(216 days)
No
The description mentions a "proprietary algorithm" and a "multi-step process" for data analysis, but there is no explicit mention of AI or ML techniques being used. The analysis appears to be based on comparing sequences to a database and quantifying known clonotypes, which is a rule-based or statistical approach rather than AI/ML.
No
The device is an in vitro diagnostic (IVD) assay designed to measure minimal residual disease (MRD) by detecting and quantifying specific gene sequences. It provides information for clinical decision-making and monitors changes in the burden of disease but does not directly treat or prevent disease.
Yes
The clonoSEQ Assay is explicitly stated to be "an in vitro diagnostic" and "measures minimal residual disease (MRD) to monitor changes in burden of disease during and after treatment," indicating its use in diagnosing and monitoring disease states.
No
The device is an in vitro diagnostic assay that involves physical sample processing (DNA extraction, PCR, NGS) and analysis, not solely software.
Yes, this device is an IVD (In Vitro Diagnostic).
The very first sentence of the "Intended Use / Indications for Use" section explicitly states: "The clonoSEQ Assay is an in vitro diagnostic..."
Furthermore, the description of the device and its intended use aligns with the definition of an IVD:
- In vitro: The test is performed on biological samples (DNA extracted from bone marrow and blood) outside of the living organism.
- Diagnostic: The test is used to identify and quantify specific genetic sequences related to certain cancers (B-cell ALL, multiple myeloma, and CLL) to monitor disease burden and aid in clinical decision-making.
N/A
Intended Use / Indications for Use
The clonoSEQ Assay is an in vitro diagnostic that uses multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to identify and quantify rearranged IgH (VDJ), IgH (DJ), IgK and IgL receptor gene sequences, as well as translocated BCL1/1gH (J) and BCL2/1gH (J) sequences in DNA extracted from bone marrow from patients with B-cell acute lymphoblastic leukemia (ALL) or multiple myeloma (MM), and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL).
The clonoSEQ Assay measures minimal residual disease (MRD) to monitor changes in burden of disease during and after treatment. The test is indicated for use by qualified healthcare professionals in accordance with professional guidelines for clinical decision-making and in conjunction with other clinicopathological features.
Product codes (comma separated list FDA assigned to the subject device)
QDC
Device Description
The clonoSEQ Assay is a next-generation sequencing (NGS) based assay that identifies rearranged IgH (VDJ), IgH (DJ), IgK, and IgL receptor gene sequences, as well as translocated BCL1/IgH (J) and BCL2/IgH (J) sequences. The assay also includes primers that amplify specific genomic regions present as diploid copies in normal genomic DNA (gDNA) to allow determination of total nucleated cell content.
Testing begins with gDNA extracted from the specimen supplied (Figure 1). Extracted gDNA quality is assessed and rearranged immune receptors are amplified using a multiplex PCR. Reaction-specific index barcode sequences for sample identification are added to the amplified receptor sequences by PCR. Sequencing libraries are prepared from barcoded amplified DNA, which are then sequenced by synthesis using NGS. Raw sequence data are uploaded from the sequencing instrument to the Adaptive analysis pipeline. These sequence data are analyzed in a multi-step process: first, a sample's sequence data are identified using the sample index sequences. Next, data are processed using a proprietary algorithm with in-line controls to remove amplification bias. When the clonoSEQ Clonality (ID) assessment is conducted, the immune repertoire of the sample is checked for the presence of DNA sequences specific to "dominant" clone(s) consistent with the presence of a lymphoid malignancy. Each sequence that is being considered for MRD tracking is compared against a B cell repertoire database and assigned a uniqueness value that, together with its abundance relative to other sequences, is used to assign the sequence to a sensitivity bin which will be used in the estimation of the reported LoD and LoO on the patient report. During clonoSEQ Tracking (MRD) assessment, the complete immunoglobulin receptor repertoire is again assessed, and the previously identified dominant clonotype sequence(s) are detected and quantified to determine the sample MRD level. The clonoSEQ Assay MRD assessment measures residual disease in a biologic sample.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Bone marrow, Blood
Indicated Patient Age Range
Not Found
Intended User / Care Setting
Qualified healthcare professionals in accordance with professional guidelines for clinical decision-making and in conjunction with other clinicopathological features.
Single-site assay performed at Adaptive Biotechnologies Corporation in Seattle, Washington.
Description of the training set, sample size, data source, and annotation protocol
Not Found
Description of the test set, sample size, data source, and annotation protocol
Not Found
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Precision Studies (Blood and Bone Marrow)
- Study Type: Main effects screening design over 21 calendar days.
- Sample Size:
- BMA: 22 patients with CLL, 360 contrived samples (340 remaining after QC failures), 7,480 MRD measurements.
- Blood: 15 patients with CLL, 320 contrived samples (319 remaining after QC failures), 4,785 MRD measurements.
- Key Results:
- Precision of MRD Frequency for CLL in BMA: Ranged from 18.5% to 60.1% CV. The precision is largely dependent on the number of malignant cells being evaluated.
- Precision of MRD Frequency for CLL in blood: Ranged from 18.7% to 54.9% CV, comparable to BMA samples.
- Precision of Malignant Cells Detected in BMA: Ranged from 59% CV at 2.14 cells to 20% CV at 612.56 cells. Tested factors (Operator, Instrument Sets, Reagent Lots, Day, and Run) minimally contributed to variability (0% to 5%).
- Precision of Malignant Cells Detected in CLL blood: Ranged from 53% CV at 3.10 cells to 19% CV at 765.70 cells. DNA input mass contributed 8% to 10% to variability, while pre-specified test factors contributed minimally (0% to 2%).
- The precision of the clonoSEQ Assay is largely dependent on the number of malignant cells that are being evaluated by the assay.
Linearity Studies (Cell Lines and Clinical Samples)
- Study Type: Evaluation of MRD frequency across different DNA inputs.
- Sample Size:
- Cell Lines: 3 CLL cell lines (HG-3, MEC-1, PGA-1).
- Clinical BMA Specimens: 22 CLL specimens (from precision study).
- Clinical Blood Specimens: 15 CLL specimens (from precision study).
- Key Results:
- Linearity was established across the entire tested range for both cell line and clinical samples.
- Demonstrated linearity of MRD frequencies across several orders of magnitude.
- Linear range was determined by finding the input range where the maximum deviation from linearity was less than 5%.
Accuracy (Concordance with mpFC in Blood Clinical Samples)
- Study Type: Comparison of clonoSEQ Assay results to multiparameter flow cytometry (mpFC).
- Sample Size: 299 matched samples.
- Key Results:
- Positive Percent Agreement (PPA): 98.9% (95% CI: 94.3%-100%).
- Negative Percent Agreement (NPA): 47.5% (95% CI: 40.5%-54.6%).
- High concordance of positive calls and higher sensitivity of clonoSEQ Assay compared to mpFC.
Analytical Sensitivity (Limit of Blank, Limit of Detection, Limit of Quantitation)
- Study Type: Determination of LoB, LoD, and LoQ.
- Sample Size:
- LoB (Bone Marrow): 22 CLL patients, healthy bone marrow samples.
- LoB (Blood): 15 CLL patients, healthy blood samples.
- LoD/LoQ (Bone Marrow): 22 CLL specimens.
- LoD/LoQ (Blood): 15 CLL specimens.
- Key Results:
- LoB: Zero for all sample types and disease indications (trackable Ig sequences are highly patient-specific).
- LoD (BMA): 1.630 malignant cells (95% CI; 1.42 - 1.87).
- LoQ (BMA): 2.427 malignant cells (95% CI; 1.63 - 8.91).
- LoD/LoQ (Blood): Comparable to previously determined LoD/LoQ in bone marrow (DEN170080) and were lower or within the 95% CI of bootstrapped prior data.
