CCAAT/enhancer-binding protein-α (C/EBPα) is a transcription factor strongly implicated in hematopoiesis through control of proliferation and differentiation of myeloid progenitors. Disruption of C/EBPα results in selective early block of granulocyte maturation, without affecting other hematopoietic lineages.1
In patients with acute myeloid leukemia, the normal function of C/EBPα can be modified by interaction with other oncogenic proteins (like those resulting from inv(16)(p13;q22))2 or more often, by mutations (in about 8% of cases).3 Those mutations are generally in-frame mutations in the C-terminal region or out-of-frame mutation in the N-terminal region, resulting in loss of C/EBPα function.3
Among these mutations, an in-frame insertion of 6 bp in the trans-activator domain 2 (TAD2) domain, resulting in a His–Pro duplication (HP196–197ins), was described in patients at diagnosis.4 Our group also found this abnormality at time of remission, suggesting a polymorphism, possibly predisposing to AML. Furthermore, this 6 bp insertion was also observed in healthy controls too. For this reason, we did not take into account this 6 bp insertion in our previous works. Other groups reported this mutation and either included it as a C/EBPα mutation,4 or like us excluded it from C/EBPα mutations.3
The Prolin–Histidine-rich region (PHR), region where this 6 bp insertion is found, is particularly interesting because in vitro studies established an interaction between C/EBPα and molecules like cdk2 and cdk4, for growth arrest and differentiation. Cdk2 and cdk4 are under the control of C/EBPα by a region in the TAD2 between amino acids 175 and 187.5 Unexpectedly, in vivo studies with transgenic mouse ΔPHR did not confirm this previous finding, showing that this region was dispensable for C/EBPα-mediated growth regulation.6 Recently, Delwel et al.7 described this 6 bp insertion as being a polymorphism. They reported it in 3.2% (9/282)–3.9% (12/305) of AML patients, while in non-leukemic blood samples the incidence was 8% (22/274). Furthermore, they found no correlation between this insertion and C/EBPα gene-expression signature.
In this study we tried to elucidate the possible impact of this 6 bp insertion in AML patients and to study its prognostic value in a prospective clinical trial.8 A total of 305 patients with de novo AML (excluding APL), aged 16–50 years, enrolled in ALFA 98-02 trial with available pretreatment blood stored, and 996 healthy controls were investigated for the presence of C/EBPα mutations, including the 6 bp insertion. Median follow-up was 3 years. In all, 17.7% of patients belonged to the favorable cytogenetic group, 15.7% to poor risk group and 56.7% to the intermediate risk group. Karyotype was a failure in the remaining 9.8% of patients.
Detection of 6 bp insertion was performed on genomic DNA by PCR and fragment analysis. To amplify the region, we used a fluorescently labeled forward primer 5′-(dyeD4) IndexTermGAGGAGGATGAAGCCAAGC-3′ (Proligo, Boulder, CO, USA) and a reverse primer 5′-IndexTermATGCACCTGCAGCCCGGT-3′ (Invitrogen, Cergy-Pontoise, France). Fluorescent PCR products were subjected to capillary electrophoresis on denaturing polyacrylamide gel and analyzed by the CEQ 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA). Data were processed using Genetic Analysis System Software (Beckman Coulter, Fullerton, CA, USA). Mutations were confirmed by direct sequencing using AB3100 (Applied Biosystems, Foster City, CA, USA) in two different reactions. Other mutations of C/EBPα gene were detected as described previously.4
Comparisons between patient characteristics were performed using the Pearson χ2 test for binary variables. Event-free survival (EFS) and overall survival (OS) were measured from trial inclusion to date of event or death respectively, regardless of cause, censoring patients alive at last follow-up. OS and EFS were estimated by the Kaplan–Meier method and compared using the log–rank test.
We first tried to compare the frequency of 6 bp insertion in healthy controls and AML patients. In healthy controls this insertion was found in 66 cases of 996 (6.6%) and in 21 of 305 (6.5%) AML patients. A similar incidence in AML patients and controls suggests that this 6 bp mutation does not predispose to AML.
We also assessed the role of this insertion in the prognosis of AML by comparing AML patients with no C/EBPα mutations, with this 6 bp insertion and with ‘true’ C/EBPα mutations. Patients who harbored the 6 bp insertion (n=21), in this AML cohort, had no other C/EBPα mutations, while most of the AML patients present two different mutations in C/EBPα gene (in C- and N-terminal regions, monoallelic or biallelic). Patients positive for the 6 bp insertion had heterogeneous cytogenetic findings (five complex, five normal, three intermediate karyotypes, four CBF-AML and four unknown), while patients with others, abnormality of C/EBPα gene (n=21) showed significant association with normal karyotype (P=0.03). Mutations of C/EBPα are most frequently associated to M1 and M2 FAB subtypes (14 of 21 C/EBPα positive patients presenting a M1–M2 FAB subtype, P=0.03), but we found an association to FAB subtype in patients with the 6 bp polymorphism (10 of 21 patients presenting the 6 bp insertion presenting a M1–M2 FAB subtype, P>0.7).
C/EBPα mutations and 6 bp insertion were rarely associated to NPM mutations (1 of 21 patients for C/EBPα versus 2 of 21 patients for 6 bp insertion). Finally, C/EBPα mutations and 6 bp insertion are mutually exclusive with FLT3-ITD mutations (2 of 21 patients for C/EBPα versus 1 of 21 patients for 6 bp insertion) (Table 1).
Median EFS was 27.4 months for the 6 bp polymorphism and 27.4 months for C/EBPα positive patients in the total population. In the intermediate cytogenetic subgroup median EFS was not reached for the 6 bp insertion and 28.8 months for C/EBPα mutated patients. Median EFS in patients with normal karyotype was 27.4 months for 6 bp insertion and not reached for C/EBPα mutations. The median EFS for others not mutated patients was 17.5, 19.3 and 22.1 months for total, intermediate and normal cytogenetic group respectively. EFS curve did not show any difference between patients with 6 bp insertion or patients with C/EBPα mutations, compared to other not mutated patients (P=0.9; P=0.6 and P=0.4 respectively).
Median OS for patients presenting the 6 bp insertion was not reached for the global population and for the intermediate group and 27.4 months for normal karyotype, compared to 60.2 months for C/EBPα-positive patients in global population and not reached for intermediate and normal karyotype. For other not mutated patients OS median value was 50.7 months in global population and 72.8 months in intermediate and normal karyotype groups (OS P-value: P=0.9; P=0.6 and P=0.4 for total population, intermediate risk group and normal karyotype respectively) (Figure 1).
C/EBPα was the only factor associated with a trend for longer survival in normal karyotypes (P=0.09, data not shown).
In conclusion, the 6 bp insertion (HP196–197ins) is a common inherited mutation spread in normal population and AML patients and it is now preferable to separate this 6 bp insertion from C/EBPα mutations and recalculate the real frequency of C/EBPα mutations, because, in several studies this insertion was considered like C/EBPα mutations.
In our cohort of AML patients, the 6 bp insertion showed no prognostic value.
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Porse BT, Pedersen TA, Hasemann MS, Schuster MB, Kirstetter P, Luedde T et al. The proline-histidine-rich CDK2/CDK4 interaction region of C/EBPalpha is dispensable for C/EBPalpha-mediated growth regulation in vivo. Mol Cell Biol 2006; 26: 1028–1037.
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Biggio, V., Renneville, A., Nibourel, O. et al. Recurrent in-frame insertion in C/EBPα TAD2 region is a polymorphism without prognostic value in AML. Leukemia 22, 655–657 (2008). https://doi.org/10.1038/sj.leu.2404926
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