PS994

CELL-FREE DNA MONITORING OF MINIMAL RESIDUAL DISEASE IN AML USING A TARGETED NGS GENE PANELZhiyi Xie, Lisa Chamberlain*, Andrew Carson, Valerie McClain, Ogeen Kiya, Bradley Patay, Martin Blankfard, Timothy Stenzel, Jeffrey Miller
Invivoscribe, Inc., San Diego, United States

Results: LOD and Linearity

Methods
Cell-free DNA was extracted from fresh, frozen, and synthetic plasma using the Qiagen Circulating
Nucleic Acid extraction kit (Qiagen, Hilden, Germany). To overcome the limitations of testing cfDNA,DNA fragments with size profiles similar to cfDNA were generated using cell line DNA for initial
feasibility studies during assay development. Genomic DNA was fragmented by sonication and the DNA
fragments were size selected using bead based methods (final mean size ~160bp). Whole genome
libraries, generated from cfDNA and DNA fragments, were hybridized with MyMRD® probes togenerate targeted sequencing libraries.

Enriched libraries were sequenced using Illumina platforms. Sequencing data was analyzed using
proprietary Invivoscribe MyInformatics™ software.

Sensitivity data comparing results fromgenomic DNA input from a previous study
versus sheared DNA input is listed in Table
3. The genomic assay has been designedwith an LOD of 0.5%, our results with thecfDNA version are demonstrating an LOD
closer to 1% for observed variants. Thisdifference likely stems from the genomic
assay having higher DNA input, and fromthe smaller insert size of cfDNA.

FLT3-ITD

20.00%
10.00%
5.00%
2.50%
2.00%
1.00%
0.50%
0.25%
0.20%
0.10%
0.05%

100%
100%
100%
100%
100%
100%
100%
80%
-Results: cfDNA Preparation
DNA from plasma, synthetic plasma, and sheared cellline DNA was analyzed for concentration and size. DNA
concentrations from extracted synthetic plasma weresignificantly higher than DNA concentrations fromextracted healthy donor plasma (p < 0.001, Figure 1).
Additionally, we have found that cfDNA from clinical
AML samples is significantly higher in concentration
than cfDNA from healthy donor plasma (data notshown). DNA from these sources cover a narrow
average size range, but are significantly different.
Sheared cell line DNA has the smallest average size,
followed by Healthy Donor cfDNA, followed bysynthetic plasma DNA (p=0.03 and <0.001,
respectively, Figure 2). Data is summarized in Table 1,
which also includes example bioanalyzer traces ofcfDNA preparations.

Figure 1: DNA Concentration (ng/uL) from cfDNA Preparations

DNMT3A100%
100%100%100%100%91%
73%
27%

NPM1
100%100%
100%
86%
100%
55%
45%
0%

TP53
100%100%100%100%100%91%
82%
55%

Figure 2: DNA Average Size (bp) from cfDNA Preparations

FLT3-ITD
100%
100%
100%100%100%
50%
0%-FLT3-TKD
100%
100%100%100%100%0%0%0%

DNMT3A
100%100%
100%
100%100%0%0%0%

NPM1
100%
100%
100%100%0%0%0%
0%

TP53
100%
100%
100%
100%
100%
100%0%0%

Synthetic plasma was purchased from SeraCare (Milford, MA, USA) and DNA was extracted using thesame methods developed for human plasma. Samples were tested with the MyMRD® cfDNA assay, VRF
data is presented in Table 4. While most variants from these samples are not detected in the wild typesample, one KRAS, one FLT3, and one TP53 variant all exhibit background reads indicating that residualcrossover will affect the potential LOD for this assay, particularly for certain variants.
Table 4: Detected VRF Values from SeraCare Synthetic Plasma Samples With Expected VAF’s
Variant Type

Cell Line DNA sheared to ~160 bp

N

20

68

Concentration(mean, ng/uL)

0.85

3.21

4.92

Average size
(BA smear from50 – 400 bp)

