Hidden DNA Copy Number Alterations and Mutations in IKZF1, TP53, CRLF2 and JAK2 Genes Are Associated with a Poor Prognosis in B-Progenitor Acute Lymphoblastic Leukemia

Abstract Background: In B-progenitor acute lymphoblastic leukemia (B-ALL) the identification of additional genetic alterations associated with treatment failure is still a challenge. Aims: 1.To identify genomic gains and losses in B-ALL at the time of diagnosis and to correlate these abnormalities with the genetic characteristics and the patients outcome. 2.To assess the prevalence and prognostic impact of genetic lesions in IKZF1, TP53, CRLF2, IL7R, PAX5, JAK2 and LEF1 genes in B-ALL. Methods: A total of 215 B-ALL patients were eligible for this study. 115 (53.5%) had less than <18 years. In all cases oligonucleotide microarrays (aCGH) were carried out. For genetic gain and losses analysis, the NimbleGen CGH 12x135K array (Roche) was used. The data analysis was performed with GISTIC and aCGHweb software. In 118 of these cases (65 children and 53 adults), an integrative analysis of aCGH and NGS was performed to identify genetic lesions in genes associated with B-ALL: JAK2 (Exon 12-Exon 16), PAX5 (E2-E3), LEF1 (E2-E3), CRLF2 (E6), IL7R (E5), IKZF1 (E2-E8) and TP53 (E4-E11). The 454 GS Junior system (Roche) was used. The variant analysis was performed using Amplicon Variant Analyzer (AVA-Roche 454) and Sequence Pilot (JSI Medical Systems) software. Results: DNA copy number aberrations (CNAs) were observed in 96.5% of cases. Gains on chromosomes 4 (10%), 6 (10%), 10 (12%), 14 (13%), 21 (31%) and X (18%) were more common in children, whereas losses in 7p (13%) and 9p21 (24%) were frequent in adults. In children, gains on chromosomes 6, 10, 14q, 17, 18, 21 and X were associated with longer overall survival (OS) while loss on 7p and 17p were associated with shorter OS in adults (Table 1). The integrative analysis of aCGH and NGS showed that 60% of patients carried at least one alteration (deletion and/or mutation) in the seven genes analyzed. Focal deletions were common in IKZF1, CRLF2, PAX5, and LEF1 genes while broad deletions were more frequent in TP53, JAK2 and IL7R. Forty-one percent of patients harbored IKZF1 alterations (IKZF1+), 22.9% in PAX5, 18.6% in JAK2 and 11% had TP53 or CRLF2 alterations. LEF1 and IL7R genomic lesions were only present in 4.2% and 2.5% of the B-ALL, respectively. JAK2 and CRLF2 mutations were associated (p=0.01). Moreover abnormalities in IKZF1+ were associated with alterations in JAK2 (p=0.004), TP53 (p=0.04) and PAX5 (p=0.03). The presence of alterations was most frequent in high-risk (HR) B-ALL (38% vs 18%, p=0.02), although 8% low-risk (LR) childhood patients showed genetic alterations. Fourteen B-ALL patients carried mutations in TP53, CRLF2 and/or JAK2 genes: all of them were Ph-, and 11 were classified as HR. Of note, 3 cases were children. In the HR group the presence of mutation in TP53, CRLF2 and/or JAK2 was related to a shorter OS (3-year OS: 56% vs. 36%; P=0.034) and event-free survival (3-year EFS: 56% vs. 11%; P<0.002) (Figure 1), due to an increased cumulative incidence of relapse (3-year CIR: 38% vs. 88%; P<0.004). The presence of IKZF1 alterations stratified HR-Ph- B-ALL cases. Thus HR-Ph- patients with normal IKZF1 were associated with longer EFS compared to HR-Ph- B-ALL IKZF1+ (3-year EFS: 60% vs. 33%; P=0.005) (Figure 2). The presence of IKZF1 deletion was associated with shorter EFS (89% vs. 67%, P= 0.04) and increased CIR (3-year CIR: 12% vs. 33%; P=0.05) in children. Summary/Conclusions: CNAs are frequent in B-ALL and are associated with genetic subtype, age and overall survival. The integrative analysis by aCGH and NGS techniques demonstrated that alterations in IKZF1 gene and mutations in TP53, CRLF2 and/or JAK2 genes could stratify high-risk Ph- B-ALL patients. Subvention:FP7/2007-13, Nº306242-NGS-PTL; HUS272U13, JCyL,Consejería de Educación; BIO/SA31/13 Gerencia Regional de Salud, SACYL, Spain Table 1. DNA copy number aberrations (CNAs) associated with overall survival in 215 B-ALL patients Association CNA n Median (months) P value Short overall survival 7p-ChildrenAdults 19613 9615 0.05 17p-ChildrenAdults 1257 694 0.029 +19ChildrenAdults 351421 Not reached10 0.001 22q+ChildrenAdults 261214 Not reached7 <0.0001 Longoverall survival +6ChildrenAdults 26197 Not reached7 <0.0001 +10ChildrenAdults 25196 Not reached7 0.001 +14qChildrenAdults 26188 Not reached4 <0.0001 +17ChildrenAdults 24177 Not reached60 0.012 +18ChildrenAdults 25187 Not reachedNot reached <0.0001 21q+ChildrenAdults 473116 Not reached8 <0.0001 XChildrenAdults 522725 Not reached9 <0.0001 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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Comprehensive Genomic and DNA Methylation Analysis in Twins with ETV6-RUNX1 Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v112.11.597.597 ◽

