Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes.

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.

UHMK1 is a novel splicing regulatory kinase.

The U2AF Homology Motif Kinase 1 (UHMK1) is the only kinase that contains the U2AF homology motif, a common protein interaction domain among splicing factors. Through this motif, UHMK1 interacts with the splicing factors SF1 and SF3B1, known to participate in the 3' splice site recognition during the early steps of spliceosome assembly. Although UHMK1 phosphorylates these splicing factors in vitro, the involvement of UHMK1 in RNA processing has not previously been demonstrated. Here, we identify novel putative substrates of this kinase and evaluate UHMK1 contribution to overall gene expression and splicing, by integrating global phosphoproteomics, RNA-seq, and bioinformatics approaches. Upon UHMK1 modulation, 163 unique phosphosites were differentially phosphorylated in 117 proteins, of which 106 are novel potential substrates of this kinase. Gene Ontology analysis showed enrichment of terms previously associated with UHMK1 function, such as mRNA splicing, cell cycle, cell division, and microtubule organization. The majority of the annotated RNA-related proteins are components of the spliceosome but are also involved in several steps of gene expression. Comprehensive analysis of splicing showed that UHMK1 affected over 270 alternative splicing events. Moreover, splicing reporter assay further supported UHMK1 function on splicing. Overall, RNA-seq data demonstrated that UHMK1 knockdown had a minor impact on transcript expression and pointed to UHMK1 function in epithelial-mesenchymal transition. Functional assays demonstrated that UHMK1 modulation affects proliferation, colony formation, and migration. Taken together, our data implicate UHMK1 as a splicing regulatory kinase, connecting protein regulation through phosphorylation and gene expression in key cellular processes.

DNA methylation epitypes highlight underlying developmental and disease pathways in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a molecularly complex disease characterized by heterogeneous tumor genetic profiles and involving numerous pathogenic mechanisms and pathways. Integration of molecular data types across multiple patient cohorts may advance current genetic approaches for improved subclassification and understanding of the biology of the disease. Here, we analyzed genome-wide DNA methylation in 649 AML patients using Illumina arrays and identified a configuration of 13 subtypes (termed "epitypes") using unbiased clustering. Integration of genetic data revealed that most epitypes were associated with a certain recurrent mutation (or combination) in a majority of patients, yet other epitypes were largely independent. Epitypes showed developmental blockage at discrete stages of myeloid differentiation, revealing epitypes that retain arrested hematopoietic stem-cell-like phenotypes. Detailed analyses of DNA methylation patterns identified unique patterns of aberrant hyper- and hypomethylation among epitypes, with variable involvement of transcription factors influencing promoter, enhancer, and repressed regions. Patients in epitypes with stem-cell-like methylation features showed inferior overall survival along with up-regulated stem cell gene expression signatures. We further identified a DNA methylation signature involving STAT motifs associated with FLT3-ITD mutations. Finally, DNA methylation signatures were stable at relapse for the large majority of patients, and rare epitype switching accompanied loss of the dominant epitype mutations and reversion to stem-cell-like methylation patterns. These results show that DNA methylation-based classification integrates important molecular features of AML to reveal the diverse pathogenic and biological aspects of the disease.

Genetics and beyond: Precision Medicine Real-World Data for Patients with Cervical, Vaginal or Vulvar Cancer in a Tertiary Cancer Center.

Advances in molecular tumor diagnostics have transformed cancer care. However, it remains unclear whether precision oncology has the same impact and transformative nature across all malignancies. We conducted a retrospective analysis of patients with human papillomavirus (HPV)-related gynecologic malignancies who underwent comprehensive molecular profiling and subsequent discussion at the interdisciplinary Molecular Tumor Board (MTB) of the University Hospital, LMU Munich, between 11/2017 and 06/2022. We identified a total cohort of 31 patients diagnosed with cervical (CC), vaginal or vulvar cancer. Twenty-two patients (fraction: 0.71) harbored at least one mutation. Fifteen patients (0.48) had an actionable mutation and fourteen (0.45) received a recommendation for a targeted treatment within the MTB. One CC patient received a biomarker-guided treatment recommended by the MTB and achieved stable disease on the mTOR inhibitor temsirolimus for eight months. Factors leading to non-adherence to MTB recommendations in other patient cases included informed patient refusal, rapid deterioration, stable disease, or use of alternative targeted but biomarker-agnostic treatments such as antibody-drug conjugates or checkpoint inhibitors. Despite a remarkable rate of actionable mutations in HPV-related gynecologic malignancies at our institution, immediate implementation of biomarker-guided targeted treatment recommendations remained low, and access to targeted treatment options after MTB discussion remained a major challenge.

