A unique role of p53 haploinsufficiency or loss in the development of acute myeloid leukemia with FLT3-ITD mutation.

With an incidence of ~50%, the absence or reduced protein level of p53 is much more common than TP53 mutations in acute myeloid leukemia (AML). AML with FLT3-ITD (internal tandem duplication) mutations has an unfavorable prognosis and is highly associated with wt-p53 dysfunction. While TP53 mutation in the presence of FLT3-ITD does not induce AML in mice, it is not clear whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML. Here, we generated FLT3-ITD knock-in; p53 knockout (heterozygous and homozygous) double-transgenic mice and found that both alterations strongly cooperated in the induction of cytogenetically normal AML without increasing the self-renewal potential. At the molecular level, we found the strong upregulation of Htra3 and the downregulation of Lin28a, leading to enhanced proliferation and the inhibition of apoptosis and differentiation. The co-occurrence of Htra3 overexpression and Lin28a knockdown, in the presence of FLT3-ITD, induced AML with similar morphology as leukemic cells from double-transgenic mice. These leukemic cells were highly sensitive to the proteasome inhibitor carfilzomib. Carfilzomib strongly enhanced the activity of targeting AXL (upstream of FLT3) against murine and human leukemic cells. Our results unravel a unique role of p53 haploinsufficiency or loss in the development of FLT3-ITD + AML.

Genome-wide DNA methylation analysis pre- and post-lenalidomide treatment in patients with myelodysplastic syndrome with isolated deletion (5q).

Myelodysplastic syndrome (MDS) with isolated deletion of chromosome 5q (MDS del5q) is a distinct subtype of MDS with quite favorable prognosis and excellent response to treatment with lenalidomide. Still, a relevant percentage of patients do not respond to lenalidomide and even experience progression to acute myeloid leukemia (AML). In this study, we aimed to investigate whether global DNA methylation patterns could predict response to lenalidomide. Genome-wide DNA methylation analysis using Illumina 450k methylation arrays was performed on n=51 patients with MDS del5q who were uniformly treated with lenalidomide in a prospective multicenter trial of the German MDS study group. To study potential direct effects of lenalidomide on DNA methylation, 17 paired samples pre- and post-treatment were analyzed. Our results revealed no relevant effect of lenalidomide on methylation status. Furthermore, methylation patterns prior to therapy could not predict lenalidomide response. However, methylation clustering identified a group of patients with a trend towards inferior overall survival. These patients showed hypermethylation of several interesting target genes, including genes of relevant signaling pathways, potentially indicating the evaluation of novel therapeutic targets.

Genome-wide DNA methylation profiling is able to identify prefibrotic PMF cases at risk for progression to myelofibrosis.

BACKGROUND: Patients suffering from the BCR-ABL1-negative myeloproliferative disease prefibrotic primary myelofibrosis (pre-PMF) have a certain risk for progression to myelofibrosis. Accurate risk estimation for this fibrotic progression is of prognostic importance and clinically relevant. Commonly applied risk scores are based on clinical, cytogenetic, and genetic data but do not include epigenetic modifications. Therefore, we evaluated the assessment of genome-wide DNA methylation patterns for their ability to predict fibrotic progression in PMF patients. RESULTS: For this purpose, the DNA methylation profile was analyzed genome-wide in a training set of 22 bone marrow trephines from patients with either fibrotic progression (n = 12) or stable disease over several years (n = 10) using the 850 k EPIC array from Illumina. The DNA methylation classifier constructed from this data set was validated in an independently measured test set of additional 11 bone marrow trephines (7 with stable disease, 4 with fibrotic progress). Hierarchical clustering of methylation ß-values and linear discriminant classification yielded very good discrimination between both patient groups. By gene ontology analysis, the most differentially methylated CpG sites are primarily associated with genes involved in cell-cell and cell-matrix interactions. CONCLUSIONS: In conclusion, we could show that genome-wide DNA methylation profiling of bone marrow trephines is feasible under routine diagnostic conditions and, more importantly, is able to predict fibrotic progression in pre-fibrotic primary myelofibrosis with high accuracy.

Combination of myeloproliferative neoplasm driver gene activation with mutations of splice factor or epigenetic modifier genes increases risk of rapid blastic progression.

