Leukemia Stem Cells (LSCs) Are Frequent in Childhood Precursor B Acute Lymphoblastic Leukemia (ALL).
Authors: Morisot Sebastien , Wayne Alan S , Bohana-Kashtan Osnat , Kaplan Ian M , Hildreth Richard , Brown Patrick , Stetler-Stevenson Maryalice , Civin Curt I 
Published: April 2020

Abstract
Several groups have reported that LSCs comprise a discrete but very rare (&lt;1 per 10,000 cells) subset of the total cancer cells in hematologic malignancies and multiple solid tumors. However, the existence of a rare cancer stem cell subset has been questioned by other studies in solid tumors. In addition, recent results using genetic models of acute myeloid leukemia (AML) indicate that LSCs may comprise a larger subset (&gt;1 per 100 cells) of the total leukemia cells. We sought to determine the frequency of LSCs in unpurified samples of primary human cells from cases of childhood precursor B ALL. We used the recently described highly immunodeficient NOD-scid-IL2γnull (NOG-scid) mouse model as an assay for LSCs. Sublethally (250 cGy) irradiated adult NOG-scid mice were transplanted by intravenous injection with either human pre B ALL cell lines (REH and KOPN8) or unpurified human primary leukemia blast cells that had been cryopreserved from pediatric patients. In preliminary studies, transplantation of both of the 2 precursor B ALL cell lines and 7 of 11 of the tested primary ALL cases generated fatal leukemia-like proliferations in mice. 5 of the 7 human primary precursor B ALL cases were further studied herein. Time to fatal (or clinically severe) leukemias in the transplanted mice was dependent on leukemia specimen, varying from 1–7 months post-transplant of ~1 million leukemia cells. At necropsy, all these mice had massive splenomegaly and hyperplastic bone marrows. Microscopic examination revealed that most organs were heavily infiltrated with morphologic blast cells. Blood, spleen, and marrow were essentially replaced with human cells with morphology and immunophenotype matching the transplanted human ALL cell lines or the primary patient samples. All mice transplanted with 1000 or more REH or KOPN8 cells developed clinically severe leukemias by ≤53 days post-transplant. 60% and 40% of mice injected with 100 REH and KOPN8 cells developed leukemias by 61 and 55 days, respectively (Table). All mice transplanted with 100 or more unpurified cells from primary case#1, and 3 of 10 mice transplanted with 10 cells, developed clinically severe leukemias by ≤76 days post-transplant, whereas 0 of 10 mice transplanted with 1 cell developed leukemia by 145 days (when the experiment was terminated and leukemia could not be detected in the spleens by FACS). Secondary transplant of 10 splenocytes from mice that had been transplanted with 10 primary cells from case#1 generated leukemias, with the same times to clinical leukemia as had been observed after transplant of the same dose of primary cells. With primary case#2, all mice transplanted with 1000 or more unpurified cells and 4 of 10 mice transplanted with 100 cells developed severe leukemias by ≤79 days post-transplant, whereas 0 of 10 mice transplanted with 10 cells or 1 cell developed leukemia by experiment termination at 125 days. Secondary transplant of splenocytes from mice that had been transplanted with 100 primary cells from case#2 generated leukemias. In primary case#3, all mice transplanted with 10 or more unpurified cells and 2 of 10 mice transplanted with 1 cell developed severe leukemias by &lt;208 days. Similar studies of cells from 2 additional primary ALL cases of childhood precursor B ALL are in progress: In results to date, the lowest cell dose to generate clinical leukemias in case#4 and case#5 was 100 cells. Transplantation into the classic NOD-scid mouse strain revealed a similar frequency of LSCs, although the onsets of the leukemias were in some cases delayed by up to 3 weeks. These results suggest that LSCs, as defined by generation of leukemia-like proliferations in immunodeficient mice, are relatively frequent (~1 per &lt;10–100 total unpurified cells) in childhood precursor B ALL samples.
Human ALL sample designation ALL cell line or patient status at time of sampling Minimum number of transplanted unpurified ALL cells that generated clinical leukemia in NOG-scid mice Time to clinical leukemias (days) REH Cell line 100 ≤55 KOPN8 Cell line 100 ≤61 Case#1 Relapse 10 ≤76 Case#2 Relapse 100 ≤79 Case#3 Relapse 1 ≤208 Case#4 Initial diagnosis (untreated) 100 ≤120 Case#5 Initial diagnosis (untreated) 100 ≤144