Core-binding factor leukemia hijacks the T-cell-prone PU.1 antisense promoter.

The blood system serves as a key model for cell differentiation and cancer. It is orchestrated by precise spatiotemporal expression of crucial transcription factors. One of the key master regulators in the hematopoietic systems is PU.1. Reduced levels of PU.1 are characteristic for human acute myeloid leukemia (AML) and are known to induce AML in mouse models. Here, we show that transcriptional downregulation of PU.1 is an active process involving an alternative promoter in intron 3 that is induced by RUNX transcription factors driving noncoding antisense transcription. Core-binding factor (CBF) fusions RUNX1-ETO and CBFß-MYH11 in t(8;21) and inv(16) AML, respectively, activate the PU.1 antisense promoter that results in a shift from sense toward antisense transcription and myeloid differentiation blockade. In patients with CBF-AML, we found that an elevated antisense/sense transcript and promoter accessibility ratio represents a hallmark compared with normal karyotype AML or healthy CD34+ cells. Competitive interaction of an enhancer with the proximal or the antisense promoter forms a binary on/off switch for either myeloid or T-cell development. Leukemic CBF fusions thus use a physiological mechanism used by T cells to decrease sense transcription. Our study is the first example of a sense/antisense promoter competition as a crucial functional switch for gene expression perturbation by oncogenes. Hence, this disease mechanism reveals a previously unknown Achilles heel for future precise therapeutic targeting of oncogene-induced chromatin remodeling.

Therapy-related myelodysplastic syndromes deserve specific diagnostic sub-classification and risk-stratification-an approach to classification of patients with t-MDS.

In the current World Health Organization (WHO)-classification, therapy-related myelodysplastic syndromes (t-MDS) are categorized together with therapy-related acute myeloid leukemia (AML) and t-myelodysplastic/myeloproliferative neoplasms into one subgroup independent of morphologic or prognostic features. Analyzing data of 2087 t-MDS patients from different international MDS groups to evaluate classification and prognostication tools we found that applying the WHO classification for p-MDS successfully predicts time to transformation and survival (both p < 0.001). The results regarding carefully reviewed cytogenetic data, classifications, and prognostic scores confirmed that t-MDS are similarly heterogeneous as p-MDS and therefore deserve the same careful differentiation regarding risk. As reference, these results were compared with 4593 primary MDS (p-MDS) patients represented in the International Working Group for Prognosis in MDS database (IWG-PM). Although a less favorable clinical outcome occurred in each t-MDS subset compared with p-MDS subgroups, FAB and WHO-classification, IPSS-R, and WPSS-R separated t-MDS patients into differing risk groups effectively, indicating that all established risk factors for p-MDS maintained relevance in t-MDS, with cytogenetic features having enhanced predictive power. These data strongly argue to classify t-MDS as a separate entity distinct from other WHO-classified t-myeloid neoplasms, which would enhance treatment decisions and facilitate the inclusion of t-MDS patients into clinical studies.

A microRNA-mediated regulatory loop modulates NOTCH and MYC oncogenic signals in B- and T-cell malignancies.

Growing evidence suggests that microRNAs (miRNAs) facilitate the cross-talk between transcriptional modules and signal transduction pathways. MYC and NOTCH1 contribute to the pathogenesis of lymphoid malignancies. NOTCH induces MYC, connecting two signaling programs that enhance oncogenicity. Here we show that this relationship is bidirectional and that MYC, via a miRNA intermediary, modulates NOTCH. MicroRNA-30a (miR-30a), a member of a family of miRNAs that are transcriptionally suppressed by MYC, directly binds to and inhibits NOTCH1 and NOTCH2 expression. Using a murine model and genetically modified human cell lines, we confirmed that miR-30a influences NOTCH expression in a MYC-dependent fashion. In turn, through genetic modulation, we demonstrated that intracellular NOTCH1 and NOTCH2, by inducing MYC, suppressed miR-30a. Conversely, pharmacological inhibition of NOTCH decreased MYC expression and ultimately de-repressed miR-30a. Examination of genetic models of gain and loss of miR-30a in diffuse large B-cell lymphoma (DLBCL) and T-acute lymphoblastic leukemia (T-ALL) cells suggested a tumor-suppressive role for this miRNA. Finally, the activity of the miR-30a-NOTCH-MYC loop was validated in primary DLBCL and T-ALL samples. These data define the presence of a miRNA-mediated regulatory circuitry that may modulate the oncogenic signals originating from NOTCH and MYC.

