Translocations involving the mixed lineage leukemia (MLL) gene, localized at 11q23, frequently occur in pediatric acute myeloid leukemia (AML). We recently reported differences in prognosis between the different translocation partners, suggesting differences in biological background. To unravel the latter, we used microarrays to generate gene expression profiles of 245 pediatric AML cases, including 53 MLL-rearranged cases. Thereby, we identified a specific gene expression signature for t(9;11)(p22;q23), and identified BRE (brain and reproductive organ expressed) to be discriminative for t(9;11)(p22;q23) (P<0.001) when compared with other MLL subtypes. Patients with high BRE expression showed a significantly better 3-year relapse-free survival (pRFS) (80±13 vs 30±10%, P=0.02) within MLL-rearranged AML cases. Moreover, multivariate analysis identified high BRE expression as an independent favorable prognostic factor within pediatric AML for RFS (HR=0.2, P=0.04). No significant differences were identified for 3-year event-free survival or for 3-year overall survival. Forced expression of BRE did not result in altered cell proliferation, apoptosis or drug sensitivity, which could explain the favorable outcome. In conclusion, overexpression of the BRE gene is predominantly found in MLL-rearranged AML with t(9;11)(p22;q23). Although further investigation for the role of BRE in leukemogenesis and outcome is warranted, high BRE expression is an independent prognostic factor for pRFS in pediatric AML.
Acute myeloid leukemia (AML) is a heterogeneous disease. Currently, initial response to therapy and cytogenetic abnormalities are the main prognostic factors.1 Translocations involving chromosome 11q23 comprise 15–20% of all pediatric AML cases. In more than 95% of the cases with 11q23 rearrangements, the mixed lineage leukemia (MLL) gene is involved. The heterogeneity of MLL-rearranged AML is reflected by the identification of more than 60 different fusion partners of this gene.2 In AML, the most common 11q23 rearrangements are t(9;11)(p22;q23)(MLL-AF9) (approximately 50% of cases), t(11;19)(q23;p13.1)(MLL-ENL), t(11;19)(q23;p13.3)(MLL-ELL), t(6;11)(q27;q23)(MLL-AF6) and t(10;11)(p12;q23)(MLL-AF10).3, 4 Most AML samples with 11q23 rearrangements are morphologically classified as FAB (French–American British morphology classification)-M4 or FAB-M5.5
We recently showed by a large retrospective international collaborative study that t(1;11)(q21;q23)(MLL-AF1q) was associated with a favorable outcome, whereas t(10;11)(p12;q23), t(10;11)(p11.2,q23)[MLL-ABI1] or t(6;11)(q27;q23) was associated with a poor outcome.6 In some studies, t(9;11)(p22;q23) had been associated with a better prognosis, which may at least partially be due to enhanced sensitivity to different drugs.7, 8, 9, 10 We found that within the t(9;11)(p22;q23) cases, prognosis was related to the cell type from which the leukemia originated, as patients with FAB-M5 showed a significantly better outcome than those with other FAB types.6 These outcome differences between the various translocation partners may point at differences in biological background.
In gene expression profiling studies in MLL-rearranged AML and acute lymphoblastic leukemia, MLL-rearranged cases clustered together as a single entity.11, 12, 13, 14 However, Stam et al.15 showed that, within MLL-rearranged infant acute lymphoblastic leukemia, each type of MLL translocation is associated with a translocation-specific gene expression signature. In this study, we performed a supervised analysis of gene expression profiles in a large cohort of pediatric AML cases (n=245) to identify and analyze differentially expressed genes between the various MLL-rearranged AML cases stratified by translocation partners, to elucidate potential genes of interest that are related to the observed differences in outcome. This led to the identification of high expression of a novel gene of interest related to t(9;11)(p22;q23), that is, BRE (brain and reproductive organ expressed), which is further described in this paper.
