(39 days)
QuantideX® qPCR BCR-ABL IS Kit
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No
The description focuses on standard quantitative PCR techniques and software for calculation and data analysis, with no mention of AI or ML algorithms.
No.
The device is an in vitro diagnostic test for monitoring BCR-ABL mRNA transcript levels in CML patients, aiding in treatment monitoring and discontinuation decisions, but it does not directly treat or prevent a disease.
Yes.
The "Intended Use / Indications for Use" section explicitly states, "The MolecularMD MRDx BCR-ABL Test is an in vitro diagnostic test for the quantitative detection of BCR-ABL 1 transcripts..." Furthermore, it describes the test's use in monitoring treatment and aiding in identifying candidates for treatment discontinuation in patients with CML, all of which fall under diagnostic purposes.
No
The device description explicitly states that the test is performed on the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument, which is a hardware component. While the device includes software for data analysis, it is an integral part of a larger in vitro diagnostic system that includes hardware.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The "Intended Use / Indications for Use" section explicitly states that the MolecularMD MRDx BCR-ABL Test is an in vitro diagnostic test.
- Purpose: The test is designed to analyze a biological sample (peripheral blood) in vitro (outside the body) to provide information about a patient's health status (monitoring BCR-ABL transcript levels in CML patients).
- Clinical Application: The results are used to aid in clinical decision-making, specifically for monitoring treatment response to TKIs and identifying candidates for treatment discontinuation in CML patients.
The description clearly aligns with the definition of an in vitro diagnostic device.
N/A
Intended Use / Indications for Use
The MolecularMD MRDx BCR-ABL Test is an in vitro diagnostic test for the quantitative detection of BCR-ABL1 transcripts (e13a2/b2a2 and/or e14a2/b3a2) and the ABL1 endogenous control mRNA in peripheral blood specimens from patients previously diagnosed with t(9;22) positive chronic myeloid leukemia (CML). The ratio of BCR-ABL1 to ABL1 is calculated and reported on the WHO International Scale. The test utilizes quantitative, real-time reverse transcription polymerase chain reaction performed on the Applied Biosystems 7500 Fast Dx instrument.
The MolecularMD MRDx BCR-ABL Test is intended to measure BCR-ABL mRNA transcript levels in patients diagnosed with t(9;22) positive CML during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).
The device is also intended to be used in the serial monitoring for BCR-ABL mRNA transcript levels as an aid in identifying CML patients in the chronic phase being treated with nilotinib who may be candidates for treatment discontinuation and for monitoring of treatment-free remission.
The test does not differentiate between e13a2 or e14a2 fusion transcripts and does not monitor other rare fusion transcripts resulting from t(9;22). The test is not intended for the diagnosis of CML.
Product codes
OYX
Device Description
The MolecularMD MRDx® BCR-ABL Test is a quantitative, real-time polymerase chain reaction test that provides quantitation of BCR-ABL1 (hereafter BCR-ABL) transcript e13a2/b2a2 or e14a2/b3a2 and ABL1 (hereafter ABL) transcript levels in RNA extracted from peripheral blood samples collected from CML patients. Peripheral blood is collected in either EDTA or PAXgene Blood RNA Tubes. Each collection tube type requires a specified RNA extraction method and RNA input amount. The detailed extraction protocol and RNA quantity requirements are provided in the device package insert and instructions for use. Total RNA is extracted from peripheral blood and serves as the template for RT-qPCR. The test is performed using a onestep RT-qPCR protocol wherein the reverse transcription and quantitative, real-time PCR reactions are performed in the same well. BCR-ABL and ABL amplicons are generated and detected in real-time using TaqMan® MGB probes. For RNA extracted from EDTA tubes, a total of 4 µg of RNA is required for each patient sample: 1 µg of extracted RNA is used in the MRDx BCR-ABL Test in each of 4 wells, 2 wells for BCR-ABL quantitation and 2 wells for ABL quantitation. For RNA extracted from PAXgene tubes, a total of 8 µg of RNA is required for each patient sample: 2 µg of extracted RNA is used in the MRDx BCR-ABL Test in each of 4 wells, 2 wells for BCR-ABL quantitation and 2 wells for ABL quantitation.
