Jan Krönke

IDH1 and IDH2 Mutations Are Frequent Genetic Alterations in Acute Myeloid Leukemia and Confer Adverse Prognosis in Cytogenetically Normal Acute Myeloid Leukemia With NPM1 Mutation Without FLT3 Internal Tandem Duplication

PURPOSE To analyze the frequency and prognostic impact of isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) mutations in acute myeloid leukemia (AML). PATIENTS AND METHODS We studied 805 adults (age range, 16 to 60 years) with AML enrolled on German-Austrian AML Study Group (AMLSG) treatment trials AML HD98A and APL HD95 for mutations in exon 4 of IDH1 and IDH2. Patients were also studied for NPM1, FLT3, MLL, and CEBPA mutations. The median follow-up for survival was 6.3 years. RESULTS IDH mutations were found in 129 patients (16.0%) -IDH1 in 61 patients (7.6%), and IDH2 in 70 patients (8.7%). Two patients had both IDH1 and IDH2 mutations. All but one IDH1 mutation caused substitutions of residue R132; IDH2 mutations caused changes of R140 (n = 48) or R172 (n = 22). IDH mutations were associated with older age (P < .001; effect conferred by IDH2 only); lower WBC (P = .04); higher platelets (P < .001); cytogenetically normal (CN) -AML (P< .001); and NPM1 mutations, in particular with the genotype of mutated NPM1 without FLT3 internal tandem duplication (ITD; P < .001). In patients with CN-AML with the latter genotype, IDH mutations adversely impacted relapse-free survival (RFS; P = .02) and overall survival (P = .03), whereas outcome was not affected in patients with CN-AML who lacked this genotype. In CN-AML, multivariable analyses revealed a significant interaction between IDH mutation and the genotype of mutated NPM1 without FLT3-ITD (ie, the adverse impact of IDH mutation [RFS]; P = .046 was restricted to this patient subset). CONCLUSION IDH1 and IDH2 mutations are recurring genetic changes in AML. They constitute a poor prognostic factor in CN-AML with mutated NPM1 without FLT3-ITD, which allows refined risk stratification of this AML subset.

Monitoring of Minimal Residual Disease in NPM1-Mutated Acute Myeloid Leukemia: A Study From the German-Austrian Acute Myeloid Leukemia Study Group

PURPOSE To evaluate the prognostic value of minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) with NPM1 mutation (NPM1(mut)). PATIENTS AND METHOD RNA-based real-time quantitative polymerase chain reaction (RQ-PCR) specific for the detection of six different NPM1(mut) types was applied to 1,682 samples (bone marrow, n = 1,272; blood, n = 410) serially obtained from 245 intensively treated younger adult patients who were 16 to 60 years old. RESULTS NPM1(mut) transcript levels as a continuous variable were significantly associated with prognosis after each treatment cycle. Achievement of RQ-PCR negativity after double induction therapy identified patients with a low cumulative incidence of relapse (CIR; 6.5% after 4 years) compared with RQ-PCR-positive patients (53.0%; P < .001); this translated into significant differences in overall survival (90% v 51%, respectively; P = .001). After completion of therapy, CIR was 15.7% in RQ-PCR-negative patients compared with 66.5% in RQ-PCR-positive patients (P < .001). Multivariable analyses after double induction and after completion of consolidation therapy revealed higher NPM1(mut) transcript levels as a significant factor for a higher risk of relapse and death. Serial post-treatment assessment of MRD allowed early detection of relapse in patients exceeding more than 200 NPM1(mut)/10(4) ABL copies. CONCLUSION We defined clinically relevant time points for NPM1(mut) MRD assessment that allow for the identification of patients with AML who are at high risk of relapse. Monitoring of NPM1(mut) transcript levels should be incorporated in future clinical trials to guide therapeutic decisions.

