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/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).

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 decisionmaking and in conjunction with other clinicopathological features.

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

Device Description

A description of required equipment, software, reagents, vendors, and storage conditions were provided, and are described in the product labeling. Adaptive Biotechnologies assumes responsibility for the device.

AI/ML Overview

Here's an analysis of the acceptance criteria and the study that proves the clonoSEQ® Assay meets those criteria, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are derived from the "Special Controls" section (S(b) Design verification and validation must include...) and from the performance study results. The reported device performance aligns with specific metrics and ranges demonstrated in the various analytical and clinical studies.

Acceptance Criteria Category	Specific Acceptance Criteria (from S.(b).1.iv)	Reported Device Performance (from L. Performance Characteristics)
Device Precision (Repeatability & Reproducibility)	Data using clinical samples covering the range of MRD frequencies and DNA inputs. Report SD/CV with 95% CI. Evaluate all sources of variability (site, operator, day, run, lot, instrument).	Precision of MRD Frequency: - MM: %CV ranged from 29.6% to 70.0% (Tables 1). - ALL: %CV ranged from 25.9% to 76.9% (Table 2). Precision of Malignant Cells Detected: - %CV ranged from 72% at 2.14 cells to 21% at 612.56 cells (Table 3). - Minimal contribution from Operator, Instrument Sets, Reagent Lots, Day, Run (0-3%). - All studies included assessment of instrument sets, operator, processing day, processing run, and reagent lot.
Device Linearity	Data generated from samples covering the device measuring range using a dilution panel created from clinical samples.	Linearity using Cell Lines (Table 5): - ALL: Linear range 0% to 100% (200ng), 3x10-5 to 30% (2µg), 0% to 10% (20µg). Average slopes close to 1 (0.95-1.01). - MM: Linear range 0% to 100% (200ng), 9.8x10-6 to 30% (2µg), 0% to 10% (20µg). Average slopes close to 1 (0.98-1.03). Linearity using Clinical Specimens (Table 6): - Linear range across several orders of magnitude for each condition (e.g., ALL 500ng: 2.8x10-5 to 8x10-3). Average slopes close to 1 (0.948-0.985). - Maximum deviation from linearity less than 5%.
Device Accuracy (Quantitative Measurement)	Comparison to flow cytometry across the measuring interval or to the predicate method.	Accuracy in Cell Mixtures Comparing to mpFC (Figure 6): Similar quantitative accuracy when comparing clonoSEQ with mpFC at frequencies above 1x10-4. Quantitation Bias on Clinical Specimens (Figure 8): Quantitative accuracy within ±25% across all tested diseased cell inputs. Modest upward bias at lower MRD and downward bias at higher MRD frequencies.
Device Analytical Sensitivity (LoB, LoD, LoQ)	Data using a dilution panel created from clinical samples.	Limit of Blank (LoB): Zero (based on 95th percentile of MRD frequencies in healthy bone marrow samples at 500 ng and 20 µg gDNA input). Limit of Detection (LoD): 1.903 malignant cells (95% CI; 1.75 - 2.07) across DNA input levels (Table 7). Limit of Quantitation (LoQ): 2.390 malignant cells (95% CI; 1.90 - 9.14) across DNA input levels (Table 7).
Analytical Specificity (Interference, Cross-Contamination)	Data including interference and cross-contamination, and index cross-contamination.	Interfering Substances (Tables 8 & 9): 5 endogenous and 3 exogenous substances tested. All conditions passed pre-specified MRD frequency equivalence margin of ±30%, concluding no substantial influence. Cross-Contamination/Sample Carryover: - Automated DNA extraction: 0 of 44 BMA/BMMC false calibrations for run-to-run; 1 of 44 BMA for well-to-well (minor, unimpactful). - gDNA contamination: No run-to-run (0 of 36); 8 of 712 well-to-well (likely vendor issue, unimpactful as <4x10-6 % contamination).
Validation of Pre-Analytical Methods (DNA Extraction)	DNA extraction methods and cell enrichment methods, as appropriate.	DNA Extraction Reproducibility: - Study 1 (healthy BMA): All samples passed pre-established acceptance criteria of ±30% MRD frequency variation; neither operator, instrument, reagent lot, or extraction run had significant effect on gDNA yields. - Study 2 (contrived ALL/MM): Met pre-established acceptance criteria of ±30% MRD frequency variation between different operators, instrument sets, and reagent lots.
Device Stability (Reagents, Specimen)	Real-time stability of reagents; specimen and prepared sample stability for each matrix, anticoagulant, storage/use conditions, and transport.	In-Use Stability: All tested conditions met pre-specified acceptance criteria. Real Time Stability of Pre-Amp and PCR Mixes: 12-month shelf life at -20±5℃ established; confirmed by equivalence of MRD frequency in clinical samples within 30% MRD frequency variation. Frozen Bone Marrow Stability: Stable at -15°C to -25°C for 12 months (±30% MRD frequency variation). Bone Marrow Stability RT/Refrigerated: Stable for 3 days at RT, 7 days refrigerated (±30% MRD frequency variation). Shipping Stability: Stable for up to 4 days (96 hours) under ambient shipping conditions (±30% MRD frequency variation). Freeze/Thaw Stability: Up to 3 freeze/thaw cycles for bone marrow samples acceptable (±30% MRD frequency variation).
Clinical Validity	Ability to measure MRD in claimed specimen type(s) from patients representative of intended use population. (Method comparison to predicate or clinical study with known outcomes.)	ALL Clinical Validation (COG AALL0232 & AALL0331): - clonoSEQ MRD negativity (<1x10-4) significantly predicts improved event-free survival (EFS) (P=0.0034), with a 2.74-fold higher event risk for MRD positive patients. - Continuous MRD levels significantly associated with EFS (P=0.0057), with 1.499-fold increase in event risk per 10-fold increase in MRD. - MRD negativity (<1x10-5) also significantly associated with EFS (P=8.4x10-4). - Differentiated EFS across three MRD bins (≤10-5, 10-5 - 10-4, ≥10-4) (P=0.00065). MM Clinical Validation (DFCI 10-106 & ALCYONE): - DFCI 10-106: Continuous clonoSEQ MRD levels modestly associated with DFS in CR patients (P=0.064). MRD status at <1x10-5 significantly predicts PFS in all evaluable patients (P=0.027). Continuous MRD associated with disease progression (P=1.9x10-4), 1.69 times higher event likelihood per 10-fold increase. - ALCYONE: MRD negative patients (≤10-5) had longer PFS compared to MRD positive patients.
DNA Extraction Equivalence	N/A (Internal study to characterize materials for other analyses)	MRD estimates on gDNA blends were comparable to MRD estimates of blended cells, demonstrating functional equivalence for analytical studies.
Precision of Sequence/Nucleotide Base Calls	N/A (Internal study to confirm reliability of reported sequences)	Overall Percent Agreement (OPA) of nucleotide sequences was high (e.g., 99.9968% for 1 allowed mutation), with low disagreement rate (approx. 3.5 parts per 100,000), corresponding to a Phred score of ~44.5. Supports reliable identification of clones.
Amplification Bias by Clonotype	N/A (Internal study to confirm robust amplification)	Data from synthetic templates and clinical samples demonstrated efficient and consistent amplification with nominal bias.
Concordance with mpFC in Clinical Samples	N/A (Comparison to existing reference standard)	ALL: NPA=93.5% (82.1-98.6%). Concordance correlation coefficient = 92.8% for MRD frequency. MM: NPA=97.9% (88.7-99.9%). Concordance correlation coefficient = 91.9% for MRD frequency.

