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Background Community‐acquired respiratory viruses (CARV) cause upper and lower respiratory tract infections (URTI/LRTI) and may be life‐threatening for recipients of an allogeneic stem cell transplantation (allo‐SCT). Methods In a prospective study encompassing 4 winter‐seasons, we collected throat gargles (TG) at random time points from allo‐SCT recipients (patients) and controls and followed them up for at least 3 weeks including repetitive sampling and documentation of symptoms. A Multiplex‐PCR system to identify 20 CARV and Mycoplasma pneumoniae was used to detect CARV. Results One hundred ninety‐four patients with 426 TG and 273 controls with 549 TG were included. There were more patients with a positive test result (25% vs 11% in the controls), and the patients had a higher number of positive TG (70 = 16%) compared to controls (32 = 6%) (P 40 years (OR 3.38, 95% CI 1.8‐6.4, P

Infectious complications such as invasive aspergillosis or infection with Stenotrophomonas maltophilia (SM) in immunocompromised patients are associated with a high mortality rate. Our report concerns a 40-year-old male newly diagnosed very severe aplastic anemia (vSAA) who in consequence of a mosquito bite was suffering from skin lesion and consecutive soft tissue phlegmon subsequent to the administration of antithymocyte globulin; a full-thickness autologous meshed skin graft successfully performed to cover skin ulcera after allogeneic stem cell transplantation (SCT). This unusual case illustrates the importance of appropriate diagnosis, anti-infective therapy and close interdisciplinary diagnostic algorithms to minimalize side effects and the selection of resistant strains and to improve patients’ outcome.

SETBP1 mutations have been established as a diagnostic marker in myeloid malignancies and are associated with inferior survival. Since there is limited data on their clinical impact and stability during disease progression, we sought to investigate the relationship between SETBP1 mutations and disease evolution. Bidirectional Sanger sequencing of the SETBP1 gene was performed for 442 unselected patients with World Health Organization (WHO) defined myeloid disorders. Follow-up analysis was performed on samples from 123/442 patients to investigate SETBP1 mutation dynamics. Targeted deep next-generation sequencing for a panel of 30 leukemia-associated genes was established to study SETBP1 cooperating mutations. 10/442 patients (2.3%) had SETBP1 hotspot mutations (MDS/MPN, n = 7, sAML, n = 3), whereas four patients (1%) had SETBP1 non-hotspot mutations (MPN, n = 1; MDS, n = 2; sAML, n = 1). The median overall survival for patients with SETBP1 hotspot mutations, SETBP1 non-hotspot mutations, and SETBP1 wild type was 14 (range 0–31), 50 (range 0–71), and 47 months (range 0–402), respectively. In Kaplan–Meier analysis, SETBP1 hotspot mutations were significantly associated with reduced overall survival compared to SETBP1 non-hotspot mutations and the SETBP1 wild type (p

Mutations in the epigenetic regulator gene EZH2 are frequently observed in patients with myelodysplastic/myeloproliferative neoplasms (MDS/MPN; 10-13%) and are associated with a poor outcome. To gain more insight into EZH2 pathology, we sought to genetically characterize a cohort of 41 EZH2-mutated MDS/MPN patients using targeted deep next-generation sequencing (NGS), colony-forming progenitor assays and transcriptome analysis. Stable short hairpin RNA (shRNA)-mediated downregulation of EZH2 was performed in MDS-derived F-36P, MOLM-13 and OCI-M2 cells to study EZH2-specific changes. Targeted NGS revealed a complex pattern of mutations with a total of 190 individual mutations. EZH2 mutations frequently co-occur with TET2 (58%), RUNX1 (40%) and ASXL1 (34%) mutations. Colony assays indicated EZH2 mutations to be mostly early events in leukemogenesis and showed a complex mutational hierarchy. Gene expression data revealed a number of differently expressed genes between EZH2 wild-type and mutant patients including known EZH2 targets. Comparison of patient transcriptome to EZH2-downregulated cell line data revealed several genes as novel EZH2 targets, showing opposite as well as unidirectional regulation between cell lines and patients. Some genes, such as CXXC5, ETS1 and VAV3 have previously been implied to have a role in leukemogenesis. Their precise role in MDS/MPN needs to be further investigated.