2008 ◽

Vol 112 (11) ◽

pp. 597-597

Author(s):

Manoo Bhakta ◽

Mathias Ehrich ◽

Eric J. Gratias ◽

James R. Downing ◽

Charles G Mullighan

Keyword(s):

Dna Methylation ◽

Acute Lymphoblastic Leukemia ◽

Copy Number ◽

Lymphoblastic Leukemia ◽

Genetic Alterations ◽

Copy Number Alterations ◽

Methylation Analysis ◽

Dna Copy Number ◽

Dna Copy Number Alterations ◽

Control Samples

Abstract DNA methylation as a source for epigenetic variability has been implicated in a variety of different cancer types. Often these studies are confounded by inter-individual differences in the epigenetic profiles. The pattern of epigenetic marks can be altered by factors like age, nutrition, behavior or other environmental factors, which are difficult to control. We had the unique opportunity to study DNA methylation profiles in a pair of monozygotic twin boys who developed ETV6-RUNX1 B-progenitor acute lymphoblastic leukemia at 2 years of age within 3 weeks of each other. ETV6-RUNX1 ALL is characterized by a high frequency of recurring genetic alterations, but the full complement of genomic and epigenetic alterations contributing to leukemogenesis is unknown. For these twin cases, environmental influences upon epigenetic variation are largely eliminated. We used a mass spectrometry-based quantitative DNA methylation analysis technique (Sequenom’s® EpiTYPER™ application) to investigate 597 amplicons covering the promoter regions of 190 genes. The genomic target regions were selected to be enriched for genes involved in transcriptional regulation (n=130) and/or genes known to be targeted by recurring DNA copy number alterations in childhood leukemia (n= 60). Methylation analysis were performed on DNA extracted from cryopreserved, Ficoll enriched bone leukemic blasts obtained from diagnostic bone marrow aspirates, and non-leukemic peripheral blood leukocytes obtained at remission. We also examined DNA copy number alterations (CNAs) and loss-of- heterozygosity (LOH) using Affymetrix single nucleotide polymorphism (SNP) 6.0 arrays, which examine over 1.8 million loci, in both tumor and normal tissue for both twins. Analysis of SNP array data identified different somatic CNAs in the tumor samples of the two twins involving 9p21.3 (the CDKN2A/B tumor suppressor locus), 12p13.2 (ETV6) and trisomy 21, indicating that the shared ETV6-RUNX1 positive pre-leukemic clone acquired different secondary genetic alterations during leukemogenesis in each twin. Despite these genetic differences, the methylation profiles of the tumor samples were remarkably similar. Unsupervised two-dimensional clustering of quantitative methylation data revealed that the tumor samples clustered separately from the control samples. Based on these findings we calculated the methylation differences in each genomic target region. A total of 51 genomic regions were significantly differentially methylated between tumor and control samples (paired t-test P<0.001, and an average methylation difference > 10%). Within the differentially methylated genomic regions, a subset of approximately 20 exhibited strong regional differences, indicating that DNA methylation changes can be limited to certain areas of the promoter. In the group of genes known to be involved in transcriptional regulation, 32% were differentially methylated, including the HOXA, HOXB, HOXC and HOXD regions, while in the remaining genes only 15% were differentially methylated. This enrichment is significant on the level of 0.05 (Fisher’s exact test, odds ratio: 2.7). This represents the first study comparing genomic and epigenetic alterations in B-precursor ALL involving monozygotic twins. Notably, different DNA copy number alterations are acquired in each twin during leukemogeneis. In contrast, the tumor samples exhibit similar methylation patterns that are strikingly different to control samples obtained from the same individuals. These results indicate that combined genomic and epigenetic analyses will be important to characterize the full repertoire of genomic alterations in acute lymphoblastic leukemia.

