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Knowledge of the molecular and clonal characteristics in the myelodysplastic syndromes (MDS) and during progression to acute myeloid leukaemia (AML) is essential to understand the disease dynamics and optimize treatment. Sequencing serial bone marrow samples of eight patients, we observed that MDS featured a median of 3 mutations. Mutations in genes involved in RNA-splicing or epigenetic regulation were most frequent, and exclusively present in the major clone. Minor subclones were distinguishable in three patients. As the MDS progressed, a median of one mutation was gained, leading to clonal outgrowth. No AML developed genetically independent of a pre-existing clone. The gained mutation mostly affected genes encoding signalling proteins. Additional acquisition of genomic aberrations frequently occurred. Upon treatment, emergence of new clones could be observed. As confirmed by single-cell sequencing, multiple mutations in identical genes in different clones were present within individual patients. DNA-methylation profiling in patients without identification of novel mutations in AML revealed methylation changes in individual genes. In conclusion, our data complement previous observations on the mutational and clonal characteristics in MDS and at progression. Moreover, DNA-methylation changes may be associated with progression in single patients. Redundancy of mutated genes in different clones suggests fertile grounds promoting clonal selection or acquisition.

Although azanucleoside DNA-hypomethylating agents (HMAs) are routinely used for the treatment of myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML), very few outcome predictors have been established. Expression of the β-like globin gene locus is tightly regulated by DNA methylation, is HMA-sensitive in vitro, and fetal haemoglobin (HbF) expression is under study as a potential biomarker for response of MDS patients to azacitidine. We determined HbF expression in 16 MDS and 36 AML patients receiving decitabine (DAC). Pre-treatment HbF was already elevated (>1·0% of total haemoglobin) in 7/16 and 12/36 patients, and HbF was induced by DAC in 81%/54% of MDS/AML patients, respectively. Elevated pre-treatment HbF was associated with longer median overall survival (OS): 26·6 vs. 8·6 months for MDS (hazard ratio [HR] 8·56, 95% confidence interval [CI] 1·74-42·49, P = 0·008, with similarly longer progression-free and AML-free survival), and 10·0 vs. 2·9 months OS for AML (HR 3·01, 95% CI 1·26-7·22, P = 0·014). In a multivariate analysis, the prognostic value of HbF was retained. Time-dependent Cox models revealed that the prognostic value of treatment-induced HbF induction was inferior to that of pre-treatment HbF. In conclusion, we provide first evidence for in vivo HbF induction by DAC in MDS/AML, and demonstrate prognostic value of elevated pre-treatment HbF, warranting prospective, randomized studies.

SummaryAlpha thalassaemia myelodysplastic syndrome (ATMDS) is an unusualcomplication of chronic myeloid malignancy that is associated with a strikingred cell phenotype. It represents an acquired form of a-thalassaemia thatmost commonly arises in the context of myelodysplasia. It has recently beenshown that this condition occurs in association with somatic mutations of aknown X-encoded trans-acting regulator of a globin gene (HBA) expression,ATRX. There is an unexplained, strong male preponderance of individualswith the ATMDS phenotype with a >5:1 male–female ratio and furthermore,all the somatic ATRX mutations described to date have been in males. Herewe report the identification, in a single centre, of two females with ATMDSand mutations in the ATRX gene, proving that ATMDS associated with suchmutations may occur, albeit rarely, in females. It seemed possible that femalesmight be less likely to develop ATMDS if the inactivated copy of the ATRXgene (ATRX) became progressively re-activated throughout life. This studyruled out this hypothesis by investigating the pattern of ATRX inactivation ina cross-sectional analysis of normal females at ages ranging from newborn to90 years.Keywords: chromatin, DNA methylation, epigenetic disorders, haemoglobinregulation.

Summary The primary objective of this study was to investigate factors associated with fatigue severity in newly diagnosed patients with higher-risk myelodysplastic syndromes (MDS). The secondary objectives were to assess symptom prevalence and to examine the relationships between fatigue, quality of life (QoL) and overall symptom burden in these patients. The analyses were conducted in 280 higher-risk MDS patients. Pre-treatment patient-reported fatigue was evaluated with the Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue scale and QoL was assessed with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30). Female gender (P = 0·018), poor performance status (i.e., ECOG of 2–4) (P

Polo-like kinases (Plks) play an important role in cell cycle checkpoint controls and are over-expressed in acute myeloid leukaemia (AML). BI 2536, a novel Plk inhibitor, induces mitotic arrest and apoptosis. In this phase I/II trial of BI 2536 in 68 elderly patients with relapsed/refractory AML, three schedules were investigated (day 1, days 1-3, and days 1 + 8). Maximum tolerated dose was 350 and 200 mg in the day 1 and days 1 + 8 schedules, respectively. The day 1-3 schedule appeared equivalent to the day 1 schedule and was discontinued early. BI 2536 exhibited multi-compartmental pharmacokinetic behaviour. The majority of patients showed an increase of bone marrow cells in G2/M with a characteristic pattern of mitotic catastrophe. The overall response rate in the day 1 and day 1 + 8 schedules was 9% (5/54) with 2 complete and 3 partial responses. The majority of drug-related adverse events grade ≥3 were haematological. Taken together, Plk inhibition induced cell cycle arrest in AML blasts in vivo and BI 2536 monotherapy showed modest clinical activity in this poor prognosis patient group.