OBJECTIVES: Myeloproliferative neoplasms (MPN) comprising polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) follow a bi-phasic course of disease with fibrotic and/or blastic progression. At presentation in the chronic phase, currently there are only insufficient tools to predict the risk of progression in individual cases. METHODS: In this study, chronic phase MPN (16 PMF, 11 PV, and 11 MPN unclassified) with blastic transformation during course of disease (n = 38, median follow-up 5.3 years) were analyzed by high-throughput sequencing. MPN cases with a comparable follow-up period and without evidence of blast increase served as control (n = 63, median follow-up 5.8 years). RESULTS: Frequent ARCH/CHIP-associated mutations (TET2, ASXL1, DNMT3A) found at presentation were not significantly associated with blastic transformation. By contrast, mutations of SRSF2, U2AF1, and IDH1/2 at first presentation were frequently observed in the progression cohort (13/38, 34.2%) and were completely missing in the control group without blast transformation during follow-up (P = .0007 for SRSF2; P = .0063 for U2AF1 and IDH1/2). CONCLUSION: Unlike frequent ARCH/CHIP alterations (TET2, ASXL1, DNMT3A), mutations in SRSF2, IDH1/2, and U2AF1 when manifest already at first presentation provide an independent risk factor for rapid blast transformation of MPN.

3D culture conditions support Kaposi's sarcoma herpesvirus (KSHV) maintenance and viral spread in endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi's sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman's disease). While in patients with late stage of Kaposi's sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. KEY MESSAGES: In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance.

Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis.

Functional contributions of individual cellular components of the bone-marrow microenvironment to myelofibrosis (MF) in patients with myeloproliferative neoplasms (MPNs) are incompletely understood. We aimed to generate a comprehensive map of the stroma in MPNs/MFs on a single-cell level in murine models and patient samples. Our analysis revealed two distinct mesenchymal stromal cell (MSC) subsets as pro-fibrotic cells. MSCs were functionally reprogrammed in a stage-dependent manner with loss of their progenitor status and initiation of differentiation in the pre-fibrotic and acquisition of a pro-fibrotic and inflammatory phenotype in the fibrotic stage. The expression of the alarmin complex S100A8/S100A9 in MSC marked disease progression toward the fibrotic phase in murine models and in patient stroma and plasma. Tasquinimod, a small-molecule inhibiting S100A8/S100A9 signaling, significantly ameliorated the MPN phenotype and fibrosis in JAK2V617F-mutated murine models, highlighting that S100A8/S100A9 is an attractive therapeutic target in MPNs.

Exposure of Patient-Derived Mesenchymal Stromal Cells to TGFB1 Supports Fibrosis Induction in a Pediatric Acute Megakaryoblastic Leukemia Model.

Bone marrow fibrosis (BMF) is a rare complication in acute leukemia. In pediatrics, it predominantly occurs in acute megakaryoblastic leukemia (AMKL) and especially in patients with trisomy 21, called myeloid leukemia in Down syndrome (ML-DS). Defects in mesenchymal stromal cells (MSC) and cytokines specifically released by the myeloid blasts are thought to be the main drivers of fibrosis in the bone marrow niche (BMN). To model the BMN of pediatric patients with AMKL in mice, we first established MSCs from pediatric patients with AMKL (n = 5) and ML-DS (n = 9). Healthy donor control MSCs (n = 6) were generated from unaffected children and adolescents ≤18 years of age. Steady-state analyses of the MSCs revealed that patient-derived MSCs exhibited decreased adipogenic differentiation potential and enrichment of proliferation-associated genes. Importantly, TGFB1 exposure in vitro promoted early profibrotic changes in all three MSC entities. To study BMF induction for longer periods of time, we created an in vivo humanized artificial BMN subcutaneously in immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, using a mixture of MSCs, human umbilical vein endothelial cell, and Matrigel. Injection of AMKL blasts as producers of TGFB1 into this BMN after 8 weeks induced fibrosis grade I/II in a dose-dependent fashion over a time period of 4 weeks. Thus, our study developed a humanized mouse model that will be instrumental to specifically examine leukemogenesis and therapeutic targets for AMKL blasts in future. IMPLICATIONS: TGFB1 supports fibrosis induction in a pediatric AMKL model generated with patient-derived MSCs. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/10/1603/F1.large.jpg.

