Hyperdiploid Childhood Acute Lymphoblastic Leukemia Research Articles

The Prognostic Effect of IKZF1 Deletions in ETV6::RUNX1 and High Hyperdiploid Childhood Acute Lymphoblastic Leukemia.

IKZF1 deletions are an established prognostic factor in childhood acute lymphoblastic leukemia (ALL). However, their relevance in patients with good risk genetics, namely ETV6::RUNX1 and high hyperdiploid (HeH), ALL remains unclear. We assessed the prognostic impact of IKZF1 deletions in 939 ETV6::RUNX1 and 968 HeH ALL patients by evaluating data from 16 trials from 9 study groups. Only 3% of ETV6::RUNX1 cases (n = 26) were IKZF1-deleted; this adversely affected survival combining all trials (5-year event-free survival [EFS], 79% versus 92%; P = 0.02). No relapses occurred among the 14 patients with an IKZF1 deletion treated on a minimal residual disease (MRD)-guided protocols. Nine percent of HeH cases (n = 85) had an IKZF1 deletion; this adversely affected survival in all trials (5-year EFS, 76% versus 89%; P = 0.006) and in MRD-guided protocols (73% versus 88%; P = 0.004). HeH cases with an IKZF1 deletion had significantly higher end of induction MRD values (P = 0.03). Multivariate Cox regression showed that IKZF1 deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in HeH ALL (hazard ratio of relapse rate [95% confidence interval]: 2.48 [1.32-4.66]). There was no evidence to suggest that IKZF1 deletions affected outcome in the small number of ETV6::RUNX1 cases in MRD-guided protocols but that they are related to higher MRD values, higher relapse, and lower survival rates in HeH ALL. Future trials are needed to study whether stratifying by MRD is adequate for HeH patients or additional risk stratification is necessary.

The Prognostic Effect of IKZF1 Deletions in ETV6::RUNX1 and High Hyperdiploid Childhood Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and recent cure rates approach 90% on first line therapy. IKZF1 deletions have been reported as an unfavorable prognostic factor and are rare in ETV6::RUNX1 and high hyperdiploid (HeH) ALL, the two cytogenetic subgroups with the most favorable prognosis. Due to its rarity, the prognostic effect of IKZF1deletions within this subset of patients remains unclear. Therefore, we assessed the prognostic impact of IKZF1deletions in 939 ETV6::RUNX1 and 968 HeH ALL patients. We performed this retrospective analysis on data of children and adolescents of 1-18 years with B-cell precursor ALL diagnosed in 1991-2016 and treated on one of 16 trials from 9 study groups, of which 6 were minimal residual disease (MRD) guided. Cytogenetic, fluorescence in-situ hybridization, and RT-PCR analyses of pre-treatment bone marrow samples to determine ploidy and fusion gene status were performed locally. Trials using MRD for risk stratification applied either PCR or flow cytometry analyses. The IKZF1 status was determined by multiplex ligation-dependent probe amplification by each individual study group. 3% of ETV6::RUNX1 (n=26) cases had an IKZF1 deletion; this affected survival adversely overall (5-year event free survival [EFS]: 79% vs. 92%, p = 0.02) (Table 1). However, in minimal residual disease (MRD) guided protocols, IKZF1 did not affect outcome in ETV6::RUNX1 cases ( 5-year EFS: 100% vs. 93%, p = 0.34) (Table 1). 9% of HeH cases had an IKZF1 deletion (n=85); this adversely affected survival overall (5-year EFS: 76% vs. 89%, p = 0.006), and in MRD-guided protocols (73% vs. 88%, p = 0.004) (Table 1). Multivariate cox regression showed that IKZF1deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in HeH ALL (Hazard ratio of EFS [95% CI]: 2.00 [1.20-3.30]) (Table 1). As the prognostic effect of IKZF1 deletions has been shown to vary by MRD risk group, we examined the prognostic effect of IKZF1deletions in HeH cases with detectable MRD <5% at end of induction in more detail. Of these cases, 10% carried an IKZF1deletion (n=34) which negatively affected outcome (5-year EFS: 78% vs. 92%, p = 0.01) Multivariate cox regression showed that IKZF1deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in this intermediate MRD HeH ALL (Hazard ratio of EFS [95% CI]: 2.90 [1.20-6.80]). However, cases with an IKZF1 deletion had significantly higher MRD values than their wild-type counterparts (p = 0.03, Figure 1). In conclusion, our analysis of a large composite cohort consisting of 16 trials shows that IKZF1deletions do not affect survival in ETV6::RUNX1 ALL when treated on MRD-guided protocols. In contrast, our data shows that in HeH ALL IKZF1deletions are associated with lower survival rates, higher relapse rates, and higher MRD values. Future results of current trials such as the ALLTogether will likely reveal whether risk stratification predominantly reliant on MRD is adequate for HeH patients or whether stratification by copy number alteration profile, including IKZF1 status, or by other methods would be more suitable. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genomic Heterogeneity and Clonal Evolution in High Hyperdiploid Childhood Acute Lymphoblastic Leukemia

