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Keywords:

  • acute promyelocytic leukaemia;
  • International Consortium on Acute Promyelocytic Leukaemia;
  • KMT2E (MLL5) ;
  • developing countries;
  • all-trans retinoic acid

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. References
  9. Supporting Information

The KMT2E (MLL5) gene encodes a histone methyltransferase implicated in the positive control of genes related to haematopoiesis. Its close relationship with retinoic acid–induced granulopoiesis suggests that the deregulated expression of KMT2E might lead acute promyelocytic leukaemia (APL) blasts to become less susceptible to the conventional treatment protocols. Here, we assessed the impact of KMT2E expression on the prognosis of 121 APL patients treated with ATRA and anthracycline-based chemotherapy. Univariate analysis showed that complete remission (= 0·006), 2-year overall survival (OS) (= 0·005) and 2-year disease-free survival (DFS) rates (= 0·037) were significantly lower in patients with low KMT2E expression; additionally, the 2-year cumulative incidence of relapse was higher in patients with low KMT2E expression (= 0·04). Multivariate analysis revealed that low KMT2E expression was independently associated with lower remission rate (odds ratio [OR]: 7·18, 95% confidence interval [CI]: 1·71–30·1; = 0·007) and shorter OS (hazard ratio [HR]: 0·27, 95% CI: 0·08–0·87; = 0·029). Evaluated as a continuous variable, KMT2E expression retained association with poor remission rate (OR: 10·3, 95% CI: 2·49–43·2; = 0·001) and shorter survival (HR: 0·17, 95% IC: 0·05–0·53; = 0·002), while the association with DFS was of marginal significance (HR: 1·01; 95% CI: 0·99–1·02; = 0·06). In summary, low KMT2E expression may predict poor outcome in APL patients.

Treatment of acute promyelocytic leukaemia (APL) with all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy results in complete remission (CR) and long-term overall survival (OS) rates greater than 90% and 80% respectively (Ades et al, 2010; Sanz et al, 2010; Avvisati et al, 2011). Nevertheless, patients with high white blood cell (WBC) counts (>10 × 109/l) at diagnosis have a significantly higher cumulative incidence of relapse (CIR) (Asou et al, 1998; Sanz et al, 2004, 2010; Ades et al, 2010; Avvisati et al, 2011). Moreover, until recently the outcome reported in developing countries was significantly poorer, with a long-term OS below 60%, mainly due to higher mortality rates during induction (Jacomo et al, 2007). In 2005, the International Consortium on Acute Promyelocytic Leukaemia (IC-APL) was created in a collaborative effort to improve the outcome of patients with APL in developing countries by adopting a common therapeutic and correlative science protocol (Rego et al, 2013). The IC-APL was conducted under the auspices of the International Members Committee of the American Society of Hematology and the protocol was initially carried out in Brazil, Mexico, Uruguay and Chile. Recently, we reported that the 2-year CIR, OS and disease-free survival (DFS) among 183 APL enrolled in the IC-APL protocol was 4·5%, 80% and 91% respectively (Rego et al, 2013), representing outcomes similar to those reported in developed countries.

The lysine (K)-specific methyltransferase 2E gene [KMT2E; previously termed human mixed lineage leukaemia 5 (MLL5)] is a member of the Trithorax-group, a family of histone-modifying proteins implicated in positive transcriptional control of a specific set of genes related to haematopoiesis (Milne et al, 2002; Nakamura et al, 2002; Ernst et al, 2004; Hughes et al, 2004). Functional analyses demonstrated that KMT2E protein is involved in terminal myeloid differentiation and facilitates retinoic acid–induced granulopoiesis in human promyelocytes (Heuser et al, 2009; Madan et al, 2009; Zhang et al, 2009). Due to its location on chromosome 7q22, a genomic region frequently deleted in myeloid malignancies (Emerling et al, 2002), the human KMT2E gene was initially identified as a candidate tumour suppressor gene. In fact, lower KMT2E transcript levels have been associated with worse prognosis in patients with cytogenetically normal acute myeloid leukaemia (CN-AML) and in core-binding factor (CBF) leukaemia (Damm et al, 2011). However, patients with APL were not included in this study and, as far as we know, the prognostic impact of KMT2E gene has not been yet explored in APL. Given that the aberrant methylation of specific gene promoters is a key feature in APL (Figueroa et al, 2010; Martens et al, 2010), it is conceivable that deregulated expression of the KMT2E gene may influence the response of APL blasts to ATRA, which, combined with anthracycline-based chemotherapy, constitutes the backbone of the modern therapy for APL.

