RESUMO
Richter's transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase-associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.
Assuntos
Leucemia Linfocítica Crônica de Células B/patologia , Linfoma Difuso de Grandes Células B/patologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptor Notch1/fisiologia , Animais , Evolução Clonal , Progressão da Doença , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Genes p53 , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/fisiopatologia , Linfócitos do Interstício Tumoral/imunologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Fosfoproteínas/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/fisiologia , Transcriptoma , Microambiente Tumoral , Proteína Supressora de Tumor p53/fisiologia , Regulação para CimaRESUMO
We have previously shown that an excess of deoxycorticosterone acetate and high sodium chloride intake (DOCA/salt) in one-renin gene mice induces a high urinary Na/K ratio, hypokalemia, and cardiac and renal hypertrophy in the absence of hypertension. Dietary potassium supplementation prevents DOCA/salt-induced pathological processes. In the present study, we further study whether DOCA/salt-treated mice progressively develop chronic inflammation and fibrosis in the kidney and whether dietary potassium supplementation can reduce the DOCA/salt-induced renal pathological process. Results showed that (1) long-term DOCA/salt-treated one-renin gene mice developed severe kidney injuries including tubular/vascular hypertrophy, mesangial/interstitial/perivascular fibrosis, inflammation (lymphocyte's immigration), proteinuria, and high serum creatinine in the absence of hypertension; (2) there were over-expressed mRNAs of plasminogen activator inhibitor-1 (PAI-1), fibronectin, collagen type I and III, interferon-inducible protein-10 (IP-10), monocyte chemotactic protein-1 (MCP1), transforming growth factor-ß (TGF-ß), tumor necrosis factor-alpha (TNF-α), osteopontin, Nuclear factor kappa B (NF-κB)/P65, and intercellular adhesion molecule (ICAM)-1; and (3) dietary potassium supplementation normalized urinary Na/K ratio, hypokalemia, proteinuria, and serum creatinine, reduced renal hypertrophy, inflammations, and fibrosis, and down-regulated mRNA expression of fibronectin, Col-I and III, TGF-ß, TNF-α, osteopontin, and ICAM without changes in the blood pressure. The results provide new evidence that potassium and sodium may modulate proinflammatory and fibrotic genes, leading to chronic renal lesions independent of blood pressure.
Assuntos
Acetato de Desoxicorticosterona , Glomerulonefrite , Hipertensão , Hipopotassemia , Camundongos , Animais , Pressão Sanguínea , Cloreto de Sódio/metabolismo , Fibronectinas/metabolismo , Osteopontina/metabolismo , Potássio na Dieta/metabolismo , Acetato de Desoxicorticosterona/efeitos adversos , Cloretos/metabolismo , Renina/metabolismo , Hipopotassemia/patologia , Fator de Necrose Tumoral alfa/metabolismo , Creatinina/metabolismo , Hipertensão/metabolismo , Rim/metabolismo , Cloreto de Sódio na Dieta/metabolismo , Glomerulonefrite/patologia , Inflamação/metabolismo , Suplementos Nutricionais , Fator de Crescimento Transformador beta/metabolismo , Proteinúria/metabolismo , Hipertrofia/metabolismo , Fibrose , Acetatos/metabolismoRESUMO
Interstitial lung diseases present clinically with unspecific respiratory symptoms and occur idiopathically or etiologically linked to various causes. The morphology of interstitial lung diseases (radiology or histopathology) may also be unspecific in the individual case, due to the limited arsenal of reaction patterns of the lungs. Only the combination of all findings assembled during multidisciplinary discussion (MDD) between pulmonologist, radiologist and pathologist, and if required other specialties, enables a highly reliable final diagnosis, permitting improved, personalized patient treatment. The necessity for histological evaluation and the means of tissue acquisition should also be decided on during MDD, considering clinical and radiological differential diagnoses, the risks involved in the procedures and patient-specific characteristics. In the current review, we discuss MDD as the diagnostic gold standard and exemplify its merit presenting a case of interstitial lung disease.
