RESUMO
Background: Primary glioblastoma cell (GC) cultures have emerged as a key model in brain tumor research, with the potential to uncover patient-specific differences in therapy response. However, there is limited quantitative information about the stability of such cells during the initial 20-30 passages of culture. Methods: We interrogated 3 patient-derived GC cultures at dense time intervals during the first 30 passages of culture. Combining state-of-the-art signal processing methods with a mathematical model of growth, we estimated clonal composition, rates of change, affected pathways, and correlations between altered gene dosage and transcription. Results: We demonstrate that GC cultures undergo sequential clonal takeovers, observed through variable proportions of specific subchromosomal lesions, variations in aneuploid cell content, and variations in subpopulation cell cycling times. The GC cultures also show significant transcriptional drift in several metabolic and signaling pathways, including ribosomal synthesis, telomere packaging and signaling via the mammalian target of rapamycin, Wnt, and interferon pathways, to a high degree explained by changes in gene dosage. In addition to these adaptations, the cultured GCs showed signs of shifting transcriptional subtype. Compared with chromosomal aberrations and gene expression, DNA methylations remained comparatively stable during passaging, and may be favorable as a biomarker. Conclusion: Taken together, GC cultures undergo significant genomic and transcriptional changes that need to be considered in functional experiments and biomarker studies that involve primary glioblastoma cells.
Assuntos
Técnicas de Cultura de Células/métodos , Aberrações Cromossômicas , Instabilidade Genômica , Glioblastoma/genética , Glioblastoma/patologia , Medicina de Precisão , Idoso , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proliferação de Células , Metilação de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células Tumorais CultivadasRESUMO
BACKGROUND: Molecular alterations are well studied in colon cancer, however there is still need for an improved understanding of their prognostic impact. This study aims to characterize colon cancer with regard to KRAS, BRAF, and PIK3CA mutations, microsatellite instability (MSI), and average DNA copy number, in connection with tumour dissemination and recurrence in patients with colon cancer. METHODS: Disease stage II-IV colon cancer patients (n = 121) were selected. KRAS, BRAF, and PIK3CA mutation status was assessed by pyrosequencing and MSI was determined by analysis of mononucleotide repeat markers. Genome-wide average DNA copy number and allelic imbalance was evaluated by SNP array analysis. RESULTS: Patients with mutated KRAS were more likely to experience disease dissemination (OR 2.75; 95% CI 1.28-6.04), whereas the opposite was observed for patients with BRAF mutation (OR 0.34; 95% 0.14-0.81) or MSI (OR 0.24; 95% 0.09-0.64). Also in the subset of patients with stage II-III disease, both MSI (OR 0.29; 95% 0.10-0.86) and BRAF mutation (OR 0.32; 95% 0.16-0.91) were related to lower risk of distant recurrence. However, average DNA copy number and PIK3CA mutations were not associated with disease dissemination. CONCLUSIONS: The present study revealed that tumour dissemination is less likely to occur in colon cancer patients with MSI and BRAF mutation, whereas the presence of a KRAS mutation increases the likelihood of disseminated disease.
Assuntos
Neoplasias do Colo/diagnóstico , Neoplasias do Colo/genética , Instabilidade de Microssatélites , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Idoso , Feminino , Seguimentos , Humanos , Masculino , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas p21(ras)RESUMO
Genetic differences among neoplastic cells within the same tumour have been proposed to drive cancer progression and treatment failure. Whether data on intratumoral diversity can be used to predict clinical outcome remains unclear. We here address this issue by quantifying genetic intratumoral diversity in a set of chemotherapy-treated childhood tumours. By analysis of multiple tumour samples from seven patients we demonstrate intratumoral diversity in all patients analysed after chemotherapy, typically presenting as multiple clones within a single millimetre-sized tumour sample (microdiversity). We show that microdiversity often acts as the foundation for further genome evolution in metastases. In addition, we find that microdiversity predicts poor cancer-specific survival (60%; P=0.009), independent of other risk factors, in a cohort of 44 patients with chemotherapy-treated childhood kidney cancer. Survival was 100% for patients lacking microdiversity. Thus, intratumoral genetic diversity is common in childhood cancers after chemotherapy and may be an important factor behind treatment failure.
