scholarly journals Identifying novel genetic alterations in pediatric acute lymphoblastic leukemia based on copy number analysis

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Jéssica Almeida Batista-Gomes ◽  
Fernando Augusto Rodrigues Mello ◽  
Edivaldo Herculano Corrêa de Oliveira ◽  
Michel Platini Caldas de Souza ◽  
Alayde Vieira Wanderley ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Copy Number Analysis ◽  
Pediatric Acute Lymphoblastic Leukemia

High Resolution Genomic Profiling Using Single Nucleotide Polymorphism Microarrays Identifies Multiple Novel Genomic Lesions in Pediatric Acute Lymphoblastic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 108-108
Author(s):  
Charles G. Mullighan ◽  
Salil Goorha ◽  
Ina Radtke ◽  
James Dalton ◽  
Jing Ma ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Acute Lymphoblastic Leukemia ◽  
High Resolution ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Chromosome 9 ◽  
Copy Number Analysis ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Pediatric Acute Lymphoblastic Leukemia

Abstract To obtain a comprehensive registry of oncogenic lesions in pediatric acute lymphoblastic leukemia (ALL), we used Affymetrix single nucleotide polymorphism (SNP) arrays to examine changes in DNA copy number and loss-of heterozygosity (LOH) in leukemic blasts and matched remission samples from 250 ALLs. We studied B-progenitor ALLs with high hyperdiploidy, n=39; ETV6-RUNX1, n=47; MLL rearranged, n=11; TCF3-PBX1, n=17; BCR-ABL1, n=9; low hyperdiploidy, n=23; hypodiploidy, n=10; unclassified cases, n=42; and 50 T-lineage ALLs. Four arrays (50K Hind and Xba, 250K Sty and Nsp) were used to interrogate over 615,000 loci at a mean inter-marker distance of 4.8 kb. Data was analyzed using dChipSNP and a modified array normalization algorithm using only SNPs from regions known to be diploid by routine karyotyping. Copy number abnormalities were confirmed by FISH and genomic quantitative PCR. Complementary methylation analysis and sequencing of candidate genes was performed. 84% of B-ALLs and 96% of T-ALLs had at least one region of somatic deletion, and excluding cases with high hyperdiploidy, 68% of B-ALLs and 50% of T-ALLs had at least one region of somatic amplification. These included previously identified abnormalities including chromosomal duplications in hyperdiploid B-ALL; 1q duplication in TCF3-PBX1 ALL; and deletions of 9p21 (harboring CDKN2A/B, 70% of T-ALLs, 34.5% of B-ALL), 12p13 (ETV6; 25.5% of B-ALLs, 10% of T-ALLs), 6q16 (22 cases) and 11q (15 cases). The resolution of the arrays enabled precise mapping of the minimal regions of deletion at 9p21 to CDKN2A, and at 12p13 to ETV6. Combined LOH and copy number analysis identified several patterns of 9p21 abnormality: focal hemizygous deletion with corresponding LOH; focal homozygous and flanking hemizygous loss with corresponding LOH, indicating two focal deletional events; and focal homozygous loss with LOH of all of 9p or chromosome 9, indicating loss of the normal 9 or 9p and duplication of the chromosome or chromosomal arm containing the focal deletion. Copy-neutral LOH without any focal deletion in the affected region was uncommon. Deletions involving other genes with potential roles in leukemogenesis were identified including BTG1 (17 cases), ERG (10), FHIT (14), mir-16/-15a (19), MYB (5), NF1 (9), the glucocorticoid receptor NR3C1 (11), PTEN (4), and RB1 (20). Furthermore, deletions, translocations, amplifications, and point mutations of genes that regulate B-cell development and differentiation, including EBF, PAX5, Ikaros and Aiolos, were identified in 40% of B-ALL. For each of the listed genes, cases were identified that contained focal deletions limited to the specific gene. Overall, 73.6% B-ALL and 88% T-ALLs harbored deletions of one of the common lesions listed above, with 48% of B-ALLs and 48.5% of T-ALLs having multiple common lesions. The average number of deletions per case was 3.8 and 5.7 for B and T-lineage ALLs respectively. By contrast, when hyperdiploid cases were excluded, it was rare to find more than 2 regions of amplification in a single case, and the majority of cases contained no amplifications. These findings show the power of high-resolution copy number analysis for the identification of new genetic lesions in cancer, and demonstrate that multiple genetic abnormalities contribute to leukemogenesis in pediatric ALL.

