Internal tandem duplications (ITDs) of the FLT3 gene have been observed in about 35% of APL cases. If FLT3-ITD is associated with a worse outcome in patients with acute myeloid leukemia (AML) in general, its prognostic value in acute promyelocytic leukemia (APL) is still a matter of debate. We investigated incidence, associated clinical features, and prognostic implication of FLT3-ITD, but also FLT3-D835 point mutation and N-Ras or K-Ras mutations in 119 APL patients, all prospectively enrolled in the two consecutive APL-93 and APL-2000 trials. Mutation incidences were 38, 20, and 4%, for FLT3-ITD, FLT3-D835, and Ras, respectively. The presence of FLT3-ITD was associated with high white blood cell count, high Sanz index, M3-variant subtype, and V/S PML-RARα isoforms. Complete remission (CR), induction death, and death in CR rates were not affected by FLT3 or Ras mutations, as well as cumulative incidence of relapse. However, a trend for a shorter overall survival (P=0.09) was observed in FLT3-ITD patients, because of a very poor postrelapse survival (P=0.02). This feature, which has been also reported in patients with AML in general, is suggestive of an underlying genetic instability in FLT3-ITD patients, leading to the acquisition of additional unknown bad-prognosis gene mutations at relapse.
Recent advances in the knowledge of molecular events leading to acute myeloid leukemia (AML) genesis include the description of activating mutations involving genes coding for class III tyrosine kinase receptors, such as c-Fms, c-Kit, and FLT3 or signal transduction molecules, such as K-Ras and H-Ras. Activating mutations of the FLT3 gene have been reported in nearly 20% of adult and pediatric AML,1 while N-Ras or K-Ras mutations are found in about 15–20% of cases tested.2, 3, 4
Usually, FLT3 mutated gene presents an internal tandem duplication (ITD) of the juxtamembrane (JM) domain-coding sequence, which frequently involve exon 14 and eventually intron 14 or exon 15.5, 6 Activating point mutations within the A-loop of FLT3 have also been reported in AML, either in full articles7, 8, 9, 10, 11 or abstracts (Thiede et al, Blood 2003; 102: 606a; abstract) (Schittenhelm et al, Blood 2003; 102: 204b; abstract), even if with lower incidences. These mutations are mainly located in D835, but also in I836, Y842, or D853. Interestingly, a higher frequency of FLT3-ITD and FLT3-D835 mutations is found in AML cases with a normal karyotype8, 12, 13, 14, 15, 16 or carrying the t(6;9) translocation,13 as well as in the acute promyelocytic leukemia (APL) subset (AML-M3 and M3-variant in the French–American–British (FAB) classification).13, 17, 18, 19 Partial duplications of the MLL gene are frequently associated with FLT3-ITDs,20, 21 suggesting a common mechanistic basis.
Activating mutations of N-Ras and K-Ras are essentially located within codons 12, 13 and 61 of these genes. These mutations have been reported as more frequent in advanced myelodysplastic syndrome and secondary AML, as well as in relatively good-risk cases with inversion of the chromosome 16 (AML-M4eo in the FAB classification).2, 22
A lot of recent retrospective studies have evaluated the prognostic significance of FLT3 mutations in childhood and adult AML in general. ITDs of the gene, usually associated with a higher white blood cell count (WBC), appear to have no great impact on the complete remission (CR) rate, but are reported as independently associated with a significant reduction either in event-free or relapse-free,8, 10, 12, 13, 14, 15, 19, 23, 24, 25, 26, 27, 28 postrelapse,16 or overall survival.8, 10, 13, 14, 15, 26, 29 The presence of D835 mutation did not seem to be associated with higher WBC and its prognostic significance remains uncertain (Thiede et al, Blood 2003; 102: 606a; abstract).7 Similarly, the prognostic value of Ras mutations in patients with AML in general is still a matter of controversy, since reported as poor,3 neutral,2, 4 or even good (Goemans et al, Blood 2003; 102: 198b; abstract),30, 31 depending of the study and the control group used.
