ipsogen JAK2 RGQ PCR Kit

K172287 · Qiagen, Inc. · PSU · Jan 12, 2018 · Medical Genetics

Device Facts

• Record ID: K172287
• Device Name: ipsogen JAK2 RGQ PCR Kit
• Applicant: Qiagen, Inc.
• Product Code: PSU · Medical Genetics
• Decision Date: Jan 12, 2018
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.6070
• Device Class: Class 2

Indications for Use

The ipsogen JAK2 RGQ PCR Kit is a qualitative in vitro diagnostic test for the detection of the JAK2 V617F/G1849T allele in genomic DNA extracted from EDTA whole blood. The ipsogen JAK2 RGQ PCR Kit is a real time PCR test performed on the QIAGEN Rotor-Gene Q MDx instrument. The test is intended for use as an adjunct to evaluation of suspected myeloproliferative neoplasms, in conjunction with other clinicopathological factors. This test does not detect less common JAK2 mutations associated with Myeloproliferative Neoplasms including mutations in exon 12 and is not intended for stand-alone diagnosis of Myeloproliferative Neoplasm.

Device Story

The ipsogen JAK2 RGQ PCR Kit is an in vitro diagnostic test for detecting the JAK2 V617F mutation. It uses allele-specific, quantitative PCR (ARMS technology) on genomic DNA extracted from K2-EDTA whole blood via the QIAsymphony instrument. The assay runs on the QIAGEN Rotor-Gene Q MDx platform. The system uses mutation-specific and wild-type reaction mixes, including standards for curve generation. The Rotor-Gene AssayManager software processes the PCR signals to determine the JAK2 mutation status. Results are used by clinicians as an adjunct to other clinicopathological factors for evaluating suspected myeloproliferative neoplasms. The device provides a percentage of mutant allele burden, though it is not intended for quantitative use. It benefits patients by aiding in the diagnostic workup of MPNs, providing higher sensitivity than traditional Sanger sequencing.

Clinical Evidence

Bench testing only. Accuracy was evaluated using 473 clinical specimens (ET, PMF, and PV) compared to bi-directional Sanger sequencing. Overall agreement was 96.8% (95% CI: 94.8%–98.2%). Discordant results (15/473) were resolved using NGS, which confirmed the kit's results. Whole blood stability was validated for 96 hours at room temperature or 4°C.

Technological Characteristics

Real-time PCR assay using ARMS technology. Components: mutation-specific/wild-type primer mixes, fluorescent probes, internal control. Instrument: Rotor-Gene Q MDx. Software: Rotor-Gene AssayManager v2.1.x with Gamma MDx plug-in v1.0.x. Sample input: gDNA from EDTA whole blood. Extraction: QIAsymphony SP with DSP DNA Mini Kit.

Indications for Use

Indicated for qualitative detection of JAK2 V617F/G1849T allele in genomic DNA from EDTA whole blood in patients suspected of having Myeloproliferative Neoplasms (MPN). Not for detection of exon 12 mutations; not for stand-alone diagnosis.

Regulatory Classification

Identification

A mutation detection test for myeloproliferative neoplasms is an in vitro diagnostic device intended for the detection of the JAK2 V617F/G1849T allele in genomic DNA extracted from whole blood. The test is intended for use as an adjunct to evaluation of suspected polycythemia vera in conjunction with other clinicopathological factors.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) Design verification and validation must include: (i) A detailed description of all components in the test, including the following: (A) A detailed description, including illustrations or photographs of non-standard equipment or methods, of the test components, including all required reagents, instrumentation, and equipment. (B) Detailed documentation of the device software, including standalone software applications and hardware-based devices that incorporate software. (C) A detailed description of methodology and assay procedures including appropriate internal and external quality controls that are recommended or provided. The description must identify those control elements that are incorporated into the testing procedure. (D) A detailed specification for sample collection, processing, and storage. (E) A description of the criteria for test result interpretation and reporting including result outputs, analytical sensitivity of the assay, and the values that will be reported. (ii) 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 study that includes at least two operators per site, two runs per operator per day over a minimum of three non-consecutive days evaluating a sample panel that contains allelic frequencies that span the claimed measuring range, and include the clinical threshold allelic frequency. Pre-specified acceptance criteria must be provided and followed. (D) Information on device traceability and a description of the value assignment process for calibrators and controls. (E) Device precision data using clinical samples and controls to evaluate the within-lot, between-lot, within-run, between-run, and total variation. (F) Device linearity data generated from samples covering the device measuring range and for any standards used in the quantitation of allelic frequencies. (G) Device analytic sensitivity data, including limit of blank and limit of detection. (H) Device specificity data, including interference and cross-contamination. (I) Device and clinical specimen stability data, including real-time stability (long-term storage and in-use stability) and stability evaluating various storage times, temperatures, and freeze-thaw conditions, as appropriate. (iii) Identification of risk mitigation elements used by the 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 the device. (2) The labeling required under § 809.10(b) of this chapter must include: (i) An intended use statement, including an indication for use that includes the variant(s) for which the assay was designed and validated, for example, JAK2 G1849T. (ii) A detailed description of the performance studies conducted to comply with paragraph (b)(1)(ii) of this section and a summary of the results.