Increased CXCL4 expression in hematopoietic cells links inflammation and progression of bone marrow fibrosis in MPN.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic-stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.

Feasibility of Combined Detection of Gene Mutations and Fusion Transcripts in Bone Marrow Trephines from Leukemic Neoplasms.

Chromosomal translocations resulting in fusion genes represent important oncogenic drivers and potential therapeutic targets in rare leukemia subtypes. Formalin-fixed, paraffin-embedded trephines are frequently used in hematologic diagnostic tests and provide relevant access to leukemic cells for further studies, for example, phenotyping in bone marrow fibrosis. However, high-throughput molecular analysis of nucleic acids obtained from this material is challenging, especially the reliable detection of RNA transcripts. Sixty-three formalin-fixed, paraffin-embedded bone marrow trephines of patients with chronic eosinophilic leukemia, chronic myeloid leukemia, acute myeloid leukemia, and myeloproliferative neoplasms were analyzed for gene mutations and the presence of fusion transcripts with a commercial amplicon-based next-generation sequencing approach. Fusion transcripts relevant for diagnosis and therapy could be detected and validated (by RT-PCR) in 25 patients (39.7%). Retrospectively selected material, up to 10 years old, was used for this purpose, and only one sample failed in the RNA analysis (1.6%). This study concludes that amplicon-based fusion transcript detection in bone marrow trephines is feasible and that bone marrow trephines taken for histologic assessment can also be applied for high-throughput molecular analysis.

Mutations associated with age-related clonal hematopoiesis in PMF patients with rapid progression to myelofibrosis.

Besides histopathological findings there are no indicators of increased risk for fibrotic progression in myeloproliferative neoplasms (MPN). Age-related clonal hematopoiesis (ARCH/CHIP) is a frequent finding in the elderly and combinations with MPN driver mutations (JAK2, MPL, and CALR) have been described. To determine the impact of ARCH/CHIP-related mutations for development of fibrosis in primary myelofibrosis (PMF), the mutational status of cases with fibrotic progression from grade 0 to grade 2/3 (n = 77) as evidenced by follow-up bone marrow biopsies (median 6.2 years) was compared with prefibrotic PMF samples without development of fibrosis (n = 27; median follow-up 7.3 years). Frequent ARCH/CHIP-associated mutations (TET2, ASXL1, and DNMT3A) demonstrable at presentation were not connected with fibrotic progression. However, mutations which are rarely found in ARCH/CHIP (SRSF2, U2AF1, SF3B1, IDH1/2, and EZH2) were present in 24.7% of cases with later development of fibrosis and not detectable in cases staying free from fibrosis (P = 0.0028). Determination of the tumor mutational burden (TMB) in a subgroup of cases (n = 32) did not show significant differences (7.68 mutations/MB vs. 6.85 mutations/MB). We conclude that mutations rarely found in ARCH/CHIP provide an independent risk factor for rapid fibrotic progression (median 2.0 years) when manifest already at first presentation.

Comprehensive mutation profiling and mRNA expression analysis in atypical chronic myeloid leukemia in comparison with chronic myelomonocytic leukemia.

Atypical chronic myeloid leukemia (aCML) and chronic myelomonocytic leukemia (CMML) represent two histologically and clinically overlapping myelodysplastic/myeloproliferative neoplasms. Also the mutational landscapes of both entities show congruencies. We analyzed and compared an aCML cohort (n = 26) and a CMML cohort (n = 59) by next-generation sequencing of 25 genes and by an nCounter approach for differential expression in 107 genes. Significant differences were found with regard to the mutation frequency of TET2, SETBP1, and CSF3R. Blast content of the bone marrow revealed an inverse correlation with the mutation status of SETBP1 in aCML and TET2 in CMML, respectively. By linear discriminant analysis, a mutation-based machine learning algorithm was generated which placed 19/26 aCML cases (73%) and 54/59 (92%) CMML cases into the correct category. After multiple correction, differential mRNA expression could be detected between both cohorts in a subset of genes (FLT3, CSF3R, and SETBP1 showed the strongest correlation). However, due to high variances in the mRNA expression, the potential utility for the clinic is limited. We conclude that a medium-sized NGS panel provides a valuable assistance for the correct classification of aCML and CMML.

