RESUMO
Genomic alterations occurring during melanoma progression and the resulting genomic heterogeneity between metastatic deposits remain incompletely understood. Analyzing 86 metastatic melanoma deposits from 53 patients with whole-exome sequencing (WES), we show a low branch to trunk mutation ratio and little intermetastatic heterogeneity, with driver mutations almost completely shared between lesions. Branch mutations consistent with UV damage indicate that metastases may arise from different subclones in the primary tumor. Selective gain of mutated BRAF alleles occurs as an early event, contrasting whole-genome duplication (WGD) occurring as a late truncal event in about 40% of cases. One patient revealed elevated mutational diversity, probably related to previous chemotherapy and DNA repair defects. In another patient having received radiotherapy toward a lymph node metastasis, we detected a radiotherapy-related mutational signature in two subsequent distant relapses, consistent with secondary metastatic seeding. Our findings add to the understanding of genomic evolution in metastatic melanomas.
Assuntos
Genômica/métodos , Melanoma/genética , Mutação , Neoplasias Cutâneas/genética , Progressão da Doença , Feminino , Heterogeneidade Genética , Genoma Humano/genética , Humanos , Masculino , Melanoma/patologia , Melanoma/terapia , Metástase Neoplásica , Recidiva Local de Neoplasia , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Sequenciamento do Exoma/métodosRESUMO
The microRNAs in the miR-34 family, consisting of miR-34a, miR-34b and miR-34c, are tumour suppressors. The annotated human miR-34b-5p has one additional base at the 5' end of the common miR-34 family seed sequence, compared to miR-34a-5p and miR-34c-5p. This extra base results in a shift of the seed sequence, which would affect the target gene repertoire and have functional consequences. During our studies of miR-34 functions, we investigated the precise sequence of mature miR-34b-5p in human cells by deep sequencing. We found that a miR-34b-5p without the extra base was the predominant form in both non-malignant and malignant cells derived from several human tissues, indicating that the miR-34b annotation is misleading. We evaluated the functional implications of the seed shift, by comparing the effect of mimics representing the alternative miR-34b-5p sequences in MDA-MB-231 cells. In contrast to the annotated miR-34b, the endogenously expressed miR-34b displayed tumour suppressive characteristics in vitro similarly to miR-34c. These data demonstrate the importance of determining the precise sequence of a mature microRNA before exploring miRNA functions.
Assuntos
Neoplasias da Mama/genética , Biologia Computacional , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Anotação de Sequência Molecular , Família Multigênica , Sequência de Bases , Linhagem Celular Tumoral , Biologia Computacional/métodos , Sequência Conservada , Feminino , Humanos , MicroRNAs/químicaRESUMO
Lung cancer is the leading cause of cancer related death, and the past years' improved insight into underlying molecular events has significantly improved outcome for specific subsets of patients. In particular, several new therapies that target protein kinases have been implemented, and many more are becoming available. We have investigated lung cancer specimens for somatic mutations in a targeted panel of 612 human genes, the majority being protein kinases. The somatic mutation profiles were correlated to profiles of immune cell infiltration as well as relapse-free survival. Targeted deep sequencing was performed on 117 tumour/normal pairs using the SureSelect Human Kinome kit (Agilent Technologies), with capture probes targeting 3.2 Mb of the human genome, including exons and untranslated regions of all known kinases, kinase receptors and selected cancer-related genes (612 genes in total). CD8 staining was determined using Ventana Benchmark. Survival analyses were performed using SPSS. The number of mutations per sample ranged from 0 to 50 (within the 612 genes tested), with a median of nine. The prognosis was worse for patients with more than the median number of mutations. A significant correlation was found between mutations in one of selected DNA-repair genes and the total number of mutations in that tumour (p < 0.001). There was a significant inverse correlation between the number of infiltrating stromal CD8+ lymphocytes and the presence of EGFR mutations.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Imunidade Celular/genética , Proteínas de Neoplasias/genética , Fosfotransferases/genética , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/patologia , Intervalo Livre de Doença , Feminino , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Fosfotransferases/antagonistas & inibidores , Prognóstico , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
In this study, we analyzed the prevalence and clone size of BRAF V600E mutation in 209 patients with multiple myeloma and related the results to clinical phenotype, response and survival. Biopsies were screened for BRAF V600E by allele-specific real-time PCR (AS-PCR). Positive results were confirmed by immunohistochemistry, Sanger sequencing and, in three patients from whom we had stored purified myeloma cells, whole-exome sequencing. Eleven patients (5.3%) were BRAF V600E mutation positive by AS-PCR and at least one other method. The fraction of mutated cells varied from 4 to 100%. BRAF V600E-positive patients had no characteristic clinical phenotype except for significantly higher levels of serum creatinine (125 versus 86 µmol/l) Seven of eleven patients responded with at least very good partial response to alkylators, immunomodulatory agents or proteasome inhibitors. Progression-free and overall survival were similar in patients with and without the mutation. By this integrated approach, we found that patients with BRAF V600E mutation responded very well to broad acting drugs and there was no relation to prognosis in early-stage myeloma. In particular, a large mutated cell fraction did not correlate with aggressive disease.