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

Blood ◽  
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.

Identification of Novel Recurring Mutations in Relapsed Acute Lymphoblastic Leukemia.

Blood ◽  
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.

Identification of distinct protein Signatures Associated with genetic Abnormalities In Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1313-1313
Author(s):  
Roland P. Kuiper ◽  
Blanca Scheijen ◽  
Agata Pastorczak ◽  
Simon V. van Reijmersdal ◽  
Deborah A. Thomas ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Chromosomal Translocation ◽  
Genetic Alterations ◽  
Chromosomal Translocations ◽  
Integrated Analysis ◽  
Copy Number Alterations ◽  
Protein Signatures

Abstract Background Treatment outcome in acute lymphoblastic leukemia (ALL) has improved over the past 30 years, with overall survival rates of ∼45% in adults and ∼85% in children. Gross cytogenetic abnormalities, including numerical changes and chromosomal translocations, are of considerable prognostic value in both pediatric and adult ALL. In addition, we and others have recently identified novel molecular markers associated with a poor outcome in ALL, including deletions of the lymphoid transcription factor IKZF1. In order to identify downstream signaling events associated with these genetic alterations, we performed an integrated analysis of genomic abnormalities, including copy number alterations, sequence mutations and chromosomal translocations, with alterations in protein expression and modification. Methods A cohort of 91 precursor B-ALL cases treated at M.D. Anderson Cancer Center in Houston, USA, including 82 newly diagnosed cases and 5 diagnosis-relapse pairs was used for this study. The cohort consisted of 6 children (age 1-6), 30 young adults (age 15-39) and 45 adults (age>39), and 20 patients carried a BCR-ABL1 chromosomal translocation. Copy number alterations in eight genes frequently deleted in ALL (IKZF1, PAX5, EBF1, RB1, CRLF2, CDKN2A/2B, BTG1, and ETV6) were determined by multiplex ligation-dependent probe amplification analysis. IKZF1 deletions were associated with relapse (Pearson's chi-square test, p=0.009), and the presence of BCR-ABL1 translocation (p=0.032). Protein expression and modification levels were determined by probing Reverse Phase Protein Arrays (RPPA) containing protein lysates of all above samples with 128 rigorously validated antibodies including 34 phospho-specific antibodies. Hierarchical clustering analysis was used to determine which (phospho)proteins are differently expressed in genetic subsets of ALL. The significance of correlations was determined using two-sample t-test, with correction for multiple testing (Beta-Uniform Mixture model). Results We identified clustering of cases with a BCR-ABL1 chromosomal translocation (p=0.01; false discovery rate (FDR)=0.1), IKZF1-deletions (p=0.01, FDR=0.072), RB1-deletions (p=0.03, FDR=0.43) and EBF1 deletions (p=0.05, FDR=0.63). As expected RB1 deletion positive cases were characterized by decreased levels of (phospho)-RB1 and increased levels of cyclin E, illustrating the validity of our approach. EBF1-deleted cases showed relatively high levels of SHIP1, SSBP2 and phospho-STAT5, and lower levels of FAK and LYN. The protein signatures of BCR-ABL1-positive cases and IKZF1-deletion positive cases largely overlapped, and were characterized by elevated levels of (phospho)PKCα, SMAD1, phospho-STAT3, and phospho-STAT5 and lower levels of LYN and cyclinD3 (Figure 1). In total 70% of the BCR-ABL1-positive cases carried an IKZF1 deletion and several BCR-ABL1-negative cases with similar RPPA signature could be identified, all of which were IKZF1-deletion positive. These cases may represent the “BCR-ABL1-like” cases that were previously identified using gene expression signatures (Mullighan et al. 2009, NEJM 360:470-480; Den Boer et al. 2009, Lancet Oncol. 10:125-134), and could reflect activation of cAbl or other cellular tyrosine kinases. Together, we conclude that integrated analysis of genetic and proteomic aberrations identified protein signatures downstream of recurrent mutational events in ALL, a strategy that promises to facilitate the discovery of novel therapeutic targets in ALL and may aid in the identification of (high risk) patients that would benefit from tyrosine kinase inhibition. Disclosures: No relevant conflicts of interest to declare.

