Analysis of Common Chromosomal Abnormalities in Hematopoietic Stem Cells by FISH in Multiple Myeloma for Determination of Early Lineage Involvement
Farid Saei Hamedani, Jean Lopategui, Raju Pillai, Kevin Baden, Robert A Vescio, Serhan Alkan. Cedars-Sinai Medical Center, Los Angeles, CA
Background: Multiple myeloma (MM) is characterized by a clonal expansion of malignant plasma cells that usually show certain cytogenetic abnormalities such as 13q del, t (11; 14), 17p del. Patients treated with autologous bone marrow transplantation eventually relapse that raise a question whether MM is a hematopoietic stem cell disorder. In order to investigate whether hematopoietic stem cells have the same cytogenetic abnormalities as the neoplastic plasma cells, we have developed a novel method to assess presence of MM associated cytogenetic abnormalities utilizing magnetically separated CD34+ hematopoietic cells.
Design: Bone marrow aspirates from patients with clinical suspicion of MM were analyzed by standard cytogenetic studies. In addition, MM samples were subject to FISH analysis using probes specific for commonly encountered cytogenetic abnormalities in MM. Mononuclear cells from bone marrow aspirate were separated using Ficoll-Paque. CD34+ cells were separated from mononuclear cells with EasySep® Human CD34+ Positive Selection Kit from Stem Cell Technologies (Vancouver, Canada). Isolated CD34+ cells were assessed for purity with flow cytometric analysis using antibodies against CD34, CD38, CD138, and CD45. Cytospin samples following CD34 selection were prepared to microscopically assess the morphology of the isolated cells that were subjected to FISH analysis using probes against the specific abnormalities detected by cytogenetic/FISH studies in the corresponding non-purified plasma cells.
Results: Magnetically separated samples yielded 1-2% CD34+ cells after selection by CD34-antibody coated beads. Common cytogenetic abnormalities were noted in unfractionated MM samples using FISH analysis in the bone marrow aspirates. Flow cytometric analysis revealed that purity of the magnetically separated CD34+ cells was more than 90%. The CD34-enriched cell population subjected to FISH assay did not show any abnormalities that were detected in plasma cells.
Conclusions: The lack of cytogenetic abnormalities in CD34+ stem cell population indicates that the chromosomal abnormalities detected in plasma cells are likely to occur in the later stages of B-cell development. Our results suggest that the hematopoietic stem cells (CD34+ cells) do not have the same chromosomal abnormalities noted in the plasma cells in MM patients. Therefore, failure in MM patients treated with autologous bone marrow transplantation is most likely due to the growth of the chemotherapy-resistant cells residing in the host.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 281, Wednesday Morning