ALBERT

Description: Key Points Mutational trajectories are defined by complex patterns of molecular heterogeneity in MDS, including lower-risk cases. Therapeutic intervention dynamically reshapes mutational patterns often resulting in branched or independent evolution of MDS clones.

Description: Malignant hematopoietic cells of myelodysplastic syndromes (MDS)/chronic myelomonocytic leukemias (CMML) and acute myeloid leukemias (AML) may be particularly vulnerable to inhibition of poly(ADP ribose) polymerase 1/2 (PARP1/2) and apurinic/apyrimidinic endonuclease 1 (APE1). PARP1/2 and APE1 are critical enzymes involved in single-strand break repair and base excision repair, respectively. Here, we investigated the cytotoxic efficacy of talazoparib and APE1 inhibitor III, inhibitors of PARP1/2 and APE1, as single-agents, combined with decitabine and combined with each other in CD34+ MDS/CMML cells and in CD34+ or CD34- AML cells in comparison to healthy CD34+ donor cells. The surviving fraction of CD34+ MDS/CMML cells (n = 8; 4 MDS and 4 CMML), CD34+ or CD34- AML cells (n = 18) and healthy CD34+ donor cells (n = 8) was analyzed using the CellTiter-Glo luminescent cell viability assay (Promega, Southampton, UK). Cell proliferation of untreated MDS/CMML and AML cells was determined by trypan blue exclusion assay (Merck, Darmstadt, Germany). PARP1/APE1 mRNA expression was evaluated using validated primer sets for PARP1 (Hs_PARP1_1_SG QuantiTect Primer Assay, NM_001618) and APE1 (Hs_APEX1_1_SG QuantiTect Primer Assay, ENST00000216714) (Qiagen, Hilden, Germany). Immunofluorescence microscopy of γH2AX foci was performed using a JBW301-derived mouse monoclonal anti-γH2AX antibody (Merck). Talazoparib and APE1 inhibitor III demonstrated critical anti-leukemic efficacy as single-agents in about 19-25% of MDS/CMML/AML cell samples (Figure 1A and B). Low doses of talazoparib and APE1 inhibitor III further increased the cytotoxic efficacy of decitabine in about 78-86% of MDS/CMML/AML cell samples. Moreover, low doses of APE1 inhibitor III increased the cytotoxic efficacy of talazoparib in about 68% of MDS/CMML/AML cell samples. In summary, talazoparib and APE1 inhibitor III demonstrated substantial anti-leukemic efficacy as single-agents, in combination with decitabine and combined with each other. Hence, our findings support further investigation of these agents in sophisticated clinical trials. Figure 1 Cytotoxic efficacy of talazoparib and APE1 inhibitor III in healthy CD34+ donor cells, in CD34+ myelodysplastic syndrome (MDS)/chronic myelomonocytic leukemia (CMML) cells and in CD34+ or CD34- acute myeloid leukemia (AML) cells after 3 days of treatment. (A) The mean IC50 of talazoparib was significantly lower (*p = 0.016) in 1 MDS (MDS#2), 1 CMML (CMML#2) and 3 AML cell samples (AML#1, AML#2, AML#3) as compared to 8 healthy donor cell samples. (B) The mean IC50 of APE1 inhibitor III was substantially lower (p = 0.059) in 1 MDS (MDS#2) and 5 AML cell samples (AML#1, AML#2, AML#3, AML#6, AML#12) as compared to 8 healthy donor cell samples. Figure 1 Disclosures Fabarius: Novartis: Research Funding.

