Point Mutations In Tyrosine Kinase Domain Research Articles

Activating point mutations in the MET kinase domain represent a unique molecular subset of lung cancer and other malignancies targetable with MET inhibitors.

Activating point mutations in the MET tyrosine kinase domain (TKD) are oncogenic in a subset of papillary renal cell carcinomas (PRCC). Here, using comprehensive genomic profiling among >600,000 patients, we identify activating MET TKD point mutations as putative oncogenic driver across diverse cancers, with a frequency of ~0.5%. The most common mutations in the MET TKD defined as oncogenic or likely oncogenic according to OncoKB resulted in amino acid substitutions at positions H1094, L1195, F1200, D1228, Y1230, M1250, and others. Preclinical modeling of these alterations confirmed their oncogenic potential, and also demonstrated differential patterns of sensitivity to type I and type II MET inhibitors. Two patients with metastatic lung adenocarcinoma harboring MET TKD mutations (H1094Y, F1200I) and no other known oncogenic drivers achieved confirmed partial responses to a type I MET inhibitor. Activating MET TKD mutations occur in multiple malignancies and may confer clinical sensitivity to currently available MET inhibitors.

A Phase 1a/1b First in Human Study of PHI-101, a Potent Small Molecule Inhibitor of FLT3 in Relapsed and Refractory Acute Myeloid Leukemia

Background: The FMS-like tyrosine kinase 3 (FLT3) is mutated in approximately 30% of acute myeloid leukemia (AML) patients either by internal tandem duplication (ITD) or by point mutations in the tyrosine kinase domain (TKD). PHI-101 is a Type I FLT3 inhibitor developed to overcome resistance in AML. PHI-101 showed potent cellular activity in vitro and in vivo for both FLT3 ITD mutations and a wide variety of FLT3 point mutations in TKD. In preclinical studies utilizing primary human FLT3-ITD leukemia cells, PHI-101 showed increased anti-leukemic activity compared to gilteritinib based on increased survival in mice transplanted with these samples.The Phase 1a and Phase 1b global clinical trial with PHI-101 (NCT04842370) is currently underway to assess the overall safety and efficacy of PHI-101 in refractory and relapsed AML.Study Design and Methods: The overall design of the AML clinical trial consists of two parts, phase 1a, the dose-escalation, and phase 1b, the dose-expansion trial utilizing oral PHI-101 tablets. Up to 5 dose levels are planned for phase 1a and a single subject enrolled per dose level until the occurrence of at least one subject with more than Grade 2 toxicity according to CTCAE5.0 criteria during the 28-day evaluation period. The dose escalation will then convert to a standard 3+3 scheme, with 3 to 6 subjects per dose level cohort over the 28-day cycle. If there are no dose-limiting toxicities (DLT), dose escalation to the next higher dose levels will proceed upon the recommendation of the Safety Monitoring Committee. Subjects with FLT3 mutations or FLT3 wild-type will be enrolled in the dose-escalation cohort, and blood samples are collected for the primary endpoint pharmacokinetics (PK) and for the exploratory pharmacodynamic (PD) endpoint evaluation, including plasma inhibitory assay and biomarker analysis.Results: The Phase 1a clinical trial of PHI-101 was initiated in June 2020 at a daily dose of 40mg for level 1. As of July 31, 2021, 8 patients with relapsed or refractory AML have been enrolled in the trial, all of whom had prior anti-leukemic treatments with intensive chemotherapy, hypomethylating agents and/or other FLT3 inhibitors. To date, 5 enrolled patients were available for safety assessment at three dose levels and have not experienced any dose-limiting toxicities (DLTs) from repeating doses of the 28-day DLT-assessment window. The leukemic blasts in marrow or peripheral blood were dramatically reduced by up to 98% with one cycle (28 days). The pharmacokinetic data of plasma PHI-101 varied in a dose-proportional manner and peak plasma concentrations (Cmax) were reached between 4 and 6 hours after once-daily dosing. The pharmacodynamic evaluation by plasma inhibitory assay showed that plasma from patients receiving at daily doses of PHI-101 inhibited more than 90% of the phosphorylation of the FLT3-ITD receptor. PHI-101 was well tolerated with these dose levels. This study is currently recruiting FLT3 mutation or wild-type AML patients at multiple sites in Korea and Australia.Conclusion: PHI-101 is a next-generation FLT3 inhibitor that showed a potent anti-leukemic activity and improved efficacy in primary AML samples harboring FLT3/ITD and FLT3/TKD mutations in preclinical studies. The current analysis of this trial indicated that PHI-101 is a very effective FLT3 inhibitor for both refractory and relapsed AML patients, including those that have relapsed on other FLT3 TKI. The assessments of safety, tolerability, and PK of PHI-101 to determine the recommended dose for expansion are ongoing in the Phase 1a clinical trial. DisclosuresLee: Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board; AbbVie: Honoraria, Other: Advisory board; Korean Society of Hematology: Membership on an entity's Board of Directors or advisory committees. Im: Pharos iBio Co., Ltd.: Current Employment. Nam: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Kim: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Han: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Yoon: Pharos iBio Co., Ltd.: Current holder of individual stocks in a privately-held company. Small: Pharos iBio Co., Ltd.: Consultancy, Other: Scientific Advisory Board. The arrangement has been reviewed and approved by Johns Hopkins University in accordance with its conflict-of-interest policies, Research Funding; InSilico Medicine: Other: Scientific Advisory Board.