Analytical Specificity (Interfering Substances)
- Study Type: Characterization of effects of endogenous and exogenous substances.
- Sample Size: 4 different donors for each endogenous and exogenous substance test.
- Key Results:
- All tested endogenous (Bilirubin conjugated, Bilirubin unconjugated, Hemoglobin, Cholesterol, Triglycerides) and exogenous (K2EDTA, K3EDTA, Heparin, Chloroform) substances did not substantially influence MRD results.
- Mean MRD frequency difference remained > -30% and 10-5).
- Continuous MRD values significantly associated with PFS (likelihood ratio P = 2.96 x 10-2); each 10-fold increase in MRD level associated with 2.35-fold increase in event risk.
- MRD level at FUM3 was a stronger predictor of PFS than other prognostic variables.
- Patients with MRD ≤ 10-6 or between 10-6 and 10-5 had longer PFS.
- NCT00759798:
- Significant association between PFS and continuous clonoSEQ MRD measurement in both blood (p = 9.66E-04) and bone marrow (p = 2.13E-04) after end of treatment.
- MRD negative patients ( 10-4) (p = .02 for blood and p = 8.17E-05 for bone marrow).
- Conclusion: MRD negativity as measured by clonoSEQ Assay was associated with improved patient outcomes in CLL, supporting its use for MRD measurement and monitoring changes in disease burden.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
Accuracy (Concordance with mpFC in Blood Clinical Samples)
- PPA: 98.9%
- NPA: 47.5%
Analytical Sensitivity (Limit of Detection/Limit of Quantitation)
- LoD (BMA): 1.630 malignant cells (95% CI; 1.42 - 1.87)
- LoQ (BMA): 2.427 malignant cells (95% CI; 1.63 - 8.91)
Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.
Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).
Not Found
§ 866.6100 DNA-based test to measure minimal residual disease in hematological malignancies.
(a)
Identification. A DNA-based test to measure minimal residual disease in hematological malignancies is a prescription in vitro diagnostic device that identifies and quantifies specific nucleic acid sequences within human tissues to estimate the percentage of cells that harbor the specific sequence(s). The test is intended to be used as an aid to measure minimal residual disease to assess the change in burden of disease during monitoring of treatment. The test is indicated for use by qualified healthcare professionals in accordance with professional guidelines for clinical decision-making, in conjunction with other clinicopathological features.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Design verification and validation must include:
(i) A detailed description of the device, including:
(A) A detailed description of all test components, reagents, instrumentation, and software, including software applications and any hardware-based devices that incorporate software.
(B) A detailed description of all genomic regions that are detected and quantified by the assay.
(C) A detailed description of the methodology and protocols for each step of the test, including description of the quality metrics, thresholds, and filters at each step of the test that are implemented for final result reporting and a description of the metrics for run-failures, specimen-failures, and invalids, as appropriate.
(D) Detailed specifications and procedures for sample collection, processing, and storage.
(E) A description of the internal and external controls that are recommended or provided. The description must identify those control elements that are incorporated into the testing procedure. If appropriate, this description must include a description of the controls and control procedures used during the sequencing and data analysis.
(ii) 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 use of the device.
(iii) As part of the risk management activities, an appropriate end user device training program must be offered as an effort to mitigate the risk of failure from user error, as appropriate.
(iv) Description of analytical and clinical studies, including:
(A) Device performance data that demonstrates the ability to measure minimal residual disease in the claimed specimen type(s) from patients that are representative of the intended use population. Data can be obtained via:
(
1 ) A method comparison study comparing the device to a predicate device with clinical data for the specified hematological neoplastic indication using the specified specimen type(s); or(
2 ) A clinical study demonstrating clinical validity using well characterized clinical specimens from patients with known clinical outcomes using a study design deemed acceptable by FDA.(B) Device precision (repeatability and reproducibility) data using clinical samples covering the range of minimal residual disease frequencies reported by the test and covering the stated range of DNA inputs that are indicated as allowable for use with the test. Results shall be reported as the standard deviation and/or percentage coefficient of variation with the 95 percent confidence interval for each level tested. The study must evaluate all sources of variability, including, as appropriate, between-site and between operator (minimum of three sites of which two must be external with a minimum of two operators per site), between-day (minimum of 3 days), between-run, within-run, between-lot (minimum of three lots), between instrument (minimum of three instruments), and total variation.
(C) Device linearity data generated from samples covering the device measuring range using a dilution panel created from clinical samples.
(D) Device accuracy by comparison to flow cytometry across the measuring interval or to the predicate method across the measuring interval.
(E) Device analytic sensitivity data, including limit of blank, limit of detection, and limit of quantitation, using a dilution panel created from clinical samples.
(F) Analytical specificity data, including interference and cross-contamination, and index cross-contamination, as appropriate.
(G) Validation of pre-analytical methods, including DNA extraction methods and cell enrichment methods, as appropriate.
(H) Device stability data, including real-time stability of reagents under various storage times and temperatures.
(I) Specimen and prepared sample stability data established for each specimen matrix in the anticoagulant combinations and storage/use conditions that will be indicated, including specimen transport, as appropriate.
(2) The intended use for the labeling required under § 809.10(a)(4) of this chapter and for the labeling required under § 809.10(b)(5)(ii) of this chapter, as applicable, must include:
(i) The clinical hematopoietic malignancy for which the assay was designed and validated (
e.g., multiple myeloma or B-cell acute lymphoblastic leukemia);(ii) Specimen type (
e.g., bone marrow);(iii) The specific DNA regions that are being identified and quantified (
e.g., rearranged IgH (VDJ), IgH (DJ), IgK, and IgL receptor gene sequences); and(iv) A statement that the results are indicated to be interpreted by qualified healthcare professionals in accordance with professional guidelines for clinical decision-making in conjunction with other clinicopathological features.
(3) The labeling required under § 809.10(b) of this chapter must include information that demonstrates the performance characteristics of the test, including a detailed summary of the performance studies conducted and their results, as described in paragraphs (b)(1)(iv)(A) through (I) of this section.
(4) The device output, including any test report, must include the estimated minimal residual disease (MRD) frequency and an appropriate range of the uncertainty of that frequency based on the amount of DNA that was evaluated by the test and the number of specific nucleic acid sequences that were detected (
e.g., “MRD = 1.2 × 10−5 [Range = 0.8 × 10−6 to 2.0 × 10−5 ]”).
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August 5, 2020
Adaptive Biotechnologies Corporation c/o Pamela Swatkowski Consultant, Regulatory Affairs 1551 Eastlake Ave E., Ste 200 Seattle, Washington 98102
Re: K200009
Trade/Device Name: Adaptive Biotechnologies clonoSEO Assay Regulation Number: 21 CFR 866.6100 Regulation Name: DNA-based test to measure minimal residual disease in hematological malignancies Regulatory Class: Class II Product Code: QDC Dated: December 30, 2019 Received: January 2, 2020
Dear Pamela Swatkowski:
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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmp/bmn.cfm identifies combination product submissions. 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) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see
1
https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.
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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.
For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). 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 (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE(@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerely,
Donna M. Roscoe -S
Donna Roscoe, Ph.D. Acting Deputy Director Division of Molecular Genetics and Pathology OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health
Enclosure
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Indications for Use
510(k) Number (if known) K200009
Device Name The clonoSEQ® Assay
Indications for Use (Describe)
The clonoSEQ Assay is an in vitro diagnostic that uses multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to identify and quantify rearranged IgH (VDJ), IgH (DJ), IgK and IgL receptor gene sequences, as well as translocated BCL1/1gH (1) and BCL2/1gH (1) sequences in DNA extracted from bone marrow from patients with B-cell acute lymphoblastic leukemia (ALL) or multiple myeloma (MM), and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL).