An endothelial cell line infected by Kaposi's sarcoma-associated herpes virus (KSHV) allows the investigation of Kaposi's sarcoma and the validation of novel viral inhibitors in vitro and in vivo.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), a tumor of endothelial origin predominantly affecting immunosuppressed individuals. Up to date, vaccines and targeted therapies are not available. Screening and identification of anti-viral compounds are compromised by the lack of scalable cell culture systems reflecting properties of virus-transformed cells in patients. Further, the strict specificity of the virus for humans limits the development of in vivo models. In this study, we exploited a conditionally immortalized human endothelial cell line for establishment of in vitro 2D and 3D KSHV latency models and the generation of KS-like xenograft tumors in mice. Importantly, the invasive properties and tumor formation could be completely reverted by purging KSHV from the cells, confirming that tumor formation is dependent on the continued presence of KSHV, rather than being a consequence of irreversible transformation of the infected cells. Upon testing a library of 260 natural metabolites, we selected the compounds that induced viral loss or reduced the invasiveness of infected cells in 2D and 3D endothelial cell culture systems. The efficacy of selected compounds against KSHV-induced tumor formation was verified in the xenograft model. Together, this study shows that the combined use of anti-viral and anti-tumor assays based on the same cell line is predictive for tumor reduction in vivo and therefore allows faithful selection of novel drug candidates against Kaposi's sarcoma. KEY MESSAGES: Novel 2D, 3D, and xenograft mouse models mimic the consequences of KSHV infection. KSHV-induced tumorigenesis can be reverted upon purging the cells from the virus. A 3D invasiveness assay is predictive for tumor reduction in vivo. Chondramid B, epothilone B, and pretubulysin D diminish KS-like lesions in vivo.

Normal and pathological erythropoiesis in adults: from gene regulation to targeted treatment concepts.

Pathological erythropoiesis with consequent anemia is a leading cause of symptomatic morbidity in internal medicine. The etiologies of anemia are complex and include reactive as well as neoplastic conditions. Clonal expansion of erythroid cells in the bone marrow may result in peripheral erythrocytosis and polycythemia but can also result in anemia when clonal cells are dysplastic and have a maturation arrest that leads to apoptosis and hinders migration, a constellation typically seen in the myelodysplastic syndromes. Rarely, clonal expansion of immature erythroid blasts results in a clinical picture resembling erythroid leukemia. Although several mechanisms underlying normal and abnormal erythropoiesis and the pathogenesis of related disorders have been deciphered in recent years, little is known about specific markers and targets through which prognosis and therapy could be improved in anemic or polycythemic patients. In order to discuss new markers, targets and novel therapeutic approaches in erythroid disorders and the related pathologies, a workshop was organized in Vienna in April 2017. The outcomes of this workshop are summarized in this review, which includes a discussion of new diagnostic and prognostic markers, the updated WHO classification, and an overview of new drugs used to stimulate or to interfere with erythropoiesis in various neoplastic and reactive conditions. The use and usefulness of established and novel erythropoiesis-stimulating agents for various indications, including myelodysplastic syndromes and other neoplasms, are also discussed.

Flow cytometric characterization of acute leukemia reveals a distinctive "blast gate" of murine T-lymphoblastic leukemia/lymphoma.

Immunophenotypic analysis using multiparameter flow cytometry is an indispensable tool for diagnosis and management of acute leukemia. Mouse models have been widely used for medical research for more than 100 years and are indispensable for leukemia research. However, immunophenotypic analysis of murine leukemia was not always performed in published studies, and blast gating for isolation of blasts was shown only in very few studies. No systemic characterization of all types of murine acute leukemia in large cohorts by flow cytometry has been reported. In this study, we used flow cytometry to comprehensively characterize murine acute leukemia in a large cohort of mice. We found that murine T-lymphoblastic leukemia/lymphoma (T-ALL) exhibits a distinctive "blast gate" (CD45bright) with CD45/side scatter gating that differs from the "blast gate" (CD45dim) of human T-ALL. By contrast, murine B-lymphoblastic leukemia and acute myeloid leukemia show the same blast region (CD45dim) as human leukemia. Using blast cell gating, we for first time detected T-ALL development in FLT3-ITD knock-in mice (incidence: 23%). These leukemic cells were selectively killed by the FLT3 inhibitors crenolanib and midostaurin in vitro. These data suggest that FLT3-ITD plays a potential role in the pathogenesis of T-ALL and that FLT3-ITD inhibition is a therapeutic option in the management of patients with T-ALL. Our gating strategy for immunophenotypic analysis can be used for leukemogenesis and preclinical gene therapy studies in mice and may improve the quality of such analyses.

