Instructions for Use

LeukoStrat CDx FLT3 Mutation Assay®
For detection of internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations in the FMS-like tyrosine
kinase 3 (FLT3) gene.
For In Vitro Diagnostic Use

Catalog #K-412-0331

Product

LeukoStrat CDx FLT3 Mutation Assay

Quantity

33 Reactions

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Table of Contents
1.

PROPRIETARY NAME .................................................................................................................................................................................. 4

2.

INTENDED USE ............................................................................................................................................................................................ 4

3.

GLOSSARY .................................................................................................................................................................................................... 44.

SUMMARY AND EXPLANATION OF THE TEST ............................................................................................................................................. 4

5.

PRINCIPLES OF THE PROCEDURE ............................................................................................................................................................... 5

6.
7.

5.1.
5.2.

Internal Tandem Duplication (ITD) Mutations of FLT3 ............................................................................................................................... 5
Tyrosine Kinase Domain (TKD) Mutations of FLT3....................................................................................................................................... 6

REAGENTS AND MATERIALS ....................................................................................................................................................................... 7
INSTRUMENTS/ACCESSORIES ................................................................................................................................................................... 10

8.

7.1.

Software (Provided) .................................................................................................................................................................................................. 10

9.

8.1.

Cyber Security Precautions .................................................................................................................................................................................... 12

10.

9.1.
9.2.
9.3.

Precautions .................................................................................................................................................................................................................... 12Interfering Substances ............................................................................................................................................................................................. 12
Specimen Requirements and Handling ............................................................................................................................................................ 12

11.

10.1.
10.2.
10.3.
10.4.
10.5.
10.6.
10.7.
10.8.
10.9.
10.10.
10.11.
10.12.
10.13.
10.14.
10.15.
10.16.
10.17.
10.18.
10.19.

Specimen Inspection ................................................................................................................................................................................................. 13
Sample Processing Preparation ........................................................................................................................................................................... 13
Diluting Clinical Samples......................................................................................................................................................................................... 13
Mononuclear Cell (MNC) Isolation ..................................................................................................................................................................... 13
Mononuclear Cell Count .......................................................................................................................................................................................... 14
Preparing Samples for DNA Extraction and Isolation Completion ...................................................................................................... 14
Preparing QIAcube Automation Station........................................................................................................................................................... 15
DNA Extraction ............................................................................................................................................................................................................ 15
Quantification and Dilution of DNA ................................................................................................................................................................... 15
Amplification ................................................................................................................................................................................................................ 16
Restriction Digest (TKD Mutation Only) .......................................................................................................................................................... 17
Capillary Electrophoresis Detection .................................................................................................................................................................. 18
Prepare Size Standard Solution, if Needed ..................................................................................................................................................... 19
Prepare Sample Plate ................................................................................................................................................................................................ 19
Set up PlateMapper with LeukoStrat CDx FLT3 Software ........................................................................................................................ 20
Set Up 3500xL Dx Software ................................................................................................................................................................................... 27
Run 3500xL Dx Genetic Analyzer........................................................................................................................................................................ 29
Data Analysis with GeneMapper Software ...................................................................................................................................................... 29
Data Analysis with LeukoStrat CDx FLT3 Software .................................................................................................................................... 31

12.

11.1.
11.2.

Run Validity ................................................................................................................................................................................................................... 36
Extraction Control and Sample Validity ........................................................................................................................................................... 36

13.

14.

WARNINGS AND PRECAUTIONS ................................................................................................................................................................ 11
SPECIMEN COLLECTION AND PREPARATION ........................................................................................................................................... 12

ASSAY PROCEDURE ...................................................................................................................................................................................13QUALITY CONTROL ...................................................................................................................................................................................36

INTERPRETATION OF RESULTS................................................................................................................................................................. 37

RETESTING ................................................................................................................................................................................................38
13.1.
13.2.
13.3.
13.4.
13.5.
13.6.

Invalid Runs .................................................................................................................................................................................................................. 38
Invalid Extraction Control within Valid Runs................................................................................................................................................ 38
Invalid Samples within Valid Runs ..................................................................................................................................................................... 38
Fail Details and Retesting ....................................................................................................................................................................................... 38
Multiple Failures within a Run ............................................................................................................................................................................. 41
Dye Shift .......................................................................................................................................................................................................................... 42

LIMITATIONS OF PROCEDURE .................................................................................................................................................................. 43

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15.

EXPECTED VALUES ....................................................................................................................................................................................43

16.

15.1.

Expected Size of Amplified Products ................................................................................................................................................................. 43

17.

16.1.
16.2.
16.3.
16.4.
16.5.
16.6.
16.7.
16.8.
16.9.
16.10.
16.11.
16.12.
16.13.
16.14.

Analytical Sensitivity – Limit of Blank (LoB) ................................................................................................................................................. 44
Analytical Sensitivity................................................................................................................................................................................................. 44
Precision ......................................................................................................................................................................................................................... 45
Operator-to-Operator Reproducibility (cell-lines) ..................................................................................................................................... 46
Operator-to-Operator Reproducibility (clinical samples) ....................................................................................................................... 46
Lot-to-Lot and Instrument-to-Instrument Reproducibility .................................................................................................................... 47
Interfering Substances – Exogenous.................................................................................................................................................................. 47
Interfering Substances – Endogenous .............................................................................................................................................................. 47
Interfering Substances – Treatment Drugs .................................................................................................................................................... 47
Carryover and Cross Contamination ................................................................................................................................................................. 47
DNA Input....................................................................................................................................................................................................................... 47
Validation of EDTA Blood Collection Tubes ................................................................................................................................................... 47
Equivalency: NEBuffer r3.1 vs NEBuffer 3.1 .................................................................................................................................................. 48
Density Gradient Media Validation .................................................................................................................................................................... 48

18.

17.1.
17.2.
17.3.
17.4.
17.5.
17.6.
17.7.
17.8.
17.9.
17.10.
17.11.
17.12.
17.13.
17.14.
17.15.
17.16.
17.17.
17.18.
17.19.

Study Overview (IVS-056-001) ............................................................................................................................................................................ 48
Study Objectives (IVS-056-001) .......................................................................................................................................................................... 48
Patient Population (IVS-056-001) ...................................................................................................................................................................... 49
Selection of Samples for Reference Method Testing (IVS-056-001) .................................................................................................. 49
Safety Analysis (IVS-056-001).............................................................................................................................................................................. 49
Effectiveness (IVS-056-001) ................................................................................................................................................................................. 49
Conclusions (IVS-056-001) .................................................................................................................................................................................... 50
Pivotal Bridging Study Overview (IVS-062-002)......................................................................................................................................... 51
Study Objectives (IVS-062-002) .......................................................................................................................................................................... 51
Patient Population (IVS-062-002) ...................................................................................................................................................................... 51
Safety Analysis (IVS-062-002).............................................................................................................................................................................. 51
Effectiveness (IVS-062-002) ................................................................................................................................................................................. 51
Conclusions (IVS-062-002) .................................................................................................................................................................................... 52
Pivotal Bridging Study Overview (IVS-062-005)......................................................................................................................................... 52
Study Objectives (IVS-062-005) .......................................................................................................................................................................... 52
Patient Population (IVS-062-005) ...................................................................................................................................................................... 52
Safety Analysis (IVS-062-005).............................................................................................................................................................................. 53
Effectiveness (IVS-062-005) ................................................................................................................................................................................. 53
Conclusions (IVS-062-005) .................................................................................................................................................................................... 53

NON-CLINICAL PERFORMANCE EVALUATION.......................................................................................................................................... 44

CLINICAL PERFORMANCE EVALUATION ................................................................................................................................................... 48

REFERENCES ..............................................................................................................................................................................................53

19.

TECHNICAL AND CUSTOMER SERVICE ...................................................................................................................................................... 54

20.

SYMBOLS....................................................................................................................................................................................................54

21.

LEGAL NOTICE ...........................................................................................................................................................................................54

LeukoStrat CDx FLT3 Mutation Assay

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1. Proprietary Name
LeukoStrat CDx FLT3 Mutation Assay

2. Intended Use
The LeukoStrat CDx FLT3 Mutation Assay is a PCR-based, in vitro diagnostic test designed to detect internal tandem duplication(ITD) mutations and tyrosine kinase domain (TKD) mutations D835 and I836 in the FLT3 gene in genomic DNA extracted frommononuclear cells obtained from peripheral blood or bone marrow aspirates of patients diagnosed with acute myelogenous leukemia.
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in the assessment of patients with AML for whom Gilteritinib Fumarate
treatment is being considered.
The LeukoStrat CDx FLT3 Mutation Assay is used as an aid in the assessment of patients with AML for whom Quizartinib
Hydrochloride treatment is being considered.

3. Glossary
LeukoStrat CDx FLT3 Software

LeukoStrat CDx FLT3 Mutation Assay data analysis software.

Internal Tandem Duplication (ITD) Mutation

The duplication and insertion of a portion of the FLT3 gene that includes
the region in and around the juxtamembrane region of the FLT3 gene.

EC

Extraction Control

NTC

No Template Control

PC

Positive Control

Signal Ratio (SR)Calculated by dividing the mutant peak area by the wild-type peak area.

Tyrosine Kinase Domain (TKD) Mutation

Nucleotide change(s) that result in changes in codon 835 and/or 836 that aredetected by inactivation of the EcoRV restriction digest site within thetyrosine kinase domain of the FLT3 gene.

AR

Allelic ratio

CR

Complete remission

CRhComplete remission with partial hematological recovery

4. Summary and Explanation of the Test
Acute myelogenous leukemia (AML) in general has a poor prognosis. Assessment of the mutation status of the FLT3 (fms related
tyrosine kinase 3) receptor gene in karyotype normal AML is the most important prognostic indicator of disease outcome, which isoften substantial, as many studies in AML have shown that the presence of FLT3 activating mutations portends a poor prognosis.1,2
The LeukoStrat CDx FLT3 Mutation Assay targets regions of the FLT3 gene to identify internal tandem duplication (ITD) mutations
and tyrosine kinase domain (TKD) mutations, such as the D835 and I836 mutations and has been validated in an international clinical
trial.
The LeukoStrat CDx FLT3 Mutation Assay includes reagents, equipment, software and procedures for isolating mononuclear cells
and extracting DNA from patient specimens to determine if FLT3 mutations are present. DNA is amplified via PCR, TKD ampliconis enzymatically digested, and the amplicons are detected via capillary electrophoresis. FLT3 mutation status is determined by the
LeukoStrat CDx FLT3 Software. A FLT3 ITD and/or TKD mutation is reported as Positive if the mutant : wild-type signal ratio meetsor exceeds the cutoff of 0.05 (see section 12: Interpretation of Results). A depiction of the workflow is shown in Figure 1.

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5. Principles of the Procedure

Figure 1:

5.1.

Workflow summary

Internal Tandem Duplication (ITD) Mutations of FLT3

FLT3 ITD or length mutations are caused by duplication and insertion of a portion of the FLT3 gene that includes the region in
and around the juxtamembrane (JM) region of the FLT3 gene. These mutations vary in both the location and the length of theinserted duplicated DNA sequence. ITD mutations result in constitutive autophosphorylation and activation of FLT3.1
The LeukoStrat CDx FLT3 Mutation Assay uses primers that are in and around the JM region. The forward and reverse PCRprimers are fluorescently labeled with different fluorophores that serve to confirm the presence of sample signal. Wild-type FLT3
alleles will amplify and produce a product measured at 327±1 bp as measured by this assay, while alleles that contain ITD
mutations will produce a product that exceeds 330±1 bp (Figure 2).

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5.2.

Tyrosine Kinase Domain (TKD) Mutations of FLT3

FLT3 TKD mutations are caused by nucleic acid substitutions and/or deletions within codon sequences for amino acids D593,
D835, I836, and Y842 that result in a change in the amino acid in this highly-conserved catalytic center. TKD mutations, such as
D835 and I836 substitutions and deletions, result in constitutive autophosphorylation and activation of FLT3.2 The LeukoStrat
CDx FLT3 Mutation Assay can detect mutation of D835 and I836 among the listed mutations.
Wild-type alleles of the FLT3 gene include an EcoRV restriction digest site. When a nucleic acid substitution or deletion occurs
at D835 or I836, the restriction digest recognition site disappears, and the EcoRV endonuclease is unable to identify and digest
the DNA at this site. The LeukoStrat CDx FLT3 Mutation Assay uses primers that lie on either side of the TKD region. The
FLT3 target region is amplified using PCR and then an EcoRV restriction digest is performed. One of the PCR primers is labeledwith a fluorophore and the other contains an engineered EcoRV restriction site, so both wild-type and mutant alleles are digested.
The digestion pattern identifies loss of the normal gene sequence and ensures that digestion occurred. Wild-type alleles of the
FLT3 gene yield digestion products of 79±1 bp whereas mutant alleles yield products of 125±1 bp or 127±1 bp from the original
undigested amplicon product of 145±1 bp or 147±1 bp as measured by this assay (Figure 2).

Figure 2:

Depicted is a representation of the FLT3 juxtamembrane (JM) region (TM = transmembrane) and theactivating loop of the tyrosine kinase (TK) domain. Black arrows represent the relative positions ofprimers that target in and around the JM region for ITD or the activating loop of the kinase domain for
TKD. Colored dots represent fluorophores on labeled primers. The yellow box has vertical black linesthat represent the position of the EcoRV restriction digest sites.

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6. Reagents and Materials
NOTE:

The LeukoStrat CDx FLT3 Mutation Assay Kit is usable until the labeled kit expiration date when stored as
described in Table 1.

Table 1: Reagent List of the LeukoStrat CDx FLT3 Mutation Assay Kit, Part Number K-412-0331

Catalog Number

Reagent Name

Storage
Temperature

Unit Quantity# of Units / Kit

R0880220**

FLT3 Extraction Control

1800 µL/Vial

1 Vial

B4120131*

FLT3 ITD Master Mix

1500 µL/Vial

1 Vial

B4120141*

FLT3 TKD Master Mix

1500 µL/Vial

1 VialR0880200**

FLT3 ITD Positive Control

100 µL/Vial

1 VialR0880210**

FLT3 TKD Positive Control

100 µL/Vial

1 Vial

R0930060**

FLT3 No Template Control

200 µL/Vial

1 Vial261604

Taq DNA Polymerase

200 µL/Vial

1 Vial261779EcoRV Enzyme

200 µL/Vial

1 Vial261985

NEBuffer r3.1

1250 µL/Vial

1 Vial*Opened vials of Master Mixes and NEBuffer r3.1 stored frozen may incur up to 4 freeze thaw cycles.
**Opened vials of Controls stored frozen may incur up to 8 freeze thaw cycles.
Table 2: Additional Reagents, Materials, and Equipment Required (Not Provided)

Reagent/Material

Recommended Reagents/Materials and Suppliers

Catalog #Notes

Hi-Di Formamide

ThermoFisher Scientific:
Hi-Di Formamide

4440752

N/A

Liz Size Standard

ThermoFisher Scientific:
GeneScan 600 LIZ® dye Size Standard v2.0 - Dx

A25794

N/A

Polymer

ThermoFisher Scientific:
POP-7TM Polymer (384 Samples) 3500 Dx Series

4393709

N/A

ThermoFisher Scientific:
Anode Buffer Container for 3500 Dx/3500xL DxGenetic Analyzers

4393925

N/A

ThermoFisher Scientific:
Cathode Buffer Container for 3500 Dx/3500xL DxGenetic Analyzers

4408258

N/A

ThermoFisher Scientific:
3500xL Dx Genetic Analyzer series
3500 Data Collection Software v3.0

A27856

Dx Fragment Analysis
Software Module Required

GeneMapper® v4.1.x Software

4366925

N/A

ThermoFisher Scientific:
3500xL Dx Genetic Analyzer 24-Capillary Array 50
cm

4404688

N/A

BufferCapillary Electrophoresis
Instrument and Software

Capillary Array

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Table 2: Additional Reagents, Materials, and Equipment Required (Not Provided)

Reagent/Material

Recommended Reagents/Materials and Suppliers

Catalog #Notes

ThermoFisher Scientific:• Septa Cathode Buffer Container for 3500
Dx/3500xL Dx Genetic Analyzers

4410716

N/A

ThermoFisher Scientific:• Septa for 3500/3500xL Dx Genetic Analyzers, 96
well4410700

N/A

Retainer & Base Set for
3500xL Dx

ThermoFisher Scientific:• 3500 Series 96 Well Standard Retainer & Base Set(Standard) for 3500 Dx/3500xL Dx Genetic
Analyzers, 96 well

4410227

N/A

Spectral Calibration Dye
Set

ThermoFisher Scientific• DS-33 Matrix Standard Set (Dye Set G5) Dx

4482974

N/A

Calibrated Pipettes

Sartorius:• eLINE® Single Channel 5 - 120µL• eLINE® 8 Channel 0.2-10 µL or Equivalent
Gilson:• P-2M, P-10N, P-20N, P100N, P-200N, and• P-1000N pipettes or Equivalent

N/A

Must be able to accurately
measure volumes between
0.5µL and 1000 µL.