2. Sample Size Used for the Test Set and Data Provenance

Precision/Reproducibility:
- MM: 23 patients
- ALL: 21 patients
- DNA inputs: 500ng, 2ug, 20ug
- MRD levels: 6 per DNA input
- Total contrived samples analyzed: 340 (from 360 initially tested due to QC failures)
- Total MRD measurements: 14,744
- Provenance: Clinical specimens from patients with MM and ALL, blended with gDNA from healthy donors (contrived samples). Not explicitly stated if retrospective or prospective, but likely retrospective collection of patient gDNA.
DNA Extraction Reproducibility:
- Study 1 (Healthy): BMA from 3 healthy subjects.
- Study 2 (Contrived): BMMCs from 3 ALL patients and 3 MM patients, diluted with BMA from healthy subjects.
- Provenance: Healthy subjects and ALL/MM patients.
Precision of Sequence/Nucleotide Base Calls:
- ID samples: 72 lymphoid malignancy samples and 9 cell lines.
- MRD samples: 20 replicates of the samples at disease inputs of ~2 to 600 malignant cells across four DNA inputs (10ng, 500ng, 2ug, 20ug).
- Provenance: Clinical samples from lymphoid malignancy patients and cell lines.
Linearity (Clinical Specimens):
- MM: 23 patients
- ALL: 21 patients
- DNA inputs: 3
- MRD frequencies: 6 per DNA input
- Provenance: Clinical specimens from patients with MM and ALL (same as precision study).
Analytical Sensitivity (LoB, LoD, LoQ):
- Trackable Ig sequences (for LoB): 66 patients (23 MM, 21 ALL, 22 other malignancy).
- Healthy bone marrow samples (for LoB): Number not specified, but likely a healthy cohort.
- Dilution series (for LoD/LoQ): 66 specimens from patients with lymphoid malignancies (23 MM, 21 ALL, 22 other malignancy).
- Provenance: Clinical specimens from patients with various lymphoid malignancies and healthy subjects.
Interfering Substances:
- Bone marrow from 4 different donors.
- Each condition replicated 8 times (4 donors x 2 replicates).
- Provenance: Bone marrow from donors.
Cross-Contamination/Sample Carryover (DNA Extraction):
- 3 ALL cancer cell lines and 3 MM cell lines.
- BMA pool of 2 healthy subjects or BMMC pool of 4 healthy subjects.
- PBS (blank) samples.
- Provenance: Cell lines and healthy subjects.
Cross-Contamination/Sample Carryover (gDNA):
- Peripheral blood from healthy subjects (MRD-negative specimens).
- Blends of cell line gDNA and gDNA from peripheral blood of healthy subjects (spiked to 5%).
- Provenance: Cell lines and healthy subjects.
Real Time Stability of Pre-Amp and PCR Mixes:
- 40 clinical samples.
- Provenance: Clinical samples.
Specimen Stability (Frozen, RT, Refrigerated, Shipping, Freeze/Thaw):
- Bone marrow samples from 4 donors.
- Provenance: Bone marrow from donors.
Concordance with mpFC in Clinical Samples:
- ALL: 273 ALL samples from Children's Oncology Group (COG) AALL0331 and AALL0232 regimens.
- MM: 91 MM samples from Dana-Farber Cancer Institute (DFCI) Study 10-106.
- Provenance: Retrospective clinical samples from ALL and MM patients enrolled in COG and DFCI studies, respectively.
Clinical Validation:
- ALL: 283 patients from COG studies AALL0232 and AALL0331 had sufficient leftover samples; final analysis used 273 specimens (10 failed QC). These were patients enrolled in previously conducted COG studies (retrospective).
- MM (DFCI Study 10-106): 365 patients originally had leftover samples; 323 patients were evaluable and passed QC. These were patients enrolled in an ongoing randomized Phase III study (retrospective use of collected samples).
- MM (ALCYONE study): Not explicitly stated how many samples were specifically used for the clonoSEQ assay, but the study enrolled 706 patients. This was a multicenter, randomized, open-label, active-controlled phase 3 trial (retrospective use of collected samples).

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The document does not explicitly state the number of experts or their specific qualifications for establishing the ground truth for the test set in most analytical studies.

For clinical validation: The clinical studies (COG for ALL, DFCI and ALCYONE for MM) rely on established clinical endpoints such as Event-Free Survival (EFS), Progression-Free Survival (PFS), and Disease-Free Survival (DFS). These outcomes are considered the ground truth and are monitored and determined by the clinical trial protocols and medical professionals involved in those trials. While not directly "experts for ground truth determination" in the sense of adjudicating individual cases for the device's test set, the clinical trial structure itself provides the gold standard for patient outcomes.
For comparison with mpFC: Multiparametric Flow Cytometry (mpFC) is described as the "reference standard for MRD." The comparison studies used a validated mpFC assay, implying that the results from this method were considered the ground truth for those comparisons. The experts operating the mpFC and interpreting its results would be specialists in flow cytometry, typically pathologists or laboratory scientists with expertise in hematological malignancies. The document does not specify the number of such experts.

4. Adjudication Method for the Test Set

The document does not describe any specific adjudication method (e.g., 2+1, 3+1) for establishing ground truth within the analytical or clinical validation studies. Instead, the ground truth is established by:

Expected concentrations: In analytical studies like precision, linearity, LoD/LoQ, and functional equivalence, "known" or "expected" MRD levels were created by blending gDNA or cells at specific, pre-determined concentrations. This creates a quantitative ground truth by experimental design.
Reference Method: For the "Comparison Studies (Accuracy of Quantitative Measurement)," particularly the concordance with mpFC, the mpFC results were considered the reference standard, acting as the ground truth.
Clinical Outcomes: For the clinical validation studies, the ground truth is the patient's clinical outcome (EFS, PFS, DFS), which are objective and determined by the clinical trial protocols rather than individual expert adjudication of the clonoSEQ results themselves.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No multi-reader multi-case (MRMC) comparative effectiveness study was done. The clonoSEQ Assay is an automated in vitro diagnostic (IVD) device. Its measurements are quantitative and are not dependent on human reader interpretation of images or complex diagnostic features in the same way an AI for medical imaging might be. Therefore, the concept of "human readers improve with AI vs. without AI assistance" is not directly applicable to this device. The device itself provides the measurement, and clinicians (healthcare professionals) then interpret these results in conjunction with other clinical features.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study

Yes, the vast majority of the performance studies presented are standalone algorithm-only performance studies. The clonoSEQ Assay, run at Adaptive Biotechnologies, is an automated system (sequencing and bioinformatics pipeline) that processes samples and generates quantitative MRD results. The precision, linearity, analytical sensitivity, specificity, and stability studies all evaluate the performance of this automated system directly, without a human-in-the-loop interacting with the algorithm's output during the measurement process. The only human involvement is at the input (DNA extraction, library preparation) and output (interpretation of the final report by qualified healthcare professionals).

7. Type of Ground Truth Used

Contrived Samples/Known Concentrations: For precision, linearity (cell lines and clinical specimens), analytical sensitivity (LoD/LoQ), DNA extraction reproducibility, and some cross-contamination studies, the ground truth was established by creating samples with "known" or "expected" concentrations of malignant cells/gDNA through precise dilutions and blending.
Reference Standard (mpFC): For accuracy and concordance studies, Multiparametric Flow Cytometry (mpFC) was used as the reference standard.
Clinical Outcomes Data: For clinical validation studies (ALL and MM), the ground truth for effectiveness was patient outcomes data, specifically Event-Free Survival (EFS), Progression-Free Survival (PFS), and Disease-Free Survival (DFS) from well-characterized clinical trials.

8. Sample Size for the Training Set

The document does not explicitly describe a separate "training set" for the clonoSEQ assay's algorithm development in the way one might for a machine learning model. The provided information focuses on analytical and clinical validation of the finalized assay.

The description of the "Adaptive analysis pipeline" mentions a "proprietary algorithm with in-line controls to remove amplification bias" and "Calibrations" where sequences are compared against a "B cell repertoire database." This database and any associated models would have been developed using a larger, likely internal, dataset for algorithm training and optimization. However, the size and nature of this "training set" are not provided in this regulatory document.

9. How the Ground Truth for the Training Set Was Established

Since an explicit "training set" is not detailed, the method for establishing its ground truth is also not provided. For an immune repertoire sequencing assay like clonoSEQ, the "ground truth" for training would likely involve:

Known Ig/TCR rearrangements: Databases of known or characterized V(D)J rearrangements.
Healthy vs. Malignant samples: Large cohorts of samples with confirmed diagnoses (healthy, various lymphoid malignancies) to learn characteristic clonal patterns and distinguish them from polyclonal backgrounds.
Synthetic sequences: Designer sequences with known properties to test and tune amplification, sequencing, and bioinformatics pipeline parameters without biological variability.

The document mentions that the "uniqueness value" of sequences (used for sensitivity bin assignment and LoD/LoQ estimation) is derived from comparison against a "B cell repertoire database." This database would constitute a form of ground truth for characterizing sequence uniqueness and prevalence.

