FLT3 Internal Tandem Duplication Mutations Research Articles

Detection of Phosphorylated and Total Flt3 and STAT5 in Whole Blood: Modulation by AC220 From Phase I and II Trials in AML

Abstract 2607Fms-like tyrosine kinase 3 (Flt3) plays a critical role in the leukemogenesis of a significant percentage of acute myeloid leukemias (AML) and acute lymphoblastic leukemias making it an attractive target for therapeutic intervention with small molecule inhibitors. To evaluate the pharmacodynamic (PD) effects of AC220, a highly selective and potent Flt3 inhibitor, in phase I and II clinical trials, we developed Flt3 immunoassays using the Meso Scale Discovery (MSD) 96-well platform. Several antibodies were screened to identify capture and detection pairs for both total (t) and phospho (p) Flt3 that were optimized for selectivity, sensitivity, and reproducibility. To validate the assays, cell lysates from SEM-K2 cells that express constitutively phosphorylated Flt3, the MSD assays reproducibly detected tFlt3 with high signal to background ratios, and consistently tracked changes in Flt3 phosphorylation when cells were subjected to Flt3 inhibitors. The functionality of these assays was minimally affected in the context of a whole blood matrix, as p/tFlt3 levels were similarly detected when SEM-K2 cells were spiked into human blood before lysis.To determine the PD effects of AC220 in humans, we tested whole blood samples from a phase I trial in predominantly relapsed or refractory AML patients (pts). AC220 was dosed once daily with escalations from 12 to 450 mg/day. Blood samples were initially examined in 6 pts taken pre-drug administration, at 2, 9, and 24 hrs, and 8 and 15 days post-administration. AC220 markedly reduced pFlt3 levels in the blood lysates at the 2 hr time point and this reduction was maintained over the 5 subsequent time points. Interestingly, the tFlt3 levels increased in all 6 pts at 2 and 9 hrs after treatment, began decreasing by 24 hrs (albeit still elevated from pre-dose levels), and was severely reduced by day 8 and day 15, consistent with elimination of Flt3-expressing peripheral blasts. Expanding the analysis to 45 patients from this phase I study, statistically significant reductions were observed in pFlt3 levels at 2 hrs, 24 hrs, and 15 days after dosing. Furthermore, pts receiving >90 mg demonstrated significantly greater reductions in pFlt3 compared to those receiving <90 mg. There was also significantly higher inhibition of pFlt3 in pts with Flt3 harboring the internal tandem duplication (ITD) activating mutation compared to those lacking this mutation. Importantly, absolute levels, as well as reductions in tFlt3 from day 1 to day 15 showed a statistically significant (p<0.001) rank correlation with the absolute levels and reductions in peripheral blast counts over the same time period. These results demonstrate that the Flt3 levels detected by the MSD assay effectively parallels the peripheral disease burden in AML pts.More recently, the PD effects of AC220 were assessed for pts from the exploratory part of an ongoing phase II trial in relapsed/refractory Flt3 ITD+ AML pts. Blood was taken at day 1 just before the first dose, and 24 hrs and 8 days following the initial dose. Phosphorylated STAT5, a marker closely associated with the Flt3 ITD mutation, was also monitored using a commercially available MSD assay. Blood samples from 52 pts were tested in both MSD assays. For Flt3, 13 pts were excluded from the analysis due to signals below the level of quantitation. Of the 39 pts remaining, a median pFlt3 inhibition of 75% (mean 70%) was observed at 24 hrs following the first dose, consistent with a direct effect of AC220 on its intended target. Similar to what was observed in the phase I trial, 52% of the pts had an increase in tFlt3 levels at 24 hrs. Importantly, 47% of pts had >90% reduction in tFlt3 levels after one week on AC220, signifying a major inhibitory effect on peripheral blast counts. Consistent with the strong linkage between Flt3 ITD autoactivation and STAT5 phosphorylation, there was a significant (p<0.006) correlation between changes in pFlt3 and pSTAT5 levels 24 hrs after dosing. These newly developed assays allow for the rapid assessment of Flt3 autophosphorylation and correlation with downstream signaling and peripheral disease burden. Disclosures:Nepomuceno:Ambit Biosciences: Employment, Equity Ownership. Gunawardane:Ambit Biosciences: Employment, Equity Ownership. Rooks:Ambit Biosciences: Employment, Equity Ownership. Armstrong:Ambit Biosciences: Employment, Equity Ownership.

Phospho-Specific Flow Cytometry of Fixed Whole Blood Demonstrates In Vivo FLT3 Inhibition in Circulating Leukemic Blasts During AC220 Therapy and Accurately Detects the Development of Therapeutic Resistance,