Description: Purpose: Numerical and structural centrosome abnormalities are hallmarks of a variety of cancers and have been implicated in chromosome missegregation, chromosomal instability, and aneuploidy. These phenomena already occur in preneoplastic lesions like oral leukoplakia, early cervical neoplasias, and small benign tumors of colon and breast. Moreover, deviations from normal karyotype seem to increase as tumors enlarge and become malignant. Genetic instability is a common feature in chronic myeloid leukemia (CML). We sought to establish a relationship between centrosome abnormalities and cytogenetic aberrations in CD34+ cells from CML patients at diagnosis (chronic phase - CP) and in blast crisis (BC). Methods: Diagnosis of CML was established by hematologic, cytogenetic and molecular parameters. Treatment was performed according to the protocols of the German CML study group (www.kompetenznetz-leukaemie.de). CD34+ cells from ten umbilical cord blood specimens served as negative controls. Centrosome number and morphology were analyzed by immunofluorescence microscopy. In brief, CD34+ cells from ficollized peripheral blood samples were concentrated by magnetic cell sorting (MACS) and cytospun onto coated slides. After methanol fixation cells were incubated with antibodies directed to centrosomal proteins Pericentrin and gamma-Tubulin. Antibody-antigen complexes were stained by incubation with FITC- and Cy3-conjugated secondary antibodies. Results: CML CP samples tested at initial diagnosis (n=20) already displayed numerical and structural centrosome aberrations (30.0% +/−2.3) as compared with corresponding normal control cells (n=10) (2.3% +/−1.1). In BC samples (n=10) an increase of centrosome aberrations was observed (58.0% +/−2.0). Conclusion: The findings suggest that centrosome defects in CML occur early and are already present at primary diagnosis. Centrosome defects may contribute to disease progression by generation of further chromosome instability leading to accumulation of alleles carrying pro-oncogenic mutations and loss of alleles containing normal tumor suppressor genes and thus accelerating complex genomic changes associated with CML BC.

Description: Introduction Deletion of chromosome 5q (del(5q)) defines a distinct clinical subtype of myelodysplastic syndromes (MDS) and qualifies patients to specific treatment with Lenalidomide (LEN). Therefore, detection and monitoring of this deletion is an important element in routine clinical diagnostics for determining molecular response. Current methodologies for performing these analyses consist of cytogenetics, fluorescence in situ hybridization (FISH) or microarrays. All of these methods have downsides due to the high demands to the input material, i.e. viable cells or necessity for large amounts of high quality genomic DNA (gDNA). To perform quantitative assessment of cytogenetic lesions in low quantity or residual material we here present the establishment of a PCR-based assay for interrogation of del(5q) in MDS, based on the allelic loss at heterozygous short tandem repeat (STR) loci within deleted regions. Methods Genomic DNA was isolated from bone marrow (BM) and peripheral blood (PB) of n=86 MDS del(5q) patients. 49 non-del(5q) MDS patients were used as controls. Serial chronological BM samples (n=95) following treatment with LEN from n=40 del(5q) patients, who were enrolled in the LEMON-5 trial from the German MDS study group, were analysed. Using 10ng DNA, 12 fluorochrome-labelled PCR amplicons of STR loci located between chromosomal bands 5q21 and 5q31 were amplified in a single optimized multiplex-PCR reaction. Subsequently, amplicon fragment analysis was carried out via capillary electrophoresis and allele size quantification of heterozygous STR loci was performed. Finally, the degree of skewing in the allelic ratios of all informative STR markers was averaged and translated into an allelic burden of del(5q). Results Paired quantitative correlation of clone sizes using STR-PCR and interphase FISH was carried out in n=34 samples and revealed highly concordant results with r²=0.924. The diagnostic accuracy of the PCR assay was evaluated by receiver operating characteristic (ROC) analysis and revealed an area under the curve of 0.989 (sensitivity and specificity of 0.977 and 0.948, respectively). Prior to treatment with LEN, clone sizes as determined by STR-PCR were heterogeneous (mean: 57%, range: 11-91%). During follow-up analysis, while cytogenetic analyses failed (e.g. metaphase failure) in 7/40 (18%) cases, our STR-PCR assay successfully generated estimates of del(5q) cell burden in all available samples. Upon LEN treatment, n=12 patients achieved major cytogenetic remission (absence of del(5q)-positive metaphases). The mean clone size carrying del(5q) determined by STR-PCR in that group was 7% (range 3 - 10%) and significantly increased compared with n=15 patients who reached minor