A Novel Multiplex NGS-Based Digital MLPA Assay for Copy Number Detection of 700 Target Sequences in Childhood Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4071-4071
Author(s):  
Anne Benard ◽  
Ilse Zondervan ◽  
Jan Schouten ◽  
Karel de Groot ◽  
Farzaneh Ghazavi ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Copy Number ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Gene Deletions ◽  
Reliable Technique ◽  
Dilution Series ◽  
Pediatric All ◽  
Target Sequences

Abstract Background: Recurrent and clonal genetic alterations are characteristic of different subtypes of pediatric acute lymphoblastic leukemia (ALL) and several of them are strong independent predictors of patient outcome. Multiplex Ligation-dependent Probe Amplification (MLPA) has become one of the standard methods for detection of common copy number alterations (CNAs), including IKZF1 deletions, which are associated with a poor outcome. However, standard MLPA assays only accommodate a maximum of 60 MLPA probes. In order to increase the number of target sequences in one assay, a next generation sequencing (NGS)-based MLPA variant has been developed. This digitalMLPA assay is based on the well-known MLPA procedure but can include up to 1000 probes in a single reaction, uses minute amounts of DNA (≥20ng) and can be analysed on all Illumina NGS platforms. Methods: A digitalMLPA assay was designed and optimised to detect CNAs of 55 key target genes and regions in childhood ALL, including deletions of B-cell differentiation and cell cycle control genes (PAX5, IKZF1/2/3, EBF1, RB1, CDKN2A/B, ETV6, and BTG1), iAMP21 and rearrangements of the PAR1 region, T-cell ALL associated aberrations (STIL-TAL1, LEF1, CASP8AP2, MYB, EZH2, MLLT3, NUP214-ABL1, PTEN, LMO1/2, NF1, SUZ12, PTPN2, PHF6), ERG and TP53 deletions. Several genetic alterations of potential prognostic and/or therapeutic relevance in ALL have also been included (NOTCH1, CD200/BTLA, VPREB1, TBL1XR1, PDGFRB-EBF1, IGHM, NR3C1/2, CREBBP, CTCF, ADD3, EPHA1, FHIT, SPRED1 and TOX). All target genes are covered by at least 3 digitalMLPA probes (~450 probes in total). Moreover, a set of 200 probes was designed for genome-wide detection of gross ploidy changes (high hyper- or hypodiploidy) and to determine the extent of CNAs, while also acting as reference probes for data normalization. Performance of all probes has been extensively tested on genomic DNA from healthy individuals and positive cell lines. Only probes with a standard deviation <0.12 were included. All probes were also tested under various experimental conditions, such as different salt, probe and polymerase concentrations. A series of 76 pediatric ALL patient samples (including both B-ALL and T-ALL samples), previously characterized for specific genetic aberrations by array CGH (aCGH) and/or SNP array, has been analysed single-blinded using the digitalMLPA assay. Results were compared to those obtained from standard MLPA assays (P335 ALL-IKZF1 and P327 iAMP21-ERG), which contain probes with different ligation sites for the respective genes. A dilution series of three B-ALL patient samples was analysed to determine the detection limit for subclonal aberrations. Results: All aberrations previously identified by other methods in 67 pediatric ALL samples, were also identified using the digitalMLPA assay. These included whole chromosome gains and losses, whole gene deletions or gains, iAMP21, fusion genes (STIL-TAL1, NUP214-ABL1 and PDGFRB-EBF1) and intragenic gene deletions (IKZF1, ERG, CDKN2A/B, ETV6, PAX5 RUNX1, RB1, LEF1, NR3C1, RAG2, VPREB1, MLLT3, BTG1 and PTEN). Of interest was a case of high hyperdiploidy, which was correctly identified by digitalMLPA, while by aCGH analysis this case was misinterpreted as having multiple deletions. Among several other intragenic deletions, a heterozygous deletion of ERG exons 5-9 was observed in one patient. In addition, several cases of homozygous single-exon deletions were observed (e.g. IKZF1 exon 8, LEF1exon 3) and confirmed by aCGH. These findings should be further investigated with respect to their clinical impact. Results from the dilution series indicated that subclonal aberrations can be reliably detected by this digitalMLPA assay if present in at least 20- 30% of tumour cells. Conclusions: This study demonstrates that digitalMLPA is a reliable technique that can be used to genetically characterize clinical samples of ALL patients. Experiments can be performed on small amounts of DNA in a high-throughput and cost-effective fashion. Because of the targeted approach, data analysis will be much easier as compared to array or other sequencing platforms. These results merit further consideration of (digital)MLPA as a valuable alternative for genetic work-up of newly diagnosed ALL patients. Disclosures No relevant conflicts of interest to declare.