Assuntos
Doenças Pulmonares Intersticiais/diagnóstico , Diagnóstico Diferencial , Humanos , Pulmão/patologia , RadiografiaRESUMO
Idiopathic pulmonary fibrosis (IPF) plays a special role within the group of interstitial lung diseases (ILDs) due to its inexorable progression and its specific medical treatment. With a median survival of only 2-3 years from the time of diagnosis, the prognosis is worse than many carcinomas.In contrast to other ILDs, IPF does not respond to anti-inflammatory treatment with corticosteroids but rather demands a specific medical therapy. Even though this cannot cure the disease, it can prolong survival. Lung transplantation is the only cure for progressive lung fibrosis. The clinical course is individual and difficult to predict. Acute exacerbations accelerate the clinical course and lead to high mortality.The underlying pathomechanisms of IPF, with its complex immunological and inflammatory processes and external impacts, have been the focus of recent research. Lifestyle and environmental influences are held responsible for much of its natural history. Smoking, pneumotoxic medications, and inhalation of dusts are well-known risk factors. Likewise, genetic and hereditary factors play a crucial role.This short review focuses on the peculiarities of IPF within the group of ILDs, especially in relation to its underlying mechanisms and clinical progression.
Assuntos
Fibrose Pulmonar Idiopática , Progressão da Doença , Humanos , Fibrose Pulmonar Idiopática/diagnóstico , Fibrose Pulmonar Idiopática/epidemiologia , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/patologia , Doenças Pulmonares Intersticiais/diagnóstico , Doenças Pulmonares Intersticiais/epidemiologia , Doenças Pulmonares Intersticiais/etiologia , Doenças Pulmonares Intersticiais/patologia , Prognóstico , Fatores de RiscoRESUMO
The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.
Assuntos
Linfócitos B/metabolismo , Transformação Celular Neoplásica/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Mutação de Sentido Incorreto , Fator 88 de Diferenciação Mieloide/biossíntese , Neoplasias Experimentais/metabolismo , Animais , Linfócitos B/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Myoepithelioma is a dimorphic neoplasm with contractile-epithelial phenotype, originally interpreted as deriving from, but not actually restricted to the salivary glands. As a novel addition to the list of exquisitely rare intracranial salivary gland-type tumors and tumor-like lesions, we report on an example of myoepithelioma encountered in the left cerebellopontine angle of a 32-year-old male. Clinically presenting with ataxia and dizziness, this extraaxial mass of 4 × 3.5 × 3 cm was surgically resected, and the patient is alive 6 years postoperatively. Histologically, the tumor exhibited a continuum ranging from compact fascicles of spindle cells to epithelial nests and trabeculae partitioned by hyalinized septa, while lacking tubular differentiation. Regardless of architectural variations, there was robust immunoexpression of S100 protein, smooth muscle actin, GFAP, cytokeratin, and vimentin. Cytologic atypia tended to be modest throughout, and the MIB1 labeling index averaged less than 1%. Fluorescent in situ hybridization indicated no rearrangement of the EWSR1 locus. We interpret these results to suggest that myoepithelioma of the posterior fossa - along with related salivary epithelial tumors in this ostensibly incongruous locale - may possibly represent analogous neoplasms to their orthotopic counterparts, ones arising within aberrant salivary anlagen. The presence of the latter lends itself to being mechanistically accounted for by either postulating placodal remnants in the wake of branchial arch development, or linking them to exocrine glandular nests within endodermal cysts. Alternatively, myoepithelioma at this site could be regarded as a non tissue-specific lesion similar to its relatives ubiquitously occurring in the soft parts.
Assuntos
Neoplasias Cerebelares/patologia , Ângulo Cerebelopontino/patologia , Mioepitelioma/patologia , Adulto , Biomarcadores Tumorais/análise , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/cirurgia , Ângulo Cerebelopontino/metabolismo , Ângulo Cerebelopontino/cirurgia , Humanos , Imuno-Histoquímica , Masculino , Mioepitelioma/metabolismo , Mioepitelioma/cirurgia , Crânio/patologiaRESUMO
Rosette-forming glioneuronal tumor (RGNT) is a recently introduced, indolent neoplasm composed of diminutive circular aggregates of neurocytic-like cells on a noninfiltrative astrocytic background, typically located in the cerebellar midline The traded concept of RGNT being derived from site-specific periventricular precursors may be questioned in the face of extracerebellar examples as well as ones occurring in combination with other representatives of the glioneuronal family. We describe a hitherto not documented example of asymptomatic RGNT discovered during autopsy of a 74-year-old male. Located in the tuberal vermis, this lesion of 6 mm diameter consisted of several microscopic nests of what were felt to represent nascent stages of RGNT, all of them centered on the internal granular layer, and ranging from mucoid dehiscences thereof to fully evolved - if small - tumor foci. Molecular genetic analysis revealed a missense mutation in Exon 20 of the PIK3CA gene involving an AâG transition at Nucleotide 3140. On the other hand, neither codeletion of chromosomes 1p/19q nor pathogenic mutations of IDH1/2 were detected. By analogy with in situ paradigms in other organs, we propose that this tumor is likely to have arisen from the internal granular layer, rather than the plate of the 4th ventricle. A suggestive departure from the wholesale argument of "undifferentiated precursors", this finding also indirectly indicates that a subset of non-classical RGNTs - in particular extracerebellar examples, whose origin cannot be mechanistically accounted for by either of the above structures - may possibly reflect an instance of phenotypic convergence, rather than a lineage-restricted entity.
