Christine Zoi

Recent evidence has demonstrated that acquired uniparental disomy (aUPD) is a novel mechanism by which pathogenetic mutations in cancer may be reduced to homozygosity. To help identify novel mutations in myeloproliferative neoplasms (MPNs), we performed a genome-wide single nucleotide polymorphism (SNP) screen to identify aUPD in 58 patients with atypical chronic myeloid leukemia (aCML; n = 30), JAK2 mutation-negative myelofibrosis (MF; n = 18), or JAK2 mutation-negative polycythemia vera (PV; n = 10). Stretches of homozygous, copy neutral SNP calls greater than 20Mb were seen in 10 (33%) aCML and 1 (6%) MF, but were absent in PV. In total, 7 different chromosomes were involved with 7q and 11q each affected in 10% of aCML cases. CBL mutations were identified in all 3 cases with 11q aUPD and analysis of 574 additional MPNs revealed a total of 27 CBL variants in 26 patients with aCML, myelofibrosis or chronic myelomonocytic leukemia. Most variants were missense substitutions in the RING or linker domains that abrogated CBL ubiquitin ligase activity and conferred a proliferative advantage to 32D cells overexpressing FLT3. We conclude that acquired, transforming CBL mutations are a novel and widespread pathogenetic abnormality in morphologically related, clinically aggressive MPNs.

Abstract Recent evidence has demonstrated that acquired uniparental disomy (aUPD) is a novel mechanism by which pathogenetic mutations in cancer may be reduced to homozygosity. As a route towards identifying novel mutations in myeloproliferative neoplasms (MPN), we performed a genome wide single nucleotide polymorphism (SNP) screen to identify aUPD in 58 patients with atypical chronic myeloid leukemia (aCML; n=30), JAK2 mutation negative myelofibrosis (MF; n=18) or JAK2 mutation negative polycythaemia vera (PV; n=10). Stretches of homozygous, copy neutral SNP calls >20Mb were seen in 10 (33%) aCML, 1 (6%) MF but absent in PV. In total seven different chromosomes were involved with 7q and 11q each affected in 3 (10%) of aCML cases and 1p, 13, 17q, 20q, 21q involved in single individuals. The 1p and 13 abnormalities were associated with homozygous mutations in MPL and FLT3, respectively. To characterize the two recurrent regions at 7q and 11q, we focused on genes encoding intracellular signal transduction components because of the known association between MPNs and deregulated tyrosine kinase signaling. No mutations were detected in MET, EPHA1, EPHB6 or BRAF but homozygous CBL missense mutations were found in all three cases with 11q aUPD. To determine the prevalence of CBL mutations, we sequenced exons 8 and 9 in an additional 574 MPN cases. A total of 27 sequence variants were identified in 26 patients of whom 3 had MF, 10 had CMML, 12 had aCML/MPD-U and one had HES/CEL. Microsatellite analysis across 11q indicated significant tracts of 11q copy neutral homozygosity in 11/26 CBL mutated cases. In two cases, CBL mutations were acquired as secondary events during progression of a pre-existing MPN. Patients with CBL mutations had a shorter overall survival and progression-free survival compared to mutation negative cases (OS: 33 months vs 39 months; PFS: 22 months vs 32 months) but the differences were not significant. Similarly there was no difference in gender, age, white cell count or percentage of eosinophils between mutation positive and mutation negative cases. CBL plays both positive and negative roles in tyrosine kinase signal transduction by acting as an adaptor and also a ubiquitin ligase. Of the 27 variants, 21 (78%) were missense substitutions (15 different mutations) in the region encoding the RING or linker domains, 5 (19%) were candidate splicing abnormalities involving exon 8 and one (3%) produced a stop codon. Functional analysis of selected mutations demonstrated that they abrogated CBL ubiquitin ligase activity and were transforming as assessed by the ability to confer a proliferative advantage in both liquid and semi-solid cultures of Ba/F3-FLT3 cells. We conclude that acquired, inactivating CBL mutations are a novel and widespread pathogenetic abnormality in morphologically-related, aggressive MPNs.

