High hyperdiploid acute lymphoblastic leukemia (HeH ALL), one of the most common childhood malignancies, is driven by nonrandom aneuploidy (abnormal chromosome numbers) mainly comprising chromosomal ...gains. In this study, we investigate how aneuploidy in HeH ALL arises. Single cell whole genome sequencing of 2847 cells from nine primary cases and one normal bone marrow reveals that HeH ALL generally display low chromosomal heterogeneity, indicating that they are not characterized by chromosomal instability and showing that aneuploidy-driven malignancies are not necessarily chromosomally heterogeneous. Furthermore, most chromosomal gains are present in all leukemic cells, suggesting that they arose early during leukemogenesis. Copy number data from 577 primary cases reveals selective pressures that were used for in silico modeling of aneuploidy development. This shows that the aneuploidy in HeH ALL likely arises by an initial tripolar mitosis in a diploid cell followed by clonal evolution, in line with a punctuated evolution model.
High-resolution genomic microarrays enable simultaneous detection of copy-number aberrations such as the known recurrent aberrations in chronic lymphocytic leukemia del(11q), del(13q), del(17p) and ...trisomy 12, and copy-number neutral loss of heterozygosity. Moreover, comparison of genomic profiles from sequential patients' samples allows detection of clonal evolution.
We screened samples from 369 patients with newly diagnosed chronic lymphocytic leukemia from a population-based cohort using 250K single nucleotide polymorphism-arrays. Clonal evolution was evaluated in 59 follow-up samples obtained after 5-9 years.
At diagnosis, copy-number aberrations were identified in 90% of patients; 70% carried known recurrent alterations, including del(13q) (55%), trisomy 12 (10.5%), del(11q) (10%), and del(17p) (4%). Additional recurrent aberrations were detected on chromosomes 2 (1.9%), 4 (1.4%), 8 (1.6%) and 14 (1.6%). Thirteen patients (3.5%) displayed recurrent copy-number neutral loss of heterozygosity on 13q, of whom 11 had concurrent homozygous del(13q). Genomic complexity and large 13q deletions correlated with inferior outcome, while the former was linked to poor-prognostic aberrations. In the follow-up study, clonal evolution developed in 8/24 (33%) patients with unmutated IGHV, and in 4/25 (16%) IGHV-mutated and treated patients. In contrast, untreated patients with mutated IGHV (n=10) did not acquire additional aberrations. The most common secondary event, del(13q), was detected in 6/12 (50%) of all patients with acquired alterations. Interestingly, aberrations on, for example, chromosome 6q, 8p, 9p and 10q developed exclusively in patients with unmutated IGHV.
Whole-genome screening revealed a high frequency of genomic aberrations in newly diagnosed chronic lymphocytic leukemia. Clonal evolution was associated with other markers of aggressive disease and commonly included the known recurrent aberrations.
The expression levels of LPL, ZAP70, TCL1A, CLLU1 and MCL1 have recently been proposed as prognostic factors in chronic lymphocytic leukemia. However, few studies have systematically compared these ...different RNA-based markers.
Using real-time quantitative PCR, we measured the mRNA expression levels of these genes in unsorted samples from 252 newly diagnosed chronic lymphocytic leukemia patients and correlated our data with established prognostic markers (for example Binet stage, CD38, IGHV gene mutational status and genomic aberrations) and clinical outcome.
High expression levels of all RNA-based markers, except MCL1, predicted shorter overall survival and time to treatment, with LPL being the most significant. In multivariate analysis including the RNA-based markers, LPL expression was the only independent prognostic marker for overall survival and time to treatment. When studying LPL expression and the established markers, LPL expression retained its independent prognostic strength for overall survival. All of the RNA-based markers, albeit with varying ability, added prognostic information to established markers, with LPL expression giving the most significant results. Notably, high LPL expression predicted a worse outcome in good-prognosis subgroups, such as patients with mutated IGHV genes, Binet stage A, CD38 negativity or favorable cytogenetics. In particular, the combination of LPL expression and CD38 could further stratify Binet stage A patients.
LPL expression is the strongest RNA-based prognostic marker in chronic lymphocytic leukemia that could potentially be applied to predict outcome in the clinical setting, particularly in the large group of patients with favorable prognosis.
The existence of multiple subsets of chronic lymphocytic leukemia expressing 'stereotyped' B-cell receptors implies the involvement of antigen(s) in leukemogenesis. Studies also indicate that ...'stereotypy' may influence the clinical course of patients with chronic lymphocytic leukemia, for example, in subsets with stereotyped IGHV3-21 and IGHV4-34 B-cell receptors; however, little is known regarding the genomic profile of patients in these subsets.
