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.

The clonoSEQ Assay is a single-site assay performed at Adaptive Biotechnologies Corporation.

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.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study details for the Adaptive Biotechnologies clonoSEQ Assay, based on the provided FDA 510(k) summary:

This device, the Adaptive Biotechnologies clonoSEQ Assay, is an in vitro diagnostic (IVD) that identifies and quantifies rearranged immune receptor gene sequences (IgH, IgK, IgL) and translocated BCL1/IgH and BCL2/IgH sequences using multiplex PCR and Next-Generation Sequencing (NGS). It measures Minimal Residual Disease (MRD) in patients with B-cell acute lymphoblastic leukemia (ALL), multiple myeloma (MM), and chronic lymphocytic leukemia (CLL) to monitor disease burden. The current submission is an expansion of indications to include blood samples from CLL patients.

1. A table of acceptance criteria and the reported device performance

The provided document doesn't explicitly list "acceptance criteria" in a single table, but rather describes the performance characteristics that were measured and the outcomes for each. I will compile these for the CLL in Blood indication, as this is the focus of the 510(k) expansion.

Acceptance Criteria & Reported Device Performance for clonoSEQ Assay (CLL in Blood)

Performance Characteristic	Acceptance Criteria (Implicit)	Reported Device Performance (CLL in Blood)
Precision (MRD Frequency)	%CV within acceptable clinical/analytical limits (not explicitly stated, but inferred from successful results)	Range: 18.7% - 54.9% CV. - At 500 ng DNA input: 21.9% - 54.9% CV - At 2 µg DNA input: 20.8% - 51.6% CV - At 20 µg DNA input: 18.7% - 49.2% CV (Comparable to BMA precision)
Precision (Malignant Cells Detected)	%CV within acceptable clinical/analytical limits, primarily influenced by cell number.	Range: 19% CV (at 765.70 cells) to 53% CV (at 3.10 cells). Primarily due to residual variability; other factors (operator, instruments, reagents, day, run) contributed 0%-10% CV.
Linearity	Maximum deviation from linearity (based on quadratic or cubic fit) less than 5%.	Met for all DNA inputs (20µg, 2µg, 500ng) across the entire tested MRD frequency range (0 to 1x10^-3 / 4x10^-3). - Slopes: 0.989 - 0.997 (indicating strong linearity) - Intercepts: -0.009 to -0.075
Accuracy (Concordance with mpFC)	High positive percent agreement (PPA) and understanding of negative percent agreement (NPA) reflecting greater sensitivity.	PPA: 98.9% (95% CI: 94.3%-100%) NPA: 47.5% (95% CI: 40.5%-54.6%) (NPA lower due to clonoSEQ's higher sensitivity detecting MRD where mpFC calls negative)
Limit of Blank (LoB)	LoB should be zero or negligible.	LoB was confirmed as zero (95th percentile of trackable sequences in healthy blood was zero).
Limit of Detection (LoD) / Limit of Quantitation (LoQ)	LoD/LoQ for blood should be comparable to or lower than previously determined values for bone marrow.	LoD and LoQ for CLL in blood were lower or within the 95% CI of bootstrapped prior BMA data, confirming comparability.
Analytical Specificity (Interfering Substances)	Mean MRD frequency difference $\pm$ 30% when comparing with and without interferent substances.	All tested endogenous and exogenous substances met acceptance criteria. - Endogenous: Bilirubin (conjugated & unconjugated), Hemoglobin, Cholesterol, Triglycerides. - Exogenous: K2EDTA, K3EDTA, Heparin, Chloroform. (MRD results not substantially influenced).
Cross-Contamination/Sample Carryover	No significant contamination events leading to false positive ID or MRD results.	- No false calibrations for run-to-run (0/44 BMA, 0/44 BMMC). - One single well-to-well false calibration (1/44 BMA) at a very low template count (83 templates), not associated with cell lines. - Blood: No contamination or disease clone-sharing events leading to false positive ID/MRD results. - PCR/Library/Sequencing: No run-to-run contamination (0/36 tests). 8/712 well-to-well events (likely vendor-related primer barcode issue) < 4x10^-6, deemed unimpactful due to patient-specific clonotypes.
Reagent Stability (In-Use)	Sequencing results meeting all QC metrics.	All conditions tested met acceptance criteria. (Pre-amp/PCR primer mix, master mix, complete reaction, process pause stability).
Reagent Stability (Real Time)	Performance adequate and consistent; pairwise equivalence test of clinical specimens within $\pm$ 30% MRD frequency.	Established 15-month shelf life for pre-amp and PCR primer mixes at -20 ± 5 ℃. Clinical sample equivalence met $\pm$ 30% MRD frequency criterion.
Sample Stability	Samples remain stable under specified storage and shipping conditions for stated durations.	Met for blood samples: - -15 ℃ to -25 ℃: up to 6 months - 2 ℃ to 8 ℃: up to 14 days - 15 ℃ to 25 ℃: up to 5 days - Freeze/Thaw: Up to 3 cycles Shipper Stability: - Ambient, Summer, Winter: up to 5 days