Genetic Profiling of Adult Acute Lymphoblastic Leukemia Cells by Single Nucleotide Polymorphism Oligonucleotide Microarray.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2807-2807
Author(s):  
Ryoko Okamoto ◽  
Seishi Ogawa ◽  
Tadayuki Akagi ◽  
Motohiro Kato ◽  
Masashi Sanada ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Acute Lymphoblastic Leukemia ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Uniparental Disomy ◽  
Genetic Alterations ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Snp Chip ◽  
Pediatric All

Abstract Acute lymphoblastic leukemia (ALL) is a malignant disease of bone marrow cells, resulting from accumulation of genetic alterations of these cells. We analyzed 74 adult ALL samples by single-nucleotide polymorphism DNA microarray (SNP-Chip) using the new algorithm AsCNAR (allele-specific copy-number analysis using anonymous references). 71 samples (96%) showed genomic abnormalities in a mean 4.5 chromosomes including duplications, deletions and loss of heterozygosity with normal copy number [we call this uniparental disomy (UPD)]. About 25% of samples had a normal karyotype but each had genomic changes detectable by SNP-Chip. Importantly, 21 cases (28%) had UPD, and 29% of these had 9p UPD. Other genomic defects included deletions of p16INK4A in 18 cases (24%), deletions of ETV6 in 7 cases (9%), and hyperdiploidy (>50 chromosomes) in 3 cases (4%). In contrast, we also analyzed 399 pediatric ALL samples and deletions occurred in p16INK4A (28%) and ETV6 (22%) and 29% cases had hyperdiploidy. Hyperdiploidy is associated with a good prognosis and occured much more frequency in pediatric ALL (29%) than adult ALL (4%) which may in part explain the better prognosis in pediatric ALL compared to adult ALL. Also, small copy number changes were detected in adult ALL including deletion of B-cell differentiation genes: EBF (4 cases, 5%), Pax5 (5 cases, 7%) and IKZF (Ikaros) (8 cases, 11%), as well as, deletion of miR-15a and miR-16-1 (2 cases, 3%), which is often found in CLL. Amplification of Rel and BCL11A occurred in one case and amplification of Akt2 occurred in another case. Moreover, we found PAX5/ETV6 fusion in one case (1%); in comparison, 14 of 399 pediatric ALL cases (4%) had PAX5 fusion genes. In summary, we discovered hidden abnormalities including small copy number change and UPD in adult ALL and identified differences between adult and pediatric ALLs. In the future, routine SNP-Chip analysis may provide novel subclassification criteria for ALL and identify unique therapeutic targets.

scholarly journals Integrated Genomic Analysis of Down Syndrome Acute Lymphoblastic Leukemia Reveals Recurrent Cancer Gene Alterations and Evidence of Frequent Subclonal Driver Events

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4083-4083
Author(s):  
Oliver A, Hampton ◽  
Enrique I. Ramos ◽  
Nipun Kakkar ◽  
Jacob J. Junco ◽  
Jacquelyn Reuther ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Acute Lymphoblastic Leukemia ◽  
Copy Number ◽  
Research Funding ◽  
Somatic Mutations ◽  
Lymphoblastic Leukemia ◽  
White Blood Count ◽  
Copy Number Analysis ◽  
Ras Pathway ◽  
Recurrent Mutations

