Studyof MRD

FLT3 ITD MRD Testing
CLIA/CAP-validated assay

Minimal residual disease (MRD) detection has proven useful in the clinical managementof leukemia, as it can be used to better monitor patients, refine treatment, and facilitate
the development of new therapies. Mutations in the fms related tyrosine kinase 3 (FLT3)
gene are the most common mutations found in acute myeloid leukemia (AML) andare characterized by an aggressive phenotype with a high prevalence of relapse.1 The
most prevalent and clinically significant type of FLT3 mutation is an internal tandem
duplication (ITD) in the juxtamembrane domain.2 The development of a sensitive andspecific assay with design-controlled software that reliably identifies FLT3 ITD mutations
represents a significant advancement in guiding treatment decisions3.
The Invivoscribe FLT3 ITD MRD test is a next-generation sequencing (NGS)-based
assay developed with cGMP reagents and bioinformatics software following an
ISO 13485 and QSR compliant design control system meeting international regulatory
requirements required to support a seamless evolution from an LDT service to aregistered IVD. The assay has been validated to detect ITDs ranging from 3bp to 126bp
in size; however, clinical performance of the assay has demonstrated that ITDs ranging
from 3bp to over 200bp in size can be detected. The Invivoscribe FLT3 ITD MRD assay
can detect ITDs even if a diagnostic sample is not available, although testing of a
primary sample is preferred in order to identify the specific ITD (length and sequence)
to be tracked in subsequent samples. This assay can track FLT3 ITDs at a sensitivity of
10-4 or greater, provided sufficient high quality DNA is available.
References
1
NEJM. 2013; 368(22): 2059-74
2
Expert Opin. Ther. Targets. 2015; 19(1): 37-54

3

Blood Adv. 2018; 2(8): 825-831

1| FLT3 ITD MRD Clinical Testing Service
The FLT3 ITD MRD Test developed by Invivoscribe is an amplicon-based NGS assay. This assay can detectmutations with a mutant cell sensitivity of 10-4 (1 mutant cell in a background of ten thousand normal cells;
equivalent to an allelic sensitivity of 5x10-5 when a single mutant allele is present).
Recommended sample types include peripheral blood, bone marrow, or high-quality extracted andpurified genomic DNA (quantified with a method specific for double-stranded DNA). It is especially
important that DNA be free of PCR amplification inhibitors when a high quantity of DNA (> 500 ng) is
required for detection of mutations at low frequencies.
The following three controls are included in every test:
1. A positive control with an FLT3 mutant allelic concentration of 5x10-5 (a mutant cell concentration of
approximately 10-4).
2. A negative control with a wild-type FLT3 gene.
3. A no template control with water in place of the DNA sample in the PCR reaction.
The sequencing output data is analyzed using an Invivoscribe developed proprietary FLT3 ITD MRD Data
Analysis software.
FLT3 ITD MRD testing is currently being provided as a CLIA-validated service through an Invivoscribe
fully-owned subsidiary, Laboratories for Personalized Molecular Medicine (LabPMM).
Service description:
Interpretation
An interpretive
report will be
issued indicating
whether FLT3 ITD
MRD was detected.

Turn - aroundTime

Specimen
Requirements

Shipping
Conditions

Storage
Conditions

7 to 10 businessdays• 5 mL of peripheral
blood in EDTA or ACD• Ambient or Cool;
Do not freeze• Room Temp up
to 72 hours• 3 mL of bone marrow in
EDTA or ACD• 1 µg of previously
isolated DNA• 4°C up to
7 daysLabPMM is a network of internationally harmonized reference laboratories, with locations in:

Invivoscribe, Inc.
San Diego, CA, USA
Manufacturing &Headquarters
LabPMM LLC
San Diego, CA, USA
ISO 15189, CLIA/CAP
accredited& NY State licensed

2

Invivoscribe, SARLLa Ciotat, FR
Distribution
LabPMM GmbH
Munich, DE
Accredited by DAkkS
to ISO 15189
CLIA/CAP
accreditation planned

Invivoscribe Diagnostic
Technologies (Shanghai) Co. Ltd.
Shanghai, CN
ISO 15189, CLIA/CAP
accreditation planned

LabPMM GK
Tokyo, JP
ISO 15189, CLIA/CAP
accreditation planned

2| Advantages of NGS-MRD Methodologies
MRD testing by NGS provides the sensitivity and specificity needed to detect the presence of residual
disease. This method offers a number of advantages over the alternatives of flow cytometry andallele–specific oligonucleotide PCR.
These advantages include the ability to:
1. Standardize the workflow and testing in a regulated environment.
2. Detect clones and newly emerging clones or subclones in follow-up samples.
3. Test at a level of sensitivity only limited by the amount of input DNA interrogated.
4. Generate concordant, internationally harmonized sequence-specific results for mutant detection,
as well as confidence lebel calling for negative specimens.

