Abstract 4856: ITD Assembler: An algorithm for internal tandem duplication discovery from short-read sequencing data

Sensitive detection of internal tandem duplication (ITD) mutation of FLT3 is very important in acute myeloid leukemia. To increase detection sensitivity of FLT3-ITD, we developed new detection algorithm using next generation sequencing (NGS) data. We validated results using nested polymerase chain reaction (PCR) methods. We compared results of NGS data, nested PCR and conventional PCR methods. First, using whole exome sequencing data of 83 AML patients, we applied calling algorithm for FLT3-ITD. Briefly, to detect ITDs with NGS data, the reads are aligned to a reference sequence (UCSC hg19), with BWA which is a read aligner allowing soft-clipping. Some reads can be an indication of the occurrence of ITD and BWA aligns those reads as soft-clipped. Second, we deigned two types of primer for Nested PCR. The first primer was targeted wildly for between exon14 and exon15 of FLT3 gene. Nested PCR primer was deigned to target previously reported regions which are frequently occurred ITD mutation. PCR reactions of two steps were performed using the PCR primers sequentially. In these 83 patients, FLT3-ITD was positive only in 7 patients when tested by conventional PCR methods. When NGS detection method was applied, this resulted in positive FLT3-ITD in 11 patients (11/83, 13%). When validation was performed using nested PCR, FLT3-ITD was confirmed in all of 11 patients. Nested PCR detected additional 4 patient positive for FLT3-ITD in this population. For 68 patients, FLT3-ITD was negative by both NGS and nested PCR method. Overall, NGS method improved sensitivity of FLT3-ITD detection by 57% in this population. And the concordance rate of NGS method and nested PCR was 95.2% (79/83). Then we investigated clinical significance of sensitive FLT3-ITD detection. For this, we performed nested PCR and conventional PCR at the same time in 238 AML patients to detect FLT3-ITD. Positive rate for FLT3-ITD was 20% (48/238) and 10% (24/238) by nested PCR and conventional PCR respectively. When survival analysis was performed, among patients with negative FLT3-ITD result by conventional PCR, patients who showed positive for FLT3-ITD by nested PCR had shorter overall survival compared to those who showed negative for FLT3-ITD by nested PCR. (p = 0.03). This implies that sensitive FLT3-ITD detection using nested PCR is clinically meaningful. Diagnosis of FLT3-ITD is very important genetic factor, leading a therapeutic direction for AML patient. Here we report that we have developed alternative more sensitive detection methods for FLT3-ITD based on nested PCR and NGS. Sensitive detection of FLT3-ITD was clinically meaningful, suggesting that these methods should be incorporated in a future clinical practice. Also, we want to note that, NGS method is capable of quantifying FLT3-ITD size and amount in AML patients. Citation Format: Daeyoon Kim, Yoojin Hong, Youngil Koh, Sung-Soo Yoon, Choong-Hyun Sun, Kwang-Sung Ahn, Seungmook Lee, Hongseok Yun, Suyeon Lee. Improved sensitive detection method of FLT3 (FMS-like tyrosine kinase) internal tandem duplication (ITD) mutation using next-generation sequencing technology and nested PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3186.