High hyperdiploid (HeH) B-cell precursor acute lymphoblastic leukemia (BCP ALL) is characterized by a very specific nonrandom gain of chromosomes; a feature distinct from other types of aneuploid tumor types that usually display very heterogeneous gains and losses of chromosomes. Gains of chromosomes X, 4, 6, 10, 14, 17, and 18 are seen in more than 75% of cases of HeH childhood ALL, and of chromosome 21 in 100% of cases. In contrast to many aneuploid malignancies, there has been little evidence of chromosomal instability (CIN) in HeH ALL and the mechanisms leading to these chromosomal gains remain unknown. The aim of this project was to determine the level of genomic heterogeneity in HeH ALL. In order to do this, we performed low-pass whole genome sequencing (WGS) of single cells isolated from diagnostic bone marrow samples from HeH ALL patients to investigate cell-to-cell heterogeneity. Single nuclei in G 1 phase from nine diagnostic childhood HeH ALL samples were isolated using fluorescence-activated cell sorting and DNA libraries were constructed for low-pass WGS. Copy number analysis for each individual cell was performed in-house using the software programs AneuFinder and Ginkgo. Homolog inheritance of chromosomes gained or lost was determined by screening for heterozygous variants and calculation of the variant allele frequencies (VAF). Sequencing 2,572 single cells showed that the nine HeH ALL patients were all relatively homogenous at the trunk of their evolution tree and that the bulk of chromosomal gains were stable and unchanging. Structural aberrations, visible as partial chromosome copy number changes, were detected at diagnosis in most cases, with a greater number of structural aberrations detected in patients with greater numbers of sub-clones (defined as three or more cells presenting with the same numerical and structural aberrations). Unique numerical chromosomal aberrations detected in two or fewer cells were relatively random and therefore indicative of nondisjunction events in recent cell divisions rather than being part of the clonal evolution of the leukemia. These results indicate very low heterogeneity in HeH ALL and suggests that the genome of these leukemias is relatively stable. DisclosuresNo relevant conflicts of interest to declare.

Sister chromatid cohesion defects are associated with chromosomal copy number heterogeneity in high hyperdiploid childhood acute lymphoblastic leukemia.

High hyperdiploid acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. The main driver event of this disease is a nonrandom aneuploidy consisting of gains of whole chromosomes but without overt evidence of chromosomal instability (CIN). Here, we investigated the frequency and severity of defective sister chromatid cohesion—a phenomenon related to CIN—in primary pediatric ALL. We found that a large proportion (86%) of hyperdiploid cases displayed aberrant cohesion, frequently severe, to compare with 49% of ETV6/RUNX1‐positive ALL, which mostly displayed mild defects. In hyperdiploid ALL, cohesion defects were associated with increased chromosomal copy number heterogeneity, which could indicate increased CIN. Furthermore, cohesion defects correlated with RAD21 and NCAPG mRNA expression, suggesting a link to reduced cohesin and condensin levels in hyperdiploid ALL. Knockdown of RAD21 in an ALL cell line led to sister chromatid cohesion defects, aberrant mitoses, and increased heterogeneity in chromosomal copy numbers, similar to what was seen in primary hyperdiploid ALL. In summary, our study shows that aberrant sister chromatid cohesion is frequent but heterogeneous in pediatric high hyperdiploid ALL, ranging from mild to very severe defects, and possibly due to low cohesin or condensin levels. Cases with high levels of aberrant chromosome cohesion displayed increased chromosomal copy number heterogeneity, possibly indicative of increased CIN. These abnormalities may play a role in the clonal evolution of hyperdiploid pediatric ALL.

Role of rs10406069 in miR-5196 in hyperdiploid childhood acute lymphoblastic leukemia.

