H e m a t o lo g y E d u c a t io n - European Hematology Association
H e m a t o lo g y E d u c a t io n - European Hematology Association
H e m a t o lo g y E d u c a t io n - European Hematology Association
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16 th Congress of the <strong>European</strong> Hemato<strong>lo</strong>gy Associat<strong>io</strong>n<br />
chemotherapy, are likely to result in activat<strong>io</strong>n of dormant<br />
HSCs. It remains to be investigated what the <strong>lo</strong>ngterm<br />
effects of this therapy will be. Several recent studies<br />
indicated that activat<strong>io</strong>n of quiescent stem cells by<br />
cytokines associated with viral and bacterial infect<strong>io</strong>n<br />
rendered them vulnerable to competit<strong>io</strong>n with HSCs<br />
resistant to stimulat<strong>io</strong>n. 54,55<br />
Conversely, in some cases, it could be clinically beneficial<br />
to activate quiescent stem cells. Certain types of<br />
stress, such as radiat<strong>io</strong>n-induced damage, are more<br />
detrimental to dormant than to cycling cells, making<br />
them prone to apoptosis. 56 Pre-stimulat<strong>io</strong>n of quiescent<br />
HSCs before rad<strong>io</strong>therapeutic treatment could possibly<br />
help to decrease the damage. 57 Moreover, use of agents<br />
activating HSCs have been proposed to break the resistance<br />
of quiescent leukemic stem cells to chemotherapeutic<br />
drugs, and consequently to increase response to<br />
chemotherapeutics (reviewed in 10 ). The first clinical<br />
study emp<strong>lo</strong>ying a combinat<strong>io</strong>n of G-CSF and imatinib<br />
mesolate is underway. 58<br />
Factors mediating the exit of HSCs from the quiescent<br />
state are mostly unidentified but are likely to be<br />
cytokines and growth factors. Current known signaling<br />
molecules include granu<strong>lo</strong>cyte co<strong>lo</strong>ny-stimulating factor<br />
(G-CSF), 59 interferon-a, 55 and interferon-g. 54 It is also likely<br />
that molecules that were used many years ago to<br />
exp<strong>lo</strong>re their potential as rad<strong>io</strong>protective agent, such as<br />
stem cell factor (SCF), 26 interleukin-1 (IL-1), 60 and FLT3ligand<br />
61 will activate dormant HSCs.<br />
HSC transplantat<strong>io</strong>n<br />
Transplantat<strong>io</strong>n of HSCs is used for treatment of a<br />
range of congenital and acquired diseases, including<br />
hematopoietic cancers (leukemias, lymphomas, and<br />
mye<strong>lo</strong>mas), mye<strong>lo</strong>dysplastic and mye<strong>lo</strong>proliferative disorders,<br />
anemias, and immunodeficiencies. The studies<br />
referred to above that document heterogeneity of bone<br />
marrow-derived HSCs al<strong>lo</strong>w speculat<strong>io</strong>n about the<br />
quality of cells derived from different sources (bone<br />
marrow, peripheral b<strong>lo</strong>od, or cord b<strong>lo</strong>od).<br />
It can be expected that cord b<strong>lo</strong>od will bear the cells<br />
with both the higher proliferative capacity 62 and lymphoid<br />
deve<strong>lo</strong>pmental potential than HSCs from mobilized<br />
b<strong>lo</strong>od or adult bone marrow, and with increased<br />
donor age, the HSC pool will <strong>lo</strong>se the ability to generate<br />
lymphoid cells and will have an overall reduced repopulating<br />
ability. Longer neutrophil and platelet recovery<br />
times in recipients of UCB graft compared with peripheral<br />
b<strong>lo</strong>od HSC or bone marrow HSCs could also reflect<br />
more quiescent, s<strong>lo</strong>wer cycling behav<strong>io</strong>r of UCB stem<br />
cells. 63 Interestingly, one of HSC activating molecules,<br />
G-CSF 59 is routinely used to mobilize HSCs for bone<br />
marrow transplantat<strong>io</strong>ns. It still remains to be investigated<br />
whether heterogeneity within the mobilized HSC<br />
pool in the b<strong>lo</strong>od reflects the bone marrow situat<strong>io</strong>n and<br />
how this could affect recipients of the HSC transplants.<br />
C<strong>lo</strong>nal behav<strong>io</strong>r of human hematopoietic<br />
cells – lessons from gene therapy<br />
Correct<strong>io</strong>n of genetic defects by introducing a therapeutic<br />
gene in a patient’s own stem cells – gene therapy<br />
– is one of the most promising applicat<strong>io</strong>ns of stem cells.<br />
HSC gene therapy has already been proven successful<br />
for correct<strong>io</strong>n of both hematopoietic and nonhematopoietic<br />
diseases, including X-linked severe-combined<br />
immunodeficiency (SCID-X), 64 b-thalassemia 65<br />
chronic granu<strong>lo</strong>matous disease (CGD), 66 adenosine<br />