Assuntos
Antineoplásicos/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Adulto , Idoso , Biomarcadores Farmacológicos , Intervalo Livre de Doença , Exoma/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Mutação , Estadiamento de NeoplasiasRESUMO
BACKGROUND: Osteosarcoma is the most common primary malignant bone tumour, predominantly affecting children and adolescents. Cancer cell line models are required to understand the underlying mechanisms of tumour progression and for preclinical investigations. METHODS: To identify cell lines that are well suited for studies of critical cancer-related phenotypes, such as tumour initiation, growth and metastasis, we have evaluated 22 osteosarcoma cell lines for in vivo tumorigenicity, in vitro colony-forming ability, invasive/migratory potential and proliferation capacity. Importantly, we have also identified mRNA and microRNA (miRNA) gene expression patterns associated with these phenotypes by expression profiling. RESULTS: The cell lines exhibited a wide range of cancer-related phenotypes, from rather indolent to very aggressive. Several mRNAs were differentially expressed in highly aggressive osteosarcoma cell lines compared with non-aggressive cell lines, including RUNX2, several S100 genes, collagen genes and genes encoding proteins involved in growth factor binding, cell adhesion and extracellular matrix remodelling. Most notably, four genes-COL1A2, KYNU, ACTG2 and NPPB-were differentially expressed in high and non-aggressive cell lines for all the cancer-related phenotypes investigated, suggesting that they might have important roles in the process of osteosarcoma tumorigenesis. At the miRNA level, miR-199b-5p and mir-100-3p were downregulated in the highly aggressive cell lines, whereas miR-155-5p, miR-135b-5p and miR-146a-5p were upregulated. miR-135b-5p and miR-146a-5p were further predicted to be linked to the metastatic capacity of the disease. INTERPRETATION: The detailed characterisation of cell line phenotypes will support the selection of models to use for specific preclinical investigations. The differentially expressed mRNAs and miRNAs identified in this study may represent good candidates for future therapeutic targets. To our knowledge, this is the first time that expression profiles are associated with functional characteristics of osteosarcoma cell lines.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , MicroRNAs/genética , Osteossarcoma/genética , Osteossarcoma/patologia , RNA Mensageiro/genética , Animais , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade NeoplásicaRESUMO
Gain of chromosome 18q and translocation t(14;18) are] frequently found in B-cell non-Hodgkin's lymphomas (B-NHL). Increased BCL2 transcription and BCL2 protein expression have been suggested to be the result of the gain. We utilized FISH, PCR and array CGH to study BCL2 and chromosome 18 copy number changes and rearrangements in 93 cases of B-NHL. BCL2 protein was expressed in >75% of the tumor cells in 92% of the cases by immunohistochemistry. Gain of BCL2 was associated with a 25% increase in BCL2 expression levels (immunoblotting), whereas t(14;18) resulted in a 55% increase in BCL2 levels compared to cases without BCL2 alterations. The tumor cell (spontaneous) apoptotic fractions were similar for the cases with different BCL2 genotypes. However, the normal cell apoptotic fractions were higher for the tumors with t(14;18) compared to the tumors without BCL2 alterations, while the tumors with gain of BCL2 only showed intermediate levels. Low-level gains of parts of chromosome 18 were found in 14 of the 38 B-NHL cases with t(14;18), with a consensus region 18pter-q21.33 that did not include the BCL2 gene. The 11 cases with 18q gain only showed a consensus region encompassing 18q21.2-18q21.32 and 18q21.33, which contain PMAIP1/MALT1 and BCL2, respectively.
