Clonal evolution is believed to be a main driver for progression of various types of cancer and implicated in facilitating resistance to drugs. However, the hierarchical organization of malignant ...clones in the hematopoiesis of myelodysplastic syndromes (MDS) and its impact on response to drug therapy remain poorly understood. Using high-throughput sequencing of patient and xenografted cells, we evaluated the intratumoral heterogeneity (n= 54) and reconstructed mutational trajectories (n = 39) in patients suffering from MDS (n = 52) and chronic myelomonocytic leukemia-1 (n = 2). We identified linear and also branching evolution paths and confirmed on a patient-specific level that somatic mutations in epigenetic regulators and RNA splicing genes frequently constitute isolated disease-initiating events. Using high-throughput exome- and/or deep-sequencing, we analyzed 103 chronologically acquired samples from 22 patients covering a cumulative observation time of 75 years MDS disease progression. Our data revealed highly dynamic shaping of complex oligoclonal architectures, specifically upon treatment with lenalidomide and other drugs. Despite initial clinical response to treatment, patients' marrow persistently remained clonal with rapid outgrowth of founder-, sub-, or even fully independent clones, indicating an increased dynamic rate of clonal turnover. The emergence and disappearance of specific clones frequently correlated with changes of clinical parameters, highlighting their distinct and far-reaching functional properties. Intriguingly, increasingly complex mutational trajectories are frequently accompanied by clinical progression during the course of disease. These data substantiate a need for regular broad molecular monitoring to guide clinical treatment decisions in MDS.
•Mutational trajectories are defined by complex patterns of molecular heterogeneity in MDS, including lower-risk cases.•Therapeutic intervention dynamically reshapes mutational patterns often resulting in branched or independent evolution of MDS clones.
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.
•Genomic deletions of CDKN2A/2B are a new independent prognostic risk factor in adult Ph+ ALL.
Introduction: Thrombocytopenia is a common complication among MDS patients. Thus, many patients are dependent on platelet (PLT) transfusions, which give short-term therapeutic relief but are also ...associated with considerable clinical risks. In this context, thrombopoietin receptor agonists (TRAs) are under investigation as alternative treatment option, albeit with the concern that these substances may promote adverse events in MDS. However, beside potential positive effects on thrombopoiesis in MDS patients the TRA Eltrombopag (EPAG) has also been shown to exert positive disease modifying effects in vitro (Roth et al., Blood 2012). Using a MDS xenograft model, we here investigate the efficacy of EPAG and its influence on clonal composition on primary patient derived MDS xenografts and present data from an ongoing study.
Methods: Currently, samples from n=18 MDS patients (MDS del(5q)=2, MDS-MLD=6, MDS-RS-MLD=1 MDS-EB-1=2, MDS-EB-2=7) have been xenografted into NSG mice by intrafemoral co-injection of CD34+ hematopoietic stem cells and mesenchymal stromal cells using a modified protocol according to Medyouf et al., Cell Stem Cell 2014. Long term engraftment is assessed 12 weeks post-transplant by intrafemoral bone marrow (BM) biopsy and mice with positive human engraftment are subsequently treated with either EPAG (50mg/kg) or vehicle control for 18 weeks. During that time, the mice are bled every two weeks and BM aspiration is performed every six weeks. Human hematopoietic cells are FACS sorted. In peripheral blood, human PLTs are specifically and absolutely counted with a FACS assay based on hCD41+ cells and beads. To track clonal composition of MDS samples upon xenografting and EPAG treatment in comparison to placebo control, the original patient sample and the final MDS xenograft sample are being whole exome sequenced (WES). Interspersed time points are analyzed with a patient individual amplicon based deep sequencing approach (Mossner et al., Blood 2016) to calculate dynamics of variant allele frequencies (VAF) in dependency of treatment.
