ABSTRACT
Heterozygous somatic mutations affecting the spliceosome gene SF3B1 drive age-related clonal hematopoiesis, myelodysplastic syndromes (MDS) and other neoplasms. To study their role in such disorders, we generated knock-in mice with hematopoietic-specific expression of Sf3b1-K700E, the commonest type of SF3B1 mutation in MDS. Sf3b1K700E/+ animals had impaired erythropoiesis and progressive anemia without ringed sideroblasts, as well as reduced hematopoietic stem cell numbers and host-repopulating fitness. To understand the molecular basis of these observations, we analyzed global RNA splicing in Sf3b1K700E/+ hematopoietic cells. Aberrant splicing was associated with the usage of cryptic 3' splice and branchpoint sites, as described for human SF3B1 mutants. However, we found a little overlap between aberrantly spliced mRNAs in mouse versus human, suggesting that anemia may be a consequence of globally disrupted splicing. Furthermore, the murine orthologues of genes associated with ring sideroblasts in human MDS, including Abcb7 and Tmem14c, were not aberrantly spliced in Sf3b1K700E/+ mice. Our findings demonstrate that, despite significant differences in affected transcripts, there is overlap in the phenotypes associated with SF3B1-K700E between human and mouse. Future studies should focus on understanding the basis of these similarities and differences as a means of deciphering the consequences of spliceosome gene mutations in MDS.
Subject(s)
Anemia, Sideroblastic/etiology , Anemia, Sideroblastic/pathology , Hematopoiesis/genetics , Myelodysplastic Syndromes/complications , Myelodysplastic Syndromes/genetics , Phosphoproteins/genetics , RNA Splicing Factors/genetics , RNA Splicing , Anemia, Sideroblastic/mortality , Animals , Disease Models, Animal , Gene Targeting , Humans , Mice , Mice, Transgenic , Mutation , Phenotype , RNA Splicing Factors/metabolismABSTRACT
Advances in sequencing technologies are giving unprecedented insights into the spectrum of somatic mutations underlying acute myeloid leukaemia with a normal karyotype (AML-NK). It is clear that the prognosis of individual patients is strongly influenced by the combination of mutations in their leukaemia and that many leukaemias are composed of multiple subclones, with differential susceptibilities to treatment. Here, we describe a method, employing targeted capture coupled with next-generation sequencing and tailored bioinformatic analysis, for the simultaneous study of 24 genes recurrently mutated in AML-NK. Mutational analysis was performed using open source software and an in-house script (Mutation Identification and Analysis Software), which identified dominant clone mutations with 100% specificity. In each of seven cases of AML-NK studied, we identified and verified mutations in 2-4 genes in the main leukaemic clone. Additionally, high sequencing depth enabled us to identify putative subclonal mutations and detect leukaemia-specific mutations in DNA from remission marrow. Finally, we used normalised read depths to detect copy number changes and identified and subsequently verified a tandem duplication of exons 2-9 of MLL and at least one deletion involving PTEN. This methodology reliably detects sequence and copy number mutations, and can thus greatly facilitate the classification, clinical research, diagnosis and management of AML-NK.
Subject(s)
Karyotype , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Molecular Diagnostic Techniques , Adult , Aged , Aged, 80 and over , Exons , Female , Gene Duplication , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Mutation , Oligonucleotide Array Sequence Analysis , Tandem Repeat SequencesABSTRACT
The objective of the present study was to elaborate a survival model that integrates anatomic factors, according to the 2010 seventh edition of the tumour, node and metastasis (TNM) staging system, with clinical and molecular factors. Pathologic TNM descriptors (group A), clinical variables (group B), laboratory parameters (group C) and molecular markers (tissue microarrays; group D) were collected from 512 early-stage nonsmall cell lung cancer (NSCLC) patients with complete resection. A multivariate analysis stepped supervised learning classification algorithm was used. The prognostic performance by groups was: areas under the receiver operating characteristic curve (C-index): 0.67 (group A), 0.65 (Group B), 0.57 (group C) and 0.65 (group D). Considering all variables together selected for each of the four groups (integrated group) the C-index was 0.74 (95% CI 0.70-0.79), with statistically significant differences compared with each isolated group (from p = 0.006 to p < 0.001). Variables with the greatest prognostic discrimination were the presence of another ipsilobar nodule and tumour size > 3 cm, followed by other anatomical and clinical factors, and molecular expressions of phosphorylated mammalian target of rapamycin (phospho-mTOR), Ki67cell proliferation index and phosphorylated acetyl-coenzyme A carboxylase. This study on early-stage NSCLC shows the benefit from integrating pathological TNM, clinical and molecular factors into a composite prognostic model. The model of the integrated group classified patients with significantly higher accuracy compared to the TNM 2010 staging.
