[Show abstract][Hide abstract] ABSTRACT: Although mast cells (MC) play an important role in allergic reactions, their physiologic role remains unknown. In mice, several models of MC-deficiency have been developed. However, no comparable human model is available. We examined the in vitro- and in vivo effects of the KIT-targeting drug imatinib on growth and development of human MC. Imatinib was found to inhibit stem cell factor (SCF)-induced differentiation of MC in long-term suspension cultures (IC50: 0.01 µM). Correspondingly, long-term treatment of chronic myeloid leukemia (CML) patients with imatinib (400 mg/day) resulted in a marked decrease in MC. In patients with continuous complete molecular response during therapy, bone marrow MC decreased to less than 5% of pre-treatment values, and also serum tryptase concentrations decreased significantly (pre-treatment: 32.0±11.1 ng/ml; post-therapy: 3.4±1.8, p<0.01). Other myeloid lineages, known to develop independently of KIT, were not affected by imatinib-therapy. Imatinib also produced a substantial decrease in MC-development in mice. However, no clinical syndrome attributable to drug-induced MC-deficiency was recorded in our CML patients. Together, imatinib suppresses MC production in vitro and in vivo. However, drug-induced MC depletion is not accompanied by adverse clinical events, suggesting that MC are less relevant to homeostasis in healthy tissues than we assumed so far.
[Show abstract][Hide abstract] ABSTRACT: The Austrian myelodysplastic syndromes (MDS) Platform was founded as a national working group in 2003 to initiate and coordinate common projects in the field. The incidence of MDS in Austria is approximately 400-500 new MDS cases per year. The overall low number of MDS patients underlines the importance of a national initiative to concentrate knowledge at certain specialized centres, where treatment of these patients mainly takes place. Clinical trials as well as basic research are facilitated by the cooperation of university and non-university hospitals. Other objectives are the generation of therapeutic standards, organization of meetings to spread this information to physicians and patients as well as promoting patient-support groups. Cooperation with international working groups is another important aim of the Platform. The 10th anniversary of the Austrian MDS Platform was organized as a meeting for all interested physicians throughout Austria providing an update on the disease and ongoing projects.
Wiener klinische Wochenschrift 11/2014; · 0.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated sinks as possible sources of a prolonged KPC-producing K. oxytoca outbreak. Seven carbapenem resistant K. oxytoca isolates were identified in sink drains in 4 patient rooms and in the medication room. Investigations for resistance genes and genetic relatedness of patient and environmental isolates revealed that all isolates harboured the blaKPC-2 and blaTEM-1 and were genetically indistinguishable. We describe a clonal outbreak caused by KPC-2-producing K. oxytoca pointing to handwashing sinks as possible reservoir.
Antimicrobial Agents and Chemotherapy 10/2014; · 4.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Familial colorectal cancer type X (FCCTX) is characterized by clinical features of hereditary non-polyposis colorectal cancer with a yet undefined genetic background. Here we identify the SEMA4A p.Val78Met germline mutation in an Austrian kindred with FCCTX, using an integrative genomics strategy. Compared with wild-type protein, SEMA4A(V78M) demonstrates significantly increased MAPK/Erk and PI3K/Akt signalling as well as cell cycle progression of SEMA4A-deficient HCT-116 colorectal cancer cells. In a cohort of 53 patients with FCCTX, we depict two further SEMA4A mutations, p.Gly484Ala and p.Ser326Phe and the single-nucleotide polymorphism (SNP) p.Pro682Ser. This SNP is highly associated with the FCCTX phenotype exhibiting increased risk for colorectal cancer (OR 6.79, 95% CI 2.63 to 17.52). Our study shows previously unidentified germline variants in SEMA4A predisposing to FCCTX, which has implications for surveillance strategies of patients and their families.