Assuntos
Progressão da Doença , Heterogeneidade Genética , Neoplasias/genética , Neoplasias/patologia , Alelos , Antineoplásicos/uso terapêutico , Criança , Pré-Escolar , Evolução Molecular , Genoma Humano , Instabilidade Genômica , Humanos , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Prognóstico , Resultado do TratamentoRESUMO
BACKGROUND: The clinical behaviour of colon cancer is heterogeneous. Five-year overall survival is 50-65% with all stages included. Recurring somatic chromosomal alterations have been identified and some have shown potential as markers for dissemination of the tumour, which is responsible for most colon cancer deaths. We investigated 115 selected stage II-IV primary colon cancers for associations between chromosomal alterations and tumour dissemination. METHODS: Follow-up was at least 5 years for stage II-III patients without distant recurrence. Affymetrix SNP 6.0 microarrays and allele-specific copy number analysis were used to identify chromosomal alterations. Fisher's exact test was used to associate alterations with tumour dissemination, detected at diagnosis (stage IV) or later as recurrent disease (stage II-III). RESULTS: Loss of 1p36.11-21 was associated with tumour dissemination in microsatellite stable tumours of stage II-IV (odds ratio = 5.5). It was enriched to a similar extent in tumours with distant recurrence within stage II and stage III subgroups, and may therefore be used as a prognostic marker at diagnosis. Loss of 1p36.11-21 relative to average copy number of the genome showed similar prognostic value compared to absolute loss of copies. Therefore, the use of relative loss as a prognostic marker would benefit more patients by applying also to hyperploid cancer genomes. The association with tumour dissemination was supported by independent data from the The Cancer Genome Atlas. CONCLUSION: Deletions on 1p36 may be used to guide adjuvant treatment decisions in microsatellite stable colon cancer of stages II and III.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 1 , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Repetições de Microssatélites/genética , Biomarcadores Tumorais/genética , Duplicação Cromossômica , Neoplasias do Colo/mortalidade , Variações do Número de Cópias de DNA , Feminino , Seguimentos , Humanos , Masculino , Instabilidade de Microssatélites , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , Carga TumoralRESUMO
AKN-028 is a novel tyrosine kinase inhibitor with preclinical activity in acute myeloid leukemia (AML), presently undergoing investigation in a phase I/II study. It is a potent inhibitor of the FMS-like kinase 3 (FLT3) but shows in vitro activity in a wide range of AML samples. In the present study, we have characterized the effects of AKN-028 on AML cells in more detail. AKN-028 induced a dose-dependent G0/1 arrest in AML cell line MV4-11. Treatment with AKN-028 caused significantly altered gene expression in all AML cell types tested (430 downregulated, 280 upregulated transcripts). Subsequent gene set enrichment analysis revealed enrichment of genes associated with the proto-oncogene and cell cycle regulator c-Myc among the downregulated genes in both AKN-028 and midostaurin treated cells. Kinase activity profiling in AML cell lines and primary AML samples showed that tyrosine kinase activity, but not serine/threonine kinase activity, was inhibited by AKN-028 in a dose dependent manner in all samples tested, reaching approximately the same level of kinase activity. Cells sensitive to AKN-028 showed a higher overall tyrosine kinase activity than more resistant ones, whereas serine/threonine kinase activity was similar for all primary AML samples. In summary, AKN-028 induces cell cycle arrest in AML cells, downregulates Myc-associated genes and affect several signaling pathways. AML cells with high global tyrosine kinase activity seem to be more sensitive to the cytotoxic effect of AKN-028 in vitro.
