ABSTRACT
Molecular classification of acute myeloid leukemia (AML) aids prognostic stratification and clinical management. Our aim in this study is to identify transcriptome-wide mRNAs that are specific to each of the molecular subtypes of AML. We analyzed RNA-sequencing data of 955 AML samples from three cohorts, including the BeatAML project, the Cancer Genome Atlas, and a cohort of Swedish patients to provide a comprehensive transcriptome-wide view of subtype-specific mRNA expression. We identified 729 subtype-specific mRNAs, discovered in the BeatAML project and validated in the other two cohorts. Using unique proteomics data, we also validated the presence of subtype-specific mRNAs at the protein level, yielding a rich collection of potential protein-based biomarkers for the AML community. To enable the exploration of subtype-specific mRNA expression by the broader scientific community, we provide an interactive resource to the public.
Subject(s)
Leukemia, Myeloid, Acute/genetics , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Transcriptome , Biomarkers, Tumor , Genes, Neoplasm , Humans , Leukemia, Myeloid, Acute/classification , Leukemia, Myeloid, Acute/metabolism , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/genetics , Proteome , RNA, Messenger/genetics , RNA, Neoplasm/genetics , RNA-Seq , Retrospective Studies , SwedenABSTRACT
Eradication of chemotherapy-resistant leukemia stem cells is expected to improve treatment outcomes in patients with acute myelogenous leukemia (AML). In a mouse model of AML expressing the MOZ-TIF2 fusion, we found that Ring1A and Ring1B, components of Polycomb repressive complex 1, play crucial roles in maintaining AML stem cells. Deletion of Ring1A and Ring1B (Ring1A/B) from MOZ-TIF2 AML cells diminished self-renewal capacity and induced the expression of numerous genes, including Glis2 Overexpression of Glis2 caused MOZ-TIF2 AML cells to differentiate into mature cells, whereas Glis2 knockdown in Ring1A/B-deficient MOZ-TIF2 cells inhibited differentiation. Thus, Ring1A/B regulate and maintain AML stem cells in part by repressing Glis2 expression, which promotes their differentiation. These findings provide new insights into the mechanism of AML stem cell homeostasis and reveal novel targets for cancer stem cell therapy.
Subject(s)
Gene Expression Regulation, Leukemic , Histone Acetyltransferases/biosynthesis , Kruppel-Like Transcription Factors/biosynthesis , Leukemia, Myeloid, Acute/metabolism , Nerve Tissue Proteins/biosynthesis , Nuclear Receptor Coactivator 2/biosynthesis , Oncogene Proteins, Fusion/biosynthesis , Polycomb Repressive Complex 1/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Differentiation , Histone Acetyltransferases/genetics , Kruppel-Like Transcription Factors/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Nuclear Receptor Coactivator 2/genetics , Oncogene Proteins, Fusion/genetics , Polycomb Repressive Complex 1/genetics , Ubiquitin-Protein Ligases/geneticsABSTRACT
Juvenile myelomonocytic leukemia (JMML), a rare and aggressive myelodysplastic/myeloproliferative neoplasm that occurs in infants and during early childhood, is characterized by excessive myelomonocytic cell proliferation. More than 80% of patients harbor germ line and somatic mutations in RAS pathway genes (eg, PTPN11, NF1, NRAS, KRAS, and CBL), and previous studies have identified several biomarkers associated with poor prognosis. However, the molecular pathogenesis of 10% to 20% of patients and the relationships among these biomarkers have not been well defined. To address these issues, we performed an integrated molecular analysis of samples from 150 JMML patients. RNA-sequencing identified ALK/ROS1 tyrosine kinase fusions (DCTN1-ALK, RANBP2-ALK, and TBL1XR1-ROS1) in 3 of 16 patients (18%) who lacked canonical RAS pathway mutations. Crizotinib, an ALK/ROS1 inhibitor, markedly suppressed ALK/ROS1 fusion-positive JMML cell proliferation in vitro. Therefore, we administered crizotinib to a chemotherapy-resistant patient with the RANBP2-ALK fusion who subsequently achieved complete molecular remission. In addition, crizotinib also suppressed proliferation of JMML cells with canonical RAS pathway mutations. Genome-wide methylation analysis identified a hypermethylation profile resembling that of acute myeloid leukemia (AML), which correlated significantly with genetic markers with poor outcomes such as PTPN11/NF1 gene mutations, 2 or more genetic mutations, an AML-type expression profile, and LIN28B expression. In summary, we identified recurrent activated ALK/ROS1 fusions in JMML patients without canonical RAS pathway gene mutations and revealed the relationships among biomarkers for JMML. Crizotinib is a promising candidate drug for the treatment of JMML, particularly in patients with ALK/ROS1 fusions.