We applied 250K single nucleotide polymorphism-arrays to study copy-number aberrations and copy-number neutral loss-of-heterozygosity in patients with stereotyped IGHV3-21 (subset #2, n=29), stereotyped IGHV4-34 (subset #4, n=17; subset #16, n=8) and non-subset #2 IGHV3-21 (n=13) and non-subset #4/16 IGHV4-34 (n=34) patients.
Over 90% of patients in subset #2 and non-subset #2 carried copy-number aberrations, whereas 75-76% of patients in subset #4 and subset #16 showed copy-number aberrations. Subset #2 and non-subset #2 patients also displayed a higher average number of aberrations compared to patients in subset #4. Deletion of 13q was the only known recurrent aberration detected in subset #4 (35%); this aberration was even more frequent in subset #2 (79%). del(11q) was more frequent in subset #2 and non-subset #2 (31% and 23%) patients than in subset #4 and non-subset #4/16 patients. Recurrent copy-number neutral loss-of-heterozygosity was mainly detected on chromosome 13q, independently of B-cell receptor stereotypy.
Genomic aberrations were more common in subset #2 and non-subset #2 than in subset #4. The particularly high frequency of del(11q) in subset #2 may be linked to the adverse outcome reported for patients in this subset. Conversely, the lower prevalence of copy-number aberrations and the absence of poor-prognostic aberrations in subset #4 may reflect an inherently low-proliferative disease, which would prevent accumulation of genomic alterations.
INTRODUCTION Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, with the high hyperdiploid (HeH) subtype accounting for approximately 25% of B-cell precursor (BCP) ALL ...cases. It has been shown that germline variants in the ARID5B gene in chromosome band 10q21.2 are associated with increased risk of BCP ALL, in particular HeH ALL, and that the risk alleles result in lower expression of ARID5B in hematopoietic cells. ARID5B codes for a protein involved in regulating gene expression and chromatin remodeling. We have previously reported somatic deletions in the ARID5B locus in two cases of HeH ALL, but the overall frequency of acquired copy number changes and rearrangements involving ARID5B in BCP ALL remains unknown. Here, we have investigated constitutional and somatic ARID5B variants in pediatric BCP ALL, with a particular focus on HeH cases. METHODS Constitutional variants We studied four known risk single nucleotide polymorphisms (SNPs) in the ARID5B locus (rs7090445, rs7089424, rs7073837 and rs10740055) in HeH ALL cases heterozygous for the risk SNP and with trisomy 10. These were investigated in three different cohorts, including a total of 92 cases informative for rs7090445, 92 cases for rs7089424, 119 cases for rs7073837 and 66 cases for rs10740055. The genotype and relative allele frequencies were ascertained from SNP array or whole genome sequencing (WGS) data. One-sided binomial tests were applied to investigate whether the risk allele was more often in the duplicated chromosome than the non-risk allele. Somatic variants For somatic variants, we ascertained copy number status based on SNP array and/or WGS analysis in the ARID5B region in a total of 466 pediatric HeH ALL cases and structural rearrangements based on WGS in 77 cases. We also studied, using SNP array analysis, somatic copy number variants in a separate cohort consisting of 590 non-HeH BCP ALL cases. To compare the proportions of deletions in the HeH cohort and in the other genetic subtypes, we used Fisher's Exact two-sided test. RESULTS Constitutional variants All four risk SNPs showed a significantly higher proportion of risk allele duplication (one-sided binominal test); rs7090445 ( P=0.009), rs7089424 ( P=0.005), rs7073837 ( P=0.03) and rs10740055 ( P=0.04). This validates that there is a clonal selection for HeH blast cells with gain of the chromosome 10 homologue carrying the risk allele as opposed to gain of the homologue that carries the non-risk allele. Somatic variants Somatic deletions targeting ARID5B were found in 9/466 cases (1.9%) of HeH cases. The deletions covered different parts of ARID5B, with no minimally deleted region, suggesting that the functional outcome was downregulation of ARID5B expression. In the cohort with non-HeH BCP ALL (other genetic subtypes), 4/590 somatic deletions were found (0.68%). There was no statistically significant difference between the frequency of copy number aberrations targeting ARID5B between HeH and the other genetic subtypes of ALL (P=0.09). WGS analysis of the HeH cases revealed one translocation and one missense mutation in 2/77 cases (2.6%). The translocation involved the PAN3 and ARID5B genes and was also present in RNA sequencing data from this case. CONCLUSIONS We show that our previous finding that HeH ALL constitutionally heterozygous for ARID5B risk alleles and with an acquired trisomy 10 more commonly duplicates the chromosome 10 homologue carrying the risk allele holds true in a much larger cohort. Furthermore, somatic deletions involving ARID5B are recurrent in pediatric BCP ALL.
