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Practical Considerations for Using RNA Sequencing in Management of B-Lymphoblastic Leukemia: Malaysia-Singapore Acute Lymphoblastic Leukemia 2020 Implementation Strategy

Ni Chin WH, Li Z, Jiang N, Lim EH, Suang Lim JY, Lu Y, et al.

J Mol Diagn, 2021 10;23(10):1359-1372.
PMID: 34365011 DOI: 10.1016/j.jmoldx.2021.07.013

Despite the immense genetic heterogeneity of B-lymphoblastic leukemia [or precursor B-cell acute lymphoblastic leukemia (B-ALL)], RNA sequencing (RNA-Seq) could comprehensively interrogate its genetic drivers, assigning a specific molecular subtype in >90% of patients. However, study groups have only started to use RNA-Seq. For broader clinical use, technical, quality control, and appropriate performance validation are needed. We describe the development and validation of an RNA-Seq workflow for subtype classification, TPMT/NUDT15/TP53 variant discovery, and immunoglobulin heavy chain (IGH) disease clone identification for Malaysia-Singapore acute lymphoblastic leukemia (ALL) 2020. We validated this workflow in 377 patients in our preceding Malaysia-Singapore ALL 2003/Malaysia-Singapore ALL 2010 studies and proposed the quality control measures for RNA quality, library size, sequencing, and data analysis using the International Organization for Standardization 15189 quality and competence standard for medical laboratories. Compared with conventional methods, we achieved >95% accuracy in oncogene fusion identification, digital karyotyping, and TPMT and NUDT15 variant discovery. We found seven pathogenic TP53 mutations, confirmed with Sanger sequencing, which conferred a poorer outcome. Applying this workflow prospectively to the first 21 patients in Malaysia-Singapore ALL 2020, we identified the genetic drivers and IGH disease clones in >90% of patients with concordant TPMT, NUDT15, and TP53 variants using PCR-based methods. The median turnaround time was 12 days, which was clinically actionable. In conclusion, RNA-Seq workflow could be used clinically in management of B-cell ALL patients.

Matched MeSH terms: Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics*
Fulltext Novel variants in NUDT15 and thiopurine intolerance in children with acute lymphoblastic leukemia from diverse ancestry

Moriyama T, Yang YL, Nishii R, Ariffin H, Liu C, Lin TN, et al.

Blood, 2017 Sep 07;130(10):1209-1212.
PMID: 28659275 DOI: 10.1182/blood-2017-05-782383

Prolonged exposure to thiopurines (eg, mercaptopurine [MP]) is essential for curative therapy in acute lymphoblastic leukemia (ALL), but is also associated with frequent dose-limiting hematopoietic toxicities, which is partly explained by inherited genetic polymorphisms in drug metabolizing enzymes (eg, TPMT). Recently, our group and others identified germ line genetic variants in NUDT15 as another major cause of thiopurine-related myelosuppression, particularly in Asian and Hispanic people. In this article, we describe 3 novel NUDT15 coding variants (p.R34T, p.K35E, and p.G17_V18del) in 5 children with ALL enrolled in frontline protocols in Singapore, Taiwan, and at St. Jude Children's Research Hospital. Patients carrying these variants experienced significant toxicity and reduced tolerance to MP across treatment protocols. Functionally, all 3 variants led to partial to complete loss of NUDT15 nucleotide diphosphatase activity and negatively influenced protein stability. In particular, the p.G17_V18del variant protein showed extremely low thermostability and was completely void of catalytic activity, thus likely to confer a high risk of thiopurine intolerance. This in-frame deletion was only seen in African and European patients, and is the first NUDT15 risk variant identified in non-Asian, non-Hispanic populations. In conclusion, we discovered 3 novel loss-of-function variants in NUDT15 associated with MP toxicity, enabling more comprehensive pharmacogenetics-based thiopurine dose adjustments across diverse populations.

Matched MeSH terms: Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics*
Fulltext Whole-transcriptome sequencing identifies a distinct subtype of acute lymphoblastic leukemia with predominant genomic abnormalities of EP300 and CREBBP

Qian M, Zhang H, Kham SK, Liu S, Jiang C, Zhao X, et al.

