scholarly journals Comprehensive Custom NGS Panel Validation for the Improvement of the Stratification of B-Acute Lymphoblastic Leukemia Patients

2020 ◽  
Vol 10 (3) ◽  
pp. 137
Author(s):  
Adrián Montaño ◽  
Jesús Hernández-Sánchez ◽  
Maribel Forero-Castro ◽  
María Matorra-Miguel ◽  
Eva Lumbreras ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Wide Spectrum ◽  
Lymphoblastic Leukemia ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Copy Number Variations ◽  
Single Step ◽  
Fusion Genes ◽  
Single Nucleotide Variants ◽  
Single Nucleotide

Background: B-acute lymphoblastic leukemia (B-ALL) is a hematological neoplasm of the stem lymphoid cell of the B lineage, characterized by the presence of genetic alterations closely related to the course of the disease. The number of alterations identified in these patients grows as studies of the disease progress, but in clinical practice, the conventional techniques frequently used are only capable of detecting the most common alterations. However, techniques, such as next-generation sequencing (NGS), are being implemented to detect a wide spectrum of new alterations that also include point mutations. Methods: In this study, we designed and validated a comprehensive custom NGS panel to detect the main genetic alterations present in the disease in a single step. For this purpose, 75 B-ALL diagnosis samples from patients previously characterized by standard-of-care diagnostic techniques were sequenced. Results: The use of the custom NGS panel allowed the correct detection of the main genetic alterations present in B-ALL patients, including the presence of an aneuploid clone in 14 of the samples and some of the recurrent fusion genes in 35 of the samples. The panel was also able to successfully detect a number of secondary alterations, such as single nucleotide variants (SNVs) and copy number variations (CNVs) in 66 and 46 of the samples analyzed, respectively, allowing for further refinement of the stratification of patients. The custom NGS panel could also detect alterations with a high level of sensitivity and reproducibility when the findings obtained by NGS were compared with those obtained from other conventional techniques. Conclusions: The use of this custom NGS panel allows us to quickly and efficiently detect the main genetic alterations present in B-ALL patients in a single assay (SNVs and insertions/deletions (INDELs), recurrent fusion genes, CNVs, aneuploidies, and single nucleotide polymorphisms (SNPs) associated with pharmacogenetics). The application of this panel would thus allow us to speed up and simplify the molecular diagnosis of patients, helping patient stratification and management.

scholarly journals Clinical application of whole transcriptome sequencing for the classification of patients with acute lymphoblastic leukemia

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Wencke Walter ◽  
Rabia Shahswar ◽  
Anna Stengel ◽  
Manja Meggendorfer ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Copy Number Variations ◽  
Current Standard ◽  
Genetic Classification ◽  
Genetic Characteristics ◽  
Standard Methods

Abstract Background Considering the clinical and genetic characteristics, acute lymphoblastic leukemia (ALL) is a rather heterogeneous hematological neoplasm for which current standard diagnostics require various analyses encompassing morphology, immunophenotyping, cytogenetics, and molecular analysis of gene fusions and mutations. Hence, it would be desirable to rely on a technique and an analytical workflow that allows the simultaneous analysis and identification of all the genetic alterations in a single approach. Moreover, based on the results with standard methods, a significant amount of patients have no established abnormalities and hence, cannot further be stratified. Methods We performed WTS and WGS in 279 acute lymphoblastic leukemia (ALL) patients (B-cell: n = 211; T-cell: n = 68) to assess the accuracy of WTS, to detect relevant genetic markers, and to classify ALL patients. Results DNA and RNA-based genotyping was used to ensure correct WTS-WGS pairing. Gene expression analysis reliably assigned samples to the B Cell Precursor (BCP)-ALL or the T-ALL group. Subclassification of BCP-ALL samples was done progressively, assessing first the presence of chromosomal rearrangements by the means of fusion detection. Compared to the standard methods, 97% of the recurrent risk-stratifying fusions could be identified by WTS, assigning 76 samples to their respective entities. Additionally, read-through fusions (indicative of CDKN2A and RB1 gene deletions) were recurrently detected in the cohort along with 57 putative novel fusions, with yet untouched diagnostic potentials. Next, copy number variations were inferred from WTS data to identify relevant ploidy groups, classifying an additional of 31 samples. Lastly, gene expression profiling detected a BCR-ABL1-like signature in 27% of the remaining samples. Conclusion As a single assay, WTS allowed a precise genetic classification for the majority of BCP-ALL patients, and is superior to conventional methods in the cases which lack entity defining genetic abnormalities.

