Letters to the Editor
804
Another pedigree with familial acute myeloid leukemia and germline CEBPA mutation
Leukemia (2009) 23, 804–806; doi:10.1038/leu.2008.294; published online 23 October 2008
The CCAAT enhancer-binding protein-a (CEBPA) is a transcription factor strongly implicated in hematopoiesis through the control of proliferation and differentiation of myeloid progenitors. The CEBPA protein consists of two N-terminal transactivating domains (TAD1 and TAD2), a basic region able to interact with specific DNA sequences and a C-terminal leucinezipper (bZIP) domain necessary for dimerization. Mutations in the CEBPA gene are observed in 5–10% of sporadic acute myeloid leukemia (AML) cases and are associated with a favorable outcome.1 Recently, four families have been independently described with several members affected by AML and carrying a germline CEBPA mutation.2–6 Here, we report the clinical and biological features of two members of a novel family in which a germline CEBPA mutation predisposed to develop AML (Figure 1a).
The mother (I:2) presented with AML in 1989, at the age of 23 years. The hemogram showed a normal white blood cells count (6.5 G/l) including 72% of blasts. Bone marrow was infiltrated by 95% of myeloperoxidase-positive blasts, some of them containing an Auer rod, corresponding to a M1-AML in the French–American–British classification. Blast cells were positive for CD33, CD13, CD34, CD7 and HLA-DR. Cytogenetic evaluation revealed no abnormalities. She achieved complete remission (CR) after induction chemotherapy and received two consolidation courses. She was in persistent CR during 14 years and gave birth to two children after 4 and 9 years of CR. She had a relapse with the same morphological and phenotypical profile in 2003. She received chemotherapy to obtain second CR and had autologous bone marrow transplantation. During her treatment for relapse, her son, a 5-year-old-boy (II:2) was diagnosed with M1-AML as well. Blast cells were positive for CD33, CD13, CD65, CD34, CD45 and CD7, but HLA-DR negative. Cytogenetic analysis showed no abnormalities.
a I 1
2
II 1
2
b
Figure 1 Family tree and sequence analysis of CEBPA showing the germline mutation. (a) The patients’ family tree. Square is for men and circle is for women. Open symbols represent unaffected individuals and blackened symbols represent individuals who developed AML. (b) Sequence analysis of CEBPA showing the germline mutation identified in patients I:2 and II:2. This mutation consists of an insertion of a cytosine residue at nucleotide 217 (c.217insC) of CEBPA. AML, acute myeloid leukemia; CEBPA, CCAAT enhancer-binding protein-a gene. Leukemia
Letters to the Editor
4 4 This study This study 216+ 24+ 228+ 60+ CR2 CR1 Third relapse CR2 No No No No Yes No Yes Yes In-frame insertion In-frame insertion c.1083_1085delAAG c.1065_1066insGGG M2 M2Eo M1 M1 M M F M 39 26 23 5
Abbreviations: AML: acute myeloid leukemia; CR: complete remission; F: female; FAB: French–American–British; M: male; ND: not determined or not mentioned; OS: overall survival. Sequence numbering is according to GenBank DNA sequence number Y11525 and Swiss-Prot protein sequence number P49715.
c.217insC
Normal at initial diagnosis Trisomy 8 at 1st relapse Trisomy 8 and 21 at 2nd relapse ND ND Normal Normal M 4
M1
c.217insC Normal M 24
ND
c.212delC c.212delC c.217insC c.217insC Normal Normal ND 6q deletion M F M M 30 18 34 25
M2Eo M2Eo ND M4Eo
c.350_351insCTAC c.350_351insCTAC c.217insC c.217insC
3 168+ CR2 No Yes
3 132+ CR1 No No
2 2 3 3 20+ 20+ 13 216+ CR1 CR1 ND CR3 No No Yes No No No ND Yes
2 348+ CR3 No Yes
36-bp duplication of nucleotides 1050 to 1085 Absence Absence ND 7-bp deletion and 10-bp insertion at position 1071 3-bp insertion at position 1071 ND c.212delC ND M 10
M1
Somatically acquired CEBPA mutation Germline CEBPA mutation Karyotype at AML diagnosis FAB subtype Sex Age at AML diagnosis (years)
Table 1
Clinical and biological features of reported AML patients with a germline CEBPA mutation
Relapse
Death
Disease OS (months) status at last followup
Selected reference
805 Physical examination of both patients (I:2 and II:2) revealed no dysmorphic features. Patient II:2 achieved a CR after the first course of chemotherapy and received three consolidation courses. He had a bone marrow relapse after 13 months of CR. He entered a second CR and was allografted with umbilical cord blood-derived stem cells in 2005. He is currently alive in second CR and is healthy. After 3 years of CR, his mother had a second relapse. She has presented a stable disease for 16 months. Bone marrow cells collected at the time of AML diagnosis and peripheral blood cells collected during CR were available for patients I:2 and II:2. In addition, peripheral blood cells collected 2 years after allogeneic stem cell transplantation were available for patient II:2. The two other family members