Differential Distribution of Clonal Hematopoiesis in Flow-Sorted Subpopulations of Patients with Myelodysplastic Syndromes (MDS)

Introduction:

Patients (pts) with MDS display characteristic morphological and cytogenetic features in the bone marrow (BM). Flow cytometry (FCM) has become a valuable tool in diagnostics e.g. allowing for the distinct identification of abnormal antigen expression (e.g. CD5, CD7, CD56). It is still unknown, whether 'phenotypically defined dysplasia' reflects the genetically abnormal clone.

Methods:

A total of 52 BM samples (IPSS-R very low/low=18, int=9, high/very high=25) of 39 MDS pts with cytogenetic aberrations [del(5q)=41; del(7q)=4; other=7] were analyzed by FCM according to ELN criteria and sorted for specific subpopulations. CD45 expression and side scatter were used to differentiate between progenitors, granulopoiesis (GP) or nucleated red cells (NRC). Myeloid progenitors (myPC) were further characterized by CD117+CD34+ and sorted according to their CD56 expression. Maturation stages of GP were differentiated according to their CD13/CD16 or CD10 expression and NRC as CD235a+ and CD71+/dim cells. Clonal distribution of FCM-defined cellular subpopulations was subsequently quantified via interphase (i)FISH. Additionally, in five selected samples with a known TP53 mutation, next generation sequencing on a 454Junior system was used to assess individual variant allele frequency (VAF).

Results:

Cytogenetic aberrations were detected in all FCM-defined subpopulations with the highest frequency in myPC (median 81%) vs. GP (67%, p=0.002) and NRC (28%, p<0.01). When focusing on del(5q) pts, there was a difference in the proportion of clonal cells between CD56+ vs. CD56- myPC (81% vs. 58%, n=7, p=0.091) as well as CD10- vs. CD10+ GP (73% vs. 67%, p=0.021). Grouping del(5q) pts by IPSS-R, myPC showed a higher amount of clonal cells than immature GP (CD13+CD16-) (82% vs. 61%, p=0.037) in the very low/low risk compared to more advanced risk stages (81% vs. 78%, ns).

Interestingly, myPC of del(5q) pts with an aberrant CD5 or CD7 expression harboured a higher clonal involvement than CD5-CD7- pts (88% vs. 73%, p=0.035). This difference was even more prominent at the GP level (88% vs. 56%, p=0.003). Furthermore, the significantly higher amount of clonal NRC in the CD5/CD7 aberrant group (72% vs. 17%, p=0.002) was associated with higher serum erythropoietin levels (median: 355 vs. 78 U/L, ns) while LDH serum levels were not different (median: 211 vs. 175 U/l, ns).

Additionally, we observed different VAF of TP53 gene mutation within FCM-defined subpopulations [median VAF myPC: 79% (47-81); GP: 29% (14-52); NRC: 49% (47-52)]. Interestingly, VAF of TP53 was lower compared to the amount of cells harbouring del(5q). In fact, there was a major difference for the GP (29% vs. 83%, p=0.006).

Conclusions:

Within this study we demonstrate that clonal cells can be detected in all FCM-defined cellular compartments with a varying distribution and changes correlate with disease stage. Aberrant CD5 or CD7 expression seems to define a subgroup with a high clonal burden in all investigated cell compartments, including the erythroid lineage. This might explain the worse response to erythropoietin compared to pts without these features. In addition, the detection of TP53 mutations in a subset of the del(5q) clone only points to the fact that this mutation is a rather late event in the evolution of MDS.