Frequent loss of RAF kinase inhibitor protein expression in acute myeloid leukemia.

RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated its role in acute myeloid leukemia (AML), an aggressive malignancy arising from hematopoietic stem and progenitor cells (HSPCs). Western blot analysis revealed loss of RKIP expression in 19/103 (18%) primary AML samples and 4/17 (24%) AML cell lines but not in 10 CD34+ HSPC specimens. In in-vitro experiments with myeloid cell lines, RKIP overexpression inhibited cellular proliferation and colony formation in soft agar. Analysis of two cohorts with 103 and 285 AML patients, respectively, established a correlation of decreased RKIP expression with monocytic phenotypes. RKIP loss was associated with RAS mutations and in transformation assays, RKIP decreased the oncogenic potential of mutant RAS. Loss of RKIP further related to a significantly longer relapse-free survival and overall survival in uni- and multivariate analyses. Our data show that RKIP is frequently lost in AML and correlates with monocytic phenotypes and mutations in RAS. RKIP inhibits proliferation and transformation of myeloid cells and decreases transformation induced by mutant RAS. Finally, loss of RKIP seems to be a favorable prognostic parameter in patients with AML.

Reduced-intensity allografting in patients with therapy-related myeloid neoplasms and active primary malignancies.

Therapy-related myeloid neoplasms (t-MNs) are severe long-term consequences of cytotoxic treatments for a primary, often, malignant disorder. So far, the majority of patients eligible for transplantation have undergone myeloablative allo haematopoietic SCT (HSCT) as a potentially curative treatment, but it has been associated with high transplantation-related mortality (TRM) rates. In this retrospective study, we analysed the outcome of patients with t-MNs undergoing HSCT with reduced-intensity conditioning (RIC). Of 55 patients, seen at a single centre over a 10-year period, 17 underwent RIC HSCT with related or unrelated donors. The estimated overall survival was 53% at 1 year and 47% at 3 years, and disease-free survival was 47% at 1 year. At 1 year, the cumulative incidence of relapse and TRM were 24% and 30%, respectively. Of five patients with active primary neoplasms who underwent transplantation, two are alive beyond 1 year and show CR of both t-MNs and the primary malignancy. These data indicate that RIC HSCT is an encouraging approach for patients with t-MNs. The issue of primary malignancies not being in remission at the time of transplantation should be explored in further studies.

Epidemiology of invasive fungal infections and rationale for antifungal therapy in patients with haematological malignancies.

Invasive fungal infections (IFIs) in patients with haematological malignancies are difficult to diagnose and outcome is often fatal. Over the 7-month study period, 117 cases with haematological malignancies receiving systemic antifungal treatment were included. Data regarding antifungal agents, dosage and reason for administration were recorded. Fungal infections in study patients were classified as possible, probable or proven according to recent European Organization for Research and Treatment of Cancer criteria. During the study period, 690 cases with haematological malignancies were admitted. A total of 117 cases received systemic antifungal therapy. Twenty-four of 117 patients (21%) had possible, six (5.1%) had probable and four (3.4%) had proven IFI. Seven of 10 probable and proven infections were caused by Candida spp., 2 by Aspergillus spp. and 1 by a fungus belonging to Zygomycetes. Fifty-two of 117 patients (44%) received antifungal prophylaxis, 81 of 117 (69%) received empirical (31/117; 26%) or pre-emptive (50/117; 43%) antifungal therapy and four of 117 patients (3.4%) directed antifungal therapy. Mostly, systemic antifungal therapy was administered empirically or pre-emptively. Twenty-nine per cent of cases receiving systemic antifungal treatment met the international consensus criteria of mostly possible IFI, whereas 71% did not. Proven invasive fungal infections were rare.

