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NOTCH1 and SF3B1 mutations have been previously reported to have prognostic significance in chronic lymphocytic leukemia but to date they have not been validated in a prospective, controlled clinical trial. We have assessed the impact of these mutations in a cohort of 494 patients treated within the randomized phase 3 United Kingdom Leukaemia Research Fund Chronic Lymphocytic Leukemia 4 (UK LRF CCL4) trial that compared chlorambucil and fludarabine with and without cyclophosphamide in previously untreated patients. We investigated the relationship of mutations in NOTCH1 (exon 34) and SF3B1 (exon 14-16) to treatment response, survival and a panel of established biologic variables. NOTCH1 and SF3B1 mutations were found in 10% and17% of patients, respectively. NOTCH1 mutations correlated with unmutated IGHV genes, trisomy 12, high CD38/ ZAP-70 expression and were associated with reduced overall (median 54.8 vs 74.6 months, P = .02) and progression-free (median 22.0 vs 26.4 months, P = .02) survival. SF3B1 mutations were significantly associated with high CD38 expression and with shorter overall survival (median 54.3 vs 79.0 months, P < .001). Furthermore, multivariate analysis, including baseline clinical variables, treatment, and adverse prognostic factors demonstrated that although TP53 alterations remained the most informative marker of dismal survival in this cohort, NOTCH1 (HR 1.58, P = .03) and SF3B1 (HR 1.52, P = .01) mutations have added independent prognostic value.

The polycomb repressive complex 2 (PRC2) is a highly conserved histone H3 lysine 27 methyltransferase that regulates the expression of developmental genes. Inactivating mutations of the catalytic component of PRC2, EZH2, are seen in myeloid disorders. We reasoned that the other 2 core PRC2 components, SUZ12 and EED, may also be mutational targets in these diseases, as well as associated factors such as JARID2. SUZ12 mutations were identified in 1 of 2 patients with myelodysplastic syndrome/myeloproliferative neoplasms with 17q acquired uniparental disomy and in 2 of 2 myelofibrosis cases with focal 17q11 deletions. All 3 were missense mutations affecting the highly conserved VEFS domain. Analysis of a further 146 myelodysplastic syndrome/myeloproliferative neoplasm patients revealed an additional VEFS domain mutant, yielding a total mutation frequency of 1.4% (2 of 148). We did not find mutations of JARID2 or EED in association with acquired uniparental disomy for chromosome 6p or 11q, respectively; however, screening unselected cases identified missense mutations in EED (1 of 148; 1%) and JARID2 (3 of 148; 2%). All 3 SUZ12 mutations tested and the EED mutation reduced PRC2 histone methyltransferase activity in vitro, demonstrating that PRC2 function may be compromised in myeloid disorders by mutation of distinct genes.

Background: The combination of treosulfan witth fludarabine was successfully introduced into toxicity-reduced conditioning regimens for hematopoietic stem cell transplantation (HCT). However, the risk of post-HCT relapse remains of concern. Here we report for the first time on the results of an individual treatment approach with treosulfan, fludarabin and cytarabine as conditioning for allogeneic HCT in patients with AML, MPN or MDS. Methods: 22 patients were treated with fludarabine 30 mg/m² given on day -6 to day -2, treosulfan 14 g/m² administered on days -4 to day -2 and cytarabin 2g/m² given on days -6 and -5. GvHD-prophylaxis consisted of cyclosporine A and methotrexate or MMF. In addition, antithymocyte globulin was applied in case of an unrelated donor. One patient received bone marrow and the remaining patients received peripheral blood stem cells from matched related donors (9%), matched unrelated donors (73%) or mismatched unrelated donors (18%). All patients were considered to have high risk of relapse because of unfavourable cytogenetic features and/or insufficient or missing response to previous treatment. Three patients (14 %) with CML after blast crisis received the combination because of the reported high relapse rate (46%) after three-day scheduled conditioning with treosulfan [1]. In addition, patients were considered to be ineligible for myeloablative standard conditioning because of multi-morbidity (n = 4; 18% with HCT-CI >2) and/or age >55 years (n = 14; 64%). Results: The median age of patients was 59 (35-68) years. Patients suffered from acute myeloid leukemia (n = 14, 64%), myeloproliferative neoplasia (n = 6, 27%) or myelodysplasic syndrome (n = 2, 9%). The conditioning regime was well tolerated and nearly all patients engrafted and achieved complete donor-type chimerism, except for one who died very early from sepsis. Another patient with underlying myelofibrosis suffered from secondary graft failure on day 100. Two patients developed aGVHD °III/ IV. None of the patients suffered from veno-occlusive disease or severe chronic GVHD. Overall survival and event-free survival at one year reached 60.2% and 59.6%, respectively. Six patients died from infectious disease, two from relapse and one patient from acute GVHD °IV. Conclusion: The combination of cytarabine with the established conditioning of treosulfan and fludarabine is feasible in patients with high risk of relapse and ineligible for myeloablative standard conditioning. Holowiecki J, Giebel S, Wojnar J et al. Treosulfan and fludarabine low-toxicity conditioning for allogeneic 334 haematopoietic stem cell transplantation in chronic myeloid leukaemia. Br J Haematol 335 2008; 142(2): 284-92. Disclosures Hilgendorf: Novartis: Other: Travel support, Research Funding; Medac: Other: Travel support, Research Funding. Frietsch:Deutsche Krebshilfe: Research Funding. Scholl:Abbivie: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Deutsche Krebshilfe: Research Funding; Alexion: Other: Travel support. Hochhaus:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Pfizer: Research Funding. Casper:Medac: Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Research Funding.

