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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.

The BCL11A gene encodes a transcriptional repressor with essential functions in multiple tissues during human development. Haploinsufficiency for BCL11A causes Dias-Logan syndrome (OMIM 617101), an intellectual developmental disorder with hereditary persistence of fetal hemoglobin (HPFH). Due to the severe phenotype, disease-causing variants in BCL11A occur de novo. We describe a patient with a de novo heterozygous variant, c.1453G>T, in the BCL11A gene, resulting in truncation of the BCL11A-XL protein (p.Glu485X). The truncated protein lacks the 3 C-terminal DNA-binding zinc fingers and the nuclear localization signal, rendering it inactive. The patient displayed high fetal hemoglobin (HbF) levels (12.1-18.7% of total hemoglobin), in contrast to the parents who had HbF levels of 0.3%. We used cultures of patient-derived erythroid progenitors to determine changes in gene expression and chromatin accessibility. In addition, we investigated DNA methylation of the promoters of the γ-globin genes HBG1 and HBG2. HUDEP1 and HUDEP2 cells were used as models for fetal and adult human erythropoiesis, respectively. Similar to HUDEP1 cells, the patient’s cells displayed Assay for Transposase-Accessible Chromatin (ATAC) peaks at the HBG1/2 promoters and significant expression of HBG1/2 genes. In contrast, HBG1/2 promoter methylation and genome-wide gene expression profiling were consistent with normal adult erythropoiesis. We conclude that HPFH is the major erythroid phenotype of constitutive BCL11A haploinsufficiency. Given the essential functions of BCL11A in other hematopoietic lineages and the neuronal system, erythroid-specific targeting of the BCL11A gene has been proposed for reactivation of γ-globin expression in β-hemoglobinopathy patients. Our data strongly support this approach.

Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein a (C/EBP alpha), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located 142 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only.

Human herpes virus 8 (HHV-8)-associated secondary hemophagocytic lymphohistiocytosis is a rare but critical immuno-hematological entity in immunocompetent patients. Establishing a diagnosis is challenging as is the monitoring of disease activity and therapeutic effects. We report a case of a HHV-8-associated hemophagocytic lymphohistiocytosis in a HIV-negative adult patient with multicentric Castleman's disease. As a novel finding, we report the use of certain inflammatory parameters, primarily interleukin-10 combined with viral load monitoring of the causative infectious agent in this case HHV-8 to monitor the clinical course of the hemophagocytic lymphohistiocytosis in the setting of bacterial septic complications.

Peptide receptor radionuclide therapy (PRRT) may induce long-term toxicity to the bone marrow (BM). The aim of this study was to analyze persistent hematologic dysfunction (PHD) after PRRT with 177Lu-DOTATATE in patients with gastroenteropancreatic neuroendocrine tumors (GEP NETs). Methods: The incidence and course of PHD were analyzed in 274 GEP NET patients from a group of 367 patients with somatostatin receptor-positive tumors. PHD was defined as diagnosis of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), myeloproliferative neoplasm (MPN), MDS/MPN, or otherwise unexplained cytopenia (for >6 mo). Using data from The Netherlands Cancer Registry, the expected number of hematopoietic neoplasms (MDS, AML, MPN, and MDS/MPN) was calculated and adjusted for sex, age, and follow-up period. The following risk factors were assessed: sex, age over 70 y, bone metastasis, prior chemotherapy, prior external-beam radiotherapy, uptake on the [111In-DTPA0]octreotide scan, tumor load, grade 3-4 hematologic toxicity during treatment, estimated absorbed BM dose, elevated plasma chromogranin A level, baseline blood counts, and renal function. Results: Eleven (4%) of the 274 patients had PHD after treatment with 177Lu-DOTATATE: 8 patients (2.9%) developed a hematopoietic neoplasm (4 MDS, 1 AML, 1 MPN, and 2 MDS/MPN) and 3 patients (1.1%) developed BM failure characterized by cytopenia and BM aplasia. The median latency period at diagnosis (or first suspicion of a PHD) was 41 mo (range, 15-84 mo). The expected number of hematopoietic neoplasms based on The Netherlands Cancer Registry data was 3.0, resulting in a relative risk of 2.7 (95% confidence interval, 0.7-10.0). No risk factors for PHD could be identified for the GEP NET patients, not even bone metastasis or estimated BM dose. Seven patients with PHD developed anemia in combination with a rise in mean corpuscular volume. Conclusion: The prevalence of PHD after PRRT with 177Lu-DOTATATE was 4% in our patient population. The median time at which PHD developed was 41 mo after the first PRRT cycle. The relative risk for developing a hematopoietic neoplasm was 2.7. No risk factors were found for the development of PHD in GEP NET patients.