Material and methods
Viably frozen diagnostic bone marrow or peripheral blood samples from 237 de novo and 8 secondary pediatric AML patients, including 53 pediatric MLL-rearranged AML cases, were provided by the Dutch Childhood Oncology Group (DCOG), the AML ‘Berlin–Frankfurt–Münster’ Study Group (AML-BFM-SG), the Czech Pediatric Hematology Group and the St Louis Hospital in Paris, France. Informed consent was obtained after Institutional Review Board approval according to local law and regulations. Each study group performed a central morphology review. In addition, the collaborative study groups also provided data on the clinical follow-up of these patients. Survival analysis was restricted to the subset of 205 pediatric AML patients who were treated according to the BFM and Dutch pediatric AML protocols (studies AML-BFM 98, AML-BFM 04, DCOG-BFM 87, DCOG 92/94 and DCOG 97). Details of the treatment protocols included in the survival analysis and overall outcome data have been previously published, with the exception of study AML-BFM 2004, which is ongoing.16, 17, 18
Leukemic cells were isolated by sucrose density centrifugation and non-leukemic cells were eliminated as previously described.19 All processed samples contained more than 80% leukemic cells, as determined morphologically using cytospins stained with May–Grünwald–Giemsa (Merck, Darmstadt, Germany). Subsequently, a minimum of 5 × 106 leukemic cells were lysed in Trizol reagent (Gibco BRL, Life Technologies, Breda, The Netherlands). Genomic DNA and total RNA were isolated according to the manufacturer's protocol, with minor modifications.20
Cytogenetic and molecular analysis
Leukemic samples were routinely investigated for MLL rearrangements by standard chromosome-banding analysis and/or fluorescent in situ hybridization. If necessary, RT–PCR was performed for the common translocations MLL-AF9, MLL-AF10, MLL-AF6, MLL-ENL and MLL-ELL (primers are described in Supplementary Table S1). Of the 53 cases, 21 harbored a t(9;11)(p22;q23), 16 a t(10;11)(p12;q23) and 5 a t(6;11) (q27;q23). The remaining 11 cases were confirmed with long-distance-inverse–PCR to have another translocation partner and were considered as MLL others.21 NPM1, CEBPA, WT1, NRAS, KRAS, PTPN11, CKIT and FLT3 hotspot mutational screening was performed as previously described.22, 23, 24, 25, 26 Overexpression of EVI1 was previously established by gene expression profiling and real-time quantitative (RQ)–PCR.27
Microarray-based gene expression profiling
Integrity of total RNA was checked using the Agilent 2100 Bio-analyzer (Agilent, Santa Clara, CA, USA). Complementary DNA and biotinylated complementary RNA were synthesized, hybridized and processed on the Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's guidelines. Data acquisition was performed using expresso (Bioconductor package Affy, www.bioconductor.org) and probe-set intensities were normalized using variance stabilization normalization (VSN) (Bioconductor package VSN) in the statistical data analysis environment R, version 220.127.116.11, 29
To find a gene expression signature characteristic for the t(9;11)(p22;q23) group, which were mostly FAB-M5 cases, an empirical Bayes linear regression model was used to compare samples from this group with all other samples.30 Moderated T-statistics P-values were corrected for multiple testing using the false discovery rate method defined by Benjamini and Hochberg.
RQ–PCR for BRE
In 45 of the 53 MLL-rearranged AML samples, the RNA expression could be validated by RQ–PCR. The relative expression of BRE was calculated using the comparative cycle time (ΔCt) method, with GAPDH as the housekeeping gene.31 Primer and probe sequences are described in Supplementary Table S1.
Protein extraction and western blot analysis
In 11 of the 53 MLL-rearranged AML samples, material for protein extraction and western blot analysis was available. Western blots were probed with mouse anti-BRE (kindly provided by Dr Y.L. Chui32). Further details on protein extraction and western blot are described in Supplementary Material and Methods.
