Genomic landscape and clonal architecture in pediatric myeloid neoplasms with chromosome 7 deletions

TO THE EDITOR:

Full or partial loss of chromosome 7 (Chr7; del7 and 7q–) is a prevalent cytogenetic abnormality in pediatric myeloid neoplasms (MNs; pMNs), including myelodysplastic syndrome (MDS), juvenile myelomonocytic leukemia (JMML), acute myeloid leukemia (AML), and therapy-related AML/MDS, and is associated with a high risk of disease progression and poor clinical outcomes.^1,2 del7 and 7q– result in haploinsufficiency of key hematopoietic regulator genes located on Chr7, including SAMD9, SAMD9L, KMT2C/MLL3, EZH2, CUX1, and DOCK4. The overall paucity of second-hit mutations in these Chr7 genes in MNs with Chr7 abnormalities supports the idea that this represents a contiguous gene syndrome.3, 4, 5, 6 Although the genomic landscape of MNs in adults with del7 and 7q– has been described,^7,8 comprehensive next-generation sequencing analyses of pMNs with similar Chr7 losses have not been reported.

To address this knowledge gap, we assembled a cohort of 108 pediatric and young adult patients (median age, 6.9 years [range, 0.4-24.6]; supplemental Table 1) with Chr7 abnormalities in the setting of AML (48%), MDS (36%), JMML (2%), acute leukemia of ambiguous lineage (2%), and unclassified MNs (12%; supplemental Figure 1A). Whole-exome sequencing (n = 63), whole-genome sequencing (n = 41), probe-based target capture sequencing (n = 64), and/or RNA sequencing (RNA-seq; n = 75)9, 10, 11, 12, 13, 14 were used to define the Chr7 status of each patient and identify single-nucleotide variants, insertion/deletion events, structural variants (SVs), and additional copy number alterations (supplemental Tables 2-4; supplemental Figure 1B). Patients with Chr7 alterations (hereafter collectively termed alt(7)) were grouped into 3 broad categories: complete loss of Chr7 (61%), full or partial loss of 7q (minimum = 0.4 kb loss; 32%), and complex changes including incomplete losses of both 7q and 7p (7%). Commonly deleted regions found in our cohort overlap with those reported by other groups (Figure 1A).^3,4,15, 16, 17, 18

Figure 1.

Genomic landscape and expression profile of pediatric patients with del7 or 7q alterations. (A) Ideogram of Chr7 summarizing deleted regions. Each vertical bar represents the portion of Chr7 lost in a single patient. Genes of interest in red font. Previously established commonly deleted regions highlighted in yellow.^3,4,15, 16, 17, 18 (B) Recurrent somatic and germ line SNVs and insertion/deletion events (indels) in patients with AML (n = 494) and MDS (n = 24) with 2 intact copies of Chr7 compared with those in patients with del7 and 7q alterations (n = 108). Genes located on Chr7 are in red font. Statistical significance was assessed by 2-sided Fisher exact test (∗P < .05). (C) Clinical and genomic features of the study cohort with each column representing a single patient. Top section depicts clinical characteristics. Heat map below highlights recurrently mutated genes sorted by functional class. Box and whisker plot representing interquartile range, median, and value range of VAFs for each gene located on the right. (D) Volcano plot of the differentially expressed genes (fold change > 1; FDR < 0.05; 2443 upregulated and 510 downregulated) resulting from a comparison of del7 patients (n = 25) with patients with 2 intact, wild-type copies of Chr7 (AML, n = 494; MDS, n = 24). (E) Top 10 significantly upregulated and downregulated GO terms from the comparison described in panel D. ALAL, acute leukemia of ambiguous lineage; FDR, false discovery rate; GO, gene ontology; ITD, Internal Tandem Duplication; mRNA, messenger RNA; SNV, single-nucleotide variant; VAF, variant allele frequency.

Pathogenic or likely pathogenic somatic and germ line single-nucleotide variants and insertion/deletion events were identified in 103 patients (95%). Most mutations involved genes in the Ras/MAPK pathway, including PTPN11 (24%), KRAS (21%), NRAS (19%), and NF1 (15%). Other commonly mutated genes (>10% of the cohort) include RUNX1 (15%), ASXL1 (13%), SETBP1 (11%), and EZH2 (10%; Figure 1B-C). Consistent with previous data, germ line alterations in SAMD9, SAMD9L, and GATA2 were common in this cohort.^10,19,20 Mutations involving EZH2 (located at 7q36) were the most commonly observed somatic mutations involving genes on Chr7. Somatic EZH2 mutations were identified in 11 cases, all of which had Chr7 copy number alterations involving 7q36. Many of these variants present at a variant allele frequency >40%, suggesting that the variant occurs on the nondeleted chromosome, potentially leading to a double hit for EZH2. In contrast to EZH2, only 1 of 5 mutations in IKZF1 (located at 7p12) occurred in a case that also harbored a deletion that extended to 7p12. We did not observe secondary CUX1 mutations.⁶ To compare our alt(7) cohort with pediatric and young adult myeloid tumors with 2 intact, wild-type copies of Chr7 (collectively termed wt(7)), we compiled recently published whole-genome sequencing, whole-exome sequencing, and RNA-seq data from pediatric patients with AML (n = 494) and MDS (n = 24; supplemental Table 5).^10,13 Mutations in 14 genes, including PTPN11, KRAS, and SETBP1, were overrepresented in alt(7) compared with wt(7), whereas mutations in CEBPA, NPM1, and IDH1 were absent in our alt(7) cohort (Figure 1B).