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset # 2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset # 2. Within subset # 2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset # 2/IGHV3-21 was enriched for IGHV-unmutated cases (P =.002). Subset # 2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset # 2/IGHV3-21 (22 vs 60 months, P =.001). No such difference was observed between non-subset # 2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset # 2 emerges as uniformly aggressive, contrasting non-subset # 2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

Peptide receptor radionuclide therapy (PRRT) may induce long-term toxicity to the bone marrow (BM). The aim of this study was to analyze persistent hematologic dysfunction (PHD) after PRRT with

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

Adult T-cell leukaemia/lymphoma (ATLL) is a rare mature T-cell malignancy that occurs in patients infected with human T-cell leukaemia virus type 1 (HTLV-1). Only a minority of HTLV-1 carriers develop neoplastic transformation to ATLL after a 40- to 60-year latency period.A 49-year-old Columbian male presented to the Emergency Department with abdominal pain, weight loss, generalized cutaneous papules and epistaxis. Extensive cytomorphological, histopathological, immunophenotypical, serological, and molecular (cyto)genetic analysis all contributed to reach the diagnosis of acute HTLV-1 associated ATLL. The patient was treated with multiagent cytotoxic chemotherapy, consisting of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP).Acute ATLL is a clinically aggressive disease and carries a poor prognosis. Treatment options include antiviral regimens, chemotherapy, monoclonal antibody-targeted therapy, and allogeneic stem-cell transplantation. The incidence of ATLL in non-endemic areas, such as the Netherlands, is increasing due to emigration of HTLV-1 carriers from endemic areas.

Purpose Adequate dosimetry is mandatory for effective and safe peptide receptor radionuclide therapy (PRRT). Besides the kidneys, the bone marrow is a potentially dose-limiting organ. The radiation dose to the bone marrow is usually calculated according to the MIRD scheme, where the accumulated activity in the bone marrow is calculated from the accumulated radioactivity of the radiopharmaceutical in the blood. This may underestimate the absorbed dose since stem cells express somatostatin receptors. We verified the blood-based method by comparing the activity in the blood with the radioactivity in bone marrow aspirates. Also, we evaluated the absorbed cross-dose from the source organs (liver, spleen, kidneys and blood), tumours and the so-called “remainder of the body” to the bone marrow. Methods Bone marrow aspirates were drawn in 15 patients after treatment with [177Lu-DOTA0,Tyr3]octreotate. Radioactivity in the bone marrow was compared with radioactivity in the blood drawn simultaneously. The nucleated cell fraction was isolated from the bone marrow aspirate and radioactivity was measured. The absorbed dose to the bone marrow was calculated. The results were correlated to the change in platelet counts 6 weeks after treatment. Results A strong linear correlation and high agreement between the measured radioactivities in the bone marrow aspirates and in the blood was found (r=0.914, p

A 41-year-old man visited his general practitioner because of upper abdominal pain. Physical examination revealed splenomegaly. Laboratory testing showed pancytopenia with a striking monocytopenia with hairy cells. Immunological and molecular analysis confirmed the diagnosis hairy cell leukemia.

In 10 JAK2V617F positive patients with early myeloproliferative essential thrombocythemia (ET) or polycythemia vera (PV) we sequentially used the Polycythemia Vera Study Group (PVSG), the Thrombocythemia Vera Study Group (TVSG) and the 2008 WHO criteria for the clinical diagnosis of essential thrombocythemia (ET) ad polycythemia vera (PV). Subsequently, we evaluated bone marrow features and laboratory and molecular markers including endogenous erythroid colony (EEC), serum erythropoietine (EPO) mutation for the classification of early MPD. Six symptomatic patients had a delayed diagnosis of slow onset latent (masked) ET and 4 patients presented with rapid onset PV associated with thrombocythemia. We could clearly distinguish three phenotypes of ET when the 2008 European clinical, molecular and pathological (2008 WHO-ECMP) criteria for ET and PV are applied. These include ET phenotype 1, ET phenotype type 2 with features of early polycythemia vera (PV) and normal red cell mass, and ET phenotype 3 associated with megakaryocytic granulocytic myeloproliferation (ET-MGM) in the absence of characteristic features of primary myelofibrosis (PMF). Through the presentation of a number of highly illustrative clinical cases during long-term follow-up we could demonstrate that diagnostic differentiation of three phenotypes of ET is important, because natural history clearly differ.