Abstract 3502 Background:AC220 (quizartinib) is a potent, highly selective inhibitor of FLT3, c-KIT, and c-FMS tyrosine kinases with promising clinical activity in AML, particularly among patients with FLT3 internal tandem duplication mutations (FLT3-ITD). However, direct confirmation of biochemical FLT3 inhibition in AML blasts in vivo by AC220 has not been previously described. We report separately that phospho-ribosomal protein S6 (pS6) by flow cytometry provides a sensitive and dynamic readout of FLT3 kinase activity during FLT3 inhibitor therapy. S6 is a downstream target of the PI3 kinase/mammalian target of rapamycin (mTOR) pathway, is constitutively phosphorylated in nearly all FLT3-ITD+ samples, and its phosphorylation shows dynamic changes in response to FLT3 ligand (FL) or FLT3 inhibitors. We hypothesized that reductions in S6 phosphorylation by flow could serve as a biomarker for FLT3 inhibition and predict response and/or resistance to AC220. Methods:Serial peripheral blood samples were collected during a phase II AC220 clinical trial (Cortes, et al. EHA 2011, abstract #1019). Samples were aliquoted into FACS tubes within four hours of collection and a subset was exposed to signaling inhibitors (ex vivo AC220, rapamycin × 30 min.) or activators (phorbol ester/PMA or FL × 10 min.) to establish dynamic controls. Following incubation, samples were formaldehyde-fixed and red cells were lysed with the permeabilizing agent triton X-100. Samples were stored at −20C in glycerol medium. Samples from all time points were simultaneously thawed, denatured with ice-cold methanol, and stained with a single antibody cocktail. Blasts were identified by using CD45 and side scatter (SSC) and confirmed by expression of multiple surface markers (CD33, CD34, CD117, HLA-DR, etc.). Positive and negative pS6 gates were created by comparing blasts in stimulated and unstimulated conditions and/or autofluorescence (FMO) controls. Results:6 subjects provided blood samples (5 FLT3-ITD, 1 FLT3-WT) and had evaluable peripheral blasts (>100/microliter) for pS6 monitoring. As previously described by our group and others using intracellular flow, pS6 is heterogeneous in primary AML samples and, at a basal state, frequently only demonstrable in a subset of blasts. The mean percentage of blasts demonstrating S6 phosphorylation prior to AC220 therapy was 29% (median 9.8%, range 2–97%). To quantify FLT3 kinase inhibition in vivo, we monitored pS6 prior to and following subjects’ initial dose of AC220. Samples obtained two hours following the initial dose consistently reduced the percentage of blasts with pS6 to a mean of 2.8% (median 1, range 0.02 to 11). Consistent with its long half-life, pS6 following a single dose of AC220 remained consistently and potently suppressed at trough concentration on day 2. Comparing clinical response and biochemical inhibition by AC220, all 5 subjects with marked reduction in pS6 to AC220 cleared peripheral blasts by day 29 and 3 subjects achieved marrow blasts <5%. By contrast, a FLT3-WT subject with no WBC reduction during AC220 therapy showed persistent constitutive S6 phosphorylation in FLT3-expressing cells. Re-activation of S6 phosphorylation also occurred in 2/2 FLT3-ITD+ subjects who relapsed during AC220. In both cases, we detected treatment-emergent FLT3-D835 mutations, which have been shown to confer potent in vitro resistance to AC220 (Smith, et al, AACR 2011, abstract #4737). Although S6 phosphorylation in relapsed patients’ blasts was not suppressed by additional ex vivo AC220, plasma obtained at relapse potently inhibited S6 phosphorylation in the FLT3-ITD+ cell line Molm14. Taken together, these data confirm that these subjects’ blasts show intrinsic resistance to AC220 and that drug failure was not secondary to pharmacokinetic changes over time. Conclusions:We demonstrate the feasibility and utility of intracellular flow cytometry for phospho-S6 to monitor the biochemical efficacy of FLT3 inhibitors. The potential of these methods to predict clinical response/resistance paired with the rapid turnaround time of flow cytometry suggests potential future application of this technology in the screening evaluation of patients offered novel signal transduction inhibitors. Studies correlating the magnitude of signal inhibition with IWG responses are ongoing in a larger cohort treated with AC220 and will be reported once clinical data mature. Disclosures:Carroll:TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding; Agios Pharmaceuticals: Research Funding.

Knock-in of a FLT3 Internal Tandem Duplication Mutation Cooperates with a NUP98-HOXD13 Fusion to Generate Acute Myeloid Leukemia

Abstract 865Acute myeloid leukemia (AML) with myelodysplastic related changes represents 24–35% of all cases of AML and has a poor response to chemotherapy and a dismal prognosis. Activating mutations of the FMS-like tyrosine kinase 3 (FLT3) receptor have been seen in 5% of myelodysplastic syndrome (MDS) cases, and an additional 10% of patients with MDS acquire FLT3 mutations during progression to AML. We have previously generated a knock-in mouse model in which an internal tandem duplication (ITD) mutation was inserted into the murine Flt3 gene, which induces a lethal myeloproliferative neoplasm, but not progression to overt leukemia. One mouse model of MDS involves the transgenic expression of a Nup98-HoxD13 (NHD13) fusion under the hematopoietic specific vav promoter. We bred the FLT3/ITD knock-in mice to the NHD13 transgenic mice to see if the two genetic alterations would cooperate. Strikingly, the FLT3/ITD-NHD13 mice on the FVB/N background developed acute leukemia with 100% penetrance and a mean survival of 95 ± 32 days (p<0.0001, n=20). This compares with a mean survival of 281 ± 94 days and 372 ± 84 days for the NHD13 alone and FLT3/ITD alone mice, respectively (both p<0.0001, n=20). FLT3/ITD-NHD13 mice generated on the C57Bl/6N background developed leukemia with a longer latency of 143 ± 37 days, but they still had a significantly shorter survival compared to the single mutants alone.Competitive repopulation experiments showed that leukemic bone marrow was able to engraft in lethally irradiated recipients, with 1:200 cells in the bulk bone marrow having the potential to generate leukemia in recipient mice. To determine the identity of the leukemic initiating cell (LIC), the bulk bone marrow was further sorted into the multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte/macrophage progenitor (GMP), and megakaryocytic/erythroid progenitor (MEP) populations and limiting dilution transplantation was performed for each group. The highest engraftment potential was found in the MPP population (1:100 cells) with much rarer LICs from the CMP and GMP. The MEP population did not engraft in any recipient mice even at the highest cell dose used of 100,000 sorted cells.RNA was extracted from the total bone marrow of mice 3 months prior to the development of disease and showed overexpression of HoxA7, HoxA9, HoxB4, HoxB6, HoxC4, and HoxC6 in the FLT3/ITD-NHD13 mice compared to the age-matched wildtype or FLT3/ITD mice. The overexpression of these genes appeared to be primarily driven by the NHD13 transgene, although there was also an increase in HoxA7 and HoxA9 expression observed in the FLT3/ITD mice. Since Hox genes are known to play an important role in stem cell self-renewal, we isolated RNA from 2-month-old littermates and stained for the cell surface markers characterizing the KSL, MPP, ST-HSC, and LT-HSC populations. FLT3/ITD-NHD13 and FLT3/ITD mice had a significant increase in the percentage of KSLs, long term HSCs, and short term HSCs. FLT3/ITD mice showed increased numbers of MPPs with a smaller but significant increase in the FLT3/ITD-NHD13 cohort.A significant number of AML patients with FLT3/ITD mutations present with loss of the wildtype allele of FLT3 and additional patients acquire this loss at the time of relapse. Hemizygosity at the FLT3 locus in FLT3/ITD mutant AML patients is associated with an even more adverse prognosis compared to patients with an intact wild-type allele. We observed spontaneous loss of heterozygosity (LOH) occurring in 100% of the FLT3/ITD-NHD13 mice and resulting in loss of the wildtype Flt3 allele. The frequent LOH seen in the FLT3/ITD-NHD13 mice provides additional evidence that this transgenic mouse serves as an accurate model to further explore the molecular pathways that underlie the development of leukemias with activating FLT3 mutations. This mouse model is also one of the first models of MDS-related AML and should provide a means to explore the molecular pathways that underlie these devastating hematopoietic neoplasms. Disclosures:Borowitz:BD Biosciences: Research Funding.

