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Core-binding factor acute myeloid leukemia in pediatric patients enrolled in the AIEOP AML 2002/01 trial: screening and prognostic impact of c-KIT mutations

Leukemia volume 28, pages 1132–1134 (2014)Cite this article

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The proto-oncogene c-KIT, which encodes a receptor for stem cell factor (SCF), belongs to the type-III receptor of the tyrosine kinase subfamily and is characterized by five extracellular immunoglobulin-like domains, a single transmembrane helix (TM), a cytoplasmic juxtamembrane domain(JMD), and a kinase domain. Abnormal activation of c-KIT/SCF growth signal has been frequently documented to occur in cancers, including hematological malignancies, and has been frequently associated with poor prognosis in adults with acute myeloid leukemia (AML) harboring aberrancies at core-binding factor genes (CBF).1, 2, 3 c-KIT mutations have been reported in pediatric CBF-rearranged AML at frequencies ranging from 15 to 54.5%; however, their prognostic significance is still debated.4, 5, 6, 7 Mutations of c-KIT occur in the extracellular portion of the receptor implicated in dimerization within exon 8, in the TM-JMD domain within exon 11 and in the activation loop of the tyrosine kinase domain within exon 17, this mediating the constitutive activation of the receptor. The AIEOP AML 2002/01 protocol allocated patients with CBF rearrangements in the standard-risk (SR) group, and although all these patients reached complete remission after the first two induction courses, they showed a higher than expected cumulative incidence of relapse (24%).8 The identification of new independent prognostic factors and therapeutic targets is desirable to optimize the outcome of this subgroup of childhood AML. In particular, our interest focused on determining whether the presence of a c-KIT mutation could have a prognostic impact and could allow refining the risk stratification for this subgroup of AML patients. We retrospectively analyzed the bone marrow at diagnosis of 88 children with CBF-AML enrolled in the SR group of the AIEOP AML 2002/01 protocol. Sixty-one patients carried t(8;21) RUNX1-RUNX1T1, 26 inv(16)(p13;q22) CBFB-MYH11 and 1 t(16;16)(p13;q22) CBFB-MYH11. Screening for mutations of c-KIT was performed on cDNA by PCR amplification followed by Sanger sequencing of exons 8 and 17 or analysis by the Genescan and Genemapper software (Applied Biosystems Inc., Foster City, CA, USA) for exon 11. The primers used are listed in Supplementary Table 1S. Denaturing, annealing and extension steps were performed at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, respectively, for a total of 40 cycles on a thermocycler. PCR products were resolved on a 2% agarose gel. After visual confirmation of amplification, 4 μl of PCR products of exon 8 or 17 were purified with a mixture of 0.5 μl Exonuclease I and 1 μl of FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, Milan, Italy) and analyzed by bidirectional sequencing on an ABI310 sequencer, using the BigDye terminator kit v3.1 (Applied Biosystems Inc.). The prognostic impact of a c-KIT mutation was assessed analyzing overall and event-free survival (OS, EFS) probabilities; the log-rank (Mantel–Cox) test was employed to detect differences between subgroups.

The prognostic significance of a c-KIT mutation in other pediatric CBF-AML cohorts has been reported to be different in previously published studies. Goemans et al.5 identified c-KIT aberrancies in 10/27 children (37%), with a higher incidence of mutation in inv(16) compared with t(8;21) (54.5% vs 31.3%). Shih et al.6 detected abnormalities of c-KIT in 17/41 (41.4%) children with CBF-AML: 12/28 (43%) were mutated in RUNX1-RUNX1T1-rearranged children as compared with 5/13 (38.5%) in inv(16). Both studies did not find any statistical influence of a c-KIT mutation on patient outcome. In addition, Pollard et al.4 analyzed the mutation status of 203 children with CBF-AML finding c-KIT mutations in 19/94 t(8;21) patients and in 19/71 carrying inv(16). Notwithstanding the large sample size, the results did not reach significance for survival parameters. These findings are in contrast with our data and with the data published by Shimada et al.7 who screened 46 t(8;21) children for c-KIT mutations. Significant differences between patients with or without c-KIT mutations were observed in the 4-year OS (50.0% versus 97.4%, P=0.001), disease-free survival (37.5% versus 94.7%, P<0.001) and relapse rate (47.0% versus 2.7%, P<0.001). In view of our data on the incidence and the prognostic impact of c-KIT mutations, we believe that t(8;21) and inv(16)/t(16;16) patients should be analyzed separately. In particular, among our c-KIT-mutated patients who experienced relapse, we observed that five out of five t(8;21) patients were dead, whereas five out of the seven RUNX1-RUNX1T1-positive non c-KIT-mutated children who relapsed were rescued by second-line treatment. These findings provide the rationale for considering c-KIT mutations as an additional genetic marker to be taken into account in patient stratification.

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Acknowledgements

This work was supported in part by grants from Cariparo, IRP-Istituto di Ricerca Pediatrica-Città della Speranza Padova (EM, VB, MP and GB), by Fondazione Umberto Veronesi (Milan) and to Fondazione Ginevra Caltagirone and Banca Popolare di Milano (RM) and by the Special Grant ‘5 × 1.000’ from AIRC (FL).

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Authors and Affiliations

  1. Clinica di Oncoematologia Pediatrica, Università degli Studi di Padova, Padova, Italy

    E Manara, V Bisio, V Beqiri, G Basso & M Pigazzi

  2. Clinica Pediatrica, Università di Bologna, Ospedale ‘S. Orsola’, Bologna, Italy

    R Masetti & R Rondelli

  3. Oncoematologia Pediatrica, Ospedale Pausilipon, Napoli, Italy

    G Menna

  4. Oncoematologia Pediatrica, IRCCS Istituto ‘Giannina Gaslini’, Genova, Italy

    C Micalizzi

  5. Clinica Pediatrica, Policlinico di Bari, Bari, Italy

    N Santoro

  6. Dipartimento di Oncoematologia Pediatrica, IRCCS Ospedale Pediatrico Bambino Gesù, Università di Pavia, Roma, Italy

    F Locatelli

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Manara, E., Bisio, V., Masetti, R. et al. Core-binding factor acute myeloid leukemia in pediatric patients enrolled in the AIEOP AML 2002/01 trial: screening and prognostic impact of c-KIT mutations. Leukemia 28, 1132–1134 (2014). https://doi.org/10.1038/leu.2013.339

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