Mutations In Chronic Myeloid Leukemia Research Articles

Imatinib-resistance without BCR/ABL Point Mutation in Chronic Myeloid Leukemia

Imatinib-resistance without BCR/ABL Point Mutation in Chronic Myeloid Leukemia

Utility of T315I mutation in Egyptian patients with chronic myeloid leukemia

ABSTRACT Chronic myeloid leukemia(CML) is a myeloproliferative neoplasm (MPN) characterized by the presence of the Philadelphia chromosome (BCR:ABL), and there is a higher risk in untreated CML to be transformed to blast crisis phase. The development of tyrosine kinase inhibitor (TKI) imatinib was a remarkable step in CML treatment. However, patients showed resistance due to some mutations including T315I which still be the most challenging mutation in CML. Therefore, the aim of our study is to screen T315I in our Egyptian CML patients to predict and determine the frequency of T315I in patients with TKIs resistance. AS-PCR performed on 50 CML patients resistant to the first-line TKIs and 50 CML patients respond to the first-line TKIs. Positive-T315I was found in 15 (30%) of TKIs resistant cases. T315I was significantly associated with higher WBC, platelet count, cellularity of BM aspiration and biopsy when compared to those with negative-T315I. Resistant positive-T315I was significantly associated with lower cumulative OS 55.9% and shorter mean OS 93.4 months when compared to resistant negative-T315I. We conclude that T315I can exist at the diagnosis before starting treatment. Therefore, screening test for T315I can be performed at the time of diagnosis to help in choosing the suitable treatment protocol.

I13 overrides resistance mediated by the T315I mutation in chronic myeloid leukemia by direct BCR-ABL inhibition.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by a BCR-ABL fusion gene. Imatinib has significantly improved the treatment of CML as a first-generation tyrosine kinase inhibitor (TKIs). The T315I mutant form of BCR-ABL is the most common mutation that confers resistance to imatinib or the second-generation TKIs, resulting in poor clinical prognosis. In this work, we assessed the effect of a potent histone deacetylase (HDAC) inhibitor, I13, on the differentiation blockade in CML cells harboring T315I-mutated and wild-type BCR-ABL by MTT assay, flow cytometery, cell colony formation assay, mRNA Sequencing, Quantitative real-time PCR and Western blotting analysis. We found that I13 possessed highly potent activity against T315I-mutated BCR-ABL mutant-expressing cells and wild-type BCR-ABL-expressing cells. I13 induced cell differentiation and significantly suppressed the proliferation of these CML cells via the cell cycle G0/G1-phase accumulation. Moreover, it was revealed that I13 triggered the differentiation of BaF3-T315I cells, which was attributed to the block of the chronic myeloid leukemia signaling pathway via the depletion of BCR-ABL that was mediated by the inhibition of HDAC activity presented by the acetylation of histones H3 and H4. Taken together, I13 efficiently depleted BCR-ABL in CML cells expressing the BCR-ABL-T315I mutation, which blocked its function, serving as a scaffold protein that modulated the chronic myeloid leukemia signaling pathway mediating cell differentiation. The present findings demonstrate that I13 is a BCR-ABL modulator for the development of CML therapy that can override resistance caused by T315I-mutated BCR-ABL.

Somatic Mutations in Myeloid Transcription Factors and in Activated Signaling Pathway, but Not in Epigenetic Modifier Pathway, Predict the Risk of Treatment Failure and Progression to Advanced Phase in Chronic Myeloid Leukemia

Background Next-generation sequencing (NGS) is currently reforming contemporary clinical practice into precision medicine incorporating genomics-based knowledge into the clinic. Recently, multiple studies showed that mutations in epigenetic modifier genes are associated with poor treatment outcomes following tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia (CML), particularly after Imatinib treatment. However, it is uncertain whether they either originated from clonal hematopoiesis or the CML clone. For example, ASXL1 mutation is the most recurrent mutation at CML diagnosis, and its presence is correlated with adverse prognosis in CML. Finding of rapid reduction in ASXL1 allele frequency following TKI therapy (Kim et al, Blood 2017) raises the question of whether it is a real driver of treatment failure in CML or not. In addition, it is of interest whether 2nd generation TKI (2G-TKI) therapies can abrogate the adverse impact of somatic mutations in CML. Accordingly, the present study attempted to evaluate the prognostic impact of mutation profiles on clinical outcomes in CML patients treated either with Imatinib or 2G-TKIs. Patients and Methods Our cohort includes a total of 394 samples including 254 samples collected at initial diagnosis and 140 samples taken during follow-up from 309 CML patients in two CML treating centers. Disease characteristics and treatment outcomes were evaluated and compared according to mutation profiles at initial diagnosis and during follow-up after TKI therapy. Treatment outcomes include any response with respect to major molecular response (MMR), molecular response (MR) with 2-log/deeper (MR2) or 4-log/deeper (MR4) with any lines of TKI therapy, failure-free survival (FFS), progression and overall survival (OS). Instead of conventional NGS, we applied smMIP-based NGS (limit of detection up to 0.2%) using our in-house CML-specific panel targeting 40 genes including epigenetic modifiers, myeloid transcription factors (TFs), tumour suppressors, genes in activated signaling, spliceosome and, cohesin complex. ResultsPatient group and treatment summary Summary of patient characteristics are as follows: median age of 54 years; male:female n=143/111 (56.3:43.7); disease status at presentation includes chronic- (n=233; 91.7%), accelerated- (n=15; 5.9%), blastic phase (n=4; 1.6%), or N/A (n=2; 0.8%); Sokal score, low (n=61, 24.0%), intermediate (n=84, 33.1%), high (n=74, 29.1%), or N/A (n=35, 13.8%). Frontline therapy includes Imatinib (n=190; 74.8%), or 2G-TKI (n=62) including Dasatinib (n=15; 6.0%), Nilotinib (n=37; 14.7%), Bosutinib (n=10; 4.0%), or other (n=2; 0.8%). Mutation profiles and their correlation with clinical outcomes following TKI therapy70 mutations were detected in 57 (22.4%) out of 254 diagnostic samples, while 64 mutations were detected in 39 (27.9%) out of 140 follow-up samples.Any mutation detected at initial diagnosis is associated with worse outcomes following TKI therapy. The adverse impact of somatic mutation was mainly from myeloid TF or activated signaling pathway mutations. Patients having those mutations at initial diagnosis showed higher risks of treatment failure (HR 2.8 95% CI [1.4-5.4], p = 0.003) and progression (HR 3.1 95% CI [1.1-9.1], p = 0.04), mainly when treated with Imatinib. On the other hand, the adverse impact of epigenetic modifiers was not apparent although a trend was observed toward a lower response rate for MR2 and MMR in patients carrying mutations in epigenetic modifiers.The adverse prognostic impact from these mutations seems to be abrogated by the use of 2G-TKI therapy, supporting the use of smMIP-based NGS for upfront TKI selection in newly diagnosed CML patients.In the cross-sectional analysis of follow-up samples, overall mutation frequency was significantly higher in the treatment failure or progression group compared to the optimal responders (p=0.012), suggesting longitudinal tracking of mutation profiles may help identify cases of impending treatment failure or progression early. Conclusion Our study shows that mutation profiling utilizing smMIP-based NGS can help identify patients with a potential adverse prognosis from initial diagnosis and may help upfront TKI selection. Further follow-up analysis is ongoing to evaluate the effect of TKI therapy on longitudinal kinetics of mutations in serially collected samples. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 15594: Decreasing the High Risk of Ponatinib-Induced Cardiovascular Toxicity in Chronic Myeloid Leukemia