The leukaemia-specific fusion oncoprotein RUNX1/RUNX1T1 (AML1/ETO), resulting from the chromosomal translocation (8;21) in acute myeloid leukaemia (AML), imposes a striking genotype-phenotype relationship upon this distinct subtype of AML, which is mediated by multiple, co-ordinate downstream effects induced by this chimeric transcription factor. We previously identified the LAT2 gene, encoding the adaptor molecule LAT2 (NTAL, LAB), which is phosphorylated by KIT and has a role in mast cell and B-cell activation, as a target of the repressor activity of RUNX1/RUNX1T1. These results were confirmed and extended by demonstrating downregulation of the LAT2 protein in response to conditional RUNX1/RUNX1T1 expression, and its absence in primary AML with the t(8;21). In contrast, in a cohort of 43 AML patients, higher levels of LAT2 were associated with myelomonocytic features. Differentiation of HL-60 and NB4 cells towards granulocytes by all trans-retinoic acid (ATRA) resulted in downregulation of LAT2; conversely, it was upregulated during phorbol ester-induced monocytic differentiation of HL-60 cells. Forced expression of LAT2 in Kasumi-1 cells resulted in a striking block of ATRA- and phorbol ester-induced differentiation, implicating disturbances of the graded expression of this adaptor molecule in the maturation block of myeloid leukaemia cells.

We demonstrate that macrophage-colony stimulating factor (M-CSF) is expressed in human fibroblasts at the mRNA and protein level. Following activation with both interleukin (IL)-1 beta and IL-4, fibroblasts synthesized M-CSF transcripts detectable by Northern blot analysis with peak expression occurring at 8 h and 12 h, respectively. Exposure of fibroblasts to both cytokines resulted in M-CSF protein release at 60 h of c. 500 U/ml (for IL-1 beta) and 1000 U/ml (for IL-4), relative to a control preparation of recombinant human M-CSF in a murine bone marrow colony assay. Both interleukins synergized to enhance M-CSF mRNA accumulation and their ability to induce M-CSF transcripts could be abolished by treatment with specific neutralizing antibodies. These observations provide support for the idea that fibroblasts may control monocyte/macrophage development and function, and that IL-1 beta and IL-4 are involved in the regulation of this process.

Decitabine (5-aza-2'-deoxycytidine) acts as a powerful demethylating agent in vitro. Clinically, low-dose decitabine ameliorates cytopenias including induction of trilineage responses in approximate to 50% of patients with high-risk myelodysplastic syndrome (MDS). We examined the incidence and kinetics of cytogenetic responses to decitabine in these patients. Of 115 successfully karyotyped patients, 61 (53%) had clonal chromosomal abnormalities prior to treatment. Major cytogenetic responses were observed in 19 patients (31% of those with abnormal cytogenetics, 17% of all patients by intention-to-treat) after a median of three courses (range, 2-6) until best cytogenetic response. Progressive decrease of the abnormal clone over time was also determined using fluorescence in situ hybridization (FISH) analysis in two patients. Median duration of cytogenetic responses was 7.5 months (range, 3-15). Analysis of response by the International Prognostic Scoring System (IPSS) cytogenetic risk groups revealed three out of five cytogenetic responses (60%) in the IPSS 'low-risk' group, 6 out of 30 with 'intermediate risk' (20%) and 10 out of 26 in the 'high-risk' group (38%). Median survival in these cytogenetic subgroups was 30, 8 and 13 months respectively. The relative risk of death in patients achieving a major cytogenetic response was 0.38 (95% confidence interval 0.17-0.88) compared with patients in whom the cytogenetically abnormal clone persisted (P = 0.0213). In conclusion, repeated courses of low-dose decitabine induce cytogenetic remissions in a substantial number of elderly MDS patients with pre-existing chromosomal abnormalites; these are associated with improved survival compared with patients in whom the cytogenetically abnormal clone persists. Patients with 'high-risk' chromosomal abnormalities may particularly benefit from this treatment.

In 140 patients with de novo acute myeloid leukaemia (AML) standard cytogenetics were compared with RT-PCR for the detection of t(8;21), t(15;17) and inv(16) and fluorescence in situ hybridization (FISH) for numerical aberrations of chromosomes 7, 8, X and Y. RT-PCR detected 18 cases with t(8;21), 12 with t(15;17) and seven with inv(16). In two cases with t(8;21), two with t(15;17) and four with inv(16) these aberrations had not been detected by standard cytogenetics. There were no false negative PCR results. In 12 patients with these chromosomal changes, standard cytogenetics revealed additional chromosomal aberrations. In 16 patients sole numerical aberrations of the chromosomes 7, 8, X or Y were found by FISH. In these patients the sensitivity of FISH and standard cytogenetics was comparable. In 87 patients no aberrations could be found by PCR and FISH whereas in 24 of these patients standard cytogenetics revealed an abnormal karyotype. These data recommend the combination of standard cytogenetics and molecular techniques to improve the sensitivity for the detection of genetic lesions in AML. Once chromosomal markers have been identified by combined analysis these markers could be used to monitor residual disease during/after chemotherapy, by RT-PCR and/or FISH.

We report analysis of allele-specific expression of N-RAS transcripts in myeloid leukaemic cells and cell lines. Expression was assessed by an assay of reverse-transcription/PCR combined with differential hybridization with mutation-specific oligonucleotides. In cells from all patients with acute myeloid leukaemia (AML) and in myeloid cell lines HL-60 and THP-1, expression of both the wild-type allele and the abnormal allele altered by a point mutation could be detected, albeit not always at comparable levels. This might be due for instance to allelic exclusion. The assay described provides a means of analysing the degree of expression of dominant oncogenes.