RESUMO
Merkel cell carcinoma is a highly aggressive cutaneous neuroendocrine tumor that has been associated with Merkel cell polyomavirus in up to 80% of cases. Merkel cell polyomavirus is believed to influence pathogenesis, at least in part, through expression of the large T antigen, which includes a retinoblastoma protein-binding domain. However, there appears to be significant clinical and morphological overlap between polyomavirus-positive and polyomavirus-negative Merkel cell carcinoma cases. Although much of the recent focus of Merkel cell carcinoma pathogenesis has been on polyomavirus, the pathogenesis of polyomavirus-negative cases is still poorly understood. We hypothesized that there are underlying human somatic mutations that unify Merkel cell carcinoma pathogenesis across polyomavirus status, and to investigate we performed whole exome sequencing on five polyomavirus-positive cases and three polyomavirus-negative cases. We found that there were no significant differences in the overall number of single-nucleotide variations, copy number variations, insertion/deletions, and chromosomal rearrangements when comparing polyomavirus-positive to polyomavirus-negative cases. However, we did find that the retinoblastoma pathway genes harbored a high number of mutations in Merkel cell carcinoma. Furthermore, the retinoblastoma gene (RB1) was found to have nonsense truncating protein mutations in all three polyomavirus-negative cases; no such mutations were found in the polyomavirus-positive cases. In all eight cases, the retinoblastoma pathway dysregulation was confirmed by immunohistochemistry. Although polyomavirus-positive Merkel cell carcinoma is believed to undergo retinoblastoma dysregulation through viral large T antigen expression, our findings demonstrate that somatic mutations in polyomavirus-negative Merkel cell carcinoma lead to retinoblastoma dysregulation through an alternative pathway. This novel finding suggests that the retinoblastoma pathway dysregulation leads to an overlapping Merkel cell carcinoma phenotype and that oncogenesis occurs through either a polyomavirus-dependent (viral large T antigen expression) or polyomavirus-independent (host somatic mutation) mechanism.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Célula de Merkel/genética , Análise Mutacional de DNA/métodos , Exoma , Genes do Retinoblastoma , Mutação , Infecções por Polyomavirus/genética , Proteína do Retinoblastoma/genética , Neoplasias Cutâneas/genética , Infecções Tumorais por Vírus/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Carcinoma de Célula de Merkel/química , Carcinoma de Célula de Merkel/virologia , Feminino , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Masculino , Poliomavírus das Células de Merkel/isolamento & purificação , Pessoa de Meia-Idade , Fenótipo , Infecções por Polyomavirus/metabolismo , Infecções por Polyomavirus/virologia , Proteína do Retinoblastoma/análise , Neoplasias Cutâneas/química , Neoplasias Cutâneas/virologia , Infecções Tumorais por Vírus/metabolismo , Infecções Tumorais por Vírus/virologiaRESUMO
The accurate diagnosis of a juvenile granulosa cell tumor (JGCT) can be challenging, as these neoplasms often exhibit morphologic features that overlap other ovarian neoplasms. In addition, the immunohistochemical profile exhibited by JGCT is fairly nonspecific and typically includes reactivity for CD99. Recently, we noted that JGCTs can show immunohistochemical expression of Fli-1, a transcription factor expressed by Ewing sarcoma, a neoplasm that is occasionally in the differential diagnosis of JGCT. We evaluated a series of JGCTs to determine whether Fli-1 is commonly expressed by these tumors and whether they demonstrate chromosomal arrangements in EWSR1. Cases diagnosed as JGCT (n=11) were immunohistochemically evaluated for expression of Fli-1 and CD99. Fluorescence in situ hybridization was performed on all cases to search for chromosomal rearrangements in EWSR1. All 11 of our cases exhibited positive immunohistochemical staining for Fli-1 and CD99. None of the cases demonstrated rearrangement in EWSR1 by fluorescence in situ hybridization. In cases of JGCT that cannot be reliably distinguished from Ewing sarcoma based on morphology and immunohistochemistry alone, fluorescence in situ hybridization testing for EWSR1 rearrangements seems to be a useful diagnostic adjunct for their separation.