FLT3 ITD, a prognostic marker and drug target, is present in ~19% of AML patients. FLT3 ITD is a tandem repeat of the entire or partial exon 13-15 region. FLT3 ITDs range in size from 3bp to above 300bp and impact RTK signaling. Recent publications have shown that FLT3 ITD can be detected at single nucleotide resolution by NGS panels. However, detection of large FLT3 ITD and accurate reporting of ITD frequency remain challenging for NGS methods. False negative ITD results or inaccurate mutant/wild type allele ratio measurements could negatively alter treatment decisions for AML patients. The Illumina TruSight Myeloid panel (TMP) is a commercial NGS assay that covers 54 genes involved in myeloid malignancies. Although the TMP assay can detect SBS and small indel, medium to large size FLT3 ITD cannot be detected. Here we present an enhanced workflow to overcome these challenges. Positive controls, dynamic range, accuracy, and inter-run reproducibility were validated for the detection of SBS, indel, and FLT3 ITD using reference standards and DNA extracted from cell lines and AML patient EDTA blood and bone marrow aspirate samples. NGS libraries were prepared following Illumina's standard protocol. 275bp X2 sequencing instead of the standard 150bp X2 was run on MiSeq flowcell v3. Data analysis was performed using a MolecularMD custom pipeline. The analysis of FLT3 ITD was conducted using three methods including two off-the-shelf programs, Pindel and ITDseek, as well as Cigar, a MolecularMD developed ITD detection algorithm. FLT3 ITDs at 126 bp and 21 bp in PL-21 and MOLM-13 cell lines, respectively, were detected by TMP assay using the three algorithms. In contrast to ITDseek which only reports ITD sequence without frequency information, Pindel and Cigar report both ITD sequence and frequency. Compared to Pindel, ITD frequency reported by the custom algorithm is closer to that determined by the fragment analysis. Serial dilution of the 126bp ITD into wild type DNA showed the 0.55% ITD (from fragment analysis) can be reproducibly detected at ~0.6% by the Cigar algorithm. FLT3 ITDs ranging in size from 6bp to 300bp were detected in clinical AML samples using the enhanced TMP workflow and confirmed by a fragment analysis method. Limit of detection, reproducibility and accuracy of SBS and indel detection in other key genes by the TMP assay have also been validated. Low-level mutations, i.e. NRAS G12D at 1.7%, NRAS G13D at 2%, and IDH2 R140Q at 1.4%, were detected by TMP panel from AML samples and further confirmed with an independent assay. A long deletion (52bp) in exon 9 of CALR was also detected by the custom pipeline. In summary, the enhanced TMP workflow allows for accurate detection and reporting of SBS, indel and FLT3 ITDs including large duplications. Optimized detection of FLT3 ITDs will improve AML patient selection for targeted therapy. Citation Format: Xiaodong Wang, Zhenyu Yan, Peng Fang, Weihua Liu, Scott Glynn, Jennifer Biroschak, Chad Galderisi, Cindy Spittle, Jin Li. An optimized NGS workflow for detection of FLT3 internal tandem duplication (ITD) in AML samples [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 4522.

Mutations in the Fms-Related Tyrosine Kinase 3 (FLT3) Gene Independently Predict Poor Outcome in Acute Myeloid Leukemia (AML) with t(8;21): A Study of the German-Austrian AML Study Group (AMLSG).

Introduction: When compared to older patients, infants with acute leukemia exhibit high frequency of MLL gene rearrangement (MLL-R), and are known to have higher vulnerability to intensive cytotoxic therapy, such as hematopoietic stem cell transplantation (HSCT). In contrast to acute lymphoblastic leukemia (ALL) in infants, there have been a few reports on acute myeloid leukemia (AML) in infants including our group (A. Shimada et al, ASH 2011). We and other group reported that MLL-R had the clinical impact in infant ALL, however, not in infant AML. Infant ALL with MLL-R expressed high FLT3 mRNA, on the other hand, the importance of FLT3 mRNA remains to be obscure in infant AML. Patients: Forty four infants with AML, aged less than 1 year at diagnosis, registered in JPLSG AML-05 from 2006-2010. Patients with Down syndrome were excluded. The most frequent FAB classification type was M7 (18/44, 40.9%), followed by M5 (15/44, 34.1%), and M4 (4/44, 9.0%). Total 16 of 44 (36.4%) infant AML patients had MLL-R, 9 patients were detected by G-banding method, and other 7 patients were by FISH analysis or RT-PCR. There was not t(8;21), t(15;17) except for one inv(16) patient. Methods: FLT3 mRNA expression and internal tandem duplication (ITD) were analyzed in the first diagnostic bone marrow samples by RQ-PCR and PCR. Results: No patients had FLT3-ITD, however, higher FLT3 mRNA expression was found in MLL-R patients (n=16) (median 623,150 copies/μgRNA) compared with MLL-wild type (WT) (n=28) (median 25,105) (p<0.005). This phenomenon was also observed in 12-23 months AML patients. Discussion: High FLT3 mRNA expression was reported in infant ALL with MLL-R and it can be a molecular target for FLT3 inhibitor. On the other hand, there was no report in infant AML. Our previous data suggested that SCT had the limited efficacy for infant AML, FLT3 inhibitor might be considered to be used for infant AML with MLL-R in future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1422. doi:1538-7445.AM2012-1422

