either one or both panels. Sequencing data was analyzed using
proprietary software developed by Invivoscribe. Eight AML clinical samples were tested for FLT3
mutations by this small panel, amplicon based NGS assay, and capillary electrophoresis (CE) assay.
Results: DNA from 24 cell lines was assessed using both panels, confirming variants previously
detected using other methods. A validation was run on the 3-gene panel using a series ofcontrived samples generated from cell lines containing between 0.5% and 25% variant allele
frequencies for expected variants. Initial validation indicates that these small panel assays candetect mutations down to 0.5% variant allele frequencies. Assay linearity for FLT3/TKD detection
from 0.25% to 12.5% or for FLT3/ITD detection from 0.5% to 25% is excellent (R2= 0.996 and 0.998,respectively). Average sequencing coverage was high, ranging from 5,265x to 7,680x. Comparisonof FLT3 analysis of the small panel to amplicon based NGS assay and CE, FLT3-ITD showed a strong
linear relationship between calculated VAFs and detected ITD sizes.
Conclusions: Small hybridization panels are cost effective in detecting low-frequency variants fromsmaller subsets of genes while using far less DNA than individual PCR-based biomarker assays
would require. Preliminary data shows great accuracy on clinical samples. These smaller assays
focus on the most pertinent genes for a targeted therapy, and have the potential to greatly assistin understanding the molecular backgrounds of responders, super-responders, and nonresponders.

Figure 1: Linearity for 4 selected variants
VAF = 0.060444 + 1.029597*Expected
VAF

VAF = 0.0590254 + 0.9853445*Expected
VAFR2 = 0.998

R2 = 0.996

Table 2: %CV of VAFs for all expected variants are below the cutoff of 30% for precision validation.
Contrived Sample(DNA:DNA%)% CV of detected VAF (≤30%)
Expected VAF (%)

Expected Insertion
1510.18

5%
VAF = 0.0696287 +0.84947*Expected VAF

VAF = -0.012821 +0.738447*Expected VAF

2%

R2 = 0.995

R2 = 0.994

1%Insert 1
SNV 2
SNV 4Expected
SNV 4Expected
SNV 3

13.52

10.19

12.44

12.31

13.55

9.56

18.35

16.90

22.13

16.28

20.46

21.93

2.528.3611

21.97

0.5
0.50.5%SNV 3

Expected
SNV 2

28.48

0.25

Figure 2: Variance analysis of P/R data indicates very little variance stemming from random sources.

Table 1: LoD is established at 0.5%, and all expected mutations are observed at that VAF.

Variance Components

Variance Components
Var

Expected Insert 1
Expected SNV 2
Expected SNV 3
Expected SNV 4
Tests
Tests
Tests
Tests
positive
positive
positive
positive
VAF
VAF
VAF
N Expected VAF
Expected
Expected
Expected
Range
forforforfor
Range
Range
Range
VAF
VAF
VAFVAF(%)
mutation(%) mutation(%) mutation(%) mutation
(%)
(%)
(%)
(%)
12.5% 8.8-10
100%
325%
25-26.3 100%
12.5% 11.6-13 100%
12.5% 10.1-10.9 100%
310%
10-11.1 100%
5.0%
4.6-4.9100%
5.0%
4.1-4.5100%
5.0% 3.47-3.52 100%28
5%
4.7-5.6100%
2.5%
2.4-2.6
100%
2.5%
2-2.7
100%
2.5%
1.8-2.1
100%
282%
1.8-2.2100%
1.0%
1.1-1.2
100%
1.0%
0.7-1
100%
1.0%
0.6-0.9100%
281%
0.9-1.2100%
0.5%
0.5-0.7100%
0.5%
0.4-0.6100%
0.5%
0.3-0.4
100%
28
0.5%
0.5-0.8
100%
0.25% 0.3-0.4 67.9%
0.25% 0.2-0.3
100%0.25% 0.2-0.4
64%% of Total

20 40 60 80

Sqrt(VarComp)Var
20 40 60 80

Sqrt(VarComp)

