Insight Into T-ALL Leukaemia Initiating Cells, Clonal Evolution and Impact of Treatment In Vivo Using Immune-Compromised Mouse Model,

Abstract 3577

T-ALL constitutes 15% of ALL in children, requires more aggressive treatment, and exhibits a 5 year cure rate of around 75%. Relapsed T-ALL has a dismal salvage rate. Understanding the leukaemic stem cells responsible for clonal evolution and the emergence of treatment resistance may facilitate the treatment.

We used immuno-compromised NOD/Shi-scid/IL-2Rγ^null (NOG) mice to study in vivo engraftment kinetics and clonal evolution of a SIL-TAL+ primary T-ALL. Initially, we compared the pre-injection immunophenotype with that after primary transplantation. While 18% of the pre-injection cells were CD34+, 14% of human cells (hCD45+) at termination of the first passage were CD34+. Although the leukaemia-specific TCRγ gene rearrangement was stable throughout, cells following transplantation exhibited greater resistance to vincristine in vitro indicating the emergence of drug resistant cells.

We next engrafted cells from the primary recipient into a second cohort of mice and evaluated the impact of vincristine in vivo on leukaemic subpopulations in different organs. Four weeks post-injection, vincristine treatment (1mg/kg once weekly by IV injection) was initiated. Control animals from this time exhibited increasing spleen weight compared with vincristine-treated mice whose spleen sizes decreased throughout treatment. One week following initiation of vincristine treatment, cells from the control animals predominantly comprised of more mature CD34-CD7+CD5+CD3- or CD34-CD7-CD5+CD3- cells in the BM, liver and spleen as well as small populations of more immature CD34+CD7-CD5-CD3-, CD34+CD7+CD5-CD3-, CD34+CD7+CD5+CD3- cells. An hCD34+CD34-CD7-CD5-CD3- subpopulation was also evident in all organs. In contrast, vincristine-treated mice revealed a reduction in the proportions of CD34-CD7+CD5+CD3- and CD34-CD7-CD5+CD3- cells in all organs, with a concomitant increase in CD34+CD7-CD5-CD3-, CD34+CD7+CD5-CD3- and CD34+CD7+CD5+CD3- as well as a dramatic increase in the proportion of hCD45+CD34-CD7-CD5-CD3- cells. After 4 weeks following treatment-initiation, cells from the control mice again significantly comprised CD34-CD7+CD5+CD3- and, to a greater proportion than week 1, CD34-CD7-CD5+CD3- cells. The proportion of hCD45+CD34-CD7-CD5-CD3- cells was also slightly elevated in all organs compared with week 1. The TCRγ gene rearrangement was easily detectable. In contrast, residual cells from the organs of vincristine-treated animals almost entirely comprised hCD45+CD34-CD7-CD5-CD3- and no TCRγ gene rearrangement could be detected, suggesting these residual cells were both drug resistant and very immature. Finally, we harvested cells from animals 6 weeks after initiation and 2 weeks after cessation of treatment which coincided with leukaemia presentation in control animals. At this time-point, cells from both the control and treated mice significantly comprised CD34-CD7+CD5+CD3-, CD34-CD7-CD5+CD3- (which was reduced in proportion compared with week 4), as well as an emergent CD34-CD7+CD5-CD3- population. The proportion of hCD45+CD34-CD7-CD5-CD3- cells was lower in all organs compared with week 4. Vincristine treated cells were, therefore, able to rapidly repopulate the organs following cessation of treatment.

At the end of the second passage (untreated), immunophenotyping of human cells revealed a further dramatic reduction in CD34 positivity to <1% compared with 18% and 14% at pre-injection and end of first engraftment, respectively. There was also significant emergence of hCD45+CD34-CD7-CD5-CD3- cells. We are currently investigating the sensitivity of these cells to vincristine in vitro.

Intriguingly, the concomitant loss of CD34 positivity, reduction in vincristine sensitivity and increase in hCD45+CD34-CD7-CD5-CD3- cells indicates the outgrowth of a very immature population of drug resistant leukaemia stem cells during serial transplantations in mice. We are currently characterising these hCD45+CD34-CD7-CD5-CD3- cells by immunophenotyping with additional stem cell markers, gene expression analysis, SIL-TAL+ PCR, and cell sorting to investigate their ability to engraft and reconstitute leukaemia in mice. We are addressing the possibility that they can give rise to relapse over a prolonged period following treatment cessation. These findings have important implications for the development of novel therapies for T-ALL.