Introduction: Cardiovascular (CV) toxicity of cancer therapeutics is a growing problem. Ponatinib is one of the most cardiotoxic Tyrosine Kinase Inhibitors (TKIs) and is used as a highly effective TKI against ABL T315I mutation in Chronic Myeloid Leukemia (CML). Long-term exposure to ponatinib increases CV events including myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. There is need of novel therapeutics to provide treatment for CML while avoiding CV toxicity. Hypothesis: Using in vitro and in vivo models of CV toxicity can make possible to reengineer ponatinib to minimize its toxicity. Methods: We developed new safer analogues of ponatinib using fragment-based approach. Their anti-tumor efficacy was tested in 2 different CML cell lines and CML patient samples. We assayed for myocardial toxicity by measuring contractile function in human iPSC-cardiomyocytes (hiPSC-CMs) using high-throughput functional imaging, for vascular toxicity by measuring vasculogenesis in human microvascular endothelial cells (HMVECs), and for bone marrow toxicity by colony formation assay in human bone marrow cells (HBMCs). Finally, we confirmed the safer CV profile and adequate anti-tumor efficacy in an in vivo xenograft mouse model of CML. Results: The new analogues inhibited T315I BCR-ABL kinase activity similar to ponatinib and suppressed T315I mutant CML tumor growth in vitro and in vivo . Compared to ponatinib, the new compounds showed markedly decreased adverse effects on contractility of hiPSC-CMs, vasculogenesis in HMVECs and bone marrow toxicity in HBMCs in vitro . The therapeutic window was increased in vivo , leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increased levels of cardiac troponin. Additionally, we identified multiple kinases, including FGFR1, that were inhibited by ponatinib but not the analogues, suggesting that there is a specific set of kinases responsible for ponatinib toxicity. Conclusions: This study demonstrates that chemical reengineering can generate novel, CV-safe TKIs that retain effective therapeutic properties against CML carrying ABL T315I mutation, but that exhibit minimal CV toxicity compared to ponatinib.

Abstract P3105: Improving Cardiovascular Toxicity Of Chronic Myeloid Leukemia Therapy.

Introduction: Ponatinib is one of the most cardiotoxic Tyrosine Kinase Inhibitors (TKIs), but continues to be used in clinical practice as it is the only TKI effective against the most common ABL T315I mutation in Chronic Myeloid Leukemia (CML). Long-term exposure to ponatinib increases cardiovascular events including myocardial infarction, heart failure, stroke, peripheral vascular disease and venous thrombosis. Novel therapeutics are needed to provide treatment for this common form of CML while avoiding cardiovascular side effects. Hypothesis: Chemical reengineering can create novel TKIs effective against T315I mutant CML but with reduced cardiovascular toxicity. Methods: Using fragment-based approach, we generated new, safer analogues of ponatinib. The anti-tumor efficacy of these analogues was tested in 2 different CML cell lines (K562 T315I and KCL22 T315I) and in CML patient samples. We assayed for myocardial toxicity by measuring contractile function in human iPSC-cardiomyocytes (hiPSC-CMs) using high-throughput functional imaging, and assayed for vascular toxicity by measuring vasculogenesis in human microvascular endothelial cells (HMVECs). Finally, we confirmed the safer cardiovascular profile and adequate anti-tumor efficacy in an in vivo xenograft mouse model of CML. Results: The new analogues inhibited T315I BCR-ABL kinase activity similar to ponatinib and suppressed T315I mutant CML tumor growth in vitro and in vivo . Compared to ponatinib, the new compounds showed markedly decreased adverse effects on contractility of hiPSC-CMs and vasculogenesis in HMVECs in vitro . The therapeutic window was increased in vivo , leading to regression of human T315I mutant CML xenografts comparable to ponatinib but without increased levels of cardiac troponin. Additionally, we identified multiple kinases, including FGFR1, that were inhibited by ponatinib but not the analogues, suggesting that there is a specific set of kinases responsible for ponatinib toxicity. Conclusions: This study demonstrates that chemical reengineering can generate novel, cardiovascular-safe TKIs that retain effective therapeutic properties against CML carrying ABL T315I mutation, but that exhibit minimal cardiovascular toxicity compared to ponatinib.