Assuntos
Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Genes myc/efeitos dos fármacos , Indóis/farmacologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazinas/farmacologia , Antineoplásicos/uso terapêutico , Pontos de Checagem do Ciclo Celular/genética , Relação Dose-Resposta a Droga , Regulação para Baixo/genética , Genes myc/genética , Células HL-60 , Humanos , Indóis/uso terapêutico , Leucemia Mieloide Aguda/enzimologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Pirazinas/uso terapêutico , Células Tumorais CultivadasRESUMO
PURPOSE: The current treatment strategies for glioblastoma have limited health and survival benefits for the patients. A common obstacle in the treatment is chemoresistance. A possible strategy to evade this problem may be to combine chemotherapeutic drugs with agents inhibiting resistance mechanisms. The aim with this study was to identify molecular pathways influencing drug resistance in glioblastoma-derived cells and to evaluate the potential of pharmacological interference with these pathways to identify synergistic drug combinations. METHODS: Global gene expressions and drug sensitivities to three chemotherapeutic drugs (imatinib, camptothecin and temozolomide) were measured in six human glioblastoma-derived cell lines. Gene expressions that correlated to drug sensitivity or resistance were identified and mapped to specific pathways. Selective inhibitors of these pathways were identified. The effects of six combinations of inhibitors and chemotherapeutic drugs were evaluated in glioblastoma-derived cell lines. Drug combinations with synergistic effects were also evaluated in non-cancerous epithelial cells. RESULTS: Four drug combinations had synergistic effects in at least one of the tested glioblastoma-derived cell lines; camptothecin combined with gefitinib (epidermal growth factor receptor inhibitor) or NSC 23766 (ras-related C3 botulinum toxin substrate 1 inhibitor) and imatinib combined with DAPT (Notch signaling inhibitor) or NSC 23766. Of these, imatinib combined with DAPT or NSC 23766 did not have synergistic effects in non-cancerous epithelial cells. Two drug combinations had at least additive effects in one of the tested glioblastoma-derived cell lines; temozolomide combined with gefitinib or PF-573228 (focal adhesion kinase inhibitor). CONCLUSION: Four synergistic and two at least additive drug combinations were identified in glioblastoma-derived cells. Pathways targeted by these drug combinations may serve as targets for future drug development with the potential to increase efficacy of currently used/evaluated chemotherapy.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Benzamidas/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Camptotecina/farmacologia , Receptores ErbB/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Piperazinas/farmacologia , Pirimidinas/farmacologia , Receptores Notch/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Antineoplásicos Alquilantes/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Biologia Computacional , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Glioblastoma/patologia , Humanos , Mesilato de Imatinib , Análise em Microsséries , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Transdução de Sinais/efeitos dos fármacos , TemozolomidaRESUMO
Chronic lymphocytic leukemia (CLL) can be divided into prognostic subgroups based on the IGHV gene mutational status, and is further characterized by multiple subsets of cases with quasi-identical or stereotyped B cell receptors that also share clinical and biological features. We recently reported differential DNA methylation profiles in IGHV-mutated and IGHV-unmutated CLL subgroups. For the first time, we here explore the global methylation profiles of stereotyped subsets with different prognosis, by applying high-resolution methylation arrays on CLL samples from three major stereotyped subsets: the poor-prognostic subsets #1 (n = 15) and #2 (n = 9) and the favorable-prognostic subset #4 (n = 15). Overall, the three subsets exhibited significantly different methylation profiles, which only partially overlapped with those observed in our previous study according to IGHV gene mutational status. Specifically, gene ontology analysis of the differentially methylated genes revealed a clear enrichment of genes involved in immune response, such as B cell activation (e.g., CD80, CD86 and IL10), with higher methylation levels in subset #1 than subsets #2 and #4. Accordingly, higher expression of the co-stimulatory molecules CD80 and CD86 was demonstrated in subset #4 vs. subset #1, pointing to a key role for these molecules in the crosstalk of CLL subset #4 cells with the microenvironment. In summary, investigation of three prototypic, stereotyped CLL subsets revealed distinct DNA methylation profiles for each subset, which suggests subset-biased patterns of transcriptional control and highlights a key role for epigenetics during leukemogenesis.