cytogenetic response (defined as 50% reduced aberrant metaphases, mean 13%, range 5 - 39%, p=0.025). Intriguingly none of n=6 patients without cytogenetic response achieved a del(5q) clone size of less than 35% as determined by STR-PCR (mean 46%, range 35 - 66%), highlighting the correlation of PCR based follow-up analysis with currently used cytogenetic methods for response evaluation. Finally in n=93 matched PB and BM samples a correlation of del(5q)-frequency in BM versus PB showed r²=0.81. Moreover, in 96% of samples in which the BM still showed clone sizes 〉10%, we reliably detected del(5q) in corresponding PB cells with a robust sensitivity of 5% deleted cells. Discussion We present a highly adaptable tool for precise measurement of large chromosomal deletions, requiring only minute amounts of genomic DNA. It shows a very good quantitative correlation with established methods and good diagnostic accuracy. Most importantly, this PCR based assay does not require dividing cells so it can be performed from PB, which shows a sufficient correlation with clone sizes in BM and rarely involves the risk of underrepresentation of del(5q)-clones in PB, possibly allowing the use of PB as a regular specimen for clone size monitoring. Thus, especially in the context of serially monitored patients this assay represents an alternative method for less invasive tracking of cytogenetically aberrant clones. Disclosures Platzbecker: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Boehringer: Research Funding. Schlenk:Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Teva: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Bug:TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: Clonal cytogenetic aberrations of the Philadelphia chromosome (Ph) positive hematopoiesis have been associated with the natural evolution of CML to advanced disease. Clonal aberrations of Ph negative metaphases have been described after treatment with interferon alpha or imatinib in a minority of patients (pts) with cytogenetic response. Conflicting data suggest selection of preexisting clones vs. induction of aneuploidy by tyrosine kinase inhibitors. The prognostic impact of aberrations in the Ph negative hematopoiesis for the individual pts remains to be determined. Dasatinib is a multitargeted agent inhibiting both ABL and SRC tyrosine kinases. The efficacy and safety of dasatinib has been demonstrated in phase I and II studies in pts with Ph positive CML after failure of imatinib therapy. We sought to evaluate the effect of dasatinib on Ph positive clones with additional cytogenetic aberrations and the frequency of novel aberrations in Ph positive and Ph negative metaphases during dasatinib therapy. 71 pts (40 m, 31 f) with Ph positive CML after failure of imatinib therapy have been evaluated. Median age was 58 years (range, 28–78), median time from diagnosis 73 months (range, 14–231). Pts were in chronic phase (CP, n=50), accelerated phase (AP, n=6), myeloid (n=8) or lymphoid (n=7) blast crisis (BC). Pretreatment consisted of imatinib (n=71), hydroxyurea (n=48), interferon alpha (n=49), cytosine arabinoside (n=11) and nilotinib (n=2). Dasatinib therapy was commenced at a dose of 100–140 mg/day, median duration of dasatinib therapy was 9 months, (range, 1–16). Prior to dasatinib therapy, 22 pts (31%) demonstrated additional chromosomal aberrations in the Ph positive clone indicating BCR-ABL independent mechanisms of resistance. Of these, 8 pts were in CP (16%), 5 in AP (83%), and 9 in BC (60%). In 35 pts (49%), BCR-ABL mutations associated indicating a BCR-ABL dependent mechanism of resistance were observed. 9 pts (13%) showed both clonal evolution and BCR-ABL mutations. Two pts (3%) had trisomy 8 as an aberration of the Ph negative clone at baseline. During dasatinib therapy, 33 pts (46%) achieved major cytogenetic remission, 26 (37%) being complete. From pts with clonal evolution, 3 (14%) achieved a major with 2 (9%) complete cytogenetic remissions. Novel aberrations of the Ph positive clone appeared during dasatinib therapy in 6 pts (8%), aberrations of the Ph negative clone in 3 pts (4%). In total, 5/71 pts (7%) showed clonal aberrations of Ph negative metaphases after consecutive imatinib/dasatinib therapies. None of these pts had morphological indications for secondary neoplastic changes, like myelodysplasia or acute leukemia. In conclusion, dasatinib was efficacious in pts with clonal cytogenetic aberrations as a marker of BCR-ABL independent imatinib resistance. However, cytogenetic response was delayed compared to pts without additional aberrations. Preexisting clonal aberrations of the Ph negative hematopoiesis were uncovered by a simultaneous gradual elimination of the Ph positive clone. In addition, a minority of pts demonstrated novel aberrations in Ph negative cells. The prognostic significance of aneuploidy of the Ph negative hematopoiesis remains to be evaluated by long term observation.