Subject(s)
Cell Proliferation , Crizotinib/pharmacology , Gene Expression Profiling , Leukemia, Myelomonocytic, Juvenile/genetics , Leukemia, Myelomonocytic, Juvenile/metabolism , Mutation , Oncogene Proteins, Fusion , Protein Kinase Inhibitors/pharmacology , Adolescent , Cell Proliferation/drug effects , Cell Proliferation/genetics , Child , Child, Preschool , DNA Methylation/drug effects , DNA Methylation/genetics , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Female , Genome-Wide Association Study , Humans , Infant , Leukemia, Myelomonocytic, Juvenile/drug therapy , Male , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/geneticsABSTRACT
BACKGROUND: Atypical small acinar proliferation (ASAP) is a precursor lesion of prostate cancer (PC), and PC develops from this suspicious focus or an unsampled malignant gland nearby. However, PC-related molecular alterations that could guide the timing of repeat biopsies and help monitor PC risk in ASAP-diagnosed patients have not been investigated. The purpose of this study was to first investigate the expression of seven different PC-related RNAs that included serine 2 (TMPRSS2): erythroblastosis virus E26 oncogene homolog (ERG) gene (TMPRSS2-ERG, T2E) fusion, alpha-methylacyl-CoA racemase (AMACR), kallikrein related peptidase 3 (KLK3), androgen receptor (AR), prostate cancer specific antigen 3 (PCA3), and matrix metalloproteinases (MMP)-2 and 9. METHODS: PC-related RNAs were evaluated using a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) system in pathologically ASAP-diagnosed prostate biopsy cores from 55 patients presenting with a normal digital rectal examination and a PSA level of 4-10 ng/mL. RESULTS: We detected that positive T2E fusion status (P = 0.013) and the expression of AMACR (P = 0.016), AR (P = 0.016) and MMP-2 (P = 0.013) were independently and significantly associated with PC risk in ASAP patients. There were also several statistically significant correlations between expression levels. Additionally, we demonstrated that T2E fusion positive ASAP patients with higher MMP-2 expression were more likely to be diagnosed with PC at a subsequent biopsy during the follow-up period (P = 0.003). CONCLUSIONS: Although, more clinical validations are needed for the stratification of PC risk in ASAP-diagnosed biopsy cores, our current results indicate that the coexistence of T2E fusion positivity with MMP-2 upregulation may help clinicians adjust their biopsy timetable and/or assessment of PC risk in ASAP-diagnosed patients with a PSA level of 4-10 ng/mL.
Subject(s)
Matrix Metalloproteinase 2/genetics , Oncogene Proteins, Fusion/genetics , Precancerous Conditions/genetics , Prostatic Neoplasms/genetics , Aged , Biomarkers, Tumor/biosynthesis , Biomarkers, Tumor/genetics , Biopsy, Large-Core Needle , Disease-Free Survival , Formaldehyde , Humans , Male , Matrix Metalloproteinase 2/metabolism , Oncogene Proteins, Fusion/biosynthesis , Precancerous Conditions/metabolism , Precancerous Conditions/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , RNA/biosynthesis , RNA/genetics , Racemases and Epimerases/biosynthesis , Racemases and Epimerases/genetics , Receptors, Androgen/biosynthesis , Receptors, Androgen/genetics , Reverse Transcriptase Polymerase Chain Reaction , Tissue Fixation , Up-RegulationABSTRACT
Essential for cell survival, the 90 kD Heat Shock Proteins (HSP90) are molecular chaperons required for conformational stabilization and trafficking of numerous client proteins. Functional HSP90 is required for the stability of AKT, a serine-threonine kinase phosphorylated in response to growth factor stimulation. AKT plays a crucial regulatory role in differentiation, cell cycle, transcription, translation, metabolism and apoptosis. Acute promyelocytic leukaemia (APL) is characterized by the presence of the promyelocytic leukaemia/retinoic acid receptor alpha (PML/RARA) fusion protein, which deregulates expression of several genes involved in differentiation and apoptosis. Here, we report inhibition of HSP90AA1 and HSP90AB1 isomer transcription in blasts isolated from patients with APL, associated with reduction of HSP90 protein expression and loss of control on AKT protein phosphorylation. We show that in vitro treatment of PML/RARA expressing cells with all-trans retinoic acid (ATRA) up-regulates HSP90 expression and stabilizes AKT. Addition of the HSP90-inhibitor 17-(allylamino)-17-demethoxygeldanamycin in combination with ATRA, blocks upregulation of AKT protein, indicating that HSP90 is necessary for ATRA action on AKT. This is the first report proving that expression of HSP90 isomers are directly and differentially repressed by PML/RARA, with critical results on cellular homeostasis of target proteins, such as AKT, in APL blasts.
