Letters to the Editor 2404 In hematopoietic cells with a germline mutation of CBL, loss of heterozygosity is not a signature of juvenile myelo-monocytic leukemia

Leukemia (2013) 27, 2404–2407; doi:10.1038/leu.2013.203 are at increased risk of developing juvenile myelo-monocytic leukemia (JMML), an aggressive myelodysplastic and myeloproliferative neoplasm of early childhood characterized by The CBL gene (11q23.3) encodes an E3 ligase clonal macrophage/monocyte proliferation.3,4 JMML cells are that negatively regulates signaling by promoting degradation of hypersensitive to granulocyte–macrophage colony-stimulating activated kinase receptors. In addition, CBL is an adaptor factor consecutively to the activation of the RAS-MAPK signaling protein that positively regulates .1 Individuals pathway through the mutation of the following genes: PTPN11, with germline heterozygous CBL mutations present with a NRAS, KRAS, NF1 or CBL. CBL-associated JMML can follow clinically variable condition that can resemble an aggressive clinical course or resolve without treatment.4–6 and will be further referred as the ‘CBL syndrome’.2 These patients The current diagnostic criteria for JMML are based on an

Table 1. Patients with CBL mutation

Hematological Gender Age at Nucleotidic Amino-acid Hematopoietic Germline Pattern of Other RAS- Endogenous Treatment, outcome phenotype diagnosis of change change CBL status CBL status inheritance related growth of (follow up in months) hematological of the CBL mutationsa myeloid disorders mutation progenitors (years)

1 JMML F 1 c.1111 T4C p.Tyr371His Homozygous Heterozygous De novo No Yes Relapse after HSCT (LOF) 2 JMML F 1.1 c.1111 T4C p.Tyr371His Homozygous Heterozygous De novo No Yes HSCT, CR (51 mo) 3 JMML F 2.2 c.1111 T4C p.Tyr371His Homozygous Heterozygous Paternally No Yes HSCT, CR (72 mo) inherited 4 JMML F 1 c.1228-2 A4G Splice site Homozygous Heterozygous De novo No Yes HSCT, CR (80 mo) 5 JMML M 0.5 c.1254 C4G p.Phe418Leu Homozygous Heterozygous Maternally No Yes No treatment, stable inherited disease (72 mo) 6 JMML F 2.1 c.1096 -1delGGb Splice site Heterozygousc Heterozygous De novo No Yes Relapse after HSCT, stable disease (26 mo) 7 JMML F 2.5 c.1111 T4C p.Tyr371His Homozygous Heterozygous De novo No Yes Relapse after HSCT, 2nd HSCT, CR (11 mo) 8 JMML M 4.2 c.1096-1G4C Splice site Heterozygous Wild-type NA NF1 (LOH) Yes JMML progression and second malignancy (rhabdomyosarcoma). Dead 9 JMML M 3.9 c.1150T4C p.Cys384Arg Heterozygous Wild-type NA PTPN11Ala72Val Yes HSCT, CR (21 mo) 10 Splenomegaly F 1.6 c.1258 C4G p.Arg420Gly Homozygous Heterozygous De novo no ND No treatment. Splenomegaly (53 mo) 11 Neutrophilic F 5.7 c.1253 T4C p.Phe418Ser Homozygous Heterozygous Maternally No Yes Mercaptopurine, dermatosis inherited Splenomegaly (34 mo) 12 Lympho-histiocytic M 2.9 c.1141 T4Gb p.Cys381Gly Homozygous Heterozygous De novo No Yes No treatment. syndrome Auto-immune manifestations (44 mo) Abbreviations: CR, complete remission; F, female; HSCT, allogeneic hematopoietic stem cell transplantation; JMML, juvenile myelo-monocytic leukemia; LOF, lost of follow-up; LOH, loss of heterozygosity; M, male; mo, month; NA, not applicable; ND, not determined. aThese mutations were absent from fibroblasts and thus considered as somatically aquired. bNovel mutation (see Figure 1 for details). cIn this patient, microsatellite analysis on serial samples confirmed the absence of LOH after 4 months of evolution of JMML (see Figure 1c).

