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Whether high-dose cytarabine-based salvage chemotherapy, administered to induce complete remission in patients with poor responsive or relapsed acute myeloid leukaemia scheduled for allogeneic haematopoietic stem-cell transplantation (HSCT) after intensive conditioning confers a survival advantage, is unclear.

Impaired proinsulin-to-insulin processing in pancreatic β-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. 1,2), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. 3–8); nonetheless, the role of specific SL species in β-cell function and demise is unclear. Here we define the lipid signature of T2D-associated β-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. β-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL–protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum–Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in β-cell function and T2D-associated β-cell failure.

Introduction:

New treatment options are needed to prevent relapses following failed antibiotic therapies of Clostridium difficileinfections (CDI) in humans. The concomitant therapy with an anti-C. difficileIgA containing whey protein concentrate can support the sustainable recovery of CDI patients. For 31 weeks, nine dairy cows were continuously vaccinated with several anti-C. difficilevaccines by certain routes of administration to produce anti-C. difficileIgA enriched milk. The study aimed at finding decisive differences between low responder (LR) and high responder (HR) cows (> 8.0 µg ml−1total milk C. difficilespecific IgA) concerning their immune response to vaccination on cellular and molecular biological levels. The results of total and differential cell counting (DCC) in blood and milk and the outcomes of the gene expression analysis of selected immune factors were assessed relating to the usage of two vaccine batches for injection (MucoCD-Ibatch A and B), marking two immunization (IM) periods, and compared to a control group (Ctr). The MucoCD-Ibatch A caused short-term leukopenia followed by leukocytosis in the blood of LR and HR. The total somatic cell counts in milk were not altered by the treatment. The DCC revealed that the leukocytes of the treated groups were partly impaired by the treatment. The gene expression analysis exposed cumulative and sustainable differences (p< 0.05) between LR and HR for the genes encoding for lactoferrin, CXCL8, IL1β, IL2, IL6, IL12β, IFNγ, CD4and CD163. The regulation of the epithelial IgA cell receptor PIGRwas not impaired by the IM. In contrast to the vaccination with MucoCD-Ibatch A, the second IM period with MucoCD-Ibatch B resulted in mitigation and synchronization of the treated groups’ immune responses. The inversely regulated cytokines in the blood and milk cells of the treated groups led to a variously directed, local T cell response resulting in their different production intensities of C. difficilespecific IgA in milk.

Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin−CD34+CD38−stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.

This review presents the latest experimental and clinical research focussing on the relationship between the intake of tomato products and lycopene supplementation and carcinogenesis, with the aim of drawing conclusions for concepts of clinical nutritional support.

Differential cloning revealed a partial mRNA sequence expressed in the mouse testis, which on further molecular characterization proved to be a member of a new family of 14 transcribed genes. Six of the genes appear to be expressed pseudogenes. The remainder indicate an open reading frame of approximately 200–220 amino acids encoding proteins with a very high proportion of alpha helical secondary structure, comprising approximately 15% glutamate residues. Because of this property, the family has been named SPErm-associated glutamate (E)-Rich protein (SPEER). Three members were chosen for more detailed characterization: SPEER-1 (pseudogene), SPEER-2, and SPEER-4D. All three are expressed tissue specifically in the testis of mice, with only very weak expression evident in the rat testis but in no other species tested. Using reverse transcription-polymerase chain reaction (RT-PCR), all three transcripts can be detected also in the epididymis, presumably due to the presence of spermatozoa. All three transcripts are expressed to high levels in haploid germ cells at the spermatocyte-spermatid transition. SPEER-1 mRNA is present in the cytoplasm as a sense transcript, SPEER-2 appears to be made mostly as an antisense transcript, whereas SPEER-4D appears to be localized within a subcellular compartment as a conventional sense transcript. Codon usage analysis suggests that all but the pseudogenes can be expressed as protein, confirmed for SPEER-2 and SPEER-4D by in vitro transcription/translation. An antibody raised against a peptide region of SPEER-4D, which probably cross-reacts with other SPEER members, immunohistochemically stains the nuclei of early round spermatids. While there are no true homologies to other proteins in the genome databases, some motifs are present that suggest a relationship to nuclear matrix proteins, implying that the SPEER family is a new group of haploid sperm-specific nuclear factors.