Summary

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EVALUATION OF ATUOMATIC CLASS II DESIGNATION FOR clonoSEQ® ASSAY DECISION SUMMARY

A. DEN Number:

DEN170080

B. Purpose for Submission:

De novo request for evaluation of automatic class III designation for the clonoSEQ® Assay

C. Measurand:

Rearranged IgH (VDJ), IgH (DJ), IgK, and IgL receptor gene sequences, as well as translocated BCL1/IgH (J) and BCL2/IgH (J) sequences.

D. Type of Test:

Multiplex polymerase chain reaction and next generation sequencing-based in vitro diagnostic to evaluate minimal residual disease

E. Applicant:

Adaptive Biotechnologies Corporation

F. Proprietary and Established Names:

Adaptive Biotechnologies clonoSEQ Assay

G. Regulatory Information:

1. Regulation section:
  21 CFR 866.6100
1. Classification:
  Class II (Special Controls)
1. Product code:
  QDC

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1. Panel:
  88 – Pathology

H. Indications For Use:

1. Indications for Use:

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

2. Special conditions for use statement(s)

For prescription use.

For in vitro diagnostic use.

3. Special instrument requirements:

Illumina NextSeq™500 System (qualified by Adaptive Biotechnologies)

I. Device Description

1. DNA Extraction

DNA extraction methods have been validated using volumes ranging from 250 µL to 1 mL bone marrow aspirate (BMA)collected in (b) (4) The extraction method isolates DNA 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

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the wash steps, the DNA is eluted from the substrate. DNA is quantified using a spectrophotometer; the measured DNA concentration is used to add up to 20ug of gDNA to the assay. The MRD test can be performed with 500ng - 20μ g gDNA. Internal controls in the PCR and sequencing steps are used to confirm that sufficient gDNA has been amplified and that amplification was successful.

2. Library preparation

Genomic DNA is amplified using locus-specific multiplex PCR using V. D. and J gene primers containing molecular barcodes to amplify IgH (V(D)J), IgH (DJ), IgK, IgL, BCL1/IgH (J), BCL2/IgH (J), 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.

3. Sequencing and data analysis

Sequencing is conducted with the Illumina NextSeq™ 500 System. The sequencing process incorporates multiple quality checks. Sequencing output is then processed by the bioinformatics pipeline software as follows:

a. Flowcell level metrics:

The analysis pipeline performs quality control (QC) 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%.

b. 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.

c. Read assignment to receptors:

The pipeline assigns reads to rearranged receptors for each sample after demultiplexing.

d. Clonal sequence determination:

After assigning reads to receptor loci, the pipeline then clusters reads into clonal receptor sequences.

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e. Sample Level OC Checks:

The pipeline performs a series of sample level QC 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 number 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 process together.

f. 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).

g. 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 LOD and LOQ values. The analysis pipeline then reports whether ID sequences were detected above the LOO, above LOD but below LOQ, below LOD, or not detected.

h. Control Materials:

The following controls are used to measure the success of DNA extraction, PCR amplification, and sequencing:

i. Synthetic Internal Controls:

Each sample includes two sets of internal synthetic controls. The controls are panels of synthetic analogues of somatically rearranged B-cell receptor (BCR) immune receptor molecules. The composition of the reference template pools

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before and after amplification is measured and used for OC. 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 used to remove residual primers.The lack of amplification of these molecules is used to confirm the success of primer removal.

ii. 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.

iii. 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 peripheral blood mononuclear cells and serves as an additional check to confirm successful product amplification. Buffer (1xTE) is used as the Amplification Negative Control.

Sequencing Process Controls: iv.

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.

i. Result Reporting:

The pipeline renders results into a PDF-formatted patient report. The 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, below LOQ, or not detected) and quantitation for the tracked sequences within the most recent sample.

J. Standards/Guidance Documents Referenced

CLSI guideline EP06-A Evaluation of the Linearity of Quantitative Measurement

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Procedures- A Statistical Approach

K. Test Principle:

The clonoSEO Assay is an in vitro diagnostic assay 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/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 bone marrow (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 generic B cell repertoire database and assigned a uniqueness value based on the observed or estimated abundance of that sequence in the database. The uniqueness value 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) 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 MRD disease in a biologic sample.

Figure 1: clonoSEQ Assay Workflow

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

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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).

L. Performance Characteristics:

1. Analytical Performance

a. Functional Equivalence of gDNA Blends to Cellular Blends

Clinical specimens from 23 patients with MM and 21 patients with ALL were used for precision, quantitative accuracy and linearity studies. An additional 22 specimens from patients with other lymphoid malignancies were used to supplement the analytical sensitivity studies. Sample types included bone marrow aspirate (BMA), bone marrow mononuclear cells (BMMCs). CD138+ bone marrow cells, peripheral blood, and peripheral blood mononuclear cells (PBMCs). gDNA was isolated from these clinical samples and blended with gDNA isolated from bone marrow to contrive specific MRD levels for the analytical studies.

In order to evaluate whether the blended DNA from clinical specimen that are described above were suitable for specific analytical studies, a study was performed to evaluate whether MRD estimates from blended gDNA (i.e., gDNA first isolated from specimens and then blended with gDNA from peripheral blood mononuclear cells to create specific MRD levels) were equivalent to MRD estimates from gDNA isolated from blended specimens (i.e., specimens were blended with peripheral blood mononuclear cells at known concentrations prior to gDNA isolation). The accuracy and linearity of the assay results were assessed across 11 MRD frequency levels, ranging from 3.3x10-1 to 3.0x103, for both blended gDNA and DNA extracted from blended cells. These dilutions included levels below the LoD and spanned the range of reportable MRD levels. The MRD estimates on gDNA blends were comparable to the MRD estimates of the blended cells they were intended to mimic. Therefore, the blended gDNA from the clinical samples was determined to be functionally equivalent to clinical specimens for use in specific analytical studies.

b. Precision/Reproducibility Studies

Precision studies tested gDNA extracted from clinical specimens from 23 patients with MM and 21 patients with ALL. 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 three DNA inputs (500ng, 2ug,

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20ug) and six MRD levels were tested at each DNA input for each patient sample. The studies were designed to test the MRD levels of 2.8x10-3, 8.0x10-3, 2.8x10-4, 8.0x104, 2.8x103 and 8.0x103 at 500 ng DNA input: 7.0X106, 2.0x105, 7.0x103. 2.0x10-4, 7.0x10-4 and 2.0x10-3 at 2 µg DNA input; and 7.0 x10-7, 2.0x10 -9, 7.0x10 °, 2.0x105, 7.0x105 and 2.0x104 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.

The precision study used a main effects screening design over 21 calendar days. This study used 10 runs, with 2 PCR plates in 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 is summarized in Figure 2.

Figure 2: Precision Study Design Schematic

Image /page/7/Figure/3 description: This 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 operator is responsible for a different set of reagent groups, thermal cycler/liquid handler sets, and NextSeq numbers. The run numbers are listed at the bottom, ranging from 1 to 10.

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 (88 MRD measurements) failed sample QC due to insufficient sequencing coverage. While normal operating procedures permit re-sequencing, for this analysis these 2 samples were classified as failures. The analysis used the remaining 340 contrived samples with up to 44 MRD measurements per sample, for a total of 14,744 MRD measurements.

The precision of the clonoSEQ assay is largely dependent upon the number of malignant cells that are being evaluated, rather than the actual MRD frequency. Consequently, the same MRD frequency is expected to have lower precision at lower DNA inputs. For this study, precision estimates were first calculated based on the MRD frequency per DNA input, followed by estimates of imprecision of the absolute number of malignant cells detected per reaction.

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i. Precision of MRD Frequency for MM and ALL

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. The analysis was done separately for MM and ALL, and the data are summarized in Tables 1 and 2. Precision ranged from 29.6% to 70.0% CV for MM and 25.9% to 76.9% CV for ALL.

In these tables, 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 the patient reports.