Subject(s)
HSP90 Heat-Shock Proteins/antagonists & inhibitors , Leukemia, Promyelocytic, Acute/drug therapy , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Benzoquinones/pharmacology , HEK293 Cells , HSP90 Heat-Shock Proteins/biosynthesis , HSP90 Heat-Shock Proteins/genetics , Histones/genetics , Histones/metabolism , Humans , Lactams, Macrocyclic/pharmacology , Leukemia, Promyelocytic, Acute/pathology , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/genetics , Promoter Regions, Genetic , Promyelocytic Leukemia Protein/biosynthesis , Promyelocytic Leukemia Protein/genetics , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Retinoic Acid Receptor alpha/biosynthesis , Retinoic Acid Receptor alpha/genetics , Transfection , Tretinoin/pharmacology , Tumor Cells, Cultured , Up-Regulation/drug effectsABSTRACT
Ninety acute myeloid leukemia (AML) patients with inv(16) were monitored CBFß/MYH11 transcript around allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 23 patients received HLA-matched sibling donor transplantation (MSDT) and 67 patients received unmanipulated haploidentical hematopoietic stem cell transplantation (haplo-HSCT) were analyzed in this study. Patients were divided into four groups based on CBFß/MYH11 expression prior to transplantation (pre-MRD): with negative (group 1)/positive (group 2) pre-MRD before MSDT; with negative (group 3)/positive (group 4) pre-MRD before haplo-HSCT. The results showed that patients in group 2 had the highest cumulative incidence of relapse (2-year CIR, 40.7%), the lowest leukemia-free survival (2-year LFS, 50.8%), and overall survival (2-year OS, 62.5%). The other three groups of patients had comparable outcomes. The patients were also classified into the other three groups according to CBFß/MYH11 value of + 1 month after transplantation: group 5: pre- and post-transplant MRD were both negative; group 6: the value of post-transplant MRD was lower than 0.2%; group 7: the value of post-transplant MRD was higher than 0.2%. Group 7 had the highest CIR and the lowest LFS. These results indicated that AML patients with inv(16) were able to be separated into high-risk and low-risk relapse groups based on peritransplant MRD determined by RQ-PCR-based CBFß/MYH11. Haplo-HSCT might overcome the negative impact of pre-MRD on patient outcomes compared to MSDT.
Subject(s)
Chromosome Inversion , Chromosomes, Human, Pair 16 , Core Binding Factor beta Subunit , Gene Expression Regulation, Leukemic , Hematopoietic Stem Cell Transplantation , Myosin Heavy Chains , Oncogene Proteins, Fusion , Adult , Allografts , Child , Child, Preschool , Chromosomes, Human, Pair 16/genetics , Chromosomes, Human, Pair 16/metabolism , Core Binding Factor beta Subunit/biosynthesis , Core Binding Factor beta Subunit/genetics , Disease-Free Survival , Female , Humans , Leukemia, Myeloid, Acute/classification , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/therapy , Male , Middle Aged , Myosin Heavy Chains/biosynthesis , Myosin Heavy Chains/genetics , Neoplasm, Residual , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/genetics , Recurrence , Risk Factors , Survival RateABSTRACT
We analyzed the clinical significance and genetic features of ASXL2 and ZBTB7A mutations, and the alternatively spliced isoform of the RUNX1-RUNX1T1 transcript, which is also called AML1-ETO9a (AE9a), in Japanese CBF-AML patients enrolled in the JALSG AML201 study. ASXL2 and ZBTB7A genes were sequenced using bone marrow samples of 41 AML patients with t(8;21) and 14 with inv(16). The relative expression levels of AE9a were quantified using the real-time PCR assay in 23 AML patients with t(8;21). We identified ASXL2 (34.1%) and ZBTB7A (9.8%) mutations in only AML patients with t(8;21). ASXL2-mutated patients had a significantly higher WBC count at diagnosis (P = 0.04) and a lower frequency of sex chromosome loss than wild-type patients (33 vs. 76%, respectively, P = 0.01). KIT mutations were the most frequently accompanied with both ASXL2 (36%) and ZBTB7A (75%) mutations. Neither ASXL2 nor ZBTB7A mutations had an impact on overall or event-free survival. Patients harboring cohesin complex gene mutations expressed significantly higher levels of AE9a than unmutated patients (P = 0.03). In conclusion, ASXL2 and ZBTB7A mutations were frequently identified in Japanese AML patients with t(8;21), but not in those with inv(16). Further analysis is required to clarify the detailed biological mechanism of AE9a regulation of the cohesin complex.