3| Linearity of the Assay
DNA from two cell lines with known FLT3 ITD mutations (30 bp and 126 bp, respectively) were seriallydiluted into background DNA from a wild-type FLT3 cell line and tested with the FLT3 ITD MRD assay. Input
DNA quantity was 700 ng per dilution point. The Invivoscribe FLT3 ITD MRD software was used to analyze
the data (Table 1). As shown below (Figure 1), the assay has excellent linearity for the mutation to total readratio in the range of 10-2 – 10-5.
FIGURE 1: LINEARITY OF THE FLT3 ITD MRD ASSAY3-2-­‐2	  y	  =	  0.999x	
  -­‐	
  0.111	  y = 0.999x
0.111
R²	
  =+
	
  0.992	
  Detected	  Mutant/Total	  Reads	
  (Log10)	
  Detected Mutation/Total Reads (Log10)-1-­‐1	  

R2 = 0.992-3-­‐3	  -­‐	
  0.540	
  
yy	
  ==	
  1.018x	
  1.018x- 0.540Cell	
  LCelline	
  A	
  Line AR²	
  =	
  0.976	
  

R2 = 0.976-4-­‐4	  (30 bp ITD)Cell	  Line	  B	
  Cell Line B(126 bp ITD)

-5-­‐5	
  -6-­‐6	
  -6-­‐6	
  -5-­‐5	
  

-4

-3

-­‐4	
  
-­‐3	
  
Expected	  Mutant/Total	  Reads	
  (Log10)	
  

-­‐2	
  
-2

-­‐1	
  
-1

Expected Mutation/Total Reads (Log10)
Reference:3Mark J. Levis et al. (2018) A next-generation sequencing–based assay for minimal residual disease assessment in AML patients with
FLT3-ITD mutations. Blood Adv 2(8):825-831.

3

TABLE 1. LINEARITY OF THE FLT3 ITD MRD ASSAYEXPECTEDMUTATION/TOTAL
READS

1.0 x 10-2

1.0 x 10-3

1.0 x 10-4

5.0 x 10-5

2.5 x 10-5

4DETECTEDCELL LINE A (30 bp ITD)

CELL LINE B (126 bp ITD)

ITD MUTATION/TOTAL READS

ITD MUTATION/TOTAL READS

8.23E-03

2.97E-03

8.10E-03

2.58E-03

7.80E-03

2.68E-03

8.31E-03

2.32E-03

7.50E-04

2.69E-04

7.82E-04

2.46E-04

8.22E-04

3.33E-04

7.39E-04

2.45E-04

7.83E-05

7.16E-06

6.28E-05

2.29E-05

7.26E-05

2.76E-05

7.16E-05

2.62E-05

4.79E-05

1.92E-05

3.30E-05

1.60E-05

2.41E-05

1.71E-05

3.34E-05

Not Detected

2.52E-05

5.24E-06

3.39E-05

5.29E-06

2.14E-05

Not Detected

1.79E-05

Not Detected

4| Sensitivity and Specificity
The sensitivity and specificity of the FLT3 ITD MRD assay was demonstrated by testing DNA from two cell lines
diluted into background DNA from a wild-type FLT3 cell line. Input DNA quantity was 700 ng per sample. The
data was generated by different operators and instruments and conducted on different days. The results show
excellent precision and reproducibility (data not shown). Sensitivities and specificity of the FLT3 ITD MRD assay
are highlighted in Table 2. This table shows that the assay sensitivity is >95% for both small (30 bp) and large(126 bp) size ITDs. In addition, we observed no false positives in any of our negative samples (cell line C), nor
any unexpected ITDs in any of the positive samples. This points to a very high specificity and positive predictive
agreement for the assay.
TABLE 2: SENSITIVITY AND SPECIFICITY OF THE OF THE FLT3 ITD MRD ASSAYSAMPLE

ITD
SIZE
(bp)