In the present study, we retrospectively determined the KMT2E transcript levels in samples from APL patients enrolled in the IC-APL trial and analysed its relationship to clinical and laboratory features, haematological recovery, relapse and survival.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. References
  9. Supporting Information

Patients

One hundred and twenty-one consecutive patients with newly diagnosed APL enrolled in the IC-APL study were included. This study was restricted to Brazilian samples due to logistic issues related to sample transport. Bone marrow (BM) samples were sent from seven centres in Brazil between October 2006 and October 2011 to the Haematology Service of the Medical School of Ribeirão Preto, University of São Paulo, for diagnostic purposes. The rapid diagnosis of APL was based on the detection of a microspeckled nuclear pattern by immunofluorescence that is characteristic of PML protein delocalization from nuclear bodies in APL (Falini et al, 1997). The diagnosis was confirmed with cytogenetic analysis for t(15;17)(q22;q21) and/or reverse transcription polymerase chain reaction (RT-PCR) for PML/RARA rearrangements on BM samples. Additionally, patients were classified according to Programa para el Estudio y Tratamiento de las Hemopatias Maligna (PETHEMA)/Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) risk criteria, which take into account the WBC and platelet counts at diagnosis (Sanz et al, 2000). Details of the treatment protocol of the IC-APL trial have been previously described (Rego et al, 2013). Briefly, the induction and post-induction therapy was identical to that adopted in the PETHEMA/Stichting Hemato-Oncologie voor Volwassenen Nederland (HOVON) LPA2005 trial (Sanz et al, 2010), except that idarubicin (12 mg/m2/d) was replaced by daunorubicin (60 mg/m2/d) because of the better availability and lower cost of the latter.

As normal controls, total BM (n = 8) and peripheral blood (PB, n = 101) cells from healthy donors (aged 18–60 years) were collected and mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation (Sigma-Aldrich, St Louis, MO, USA). Additionally, 28 samples from patients with CBF-leukaemia (aged 23–54 years), randomly selected based on the presence of translocations and/or gene rearrangements of inv(16)(p13.1;q22)/t(16;16)(p13.1;q22)/CBFB-MYH11 and t(8;21)(q22;q22)/RUNX1-RUNX1T1 at diagnosis, were included. According to the Declaration of Helsinki, informed consent was obtained from all patients and healthy donors and the study was approved by the by the Research Ethics Board of each participating hospital.

Expression analysis of KMT2E transcript levels

Total RNA from leukaemic and healthy donor's samples was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Real time quantitative polymerase chain reaction (RQ-PCR) assays with sample-derived cDNA were performed in duplicate with appropriate water controls on MicroAmp optical 96-well plates using a 7500 Real-Time PCR System (Applied BioSystems, Foster City, CA, USA) and using the ABL1 FusionQuant Standard Kit as an endogenous control (Ipsogen, Stamford, CT, USA). KMT2E transcript levels were quantified by the TaqMan Gene Expression Assay, following the manufacturer's instructions (Assay ID KMT2E: Hs00218773_m1; Applied Biosystems). KMT2E transcripts were normalized to ABL1 (KMT2E/ABL1) by using the respective plasmid standards to generate a reference standard curve. To obtain a standard curve for KMT2E, RQ-PCRs were carried out with fivefold dilutions covering the expected detection range with a reference cDNA (NB4 cell line). Additionally, the same reference cDNA (NB4 cell line) was used as internal control and included in each assay run, in order to guarantee that the results would be fully comparable amongst experiments.

