The QuantideX qPCR BCR-ABL IS Kit is an in vitro nucleic acid amplification test for the quantitation of BCR-ABL1 and ABL1 transcripts in total RNA from whole blood of diagnosed t(9,22) positive Chronic Myeloid Leukemia (CML) patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The QuantideX qPCR BCR-ABL IS Kit is a reverse transcription-quantitative PCR performed on the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument and is intended to measure BCR-ABL1 to ABL1, expressed as a log molecular reduction (MR value) from a baseline of 100% on the International Scale, in t(9;22) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).

The test does not differentiate between e13a2 or e14a2 fusion transcripts and does not monitor other rare fusion transcripts resulting from t(9:22). This test is not intended for the diagnosis of CML.

Device Description

The QuantideX qPCR BCR-ABL IS Kit reagents are adapted for use on the ABI 7500 Fast Dx Real-Time PCR Instrument. The assay includes reagents sufficient for 60 reactions. A description of the reagents provided is described below in Table 1.

AI/ML Overview

Here's a summary of the acceptance criteria and study that proves the device meets them, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document describes both analytical and clinical performance acceptance criteria.

Analytical Performance - Precision/Reproducibility

Performance Metric	Acceptance Criteria (SD)	Reported Device Performance (Max Total SD)	Met?
Within-lab Precision
MR Value < 3.5	≤ 0.21	0.128 (at MR 1)	Yes
MR Value 3.5-4.25	≤ 0.29	0.111 (at MR 3.5)	Yes
MR Value > 4.25	≤ 0.36	0.134 (at MR 4)	Yes
Site-to-Site Reproducibility
MR Value < 3.5	≤ 0.21	0.117 (at MR 3)	Yes
MR Value 3.5-4.25	≤ 0.29	0.169 (at MR 3.5)	Yes
MR Value > 4.25	≤ 0.36	0.167 (at MR 4)	Yes

Analytical Performance - Linearity

Performance Metric	Acceptance Criteria (Slope/Intercept for Regression)	Reported Device Performance (Slope/Intercept/Max SD)	Met?
Linear Regression	Slope close to 1 (not explicitly defined but implied typical regression expectations), intercept close to 0 (implied)	e13a2: Slope 1.01, Intercept -0.11, Max SD 0.17 e14a2: Slope 1.01, Intercept -0.05, Max SD 0.17	Yes
Range of Linearity	(Implied based on clinical use range)	MR 0.3 (50% IS) to MR 4.7 (0.002% IS) for both transcripts	Yes

Analytical Performance - Limit of Quantitation (LoQ)

Performance Metric	Acceptance Criteria (SD)	Reported Device Performance (MR Range/SD Range)	Met?
LoQ	≤ 0.36 at MR 4.5 or greater	MR values 4.6 to 4.87, SD values 0.23 to 0.34	Yes

Analytical Performance - Interfering Substances

Performance Metric	Acceptance Criteria (Mean difference from control)	Reported Device Performance	Met?
Interference	Mean of test samples ± 0.5 MR of the control	All met acceptance criteria	Yes

Analytical Performance - Primer Specificity

Performance Metric	Acceptance Criteria	Reported Device Performance	Met?
Specificity	Specimens without a major BCR-ABL1 breakpoint reported as negative or below LoD in > 8/9 replicates	Met acceptance criteria (e.g., cell lines and IVT controls without e13a2/e14a2 were <LOD in 8/9 or 9/9 replicates)	Yes

Analytical Performance - Specimen Carryover Contamination

Performance Metric	Acceptance Criteria	Reported Device Performance	Met?
Carryover	> 95% of CML-negative samples tested negative or below LoD	All negative specimens (25/25) reported as "Negative (sufficient ABL1)"	Yes

Analytical Performance - RNA Input

Performance Metric	Acceptance Criteria (SD criteria from Table 3)	Reported Device Performance (RNA input range with met criteria)	Met?
RNA Input Range	Same as Table 3 for corresponding MR values	750 to 6000 ng from MR 1 to MR 4.5	Yes

Analytical Performance - Traceability

Performance Metric	Acceptance Criteria (Regression to WHO panel)	Reported Device Performance (Slope/Intercepts for 3 lots)	Met?
Traceability	Slope 1.0 to 1.1, Intercept 0.02 - 0.11	Lot1: y=-0.11+1.1x; Lot2: y=0.02+1x; Lot3: y=-0.059+1.1x	Yes

Analytical Performance - Specimen Stability (Whole Blood)

Performance Metric	Acceptance Criteria (MR unit difference from baseline)	Reported Device Performance (Duration and storage conditions)	Met?
Whole Blood Stability	All results within 0.5 MR units from baseline	Up to 72 hours at 2-8°C	Yes

Clinical Performance

Performance Metric	Acceptance Criteria	Reported Device Performance	Met?
Event-Free Survival (EFS) Difference at 36 months	Statistically significantly different AND point estimates differing by at least 10 percentage points (for MR < 3 vs MR ≥ 3 groups)	EFS difference: 22.2% (95% CI: 2.0%-42.4%), p = 0.0279	Yes
Event Status Known at 36 months	95% Wilson score confidence interval for the difference > 0	Met	Yes

2. Sample Size Used for the Test Set and Data Provenance

The primary test set for clinical performance involved a retrospective study with:

Sample Size: 137 evaluable samples from 96 subjects (out of 139 samples from 98 patients initially collected).
Data Provenance: Retrospective, collected at 2 clinical sites in the US (OHSU and Hospital of the University of Pennsylvania).

For analytical performance studies, various sample compositions and quantities were used:

Precision (within-lab & site-to-site): 25 samples formulated from 5 human RNA specimens positive for BCR-ABL1 diluted into RNAs from CML-negative blood. The site-to-site reproducibility study involved 1200 measurements from this 25-sample pool.
Linearity/Reportable Range: 2 separate RNA specimens (one e13a2, one e14a2) diluted into RNAs from CML-negative whole blood (ranging from MR 0.1 to MR 4.8).
Limit of Blank (LoB): 30 non-leukemic human RNA specimens.
Limit of Detection (LoD): 4 separate human RNA specimens positive for BCR-ABL1, serially diluted to 28 levels.
Limit of Quantitation (LoQ): 6 specimens derived from 6 human RNA positive for BCR-ABL (diluted to target MR 4.7).
Interfering Substances: Residual CML-positive blood diluted to approximately MR 4.0 into RNA from CML-negative whole blood (tested in 9 replicates).
Primer Specificity: 11 leukemic specimens (CML, AML, ALL) and 2 non-leukemic RNA specimens.
Specimen Carryover Contamination: Serially diluted total RNA from residual clinical CML-positive blood into RNA from CML-negative whole blood in a checkerboard pattern (25 high positive, 25 negative).
RNA Isolation: CML-positive white blood cells serially diluted across 4 levels (MR 1-4) into CML-negative human anti-coagulated whole blood.
RNA Input: 30 samples derived from 2 primary RNA samples diluted into non-leukemic human RNA (total 216 evaluable observations).
Traceability: WHO Reference Panel (4 panel members tested in duplicate).
Reagent Stability: 5 samples diluted to approximately MR 1, 2, 3, 4, and < LoD (15 replicates per time point).
Specimen Stability (Whole Blood): 13 CML positive specimens and 3 CML negative specimens.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The document does not explicitly state the number of experts or their specific qualifications (e.g., radiologist with X years of experience) used to establish the ground truth for the test set.

For the clinical study, the "ground truth" for event-free survival (EFS) was based on clinical outcomes defined by specific criteria:

Death by any cause
Development of accelerated-phase or blast crisis CML
Loss of complete hematologic response
Loss of complete cytogenetic response
Appearance of mutation
Change in TKI treatment not due to toxicity and not due to the results of another BCR-ABL assay

These outcomes would typically be determined by treating physicians and medical records, often relying on established clinical guidelines and diagnostic criteria. While not explicitly stated as "experts establishing ground truth," the medical documentation and clinical assessments would serve this role.