Aim: To determine the role of single nucleotide polymorphisms (SNPs)in noncoding RNAs in childhood acute lymphoblastic leukemia (ALL) subtypes. Materials & methods: We screened all SNPs in 130 pre-miRNA genes to assess their role in the susceptibility of the most common subtypes of ALL: hyperdiploid and ETV6-RUNX1. Results: In two independent cohorts, we found a significant association between rs10406069 in miR-5196 and the risk of developing hyperdiploid ALL. This observation could be explained by the impact of the SNP on miR-5196 expression and in turn, in its target genes. Indeed, rs10406069 was associated with expression changes in SMC1A, a gene involved in sister chromatin cohesion. Conclusion: rs10406069 in miR-5196 may have a relevant role in hyperdiploid ALL risk.

Proteogenomics and Hi-C reveal transcriptional dysregulation in high hyperdiploid childhood acute lymphoblastic leukemia

Hyperdiploidy, i.e. gain of whole chromosomes, is one of the most common genetic features of childhood acute lymphoblastic leukemia (ALL), but its pathogenetic impact is poorly understood. Here, we report a proteogenomic analysis on matched datasets from genomic profiling, RNA-sequencing, and mass spectrometry-based analysis of >8,000 genes and proteins as well as Hi-C of primary patient samples from hyperdiploid and ETV6/RUNX1-positive pediatric ALL. We show that CTCF and cohesin, which are master regulators of chromatin architecture, display low expression in hyperdiploid ALL. In line with this, a general genome-wide dysregulation of gene expression in relation to topologically associating domain (TAD) borders were seen in the hyperdiploid group. Furthermore, Hi-C of a limited number of hyperdiploid childhood ALL cases revealed that 2/4 cases displayed a clear loss of TAD boundary strength and 3/4 showed reduced insulation at TAD borders, with putative leukemogenic effects.

High hyperdiploid childhood acute lymphoblastic leukemia: Chromosomal gains as the main driver event

ABSTRACTHigh hyperdiploid childhood acute lymphoblastic leukemia is characterized by multiple chromosomal gains. Recent results show that this subtype harbors relatively few genetic abnormalities besides the extra chromosomes, which appear to arise early and are likely the main driver event. Secondary hits primarily target genes in the rat sarcoma (RAS) signaling pathway and histone modifiers.

The genomic landscape of high hyperdiploid childhood acute lymphoblastic leukemia.

High hyperdiploid (51-67 chromosomes) acute lymphoblastic leukemia (ALL) is one of the most common childhood malignancies, comprising 30% of all pediatric B cell-precursor ALL. Its characteristic genetic feature is the nonrandom gain of chromosomes X, 4, 6, 10, 14, 17, 18 and 21, with individual trisomies or tetrasomies being seen in over 75% of cases, but the pathogenesis remains poorly understood. We performed whole-genome sequencing (WGS) (n = 16) and/or whole-exome sequencing (WES) (n = 39) of diagnostic and remission samples from 51 cases of high hyperdiploid ALL to further define the genomic landscape of this malignancy. The majority of cases showed involvement of the RTK-RAS pathway and of histone modifiers. No recurrent fusion gene-forming rearrangement was found, and an analysis of mutations on trisomic chromosomes indicated that the chromosomal gains were early events, strengthening the notion that the high hyperdiploid pattern is the main driver event in this common pediatric malignancy.

FLT3 mutations in patients with childhood acute lymphoblastic leukemia (ALL)

