Longterm Serial Transplantability of BCR-ABL+ Chronic Phase CML Cells Is Predicted by a 6-Week LTC-IC Assay.

Longterm culture-initiating cells (LTC-ICs) were assayed by coculturing immunomagnetically isolated CD34⁺ cells for 5-weeks in LTCs containing murine stromal cells (n=10) and/or for 6-weeks in LTCs containing murine stromal cells secreting human SCF, IL-3 and G-CSF (n=33). Genotyping of the LTC-ICs was based on genotyping individual colonies generated in methylcellulose assays of cells harvested at the end of the 5 or 6 weeks of coculture, either by karyotyping G-banded metaphases (n=16) or by qRT-PCR of extracted RNA (n=17). In vivo assays were performed on 2 samples by injecting 10⁶ CD34+ cells (>97% and 44% Ph+ 6-week LTC-ICs) IV into primary sublethally irradiated NOD/SCID IL2Rγc−/− (NSG) mice and after 30–35 weeks further into secondary NSG mice. All mice were analyzed periodically for human hematopoietic cells by flow cytometry of marrow aspirates. CD34⁺CD38⁻ cells isolated by FACS from primary CML samples (n=17) were spotted on slides and examined by FISH for the presence of the BCR-ABL gene.

The proportion of 6-week LTC-ICs that was Ph+/BCR-ABL+ ranged from <5% to 100%. Although the CML LTC-ICs represented >80% of the LTC-ICs in 36% of the 33 cases studied, these represented <50% in 45% of these cases. Ph+/BCR-ABL+ LTC-ICs were more prevalent when measured with the 5-week LTC-IC assay (86±7% Ph+/BCR-ABL+) than with the 6-week LTC-IC assay (11±8% Ph+/BCR-ABL+, n=10). FISH analysis of the initial CD34+CD38- cells showed that >80% of these were BCR-ABL+ in 70% of the 17 cases studied. Notably, these latter values were not correlated with the proportion of leukemic 6-week LTC-ICs in the same samples (Spearman rank correlation r = 0.43, p = 0.08). Primary NSG mice transplanted with CD34+ cells from the patient with no detectable normal LTC-ICs regenerated almost exclusively differentiated human myeloid cells for up to 35 weeks and at increasing levels at the later time points (35% of total marrow cells after 35 weeks). Cells obtained 8 and 35 weeks post-transplant showed these contained readily detectable clonogenic cells which were exclusively BCR-ABL+ (84 genotyped colonies). Secondary recipients were again repopulated with exclusively BCR-ABL+ myeloid cells. Recipients of the second sample showed a transient early peak of myeloid cells followed by a peak of B lymphoid cells at 8 weeks, at which time only 20% of the human CFCs present were BCR-ABL+. At 35 weeks post-transplant, human cells were still detectable in these mice (5±3% of the marrow) and myeloid cells had again become the predominant lineage with BCR-ABL+ cells detectable by qRT-PCR but no CFCs were identified. Secondary recipients of these cells were reconstituted with myeloid cells but only transiently.

Functionally defined chronic phase CML stem cells represent a very minor subset of the CD34⁺CD38- compartment and assessment of these cells, like assessment of the most commonly used 5-week LTC-IC assay, overestimates the BCR-ABL+ stem cell compartment. Both of these endpoints also fail to provide a reliable indicator of the prevalence of BCR-ABL+ stem cells defined by more stringent functional assays. We also show for the first time that BCR-ABL+ CML stem cells are capable of serial transplantability spanning one and a half years in NSG mice and this may be anticipated by results from the 6-week LTC-IC assay.

No relevant conflicts of interest to declare.