(44 days)
The Invader® UGTIA1 Molecular Assay is an in vitro diagnostic test for the detection and genotyping of the *1 (TA6) and *28 (TA7) alleles of the UDP glucuronosyltransferase 1A1 (UGTIA1) gene in genomic DNA from whole peripheral blood as an aid in the identification of patients with greater risk for decreased UDP-glucunorosyltransferase activity.
The Invader® UGTIA1 Molecular Assay is an in vitro diagnostic test which utilizes sequence specific Invader DNA probes, a structure-specific cleavage enzyme and a fluorescent resonance energy transfer (FRET) system combined with universal interpretative software and third party microtiter plate reader instrumentation. Invader® is the term used to generically refer to the patented chemistry on which the Invader® UGT A 1 Molecular Assay is based. The assay is designed to identify specific nucleic acid sequences and query for the presence of known sequence polymorphisms through the structurespecific cleavage of a series of probes that are specifically complementary to TA repeat sequences in in the "TATA Box" of of the UGT1A1 promoter region.
In the Invader® UGT1A1 Molecular Assay, two oligonucleotides (a discriminatory Primary Probe and an Invader® Oligo) hybridize in tandem to the target DNA to form an overlapping structure. The 5'-end of the Primary Probe includes a 5'-flap that does not hybridize to the target DNA. The 3'-nucleotide of the bound Invader® Oligo overlaps the Primary Probe, but need not hybridize to the target DNA. The Cleavase® enzyme recognizes this overlapping structure and cleaves off the unpaired 5'-flap of the Primary Probe, releasing it as a target-specific product. The Primary Probe is designed to have a melting temperature close to the reaction temperature. Therefore, under the isothermal assay conditions, Primary Probes, which are provided in excess, cycle on the target DNA. This allows for multiple rounds of Primary Probe cleavage for each target DNA, and amplification of the number of released 5'-flaps.
In the secondary reaction, each released 5'-flap can serve as an Invader® Oligo on a fluorescence resonance energy transfer (FRET) Cassette to create another overlapping structure that is recognized and cleaved by the Cleavase® enzyme. When the FRET Cassette is cleaved, the fluorophore and quencher are separated, generating detectable fluorescence signal. Similar to the initial reaction, the released 5'-flap and the FRET Cassette cycle, resulting in amplified fluorescence signal. The initial and secondary reactions run concurrently in the same well.
The biplex format of the Invader® UGT1A1 Molecular Assay enables simultaneous detection of two DNA sequences, a non-varying segment of the human alpha actin (ACTAI) gene and the TA repeat in the TATA box of the human UGTIA1 gene, in a single well. The biplex format uses two different discriminatory Primary Probes, each with a unique 5'-flap, and two different FRET Cassettes, each with a spectrally distinct fluorophore. By design, the released 5'-flaps will bind only to their respective FRET Cassettes to generate a target-specific signal.
The Invader® UGT1A1 Molecular Assay utilizes four independent wells per sample (one well for each of the TA Oligo mix reactions), to make a single genotype call. Each well contains a TATA box specific probe and an alpha actin probe. The alpha actin probe serves as an internal control to confirm the validity of a given result when a particular TATA box polymorphism is absent.