Mutations in HID1 Cause Syndromic Infantile Encephalopathy and Hypopituitarism.

OBJECTIVE: Precursors of peptide hormones undergo posttranslational modifications within the trans-Golgi network (TGN). Dysfunction of proteins involved at different steps of this process cause several complex syndromes affecting the central nervous system (CNS). We aimed to clarify the genetic cause in a group of patients characterized by hypopituitarism in combination with brain atrophy, thin corpus callosum, severe developmental delay, visual impairment, and epilepsy. METHODS: Whole exome sequencing was performed in seven individuals of six unrelated families with these features. Postmortem histopathological and HID1 expression analysis of brain tissue and pituitary gland were conducted in one patient. Functional consequences of the homozygous HID1 variant p.R433W were investigated by Seahorse XF Assay in fibroblasts of two patients. RESULTS: Bi-allelic variants in the gene HID1 domain-containing protein 1 (HID1) were identified in all patients. Postmortem examination confirmed cerebral atrophy with enlarged lateral ventricles. Markedly reduced expression of pituitary hormones was found in pituitary gland tissue. Colocalization of HID1 protein with the TGN was not altered in fibroblasts of patients compared to controls, while the extracellular acidification rate upon stimulation with potassium chloride was significantly reduced in patient fibroblasts compared to controls. INTERPRETATION: Our findings indicate that mutations in HID1 cause an early infantile encephalopathy with hypopituitarism as the leading presentation, and expand the list of syndromic CNS diseases caused by interference of TGN function. ANN NEUROL 2021;90:149-164.

Fusion gene detection by RNA-sequencing complements diagnostics of acute myeloid leukemia and identifies recurring NRIP1-MIR99AHG rearrangements.

Identification of fusion genes in clinical routine is mostly based on cytogenetics and targeted molecular genetics, such as metaphase karyotyping, fluorescence in situ hybridization and reverse-transcriptase polymerase chain reaction. However, sequencing technologies are becoming more important in clinical routine as processing time and costs per sample decrease. To evaluate the performance of fusion gene detection by RNAsequencing compared to standard diagnostic techniques, we analyzed 806 RNA-sequencing samples from patients with acute myeloid leukemia using two state-of-the-art software tools, namely Arriba and FusionCatcher. RNA-sequencing detected 90% of fusion events that were reported by routine with high evidence, while samples in which RNA-sequencing failed to detect fusion genes had overall lower and inhomogeneous sequence coverage. Based on properties of known and unknown fusion events, we developed a workflow with integrated filtering strategies for the identification of robust fusion gene candidates by RNA-sequencing. Thereby, we detected known recurrent fusion events in 26 cases that were not reported by routine and found discrepancies in evidence for known fusion events between routine and RNA-sequencing in three cases. Moreover, we identified 157 fusion genes as novel robust candidates and comparison to entries from ChimerDB or Mitelman Database showed novel recurrence of fusion genes in 14 cases. Finally, we detected the novel recurrent fusion gene NRIP1- MIR99AHG resulting from inv(21)(q11.2;q21.1) in nine patients (1.1%) and LTN1-MX1 resulting from inv(21)(q21.3;q22.3) in two patients (0.25%). We demonstrated that NRIP1-MIR99AHG results in overexpression of the 3' region of MIR99AHG and the disruption of the tricistronic miRNA cluster miR-99a/let-7c/miR-125b-2. Interestingly, upregulation of MIR99AHG and deregulation of the miRNA cluster, residing in the MIR99AHG locus, are known mechanisms of leukemogenesis in acute megakaryoblastic leukemia. Our findings demonstrate that RNA-sequencing has a strong potential to improve the systematic detection of fusion genes in clinical applications and provides a valuable tool for fusion discovery.