[Show abstract][Hide abstract] ABSTRACT: Please indicate your presentation preference: No Preference Background: Therapy-related myeloid neoplasms (t-MNs) are thought to arise due to mutational events in hematopoietic stem and precursor cells induced by cytotoxic treatments for a primary disorder. However, no consistent biomarker has been identified yet that unambiguously classifies a particular neoplasm as "therapy-related". This raises the possibility that other mechanisms may also be operational in their pathogenesis. Aims: We postulated that leukemia-specific mutations were pre-existing in some of these patients and focused on the TP53 gene which is frequently mutated in t-MNs. Methods: We used Sanger sequencing of paired samples -t-MN and constitutional material -for initial mutation identification and rearrangement-specific PCR for the detection of a TP53 duplication in pre-leukemic specimens, respectively. Quantitative PCR was performed to semi-quantify the mutated clone and Ion torrent deep sequencing (Life Technologies, Carlsbad, CA) to search for co-operating mutations. Results: We screened 53 t-MN specimens for TP53 mutations and identified one patient with a somatic heterozygous 64-base pair duplication (NM_000546.5:c.276_339dup:p.Leu114Profs*31) who developed therapy-related acute myeloid leukemia (t-AML) with complex karyotype (46-50,XY,del(5)(q12q33),?r(7)(p22q11)[cp20]) 13 years after combined modality treatment for Hodgkin's lymphoma. This duplication was particularly amenable for detection by a highly sensitive PCR assay enabling the detection of 0.01% rearranged cells. We could not only unambiguously detect the presence of TP53 mutated cells in the patient´s pre-treatment bone marrow but also in a lymphadenitis sample obtained seven years before lymphoma diagnosis. Quantitative PCR estimated the amount of affected bone marrow cells as <1% as compared to the t-AML specimen. Analysis of the PTEN and PHF6 genes by deep sequencing using bone marrow and leukemia specimens did not show evidence of co-operating mutations. Summary/Conclusion: The fact that a leukemia-specific TP53 mutation is already present before any chemo-or radiotherapy has been administered suggests a different mode of therapy-related leukemogenesis than currently assumed. Instead of inducing a leukemia-specific mutation, cytotoxic treatments have facilitated the expansion of a pre-leukemic clone in this patient.
19th Congress of the European Hematology Association, Milan, Italy; 06/2014
[Show abstract][Hide abstract] ABSTRACT: Acute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations.
The expression of 636 human miRNAs was compared between samples from 52 patients with AML and 13 healthy individuals by highly specific locked nucleic acid (LNA) based microarray technology. The levels of individual mature miRNAs and of primary miRNAs (pri-miRs) were determined by quantitative reverse transcriptase (qRT) PCR. Transfections and infections of human cell lines were performed using standard procedures.
64 miRNAs were significantly differentially expressed between AML and controls. Further studies on the clustered miRNAs 221 and 222, already known to act as oncogenes in other tumor types, revealed a deficiency of human myeloid cell lines to process vector derived precursor transcripts. Moreover, endogenous pri-miR-221/222 was overexpressed to a substantially higher extent than its mature products in most primary AML samples, indicating that its transcription was enhanced, but processing was rate limiting, in these cells. Comparison of samples from the times of diagnosis, remission, and relapse of AML demonstrated that pri-miR-221/222 levels faithfully reflected the stage of disease.
Expression of some miRNAs is strongly regulated at the posttranscriptional level in AML. Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease.
BMC Cancer 07/2013; 13(1):364. · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Circulating tumor cells (CTCs) released into blood from primary cancers and metastases reflect the current status of tumor genotypes, which are prone to changes. Here we performed the first comprehensive genomic profiling of CTCs using array-CGH and next-generation sequencing. We used the FDA-cleared CellSearchTM system, which detected CTCs in 21 of 37 patients (range 1-202 per 7.5 ml sample) with stage IV colorectal carcinoma (CRC). In total, we were able to isolate 37 intact CTCs from 6 patients and identified in those multiple CRC-associated copy number changes, many of which were also present in the respective primary tumor. We then used massive parallel sequencing of a panel of 68 CRC-associated genes to compare the mutation spectrum in the primary tumors, metastases, and the corresponding CTCs from two of these patients. Mutations in known driver genes (e.g., APC, KRAS, or PIK3CA) found in the primary tumor and metastasis were also detected in corresponding CTCs. However, we also observed mutations exclusively in CTCs. To address whether these mutations were derived from a small subclone in the primary tumor or represented new variants of metastatic cells, we performed additional deep sequencing of the primary tumor and metastasis and applied a customized statistical algorithm for analysis. We found that most mutations initially found only in CTCs were also present at subclonal level in the primary tumors and metastases from the same patient. Our study paves the way to use CTCs as a liquid biopsy in patients with cancer.
[Show abstract][Hide abstract] ABSTRACT: Background: Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy with a dismal outcome. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet. Also, little is known about the regulation of miRNA expression. Further investigations are therefore warranted.
Methods: The expression of 636 human miRNAs was compared between samples from 52 patients with AML and 13 healthy individuals by highly specific locked nucleic acid (LNA) based microarray technology. The levels of individual mature miRNAs and of primary miRNAs (pri-miRs) were determined by quantitative reverse transcriptase (qRT) PCR. Transfections and infections of human cell lines were performed using standard procedures.