The clonoSEQ Assay measures minimal residual disease (MRD) to monitor changes in burden of disease during and after treatment. The test is indicated for use by qualified healthcare professionals in accordance with professional guidelines for clinical decision-making and in conjunction with other clinicopathological features.
The clonoSEQ Assay is a single-site assay performed at Adaptive Biotechnologies Corporation.
| 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 Adaptive Biotechnologies. The logo features the word "Adaptive" in a bold, sans-serif font, with a stylized DNA helix incorporated into the letter "A". Below the word "Adaptive" is the word "biotechnologies" in a smaller, lighter font. The logo is simple and modern, and the use of the DNA helix suggests that the company is involved in the field of biotechnology.
1551 Eastlake Ave E, Ste 200 Seattle, WA 98102
206.659.0067 adaptivebiotech.com
510(k) Summary
Submitted By:
Adaptive Biotechnologies Corporation 1551 Eastlake Ave E, Ste 200 Seattle, WA 98102 Ph: 888-552-8988 Fax: 866-623-4408
Company Contact:
Pamela Swatkowski Consultant, Regulatory Affairs (630).430.5095 pswatkowski@adaptivebiotech.com
Date Prepared: August 5, 2020
Predicate Trade/Device Name:
Adaptive Biotechnologies clonoSEQ® Assay
Regulation Number:
21 CFR 866.6100
Classification and Regulation Name:
DNA-based test to measure minimal residual disease in hematological malignancies (21 CFR 866.6100, Product Code QDC)
Purpose of the Submission:
Traditional 510(k) request for expansion of indications for use for the Adaptive clonoSEQ® Assay
Manufacturer:
Adaptive Biotechnologies is the legal manufacturer of the Adaptive clonoSEQ® Assay. The Adaptive clonoSEO® Assay is intended as a single laboratory service, performed at Adaptive Biotechnologies' single site located at 1551 Eastlake Ave E, Ste 200, Seattle, WA 98102.
Common Name:
DNA-based test to measure minimal residual disease in hematological malignancies
Predicate Device:
Adaptive Biotechnologies clonoSEQ® Assay
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DEVICE DESCRIPTION
Sample Preparation
The minimum gDNA sample input requirement is 500 ng. Shipment of 1 mL of bone marrow aspirate (BMA) is requested to obtain sufficient extracted gDNA and extraction methods have been validated using volume ranging from 250 µL to 1 mL. Shipment of 2 mL of whole blood is requested to obtain sufficient extracted gDNA and extraction methods have been validated using volumes ranging from 500 uL to 2 mL. For both sample types, the extraction method isolates gDNA by first lysing cells and denaturing proteins after which the DNA is bound to a substrate. Once the DNA is bound, a series of wash steps removes impurities. Following the wash steps the DNA is eluted from the substrate. DNA is quantified using a spectrophotometer; the measured DNA concentration is used to add a target of 20 ug of amplifiable gDNA to the assay. The MRD test can be performed with 500 ng – 40 µg gDNA. Internal controls in the PCR and sequencing steps are used to confirm that sufficient gDNA has been amplified and that amplification was successful.
Library Preparation
Genomic DNA is amplified using locus-specific multiplex PCR using V, D and J gene primers containing molecular barcodes to amplify IgH (VDJ), IgH (DJ), IgK, IgL, BCL1/IgH (J), BCL2/IgH (I) and housekeeping gene (HKG) sequences. Reaction-specific index barcodes for sample identification are added to the amplified receptor sequences by PCR. Sequencing libraries are prepared by pooling barcoded amplified DNA. qPCR is used to verify the adequacy of the pooled amplified DNA library concentration.
Sequencing and Data Analysis
Sequencing is conducted with the Illumina NextSeq™ 500 or 550 Systems. The sequencing process incorporates multiple quality checks. Sequencing output is then processed by the bioinformatics pipeline software as follows:
Flowcell Level Metrics
The analysis pipeline performs quality control (OC) checks on the flowcell data. The pipeline evaluates the percentage of reads that pass the Illumina quality filter (%PF), which must be greater than 70% of reads. The system uses spike-in PhiX templates to evaluate the error rate. The pipeline evaluates the proportion of PhiX reads, which must be greater than 2%, and the associated error rate as computed by the Illumina RTA software, which must be less than 3%.
Demultiplexing and FASTQ Generation
The pipeline uses Illumina software to demultiplex reads from the instrument output run folder. The analysis pipeline performs a QC check to evaluate whether unexpected barcodes are observed and raises a flag if more than 30,000 reads carry a barcode not specified in the input sample sheet.
Read Assignment to Receptors
The pipeline assigns reads to rearranged receptors for each sample after demultiplexing.
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Clonal Sequence Determination
After assigning reads to receptor loci, the pipeline then clusters reads into clonal receptor sequences.
Sample Level QC Checks
The pipeline performs a series of sample level OC checks: assessment that sequencing data is sufficient and acceptable based on amplification of sets of internal synthetic controls, assessment that sufficient gDNA is sampled, and a final screen of the calculated values for biologic relevance. One set of internal synthetic controls are evaluated for sufficient read quantity per molecule and read coverage across receptor loci. Another set of internal controls' presence or absence is used to screen for the expected degradation of residual primers. The estimated mass of input gDNA based on an optical density measurement and the estimated number of sampled nucleated cells based on amplification of a set of internal reference genes are used as metrics to check if sufficient material is sampled. The pipeline also checks that the detected numbers of total and B cells are within a biologically relevant range, and screens for clone sharing by evaluating if sequences are shared across samples that are processed together.
Calibrations
Clonal sequences are assessed for their suitability as ID sequences (to be used for subsequent tracking) by first aggregating highly similar sequences and requiring that the frequency of the sequence is at least 3% as a percentage of all sequences in the locus. The clone must also have a frequency of at least 0.2% of all nucleated cells in the sample and must have sufficient abundance and differentiation from a polyclonal background. Each sequence that is being considered for MRD tracking is compared against a B cell repertoire database and assigned a uniqueness value that, together with its abundance relative to other sequences, is used to assign the sequence to a sensitivity bin which will be used in the estimation of the reported limit of detection (LoD) and limit of quantitation (LoQ).
Tracking
When a previous calibration test has identified suitable ID sequences for tracking, they are compared to sequences in the most recent tracking sample in order to assess residual disease. After approximate matching, which allows for mutations in the sample clones as compared to the ID sequences, sequence proportions in the sample are assessed and compared to the LoQ values. The analysis pipeline then reports whether ID sequences were detected above the LoQ, above LoD but below LoQ, below LoD, or not detected.
Control Materials
The following controls are used to measure the success of DNA extraction, PCR amplification and sequencing:
Synthetic Internal Controls
Each sample includes two sets of internal synthetic controls are panels of synthetic analogues of somatically rearranged B-cell receptor (BCR) immune receptor molecules. The composition of the reference template pools before and after amplification is measured and used for QC. One set of synthetic templates is added to every pre-amp PCR well as a positive control; these synthetic templates are used to measure primer performance, including identification and correction of amplification bias, and to screen for sufficient sequencing coverage. Another set is added after a step
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used to remove residual primers; the lack of amplification of these molecules is used to confirm the success of primer removal.
DNA Extraction Process Controls
Each extraction is performed with Positive and Negative Extraction Controls. The Extraction Negative Control is used to confirm lack of contamination during the extraction process. The Extraction Negative Control is subsequently amplified and sequenced in the same fashion as test samples. The Extraction Positive Control is included to assess effectiveness of the extraction process (it is required to be above a pre-set threshold for DNA recovery). If readily available, source material for Extraction Positive Controls is matched to the specimen source type. Exception: The Extraction Positive Control for bone marrow specimens consists of frozen human whole blood.