declined testing. Informed consent was obtained for both patients to use their samples for molecular analysis. Genomic DNA was extracted using standard procedures. Screening for FLT3 internal tandem duplication (FLT3-ITD), FLT3-D835/I836, NPM1, N- and K-RAS, c-KIT, RUNX1 and CEBPA mutations were performed on genomic DNA. CEBPA mutations were detected by direct sequencing as described previously7 (referenced to GenBank accession number Y11525). For both patients, two different CEBPA mutations were identified at AML diagnosisFone in the N-terminal domain and the other in the C-terminal domain. The N-terminal mutation was identical for both patients and consisted of an insertion of a cytosine residue at nucleotide 217 (c.217insC) of CEBPA (Figure 1b). This mutation creates a frameshift leading to the truncation of the CEBPA 42-kD protein (p42) at amino acid 106 (p.His24AlafsX83) and potentially increasing translation of the alternative 30-kD isoform (p30) with dominant-negative activity on the full-length p42. In the C-terminal domain, a 3-bp deletion (c.1083_1085delAAG, p.Lys313del) and a 3-bp insertion (c.1065_1066insGGG, p.Arg306_Asn307insGly) were identified in patients I:2 and II:2, respectively (Table 1). Both in-frame C-terminal mutations are predicted to cause an alteration of the dimerization domain and therefore a loss-offunction of the normal allele. The same c.217insC mutation was present in remission samples from patients I:2 and II:2, indicating that this mutation is a germline mutation. In contrast, C-terminal mutations were not detected in remission samples from both patients, suggesting that these mutations are somatically acquired. Besides, the N-terminal CEBPA mutation was not found in the peripheral blood sample from patient II:2 collected 2 years after allogeneic stem cell transplantation. Remarkably, the site of the germline mutation reported here is similar to those described previously.2,3 Moreover, the germline mutation reported here is strictly identical to the germline mutation reported by Sellick et al.,3 suggesting that this region of CEBPA is probably unstable (Table 1). However, acquired mutations in this region have been rarely described and, to our knowledge, remission samples were not tested to exclude a germline mutation. Except for the absence of bone marrow eosinophilia, the biological features of these two patients are similar to those of previously reported casesFmajority of M1 French–American–British subtype, presence of Auer rods in some blasts, aberrant CD7 expression, and normal karyotype. Strikingly, these features are common with sporadic AML cases with acquired CEBPA mutations. Although CEBPA mutations have been found to be an independent favorable prognostic factor in sporadic AML cases,1 the prognostic value associated with inherited CEBPA mutation is difficult to assess because of the small number of reported cases, the heterogeneity of treatment and the short follow-up period. Nevertheless, the clinical course of AML patients with germline CEBPA mutation does not appear Leukemia
Letters to the Editor
806 to be particularly aggressive since 9 out of 11 reported patients were alive in CR at the last follow-up (Table 1).2–4 Like in most of the previously reported patients with germline CEBPA mutation that could have been analyzed at somatic level,2–6 patients I:2 and II:2 exhibited a second CEBPA mutation without other detectable molecular abnormality in the leukemic cells. These findings suggest that the N-terminal germline CEBPA mutation seems to promote the occurrence of an additional C-terminal mutation in CEBPA, which may represent the second genetic event in AML pathogenesis. Furthermore, these data support the hypothesis that biallelic alteration of CEBPA may be sufficient to induce AML. Recently, Kirstetter et al.8 showed that the p42 CEBPA protein was required for control of myeloid progenitor proliferation and that p42-deficient mice developed AML with complete penetrance, showing that, in a mouse model, patient-derived CEBPA mutations are AML-initiating mutations. In human patients with AML, acquired biallelic alteration of CEBPA is observed in approximately 20% of CEBPA-mutated cases. Testing germline material in such cases seems to be important to check whether one of the two CEBPA mutations is a germline mutation. However, not all germline sequence variants are important to consider. We and others previously reported another germline CEBPA alteration, a 6-bp duplication in the TAD2 domain (c.730_735dupACCCGC, p.His195_Pro196dup), which has no clinical implication. In contrast with the germline mutation reported here, this 6-bp duplication was detected at the same frequency in AML patients and in control cases (6.6%), suggesting that it is a polymorphism, rather than a mutation.9
Acknowledgements This work was supported by the North West Canceropole (OncoHematology axis).