Results: To date, n=12 patient samples have been analyzed for human engraftment after 12 weeks post-transplant. Of these, n=7 (58%) have shown positive human engraftment and are being treated with EPAG versus placebo. To this end, one case has been completely followed up, including final molecular analysis. This MDS high risk case (MDS-EB-2) with a clinical PLT count of 29x109 PLT/L was transplanted into n=3 NSG mice. While two mice treated with EPAG survived the complete duration of the experiment, the placebo mouse died prematurely due to severe weight loss after 6 weeks of treatment. Further, EPAG treatment led to an initial rise of human PLT levels, while the placebo treated mouse presented a continuous decline of human PLTs, showing the efficacy of EPAG on human xenografts in the model. This observation has been confirmed in another case currently still under treatment. Molecular tracking by WES confirmed MDS patient specific molecular lesions in the MDS xenograft such as monosomy 7 and the disease related mutations CBL, DNMT3A and EZH2 with VAFs of 83%/43%/23% respectively. The monosomy 7 was detectable in all mice. CBL and DNMT3A exhibited similar VAFs in mouse EPAG1 (VAF=100%/54%), EPAG2 (VAF=100%/34%) and placebo (VAF=100%/50%). The EZH2 mutation was only detected in mouse EPAG2 (VAF=11%). Interestingly, the placebo mouse acquired a de novo mutation of U2AF1 (VAF=10%), which was not detectable in the initial patient sample or the EPAG treated mice. This spliceosomal mutation is associated with a higher risk of transformation to AML and shorter survival (Graubert et al., Nat Genet 2012; Makishima et al., Blood 2012).
Conclusions: Our data show first proof of principle results that new treatment options can be tested successfully in a preclinical murine xenograft model of primary MDS patient samples in a placebo controlled experimental setting. This approach allows the performance of patient individual substance testing that can segregate substance specific effects from natural disease progression in the same patient. Clinical parameters such as human PLT production and molecular clonal composition can be measured with a high confidence in vivo. Our current data show preliminary support for the hypothesis that EPAG may be efficacious in increasing PLT production in MDS patients without adversely influencing the underlying clonal composition.
Nowak:Novartis: Research Funding.
Introduction: Myelodysplastic Syndrome (MDS) can occur in young people but it is mainly a disease of the elderly with a dramatic increase of incidence in the decades above 60 years. Accordingly, the ...factor age may be an important gateway to the understanding of the molecular pathogenesis of MDS. Insights into the molecular changes of aging hematopoiesis in healthy organisms have found molecular changes, which often parallel the observations in MDS such as increase of clonality with age, change of epigenetic profiles, skewed lineage commitment toward the myeloid compartment and reduced regenerative capacity after stress. The development of MDS is often suggestive of an accelerated extrapolation of molecular changes, which also occur in normal aging hematopoiesis. Beyond this, increasing evidence is suggesting that MDS hematopoiesis is highly dependent on support of the bone marrow (BM) stroma, which has been shown to display aberrant transcriptomic profiles as compared to healthy BM stroma. To this end, we aimed to test the hypothesis whether the emergence of MDS may be associated with a continuity of molecular changes in BM stroma cells during aging. Therefore, we performed explorative RNA sequencing in a set of MSCs collected from healthy young, healthy old and patients with MDS with a highly standardized pre-analytical work-up algorithm.
Methods: We collected BM samples from voluntary healthy young adults (age = 24 - 25 years, female n=3, male n=3), healthy old adults (age 66 - 79 years, female n=3, male n=3) and patients with very low - intermediate risk MDS (age 51 - 87 years, female n=3, male n=3). After isolation of BM mononuclear cells by Ficoll gradient centrifugation, 5x106 mononuclear BM cells were seeded into 25cm² flasks and cultured using StemMACS human MSC Expansion Media (Miltenyi Biotec) with weekly media exchange to select for MSCs. These were expanded and harvested in passage 2. Absence of residual hematopoietic cells was controlled by FACS with anti CD45, CD31, and CD146. Whole transcriptome RNA-sequencing on all samples was carried out from 150ng of high quality RNA using the TruSeq stranded total RNA protocol and 100bp paired end sequencing (Illumina). The bio-informatical pipeline consisted of mapping using hisat2 and cufflinks for calculation of differentially expressed genes.
Results: RNA-sequencing generated a mean of 94 million reads per sample. Between the groups "healthy young" and "healthy old" 331 differentially regulated genes were identified. Between "healthy old" and "MDS" 514 genes were differentially regulated (fold change > 1.5, false discovery rate, FDR < 0.05). Among these, 197 genes were differently expressed between all three groups. With these parameters, a total of 17 genes showed a continuous and significant increase of expression from healthy young over healthy old toward MDS. Among these were Kit ligand (KITLG) but also a cluster of membrane based cell adhesion molecules such as Cadherin-6 (CDH6), Laminin Subunit Alpha 2 (LAMA2) and Laminin Subunit Gamma 2 (LAMC2) and others. Conversely, 5 genes showed a continuous and significant decrease of expression from healthy young over healthy old toward MDS, among these Leukocyte-specific protein 1 (LSP1), a gene implicated in regulation of T-cell migration. Gene set enrichment analysis revealed that MDS MSCs exhibited a significant depletion of genes involved in early adipogenic differentiation and enrichment of gene sets associated with extracellular matrix remodeling (FDR < 0.05, normalized enrichment score > 1.7). Although cells were cultured under normoxic conditions, MDS-MSCs displayed marked intrinsic feature of hypoxia.