Subject(s)
Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Neoplasm Staging/methods , Aged , Algorithms , Area Under Curve , Carcinoma, Non-Small-Cell Lung/therapy , Cohort Studies , Humans , Ki-67 Antigen/biosynthesis , Lung Neoplasms/therapy , Medical Oncology/methods , Middle Aged , Neoplasm Metastasis , Probability , Prognosis , Time FactorsABSTRACT
In acute myeloid leukaemia (AML), nucleophosmin-1 (NPM1) mutations create a nuclear export signal (NES) motif and disrupt tryptophans at NPM1 C-terminus, leading to nucleophosmin accumulation in leukaemic cell cytoplasm. We investigated how nucleophosmin NES motifs (two physiological and one created by the mutation) regulate traffic and interaction of mutated NPM1, NPM1wt and p14(ARF). Nucleophosmin export into cytoplasm was maximum when the protein contained all three NES motifs, as naturally occurs in NPM1-mutated AML. The two physiological NES motifs mediated NPM1 homo/heterodimerization, influencing subcellular distribution of NPM1wt, mutated NPM1 and p14(ARF) in a 'dose-dependent tug of war' fashion. In transfected cells, excess doses of mutant NPM1 relocated completely NPM1wt (and p14(ARF)) from the nucleoli to the cytoplasm. This distribution pattern was also observed in a proportion of NPM1-mutated AML patients. In transfected cells, excess of NPM1wt (and p14(ARF)) relocated NPM1 mutant from the cytoplasm to the nucleoli. Notably, this distribution pattern was not observed in AML patients where the mutant was consistently cytoplasmic restricted. These findings reinforce the concept that NPM1 mutants are naturally selected for most efficient cytoplasmic export, pointing to this event as critical for leukaemogenesis. Moreover, they provide a rationale basis for designing small molecules acting at the interface between mutated NPM1 and other interacting proteins.
Subject(s)
Active Transport, Cell Nucleus/physiology , Leukemia, Myeloid/genetics , Neoplasm Proteins/genetics , Nuclear Export Signals/genetics , Nuclear Proteins/genetics , Protein Interaction Mapping , Tumor Suppressor Protein p14ARF/chemistry , Active Transport, Cell Nucleus/genetics , Acute Disease , Animals , Cell Nucleolus/metabolism , Cell Transformation, Neoplastic/genetics , Cytoplasm/metabolism , Dimerization , Drug Delivery Systems , Humans , Leukemia, Myeloid/metabolism , Mice , NIH 3T3 Cells/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Nuclear Export Signals/physiology , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Nucleophosmin , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Structure-Activity Relationship , Transfection , Tumor Suppressor Protein p14ARF/metabolismSubject(s)
B-Lymphocytes/metabolism , Leukemia, Myeloid/genetics , Mutation , Nuclear Proteins/genetics , T-Lymphocytes/metabolism , Active Transport, Cell Nucleus , Acute Disease , B-Lymphocytes/pathology , Biopsy , Bone Marrow/metabolism , Cell Lineage , Cytoplasm/metabolism , Fluorescent Antibody Technique , Humans , Immunoenzyme Techniques , Leukemia, Myeloid/diagnosis , Myeloid Cells , Nucleophosmin , Phosphoproteins/genetics , Subcellular Fractions , T-Lymphocytes/pathologyABSTRACT
A simple and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) method is described for the detection of anabolic steroids, usually found in keratin matrix at very low concentrations. Hair samples from seven athletes who spontaneously reported their abuse of anabolic steroids, and in a single case cocaine, were analyzed for methyltestosterone, nandrolone, boldenone, fluoxymesterolone, cocaine and its metabolite benzoylecgonine. Anabolic steroids were determinate by digestion of hair samples in 1 m NaOH for 15 min at 95 degrees C. After cooling, samples were purificated by solid-phase and liquid-liquid extraction, then anabolic steroids were converted to their trimethylsilyl derivative and finally analyzed by GC/MS/MS. For detection of cocaine and benzoylecgonine, hair samples were extracted with methanol in an ultrasonic bath for 2 h at 56 degrees C then overnight in a thermostatic bath at the same temperature. After the incubation, methanol was evaporated to dryness, and benzoylecgonine was converted to its trimethylsilyl derivative prior of GC/MS/MS analysis. Results obtained are in agreement with the athletes' reports, confirming that hair is a valid biological matrix to establish long-term intake of drugs.