Loss of the wild-type allele contributes to myeloid expansion and disease aggressiveness in FLT3/ITD knockin mice

AbstractClinical evidence has shown that FLT3 internal tandem duplication (ITD) mutation confers poor prognosis in acute myeloid leukemia. Loss of the FLT3 wild-type (WT) allele is associated with even worse prognosis. We have previously reported that heterozygous FLT3wt/ITD “knockin” mice develop a slowly fatal myeloproliferative neoplasm (MPN). To study the roles of the WT FLT3 and ITD alleles in the development of MPNs, we generated FLT3/ITD homozygous (FLT3ITD/ITD) and hemizygous (FLT3−/ITD) mice. FLT3−/ITD mice, with the loss of WT allele, display a more severe MPN, as evidenced by even larger spleen, higher white blood cell counts, and shorter survival, compared with FLT3wt/ITD mice. Reintroduction of the WT FLT3 allele into FLT3−/ITD BM slowed the progression of MPN in recipient mice. FLT3ITD/ITD mice had an even severe MPN compared with the FLT3−/ITD and FLT3wt/ITD mice. Spontaneous leukemia developed in a small fraction of the FLT3ITD/ITD mice but was never observed in the FLT3−/ITD and FLT3wt/ITD mice. Our results suggest that loss of the WT allele contributes to the development of a more severe phenotype. Thus, the WT FLT3 allele seemingly functions as a tumor suppressor, attenuating the function of the FLT3/ITD allele in leukemia harboring FLT3/ITD mutations.

Sorafenib monotherapy gives sustainable suppression of FLT3 clone in untreated patients with FLT3‐internal tandem duplication positive acute myeloid Leukaemia

Sorafenib monotherapy gives sustainable suppression of FLT3 clone in untreated patients with FLT3‐internal tandem duplication positive acute myeloid Leukaemia

Distinct immunophenotype of early T-cell progenitors in T lymphoblastic leukemia/lymphoma may predict FMS-like tyrosine kinase 3 mutations

FMS-like tyrosine kinase 3 (FLT3) mutation in T lymphoblastic leukemia/lymphoma (T-LL) is rare (∼4%) and reported only in cases with CD117 expression. This study aimed to identify the immunophenotypic features that may predict FLT3 mutations. We report 3 (43%) of 7 CD117(+) T-LL cases harboring FLT3-internal tandem duplication mutation. Compared with 4 FLT3-unmutated cases, all 3 FLT3-mutated cases had a distinct immunophenotype (CD1a(-)/CD2(+)/CD7(+)/CD34(+)/CD117(uniform+)/Tdt(+)) corresponding to the stage of earliest thymic T-cell progenitors possessing myeloid lineage potential. Indeed, all FLT3-mutated T-LL cases expressed myeloperoxidase on a very small subset of blasts and, thus, may be further considered a mixed phenotype acute leukemia, T/myeloid, by the 2008 World Health Organization classification scheme. We conclude that this unique immunophenotype (CD1a(-)/CD2(+)/CD7(+)/CD34(+)/CD117(+)/Tdt(+)) is a better predictor of FLT3 mutation than sole CD117 expression.

A potential therapeutic target for FLT3-ITD AML: PIM1 kinase

Patients with acute myeloid leukemia (AML) and a FLT3 internal tandem duplication (ITD) mutation have a poor prognosis, and FLT3 inhibitors are now under clinical investigation. PIM1, a serine/threonine kinase, is up-regulated in FLT3-ITD AML and may be involved in FLT3-mediated leukemogenesis. We employed a PIM1 inhibitor, AR00459339 (Array Biopharma Inc.), to investigate the effect of PIM1 inhibition in FLT3-mutant AML. Like FLT3 inhibitors, AR00459339 was preferentially cytotoxic to FLT3-ITD cells, as demonstrated in the MV4-11, Molm-14, and TF/ITD cell lines, as well as 12 FLT3-ITD primary samples. Unlike FLT3 inhibitors, AR00459339 did not suppress phosphorylation of FLT3, but did promote the de-phosphorylation of downstream FLT3 targets, STAT5, AKT, and BAD. Combining AR00459339 with a FLT3 inhibitor resulted in additive to mildly synergistic cytotoxic effects. AR00459339 was cytotoxic to FLT3-ITD samples from patients with secondary resistance to FLT3 inhibitors, suggesting a novel benefit to combining these agents. We conclude that PIM1 appears to be closely associated with FLT3 signaling, and that inhibition of PIM1 may hold therapeutic promise, either as monotherapy, or by overcoming resistance to FLT3 inhibitors.