FLT-3 and MER tyrosine kinases have been previously identified as potential targets in the treatment of acute myeloid leukemia (AML). Expression of FLT-3 internal tandem duplication (ITD) occurs in ∼30-40% of AML patient samples and MER overexpression has been detected in ∼80-100%. Here we describe a novel first-in-class small molecule inhibitor that has potent activity against both of these kinases and mediates growth inhibition or apoptosis of cell lines and patient myeloblasts. UNC1666 is an ATP-competitive reversible small molecule inhibitor that potently inhibits FLT-3 and MER, preventing phosphorylation of these kinases and resultant downstream signaling. In these studies, the effects of treatment with UNC1666 were analyzed in FLT3-ITD-positive (Molm-13 and MV4;11) and MER-positive (Kasumi-1 and U937) AML cell lines and in primary AML patient samples with variable expression of FLT3-ITD and MER. Short term exposure to UNC1666 in cell lines that express either a FLT3-ITD or MER resulted in a dose-dependent decrease in AKT and STAT6 activation compared to cells treated with vehicle, confirming that UNC1666 inhibits both targets in cell-based assays. AML cell lines were also stained with Yo-Pro-1 iodide and propidium iodide and analyzed by flow cytometry to determine induction of apoptosis in response to treatment with UNC1666. Treatment of MER-positive cell lines with UNC1666 resulted in a two to five-fold induction of apoptosis relative to vehicle-treated cells (66±10% and 20±10% apoptotic cells respectively; p<0.01). Treatment of FLT3-ITD cell lines with UNC1666 resulted in an even more dramatic nine-fold induction of apoptosis (90±6% verses10±2% in vehicle-treated cultures, p<0.001). When AML cell lines were cultured in soft agar, treatment with the dual inhibitor resulted in decreased colony formation compared to cells treated with vehicle (relative colony counts were 100 for vehicle-treated cultures versus, 34±15 for MER-positive cell lines and 15±12 for FLT3-ITD cell lines treated with UNC1666, p<0.01). Six primary patient samples that were MER and/or FLT3-ITD positive were analyzed in similar assays and exhibited dose-dependent induction of apoptosis and near complete inhibition of colony formation in methylcellulose after treatment with UNC1666. In summary, UNC1666 is a novel first-in-class small molecule with ability to inhibit activation of both FLT-3 and MER tyrosine kinases. Treatment with UNC1666 blocked activation of pro-survival signaling, including AKT and STAT6, induced apoptosis, and decreased myeloblast colony-forming potential in FLT3-ITD positive and MER-expressing AML cell lines. Moreover, similar anti-leukemia effects were observed in primary samples from patients with AML. These data validate dual FLT-3/MER inhibition as a new and attractive approach for treatment of AML with potential for clinical application. Citation Format: Alisa Lee Sherick, Kelly Menachof, Amanda Hill, Sean Rinella, Deborah DeRyckere, Jing Liu, Xiaodong Wang, Stephen Frye, H. Shelton Earp, Douglas Graham. A dual FLT-3 and MER tyrosine kinase small molecule inhibitor in acute myeloid leukemia cell lines and patient samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1743. doi:10.1158/1538-7445.AM2014-1743

Patient Specific Droplet Digital PCR Targeting FLT3-ITD Allows to Detect Small Clones in Acute Leukemias at Diagnosis and during Follow-up