Subject(s)
Chromosomes, Human, Pair 21/genetics , Chromosomes, Human, Pair 8/genetics , Core Binding Factor Alpha 2 Subunit , DNA-Binding Proteins , Gene Expression Regulation, Leukemic , Leukemia, Myeloid, Acute , Oncogene Proteins, Fusion , RUNX1 Translocation Partner 1 Protein , Repressor Proteins , Transcription Factors , Translocation, Genetic , Adolescent , Adult , Aged , Child , Core Binding Factor Alpha 2 Subunit/biosynthesis , Core Binding Factor Alpha 2 Subunit/genetics , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Disease-Free Survival , Female , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/mortality , Male , Middle Aged , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/genetics , RUNX1 Translocation Partner 1 Protein/biosynthesis , RUNX1 Translocation Partner 1 Protein/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Survival Rate , Transcription Factors/biosynthesis , Transcription Factors/geneticsABSTRACT
A network of lineage-specific transcription factors and microRNAs tightly regulates differentiation of hematopoietic stem cells along the distinct lineages. Deregulation of this regulatory network contributes to impaired lineage fidelity and leukemogenesis. We found that the hematopoietic master regulator RUNX1 controls the expression of certain microRNAs, of importance during erythroid/megakaryocytic differentiation. In particular, we show that the erythorid miR144/451 cluster is epigenetically repressed by RUNX1 during megakaryopoiesis. Furthermore, the leukemogenic RUNX1/ETO fusion protein transcriptionally represses the miR144/451 pre-microRNA. Thus RUNX1/ETO contributes to increased expression of miR451 target genes and interferes with normal gene expression during differentiation. Furthermore, we observed that inhibition of RUNX1/ETO in Kasumi1 cells and in RUNX1/ETO positive primary acute myeloid leukemia patient samples leads to up-regulation of miR144/451. RUNX1 thus emerges as a key regulator of a microRNA network, driving differentiation at the megakaryocytic/erythroid branching point. The network is disturbed by the leukemogenic RUNX1/ETO fusion product.
Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Leukemia, Myeloid, Acute/genetics , MicroRNAs/biosynthesis , Oncogene Proteins, Fusion/genetics , Cell Differentiation/genetics , Cell Lineage , Core Binding Factor Alpha 2 Subunit/biosynthesis , Gene Expression Regulation, Leukemic , Gene Regulatory Networks/genetics , Humans , Leukemia, Myeloid, Acute/pathology , Megakaryocytes/cytology , MicroRNAs/genetics , Oncogene Proteins, Fusion/biosynthesisABSTRACT
Epithelioid fibrous histiocytoma is a rare and distinctive cutaneous neoplasm. Most cases harbor ALK rearrangement and show ALK overexpression, which distinguish this neoplasm from conventional cutaneous fibrous histiocytoma and variants. SQSTM1 and VCL have previously been shown to partner with ALK in one case each of epithelioid fibrous histiocytoma. The purpose of this study was to examine a large cohort of epithelioid fibrous histiocytomas by next-generation sequencing to characterize the nature and prevalence of ALK fusion partners. A retrospective archival review was performed to identify cases of epithelioid fibrous histiocytoma (2012-2016). Immunohistochemistry was performed to confirm ALK expression. Targeted next-generation sequencing was applied on RNA extracted from formalin-fixed paraffin-embedded tissue to identify the fusion partners. Twenty-three cases fulfilled inclusion criteria. The mean patient age was 39 years (range, 8-74), there was no sex predilection, and >75% of cases involved the lower extremities. The most common gene fusions were SQSTM1-ALK (N=12; 52%) and VCL-ALK (N=7; 30%); the other four cases harbored novel fusion partners (DCTN1, ETV6, PPFIBP1, and SPECC1L). The pattern of ALK immunoreactivity was usually granular cytoplasmic (N=12; 52%) or granular cytoplasmic and nuclear (N=10; 43%); the case containing an ETV6 fusion partner showed nuclear staining alone. There was no apparent relationship between tumor morphology and the ALK fusion partner. In summary, SQSTM1 and VCL are the most common ALK fusion partners in epithelioid fibrous histiocytoma; DCTN1, ETV6, PPFIBP1, and SPECC1L represent rare fusion partners. The proteins encoded by these genes play diverse roles in scaffolding, cell adhesion, signaling, and transcription (among others) without clear commonalities. These findings expand the oncogenic promiscuity of many of these ALK fusion genes, which drive neoplasia in tumors of diverse lineages with widely varied clinical behavior. This is the first documented account of ETV6-ALK and SPECC1L-ALK translocations in neoplasms.