Component
0.00074577

OperatorDay

0.00061351

0.10980.024770.04080.03210.22850.03572

OperatorDay

0.0010328

0.00127621

0.0089603

0.35390.0947

DNA:DNA%

0.52152887

93.4

0.72217

DNA:DNA%

2.4533482

96.9

1.5663

Within0.034408486.20.18550

Within0.0675432

2.70.2599

Total

0.55857285

100.00.74738

Total

2.5317916

100.0

1.59120.13350.02731

Component
Instrument% of Total

Component
Instrument

Component
0.0009070

0.03580.0301

Variance Components

Variance Components
Var% of Total

20 40 60 80

Sqrt(VarComp)Var% of Total

20 40 60 80

Sqrt(VarComp)

Component
Instrument

Component
0.00031962

OperatorDay

0.00028378

0.08180.016850.020670.00077069

0.22230.02776

0.73300

DNA:DNA%

0.33140204

95.60.57568

5.00.16755

Within0.01397989

4.00.11824

100.00.75269

Total

0.34675602

100.00.58886

Component
Instrument

Component
0.00038271

OperatorDay

0.00037567

0.06630.019380.00042741

0.0754

DNA:DNA%

0.53728328

94.8

Within0.02807306

Total

0.56654213

0.06760.01956

0.09220.01788

Results: Small Target Capture Panel is Well Correlated With Other Assays for FLT3-ITD
Materials and Methods
Library Preparation, Hybridization, Capture, and Washing: Whole-genome libraries wereprepared and hybridized to probe sets.
Contrived Samples: 5 cell lines were used to generate contrived mixes of DNA with 4 expectedvariants against background DNA. Contrived mixes containing 25%, 10%, 5%, 2%, 1%, and 0.5%
variant DNA were diluted in background DNA (DNA:DNA). Prior to dilution, input cell lines had
expected mutations with variant allele frequencies (VAF) of 1 (Insertion 1), 0.5 (SNV 2), 0.5 (SNV3), and 0.5 (SNV 4).
LoD, LoB, and Linearity: 3 replicates of 25% and 10% DNA:DNA dilutions, 4 replicates of 5%, 2%,1%, and 0.5% DNA:DNA dilutions, and 2 replicates of background DNA were sequenced.
Precision and Reproducibility: 24 replicates of 5%, 2%, 1%, and 0.5% DNA:DNA dilution contrived
samples were run through the 3-gene assay by 2 operators on 2 different days on 2 different
instruments.
Comparison of Small Scale Hybridization Panel Results to Capillary Electrophoresis and FLT3-ITDMRD Results: DNA from 8 subjects that had been previously analyzed by other methods wereassayed using our small-panel hybridization assay. All 8 DNA samples had previously beenanalyzed using our capillary electrophoresis (CE) FLT3-ITD assay and FLT3-ITD-MRD assay. Assay
results were compared for agreement.

FLT3-ITD results from oursmall panel target capture
Table 3: ITD calls are consistentpanel were compared to other among the 3 assays tested
proprietary assays, ourSmall NGS-ITDcapillary electrophoresis, and Sample CE ITD Panel - MRD our FLT3-ITD-MRD assay. WeITD
ITD
found 100% agreement among AML-03
AML-04+++panels for 8 clinical samples
AML-05+++tested for the FLT3-ITD
AML-08insertion (Table 3).
+++
Additionally, the VAF and ITD AML-09
AML-12+++size (bp) measured by both
AML-18+++the CE assay and the MRD
AML-58assay are highly correlated(Figures 3 and 4, respectively).

Figure 3: VAF is highly correlated among assays
R2 = 0.91

R2 = 0.99

Figure 4: Insert size is highly correlated among assays
R2 = 1R2 = 1

Conclusions
Small hybridization panels are cost effective in detecting low-frequency variants from smaller subsets of genes while using far less DNA than individual PCR-based biomarker assays. Small hybridization assays focus on themost pertinent genes for a targeted therapy and have the potential to greatly assist in understanding the molecular backgrounds of therapeutic monitoring. These panel based assays have high sensitivity and give highly
comparable results to other NGS and CE based assays.

Data was generated for Research Purposes only.