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Low hypodiploidy defines a rare subtype of B-cell acute lymphoblastic leukemia (B-ALL) with a dismal outcome. To investigate the genomic basis of low-hypodiploid ALL (LH-ALL) in adults, we analyzed copy-number aberrations, loss of heterozygosity, mutations, and cytogenetics data in a prospective cohort of Philadelphia (Ph)-negative B-ALL patients (n = 591, ages 18–84 years), allowing us to identify 80 LH-ALL cases (14%). Genomic analysis was critical for evidencing low hypodiploidy in many cases missed by cytogenetics. The proportion of LH-ALL within Ph-negative B-ALL dramatically increased with age, from 3% in the youngest patients (under 40 years old) to 32% in the oldest (over 55 years old). Somatic TP53 biallelic inactivation was the hallmark of adult LH-ALL, present in virtually all cases (98%). Strikingly, we detected TP53 mutations in posttreatment remission samples in 34% of patients. Single-cell proteogenomics of diagnosis and remission bone marrow samples evidenced a preleukemic, multilineage, TP53-mutant clone, reminiscent of age-related clonal hematopoiesis.Significance:We show that low-hypodiploid ALL is a frequent entity within B-ALL in older adults, relying on somatic TP53 biallelic alteration. Our study unveils a link between aging and low-hypodiploid ALL, with TP53-mutant clonal hematopoiesis representing a preleukemic reservoir that can give rise to aneuploidy and B-ALL.See related commentary by Saiki and Ogawa, p. 102.This article is highlighted in the In This Issue feature, p. 101

Supplementary figure 1. Sequencing-based analysis of CNA and LOH in adult LH-ALL. Supplementary figure 2. Representation of mutations in the frequent targeted genes in adult LH-ALL. Supplementary figure 3. Longitudinal assessment of TP53-mutant cell fraction as determined by ddPCR along with clonal IG/TR-based MRD quantification in three patients with undetectable TP53 mutation at remission. Supplementary figure 4. Merged single-cell analysis of cell-surface markers by ADT-sequencing of remission samples from three LH-ALL patients. Supplementary figure 5. Individual single-cell analyses of cell-surface markers by ADT-sequencing of remission samples from three LH-ALL patients. Supplementary figure 6. Individual analyses of single-cell immunophenotyping and genotyping of remission samples from three LH-ALL patients. Supplementary figure 7. Evaluation of allelic dropout rate on heterozygous single nucleotide polymorphism (SNPs) in remission samples. Supplementary figure 8. Individual single-cell analysis of cell-surface markers by ADT-sequencing of diagnosis samples from three LH-ALL patients. Supplementary figure 9. Single-cell genotyping of heterozygous SNPs allowing LOH assessment in diagnosis samples from three LH-ALL patients. Supplementary Figure 10. Distribution of single-cell genotypes of two LH-ALL patients with persistent TP53 and JAK2 (EI_046) or DNMT3A (EI_035) mutations in remission samples.

Supplementary Table 1. Karyotypes of LH-ALL patients at diagnosis. Supplementary Table 2. Somatic variants detected in LH-ALL patients at diagnosis. Supplementary Table 3. ARCH related variants detected in LH-ALL patients at remission. Supplementary Table 4. Minimal residual disease values of remission samples used for mutation analysis. Supplementary Table 5. Somatic alterations in cell populations from diagnostic samples (BMMC) based on single-cell analyses. Supplementary Table 6. Somatic alterations in FACS-sorted cell populations from diagnostic samples. Supplementary Table 7. Panel of genes for targeted sequencing. Supplementary Table 8. Single cell DNA amplicons for genotyping and LOH analyses. Supplementary Table 9. Single cell DNA amplicons for B-ALL clono-specific IG/TR detection. Supplementary Table 10. ADT-seq panel and spike-in antibodies. Supplementary Table 11. Single cell sequencing metrics.

Low hypodiploidy defines a rare subtype of B-cell acute lymphoblastic leukemia (B-ALL) with a dismal outcome. To investigate the genomic basis of low-hypodiploid ALL (LH-ALL) in adults, we analyzed copy-number aberrations, loss of heterozygosity, mutations, and cytogenetics data in a prospective cohort of Philadelphia (Ph)-negative B-ALL patients (n = 591, ages 18–84 years), allowing us to identify 80 LH-ALL cases (14%). Genomic analysis was critical for evidencing low hypodiploidy in many cases missed by cytogenetics. The proportion of LH-ALL within Ph-negative B-ALL dramatically increased with age, from 3% in the youngest patients (under 40 years old) to 32% in the oldest (over 55 years old). Somatic TP53 biallelic inactivation was the hallmark of adult LH-ALL, present in virtually all cases (98%). Strikingly, we detected TP53 mutations in posttreatment remission samples in 34% of patients. Single-cell proteogenomics of diagnosis and remission bone marrow samples evidenced a preleukemic, multilineage, TP53-mutant clone, reminiscent of age-related clonal hematopoiesis.Significance:We show that low-hypodiploid ALL is a frequent entity within B-ALL in older adults, relying on somatic TP53 biallelic alteration. Our study unveils a link between aging and low-hypodiploid ALL, with TP53-mutant clonal hematopoiesis representing a preleukemic reservoir that can give rise to aneuploidy and B-ALL.See related commentary by Saiki and Ogawa, p. 102.This article is highlighted in the In This Issue feature, p. 101

Oncogenic alterations underlying B-cell acute lymphoblastic leukemia (B-ALL) in adults remain incompletely elucidated. To uncover novel oncogenic drivers, we performed RNA sequencing and whole-genome analyses in a large cohort of unresolved B-ALL. We identified a novel subtype characterized by a distinct gene expression signature and the unique association of 2 genomic microdeletions. The 17q21.31 microdeletion resulted in a UBTF::ATXN7L3 fusion transcript encoding a chimeric protein. The 13q12.2 deletion resulted in monoallelic ectopic expression of the homeobox transcription factor CDX2, located 138 kb in cis from the deletion. Using 4C-sequencing and CRISPR interference experiments, we elucidated the mechanism of CDX2 cis-deregulation, involving PAN3 enhancer hijacking. CDX2/UBTF ALL (n = 26) harbored a distinct pattern of additional alterations including 1q gain and CXCR4 activating mutations. Within adult patients with Ph− B-ALL enrolled in GRAALL trials, patients with CDX2/UBTF ALL (n = 17/723, 2.4%) were young (median age, 31 years) and dramatically enriched in females (male/female ratio, 0.2, P = .002). They commonly presented with a pro-B phenotype ALL and moderate blast cell infiltration. They had poor response to treatment including a higher risk of failure to first induction course (19% vs 3%, P = .017) and higher post-induction minimal residual disease (MRD) levels (MRD ≥ 10−4, 93% vs 46%, P < .001). This early resistance to treatment translated into a significantly higher cumulative incidence of relapse (75.0% vs 32.4%, P = .004) in univariate and multivariate analyses. In conclusion, we discovered a novel B-ALL entity defined by the unique combination of CDX2 cis-deregulation and UBTF::ATXN7L3 fusion, representing a high-risk disease in young adults.