Statistical analysis and clinical end points

Patient baseline characteristics were reported descriptively. The follow-up was last updated in January 2013 and the median follow-up time among survivors was 33 months (range, 1–72 months). Early mortality was defined as death within 30 d of the initiation of induction therapy. OS was defined as the time from the initiation of induction therapy to death from any cause; those alive or lost to follow-up were censored at the date last known alive. DFS was defined as the time from CR to disease relapse or death from any cause, whichever occurred first. Patients who were alive without disease relapse were censored at the time last seen alive and disease-free. Cumulative incidence curves for relapse with or without death were constructed reflecting time to relapse as competing risks (Gray, 1998). Time to relapse was measured from the date of CR. Survival curves were generated by the Kaplan–Meier method, and the log-rank test was used for comparisons of Kaplan–Meier curves. The CIR was calculated in the competing risk framework, and the Gray test was used for comparison of CIR curves. Univariate and multivariate proportional hazards (PH) regression analysis was performed for potential prognostic factors for overall survival. Potential prognostic variables considered for model inclusion were age at diagnosis, gender, WBC and platelet counts, PML breakpoint and KMT2E transcript levels, which were explored as a continuous variable, using the logarithmic levels of the KMT2E/ABL1 expression. Multivariate models were not explored for DFS and CIR due to the small number of events. PH assumption for each variable of interest was tested. Statistical analyses were performed using r 2.10.1 (The CRAN project) (www.R-project.org, Vienna, Austria) and Stata Statistic/Data Analysis version 12 (Stata Corporation, College Station, TX, USA). All P-values were two sided with significance set to 5%.

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. References
  9. Supporting Information

Clinical and laboratory features

Initially, we compared the values of KMT2E/ABL1 expression in samples of newly diagnosed APL (n = 121), de novo CBF-leukaemia patients (n = 28) and healthy donors (n = 109). KMT2E/ABL1 expression was lower in CBF-leukaemia groups (< 0·05; Fig 1). Next we analysed only the patients with APL. The main clinical and laboratorial features are presented in Table 1. There was no ideal KMT2E transcript cut-off level that allowed the dichotomization of the cohort. Moreover, the effect of KMT2E transcript levels on the OS was gradual; therefore this variable was explored as a continuous variable, using the logarithmic levels of the KMT2E/ABL1 expression. Nevertheless, in order to compare the clinical and laboratory features of APL patients grouped according to KMT2E transcript levels, we adopted the median value of KMT2E/ABL1 expression as the cut-off level. Details of dichotomization can be found in the Data S1 and Figures S1 and S2. There was no significant difference between patients with low (n = 60) and high (n = 61) KMT2E/ABL1 expression with respect to clinical and laboratory features, except for a higher frequency of females in the high expression group (= 0·029) (Table 1). Although more patients in the low expression group were classified as high-risk according to the PETHEMA-GIMEMA criteria (Sanz et al, 2000) (43·3% vs. 26·2%) the difference was not significant (= 0·074).

Table 1. Comparison of clinical and laboratory features of APL patients according to KMT2E gene expression.
Characteristics patientsAll patients (n = 121)Low KMT2E expression (n = 60)High KMT2E expression (n = 61)P-valueb
No.%No.%No.%
  1. APL, acute promyelocytic leukaemia; KMT2E, lysine (K)-specific methyltransferase 2E gene; ECOG, Eastern Cooperative Oncology Group; WBC, white blood cell count; PLT, platelet count; BCR, breakpoint cluster region; PT, prothrombin time; aPTT, activated partial thromboplastin time.

  2. a

    Statistically significant difference.

  3. b

    Missing values were excluded in the calculation of P-values.

  4. c

    Classification according to PETHEMA-GIMEMA criteria (Sanz et al, 2000).

  5. d

    BCR1 and BCR2 were combined.

