Bone marrow cytomorphology and histopathology are the cornerstones for the initial diagnosis of myelodysplastic syndromes (MDS) and other related myeloid disorders. They provide a rapid first insight into diagnostic categories and thus help in clinical decision making. However, difficulties in the morphologic assessment of MDS exist due to inter- and intra-observer variability. In this study, we directly compared the results of cytomorphology and histopathology obtained in a real-world diagnostic scenario in 90 patients with myeloid malignancies aiming to evaluate their validity for diagnosing and classifying various myeloid malignancies. While both techniques placed 80% of our bone marrow samples into the same diagnostic category and thus showed a good correlation, our study also demonstrates the limitations in correlating marrow cytomorphology and histopathology, even following stringent and repetitive diagnostic assessments. This was particularly true for CMML, where not only additional diagnostic tools such as molecular genetics or clinical evaluation but also the analysis of the peripheral blood smears aided in finding the correct diagnosis. Overall, our data emphasize the need for a comprehensive diagnostic review in a patient-for-patient setting when a myeloid malignancy is suspected or confirmed. We propose that the combination of cytomorphologic and histopathologic assessment with clinical, laboratory, and genetic parameters is essential in achieving high diagnostic accuracy in an interdisciplinary setting.

MDS is a heterogeneous group of myeloid neoplasms caused by genetic and epigenetic alterations. During the past decade, the major driver mutations in MDS have been fully investigated. However, the role of epigenetic alterations, particularly those of DNA methylation, has less intensively been studied, even though abnormal DNA methylation has long been implicated in the pathogenesis of MDS. In this study, we analyzed DNA methylation status of bone marrow mononuclear cells from 320 cases with MDS-SLD (n = 7), MDS-RS (n = 63), MDS-MLD (n = 51), MDS-EB (n = 186), MDS-U (n = 1), and MDS with isolated del(5q) (n = 12), using Illumina 450K methylation array. Mutations in major driver genes (51 genes) and abnormal genomic copy numbers were also interrogated using targeted-capture sequencing. Using unsupervised consensus clustering, we identified 3 subgroups showing unique DNA methylation profiles. Subsequently, we assessed differentially methylated positions (DMPs) associated with each subgroup. Differentially hypermethylated positions (hyper-DMPs) were significantly more enriched in Group 3 (n = 82) (P < 0.001), while differentially hypomethylated positions (hypo-DMPs) were more prominent in Group 1 (n = 125). Group 1 was significantly enriched for SF3B1 (46%) mutations (q < 0.01), while Group 2 (n = 131) was characterized by the enrichment of ASXL1 (38%), RUNX1 (30%), TP53 (26%), STAG2 (15%), and SETBP1 (6.7%) mutations (q < 0.01). In contrast, Group 3 (n = 64) was significantly enriched for TET2 (67%) and IDH1/2 (12% and 15%, respectively) mutations (q < 0.01), suggesting a strong association between DNA methylation and gene mutations. To further elucidate mutation-specific DNA methylation patterns, supervised analysis was performed for each mutation. As expected from their enrichment in Group 3 (q < 0.01), TET2 and IDH1/2 mutations were significantly associated with hyper-DMPs (P < 0.001) involving 1891 and 8330 promotor sites, respectively. Conspicuously, among these hypermethylated promoter sites, >1616 were commonly hypermethylated, strongly supporting the common impact of TET2 and IDH1/2 mutations on deregulated DNA methylation. To clarify prognostic impact of abnormal DNA methylation, we first interrogated the correlation between unique methylation subgroups and revised IPSS. Patients with very low or low risk were significantly dominant (74%) in Group 1 (q < 0.01), and very high or high risk cases were significantly enriched (68%) in Group 2 (q < 0.01). In accordance with this finding, patients in Group 3 showed significantly shorter overall survival (OS) compared to Group 1 (HR: 1.94, 95%CI: 1.11-3.4, P < 0.05) and OS was even worse in Group 2 patients (vs. Group 1: HR: 5.18, 95%CI: 3.21-8.36, P < 0.001). Strong correlations between epigenetic and