FLT3 (Fms-like tyrosine kinase 3) is the most frequently mutated gene in acute myeloid leukemia (AML). Activating Flt3 mutations occur in about 30% of patients with AML, often as internal tandem duplication(ITD) mutations. These mutations are associated with the poor prognosis. Here, we report the Denovo preclinical candidate SC110237, a novel, selective, highly potent, and orally active FLT3 inhibitor which has very similar antitumor efficacy in MV4-11 xenograft models, but has better cardiac safety, and more favorable pharmacokinetic profiles compared with AC220, the first-in-class FLT3 inhibitor in clinical trial. SC110237 showed potent activity against FLT3 enzyme (IC50 = 91 nM) and anti-proliferative activity against MV4-11 tumor cells (SC110237 IC50 = 0.09 nM, AC220 IC50 = 0.56 nM). Unlike most FLT3 inhibitors in clinical trials, SC110237 showed good selectivity within the Class III RTK family. In vivo, SC110237showed excellent efficacy in the subcutaneous models of AML and CEL (Chronic Eosinophilic Leukemia): MV4-11, MOLM-13 and EOL-1 model. MV4-11, MOLM-13 human AML cells have FLT3-ITD mutations, EOL-1 human CEL (Chronic Eosinophilic Leukemia) cells have FIP1L1-PDGFRA and over-expression of wild-type FLT3 receptor. In s.c. MV4-11 AML model, SC110237-2 (2, 6, 18 mg/kg) demonstrated potent activity. Tumor is completely eradicated in all groups even at the lowest dose of 2 mg/kg, with prolonged inhibition effect on tumor growth after drug withdrawal. At the lowest dose of 2 mg/kg for SC110237-2, there is 1 tumor re-growth until day 55 (12 days post last dose) compared to 3 tumors re-growth (one of the mice died) on the day 55 (12 days post dose) for AC220 at the same dose of 2 mg/kg. At higher dose of 6 mg/kg for SC110237-2, there is no sign of tumor re-growth until day 82 (39 days post last dose) comparing with 1 tumor re-growth on the day 76 (33 days post dose) for AC220 at the same dose of 6 mg/kg. In s.c. MOLM-13 and EOL-1 models, Tumor is completely eradicated in 4/8 mg/kg groups after 5 or 8 days dosing. SC110237-2 has no inhibition on CYP450 2C9, 2D6, 3A4 (Midazolam). Exposure of SC110237 is similar to that of AC220 which has the best PK profile among FLT3 inhibitors, while Cmax of SC110237 is lower than that of AC220. SC110237-2 exhibits linear dose-proportional exposure in mice and dog, and easier to adjust the dosage. Bioavailability of SC110237-2 in dog is 35%. To evaluate the effects of SC110237-2 and AC220 on the cardiovascular system, real time ECG was observed in conscious monkeys by a telemetry system. No abnormal phenomena have been observed in the 20 and 40 mg/kg SC110237 group, only with faster heart rate in the 80 mg/kg SC110237-2. On the other hand, 40 mg/kg AC220 can obviously prolong the QTcB interval. In conclusion, SC110237 exhibited a remarkable anti-tumor activity with favorable pharmacokinetic profiles and showed promise as a novel oral drug for the treatment of acute myeloid leukemia. Citation Format: Norman Kong, Daxin Gao, Heping Yang, Yajun Yu. Discovery of SC110237, a highly potent, selective and orally active FLT3 inhibitor for the treatment of acute myeloid leukemia. [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 2631. doi:10.1158/1538-7445.AM2015-2631

Allogeneic Stem Cell Transplant in First Complete Remission Overcomes the Poor Prognosis Associated with the FLT-3 Internal Tandem Duplication in Acute Myeloid Leukemia.

Comprehensive Molecular Profiling of FLT3-Mutated Acute Myeloid Leukemia (AML) Patients Treated within the Ratify Trial (Alliance C10603)

Mutations in the FLT3 gene are the most common genetic alteration found in AML patients. FLT3 internal tandem duplication (ITD) mutations result in constitutive activation of FLT3 tyrosine kinase activity. The consequences of this activation are an increase in total phosphotyrosine content, persistent downstream signaling, and ultimately transformation of hematopoietic cells to factor-independent growth. The Src homology (SH)2 domain-containing protein-tyrosine phosphatase (SHP)-1 is involved in the down-regulation of a broad range of growth factor and cytokine-driven signaling cascades. Loss-of-function or deficiency of SHP-1 activity results in a hyperproliferative response of myelomonocytic cell populations to growth factor stimulation. In this study, we examined the possible role of SHP-1 in regulating FLT3 signaling. We found that transformation of TF-1 cells with FLT3/ITD mutations suppressed the activity of SHP-1 by approximately 3-fold. Suppression was caused by decreased SHP-1 protein expression, as analyzed at both the protein and RNA levels. In contrast, protein levels of SHP-2, a phosphatase that plays a stimulatory role in signaling through a variety of receptors, did not change significantly in FLT3 mutant cells. Suppressed SHP-1 protein levels in TF-1/ITD cells were partially overcome after cells were exposed to CEP-701, a selective FLT3 inhibitor. SHP-1 protein levels also increased in naturally occurring FLT3/ITD expressing AML cell lines and in primary FLT3/ITD AML samples after CEP-701 treatment. Furthermore, a small but reproducible growth/survival advantage was observed in both TF-1 and TF-1/ITD cells when SHP-1 expression was knocked down by RNAi. Taken together, these data provide the first evidence that suppression of SHP-1 by FLT3/ITD signaling may be another mechanism contributing to the transformation by FLT3/ITD mutations.

