Karin Lind

Frequency, onset and clinical impact of somatic DNMT3A mutations in therapy-related and secondary acute myeloid leukemia

The recent identification of DNMT3A mutations in de novo acute myeloid leukemia prompted us to determine their frequency, patterns and clinical impact in a cohort of 98 patients with either therapy-related or secondary acute myeloid leukemia developing from an antecedent hematologic disorder. We identified 24 somatic mutations in 23 patients with a significantly higher frequency in secondary acute myeloid leukemia (35.1%) as compared to therapy-related acute myeloid leukemia (16.4%, P=0.0486). DNMT3A mutations were associated with a normal karyotype and IDH1/2 mutations, but did not affect survival. In contrast to de novo acute myeloid leukemia, most mutations did not affect arginine on position 882, but were predominantly found in the methyltransferase domain. All DNMT3A mutations identified in secondary acute myeloid leukemia were already present in the antecedent disorders indicating an early event. Reduction to homozygosity by uniparental disomy was observed in 2 patients with secondary acute myeloid leukemia during disease progression.

Germline mutations in the DNA damage response genes BRCA1, BRCA2, BARD1 and TP53 in patients with therapy related myeloid neoplasms

Background Therapy related myeloid neoplasms (t-MNs) are complex diseases originating from an interplay between exogenous toxicities and a susceptible organism. It has been hypothesised that in a subset of cases t-MNs develop in the context of hereditary cancer predisposition syndromes. Methods The study systematically evaluated pedigrees of patients with t-MNs for cancer incidences and the possibility of a hereditary cancer predisposition syndrome. In addition, mutational analyses were performed using constitutional DNA from index patients, and deleterious heterozygous germline mutations were assessed for loss of heterozygosity in sorted leukaemic cells by single nucleotide polymorphism array. Results A nuclear pedigree was obtained in 51/53 patients with t-MNs resulting in a total of 828 individuals analysed. With a standardised incidence ratio of 1.03 (95% CI 0.74 to 1.39), the tumour incidence of first- degree relatives was not increased. However, six pedigrees were suggestive for a hereditary breast and ovarian cancer syndrome, three of a Li-Fraumeni like syndrome, and three index patients showed multiple primary neoplasms. Mutational analysis revealed two BRCA1 (c.3112G→T, c.5251C→T), one BRCA2 (c.4027A→G), two BARD1 (C557S) and four TP53 germline mutations (g.18508_18761delinsGCC, c.847C→T, c.845_848dupGGCG, c.1146delA) in nine of 53 (17%) index patients with t-MNs. Loss of heterozygosity in leukaemic cells was demonstrated for the BRCA1c.3112G→T and TP53c.845_848dupGGCG mutations, respectively. Conclusion It is concluded that a proportion of patients with t-MNs carry cancer susceptibility mutations which are likely to contribute to therapy related leukaemogenesis.

Frequent loss of RAF kinase inhibitor protein expression in acute myeloid leukemia

RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated its role in acute myeloid leukemia (AML), an aggressive malignancy arising from hematopoietic stem and progenitor cells (HSPCs). Western blot analysis revealed loss of RKIP expression in 19/103 (18%) primary AML samples and 4/17 (24%) AML cell lines but not in 10 CD34+ HSPC specimens. In in-vitro experiments with myeloid cell lines, RKIP overexpression inhibited cellular proliferation and colony formation in soft agar. Analysis of two cohorts with 103 and 285 AML patients, respectively, established a correlation of decreased RKIP expression with monocytic phenotypes. RKIP loss was associated with RAS mutations and in transformation assays, RKIP decreased the oncogenic potential of mutant RAS. Loss of RKIP further related to a significantly longer relapse-free survival and overall survival in uni- and multivariate analyses. Our data show that RKIP is frequently lost in AML and correlates with monocytic phenotypes and mutations in RAS. RKIP inhibits proliferation and transformation of myeloid cells and decreases transformation induced by mutant RAS. Finally, loss of RKIP seems to be a favorable prognostic parameter in patients with AML.