Assuntos
Neoplasias Cerebelares/patologia , Quarto Ventrículo/patologia , Neurônios/patologia , Idoso , Cerebelo/patologia , Humanos , MasculinoRESUMO
We previously reported that excess of deoxycorticosterone-acetate (DOCA)/salt-induced cardiac hypertrophy in the absence of hypertension in one-renin gene mice. This model allows us to study molecular mechanisms of high-salt intake in the development of cardiovascular remodeling, independently of blood pressure in a high mineralocorticoid state. In this study, we compared the effect of 5-wk low- and high-salt intake on cardiovascular remodeling and cardiac differential gene expression in mice receiving the same amount of DOCA. Differential gene and protein expression was measured by high-density cDNA microarray assays, real-time PCR and Western blot analysis in DOCA-high salt (HS) vs. DOCA-low salt (LS) mice. DOCA-HS mice developed cardiac hypertrophy, coronary perivascular fibrosis, and left ventricular dysfunction. Differential gene and protein expression demonstrated that high-salt intake upregulated a subset of genes encoding for proteins involved in inflammation and extracellular matrix remodeling (e.g., Col3a1, Col1a2, Hmox1, and Lcn2). A major subset of downregulated genes encoded for transcription factors, including myeloid differentiation primary response (MyD) genes. Our data provide some evidence that vascular remodeling, fibrosis, and inflammation are important consequences of a high-salt intake in DOCA mice. Our study suggests that among the different pathogenic factors of cardiac and vascular remodeling, such as hypertension and mineralocorticoid excess and sodium intake, the latter is critical for the development of the profibrotic and proinflammatory phenotype observed in the heart of normotensive DOCA-treated mice.
Assuntos
Doença da Artéria Coronariana/fisiopatologia , Desoxicorticosterona , Hipertrofia Ventricular Esquerda/induzido quimicamente , Hipertrofia Ventricular Esquerda/fisiopatologia , Miocárdio/metabolismo , Proteoma/metabolismo , Cloreto de Sódio na Dieta/metabolismo , Animais , Doença da Artéria Coronariana/complicações , Citocinas/metabolismo , Regulação da Expressão Gênica , Hipertensão/complicações , Hipertensão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MineralocorticoidesRESUMO
Host-modulating therapies have become an important focus in the development of novel concepts for improved management of tuberculosis (TB). Previous in vitro studies revealed that the p38 MAP kinase signaling pathway coordinates several inflammatory and stress responses in Mycobacterium tuberculosis (Mtb)-infected host cells. Here we extend these findings and show that in vivo treatment of Mtb-infected C57BL/6 mice with doramapimod, a p38 MAP-kinase inhibitor, results in reduced inflammation, granuloma formation and lung pathology. Moreover, doramapimod, together with standard antibiotic treatment, significantly reduced lung and spleen mycobacterial loads compared to antibiotic treatment alone. Our in vivo data suggest the opportunity to repurpose p38 MAPK inhibitors for adjunct host directed therapies. We also provide first data on safety of p38 MAPK inhibition which is of relevance for future application of these substances in inflammatory diseases and concomitant TB.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Pneumonia/prevenção & controle , Tuberculose/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/imunologia , Pneumonia/imunologia , Tuberculose/microbiologiaRESUMO
BACKGROUND: Most molecular techniques currently require fresh frozen tumor tissue, which in the case of prostatectomy specimen is a challenge to obtain for a variety of intrinsic reasons. Prostate cancers are usually located in the organ periphery and hence meticulous attention has to be paid to the relation between the tumor and the surgical margin. In this article we describe a new technique that allows to obtain fresh frozen tumor material in rather large quantities and without jeopardizing diagnostic accuracy. METHOD: An inner triangle, representing roughly 50% of the entire prostate tissue, is removed from native prostatectomy specimen and cryopreserved, leaving the periphery of the organ for routine histomorphological analysis. We have validated the technique using a series of 180 archived radical prostatectomy specimen that had been studied by histology in their entirety, as well as 42 prostatectomy specimen worked-up by the new technique. RESULTS: The described technique is effective, yielding frozen tumor tissue in 84.2% of unilateral (
Assuntos
Criopreservação/métodos , Neoplasias da Próstata/patologia , Artefatos , Criopreservação/normas , Humanos , Masculino , Prostatectomia , Neoplasias da Próstata/cirurgia , Reprodutibilidade dos TestesRESUMO