Description: Abstract 1694 Introduction: Myelodysplastic syndromes are a heterogeneous group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis, peripheral cytopenias and dysplastic bone marrow cells, with frequent progression to acute myeloid leukemia. Because of its heterogeneous nature, modeling of this disease has proven to be very difficult in cell culture systems as well as mice. In addition, attempts to generate a xenotransplant model in immuno-compromised mice have only achieved very low levels of engraftment that are often transient, making it very difficult to study the biology of this disease in vivo. Recent studies in mice have shown that conditional impairment of the small RNA processing enzyme Dicer in mouse osteolineages induced a stromal niche that promoted myelodysplasia, leading to the hypothesis that abnormal bone marrow stromal cells might provide a “fertile soil“ for the expansion of the malignant clone. Patients and Methods: To the date of writing, a total of 12 primary hematopoietic stem cell- and mesenchymal stroma cell (MSCs) samples selected from patients with MDS have been isolated and xenotransplanted into NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) mice: MDS 5q- (n=7), MDS RCMD (n=3), MDS RAEB I (n=1), MDS-U (n=1). Engraftment was monitored by FACS using human specific antibodies to CD45, CD34 and CD38. In addition cell cycle behavior was analyzed by Ki67/Hoechst staining. Mesenchymal stromal cells were characterized using previously described stromal markers: CD105, CD271, CD73, CD166, CD90, CD146 and CD44. To isolate genomic DNA and RNA for molecular analyses, MDS xenografts were flow sorted based on human CD45 expression. Molecular characterization of primary MDS samples and xenotransplants was carried out by serial copy number analysis using Affymetrix SNP 6.0 Arrays, metaphase cytogenetics and direct sequencing of known mutations in the transplanted MDS samples. Results: We show, that the concomitant transplantation of MDS-derived mesenchymal stromal cells with the corresponding hematopoietic patient stem/progenitor cells leads to significant and long-term engraftment (0.1 – 15% for up to 23 weeks) of cells isolated from IPSS low and intermediate risk MDS patients. In addition to the bone marrow, MDS hematopoietic cells also infiltrate other hematopoietic compartments of the mouse including the spleen. Significant engraftment of cells with progenitor (CD34+CD38+) as well as stem cell phenotype (CD34+CD38-) was observed, which is consistent with engraftment of an MDS stem cell that sustains long-term hematopoiesis. SNP array analysis confirmed the clonal origin of the engrafted cells as MDS xenografts harboring the identical genomic lesions as present in the patient disease. Conclusion: We present a robust MDS xenograft model of low risk MDS entities based on the concomitant transplantation of primary MDS hematopoietic cells with MSCs from the same patients. This model does not only allow to study the biology of this disease in vivo but also the molecular and cellular interactions between MSCs and hematopoietic MDS cells. In addition it provides a useful platform to study the effects of new experimental therapeutic agents for the treatment of MDS in molecularly defined MDS cells. Disclosures: No relevant conflicts of interest to declare.