172

188

166

SNV

Del
Ins

Variant Info
Gene
Mutation
Ref/Alt
MPL
p.W515L
G/T
NRAS
p.Q61R
T/C
IDH1
p.R132C
G/A
KIT
p.D816V
A/T
BRAF
p.V600E
A/T
JAK2p.V617F
G/T
KRAS
p.G12DC/T
FLT3
p.D835Y
C/A
TP53
p.R273H
C/T
TP53
p.R248Q
C/T
TP53
p.R175H
C/T
TP53
p.C242fs*5
AG/A
NPM1 p.W288fs*12 C/CTCTG

2
2.39
2.36
2.79
2.16
2.25
1.57
2.22
2.08
2.93
2.12
2.36
1.96
2.26

1
1.01
1.57
1.12
0.84
0.86
0.44
0.7
0.85
1.29
1.28
1.21
1.17
0.57

0.5
0.7
0.75
0.5
0.45
0.4
0.21
0.48
0.58
0.68
0.54
0.74
0.51
0.42

Overall linearity is excellent
for Seracare samples, withgood detection below
0.25%, likely due toincreased sequencing depthfor these samples
compared to previous LOD
studies as well as the use ofmolecular barcodes toreduce sequencing errors.

cfDNA Input (N = 1 to 2)

Results: Variant Detection of SeraCare samples

Table 1: Summary of Concentration, Average Size, and Bioanalyzer Traces from cfDNA Preparations
cfDNA from healthy donors
DNA from synthetic plasma

Bioanalyzer
Trace

FLT3-TKD
100%
100%100%100%100%100%
91%64%

Linearity data for SeraCaresamples is presented inFigure 4 and Table 5. VRF’sfrom samples withexpected VAF’s of 0.125%,0.25%, 0.5% (N=3), 1%, and2% are plotted for all
expected variants. Linearity
is highly dependent on theparticular variant, with R2values ranging from 0.888
to 0.993.

Table 2: R2 values and Linear Fit Equations for Sheared Contrived Samples
VariantR^2GeneTypevalue
Linear Fit
ASXL1
SNV0.994
VRF = 0.00053 + 1.21*Expected VAF
DNMT3A
SNV0.975
VRF = 0.00004 + 0.83*Expected VAF
FLT3 ITD
30 bp Ins 0.991
VRF = 0.00585 + 1.04*Expected VAF
FLT3 TKD
SNV
0.993
VRF = 0.00036 + 1.39*Expected VAF
KIT
SNV0.986
VRF = 0.00740 + 1.44*Expected VAF
NPM14 bp Ins0.981
VRF = -0.00044 + 0.69*Expected VAF
NRAS
SNV
0.983
VRF = 0.00195 + 1.02*Expected VAF
TP53
SNV
0.998
VRF = 0.00110 + 0.74*Expected VAF

Table 3: Sensitivity of Contrived Samples Compared to Standard Genomic Input
Genomic DNA Input (N = 8 to 11)
Expected VAF

Figure 4: SeraCare Synthetic Plasma Samples Exhibit Excellent Linearity

Figure 3: Sheared contrived samples demonstrate excellent linearity

Expected VAF (%)
0.5
0.5
0.59
0.83
0.5
0.6
0.63
0.47
0.41
0.41
0.38
0.48
0.14
0.28
0.35
0.7
0.65
0.53
0.65
0.62
0.52
0.68
0.77
0.64
0.51
0.66
0.63
0.42