Here's a summary of the acceptance criteria and study details for the Invader® UGT1A1 Molecular Assay, based on the provided text:
Acceptance Criteria and Reported Device Performance
| Acceptance Criteria Category | Specific Metric | Acceptance Criteria (Implied) | Reported Device Performance |
|---|---|---|---|
| Limit of Detection | Lower Limit (Correct Detection) | 100% correct detection | 100% correct detection at 50 ng/reaction (95% CI: 92.8%) |
| Upper Limit (Genotype Call Agreement) | 100% agreement | 100% agreement at 80 ng/µL (95% CI: 97.5%) | |
| Genotype Detection | Agreement with Bi-directional DNA Sequencing | 100% agreement | 100% agreement for all genotypes (95% CI: 90.5% - 98.95%) |
| Repeat Rate | On first attempt | Low (quantitative not specified) | 0% (in initial study); 5.4% (in external reproducibility study) |
| Stability (Freeze-Thaw) | Agreement with known genotype | 100% agreement | 100% agreement for all samples after 1, 3, 5, 10 freeze-thaw cycles |
| Stability (Storage) | Agreement with bi-directional DNA sequencing | 100% agreement | 100% agreement at -20°C and simulated shipping stress (ongoing for 18 months) |
| Lot-to-Lot Reproducibility | Agreement with bi-directional DNA sequencing | 100% agreement | 100% agreement across three lots (95% CI: 92.8%) |
| Sample Preparation Equivalency | Agreement in genotype calls across different kits | 100% agreement | 100% agreement with two different Qiagen DNA purification kits |
| Reproducibility | Total Correct Genotype Call Rate (after 1st run) | High (quantitative not specified) | 93.3% (840/900) - 1st run (95% CI: 91.8%) |
| Total Correct Genotype Call Rate (combined 1st & 2nd run) | High (quantitative not specified) | 98.1% (883/900) - combined (95% CI: 97.2%) | |
| Interference Studies | Genotype Call Agreement (spiked vs. non-spiked samples) | 100% agreement | 100% agreement for bilirubin, lipids, EDTA, hemoglobin, 1% AW2; 1 incorrect call and 1 low signal for 5% AW2. |
Study Details:
-
Sample sizes used for the test set and the data provenance:
- Limit of Detection (Lower): 3 genomic DNA samples (TA6/6, TA6/7, TA7/7) concentrated at 50, 100, 150 ng DNA/µL. Total genotype calls for calculation: 120 (40 at each concentration for 3 genotypes).
- Limit of Detection (Upper): 3 genomic DNA samples (TA6/6, TA6/7, TA7/7) concentrated at 80 ng DNA/µL. Total genotype calls: 120.
- Genotype Detection: 285 blood samples.
- Repeat Rate (Initial): 285 blood samples.
- Repeat Rate (External Study): 20 samples, tested in triplicate at 3 sites on 5 separate days (900 possible genotype calls).
- Stability (Freeze-Thaw): 20 genomic DNA samples.
- Stability (Storage): 3 genomic DNA samples.
- Lot-to-Lot Reproducibility: 40 whole blood samples, analyzed with 3 different lots of reagents (120 data points).
- Sample Preparation Equivalency: 60 human genomic DNA samples.
- Reproducibility (Multi-site): 20 blood samples, tested across 3 sites (900 sample points generated).
- Interference Studies: 16 whole blood samples (for bilirubin, lipids, EDTA); 16 whole blood samples (for hemoglobin, AW2 buffer).
The data provenance is not explicitly stated as retrospective or prospective, nor is the country of origin mentioned for the samples. It is implied to be clinical samples, likely from a patient population relevant to UGT1A1 genotyping.
-
Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
- The ground truth for all performance studies (Limit of Detection, Genotype Detection, Stability, Lot-to-Lot Reproducibility, Sample Preparation Equivalency, Interference, Reproducibility) was established using bi-directional DNA sequence analysis.
- The document does not specify the number or qualifications of experts who performed or interpreted the bi-directional DNA sequencing. It is presented as a gold standard laboratory method.
-
Adjudication method for the test set:
- The document does not describe an adjudication method involving multiple human readers for discrepancies. The genotype calls from the Invader assay were directly compared to the results of bi-directional DNA sequencing.
- For the reproducibility study, where "misidentified" samples occurred at one site, it states "Discrepancy resolution by resequencing of the template in the original Invader assay confirmed that the genotypes present in the assay wells were consistent with the reported genotypes of the assay." This suggests a re-analysis by the measurement method itself rather than a formal expert adjudication panel.
-
If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:
- No, an MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic molecular assay, not an AI-powered diagnostic imaging tool that would typically involve human readers. The performance is evaluated against a gold standard molecular method (bi-directional DNA sequencing), not against human interpretation of images.
