Massive Parallel Sequencing and Mutations Analysis of Mismatch Repair Genes in Recurrent Childhood Acute Lymphoblastic Leukemia Cases-Identification of Possible Molecular Signature of Poor Clinical Outcome

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5266-5266
Author(s):  
Keisuke Kato ◽  
Ai Yoshimi ◽  
Satoru Matsushima ◽  
Chie Kobayashi ◽  
Kunio Fukuda ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Mismatch Repair ◽  
Lymphoblastic Leukemia ◽  
Clinical Samples ◽  
Established Cell Line ◽  
Chimeric Transcript ◽  
Whole Exome ◽  
Established Cell ◽  
Exome Analysis ◽  
Gene Status

Abstract [Introduction] Mechanism of recurrence is fully characterized and is significant to improve prognosis in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and T lymphoblastic leukemia (T-ALL).. We have analyzed recurrent cases of childhood BCP-ALL and T-ALL. [Procedure] We analyzed clinical samples of 18 cases with BCP-ALL and 3 cases with T-ALL, who had been treated in our institution. We have investigated gene status of IKZF1, CRLF2, CDKN2A/2B, JAKs, IL7RA, and TP53 using RT-PCR and MLPA methods and IGH/TCR on genomic PCR. Whole exome analysis was done using Ion AmpliSeq Exome in seven cases. [Result] We analyzed clinical samples of 21 cases with ALL, who had been treated in our institution. The cases were 18 cases with BCP-ALL and 3 cases with T-ALL. We have investigated gene status of IKZF1, CRLF2, CDKN2A/2B,JAKs,IL7RA, NOTCH (for T-ALL), and TP53. Whole exome analysis was done in two cases, particularly for a case from which we have established cell line. < Result > Eight BCP-ALL cases with P2RY8-CRLF2 chimeric transcript: four cases of which had P2RY8-CRLF2 chimeric transcript only at initial diagnosis; two cases obtained transcript at recurrence. This was confirmed employing LD-PCR genomic analysis. Ten BCP-ALL cases had deletion of IKZF1; two showed deletion at relapse and a case demonstrated deletion at diagnosis only. Two BCP-ALL cases showed mutation of IL7RA. One of T-ALL cases from which we have established cell line showed mutation of MSH2 and more than 200 non-synonymous mutations on whole exome analysis. One T-ALL cases showed mutation of JAK3 at diagnosis. One BCP-ALL case had MLH1 mutation < Discussion > The present study has suggested acquisition of complex genetic change at different point of evolution may work in recurrence of ALL. The present study has indicated P2RY8-CRLF2 works not as simple growth advantage but rather as manifestation of genomic instability. This may be also illustrated by recurrent cases with T-ALL having mutation of MSH2 and BCP-ALL having mutation of MLH1. Mutation of mismatch repair gene may be driver of gene mutation acquisition and consequently alterations of CREBBP and RAS or IKZF1, TP53, mismatch repair genes, or emergence of P2RY-CRLF2 chimeric transcript may be prognostically relevant in childhood acute lymphoblastic leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Detection of the Philadelphia chromosome in acute lymphoblastic leukemia by pulsed-field gel electrophoresis

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 1101-1107 ◽  
Author(s):  
AL Hooberman ◽  
CM Rubin ◽  
KP Barton ◽  
CA Westbrook
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Gel Electrophoresis ◽  
Chromosomal Abnormalities ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Pulsed Field Gel Electrophoresis ◽  
Myelogenous Leukemia ◽  
Clinical Samples ◽  
Pulsed Field ◽  
Translocation Breakpoints

Abstract The Philadelphia (Ph1) chromosome is an acquired abnormality in the malignant cells of 10% to 25% of patients with acute lymphoblastic leukemia (ALL). Unlike chronic myelogenous leukemia (CML), where the molecular detection of the Ph1 chromosome is relatively straightforward using conventional Southern hybridization analysis, the detection of the Ph1 chromosome in ALL is complicated by the existence of several molecular subtypes, and the fact that translocation breakpoints are dispersed over a large genomic area. To circumvent these difficulties, we investigated pulsed-field gel electrophoresis (PFGE) to determine if this method could be used directly on clinical samples to detect the Ph1 chromosome in ALL. We report that, in a study of seven patients with Ph1-positive ALL, we could easily detect the Ph1 using only a single PFGE analysis, regardless of the Ph1 subtype, and we could confirm that the translocations occur either within or very near the BCR gene in all seven. We conclude that PFGE is a useful technique for the detection of the Ph1 in ALL, which ultimately may find wide applicability in the detection of other chromosomal abnormalities in other malignancies.