PCR Amplification Process Controls
Each PCR amplification is performed with an Amplification Positive and Negative Control and subsequently sequenced in the same manner as test samples. The Amplification Positive Control consists of gDNA derived from blood mononuclear cells (PBMCs) and serves as an additional check to confirm successful product amplification. Buffer (1x TE) is used as the negative control.
Sequencing Process Controls
To every sequencing flow cell, two sequencing controls are added. Both a PhiX control purchased from Illumina and a well-characterized amplified library (Sequencing Positive Control) are loaded with test samples.
Result Reporting
The pipeline renders results into a PDF-formatted patient report displays any ID sequences identified in the sample that can be used for tracking with their quantitation and sample-level metrics. For tracking tests, the report includes a result (ID sequences detected above LoD, below LoD, or not detected) and quantitation for the tracked sequences within the most recent sample.
Test Components
All reagents, materials, and equipment needed to perform the assay, with the exception of sample collection materials, are used exclusively at the Adaptive Biotechnologies single laboratory site. The clonoSEO Assay is intended to be performed with serial number-controlled instruments. An ambient temperature sample shipper kit is available for use through Adaptive Clinical Services Team if requested by the ordering healthcare provider.
Test Principle
The clonoSEQ Assay is a next-generation sequencing (NGS) based assay that identifies rearranged IgH (VDJ), IgH (DJ), IgK, and IgL receptor gene sequences, as well as translocated BCL1/IgH (J) and BCL2/IgH (J) sequences. The assay also includes primers that amplify specific genomic regions present
7
as diploid copies in normal genomic DNA (gDNA) to allow determination of total nucleated cell content.
Testing begins with gDNA extracted from the specimen supplied (Figure 1). Extracted gDNA quality is assessed and rearranged immune receptors are amplified using a multiplex PCR. Reaction-specific index barcode sequences for sample identification are added to the amplified receptor sequences by PCR. Sequencing libraries are prepared from barcoded amplified DNA, which are then sequenced by synthesis using NGS. Raw sequence data are uploaded from the sequencing instrument to the Adaptive analysis pipeline. These sequence data are analyzed in a multi-step process: first, a sample's sequence data are identified using the sample index sequences. Next, data are processed using a proprietary algorithm with in-line controls to remove amplification bias. When the clonoSEQ Clonality (ID) assessment is conducted, the immune repertoire of the sample is checked for the presence of DNA sequences specific to "dominant" clone(s) consistent with the presence of a lymphoid malignancy. Each sequence that is being considered for MRD tracking is compared against a B cell repertoire database and assigned a uniqueness value that, together with its abundance relative to other sequences, is used to assign the sequence to a sensitivity bin which will be used in the estimation of the reported LoD and LoO on the patient report. During clonoSEQ Tracking (MRD) assessment, the complete immunoglobulin receptor repertoire is again assessed, and the previously identified dominant clonotype sequence(s) are detected and quantified to determine the sample MRD level. The clonoSEQ Assay MRD assessment measures residual disease in a biologic sample.
Figure 1: clonoSEQ Assay Workflow
Image /page/7/Figure/3 description: The image shows a flowchart of a process. The process starts with "Test ordered" and then splits into two parallel paths. The top path includes "Accessioning", "Extraction (if Applicable) & Sample Prep Workflow", "Specific Assay Method", and "Pipeline Analysis". The bottom path includes "Data QC", "Matching of Calibrated Clones & Report Generation", and "Report Review", which leads to "Report Delivery".
Following completion of these data processing steps, a report is issued. A Clonality (ID) report indicates the presence of dominant sequences residing within a presumed malignant lymphocyte clonal population, as identified in the baseline (diagnostic or high disease burden) sample from a patient. After one or more dominant sequence(s) have been identified in a baseline sample, subsequent samples from the same patient can be assessed for MRD after which a Tracking (MRD) report is generated. The MRD is expressed as a frequency that quantifies the level of residual disease based on the number of remaining copies of the initially dominant sequence(s) relative to the total number of nucleated cells in the sample.
8
Intended Use
The clonoSEO Assay is an in vitro diagnostic that uses multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to identify and quantify rearranged IgH (VDJ), IgH (DJ), IgK and IgL receptor gene sequences, as well as translocated BCL1/1gH (J) and BCL2/1gH (J) sequences in DNA extracted from bone marrow from patients with B-cell acute lymphoblastic leukemia (ALL) or multiple myeloma (MM), and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL).
The clonoSEQ Assay measures minimal residual disease (MRD) to monitor changes in burden of disease during and after treatment. The test is indicated for use by qualified healthcare professionals in accordance with professional guidelines for clinical decision-making and in conjunction with other clinicopathological features.
The clonoSEQ Assay is a single-site assay performed at Adaptive Biotechnologies Corporation in Seattle, Washington.
Differences in Indications Statement
The clonoSEQ Assay claims are expanding to include blood and bone marrow as a specimen type from patients with CLL. Analytical validations (including LOD and precision) using gDNA extracted from blood were compared with validations using gDNA extracted from bone marrow; these validations yielded comparable results between the two sample types. There are no differences that affect the safety or effectiveness of the device when used as labeled.
| Similarities and Differences | ||
|---|---|---|
| Item | Device | Predicate (DEN170080) |
| Device | Same | Adaptive clonoSEQ® Assay |
| Intended Use | The clonoSEQ Assay is an in vitro diagnostic that uses multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to identify and quantify rearranged IgH (VDJ), IgH (DJ), IgK and IgL receptor gene sequences, as well as translocated BCL1/IgH (J) and BCL2/IgH (J) sequences in DNA extracted from bone marrow from patients with B-cell acute lymphoblastic leukemia (ALL) or multiple myeloma (MM), and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL). | The clonoSEQ Assay is an in vitro diagnostic that uses multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to identify and quantify rearranged IgH (VDJ), IgH (DJ), IgK, and IgL receptor gene sequences as well as translocated BCL1/IgH (J) and BCL2/IgH (J) sequences in DNA extracted from bone marrow from patients with B-Cell acute lymphoblastic leukemia (ALL) and multiple myeloma (MM). |
| Similarities and Differences | ||
| Item | Device | Predicate (DEN170080) |
| The clonoSEQ Assay measures | ||
| minimal residual disease | ||
| (MRD) to monitor changes in | ||
| burden of disease during and | ||
| after treatment. The test is | ||
| indicated for use by qualified | ||
| healthcare professionals in | ||
| accordance with professional | ||
| guidelines for clinical decision- | ||
| making and in conjunction with | ||
| other clinicopathological | ||
| features. | The clonoSEQ Assay measures | |
| minimal residual disease | ||
| (MRD) to monitor changes in | ||
| burden of disease during and | ||
| after treatment. The test is | ||
| indicated for use by qualified | ||
| healthcare professionals in | ||
| accordance with professional | ||
| guidelines for clinical decision- | ||
| making and in conjunction with | ||
| other clinicopathological | ||
| features. | ||
| The clonoSEQ Assay is a | ||
| single-site assay performed at | ||
| Adaptive Biotechnologies | ||
| Corporation in Seattle, | ||