In this study, we evaluated the clinical features and the prognostic significance associated with FLT3 and Ras mutations in 119 adults with APL, all prospectively enrolled in two prospective randomized trials conducted from 1993 by the European cooperative APL Group. We confirmed the prevalence of these mutations in this AML subset and the profile of associated clinical features and found that FLT3-ITDs are associated with a worse postrelapse survival, even if not responsible for a higher incidence of relapse.
Patients and methods
Patients and treatments
Between April 1993 and December 1996, 439 adult patients with newly diagnosed APL have been enrolled in the multicenter APL-93 trial.32 Diagnosis had to be subsequently confirmed by the presence of t(15;17) or PML-RARα gene rearrangement. Other inclusion criteria were of age 75 years or less and written informed consent. The principal objectives of this trial were to assess the optimal timing of all-trans retinoic acid (ATRA) treatment and the role of maintenance therapy. Induction treatment was stratified by age and initial WBC count. Briefly, patients ⩽65 years of age with a WBC count less than 5 × 109/l were randomized to receive the reference ATRA treatment of the previous trial APL-91 trial,33 that is, 45 mg/m2/day ATRA followed by CT (ATRA → CT group=arm A) or ATRA plus CT (ATRA+CT group=arm B). In the ATRA → CT group, patients received 45 mg/m2/day ATRA orally until CR, with a maximum of 90 days. After CR achievement, they received a course of 60 mg/m2/day daunorubicin (DNR) for 3 days and 200 mg/m2/day cytarabine (AraC) for 7 days (course I). However, course I was added to ATRA if the WBC count was increased to greater than 6 × 109/l, 10 × 109/l, or 15 × 109/l by day 5, 10, and 15 of ATRA treatment, respectively, because, from our experience, patients were at risk of ATRA syndrome above those thresholds. Patients randomized to the ATRA+CT group received the same combination of ATRA and CT, with course I of CT starting on day 3 of ATRA treatment. This 48-h interval before onset of CT was also based on our previous report, because it allowed correction of coagulopathy. Patients with a WBC count greater than 5 × 109/l at presentation (irrespective of their age) and patients 66–75 years of age with a WBC count ⩽5 × 109/l were not randomized but received ATRA plus CT course I from day 1 (high WBC group=arm C) and the same schedule as in the ATRA → CT group (elderly group=arm D), respectively. Patients reaching CR received then two CT consolidation courses, including course II (identical to course I) and course III, consisting of 45 mg/m2/day DNR for 3 days and 1 g/m2 AraC every 12 h for 4 days. The elderly group only received course II. At 3 to 4 weeks after hematological recovery from this consolidation CT, patients who were still in CR were randomized both to receive or not receive intermittent ATRA (45 mg/m2/day, 15 days every 3 months) and to receive or not receive continuous CT with 6-mercaptopurine (6MP, 90 mg/m2/day orally) and methotrexate (MTX, 15 mg/m2/week orally), according to a 2-by-2 factorial design stratified on the initial induction treatment group. Maintenance treatment was scheduled for 2 years. Randomizations for induction and maintenance were performed through a centralized telephone assignment procedure.
Between July 2000 and June 2004, 423 adult patients with newly diagnosed APL have been enrolled in the multicenter APL-2000 trial. In this ongoing trial, all standard-risk patients (⩽60 years of age with a WBC⩽10 × 109/l) were treated according to the ATRA+CT induction schedule with a front-line randomization between chemotherapy with (arm A) or without AraC (arm B) for the first induction course as well as for the two consolidation courses. Patients aged more than 60 years were systematically treated according to the treatment arm without AraC if the WBC was ⩽10 × 109/l (arm D) or with AraC (arm E) if the WBC was >10 × 109/l. Younger patients with WBC>10 × 109/l also received the treatment arm containing AraC for the first two courses. In these younger high count patients (arm C), AraC doses were increased for the second consolidation cycle up to 1 g/m2/12 h for 4 days (eight bolus infusions). In addition, these patients received a central nervous system (CNS) prophylaxis based on five intrathecal injections of 15 mg MTX, 50 mg AraC, and depomedrol. As maintenance, all patients received the triple ATRA–6MP–MTX treatment according to the APL-93 schedule. Both trials have been approved by ethical committees in all participating countries.