Fusion genes involving the catalytic domain of tyrosine kinases (TKs) play an important role in the pathogenesis of hematological malignancies and solid tumors. In BCR-ABL1-negative myeloproliferative neoplasms (MPNs) several different tyrosine kinase fusion events have been described, most commonly involving the genes encoding the platelet-derived growth factor receptor alpha (PDGFRA) or beta (PDGFRB). Since the introduction of small molecule kinase inhibitors, TK fusions have emerged as prime therapeutic targets. Here, we report a recurrent CEP85L-PDGFRB fusion in a patient with eosinophilia and an MPN. The fusion was confirmed by specific amplification of the genomic breakpoints and reverse transcription polymerase chain reaction (PCR). The patient was treated with imatinib and achieved hematologic and cytogenetic remission. Minimal residual disease screening over 3 years with nested PCR failed to detect CEP85L-PDGFRB mRNA or genomic DNA, confirming a long-term molecular remission on imatinib.

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.

Even in the era of proteasome inhibitors and immunomodulatory drugs, the autologous stem cell transplantation after high-dose melphalan continues to represent a standard approach for myeloma patients in first-line therapy. Different mobilization chemotherapies before stem cell apheresis have been published while cyclophosphamide at a dose level of up to 4 g/m2 has been evaluated and is commonly applied. In contrast, lower dose levels of cyclophosphamide (e.g., 1.5 g/m2) did not result in a sufficient collection of stem cells. We retrospectively analyzed the impact of “intermediate-dose” (ID-CY, 2.5 g/m2) versus “high-dose” (HD-CY, 4 g/m2) cyclophosphamide in 101 (48 vs. 53) consecutively evaluable myeloma patients (median age 59 years, range 32–72 years) who underwent stem cell mobilization after induction chemotherapy. Successful stem cell harvest was defined as a stem cell yield of at least 5 million CD34 cells per kg bodyweight. Evaluation of toxicity especially considered infectious complications and hematological toxicity in both subgroups. Successful stem cell mobilization was achieved in 40 of 48 (83 %) and 44 of 53 (83 %) patients, respectively. The median time to apheresis (11 vs. 12 days) and the median CD34 content of stem cell harvest (8.3 vs. 7.6 million CD34 cells per kg bodyweight) did not differ significantly between both groups. There was a significant difference of WBC nadir in favor of the cyclophosphamide regimen with 2.5 g/m2 (0.8 vs. 0.3 Gpt/L, p = 0.021), and neutropenic fever was more often observed in patients who received 4 g/m2 cyclophosphamide (34 vs. 15 %, p = 0.078). Importantly, after induction chemotherapy using the VCD regimen (bortezomib, cyclophosphamide, dexamethasone), successful stem cell mobilization was achieved in 26 of 29 (90 %) patients treated with 2.5 g/m2 and 21 of 25 (84 %) patients receiving 4 g/m2 cyclophosphamide, respectively. ID-CY is safe and highly effective for stem cell mobilization in patients with newly diagnosed myeloma and associated with a reduced toxicity compared to HD-CY.

Summary: Whole exome sequencing was performed in a patient with myelodysplastic syndrome before and after progression to acute myeloid leukaemia. Mutations in several genes, including SETBP1, were identified following leukaemic transformation. Screening of 328 patients with myeloid disorders revealed SETBP1 mutations in 14 patients (4·3%), 7 of whom had -7/del(7q) and 3 had i(17)(q10), cytogenetic markers associated with shortened overall survival and increased risk of leukaemic evolution. SETBP1 mutations were frequently acquired at the time of leukaemic evolution, coinciding with increase of leukaemic blasts. These data suggest that SETBP1 mutations may play a role in MDS and chronic myelomonocytic leukaemia disease progression. © 2013 John Wiley and Sons Ltd.

