Detection of Differential Mitotic Cell Ages in Bone Marrow CD34+ Cells Selected from Patients with Myelodysplastic Syndrome and Acute Leukemia by Pyrosequencing Analysis of An Epigenetic Molecular Clock DNA Signature
Authors: Mossner Maximilian , Hopfer Olaf , Baldus Claudia D , Neumann Uwe , Kmetsch Anke , Benlasfer Ouidad , Thiel Eckhard , Hofmann Wolf-Karsten 
Published: April 2020

Abstract
Introduction: Disturbed proliferation and differentiation are the most crucial oncogeneic factors leading to malignant turnover of hematopoiesis in myeloid malignancies. Therefore, estimating the lifetime proliferation status of malignant hematopoietic cells is critical. Recently the hypothesis of an epigenetic molecular clock has been corroborated. Depending on the accumulation of CpG methylation errors throughout life after each cell division it is possible to measure an increased DNA methylation of formerly unmethylated CpG islands and subsequently relate it to the mitotic cell age. In order to elucidate the importance of disturbed proliferation in hematologic diseases we initiated a novel approach for profiling mitotic ages of hematopoietic cells in myelodysplastic syndrome and acute leukemia.
Patients & Methods: Bone marrow (BM) cells of patients with myelodysplastic syndrome (MDS, IPSS-low/int-1-risk n=23, IPSS-int-2/high-risk n=27), acute myeloid leukemia (AML, n=55), acute lymphoblastic leukemia (ALL, T-lineage n=40, B-lineage n=8), and of age matched healthy individuals (n=24) were analyzed. In addition, selection of CD34+ cells was performed in MDS (n=43), in AML (n=10) as well as in healthy BM samples (n=31). CD19+ peripheral blood cells from healthy donors (n=13) served as an additional control. Genomic DNA was isolated and bisulfite converted using standard TRIZOL technique (Invitrogen, Carlsbad/CA, USA) followed by EpiTect-Bisulfite-Kit conversion (Qiagen, Hilden, Germany). PCR amplification of a CpG rich 3′ site of the Cardiac Specific Homeobox gene (CSX), considered as an epigenetic molecular clock locus, was performed as previously reported. DNA methylation was quantitative measured using the PyroMark ID Pyrosequencing system (Biotage, Uppsala, Sweden). Quantitative DNA methylation data are presented with mean ± S.E.M.
Results: In MDS int-2/high-risk specific DNA methylation of BM (26.6 ± 1.8 %) and CD34+ (28.6 ± 2.7 %) was significant higher compared to low/int-1-risk MDS (BM: 19.2 ± 1.6 %, p=0.0047, CD34+: 18.7 ± 2.4 %, p=0.0093) and healthy donors (BM: 17.8 ± 0.5 %, CD34+: 17.0 ± 0.4 %, p&lt;0.0001). Furthermore, AML BM samples showed significant higher methylation of 34.2 ± 1.7 % compared to MDS BM int-2/high-risk samples (p=0.0081). Interestingly we could detect significant higher differences in CSX methylation between paired BM/CD34+ samples in MDS low/int-1-risk, but not in MDS int-2/high-risk or AML compared to age matched healthy individuals (p=0.0063). Notably, T-lineage ALL samples did show a remarkable high mean methylation of 61.7 ± 3.1 %. However, B-lineage ALL analysis revealed a similar methylation pattern in comparison to healthy CD19+ cells (26.1 ± 1.4 % and 25.1 ± 1.4% respectively).
Discussion: The significant higher CSX methylation in AML compared to int-2/high-risk and in int-2/high-risk compared to low/int-1-risk MDS or healthy individuals could possibly be considered as a disease stage related molecular marker. The intra-individual similarity of CSX methylation levels between BM and CD34+ cells in int-2/high-risk MDS patients supports the theory of a stem cell origin of this disease subgroup, whereas low/int-1-risk MDS samples reveal higher differences possibly pointing to an origin in a more differentiated progenitor cell. However, the observation of higher mitotic ages in T-lineage but not B-lineage ALL raises questions about the role of cell proliferation in distinct lymphoblastic leukemias. In summary, the determination of mitotic cell ages by quantitative DNA methylation analysis could contribute to the molecular classification of hematological malignancies and may further be used for riskassessment in patients with MDS.