Up-Regulation of Genes Involved in Folate Metabolism Characterize Late but Not Early Relapse in Childhood Acute Lymphoblastic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1733-1733
Author(s):  
Laura Hogan ◽  
Deepa Bhojwani ◽  
Jinhua Wang ◽  
Debra Morrison ◽  
Jun J Yang ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Real Time ◽  
Quantitative Pcr ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Nucleotide Metabolism ◽  
Clinical Samples ◽  
Late Relapse ◽  
Early Relapse ◽  
Real Time Quantitative Pcr

Abstract Abstract 1733 Poster Board I-759 Introduction Outcomes for relapsed pediatric acute lymphoblastic leukemia (ALL) are poor overall but depend on the timing of relapse, with lower survival rates for patients that relapse early (defined as < 36 months from diagnosis). Previously we sought to discover underlying biological pathways that mediate relapse by analyzing gene expression profiles in a large cohort of diagnosis/relapse paired bone marrow samples on the Affymetrix U133A arrays (Bhojwani et al, ASH 2008). We determined that early relapse was characterized by over-expression of cell cycle genes reflective of a proliferative state but late relapse was characterized by genes involved in nucleotide biosynthesis including targets of anti-folate agents. Given the potential therapeutic implications of these results we sought to validate these findings in an independent set of samples. Patients and Methods Validation of the first 60 pairs was performed using 26 additional pairs analyzed on Affymetrix U133Plus2 arrays. This validation set consisted of 17 early and 9 late diagnosis/relapse pairs. All patients had B precursor ALL and were at first relapse. Probesets differentially expressed between early and late relapse were identified in a supervised pair-wise analysis using an FDR cutoff of <10%. Expression of several genes found to be up-regulated at relapse by array expression were validated using real-time quantitative PCR. Results Evaluation of the last 26 pairs once again revealed distinct gene signatures that could distinguish between early and late relapse and many genes that were significant in the original 60 pairs were validated. Overall, thirty percent of the genes that distinguish diagnosis from relapse were validated in this smaller cohort. Importantly, genes involved in nucleotide metabolism that are targets of anti-folates such as thymidylate synthetase (TYMS), dihydrofolate reductase (DHFR) and methylenetetrahydrofolate dehydrogenase (MTHFD1) were validated as up-regulated at late relapse. TYMS was elevated at late relapse in the original (p=0.004) and validation (p=0.02) cohorts. MTHFD1 and DHFR also showed increased expression at late relapse (p=0.01 and p=0.09 for MTHFD1; and p=0.01and p=0.07 for DHFR) in both the discovery and validation cohorts respectively. Increased expression of DHFR and TYMS in late relapse was also confirmed by real-time quantitative PCR comparing 12 early and 12 late relapse pairs. Median expression at late relapse was 2 fold higher than diagnosis for both DHFR (p= 0.03) and TYMS (p= 0.01). Conclusion Increased expression of anti-folate target genes is seen in clinical samples from patients whose disease recurs late, indicating possible selection during maintenance where the bulk of anti-folate exposure occurs. Therapeutic strategies to augment folate antagonism may prevent late recurrences of ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Prospective Analysis of TEL Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia: A Children's Oncology Group Study

2008 ◽  
Vol 26 (13) ◽  
pp. 2186-2191 ◽  
Author(s):  
Jeffrey E. Rubnitz ◽  
David Wichlan ◽  
Meenakshi Devidas ◽  
Jonathan Shuster ◽  
Stephen B. Linda ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Prognostic Significance ◽  
Oncology Group ◽  
Free Survival ◽  
Pediatric Oncology Group ◽  
Gene Status ◽  
Group Sex ◽  
Cure Rates ◽  
Standard Risk

Purpose To prospectively determine the prognostic significance of the TEL-AML1 fusion in children with acute lymphoblastic leukemia (ALL). Patients and Methods TEL gene status was determined for 926 patients with B-precursor ALL enrolled on the Pediatric Oncology Group ALinC 16 trials and patients were observed for a median time of 8 years. Results Rearrangements of the TEL gene were detected in 244 patients (26%). The estimated 5-year event-free survival rate (± SE) for patients with TEL rearrangements was 86% ± 2%, compared with 72% ± 2% for those with germline TEL (P < .0001). TEL rearrangements were associated with a superior outcome among patients with standard-risk ALL, high-risk ALL, and rapid early responses to therapy. In a multivariate analysis that included risk group, sex, and day 15 marrow status, TEL status was an independent predictor of outcome (P = .0002). Conclusion We conclude that TEL gene status should be incorporated into risk classification schemes and suggest that patients who have standard-risk features, the TEL-AML1 fusion, and rapid early responses to therapy, should be treated with antimetabolite-based therapy designed to maintain their high cure rates and avoid late effects.