FLT3/ITD AML and the law of unintended consequences

Acute myeloid leukemia with a FLT3 internal tandem duplication (FLT3/ITD) mutation is an aggressive hematologic malignancy with a generally poor prognosis. It can be successfully treated into remission with intensive chemotherapy, but it routinely relapses. At relapse, the blasts tend to have higher mutant allelic ratios and, in vitro, are more addicted to the aberrant signaling from the FLT3/ITD oncoprotein. They remain highly responsive to FLT3 ligand, the levels of which rise several-fold during the course of chemotherapy. The question now arises as to whether these high levels of FLT3 ligand are actually promoting relapse, and, if so, how we can use this information to adjust our therapeutic approach and improve the cure rate for acute myeloid leukemia with FLT3/ITD.

Outcome of older adults with cytogenetically normal AML (CN-AML) and FLT3 mutations

AML patients under the age of 60 whose blasts harbor a FLT3 internal tandem duplication (ITD) mutation have a higher relapse rate and inferior survival compared to those without this mutation. To determine if FLT3ITD also carries a negative prognostic impact in older adults receiving therapies commonly used in this age group, we retrospectively analyzed outcomes of patients ≥60 years with CN-AML according to FLT3 mutation status. We identified 91 newly diagnosed CN-AML patients, 55 with wild-type FLT3 and 36 with FLT3ITD. Of the 91 patients, 36 received supportive care and/or experimental therapies while the remaining 55 received induction chemotherapy, followed by allogeneic SCT in 17 of these patients. Based on univariate analysis, advanced age at diagnosis was significantly associated with shorter overall survival (OS) ( p < .0001) while intensive therapies were associated with improved OS ( p < .0001). In a multivariate analysis that accounted for type of treatment, patient age, gender, and WBC count, FLT3ITD was significantly associated with shorter OS compared to wtFLT3 [ p = .001; hazard ratio (HR) = 2.23; 95% CI: 1.35–3.70]. Our data support the negative prognostic impact of FLT3ITD in older adults with CN-AML.

Tyrosine kinase inhibitor insensitivity of non‐cycling CD34+ human acute myeloid leukaemia cells with FMS‐like tyrosine kinase 3 mutations

The efficacy of tyrosine kinase (TK) inhibitors on non-cycling acute myeloid leukaemia (AML) cells, previously shown to have potent tumourigenic potential, is unknown. This pilot study describes the first attempt to characterize non-cycling cells from a small series of human FMS-like tyrosine kinase 3 (FLT3) mutation positive samples. CD34+ AML cells from patients with FLT3 mutation positive AML were cultured on murine stroma. In expansion cultures, non-cycling cells were found to retain CD34+ expression in contrast to dividing cells. Leukaemic gene rearrangements could be detected in non-cycling cells, indicating their leukaemic origin. Significantly, the FLT3-internal tandem duplication (ITD) mutation was found in the non-cycling fraction of four out of five cases. Exposure to the FLT3-directed inhibitor TKI258 clearly inhibited the growth of AML CD34+ cells in short-term cultures and colony-forming unit assays. Crucially, non-cycling cells were not eradicated, with the exception of one case, which exhibited exquisite sensitivity to the compound. Moreover, in longer-term cultures, TKI258-treated non-cycling cells showed no growth impairment compared to treatment-naive non-cycling cells. These findings suggest that non-cycling cells in AML may constitute a disease reservoir that is resistant to TK inhibition. Further studies with a larger sample size and other inhibitors are warranted.

Prognostic value of FLT3 mutations in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline monochemotherapy

Fms-like tyrosine kinase-3 (FLT3) gene mutations are frequent in acute promyelocytic leukemia but their prognostic value is not well established. We evaluated FLT3-internal tandem duplication and FLT3-D835 mutations in patients treated with all-trans retinoic acid and anthracycline-based chemotherapy enrolled in two subsequent trials of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups between 1996 and 2005. FLT3-internal tandem duplication and FLT3-D835 mutation status was available for 306 (41%) and 213 (29%) patients, respectively. Sixty-eight (22%) and 20 (9%) patients had internal tandem duplication and D835 mutations, respectively. Internal tandem duplication was correlated with higher white blood cell and blast counts, lactate dehydrogenase, relapse-risk score, fever, hemorrhage, coagulopathy, BCR3 isoform, M3 variant subtype, and expression of CD2, CD34, human leukocyte antigen-DR, and CD11b surface antigens. The FLT3-D835 mutation was not significantly associated with any clinical or biological characteristic. Univariate analysis showed higher relapse and lower survival rates in patients with a FLT3-internal tandem duplication, while no impact was observed in relation to FLT3-D835. The prognostic value of the FLT3-internal tandem duplication was not retained in the multivariate analysis. FLT3-internal tandem duplication mutations are associated with several hematologic features in acute promyelocytic leukemia, in particular with high white blood cell counts, but we were unable to demonstrate an independent prognostic value in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens.