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.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and young adults. The polycomb repressive complex 2 (PRC2) has been identified as one of the most frequently mutated epigenetic protein complexes in hematologic cancers. PRC2 acts as an epigenetic repressor through histone H3 lysine 27 trimethylation (H3K27me3), catalyzed by the histone methyltransferase enhancer of zeste homolog 2 protein (EZH2). To study the prevalence and clinical impact of PRC2 aberrations in an unselected childhood ALL cohort (n = 152), we performed PRC2 mutational screenings by Sanger sequencing and promoter methylation analyses by quantitative pyrosequencing for the three PRC2 core component genes EZH2, suppressor of zeste 12 (SUZ12), and embryonic ectoderm development (EED). Targeted deep next-generation sequencing of 30 frequently mutated genes in leukemia was performed to search for cooperating mutations in patients harboring PRC2 aberrations. Finally, the functional consequence of EZH2 promoter hypermethylation on H3K27me3 was studied by Western blot analyses of primary cells. Loss-of-function EZH2 mutations were detected in 2/152 (1.3 %) patients with common-ALL and early T-cell precursor (ETP)-ALL, respectively. In one patient, targeted deep sequencing identified cooperating mutations in ASXL1 and TET2. EZH2 promoter hypermethylation was found in one patient with ETP-ALL which led to reduced H3K27me3. In comparison with healthy children, the EZH2 promoter was significantly higher methylated in T-ALL patients. No mutations or promoter methylation changes were identified for SUZ12 or EED genes, respectively. Although PRC2 aberrations seem to be rare in childhood ALL, our findings indicate that EZH2 aberrations might contribute to the disease in specific cases. Hereby, EZH2 promoter hypermethylation might have functionally similar consequences as loss-of-function mutations.

In patients undergoing allogeneic peripheral blood stem cell (PBSC) transplantation after reduced-intensity conditioning (RIC), graft-versus-host disease (GVHD) represents a major cause of morbidity and mortality. T-cell depletion (TCD) prevents GVHD but carries potential risks of graft failure, opportunistic infections, and disease relapse. We explored ex vivo TCD of stem cell allografts that were administered after RIC treatment. Thirteen high-risk patients with hematological malignancies were treated with a fludarabine/melphalan-based RIC regimen followed by transplantation of immunomagnetically selected CD34(+) PBSC from HLA-identical sibling or matched unrelated donors. Patients were sequentially enrolled in two cohorts: group A (n=6) received antithymocyte globulin (ATG) during conditioning and 10(5) donor T cells/kg at transplantation, while group B (n=7) received 10(6) donor T cells/kg without ATG pretreatment. Primary graft failure occurred in two patients of group A and in one patient of group B. Complete donor chimerism persisting more than 1 year was achieved in two cases per cohort. Acute grade II to IV or chronic extensive GVHD were observed in a total of six patients (group A, 2; group B, 4). Procedure-related deaths were mainly due to severe pneumonia occurring in two patients of group A and in five patients of group B. These results suggest that CD34 selection of reduced-intensity PBSC allografts may cause adverse effects upon specific antimicrobial immunity which can lead to fatal infections, particularly in high-risk patients. In our study, simultaneous add-back of < or =10(6)/kg donor T cells was unable to compensate for this deficiency.

Diffuse large B-Cell Lymphoma (DLBCL) is the most common subtype of maligant lymphoma (ML). The incidence rate is about 10–15 of 100,000 people in the US and in Europe per year. Men are more frequently affected than women [1]. The incidence of DLBCL in people over the age of 65 years is rapidly rising. In the elderly (75 years or older), rates of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma increased 1.4 %. According to the SEER cancer statistics review 2000–2011, 9 per 100,000 of those younger than 65 years develop the disease, compared to 90 per 100,000 in those aged older than 65 years. The 5-years relative survival rates decreases from 78 % in those younger than 65 years to 62 % in those older than 65 years. The occurrence of all Non-Hodkgin’s Lymphomas (NHL) has been rising from 10 to over 20 newly diagnosed patients per 100,000 from 1975 to 2010. For patients over 75 years of age, incidence rates have doubled (50–100 per 100,000) since 1975. Thus DLBCL is predominantly a disease of older individuals, with a median age of diagnosis at approximately 70 years of age. As demographic changes result in an increasing number of older people the occurrence of NHL in this older patient population will pose an increasing problem [2].

To study clonal evolution in chronic myeloid leukemia (CML), we searched for BCR-ABL-independent gene mutations in both Philadelphia chromosome (Ph)-negative and Ph-positive clones in 29 chronic-phase CML patients by targeted deep sequencing of 25 genes frequently mutated in myeloid disorders. Ph-negative clones were analyzed in 14 patients who developed clonal cytogenetic abnormalities in Ph-negative cells during treatment with tyrosine kinase inhibitors (TKI). Mutations were detected in 6/14 patients (43%) affecting the genes DNMT3A, EZH2, RUNX1, TET2, TP53, U2AF1 and ZRSR2. In two patients, the mutations were also found in corresponding Ph-positive diagnostic samples. To further investigate Ph-positive clones, 15 randomly selected CML patients at diagnosis were analyzed. Somatic mutations additional to BCR-ABL were found in 5/15 patients (33%) affecting ASXL1, DNMT3A, RUNX1 and TET2. Analysis of individual hematopoietic colonies at diagnosis revealed that most mutations were part of the Ph-positive clone. In contrast, deep sequencing of subsequent samples during TKI treatment revealed one DNMT3A mutation in Ph-negative cells that was also present in Ph-positive cells at diagnosis, implying that the mutation preceded 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 clonal evolution of CML.