genetic profiles were further interrogated using a Bayesian statistical model; on the basis of DNA methylation and gene mutations, the original 3 clusters were re-classified into 5 discrete clusters, clusters A, B, C, D, and E (n = 124, 17, 74, 46, and 59, respectively); patients in Group 1 and 3 largely clustered into Cluster A and E, respectively, while Group 2 was further subclassified into clusters B, C, and D. Clusters B and D were characterized by a conspicuos enrichment of DNMT3A (88%) and TP53 (69%) mutations (q < 0.001), while Cluster C was characterized by higher frequency of ASXL1 (71%), RUNX1 (54%), STAG2 (27%), and EZH2 (21%) mutations (q < 0.001). In contrast to significant associations between epigenetic regulators and unique methylation clusters, splice factor mutations tended to be clustered into multiple clusters, depending on type of co-occurring mutations. For example, combined SF3B1 and TET2 mutations (n = 20) were enriched in Cluster A, where highly associated with MDS-RS, while patients with SF3B1 and RUNX1 mutations (n = 9) were more grouped in Cluster C, mostly showing MDS-EB phenotype (89%). Similarly SRSF2 mutations with RUNX1 and/or ASXL1 mutations (n = 36) were enriched in Cluster C, largely associated with MDS-EB phenotype (80%), while those with TET2 or IDH1/2 (n = 39) were mainly grouped into Cluster C, many of which showed MDS-EB phenotype (74%). These findings highlight differential roles of mutated epigenetic regulators and splicing factors in abnormal DNA methylation. In conclusion, we elucidated the collaborative impact of DNA methylation profiles and mutation status on heterogeneous pathogenesis and prognosis in MDS. Figure. Figure. Nadarajah: MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract In this study, we performed whole-exome and targeted sequencing on 85 MLL-PTD AML patients. These AMLs have oncogenic tandem duplication of the MLL gene. At least one well-known oncogenic driver mutation was identified in over 90% of the MLL-PTD patients. In line with earlier sequencing studies of other AML subtypes and the TCGA-AML-sequencing project, DNMT3A was the most often mutated epigenetic regulator (25%); IDH1/2 hotspot mutations were identified in 31% of patients. TET family was the third most prominently mutated epigenetic regulator (TET1 (5%), TET2 (16.3%). Mutations of epigenetic regulators also occurred in polycomb-associated proteins (EZH2, ASXL family members), chromatin remodelers (ARID2, ARID1A), genes associated with histone acetylation (CREBBP, EP300, KAT6A, KAT6B) and histone methylation (MLL2, MLL3). Proliferation-related pathway was extensively mutated, with 54 of 80 MLL-PTD patients (67.5%) carrying at least one mutation of proliferative genes. Specifically, FLT3 mutations were found in 46% of patient samples. Notably, some FLT3-ITD patients had more than one type of internal tandem duplication (ITD) insertion, probably reflecting existence of multiple subclones in these leukemias. We found highly prevalent mutations of cohesin genes: STAG2 (16%), SMC1A (6%), SMC3 (1%), RAD21 (1%) and CTCF (6%). Cohesin pathway is more frequently mutated in MLL-PTD patients (26%) than the AML samples from either TCGA (13%) or a meta-analysis of 1000 AML (9.1%). Remarkably, an extremely high proportion of the mutations had a strong tendency to disrupt the coding sequence in STAG2, emphasizing their crucial tumor-suppressor role in this AML subtype (16% in MLL-PTD vs 3% in TCGA-AML. RNA processing pathway was also strikingly altered in MLL-PTD patients. The most prominently mutated genes within this category were the splicing factors. They included U2AF1 (13%, S34F/Y), SRSF2 (3%), SF3A1 (5%), ZRSR2 (3%), DHX15 (1%) and CWC22 (1%). Multiple mutations co-occur with MLL-PTD which are usually acquired in a sequential manner. A potential ordering for acquisition of many mutations include IDH2/DNMT3A/U2AF1/TET2→MLL-PTD→RAS-receptor tyrosine kinase based on the following reasons: #1, real-time-PCR showed that MLL-PTD was absent in remission while mutations of IDH2, DNMT3A, TET2 and U2AF1 were still retained with a high VAF. This suggests that MLL-PTD was acquired after mutations of IDH2, DNMT3A, TET2 and U2AF1; #2, MLL-PTD is highly stable during disease progression as compared with mutations of the RAS-RTK. On the other hand, RAS-RTK