Assuntos
Biomarcadores Tumorais/análise , Tumor de Células da Granulosa/genética , Tumor de Células da Granulosa/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Antígeno 12E7 , Antígenos CD/biossíntese , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação a Calmodulina/genética , Moléculas de Adesão Celular/biossíntese , Criança , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Proteínas dos Microfilamentos/biossíntese , Proteína EWS de Ligação a RNA , Proteínas de Ligação a RNA/genética , Receptores Citoplasmáticos e Nucleares/biossíntese , Transativadores , Translocação GenéticaRESUMO
The serine/threonine-protein kinase B-raf (BRAF) is an oncogene mutated in various neoplasms, including 5-15% of colorectal carcinomas. The T1799A point mutation, responsible for a large majority of these alterations, results in an amino acid substitution (V600E) causing the constitutive activation of a protein kinase cascade. BRAF V600E in MLH1 deficient tumors implicates somatic tumor-only methylation of the MLH1 promoter region instead of a germline MLH1 mutation. BRAF V600E also predicts poor prognosis in microsatellite stable colorectal cancers and may be a marker of resistance to anti-EGFR therapy in metastatic disease. Currently, only molecular methods are available for assessing BRAF mutational status. An immunohistochemical approach is evaluated here. Colon cancers from 2008 to 2012 tested by pyrosequencing for BRAF V600E mutation were selected. A total of 31 tumors with (n = 14) and without (n = 17) the BRAF V600E mutation were analyzed by immunohistochemistry using a commercially available antibody specific to the V600E-mutated protein. All 14 colorectal carcinomas with the BRAF V600E mutation demonstrated cytoplasmic positivity in tumor cells with the anti-BRAF antibody. In a minority of cases, staining intensity for the mutated tumor samples was weak (n = 2) or heterogeneous (n = 4); however, the majority of cases showed diffuse, strong cytoplasmic positivity (8 of 14 cases). None of the 17 BRAF wild-type colorectal cancers showed immunoreactivity to the antibody. The overall sensitivity and specificity of the immunohistochemical BRAF V600E assay was 100%. Detection of the BRAF V600E mutation in colorectal cancer by immunohistochemistry is a viable alternative to molecular methods.
Assuntos
Neoplasias Colorretais/genética , Imuno-Histoquímica/métodos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Anticorpos Monoclonais/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/isolamento & purificação , Neoplasias Colorretais/patologia , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteína 1 Homóloga a MutL , Proteínas Nucleares/genética , Mutação Puntual/genética , Proteínas Proto-Oncogênicas B-raf/isolamento & purificaçãoRESUMO
The TP53 (p53) pathway can be inhibited by TP53 mutation or deletion or by MDM2 overexpression. Both occur in Burkitt lymphoma (BL), but many cases lack either abnormality. Expression patterns of the TP53 inhibitor MDM4 have not been reported in BL, and increased MDM4 could deregulate the TP53 pathway in cases without TP53 or MDM2 abnormalities. We investigated TP53 pathway disruption in paediatric BL patient samples (n = 30) by studying MDM4, MDM2, and CDKN1A (p21) protein and mRNA expression; TP53 mutations; TP53 protein expression; and gene copy number abnormalities. MDM4 protein was expressed in 30/30 tumours, and MDM2 protein was weakly expressed in 7/30 (23%). All cases were negative for CDKN1A protein, and CDKN1A mRNA levels were decreased. TP53 mutations were detected in 5/28 (18%) cases and confirmed by sequencing. TP53 protein was expressed in 15/30 (50%) cases, including 7/8 with TP53 genetic alterations. MDM2 protein and mRNA expression levels did not correlate with lack of TP53 genetic changes or TP53 protein expression; however, there was an inverse relationship between detectable TP53 protein expression and MDM4 copy number gains and mRNA expression. The TP53 pathway is deregulated in paediatric BL cases, and increased MDM4 expression may be the primary mechanism in some cases.
