Navigate BioPharma Services AML-MRD Method Summary

Intended Use

The Acute Myeloid Leukemia Measurable Residual Disease by Multiparametric Flow Cytometry (AML MRD MFC) Test is a quasiquantitative, laboratory developed, in vitro test that utilizes consensus cell differentiation markers recommended by European Leukemia Network (ELN) to evaluate the presence of Leukemia Associated Immunophenotypes (LAIP) in the bone marrow (primarily) and peripheral blood (alternative when no bone marrow can be collected), of AML patients. AML-MRD MFC Test data collected from global clinical trials is interpreted centrally at Navigate BP CLIA Laboratory (a Novartis subsidiary), located in Carlsbad, CA. Navigate’s validation package fulfills Clinical and Laboratory Standards Institute (CLSI H62) guidelines commensurate with Secondary Clinical Trial Endpoint Data Submission and Patient Selection under CLIA (Tettero et al., 2023). AML-MRD MFC Test results in conjunction with standard clinical response criteria could be utilized to interpret the efficacy of investigational drugs for AML or High Risk-Myeloid Dysplastic Syndrome patients enrolled in clinical trials.

Disease Background & Method Rationale

AML is a cancer of the blood and bone marrow characterized by the rapid growth of abnormal white blood cells, known as myeloid blasts, which outgrow healthy blood cells resulting in bone marrow failure (Dohner et al., 2015). AML is often considered to be the most complex type of blood cancer due to its heterogenous morphology, immunophenotype, cytogenetics, molecular aberrations and poor survival outcomes following current approved treatment modalities (Hou & Tien, 2020). Indeed, the five-year survival rate is less than 30% for older patients (over 60 years) predominantly due to relapse highlighting the need for early detection of minimal residual disease (MRD) to guide therapy (Ossenkoppele and Schuurhuis, 2014; Short et al., 2018).

The most popular method for monitoring MRD in AML is the use of multiparametric flow cytometry (MFC), which can identify and quantify residual leukemic cells in the bone marrow or peripheral blood sample at a sensitivity of 0.1% or 1 in 1000 white blood cells (Buccisano et al., 2017). MRD status is prognostic for overall survival (OS) and relapse-free survival (RFS) (Schuurhuis et al., 2018; Short et al., 2020). MRD has gained importance not only in treatment decision-making but also in patient selection for clinical trials (Hu et al., 2020; Lowenberg et al., 2021). More importantly, MRD is expected to become a surrogate endpoint for survival based on industry wide meta-analyses, potentially expediting new drug approvals in the future. There is a growing emphasis on obtaining accurate MRD results and robust validation of MFC-based monitoring methods. Currently, MFC-MRD assays vary among different centers and depend on extensive scientific expertise and knowledge of inter-patient differences for accurate interpretation of data (Keeney et al., 2015). To harmonize interpretation and reporting, the European Leukemia Network (ELN) MRD working party on AML has recommended the utilization of a single consensus panel of markers for MRD monitoring (Heuser et al., 2021).

MFC Method Overview 

Navigate’s AML-MRD MFC Assay is a 4-tube, 8-parameter assay incorporating cell differentiation markers for identification of the broadest group (>90%) of AML LAIPs (see Table 2). These markers are aligned with consensus recommendations from ELN AML MRD working party (Heuser et al., 2021; Schuurhuis et al., 2018), while specimen testing and interpretation principles are described in Cloos et al., 2018. Real world performance of this method has been evaluated in a multicenter clinical trial (Terwijn et al., 2013). A minimum of 500,000 white blood cells (WBCs) are acquired for each specimen on a Flow Cytometer System. LAIPs are identified utilizing a consistent DfN (different from normal) approach at baseline and tracked through the course of the treatment by trained and qualified scientists with extensive prior clinical experience in diagnosing myeloid neoplasms. Utilizing this approach, all the data from multi-center clinical studies are interpreted centrally at Navigate BP CLIA laboratory and test results are reported per ELN AML working party guidelines (Schuurhuis et al., 2018).  

Table 2: AML-MRD MFC Assay Design

Patient Specimen Handling 

Bone marrow aspirates and peripheral blood are collected in sodium-heparin tubes and shipped to the Navigate BP clinical testing laboratory at ambient (18-22 ^OC) temperature. All specimens received are visually inspected and a description of sample integrity, including clotting and/or hemolysis are documented. Hemodilution is monitored using CD13 versus CD16 stain in the granulocyte gate outlined in Table 2 above. Ambient specimen stability is 3 days; specimens received outside the stability window are evaluated by a board-certified pathologist and results are reported with a “disclaimer”. Specimen handling workflow is illustrated in Figure 1 and representative patient results are presented in Table 3. 