Subject(s)
Anaplastic Lymphoma Kinase/biosynthesis , Histiocytoma, Benign Fibrous/metabolism , Histiocytoma, Benign Fibrous/pathology , Adolescent , Adult , Aged , Anaplastic Lymphoma Kinase/genetics , Child , Cohort Studies , Epithelioid Cells/pathology , Female , Humans , Male , Middle Aged , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/genetics , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Retrospective Studies , Sequestosome-1 Protein/biosynthesis , Sequestosome-1 Protein/metabolism , Young Adult , von Willebrand Factor/biosynthesis , von Willebrand Factor/geneticsABSTRACT
Genetic aberrations in leukemia often lead to the formation of expressed chimeric genes, which should be assessed for proper diagnosis and therapy. Modern methods of molecular diagnostic mainly allow to identify already known fusion genes. RNAseq is an efficient tool for identification of rare and novel chimeric transcripts. Here we present the results of the whole transcriptome analysis of bone marrow samples from five patients with acute myeloblastic leukemia and one, with myelodysplastic syndrome. The whole-transcriptome analysis was performed using Illumina/Solexa approach. We found rare or unknown chimeric transcripts including ETV6-MDS1, MN1-ETV6, OAZ1-PTMA, and MLLT10-GRIA4. Each of these transcripts was confirmed by RT-PCR and Sanger sequencing.
Subject(s)
High-Throughput Nucleotide Sequencing , Leukemia , Oncogene Proteins, Fusion , RNA, Messenger , RNA, Neoplasm , Transcriptome , Adolescent , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Leukemia/genetics , Leukemia/metabolism , Male , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/geneticsABSTRACT
AIMS: Pulmonary adenofibromas are rare benign fibroepithelial tumours of the lung with unknown histogenesis and an indolent clinical behaviour. Their stroma resembles that of solitary fibrous tumours, whereas the glands are composed of respiratory epithelium organized in a phyllodes-like architecture. Differentiation of pulmonary adenofibromas from other more aggressive intrathoracic tumours is clinically relevant. However, their biology is unknown. Here, we sought to characterize pulmonary adenofibromas at a clinicopathological level and to define whether they could be underpinned by a highly recurrent somatic genetic alteration akin to tumours with similar morphology. METHODS AND RESULTS: Seven pulmonary adenofibromas were subjected to immunohistochemical analysis for thyroid transcription factor 1 (TTF1), napsin A, cytokeratin 7, E-cadherin, CD99, CD34, CD31, STAT6, oestrogen receptor (ER), progesterone receptor, androgen receptor, bcl-2, and vimentin, as well as electron microscopy and capillary sequencing on microdissected samples to evaluate the presence of NAB2-STAT6 fusion genes and MED12 exon 2 mutations in their discrete components. A control group comprising pulmonary solitary fibrous tumours, pulmonary hamartomas and breast fibroadenomas was also analysed. We confirmed that the stromal elements of pulmonary adenofibromas pertain to the fibroblastic lineage, and show ER overexpression in 71% of cases, whereas the epithelium consists of TTF1-positive, E-cadherin positive bronchiolar elements. A highly recurrent NAB2-STAT6 fusion variant (exon 4-exon 2) was detected in the stroma but not in the epithelium. No MED12 mutations were identified. CONCLUSIONS: Here, we demonstrate that pulmonary adenofibromas are neoplastic lesions harbouring the molecular hallmark of solitary fibrous tumours.
Subject(s)
Adenofibroma/genetics , Estrogen Receptor alpha/biosynthesis , Lung Neoplasms/genetics , Repressor Proteins/genetics , STAT6 Transcription Factor/genetics , Adenofibroma/metabolism , Adenofibroma/pathology , Aged , Biomarkers, Tumor/analysis , Female , Humans , Immunohistochemistry , Laser Capture Microdissection , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Microscopy, Electron, Transmission , Middle Aged , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/geneticsABSTRACT
BACKGROUND: A high-level expression of the CRLF2 gene is frequent in precursor B-cell acute lymphoblastic leukemia (pB-ALL) and can be caused by different genetic aberrations. The presence of the most frequent alteration, the P2RY8/CRLF2 fusion, was shown to be associated with a high relapse incidence in children treated according to ALL-Berlin-Frankfurt-Münster (BFM) protocols, which is poorly understood. Moreover, the frequency of other alterations has not been systematically analyzed yet. PROCEDURE: CRLF2 mRNA expression and potential genetic aberrations causing a CRLF2 high expression were prospectively assessed in 1,105 patients treated according to the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP)-BFM ALL 2009 protocol. Additionally, we determined copy number alterations in selected B-cell differentiation genes for all CRLF2 high-expressing pB-ALL cases, as well as JAK2 and CRLF2 mutations. RESULTS: A CRLF2 high expression was detected in 26/178 (15%) T-cell acute lymphoblastic leukemia (T-ALL) cases, 21 of them (81%) had been stratified as high-risk patients by treatment response. In pB-ALL, a CRLF2 high expression was determined in 91/927 (10%) cases; the P2RY8/CRLF2 rearrangement in 44/91 (48%) of them, supernumerary copies of CRLF2 in 18/91 (20%), and, notably, the IGH/CRLF2 translocation was detected in 16/91 (18%). Remarkably, 7 of 16 (44%) patients with IGH/CRLF2 translocation had already relapsed. P2RY8/CRLF2- and IGH/CRLF2-positive samples (70 and 94%, respectively) were characterized by a high frequency of additional deletions in B-cell differentiation genes such as IKZF1 or PAX5. CONCLUSION: Our data suggest that this high frequency of genetic aberrations in the context of a high CRLF2 expression could contribute to the high risk of relapse in P2RY8/CRLF2- and IGH/CRLF2-positive ALL.