In systemic mastocytosis (SM), clinical phenotype, response to treatment and prognosis are influenced by the KITD816V mutation (80-90% of patients positive), additional mutations in SRSF2, ASXL1or RUNX1(S/A/Rpos) and chromosomal aberrations. In core-binding factor acute myeloid leukemias (CBF-AML), KITmutations are identified in about 30% of cases, conferring an adverse prognostic impact on survival. However, patients with AML are not routinely checked for the contemporaneous presence or absence of SM. We sought to evaluate a) clinical and molecular characteristics, b) responses to treatment and survival, and c) prognostic factors in 44 patients with SM and associated AML (SM-AML). The median age was 64 years (range 28-83), 64% were male. In the majority of patients (30/44, 68%), AML evolved a median of 24 months (range 2-153) after diagnosis of SM without (6/30, 20%) or with (24/30, 80%) an associated hematologic neoplasm (AHN) such as myelodysplastic syndrome (SM-MDS), myeloproliferative neoplasm (SM-MPN) or MDS/MPN (SM-MDS/MPN). Based on histopathology, SM was contemporaneously diagnosed with AML in 14/44 (32%) patients. At diagnosis of SM-AML, the median bone marrow infiltration by mast cells and myeloid blasts was 15% (range 5-65; ≥20% in 45% of patients) and 39% (range 20-95), respectively, and the median serum tryptase level was 92 μg/l (range 2-885; ≥150 in 39% of patients). In the majority of patients, material was available for molecular analyses and revealed the following markers at diagnosis of SM-AML: a) KITD816V in 36/41 (88%) patients, b) additional mutations by targeted next-generation sequencing (NGS, 28 myeloid gene panel) in 32/33 (97%) patients, the most frequent mutations were: RUNX1(n=12, 36%), TET2(n=11, 33%), SRSF2(n=11, 33%), ASXL1(n=9, 27%), IDH1/ 2(n=6, 18%), NPM1(n=6, 18%), BCOR(n=5, 15%), DNMT3A(n=4, 12%), and, c) an aberrant karyotype in 25/35 (71%) patients (in analogy to related myeloid neoplasms, e.g. MDS or AML, patients were classified according to their karyotype into three groups: favorable-, intermediate- and poor-risk; only 1 patient with a CBF translocation). Patients with a known history of SM were frequently S/A/Rpos(19/22, 86%) and acquisition of additional mutations (NPM1, n=2; IDH1, n=2; BCOR/ JAK2/ TP53/ PHF6/ RUNX1, n=1) or karyotype evolution at time of SM-AML was observed in 8/15 (53%) or 9/13 (69%) patients, respectively. NGS on DNA derived from CD34+ myeloid blasts of 6 KITD816V positive patients revealed the presence of KITD816V positive blasts in only 1/6 (17%) patients while additional mutations were found in blast cells of all 6 patients. Eight patients received supportive care due to age and/or comorbidity. In 36 patients, disparate combinations of AML-type chemotherapy ± hypomethylating agents (HMA) ± cladribine (n=16) were administered. Cytarabine-based consolidation treatment was administered in 4/36 (11%) patients in complete remission (CR). Allogeneic stem cell transplantation (SCT) was performed in 15/36 (42%) patients, only 6/15 (40%) patients were in CR prior to allogeneic SCT. Overall, 5/10 (50%) patients in CR of AML showed persistence of SM. From diagnosis of SM-AML, the median observation was 10 months (range 0-200); 32/44 (73%) patients died during observation. The median overall survival (OS) was 11 months (95% confidence interval [CI], 4-17) with median OS of 2 (range 0-5), 4 (range 0-12) and 74 (range 0-149) months for patient cohorts who were treated with supportive care, intensive chemotherapy/HMA and intensive chemotherapy followed by allogeneic SCT, respectively (P=0.0002). On multivariate analysis, the only independent prognostic marker for OS was favorable-risk karyotype (n=14) vs. intermediate-/poor-risk karyotype (n=21; median OS 21 vs. 7 months; HR 3.2 [1.3-7.8], P=0.007). We conclude that a) SM-AML seems to be more frequent than commonly believed, b) SM-AML should be regarded as secondary AML evolving from multimutated SM-AHN, c) KITmutations are not restricted to CBF-AML, d) progression to SM-AML is often triggered by acquisition of new mutations and/or karyotype evolution, e) SM-AML has an aggressive phenotype and a poor prognosis, f) treatment of SM-AML should include a combination of intensive chemotherapy and allogeneic SCT for eligible patients and g) the disease frequency and poor prognosis warrant routine screening of all AML cases for serum tryptase and KITD816V.