Atrial natriuretic peptide (ANP) regulates arterial blood pressure and volume. Its guanylyl cyclase-A (GC-A) receptor is expressed in vascular endothelium and mediates increases in cGMP, but the functional relevance is controversial. Notably, mice with endothelial-restricted GC-A deletion [EC GC-A knockout (KO) mice] exhibit significant chronic hypervolemic hypertension. The present study aimed to characterize the endothelial effects of ANP and their relevance for the acute regulation of intravascular fluid volume. We studied the effect of ANP on microvascular permeability to fluorescein isothiocyanate-labeled albumin (BSA) using intravital microscopy on mouse dorsal skinfold chambers. Local superfusion of ANP (100 nm) increased microvascular fluorescein isothiocyanate-BSA extravasation in control but not EC GC-A KO mice. Intravenous infusion of synthetic ANP (500 ng/kg x min) caused immediate increases in hematocrit in control mice, indicating intravascular volume contraction. In EC GC-A KO mice, the hematocrit responses were not only abolished but even reversed. Furthermore, acute vascular volume expansion, which caused release of endogenous cardiac ANP, did not affect resting central venous pressure of control mice but rapidly and significantly increased central venous pressure of EC GC-A KO mice. In cultured lung endothelial cells, ANP provoked cGMP-dependent protein kinase I-mediated phosphorylation of vasodilator-stimulated phosphoprotein. We conclude that ANP, via GC-A, enhances microvascular endothelial macromolecule permeability in vivo. This effect might be mediated by cGMP-dependent protein kinase I-dependent phosphorylation of vasodilator-stimulated phosphoprotein. Modulation of transcapillary protein and fluid transport may represent one of the most important hypovolemic actions of ANP.
Assuntos
Volume Sanguíneo/fisiologia , Comunicação Celular/fisiologia , Endotélio Vascular/fisiologia , Coração/fisiologia , Receptores do Fator Natriurético Atrial/fisiologia , Animais , Fator Natriurético Atrial/sangue , Fator Natriurético Atrial/farmacologia , Volume Sanguíneo/genética , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Hematócrito , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Miocárdio/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismo , Pressão Venosa/genéticaRESUMO
Lung adenocarcinoma (LUAD) is the most prevalent subtype of non-small cell lung cancer. Despite the development of novel targeted and immune therapies, the 5-year survival rate is still only 21%, indicating the need for more efficient treatment regimens. Lysine-specific demethylase 1 (LSD1) is an epigenetic eraser that modifies histone 3 methylation status, and is highly overexpressed in LUAD. Using representative human cell culture systems and two autochthonous transgenic mouse models, we investigated inhibition of LSD1 as a novel therapeutic option for treating LUAD. The reversible LSD1 inhibitor HCI-2509 significantly reduced cell growth with an IC50 of 0.3-5 µmin vitro, which was linked to an enhancement of histone 3 lysine methylation. Most importantly, growth arrest, as well as inhibition of the invasion capacities, was independent of the underlying driver mutations. Subsequent expression profiling revealed that the cell cycle and replication machinery were prominently affected after LSD1 inhibition. In addition, our data provide evidence that LSD1 blockade significantly interferes with EGFR downstream signaling. Finally, our in vitro results were confirmed by preclinical therapeutic approaches, including the use of two autochthonous transgenic LUAD mouse models driven by either EGFR or KRAS mutations. Importantly, LSD1 inhibition resulted in significantly lower tumor formation and a strong reduction in tumor progression, which were independent of the underlying mutational background of the mouse models. Hence, our findings provide substantial evidence indicating that tumor growth of LUAD can be markedly decreased by HCI-2509 treatment, suggesting its use as a single agent maintenance therapy or combined therapeutical application in novel concerted drug approaches.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Adenocarcinoma de Pulmão/enzimologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologiaRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to understand the mechanisms that lead to treatment resistance. To this end, we analyzed whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent mutations in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that developed during treatment, as well as a mutation in BRAF and a high-level focal amplification of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further therapeutic interventions.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Sulfonamidas/uso terapêutico , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p18/genética , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMO
Any academic pathologist will sooner or later be confronted with the need to use human tissues for quality control purposes or for research projects or else with the demand for human tissue samples for research projects of external researchers. Over the last 10 years, the use of human tissues for such non-diagnostic purposes has been the subject of extensive debate and wide-reaching regulations. In particular, questions of medical secrecy, safety, autonomy and anonymization have been addressed, and the role of ethical review boards defined. However, these guidelines are not uniform in all countries and unfortunately tend to suffer from a certain lack of precision, which may in part be due to the fact that they are usually edited by multiple authors with quite diverse backgrounds (physicians, lawyers, ethicists, philosophers, anthropologists, theologists or lay persons, for example). The wide spectrum of interpretations of such regulations may be embarrassing to such an extent that a continuation of academic activities with human tissues appears to many pathologists as almost impossible or even outright illegal. This paper describes a set of characteristic, recurrent situations to which an academic pathologist may be confronted and proposes simple, realistic solutions that are nevertheless in line with most current regulations.