Conclusion: By integrating transcriptomic data from BM stroma cells from healthy individuals during aging and comparison to BM stroma cells from MDS patients we have identified gene sets that are significantly differentially expressed per continuitatem. On the background of the hypothesis that molecular changes in the microenvironment of MDS are an exacerbation of changes also taking place during normal aging in the bone marrow, these genes, which are accumulated in the context of extracellular matrix and cell adhesion are promising candidates to further elucidate a BM stroma based pathogenesis of MDS.
No relevant conflicts of interest to declare.
Cytogenetic aberrations such as deletion of chromosome 5q (del(5q)) represent key elements in routine clinical diagnostics of haematological malignancies. Currently established methods such as ...metaphase cytogenetics, FISH or array-based approaches have limitations due to their dependency on viable cells, high costs or semi-quantitative nature. Importantly, they cannot be used on low abundance DNA. We therefore aimed to establish a robust and quantitative technique that overcomes these shortcomings.
For precise determination of del(5q) cell fractions, we developed an inexpensive multiplex-PCR assay requiring only nanograms of DNA that simultaneously measures allelic imbalances of 12 independent short tandem repeat markers.
Application of this method to n=1142 samples from n=260 individuals revealed strong intermarker concordance (R²=0.77-0.97) and reproducibility (mean SD: 1.7%). Notably, the assay showed accurate quantification via standard curve assessment (R²>0.99) and high concordance with paired FISH measurements (R²=0.92) even with subnanogram amounts of DNA. Moreover, cytogenetic response was reliably confirmed in del(5q) patients with myelodysplastic syndromes treated with lenalidomide. While the assay demonstrated good diagnostic accuracy in receiver operating characteristic analysis (area under the curve: 0.97), we further observed robust correlation between bone marrow and peripheral blood samples (R²=0.79), suggesting its potential suitability for less-invasive clonal monitoring.
In conclusion, we present an adaptable tool for quantification of chromosomal aberrations, particularly in problematic samples, which should be easily applicable to further tumour entities.
Introduction: Recently we identified a recurrent acquired genomic deletion on chromosome 1q as a potential new marker in approximately 14% of APL patients predicting a significantly increased risk of ...relapse (Nowak D et al., Genes Chromosomes and Cancer 2012). The deleted region contains the coding sequences for the microRNAs hsa-mir-181a1 and hsa-mir-181b1, which have been implicated as prognostic factors in Acute Myeloid Leukemia (AML) and a corresponding host gene (MIR181A1HG). To elucidate biologic mechanisms associated with the described genomic deletion we performed targeted sequencing of the affected region and RNA sequencing of APL samples carrying the deletion versus samples not carrying the deletion with subsequent validation of novel variants of MIR181A1HG.
Methods: Explorative sequencing of genomic DNA in the chromosomal subband 1q31.3, pos. 197073900-197196158 (hg18) was performed using the amplicon sequencing workflow of the Roche 454 platform sequencing 5000 bp fragments tiling a region of approximately 120 kb on n=3 APL samples. Corresponding patient samples from molecular remission were used as germline controls. Whole transcriptome sequencing of poly-A enriched RNA was performed on n=6 samples of bone marrow blasts of APL patients either carrying a deletion of the mir181a1/b1 coding region (n=3) or not carrying a deletion (n=3). RNA Sequencing was performed using the HiSeq2000 platform. Data analysis was carried out using Bowtie vers. 2.2.30, TopHat vers. 2.0.12 for alignment and mapping and the Cufflinks package vers. 2.2.1 for transcriptome assembly and expression analysis all using default settings and hg19 as reference genome. Validation of newly identified variants and differential expression of MIR181A1HG was carried out by RACE PCR and qRT-PCR on cDNA from primary leukemic blasts of APL patients (n=45), CD34+ cells from healthy donors (n=29). In vitro differentiation assays with concomitant gene expression analysis of MIR181A1HG variants were performed with CD34+ cells from healthy donors.