4. Adjudication Method for the Test Set

The document does not describe an explicit adjudication method (e.g., 2+1, 3+1, none) for a test set, particularly in the context of expert review. The ground truth for the clinical study was based on observed clinical outcomes and treatment changes, which are derived from patient records and physician decisions rather than expert consensus on image or assay interpretation.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No, an MRMC comparative effectiveness study was not done. This device is a quantitative in vitro diagnostic assay, not an imaging device or a device requiring human interpretation in the loop in the same way an MRMC study evaluates. The clinical study assessed the device's ability to predict clinical outcomes (EFS) based on its quantitative output (MR value), rather than comparing human reader performance with and without AI assistance.

6. If a Standalone (Algorithm Only Without Human-in-the-loop Performance) Was Done

Yes, the primary clinical performance and all analytical performance studies represent a standalone assessment of the device. The QuantideX qPCR BCR-ABL IS Kit is an in vitro diagnostic test that provides quantitative results (MR values) directly. There is no human-in-the-loop interpretation of the assay's raw output described; the device itself calculates the BCR-ABL1 to ABL1 ratio and MR value. The clinical study then correlated these device-generated MR values with patient outcomes.

7. The Type of Ground Truth Used

Analytical Ground Truth: For many analytical performance studies (precision, linearity, LoD, LoQ, interference), the ground truth was established by known concentrations or dilutions of BCR-ABL1 and ABL1 transcripts, or by using characterized RNA specimens (e.g., CML-positive or CML-negative clinical samples, or cell lines with specific translocations) to create spiked samples or panels. Traceability was established against the First (1st) WHO International Genetic Reference Panel.
Clinical Ground Truth: For the clinical performance study, the ground truth was patient outcomes data, specifically "event-free survival" at 32-40 months after TKI treatment. "Events" were defined by specific clinical criteria related to disease progression, death, or changes in therapy.

8. The Sample Size for the Training Set

The document does not specify a separate training set size. As an in vitro diagnostic kit, the "training" analogous to machine learning would be the extensive analytical validation and optimization performed by the manufacturer during product development, which informs the assay's design, protocols, and reagent formulations. The clinical and analytical studies described are primarily for validation and verification of the final device, not for training a specific algorithm.

9. How the Ground Truth for the Training Set Was Established

Since a distinct "training set" in the machine learning sense is not explicitly mentioned or applicable for this type of IVD kit, the concept of establishing ground truth for a training set does not directly apply. Instead, the analytical characteristics (like limit of detection, linearity, precision) are established using controlled experiments with known concentrations and reference materials, and clinical utility is demonstrated by correlating the device's measurements with observed patient clinical outcomes. These outcomes in the clinical study are determined by medical definitions and patient records, as described in point 3.

Summary

{0}------------------------------------------------

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR QUANTIDEX QPCR BCR-ABL IS KIT DECISION SUMMARY

A. DEN Number:

DEN160003

B. Purpose for Submission:

De novo request for evaluation of automatic class III designation of the QuantideX qPCR BCR-ABL IS Kit

C. Measurand:

BCR-ABL1 and ABL1 transcripts

D. Type of Test:

Reverse transcription, quantitative, polymerase chain reaction (qPCR) based nucleic acid amplification

E. Applicant:

Asuragen

F. Proprietary and Established Names:

Trade Name: QuantideX qPCR BCR-ABL IS Kit Common Name: BCR-ABL1 RT-qPCR Test

G. Regulatory Information:

1. Regulation section:
21 CFR 866.6060
1. Classification:

Class II (Special Controls)

1. Product code:
  OYX
1. Panel:
88 Pathology

{1}------------------------------------------------

H. Indications For Use:

1. Indications for Use

1. Special conditions for use statement(s):
  For in vitro diagnostic use only.

For prescription use only.

1. Special instrument requirements:
  Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument using System Sequence Detection Software v1.4.1.

I. Device Description:

Item	Description	Use
QuantideX qPCR BCR-ABL IS Reagents (Box 1 of 2)
BCR-ABL IS Diluent	Nuclease-free water	Adjust volume of RT reactions
BCR-ABL IS RT Buffer	Deoxynucleotides (dNTPs) and degeneratedeoxyoligonucleotides in abuffered salt solution	Random priming of RNA templates and primer extensionto generate cDNA from RNAtemplates
BCR-ABL IS RT Enzyme Mix	Reverse transcriptase	Enzyme component of the RTreaction to generate cDNA fromRNA template
BCR-ABL IS qPCR Buffer	Deoxynucleotides (dNTPs) in abuffered salt solution	Provides dNTPs for primerextension to generate DNAamplicons from cDNAtemplates
BCR-ABL IS Primer Probe Mix	Deoxyoligonucleotide primers, dye- and quencher- conjugated deoxyoligonucleotide probes, and passive reference dye	Provides primers and probes for PCR amplification and detection of target sequences. Passive reference dye is used by the instrument software to normalize fluorescence throughout the PCR
BCR-ABL IS Enzyme Mix	Thermostable DNA polymerase	Catalyzes the amplification of templates hybridized to templates in the cDNA. Enzyme exonuclease activity degrades hybridized probes to release fluorescence for detection of amplicons in each PCR cycle.
QuantideX qPCR BCR-ABL IS Calibrators and Controls (Box 2 of 2)
BCR-ABL IS CAL1	BCR-ABL and ABL armored RNA formulated to approximately MR 1.0	Generate calibration curve for calculation of MR values for controls and patients specimens
BCR-ABL IS CAL2	BCR-ABL and ABL armored RNA formulated to approximately MR 2.0
BCR-ABL IS CAL3	BCR-ABL and ABL armored RNA formulated to approximately MR 3.0
BCR-ABL IS CAL4	BCR-ABL and ABL armored RNA formulated to approximately MR 4.0
BCR-ABL IS CONH	BCR-ABL and ABL armored RNA formulated to approximately MR 1.5	Control used to ensure that RT and PCR steps performed properly by generating expected MR value
BCR-ABL IS CONL	BCR-ABL and ABL armored RNA formulated to approximately MR 3.5	Control used to ensure that RT and PCR steps performed properly and protect against contamination and falsely positive samples to due contamination by generating a results of "Negative (Sufficient ABL1)"
BCR-ABL IS CONN	ABL armored RNA

Table 1: Components of the QuantideX Assay

{2}------------------------------------------------

Additional materials required but not provided with the QuantideX qPCR BCR-ABL IS Kit:

1. Reagents for total RNA isolation
1. Applied Biosystems 7500 Fast Dx Real-Time PCR instrument

J. Substantial Equivalence Information:

1. Predicate device name(s) and DEN number(s): Not applicable.

{3}------------------------------------------------

1. Comparison with predicate: Not applicable.

K. Standards/Guidance Documents Referenced:

Guidance for Industry and FDA Staff: Guidance for the Content of Premarket Submission for . Software Contained in Medical Devices
General Principles of Software Validation; Final Guidance for Industry and FDA Staff .
. Guidance for Industry, FDA Reviewers and Compliance on Off-the-Shelf Software Use in Medical Devices
CLSI EP05-A3, Evaluation of Precision Performance of Quantitative Measurement Methods
CLSI EP06-A, Evaluation of the Linearity of Quantitative Measurement Procedures, A ● Statistical Approach
CLSI EP07-A2, Interference Testing in Clinical Chemistry ●
CLSI EP25-A. Evaluation of Stability on In Vitro Diagnostic Reagents .