Description: Multitargeted ABL inhibitors have been developed to simultaneously inhibit various pathways associated with proliferation in BCR-ABL+ diseases. Dasatinib (Bristol Myers Squibb) is a potent inhibitor targeting ABL, SRC, and other tyrosine kinases. SRC kinases are required for progression through the initial phase of mitosis. Centrosomes play a fundamental role in mitotic spindle organization, chromosome segregation and genetic stability. We sought to evaluate the activity of dasatinib on proliferation, centrosome status, spindle formation, and cell cycle progression in vitro and in vivo. Normal human dermal fibroblasts (NHDF), Chinese hamster embryonal fibroblasts (CHE), and the human osteosarcoma cell line U2OS were treated with serial concentrations (1nM-10μM) of dasatinib for 3 weeks. Effects of dasatinib were compared with data achieved with the ABL inhibitors imatinib (Novartis, 5–20μM) and nilotinib (Novartis, 0.5–20μM), the specific SRC inhibitor PP2 (Calbiochem-Novabiochem, 0.1–2μM), the ABL/LYN inhibitor INNO-406 (Innovive, 0.1–2μM), and solvent control. Bone marrow and peripheral blood samples from 18 patients (pts, 10 m, 8 f; median age 57 yrs, range 26–75) treated with dasatinib (70mg bid) after imatinib failure for a median of 11 mo (range, 3–16) were investigated. 17 pts had chronic myeloid leukemia (CML) in chronic phase. One patient suffered from a gastrointestinal stromal tumor. For comparison, 3 untreated CML pts and 3 healthy individuals were evaluated. Cell proliferation was determined in liquid culture incubated with serial dilutions of the inhibitor. Centrosome morphology and spindle formation were evaluated after pericentrin and α-tubulin staining, respectively. Cell cycle progression was analyzed by FACS and expression of EG5 by immunofluorescence microscopy. Dasatinib induced a G1 cell cycle arrest in all cell lines tested and in pts associated with a shift to 1n DNA ploidy and absence of EG5 as a marker for G2 phase/mitosis. In vitro, centrosomal aberrations and delay of spindle formation were observed in a dose dependent fashion. In pts, centrosome alterations were found in a median of 17% (range, 10–15) of cells. Disturbed spindle formation was observed in 9/18 pts. In comparison, incubation with imatinib and nilotinib was associated with centrosome aberrations but not with defects of spindle formation and G1 arrest. PP2 induced S-phase arrest; centrosome aberrations were observed at higher dosages (1–2 μM) only, spindle formation was not affected. INNO-406 was associated with both centrosome aberrations and disturbed spindle formation. In pharmacological doses, proliferation of BCR-ABL neg. cell lines was inhibited after dasatinib treatment, but not after incubation with imatinib, nilotinib, PP2, or INNO-406. In conclusion, dasatinib blocks the G1/S transition and thereby inhibits cell growth in normal and neoplastic cells. In addition, it induces both centrosomal and spindle aberrations. Effects of dasatinib are not based on SRC inhibition alone but may be associated with the combination of SRC and ABL inhibition or with non-specific effects on multiple kinases. Therefore, dasatinib should be defined as a cytostatic drug with a strong targeted component resulting in a preferential inhibition of cells harboring a specific target, like BCR-ABL.

Description: Background Myelodysplastic Syndromes (MDS) likely arise from an evolutionary process involving accumulation of somatic mutations and selection that occurs at the level of hematopoietic stem cells. Using next generation sequencing (NGS), several groups found recurrent somatic mutations to be associated with MDS. However, the history of stepwise molecular progression is still poorly defined. Likewise, little is known about patient-specific subclonal compositions, which may well contribute to the tremendous heterogeneity observed both in terms of clinical manifestations and response to treatment. Therefore, we sought to reconstruct patient-specific clonal hierarchies and decipher their dynamic evolution during long-term disease monitoring in order to better understand MDS pathogenesis and aid in adapting targeted therapeutic options in the future. Methods Bone marrow (BM) or CD34+ cells from patients with MDS were subjected to mutational screening by whole exome sequencing (WES, n=44) or targeted NGS (n=28) interrogating up to 17 recurrently mutated genes. Mesenchymal stromal cells (MSCs) were used as germline control. Allelic burdens were quantified with custom pyrosequencing assays and interphase-FISH in FACS purified myeloid, erythroid, lymphoid and stem cells isolated from both patients’ BM and corresponding xenografts in NSG mice. For unbiased follow up analysis, BM or CD34+ cells from two distant time points (median: 3 years, range 0.5-4.0 years) were analyzed for 13 patients using WES and these data were used to design patient-specific mutational panels. These panels contained amplicons representing mutational events uniquely present at one time point as well as others present at both time