Cell culture, transfection and cell-cycle proliferation
Different cell lines with an MLL rearrangement, that is, Monomac-1, THP1, NOMO1, MV4;11 and ML2 (DSMZ, Braunschweig, Germany), were tested for BRE expression. However, none of them showed overexpression of BRE. As a cell line model was not available, the Monomac-1 cell line was transfected to overexpress BRE, and cell viability, transfection efficiency and cell-cycle proliferation were measured as described in Supplementary Material and Methods.
In vitro drug resistance assay
In vitro drug resistance for the different cytostatic agents as described in Supplementary Material and Methods was determined using the 4-day 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay as described previously.33
Additional statistical analysis
Statistical analysis was performed with SPSS 15.0 (SPSS Inc., Chicago, IL, USA). Different variables were compared with the χ2-test, t-statistics test or the Mann–Whitney U-test. Probabilities of overall survival (pOS), event-free survival (pEFS, events: no CR, relapse, secondary malignancy or death from any cause) and relapse-free survival (pRFS, events: relapse) were estimated by the method of Kaplan and Meier. The Cox proportional hazards model analysis was applied to determine the association of overexpression of BRE with pOS, pEFS and pRFS adjusted for prognostic factors as described for pediatric AML (white blood cell count >50 × 109/l, age >10 years, favorable karyotype, that is, t(8;21), inv(16) and t(15;17), and t(9;11) (p22;q23) ). All tests were two-tailed and a P-value of less than 0.05 was considered significant.
High BRE expression in t(9;11)(p22;q23)
From the microarray-based gene expression profiles of 245 pediatric AML cases, the 21 t(9;11)(p22;q23) cases were compared with the 32 other MLL-rearranged cases, and a specific gene expression signature for t(9;11)(p22;q23) was identified (Figure 1). Interestingly, 5 of the top 50 most discriminative probe sets for the t(9;11)(p22;q23) group were probe sets for the BRE gene (Supplementary Table S2). The VSN-normalized mean average intensity of three of these probe sets (205550_s_at, 211566_x_at and 212645_x_at) was 3.7-fold higher for t(9;11)(p22;q23) compared with the other MLL-rearranged AML cases (P<0.001) (Figure 2a). In the total cohort of pediatric AML cases (n=245), high expression of BRE was only identified in MLL-rearranged AML cases, and in one infant AML harboring a t(8;16)(p11;p13) (Figure 2b).
The gene expression data were validated with RQ–PCR in 45 of 53 MLL-rearranged AML cases. A correlation between the log-transformed gene expression profiling data and the log-transformed RQ–PCR data was found (r2=0.6, P<0.001) (Supplementary Figure S1). The median relative BRE expression to GAPDH for t(9;11)(p22;q23) was 56% compared with 8% for the other MLL-rearranged AML cases (P<0.001) (Figure 2c).
In a previous pediatric AML study by Ross et al., gene expression profiling was performed with the Affymetrix Human Genome U133A microarray, which had one probe set representing the BRE gene. We reanalyzed their MLL-rearranged AML cases (n=23) for validation purposes and confirmed that high BRE expression was predominantly found in the t(9;11)(p22;q23) cases (Supplementary Figure S2).
Clinical and genetic characteristics of patients with high BRE expression
On the basis of the VSN-normalized mean average intensity (712 AU) of the three most significant differentially expressed probe sets within the MLL-rearranged AML cases, a distinction was made into a group with high BRE expression (>712 AU) and a group with low expression (<712 AU).
Within the total cohort of pediatric AML, high BRE expression was found in 19 patients, including 18 of 53 with MLL-rearranged AML, and one case with a t(8;16)(p11;p13). In all, 17 of the 19 cases with a high BRE expression were FAB-M5. The remaining two cases were FAB-M4 and an unknown FAB type (P<0.001). In addition, patients with a high BRE expression were significantly younger than those with a low BRE expression (2.8 vs 9.8 years, P=0.007). None of the cases with high BRE expression had an FLT3-ITD (P=0.03) (Table 1a). However, the differences in morphology, age and FLT3-ITD were determined by the significantly high frequency of MLL-rearranged AML in patients with high BRE expression.