Activation of TRKA receptor elicits mastocytosis in mice and is involved in the development of resistance to KIT-targeted therapy.

The neurotrophins (NTs) play a key role in neuronal survival and maintenance. The TRK (tropomyosin-related kinase) tyrosine kinase receptors (TRKA, TRKB, TRKC) are high affinity receptors for NTs. There is increasing data demonstrating an important role of the TRK family in cancer initiation and progression. NTs have been known for many years to promote chemotaxis, maturation, and survival of mast cells. However, the role of NT signaling in the pathogenesis of mastocytosis is not well understood. In this study, we demonstrate that activation of TRKA by its ligand nerve growth factor (NGF) is potent to trigger a disease in mice with striking similarities to human systemic mastocytosis (SM). Moreover, activation of TRKA by NGF strongly rescues KIT inhibition-induced cell death of mast cell lines and primary mast cells from patients with SM, and this rescue effect can be efficiently blocked by entrectinib (a new pan TRK specific inhibitor). HMC-1 mast cell leukemia cells that are resistant to KIT inhibition induced by TRKA activation show reactivation of MAPK/ERK (extracellular signal-regulated kinase) and strong upregulation of early growth response 3 (EGR3), suggesting an important role of MAPK-EGR3 axis in the development of resistance to KIT inhibition. Targeting both TRK and KIT significantly prolongs survival of mice xenotransplanted with HMC-1 cells compared with targeting KIT alone. Thus, these data strongly suggest that TRKA signaling can improve neoplastic mast cell fitness. This might explain at least in part why treatment with KIT inhibitors alone so far has been disappointing in most published clinical trials for mastocytosis. Our data suggest that targeting both KIT and TRKs might improve efficacy of molecular therapy in SM with KIT mutations.

The Kaposi's sarcoma-associated herpesvirus (KSHV) non-structural membrane protein K15 is required for viral lytic replication and may represent a therapeutic target.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of the highly vascularized tumor Kaposi's sarcoma (KS), which is characterized by proliferating spindle cells of endothelial origin, extensive neo-angiogenesis and inflammatory infiltrates. The KSHV K15 protein contributes to the angiogenic and invasive properties of KSHV-infected endothelial cells. Here, we asked whether K15 could also play a role in KSHV lytic replication. Deletion of the K15 gene from the viral genome or its depletion by siRNA lead to reduced virus reactivation, as evidenced by the decreased expression levels of KSHV lytic proteins RTA, K-bZIP, ORF 45 and K8.1 as well as reduced release of infectious virus. Similar results were found for a K1 deletion virus. Deleting either K15 or K1 from the viral genome also compromised the ability of KSHV to activate PLCγ1, Erk1/2 and Akt1. In infected primary lymphatic endothelial (LEC-rKSHV) cells, which have previously been shown to spontaneously display a viral lytic transcription pattern, transfection of siRNA against K15, but not K1, abolished viral lytic replication as well as KSHV-induced spindle cell formation. Using a newly generated monoclonal antibody to K15, we found an abundant K15 protein expression in KS tumor biopsies obtained from HIV positive patients, emphasizing the physiological relevance of our findings. Finally, we used a dominant negative inhibitor of the K15-PLCγ1 interaction to establish proof of principle that pharmacological intervention with K15-dependent pathways may represent a novel approach to block KSHV reactivation and thereby its pathogenesis.

Gli1+ Mesenchymal Stromal Cells Are a Key Driver of Bone Marrow Fibrosis and an Important Cellular Therapeutic Target.

