Maximilian Mossner

Long-Term Control of HIV by CCR5 Delta32/Delta32 Stem-Cell Transplantation

Infection with the human immunodeficiency virus type 1 (HIV-1) requires the presence of a CD4 receptor and a chemokine receptor, principally chemokine receptor 5 (CCR5). Homozygosity for a 32-bp deletion in the CCR5 allele provides resistance against HIV-1 acquisition. We transplanted stem cells from a donor who was homozygous for CCR5 delta32 in a patient with acute myeloid leukemia and HIV-1 infection. The patient remained without viral rebound 20 months after transplantation and discontinuation of antiretroviral therapy. This outcome demonstrates the critical role CCR5 plays in maintaining HIV-1 infection.

Myelodysplastic Cells in Patients Reprogram Mesenchymal Stromal Cells to Establish a Transplantable Stem Cell Niche Disease Unit

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.

SNP array analysis of tyrosine kinase inhibitor-resistant chronic myeloid leukemia identifies heterogeneous secondary genomic alterations

To elucidate whether tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia is associated with characteristic genomic alterations, we analyzed DNA samples from 45 TKI-resistant chronic myeloid leukemia patients with 250K single nucleotide polymorphism arrays. From 20 patients, matched serial samples of pretreatment and TKI resistance time points were available. Eleven of the 45 TKI-resistant patients had mutations of BCR-ABL1, including 2 T315I mutations. Besides known TKI resistance-associated genomic lesions, such as duplication of the BCR-ABL1 gene (n = 8) and trisomy 8 (n = 3), recurrent submicroscopic alterations, including acquired uniparental disomy, were detectable on chromosomes 1, 8, 9, 17, 19, and 22. On chromosome 22, newly acquired and recurrent deletions of the IGLC1 locus were detected in 3 patients, who had previously presented with lymphoid or myeloid blast crisis. This may support a hypothesis of TKI-induced selection of subclones differentiating into immature B-cell progenitors as a mechanism of disease progression and evasion of TKI sensitivity.

Diagnosis of invasive fungal infections in haematological patients by combined use of galactomannan, 1,3-β-D-glucan, Aspergillus PCR, multifungal DNA-microarray, and Aspergillus azole resistance PCRs in blood and bronchoalveolar lavage samples: results of a prospective multicentre study

High mortality rates of invasive fungal disease (IFD), especially invasive aspergillosis (IA), in immunocompromised haematological patients and current diagnostic limitations require improvement of detection of fungal pathogens by defining the optimal use of biomarkers and clinical samples. Concurrent bronchoalveolar lavage (BAL) and peripheral blood samples of 99 haematological patients with suspected IFD were investigated within a multicentre prospective study. Diagnostic performance of a galactomannan (GM) enzyme immune assay (EIA), a 1,3-β-D-glucan assay (BDG), an Aspergillus PCR, and a multifungal DNA-microarray (Chip) alone or in combination were calculated. IFD were classified as proven (n=3), probable (n=34), possible (n=33), and no IFD (n=29) according to EORTC/MSG criteria. GM, PCR, and Chip showed superior diagnostic performance in BAL than in blood, whereas specificity of BDG in BAL was poor (48% (14/29)). The combination of GM (BAL) with BDG (blood) showed sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and DOR (diagnostic odds ratio) of 92% (34/37), 93% (27/29), 94%, 90%, and 153.0, respectively. Combining GM (BAL) with PCR (BAL) showed convincing diagnostic potential for diagnosing IA with sensitivity, specificity, PPV, NPV, and DOR of 85% (17/20), 97% (28/29), 94%, 90%, and 158.7. Addition of the DNA-microarray resulted in further detection of two mucormycetes infections. In 1 out of 15 Aspergillus DNA-positive samples a triazole resistance-mediating Cyp51A mutation was found. Combination of biomarkers is superior to their sole use in diagnosing IFD, particularly IA. Integrating blood and BAL samples into a diagnostic algorithm is an advantageous approach.