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.

The prevalence of elderly and comorbid patients (pts) with malignant lymphoma (ML) will steadily increase in future. Elderly patients comprise a heterogeneous population. Comprehensive geriatric assessment (CGA) is an established diagnostic tool in geriatric medicine. However, the prognostic value in patients with ML is unclear. We sought to establish a relationship between results of CGA and survival time in patients with ML. Newly diagnosed patients with ML and indication for chemotherapeutical treatment were prospectively recruited in an observational trial. In addition to usual diagnostic work up, a CGA including activities of daily living (ADL), instrumental activities of daily living (IADL) and comorbidities was performed. Association of patients’ characteristics and results of CGA with survival were analysed according to Kaplan–Meier method and in a multivariate Cox-regression analysis. About 143 patients were included, median age was 63 years, 63 patients were women. Median follow-up of surviving patients was 62 months. Sixty-six patients died within this time. Advanced age, poor Karnofsky performance status, dependence in ADL and IADL and presence of severe comorbidity were significantly associated with shorter survival time. In a Cox-regression analysis, IADL (HR 2.1; 95% CI 1.1–3.9) and comorbidity (HR 1.9; 95% CI 0.9–3.9) were independent and strongest associated with survival time. Results of CGA, such as IADL and comorbidities, are prognostic variables for survival of patients with ML. Results should be validated in homogeneous clinical groups and if confirmed included in diagnostic and therapeutic algorithm.

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.

Atypical Chronic Myeloid Leukemia (aCML) is a heterogeneous disorder belonging to the group of myelodysplastic/myeloproliferative (MDS/MPN) syndromes. The molecular pathogenesis of this disease is still unclear and the outcome is poor with no improvement over the last 20 years. We applied whole exome sequencing approach in 9 aCML patient samples in order to identify possible recurrent alterations. The analysis revealed the presence of unique mutations in 70 genes with 3 cases of SETBP1 alterations. Some of the genes identified as mutated in the initial set of 9 patients (IDH2, MTA2, EPHB3, ETNK1, GATA2, IRAK4) and having a score higher than 1 in the oncogenic GeneRanker database were resequenced in a cohort of 40 aCML patients (15 with and 25 without SETBP1 mutations). With the exception of IDH2, no other gene was found mutated in any case apart from the index patient. Evaluation on a larger cohort of 70 aCML samples revealed recurrent SETBP1 mutations in 24.3% of cases (see designated abstract). To test the relationship between SETBP1 variants and mutations in oncogenes known to be involved in myeloid malignancies, mutations in ASXL1, CBL, CEBPA, DNMT3A, EED, EZH2, FLT3, IDH1/2, JAK2, JARID2, NPM1, N/KRAS, RBBP4, RUNX1, SF3B1, SUZ12, TET2 and WT1 were evaluated in a population of 61 aCML patients (14 with and 47 without SETBP1 mutations) by Sanger sequencing. Overall we identified 60 mutations in 14 genes: 28 were missense point mutations, 13 nonsense point mutations, 15 missense ins/del and 4 ins/del leading to a premature stop codon. No mutations were found in IDH1, RBBP4, NPM1, JAK2, FLT3, DNMT3A. Mutations in ASXL1 were present in 14 patients and appeared more frequent in patients with mutated SETBP1 (36% vs 19%) while the 15 TET2 mutations were more prevalent in patients with SETBP1 WT than in mutated samples(28% vs. 14%). Further associations will be presented at the meeting, although further analysis on larger cohorts of patients will be necessary to determine the significance of this differences. Additional data on epigenetic signature of aCML will clarify the role of epigenetic dysregulation in aCML and related diseases. Citation Format: Sara Redaelli, Simona Valletta, Rocco Piazza, Nils Winkelmann, Roberta Spinelli, Alessandra Pirola, Laura Antolini, Luca Mologni, Carla Donadoni, Elli Papaemmanuil, Susanne Schnittger, Kim Dong-Wook, Jacqueline Boultwood, Fabio Rossi, Giuseppe Gaipa, Greta De Martini, Paola Francia di Celle, Hyun Gyung Jang, Valeria Fantin, Graham R. Bignell, Vera Magistroni, Torsten Haferlach, Enrico Maria Pogliani, Peter Campbell, Andrew J. Chase, William J. Tapper, Nick C.P. Cross, Carlo Gambacorti Passerini. Patterns of recurrent mutations in SETBP1 mutated and wild-type atypical Chronic Myeloid Leukemia patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2993. doi:10.1158/1538-7445.AM2013-2993

