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Acute myeloid leukemia (AML) is a heterogeneous disease with a great variety of somatic driver mutations. Each AML carries specific (combinations of) acquired mutations, which enables minimal residual disease (MRD) detection in virtually every patient with next-generation sequencing (NGS). In recent years there has been an increasing interest to conduct longitudinal molecular MRD detection to better estimate prognosis, monitor treatment efficacy, as well as prediction of an impending relapse. The development of molecular monitoring on archived samples has been slow because only limited numbers of samples collected during the course of the disease were biobanked. We have systematically investigated whether archived May-Giemsa Grünwald (MGG)-stained bone marrow slides could be an alternative source of material to conduct NGS-based molecular MRD detection in AML. Since MGG-stained bone marrow evaluations are performed at high frequency to evaluate the response on therapy and repopulation of the bone marrow, we focused on these bone marrow slides taken at regular intervals during treatment. We initial tested DNA isolation protocol on MGG-stained and unstained morphology slides, which were archived over the years. The DNA could be isolated from the MGG-stained (n=16) and unstained bone marrow slides (n=16). However, we were unable to isolate genomic DNA from all of the unstained slides (11 out of 16). Furthermore, the DNA concentration from the unstained slides was much lower than the MGG-stained slides. We next isolated DNA from MGG-stained bone marrow slides of 43 AML samples at diagnosis and relapse of which the mutation status of genes frequently mutated in myeloid diseases was known. DNA isolated from Ficoll-purified mononuclear cells of the same pairs was previously sequenced by NGS using the Illumina Trusight Myeloid panel. NGS-based mutation screening with the same panel on DNA obtained from morphology slides revealed that detection of the driver mutations was feasible. In fact, all known mutations were detectable at diagnosis and relapse. Interestingly, in 91% (39 out of 43) of cases, the variant allele frequency (VAF) of the mutations were virtually identical to those detected using the DNA from MNCs. Representative examples are shown in Figure 1 (A and B). In a minority of samples, the VAFs differed between MNCs and bone marrow slides (3 cases VAF higher in MNCs and 1 case VAF lower in MNCs). These findings indicate that DNA isolated from bone marrow slides are a rich source for NGS-based mutation detection in AML at diagnosis and relapse. We next sought to evaluate the use of MGG-stained bone marrow slides to study the mutations kinetics in time from AML diagnosis to relapse. To perform this analysis 30 paired-AML (diagnosis - relapse) with variable time to relapse (range: 200 to 4004 days) were selected. We collected the archived bone marrow slides at different time points between diagnosis and relapse, isolated DNA and performed NGS-based mutation detection. The NGS analysis of the 30 paired-AML cases showed different patterns of mutation dynamics and clonal evolution. Representative examples are shown in Figure 1 (C and D). Interestingly, by this systematic analyses of somatic mutations at various time points clear distinction could be made between leukemia-specific mutations and mutations associated with clonal hematopoiesis at the time of complete remission in the individual AML cases. Our study uncovers an unutilized source of material for mutation detection at diagnosis and relapse in AML. These materials are widely available and well archived. Furthermore, our study reveals the potential use of MGG stained bone marrow morphology slides to track mutations and (sub)clones in AML, which is applicable to virtually every AML patient and possibly valuable for other hematological malignancies as well. Disclosures No relevant conflicts of interest to declare.

T-cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small- to medium-sized prolymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, and no clear T-cell receptor a or β CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of microRNA (miRNA) is aberrant and often heterogeneous in T-PLL. We identified 35 miRNA that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR- 200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFβR3 and aberrant TGFβ1- induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFβ pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNA in T-PLL and pave the way for new therapeutic targets in this disease.

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are heterogeneous malignancies characterized by a variety of acquired genetic abnormalities and variable response to treatment.[1][1],[2][2] In the last decade, a number of novel molecular genetic abnormalities have been revealed in MDS