CELL LINEA

30

CELL LINE
B
CELL LINEC

EXPECTEDFREQUENCY

SAMPLESIZE

TRUE
POSITIVE

FALSE
POSITIVE

TRUE
FALSE
SENSITIVITY SPECIFICITY
NEGATIVE NEGATIVE

1.0 x 10-4

36

36

0

N/A

0

100.00%N/A

5.0 x 10-5

68

68

0

N/A

0

100.00%N/A

126

1.0 x 10-4

36

35

0

N/A

1

97.22%

N/A

N/A

0.0

38

N/A

0

38

N/A

N/A

100.00%5

5| Feasibility Study
A group of samples with clinical outcome information were tested with the FLT3 ITD MRD assay and a capillaryelectrophoresis (CE) method by John Hopkins3. A summary of clinical samples tested by standard CE and the
FLT3 ITD MRD assays is shown in Table 3. The MRD assay correctly detected the FLT3 ITD mutations in follow-up
samples of patients who were not disease free. Patients without detectable FLT3 ITDs by the MRD assay weredisease free. These results demonstrate that the FLT3 ITD MRD assay is highly specific, and at least two orders ofmagnitude more sensitive than current commercially available capillary electrophoresis assays.

TABLE 3: SUMMARY OF CLINICAL SAMPLES TESTED BY A STANDARD METHOD AND THE FLT3 ITD MRD ASSAYSAMPLENUMBER

STANDARD FLT3/ITD PCR ASSAYDIAGNOSTIC SAMPLESITD SIZE (bp)

FOLLOW-UP
SAMPLESALLELIC RATIO

FLT3/ITD MRD ASSAY
OF FOLLOW-UP SAMPLES
DETECTED
ITD SIZE (bp)

DETECTEDITD FREQUENCYFOLLOW-UP
SAMPLES

1

33

1490%

Neg

33

1.4 x 10-6

On treatment

2

48

245%

Neg

48

1.7 x 10-4

Unavailable

3

69

1%

Neg

69

1.1 x 10-4

Died

4

24

59%

Neg

24

2.0 x 10-4

On treatment

5

72

17%

Neg

72

2.8 x 10On treatment

6

21

1%

Neg

21

4.0 x 10

15

11%

15

1.4 x 10-5

39

124%

39

3.3 x 10-4

842Unavailable

Neg

N/A

0.0

Disease Free

936

Unavailable

Neg

No PCR amplification

Disease Free

10

78

110%

Neg

N/A

0.0

Disease Free

11

96

Unavailable

Neg

N/A

0.0

Disease Free

12

30

9%

Neg

N/A

0.0

Disease Free

13

30

646%

Neg

N/A

0.0

Disease Free

7

Neg

-5
-6

RelapsedOn treatment

14

Detected*Neg

24

3.7 x 10-3

Relapsed and
Died

15

Detected*Neg

18

1.0 x 10-4

Relapsed andDied*Size & ratio unavailable

Reference:3Mark J. Levis et al. (2018) A next-generation sequencing–based assay for minimal residual disease assessment in AML patients with
FLT3-ITD mutations. Blood Adv 2(8):825-831.

6

6| FLT3 ITD MRD Assay as predictor of molecular response
FLT3 ITD mutated patients enrolled in the CHRYSALIS study, who were treated with FLT3-inhibitory oral dosesof 120mg/day or 200 mg/day gilteritinib, had their molecular response assessed from bone marrow aspirates
obtained at baseline and at ≥1 additional time point. FLT3 ITD and total FLT3 alleles were quantified using theInvivoscribe FLT3 ITD MRD assay and used to determine molecular response3. A Cox regression model of overall
survival (OS) by Kaplan-Meier estimation was used to evaluate the impact of ITD variant allele frequency (VAF)
on overall survival. Molecular response was defined as follows:
defined by an FLT3-ITD VAF #1022, 1023, or 1024, hadable 2. OS for subjects in the CHRYSALIS study (ASP2215-CL-0101)−2• Achieved
Molecular= ITD
VAF (FLT3significantly
mutantlonger
reads:FLT3
total
median OS
than reads)
those whoof
did≤10not (P , .001,
a molecularresponse
Did not achievearesponse

Molecular response

Table 2; Figure 3).

molecular response

Median OS(95% CI), d

Median OS(95% CI), dnn• Majormolecular
response = PITD VAFof ≤10−3
Discussion

TD VAF #1022

20

417 (246–NA)

60

199 (142–234),.001

TD VAF #1023

18417 (228–NA)