A Renneville1,6, V Mialou2, N Philippe1, S Kagialis-Girard3, V Biggio1, M-T Zabot4, X Thomas5, Y Bertrand2 and C Preudhomme1,6 1 Laboratoire d’He´matologie, Centre de Biologie-Pathologie, CHRU de Lille, Lille, France; 2 Institut d’He´matologie et Oncologie Pe´diatrique, Hospices civils de Lyon, Lyon, France;
3
Laboratoire d’He´matologie, Hoˆpital Edouard Herriot, Lyon, France; 4 Laboratoire de Biologie Cellulaire, Centre de Biologie Est, Lyon, France; 5 Service d’He´matologie Clinique, Hoˆpital Edouard Herriot, Lyon, France and 6 INSERM, Unite´ 837, Institut de Recherche sur le Cancer de Lille, Lille, France E-mail:
[email protected]
References 1 Leroy H, Roumier C, Huyghe P, Biggio V, Fenaux P, Preudhomme C. CEBPA point mutations in hematological malignancies. Leukemia 2005; 19: 329–334. 2 Smith ML, Cavenagh JD, Lister TA, Fitzgibbon J. Mutation of CEBPA in familial acute myeloid leukemia. N Engl J Med 2004; 351: 2403–2407. 3 Sellick GS, Spendlove HE, Catovsky D, Pritchard-Jones K, Houlston RS. Further evidence that germline CEBPA mutations cause dominant inheritance of acute myeloid leukaemia. Leukemia 2005; 19: 1276–1278. 4 Nanri T, Uike N, Kawakita T, Iwanaga E, Hoshino K, Mitsuya H et al. A pedigree harboring a germ-line N-terminal C/EBPa mutation and development of acute myeloblastic leukemia with a somatic Cterminal C/EBPa mutation. Blood 2006; 108: 543a (abstract 1916). 5 Corbacioglu A, Fro¨hling S, Mendla C, Eiwen K, Habdank M, Do¨hner H et al. Germline mutation screening in cytogenetically normal acute myeloid leukemia with somatically acquired CEBPA mutations. Blood 2007; 110: 114a (abstract 363). 6 Owen C, Barnett M, Fitzgibbon J. Familial myelodysplasia and acute myeloid leukaemia-a review. Br J Haematol 2008; 140: 123–132. 7 Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S, Schnittger S et al. Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nat Genet 2001; 27: 263–270. 8 Kirstetter P, Schuster MB, Bereshchenko O, Moore S, Dvinge H, Kurz E et al. Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells. Cancer Cell 2008; 13: 289–291. 9 Biggio V, Renneville A, Nibourel O, Philippe N, Terriou L, Roumier C, et al., French ALFA group. Recurrent in-frame insertion in C/EBPalpha TAD2 region is a polymorphism without prognostic value in AML. Leukemia 2008; 22: 655–657.
Simultaneous study of five candidate target antigens (CD20, CD22, CD33, CD52, HER2) for antibody-based immunotherapy in B-ALL: a monocentric study of 44 cases
Leukemia (2009) 23, 806–807; doi:10.1038/leu.2008.303; published online 30 October 2008
In the last few years, targeted immunotherapy for haematological diseases has been developed to reduce the toxicity of chemotherapy and to improve treatment efficacy.1 As the prognosis of B-acute lymphoblastic leukaemia (B-ALL) in adults and in some children remains poor, we have investigated the simultaneous expression of five target surface antigens (sAg) (CD20, CD22, CD33, CD52, HER2) for which monoclonal antibodies are currently available for immunotherapy (rituximab, epratuzumab, gemtuzumab, alemtuzumab, trastuzumab). Leukemia
We have identified a series of 44 B-ALL (22 males and 22 females) at diagnosis (n ¼ 40) or at relapse (n ¼ 4) between March 2000 and June 2008. The median age was 51 (range 5–92) years. There were 13 Philadelphia positive B-ALL. CD20, CD22, CD33, CD52 and HER2 expression was assessed using multicolour flow cytometry with the following phycoerythrinconjugated monoclonal antibodies directed against: CD20 (L27), CD22 (SHCL1), CD33 (P67.6), HER2 (Neu 24.7) purchased from BD (San Jose, CA, USA) and CD52 (CF1.D12) purchased from Caltag (Buckingham, UK). A CD19 þ CD45 þ low blast cell gating strategy was also used. The mean fluorescence intensity ratio was obtained by dividing the mean fluorescence intensity of the considered antibody by