deaminase deficiency, 67 and adrenoleukodystrophy. 68<br />
The essence of the treatment is the isolat<strong>io</strong>n of patients’<br />
own HSCs, which are corrected by (retroviral) gene<br />
transfer ex vivo and infused in the b<strong>lo</strong>od stream.<br />
However, the wide-spread usage of this technique has<br />
been hampered by the observat<strong>io</strong>n of a high incidence<br />
of leukemia in a severe combined immunodeficiency<br />
gene therapy trial – 4 of 10 patients in a French SCID-X<br />
study deve<strong>lo</strong>ped T-cell acute lymphoid leukemia (T-<br />
ALL). 64 One addit<strong>io</strong>nal case of leukemia was registered<br />
in a similar trial of 10 patients in the United Kingdom. 69,70<br />
Leukemia deve<strong>lo</strong>pment was associated with activat<strong>io</strong>n<br />
of pro-oncogenes (including LMO2 and CCND2) after<br />
integrat<strong>io</strong>n of the virus that was used for gene delivery<br />
in c<strong>lo</strong>se proximity to these genes, resulting in expans<strong>io</strong>n<br />
of c<strong>lo</strong>nes marked by these integrat<strong>io</strong>n sites. Similarly,<br />
c<strong>lo</strong>nal dominance preceded the deve<strong>lo</strong>pment of<br />
mye<strong>lo</strong>dysplasia and leukemia in two patients with Xlinked<br />
CDG treated with gene therapy. 66 C<strong>lo</strong>nal restrict<strong>io</strong>n<br />
was triggered by retroviral integrat<strong>io</strong>n in proximity<br />
to EVI1 and MDS-EVI1 <strong>lo</strong>ci. 66<br />
The deve<strong>lo</strong>pment of safer therapeutic vectors and the<br />
establishment of methods to control dynamics of retrovirally<br />
marked c<strong>lo</strong>nes was a prerequisite for the restart<br />
of gene therapy trials. Several methods for tracking individual<br />
HSC c<strong>lo</strong>nes by analysis of integrat<strong>io</strong>n sites have<br />
been reported. The most advanced methods combine<br />
detect<strong>io</strong>n of insert<strong>io</strong>n sites with high-throughput<br />
sequencing. Integrat<strong>io</strong>n site analysis is based on the cutting<br />
of genomic DNA isolated from transduced cells by<br />
restrict<strong>io</strong>n enzymes. Subsequently, the fragments containing<br />
parts of viral sequences are amplified by polymerase<br />
chain react<strong>io</strong>n (PCR) and analyzed by sequencing,<br />
al<strong>lo</strong>wing identificat<strong>io</strong>n of viral insert<strong>io</strong>n site. If the<br />
relative contribut<strong>io</strong>n of an integrat<strong>io</strong>n site over time<br />
increases, such c<strong>lo</strong>nal expans<strong>io</strong>n can mark deve<strong>lo</strong>pment<br />
of a mye<strong>lo</strong>proliferative disease. Coupling of integrat<strong>io</strong>n<br />
site analysis with high-throughput sequencing al<strong>lo</strong>ws<br />
simultaneous detect<strong>io</strong>n of multiple c<strong>lo</strong>nes in a single<br />
sample. For instance, a <strong>lo</strong>ngitudinal study of c<strong>lo</strong>nal fluctuat<strong>io</strong>ns<br />
in the French SCID-X study reported variat<strong>io</strong>n<br />
in 9767 integrat<strong>io</strong>n sites in the b<strong>lo</strong>od of eight patients. 71<br />
However, these data need careful interpretat<strong>io</strong>n.<br />
Aspects, such as efficiency of integrat<strong>io</strong>n site recovery,<br />
sensitivity of the method, robustness against sequencing<br />
noise, and statistical analysis can influence the scope<br />
of reported integrat<strong>io</strong>n sites.<br />
Importantly, c<strong>lo</strong>nal tracking in gene therapy studies<br />
would provide a unique opportunity to assess the heterogeneity<br />
in the human HSC pool. By analyzing the<br />
presence of different HSC c<strong>lo</strong>nes in subtypes of mature<br />
b<strong>lo</strong>od cells (granu<strong>lo</strong>cytes, B and T cells) over time, it<br />
would be possible to distinguish different human HSC<br />
types. Remarkably, a study of a patient who underwent<br />
HSC gene therapy for treatment of b-thalassemia,<br />
demonstrated <strong>lo</strong>ng-term (33 months) labeling of erythroid<br />
and mye<strong>lo</strong>id, but not lymphoid cells with vector<br />
insert<strong>io</strong>n in the HMGA2 gene, indicating possible gene<br />
transfer into mye<strong>lo</strong>id-biased stem cell. 65<br />
| 136 | Hemato<strong>lo</strong>gy Educat<strong>io</strong>n: the educat<strong>io</strong>n programme for the annual congress of the <strong>European</strong> Hemato<strong>lo</strong>gy Associat<strong>io</strong>n | 2011; 5(1)
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