H04

Assessment of Minimal Residual Disease in Pa3ents with Acute Myeloid
Leukemia by Monitoring FLT3 and NPM1 Muta3onsZhiyi Xie1, Andrew R. Carson1, Valerie McClain1, Chris Chander1, Selena Zheng1, Tim Stenzel1, Mark J. Levis3, and Jeﬀrey E. Miller1,2,1Invivoscribe, San Diego, USA, 2LabPMM, San Diego, USA, 3Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, BalQmore, USA

Introduc3onMaterial and Methods

Current understanding of the soma3c driver muta3ons in acute myeloid leukemia (AML)
make gene3c-based classiﬁca3on of AML essen3al to properly stra3fy pa3ents, assess risk,
and op3mize treatments. Growing evidence supports that the presence of speciﬁc-driver
muta3ons at residual disease levels impacts pa3ent outcomes. In order to op3mizetreatments and intervene early, it is important to iden3fy the emergence or reemergence of
these soma3c muta3ons throughout the course of the disease. The most commonly mutatedsoma3c biomarkers helpful in stra3ﬁca3on of AML are muta3ons in the fms related tyrosine
kinase 3 (FLT3) and the nucleophosmin (NPM1) genes. The development of interna3onallystandardized, regulatory-compliant assays that detect FLT3 and NPM1 muta3ons at MRD
levels represents a signiﬁcant advancement in guiding the treatment of AML.

The NGS FLT3/ITD and NPM1 MRD assays were designed so they could be run separately ortogether. The FLT3/ITD assay targets exons 14 and 15; the NPM1 assay targets exon 12 of
their respec3ve genes. The DNA input for the assays was 700 ng (>100,000 cell equivalents)for MRD detec3on. Less DNA input (as low as 50 ng) is feasible to detect rela3vely highmuta3on frequencies in diagnos3c samples. Three controls: posi3ve, nega3ve, and notemplate, were included in every test. Up to 24 amplicons from either one or both assays
were puriﬁed, pooled, and sequenced. The sequencing data was analyzed using proprietaryso[ware developed by Invivoscribe. In the combo linearity assay, a FLT3 library and a NPM1
library were combined and loaded onto two MiSeq instruments. DNA isolated from bonemarrow aspirates and peripheral blood from AML pa3ents was tested by both assays.

Results: LoD, LoB, and Linearity of Contrived Samples

Results: Clinical Sample Tes3ng

Prac3cal, cost-eﬀec3ve, standardized tests were developed to simultaneously detect andmonitor FLT3/ITD and NPM1 muta3ons in AML pa3ents in a clinical seing. The LoD, LoB, andlinearity of these assays were accessed by running the libraries alone (single assay) or
together (combo assay).
For the FLT3/ITD MRD assay, DNA from two cell lines with known ITD (30 bp homozygous and126 bp heterozygous, respec3vely) were serially diluted into background DNA from a wildtype FLT3/NPM1 cell line and tested. For the NPM1 MRD assay, DNA from a cell line with aknown 4 bp inser3on (heterozygous) was serially diluted into background DNA from the samewild-type FLT3/NPM1 cell line and tested.
The experimental data for the NPM1 MRD assay is presented in Table 1 and ploked inFigure 1. As shown in Figure 1, the linearity of the assay is excellent repor3ng in the range of10-2 – 10-5. There is no signiﬁcant diﬀerence between NPM1 linearity detected by the single
assay or the combo assay. The experimental data for the FLT3/ITD MRD assay is presented inTable 2 and ploked in Figure 2. As shown in
Table 1. Linearity determina-on of the NPM1
Figure 2, the linearity of the assay is excellent
MRD assay in single and combo format-2-5
repor3ng in the range of 10 – 10 . There is no
Detected
Muta-on/
Expectedsigniﬁcant diﬀerence between FLT3/ITDTotal Reads
linearity detected by the single assay or the Muta-on/Total Reads
SingleCombo
4.83E-01
4.95E-01
combo assay.
5.00E-01-5
4.84E-01
4.98E-01
The limit of detec3on (LoD) is 5x10 for both
9.57E-03
9.29E-03
assays. No muta3on is detected in the nega3ve
1.00E-02
8.35E-03
7.93E-03
sample in either of the assays (data not
5.00E-03
3.93E-03
3.67E-03
shown), indica3ng that the limit of blank (LoB)
5.81E-04
5.61E-04
1.00E-03
6.05E-04
5.03E-04
is zero.
5.00E-04
3.51E-04
3.77E-04
Figure 1. Linearity of the NPM1 MRD assay
5.34E-05
3.44E-05
1.00E-04
in single and combo format
1.08E-04
1.19E-04

MRD tests are more sensi3ve and speciﬁc than commercially available capillaryelectrophoresis (CE) assays. As shown in Tables 3, seven FLT3/ITD and three NPM1 muta3ons
were detected in sample AML-04 by the MRD assays. Mul3ple muta3ons were also detectedin samples AML-05 and AML-12. In addi3on, nucleo3de sequence informa3on was obtained,
providing accurate informa3on for tracking and pa3ent follow-up tes3ng.