Subject(s)
Anabolic Agents/analysis , Gas Chromatography-Mass Spectrometry/methods , Hair/chemistry , Steroids/analysis , Substance Abuse Detection/methods , Tandem Mass Spectrometry/methods , Humans , Reference Standards , SportsABSTRACT
OBJECTIVE: To quantify changes in tumor-node-metastasis (TNM) staging (numerical migration) and survival (prognostic migration) that arise when certainty criteria are applied to a patient population with non-small cell lung cancer (NSCLC) treated surgically. METHODS: The population consisted of 1,844 patients with NSCLC who underwent surgery between 1993 and 1996 at hospitals participating in the Bronchogenic Carcinoma Co-operative Group of the Spanish Society of Pneumology and Thoracic Surgery (GCCB-S). For every patient, surgical-pathological TNM staging (p) was based on two classifications: initial staging by each participating GCCB-S center (pTNM-i) and a second classification bearing greater classificatory certainty (pTNM-cc) resulting from the application of stricter criteria. Numerical migration was said to have occurred in cases where the two classifications did not coincide, and the possible prognostic migration under the new staging was then assessed. RESULTS: The results revealed great numerical migration in the pN0 classification (from 1,091 cases to 665). The changes did not result in prognostic migration either for the group as a whole or for pT1-2N0M0 cases. However, for pT3N0M0 cases, median survival increased by 13 months. The difference in three-year survival (S3) for pT3N0M0-i without certainty confirmation [S3 = 0.30 (95%CI 0.18-0.42), n=59] and pT3N0M0-cc [S3=0.54 (95%CI = 0.44-0.64), n = 92] was significant (log-rank, p = 0.035). Such behavior was not observed for pT1-2N0M0. CONCLUSIONS: The numerical migration observed as a result of applying surgical-pathological classificatory certainty criteria is relevant but the prognostic repercussion is scarce, except in cases classified as pT3N0M0, in which a significant positive prognostic migration is observed (the "Will Rogers phenomenon").
Subject(s)
Carcinoma, Non-Small-Cell Lung/pathology , Lung Neoplasms/pathology , Neoplasm Staging/statistics & numerical data , Treatment Outcome , Carcinoma, Non-Small-Cell Lung/surgery , Disease Progression , Humans , Lung Neoplasms/surgery , Lymph Nodes/pathology , PrognosisABSTRACT
OBJETIVO: Cuantificar el cambio en la clasificación TNMestadios (migración numérica) y la modificación en supervivencia (migración pronóstica) al aplicar criterios de certeza clasificatoria a una población de carcinoma broncogénico no microcítico (CBNM) con tratamiento quirúrgico. MÉTODOS: La población está formada por 1.844 casos de CBNM operados entre 1993 y 1996 por los hospitales del Grupo Cooperativo de Carcinoma Broncogénico de la Sociedad Española de Neumología y de Cirugía Torácica (GCCB-S). La estadificación TNM quirúrgico-patológica (p) de cada paciente tiene dos clasificaciones; una inicial efectuada por cada centro del GCCB-S (TNMp-i) y otra, con mayor certeza clasificatoria (TNMp-cc), que se obtiene tras la aplicación de criterios más exigentes. En los casos en que ambas clasificaciones no coincidan se produce una migración numérica, evaluando la posible migración pronóstica con las nuevas estadificaciones. RESULTADOS: Se detecta una gran migración numérica en la clasificación N0p (de 1.091 a 665 casos). Estos cambios no producen migraciones pronósticas en el grupo considerado globalmente, ni tampoco en las categorías T1-2N0M0p; sin embargo, en la clasificación T3N0M0p se incrementa en 13 meses la mediana de supervivencia. La diferencia de supervivencia a 3 años (S3) entre T3N0M0p-i sin confirmación de certeza (S3 = 0,30; intervalo de confianza [IC] del 95 por ciento; 0,18-0,42; n = 59) y T3N0M0p-cc (S3 = 0,54; IC del 95 por ciento = 0,44-0,64; n = 92) era significativa (rangos logarítmicos; p = 0,035); este comportamiento no se observa en T1-2N0M0p.CONCLUSIÓN: La migración numérica observada por la aplicación de criterios de certeza clasificatoria quirurgicopatológica es relevante, aunque la repercusión pronóstica es pequeña, salvo en T3N0M0p, en donde se detecta una mejora significativa del pronóstico (migración tipo fenómeno "WillRogers"). (AU)
Subject(s)
Humans , Treatment Outcome , Disease Progression , Prognosis , Lymph Nodes , Neoplasm Staging , Carcinoma, Non-Small-Cell Lung , Lung NeoplasmsABSTRACT
The present review addresses recent advances in research into a family of bifunctional enzymes that are responsible for the twofold task of synthesizing and hydrolyzing fructose-2,6-bisphosphate (Fru-2,6-P2), which in turn regulates the rate of glycolysis in most cells. The structure of the synthetic kinase, conjoined at its carboxyl-terminus to the phosphatase, is very highly conserved throughout evolution and differentiation, with isotypic expression arising from highly variable amino-terminal and carboxyl-terminal regulatory domains. These domains, which frequently contain protein-kinase-catalyzed phosphorylation motifs, are responsible for the widely divergent kinetics observed in various tissues and species, and for the hormonal modulation that alters intracellular levels of Fru-2,6-P2. The present review discusses recent advances in relating structure to function, and the identification of new pathways of transcriptional regulation of this important family of regulatory enzymes.
