Low frequency of type-I and type-II aberrations in myeloid leukemia of Down syndrome, underscoring the unique entity of this disease

We recently published in this journal an overview of the currently known genetic events required for the development of pediatric acute myeloid leukemia (AML).1 These aberrations can be subdivided into type-I aberrations that result in uncontrolled proliferation, and type-II aberrations that lead to the impaired differentiation of the leukemic cells.1–2

Recent advances in technology have allowed many novel genetic and molecular abnormalities to be detected, including cryptic translocations (such as NUP98-NSD1), and single gene mutations, occurring for instance in the NPM1, CEBPA, WT1 and MLL-gene (MLL-PTD) which are predominantly found in patients with cytogenetically normal (CN)-AML.1,3 Newly discovered mutations identified by whole genome sequencing include mutations in the genes encoding for IDH1/ IDH2 and the DNA methyl-transferase (DNMT3A) gene, which are rare in pediatric AML.4–5

Children with Down syndrome have an increased risk of developing acute myeloid leukemia (ML-DS).6 ML-DS is a unique disease entity, and differs in clinical characteristics and biology from AML in non-DS children. It is characterized by somatic mutations in the GATA-1 gene7 which are unique for every patient. The role of the well-known and newly discovered type-I/II aberrations in myeloid leukemia of Down syndrome (ML-DS) has not yet been systematically investigated.

Therefore, we screened 34 newly diagnosed ML-DS patients for the presence of the above mentioned type-I and type-II aberrations. Samples were provided by the Dutch Childhood Oncology Group, the AML-’Berlin-Frankfurt-Munster’ Study Group, and the Nordic Society for Pediatric Hematology and Oncology. Of the 34 patients, 12 ML-DS patients had a normal karyotype; this is important to note since some of the novel aberrations in non-DS AML are highly associated with a normal karyotype.

Screening of gene mutations was carried out according to availability of material. Mutation analysis was performed for the hotspot regions of the NPM1, CEPBA, MLL (i.e. partial tandem duplications, PTD), WT1, FLT3 (i.e. internal tandem duplications, ITD) and tyrosine kinase domain mutations (TKD), N-RAS and K-RAS, PTPN11, KIT, IDH1/ IDH2, and the DNMT3A genes, as previously described.1,5 In addition, we investigated the presence of the cryptic translocation NUP98/NSD1 by reverse transcriptase-polymerase chain reaction (RT-PCR).3 A complete list of investigated regions, primers and PCR conditions is provided in the Online Supplementary Table S1. Purified PCR products were bi-directionally sequenced on an ABI Prism 3100 genetic analyzer (Applied Biosystems Inc., Foster City, CA, USA). The sequence data were assembled and analyzed for mutations using CLC Workbench version 3.5.1 (CLC Bio, Aarhus, Denmark).

Table 1.Characteristics of ML-DS patients.

Median age of the ML-DS patients was 1.8 years (range 0.7–2.7 years). Median WBC was 7.1×10/L (range 3–168×10/L) and 42% were male. Patients’ characteristics are described in detail in Table 1.

In our cohort, only mutations were found in the RAS-pathway, i.e. 2 of 34 (7%) of the patients (ID5 and ID30) carried the G12D mutation in the K-RAS gene, and in one patient (ID 32) (3%) the G12D mutation in N-RAS was found. These RAS-mutations have been described in approximately 20% of non-DS pediatric AML patients.1 In a previous report by Chen et al., N-RAS mutations were reported in one of 9 ML-DS and in none of 11 TMD patients.8

No WT1 mutations were found, but 4 of 34 (17%) ML-DS patients carried the rs16754 single nucleotide polymorphism (SNP) in the WT1 gene. WT1 expression is used as a marker for minimal residual disease (MRD) detection in TMD9 and non-DS pediatric AML.10 To date, no WT1 SNPs have been described in ML-DS patients. There is still some controversy about the prognostic impact of this SNP in non-DS pediatric AML; Hollink et al. did not find any prognostic significance11 whereas Ho et al. identified this WT1 SNP as an independent predictor of favorable outcome.12 None of our patients with the WT1 SNP had an event; however, one patient died due to resistant disease. Two patients with the WT1 rs16754 SNP simultaneously had an RAS-mutation.

Mutations in FLT3, KIT, CEBPA, NPM1, MLL-PTD, DNMT3A and IDH1/2 were not found. Two patients (7%) carried the IDH1 rs11554137 SNP. In non-DS pediatric AML, this IDH1 SNP was found in 47 of 460 cases (10%).4 The NUP98/NSD1 transcript was not detected in any of our samples.

Table 2.Frequency of aberrations in ML-DS patients compared to non-DS AML patients.

When we examined the expected frequency of the aberrations in our ML-DS cohort compared to the observed frequency in non-DS AML pediatric AML patients as a reference cohort (calculated from a binomial distribution), only the frequency of WT1, FLT3-ITD, and nRAS-mutations appeared to be significantly lower. The lack of a statistically significant result in the other aberrations may be due to the low frequencies in non-DS AML. An overview of the frequencies and aberrations is provided in Table 2.

We conclude that the (molecular) type-I/type-II aberrations, relevant in pediatric non-DS AML, are absent or rare in ML-DS patients. Except for mutations in the RAS-gene (and SNPs in the WT1 and IDH1 genes), we did not detect any aberrations. Our study underscores the unique signature of ML-DS, and stresses the fact that further research is needed to unravel the molecular abnormalities involved in the leukemogenesis of this specific disease.

Footnotes

• The online version of this article has a Supplementary Appendix.
• The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www.haematologica.org.
• Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are also available at www.haematologica.org.