scholarly journals Molecular Genetics of Pre-B Acute Lymphoblastic Leukemia Sister Cell Lines during Disease Progression

2021 ◽  
Vol 43 (3) ◽  
pp. 2147-2156
Author(s):  
Hilmar Quentmeier ◽  
Claudia Pommerenke ◽  
Hans G. Drexler
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Tumor Suppressor Genes ◽  
Common Ancestor ◽  
Lymphoblastic Leukemia ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Sister Cell ◽  
Dna And Rna ◽  
Clonal Development

For many years, immortalized tumor cell lines have been used as reliable tools to understand the function of oncogenes and tumor suppressor genes. Today, we know that tumors can comprise subclones with common and with subclone-specific genetic alterations. We sequenced DNA and RNA of sequential sister cell lines obtained from patients with pre-B acute lymphoblastic leukemia at different phases of the disease. All five pairs of cell lines carry alterations that are typical for this disease: loss of tumor suppressors (CDKN2A, CDKN2B), expression of fusion genes (ETV6-RUNX1, BCR-ABL1, MEF2D-BCL9) or of genes targeted by point mutations (KRAS A146T, NRAS G12C, PAX5 R38H). MEF2D-BCL9 and PAX R38H mutations in cell lines have hitherto been undescribed, suggesting that YCUB-4 (MEF2D-BCL9), PC-53 (PAX R38H) and their sister cell lines will be useful models to elucidate the function of these genes. All aberrations mentioned above occur in both sister cell lines, demonstrating that the sisters derive from a common ancestor. However, we also found mutations that are specific for one sister cell line only, pointing to individual subclones of the primary tumor as originating cells. Our data show that sequential sister cell lines can be used to study the clonal development of tumors and to elucidate the function of common and clone-specific mutations.

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.

Adult B-Cell Precursor Acute Lymphoblastic Leukemia (BC-ALL) Negative For Recurrent Fusion Genes Are Characterized By a High Complex Genetic Heterogeneity Influencing Prognosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2622-2622
Author(s):  
Ilaria Iacobucci ◽  
Anna Ferrari ◽  
Margherita Perricone ◽  
Valentina Robustelli ◽  
Cristina Papayannidis ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Oligonucleotide Primer ◽  
Fusion Genes ◽  
Cell Precursor ◽  
Snp Arrays ◽  
Molecular Features ◽  
Therapeutic Decisions