Genome Res, 2017 02;27(2):185-195.
PMID: 27903646 DOI: 10.1101/gr.209163.116

Chromosomal translocations are a genomic hallmark of many hematologic malignancies. Often as initiating events, these structural abnormalities result in fusion proteins involving transcription factors important for hematopoietic differentiation and/or signaling molecules regulating cell proliferation and cell cycle. In contrast, epigenetic regulator genes are more frequently targeted by somatic sequence mutations, possibly as secondary events to further potentiate leukemogenesis. Through comprehensive whole-transcriptome sequencing of 231 children with acute lymphoblastic leukemia (ALL), we identified 58 putative functional and predominant fusion genes in 54.1% of patients (n = 125), 31 of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature predominantly driven by chromosomal rearrangements of the ZNF384 gene with histone acetyltransferases EP300 and CREBBP ZNF384-rearranged ALL showed significant up-regulation of CLCF1 and BTLA expression, and ZNF384 fusion proteins consistently showed higher activity to promote transcription of these target genes relative to wild-type ZNF384 in vitro. Ectopic expression of EP300-ZNF384 and CREBBP-ZNF384 fusion altered differentiation of mouse hematopoietic stem and progenitor cells and also potentiated oncogenic transformation in vitro. EP300- and CREBBP-ZNF384 fusions resulted in loss of histone lysine acetyltransferase activity in a dominant-negative fashion, with concomitant global reduction of histone acetylation and increased sensitivity of leukemia cells to histone deacetylase inhibitors. In conclusion, our results indicate that gene fusion is a common class of genomic abnormalities in childhood ALL and that recurrent translocations involving EP300 and CREBBP may cause epigenetic deregulation with potential for therapeutic targeting.

Matched MeSH terms: Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics*
Fulltext Effect of polymorphisms within methotrexate pathway genes on methotrexate toxicity and plasma levels in adults with hematological malignancies

Suthandiram S, Gan GG, Zain SM, Bee PC, Lian LH, Chang KM, et al.

Pharmacogenomics, 2014 Aug;15(11):1479-94.
PMID: 25303299 DOI: 10.2217/pgs.14.97

Pharmacogenetics of methotrexate (MTX) contributes to interindividual differences in toxicity. We aimed to evaluate the impact of SNPs within the MTX pathway genes on MTX-induced toxicity and MTX plasma levels at 48 h following treatment in Asian adults with acute lymphoblastic leukemia or non-Hodgkin lymphoma.

Matched MeSH terms: Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics
Detection of immunoglobulin gene rearrangement in B-cell malignancies

Ainoon O, Hamidah AB, Cheong SK, Hamidah HN

Malays J Pathol, 2000 Jun;22(1):5-11.
PMID: 16329531

Rearrangement of the immunoglobulin heavy chain (IgH) gene has been used as a marker of lineage and clonality in the diagnosis of B lymphoproliferative disorders. A number of PCR-based techniques have been developed to overcome the disadvantages of Southern blotting, the standard technique in detecting IgH gene rearrangement. Using an established seminested PCR technique with consensus primers to the V and J regions of the IgH gene, we analysed DNA prepared from peripheral blood and/or bone marrow specimens from 30 cases of known B cell malignancies (16 chronic lymphocytic leukemia, 11 acute lymphoblastic leukemia and 3 Non-Hodgkin Lymphoma), 3 cases of T lymphoproliferative disease and 3 cases of reactive lymphocytosis diagnosed in Hospital UKM to detect rearranged IgH gene. We found that monoclonality as represented by the presence of rearranged IgH gene were demonstrated in all the 30 cases. The PCR findings showed 100% concordance with the Southern blot analysis results which also showed rearranged IgH bands in all the 30 cases. We also found that none of the cases of T lymphoproliferative diseases and reactive lymphocytosis showed presence of rearranged IgH band, suggesting that the amplification using the IgH primers is lineage-specific. In conclusion, we find the PCR a useful method to detect IgH gene rearrangement in peripheral blood and bone marrow specimen. Since the PCR results are comparable to that of the Southern blotting in demonstrating B cell monoclonality and owing to its many advantages we feel that it can replace the Southern blot technique for the diagnosis of B cell malignancies.

Matched MeSH terms: Burkitt Lymphoma/genetics*
High frequency of EBV association and 30-bp deletion in the LMP-1 gene in CD56 lymphomas of the upper aerodigestive tract

Tai YC, Kim LH, Peh SC

Pathol. Int., 2004 Mar;54(3):158-66.
PMID: 14989738

Natural killer (NK)/T-cell lymphomas are frequently associated with Epstein-Barr virus (EBV), and usually lack TCR gene rearrangement. Studies from Asia have reported frequent deletion in the LMP-1 gene in EBV-associated nasopharyngeal carcinoma (NPC). The present study aims to investigate LMP-1 and TCRgamma gene status in upper aerodigestive tract lymphomas. A total of 43 cases were classified into T-, B-, and NK/T-cell tumors based on the phenotype expressions of CD3(+)/CD20(-)/CD56(-), CD3(-)/CD20(+)/CD56(-), and CD3(+)/CD20(-)/CD56(+), respectively. The presence of EBV in the tumor was confirmed by EBV early RNA-in situ hybridization. LMP-1 gene deletion and TCR gamma gene rearrangement were analyzed by polymerase chain reaction on paraffin-embedded tissues. There were 20 NK/T-, eight T-, and 15 B-cell phenotype lymphomas in the present series, and EBV was detected in 19 (95%), two (25%), and three (20%) cases in the respective groups. All EBV+ cases carried 30-bp deletion in the LMP-1 gene, and two of the NK/T-cell cases were infected by both the wild type and deleted strains. Five (25%) of the NK/T-cell phenotype lymphomas showed rearranged TCR gamma gene. The present study revealed a high frequency of EBV association, and a high frequency of 30-bp deletion in the LMP-1 gene in the virus in the present series of lymphoma. The NK/T-phenotype lymphomas are comprised of both NK-cell and cytotoxic T-lymphocyte-derived tumors.

Matched MeSH terms: Lymphoma/genetics*
Fulltext Role of exosomes as a proinflammatory mediator in the development of EBV-associated lymphoma

Higuchi H, Yamakawa N, Imadome KI, Yahata T, Kotaki R, Ogata J, et al.

Blood, 2018 06 07;131(23):2552-2567.
PMID: 29685921 DOI: 10.1182/blood-2017-07-794529

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma such as Hodgkin's lymphoma and diffuse large B-cell lymphoma. Here, we show that EBV acts in trans on noninfected macrophages in the tumor through exosome secretion and augments the development of lymphomas. In a humanized mouse model, the different formation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8) was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity, possibly through the regulation of macrophage phenotype in vivo. Exosomes collected from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAs such as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBV-encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro, BART miRNAs could induce the immune regulatory phenotype in macrophages characterized by the gene expressions of interleukin 10, tumor necrosis factor-α, and arginase 1, suggesting the immune regulatory role of BART miRNAs. The expression level of an EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients with diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV+ B-cell lymphoma.

Matched MeSH terms: Lymphoma/genetics
Rapid detection of prognostically important childhood acute lymphoblastic leukemia chimeric transcripts using multiplex SYBR green real-time reverse transcription PCR

Ibrahim K, Daud SS, Seah YL, Yeoh AE, Ariffin H, Malaysia-Singapore Leukemia Study Group

Ann Clin Lab Sci, 2008;38(4):338-43.
PMID: 18988926

Childhood acute lymphoblastic leukaemia (ALL) is a heterogenous disease in which oncogene fusion transcripts are known to influence the biological behaviour of the different ALL subtypes. Screening for prognostically important transcripts is an important diagnostic step in treatment stratification and prognostication of affected patients. We describe a SYBR-Green real-time multiplex PCR assay to screen for transcripts TEL-AML1, E2A-PBX1, MLL-AF4, and the two breakpoints of BCR-ABL (p190 and p210). Validation of the assay was based on conventional karyotyping results. This new assay provides a rapid, sensitive, and accurate detection method for prognostically important transcripts in childhood ALL.

Matched MeSH terms: Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics*
Fulltext Multiplexed automated digital quantification of fusion transcripts: comparative study with fluorescent in-situ hybridization (FISH) technique in acute leukemia patients

Akhter A, Mughal MK, Elyamany G, Sinclair G, Azma RZ, Masir N, et al.

Diagn Pathol, 2016 Sep 15;11(1):89.
PMID: 27632978 DOI: 10.1186/s13000-016-0541-z

The World Health Organization (WHO) classification system defines recurrent chromosomal translocations as the sole diagnostic and prognostic criteria for acute leukemia (AL). These fusion transcripts are pivotal in the pathogenesis of AL. Clinical laboratories universally employ conventional karyotype/FISH to detect these chromosomal translocations, which is complex, labour intensive and lacks multiplexing capacity. Hence, it is imperative to explore and evaluate some newer automated, cost-efficient multiplexed technologies to accommodate the expanding genetic landscape in AL.

Matched MeSH terms: Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics*
Differential attenuation of Marek's disease virus-induced tumours and late-Marek's disease virus-induced immunosuppression

Faiz NM, Cortes AL, Guy JS, Reddy SM, Gimeno IM

J Gen Virol, 2018 07;99(7):927-936.
PMID: 29767614 DOI: 10.1099/jgv.0.001076

Marek's disease virus (MDV) is a herpesvirus that induces lymphoma and a variety of non-neoplastic syndromes in chickens. Furthermore, very virulent plus (vv+) MDVs induce a form of immunosuppression (late-MDV-IS) that might involve both neoplastic and non-neoplastic mechanisms. The objective of this study was to evaluate whether the attenuation of MDV-induced tumours and late-MDV-IS occurs simultaneously or can be dissociated. The immunosuppressive ability of three viruses derived from vv+ MDV strain 686 (wild-type 686, the somewhat attenuated molecular clone 686-BAC, and the nononcogenic molecular clone lacking the two copies of the oncogene meq 686-BACΔMEQ) was evaluated. Late-MDV-IS was evaluated indirectly by assessing the negative effect of MDV strains on the protection conferred by infectious laryngotracheitis (ILT) vaccines. Our results showed that the ability to induce late-MDV-IS was attenuated before the ability to induce tumours. Strain 686 induced both tumours and late-MDV-IS, 686-BAC induced tumours but did not induce late-MDV-IS and 686-BACΔMEQ did not induce either tumours or late-MDV-IS. Further comparison of strains 686 and 686-BAC revealed that strain 686 reduced the humoral immune responses to ILTV (1132 vs 2167) more severely, showed higher levels of meq transcripts (2.1E+09 vs 4.98E+8) and higher expression of MDV microRNAs (mdv1-miR-M4-5p and mdv1-miR-M2-3p) in the spleen, and further reduced the percentage of CD45+-MHC-I+splenocytes (13 vs32 %) compared to molecular clone 686-BAC. This study suggests that the immunosuppressive ability of MDV might follow a continuous spectrum and only the most virulent MDVs can overcome a certain threshold level and induce clinical MDV-IS in the ILT model.

Matched MeSH terms: Lymphoma/genetics
Contributions of IKZF1, DDC, CDKN2A, CEBPE, and LMO1 Gene Polymorphisms to Acute Lymphoblastic Leukemia in a Yemeni Population

Al-Absi B, Razif MFM, Noor SM, Saif-Ali R, Aqlan M, Salem SD, et al.

Genet Test Mol Biomarkers, 2017 Oct;21(10):592-599.
PMID: 28768142 DOI: 10.1089/gtmb.2017.0084

BACKGROUND: Genome-wide and candidate gene association studies have previously revealed links between a predisposition to acute lymphoblastic leukemia (ALL) and genetic polymorphisms in the following genes: IKZF1 (7p12.2; ID: 10320), DDC (7p12.2; ID: 1644), CDKN2A (9p21.3; ID: 1029), CEBPE (14q11.2; ID: 1053), and LMO1 (11p15; ID: 4004). In this study, we aimed to conduct an investigation into the possible association between polymorphisms in these genes and ALL within a sample of Yemeni children of Arab-Asian descent.
METHODS: Seven single-nucleotide polymorphisms (SNPs) in IKZF1, three SNPs in DDC, two SNPs in CDKN2A, two SNPs in CEBPE, and three SNPs in LMO1 were genotyped in 289 Yemeni children (136 cases and 153 controls), using the nanofluidic Dynamic Array (Fluidigm 192.24 Dynamic Array). Logistic regression analyses were used to estimate ALL risk, and the strength of association was expressed as odds ratios with 95% confidence intervals.
RESULTS: We found that the IKZF1 SNP rs10235796 C allele (p = 0.002), the IKZF1 rs6964969 A>G polymorphism (p = 0.048, GG vs. AA), the CDKN2A rs3731246 G>C polymorphism (p = 0.047, GC+CC vs. GG), and the CDKN2A SNP rs3731246 C allele (p = 0.007) were significantly associated with ALL in Yemenis of Arab-Asian descent. In addition, a borderline association was found between IKZF1 rs4132601 T>G variant and ALL risk. No associations were found between the IKZF1 SNPs (rs11978267; rs7789635), DDC SNPs (rs3779084; rs880028; rs7809758), CDKN2A SNP (rs3731217), the CEBPE SNPs (rs2239633; rs12434881) and LMO1 SNPs (rs442264; rs3794012; rs4237770) with ALL in Yemeni children.
CONCLUSION: The IKZF1 SNPs, rs10235796 and rs6964969, and the CDKN2A SNP rs3731246 (previously unreported) could serve as risk markers for ALL susceptibility in Yemeni children.

Matched MeSH terms: Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics*
Miltefosine suppression of Pten null T-ALL leukemia via β-catenin degradation through inhibition of pT308-Akt and TGFβ1/Smad3

Zhang Y, Lee S, Xu W

Biochem Biophys Res Commun, 2020 04 16;524(4):1018-1024.
PMID: 32063363 DOI: 10.1016/j.bbrc.2020.02.021

Pten deletion in the hematopoietic stem cells (HSC) causes a myeloproliferative disorder, which may subsequently develop into a T-cell acute lymphoblastic leukemia (T-ALL). β-catenin expression was dramatically increased in the c-KitmidCD3+Lin- leukemia stem cells (LSC) and was critical for T-ALL development. Therefore, the inactivation of β-catenin in LSC may have a potential to eliminate the LSC. In this study, we investigated the mechanism of enhancement of the β-catenin expression and subsequently used a drug to inactivate β-catenin expression in T-ALL. Western blot (WB) analysis revealed an increased level of β-catenin in the leukemic cells, but not in the pre-leukemic cells. Furthermore, the WB analysis of the thymic cells from different stages of leukemia development showed that increased expression of β-catenin was not via the pS9-GSK3β signaling, but was dependent on the pT308-Akt activation. Miltefosine (Hexadecylphosphocholine) is the first oral anti-Leishmania drug, which is a phospholipid agent and has been shown to inhibit the PI3K/Akt activity. Treatment of the PtenΔ/Δ leukemic mice with Miltefosine for different durations demonstrated that the pT308-Akt and the β-catenin expressions were inhibited in the leukemia blast cells. Miltefosine treatment also suppressed the TGFβ1/Smad3 signaling pathway. Analysis of TGFβ1 in the sorted subpopulations of the blast cells showed that TGFβ1 was secreted by the CD3+CD4- subpopulation and may exert effects on the subpopulations of both CD3+CD4+ and CD3+CD4- leukemia blast cells. When a TGFβR1 inhibitor, SB431542 was injected into the PtenΔ/Δ leukemic mice, the Smad3 and β-catenin expressions were down-regulated. On the basis of the results, we conclude that Miltefosine can suppress leukemia by degrading β-catenin through repression of the pT308-Akt and TGFβ1/Smad3 signaling pathways. This study demonstrates a possibility to inhibit Pten loss-associated leukemia genesis via targeting Akt and Smad3.

Matched MeSH terms: Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics
Fulltext Expression of p53 N-terminal isoforms in B-cell precursor acute lymphoblastic leukemia and its correlation with clinicopathological profiles

Oh L, Hainaut P, Blanchet S, Ariffin H

BMC Cancer, 2020 Feb 10;20(1):110.
PMID: 32041553 DOI: 10.1186/s12885-020-6599-8

BACKGROUND: TP53 mutations occur in only about 3% of primary and 10-20% of relapse B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). However, alternative mechanisms may contribute to functionally impairing the p53 pathway in the absence of a mutation. Candidate mechanisms include overexpression of p53 mRNA variants encoding either dominant-negative p53 protein isoforms such as Delta40p53 and Delta133p53, or modulatory isoforms such as p53beta, which counteract the effects of Delta133p53 on replicative senescence in T-lymphocytes.
METHODS: We used semi-quantitative reverse-transcriptase PCR (RT-PCR) and Western blot to investigate the expression of full length p53 (TAp53), Delta40p53, Delta133p53 or p53beta in diagnostic marrow from a clinical cohort of 50 BCP-ALL patients without TP53 mutation (29 males and 21 females, age range 2-14 years) and in the bone marrow cells of 4 healthy donors (used as controls).
RESULTS: Irrespective of isoforms, levels of p53 mRNA were low in controls but were increased by 2 to 20-fold in primary or relapse BCP-ALL. TAp53 was increased in primary BCP-ALL, Delta40p53 was elevated in relapse BCP-ALL, whereas Delta133p53 and p53beta were increased in both. Next, mRNA levels were used as a basis to infer the ratio between protein isoform levels. This inference suggested that, in primary BCP-ALL, p53 was predominantly in active oligomeric conformations dominated by TAp53. In contrast, p53 mostly existed in inactive quaternary conformations containing ≥2 Delta40 or Delta133p53 in relapse BCP-ALL. Western blot analysis of blasts from BCP-ALL showed a complex pattern of N-terminally truncated p53 isoforms, whereas TAp53beta was detected as a major isoform. The hypothesis that p53 is in an active form in primary B-ALL was consistent with elevated level of p53 target genes CDKN1A and MDM2 in primary cases, whereas in relapse BCP-ALL, only CDKN1A was increased as compared to controls.
CONCLUSION: Expression of p53 isoforms is deregulated in BCP-ALL in the absence of TP53 mutation, with increased expression of alternative isoforms in relapse BCP-ALL. Variations in isoform expression may contribute to functional deregulation of the p53 pathway in BCP-ALL, specifically contributing to its down-regulation in relapse forms.

Matched MeSH terms: Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics*

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