L. Test Principle:

The QuantideX qPCR BCR-ABL IS Kit is a nucleic acid amplification test for the quantitation of BCR-ABL1 RNA. The assay provides simultaneous amplification and detection of two BCR-ABL1 fusion transcripts (e13a2 and e14a2) and ABL1 (an endogenous control) using total RNA extracted from human white blood cells enriched from EDTA whole blood. The test uses multiplex reverse transcription-PCR (RT-PCR) in combination with real-time hydrolysis probe technology. BCR-ABL1 translocations and ABL1 mRNA are simultaneously reverse-transcribed, amplified, detected, and quantified in a single reaction well. b(4)

This design is diagrammed below.

Figure 1: Primer and Probe Design for the QuantideX Assay b(4)

{4}------------------------------------------------

The RNA quantity of BCR-ABL is assessed relative to ABL1 expression within each reaction well, and is computed two ways: (1) as the specimen percent IS value (the percent ratio of BCR-ABLI to ABL1 expressed on the International Scale), and (2) as a molecular reduction value (MR, a logarithmic decrease from the common baseline of 100% IS or MR 0). ABL1 also serves as a control for assessment of RNA quality for the specimen.

The Assay Calibrator set includes external calibrators built with Armored RNA® Quant (ARQ) technology to generate traceable %IS values (transformed into the primary measurement of a molecular reduction (MR) from the common baseline of 100%IS) from a single calibration curve (provided). Calibrators are run in duplicate and all other specimens and controls may be run in singlicate. The time required from the RT set-up step through the generation of the last test result is $\n[ \leq ]\n$ 4 hours for a full kit.

M. Interpretation of Results

The numerical value of the World Health Organization (WHO) International Scale is % IS, the ratio expressed as a percentage of BCR-ABL1 expression to the expression of a control gene (ABL1 in this instance). The International Scale (%IS) is a geometric progression and therefore repeated measurements of a sample are non-normally distributed about the mean. %IS values require logtransformation prior to performing any statistical analyses that require normally-distributed data.

Another value commonly reported in the literature is the Molecular Reduction, or MR value. The MR value is traditionally written as $MR^{x.x}$. However, for simplicity and legibility, the QuantideX assay will report the value as MRx.x. The MR value is the logio reduction from the internationally standardized baseline, defined as 100% IS. Therefore,

$$MR\ge\colon \pi = \log_{10}\left(\frac{100}{% \delta IS}\right) = \log_{10}\left(100\right) - \log_{10}\left(% \delta IS\right) = 2 - \log_{10}\left(% \delta IS\right)$$

The test uses MR values for the calibration standards as well as the primary specimen output, with %IS also reported. MR values with their corresponding %IS values are shown below in Table 2.

MR	%IS
0.0	100
0.5	32
1.0	10
1.5	3.2
2.0	1
2.5	0.32
3.0	0.1
3.5	0.032
4.0	0.01
4.5	0.0032
4.7	0.002
5.0	0.001

Table 2: MR Values and Corresponding %IS Values

N. Performance Characteristics:

{5}------------------------------------------------

1. Analytical Performance:

a. Precision:
- i. Within-laboratory Precision: Five human RNA specimens positive for BCR-ABL1 were diluted into RNAs from human CML-negative blood to formulate 25 samples ranging from MR 1.0 to MR 4.0, encompassing both transcript types (e13a2 and e14a2). Testing spanned 3 lots, 3 operators, 20 runs, and 3 instruments. The acceptance criteria for this study were that the standard deviations satisfy the criteria in Table 3. The results of the precision study are shown in Table 4. The range of observed standard deviations at each MR level across the 5 specimens is summarized in Table 5. The maximum observed standard deviation was 0.134. The acceptance criteria for this study were met supporting the conclusion that the assay has acceptable repeatability < MR 4.0.

Table 3: Acceptance Criteria for Reproducibility/Precision Studies

Log Scale		%IS
MR Value	SD Criteria	%IS Value	%CV Criteria
< 3.5	≤0.21	> 0.0316	≤ 50
3.5-4.25	≤0.29	0.0316-0.0056	≤ 75
> 4.25	≤0.36	< 0.0056	≤ 100

Table 4: Precision Study Results

TargetMR	Sample	LotSD	OperatorSD	InstrumentSD	RunSD	WithinSD	TotalSD
		1	PA1S01	0.049	0.000	0.000	0.000
1	PA1S06	0.003	0.028	0.000	0.046	0.016	0.093
1	PA1S11	0.000	0.020	0.057	0.000	0.014	0.091
1	PA1S16	0.036	0.000	0.065	0.000	0.027	0.128
1	PA1S21	0.000	0.026	0.023	0.000	0.027	0.076
2	PA1S02	0.033	0.010	0.000	0.000	0.011	0.054
2	PA1S07	0.015	0.015	0.033	0.021	0.022	0.105
2	PA1S12	0.027	0.000	0.000	0.012	0.019	0.058
2	PA1S17	0.000	0.000	0.051	0.000	0.023	0.074
2	PA1S22	0.000	0.000	0.021	0.000	0.010	0.031
3	PA1S03	0.006	0.025	0.000	0.000	0.025	0.056
3	PA1S08	0.000	0.000	0.017	0.000	0.018	0.035
3	PA1S13	0.032	0.000	0.000	0.000	0.038	0.070
3	PA1S18	0.000	0.000	0.022	0.000	0.028	0.050
3	PA1S23	0.000	0.028	0.000	0.000	0.019	0.047
3.5	PA1S04	0.000	0.000	0.000	0.000	0.046	0.046
3.5	PA1S09	0.000	0.000	0.000	0.000	0.047	0.047
3.5	PA1S14	0.000	0.000	0.000	0.053	0.058	0.111

{6}------------------------------------------------

TargetMR	Sample	LotSD	OperatorSD	InstrumentSD	RunSD	WithinSD	TotalSD
	PA1S19	0.016	0.000	0.000	0.000	0.035	0.052
PA1S24	0.018	0.000	0.053	0.000	0.031	0.103
4	PA1S05	0.000	0.000	0.000	0.000	0.074	0.074
	PA1S10	0.037	0.021	0.024	0.000	0.052	0.134
	PA1S15	0.000	0.000	0.000	0.000	0.071	0.071
	PA1S20	0.000	0.000	0.000	0.000	0.040	0.040
	PA1S25	0.000	0.067	0.020	0.000	0.024	0.111

Table 5: Minimum and Maximum Standard Deviations Observed by MR Level

Target MR	SD Range
	Min	Max
MR1	0.059	0.128
MR2	0.031	0.105
MR3	0.035	0.070
MR3.5	0.046	0.111
MR4	0.040	0.134

ii. Site-to-Site Reproducibility: The same 25 sample pool described above was evaluated at 4 sites by multiple operators over multiple nonconsecutive days for a total of 1200 measurements. This dataset was analyzed using a nested random effect analysis of variance. The acceptance criteria were the same as those described in Table 3 above. The results are summarized in Table 6 below. The acceptance criteria were met supporting the overall conclusion that the assay is reproducible within all variables tested >MR 1.0 $\n[ \leq ]\n$ 4.0.