-
If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
- Yes, this entire submission focuses on the standalone performance of the Invader® UGT1A1 Molecular Assay, which is an algorithm-driven automated test. The "algorithm" here refers to the sequence-specific Invader DNA probes, cleavage enzyme, FRET system, and universal interpretative software that automates genotype calling. There is no human-in-the-loop performance described beyond standard laboratory handling and setup. The assay signal results are interpreted by a software program and assigned a genotype.
-
The type of ground truth used:
- The primary ground truth used for all performance evaluations was bi-directional DNA sequence analysis. This is a highly accurate molecular method for determining DNA sequences.
-
The sample size for the training set:
- The document does not mention a separate training set or training data used to develop the assay. The focus is on the performance data from non-clinical studies (validation studies) comparing the Invader assay results to bi-directional DNA sequencing. This suggests the assay's underlying chemistry and interpretive software were already developed and are being validated, rather than being developed using a specific training dataset in the context of machine learning.
-
How the ground truth for the training set was established:
- As no training set is explicitly described, the method for establishing its ground truth is not applicable or detailed in this document. The assay's fundamental design relies on established molecular biology principles and specific probe-target recognition, rather than learning from a large, pre-labeled training dataset in the way a machine learning algorithm would.
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AUG 1 8 2005
Invader® UGT1A1 Molecular Assay 510(k) Summary
Premarket Notification 510(k) Summary July 1, 2005
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TABLE OF CONTENTS
| 1. | I ABLE UI CUNTENTSSubmitter's Name, Address, Telephone Number, Contact Person, | |
|---|---|---|
| and date Summary was prepared | ||
| 2. Device Name | ||
| 3. Identification of the legally marketed device to which equivalence is claimed3 | ||
| 4. | Description of the Device | |
| 5. | Statement of the Intended Use of the Device | |
| 6(a) Summary of the Technological Characteristics of the Device Compared to the | ||
| Predicate Device | ||
| 6(b) Performance Data from the Non-Clinical Studies | ||
| 6(b)(1)(i) Limit of Detection | ||
| 6(b)(1)(ii)Genotype Detection | ||
| 6(b)(1)(iii) Repeat Rate | ||
| 6(b)(1)(iv) Stability (Freeze-Thaw) | ||
| 6(b)(1)(v) Stability (Storage) | ||
| 6(b)(1)(vi) Lot-to-Lot Reproducibility | ||
| 6(b)(1)(vii) Sample Preparation Equivalency | ||
| 6(b)(1){viii) Reproducibility | ||
| 6(b)(2) Interference Studies | ||
| 6(b)(3) Conclusions | ||
| 7. Clinical Validity |
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1. SUBMITTER'S NAME, ADDRESS, TELEPHONE NUMBER, CONTACT PERSON, AND DATE SUMMARY WAS PREPARED
Submitted by Third Wave Technologies, Inc.
502 S. Rosa Rd. Madison, WI 53719 888-898-2357 x2966
Contact Person: Sam Rua, RAC, Director of Regulatory Affairs and Quality Assurance
2. DEVICE NAME
Trade or Proprietary Name: Invader® UGT1A1 Molecular Assay Common or Usual Name: UGT1A1 Molecular Assay Classification Name: 21 CRF 862.3360 - Drug Metabolizing Enzyme Genotyping System
3. IDENTIFICATION OF THE LEGALLY MARKETED DEVICE TO WHICH IS CLAIMED EQUIVALENCE
The Invader® UGT1A1 Molecular Assay is equivalent to the AmpliChip CYP450 Test for CYP2C19 (510(k) Premarket Notification K043576, submitted December 03, 2004.