The MolecularMD MRDx BCR-ABL Test is performed on the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument. The instrument integrates a thermal cycler, a fluorometer and application specific software. The instrument houses the thermal cycler and the fluorometer, while the application software is installed on a computer that is attached to the instrument. The ABI 7500 Fast Dx instrument software v1.4.1 calculates the BCR-ABL and ABL copy numbers using a standard curve generated with calibrators on each individual plate.
Quantitation is achieved using RNA calibration standards and linear regression analysis provided by the ABI 7500 Fast Dx PCR Instrument Software. BCR-ABL transcript levels are measured in relation to the ABL transcript as an endogenous reference. The data are exported as a CSV file for further analysis in the MRDx® BCR-ABL Test Software. The BCR-ABL/ABL ratio is calculated and converted to the International Scale by the MRDx BCR-ABL Test Software.
The MRDx BCR-ABL Test Software is used to analyze all test results. This software, provided with the MRDx BCR-ABL Test, is used to calculate the BCR-ABL/ABL % IS and MR value for patient sample using the conversion factor for the MRDx BCR-ABL Test after validating each MRDx BCR-ABL Test result against the run acceptance criteria and sample acceptance criteria.
Calibrators are analyzed for both BCR-ABL and ABL in every run simultaneously with patient samples. RNA controls provided at MMR (MR3.0) and MR4.5 allow for on-plate verification of test accuracy and reproducibility over this critical transcript range. The integrated conversion factor provides test results on the International Scale (IS) harmonized to the WHO.
Mentions image processing
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Mentions AI, DNN, or ML
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Input Imaging Modality
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Anatomical Site
peripheral blood
Indicated Patient Age Range
Adult patients
Intended User / Care Setting
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Description of the training set, sample size, data source, and annotation protocol
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Description of the test set, sample size, data source, and annotation protocol
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Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Pre-Clinical Analytical Studies:
- Traceability to the International Scale: The assay calibrators are traceable to the First (1st) WHO International Genetic Reference Panel. Deming Regression analyses performed comparing expected and observed MR values for three different lots showed a consistent conversion factor of 1.1 for all lots, with a correlation of 1.0, indicating high traceability.
- Correlation to a Reference Method (Accuracy): Evaluated by comparing the MRDx BCR-ABL Test to a validated BCR-ABL reverse transcription droplet digital PCR (ddPCR) assay.
- Sample Size: 217 samples of blood from CML patients, ranging from 10% BCR-ABL/ABL IS (MR1.0) to 0.001% BCR-ABL/ABL IS (MR5.0). 119 evaluable EDTA samples and 98 evaluable PAXgene samples.
- Results (Deming Regression):
- EDTA: Slope = 0.96 (95% CI [0.94, 0.99]), Y-Intercept = 0.077 (95% CI [0.0027, 0.17]), Pearson Coefficient = 0.984.
- PAXgene: Slope = 0.98 (95% CI [0.95, 1.0]), Y-Intercept = -0.021 (95% CI [-0.11, 0.056]), Pearson Coefficient = 0.985.
- High concordance observed for both tube types, supporting detection accuracy.
- Detection Capability:
- Limit of Blank (LoB): Determined by testing 30 BCR-ABL negative samples in duplicate using 3 assay kit lots (180 replicates). 177 measurements had no measurable BCR-ABL values, and 3 (1.7%) had measurements below the LoD.
- Limit of Detection (LoD): Determined using 6 CML patient specimens (3 each for e13a2 and e14a2 transcripts) serially diluted into non-diseased subject blood.
- Sample Size: 1725 valid EDTA samples and 1595 valid PAXgene samples.
- Results:
- EDTA: 0.00029% IS (MR5.5) for both transcripts.
- PAXgene: 0.00039% IS (MR5.4) for both transcripts.
- Limit of Quantitation (LoQ):
- Results:
- EDTA: 0.0016% IS (MR4.8).
- PAXgene: 0.0025% IS (MR4.6).
- The MRDx BCR-ABL Test Software limits reported quantifiable results to 0.0032% IS (MR4.5) for both tube types.
- Results:
- Analytical Specificity - Interfering Substances:
- Endogenous: CML samples at 2 levels (MR3.0 and MR4.5) from EDTA and PAXgene tubes spiked with hemoglobin, bilirubin, and triglycerides. No clinically significant difference observed compared to matrix controls.