Therapy-related myeloid neoplasms: pathobiology and clinical characteristics.

Therapy-related myeloid neoplasms (t-MNs) are serious long-term consequences of cytotoxic treatments for an antecedent disorder. t-MNs are observed after ionizing radiation as well as conventional chemotherapy including alkylating agents, topoisomerase-II-inhibitors and antimetabolites. In addition, adjuvant use of recombinant human granulocyte-colony stimulating factor may also increase the risk of t-MNs. There is clinical and biological overlap between t-MNs and high-risk de novo myelodysplastic syndromes and acute myeloid leukaemia suggesting similar mechanisms of leukaemogenesis. Human studies and animal models point to a prominent role of genetic susceptibilty in the pathogenesis of t-MNs. Common genetic variants have been identified that modulate t-MN risk, and t-MNs have been observed in some cancer predisposition syndromes. In either case, establishing a leukaemic phenotype requires acquisition of somatic mutations - most likely induced by the cytotoxic treatment. Knowledge of the specific nature of the initiating exposure has allowed the identification of crucial pathogenetic mechanisms and for these to be modelled in vitro and in vivo. Prognosis of patients with t-MNs is dismal and at present, the only curative approach for the majority of these individuals is haematopoietic stem cell transplantation, which is characterized by high transplant-related mortality rates. Novel transplantation strategies using reduced intensity conditioning regimens as well as novel drugs - demethylating agents and targeted therapies - await clinical testing and may improve outcome. Ultimately, individual assessment of genetic risk factors may translate into tailored therapies and establish a strategy for reducing t-MN incidences without jeopardizing therapeutic success rates for the primary disorders.

Loss of RAF kinase inhibitor protein is a somatic event in the pathogenesis of therapy-related acute myeloid leukemias with C-RAF germline mutations.

We recently described oncogenic and anti-apoptotic C-RAF germline mutations in patients with therapy-related acute myeloid leukemia (t-AML). Activation of the RAF effector ERK was restricted to transformed cells, suggesting the requirement for cooperating events in leukemogenesis. Western blot analysis of blast cells from patients with C-RAF germline mutations revealed loss of the tumor and metastasis suppressor RAF kinase inhibitor protein (RKIP). Immunohistochemistry of the patients' primary tumors revealed normal RKIP expression levels, indicating that the loss of RKIP is a somatic, t-AML-specific event. In focus formation assays, the oncogenic potential of human mutant C-RAF was strongly influenced by expression levels of RKIP. Although the number of colonies formed by C-RAF(S427G) was significantly increased by RKIP silencing, the opposite was observed after RKIP overexpression. These results show that the loss of RKIP is a functional somatic event in carriers of C-RAF germline mutations, which contributes to the development of t-AML.

Enhanced thrombin generation in plasma of severe thrombocytopenic patients due to rFVIIa.

UNLABELLED: RFVIIa-enhanced thrombin generation has been shown to be dependent on platelets. In previous work we have shown that addition of monocytes and rFVIIa to microparticle free plasma causes a distinct thrombin generation. The aim of our study has been to examine whether there is enough surface provided by microparticles in thrombocytopenic plasma to allow an effect of rFVIIa. PATIENTS, METHODS: Thrombin generation was measured in platelet rich plasma (PRP) and microparticle free plasma (MFP) of thrombocytopenic haemato-oncological patients with and without addition of rVIIa by means of calibrated automated thrombography. Microparticles were analyzed in PRP by FACS flow cytometry. RESULTS: Microparticle free plasma showed no thrombin generation with or without addition of rFVIIa. Addition of rFVIIa to PRP of thrombocytopenic patients led to a significant shortening of lag time and time to peak in thrombin generation, while ETP and peak remained unchanged. CONCLUSION: Our results show that even in plasma of severe thrombocytopenic patients enough surface may be provided by microparticles to allow an enhancement of thrombin generation by rFVIIa.

