IGHcytogenetic abnormalities can be detected in multiple myeloma by imaging flow cytometry

Abstract

AimsCytogenetic abnormalities involving the IGH gene are seen in up to 55% of patients with multiple myeloma. Current testing is performed manually by fluorescence in situ hybridisation (FISH) on purified plasma cells. We aimed to assess whether an automated imaging flow cytometric method that uses immunophenotypic cell identification, and does not require cell isolation, can identify IGH abnormalities.MethodsAspirated bone marrow from 10 patients with multiple myeloma were studied. Plasma cells were identified by CD38 and CD138 coexpression and assessed with FISH probes for numerical or structural abnormalities of IGH. Thousands of cells were acquired on an imaging flow cytometer and numerical data and digital images were analysed.ResultsUp to 30 000 cells were acquired and IGH chromosomal abnormalities were detected in 5 of the 10 marrow samples. FISH signal patterns seen included fused IGH signals for IGH/FGFR3 and IGH/MYEOV, indicating t(4;14) and t(11;14), respectively. In addition, three IGH signals were identified, indicating trisomy 14 or translocation with an alternate chromosome. The lowest limit of detection of an IGH abnormality was in 0.05% of all cells.ConclusionsThis automated high-throughput immuno-flowFISH method was able to identify translocations and trisomy involving the IGH gene in plasma cells in multiple myeloma. Thousands of cells were analysed and without prior cell isolation. The inclusion of positive plasma cell identification based on immunophenotype led to a lowest detection level of 0.05% marrow cells. This imaging flow cytometric FISH method offers the prospect of increased precision of detection of critical genetic lesions involving IGH and other chromosomal defects in multiple myeloma.

Imaging flow cytometric detection of del(17p) in bone marrow and circulating plasma cells in multiple myeloma

“…This was achieved as many thousands of cells were analyzed, and FISH signals were assessed in gated cells with the phenotype of interest (i.e., CD38/CD138‐positive) ensuring plasma cell specificity. This adds to the literature of imaging flow cytometry which had previously shown to be able to identify IgH rearrangements and hyperdiploidy in myeloma 14–16 …”

“…This adds to the literature of imaging flow cytometry which had previously shown to be able to identify IgH rearrangements and hyperdiploidy in myeloma. [14][15][16] Most FISH methods require positive isolation or enrichment of plasma cells to increase the yield of cells of interest prior to chromosomal analysis. In the present study, the only prior sample handling was red cell lysis or density gradient separation to remove erythrocytes and granulocytic cells.…”

“…Samples were processed using the previously published immuno‐flowFISH protocol, with minor modifications 16,18,19 . Cells were incubated for 30 min at 4°C with CD38 (clone HB‐7) conjugated to BV605 or AF647 fluorophores and CD138‐BV480 (clone MI15) monoclonal antibodies (BD Biosciences) at the manufacturer's recommended concentration.…”

“…The thawed samples were stained with eBiosciences Fixable Viability Dye (FVD) conjugated to the fluorophore eFluor780 (Thermo Fisher Scientific, Sydney, Australia) as per manufacturer recommendations. Samples were heated to 78°C for 5 min to denature DNA (Veriti Thermal Cycler, Thermo Fisher Scientific) followed by probe hybridization at 42°C for 24 h 16 . Two FISH probe panels were used, each consisting of a centromeric chromosome 17 and locus specific 17p probe.…”

“…[13][14][15] This technique, called 'immuno-flowFISH' combines immunophenotyping and FISH with analysis on an imaging flow cytometer and has been shown to be able to identify numerical (e.g., hyperdiploidy) and structural chromosomal abnormalities (e.g., IGH translocations) in antigen-positive cells in hematological malignancies. 14,16,17 In the present study, we aimed to determine the potential of immuno-flowFISH to identify abnormalities of chromosome 17, and specifically del(17p), in phenotypically identified plasma cells in both bone marrow and blood of patients with myeloma. We hypothesized that this approach would give greater specificity than standard FISH and be able to assess del(17p) with lower-level plasma cell burden.…”