Clonal evolution in relapsed NPM1-mutated acute myeloid leukemia

Mutations in the nucleophosmin 1 (NPM1) gene are considered a founder event in the pathogenesis of acute myeloid leukemia (AML). To address the role of clonal evolution in relapsed NPM1-mutated (NPM1mut) AML, we applied high-resolution, genome-wide, single-nucleotide polymorphism array profiling to detect copy number alterations (CNAs) and uniparental disomies (UPDs) and performed comprehensive gene mutation screening in 53 paired bone marrow/peripheral blood samples obtained at diagnosis and relapse. At diagnosis, 15 aberrations (CNAs, n = 10; UPDs, n = 5) were identified in 13 patients (25%), whereas at relapse, 56 genomic alterations (CNAs, n = 46; UPDs, n = 10) were detected in 29 patients (55%) indicating an increase in genomic complexity. Recurrent aberrations acquired at relapse included deletions affecting tumor suppressor genes (ETV6 [n = 3], TP53 [n = 2], NF1 [n = 2], WT1 [n = 3], FHIT [n = 2]) and homozygous FLT3 mutations acquired via UPD13q (n = 7). DNMT3A mutations (DNMT3Amut) showed the highest stability (97%). Persistence of DNMT3Amut in 5 patients who lost NPM1mut at relapse suggests that DNMT3Amut may precede NPM1mut in AML pathogenesis. Of note, all relapse samples shared at least 1 genetic aberration with the matched primary AML sample, implying common ancestral clones. In conclusion, our study reveals novel insights into clonal evolution in NPM1mut AML.

Commonly altered genomic regions in acute myeloid leukemia are enriched for somatic mutations involved in chromatin remodeling and splicing

Acute myeloid leukemia (AML) is characterized by molecular heterogeneity. As commonly altered genomic regions point to candidate genes involved in leukemogenesis, we used microarray-based comparative genomic hybridization and single nucleotide polymorphism profiling data of 391 AML cases to further narrow down genomic regions of interest. Targeted resequencing of 1000 genes located in the critical regions was performed in a representative cohort of 50 AML samples comprising all major cytogenetic subgroups. We identified 120 missense/nonsense mutations as well as 60 insertions/deletions affecting 73 different genes (∼ 3.6 tumor-specific aberrations/AML). While most of the newly identified alterations were nonrecurrent, we observed an enrichment of mutations affecting genes involved in epigenetic regulation including known candidates like TET2, TET1, DNMT3A, and DNMT1, as well as mutations in the histone methyltransferases NSD1, EZH2, and MLL3. Furthermore, we found mutations in the splicing factor SFPQ and in the nonclassic regulators of mRNA processing CTCF and RAD21. These splicing-related mutations affected 10% of AML patients in a mutually exclusive manner. In conclusion, we could identify a large number of alterations in genes involved in aberrant splicing and epigenetic regulation in genomic regions commonly altered in AML, highlighting their important role in the molecular pathogenesis of AML.

High-resolution genomic profiling of adult and pediatric core-binding factor acute myeloid leukemia reveals new recurrent genomic alterations

To identify cooperating lesions in core-binding factor acute myeloid leukemia, we performed single-nucleotide polymorphism-array analysis on 300 diagnostic and 41 relapse adult and pediatric leukemia samples. We identified a mean of 1.28 copy number alterations per case at diagnosis in both patient populations. Recurrent minimally deleted regions (MDRs) were identified at 7q36.1 (7.7%), 9q21.32 (5%), 11p13 (2.3%), and 17q11.2 (2%). Approximately one-half of the 7q deletions were detectable only by single-nucleotide polymorphism-array analysis because of their limited size. Sequence analysis of MLL3, contained within the 7q36.1 MDR, in 46 diagnostic samples revealed one truncating mutation in a leukemia lacking a 7q deletion. Recurrent focal gains were identified at 8q24.21 (4.7%) and 11q25 (1.7%), both containing a single noncoding RNA. Recurrent regions of copy-neutral loss-of-heterozygosity were identified at 1p (1%), 4q (0.7%), and 19p (0.7%), with known mutated cancer genes present in the minimally altered region of 1p (NRAS) and 4q (TET2). Analysis of relapse samples identified recurrent MDRs at 3q13.31 (12.2%), 5q (4.9%), and 17p (4.9%), with the 3q13.31 region containing only LSAMP, a putative tumor suppressor. Determining the role of these lesions in leukemogenesis and drug resistance should provide important insights into core-binding factor acute myeloid leukemia.

Genome-wide genotyping of acute myeloid leukemia with translocation t(9;11)(p22;q23) reveals novel recurrent genomic alterations

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene are common genetic events in acute leukemias. In acute myeloid leukemia (AML), the translocation t(9;11)(p22;q23) [subsequently referred to as t(9;11)], resulting in the MLL-MLLT3-fusion protein, is the most common translocation