TargetedMR	Sample	SiteSD	DaySD	OperatorSD	WithinSD	TotalSD
MR1	pAls01	0.008	0.000	0.012	0.028	0.049
	pAls06	0.009	0.014	0.000	0.021	0.044
	pAls11	0.024	0.017	0.014	0.022	0.077
	pAls16	0.017	0.020	0.000	0.019	0.056
	pAls21	0.013	0.021	0.011	0.020	0.064
MR2	pAls02	0.000	0.021	0.000	0.068	0.089
	pAls07	0.000	0.022	0.004	0.017	0.044
	pAls12	0.032	0.009	0.026	0.021	0.087
	pAls17	0.012	0.016	0.009	0.019	0.055
	pAls22	0.012	0.014	0.000	0.031	0.057
MR3	pAls03	0.039	0.024	0.005	0.036	0.104
	pAls08	0.000	0.023	0.009	0.028	0.059
	pAls13	0.039	0.026	0.000	0.052	0.117
	pAls18	0.018	0.000	0.009	0.033	0.059

Table 6: Summarized Variance for all Samples and Pools in Multi-Site Reproducibility

{7}------------------------------------------------

MR3.5	pA1s23	0.036	0.019	0.000	0.032	0.087
	pA1s04	0.061	0.023	0.026	0.037	0.147
	pA1s09	0.013	0.042	0.010	0.043	0.108
	pA1s14	0.038	0.039	0.041	0.051	0.169
	pA1s19	0.026	0.010	0.000	0.035	0.071
	pA1s24	0.050	0.027	0.000	0.032	0.109
MR4	pA1s05	0.054	0.027	0.032	0.053	0.167
	pA1s10	0.029	0.000	0.000	0.067	0.095
	pA1s15	0.014	0.037	0.000	0.070	0.122
	pA1s20	0.042	0.000	0.000	0.079	0.121
	pA1s25	0.026	0.028	0.005	0.063	0.123

b.Linearity/Reportable Range:

Linearity was estimated by testing 2 separate RNA specimens that were positive for BCR-ABL (one e13a2 and one e14a2). Because an appropriate reference method could not be identified, transcript quantity is relative to the QuantideX assay. Each was diluted into RNAs from CML-negative whole blood to a range of MR 0.1 to MR 4.8. Testing was conducted across 2 lots of assay kits. The acceptance criteria for this testing were the same as those described in Table 3 above.

The linear regression curves demonstrated slopes of 1.01 for both transcripts and intercepts of -0.11 and -0.05 for e13a2 and e14a2, respectively. Transcript e13a2 was linear from MR 0.12 to MR 4.84 with a maximum SD of 0.17 and transcript e14a2 was linear from MR 0-.22 to MR 4.78 with a maximum SD of 0.17.

Second and 3th order polynomial regressions were also assessed and the analysis supports linearity from at least MR 0.3 (50% IS) to MR 4.7 (0.002% IS). These results support the conclusion that the assay is linear for both transcripts from MR 0.3 (50%IS) to MR4.7 (0.002%IS).

{8}------------------------------------------------

Image /page/8/Figure/0 description: The image contains two scatter plots, labeled 'e13a2' and 'e14a2'. Both plots show a strong positive correlation between 'Targeted MR' on the x-axis and 'MR' on the y-axis. The data points are clustered tightly around a diagonal line, indicating a close agreement between the targeted and actual MR values, ranging from 0 to 5 on both axes.

Figure 2: Linearity of e13a2 (left) and e14a2 (right) transcripts

c. Detection Limit:

i. Limit of Blank (LoB):
The LoB was determined by testing 30 non-leukemic human RNA specimens that were presumed negative for BCR-ABL. Testing spanned 3 assay kit lots, 4 operators, 9 runs, 4 calendar days and 4 qPCR instruments for a total of 265 valid measurements.

Out of 265 valid measurements, 263 were undetectable.

ii. Limit of Detection (LoD):
The LoD was determined using 4 separate human RNA specimens that were positive for BCR-ABL1, each serially diluted into RNAs from human CML-negative whole blood to 28 dilution levels. Testing spanned 2 assay kit lots, 4 operators, 15 calendar days, and 4 qPCR instruments. Both e13a2 and e14a2 transcripts were evaluated.

The non-parametric method to determine LoD for quantitative devices was used, keeping the type II error under 5%. The median value of the tested %IS values across all included panel member replicates was determined and defined as the LoD. This analysis vielded a LoD for each transcript of 0.002% IS/MR 4.7. The results are pictured below in Table 7.

Transcript	Specimens	TotalResults	TotalPositive	PercentUndetected	Median%IS	LOD(MR)
e13a2	pC1s02, pC1s24, pC1s25	179	172	3.9	0.002	4.70
e14a2	pC1s09, pC1s10, pC1s11,pC1s12, pC1s17, pC1s18,pC1s19	420	400	4.8	0.002	4.70

Table 7: Determination of LoD for both BCR-ABL Transcripts

{9}------------------------------------------------

iii. Limit of Quantitation (LoO):

The LoQ was determined by testing 6 specimens that were derived from 6 human RNA that were positive for BCR-ABL (each diluted into RNA from human CML-negative whole blood to the target of MR 4.7).The results were evaluated against the acceptance criteria of SD ≤ 0.36 at MR 4.5 or greater. Testing generated 120 measurements across 2 lots of kit. MR values ranged from 4.6 to 4.87 with SD values ranging from 0.23 to 0.34. The results indicate that the LoQ is equivalent to the LoD (MR 4.7).

d.Analytical Specificity:

i. Interfering Substances:
Potential interfering substances from both blood sources and RNA extraction sources were tested with specimens at the LoQ in the following concentrations:
Hemoglobin 200 g/L
Lipid 5.6 mM ●
Albumin 50 g/L
Conjugated bilirubin 86 mM
Unconjugated bilirubin 257 µM ●
Guanidinium containing lysis buffer 1%
Ethanol 7% ●
Phenol 0.1%
Assay wash buffer 10%
Genomic DNA 50 ng/RT ●

The RNA specimens tested were residual CML-positive blood diluted to approximately MR4.0 into RNA from CML-negative whole blood. Testing was performed in 9 replicates and compared to control samples. The acceptance criteria were that the mean of the test samples was ± 0.5 MR of the control. The acceptance criteria were met, supporting the conclusion that none of the tested agents interfered with the assay at the concentrations listed.

ii. Primer Specificity:
Analytical specificity (i.e., exclusivity) for the BCR-ABL breakpoints e13a2 and e14a2 was tested using 11 leukemic specimens (CML, Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL)) and 2 non-leukemic RNA specimens. Testing generated 117 measurements across 3 lots of assay kit. The acceptance criteria were met in that specimens without a major BCR-ABL1 break point were reported as negative for BCR-ABL1 or below LoD in > 8/9 replicates. The results across the various samples are shown in Table 8. The results support supported the conclusion that the test detects the BCR-ABL1 e13a2 and e14a2 fusion transcripts exclusively.

{10}------------------------------------------------

LeukemiaType	Fusion Transcript	Sample type	Result2
CML or ALL	t(9;22)minor breakpoint e1a2 (p190)	Cell line RNA	<LOD (9/9 Und)
CML	t(9;22)micro breakpoint e19a2 (p230)	IVT1 in Non-leukemic RNA	<LOD (9/9 Und)
AML	t(8;21)AML1/ETO	Cell line RNA	<LOD (9/9 Und)
AML M3 /APL	t(15;17)PML/RARA	Cell line RNA	<LOD (9/9 Und)
AML M4	inv(16)CBFB/MYH11	Cell line RNA	<LOD (9/9 Und)
AML M5	t(9;11)MLLT3/MLL	Cell line RNA	<LOD (9/9 Und)
AML M7	t(1;22)Megakaryoblastic	IVT in Non-leukemic RNA	<LOD (9/9 Und)
ALL	t(12;21)TEL/AML1	Cell line RNA	<LOD (9/9 Und)
ALL	t(1;19)E2A/PBX1	Cell line RNA	<LOD (9/9 Und)
ALL	t(4;11)MLL/AF4 (e9e5)	Cell line RNA	<LOD (8/9 Und)
ALL	t(4;11)MLL/AF4 (e10e4)	Cell line RNA	<LOD (9/9 Und)
Non-leukemic	N/A	Donor blood	<LOD (9/9 Und)across 2 specimens

Table 8: Specificity in the Presence of other Fusion Transcripts

1The in vitro transcript (IVT) for e19a2 did not contain BCR exons e13 or e14. Specimens harboring the native e19a2 breakpoint are expected to contain these exons and therefore may generate some low level positivity.

