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FLT3–internal tandem duplications (FLT3-ITDs) are prognostic driver mutations found in acute myeloid leukemia (AML). Although these short duplications occur in 25% of AML patients, little is known about the molecular mechanism underlying their formation. Understanding the origin of FLT3-ITDs would advance our understanding of the genesis of AML. We analyzed the sequence and molecular anatomy of 300 FLT3-ITDs to address this issue, including 114 ITDs with additional nucleotides of unknown origin located between the 2 copies of the repeat. We observed anatomy consistent with replication slippage, but could only identify the germline microhomology (1-6 bp) anticipated to prime such slippage in one-third of FLT3-ITDs. We explain the paradox of the “missing” microhomology in the majority of FLT3-ITDs through occult microhomology: specifically, by priming through use of nontemplated nucleotides (N-nucleotides) added by terminal deoxynucleotidyl transferase (TdT). We suggest that TdT-mediated nucleotide addition in excess of that required for priming creates N-regions at the duplication junctions, explaining the additional nucleotides observed at this position. FLT3-ITD N-regions have a G/C content (66.9%), dinucleotide composition (P < .001), and length characteristics consistent with synthesis by TdT. AML types with high TdT show an increased incidence of FLT3-ITDs (M0; P = .0017). These results point to an unexpected role for the lymphoid enzyme TdT in priming FLT3-ITDs. Although the physiological role of TdT is to increase antigenic diversity through N-nucleotide addition during V(D)J recombination of IG/TCR genes, here we propose that illegitimate TdT activity makes a significant contribution to the genesis of AML.

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML). AML with mutated NPM1 is recognized as a separate entity in the World Health Organization 2016 classification and carries a relatively favorable prognosis. NPM1 mutations are predominantly 4-bp duplications or insertions in the terminal exon that arise through an unknown mechanism. Here we analyze 2430 NPM1 mutations from 2329 adult and 101 pediatric patients to address their origin. We show that NPM1 mutations display the hallmarks of replication slippage, but lack suitable germline microhomology available for priming. Insertion mutations display G/C-rich N-nucleotide tracts, with a significant bias toward polypurine and polypyrimidine stacking (P < .001). These features suggest terminal deoxynucleotidyl transferase (TdT) primes replication slippage through N-nucleotide addition, with longer syntheses manifesting as N-regions. The recurrent type A, type D, and type B mutations require 1, 2, and 3 N-nucleotide extensions of T, CC, and CAT, respectively, with the last nucleotide used as occult microhomology. This TdT-mutator model successfully predicts the relative incidence of the 256 potential 4-bp insertion/duplication mutations at position c.863_864 over 4 orders of magnitude (? = 0.484, P < .0001). Children have a different NPM1 mutation spectrum to adults, including a shift away from type A mutations and toward longer N-regions, consistent with higher TdT activity in pediatric myeloid stem cells. These findings complement our FLT3-ITD data, suggesting illegitimate TdT activity contributes to around one-half of AMLs. AML may therefore reflect the price for adaptive immunity.

FLT3–internal tandem duplications (FLT3-ITDs) are prognostic driver mutations found in acute myeloid leukemia (AML). Although these short duplications occur in 25% of AML patients, little is known about the molecular mechanism underlying their formation. Understanding the origin of FLT3-ITDs would advance our understanding of the genesis of AML. We analyzed the sequence and molecular anatomy of 300 FLT3-ITDs to address this issue, including 114 ITDs with additional nucleotides of unknown origin located between the 2 copies of the repeat. We observed anatomy consistent with replication slippage, but could only identify the germline microhomology (1-6 bp) anticipated to prime such slippage in one-third of FLT3-ITDs. We explain the paradox of the “missing” microhomology in the majority of FLT3-ITDs through occult microhomology: specifically, by priming through use of nontemplated nucleotides (N-nucleotides) added by terminal deoxynucleotidyl transferase (TdT). We suggest that TdT-mediated nucleotide addition in excess of that required for priming creates N-regions at the duplication junctions, explaining the additional nucleotides observed at this position. FLT3-ITD N-regions have a G/C content (66.9%), dinucleotide composition (P< .001), and length characteristics consistent with synthesis by TdT. AML types with high TdT show an increased incidence of FLT3-ITDs (M0; P= .0017). These results point to an unexpected role for the lymphoid enzyme TdT in priming FLT3-ITDs. Although the physiological role of TdT is to increase antigenic diversity through N-nucleotide addition during V(D)J recombination of IG/TCR genes, here we propose that illegitimate TdT activity makes a significant contribution to the genesis of AML.

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML). AML with mutated NPM1is recognized as a separate entity in the World Health Organization 2016 classification and carries a relatively favorable prognosis. NPM1mutations are predominantly 4-bp duplications or insertions in the terminal exon that arise through an unknown mechanism. Here we analyze 2430 NPM1mutations from 2329 adult and 101 pediatric patients to address their origin. We show that NPM1mutations display the hallmarks of replication slippage, but lack suitable germline microhomology available for priming. Insertion mutations display G/C-rich N-nucleotide tracts, with a significant bias toward polypurine and polypyrimidine stacking (P< .001). These features suggest terminal deoxynucleotidyl transferase (TdT) primes replication slippage through N-nucleotide addition, with longer syntheses manifesting as N-regions. The recurrent type A, type D, and type B mutations require 1, 2, and 3 N-nucleotide extensions of T, CC, and CAT, respectively, with the last nucleotide used as occult microhomology. This TdT-mutator model successfully predicts the relative incidence of the 256 potential 4-bp insertion/duplication mutations at position c.863_864 over 4 orders of magnitude (ρ = 0.484, P< .0001). Children have a different NPM1mutation spectrum to adults, including a shift away from type A mutations and toward longer N-regions, consistent with higher TdT activity in pediatric myeloid stem cells. These findings complement our FLT3-ITD data, suggesting illegitimate TdT activity contributes to around one-half of AMLs. AML may therefore reflect the price for adaptive immunity.

Acute myeloid leukemia (AML) is characterized by recurrent mutations that affect the epigenetic regulatory machinery and signaling molecules, leading to a block in hematopoietic differentiation. Constitutive signaling from mutated growth factor receptors is a major driver of leukemic growth, but how aberrant signaling affects the epigenome in AML is less understood. Furthermore, AML cells undergo extensive clonal evolution, and the mutations in signaling genes are often secondary events. To elucidate how chronic growth factor signaling alters the transcriptional network in AML, we performed a system-wide multi-omics study of primary cells from patients suffering from AML with internal tandem duplications in the FLT3 transmembrane domain (FLT3-ITD). This strategy revealed cooperation between the MAP kinase (MAPK) inducible transcription factor AP-1 and RUNX1 as a major driver of a common, FLT3-ITD-specific gene expression and chromatin signature, demonstrating a major impact of MAPK signaling pathways in shaping the epigenome of FLT3-ITD AML.

Objective It is debatable whether BCR-ABL1 transcript type has an impact on outcome of treatment of patients with CML, and it is not widely studied whether body weight influences response to treatment. In this study, we tried to find out if any of these factors has an impact on response to treatment and outcome.Methodology We conducted a retrospective analysis of the files of 79 patients being treated in our center for CML with known BCR-ABL1 breakpoints, and patients’ management and response assessment was done based on ELN 2013 guidelines. The analysis was performed based on two main groups, obese vs. normal BMI, and then based on BCR-ABL1 transcripts: e13a2 vs. e14a2. Cumulative incidence of MMR, CCyR, and DMR were estimated using the Kaplan–Meier survival curve method, and comparisons between groups were performed by the Log-rank/Gray test methods.Results/conclusion In the patient-cohort studied, there was no statistically significant difference in molecular response between patients with CML based on body weight or transcript type although patients in the obesity group achieved higher and faster MMR with no statistical significance.

The overall survival of patients with Chronic Myeloid Leukemia (CML) treated by using tyrosine kinase inhibitors (TKIs) is very close to that of the healthy population. However, little is known about the effect of specific measures such as intermittent fasting, especially during Ramadan period. A 3-year retrospective study was conducted to evaluate the effect of fasting on patients with CML receiving TKIs by evaluating certain clinical, hematological, and molecular parameters. A total of 49 patients were eligible, with a median age of 46 years (range: 22-86), of these 36 (73.5%) were males and 13 (26.5%) were females. Twenty-seven (55%) patients are Middle Eastern, while 16 (32.7%) from the Indian subcontinent, and 6 (12.3%) Africans. Imatinib was the most common TKI; used in 25 patients (51%). The mean White blood cells (WBCs), neutrophils, and BCR-ABL were found to be reduced after fasting compared to before and during with statistical difference. The use of TKIs while fasting did not result in significant changes in hematological nor BCR-ABL levels in our study. Patients who wish to practice intermittent fasting may be reassured in this regard, yet physicians can adopt the safe trial approach, where they allow the patients to fast, but with instructions such as when to break fasting.

Background

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Background and Objectives:

Background

Introduction:

Introduction

According to World Health Organization (WHO) classification (2008), B-cell neoplasms are classified into precursor B-cell or a mature B-cell phenotype and this classification was also kept in the latest WHO revision (2016). We are reporting a male patient in his fifties, with tonsillar swelling diagnosed as diffuse large B-cell lymphoma (DLBCL), germinal center. He received 6 cycles of RCHOP and showed complete metabolic response. Two months later, he presented with severe CNS symptoms. Flow cytometry on bone marrow (BM) showed infiltration by CD10-positive Kappa-restricted B-cells with loss of CD20 and CD19, and downregulation of CD79b. Moreover, the malignant population showed Tdt expression. BM Cytogenetics revealed t(8;14)(q24;q32) within a complex karyotype. Retrospectively, MYC and Tdt immunostains performed on original diagnostic tissue and came negative for Tdt and positive for MYC. It has been rarely reported that mature B-cell neoplasms present with features of immaturity; however the significance of Tdt acquisition during disease course was not addressed before. What is unique in this case is that the emerging disease has acquired an immaturity marker while retaining some features of the original mature clone. No definitive WHO category would adopt high-grade neoplasms that exhibit significant overlapping features between mature and immature phenotypes.

Griffiths: Affymetrix: Research Funding. Apperley:Bristol Myers Squibb: Honoraria, Speakers Bureau; Incyte: Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Ariad: Honoraria, Speakers Bureau.

BACKGROUND. Tyrosine Kinase Inhibitors (TKIs) are part of the successful clinical management of patients with Chronic Myeloid Leukaemia (CML). However, optimal clinical management of CML requires a robust, standardized laboratory assay used at key clinical milestones to ensure a successful outcome for patients on TKIs. Quantitative monitoring of %BCR-ABL1ISby reverse transcription quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to therapy and classification into prognostic subgroups. However, it can be challenging to perform in a reproducible manner. Reverse-Transcription Digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for patient stratification.

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FLT3 internal tandem duplication (itd) mutations are found in 25% of adult patients with acute myeloid leukaemia (AML) and are associated with an adverse prognosis. This mutation results in constitutive activation of downstream pathways.Distinct biological subgroups can be identified based on FLT3-itd mutation type: clones with heterozygous FLT3-itd mutated/wildtype; homozygous mutated FLT3 allele; FLT3 heterozygous biallelic mutant and clones with evidence of overexpression of FLT3-itd. There is evidence to suggest that these mechanisms are important in the clonal evolution of AML cells. We sought to investigate their clinical impact.Itd mutation analysis within exon 14 and/or exon 15 of the FLT3 gene was carried out at diagnosis by PCR of genomic DNA (gDNA) and cDNA for all new AML referrals in the region over 8 years. PCR products were identified and sized using fluorescent based fragment analysis. Allelic ratio (AR) and expression ratio (ER) (mutation: wild type ratio in gDNA and cDNA respectively) was determined from the relative peak heights. High relative expression was defined as 10 fold difference of ER/AR. Copy neutral loss of heterozygosity for the wild type allele resulting in homozygosity of the FLT3-itd mutation (acquired isodisomy (AID)) was determined by analysis of microsatellite markers along chromosome 13. Overall survival (OS) (time of diagnosis to death), event free survival (EFS) (time from diagnosis to induction failure, relapse or death) was calculated. Survival rates were estimated by the Kaplan-Meier method. Differences between the survival distributions were compared with the log-rank test.177 patients positive for the FLT3-itd mutation were identified. Median follow-up for patients alive was 3.4 years. A separate group of 49 patients tested negative for this mutation during this period had better OS and EFS (p=0.02) compared to the patients who were FLT3-itd positive (median survival 1715 and 307 days respectively).Patients who were FLT3-itd positive had statistically significant (p<0.05) differences in outcomes based on age, presenting white cell count, treatment intensity and cytogenetic risk. The characteristics of this group of patients are described below.Patients with lower AR (less than/equal to 0.3) as compared to higher AR (greater than 0.3) had an improved OS (p=0.018) and EFS (p=0.02). The impact of AR on OS had borderline significance (p=0.05) when only patients treated with intensive chemotherapy were considered.AID provides true evidence of FLT3 mutant homozygosity and was detected in 15 (142 tested) patients. In 38 patients who relapsed and had samples at these stages, 5 had developed AID, but were heterozygous (mutant/wildtype) at diagnosis. 6 patients with multiple FLT3-itd products may comprise patients who have multiple different mutant/wildtype clones but may include patients with a second, independent FLT3-itd mutation resulting in biallelic heterozygous mutations, although this cannot be confirmed. A high relative expression level of FLT3-itd was seen in 12 patients. The clinical significance of these findings is uncertain due to the small numbers.The impact of FLT3-itd mutation and other known prognostic factors has been confirmed in a heterogeneous, real life cohort of patients.An AR over 1 provides firm evidence of loss of the wild type allele (9 patients). AID also occurs at an AR of less than 1 because of the presence of normal cells or due to preferential amplification of the wildtype allele. Direct testing for AID is a more sensitive measure of FLT3 mutant homozygosity (detected in 14% of tested patients). The development of AID in patients who relapse may be an important mechanism by which an AML clone gains a further advantage.Lower AR was associated with improved survival. In the context of a high blast count (median bone marrow blast 80%), this implies having a small sub-clone of FLT3-itd positive cells is advantageous to having a larger FLT3-itd clone in their population of AML cells.No relevant conflicts of interest to declare.

Abstract 1460