Results: 64 miRNAs were significantly differentially expressed between AML and controls. Further studies on the clustered miRNAs 221 and 222, already known to act as oncogenes in other tumor types, revealed a deficiency of human myeloid cell lines to process vector derived precursor transcripts. More importantly, endogenous pri-miR-221/222 was overexpressed to a substantially higher extent than its mature products in most primary AML samples, indicating that its transcription was enhanced, but processing was rate limiting, in these cells. Comparison of samples from the times of diagnosis, remission, and relapse of AML demonstrated that pri-miR-221/222 levels faithfully reflected the stage of disease.
Conclusions: Expression of certain miRNAs is strongly regulated at the posttranscriptional level in AML. Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease.
[Show abstract][Hide abstract] ABSTRACT: Mutations in DNA methyltransferase 3A (DNMT3A) have been described at high frequency in acute myeloid leukemia (AML) particularly in subgroups with a monocytic phenotype. It has been shown that DNMT3A can alter gene expression by inducing hyper- and hypometylation of distinct gene loci including non-promoter regions. We recently demonstrated that expression of the tumor and metastasis suppressor RAF kinase inhibitor protein (RKIP) is frequently lost in AML and - similar to mutated DNMT3A - correlates with a monocytic phenotype. As the mechanisms behind RKIP silencing could not be elucidated so far, we investigated a possible correlation between loss of RKIP and mutations in DNMT3A in monocytic AML. In this study we detected one novel somatic DNMT3A substitution (R676W) and could confirm the high frequency of DNMT3A mutations in monocytic AML. Importantly, mutations in DNMT3A occurred independently of RKIP loss, suggesting that RKIP silencing is not mediated by mutations in DNMT3A.
[Show abstract][Hide abstract] ABSTRACT: Therapy related myeloid neoplasms (t-MNs) are complex diseases originating from an interplay between exogenous toxicities and a susceptible organism. It has been hypothesised that in a subset of cases t-MNs develop in the context of hereditary cancer predisposition syndromes.
The study systematically evaluated pedigrees of patients with t-MNs for cancer incidences and the possibility of a hereditary cancer predisposition syndrome. In addition, mutational analyses were performed using constitutional DNA from index patients, and deleterious heterozygous germline mutations were assessed for loss of heterozygosity in sorted leukaemic cells by single nucleotide polymorphism array.
A nuclear pedigree was obtained in 51/53 patients with t-MNs resulting in a total of 828 individuals analysed. With a standardised incidence ratio of 1.03 (95% CI 0.74 to 1.39), the tumour incidence of first- degree relatives was not increased. However, six pedigrees were suggestive for a hereditary breast and ovarian cancer syndrome, three of a Li-Fraumeni like syndrome, and three index patients showed multiple primary neoplasms. Mutational analysis revealed two BRCA1 (c.3112G→T, c.5251C→T), one BRCA2 (c.4027A→G), two BARD1 (C557S) and four TP53 germline mutations (g.18508_18761delinsGCC, c.847C→T, c.845_848dupGGCG, c.1146delA) in nine of 53 (17%) index patients with t-MNs. Loss of heterozygosity in leukaemic cells was demonstrated for the BRCA1c.3112G→T and TP53c.845_848dupGGCG mutations, respectively.
It is concluded that a proportion of patients with t-MNs carry cancer susceptibility mutations which are likely to contribute to therapy related leukaemogenesis.
Journal of Medical Genetics 05/2012; 49(7):422-8. · 5.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Posaconazole (PCZ) is a triazole antifungal agent that has broad activity against pathogenic fungi and is increasingly used for prophylaxis and treatment of invasive mould infections (IMIs). PCZ is only available as an oral formulation, with varying absorption from the gastrointestinal tract. However, reports correlating PCZ plasma concentrations (PPCs) with breakthrough IMIs are rare. In this study, PPCs were analysed in a prospective, observational, single-centre study and the correlation of PPCs with breakthrough IMIs in patients with haematological malignancies was evaluated. Risk factors associated with low PPCs were further evaluated. A total of 109 PPCs were measured in 34 cases receiving PCZ prophylaxis (n=31) or treatment (n=3). Levels below the target of 0.5 μg/mL were detected in 24 (71%) of the 34 cases; in 15 (63%) of these 24 cases concentrations were found to be <0.20 μg/mL. Three patients receiving PCZ prophylaxis met the criteria of breakthrough infection. Notably, prior to development of IMI, PPCs were below the target in all three individuals. Associated risk factors for insufficient PPCs varied from previous reports. In conclusion, these data demonstrate that therapeutic drug monitoring of PCZ is mandatory in all patients with haematological malignancies as low PPCs are common and may be associated with development of IMIs.
International journal of antimicrobial agents 04/2012; 39(6):510-3. · 3.03 Impact Factor