Assuntos
Centros Médicos Acadêmicos , Pesquisa Biomédica/métodos , Patologia/métodos , Bancos de Tecidos/organização & administração , Pesquisa Biomédica/educação , Pesquisa Biomédica/ética , Guias como Assunto , Humanos , Patologia/educação , Patologia/ética , Bancos de Tecidos/éticaRESUMO
The improvement in sensitive techniques has allowed the detection of tumor-specific aberrations in circulating tumor (ct) DNA. Amplification-refractory mutation system PCR has been used for the analysis of ctDNA to detect resistance-causing mutations in the epidermal growth factor receptor gene (eg, EGFR T790M) in lung cancer patients. So far, Streck tubes have been widely used as standard blood collection tubes. Here, we compared blood collection tubes manufactured by Streck with tubes from Roche and Qiagen regarding their utility in stabilizing ctDNA in plasma samples. Venous blood from healthy donors was collected in tubes from Streck, Roche, and Qiagen. Samples were spiked with artificially fragmented EGFR T790M-mutated DNA and stored for different periods of time or spiked with different DNA amounts before plasma preparation. Extracted ctDNA was analyzed by amplification-refractory mutation system PCR. Mutant DNA, spiked into blood samples from healthy donors at quantities of 1 or 3 ng, was still reliably detectable in all samples after 7 days. EGFR T790M could be detected when spiking was performed with an amount of artificial ctDNA of 0.5 ng when tubes from Roche and Qiagen were used. Blood collection tubes from Roche and Qiagen are highly suitable for ctDNA stabilization and subsequent liquid biopsy testing. Even low ctDNA concentrations allow the detection of somatic mutations.
Assuntos
Coleta de Amostras Sanguíneas/instrumentação , Coleta de Amostras Sanguíneas/normas , Ácidos Nucleicos Livres , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/normas , Biópsia Líquida/instrumentação , Biópsia Líquida/normas , Receptores ErbB/genética , Marcadores Genéticos , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , MutaçãoRESUMO
The epigenetic writer lysine-specific demethylase 1 (LSD1) is aberrantly upregulated in many cancer types and its overexpression correlates with poor survival and tumor progression. In this study, we analysed LSD1 function in non-small cell lung cancer adenocarcinomas. Expression profiling of 182 cases of lung adenocarcinoma proved a significant correlation of LSD1 overexpression with lung adenocarcinoma progression and metastasis. KRAS-mutated lung cancer cell clones were stably silenced for LSD1 expression. RNA-seq and comprehensive pathway analysis revealed, that genes related to a recently described non-canonical integrin ß3 pathway, were significantly downregulated by LSD1 silencing. Hence, invasion and self-renewal capabilities were strongly decreased. Notably, this novel defined LSD1/integrin ß3 axis, was also detected in human lung adenocarcinoma specimens. Furthermore, the linkage of LSD1 to an altered expression pattern of lung-lineage specific transcription factors and genes, which are involved in alveolar epithelial differentiation, was demonstrated. Thus, our findings point to a LSD1-integrin ß3 axis, conferring attributes of invasiveness and tumor progression to lung adenocarcinoma.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Histona Desmetilases/metabolismo , Integrina beta3/metabolismo , Neoplasias Pulmonares/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Histona Desmetilases/genética , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Mutação , NF-kappa B/metabolismo , Gradação de Tumores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Mensageiro/genética , Transdução de SinaisRESUMO
The human gene, human giant larvae (Hugl-1/Llg1/Lgl1) has significant homology to the Drosophila tumour suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that binds Myosin II and is involved in maintaining cell polarity and epithelial integrity. The human protein, Hugl-1 contains several conserved functional domains found in Lgl, suggesting that these proteins may have closely related functions. Whether loss of Hugl expression plays a role in human tumorigenesis has so far not been extensively investigated. Thus, we evaluated tumour tissues from 94 patients undergoing surgery for colorectal cancer (CRC) for loss of Hugl-1 transcription and compared our findings with the clinical data from each of these patients. We found that Hugl-1 was lost in 75% of tumour samples and these losses were associated with advanced stage and particularly with lymph node metastases. Reduced Hugl-1 expression during the adenoma-carcinoma sequence occurring as early as in colorectal adenomas was detected by both immunohistochemical and reverse transcription-polymerase chain reaction analysis. Functional assays with ecdysone-inducible cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Our studies thus indicate that downregulation of Hugl-1 contributes to CRC progression.