Abstract 1609: In vivo and ex vivo comparison of two human AML bone marrow engraftment mouse models using fire fly luciferase expressing MOLM-13 and MV4-11 cells

Abstract Acute myeloid leukemia (AML) is the most common leukemia affecting adults. In 70-90% of all AML patients FLT3, a receptor tyrosine kinase which plays an important role in normal hematopoiesis, is highly expressed. Furthermore, in about 30% of all AML patients activating internal tandem duplications (ITDs) or point mutations within the FLT3 gene have been identified. FLT3, therefore, is one of the most promising drug targets for the treatment of AML. Most often novel FLT3 protein kinase inhibitors are tested in subcutaneous xenograft mouse models using various human AML cell lines. To generate more relevant, orthotopic AML in vivo models, we stably transduced the human AML cell lines MOLM-13 and MV4-11 with fire fly luciferase (MOLM-13-Luci and MV4-11-Luci) suitable for in vivo bioluminescence imaging. MOLM-13 carries a heterozygous and MV4-11 a homozygous FLT3 internal tandem duplication (ITD) mutation. In vivo growth of MOLM-13-Luci and MV4-11-Luci cells after subcutaneous implantation in NMRI nude mice proofed that stable expression of luciferase did not alter growth characteristics of these cells. To initiate orthotopic growth, both AML cell lines were implanted intravenously via the tail vein into NOD SCID mice after Cyclophosphamide treatment. Cyclophosphamide facilitates engraftment of the human cells by reducing the endogenous bone marrow cell population. Approximately two (MOLM-13-Luci), or three (MV4-11-Luci) weeks after implantation, luciferase activity was detectable in the pelvic region of the mice indicating growth of both tumor cell lines. Within the next weeks luciferase activity was also seen in additional parts of the mouse at different levels. After necropsy a set of different organs and tissues was analysed ex vivo for luciferase activity. The pattern of metastasising tumor cells was compared in both models. Furthermore, detection of human Ki-67 protein using appropriate human specific antibodies allowed verification of growth of MOLM-13-Luci and MV4-11-Luci cells in different tissues. Sutent, a small-molecule kinase inhibitor, totally abolished tumor growth of both orthotopically growing tumors, indicating that both in vivo models are highly suitable for the characterisation of novel drugs directed against AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1609. doi:10.1158/1538-7445.AM2011-1609

Pre-natal, clonal origin of t(1;11)(p32;q23) acute lymphoblastic leukemia in monozygotic twins

Aim Observation of identical genetic changes in leukemia cells from monozygotic twin pairs has provided evidence for the in utero single clonal origin hypothesis of leukemia, with intraplacental metastasis the basis for concordance. Investigation of this rare mixed lineage leukemia ( MLL) cytogenetic abnormality aims to provide further evidence of the genetic changes that underpin this aggressive form of leukemia in infants. Method The clinical features of a monozygotic infant twin pair with acute lymphoblastic leukemia (ALL) are reported. Banded chromosomal analysis and fluorescent in situ hybridization were used for cytogenetic characterization of the leukemic cells. Immunophenotype was determined by flow cytometry and polymerase chain reaction was used to determine the presence of FLT3-D835/I836 and FLT3-internal tandem duplication (ITD) mutations. Results The twins were seven weeks of age at diagnosis. Both had cytogenetic evidence for the t(1;11)(p32;q23) translocation. Trisomy X was present in a subpopulation of cells in one twin. Immunophenotypic profile at diagnosis was consistent with B precursor ALL (CD19, CD24, CD33 positive, weak CD13 positivity, CD10 negative) and both were negative for FLT3-D835/I836 and FLT3-ITD mutations. Conclusions This is the first report of monochorionic monozygotic twins harboring the t(1;11)(p32;q23) translocation. Identification of this rare translocation in both twins, indicates a common stem line and provides further evidence for the intrauterine monoclonal origin for infant ALL with concordance explained by the shared circulation. Genetic diversity was observed in a subpopulation of cells from one twin at diagnosis. We must now utilize the sophisticated molecular biology tools available to capture changes at the genome-wide level to gain further insight into the complex events contributing to MLL leukemogenesis in infants.

Up-regulation of homeodomain genes, DLX1 and DLX2, by FLT3 signaling

Activating mutations in fms-like tyrosine kinase-3 (FLT3) are frequent in acute myeloid leukemia and represent both a poor prognostic feature and a therapeutic target. We have identified a previously unrecognized downstream effect of FLT3 activation, namely up-regulation of the homeodomain genes, DLX1 and DLX2. MV4;11 cells with FLT3-internal tandem duplication mutation, RS4;11 cells with wild-type FLT3 and blasts from patients with acute myeloid leukemia were used to pursue the relation between FLT3, DLX1/2 and transforming growth factor-β (TGFβ). Real-time quantitative reverse transcriptase polymerase chain reaction, western blot and reverse-phase protein array were performed to detect changes in gene and protein expression. RNA interference and MTS assays were used to study the interaction of PKC412, FLT3 inhibitor and TGFβ1. A direct relationship between FLT3 activity and DLX1/2 expression was revealed by both inhibition and up-regulation of FLT3 signaling in MV4;11 and RS4;11 cell lines, respectively, in isolated blast cells from patients with acute myeloid leukemia, and in reverse-phase protein array assays of samples from patients with acute myeloid leukemia. Mechanistically, the link between FLT3 and DLX1 expression appears to involve MAPK signaling through the ERK and JNK pathways. To determine whether elevated DLX1 had a functional consequence, we explored the reported inhibition by DLX1 on TGFβ/Smad signaling. Indeed, TGFβ responses were blunted by FLT3 activation in a DLX1-dependent manner and FLT3 inhibition resulted in a time-dependent increase in nuclear phospho-Smad2. These findings suggest that alterations in DLX1/2 contribute to the biological consequences of FLT3 activation.