Predicate Devices

• ipsogen® JAK2 RGQ PCR Kit (DEN160028)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
K172287

B. Purpose for Submission:
Expansion of the Indications for Use.

C. Measurand:
Janus Tyrosine Kinase 2 (JAK2) gene mutation G1849T (V617F)

D. Type of Test:
Allele-specific, quantitative, polymerase chain reaction (PCR) using an amplification refractory mutation system (ARMS)

E. Applicant:
QIAGEN

F. Proprietary and Established Names:
Trade Name: QIAGEN ipsogen® JAK2 RGQ PCR Kit

G. Regulatory Information:
1. Regulation section:
21 CFR 866.6070
2. Classification:
Class II
3. Product code:
PSU
4. Panel:
88 – Pathology

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# H. Intended Use:

# 1. Intended use(s):

The ipsogen JAK2 RGQ PCR Kit is a qualitative in vitro diagnostic test for the detection of the JAK2 V617F/G1849T allele in genomic DNA extracted from EDTA whole blood. The ipsogen JAK2 RGQ PCR Kit is a real time PCR test performed on the QIAGEN Rotor-Gene Q MDx instrument. The test is intended for use as an adjunct to evaluation of suspected myeloproliferative neoplasms, in conjunction with other clinicopathological factors.

This test does not detect less common JAK2 mutations associated with Myeloproliferative Neoplasms including mutations in exon 12 and is not intended for stand-alone diagnosis of Myeloproliferative Neoplasm.

# 2. Indication(s) for use:

Same as Intended use

# 3. Special conditions for use statement(s):

For prescription use only.

For in vitro diagnostic use only.

# 4. Special instrument requirements:

QIAGEN Rotor-Gene Q MDx platform using Rotor-Gene AssayManager software version 2.1.

# I. Device Description:

The ipsogen JAK2 RGQ PCR Kit employs allele-specific, quantitative, polymerase chain reaction (PCR) using an amplification refractory mutation system (ARMS). DNA is extracted from K2-EDTA anti-coagulated whole blood using the QIAsymphony instrument (QSSP) and QIAsymphony® DSP DNA Mini Kit. Purified DNA must be diluted to $10\mathrm{ng} / \mu \mathrm{l}$ using the TE buffer provided in the JAK2 Kit. Each PCR reaction of the Rotor-Gene Q MDx is optimized for $50\mathrm{ng}$ of purified gDNA diluted in a final volume of $5\mu \mathrm{l}$ . A total of $100\mathrm{ng}$ per tested sample (50 ng for each reaction) is needed. The Kit contains sufficient reagents to test 24 reactions. Table 1 describes the components of the ipsogen JAK2 RGQ PCR Kit.

Table 1. Components of the ipsogen JAK2 RGQ Assay

| Item | Description | Use |
| --- | --- | --- |
| JAK2 Mutant Control | 100% V617F allele | Assay Positive Control |
| JAK2 WT Control | 100% WT allele | Assay Negative Control |