DNA Input	MRD Frequency	Measurements	Patients*	%CV	Frequency Range(95% CI)
500 ng	3.5x10-5	378	21	70.0	0 - 9.0x10-5
	8.9x10-5	378	21	52.6	1.9x10-5 - 2.0x10-4
	3.0x10-4	414	23	37.6	1.2x10-4 - 5.7x10-4
	8.4x10-4	460	23	34.8	3.9x10-4 - 1.6x10-3
	2.7x10-3	460	23	32.3	1.4x10-3 - 5.1x10-3
	6.5x10-3	460	23	30.1	3.6x10-3 - 1.2x10-2
2 µg	8.1x10-6	378	21	69.2	0 - 2.1x10-5
	2.3x10-5	378	21	52.1	5.7x10-6 - 5.4x10-5
	7.8x10-5	414	23	38.8	3.1x10-5 - 1.5x10-4
	2.1x10-4	460	23	34.4	1.0x10-4 - 4.0x10-4
	7.4x10-4	460	23	32.4	3.7x10-4 - 1.4x10-3
	2.0x10-3	460	23	30.2	1.1x10-3 - 3.8x10-3
20 µg	8.6x10-7	378	21	65.6	0 - 2.1x10-6
	2.3x10-6	378	21	51.7	5.1x10-7 - 5.0x10-6
	8.2x10-6	414	23	37.2	3.5x10-6 - 1.5x10-5
	2.3x10-5	460	23	33.3	1.2x10-5 - 4.5x10-5
	7.6x10-5	460	23	31.1	4.1x10-5 - 1.4x10-4
	2.1x10-4	414	23	29.5	1.2x10-4 - 3.7x10-4

Table 1: Precision of the clonoSEQ Assay in MM Samples

Note: Some contrived samples included a subset of patient samples

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DNA Input	MRD Frequency	Measurements	Patients	%CV	Frequency range(95% CI)
500 ng	3.2x10-5	378	21	76.9	0 - 9.3x10-5
	8.6x10-5	378	21	54.1	1.0x10-5 - 1.9x10-4
	2.8x10-4	378	21	33.0	1.0x10-4 - 4.8x10-4
	7.6x10-4	420	21	29.3	3.4x10-4 - 1.2x10-3
	2.5x10-3	420	21	26.5	1.0x10-3 - 4.0x10-3
	6.0x10-3	420	21	25.9	2.2x10-3 - 9.8x10-3
2 µg	7.6x10-6	378	21	74.1	0 - 2.0x10-5
	2.1x10-5	378	21	47.4	3.8x10-6 - 4.0x10-5
	6.8x10-5	378	21	33.3	2.2x10-5 - 1.1x10-4
	1.9x10-4	420	21	29.1	7.0x10-5 - 3.1x10-4
	6.8x10-4	420	21	27.1	2.5x10-4 - 1.1x10-3
	1.9x10-3	420	21	25.9	6.9x10-4 - 3.1x10-3
20 µg	8.5x10-7	378	21	74.8	0 - 2.2x10-6
	2.3x10-6	378	21	49.8	4.0x10-7 - 4.7x10-6
	7.5x10-6	378	21	34.5	2.6x10-6 - 1.3x10-5
	2.1x10-5	420	21	27.9	8.5x10-6 - 3.4x10-5
	6.9x10-5	420	21	26.7	2.5x10-5 - 1.1x10-4
	2.0x10-4	378	21	26.5	7.1x10-5 - 3.3x10-4

Table 2: Precision of the clonoSEQ Assay in ALL Samples

ii. Precision of Malignant Cells Detected

The precision for detection of malignant cells detected was evaluated across a range of tested malignant cells (2.14 - 612.56). For this analysis, the results from all of the DNA inputs across MM and ALL were pooled into a single analysis that is summarized in Table 3. As expected, the precision was primarily influenced by the number of cells being evaluated. Precision ranged from %CV = 72% at 2.14 cells to %CV = 21% at 612.56 cells. The tested factors (Operator, Instrument Sets, Reagent Lots, Day, Run) minimally contributed to variability, with attributable %CV ranging from 0% to 3% (Table 3). The majority of the observed variation is due to the residual variability. Residual variability refers to the amount of variability remaining after accounting for the variability explained by the tested factors.

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		%CV Attributed to Each Variable at CellInputs*
	# of Input CancerCells	2.14	6.13	21.44	61.26	214.4	612.56
	Instrument Set	0%	1%	0%	1%	1%	0%
	Operator		1%	0%	2%	0%	0%
	Processing Day	0%	0%	1%	0%	0%	3%
	Processing Run	0%	0%	1%	0%	0%	0%
Lot-to-Lot	Reagent Lot	2%	0%	0%	2%	2%	1%
Variability
	Residual Variability	72%	ર્ 1 %	30%	24%	20%	19%
	Total MRD	2268	2268	2376	2640	2640	2552
N	Measurements

Table 3: Summary of the clonoSEQ Assay Precision of Malignant Cells Detected

These values were aggregated across diseases (ALL and MM) and 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 3). 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.

Figure 3: Sadler's Precision Profile (Coefficient of Variation) of the clonoSEQ Assay as a Function of Input Cancer Cells

Disease Indication · ALL A MM

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c. DNA Extraction Reproducibility

Precision studies (described above) were conducted with extracted, pooled and aliquoted genomic DNA from patient specimens. In order to determine the impact of the DNA extraction step on the reproducibility of the clonoSeq assay, two reproducibility studies were conducted beginning with bone marrow aspirates. For the first study. BMA from 3 heathy subjects were extracted using 250 uL or 500 uL of BMA. Two (2) replicate aliquots of each sample were extracted in each of 4 separate extraction runs using 2 operators, 2 instruments, and 2 extraction reagent lots. DNA yields from the 250 uL input ranged from 9.6 ug to 21.7 ug of DNA. DNA yields from the 500 µL input of BMA ranged from 18.7 µg to 45.9 µg of DNA. Neither operator, instrument, reagent lot, or extraction run had a significant effect on gDNA yields. All samples had acceptable A260/A280 measurements within the required range of 1.7 to 2.3. The sequencing data were assessed for the impact of the different extraction conditions on the ratio of rearranged B cell receptors to the total number of cells as measured by the clonoSEQ assay and all samples passed pre-established acceptance criteria of ± 30% MRD frequency.

A second study was conducted to evaluate DNA extraction on MRD estimation. Samples were created by diluting bone marrow mononuclear cells (BMMCs) from 3 ALL and 3 MM patients with BMA from healthy subjects to a concentration of ~50 cells per disease sample per 1 million BMA cells. The resulting simulated MRD levels ranged from 4.9 x 106 to 1.5 x 10- malignant cells. Four (4) extraction runs were performed on 500 µL BMA and on 1.0 mL BMA, each in duplicate using 2 operators, 2 instruments, and 2 lots. The study met the pre-established acceptance criteria of ± 30% MRD frequency between different operators, instrument sets, and reagent lots.

d. Precision of Sequence/Nucleotide Base Calls

The clonoSEO assay reports out the sequence information for dominant clones which can then be tracked in future measurements. Therefore, the repeatability of sequences generated by of the clonoSEO Assay was assessed using a two-step process. First, ID samples from 72 lymphoid malignancy samples and 9 cell lines were processed to determine the baseline calibrating clonotype nucleotide sequences. Next, 20 replicates of the samples were run at disease inputs of ~2 to 600 malignant cells across four DNA inputs (10ng, 500ng, 2ug, 20ug). The replicates were tested using 3 operators, 2 instrument sets, and 4 reagent lots. These data were used to assess the observed rate of agreement between the nucleotide sequences chosen for tracking and the nucleotide sequences observed in contrived samples from the same biological specimens.

For each calibrating clonotype sequence in an ID sample, all sequences in the corresponding MRD samples within N bp were included for assessment of overall percent agreement (OPA), where N is defined for each sequence as the number of allowable mutations determined during specimen characterization by the calibration algorithm. N is chosen to capture somatic variation among B cells from the same

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clonal lineage without incorrectly grouping sequences from different clonal lineages. Once this population was established, the OPA between the original calibrating clonotype sequence and the sequences identified in the MRD assessment was calculated.

Table 4 reports the number of nucleotides assessed, the OPA, the lower and upper 95% confidence limits, and the OPA restated in the same terms as a Phred quality score (i.e., -10 x log10 disagreement rate). This test assessed approximately 442.5 million nucleotides for sequence agreement, with an overall disagreement rate of approximately 3.5 parts per 100.000 (corresponding to a Phred score of about 44.5; in typical NGS applications a Phred score of 30 or higher constitutes a high-quality base call).