Living without treatment has become a realistic target for patients with chronic myeloid leukemia (CML) who achieved durable deep molecular remission under treatment with tyrosine kinase inhibitors (TKI). Recently, we have identified novel BCR-ABL-independent gene mutations in newly diagnosed CML patients that may function as important cofactors in the evolution of CML and persistence of the disease (Schmidt M, Rinke J, et al. Leukemia 2014; 28(12): 2292-9). Here, we sought to investigate the dynamics of such mutations during a 2-year treatment with the second generation TKI nilotinib. To check if BCR-ABL independent mutations are an age associated phenomenon we also screened a cohort of children and adolescents with CML. A total of 51 chronic phase CML patients were analyzed including 31 adults (male, n=23; median age 57 years, range 42 to 73 years) and 20 children/adolescents (male, n= 14; median age 14 years, range 10 months to 27 years). For each adult patient, samples were analyzed at diagnosis, in subsequent complete cytogenetic remission (CCyR) after 3 months and in deep molecular remission (≤ MMR) after 24 months of nilotinib therapy. Children/adolescents samples were investigated at diagnosis only. Targeted deep next-generation sequencing (NGS) was used to analyze a panel of 30 commonly mutated genes in myeloid disorders. Constitutional DNA obtained from buccal swabs at diagnosis was investigated to check whether mutations are of somatic origin. BCR-ABL independent gene mutations were found in 8/31 (26%) adult CML patients at diagnosis affecting the genes ASXL1 (n=3), DNMT3A (n=3), RUNX1 (n=1) and RUNX1 plus TET2 (n=1). No mutation was recognized in corresponding constitutional DNA specimens indicating that all mutations were somatically acquired. Analysis of individual hematopoietic colonies from three adult patients revealed that most mutations were part of the BCR-ABL-positive clone. NGS of subsequent samples obtained after three months of TKI therapy identified two DNMT3A mutations in BCR-ABL-negative cells that were also present in BCR-ABL-positive cells at diagnosis, implying that this mutation preceded the BCR-ABL rearrangement. After 24 months of TKI treatment, NGS revealed these two DNMT3A mutations again as well as a novel low-level EZH2 mutation in a patient with deep molecular remission (MR4.5) that was neither detectable at diagnosis nor after 3 months of treatment indicating the emergence of a BCR-ABL-independent subclone in BCR-ABL-negative cells during long-term nilotinib treatment. Within the children/adolescents cohort BCR-ABL independent gene mutations were identified in 5/20 (25%) subjects all affecting the ASXL1 gene. All mutations were stop codon or frameshift mutations implying loss of ASXL1 gene function. We conclude that BCR-ABL independent gene mutations can be found frequently in CML patients and affect predominately epigenetic modifier genes. The finding of frequent mutations also in children/adolescents implicates that BCR-ABL independent gene mutations are not just age-related events. Such molecular aberrations may play an important role in the evolution and persistence of the disease and may affect therapy in both children and adults with CML. These findings may provide important novel genetic information regarding CML biology and for the design and performance of discontinuation trials. Disclosures Hochhaus: Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding.