Droplet digital PCR for the detection of second-generation tyrosine kinase inhibitor-resistant BCR::ABL1 kinase domain mutations in chronic myeloid leukemia.

One of the indications for BCR::ABL1 mutation testing in chronic myeloid leukemia (CML) is when tyrosine kinase inhibitor therapy (TKI) needs to be changed for unsatisfactory response. In this study, we evaluated a droplet digital PCR (ddPCR)-based multiplex strategy for the detection and quantitation of transcripts harbouring mutations conferring resistance to second-generation TKIs (2GTKIs). Parallel quantitation of e13a2, e14a2 and e1a2 BCR::ABL1 fusion transcripts enables to express results as percentage of mutation positive- over total BCR::ABL1 transcripts. We determined the limit of blank in 60 mutation-negative samples. Accuracy was demonstrated by further analysis of 48 samples already studied by next generation sequencing (NGS). Mutations could be called down to 0.5% and across 3-logs of BCR::ABL1 levels. Retrospective review of BCR::ABL1 NGS results in 513 consecutive CML patients with non-optimal response to first- or second-line TKI therapy suggested that a ddPCR-based approach targeted against 2GTKI-resistant mutations would score samples as mutation-negative in 22% of patients with warning response to imatinib but only in 6% of patients with warning response to 2GTKIs. We conclude ddPCR represents an attractive method for easy, accurate and rapid screening for 2GTKI-resistant mutations impacting on TKI selection, although ddPCR cannot identify compound mutations.

The Clinical Significance of BCR-ABL1 Mutations in Patients With Philadelphia Chromosome–Positive Chronic Myeloid Leukemia Who Underwent Allogeneic Hematopoietic Cell Transplantation

The global standard therapy for chronic myeloid leukemia (CML) is tyrosine kinase inhibitors (TKIs). One of the causes of therapeutic resistance to some TKIs corresponds to point mutations in the BCR-ABL1 fusion gene. Allogeneic hematopoietic cell transplantation (HCT) is a treatment option for high-risk CML, including TKI resistance. Although BCR-ABL1 point mutations comprise a major factor in the assessment of the indications for HCT, there is limited evidence for their significance in relation to transplant outcomes. This study aimed to evaluate the profiles and transplant outcomes of BCR-ABL1 mutations in allografted patients with CML. The retrospective study used a nationwide registry data including adult patients with CML who underwent their first HCT between 2006 and 2016. The inclusion criterion was the evaluation of the status of the BCR-ABL1 mutation before HCT. The cohort included 315 patients with a median age of 44 years (range 16-70 years). Point mutations were detected in 152 patients, of which 101 (66%) harbored T315I mutations and 51 harbored mutations other than T315I (non-T315I). With a median follow-up period of 38 months (range 2-114 months), overall survival (OS) at 3 years was worse in the mutation group than in the no-mutation group (53% versus 71%; P=.002), which was validated by multivariate analysis (hazard ratio [HR]=1.50; 95% confidence interval [CI], 1.0-2.2; P=.038); this difference was remarkable in the chronic phase of CML. OS in the non-T315I group was significantly worse than that in the no-mutation group (HR=1.69; 95% CI, 1.0-2.8; P=.035). The nationwide study has successfully evaluated the BCR-ABL1 mutational profile and its outcomes in patients with CML who received HCT. The mortality risk was significantly higher in patients with the BCR-ABL1 mutation than in patients without the mutation. These findings would be useful to understand the clinical significance of various BCR-ABL1 mutations in CML and provide insight into the on mid need for treatment strategies for cases of CML with BCR-ABL1 mutations.

High Expression of TWIST-1 in the Chronic Myeloid Leukemia Patients with T315I Mutation

Background: Among the known ABL mutations in chronic myeloid leukemia (CML), T315I is of particular importance. The T315I mutation may develop resistant cells that increase disease progression. TWIST-1 expression is impaired in patients with increased drug resistance. Objectives: The current study aimed to measure the expression of TWIST-1 gene in CML patients to investigate its association with T315I mutation. Methods: Peripheral blood samples were taken from 40 CML patients. The expression of TWIST-1 and BCR-ABL1 genes was quantified by real-time polymerase chain reaction (PCR). The gene expression was evaluated by REST software. cDNA was used for amplification refractory mutation system (ARMS)-PCR reaction. Results: Of the 40 patients (age range: 19 - 72 years) participating in the study, 23 (57.7%) were female, and 17 (42.5%) were male. The expression of TWIST-1 gene was 43 ± 184.09-fold. The T315I mutation was detected in 3 (7.5%) patients. Conclusions: According to our results, the TWIST-1 gene expression in patients with T315I mutation was significantly higher than patients without that mutation.

