ALBERT

Beschreibung: Abstract 2173 Poster Board II-150 Loss of heterozygosity (LOH) due to acquired uniparental disomy (UPD) is a commonly observed chromosomal lesion in myeloproliferative neoplasms (MPN) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) including chronic myelomonocytic leukemia (CMML). Most recurrent areas of LOH point towards genes harboring mutations. For example, UPD11q23.3 and UPD4q24 were found to be associated with c-Cbl and TET2 mutations, respectively. Cbl family mutations (c-Cbl and Cbl-b) have been associated with atypical MDS/MPN including CMML and juvenile myelomonocytic leukemia (JMML) as well as more advanced forms of MDS and secondary AML (sAML). Ring finger mutants of Cbl abrogate ubiquitination and thereby tumor suppressor function related to inactivation of phosphorylated receptor tyrosine kinases, Src and other phosphoproteins. TET2 mutations are present in a similar disease spectrum. The TET family of proteins is involved in conversion of methylcytosine to methylhydroxycytosine which cannot be recognized by DNMT1. Thereby, the proteins seem to counteract maintenance hypermethylation. In our screen of MDS/MPN, we found c-Cbl and Cbl-b ring finger mutations in 5/58 (9%) of CMML and AML derived from CMML, 2/39 (5%) MDS/MPNu, 4/21 (19%) JMML and 14/148 (9%) RAEB/sAML. In the same cohort, TET2 mutations were present in 37% and 14% of patients with MDS/MPN and MDS, respectively. Of note we did not find any TET2 mutations in JMML. We and others have also noted that TET2 and c-Cbl mutations were also detected in atypical chronic myeloid leukemia. While translocations resulting in BCR/ABL fusion characterize CML, we stipulated that in analogy to other chronic myeloproliferative diseases, TET2 and c-Cbl mutations may be also present in CML and contribute to phenotypic heterogeneity within BCR/ABL associated disorders. In particular, progression of CML to accelerated phase (AP) or blast crisis (BC) could be associated with acquisition of additional lesions. When 22 patients with CML chronic phase (CP) were screened, no TET2 and c-Cbl mutations were found. However, we identified 1 c-Cbl, 2 Cbl-b (6%) and 6 TET2 (12%) mutations in 51 patients with CML-AP (N=18) and CML-BC (N=33) with myeloid and lymphoid/mix 24 and 9 phenotype, respectively. These mutations were mutually exclusive. We also noted that TET2 mutations were present in 1/9 CML in BC with lymphoid phenotype. We subsequently screened Ph+ ALL cases (N=9) and found a TET2 mutation in 1 case but no Cbl family mutations. In contrast when 9 Ph- ALL cases were screened as controls, neither TET2 or Cbl mutations were found. SNP-A analysis revealed 2 cases of LOH involving chromosome 4 (UPD4q24 and del4) in a patient with lymphoid blast crisis and Ph+ ALL, respectively. However, UPD was not found in Cbl family gene regions (11q23.3 or 3q13.11). A homozygous deletion of Cbl-b region was seen in a CP patient. Cbl family mutations were associated with a more complex karyotype than TET2 mutations (67% vs. 17% cases with abnormal phenotype). Patients with Cbl family mutations were resistant to imatinib which was effective in only 2 out of 6 patients with TET2mutations. Dasatinib was effective in 2 patients with TET2 mutation. Median over all survival of progressed CML was 47, 49 and 48 months in patients with Cbl, TET2 or no mutations, respectively. In conclusion, our results indicate that Cbl family mutations can occur as secondary lesions in myeloid type aggressive CML (AP and myeloid BC), but not in lymphoid types. TET2 mutations were identified in both lymphoid BC and Ph1+ALL, as well as myeloid BC and AP. In contrast to CMML or JMML in which a vast majority of mutations are homozygous, all Cbl family mutations were heterozygous (no LOH). Similarly, all but two TET2 mutations were heterozygous (1 hemizygous in del4 and 1 homozygous case in UPD4q), suggesting that additional cooperating lesions affecting corresponding pathways may be present. These mutations likely represent secondary lesions which contribute to more either progression (CML) or more aggressive features (Ph+ ALL) and characterize disease refractory to therapy with imatinib. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 3396 Chronic myeloid leukemia (CML) is characterized by the BCR/ABL fusion gene. However, secondary molecular events leading to accelerated (AP) or blast phase (BP) have not been sufficiently clarified. We hypothesized that, in analogy to other MDS/MPN or MPN, TET2, ASXL1, CBL and IDH family mutations may also occur in CML and contribute as secondary events leading to progression to AP or BP. Similarly, higher resolution of cytogenetic testing by single nucleotide polymorphism array (SNP-A)-based karyotyping may reveal additional chromosomal abnormalities associated with stepwise progression. This study is focused on the combined analysis of chromosomal lesions and mutations associated with AP, BP and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (ALL) and the association of these defects with clinical features. We screened TET2, ASXL1, CBL and IDH for mutations in AP (N=14) and BP (N=26) and Ph+ ALL (N=9). Chronic phase (CP) (N=14) and Ph negative ALL (N=9) served as controls. We identified 3 CBL family (9%), 7 TET2 (21%), 2 ASXL1 (6%) and 2 IDH family (6%) mutations in patients with AP and myeloid BP. Subsequently, we also detected a TET2 mutation in a case of Ph+ ALL. None of these mutations were found in patients with CP or Ph negative ALL. We also performed SNP-A-based karyotyping and only included lesions which did not overlap with copy number variations (CNVs) or germ line regions of homozygosity present in any of the controls. 23 gains, 21 losses and 4 regions of somatic UPD lesions were identified. By SNP-A, additional copy number abnormalities, including microdeletions were found in 67% and 50% of patients with AP and BP, respectively. Recurrent lesions were detected on chromosome 1, 8, 9, 17 and 22. Microdeletions on chromosome 17 and 21 involved tumor associated genes NF1 and RUNX1. Deletions flanking the ABL1 and BCR genes were observed in 3 cases with der(22)t(9;22) or der(9)t(9;22) by metaphase cytogenetics. Gains including 1q25.3q41, chromosome 8 and 17q24.3 were found in 3 cases. Regions of UPD included UPD5q, 8q, 11p and 17q but no UPD involving 11q (CBL) and 4q (TET2) regions were found confirming heterozygous nature of the corresponding mutations. Newly detected molecular lesions associated with AP and BP may change the biology and thereby clinical features of affected cases. Overall survival of patients with mutations did not differ from those without mutations. Of note is that BCR/ABL1 kinase domain mutations were detected in 9/10 patients with imatinib resistance. In these 9 cases, 3 TET2 and 2 CBLB mutations were detected (but no mutations in the other genes). In an imatinib-resistant patient without BCR/ABL1 kinase domain mutation, CBL mutation was present. In the patients with TET2 mutations, additional chromosomal lesions were found by SNP-A, significantly more frequently when compared with WT cases (P=0.017). Of the 9 TET2 variants in 8 cases, 7 (78%) were missense substitutions, 1 (11%) was frame shift and 1 (11%) produced a stop codon and were located within the N-terminus as well as in a conserved DSBH 2OG-Fe(II)-dependent dioxygenase domain. The presence of nonsense and frameshift mutations suggests that mutated lesions result in inactivation, consistent with putative tumor suppressor functions, while heterozygous mutations indicate that the wild type allele is not completely protective. Since no TET2 mutations were identified in chronic phase CML, these mutations might represent an additional pathogenic event and contribute to progression. In 3 cases we observed a combination of 2 mutations. Coincidence of CBLB and TET2 mutations in 2 cases suggests that these might cooperate in the evolution of advanced phase of CML. We also found a combination of IDH1 and ASXL1 mutations in a patient with BP, suggesting that both mutations contribute to clonal advantage. In conclusion, while CBL family, ASXL1 and IDH family mutations as well a additional unbalanced chromosomal abnormalities not seen by metaphase cytogenetics can occur in myeloid type advanced phase CML, TET2 mutations were identified in Ph+ ALL, as well as myeloid BP and AP. These mutations likely represent secondary lesions which contribute to either disease progression or more aggressive features and commonly occur in association with imatinib-resistant BCR/ABL mutations. Disclosures: No relevant conflicts of interest to declare.