mutations frequently exist as subclonal mutations and tend to be unstable during disease progression. These observations support a notion that MLL-PTD was acquired prior to RAS-RTK. Taken together, MLL-PTD is acquired after those remission-persisting, initiating mutations (IDH2, DNMT3A, TET2 and U2AF1), but prior to lesions of the proliferation-related drivers. Citation Format: Lingwen Ding, Qiaoyang Sun, Kar-Tong Tan, Wenwen Chien, Anand Mayakonda, Dechen Lin, Xinyi Loh, Jinfen Xiao, Manja Meggendorfer, Tamara Alpermann, Manoj Garg, Su-Lin Lim, Vikas Madan, Norimichi Hattori, Yasunobu Nagata, Satoru Miyano, Allen Yeoh Eng Juh, Hsin-An Hou, Yan-Yi Jiang, Yan-Yi Jiang, Sumiko Takao, Li-Zhen Liu, Siew-Zhuan Tan, Siew-Zhuan Tan, Michael Lill, Mutsumi Hayashi, Akitoshi Kinoshita, Hagop M. Kantarjian, Steven M. Kornblau, Seishi Ogawa, Torsten Haferlach, Henry Yang, H. Phillip Koeffler. Mutational profiling of MLL-PTD acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2450. doi:10.1158/1538-7445.AM2017-2450

BCOR is a component of a variant Polycomb group repressive complex 1 (PRC1). Recently, we and others reported recurrent somatic BCOR loss-of-function mutations in myelodysplastic syndrome and acute myelogenous leukemia (AML). However, the role of BCOR in normal hematopoiesis is largely unknown. Here, we explored the function of BCOR in myeloid cells using myeloid murine models with Bcor conditional loss-of-function or overexpression alleles. Bcor mutant bone marrow cells showed significantly higher proliferation and differentiation rates with upregulated expression of Hox genes. Mutation of Bcor reduced protein levels of RING1B, an H2A ubiquitin ligase subunit of PRC1 family complexes and reduced H2AK119ub upstream of upregulated HoxA genes. Global RNA expression profiling in murine cells and AML patient samples with BCOR loss-of-function mutation suggested that loss of BCOR expression is associated with enhanced cell proliferation and myeloid differentiation. Our results strongly suggest that BCOR plays an indispensable role in hematopoiesis by inhibiting myeloid cell proliferation and differentiation and offer a mechanistic explanation for how BCOR regulates gene expression such as Hox genes.

Partial tandem duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated, whereas NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS), typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.

Moreover, forced expression of Egr2 reversed the inhibitory effect of stabilized β-catenin on monocyte/macrophage differentiation of primary BM cells (Figure 2h).Together, stabilized β-catenin inhibits Egr2 expression in mouse primary BM cells, PUER and human U937 cells whereas forced expression of Egr2 released the blockage of monocyte-macrophage differentiation induced by stabilized β-catenin in these cells, suggesting an important role of Egr2 in mediating the negative effect of β-catenin overexpression in monocyte-macrophage differentiation.β-Catenin overexpression is frequently detected in AML samples and it is associated with an adverse prognosis. 10,11 By analysis of a published set of microarray data from BM cells from 69 AML patients and 18 control healthy individuals, 12 we found that a group of AML patients (15 out of 69) had a markedly increased β-catenin expression as compared to healthy individuals (P = 0.001) and the other AML patients (P = 5.65E -10).Of interest, this group of AML patients with a high β-catenin expression displayed a reduced Egr2 expression in BM cells as compared with healthy individuals (P = 0.0156) and the other AML patients (P = 0.034; Figures 2i andj).These data suggest that an increased expression of β-catenin likely inhibits Egr2 expression in BM cells in AML patients.In summary, we have shown that β-catenin overexpression induced blockage of monocyte-macrophage differentiation by inhibiting PU.1-targeted gene transcription including Egr2 expression in myeloid progenitor cells.A recent study showed that minimal PU.1 reduction induces myeloid-biased preleukemic stem cells and promotes subsequent transformation to AML in the context of Msh2 deficiency. 13 Therefore, compromised PU.1-targeted gene transcription induced by β-catenin overexpression, at least partially, may mediate a pathogenic role of β-catenin in myeloid leukemia.