Assuntos
Linfoma de Burkitt/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Proteínas de Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , DNA de Neoplasias/genética , Genes p53 , Humanos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas Nucleares , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-mdm2/biossíntese , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/biossínteseRESUMO
Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene, which results in the constitutive activation of the hypoxia inducible factors, HIF-1α and HIF-2α. Using a high throughput screen, we identify novel compounds that decrease HIF-1/2α levels and induce ferroptosis by targeting Iron Sulfur Cluster Assembly 2 (ISCA2), a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex. ISCA2 inhibition either pharmacologically or using siRNA decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation through unknown mechanisms. Additionally, ISCA2 inhibition triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 levels are decreased in ccRCC compared to normal kidney, and decreased ISCA2 levels are associated with pVHL loss and with sensitivity to ferroptosis induced by ISCA2 inhibition. Strikingly, pharmacological inhibition of ISCA2 using an orally available ISCA2 inhibitor significantly reduced ccRCC xenograft growth in vivo, decreased HIF-α levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Thus, the targeting of ISCA2 may be a promising therapeutic strategy to inhibit HIF-1/2α and to induce ferroptosis in pVHL deficient cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Carcinoma de Células Renais , Ferroptose , Proteínas Ferro-Enxofre , Neoplasias Renais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , RNA Interferente Pequeno , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
The tumor suppressor gene PTEN is the second most commonly deleted gene in cancer. Such deletions often include portions of the chromosome 10q23 locus beyond the bounds of PTEN itself, which frequently disrupts adjacent genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here, we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis triggered by proteasome dysfunction and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic protein BIM from mitochondria to inactivate it. Cultured cells and mouse xenografts lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which activate BIM and trigger apoptosis. This work furthers our understanding of mitochondrial protein homeostasis and could lead to new therapeutic options for the hundreds of thousands of cancer patients who have tumors with chromosome 10q23 deletion.
Cancer cells have often lost genetic sequences that control when and how cell division takes place. Deleting these genes, however, is not an exact art, and neighboring sequences regularly get removed in the process. For example, the loss of the tumor suppressor gene PTEN, the second most deleted gene in cancer, frequently involves the removal of the nearby ATAD1 gene. While hundreds of thousands of human tumors completely lack ATAD1, individuals born without a functional version of this gene do not survive past early childhood. How can tumor cells cope without ATAD1 and could these coping strategies become the target for new therapies? Winter et al. aimed to answer these questions by examining a variety of cancer cells lacking ATAD1 in the laboratory. Under normal circumstances, the enzyme that this gene codes for sits at the surface of mitochondria, the cellular compartments essential for energy production. There, it extracts any faulty, defective proteins that may otherwise cause havoc and endanger mitochondrial health. Experiments revealed that without ATAD1, cancer cells started to rely more heavily on an alternative mechanism to remove harmful proteins: the process centers on MARCH5, an enzyme which tags molecules that require removal so the cell can recycle them. Drugs that block the pathway involving MARCH5 already exist, but they have so far been employed to treat other types of tumors. Winter et al. showed that using these compounds led to the death of cancerous ATAD1-deficient cells, including in human tumors grown in mice. Overall, this work demonstrates that cancer cells which have lost ATAD1 become more vulnerable to disruptions in the protein removal pathway mediated by MARCH5, including via already existing drugs. If confirmed by further translational work, these findings could have important clinical impact given how frequently PTEN and ATAD1 are lost together in cancer.