2. Sample sizes used for the test set and the data provenance

Precision Studies (Test Set):
- CLL in BMA: 22 patients. Contrived samples (blending patient gDNA with healthy donor BMA gDNA). 360 contrived samples tested, yielding ~7,480 MRD measurements.
- CLL in Blood: 15 patients. Contrived samples (blending patient gDNA with healthy donor blood gDNA). 320 contrived samples tested, yielding ~4,785 MRD measurements.
Linearity Studies (Test Set):
- Cell Lines in BMA: 3 CLL cell lines (HG-3, MEC-1, PGA-1). Blended cell line gDNA with healthy subject gDNA. Data shown for multiple DNA inputs and 11 MRD frequencies.
- Clinical BMA Specimens: Re-analysis of data from the 22 CLL patients in the precision study.
- Clinical Blood Specimens: Re-analysis of data from the 15 CLL patients in the precision study.
Accuracy (Concordance with mpFC in Blood Clinical Samples): 299 matched clinical samples.
Limit of Blank (LoB): 22 CLL patient samples (for trackable sequences) and healthy bone marrow samples. For blood, 15 CLL patient samples and healthy blood samples.
Limit of Detection/Quantitation (LoD/LoQ): 22 CLL patient specimens (BMA) and 15 CLL patient specimens (Blood). Contrived dilution series.
Interfering Substances: 4 different donors for both BMA and blood samples. Each condition replicated 8 times. Additional assessment on 4 CLL clinical blood specimens.
Cross-Contamination/Carryover:
- Automated DNA Extraction (BMA/BMMC): Panel of 6 lymphoid malignancy cell lines (3 ALL, 3 MM), 10% spiked into healthy BMA/BMMC pool + PBS blanks.
- Automated DNA Extraction (Blood): Panel of 6 lymphoid malignancy cell lines, 10% spiked into normal healthy blood + PBS blanks.
- PCR, Library Pooling, Sequencing: gDNA from blood from healthy subjects (MRD-negative) and 5% spiked cell line gDNA blends.
Clinical Studies (to support prognostic utility):
- NCT02242942 (Primary CLL study): Samples and outcomes data from 445 patients initially. For primary analysis, 337 patients had usable clonoSEQ Assay MRD data and clinical outcomes (after QC and excluding early progression).
- NCT00759798 (Secondary CLL study): 111 front-line CLL patients with clonoSEQ ID samples, 137 clonoSEQ MRD samples (also evaluated by 4-color flow cytometry). Bone marrow available for 75 patients, blood for 62 patients (26 had both). 3 patients excluded due to missing clinical covariates.

Data Provenance:
The data provenance is largely implied as originating from Adaptive Biotechnologies Corporation as a single-site assay performed in Seattle, Washington. The studies involve clinical specimens from patients with CLL.