4452300(VRTI DX 200-Q)

N/A

Septa

Thermal Cycler

ThermoFisher Scientific:• Veriti™ Dx 96-Well Thermal Cycler

Vortex Mixer

N/A

N/A

N/A

PCR Plates or Tubes

N/A

N/A

Sterile, Skirted Plates

Filter Barrier Pipette Tips

N/A

N/A

Sterile,RNase/DNase/Pyrogen-free

Microcentrifuge

N/A

N/A

N/A

96-well Aluminum Foil
Sheet

N/A

N/A

N/A

96-well 8-cap Strips

N/A

N/A

N/A

Glass Distilled
De-ionized Molecular
Biology Grade or USP
Water

N/A

N/A

Water should be sterile andfree of DNase and RNase.

DNA Extraction

Qiagen:• QIAamp® DNA Blood Mini Kit

51104

Includes AL buffer, AW1
buffer, AW2 buffer, AE buffer,protease solvent, protease,elution tubes, lysis tubes, andspin columns

Mononuclear Cell
Isolation

Density Gradient Medium

N/A

Density: 1.077 g/mL

Mediatech Inc. (Corning):• Dulbecco’s Phosphate-Buffered Saline (DPBS)

21-031-CVR

N/AMediatech Inc. (Corning):• RPMI 1640 with L-glutamine

10-040-CVR

N/A

Buffered Saline Solution

Growth Medium

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Table 2: Additional Reagents, Materials, and Equipment Required (Not Provided)

Reagent/Material

Recommended Reagents/Materials and Suppliers

Catalog #Notes

Cell Counter

N/A

N/A

N/A

N/A

Capable of measuring
absorbance at 260 nm fornucleic acid concentration
calculation

N/A

Ethanol should be 200 proof
and absolute anhydrous
ACS/USP grade

Microvolume UVSpectrophotometer

Microvolume UV Spectrophotometer

Ethyl Alcohol/Ethanol

N/A

DNA Extractor

QIAgen:• QIAcube System (230 V)

9001882

N/A

DNA Extractor Reagent
Bottles

QIAGEN:• Reagent Bottles, 30 mL (6)990393

N/A

DNA Extractor Reagent
Bottle Rack

QIAGEN:• Reagent Bottle Rack

990390N/A

DNA Extractor Rotor
Adapter and its Holder

QIAGEN:• Rotor Adapters (10 x 24)• Rotor Adapter Holder

990394
990392

N/A

Table 3: General Laboratory Materials (Not Provided)

Material Description
15 mL conical tubes
50 mL conical tubes
Serological pipettes – 5 mL, 10 mL, 25 mL
Lint free wipes
Calibrated timer
QIAcube disinfectant, such as Steris Coverage Spray TB
Ice and ice bucket
Container for liquid waste
Volume appropriate non-binding surface tubes for DNA dilutions and aliquots
Volume appropriate tubes for DPBS, PCR, and Digestion Master Mix solutions
QIAcube sample tubes
Screw caps for QIAcube sample tubes
Disposable transfer pipettes
Pipette tips
96-Well Capillary Electrophoresis Plates, Skirted

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7. Instruments/Accessories
NOTE:
NOTE:









7.1.

This assay is for use on the 3500xL Dx Genetic Analyzer and the associated Data Collection Software installed oneach instrument.
Properly maintain all equipment according to the manufacturer’s instructions.

Refrigerator capable of 2°C to 8°C storage
Freezer capable of -30°C to -15°C storage
Dead air box
Pipette aid
Repeat pipettes
Multichannel pipettes, manual and electronic
Centrifuge capable of 1000 x g with a swing-out rotor and refrigeration
Centrifuge capable of 1400 x g with a swing-out rotor
The above instrumentation and accessories are not provided

Software (Provided)


NOTE:

LeukoStrat CDx FLT3 Software v1.1.x.IVD (K-412-0341)

Validation of the LeukoStrat software application was performed on display set to 1920 x 1200 resolution, with the“Smaller – 100%” display setting. Display issues may occur at other resolutions.
○

Computer Requirements:







Operating System: Windows™ 10 or Windows™ 11 Pro
Processor: Intel Core 2 Duo or newer CPU recommended
RAM: 4 GB minimum
Available Disk Space: 5 GB minimum
A CD-ROM drive
A PDF viewing software (such as Adobe Reader) is required to examine report outputs

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8. Warnings and Precautions


Please read the Instructions for Use carefully prior to starting the assay procedure and follow each step closely.

This product is for In Vitro Diagnostic Use.
The assay is only validated for use on the 3500xL Dx Genetic Analyzer and the associated Data Collection Software
installed on the instrument.
The assay must be used as a system. Do not substitute other manufacturers’ reagents.
Dilution, reducing amplification reaction volumes, or other deviation in this protocol may affect the performance of thistest and/or nullify any limited sublicense that comes with the purchase of this testing kit.
Do not mix or combine reagents from kits with different lot numbers.
Materials are stable until the labeled expiration date when stored and handled as directed. Do not use kits beyond their
expiration date.
Dispose of unused reagents and waste in accordance with country, federal, state and local regulations.
Track the number of freeze thaw cycles.
Perform all laboratory procedures with standard personal protective equipment (gloves, laboratory coats and protective eyewear). Follow good laboratory practices and universal precautions when working with specimens. Do not pipette bymouth. Do not eat, drink, or smoke in laboratory work areas. Wash hands thoroughly after handling specimens and assay
reagents. Handle specimens in approved biological safety containment facilities and open only in certified biological safetycabinets.
Due to the analytical sensitivity of this test, use extreme care to avoid the contamination of reagents or amplification
mixtures with samples, controls, or amplified materials. Use fresh, aerosol-resistant pipette tips between samples andbetween dispensing reagents. Closely monitor all reagents for signs of contamination (e.g., negative controls giving
positive signals). Discard reagents suspected of contamination.
To minimize contamination, wear clean gloves when handling samples and reagents and routinely clean work areas andpipettes prior to performing PCR.
Autoclaving does not eliminate DNA contamination. Always follow a uni-directional workflow in the PCR laboratory
between separate work areas; begin in specimen preparation, then amplification, and finally detection. Do not bring
amplified DNA into the areas designated for specimen preparation.
Dedicate all pipettes, pipette tips, and any equipment used in a particular area to that area of the laboratory.
Use sterile, disposable plastic ware whenever possible to avoid RNase, DNase, or cross-contamination.
All instruments and equipment must be maintained and calibrated according to the manufacturers’ recommendations.
Once the pouch has been brought to room temperature, examine inside the neck of each POP-7 Polymer pouch at point of
installation. Ensure the pouch attachment is free of dried or crystallized polymer. Do not install the pouch on the 3500xL
Dx instrument if crystallization is observed, as crystallization may impact the performance of the LeukoStrat CDx FLT3
Mutation Assay and 3500xL Dx instrument. If crystallization is observed, do not install the pouch on the 3500xL Dx
instrument and contact Thermo Fisher customer support.
Failure of the device to perform as expected or failure to correctly interpret test results may lead to incorrect FLT3 mutation
results and subsequently, improper patient management decisions in AML treatment.




















A false negative assay result could cause an AML patient not to experience any potential benefit that might beassociated with receiving gilteritinib fumarate or quizartinib hydrochloride treatments. However, the patient would
receive intense chemotherapy as the standard therapy for AML.
 Patients with a false positive assay result may receive treatment with gilteritinib fumarate or quizartinibhydrochloride for which there is no expectation of benefit. For adverse events related to these treatments, refer tothe relevant pharmaceutical manufacturer labeling.
NOTE:
If incorrect specimens, or reagents are used and/or these instructions are not followed properly, there is arisk of delayed results, which may lead to delay in treatment.


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8.1.

Cyber Security Precautions








Computers and networks are susceptible to security risk if not secured and actively updated. Proper computer andnetwork security helps ensure data is not compromised, lost or damaged due to preventable cyber risks. Equip allcomputers with up to date and active antivirus software.
Filter and secure network traffic with a firewall.
Keep data on local computers to reduce cyber security risks that may be present in transferring sensitive data over a
network.
Install software only for the local user to prevent unauthorized use of the software.
Ensure Windows and Adobe Acrobat Reader are always updated to the latest available security patches.
Ensure the default PDF reader in Windows is set to Adobe Acrobat Reader. Opening sample and run reports in aninternet browser may lead to cybersecurity risks of patient data.
The LeukoStrat CDx FLT3 Software has been validated with the following Antivirus software:○○○

Symantec Endpoint Protection Version 14.3
McAffee Endpoint Security Version 10.7
ESET Endpoint Security Version 10.0

9. Specimen Collection and Preparation
9.1.

Precautions



9.2.

Biological specimens from humans may contain potentially infectious materials. Handle all specimens according
to your institute’s Bloodborne Pathogen program and/or Biosafety Level 2.
The assay is validated for blood and bone marrow anti-coagulated with sodium heparin or EDTA.

Interfering Substances

The following substances are known to interfere with PCR:

Divalent cation chelators
Low retention pipette tips
EDTA (not significant at low concentrations)

The following interfering substances were tested and determined not to negatively impact the performance of the LeukoStrat CDx
FLT3 Mutation Assay:



9.3.

For exogenous interference substances, 0.8 mg/mL Na-heparin and 10% residual DNA extraction wash buffer.
For endogenous interference substances, 60 mg/mL human serum albumin, 2 mg/mL hemoglobin, 0.19 mg/mL (342µM) bilirubin and approximately 37 mM lipids/ triglycerides.
For common drug interference, 24 μg/mL cytarabine and 180 ng/mL daunorubicin.

Specimen Requirements and Handling
9.3.1.

At least 1 mL of peripheral blood and 0.25 mL of bone marrow anti-coagulated with sodium heparin or EDTA
are required for the LeukoStrat CDx FLT3 Mutation Assay.

9.3.2.

Specimens can be stored at 2°C to 8°C for up to 7 days prior to testing.

9.3.3.

Specimen tube integrity and contents must not be compromised (i.e. frozen during shipment).

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10. Assay Procedure
10.1.

Specimen Inspection


10.2.

Unpack peripheral blood (PB) and/or bone marrow aspirate (BM) specimens and reject any specimens that do notmeet the requirements listed in section 9.3.

Sample Processing Preparation

10.2.1.

Perform sample processing in the assigned sample processing work space.

10.2.2.

Transfer approximately 14 mL of RPMI-1640 media per specimen into labeled 50 mL conical tubes. Allow themedia to warm at room temperature (15°C to 30°C) for at least 1.75 hours.

10.2.2.1.
10.2.3.

For each specimen, aliquot 3 mL of density gradient medium into a labeled 15 mL conical tube.

10.2.3.1.
10.2.4.
10.3.

If chilled RPMI-1640 media is aliquoted into 15 mL conical tubes, warm at room temperature (15°C to30°C) for at least 45 minutes.
If the density gradient medium was stored at 2°C to 8°C, warm the density gradient medium aliquots atroom temperature for 1 hour before use.

Transfer approximately 200 µL of DPBS per specimen into a labeled, volume appropriate tube and allow it towarm at room temperature (15°C to 30°C) for at least 45 minutes prior to use.

Diluting Clinical Samples

NOTE:

Instructions for using a QIAcube for DNA extraction are included in this manual. A QIAcube is recommended but
not required. If a QIAcube is used, ensure that one space is reserved for the Extraction Control.

10.3.1.

Mix the specimen tubes by inverting 4-6 times. Add specimen aliquots (1-3 mL peripheral blood or 0.25-0.75 mL
bone marrow) to uniquely labeled 15 mL conical tubes.

10.3.2.

Add RPMI-1640 media to each specimen aliquot to bring the total volume to 6 mL. Cap the tubes tightly andgently mix by inverting 3-5 times or pipette up and down until the mixture has a uniform consistency.


10.4.

Any remaining specimen may be stored at 2°C to 8°C.

Mononuclear Cell (MNC) Isolation

10.4.1.

Using a transfer pipette, gently overlay the diluted peripheral blood or bone marrow sample on top of the density
gradient medium. Tilt the tube containing the density gradient medium while very slowly pipetting the sample on
top to prevent layer mixing.

10.4.2.

After pipetting the entire sample, gently straighten the tube to a vertical position and cap the tube tightly.

10.4.3.

Centrifuge the 15 mL conical tubes under the following conditions, ensuring that the brake is completely turned
off:





Force = 400 x g (rcf)Time = 30 minutes
Temperature = 20°C
Accel/Decel = minimum

10.4.4.

For each sample to be processed, aliquot 6 mL of RPMI-1640 media into a new labeled 15 mL conical tube.

10.4.5.

After centrifugation, use a transfer pipette to slowly aspirate the MNC layer or until no more than 3 mL has been
removed. Three milliliters has been removed when the total volume in the tube has decreased by 3 mL according
to the demarcations on the 15 mL conical tube.

10.4.6.

Dispense the collected MNC layer suspension into the appropriately labeled 15 mL conical tube containing 6 mLof RPMI-1640 media. Cap the tube and gently mix by inverting 3-5 times.

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10.4.7.

Centrifuge the conical tubes under the following conditions:Force = 355-364 x g (rcf)Time = 10 minutes
Temperature = 20°C
Accel/Decel = maximum

10.4.8.

Pour the supernatant off the cell pellet by only inverting the tube once before returning it to the vertical position.
Resuspend the pellet in the remaining liquid by tapping the tube 10-15 times or until the pellet is resuspended.

10.4.9.

Add 1 mL of RPMI-1640 media to the resuspended cell pellet. Cap the tube and gently mix by tapping the tube
6-8 times.

10.4.10.

Place the sample tubes in an ice water bath until the mononuclear cell counts have been completed.

10.5.

Mononuclear Cell Count

10.5.1.

Obtain mononuclear cell counts using a suitable cell counting system.

10.5.2.

Minimize the volume consumed for cell counts to ensure an adequate amount of DNA is retained for the assay.

10.5.3.

Discard the sample used for cell counting.

10.6.

Preparing Samples for DNA Extraction and Isolation Completion

10.6.1.

If the reported concentration is ≤ 5 million cells/mL, the entire cell suspension volume is processed. Proceed tostep 10.6.3.

10.6.2.

If the reported concentration is > 5 million cells/mL, calculate the volume of sample that contains 5 million live
cells (Vi) since the QIAcube spin columns can only accommodate ≤ 5 million cells.

10.6.2.1.

Use the equation CiVi = CfVf to solve for Vi for each of these samples.
Ci = cell concentration (cells/mL) from the MNC countCf = final concentration (5 million cells/mL)
Vf = final volume (1 mL)(5,000,000 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 ⁄𝑚𝑚𝑚𝑚 ) × 1 𝑚𝑚𝑚𝑚
𝑽𝑽𝒊𝒊 =
𝑪𝑪𝒊𝒊

10.6.2.2.

Use the equation Vf - Vi to solve for the volume of RPMI-1640 media to add to Vi to bring the volume up
to 1000 µL.

10.6.2.3.

Gently mix the > 5 million cells/mL samples by tapping the tubes 6-8 times.

10.6.2.4.

Transfer the calculated volumes to a labeled 15 mL conical tube for each sample.

10.6.3.

Centrifuge the 15 mL conical sample tubes containing the cell suspensions under the following conditions:Force = 355-364 x g (rcf)Time = 10 minutes
Temperature = 20°C
Accel/Decel = maximum

10.6.4.