4. DESCRIPTION OF THE DEVICE
The Invader® UGTIA1 Molecular Assay is an in vitro diagnostic test which utilizes sequence specific Invader DNA probes, a structure-specific cleavage enzyme and a fluorescent resonance energy transfer (FRET) system combined with universal interpretative software and third party microtiter plate reader instrumentation. Invader® is the term used to generically refer to the patented chemistry on which the Invader® UGT A 1 Molecular Assay is based. The assay is designed to identify specific nucleic acid sequences and query for the presence of known sequence polymorphisms through the structurespecific cleavage of a series of probes that are specifically complementary to TA repeat sequences in in the "TATA Box" of of the UGT1A1 promoter region.
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In the Invader® UGT1A1 Molecular Assay, two oligonucleotides (a discriminatory Primary Probe and an Invader® Oligo) hybridize in tandem to the target DNA to form an overlapping structure. The 5'-end of the Primary Probe includes a 5'-flap that does not hybridize to the target DNA. The 3'-nucleotide of the bound Invader® Oligo overlaps the Primary Probe, but need not hybridize to the target DNA. The Cleavase® enzyme recognizes this overlapping structure and cleaves off the unpaired 5'-flap of the Primary Probe, releasing it as a target-specific product. The Primary Probe is designed to have a melting temperature close to the reaction temperature. Therefore, under the isothermal assay conditions, Primary Probes, which are provided in excess, cycle on the target DNA. This allows for multiple rounds of Primary Probe cleavage for each target DNA, and amplification of the number of released 5'-flaps.
In the secondary reaction, each released 5'-flap can serve as an Invader® Oligo on a fluorescence resonance energy transfer (FRET) Cassette to create another overlapping structure that is recognized and cleaved by the Cleavase® enzyme. When the FRET Cassette is cleaved, the fluorophore and quencher are separated, generating detectable fluorescence signal. Similar to the initial reaction, the released 5'-flap and the FRET Cassette cycle, resulting in amplified fluorescence signal. The initial and secondary reactions run concurrently in the same well.
The biplex format of the Invader® UGT1A1 Molecular Assay enables simultaneous detection of two DNA sequences, a non-varying segment of the human alpha actin (ACTAI) gene and the TA repeat in the TATA box of the human UGTIA1 gene, in a single well. The biplex format uses two different discriminatory Primary Probes, each with a unique 5'-flap, and two different FRET Cassettes, each with a spectrally distinct fluorophore. By design, the released 5'-flaps will bind only to their respective FRET Cassettes to generate a target-specific signal.
The Invader® UGT1A1 Molecular Assay utilizes four independent wells per sample (one well for each of the TA Oligo mix reactions), to make a single genotype call. Each well contains a TATA box specific probe and an alpha actin probe. The alpha actin probe serves
Premarket Notification 510(k) Summary July 1, 2005
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as an internal control to confirm the validity of a given result when a particular TATA box polymorphism is absent.
5. INTENDED USE OF THE DEVICE
The Invader® UGTIA1 Molecular Assay is an in vitro diagnostic test for the detection and genotyping of the *1 (TA6) and *28 (TA7) alleles of the UDP glucuronosyltransferase 1A1 (UGTIA1) gene in genomic DNA from whole peripheral blood as an aid in the identification of patients with greater risk for decreased UDP-glucunorosyltransferase activity.