The clinical validation studies reference specific US clinical trials (NCT02242942, NCT00759798), indicating prospective data collection from multi-center clinical trials, likely including locations within the US.
The analytical studies (precision, linearity, etc.) used both contrived samples (blending patient/cell line gDNA with healthy donor gDNA) and clinical specimens, processed and analyzed at the Adaptive Biotechnologies lab. These are described as analytic validation studies.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

The document describes an in vitro diagnostic (IVD) test that quantifies specific DNA sequences. The "ground truth" for the analytical performance studies (precision, linearity, LoD/LoQ) is established by the known concentrations of the contrived samples, where specific amounts of malignant cells or gDNA from patient samples (with known clonal sequences) are blended into a background of healthy donor gDNA. This is a common and appropriate method for analytical validation of quantitative IVD assays.

For the Clinical Studies, the "ground truth" for patient outcomes is clinical progression-free survival (PFS) data and disease assessment from the clinical trials (NCT02242942 and NCT00759798). These outcomes are established by the clinical trial investigators, who would be qualified healthcare professionals (e.g., oncologists, hematologists) following standard clinical practice and trial protocols. The document does not specify a number of "experts" to establish ground truth in the sense of independent expert review of images or clinical cases, as would be common for an imaging AI device. The ground truth for this device is based on quantifiable molecular levels and patient outcomes.

4. Adjudication method for the test set

Not applicable in the typical sense for this type of IVD device. "Adjudication" usually refers to a process for resolving discrepancies in expert interpretations (e.g., radiologist reads).

For the analytical studies, the ground truth is established by the known input concentrations of contrived samples or the re-analysis of patient data to show linearity. Reproducibility and accuracy are assessed by repeatedly measuring these known inputs across different conditions (operators, instruments, reagents, etc.).
For the clinical studies, the ground truth is patient outcome data (PFS) collected as part of the clinical trials, which would be managed and reviewed according to standard clinical trial protocols, not a specific "adjudication method" as seen in image reading studies.

5. 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 is an in vitro diagnostic (IVD) assay, not an AI imaging device where human readers interact with AI. The clonoSEQ Assay is a laboratory test that provides quantitative MRD measurements. There is no "human reader" in the sense of interpreting an AI output directly influencing their decision-making for a specific case (like interpreting a radiology image with or without AI assistance). The output is a numerical MRD value reported to qualified healthcare professionals for use in clinical decision-making.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the "standalone" performance is the core of the studies presented. The clonoSEQ Assay is an algorithm-driven test that provides a quantitative MRD value. The precision, linearity, LoD/LoQ, and analytical specificity studies directly evaluate the "algorithm only" performance (i.e., the performance of the assay system independent of clinical interpretation for a specific patient). The output (MRD value) is generated by the assay system and its bioinformatics pipeline, which includes proprietary algorithms. The clinical studies then demonstrate the prognostic utility of this standalone quantitative output.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)

The ground truth for the different aspects of the study include:

Analytical Studies (Precision, Linearity, LoD/LoQ, Interfering Substances, Cross-Contamination): Known input concentrations of contrived samples (blending gDNA from patient/cell lines with healthy donor gDNA). This is a quantitative ground truth.
Accuracy (Concordance with mpFC): Flow Cytometry (mpFC) results served as a comparator method. While not a "gold standard" pathology ground truth, it's a widely accepted method for MRD detection, and the study highlights clonoSEQ's greater sensitivity.
Clinical Studies: Patient Outcomes Data, specifically Progression-Free Survival (PFS), as recorded during controlled clinical trials.

8. The sample size for the training set

The document does not explicitly state a "training set" size for the clonoSEQ assay's algorithmic development. For IVD assays based on NGS and bioinformatics pipelines, the "training" (or development/optimization) often involves extensive analytical validation, optimization using synthetic controls, and iterations on algorithms rather than a distinct "training set" like in machine learning for image classification.

The document states:

"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..."

This suggests the algorithms were likely developed and refined using a combination of synthetic data, known biological samples, and potentially retrospective patient data to build the "B cell repertoire database" and optimize the bias correction and clonality assessment. However, the exact sample sizes for this development phase are not provided in this 510(k) summary, as it primarily focuses on the validation of the finalized assay for regulatory approval.