points. The panels were then subjected to ultra-deep-sequencing (UDS) to accurately quantify mutational burdens in all follow up (FU) samples. Results Integrative mutational data allowed us to reconstruct patient-specific mutational hierarchies in 35 cases, which revealed both linear and branching evolution in MDS. The data is also in support of the notion that potential founder lesions are highly enriched in genes involved in RNA splicing and epigenetic regulators, which we further show to be frequently detected in primary and/or xenografted lymphocytes. In contrast, we clearly demonstrate that large scale cytogenetic lesions (e.g. monosomy 7, trisomy 8, del(5q)) occur as late mutational events in at least 85% of the cases analyzed (n=17/20, 3 unresolved cases). Moreover, we could readily demonstrate the existence of subclonal heterogeneity in the patients’ BM, with variable contribution to different lineages and also cases showing lineage restriction of specific sub-clones. Most importantly, UDS analysis of FU samples from 13 independent patients (median FU 3.3 years, range FU 0.5-11.8 years and median of 5 samples per patient) with detailed clinical data revealed a highly dynamic clonal evolution during the course of the disease and dramatic shifts in the composition of mutational (sub-)clusters during therapy. Treatment often resulted in complete disappearance of specific sub-clones and most importantly, simultaneous outgrowth of previously undetectable subclones that subsequently dominated the marrow. Conclusion By reconstructing patient-specific mutational hierarchies we gained invaluable insights into the dynamic clonal composition and the molecular progression of human MDS. Our study shows that acquisition of large scale cytogenetic lesions appears to be a rather late event, which likely indicates that such lesions might not be tolerated by healthy stem cells. Most importantly, WES and subsequent deep sequencing analysis of FU samples demonstrated that patient-specific clonal diversity is highly dynamic and modulated during the course of the disease. This is especially true upon response to treatment, where concomitant disappearance and emergence of new pathogenic subclones was observed. Our work unravels two major paths of mutational acquisition by which MDS cells evade therapeutic pressure: (1) linear evolution of the previously sensitive subclone or (2) by branching evolution of an earlier independent founder clone. Our findings therefore strongly emphasize the importance of a genetically unbiased disease monitoring and the development of novel therapeutic strategies aiming at targeting founder lesions that are present in all ensuing subclones. Disclosures Nolte: Celgene Corp., Novartis Pharma: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: Background: In the current ELN recommendations (Baccarani et al., Blood 2013) the optimal time point to achieve major molecular remission (MMR) is defined at 12 months after diagnosis of CML. MMR is not a failure criterion at any time point leading to uncertainties when to change therapy in CML patients not reaching MMR after 12 months. Aims: We sought to evaluate a failure time point for MMR using data of the CML-Study IV, a randomized five-arm trial designed to optimize imatinib therapy alone or in combination. In addition the optimal time-point to achieve a MMR should be evaluated. Methods: Patients with valid molecular analysis on MR4 level were divided randomly into a learning (LS) and a validation sample (VS). For the LS, MR2 (defined as BCR-ABL

Description: Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by abnormal accumulation of mast cells in various tissues, predominantly skin, bone marrow and visceral organs. The morphologic phenotype and extent of organ infiltration/dysfunction are basis for the subclassification of SM into indolent SM (ISM), smoldering SM (SSM), SM with associated hematologic non-mast cell disease (SM-AHNMD), aggressive SM (ASM) and mast cell leukemia (MCL). A somatic point mutation in the kinase domain of the receptor tyrosine kinase (TK) KIT at position 816 (KIT D816V) is present in 〉95% of patients and plays a central role in the pathogenesis and diagnosis of SM. To further explore mechanisms contributing to the clinical diversity of SM, we analyzed 39 KIT D816V mutated patients with different SM subtypes [ISM, n=10; SSM, n=2; ISM-AHNMD, n=5 (CMML, n=2; MDS/MPNu, n=3); ASM, n=1; ASM-AHNMD, n=14 (CMML, n= 5, MDS/MPNu, n=4, HES/CEL, n=2; AML, n=3); MCL, n=3; MCL-AHNMD n=4 (MDS/MPNu, n=2; HES/CEL, n=1; MDS, n=1)] for the presence of additional mutations. We applied next-generation sequencing to investigate ASXL1, CBL, IDH1/2, JAK2, KRAS, MLL-PTD, NPM1, NRAS, TP53, SRSF2, SF3B1, SETBP1, U2AF1 at mutational hotspot regions, and analyzed the complete coding regions of EZH2, ETV6, RUNX1, and TET2. Additional molecular aberrations were identified in 24/27 (89%) patients with advanced SM (SM-AHNMD, 5/5; ASM/MCL, 19/23) while only 3/12 (25%) ISM/SSM patients carried one additional mutation each (U2AF1, SETBP1, CBL) (p