Figure 1. CBL mutations. (a) Sequence electropherograms documenting CBL mutations in the three patients with ‘CBL syndrome’ and atypical immuno-hematological manifestations (patients 10, 11, 12), and in the patient with the novel mutation that remains heterozygous in JMML cells (patient 6). Asterisks indicate heterozygous mutations, black triangles indicate homozygous mutations. Subcloning of mutation c.1096-1delGG (patient 6) in Escherichia coli permitted to sequence separately each allele and confirmed that both deleted bases lied on a single allele. Sequencing of the complete coding sequence of CBL revealed no other mutation in JMML cells (data not shown). Pedigrees of the patients are indicated below. Black boxes: patients with homozygous CBL mutation in peripheral blood; gray boxes: patients with heterozygous CBL mutations in peripheral blood; white boxes: patients with wild-type CBL in peripheral blood. (b) Complementary DNA (cDNA) analysis shows that mutation c.1096-1delGG (patient 6) has the same consequences on splice than mutation c.1096-1G4C (patient 8) previously described.4 Left: reverse transcription PCR using an exon 6 forward primer and an exon 10 reverse primer on RNA and electrophoretic migration in agarose gel. Lane 1: 100–1000 bp size ladder; lane 2: no template control; lane 3: CBL wild-type control; lane 4: patient 8 (c.1096-1G4C); lane 5: patient 6 (c.1096-1delGG). Right: schematic representation of the mRNA variants detected in patients 6 and 8 (D: deletion; ins: insertion). The length of each amplicon is indicated. (c) Analysis of microsatellite markers covering the 11q23 region in patient 6. Loss of heterozygosity (LOH) at CBL locus was assessed by PCR amplification of seven microsatellite markers covering the 11q arm: D11S4206 (11q22.3), D11S4129 (11q22.3), D11S924 (11q22.3), D11S1774 (11q22.3), D11S925 (11q23), D11S934 (11q23-24) and D11S968 (11q25). Fluorescent PCR products were separated by capillary electrophoresis. Allelic sizes are indicated in base pairs (bp) between brackets for each marker. Allelic ratios of JMML cells DNA over germline DNA were calculated as follows: R ¼ [(A1/A2) JMML]/[(A1/A2) Germline]. They were comprised between 0.9 and 1.1, showing no LOH both at diagnosis and 4 months after diagnosis. WT, wild type.

Accepted article preview online 4 July 2013; advance online publication, 26 July 2013

Leukemia (2013) 2376 – 2424 & 2013 Macmillan Publishers Limited Letters to the Editor 2405 international consensus that recently incorporated NF1, RAS and protein with defective E3 ligase activity that constitutively PTPN11 mutational status and monosomy 7.7 CBL mutations have activates key RAS effector pathways.4 Importantly, in virtually since been discovered and are also screened for in the workup of all patients described so far, the wild-type CBL allele is lost in patients with suspected JMML.8,9 CBL mutations reported so far leukemic cells and replaced with the mutant allele by acquired are missense mutations or splice site variants clustered in the uniparental isodisomy of the 11q23 chromosomal region leading linker region, and at or near the zinc-coordinating amino acids of to loss of heterozygosity (LOH) of the mutated CBL.4,3,6 Hence, LOH the RING finger domain.1 They all result in the expression of a CBL of the mutated CBL in hematopoietic cells is usually considered as

Patient 10 Patient 11 Patient 12 Patient 6 (p.Arg420Gly) (p.Phe418Ser) (p.Cys381Gly) (c.1096 -1delGG)

Intron 7

Mutated allele Germline DNA Subcloning (fibroblasts) * into E. coli * *

DNA from Wild type allele hematopoietic cells

WT 12345612 bp Exon 6 Exon 7 Exon 8 Exon 9 1 Kb 800 bp

600 bp 592 bp Exon 6 Exon 7 ExonExon 88 Exon 9

400 bp 24 bp

200 bp 544 bp Exon 6 Exon 7 ExonExon 88 Exon 9

72 bp

484 bp Exon 6 Exon 7 Exon 9

Exon8 (132 bp)