Molecularly defined clonal evolution has been identified as a key phenomenon in the biology of acute myeloid leukemia. Molecular aberrations may be markers of individual subclones of the disease, but also responsible for treatment resistance. In most patients with chronic myeloid leukemia (CML), treatment with tyrosine kinase inhibitors (TKI) induces complete cytogenetic remissions (CCyR) characterized by polyclonal hematopoiesis. However, some CML patients show emergence of clonal cytogenetic abnormalities (CCA) in Philadelphia (Ph) negative cells during treatment indicating clonal hematopoiesis. We searched for BCR-ABL independent gene mutations in both Ph-negative and Ph-positive clones in 29 chronic phase CML patients (male, n=16; median age 58 years, range 29-73 years) using targeted deep next-generation sequencing of 25 genes frequently mutated in myeloid disorders: ASXL1, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, LNK, MPL, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TP53, TET2, U2AF1, UTX, WT, and ZRSR2. Ph-negative clones were analyzed in 14 CML patients who developed CCA in Ph-negative cells (trisomy 8, n=10; nonrecurrent reciprocal translocations, n=2; monosomy 7, n=2). At time of analysis, patients were in CCyR (n=7), partial CyR (n=6), or minimal CyR (n=1) after a median of 21 months (range 7-114 months) of TKI treatment with imatinib, and/or nilotinib, and/or dasatinib. After exclusion of known polymorphisms, mutations were detected in 6/14 patients (43%) affecting the genes DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1, and ZRSR2. These patients were in CCyR (n=2), partial CyR (n=3), or minimal CyR (n=1), respectively, indicating BCR-ABL independent mutations in both Ph-negative and Ph-positive subclones. In two patients, the mutations were also found in corresponding diagnostic samples at higher or lower mutation level. To further investigate BCR-ABL independent gene mutations in Ph-positive clones, 15 randomly selected CML patients at diagnosis were analyzed. Mutations additional to BCR-ABL were found in 5/15 CML patients (33%) affecting ASXL1, DNMT3A, RUNX1, and TET2. None of the mutations were recognized in corresponding constitutional DNA specimens indicating that all mutations had been somatically acquired. Deep-sequencing of subsequent samples obtained in early CCyR after three months of TKI treatment revealed one DNMT3A mutation in Ph-negative cells which was also present in Ph-positive cells at diagnosis. Follow-up investigation showed that the mutation persisted in Ph-negative cells throughout CCyR and deep molecular remission (MR4.5) up to month 36 thereby not significantly changing its mutation level of approximately 15% implying a clonal hematopoiesis before the acquisition of the BCR-ABL rearrangement. In summary, BCR-ABL independent gene mutations were frequently found in Ph-negative and Ph-positive clones of CML patients and may be considered as important cofactors in the evolution of CML. Additional mutations acquired in the Ph-positive clone may impact on response to TKI treatment. Mutations preexisting to the occurrence of the BCR-ABL rearrangement may predispose patients to secondary hematological neoplasms. Our findings provide novel genetic information regarding CML biology and warrant further studies and impact on the design and performance of discontinuation trials. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Other. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; ARIAD: Honoraria, Research Funding; Pfizer: Consultancy, Research Funding.

Abstract 617 Single nucleotide polymorphism (SNP) arrays have revealed regions of acquired uniparental disomy (aUPD) as recurrent events in hematological malignancies. Some of these regions are associated with the acquisition of somatic mutations in specific genes but the presumptive targets of many stretches of aUPD remain to be identified. We and others recently identified EZH2 as a target for 7q aUPD in myeloid malignancies, specifically myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and the overlap myelodysplastic/myeloproliferative neoplasms (MDS/MPN). EZH2 is the catalytic component of the polycomb repressive complex 2 (PRC2), a highly conserved histone H3 lysine 27 methyltransferase that regulates the expression of developmental genes. We reasoned that the other two core PRC2 components SUZ12 and EED may also be mutational targets in these diseases, as well as associated factors such as Jarid2. We identified SUZ12 mutations in 1 of 2 cases with MDS/MPN with 17q aUPD and 2 of 2 myelofibrosis cases with focal 17q11 deletions. All three were missense mutations affecting the highly conserved VEFS domain, necessary for the interaction between SUZ12 and EZH2 and thought to help stabilize the PRC2 complex. Analysis of a further 146 MDS/MPN cases revealed an additional VEFS domain mutant, yielding a total mutation frequency of 2/148 (1.4%). We did not find mutations of Jarid2 or EED in association with aUPD for chromosome 6p or 11q, respectively, however screening additional cases identified missense mutations in EED (1/87; 1%) and Jarid2 (3/148; 2%). All three SUZ12 mutations tested and the EED mutation reduced PRC2 histone methyltransferase (HMT) activity as assessed by an in vitro baculovirus expression assay, thus demonstrating that PRC2 function may be compromised in myeloid disorders by mutation of distinct genes. SUZ12 is located at 17q11.2 close to NF1, a gene that encodes a negative regulator of Ras signalling that has been strongly implicated in the pathogenesis of myeloid neoplasms. Of the cases with SUZ12 mutations, one with del(17q11.2) had mutations of both NF1 and SUZ12. Both of these mutations were clearly inactivating: a frameshift in exon 5 of NF1 and a D605V change in SUZ12 that greatly reduced PRC2 HMT activity. In summary, we have shown that inactivation of the PRC2 can occur through mutation of any of the 3 core components, and that inactivating mutations of EZH2, SUZ12 or EED are seen in 15% of cases of MDS/MPN. These findings emphasize the importance of PRC2 function in hemopoiesis and suggest that deregulation of the epigenetic control of gene expression plays an important role in the development of myeloid disorders. Disclosures: No relevant conflicts of interest to declare.