Using a transfer pipette, aspirate the supernatant from the cell pellets. Some small volume of media may remain.

10.6.5.

Tap the 15 mL conical tubes 10-15 times or until the pellets are loosened from the tubes.

10.6.6.

Add 200 µL of DPBS and gently mix by tapping the tube 10-15 times to resuspend the cells. Place these cappedsamples in the ice water bath.

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10.7.

Preparing QIAcube Automation Station

NOTE:

Instructions for use of a QIAcube for DNA extraction are included in this manual. A QIAcube is recommended but
not required. DNA extraction can be performed with the Qiagen DNA Blood Mini Kit without a QIAcube.

10.7.1.

All QIAcube Automation Station steps including installation, operation, calibration, cleaning and maintenanceprocedures are performed according to the manufacturer’s instructions unless stated otherwise below.

10.7.2.

Follow the QIAgen guidelines for performing maintenance on the QIAcube Automation Station, with one
exception. Perform the Tightness Test monthly instead of every 6 months.

10.7.3.

Prepare the QIAcube Automation Station for use, loading materials and reagents into the instrument.

10.7.3.1.

A QIAcube is capable of processing up to 12 tubes; however, 1 of the spaces is reserved for the Extraction
Control (used as the extraction contamination control and PCR negative control). It is not possible toprocess 1 or 11 tubes due to centrifuge imbalance.

10.7.3.2.

Blank tubes, using DPBS, may be used if the number of required extractions including Extraction Control
is 11 tubes.

10.7.4.

Remove a tube of Extraction Control (EC) from -30°C to -15°C storage and thaw at room temperature (15°C to30°C). Control EC tubes may be returned to the freezer after use. Track the number of freeze thaw cycles.

10.7.5.

Vortex the EC tube at MAX speed for 5-15 seconds. Centrifuge the tube for 2-5 seconds if liquid is present in thelid. Add 200 µL of the Extraction Control to a sample tube. This EC tube may be capped and stored at 2°C to
8°C until the run is ready.

10.8.

DNA Extraction

NOTE:

Instructions for using a QIAcube for DNA extraction are included in this manual. A QIAcube is recommended but
not required. DNA extraction can be performed with the Qiagen DNA Blood Mini Kit without a QIAcube.

10.8.1.

Pipette the cell suspensions (from 10.6.6) up and down 4-6 times to resuspend the cells. Transfer the entire volumeof the cell suspensions in DPBS to sample tubes. Ensure that the majority of the solution is at the bottom of thetube.

10.8.2.

Place the Extraction Control sample tube in the last position of the run.

10.8.3.

Load all of the remaining sample tubes, the reagents, and the aliquoted Protease solution into the instrument.

10.8.4.

Start the run, ensuring the following selections are made.

10.8.4.1.

Use the QIAamp DNA Blood Mini protocol.

10.8.4.2.

Select Blood or body fluid as the starting material.

10.8.4.3.

Set the Elution volume to 100 µL.

10.8.5.
10.9.

When the extraction is complete, cap the DNA sample tubes and store them at 2°C to 8°C until quantification is
performed.

Quantification and Dilution of DNA

10.9.1.

All microvolume UV spectrophotometer steps including installation, operation, calibration, cleaning andmaintenance procedures are performed according to the manufacturer’s instructions unless stated otherwise below.

10.9.2.

Vortex the DNA sample tubes at MAX speed for 5 to 15 seconds. Using a microcentrifuge, centrifuge the DNA
sample tubes for 2 to 5 seconds to remove liquid from the lids.

10.9.3.

Blank the instrument using 2 µL of AE buffer.

10.9.4.

Read 2 µL of each DNA sample in singlicate.

10.9.5.

If the concentration of a DNA sample has a reading ≤9.4 ng/µL, re-quantify the DNA sample twice more using
fresh 2 µL aliquots. Ensure the sample is well mixed to avoid inaccurate microvolume UV spectrophotometerreadings. The average of these three readings is considered the final DNA concentration.

NOTE:

If the final quantification value is ≤9.4 ng/µL, the DNA sample cannot be tested in the LeukoStrat CDx FLT3
Mutation Assay. Reprocess the specimen in order to obtain adequate DNA.
If the final quantification value of the extraction control is ≤9.4 ng/µL, the associated DNA samples cannot be tested
in the LeukoStrat CDx FLT3 Mutation Assay. Reprocess these specimens in order to obtain adequate DNA.

NOTE:

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10.9.6.
NOTE:
10.9.7.

DNA samples may be stored undiluted at -30°C to -15°C for up to one year. Alternatively, DNA samples,
undiluted or diluted to 10 ng/µL may be stored at 2°C to 8°C for up to 7 days.
Undiluted DNA may be exposed to up to 5 freeze/thaw cycles.
DNA samples ≥10.5 ng/µL must be diluted to 10 ng/µL in AE buffer using non-binding surface tubes. Using theequation CiVi = CfVf, solve for Vi after selecting the final volume (Vf) from Table 4.







𝑽𝑽𝒊𝒊 =

𝑉𝑉𝑓𝑓 × 10 𝑛𝑛𝑛𝑛/µ𝐿𝐿
𝑪𝑪𝒊𝒊

Ci = DNA concentration from microvolume UV spectrophotometer reading
Cf = final DNA concentration (10 ng/µL)
Vi = volume of undiluted DNA to dilute
Vf = final volume of diluted DNA (from Table 4)
Vf - Vi = amount of AE buffer to add to Vi
Table 4: Final Volumes Determination by Quantification ValueDNA Concentration from microvolume UV
spectrophotometer (Ci)10.10.

Final Volume (V f )Ci ≤ 9.4 ng/µL

Not testable

9.5 ≤ Ci ≤ 10.4 ng/µL

Test as is10.5 ≤ Ci ≤ 50.4 ng/µL

35 µL

50.5 ≤ Ci ≤ 200.4 ng/µL

100 µLCi ≥ 200.5 ng/µL

180 µL

Amplification

NOTE:
NOTE:
NOTE:

Complete all steps in this section on the same day for an ITD or TKD run.
Minimize the amount of light exposure to the Master Mixes.
Minimize the amount of time Taq is out of -30°C to -15°C storage.

10.10.1.

Perform all Veriti Dx thermal cycler steps including installation, operation, calibration, cleaning and maintenanceprocedures according to the manufacturer’s instructions unless stated otherwise below.

10.10.2.

Allow Master Mixes (ITD Master Mix and TKD Master Mix) to thaw at room temperature (15°C to 30°C).
Remove Control tubes (ITD Positive Control, TKD Positive Control, Extraction Control, and No Template
Control) from appropriate storage and allow to thaw at room temperature (15°C to 30°C). Control tubes may bereturned to the freezer after use. Track the number of freeze thaw cycles. While reagents are warming to room
temperature (15°C to 30°C), label separate 96-well plates with ITD PCR or TKD PCR, as applicable, and a unique
identifier.

NOTE:
10.10.3.

Run all samples on the same PCR plate as the associated Extraction Control.
Determine the number of plate wells (samples, TKD Positive Controls, ITD Positive Controls, Extraction Controls,and No Template Control) to be tested on the ITD and TKD plates. The total number of plate wells to be tested
per ITD or TKD plate = X. To prevent variation when pipetting small volumes of reagent, the minimal value of
X is 2.

10.10.3.1.

Calculate the volume of master mix plus Taq needed. This calculation is 45 µL of ITD or TKD MasterMix multiplied by (X+3) and 0.2 µL Taq multiplied by (X+3). The additional 3 samples added to X
compensates for pipetting error.

10.10.4.

Vortex the Master Mix, Controls, and DNA sample tubes at MAX speed for 5 to 15 seconds.

10.10.5.

Remove Taq from storage at -30°C to -15°C. Do not vortex.

10.10.6.

Using a microcentrifuge, centrifuge all tubes (including Taq) for 2 to 5 seconds to remove liquid from the lids.

10.10.7.

Add the calculated volumes of Master Mix and Taq to labeled tubes of the appropriate volume for the ITD and
TKD plates.

10.10.8.

Cap and vortex the tubes at MAX 5 to 15 seconds to mix. Use a microcentrifuge to centrifuge for 2 to 5 seconds,
when possible. Place Taq back in storage at -30°C to -15°C.

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10.10.9.

Aliquot 45 µL of the mixture of Master Mix and Taq to the appropriate wells of the PCR plate layout.

10.10.10. Add 5 µL of the DNA samples at 10 ng/µL and Controls to the appropriate wells of the 96-well plate according
to the PCR plate layout.
10.10.11. Seal the columns of the PCR plate with well strips. Centrifuge the 96-well plate at 1400×g for 1 minute.
10.10.12. Place the PCR plate in a Veriti Dx thermal cycler and close the lid. Program the thermal cycler with the steps
listed in Table 5.
Table 5: PCR Amplification Thermal Cycler Programs

Step

FLT3 ITD CDx Program

FLT3 TKD CDx Program

1

95°C for 11 minutes

94.5°C for 11 minutes

2

94°C for 30 seconds

93.5°C for 30 seconds

3

57°C for 60 seconds

56.5°C for 60 seconds

4

72°C for 2 minutes

71.5°C for 2 minutes

5

Repeat Steps 2 to 4, 24 times

Repeat Steps 2 to 4, 28 times

694°C for 30 seconds

93.5°C for 30 seconds

7

60°C for 45 minutes

59.5°C for 45 minutes

8

4°C for ∞4°C for ∞Ramp rate 75%.

10.10.13. Press Run to proceed to the next screen. Ensure that the reaction volume setting is 50 µL, the cover temperature
setting is 105.0 °C, and that the cover will be heated for the run. Press Start Run Now to begin the run.
10.10.14. Store leftover reagents and DNA. Store opened Master Mixes at -30°C to -15°C. Track the number of freeze
thaw cycles.
10.10.15. After completion of the PCR protocol, the PCR plate may be stored at 2°C to 8°C for up to 72 hours. Otherwise,
for TKD plates, continue to the Restriction Digest section and for ITD plates, continue to the Capillary
Electrophoresis Detection section.
10.11.

Restriction Digest (TKD Mutation Only)

NOTE:
NOTE:
NOTE:

Complete all steps in this section in the same day.
Perform the restriction enzyme digest on the TKD amplicons only.
Minimize the amount of time EcoRV is out of -30°C to -15°C storage.

10.11.1.

Allow a NEBuffer r3.1 tube to thaw at room temperature (15°C to 30°C).

10.11.2.

While reagents are warming to room temperature (15°C to 30°C), label a 96-well plate with TKD digestion and aunique identifier.

10.11.3.

Determine the number of plate wells (samples and Controls) to be digested on the plate. The total number ofsamples to be digested = Y. To prevent variation when pipetting small volumes of reagent, the minimum valueof Y is 4.

10.11.3.1.

Calculate the volume of digestion mix needed. The calculation is 1.1 µL of NEBuffer r3.1 multiplied by(Y+6) and 0.5 µL of EcoRV multiplied by (Y+6). The additional 6 samples added to Y compensates for
pipetting error.

10.11.4.

Vortex the NEBuffer r3.1 tube at MAX for 5 to 15 seconds.

10.11.5.

Remove the EcoRV from the -30°C to -15°C storage. Do not vortex.

10.11.6.

Using a microcentrifuge, centrifuge all tubes (including EcoRV) for 2 to 5 seconds to remove liquid from the lids.

10.11.7.

Add the calculated volumes of NEBuffer r3.1 and EcoRV to a labeled tube of appropriate volume.

10.11.8.

Mix the solution by pipetting up and down 5 to 10 times. Place EcoRV back in storage at -30°C to -15°C.

10.11.9.

Aliquot 1.5 µL of the digestion mix solution to the appropriate wells of the digestion plate.

10.11.10. Remove the TKD PCR plate from the thermal cycler or 2°C to 8°C storage (plate does not need to warm to room
temperature) and centrifuge the plate at 1400×g for 1 minute.
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10.11.11. Add 8.5 µL of the samples from the PCR plate to the appropriate wells of the digestion plate. Seal the digestion
plate columns with cap strips.
10.11.12. Centrifuge the plate at 1400×g for 1 minute.
10.11.13. Place the digestion plate in a Veriti Dx thermal cycler and close the lid.
10.11.14. Program the thermal cycler with the steps listed below (ramp rate 75%).
Step 1: 37°C for 1 hour
Step 2: 65°C for 10 minutes
Step 3: 4°C forever

10.11.15. Press Run to proceed to the next screen. Ensure that the reaction volume setting is 10 µL, the cover temperature
setting is 105.0°C, and that the cover will be heated for the run. Press Start Run Now to begin the run.
10.11.16. After completion of the digestion protocol, the digestion plate may be stored at 2°C to 8°C for up to 72 hours,
keeping the light exposure to a minimum. Otherwise, continue to the Capillary Electrophoresis Detection section.
10.12.

Capillary Electrophoresis Detection

NOTE:
NOTE:

Minimize the amount of time the Liz Size Standard tube is out of 2°C to 8°C storage.
The 3500xL Dx runs in sets of 24 capillaries, called an injection, which comprises 3 columns by 8 rows on a 96well plate. Each capillary corresponds with one well. There are no partial injections although the injections can be
independently programmed.

10.12.1.

All 3500xL Dx steps including installation, operation, calibration, cleaning and maintenance procedures are
performed according to the manufacturer’s instructions unless stated otherwise below.

10.12.2.

ITD and TKD assays must be run on different injections and with different injection conditions. The 3500xL Dx
ITD and TKD conditions are listed in Table 6 below. These settings may be saved in the 3500xL Dx for futureuse.
Table 6: 3500xL Dx Genetic Analyzer Conditions

10.12.3.

Parameter

ITD CDx Assay
Parameters

TKD CDx Assay
Parameters

Injection Time

12 sec.

7 sec.

Injection Voltage

1.2 kVolts

1.0 kVolts

Capillary Length

50 cm

Polymer

POP-7

Dye SetG5Oven Temp

60°C

Run Time

1630 sec.

Run Voltage

19.5 kVolts

PreRun Time180 sec.

PreRun Voltage

15 kVolts

Data Delay

1 sec.

Click Refresh to update the consumables’ time on the instrument and number of injections performed in the
3500xL Dx Dashboard. Check the 3500xL Dx dashboard to ensure that the buffers, polymer, and capillary havenot exceeded their maximum allowable time on the instrument for this assay, listed in Table 7. Check that the
number of samples (not just injections) remaining for POP-7 is sufficient for the run. If a consumable component
needs to be replaced, perform the necessary maintenance before proceeding.
Table 7: Maximum Allowable On-Board Times, 3500xL Dx Materials

3500xL Dx Material

Maximum Allowable Time on Instrument

POP-7 Polymer

7 daysAnode Buffer

7 daysCathode Buffer

7 days3500xL Dx Capillary Array

160 injections

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10.13.

Prepare Size Standard Solution, if Needed

10.13.1.

The size standard solution consists of a mixture of Liz Size Standard and HIDI Formamide.

10.13.2.

Remove a tube of size standard solution from 2°C to 8°C storage, if available, and proceed to Step 10.13.6. If not,make a size standard solution tube by performing the next three steps.

10.13.3.

Allow a bottle of HIDI Formamide to thaw at room temperature (15°C to 30°C). Remove a tube of Liz Size
Standard from storage at 2°C to 8°C. Label newly opened tubes of Liz Size Standard with the date.

10.13.4.

Vortex the tubes at MAX for 5 to 15 seconds. Centrifuge the tubes for 2 to 5 seconds in a microcentrifuge.

10.13.5.

Add 59 µL of Liz Size Standard to 1050 µL of HIDI Formamide. Label the size standard solution tube with thedate and lot number of Liz Size Standard. Store opened Liz Size Standard in 2°C to 8°C storage.

10.13.6.

Vortex the size standard solution tube at MAX for 5 to 15 seconds. Centrifuge the mixture tube for 2 to 5 seconds
in a microcentrifuge. Any unused solution can be stored at 2°C to 8°C for up to 7 days. Discard after 7 days.

10.14.

Prepare Sample Plate

10.14.1.

Centrifuge the 96-well ITD PCR and/or TKD digestion plate at 1400×g for 1 minute.

10.14.2.