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Gene Expression Regulation, Leukemic/drug effects , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Receptors, Cytokine/biosynthesis , Adolescent , Asparaginase/administration & dosage , Child , Child, Preschool , Daunorubicin/administration & dosage , Female , Gene Rearrangement , Humans , Ikaros Transcription Factor/biosynthesis , Ikaros Transcription Factor/genetics , Infant , Male , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/genetics , PAX5 Transcription Factor/biosynthesis , PAX5 Transcription Factor/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Prednisone/administration & dosage , Receptors, Cytokine/genetics , Receptors, Purinergic P2Y/biosynthesis , Receptors, Purinergic P2Y/genetics , Vincristine/administration & dosageABSTRACT
Rhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in childhood. Most rhabdomyosarcoma falls into one of two biologically distinct subgroups represented by alveolar or embryonal histology. The alveolar subtype harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. However, tumor cells have heterogeneous expression for the fusion gene. Using a conditional genetic mouse model as well as human tumor cell lines, we show that that Pax3:Foxo1a expression is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation under stress conditions such as irradiation in vitro and in vivo. Pax3:Foxo1a also tolerizes tumor cells to clinically-established chemotherapy agents and emerging molecularly-targeted agents. Thus, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer cells.
Subject(s)
Forkhead Transcription Factors/biosynthesis , Oncogene Proteins, Fusion/genetics , Paired Box Transcription Factors/biosynthesis , Rhabdomyosarcoma/genetics , Adaptation, Physiological/radiation effects , Animals , Cell Cycle Checkpoints/genetics , Cell Cycle Checkpoints/radiation effects , Cell Line, Tumor , Forkhead Box Protein O1 , Forkhead Transcription Factors/genetics , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Mice , Oncogene Proteins, Fusion/biosynthesis , PAX3 Transcription Factor , Paired Box Transcription Factors/genetics , Radiation , Rhabdomyosarcoma/pathology , Translocation, GeneticABSTRACT
BACKGROUND & AIMS: Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. METHODS: Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. RESULTS: Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. CONCLUSIONS: A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. LAY SUMMARY: HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC.
Subject(s)
Carcinoma, Hepatocellular , Hepatitis B virus , Liver Neoplasms , Oncogene Proteins, Fusion , RNA Precursors , RNA Splicing , Virus Integration , Adult , Aged , Female , Humans , Male , Middle Aged , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/virology , Cyclin A2/genetics , Hepatitis B virus/genetics , Introns , Liver Neoplasms/genetics , Liver Neoplasms/virology , Oncogene Proteins, Fusion/biosynthesis , RNA Precursors/genetics , TranscriptomeABSTRACT
AIMS: Solitary fibrous tumour (SFT) harbours recurrent inv12(q13q13)-derived NAB2-STAT6 fusions, resulting in STAT6 nuclear expression. SFTs affecting the head and neck are rare, for which we reported their clinicopathological, immunohistochemical, and genetic features. METHODS AND RESULTS: With 19 cases assessable for NAB2-STAT6 fusions, 36 head and neck SFTs (18 males; 18 females) diagnosed between 13 and 79 years (median, 47 years) of age were analysed for clinicopathological features and STAT6 immunoexpression. These SFTs, ranging from 5 to 80 mm (median, 25 mm), affected the oral cavity/pharynx (12), orbit (11), sinonasal structures (seven), and somatic soft tissues or skull (six). Histologically, 20 SFTs were conventional, six were giant-cell angiofibroma-like, one was fat-forming, four were cellular/atypical, and five were malignant (two developing metastases). STAT6 distinctively decorated the tumoral nuclei in 35 (97.2%) SFTs, but not in 29 site-relevant histological mimics categorized into 12 entities. Sixteen (84.2%) SFTs showed NAB2-STAT6 fusions with highly heterogeneous exon compositions, including NAB2ex6-STAT6ex17 in four cases, NAB2ex4-STAT6ex2 in three cases, NAB2ex2-STAT6ex2, NAB2ex4-STAT6ex4, NAB2ex6-STAT6ex16 and NAB2ex6-STAT6ex18 in two cases each, and NAB2ex3-STAT6ex19 in one case. CONCLUSIONS: Nuclear STAT6 immunoexpression is sensitive and specific for distinguishing SFT from mimics. However, considerable heterogeneity exists in the head and neck SFTs regarding the locations, histological patterns, and NAB2-STAT6 fusion variants.