Assuntos
Neoplasias , Complexo de Endopeptidases do Proteassoma , Humanos , Animais , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Mitocôndrias/metabolismo , Neoplasias/genéticaRESUMO
KIT mutations are known to occur in ~15% of chronic sun damaged cutaneous, mucosal, and acral melanomas. Melanomas with demonstrated activating mutations in KIT or platelet-derived growth factor receptor A (PDGFRA) may benefit from treatment with tyrosine kinase inhibitors. Currently, the limited data regarding KIT mutational status in ocular melanoma suggest that activating mutations are extremely rare. PDGFRA mutational status in ocular melanoma has not been determined. Seventy-five ocular melanomas (53 choroidal, 6 iris, 11 ciliary body, and 5 conjuctival) were selected from the files of the Department of Ophthalmology. High-resolution melting curve analysis and sequencing were performed to detect mutations in KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18. Results of mutational analysis were correlated with anatomical site and KIT (CD117) immunohistochemistry. Eight of 75 (11%) ocular melanomas contained mutations in either the KIT or PDGFRA gene. Five of 53 (9%) choroidal melanomas were associated with mutations (KIT exon 11=3; KIT exon 17=1; PDGFRA intron 18=1). Two of six (33%) iris melanomas and a single (9%) ciliary body melanoma harbored KIT exon 11 mutations. No mutations were identified in conjunctival melanomas. The distribution of KIT and PDGFRA mutations by ocular melanoma anatomical site did not reach statistical significance (P=0.393) CD117 positivity was not predictive of KIT mutational status as only 6 of 58 (10%) CD177-positive tumors harbored KIT mutations. In addition, a KIT exon 17 mutation was identified in one CD117-negative tumor. KIT and PDGFRA mutations do occur in ocular melanomas at a frequency (11%) that is similar to acral and mucosal melanomas. Limited correlation of CD117 positivity with mutational status suggests that all ocular melanomas should undergo mutational analysis to determine if imatinib therapy is appropriate.
Assuntos
Neoplasias Oculares/genética , Melanoma/genética , Mutação , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Fator de Células-Tronco/genética , Análise Mutacional de DNA , Neoplasias Oculares/metabolismo , Neoplasias Oculares/patologia , Humanos , Imuno-Histoquímica , Melanoma/metabolismo , Melanoma/patologia , Microdissecção , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
Constitutive expression of the chimeric NPM/ALK fusion protein encoded by the t(2;5)(p32;q35) is a key oncogenic event in the pathogenesis of most anaplastic large cell lymphomas (ALCLs). The proteomic network alterations produced by this aberration remain largely uncharacterized. Using a mass spectrometry (MS)-driven approach to identify changes in protein expression caused by the NPM/ALK fusion, we identified diverse NPM/ALK-induced changes affecting cell proliferation, ribosome synthesis, survival, apoptosis evasion, angiogenesis, and cytoarchitectural organization. MS-based findings were confirmed using Western blotting and/or immunostaining of NPM/ALK-transfected cells and ALK-deregulated lymphomas. A subset of the proteins distinguished NPM/ALK-positive ALCLs from NPM/ALK-negative ALCLs and Hodgkin lymphoma. The multiple NPM/ALK-deregulated pathways identified by MS analysis also predicted novel biologic effects of NPM/ALK expression. In this regard, we showed loss of cell adhesion as a consequence of NPM/ALK expression in a kinase-dependent manner, and sensitivity of NPM/ALK-positive ALCLs to inhibition of the RAS, p42/44ERK, and FRAP/mTOR signaling pathways. These findings reveal that the NPM/ALK alteration affects diverse cellular pathways, and provide novel insights into NPM/ALK-positive ALCL pathobiology. Our studies carry important implications for the use of MS-driven approaches for the elucidation of neoplastic pathobiology, the identification of novel diagnostic biomarkers, and pathogenetically relevant therapeutic targets.