6(a). SUMMARY OF THE TECHNOLOGICAL CHARACTERISTICS OF THE DEVICE COMPARED TO THE PREDICATE DEVICE
DE VICE Coint Averacteristics of the Invader® UGTIA1 Molecular Assay are
summarized in Table 1 (below):
| Technological Characteristics | ||
|---|---|---|
| AmpliChip CYP450 Test forCYP2C19 (K043576) | Invader® UGT1A1 MolecularAssay | |
| DNA sequencedetection | 1) Detects specific DNA sequencesthrough recognition of DNAtargets | 1) Same as predicate |
| Reactionconditions | 1) Utilizes thermal cycling | 1) No thermal cycling;isothermal reaction at 63° C |
| 2) Utilizes target DNAamplification | 2) Utilizes signal amplification | |
| 3) Reactions occur on a single glassslide | 3) Reactions occur in multipleplastic microtiter wells | |
| Assay results | 1) Assay signal results areinterpreted by a softwareprogram and are assigned agenotype that is presented to theend user in a report format. | 1) Same as predicate |
Table 1 Comparison of Technological Characteristics
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6(b). PERFORMANCE DATA FROM THE NON-CLINICAL STUDIES 6(b)(1), DISCUSSION OF NON-CLINICAL TESTS
6(b)(1)(i) Limit of Detection
The lower limit of detection of the Invader® UGTIA1 Molecular Assay was determined by analysis of dilutions of 3 genomic DNA samples at 50, 100 and 150 ng DNA/µL. The concentration of the DNA samples was determined by use of a PicoGreen® double stranded DNA Quantitation kit. The UGT1A1 genotypes in the DNA samples were 6/6, 6/7, and 7/7 as determined through bi-directional DNA sequence analysis. The lowest level of genomic DNA at which there was 100% correct detection of the UGTIA1 TATA box polymorphisms, as confirmed through the bi-directional DNA sequencing, was 50 ng/reaction. The 95% one-sided lower confidence limit was 92.8% (120/120 genotype calls).
The upper limit of detection of the Invader® UGT1A1 Molecular Assay was determined by analysis of three genomic DNA samples at 80 ng DNA/μL. The concentration of the DNA samples was determined by use of a PicoGreen® double stranded DNA Quantitation kit. The UGT1A1 genotypes in the DNA samples were 6/6, 6/7, and 7/7 as determined through bi-directional DNA sequence analysis. The genotype call agreement between the Invader® UGTIA1 Molecular Assay and the bi-directional sequencing result was 100%, (120/120 genotype calls) with the 95% one-sided lower confidence limit of 97.5%.
6(b)(1)(ii) Genotype Detection
Method comparison studies were performed using bi-directional DNA sequencing as the comparator for the Invader UGT1A1 Molecular Assay. Genotype detection was evaluated using genomic DNA samples at approximately 300 ng/reaction. The percent agreement for genotype detection of the Invader® UGT1A1 Molecular Assay was calculated by determining the percentage of tested samples with the correct genotype assigned as compared to the total number of samples tested of that genotype. The results for each UGT1A1 allele analyzed in this manner are presented in Table 2.
Premarket Notification 510(k) Summary July 1, 2005
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| UGT1A1Genotype* | Numbertested | Replicatesper sample | Number ofCorrectgenotypecalls | Number ofInvalidcalls | Agreement | 95% One-SidedConfidenceLower Limit |
|---|---|---|---|---|---|---|
| *1 (TA6/6) | 73 | 1 | 73 | 0 | 100% | 96.0% |
| *28 heterozygous(TA6/7) | 109 | 1 | 109 | 0 | 100% | 97.3% |
| *28 homozygous(TA7/7) | 30 | 1 | 30 | 0 | 100% | 90.5% |
| Others | 73 | 1 | 73 | 0 | 100% | 96.0% |
| Total | 285 | 1 | 285 | 0 | 100% | 98.95% |
® TICT1 A1 Molecular Assay and Bi-directional
▼ Calls produced on first run
6(b)(1)(iii). Repeat Rate
Two hundred eighty five (285) blood samples containing various UGT1A1 TATA box promoter region polymorphisms were tested (see Table 2). A correct genotype was obtained on the first attempt for all samples, yielding a repeat rate of 0%. Repeat rate performance results were also derived from the external study in which three sites extracted (in triplicate) the same 20 samples and analyzed each replicate on each of 5 separate days. Of the 900 possible genotype calls, valid calls were obtained on 851 sample points on the first attempt. This vields a repeat rate of 5.4% (see Table 3).