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IJS, IMTLJ, KILJ, NLZOH, NUK, SAZU, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Introduction. Pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) is the most common pediatric hematological malignancy and it remains an important cause of morbidity and mortality in ...children. In this study, we performed an allele-specific expression (ASE) analysis of pediatric BCP ALL with the aim to investigate the relationship between cis-regulatory mutations and gene expression patterns.
Materials and methods. Twenty-two high hyperdiploid ALL, twenty ETV6/RUNX1-positive ALL, seven TCF3/PBX1-positive ALL and twenty-eight genetically unclassified BCP ALL (“B-other”) were subjected to whole genome sequencing, SNP array analysis and RNA sequencing. The binomial test was applied to estimate the allelic bias of heterozygous exonic single nucleotide variants (SNVs) in the RNA sequencing data against the genomic data. Allelic ratios >2 or <0.5, and P values <0.05 were used to identify allele-specific expression protein-coding genes.
Results. We identified 12,693 expressed genes, of which 9,672 (76%) had heterozygous exonic SNVs (informative genes), in multiple BCP ALL samples (n>2) in 77 of the investigated samples. Genes with ASE were distributed evenly across the autosomal chromosomes in the different subtypes with a range of 30 - 165 ASE genes per case (median number, 86). We found that 630 (6.5%) genes displayed ASE in multiple BCP ALL samples (n>2), of which only eight autosomal genes had monoallelic expression in more than two investigated samples. This suggests that ASE and monoallelic expression are relatively rare in BCP ALL. Gene enrichment analyses showed that pathways involving negative regulation of natural killer cell-mediated cytotoxicity and cell proliferation were enriched, indicating that ASE events possibly were associated with the cell proliferation and leukemia progression in BCP ALL. Furthermore, the hematopoiesis pathway was also enriched in ASE genes that showed high allelic expression bias (allelic ratios >2.5), suggesting that ASE genes might be associated with leukemia development. Somatic genomic aberrations that could cause ASE were also investigated in this study. All informative cases with TCF3/PBX1 rearrangement (n=4) showed monoallelic expression of the PBX1 gene, likely associated with the PBX1 truncation caused by the fusion. Additionally, CHP1, located in 15q15.1, displayed ASE in one case with an inversion involving that chromosome band, indicating a potential cis-acting element in the inversion region that regulated the CHP1 gene expression. Notably, PAX5 displayed various patterns of ASE in BCP ALL. One of three cases with PAX5/ZCCHC7 gene rearrangements displayed PAX5 ASE while the other two did not, indicating a potential uncovered cis-regulatory element around the PAX5/ZCCHC7 breakpoints. Furthermore, two cases with no PAX5 gene rearrangement displayed monoallelic expression of the PAX5 gene, suggesting that there are additional epigenetic alterations were also involved in the regulation of PAX5 gene expression in BCP ALL.
Conclusions. In this study, we have characterized genes displaying ASE in childhood BCP ALL. Our data provide new insight into pathogenesis of BCP ALL and may be used to identify novel targets for treatment.
No relevant conflicts of interest to declare.
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IJS, IMTLJ, KILJ, NLZOH, NUK, SAZU, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Complex karyotype (CK) identified by chromosome-banding analysis (CBA) has shown prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genome-wide detection of ...copy-number alterations (CNAs) and could therefore be well equipped to detect the presence of a CK. Current knowledge on genomic arrays in CLL is based on outcomes of single center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2293 arrays from 13 diagnostic laboratories according to established standards. CNAs were found outside regions captured by CLL FISH probes in 34% of patients, and several of them including gains of 8q, deletions of 9p and 18p (p<0.01) were linked to poor outcome after correction for multiple testing. Patients (n=972) could be divided in three distinct prognostic subgroups based on the number of CNAs. Only high genomic complexity (high-GC), defined as ≥5 CNAs emerged as an independent adverse prognosticator on multivariable analysis for time to first treatment (Hazard ratio: 2.15, 95% CI: 1.36-3.41; p=0.001) and overall survival (Hazard ratio: 2.54, 95% CI: 1.54-4.17; p<0.001; n=528). Lowering the size cutoff to 1 Mb in 647 patients did not significantly improve risk assessment. Genomic arrays detected more chromosomal abnormalities and performed at least as well in terms of risk stratification compared to simultaneous chromosome banding analysis as determined in 122 patients. Our findings highlight genomic array as an accurate tool for CLL risk stratification.
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