Prognostic impact, concurrent genetic mutations, and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients: further evidence for CEBPA double mutant AML as a distinctive disease entity

AbstractWe evaluated concurrent gene mutations, clinical outcome, and gene expression signatures of CCAAT/enhancer binding protein alpha (CEBPA) double mutations (CEBPAdm) versus single mutations (CEBPAsm) in 1182 cytogenetically normal acute myeloid leukemia (AML) patients (16-60 years of age). We identified 151 (12.8%) patients with CEBPA mutations (91 CEBPAdm and 60 CEBPAsm). The incidence of germline mutations was 7% (5 of 71), including 3 C-terminal mutations. CEBPAdm patients had a lower frequency of concurrent mutations than CEBPAsm patients (P < .0001). Both, groups were associated with a favorable outcome compared with CEBPAwt (5-year overall survival [OS] 63% and 56% vs 39%; P < .0001 and P = .05, respectively). However, in multivariable analysis only CEBPAdm was a prognostic factor for favorable OS outcome (hazard ratio [HR] 0.36, P < .0001; event-free survival, HR 0.41, P < .0001; relapse-free survival, HR 0.55, P = .001). Outcome in CEBPAsm is dominated by concurrent NPM1 and/or FLT3 internal tandem duplication mutations. Unsupervised and supervised GEP analyses showed that CEBPAdm AML (n = 42), but not CEBPAsm AML (n = 18), expressed a unique gene signature. A 25-probe set prediction signature for CEBPAdm AML showed 100% sensitivity and specificity. Based on these findings, we propose that CEBPAdm should be clearly defined from CEBPAsm AML and considered as a separate entity in the classification of AML.

Clonal Markers In Relapsed Acute Promyelocytic Leukemia (APL): Clinicopathological Associations and Relation to All-Trans Retinoic Acid (ATRA) Treatment on Intergroup Phase III Trial C9710.

AbstractAbstract 1038The relationships of inherent disease-related characteristics and of treatment to the nature of APL cell clones that emerge at disease relapse are poorly understood. We studied these relationships in 45 patients who relapsed after ATRA/chemotherapy treatment on the non-arsenic trixoxide arm of intergroup phase III trial C9710 (Powell, et al. Blood, Epub). Four variably-expressed APL cell clonal markers were assessed: PML-RARA ligand binding domain mutations (LBD-M), FLT3 internal tandem duplication mutations (ITD), FLT3 receptor tyrosine kinase mutations (D835; RTK-M) and additional chromosome abnormalities (ACA), i.e., in addition to the hallmark t(15;17). The methods for mutation analysis of PML-RARA and FLT3 have been reported and were RNA transcript-initiated supplemented by selected quantitative FLT3 mutation analyses initiated from DNA. Karyotype data were derived by standard cytogenetic methods. Four patients were excluded from analysis because the sample level of PML-RARA, as determined by quantitative RT-PCR, was insufficient to exclude possible false-negative mutation results. In 41 evaluable patients, the marker incidences were: LBD-M, 44%; ITD, 37%; RTK-M, 12%; ACA (22 tested), 59%. At presentation, the corresponding incidences were: LBD-M, 0% (by high-sensitivity testing a minor subclone of the relapse mutation was found in 2/7 patients tested); ITD, 43% (37 tested); RTK-M, 22% (37 tested); ACA, 24% (34 tested). The low frequency of RTK-M impeded further analysis. The other markers, using relapse determinations, were assessed for potential associations between the markers and with the following parameters: age, sex, presenting WBC count, PML-RARA type, time to relapse, relapse on or off ATRA treatment (on = taking or within 30 days of discontinuing) and post-relapse survival. The most essential positive findings are summarized below:Association of Marker with Parameter or Other MarkerCases with ParameterCases without Parameterp-valuePretreatment parameters:LBD-M with WBC count < 5,000/uL12/18 (67%)7/23 (30%)0.030ITD with WBC count >10,000/uL10/15 (67%)5/26 (19%)0.006ITD with S-form PML-RARA15/15 (100%)10/26 (38%)<0.0001Relapse parameters:ITD with lack of ACA1/7 (14%)12/15 (80%)0.007LBD with relapse On-ATRA11/18 (61%)7/23 (30%)0.064Off-ATRA only: LBD with lack of ITD0/7 (0%)9/16 (56%)0.019The results confirm reports, which tested pretreatment samples, of a strong association between FLT3ITD mutations present at relapse and high WBC count and S-form PML-RARA. At relapse, there was, also, divergent selection of ITD-harboring vs ACA-harboring clones. LBD-M occurred more frequently in patients with low presenting WBC counts. They, also, occurred in patients who relapsed while on ATRA, consistent with an ATRA selective role for clones harboring LBD mutations. A significant segregation of patients with LBD-M or ITD was observed after relapse off ATRA treatment (p = 0.019), while no segregation was observed in overall relapse patients (p = 0.346) or after relapse on ATRA (p = 0.316). In 2 patients who relapsed on ATRA, quantitative analysis indicated that LBD-M and ITD mutations must be present in the same APL cell clone. There were no significant survival differences related to any clonal marker (post-relapse survival = 66%). Although the small number of cases is cautionary, the overall data suggest that LBD-M may have an overriding, dominant APL clone selection role in relapse that occurs on ATRA therapy. Other inherent clinicopathologic characteristics of APL may predispose to divergent molecular pathways of disease progression and relapse in patients alternatively harboring clones with LBD-M or ITD after the termination of ATRA selection pressure. Disclosures:No relevant conflicts of interest to declare.