Assuntos
Apoptose/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 18 , Linfoma de Células B/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Análise Citogenética , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Humanos , Linfonodos/patologia , Linfoma de Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Translocação GenéticaRESUMO
Soft tissue sarcomas represent a heterogeneous group of tumors and include over 50 histotypes. Some of these tumor types are characterized by specific chromosomal translocations, whereas other types show complex genetic aberrations. The recent developments within gene expression technologies have now been applied to studies of soft tissue sarcomas (STS) and the first results indicate that genetic signatures are useful for classification and diagnosis. Distinctive expression profiles have been found in e.g. gastrointestinal stromal tumors (GISTs), synovial sarcomas, malignant peripheral nerve sheath tumors (MPNSTs), and in subsets of liposarcomas. The more pleomorphic tumor types, such as high-grade variants of leiomyosarcomas, malignant fibrous histiocytomas (MFHs), fibrosarcomas, and subtypes of liposarcomas, show a greater variability among the expression profiles, but interestingly subsets with distinctive expression profiles can be identified also among these tumors. The data available place many of the genes hypothesized to be involved in the development of a certain type of STS, such as the KIT gene in GIST development, among the top discriminating genes. Thereby expression profiling provides novel insights into the pathogenesis of STS. Although much work remains to be done to validate the data and to define optimal discriminating gene lists, the current lessons from gene expression studies in STS are encouraging and imply that genetic signatures may serve as diagnostic and prognostic markers and may help identify novel therapeutic strategies.
Assuntos
Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Sarcoma/genética , Neoplasias de Tecidos Moles/genética , Aberrações Cromossômicas , Biologia Computacional , Humanos , Processamento de Imagem Assistida por Computador , Hibridização de Ácido Nucleico/métodos , Translocação GenéticaRESUMO
Well-differentiated liposarcomas (WDLPS) are cytogenetically characterized by the presence of supernumerary ring or giant rod marker chromosomes. These supernumerary chromosomes are composed of amplified sequences from chromosome 12 (12q14 approximately 15) in association with amplified segments from various other chromosomes, and contain alterations of the alpha satellite sequences. We report a case of WDLPS of the lipoma-like and sclerosing subtype that contains a novel type of supernumerary marker chromosome. Instead of rings or giant rods, these cells had three apparently identical copies of a subtelocentric supernumerary marker with a size and shape similar to C-group chromosomes. Fluorescence in situ hybridization analysis revealed that the markers were composed of amplified material from 12q14 approximately 15, including the genes MDM2 and CDK4. Similar to the rings and giant rods observed in other WDLPS cases, these unusual markers had no alpha satellite repeats at the primary constriction site, but centromeric activity could be demonstrated by using anti-centromere protein C antibodies. These findings show that the supernumerary markers of WDLPS may be variable in size and shape, but consistently share the same genomic structure, specifically 12q amplified sequences together with centromere alterations, and underline the importance of molecular methods in the diagnosis of adipose tissue tumors.