Hypereosinophilic syndrome (HES) is a heterogeneous group of disorders characterized by unexplained persistent primary eosinophilia causing end-organ damage. We conducted a prospective cohort study of patients fulfilling the diagnostic criteria for HES. Of 20 patients considered eligible for the study, 2 were found to have clonal myeloid disorders, limiting the diagnosis of "true" HES to 18 patients. No patient carried the FIP1L1-PDGFRA fusion gene or other imatinib-responsive translocations. A clonal interleukin-5-producing T-cell population was not detected in any patient. Common manifestations at presentation were pulmonary, cutaneous, and neurologic involvement; serositis; and gastrointestinal involvement. Only 3 patients developed cardiac involvement. Fifteen of the HES patients were administered first-line combined treatment with steroids and hydroxyurea. Nine patients achieved complete response, while 6 attained only partial response. Imatinib was administered to 3HES patients who had been pretreated with steroids, resulting in complete hematologic and clinical response in 2 patients and no response at all in 1. Further treatment of the latter patient with steroids and hydroxyurea also proved ineffective. We conclude that the therapeutic approach should be individualized according to molecular findings. We consider the coadministration of corticosteroids and hydroxyurea to be an effective combination for the treatment of FIP1L1-PDGFRA-negative HES. Abbreviations: ABL = Abelson murine leukemia viral oncogene homolog, ANCA = anti-neutrophil cytoplasmic antibodies, ARMS = amplification refractory mutation system, CML = chronic myeloid leukemia, CT = computerized tomography, ELISA = enzyme-linked immunosorbent assay, HES = hypereosinophilic syndrome, IL-5 = interleukin-5, MPD = myeloproliferative disorder, PCR = polymerase chain reaction, PDGFRA = platelet-derived growth factor alpha, RT-PCR = reverse transcription-PCR, SM = systemic mastocytosis, TCR = T-cell receptor, WBC = white blood cells.

Following the identification in 2005 of the recurrent V617F mutation in exon 14 of JAK2 in Myeloproliferative Disorder (MPD) patients, many studies have confirmed the frequency of this mutation in distinct MPD subclasses. The presence of the JAK2 V617F mutation has proved even more useful to distinguish, in patients with true polycythaemia, those with Polycythaemia Vera (PV, 90%JAK2 V617F positive) from those with Idiopathic Erythrocytosis (IE, V617F-negative) (Percy et al, 2006; unpublished observations). The JAK2 V617F mutation is also present in about half of the patients with splanchnic vein thrombosis (Patel et al, 2006), suggesting that occurrence of thrombosis in these patients is caused by an undiagnosed MPD. However, there still exists patients in whom the diagnosis of MPD is highly suspected but the absence of the JAK2 V617F mutation does not allow correct classification. Novel recurrent mutations clustered in a highly conserved region in exon 12 of JAK2 have been described in patients with erythrocytosis (Scott et al, 2007). We took advantage of our cohort of JAK2 V617F-negative patients and analyzed three distinct groups: two groups of patients with increased red cell mass, either with (PV) or without (IE) sufficient criteria for a diagnosis of PV, and one group of patients with splanchnic vein thrombosis. All patients had been previously found negative for the JAK2 V617F mutation using allele-specific real-time polymerase chain reaction (AS-PCR) with a sensitivity of 1–2% as previously described (Kiladjian et al, 2006). Identification of JAK2 exon 12 mutations was performed on DNA extracted from peripheral blood using the AS-PCR assays previously described (Scott et al, 2007). Serum erythropoietin (Epo) levels were measured using a radioimmunoassay (DiaSorin, France) (Schlageter et al, 1990) and Endogenous Erythroid Colonies (EECs) analysed on semi solid media (StemCell Technologies, Vancouver, BC, Canada). Among 47 patients with an increased red cell mass (>25% excess), 26 fulfilled the Polycythaemia Vera Study Group (PVSG) or World Health Organization (WHO) criteria for PV at diagnosis, while 21 were classified as having IE on the basis of a raised red cell mass, with no identifiable cause of secondary erythrocytosis and absence of PV according to