Effective Targeting of Acute Myeloid Leukemia (AML) Harboring the FLT3 ITD Mutation through the Axl/Gas6 Pathway

AbstractAbstract 500Approximately 30% of AML patients harbor activating mutant forms of Fms-like tyrosine kinase-3 (FLT3) such as internal tandem duplications (ITD). To date, targeting FLT3 ITD+ AML patients with FLT3 inhibitors has not improved outcomes, suggesting alternative strategies should be pursued. Axl is a type III receptor tyrosine kinase (RTK) which is, along with its ligand Gas6, over-expressed in AML leukemic blasts and correlated with a poor prognosis. In the present study, we asked if blocking the autocrine Axl/Gas6 pathway could impact aberrant proliferation and survival of FLT3 ITD+ AML cells. We used a soluble Axl receptor, termed Axl-Fc, to block autocrine binding of Gas6 to Axl. The FLT3 ITD+ AML cell line MV4;11 was treated with Axl-Fc in vitro for 4 days and showed a significant reduction in cell proliferation (4.1 ± 0.73 × 106 cells for isotype control (IC) Fc vs. 2.1 ± 0.56 × 106 cells for Axl-Fc, p < 0.05). This growth inhibition resulted from both G1/S cell cycle arrest (51.5 ± 9.5 % at the S phase for IC Fc vs. 2.75 ± 1.5 % for Axl-Fc, p < 0.01), as well as increased apoptosis over control (63.2 ± 8.5 % Annexin V-positive for Axl-Fc vs. 25.7 ± 4.0 % for IC Fc, p < 0.01). Axl-Fc-treated MV4;11 and primary FLT3 ITD+ AML blasts showed elevated expression of granulocyte-specific genes compared to IC Fc-treated cells ([MV4;11: lysozyme (48.6 ± 9.5-fold), myeloperoxidise (6.8 ± 0.26-fold), and elastase 2 (2.7 ± 0.45-fold); p < 0.05] [primary FLT3 ITD+ AML blasts: elastase 2 (2.4 ± 0.46-fold) and lysozyme (2.7 ± 0.56-fold); p < 0.05]). Morphological analysis showed Axl-Fc could relieve the block in myeloid differentiation for the MV4;11 AML cell line. Treatment with Axl-Fc increased the expression levels of two myeloid transcription factors in MV4;11 (C/EBPα: 16.5 ± 2.6-fold over that seen with IC Fc, p < 0.01; PU.1: 3.9 ± 0.4-fold over that seen with IC Fc, p < 0.01) and in primary FLT3 ITD+ AML blasts (C/EBPα: 2.2 ± 0.31-fold over that seen with IC Fc, p < 0.05; PU.1: 2.1 ± 0.36-fold over that seen with IC Fc, p < 0.01). A co-immunoprecipitation study demonstrated that Axl associated with phosphorylated FLT3 in MV4;11 cells. Treatment with Axl-Fc abrogated both the FLT3 phosphorylation and co-precipitation. These in vitro data suggest that the Axl/Gas6 pathway contributes to AML, at least in part, through positively regulating the phosphorylation of FLT3, and that targeting the Axl/Gas6 pathway may diminish phosphorylation of both of these RTKs in AML. In vivo, using a subcutaneous tumor xenograft model, administration of Axl-Fc into MV4;11 tumor-bearing SCID mice significantly suppressed the growth of tumor mass (0.52 ± 0.1 g for PBS-treated mice (n=7); 0.26 ± 0.04 g for IC Fc (n=7) vs. 0.08 ± 0.07 g for Axl-Fc (n=10), p < 0.01). Axl-Fc also diminished the engraftment of primary FLT3 ITD+ AML blasts into SCID mice at 8 weeks (WBC: 4.75 ± 0.59 × 106 cells per ml for IC Fc (n=4) vs. 1.6 ± 0.16 × 106 for Axl-Fc (n=5), p < 0.001; 6.9 ± 0.67 % human CD45+ cells for IC Fc vs. 2.5 ± 0.59 % for Axl-Fc, p < 0.001). Finally, we assessed Axl-Fc against the FLT3 inhibitor PKC412 (LC Laboratories, Woburn, MA). In vitro treatment of both MV4;11 and primary FLT3 ITD+ AML blasts with PKC412 induced phosphorylation of Axl and its downstream signaling molecules, such as ERK and AKT, in both. PKC412-treated MV4;11 cells harboring low level of cleaved caspase 3 (i.e., apoptotic resistant) showed a higher level of phospho-Axl, while MV4;11 cells with high level of cleaved caspase 3 possessed less phospho-Axl (39.6 ± 1.4 % phospho-Axl+ for cleaved caspase 3low population vs. 9.7 ± 3.8 % phospho-Axl+ for cleaved caspase 3high population). These results suggest that activation of the Axl/Gas6 pathway could be, at least in part, responsible for drug resistance against FLT3 inhibition. In in vivo experiment, Axl-Fc was more effective than PKC412 (75 mg/kg) at inhibiting the engraftment of primary FLT3 ITD+ AML blasts into SCID mice at 8 weeks (WBC: 2.66 ± 0.36 × 106/ml for PBS (n=5); 1.74 ± 0.19 × 106/ml for PKC412 (n=5) vs. 0.9 ± 0.11 × 106/ml for Axl-Fc (n=5), p = 0.003), while SCID mice treated with Axl-Fc plus PKC412 showed no further reduction of engraftment compared to mice treated with Axl-Fc alone. Survival studies are ongoing. In summary, our study identifies the Axl/Gas6 pathway as an important player for human FLT3 ITD+ AML cell growth and survival. Targeting the Axl/Gas6 pathway may prove as effective or superior to small molecule FLT3 inhibitors for the treatment of this subgroup of AML patients. Disclosures:No relevant conflicts of interest to declare.