| Site | Number ofRepeats | Repeat Rate out of300 sample points |
|---|---|---|
| 1 | 28 | 9.3% |
| 2 | 0 | 0% |
| 3 | 21 | 7.0% |
| Total | 49 | 5.4% (n=900) |
Table 3. Invader® UGT1A1 Molecular Assay Repeat Rate for the Reproducibility External Study
6(b)(1)(iv). Stability (Freeze-Thaw)
The stability of the Invader UGT1A1 Molecular Assay was evaluated using identical sets of assay components that were subjected to multiple freeze-thaw cycles. All testing was performed using the same lots of reagents in the manufactured kit format, which includes all oligonucleotide mixtures, reaction buffer, and enzyme. Kits were subjected to differing numbers of freeze thaw cycles (1, 3, 5, 10) and evaluated using 20 genomic DNA samples Page 7 of 11 Premarket Notification 510(k) Summary July 1, 2005
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of known genotype, as determined though bi-directional DNA sequence analysis. There was 100% agreement between each freeze-thaw cycle for all samples tested.
6(b)(1)(v). Stability (storage)
The stability of the Invader® UGT1A1 Molecular Assay was evaluated using three (3) different genomic DNA samples with three different lots of assay components. The Invader® UGTIA1 Molecular Assay accurately identified the UGT1A1 genotypes as compared to bi-directional DNA sequencing when stored under the storage conditions specified on the label (-20°C). The assay also demonstrated 100% agreement with bidirectional DNA sequencing genotypes when stored outside of the recommended storage conditions, including a simulated shipping stress condition (37° C for 48 hours / -20° C for 19 days). The one month time point for intended storage conditions (-20℃) was successfully completed with 100% agreement between the Invader® UGT1A1 Molecular Assay and bi-directional DNA sequencing. Testing is ongoing to establish stability dating out to 18 months.
6(b)(1)(vi). Lot-to-lot reproducibility
Whole blood samples (n = 40) underwent DNA extraction and subsequent bi-directional DNA sequence analysis. The same DNA samples were then analyzed using the Invader® UGT1A1 Molecular Assay using three different lots of the reagents. The observed agreement between all three lots of the Invader® UGTIA1 Molecular Assay and bidirectional DNA sequencing was 100% (120/120). The 95% one-sided lower confidence limit was 92.8%.
6(b)(1)(vii). Sample preparation equivalency
The performance of the Invader® UGT1A1 Molecular Assay using genomic DNA samples extracted from two differing Qiagen DNA purification kits was evaluated. Sixty human genomic DNA samples of known genotype (confirmed through bi-directional DNA sequencing) were isolated from whole blood using both the Qiagen® QIAamp® 96 DNA Blood Kit and the Oiagen OlAamp DNA Blood Mini Kit and were tested with the Invader UGT1A1 Molecular Assay. There was 100% agreement in genotype calls between the two data sets.
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6(b)(1)(viii) Reproducibility
The reproducibility of the Invader® UGTIA1 Molecular Assay was assessed using twenty (20) blood samples comprised of the UGT1A1 genotypes listed in Table 4.
| UGT1A1Genotype¹ | # tested | 1st runtests per site | Site | 1st Run Results | # Invalidafter 2ndrun² | # Incorrectfor allruns | CorrectCall Ratefor allruns | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| GenotypeCalls | CorrectCalls | IncorrectCalls | #Invalid² | CorrectCall Rate | |||||||
| *1(TA6/6) | 6 | 90 | 1 | 82 | 793 | 33 | 8 | 87.8%3 | 2 | 33 | 94.4%3 |
| 2 | 90 | 90 | 0 | 0 | 100% | 0 | 0 | 100% | |||
| 3 | 84 | 84 | 0 | 6 | 93.3% | 1 | 0 | 98.9% | |||
| *28 hetero-zygous(TA6/7) | 5 | 75 | 1 | 69 | 69 | 0 | 6 | 92% | 0 | 0 | 100% |
| 2 | 75 | 75 | 0 | 0 | 100% | 0 | 0 | 100% | |||
| 3 | 70 | 70 | 0 | 5 | 93.3% | 1 | 0 | 98.7% | |||
| *28 homo-zygous(TA7/7) | 4 | 60 | 1 | 53 | 503 | 33 | 7 | 83.3%3 | 1 | 33 | 93.3%3 |
| 2 | 60 | 60 | 0 | 0 | 100% | 0 | 0 | 100.0% | |||
| 3 | 55 | 55 | 0 | 5 | 91.7% | 0 | 0 | 100.0% | |||
| Other | 5 | 75 | 1 | 68 | 633 | 53 | 7 | 84.0%3 | 1 | 53 | 92.0%3 |
| 2 | 75 | 75 | 0 | 0 | 100% | 0 | 0 | 100.0% | |||
| 3 | 70 | 70 | 0 | 5 | 93.3% | 0 | 0 | 100.0% | |||
| Total | 900 | 851 | 8403 | 113 | 49 | 93.3%3 | 6 | 113 | 98.1%4 |
Genotype determined using bi-directional sequencing
2 Invalid = reported to user as "Low Signal". Insufficient signal generated to make a genotype call
3 Nine samples were misidentified at site 1 on day 5. The data are reported using generype calls based on sample identification error.