0.25
0.09
0.23
0.36
0.37
0.19
0.28
0.34
0.16
0.15
0.17
0.46
0.18
0.21

0.125
0.1
0.08
0.25
0.09
0.11
0.2
0.29
0.11
0.19
0.13
0.14
0.03
ND

WT
ND
ND
ND
ND
ND
ND
0.06
0.06
ND
ND
0.11
ND
ND

Table 5: R2 values and Linear Fit Equations for SeraCare Samples
VariantR^2Gene Mutation Ref/Alt
Linear Fit
TypeValue
MPL
p.W515L
G/T
0.961
VRF = -0.0168 + 1.20*Expected VAF
NRAS
p.Q61R
T/C
0.958
VRF = -0.00052 + 1.25*Expected VAF
IDH1
p.R132C
G/A
0.977
VRF = -0.08722 + 1.38*Expected VAF
KIT
p.D816V
A/T
0.975
VRF = -0.06984 + 1.07*Expected VAF
BRAF
p.V600E
A/T
0.985
VRF = -0.13402 + 1.15*Expected VAF
SNV
JAK2
p.V617F
G/T
0.888
VRF = -0.07442 + 0.75*Expected VAF
KRAS
p.G12DC/T
0.917
VRF = 0.01344 + 1.02*Expected VAF
FLT3p.D835Y
C/A
0.975
VRF = -0.00401 + 1.02*Expected VAF
TP53
p.R273H
C/T
0.993
VRF = -0.11549 + 1.50*Expected VAF
TP53
p.R248Q
C/T
0.98
VRF = 0.01570 + 1.09*Expected VAF
TP53
p.R175H
C/T
0.99
VRF = 0.11969 + 1.12*Expected VAF
Del
TP53 p.C242fs*5 AG/A
0.974
VRF = 0.00651 + 1.02*Expected VAF
Ins
NPM1 p.W288fs*12 C/CTCTG 0.914
VRF = -0.13838 + 1.12*Expected VAF

Results: Depth of Coverage of Synthetic Plasma Samples
Coverage data of coding bases
for synthetic plasma samples ispresented in Figure 5 and Table
6. These samples weresequenced much deeper thanthose utilized for LOD studies
and employed proprietary
molecular barcodes to generatefamilies to correct forbackground (average depth after
removing duplicates 33702).
Overall coverage data indicatesthe potential for LOD values of
0.1% for 96% of bases, however
background reads in wildtypesamples (Table 5) demonstratethat this LOD is not global acrossthe panel.

Figure 5: Synthetic plasma sample depth of coverage

0.1% LOD – 96.34%

pathogenic variants in acute myeloid leukemia (AML) establishing remission status. This panel targets
mutation hotspots in 23 genes associated with AML. It identifies driver mutations that cause relapse in>90% of all AML patients, as well as common drivers in other myeloid neoplasms (MPN) andmyelodysplasic syndromes (MDS). We have leveraged data from literature that suggest cell-free DNA(cfDNA) isolated from blood plasma of cancer patients contains tumor-derived DNA fragments with amolecular profile similar to that of bone-marrow tumor cells, and that DNA obtained from plasma
provided a more accurate assessment of disease burden than testing circulating leukocytes, and resultscorrelated with disease burden. Therefore we set out to investigate whether the MyMRD® assay,
originally developed for genomic DNA analysis, could be applied to cfDNA to assess mutations at a levelcomparable to testing of genomic DNA and developed the MyMRD® cfDNA assay for characterization
and residue disease monitoring of targeted variants.

A linearity panel was generated by diluting
DNA from 5 cell lines containing known
variants into a background of genome in abottle (NA12878) DNA from 20% to 0.1%.
Contrived samples were then sheared togenerate fragments of approximately 160
bp to mimic cfDNA. Samples weresequenced to an average depth of 1372.
Expected variant allele frequency (VAF) isplotted against observed variant readfrequency (VRF) in Figure 3. R2 values andlinear fit equations are listed in Table 2.

0.5% LOD – 99.98%

We have developed a sensitive NGS gene panel (MyMRD®, Research Use Only), which identifies

Results: Linearity of SeraCare Samples

1% LOD – 100%

Background

Table 6: Percent of Bases Covered for various LOD’s
LOD
mean% CVHigh
5%
100.00%0.0%
100.00%2.50%
100.00%0.0%
100.00%1%
100.00%0.0%
100.00%0.50%
99.98%
0.1%
100.00%0.25%
99.76%
0.2%
100.00%0.10%
96.34%
1.8%
98.49%

Low
100.00%100.00%100.00%99.82%
99.25%
92.59%

Conclusions
This MyMRD® gene panel is a sensitive, reliable assay that provides monitoring of residual disease using
cfDNA. The assay is shown to detect clinically important driver variants and has excellent linearity andLOD for targeted variant sites. This assay can potentially replace invasive BM sampling and provide analternative test for longitudinal genetic monitoring of patients receiving targeted therapy. Additionally,
higher sensitivity can be obtained through deeper sequencing, but is limited by background noise atcertain bases.

EUROPEAN HEMATOLOGY ASSOCIATION, JUNE 14-17, 2018, STOCKHOLM, SWEDEN – INVIVOSCRIBE BOOTH #1210