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Plasmacytoid dendritic cells in AML
Introduction
Measurable residual disease (MRD) is associated with inferior outcomes in patients with acute myeloid leukemia (AML).1-4 Post-induction MRD independently predicts overall survival (OS) and relapse-free survival (RFS).5-10 MRD after consolidation is also associated with a higher risk of relapse and shorter OS and RFS.6,11-17 Patients with AML who undergo hematopoietic stem cell transplant (HSCT) with any level of MRD by flow cytom- etry prior to HSCT are at increased risk of relapse and death.18-21
Given the prognostic significance of MRD, the 2017 European LeukemiaNet (ELN) recommends comprehen- sive MRD monitoring following induction and consolida- tion courses to assess kinetics of disease response, and then monitoring sequentially after consolidation to detect impending relapse.22 Dynamic MRD assessment for patients with AML complements baseline patient risk assessment factors, such as age, karyotype, and molecular alterations, in determining patient prognosis. More impor- tantly, MRD status may be helpful in informing therapeu- tic decisions.6,23
Currently, the most commonly used methods of MRD assessment are multi-color flow cytometry (MFC) and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) of specific mutations or gene fusions detected at diagnosis. Recently, digital PCR and next generation sequencing (NGS) have emerged as prom- ising technologies to improve upon these methodolo- gies.1,3,4 The major advantages of MFC are its wide applica- bility, assay availability, relative affordability, and rapid turnaround time, the latter facilitating prompt decision- making for therapeutic intervention.1 It also allows for simultaneous evaluation of expression levels of multiple antigens on the leukemic blasts, which are relevant for potential targeted immunotherapy.
Multi-color flow cytometry techniques are based on the expression of antigens that characterize diverse lineages of hematopoietic cells. AML blasts may either be defined by expression of distinct leukemia-associated immunopheno- types (LAIPs),16,24,25 or by manifesting an immunopheno- typic maturation profile detected using a fixed antibody panel that is “different from normal” (DfN).1 An integrated LAIP-based DfN approach has recently been recommend- ed by the ELN working party.4 However, this approach requires a high level of expertise for data interpretation.1 Additionally, the DfN approach has considerable variabil- ity in the equipment, reagents, data analysis methods, and reporting used in MRD evaluation, confounding repro- ducibility and applicability outside of a limited number of expert centers with a high level of analytical expertise.26-28 Numerous analytical variables and technical obstacles are evident in the process of standardizing this MRD approach. A simpler, but nonetheless robust, approach may be beneficial in expanding risk stratification by post- treatment disease analysis outside of a few large centers.
Plasmacytoid dendritic cells (PDC) are the major natural type I interferon-producing dendritic cells that play critical roles in the immune response.29,30 PDC are derived from hematopoietic stem cells and can be easily identified by flow cytometry due to high-level expression of CD123, HLA-DR and/or CD303/BDCA2 in the context of lack of lineage markers, low side scatter, and moderate expres- sion of CD45.31 In healthy subjects, PDC are <1% of total
nucleated cells in both marrow and blood, but are nonetheless present in a relatively narrow and repro- ducible range of proportions.32-34 Loss of PDC is observed in germline GATA-2 deficiency and increased PDC have been reported in chronic myelomonocytic leukemia.35-39 Very few studies have attempted to characterize PDC in AML and the results are inconclusive due to a small num- ber of patients.32,33 One study suggests slightly higher lev- els of peripheral blood PDC at time of diagnosis in patients with FLT3-ITD+ (vs. FLT3-ITD–) AML.33 We observed a dramatic loss of PDC in most AML patients at diagnosis and investigated if the loss of PDC could be used as surrogate marker for disease persistence and risk strati- fication. Here we report a simple, objective, widely appli- cable and quantitative MFC approach using quantitation of PDC and blasts. We show that blast/PDC ratio not only correlates with MRD status but also predicts MRD clear- ance and outcomes in AML.
Methods
Patients
A cohort of 163 adult patients with a confirmed diagnosis of AML who underwent induction therapy with “7+3” at Memorial Sloan Kettering Cancer Center (MSKCC) between April 2014 and September 2017 was initially included. Of these, 27 had no post- induction follow-up flow cytometric data within the time frame of days 28 to 60 post induction at MSKCC and were excluded from further analysis. Post induction, 34 of 136 (25%) patients had >20% blasts and 33 of 136 (24%) patients >5% but <20% blasts (named “residual AML”); 69 of 136 (51%) patients achieved mor- phological remission with <5% blasts in the marrow: 36 were MRD-negative (“MRD-neg.”) and 33 MRD-positive (“MRD- pos.”). Complete remission (CR), CR with incomplete hematolog- ic recovery (CRi), and morphological leukemia-free state (MLFS) were defined by the ELN criteria.22 As a control, we analyzed MFC data from two cohorts of patients (age >18 years old) who underwent bone marrow evaluation for mild cytopenia (absolute neutrophil counts >1 but <1.5 x109/L, hemoglobin >9 but <12 g/dL, or platelets >90 but <150 x109/L): 20 patients had a history of non-hematologic solid malignancy and another 11 patients had no history of malignancy, but had cytopenias attributable to other etiologies, such as autoimmune diseases or aplastic anemia. Morphological, cytogenetic and molecular (by a NGS platform panel composed of 28 genes frequently mutated in AML40) studies in the bone marrow were negative for myeloid neoplasm or clonal hematopoiesis in these control subjects. This study was approved by the Institutional Review Board of MSKCC.
Measurable residual disease detection and plasmacytoid dendritic cell quantitation by flow cytometry
Multi-color flow cytometry was performed on bone marrow aspirates at diagnosis and/or relapse and prior to induction chemotherapy.41 Briefly, up to 1.5 million cells from freshly drawn bone marrow aspirate were stained with 3-4 10-color panels (Online Supplementary Table S1), washed, and acquired on FACS Canto-10 cytometer (BD Biosciences, San Jose, CA, USA). The results were analyzed with custom Woodlist software (a generous gift from BL Wood, University of Washington, USA). An abnormal population was identified by visual assessment of populations with antigen expression that was ‘different-from-normal’, as described in prior publications.9,41 The assay is able to detect abnormal populations to a sensitivity of approximately 1 in 1000
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