Gender
Male5646·33456·72236·10·029a
Female6553·72643·33963·9
Age (years), median34·534·534·70·992
Range15·9–73·615·9–73·616–65·4 
Age
<18 years32·523·311·60·869
≥18 & <40 years7259·53456·73862·3
≥40 & <60 years3932·22033·31931·1
≥60 years75·846·734·9
ECOG performance score
0–196805186·44573·80·191
21411·746·81016·4
≥3108·346·869·8
Missing11
WBC (×109/l), median4·96·63·30·093
Range0·3–128·50·2–128·50·2–73·5 
Haemoglobin (g/l), median8888860·93
Range34–14150–14134–133 
PLT (×109/l), median2827·5280·797
Range3–1283–1104–128 
Relapse risk categoryc
Low-risk2419·8813·41626·20·074
Intermediate-risk5545·52643·32947·5
High-risk4234·72643·31626·2
PML breakpoint regiond
BCR1/27063·63465·43662·10·843
BCR34036·41834·62237·9
Missing1183
Creatinine (μmol/l), median70·774·270·20·227
Range30–380·130–380·135·3–109·6 
Uric acid (μmol/l), median220231·9208·10·056
Range65·4–249883·2–249871·3–547·2 
Albumin (g/l), median4139410·628
Range23–41023–41022–340 
Fibrinogen (g/l), median1·631·631·630·582
Range0·01–6·050·12–6·050·01–5·49 
PT (s), median1414150·182
Range10–2612–2211·5–26 
aPTT (s), median2726280·103
Range5–5214–4614–52 
Morphological subtype
Hypergranular115955693·35996·70·439
Microgranular6546·723·3
image

Figure 1. Quantitative analysis of KMT2E gene expression in samples from APL patients, normal bone marrow (BM)/normal peripheral blood (PB) and core binding factor (CBF) leukaemia. The expression of KMT2E gene was quantified by Real-Time Quantitative PCR (RQ-PCR). The horizontal bars represent the median value of KMT2E expression relative to ABL1 gene. KMT2E/ABL1 expression was lower in CBF-leukaemia groups (anova test followed by a Dunn's post-test).

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Induction outcome

Overall, 102 (84%) of the 121 APL patients achieved CR. The median time to achieve haematological CR was 33 d (range, 16–67 d). Patients with low KMT2E transcript levels had a significantly lower CR rate than patients with high KMT2E transcript levels (75% vs. 93%; = 0·006). In multivariate logistic regression analysis, low KMT2E transcript levels were independently associated with a lower CR rate [odds ratio (OR): 7·18, 95% confidence interval (CI): 1·71–30·1; = 0·007] (Table SI; Data S1). Evaluated as a continuous variable, KMT2E transcript levels retained the association with poor remission rate in an independent manner (OR: 9·3, 95% CI: 1·75–49·3; = 0·009) (Table 2).

Table 2. Univariate and multivariate Cox analysis for complete remission, overall survival and disease-free survival according to KMT2E transcript levels (continuous variable).
End pointModel variableUnivariate analysisMultivariate analysis
ORHR95% CIP-valueORHR95% CIP-value
  1. OR, odds ratio; HR, hazard ratio; 95% CI, 95% confidence interval; CR, complete remission; OS, overall survival; DFS, disease-free survival; KMT2E, lysine (K)-specific methyltransferase 2E gene; WBC, white blood cell; PML, promyelocytic leukaemia gene; BCR, breakpoint cluster region.

  2. A HR >1 or <1 indicates an increased or decreased risk, respectively, of an event for the first category listed.

  3. a

    BCR1 and BCR2 were combined.

  4. b

    Due to the small number of events, multivariate model for DFS was not conducted.