scholarly journals Detection of the Genomic Variants in the Paired De Novo-Relapsed Samples with Pan-Cancer Panel and Whole Exome-Seq in Adult B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5206-5206
Author(s):  
Zheng Ge ◽  
Yuka Imamura Kawasawa ◽  
Yan Gu ◽  
Qi Han ◽  
Jie Zi ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Amino Acid ◽  
Amino Acid Change ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Genomic Variants ◽  
Whole Exome ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Pan Cancer ◽  
Cancer Panel

Background: The relapse rate is around 20-40% in adult B-cell acute lymphoblastic leukemia (B-ALL). The genetic defects are the major reasons for the relapse and poor outcome. We screened the genomic variants with Pan-cancer panel in B-ALL patients and whole exome-seq (WES) in the paired de novo-relapsed B-ALL samples. Methods: The xGen Pan-Cancer Panel (IDT), which has been designed with the probes targeting 127 significantly mutated genes from the TCGA database across 12 tumor tissue types (Nature, 502:333-339) was used. Agilent SureSelect Human All Exon V4+UTRs (Agilent) was used to target coding exons plus UTRs for the WES analysis. The genomic DNA from 81 Philadelphia chromosome positive (Ph+) B-ALL patient samples (71 de novo and 10 relapsed, 14 to 77 years old) collected between June 2008 and June 2016 at Zhongda Hospital Southeast University were used for the Pan-cancer panel screening. All DNA samples were sheared and generated approximately 260bp DNA fragments. The fragmented DNA was processed into Illumina compatible sequencing libraries using the Kapa Hyper Prep Kit. Each library was uniquely barcoded and captured by either the Pan-cancer panel or WES probes, followed by PCR amplification and sequencing on a HiSeq 2500 (Illumina) with 2x100 bp reads. The sequencing reads were aligned to the human genome (hg19) by following Broad Institute's GATK best practice pipeline to call germline short variants (SNPs and Indels). Called variants were annotated using ANNOVAR (version 2.3). Variants with exonic, nonsynonymous, stopgain, or stoploss, novel SNPs (not covered by dbSNP138), and with predicted deleterious/damaging functions were manually surveyed by IGV to confirm. Two paired de novo-relapse samples from Ph(+) B-ALL patients were performed the WES analysis. Results: We identified a total of 3945 single nucleotide variants (SNVs), 2222 insertions and deletions (INDELs) in the Pan-cancel panel analysis in 81 Ph(+) B-ALL patients. Among these, 101 genomic variants are with amino acid change, 8 are with stopgain, and 5944 have not been previously reported. We evaluated the frequency and distribution of likely pathogenic variants (PVs) detected in the cohort. Likely PVs were defined by SIFT algorithm which predicts whether an amino acid substitution is likely to affect protein function. Defined by the SIFT's qualitative score 'deleterious', we detected 46 PVs. Among these, PVs were commonly detected in KMT2C, APC, CDKN1A, NSD, BRCA1, EPHA3, and PIK3CG. The PVs were also validated with the Sanger DNA sequencing in the patients. The patients with the likely PVs have significantly higher WBC count (61*10^9/L vs. 24.45*10^9/L, P=0.004). Survival analysis showed that the patients with likely PVs had a worse event-free survival (EFS) and overall survival (OS), the difference was statistically significant (8 months vs. 15 months, P=0.017 and 14 months vs. 25 months, P=0.021). In order to gain an insight to the gene mutations contributing to the disease relapse, we compared the mutants spectrum between the de novo and relapsed samples. We found the genomic variants in NF1, CDK12, mTOR, and USP9X genes appeared mostly in relapsed samples, indicating their roles in the relapse. Using the WES, we further analyzed the genomic variants in two paired de novo and relapsed samples. We detected totally 40354 (de novo) and 16822 (relapsed) genomic variants, and among these 10415 (de novo) and 3082 (relapsed) are with amino acid change in patient 1; likewise, 30130 (de novo) and 14003 (relapse), and among these 7200 (de novo) 2534 (relapsed) with amino acid changes in patient 2. Totally 216 genomic variants with amino acid changes in 162 genes appeared only in the two relapsed samples, among which 10 genomic variants in ADAMTS8, CDK11B, EFCAB4B, FBXL21, HYDIN, IRF2BPL, MIR205HG, PRIM2, ZNF717, ZNF880 appeared in both relapsed samples, revealing their driver roles in relapse. Also, 110 of the 216 genomic variants are not previously reported. Conclusion: Genomic variants in common human cancer driver genes were also detected in B-ALL patients. The new genomic variants detected in the relapse samples may be involved in the oncogenesis of the relapse, which will be further verified with functional analysis. Our data also suggested the significance of developing more efficient new therapies to prevent the relapse in hematological malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of functional cooperative mutations of GNAO1 in human acute lymphoblastic leukemia