Assuntos
Linfoma Anaplásico de Células Grandes/metabolismo , Redes e Vias Metabólicas , Proteínas Tirosina Quinases/metabolismo , Proteoma/análise , Sequência de Aminoácidos , Regulação Neoplásica da Expressão Gênica , Humanos , Células Jurkat , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Tirosina Quinases/genética , Proteoma/metabolismo , Proteômica , Proteínas Quinases S6 Ribossômicas/química , Proteínas Quinases S6 Ribossômicas/metabolismo , Análise Serial de Tecidos , Transfecção , Células Tumorais CultivadasRESUMO
PURPOSE: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel-Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC. EXPERIMENTAL DESIGN: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC. RESULTS: HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs. CONCLUSIONS: These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/mortalidade , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/mortalidade , Macrófagos Associados a Tumor/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/análise , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores Tumorais/análise , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/terapia , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , Neoplasias Renais/terapia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Nefrectomia , Prognóstico , RNA-Seq , Estudos Retrospectivos , Análise de Célula Única , Análise de Sobrevida , Macrófagos Associados a Tumor/imunologiaRESUMO
OBJECTIVE: High levels of microsatellite instability (MSI-H) have been associated in many studies with improved prognosis in colon cancer. Very few studies have evaluated the effect of MSI-H on rectal cancer survival. We assessed MSI-H and other genetic and epigenetic changes on survival of 990 individuals diagnosed with first primary rectal cancer. METHODS: MSI was assessed primarily by instability in the mononucleotide repeat BAT-26. The BRAF V600E mutation was assessed by TaqMan assay. The CpG island methylator phenotype (CIMP) was determined by methylation-specific PCR of CpG islands in MLH1, methylated in tumors (MINT)1, (MINT)2, (MINT)31 and CDKN2A. KRAS2 codons 12 and 13 mutations, and TP53 mutations in exons 5-8 were determined by sequencing. RESULTS: Multivariate analysis revealed that MSI-H (HRR 2.47, 95% CI 1.13-5.40) and KRAS2 mutations (HRR 1.37, 95% CI 1.04-1.81) were associated with a significantly higher risk of dying of rectal cancer. Only one of 22 MSI-H tumors showed a BRAF V600E mutation. Of 15 MSI-H rectal cancers evaluated for methylation, two exhibited MLH1 methylation and four exhibited CIMP. CONCLUSION: The genetic and epigenetic characteristics of MSI-H rectal cancers suggest that they are enriched for Lynch-associated tumors; adverse prognosis associated with MSI-H in these tumors may reflect the relatively high frequency of Lynch-associated cancers and/or the effect of radiation or chemotherapy on Lynch-associated rectal cancers or MSI tumors in general.
Assuntos
Instabilidade de Microssatélites , Neoplasias Retais/genética , Neoplasias Retais/mortalidade , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Caderinas/genética , Proteínas de Transporte/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Ilhas de CpG , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Neoplasias Retais/patologia , Proteína Supressora de Tumor p53/genética , Proteínas ras/genéticaRESUMO
Higher-grade gliomas are distinguished by increased vascular endothelial cell proliferation and peritumoral edema. These are thought to be instigated by vascular endothelial growth factor, which in turn is regulated by cellular oxygen tension. Hypoxia inducible factor-1alpha (HIF-1alpha) is a main responder to intracellular hypoxia and is overexpressed in many human cancers, including gliomas. Here we present methods for investigating the role of HIF-1alpha in glioma growth in vivo and in vitro using RNA interference in U251, U87, and U373 glioma cells.
Assuntos
Neoplasias Encefálicas/terapia , Glioma/terapia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Animais , Western Blotting , Neoplasias Encefálicas/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Glioma/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Hipóxia , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase , Transfecção , Ubiquitina-Proteína Ligases/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The aberrant expression of T-cell antigens on B-cell-derived non-Hodgkin lymphomas has been described. However, the expression of the lineage-specific T-cell antigen, CD3, in hematologic malignancies is exceedingly rare and to the best of our knowledge has not been reported in pediatric patients. Here we describe the first case of a CD3+ diffuse large B-cell lymphoma in a 9-year-old male patient that is well documented by immunohistochemistry. In addition, results of a tissue microarray study composed of B-cell-derived non-Hodgkin lymphomas (n=77) and reactive lymphoid hyperplasia (n=13) dual stained for PAX5/CD3 are also reported.