- Exogenous: Extraction kit reagents tested for interference. No clinically significant difference observed.
- Key Results: Data support no significant interference from evaluated substances.
- Analytical Specificity - Primer Specificity:
- PCR products from 6 CML patient samples (e13a2 and e14a2) were sequenced, confirming agreement with Genbank sequences.
- No cross-reactivity with ABL2 IVT RNA. Cross-reactivity observed with e19a2 BCR-ABL transcript, mitigated by a precaution statement in labeling.
- Analytical Specificity - Specimen Cross Contamination (Carryover):
- Tested high positive samples alternately juxtaposed with negative samples.
- Results: No negative samples showed detectable BCR-ABL/ABL % IS values above the LoD. Data support no significant carryover.
- Precision - Repeatability:
- Contrived patient samples (MR3.0, MR4.0, MR4.5 for e13a2 and e14a2) from both tube types were tested in 17 replicates.
- Key Results: All levels for both transcripts passed acceptance criteria (SD = 2 years who achieved MR4.5. 190 patients entered TFR phase.
- Key Results:
- After 96 weeks, 93 patients (48.9%) remained in TFR phase.
- 91 patients (47.9%) discontinued TFR due to loss of MMR.
- Of 88 patients who restarted treatment due to loss of MMR, 87 (98.9%) regained MMR, and 81 (92.0%) regained MR4.5.
- Cumulative rate of MMR regained at 24 weeks post-reinitiation: 97.7%. MR4.5 regained: 86.4%.
- ENESTop (CAMN107A2408):
- Study Type: Open-label, multicenter, single-arm study.
- Sample Size: 163 adult patients with Ph+ CML-CP on TKIs for >= 3 years (imatinib then nilotinib). 126 patients entered TFR phase.
- Key Results:
- After 96 weeks, 67 patients (53.2%) remained in TFR phase.
- 58 patients (46.0%) discontinued TFR due to loss of MMR or confirmed loss of MR4.0.
- Of 56 patients who restarted treatment, 52 (92.9%) regained MR4.0 and MR4.5.
- Cumulative rate of MR4.0 and MR4.5 regained by 48 weeks post-reinitiation: 92.9% and 91.1%, respectively.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
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Predicate Device(s)
QuantideX® qPCR BCR-ABL IS Kit
Reference Device(s)
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Predetermined Change Control Plan (PCCP) - All Relevant Information
Not Found
§ 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).
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December 22, 2017
MolecularMD Corporation Kevin Hawkins Director, Quality and Regulatory Affairs 1341 SW Custer Drive Portland, OR 97219
Re: K173492
Trade/Device Name: MolecularMD MRDx BCR-ABL Test Regulation Number: 21 CFR 866.6060 Regulation Name: BCR-ABL Quantitation Test Regulatory Class: Class II Product Code: OYX Dated: November 9, 2017 Received: November 13, 2017
Dear Kevin Hawkins:
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.
If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
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Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerely,
Reena Philip -S
Reena Philip, Ph.D. Director Division of Molecular Genetics and Pathology Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known) K173492
Device Name MRDx® BCR-ABL Test
Indications for Use (Describe)
The MolecularMD MRDx BCR-ABL Test is an in vitro diagnostic test for the quantitative detection of BCR-ABL 1 transcripts (e13a2/b2a2 and/or e14a2/63a2) and the ABL1 endogenous control mRNA in peripheral blood specimens from patients previously diagnosed with t(9:22) positive chronic myeloid leukemia (CML). The ratio of BCR-ABL1 is calculated and reported on the WHO International Scale. The test utilizes quantitative, real-time reverse transcription polymerase chain reaction performed on the Applied Biosystems 7500 Fast Dx instrument.
The MolecularMD MRDx BCR-ABL Test is intended to measure BCR-ABL mRNA transcript levels in patients diagnosed with t(9;22) positive CML during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).
The device is also intended to be used in the serial monitoring for BCR-ABL mRNA transcript levels as an aid in identifying CML patients in the chronic phase being treated with nilotinib who may be candidates for treatment discontinuation and for monitoring of treatment-free remission.