Assuntos
Metilação de DNA , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Ativação Linfocitária , Masculino , Prognóstico , Transcrição GênicaRESUMO
PURPOSE: Although the central role of the immune system for tumor prognosis is generally accepted, a single robust marker is not yet available. EXPERIMENTAL DESIGN: On the basis of receiver operating characteristic analyses, robust markers were identified from a 60-gene B cell-derived metagene and analyzed in gene expression profiles of 1,810 breast cancer; 1,056 non-small cell lung carcinoma (NSCLC); 513 colorectal; and 426 ovarian cancer patients. Protein and RNA levels were examined in paraffin-embedded tissue of 330 breast cancer patients. The cell types were identified with immunohistochemical costaining and confocal fluorescence microscopy. RESULTS: We identified immunoglobulin κ C (IGKC) which as a single marker is similarly predictive and prognostic as the entire B-cell metagene. IGKC was consistently associated with metastasis-free survival across different molecular subtypes in node-negative breast cancer (n = 965) and predicted response to anthracycline-based neoadjuvant chemotherapy (n = 845; P < 0.001). In addition, IGKC gene expression was prognostic in NSCLC and colorectal cancer. No association was observed in ovarian cancer. IGKC protein expression was significantly associated with survival in paraffin-embedded tissues of 330 breast cancer patients. Tumor-infiltrating plasma cells were identified as the source of IGKC expression. CONCLUSION: Our findings provide IGKC as a novel diagnostic marker for risk stratification in human cancer and support concepts to exploit the humoral immune response for anticancer therapy. It could be validated in several independent cohorts and carried out similarly well in RNA from fresh frozen as well as from paraffin tissue and on protein level by immunostaining.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Colorretais/genética , Perfilação da Expressão Gênica , Imunoglobulinas/genética , Neoplasias Ovarianas/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Estudos de Coortes , Neoplasias Colorretais/metabolismo , Feminino , Seguimentos , Humanos , Técnicas Imunoenzimáticas , Imunoglobulinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Ovarianas/metabolismo , Inclusão em Parafina , Prognóstico , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
INTRODUCTION: Non-small cell lung cancer (NSCLC) is characterized by a multitude of genetic aberrations with unknown clinical impact. In this study, we aimed to identify gene copy number changes that correlate with clinical outcome in NSCLC. To maximize the chance to identify clinically relevant events, we applied a strategy involving two prognostically extreme patient groups. METHODS: Short-term (<20 month; n = 53) and long-term survivors (>58 month; n = 47) were selected from a clinically well-characterized NSCLC patient cohort with available fresh frozen tumor specimens. The samples were analyzed using high-resolution single-nucleotide polymorphism array technology to assess gene copy number variations and array-based gene expression profiling. The molecular data were combined with information on clinical parameters. RESULTS: Genetic aberrations were strongly associated with tumor histology. In adenocarcinoma (n = 50), gene copy number gains on chromosome 8q21-q24.3 (177 genes) were more frequent in long-term than in short-term survivors. In squamous cell carcinoma (n = 28), gains on chromosome 14q23.1-24.3 (133 genes) were associated with shorter survival, whereas losses in a neighboring region, 14q31.1-32.33 (110 genes), correlated with favorable outcome. In accordance with copy number gains and losses, messenger RNA expression levels of corresponding genes were increased or decreased, respectively. CONCLUSION: Comprehensive tumor profiling permits the integration of genomic, histologic, and clinical data. We identified gene copy number gains and losses, with corresponding changes in messenger RNA levels that were associated with prognosis in adenocarcinoma and squamous cell carcinoma of the lung.