AllowedMutations(N)	NucleotidesAssessed	OPA	LCL	UCL	Phred
1	135,025,044	99.9968	99.9967	99.9969	44.9
2	57,248,770	તેવે તેતે રિજેવર	99.9964	99.9967	44.6
3	151,018,837	dd dde 2	99.9965	99.9966	44.6
ব	82,780,612	99.9960	ਰੇਰੇ ਰੇਰੇਟਰੇ	99.9962	44.0
റ	13,918,166	99.9966	99.9963	dd 9969	44.6
6	2,587,014	99.9961	99.9953	99.9968	44.1

Table 4: Summary of Sequence Agreement Metrics

The data support that the sequences reported by clonoSEQ assay are reproducible with low sequencing error rates. This demonstrates that the sequences identified in the dominant clones during the clonoSEQ Clonality ID assessment can be reliably identified in the clonoSEQ MRD tracking assessment.

a. Amplification Bias by Clonotype

Two types of studies were executed to assess amplification bias. One study used a comprehensive panel of synthetic double-stranded molecules representing rearrangements of the targeted exons, while the other used clinical samples. Data from amplification of the synthetic templates demonstrate that the clonoSEQ Assay amplifies the targeted exon segments efficiently and consistently with nominal bias. These conclusions were supported by data from clinical samples, which show that patients who carry certain exons in their malignant clonotypes do not have biased precision profiles.

e. Linearity

Linearity using Cell Lines i.
Linearity of the clonoSEQ Assay using 3 ALL cell lines (SUP-B15, GM14952,

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and GM20930) and 3 MM cell lines (IM9, U266, and L363) was evaluated by blending cell line gDNA with gDNA from healthy subjects, using DNA inputs of 200 ng, 2 µg and 20 µg gDNA, and tested at zero and across 11 MRD frequencies at each DNA input. The frequency range of 6.0x10 to 1 was tested at the 200 ng DNA input. The frequency range of 6.5x100 to 1 was tested at the 2 µg DNA input. The frequency range of 6.6x107 to 0.1 was tested at 20 µg 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 and disease type across the entire tested range (Table 5 and Figure). This study demonstrated linearity of MRD frequencies across several orders of magnitude for each condition tested.

Table 5: Linearity of the clonoSEO assay using cell lines

DiseaseIndication	InputDNA	TestedRange	Linear Range	Average Slope(Range)*	AverageIntercept(Range)*
ALL celllines	200ng	0 to100%	0% to 100%	1.01(0.98 to 1.05)	-0.11(-0.31 to 0.16)
ALL celllines	2µg	0 to100%	$3 x 10^{-5}$ to 30%	0.98(0.98 to 1.00)	-0.17(-0.39 to -0.04)
ALL celllines	20µg	0 to 10%	0% to 10%	0.95(0.88 to 0.99)	-0.26(-0.63 to -0.05)
MM celllines	200ng	0 to100%	0% to 100%	1.03 (0.98 to1.11)	-0.07 (-0.08 to -0.04)
MM celllines	2µg	0 to100%	$9.8 x 10^{-6}$ to 30%	1.02(0.98 to 1.06)	-0.03(-0.08 to 0.02)
MM celllines	20µg	0 to 10%	0% to 10%	0.98(0.96 to 0.99)	-0.15(-0.22 to -0.04)

*Range represents the range of values across the three cell lines tested.

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Figure 4: Linearity of the clonoSEQ Assay. Expected (x-axis) and Observed (y-axis) MRD Frequency of Six Cell Lines.

ii. Linearity using Clinical Specimens

The precision study described in section (L)(1)(b) evaluated blended gDNA extracted from 23 MM and 21 ALL specimens at three DNA inputs and 6 MRD

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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 6. 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. This study demonstrated linearity across a wide range of MRD frequencies for each condition tested using clinical specimens. Results from 3 representative specimens for each ALL and MM are visualized in Figure 5.

		Combined Analysis			Summary of individual patient analyses
DiseaseIndication	InputDNA	TestedRange	LinearRange	Slope	Intercept	AverageSlope	AverageIntercept	SlopeRange	InterceptRange
ALL	500ng	2.8x10-5 to8x10-3	2.8x10-5 to8x10-3	0.948	-0.214	0.948	-0.214	0.853 to1.073	-0.461 to0.018
	2 μg	7x10-6 to2x10-3	7x10-6 to2x10-3	0.985	-0.074	0.985	-0.074	0.909 to1.076	-0.757 to0.233
	20 ug	7x10-7 to2x10-4	7x10-7 to2x10-4	0.978	-0.101	0.978	-0.101	0.859 to1.029	-1.018 to0.336
MM	500ng	2.8x10-5 to8x10-3	2.8x10-5 to8x10-3	0.962	-0.143	0.956	-0.158	0.853 to1.148	-0.462 to0.183
	2 μg	7x10-6 to2x10-3	7x10-6 to2x10-3	0.986	-0.040	0.983	-0.049	0.924 to1.068	-0.341 to0.246
	20 ug	7x10-7 to2x10-4	7x10-7 to2x10-4	0.985	-0.034	0.981	-0.052	0.933 to1.075	-0.419 to0.611

Table 6: Linearity of clonoSEQ assay using clinical specimens

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Image /page/16/Figure/0 description: The image contains six scatter plots arranged in a 3x2 grid. Each plot shows the relationship between 'Observed MRD Frequency' on the y-axis and 'Expected MRD Frequency' on the x-axis, both on a logarithmic scale. The plots are grouped by 'ALL' and 'MM' with varying DNA input amounts: 500 ng, 2 µg, and 20 µg. Each plot is labeled with '(b) (4)' in the top left corner, and a legend at the bottom indicates different data points represented by symbols such as circles, triangles, and plus signs, corresponding to specific samples like 'ALL-25', 'ALL-36', 'MM-16', and 'MM-3'.

Figure 5: Linearity of the clonoSEQ Assay. The Expected (x-axis) and Observed (y-axis) MRD Frequency of Six Representative Clinical Samples.

Analytical Sensitivity f.

i. Limit of Blank (LoB)
The LoB was determined by measuring the specificity of a patient's trackable immunoglobulin (Ig) sequences. These sequences were identified from samples

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from 66 patients diagnosed with a lymphoid malignancy (23 MM, 21 ALL, and 22 other malignancy). 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 and 20 ug gDNA input from DNA extracted from healthy bone marrow. Therefore, the LoB was zero, demonstrating that trackable Ig sequences are highly patient-specific.

ii. Limit of Detection (LoD)/Limit of Quantitation (LoQ)

The LoD and LoO were determined by blending gDNA extracted from 66 specimens from patients with lymphoid malignancies (23 MM, 21 ALL, and 22 with other malignancy) into 500 ng and 20 ug of gDNA extracted from bone marrow of healthy subjects. A dilution series of 22.97, 10.72, 4.59, 2.14, and 0.94 malignant cell equivalents for each patient was made for each DNA input level. Each sample was tested in duplicate for each of four reagent lots, resulting in 8 results for each of the 66 samples at each dilution condition. A probit approach was used to determine the LoD to be 1.903 malignant cells (95% CI; 1.75 - 2.07) based on the combined data from both DNA inputs (Table 7). 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 2.390 malignant cells (95% CI; 1.90 - 9.14) (Table 7).

Table 7: LoD/LoQ by MRD Cell Counts and MRD Frequency

Measure	Malignant Cells*	500 ng DNA InputFrequency (95% CI)	20 µg DNA InputFrequency (95% CI)
LoD	1.903 (1.75 - 2.07)	2.26x10-5(2.01x10-5 - 2.53x10-5)	6.77x10-7(6.02x10-7 - 7.61x10-7)
LoQ	2.390 (1.90 - 9.14)	2.39 x 10-5(2.26x10-5 - 7.01x10-5)	1.76x10-6(6.77x10-7 - 4.09x10-6)

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

The clonoSEO Assay can use a range of DNA inputs from 500 ng to 20 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/LoO at 500 ng and 20 µg of DNA input are shown in Table 7.

To confirm the LoD and LoQ, 1.903 and 2.390 malignant cell equivalents were spiked into 200ng, 500ng, 1μg, 2μg, 5μg, 10μg, 20μg, and 40μg of gDNA extracted from bone marrow of healthy subjects. The results showed that the LoD and LoQ of malignant cells detected remained consistent across all DNA input levels.

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g. Analytical Specificity

i. Interfering Substances

Testing was performed to characterize the effects of five endogenous (Table 8) and three exogenous (Table 9) substances at the concentrations listed below. The potential exogenous and endogenous substances were spiked separately into 250 uL aliquots of bone marrow from 4 different donors. Each condition was replicated for a total of 8 times (4 donors x 2 replicates) 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.