The test does not differentiate between e13a2 or e14a2 fusion transcripts and does not monitor other rare fusion franscripts resulting from t(9:22). The test is not intended for the diagnosis of CML.
| Type of Use ( Select one or both, as applicable ) | |
|---|---|
| Prescription Use (Part 21 CFR 801 Subpart D) | Over-The-Counter Use (21 CFR 801 Subpart C) |
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Image /page/3/Picture/0 description: The image shows the logo for MolecularMD. The logo consists of a red medical symbol on the left, followed by the text "MolecularMD" in gray and red. The "Molecular" part of the text is in gray, while the "MD" part is in red.
510(k) SUMMARY
l. COMPANY AND CONTACT INFORMATION
| Company Name: | MolecularMD Corporation |
|---|---|
| Address: | 1341 SW Custer Drive |
| Portland, OR 97219 | |
| Telephone: | 503.459.4974 |
| Fax: | 503.459.4976 |
| Contact Person: | Kevin Hawkins |
| Director, Quality and Regulatory Affairs | |
| Date of Summary: | 20 December 2017 |
II. DEVICE IDENTIFICATION
| Trade (Proprietary) Name: | MolecularMD MRDx® BCR-ABL Test |
|---|---|
| Common (Usual) Name: | BCR-ABL RT-qPCR Test |
| Classification Name: | BCR-ABL Quantitation Test |
| Product Code: | OYX |
| Regulation Number: | 866.6060 |
| Regulatory Classification: | Class II |
| Panel: | 88 – Pathology |
III. PREDICATE DEVICE
QuantideX® qPCR BCR-ABL IS Kit
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IV. DEVICE DESCRIPTION
A. Principle of the Procedure - Test Methodology
The MolecularMD MRDx® BCR-ABL Test is a quantitative, real-time polymerase chain reaction test that provides quantitation of BCR-ABL1 (hereafter BCR-ABL) transcript e13a2/b2a2 or e14a2/b3a2 and ABL1 (hereafter ABL) transcript levels in RNA extracted from peripheral blood samples collected from CML patients. Peripheral blood is collected in either EDTA or PAXgene Blood RNA Tubes. Each collection tube type requires a specified RNA extraction method and RNA input amount. The detailed extraction protocol and RNA quantity requirements are provided in the device package insert and instructions for use. Total RNA is extracted from peripheral blood and serves as the template for RT-qPCR. The test is performed using a onestep RT-qPCR protocol wherein the reverse transcription and quantitative, real-time PCR reactions are performed in the same well. BCR-ABL and ABL amplicons are generated and detected in real-time using TaqMan® MGB probes. For RNA extracted from EDTA tubes, a total of 4 µg of RNA is required for each patient sample: 1 µg of extracted RNA is used in the MRDx BCR-ABL Test in each of 4 wells, 2 wells for BCR-ABL quantitation and 2 wells for ABL quantitation. For RNA extracted from PAXgene tubes, a total of 8 µg of RNA is required for each patient sample: 2 µg of extracted RNA is used in the MRDx BCR-ABL Test in each of 4 wells, 2 wells for BCR-ABL quantitation and 2 wells for ABL quantitation.
The MolecularMD MRDx BCR-ABL Test is performed on the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument. The instrument integrates a thermal cycler, a fluorometer and application specific software. The instrument houses the thermal cycler and the fluorometer, while the application software is installed on a computer that is attached to the instrument. The ABI 7500 Fast Dx instrument software v1.4.1 calculates the BCR-ABL and ABL copy numbers using a standard curve generated with calibrators on each individual plate.
Quantitation is achieved using RNA calibration standards and linear regression analysis provided by the ABI 7500 Fast Dx PCR Instrument Software. BCR-ABL transcript levels are measured in relation to the ABL transcript as an endogenous reference. The data are exported as a CSV file for further analysis in the MRDx® BCR-ABL Test Software. The BCR-ABL/ABL ratio is calculated and converted to the International Scale by the MRDx BCR-ABL Test Software.
The MRDx BCR-ABL Test Software is used to analyze all test results. This software, provided with the MRDx BCR-ABL Test, is used to calculate the BCR-ABL/ABL % IS and MR value for patient sample using the conversion factor for the MRDx BCR-ABL Test after validating each MRDx BCR-ABL Test result against the run acceptance criteria and sample acceptance criteria.
