The characterization of the cells at the origin of Hodgkin and Reed Sternberg cells in Hodgkin lymphoma (HL) is hampered mostly because of the poor growth of HL cells in vitro and in vivo. Investigation of cell markers and signaling pathways specific to HL clonogenic cells may lead to progress in therapy and improve the prognosis of patients with HL. Using a newly developed murine in vivo L428 xenograft model and circulating lymphocytes of HL patients before treatment, we attempted to determine the immunophenotype and cytogenetic profile of the clonogenic cells.
Material and methods:
L428 HL cell line was used to establish an in vivo xenograft model in immunodeficient NOD/SCID-gammac-/- mice. In vitro and in vivo clonogenic cells derived from L428 cell line and circulating lymphocytes of 50 HL patients were characterized using immunofluorescence, immunochemistry and cytogenetic. Chromosomal instability was also investigated. Telomere maintenance mechanisms (telomerase activity (TA) and Alternative Lengthening Telomere (ALT)) were explored.
Results:
We confirmed previous data that L428 cell line contained small (<2%) subpopulations that lacked CD30 and CD15 expression and had greater clonogenic potential in vitro than the corresponding CD30+ and CD15+ cells. These cells were also clonogenic in vivo in NOD/SCID gamma -/- mice. Immunophenotype characterization of the first cells harvested from the mice demonstrated a CD30, CD15 and CD14 negative profile. Over time, the cells acquired CD14 followed by CD15 and finally CD30 surface markers. Cytogenetic analysis confirmed the diploid origin of the cells that grow in the mice. Cell sorting of negative cells in HL cell lines was performed to validate this hypothesis and demonstrate their clonogenic potential to recover the parental cell line. These cells were characterized by a higher telomere instability associated to the presence of higher frequency of dicentric chromosome. A high TA was detected in these cells as well as in the circulating lymphocytes in 10 out of 50 HL patients.
Conclusion:
We better characterized the origin of the cells which grow in the L428 xenograft model, and their different steps of transformation from “clonogenic cells” to monocytes/macrophages and finally to Hodgkin and HRS cells due to the unique microenvironment of HL. The assessment of TA could be used as a biomarker of the prevalence of clonogenic cells in circulating lymphocytes.