937 bp Exon 6 Exon 7 Intron 7 Exon 8 Exon 9

Ins intron 7 (321 bp)

Size (bp)

Germline DNA (fibroblasts)

DNA from hematopoietic cells at diagnosis of JMML

DNA from hematopoietic cells 4 months after diagnosis

D11S4129 D11S968 D11S4206 D11S925 D11S934 D11S1774 D11S924 (114-116) (143-147) (163-167) (175-196) (180-202) (206) (245-249)

& 2013 Macmillan Publishers Limited Leukemia (2013) 2376 – 2424 Letters to the Editor 2406 a signature of JMML in patients with ‘CBL syndrome.’ However, to birth. At the age of 5 years, she developed acute febrile date, consequences induced by LOH of CBL mutant proteins neutrophilic dermatosis with multivisceral failure due to neutrophil remain partly unknown. invasion. White blood cell count showed increased circulating CBL mutations were screened in JMML patients of the French neutrophils and monocytes up to 25 Â 109/l and 11 Â 109/l, cohort (n ¼ 102) and in patients referred to our lab with a respectively. Bone marrow aspiration revealed a granular hyper- borderline JMML phenotype. The diagnosis of JMML was based on plasia. A CBL c.1253T4C (p.F418S) homozygous mutation was consensus criteria as described above and included centralized identified in peripheral blood cells and in neutrophils infiltrating a cytomorphological review of bone marrow and blood as well as a cutaneous lesion. An inherited heterozygous mutation was found comprehensive genetic testing by bidirectional Sanger sequen- in her fibroblasts. Treatment with corticosteroid and mercapto- cing of tumoral DNA.5,7 CBL mutations were identified in a total of purine improved her condition and she remains healthy 2 years 12 children. The germline origin of mutations was tested on after with normal white blood cell, although LOH is still detected constitutional DNA (fibroblasts and/or nails). 11q23 LOH was in her hematopoietic cells. explored on tumor cells DNA paired with germline DNA by PCR The third patient (P12) is a young boy who presented with a analysis of microsatellite markers covering the 11q23 region and/ severe hemophagocytic syndrome secondary to an Epstein–Barr or genome-wide single-nucleotide polymorphism (SNP)-array virus infection. Whole-exome sequencing of this patient and his analysis (GeneChip Human SNP-Array 6.0, Affimetrix, Santa Clara, two parents revealed the presence of a de novo c.1141T4G CA, USA).5 In vitro growth of myeloid progenitors from bone (p.C381G) CBL mutation, which was confirmed by Sanger marrow was found positive in all CBL mutated patients who were sequencing. Here again, the CBL mutation was heterozygous in tested (Table 1).10 fibroblasts, with LOH in hematopoietic cells. Notably, alike the A CBL mutation was identified in 9 out of 102 (9%) patients with other patients, LOH of CBL persisted on several years although JMML fulfilling consensus criteria.7 Although the overall M/F sex peripheral blood counts returned to normal. ratio of our cohort was 1.94, showing a large predominance These data show that, unlike what was previously thought, CBL of males in accordance with previous reports,8 it was inverted mutation is not invariably associated with LOH in JMML. Notably, to 0.5 in patients with CBL-mutated JMML (Table 1). In six out of two patients with heterozygous mutations in JMML had mutations nine patients, the mutation was homozygous in JMML cells. All six of the same splice acceptor site in intron 7 (Table 1). The only patients had a germline CBL mutation. SNP array analysis heterozygous CBL mutation reported so far in a JMML patient was confirmed the presence of an 11q23 LOH encompassing CBL,in a deletion.6 This is consistent with previous observations in other line with the classical model described for CBL-driven JMML. myeloid malignancies showing that 80% of the deletion mutations However, in the three remaining patients, the JMML clone that arise from CBL splicing mutations are heterozygous versus harbored a heterozygous CBL mutation. The absence of 11q23 less than 20% of the missense mutations.1 This finding raises an LOH was confirmed in these patients by examination of issue of whether the proteins containing deletions are more alike data obtained by SNP array hybridization and/or microsatellites to induce transformation than those with point