Bone marrow fibrosis (BMF) develops in various hematological and non-hematological conditions and is a central pathological feature of myelofibrosis. Effective cell-targeted therapeutics are needed, but the cellular origin of BMF remains elusive. Here, we show using genetic fate tracing in two murine models of BMF that Gli1+ mesenchymal stromal cells (MSCs) are recruited from the endosteal and perivascular niche to become fibrosis-driving myofibroblasts in the bone marrow. Genetic ablation of Gli1+ cells abolished BMF and rescued bone marrow failure. Pharmacological targeting of Gli proteins with GANT61 inhibited Gli1+ cell expansion and myofibroblast differentiation and attenuated fibrosis severity. The same pathway is also active in human BMF, and Gli1 expression in BMF significantly correlates with the severity of the disease. In addition, GANT61 treatment reduced the myofibroblastic phenotype of human MSCs isolated from patients with BMF, suggesting that targeting of Gli proteins could be a relevant therapeutic strategy.

Peripheral blood cytogenetics allows treatment monitoring and early identification of treatment failure to lenalidomide in MDS patients: results of the LE-MON-5 trial.

Transfusion-dependent patients with low- or intermediate-1-risk myelodysplastic syndrome, <5% bone marrow (BM) blasts and isolated 5q-deletion received lenalidomide within the German MDS study group phase-II clinical trial LE-MON-5 (EudraCT:2008-001866-10) of the University of Duesseldorf, Germany. Cytogenetic monitoring was performed by chromosome banding analyses (CBA) of BM cells and fluorescence in situ hybridization (FISH) analyses of peripheral blood (PB) mononuclear CD34+ cells using extended probe panels. Out of 144 patients screened for study enrollment, 24% failed to meet inclusion criteria due to cytogenetic findings. Eighty-seven patients were followed with a median observation time of 30 months. Cytogenetic response detected by FISH and CBA in 74 and 66% of patients, respectively, was predictive for hematologic response as well as of high prognostic relevance. After 2 years, AML rate was 8% for all patients. Karyotype evolution was detected in 21 (FISH)-34% (CBA) of patients associated with significantly shorter AML-free survival. Disease progression was first detectable on the cytogenetic level on average 5-6 months before recurrence of transfusion dependence. Our data show for the first time in a prospective setting that a cytogenetic monitoring from the PB helps to early identify treatment failure and progressive disease in lenalidomide-treated patients to improve clinical management. TRIAL REGISTRATION: EudraCT:2008-001866-10.

Prognostic factors in the myoepithelial-like spindle cell type of metaplastic breast cancer.

Metaplastic breast carcinoma (MBC) comprises a heterogeneous group of tumors with difficult to predict biological behavior. A subset of MBC, characterized by spindle-shaped tumor cells with a myoepithelial-like immunophenotype, was entered into a retrospective study (n = 42, median follow-up time 43 months). Molecular parameters (DNA sequences of mutation hot spots in AKT1, ALK, APC, BRAF, CDH1, CTNNB1, EGFR, ERBB2, FBXW7, FGFR2, FOXL2, GNAQ, GNAS, KIT, KRAS, MAP2K1, MET, MSH6, NRAS, PDGFRA, PIK3CA, PTEN, SF3B1, SMAD4, SRC, SRSF2, STK11, TP53, and U2AF1; copy numbers for EGFR, c-myc, FGFR, PLAG, c-met) were assessed. None of the patients had axillary lymph node involvement. In 13 cases, local recurrence developed after surgery (30.9 %). Distant metastasis occurred in seven patients (17 %; four after local recurrence). The most frequent genetic alteration was PIK3CA mutation (50 % of cases). None of the pathological parameters (size, grade, stage, Ki-67 labeling index) was significantly associated with disease-free survival (DFS) or overall survival (OS). PIK3CA mutation, especially the H1047R type, tended to adversely affect OS. Type of resection (mastectomy vs. breast-conserving therapy, width of margins) or adjuvant radiotherapy had no influence on DFS or OS, whereas in the group treated with radio-/chemotherapy, no local recurrence or metastasis and no death occurred. We conclude that the spindle cell type of MBC with myoepithelial features exhibits a higher frequency of PIK3CA mutation than other types of metaplastic or basal-like breast cancer and may benefit from combined radio-/chemotherapy. Classical pathological parameters are not helpful in identifying the high-risk tumors among this subgroup of MBC.