Abstract 804: Antitumor activity of the potent and novel FLT3 inhibitor HM43239 in acute myeloid leukemia

Abstract Introduction: Acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 (FLT3) mutations is associated with poor prognosis with a high risk of relapse after therapy and reduced overall survival. Activating mutations of internal tandem duplication (ITD) and tyrosine kinase domain point mutations (TKD) of FLT3 have been reported in approximately 30% of AML as oncogenic driver mutations. Currently, FLT3 inhibitors showed clinical benefits in the corresponding AML patients. In this study, we have characterized a novel FLT3 inhibitor, HM43239, and assessed the potential as a novel therapy for AML patients. Materials and Methods: HM43239 is a novel, highly potent FLT3 kinase inhibitor. This compound showed tight binding to FLT3 kinase in in silico docking model as a reversible Type I inhibitor. In vitro kinase assay was performed to identify kinase selectivity of HM43239. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM43239 in AML cell lines. HM43239 was evaluated in MV-4-11 and MOLM-13 xenograft mice models. Tumor sizes were measured and tumor samples were analyzed of the mechanisms of action. Results: Among 191 kinases biochemically assayed, HM43239 showed the high selectivity toward FLT3 and AML associated other kinases (e.g. SYK, JAK and TAK1). IC50s' of HM43239 against FLT3 WT, FLT3 ITD and FLT3 D835Y kinases were 1.1 nM, 1.8 nM and 1.0 nM, respectively. HM43239 potently inhibited the growth of AML cell lines harboring FLT3 ITD mutation, such as MV4-11 (IC50: 1.3 nM), MOLM-13 (5.1 nM) and MOLM-14 (2.9 nM). Furthermore, HM43239 effectively inhibited the phosphorylation levels of FLT3 and of downstream kinases related with cell proliferation. In addition, caspase 3/7-dependent apoptosis was induced by HM43239 in AML cell lines expressing FLT3 ITD mutant. And HM43239 inhibited proliferation and induced apoptosis of leukemic stem cell (LSC) marker-expressing KG1a cells (CD34+/CD38- cells) suggesting that the possibility for targeting LSC. HM43239 showed the excellent dose proportional antitumor activity in mouse models xenografted with both MV4-11 and MOLM-13 cell line without any significant toxicity. Moreover, we identified in vivo modulation of related targets (p-FLT3 & p-STAT5) in AML cell with FLT3 mutant. Conclusion: Taken together, HM43239 has demonstrated the potential therapeutic efficacy for the treatment of AML patients and implicated the mechanism of overcoming resistance and preventing relapse. Citation Format: Miyoung Lee, Young Eun Ha, Mi Jin Moon, Joo-Yun Byun, HyunKyung Yu, SeokJong Kang, JaeHo Lee, Kyuhang Lee, Eunkyung Kim, Eunyoung Kim, Ho Jeong Lee, YoungHoon Kim, YoungGil Ahn, KweeHyun Suh, Sun-Jin Kim. Antitumor activity of the potent and novel FLT3 inhibitor HM43239 in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 804.

High expression of FLT3 is a risk factor in leukemia.