Seventy-seven patients were identified with Rare recurring (excluding 11q23, 21q22, inv(16), and t(15; 17)) chromosome abnormalities among 511 patients with treatment-related myelodysplastic syndromes and acute leukemia accepted from centers in the United States, Europe, and Japan. The abnormality subsets included 3q21q26 (17 patients), 11p15 (17 patients), t(9;22)(q34;q 11) (10 patients), 12p13 (9 patients), t(8; I 6)(p11;p13) (9 patients), and an "other" subset, which included t(6;9)(p23;q34) (3 patients), t(10;11)(p13;q13similar toq21) (3 patients), t(1;17)(p36;q21) (2 patients), t(8; 14)(q24;q32) (2 patients), t(11;19)(q13;q13) (2 patients), t(1;3)(p36;q21) (2 patients), and t(3;5)(q21;q31) (1 patient). Increased karyotypic complexity with additional balanced and unbalanced rearrangements was observed in 70% of cases. Among 54 cases with secondary abnormalities, chromosome 5 and/or 7 abnormalities were observed in 59%. The most frequent primary diseases were breast cancer (24 cases), Hodgkin disease (14 cases), non-Hodgkin lymphoma (10 cases), and De novo ALL (5 cases). Thirty-seven patients received alkylating agents plus topoisomerase 11 inhibitors with or without radiation therapy, The presenting diagnosis was t-AML in 47 cases, t-MDS in 23 cases (10 progressed to t-AML), and t-ALL in seven cases, five of whom had a t(9;22). The median latency time from initiation of original therapy to therapy-related disease diagnosis was quite long (69 months), and the overall median survival from the date of therapy-related disease diagnosis was very short (7 months). The 1-year survival rate was 34 +/- 7%, with no significant differences among subsets. Comparison with previously reported cases showed increased karyotypic complexity and adult presentation of pediatric-associated chromosome abnormalities. (C) 2002 Wiley-Liss, Inc.

B-cell prolymphocytic leukemia (B-PLL) is a rare mature B-cell malignancy that may be hard to distinguish from mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). B-PLL cases with a t(11;14) were redefined as MCL in the World Health Organization 2008 classification. We evaluated 13 B-PLL patients [7 being t(11;14)-positive (B-PLL+) and 6 negative (B-PLL-)] and compared them with MCL and CLL patients. EuroFlow-based immunophenotyping showed significant overlap between B-PLL+ and B-PLL-, as well as between B-PLL and MCL, whereas CLL clustered separately. Immunogenotyping showed specific IGHV gene usage partly resembling MCL. Gene expression profiling showed no separation between B-PLL+ and B-PLL- but identified 3 subgroups. One B-PLL subgroup clustered close to CLL and another subgroup clustered with leukemic MCL; both were associated with prolonged survival. A third subgroup clustered close to nodal MCL and was associated with short survival. Gene expression profiles of both B-PLL+ and B-PLL- showed best resemblance with normal immunoglobulin M-only B-cells. Our data confirm that B-PLL+ is highly comparable to MCL, indicate that B-PLL- also may be considered as a specific subgroup of MCL, and suggest that B-PLL is part of a spectrum, ranging from CLL-like B-PLL, to leukemic MCL-like B-PLL, to nodal MCL-like B-PLL.

SummaryChromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

Mutations in splice factor (SF) genes occur more frequently in myelodysplastic syndromes (MDS) than in acute myeloid leukemias (AML). We sequenced complementary DNA from bone marrow of 47 refractory anemia with excess blasts (RAEB) patients, 29 AML cases with low marrow blast cell count, and 325 other AML patients and determined the presence of SF-hotspot mutations in SF3B1, U2AF35, and SRSF2. SF mutations were found in 10 RAEB, 12 AML cases with low marrow blast cell count, and 25 other AML cases. Our study provides evidence that SF-mutant RAEB and SF-mutant AML are clinically, cytologically, and molecularly highly similar. An integrated analysis of genomewide messenger RNA (mRNA) expression profiling and DNA-methylation profiling data revealed 2 unique patient clusters highly enriched for SF-mutant RAEB/AML. The combined genomewide mRNA expression profiling/DNA-methylation profiling signatures revealed 1 SF-mutant patient cluster with an erythroid signature. The other SF-mutant patient cluster was enriched for NRAS/KRAS mutations and showed an inferior survival. We conclude that SF-mutant RAEB/AML constitutes a related disorder overriding the artificial separation between AML and MDS, and that SF-mutant RAEB/AML is composed of 2 molecularly and clinically distinct subgroups. We conclude that SF-mutant disorders should be considered as myeloid malignancies that transcend the boundaries of AML and MDS.