Label a 96-well plate with ITD CE and/or TKD CE, as applicable, and a unique identifier.

NOTE:

Both ITD and TKD assays may be run on the same capillary electrophoresis (CE) plate, but they must be separated
into different injections.

10.14.3.

Determine the number of injections needed for a run, where x = the number of injections. Multiply the total
number of injections by 24 and add 4 (24x + 4). The maximum volume of size standard solution needed is 9.5 µL
multiplied by (24x + 4). The additional 4 samples added to x compensates for pipetting error.

10.14.4.

Add 9.5 µL of size standard solution to the wells in the CE plate that contain samples. Add 9.5 µL of size standard
solution or HIDI Formamide only to any remaining wells that will be injected (multiple of 24) but do not containsamples.

NOTE:
10.14.5.
NOTE:

All 24 wells within an injection must contain sample mixed with size standard solution, size standard solution alone,or HIDI Formamide alone.
From each PCR well (ITD only) or digestion well (TKD only), transfer 0.5 µL of PCR or digested product to thecorresponding well on the CE plate using an electronic multichannel pipette.
A single channel pipette may be used to transfer PCR/digested product during retests of individual wells.

10.14.6.

Seal the CE plate with a foil seal and centrifuge the plate at 1400×g for 1 minute.

10.14.7.

Place the CE plate in a Veriti Dx thermal cycler and close the lid.

10.14.8.

Program the thermal cycler with the steps listed below (ramp rate 75%).


10.14.9.

Step 1: 95°C for 3 minutes
Step 2: 4°C for 5 minutes

Press Run to proceed to the next screen. Ensure that the reaction volume setting is 10 µL, the cover temperature
setting is 105.0°C, and that the cover will be heated for the run. Press Start Run Now to begin the run.

10.14.10. After the run is complete, confirm no bubbles are present by visually inspecting the plate wells. Remove any
bubbles by centrifuging the CE plate at 1400×g for 1 minute.
10.14.11. Prepare each plate assembly by placing the CE plate on a 3500xL Dx 96-well plate base, confirming the notchedcorners align. Remove the foil seal and place a new 96-well plate septa on the plate, ensuring the septa is flat andall septa openings are unobstructed. Snap on a 3500xL Dx 96-well plate retainer.

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10.15.

Set up PlateMapper with LeukoStrat CDx FLT3 Software

NOTE:
10.15.1.

Administrator permission is required to install the LeukoStrat® CDx FLT3 Software.
Install the LeukoStrat CDx FLT3 Software.

10.15.1.1.

Copy LeukoStratCDx-1.1.x.IVD.msi installer from the software CD to a local drive on your computer.

10.15.1.2.

Double-click the LeukoStratCDx-1.1.x.IVD.msi file.

10.15.1.2.1.
If a Microsoft Defender SmartScreen message appears after double-clicking the msi file, Click
More info.
10.15.1.2.2.

Verify the publisher is Invivoscribe, Inc. To proceed with installation, click Run anyway.

10.15.1.3.

The LeukoStratCDx-1.1.x.IVD.msi setup wizard box will appear. Click Next.

10.15.1.4.

The default installation location is C:\Invivoscribe\LeukoStratCDx-1.1.x.IVD\. Click Next.

10.15.1.5.

Click Install. The installation will begin.

10.15.1.6.

A User Account Control dialog box will appear. Click Yes.

10.15.1.7.

Click Finish to exit the setup wizard.

10.15.2.

Open the LeukoStrat CDx FLT3 Software. Click Accept (LeukoStrat CDx FLT3 Mutation Assay) to agree to terms of service.

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10.15.3.

Within the PlateMapper Setup, fill in the three required fields located above the plate map. These required fields
are Plate Name, Results Group and File Name Convention (circled below).

10.15.3.1.

Plate map names may only contain 50 characters or less, made up of [A-Z, a-z, 0-9], single spaces, andhyphens.

10.15.3.2.

Results Group and File Name Convention entries must match the names of corresponding user-programmed
entries on the 3500xL Dx (selected in step 10.16.13).

10.15.4.

The plate map has four Assays allowed per plate (3 columns per assay). Each assay corresponds with the injection
which will occur during the 3500xL Dx run. Only one assay may be run per injection (ITD or TKD).

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10.15.5.

Select the assay from the drop down menu (located above it on the PlateMapper Setup screen.

10.15.6.

Within the plate map, enter information for each well that will have a sample or control to be analyzed.

NOTE:) which corresponds with the samples that are

When entering well information, the Extraction Control (EC), Positive Control (PC), and No Template Control(NTC) should be entered first. Controls may be placed anywhere on the plate, not necessarily in the first three wells.
The SAMPLE wells should be entered afterwards as they will require linking with their corresponding Extraction
Control. Positive Controls and No Template Controls are not linked to Extraction Controls.

10.15.6.1.

To enter information, click on the respective well (e.g. A01) and the following box will open:

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10.15.7.

Enter a Sample Name which describes the well. Sample names may only contain 50 characters or less, made upof [A-Z, a-z, 0-9], single spaces, and hyphens.

10.15.7.1.

10.15.8.

The user may also import sample names to the plate map using the 3500 Plate Layout File Version 1.0 by
Thermo Fisher Scientific. Enter sample names into the 3500 Plate Layout File and import with the Import
ABI button.

Select the Sample Type of the well from the drop down menu. The options to select are:


SAMPLE = unknown
EC = Extraction Control
NTC = No Template Control
PC= Positive Control

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10.15.8.1.
NOTE:

A “run” is defined by all samples, one replicate of Positive Control, all Extraction Controls associated with thesamples under test, and one replicate of No Template Control. Runs may span multiple injections and multiple runs
may be tested on one plate.

10.15.8.2.

10.15.9.

Select the Run number from the drop down menu. To add a new run number, click the + sign next to thedrop down menu.

Select the associated EC from the drop down menu (only required if the Sample Type is SAMPLE). Up to
11 samples may be associated with a single Extraction Control.

Additional comments about the sample or control may be entered in the Sample Notes field. These comments willappear in the Sample Report.

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10.15.10. Once all the information for the well has been entered, click Save Well to save. To clear the contents of the well,
click Clear Well.

10.15.11. Once the well has been saved, the plate map color display will change for that well. If the well has been set up
correctly, the well will display as green (as seen below). If there is something missing or wrong for the well setup,
the well will display as red (as seen below).
NOTE:

If correct, the well color for the Extraction Control will not change to green until the cursor hovers over that well.

10.15.12. Continue entering each well on the PlateMapper Setup screen until all wells in the plate which need to be analyzed
are highlighted green.
10.15.13. Once all wells have been entered, click Save Plate and the user will be prompted for a location to save the ABI
file (3500 Plate Layout File Version 1.0) and LIVS file generated by the software. One ABI file and one LIVS
file will be generated for every plate setup.
NOTE:
NOTE:

Do not modify the ABI file generated by the LeukoStrat CDx FLT3 Software. Doing so will result in an error upon
upload to the 3500xL Dx.
If the LeukoStrat CDx FLT3 Software is not closed once a plate map is generated, the automatically assigned Run
IDs in the output files will not be unique and will repeat across multiple runs.

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10.15.13.1. The user may review the LIVS file created by clicking on Import LIVS and navigating to the file location
where it was saved.
NOTE:

The import LIVS feature is only to review plate setup. The LIVS file cannot be modified to create a new plate map
for use in another run. Doing so will result in an error.

10.15.14. The user will continue with the software after the 3500xL Dx run has completed.
10.15.15. Use the ABI file which was generated by the LeukoStrat CDx FLT3 Software to upload the plate onto the 3500xL
Dx.
10.15.16. If saving the plate fails, follow the recommendations in Table 8. If further assistance is required, please contact
Invivoscribe Technical Support at support@invivoscribe.com.
Table 8: Save Plate Error Messages and Corrective Actions

Save Plate Error Message [code]

Potential Cause(s)Corrective Action(s)-Corrupted sample detected. [PM01]-Could not detect well for object UUID. [PM02]-Control detected unknown links for well (A-H, 0112). [PM03]

Attempt to upload modified .LIVS
file.

Do not modify the .LIVS file. If the file iscorrupted, a new .LIVS file must be created.

-Missing required field “Plate Name”. [PM04]-Illegal character detected in “Plate Name”.
[PM05]-Multiple spaces detected in “Plate Name”.
[PM06]-Plate Name must be 50 characters or less. [PM28]

Not following IFU directions fornaming a plate.

A plate map name may only contain 50
characters or less, made up of [A-Z, a-z, 09], single spaces, and hyphens.

-Missing required field “Result Group”. [PM07]

Not following IFU directions fornaming a Result Group.

The Result Group is defined on the 3500xL
Dx.

-Missing required field “File Naming Convention”.
[PM08]

Not following IFU directions fornaming a File Naming Convention

The File Naming Convention is defined on
the 3500xL Dx.

-Assay not selected for all samples. [PM09]-Run contains more than 1 Assay type. [PM10]

Not following IFU directions for
assigning assay type.

All wells must be assigned an assay type,and a run may not contain more than one
assay type.

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Table 8: Save Plate Error Messages and Corrective Actions

Save Plate Error Message [code]

Potential Cause(s)Corrective Action(s)-Sample name not detected for well (A-H, 01-12).
[PM11]-Illegal character detected in Sample Name.
[PM12]-Multiple spaces detected in Sample Name.
[PM13]-Sample name must be 50 characters or less.
[PM14]

Not following IFU directions fornaming a sample.

A sample name may only contain 50
characters or less, made up of [A-Z, a-z, 09], single spaces, and hyphens.

-Sample Type not selected for well (A-H, 01-12)[PM15].

Not following IFU directions forselecting a sample type.

All wells must have a sample type assigned.
The choices are PC, NTC, EC, and SAMPLE.

-Run not selected for well (A-H, 01-12). [PM16]-No Runs created for Plate. [PM17]

Not following IFU directions forselecting runs.

All wells must have a run assigned. The
first well assigned a run requires the user toincrement the run count (“+” button next
to Run selection). Subsequent wells caneither increment the run count, or select apreviously used run count.

-EC not selected for well (A-H, 01-12). [PM18]-Sample attached to unknown EC for well (A-H,
01-12). [PM19]-EC selected on control for well (A-H, 01-12).
[PM20]-No samples linked to EC for well (A-H, 01-12).
[PM21]-Not following IFU directions for
assigning ECs.
-Attempt to upload a modified.LIVS file

All “SAMPLE” wells must be assigned to an
EC. A control well should not be assigned
to an EC. Each EC must be linked to atleast one sample.

-Run missing PC, NTC, EC. [PM22]-Run detected control in sample list. [PM23]-Run missing samples. [PM24]-Run contains more than 1 Assay type. [PM25]-Not following IFU directions for
assigning runs.
-Attempt to upload a modified.LIVS file.

Each run must contain one of each control
type (PC, NTC, EC). A run must contain atleast one well with type “SAMPLE”. A run
must contain exactly one Assay type.

-Too many samples linked to EC for well (A-H, 0112). [PM26]-EC linked to more than one run for well (A-H, 0112). [PM27]

Not following IFU directions for
assigning ECs.

A single EC may not be linked to more than
11 samples. A single EC may not be linked
to samples across more than one run.

10.16.

Set Up 3500xL Dx Software

NOTE:

The LeukoStrat CDx FLT3 Software creates a file for import into the 3500xL Dx (ABI file) which appends
information to the Sample Name. The 3500xL Dx software may append additional information.

10.16.1.

All 3500xL Dx steps including installation, operation, calibration, cleaning and maintenance procedures are
performed according to the manufacturer’s instructions unless stated otherwise below.

10.16.2.

From the 3500xL Dx dashboard screen, click on the Create New Plate icon.

10.16.3.

For the Plate Name, enter a short descriptor.

10.16.4.

Ensure the number of wells is set to 96.

10.16.5.

For the plate type, select Fragment from the drop down menu.

10.16.6.

Ensure the capillary length is 50 cm, and the polymer is POP7.

10.16.7.

Enter the operator initials in the Owner section.

10.16.8.

Click Assign Plate Contents.

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10.16.9.

Click the Import button at the top of the screen and a popup window appears. Navigate to the 3500xL Dx import
file (the ABI file) created by the LeukoStrat CDx FLT3 Software. Click OPEN in the popup window and click
OK in the import confirmation popup window.
10.16.9.1.

If there is no match in the 3500xL Dx library for the assay name in the ABI file, click Proceed in the
popup that appears:

10.16.10. Click OK in the following popup window.

10.16.11. Once the import is complete, the Sample IDs populate the onscreen plate layout. Verify that the onscreen platelayout is correct by reviewing the Sample IDs on screen. If samples do not match the intended setup, a new ABIFile needs to be created in the LeukoStrat CDx FLT3 Software and re-imported into the ABI 3500xL Dx.
NOTE:

Do not change the Sample IDs on the 3500xL Dx plate map. Doing so will result in an error.

10.16.12. If needed, program the 3500xL Dx using the parameters listed in Table 6 for the ITD CDx Assay or TKD CDx
Assay. Programs can be saved in the 3500xL Dx assay library for future use.
10.16.13. Assign the Assay, Results Group, and File Name Convention to all wells containing samples and controls, if
necessary.
NOTE:

File Name Convention must include the Sample Name as the first attribute.

10.16.14. Load the plate(s) on the 3500xL Dx.
10.16.15. Click Link Plate for Run. Operator may save changes to plate if prompted. If a second plate is to be run, repeat
steps 10.16.2 through 10.16.15.

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10.17.

Run 3500xL Dx Genetic Analyzer

10.17.1.

Check for bubbles in the POP-7 tubing. Remove bubbles, if needed.

10.17.2.

Click Start Run to start the run on the 3500xL Dx.

10.17.3.

After the run is complete, remove and discard the septa and discard the CE plate.

NOTE:
10.18.

In case of a connectivity error between the 3500xL or 3500xL Dx instrument and computer running Data Collection
Software, follow the instrument manufacturer’s troubleshooting instructions.

Data Analysis with GeneMapper Software

NOTE:

Do not override a size standard error for a well in the GeneMapper software.

10.18.1.

Open the GeneMapper v4.1.x software.

10.18.2.

Under the File Menu, choose New Project and select Microsatellite. Click OK. Return to the File Menu andchoose Add Samples to Project.

10.18.3.

In the left panel, navigate to the data files in the 3500xL Dx data folder (designated by the Results Group) andclick Add to List to transfer them to the right panel. Click the Add or Add & Analyze button.

10.18.4.

Ensure that the Analysis Method is set to a Microsatellite method and that the Size Standard is set toGS600LIZ+Normalization for all samples.

NOTE:
10.18.5.

If there are multiple assay types in one plate, set the Analysis Method and Size Standard options per injection forease of workflow. Injections can be selected in the Project window.
Set up Analysis Method according to Figure 3.

10.18.5.1.

Click Analysis and then Analysis Method Editor from the menu at the top of the screen.

10.18.5.2.

In the Peak Detector tab, ensure that the Peak Detection Algorithm is set to Advanced.

10.18.5.3.

Ensure under Peak Amplitude Thresholds that 100 is entered for the B (blue) and G (green) dye channels,
and 50 for the remaining Y (yellow), R (red), P (purple), and O (orange) dye channels. Yellow and purple
dye channels are not used in the LeukoStrat CDx FLT3 Mutation Assay.

10.18.5.4.

Ensure that Polynomial Degree is set to 3 for ITD and set to 5 for TKD.

10.18.5.5.

Click OK at the bottom of the window.

NOTE:

ITD and TKD specific Analysis Methods may be setup and used in GeneMapper by going to Tools and selecting the
GeneMapper Manager. From the Analysis Methods tab, click the New… button and select Microsatellite as theanalysis type. Click OK. Provide a Name, Description and Instrument in the General tab, set up the Peak Detector
tab as described above and in Figure 3, and leave the Allele, Peak Quality and Quality Flags tabs set to default
Microsatellite settings. Select Done and the new Analysis Method is selectable.

10.18.6.

Click the green play button to start analysis. This will prompt the file to be saved. Save the GeneMapper projectwith an appropriate name. The file pathname must be 256 characters or less.

10.18.7.

In the GeneMapper software, highlight the specimens and controls to be analyzed and click the Display Plots
button.