Iron overload in myelodysplastic syndromes (MDS) - diagnosis, management, and response criteria: a proposal of the Austrian MDS platform.

Transfusion-related morbidity is an emerging challenge in chronically transfused patients with low-risk myelodysplastic syndromes (MDS). In these patients, transfusion-induced iron overload may represent a leading medical problem. However, although iron-chelating drugs are available, little is known about optimal diagnostic tools, predisposing factors, and the optimal management of these patients. In the current article, we provide recommendations for the diagnosis, prevention and treatment of iron overload in MDS and propose treatment response criteria. Consensus criteria and resulting recommendations were discussed and formulated by members of the MDS platform of the Austrian Society of Haematology and Oncology in a series of meetings and conferences in 2006 and 2007. These recommendations should facilitate and assist in recognition of iron overload, selection of patients, timing of treatment, drug selection and the measurement of treatment responses.

Evaluation of potential risk factors for early infectious complications after autologous peripheral blood stem cell transplantation in patients with lymphoproliferative diseases.

A number of risk factors for the occurrence of neutropaenic fever after haematopoietic stem cell transplantation (HSCT) have been proposed. We were interested in whether these factors remain valid for several early infection-related outcomes when applied to a homogeneous group of patients in uni- and multivariate analyses. Therefore, we analysed 144 consecutive patients with lymphoproliferative disorders receiving autologous peripheral blood HSCT. Variables tested as potential risk factors for the occurrence of fever, documented infection (DI), microbiologically documented infection (MDI) or failure of first-line antimicrobial therapy were sex, conditioning regimen, prolonged neutropaenia, low number of CD34+ cells transplanted, purging, lack of selective gut decontamination, higher age and increased body mass index. In uni- and multivariate analyses, conditioning including total body irradiation was the only risk factor for the occurrence of fever, and neutropaenia >or=10 days was the only factor associated with failure of first-line antimicrobial therapy. None of the variables tested was associated with an increased risk for DI or MDI. This analysis suggests that a number of previously proposed risk factors actually are of minor clinical relevance for early infections in the majority of patients receiving autologous HSCT.

Pulmonary infiltrates in patients with haematologic malignancies: transbronchial lung biopsy increases the diagnostic yield with respect to neoplastic infiltrates and toxic pneumonitis.

We retrospectively evaluated 107 fiberoptic bronchoscopies with and without transbronchial lung biopsy (TBLB) in 98 consecutive patients with haematologic malignancies and pulmonary infiltrates. Bronchoalveolar lavage (BAL) was performed in 45 and BAL plus TBLB in 62 procedures. There was no procedure-related severe haemorrhage, pneumothorax or death. Infectious aetiology was identified in 26 of 107 (24%), toxic pneumonitis in 17 of 107 (16%) and neoplastic infiltration in 9 of 107 (8.5%) episodes. Combined BAL and TBLB was significantly superior to BAL alone with respect to the diagnosis of neoplastic infiltrates (p=0.008) and toxic pneumonitis (p<0.001) and should therefore be included in the diagnostic work-up of this patient cohort.

RT-PCR and FISH analysis of acute myeloid leukemia with t(8;16)(p11;p13) and chimeric MOZ and CBP transcripts: breakpoint cluster region and clinical implications.