Results: Genomic sequencing of the recurrently deleted region revealed no somatically acquired mutations in the analyzed APL samples. Differential gene expression analysis using FPKM values (Fragments Per Kilobase Of Exon Per Million Fragments Mapped) inferred from RNA sequencing data of APL samples carrying a genomic deletion of 1q31.3 versus non-deleted samples identified n=58 genes significantly downregulated in deleted samples and n=31 upregulated genes. Interestingly, among the differentially regulated genes, BAALC, a factor recently shown to be prognostically relevant in APL was significantly upregulated 13 fold in the unfavourable group of samples with 1q31.3 deletions. Furthermore, RNA sequencing revealed numerous new isoforms of known transcripts as well as novel long non-conding RNA (lncRNA) sequences. Among these were a total of 6 new transcript variants of the MIR181A1HG gene in the recurrently deleted region on chromosome 1q31.3. One novel 5600bp lncRNA covering the coding regions for the hsa-mir-181a1/b1 was 24 fold overexpressed in samples carrying the recurrent 1q31.3 deletions. Expression analysis of MIR181A1HG in blasts of APL patients, CD34+ cells, unselected bone marrow cells and granulocytes of healthy donors revealed significantly elevated levels of MIR181A1HG in APL cells as compared to healthy CD34+ cells and almost absent expression in unselected bone marrow and granulocytes. This indicated a possible role for MIR181A1HG in APL blasts and hematopoietic stem cells. Subsequent in vitro differentiation experiments of primary healthy CD34+ cells showed that MIR181A1HG is downregulated 7 fold within 14 days of cytokine induced myeloid differentiation. Furthermore, MIR181A1HG was downregulated 5 fold during ATRA induced differentiation of NB4 cells.
Conclusion: RNA sequencing of APL cells demonstrated numerous novel uncharacterized lncRNAs whose expression is associated with clinical risk and which merit further investigation. Identification of novel isoforms of MIR181A1HG, which are highly expressed in APL blasts and purified CD34+ cells suggest a potential role for this lncRNA in hematopoietic stem cells and response to ATRA induced differentiation of APL cells.
No relevant conflicts of interest to declare.
Introduction
The acquisition of large-scale chromosomal lesions is a frequent event in malignant disorders. One example is the recurrent deletion of chromosome 5q (del(5q)) in myelodysplastic ...syndromes (MDS). The detection and monitoring of such deletions are important elements in routine clinical diagnostics and cancer genomic studies. Currently established methods for their assessment are metaphase cytogenetics (MC), fluorescence in situ hybridization (FISH) and micro-array based techniques. However, each of these methods harbours specific disadvantages as they depend on (viable) cells, are expensive, labour-intensive or only semi-quantitative. One possible approach to interrogate chromosomal deletions constitutes the assessment of allelic loss at heterozygous short tandem repeat (STR) loci within deleted regions. Therefore, we aimed to establish a robust, quick and inexpensive PCR assay that measures allelic imbalance at such STR loci in order to reliably estimate frequencies of cells carrying del(5q) from only minute amounts of DNA.
Methods
Genomic DNA (gDNA) was isolated from bone marrow (BM) or blood cells of MDS and acute myeloid leukemia (AML) patients with cytogenetically confirmed del(5q). Based on NCBI UniSTS database, we designed 12 fluorochrome-labelled PCR amplicons with size ranges of 100-400 bp that surround STR loci between chromosomal bands 5q21 and 5q31. Using only 10 ng gDNA, all 12 PCR amplicons were amplified in a single optimized multiplex-PCR reaction. Subsequently, amplicon fragment analysis was carried out via capillary electrophoresis on an ABI 3130 Genetic Analyzer. Allele size quantification of informative heterozygous loci was performed using ABI Genemapper software. Furthermore, size calculations of individual alleles were corrected for PCR-stutter, which was estimated from corresponding loci in homozygous samples. Finally, using mesenchymal stromal cells as a germline control, the degree of skewing in the allelic ratios of all informative STR markers in the tumor sample relative to the corresponding allelic ratios in the control was averaged and subsequently translated into fractions of cells carrying the del(5q) lesion.