10.18.7.1.

For ITD, ensure the Sizing Table icon is selected and that the blue, green, and red dyes are selected in the
Samples Plot window.

10.18.7.2.

For TKD, ensure the Sizing Table icon is selected and that the blue, green, and red dyes are selected.

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Figure 3:

Analysis method settings for ITD. For TKD, settings are identical except that Polynomial
Degree is set to “5.”10.18.8.

Ensure that the table below the electropherogram contains the following columns: Dye/Sample Peak, Sample
File Name, Size, Height, and Area

10.18.9.

To export the sizing table information, select File and then Export Table from the Samples Plot menu.

10.18.9.1.

Enter a file name and select the location to save the file.

10.18.9.2.

In the Export File As dropdown menu, select Comma-separated values (.csv).

10.18.9.3.

Click Export.

NOTE:

Do not edit the CSV file in any way.

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10.19.

Data Analysis with LeukoStrat CDx FLT3 Software

10.19.1.

Open the LeukoStrat CDx FLT3 Software, accept the licensing agreement, and click on the CDx Analysis tab of
the LeukoStrat CDx FLT3 Software. Click Select Platemap File (*.livs) and navigate to the LIVS file generated
from the PlateMapper Setup tab.

10.19.2.

Click the Select Results Data File (*.csv) and select an exported CSV file from step 10.18.9 for analysis.

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10.19.3.

Click the Select Results Output Directory and choose a destination folder for the results.

10.19.3.1.
NOTE:

10.19.4.

Additional comments about the run, samples or controls can be entered in the Report Comments field.
These comments will appear in the Run Report.

Once all files are selected, do not create or import a new Plate Map into the PlateMapper Setup tab prior to analyzing
the current run/data. Alterations to the PlateMapper Setup tab prior to selecting the Analyze button will cause thereport to display the incorrect Plate Name. Close the LeukoStrat CDx FLT3 Software before switching between
PlateMapper Setup tab and the CDx Analysis tab.
Once all three have been selected the analyze button will become selectable. Click Analyze and reports will be
generated in the destination folder. Three report types will be generated – a PDF Run Report, PDF Sample
Report(s), and a CSV run export file (see Figure 4, Figure 5, and Figure 6). The Run Report will contain asummary of results for all controls and samples. The Sample Report will contain results for the controls anddetails for the sample results. The CSV run export file will contain all of the run results in a spreadsheet format.
IDs in the LeukoStrat CDx FLT3 Software reports are the last 12 characters of the ID generated by the software.

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Figure 4: Example Run Report

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Figure 5: Example Sample Report

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Figure 6: Example CSV Run Export File

10.19.5.

Click Clear to reset all fields.

10.19.6.

If results are not obtained, verify all steps were completed correctly. Refer to Table 9 for troubleshooting data
results errors.
If further assistance is required, please contact Invivoscribe Technical Support at
support@invivoscribe.com.

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Table 9: Data Results Error Messages and Corrective Actions

Data Results Upload Error Message

Potential Cause(s)Corrective Action(s)-Unrecognized dye: <dye letter> [AD01]

Selection of unused dyes during
GeneMapper analysis step.

Ensure that only Red, Green, and Blue dyes
are selected during the GeneMapper software
analysis step.

-No red dye detected. Please make sure red
dye is selected during previous signal analysis
step. [AD02]

Did not select red dye during
GeneMapper analysis step

Make sure to select red dye during
GeneMapper analysis step.

-Unrecognized data results file format. [AD03]

GeneMapper file is corrupted

Do not edit the GeneMapper file in any way.

-Unable to load LIVS platemap file; incorrect
format. [AD04]

LIVS file is corrupted

Do not edit the LIVS file in any way.

-Did not find run for runId <runId> [AD05]-Did not find sample for sample name<sampleName> [AD06]

Incorrect LIVS file selected, or LIVS file
is corrupted.

Make sure to select the correct LIVS fileassociated with the experiment analyzed.

-General error loading results data file; pleasecontact technical support. [AD07]

Unknown error has occurred.

Contact Technical Support.

-String index out of range: -1<Sample Name> was not the first
attribute selected when setting up
File Name Convention (step 10.16.13
this is done).

Repeat run beginning with step 10.12
Capillary Electrophoresis Detection withcorrect File Name Convention-String index out of range: 15

CSV results file have been edited.

Repeat export of CSV results file from
GeneMapper. Do not edit the CSV file in anyway.

11. Quality Control
11.1.

Run Validity

11.1.1.

The LeukoStrat CDx FLT3 Software automatically evaluates the results.

11.1.2.

If the run status is Fail, all test results in the same run are invalid. Depending on the Fail Detail, the run must be
repeated at different starting points within the assay (see section 13. Retesting).

11.2.

Extraction Control and Sample Validity

11.2.1.

Within a valid run, individual samples may be invalid (Fail). If an Extraction Control does not meet validity
criteria, all samples associated with that Extraction Control will be labeled Fail.

11.2.2.

Samples where all controls are valid may fail if they individually do not meet specifications. Depending on the
Fail Detail in the LeukoStrat CDx FLT3 Software, the sample(s) must be repeated at different starting points
within the assay (see section 13. Retesting).

NOTE:

If multiple failures of the same Fail Detail Type are observed in a run, the retest strategy is different from isolated
control or sample failures (see section 13. Retesting).

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12. Interpretation of Results
12.1.

AML patients with a detectable FLT3 ITD or TKD mutation at or above the clinical cut-off are indicated for gilteritinib
fumarate therapy.

12.2.

AML patients with a detectable FLT3 ITD mutation at or above the clinical cut-off are indicated for quizartinibhydrochloride therapy. AML patients with only a detectable FLT3 TKD mutation at or above the clinical cut-off are notindicated for quizartinib hydrochloride therapy.

12.3.

The mutant : wild-type signal ratio is calculated by the LeukoStrat CDx FLT3 Software and automatically evaluatedagainst the clinical cut-off (medical decision point) of 0.05. The signal ratio is the peak area of the mutant signal, if
present, divided by the peak area of the wild-type signal, if present. The mutant : wild-type signal ratio is displayed totwo decimal places.

12.4.

To note, ITD mutations may carry multiple mutations; the peak areas of the mutations are summed to calculate the total
mutant signal. Furthermore, a sample may contain no wild-type signal (pure mutant). In this case the mutant : wild-typesignal ratio is reported by the LeukoStrat CDx FLT3 Software as 100; it is not intended to convey a ratio value.

12.5.

For determination of overall FLT3 mutation status or whether gilteritinib fumarate is indicated:

12.5.1.

If the mutant : wild-type signal ratio for either ITD or TKD in a valid sample result is at or above the clinical cutoff of 0.05, the result will be interpreted and reported as Positive and gilteritinib fumarate is indicated.

12.5.2.

If the mutant : wild-type signal ratios for both ITD and TKD in a valid sample result are below the clinical cutoff of 0.05, the result will be interpreted and reported as Negative and gilteritinib fumarate is not indicated.

12.5.3.

The mutation status of a sample is defined by the rules included in Table 10.

Table 10: Determining Sample Mutation Status (applicable for overall FLT3 mutation status reporting or gilteritinib
fumarate indication)

12.6.

Scenario

ITD Software
ResultITD Signal
Ratio

TKD Software
ResultTKD Signal
Ratio

Final Assay Result

1

Positive≥0.05

Positive≥0.05

Positive

2

Negative<0.05

Negative<0.05

Negative

3

Invalid

N/A

Invalid

N/A

Invalid4

Positive≥0.05

Negative<0.05

Positive

5

Negative<0.05

Positive≥0.05

Positive

6

Positive≥0.05

Invalid

N/A

Positive

7

Negative<0.05

Invalid

N/A

Invalid8

Invalid

N/A

Positive≥0.05

Positive

9

Invalid

N/A

Negative<0.05

Invalid

For determination of whether quizartinib hydrochloride is indicated:

12.6.1.

If the mutant : wild-type signal ratio for ITD in a valid sample result is at or above the clinical cut-off of 0.05, theresult will be interpreted and reported as ITD Positive and quizartinib hydrochloride is indicated.

12.6.2.

If the mutant : wild-type signal ratio for ITD in a valid sample result is below the clinical cut-off of 0.05, the result
will be interpreted and reported as ITD Negative and quizartinib hydrochloride is not indicated.

12.6.3.

If the ITD result is invalid, the result will be interpreted and reported as ITD Invalid and quizartinib hydrochloride
is not indicated.

12.7.

Fail Details are provided in the LeukoStrat CDx FLT3 Software report; repeat the run or retest samples according to
instructions in the Retesting section.

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13. Retesting
13.1.

Invalid Runs

13.1.1.

A run in which either Positive Control or No Template Control, or both, does not meet validity criteria is an invalid
run. Repeat the run including all samples, Positive Control, all associated Extraction Controls, and No Template
Control. ITD and TKD runs are independent of each other.

13.1.2.

Repeat the run according to Table 11 or Table 12, based on the assay and the specific Fail Detail(s) listed in theControls section of the LeukoStrat CDx FLT3 Software reports. The Fail Detail(s) listed for the failed Positive
Control or No Template Control supersedes all Extraction Control and sample Fail Details.

13.2.

Invalid Extraction Control within Valid Runs

13.2.1.

13.3.

Invalid Samples within Valid Runs

13.3.1.

13.4.

For Extraction Control failures within a valid run which may contain multiple Extraction Controls, retest all thefailed Extraction Controls, associated samples, Positive Control, and No Template Control for the appropriate ITD
or TKD run. Retest according to Table 11 or Table 12, based on the assay and the specific Fail Detail(s) listed in
the Controls section of the LeukoStrat CDx FLT3 Software reports. The Fail Detail(s) listed for the failed
Extraction Control supersedes all sample Fail Details.

For sample failures within a valid run, retest the sample(s), Positive Control, Extraction Control(s) associated withthe failed sample(s), and No Template Control for the appropriate ITD or TKD run. Retest according to Table 11
or Table 12, based on the assay and the specific Fail Detail(s) listed in the Samples section of the LeukoStrat CDx
FLT3 Software reports. Retesting of a sample must include retesting of the associated Extraction Control.

Fail Details and Retesting

13.4.1.

Table 11 and Table 12 summarize retesting based on the Fail Detail by sample type for ITD and TKD, respectively.
Consult Table 13 for the retest codes listed in Table 11 and Table 12.

13.4.2.

The hierarchy of retesting is 1) invalid ITD or TKD Positive Control (PC) or No Template Control (NTC) (seeSection 13.1); 2) invalid Extraction Control (EC) within a valid run (see Section 13.2); and 3) invalid samples
within a valid run (see section 13.3). Figure 7 diagrams the hierarchy of retesting.

13.4.3.

If more than one failure within a single sample or control has occurred, perform the retest that returns the operator
to the step closest to the beginning of the assay procedure.

13.4.3.1.

If the same Fail Detail occurs in the same control/sample, proceed to the next Retest Starting Point, if one
is listed. If the same Fail Detail occurs again after all the troubleshooting actions have been completed, thecontrol/sample result is invalid.

13.4.3.2.

If the results of the retest are a different failure mode than the initial results, follow the troubleshooting
action described for the new retest failure mode.

NOTE:
13.4.4.
NOTE:

No more than four retests are allowed for a single control/sample.
Invalid samples are assessed independently; thus, if multiple samples with different Fail Details for each sample are
identified within a single run, perform the retest that is appropriate for each sample.
If further assistance is required, please contact Invivoscribe Technical Support at support@invivoscribe.com.

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Figure 7: Retest hierarchy diagram.
Table 11: Retesting, ITD Controls and Samples

ITD Fail Detail

Controls
PC

IR05: Sample or Control Failed.

Amp

IR06: Sample or Control Failed.

Amp

NTC

SamplesEC

Pos Sample

Amp

Neg Sample
Quant/Proc
Quant/Proc

IR07: Sample Failed.

CE-DS

CE-DS

CE

CE/Proc

CE/Proc

Q-Amp

Quant/Proc

Quant/Proc

Ctrl

Ctrl

Ctrl

Ctrl

IR53: Control Failed.

Ctrl

Ctrl

IR70: Sample Failed.

CE/Proc

IR09: Sample or Control Failed.

CE

IR12: Sample or Control Failed.

Amp

IR13: Control Failed.

CE

CE

IR20: Control Failed.
IR21: Control Failed.
IR31: Control Failed.

Amp

IR32: Control Failed.

CE/AmpIR33: Control Failed.

CE/AmpIR34: Control Failed.

Amp

IR40: Control Failed.

Amp

IR51: Control Failed.

Q-Amp

IR52: Control Failed.

CE/Q-Amp

IR80: Sample Failed.
IR91: Sample or Control Failed.

LeukoStrat CDx FLT3 Mutation Assay

Quant/Proc
CE

CE

CE

CE

CE

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Table 12: Retesting, TKD Controls and Samples

TKD Fail DetailTR07: Sample or Control Failed.

Controls
PC

Samples

NTC

EC

Dig

TR09: Sample or Control Failed.

CE

TR12: Sample or Control Failed.

Amp

Pos Sample

Neg Sample

Dig
CE

CE/Proc

CE/Proc

Q-Amp

Quant/Proc

Quant/Proc

TR20: Control Failed.

Ctrl

Ctrl

TR21: Control Failed.

Ctrl

Ctrl

TR53: Control Failed.

Ctrl

Ctrl

TR70: Sample Failed.

Xtalk/Quant/Proc

TR71: Sample Failed.

CE/Proc

TR72: Sample Failed.

Dig/Proc

TR30: Control Failed.

Xtalk/Amp

TR31: Control Failed.

CE/AmpTR32: Control Failed.

CE/AmpTR33: Control Failed.

Amp

TR40: Control Failed.

CE

Dig/Proc

Amp

TR50: Control Failed.

Xtalk/Q-Amp

TR51: Control Failed.

CE/Q-Amp

TR52: Control Failed.

Dig

TR80: Sample Failed.

Xtalk/Quant/Proc

TR81: Sample Failed.

Quant/Proc

TR93: Sample or Control Failed.

CE

CE

CE

CE

CE

Table 13: Retest Codes, Controls and Sample Retesting

Retest CodeDescription

Amp

Repeat beginning at amplification, using previously made DNA sample
dilutions.

CE

Repeat beginning at capillary electrophoresis. Make a new CE plate with fresh
amplicon (from the stored ITD PCR or TKD digestion plate) ensuring the
Positive Control, No Template Control, and associated Extraction Control(s)are also present on the plate with a failed sample. Retesting due to artifacts
must stop if the artifact appears at the same size for two runs in a row;otherwise, artifacts at different sizes are considered unique failure modes.

Retest Starting Point
10.10. Amplification

10.12. Capillary Electrophoresis

Detection

10.12. Capillary Electrophoresis
CE/AmpCE/Q-Amp

Retest following the instructions for the CE retest code. If the retest results
yield the same Fail Detail, retest again following the instructions for Amp or
Q-Amp, as prescribed.

Detection
10.9. Quantification and Dilution ofDNA
10.10. Amplification

CE/Proc

CE-DS

Retest following the instructions for the CE retest code. If the retest results
yield the same Fail Detail, reprocess the sample beginning from peripheral
blood or bone marrow aspirate.
Repeat beginning at capillary electrophoresis. Make a new CE plate with fresh
amplicon (from the stored ITD PCR plate) ensuring the Positive Control, No
Template Control, and the associated Extraction Control(s) are also present
on the plate. If the same Fail Detail (IR07) occurs a second time, report the10.12. Capillary Electrophoresis

Detection
10.2. Sample Processing

Preparation
10.12. Capillary Electrophoresis

Detection

sample as Invalid.

Ctrl

Retest following the instructions for the control that failed.

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Table 13: Retest Codes, Controls and Sample Retesting

Retest CodeDescription

Retest Starting PointDig

Repeat beginning at digestion, using fresh amplicon (from stored TKD PCR
plate) of the sample(s), associated Extraction Control(s) and controls.

Dig/Proc

Retest following the instructions for the Dig retest code. If the retest results
yield the same Fail Detail, reprocess the sample beginning from peripheral
blood or bone marrow aspirate.

Repeat beginning at quantification of Extraction Control(s), using previously
made DNA sample dilutions.