Calibrators are analyzed for both BCR-ABL and ABL in every run simultaneously with patient samples. RNA controls provided at MMR (MR3.0) and MR4.5 allow for on-plate verification of
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test accuracy and reproducibility over this critical transcript range. The integrated conversion factor provides test results on the International Scale (IS) harmonized to the WHO.
Interpretation of Results
Test results are reported in both IS scale and the corresponding molecular response (MR) value. The BCR-ABL/ABL ratio percent IS result and MR value is calculated by the MRDx BCR-ABL Test Software using the following equations.
$$\frac{BCR - ABL}{ABL} ,% ,% , IS = \left(\frac{Mean,BCR - ABL,,Copy,Number}{Mean,,ABL,,Copy,Number}\right) \ast 100 ,*,Conversion,,Factor$$
$$MRx.x = \log_{10}\left(\frac{100}{9% IS}\right) = \log_{10}(100) - \log_{10}(9% IS) = 2 - \log_{10}(9% IS)$$
MR Values with the corresponding IS values are show in the table below:
| MR | IS (%) |
|---|---|
| 0.0 | 100 |
| 0.5 | 32 |
| 1.0 | 10 |
| 1.5 | 3.2 |
| 2.0 | 1.0 |
| 2.5 | 0.32 |
| 3.0 | 0.10 |
| 3.5 | 0.032 |
| 4.0 | 0.010 |
| 4.5 | 0.0032 |
| 5.0 | 0.0010 |
The assay reporting is shown in the table below. The assay will only report quantified results for BCR-ABL levels within the range of MR4.5 to MR1.0.
| Collection Tube | BCR-ABL% (MR) | MRDx Result |
|---|---|---|
| EDTA | 10% IS) | BCR-ABL detected, not quantifiable |
| EDTA | 1.0-4.5 | BCR-ABL detected |
| EDTA | 4.5-5.3 | BCR-ABL detected, not quantifiable |
| EDTA | >5.3 (10% IS) | BCR-ABL detected, not quantifiable |
| PAXgene | 1.0-4.5 | BCR-ABL detected |
| PAXgene | 4.5-4.7 | BCR-ABL detected, not quantifiable |
| PAXgene | >4.7 ( 0.05). When significant non-linear coefficients were observed, the Degree of Nonlinearity was calculated per CLSI EP6-A. The linear regression curves for both transcripts in the two collection tube types are shown in below followed by the estimated regression intercepts and slopes from the linear model. |
Image /page/19/Figure/6 description: The image contains two scatter plots titled "Linearity - EDTA Tubes". The left plot shows data for "1.0 ug RNA input EDTA, e13a2", while the right plot shows data for "1.0 ug RNA input EDTA, e14a2". Both plots depict "Molecular Response" on the y-axis and "Nominal Molecular Response" on the x-axis, with data points scattered along a generally linear trend.
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Image /page/20/Figure/1 description: The image contains two scatter plots comparing nominal molecular response to molecular response. Both plots show a linear relationship between the two variables. The left plot is labeled "2.0 µg RNA input PAXgene,e13a2", while the right plot is labeled "2.0 µg RNA input PAXgene,e14a2". Both plots show data points clustered around a line, indicating a strong correlation.
Regression Coefficients from Linear Model
| Transcript | Intercept | Slope | |||
|---|---|---|---|---|---|
| PAXgene | e14a2 | -0.045 | 0.054 | 0.98 | 0.017 |
| e13a2 | 0.012 | 0.026 | 0.93 | 0.008 | |
| EDTA | e14a2 | -0.016 | 0.037 | 0.96 | 0.011 |
| e13a2 | 0.059 | 0.046 | 0.92 | 0.015 |
Transcript e13a2 was linear from MR0.93 to MR5.1 with a maximum SD of 0.22 using EDTA tubes and was linear from MR0.78 to MR4.8 with a maximum SD of 0.13 using PAXgene tubes. Transcript e14a2 was linear from MR0.99 to MR5.0 with a maximum SD of 0.26 using EDTA tubes and was linear from MR0.93 to MR4.9 with a maximum SD of 0.31 using PAXgene tubes. The data support the assay's linearity across the indicated detection range.