Abstract Introduction High-resolution genome-wide profiling analysis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) samples has identified many novel somatic genetic alterations, several of which have clear implications for risk stratification or future therapeutic targeting. However, most of the studies focused on children and therefore a deep molecular characterization of adults is still challenging, especially for those cases lacking recurrent fusion genes. Subjects and Methods In order to shed light on the molecular features of this ALL subgroup, we retrospectively analyzed 28 newly diagnosed BCR-ABL1-negative BCP-ALL subjects (19 males/9 females; median age 41.5 years; negative for known fusion genes) and 28 BCR-ABL1-positive BCP-ALL subjects as a comparison group, since it represents the most frequent genetic subgroup in adults with ALL. In BCR-ABL1-negative ALL karyotype was normal in 10/28 (36%), showed abnormalities in 5/28 (18%) and failed or was not available in 13/28 (46%) cases. The overall survival rate was very poor with a median of 14 months (range, 1-75). We analyzed copy number alterations (CNA) of IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, and genes within PAR1: CRLF2, CSF2RA, IL3RA by the SALSA MLPA kit P335 IKZF1 (MRC Holland). In addition, mutation status was assessed for TP53, CRLF2, JAK2, LEF1, PAX5 and IL7R by next-generation deep-sequencing (NGS) (Roche Applied Science; IRON-II study oligonucleotide primer plates). Positivity for newly described BCR-JAK2, PAX5-JAK2, ETV6-ABL1, EBF1-PDGFRB, NUP-ABL1 gene fusions occurring in BCR-ABL1-like ALL (Roberts KG et al., Cancer Cell. 2012) was assessed by PCR amplification and sequencing. Finally, SNP arrays (SNP 6.0, Affymetrix) and gene expression profile analyses (GeneChip® Human Transcriptome Array 2.0) were performed to more fully assess genomic complexity. Results Overall, 76% of BCR-ABL1-negative subjects showed an abnormality of at least one of the analyzed genes: 7 (25%) had one, 4 (14%) had two, 6 (21%) had three, and 6 (21%) had four or more alterations. In subjects showing no abnormalities, SNP arrays analysis revealed amplifications of chromosome 1q in 2/6 cases (33%). Deletions of CDKN2A/B were the most frequent (39%) and in 73%, they occurred together with other abnormalities, suggesting that multiple events are needed to induce the full leukemia phenotype. Other common CNA included: deletions of IKZF1 (25%), ETV6 (25%), PAX5 (14%), EBF1 (11%), PAR1 region (11%) and RB1 (7%). NGS showed mutations of TP53 in 18% of cases (W147*, V172L/G, G245C, Del244-246, D259Y), while JAK2 and CRLF2 were mutated in 7% (R683S/G) and 4% (F232C), respectively. No positivity for newly described fusion genes activating tyrosine kinase was confirmed. Importantly, subjects with no abnormalities showed better survival rates compared to those with one or more molecular alterations (p < 0.01). The BCR-ABL1-positive subgroup shared the same CNA of BCR-ABL1-negative cases, such as deletions of IKZF1 (71%), CDKN2A/B (21%), PAX5 (14%), BTG1 (11%), EBF1 (11%), and ETV6 (4%), but they did not show mutations in the analyzed genes. Conclusions BCP-ALL lacking recurrent fusion genes is a highly heterogeneous and complex disease. Current diagnostic procedures need to be revised to improve risk assessment and to guide therapeutic decisions. Supported by AIL, AIRC, PRIN 2010-2011, Programma Ricerca Regione-Università 2010-2012, FP7 “NGS-PTL” project. Disclosures: Soverini: Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; ARIAD: Consultancy. Chiaretti:Roche Diagnostics: Research Support Other. Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Martinelli:Novartis: Consultancy, Speaker fees Other; Bristol-Myers Squibb: Consultancy, Speaker fees, Speaker fees Other; Pfizer: Consultancy, Speaker fees, Speaker fees Other; Ariad: Consultancy, Speaker fees, Speaker fees Other.

Genetic Abnormalities and Prognosis in Pediatric B-Precursor Acute Lymphoblastic Leukemia without Conventional Genetic Abnormalities; Tokyo Children's Cancer Study Group

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4079-4079
Author(s):  
Kenro Ohki ◽  
Shinsuke Hirabayashi ◽  
Akinori Yaguchi ◽  
Akinori Yaguchi ◽  
Kazuki Terada ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Study Group ◽  
Gene Rearrangements ◽  
Fusion Genes ◽  
Cancer Study ◽  
Cancer Study Group ◽  
Genetic Abnormalities