Role of Somatic Mutations in Clonal Evolution of Chronic Myeloid Leukemia from Chronic Phase to Blast Phase

BackgroundRole of somatic mutations in Chronic Myeloid Leukemia (CML) is fairly underexplored. We hypothesize that clonal evolution of chronic phase CML (CP-CML) to blast phase CML (BP-CML) is molecularly driven thus augmenting on previous findings of ASXL1 being a commonly recurrent mutation in CML. Therefore, early detection of specific somatic mutations can predict progression of disease.MethodsWe performed a retrospective chart review of clinically and molecularly annotated 60 cases of CML from Moffitt Cancer Center. Genotyping was performed using next generation targeted DNA sequencing detecting for common recurrent mutations in hematological malignancies. Fisher's Exact and Student T-test were used to determine statistical significance (p-value of <0.05). All calculated p values were two sided.ResultsTotal 48 patients with CP-CML and 12 patients with BP-CML were included. Median age was 56 and 53.5 years in CP-CML and BP-CML cohort, respectively. Among CP-CML cohort, 37.5% (n=18) had at least 1 mutation with ASXL1 being the most frequently mutated gene (27.7%, n=5) where 80% (n=4) were isolated and only 20% (n=1) had comutation in RUNX1 (Figure 1A). In BP-CML patients, at least 1 mutation was detected in 100% of the cases (n=12) with ASXL1 being the most frequently mutated gene (67%, n=8) (Figure 1B). In contrast to the CP-CML cohort, ASXL1 mutation co-occurred with other mutations in 87.5% of the cases (n=7). The most frequent co-occurring mutations were TET2 (37.5%, n=3) and CUX1 (37.5%, n=3) followed by RUNX1 (25%, n=2), SETBP1 (25%, n=2), TP53 (25%, n=2) and PHF6 (12.5%, n=1). Majority of the BP-CML comprised of myeloid BP (83%) where ASXL1 mutations were exclusively found. Lymphoid BP (16%, n=2) cases revealed lymphoid specific mutations in CDKN2A and IKZF1 genes.In BP-CML compared to CP-CML, presence of ASXL1 mutation was more frequent (OR 17.2, 95% CI 3.77-78.32, p=0.002) with increased frequency of co-occurrence with at least one other mutation (OR 28.0, 95% CI 1.35-580.0, p=0.032). In BP-CML cohort, 83% (n=10) of the patients had additional cytogenetic abnormalities (ACAs) and only 16.5% (n=2) had isolated standard Philadelphia chromosome. In contrast to BP-CML, there was no correlation between presence of ACAs and presence of mutations in CP-CML cohort.ConclusionsAlthough this study is comprised of a small sample size, the findings suggest that the presence of ASXL1 in CP-CML may represent an early event or “first hit”, which then acquires an additional mutation or “second hit”, contributing to progression to advanced phase CML. These findings need to be validated in a large prospective cohort study. [Display omitted] DisclosuresChavez:Abbvie: Speakers Bureau; Janssen: Speakers Bureau; Kite: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees. Pinilla-Ibarz:Pfizer: Honoraria, Speakers Bureau; ARIAD: Consultancy, Honoraria; BMS: Honoraria, Speakers Bureau.

Identification of Progression-Associated Mutations in Chronic Myeloid Leukemia (CML) By Comparative Genomic Analysis

Introduction: CML progression from chronic phase (CP) to blast crisis (BC) has been related to the acquisition of additional mutations, but detailed information on the genes involved is lacking. Here we aimed to identify progression-associated driver mutations and potentially targetable pathways.Patients and methods: Bone marrow samples from 58 patients (14 BC-CML, 2 AP and 42 newly diagnosed CP) were collected from Helsinki University Hospital and National Cancer Institute, Cairo University. For CML-CP patients, CD34+ sorted cells were used to facilitate the detection of low variant allele frequency mutations. Genetic alterations were identified by whole exome sequencing (WES) (8 BC, 1 AP and 15 CP samples) and deep targeted sequencing (Nimblegen gene panel, SeqCap EZ comprehensive cancer Design) (10 BC, 1 AP, and 32 CP diagnostic samples and 15 follow up samples) comprising target regions for 578 driver genes with a documented association with cancer. RNA sequencing was used to identify progression-associated gene expression profiles (8 BC and 5 CP diagnostic CD34+ samples).Results: Analysis of data from all 16 advanced patients and 24 CP-diagnostic samples revealed 80 mutations in 62 driver genes in BC samples compared to 69 mutations in 54 driver genes in CP samples. All variants had high oncogenic potential (score >28) as measured by OncoScore (Piazza, R. et al . Sci. Rep. 2017). The median number of mutations per patient (WES data only) was two-fold higher in BC-CML (10, range: 7-14, n=9) than in CP samples (5, range: 1-10, n=11) (p=0.0027). In BC-CML patients, core-binding factor aberrations (RUNX1 mutations and inv(16)) were the most recurrent (n=6) followed by ABL1 (n=5) and BCOR mutations (n=4). Mutations were also discovered in ASXL1 (n=3), NF1, ALK, SETD2, SF3B2, MSH2 (n=2 each), FLT3-TKD, IKZF1 and IDH2 (n=1 each) . In CP-CML, recurrent mutations were detected in ASXL1, TET2 (n=4 each), NOTCH3 (n=3), TET1, KMT2C and PTPRD (n=2 each). CNV data analysis of BC samples identified progression-associated events including +8, inv(16) (n=2 each), i(17q) with P53 loss, LOH in 21q and IKZF1 deletion (n=1 each). Driver mutations known to be associated with hematological malignancies (RUNX1, FLT3 and BCOR) were almost exclusively limited to BC (RUNX1 mutation in one CP-patient who progressed to BC within a year). Epigenetic modifiers (ASXL1, TET1, TET2, and IDH2) represented a major subset of mutated genes in both phases. Mutations in protein tyrosine phosphatase (PTP) genes (putative negative regulators of JAK-STAT signaling) were common in CP patients (n=5), and they were associated with poor response or progression as none of the patients carrying these mutations achieved major molecular remission within 12 months. In CP patients, higher mutation burden (median mutation number from WES data) was associated with poor treatment responses (failure to achieve MMR at 12 months). The median mutation number was almost 2-fold higher in poor responders (8, range: 3-10, n= 5) than in good responders (4.5, range: 2-5, n=4). Variant allele frequencies of putative driver mutations (ex; RUNX1, MECOM, PTP genes) in poorly responding CP patients were tracked in follow up samples, and were found to correlate with BCR-ABL1 levels confirming their presence in the leukemic clone.Conclusion:Comparative analysis of CP-CML and BC-CML patients revealed a group of genes associated with a particular disease phase (e.g. RUNX1, FLT3, and BCOR) of which some could represent therapeutic targets for future studies. In CP-CML patients, mutations in PTP genes were found to correlate with poor response warranting their screening at the time of diagnosis. DisclosuresHeckman:Orion Pharma: Research Funding; IMI2 project HARMONY: Research Funding; Pfizer: Research Funding; Celgene: Research Funding; Novartis: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Mustjoki:Ariad: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Celgene: Honoraria.