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.

The IVS‐I‐116 (A→G) (α2) point mutation was previously observed in 11 carriers of two unrelated Dutch families, in association with a very mild α‐thalassemia (thal) phenotype [[1]]. The mutation, a...

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)

Summary. The diagnostic findings of malignant histiocytosis (MH) were analysed in 12 consecutive patients in a single institution. Most patients presented with systemic symptoms and lymphadenopathy (92%), splenomegaly (100%) and hepatomegaly (67%). Neurologic symptoms were present in three patients, while involvement of other organs was present in five patients. The incidence of severe thrombocytopenia was 92%, of anaemia 92% and of leucocytopenia 67%. Serum angiotensin converting enzyme, α1-antitrypsin and lysozyme were independently increased in 6/9, 3/10 and 1/9 patients respectively. High serum levels of tumour necrosis factor (TNF) were present in 3/10 patients, while serum levels of interleukin-1 were normal in 10/10 patients. Histologic evidence of MH was obtained in all patients by repeated biopsies of involved tissues. Four patients died prior to treatment. Seven patients were treated with combination chemotherapy, consisting of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) or MOPP (chloromethine, vincristine, procarbazine, prednisone), in some cases followed by non-cross-resistant second line chemotherapy, if no complete response was attained. The response rate of treated patients was 5 7%, and progression was observed in two patients. The median duration of response was 38 months. Three patients are alive without evidence of disease and off therapy (30+, 83+, 85+ months). Although MH is a potentially lethal disease, combination chemotherapy may offer a chance for cure in some patients.

Prolymphocytic leukemia (PLL) is a rare clonal lymphoproliferative disorder of mature lymphocytes. The disease originates from B lymphocytes in approximately 80% of cases and from T lymphocytes in 20% of cases.[1][1] B-PLL and T-PLL are both characterized by an aggressive clinical course with

The mutational status of the immunoglobulin heavy chain variable region genes (IGVH) is a strong indicator of prognosis in B-cell chronic lymphocytic leukaemia (CLL). Since the determination of the IGVH mutation status is very labor-intensive, alternative prognostically relevant markers would facilitate CLL diagnostics.Ten genes were selected from previously published gene expression profiling studies based on their differential expression in IGVH mutated versus unmutated cases of CLL, and tested with real-time quantitative polymerase chain reaction (RQ-PCR) in unpurified samples from 130 CLL patients. To ascertain potential contaminating effects by normal hematopoietic cells, the expression levels of the selected genes were determined in normal monocytes, B cells, T cells, NK cells and granulocytes.The selected genes, i.e., ZAP70, LPL, SPG20, ADAM29, NRIP1, AKAP12, DMD, SEPT10, TPM2 and CLECSF2, showed prognostic significance. In multivariate logistic regression analysis expression levels of LPL, ZAP70, ADAM29 and SEPT10 were the most predictive for IGVH mutational status. In univariate analysis the expression of LPL was the best predictor. For survival, expression of LPL was the strongest prognostic factor. In combination with the three cytogenetic markers associated with a poor prognosis, i.e., deletions 17p13, 11q22 and trisomy 12, expression of LPL and IGVH mutational status performed equally well with regard to their predictive value for survival, both being more predictive than ZAP70.This study demonstrates that LPL expression is a predictor for survival in CLL, and for this purpose is as good as IGVH mutational status and more reliable than ZAP70 expression when tested in unpurified CLL samples.

Increased CD38 expression by leukemic cells has been suggested as an adverse prognostic factor in B-CLL. Several approaches have been proposed to quantify its level of expression by flow cytometry.We compared the use of (i) the percentage of CD38 positive cells, (ii) CD38 antibodies bound per cell (ABC), and (iii) a semi-quantitative method based on the shape of the CD38 histogram, within a cohort of 78 B-CLL patients.A decreased overall survival was seen with30% CD38 positivity among B-CLL cells, with CD38 ABC100, and with bimodal or unimodal, strongly positive CD38 histograms. However, patients with unimodal weakly positive CD38 histograms also showed a significantly reduced survival as did patients with intermediate proportions (i.e. 5-30%) of CD38+ cells. Furthermore, within the group with5% CD38 positivity among their B-CLL cells, 84% of patients showed prognostically favourable mutated IGVH gene segments and 100% had low ZAP70 gene expression. For 5-30% CD38 positivity, these proportions were 50 and 83%, while for30% CD38 positivity, these proportion were only 28 and 56%, respectively.We found a simple method of quantitation of CD38 expression (i.e.,5% CD38 positivity among B-CLL cells) to be sufficient to identify patients with an unfavourable prognosis. The level of CD38 expression as defined with this method correlated well with the IGVH mutation status and ZAP70 gene expression.