Several studies have shown that internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) can result in the failure of leukemia treatment and contribute to a poor prognosis. However, the role of the overexpression of FLT3 in leukemia remains to be fully elucidated. By mining public database, the present study first identified that the expression of FLT3 in leukemia was markedly higher, compared with that in other types of tumor and cell lines, indicating that FLT3 is important in leukemia. In leukemia, FLT3 was found to be significantly upregulated in acute myeloid leukemia and acute lymphoblastic leukemia, and a high expression of FLT3 contributed to reduced survival rates. By analyzing Gene Expression Omnibus and The Cancer Genome Atlas data, it was found that genetic alterations and modification of DNA methylation increased the expression of FLT3 in leukemia. FLT3-ITD and FLT3 tyrosine kinase domain point mutations increased the expression of FLT3 in four independent datasets. In addition, the status of FLT3 gene methylation was negatively correlated with the expression of FLT3, and haploinsufficiency of DNA methyltransferase 1 increased the expression of Flt3 in mouse leukemia cells. By analyzing the enrichment of differentially-expressed genes in chemical and genetic perturbation datasets, it was found that genes, which were upregulated in the FLT3 high expression group had myeloid lymphoid leukemia- and nucleophosmin 1-like signatures, indicating that the overexpression of FLT3 may use the same mechanism to promote leukemia. Collectively, the results of the present study showed that the overexpression of FLT3 is a potential risk factor in leukemia.

A randomized assessment of adding the kinase inhibitor lestaurtinib to first-line chemotherapy for FLT3-mutated AML

AbstractThe clinical benefit of adding FMS-like tyrosine kinase-3 (FLT3)-directed small molecule therapy to standard first-line treatment of acute myeloid leukemia (AML) has not yet been established. As part of the UK AML15 and AML17 trials, patients with previously untreated AML and confirmed FLT3-activating mutations, mostly younger than 60 years, were randomly assigned either to receive oral lestaurtinib (CEP701) or not after each of 4 cycles of induction and consolidation chemotherapy. Lestaurtinib was commenced 2 days after completing chemotherapy and administered in cycles of up to 28 days. The trials ran consecutively. Primary endpoints were overall survival in AML15 and relapse-free survival in AML17; outcome data were meta-analyzed. Five hundred patients were randomly assigned between lestaurtinib and control: 74% had FLT3-internal tandem duplication mutations, 23% FLT3–tyrosine kinase domain point mutations, and 2% both types. No significant differences were seen in either 5-year overall survival (lestaurtinib 46% vs control 45%; hazard ratio, 0.90; 95% CI 0.70-1.15; P = .3) or 5-year relapse-free survival (40% vs 36%; hazard ratio, 0.88; 95% CI 0.69-1.12; P = .3). Exploratory subgroup analysis suggested survival benefit with lestaurtinib in patients receiving concomitant azole antifungal prophylaxis and gemtuzumab ozogamicin with the first course of chemotherapy. Correlative studies included analysis of in vivo FLT3 inhibition by plasma inhibitory activity assay and indicated improved overall survival and significantly reduced rates of relapse in lestaurtinib-treated patients who achieved sustained greater than 85% FLT3 inhibition. In conclusion, combining lestaurtinib with intensive chemotherapy proved feasible in younger patients with newly diagnosed FLT3-mutated AML, but yielded no overall clinical benefit. The improved clinical outcomes seen in patients achieving sustained FLT3 inhibition encourage continued evaluation of FLT3-directed therapy alongside front-line AML treatment. The UK AML15 and AML17 trials are registered at www.isrctn.com/ISRCTN17161961 and www.isrctn.com/ISRCTN55675535 respectively.

Mechanism-Based Combinatorial Strategy for Overcoming FLT3-Inhibitor Resistance in Dual FLT3 ITD and TKD Point Mutations