Discovery and Protein Modeling Studies of Novel Compound Mutations Causing Resistance to Multiple Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia.

Objective:BCR-ABL fusion oncogene is the hallmark of chronic myeloid leukemia (CML), causing genomic instability which leads to accumulation of mutations in BCR-ABL as well as other genes. BCR-ABL mutations are the cause of tyrosine kinase inhibitors (TKIs) resistance in CML. Recently, compound BCR-ABL mutations have been reported to resist all FDA approved TKIs. Therefore, finding novel compound BCR-ABL mutations can help and clinically manage CML. Therefore, our objective was to find out novel drug-resistant compound BCR-ABL mutations in CML and carry out their protein modelling studies. Methodology:Peripheral blood samples were collected from ten imatinib resistant CML patients receiving nilotinib treatment. BCR-ABL transcript mutations were investigated by employing capillary sequencing. Patient follow-up was carried out using European LeukemiaNet guidelines. Protein modeling studies were carried out for new compound mutations using PyMol to see the effects of mutations at structural level. Results:A novel compound mutation (K245N mutation along with G250W mutation) and previously known T351I utation was detected in two of the nilotinib resistance CML patients respectively while in the rest of 8 nilotinib responders, no resistant mutations were detected. Protein modelling studies indicated changes in BCR-ABL mutant protein which may have negatively impacted its binding with nilotinib leading to drug resistance. Conclusion:We report a novel nilotinib resistant BCR-ABL compound mutation (K245N along with G250W mutation) which impacts structural modification in BCR-ABL mutant protein leading to drug resistance. As compound mutations pose a new threat by causing resistance to all FDA approved tyrosine kinase inhibitors in BCR-ABL+ leukemias, our study opens a new direction for in vitro characterization of novel BCR-ABL compound mutations and their resistant to second generation and third generation TKIs.

Expert Curation of Somatic Variants in Hematological Malignancies By the Clingen Somatic Hematological Cancer Taskforce (ClinGen HCT)

Hematological malignancies comprise a genetically heterogeneous spectrum of diseases caused by abnormal proliferation or maturation of a variety of hematological cell lineages. Genomic abnormalities including chromosomal translocations, copy number variations and sequence level gene mutations underlie the pathogenesis of these disorders and frequently serve as important diagnostic, prognostic and/or therapeutic markers. However, the substantial discrepancy in interpretation and reporting of these genomic abnormalities among testing labs creates challenges for patient management. Therefore, standardizing the curation, clinical interpretation and reporting of somatic alterations within the context of their diagnostic, prognostic and therapeutic significance in hematological cancers is critical. In January 2020, the ClinGen Somatic Cancer Clinical Domain working group formed the Hematological Cancer Taskforce (HCT) with a goal to undertake systematic curation and evidence-based clinical interpretation of genes/somatic variants associated with hematological malignancies. The HCT has recruited 32 multi-disciplinary experts including oncologists, molecular pathologists, clinical lab directors, genomic scientists and biocurators with expertise in hematological malignancies. In collaboration with the Clinical Interpretation of Variants in Cancer (CIViC) (civicdb.org) knowledgebase, variants from peer-reviewed publications are curated with editor review for clinical utility as evidence items. Monthly discussions based on these evidence items lead to the creation of summary variant assertions using the AMP/ASCO/CAP guidelines (Li M, et al., Journal of Molecular Diagnostics, 2017). The HCT is currently focused on expert curation and clinical interpretation of somatic variants in FLT3 (internal tandem duplication, tyrosine kinase domain and non tyrosine kinase domain variants) in acute myeloid leukemia (AML). Expert curation of gene fusions in Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) in collaboration with the ClinGen somatic pediatric cancer taskforce is currently underway. To date, the HCT has curated 45 evidence items from clinical and pre-clinical studies on the aforementioned genes/variants. In addition, three AMP Tier I, level A variant assertions of FLT3-ITD, D835 and I836, which predict response to Gilteritinib, an FDA-approved drug for relapsed or refractory AML, have been curated. In the future, the HCT plans to extend its focus on curation of BCR-ABL1 kinase domain mutations in chronic myeloid leukemia (CML). Based on the initial pilot curation phase, the HCT will develop gene-specific recommendations to standardize the reporting and interpretation of somatic variants to better assist clinical decisions and apply to become official ClinGen Somatic Expert Panels in each of these gene-disease domains. Disclosures Blombery: Novartis: Consultancy; Invivoscribe: Honoraria; Amgen: Consultancy; Janssen: Honoraria.