Assuntos
Complexo CD3 , Linfoma Difuso de Grandes Células B/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/patologia , Criança , Pré-Escolar , Feminino , Humanos , Imunofenotipagem , Lactente , Linfoma de Células B/patologia , Masculino , Pessoa de Meia-Idade , Pseudolinfoma/patologia , Análise Serial de Tecidos , Adulto JovemRESUMO
BACKGROUND: T-lymphoblastic lymphoma (T-LBL) accounts for 25-30% of childhood non-Hodgkin's lymphoma and is closely related to T-lymphoblastic leukemia (T-ALL). Recently, we demonstrated distinct differences in gene expression between childhood T-LBL and T-ALL, but molecular pathogenesis and relevant protein expression patterns in T-LBL remain poorly understood. PROCEDURE: Children with T-LBL with disseminated disease were registered and treated on COG protocol 5971. Paraffin-embedded tumor tissue was obtained at diagnosis for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) studies. We determined the pattern and intensity of staining for c-Myc, Skp2, Mib-1, p53, TCL-1, bcl-2, and bcl-6 proteins by IHC and c-Myc, p53, bcl-2, bcl-6, and TCR alpha/delta molecular alterations by FISH in 22 pediatric T-LBL cases. RESULTS: The majority of T-LBL samples expressed Mib-1 (59%) and c-Myc (77%) proteins in greater than 50% of the cells, but Skp2 (14%), p53 (14%), and bcl-2 (23%) expression was less common. FISH studies demonstrated 18% gains and 10% losses in c-Myc, 16% gains in p53, 12% gains and 6% losses in bcl-2, and 6% gains and 19% losses in bcl-6 with little direct correlation between the IHC and FISH studies. CONCLUSIONS: Childhood T-LBL is a highly proliferative tumor associated with enhanced expression of c-Myc protein, but without detectable c-Myc molecular alterations. FISH studies did not identify consistent etiologies of molecular dysregulation, and future studies with other molecular approaches may be required to elucidate the molecular pathogenesis of childhood T-LBL.
Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras/classificação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Criança , Humanos , Imuno-Histoquímica , Imunofenotipagem , Hibridização in Situ Fluorescente , Oncologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismoRESUMO
OBJECTIVE: Constitutive overexpression of nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) is a key oncogenic event in anaplastic large cell lymphomas (ALCL) that carry the t(2;5)(p23;q35) translocation. Global proteomic analysis of NPM/ALK-positive ALCL would improve understanding of the disease pathogenesis and yield new candidate targets for novel treatment and diagnostic strategies. MATERIALS AND METHODS: To comprehensively determine the inventory of proteins from NPM/ALK-positive ALCL SUDHL-1 cells, the membrane, cytoplasm, and nuclear subcellular fractions were resolved by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS spectra were interpreted using SEQUEST to search the electronic UniProt protein database, and analyzed by ProteinProphet and INTERACT. RESULTS: A total of 623 proteins consisting of 210 membrane, 229 cytoplasm, and 184 nuclear proteins were identified with a Assuntos
Perfilação da Expressão Gênica
, Linfoma Difuso de Grandes Células B/genética
, Proteínas de Fusão Oncogênica/genética
, Proteínas Tirosina Quinases/genética
, Proteoma
, Sequência de Aminoácidos
, Linhagem Celular Tumoral
, Humanos
, Linfoma Difuso de Grandes Células B/patologia
, Dados de Sequência Molecular
, Proteínas de Neoplasias/genética
RESUMO
Recurrent heterozygous mutation of isocitrate dehydrogenase 1 gene (IDH1), predominantly resulting in histidine substitution at arginine 132, was first identified in glioma. The biological significance of IDH1R132H, however, has been controversial, and its prevalent association with glioma remains enigmatic. Although recent studies indicate that IDH1R132H is nonessential to tumor growth or even anti-tumor growth, whether IDH1R132H initiates gliomagenesis remains obscure. In this study, we report that IDH1R132H is intrinsically tumor-suppressive but the activity can be attenuated by glutamate-the cerebral neurotransmitter. We observed that IDH1R132H was highly suppressive of subcutaneous tumor growth driven by platelet-derived growth factor B (PDGFB), but IDH1R132H tumor growth and glioma penetrance were virtually indistinguishable from those of IDH1-wildtype tumors in orthotopic models. In vitro, addition of glutamate compromised IDH1R132H inhibition of neurosphere genesis, indicating glutamate promotion of oncogenic dominance. Furthermore, we observed that IDH1R132H expression was markedly decreased in