Atypical Chronic Myeloid Leukemia (aCML) shares clinical and laboratory features with Chronic Myeloid Leukemia (CML), but it lacks the pathognomonic BCR-ABL1 fusion. The molecular pathogenesis of this disease has remained elusive and the outcome dismal. To investigate the molecular pathogenesis of aCML, we applied exome sequencing and RNA-SEQ to aCML, with the aim of identifying novel recurrent driver mutations. Whole-exome sequencing of 9 aCML patients(pts) revealed the presence of 70 unique mutations, including recurrent alterations of SETBP1 in 3 cases: 2 cases with a G870S and a case with a D868N alteration were found. Targeted resequencing in 70 aCMLs, 574 pts with different hematological malignancies and 344 cell lines, identified SETBP1 mutations in 17/70 aCML (24.3%; 95% CI: 16-35%), 4/30 (13%) MDS/MPN-u, 3/82 (3.6%) CMML and 0/100 MDS. aCML pts with SETBP1 mutations had higher white blood cell counts (p=0.008) and worse prognosis (p=0.01) when tested in multivariate analysis. The SETBP1 gene encodes for a predominantly nuclear protein with a predicted MW of 170kDa. Germline mutations of SETBP1 were previously described in pts affected by the Schinzel-Giedion syndrome (SGS), a rare disease characterized by bone, muscle and cardiac abnormalities and neuroepithelial neoplasms. The vast majority of aCML SETBP1 mutations (85%) was located between residues 858 and 871 and were identical to the germline changes seen in pts with SGS. This region may be critical for ubiquitin binding and for subsequent protein degradation, since the Eukaryotic Linear Motif identified a putative functional site (aa. 868-873) for beta-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase. The prediction was experimentally validated using biotinylated, phosphorylated peptides encompassing this region (aa 859-879): while the wild type (wt) peptide could efficiently bind beta-TrCP, a peptide presenting G870S was incapable of binding this subunit, indicating a possible alteration in SETBP1 protein stability caused by the mutation. In line with the known binding model of beta-TrCP, dephosphorylated peptides failed to bind beta-TrCP, therefore confirming the specificity of the interaction. In agreement with these findings, TF1 cells transduced with SETBP1 G870S showed increased SETBP1 and SET protein levels, decreased PP2A activity and increased proliferation rates when compared to cells expressing the wt SETBP1 gene. Mutated SETBP1 represents a novel oncogene specifically present in aCML and closely related diseases. These data allow for a better understanding of the molecular pathogenesis of this disease; they provide evidence that SETBP1 mutations might be a new biomarker for future diagnosis and classification of aCML and related diseases, and indicate a potential strategy to develop new treatment modalities for malignancies caused by mutated SETBP1. Citation Format: Rocco Piazza, Simona Valletta, Nils Winkelmann, Sara Redaelli, Roberta Spinelli, Alessandra Pirola, Laura Antolini, Luca Mologni, Carla Donadoni, Elli Papaemmanuil, Susanne Schnittger, Kim Dong-Wook, Jacqueline Boultwood, Fabio Rossi, Giuseppe Gaipa, Greta De Martini, Paola Francia di Celle, Hyun Gyung Jang, Valeria Fantin, Graham R. Bignell, Vera Magistroni, Torsten Haferlach, Enrico Maria Pogliani, Peter Campbell, Andrew J. Chase, William J. Tapper, Nick C.P. Cross, Carlo Gambacorti Passerini. Recurrent SETBP1 mutations in atypical chronic myeloid leukemia abrogate an ubiquitination site and dysregulate SETBP1 protein levels. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3176. doi:10.1158/1538-7445.AM2013-3176

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.