FLT3-activating mutations in acute promyelocytic leukaemia: a rationale for risk-adapted therapy with FLT3 inhibitors

Our understanding of the genetic basis of acute myeloid leukaemias has been enhanced through cloning of recurring chromosomal translocation breakpoints. However, the remarkable observation, more than a decade ago, that all-trans retinoic acid (ATRA) induced remission in patients with t(15;17) acute promyelocytic leukaemia (APL) was a driving force in the subsequent cloning and characterization of the PML-RARalpha fusion that is causally implicated in the pathogenesis of this disease. Major improvements in treatment and outcome of APL patients have been made since that time by incorporating ATRA in conventional chemotherapy but 30% of APL patients still succumb to complications of their disease or their therapy. Recent information that the haematopoietic receptor tyrosine kinase FLT3 is mutated in about 30% of APL patients suggests strategies for further improving treatment and outcome in this subset of APL patients using small-molecule inhibitors of FLT3. The role of FLT3 mutations in APL and other AML will be discussed.     

Calcitonin-related peptides in patients with acute leukemia: association of human calcitonin with poor prognosis

Elevated serum levels of peptides hormones in patients with acute leukemia and production of these agents by the leukemic blasts have been described. In 77 patients with acute leukemia the influence of common risk factors and elevations of serum levels of calcitonin-related peptides on clinical outcome was evaluated. By multivariate analysis, only age and elevated serum level of h-CT were found to be significantly correlated to survival. CGRP and s-CT showed no influence on outcome. Closer inspection of the clinical course of these patients showed that patients with elevated h-CT are not likely to survive the first 4 weeks after diagnosis. The possibility that this hormone may influence the biological behavior of the leukemic cells is discussed.     

FLT3 mutations in childhood acute lymphoblastic leukemia

Activating mutations of the FLT3 receptor tyrosine kinase are common in acute myelogenous leukemia (AML) but are rare in adult acute lymphoblastic leukemia (ALL). We have recently shown that FLT3 is highly expressed and often mutated in ALLs with rearrangement of the mixed lineage leukemia (MLL) gene on chromosome 11q23. Because hyperdiploid ALL samples also show high-level expression of FLT3, we searched for the presence of FLT3 mutations in leukemic blasts from 71 patients with ALL. The data show that approximately 25% (6 of 25) of hyperdiploid ALL samples possess FLT3 mutations, whereas only 1 of 29 TEL/AML1-rearranged samples harbored mutations (P =.04, Fisher exact test). Three mutations are novel in-frame deletions within a 7-amino acid region of the receptor juxtamembrane domain. Finally, 3 samples from patients whose disease would relapse harbored FLT3 mutations. These data suggest that patients with hyperdiploid or relapsed ALL might be considered candidates for therapy with newly described small-molecule FLT3 inhibitors.     

Biological characteristics of newly diagnosed poor prognosis acute myelogenous leukemia

A pilot study was conducted of the biological characteristics of the leukemia cells of newly diagnosed patients with poor prognosis acute myelogenous leukemia (AML). This study included measurements of the pretherapy proliferative rate of the leukemia cells in vivo, assessment of differentiation in vivo during remission induction therapy, and the level of expression of the fms, myc, and IL1β genes in pretherapy leukemia cells. Short cell cycle times were characteristic of the best prognostic category and were associated with a rapid reduction in marrow leukemia cells in cytosine arabinoside (araC)-sensitive patients. Expression of c-fms was associated with rapid reduction in marrow leukemia cells during araC therapy and with a successful treatment outcome. Expression of the IL1β gene was associated with short remissions. These studies suggest that when compared to newly diagnosed standard prognosis AML, the leukemia of poor prognosis patients is more likely to exhibit long cell cycle times, low levels of fms expression, and is less likely to be associated with myeloid differentiation during remission induction therapy. © 1993 Wiley-Liss, Inc.     

