CBL Mutations Are Correlated with CMML, Frequently Associated with RUNX1 but Mutually Exclusive of JAK2V617F Mutations.

Abstract 962

Mutations in exons 8 and 9 of the CBL gene containing the RING and linker domains were recently described in 10 % of CMML , 8% of atypical CML and single cases of PMF or HES (Grand et al., Blood 2009). However, the number of cases is still limited and further biological characterization scarce. We therefore performed CBL mutation analysis in a large cohort of well characterized myeloproliferative neoplasms (MPN). This cohort was selected according to the availability of cytomorphology, cytogenetics and further molecular genetic characterization. In total, 418 MPN patients were analysed. The cohort was composed of 182 males and 236 females with a median age of 68.3 years (range 16.3-93.3). Diagnoses according to cytomorphology were as follows: ET (n=88), PMF (n=33), PV (n=59), HES (n=14), CMML-1 (n=73), CMML-2 (n=26), MDS/MPN overlap (n=8), RARS-T (n=18), MPN unclassifiable (MPNu) (n=99). Cytogenetics was available in 382/418 cases (91.4%) and revealed a normal karyotype in 312 cases (81.7%) whereas 70 (18.3%) had chromosomal aberrations. A BCR-ABL rearrangement was excluded in all cases. Further molecular characterization revealed a JAK2V617F-mutation in 120/391 (30.7%), diverse JAK2exon12 mutations in 7/37 (18.9%) (all these cases were JAK2V617F unmutated PV), a MPLW515 mutation in 12/160 (7.5%), and TET2 mutations in 26/110 (23.6%) cases. Within the total cohort, CBL mutations were detected in 26/418 cases (6.2%). As recently described, all mutations were missense mutations and were clustered in a region spanning 50 amino acids around the RING and linker domains. Two of 28 mutations were detected in two cases each (C419Y and A420G), all others were single and as follows (L380P, L381G, L381P, I383M, C384T, I384T, D388G, D390V, C396Y, G397V, H398A, C404Y, W408C, C416R, G415S, C416S, C416Y, P417H, R420L, R421L, I423N, I429F, I429N, V430M) (according to ENST00000264033). In 12 /26 cases the detection of the CBL wildtype (wt) only indicated allelic loss, and two patients had two different mutations. The mutations were most frequently found in CMML-1 (14/73; 19.2%), CMML-2 (2/26; 7.9%), PMF (4/32; 12.5%) and MPN unclassifiable (7/99; 7.1%). In contrast, CBL mutations were not detected in PV, ET, HES, RARS-T and MDS/MPN overlap. The presence of CBL mutations was not correlated to age, gender, chromosomal aberrations or WBC in comparison to CBL unmutated cases within the respective subcohorts. In addition, all 26 CBL mutated cases were analysed for JAK2V617F, JAK2exon12, MPLW515, FLT3-ITD, MLL-PTD, NPM1, NRAS and RUNX1 mutations. TET2 sequence analysis was available in two cases. The CBL mutations were exclusive of all these mutations with the exception of RUNX1 and TET2. Four of 16 CMML cases (25%) with CBL mutations had a RUNX1 mutation and one MPNu case revealed a TET2 mutation. In CMML-1 11/14 (78.6%) cases showed loss of the wt allele or two different mutations and thus lack a functionally intact CBL allele. In contrast, nearly all CBL mutations in MPNu and all PMF had a low allelic burden. From four of the CBL mutated cases also a sample at an earlier time point (3 to 14 months) was available. In three cases the mutation was already present two to three months before when MPN was suspected but not diagnosed. One case clearly gained a D388G within 14 months between first appearance of leukocytosis and diagnosis of MPN which indicates that CBL mutation is a later event in this case. In conclusion, these data show that CBL mutations i) are highly correlated with CMML, ii) in CMML are associated with high allelic burden or two CBL mutations, iii) in CMML are frequently associated with RUNX1 mutations, iv) are rarely found in PMF and MPNu and are rare or absent in all other MPN, v) seem to be mutually exclusive of other mutations typical for MPN such as JAK2 and MPLW515 mutations. Thus patients with suspected MPN who lack other MPN typical mutations should be routinely screened for CBL mutations.