Assuntos
Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas/metabolismo , Adenoma/metabolismo , Adulto , Animais , Western Blotting , Células CACO-2 , Carcinoma/metabolismo , Adesão Celular , Ciclo Celular , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Proteínas do Citoesqueleto , Citoesqueleto/metabolismo , Progressão da Doença , Regulação para Baixo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Neoplasias/metabolismo , Estrutura Terciária de Proteína , Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismoRESUMO
A global understanding of miRNA function in EGFR signaling pathways may provide insights into improving the management of KRAS-mutant lung cancers, which remain relatively recalcitrant to treatment. To identify miRNAs implicated in EGFR signaling, we transduced bronchial epithelial BEAS-2B cells with retroviral vectors expressing KRAS(G12V) and monitored miRNA expression patterns by microarray analysis. Through this approach, we defined miR-29b as an important target for upregulation by mutant KRAS in non-small cell lung cancers. Cell biologic analyses showed that pharmacologic inhibition of EGFR or MEK was sufficient to reduce levels of miR-29b, while PI3K inhibition had no effect. In KRAS(G12V)-transduced BEAS-2B cells, introduction of anti-miR-29b constructs increased the sensitivity to apoptosis, arguing that miR-29b mediated apoptotic resistance conferred by mutant KRAS. Mechanistic investigations traced this effect to the ability of miR-29b to target TNFAIP3/A20, a negative regulator of NF-κB signaling. Accordingly, overexpression of an miR-29b-refractory isoform of TNFAIP3 restored NF-κB and extrinsic apoptosis, confirming that TNFAIP3 is a functionally relevant target of miR-29b. We also noted that miR-29b could confer sensitivity to intrinsic apoptosis triggered by exposure to cisplatin, a drug used widely in lung cancer treatment. Thus, miR-29b expression may tilt cells from extrinsic to intrinsic mechanisms of apoptosis. Overall, our results reveal a complexity in cancer for miR-29b, which can act as either an oncogene or tumor suppressor gene depending on signaling context. Cancer Res; 76(14); 4160-9. ©2016 AACR.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , MicroRNAs/fisiologia , NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/fisiologia , Apoptose , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases , Mutação , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genéticaRESUMO
Lung adenosquamous carcinoma is a particular subtype of non-small cell lung carcinoma that is defined by the coexistence of adenocarcinoma and squamous cell carcinoma components. The aim of this study was to assess the mutational profile in each component of 16 adenosquamous carcinoma samples from a Caucasian population by a combination of next generation sequencing using the cancer hotspot panel as well as the colon and lung cancer panel and FISH. Identified mutations were confirmed by Sanger sequencing of DNA from cancer cells of each component collected by Laser Capture microdissection. Mutations typical for adenocarcinoma as well as squamous cell carcinoma were identified. Driver mutations were predominantly in the trunk suggesting a monoclonal origin of adenosquamous carcinoma. Most remarkably, EGFR mutations and mutations in the PI3K signaling pathway, which accounted for 30% and 25% of tumors respectively, were more prevalent while KRAS mutations were less prevalent than expected for a Caucasian population. Surprisingly, expression of classifier miR-205 was intermediate between that of classical adenocarcinoma and squamous cell carcinoma suggesting that adenosquamous carcinoma is a transitional stage between these tumor types. The high prevalence of therapy-relevant targets opens new options of therapeutic intervention for adenosquamous carcinoma patients.