The translocation t(8;16)(p11;p13) is associated with acute myeloid leukemia displaying monocytic differentiation (AML FAB M4/5) and fuses the MOZ (also named MYST3) gene (8p11) with the CBP (also named CREBBP) gene (16p13). Detection of the chimeric RNA fusions has proven difficult; only three studies have described successful amplification of the chimeric MOZ-CBP and CBP-MOZ fusions by reverse transcriptase-polymerase chain reaction (RT-PCR). We analyzed four cases of AML M4/5 with t(8;16)(p11;p13) by RT-PCR and fluorescence in situ hybridization (FISH) and characterized the reciprocal RNA fusions from three cases. We cloned both genomic translocation breakpoints from one case by long-range PCR and successfully applied RT-PCR to monitor minimal residual disease (MRD) between clinical complete remission and relapse. In three cases, the genomic breakpoints occurred in MOZ intron 16 and CBP intron 2. In one case, no fusion transcript was detected. The available data suggest clustering of t(8;16)(p11;p13) breakpoints in these introns leading to reciprocal in-frame MOZ exon 16/CBP exon 3 and in-frame CBP exon 2/MOZ exon 17 chimeric transcripts in the majority of cases. The described RT-PCR strategy may be valuable both for the routine detection of the t(8;16)(p11;p13) as well as for monitoring of MRD in this prognostically unfavorable patient group.

High expression of the sister-chromatid separation regulator and proto-oncogene hSecurin occurs in a subset of myeloid leukaemias but is not implicated in the pathogenesis of aneuploidy.

Aneuploidy is considered to play an important role in the pathogenesis of malignancies. We were interested whether abnormalities of the sister-chromatid separation regulator and proto-oncogene hSecurin occurred in myeloid leukaemias, and whether such abnormalities correlated with aneuploidy. The expression of hSecurin was assessed by real-time quantitative PCR in samples from patients with acute myeloid leukaemia (AML, n=70), chronic myeloid leukaemia (CML) in chronic phase (CP, n=20) or blast phase (BP, n=12), and granulocytes as well as mononuclear cells (MNCs) from healthy donors (n=21). Median hSecurin expression in AML with normal karyotypes was not significantly different from AML showing aneuploidy, CML BP or cells from healthy donors. However, hSecurin expression in CML CP was significantly increased compared to AML with normal karyotypes (1.82-fold; P<0.001), CML BP (3.18-fold; P<0.001), MNCs (3.17-fold; P<0.001) and granulocytes (2.69 fold; P<0.001) from healthy donors. Mutations in the coding region of hSecurin were not detected. These results do not support a major role of hSecurin in the development of aneuploidy in myeloid leukaemias. However, high expression of hSecurin may be of pathogenetic relevance in a subset of patients with regard to its potential to stimulate angiogenesis and to interact with the DNA-damage response pathway.

High-risk AML complicated by pulmonary aspergillosis: successful treatment with nonmyeloablative stem cell transplantation and long-term administration of voriconazole.

Acute myeloid leukemia (AML) associated with central diabetes insipidus (DI) and chromosomal aberrations is characterised by a very poor prognosis. We present a 28-year-old female with AML FAB M0, preceding DI and cytogenetic abnormalities (monosomy 7 and inversion of chromosome 9). Complete remission was achieved with FLAG after she was refractory to two different induction regimens. Prolonged neutropenia resulted in invasive pulmonary aspergillosis. Allogeneic stem cell transplantation from a matched unrelated donor was performed using a reduced-intensity conditioning regimen. Desmopressin substitution for DI was withdrawn after transplant without recurrence of symptoms. Initial antifungal treatment, including liposomal amphotericin B, caspofungin and itraconazole, was replaced by voriconazole after deterioration of pulmonary aspergillosis, resulting in improvement, stabilisation and finally, also as the combined effect of discontinuation of the immunosuppressive therapy, in disappearance of signs and symptoms. Thirteen months after transplant, the patient is in continuous complete remission. The presented case study thus demonstrates that high-risk AML with concomitant invasive fungal infection may be safely and effectively treated by nonmyeloablative stem cell transplantation and long-term administration of voriconazole.