We previously reported that bone marrow mononuclear cells grown in a high concentration of G-CSF (400 ng/mL) exhibit expansion of occult monosomy 7 clones in patients with MDS, but G-CSF has no effect on bone marrow mononuclear cells from patients with severe aplastic anemia or healthy donors15
. In our prior studies healthy donor controls exhibited aneuploidy levels of up to 4% due to technical artifacts such as incomplete probe hybridization. 15
. Previous studies have reported that G-CSF mobilization induces tetraploidy, aneuploidy, and alterations in replication timing in the peripheral blood of healthy HSCT donors6–8
. However, these studies did not assess the effects of G-CSF on monosomy 7 aneuploidy. To examine the effects of G-CSF mobilization on chromosomes 7 and 8 (two MDS-associated chromosomal abnormalities) in healthy SCT donors, FISH studies were performed using centromere probes for chromosomes 7 (orange) and 8 (green) in 35 healthy G-CSF mobilized, CD34+ selected PBSC grafts (). As shown in , the percentage of monosomy 7 aneuploidy in CD34+- selected PBSCs was below the 4% detection threshold both before and after stimulation with G-CSF in culture for two weeks. Trisomy 8 aneuploidy was also undetectable in both unstimulated and stimulated CD34+ PBSCs (not shown). Previous studies by Nagler et al and Marmier-Savet et al reported that G-CSF mobilization is associated with aneuploidy of chromosomes 8 and 17 in healthy HSC donor PBSCs cultured with phytohemagglutinin to stimulate lymphocytes7–8
. While neither study examined monosomy 7 aneuploidy, the lack of trisomy 8 in our study contrasts their findings. Our study differs from theirs in that we preferentially stimulated the myeloid and stem cell pool rather than lymphocytes prior to performing FISH. The presence of chromosomal aneuploidy in mitogen-stimulated effector lymphocytes is less concerning than aneuploidy within the CD34+ stem cell compartment. Indeed, Marmier-Savet et al demonstrated that loss of chromosome 17 is detectable in lymphocytes, but not in the CD34+ fraction of G-CSF mobilized donors8
Figure 1A. FISH image of CD34+ selected PBMCs from a normal donor treated with G-CSF for 5 days. Orange is a chromosome 7 specific probe, green is chromosome 8 specific.
To further assess for chromosomal aneuploidy or rearrangements in G-CSF mobilized allografts, we performed spectral karyotype analysis (SKY) on PBSCs after G-CSF mobilization and CD34+ selection in samples from four randomly selected individual donors. A minimum of 20 metaphase spreads were analyzed for each donor sample. Chromosomal rearrangements were detected, including breakage on the short arm of chromosome 1 (depicted in ), centromeric breakage of chromosome 9, breakage on the long arm of chromosome 4, and centromeric breakage of chromosome 2. However, none met the International System for Human Cytogenetics Nomenclature definition of clonal abnormalities, nor did we detect any clonal aneuploidy in CD34+ cells by SKY.
Spectral karyotype of PBSCs obtained from G-CSF mobilized healthy allograft donors
G-CSF is administered extensively to granulocyte donors who may receive serial doses of this cytokine over a lifetime. No previous report has examined the late effects of G-CSF on monosomy 7 aneuploidy in healthy donors. We quantitated monosomy 7 and trisomy 8 aneuploidy in 38 healthy granulocyte donors who had received serial doses of 5 mcg/kg G-CSF (median= 12 doses; range 3–42) and dexamethasone (8 mg), and in 36 healthy controls. This cohort afforded us the opportunity to examine the steady state effects of G-CSF and Dex on monosomy 7 aneuploidy in healthy individuals. A single 300 mcg dose of G-CSF raises serum levels as high as 1000-fold above physiologic levels17
. As shown in , the percentage of monosomy 7 aneuploidy in G-CSF/Dex treated donors was the same as in untreated controls (p= 0.5954), and both were below the threshold of detection for FISH. Similarly, trisomy 8 aneuploidy was not detectable in control or G-CSF/Dex treated donors (not shown). To our knowledge, this is the first study examining the effects of G-CSF/Dex on monosomy 7 aneuploidy in healthy granulocyte donors.
Effect of serial doses of G-CSF/Dex on monosomy 7 aneuploidy in healthy granulocyte donors
Some imitations of our study design are that it does not allow us to assess the effects of G-CSF on chromosomal aneuploidy in the bone marrow, and our HSCT donor studies were performed immediately after a first or second round of G-CSF mobilization, so they may not be applicable to donors who undergo G-CSF mobilization on a more frequent basis. It is conceivable that small monosomy 7 clones may be induced by G-CSF in the marrow which we could not detect in circulating CD34+ HSCs. However, the chromosomal changes seen in previous studies were readily detectable in peripheral blood cells at the time of G-CSF mobilization6–8
. Furthermore, monosomy 7 MDS has a short latency from initial diagnosis to progression to acute leukemia2
, and emerging data from observational studies do not indicate an increased incidence of monosomy 7 AML in unrelated HSCT donors18;19
In summary, we observed neither monosomy 7 aneuploidy nor clonal loss of chromosome integrity in G-CSF mobilized, CD34+ selected PBMCs in unmanipulated cells, or after culturing the allografts in high concentrations of G-CSF in order to promote the expansion of occult aneuploid myeloid or stem cell clones.15
We also did not detect monosomy 7 or trisomy 8 aneuploidy in a cohort of granulocyte donors treated serially with as many as 42 doses of G-CSF/Dex. Our findings should be reassuring to healthy granulocyte and HSCT donors, and they are consistent with recent reports of no increase in myeloid malignancies in cohorts of unrelated stem cell18;19
and granulocyte donors20