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Upon detecting pathogens or cell stress, several NOD-like receptors (NLRs) form inflammasome complexes with the adapter ASC and caspase-1, inducing gasdermin D (GSDMD)-dependent cell death and maturation and release of IL-1ß and IL-18. The triggers and activation mechanisms of several inflammasome-forming sensors are not well understood. Here we show that mitochondrial damage activates the NLRP10 inflammasome, leading to ASC speck formation and caspase-1-dependent cytokine release. While the AIM2 inflammasome can also sense mitochondrial demise by detecting mitochondrial DNA (mtDNA) in the cytosol, NLRP10 monitors mitochondrial integrity in an mtDNA-independent manner, suggesting the recognition of distinct molecular entities displayed by the damaged organelles. NLRP10 is highly expressed in differentiated human keratinocytes, in which it can also assemble an inflammasome. Our study shows that this inflammasome surveils mitochondrial integrity. These findings might also lead to a better understanding of mitochondria-linked inflammatory diseases.
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Citocinas , Inflamassomos , Humanos , Inflamassomos/metabolismo , Caspase 1/metabolismo , Citocinas/metabolismo , Morte Celular , DNA Mitocondrial/genética , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismoRESUMO
BACKGROUND: The long-term outcomes following surgical resection for pancreatic ductal adenocarcinoma (PDAC) remains poor, with only 20% of patients surviving 5 years after pancreatectomy. Patient selection for surgery remains suboptimal largely due to the absence of consideration of aggressive tumor biology. OBJECTIVE: The aim of this study was to evaluate traditional staging criteria for PDAC in the setting of molecular subtypes. METHODS: Clinicopathological data were obtained for 5 independent cohorts of consecutive unselected patients, totaling n = 1298, including n = 442 that underwent molecular subtyping. The main outcome measure was disease-specific survival following surgical resection for PDAC stratified according to the American Joint Commission for Cancer (TNM) staging criteria, margin status, and molecular subtype. RESULTS: TNM staging criteria and margin status confers prognostic value only in tumors with classical pancreatic subtype. Patients with tumors that are of squamous subtype, have a poor outcome irrespective of favorable traditional pathological staging [hazard ratio (HR) 1.54, 95% confidence interval (CI) 1.04-2.28, P = 0.032]. Margin status has no impact on survival in the squamous subtype (16.0 vs 12.1 months, P = 0.374). There were no differences in molecular subtype or gene expression of tumors with positive resection margin status. CONCLUSIONS: Aggressive tumor biology as measured by molecular subtype predicts poor outcome following pancreatectomy for PDAC and should be utilized to inform patient selection for surgery.
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Carcinoma Ductal Pancreático , Carcinoma de Células Escamosas , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patologia , Prognóstico , Carcinoma Ductal Pancreático/patologia , Pancreatectomia , Estadiamento de Neoplasias , Carcinoma de Células Escamosas/cirurgia , Estudos Retrospectivos , Taxa de Sobrevida , Neoplasias PancreáticasRESUMO
BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.
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Adenocarcinoma/patologia , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Neoplasias Pancreáticas/patologia , Adenocarcinoma/genética , Adenocarcinoma/terapia , Biomarcadores , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Humanos , Terapia de Alvo Molecular , Organoides , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: We aimed to define preoperative clinical and molecular characteristics that would allow better patient selection for operative resection. BACKGROUND: Although we use molecular selection methods for systemic targeted therapies, these principles are not applied to surgical oncology. Improving patient selection is of vital importance for the operative treatment of pancreatic cancer (pancreatic ductal adenocarcinoma). Although surgery is the only chance of long-term survival, 80% still succumb to the disease and approximately 30% die within 1 year, often sooner than those that have unresected local disease. METHOD: In 3 independent pancreatic ductal adenocarcinoma cohorts (total participants = 1184) the relationship between aberrant expression of prometastatic proteins S100A2 and S100A4 and survival was assessed. A preoperative nomogram based on clinical variables available before surgery and expression of these proteins was constructed and compared to traditional measures, and a postoperative nomogram. RESULTS: High expression of either S100A2 or S100A4 was independent poor prognostic factors in a training cohort of 518 participants. These results were validated in 2 independent patient cohorts (Glasgow, n = 198; Germany, n = 468). Aberrant biomarker expression stratified the cohorts into 3 distinct prognostic groups. A preoperative nomogram incorporating S100A2 and S100A4 expression predicted survival and nomograms derived using postoperative clinicopathological variables. CONCLUSIONS: Of those patients with a poor preoperative nomogram score, approximately 50% of patients died within a year of resection. Nomograms have the potential to improve selection for surgery and neoadjuvant therapy, avoiding surgery in aggressive disease, and justifying more extensive resections in biologically favorable disease.