CRKMT2E transcript level (continuous variable)10·3 2·4943·20·0019·3 1·7549·30·009
WBC count (<10 × 109/l vs. ≥10 × 109/l)0·59 0·241·430·2441·03 0·273·840·959
Platelet count (<40 × 109/l vs. ≥40 × 109/l)0·68 0·261·740·4220·63 0·182·230·478
Age at diagnosis (continuous variable)0·99 0·961·020·6740·98 0·941·020·415
PML breakpoint regiona (BCR1/2 vs. BCR3)0·39 0·141·040·0610·54 0·151·840·325
Gender (female vs. male)1·19 0·492·890·6341·73 0·496·020·387
OSKMT2E transcript levels (continuous variable) 0·170·050·530·002 0·210·050·830·026
WBC count (<10 × 109/l vs. ≥10 × 109/l) 2·361·095·10·029 1·970·655·950·228
Platelet count (<40 × 109/l vs. ≥40 × 109/l) 1·220·532·820·629 1·010·333·060·985
Age at diagnosis (continuous variable) 1·090·981·030·517 1·010·971·050·403
PML breakpoint regiona (BCR1/2 vs. BCR3) 1·730·734·080·208 0·940·312·830·921
Gender (female vs. male) 0·740·331·630·455 0·520·171·540·243
DFSbKMT2E transcript levels (continuous variable) 1·010·991·020·06     
WBC count (<10 × 109/l vs. ≥10 × 109/l) 4·140·9817·40·05     
Platelet count (<40 × 109/l vs. ≥40 × 109/l) 1·080·215·380·92     
Age at diagnosis (continuous variable) 0·950·891·020·172     
PML breakpoint regiona (BCR1/2 vs. BCR3) 1·820·457·310·394     
Gender (female vs. male) 3·060·6115·20·17     

Of the 20 patients who failed to reach CR, 14 (70%) experienced early mortality (i.e., death within 30 d of the induction therapy), due mainly to haemorrhage (n = 10; 71%), followed by infection (n = 3; 21%) and differentiation syndrome (n = 1; 7%). Although the early mortality rate was higher in patients with low KMT2E transcript levels (18% vs. 8%), this difference was not significant (= 0·155).

Post-remission outcomes

The prognostic impact of KMT2E expression was first evaluated in the whole cohort (121 patients). The estimated 2-year OS and DFS rates were 84% and 93%, respectively. Patients with low KMT2E transcript levels had a significantly lower 2-year OS rate (74%) compared with those with high KMT2E transcript levels (93%) (= 0·005; Fig 2A). Multivariate analysis demonstrated that KMT2E transcript levels were independently associated with shorter survival [hazard ratio (HR): 0·27, 95% CI: 0·08–0·87; = 0·029] (Table SI). The 2-year DFS rate of patients in the low KMT2E transcript levels group was significantly lower (86%) compared with patients in the high group (97%) (= 0·037; Fig 2B). Evaluated as a continuous variate, KMT2E transcript levels were associated with poor OS rate in both univariate (HR: 0·17, 95% CI: 0·05–0·53; = 0·002) and multivariate analysis (2-year OS: HR: 0·21, 95% CI: 0·05–0·83; = 0·026), while the association between KMT2E transcript level and DFS was of marginal significance (2-year DFS rate: HR: 1·01; 95% CI: 0·99–1·02; = 0·06) (Table 2). Up to January 2013, a total of six relapses had been recorded. The 2-year CIR rate was 4% (95% CI: 2–9%). The 2-year CIR among patients with low and high KMT2E transcript levels was 14% (95% CI: 5–27%) and 2% (95% CI: 0·1–11%), respectively (= 0·04). Due to the small number of events, multivariate analysis of DFS and CIR was not conducted.

image

Figure 2. Overall survival (A) and disease-free survival (B) in APL patients according to KMT2E transcript levels. (C) Overall survival in APL patients who remain alive after induction therapy (106 patients).

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We further evaluated the prognostic impact of KMT2E transcript levels in those patients who remain alive after induction therapy (106 patients). The DFS and CIR rates were the same as the whole cohort (data not shown), while the CR and 2-year OS rates were 95% and 94%, respectively. The association between the logarithmic levels of the KMT2E/ABL1 expression and OS remained significant after the exclusion of the patients who died during induction therapy (HR: 0·04, 95% CI: 0·004–0·43; = 0·007) (Fig 2C).