Blood ◽  
2020 ◽  
Author(s):  
Lili Song ◽  
Bo Yu ◽  
Yi Yang ◽  
Jianwei Liang ◽  
Yingwen Zhang ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Chromosomal Rearrangements ◽  
Ectopic Expression ◽  
Monozygotic Twins ◽  
Human Leukemia ◽  
Missense Mutations ◽  
Protein Subunit ◽  
Whole Exome ◽  
Human Acute Lymphoblastic Leukemia

Leukemogenesis is characterized by chromosomal rearrangements with additional molecular disruptions, yet the cooperative mechanisms are still unclear. Using whole-exome sequencing of a pair of monozygotic twins discordant for childhood acute lymphoblastic leukemia (ALL) with ETV6-RUNX1 (E/R) gene fusion successively after birth, we identified the R209C mutation of G protein subunit alpha o1 (GNAO1) as a new ALL risk loci. Moreover, GNAO1 missense mutations are only recurrent in ALL patients and are associated with E/R fusion. Ectopic expression of the GNAO1 R209C mutant increased its GTPase activity and promoted cell proliferation and cell neoplastic transformation. Combined with the E/R fusion, the GNAO1 R209C mutant promoted leukemogenesis through activating PI3K/Akt/mTOR signaling. Reciprocally, activated mTORC1 phosphorylated p300 acetyltransferase, which acetylated E/R and thereby enhanced the E/R transcriptional activity of GNAO1 R209C. Thus, our study provides clinical evidence for the functional cooperation of GNAO1 mutants and E/R fusion, suggesting GNAO1 as a potential therapeutic target in human leukemia.

scholarly journals Case-Control Study Suggests a Favorable Impact of TEL Rearrangement in Patients With B-Lineage Acute Lymphoblastic Leukemia Treated With Antimetabolite-Based Therapy: A Pediatric Oncology Group Study

Blood ◽  
1997 ◽  
Vol 89 (4) ◽  
pp. 1143-1146 ◽  
Author(s):  
Jeffrey E. Rubnitz ◽  
Jonathan J. Shuster ◽  
Vita J. Land ◽  
Michael P. Link ◽  
D. Jeanette Pullen ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Pediatric Oncology ◽  
Leukocyte Count ◽  
Lymphoblastic Leukemia ◽  
Intensive Therapy ◽  
Oncology Group ◽  
Pediatric Oncology Group ◽  
B Lineage ◽  
And Gender ◽  
Gene Status

Abstract TEL gene rearrangement is the most common genetic lesion in pediatric acute lymphoblastic leukemia (ALL), occurring in about 25% of B-lineage cases. We previously showed that, among patients treated on St Jude protocols, TEL rearrangement independently conferred an excellent prognosis. To extend these results to patients treated with antimetabolite-based therapy, we performed Southern blot analysis to determine the TEL gene status of 104 cases of B-lineage ALL treated on Pediatric Oncology Group 8602, matched on age, gender, and leukocyte count. There were 52 failures among the 77 patients with germline TEL, compared with only 8 failures among 27 patients in the rearranged group. Based on a two-sided logistic regression analysis, stratified for age (subdivided at 10 years), leukocyte count (subdivided at 50,000), and gender, the estimated odds of failing by 4 years in the germline TEL group is 5.4 times that of the rearranged TEL group, with 95% confidence from 1.9 to 15.6, two-sided P = .0009. Thus, the presence of a rearranged TEL gene is also associated with an improved survival among patients treated with antimetabolite-based therapy. Our results indicate that all newly diagnosed ALL patients should be screened for TEL gene rearrangements and suggest that these patients are candidates for less intensive therapy.

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