9. How the ground truth for the training set was established

As inferred above, if there was a "training set" in the context of algorithm development, the ground truth would have been established through:

Known molecular constructs/synthetic controls: For optimizing sequencing and amplification bias correction.
Well-characterized cell lines and patient samples: Where the presence and frequency of specific clonal rearrangements are either known or confirmed by orthogonal methods (e.g., flow cytometry, Sanger sequencing, or other molecular techniques) to build the B cell repertoire database or tune the clonality determination.
Expert knowledge of immunology and genetics: To design the algorithms that identify and quantify rearranged gene sequences and interpret their significance in the context of hematological malignancies.

The document implicitly refers to this through descriptions of the "proprietary algorithm with in-line controls" and comparison against a "B cell repertoire database," indicating an internally developed and optimized system for which ground truth would have been internally established during its development phase.

Summary

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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

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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 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

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as diploid copies in normal genomic DNA (gDNA) to allow determination of total nucleated cell content.

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.

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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 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 measuresminimal residual disease(MRD) to monitor changes inburden of disease during andafter treatment. The test isindicated for use by qualifiedhealthcare professionals inaccordance with professionalguidelines for clinical decision-making and in conjunction withother clinicopathologicalfeatures.	The clonoSEQ Assay measuresminimal residual disease(MRD) to monitor changes inburden of disease during andafter treatment. The test isindicated for use by qualifiedhealthcare professionals inaccordance with professionalguidelines for clinical decision-making and in conjunction withother clinicopathologicalfeatures.
	The clonoSEQ Assay is asingle-site assay performed atAdaptive BiotechnologiesCorporation in Seattle,Washington.	The clonoSEQ Assay is asingle-site assay performed atAdaptive BiotechnologiesCorporation.
Special conditions foruse statement(s)	Same	For prescription use only
Device Description	Same	DNA-based test for minimalresidual disease for hematologicmalignancies
Test Sample	Blood or bone marrow aspirate	Bone marrow aspirate
Extraction/amplificationreagents/amplificationprocedures	Methods to isolate gDNA frombone marrow, bone marrowmononuclear cells (BMMCs),blood, PBMCs, formalin fixedparaffin embedded (FFPE) bonemarrow clot slides, and bonemarrow smear slides wereevaluated for performance in theclonoSEQ Assay. Studies wereperformed to determineextraction equivalence acrossmultiple extraction runs with 3variables (automated extractioninstrument, operator, andreagent lot). Based on theresults of all extractions, thetested gDNA extractionvariables (operator, instrument,	Methods to isolate gDNA frombone marrow, bone marrowmononuclear cells (BMMCs),PBMCs, formalin fixed paraffinembedded (FFPE) bone marrowclot slides, and bone marrowsmear slides were evaluated forperformance in the clonoSEQAssay. Studies were performedto determine extractionequivalence across multipleextraction runs with 3 variables(automated extractioninstrument, operator, andreagent lot). Based on theresults of all extractions, thetested gDNA extractionvariables (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

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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

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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.

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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.

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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	TargetMRD Frequency	Measurements	Patients*	%CV	MeanMRDFrequency	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

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DNA Input	TargetMRD Frequency	Measurements	Patients*	%CV	MeanMRDFrequency	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.

DNAInput	Target MRDFrequency	Measurements	Patients	%CV	Mean MRDFrequency	Frequency Range(95% CI)
500ng	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

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Target MRDFrequency	Measurements	Patients	%CV	Mean MRDFrequency	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 CancerCells	%CV Attributed to Each Variable at CellInputs*
		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-LotVariability	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.

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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).

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	# of InputCancer 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-Lotvariability	Reagent Lot	2%	0%	2%	0%	0%	0%
	Residualvariability	52%	37%	27%	23%	21%	19%
Precision		53%	39%	29%	25%	22%	19%
N	Total MRDMeasurements	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.

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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.

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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.

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InputDNA	Tested		Combined Analysis		Summary of IndividualPatient Analyses
	Range	LinearRange	Slope	Intercept	SlopeRange	InterceptRange
200ng	0 to 1	0 to 1	0.994	0.018	0.978 to1.011	-0.045 to0.129
2 ug	0 to 1	0 to 1	1.004	0.034	0.998 to1.016	-0.045 to0.161
20 ug	0 to0.1	0 to 0.1	0.994	-0.033	0.974 to1.019	-0.159 to0.111

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.