2The specimens listed in the table as "Und" were reported as "Undetected (sufficient ABL1)".

iii. Specimen Carryover Contamination:

Carryover contamination studies were performed on a specimen panel composed of total RNA derived from residual clinical CML-positive blood serially diluted into RNA from CML-negative whole blood. Samples were added to the plate such that 25 wells of high positive (MR 0.8) were alternated with 25 wells of CML-negative specimen in a checkerboard pattern. The acceptance criteria were that > 95% of CML- negative samples tested negative or below LoD.

Testing found that all negative specimens (25/25) were reported as "Negative (sufficient ABL1)". All high positive samples gave an appropriate MR value (mean MR value of 0.79 [16%IS], range of MR0.73 to 0.83 [19 to 15%IS, respectively]). The acceptance criteria were met supporting the conclusion that the test generates no significant carryover between wells on the plate.

{11}------------------------------------------------

e.RNA Isolation and RNA Input:

i. RNA Isolation:

Three commonly used RNA extraction methods were tested using CML-positive white blood cells serially diluted across 4 levels (MR 1-4) into CML-negative human anti-coagulated whole blood. Testing incorporated both transcripts (e13a2 and e14a2). Two lots of assay kits were used in this testing. The acceptance criteria were that each MR value would conform to the SD criteria described in Table 3 above.

All 3 isolation methods gave equivalent results with the standard deviation within each method being less than 0.1 for all levels and lots. Therefore, it is concluded that the test is compatible with generic methods of RNA isolation, and any validated method of RNA extraction and isolation that yields unbiased isolation of total RNA in sufficient quantity and quality may be used with the assay.

ii. RNA Input:

The assay uses an RNA input concentration of 1000 to 5000 ng. To validate that the performance of the assay across the RNA concentrations, an RNA input study was conducted. A total of 30 samples derived from 2 primary RNA samples diluted into nonleukemic human RNA were tested. Samples were set to target MR values of 1, 3, or 4 with target RNA inputs ranging from 250 ng to 6000 ng, and were run in 9 replicates per sample giving a total of 216 evaluable observations. Acceptance criteria were the same as those described in Table 3 above. The results are shown in Table 9 below. The acceptance criteria were met for RNA inputs ranging from 750 to 6000 ng from MR 1 to MR 4.5, supporting the recommended RNA input range of 1000-5000 ng.

{12}------------------------------------------------

DilutionSeries	Specimen	RNAInput(ng)	Reps	PercentPositive	MeanMR	SD(MR)	Mean%IS	%CV(%IS)	SeriesMeanMR	SeriesSD(MR)	BatchMean%IS	Batch%CV(%IS)
1	pR1s01	6000	9	100	0.99	0.06	10.2263	13.3	1.00	0.04	10.1028	9.0
	pR1s02	5000	9	100	1.00	0.03	10.0471	7.9
	pR1s03	3000	9	100	1.00	0.04	10.0171	9.7
	pR1s04	1000	9	100	0.98	0.02	10.5391	5.7
	pR1s05	750	9	100	1.00	0.05	10.0122	10.4
	pR1s06	250	9	100	1.01	0.02	9.7939	5.1
2	pR1s07	6000	9	100	3.05	0.06	0.0905	13.6	3.06	0.07	0.0886	16.0
	pR1s08	5000	9	100	3.08	0.06	0.0844	13.5
	pR1s09	3000	9	100	3.07	0.03	0.0859	6.8
	pR1s10	1000	9	100	3.05	0.03	0.0897	7.8
	pR1s11	750	9	100	3.06	0.08	0.0892	17.3
	pR1s12	250	9	100	3.05	0.13	0.0928	30.0
3	pR1s13	6000	9	100	4.67	0.23	0.0025	55.7	4.70	0.27	0.0024	68.4
	pR1s14	5000	9	100	4.69	0.27	0.0024	69.7
	pR1s15	3000	9	100	4.73	0.29	0.0023	72.5
	pR1s16	1000	9	89	4.67	0.32	0.0028	84.3
	pR1s17	750	9	89	4.75	0.22	0.0020	54.1
	pR1s18	250	9	33	4.66	0.52	0.0044	173.1
5	pR2s01	6000	9	100	4.30	0.09	0.0051	19.8	4.28	0.21	0.0059	50.6
	pR2s02	5000	9	100	4.32	0.21	0.0053	53.0
	pR2s03	3000	9	100	4.27	0.15	0.0057	35.5
	pR2s04	1000	9	100	4.32	0.29	0.0060	75.4
	pR2s05	750	9	100	4.36	0.17	0.0047	40.2
	pR2s06	250	9	100	4.11	0.22	0.0089	54.7

Table 9: RNA Input Study Results

f. Traceability and Stability (Reagent and Specimen):

i. Traceability:

The assay calibrators are traceable to the First (1ªt) WHO International Genetic Reference Panel. White HE, Matejtschuk P, Rigsby P, et al. Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA. Blood. 2010;116(22):e111-7. The Value Assignment process for the calibrators is depicted below in Figure 3. Traceability to the 1st WHO International Genetic Reference Panel for quantitation of BCR-ABL translocation by RQ-PCR was demonstrated by measuring the WHO Reference Panel with 3 assay kit lots and comparing the measured values to the published values of the panel. Each of the 4 panel members was tested in duplicate across 3 runs (1 run per lot). The MR values for each level of the reference panel were calculated by regression to each lot of the kit calibrator. Measured MR values were compared to published MR values through an additional

{13}------------------------------------------------

regression analysis to determine the slope and intercept. The acceptance criteria for this testing were that the regression model have a slope close to 1 (1.0 to 1.1.) and an intercept close to 0 (0.02-0.11). The results are pictured below in Figure 4. The acceptance criteria were met supporting the conclusion that traceability of the assay calibrators to the WHO Reference Panel has been established.

Image /page/13/Figure/1 description: The image is a title for a figure. The title reads "Figure 3: Depiction of the Traceability of the QuantideX Calibrators to the WHO Reference Standard Panel." The title is written in a clear, sans-serif font and is centered on the page. The text provides context for the figure.

Image /page/13/Figure/2 description: The image shows a diagram of the WHO IS Panel (1RM), Asuragen Secondary Reference (2RM), and QuantideX Kit Calibrators (3RM). The WHO IS Panel (1RM) includes an international standard, cell line blends, limited supply, and values assigned by international consensus. The Asuragen Secondary Reference (2RM) is used to assign IS values to each lot of kit calibrators and has an unlimited supply. The QuantideX Kit Calibrators (3RM) include routine lots each aligned to WHO Primary using 2RM.

Figure 4: Measured vs. Published Values for WHO Standards by Lot

Image /page/13/Figure/4 description: The image shows three scatter plots comparing Kit-determined MR values to published MR values for WHO Primary, separated by LOT number. Each plot displays a linear regression line with its equation and R-squared value. For LOT1, the equation is y = -0.11 + 1.1x with r^2 = 0.995; for LOT2, y = 0.02 + 1x with r^2 = 0.996; and for LOT3, y = -0.059 + 1.1x with r^2 = 0.999. The data points for each LOT are represented by different colors: LOT1 in black, LOT2 in blue, and LOT3 in orange.