Q-Amp

Quant

Repeat beginning at quantification of all sample(s) and associated Extraction
Control(s).

Quant/Proc

Retest following the instructions for the Quant retest code. If the retest
results yield the same Fail Detail, reprocess the sample beginning fromperipheral blood or bone marrow aspirate.
Make a new CE plate such that all samples are separated by 5 empty
capillaries (i.e. only load samples in wells A01, C01, E01, and G01 for injection
1. Load samples in the equivalent wells for the remaining injections). If theretest results yield the same Fail Detail, retest following the instructions for
Amp, Q-Amp, or Quant, as prescribed. If the Quant retest results yield thesame Fail Detail, reprocess the sample beginning from peripheral blood orbone marrow aspirate.

Xtalk/Amp
Xtalk/Q-Amp
Xtalk/Quant/Proc

13.5.

10.11. Restriction Digest (TKD

Mutation Only)
10.11. Restriction Digest (TKD

Mutation Only)
10.2. Sample Processing

Preparation
10.9. Quantification and Dilution of

DNA (Extraction Control(s))
10.10. Amplification (Samples)
10.9. Quantification and Dilution ofDNA
10.9. Quantification and Dilution of

DNA
10.2

Sample Processing
Preparation

10.12. Capillary Electrophoresis

Detection
10.9. Quantification and Dilution of

DNA (Extraction Control(s))
10.10. Amplification (Samples, PC)
10.2. Sample Processing

Preparation

Multiple Failures within a Run

13.5.1.

In contrast to isolated invalid sample or Control Results, some Fail Details can be observed in several to all
reaction wells. When this type of failure occurs, repeat the run including all samples, Positive Control, allassociated Extraction Controls, and No Template Control according to Table 14; retest codes are listed in Table
15.

13.5.2.

Additional troubleshooting actions may include the following items:

13.5.2.1.
13.5.2.2.
13.5.2.3.
13.5.2.4.
13.5.2.5.

Confirm that the Analysis Method settings, Size Standard settings, and other GeneMapper softwaresettings, are correct.
Ensure that all steps were followed in the GeneMapper section, as forgetting a step, like not pressing thegreen play button, can lead to erroneous results.
Open the CSV file to confirm it contains results for all sample and control wells that have an associated3500xL Dx *.fsa file.
In the CSV file, ensure the proper columns are present, the peak thresholds are correct (e.g., no peaks lessthan 100 in Blue and Green or less than 50 in Red), and that the columns are populated with non-zero
numbers.
Consult the electropherogram provided by the GeneMapper software to visualize peak presence, shape, andsize.
Table 14: Retesting, Multiple Failures within a Run

LeukoStrat CDx FLT3 Mutation Assay

Sample Type

Fail Code

ITD PCIR31

ITD NTCIR40

ITD ECIR51

TKD PCTR30

TKD NTC

TR40

TKD EC

TR50

Retest Code

Amp

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Table 14: Retesting, Multiple Failures within a Run

Sample Type

Fail Code

ITD PCIR33

ITD Sample

IR70

TKD PCTR32

TKD Sample

TR71

ITD PCIR32

ITD ECIR52

ITD Sample

IR80

TKD PCTR31

TKD ECTR51

TKD Sample

TR81

All ITD within an injection

IR91

All TKD within an injection

TR93

All ITD within a run

IR04

All TKD within a run

TR04

Retest Code

CE/AmpAnalysis/Amp

CE-SS
GM

Table 15: Retest Codes, Multiple Failure Retesting

Retest CodeDescription
Repeat analysis in GeneMapper, ensuring the green play button is pushed
to analyze the data (step 10.18.6).

Retest Starting Point

10.18. Data Analysis with

If the repeat analysis in GeneMapper produces the same result, repeat
beginning at amplification, using previously made test sample DNA
dilutions. Ensure all tubes are vortexed per instructions and that Taq wasadded.

10.10. Amplification

Amp

Repeat beginning at amplification, using previously made test sample DNA
dilutions. Ensure all tubes are vortexed per instructions and that Taq wasadded.

10.10. Amplification

CE

Repeat beginning at capillary electrophoresis. Make a new CE plate withfresh amplicon (from the stored ITD PCR or TKD digestion plate) and fresh
size standard solution. Ensure the Positive Control, No Template Control,
and associated Extraction Control(s) are also present on the plate with thefailed sample.

CE/AmpRetest following the instructions for the CE retest code. If the retest results
yield the same Fail Detail, retest again following the instructions for Amp.

Analysis/Amp

13.6.

GeneMapper Software

10.12. Capillary Electrophoresis

Detection

10.12. Capillary Electrophoresis

Detection
10.10. Amplification

CE-SS

Repeat beginning at capillary electrophoresis, using a new preparation ofsize standard solution.

10.12. Capillary Electrophoresis

GM

Repeat creation of export data files from GeneMapper software, ensuring
the threshold(s) are set to 100 RFU.

10.18. Data Analysis with

Detection
GeneMapper Software

Dye Shift13.6.1.

In rare instances with some large ITD inserts, the LeukoStrat CDx FLT3 Software may misidentify confirmationof a mutant peak. To confirm the dye shift, repeat capillary electrophoresis by making a new CE plate with fresh
amplicon from the stored ITD PCR plate.

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14. Limitations of Procedure
14.1.

Test only the indicated specimen types, as the LeukoStrat CDx FLT3 Mutation Assay has been validated for use onlywith peripheral blood and bone marrow aspirate. Reliable results are dependent on appropriate storage and processingof the specimens; therefore, follow the procedures in this Package Insert.

14.2.

The LeukoStrat CDx FLT3 Mutation Assay has been validated using only QIAamp DNA Blood Mini Kit to extract
genomic DNA.

14.3.

The LeukoStrat CDx FLT3 Mutation Assay will detect ITD mutations sized 3 bp to 323 bp; however, the assay is only
validated to detect mutations sized 30 bp to 279 bp.

14.3.1.

ITD insertions sized between 3 bp and 30 bp will be reported as ITD mutations.

14.3.2.

ITD insertions sized between 279 bp and 323 bp will be reported as ITD mutations.

14.3.3.

ITD insertions sized greater than 323 bp, will not be reported as insertions.

14.4.

This assay may not detect FLT3 mutations that present below the sensitivity level of the assay.

14.4.1.

For ITD insertions sized 30 bp to 126 bp, inclusive, an allelic ratio of 0.08 will yield a positive LeukoStrat CDx
FLT3 Mutation Assay result.

14.4.2.

For ITD insertions sized 129 bp to 279 bp, inclusive, an allelic ratio of 1 will yield a positive LeukoStrat CDx
FLT3 Mutation Assay result.

14.4.3.

For TKD mutations that modify the EcoRV site, an allelic ratio of 0.18 will yield a positive LeukoStrat CDx FLT3
Mutation Assay result.

14.5.

The results of the assay should always be interpreted in the context of clinical data and other tests performed for thepatients.

14.6.

Detection of a mutation is dependent on the number of mutant sequence copies present in the specimen and may beaffected by specimen integrity, amount of DNA isolated, and the presence of interfering substances. PCR-based assays
are subject to interference by degradation of DNA or to inhibition of PCR due to EDTA and other agents.

14.7.

Use of this product must be limited to personnel trained in the techniques of PCR and the use of the LeukoStrat CDx
FLT3 Mutation Assay.

14.8.

The LeukoStrat CDx FLT3 Mutation Assay is a qualitative test. The test is not for quantitative measurements of ITD or
TKD mutations.

14.9.

The allelic ratio of a specimen cannot be calculated, measured, or determined using this assay.

15. Expected Values
15.1.

Expected Size of Amplified Products

15.1.1.
NOTE:

The amplicon sizes listed were determined using a 3500xL Dx instrument (Table 16).
“Dye Channel” indicates the color of products generated with the master mix when using the default color
assignment on ABI fluorescence detection systems.

Table 16: Expected Amplicon Sizes

Master Mix

FLT3 ITD

FLT3 TKDaPart #

TargetDye Channel

R0880200

Exon 14

Blue &

R0880220

and 15

Green

R0880210
R0880220

Exon 20

Blue

Control DNA

Product Size in Nucleotides

Valid Size Range

326-650

FLT3 ITD Positive Control DNA

327±1, 357±1

FLT3 Extraction Control DNA

327±1

Valid Size Range

78-80, 124-128

FLT3 TKD Positive Control DNA

79±1, 127±1

FLT3 Extraction Control DNA

79±1, 127±1a

A small 127 bp product peak may or may not be present in the extraction control.

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16. Non-Clinical Performance Evaluation
16.1.

Analytical Sensitivity – Limit of Blank (LoB)

16.1.1.

16.2.

When samples containing wild-type DNA only (i.e. a mutant blank) were tested in the LeukoStrat CDx FLT3
Mutation Assay, the SR was 0.00 in the ITD assay and 0.00 to 0.01 in the TKD assay. This limit of blank is well
below the clinical cutoff SR of 0.05.

Analytical Sensitivity

16.2.1.

LoD of the assay was evaluated in two studies. The first study used contrived samples created by blending celllines with leukocyte-depleted whole blood. Cell line samples were used to represent three ITD insert sizes: 21 bp,
30 bp insert, 126 bp insert, and a 279 bp insert. An additional cell line containing the D835 mutation was also
assessed. DNA was diluted to 5 ng/μL, 10 ng/μL, and 15 ng/μL and tested at multiple allelic ratios for each cellline. A second study with clinical specimens was conducted to confirm the LoD observations obtained with celllines. Five clinical samples were diluted with clinical negative samples in order to yield a targeted signal ratio(TSR) within the linear range of an appropriate cell line standard (Table 17). Each specimen was diluted to 5
levels representing a low negative (LN), high negative (HN), near the cut-off (CO), a low positive (LP), and amoderate positive (MP). These linear range samples were tested in the LeukoStrat CDx FLT3 Mutation Assayand an average SR value was determined. Each clinical LoD sample dilution was tested 20 times for each dilution
level over four nonconsecutive days (5 replicates per day) by one operator using one equipment set. The AR ofeach clinical LoD sample dilution was calculated using the AR estimated from the cell line standard curves. The
ARs of the clinical LoD samples were estimated based on the study meeting the following acceptance criteria:The SR and AR where FLT3 mutations can be detected above the limit of blank (LoB) in ≥95% ofreplicates (Analytical LoD).
The AR near the clinical cut-off, a SR of 0.04 – 0.06 (Cut-off).
The AR and SR that is detected equal to or above the clinical cut-off in ≥95% of replicates (Above Cut-off).

Table 17: SR, AR and LoD per each Sample and Dilution Level

Sample ID

TKD CS1TKD CS2

ITD CS1

ITD CS2

Mutation

Level

TSR

SR
Mean

AR of
Blend

Valid NN (%)
SR > LoBN (%)
SR ≥ 0.05*Classification

LN

0.02

0.02

0.039

20

20 (100.0)0

Analytical LoD

HN

0.03

0.03

0.057

20

20 (100.0)

0

-

CO

0.05

0.05

0.094

20

20 (100.0)

16 (80.0%)

Cut-off

LP

0.08

0.07

0.144

20

20 (100.0)

20 (100.0)

Above Cut-off

MP

0.13

0.12

0.224

20

20 (100.0)

20 (100.0)-

TKD I836

TKD D835

ITD
24 bp

ITD
66 bp

LeukoStrat CDx FLT3 Mutation Assay

LN

0.01

0.02

0.023

20

20 (100.0)0

Analytical LoD

HN

0.02

0.03

0.047

20

20 (100.0)

0

-

CO

0.04

0.05

0.089

20

20 (100.0)

19 (95.0)

Cut-off
Above Cut-off

LP

0.07

0.08

0.152

20

20 (100.0)

20 (100.0)

-

MP

0.13

0.15

0.269

20

20 (100.0)

20 (100.0)

-

LN

0.02

0.02

0.044

20

20 (100.0)0

Analytical LoD

HN

0.03

0.03

0.065

20

20 (100.0)

0

-

CO

0.05

0.05

0.107

20

20 (100.0)

20 (100.0)

Cut-off
Above Cut-off

LP

0.08

0.08

0.165

20

19 (95.0)

19 (95.0)

-

MP

0.13

0.13

0.257

20

20 (100.0)

20 (100.0)

-

LN

0.02

0.02

0.045

20

20 (100.0)0

Analytical LoD

HN

0.03

0.03

0.066

20

20 (100.0)

0

-

CO

0.05

0.05

0.110

20

20 (100.0)

18 (90.0)

Cut-off

LP

0.09

0.08

0.189

20

20 (100.0)

20 (100.0)

Above Cut-off

MP

0.14

0.13

0.280

20

20 (100.0)

20 (100.0)-

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Table 17: SR, AR and LoD per each Sample and Dilution Level

Sample ID

Mutation

Level

TSR

SR
Mean

AR of
Blend

Valid NN (%)
SR > LoBN (%)
SR ≥ 0.05*Classification

LN

0.01

0

0.073

20

2 (10.0)

0

-

HN

0.02

0.02

0.147

20

15 (75.0)

0

-

CO

0.04

0.04

0.276

20

20 (100.0)

9 (45.0)

Analytical LoD
Cut-off

LP

0.08

0.08

0.539

20

19 (95.0)

19 (95.0)

Above Cut-off

ITD
217 bp

ITD CS3

MP

0.13

0.13

0.838

20

20 (100.0)

20 (100.0)-

True Neg ITD

None

TN

N/A

0

0

20

0

0

N/A

TrueNegTKD

None

TN

N/A

0

0

20

0

0

N/A

*Classifications are defined as 1: Analytical LoD = lowest AR where samples were detected 95% of the time above the LoB, 2: Cut-off is AR where samples
were near SR 0.05, and 3: Above Cut-off = lowest AR where samples could be detected 95% of the time equal to or above SR 0.05.

16.2.2.

The LeukoStrat CDx FLT3 Mutation Assay is capable of detecting the following mutant/wild-type allelic ratios
above the clinical cut-off of the following mutation types:

16.2.3.

For ITD insertions sized at 24 bp, an allelic ratio of 0.107 was detected above the cut-off SR in more than 95% of
samples. The SR %CV for these samples was 7.1%.

16.2.3.1.

For ITD insertions sized at 66 bp, an allelic ratio of 0.189 was detected above the cut-off SR in more than
95% of samples. The SR %CV for these samples was 7.1%.

16.2.3.2.

For ITD insertions sized at 217 bp, an allelic ratio of 0.539 was detected above the cut-off SR in more than
95% of samples. The SR %CV for these samples was 25.6%.

16.2.3.3.

For D835 TKD mutations that destroy the EcoRV site, an allelic ratio of 0.089 was detected above the cutoff SR in more than 95% of samples. The SR %CV for these samples was 4.5%.

16.2.3.4.

For I836 TKD mutations that destroy the EcoRV site, an allelic ratio of 0.144 was detected above the cutoff SR in more than 95% of samples. The SR %CV for these samples was 5.7%.

16.2.3.5.

Conversion of AR values to % Mutant is shown in Table 18.

Table 18: Analytical Sensitivity Allelic Ratio and % Mutant

Sample ID

16.3.

Mutation

Mutation Classification

Above Cut-off 95% SR≥ 0.05
AR

SR

%Mut

TKD CS1TKD I836

TKD I836 Deletion

0.144

0.07

12.6

TKD CS2TKD D835

TKD D835 Substitution

0.089

0.05

8.2

ITD CS1

ITD 24 bp

Small ITD Insert <30 bp

0.107

0.05

9.7

ITD CS2

ITD 66 bp

Medium ITD Insert 30-100 bp

0.189

0.08

15.9

ITD CS3

ITD 217 bp

Large ITD Insert ~200 bp

0.539

0.08

35.0

Precision

16.3.1.

The precision of the LeukoStrat CDx FLT3 Mutation Assay was determined by three operators independently
testing 10 replicates each of ITD mutation samples with inserts ranging in size from 21 bp to 126 bp and TKD
mutation samples. The 10 replicates were tested in batches of two a total of 5 separate times.

16.3.2.

For the ITD mutation samples, the SR %CV ranges for the 3 operators were 7.4% to 15.0%, 3.7% to 13.0%, and4.2% to 8.8%.