62

213 (143–264).003

We have described the development of a novel NGS-based sensitiveand −4highly specific MRD assay to detect FLT3-ITD mutations in• 13Negative
MRD
status= ITD VAFof ≤10417 (228–NA)
67
213 (144–264).002

patients with AML. This assay differs from other widely used NGS
assays for AML mutations in that it starts with a PCR amplification step
Pre- and posttreatment bone marrow samples were available from 80 patients withand isresponse
followed by NGS
using
a uniqueoverall
software program.
Unlikemostthose without a molecularAs
shownbelow,
patients
withmolecular
hadlongersurvival
than
LT3-ITD AML treated with FLT3-inhibitory doses of gilteritinib (ASP2215; 120 or 200 mg/d).
AML driver mutations, FLT3-ITD mutations consist of variable-lengthcomparison was made between patients achieving a molecular response (FLT3-ITD
response. This is the first demonstrationof molecularresponse
toare
a FLT3
inhibitor
intoAML3.
insertsof nucleotide sequences,which
not readily
amenableAF #1022, #1023, or negative as defined by ITD VAF #1024) by the MRD assay and thoseot achieving a molecular response by the MRD assay. The P values were determined by the
analysis by the conventional NGS algorithms. PCR, by itself, lacks theg-rank test.
necessary sensitivity because of competition from the wild-type allele
TABLE 4: SUBJECTS, OVERALL SURVIVAL
patient-specificprimers arestudyused).(ASP2215-CL-0101)
This NGS-based MRD
Table 2. OS for(unless
subjects
in the CHRYSALIS
defined by an FLT3-ITD VAF #10
assay detected FLT3-ITD mutations in patients with AML who were insignificantly longer median OS than thAchieved a molecular
Did not achieve aremission, including patients with FLT3-ITD allele burdens that fell
response
molecular response
Table 2; Figure 3).
Relationship between FLT3 variant allele frequency
below the range of linearity of the assay. It should also be noted thatMedianMedianOS
the assay was foundto beOSmore sensitive thanconventional
reversend survival
Molecular responsen PCR–based(95% CI), dassays nfor NPM1
(95%performed
CI), dtranscription
at PJohns
he NGS-based MRD assay for FLT3-ITD mutations was used to19
22 Hopkins Hospital using a published methodology.
20
417 (246–NA)
60
199 (142–234),.001
ITD treated
VAF #10urther characterize the molecular responses of patients
We have described the development ofwith gilteritinib by examining the correlation between FLT3-ITD VAF 23 The MRD assay described here fulfills the requirements for a test
18
417 (228–NA)
62
213 (143–264).003
ITD VAF #10
and highly specific MRD assay to dethat can be used by any clinician treating an AML patient withnd survival. Of the 80 patients from whom pre- and posttreatment
24
VAF #1013417 (228–NA)
67 assay213 (144–264).002
an FLT3-ITD
mutation.
This MRD
is sensitive, specific,
amples were obtained, 20 (25%) had an FLT3-ITD VAF ITD(mutant
topatients with AML. This assay differs fr(MRD
negative)standardized, and is commercially available and accessible to any
otal reads) #1022. When we empirically examined multiple
cutoffassays for AML mutations in that it starts
21
24
23
practitioner. Although there have been a number of other reported
oints from 10 to 10 , we noted that a reduction in FLT3-ITD
Pre- and posttreatment bone marrow samples were available
80 patients withand is followed by NGS using a unique s
22−2 from−3
AF (mutant to total
reads) downbetween
to #10 patientsassays response
for detecting
andmonitoringthese
was associatedwithaa molecularComparison
achieving(FLT3
ITD
VAF ≤10 FLT3-ITD, ≤10 , ormutations,
negative as
defined by ITD VAF ≤10−4) by the
FLT3-ITD AML treated with FLT3-inhibitory doses of gilteritinib (ASP2215; 120 or 200 mg/d).
22
AML
driver mutations,
FLT3-ITD mutatio
havethedisadvantagesoflackingsensitivity,
being
overlycumberonger median OS.MRD
Therefore,
wedefined#10asamolecular
assay and those not achieving
a molecularresponse
the MRD
assay.
The Presponse
values(FLT3-ITD
were determined by
the log-rank
test.
A comparison
was madebetween bypatients
achievinga molecular
17,24-26
inserts of nucleotide sequences, whichsome,or not as
being
publicly
Midostaurin,
TKIthosewithesponse. This level also approximates the lower limit of detectability
VAF #1022, #1023, or negative
definedby ITDavailable.
VAF #1024) by the
MRD assay aand27
activityresponse
against by
FLT3,
was
recently
regulatory
approvalor the CE assay, Reference:as performed at different institutionsnot
throughout
achieving a molecular
the MRD
assay.
The Pgranted
values were
determinedby the inanalysis by the conventional NGS algorit
3
23
M20
arkpatients,
J. Levis 18
et hadal. (2018)
A next-generation
sequencing–based
for minimal
residual disease
in AML patients with
the United States forassay
the treatmentof FLT3-mutated
AML,28assessment
he world.16 Of thesean FLT3-ITD
VAF
#10
and
log-rank
test.
necessary sensitivity because of compe24
FLT3-ITDandmutations.
AdvVAF2(8):825-831.
major molecular response),
13 had anBlood
FLT3-ITD#10several FLT3 inhibitors have entered pivotal trials. Therefore, the(unless patient-specific primers are us
MRD-negative status). The median time to achieve the minimum
introduction of this assay is timely, because the future use of theseassay detected FLT3-ITD mutations in pa
AF was 57 days. Patients who had a molecular response, as
agents could be guided by the presence or absence of detectable