Figure 1. AML-MRD MFC Assay Workflow

Interpretation Principles

The general principles of our gating strategy are illustrated in Figure 2 below and described in Cloos 2018.  The first three gates in the analysis scheme (Cells, Single Cells and WBC) help in eliminating cell debris, dead cells, erythrocytes and gate on white blood cell population. Lymphocyte, Monocyte and Granulocyte gates are placed by backgating cell lineage specific markers including CD7, CD19, CD13, HLADR and CD33. CD45dim gate is a flexible gate positioned by carefully backgating blast cells for their primitive marker (CD34, CD117) expression. AML blasts may be positive or sometimes negative for CD34, CD117 expression.  AML blasts are then scanned for aberrant LAIPs using DfN approach. LAIPs are identified using the principles of cross-lineage expression, asynchronous expression or over/under expression of markers.

Figure 2. AML-MRD MFC Analysis (Gating) Paradigm

Method Performance Summary

The analytical validation focused on evaluation of sensitivity (lower limit of quantitation (LLOQ), MRD cut point), reproducibility, repeatability, and accuracy (orthogonal verification by morphological examination) as key criteria for assessing MFC method performance. The use of Bone Marrow Mononuclear Cell (BMMC) specimens was necessitated by difficulties with procuring adequate native samples with defined tumor burden, however, the BMMC experimental design is supported by clinical cytometry consortia (Wood et al., 2013) and represents a more challenging (worst-case) scenario due to potential antigen decay and cell loss. Specimen numbers were chosen based on recommendations by clinical cytometry consortia (Wood et al., 2013) and CLSI H62 for MFC methods (Wayne, 2020). The results of the AML-MRD MFC assay validation activities were found to be acceptable, with the MRD cutoff defined at 0.1%. Complete details are described in an independent validation report. We are currently supplementing the validation package with native specimen testing to meet evolving health authority requirements and obtain Investigational Use Only (IUO) label. 

Discussion & Clinical Experience

Utilization of a core set of markers previously proven to correlate with survival outcomes indicate that the MRD results generated by Navigate BioPharma are suitable for interpretation of treatment efficacy. The sensitivity (MRD cut point) results indicate a lower limit of quantification of at least 0.1% LAIP/WBC across five independent patient LAIPs. The presence of non-leukemic hematopoietic precursors in healthy individuals with phenotypes overlapping LAIPs precluded reliable quantitation of disease burden below 0.1% except for one patient, whose LAIP could be reliably quantified at 0.01% due to absence of overlapping healthy donor phenotypes. Both the frequencies and the nature of LAIPs observed in healthy donors are within the range reported by others (Feller et al., 2004; Kern et al., 2003). As highlighted in Schuurhuis 2018, based on the large numbers of LAIPs that can be defined in AML (up to 100) and their inherent differences in specificity, cutoff levels that capture MRD positivity and are applicable to all LAIPs must be relatively high (i.e., 0.035% to 0.2%). A cutoff of 0.1% was found clinically relevant in published studies and is the recommended clinical cutoff by the ELN AML-MRD working party (Heuser et al., 2021). Thus, a 0.1% threshold to distinguish MRD-positive from MRD-negative patients was implemented with our method for clinical trials.

Table 3: Summary of Representative Patient Results

Over the past decade, this validated method has been utilized for understanding the depth of tumor clearance and length of remission in multiple Phase 2 and Phase 3 clinical trials assessing the effect of immune checkpoint inhibitors in combination with venetoclax and azacitidine on MRD (see Table 4). MRD assessments are proposed to complement the efficacy results in these clinical studies (e.g., MRD negative CR rate), providing a measure of deeper responses with reported prognostic value of long-term outcome in AML (Freeman et al., 2018; Ivey et al., 2016; Jongen-Lavrencic et al., 2018; Terwijn et al., 2013).  We believe incorporating MRD monitoring by MFC method in AML and High Risk MDS clinical trial programs can provide valuable insights into treatment response, patient risk stratification, and personalized therapeutic approaches, ultimately leading to improved patient outcomes.

Table 4: Summary of Clinical Trial Experience with AML-MRD MFC Assay

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