| Washington. | The clonoSEQ Assay is a | |
| single-site assay performed at | ||
| Adaptive Biotechnologies | ||
| Corporation. | ||
| Special conditions for | ||
| use statement(s) | Same | For prescription use only |
| Device Description | Same | DNA-based test for minimal |
| residual disease for hematologic | ||
| malignancies | ||
| Test Sample | Blood or bone marrow aspirate | Bone marrow aspirate |
| Extraction/amplification | ||
| reagents/amplification | ||
| procedures | Methods to isolate gDNA from | |
| bone marrow, bone marrow | ||
| mononuclear cells (BMMCs), | ||
| blood, PBMCs, formalin fixed | ||
| paraffin embedded (FFPE) bone | ||
| marrow clot slides, and bone | ||
| marrow smear slides were | ||
| evaluated for performance in the | ||
| clonoSEQ Assay. Studies were | ||
| performed to determine | ||
| extraction equivalence across | ||
| multiple extraction runs with 3 | ||
| variables (automated extraction | ||
| instrument, operator, and | ||
| reagent lot). Based on the | ||
| results of all extractions, the | ||
| tested gDNA extraction | ||
| variables (operator, instrument, | Methods to isolate gDNA from | |
| bone marrow, bone marrow | ||
| mononuclear cells (BMMCs), | ||
| PBMCs, formalin fixed paraffin | ||
| embedded (FFPE) bone marrow | ||
| clot slides, and bone marrow | ||
| smear slides were evaluated for | ||
| performance in the clonoSEQ | ||
| Assay. Studies were performed | ||
| to determine extraction | ||
| equivalence across multiple | ||
| extraction runs with 3 variables | ||
| (automated extraction | ||
| instrument, operator, and | ||
| reagent lot). Based on the | ||
| results of all extractions, the | ||
| tested gDNA extraction | ||
| variables (operator, instrument, | ||
| Similarities and Differences | ||
| Item | Device | Predicate (DEN170080) |
| Software | Same | See DEN170080 |
| Reagents | Same | See DEN170080 |
| Principle of the assay method | Same | MET-00072 |
| Stability/Shelf Life (if applicable) | extraction run) met acceptance criteria. gDNA isolated from FFPE bone marrow clot slides and bone marrow smear slides was only assessed for utility in identifying sequences and not MRD tracking. | extraction run) met acceptance criteria. gDNA isolated from FFPE bone marrow clot slides and bone marrow smear slides was only assessed for utility in identifying sequences and not MRD tracking. |
| Specimens assessed by the clonoSEQ Assay were stable for the following storage conditions: | ||
| • At -15 °C to -25 °C for up to 18 months for BMA and up to 6 months for blood | ||
| • At 2 °C to 8 °C for up to 7 days for BMA and up to 14 days for blood | ||
| • At 15 °C to 25 °C for up to 3 days for BMA and up to 5 days for blood | ||
| • Up to 3 freeze/thaw cycles for BMA and blood | Specimens assessed by the clonoSEQ Assay were stable for the following storage conditions: | |
| • At -15 °C to -25 °C for up to 18 months for BMA | ||
| • At 2 °C to 8 °C for up to 7 days for BMA | ||
| • At 15 °C to 25 °C for up to 3 days for BMA | ||
| • Up to 3 freeze/thaw cycles for BMA | ||
| Specimens assessed by the clonoSEQ Assay were stable within the clonoSEQ shipper for ambient, summer and winter shipping conditions: | ||
| • For up to 4 days for BMA | ||
| • For up to 5 days for blood | Specimens assessed by the clonoSEQ Assay were stable within the clonoSEQ shipper for ambient, summer and winter shipping conditions: | |
| • For up to 4 days for BMA | ||
| Instrument | NextSeq 500 and 550 series | NextSeq 500 |
Table 1: Comparison with Predicate
9
10
Performance Characteristics
Precision
Precision studies in BMA-derived samples tested gDNA extracted from clinical specimens from 22 patients with CLL. The gDNA from these specimens was used to contrive specific MRD levels by pooling and blending them into gDNA extracted from the BMA of healthy donors. The study included 3
11
DNA inputs (500 ng, 2 µg, 20 µg) and 6 MRD levels were tested at each DNA input for each patient sample. The studies were designed to test the target MRD levels of: 2.8x10-5, 8.0x10-2, 2.8x10-4, 8.0x10-3 and 8.0x10-3 at 500 ng DNA input; 7.0x10-6, 2.0x10-5, 7.0x10-5, 2.0x10-4, 7.0x10-4 and 2.0x10-3 at 2 µg DNA input; and 7.0x10-7, 2.0x10-6, 7.0x10-6, 2.0x10-5, 7.0x10-5 and 2.0x10-4 at 20 µg DNA input. These frequencies correspond to an estimated 2.14, 6.13, 21.44, 61.26, 214.40 and 612.56 malignant cells tested at each DNA dilution.
Precision studies in blood-derived samples tested gDNA extracted from clinical specimens from 15 patients with CLL. The gDNA from these specimens was used to contrive specific MRD levels by pooling and blending them into gDNA extracted from the blood of healthy donors. The study included 3 DNA inputs (500 ng, 2 µg, 20 µg) and multiple MRD levels were tested at each DNA input for each patient sample. The studies were designed to test the target MRD levels of: 4.0x10-5, 1.0x10-4, 4.0x10-4, 1.0x10-3 at 500 ng DNA input; 1.0x10-5, 2.5x10-5, 1.0x10-4, 2.5x10-4, 1.0x10-3 for 2 µg DNA input; and 1.0x10-6, 2.5x10-6, 1.0x10-5, 2.5x10-5, 1.0x10-4, 2.5x10+ at 20 µg DNA input. These frequencies correspond to an estimated 3.06, 7.667, 306.28, and 765.70 malignant cells tested at each DNA dilution.
Both precision studies used a main effects screening design over 21 calendar days. This study used 10 runs, with 2 PCR plates each run, using 3 operator sets, 4 reagent lots, and 4 instrument sets (2 thermal cycler/liquid handlers and 2 NextSeq™ instruments). The study design for blood used both NextSeq 500 and 550s. The study design for BMA is summarized in Figure 2 and for blood in Figure 3.
12
Image /page/12/Figure/1 description: The image shows a diagram of 10 PCR runs, with each run consisting of 2 PCR plates. The runs are divided among three operators, labeled #1, #2, and #3. Each run is associated with a reagent group number, a thermal cycler/liquid handler set number, and a NextSeq number. The run numbers are 2, 3, 5, 8, 1, 4, 6, 7, 9, and 10.
clonoSEQ Assay: Precision Study PCR Run Execution Map
Figure 3: Blood Precision Study Design Schematic
Image /page/12/Figure/4 description: This image shows a flowchart of 10 PCR runs, with each run having an A and B component. The flowchart starts with the 'Start Day', which is divided into five pairs of days: 1 & 2, 3 & 4, 5 & 6, 7 & 8, and 9 & 10. Each of these pairs is further divided into two 'Sample Sets', labeled A and B, which are then assigned to different 'Operators' (OP 1, OP 2, OP 3). The flowchart continues with 'Reagent Group', 'TC/Liquid Handler Set', and 'NextSeq', each having numerical assignments, and concludes with the 'Run #' which lists the runs as 1A & 1B through 10A & 10B.
13
For the BMA study, each run of the assay tested 18 combinations of DNA input and MRD frequency in duplicate. In all, 360 contrived samples were tested. Of these, one plate with 18 samples was invalid due to sample QC failures; the plate-level failure rate was therefore 1 / 20 = 0.05. An additional two contrived samples (44MRD measurements) failed sample OC due to insufficient sequencing coverage. While normal operating procedures permit re-sequencing, for this analysis these two samples were classified as failures. The analysis used the remaining 340 contrived samples with up to 22 MRD measurements per sample, for a total of 7,480 MRD measurements.
For the blood precision study, each run of the assay tested 16 combinations of gDNA input and MRD frequency, in duplicate, for a total of 320 contrived samples tested. There were no plate failures. One sample replicate was invalid due to sample QC failure, leaving 319 contrived samples and a total of 4,785 MRD measurements in the final analysis (15 MRD measurements x 319 samples).
In both BMA and blood sample types, the precision of the clonoSEQ Assay is largely dependent upon the number of malignant cells that are being evaluated rather than the MRD frequency. Consequently, the same MRD frequency is expected to have lower precision at lower gDNA inputs. For these studies, precision estimates were calculated based on the MRD frequency per gDNA input and estimates of imprecision of the absolute number of malignant cells detected were calculated.