A gene expression profile associated with relapse of cytogenetically normal acute myeloid leukemia is enriched for leukemia stem cell genes

Some 50–80% of patients with acute myeloid leukemia (AML) achieve a complete remission with contemporary chemotherapy protocols, yet the majority of them eventually relapse with resistant disease: some patients no longer respond to chemotherapy at disease recurrence; others accomplish second and even third remissions whose decreasing duration nevertheless indicates that the pool of residual leukemic cells, i.e. of cells that persisted during treatment with cytotoxic drugs, increases with every round of therapy [1]. Either of these clinical courses therefore reflects an enhanced chemotherapy resistance of leukemic cells at relapse as compared to the cell population at diagnosis. Molecular changes enabling malignant cells to survive exposure to cytotoxic drugs may already have been present in a subset of the leukemic cell population at presentation, or may emerge during treatment [2,3], but in any case are thought to be selected as a consequence of drug therapy, and to play a major role in therapy resistance at relapse. Remarkably, however, even though various types of molecular alterations may be acquired at relapse, neither specific cytogenetic alterations nor functionally relevant point mutations as identified by whole genome sequencing were associated with relapse in a recurrent manner [2,3]. Certain copy number variations and known AML associated point mutations were newly present at relapse in small proportions of patients (usually < 10%), but the latter were lost in other patients, indicating that they are unlikely to represent drivers of therapy resistance at disease recurrence [4]. These findings could either indicate that chemotherapy resistance at relapse is acquired through a large variety of different mechanisms, or that molecular changes of other types than those mentioned above are of more general relevance in this context. Indeed, an earlier study has suggested that the expression of specific genes may change in a consistent manner between diagnosis and relapse of AML [5]. However, only a limited number of genes and mostly unpaired samples were probed in this investigation. Therefore, in the present study, genes whose expression changed in a relapse-specific manner were sought in a set of paired AML samples and on a genome-wide scale. To limit the genetic heterogeneity of the study population, only samples from patients with cytogenetically normal (CN) AML were used. Clinical characteristics of 11 patients with CN AML from whom samples had been obtained at the time of diagnosis and of relapse are summarized in Supplementary Table I available online at http://informahealthcare.com/doi/abs/10.3109/10428194.2014.944523. Patients provided written informed consent prior to sample collection, and the reported studies were approved by the ethics committee of the Medical University of Vienna (EK 179/2011). Mononuclear cells were enriched through Ficoll gradient centrifugation, and RNA was extracted and hybridized to human ST1.1 microarrays (Affymetrix). Primary data analysis was performed using the Robust Multi-array Average algorithm. The levels of 4679 genes that displayed variable expression (i.e. an interquartile range of the log2 transformed data of > 0.65 across all samples) were compared between diagnosis and relapse samples using a paired moderated t-test (R-package limma), followed by multiple hypothesis correction according to Benjamini and Hochberg [6]. These analyses revealed that 536 unique genes were up- and 551 down-regulated at relapse at a false discovery rate (FDR) < 10% (Figure 1, Supplementary Table II available online at http://informahealthcare.com/doi/abs/10.3109/10428194.2014.944523). Figure 1. Genes differentially expressed between diagnosis and relapse of CN AML. Log2 fold changes compared to the mean of all samples are displayed for the 30 most up- and the 30 most down-regulated genes (i.e. significantly differentially expressed genes with ... Because relapse of AML is considered to result from the outgrowth of usually largely quiescent, chemotherapy resistant leukemic stem cells (LSCs), a possible relationship between LSC and relapse-associated gene expression signatures was investigated. Gene expression profiles of LSC enriched versus LSC depleted human AML cell populations, functionally defined based on their engraftment ability in an optimized xenotransplant assay, were recently reported [7]. Of the 163 genes up-regulated in LSC enriched cell populations at an FDR < 10%, 19 were also up-regulated at relapse (p = 6.3 × 10− 7, odds ratio 4.29; Fishers exact test). Similarly, 14 of the 41 genes down-regulated in LSCs were also down-regulated at relapse (p = 1.2 × 10− 11, odds ratio 16.36; Fishers exact test), but no genes were regulated in an opposite manner in the two conditions (Supplementary Table II available online at http://informahealthcare.com/doi/abs/10.3109/10428194.2014.944523). To further explore relations between the relapse-associated gene expression profile and gene expression patterns associated with LSCs, as well as with normal hematopoietic stem cells (HSCs) and with prognosis in AML, gene set enrichment analysis (GSEA) [8] was performed. The 4679 genes whose expression had been compared between diagnosis and relapse of CN AML were ranked according to their associated t-statistic. The following gene lists were then probed against this relapse-associated gene expression profile: (i) genes up-regulated in functionally defined LSC enriched versus LSC depleted human AML cell populations [7]; (ii) genes up- or down-regulated in LSCs versus other leukemic cells as defined by the expression of cell surface markers [9]; (iii) genes up-regulated in HSCs versus progenitor and differentiated hematopoietic cells defined by specific cell surface markers [7]; and (iv) genes whose increased or decreased expression was associated with poor outcome in AML [10,11], or in the subgroup of CN AML [12]. Of note, the LSC and HSC signatures were related to each other [7], and, even though not defined on this basis, were able to predict chemotherapy responsiveness in AML [7,9]. All gene lists were used as reported, without any modifications. Where available, the corresponding lists of down-regulated genes were also probed, but in several cases these were either not reported, or too short to be useful for GSEA. In agreement with relapse representing a chemotherapy resistant state, the functionally defined LSC signature [7] and the HSC signature [7], as well as the three gene expression signatures linked to poor outcome in AML [10–12], were significantly enriched in the relapse-associated gene expression profile (Figure 2). Conversely, the list of genes down-regulated in patients with poor response to chemotherapy [10] was significantly negatively enriched in the relapse profile (while only small numbers of genes were down-regulated in poor responders in [11,12]), as was the list of genes down-regulated in surface-marker defined LSCs [9] (Figure 2). Figure 2. Gene signatures associated with LSCs, HSCs and poor therapy response are enriched in the CN AML relapse profile. Lists of genes associated with functionally defined LSCs [7], cell surface marker-defined HSCs [7], poor response to chemotherapy [10–12 ... The data presented in this report show that, in contrast to other investigated molecular alterations, changes in the expression of specific genes are associated with relapse of CN AML in a recurrent and significant manner. Corroborating the assumption that these changes indeed reflect, and possibly contribute to, a state of increased therapy resistance, the relapse-associated gene expression profile was enriched for gene signatures connected to poor outcome. Furthermore, a significant enrichment for gene expression signatures associated with LSCs was observed, thereby supporting the concept that relapse of AML results from the outgrowth of chemotherapy resistant LSCs and is associated with increased “stemness.” At the intersection of the relapse and the LSC signatures, a number of genes with potential roles in chemotherapy resistance were uncovered. For example, the gene coding for integrin α6, ITGA6, was expressed at elevated levels at relapse and in LSCs (Supplementary Table II available online at http://informahealthcare.com/doi/abs/10.3109/10428194.2014.944523). It was also up-regulated in AML cells with high levels of EVI1, which itself is a harbinger of a poor prognosis, and contributed to their therapy resistance [13]. Similarly, targeted deletion of interferon regulatory factor 8 (IRF8), which was down-regulated both at relapse of CN AML and in LSCs (Supplementary Table II available online at http://informahealthcare.com/doi/abs/10.3109/10428194.2014.944523), increased proliferation and reduced apoptosis of myeloid cells in vitro, and promoted leukemogenesis in a mouse model [14]. In metastatic colon cancer cell lines, its methylation mediated down-regulation also contributed to apoptosis resistance [15]. Functional analyses of these and other genes at the intersections of LSCs, chemotherapy resistance, and relapse can be expected to yield novel insights into the biology of AML, and may lead to the discovery of novel targets for rationally designed therapies.