The presence of FLT3 and Ras mutations was assessed on available diagnosis material. Bone marrow samples from 69 patients included in the APL-93 and 50 patients included in the APL-2000 trial were retrospectively analyzed.
Detection of FLT3 and Ras mutations
RNA was extracted by centrifugation on cesium chloride gradient. Reverse transcription was performed as described.34 FLT3 mutations detection were performed on cDNA. ITDs were detected after PCR amplification using primers 11F and 12R.3 Primer 12R was labelled with Cy5. PCR products were analyzed on an ALF Express (Amersham Pharmacia Biotech, Orsay, France). Cases displaying faint abnormal sized bands were confirmed after hybridization with an internal oligonucleotide probe IndexTerm5′-TGAGACTCCTGTTTTGCTAAT-3′, located on exon 12. D835 and I836 mutations were detected by EcoRV restriction enzyme digestion after PCR amplification, using forward primer 20A35 and reverse primer FLTEX18: IndexTerm5′-ATGCCAGGGTAAGG ATTCACA-3′. Mutations affecting codons 12, 13 and 61 of N-Ras and K-Ras were searched, by direct sequencing of N-Ras or K-Ras PCR products, obtained with the following primers. NRASEX1S IndexTerm5′-TACAAACTGGTGGTGGTTGG-3′; NRASEX2R IndexTerm5′-CAAATGACTTGCTATTAT TGATG-3′; KRASEX1S IndexTerm5′-GGCCTGCTGAAAATGACTGAATAT-3′; KRASEX2R IndexTerm5′-ACTGGTCCCTCATTGCACT-3′.
Patient characteristics and CR rate comparisons were performed using the Fisher's exact test for discrete variables and the Mann–Whitney test for continuous variables. Failure time data but cumulative incidence of relapse were estimated by the Kaplan–Meier method,36 then compared by the log-rank test.37 Proportional hazards assumptions were graphically checked. By contrast, in estimating cumulative incidence of relapse, we took into account for competing risks deaths and allogeneic stem cell transplantations (SCT) in first CR (although not planned in both trial schedules, two patients actually received allogeneic SCT in first CR) using the cumulative incidence curves, then compared by the Gray test while the Fine and Gray model was used to estimate specific hazard ratio (SHR).38, 39 Type I error was fixed at the 5% level. Reference date of analysis was June, 2004. All tests were two-tailed. Statistical analysis was performed on SAS 8.2 (SAS, Inc., Cary, NC, USA) and R 1.9.1 (R Foundation for Statistical Computing, Vienna, Austria) software packages.
The main characteristics of the 119 patients studied for FLT3 and Ras mutations are shown in Table 1. These characteristics did not appear to significantly differ from a standard population of patients with APL. Moreover, no major selection biases were found when comparing baseline characteristics of these 119 patients with the 743 patients included in APL-93 and APL-2000 trials during the same period but not studied for FLT3 and Ras mutations. Patients from the former group were slightly younger (median age, 41.9 vs 45.5 years) and had lower median baseline fibrinogen level (0.95 vs 1.41 g/l), but WBC distribution was similar in both groups.
Incidence of FLT3 and Ras mutations
Overall, 117, 112, and 97 of these 119 patients were studied for FLT3-ITD, FLT3-D835, and Ras mutations, respectively. Results are given in Table 2. Incidences of FLT3-ITD, FLT3-D835, and Ras mutations were 38% (45/117 patients), 20% (22/112 patients) and 4% (4/97 patients), respectively. In total, 96 patients only were tested for the three FLT3-ITD, FLT3-D835, and Ras mutations. Eight patients (7%) presented a FLT3-D835 mutation associated with an FLT3-ITD, two of them in association with a Ras mutation. In the subset of 101 patients studied for both FLT3-ITD and Ras mutations, the incidence of at least one of these mutations was 47%.