Assuntos
Cromossomos Humanos Par 12/genética , Análise Citogenética/métodos , Lipossarcoma/genética , Lipossarcoma/patologia , Idoso , Feminino , Marcadores Genéticos/genética , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Metáfase , Hibridização de Ácido Nucleico , Neoplasias Retroperitoneais/genéticaRESUMO
PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Proteínas de Transporte/genética , Proteínas de Drosophila , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Proteínas de Insetos/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Núcleosídeo-Difosfato Quinase , Sarcoma/genética , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Neoplasias da Mama/patologia , Células COS , Carcinoma/patologia , Proteínas de Transporte/fisiologia , Divisão Celular , Feminino , Humanos , Proteínas de Insetos/fisiologia , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Nucleosídeo NM23 Difosfato Quinases , Metástase Neoplásica , Monoéster Fosfórico Hidrolases , RNA Neoplásico/biossíntese , Sarcoma/patologia , Fatores de Transcrição/genéticaRESUMO
The HMGIC gene codes for an architectural transcription factor frequently rearranged by translocation in lipomas and other benign mesenchymal tumors. In sarcomas, malignant tumors of mesenchymal origin, the gene is also found to be rearranged, but in addition amplified and overexpressed. Here we report the sequence, chromosomal localization, and expression patterns of 11 novel ectopic sequences fused to exons 2 and 3 of HMGIC in seven different sarcoma samples. In addition, we identified a number of variant transcripts observed previously in benign tumors. Consistent with the suggested role of HMGIC in adipocytic differentiation, most of the novel ectopic sequences were observed in well-differentiated liposarcomas. These tumors are known to have complex marker chromosomes containing amplified segments from several chromosomes. Five novel sequences were derived from 12q14-q15, where HMGIC resides, two from 1q24, a region frequently amplified in these types of tumors, two from 11q14, and one from chromosome 2. All except one of the aberrant transcripts encoded truncated proteins with intact DNA-binding domains (AT hooks) but lacking the C-terminal acidic region, a target for constitutive phosphorylation by protein kinase CK2. Some of the ectopic sequences were transcribed in other tissues, and most of the ectopic sequences also showed recurrent amplification in liposarcomas.
Assuntos
Amplificação de Genes , Proteínas de Grupo de Alta Mobilidade/genética , Lipossarcoma/genética , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Northern Blotting , Southern Blotting , Mapeamento Cromossômico , Dosagem de Genes , Proteína HMGA2 , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Translocação Genética/genética , Células Tumorais CultivadasRESUMO
Well-differentiated liposarcomas (WDLPS), especially those located in the retroperitoneum, may occasionally undergo dedifferentiation. Although this process is associated with a more aggressive clinical course, dedifferentiated liposarcomas rarely produces metastases. The case reported here is rather uncommon: A retroperitoneal WDLPS gave lung metastases that were diagnosed as highly malignant osteosarcomas. We used comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and Southern blot analyses to characterize the copy number changes and genetic aberrations occurring at different stages of the disease. In the primary tumor, the only detectable aberration was amplification of 12q13-q14, which was present only in a fraction of the cells and revealed by FISH analysis. High-level amplification of 12q13-q14, involving CDK4, MDM2, and HMGIC, was seen both in the relapse and the metastases. The second most common change, gain or high-level amplification of 1q22-q24, was detectable by CGH only in the osteogenic metastases, as was loss of the distal 2q. FISH analyses revealed considerable heterogeneity in the samples, and the percentage of cells showing aberrations was significantly higher in the metastatic samples. In particular, increased copy numbers of 789f2, a marker for 1q21 amplification in sarcomas, was observed in more than 65% of the cells in the metastatic samples, but in less than 10% of the cells from the recurrent samples. These observations could indicate that 1q amplification, in particular, may be indicative of a more malignant phenotype and ability of metastasis in WDLPS, as has also been suggested by others.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 12/ultraestrutura , Cromossomos Humanos Par 1/ultraestrutura , Lipossarcoma/patologia , Neoplasias Pulmonares/secundário , Metástase Neoplásica/genética , Recidiva Local de Neoplasia/patologia , Osteossarcoma/secundário , Neoplasias Retroperitoneais/patologia , Adulto , Northern Blotting , Southern Blotting , Diferenciação Celular/genética , Centrômero/ultraestrutura , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 12/genética , Terapia Combinada , Evolução Fatal , Feminino , Seguimentos , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Lipossarcoma/genética , Lipossarcoma/terapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Recidiva Local de Neoplasia/genética , Oncogenes , Osteossarcoma/genética , Osteossarcoma/patologia , Neoplasias Retroperitoneais/genética , Neoplasias Retroperitoneais/terapiaRESUMO
Clinical and experimental evidence suggests that tumor cells shed into the circulation from solid cancers are ineffective in forming distant metastasis unless the cells are able to respond to growth conditions offered by the secondary organs. To identify the phenotypic properties that are specific for such growth response, we injected carcinoma cells, which had been recovered from bone marrow micrometastases in a breast cancer patient who was clinically devoid of overt metastatic disease and established in culture, into the systemic circulation of immunodeficient rats. The animals developed metastases in the central nervous system, and metastatic tumor cells were isolated with immunomagnetic beads coated with an antibody that was reactive with human cells. The segregated cell population was compared with the injected cells by means of differential display analysis, and two candidate fragments were identified as up-regulated in the fully metastatic cells. The first was an intracellular effector molecule involved in tyrosine kinase signaling, known to mediate nerve growth factor-dependent promotion of cell survival. The second was a novel gene product (termed candidate of metastasis-1), presumably encoding a DNA-binding protein of helix-turn-helix type. Constitutive expression of candidate of metastasis-1 seemed to distinguish breast cancer cells with metastatic potential from cells without metastatic potential. Hence, our experimental approach identified factors that may mediate the growth response of tumor cells upon establishment in a secondary organ and, thereby, contribute to the metastatic phenotype.