One-third of acute myeloid leukemias (AML) harbor fms-like tyrosine kinase 3 (FLT3) gene mutations including internal tandem duplication (ITD) and D835 mutations (1). ITD mutations are associated with very poor prognosis (2). Targeted therapy directed against FLT3-ITD mutations has been shown to induce responses in AML patients, albeit only transiently (3). We and others have reported that secondary point mutations, in addition to ITD mutations and aberrant pro-survival signaling pathways such as MAPK or PI3K, are related to the development of resistance (4). To further address the mechanism of FLT3 inhibitor resistance, we generated several murine AML cell lines by introducing dual ITD and point mutations of the tyrosine kinase domain (TKD) 2 of FLT3. We hypothesized that concomitantly targeting FLT3 and MAPK/PI3K may act as a promising approach for overcoming resistance.Previously we showed that E6201, a dual FLT3/MEK inhibitor, demonstrated encouraging anti-leukemia effectiveness in FLT3-ITD and D835G mutated cell lines (5). However, it was less sensitive in triggering cell death in D835Y and ITD/TKD2 dual mutation cells (e.g. ITD plus F691L, Y842C or D835Y etc.). Further investigations demonstrated that those mutated cells have high basal levels of either phospho-FLT3, -ERK, or -AKT. Interestingly, the phosphorylation levels of these proteins were persistently high during long-term exposure to low doses of E6201 (up to 7 to 11 days). As result, these cells became more resistant to subsequent E6201 treatment compared to naïve cells (EC50s were 0.92 vs 0.57 µM and 3.74 vs 1.08 µM in ITD+691 and D835Y mutated cells, respectively). However, concomitant targeting of FLT3/MEK and PI3K/mTOR with E6201 and voxtalisib (XL765) triggered pronounced synergistic pro-apoptotic effects in these pretreated cells (CIs were 0.83 and 0.16 in pretreated ITD+691 and D835Y mutated cells vs. 0.39 and 0.61 in naïve ITD+691 and D835Y mutated cells, respectively; suggesting that this combinatorial strategy may eliminate leukemic cells which have developed resistance during long-term E6201 monotherapy ..Importantly, this synergistic effect was observed even under physiological hypoxic conditions (CI was 0.06 ± 0.01) and in the presence of mesenchymal stromal cells (MSC) (CI was 0.06 ± 0.04) as well, which are considered hallmark conditions of the bone marrow microenvironment. Since CXCR4-SDF-1a signaling axis is associated with MSC-mediated leukemia cell protection, we further tested the combination regime in the presence of CXCR4 antagonist plerixafor. Results demonstrated massive apoptosis induction in MSC co-culture systems (apoptosis induction was 23.9% vs. 91.8% in the absence vs. presence of plerixafor).To minimize combination regime-induced toxicity, we further tested sequential treatment by E6201 and voxtalisib individually. Treatment with voxtalisib followed by E6201 demonstrated identical efficacy as concomitant voxtalisib/E6201 treatment, suggesting that this sequential approach may be beneficial by reducing putative toxicity and enhancing tolerance of the targeted drugs in clinical FLT3-targeted therapy. Conclusion: targeting FLT3/MEK and PI3K/mTOR with E6201 and voxtalisib has potential of preventing the development of FLT3-inhibitor resistance. DisclosuresNo relevant conflicts of interest to declare.

Computer-guided design, synthesis, and biological evaluation of quinoxalinebisarylureas as FLT3 inhibitors.

Activating mutations of FMS-like tyrosine kinase 3 (FLT3) are present in ∼30 % of patients with acute myeloid leukemia (AML) and are associated with poor prognosis. Point mutations in the tyrosine kinase domain (TKD) are observed as primary mutations or are acquired as secondary mutations in FLT3 with internal tandem duplications (ITDs) after treatment with tyrosine kinase inhibitors (TKIs). Although dozens of potent inhibitors against FLT3 ITD have been reported, activating TKD point mutations, especially at residues F691 and D835, remain the leading cause for therapy resistance, highlighting the consistent need for new potent inhibitors. Herein we report the identification and characterization of novel quinoxaline-based FLT3 inhibitors. We used the pharmacophore features of diverse known inhibitors as a starting point for a new optimization algorithm for type II TKIs, starting from an in silico library pharmacophore search and induced-fit docking in the known FLT3 structure. This led to the design of a set of diverse quinoxalinebisarylureas, which were profiled in an FLT3 kinase activity assay. The most promising compounds were further evaluated in a zebrafish embryo phenotype assay.

ASP2215, a novel FLT3/AXL inhibitor: Preclinical evaluation in acute myeloid leukemia (AML).

7070 Background: Activating mutations in FLT3 receptor tyrosine kinase, characterized by internal tandem duplication (ITD) and tyrosine kinase domain point mutations near position D835, have been i...