Importance of early detection and follow-up of FLT3 mutations in patients with acute myeloid leukemia

Mutations in the fms-like tyrosine kinase 3 (FLT3) gene, such as internal tandem duplication (FLT3/ITD) in the juxtamembrane domain and point mutations in the tyrosine kinase domain, are the most common abnormalities in acute myeloid leukemia (AML). FLT3/ITD and FLT3/D835 mutations were analyzed in 113 Serbian adult AML patients using polymerase chain reaction. Twenty patients were found to be FLT3/ITD positive (17.7%). The mutations occurred most frequently in M5 and M0 subtypes of AML. They were mainly associated with the normal karyotype. All patients harboring FLT3/ITD had a higher number of white blood cells than patients without it (p = 0.027). FLT3/ITD mutations were associated with lower complete remission (CR) rate (χ ²= 5.706; p = 0.017) and shorter overall survival (OS; Log rank = 8.76; p = 0.0031). As for disease-free survival, the difference between FLT3/ITD-positive and FLT3/ITD-negative patients was not statistically significant (Log rank = 0.78; p = 0.3764). In multivariate analysis, the presence of FLT3/ITD mutations was the most significant prognostic factor for both OS and CR rate (p = 0.0287; relative risk = 1.73; 95% CI = 1.06–2.82). However, in the group of patients with the intermediate-risk karyotype, the mere presence of FLT3/ITD was not associated with inferior clinical outcome. FLT3/D835 point mutation was found in four patients (3.5%) only. Follow-up of the FLT3/ITD-positive patients revealed stability of this mutation during the course of the disease. However, changes in the pattern of FLT3/D835 mutations in initial and relapsed AML were observed. Our results indicate an association of FLT3/ITD with the adverse outcome in AML patients treated with standard induction chemotherapy. Because FLT3/ITD mutation is a target for specific therapeutic inhibition, its early detection could be helpful in clinical practice. Ms. Colovic and Ms. Tosic contributed equally to this work.     

FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia

The prevalence and significance of genetic abnormalities in older patients with acute myeloid leukemia (AML) are unknown. Polymerase chain reactions and single-stranded conformational polymorphism analyses were used to examine 140 elderly AML patients enrolled in the Southwest Oncology Group study 9031 for FLT3, RAS, and TP53 mutations, which were found in 34%, 19%, and 9% of patients, respectively. All but one of the FLT3 (46 of 47) mutations were internal tandem duplications (ITDs) within exons 11 and 12. In the remaining case, a novel internal tandem triplication was found in exon 11. FLT3 ITDs were associated with higher white blood cell counts, higher peripheral blast percentages, normal cytogenetics, and less disease resistance. All RAS mutations (28 of 28) were missense point mutations in codons 12, 13, or 61. RAS mutations were associated with lower peripheral blast and bone marrow blast percentages. Only 2 of 47 patients with FLT3 ITDs also had a RAS mutation, indicating a significant negative association between FLT3 and RAS mutations (P =.0013). Most TP53 mutations (11 of 12) were missense point mutations in exons 5 to 8 and were associated with abnormal cytogenetics, especially abnormalities in both chromosomes 5 and 7. FLT3 and RAS mutations were not associated with inferior clinical outcomes, but TP53 mutations were associated with a worse overall survival (median 1 versus 8 months, P =.0007). These results indicate that mutations in FLT3, RAS, or TP53 are common in older patients with AML and are associated with specific AML phenotypes as defined by laboratory values, cytogenetics, and clinical outcomes. (Blood. 2001;97:3589-3595)     

FLT3 tyrosine kinase domain mutations are biologically distinct from and have a significantly more favorable prognosis than FLT3 internal tandem duplications in patients with acute myeloid leukemia

The prognostic impact of tyrosine kinase domain (TKD) mutations of the fms-like tyrosine kinase-3 (FLT3) gene in acute myeloid leukemia (AML) is currently uncertain. To resolve this issue we screened 1107 young adult nonacute promyelocytic leukemia AML patients with known FLT3 internal tandem duplication (ITD) status for FLT3/TKDs; they were detected in 127 (11%) cases. Mutations were associated with a high white cell count (P =.006) and patients with inv(16) (P = .005) but were infrequent in patients with adverse cytogenetics and secondary AML. Overall survival (OS) at 5 years was 53% and 37% for FLT3/TKD mutant and wild-type patients respectively (odds ratio, 0.72; 95% confidence interval, 0.58 to 0.89; P = .002). For both the cumulative incidence of relapse and OS the difference in outcome between FLT3/ITDs and FLT3/TKDs was highly significant (P < .001). In multivariate analysis, impact of FLT3/TKDs on OS when including all mutant-positive patients was not significant, but patients with high-level mutations (more than 25% mutant) had a significantly improved outcome (P = .004). The novel finding that biologically distinct activating mutations of the same gene can be associated with markedly different clinical outcomes has implications for risk stratification and therapy and is significant to the understanding of chemoresistance in AML.     

Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics,FAB subtype,and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease

FLT3 length mutation (FLT3-LM) is a molecular marker potentially useful for the characterization of acute myeloid leukemia (AML). To evaluate the distribution of FLT3-LM within biologic subgroups, we screened 1003 patients with AML at diagnosis for this mutation. FLT3-LM was found in 234 (23.5%) of all patients and thus is the most frequent mutation in AML described so far. Of all positive patients, 165 (70.5%) revealed a normal karyotype. Of the 69 patients with chromosome aberrations, 24 (34.8%) had a t(15;17). The mutation was rare in AML with t(8;21), inv(16) 11q23 rearrangements, and complex karyotypes. FLT3-LM was not distributed equally within different French-American-British (FAB) subtypes and was correlated with a high peripheral blood count in FAB M1, M2, and M4 (P <.0001). In addition, the median age of patients with the mutation was lower (54.9 vs 57.6 years; P =.043), and, at a ratio of 1.36:1 (P =.023), the mutation was more frequent in females than in males. Within the AMLCG study, FLT3-LM was of intermediate prognostic significance. The complete remission rate of 70.3% in patients with FLT3-LM was similar to that (70.4%) in patients without FLT3-LM. Overall survival was not different between patients with or without FLT3-LM. In contrast, patients with FLT3-LM had a significantly shorter event-free survival (7.4 vs 12.6 months; P =.0072) because of a higher relapse rate. Besides the importance of FLT3-LM for biologic and clinical characterization of AML, we show its value as a marker for disease monitoring based on 120 follow-up samples of 34 patients.     

FLT3突变与白血病预后的研究进展

FLT3又称FLK-2、STK-1,为Ⅲ型受体酪氨酸激酶成员之一,与其配体在正常造血及免疫系统的发育中起重要调节作用.FLT3在急性髓细胞白血病(AML)及急性B淋巴细胞白血病(B-ALL)中有过度表达,其突变是AML中最常见的基因异常.最近研究表明,FLT3过度表达与其突变均可作为预后不良因素,FLT3及其突变可能作为微小残留病灶(MRD)的标志.本文就FLT3及其突变在各型白血病中对预后的意义作一综述.     

Prospective study of 174 de novo acute myelogenous leukemias according to the WHO classification: subtypes, cytogenetic features and FLT3 mutations

We report a prospective study of 174 unselected adult de novo acute myeloid leukemia (AML) cases diagnosed using the WHO classification. Of those, 57 (33%) were AML with recurrent cytogenetic abnormalities, 41 were (24%) AML with multilineage dysplasia, 74 (42%) were AML not otherwise categorized, and two were acute leukemias of ambiguous lineage. Clonal cytogenetic abnormalities were detected in 64% of the WHO AML cases with t(15;17) (15%), t(8;21) (12%), +8 (11%), -7/del7q (8%) and del9q (5%) being the most common ones. The FLT3/ITD mutations (FMS-like tyrosine kinase 3/internal tandem duplication) were observed in 12% of the WHO AML cases, which is much lower than ones in the literature, while the 6% incidence of the FLT3-activating loop mutations (either FLT3/D835 or FLT3/I836) was comparable with others. Both mutations were associated with leukocytosis. Our study also suggests that the FLT3 mutations are biomarkers independent of cytogenetic characteristics.     

Simultaneous activation of multiple signal transduction pathways confers poor prognosis in acute myelogenous leukemia

Deregulation of signal transduction pathways (STPs) may promote leukemogenesis by conferring cell proliferation and survival advantages in acute myelogenous leukemia (AML). Several agents targeting STPs are under development; however, redundancy and cross-talk between STPs could activate multiple downstream effectors and this could negate the effect of single-target inhibition. The frequency of concurrent activation of multiple STPs in AML and the prognostic relevance of STP activation in AML are unknown. STP protein expression (PKCalpha, ERK2, pERK2, AKT, and pAKT) was measured by Western blot in samples from 188 patients with newly diagnosed, untreated AML. In univariate and multivariate analysis high levels of PKCalpha, ERK, pERK, and pAKT, but not AKT, were adverse factors for survival as was the combination variable PKCalpha-ERK2&pERK2-pAKT. Survival progressively decreased as the number of activated pathways increased. Patients were more likely to have none or all 3 pathways activated than was predicted based on the frequency of individual pathway activation, strongly suggesting that cross-activation occurred. Simultaneous activation of multiple STPs is common in AML and has a progressively worse adverse effect on prognosis. It is thus likely that only combinations of agents that target the multiply activated STPs will be beneficial for patients with AML.     