Platzbecker: Celgene Corp.: Honoraria, Research Funding. Götze:Celgene Corp, Novartis Pharma: Honoraria. Schlenk:Celgene Corp.: Research Funding, Speakers Bureau. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Bug:Celgene: Honoraria, Research Funding. Germing:Celgene Corp.: Honoraria, Research Funding. Nolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding.

Schnittger: MLL Munich Leukemia Laboratory: Other. Horny:Novartis: Consultancy, Honoraria. Haferlach:MLL: Equity Ownership. Valent:Novartis: Consultancy. Reiter:Novartis: Consultancy, Honoraria.

Systemic mastocytosis (SM) is characterized by abnormal proliferation and accumulation of mast cells (MC) in various tissues and organs, predominantly skin, bone marrow (BM) and visceral organs. The extent of organ infiltration and subsequent organ damage is the basis for the classification of SM into indolent SM (ISM), smoldering SM (SSM), SM with associated clonal hematologic non-MC lineage disease (SM-AHNMD), aggressive SM (ASM) and MC leukemia (MCL). Depending on the subtype of SM, cell source (BM or peripheral blood) and assay sensitivity, an acquired mutation in the receptor tyrosine kinase KIT, usually KITD816V, is detectable in 80-90% of patients. Next-Generation Deep Amplicon Sequencing (NGS) was performed to investigate 18 candidate genes at known mutational hotspot regions as previously described. Additional mutations in genes encoding for signaling molecules (JAK2, CBL, KRAS, NRAS), transcription factors (RUNX1), epigenetic regulators (ASXL1, DNMT3A, EZH2, TET2) or splicing factors (SRSF2, SF3B1, U2AF1) are detectable in the vast majority of patients KITD816V+ advanced SM. In order to gain more insight into clonal evolution of myeloid progenitors in indolent and advanced SM with or without AHNMD, we explored the mutation profile of single-cell-derived CFU-GM colonies - by using Sanger sequencing - in 19 KITD816V+ SM patients (investigated colonies, n=285; median per patient, n=15; range 10-30). The study included 7 patients with ISM/SSM/ASM (0 additional mutations), 4 patients with SM-AHNMD (median 1 additional mutation, range 1-4) and 8 patients with ASM-AHNMD (median 3 additional mutations, range 1-4). KITD816V+ CFU-GM could be identified in all 8 patients with ASM ± AHNMD but in only 20% (1/5) of patients with SM-AHNMD, while all CFU-GM colonies derived from ISM patients were completely KITD816V negative. On the other hand, CFU-GM colonies from individual ASM ± AHNMD patients were never entirely KITD816V+ (median 60%, range 0-95). In contrast to KITD816V, additional mutations were identified in CFU-GM colonies from all 12 multi-mutated (A)SM-AHNMD patients and many of these mutations were present in 100% of the CFU-GM-derived colonies analyzed. In 8 patients, different subclones with variable proportions of the number of mutated genes were identified and allowed to generate putative evolutionary trees. These mutations included TET2and SRSF2mutations in 6/6 and 4/4 patients with (A)SM-AHNMD. In contrast, ASXL1mutations were not identified in all TET2/SRSF2positive CFU-GM colonies suggesting that they are likely to occur later than TET2and SRSF2. When additional mutations and KITD816V were detected concomitantly in individual single-cell-derived CFU-GM colonies, the overall frequency of CFU-GM colonies positive for additional mutations was always higher than those that were KITD816V positive, indicating that the KITD816V mutation was a secondary event. In contrast to advanced SM, all ISM patients were negative for additional mutations. CFU-GM colonies and also microdissected CD15+ cells derived from BM biopsies were entirely KITD816V negative in these patients, highlighting that the KITD816V mutation may be restricted to other (probably later) stages of stem cell development and possibly only to the MC lineage. In general, the relative proportion of KITD816V+ progenitors correlated well with established parameters for quantification of disease burden, e.g. BM MC infiltration, serum tryptase levels and KITD816V allele burden in individual patients. In conclusion, the presence of multi-mutated myeloid non-MC lineage progenitors of the CFU-GM-type suggests an initial clonal expansion at an early stage of hematopoietic development due to mutations other than KITD816V with a subsequent phenotype modification towards SM due to a later acquisition of KITD816V. In contrast, ISM/SSM is not affected by mutations at the CFU-GM level which may at least in part explain its excellent prognosis.

