Standardization of Minimal Residual Disease Testing in Multiple Myeloma

Transcription

1 Standardization of Minimal Residual Disease Testing in Multiple Myeloma Linda B. Baughn 1 and Michael A. Linden 2 * Multiple myeloma (MM) 3 is an incurable neoplasm of the bone marrow characterized by a clonal expansion of plasma cells with presence of a monoclonal spike (M-spike) in the serum and/or urine (1). However, there have been major improvements in the way that MM is treated, prolonging overall survival (OS). As OS increases, clinicians and researchers have been increasingly investigating the utility of minimal residual disease (MRD) testing by flow cytometry and molecular diagnostics. Simultaneously, clinical hematology and hematopathology teams are driving efforts to achieve standardization of these laboratory-developed tests (LDTs). MINIMAL RESIDUAL DISEASE Historically, when a patient has a hematologic malignancy such as MM, residual disease is detected by morphologic examination of bone marrow by light microscopy as well as serum/urine protein electrophoresis. However, as technology has improved, we are now able to detect very small populations of clonal cells that cannot be seen by light microscopy this is termed MRD. Some have suggested that the word measurable be substituted for minimal, because as our analytical approaches improve, the lower limit of detection changes (2). FLOW CYTOMETRIC MRD TESTING Multicolor flow cytometry has facilitated the relatively widespread adoption of flow cytometric MRD (FCMRD) testing. A recent survey estimates that there are more than 90 predominantly North American laboratories that perform FCMRD (3). Most laboratories use similar markers to identify plasma cells, including CD45, CD38, and CD138 (4). These markers will identify plasma cells, but do not distinguish normal from neoplastic plasma cells (Fig. 1). Additional cell surface markers include CD19, CD27, CD56, CD81, and CD117, which are differentially expressed among normal and neoplastic plasma cells (4). Cytoplasmic light chain restriction is helpful to confirm aberrant immunophenotype and residual disease (4). Typical Boolean gating strategies to differentiate neoplastic from normal plasma cells are depicted in Fig. 1. There is no Food and Drug Administration approved FCMRD test; therefore, they are all considered LDTs. However, the International Myeloma Working Group (IMWG) has encouraged researchers 1 Department of Laboratory Medicine and Pathology, Division of Laboratory Genetics and Genomics, Mayo Clinic, Rochester, MN; 2 Department of Laboratory Medicine and Pathology, Division of Hematopathology, University of Minnesota, Minneapolis, MN. *Address correspondence to this author at: 420 Delaware St. SE, Minneapolis, MN Fax ; linde013@umn.edu. DOI: /jalm American Association for Clinical Chemistry 3 Nonstandard abbreviations: MM, multiple myeloma; OS, overall survival; LDT, laboratory-developed test; MRD, minimal residual disease; FCMRD, flow cytometric minimal residual disease; IMWG, International Myeloma Working Group; ASO-qPCR, allele-specific oligonucleotide quantitative PCR; NGS, next-generation sequencing; LOD, limit of detection; CAP, College of American Pathologists. 118 JALM :01 July 2017

2 Standardization Myeloma MRD OPINION Fig. 1. Representative flow plots from validation of a standard sensitivity flow cytometry based MRD assay. Row A represents a patient that has 0.8% clonal plasma cells. In the left column, plasma cells are first selected based on their bright expression CD38 and slightly dimmer CD45 expression. Plasma cells are then analyzed for CD19 and CD56 expression (middle column). The clonal/neoplastic plasma cells have dimmed in absence of CD19 expression, with bright CD56 expression (middle column, circles). When querying for light chain restriction among this CD56-positive subset, the abnormal plasma cells uniformly express cytoplasmic kappa light chain (right column). When the sample in row A is serially diluted into normal matrix (marrow), the MRD can be detected at 0.1% (row B) and 0.01% (row C). Scatterplots courtesy of Paula Shivers, University of Minnesota Medical Center Immunophenotyping and Flow Cytometry Laboratory. pckappa, plasma cells expressing cytoplasmic kappa light chain; pclambda, plasma cells expressing cytoplasmic lambda light chain; FITC, fluorescein isothiocyanate; FITC-A, fluorescein isothiocyanate area; PE-Cy7, phycoerythrin-cyanin 7; PE-A IC, phycoerythrin area intracytoplasmic; PE, phycoerythrin; APC, allophycocyanin; V450, Becton Dickinson (BD) Horizon V450; V500, BD Horizon V500. and clinicians to use similar/identical methods. The EuroFlow Consortium has developed an 8-color, 2-tube, 10-antigen (including CD19, CD27, CD38, CD45, CD56, CD81, CD117, CD138, cytoplasmic kappa, and cytoplasmic lambda) FCMRD test that has been widely studied/validated (5). Within the US, Memorial Sloan Kettering Cancer Center has developed a 10-color, 1-tube, 10-antigen FCMRD assay (same markers as EuroFlow but with different clones/fluorochrome combinations) that proposes to have a favorable workflow for busy clinical laboratories (6). July : JALM 119