Within the MLL-rearranged cases, the group with high BRE expression consisted of 15 of 21 with t(9;11)(p22;q23) and 3 of 16 with t(10;11)(p12;q23). No differences for age, sex, white blood cell count and FAB morphology, nor for mutations in WT1, FLT3-ITD, CKIT, N/K-RAS, were found between cases with high and low BRE expression. In contrast, cases with high BRE expression showed an inverse correlation with EVI1 overexpression (P=0.02) (Table 1b). In fact, four of the five patients with EVI1 overexpression and a t(9;11)(p22;q23) had the lowest expression of BRE.
Within the patients with a t(9;11)(p22;q23), 13 of 15 patients with a high BRE expression had a FAB-M5, which was not significantly different compared with those with a low BRE expression (P=0.31). Within the patients with a t(10;11)(p12;q23), numbers were too small to identify significant correlations between cases with high and low BRE expression.
Favorable disease-free survival in pediatric AML patients with high BRE expression
Within the total cohort of pediatric AML, follow-up data were available for 205 patients, including 17 cases with high BRE expression. No differences were found for 3-year pEFS between cases with high and low BRE expression (53±12 vs 43±4%, P=0.61) and for 3-year pOS (64±12 vs 62±4%, P=0.77). However, the cases with a high BRE expression had a higher 3-year pRFS as compared with cases with a low expression (82±12 vs 52±4%, P=0.01) (Figure 3a).
Within the MLL-rearranged cases, high BRE expression suggested a better 3-year pEFS compared with those with a low BRE expression (50±13 vs 21±8%, P=0.12) and a better 3-year pOS (62±13 vs 32±9%, P=0.19), but the results were not statistically significant. However, again, cases with high BRE expression had a significantly better 3-year pDFS compared with cases with a low expression (80±13 vs 32±11%, P=0.03) (Figure 3b).
Multivariate analysis within the total cohort of pediatric AML, including prognostic factors such as age, white blood cell count, favorable karyotype and t(9;11)(p22;q23), showed that next to favorable karyotype, high BRE expression was an independent favorable prognostic factor for RFS (HR 0.2, P=0.04), but not for EFS (HR 0.4, P=0.06) and OS (HR 0.5, P=0.29) (Table 2).
Functional assays after transfection
Functional studies were performed to find an explanation for the association of high BRE expression and favorable outcome. To explore a possible role for BRE in cell proliferation, the Monomac-1 cell line, harboring a t(9;11)(p22;q23), was transiently transfected with pLNCX-BRE by means of electroporation. However, a maximum transfection efficiency of only 25–30% was achieved with pLNCX-EGFP. At different time points, an increased expression of BRE on messenger (m) RNA was measured (Supplementary Figure 3A), but not evidently on protein level (Supplementary Figure 3B), which could be explained by the low transfection efficiency. This could explain why we did not detect significant differences in cell-cycle proliferation between BRE-transfected cells and those transfected with an empty vector (Supplementary Figure 3C), nor an apoptotic effect of BRE (Supplementary Figure 3D). In addition, in vitro drug sensitivity for cells overexpressing BRE showed no significant differences in drug sensitivity compared with cell transfected with an empty vector or non-transfected cell line (Supplementary Table 3).
BRE protein expression in MLL-rearranged AML
To identify a correlation between protein and mRNA expression levels, we performed western blot analysis on 11 MLL-rearranged AML cases, for whom protein was available in our cell bank. No differences in BRE protein expression were found between t(9;11)(p22;q23) (n=6) and other MLL-rearranged AML cases (n=5) (Figure 2d). The BRE-transfected Monomac-1 cell line was used as positive control (Supplementary Figure 3B), indicating the specificity of the antibody against BRE.