Table 8: Endogenous Interfering Substances Tested

Substance Name	Reference Level(Low)	RecommendedConcentration (High)	Acceptance Criteria:Pass/Fail
Bilirubin conjugated	3.4 µmol/L	342 µmol/L	Pass
Bilirubin unconjugated*	21 µmol/L	342 µmol/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

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

Table 9: Exogenous Interfering Substances Tested

Substance Name	Concentration(Low)	Concentration(High)	Acceptance Criteria:Pass/Fail
(b) (4)	1.8 mg/mL	3.6 mg/mL	Pass
(b) (4)	15 USP U/mL	30 USP U/mL	Pass
(b) (4)	N/A	N/A	Pass

The BMA samples were shipped to Adaptive containing 1.8 mg/ml (b) (4) ("Low" concentration) for anti-coagulation purposes. Additional (b) (4) was spiked in to achieve the High level
(b) (4)

inhibition was tested at a single spiked-in volume (2.5 ul).

ii. Cross-Contamination/Sample Carryover
The assessment of cross-contamination included two studies: one study to measure contamination of ID samples during automated DNA extraction of BMA and BMMC and one study to measure contamination of gDNA 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 3 ALL cancer cell lines and 3 MM cell lines 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

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included in this study. Samples were evaluated as to whether they correctly calibrated. There were no false calibrations for run-to-run with 0 of 44 BMA and 0 of 44 BMMC false calibrations. There was 1 false calibration for the well-towell study with 1 of 44 BMA and 0 of 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 provides 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 of incorrectly calling samples MRD positive was assessed using gDNA from peripheral blood from healthy subjects as MRD-negative specimens and blends of cell line gDNA and gDNA from peripheral 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 (0 of 36). Well-to-well cross contamination was observed in 8 of 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 to a final MRD call because tracked clonotype sequences are highly specific to each patient, so this level of 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.

h. Traceability (controls, calibrators, or methods)
The clonoSEQ Assay is not traceable to any known standard. Controls and quality metrics are described in the device description section.
i. Reagent Stability
- i. In-Use 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. (0) (4) gDNA was tested using seven replicates for all conditions tested. Acceptance criteria were based the on sequencing results meeting all QC metrics and all of the conditions tested met the pre-specified acceptance criteria and the clonoSEQ Assay in-use stability needs.

ii. 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

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set of synthetic double-stranded molecules representing rearrangements of the targeted exons to determine whether each manufactured lot of pre-amp 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 pre-amp and PCR primers was adequate and consistent with previous primer lots.

This real-time reagent stability study established a 12-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 of acceptance criteria of pairwise equivalence test of clinical specimens to be within 30% MRD frequency.

j. Specimen Stability
- i. Frozen Bone Marrow Stability at -15° C to -25° C

To demonstrate frozen bone marrow stability, four bone marrow samples from donors were aliquoted and stored frozen (-15°C to -25°C). Samples were tested after freezing and specific values to clones within the specimen were compared to baseline. Frozen bone marrow is stable at -15°C to -25°C for 12 months within the prespecified ±30% MRD frequency variation.

ii. Bone Marrow Stability at Room Temperature and Refrigerated

To demonstrate bone marrow stability, four bone marrow samples from donors were aliquoted and stored at room temperature (19°C to 25°C) or refrigerated (2°C to 8°C) for up to seven days. Bone marrow samples stored at room temperature (19℃ to 25℃) remained stable for three days. For bone marrow samples stored refrigerated (2°C to 8°C), MRD measurements for the clonoSEQ Assay remained stable for seven days. Acceptance criteria were based on the prespecified ±30% MRD frequency variation.

iii. Shipping Stability

Sample stability of bone marrow samples stored in Adaptive shipping containers at ambient temperature was tested under summer and winter shipping conditions. Study results demonstrated that samples are stable for up to 4 days (96 hours) under ambient shipping conditions based on an allowable 30% MRD frequency variation.

Freeze/Thaw Stability of Bone Marrow Samples iv.

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The stability of bone marrow samples was evaluated using four bone marrow samples from donors split into aliquots (0.25 ml) with one aliquot extracted upon receipt. The remaining aliquots were subjected to up 5 to freeze/thaw cycles. gDNA was extracted and the concentration was determined using a spectrophotometer. Each sample was processed using the clonoSEQ Assay. Bone marrow samples subjected to up to three freeze/thaw cycles continued to report acceptable sample MRD frequency within the prespecified ± 30% frequency variation.

2. Comparison Studies (Accuracy of Quantitative Measurement)

a. Assessment of clonoSEQ Assay Accuracy in Cell Mixtures Comparing to Multiparametric Flow Cytometry (mpFC)
The quantitative accuracy of the clonoSEQ Assay was compared to measurements obtained with a multiparametric flow cytometry (mpFC) method. Flow cytometry is considered the reference standard for MRD. Results were assessed using cell line blends. Measured MRD frequencies were compared against known frequencies based on diluting cell lines into background mononuclear cells at specific MRD levels. This study evaluated 2 MM cell lines (U266B1 and NCI-H929) and 2 ALL cell lines (SUP-B15 and GM20390). Each cell line was tested at 5 dilutions, from 5x107 to 1x102. Two replicates of each sample were assessed by the clonoSEQ assay and a validated mpFC assay. A pairwise comparison of MRD frequency measurements is shown in Figure 6. This study demonstrated similar quantitative accuracy when comparing clonoSEQ with mpFC at frequencies above 1x104.

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Image /page/22/Figure/0 description: This scatter plot compares MRD clonoSEQ to MRD Flow, with both axes displayed on a logarithmic scale ranging from 10^-6 to 10^-2. A dashed line represents the ideal correlation between the two methods. Data points are grouped by cell line, including GM20390, NCI-H929, SUP-B15, and U266B1, each represented by a different color and symbol.

Figure 6: Pairwise comparison of MRD frequency measurements with mpFC and the clonoSEQ Assay

Concordance with mpFC in clinical samples b.

Two concordance studies between mpFC and the clonoSEQ Assay were performed using clinical samples. For both studies, concordance was assessed two ways: concordance of MRD positive or negative calls and concordance of reported MRD frequency. One study used 273 ALL samples from the Children's Oncology Group (COG) AALL0331 (standard risk) and AALL0232 (high risk) regimens and compared the clonoSEO Assay to a validated mpFC assay. The other study performed a similar comparison using 91 MM samples from the Dana-Farber Cancer Institute (DFCI) Study 10-106 that were measured by both the clonoSEQ Assay and mpFC. MRD negativity was defined as < 1x104 for mpFC in ALL (a commonly used threshold in that patient population) and <1x103 for mpFC in MM. For the clonoSEO Assay, MRD calls were assessed at the LOD in both studies. The negative percent agreement (NPA) between the clonoSEQ Assay and mpFC was 93.5% for ALL and 97.9% for MM (Table 10).

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	clonoSEQ+	clonoSEQ+	clonoSEQ-	clonoSEQ-	mpFC reference
	mpFC+	mpFC-	mpFC+	mpFC-	PPA	NPA
ALL	43	117	3	110	48.5%(41.8-55.2%)	93.5%(82.1-98.6%)
MM	46	23	1	21	47.7%(32.5-63.3%)	97.9%(88.7-99.9%)

Table 10: Summary of mpFC vs. the clonoSEQ Assay Concordance Data for ALL and MM

Concordance of MRD frequency was visualized by plotting reported MRD frequency of mpFC against the clonoSEQ Assay for both MM and ALL (Figure 7). Concordance of MRD call is indicated by color/shape; blue circles indicate samples had concordant MRD positive calls, while orange triangle and red squares denote discordant calls, with orange triangles indicating that clonoSEQ identified the sample as MRD positive and red squares indicating that mpFC identified the sample as MRD positive. To simplify the plot, samples with concordant MRD negative calls were not plotted. To quantify the similarity of reported MRD frequencies, correlations were calculated for samples with either concordant calls or mpFC positive calls; MRD frequencies were highly concordant (ALL, concordance correlation coefficient = 92.8%; MM, concordance correlation coefficient = 91.9%). These data demonstrate that at high disease burdens mpFC and clonoSEQ report similar MRD levels, while clonoSEQ continues to detect MRD at lower frequencies.

Figure 7: Measurements of the clonoSEQ Assay Compared to Flow Cytometry Measurements from ALL (left) and MM (right) Clinical Studies.