TD VAF #10(MRD negative)

Discussion

FIGURE 2: SUBJECTS, OVERALL SURVIVAL STRATIFIED BY MOLECULAR RESPONSE, remission,
USING THE
including patients with FLT3
INTERNATIONALLY-HARMONIZEDFLT3
ITD MRD
ASSAY(10-2)allele frequency
Relationshipbetween
FLT3
variant
below the range of linearity of the assay
the assay was found to be more sensitiv
and survival
Median OS, days (95% CI)-2
The NGS-based MRD assay for FLT3-ITD
mutationsto
FLT3-ITD VAF 10417was(246, usedNA)
VAF 10-2 of patients
199 (142, 234)
further characterize the molecularFLT3-ITD
responses
treated
Log-Rank Test: P  0.001
with gilteritinib by examining the correlation
between FLT3-ITD VAF
Hazard Ratio: 0.272 (95% CI: 0.12, 0.61)
100
and survival. Of the 80 patients from whom pre- and posttreatment
samples were90obtained, 20 (25%) had an FLT3-ITD VAF (mutant to80 22
total reads) #10. When we empirically examined multiple cutoff
7021
points from 10
to1024, we noted that a reduction in FLT3-ITD
60
22
VAF (mutant towas associated with a
50 total reads) down to #10
longer medianOS.
Therefore,
wedefined#1022 as a molecular40
response. This30level also approximates the lower limit of detectability
20
for the CE assay,
as performed at different institutions throughout
10
the world.16 Of
these 20 patients, 18 had an FLT3-ITD VAF #1023
024(major molecularresponse), and13 had an FLT3-ITD
VAF #10600
200
400
0
(MRD-negative status). The median timeto achieve the minimum
Time (days)
Number of patients at risk
VAF
was 57-2 days. Patients who had a molecular response, as

Overall survival (%)

24

igure 3. OS for subjects in the CHRYSALIS study

ASP2215-CL-0101) according to molecular response.

aplan-Meier analysis of survival for subjects treated with FLT3-

hibitory doses of gilteritinib (ASP2215), according to ITD VAF

etected by the NGS-MRD assay after treatment.

FLT3-ITD VAF 10
20
FLT3-ITD VAF 10-2 60

15
21

4
3

0
0

transcription PCR–based assays for
Hopkins Hospital using a published met

The MRD assay described here fulfills
that can be used by any clinician tre
an FLT3-ITD mutation. This MRD astandardized, and is commercially avai
practitioner.23 Although there have bee
assays for detecting and monitoring
have the disadvantages of lacking sens
some, or not being publicly available.17
activity against FLT3,27 was recently gr
the United States for the treatment ofseveral FLT3 inhibitors have enteredintroduction of this assay is timely, bec
agents could be guided by the presenc

Reference:3Mark24J. APRIL
Levis2018
et al.
(2018) 2,ANUMBER
next-generation
sequencing–based assayNGS
for ASSAY
minimaldiseasein AML patients withx VOLUME
8
FORresidual
MRD IN FLT3
-ITD AMLassessment
829
FLT3-ITD mutations. Blood Adv 2(8):825-831.

FLT3-ITD VAF 
FLT3-ITD VAF 

7 P
Log-Rank Test:

The Invivoscribe FLT3 ITD MRD Assay is a sensitive, specific, and prognostically relevant assay for thedetection of MRD in FLT3 ITD mutated AML patients. It can be used as a surrogate endpoint - reducing
clinical trial duration and cost and, most importantly, giving patients access to drugs more quickly.

029 Rev. E May 2018 280370©2018 Invivoscribe Technologies, Inc. (Invivoscribe®). All rights reserved. The trademarks mentioned herein are the property of
Invivoscribe® and/or its affiliates, or (as to the trademarks of others used herein) their respective owners.
FLT3 Mutation Testing is performed pursuant to patents licensed from Takara Bio Inc. of Kusatsu (Shiga prefecture), Japan.
For additional patent information, contact our legal department by emailing legal@invivoscribe.com, or by telephone in the
United States: 1 (858) 224-6600.

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