Pediatric AML is a heterogeneous disease, and currently, response to therapy and cytogenetic abnormalities are the main prognostic factors. Interestingly, for the MLL-rearranged group, we previously showed in a large international retrospective study that prognosis mainly depends on the translocation partner of MLL.6 AML patients with t(1;11)(q21;p23) and t(9;11)(p22;q23) with FAB-M5 were found to have a favorable prognosis, whereas patients with t(10;11)(p12;q23) and t(6;11)(q27;q23) were found to have a unfavorable prognosis. This diversity in outcome indicates differences in leukemogenesis within MLL-rearranged AML. However, to date, the factors that have a role in these biological differences are largely unknown.
In this study, we performed gene expression profiling on a large cohort of pediatric AML samples, with the aim to detect such differences in biology. Supervised clustering analysis identified a specific gene expression signature for t(9;11)(p22;q23), the most common translocation partner in MLL-rearranged AML. High expression of the BRE gene was one of the strongest components of this signature. High BRE expression was not restricted to t(9;11)(p22;q23) only; three cases with a t(10;11)(p12;q23) and one case with a t(8;16)(p11;p13) also showed high BRE expression. Interestingly, t(8;16)(p11;q23) has been linked to MLL-rearranged AML on the basis of gene expression profiles in adult AML.34 This particular t(8;16)(p11;p13) case occurred in an infant with a FAB-M5 AML. None of the other pediatric AML cases showed a high BRE expression, indicating that particularly MLL fusions may lead to high BRE expression and hence have a role in leukemogenesis in AML. Interestingly, high BRE expression was not found in t(9;11)(p22;q23) (n=10) or in other MLL-rearranged precursor B or infant acute lymphoblastic leukemia (n=71) cases in two large GEP studies conducted at our center, indicating its specific role in AML.15, 35
Recently, overexpression of BRE has been described in hepatocellular and esophageal carcinomas.32, 36 However, to date BRE has never been associated with hematological malignancies. This study shows that BRE has a role in pediatric AML and mainly in patients with t(9;11)(p22;q23). Moreover, high BRE expression was an independent favorable prognostic factor because of a reduced relapse rate in remission. To explain the observed difference in relapse risk, we examined the effect of BRE expression in vitro on altered cell proliferation, apoptosis and drug sensitivity in AML.
The BRE protein is mainly localized in the cytoplasm, but nuclear localization has also been reported as a subunit of the holoenzyme complex BRCC, which contains BRCA1, BRCA2, RAD51 and BARD1.37 BRCC enhances cellular survival after DNA damage by ubiquitin E3 ligase activity. Interestingly, association of BRE to this complex further enhances the E3 ligase activity. After death-receptor stimulation an increased binding of cytosolic BRE to ubiquitinated proteins was found, suggesting that BRE has an important role in post-translational modification of proteins. Intriguingly, BRE transcription has been shown to be downregulated after DNA damage and retinoic acid treatment.38 Li et al.39 showed that BRE is a death receptor-associated antiapoptotic protein, inhibiting the mitochondrial apoptotic pathway. We could not find evidence that overexpression of BRE influenced apoptosis in an MLL-rearranged AML with the current cell line studies. This may be because of the fact that only a transfection rate of 30% was achieved despite rigorous efforts. Alternatively, other mechanisms could have a role, and the role in apoptosis may be tissue dependent. Alternative approaches, such as stable transduction with a selectable vector and manipulation of primary hematopoietic progenitors, could elucidate this.
Previous studies demonstrated that overexpression of BRE enhances tumor growth, rather than initiating tumor formation in vivo.40 However, BRE overexpression did not lead to cell proliferation in vitro.41 Tang et al.42 showed conflicting data, that is, a decreased in vitro cell proliferation by overexpression of BRE and upregulation of p53, prohibitin and proteins involved in the nuclear factor-kB signaling. In an MLL-rearranged AML cell line, we could not show such a role for BRE in cell proliferation. It is conceivable that stable transfection cell line models or in vivo models could give more insight into whether BRE overexpression influences proliferation or apoptosis of AML cells similar to what was previously shown in hepatocellular carcinoma models.