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DiseaseIndication	InputDNA	TestedRange	Combined Analysis			Summary ofIndividual PatientAnalyses
			LinearRange	Slope	Intercept	SlopeRange	InterceptRange
	500ng	$2.8x10^{-5}$to 0.008	$2.8x10^{-5}$ to0.008	0.916	-0.216	0.847to1.004	-0.450 to0.011
CLL	2ug	$7.0x10^{-6}$to 0.002	$7x10^{-6}$ to0.002	0.964	-0.057	0.877to1.043	-0.358 to0.248
	20ug	$7.0x10^{-7}$to $2.0x10^{4}$	$7.0x10^{-7}$ to$2.0x10^{-4}$	0.984	0.013	0.924to1.048	-0.417 to0.272

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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DiseaseIndication	InputDNA	TestedRange	Combined Analysis			Summary of IndividualPatient Analyses
			LinearRange	Slope	Intercept	SlopeRange	InterceptRange
CLL	20µg	0 to1x10-3	0 to1x10-3	0.997	-0.009	0.942 to1.044	-0.515 to0.51
	2µg	0 to1x10-3	0 to1x10-3	0.995	-0.030	0.93 to1.049	-0.78 to 0.53
	500ng	0 to4x10-3	0 to4x10-3	0.989	-0.075	0.941 to1.092	-0.628 to0.872

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 < 104 for flow and MRD < LOD for the clonoSEQ Assay. The positive percent agreement (PPA) between the clonoSEQ Assay and flow was 98.9%, with a 95% confidence bound of 94.3%-100%; the negative percent agreement (NPA) was 47.5%, with a 95% confidence bound of 40.5%-54.6% (Table 9). A PPA > 98% demonstrates the high concordance of positive calls between flow

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and the clonoSEQ Assay, while an NPA < 50% reflects the greater sensitivity of the clonoSEQ Assay, with 107 samples being called MRD positive by clonoSEQ and MRD negative by flow.). These results demonstrate the high concordance between the MRD calls of these 2 technologies.

	flow MRD+	flow MRD-	PPA(95% CI)	NPA(95% CI)
clonoSEQ MRD+	94	107	98.9%	47.5%
clonoSEQ MRD-	1	97	(94.3-100%)	(40.5-54.6%)
clonoSEQ MRD-	2	238

Table 9: Summary of mpFC vs. the clonoSEO Assay Concordance Data for CLL

Analytical Sensitivity

Limit of Blank (LOB) was measured to be 0 for all sample types and disease indications. Limit of Detection (LOD) and Limit of Quantitation (LOQ) for blood were comparable to the previously determined LOD and LOQ in bone marrow (DEN170080).

Limit of Blank

The LoB was determined by measuring the specificity of a patient's trackable immunoglobulin (Ig) sequences. These sequences were identified from 22 samples from patients diagnosed CLL. The LoB was determined by searching for the presence and abundance of these trackable sequences in healthy bone marrow samples. The 95th percentile of sample MRD frequencies for these trackable sequences was zero at 500 ng, 20 µg, and 40 µg of gDNA input. Therefore, the LoB was zero, demonstrating that trackable Ig sequences are highly patient-specific.

Similarly, the LoB in samples derived from blood was verified by searching for the presence and abundance of the trackable sequences from 15 samples from patients with CLL. The presence and abundance of these trackable sequences in healthy blood was assessed. The LoB was confirmed as zero in blood, based on the 95th percentile of sample MRD frequencies at 500 ng, 20 ug and 40 ug inputs.

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Limit of Detection/Limit of Quantitation

For bone marrow samples, the LoD and LoQ were determined by blending gDNA extracted from 22 specimens from patients with CLL into 500 ng and 20 ug of gDNA from bone marrow. A dilution series of 22.97, 10.72, 4.59, 2.14 and 0.94 malignant cell equivalents was made for each patient at each DNA input level. Each sample was tested in duplicate for each of 4 reagent lots resulting in eight results for each of the 22 samples at each dilution condition. A probit approach was used to determine the LoD to be 1.630 malignant cells (95% CI; 1.42 - 1.87) based on the combined data from both DNA inputs (Table 11). The LoQ was defined as the lowest absolute number of malignant cells whose frequency can be quantitatively determined with an accuracy of 70% relative total error. The LoQ was found to be 2.427 malignant cells (95% CI; 1.63 - 8.91) (Table 11).

