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In Reply

Department of Internal Medicine III, University Hospital of Ulm, Ulm, Germany

Using DNA sequence analysis of the gene encoding the myeloid transcription factor CEBPA, Resende and colleagues demonstrate that an in-frame insertion (c.1175_1180dup; p.P194_H195dup) in the second transactivation domain (TAD2) of CEBPA that was first described in patients with acute myeloid leukemia (AML)¹ represents a germline polymorphism rather than a somatic mutation implicated in leukemogenesis. This observation confirms previous data by Lin et al² who detected specific TAD2 alterations, including the one discussed by Resende et al, in normal individuals, remission bone marrow samples from AML patients, or both. Furthermore, Wouters et al³ recently reported that the gene-expression profiles derived from AML patients with the 1175_1180dup variant do not cluster together with those of other CEBPA mutant cases, thus reinforcing the notion that this insertion is unlikely to be linked to AML pathogenesis.

Collectively, these findings highlight a major challenge of large-scale sequencing studies in cancer, that is, to distinguish mutations that contribute to malignant transformation from germline variants or nonfunctional passenger alterations that arise due to an increased mutation rate and accumulate during repeated rounds of cell division. Ideally, the following steps are employed to identify candidate oncogenic alleles. First, any silent sequence variations, annotated single-nucleotide polymorphisms, or changes that are also present in normal control samples are excluded. Second, the genomic regions containing putative mutations are sequenced in matched normal DNA samples to determine whether the alterations are truly somatic. And finally, the functional consequences of candidate alleles are experimentally tested.

We recently reported that mutant CEBPA was associated with favorable clinical outcome in 236 younger AML patients with normal cytogenetics.¹ One limitation of our study was that matched germline DNA was not available for this set of AML samples and that no normal control population could be analyzed. We therefore decided to include all DNA sequence variations that were predicted to alter the amino acid sequence of the CEBPA protein, including the 1175_1180dup variant, in our analysis. Importantly, our study focused on the clinical relevance of mutant CEBPA and was not designed to determine the functional consequences of individual CEBPA mutations, thereby precluding any conclusions as to their potential contribution to AML pathogenesis.

To examine if the main conclusion of our article was substantially influenced by the miscategorization of patients with germline polymorphisms as CEBPA-mutated patients, we classified the three patients that had the 1175_1180dup variant as the sole CEBPA abnormality as wild-type CEBPA and repeated the analyses of remission duration and overall survival (OS) according to CEBPA mutation status. As shown in Figure 1, patients with mutated CEBPA had a significantly longer remission duration (P = .02 by the log-rank test) and showed a strong trend toward a longer overall survival (P = .09) as compared with patients with wild-type CEBPA.

View larger version (15K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Long-term outcome of 236 younger acute myeloid leukemia patients with normal cytogenetics according to CEBPA mutation status. (A) Remission duration; (B) overall survival.

Specific patterns of gene mutations have emerged as highly significant prognostic factors for response to induction therapy and survival in AML patients with normal karyotype,^5,6 although the molecular mechanisms underlying responsiveness or resistance to antileukemic therapy remain elusive. As illustrated by the example of mutant CEBPA, our ability to systematically compile mutational data in large patient cohorts has been substantially improved by the increasing throughput of DNA sequencing technologies. However, the interpretation of the results from large-scale sequencing studies in patients with cancer will be greatly facilitated by the availability of matched germline samples or adequately sized normal control populations, as pointed out by Resende et al.

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The author(s) indicated no potential conflicts of interest.

REFERENCES

1. Frohling S, Schlenk RF, Stolze I, et al: CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: Prognostic relevance and analysis of cooperating mutations. J Clin Oncol 22:624-633, 2004[Abstract/Free Full Text]

2. Lin LI, Chen CY, Lin DT, et al: Characterization of CEBPA mutations in acute myeloid leukemia: Most patients with CEBPA mutations have biallelic mutations and show a distinct immunophenotype of the leukemic cells. Clin Cancer Res 11:1372-1379, 2005[Abstract/Free Full Text]

3. Wouters BJ, Louwers I, Valk PJ, et al: A recurrent in-frame insertion in a CEBPA transactivation domain is a polymorphism rather than a mutation that does not affect gene expression profiling-based clustering of AML. Blood 109:389-390, 2007[Free Full Text]

4. Pabst T, Mueller BU, Zhang P, et al: Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nat Genet 27:263-270, 2001[CrossRef][Medline]

5. Estey E, Dohner H: Acute myeloid leukaemia. Lancet 368:1894-1907, 2006[CrossRef][Medline]

6. Mrozek K, Dohner H, Bloomfield CD: Influence of new molecular prognostic markers in patients with karyotypically normal acute myeloid leukemia: Recent advances. Curr Opin Hematol 14:106-114, 2007[Medline]

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