Validation of the LeukoStrat® CDx FLT3 Mutation Assay: Used to Detect both Internal Tandem Duplication (ITD)
and Tyrosine Kinase Domain (TKD) Mutations and Response to Midostaurin in 1058 Patients with AML

H53April Osgood1, Erica Shakeri1, Veronika Atkinson1, Reginald Navarrete1, Keri Garcia1, Dariel Caguioa2, Stephanie Ferguson2, Nelson Kha2, Christophe Simon4, Jordan
Thornes2,3,4, Timothy Stenzel 1, Jeffrey E. Miller1,2,3,4
1Invivoscribe, San Diego, USA, 2 LabPMM, LLC, San Diego, CA, 3LabPMM GmbH, Martinsried, Germany, and 4LabPMM GK, Kawasaki, Kanagawa, Japan

Abstract
Introduction: Acute myeloid leukemia (AML) in general has a poor prognosis, but AML patients with mutations in thefms-related tyrosine kinase 3 (FLT3), have a particularly poor prognosis, as ITD and TKD FLT3 mutations result inconstitutive autophosphorylation and activation of FLT3 - an important driver mutation in AML. The Invivoscribe®
LeukoStrat® CDx FLT3 Mutation Assay (CDx), recently approved by the FDA, targets regions of the FLT3 gene to detect
internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations to identify patients for whom
midostaurin treatment might be beneficial. It is the only internationally standardized signal ratio test used to detect
FLT3 ITD and TKD mutations.
Methods: Mononuclear cells were isolated from peripheral blood and bone marrow aspirate by a proprietary method.
DNA was extracted and amplified by PCR. TKD amplicon was enzymatically digested and along with the ITD amplicon
were analyzed by capillary electrophoresis on serialized ABI 3500 xL instruments (Thermo Fisher). The assay measured
the mutation:wild type signal using data from GeneMapper® analysis software. FLT3 Mutation Analysis Software(FMAS) developed by Invivoscribe calculated the signal ratio (SR) for both ITD and TKD and reported “Positive” or“Negative” for valid sample results or “Fail” for invalid results. Invalid results were generated when runs did not meet
validity criteria for analysis with FMAS. When FLT3 mutations were detected, the results were reported as positive if
the mutant:wild type signal ratio (SR) met or exceeded the clinical cut-off of 0.05. A validated Roche 454 Next
Generation DNA Sequencing method (454 Sequencing) was used for comparison in which DNA was amplified, and theamplicons were inspected, purified, quantified, normalized, pooled and sequenced. Specimens tested were collectedfrom AML patients during the ten year international RATIFY drug trial and from residual clinical samples tested atLabPMM in San Diego.
Results: The clinical performance of the CDx was evaluated using data from 1058 AML patient specimens from theRATIFY trial. The RATIFY trial used laboratory developed tests as clinical trial assays (CTAs), screening patients forenrollment at 8 different sites. The CTAs to CDx concordance for FLT3 status was 97.2% for positive percent agreement(PPA), 97.3% negative percent agreement (NPA) and 97.3% for overall percent agreement (OPA). Further, the hazard
ratio for the CTA+/CDx+ population was 0.67 versus 0.77 for patients stratified using the CTA+ assays. CDx agreement
to 454 Sequencing (PPA, NPA and OPA) was 94.0%, 97.5% and 95.1%, respectively, based on 764 patient results.
Peripheral blood and bone marrow concordance (PPA and NPA) was 98.4%, indicating both specimen types can beused for patient diagnosis (N=184 pairs). Additionally, clinical samples tested at 3 independent clinical LabPMM
laboratory sites on 3 continents showed both 100% positive and negative percent agreement, and high concordance
for signal ratios.
Conclusion: Overall, there was high concordance between the CDx, the RATIFY CTAs, and 454 Sequencing with respect
to FLT3 ITD and TKD gene mutation detection. In addition, analysis of patients stratified as positive by the CDx assaydemonstrated superior prediction for response to midostaurin relative to the CTAs - 13% better drug efficacy
compared with the CTAs used for patient enrollment (HR 0.67 vs 0.77). The high reproducibility between the 3
LabPMM clinical laboratories provides evidence that the CDx is a highly reproducible and reliable internationallyharmonized test useful for patient enrollment on multiple continents. It is currently being used in more than a dozen
large scale international clinical trials.