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c. Analysis of Ouantitation Bias on Clinical Specimens
The precision study described in section (L)(1)(b) evaluated blended gDNA extracted from 23 MM and 21 ALL specimens at 3 DNA inputs and 6 MRD frequencies per DNA input. These data were reanalyzed to evaluate if there was a quantitation bias for clonoSEQ. Sample MRD frequencies measured with the clonoSEQ Assay were compared to the expected MRD, value as calculated using flow cytometry on the original clinical sample and applying the appropriate dilution factor.

For ALL and MM, the quantitative accuracy of the clonoSEO Assay was within ±25% across all tested diseased cell inputs (Figure 8). The assay tended to have a modest upward bias in MRD estimation at lower MRD frequencies and a modest downward bias at higher MRD frequencies.

Image /page/24/Figure/3 description: The image contains two plots comparing Bootstrap Bias Estimate vs Input Cancer Frequency for ALL and MM specimens. The x-axis represents the Input Cancer Frequency, ranging from 10^-6 to 10^-2, while the y-axis represents the Bootstrap Bias Estimate, ranging from -30% to 30%. Each plot shows data points for different DNA inputs: 500 ng, 2 μg, and 20 μg, represented by red circles, green triangles, and blue squares, respectively.

Figure 8: Estimates of Bias the clonoSEQ assay by MRD Frequency using clinical specimens

1. Clinical Performance
  Clinical validation for the measurement of MRD in ALL and MM was demonstrated using an analysis of samples obtained from two clinical studies for each disease indication.
Clinical Validation of the clonoSEQ Assay for Acute Lymphoblastic Leukemia in a. Children's Oncology Group (COG) Studies AALL0232 and AALL0331
The primary objective of this analysis was to establish the ability of the clonoSEQ Assay to predict event-free survival (EFS) at the MRD threshold of 10ª using available bone marrow samples from patients who were enrolled in previously conducted COG studies AALL0232 and AALL0331.

COG study AALL0331 is a Phase III randomized study of different combination

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chemotherapy regimens in pediatric patients with newly diagnosed standard risk Bprecursor acute lymphoblastic leukemia. COG study AALL0232 is a Phase III randomized study of dexamethasone versus prednisone during induction and highdose methotrexate with leucovorin rescue versus escalating-dose methotrexate without leucovorin rescue during interim maintenance I in patients with newly diagnosed high-risk acute lymphoblastic leukemia. Within these studies, bone marrow was collected at six separate time points to assess response to treatment, however, only the post induction marrow was used for MRD determination for these analyses.

Clinical samples (pre-treatment BMA and day 29 post-induction BMA) were collected from 619 individuals, with samples from 315 patients that were enrolled as part of the "high risk" COG protocol AALL0232 and samples from 304 patients enrolled as part of the "standard risk" COG protocol AALL0331. Available specimens from these trials were tested with the clonoSEQ assay and results from both studies were pooled into a single analysis. Specimens were selected based on having a sufficient quantity of gDNA, available MRD flow cytometry results, and patients with study related endpoints for EFS.

A subset of 283 of the 619 patients originally enrolled in COG studies AALL0232 and AALL0331 had leftover samples of sufficient amount that could be tested with the clonoSEO Assay. The population characteristics between these 283 patients were compared against the remaining 336 that were not tested and there were no significant differences in any characteristic that was evaluated, including age, gender, presence of specific genetic fusions, trisomy, and progression free survival. The 283 specimens were tested to evaluate the clinical performance of the clonoSEQ Assay and to demonstrate concordance in MRD measurements between the clonoSEQ Assay and results of original testing with a previous version of the clonoSEQ Assay and mpFC. Ten specimens did not pass QC, leaving results from 273 specimens available for the final analysis.

The clonoSEO Assay MRD negativity of < 1x10 + was found to predict improved event free survival (EFS) irrespective of age (P=0.0034; Figure 9). Results demonstrate a 2.74-fold higher event risk in MRD positive patients (MRD>10-10-1) compared to MRD negative patients (95% CI: 1.330-5.656). Similar findings were published in a broader COG analysis of the relationship between EFS and MRD negativity by an earlier version of the clonoSEO Assay in pediatric ALL (Wood et al. 2018).1

1 Wood B. Crossley B. Dai Y. et.al., Measurable residual disease detection by high-throughput sequencing improves risk stratification for pediatric B-ALL. Blood 2018; 131:1350-1359.

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Figure 9: Kaplan-Meier Survival Curve for EFS using the clonoSEQ Assay at an MRD cutoff of 1x10-4 in ALL

Image /page/26/Figure/1 description: This image is a survival probability graph that compares two strata: MRD less than or equal to 10^-4 and MRD greater than 10^-4. The x-axis represents time in days, ranging from 0 to 4000, while the y-axis represents survival probability, ranging from 0.00 to 1.00. The graph shows that the MRD less than or equal to 10^-4 strata has a higher survival probability than the MRD greater than 10^-4 strata.

The study was also designed to evaluate the clinical validity of the clonoSEQ Assay using alternative MRD thresholds and continuous MRD measures. Cox regression analysis of MRD and EFS using continuous MRD values demonstrates that the clonoSEQ Assay is significantly associated with EFS after adjusting for age (P=0.0057), and that each 10-fold increase in MRD level is associated with a 1.499fold increase in event risk (95% CI: 1.139-1.974). These data further demonstrate that the MRD level remains a significant predictor of EFS, even after accounting for age, gender, and genetic abnormalities, which demonstrates the validity of MRD measurement in ALL.

A qualitative assessment of MRD was also evaluated with MRD negativity defined as < 1X103 (Figure 10). This threshold is significantly associated with EFS (P=8.4x10 4).

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Figure 10: Kaplan-Meier Survival Curve for EFS using the clonoSEQ Assay at an MRD cutoff of 1x10-5 in ALL

Image /page/27/Figure/1 description: This image is a Kaplan-Meier survival plot comparing two strata: MRD <= 10^-5 and MRD > 10^-5. The x-axis represents time in days, ranging from 0 to 4000, while the y-axis represents survival probability, ranging from 0.00 to 1.00. The plot shows that the MRD <= 10^-5 strata has a higher survival probability than the MRD > 10^-5 strata. The number of individuals at risk at different time points is also displayed for each strata.

The clonoSEQ Assay was used to assess MRD at various disease burden thresholds to determine the correlation of MRD level with EFS. Patients who are clonoSEQ MRD negative (<1x10~) have longer EFS, followed by patients with MRD between 10-5 - 10-4 and, patients with MRD >10-4 (P=0.00065; Figure 11). These data demonstrate that patients with the lowest levels of MRD have better outcomes than patients with higher disease burden, regardless of risk stratification.

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Figure 11: Non-parametric Kaplan-Meier survival curve for EFS in clonoSEQ MRD positive and MRD negative patients using three MRD bins: ≤10°, 10° - 10°4 ≥10-4.

Image /page/28/Figure/1 description: This image is a survival probability graph. The x-axis is time in days from 0 to 4000, and the y-axis is survival probability from 0.00 to 1.00. There are three lines on the graph, each representing a different strata: MRD less than or equal to 10^-5, 10^-5 < MRD < 10^-4, and MRD greater than or equal to 10^-4.

These analyses demonstrated that MRD estimation by the clonoSEQ assay is associated with patient outcomes for B-cell precursor ALL.

b. Clinical Validation of the clonoSEO Assay for Multiple Myeloma

Two separate studies were analyzed to support that MRD as estimated with the clonoSEQ Assay is associated with patient outcomes in MM. Samples for the analysis of the clonoSEQ Assay performance in MM were obtained from an ongoing randomized, open label, Phase III study of lenalidomide and bortezomib in a combination therapy regimen (DFCI Study 10-106). Multiple timepoints were assessed in this two-arm analysis and not all patients have the same number of MRD assessments (see data in section L.3.b). Patients on Arm A (blinded to Adaptive Biotechnologies) had assessments after eight cycles of RVD (lenalidomide, bortezomib, and dexamethasone), and then after lenalidomide maintenance. Patients on Arm B (blinded to Adaptive Biotechnologies) were assessed following 3 cycles of RVD, following auto transplant, and again after two more cycles of RVD consolidation, and then following lenalidomide maintenance.

i. DFCI Study 10-106
The objective of this study was to establish that the clonoSEQ Assay is predictive

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of progression-free survival (PFS) and disease-free survival (DFS) in MM. Patient samples were accrued under DFCI Study 10-106, "A Randomized Phase III Study Comparing Conventional Dose Treatment Using a Combination of Lenalidomide, Bortezomib, and Dexamethasone (RVD) to High-Dose Treatment with Peripheral Stem Cell Transplant in the Initial Management of Myeloma in Patients up to 65 Years of Age."