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| JAK2 MT Quant Standard 1 | 5x10^1 V617F copies in 5 μL | Mutation Standard Curve |
| --- | --- | --- |
| JAK2 MT Quant Standard 2 | 5x10^2 V617F copies in 5 μL | Mutation Standard Curve |
| JAK2 MT Quant Standard 3 | 5x10^3 V617F copies in 5 μL | Mutation Standard Curve |
| JAK2 MT Quant Standard 4 | 5x10^4 V617F copies in 5 μL | Mutation Standard Curve |
| JAK2 WT Quant Standard 1 | 5x10^1 wild-type copies in 5 μL | Wild-type Standard Curve |
| JAK2 WT Quant Standard 2 | 5x10^2 wild-type copies in 5 μL | Wild-type Standard Curve |
| JAK2 WT Quant Standard 3 | 5x10^3 wild-type copies in 5 μL | Wild-type Standard Curve |
| JAK2 WT Quant Standard 4 | 5x10^4 wild-type copies in 5 μL | Wild-type Standard Curve |
| JAK2 MT Reaction Mix | Primers, probes, and necessary components for the mutation-specific and internal control PCR reaction | Mutation Specific PCR Reaction |
| JAK2 WT Reaction Mix | Primers, probes, and necessary components for the wild-type and internal control PCR reaction | Wild-type Specific PCR Reaction |
| Taq DNA Polymerase | PCR reaction enzyme | Mutation and Wild-type PCR |
| TE buffer | Tris-EDTA | Sample Dilution |
| Nuclease-Free Water | Water | No Template Control |
| Rotor-Gene AssayManager JAK2 plug-in | JAK2 Assay-specific software | Results Acquisition and Analysis |
| JAK2 Assay Profile | JAK2 Assay-specific software parameters | Results Acquisition and Analysis |

Additional materials required but not provided with the JAK2 RGQ PCR Kit:

- QIAsymphony® DSP DNA Mini Kit
- QIAsymphony Sample Preparation instrument and accessories
- QIAsymphony software version 4.0 that operates the QIAsymphony instrument
- QIAGEN Rotor-Gene Q MDx Platform
- Rotor-Gene AssayManager® software version 2.1 that operates the Rotor Gene Q MDx

# J. Substantial Equivalence Information:

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1. Predicate device name(s): ipsogen® JAK2 RGQ PCR Kit
2. Predicate 510(k) number(s): DEN160028
3. Comparison with predicate:

| Characteristic | Device | Predicate |
| --- | --- | --- |
| Similarities | | |
| Intended Use | The ipsogen JAK2 RGQ PCR Kit is a qualitative in vitro diagnostic test for the detection of the JAK2 V617F/G1849T allele in genomic DNA extracted from EDTA whole blood. The ipsogen JAK2 RGQ PCR Kit is a real-time PCR test performed on the QIAGEN Rotor-Gene Q MDx instrument. | Same |
| Specimen Type | Genomic DNA extracted from EDTA whole blood | Same |
| Assay Targets | JAK2 V617F/G1849T allele | Same |
| Genomic DNA Extraction | DNA should be extracted using the QIAsymphony SP instrument in combination with the QIAsymphony DSP DNA Mini Kit | Same |
| Amplification and Detection Technology | Real-time PCR DNA amplification | Same |
| Amplification and Detection Instrument System | Assay uses the Rotor-Gene Q MDx | Same |
| Assay Controls | Positive Control, Negative Control and Internal Control included in the kit | Same |
| Characteristic | Device | Predicate |
| --- | --- | --- |
| Differences | | |
| Indications for Use | The test is intended for use as an adjunct to evaluation of suspected Myeloproliferative | The test is intended for use as an adjunct to evaluation of suspected Polycythemia Vera, |
| | drugs, drugs, and other drugs | drugs, drugs, and other drugs |
| Indications for Use in the Treatment of Myeloproliferative Diseases | drugs, drugs, and other drugs | drugs, drugs, and other drugs |

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| | Neoplasms, in conjunction with other clinicopathological factors.
This test does not detect less common JAK2 mutations associated with Myeloproliferative Neoplasms including mutations in exon 12 and is not intended for stand-alone diagnosis of Myeloproliferative Neoplasms. | in conjunction with other clinicopathological factors.
This test does not detect less common mutations associated with Polycythemia Vera including mutations in exon 12 and is not intended for stand-alone diagnosis of Polycythemia Vera. |
| --- | --- | --- |
| Amplification and Detection Instrument System Software | Rotor-Gene AssayManager® software version 2.1 | Rotor-Gene AssayManager® software version 1.04 |

K. Standard/Guidance Document Referenced (if applicable):
CLSI EP25-A: Evaluation of stability of in vitro diagnostic reagents

L. Test Principle:
Refer to DEN160028.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

Overall analytical performance of the QIAGEN ipsogen® JAK2 RGQ PCR Kit was previously demonstrated using specimens from patients with suspected polycythemia vera (PV). Refer to decision summary for DEN160028 available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/denovo.cfm?ID=DEN160028 for a description of the following analytical performance studies:

- Repeatability/Reproducibility/Lot-to-Lot
- Linearity/assay reportable range and DNA input
- Traceability/Calibration
- Detection limit (Limit of Blank and Limit o Detection)
- Analytical specificity (including Interfering Substances)
- Reagent Stability
- Extracted DNA stability

Analytical performance studies provided to support expansion of the indications for use claim to include additional JAK2 positive myeloproliferative neoplasms included accuracy study using specimens from essential thrombocytopenia (ET) and primary

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myelofibrosis (PMF) patients, and specimen stability study. The studies are described below.

a. Accuracy study – Comparison to a Reference Method

A study was conducted to demonstrate the accuracy for detecting JAK2 V617F/G1849T allele in clinical specimens from patients representing the target population. Because the original study included suspected MPNs, specimens in this study were selected based on diagnosis which included both JAK2 positive and negative specimens. A total of 197 specimens (98 ET and 99 PMF) was tested with the ipsogen JAK2 RGQ PCR Kit and compared with validated bi-directional Sanger Sequencing (BDS).

All specimens were concordant except for 9 ET and 5 PMF samples, all of which were JAK2 V617F positive with the ipsogen JAK2 RGQ PCR Kit and negative with BDS. Concordance between the two methods is shown in Table 2 for ET specimens and Table 3 for PMF specimens.

Table 2. Concordance table between ipsogen JAK2 RGQ PCR Kit and Sanger Bidirectional Sequencing in ET population

| | Sanger bi-directional sequencing | | | |
| --- | --- | --- | --- | --- |
| | | JAK2 V617F positive | JAK2 V617F negative | Total |
| ipsogen JAK2 RGQ PCR Kit | JAK2 V617F positive | 43 | 9 | 52 |
| | JAK2 V617F negative | 0 | 46 | 46 |
| | Total | 43 | 55 | 98 |

The overall agreement is 90.8% (89/98 subjects; 95% CI: [83.3% – 95.7%]), the positive agreement is 100% (43/43 subjects; 95% CI: [91.8% – 100%]), and the negative agreement is 83.6% (46/55 subjects; 95% CI: [71.2% – 92.2%]).

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Table 3. Concordance table between ipsogen JAK2 RGQ PCR Kit and Sanger Bidirectional Sequencing in PMF population

| | Sanger Bi-directional sequencing | | | |
| --- | --- | --- | --- | --- |
| | | JAK2 V617F positive | JAK2 V617F negative | Total |
| ipsogen JAK2 RGQ PCR Kit | JAK2 V617F positive | 51 | 5 | 56 |
| | JAK2 V617F negative | 0 | 43 | 43 |
| | Total | 51 | 48 | 99 |

The overall agreement is $94.9\%$ (94/99 subjects; $95\%$ CI: $[88.6\% - 98.3\%]$ ), the positive agreement is $100\%$ (51/51 subjects; $95\%$ CI: $[93.0\% - 100\%]$ ), and the negative agreement is $89.6\%$ (43/48 subjects; $95\%$ CI: $[77.3\% - 96.5\%]$ ).

Previously in DEN160028, a clinical study was conducted in which 276 PV samples were evaluated with the ipsogen JAK2 RGQ PCR Kit and the results were compared to results obtained with BDS. One PV sample was found JAK2 V617F positive with the ipsogen JAK2 RGQ PCR Kit and negative with BDS. Concordance between the two methods is shown in Table 4 for PV specimens. The overall agreement is $99.6\%$ (275/276 subjects; $95\%$ CI: $[98.0\% - 100\%]$ ), the positive agreement is $100\%$ (71/71 subjects; $95\%$ CI: $[94.9\% - 100\%]$ ), and the negative agreement is $99.5\%$ (204/205 subjects; $95\%$ CI: $[97.3\% - 100\%]$ ).

Table 4. Concordance table between ipsogen JAK2 RGQ PCR Kit and Sanger Bidirectional Sequencing in PV population

| | Sanger Bi-directional sequencing | | | |
| --- | --- | --- | --- | --- |
| | | JAK2 V617F positive | JAK2 V617F negative | Total |
| ipsogen JAK2 RGQ PCR Kit | JAK2 V617F positive | 71 | 1 | 72 |
| | JAK2 V617F negative | 0 | 204 | 204 |
| | Total | 71 | 205 | 276 |

Accuracy with all MPN specimens evaluated combined

The overall accuracy of the test in MPN specimens was assessed by combining the

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data obtained from each specimen cohort. A total of 473 MPN specimens were evaluated and a total of 15 samples were discordant. The overall agreement is $96.8\%$ (458/473 subjects; $95\%$ CI: $[94.8\%; 98.2\%]$ ). The positive agreement was $100\%$ (165/165 subjects; $95\%$ CI: $[97.8\%; 100\%]$ and the negative agreement was $95.1\%$ (293/308 subjects; $95\%$ CI: $[92.1\%; 97.2\%]$ ). The results are shown in Table 5.