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Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Fatores Quimiotáticos/genética , Pancreatectomia/métodos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Proteínas S100/genética , Idoso , Carcinoma Ductal Pancreático/cirurgia , Causas de Morte , Estudos de Coortes , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Nomogramas , Pancreatectomia/mortalidade , Neoplasias Pancreáticas/cirurgia , Seleção de Pacientes , Prognóstico , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Medição de Risco , Análise de SobrevidaRESUMO
Structure determination of biomacromolecules under in-cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site-directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well-suited for in-cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in-cell trityl-iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal-to-noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in-cell measurement.
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Sistema Enzimático do Citocromo P-450/análise , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Pseudomonas putida/enzimologia , Marcadores de Spin/síntese química , Compostos de Tritil/síntese química , Proteínas de Xenopus/análise , Xenopus laevis , Animais , Ferro/análise , Oócitos/enzimologia , Razão Sinal-Ruído , Xenopus laevis/metabolismoRESUMO
The ferrous iron transporter FeoB is an important factor in the iron metabolism of many bacteria. Although several structural studies have been performed on its cytosolic GTPase domain (NFeoB), the full-length structure of FeoB remains elusive. Based on a crystal packing analysis that was performed on crystals of NFeoB, a trimeric structure of the FeoB channel was proposed, where the transport pore runs along the trimer axis. Because this trimer has not been observed in some subsequently solved structures of NFeoB homologs, it remains unclear whether or not the trimer is indeed functionally relevant. Here, pulsed electron-electron double resonance spectroscopy, negative stain electron microscopy, and native mass spectrometry are used to analyze the oligomeric state of different soluble and full-length FeoB constructs. The results show that the full-length protein is predominantly monomeric, whereas dimers and trimers are formed to a small percentage. Furthermore, the solution structure of the switch I region is analyzed by pulsed electron-electron double resonance spectroscopy and a new, to our knowledge, crystal structure of NFeoB from Escherichia coli BL21 is presented.
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Proteínas de Transporte de Cátions/química , Proteínas de Escherichia coli/química , Dicroísmo Circular , Cristalografia por Raios X , Escherichia coli , Espectrometria de Massas , Microscopia Eletroquímica de Varredura , Domínios Proteicos , Multimerização Proteica , SoluçõesRESUMO
The ferrous iron transporter FeoB is an important factor in the iron metabolism of various bacteria. As a membrane bound GTPase it also represents an interesting evolutionary link between prokaryotic and eukaryotic membrane signalling pathways. To date, structural information for FeoB is limited to the cytosolic GTPase domain and structural features such as the oligomeric state of the transporter in the membrane, and thereby the nature of the transport pore are a matter of constant debate. Recently, EPR distance measurements have become an important tool to investigate such questions in frozen solution. As a prerequisite for these experiments, we designed protocols to express and purify both the cytosolic domain of FeoB (NFeoB) and full-length FeoB from Escherichia coli BL21 in purity, quantity and quality needed for EPR studies. Since FeoB from E. coli contains 12 native cysteines, we incorporated the unnatural amino acid para-acetylphenylalanine (pAcF) into the protein. We spin labelled the mutant protein using the HO4120 spin label and performed preliminary EPR experiments using cw-X-band EPR spectroscopy. Our results provide new insights concerning the oligomeric state of full-length FeoB.