NGS-guided precision oncology in metastatic breast and gynecological cancer: first experiences at the CCC Munich LMU.

PURPOSE: Comprehensive genomic profiling identifying actionable molecular alterations aims to enable personalized treatment for cancer patients. The purpose of this analysis was to retrospectively assess the impact of personalized recommendations made by a multidisciplinary tumor board (MTB) on the outcome of patients with breast or gynecological cancers, who had progressed under standard treatment. Here, first experiences of our Comprehensive Cancer Center Molecular Tumor Board are reported. METHODS: All patients were part of a prospective local registry. 95 patients diagnosed with metastatic breast cancer or gynecological malignancies underwent extended molecular profiling. From May 2017 through March 2019, the MTB reviewed all clinical cases considering tumor profile and evaluated molecular alterations regarding further diagnostic and therapeutic recommendations. RESULTS: 95 patients with metastatic breast or gynecological cancers were discussed in the MTB (68% breast cancer, 20% ovarian cancer, 5% cervical cancer, 3% endometrial cancer and 4% others). Genes with highest mutation rate were PIK3CA and ERBB2. Overall, 34 patients (36%) received a biomarker-based targeted therapy recommendation. Therapeutic recommendations were implemented in nine cases; four patients experienced clinical benefit with a partial response or disease stabilization lasting over 4 months. CONCLUSION: In the setting of a multidisciplinary molecular tumor board, a small but clinically meaningful group of breast and gynecological cancer patients benefits from comprehensive genomic profiling. Broad and successful implementation of precision medicine is complicated by patient referral at late stage disease and limited access to targeted agents and early clinical trials. TRIAL REGISTRATION NUMBER: 284-10 (03.05.2018).

GATA2 mutations in myeloid malignancies: Two zinc fingers in many pies.

The GATA family of transcription factors are zinc finger (ZF) DNA-binding proteins that regulate transcription during development and cell differentiation. GATA2 plays an essential role in the regulation of hematopoiesis. As a result, mutations in this gene or alterations in its expression level or function have been linked to a variety of human hematologic disorders. In this review, we summarize the findings and developments over the recent years regarding the clinical correlations and functional properties of distinct GATA2 mutations in hematopoietic malignancies, with particular focus on the mutational hotspots in the ZF domains.

Loss of ISWI ATPase SMARCA5 (SNF2H) in Acute Myeloid Leukemia Cells Inhibits Proliferation and Chromatid Cohesion.

ISWI chromatin remodeling ATPase SMARCA5 (SNF2H) is a well-known factor for its role in regulation of DNA access via nucleosome sliding and assembly. SMARCA5 transcriptionally inhibits the myeloid master regulator PU.1. Upregulation of SMARCA5 was previously observed in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Since high levels of SMARCA5 are necessary for intensive cell proliferation and cell cycle progression of developing hematopoietic stem and progenitor cells in mice, we reasoned that removal of SMARCA5 enzymatic activity could affect the cycling or undifferentiated state of leukemic progenitor-like clones. Indeed, we observed that CRISPR/cas9-mediated SMARCA5 knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the SMARCA5 deletion induced karyorrhexis and nuclear budding as well as increased the ploidy, indicating its role in mitotic division of AML cells. The cytogenetic analysis of S5KO cells revealed the premature chromatid separation. We conclude that deleting SMARCA5 in AML blocks leukemic proliferation and chromatid cohesion.

Clonal evolution of acute myeloid leukemia from diagnosis to relapse.