Subject(s)
Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Repressor Proteins/genetics , STAT6 Transcription Factor/genetics , Solitary Fibrous Tumors/genetics , Solitary Fibrous Tumors/pathology , Adolescent , Adult , Aged , Biomarkers, Tumor/analysis , Cell Nucleus/metabolism , Diagnosis, Differential , Female , Humans , Immunohistochemistry , Male , Middle Aged , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/genetics , Reverse Transcriptase Polymerase Chain Reaction , STAT6 Transcription Factor/biosynthesis , Young AdultABSTRACT
When murine fetal liver cells were transduced with either of the human acute myeloid leukemia fusion oncogenes MLL-ENL or MLL-AF9 and then transplanted to irradiated recipient mice, myelomonocyte leukemias rapidly developed from the transplanted cells. Analysis of initial events following transduction showed that both oncogenes immediately induced a wide range of enhanced proliferative states, the most extreme of which could generate continuous lines of cells. Maturation defects accompanied the enhanced proliferative states. At all times, the transformed cells exhibited complete dependency on hematopoietic growth factors, particularly GM-CSF and IL-3. Myelomonocytic leukemic cells from primary or transplanted mice formed colonies in semisolid agar. The large majority were dependent on hematopoietic growth factors, but a low frequency of autonomous colonies was also detected. Unexpectedly, reculture of autonomous leukemic colonies generated large numbers of growth factor-dependent clonogenic progeny. Similarly, transplanted clonal autonomous leukemic cells produced leukemias containing a majority of factor-dependent cells. Conversely, recultures of factor-dependent colonies in vitro always produced small numbers of autonomous colonies among the dependent progeny. The reversible relationship between factor dependency and autonomy is surprising because autonomy would have been presumed to represent the final, irreversible, leukemic state.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Interleukin-3/pharmacology , Leukemia, Myeloid/metabolism , Myeloid-Lymphoid Leukemia Protein/biosynthesis , Oncogene Proteins, Fusion/biosynthesis , Animals , Cell Line , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Interleukin-3/metabolism , Leukemia, Myeloid/genetics , Leukemia, Myeloid/pathology , Mice , Myeloid-Lymphoid Leukemia Protein/genetics , Oncogene Proteins, Fusion/geneticsABSTRACT
MLL is involved in fusion genes with more than 100 partner genes, approximately 80 of which have been characterized at the molecular level. MLL fusion genes are often found in infants (60-80% of acute lymphoblastic leukemia (ALL) cases and 40-50% of acute myeloblastic leukemia (AML) cases) and are appreciably rarer (8-10%) in children older than 1 year of age. MLL rearrangements are important markers in diagnosis and treatment choice. To identify the partner gene is of primary importance for prognosis and minimal residual disease monitoring. The structure of the fusion gene, including localization of the MLL breakpoints, is also informative. A method was developed to examine the fusion transcripts in order to identify the partner gene among the six most common ones and to establish the exon structure of the rearranged MLL. The method includes a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify and to fluorescently label a fusion transcript fragment and subsequent hybridization of the product on a biological microchip with immobilized oligonucleotides complementary to exons of MLL and its partner genes AFF1, MLLT1, MLLT3, MLLT4, MLLT10, and ELL. Hybridization results were verified by sequencing the RT-PCR products and, in some cases, performing long-distance inverse PCR (LDI-PCR). The study involved 38 bone marrow samples from ALL patients (including 33 children younger than 1 year of age) and 15 samples from AML patients (including 10 from children younger than 1 year of age). The main partner genes were AFF1 (49%), MLLT1 (27%), MLLT3 (12%), and MLLT10 (12%) in ALL and MLLT3 (80%), MLLT10 (10%), and MLLT4 (10%) in AML. Fusion gene transcripts most commonly included MLL exon 11 (58% of ALL cases and 50% of AML cases), suggesting a breakpoint in MLL intron 11.
