Expires: October 12, 2023
Dragan Jevremovic, M.D., Ph.D.
Assistant Professor of Laboratory Medicine and Pathology
Division of Hematopathology
Mayo Clinic, Rochester, Minnesota
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My name is Dragon Jevremovic I am an assistant professor in the Division of Hematopathology at Mayo Clinic in Rochester, Minnesota.
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Multiple myeloma is a neoplasm of malignant plasma cells in the bone marrow. Diagnostic criteria from WHO and International Myeloma Working Group classifications for active or symptomatic myeloma include more than 10% of clonal bone marrow plasma cells (or extramedullary plasmacytoma), and the presence of a so-called “myeloma-defining event,” which includes end-organ damage, more than 60% bone marrow plasma cells, high serum free light chain ratio, or at least 2 focal lesions on MRI. Smoldering myeloma is defined by clonal bone marrow plasmacytosis between 10 and 60% or any percentage if there is high M-protein level, and absence of myeloma-defining events.
Mayo Clinic Multiple Myeloma group has developed a stratification of active myeloma into standard risk, intermediate risk, and high risk, based on immunophenotypic, cytogenetic, and molecular genetic findings. This classification is used to guide patient therapy. For more information on the classification and therapeutic recommendations, please visit the site mSMART.org.
The Mayo Clinic Department of Laboratory Medicine and Pathology and Mayo Clinic Laboratories provide myeloma-specific tests that are used for stratification and therapy guidance of myeloma patients. There are several immunophenotyping tests to identify, quantify, and characterize clonal plasma cells. PCPRO assay determines clonality of bone marrow plasma cells, as well as their proliferation rate and the percentage of polyclonal plasma cells. PBLI test measures the frequency of circulating plasma cells in the peripheral blood. Finally, MRDMM assay identifies rare clonal plasma cells with high sensitivity.
Plasma cell fluorescent in situ hybridization is performed by the cytogenetics laboratory. The probes are designed to identify clinically significant rearrangements. The full panel is usually performed at diagnosis, and subsequently only a subset of probes are used to check for the disease progression. A combined test called mSMRT includes an algorithmic approach in which FISH analysis is performed only if PCPRO identifies more than 0.1% of clonal plasma cells, to avoid unnecessary testing and reduce cost.
This is the example of the PCPRO in which clonal plasma cells had a high proliferative rate. In general, the higher the proliferation rate, the worse survival of myeloma patients. In the lower right corner there is an example of survival split between low and high proliferation rates. With the current version of the test, the best cutoff has been established at the S-phase of 2%. Importantly, PCPRO can be used to detect small clones, with the sensitivity of 10-4 to 2 x 10-5 , depending on the immunophenotype of the abnormal plasma cells.
As new myeloma therapies provide much longer remissions and deeper responses, there is a need for a more sensitive testing in patients with complete response, to determine which patients have to continue on therapy, and which patients can be safely taken off therapy. The higher sensitivity of identification of abnormal plasma cells is obtained by collecting more events, and by using more antibodies to detect aberrant antigen expression. Abnormal plasma cells show one or more of the phenotypic aberrancies listed on this slide.
A recent publication from international collaborative group shows the importance of high-sensitivity MRD testing in myeloma patients with complete response to therapy. Patients who were MRD positive by the next-generation flow cytometry method showed faster disease progression.
Our new myeloma MRD test uses the methodology recommended by the International Myeloma Working Group. We employ a 2-tube analysis with 8 antibodies and 5 million cells per tube. The sensitivity of our assay is 10-5, as recommended by IMWG guidelines.
This is an example of the histograms generated by the Infinicyte software which performs semi-automated gating to identify abnormal cells.
MRD test should be ordered only in patients who have undergone therapy and have a complete response or even better, stringent complete response. Complete response is defined by negative serum and urine immunofixation, less than 5% of bone marrow plasma cells, and no extramedullary plasmacytoma. In patients who do not meet this criteria, such as patients at diagnosis, progression, or stable disease, the appropriate test to order is PCPRO or MSMRT if FISH analysis is required as well.
For this myeloma MRD test there is a need for a minimum of 2 mL of bone marrow aspirate. It should be indicated on the requisition form whether the patient has been recently treated with anti-CD38 antibody.
In addition to MRD testing, at Mayo Clinic we have recently developed a next-generation sequencing (NGS) assay to detect smaller genetic changes, such as point mutations, important for plasma cell signaling, proliferation, survival, and sensitivity to chemotherapy. The assay covers 61 genes and 7 intronic regions which have been shown to have a potential clinical impact. Plasma cells are first sorted from the bone marrow aspirate, and isolated DNA is sequenced on the Illumina platform, with minimum 250x coverage.
This is the list of genes and regions covered by the assay. Many of the abnormalities found in myeloma are shared with other hematologic neoplasms, such as lymphoma and myeloid disorders.
The main indication for performing mutational analysis is a case of therapy-resistant myeloma. In addition, clinically useful information could be obtained at initial diagnosis, for prognostic purposes. The NGS report will identify the mutations, estimate their percentage, and describe their potential clinical importance and therapeutic implications.
Mayo Algorithm for Multiple Myeloma Testing
This is the recommended algorithm for myeloma testing. At diagnosis or relapse, MSMRT will incorporate PCPRO data with FISH results for adequate stratification of patients. For patients with complete response, MRDMM can identify rare clonal plasma cells with high sensitivity.