Table 5. Concordance table between ipsogen JAK2 RGQ PCR Kit and Sanger Bidirectional Sequencing in MPN population (combined ET, PMF and PV populations)

| | Sanger bi-directional sequencing | | | |
| --- | --- | --- | --- | --- |
| | | JAK2 V617F positive | JAK2 V617F negative | Total |
| ipsogen JAK2 RGQ PCR Kit | JAK2 V617F positive | 165 | 15 | 180 |
| | JAK2 V617F negative | 0 | 293 | 293 |
| | Total | 165 | 308 | 473 |

The specimens yielding discordant results appeared to have mutation levels below the BDS detection capability (around $10\%$ ). Because Sanger sequencing is not as sensitive as the JAK2 assay, and the JAK2 assay sensitivity is $1\%$ , a separate study was conducted using a validated next generation sequencing (NGS) method to detect JAK2 V617F allele in the 15 discordant samples (9 ET, 5 PMF, and 1 PV), as well as a randomly selected set of 22 JAK2 V617F concordant specimens (11 positive and 11 negative specimens). All 15 discordants tested positive by NGS, agreeing with the ipsogen JAK2 RGQ PCR Kit. All concordant samples tested the same with NGS.

# b. Whole blood stability

A specimen stability was conducted with 6 PV specimens (3 JAK2 V617F positive and 3 JAK2 V617F negative samples in K2-EDTA tubes) collected and stored at $4^{\circ}\mathrm{C}$ or room temperature (RT) before proceeding to gDNA extraction. Specimen stability was tested by assessing the agreement between qualitative results, and separately, for trends in changes in the percent mutation measured with the JAK2 Kit at each time point (i.e., baseline, day 2, 3, 4 and day $4 + 2\mathrm{h}$ ) and storage condition (RT or $2 - 8^{\circ}\mathrm{C}$ ). At each extraction, 3 aliquots per sample were extracted using QIAsymphony. The results passed acceptance criteria and support the claim that whole blood can be stored at RT and $2 - 8^{\circ}\mathrm{C}$ for 96 hours before being processed for gDNA extraction to assess the JAK2 V617F status with the JAK2 Kit.

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2. Comparison studies:

a. Method comparison with predicate device:
Not applicable.

b. Matrix comparison:
Not applicable.

3. Clinical studies:
Not applicable.

4. Clinical cut-off:

Specimens < 1% are considered negative and no value is generated. Specimens ≥1% are considered positive. The assay reports the JAK2 percentage because the additional information on potential mutant allele burden enhances diagnostic evaluation, however the assay is not intended for quantitative use. There is currently no consensus on the clinical value of extremely low JAK2 V617F loads, however a 1% mutation load is considered by experts and literature (Tefferi, 2011 and Martinaud, 2010) as a meaningful cut-off for reporting JAK2 V617F positivity.

5. Expected values/Reference range:

The assay is not intended for quantitative use. Low JAK2 allele has been detected in subjects without MPNs.

N. Instrument Name:

QIAGEN Rotor-Gene Q MDx platform using Rotor-Gene AssayManager software version 2.1. The instrument was cleared by FDA under K113319 on February 06, 2012.

O. System Descriptions:

1. Modes of Operation:

Does the applicant’s device contain the ability to transmit data to a computer, webserver, or mobile device?
Yes ☐ X ☐ or No ☐

Does the applicant’s device transmit data to a computer, webserver, or mobile device using wireless transmission?
Yes ☐ or No ☐ X ☐

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2. Software:

FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types:

Yes ☐ X ☐ or No ☐

3. Specimen Identification:

Whole blood.

4. Specimen Sampling and Handling:

4C or RT for 96 hours.

5. Calibration and Quality Control:

Installation and calibration are performed by the manufacturer. The assay uses standards for generation of a curve by which the % mutation is assessed. The Instrument and assay employ both in-process QC checks and array QC metrics to assist in identifying problems in the assay and instances in which the assay has failed.

P. Other Supportive Instrument Performance Characteristics Data Not Covered In The “Performance Characteristics” Section above:

None.

Q. 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.

R. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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