Abstract Introduction: Children with Down syndrome (DS) have a 10-20 fold increased risk of acute lymphoblastic leukemia (ALL) and suffer significantly poorer outcomes due to increased relapse and treatment-related mortality. DS-ALL exhibits a distinct spectrum of cytogenetic alterations, with a lower incidence of common recurrent ALL alterations, a higher incidence of alterations involving CRLF2 and JAK2, and a higher proportion of cases lacking a known oncogenic driver compared to non-DS ALL. We hypothesized that identifying genetic drivers in DS-ALL will facilitate improved risk-based treatment stratification and lead to novel therapeutic targets that may be harnessed to improve outcomes in this vulnerable population. Methods: We interrogated diagnostic tumor (bone marrow or peripheral blood) and matched remission ("normal") blood samples from a cohort of 63 DS-ALL cases (65% male; median age 5 years, range 1-21 years) obtained from Texas Children's Cancer Center and the Children's Oncology Group. Samples were selected to include ~1/3 CRLF2-rearranged (CRLF2-R) and ~2/3 CRLF2-wild-type (CRLF2-WT) cases. Whole exome sequencing (WES) and copy number analysis using the OmniExpress array were performed on both tumor and normal DNA and transcriptome sequencing (RNAseq) was conducted on tumor RNA, all using the Illumina platform and existing informatic pipelines at the Baylor College of Medicine Human Genome Sequencing Center. A median of >97% of targeted bases with at least 20x coverage (range 93.5-98.1%) was observed for WES, and an average of 55.4 million paired reads were generated per RNAseq. Results: Analysis of RNAseq data (n=60) using deFuse and SOAPfuse algorithms revealed known ALL-associated gene rearrangements in 37/60 (62%) of cases, including CRLF2-R (21/60, 35%) involving P2RY8 (n=17) or IGH (n=4); ETV6-RUNX1 fusions (14/60, 23%) and TCF3 fusions (PBX1 x 2 and a novel in-frame fusion with FLI1 x 1). WES (n=62) identified a median of 19 nonsilent somatic mutations per case (range 6-228). We did not find evidence of somatic or germline mismatch repair gene mutations in the 3 cases with >50 somatic mutations. WES findings included recurrent activating hotspot mutations in JAK2 (predominantly p.R683G/S) in 14 cases (23%) and CRLF2 (p.F232C) in 2 cases, all of which also had CRLF2-R. Somatic RAS pathway mutations (13 KRAS, 7 NRAS, and 2 PTPN11) were found in 20/62 cases (32%), 4 CRLF2-R and 16 CRLF2-WT, including 2 cases also harboring JAK2 activating mutations. Recurrent mutations in epigenetic or chromatin remodeling genes (e.g. KMT2C, SETD2, KDM6A, WHSC1, DOT1L) were observed in ~25% of patients, as were mutations in other known ALL genes (e.g. FLT3, IL7R, IKZF1, PAX5). Interestingly, inactivating mutations in the deubiquitinase USP9X, also recently reported in acute promyelocytic leukemia, were identified in 5/62 (8%) cases. Evaluation for internal tandem duplications (ITDs) demonstrated typical in-frame events in FLT3 (n=2) and an out-of-frame ITD in SETD2. Copy number analysis revealed frequent losses in cell cycle genes (CDKN2A/B, BTG1, RB1) and other known B-cell development genes (IKZF1, PAX5). Other putative candidate alterations detected by RNAseq, WES, and array are currently being validated by orthogonal platforms. We observed multiple subclonal driver events in >10% of patients, including one case with CRLF2-R and mutations in CRLF2, JAK2, KRAS, and NRAS; and a second case with CRLF2 and ETV6-RUNX1 rearrangements and a PTPN11 hotspot mutation. Six cases exhibited 2 different JAK2, KRAS, or NRAS mutations: in all four cases where evaluation was feasible, these mutations were present on different sequencing reads, suggesting the existence of distinct subclones. Clinical features (gender, age, initial white blood count) did not appear to co-cluster with any of the recurrent alterations. Conclusions: These findings detail the landscape of genomic and transcriptomic alterations in the largest cohort of DS-ALL comprehensively characterized to date. Our data confirm the frequency and occasional polyclonal nature of JAK2 and RAS pathway mutations, and demonstrate that mutations in these pathways are not mutually exclusive. We also identified several novel, recurrent mutations and fusions. Future analyses of additional cohorts will be needed to assess recurrence frequency and prognostic impact of these alterations. Disclosures Schore: Baxalta: Honoraria; Millennium Pharmaceuticals, Inc: Research Funding; Onyx/Amgen: Research Funding; Merck: Research Funding.

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