The pan-BCL-2-blocker obatoclax (GX15-070) and the PI3-kinase/mTOR-inhibitor BEZ235 produce cooperative growth-inhibitory effects in ALL cells

Acute lymphoblastic leukemia (ALL) is characterized by leukemic expansion of lymphoid blasts in hematopoietic tissues. Despite improved therapy only a subset of patients can be cured. Therefore, current research is focusing on new drug-targets. Members of the BCL-2 family and components of the PI3-kinase/mTOR pathway are critically involved in the regulation of growth and survival of ALL cells. We examined the effects of the pan-BCL-2 blocker obatoclax and the PI3-kinase/mTOR-inhibitor BEZ235 on growth and survival of ALL cells. In 3H-thymidine uptake experiments, both drugs suppressed the in vitro proliferation of leukemic cells in all patients with Philadelphia chromosome-positive (Ph+) ALL and Ph- ALL (obatoclax IC50: 0.01-5 μM; BEZ235, IC50: 0.01-1 μM). Both drugs were also found to produce growth-inhibitory effects in all Ph+ and all Ph- cell lines tested. Moreover, obatoclax and BEZ235 induced apoptosis in ALL cells. In drug-combination experiments, obatoclax and BEZ235 exerted synergistic growth-inhibitory effects on ALL cells. Finally, we confirmed that ALL cells, including CD34+/CD38- stem cells and all cell lines express transcripts for PI3-kinase, mTOR, BCL-2, MCL-1, and BCL-xL. Taken together, this data shows that combined targeting of the PI3-kinase/mTOR-pathway and BCL-2 family-members is a potent approach to counteract growth and survival of ALL cells.Acute lymphoblastic leukemia (ALL) is characterized by leukemic expansion of lymphoid blasts in hematopoietic tissues. Despite improved therapy only a subset of patients can be cured. Therefore, current research is focusing on new drug-targets. Members of the BCL-2 family and components of the PI3-kinase/mTOR pathway are critically involved in the regulation of growth and survival of ALL cells. We examined the effects of the pan-BCL-2 blocker obatoclax and the PI3-kinase/mTOR-inhibitor BEZ235 on growth and survival of ALL cells. In 3H-thymidine uptake experiments, both drugs suppressed the in vitro proliferation of leukemic cells in all patients with Philadelphia chromosome-positive (Ph+) ALL and Ph− ALL (obatoclax IC50: 0.01-5 μM; BEZ235, IC50: 0.01-1 μM). Both drugs were also found to produce growth-inhibitory effects in all Ph+ and all Ph− cell lines tested. Moreover, obatoclax and BEZ235 induced apoptosis in ALL cells. In drug-combination experiments, obatoclax and BEZ235 exerted synergistic growth-inhibitory effects on ALL cells. Finally, we confirmed that ALL cells, including CD34+/CD38− stem cells and all cell lines express transcripts for PI3-kinase, mTOR, BCL-2, MCL-1, and BCL-xL. Taken together, this data shows that combined targeting of the PI3-kinase/mTOR-pathway and BCL-2 family-members is a potent approach to counteract growth and survival of ALL cells.

