“Double Hit” Myeloma: Detection By FISH and Flow Cytometry in Circulating Myeloma Plasma Cells

“Double hit” myeloma is defined as the presence of 2 of the known high-risk genetic abnormalities in myeloma. This includes amp(1q), del(17p) and IGH translocations involving chromosomes 4, 16 and 20. Given the prognostic significance of these abnormalities, with poor overall survival, identification is crucial. Testing is traditionally by fluorescence in situ hybridization (FISH) on a single-site bone marrow sample at baseline or at specific disease timepoints. We explored whether amp(1q) and del(17p) “double hit” abnormalities could be detected in circulating myeloma plasma cells (CMPC). Aims: We assessed our novel imaging flow cytometry method that simultaneously integrates immunophenotyping and FISH. Called “immuno-flowFISH” this single cell method combines antigenic detection of neoplastic plasma cells, FISH for DNA loci of interest and imaging flow cytometry for automated assessment. Methods: Blood from 16 myeloma patients, at diagnosis or on therapy was studied. Following density gradient centrifugation, cells were incubated with CD38/CD138/CD319-AF647-conjugated antibodies to identify plasma cells, CD19-BV480 (to differentiate normal from neoplastic plasma cells) and CD3-BV510 (T-lymphocyte control). After fixation and cell membrane permeabilization, DNA was denatured (78 oC for 5 minutes). The cells were then incubated with FISH probes to 1q21 ( CKS1B) and 17p13 loci, and to the centromere of chromosome 17 (C17), and hybridized (24 hours for 37 oC). The nuclei were stained with SYTOX AADvanced and 50,000 to 500,000 cells acquired on the Amnis ® ImageStream ®X Mk II imaging flow cytometer (Cytek Biosciences). Digital images and quantitative data derived from computational algorithms (IDEAS software) were used to assess FISH signals overlying the counterstained nuclei. Results: A mean of 264,937 cells were acquired and 110,696 analysed. Cells with a neoplastic plasma cell phenotype (CD38/CD138/CD319-positive, CD19-negative) were present in all samples. The number of these CMPC ranged from 7 to 797, amounting to 0.011- 0.546% of all cells. FISH abnormalities were present in CMPC of 10 of the 16 cases (see Table). Isolated amp(1q) (three CKS1B FISH signals) was present in 4 cases, and isolated del(17p), (one 17p13 and two C17 FISH spots) in CMPC in 3 cases. “Double hit” abnormalities, with both amp(1q) and del(17p), were present in another 3 cases, with the following cytogenetic patterns (illustrated in the Figure): 1. amp(1q) and del(17p) in differen