Novel Illumina-based next generation sequencing approach with one-round amplification provides early and reliable detection of BCR-ABL1 kinase domain mutations in chronic myeloid leukemia.

The occurrence of mutations in the BCR-ABL1 kinase domain (KD) can lead to treatment resistance in chronic myeloid leukaemia patients. Nowadays, next-generation sequencing (NGS) is an alternative method for the detection of kinase domain mutations, compared to routinely used Sanger sequencing, providing a higher sensitivity of mutation detection. However, in the protocols established so far multiple rounds of amplification limit reliable mutation detection to approximately 5% variant allele frequency. Here, we present a simplified, one-round amplification NGS protocol for the Illumina platform, which offers a robust early detection of BCR-ABL1 KD mutations with a reliable detection limit of 3% variant allele frequency.

Detection of Actionable BCR-ABL1 Kinase Domain (KD) Mutations in Chronic Myeloid Leukemia (CML) Patients with Failure and Warning Response to Tyrosine Kinase Inhibitors (TKIs): Potential Impact of Next-Generation Sequencing (NGS) and Droplet Digital PCR (ddPCR) on Clinical Decision Making

A variety of mechanisms underlie the lack or loss of response to TKIs in patients (pts) with CML, but acquisition of point mutations in the BCR-ABL1 KD is, at present, the only actionable one. Detection of a TKI-resistant mutation by Sanger Sequencing (seq) should trigger a change of therapy and, in some cases, guide TKI selection. Although research studies have shown that NGS may hold added value over Sanger seq for BCR-ABL1 KD mutation screening, routine NGS is technically demanding and expensive and is not yet widely available. Newer methods, like ddPCR, might represent an attractive alternative. Here we set out to 1) assess the actionability of results, hence the potential clinical benefit, of more sensitive NGS-based testing vs Sanger seq in a consecutive series of CML pts who had non-optimal response to TKI therapy according to the 2013 ELN recommendations, and 2) test a novel ddPCR-based multiplex assay for rapid screening for a panel of BCR-ABL1 KD mutations relevant for TKI selection. Between January 2015 and May 2019, samples from 712 CML pts followed at one of 66 GIMEMA CML Working Party hematology centers were referred to our laboratory for BCR-ABL1 KD mutation testing because of a Failure (n=251 pts) or Warning (n=461 pts) response to TKI therapy. In parallel to Sanger seq, NGS of amplicons generated by nested reverse transcription(RT)-PCR was performed on a Roche GS Junior instrument until April 2017, and on an Illumina MiSeq instrument from May 2017 on. Read alignment and variant calling was done using AmpSuite software (SmartSeq). Variants detected in &amp;lt;3% of BCR-ABL1 transcripts were filtered out. ddPCR was performed on a QX200 instrument using a single-tube assay (BioRad) containing primers and probes for the simultaneous detection and discrimination of 2nd generation TKI (2GTKI)-resistant mutations: T315I/A, Y253H, E255K/V, F359V/I/C, V299L and F317L/V/I/C. Data analysis was performed using QuantaSoft software. Among Failures, pts positive for BCR-ABL1 KD mutations by Sanger seq were 88/251 (35%). NGS detected low level mutations in 38/251 (15%) additional pts who were negative for mutations by Sanger seq. Moreover, 31/251 (12%) Failure pts were found to have low level mutations additional to those detectable by Sanger seq. Among low level mutations detected by NGS, those actually relevant for TKI selection (2GTKI-resistant) were identified in 22/251 (9%) pts. Compound mutations were found in 10 pts (4%; all progressed to blastic phase). The average turnaround time (TAT) of routine NGS-based analysis was 15 working days (range, 11-32). Thus, in the setting of Failure, NGS-based mutation scanning of the whole KD is of real clinical benefit in a minority of pts while increasing the TAT. Among Warnings, pts positive for BCR-ABL1 KD mutations by Sanger seq were 65/461 (14%). NGS detected low level mutations in 97/461 (21%) additional pts who were negative for mutations by Sanger seq. All Warning pts positive for low level resistant mutations who were not switched to another TKI turned their response into Failure after 3 to 8 months. The multiplex digital PCR assay proved capable to accurately identify and separate the T315I/A, F317L/V/I/C, Y253H, E255K, F359V/I/C, E255V, V299L mutations in five spatially distinct clusters (pan-resistant, Dasatinib-resistant, Nilotinib-resistant, Nilotinib- and Bosutinib-resistant, Dasatinib- and Bosutinib-resistant, respectively) starting from as little as 30ng of total cDNA or 1ng of the 1.7kb 1st PCR product. Very good concordance was observed between ddPCR- and NGS-identified mutations irrespective of mutation frequency or cluster proximity, even for compound mutations. Extensive assay assessment will be presented. TAT of ddPCR was 1 day. In conclusion: - Mutation testing in the Failure setting aims to a timely and rational TKI switch. The incidence of low level mutations relevant to the selection of subsequent-line therapy and the TAT of routine NGS in our cohort suggest that multiplex ddPCR would be an easier and faster alternative. - Mutation testing in the Warning setting may identify pts who need a change in therapy rather than a 'watch and wait' approach. In our cohort, approx 1/5 of the Warning pts negative by Sanger seq had low level mutations resistant to the TKI they were receiving, that ultimately led to Failure. Earlier detection of emerging resistant mutations enabled by NGS (or by ddPCR in pts receiving 2GTKIs) should support proactive TKI switch. Disclosures Soverini: Incyte: Consultancy. Iurlo:Incyte: Other: Speaker Honoraria; Novartis: Other: Speaker Honoraria; Pfizer: Other: Speaker Honoraria. Pregno:Bristol Myers Squibb: Honoraria; Incyte: Consultancy, Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Bonifacio:BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Amgen: Honoraria. Lunghi:Pfizer: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Castagnetti:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; Bristol Myers Squiib: Consultancy, Honoraria. Gugliotta:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria. Rosti:BMS: Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Tiribelli:Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Stagno:Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria; BMS: Honoraria. D'Adda:Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Crugnola:Incyte: Honoraria; Novartis: Honoraria. Corner:BioRad: Employment. Maar:BioRad: Employment. Pane:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: research founding; Janssen: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Saglio:BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy. Baccarani:Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Takeda: Consultancy. Martinelli:Pfizer: Consultancy; ARIAD: Consultancy; Roche: Consultancy; BMS: Consultancy; Novartis: Consultancy. Cavo:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria.