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Identification of Novel Recurring Mutations in Relapsed Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v114.22.703.703 ◽

2009 ◽

Vol 114 (22) ◽

pp. 703-703

Author(s):

Charles G. Mullighan ◽

Jinghui Zhang ◽

Letha A Phillips ◽

Ching-Hon Pui ◽

James R. Downing

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Copy Number ◽

Lymphoblastic Leukemia ◽

Transcriptional Regulators ◽

Genetic Alterations ◽

Copy Number Alterations ◽

Dna Copy Number ◽

Sequence Variations ◽

Dna Copy Number Alterations ◽

Risk Of Relapse

Abstract Abstract 703 Relapse occurs across the spectrum of cytogenetic subtypes of acute lymphoblastic leukemia (ALL), and the biologic factors influencing risk of relapse are poorly understood. Previous studies have demonstrated substantial evolution in the complement of DNA copy number alterations from diagnosis to relapse, with the majority of cases acquiring new lesions at relapse that are not present at diagnosis, and also losing lesions present in the predominant clone at diagnosis. Moreover, fingerprinting and backtracking of deletions indicate a common ancestral origin of the diagnosis and relapse clones in most cases, suggesting that multiple, genetically distinct clones are present at diagnosis, and that specific genetic alterations influence risk of relapse. At present, detailed examination of DNA sequence variations in relapsed ALL has not been performed. In this study, we have performed genomic resequencing of 238 genes in leukemic samples obtained at diagnosis and relapse in 23 childhood ALL cases. The cohort comprised cases with high hyperdiploidy (N=3), TCF3-PBX1 (N=1), ETV6-RUNX1 (N=3), MLL-rearrangement (N=3), BCR-ABL1 (N=3), and cases with low hyperdiploid, pseudodiploid, normal, and miscellaneous karyotypes (N=10). All samples had over 80% blasts or were flow sorted to high purity prior to DNA extraction. Whole genome amplification of DNA was performed prior to sequencing. We selected genes targeted by recurring copy number alterations in ALL, genes in key pathways targeted by genetic alterations in ALL (e.g. lymphoid development, tumor suppression, cell cycle regulation and apoptosis), known cancer genes and tyrosine kinases. The complete coding region of each gene was sequenced in both diagnosis and relapse sample in all cases. Validation of putative variants was performed by sequencing of matched normal DNA. 248 putative protein changing variations were identified. After removal of variants also identified in matched normal DNA, 55 variants in 32 genes were identified in 20 cases (mean 2.5 variants per case, range 0-5). Eleven genes were mutated in multiple patients (ASMTL, CREBBP, ERG, FLT3, KRAS, NF1, NRAS, PAX5, PTPN11, TP53 and TUSC3). We identified tumor-acquired variations in genes previously known to be mutated in acute leukemia, including ETV6 (1 case) JAK1 (1), NRAS (5), KRAS (2), NF1 (3) PTPN11 (2), PAX5 (2), FBXW7 (1), and TP53 (2). In addition, we identified recurring mutations in genes not previously known to be mutated in cancer, including the transcriptional regulators CREBBP (N=4), NCOR1 (N=2), the tumor suppressor candidate gene TUSC3 (N=2), and the ETS family transcription factor ERG (N=2). Single mutations were also identified in transcriptional regulators (THADA, SPI1 (PU.1), TCF4, TCF7L2), the histone gene HIST1H2BG, and additional genes also targeted by copy number alterations in ALL (ARMC2, ATP10A, PLEKHG1, STIM2). The patterns of evolution of sequence variations between diagnosis and relapse were similar to those previously reported for DNA copy number alterations. Four patients had identical sequence mutations at diagnosis and relapse. Twelve cases had the some sequence variations identified at both diagnosis and relapse, but either acquired additional mutations at relapse (N=9), lost mutations present at diagnosis (N=2), or both acquired new lesions at relapse and lost variants at diagnosis (N=1). An additional three cases had variants detected at either diagnosis or relapse with no commonality between the two samples. Notably, eight (35%) cases had lesions resulting in constitutive RAS activation at diagnosis or relapse (NRAS in 3 cases, FLT3 in 2, PTPN11 in 2, NF1 in 2, and KRAS in 1), with 5 cases harboring mutations at diagnosis only (NF1 in 2 cases, NRAS in 2, and FLT3 in 1), and three cases harboring mutations at relapse only (FLT1, PTPN11 and NRAS 1 case each). In several cases, relapse-acquired mutations were identified as minor subclones at diagnosis. These data have identified novel targets of somatic mutation in ALL, and suggest that sequence variation is important determinant of risk of relapse. Identification of mutations in multiple cases suggests that several of these variants are driver mutations. These findings also indicate that resequencing of the entire coding genome of relapsed ALL will be essential to identify all lesions influencing response to therapy. Disclosures: No relevant conflicts of interest to declare.

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From DNA Copy Number Gains and Tumor Dependencies to Novel Therapeutic Targets for High-Risk Neuroblastoma

Journal of Personalized Medicine ◽

10.3390/jpm11121286 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1286

Author(s):

Bieke Decaesteker ◽

Kaat Durinck ◽

Nadine Van Roy ◽

Bram De Wilde ◽

Christophe Van Neste ◽

...

Keyword(s):

High Risk ◽

Copy Number ◽

Current Status ◽

Dna Copy Number ◽

Direct Role ◽

Pediatric Tumor ◽

Copy Number Aberrations ◽

Entry Points ◽

Dna Copy Number Aberrations ◽

Novel Therapeutic

Neuroblastoma is a pediatric tumor arising from the sympatho-adrenal lineage and a worldwide leading cause of childhood cancer-related deaths. About half of high-risk patients die from the disease while survivors suffer from multiple therapy-related side-effects. While neuroblastomas present with a low mutational burden, focal and large segmental DNA copy number aberrations are highly recurrent and associated with poor survival. It can be assumed that the affected chromosomal regions contain critical genes implicated in neuroblastoma biology and behavior. More specifically, evidence has emerged that several of these genes are implicated in tumor dependencies thus potentially providing novel therapeutic entry points. In this review, we briefly review the current status of recurrent DNA copy number aberrations in neuroblastoma and provide an overview of the genes affected by these genomic variants for which a direct role in neuroblastoma has been established. Several of these genes are implicated in networks that positively regulate MYCN expression or stability as well as cell cycle control and apoptosis. Finally, we summarize alternative approaches to identify and prioritize candidate copy-number driven dependency genes for neuroblastoma offering novel therapeutic opportunities.