New Strategies in Neuroblastoma: Therapeutic Targeting of MYCN and ALK

Clinical outcome remains poor in patients with high-risk neuroblastoma, in which chemoresistant relapse is common following high-intensity conventional multimodal therapy. Novel treatment approaches are required. Although recent genomic profiling initiatives have not revealed a high frequency of mutations in any significant number of therapeutically targeted genes, two exceptions, amplification of the MYCN oncogene and somatically acquired tyrosine kinase domain point mutations in anaplastic lymphoma kinase (ALK), present exciting possibilities for targeted therapy. In contrast with the situation with ALK, in which a robust pipeline of pharmacologic agents is available from early clinical use in adult malignancy, therapeutic targeting of MYCN (and MYC oncoproteins in general) represents a significant medicinal chemistry challenge that has remained unsolved for two decades. We review the latest approaches envisioned for blockade of ALK activity in neuroblastoma, present a classification of potential approaches for therapeutic targeting of MYCN, and discuss how recent developments in targeting of MYC proteins seem to make therapeutic inhibition of MYCN a reality in the clinic.

Acquired Point Mutations of TKD Are Responsible for Sorafenib Resistance in FLT3-ITD Mutant AML,

Abstract 3505Activating mutations of fms-like tyrosine kinase 3 (FLT3), including Internal tandem duplications (ITD) mutations and point mutations of tyrosine kinase domain (TKD), can be detected in 1/3 of acute myeloid leukemia (AML) patients, and are associated with higher risk of relapse. (1) We reported that sorafenib, by directly targeting mutant FLT3-ITD, exerted anti-leukemia effects in AML with FLT3-ITD mutations in Phase I/II clinical trial and increased the complete remission rate to 100% (CR and CRp) in combination with Idarubicin/cytarabine.(2, 3) However, resistance to sorafenib develops in most AML patients with ITD mutations during prolonged therapy and leads to relapse.(3) Therefore, understanding the mechanisms of sorafenib resistance remains a challenge that may aid approaches to overcoming drug resistance in this dismal prognostic group of AML patients. The present study found that acquired point mutations in the TKD1 and TKD2 domains of the FLT3 gene play a crucial role by elevating levels of phosphorylated FLT3 and its downstream signaling proteins.cDNA-based mutation analysis of the FLT3 gene was performed in blood/bone marrow samples from AML patients who showed sensitivity to sorafenib during the first cycles of therapy in Phase I/II trials and then became resistant. Results showed that 50% (3 of 6 cases) developed single or multiple acquired point mutations of the FLT3 gene, which included mutations of D651G(H), G619C and I687F in TKD1 and/or E858K in TKD2. Further, a murine sorafenib-resistant AML cell line (Ba/F3-ITD-Res) was developed by long-term/low-dose exposure to sorafenib in vitro. Sequence analysis identified acquired point mutations of N676D and Y842C. To further investigate the correlation of these point mutations with drug resistance, selected single point mutations were individually introduced into Ba/F3-ITD cells by lentiviral infection. Apoptosis induction was measured after exposure to sorafenib. Results showed varying degrees of resistance in sublines with different point mutations and their EC50s for apoptosis induction were 0.69, 0.61 and 19 μM in D651G, N676D and Y842C mutation-containing cells, respectively. By comparison, EC50 for Ba/F3-ITD was 0.16 μM. However, cells with multiple concomitant point mutations (such as N676D plus Y842C) displayed impressive resistance (EC50 = 32μM). These results suggest that single mutations in either TKD1 or TKD2 are associated with resistance of sorafenib-induced apoptosis, and that structural alterations of TKD2 are more critical than those of TKD1. Further, concomitant mutations in both TKDs strongly impair response to sorafenib, suggesting a pivotal role for the structural integrity of both TKDs in maintaining sensitivity of FLT3-ITD AML cells to sorafenib.To better understand mechanisms of resistance, relevant biomarkers were investigated by Western blot in these cells with point mutations. The results showed that TKD1 mutations (D651G and/or N676D) increased basal levels of phospho–FLT 3 and its downstream targets phospho–ERK, –Stat5, –AKT and –S6K, suggesting that mutations of TKD1 result in increased FLT3 activation. However, one TKD2 mutation (Y842C) which does not result in increased basal levels of phospho-FLT3, was nevertheless resistant to sorafenib-induced suppression of FLT3 targets, implying that Y842C may interfere with the binding of sorafenib to FLT3 by a conformational shift of the adenosine triphosphate (ATP)–binding pocket, which reduces the accessibility for sorafenib and mediates resistance. Notably, cells with concomitant N676D and Y842C mutations in both TKDs displayed higher basal phosphorylation levels of FLT3 and its downstream targets, resulting in increased resistance to sorafenib-induced suppression of phosphorylation, suggesting that synergistic resistance to sorafenib may result from multiple mutations in by both, TKD1 and TKD2.Our findings provide an improved understanding of acquired FLT3-ITD/TDK point mutations associated with sorafenib resistance in FLT3-ITD-mutant AML, and might be useful in developing agents that have the potential of overcoming resistance of FLT3-ITD inhibitor in AML. Disclosures:Ravandi:Bayer: Research Funding; Onyx: Research Funding.