Precision of MRD Frequency for CLL in BMA
Precision analysis, including variation from instrument set, operator, processing day, processing run, and reagent lot, is reported as %CV for each tested MRD frequency at each DNA input. This analysis is summarized in Table 2. Precision ranged from 18.5% to 60.1% CV.
MRD frequency range refers to the central 95% range of MRD estimates that were observed across all of the patient samples tested at each DNA input and frequency condition. These data were used to define the 95% confidence intervals that are used in patient reports.
| DNA Input | Target
MRD Frequency | Measurements | Patients* | %CV | Mean
MRD
Frequency | Frequency Range
(95% CI) |
|-----------|-------------------------|--------------|-----------|------|--------------------------|-----------------------------|
| 500 ng | 2.8x10-5 | 396 | 22 | 57.7 | 4.4 x 10-5 | 0 - 1.0 x 10-4 |
| | 8.0x10-5 | 396 | 22 | 42.6 | 1.0 x 10-4 | 3.4 x 10-5 - 2.0 x 10-4 |
| | 2.8x10-4 | 396 | 22 | 27.8 | 3.4 x 10-4 | 1.7 x 10-4 - 5.6 x 10-4 |
| | 8.0x10-4 | 440 | 22 | 24.2 | 9.2 x 10-4 | 5.6 x 10-4 - 1.5 x 10-3 |
| | 2.8x10-3 | 440 | 22 | 21.2 | 2.9 x 10-3 | 1.9 x 10-3 - 4.4 x 10-3 |
| | 8.0x10-3 | 440 | 22 | 19.6 | 7.1 x 10-3 | 4.5 x 10-3 - 1.0 x 10-2 |
| 2 µg | 7.0x10-6 | 396 | 22 | 60.1 | 1.0 x 10-5 | 0 - 2.4 x 10-5 |
| | 2.0x10-5 | 396 | 22 | 44.5 | 2.6 x 10-5 | 9.3 x 10-6 - 5.4 x 10-5 |
| | 7.0x10-5 | 396 | 22 | 28.9 | 8.5 x 10-5 | 4.5 x 10-5 - 1.4 x 10-4 |
| | 2.0x10-4 | 440 | 22 | 23.5 | 2.3 x 10-4 | 1.4 x 10-4 - 3.8 x 10-4 |
Table 2: Precision of the clonoSEO Assav in CLL BMA Samples
14
| DNA Input | Target
MRD Frequency | Measurements | Patients* | %CV | Mean
MRD
Frequency | Frequency Range
(95% CI) |
|-----------|-------------------------|--------------|-----------|------|--------------------------|-----------------------------|
| | 7.0x10-4 | 440 | 22 | 21.4 | 8.2 x 10-4 | 5.1 x 10-4 - 1.3 x 10-3 |
| | 2.0x10-3 | 440 | 22 | 19.2 | 2.2 x 10-3 | 1.4 x 10-3 - 3.2 x 10-3 |
| 20 µg | 7.0x10-7 | 396 | 22 | 59.8 | 1.1 x 10-6 | 0 - 2.6 x 10-6 |
| | 2.0x10-6 | 396 | 22 | 40.7 | 2.9 x 10-6 | 1.0 x 10-6 - 5.6 x 10-6 |
| | 2.8x10-5 | 396 | 22 | 27.2 | 9.1 x 10-6 | 5.4 x 10-6 - 1.5 x 10-5 |
| | 8.0x10-5 | 440 | 22 | 22.1 | 2.6 x 10-5 | 1.6 x 10-5 - 3.9 x 10-5 |
| | 2.8x10-4 | 440 | 22 | 19.9 | 8.4 x 10-5 | 5.2 x 10-5 - 1.2 x 10-4 |
| | 8.0x10-4 | 396 | 22 | 18.5 | 2.3 x 10-4 | 1.6 x 10-4 - 3.3 x 10-4 |
Precision of MRD Frequency for CLL in blood
Precision analysis, including variation from instrument set, operator, processing run, and reagent lot, is reported as %CV for each tested MRD frequency at each DNA input. This analysis is summarized in Table 3. Precision ranged from 18.7% to 54.9% CV.
The measured %CV of the assay for CLL blood samples was comparable to the %CV in CLL bone marrow samples.
| DNA
Input | Target MRD
Frequency | Measurements | Patients | %CV | Mean MRD
Frequency | Frequency Range
(95% CI) |
|--------------|-------------------------|--------------|----------|------|-----------------------|-----------------------------|
| 500
ng | 4.0x10-5 | 300 | 15 | 54.9 | 5.3x10-5 | 1.1x10-5 - 1.2x10-4 |
| | 1.0x10-4 | 300 | 15 | 38.3 | 1.2x10-4 | 4.1x10-5 - 2.2x10-4 |
| | 4.0x10-4 | 300 | 15 | 28.9 | 4.4x10-4 | 2.3x10-4 - 7.1x10-4 |
| | 1.0x10-3 | 285 | 15 | 24.4 | 1.1x10-3 | 6.4x10-4 - 1.7x10-3 |
| | 4.0x10-3 | 300 | 15 | 21.9 | 4.3x10-3 | 2.8x10-3 - 6.3x10-3 |
| 2 µg | 1.0x10-5 | 300 | 15 | 51.6 | 1.2x10-5 | 2.4x10-6 - 2.7x10-5 |
| | 2.5x10-5 | 300 | 15 | 37.3 | 2.9x10-5 | 1.1x10-5 - 5.2x10-5 |
| | 1.0x10-4 | 300 | 15 | 26.5 | 1.1x10-4 | 6.1x10-5 - 1.7x10-4 |
| | 2.5x10-4 | 300 | 15 | 23.0 | 2.7x10-4 | 1.7x10-4 - 4.0x10-4 |
| | 1.0x10-3 | 300 | 15 | 20.8 | 1.1x10-3 | 6.8x10-4 - 1.6x10-3 |
| 20 µg | 1.0x10-6 | 300 | 15 | 49.2 | 1.3x10-6 | 2.9x10-7 - 2.8x10-6 |
| | 2.5x10-6 | 300 | 15 | 36.4 | 2.9x10-6 | 1.2x10-6 - 5.3x10-6 |
Table 3: Precision of the clonoSEQ Assay in CLL Blood
15
| DNA
Input | Target MRD
Frequency | Measurements | Patients | %CV | Mean MRD
Frequency | Frequency Range
(95% CI) |
|--------------|-------------------------|--------------|----------|------|-----------------------|-----------------------------|
| | 1.0x10-5 | 300 | 15 | 25.9 | 1.1x10-5 | 6.6x10-6 - 1.8x10-5 |
| | 2.5x10-5 | 300 | 15 | 23.0 | 2.7x10-5 | 1.7x10-5 - 4.3x10-5 |
| | 1.0x10-4 | 300 | 15 | 19.5 | 1.1x10-4 | 7.5x10-5 - 1.6x10-4 |
| | 2.5x10-4 | 300 | 15 | 18.7 | 2.7x10-4 | 1.9x10-4 - 3.9x10-4 |
Precision of Malignant Cells Detected in BMA
The precision of malignant cells detected was evaluated across the range of tested malignant cells (2.14 - 612.56). For this analysis, the results from all of the DNA inputs were pooled into a single analysis that is summarized in Table 4. As expected, the precision was primarily influenced by cell numbers being evaluated. Precision ranged from 59% CV at 2.14 cells to 20% CV at 612.56 cells. The majority of the observed variation is due to residual variability; the tested factors (Operator, Instrument Sets, Reagent Lots, Dav, and Run) minimally contributed to variability with attributable %CV ranging from 0% to 5% (Table 4).