PB1920 BCR‐ABL GENE ABL KINASE DOMAIN MUTATIONS IN IMATINIB RESISTANT CHRONIC MYELOID LEUKEMIA PATIENTS FROM AZERBAIJAN

Background:While chronic myeloid leukemia (CML) can successfully be treated with tyrosine kinase inhibitors, point mutations within ABL kinase domain of the BCR‐ABL gene are the most common cause of resistance to Imatinib Mesylate. More than 100 BCR‐ABL1 kinase domain point mutations with various frequencies of incidence, domain positions and implications on tyrosine kinase inhibitors (TKI) response in CML are associated with TKI resistance.Aims:To assess the frequency and type of ABL kinase domain mutations in Imatinib resistant CML patients.Methods:115 Imatinib resistant CML patients having been observed during the period from 2014 to 2018. 90 patients at the time of diagnosis were in the chronic phase (78.3%), 19 patients in the acceleration phase (16.5%) and 6 patients in the blast crisis phase (5.2%). By risk groups by Socal et al., the patients were distributed as: low risk ‐ 35 patients; the average risk is 67 patients; high risk ‐ 13 patients. Total RNA was isolated from whole blood and reverse‐transcribed. The resulting cDNA was subjected to an initial round of RT‐PCR across the BCR‐ABL1 breakpoint followed by a second round to amplify the sequence. The final PCR product was pyrosequenced. A total of 14 mutations were identified. Statistical analysis was performed using SPSS 25.0 (IBM, USA) with Chi‐Square. Critical p‐value was set to 0.05.Results:115 Imatinib resistant CML patients were analyzed, 44.3% men (n = 51) and 55.7% women (n = 64), average age – 45.3 ± 14.8 (from 16 to 80). BCR‐ABL1 mutations were found in 16.5% (19/115) CML patients. 7 different mutation variants were identified in 19 patients (Y253H – 2, E255K – 1, F317L (TTG) – 2, F317L (TTA) – 1, T315I – 7, F359 V – 3, F359C ‐ 3). Mutations Y253F, E255 V, V299L(CTG), V299L(TTG), F317 V, F317L(CTC), T315A didn’t identified. Mutation associated resistance rate was higher in men than in women (24.5% against 7.8%, p = 0,025). Also these mutations were analyzed between the group of patients aged ≥50 and &lt;50. The results were 20.45 % and 14.08 %, respectively, p = 0.371. In men and women, T315I mutation prevailed, which occurred in 9.8% and 3.1% of cases, respectively, p = 0.136. In addition, the mutations identified in men were Y253H ‐ 2 (3.92%), F317L (TTG) ‐ 2 (3.92%), F359 V ‐ 2 (3.92%), F359C ‐ 2 (3.92%), E255K ‐ 1 (1.96%), and in women, F317L (TTG) ‐ 1 (1.56%), F359 V ‐ 1 (1.56%), F359C ‐ 1 (1.56%). Studying the frequencies of mutations in the different phases of the disease allowed us to establish that mutations were detected in 10 patients in the chronic phase, which accounted for 11.1% of the total number of patients in this phase, and they were detected in 5 patients in the acceleration phase (26.3%), and in 4 patients in the blast crisis phase (66.7%), p = 0.0008. Studying the distribution of mutations by risk groups showed that mutations were detected in 2 patients in the high‐risk group, which accounted for 15.4% of the total number of patients in this group, in 12 patients in the medium‐risk group, (17.9%), and in 5 patients in the low‐risk group (14.3%), p = 0.890.Summary/Conclusion:Since different mutations of the kinase domain of BCR‐ABL1 are associated with different types of resistance to TKI, identifying trends in the distribution of mutations in CML patients receiving TKI treatment is very important for choosing treatment strategy and early prediction of therapy resistance.

ID: 2036 Next-generation sequencing to evaluate frequency of BCR-ABL1 kinase domain Imatinib-resistance mutations

Chronic myeloid leukemia is a clonal myeloproliferative neoplasm, characterized by the presence of chromosomal translocation t(9; 22)(q34; q11). This is found in over 95% of the cases and results in the BCR-ABL1 fusion gene with high tyrosine kinase activity. During the last decades, imatinib and other generation of tyrosine kinase inhibitors have been used effectively for target therapy of the disease. However, many of the drug resistance cases have been reported recently, due to the mutation within kinase domain of the BCR-ABL1 fusion gene. In this work, we performed a retrospective study of 141 imatinib-resistance chronic myeloid leukemia patients to analyze kinase domain mutation by deep sequencing. Another group of 20 untreated patients were added as control. RNA from bone marrow cells was extracted and deeply sequenced utilizing Illumina MiSeq. Bioinformatics pipeline was applied for variant calling and annotation. And the Sanger sequencing was used to validate those mutations. The results showed that nearly one-fourth of patients harboring mutations that resist to Imatinib, among those, Y253F/H, M351T, G250E, F359V/I and M244V were the most frequent mutations. There were also a number of samples harboring multiple substitutions and new variations. Thus, Next-generation sequencing could be the sensitive and effective method to detect kinase domain mutation and our results could provide further information about the drug-resistance mutation in chronic myeloid leukemia.