mutations, analyses, which is more sensitive. Of course, the presence of a possibly via a dominant negative effect. Another surprising minor subclone with LOH, which is not fully expanded at the finding is that, in contrast with homozygous mutations, time of JMML diagnosis but subsequently selected for during heterozygous CBL mutations were somatically acquired and tumor evolution, is still possible. However, repeated analysis 4 associated with the presence of another RAS-activating lesion in months after diagnosis did not evidence the emergence of such a two patients, suggesting that the CBL mutation can also represent clone in patient 6 (Figure 1c). Interestingly, two out of three a secondary event in JMML. Noteworthy, these two patients patients had a mutation affecting exon 7 splice (Figure 1; Table 1). underwent a more aggressive course than other CBL-related JMML One of these patients had a ‘CBL syndrome’ but in the two others, of our cohort. the CBL mutation was somatically acquired and selected for in On the other hand, LOH of mutated CBL has been evidenced in JMML cells. Noteworthy, another somatic mutation activating RAS some of our patients with ‘CBL syndrome’ and non-tumoral pathway was present in the JMML cells of these two patients (that hematological phenotypes. Clonal hematopoiesis observed in is, PTPN11 and LOH encompassing NF1), suggesting that CBL these patients reflects an overgrowth of the hematopoietic clone mutation may represent a secondary event in these cases. In one harboring LOH of the mutated CBL, in line with experimental data patient, this could be confirmed by subsequent analysis of a in mice showing that mutant Cbl proteins confer growth second tumor showing wild-type CBL but the presence of the NF1 advantage when the normal Cbl is lost.11 More unexpected is LOH. Of note, the presence of the NF1 loss in rhabdomyosarcoma the observation that long-term persistence of these mutated cells of this patient suggests that NF1 loss occurred very early in clones can be hematologically and clinically silent. Indeed, LOH development and might be considered as a mosaic. was found in one patient with virtually no hematological Intriguingly, LOH was also evidenced in hematopoietic cells of abnormalities and persisted in other patients several years after three additional patients with ‘CBL syndrome’ and a borderline regression of hematological alterations. Interestingly, long-term hematological phenotype that did not meet consensus criteria for persistence of LOH has been also reported in some patients with JMML, that is, isolated splenomegaly, Sweet syndrome with multi- bona fide JMML who continue to show homozygous CBL organ invasion by granulocytes and hemophagocytic syndrome mutations in their peripheral blood despite having improved (Table 1; Figure 1). blood counts.4 Altogether, these observations suggest that CBL The first patient (P10) was referred initially for persistent mutation is not always sufficient to drive and/or support splenomegaly and xanthogranuloma. At the age of 14 years, she leukemogenesis even in the absence of the normal CBL allele developed a cerebral hemorrhage with a moyamoya arteriopathy and despite persistence of a clonal hematopoiesis over years. This on magnetic resonance imaging. A heterozygous c.1258C4G may be due to a lower oncogenic potential of some CBL (p.R420G) mutation was found in germline DNA. LOH with mutations. However, mutations found in patients 10 and 11 homozygous CBL mutation was identified in blood cells, which have been reported in adult myeloid malignancies.12,13 was unexpected considering she had no peripheral blood Alternatively, a specific background and/or additional oncogenic anomalies but a mild monocytosis (1.3 Â 109/l). Retrospective lesions may be needed in addition to CBL mutation to drive study showed that LOH was already present at the age of 6 years. leukemogenesis. Interestingly, this is reminiscent of what has In the second patient (P11), the hematopoietic disorder been reported for some patients with acquired RAS mutations and consisted in a dramatic neutrophil proliferation. This young girl JMML with spontaneous remisson or non-malignant had unexplained growth delay and dysmorphic syndrome since lymphoproliferative disease.14