Differences in prognosis of stereotyped IGHV3-21 chronic lymphocytic leukaemia according to additional molecular and cytogenetic aberrations

BACKGROUNDTo obtain better insight into the biology of acute myeloid leukemia (AML) in various age groups, this study focused on the genetic changes occurring during a lifetime.METHODSThis study analyzed the relation between age and genetics from birth to 100 years in 5564 patients with de novo AML diagnosed from 1998 to 2012 (1192 patients from nationwide pediatric studies [AML Berlin‐Frankfurt‐Münster studies 98 and 2004] and 4372 adults registered with the Munich Leukemia Laboratory).RESULTSThe frequencies of cytogenetic subgroups were age‐dependent. Favorable subtypes (t(8;21), inv(16)/t(16;16), and t(15;17)) decreased in general from the pediatric age group (2 to < 18 years; 33%) to the oldest groups (<5% for > 70 years; P < .0001). Unfavorable cytogenetics (–7/del(7), –5/del(5q) or 5p, inv(3)/t(3;3), t(6;9), complex karyotype, 12p, 17p, and 11q23/mixed‐lineage leukemia aberrations, excluding t(9;11)) were frequent (42%) in infants (<2 years), had a low frequency in children and young adults (<22%), and increased in frequency up to 36% in patients older than 85 years (P = .01). This was even more significant for complex karyotypes (P ≤ .0001), which also showed a strong increase in the absolute age‐specific incidence with age. Interestingly, the frequency of 11q23 abnormalities decreased from infants to older patients. The proportion of clinically relevant molecular aberrations of CCAAT/enhancer binding protein α, nucleophosmin (NPM1), and NPM1/fms‐related tyrosine kinase 3–internal tandem duplication increased with age.CONCLUSIONSAltogether, with the exclusion of infants, a significant decrease in the proportion of favorable cytogenetic subtypes and an increase in unfavorable cytogenetics were observed with increasing age. These findings indicate different mechanisms for the pathogenesis of AML; these different mechanisms also suggest directions for etiological research and contribute to the more unfavorable prognosis with increasing age. Cancer 2016;122:3821–3830. © 2016 American Cancer Society.

Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by differentiation block at the promyelocyte stage. Besides the presence of chromosomal rearrangement t(15;17), leading to the formation of PML-RARA (promyelocytic leukemia-retinoic acid receptor alpha) fusion, other genetic alterations have also been implicated in APL. Here, we performed comprehensive mutational analysis of primary and relapse APL to identify somatic alterations, which cooperate with PML-RARA in the pathogenesis of APL. We explored the mutational landscape using whole-exome ( n =12) and subsequent targeted sequencing of 398 genes in 153 primary and 69 relapse APL. Both primary and relapse APL harbored an average of eight non-silent somatic mutations per exome. We observed recurrent alterations of FLT3 , WT1 , NRAS and KRAS in the newly diagnosed APL, whereas mutations in other genes commonly mutated in myeloid leukemia were rarely detected. The molecular signature of APL relapse was characterized by emergence of frequent mutations in PML and RARA genes. Our sequencing data also demonstrates incidence of loss-of-function mutations in previously unidentified genes, ARID1B and ARID1A , both of which encode for key components of the SWI/SNF complex. We show that knockdown of ARID1B in APL cell line, NB4, results in large-scale activation of gene expression and reduced in vitro differentiation potential.

