Pathophysiologic and clinical implications of molecular profiles resultant from deletion 5q

Abstract

Background: Haploinsufficiency (HI) resulting from deletion of the long arm of chromosome 5 [del(5q)] and the accompanied loss of heterozygosity are likely key pathogenic factors in del(5q) myeloid neoplasia (MN) although the consequences of del(5q) have not been yet clarified.

Methods: Here, we explored mutations, gene expression and clinical phenotypes of 388 del(5q) vs. 841 diploid cases with MN [82% myelodysplastic syndromes (MDS)].

Findings: Del(5q) resulted as founder (better prognosis) or secondary hit (preceded by TP53 mutations). Using Bayesian prediction analyses on 57 HI marker genes we established the minimal del(5q) gene signature that distinguishes del(5q) from diploid cases. Clusters of diploid cases mimicking the del(5q) signature support the overall importance of del(5q) genes in the pathogenesis of MDS in general. Sub-clusters within del(5q) patients pointed towards the inherent intrapatient heterogeneity of HI genes.

Interpretation: The underlying clonal expansion drive results from a balance between the "HI-driver" genes (e.g., CSNK1A1, CTNNA1, TCERG1) and the proapoptotic "HI-anti-drivers" (e.g., RPS14, PURA, SIL1). The residual essential clonal expansion drive allows for selection of accelerator mutations such as TP53 (denominating poor) and CSNK1A1 mutations (with a better prognosis) which overcome pro-apoptotic genes (e.g., p21, BAD, BAX), resulting in a clonal expansion. In summary, we describe the complete picture of del(5q) MN identifying the crucial genes, gene clusters and clonal hierarchy dictating the clinical course of del(5q) patients.

Funding: Torsten Haferlach Leukemia Diagnostics Foundation. US National Institute of Health (NIH) grants R35 HL135795, R01HL123904, R01 HL118281, R01 HL128425, R01 HL132071, and a grant from Edward P. Evans Foundation.

Keywords: 5q deletion; CSNK1A1; Haploinsufficiency; Myelodysplastic syndromes; TP53.

Figure 1

Cytogenetic and molecular characterization of 5q: (a) CNV analysis based on the cytogenetic break points was used to determine the most commonly deleted region in del(5q). (b) Mutation distribution in isolated del(5q), compound del(5q), and diploid patients. (c) Percentage of mutant patients in the most common mutated genes (mutations on chromosome 5 are not included).

Figure 2

Clonal architecture and subclonal hierarchy: reconstruction of the clonal hierarchy using an allelic imbalance method for WES samples (with available paired sample) and by CNV analyses for WGS, were compared to the VAF of most common mutations adjusted by zygosity and copy number. (a) Distribution of ancestral (navy blue), co-dominant (yellow) or subclonal (light purple) del(5q) in isolated del(5q) and compound del(5q). (b) Kaplan-Meier curves for ancestral, secondary and co-dominant del(5q) event with significant differences (Log rank test). (c) Mutational distribution of the most representative mutations in dominant, co-dominant or subclonal del(5q) in patients with isolated del(5q) or compound del(5q). (d) Exemplary cases of clonal architecture of isolated del(5q) patients (left) and in compound del(5q) patients (right). (e) Percentage of clonal burden of del(5q) (red), increased subclonal mutations (yellow), and decreased subclonal mutations (blue). Lines connect paired samples. Doted lines indicate undetectable mutation after treatment.

Figure 3

Haploinsufficiency (HI) analysis of selected genes on 5q. (a) Box plot showing the definition of HI expression defined when the expression in del(5q) was lower than the percentile 25th of the diploid expression level. (b) Gene expression (Log₂ CPM) in del(5q) cases (pink) and diploid cases (blue). Dashed lines show median values. (c) Exemplary cases of different correlation types between expression and clonality in isolated del(5q) and compound del(5q). Green error bar shows the expression of the given gene in diploid cases. Black error bar shows the expected expression at 50% and 100% clonality of del(5q). (d) Heatmap using the spares model-based clustering of del(5q) and diploid patients (see methods), including the 57 HI genes. Karyotype group, TP53 mutational status, BM Blast % group and % of clonality are also depicted.

Figure 4

Minimal gene signature of del(5q). (a) Heat map including the del(5q) 5 gene-minimal signature according to a sparse Bayesian prediction analysis that differentiates between del(5q) and diploid patients. Expression of del(5q) patients was adjusted to a 50% del(5q) clonality representing the expression of del(5q) and diploid patients. Blue colours represent a low expression while red colours represent up-regulation of the expression (white means neutral). (b) Heat map including the del(5q) 9 gene-minimal signature within del(5q) patients. Blue colours represent a low expression while red colours represent up-regulation of the expression (white means neutral).

Figure 5

Pathogenic mutational interactions in del(5q): (a) Expression of selected pro-apoptotic genes showing a significant higher expression in del(5q) compared to diploid patients. (b) Frequency of mutant CSNK1A1 that are co-mutated with TP53 (left) and frequency of TP53 mutants that include CSNK1A1 mutations (right). (c) Expression (Log₂ CPM) of CSNK1A1 in del(5q) patients and diploid patients. (d) RPS14 expression (Log₂ CPM) comparison between diploid patients with low expression (le) of CSNK1A1 and diploid patients with normal expression of CSNK1A1. (e) CSNK1A1 expression comparison between del(5q) patients WT (solid colour) or MT (grey stripes) for CSNK1A1, TP53. Diploid WT patient expression is also depicted. (f) CDKN1A expression comparison between del(5q) patients WT (solid colour) or MT (grey stripes) for the CSNK1A1 and TP53 genes. Diploid WT patient expression is also depicted. (g) CDKN1A expression in del(5q) vs. del(5q) low RPS14 expressors and diploid patients vs. diploid low RPS14 expressors (cluster-2). (h) Correlation between the percentage of del(5q) (according to CNV analysis) and expression (Log₂ CPM) of excluded genes located on 5q. Green error bar represents the expression of the given gene in diploid patients, TP53 wild type, and <5% bone marrow blasts. Black error bar represents the estimated expression of the given gene at 50% and 100% of del(5q) clonality. [Isolated del(5q) patients (blue line), compound del(5q) (pink line)]. (i) EGR1 expression in del(5q) and diploid cases TP53 mutant (solid colour) or wild type (grey stripes). (j) Correlation between % of del(5q) (according to CNV analysis) and expression (log₂ CPM). Dots represents expression and clonality of patients without deletion involving APC or DDX41. (k) APC expression in del(5q) and diploid cases. On the right frequency of CSNK1A1 mutants and wild type in del(5q) APC low expressors. (l) DDX41 expression in patients with del(5q) and DDX41 deleted, del(5q), and diploid cases.