Based on the individual genetic profile, acute myeloid leukemia (AML) patients are classified into clinically meaningful molecular subtypes. However, the mutational profile within these groups is highly heterogeneous and multiple AML subclones may exist in a single patient in parallel. Distinct alterations of single cells may be key factors in providing the fitness to survive in this highly competitive environment. Although the majority of AML patients initially respond to induction chemotherapy and achieve a complete remission, most patients will eventually relapse. These points toward an evolutionary process transforming treatment-sensitive cells into treatment-resistant cells. As described by Charles Darwin, evolution by natural selection is the selection of individuals that are optimally adapted to their environment, based on the random acquisition of heritable changes. By changing their mutational profile, AML cell populations are able to adapt to the new environment defined by chemotherapy treatment, ultimately leading to cell survival and regrowth. In this review, we will summarize the current knowledge about clonal evolution in AML, describe different models of clonal evolution, and provide the methodological background that allows the detection of clonal evolution in individual AML patients. During the last years, numerous studies have focused on delineating the molecular patterns that are associated with AML relapse, each focusing on a particular genetic subgroup of AML. Finally, we will review the results of these studies in the light of Darwinian evolution and discuss open questions regarding the molecular background of relapse development.

Controlled stem cell amplification by HOXB4 depends on its unique proline-rich region near the N terminus.

There is high interest in understanding the mechanisms that drive self-renewal of stem cells. HOXB4 is one of the few transcription factors that can amplify long-term repopulating hematopoietic stem cells in a controlled way. Here we show in mice that this characteristic of HOXB4 depends on a proline-rich sequence near the N terminus, which is unique among HOX genes and highly conserved in higher mammals. Deletion of this domain substantially enhanced the oncogenicity of HOXB4, inducing acute leukemia in mice. Conversely, insertion of the domain into Hoxa9 impaired leukemogenicity of this homeobox gene. These results indicate that proline-rich stretches attenuate the potential of stem cell active homeobox genes to acquire oncogenic properties.

Spectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia.

The clinical and prognostic relevance of many recently identified driver gene mutations in adult acute myeloid leukemia (AML) is poorly defined. We sequenced the coding regions or hotspot areas of 68 recurrently mutated genes in a cohort of 664 patients aged 18 to 86 years treated on 2 phase 3 trials of the German AML Cooperative Group (AMLCG). The median number of 4 mutations per patient varied according to cytogenetic subgroup, age, and history of previous hematologic disorder or antineoplastic therapy. We found patterns of significantly comutated driver genes suggesting functional synergism. Conversely, we identified 8 virtually nonoverlapping patient subgroups, jointly comprising 78% of AML patients, that are defined by mutually exclusive genetic alterations. These subgroups, likely representing distinct underlying pathways of leukemogenesis, show widely divergent outcomes. Furthermore, we provide detailed information on associations between gene mutations, clinical patient characteristics, and therapeutic outcomes in this large cohort of uniformly treated AML patients. In multivariate analyses including a comprehensive set of molecular and clinical variables, we identified DNMT3A and RUNX1 mutations as important predictors of shorter overall survival (OS) in AML patients <60 years, and particularly in those with intermediate-risk cytogenetics. NPM1 mutations in the absence of FLT3-ITD, mutated TP53, and biallelic CEBPA mutations were identified as important molecular prognosticators of OS irrespective of patient age. In summary, our study provides a comprehensive overview of the spectrum, clinical associations, and prognostic relevance of recurrent driver gene mutations in a large cohort representing a broad spectrum and age range of intensively treated AML patients.

A 29-gene and cytogenetic score for the prediction of resistance to induction treatment in acute myeloid leukemia.