Measure	Malignant Cells*	500 ng DNA InputFrequency (95% CI)	20 µg DNA InputFrequency (95% CI)
LoD	1.630 (1.42 - 1.87)	2.13x10-5(1.74x10-5 - 2.61x10-5)	5.28x10-7(4.39x10-7 - 6.3x10-7)
LoQ	2.427 (1.63 - 8.91)	2.63x10-5(2.13x10-5 - 6.07x10-5)	1.58 x 10-6(5.28x10-7 - 4.59x10-6)

Table 11: LoD/LoO in BMA by MRD Cell Counts and by MRD Frequency for CLL

*Calculated from samples with 500 ng and 20 µg of DNA input.

The clonoSEQ Assay can use a range of DNA inputs from 500 ng to 40 ug of DNA. The LoD/LoQ by MRD frequency will vary based on the DNA input and the total nucleated cells that are evaluated by the assay. The estimated LoD/LoQ at 500 ng and 20 ug of DNA input are shown in Table 11.

The LoD and LoQ were similarly determined for the background matrix of blood. The gDNA extracted from 15 specimens from patients with CLL was mixed into 500 ng and 20 ug of gDNA from healthy donors of blood to achieve targeted disease levels. A dilution series of 30.63, 7.66, 3.06, 1.91 and 0.77 malignant cell equivalents was made for each patient at each DNA input level. Each sample was tested in duplicate in each run, for each of 4 reagent lots, 3 operators and 2 sequencing instruments, resulting in twenty results for each of the 15 samples at each dilution condition. A probit approach was used to determine the LoD to be based on the combined data for all indications from both DNA inputs. The LoQ was calculated by applying the Sadler's precision profile model to all non-zero MRD frequency results for each indication.

The LoD and LoO for MRD measurements of CLL in the background matrix of blood was compared to the LoD and LoO for measurements in BMA specimens for ALL. MM and CLL using bootstrapping of BMA data. The LoD and LoQ of CLL in blood were lower or within the 95% CI of bootstrapped prior data and thus were not significantly higher than the prior established values.

Analytical Specificity

Interfering Substances

All acceptance criteria for this study were met. For each endogenous and exogenous substance, two-one sided t-tests demonstrated a mean MRD frequency difference > -30% and < 30%, when comparing MRD frequency with and without interferent substances. These results support the claim that each

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interfering substance tested does not have an effect on the clonoSEQ output for a given sample.

Testing was performed to characterize the effects of 5 endogenous (Table 12) and 3 exogenous (Table 13) substances in BMA and blood samples on the clonoSEQ Assay to identify potential interfering substances. BMA was tested with K2EDTA, Chloroform and blood was tested with K2EDTA, K3EDTA, and Heparin.

Substance Name	Reference Level(Low)	RecommendedConcentration (High)	Acceptance Criteria:Pass/Fail
Bilirubin conjugated	3.4 $\mu$ mol/l	342 $\mu$ mol/l	Pass
Bilirubin unconjugated*	21 $\mu$ mol/l	342 $\mu$ mol/l	Pass
Hemoglobin	1 g/l	2 g/l	Pass
Cholesterol*	5.2 mmol/l	13 mmol/l	Pass
Triglycerides	3.7 mmol/l	37 mmol/l	Pass

Table 12: Endogenous Interfering Substances Tested

Chloroform was used as solvent to resuspend bilirubin (unconjugated) and cholesterol.