Background: C/EBPa is a lineage determining transcription factor, critical for terminal cell differentiation in different tissues including the bone marrow (BM), lung, liver, and adipocytes. Adequate CEBPA levels are needed to maintain the haematopoietic stem cell (HSC) pool and to promote neutrophilic differentiation. This knowledge points towards the importance of CEBPA dosage at different stages of differentiation. Aim: To investigate how CEBPA dosage is regulated at the transcriptional level by specific enhancer(s) and to study their role in haematopoiesis. Results: Chromatin immunoprecipitation for the active mark H3K27ac followed by deep sequencing (ChIP-seq), revealed eight putative regulatory elements within the CEBPA locus. One enhancer showed marked H3K27ac enrichment at +42Kb downstream of CEBPA particularly in CD34+ haematopoietic stem cells (HSCs), implying a role for CEBPA regulation at earlier stages of haematopoiesis. ChIP-Seq experiments revealed binding of RUNX1, ERG, PU.1, FLI1 and GATA2 to this +42Kb enhancer in CD34+ BM cells. Moreover, myeloid cell lines MOLM1 and U937 also showed H3K27ac enrichment at this enhancer, indicating that its activity is maintained upon myeloid differentiation. In contrast, active histone marks were completely devoid at this element in CEBPA expressing lung (A549) and liver (HepG2) cell lines, indicating hematopoietic specificity of this enhancer. Chromatin looping between the +42Kb enhancer and the CEBPA promoter was demonstrated by 4C-Seq, highlighting the specificity of this enhancer in CEBPA regulation. Furthermore, using CRISPR/Cas9 technology we deleted the +42Kb enhancer in the myeloid cell line THP-1 and showed 70% reduction of CEBPA mRNA levels. The +42Kb enhancer is conserved and located at +37Kb near Cebpa in mice. Using CRISPR/Cas9 system, we deleted the +37Kb enhancer in one-cell stage zygotes. Heterozygous mice were inter-crossed and F1 generation mice were born at normal mendelian ratios. Morphological and flow-cytometric analysis of peripheral blood and BM at 8-10 weeks of age, showed 10-20 fold decrease in (MAC1+/GR1+) neutrophil counts in homozygous +37Kb-/- mice, compared to +37Kbwt/wt and +37Kb-/wt controls. In line with the block of neutrophil development, flow-cytometric analysis revealed an increase (2 fold) in CD34+CD16/32low common myeloid progenitors and a decrease (2 fold) in the CD34+CD16/32high granulocyte/ monocyte progenitorsof +37Kb-/- mice. From these findings we hypothesized that the absence of the +37Kb enhancer disturbs the myeloid differentiation program via reduced Cebpa levels. In fact, Cebpa expression levels were reduced by 60-80% in bone marrow of +37Kb-/- mice, but unchanged in other Cebpa -expressing tissues such as lung and liver, indicating tissue specificity of this enhancer. Diminished Cebpa expression levels were accompanied by decreasedexpression of Cebpa target genes, including Csf3r. In line with this, bone marrow progenitor cells from +37Kb-/- mice were completely unresponsive to Csf3 in a colony forming assay. Given the importance of Cebpa in HSCs maintenance, we investigated the HSC population and found that long-term HSCs (CD48- CD150+) and short-term HSCs (CD48- CD150-) were depleted in the bone marrow of the +37Kb-/- mice. HSC depletion was accompanied by an increase in the CD48+/CD150- multipotent progenitors (MPPs). The +37Kb-/- MPPs, unlike controls, were able to serially replate in vitro under IL-3, GM-CSF, IL-6, SCF growth factor conditions with minimal evidence of differentiation, suggesting a leukemogenic potential. Reintroduction of Cebpa cDNA into +37Kb-/- MPPs fully recovered neutrophil development. Conclusion: We conclude that the +37Kb enhancer is tissue-specific and plays a central role in haematopoiesis regulating Cebpa dosage. Our study reveals that the bone marrow maintains its integrity through the activity of the +37Kb enhancer, which (1) prevents HSC exhaustion and (2) preserves neutrophilic development. The in vitro replating capacity of MPPs isolated from +37Kb-/- animals suggests that aberrant control of this enhancer may be a primary leukaemogenic event. In line with this, it is important to note that the conserved enhancer in humans (+42 KB) is a frequent target for oncogenic transcription factors such as AML1-ETO or EVI1, two oncogenes which are found in two distinct subtypes of AMLs with very low C/EBPa expression. Disclosures No relevant conflicts of interest to declare.

An estimated 6% to 7% of the earth's population carries a mutation affecting red blood cell function. The beta-thalassemias and sickle cell disease are the most common monogenic disorders caused by these mutations. Increased levels of gamma-globin ameliorate the severity of these diseases because fetal hemoglobin (HbF; alpha 2 gamma 2) can effectively re-place adult hemoglobin (HbA; alpha 2 beta 2) and counteract polymerization of sickle hemoglobin (HbS; alpha 2 beta(S)2). Therefore, understanding the molecular mechanism of globin switching is of biologic and clinical importance. Here, we show that the recently identified chromatin factor Friend of Prmt1 (FOP) is a critical modulator of gamma-globin gene expression. Knockdown of FOP in adult erythroid progenitors strongly induces HbF. Importantly, gamma-globin expression can be elevated in cells from beta-thalassemic patients by reducing FOP levels. These observations identify FOP as a novel therapeutic target in beta-hemoglobinopathies. (Blood. 2010;116(20):4349-4352)