PVSG or WHO criteria. Eight cases were found to carry an exon 12 mutation; all of them fulfilled the WHO or PVSG criteria for PV. As shown in Table I, most of these patients were characterized by a young age at diagnosis, a trend that was also observed in the first report of exon 12 mutations (Scott et al, 2007). One patient (Patient 2 in Table I), diagnosed in childhood, was the youngest patient reported to date with an exon 12 mutation at diagnosis. All mutation-positive patients tested (7/7) had a low serum Epo level, a feature frequently reported in this group of patients (5/8 (Scott et al, 2007), 2/2 (Butcher et al, 2007), 5/5 (Pardanani et al, 2007) and 16/17 (Pietra et al, 2007) respectively). Similarly, 4/4 patients tested had EECs, a constant feature of patients with exon 12 mutations (Butcher et al, 2007; Percy et al, 2007; Scott et al, 2007). Interestingly, in previous reports, exon 12 JAK2 mutated patients had either normal levels of other blood cells (Pardanani et al, 2007; Pietra et al, 2007) or occasionally increased levels (Butcher et al, 2007; Scott et al, 2007). In our study, increased white blood cell or platelet counts were noted in four out of the eight cases (Table I). The presence of exon 12 mutations is therefore not restricted to patients with an isolated increase in red blood cells, and an increase in other additional blood cell lineages may be observed. In 7/8 exon 12 mutated cases, the N542-E543del allele was the mutation detected (Table I). This was also the most frequent mutant reported in the other published series: 4/10 (Scott et al, 2007), 2/5 (Pardanani et al, 2007) and 10/17 (Pietra et al, 2007) patients. Therefore, 57% (23 of 40 cases, including our present data) of patients with exon 12 mutations have been reported to carry the N542-E543del mutant. None of the 21 JAK2 V617F-negative patients with increased red cell mass that were diagnosed with IE (unpublished observations) carried a mutation (see Table II for clinical and biological characteristics). In two recent reports, 2/44 and 8/58 patients classified as IE were found to carry exon 12 mutations, six out of the 10 positive patients having the N542-E543del mutant (Percy et al, 2007; Williams et al, 2007). Finally, we analysed DNA from 23 patients with splanchnic venous thrombosis, eight with Budd-Chiari syndrome and 15 with portal venous thrombosis. No exon 12 mutations were detected in these patients (sex: eight females and 14 males; median age: 53 years, range 33–70; median hematocrit: 40% range 27–50). Thus, JAK2 exon 12 mutations were detected in 31% of our JAK2 V617F negative patients with increased red cell mass and PV criteria, with a preponderance of the N542-E543del mutant. This quite low percentage suggests that other genetic alterations may cause PV, but could also be explained by the recent description of new JAK2 exon 12 mutants (Butcher et al, 2007; Pietra et al, 2007) that could be overlooked by the AS-PCR assays we used. One important concern for a diagnostic laboratory will be the choice of a method for detecting these mutations, as the complexity of the several mutations reported does not allow the development of simple high throughput assays as used for the JAK2 V617F mutant. In addition, using a sequencing approach, Butcher et al (2007) detected JAK2 exon 12 mutations only on isolated endogenous erythroid burst-forming units, suggesting that the mutated allele burden could be too small to allow the identification of these mutations by the relatively insensitive sequencing method (sensitivity, approximately 20%), as frequently observed for the JAK2 V617F mutation. Furthermore, in our study, AS-PCR identified two mutated patients that had previously been negative using a sequencing approach (Patients 1 and 3). In conclusion, our data and recently published literature show that exon 12 mutations are very useful markers to identify MPD patients. Their frequency, unlike the JAK2 V617F mutation, is low, suggesting that the search for JAK2 exon 12 mutations should be restricted to patients who fulfil PV diagnostic criteria, have low serum Epo levels and EECs and do not carry the JAK2 V617F mutation. We are very grateful to Marie-Laurence Menot and Nadine Bonnin for technical assistance and sample management and Rose Ann Padua for correcting the manuscript.