{14}------------------------------------------------

ii. Reagent Stability:

Real-time Stability: a.
Reagent shelf life stability studies were conducted using 3 lots with testing ongoing at T0, T3, T6, T9, T12, and T13 months. At each time point 15 replicates are tested (5 replicates/lot). Samples are total RNA derived from residual CML positive whole blood serially diluted into total RNA from human CML negative whole blood. The 5 samples are diluted to approximately MR 1, 2, 3, 4, and < LoD. Acceptance criteria for each time point and each panel member are results within ± 0.5 MR of the mean MR value of the same panel member from T0. Controls and calibrators must pass acceptance criteria at each time point. Current stability is 6 months.
b. Freeze-thaw Stability:
Total RNA derived from residual BCR-ABL positive whole blood serial diluted into RNA from BCR-ABL negative was used to evaluate the impact of freeze-thaw on reagent performance. Stability testing was performed on 3 assay kit lots, 5 kits from each lot, 1 kit tested per cycle (per calendar week). The acceptance criteria were results within $\n[ \pm ]\n$ 0.5 MR value of time 0 (testing began from a frozen reagent) for up to 5 freeze-thaw cycles for all panel members (MR 1, 2, 3, 4, and negative). The testing met the acceptance criteria and will be labeled for stable up to 4 uses (including freeze-thaw between uses).
Shipping Stability: c.
Three shipping conditions were examined, (1) stable shipping at -15 to -30°C (2) shipping stress with minimum load with temperature recorders and (3) shipping stress with maximum load with temperature recorders. Samples were the same panel as described in the freeze-thaw study. Shipping stress conditions were as follows:
22°C$\n[ \pm ]\n$ 3°C, 4 hrs then .
35°C± 3°C, 6 hrs then ●
30°C± 3°C, 56 hrs then ●
35°C± 3°C, 6 hrs then ●
Returned to -15 to -30°C until testing. ●

Acceptance criteria were the average MR value of each panel member from the shipping stress conditions were within ± 0.5 MR of the same panel member from the stable storage condition. All acceptance criteria were met. The assay is stable following shipment of up to 72 hr on dry ice.

iii. Specimen Stability (Whole Blood Stability):
Testing was performed on human CML positive clinical whole blood specimens. Time points were calculated from time of blood draw.

{15}------------------------------------------------

Receipt time of specimens was used as the baseline time point. RNA was isolated after receipt and accessioning and at T48, T60, and T≥72 hrs from whole blood stored at 2-8°C. Additionally RNA from CMLnegative blood was isolated at T4 and again at T24, T48, T60, and T72. Multiple replicates of 13 CML positive specimens were tested at 4 time points. Three CML negative specimens were tested at 5 time points. The acceptance criteria were that all results be within 0.5 MR units from the baseline result. The negative sample was evaluated in terms of the variability of the ABL1 transcript. The acceptance criteria were met supporting that the test may be used with whole blood collected in EDTA and stored at 2-8°C for up to 72 hrs.

1. Clinical Performance:
  To support the indicated use of measuring BCR-ABL1 to ABL1, expressed as a log molecular reduction (MR value) from a baseline of 100% on the International Scale, the sponsor performed a study demonstrating the clinical performance of the QuantideX qPCR BCR-ABL IS Kit in t(9;22) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs). This approach is consistent with multiple studies cited in guidelines reporting the significance of MR status after TKI treatment for the monitoring of CML.

A retrospective, multi-center clinical outcome study was conducted at 3 US sites to provide evidence of the clinically validity of MR3.0 (0.1% IS) as a threshold for event status at 36 months in CML patients. A total of 139 samples from 98 patients were collected at 2 clinical sites according to protocols approved by the Institutional Review Boards (IRBs). Of those enrolled, a total of 137 evaluable samples were available from 96 subjects. Sample inclusion criteria included the following:

Samples must be in the 12-18 month time frame after the patient started on ● original or new TKI therapy
Samples from be from adults diagnosed with CML and started with first line ● or new TKIs
After the date of the sample and through to 32-40 months clinical treatment ● failure and disease progression status information for event determination is required.

The demographic information regarding the 96 subjects is shown in Table 10 below.

Statistic	Value
Number of Samples in Analysis Population	137
Age(years)
N	96
Mean (SD)	46.5 (13.8)
Minimum	19
Median	47.5
Maximum	75
Statistic	Value
Race (n[%])
American Indian or Alaska Native	1 (1.0%)
Asian	5 (5.2%)
Black or African American	3 (3.1%)
White	83 (86.5%)
Other	1 (1.0%)
Unknown	3 (3.1%)
Gender (n[%])
Male	53 (55.2%)
Female	43 (44.8%)
Ethnicity (n[%])
Hispanic or Latino	3 (3.1%)
Not Hispanic or Latino	92 (95.8%)
Unknown	1 (1.0%)
State or Province of Residency
AK	1 (1.0%)
AZ	1 (1.0%)
BC	1 (1.0%)
CA	6 (6.3%)
CO	1 (1.0%)
FL	3 (3.1%)
GA	1 (1.0%)
IA	1 (1.0%)
ID	4 (4.2%)
MD	1 (1.0%)
MO	1 (1.0%)
NJ	7 (7.3%)
NY	1 (1.0%)
OR	42 (43.8%)
PA	9 (9.4%)
TN	1 (1.0%)
WA	15 (15.6%)
Total	96 (100%)

Table 10: Demographics of the Clinical Study Population

{16}------------------------------------------------

Outcome evaluation was based on determination of event-free survival at 32-40 months on CML patients tested 12-18 months after starting TKI medication.

The device performance was assessed by the probability of at least one event by the endpoint 32-40 months after initiation of TKI treatment as estimated from the Kaplan-Meier survival function. Acceptance criteria were that these probabilities be statistically significantly different and have point estimates differing by at least 10 percentage points.

The definition of an event included:

Death by any cause ●
The development of accelerated-phase or blast crisis CML .

{17}------------------------------------------------

Loss of complete hematologic response
Loss of complete cytogenetic response
Appearance of mutation
Change in TKI treatment not due to toxicity and not due to the results of ● another BCR-ABL assay

Results are shown below. Figure 5 shows the event free survival (EFS) curves of subjects by MR status based on clinical decision point: MR < 3 (top line) and MR ≥ 3 (bottom line). Figure 6 shows the EFS curves for all subjects < MR 3 (solid line) with the 95% confidence intervals captured in dotted lines. Figure 7 shows the EFS curves for all subjects ≥ 3 (solid line) with the 95% confidence intervals captured in dotted lines.

Figure 5: EFS by MR Status - MR <3 (top) and MR ≥3 (bottom)

Image /page/17/Figure/6 description: This image shows a survival plot with two survival curves. The x-axis represents time in months, ranging from 0 to 40. The y-axis represents EFS (event-free survival), ranging from 0.0 to 1.0. The top curve starts at 1.0 and decreases to approximately 0.8 at 40 months, while the bottom curve starts at 1.0 and decreases to approximately 0.45 at 40 months.

Figure 6: EFS for subjects with MR < 3 (solid line) and 95% CI (dotted lines)

Image /page/17/Figure/8 description: The image is a survival plot showing event-free survival (EFS) over time in months. The x-axis represents time in months, ranging from 0 to 40. The y-axis represents EFS, ranging from 0.0 to 1.0. The plot shows a decreasing trend in EFS over time, with a solid line representing the estimated survival probability and dashed lines representing the confidence intervals.

{18}------------------------------------------------

Figure 7: EFS for subjects with MR ≥ 3 (solid line) and 95% CI (dotted lines)

Image /page/18/Figure/1 description: The image shows a survival plot with the x-axis labeled 'Time (months)' ranging from 0 to 40, and the y-axis labeled 'EFS' ranging from 0.0 to 1.0. The plot shows a survival curve that starts at 1.0 and gradually decreases over time. There are also dashed lines above and below the survival curve, representing confidence intervals. The survival rate drops to around 0.8 at 25 months.

Table 11 below summarizes the EFS estimate difference around MR 3, and demonstrates that the acceptance criteria for the study were met.

Table 11: EFS Estimate Difference by MR 3

	Estimate	se	Z	P	LowCI	HighCI
EFSdifference	22.2%	10.1%	2.20	0.0279	2.0%	42.4%

An analysis was conducted to compare patients whose event status was known at 36 months versus those who were not. The acceptance criteria were that a 95% Wilson score confidence interval for the difference would be above 0, which were met.

To support the quantitative claim, the association of event hazard was also assessed using the Cox Proportional Hazards (PH) model. The 5 specimens with non-detectable MR readings were assigned either the value of MR 5 or a random value. Both analyses yielded the same results. Table 12 below summarizes the results obtained.