4 Correct call rate for all runs is based on 883/900 genotype calls.
A total of 900 sample points were generated, 300 per site, with genotype results reported for 894 of the 900 sample points (99.3%). Correct genotype calls for UGT1A1 were obtained for 840/900 (93.3%) samples after the 15 run. The one-sided 95% confidence lower limit is 91.8%. After retesting 49 samples, 43/49 were correctly identified after the 2nd run. The total correct genotype calls for the combined first and second runs was 883/900 (98.1%, with a 1-sided 95% confidence lower limit of 97.2%). Nine samples were misidentified at site 1 on day 5. As a result, the 3 replicates for each sample produced genotype results that were inconsistent with the bi-directional sequencing result for those samples. Discrepancy resolution by resequencing of the template in the original Invader
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assay confirmed that the genotypes present in the assay wells were consistent with the reported genotypes of the assay.
From the 49 repeated genotype tests, 20 were due to an invalid Positive Control result, 20 were due to an invalid Negative Control result, and the remaining nine were due to "Low Signal" as a result in the 1st run. After retesting, 6 genotype reactions were still reported as "Low Signal".
6(b)(2). Interference Studies
Sixteen (16) whole blood samples were spiked with bilirubin (8mg/dL), lipids (mono-, di-, and triglycerides) (150mg/dL), and dipotassium EDTA (0.36mg/dL), underwent DNA extraction, and were tested with the Invader® UGTIA1 Molecular Assay. Genotype results were compared to those obtained from non-spiked samples, with 100% genotype call agreement between spiked vs. non-spiked samples. All genotype results were verified using bi-directional DNA sequencing.
Additionally, DNA extracts from 16 whole blood samples were spiked with hemoglobin (0.0125%), 1% Oiagen® AW2 buffer, and 5% Qiagen® AW2 buffer and tested with the Invader® UGTIA1 Molecular Assay. Genotype results were compared to those obtained from non-spiked samples. There was 100% genotype call agreement between spiked vs. non-spiked samples with the hemoglobin and 1% AW2 buffer interferants; 5% AW2 buffer resulted in 1 incorrect genotype call and 1 "low signal" result. All genotype results were verified using bi-directional DNA sequencing.
6(b)(3) Conclusions
The Invader® UGT1A1 Molecular Assay accurately identifies the UGT1A1 genotype of clinical specimens using DNA purified from human whole blood samples.
7. CLINICAL VALIDITY
Individual differences in drug response are common and variability in drug response among patients affects the disposition (absorption, distribution, metabolism and excretion) of a given drug. This is particularly true of drugs with a narrow therapeutic index. Sequence variations in genes involved in drug response have been shown to produce differences in
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drug disposition that alters the expected relationship between the dose of a drug and its concentration in the blood or the length of time it stays in the blood. A polymorphism of seven TA repeats TA(7) in the TATA box of the UGT1A1 promoter region has been found to produce reduced gene expression and reduced glucuronidation in human liver microsomes resulting in decreased drug metabolism and increased toxicity (see Table 6). Adjustment of drug dosage may be beneficial based upon knowledge of these differences in metabolism, particularly for individuals possessing the TA(7) genotype.