Results
Application of our novel assay for quantification of del(5q) burden in n=559 samples from n=67 patients revealed a high frequency of informative markers with an average of 7 heterozygous STR loci per patient. The data shows a strong inter-marker concordance with a standard deviation (SD) of 2.3% for del(5q) cell frequencies. Moreover, duplicate analysis of 328 samples revealed an average SD of 0.86%. Most importantly, paired analysis of the proportion of del(5q) cells estimated using interphase-FISH and our PCR assay was carried out for n=9 samples and resulted in strong correlation with r²=0.93. A serial dilution series with deleted and non-deleted gDNA also revealed highly concordant results with r²=0.96. When comparing matched germline and tumor STR profiles, no case of microsatellite instability was detectable in our MDS/AML cohort thus highlighting the suitability of STR based lesion quantification in this disease entity. Furthermore, we could use our large dataset to calculate amplification efficiencies for each locus in order to predict “surrogate” germline profiles eventually allowing us to calculate del(5q) frequencies in tumor samples that lack germline controls. Finally, our assay reliably confirmed molecular remission in BM from del(5q) MDS patients after Lenalidomide treatment, in agreement with concomitant MC analyses.
Conclusion
Using a multiplexed PCR assay for measurement of STRs in deleted chromosomal regions we present a highly adaptable tool for precise quantification of large scale lesions. We show a very good correlation with established methods exemplarily for del(5q) lesions. Even without available germline control our assay provides robust results. Requiring only minute amounts of gDNA, this assay is ideally suitable for copy number quantification in samples for which only residual archival gDNA and no cells for FISH analysis are available. Due to the small amplicon sizes it should also be useful for investigation of fragmented gDNA from formalin-fixed archival specimen. In summary, our newly developed assay offers a mean to obtain quantitative data for basically any large scale chromosomal deletion which contains STRs and should be easily applicable to other clonal diseases.
Nolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
Introduction
Recently, Erythroferrone (ERFE) was discovered as a new regulator of hepcidin in the context of hematopoietic stress and erythropoietin (EPO) stimulation (Kautz et al., Nature Genetics ...2014). ERFEhas been shown to be expressed by erythroprogenitor cells of the bone marrow in response to increased erythroid activity induced by phlebotomy, EPO treatment or simulation of infectious situations in mice. It induces increased iron availability by downregulation of hepcidin in the liver and therefore represents an important new factor in iron homeostasis to be explored as a potential diagnostic or therapeutic target in the context of anemia and iron overload. Myelodysplastic Syndromes (MDS) are a group of heterogeneous malignant hematologic diseases characterized by inefficient hematopoiesis, severe anemia and deregulated iron homeostasis. In order to determine the specific role of ERFE in MDS, we analyzed the gene expression of ERFE in different hematopoietic compartments of MDS patients and healthy controls and correlated the differential expression data with clinical parameters and survival.
Methods
CD71+ erythroprogenitor cells (n=198 samples) were immunomagnetically purified from mononuclear bone marrow (BM) cells of a total of n=148 MDS and n=18 sAML patients. Chronological samples were available in n=21 cases. For controls, CD71+ BM cells were analyzed from n=35 healthy donors. In addition to CD71+ cells, CD61+, CD15+ , CD34+, selected from BM, as well as CD3+ selected peripheral blood (PB) cells were immunomagnetically collected from three MDS patients as well as two healthy young and two healthy old volunteers. After total RNA extraction using the AllPrep DNA/RNA Mini kit (Qiagen), cDNA was transcribed from RNA via Quantitect cDNA synthesis kit (Qiagen). Subsequently, ERFE expression was quantified from cDNA by quantitative PCR.
Results
In comparative expression analyses of different hematopoietic BM progenitor fractions (CD34+, CD15+, CD61+ and CD71+), ERFE was almost exclusively expressed in the erythropoietic CD71+ compartment. ERFE expression profiles in the CD71+ subset revealed a highly significant overexpression of this gene in MDS IPSS-low/int-1-risk (fold change (FC)=4.3, p<0.0001), IPSS-int-2/high-risk (FC=6.23, p<0.0001) and sAML (FC=6.69, p<0.0001) relative to healthy controls. ERFE expression profiles in MDS and sAML did not correlate with clinical laboratory parameters such as hemoglobin, EPO levels, ferritin, cobalamine, folic acid, transferrin, transferrin saturation, soluble transferrin receptor, reticulocytes, zinc protoporphyrin and lactate dehydrogenase. A negative correlation was observable for c-reactive protein levels (p=0.0053, Spearman r=-0.29) suggesting a possible link between an inflammatory environment and ERFE regulation. In exemplary chronological time course samples, ERFE expression was upregulated subsequent to clinical therapies such as 5-Azacytidine or Lenalidomide. Interestingly, in the total cohort of MDS patients with survival data follow up (n=90), low ERFE expression was associated with a significantly worse survival than high ERFE expression (median survival 2.1 years versus not reached, HR: 4.4, p=0.0007). This observation was even more pronounced in the subgroup analysis of MDS IPSS low/int-1 risk patients (n=54, median survival 2.1 years versus not reached, HR: 22, p<0.0001).