tumors but became more permissible upon the deletion of tumor-suppressor gene Cdkn2a. To provide direct evidence for the opposing effect of IDH1R132H on PDGFB-driven glioma development, we explored tandem expression of the two molecules from a single transcript to preclude selection against IDH1R132H expression. Our results demonstrate that when juxtaposed with oncogenic PDGFB, IDH1R132H overrides the oncogenic activity and obliterates neurosphere genesis and gliomagenesis even in the glutamate-rich microenvironment. We propose therefore that IDH1R132H is intrinsically suppressive of glioma initiation and growth but such tumor-suppressive activity is compromised by the glutamate-rich cerebral cortex, thereby offering a unifying hypothesis for the perplexing role of IDH1R132H in glioma initiation and growth.
RESUMO
Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated gene in grade II-III glioma and secondary glioblastoma (GBM). A causal role for IDH1R132H in gliomagenesis has been proposed, but functional validation in vivo has not been demonstrated. In this study, we assessed the role of IDH1R132H in glioma development in the context of clinically relevant cooperating genetic alterations in vitro and in vivo. Immortal astrocytes expressing IDH1R132H exhibited elevated (R)-2-hydroxyglutarate levels, reduced NADPH, increased proliferation, and anchorage-independent growth. Although not sufficient on its own, IDH1R132H cooperated with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote glioma development in vivo. These tumors resembled proneural human mutant IDH1 GBM genetically, histologically, and functionally. Our findings support the hypothesis that IDH1R132H promotes glioma development. This model enhances our understanding of the biology of IDH1R132H-driven gliomas and facilitates testing of therapeutic strategies designed to combat this deadly disease.
Assuntos
Astrócitos/enzimologia , Carcinogênese/metabolismo , Glioma/enzimologia , Isocitrato Desidrogenase/metabolismo , Mutação de Sentido Incorreto , Proteínas de Neoplasias/metabolismo , Substituição de Aminoácidos , Animais , Astrócitos/patologia , Carcinogênese/genética , Carcinogênese/patologia , Glioma/genética , Glioma/patologia , Humanos , Isocitrato Desidrogenase/genética , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genéticaRESUMO
Mass spectrometry-based proteomics in conjunction with liquid chromatography and bioinformatics analysis provides a highly sensitive and high-throughput approach for the identification of proteins. Hodgkin lymphoma is a form of malignant lymphoma characterized by the proliferation of Reed-Sternberg cells and background reactive lymphocytes. Comprehensive analysis of proteins expressed and released by Reed-Sternberg cells would assist in the discovery of potential biomarkers and improve our understanding of its pathogenesis. The subcellular proteome of the three cellular compartments from L428 and KMH2 Hodgkin lymphoma-derived cell lines were fractionated, and analyzed by reverse-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Additionally, proteins released by Hodgkin lymphoma-derived L428 cells were extracted from serum-free culture media and analyzed. Peptide spectra were analyzed using TurboSEQUEST against the UniProt protein database (5.26.05; 188 712 entries). A subset of the identified proteins was validated by Western blot analysis, immunofluorescence microscopy and immunohistochemistry. A total of 1945 proteins were identified with 785 from the cytosolic fraction, 305 from the membrane fraction, 441 from the nuclear fraction and 414 released proteins using a minimum of two peptide identifications per protein and an error rate of <5.0%. Identification of proteins from diverse functional groups reflected the functional complexity of the Reed-Sternberg proteome. Proteins with previously reported oncogenic function in other cancers and from signaling pathways implicated in Hodgkin lymphoma were identified. Selected proteins without previously demonstrated expression in Hodgkin lymphoma were validated by Western blot analysis (B-RAF, Erb-B3), immunofluorescence microscopy (Axin1, Tenascin-X, Mucin-2) and immunohistochemistry using a tissue microarray (BRAF, PIM1). This study represents the first comprehensive inventory of proteins expressed by Reed-Sternberg cells of Hodgkin lymphoma and demonstrates the utility of combining cellular subfractionation, protein precipitation, tandem mass spectrometry and bioinformatics analysis for comprehensive identification of proteins that may represent potential biomarkers of the disease.