| Linear Range | |||||
|---|---|---|---|---|---|
| Tube | |||||
| Type | BCR-ABL | ||||
| Transcript | Nominal | ||||
| Lower Limit | |||||
| BCR-ABL/ABL | |||||
| % IS | Nominal | ||||
| Lower Limit | |||||
| Molecular | |||||
| Response | Nominal | ||||
| Upper Limit | |||||
| BCR-ABL/ABL | |||||
| % IS | Nominal Upper | ||||
| Limit | |||||
| Molecular | |||||
| Response | |||||
| EDTA | e13a2 | 0.00080 | 5.1 | 12 | 0.93 |
| EDTA | e14a2 | 0.0010 | 5.0 | 10 | 0.99 |
| PAXgene | e13a2 | 0.0017 | 4.8 | 17 | 0.78 |
| PAXgene | e14a2 | 0.0012 | 4.9 | 12 | 0.93 |
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The linear range was truncated by the LoQ to determine the assay range. The data support the conclusion that the assay is linear across the reportable range of MR1.0 to MR4.5. The assay range for the MRDx BCR-ABL Test for each blood collection tube type are:
| Assay Range | ||
|---|---|---|
| Tube Type | Linear Range | |
| BCR-ABL/ABL % IS | Linear Range | |
| Molecular Response | ||
| EDTA | 10% to 0.0032% | MR1.0 to MR4.5 |
| PAXgene | 10% to 0.0032% | MR1.0 to MR4.5 |
10. Stability Studies
Kit Real-Time and Freeze-Thaw Stability:
The real-time stability of the MRDx BCR-ABL Test was evaluated at two storage temperatures (-30 to -15°C and -80 to -65°C) at periodic intervals with three lots. Six kits from each lot for each time point were tested using the final product release testing procedure. After use, the kits were frozen, thawed, and refrozen without testing to achieve an additional freeze/thaw cycle (Cycle 2). These six kits were then open-vial tested at one month (Test 2 and Cycle 3), and two months (Test 3 and Cycle 4) post-reconstitution using the WHO BCR-ABL Reference Panel Secondary Standards to ensure the kits were stable for three kit uses with three freeze/thaw cycles with 2 month stability. Kits were stored at -30 to -15°C after initial use for open-vial stability, regardless the original storage condition.
Image /page/21/Figure/7 description: The image shows a flowchart that describes the process of using manufactured kits. The process starts with manufactured kits, which are then thawed and used for reconstitution and initial use, labeled as "Test 1" and "Cycle 1". After this, the kits undergo a freeze-thaw cycle and are then labeled as "Cycle 2" without use of the kit. The kits then undergo another freeze-thaw cycle and are used a second time, labeled as "Test 2" and "Cycle 3", followed by a final freeze-thaw cycle and a third use, labeled as "Test 3" and "Cycle 4".
For each time point, the MRDx kit was tested for performance of the calibration curve criteria, the control results, and the WHO secondary standards. Passing test results data exist for at least 16 months for 3 lots of MRDx BCR-ABL Test stored at the two separate temperatures.
The data support a shelf life of 15 months at both storage temperatures (-30 to -15°C and -80 to -65°C) and an open vial stability of 3 freeze-thaw cycles and up to 2 months following initial use.
Reagent Preparation and Reaction Intermediate Stability (In Use):
This study demonstrated the MRDx BCR-ABL Test reagent stability during PCR plate preparation and the post-plate-assembly PCR reaction stability at 2 to 8°C before evaluation on the ABI 7500 Fast Dx instrument.
Both standard and extreme conditions were tested. The standard condition used a newly opened kit immediately after thawing to setup a PCR plate. The PCR plate was processed
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immediately after setup on the ABI 7500 Fast Dx. The extreme condition used a kit that was thawed and refrozen twice to simulate the third use of the kit. After the reagents were thawed, components were stored at 2 to 8°C for eight hours, and then the PCR plate was assembled and placed at 2 to 8 °C for 1.5 hours before evaluation on the ABI 7500 Fast Dx instrument. Two kits were run for each condition on each of 2 days with 2 sets of calibration curves, RNA controls, and samples (total n=8 per condition).