As compared to nonmutated patients, FLT3-ITD was associated with significantly higher frequencies of M3-variant subtype and V (bcr2) or S (bcr3) PML-RARα isoforms (Table 1). Patients with FLT3-ITD had significantly higher WBC and were thus more frequently classified in the high-risk subset of the Sanz prognostic index40 (Table 1). Such differences were not observed for patients with FLT3-D835 mutation, who were just more frequently males than females (Table 1).
Response to induction therapy
In total, 118 patients reached CR after induction (CR rate, 99%). The sole remaining patient had an M3-variant APL with a WBC of 53 G/l and FLT3-ITD (without FLT3-D835 or Ras mutation) and died very early. No significant differences in CR rate were thus noted between FLT3-ITD and non-FLT3-ITD patients or between FLT3-D835 and non-FLT3-D835 patients.
Prognostic factors for outcome
In this population of 119 APL patients studied for mutations, identified poor-prognostic factors for cumulative incidence of relapse were male gender and the trial itself, at least in univariate analyses. Among the 25 patients who experienced a hematological relapse, 20 were males (P=0.0008 by the Gray test) and 20 were included in APL-93 trial (P=0.035 by the Gray test). In multivariate analysis, both variables had poor-prognostic value, either male gender (SHR=5.25, 95% confidence interval (CI): 2.0–13.8; P=0.00075) and enrollment in APL-93 trial (SHR=3.36, 95% CI: 1.3–8.9; P=0.015). The same two factors, but also WBC>10 G/l, were identified as poor-prognostic factors for overall survival. Among the 23 patients who died, 16 were males (P=0.036 by the log-rank test), 13 had WBC>10 G/l (P=0.006 by the log-rank test), and 20 were included in APL-93 trial (P=0.025 by the log-rank test). In multivariate analysis, male gender and WBC>10 G/l remained independently predictive of shorter overall survival (HR=2.8 and 2.5, respectively; P=0.02 and 0.03, respectively).
Prognostic impact of FLT3 and Ras mutations
Neither FLT3-ITD nor FLT3-D835 influenced the cumulative incidence of relapse. As indicated in Figure 1, relapse incidence curves looked very similar for FLT3 mutated and FLT3 nonmutated patients. However, at least in univariate analyses, a trend for a shorter overall survival was observed in FLT3-ITD as compared to non-FLT3-ITD patients (Table 3 and Figure 1). Among the 23 patients who died, 13 had FLT3-ITD and 10 had not (P=0.09 by the log-rank test). This was not observed for FLT3-D835 patients (Figure 1). When evaluated in the subset of 96 patients tested for the three FLT3-ITD, FLT3-D835 and Ras mutations, this trend for shorter survival was less marked (P=0.12 by the log-rank test).
This trend for worse overall survival despite similar relapse incidence was not explained by an increased rate of induction deaths or deaths in first CR in FLT3-ITD patients (Table 3), but once relapse occurred the postrelapse survival was significantly shorter in FLT3-ITD patients as compared to non-FLT3-ITD patients (Figure 2). Among the 25 patients who eventually relapsed, 11 had FLT3-ITD while the 14 other did not. Second CR rate was not significantly different between these two subsets of patients, but among the 13/25 patients who died after relapse, 11 had M3-variant APL (P=0.0075 by the log-rank test) and nine had FLT3-ITD (P=0.02 by the log-rank test). After adjustment, FLT3-ITD was the only identified prognostic factor for worse postrelapse survival (HR=3.6, 95% CI: 1.0–12.4; P=0.04). As the therapy received for reinduction may have influenced patients outcome, we retrospectively verified that the proportion of patients salvaged by either ATRA and CT or arsenic trioxide was similar in FLT3-ITD and non-FLT3-ITD patients (not shown).