Introduction: MDS with isolated deletion 5(q) accounts for approximately 5% of all MDS cases. A recent retrospective analysis has found a cumulative progression rate of 18% after 5 years in patients with MDS deletion 5(q) without an increased medullary blast count[1]. Retrospective analyses have indicated that mutations in TP53have an adverse impact on the clinical course of affected patients and their response to Len treatment. Here we report on the results of the German multi-center, prospective Le-Mon-5 trial that investigated the safety and efficacy of lenalidomide (Len) in patients with MDS and isolated deletion 5(q).

AIM. Expression changes of D-type cyclins are thought to be an early event in the genesis of Multiple Myeloma and are associated with distinct cytogenetic aberrations. These aberrations appear with different percentages (“clonal” or “subclonal”) in a given patient. We assessed whether the height of CCND expression assessed by gene expression profiling and quantitative RT-PCR (qRT-PCR) correlates with the presence of clonal or subclonal aberrations of 11q13, t(11;14) and t(4;14). PATIENTS AND METHODS. 128 newly diagnosed MM-patients (65 training (TG)/63 independent validation group (VG)) and 14 normal donors (ND) were included. Bone marrow aspirates were CD138-purified by activated magnetic cell sorting. RNA was in-vitro transcribed and hybridised to Affymetrix HG U133 A+B GeneChip (TG) and HG U133 2.0 plus array (VG). CCND1 and CCND2 expression was verified by real time RT-PCR and western blotting. iFISH was performed on purified MM-cells using probesets for chromosomes 1q21, 9q34, 11q23, 11q13, 13q14, 15q22, 17p13, 19q13, 22q11 and the translocations t(4;14) and t(11;14). Clonal aberrations were defined as being present in >60%, subclonal aberrations in 20 to 60% of MMC in a given patient. Expression data were gcrma normalised and a Kruskal-Wallis rank sum test used (Bioconductor). RESULTS. 11q13+. CCND1 (208711_s_at, 208712_at) is significantly higher (p<0.0001), CCND2 (200953_s_at, 200951_s_at) significantly lower (p<0.0001) expressed in MMC harbouring clonal, compared to subclonal, or no gain of 11q13. t(11;14). CCND1 is significantly higher (p<0.0001), CCND2 significantly lower (p<0.0001) expressed in MMC harbouring clonal, compared to subclonal, or no t(11;14). t(4;14). CCND1 is significantly lower (p<0.0001), CCND2 significantly higher (p<0.0001) expressed in MMC harbouring clonal compared to subclonal, or no t(4;14). The expression of CCND3 (201700_at) is not significantly different between the 3 groups for all aberrations investigated. CCND2 and CCND3, but not CCND1 are expressed by normal plasma cells. Results have been verified by qRT-PCR (n=40) for CCND1 and CCND2. Expression of CCND1, CCND2 and CCND3 has been verified by western blotting on selected samples. The expression of CCND2 correlates with short EFS, but not if patients with t(4;14) are excluded. There is no significant difference in EFS for patients harbouring the respective aberrations in a clonal or subclonal pattern. CONCLUSION. An additional copy of 11q13 or t(11;14) correlates with increased CCND1 and decreased CCND2 expression, a t(4;14) is associated with an increase of CCND2 and a decrease of CCND1 expression. In each case, the height of the CCND-expression is significantly different whether the respective aberration is clonal or subclonal. Thus, when interpreting expression data in the context of cytogenetic aberrations, it is important to consider if plasma cells carry a respective aberration in a subclonal/clonal pattern.

AIM. Expression changes of D-type cyclins are thought to be an early event in the genesis of Multiple Myeloma and are associated with distinct cytogenetic aberrations. These aberrations appear with different percentages (“clonal” or “subclonal”) in a given patient. We assessed whether the height of CCND expression assessed by gene expression profiling and quantitative RT-PCR (qRT-PCR) correlates with the presence of clonal or subclonal aberrations of 11q13, t(11;14) and t(4;14).