Leukemia (2013) 2376 – 2424 & 2013 Macmillan Publishers Limited Letters to the Editor 2407 In conclusion, LOH of the mutated CBL allele can be absent in 8Department of Pediatric Hematology, Assistance Publique-Hoˆpitaux children with bona fide JMML and CBL mutations. Conversely, de Paris (AP-HP) Trousseau Hospital, Paris, France; although the occurrence of a mild transient myeloproliferation in 9Department of Pediatric Hematology, La Timone Hospital, Marseille, the neonatal period cannot be absolutely ruled out in our patients, France and our data show that homozygous CBL mutations can be associated 10Department of Pediatric Hematology, with non-malignant clonal hematological phenotypes different Assistance Publique-Hoˆpitaux de Paris (AP-HP) Robert Debre´ Hospital, from JMML. Hence, LOH of the mutated CBL allele cannot be Paris, France considered as a signature of JMML. Besides raising some questions E-mail: [email protected] about CBL-driven leukemogenesis, this is an important issue for patients’ management. Our observations also highlight the variety of immuno-hemato- logical phenotypes induced by CBL mutations. Besides JMML, a REFERENCES heterogenous spectrum of atypical immuno-hematological presen- 1 Kales S, Ryan PE, Nau MM, Lipkowitz S. Cbl and human myeloid neoplasms: the tations can be observed in patients with ‘CBL syndrome’, ranging Cbl comes of age. Cancer Res 2010; 70: 4789–4794. from very mild hematological symptoms to more aggressive 2 Martinelli S, ADe Luca, Stellacci E, Rossi C, Checquolo S, Lepri F et al. Heterozygous presentations. The importance of Cbl in hematopoiesis has been germline mutations in the CBL tumor-suppressor gene cause a Noonan demonstrated in knockout mice that show hyper responsiveness to syndrome-like phenotype. Am J Hum Genet 2010; 87: 250–257. ´ hematopoietic growth factors, expansion of the progenitor and 3Perez B, Mechinaud F, Galambrun C, Romdhane NBen, Isidor B, Philip N et al. 11 Germline mutations of the CBL gene define a new genetic syndrome with stem cell pool, and mild myeloproliferative features. The incidental predisposition to juvenile myelomonocytic leukaemia. J Med Genet 2010; 47: finding of CBL mutations with LOH in disorders involving monocyte/ 686–691. macrophages, neutrophils and/or the immune system underlines 4 Niemeyer CM, Kang MW, Shin DH, Furlan I, Erlacher M, Bunin NJ et al. Germline the multilineage impact of CBL mutations on hematopoiesis. CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia. Nat Genet 2010; 42: 794–800. 5Pe´rez B, Kosmider O, Cassinat B, Renneville A, Lachenaud J, Kaltenbach S et al. CONFLICT OF INTEREST Genetic typing of CBL, ASXL1, RUNX1, TET2 and JAK2 in juvenile myelomonocytic The authors declare no conflict of interest. leukaemia reveals a genetic profile distinct from chronic myelomonocytic leukaemia. Br J Haematol 2010; 151: 460–468. 6 Muramatsu H, Makishima H, Jankowska AM, Cazzolli H, O’Keefe C, Yoshida N et al. ACKNOWLEDGEMENTS Mutations of an E3 c-Cbl but not TET2 mutations are pathogenic in juvenile myelomonocytic leukemia. Blood 2010; 115: 1969–1975. SNP arrays were performed on the Plateforme de Ge´nomique du GHU Nord and 7 Chan RJ, Cooper T, Kratz CP, Weiss B, Loh ML. Juvenile myelomonocytic leukemia: analyzed with the help of Steven Gazal. We thank Dr Franck Bourdeault (Institut Curie, a report from the 2nd International JMML Symposium. Leuk Res 2009; 33: Paris, France) for analysis of the rhabdomyosarcoma sample. 