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Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/isolamento & purificação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Transporte de Cátions/análise , Proteínas de Transporte de Cátions/metabolismo , Clonagem Molecular , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Marcadores de SpinRESUMO
BACKGROUND: Pancreatico-biliary adenocarcinomas (PBA) have a poor prognosis. Diagnosis is usually achieved by imaging and/or endoscopy with confirmatory cytology. Cytological interpretation can be difficult especially in the setting of chronic pancreatitis/cholangitis. Immunohistochemistry (IHC) biomarkers could act as an adjunct to cytology to improve the diagnosis. Thus, we performed a meta-analysis and selected KOC, S100P, mesothelin and MUC1 for further validation in PBA resection specimens. METHODS: Tissue microarrays containing tumour and normal cores in a ratio of 3:2, from 99 surgically resected PBA patients, were used for IHC. IHC was performed on an automated platform using antibodies against KOC, S100P, mesothelin and MUC1. Tissue cores were scored for staining intensity and proportion of tissue stained using a Histoscore method (range, 0-300). Sensitivity and specificity for individual biomarkers, as well as biomarker panels, were determined with different cut-offs for positivity and compared by summary receiver operating characteristic (ROC) curve. RESULTS: The expression of all four biomarkers was high in PBA versus normal ducts, with a mean Histoscore of 150 vs. 0.4 for KOC, 165 vs. 0.3 for S100P, 115 vs. 0.5 for mesothelin and 200 vs. 14 for MUC1 (p < .0001 for all comparisons). Five cut-offs were carefully chosen for sensitivity/specificity analysis. Four of these cut-offs, namely 5%, 10% or 20% positive cells and Histoscore 20 were identified using ROC curve analysis and the fifth cut-off was moderate-strong staining intensity. Using 20% positive cells as a cut-off achieved higher sensitivity/specificity values: KOC 84%/100%; S100P 83%/100%; mesothelin 88%/92%; and MUC1 89%/63%. Analysis of a panel of KOC, S100P and mesothelin achieved 100% sensitivity and 99% specificity if at least 2 biomarkers were positive for 10% cut-off; and 100% sensitivity and specificity for 20% cut-off. CONCLUSION: A biomarker panel of KOC, S100P and mesothelin with at least 2 biomarkers positive was found to be an optimum panel with both 10% and 20% cut-offs in resection specimens from patients with PBA.
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Promoting brown adipose tissue (BAT) activity innovatively targets obesity and metabolic disease. While thermogenic activation of BAT is well understood, the rheostatic regulation of BAT to avoid excessive energy dissipation remains ill-defined. Here, we demonstrate that adenylyl cyclase 3 (AC3) is key for BAT function. We identified a cold-inducible promoter that generates a 5' truncated AC3 mRNA isoform (Adcy3-at), whose expression is driven by a cold-induced, truncated isoform of PPARGC1A (PPARGC1A-AT). Male mice lacking Adcy3-at display increased energy expenditure and are resistant to obesity and ensuing metabolic imbalances. Mouse and human AC3-AT are retained in the endoplasmic reticulum, unable to translocate to the plasma membrane and lack enzymatic activity. AC3-AT interacts with AC3 and sequesters it in the endoplasmic reticulum, reducing the pool of adenylyl cyclases available for G-protein-mediated cAMP synthesis. Thus, AC3-AT acts as a cold-induced rheostat in BAT, limiting adverse consequences of cAMP activity during chronic BAT activation.
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Adenilil Ciclases , Tecido Adiposo Marrom , Temperatura Baixa , Adenilil Ciclases/metabolismo , Adenilil Ciclases/genética , Tecido Adiposo Marrom/metabolismo , Animais , Camundongos , Masculino , Humanos , Termogênese/genética , Metabolismo Energético , AMP Cíclico/metabolismo , Camundongos KnockoutRESUMO
Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.