The TP53 Pro72Arg SNP in de novo acute myeloid leukaemia – results of two cohort studies involving 215 patients and 3759 controls

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Detection of prognostically relevant mutations and translocations in myeloid sarcoma by next generation sequencing

Myeloid sarcoma (MS) is a subgroup of acute myeloid leukemia (AML) where myeloid blasts form a tumoral mass in extramedullary tissues [1]. MS may occur at any point during the disease course and almost every site of the body can be affected. Although MS is often diagnosed as an isolated event without concomitant bone marrow involvement, virtually all of these patients will develop overt hematologic disease if left untreated [2,3], which further highlights the systemic nature of this disease. Beside isolated MS, extramedullary manifestations might also occur simultaneously with leukemic bone marrow (BM) infiltration. The incidence rate of this situation in newly diagnosed AML patients is estimated to be around 9%, however, some authors report incidence rates of up to 20–40% [2,4]. MS is usually treated with standard AML induction regimens, even if it occurs as isolated event. However, little is known about optimal consolidation strategies after achievement of complete remission [5,6]. This is particularly true for cases with isolated MS, which is due to the fact that material from MS biopsies is often sparse and usually Formalin-Fixed-ParaffinEmbedded (FFPE), thereby often precluding comprehensive AML risk stratification. In this study analyzing 18 cases of MS, we developed a next-generation sequencing (NGS) based approach, which enables the detection of both mutations and translocations with prognostic relevance from FFPE tissues of MS. Additionally, we analyzed the effects of this MS tissue based NGS profiling on AML risk stratification in cases where MS coincides with systemic AML and where cytogenetic/molecular analyses have already been performed from leukemic BM. All patient specimens of MS and corresponding BM biopsies were collected at the Division of Hematology, Medical University of Graz (MUG), between June 2003 and December 2016 and stored as FFPE samples. Patient characteristics are presented in Table 1. Briefly, MS coincided with systemic AML in 11 patients, whereas isolated MS was present in seven cases. The study was approved by the MUG-ethical committee (vote number 24-036 ex 11/12) and performed in accordance with the Declaration of Helsinki. In a first step, we focused on the detection of translocations with relevance for AML risk stratification [5]. This is of relevance, as complete cytogenetic analysis from MS specimens is usually not possible, which is due to the frequent unavailability of fresh material. Additionally, although fluorescence in situ hybridization (FISH) was successfully applied to MS specimens previously [7], this approach is limited by the fact that the amount of MS material is often sparse and that every abnormality tested requires at least one FFPE-section slide. Significant progress in this area came from Mirza et al., who recently analyzed six cases of MS by chromosomal microarray analysis (CMA) [8]. Without the need to target their analyses to selected abnormalities, they successfully detected unbalanced chromosomal aberrations and complex karyotypes. However, a limitation of this approach was the fact that prognostically relevant balanced chromosomal rearrangements could not be detected. To circumvent this limitation, we now performed NGS-based translocation analysis using only 100 ng of RNA extracted from FFPE-MS specimens discovering CBFB-MYH11, DEK/CANNUP214, DEK-NUP214, MLL-MLLT3, PML-RARA, RBM15MKL1, RPN1-MECOM, and RUNX1-RUNX1T1 (for details see also the Supplementary information). By studying 18MS specimens, we were able to unambiguously detect the presence of CBFB-MYH11 in three of them (Figure 1). In two of these cases, MS presented as an isolated event without any evidence of systemic disease. While the follow up of these patients was too short to allow any conclusions about a prognostic relevance of this lesion in MS, previous reports suggested a potential correlation of