Abstract Introduction ; Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease that can be subdivided according to primary recurrent genetic abnormalities that are strongly associated with characteristic biological and clinical features. In approximately two-thirds of pediatric patients with BCP-ALL, well-characterized genetic abnormalities can be detected by standard genetic analyses. In the remaining BCP-ALL patients, major pathogenic or driver cytogenetic abnormalities have yet to be clarified and they are called B-others. Recent studies using advanced analytical approaches have stratified a variety of subgroups harboring novel genetic abnormalities. For example, Ph-like ALL, iAMP21, deletion of ERG and rearrangements of ZNF384 and MEF2D have been identified in B-others. However, a certain portion of B-others still remains as genetically unclassified. Patients and Methods; 291 children with de novo BCP-ALL were enrolled on Tokyo Children Cancer Study Group (TCCSG) ALL L0416 and L0616 trials (between 2004-2007). Conventional genetic abnormalities, including BCR-ABL1, MLL-AF4, MLL-AF9, TCF3-PBX1, ETV6-RUNX1, hyperdiploidy and hypodiploidy were detected in 16 (5.5%), 3 (1.0%), 2 (0.7%), 18 (6.2%), 54 (18.5%), 65 (22.3%) and 3 (1.0%) cases by G-banding, fluorescent in situ hybridization and molecular analyses. 131 cases (44.9%) were classified B-others. We studied 121 (93.1%) out of 131 B-others ALL of the L0416/L0616 cohort. We performed whole transcriptome sequencing (WTS) of 35 pediatric B-others cases. For the confirmation and screening of the fusion transcripts we performed RT-PCR in a total of 121 B-others cases. Alterations of ERG and iAMP21 were examined by Multiplex Ligation-dependent Probe Amplification (MLPA) kit P327. We also analyzed the known genetic mutations or copy number alterations associated with these patients using the data from WTS and MLPA kit P335 and P200. Results; A total of 47 candidate in-frame gene rearrangements were identified in 29/35 (82.8%) B-others samples. The fusion genes including ZNF384 (n=12, 4.1%) or MEF2D (n=6, 2.1%) were frequently found in this cohort. All of patients with ZNF384-related fusion genes were found to have a weak or negative expression of CD10. In addition, 7 and 17 patients out of 22 patients with ZNF384-related fusion genes exhibited more than 20% expression of CD13 and CD33. We identified 6 recurrently mutated genes, including NRAS, KRAS, PTPN11, EZH2, MLL2 and ASH1L. Especially, activating mutations in RAS signaling pathway genes, including NRAS, KRAS and PTPN11 were detected in 4 out of 10 ZNF384-translocated patients. Mutations in B-cell developmental genes such as PAX5, BTG1 and IKZF1 were not detected at all in DNA samples with ZNF384-related fusion genes. We identified a total of 6 (1.8%) patients with the fusion genes related with Ph-like ALL, including IGHM-CRLF2, P2RY8-CRLF2, IGH@-EPOR and PAX5-JAK2. Three of 6 patients with Ph-like ALL related fusion gene relapsed and two patients died. We identified iAMP21 in 6 (2.1%) patients and deletion of ERG in 7 (2.4%). One of 6 patients with iAMP21 and 3 of 8 patients with ERG deletion had IKZF1 deletion. No mutations of CRLF2 and JAK2 were identified in patients with iAMP21 and ERG deletion. Among 6 patients with iAMP21, three patients relapsed, and two patients died. No patients with ERG deletion relapsed or died. Conclusions; WTS analysis unmasked a complexity of gene rearrangements in pediatric BCP-ALL without conventional genetic abnormalities. In this study, 10.6% of genetic alterations were revealed in Japanese pediatric B-others cohort. Apart from increasing our understanding of the pathogenesis of pediatric BCP-ALL, this may help improve risk stratification and eventually increase the therapeutic option. Disclosures No relevant conflicts of interest to declare.

scholarly journals Predicting interactome network perturbations in human cancer: application to gene fusions in acute lymphoblastic leukemia

2014 ◽  
Vol 25 (24) ◽  
pp. 3973-3985 ◽  
Author(s):  
Leon Juvenal Hajingabo ◽  
Sarah Daakour ◽  
Maud Martin ◽  
Reinhard Grausenburger ◽  
Renate Panzer-Grümayer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Human Cancer ◽  
Lymphoblastic Leukemia ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Gene Fusions ◽  
Novel Approach ◽  
Interactome Network ◽  
Cell Acute Lymphoblastic Leukemia

Genomic variations such as point mutations and gene fusions are directly or indirectly associated with human diseases. They are recognized as diagnostic, prognostic markers and therapeutic targets. However, predicting the functional effect of these genetic alterations beyond affected genes and their products is challenging because diseased phenotypes are likely dependent of complex molecular interaction networks. Using as models three different chromosomal translocations—ETV6-RUNX1 (TEL-AML1), BCR-ABL1, and TCF3-PBX1 (E2A-PBX1)—frequently found in precursor-B-cell acute lymphoblastic leukemia (preB-ALL), we develop an approach to extract perturbed molecular interactions from gene expression changes. We show that the MYC and JunD transcriptional circuits are specifically deregulated after ETV6-RUNX1 and TCF3-PBX1 gene fusions, respectively. We also identified the bulk mRNA NXF1-dependent machinery as a direct target for the TCF3-PBX1 fusion protein. Through a novel approach combining gene expression and interactome data analysis, we provide new insight into TCF3-PBX1 and ETV6-RUNX1 acute lymphoblastic leukemia.