		Table 13: Exogenous Interfering Substances Tested

Substance Name	Concentration(Low)	Concentration(High)	Acceptance Criteria:Pass/Fail
K2EDTA*	1.8 mg/ml	3.6 mg/ml	Pass
K3EDTAY	1.8 mg/ml	3.6 mg/ml	Pass
K3EDTA¥	1.8 mg/ml	5.4 mg/ml	N/A¥
Heparin	15 USP U/μ	30 USP U/ml	Pass
Chloroform (solvent)†	2.5 μl	N/A	Pass

The BMA samples were shipped to Adaptive containing 1.8 mg/ml EDTA ("Low" concentration) for anti-

coagulation purposes. Additional EDTA was spiked in to achieve the High level.

Chloroform inhibition was tested at a single spiked-in volume (2.5 ul) only in bone marrow.

4 Assessed only for blood samples.

¥Assessed only for CLL patient blood samples. Assay performance with interferent was within assay variation at the baseline measurement.

The potential exogenous and endogenous substances were spiked separately into 250 uL aliquots of bone marrow or 2 µL blood from 4 different donors. Each condition was replicated for a total of eight times (4 donors with 2 replicates each) and all conditions passed the pre-specified MRD frequency equivalence margin of ± 30%. This study concluded that MRD results were not substantially influenced by the presence of the tested interfering substances in either blood or BMA.

An additional assessment of four CLL clinical blood specimens was performed with inclusion of K3EDTA at low and high (3x) concentrations. The MRD results of both high and low K3EDTA were within the confidence intervals of the baseline MRD measurement for all four CLL specimens. The assay performance with K3EDTA in clinical samples was within assay variation at the baseline measurement.

Cross-Contamination/Sample Carryover

The assessment of cross-contamination included multiple studies; one study to measure contamination of ID samples during automated DNA extraction of BMA and BMMCs, one study to measure

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contamination causing false ID or false MRD results in samples during automated DNA extraction of blood, and one study to measure contamination of DNA from MRD samples during PCR, library pooling, and sequencing with the clonoSEQ Assay.

Cross-contamination of ID samples during automated DNA extraction was assessed using a panel of lymphoid malignancy cell lines (3 ALL and 3 MM) each spiked to 10% of total cells in a BMA pool of 2 healthy subjects or a bone marrow mononuclear cell (BMMC) pool of 4 healthy subjects. PBS (blank) samples were included in this study. Samples were evaluated as to whether they correctly calibrated. There were no false calibrations for run-to-run with 0/44 BMA and 0/44 BMMC false calibrations. There was one false calibration for the well study with 1/44 BMA and 0/44 BMMC samples falsely calibrating. The falsely calibrated sequence was found in a PBS sample with 83 total templates and the sequence was not associated with any of the 6 cell lines. The PBS sample provided a sensitive test for contamination since there was no background DNA and a contamination of 83 templates would not be expected to cause false calibration of a clinical specimen.

Cross contamination during extraction of blood samples leading to incorrectly calling samples ID calibrated or MRD positive was determined using a panel of lymphoid malignancy cell lines (N=6), either individually spiked into background normal healthy blood at a 10% frequency, or by pooling the clinical cell line samples and spiking this pool into a background normal blood sample at a target of 105 PBS (blank) samples. Samples were evaluated for ID calibration results and MRD results. There were no contamination or disease clone-sharing events that resulted in false positive ID or MRD results.

Cross contamination of incorrectly calling samples MRD positive was assessed using gDNA from blood from healthy subjects as MRD-negative specimens and blends of cell line gDNA and gDNA from blood of healthy subjects spiked to a concentration of 5%. The 5% level was used to simulate a patient with clinical relapse. This study evaluated for the presence of a clonal sequence and molecular barcode simultaneously. There were no run-to-run contamination events observed in 0/36 tests. Well-to-well cross contamination was observed in 8/712 comparisons: this was likely caused by contamination of a primer barcode plate sourced from a vendor. All contamination events were below 4x10°. This low level of contamination is unimpactful because tracked clonotype sequences are highly specific to each patient, so contamination between samples from different patients would not affect the reported MRD result. Cross contamination between samples from the same patient is prevented by process controls that disallow co-processing of samples from the same patient.