Table 12: Cox PH Model

Coefficient	SE	Hazardrate	Z	P	LR
-0.642	0.175	0.526	-3.68	0.0002	14.58(P=0.0001)

Finally, results were analyzed using all of the cut-points in addition to the clinical decision cut-point of MR3. Table 13 below summarizes the results for MR 1, 2, 4, and 4.5, in addition to the cut-point MR3. All but the 2 highest thresholds were significant. In summary, the data support the conclusion that the assay is appropriate for use in BCR-ABL (e13a2 and e14a2) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs) around a threshold of MR3 (the pre-specified threshold for clinical use). The clinical validity of thresholds other than MR3 has not been established.

{19}------------------------------------------------

Split	Chi-squared	P	n	EFS	SE
MR < 1	39.9	0.0000	12	15.6%	13.7%
MR ≥ 1			84	74.4%	5.2%
MR < 2	11.6	0.0007	28	50.1%	9.9%
MR ≥ 2			68	76.0%	5.7%
MR < 3	6.0	0.0139	51	57.5%	7.8%
MR ≥ 3			45	79.7%	6.4%
MR < 4	2.1	0.1520	79	64.8%	6.0%
MR ≥4			17	82.4%	9.3%
MR < 4.5	1.05	0.3050	86	66.9%	5.6%
MR ≥ 4.5			10	80.0%	12.7%

Table 13: EFS Rate by MR Threshold

O. Instrument Name: Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument

P. System Descriptions:

1. Modes of Operation:
  The Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument using System Sequence Detection Software v1.4.1is designed to accept 96-well microtiter plates. The instrument outputs quantitative results based on the measured fluorescence signals.
1. Software:
  FDA has reviewed the applicant's Hazard Analysis and software development processes for this line of product types:

Yes

1. Calibration and Quality Controls: See discussion of Traceability in section N.1.f.i above

Q. Other Supportive Performance Characteristics Data Not Covered in the "Performance Characteristics" Section above:

1. Evidence Supporting the Prognostic Significance of MR 3.0 (0.1% IS) in CML: The evidence supporting the prognostic significance of molecular response to TKI therapy in CML is discussed in the NCCN Guidelines Version 1.2016 Chronic Myelogenous Leukemia
  (https://www.nccn.org/patients/guidelines/cml/files/assets/common/downloads/files/c ml.pdf) as outlined below:
. Page MS-23 Prognostic Significance of Molecular Response to First-line TKI Therapy - Several studies have reported that achievement of a major

{20}------------------------------------------------

molecular response (MMR) after treatment with imatinib is associated with durable long-term cytogenetic remission and a lower rate of disease progression.

Page MS-26 Prognostic Significance of Molecular Response to Second-line ● TKI Therapy - The 3-month molecular response after initiation of secondline TKI therapy has also been reported to be a predictor of OS and EFS in patients who are still in chronic phase resistant to imatinib.
1. Verification of ABL1 as Endogenous Control:

Two human RNA specimens serially were diluted 2-fold into water to create a sample set ranging from ~8-2,000 ng/RT. Two lots of assay were used in this testing. Data showed that RNA at approximately 125 ng was the inflection point for assay reporting "Negative (sufficient ABL)" and "Fail (low ABL1)". The acceptance criteria were that the results of testing would all fall into 1 of these 2 categories. The data support the claim that samples with undetectable BCR-ABL1 but with acceptable ABL1 levels may be reported as "Negative (sufficient ABL1)" and that ABL1 can serve as an endogenous internal control.

	PredominantBCR-ABL1Breakpoint	RNA ng/RT	Result of"Negative(sufficientABL1)" (n)	Result of "Fail(low ABL1)" (n)
Member #
1	RNA 1:N/A	2000	6	0
2		1000	6	0
3		500	6	0
4		250	6	0
5		125	3	3
6		63	0	6
7		31	0	6
8		16	0	6
9		8	0	6
10	RNA 2:N/A	2000	6	0
11		1000	6	0
12		500	6	0
13		250	6	0
14		125	4	1
15		63	0	6
16			31	0
17			16	0
18			8	0

Table 14: ABL Control Verification

R. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Parts 801 and 809 and the special controls for this device type.

{21}------------------------------------------------

S. Patient Perspectives:

This submission did not include specific information on patient perspectives for this device.

T. Identified Risks and Required Mitigations:

Identified Risks to Health	Required Mitigations (See Section V belowfor Special Controls)
False negative results	Special Controls (1) and (2)
False positive results	Special Controls (1) and (2)
Lack of traceability of results	Special Control (3)

U. Benefit/Risk Analysis:

	Summary
	The QuantideX qPCR BCR-ABL IS Kit is an in vitro nucleic acidamplification test for the quantitation of BCR-ABL1 and ABL1transcripts in total RNA from whole blood of diagnosed t(9;22)positive Chronic Myeloid Leukemia (CML) patients expressingBCR-ABL1 fusion transcripts type e13a2 and/or e14a2. TheQuantideX qPCR BCR-ABL IS Kit is a reverse transcription-quantitative PCR performed on the Applied Biosystems 7500 FastDx Real-Time PCR Instrument and is intended to measure BCR-ABL1 to ABL1, expressed as a log molecular reduction (MR value)from a baseline of 100% on the International Scale, in t(9;22)positive CML patients during monitoring of treatment withTyrosine Kinase Inhibitors (TKIs).The test does not differentiate between e13a2 or e14a2 fusiontranscripts and does not monitor other rare fusion transcriptsresulting from t(9;22). This test is not intended for use in thediagnosis of CML.This kit has significant benefit in that it allows for quantification ofthe BCR-ABL1 transcript in CML patients with the e13a2 or e14a2fusion during treatment.Performance was assessed by the probability of at least one eventby the endpoint 36 months after initiation of their current TKItreatment as estimated from the Kaplan Meier survival function. Atotal of 139 samples from 98 patients were collected and enrolledin the clinical trial at 2 clinical sites (OHSU and Hospital of theUniversity of Pennsylvania). Performance of the test was assessedby the probability of at least one event by the endpoint 36 monthsafter initiation of TKI treatment as estimated from the KaplanMeier survival function. The primary endpoint set the nullhypothesis as the event probabilities of the group attaining MR3.0and of the group not attaining MR3.0 is equal. The alternative isthat the 36-month event-free probability of the group attainingMR3.0 is greater than those not attaining MR3.0. The successcriteria for the primary endpoint were a difference whose estimate
Summary of the Benefit(s)	exceeded 10 percent, and which was significantly different from
	zero at the one-sided 2½ % level. Both requirements have beenmet: the EFS difference was 22.2% (95% CI: 2.0%-42.4%) and thep-value was 0.0279.
Summary of Risk(s)	There is minimal potential risk associated with use of this devicegiven the combination of required general controls and specialcontrols. This device is not a qualitative device, but a quantitativedevice for the levels of BCR-ABL1 and ABL1 transcripts.
	There is a possibility that an individual with a low MR value (i.e.MR 4.5) would be assigned a higher MR value (i.e. MR 1.0). Falseresults would lead to inappropriate conclusions about the patientstatus; however, in this case, it is likely that the patient would getrepeat testing for BCR-ABL1, either immediately or duringlongitudinal monitoring; in either case, this scenario would notaffect the patient receiving therapy. Additionally, the patientwould possibly get testing for mutations in BCR-ABL1 todetermine if increasing BCR-ABL transcripts is due thedevelopment of resistance markers associated poor response to theTKI therapy.
	Another possibility is that an individual with a high MR value(MR 1.0) would be assigned a low MR value (i.e. MR 4.0). In thiscase, false results may lead to conclusions that the patient isresponding to therapy. However, patients are retested at regularintervals. Additionally, the patient may miss the opportunity to gettested for mutations in BCR-ABL1. In addition, there are specialcontrols in place based on the analytical performance of the deviceand traceability to international standards to mitigate thesepossibilities.
	Finally, the test may yield no result due to presence of the wrongtranslocation for this assay. In this case, the clinician would ordera FISH assay and the translocation would be appropriately typedand the disease would be followed by other assays. In addition,special controls, in particular labeling requirements, mitigate thesepossibilities.
Summary of Other Factors	The ordering physician should be certain that the patient has thee13a2 or e14a2 fusion transcripts before ordering this test.
ConclusionsDo the probable benefitsoutweigh the risks?	Yes, the probable benefits of this device, which allows forquantitation of the BCR-ABL1 and ABL1 transcripts, outweighthe potential risks, given that the combination of required generalcontrols and special controls established for this device.