Conclusion
The observed highly aberrant overexpression of ERFE in CD71+ erythropoietic progenitor cells suggests an important role for this gene in the dysfunctional erythropoiesis of MDS. The observation of a correlation between ERFE expression and survival, especially in low risk MDS patients with no apparent coherence to other established clinical markers warrants further pursuit of ERFE expression profiles in CD71+ BM cells of MDS patients as a possible independent prognostic marker. Moreover, aberrant levels of ERFE could provide a promising target for novel therapeutic avenues that mechanistically address dysfunctional erythropoiesis in MDS.
No relevant conflicts of interest to declare.
Introduction: Myelodysplastic syndromes (MDS) are heterogeneous diseases characterized by clonal, ineffective hematopoiesis resulting in peripheral cytopenia and an increased risk of progression to ...acute myeloid leukemia. Particularly in low risk MDS a pro-apoptotic milieu has been found with increased levels of apoptosis-promoting factors such as tumor necrosis factor and fas ligand (CD95 ligand). This is thought to be a major pathomechanism in low risk MDS resulting in increased apoptosis of medullary erythroid progenitors leading to peripheral cytopenia. APG101 is a fusion protein consisting of the extracellular domain of human CD95 and the Fc domain of human IgG1. APG101 binds to CD95 ligand expressed on effector cells as well as to functionally active ligand in solution, thus blocking the interaction between CD95 and its ligand. Here, we report on interim results of a phase I study in transfusion dependent low risk MDS patients treated with APG101.
Methods: A total of 15 patients have been enrolled by July 30th 2014. All patients gave their written informed consent. Patients were treated for 12 weeks with weekly intravenous infusions of 100 mg or 400 mg APG101, respectively. Thereafter the treatment was discontinued and the patients were followed for another 6 months. Bone marrow biopsies were taken prior to the first APG101 dosing (baseline), at the end of treatment (EOT, week 12), at week 25 (follow up) and at week 37 (follow up). Methylcellulose assays on isolated CD34+ cells from the bone marrow aspirates at the different time points were performed. In addition, bone marrow samples at each time point were subjected to amplicon deep sequencing to detect gene mutations prior to APG101 dosing and to follow the mutational allele burden during and after treatment. This was done by sequencing of genomic DNA from ficollized mononuclear bone marrow cells on a Roche 454 GS Junior system.
Results: At data cut-off, 5 patients (3 male) had completed the study. Median age was 77 (range 65-78). WPSS categories were low in 2 patients and intermediate in 3 patients, respectively. According to the exclusion criteria none of the patients showed a medullary blast count ≥ 5%. Two patients received 400 mg weekly while 3 patients received 100 mg APG101 weekly, respectively. All patients stayed on treatment as scheduled and no adverse events were reported so far. Careful evaluation of bone marrow smears as well as immune phenotypic analysis of bone marrow aspirates showed no increase in bone marrow blasts at any time point. Moreover, quantification of the allele burden of pre-existing mutations (mutations of ASXL1 and SF3B1 in one patient and DNMT3A in the other patient) showed no significant expansion of the mutated clones. Ex vivo differentiation analyses revealed an increase in granulocytic colony forming as well as CFU-E/BFU-E forming capacity. With regard to efficacy, 4 of 5 patients showed a decrease in transfusion frequencies, which so far did not fulfill the IWG 2006 criteria for hematologic improvement (HI).
Conclusions: APG101 is a well tolerable compound in transfusion-dependent low risk MDS patients, particularly in elderly patients. None of the patients discontinued treatment due to toxicity. Monitoring did not reveal any signs of progress i.e. increase in medullary blasts in any of the patients. Deep amplicon sequencing did not show any signs of expansion of preexisting clones during therapy, i.e. APG101 did not provide malignant clones with a survival benefit. Although no major or minor HI (IWG 2006) was seen in 5 analyzed patients, the majority of patients showed a slight reduction in transfusion need which makes APG101 an interesting target for further investigation.