Abstract 4158: Targeting the imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia through a novel BCR-ABL inhibitor

Abstract Chronic Myeloid Leukemia (CML), a hematological malignancy, is largely caused by the Philadelphia (Ph) chromosome carrying the Break point Cluster Region-Abelson (BCR-ABL) oncogene. Imatinib is a BCR-ABL-targeted therapy and considered the standard of care in CML management. Resistance to imatinib therapy often develops because of mutations in the BCR-ABL kinase domain. In this study, we evaluated PBA2, a novel BCR-ABL inhibitor, for its anti-cancer activity against BCR-ABL expressing BaF3 cells. PBA2 shows potent activity against wild-type and T315I mutated BaF3 cells as compared with imatinib. PBA2 inhibited the phosphorylation of BCR-ABL and its downstream signaling in BaF3/WT and BaF3/T315I cells. PBA2 inhibited the mRNA expression of BCR-ABL in BaF3/WT and BaF3/T315I cells. Mechanistically, PBA2 increased the cell population in sub G1 phase of the cell cycle, induced apoptosis and elevated ROS production in both BaF3/WT and BaF3/T315I cells. Taken together, our results indicate that PBA2 exhibits anti-proliferative effects and inhibits the imatinib-resistant T315I BCR-ABL mutation. PBA2 may be a novel drug candidate for overcoming the resistance to imatinib in CML patients. Note: This abstract was not presented at the meeting. Citation Format: Pranav Gupta, Rishil Kathawala, Liuya Wei, Fang Wang, XiaoKun Wang, Brian J. Druker, Li-Wu FU, Zhe-Sheng Chen. Targeting the imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia through a novel BCR-ABL inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4158. doi:10.1158/1538-7445.AM2017-4158

Utilization of Next-Generation Deep Sequencing to Analyze BCR-ABL1 Kinase Domain Mutation for Imatinib-Resistant Chronic Myeloid Leukemia Patients in Vietnam

Background: Chronic myeloid leukemia is a clonal myeloproliferative neoplasm, characterized by the presence of chromosomal translocation t(9; 22)(q34; q11). This is found in over 95% of the cases and results in the BCR-ABL1 fusion protein with high tyrosine kinase activity. During the last decades, imatinib and other generations of tyrosine kinase inhibitor have been used effectively for target therapy of the disease. However, many of the drug resistant cases have been reported recently, due to the mutation within kinase domain of the BCR-ABL1 fusion gene. In order to provide further information about this incidence, we performed a retrospective study of 141 imatinib-resistance chronic myeloid leukemia patients to analyze kinase domain mutation by deep sequencing. Another group of 20 untreated patients were added as control. Methods: RNA from bone marrow cells were extracted and followed by cDNA synthesis. Nested polymerase chain reaction was performed to amplify kinase domain of the BCR-ABL1 fusion gene. The amplified products were monitored size, concentration and prepared DNA sequencing library. Sequence analysis was performed using Illumina MiSeq sequencer and Sequence Pilot software. The sequencing results were randomly chose for Sanger sequencing. Results: None of the control group was positive with kinase domain mutation. There were 47 out of 141 patients (33%) detected with at least one nucleotide substitution. The sequencing results were also confirmed by Sanger sequencing. Of those 47 samples, 72 nucleotide substitutions of 28 types, altered 24 codons were identified. Among those, Y253F/H, M351T, G250E, F359V/I and M244V were the most frequent mutations, while T315I took only 4.1%. There were also a number of samples harboring multiple substitutions and new variations. Conclusion: Nextgeneration deep sequencing is a sensitive and effective method to detect kinase domain mutation and our results could provide further information about the drug-resistance mutation in chronic myeloid leukemia.

Aurora A Kinase Inhibitor AKI603 Induces Cellular Senescence in Chronic Myeloid Leukemia Cells Harboring T315I Mutation.

The emergence of resistance to imatinib mediated by mutations in the BCR-ABL has become a major challenge in the treatment of chronic myeloid leukemia (CML). Alternative therapeutic strategies to override imatinib-resistant CML are urgently needed. In this study, we investigated the effect of AKI603, a novel small molecule inhibitor of Aurora kinase A (AurA) to overcome resistance mediated by BCR-ABL-T315I mutation. Our results showed that AKI603 exhibited strong anti-proliferative activity in leukemic cells. AKI603 inhibited cell proliferation and colony formation capacities in imatinib-resistant CML cells by inducing cell cycle arrest with polyploidy accumulation. Surprisingly, inhibition of AurA by AKI603 induced leukemia cell senescence in both BCR-ABL wild type and T315I mutation cells. Furthermore, the induction of senescence was associated with enhancing reactive oxygen species (ROS) level. Moreover, the anti-tumor effect of AKI603 was proved in the BALB/c nude mice KBM5-T315I xenograft model. Taken together, our data demonstrate that the small molecule AurA inhibitor AKI603 may be used to overcome drug resistance induced by BCR-ABL-T315I mutation in CML.