The type III receptor tyrosine kinase FLT3 is of importance in hematopoiesis. FLT3 is a frequently mutated gene in acute myeloid leukemia (AML) as well as prognostic marker. The most common mutation that occurs in patients includes internal tandem duplication (ITD) in the juxtamembrane domain. Point mutations in the kinase domain are also identified in many patients. These mutations result in constitutive activation of receptor as well as uncontrolled activation of survival pathways. Although 30 % of AML patients carry a mutation in FLT3 [2], its expression is not limited to only those patients. It has also been found to be expressed in acute lymphoblastic leukemia (ALL) and several studies identified FLT3 mutations in a portion of ALL patients. This study will identify possible correlation in between FLT3 and childhood ALL patients. With the search criteria depicted in Fig. 1a, we retrieved 12 references that included 1,213 childhood ALL patients with 76 patients (6.3 %) carrying FLT3 mutations (Table S1). All 12 studies described FLT3 mutations. Further review of those references identified 89 patients with MLL-rearrangement and 196 patients with hyperdiploid ALL. Although 6.3 % childhood ALL patients carried FLT3 mutations, 15.7 % of MLL-rearrangement and 14.4 % of hyperdiploid childhood ALL patients carried FLT3 mutations (data not shown). Only 1 % patients carried FLT3-ITD mutations (12/1,213). Childhood ALL patients with MLL-rearrangement displayed an overall odds ratio of 4.17 (95 % Cl = 2.60–9.88, p 0.0001) (Fig. 1b) and patients with hyperdiploid ALL showed an overall odds ratio of 4.03 (95 % Cl = 2.18–7.95, p 0.0001) (Fig. 1c). Studies with FLT3-ITD mutation (Fig. 1d) and FLT3-TKD mutations (Fig. 1e) showed that TKD mutations happened more frequently than ITD mutations. An increased rate of FLT3 mutations was observed in American population (odds ratio = 1.4, 95 % CI = 0.85–2.29), while in Japanese and European population, comparatively lower FLT3 mutations were identified (Fig. 1f). The receptor tyrosine kinase FLT3 is frequently mutated in AML and activating FLT3 mutations cause poor prognosis in this disease [2]. Several FLT3 inhibitors display promising results in clinical trials. Although several studies described presence of FLT3 mutations in childhood ALL, still we have limited knowledge in defining roles of FLT3 mutations. In this study, we carried out a meta-analysis to define the role of FLT3 mutations in childhood ALL. All 12 studies analyzed in this study were mainly from three different populations that cover 1,213 childhood ALL patients. The identification of 76 mutations in these populations suggests that FLT3 mutations in childhood ALL is less frequent than that of in AML. Although about 30 % of FLT3-ITD mutations were reported in AML [2], childhood ALL patients carry only 1 % FLT3-ITD mutations suggesting that targeting FLT3-ITD will not be beneficial in pediatric ALL. It is likely that mutations other than FLT3ITD mutations are more frequent, e. g. FLT3-TKD mutations. However, it is quite difficult to suggest a correlation between FLT3 mutations and ALL prognosis due to the Electronic supplementary material The online version of this article (doi:10.1007/s12032-013-0462-6) contains supplementary material, which is available to authorized users.

Partial 17q gain resulting from isochromosomes, unbalanced translocations and complex rearrangements is associated with gene overexpression, older age and shorter overall survival in high hyperdiploid childhood acute lymphoblastic leukemia

Partial 17q gain resulting from isochromosomes, unbalanced translocations and complex rearrangements is associated with gene overexpression, older age and shorter overall survival in high hyperdiploid childhood acute lymphoblastic leukemia

CREBBP HAT domain mutations prevail in relapse cases of high hyperdiploid childhood acute lymphoblastic leukemia

Despite their apparently good prognosis ∼15% of high hyperdiploid (HD) childhood acute lymphoblastic leukemia (ALL) cases relapse. To search for responsible risk factors we determined copy number aberrations as well as copy neutral loss of heterozygosity (LOH) in 13 matched diagnosis and relapse samples and added the data of the only three available cases from the literature. Deletions and copy neutral LOH in 3 and 2 of the 16 cases directed us to the histone-modifying CREB-binding protein (CREBBP) gene, whose functional impairment is implicated in drug resistance. We therefore screened all samples for mutations in this gene and discovered 9 acquired sequence mutations in 7/16 cases, leading to an overall frequency of somatic CREBBP aberrations in HD ALL relapse cases of 63% that is considerably higher than that of the reported, mainly non-HD ALL (18.3%). Moreover, mutations in HD cases occur almost exclusively in the HAT domain (8/9; 89%). Hot spot mutations are present at diagnosis in 18.8% of relapsing HD ALL cases but in none of 40 respective cases remaining in long-term remission. Thus, the particular high incidence of CREBBP mutations in relapse-prone HD ALL cases could eventually be exploited for refined risk stratification and customized treatment in this genetic subgroup.