355–362. 8 Loh ML. Recent advances in the pathogenesis and treatment of juvenile M Strullu1,2, A Caye1,2, B Cassinat2,3, O Fenneteau4, myelomonocytic leukaemia. Br J Haematol 2011; 152: 677–687. F Touzot5, T Blauwblomme6, R Rodriguez7, S Latour7, A Petit8, 9 Loh ML, Sakai DS, Flotho C, Kang M, Fliegauf M, Archambeault S et al. Mutations in V Barlogis9, C Galambrun9, T Leblanc10, A Baruchel10, CBL occur frequently in juvenile myelomonocytic leukemia. Blood 2009; 114: C Chomienne2,3 and H Cave´ 1,2 1859–1863. 1Department of Genetics, Assistance Publique-Hoˆpitaux 10 Cambier N, Menot ML, Schlageter MH, Balitrand N, Leblanc T, Bordigoni P et al. All trans retinoic acid abrogates spontaneous monocytic growth in juvenile de Paris (AP-HP) Robert Debre´ Hospital, Paris, France; 2 chronic myelomonocytic leukaemia. Hematol J 2001; 2: 97–102. INSERM UMR_S940, Institut Universitaire d’He´matologie (IUH), 11 Sanada M, Suzuki T, Shih LY, Otsu M, Kato M, Yamazaki S et al. Gain-of-function Universite´ Paris-Diderot Sorbonne-Paris-Cite´, Paris, France; of mutated C-CBL tumour suppressor in myeloid neoplasms. Nature 2009; 460: 3 Cellular Biology Unit, Assistance Publique-Hoˆpitaux de Paris (AP-HP) 904–908. Saint-Louis Hospital, Paris, France; 12 Reindl C, Quentmeier H, Petropoulos K, Greif PA, Benthaus T, Argiropoulos B et al. 4Biological Hematology Laboratory, (AP-HP) Robert Debre´ Hospital, CBL exon 8/9 mutants activate the FLT3 pathway and cluster in core binding Paris, France; factor/11q deletion /myelodysplastic syndrome subtypes. 5Department of Biotherapy, Assistance Publique-Hoˆpitaux de Paris Clin Cancer Res Off J Am Assoc Cancer Res 2009; 15: 2238–2247. (AP-HP) Necker Hospital, Paris, France; 13 Kohlmann A, Klein HU, Weissmann S, Bresolin S, Chaplin T, Cuppens H et al. The 6 Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep Department of Pediatric Neurosurgery, Assistance Publique- sequencing investigation of TET2, CBL and KRAS mutations by an international Hoˆpitaux de Paris (AP-HP) Necker Hospital, consortium involving 10 laboratories. Leukemia 2011; 25: 1840–1848. Paris, France; 14 Takagi M, Piao J, Lin L, Kawaguchi H, Imai C, Ogawa A et al. Autoimmunity and 7 INSERM U768, Necker Hospital, University Paris-Descartes persistent RAS-mutated clones long after the spontaneous regression of JMML. Sorbonne-Paris-Cite´, Institut Imagine, Paris, France; Leukemia 2013; 27: 1926–1928. Granulocyte colony-stimulating factor T595I (T618I) mutation confers independence and enhanced signaling

Leukemia (2013) 27, 2407–2410; doi:10.1038/leu.2013.164 receptor (GCSFR, gene name CSF3R) occur in patients with severe congenital neutropenia (SCN), these and other missense muta- tions have rarely been reported in other disorders. Two recent reports have identified CSF3R T595I as a recurrent mutation Although acquired nonsense mutations occurring in the in patients with chronic neutrophilic leukemia (CNL) and intracellular domain of the granulocyte colony-stimulating factor atypical (BCR-ABL1-negative) chronic myeloid leukemia (aCML).

Accepted article preview online 6 June 2013; advance online publication, 25 June 2013

& 2013 Macmillan Publishers Limited Leukemia (2013) 2376 – 2424