Childhood myelodysplastic syndromes (MDS) account for less than 5% of pediatric hematologic malignancies and differ from their adult counterpart in terms of biology, genetics, and cure rates. Complete (-7) or partial loss (del7q) of chromosome 7 constitutes the most common cytogenetic abnormality and is associated with more advanced disease typically requiring timely hematopoietic stem cell transplantation (HSCT). Previously, we and others established a link between -7 and germline GATA2 mutations in pediatric MDS (37% of MDS/-7 cases are GATA2-deficient) as well as constitutional SAMD9/9L disorders where -7 is utilized as an escape mechanism from the growth-restrictive effect of SAMD9/9L mutations. To date, comprehensive sequencing studies have been performed in 96 children with primary MDS, as reported by Pastor et al, Leukemia 2017 and Schwartz et al, Nature Comm 2017. This work established mutations in SETBP1, ASXL1, PTPN11, RUNX1 and RAS pathway genes as common somatic drivers. However, little is known about the clonal development of -7 and the role of additional somatic mutations. The knowledge about clonal hierarchies is essential for the understanding of disease progression on molecular level and for mapping potential drug targets. The rationale for the current study was to i) define the most common somatic drivers in a large cohort of patients with childhood MDS, ii) identify clonal/subclonal mutations, iii) infer clonal architecture of monosomy 7 and track the changes over time.

We studied a cohort of 576 children and adolescents with primary MDS diagnosed between 1998 and 2016 in Germany, consisting of 482 (83%) patients with refractory cytopenia of childhood (RCC) and 94 (17%) MDS with excess blasts (EB). All patients underwent deep sequencing for 30 genes relevant to pediatric MDS and additional WES was performed in 150/576 patients. Using 20 computational predictors (including CADD and REVEL), population databases and germline testing, we identified the most likely pathogenic mutations.

First, we excluded germline predisposing mutations in GATA2, SAMD9/SAMD9L and RUNX1 detected in 7% (38/576), 8% (43 of 548 evaluable) and 0.7% (4/576) of patients, respectively. Then we focused on the exploration of somatic aberrations. Most common karyotype abnormalities were monosomy 7 (13%, 77/576) and trisomy 8 (3%, 17/576). A total of 104 patients carried somatic mutations, expectedly more prevalent in the MDS-EB group as compared to RCC (56%, 53/94 vs 10.6%, 51/482; p<0.0001). The most recurrent somatic hits (≥ 1% frequency within 576 cases) were in SETBP1 (4.2%), ASXL1 (3.8%), RUNX1 (3.3%), NRAS (2.9%), KRAS (1.6%), PTPN11 (1.4%) and STAG2 (1%). We next focused on the -7 karyotype as a common denominator for the mutated group. Mutations were found in 54% (43/79), and the mutational load was significantly higher in -7 vs. non-7 (1.1 vs. 0.1 mutations per patient; p<0.001). In 11 patients with -7 and concomitant SETBP1/ASXL1 driver mutations, SETBP1 surpassed ASXL1 hits (median allelic frequency: 38% vs. 24%, p<0.05), while mutations in other genes were subclonal. Notably, these clonal patterns were independent of the underlying hereditary predisposition (4/11 GATA2; 3/11 SAMD9L). To explore the clonal hierarchy in MDS/-7 we performed targeted sequencing of several hundreds of single bone marrow derived colony forming cells (CFC) in 7 patients with MDS/-7. In all cases, the -7 clone was the founding clone followed by stepwise acquisition of mutations (i.e. -7>SETBP1>ASXL1; -7>SETBP1>ASXL1>PTPN11; -7>SETBP1>ASXL1>CBL, -7>EZH2>PTPN11). Finally, we tracked clonal evolution over time in 12 cases with 2-12 available serial samples using deep sequencing complemented by serial CFC-analysis. This confirmed that SETBP1 clones are rapidly expanding, while ASXL1 subclones exhibit an unstable pattern with clonal sweeping, while additional minor clones are acquired as late events. In 2 of 11 transplanted patients who experienced relapse, the original clonal architecture reappeared after HSCT.

In summary, the hierarchy of clonal evolution in pediatric MDS with -7 follows a defined pattern with -7 aberrations arising as ancestral event followed by the acquisition of somatic hits. SETBP1 mutations are the dominant driver while co-dominant ASXL1 mutations are unstable. The functional interdependence and potential pharmacologic targetability of such somatic lesions warrants further studies.


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Author notes


Asterisk with author names denotes non-ASH members.