Using in vitro assays, we were unable to determine that BRE overexpression was related to higher drug sensitivity, although the low transfection rate could have influenced these results. Li et al.39 showed that knockdown of endogenous BRE has little, if any, modulatory effect on apoptosis induced by etoposide in contrast to tumor necrosis factor-α-induced apoptosis. This seems to be in line with our results, as etoposide-induced apoptosis was not influenced by overexpression of BRE in AML. As we could not explain the observed difference in relapse risk in our series, further investigation is required to elucidate the role of BRE overexpression in the leukemogenesis of t(9;11)(p22;q23). MLL-fusion proteins are involved in inappropriate transcriptional activation, and a specific role for MLL-AF9 in the transcription of BRE is most likely.
In this study, protein analysis of BRE did not correlate with mRNA expression and differences in outcome were, therefore, only related to mRNA expression levels. However, differences between mRNA and BRE expression are commonly observed, and Greenbaum et al.43 suggested that, next to translation regulation and differences of in vivo protein half-lives, a significant amount of experimental error could be the lack of perfect correlation between protein and mRNA. This could have influenced the results, as we used cell-bank material instead of fresh samples. We did not further investigate other translation or post-translation regulations of BRE, as this was beyond the scope of this study. However, further research is warranted, as the differences in BRE expression with microarray data were confirmed with RQ–PCR. Moreover these differences in the transcription of BRE seem to influence outcome in pediatric AML.
This study uncovers a small part of the biological background that could explain the recently discovered clinical relevant heterogeneity of MLL-rearranged AML based on translocation partner. The majority of cases with high BRE expression were discovered in the prognostically favorable group with a t(9;11)(p22;q23) and FAB-M5. Within the unfavorable prognostic group t(10;11)(p12;q23), only 3 of 16 cases had a high BRE expression, whereas in the most unfavorable subtype t(6;11)(q27;q23), no cases were found.
Furthermore, high BRE expression is inversely correlated with the overexpression of EVI1 in MLL-rearranged AML. Overexpression of EVI1 is a poor prognostic factor in adult AML. In pediatric AML, it was predominantly found in groups with a poor outcome, including FAB-M7 and t(6;11)(q27;q23).27 Therefore, high BRE expression seems to be part of a favorable signature in MLL-rearranged AML.
In conclusion, our study shows that overexpression of BRE is predominantly found in MLL-rearranged AML with a t(9;11)(p22;q23). Moreover, high BRE expression is an independent favorable prognostic factor because of a reduced relapse rate in remission in pediatric AML. So far, we could not elucidate the exact underlying mechanism. Further research is warranted to explore the role of BRE in AML and to identify the link between MLL-AF9 and the transcription of BRE.
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We thank Dr Yiu-Loon Chui, Department of Chemical Pathology and Sir YK Pao Center for Cancer, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China, for providing the BRE antibody. Furthermore, we thank Dr E Hulleman for her input in the transfection experiments. This work was funded by the NWO ‘Netherlands Organization for Scientfic Research’ (BVB) and KOCR ‘Kinder-Oncologisch Centrum Rotterdam’ (BVB, IHIM).
BVB designed and performed research and wrote the paper; STJCMP and IHIM performed research. CM and RM performed LDI–PCR to identify the MLL rearrangements. VH, GJK, ESJMB, DR, UC, AB, and JS and JT made this research possible by collecting patient samples and characteristics in their own study groups and providing additional information; MMH-E, CMZ and RP designed and supervised research and wrote the paper.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website
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Balgobind, B., Zwaan, C., Reinhardt, D. et al. High BRE expression in pediatric MLL-rearranged AML is associated with favorable outcome. Leukemia 24, 2048–2055 (2010). https://doi.org/10.1038/leu.2010.211
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