Given the very low number of mutated cases (n=4), the prognostic impact of Ras mutation could not be evaluated by itself. However, when considering the 47 patients with FLT3-ITD and/or Ras mutation in a single subgroup, the shorter postrelapse survival mentioned above (P=0.01 by the log-rank test) translated into a significantly worse overall survival (HR=2.6, 95% CI: 1.0–6.9; P=0.04 by the log-rank test), at least in univariate analyses (Figure 2). This impact on overall survival became nonsignificant after adjustment on male gender, WBC>10 G/l, and trial (HR=1.9, 95% CI: 0.7–5.8; P=0.23).
As FLT3-ITD incidence was more frequent in patients from the APL-93 than in those from the APL-2000 trial (Table 1) and studied patients from the APL-2000 trial had a better outcome than those from the APL-93 trial, we looked at FLT3-ITD prognostic impact on survival according to trial and treatment arms. Results are shown in Table 4. Limited numbers of patients within each trial subset did not allow, however, to draw any significant conclusion (Table 4). Of interest, the poor postrelapse survival observed in FLT3-ITD patients (Figure 2) came from patients enrolled in the APL-93 (P=0.03) rather than from those enrolled in the APL-2000 trial (nonsignificant difference), suggesting that FLT3-ITD patients might benefit from our current therapeutic approach.
In patients with APL, reported incidences of FLT3-ITDs and FLT3-D835 mutations vary between 20 and 38% and between 8 and 19%, respectively. Results of studies reported either as full article6, 41, 42, 43, 44 or abstract (Grimwade et al, Blood 2003; 102: 98a; abstract) are presented in Table 5. With respective incidences of 38 and 20%, the present study does not significantly differ from these previous reports. No large study evaluating Ras mutations in APL patients is available, even if it has been reported that only a minority of APL patients have Ras mutations.45 The 4% incidence reported here is in accordance with this statement.
The presence of FLT3-ITD, but not FLT3-D835, has been related to higher WBC, lower fibrinogen level, high-risk prognostic index according to Sanz grouping, M3-variant subtype in the FAB classification, and short PML-RARα isoform (bcr3 PML breakpoint), all features previously associated with a poor outcome in APL patients (Table 5). We report here similar findings.
Conversely to its widely reported bad-prognostic impact in AML in general, the presence of FLT3-ITD appears to have only mild impact in APL patients, at least when treated with a combination of ATRA and CT, as in the present report (Table 5). As prognostic factor, its independence from the WBC itself remains questionable. In the largest British study (Grimwade et al, Blood 2003; 102: 98a; abstract), a higher induction death rate, translating into lower CR rate and shorter overall survival, was observed in patients with FLT3-ITD and/or FLT3-D835, raising the question of whether these induction deaths occurred in high count patients with more severe coagulopathy.
We did not find such a correlation with induction mortality in the present study, as almost all FLT3-ITD and non-FLT3-ITD patients reached CR. We also did not find a higher incidence of relapse in FLT3-ITD patients. However, as in our previous report in non-M3 AML,16 we found here again that the trend towards shorter overall survival in FLT3-ITD patients was related to very poor outcome after relapse in the relatively rare patients who eventually relapsed. Given the fact that FLT3-ITDs are considered as secondary events, which may occur in a subset of the leukemic cell population only, two distinct explanations may be proposed. First, the progression towards a more resistant phenotype at relapse may simply be related to an increasing mutant/wild-type ratio.12 Conversely, FLT3-ITDs might be considered as a marker of underlying genetic instability, leading to the acquisition of other unknown poor-prognosis gene mutations at relapse. The reports of the loss of FLT3-ITD or changes in FLT3-ITD at relapse are clearly in favor of the latter hypothesis.46, 47