An FDA advisory committee assessed the scientific and clinical evidence correlating the UGTIA128 genotype with greater risk for irinotecan induced toxicity. Table 5 shows the prevalence of the UGTIA11 and UGT1A128 alleles in Caucasians and the associated risk of toxicity, based on a prospective study of 66 patients receiving irinotecan treatment (adapted from FDA's presentation to the advisory committee). Patients who are homozygous for the UGT1A128 allele are at 50% risk of experiencing toxicity.
| Group | Prevalence | Risk of Toxicity |
|---|---|---|
| All Patients | --- | 10% |
| Patients that are 7/7 | 10% | 50% |
| Patients that are 6/7 | 40% | 12.5% |
| Patients that are 6/6 | 50% | 0% |
Table 5. Summary of Allele Prevalence and Risk of Toxicity
The committee concluded that there is enough clinical and scientific evidence to correlate UGT1A128 genotype with increased risk for irinotecan toxicity. The updated CAMPTOSAR® (irinotecan) package insert recommends that individuals who are homozygous for the UGT1A128 allele are at an increased risk for neutropenia following initiation of CAMPTOSAR® treatment. A reduced initial dose should be considered for patients known to be homozygous for the UGT1A1*28 allele.
Table 6. UGT1A1 Aliele Frequency
| Allele | Population | ||
|---|---|---|---|
| Caucasian | Asian | African | |
| (TA)6 | 61.3% | 84% | 47% |
| (TA)7 | 38.7% | 16% | 42.6% |
Premarket Notification 510(k) Summary July 1, 2005
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Public Health Service
Food and Drug Administration 9200 Corporate Boulevard Rockville MD 20850
Mr. Sam Rua Third Wave Technologies, Inc. 502 South Rosa Road Madison, WI 53719-1256
AUG 18 2005
Re: K051824
Trade/Device Name: Invader® UGT1A1 Molecular Assay Regulation Number: 21 CFR 862.3360 Regulation Name: Drug metabolizing enzyme genotyping system Regulatory Class: Class II Product Code: NTI Dated: August 11, 2005 Received: August 15, 2005
Dear Mr. Rua:
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.
If vour device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. 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 Parts 801 and 809); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820).
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This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.
If you desire specific information about the application of labeling requirements to your device, or questions on the promotion and advertising of your device, please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 594-3084. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 443-6597 or at its Internet address http://www.fda.gov/cdrh/dsma/dsmamain.html.
Sincerely yours,
Carol C. Benson
Carol C. Benson, M.A. Acting Director Division of Chemistry and Toxicology Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known): K051824
Device Name: Invader® UGT1A1 Molecular Assay
Indications For Use:
The Invader® UGT1A1 Molecular Assay is an in vitro diagnostic test for the detection and genotyping of the *1 (TA6) and *28 (TA7) alleles of the UDP glucuronosyltransferase 1A1 (UGT1A1) gene in genomic DNA from whole peripheral blood as an aid in the identification of patients with greater risk for decreased UDPglucunorosyltransferase activity.
Prescription Use (Part 21 CFR 801 Subpart D) AND/OR
Over-The-Counter Use (21 CFR 801 Subpart C)
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Office of In Vitro Diagnostic Device Evaluation and Safety
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510(k) K051824
§ 862.3360 Drug metabolizing enzyme genotyping system.
(a)
Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA) extracted from clinical samples to identify the presence or absence of human genotypic markers encoding a drug metabolizing enzyme. This device is used as an aid in determining treatment choice and individualizing treatment dose for therapeutics that are metabolized primarily by the specific enzyme about which the system provides genotypic information.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: Drug Metabolizing Enzyme Genotyping Test System.” See § 862.1(d) for the availability of this guidance document.