Identification of Highly Clonal Cells in CD34+ and Unselected Bone Marrow Samples From Patients with Myelodysplastic Syndrome Using a Sensitive Quantitative PCR Approach.
Authors: Mossner Maximilian , Nowak Daniel , Benlasfer Ouidad , Reins Jana , Hopfer Olaf Joachim , Baldus Claudia D , Thiel Eckhard , Hofmann Wolf-Karsten 
Published: October 2019

Abstract
Abstract 1760
Poster Board I-786
Myelodysplastic syndromes (MDS) are clonal hematologic malignancies with molecular defects most probably arising in the hematopoietic stem or progenitor compartment. However, due to a frequent lack of trackable cytogenetic aberrations in a large proportion of MDS patients the capability to monitor the manifestation and origin of malignant MDS clones remains limited. To elucidate clonal dominance in a given cell population, the analysis of skewed X-chromosome inactivation patterns in females, based on the methylation analysis of X-chromosomal HUMARA alleles has been used widely. However, this method has several technical and biological drawbacks as methylation changes can be induced with increasing age leading to inaccuracy of the method in this context. Recently, the application of a quantitative PCR-based method to accurately detect single nucleotide polymorphism (SNP) allele-specific RNA transcription from the X-chromosome (Swierczek et al, Blood, 2008) has shown robust results for reliable calculation of X-chromosome allelic ratios. In our study we employed this method to assess clonality in CD34+ selected and unselected bone marrow cells derived from MDS patients and provide evidence for distinct clonal manifestations of MDS clones in hematopoietic progenitor cells of all analysed MDS samples as compared to healthy controls.
Bone marrow (BM) cells were obtained from patients with MDS (IPSS-low/int-1-risk n=9, IPSS-int-2/high-risk n=9) after informed consent. Immunomagnetic selection of CD34+ cells was performed from the BM samples of MDS patients (IPSS-low/int-1-risk n=8, IPSS-int-2/high-risk n=10) and age related healthy donors (n=6) served as controls for normalization. Genomic DNA SNP genotyping using Taqman SNP Genotyping Assays (Applied Biosystems, Foster City, CA) was carried out in order to screen for informative clonality marker genes located on the X-chromosome, namely BTK, FHL1, IDS and MPP1. RNA transcripts from different alleles were quantified using SNP/allele-specific primers in a Taqman based quantitative PCR approach. Individual allelic ratios were calculated as previously described. Clonality values were assigned to 0 % according to an allelic ratio of 50/50 (polyclonal) up to 100 % for a ratio of 100/0 (clonal). All clonality values are presented as mean.
Our analyses revealed a remarkably elevated proportion of clonal cells in all purified CD34+ cells from MDS low/int-1-risk (88 %) and MDS int-2/high-risk patients (98 %) compared to the cells from healthy donors (16 %, p<0.001). The degree of clonality in unselected BM samples was similarly increased in MDS low/int-1-risk (74 %) and MDS int-2/high-risk specimen (87 %, both p<0.001 as compared to controls). However, whereas all purified CD34+ samples from MDS patients appeared to be highly clonal, 2 of 9 (22 %) of the MDS low/int-1-risk samples exhibited distinctively lower clonality in unselected BM cells with values comparable to the healthy control group. Furthermore, we observed nearly identical high clonality values in 12 paired BM/CD34+ MDS samples, except for 1 of 6 MDS low/int-1-risk samples with significantly lower clonality in the unselected bone marrow as compared to purified CD34+ cells of the same patient.
Our observation of specific clonality in both unselected bone marrow and purified CD34+ cells of MDS patients as compared to healthy controls underlines the proliferative manifestation of malignant MDS clones even in early hematopoietic progenitor cells. Furthermore, the high degree of clonality in all purified CD34+ cells suggests a clonal involvement of not only myeloid but also lymphoid lineages. Interestingly, we also identified 2 patients harboring polyclonal cells in the unselected bone marrow. In these cases differentiating cells in the bone marrow may be sustained by residual healthy hematopoietic progenitor cells. The determination whether patients with this constellation have a different clinical prognosis from patients with clonality of the complete bone marrow may be interesting to pursue. In summary, determination of clonality levels in distinct cell populations of hematologic malignancies using quantitative PCR appears to be highly suitable for monitoring the manifestation and origin of a malignant hematopoietic stem/precursor cell.

Disclosures
No relevant conflicts of interest to declare.