Primary therapy resistance is a major problem in acute myeloid leukemia treatment. We set out to develop a powerful and robust predictor for therapy resistance for intensively treated adult patients. We used two large gene expression data sets (n=856) to develop a predictor of therapy resistance, which was validated in an independent cohort analyzed by RNA sequencing (n=250). In addition to gene expression markers, standard clinical and laboratory variables as well as the mutation status of 68 genes were considered during construction of the model. The final predictor (PS29MRC) consisted of 29 gene expression markers and a cytogenetic risk classification. A continuous predictor is calculated as a weighted linear sum of the individual variables. In addition, a cut off was defined to divide patients into a high-risk and a low-risk group for resistant disease. PS29MRC was highly significant in the validation set, both as a continuous score (OR=2.39, P=8.63·10-9, AUC=0.76) and as a dichotomous classifier (OR=8.03, P=4.29·10-9); accuracy was 77%. In multivariable models, only TP53 mutation, age and PS29MRC (continuous: OR=1.75, P=0.0011; dichotomous: OR=4.44, P=0.00021) were left as significant variables. PS29MRC dominated all models when compared with currently used predictors, and also predicted overall survival independently of established markers. When integrated into the European LeukemiaNet (ELN) 2017 genetic risk stratification, four groups (median survival of 8, 18, 41 months, and not reached) could be defined (P=4.01·10-10). PS29MRC will make it possible to design trials which stratify induction treatment according to the probability of response, and refines the ELN 2017 classification.

Acute myeloid leukemia with del(9q) is characterized by frequent mutations of NPM1, DNMT3A, WT1 and low expression of TLE4.

Deletions of the long arm of chromosome 9 [del(9q)] are a rare but recurring aberration in acute myeloid leukemia (AML). Del(9q) can be found as the sole abnormality or in combination with other cytogenetic aberrations such as t(8;21) and t(15;17). TLE1 and TLE4 were identified to be critical genes contained in the 9q region. We performed whole exome sequencing of 5 patients with del(9q) as the sole abnormality followed by targeted amplicon sequencing of 137 genes of 26 patients with del(9q) as sole or combined with other aberrations. We detected frequent mutations in NPM1 (10/26; 38%), DNMT3A (8/26; 31%), and WT1 (8/26; 31%) but only few FLT3-ITDs (2/26; 8%). All mutations affecting NPM1 and DNMT3A were exclusively identified in patients with del(9q) as the sole abnormality and were significantly more frequent compared to 111 patients classified as intermediate-II according to the European LeukemiaNet (10/14, 71% vs. 22/111, 20%; P < 0.001, 8/14, 57% vs. 26/111, 23%; P = 0.02). Furthermore, we identified DNMT3B to be rarely but recurrently targeted by truncating mutations in AML. Gene expression analysis of 13 patients with del(9q) and 454 patients with normal karyotype or various cytogenetic aberrations showed significant down regulation of TLE4 in patients with del(9q) (P = 0.02). Interestingly, downregulation of TLE4 was not limited to AML with del(9q), potentially representing a common mechanism in AML pathogenesis. Our comprehensive genetic analysis of the del(9q) subgroup reveals a unique mutational profile with the frequency of DNMT3A mutations in the del(9q) only subset being the highest reported so far in AML, indicating oncogenic cooperativity. © 2016 Wiley Periodicals, Inc.

In-depth mutational analyses of colorectal neuroendocrine carcinomas with adenoma or adenocarcinoma components.

Neuroendocrine carcinomas (NECs) of the colorectum are rare but highly aggressive neoplasms. These tumors show some shared genetic alterations with colorectal adenocarcinomas, and most of them have adjacent glandular adenoma or adenocarcinoma components. However, genetic data on colorectal NECs still are sparse and insufficient for definite conclusions regarding their molecular origin. Based on morphological characterization, panel and whole-exome sequencing, we here present results from an in-depth analysis of a collection of 15 colorectal NECs with glandular components, 10 of which by definition were mixed adenoneuroendocrine carcinomas (MANECs). Among shared genetic alterations of both tumor components, we most frequently found TP53, KRAS and APC mutations that also had highest allele frequencies. Mutations exclusive to glandular or neuroendocrine components outnumbered shared mutations but occurred at lower allele frequencies. Our findings not only provide additional evidence for a common clonal origin of colorectal NECs and adjacent glandular tumor components, but strongly suggest their development through the classical adenoma-carcinoma sequence. Moreover, our data imply early separation of glandular and neuroendocrine components during malignant transformation with subsequent independent mutational evolution.