Circular RNAs of the nucleophosmin (NPM1) gene in acute myeloid leukemia

In acute myeloid leukemia, there is growing evidence for splicing pattern deregulation, including differential expression of linear splice isoforms of the commonly mutated gene nucleophosmin (NPM1). In this study, we detect circular RNAs of NPM1 and quantify circRNA hsa_circ_0075001 in a cohort of NPM1 wild-type and mutated acute myeloid leukemia (n=46). Hsa_circ_0075001 expression correlates positively with total NPM1 expression, but is independent of the NPM1 mutational status. High versus low hsa_circ_0075001 expression defines patient subgroups characterized by distinct gene expression patterns, such as lower expression of components of the Toll-like receptor signaling pathway in high hsa_circ_0075001 expression cases. Global evaluation of circRNA expression in sorted healthy hematopoietic controls (n=10) and acute myeloid leukemia (n=10) reveals circRNA transcripts for 47.9% of all highly expressed genes. While circRNA expression correlates globally with parental gene expression, we identify hematopoietic differentiation-associated as well as acute myeloid leukemia subgroup-specific circRNA signatures.

MicroRNA expression based outcome prediction in acute myeloid leukemia - novel insights through cross-platform integrative analyses

In the last decade, the non-coding transcriptome in normal and pathological conditions has been a focus of intensive research.[1][1] The most well-studied non-coding RNAs, microRNAs, are of critical importance in the post-transcriptional regulation in the cell and were shown to play a role in

Prognostic impact of Ikaros expression in lenalidomide-treated multiple myeloma

Multiple Myeloma is a malignant plasma cell disorder characterized by the development of end organ damages resulting in hypercalcemia, renal insufficiency, anemia and osteolytic lesions. The therapeutic options for multiple myeloma have markedly increased over the last decade. In particular, the development of proteasome inhibitors like bortezomib and carfilzomib and the immunomodulatory agents (IMiDs) thalidomide, lenalidomide and pomalidomide have become the cornerstones of current treatment strategies. In the past years, a distinct mode of action for lenalidomide and its analogs thalidomide and pomalidomide has been revealed. These compounds induce degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3), that are essential for myeloma cell proliferation, via the E3 ubiquitin ligase complex CRL4 (CRBN) [1-3]. More recently, another CRBN-dependent mechanism that results in destabilization of Basigin (BSG) and monocarboxylate transporter 1 (MCT-1) has been described for the activity of lenalidomide in multiple myeloma [4]. In order to determine whether expression of genes involved in the mechanism of lenalidomide has an impact on outcome, we measured RNA expression levels of CRBN, IKZF1, IKZF3 and BSG in pretreatment samples of patients treated with a lenalidomide-comprising regimen [5]. Patients up to the age of 65 years where treated on one of the up-front protocols of the German multiple myeloma study group (DSMM XII protocol) to receive 4 cycles of a combination of leanlidomide, doxorubicin and dexamethasone (RAD) followed by an intensive consolidation strategy including an autologous stem cell transplantation and in selected high risk cases an allogenic transplantation. All patients were scheduled to also receive a lenalidomide maintenance therapy. We observed a trend for higher IKZF1 expression levels in patients with International Staging System (ISS) stage III compared with ISS stage I and II. Patients who achieved a complete response or very good partial response at the time point of best response showed a trend towards lower pretreatment IKZF1 expression levels compared with patients with a partial response or stable disease. Using mRNA expression levels as continuous variables in univariate Cox regression analysis, we found that high IKZF1 levels were associated with an adverse PFS. Multivariable Cox regression analysis including genetic aberrations of known prognostic impact like t(4;14) and del(17p) revealed that pretreatment IKZF1 expression levels are an independent marker for PFS. When we divided the groups by quartiles of IKZF1 expression, patients within the lowest quartile had an excellent outcome with an estimated 3-year PFS of 86% and an estimated 3-year OS rate of 100% as compared to patients in the higher quartiles (3-year PFS 51%, OS 74%). In the subgroup of cytogenetically defined standardrisk patients IKZF1, IKZF3, and BSG expression levels had a significant impact on PFS. Our results suggest that multiple myeloma cells with low Ikaros expression are more sensitive to therapy. This may be counterintuitive on the first sight when considering a model where high expression of a gene indicates that it is relevant for the tumor cell and driving proliferation. However, almost all treatment-naïve multiple myeloma patients are primarily sensitive to lenalidomide and dexamethasone, suggesting that myeloma cells are in general Ikaros-dependent. Consistent with in vitro data in multiple myeloma cell lines, high expression of Ikaros, Aiolos and BSG may prevent lenalidomide-induced degradation of these proteins to critical low levels in those cells with high expression [1, 2, 4] (Figure 1). However, since the patients in our cohort received dexamethasone as well as doxorubicin and high-dose melphalan, we cannot Editorial