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The clonoSEQ Assay is an in vitro diagnostic that uses multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to identify and quantify rearranged IgH (VDJ), IgH (DJ), IgK and IgL receptor gene sequences, as well as translocated BCL1/1gH (J) and BCL2/1gH (J) sequences in DNA extracted from bone marrow from patients with B-cell acute lymphoblastic leukemia (ALL) or multiple myeloma (MM), and blood or bone marrow from patients with chronic lymphocytic leukemia (CLL).

The clonoSEQ Assay measures minimal residual disease (MRD) to monitor changes in burden of disease during and after treatment. The test is indicated for use by qualified healthcare professionals in accordance with professional guidelines for clinical decision-making and in conjunction with other clinicopathological features.

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

The clonoSEQ Assay is a next-generation sequencing (NGS) based assay that identifies rearranged IgH (VDJ), IgH (DJ), IgK, and IgL receptor gene sequences, as well as translocated BCL1/IgH (J) and BCL2/IgH (J) sequences. The assay also includes primers that amplify specific genomic regions present as diploid copies in normal genomic DNA (gDNA) to allow determination of total nucleated cell content.

Testing begins with gDNA extracted from the specimen supplied (Figure 1). Extracted gDNA quality is assessed and rearranged immune receptors are amplified using a multiplex PCR. Reaction-specific index barcode sequences for sample identification are added to the amplified receptor sequences by PCR. Sequencing libraries are prepared from barcoded amplified DNA, which are then sequenced by synthesis using NGS. Raw sequence data are uploaded from the sequencing instrument to the Adaptive analysis pipeline. These sequence data are analyzed in a multi-step process: first, a sample's sequence data are identified using the sample index sequences. Next, data are processed using a proprietary algorithm with in-line controls to remove amplification bias. When the clonoSEQ Clonality (ID) assessment is conducted, the immune repertoire of the sample is checked for the presence of DNA sequences specific to "dominant" clone(s) consistent with the presence of a lymphoid malignancy. Each sequence that is being considered for MRD tracking is compared against a B cell repertoire database and assigned a uniqueness value that, together with its abundance relative to other sequences, is used to assign the sequence to a sensitivity bin which will be used in the estimation of the reported LoD and LoO on the patient report. During clonoSEQ Tracking (MRD) assessment, the complete immunoglobulin receptor repertoire is again assessed, and the previously identified dominant clonotype sequence(s) are detected and quantified to determine the sample MRD level. The clonoSEQ Assay MRD assessment measures residual disease in a biologic sample.

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

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

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

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

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

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

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

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

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

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

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

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

4. Adjudication method for the test set

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

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

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

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

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

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

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

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

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

8. The sample size for the training set

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

The document states:

• "Raw sequence data are uploaded from the sequencing instrument to the Adaptive analysis pipeline. These sequence data are analyzed in a multi-step process: first, a sample's sequence data are identified using the sample index sequences. Next, data are processed using a proprietary algorithm with in-line controls to remove amplification bias."
• "When the clonoSEQ Clonality (ID) assessment is conducted, the immune repertoire of the sample is checked for the presence of DNA sequences specific to "dominant" clone(s) consistent with the presence of a lymphoid malignancy. Each sequence that is being considered for MRD tracking is compared against a B cell repertoire database and assigned a uniqueness value..."

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

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

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

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

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

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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.

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.

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.

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.

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