Differential gene expression and gene ontology term analyses of RNA-seq data were performed to evaluate differences between patients with complete loss of Chr7 (del7; n = 25) and wt(7) samples (n = 518). As expected, expression of genes on Chr7 was low in del7 compared with wt(7), whereas MECOM and other genes associated with MECOM-dysregulated leukemias (eg, PAWR and PREX2)²¹ and poor prognosis were high (Figure 1D). Gene ontology term analysis revealed that cell migration and immune response–related genes were enriched in del7 samples, whereas RNA splicing and processing genes were enriched in wt(7) samples (Figure 1E).

To further understand the clonal structure and cell lineage of pMN with alt(7), we subjected a representative subset of 8 alt(7) samples for single-cell DNA sequencing with cellular indexing of transcriptomes and epitopes (MissionBio Tapestri, San Francisco, CA), which enables us to phenotypically define cell populations and determine pathogenic trajectories and clonal evolution at the single-cell level. Using data from our previous publications,^10,13 we designed a custom DNA panel to detect recurrent mutations in pMNs (supplemental Table 6) and determine Chr7 ploidy status. Commercially available barcoded-tagged antibodies were used to detect cell surface proteins. These single-cell DNA sequencing and cellular indexing of transcriptomes and epitopes data showed that clonal evolution in alt(7) pediatric AML/MDS followed a consistent pattern, with the proceeding full or partial loss of Chr7 in the myeloid compartment, followed by the acquisition of leukemia-associated mutations including those in the Ras/MAPK and epigenetic pathways. JMML cases were a notable exception to this pattern, with the Ras/MAPK mutation preceding Chr7 loss, as previously described (Figure 2A-C; supplemental Figure 2).22, 23, 24

Figure 2.

Single-cell multiomics to define clonal evolution. (A) Stacked heat maps representing genotype (top), cell surface markers (middle), and Chr7 ploidy (bottom). Each column is a single analyzed cell. (B) Centered log ratio count histograms showing relative abundance of relevant cell surface markers by cell genotype. (C) Inferred clonal evolution trees using single-cell DNA-sequencing data and cellular indexing of transcriptomes and epitopes-sequencing data. Disease type and the number of cells analyzed are listed below the patient identifier. The first population (WT; purple) refers to the percentage of myeloid cells without a driving mutation or alt(7). Each following population (orange and/or green) depicts a sequential, additional modification to the initial myeloid population. Adj, Adjusted.

These molecular data highlight key differences between pediatric myeloid tumors with and without Chr7 alterations. Our data demonstrate that pediatric tumors with deletions involving Chr7 are more likely to obtain secondary mutations in PTPN11, KRAS, and NF1 than other myeloid tumors, which suggests a synergy between specific Ras/MAPK genes and Ch7 loss.^13,25 Alterations in SETBP1, which are more frequent in pediatric MDS¹⁰ and JMML²² than other pediatric myeloid tumors,¹³ are another major cooperating mutation in pediatric alt(7) cases. In contrast, secondary mutations in genes on Chr7, such as CUX1, are rare, except for EZH2. Coupled with the recurrent nature of Chr7 deletions in pediatric myeloid malignancies, these findings further highlight that this likely represents a contiguous gene syndrome driven by haploinsufficiency of multiple genes on Chr7. Unfortunately, this represents a significant challenge for disease modeling, considering the lack of synteny between whole human and mouse chromosomes. For example, our 5G2 mouse model harboring a germ line 1.5-MB deletion syntenic to human Chr 7q22 failed to result in overt hematopoietic neoplasms,^4,26 despite more profound alterations being observed with isolated knockdown of Cux1.²⁷

These data also implicate the dysregulation of specific transcriptional networks, such as MECOM and associated EVI1 pathways, as contributing to disease phenotypes in pMNs with alt(7). SVs resulting in aberrant MECOM/EVI1 expression occur in pediatric and adult MNs and are commonly associated with Chr7 deletions and poor outcomes.^11,28 There are 9 cases with MECOM SVs in this cohort, confirming that these alterations are relatively uncommon compared with monosomy 7 in general. Our findings suggest that MECOM dysregulation can occur through other mechanisms, and its expression is likely important for disease development or progression.^29,30

The involvement of inflammatory pathways is consistent with previous studies³¹ and was recently observed in our 5G2 mouse model.²⁶ Perturbation of cellular response to inflammation is likely an important cooperating event. Supporting this argument is the observation that germ line mutations in SAMD9 and SAMD9L, interferon-inducible genes located at human Chr7q21, are known predisposing factors to myeloid diseases with Chr7 deletions and that gain-of-function pathogenic mutations are lost in the resulting alt(7) clones.^19,20 Similar findings were observed in our murine Samd9l mouse model, in which an in vivo interferon challenge resulted in chromosomal deletions lacking the mutant Samd9l allele.³²

The role of Chr7 deletions in the pathogenesis and poor clinical outcomes of pMNs remains enigmatic. In contrast to many adult MNs with alt(7), Chr7 deletions are early/initiating events in pMNs. The genetic analyses of primary patient specimens in this study and our adjacent prior preclinical data support a model in which alt(7) cooperates with secondary mutations and inflammatory stress. Future studies focused on pMNs and mouse models of these disorders should advance our understanding of how Chr7 deletions perturb hematopoietic growth control and contribute to leukemogenesis.

Conflict-of-interest disclosure: M.L.L. reports serving on the advisory board for Jazz Pharmaceuticals. The remaining authors declare no competing financial interests.