3 Standardization Myeloma MRD MOLECULAR MRD TESTING Assessment of MRD can also be achieved using quantitative molecular technologies that identify patient-specific clonotypic MM cells, and the IMWG recommends the use of a molecular method that achieves a sensitivity of at least 1 in 10 5 cells (0.001%) (7). However, many of these assays are even more sensitive than FCMRD and can detect down to 1 in 10 6 cells (0.0001%). These sensitive and specific molecular methods include allelespecific oligonucleotide quantitative PCR (ASOqPCR) and next-generation sequencing (NGS) assays using the LymphoSIGHT platform (7). Both assays are designed to detect myeloma-specific clonal immunoglobulin heavy and kappa light chain gene rearrangements, either with the use of clonotypic/patient-specific primer sets for ASOqPCR or by amplification and sequencing using a combination of locus-specific primers for NGS. Both methods require a baseline diagnostic (or prior positive) sample to identify which clonotypic immunoglobulin gene rearrangements will be later tracked (7). Further, these assays are technically more complex and more time-consuming compared to FCMRD. Additional limitations to ASOqPCR include the inability to detect a trackable clone in some patients; occasional poor PCR performance as a result of multiple somatic hypermutations; and the requirement of custom, clonotypic/patient-specific primer sets (7). CD138-positive selected samples can improve the performance of these molecular assays, and some studies indicate that NGS may achieve a sensitivity of less than1in10 6 cells (0.0001%) without the use of custom primers (7). HETEROGENEITY IN FLOW CYTOMETRY MRD TESTING Prior small surveys identified that there was significant heterogeneity in FCMRD methodology and analytical limit of detection (LOD) (8). We expanded on this information by surveying 549 predominantly North American flow cytometry laboratories; of the 500 respondents, 18.2% of these laboratories performed FCMRD testing (3). Not surprising, the LOD varied from laboratory to laboratory, with LODs ranging from 1% to 0.001%; the most common LOD reported was 0.1%. A small follow-up study in 2016 suggests that the best laboratories have improved and/or established a lower LOD (9), but clearly there is marked variability in the analytical performance of MRD testing. RESPONSE AND RECOMMENDATIONS Because an ordering provider may make the assumption that all FCMRD tests have similar analytical performance, the College of American Pathologists (CAP) made changes to its requirements for CAP-accredited laboratories (referred to as Rare Events Checklist items). Similar to dilution/ recovery experiments in a clinical chemistry laboratory, the CAP now requires that all laboratories performing FCMRD validate their FCMRD assays by diluting positive samples into negative samples to verify the LOD (Fig. 1) (3). In addition, laboratories that perform FCMRD testing must report their analytical LOD within the diagnostic report (3). The main goal of these changes is to help communicate the analytical performance of these LDTs to pathologists and clinicians as they select tests that meet the IMWG guidelines. A summary of methods used to detect MRD is depicted in Table 1. Simultaneously, the IMWG has provided expectations for the analytical performance of nextgeneration flow cytometric and sequencing-based MRD detection. Testing should detect down to 1 in 10 5 cells (an LOD of 0.001%) (7). Moreover, using this established analytical LOD by either method, the IMWG has defined a bone marrow MRD-negative response category (7). Arguably, there will be heterogeneity in terms of the timing and frequency of bone marrow MRD assessment in the MM patient. The IMWG consensus authors have proposed that 120 JALM :01 July 2017

4 Standardization Myeloma MRD OPINION Table 1. Summary of methods used to detect myeloma MRD. a MRD technique Example methods Target LOD First-generation flow Next-generation flow cytometry Molecular PCR Molecular sequencing MFC (FCMRD) 4- to 8+-color flow EuroFlow 2 8-color combinations MSKCC 1 10-color combination ASO-qPCR NGS Cell surface and intracellular antigens Cell surface and intracellular antigens Immunoglobulin gene rearrangements Immunoglobulin gene rearrangements Varies, but typically not <0.01% CAP Rare Events Checklist items Applicable 0.001% Applicable 0.001% % a All flow-based methods, whether first or next generation, require compliance with CAP checklist items pertaining to rare events, including establishing and reporting the assay's LOD. IMWG guidelines endorse using a next-generation flow method that has an LOD of 0.001% or lower. For molecular methods, the IMWG also recommends an assay that has an LOD of 0.001% or lower, but it should be noted that using some molecular methods such as NGS may detect MRD down to %. MFC, multicolor flow cytometry. the assessment of MRD kinetics over the course of the disease, rather than at a single time point after complete response is achieved, could provide additional information about disease control (7). Certainly, retrospective and prospective studies will help guide the way that the clinical team uses MRD testing to inform and guide treatment decisions. In summary, though MM is considered an incurable disease, there have been dramatic improvements in OS. Clinical teams are readily incorporating MRD testing into clinical practice and decision-making. Professional societies such as the IMWG have set clear expectations/guidelines for the analytical LOD for FCMRD and molecular MRD testing in patients with MM. Finally, CAP guidelines have affirmed the importance of the FCMRD method validation to measure analytical LOD as well as including the LOD in patient reports. Collectively, these efforts will improve the way that testing for the MM patient is standardized, contributing positively to patient care. Additional Content on this Topic Serum Free Light Chain (FLC) Analysis: A Guiding Light in Monoclonal Gammopathy Management Ellen L. Jenner, Josie A.R. Evans, Stephen J. Harding. J Appl Lab Med 2017;2: Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Employment or Leadership: M.A. Linden, CAP. Consultant or Advisory Role: M.A. Linden, Bristol-Myers Squibb. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. Patents: None declared. July : JALM 121

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