Cytogenetic and molecular genetic characterization of the ‘high hyperdiploid’ B‐cell precursor acute lymphoblastic leukaemia cell line MHH‐CALL‐2 reveals a near‐haploid origin

Cytogenetic and molecular genetic characterization of the ‘high hyperdiploid’ B‐cell precursor acute lymphoblastic leukaemia cell line MHH‐CALL‐2 reveals a near‐haploid origin

Genetic landscape of high hyperdiploid childhood acute lymphoblastic leukemia

High hyperdiploid acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. It is characterized by gain of chromosomes, typically +X, +4, +6, +10, +14, +17, +18, and +21,+21; little is known about additional genetic aberrations. Approximately 20% of the patients relapse; therefore it is clinically important to identify risk-stratifying markers. We used SNP array analysis to investigate a consecutive series of 74 cases of high hyperdiploid ALL. We show that the characteristic chromosomal gains are even more frequent than previously believed, indicating that karyotyping mistakes are common, and that almost 80% of the cases display additional abnormalities detectable by SNP array analysis. Subclonality analysis strongly implied that the numerical aberrations were primary and arose before structural events, suggesting that step-wise evolution of the leukemic clone is common. An association between duplication of 1q and +5 was seen (P = 0.003). Other frequent abnormalities included whole-chromosome uniparental isodisomies (wUPIDs) 9 and 11, gain of 17q not associated with isochromosome formation, extra gain of part of 21q, deletions of ETS variant 6 (ETV6), cyclin-dependent kinase inhibitor 2A (CKDN2A) and paired box 5 (PAX5), and PAN3 poly(A) specific ribonuclease subunit homolog (PAN3) microdeletions. Comparison of whole-chromosome and partial UPID9 suggested different pathogenetic outcomes, with the former not involving CDKN2A. Finally, two cases had partial deletions of AT rich interactive domain 5B (ARID5B), indicating that acquired as well as constitutional variants in this locus may be associated with pediatric ALL. Here we provide a comprehensive characterization of the genetic landscape of high hyperdiploid childhood ALL, including the heterogeneous pattern of secondary genetic events.

High hyperdiploid childhood acute lymphoblastic leukemia

High hyperdiploidy (51-67 chromosomes) is the most common cytogenetic abnormality pattern in childhood B-cell precursor acute lymphoblastic leukemia (ALL), occurring in 25-30% of such cases. High hyperdiploid ALL is characterized cytogenetically by a nonrandom gain of chromosomes X, 4, 6, 10, 14, 17, 18, and 21 and clinically by a favorable prognosis. Despite the high frequency of this karyotypic subgroup, many questions remain regarding the epidemiology, etiology, presence of other genetic changes, the time and cell of origin, and the formation and pathogenetic consequences of high hyperdiploidy. However, during the last few years, several studies have addressed some of these important issues, and these, as well as previous reports on high hyperdiploid childhood ALL, are reviewed herein.

Mutations of FLT3, NRAS, KRAS, and PTPN11 are frequent and possibly mutually exclusive in high hyperdiploid childhood acute lymphoblastic leukemia

Although it has been suggested that mutations of the FLT3, NRAS, KRAS, and PTPN11 genes are particularly frequent in high hyperdiploid (>50 chromosomes) pediatric acute lymphoblastic leukemias (ALLs), this has as yet not been confirmed in a large patient cohort. Furthermore, it is unknown whether mutations of these genes coexist in hyperdiploid cases. We performed mutation analyses of FLT3, NRAS, KRAS, and PTPN11 in a consecutive series of 78 high hyperdiploid ALLs. Twenty-six (33%) of the cases harbored a mutation, comprising six activating point mutations and one internal tandem duplication of FLT3 (7/78 cases; 9.0%), eight codon 12, 13, or 61 NRAS mutations (8/78 cases; 10%), five codon 12 or 13 KRAS mutations (5/78 cases, 6.4%), and seven exon 3 or 13 PTPN11 mutations (7/78 cases; 9.0%). No association was seen between the presence of a mutation in FLT3, NRAS, KRAS, or PTPN11 and gender, age, white blood cell count, or relapse, suggesting that they do not confer a negative prognostic impact. Only one case harbored mutations in two different genes, suggesting that mutations of these four genes are generally mutually exclusive. In total, one third of the cases harbored a FLT3, NRAS, KRAS, or PTPN11 mutation, identifying the RTK-RAS signaling pathway as a potential target for novel therapies of high hyperdiploid pediatric ALLs.