Adults with Philadelphia chromosome-like acute lymphoblastic leukemia frequently have IGH-CRLF2 and JAK2 mutations, persistence of minimal residual disease and poor prognosis.

Philadelphia-like B-cell precursor acute lymphoblastic leukemia (Ph-like ALL) is characterized by distinct genetic alterations and inferior prognosis in children and younger adults. The purpose of this study was a genetic and clinical characterization of Ph-like ALL in adults. Twenty-six (13%) of 207 adult patients (median age: 42 years) with B-cell precursor ALL (BCP-ALL) were classified as having Ph-like ALL using gene expression profiling. The frequency of Ph-like ALL was 27% among 95 BCP-ALL patients negative for BCR-ABL1 and KMT2A-rearrangements. IGH-CRLF2 rearrangements (6/16; P=0.002) and mutations in JAK2 (7/16; P<0.001) were found exclusively in the Ph-like ALL subgroup. Clinical and outcome analyses were restricted to patients treated in German Multicenter Study Group for Adult ALL (GMALL) trials 06/99 and 07/03 (n=107). The complete remission rate was 100% among both Ph-like ALL patients (n=19) and the "remaining BCP-ALL" cases (n=40), i.e. patients negative for BCR-ABL1 and KMT2A-rearrangements and the Ph-like subtype. Significantly fewer Ph-like ALL patients reached molecular complete remission (33% versus 79%; P=0.02) and had a lower probability of continuous complete remission (26% versus 60%; P=0.03) and overall survival (22% versus 64%; P=0.006) at 5 years compared to the remaining BCP-ALL patients. The profile of genetic lesions in adults with Ph-like ALL, including older adults, resembles that of pediatric Ph-like ALL and differs from the profile in the remaining BCP-ALL. Our study is the first to demonstrate that Ph-like ALL is associated with inferior outcomes in intensively treated older adult patients. Ph-like adult ALL should be recognized as a distinct, high-risk entity and further research on improved diagnostic and therapeutic approaches is needed. (NCT00199056, NCT00198991).

Close correlation of copy number aberrations detected by next-generation sequencing with results from routine cytogenetics in acute myeloid leukemia.

High throughput sequencing approaches, including the analysis of exomes or gene panels, are widely used and established to detect tumor-specific sequence variants such as point mutations or small insertions/deletions. Beyond single nucleotide resolution, sequencing data also contain information on changes in sequence coverage between samples and thus allow the detection of somatic copy number alterations (CNAs) representing gain or loss of genomic material in tumor cells arising from aneuploidy, amplifications, or deletions. To test the feasibility of CNA detection in sequencing data we analyzed the exomes of 25 paired leukemia/remission samples from acute myeloid leukemia (AML) patients with well-defined chromosomal aberrations, detected by conventional chromosomal analysis and/or molecular cytogenetics assays. Thereby, we were able to confirm chromosomal aberrations including trisomies, monosomies, and partial chromosomal deletions in 20 out of 25 samples. Comparison of CNA detection using exome, custom gene panel, and SNP array analysis showed equivalent results in five patients with variable clone size. Gene panel analysis of AML samples without matched germline control samples resulted in confirmation of cytogenetic findings in 18 out of 22 cases. In all cases with discordant findings, small clone size (<33%) was limiting for CNA detection. We detected CNAs consistent with cytogenetics in 83% of AML samples including highly correlated clone size estimation (R = 0.85), while six out of 65 cytogenetically normal AML samples exhibited CNAs apparently missed by routine cytogenetics. Overall, our results show that high throughput targeted sequencing data can be reliably used to detect copy number changes in the dominant AML clone. © 2016 Wiley Periodicals, Inc.

Isolated trisomy 13 defines a homogeneous AML subgroup with high frequency of mutations in spliceosome genes and poor prognosis.