The preferentially expressed antigen in melanoma (PRAME) inhibits myeloid differentiation in normal hematopoietic and leukemic progenitor cells

AbstractThe preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications.

Clinical Relevance of a Pharmacogenetic Approach Using Multiple Candidate Genes to Predict Response and Resistance to Imatinib Therapy in Chronic Myeloid Leukemia

Imatinib resistance is major cause of imatinib mesylate (IM) treatment failure in chronic myeloid leukemia (CML) patients. Several cellular and genetic mechanisms of imatinib resistance have been proposed, including amplification and overexpression of the BCR/ABL gene, the tyrosine kinase domain point mutations, and MDR1 gene overexpression. We investigated the impact of 16 single nucleotide polymorphisms (SNP) in five genes potentially associated with pharmacogenetics of IM, namely ABCB1, multidrug resistance 1; ABCG2, breast-cancer resistance protein; CYP3A5, cytochrome P450-3A5; SLC22A1, human organic cation transporter 1; and AGP, alpha1-acid glycoprotein. The DNAs from peripheral blood samples in 229 patients were genotyped. The GG genotype in ABCG2 (rs2231137), AA genotype in CYP3A5 (rs776746), and advanced stage were significantly associated with poor response to IM especially for major or complete cytogenetic response, whereas the GG genotype at SLC22A1 (rs683369) and advanced stage correlated with high rate of loss of response or treatment failure to IM therapy. We showed that the treatment outcomes of imatinib therapy could be predicted using a novel, multiple candidate gene approach based on the pharmacogenetics of IM.

Absolute quantitative detection of ABL tyrosine kinase domain point mutations in chronic myeloid leukemia using a novel nanofluidic platform and mutation-specific PCR

Absolute quantitative detection of ABL tyrosine kinase domain point mutations in chronic myeloid leukemia using a novel nanofluidic platform and mutation-specific PCR

Rapid detection of Flt3 mutations in acute myeloid leukemia patients by denaturing HPLC.

fms-related tyrosine kinase 3 (Flt3) is the most commonly mutated gene in human acute myeloid leukemia (AML) and has been implicated in its pathogenesis. Because screening of Flt3 in AML patients by PCR followed by gel electrophoresis is time-consuming and fails to detect some very small internal tandem duplications (ITDs), we developed a method for screening of FLT3 receptor mutations with PCR plus denaturing HPLC (D-HPLC). Total mRNAs extracted from 34 AML patients were first analyzed for the presence of juxtamembrane length mutations and tyrosine kinase domain point mutations by a conventional method involving PCR amplification, restriction enzyme digestion, and agarose gel electrophoresis (PCR-RED-AGE). Subsequently, the same patient panel was analyzed by D-HPLC, using specifically designed primers and optimized running temperatures for the length and point mutation analysis. Thirty-four patients were analyzed by PCR-RED-AGE; 9 were positive for known Flt3 mutations: 6 of 34 (18%) for ITDs in exon 14 and 3 of 34 (9%) for point mutations in exon 20. The same patient panel was analyzed by D-HPLC, and additional nucleotide changes were discovered; in total, 14 sequence variations were identified: 7 of 34 (21%) for ITDs in exon 14; 2 of 34 (6%) for point mutations in exon 20; 1 of 34 (3%) for a new point mutation in exon 16; and 4 of 34 (12%) for polymorphisms in exons 13 and 14. Direct sequencing analysis identified nucleotide alterations in each of the "D-HPLC positives" but in none of the "D-HPLC negatives", yielding a specificity and sensitivity of 100% for D-HPLC-based screening. This novel D-HPLC-based procedure, which is optimized for identification of new point mutations in the catalytic and regulatory domains of FLT3 receptor, could potentially be useful for studies involving precise genotype determination, which could be critical for selection of innovative AML therapies targeting the FLT3 protein.