Reagent Stability

In-Use Reagent Stability

An in-use stability study was executed to determine stability needs of the clonoSEQ Assay for reaction mixes and intermediate steps. The following critical steps were evaluated: pre-amp and PCR primer mix stability, master mix stability, complete reaction stability, and process pause stability. gDNA was tested using seven replicates for all conditions tested. Acceptance criteria were based on sequencing results meeting all OC metrics; all of the conditions tested met the pre-specified acceptance criteria and the clonoSEQ Assay in-use stability needs.

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Real Time Stability of Pre-Amp and PCR Mixes

The real-time reagent stability studies used the primer QC processes to assess primer performance and determine primer stability. The primer QC process uses a set of synthetic double-stranded molecules representing rearrangements of the targeted exons to determine whether each manufactured lot of preamp PCR primers and PCR primers are performing within specification. The priming sites on synthetic molecules are identical to biologic priming sites on targeted exons. Data from these molecules were analyzed and assessed for the ability of the primers to amplify each identified exon at acceptable levels and the presence of primer sequences. These data were used to confirm that the performance of the preamp and PCR primers was adequate and consistent with previous primer lots. The performance of the amplification of the synthetic molecules met the pre-specified acceptance criteria.

This real-time reagent stability study established a 15-month shelf life of pre-amp and PCR primer mixes when stored at -20 ± 5 ℃. These data were confirmed by assessing the equivalence of MRD frequency in 40 clinical samples amplified with primer lots of different ages, and by tracking the stability of MRD measurements of synthetic molecules over time. The conditions tested in the real time stability study met the pre-specified acceptance criteria of a pairwise equivalence test of clinical specimens to be within ± 30% MRD frequency.

Sample Stability

Sample Storage and Shipment Stability

Specimens assessed by the clonoSEQ Assay were stable for the following storage conditions:

. At -15 ℃ to -25 ℃ 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 ℃ to 25 ℃ 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 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 .

Clinical Studies

Clinical Validation of the clonoSEO Assay for Chronic Lymphocytic Leukemia

Two separate studies were analyzed to support that MRD as estimated with the clonoSEQ Assay is prognostic of patient outcomes in CLL, including data from clinical trials NCT02242942 and NCT00759798.1,2,3

The primary objective of the first study was to evaluate the ability of the clonoSEQ Assay to predict progression-free survival (PFS) at the MRD threshold of 105 using available blood samples from patients accrued under clinical trial NCT02242942, protocol BO25323, "A Prospective, Open-Label, Multicenter Randomized Phase III Trial to Compare The Efficacy and Safety of A Combined Regimen of Obinutuzumab and Venetoclax (GDC-0199/ABT-199) Versus Obinutuzumab and Chlorambucil in Previously Untreated Patients With CLL and Coexisting Medical Conditions." The study was also

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designed to evaluate the clinical utility of the clonoSEO Assay using continuous MRD measures and monitoring MRD across multiple time points.

Clinical trial NCT02242942 is a Phase III randomized trial to compare, in previously untreated patients, the efficiency and safety of a combined regimen of obinutuzumab and venetoclax (GDC-0199/ABT-199) versus obinutuzumab and chlorambucil. Samples and outcomes data were collected from 445 patients. For the clinical trial, blood was collected at multiple timepoints during and after treatment; for this study, only samples collected three months following treatment (FUM3) or later were included in analyses. While all available specimens from this trial were tested with the clonoSEO Assay. 359 of the 445 patients originally enrolled in clinical trial NCT02242942 had both clinical outcomes data and sample material from the FUM3 timepoint available for analysis. Of these, one patient's sample failed QC, leaving 358 with usable clonoSEQ Assay MRD data. Twenty-one patients progressed prior to the FUM3 timepoint, leaving 337 patients for primary analysis.

The ability of MRD measurements from the clonoSEO Assay at FUM3 to predict PFS was evaluated in 337 patients. MRD negativity at < 105 by the clonoSEO Assay significantly predicted PFS (likelihood ratio P = 3.075 x 10-13; log-rank P = 7.38 x 10-12), with a 6.64-fold higher event risk in MRD positive patients (MRD > 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 < 10 had superior progression-free survival compared to patients with MRD > 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

1. 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
1. 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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Regulation Number and Section

§ 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 ]”).