| | # of Input Cancer
Cells | %CV Attributed to Each Variable at Cell
Inputs* | | | | | |
|---------------------------|----------------------------|----------------------------------------------------|------|-------|-------|-------|--------|
| | | 2.14 | 6.13 | 21.44 | 61.26 | 214.4 | 612.56 |
| | Instrument Set | 0% | 0% | 1% | 0% | 0% | 1% |
| | Operator | 5% | 1% | 0% | 0% | 0% | 0% |
| | Processing Day | 0% | 0% | 0% | 0% | 0% | 3% |
| | Processing Run | 0% | 0% | 0% | 0% | 0% | 0% |
| Lot-to-Lot
Variability | Reagent Lot | 1% | 0% | 1% | 0% | 1% | 1% |
| | Residual Variability | 59% | 43% | 28% | 23% | 21% | 19% |
| Precision | | 59% | 43% | 28% | 24% | 21% | 20% |
| | Total MRD | 1188 | 1188 | 1188 | 1320 | 1320 | 1276 |
| N | Measurements | | | | | | |
Table 4: Summary of the clonoSEQ Assay Precision in BMA
- These values were aggregated across total DNA input levels
The precision for each sample at each tested condition across all DNA inputs is summarized in a Sadler's precision profile (Figure 4). The Sadler's precision profile visualizes the relationship between the number of sampled malignant cells and precision as measured by %CV. This analysis demonstrates that the precision of the clonoSEQ Assay is largely dependent on the number of malignant cells that are being evaluated by the assay.
16
Image /page/16/Figure/0 description: The figure is a scatter plot showing the relationship between the coefficient of variation and the number of input cancer cells. The x-axis represents the number of input cancer cells, ranging from 1 to 500. The y-axis represents the coefficient of variation, ranging from 0% to 100%. The plot shows that as the number of input cancer cells increases, the coefficient of variation decreases, with a red dashed line at 75%.
Figure 4: Sadler's Precision Profile (Coefficient of Variation) of the clonoSEO Assay as a Function of Input Cancer Cells in CLL BMA
Precision of Malignant Cells Detected in CLL blood
The precision of malignant cells detected in blood was evaluated across the range of tested malignant cells (3.10 - 765.70). The results from all of the DNA inputs are summarized in Figure 5.
Precision ranged from 53% CV at 3.10 cells to 19% CV at 765.70 cells. The majority of the observed variation is due to residual variability. DNA input mass contributed to variability with attributable %CV ranging from 8% to 10%, and the pre-specified test factors (Operator, Instrument Sets, Reagent Lots, Day, and Run) minimally contributed to variability, with attributable %CV ranging from 0% to 2% (Table 5).
17
| | # of Input
Cancer Cells | 3.06 | 7.66 | 30.63 | 76.57 | 306.28 | 765.70 |
|-------------------------------------------------|----------------------------|------|------|-------|-------|--------|--------|
| | Instrument Set | 0% | 0% | 0% | 0% | 0% | 0% |
| | Operator | 0% | 0% | 0% | 0% | 0% | 0% |
| | Processing Day | 0% | 3% | 2% | 0% | 0% | 0% |
| | Processing Run | 0% | 0% | 1% | 0% | 0% | 0% |
| | DNA Input | 10% | 10% | 10% | 8% | 8% | NA |
| Lot-to-Lot
variability | Reagent Lot | 2% | 0% | 2% | 0% | 0% | 0% |
| | Residual
variability | 52% | 37% | 27% | 23% | 21% | 19% |
| Precision | | 53% | 39% | 29% | 25% | 22% | 19% |
| N | Total MRD
Measurements | 900 | 900 | 900 | 885 | 900 | 300 |
| %CV Attributed to Each Variable at Cell Inputs* | | | | | | | |
Table 5: Summary of the clonoSEQ Assay Precision in CLL Blood
The precision for each sample at each tested condition across all DNA inputs is summarized in a Sadler's precision profile (Figure 5). The Sadler's precision profile visualizes the relationship between the number of sampled malignant cells and precision as measured by %CV (note the inclusion of a lower input to align with the BMA plot). This analysis demonstrates that, like BMA, the precision of the clonoSEQ Assay in CLL blood is largely dependent on the number of malignant cells that are being evaluated by the assay.
18
Image /page/18/Figure/0 description: The figure shows the coefficient of variation as a function of input cancer cells. The x-axis shows the input cancer cells, with values ranging from 1.91 to 306.28. The y-axis shows the coefficient of variation, with values ranging from 0% to 125%. The coefficient of variation decreases as the number of input cancer cells increases, and there is a red dashed line at 75%.
Image /page/18/Figure/1 description: The image is titled "Figure 5: Sadler's Precision Profile (Coefficient of Variation) of the clonoSEQ Assay as a Function of Input CLL Cancer Cells in Blood". The image is a title for a figure. The title describes a precision profile of the clonoSEQ Assay.
Precision of the clonoSEO Assay is comparable in blood and bone marrow in CLL.
Linearity
The acceptance criterion for this study was met.
Linearity of the clonoSEQ Assay using 3 CLL cell lines (HG-3, MEC-1, and PGA-1) was evaluated by blending cell line gDNA with gDNA from healthy subjects using DNA inputs of 200 ng, 2 ug and 20 ug gDNA and tested at zero and across 11 MRD frequencies at each DNA input. This study was performed to measure the linearity of the clonoSEQ Assay at depths beyond the sensitivity of conventional tools. The frequency range of 6.0x10-to 1.0 was tested at the 200 ng DNA input. The frequency range of 6.5x10-6 to 1.0 was tested at the 2 ug DNA input. The frequency range of 6.6x107 to 0.1 was tested at 20 ug DNA input. The linear range of the assay was determined by finding the input range where the maximum deviation from linearity (based on a quadratic or cubic fit to the data) was less than 5%. Linearity was established for each sample input across the entire tested range (Table 6), with data shown in Figure 6. This study demonstrated linearity of MRD frequencies across several orders of magnitude.
19
Figure 6: Linearity of the clonoSEQ Assay in BMA. Expected (x-axis) and Observed (y-axis) MRD Frequency of 3 Cell Lines.
Image /page/19/Figure/1 description: The image contains three scatter plots comparing different DNA inputs. The first plot shows CLL 200 ng DNA Input, the second plot shows CLL 2 ug DNA Input, and the third plot shows CLL 20 ug DNA Input. Each plot displays data points for HG-3, MEC-1, and PGA-1, represented by circles, triangles, and plus signs, respectively. A dashed blue line runs diagonally across each plot, indicating a trend or correlation between the variables.
20
| Input
DNA | Tested | | Combined Analysis | | Summary of Individual
Patient Analyses | |
|--------------|-------------|-----------------|-------------------|-----------|-------------------------------------------|--------------------|
| | Range | Linear
Range | Slope | Intercept | Slope
Range | Intercept
Range |
| 200
ng | 0 to 1 | 0 to 1 | 0.994 | 0.018 | 0.978 to
1.011 | -0.045 to
0.129 |
| 2 ug | 0 to 1 | 0 to 1 | 1.004 | 0.034 | 0.998 to
1.016 | -0.045 to
0.161 |
| 20 ug | 0 to
0.1 | 0 to 0.1 | 0.994 | -0.033 | 0.974 to
1.019 | -0.159 to
0.111 |
Table 6: Linearity of the clonoSEQ Assay in BMA using Cell Lines
Linearity using Clinical BMA Specimens
Linearity was confirmed using clinical samples from the precision study (Section 2.4), which evaluated blended gDNA extracted from 22 CLL specimens at 3 DNA inputs and 6 MRD frequencies per DNA input. These data were re-analyzed to confirm linearity at the lower frequency range of the assay. The linear range of the assay was determined by finding the input range where the maximum deviation from linearity (based on a quadratic or cubic fit to the data) was less than 5%. Results are summarized in Table 7. The slopes and intercepts are reported as the average and range of values across all clinical specimens that were tested at each DNA input by disease indication. Results from 3 representative specimens for CLL are shown in Figure 7. This study demonstrated linearity across a wide range of MRD frequencies using clinical specimens.