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Integrative analysis reveals the direct and indirect interactions between DNA copy number aberrations and gene expression changes

Bioinformatics ◽

10.1093/bioinformatics/btn034 ◽

2008 ◽

Vol 24 (7) ◽

pp. 889-896 ◽

Cited By ~ 57

Author(s):

Hyunju Lee ◽

Sek Won Kong ◽

Peter J. Park

Keyword(s):

Gene Expression ◽

Copy Number ◽

Indirect Interactions ◽

Integrative Analysis ◽

Dna Copy Number ◽

Copy Number Aberrations ◽

Dna Copy Number Aberrations

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Faculty Opinions recommendation of Outcome prediction in pediatric medulloblastoma based on DNA copy-number aberrations of chromosomes 6q and 17q and the MYC and MYCN loci.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1157546.617731 ◽

2009 ◽

Author(s):

Eric Bouffet

Keyword(s):

Copy Number ◽

Outcome Prediction ◽

Dna Copy Number ◽

Copy Number Aberrations ◽

Pediatric Medulloblastoma ◽

Dna Copy Number Aberrations

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Genetic Alterations Activating Kinase and Cytokine Receptor Signaling in High-Risk Acute Lymphoblastic Leukemia

Cancer Cell ◽

10.1016/j.ccr.2012.06.005 ◽

2012 ◽

Vol 22 (2) ◽

pp. 153-166 ◽

Cited By ~ 409

Author(s):

Kathryn G. Roberts ◽

Ryan D. Morin ◽

Jinghui Zhang ◽

Martin Hirst ◽

Yongjun Zhao ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

High Risk ◽

Lymphoblastic Leukemia ◽

Genetic Alterations ◽

Cytokine Receptor ◽

Receptor Signaling

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BCR/ABL1-Like Acute Lymphoblastic Leukemia: From Diagnostic Approaches to Molecularly Targeted Therapy

Acta Haematologica ◽

10.1159/000519782 ◽

2021 ◽

pp. 1-9

Author(s):

Anna Płotka ◽

Krzysztof Lewandowski

Keyword(s):

Flow Cytometry ◽

Targeted Therapy ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

Lymphoblastic Leukemia ◽

Genetic Alterations ◽

Diagnostic Analysis ◽

Cell Precursor ◽

Molecularly Targeted Therapy ◽

Diagnostic Approaches

Background: BCR/ABL1-like acute lymphoblastic leukemia is a newly recognized high-risk subtype of ALL, characterized by the presence of genetic alterations activating kinase and cytokine receptor signaling. This subtype is associated with inferior outcomes, compared to other B-cell precursor ALL. Summary: The recognition of BCR/ABL1-like ALL is challenging due to the complexity of underlying genetic alterations. Rearrangements of CRLF2 are the most frequent alteration in BCR/ABL1-like ALL and can be identified by flow cytometry. The identification of BCR/ABL1-like ALL can be achieved with stepwise algorithms or broad-based testing. The main goal of the diagnostic analysis is to detect the underlying genetic alterations, which are critical for the diagnosis and targeted therapy. Key Messages: The aim of the manuscript is to review the available data on BCR/ABL1-like ALL characteristics, diagnostic algorithms, and novel, molecularly targeted therapeutic options.

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Role of allogeneic stem cell transplantation in adult patients with Ph-negative acute lymphoblastic leukemia

Blood ◽

10.1182/blood-2014-09-599894 ◽

2015 ◽

Vol 125 (16) ◽

pp. 2486-2496 ◽

Cited By ~ 134

Author(s):

Nathalie Dhédin ◽

Anne Huynh ◽

Sébastien Maury ◽

Reza Tabrizi ◽

Kheira Beldjord ◽

...

Keyword(s):

Overall Survival ◽

Stem Cell ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

Complete Remission ◽

Lymphoblastic Leukemia ◽

Adult Patients ◽

Key Points ◽

First Complete Remission

Key Points SCT in first complete remission is associated with 69.5% 3-year overall survival in high-risk ALL adult patients treated with intensified pediatric-like protocol. Poor early MRD response is a powerful tool to select patients who may benefit from SCT in first complete remission.

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