Clonal Evolution of FLT3 -ITD Positive AML in Patients Treated with Midostaurin in Combination with Chemotherapy within the Ratify (CALGB 10603) and AMLSG 16-10 Trials

Somatic internal tandem duplications (ITDs) are known to play important roles in cancer pathogenesis. Although recent advances in high-throughput sequencing technologies have enabled genome-wide detection of various types of genomic mutations, including single nucleotide variants, indels and structural variations, only a few studies have focused on ITDs. We have developed an analytical tool called 'Genomon ITDetector' for genome-wide detection of somatic ITDs. After evaluating the sensitivity and precision of the proposed approach using synthetic data, we have demonstrated that it can successfully detect not only common ITDs involving FLT3, but also a number of ITDs affecting other putative driver genes in acute myeloid leukemia exome sequencing data. Availability and implementaion: Genomon ITDetector is freely available at https://github.com/ken0-1n/Genomon-ITDetector.

Gene fusions are important biomarkers for cancer diagnosis, subtype classification and therapeutic decision-making. While fusion detection using RNA-seq data has become a standard practice, existing computational methods primarily focus on identifying canonical exon-to-exon fusions. However, more complex events such as multi-partner fusions, truncations, enhancer hijacking and internal tandem duplications (ITD) can also lead to abnormal function or aberrant transcription of cancer driver genes. To aid discovery of complex and diverse driver fusions, we developed CICERO (CICERO Is Clipping Extended for RNA Optimization), a local assembly-based algorithm that integrates RNA-seq reads bearing aberrant mapping signatures with extensive annotation for ranking candidate fusions. Our benchmark data set, designed to support the main application of RNA-seq fusion analysis, consists of 184 driver fusions from 170 pediatric leukemia, solid tumor and brain tumor detected by paired tumor-normal WGS and orthogonally validated by capture sequencing, RT-PCR and/or FISH. CICERO detected 95% of these fusions with an average ranking of 1.9, whereas ChimeraScan, deFuse, FusionCatcher and STAR-Fusion detected only 63%, 66%, 77% and 63% with an average ranking of 37.0, 9.0, 18.1 and 4.4, respectively. Notably, events such as ITD and rearrangements involving the highly repetitive IGH locus were detected almost exclusively by CICERO. Our re-analysis of 167 RNA-seq data from the TCGA Glioblastoma Multiforme (GBM) cohort unveiled 158 fusions of cancer genes that were not reported previously. These include kinase fusions (KLHL7-BRAF), ITD of EGFR kinase domain and a 13% prevalence of EGFR C-terminal truncation compared to the 6% reported by the TCGA Network. CICERO has greatly improved our ability to discover non-canonical fusions which are overlooked by existing fusion detection methods, and has been used to analyze >2,000 RNA-seq samples generated by the two largest pediatric cancer genomics initiatives: the St. Jude/Washington University Pediatric Cancer Genome Project (PCGP) and the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project. We anticipate that CICERO will also improve fusion analysis for adult cancer RNA-seq data, as demonstrated through our re-analysis of TCGA-GBM and our recent discovery of MAP3K8 C-terminal truncation fusion in 2% of TCGA melanoma samples. CICERO is accessible via standard (https://github.com/stjude/Cicero) or cloud-based (https://platform.stjude.cloud/tools/rapid_rna-seq) implementation. To further improve accuracy, fusions predicted by CICERO can be curated by FusionEditor (https://proteinpaint.stjude.org/FusionEditor/), an interactive viewer allowing inspection of protein domains involved in the fusion and the gene expression status of fusion-positive samples. Citation Format: Liqing Tian, Yongjin Li, Michael N. Edmonson, Xin Zhou, Scott Newman, Clay McLeod, Yu Liu, Bo Tang, Michael C. Rusch, John Easton, Jing Ma, Austyn Trull, J. Robert Michael, Andrew Thrasher, Charles Mullighan, Suzanne J. Baker, James R. Downing, David W. Ellison, Jinghui Zhang. CICERO: An accurate method for detecting complex and diverse driver fusions using cancer transcriptome sequencing (RNA-seq) data [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5478.

MAPKAP1 (Sin1), a Key Component of the MTORC2 Complex, Confers Resistance to Sorafenib and Correlates with Adverse Clinical Outcomes in Acute Myeloid Leukemia

Characterization of NPM1-Mutated/FLT3 ITD-Negative Acute Myeloid Leukemia with Normal Karyotype by Gene Expression Profiling.