21
| Disease
Indication | Input
DNA | Tested
Range | Combined Analysis | | | Summary of
Individual Patient
Analyses | |
|-----------------------|--------------|----------------------------------|-----------------------------------|-------|-----------|----------------------------------------------|--------------------|
| | | | Linear
Range | Slope | Intercept | Slope
Range | Intercept
Range |
| | 500ng | $2.8x10^{-5}$
to 0.008 | $2.8x10^{-5}$ to
0.008 | 0.916 | -0.216 | 0.847
to
1.004 | -0.450 to
0.011 |
| CLL | 2ug | $7.0x10^{-6}$
to 0.002 | $7x10^{-6}$ to
0.002 | 0.964 | -0.057 | 0.877
to
1.043 | -0.358 to
0.248 |
| | 20ug | $7.0x10^{-7}$
to $2.0x10^{4}$ | $7.0x10^{-7}$ to
$2.0x10^{-4}$ | 0.984 | 0.013 | 0.924
to
1.048 | -0.417 to
0.272 |
Table 7: Linearity of clonoSEQ Assay in BMA using Clinical Specimens
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Image /page/22/Figure/0 description: The image contains three scatter plots comparing different DNA inputs. The first plot shows CLL 500 ng DNA Input, the second plot shows CLL 2 µg DNA Input, and the third plot shows CLL 20 µg DNA Input. Each plot contains data for CLL-35, CLL-41, and CLL-46, represented by circles, triangles, and plus signs, respectively. The x and y axes are on a log scale, ranging from 10^-6 to 10^-2.
Figure 7: Linearity of clonoSEQ Assay in BMA. The Expected (x-axis) and Observed (y-axis) MRD Frequency of 3 Clinical Samples
Linearity using Clinical Blood Specimens
Linearity was confirmed in blood using clinical samples from the precision study (Section 2.4), which evaluated blended gDNA extracted from 15 CLL specimens at 3 DNA inputs and 6 MRD frequencies per DNA input. The linear range of the assay was determined by finding the input range where the maximum deviation from linearity (based on a quadratic or cubic fit to the data) was less than 5%. Results are summarized in Table 8. The slopes and intercepts are reported as the average and range of values across all clinical specimens that were tested at each DNA input. Results are shown in Figure 8. This study demonstrated linearity across a wide range of MRD frequencies using clinical blood specimens.
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| Disease
Indication | Input
DNA | Tested
Range | Combined Analysis | | | Summary of Individual
Patient Analyses | |
|-----------------------|--------------|-----------------|-------------------|-------|-----------|-------------------------------------------|--------------------|
| | | | Linear
Range | Slope | Intercept | Slope
Range | Intercept
Range |
| CLL | 20µg | 0 to
1x10-3 | 0 to
1x10-3 | 0.997 | -0.009 | 0.942 to
1.044 | -0.515 to
0.51 |
| | 2µg | 0 to
1x10-3 | 0 to
1x10-3 | 0.995 | -0.030 | 0.93 to
1.049 | -0.78 to 0.53 |
| | 500ng | 0 to
4x10-3 | 0 to
4x10-3 | 0.989 | -0.075 | 0.941 to
1.092 | -0.628 to
0.872 |
Table 8: Linearity using Clinical Blood Specimens.
Figure 8: Linearity of the clonoSEQ Assay in CLL Blood. The Expected (x-axis) and Observed (y-axis) MRD Frequency of 15 Clinical Samples
Image /page/23/Figure/3 description: The image contains three scatter plots comparing observed MRD frequency to expected MRD frequency for different DNA inputs. The plots are titled "CLL 500ng DNA Input", "CLL 2ug DNA Input", and "CLL 20ug DNA Input". Each plot shows data points scattered around a diagonal line, indicating a correlation between observed and expected frequencies. The axes are logarithmically scaled, ranging from 10^-6 to 10^-2 for the y-axis (Observed MRD Frequency) and a similar range for the x-axis (Expected MRD Frequency).
Accuracy
Concordance with mpFC in Blood Clinical Samples
This study assessed concordance in two ways: concordance of MRD positive or negative calls and concordance of quantitative MRD frequency. This study compared MRD results reported by the clonoSEQ Assay to mpFC. The flow study included 299 matched samples. In a comparison of qualitative calls between mpFC and the clonoSEQ Assay, MRD negativity was defined as 98% demonstrates the high concordance of positive calls between flow
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and the clonoSEQ Assay, while an NPA -30% and 10-5) compared to MRD negative patients (95% CI: 3.65-12.1). Cox regression analysis of PFS using continuous MRD values demonstrated that the clonoSEQ Assay is significantly associated with PFS (likelihood ratio P = 2.96 x 10-2) and that each 10-fold increase in MRD level is associated with a 2.35-fold increase in event risk (95% CI: 1.86-2.48). The results also show that the MRD level at FUM3 is a stronger predictor of PFS than than other prognostic variables identified as clinically relevant covariates or treatment arm of the clinical trial. Together, these results demonstrate the utility of MRD measurement in CLL.
The clonoSEQ Assay was also used to assess MRD at various disease burden thresholds to determine the correlation of MRD level with PFS. Patients with clonoSEQ MRD ≤ 10 o or between 10 º and 10 º had longer PFS, followed by patients with MRD between 105 and 104 and patients with MRD ≥ 104 (log-rank P = 4.902 x 10-3). These data demonstrate that patients with MRD ≤ 10-5 have better outcomes than patients with MRD > 102, and that increasing MRD levels above 105 are associated with an increased risk of progression within the follow-up time of this study.
The second study. Thompson et al. was a prospective, phase 2 clinical trial that evaluated six cycles of fludarabine, cyclophosphamide, and rituximab (FCR) in 111 front-line chronic lymphocytic leukemia (CLL) patients with clonoSEQ ID samples and a corresponding 137 clonoSEQ MRD samples also evaluated by 4-color flow cytometry at an MRD threshold of 10+ (NCT00759798) and with pertinent co-variate data. Within this cohort of 111 patients with flow MRD results, bone marrow was available for 75 patients and blood was available for 62 patients, of which 26 patients provided both blood and bone marrow. Due to some missing clinical covariates, 3 patients that provided bone marrow only were excluded from analyses requiring these covariates.
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There is a significant association between PFS and continuous clonoSEO MRD measurement in both blood and bone marrow, after end of treatment, where PFS is defined as the time from start of treatment until death, disease progression, or last time of disease assessment (p = 9.66E-04 for blood, p = 2.13E-04 for bone marrow). Additionally, patients who were MRD negative at a threshold 105 (p = .02 for blood and p = 8.17E-05 for bone marrow).
In summary, MRD negativity as measured by the clonoSEQ Assay was associated with improved patient outcomes in CLL. These data support the use of the clonoSEQ Assay in patients diagnosed with CLL to measure MRD and to monitor changes in burden of disease during and after treatment.
References
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- Thompson P. et al, Undetectable MRD Using Next Generation Sequencing is Associated With Improved PFS After Treatment with FCR for CLL. Blood 2019 28:134(22):1951-1959
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- Fischer K. et al. Venetoclax and Obinutuzumab in Patients with CLL and Coexisting Conditions. New England Journal of Medicine.2019; 380:2225-2236.
- Prospective Identification of Significant Prognostic Factors Treated With Fludarabine, 3. Cyclophosphamide, and Rituximab (FCR) as Initial Therapy for Chronic Lymphocytic Leukemia (MDA2008-0431), NCT00759798
Conclusion: Adaptive clonoSEQ is substantially equivalent to the predicate.
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