Cb2, the gene encoding the peripheral cannabinoid receptor, is located in a common virus integration site and is overex-pressed in retrovirally induced murine myeloid leukemias. Here we show that this G protein-coupled receptor (GPCR) is also aberrantly expressed in a high percentage of human acute myeloid leukemias. We investigated the mechanism of transformation by Cb2 and demonstrate that aberrant expression of this receptor on hematopoietic precursor cells results in distinct effects depending on the ligand used. Cb2-expressing myeloid precursors migrate upon stimulation by the endocannabinoid 2-arachidonoylglycerol and are blocked in neutrophilic differentiation upon exposure to another ligand, CP55940. Both effects depend on the activation of Gαi proteins and require the mitogen-induced extracellular kinase/extracellular signal-regulated kinase (MEK/ERK) pathway. Down-regulation of cyclic adenosine monophosphate (cAMP) levels upon Gαi activation is important for migration induction but is irrelevant for the maturation arrest. Moreover, the highly conserved G protein-interacting DRY motif, present in the second intracellular loop of GPCRs, is critical for migration but unimportant for the differentiation block. This suggests that the Cb2-mediated differentiation block requires interaction of Gαi proteins with other currently unknown motifs. This indicates a unique mechanism by which a transforming GPCR, in a ligand-dependent manner, causes 2 distinct oncogenic effects: altered migration and block of neutrophilic development. (Blood. 2004;104:526-534)

Item does not contain fulltext We describe 2 immunocompetent adolescents with fulminant infectious mononucleosis and virus-associated hemophagocytosis. A new quantitative polymerase chain reaction revealed high serum Epstein-Barr virus DNA levels in these patients. One patient died with an increasing viral load not responding to corticosteroids followed by antiviral and intensified immunomodulatory treatment. The other patient received corticosteroids and acyclovir at diagnosis; her rapid recovery was heralded by a steep decline of viral load. We propose monitoring the clinical course of fulminant infectious mononucleosis in immunocompetent patients by Epstein-Barr virus DNA quantification and prompt corticosteroid and antiviral therapy when viral load is high.

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.

Background: Chronic Lymphocytic Leukemia (CLL) has a variable clinical course, of which IgVH mutational status of the malignant B cells is a strong independent predictor for outcome. Objective: We studied the expression of 5 different genes for their value to predict IgVH mutational status and overall survival (OS). Methods: mRNA levels were quantified by real time PCR in lymphoprep separated but unsorted blood samples from 110 morphologically and fenotypically characterised CLL patients for the following genes: ZAP70, lipoprotein lipase (LPL), ADAM29, KIAA0610 and nRIP1. Expression levels were compared wih respect to IgVH mutational status (mutation = % germline < 98%), using logistic regression and with respect to OS (= time from day of laboratory tests to death due to any cause), using Cox regression analysis. Results: The predictive value of the expression of these 5 genes for IgVH mutational status is shown in table 1. Each of these genes showed to be an independent marker for the prediction of IgVH mutational status. LPL as a single marker was the best predictor for mutation. These factors taken together had a high predictive value towards mutational IgVH status (area under ROC curve = 0.93). table 1 N Odds ratio P value ZAP70 no 85 1 (>5.2) yes 25 0.098 0.002 LPL no 73 1 (>0.04) yes 37 0.029 30) yes 37 6.61 0.012 KIAA0610 no 81 1 (>0.25) yes 29 0.19 0.045 NRIP1 no 88 1 (>10) yes 22 48.73 0.008 The predictive value of IgVH mutation and the expression of these 5 genes for OS is shown in table 2. From the six parameters tested, IgVH mutational status and LPL were predictors for OS at a significant level of 5%, while ZAP70 was of borderline significance. table 2 Conclusion: measured in unsorted CLL samples, all genes, and especially LPL, had predictive value towards the presence of IgVH mutation. Expression of LPL and absence of mutation were adverse prognostic factors for survival. Thus, the replacement of the assessment of both IgVH mutational status and ZAP70 by assessment of LPL levels for the prediction of prognosis in CLL patients may be considered. N survival at 24months P value mutation no 36 70% yes 44 93% 0.041 ZAP70 no 66 87% yes 18 70% 0.078 LPL no 55 94% yes 29 66% 0.001 ADAM29 no 57 84% yes 27 84% 0.486 KIAA0610 no 63 88% yes 21 71% 0.133 NRIP no 71 81% yes 13 100% 0.167

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.