Kunz:Apogenix GmbH: Employment. Fricke:Apogenix GmbH: Employment. Nolte:Apogenix GmbH: Consultancy, Research Funding.
Introduction
Myelodysplastic Syndromes (MDS) are clonal hematologic diseases that are characterized by inefficient hematopoiesis, severe anemia and resulting deregulated iron homeostasis. Apart from ...supportive therapy with red blood cell transfusions some MDS patients with anemia respond to treatment with erythropoiesis stimulating agents such as Erythropoietin (EPO). However, the majority of these patients also become refractory to EPO treatment during the course of disease, suggesting a dysfunctional regulation of erythropoiesis downstream of EPO signaling in MDS. Most recently, a crucial erythroid regulator of iron metabolism named Erythroferrone (ERFE) was newly discovered, which is selectively produced by bone marrow (BM) erythroprogenitor cells during hematopoietic stress and EPO stimulation (Kautz et al. ASH plenary session 2013 and Kautz et al., Nature Genetics 2014). Aberrant expression of ERFE has been shown to directly result in critical impairment of erythropoiesis. We therefore sought to examine the role of ERFEexpression in CD71+ erythroprogenitor cells derived from patients with MDS and secondary acute myeloid leukemia (sAML).
Methods
CD71+ erythroprogenitor cells were immunomagnetically isolated from ficollized mononuclear BM cells of patients suffering from MDS (n=86, IPSS-low/int-1-risk n=69, IPSS-int-2/high-risk n=17), sAML (n=18) and age-matched healthy donors (n=17). In addition to CD71+ cells, CD34+, CD61+, CD15+ selected BM as well as CD3+ selected peripheral blood (PB) cells were immunomagnetically collected from three MDS patients as well as two healthy young and two healthy old donors. After total RNA was extracted using the AllPrep DNA/RNA Mini kit (Qiagen), cDNA was transcribed from RNA via Quantitect cDNA synthesis kit (Qiagen). Subsequently, ERFE expression was quantified from cDNA by quantitative PCR and normalized to corresponding GPIhousekeeping gene expression levels. Patient follow up (FU) data was available for n=55 MDS and n=14 sAML samples.
Results
Analysis of ERFE expression in CD34+, CD15+, CD61+ and CD71+ BM as well as CD3+ and unselected mononuclear PB cells from MDS patients and healthy donors revealed almost exclusive expression of ERFE in CD71+ erythroprogenitor cells irrespective of disease state. Our analysis of ERFE expression profiles in this specific cell subset revealed a highly significant overexpression of this gene in MDS IPSS-low/int-1-risk (fold change (FC)=4.1, p<0.0001), IPSS-int-2/high-risk (FC=4.6, p=0.0003) and sAML (FC=6.5, p<0.0001) relative to age-matched healthy controls. Despite this marked profile of aberrantly regulated ERFE we identified a distinct fraction of patients with expression levels similar or even lower than those measured in healthy donors in 20% (11/55) and 36% (5/14) of analyzed MDS and sAML cases with FU. Univariate analysis revealed that low abundance of CD71+ ERFE transcripts was significantly associated with inferior overall survival (OS) in MDS patients (median survival 1.7 years vs. not reached, p=0.0066) and also sAML (median survival 0.1 vs. 0.8 years, p=0.031).
Conclusion
The recent identification of the novel key regulatory gene ERFE in mouse models has greatly improved the understanding of the dynamic regulation of erythropoiesis. Our observation of almost exclusive ERFE expression in human BM erythroprogenitor cells further underlines its important role in human erythropoietic regulation both in healthy and myelodysplastic hematopoiesis. Moreover, strong upregulation of erythropoiesis stimulating ERFE in a large proportion of MDS patients usually suffering from anemia likely indicates its involvement in perturbed mechanisms of feedback signaling in MDS erythropoiesis. Pending integration with further clinical data, the current observation of significantly inferior survival probability for MDS and sAML patients with low ERFE expression levels indicates the potentially important biologic and clinical relevance of this novel regulatory gene in the pathogenesis of MDS. Consequently, aberrant levels of the erythroid hormone ERFEin MDS erythroprogenitor cells might provide a promising target for novel therapeutic avenues that mechanistically address dysfunctional erythropoiesis in MDS.
Nolte:Celgene Corp., Novartis Pharma: Honoraria, Research Funding.
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