scholarly journals Targeted sequencing to identify genetic alterations and prognostic markers in pediatric T-cell acute lymphoblastic leukemia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ya-Hsuan Chang ◽  
Chih-Hsiang Yu ◽  
Shiann-Tarng Jou ◽  
Chien-Yu Lin ◽  
Kai-Hsin Lin ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Signaling Pathway ◽  
Prognostic Markers ◽  
Lymphoblastic Leukemia ◽  
Survival Rates ◽  
Genetic Alterations ◽  
Targeted Sequencing ◽  
Single Nucleotide Variants ◽  
Cell Acute Lymphoblastic Leukemia

AbstractT-cell acute lymphoblastic leukemia (T-ALL) is caused by the accumulation of multiple genetic alterations. To determine the frequency of common genetic mutations and possible prognostic markers in childhood T-ALL, we performed targeted sequencing of 67 genes across 64 cases treated according to Taiwan Pediatric Oncology Group protocols between January 2002 and December 2015. Together, 302 variants were identified in 60 genes including 233 single nucleotide variants and 69 indels. Sixty-four samples had a median number of six genetic lesions each (range 1–17). Thirteen genes had mutation frequencies > 10%, and 5 were > 20%, with the highest being NOTCH1 (70.31%). Protocadherins FAT1 (32.81%) and FAT3 (17.19%), and the ubiquitin ligase component FBXW7 (28.13%) had higher mutation frequencies than previously reported. Other mutation frequencies (PHF6, DNM2, DNMT3A, CNOT3, and WT1) were within previously reported ranges. Three epigenetic-related genes (KMT2D, DNMT3A, and EZH2) were mutated in our cohort. JAK-STAT signaling pathway genes had mutation frequencies of 3–13% and were observed in 23 cases (35.94%). Changes to genes in the ErbB signaling pathway were detected in 20 cases (31.25%). Patients with NOTCH1/FBXW7 mutations and RAS/PTEN germline exhibited better 5-year overall survival rates.

scholarly journals Association Analysis Between the Functional Single Nucleotide Variants in miR-146a, miR-196a-2, miR-499a, and miR-612 With Acute Lymphoblastic Leukemia

2021 ◽  
Vol 11 ◽  
Author(s):  
Silvia Jiménez-Morales ◽  
Juan Carlos Núñez-Enríquez ◽  
Jazmín Cruz-Islas ◽  
Vilma Carolina Bekker-Méndez ◽  
Elva Jiménez-Hernández ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Gene Interaction ◽  
Control Group ◽  
Dependent Manner ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Mexican Children

BackgroundAcute lymphoblastic leukemia (ALL) is characterized by an abnormal proliferation of immature lymphocytes, in whose development involves both environmental and genetic factors. It is well known that single nucleotide polymorphisms (SNPs) in coding and noncoding genes contribute to the susceptibility to ALL. This study aims to determine whether SNPs in miR-146a, miR-196a-2, miR-499a, and miR-612 genes are associated with the risk to ALL in pediatric Mexican population.MethodsA multicenter case-control study was carried out including patients with de novo diagnosis of ALL and healthy subjects as control group. The DNA samples were obtained from saliva and peripheral blood, and the genotyping of rs2910164, rs12803915, rs11614913, and rs3746444 was performed using the 5′exonuclease technique. Gene-gene interaction was evaluated by the multifactor dimensionality reduction (MDR) software.ResultsmiR-499a rs3746444 showed significant differences among cases and controls. The rs3746444G allele was found as a risk factor to ALL (OR, 1.6 [95% CI, 1.05–2.5]; p = 0.028). The homozygous GG genotype of rs3746444 confers higher risk to ALL than the AA genotype (OR, 5.3 [95% CI, 1.23–23.4]; p = 0.01). Moreover, GG genotype highly increases the risk to ALL in male group (OR, 17.6 [95% CI, 1.04–298.9]; p = 0.00393). In addition, an association in a gender-dependent manner among SNPs located in miR-146a and miR-196a-2 genes and ALL susceptibility was found.ConclusionOur findings suggest that SNP located in miR-499a, miR-146a, and miR-196a-2 genes confer risk to ALL in Mexican children. Experimental analysis to decipher the role of these SNPs in human hematopoiesis could improve our understanding of the molecular mechanism underlying the development of ALL.