High BAALC gene expression has been associated with poor prognosis in cytogenetically normal acute myeloid leukaemia (CN-AML) and has been suggested as a suitable marker for assessing minimal residual disease (MRD). The purpose of this study was to substantiate these findings by the analysis of a large data set of 632 diagnostic and follow-up samples in 142 intensively treated CN-AML patients. Paired diagnostic/relapse samples of 35 patients revealed stable high BAALC expression in 89%, irrespective of a high proportion of clonal evolution found in 49% of these cases. High BAALC expression, both directly after induction chemotherapy and within 3-6 months after induction chemotherapy, correlated significantly with shorter event-free survival and overall survival. Moreover, 8 of 10 patients displaying high BAALC expression levels after completion of induction therapy as well as 5 of 5 patients exhibiting high BAALC expression levels within 3-6 months after induction chemotherapy experienced relapse with a median of 197 and 101 days, respectively, from sampling to relapse. Thus, BAALC expression-based MRD detection during therapy may be considered a strategy to identify patients at high risk of relapse.

Citation: Alpermann T, Schnittger S, Eder C, Dicker F, Meggendorfer M, Kern W, Schmid C, Aul C, Staib P, Wendtner CM, Schmitz N, Haferlach C, and Haferlach T. Molecular subtypes of NPM1 mutations have different clinical profiles, specific patterns of accompanying molecular mutations and varying outcome in intermediate risk acute myeloid leukemia. Haematologica. 2015; 100:xxx doi:10.3324/haematol.2015.133819

Abstract Introduction Ring sideroblasts (RS) are common findings in myeloid malignancies as MDS or MDS/MPN, especially in RARS-T, and are associated with SF3B1 mutations. However, the incidence of both RS and SF3B1 mutations have not yet been assessed properly in acute myeloid leukemia (AML). Aim Determine the frequency of RS and SF3B1 mutations in 1857 patients with de novo and therapy-related AML (t-AML). Define their impact on survival and association with other frequently mutated genes, as well as with cytogenetic abnormalities. Patients and Methods From a total of 1857 AML patients (excluding those with cytogenetically-defined entities according to WHO), bone marrow assessment revealed 473 (25%) with RS ≥ 1% and thereof 183 (10% of all) with ≥15 RS. Sequencing or melting curve analysis were performed in a subcohort of 340/473 patients for the detection of mutations in: SF3B1, ASXL1, DNMT3A, FLT3- TKD, IDH1 R132, IDH2 R140, IDH2 R172, KRAS, NPM1, NRAS, RUNX1, TET2, FLT3 -ITD and MLL -PTD. These 340 cases were subject to the study. Out of these 340, 141 cases (42%) had RS ≥ 15% and the remaining 199/340 (59%) had RS ≥1 to <15%. The cohort consisted of 303 (89%) de novo AML (FAB: M0 n=18, M1 n=67, M2 n=165, M4 n=30, M5 n=3, M6 n=20) and 37 (11%) t-AML. 148 were female and 192 were male, with median age 74 years, range: 20-93 years. Chromosome banding analysis (assisted by FISH if needed) was performed in all 340 cases. Results The percentage of bone marrow blasts correlated inversely with the percentage of RS present (r 0.213, p<0.001). 136 (40%) patients had a normal karyotype. Intermediate cytogenetics according to MRC criteria were found in 193 (57%), and adverse in 147 (43%). Patients with RS ≥15% more frequently had adverse cytogenetics in comparison to those with RS between 1-14% (54% vs 36%, p=0.001). The frequencies of gene mutations were as follows: TP53 103/331 (31%), RUNX1 84/315 (26%), DNMT3A 86/337 (25%), TET2 68/330 (20%), ASXL1 58/334 (17%), IDH2 R140 53/338 (15%), NPM1 43/340 (12%), SF3B1 34/334 (10%), FLT3 -ITD 33/340 (10%), NRAS 29/340 (8%), IDH1 R132 21/339 (6%), MLL -PTD 22/337 (6%), FLT3 -TKD 18/333 (5%), KRAS codon 12 13/299 (4%), IDH2 R172 13/338 (3%) and KRAS codon 61 3/299 (1%). Moreover, in total 30 variants in 28 patients were identified in DNMT3A, RUNX1, TET2 and TP53, which according to current knowledge cannot be assigned to mutations or SNPs yet. Patients with ≥15% more frequently had TP53 mutations (mut) (44% vs 22%, p<0.001) and