Transcriptional down-regulation of the Wnt antagonist SFRP1 in haematopoietic cells of patients with different risk types of MDS

Secreted frizzled related protein 1 (SFRP1) is an extracellular antagonist of the Wnt signalling pathway that plays an important role in the pathogenesis of solid tumours and haematopoietic malignancies. SFRP1 has been observed to be transcriptionally down-regulated due to hypermethylation in acute and chronic leukaemia, but so far not in myelodysplastic syndrome (MDS). Moreover, it has been shown that the epigenetic inactivation of SFRP1 correlates with an overexpression of the Wnt receptor Frizzled 3 (Fzd3) in acute leukaemia. Using real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) we examined mRNA expression of SFRP1 and Fzd3 in bone marrow cells derived from 121 patients with different risk types of MDS, acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL). We employed pyrosequencing to quantify promoter DNA methylation in MDS and acute leukaemia. We detected significant lower mRNA transcription of SFRP1 in MDS compared to healthy individuals. However, DNA sequence mutations or frequent elevated DNA methylation levels of the SFRP1 promoter could not be observed in MDS but in AML and ALL as previously reported. The expression levels of Fzd3 were up-regulated in both acute leukaemia and MDS. Our data show a significant transcriptional down-regulation of SFRP1 as a common event in AML, ALL and - as demonstrated for the first time - in MDS. An inactivation of SFRP1 and the transcriptional up-regulation of Fzd3 seem to be associated with an activation of the Wnt signalling pathway in these haematopoietic diseases.

Expression of IGFBP7 in acute leukemia is regulated by DNA methylation

The important role of insulin‐like growth factor binding protein 7 (IGFBP7) as a tumor suppressor in solid tumors has been revealed in several studies. Interestingly, in a recent study IGFBP7 was also shown to be aberrantly expressed in acute leukemia. Moreover, in acute T‐lymphoblastic leukemia (T‐ALL), high IGFBP7 expression predicts primary therapy resistance. In order to elucidate the mechanisms underlying aberrant IGFBP7 expression, we used pyrosequencing technology to investigate the DNA methylation of IGFBP7 in 109 T‐ALL patient samples. Aberrant methylation was shown and hypomethylation was associated with an early immunophenotype and co‐expression of the stem cell markers CD117 (P < 0.001) and CD34 (P < 0.001). In concordance, gene expression profiles of 86 T‐ALL patients revealed upregulation of stem cell markers (CD34 and CD133) as well as genes associated with poor outcome and pathogenesis of leukemia (MN1, BAALC, FLT3) in the high IGFBP7 expression group. In conclusion, aberrant IGFBP7 expression is regulated by DNA methylation in acute leukemia. Hypomethylation of the gene is likely to characterize an immature and a more malignant subtype of the disease. (Cancer Sci 2011; 102: 253–259)

Skewed X-inactivation patterns in ageing healthy and myelodysplastic haematopoiesis determined by a pyrosequencing based transcriptional clonality assay

Background Investigation of X-chromosome inactivation patterns (XCIP) by determination of differential CpG-methylation has been widely applied for investigation of female cell clonality. Using this approach the clonal origin of various tumours has been corroborated. Controversially, strong age-related increase of peripheral blood (PB) cell clonality in haematologically healthy female subjects was reported. Recently, transcriptional XCIP ratio analysis challenged these results and questioned the suitability of methylation based clonality assays. Methods To reinvestigate XCIP-skewing in CD34, low-density mononuclear bone marrow (BM) as well as PB cells from healthy female subjects and patients with myelodysplastic syndromes (MDS), we established a transcriptional assay using pyrosequencing technique for quantification of single nucleotide polymorphism allele frequencies, representative for XCIP ratios. Results Our assay provides high sensitivity for XCIP ratio assessment as determined by standard curves, reproducibility, inter-marker correlation as well as correlation with the DNA-methylation based human androgen receptor (HUMARA) assay. Notably, in agreement with most studies investigating this issue, significant age-related increase of XCIP skewing in PB cells from healthy elderly female subjects was confirmed. Moreover, XCIP ratio analysis suggests even stronger clonal manifestation in BM and CD34 cells. In MDS, XCIP skewing levels were distinctively elevated as compared with controls of similar age and higher degrees were associated with poor clinical outcome. Conclusions Transcriptional clonal profiling via pyrosequencing allows accurate assessment of XCIP ratios, confirms the validity of the DNA-methylation based HUMARA assay and reveals important insights into ageing healthy and myelodysplastic haematopoiesis.