Because there was a trend toward higher leucocyte counts in patients with low KMT2E expression (Table 1), we analysed the association between KMT2E transcript levels and CR rates and OS according to the WBC counts at diagnosis, using 10 × 109 leucocytes/l as the cut-off value. In patients with WBC counts ≥10 × 109/l (n = 42), the CR rate (77% vs. 93%; = 0·222) and the 2-year OS (68% vs. 87%, P = 0·184) in the low and high KMT2E expression groups were not significantly different. In patients with WBC <10 × 109/l (n = 79), KMT2E transcript levels were not associated with CR rate (data not shown), but there was a trend towards a shorter 2-year OS of patients in the low KMT2E expression group (79% vs. 93%; = 0·07). In addition, considering the prognostic relevance of age in APL (de la Serna et al, 2008), we opted to further analyse the association between KMT2E expression levels and treatment outcome in patients younger or older than 50 years, and no significant difference was detected (Figure S3; Data S1).

Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. References
  9. Supporting Information

The present study demonstrated that lower levels of KMT2E expression were associated with a reduced remission rate, shorter survival and higher risk of relapse in a large series of patients with APL treated with ATRA and anthracycline-based chemotherapy. Although we have not tested the functional effect of KMT2E expression in primary APL blasts, the close relationship between KMT2E function and retinoic acid–induced granulopoiesis (Heuser et al, 2009; Madan et al, 2009; Zhang et al, 2009) suggests that the deregulated expression of this histone methyltransferase might lead leukaemic cells to become less susceptible to differentiation-inducing drug effects, specifically the effect of ATRA upon APL blasts. Nevertheless, it must keep in mind that KMT2E gene expression also has prognostic significance in other types of AML (Damm et al, 2011), which suggests that the effect of this histone methyltransferase in APL is unrelated to ATRA, but rather related to the chemotherapy unresponsiveness.

KMT2E expression has been described as an independent prognostic marker in 509 patients with AML treated in the AMLSHG 0199 and AMLSHG 0295 trials (Damm et al, 2011). The authors showed that KMT2E transcript levels were particularly relevant as an independent prognostic marker in CN-AML and CBF-AML. Patients with lower KMT2E transcript levels had significantly shorter relapse-free and overall survivals (Damm et al, 2011). Despite some methodological differences in measuring KMT2E expression between the study reported by Damm et al (2011) and the present study (i.e., we used the median value of KMT2E/ABL1 expression as a cut-off), we demonstrated a similar prognostic impact of KMT2E transcript levels on treatment outcome of APL patients.

Another difference between the German study and this one is that we evaluated the transcript levels of KMT2E gene in normal BM samples. Although the sample size is limited, our results showed that the normal BM samples presented a wide distribution of KMT2E gene expression and 6/8 (75%) of the samples had KMT2E transcript levels above the cut-off. A possible explanation for this finding is that immature cell subpopulations in normal BM express lower levels of KMT2E gene compared to the mature cells in PB. In fact, previous studies reported that Kmt2e-deficient mice have impairment of terminal myeloid differentiation (Madan et al, 2009; Zhang et al, 2009) and defects of the oxidative function of mature neutrophils (Heuser et al, 2009) in comparison with wild-type mice. Therefore, it is conceivable that lower KMT2E levels might be present in less differentiated and functionally active haematopoietic cells.

The mechanism underlying the differential expression of KMT2E in patients with APL is presently unknown. Although PML/RARA is necessary, it is not sufficient for leukemogenesis and other genetic lesions have been identified in APL animal models and patients (Le Beau et al, 2003; Wartman et al, 2012; Welch et al, 2012). Moreover, distinct DNA methylation and microRNA expression profiles have been described in APL cells, thus underlying the heterogeneity of the disease (Zentz, 1990; De Marchis et al, 2009; Saumet et al, 2009).