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Appendix A APL-93 and/or APL-2000 investigators
Appendix A APL-93 and/or APL-2000 investigators
French group: Caillères (Aix-en-Provence), Desablens (Amiens), Gardembas, Hunault, Ifrah (Angers), Martin, Corront (Annecy), Dor (Antibes), Sutton, Pulik (Argenteuil), Lepeu (Avignon), Renoux (Bayonne), Deconinck (Besancon), Ades, Gardin, Casassus, Fenaux (Bobigny), Pigneux, Boiron, Reiffers (Bordeaux), Abgrall, Berthou (Brest), Reman, Leporrier (Caen), Deveaux, Salles (Chalon-sur-Saone), de Revel, Nedellec (Clamart), Plagne, Legros, Travade (Clermont-Ferrand), Gardin (Clichy), Audhuy (Colmart), Decaudin, Dutel (Compiegne), Pautas, Cordonnier, Reyes (Creteil), Caillot, Guy (Dijon), Cahn, Sotto (Grenoble), Durand (Le Havre), Solal-Celigny (Le Mans), Tertian (Kremlin-Bicetre), Morel (Lens), Nelken, de Botton (Lille), Turlure, Bordessoule (Limoges), Thomas, Archimbaud, Bastion, Michallet, Fière, Coiffier, Philippe (Lyon), Stoppa, Bouabdallah, Vey, Blaise (Marseille), Benothman, Allard (Meaux), Christian (Metz), Margueritte, Fegueux, Rossi Donadio (Montpellier), Ojeda, Henon (Mulhouse), Guerci, Witz (Nancy), Harousseau (Nantes), Pesce, Gratecos, Cassuto (Nice), Schoenwald (Orleans), Dreyfus (Paris Cochin), Vilmer (Paris Robert-Debre), Marie, Vekhoff (Paris Hotel Dieu), Buzyn, Lefrère, Varet (Paris Necker), Vernant (Paris Pitie-Salpetriere), Isnard, Gorin, Najman (Paris Saint-Antoine), Dombret, Raffoux, Gisselbrecht, Marolleau, Baruchel, Degos (Paris Saint-Louis), Leverger (Paris Trousseau), Vaque, Quetin (Point-a-Pitre), Guilhot (Poitiers), Pignon (Reims), Grosbois, Lamy, Le Prisé (Rennes), Stamatoulas, Tilly (Rouen), Bourquard (Saint-Brieuc), Janvier (Saint-Cloud), Guyotat (Saint-Etienne), Maloisel (Strasbourg), Jaubert (Toulon), Huguet, Attal, Pris, Robert, Despax (Toulouse), Colombat (Tours), Simon, Pollet (Valenciennes), Castaigne (Versailles), Bourhis, Machover (Villejuif).
Spanish group: Villegas (Almeria), Zuazu (Barcelona), Torres Carrete, Deben (Juan Canalejo), Javier de la Serna, Odriozola, Gomez-Sanz (Madrid), Rayon (Oviedo), Besalduch (Palma de Mallorca), San Miguel (Salamanca), Condé (Santander), Perez Encinas (Santiago), Sanz, Martin, Montagud (Valencia), Palomera (Zaragoza).
German group: Soucek (Dresden), Strobel (Erlangen), Ganser (Frankfurt), Brennscheidt (Freiburg), Link (Hanover), Wandt (Nurnberg), Breitenbach (Stuttgart), Herrmann (Ulm).
Swiss group: Wernli, Bargetzi (Aarau), Meyer-Monard, Ticheli, Gratwohl (Basel), Pabst, Fey, Zenhäusern (Bern), Wuillemin, Gregor (Luzern), Hess, Hitz (St Gallen), Fehr, Jacky, Taverna (Zürich), Chapuis, Starobinski (Genève), Cavalli, Ghielmini (Bellinzona), Schapira, Grob, von Fliedner (Lausanne).
Belgian group: Meeus (Anvers), Ferrant (Brussels), Boulet (Mons), Bosly (Yvoir).
Dutch group: Daenen (Griningen), Muus (Nijmegen).
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Callens, C., Chevret, S., Cayuela, JM. et al. Prognostic implication of FLT3 and Ras gene mutations in patients with acute promyelocytic leukemia (APL): a retrospective study from the European APL Group. Leukemia 19, 1153–1160 (2005). https://doi.org/10.1038/sj.leu.2403790
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