A subset of 365 of the 720 patients originally enrolled in DFCI Study 10-106 had leftover samples of sufficient amount to be tested with the ClonoSEO Assay. The populations characteristics between these 365 patients were compared against the remaining 355 patients that were not tested and there were no significant differences in any characteristic that was evaluated, including age, gender, ISS staging, cytogenetic status and progression free survival. Samples from 365 patients were tested and results from 323 patients were evaluable and passed QC. Seventy-five (75) of these samples were from patients in complete response (CR) at the time of first MRD assessment. This study aimed to demonstrate the association of the first MRD measurement with PFS in patients who achieved CR and with PFS in all evaluable patients.

Samples from 75 patients who had achieved CR were evaluable for analysis. Continuous clonoSEO MRD levels were modestly associated with DFS in patients who have achieved CR (P=0.064), such that patient with lower MRD levels were less likely to progress.

The ability of the clonoSEQ Assay MRD measurements to predict PFS in all 323 evaluable patients was also assessed. clonoSEO measurements demonstrated that MRD status at a threshold of 1x105 at the time of first MRD measurement significantly predicts PFS in all patients (P= 0.027, Figure 12).

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Figure 12: Kaplan-Meier Survival Curve for PFS using the clonoSEQ Assay at an MRD cutoff of 10-3 in MM

Image /page/30/Figure/1 description: This image is a survival probability graph that compares two strata: MRD <= 10^-5 and MRD > 10^-5. The x-axis represents time in months, ranging from 0 to 80, while the y-axis represents survival probability, ranging from 0.00 to 1.00. The graph shows that the MRD <= 10^-5 strata has a higher survival probability than the MRD > 10^-5 strata. At time 0, the number of patients in the MRD <= 10^-5 strata is 99, while the number of patients in the MRD > 10^-5 strata is 224.

Cox regression analysis using a continuous measure of MRD was also associated with disease progression (P=1.9x10''). For every 10-fold increase in continuous clonoSEQ MRD measurement, the likelihood of an event is 1.69 times higher (95% CI:1.071-2.67).

ii. ALCYONE study

The ALCYONE Trial was a multicenter, randomized, open-label, activecontrolled phase 3 trial that evaluated daratumumab plus bortezomib, melphan and prednisone (D-VMP) versus bortezomib, melphan and prednisone (VMP) in 706 patients with newly diagnosed multiple myeloma who were ineligible for stem-cell transplantation. The result of this study was reported in Mateos et al. 2018.2

Within this trial, minimal residual disease was assessed by means of the clonoSEQ assay using bone marrow aspirate collected at screening, at the time of confirmation of complete response or stringent complete response, and at 12, 18, 24, and 30 months after the first dose in patients having a complete response or stringent complete response. Patients who did not achieve a CR were considered to be MRD positive. An MRD threshold of 10- was used for analysis.

Regardless of treatment group, patients who were MRD negative by the clonoSEQ Assay at ≤105 had longer progression-free survival (PFS) compared to

2 Mateos MV, Dimopoulos MA, Cavo M, et al., Daratumumab plus Bortezomib, Melphalan, and Prednisone for Untreated Myeloma. N Engl J Med 2018; 378:518-528.

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MRD positive patients (Figure 13).

Figure 13: Analysis of MRD with Progression-Free Survival. Patients who were MRD negative by the clonoSEQ Assay had longer PFS compared to MRD positive patients.

Image /page/31/Figure/2 description: This image is a Kaplan-Meier plot showing the proportion of patients surviving without progression over time, measured in months. There are four curves on the plot, representing different groups of patients based on their VMP MRD status: VMP MRD negative, D-VMP MRD negative, D-VMP MRD positive, and VMP MRD positive. The plot shows that patients who are VMP MRD negative have the highest proportion of survival without progression, while patients who are VMP MRD positive have the lowest.

c. Clinical Cutoff:
Not Applicable

M. Instrument Name

Illumina NextSeq™ 500 System (qualified by Adaptive Biotechnologies, Inc.)

N. System Descriptions

1. Modes of operation

The Illumina NextSeq™ 500 System is a high throughput sequencing system using Sequencing-By-Synthesis chemistry.

2. Software

The Adaptive clonoSEQ assay utilizes multiple software applications for performing and

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(

analyzing sequencing results. The principle software applications used with the instrument include Illumina's (b) Fastq (b) (4) NextSeq Software System (b) (4) , and the Adaptive clonoSEQ bioinformatics pipeline (b) (4)

1. Level of concern Moderate
1. Specimen Handling Refer to Device Description section above.
1. Calibration and Quality Controls Refer to Device Description section above.

O. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section Above:

The labeling supports the decision to grant the De Novo request for this device.

P. Patient Perspective

This submission did not include specific information on patient perspectives for this device.

Q. Identified Risks to Health and Identified Mitigations:

Identified Risks to Health	Identified Mitigations
Incorrect test results	General controls and special controls(1), (2) and (3)
Incorrect interpretation of test results	General controls and special controls(1), (2), (3) and (4)

R. Benefit Risk Summary

Summary of Assessment of Benefit

The Adaptive Biotechnologies clonoSEQ Assay is a Next generation sequencing-based in vitro diagnostic to evaluate minimal residual disease, evaluating rearranged IgH (VDI), IgK. and IgL receptor gene sequences, as well as translocated BCL1/1gH(J) and BCL2/IgH(J) sequences. This assay measures minimal residual disease (MRD) to monitor changes in burden of disease during and after treatment. There is significant potential benefit associated with the use of this device in the clinical setting to assist clinicians manage patients with ALL or MM in accordance with professional guidelines and in conjunction with other clinicopathological features.

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Summary of the Assessment of Risk For the Proposed Indications for Use:

There is potential risk associated with the use of this device, mainly due to 1) false positives, false negatives, and failure to provide a result and 2) incorrect interpretation of test results by the user.

The probable risk associated with the use of this device is that false positives, false negatives, and failure to provide a result or incorrect interpretation of test results by the user can result in mismanagement of patients, resulting in significant consequences. Mismanagement of patients can occur because the marker measurement from the device is erroneous or interpreted erroneously, diverting patients away from the correct management as per professional guidelines. The risk of mismanagement is partially attenuated because the results of this device are to be use with other clinicopathological factors. In addition, the analytical validation of this device, in part mitigates the risks associated with this device.

There is moderate uncertainty about risk due to insufficient experience with the use of this device.

Summary of the Assessment of Benefit-Risk, considering risk mitigation strategies For the Proposed Indications for Use:

In summary, the probable benefits of this device outweigh the probable risks associated with the device, when considering the mitigations provided by the limitations and special controls, beyond general controls.

S. Conclusion:

The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 866.6100. FDA believes that the class II device's stated special controls, in combination with the applicable general controls, provide a reasonable assurance of the safety and effectiveness of the device is classified under the following:

Product Code:	QDC
Device Type:	DNA-based test to measure minimal residual disease inhematological malignancies
Class:	II (Special Controls)
Regulation:	21 CFR 866.6100

(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

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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). A DNA-based test to measure minimal residual disease in hematological malignancies must comply with the following special controls:
- (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, but not limited to, 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:

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(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 wellcharacterized clinical specimens from patients with known clinical outcomes using a study design deemed acceptable by the 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% confidence interval for each level tested. The study must evaluate all sources of variability including, as appropriate, between-site and between operator (minimum of 3 sites of which 2 must be external with a minimum of 2 operators per site), between-day (minimum of 3 days), between-run, withinrun, between-lot (minimum of 3 lots), between instrument (minimum of 3 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.
Specimen and prepared sample stability data established for each specimen (I) matrix in the anticoagulant combinations and storage/use conditions that will be indicated, including specimen transport, as appropriate.

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(2) The intended use required on the label under 21 CFR 809.10(a)(4) and on the labeling under 21 CFR 809.10(b)(5)(ii), 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 morrow);
- (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 decisionmaking in conjunction with other clinicopathological features.
(3) The 21 CFR 809.10(b) labeling 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 (b)(1)(iv)(A) through (b)(1)(iv)(I).
(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 X 103 [Range = 0.8 X 10to to 2.0 X 10-3]").

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 ]”).