Assuntos
Biomarcadores Tumorais/metabolismo , Doença de Hodgkin/metabolismo , Proteoma/metabolismo , Células de Reed-Sternberg/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Imuno-Histoquímica , Frações Subcelulares/metabolismo , Espectrometria de Massas em Tandem , Análise Serial de TecidosRESUMO
We studied 20 granuloma annulare (GA) cases (10 interstitial and 10 palisaded) and 19 Kaposi sarcoma (KS) cases (9 "early" and 10 typical). Tissue sections were stained for iron, Hale colloidal iron, human herpesvirus 8 (HHV-8), CD31, CD34, CD68, collagen IV, factor XIIIa, and MIB-1. Iron staining of dermal tissue associated with the lesion was confirmed in all KS cases and no GA cases. Immunohistochemical stains for HHV-8 were positive in all 9 cases of early KS and most cases (9/10) of typical KS. All 20 cases of GA were HHV-8-. CD31, CD34, CD68, factor XIIIa, and MIB-1 were also stained but were difficult to interpret and did not seem specific for GA or KS. Iron staining and immunohistochemical HHV-8 staining in combination were reliable markers for KS compared with interstitial GA. MIB-1 fractions of less than 5% favored a diagnosis of GA, whereas fractions greater than 10% favored a diagnosis of KS. This study provides novel data characterizing iron staining in KS and details the use of iron staining, HHV-8, and MIB-1 to distinguish KS from GA.
Assuntos
Granuloma Anular/diagnóstico , Herpesvirus Humano 8/isolamento & purificação , Ferro/análise , Sarcoma de Kaposi/diagnóstico , Granuloma Anular/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Sarcoma de Kaposi/patologia , Coloração e Rotulagem/métodosRESUMO
Heterotopic ossification (HO) within tissues involved by a pathologic process is a well-recognized phenomenon. It is most frequently observed in atherosclerotic plaques, in soft tissue around joints, and in the central nervous system. Less frequently, carcinomas and some benign neoplasms will undergo heterotopic ossification. We performed a retrospective review of our experience with HO over a 10-year period to determine the frequency and tissue site distribution of heterotopic ossification. A computerized review of surgical pathology records of approximately 126,000 reports revealed 85 cases in which heterotopic ossification, ectopic bone or metaplastic bone was specifically mentioned in the surgical pathology diagnosis. Twenty-two cases were neoplasms of non-osseous tissues, and 63 cases were non-neoplastic lesions. Immunohistochemical staining for bone morphogenic proteins (BMP) 1, 4, and 6 was performed. Fourteen cases showed staining for BMP-1, 22 cases showed staining for BMP-4, and five cases showed weak staining for BMP-6. HO is a relatively infrequent finding and is more commonly seen in degenerative and reparative conditions than in neoplasms.