In acute myeloid leukemia (AML), isolated trisomy 13 (AML+13) is a rare chromosomal abnormality whose prognostic relevance is poorly characterized. We analyzed the clinical course of 34 AML+13 patients enrolled in the German AMLCG-1999 and SAL trials and performed exome sequencing, targeted candidate gene sequencing and gene expression profiling. Relapse-free (RFS) and overall survival (OS) of AML+13 patients were inferior compared to other ELN Intermediate-II patients (n=855) (median RFS, 7.8 vs 14.1 months, P = .006; median OS 9.3 vs. 14.8 months, P = .004). Besides the known high frequency of RUNX1 mutations (75%), we identified mutations in spliceosome components in 88%, including SRSF2 codon 95 mutations in 81%. Recurring mutations were detected in ASXL1 (44%) and BCOR (25%). Two patients carried mutations in CEBPZ, suggesting that CEBPZ is a novel recurrently mutated gene in AML. Gene expression analysis revealed a homogeneous expression profile including upregulation of FOXO1 and FLT3 and downregulation of SPRY2. This is the most comprehensive clinical and biological characterization of AML+13 to date, and reveals a striking clustering of lesions in a few genes, defining AML+13 as a genetically homogeneous subgroup with alterations in a few critical cellular pathways. Clinicaltrials.gov identifiers: AMLCG-1999: NCT00266136; AML96: NCT00180115; AML2003: NCT00180102; and AML60+: NCT00893373.

Exome sequencing identifies recurring FLT3 N676K mutations in core-binding factor leukemia.

The t(8;21) and inv(16)/t(16;16) rearrangements affecting the core-binding factors RUNX1 and CBFB, respectively, are found in 15% to 20% of adult de novo acute myeloid leukemia (AML) cases and are associated with a favorable prognosis. Since the expression of the fusion genes CBFB/MYH11 or RUNX1/RUNX1T1 alone is not sufficient to cause leukemia, we performed exome sequencing of an AML sample with an inv(16) to identify mutations, which may collaborate with the CBFB/MYH11 fusion during leukemogenesis. We discovered an N676K mutation in the adenosine triphosphate (ATP)-binding domain (tyrosine kinase domain 1 [TKD1]) of the fms-related tyrosine kinase 3 (FLT3) gene. In a cohort of 84 de novo AML patients with a CBFB/MYH11 rearrangement and in 36 patients with a RUNX1/RUNX1T1 rearrangement, the FLT3 N676K mutation was identified in 5 and 1 patients, respectively (5 [6%] of 84; 1 [3%] of 36). The FLT3-N676K mutant alone leads to factor-independent growth in Ba/F3 cells and, together with a concurrent FLT3-ITD (internal tandem duplication), confers resistance to the FLT3 protein tyrosine kinase inhibitors (PTKIs) PKC412 and AC220. Gene expression analysis of AML patients with CBFB/MYH11 rearrangement and FLT3 N676K mutation showed a trend toward a specific expression profile. Ours is the first report of recurring FLT3 N676 mutations in core-binding factor (CBF) leukemias and suggests a defined subgroup of CBF leukemias.

Whole-exome sequencing in adult ETP-ALL reveals a high rate of DNMT3A mutations.

Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a high-risk subgroup of T-lineage ALL characterized by specific stem cell and myeloid features. In adult ETP-ALL, no comprehensive studies on the genetic background have been performed to elucidate molecular lesions of this distinct subgroup. We performed whole-exome sequencing of 5 paired ETP-ALL samples. In addition to mutations in genes known to be involved in leukemogenesis (ETV6, NOTCH1, JAK1, and NF1), we identified novel recurrent mutations in FAT1 (25%), FAT3 (20%), DNM2 (35%), and genes associated with epigenetic regulation (MLL2, BMI1, and DNMT3A). Importantly, we verified the high rate of DNMT3A mutations (16%) in a larger cohort of adult patients with ETP-ALL (10/68). Mutations in epigenetic regulators support clinical trials, including epigenetic-orientated therapies, for this high-risk subgroup. Interestingly, more than 60% of adult patients with ETP-ALL harbor at least a single genetic lesion in DNMT3A, FLT3, or NOTCH1 that may allow use of targeted therapies.