The IC-APL study has previously demonstrated that the development of a network led to an approximately 50% decrease in mortality during induction treatment compared to historical controls, and to improved OS, from about 52% to 80% (Rego et al, 2013). In the present report, we show that the positive outcome results of IC-APL have been maintained after a median follow up of 33 months, with OS and DFS rates of 84% and 93%, respectively. Additionally, a further reduction in early mortality was observed, suggesting that the learning curve has not (yet) reached the plateau. The IC-APL protocol was based on the Spanish PETHEMA 2005 (Sanz et al, 2010) protocol that reported a higher CR, but similar DFS, reflecting the higher mortality during induction in the present study. Nevertheless, the early death rate was not significantly different between patients with low and high KMT2E transcripts levels. Only prothrombin time, activated partial thromboplastin time and fibrinogen levels were routinely evaluated in the protocol and no significant difference was detected between patients with low and high KMT2E transcript levels. This is not surprising, considering the complexity of pathogenesis of coagulopathy associated with APL (Kwaan & Rego, 2010; Jacomo et al, 2012). The fact that only Brazilian patients with APL were analysed here did not represent a bias because no significant difference in treatment outcome was detected among the four participating countries (Rego et al, 2013). Therefore, our results reinforce the idea that, by developing a network and adopting guidelines covering prompt diagnosis, treatment, supportive care and disease follow-up measures, it is possible to reduce the gap between developing and developed countries with regard to the treatment outcome of APL patients.

One of the secondary aims of the IC-APL was to establish a biobank and foster research in the participating countries. The present study is a result of this effort, adding the KMT2E gene to a list of prognostic factors associated with clinical outcome in APL, which also includes the internal tandem duplication of the FLT3 gene (Noguera et al, 2002; Beitinjaneh et al, 2010; Barragan et al, 2011; Schnittger et al, 2011), CD56 expression (Murray et al, 1999; Ferrara et al, 2000; Montesinos et al, 2011), and a polymorphic variant involving the promoter of the FAS gene, encoding the CD95 cell death receptor (or FAS receptor) (Sunter et al, 2012). It is noteworthy that the analysis of KMT2E gene expression was successful in 81% patients, a rate that we considered satisfactory, given that samples were shipped to the National Reference Laboratory, involving large distances in many cases.

Taken together, our results and those reported by others (Damm et al, 2011) suggest that low KMT2E transcript levels are associated with poorer clinical outcome of patients with either APL or non-APL AMLs. Specifically in APL, our data suggest that patients with low KMT2E transcript levels may be considered as high risk regardless of WBC counts. Furthermore, it would be important to test whether APL treatment based on ATRA in combination with arsenic trioxide (ATO) (Lo-Coco et al, 2013) would overcome the adverse prognosis of low KMT2E transcript levels, because the effect of ATO is independent of retinoic acid signalling pathway (Rego et al, 2000; de Thé et al, 2012).

Acknowledgements

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. References
  9. Supporting Information

We gratefully acknowledge all members of the International Consortium on Acute Promyelocytic Leukaemia of the American Society of Hematology. This investigation was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, Grant #2013/08135-2) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNpQ, Grant #573754/2008-0). A.R.L.A. received fellowship from FAPESP (#2007/55067-1).

Author contributions

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. References
  9. Supporting Information

A.R.L.A. performed experiments and statistical analyses, analysed and interpreted data and drafted the manuscript. H.T.K performed statistical analyses. R.H.J., R.A.M., R.B., R.P., K.P., E.M.F., M.L.C., C.S.C., A.S.L. performed research. H.C.K., R.G., C.M.N., S.L.S., M.S.T., D.G., A.G., N.B., R.C.R., F.L-C., B.L. and M.A.S.performed research, interpreted data and reviewed the manuscript. E.M.R. performed the conception and design of the study, reviewed the manuscript and gave final approval of the version to be submitted.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Author contributions
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
bjh12921-sup-0001-FigS1-S3-TableS1.docWord document2081K

Data S1. Statistical analysis of the supplementary data.

Fig S1. Overall survival in APL patients divided into quartiles (Q) according to KMT2E/ABL1 expression.

Fig S2. Receiver operating characteristic (ROC) curve analysis to define the optimal cut-off point for KMT2E expression.

Fig S3. Overall survival in APL patients according to age (younger than 50 years vs. older than 50 years).

Table SI. Univariable and multivariable Cox analysis for complete remission, overall survival and disease-free survival according to KMT2E transcript levels (categorical variable).

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