16.3.3.

For the TKD mutation samples, the SR %CV ranges for the 3 operators were 6.3% to 11.2%, 5.8% to 9.3%, and5.5% to 8.3%.

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16.4.

Operator-to-Operator Reproducibility (cell-lines)

16.4.1.

Samples consisted of ITD cell lines containing inserts of 21 bp, 30 bp, and 126 bp and the D835 TKD mutation.
Samples represented low (near cutoff), mid, and high (100% mutant cell line) mutant : wild-type SRs for smallinternal tandem duplication (ITD) insert, large ITD insert, and tyrosine kinase domain (TKD) mutation. Three
operators using one reagent lot and one instrument over 15 runs tested 10 replicates each. The SR %CV ranged
from 6.6% to 13.3%.

16.4.2.

For TKD mutation samples, the SR %CV ranged from 7.9% to 9.3%.

16.4.3.

For ITD mutation samples up to and including 30 bp inserts, the SR %CV ranged from 6.6% to 9.4%.

16.4.4.

For ITD mutation samples at 126 bp inserts, the SR %CV ranged from 9.0% to 13.3%.

16.5.

Operator-to-Operator Reproducibility (clinical samples)

16.5.1.

In a second study, precision was assessed using clinical DNA samples from 7 positive clinical samples (5 PB and
2 BM) with ITD lengths of 21 bp, 24 bp, 66 bp, 90 bp, and 217 bp, TKD D835 substitution, TKD I836 deletion,and 8 (4 PB and 4 BM) FLT3 negative samples. DNA from FLT3 negative clinical specimens was pooled andused to dilute the FLT3 positive samples in order to achieve three target SR levels near the assay’s clinical cut-off(i.e., high negative, low positive, and moderate positive). Five FLT3 positive clinical samples originated from PB
and two from BM. Three replicates of 5 ITD positive, 2 TKD positive and one pooled true negative sample were
tested by three different operators/instrument sets using 1 reagent lot over five non-consecutive days at three
dilution levels for positive samples and neat for the negative. Each operator tested 15 replicates total per level fora total of 45 replicates per dilution level.

16.5.2.

The total %CV of all mutation types and levels are shown in table below and the %CV for all mutation types,
except the long ITD insert (217 bp) sample, ranged from 4.2% to 16.1%. The sample with a 217 bp mutation %CV
ranged from 26.9% to 27.2%. The low positive (LP) dilution level %CV was 26.9% for 217 bp, therefore failingthe study acceptance criteria of ≤25% CV for SR. Results show that acceptance criteria were met for both D835
and I836 TKD mutations and for ITD mutations up to 217 bp. Variation for the 217 bp ITD mutation exceeded
25%, thus indicating greater imprecision around the largest ITDs.

Table 19: Components of Variance per Mutation Type and Dilution Level

Sample ID

S1

S2

S3

S4

S5

S6

S7

Mut Type

TKD I836

TKD D835

ITD
21 bp

ITD
24 bp

ITD
66 bp

ITD
90 bp

ITD
217 bp

SR Variation Due toTotal Variation

Dilution
Level

Mean SR

Operator /Instrument SD (%)

Run Day SD (%)

Random Error SD
(%)

SD

%CV

HN

0.03

0.000 (3.22%)

0.000 (0.00%)

0.002 (96.78%)

0.002

7.1

LP

0.077

0.001 (2.60%)

0.000 (0.00%)

0.005 (97.40%)

0.005

5.9

MP

0.132

0.002 (6.67%)

0.003 (17.43%)

0.005 (75.90%)

0.006

4.6

HN

0.04

0.001 (7.13%)

0.000 (0.00%)

0.002 (92.87%)

0.002

5.3

LP

0.08

0.002 (14.02%)

0.001 (2.47%)

0.004 (83.51%)

0.004

5.3

MP

0.165

0.003 (16.28%)

0.000 (0.00%)

0.007 (83.72%)

0.007

4.2

HN

0.03

0.000 (0.00%)

0.000 (0.00%)

0.001 (100.0%)

0.001

5

LP

0.074

0.000 (0.00%)

0.002 (8.08%)

0.005 (91.92%)

0.005

7.2

MP

0.133

0.002 (14.46%)

0.000 (0.00%)

0.005 (85.54%)

0.006

4.4

HN

0.029

0.000 (0.00%)

0.000 (0.00%)

0.004 (100.0%)

0.004

15.2

LP

0.07

0.000 (0.00%)

0.000 (0.92%)

0.004 (99.08%)

0.004

5.3

MP

0.147

0.002 (8.20%)

0.001 (3.28%)

0.006 (88.52%)

0.007

4.5

HN

0.029

0.001 (4.28%)

0.000 (0.00%)

0.005 (95.72%)

0.005

16.1

LP

0.083

0.000 (0.00%)

0.001 (1.13%)

0.007 (98.87%)

0.007

8

MP

0.185

0.000 (0.00%)

0.000 (0.00%)

0.010 (100.0%)

0.01

5.3

HN

0.03

0.001 (5.15%)

0.000 (0.00%)

0.003 (94.85%)

0.003

10.1

LP

0.091

0.004 (25.23%)

0.002 (8.42%)

0.007 (66.35%)

0.008

8.5

MP

0.206

0.013 (44.26%)

0.005 (7.34%)

0.013 (48.40%)

0.019

8.5

HN

0.032

0.001 (0.90%)

0.002 (7.20%)

0.008 (91.90%)

0.009

27.2

LP

0.079

0.013 (31.42%)

0.009 (14.86%)

0.017 (53.71%)

0.023

26.9

MP

0.162

0.029 (36.75%)

0.015 (9.86%)

0.035 (53.39%)

0.047

27.2

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16.6.

Lot-to-Lot and Instrument-to-Instrument Reproducibility

16.6.1.

The lot-to-lot and instrument-to-instrument reproducibility was determined by a single operator testing the same
set of samples using 3 lots of reagents on 3 sets of instruments. Cell line samples consisted of ITD samples
containing inserts ranging in size from 21 bp to 126 bp and TKD mutation samples.

16.6.2.

For the ITD mutation samples, the SR %CV ranged from 3.0% to 8.4%.

16.6.3.

For the TKD mutation samples, the SR %CV ranged from 5.4% to 10.6%.

16.7.

Interfering Substances – Exogenous

16.7.1.
16.8.

Interfering Substances – Endogenous

16.8.1.
16.9.

The LeukoStrat CDx FLT3 Mutation Assay is capable of detecting ITD mutations sized 18 bp to 114 bp and TKD
mutations in the presence of lipids/triglycerides, hemoglobin, protein, and bilirubin.

Interfering Substances – Treatment Drugs

16.9.1.
16.10.

The LeukoStrat CDx FLT3 Mutation Assay is capable of detecting ITD mutations sized 18 bp to 114 bp and TKD
mutations in the presence of Na-Heparin and the wash buffer used during the DNA isolation process.

The LeukoStrat CDx FLT3 Mutation Assay is capable of detecting ITD mutations sized 18 bp to 114 bp and TKD
mutations in the presence of cytarabine and daunorubicin.

Carryover and Cross Contamination

16.10.1.

When challenged via the typical checkerboard plate map set ups it was shown that carryover and crosscontamination were not problematic for the LeukoStrat CDx FLT3 Mutation Assay:

16.10.2.

Carryover / Cross Contamination detected was 0%.

16.10.3.

ITD and TKD No Template Control failure rate was 0%.

16.11.

DNA Input

The purpose of this study was to provide evidence that demonstrated equivalency when using DNA inputs at 10±3 ng/µL in theassay. Extracted DNA replicates from the Limit of Detection and Dynamic Range study with contrived samples were used by
testing only the lowest allelic ratio sample panel members. DNA samples, listed below, were diluted to 7, 10, and 13 ng/µL andtested with the assay along with a single replicate of Negative Control.


AR 0.03 30 bp ITD (33 replicates at each DNA input level)
AR 0.05 D835 TKD (33 replicates)
AR 0.05 126 bp ITD (22 replicates)
AR 1 279 bp ITD (11 replicates)

Acceptance criteria were met for 30 bp ITD, 126 bp ITD, and D835 TKD cell line samples: 1) >93.9% of replicates met sample
validity criteria for every sample type and DNA input; 2) overall coefficient of variation (CV) was <20.5% for every sample type;and 3) CV was <21.0% for every sample type when replicates were pooled between 7 and 10 ng/µL and between 13 and 10 ng/µL
DNA input. Acceptance criteria were not met for long ITD cell line. While 100% of replicates met sample validity criteria, theoverall CV and CV among pooled DNA inputs exceeded 25%.
The difference in mean mutant : wild-type SRs among DNA inputs did not exceed 0.022, and the differences between means were
not significantly different. The assay is able to provide consistent results when challenged with DNA inputs at 10±3 ng/µL.
16.12. Validation of EDTA Blood Collection Tubes
16.12.1. The purpose of this study was to validate EDTA Blood collection tubes. This study used contrived samples
consisting of ITD cell lines containing inserts of 21 bp, 126 bp, and 279 bp and the D835 TKD Mutation cell line
spiked into peripheral blood collected in Na-Heparin or EDTA. Samples represented high negative, low positive(near cut-off), and moderate positive mutant : wild-type SRs. Peripheral blood alone was used as the true negative
samples.
16.12.2. Low positive and moderate positive samples resulted in 100% positive replicates in both EDTA and NaHeparin. High negative and true negative samples resulted in 100% negative replicates in both EDTA and NaHeparin. Therefore the acceptance criteria were met.
16.12.3. The individual %CVs of SRs in EDTA and Na-Heparin ranged from 6.7% to 17.8% and 7.5% to 16.3%,
respectively. The combined overall SR %CV ranged from 8% to 24.6%, meeting the study acceptance criterion.
16.12.4. All validation acceptance criteria were met and EDTA Blood Collection Tubes are validated for use with the
LeukoStrat CDx FLT3 Mutation Assay.
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16.13.

Equivalency: NEBuffer r3.1 vs NEBuffer 3.1

16.13.1.

The study was to provide objective evidence that NEBuffer r3.1 is equivalent to NEBuffer 3.1 for LeukoStrat CDx
FLT3 Mutation Assay. NEBuffer (3.1 or r3.1) is used with the restriction endonuclease enzyme EcoRV to digest
TKD amplicons in order to detect two TKD mutations (D835 and I836) by LeukoStrat CDx FLT3 Mutation Assay.
The only difference between NEBuffer 3.1 and NEBuffer r3.1 is that the bovine serum albumin within NEBuffer
3.1 is replaced by the recombinant albumin within NEBuffer r3.1. The study was designed to test 8 TKD positive
clinical DNA samples (containing at least one sample with I836 mutation) and 8 TKD negative clinical DNA
samples in triplicates using 3 lots of NEBuffer r3.1 to compare with 1 lot of NEBuffer 3.1.

16.13.2.

There was 100% agreement between NEBuffer r3.1 and 3.1 for all samples. All TKD positive samples werecorrectly called as positive and all TKD negative samples were correctly called as negative.

16.13.3.

NEBuffer r.31 has been validated for use with the LeukoStrat® CDx FLT3 Mutation Assay as all acceptancecriteria were met.

16.14.

Density Gradient Media Validation

16.14.1.

The purpose of this study was to validate the use of any density gradient media (with a 1.077 g/mL density) in the
LeukoStrat CDx FLT3 Mutation Assay. The assay was challenged by testing the ability of the density gradient
media (DGM) to isolate small populations (or percentages) of mutant cells. Cell lines (21 bp insert, 279 bp insert,
and TKD D835) were mixed with healthy peripheral blood at three low mutant cell fractions per cell line (resulting
in nine panel members). The contrived panel members for the 21bp ITD insert and the TKD cell lines contained
a mutant content up to 5% cells. The long ITD insert had a mutant content up to 30% cells, due to a known Assay
limitation as a result of PCR bias for long insert lengths. Healthy peripheral blood was also tested as the FLT3
negative sample (resulting in one panel member). Mononuclear cells were isolated from two replicates using threedensity gradient media (DGM) manufacturers by two operators on two days yielding a total of eight isolation
replicates per panel member per density gradient media.

16.14.2.

The percent of overall positive calls of two additional DGM manufacturers (DGM2 and DGM3) was comparedto the DGM that was initially validated for use with the LeukoStrat CDx FLT3 Mutation Assay (DGM1). The
mutant content was low for all panel members and therefore only a small portion of the panel member replicateswere expected to yield positive SRs (above the clinical cutoff). Positive calls per DGM2 were within 2.5% of
positive calls by DGM1 and positive calls per DGM3 were within 1.2% of positive calls by DGM1; therefore
meeting the requirement of the overall positive calls being within 10% of DGM1.

16.14.3.

Using DGM1 as the reference, the point estimates for PPA were 93.3% and 96.7% for DGM2 and DGM3,
respectively. The point estimates for NPA using DGM1 as reference were 100% for both DGM2 and DGM3.

16.14.4.

All study acceptance criteria were met which validates the use of any 1.077 g/mL density gradient media in the
LeukoStrat CDx FLT3 Mutation Assay.

17. Clinical Performance Evaluation
17.1.
Study Overview (IVS-056-001)
17.1.1.
The LeukoStrat CDx FLT3 Mutation Assay has been developed by Invivoscribe (IVS) and is FDA approved as acompanion diagnostic to be used as an aid in the assessment of Acute Myelogenous Leukemia (AML). In order todemonstrate clinical utility of the Companion Diagnostic (CDx) test, patients provided informed consent to have theirsample tested with the LeukoStrat CDx FLT3 Mutation Assay for enrollment in a pivotal clinical study (Phase III Study
2215-CL-0301 evaluating ASP2215 efficacy). The two types of mutations in the FLT3 gene detected by the FLT3 CDx
test are internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations.
17.1.2.
To assess the accuracy of the LeukoStrat CDx FLT3 Mutation Assay, a next-generation sequencing method utilizing the
MiSeq platform from Illumina served as an independent source of sequence information for the ITD and TKD mutations.
The reference method testing was developed and validated by Invivoscribe for the ability to assess the presence or absenceof FLT3 ITD and TKD mutations. The assay was then used to assess the accuracy of the LeukoStrat CDx FLT3 Mutation
Assay using the extracted DNA of biological specimens collected during screening and enrollment of the 2215-CL-0301
study.

Study Objectives (IVS-056-001)
17.2.
17.2.1.
At the interim analysis, the co-primary objective of the study was to determine the efficacy of gilteritinib fumarate(ASP2215) therapy as assessed by the rate of complete remission and complete remission with partial hematologicalrecovery (CR/CRh) in subjects with FLT3-mutated AML who are refractory to or have relapsed after first-line AML
therapy.
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17.2.2.
17.2.3.

At the final analysis, the co-primary objective of the study was to estimate gilteritinib fumarate efficacy in the LeukoStrat
CDx FLT3 Mutation Assay positive population through application of a stratified log-rank test on overall survival.
The reference method study objective is to assess independently the presence or absence of FLT3 mutations using the
MiSeq next-generation sequencing platform from Illumina in order to confirm the accuracy of the LeukoStrat CDx FLT3
Mutation Assay. The objective of this study is described in the Secondary Objective section of the protocol, Pivotal Study
for the LeukoStrat FLT3 Mutation Assay CDx for the ASP2215 Compound.

Patient Population (IVS-056-001)
17.3.
17.3.1.
At the interim analysis, over 594 specimens from 485 subjects were screened with the LeukoStrat CDx FLT3 Mutation
Assay as of the cut-off date. One hundred forty-two patients in the gilteritinib/ASP2215 arm were included in the firstinterim analysis. Subjects were enrolled in the study based on identification of FLT3 mutations in a BM or PB sample.
The clinical cut-off of the test for the study was set at 0.05 (mutant : WT signal ratio).
17.3.2.
At the final analysis, 771 specimens from 633 subjects were screened with the LeukoStrat CDx FLT3 Mutation Assay.
371 subjects were included in the final Intent to Treat (ITT). Five subjects that tested negative by the LeukoStrat CDx
FLT3 Mutation Assay and enrolled based on local FLT3 testing were excluded from the Full Analysis Set (FAS). Therefore,
366 subjects randomized to the study were used in the FAS for final analysis.
Selection of Samples for Reference Method Testing (IVS-056-001)
17.4.
17.4.1.
One sample per subject was selected for reference method testing. Samples with insufficient volume for the reference
method test were excluded from the study. A total of 467 samples were tested by the reference method.
Safety Analysis (IVS-056-001)
17.5.
17.5.1.
The LeukoStrat CDx FLT3 Mutation Assay is not expected to directly cause actual or potential adverse effects, but test
results may directly impact patient treatment risks.
Effectiveness (IVS-056-001)
17.6.
17.6.1.
Median duration of response in subjects with response of CR/CRh was 148 days (Events=16; Censored=40). In the groupof subjects with best response of CR, the median duration of response was 421 days (Events=9; Censored=27). In thegroup of subjects with response of CRh, the median duration of response was 122 days (Events=7; Censored=13). The
Kaplan-Meier plot is provided in Figure 8.