Assuntos
Medula Óssea/patologia , Neoplasias da Mama/patologia , Carcinoma Lobular/patologia , Proteínas de Ligação a DNA , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Neoplasias da Mama/genética , Carcinoma Lobular/genética , Clonagem Molecular , Feminino , Humanos , Separação Imunomagnética , Dados de Sequência Molecular , Invasividade Neoplásica , Metástase Neoplásica , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Nus , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transcrição Gênica , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
In a recent comparative genomic hybridization (CGH) study of a panel of sarcomas, we detected recurrent amplification of 1q21-q22 in soft tissue and bone tumours. Amplification of this region had not previously been associated with sarcoma development, but occasional amplification of CACY/S100A6 and MUC1 in 1q21 had been reported for melanoma and breast carcinoma respectively. Initial screening by Southern blot analysis showed amplification of S100A6, FLG and SPRR3 in several sarcomas and, in a first attempt to characterize the 1q21-q22 amplicon in more detail, we have now investigated the amplification status of these and 11 other markers in the region in 35 sarcoma samples. FLG was the most frequently amplified gene, and the markers located in the same 4.5-Mb region as FLG showed a higher incidence of amplification than the more distal ones. However, for most of the 14 markers, amplification levels were low, and only APOA2 and the anonymous marker D1S3620 showed high-level amplifications (> tenfold increases) in one sample each. We used fluorescence in situ hybridization (FISH) to determine the amplification patterns of two overlapping yeast artificial chromosomes (YACs) covering the region between D1S3620 and FLG (789f2 and 764a1), as well as two more distally located YACs in nine selected samples. Six samples had amplification of the YAC containing D1S3620 and, in three, 764a1 was also included. Five of these tumours showed normal copies of the more distal YACs; thus, it seems likely that an important gene may be located within 789f2, or very close. Two samples had high copy numbers of the most distal YACs. Taken together, FISH and molecular analyses indicate complex amplification patterns in 1q21-q22 with at least two amplicons: one located near D1S3620/789f2 and one more distal.
Assuntos
Biomarcadores , Cromossomos Humanos Par 1 , Amplificação de Genes , Sarcoma/genética , Southern Blotting , Cromossomos Artificiais de Levedura , Proteínas Filagrinas , Humanos , Hibridização in Situ Fluorescente , Proteínas de Filamentos Intermediários/genética , Mucina-1/genética , Precursores de Proteínas/genética , Proteínas S100/genéticaRESUMO
Amplified segments of the long arm of chromosome 12 are frequently observed in human sarcomas. In most cases there are separate amplified regions around the MDM2 and CDK4 genes. Here we show recurrent amplification of a third region encompassing HMGIC, a human architectural transcription factor gene. Reduced amplification frequency of sequences flanking the gene was observed, indicating that inclusion of this third region in the amplicons is also selected for. In three samples only the 5' part of HMGIC was amplified, suggesting preferential loss of the 3' part of the gene preceding or during amplification. In several other samples rearrangement of the gene was observed. Expression analysis showed transcripts of aberrant sizes, lacking 3' sequences, and 3' RACE of one sample revealed replacement of exons 4 and 5 with ectopic sequences. This truncation of HMGIC resembles that reported for translocations of HMGIC in benign tumors, including lipomas, and it is striking that the gene was frequently amplified or rearranged in well differentiated liposarcomas, the malignant counterpart of lipomas. It seems conceivable that high levels of either full length or truncated hmgic could be relevant for the etiology of these tumors.