In the myelodysplastic syndrome (MDS), cytogenetic abnormalities are often present and can be used as markers in studies for cell lineage involvement. Little is known of the involvement of the megakaryocytic lineage due to the variable ploidy of these cells. We applied dual-color fluorescence in situ hybridization (FISH) to routinely prepared bone marrow (BM) smears of cytogenetically normal patients and seven patients with MDS and monosomy 7 or trisomy 8. Probes specific for the centromeric regions of chromosomes 7 and 8 were detected with fluorescein isothiocyanate (FITC) and Texas Red, respectively. This enabled us to assess the ratio between the numbers of chromosomes 7 and 8 in the polyploid cells. We utilized confocal laser scanning microscopy to count the FITC and Texas Red FISH signals in the different focal layers of the megakaryocytes. Fifty-six megakaryocytes in six normal BM smears were analyzed, giving a mean ratio of 1.0, a standard deviation (SD) of 0.12, and a range of 0.8–1.33. This ratio was applied to evaluation of clonal involvement of individual megakaryocytes in the patients with MDS. In two patients with monosomy 7, the majority of the megakaryocytes were monosomic. In the five patients with trisomy 8, all or a majority of the analyzed megakaryocytes were trisomic. These results add direct evidence that in MDS megakaryocytes are involved in the malignant clone. Genes Chromosomes Cancer 25:332–338, 1999. © 1999 Wiley-Liss, Inc.

Patients with myelodysplastic syndrome (MDS) have ineffective in vivo and in vitro erythropoiesis, characterized by an impaired response to erythropoietin (Epo). We examined proliferation and maturation of MDS marrow cells in response to Epo in more detail. Epo-dependent DNA synthesis as well as induction of GATA-1 binding activity in marrow cells from 15 MDS cases were severely reduced as compared with normal bone marrow (NBM). Additionally, the appearance of morphologically identifiable erythroid cells was decreased in MDS cell cultures. These data indicate that both the Epo-dependent proliferation as well as the differentiation induction by Epo is suppressed. To study more upstream events of the Epo signal transduction route we investigated activation of the signal transducer and activator of transcription (STAT) 5. In all 15 MDS samples tested, STAT5 activation was absent or greatly suppressed in response to Epo. In contrast, interleukin-3 induced a normal STAT5 response in MDS cells. Further, in MDS the subset of CD71+ BM cells that is phenotypically similar to Epo-responsive cells in normal marrow, was present. We conclude that the Epo response in MDS is disturbed at an early point in the Epo receptor (EpoR) signal transduction pathway.

Interleukin-1 (Il-1) and Interleukin-6 (Il-6) have been reported to enhance the growth factor dependent colony formation of normal primitive haematopoietic progenitor cells as well as of leukaemic blast-cell progenitors. We investigated the effects of Il-1 beta and Il-6 in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) on the in vitro colony formation of myeloid progenitors from 23 patients with a myelodysplastic syndrome (MDS). Neither Il-1 beta nor Il-6 were found to have colony stimulating activity on their own. In normal bone marrow cultures, either stimulated with optimal or suboptimal doses of GM-CSF, no enhancing or antagonistic effect of Il-6 or Il-1 beta was detected. In a majority of the MDS cases, however, an enhancing effect of Il-6 and Il-1 beta in combination with GM-CSF was observed (20/23 and 10/21 cases respectively). In three cases of the Il-6 and GM-CSF combination an antagonistic effect was observed as well as in four cases of the Il-1 beta and GM-CSF combination. A delayed addition of Il-6 to the cultures did not result in an abrogation of the effect, indicating that Il-6 is not required immediately at the initiation of the culture. These results indicate that costimulation with Il-6 or Il-1 beta is able to augment the GM-CSF activity on MDS myeloid progenitor cells.

Exposure to nitrous oxide inactivates the cobalamin coenzyme of methionine synthetase, an essential enzyme in folate metabolism. Hemopoietic cells are especially dependent on the function of cobalamin for the folate-dependent synthesis of thymidylate (dTMP). Inhibition of methionine synthetase may therefore be of potential value in the treatment of hematological malignancies. In the present study we investigated the effect of nitrous oxide induced cobalamin inactivation on folate metabolism of fresh leukemic cells and the human myelomonocytic cell line U937. Cells were exposed to nitrous oxide for 20 h. Subsequently they were subjected to the deoxyuridine suppression test (dU test), which measures the disturbance of folate-dependent dTMP synthesis. In all bone marrow samples, cobalamin inactivation resulted in a 200% increase of the dU test value, implicating a decreased de-novo synthesis of dTMP. Incubation of leukemic cells with methotrexate, 5-fluorouracil or cycloleucine induced similar increases of the dU test values which could be further raised to 400% with the addition of N 2 0 exposure. Prolonged experiments with U937 cells revealed that the disturbance of folate metabolism aggravated up to 48 h of nitrous oxide exposure. It can be concluded that cobalamin inactivation in human leukemic cells results in disturbed folate-dependent dTMP synthesis. Moreover, effects of several drugs interfering with folate metabolism can be enhanced.