Figure 8 - Kaplan-Meier plot of duration of CR/CRh

17.6.2.

At the final analysis, the median OS in the gilteritinib fumarate arm was longer (9.3 months) compared to that in the salvage
chemotherapy arm (5.6 months) in the CDx+ population. The stratified hazard ratio (HR) by Cox regression was estimatedto be 0.637 (95%CI 0.488, 0.830) relative to salvage chemotherapy, p-value (1-sided, stratified log rank) = 0.0004,
corresponding to a relative risk reduction of death in favor of gilteritinib fumarate. The Kaplan-Meier plot is provided in
Figure 9.

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Figure 9 - Kaplan-Meier plot of overall survival.

17.6.3.

The LeukoStrat CDx FLT3 Mutation Assay demonstrated agreement to the Reference Method. The overall agreement washigh (97.2%). The lower bound of the OPA 95% confidence interval is above 90%, demonstrating agreement between the
FLT3 Mutation CDx and the MiSeq Sequencing assay.
Table 20: Agreement between CDx and MiSeq Sequencing

Percent
Agreement (N)Agreement

95% CI(1)PPA

100% (300 / 300)

(98.8%, 100%)NPA

92.0% (150 / 163)

(86.7%, 95.7%)

97.2% (450 / 463)

(95.2%, 98.5%)

OPA(1) The 95% CI is calculated using Exact (Clopper Pearson) method.

The point estimates of PPA, NPA, and OPA for ITD are 100%, 92.8%, and 97% respectively.
Table 21: Contingency Table between ITD CDx and MiSeq Sequencing

CDx
CDx+

MiSeq
MiSeq+

MiSeq-

270

14

Total
284

CDx-

0

180

180

Total

270

194

464

The point estimates of PPA, NPA, and OPA for TKD are 100%, 99.3%, and 99.4% respectively.
Table 22: Contingency Table between TKD CDx and MiSeq Sequencing

MiSeq
MiSeq+

MiSeq-

Total

CDx+

32

3

35

CDx-

0

431

431

Total

32

434

466

17.7.
Conclusions (IVS-056-001)
17.7.1.
At the first interim analysis, forty of 142 patients achieved a best overall response of CR/CRh (CR/CRh rate: 28.2%, 95%
CI: 20.9%, 36.3%). The lower bound of the 95% CI for the CR/CRh rate exceeded the pre-specified threshold of 12% forLeukoStrat CDx FLT3 Mutation Assay

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17.7.2.

17.7.3.

this co-primary endpoint. The evaluation of the CR/CRh rate for patients who were FLT3 positive by the LeukoStrat CDx
FLT3 Mutation Assay showed a comparable result. The lower bound of the 95% CI for the CR/CRh rate was calculated
at 20.3%. The study met the acceptance criteria, notwithstanding the exclusion of four patients from the analysis set due
to an unknown/missing/other FLT3 mutation status.
At the final analysis, 366 subjects were included in the full analysis set. The median OS in the gilteritinib fumarate arm
was longer (9.3 months) compared to that in the salvage chemotherapy arm (5.6 months) in the CDx+ population. The
stratified hazard ratio (HR) by Cox regression estimated to be 0.637 (95%CI 0.488, 0.830) relative to salvage chemotherapy,
p-value (1-sided, stratified log rank) = 0.0004, corresponding to a relative risk reduction of death in favor of gilteritinib
fumarate.
For the reference method testing, the study acceptance criterion was met: The lower limit of the overall percent agreement(OPA) 95% two-sided exact (Clopper-Pearson) confidence interval exceeded 90%. The agreement between the LeukoStrat
CDx FLT3 Mutation Assay and the MiSeq next-generation sequencing reference method could be established.

17.8.
Pivotal Bridging Study Overview (IVS-062-002)
17.8.1.
To support the safety and efficacy assessment of the LeukoStrat CDx FLT3 Mutation Assay (CDx), clinical agreement was
required to be demonstrated between samples with FLT3-ITD status determined from the AC220-007 Clinical Trial Assay(CTA) and the LeukoStrat CDx FLT3 Mutation Assay in the intent to treat population. This pivotal bridging study
corresponds to the Phase III AC220-007 clinical study of quizartinib hydrochloride in FLT3-ITD positive Acute Myeloid
Leukemia (AML) refractory to or relapsed within 6 months after first-line AML therapy subjects. The LeukoStrat CDx
FLT3 Mutation Assay is intended to assist physicians in making treatment decisions for their AML patients with FLT3ITD Mutations.
17.8.2.
The LeukoStrat CDx FLT3 Mutation Assay has been developed by Invivoscribe as a companion diagnostic to be used as
an aid in the assessment of AML patients for whom quizartinib hydrochloride treatment is being considered. Agreement
to the CTA and drug efficacy when stratified by the LeukoStrat CDx FLT3 Mutation Assay was evaluated in this Bridging
Study.

Study Objectives (IVS-062-002)
17.9.
17.9.1.
Establish agreement with respect to selection of FLT3 mutant patients between the Daiichi Sankyo Clinical Trial
Assay (CTA) and the LeukoStrat CDx FLT3 Mutation Assay (ITD Portion Only) by assessing overall, positive
and negative percent agreement between the two assays.
17.9.2.
Determine whether quizartinib hydrochloride monotherapy prolongs overall survival (OS) compared to salvage
chemotherapy in subjects with FLT3-ITD positive AML as determined by the LeukoStrat CDx FLT3 Mutation
Assay and who are refractory to or have relapsed within 6 months, after first-line AML therapy.
17.10. Patient Population (IVS-062-002)
17.10.1. 535 patients were screened with the Daiichi Sankyo Clinical Trial Assay for enrollment into the AC220-007. The
subject set for the bridging study was selected from available banked samples with informed consent that had been
screened in the AC220-007 trial. Samples from 531 subjects were available for testing with the LeukoStrat CDx
FLT3 Mutation Assay. Of these 531 subjects, 431 were CTA+ and 99 were CTA-. One subject tested by the CDx
was not available in the CTA dataset.
17.10.2. Once testing of the banked samples with the LeukoStrat CDx FLT3 Mutation Assay was completed, 528 subjects
produced valid results in both the CTA and the CDx and were available for concordance analysis.
17.11.

Safety Analysis (IVS-062-002)

17.11.1.

The LeukoStrat CDx FLT3 Mutation Assay is not expected to directly cause actual or potential adverse effects,
but test results may directly impact patient treatment risks.

17.12. Effectiveness (IVS-062-002)
17.12.1. The LeukoStrat CDx FLT3 Mutation Assay demonstrated agreement to the CTA and similar efficacy to theAC220-007 study.
17.12.1.1. The primary analysis demonstrated:
Agreement between the LeukoStrat CDx FLT3 Mutation Assay and the Daiichi Sankyo CTA wasdemonstrated based on a subset of 520 subjects with valid CDx and CTA results. The point estimates for
positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement(OPA) were above 90%.

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Table 23: Primary analysis of ITD CTA comparison.

ITD CDx

ITD CTA

Total

CTA+

CTA-

CDx+

412

0

412

CDx-

10

98

108

Invalid

0

0

0

Total

422

98

520

Invalid means that a sample was tested on the CDx assay but failed to return a valid result

Agreement (95% CI) are:

17.12.1.2.

17.13.

Effectiveness in the (CTA+, CDx+) population (351 subjects):
Quizartinib hydrochloride efficacy on overall survival in the CDx-positive population was evaluated.
Quizartinib hydrochloride treatment resulted in a statistically significant and clinically relevant
improvement in OS compared to salvage chemotherapy. The median OS in the quizartinib hydrochloride
arm was 26.9 weeks compared to 20.0 weeks for salvage chemotherapy resulting in 6.9-week prolongationof median OS; p-value (1-sided, stratified log rank) = 0.0187. The stratified hazard ratio (HR), estimatedby Cox regression, was estimated to be 0.757 (0.580, 0.956) relative to salvage chemotherapy
corresponding to a 24.3% relative risk reduction of death in favor of quizartinib hydrochloride.

Conclusions (IVS-062-002)

17.13.1.
17.14.

PPA 97.6% (95.7%, 98.9%)
NPA 100% (96.3%, 100%)
OPA 98.1% (96.5%, 99.1%)

Overall these results support that the LeukoStrat CDx FLT3 Mutation Assay identifies the same AML patient
population as enrolled in the AC220-007 clinical trial with respect to FLT3 ITD gene mutations.

Pivotal Bridging Study Overview (IVS-062-005)

17.14.1.

To support the safety and efficacy of the LeukoStrat CDx FLT3 Mutation Assay (CDx), clinical agreement was
required to be demonstrated between samples with FLT3-ITD status determined from the AC220-A-U302 Clinical
Trial Assay (CTA) and the LeukoStrat CDx FLT3 Mutation Assay in the intent to treat population. This pivotal
bridging study corresponds to the Phase III AC220-A-U302 clinical trial of quizartinib hydrochloride in newlydiagnosed AML patients with FLT3-ITD mutations (QuANTUM-First). This bridging study demonstrates thedevice and drug safety and efficacy when AML patients are stratified with the LeukoStrat CDx FLT3 Mutation
Assay and treated with the quizartinib hydrochloride, which is likely to provide a benefit to subject outcomes
without exposing the subjects to unreasonable risks. The LeukoStrat CDx FLT3 Mutation Assay is intended toassist physicians in making treatment decisions for their AML patients with FLT3-ITD Mutations.

17.14.2.

The LeukoStrat CDx FLT3 Mutation Assay has been developed by Invivoscribe as a companion diagnostic to be
used as an aid in the assessment of AML patients for whom quizartinib hydrochloride treatment is being
considered. Agreement to the CTA and drug efficacy when stratified by the LeukoStrat CDx FLT3 Mutation
Assay was evaluated in this Bridging Study.

17.15.

Study Objectives (IVS-062-005)

17.15.1.

Demonstrate agreement with respect to selection of subjects that are FLT3-ITD positive between the Clinical Trial
Assay and LeukoStrat CDx FLT3 Mutation Assay by assessing the positive and negative percent agreement (PPAand NPA, respectively) between the two assays.

17.15.2.

Determine whether quizartinib prolongs overall survival (OS) compared to placebo in newly diagnosed AML
subjects with FLT3-ITD mutation as determined by the LeukoStrat CDx FLT3 Mutation Assay (CTA(+), CDx(+)
population).

17.16.

Patient Population (IVS-062-005)

17.16.1.

3468 patients were screened with the Daiichi Sankyo Clinical Trial Assay for enrollment into AC220-A-U302,
with 1033 provided by the drug sponsor for the bridging study. Samples from 1029 subjects met the inclusion
criteria for testing with the LeukoStrat CDx FLT3 Mutation Assay. Of these 1029 subjects, 513 were CTA(+) and
516 were CTA(-). Once testing of the samples with the LeukoStrat CDx FLT3 Mutation Assay was completed,

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1023 subjects produced valid results in both the CTA and the CDx and six subjects produced invalid results with
the CDx.
17.17.

Safety Analysis (IVS-062-005)

17.17.1.
17.18.

The LeukoStrat CDx FLT3 Mutation Assay is not expected to directly cause actual or potential adverse effects,
but test results may directly impact patient treatment risks.

Effectiveness (IVS-062-005)

17.18.1.

The LeukoStrat CDx FLT3 Mutation Assay demonstrated agreement to the CTA and comparable efficacy to theAC220-A-U302 study.

17.18.1.1.

The primary analysis demonstrated:
Agreement between the LeukoStrat CDx FLT3 Mutation and the CTA was demonstrated based on a subset
of 1029 results. The point estimates for PPA and NPA were above 90%.

Table 24: Contingency between CDx and CTA.

ITD CTAITD CDx

CTA(+)CTA(-)

Total

CDx(+)

483

0

483

CDx(-)

27

513

540

Invalid

3

3

6

Total

513

516

1029

Invalid means that a sample was tested on the CDx assay but failed to return a valid result

Agreement with CDx invalids (95% CI) are:••17.18.1.2.

PPA 94.2% (91.8%, 96.0%)
NPA 99.4% (98.3%, 99.9%)

Effectiveness in the (CTA(+), CDx(+)) population (483 subjects):
Quizartinib hydrochloride efficacy on overall survival (OS) in the CDx-positive population was
evaluated. In the (CTA(+), CDx(+)) population, quizartinib treatment in combination with standard
chemotherapy resulted in a clinically relevant improvement in OS compared to placebo. The median OS
in the quizartinib arm was 29.4 months (19.1, NE) compared to 14.8 months (13.1, 26.2) for placebo
resulting in 14.6 months prolongation of median OS (2-sided, stratified log rank p-value= 0.0640). The
stratified hazard ratio (HR) was estimated to be 0.794 (0.621, 1.014) relative to placebo corresponding toa 20.6% relative risk reduction of death in favor of quizartinib. The results were comparable to thoseobserved in the QuANTUM-First (AC220-A-U302) study.

17.19.

Conclusions (IVS-062-005)

17.19.1.

Overall these results support that the LeukoStrat CDx FLT3 Mutation Assay identifies the same AML patient
population as enrolled in the AC220-A-U302 clinical trial with respect to FLT3 ITD gene mutations.

18. References1.

Murphy KM, Levis M, Hafez MJ, Gieger T, Copper LC, Smith BD, Small D and Berg KD. Detection of FLT3 Internal
Tandem Duplication and D835 Mutations by a Multiplex Polymerase Change Reaction and Capillary
Electrophoresis Assay. Journal of Molecular Diagnostics 2003, 5:96-102.

2.

Yamamoto, Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, Miyawaki S, Asou N, Kuriyama K, Yagasaki F, Shimazaki
C, Akiyama H, Saito K, Nishimura M, Motoji T, Shinagawa K, Takeshita A, Saito H, Ueda R, Ohno R, Naoe T.
Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood,
2001, 97(8):2434-9.

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19. Technical and Customer Service
Contact Information

Japan MAH and Technical Assistance

Invivoscribe, Inc
10222 Barnes Canyon Road, Bldg. 1
San Diego, California 92121-2711
USA

LabPMM, GK
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Kanagawa 210-0821
Japan

Phone:
Fax:
Technical Service:
Customer Service:
Website:Business Hours:

Phone:
Fax:
Technical Service:
Customer Service:
Website:Business Hours:+1 858 224-6600+1 858 224-6601
support@invivoscribe.com
sales@invivoscribe.com
www.invivoscribe.com
7:00AM – 5:00PM PST/PDT+81 44 281.1500+81 3 6745.9346
support@invivoscribe.com
services@labpmm.co.jp
www.invivoscribe.com/japan-cdxflt3/
9:00AM – 5:00PM JST

Technical and Customer Service Representatives are available Monday through Friday to answer phone, e-mail, or website inquiries.

20. Symbols
The following symbols are used in Invivoscribe diagnostic product labeling.
For In Vitro Diagnostic Use

Storage Conditions

Catalog Number

Expiration Date

Reagent Volume

Manufacturer

Lot Number

Consult Instructions for Use

21. Legal NoticeThis product is an in vitro diagnostic product.
Many of these products require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). No license under thesepatents to use amplification processes or enzymes is conveyed expressly or by implication to the purchaser by the purchase of thisproduct.
©2023 Invivoscribe, Inc. All rights reserved. The trademarks mentioned herein are the property of Invivoscribe, Inc. and/or its
affiliates, or (as to the trademarks of others used herein) their respective owners.

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