Genome-wide DNA-mapping of CD34+ cells from patients with myelodysplastic syndrome using 500K SNP arrays identifies significant regions of deletion and uniparental disomy

OBJECTIVE Identification of genomic lesions in progenitor cells of patients with myelodysplastic syndrome (MDS) could lead to the discovery of new disease-specific genes and may be of prognostic value. MATERIALS AND METHODS We carried out a genome-wide mapping of DNA from CD34+ cells of MDS patients with high-resolution 500K single nucleotide polymorphism arrays and a concomitant integration with global gene expression analysis. Thirteen MDS patients were analyzed. RESULTS Copy number and loss of heterozygosity analyses detected heterozygous deletions on chromosomes 2, 9, 13, 16, 17, and 20 ranging in size from 0.1 megabases (Mba) to 2.1 Mba. Additionally, numerous regions with significant uniparental disomy were detected. Integration of the genomic data with gene expression analysis showed that genes, which were downregulated at least 1.5-fold in regions of significant deletion and uniparental disomy were exclusively downregulated in those samples displaying the aberration. Genomics and gene expression data were confirmed by real-time polymerase chain reaction and variable number tandem repeat analysis. CONCLUSION High-density genomic mapping of CD34+ bone marrow cells from patients with MDS identifies cryptic genetic lesions and offers new opportunities for the discovery of target genes in MDS by integration with gene expression analysis.

Prevalence, clonal dynamics and clinical impact of TP53 mutations in patients with myelodysplastic syndrome with isolated deletion (5q) treated with lenalidomide: results from a prospective multicenter study of the german MDS study group (GMDS).

Prevalence, clonal dynamics and clinical impact of TP53 mutations in patients with myelodysplastic syndrome with isolated deletion (5q) treated with lenalidomide: results from a prospective multicenter study of the german MDS study group (GMDS)

Variegated clonality and rapid emergence of new molecular lesions in xenografts of acute lymphoblastic leukemia are associated with drug resistance

The use of genome-wide copy-number analysis and massive parallel sequencing has revolutionized the understanding of the clonal architecture of pediatric acute lymphoblastic leukemia (ALL) by demonstrating that this disease is composed of highly variable clonal ancestries following the rules of Darwinian selection. The current study aimed to analyze the molecular composition of childhood ALL biopsies and patient-derived xenografts with particular emphasis on mechanisms associated with acquired chemoresistance. Genomic DNA from seven primary pediatric ALL patient samples, 29 serially passaged xenografts, and six in vivo selected chemoresistant xenografts were analyzed with 250K single-nucleotide polymorphism arrays. Copy-number analysis of non-drug-selected xenografts confirmed a highly variable molecular pattern of variegated subclones. Whereas primary patient samples from initial diagnosis displayed a mean of 5.7 copy-number alterations per sample, serially passaged xenografts contained a mean of 8.2 and chemoresistant xenografts a mean of 10.5 copy-number alterations per sample, respectively. Resistance to cytarabine was explained by a new homozygous deletion of the DCK gene, whereas methotrexate resistance was associated with monoallelic deletion of FPGS and mutation of the remaining allele. This study demonstrates that selecting for chemoresistance in xenografted human ALL cells can reveal novel mechanisms associated with drug resistance.