Materials

Measure ofAgreement

Without CDx Invalid
95% CI (1)95% CI (1)98.2% (489/498)(96.6%, 99.2%)

97.2% (489/503)(95.4%, 98.5%)

NPA

98.5% (540/548)(97.1%, 99.4%)

97.3% (540/555)(95.6%, 98.5%)

OPA

98.4% (1029/1046)(97.4%, 99.1%)

97.3% (1029/1058)(96.1%, 98.2%)

PPA•

DNA was extracted from CTA(+) and CTA(-) RATIFY study patient samples containing peripheral blood or bone marrow
with a quantity of at least 50 ng at ≥10 ng/µL.
Identical lots of cGMP reagents were used across all clinical enrollment sites.

Methods
EcoRV
Digestion

Percent Agreement

95% CI (1)

APA

98.4%

(96.2%, 100.0%)

ANA

98.4%

(96.2%, 100.0%)(1) The 95% CI was calculated using a non-parametric bootstrapping method

Deming Regression Analysis of ITD and TKD Signal Ratios Measured by the CDx
Assay Between Peripheral Blood and Bone Marrow in the Overall Population(Samples with extremely high signal ratios were not displayed in order to focus on the spread ofvalues around the Deming line.)(1) The 95% CI was calculated using the Exact (Clopper-Pearson) method- Invalid means that a sample was tested in the CDx assay but failed to return a valid result.

Agreement Table Between CDx and 454 Sequencing
Measure ofAgreement

Without CDx InvalidWith CDx Invalid

Percent Agreement(N)

95% CI (1)Percent Agreement(N)

95% CI (1)PPA

94.0% (487/518)(91.6%, 95.9%)

92.8% (487/525)(90.2%, 94.8%)

NPA

97.5% (230/236)(94.5%, 99.1%)

96.2% (230/239)(93.0%, 98.3%)

OPA

95.1% (717/754)(93.3%, 96.5%)

93.8% (717/764)(91.9%, 95.4%)

ITD

TKD

Kaplan-Meier Curve for Overall Survival in the (CTA+, CDx+) Population –Model 2, Non-censored for Stem Cell Transplantation
Overall Survival

Deming Regression Analysis of ITD and TKD Signal Ratios Between
CTAs and CDx in the Overall Population(Samples with extremely high signal ratios were not displayed in order to focus on the spreadof values around the Deming line.)

Capillary
Electrophoresis(TKD)

With Number of Subjects at Risk

1.0- Invalid means that a sample was tested in the CDx assay but failed to return a valid result.

LeukoStrat CDx FLT3 Mutation Assay workflow
PCR
Amplification(TKD)

Measure of Agreement

With CDx Invalid
Percent Agreement(N)

Percent Agreement(N)+ Censored

0.8
Survival Probability

Kit components for the LeukoStrat® CDx FLT3 Mutation Assay were manufactured under cGMP and QC tested under a
QSR compliant regulatory system prior to use in cross validation studies.

•Agreement Table Between CDx and CTAs Based on CDx Results(1) The 95% CI was calculated using the Exact (Clopper-Pearson) method••

Agreement Table Between Peripheral Blood and Bone Marrow in93 FLT3 Positive Patients and 91 FLT3 Negative PatientsResults

0.60.40.20.0BM or PB
Samples

MNCCollection

DNA
Preparation

DataAnalysis
SoftwareMidostaurin 1
Placebo 2

Results &Report

360
357

221
172

178
14377710002040
Survival Time (Months)
1 : Midostaurin

60

80

Arm
PCR
Amplification(ITD)

Capillary
Electrophoresis(ITD)•DNA extraction was conducted by Covance in Indianapolis, IN.
•Clinical testing with the CDx FLT3 Mutation Assay was conducted by LabPMM in San Diego, CA.
•FLT3 Mutation Detection by 454 Next Generation Sequencing was conducted by SeqWright in Houston, TX.
• The statistical program SAS was used to perform the statistical analysis.

ITDTKD

2: PlaceboConclusions
The study showed a high degree of concordance between the CDx and the clinical trial assay withsuperior prediction for midostaurin response by (CDx+, CTA+) patients. This assay is the onlyinternationally harmonized FLT3 signal ratio assay whose performance has been defined and isbeing used in more than a dozen large scale international clinical trials.

AMP, November 16 –18 2017, Salt Lake City, USA