less frequently IDH2 R140 mut (11% vs 19%, p=0.094) and MLL -PTD (2% vs 6%, p=0.006). Accordingly, patients with TP53 mutations had higher percentages of RS as compared to those without (28% vs 16%, p<0.001) and patients with IDH2 R140 mut and MLL -PTD, respectively, had lower percentages of RS as compared to those without (15% vs 21%, p=0.043 and 11% vs 21%, p=0.025, respectively). Furthermore, patients with mutations in the following genes had fewer RS than patients with the respective wild-types: ASXL1 (15% vs 21%, p=0.040), FLT3 -ITD (14% vs 21%, p= 0.049), IDH2 R140 (15% vs 21%, p=0.043), MLL -PTD (11% vs 21%, p=0.025), NPM1 (13% vs 21%, p=0.018) and KRAS codon 61 (3% vs 20%, p<0.001). Conversely, patients with mutated SF3B1 had more RS than patients with wild type (27% vs 19%, p=0.054). However, the number of RS did not translate into an increase in the mutational burden of SF3B1. Given the limited degree of overlap between mutations of the four most frequently mutated genes, we hierarchically separated the patients into 5 groups: TP53 mut, ASXL1 mut, NPM1 mut, SF3B1 mut and patients without any of these mutations. Interestingly, the percentage of RS was very similar in the two groups, TP53 mut and SF3B1 mut, and significantly higher as compared to all other groups (TP53 mut/SF3B 1mut: 28% vs ASXL1 mut/NPM1 mut/others 14%, p<0.001). The number of RS did not have an impact on the overall survival (OS) and event free survival (EFS) of patients. Conclusion 1. Ring sideroblasts ≥ 15% are present in 10% of de novo and t-AML. 2. The blast count correlates inversely with the number of RS. 3. Patients with ≥ 15% RS more frequently carry TP53 mutations and adverse cytogenetics. 4. Although patients with ≥ 15% RS have worse molecular (TP53 mut) and cytogenetic features, there is no statistically significant impact on survival when compared to patients with <15% RS. 5. Patients with mutated ASXL1, FLT3 -ITD, IDH2 R140, MLL -PTD and NPM1 have less RS than wild type patients while those with TP53 or SF3B1 mutations have higher RS. Figure 1. Figure 1. Disclosures Martin-Cabrera: MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract Background: MECOM-rearrangements (formerly named EVI1 -rearrangements) are known to be prognostically adverse. Within the WHO classification of 2008, only inv(3)(q21q26.2)/t(3;3)(q21;q26.2)/RPN1-EVI1 were classified as separate entity ("classic"), whereas all other MECOM-rearrangements ("other") were not. In addition, patients (pts) have to be assigned to MDS or AML according to percentage of blasts. As progression from MDS to AML is a continuous process this arbitrary border can be questioned. Aim: To evaluate 1) whether MECOM-rearranged pts, regardless of blast count, show a "secondary-type" mutational profile and whether this differs between different types of MECOM-rearrangements. 2) the prognostic impact of the biological and genetic parameters. Patients and Methods: We here focus on a cohort of 116 newly diagnosed adult pts with MECOM-rearrangements all investigated by cytogenetics, fluorescence in situ hybridization and cytomorphology. In addition, all pts were analyzed for molecular mutations by next generation sequencing using a 18-gene panel including genes frequently mutated in MDS and AML encompassing also those defined by Lindsley et al. as "secondary-type" mutations (mut) in AML, which are highly specific for secondary AML (Blood 2015). Data was available for: ASXL1 (n=104), BCOR (n=101), CBL (n=112), DNMT3A (n=114), EZH2 (n=111), FLT3 -TKD (n=115), IDH1 (n=116), IKZF1 (n=106), KIT (n=115), NRAS (n=113), PTPN11 (n=111), RUNX1 (n=109), SF3B1 (n=113), SRSF2 (n=113), TP53 (n=116), U2AF1 (n=116), WT1 (n=116), and ZRSR2 (n=113). Variants of unknown significance were excluded from statistical analyses (n=16). Results: In our cohort of 116 pts (median age: 66 years, range 21-93) 38 (33%) pts harbored <20% bone marrow blast and therefore were classified as MDS and 78 (67%) ≥20% and therefore were AML. According to WHO classification, 45 (39%) carried "classic" and 71 (61%) "other" MECOM-rearrangements. Most common was inv(3)(q21q26) (n=35; 