Identification of cryptic aberrations and characterization of translocation breakpoints using array CGH in high hyperdiploid childhood acute lymphoblastic leukemia

High hyperdiploidy, characterized by non-random trisomies, is the largest cytogenetic subgroup in childhood acute lymphoblastic leukemia (ALL). It is not known whether the gained chromosomes are sufficient for leukemogenesis or if additional genetic aberrations are necessary. However, the suboptimal chromosome morphology of hyperdiploid ALLs makes detection of structural abnormalities difficult if using cytogenetic techniques; alternative methods are, therefore, needed. We performed array comparative genome hybridization (CGH) analyses, with a resolution of 100 kb, of eight cases of high hyperdiploid childhood ALL to characterize structural abnormalities found with G-banding/multicolor fluorescence in situ hybridization (FISH) and to detect novel changes. The non-centromeric breakpoints of four rearrangements, including three translocations and one 1q duplication, were narrowed down to <0.2 Mb. Furthermore, four submicroscopic imbalances involving 0.6-2.7 Mb were detected, comprising two segmental duplications involving 1q22 and 12q24.31 in one case and two hemizygous deletions in 12p13.2-31 - including ETV6 - and in 13q32.3-33.1 in another case. Notably, FISH analysis of the latter revealed an associated reciprocal t(3;13)(q?;32.2-33.1). In conclusion, the array CGH analyses revealed putative leukemia-associated submicroscopic imbalances and rearrangements in 2/8 (25%) hyperdiploid ALLs. The detection and characterization of these additional genetic aberrations will most likely increase our understanding of the pathogenesis of high hyperdiploid childhood ALL.

Evidence for a single‐step mechanism in the origin of hyperdiploid childhood acute lymphoblastic leukemia

High hyperdiploidy (>50 chromosomes) in childhood acute lymphoblastic leukemia (ALL) is characterized by nonrandom multiple trisomies and tetrasomies involving in particular chromosomes X, 4, 6, 8, 10, 14, 17, 18, and 21. This characteristic karyotypic pattern, the most common in pediatric ALL, may arise via a tetraploid state with subsequent loss of chromosomes, by sequential gains of chromosomes in consecutive cell divisions, or by simultaneous gain of chromosomes in a single mitosis. These alternatives may be distinguished by investigation of the allelic ratios of loci on the tetrasomic and disomic chromosomes. Previous studies of tetrasomy 21 and of the occurrence of uniparental disomies (UPDs) have suggested that the most likely mechanism is simultaneous gain. However, the other pathways have not been definitely excluded because complete analyses of all disomies and tetrasomies have never been performed. In the present study, we investigated 27 hyperdiploid ALLs by using 58 polymorphic microsatellite markers mapped to 23 of the 24 human chromosomes. Twenty-six tetrasomies were analyzed (involving chromosomes X, 8, 10, 14, 18, and 21), and the frequency of UPDs was determined in 10 cases. In total, 200 chromosomes were studied. Equal allele dosage was observed in 24 of 26 tetrasomies, and only 7 UPDs were found. These data strongly suggest that hyperdiploidy in childhood ALL generally arises by a simultaneous gain of all additional chromosomes in a single abnormal mitosis.

Evidence for a Single Step Mechanism in the Origin of Hyperdiploid Childhood Acute Lymphoblastic Leukemia.

