The aim of this feasibility study was to determine whether del(17p), the most significant chromosomal abnormality in myeloma, could be identified in CTPC identified by immuno-flowFISH.
Blood samples were obtained from 21 myeloma patients. After red cell lysis or Ficoll separation, cells were incubated with monoclonal antibodies (CD38-BV605, CD138-BV480) to identify plasma cells. Following fixation, cell membranes were permeabilised and DNA denatured. FISH was performed by hybridisation of fluorophore-conjugated probes to the chromosome 17 centromere (C17) and a locus specific 17p13 region. Nuclei were stained with SYTOX AADvanced and cells acquired on the Amnis ImageStream®X MkII imaging flow cytometer. Digital images captured at x60 magnification and quantitative data were used to assess FISH signals overlying nuclei of CD38/CD138-positive plasma cells.
10,000-786,980 (Mean 238,521) cells were acquired and CD38/CD138-dual positive CTPC could be identified in all samples (0.003-89.83% of cells). FISH probe binding for both C17 and 17p region was evident by fluorescing “spots” overlying the nucleus. In three cases there was only one FISH signal with the 17p13 probe but diploid centromeric FISH spots, indicating del(17p). Another case had a single FISH spot for 17p13 and centromeric probes indicating loss of the entire chromosome 17 (i.e. monosomy). One case had 4 copies of C17 and 3 copies of the 17p13 locus, indicating a double genome that had then lost one copy of 17p13. In 16/21 samples there were 2 FISH signals for both C17 and 17p13, a normal pattern.
Doubled genomes, loss of 17p13 and monosomy 17, are detectable in CTPC in myeloma using the immuno-flowFISH method. This provides a novel approach for assessing chromosome 17 aberrations in myeloma in blood, something currently unachievable with standard FISH. This technique has potential as a highly sensitive non-invasive blood-based tool for assessing the most significant chromosomal abnormalities in myeloma.