30%), followed by t(3;21)(q26;q22) (13; 11%), t(3;8)(q26;q24) (12; 10%) and t(2;3)(p14~p21;q26) (11; 10%), t(3;3)(q21;q26) (10; 9%), inv(3)(p24q26) (9; 8%), and t(3;21)(q26;q11) (7; 6%). All others were present in less than 5 cases. We found mut in SF3B1 (27%), NRAS (22%), ASXL1 (18%). PTPN11 and SRSF2 (17% each), RUNX1 (15%), DNMT3A (11%), and TP53 (10%). Mutations in the remaining genes were present in less than 10%. In total, 60% of the pts showed at least one of the "secondary-type" mut. Only nine pts (8%) carried no mut. MDS pts harbored more frequently SF3B1 mut than AML pts (45% vs 19%, p=0.007). Overall, "secondary-type" mut were much more frequent in MDS than in AML (82% vs 49%; p=0.001). No differences with respect to clinical parameters or survival were seen. Separating the cohort into "classic" and "other" rearrangements we found "classic" harboring more often SF3B1 mut (40% vs 19%, p=0.019) and PTPN11 mut (28 vs 11%; p=0.037) and less frequent SRSF2 mut (7% vs 23%, p=0.037) compared to "other" pts. No other differences were seen. However, diverse mutation patterns were detected between the distinct rearrangements (Figure 1). Pts with t(3;8) carried 60% CBL and 25% TP53 mut, whereas t(3;3), t(2;3), and t(3;12) yielded none of these mut. Furthermore, t(3;21)(q26;q22) harbored 54% SRSF2 and 39% ASXL1 mut, whereas inv(3)(q21q26) carried only 6 and 7%, respectively, and t(3;21)(q26;q11) carried none of these. Moreover, PTPN11 mut was present in 30% of inv(3)(q21q26) but not in t(3;8), t(2;3), t(3;6), and t(3;12). Further, IDH1 mut were carried by 50% of t(3;21)(q26;q22) and inv(3)(p24q26), but in none of the other pts. An extremely poor prognosis was observed in patients with NRAS mut and TP53 mut compared to pts without these mut (8 vs 23 months; p=0.002, 6 vs 16 months, p<0.001, respectively). Of note, the cumulative number of mutations had a negative impact on survival: pts with 0-1 vs. ≥2 mut, median OS 41 versus 9 months, p=0.001. Conclusions 1. A threshold of 20% bone marrow blasts does not separate patients with MECOM-rearrangements according to clinical or genetic parameters or outcome. 2. "Classic" MECOM-rearrangements do not differ from "other" ones according to clinical parameters or outcome. 3. However, "classic" and "other" MECOM-rearrangements show different profiles of additional mutations. 4. Furthermore, additional molecular markers lead to better separation of MECOM-rearranged patients with regard to outcome. Figure 1. Figure 1. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Fasan:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

In acute myeloid leukemia (AML), acquired genomic gains and losses are common and lead to altered expression of genes located within or nearby the affected regions. Increased expression of the ETS‐related transcription factor gene ERG has been described in myeloid malignancies with chromosomal rearrangements involving chromosome band 21q22, but also in cytogenetically normal AML, where it is associated with adverse prognosis. In this study, fluorescence in situ hybridization on interphase nuclei disclosed an amplification of the ERG gene (more than six copies) in 33 AML patients with structural rearrangements of 21q22. Array comparative genomic hybridization of these cases disclosed a minimal amplified region at the position 39.6–40.0 Mbp from pter that harbors ERG as the only gene. Analysis by quantitative real‐time reverse transcription polymerase chain reaction revealed significantly higher ERG mRNA expression in these patients and in a group of 95 AML patients with complete or partial gain of chromosome 21 (three to six copies) compared with 351 AML patients without gain of chromosome 21. Quantification of ERG DNA copy numbers revealed a strong correlation with ERG mRNA expression. Furthermore, in patients with gain of chromosome 21, higher ERG expression was found to be associated with RUNX1 mutations. Our results suggest that acquired gain of chromosome 21 or amplification of chromosome arm 21q is one mechanism contributing to increased ERG expression in AML. © 2015 Wiley Periodicals, Inc.