In chronic lymphocytic leukemia (CLL), the molecular features of the clonotypic B-cell receptor immunoglobulins (BcR IG) are set from the birth of the clone and in contrast to genetic aberrations, remain stable overtime, rendering the BcR IG a reference biomarker that is usually not significantly affected by clonal evolution. Approximately 30% of CLL cases carry quasi-identical BcR IGs and can be assigned to distinct stereotyped subsets. While preliminary evidence alludes to BcR IG stereotypy being relevant from a clinical viewpoint, this aspect has never been explored systematically or in a cohort of adequate size to enable meaningful conclusions. In order to assess the clinical implications of BcR IG stereotypy, we evaluated clinicobiological data from 8593 CLL patients, particularly focusing on 14 major stereotyped subsets of cases with unmutated (U-CLL) or mutated IGHV genes (M-CLL). The largest subset was #2 (n=254, 3%), within which 156 and 98 cases were M-CLL and U-CLL, respectively, followed by U-CLL subsets #1 (n=173, 2%) and #7 (n=123, 1.4%). Amongst M-CLL, the largest subsets were #4 (n=94, 1.1%) and #148 (n=92, 1%). Stereotyped subsets, even of the same mutational category i.e. U-CLL or M-CLL, exhibited significant clinicobiological differences regarding: age at diagnosis (median age ranging from 53-68 years, p75% in M-CLL subsets #77 and #148 (p Disclosures Montillo: Janssen: Honoraria.

Summary Background About 30% of cases of chronic lymphocytic leukaemia (CLL) carry quasi-identical B-cell receptor immunoglobulins and can be assigned to distinct stereotyped subsets. Although preliminary evidence suggests that B-cell receptor immunoglobulin stereotypy is relevant from a clinical viewpoint, this aspect has never been explored in a systematic manner or in a cohort of adequate size that would enable clinical conclusions to be drawn. Methods For this retrospective, multicentre study, we analysed 8593 patients with CLL for whom immunogenetic data were available. These patients were followed up in 15 academic institutions throughout Europe (in Czech Republic, Denmark, France, Greece, Italy, Netherlands, Sweden, and the UK) and the USA, and data were collected between June 1, 2012, and June 7, 2013. We retrospectively assessed the clinical implications of CLL B-cell receptor immunoglobulin stereotypy, with a particular focus on 14 major stereotyped subsets comprising cases expressing unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain variable genes. The primary outcome of our analysis was time to first treatment, defined as the time between diagnosis and date of first treatment. Findings 2878 patients were assigned to a stereotyped subset, of which 1122 patients belonged to one of 14 major subsets. Stereotyped subsets showed significant differences in terms of age, sex, disease burden at diagnosis, CD38 expression, and cytogenetic aberrations of prognostic significance. Patients within a specific subset generally followed the same clinical course, whereas patients in different stereotyped subsets—despite having the same immunoglobulin heavy variable gene and displaying similar immunoglobulin mutational status—showed substantially different times to first treatment. By integrating B-cell receptor immunoglobulin stereotypy (for subsets 1, 2, and 4) into the well established Dohner cytogenetic prognostic model, we showed these, which collectively account for around 7% of all cases of CLL and represent both U-CLL and M-CLL, constituted separate clinical entities, ranging from very indolent (subset 4) to aggressive disease (subsets 1 and 2). Interpretation The molecular classification of chronic lymphocytic leukaemia based on B-cell receptor immunoglobulin stereotypy improves the Dohner hierarchical model and refines prognostication beyond immunoglobulin mutational status, with potential implications for clinical decision making, especially within prospective clinical trials. Funding European Union; General Secretariat for Research and Technology of Greece; AIRC; Italian Ministry of Health; AIRC Regional Project with Fondazione CARIPARO and CARIVERONA; Regione Veneto on Chronic Lymphocytic Leukemia; Nordic Cancer Union; Swedish Cancer Society; Swedish Research Council; and National Cancer Institute (NIH).