Genomic landscape of CD34+ hematopoietic cells in myelodysplastic syndrome and gene mutation profiles as prognostic markers

Abstract

Myelodysplastic syndrome (MDS) includes a group of diseases characterized by dysplasia of bone marrow myeloid lineages with ineffective hematopoiesis and frequent evolution to acute myeloid leukemia (AML). Whole-genome sequencing was performed in CD34(+) hematopoietic stem/progenitor cells (HSPCs) from eight cases of refractory anemia with excess blasts (RAEB), the high-risk subtype of MDS. The nucleotide substitution patterns were found similar to those reported in AML, and mutations of 96 protein-coding genes were identified. Clonal architecture analysis revealed the presence of subclones in six of eight cases, whereas mutation detection of CD34(+) versus CD34(-) cells revealed heterogeneity of HSPC expansion status. With 39 marker genes belonging to eight functional categories, mutations were analyzed in 196 MDS cases including mostly RAEB (n = 89) and refractory cytopenia with multilineage dysplasia (RCMD) (n = 95). At least one gene mutation was detected in 91.0% of RAEB, contrary to that in RCMD (55.8%), suggesting a higher mutational burden in the former group. Gene abnormality patterns differed between MDS and AML, with mutations of activated signaling molecules and NPM1 being rare, whereas those of spliceosome more common, in MDS. Finally, gene mutation profiles also bore prognostic value in terms of overall survival and progression free survival.

Keywords: clonal evolution; gene mutation pattern; prognostic stratification.

Fig. 1.

Genomic features of SNVs and gene fusions in RAEB. (A) Eight cases showed similar proportions of each base substitution class. (B) Circos plot of gene fusions in RAEB cases A2, A3, and A6. Green, gene fusions within the same chromosome, apart from ≤ 500Kb; blue, gene fusions within the same chromosome, apart from >500 Kb; red, fusion genes between different chromosomes. These fusions were validated by RNAseq. Gray, fusion genes not present in transcripts detected by RNAseq. (C) Genomic heat map of the frequencies of distinct trinucleotide patterns harboring each class of nucleotide substitutions among eight RAEB cases (this work) and AML subtypes. (Original data were from the Cancer Genome Atlas Research Network. For each of the AML-M1 through -M5 subtypes, data were chosen from three cases; for M0 subtype, data were from 2 cases.) Log-transformed values of the ratios are marked in the heat map. The 5′ base to each substituted base is shown on the vertical axis and the 3′ base on the horizontal axis.

Fig. 2.

Clonality analysis and distinct models of HSPC expansion defect in RAEB. (A) VAF of SNVs in eight RAEB cases. In each plot, the density curve depict the clustered VAF to determine the number of clusters. One major clone and one subclone were found in patients A1 to A6 (with VAFs varying from 37.62% to 48.86% for major peaks and those from 15.69% to 19.88% for minor peaks), whereas only one major clone was found in patients A7 and A8. (B, Left) HSPC clonal expansion status in RAEB patients. In cases A1, A2, and A7 (Upper), the same peak heights of mutated base were detected in the two populations. In case A3 to A6 and A8 (Lower), mutations were detected with high intensity in CD34⁺ but weakly or not detectable in CD34⁻ cells. Estimated mutant allele frequency: +, between 1% and 15%; ++, between 16% and 30%; +++, between 31% and 50%. (B, Right) It has been suggested that in addition to the abnormalities of eight gene categories detected in MDS, mutations involving activated signaling molecules (FLT3, KIT) or IDH1/IDH2 or NPM1 genes could contribute to the progression to AML.

Fig. 3.

Mutations of functional gene categories in MDS. Distribution of gene mutations in 145 MDS patients with at least one identified mutation of 38 marker genes. Cytogenetic risk categories are listed below mutation distribution according to IPSS-R.

Fig. 4.

Kaplan–Meier estimates of OS using the molecular marker (M) or IPSS-R-M–based stratification systems. (A) Kaplan–Meier estimates of OS according to M-based system; 3-y OS rates for low, intermediate-1, intermediate-2, and high-risk cases were 93.2 ± 3.3%, 69.4 ± 11.2%, 53.0 ± 13.9%, and 49.7 ± 8.3%, respectively (P < 0.001). n, number of cases. (B) OS according to the IPSS-R-M system; 3-y OS rates for very low, low, intermediate, high, and very high subgroups were 100%, 89.5 ± 5.9%, 83.7 ± 5.7%, 64.9 ± 9.7%, and 41.7 ± 8.4%, respectively (P < 0.001).