N-ras gene point mutations in childhood acute lymphocytic leukemia correlate with a poor prognosis

Ras genes can be altered by point mutations at critical portions of their coding regions to acquire transforming ability in vitro. These point mutations have been detected in a variety of human malignancies. However, their relevance for the clinical and biologic behavior of the subgroups of patients exhibiting these mutations in unclear. We analyzed 100 patients with childhood acute lymphocytic leukemias (ALLs) for point mutations of exons 1 and 2 of all three ras genes (H-ras, K- ras, and N-ras) by polymerase chain reaction and a combination of oligonucleotide hybridization and direct DNA sequencing. A 6% incidence of N-ras gene mutations was detected, all of which occurred at different nucleotides of codons 12 or 13 of N-ras. When correlating presence of ras mutations with the clinical and biologic features and the clinical outcome of these cases, a significantly higher risk for hematologic relapse (P = .01) and a trend toward a lower rate of complete remission (P = .07) was noted. The two groups did not differ in any of the known high-risk factors of ALL. These results suggest that presence of an N-ras mutation in children with ALL may be an independent predictor for worse clinical outcome and therefore may have therapeutic implications; further studies to confirm these findings are required because of the small number of patients with N-ras mutations.     

Clinical implications of non-A-type NPM1 and FLT3 mutations in patients with normal karyotype acute myeloid leukemia

Mutations in the nucleophosmin (NPM1) and fms-like tyrosine kinase-3 (FLT3) genes are the most commonly observed mutations in patients with normal-karyotype acute myeloid leukemia (AML-NK). We analyzed the prognostic effects and interactions of these mutations in 201 AML-NK patients. NPM1 and FLT3 mutations were found in 38.3 and 24.9% of AML-NK patients, respectively. NPM1 mutations (NPM1mut), especially in patients without FLT3 mutations (FLT3mut), were associated with a favorable outcome. However, NPM1mut did not affect survival. FLT3mut tended to be associated with a poor survival outcome. FLT3mut showed no prognostic effects in patients with A-type NPM1mut. However, FLT3mut were associated with a significantly worse prognosis in patients with non-A-type NPM1mut. The prognostic interaction between the NPM1 and FLT3 mutations was significant in patients with non-A-type NPM1mut.     

Nucleophosmin mutations in Chinese adults with acute myelogenous leukemia

Recently, mutations in the nucleophosmin (NPM1) gene were detected in 50–60% of adult acute myelogenous leukemia (AML) patients, mainly with a normal karyotype. In this study, we detected typical NPM1 mutations (types A, B, D) in untreated Chinese AML patients using real-time quantitative polymerase chain reaction (RQ-PCR) followed by sequence analysis. The detection rate of NPM1 mutations in 220 AML patients was 16.4%, including 107 (14.2%) with the French–American–British (FAB) subtype M2, 43 (2.3%) with M3, and 52 (30.8%) with M4/M5. Only one case each with an NPM1 mutation was detected in four M0, seven M1, five M6, and two M7 cases. Eight patients were followed up after treatment, and five patients in hematologic remission continued to test negative for NPM1 mutations within 2–14 months of follow-up. Sequence analysis revealed that all the 36 positive cases were heterozygous for the mutation with 4-bp insertions at nt 959; the 36 cases included 29 (80.6%) cases with type A, four (11.1%) cases with type B, and one rare DD-3 mutation. We also detected two new mutations, namely, CTCG and CAAG insertions, named BJ-01 and BJ-02, respectively. Further, 38.9% (14/36) patients with NPM1 mutations simultaneously exhibited internal tandem duplications in the FLT3 gene, and 66.7% (22/33) patients did not express CD34. The results demonstrated that RQ-PCR was a reliable and sensitive method for detecting NPM1 mutations, for screening AML, and for the quantitative analysis of minimal residual diseases.     

Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia

Internal tandem duplication of the FLT3 gene and point mutations of the N-RAS gene are the most frequent somatic mutations causing aberrant signal-transduction in acute myeloid leukemia (AML). However, their prognostic importance is unclear. In this study, their prognostic significance was analyzed in 201 newly diagnosed patients with de novo AML except acute promyelocytic leukemia. Three patients had mutations in both genes, 43 had only the FLT3 gene mutation, 25 had only the N-RAS gene mutation, and 130 had neither. These mutations seemed to occur independently. Both mutations were related to high peripheral white blood cell counts, and the FLT3 gene mutation was infrequently observed in the French-American-British (FAB)-M2 type. AML cases with wild FLT3/mutant N-RAS had a lower complete remission (CR) rate than those with wild FLT3/wild N-RAS, whereas the presence of mutant FLT3 did not affect the CR rate. Univariate analysis showed that unfavorable prognostic factors for overall survival were age 60 years or older (P =.0002), cytogenetic data (P =.002), FAB types other than M2 (P =.002), leukocytosis over 100 +/- 10(9)/L (P =.003), and the FLT3 gene mutation (P =.004). However, the N-RAS gene mutation was only a marginal prognostic factor (P =.06). For the subjects under 60 years old, multivariate analysis showed that the FLT3 gene mutation was the strongest prognostic factor (P =.008) for overall survival. The FLT3 gene mutation, whose presence is detectable only by genomic polymerase chain reaction amplification and gel electrophoresis, might serve as an important molecular marker to predict the prognosis of patients with AML.     

FLT3-activating mutations are associated with poor prognostic features in AML at diagnosis but they are not an independent prognostic factor

FLT3: gene alterations (internal tandem duplications - ITDs - and D835 mutations) are thought to be associated with poor-risk acute myeloid leukemia (AML). However, not all studies confirm this association, so it is still a matter of debate. Moreover, their association with other molecular abnormalities is less studied. We have investigated the presence of FLT3-ITD and D835 mutations in AML patients and their correlation with clinical and biological disease characteristics. The presence of ITD was analyzed in diagnostic samples of 176 AML patients and the D835 mutation in 135 of these patients. In all these patients, the presence of four well-known molecular abnormalities were also simultaneously characterized: PML/RARalpha, AML1/ETO, CBFbeta/MYH11 and MLL rearrangements. In all, 41 (23%) patients harbored FLT3 mutations, with 34 (19.3%) of them positive for the ITD, and seven (5%) positive for the D835 mutation. Of the acute promyelocytic leukemia (APL) patients, 16 (27%) showed FLT3 mutations, more frequently in M3 hypogranular cases (62% versus 17%, P=0.001) and cases with the short (bcr3) PML-RARalpha isoform (69%, P=0.002). In contrast, FLT3 was never altered in patients with inv(16), t(8;21) or 11q23 abnormalities. FLT3 mutations were significantly associated with some negative prognostic features at diagnosis (leukocytosis, high blast-cell percentage, and elevated LDH values), but they were not associated with different disease-free or overall survival. Therefore, we confirm a high frequency of FLT3 mutations in APL and in adult AML without recurrent cytogenetic translocations. In addition, they were not found as independent prognostic factors although associated with several adverse features at diagnosis.     

Studies of FLT3 mutations in paired presentation and relapse samples from patients with acute myeloid leukemia: implications for the role of FLT3 mutations in leukemogenesis,minimal residual disease detection,and possible therapy with FLT3 inhibitors

FLT3 mutations, either internal tandem duplications (ITDs) or aspartate residue 835 (D835) point mutations, are present in approximately one third of patients with acute myeloid leukemia (AML) and have been associated with an increased relapse rate. We have studied FLT3 mutations in paired presentation and relapse samples to ascertain the biology of these mutations and to evaluate whether they can be used as markers of minimal residual disease. At diagnosis, 24 patients were wild-type FLT3, and 4 acquired a FLT3 mutation at relapse (2 D835(+), 2 ITD(+)), with a further patient acquiring an ITD at second relapse. Of 20 patients positive at diagnosis (18 ITD(+), 2 D835(+)), 5 who were all originally ITD(+) had no detectable mutation at relapse, as determined by a sensitive radioactive polymerase chain reaction. One of these patients had acquired an N-Ras mutation not detectable at presentation. Furthermore, another patient had a completely different ITD at relapse, which could not be detected in the presentation sample. These results indicate that FLT3 mutations are secondary events in leukemogenesis, are unstable, and thus should be used cautiously for the detection of minimal residual disease.