{22}------------------------------------------------

{23}------------------------------------------------

V. Conclusion:

The information provided in this de novo submission to classify this device into class II under regulation 21 CFR 866.6060. FDA believes that the stated special controls, and applicable general controls, including design controls, provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:

Product Code:	OYX
Device Type:	BCR-ABL Quantitation Test
Class:	II (special controls)
Regulation:	21 CFR 866.6060

Identification. A BCR-ABL Quantitation Test is an reverse transcription-quantitative polymerase a. chain reaction (RT-qPCR) test for the quantitation of BCR-ABL1 expressed on the International Scale and control transcripts in total RNA from whole blood of diagnosed t(9;22) positive Chronic Myeloid Leukemia (CML) patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs). This test is not intended for the diagnosis of CML.
b. Classification. Class II (special controls). A BCR-ABL Quantitation Test must comply with the following special controls:
- 1. Premarket notification submissions must include the following information:
  - The indication for use must indicate the variant(s) for which the assay was i. designed and validated, for example BCR-ABL e13a2 and/or e14a2.
  - ii. A detailed description of all components in the test, including the following:
    - (A) A detailed description of the test components, all required reagents, instrumentation and equipment, including illustrations or photographs of non-standard equipment or methods.
    - (B) Detailed documentation of the device software including, but not limited to, standalone software applications and hardware-based devices that incorporate software.
    - (C) Methodology and protocols for control procedures for the assay to allow reporting on the International Scale.
    - (D) A description of the result outputs, analytical sensitivity of the assay, and the range of values that will be reported.
    - (E) A description of appropriate internal and external controls that are recommended or provided. The description must identify those control elements that are incorporated into the testing procedure.
  - iii. Information that demonstrates the performance characteristics of the test, including:
    - (A) For indications for use based on a threshold established in a predicate device of this generic type, device performance data from either a method comparison study to the predicate device or through a clinical study demonstrating clinical validity using well-characterized prospectively or retrospectively obtained clinical specimens, as appropriate, representative of the intended use population.
    - (B) For indications for use based on a threshold not established in a predicate device of this generic type, device performance data from a clinical study demonstrating clinical validity using well characterized prospectively or

{24}------------------------------------------------

retrospectively obtained clinical specimens, as appropriate, representative of the intended use population.

(C) Device reproducibility data generated, using a minimum of three sites, of which at least two sites must be external sites, with two operators at each site. Each site must conduct a minimum of 3 runs per operator over nonconsecutive days evaluating a minimum of 5 different BCR-ABL concentrations that span and are well distributed over the measuring range and include MR3 (0.1% IS). Results shall be reported as the standard deviation and percentage coefficient of variation for each level tested. Prespecified acceptance criteria must be provided and followed.
(D) Device precision data using clinical samples to evaluate the within-lot, between-lot, within-run, between run, and total variation.
(E) Device linearity data using a dilution panel created from clinical samples.
(F) Device analytic sensitivity data, including limit of blank, limit of detection, and limit of quantification.
(G) Device specificity data, including interference and cross-contamination.
(H) Device stability data, including real-time stability of samples under various storage times, temperatures, and freeze-thaw conditions
iv. Identification of risk mitigation elements used by your device, including a detailed description of all additional procedures, methods, and practices incorporated into the instructions for use that mitigate risks associated with testing using your device.
1. Your 21 CFR 809.10 compliant labeling must include the following:
- i. The intended use in your 21 CFR 809.10(a)(2) and 21 CFR 809.10(b)(2) complaint labeling must include an indication for use statement that reads "This test is not intended for the diagnosis of CML."
- ii. A detailed description of the performance studies conducted to comply with section b.1.iii. and a summary of the results.
1. Your device output must include results on the International Scale (%1S) and your assay must include multi-point calibration controls traceable to a relevant international reference panel (e.g., the World Health Organization (WHO) International Genetic Reference Panel for quantitation of BCR-ABL mRNA).

Regulation Number and Section

§ 866.6060 BCR-ABL quantitation test.

(a)
Identification. A BCR-ABL quantitation test is identified as a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) test for the quantitation of BCR-ABL1 expressed on the International Scale (IS) and control transcripts in total RNA from whole blood of diagnosed t(9;22) positive chronic myeloid leukemia (CML) patients during monitoring of treatment with tyrosine kinase inhibitors. This test is not intended for the diagnosis of CML.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the following information:
(i) The indication for use must indicate the variant(s) for which the assay was designed and validated, for example BCR-ABL e13a2 and/or e14a2.
(ii) A detailed description of all components in the test, including the following:
(A) A detailed description of the test components, all required reagents, instrumentation and equipment, including illustrations or photographs of non-standard equipment or methods;
(B) Detailed documentation of the device software including, but not limited to, standalone software applications and hardware-based devices that incorporate software;
(C) Methodology and protocols for control procedures for the assay to allow reporting on the International Scale;
(D) A description of the result outputs, analytical sensitivity of the assay, and the range of values that will be reported; and
(E) A description of appropriate internal and external controls that are recommended or provided. The description must identify those control elements that are incorporated into the testing procedure.
(iii) Information that demonstrates the performance characteristics of the test, including:
(A) For indications for use based on a threshold established in a predicate device of this generic type, device performance data from either a method comparison study to the predicate device or through a clinical study demonstrating clinical validity using well-characterized prospectively or retrospectively obtained clinical specimens, as appropriate, representative of the intended use population;
(B) For indications for use based on a threshold not established in a predicate device of this generic type, device performance data from a clinical study demonstrating clinical validity using well-characterized prospectively or retrospectively obtained clinical specimens, as appropriate, representative of the intended use population;
(C) Device reproducibility data generated, using a minimum of three sites, of which at least two sites must be external sites, with two operators at each site. Each site must conduct a minimum of three runs per operator over non-consecutive days evaluating a minimum of five different BCR-ABL concentrations that span and are well distributed over the measuring range and include MR3 (0.1 percent IS). Results shall be reported as the standard deviation and percentage coefficient of variation for each level tested. Prespecified acceptance criteria must be provided and followed;
(D) Device precision data using clinical samples to evaluate the within-lot, between-lot, within-run, between run, and total variation;
(E) Device linearity data using a dilution panel created from clinical samples;
(F) Device analytic sensitivity data, including limit of blank, limit of detection, and limit of quantification;
(G) Device specificity data, including interference and cross-contamination; and
(H) Device stability data, including real-time stability of samples under various storage times, temperatures, and freeze-thaw conditions.
(iv) Identification of risk mitigation elements used by your device, including a detailed description of all additional procedures, methods, and practices incorporated into the instructions for use that mitigate risks associated with testing using your device.
(2) Your 21 CFR 809.10 compliant labeling must include the following:
(i) The intended use in your 21 CFR 809.10(a)(2) and (b)(2) complaint labeling must include an indication for use statement that reads “This test is not intended for the diagnosis of CML”; and
(ii) A detailed description of the performance studies conducted to comply with paragraph (b)(1)(iii) of this section and a summary of the results.
(3) Your device output must include results on the International Scale (IS) and your assay must include multipoint calibration controls traceable to a relevant international reference panel (
e.g., the World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA).