Leukemic DNA Research Articles

Minimal Residual Disease Detection Using Gene Scanning Analysis, Fluorescent Fragment Analysis, and Capillary Electrophoresis for IgH Rearrangement in Adult B-Lineage Acute Lymphoblastic Leukemia: A Cross-Sectional Study.

Minimal residual disease (MRD) is considered the greatest prognostic factor in acute lymphoblastic leukemia (ALL). MRD is a valuable tool for anticipating impending relapse and treatment response assessment. The objective of the present study was to investigate whether the detection of IgH gene rearrangement using polymerase chain reaction (PCR)-based GeneScan analysis could be a complementary method to monitor MRD along with the quantitative realtime PCR (qPCR). In this cross-sectional study, we valued the MRD levels, based on the GeneScanning analysis (GSA), and then compared the data with quantitative real-time polymerase chain reaction at different time points in peripheral blood (PB) samples of adult B-lineage ALL patients (n=35). The specific polymerase chain reaction (PCR) primers for IGH gene FR-1 and fluorescence-labeled J-primer were used and analyzed by capillary gel electrophoresis on a sequencer. The results of this study were compared with the previously reported MRD results obtained by the IGH rearrangements allele-specific oligonucleotide (ASO) -qPCR methods. The total concordance rate was 86.7%, with a P<0.001. MRD results obtained by GSA and ASO-qPCR methods were concordant in all diagnostic samples and samples on the 14th and 28th days of induction therapy. The results of these 2.5 years' follow-ups demonstrated a significant correlation between the two techniques (r=0.892, P<0.001). It seems that the PCR-based GeneScan analysis of IGH gene rearrangement detection may be a valuable molecular technique to distinguish monoclonality from polyclonality. And also, it may be a precise tool to detect the residual leukemic DNA in the PB follow-up samples of patients.

Immunoglobulin Gene Mutational Status Assessment by Next Generation Sequencing in Chronic Lymphocytic Leukemia.

B cell receptor (BcR) immunoglobulins (IG) display a tremendous diversity due to complex DNA rearrangements, the V(D)J recombination, further enhanced by the somatic hypermutation process. In chronic lymphocytic leukemia (CLL), the mutational load of the clonal BcR IG expressed by the leukemic cells constitutes an important prognostic and predictive biomarker. Here, we provide a reliable methodology capable of determining the mutational status of IG genes in CLL using high-throughput sequencing, starting from leukemic cell DNA or RNA.

DNA-Based Methods for Measurable Residual Disease Detection in NPM1-Mutated Acute Myeloid Leukemia; Establishment of Cut-Offs for qPCR, Digital Droplet PCR and Targeted Deep Sequencing

For detection of measurable residual disease (MRD) in acute myeloid leukemia with NPM1 mutations, RT-qPCR with quantification of leukemic transcripts is currently considered the method of choice; however, MRD can also be determined with DNA-based methods, offering certain advantages. For example, digital droplet PCR (ddPCR) and targeted deep sequencing (deep seq) do neither require standard curves nor reference genes and are thus less labor intense than RT-qPCR. Also, deep seq allows for quantification independently of type of NPM1 mutation. In addition, DNA-based techniques enable MRD assessment of other mutations, beyond the reach of RT-qPCR, which is limited to analyses of highly expressed genes or fusion transcripts (e.g. core binding factor leukemias). With the rapid development of highly sensitive DNA-based techniques for MRD detection, there is a need to establish clinically relevant cut-offs for accurate interpretation of MRD results and risk stratification. Here, we compare and provide MRD cut-offs for three different DNA-based MRD methods for NPM1 mutations: quantitative PCR (qPCR), ddPCR and deep seq. To compare the DNA-based methods with RT-qPCR, we analyzed 110 follow-up peripheral blood (PB) or bone marrow (BM) samples from 32 AML patients harboring NPM1 mutation type A. First, we compared the mere detectability of leukemic signals (without reference to specific MRD cut-off points). We found a high correlation between results from RT-qPCR and the three DNA-based methods (Rs=0.936 for RT-qPCR vs qPCR, Rs=0.774 for RT-qPCR vs ddPCR and Rs=0.743 for RT-qPCR vs deep seq, p&amp;lt;0.001). As expected, RT-qPCR was the most sensitive method. Among the DNA-based methods, qPCR was the most sensitive, detecting leukemic DNA in 95% (55/58) of the RT-qPCR positive samples, compared to 72% (42/58) and 62% (36/58) for ddPCR and deep seq, respectively. Interestingly, the transcript level for a given amount of measurable leukemic DNA (RNA copy number per leukemic DNA molecule) fluctuated considerably between different follow-up samples for certain patients. In some cases, the RNA/DNA ratio exceeded a hundredfold difference between different follow-up time points in both PB and BM. Hence, transcript analysis may be more complex than just a simple measurement of leukemic cell burden, which in turn may influence accurate risk stratification and treatment decisions, if relying on RT-qPCR measurements alone. To select adequate DNA MRD cut-offs, we performed ROC curve analyses for each method at various DNA cut-offs, comparing them with the gold standard RT-qPCR cut-off. In BM, this cut-off can be defined as a less than 3 log reduction of mutated NPM1 transcripts vs diagnosis, separating MRDhigh from MRDlow/undetectable (sometimes inaccurately termed "MRD-positivity" and "MRD-negativity", respectively). In PB, the mere detectability of mutated NPM1 transcripts is considered the relevant cut-off. DNA cut-offs were chosen based on the area under the curve (AUC) for the ROC analyses (Table 1), and influenced by available literature including recommendations of prognostically relevant MRD levels. For qPCR, a cut-off at 0.1% leukemic DNA was judged relevant in BM. For ddPCR and deep seq, 0.05% was chosen to adjust for measuring allelic ratio (variant allele frequency (VAF)) rather than mutant DNA alone. In PB, the selected cut-off was detectable leukemic signal irrespective of DNA method. We next determined the accuracy of the selected cut-offs, for identification of samples with clinically relevant MRD, by comparing them with the gold standard RT-qPCR. In general, the selected DNA cut-off values generated high specificity as well as high positive and negative predictive values (Table 1). The vast majority of all MRD analyses (93% (368/395)) showed concordant results irrespective of MRD method. In BM samples, MRD assessment by the DNA based methods agreed with MRD status as determined by RT-qPCR (MRDhigh high vs MRDlow/undetectable) in 93% (62/67) of the analyses for qPCR, 96% (64/67) for ddPCR, and 97% (65/67) for deep seq. In PB, the agreement was 95% (41/43), 88% (38/43) and 86% (37/43), respectively. In summary, we found strong agreement between different MRD methods and based on this could provide clinically relevant cut-offs for risk stratification. Thus, in BM follow-up samples from AML patients with NPM1 mutation, we propose 0.1% leukemic DNA as cut-off for qPCR and 0.05% VAF for ddPCR and deep seq. Disclosures No relevant conflicts of interest to declare.

Successful Therapy Reduction in NCI Standard Risk (SR) Highly Favorable Subgroup of Childhood Acute Lymphoblastic Leukemia

Background: Acute Lymphoblastic Leukemia (ALL) is the most common cancer in childhood; it is curable in 80-90% of patients. Current efforts focus on therapy guided by sensitive and specific methods of minimal residual disease (MRD) monitoring, tailoring the type and intensity of chemotherapeutic agents to improve cure rates, quality of life and decrease morbidity. We sought to study the outcome of low risk (LR) ALL treated per modified St Jude total XV Study. Methods: We adopted the modified St Jude total XV study ,B-cell precursor patients with age between 1 and 10 years and presenting leukocyte count &amp;lt;50×10^9/L, leukemic cell DNA index ≥1.16, or TEL-AML1 fusion are classified to have low-risk ALL, provided that they do not have testicular or central-nervous-system (CNS-3) leukemia, hypodiploidy (&amp;lt;45 chromosomes), E2A-PBX1 fusion, or MLL rearrangement, and have a treatment response at day 15 by minimal residual disease (MRD) of &amp;lt;5% and at day 46 of remission-induction &amp;lt;0.01%. We further identified a subgroup of (LR) ALL; defined by presenting age between 2 and 6 years, leukocyte count of &amp;lt;10x 10^9/L and MRD of &amp;lt;0.01% at day 15 of remission induction as Very Low Risk ALL (VLR). Patients with St Jude Low Risk (LR) ALL were treated per St Jude total XV study with several modifications: On 2007; doxorubicin was omitted from reinduction II to lower the total cumulative dose of anthracycline to 80 mg/m2, pulses of dexamethasone and vincristine were stopped at week 72 of maintenance instead of 100. On 2012; for patients identified as VLR; pulses of vincristine /dexamethasone were stopped at week 52 and HDMTX given at week 10 and 12 of maintenance therapy was omitted, frequency of triple Intrathecal chemotherapy remained unchanged (13-18) for LR ALL based on CNS status. Results: We reviewed 184 patients with (LR) between May 2006 to Feb 2018, (the median age at diagnosis, 7.3 years; range, 1.5-9.8). Eighty-two patients (45%) and 102 patients (55%) were treated as VLR and LR ALL respectively. The only significant difference in patients of two groups were age at diagnosis 2-6 years(VLR, 100%; LR, 46%; p&amp;lt;0.001), initial count &amp;lt;10x 10^9/L (VLR, 100%; LR, 65%; p&amp;lt;0.001) and MRD at day 15&amp;lt;0.01%(VLR, 100%; LR, 61%; p&amp;lt;0.001). At median follow up of (48 months; range, 17 to 157), in the subgroups of NCI standard risk defined above; as very low risk(VLR) ALL and low risk (LR) ALL, the 5-year EFS were 92%±3.4; 85%±4 (p=0.12), and the 5-year OS were 98%±1.7; 93%±3.3 (p=0.2) respectively. Conclusion: Chemotherapy was substantially reduced safely in subgroup of children with low risk ALL who were selected on the basis of presenting age, leukocyte count and undetectable MRD levels early at day 15 and at day 46 of remission induction without jeopardizing the survival rate. Patients with St Jude LR ALL maintained outstanding outcomes despite reduction of chemotherapy. Disclosures No relevant conflicts of interest to declare.

PS1040 DETECTION OF MRD MAY PREDICT THE OUTCOME OF PATIENTS WITH ACUTE MYELOID LEUKEMIA WITH BIALLELIC CEBPA MUTATIONS

Background: The somatic mutations that drive acute myeloid leukaemia (AML) are highly heterogenous. Identifying the mutations that drive individual cases can help determine patient prognosis and therapy. For this reason, genetic testing for prognostically important mutations in leukaemic DNA is now routine in many diagnostic laboratories. Also, the analysis of gene-expression profiles from AML RNA can provide additional clinically useful information that cannot be inferred from DNA sequencing. Whilst it is both expensive and impractical to carry out both types of sequencing and analyses, we hypothesised that the two could be combined more cheaply and effectively if the presence of mutations in prognostically important gene mutations could be identified from RNA-seq data. However, computational methodologies for robust detection of the diverse types of somatic mutations found in AML such as substitutions, indels, tandem duplications and translocations, are not currently available. Aims: To develop an stand-alone, lightweight and use-friendly software for the identification of clinically relevant mutations from AML RNA-seq data. Methods: To ensure efficient mapping of RNA-seq reads, we hash-indexed the DNA sequences of target genes using 10-mer sliding windows and implemented the ”seed and extend“ algorithm for read alignment. Point mutations and small indels were detected from reads with imperfect alignments and tandem duplications were detected from reads spanning the duplication junction. Translocations were detected from reads whose ends belonged to preselected fusion partner genes. Results: To benchmark our software we used RNA-seq data from 151 whole-exome/genome-sequenced AML samples studied by The Cancer Genome Atlas Research Network. We show that our software reliably calls clinically important mutations affecting the genes NPM1 (4-nt insertion), FLT3 (substitutions and internal tandem duplications, ITD), MLL partial tandem duplications (PTD), as well as substitutions in CEBPA, IDH1/2, TP53 and RUNX1. Furthermore, we identified gene fusions including PML-RARA, MYH11-CBFB, RUNX1-RUNX1T1, BCR-ABL1 and NUP98-NSD1. Our software is fast and memory efficient and is able to identify the above mutations in less than 20 minutes starting with RNA-seq FastQ files of 100 million 50 bp paired-end reads, using a standard modern laptop computer. In addition, the software operates through a graphical user interface making it accessible to users without programming knowledge. Summary/Conclusion: We demonstrated that clinically important somatic mutations that drives AML can be reliably detected from RNA-seq data alone using our software. As our approach can be readily combined with conventional gene expression analyses of the same RNA-seq dataset, it can be used to generate data with enhanced clinical utility that can improve prognostication and guide patient treatment. As RNA sequencing is a straightforward procedure, our approach can readily enter clinical laboratories, where it can significantly reduce experimental costs and accelerate diagnostic work-ups.

PS1039 ROBUST IDENTIFICATION OF CLINICALLY RELEVANT MUTATIONS IN AML USING RNA‐SEQ

Background:The somatic mutations that drive acute myeloid leukaemia (AML) are highly heterogenous. Identifying the mutations that drive individual cases can help determine patient prognosis and therapy. For this reason, genetic testing for prognostically important mutations in leukaemic DNA is now routine in many diagnostic laboratories. Also, the analysis of gene‐expression profiles from AML RNA can provide additional clinically useful information that cannot be inferred from DNA sequencing. Whilst it is both expensive and impractical to carry out both types of sequencing and analyses, we hypothesised that the two could be combined more cheaply and effectively if the presence of mutations in prognostically important gene mutations could be identified from RNA‐seq data. However, computational methodologies for robust detection of the diverse types of somatic mutations found in AML such as substitutions, indels, tandem duplications and translocations, are not currently available.Aims:To develop an stand‐alone, lightweight and use‐friendly software for the identification of clinically relevant mutations from AML RNA‐seq data.Methods:To ensure efficient mapping of RNA‐seq reads, we hash‐indexed the DNA sequences of target genes using 10‐mer sliding windows and implemented the ”seed and extend“ algorithm for read alignment. Point mutations and small indels were detected from reads with imperfect alignments and tandem duplications were detected from reads spanning the duplication junction. Translocations were detected from reads whose ends belonged to preselected fusion partner genes.Results:To benchmark our software we used RNA‐seq data from 151 whole‐exome/genome‐sequenced AML samples studied by The Cancer Genome Atlas Research Network. We show that our software reliably calls clinically important mutations affecting the genes NPM1 (4‐nt insertion), FLT3 (substitutions and internal tandem duplications, ITD), MLL partial tandem duplications (PTD), as well as substitutions in CEBPA, IDH1/2, TP53 and RUNX1. Furthermore, we identified gene fusions including PML‐RARA, MYH11‐CBFB, RUNX1‐RUNX1T1, BCR‐ABL1 and NUP98‐NSD1. Our software is fast and memory efficient and is able to identify the above mutations in less than 20 minutes starting with RNA‐seq FastQ files of 100 million 50 bp paired‐end reads, using a standard modern laptop computer. In addition, the software operates through a graphical user interface making it accessible to users without programming knowledge.Summary/Conclusion:We demonstrated that clinically important somatic mutations that drives AML can be reliably detected from RNA‐seq data alone using our software. As our approach can be readily combined with conventional gene expression analyses of the same RNA‐seq dataset, it can be used to generate data with enhanced clinical utility that can improve prognostication and guide patient treatment. As RNA sequencing is a straightforward procedure, our approach can readily enter clinical laboratories, where it can significantly reduce experimental costs and accelerate diagnostic work‐ups.

Identification of a Novel Mutation Predisposing to Familial AML and MDS Syndrome By a NGS Approach

IntroductionIn AML and MDS cases, the genetic lesions inherited or acquired by the hematopoietic stem cells are considered as starting events. Familial AML and MDS, recently recognized in the revised WHO classification (2016) provide a useful model for investigation of predisposing genetic mutations. Genetic analysis of several pure familial leukemia pedigrees led to the discovery of well defined syndromes associated with inherited de novo mutations on germline DNA. Growing clinical awareness as well as a widespread use of NGS have led to an enlarged description of familial MDS/AML cases, and the number of mutations involved, suggesting they are more frequent than those previously recognized. Despite the recent discovery of well-established causative gene mutations (RUNX1, GATA2, ETV6, TERT, TERC, SRP72, ANKRD26, DDX41, CEBPA), many cases remain unexplained (about 80%), suggesting that other inherited mutations could predispose to MDS/AML. It is expected that new sequencing approaches will help to the identification of more cases, more genes as well as novel syndromes.In 2017, we started a multicentric prospective study (Clinical trial.gov NCT03058588) aiming to look for predisposing mutations in patients and relatives affected by Familial AML and MDS syndromes (FAMS) by NGS and to screen for old and new mutations potentially associated with the disease.MethodsAt present, 12 AML/MDS patients have been enrolled. Leukemic (bone marrow) and germline (buccal swab) DNA were analyzed by NGS gene panel approach based on a 28 genes associated to myeloid leukemogenesis, including the 9 above mentioned genes associated to FAMS. NGS libraries were performed by a Nimblegen (Roche) custom panel based on gene capture strategy and the sequencing was performed by MiSeq (Illumina).ResultsTen patients did not reveal any germline mutations and the candidates are undergoing to whole exome sequencing. One presented a germline mutation on RUNX1, and the analysis of the affected relatives is on going. One revealed a new mutation. She was a 70 years old woman affected by RARS and her pedigree was characterized by 9 relatives affected by hematologic and solid neoplasia and trombocytopenia (fig 1).The NGS analysis revealed the mutation c.*514C>T in 3'UTR of ETV6 with VAF of 50% on tumor DNA. The variant has never been described before, while ETV6 has been already associated with FAMS. Sanger sequencing confirmed the mutation on the germline DNA in heterozygosis. The screening of 2 affected relatives still alive confirmed the presence of the variant in heterozygosis. In silico analysis performed on PolymiRST Database revealed that c.*514C>T in 3'UTR of ETV6 results in a gain of miRNA binding site: hsa-miR- 4717-3p and hsa-miR- 942-3p.DiscussionThe variant c.*514C>T in 3'UTR of ETV6 seems to repress ETV6 due to RNA interference. The new binding miRNAs have been already described as over-expressed in solid and hematologic tumors. Moreover, the down-regulation of ETV6 is associated with alteration of cell growth and hematopoiesis. Due to these evidences, c.*514C>T in 3'UTR of ETV6 could be considered as a new mutation involved in FAMS predisposition. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High-Annealing-Temperature PCR (HAT-PCR) Enables Sensitive Quantification of Minimal Residual Disease (MRD) in Blood in Acute Lymphoblastic Leukaemia (ALL)

Measurement of MRD in samples of marrow is widely used for managing patients with ALL but the clinical utility of current methods is limited by their lack of sensitivity. The limit of detection of current quantification by PCR or flow cytometry is 10-4 - 10-5 and quantification is less precise at lower levels. Improving the sensitivity of MRD assays could enable use of peripheral blood instead of marrow, facilitate decisions on whether to change therapy and improve assessment of novel therapies.We have developed a new, highly specific and hence highly sensitive PCR method to quantify MRD in ALL. Blood and marrow samples were obtained at diagnosis and during treatment from adolescents and young adults aged 15 - 40 who entered study ALL06 of the Australasian Leukaemia and Lymphoma Study Group. The Sutton laboratory measured MRD in 1.5 µg of marrow DNA using RQ-PCR according to Euro MRD guidelines and provided the sequences for the VDJ regions of the rearranged immunoglobulin heavy chain (IGH) and T-cell receptor β-chain (TRB) genes. The Morley laboratory developed HAT-PCR and used it to quantify the IGH or TRB rearrangement in blood samples. The forward PCR primer was directed to the VDJ region, had a Tm of 69 -75 oC, and the sequence was designed to minimise non-specific priming. The reverse primer was directed to the J region and was also designed to minimise non-specific priming. The PCR was performed using an annealing temperature of 72 - 77 oC. Specificity for each primer was tested using at least 20 µg of non-leukemic template DNA. Quantification of patient samples was performed using up to 30 ug of template DNA, the amount being determined by a preliminary assay and by the amount of DNA available.Using DNA from 14 patients, the accuracy of HAT-PCR was assessed by assaying tenfold dilutions, between 10-3 and 10-6 , of each leukemic DNA in nonleukemic DNA. The correlation between observed and expected values was 0.98 and the regression line had a slope of 1.00.The precision of HAT-PCR was measured by using DNA from 10 patients, making multiple dilutions in nonleukemic DNA, assaying up to 30 µg of DNA from each dilution twice, on 2 separate days, and calculating the standard deviation (SD) of the assay. For 69 paired assays the SD ranged from 0.05 logs at a dilution of 10-3 to 0.31 logs at a dilution of less than 10-6. The increase in SD at low levels reflects the stochastic Poisson effect observed when the number of target molecules is low.Primers were designed for 47 patients. Non-specificity was not seen with primers for 43 patients, a negative finding which indicated that non-specificity was below 3.35 X 10-7. Non-specificity was seen with primers for 4 patients, with levels corresponding to 3.35 X 10-7, 6.70 X 10-7, 6.70 X 10-7 and 4.36 x 10-6. The rearrangement in all 4 was a partial DNJ rearrangement with only 3 or 4 bases in the N region. To date,141 paired blood and marrow samples from 40 patients with ALL have been studied. Results are shown in the Figure. HAT-PCR quantified MRD down to 3.35 X 10-7 and for IGH rearrangements there was good correlation between levels in blood and marrow. Much of the variation between blood and marrow is likely to be due to the sampling error of marrow aspiration. For IGH rearrangements, the mean ratio between marrow and blood levels was 20 but, as marrow assays were done on separated mononuclear cells whereas blood assays were done on total cells, the true ratio is probably closer to 10. The greater sensitivity of HAT-PCR appeared to compensate for the lower levels in blood and, in quantitative terms, assay of blood appeared to be non-inferior to assay of marrow. Data on patient outcomes are not yet available.ConclusionsMeasurement of MRD by HAT-PCR is approximately 2 logs more sensitive than measurement by conventional RQ-PCR or flow cytometry.When applied to marrow, HAT-PCR has the potential to improve assay precision for some patients with currently detectable MRD and to detect and quantify MRD levels in some patients with currently undetectable MRD. These features should facilitate identification of patients who are candidates for intensification or reduction of therapy and should improve assessment of new therapies.When applied to blood, HAT-PCR has the potential to predict patient outcome and to follow patients to detect impending relapse.HAT-PCR should be applicable to quantification of MRD in other lymphoproliferative disorders. [Display omitted] DisclosuresMorley:Monoquant P/L: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Latham:Monoquant P/L: Equity Ownership, Research Funding. Hughes:Monoquant P/L: Equity Ownership, Research Funding. Budgen:Monoquant P/L: Equity Ownership, Research Funding.

Integration of Next-Generation Sequencing to Treat Acute Lymphoblastic Leukemia with Targetable Lesions: The St. Jude Children's Research Hospital Approach.

Acute lymphoblastic leukemia (ALL) is the most common type of cancer in children. In recent Total Therapy studies conducted at St. Jude Children’s Research Hospital, children with ALL had a 5-year overall survival of around 94%. This is the result of a combination of risk stratification based on the biological features of the leukemic cells and the response to treatment (as assessed by the detection of minimal residual disease), treatment modification based on pharmacodynamic and pharmacogenomic data, and improved supportive care. However, innovative approaches are required to further improve survival to as close to 100% as possible and to reduce the adverse effects of treatment. Next-generation sequencing of leukemic cell DNA and RNA, as well as of germline DNA, can identify submicroscopic genetic structural changes and sequence alterations that contribute to leukemogenesis. Next-generation sequencing data can be used to define new ALL subtypes, to help improve treatment response and reduce adverse effects, and to identify novel prognostic markers and therapeutic targets to facilitate personalized precision medicine. In this article, we describe our approach to detecting targetable lesions in patients with ALL by next-generation sequencing and explain how we integrate the sequencing data into the treatment of these patients.

Epigenetic Identity in AML Depends on Disruption of Nonpromoter Regulatory Elements and Is Affected by Antagonistic Effects of Mutations in Epigenetic Modifiers.

We performed cytosine methylation sequencing on genetically diverse patients with acute myeloid leukemia (AML) and found leukemic DNA methylation patterning is primarily driven by nonpromoter regulatory elements and CpG shores. Enhancers displayed stronger differential methylation than promoters, consisting predominantly of hypomethylation. AMLs with dominant hypermethylation featured greater epigenetic disruption of promoters, whereas those with dominant hypomethylation displayed greater disruption of distal and intronic regions. Mutations in IDH and DNMT3A had opposing and mutually exclusive effects on the epigenome. Notably, co-occurrence of both mutations resulted in epigenetic antagonism, with most CpGs affected by either mutation alone no longer affected in double-mutant AMLs. Importantly, this epigenetic antagonism precedes malignant transformation and can be observed in preleukemic LSK cells from Idh2R140Q or Dnmt3aR882H single-mutant and Idh2R140Q/Dnmt3aR882H double-mutant mice. Notably, IDH/DNMT3A double-mutant AMLs manifested upregulation of a RAS signaling signature and displayed unique sensitivity to MEK inhibition ex vivo as compared with AMLs with either single mutation.Significance: AML is biologically heterogeneous with subtypes characterized by specific genetic and epigenetic abnormalities. Comprehensive DNA methylation profiling revealed that differential methylation of nonpromoter regulatory elements is a driver of epigenetic identity, that gene mutations can be context-dependent, and that co-occurrence of mutations in epigenetic modifiers can result in epigenetic antagonism. Cancer Discov; 7(8); 868-83. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 783.

Human AML-iPSCs Reacquire Leukemic Properties after Differentiation and Model Clonal Variation of Disease

Understanding the relative contributions of genetic and epigenetic abnormalities to acute myeloid leukemia (AML) should assist integrated design of targeted therapies. In this study, we generated induced pluripotent stem cells (iPSCs) from AML patient samples harboring MLL rearrangements and found that they retained leukemic mutations but reset leukemic DNA methylation/gene expression patterns. AML-iPSCs lacked leukemic potential, but when differentiated into hematopoietic cells, they reacquired the ability to give rise to leukemia invivo and reestablished leukemic DNA methylation/gene expression patterns, including an aberrant MLL signature. Epigenetic reprogramming was therefore not sufficient to eliminate leukemic behavior. This approach also allowed us to study the properties of distinct AML subclones, including differential drug susceptibilities of KRAS mutant and wild-type cells, and predict relapse based on increased cytarabine resistance of a KRAS wild-type subclone. Overall, our findings illustrate the value of AML-iPSCs for investigating the mechanistic basis and clonal properties of human AML.

5-Azacytidine Reduces Leukemic Burden in a Xenograft Model of Juvenile Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative disorder of early childhood with often fatal outcome. Despite many attempts to develop alternative treatment options allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative modality. In the past our group has linked the prognosis of JMML to differential DNA methylation patterns (Olk-Batz, Blood 2011;117:4871-80 and Poetsch, Epigenetics 2014;9:1252-60), suggesting a key role of epigenetic modifications in JMML pathophysiology.To overcome the lack of suitable preclinical JMML research models we have developed an ex vivo JMML xenotransplantation system using neonatal Rag2-/- gamma-c-/- mice. Transplantation of 1x106 primary JMML cells resulted in stable xenologous engraftment and reproduced a characteristic JMML phenotype including myelomonocytic expansion; infiltration of spleen, liver and, notably, lung; splenomegaly; and reduced survival (median 26 weeks). Persistent human engraftment and leukemic organ infiltration was confirmed by both flow cytometry and immunohistology. Ras pathway mutations present in xenotransplanted patient samples were invariably confirmed in engrafted tissues. In addition, the model sustained serial transplantations and can therefore be used to amplify scarce patient material.We first tested if DNA methylation patterns in JMML cells were stable even after xenologous engraftment because such stability would be a prerequisite if the model were to be used for preclinical investigation of DNA methyltransferase inhibitors. JMML cells before xenotransplantation and those retrieved from the bone marrow of engrafted mice were profiled for global CpG methylation using Illumina 450K arrays. DNA methylation patterns in JMML were patient-specific and surprisingly robust in functional regions over several months of engraftment time (on average, 0.29% of 30877 promoters and 0.25 % of 30725 intragenic regions were called as "differentially methylated" between source and xenograft; 0.2 β-value change cutoff). These findings confirm the suitability of the xenograft model to investigate JMML epigenetics and, more importantly, indicate that patient-specific epigenetic profiles originate in leukemia-initiating stem cells, reinforcing a fundamental role of these alterations in JMML biology.Our group recently published a retrospective case series demonstrating unprecedented clinical efficacy of the DNA methyltransferase inhibitor 5-azacytidine (5AC) to induce partial or complete remissions in JMML before allogeneic HSCT (Cseh, Blood 2015;125:2311-3). To further investigate the drug on the preclinical level we administered 5AC to Rag2-/- gamma-c-/- mice xenografted with primary JMML cells. After a leukemia establishment phase the mice were divided into treatment or mock groups and treated with 5AC (3mg/kg body weight i.p., N=6) or saline (N=6) for 2 cycles (1 dose daily for 5 days; 9 days of recovery). This regimen was tolerated well by the animals. We found that 5AC reduced JMML infiltration in all organs analyzed, with most pronounced effects in spleen (human CD45+ fraction of all CD45+ cells, 0.24% +/- 0.04% vs 39.78% +/- 10.72%; p<0.01) and lung (0.41% +/-0.18% vs 42.88% +/-8.42%; p<0.01). The proportion of early progenitor cells (CD34+) within the human leukemia population in murine bone marrow was dramatically reduced after 5AC treatment (7.89% +/-0.74% vs 32.65% +/-3.76%; p<0.01) while the amount of granulocytes increased simultaneously (44.90% +/-1.74% vs 9.35% +/-1.95%; p<0.01). These findings suggest a loss of JMML cells induced by forced differentiation of more immature cells into mature myelomonocytic cells with reduced proliferation potential. Bisulfite pyrosequencing of the human BMP4 promoter CpG island, a locus frequently hypermethylated in JMML, showed significantly reduced DNA methylation in JMML cells retrieved from 5AC-treated mice (31.32% +/-2.66% vs 52.46% +/-1.39%; p<0.001).In summary we created an ex vivo JMML xenograft model in immunodeficient mice that reflects many important aspects of this disorder and proved its usefulness for preclinical research of DNA methyltransferase inhibition because of extraordinary stability of leukemic DNA methylation patterns. 5AC showed clear preclinical efficacy in this model, supporting its further development in clinical treatment strategies for JMML. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5464: Host variation in OATP1B1 is associated with treatment outcome in pediatric AML

Abstract Improvements in survival have been achieved for children and adolescents with acute myeloid leukemia (AML), with the 5-year survival rate increasing between 1975 and 2010 from &amp;lt;20% to 70%. However in the past decade, outcome has not improved. Therapeutic approach in AML consists of intensive combination chemotherapy and hematopoietic stem cell transplantation. Although several prognostic factors have been identified, current predictive analysis remains inadequate. We performed a pharmacogenetic-association study on leukemic and/or germline DNA of 164 pediatric patients with AML receiving cytarabine (Ara-C), daunorubicin (Dauno), etoposide (VP16) and mitoxantrone (MTZ) using the DMET platform involving 1,936 variants in 225 genes related to drug metabolism and transport. After adjusting for age, risk group and treatment arm, among the top-ranking variants associated with event-free survival (P = 0.0060) and overall survival (P = 0.012) was a silent single-nucleotide polymorphism (SNP) in SLCO1B1 (c.597C&amp;gt;T), a gene encoding the hepatocellular uptake transporter OATP1B1. This SNP was not correlated with gene expression in leukemic blasts (P = 0.58), suggesting the association might be due to a host rather than a leukemia effect. Since this SNP is in strong linkage disequilibrium (P&amp;lt;0.0001) with two non-synonymous coding variants in SLCO1B1 that encode known functional variants OATP1B1*1B (Asn130Asp) and OATP1B1*5 (Val174Ala), we hypothesized that one or more of the AML drugs may be a substrate of OATP1B1. In vitro transport studies using mammalian cells stably transfected with OATP1B1*1A (wild-type) revealed that the intracellular uptake of Ara-C, Dauno, VP16 and MTZ was enhanced compared with control cells in a time- and concentration-dependent manner. Moreover, the efficiency of drug transport was impaired in cells expressing either OATP1B1*1B or OATP1B1*5, or both (OATP1B1*15) compared with OATP1B1*1A. To test whether Ara-C, Dauno, VP16 and/or MTZ are transported by OATP1B-type carriers in vivo, we determined the pharmacokinetic profile of these agents (1-100 mg/kg; i.v. or i.p.) in mice deficient in the ortholog transporter Oatp1b2 [Oatp1b2(-/-) mice]. We found the concentrations of Dauno, VP16 and MTZ in livers of Oatp1b2(-/-) mice were reduced (1.4-2.8 fold) compared to wild-type mice, and this phenomenon was accompanied by significant increases in plasma area under the curve. Despite being a transported substrate of OATP1B1*1A and Oatp1b2 in vitro (4.7-fold vs control), plasma concentrations of Ara-C were not affected by Oatp1b2 deficiency, which may reflect a differential contribution of the liver in drug elimination between different species. Collectively, these findings indicate an important role for OATP1B1 in the systemic pharmacokinetics of multiple drugs used in the treatment of pediatric AML and suggest that inherited variability in host transporter function may indirectly regulate the effectiveness of therapy. Citation Format: Christina D. Drenberg, Stanley Pounds, Lei Shi, Shelley Orwick, Lie Li, Shuiying Hu, Alice Gibson, Raul Ribeiro, Jeffrey Rubnitz, Alex Sparreboom, Sharyn D. Baker. Host variation in OATP1B1 is associated with treatment outcome in pediatric AML. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5464. doi:10.1158/1538-7445.AM2015-5464

Abstract 3428: Aging, telomere and TP53: a potential biological insight into the mechanistic origin of acute lymphoblastic leukemia (ALL) in the elderly

Abstract Telomere attrition is a recognized feature of genomically unstable cancer genomes prevalent in older patients. Unchecked genomic instability secondary to loss of TP53 fosters telomere attrition. The relevance of this in older ALL patients has not been studied. Population based cytogenetic study of adults with ALL demonstrated that aneuploidy - reflecting genomic instability - was almost 4 fold more common in those ≥60 years than in younger counterparts. Preliminary data from a UK cohort of patients with ALL aged ≥60 years revealed a significantly higher rate of copy number alterations (CNA) (mostly comprising of deletions of key B-cell differentiation/cell-cycle genes, recognized for their proximity to recombination sequence motifs) when compared to their younger counterparts. Correlative cytogenetic analysis notably revealed 28% of such alterations were attributed to gross chromosomal losses rather than true intragenic deletions in the over 60s versus 10% in the under 35s suggesting an alternative, potentially age-related, non-RAG-mediated driver for some of such aberrations. We hypothesize that permissive telomere shortening is a driver. We assessed relative telomere length (RTL) using monochrome multiplex quantitative PCR of remission DNA samples of ALL patients aged ≥60 and compared it with younger counterparts aged &amp;lt;35. There was a significant age-related reduction in RTL; mean RTL for those aged &amp;lt;35 and ≥60 years with ALL was 1.38 and 0.6. RTL of remission DNA from ≥60 years ALL patients was significantly shorter than that of age-matched controls without ALL; mean 0.6 versus 1.07, p&amp;lt;0.01. We then assessed leukemic DNA RTL and compared it to that of matched remission DNA to define leukemic telomere state, expressed as log2 ratio, in patients aged ≥60 (N = 29) and &amp;lt;35 years (N = 35). Mean log2 ratio was significantly higher in the ≥60s than in the &amp;lt;35s (1.009 and -0.7498, p = 0.0002). Based on a log2 ratio cut-off of 3.18 derived from expectation maximization-mixture guided model to define leukemic telomere gain or loss, 8 patients (27.6%) aged ≥60 had leukemic telomere gain whereas none of those aged &amp;lt;35 years had gain. Leukemic telomere gain was statistically associated with more CNA of 8 key B-cell differentiation/cell-cycle genes assessed by multiplex ligation probe amplification P335-ALL IKZF1 kit (median 4 versus 1 with loss, p&amp;lt;0.01) and hypodiploid/near triploid or complex karyotypes (5/8 versus 1/13 with loss, p&amp;lt;0.01). TP53 alteration assessed by cytogenetics, FISH +/- direct sequencing of exons 5-8 in the ≥60 ALL cohort was noted in 5/8 patients with gain versus 4/17 with loss; p = 0.037. Whilst our study is currently limited by sample size integration of CNA with telomere features of the ALL genome and TP53 state has revealed a potential interactive link between aging, telomere attrition and TP53-dependent adaptive response loss which provides the first insight into disease biology of elderly ALL. Citation Format: Ghada Zakout, Lena Rai, Adele K. Fielding. Aging, telomere and TP53: a potential biological insight into the mechanistic origin of acute lymphoblastic leukemia (ALL) in the elderly. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3428. doi:10.1158/1538-7445.AM2015-3428

Integrated Genomic Profiling, Therapy Response And Survival in Adult Acute Myelogenous Leukemia

Purpose: Recurrent gene mutations, chromosomal translocations, and acquired genomic copy number aberrations (aCNA) have been variously associated with acute myelogenous leukemia (AML) patient outcome. However, knowledge of the co-occurrence of such lesions and the relative influence of different types of genomic alterations on clinical outcomes in AML is still evolving. Experimental Design: We performed SNP 6.0 array-based genomic profiling of aCNA/copy neutral loss-of-heterozygosity (cnLOH) along with sequence analysis of 13 commonly mutated genes on purified leukemic blast DNA from 156 prospectively enrolled non-FAB-M3 AML patients across the clinical spectrum of de novo , secondary, and therapy-related AML. Results: TP53 and RUNX1 mutations are strongly associated with the presence of SNP-A–based aCNA/cnLOH, while FLT3 and NPM1 mutations are strongly associated with the absence of aCNA/cnLOH. The presence of mutations in RUNX1 , ASXL1 , and TP53 , elevated SNP-A–based genomic complexity, and specific recurrent aCNAs predicted failure to achieve a complete response to induction chemotherapy. The presence of ≥1 aCNA/cnLOH and higher thresholds predicted for poor long-term survival irrespective of TP53 status, and the presence of ≥1 aCNA/cnLOH added negative prognostic information to knowledge of mutations in TET2 , IDH1 , NPM1 , DNMT3A , and RUNX1 . Results of multivariate analyses support a dominant role for TP53 mutations and a role for elevated genomic complexity as predictors of short survival in AML. Conclusions: Integrated genomic profiling of a clinically relevant adult AML cohort identified genomic aberrations most associated with SNP-A–based genomic complexity, resistance to intensive induction therapies, and shortened overall survival. Identifying SNP-A–based lesions adds prognostic value to the status of several recurrently mutated genes. Clin Cancer Res; 1–12. ©2015 AACR.

Integrated genomic profiling, therapy response, and survival in adult acute myelogenous leukemia.

Recurrent gene mutations, chromosomal translocations, and acquired genomic copy number aberrations (aCNA) have been variously associated with acute myelogenous leukemia (AML) patient outcome. However, knowledge of the co-occurrence of such lesions and the relative influence of different types of genomic alterations on clinical outcomes in AML is still evolving. We performed SNP 6.0 array-based genomic profiling of aCNA/copy neutral loss-of-heterozygosity (cnLOH) along with sequence analysis of 13 commonly mutated genes on purified leukemic blast DNA from 156 prospectively enrolled non-FAB-M3 AML patients across the clinical spectrum of de novo, secondary, and therapy-related AML. TP53 and RUNX1 mutations are strongly associated with the presence of SNP-A-based aCNA/cnLOH, while FLT3 and NPM1 mutations are strongly associated with the absence of aCNA/cnLOH. The presence of mutations in RUNX1, ASXL1, and TP53, elevated SNP-A-based genomic complexity, and specific recurrent aCNAs predicted failure to achieve a complete response to induction chemotherapy. The presence of ≥1 aCNA/cnLOH and higher thresholds predicted for poor long-term survival irrespective of TP53 status, and the presence of ≥1 aCNA/cnLOH added negative prognostic information to knowledge of mutations in TET2, IDH1, NPM1, DNMT3A, and RUNX1. Results of multivariate analyses support a dominant role for TP53 mutations and a role for elevated genomic complexity as predictors of short survival in AML. Integrated genomic profiling of a clinically relevant adult AML cohort identified genomic aberrations most associated with SNP-A-based genomic complexity, resistance to intensive induction therapies, and shortened overall survival. Identifying SNP-A-based lesions adds prognostic value to the status of several recurrently mutated genes.

TP53 Alterations Are Frequent in Patients over 60 Years with B-Precursor Acute Lymphoblastic Leukemia (ALL) and Low Hypodiploid/Near Triploid (HoTr) Karyotype; They Correlate with RB1 Deletion and Leukemic Telomere Gain

IntroductionTP53 is the most frequently altered gene in cancer. Until recently data on its frequency and prognostic impact in ALL has been scant, particularly in the elderly. TP53 alteration in the context of telomere shortening (the hallmark of aging) results in telomere attrition and genomic instability with resultant overexpression of telomerase to overcome the genomic crisis.ObjectiveTo determine TP53 alteration frequency and its association with cytogenetic subgroups, key B cell differentiation/cell cycle genes abnormalities and induction therapy response in ALL patients aged ≥60 and its impact on leukemic telomere stateMethodsPatients enrolled in the UK NCRI UKALL14 and UKALL60+ trials aged ≥60 years were assessed at diagnosis for 17p deletions by cytogenetics, TP53 alterations by FISH and/or direct sequencing of hot spots exons 5-8. Data were correlated with IKZF1, CDKN2A/B, PAR1, BTG, RB1, ETV6, EBF and PAX5 gene copy number status, assessed by MLPA (P335-B1). MYC-rearrangement was an exclusion criterion. Minimal residual disease (MRD) was assessed by quantitative PCR (qPCR) for Ig/TCR rearrangements or BCR-ABL1, where applicable. Leukemic DNA telomere length relative to that of remission DNA was assessed by monochrome multiplex (MM) qPCR and expressed as a log2 ratio. Using expectation maximization mixture model a log2 ratio >3.18 defined gain (reflecting telomerase overexpression) and values below this were defined as loss.ResultsThe cohort included 63 patients; characteristics are presented in table 1. Median age was 63 years (range 60–83). TP53 state by cytogenetics and/or sequencing was available in all. Eleven had TP53 alteration (17%); TP53 mutation only, n=3; TP53 deletion only, n=6; TP53 deletion and mutation, n=2. Cytogenetics was available in 60 patients. Nine patients had HoTr (14%). Seven of 9 patients with HoTr had TP53 alteration (78%) versus 4/51 without HoTr (4%); p<0.001. MLPA was done in 57 patients. Those with TP53 alteration had more copy number alterations (CNA)(median 4 versus 1; p<0.01). Twelve (21%) had RB1 deletion; 3 were due to cytogenetically visible 13q abnormalities rather than ‘true’ intragenic aberration. Four of 9 patients with TP53 alteration had RB1 deletion versus 8/51 without TP53 alteration, p=0.07. All other microaberrations were not significantly associated with TP53 alteration. Sixty-two achieved complete remission after 2 cycles of induction. MRD state (n=32) was not impacted by TP53 (2/5 with TP53 alteration and 13/27 without were MRD positive after 2 cycles of induction; p=0.563). Fourteen of 59 (24%) patients relapsed; median of four months from diagnosis (range 2–21), three had TP53 alteration versus 11 without TP53 alteration; p=0.65. Relapse occurred significantly earlier in patients with TP53 alteration (median two months versus eight months; p=0.03). Leukemic telomere state was assessed in 28 patients. Three of 4 patients with telomere gain had TP53 alteration versus 3/24 without; p=0.02ConclusionsThis is one of the largest reports of TP53 frequency in older patients with non-MYC-rearranged ALL. TP53 was deleted and/or mutated in 17%, with highest frequency in those with HoTr (7/9). RB1 deletion was the commonest associated microaberration, noted in over a third of patients. TP53 alteration was associated with earlier relapse but had no impact on induction response. TP53 alteration was notably associated with frequent CNA and telomere gain suggesting a putative potential role of TP53 in genomic instability though sample size for the latter limits assertion of this hypothesis. Mechanistic work to shed insight into this model is currently underway.Table 1: Patient characteristicsTP53 deleted and/or mutated n=11TP53 wild type n=52Age at Diagnosis, median(range)63(60-75)63(60-83)Gender; M:F06:0523:29Phenotype, n (%)· Precursor B cell11 (100)51 (98)· Precursor T cell0 (0)1 (2)Cytogenetics (risk stratification as per Moorman et al. Blood 2007; 109:3189–97), n (%)Standard Risk0 (0)23 (44)t(9;22)1 (9)21 (40)t(4;11)0 (0)2(4)Ho/Tri7 (64)2(4)Complex2 (18)2 (4)Unknown1 (9)2 (4)Morphological Remission after Cycle 2 Induction, n (%)11 (100)51(98)Induction Deaths, n (%)2 (18)15 (29)CNA by MLPA, median (range)4 (2-7)1 (0-2)RB1 deletion, n (%)· Present4 (36)8 (15)· Absent5 (45)43 (83)· Unknown2 (18)1 (2)Leukemic Telomere State· Gain3 (27)3 (6)· Loss1 (9)21 (40)· Unknown7 (64)28 (54) DisclosuresNo relevant conflicts of interest to declare.

G244(P) “Blessing in Disguise”: Noonan Syndrome with JMML

AimsDr Jacqueline Noonan (1962) identified a cohort of patients with typical facial features, short stature, pulmonary artery narrowing and chest deformities. Subsequently they were recognised as having Noonan syndrome (NS);...

Copy number genome alterations are associated with treatment response and outcome in relapsed childhood ETV6/RUNX1-positive acute lymphoblastic leukemia

The clinical heterogeneity among first relapses of childhood ETV6/RUNX1-positive acute lymphoblastic leukemia indicates that further genetic alterations in leukemic cells might affect the course of salvage therapy and be of prognostic relevance. To assess the incidence and prognostic relevance of additional copy number alterations at relapse of the disease, we performed whole genome array comparative genomic hybridization of leukemic cell DNA from 51 patients with first ETV6/RUNX1-positive relapse enrolled in and treated according to the relapse trials ALL-REZ of the Berlin-Frankfurt-Münster Study Group. Within this cohort of patients with relapsed ETV6/RUNX1-positive acute lymphoblastic leukemia, the largest analyzed for genome wide DNA copy number alterations to date, alterations were present in every ETV6/RUNX1-positive relapse and a high proportion of them occurred in recurrent overlapping chromosomal regions. Recurrent losses affected chromosomal regions 12p13, 6q21, 15q15.1, 9p21, 3p21, 5q and 3p14.2, whereas gains occurred in regions 21q22 and 12p. Loss of 12p13 including CDKN1B was associated with a shorter remission duration (P=0.009) and a lower probability of event-free survival (P=0.001). Distribution of X-chromosomal copy number alterations was gender-specific: whole X-chromosome loss occurred exclusively in females, gain of Xq only in males. Loss of the glucocorticoid receptor gene NR3C1 (5q31.3) was associated with a poor response to induction treatment (P=0.003), possibly accounting for the adverse prognosis of some of the ETV6/RUNX1-positive relapses.

Rapid detection of IDH2 (R140Q and R172K) mutations in acute myeloid leukemia

NADP-dependent enzyme isocitrate dehydrogenase (IDH) mutations, IDH1 and IDH2, have been described in acute myeloid leukemia (AML) using next generation sequencing approaches. IDH2 mutations are heterozygous; they alter a single arginine residue at position 140 or 172 and have distinct prognostic significance. The current detection methods of IDH2 mutations are laborious and time consuming as they require DNA sequencing. Herein, we report a new allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) method to detect the IDH2 mutations. Analysis of leukemic DNA samples from 120 AML patients enabled to identify IDH2 mutations in 22 cases which were confirmed by direct DNA sequencing. Of these, 17 harbored IDH2 (R140Q) and 5 IDH2 (R172K) mutations. Serial dilution experiments showed that the assay enable to detect mutations in 10⁻³ dilutions. Our ASO-PCR method appears useful for routine diagnostic screening of these prognostically relevant alterations in AML and may be conveniently included in the diagnostic workup.