High hyperdiploidy (>50 chromosomes) in childhood acute lymphoblastic leukemia (ALL) is characterized by non-random multiple trisomies and tetrasomies, involving in particular chromosomes X, 4, 6, 8, 10, 14, 17, 18, and 21. It is the most common cytogenetic subgroup in pediatric ALL, but in spite of this, the mechanisms behind its formation remain elusive. Four different pathways are possible: (1) initial near-haploidy followed by doubling of the chromosomes, (2) prior polyploidization with subsequent losses of chromosomes, (3) sequential gains of chromosomes in consecutive cell divisions, and (4) a simultaneous gain of all additional chromosomes in a single abnormal mitosis. Although these alternatives are difficult to distinguish experimentally, investigations of possible uniparental disomies (UPDs) and of the allelic ratios of loci located on tetrasomic chromosomes may provide some clues. In a previous study of 10 cases of hyperdiploid childhood ALL, we could exclude the first pathway as a general mechanism. Furthermore, the results did not favor the second alternative. Finally, our findings of equal allele dosage for chromosome 21 loci in cases with tetrasomy 21 suggested that the hyperdiploidy arose by a simultaneous gain of chromosomes. However, because all disomies and tetrasomies were not investigated, formation via a polyploid state or by a sequential gain could not be definitely excluded. In the present study, we have addressed this issue further by investigating 15 new cases of hyperdiploid childhood ALL using a total of 57 polymorphic microsatellite markers mapped to 23 of the 24 human chromosomes. Ten of the cases were analyzed with multicolor fluorescence in situ hybridization (M-FISH) and the remaining five with interphase FISH using probes for X, 4, 6, 8, 10, 14, 17, 18, and 21. Markers localized to all disomic and all tetrasomic chromosomes were then applied. One case displayed multiple UPDs and a non-typical pattern with only tetrasomies; this ALL probably arose via a near-haploid pathway. Two other cases had UPDs for 4/9 and 2/14 disomic chromosomes, respectively, and may have originated via a polyploid state. However, in the vast majority of the cases (12/15) there was no evidence for any UPDs, excluding that the hyperdiploidy originated by the near-haploid or polyploid mechanisms. Investigating the tetrasomies, 25 of a total of 27 tetrasomies present among the 15 cases displayed equal allele dosages, indicating a simultaneous gain of chromosomes as opposed to sequential gains. The two tetrasomies with unequal allele dosages were present in the same case, suggesting that this ALL constituted an exception. In conclusion, the results of the present study strongly suggest that hyperdiploidy in childhood ALL generally arises by a simultaneous gain of all additional chromosomes in a single abnormal mitosis.

Formation of trisomies and their parental origin in hyperdiploid childhood acute lymphoblastic leukemia

AbstractHigh hyperdiploidy, common in childhood acute lymphoblastic leukemia (ALL) with a favorable prognosis, is characterized by specific trisomies. Virtually nothing is known about its formation or pathogenetic impact. We evaluated 10 patients with ALL using 38 microsatellite markers mapped to 18 of the 24 human chromosomes to investigate the mechanisms underlying hyperdiploidy and to ascertain the parental origin of the trisomies. Based on the results, doubling of a near-haploid clone and polyploidization with subsequent losses of chromosomes could be excluded. The finding of equal allele dosage for tetrasomy 21 suggests that hyperdiploidy originates in a single aberrant mitosis, though a sequential gain of chromosomes other than 21 in consecutive cell divisions remains a possibility. Our study, the first to address experimentally the parental origin of trisomies in ALL, revealed no preferential duplication of maternally or paternally inherited copies of X, 4, 6, 9, 10, 17, 18, and 21. Trisomy 8 was of paternal origin in 4 of 4 patients (P = .125), and +14 was of maternal origin in 7 of 8 patients (P = .0703). Thus, the present results indicate that imprinting is not pathogenetically important in hyperdiploid childhood ALL, with the possible exception of the observed parental skewness of +8 and +14.

Increased incidence of spontaneous apoptosis in the bone marrow of hyperdiploid childhood acute lymphoblastic leukemia

Hyperdiploidy of 51-65 chromosomes is associated with a good prognosis in childhood B-lineage acute lymphoblastic leukemia (ALL). Blasts from childhood ALL patients with a hyperdiploid karyotype have a tendency to apoptosis when cultured on stromal layers in vitro. In this study, we apply a novel method to investigate the relationship between apoptosis and hyperdiploidy in lymphoblasts of childhood ALL. The DNA content of individual ALL blasts in Feulgen-stained archival bone marrow smears can be determined by static cytometry. TUNEL (TdT-mediated dUTP-biotin nick end labeling) detects the DNA degradation associated with apoptosis. We performed TUNEL in situ sequential to DNA ploidy analysis in archival bone marrow smears from 12 patients with childhood ALL. Five patients were diploid and seven were hyperdiploid (51-65 chromosomes) by conventional cytogenetic analysis. In the five diploid cases, the percentage of TUNEL-positive blasts ranged from 1.0% to 1.3%; in the seven hyperdiploid cases, the percentage of TUNEL-positive blasts ranged from 3.6% to 9.0%. Comparing TUNEL and corresponding Feulgen images, we found that apoptotic blasts were predominantly of high DNA ploidy in both diploid and hyperdiploid cases. The mean DNA value of apoptotic blasts was larger than that of the total blast population in each case. The results demonstrate an increased incidence of spontaneous apoptosis in situ of hyperdiploid blasts in ALL bone marrow and indicate that this phenomenon is not restricted to in vitro cultures. The findings provide a possible rationale for the good prognosis associated with hyperdiploid childhood ALL.