Subject(s)
Gene Expression Profiling/instrumentation , Gene Expression Regulation, Leukemic , Histone-Lysine N-Methyltransferase/biosynthesis , Leukemia, Myeloid, Acute/metabolism , Myeloid-Lymphoid Leukemia Protein/biosynthesis , Oligonucleotide Array Sequence Analysis/instrumentation , Oncogene Proteins, Fusion/biosynthesis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Transcription, Genetic , Child , Child, Preschool , Female , Gene Expression Profiling/methods , Histone-Lysine N-Methyltransferase/genetics , Humans , Infant , Infant, Newborn , Leukemia, Myeloid, Acute/genetics , Male , Myeloid-Lymphoid Leukemia Protein/genetics , Oligonucleotide Array Sequence Analysis/methods , Oncogene Proteins, Fusion/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/geneticsABSTRACT
BACKGROUND: Mucoepidermoid carcinoma (MEC) arises from multiple organs and accounts for the most common types of salivary gland malignancies. Currently, patients with unresectable and metastatic MEC have poor long-term clinical outcomes and no targeted therapies are available. The majority of MEC tumors contain a t(11;19) chromosomal translocation that fuses two genes, CRTC1 and MAML2, to generate the chimeric protein CRTC1-MAML2. CRTC1-MAML2 displays transforming activity in vitro and is required for human MEC cell growth and survival, partially due to its ability to constitutively activate CREB-mediated transcription. Consequently, CRTC1-MAML2 is implicated as a major etiologic molecular event and a therapeutic target for MEC. However, the molecular mechanisms underlying CRTC1-MAML2 oncogenic action in MEC have not yet been systematically analyzed. Elucidation of the CRTC1-MAML2-regulated transcriptional program and its underlying mechanisms will provide important insights into MEC pathogenesis that are essential for the development of targeted therapeutics. METHODS: Transcriptional profiling was performed on human MEC cells with the depletion of endogenous CRTC1-MAML2 fusion or its interacting partner CREB via shRNA-mediated gene knockdown. A subset of target genes was validated via real-time RT-PCR assays. CRTC1-MAML2-perturbed molecular pathways in MEC were identified through pathway analyses. Finally, comparative analysis of CRTC1-MAML2-regulated and CREB-regulated transcriptional profiles was carried out to assess the contribution of CREB in mediating CRTC1-MAML2-induced transcription. RESULTS: A total of 808 differentially expressed genes were identified in human MEC cells after CRTC1-MAML2 knockdown and a subset of known and novel fusion target genes was confirmed by real-time RT-PCR. Pathway Analysis revealed that CRTC1-MAML2-regulated genes were associated with network functions that are important for cell growth, proliferation, survival, migration, and metabolism. Comparison of CRTC1-MAML2-regulated and CREB-regulated transcriptional profiles revealed common and distinct genes regulated by CRTC1-MAML2 and CREB, respectively. CONCLUSION: This study identified a specific CRTC1-MAML2-induced transcriptional program in human MEC cells and demonstrated that CRTC1-MAML2 regulates gene expression in CREB-dependent and independent manners. Our data provide the molecular basis underlying CRTC1-MAML2 oncogenic functions and lay a foundation for further functional investigation of CRTC1-MAML2-induced signaling in MEC initiation and maintenance.
Subject(s)
Carcinoma, Mucoepidermoid/genetics , DNA-Binding Proteins/genetics , Gene Expression Profiling/methods , Nuclear Proteins/genetics , Oncogene Proteins, Fusion/genetics , Transcription Factors/genetics , Carcinoma, Mucoepidermoid/metabolism , Cell Line, Tumor , DNA-Binding Proteins/biosynthesis , HEK293 Cells , Humans , Nuclear Proteins/biosynthesis , Oncogene Proteins, Fusion/biosynthesis , Trans-Activators , Transcription Factors/biosynthesisABSTRACT
BACKGROUND: Ewing sarcoma is a rare tumor that occurs commonly in the long bones of children or adolescents that can also arise in soft tissues including the extremities, retroperitoneum, chest wall, and rarely in the liver as primary sites. We report a case of Ewing sarcoma arising primarily in the liver and, to our knowledge, this is the fourth reported case of Ewing sarcoma occurring in the liver. CASE PRESENTATION: A 27-year-old Japanese woman was admitted with sudden onset right upper abdominal pain. Clinical examination revealed a multilocular cystic mass consisting of thickened, irregular septa and nodal walls in the right hepatic lobe. Ultrasound-guided aspiration biopsy of the liver mass showed clusters of small atypical round cells and the clinical preoperative diagnosis was mucinous cystadenoma of the liver. The patient underwent an extended right hepatectomy and histopathological findings revealed sheet-like proliferation of small- to medium-sized round cells. Tumor cells were positive for periodic acid-Schiff reaction and immunoreactive for glycoprotein C99 and gene NKX2.2, as well as the neuroendocrine markers, CD56 and synaptophysin. EWS-FLI-1 fusion transcript type 1 was detected by reverse transcriptase polymerase chain reaction. Pathological and molecular analysis confirmed the diagnosis of Ewing sarcoma arising primarily in the liver and the patient received adjuvant systemic chemotherapy with vincristine, doxorubicin, and cyclophosphamide, alternating with ifosfamide and etoposide. We found no evidence of recurrence 15 months after completing chemotherapy. CONCLUSION: We present an extremely rare case of Ewing sarcoma arising primarily in the liver. To our knowledge, this is the fourth reported case of Ewing sarcoma occurring in the liver, and the first case with a multilocular cystic liver mass. Imaging examinations of the other three reported cases showed solid tumors and a diffuse enlarged liver without mass lesion. Clinicians should consider the possibility of Ewing sarcoma in young patients with a multilocular cystic mass with thick and/or irregular cyst walls in the liver.