Whole Transcriptome Sequencing of a Philadelphia-Positive Acute Lymphoblastic Leukemia (ALL) with “Next Generation Sequencing” Technology Revealed Novel Point Mutations Associated with Disease-Progression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3074-3074
Author(s):  
Ilaria Iacobucci ◽  
Alberto Ferrarini ◽  
Marco Sazzini ◽  
Enrico Giacomelli ◽  
Annalisa Lonetti ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Rna Seq ◽  
Sequencing Technology ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Next Generation Sequencing Technology ◽  
Whole Transcriptome ◽  
Generation Sequencing

Abstract Abstract 3074 Poster Board III-11 Background BCR-ABL1-positve Acute Lymphoblastic Leukemia (ALL) is the most common ALL subtype in adults and is associated with poor prognosis. The pathogenesis of this leukemia is related to the expression of the BCR-ABL1 fusion transcript, but additional recurrent genetic lesions are suspected to be involved in its development and progression. Aim A Next-Generation Sequencing Technology was used to sequence the whole transcriptome of leukemia cells from a BCR-ABL1-positive ALL patient at diagnosis and at relapse following tyrosine kinase inhibitor (TKI) therapy with the aim to detect acquired mutations cooperating with BCR-ABL1 in leukemia manifestation and drug-resistance. Methods Poly(A) RNA was extracted from leukemia cells and used to prepare double-stranded cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained 36 base-pair (bp) sequence reads were mapped to the reference sequence of the human genome (UCSC hg18, NCBI build 36.1) to identify single nucleotide variants (SNVs) and to estimate reads density corresponding to RNA from each known exon, canonical splice event or new candidate gene. This approach allowed us to define a detailed Digital Gene Expression (DGE) profile. Reads that showed no match to the reference genome were subsequently mapped to a dataset of all possible splice junctions created by in silico pairwise combination of the exons of all annotated genes (UCSC knownGene file) to identify alternative splicing (AS) events. Results Whole Transcriptome Shotgun Sequencing (RNA-seq) analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples respectively, achieving approximately 90% diploid coverage and detecting transcripts from 62% and 64% of human annotated genes. The great majority of these active genes (78% at diagnosis and 73% at relapse) showed very low expression levels, with a number of reads per kilobase of exon model per million mapped reads (RPKM value) from 0.01 to 10, whereas 20% and 24% showed moderate expression levels (RPKM 10-100), as well as only 2% and 3% resulted highly expressed (RPKM 100-8000). Moreover, 6,390 and 4,671 AS events were also identified within 4,334 diagnosis and 3,651 relapse annotated transcripts, with the already described ALL-related Ik6 Ikaros isoform observed in both samples. Finally, 2,011 and 2,103 single nucleotide variants (SNVs) were found at diagnosis and relapse respectively, about 94% of which have been already reported in the dbSNP. Of greater interest as potential ALL-related mutations, 124 and 115 non annotated SNVs were also found at diagnosis and relapse, respectively. Of these, 43 affected both samples, while 81 and 72 resulted diagnosis and relapse private variants. In particular, the analysis was focused to the coding sequences of annotated genes, finding that non-synonymous changes were one out of the 19 shared between the two samples and affected a transmembrane receptor gene (PLXNB2). Six out of the 12 diagnosis private variants, affecting genes involved in metabolic process (PDE4DIP, EIF2S3, DPEP1, ZC3H12D, TMEM46) or transport (MVP) and 5 out of the 30 relapse private variants, affecting genes involved in cell cycle regulation (ABL1, CDC2L1), catalytic activity (CTSZ, CXorf21) or with unknown function (FAM116B). Most of these diagnosis and relapse non-synonymous private mutations resulted highly expressed, showing frequencies of mutated unique reads higher than 50%. According to this pattern, diagnosis private mutations may be carried by primary leukemic clones that did not develop again at relapse, whereas relapse private mutations have greater probability to be variants acquired during the disease progression. Interestingly, the T315I point mutation in the Abl kinase domain, that confers resistance to many TKIs, was found at relapse but not at diagnosis. Conclusions An accurate expression profile was obtained for the leukemia cells of the examined ALL patient, as well as the discovery of several new non-synonymous mutations affecting genes from different pathways and for which no correlation was previously found with ALL pathogenesis. These findings demonstrate that RNA-Seq represents a suitable and cost-efficient approach for identifying new genes potentially involved in ALL development and progression. Acknowledgments AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo 60% grants, European LeukemiaNet. Disclosures No relevant conflicts of interest to declare.

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