The acceptance criteria were that the mean of the test samples was ± 0.5 MR of the control. The acceptance criteria were met for the standard and the extreme conditions. The differences between the standard condition and the extreme condition were less than 0.25 logio for all levels of samples tested. The largest difference observed between conditions was -0.063 (95% Cl: -0.24 to 0.12) for the MR4.5 sample. These results support the stability conclusion that after 2 freeze-thaw cycles, the reagent is stable at 2 to 8°C for up to eight hours before PCR reagent assembly and up to 1.5 hours after PCR plate assembly.
Specimen Stability:
For PAXgene Blood RNA Tubes, samples at three levels (MR3.0, MR4.0, and MR4.5) were created, stored at 2 to 8°C and tested in quadruplicate at 0, 24, 48, and 60 hours. An additional series of samples was created, stored at -30 to -15°C, and tested in quadruplicate at 0, 14, 28, and 30 days. For EDTA blood collection tubes, 17 CML patient specimens covering the clinical decision range were collected and stored at 2 to 8°C and tested in quadruplicate at 0, 24, 48, and 60 hours.
All tested samples passed the acceptance criteria of 0.01% IS were considered having a confirmed loss of MR4.0, triggering re-initiation of nilotinib treatment. Patients with loss of MMR in the TFR phase immediately restarted nilotinib treatment without confirmation. All patients who restarted nilotinib therapy had BCR-ABL transcript levels monitored every 4 weeks for the first 24 weeks, then once every 12 weeks.
Efficacy was based on the 96-week analysis data cut-off date, by which time, 61 patients (48.4%) had discontinued from the TFR phase: 58 patients (46.0%) due to loss of MMR or confirmed loss of MR4.0, 2 patients (1.6%) due to subject/guardian decision and one patient (0.8%) due to pregnancy. Among the 58 patients who discontinued from the TFR phase due to confirmed loss of MR4.0 or loss of MMR, 56 patients restarted nilotinib therapy and 2 patients permanently discontinued from the study. The efficacy results are summarized in the table below.
| Patients who entered TFR phase (Full Analysis Set, N=126) | ||||
|---|---|---|---|---|
| Patients in TFR phase | Discontinuations due to | |||
| loss of MMR or confirmed | ||||
| loss of MR4 | Patients in | |||
| NTRI phase | ||||
| Time | n (%) | 95% CI | n (%) | n (%) |
| 24 weeks | 76 (60.3%) | (51.2, 68.9) | 49 (38.9%) | 48 (38.1%) |
| 48 weeks | 73 (57.9%) | (48.8, 66.7) | 53 (42.1%) | 51 (40.5%) |
| 96 weeks | 67 (53.2%) | (44.1, 62.1) | 58 (46.0%) | 56 (44.4%) |
Efficacy Results for ENESTop Study
Of the 56 patients who restarted nilotinib treatment due to confirmed loss of MR4.0 or loss of MMR in the TFR phase, 52 patients (92.9%) regained MR4.0 and MR4.5 and 4 patients (7.1%) did not regain MR4.0 by the time of the cut-off date. The cumulative rate of MR4.0 and MR4.5 regained by 48 weeks since treatment reinitiation, was 92.9% (52/56 patients) and 91.1% (51/56 patients), respectively.
Among the 126 patients in the TFR phase, 61 patients (48.4%) had a treatment-free survival (TFS) event (defined as discontinuation from TFR phase due to any reason, loss of MMR, confirmed loss of MR4.0, death due to any cause, progression to AP/BC up to the end of TFR phase, or reinitiation of treatment due to any cause in the study) on or before the 96-month cut-off date.
C. Conclusions Drawn from Preclinical and Clinical Studies
The effectiveness and clinical benefit of the MRDx® BCR-ABL Test was demonstrated in two prospective clinical trials utilizing the monitoring of BCR-ABL mRNA transcript levels in patients MolecularMD MRDx® BCR-ABL Test 510(k) Summary Page 24 of 25
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diagnosed with CML as an aid in identifying patients being treated with nilotinib as candidates for initiating treatment-free remission. Further, the monitoring of patients in treatment-free remission successfully met the intended use of identifying patients requiring reinitiation of nilotinib treatment. Analytical performance studies provide assurance of the accuracy and reliability of the quantitation of BCR-ABL transcript levels.
The data from these studies support the reasonable assurance of safety and effectiveness of the MRDx® BCR-ABL Test when used in accordance with the indications for use.