Verena Nowak

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.

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)

Prognostic importance of expression of the Wilms’ tumor 1 gene in newly diagnosed acute promyelocytic leukemia

Wilms’ tumor 1 gene (WT1) is known to be highly expressed in acute promyelocytic leukemia (APL) but information on its impact on prognosis is lacking. WT1 expression was analyzed in bone marrow samples of 79 patients with APL at initial diagnosis. Patients had a differing outcome according to their level of WT1 expression. In patients who achieved a complete remission (CR), low or high WT1 expression was significantly associated with inferior overall survival (OS) compared to intermediate WT1 expression (49% for WT1high vs. 63% for WT1low vs. 93% for WT1int; p = 0.008). Moreover, there were significant differences in relapse-free survival (RFS) between the three expression groups (42% for WT1high vs. 63% for WT1low vs. 83% for WT1int; p = 0.047). In multivariable analysis WT1 expression showed an independent prognostic impact on OS of responders to induction therapy. In conclusion, the level of WT1 expression can add prognostic information in APL risk stratification.

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.

In acute promyelocytic leukemia (APL) low BAALC gene expression identifies a patient group with favorable overall survival and improved relapse free survival

We evaluated the prognostic value of BAALC expression in 86 patients with acute promyelocytic leukemia (APL). At 10 years, the overall survival (OS) was 66% in all patients and 75% in patients who achieved a complete remission (CR). Patients in the BAALC(low) group showed an OS of 87% as compared to 60% in the BAALC(high) group (p=0.019). This difference was more pronounced in treatment responders (92% vs. 70%; p=0.035). In multivariate analyses low BAALC expression retained its prognostic relevance. In conclusion, BAALC expression analysis might be useful in further risk stratification in APL patients.

A molecular risk score integrating BAALC, ERG and WT1 expression levels for risk stratification in acute promyelocytic leukemia

To date risk stratification in acute promyelocytic leukemia (APL) is based on highly dynamic leukocyte and platelet counts only. To identify a more robust risk stratification model, a molecular risk score was developed based on expression levels of the genes BAALC, ERG and WT1. Hereby, the main focus was on prediction of relapse. The integrative risk score divided patients into two groups with highly significant differences in outcome. It discriminated a high risk group with a high incidence of relapse successfully from a low risk group with no APL-related events after achievement of first remission. Especially the concurrent presence of molecular risk factors showed to be a negative prognostic factor in APL. The molecular risk score might be a promising approach to guide monitoring of APL patients and therapeutic decisions in the future.

Genomic CDKN2A/2B deletions in adult Ph+ ALL are adverse despite allogeneic stem cell transplantation

We investigated the role of copy number alterations to refine risk stratification in adult Philadelphia chromosome positive (Ph)+ acute lymphoblastic leukemia (ALL) treated with tyrosine kinase inhibitors (TKIs) and allogeneic stem cell transplantation (aSCT). Ninety-seven Ph+ ALL patients (median age 41 years; range 18-64 years) within the prospective multicenter German Multicenter ALL Study Group studies 06/99 (n = 8) and 07/2003 (n = 89) were analyzed. All patients received TKI and aSCT in first complete remission (CR1). Copy number analysis was performed with single nucleotide polymorphism arrays and validated by multiplex ligation-dependent probe amplification. The frequencies of recurrently deleted genes were: IKZF1, 76%; CDKN2A/2B, 45%; PAX5, 43%; BTG1, 18%; EBF1, 13%; ETV6, 5%; RB, 14%. In univariate analyses, the presence of CDKN2A/2B deletions had a negative impact on all endpoints: overall survival (P = .023), disease-free survival (P = .012), and remission duration (P = .036). The negative predictive value of CDKN2A/2B deletions was retained in multivariable analysis along with other factors such as timing of TKI therapy, intensity of conditioning, achieving remission after induction phase 1 and BTG1 deletions. We therefore conclude that acquired genomic CDKN2A/2B deletions identify a subgroup of Ph+ ALL patients, who have an inferior prognosis despite aSCT in CR1. Their poor outcome was attributable primarily to a high relapse rate after aSCT.