6.23E-05
4.15E-05
2.15E-05
3.63E-05
2.04E-05
9.49E-06
1.29E-05
1.09E-05
3.38E-05
2.93E-05
1.02E-05
1.10E-05

5.00E-05

2.50E-05

1.00E-05

6.15E-05
2.98E-05
1.57E-05
2.42E-05
2.22E-05
1.57E-05
1.33E-05
1.03E-05
2.81E-05
1.53E-05
1.87E-05
1.30E-05

Table 2. Linearity determina-on of the FLT3/ITD MRD assay in single and combo format
Expected
Muta-on/ Total Reads-2

1.0x10-3

1.0x10-4

1.0x10-5

5.0x10-5

2.5x10CESample ID
Size

AML-04

AML-05
AML-12SR1930.24

24.08

0.03

24

0.22

FLT3/ITD
NGS- MRD
Muta-on/Total
Size
Reads
192
1.48E-01
51
5.17E-04
24
1.55E-04
99
1.42E-04
27
4.97E-05
81
2.21E-05
75
5.52E-06
24
2.97E-02
33
8.41E-05
42
7.51E-06
24

1.78E-01

NPM1
NGS- MRD
Muta-on/Total
Muta-on
Reads

CE

insTGTA

4.69E-01

insTGCA

3.48E-04

insTCTG

4.30E-06

Not
TestedinsTCTG

4.06E-01

Pos

insTGCA
insTGTA

4.58E-01
2.31E-04

Pos

A group of samples with clinical outcome informa3on were tested with FLT3/ITD and NPM1
MRD assays. A summary of clinical samples tested by standard CE and MRD assays is shownin Table 4. The MRD assay correctly detected the FLT3/ITD and NPM1 muta3ons in followup samples of pa3ents who were not disease free. Pa3ents without detectable FLT3/ITD by
the MRD assay were disease free.
Table 4. Summary of clinical sample tes-ng by standard PCR assay and the MRD assay
FLT3 /ITDSample
ID

JH standard FLT3/ITD CE assay
Diagnos-c sample
ITD size(bp)Allelicra-oFollow-upsample

NPM1
IVS FLT3/ITD MRD
assay of follow-up
samplesIVS NPM1 MRD
Pa-ent
JH NPM1 assay of followStatus
CE assay ofup sample(As of June
(DetectedDetected Diagnos-c2016)
ITD Sizemutant /sample
mutant /Total(bp)Total reads
reads)

JH-1

33

1490%

Neg

33

1.38E-06

Pos

0JH-3

69~1%

Neg

69

1.11E-04

Pos

1.22E-04

On
treatment
Deceased

JH-6

21~1%

Neg

21

3.96E-06

Pos

1.57E-05

Relapsed

1511%

15

1.35E-05

Pos

39124%

39

3.30E-04

Pos

2.77E-04

On
treatment

JH-7

Detected Muta-on/Total Reads
Cell Line A

Table 3. MRD assays are sensi-ve and speciﬁc

Cell Line B

Neg

SingleCombo

SingleCombo

JH-1196

Unavail.

Neg

N/A0

Pos0

Disease free

8.12E-03

8.40E-03

2.82E-03

2.38E-03

8.02E-03

8.24E-03

2.50E-03

2.17E-03

JH-12

309%

Neg

N/A0

Pos

0

Disease free

7.50E-04

7.40E-04

2.52E-04

3.00E-04

JH-13

30

646%

Neg

N/A0Neg

0Disease free

7.48E-04

7.72E-04

2.44E-04

1.96E-04

7.48E-05

8.95E-05

7.24E-06

7.67E-06

Neg

24

3.66E-03Pos

2.03E-03

Deceased

6.38E-05

5.76E-05

2.05E-05

3.05E-05

NegN/A0

N/A

0

Disease free

4.83E-05

4.28E-05

1.62E-05

1.46E-05

3.34E-05

2.25E-05

1.48E-05

9.61E-06

2.31E-05

1.47E-05

0.00E+00

0.00E+00

3.16E-05

2.47E-05

5.30E-06

1.83E-06

Figure 2. Linearity of the FLT3/ITD MRD assay in single and combo format

JH-14
JH-16

Detected (size & ra3ounavailable)
Detected (size & ra3ounavailable)

Conclusions
These standardized NGS FLT3/ITD and NPM1 MRD assays are highly speciﬁc and at least twoorders of magnitude more sensi3ve than current commercially available capillaryelectrophoresis assays. Results of clinical samples tested by these MRD assays showedconcordance with capillary-electrophoresis assay results and clinical outcomes. Theses LDT
assay services provide reliable and cost-eﬀec3ve tools to assess MRD in AML pa3ents.
Developing FLT3 and NPM1 MRD muta3on assays with bioinforma3cs in compliance with ISO
13485 and QSR design control requirements makes these assays suitable for pre-market
submissions to worldwide regulatory authori3es.

AMP Global, April 3–5 2017, Berlin, Germany