Next generation sequencing technologies have provided insights into the molecular heterogeneity of various myeloid neoplasms, revealing previously unknown somatic genetic events. In our cohort of 1444 cases analyzed by next generation sequencing, somatic mutations in the gene BRCA1-BRCA2-containing complex 3 (BRCC3) were identified in 28 cases (1.9%). BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases capable of cleaving Lys-63 linked polyubiquitin chains, and is implicated in DNA repair. The mutations were located throughout its coding region. The average variant allelic frequency of BRCC3 mutations was 30.1%, and by a serial sample analysis at two different time points a BRCC3 mutation was already identified in the initial stage of a myelodysplastic syndrome. BRCC3 mutations commonly occurred in nonsense (n=12), frameshift (n=4), and splice site (n=5) configurations. Due to the marginal male dominance (odds ratio; 2.00, 0.84-4.73) of BRCC3 mutations, the majority of mutations (n=23; 82%) were hemizygous. Phenotypically, BRCC3 mutations were frequently observed in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms and associated with -Y abnormality (odds ratio; 3.70, 1.25-11.0). Clinically, BRCC3 mutations were also related to higher age (P=0.01), although prognosis was not affected. Knockdown of Brcc3 gene expression in murine bone marrow lineage negative, Sca1 positive, c-kit positive cells resulted in 2-fold more colony formation and modest differentiation defect. Thus, BRCC3 likely plays a role as tumor-associated gene in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.

Karyotype evolution and acquisition of FLT3 or RAS pathway alterations drive progression of myelodysplastic syndrome to acute myeloid leukemiaMyelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms associated with aberrant myeloid differentiation and ineffective hematopoiesis leading to cytopenias.In more than 20% of affected patients the MDS transforms into secondary acute myeloid leukemia (s-AML).To date, information on the molecular and cytogenetic bases of leukemic transformation into AML are rare and there is a compelling need to identify the specific molecular events potentially driving this process.We, therefore, analyzed a cohort of 38 patients with paired samples from when they had MDS and s-AML and applied cytogenetics and a 33-gene panmyeloid panel for comparison of mutation frequencies and patterns within these two groups of samples.We further compared these findings with those in a large cohort of 494 MDS patients, showing no transformation to s-AML.We identified that mutations in genes of cellular signal transduction drive leukemic transformation, and found that several MDS typical mutations predispose to s-AML transformation.In addition, karyotype evolution was detected in more than one third of patients underlining its important impact on s-AML transformation.We identified 38 patients (11 female, 27 male) who were analyzed in our laboratory by cytomorphology and cytogenetics both at diagnosis of MDS and later at progression to s-AML.The median age at diagnosis of MDS was 71 years (range, 59-86 years).The median time to progression was 18 months (range, 2-72 months).The diagnosis was made according to World Health Organization (WHO) criteria. 1Chromosome banding analysis was performed for all patients according to standard procedures in combination with fluorescence in situ hybridization, if needed.All 76 samples were analyzed by next-generation sequencing or polymerase chain reaction with a 33-gene panel targeting ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (FLT3-ITD and FLT3-TDK), GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, MPL, NPM1, NRAS, PHF6, RAD21, RUNX1, SETBP1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1, WT1, and ZRSR2.Further details on the characterization of the patients and the study methodology are provided in the Online Supplementary Material.Cytogenetically, a total of 14/38 (37%) MDS and 22/38 (58%) s-AML samples had an aberrant karyotype and 13 patients (34%) gained chromosome abnormalities during progression to s-AML.One of these patients showed in parallel clonal evolution in one cytogenetic clone but lost another cytogenetic clone completely.These findings again confirm that cytogenetic evolution in MDS has been associated with progression to AML. 2,3 The most