Prognostic factors in normal karyotype acute myeloid leukemia in the absence of the FLT3-ITD mutation

Patients with normal karyotype acute myeloid leukemia (NK-AML) without the FLT3 internal tandem duplication ( FLT3-ITD) mutation account for approximately 30% of all AML cases, and exhibit a heterogeneous clinical outcome. Except for NPM1 mutations, prognostic factors in this subgroup of AML are still unclear. Here we explored the impact of immunophenotypic markers along with NPM1 mutations and clinical features on the outcome of 133 FLT3-ITD negative NK-AML patients. CD34 expression was associated with poorer complete remission (CR) rate, disease-free survival (DFS), event-free survival (EFS), and overall survival (OS), whereas CD56 expression adversely affected EFS and OS. In contrast, NPM1 mutations correlated with an improved CR rate, DFS, and EFS. Moreover, males experienced shorter DFS and EFS, while older patients (≥60 years) had shorter EFS. Multivariate analysis of age, gender, NPM1, CD34, and CD56 showed NPM1 mutation was an independent predictor of better CR rate, DFS, and EFS ( P < 0.001, P = 0.003, and P = 0.006, respectively). In addition, older age was associated with shorter DFS and EFS ( P = 0.045 and P = 0.028, respectively), and CD56 positivity predicted shorter EFS ( P = 0.012). Our results confirm the favorable impact of NPM1 mutations and identify the adverse prognostic relevance of CD56 expression in this subgroup of AML.

FLT3 Mutations at Diagnosis and Relapse in Acute Myeloid Leukemia: Cytogenetic and Pathologic Correlations, Including Cuplike Blast Morphology

Acquired mutations in the fms-like tyrosine kinase 3 gene (FLT3) adversely impact relapse risk after chemotherapy in patients with acute myeloid leukemia (AML). The FLT3 mutation status may differ at diagnosis and relapse, suggesting a potential role in chemoresistance, yet few reports have addressed the cytogenetic and pathologic correlates of FLT3 mutations in relapsed AML. To determine FLT3 mutations at diagnosis and relapse in a cohort of adult patients with chemoresistant AML and to correlate mutation status with multiple variables. We retrospectively determined FLT3 internal tandem duplication (FLT3/ITD) and FLT3 tyrosine kinase domain mutations in 50 diagnosis/relapse pairs. We correlated FLT3 status with karyotype, World Health Organization 2008 subtype, white blood cell count, biopsy cellularity, blast percentage, and the presence of invaginated ("cuplike") blast nuclei. In 11 of 50 patients (22%) the FLT3 mutation status differed at relapse and diagnosis, with a trend toward gain of FLT3/ITD (n = 7) and loss of FLT3 tyrosine kinase domain (n = 5) mutations. FLT3-mutated AMLs correlated with the World Health Organization 2008 subtype, AML, not otherwise specified, hyperproliferative features at diagnosis and relapse, and cytogenetic evolution. FLT3-wild type AMLs correlated with the subtype AML with myelodysplasia-related changes and frequently had adverse presentation karyotypes. Cuplike blast morphology was associated with FLT3/ITD+ status and with high mutation levels. Four of 7 patients with relapse-only FLT3/ITD mutations exhibited cuplike blasts at relapse after being noncuplike at diagnosis. In addition to well-known correlates in pretreatment specimens, FLT3 mutation status has pathologic and cytogenetic significance at relapse. A shift to cuplike blast morphology at relapse may herald emergence of a previously undetected FLT3/ITD mutation.

Abstract 4492: BEZ235, a dual PI3K/mTOR inhibitor, enhances the activity of the multikinase inhibitor sorafenib in acute myeloid leukemia (AML)

Abstract The PI3K/Akt/mTOR and RAS/RAF/MEK/ERK signaling pathways are activated in 50 to 75% of primary AML cases, which have been associated with poor long-term outcome. The class I PI3K isoforms α, β, δ are expressed in nearly all primary childhood AML blast samples (Ross, Blood 2004), providing a potential target to circumvent aberrant signaling in this pathway. However, because of cross-talk and redundancy of signal transduction pathways, rational combinations of inhibitors may represent an advantageous treatment strategy for AML. We have previously shown that sorafenib, a multikinase inhibitor in development for the treatment of AML, effectively inhibits phosho-ERK in AML cells, with limited inhibition of AKT signaling (Hu, Mol Cancer Ther, 2008). Here we evaluated the anti-leukemic activity of the dual PI3K/mTOR inhibitor BEZ235 as a single agent and in combination with sorafenib at in vivo relevant concentrations (≤ 1 µM BEZ235 and ≤ 10 µM sorafenib) in four AML cell lines (HL60, U937, THP-1, and OCI-AML3), and 9 primary childhood AML blast samples (7 with wild-type FLT3 and 2 with FLT3 internal tandem duplication mutations). We first evaluated the single-agent activity of BEZ235 in AML cells in suspension culture. After exposure to increasing concentrations of BEZ235 for 72h, IC50 values in a MTT assay ranged from 0.125 to 0.5 µM, but maximal growth inhibition did not exceed 40% of untreated controls. After exposure to BEZ235 1 µM for 72 h, an increase in apoptosis (Annexin V positive cells) to 30% was observed in U937 and OCI-AML3 cells, and inhibition of proliferation (EdU incorporation) was observed in U937 and THP-1 cells; no change in cell cycle distribution was observed. Phospho-Akt was inhibited by 50% to 80% of untreated controls in 3 cell lines (U937, THP-1, and HL-60) treated with BEZ235 1 µM for 1 h. We next examined the anti-leukemic activity of the combination of BEZ235 and sorafenib (simultaneous exposure for 72 h) in AML cell lines and primary blast samples that were co-cultured in direct contact with bone-marrow-derived mesenchymal stromal cells. In AML cell lines, sorafenib 10 µM induced apoptosis in twice as many cells (39% to 54%) than BEZ235 1.0 µM (17% to 23%); however, the combination increased apoptosis to 50 to 68% in all cell lines. Enhanced apoptosis was also observed in all primary childhood AML blasts samples treated with the drug combination compared to single agent treatment. In conclusion, BEZ235 has limited single-agent activity but enhances the activity of sorafenib in vitro in AML cell lines and primary blast samples. Studies are ongoing to evaluate the drug combination in a xenograft model of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4492.