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Proteínas de Bactérias/química , Pseudomonas aeruginosa/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Domínio Catalítico , Cristalografia por Raios X , Descoberta de Drogas , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
Inflammasomes sense intracellular clues of infection, damage, or metabolic imbalances. Activated inflammasome sensors polymerize the adaptor ASC into micron-sized "specks" to maximize caspase-1 activation and the maturation of IL-1 cytokines. Caspase-1 also drives pyroptosis, a lytic cell death characterized by leakage of intracellular content to the extracellular space. ASC specks are released among cytosolic content, and accumulate in tissues of patients with chronic inflammation. However, if extracellular ASC specks contribute to disease, or are merely inert remnants of cell death remains unknown. Here, we show that camelid-derived nanobodies against ASC (VHHASC ) target and disassemble post-pyroptotic inflammasomes, neutralizing their prionoid, and inflammatory functions. Notably, pyroptosis-driven membrane perforation and exposure of ASC specks to the extracellular environment allowed VHHASC to target inflammasomes while preserving pre-pyroptotic IL-1ß release, essential to host defense. Systemically administrated mouse-specific VHHASC attenuated inflammation and clinical gout, and antigen-induced arthritis disease. Hence, VHHASC neutralized post-pyroptotic inflammasomes revealing a previously unappreciated role for these complexes in disease. VHHASC are the first biologicals that disassemble pre-formed inflammasomes while preserving their functions in host defense.
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Inflamassomos , Anticorpos de Domínio Único , Animais , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Humanos , Inflamassomos/metabolismo , Inflamação/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , PiroptoseRESUMO
Despite progress in genomic characterization, no single prognostic marker that can be evaluated using an easy-to-perform and relatively inexpensive method is available for pancreatic ductal adenocarcinoma (PDAC). MicroRNAs, which are stable, tumor- and tissue-specific molecules, are potentially ideal biomarkers, and we established an inter-laboratory validated method to investigate miR-21 as a prognostic biomarker in PDAC. The study samples of PDAC patients were recruited from a test cohort of Glasgow (n = 189) and three validation cohorts of Pisa (n = 69), Sydney (n = 249), and International Cancer Genome Consortium (ICGC) (n = 249). Tissue microarrays were used for miR-21 staining by chromogenic in situ hybridization (CISH). The patients were subdivided into no/low and high miR-21 staining groups using a specific histoscore. Furthermore, miR-21 staining was evaluated against clinicopathological variables and follow-up data by Fisher/log-rank test and Cox proportional models. The prognostic variables found to be significant in univariate analysis (P value < 0.10) were included in multivariate analysis in a backward-stepwise fashion. MiR-21 expression was cytoplasmic, with more consistent staining in the malignant ductal epithelium than in the stroma. The expression of miR-21 was significantly associated with tumor size and lymph node metastasis, whereas no association was observed with other clinicopathological variables. High miR-21 staining (histoscore ≥ 45 [median score]) was an independent predictor of survival in the Glasgow test cohort (HR 2.37, 95% CI: 1.42-3.96, P < 0.0001) and three validation cohorts (Pisa, HR 2.03, 95% CI: 1.21-3.39, P = 0.007; Sydney, HR 2.58, 95% CI (1.21-3.39), P < 0.0001; and ICGC, HR 3.34, 95% CI: 2.07-5.84, P = 0.002) when adjusted for clinical variables in a multivariate model. In comparison to the patients with low miR-21, the patients with high miR-21 expression had significant increase in OS as they benefit from gemcitabine-based adjuvant chemotherapy (Glasgow 16.5 months [with chemotherapy] vs 10.5 months [without chemotherapy]); Sydney 25.0 vs 10.6; ICGC 25.2 vs 11.9. These results indicated that miR-21 is a predictor of survival, prompting prospective trials. Evaluation of miR-21 offers new opportunities for the stratification of patients with PDAC and might facilitate the implementation of clinical management and therapeutic interventions for this devastating disease.
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Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs.
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Biomarcadores Tumorais/genética , Carcinoma Neuroendócrino/genética , Metilação de DNA , Epigênese Genética , Epigenoma , Neoplasias Pancreáticas/genética , Carcinoma Neuroendócrino/metabolismo , Epigenômica , Predisposição Genética para Doença , Humanos , Gradação de Tumores , Neoplasias Pancreáticas/patologia , Fenótipo , Carga TumoralRESUMO
Introduction The aim of this study was to compare the detection of lymphatic invasion using haematoxylin and eosin (H&E) staining versus D2-40 immunostaining on specimens from a retrospective cohort of patients with colorectal polyp cancer and to investigate the association of lymphatic invasion, detected by either method, with survival. Methods Specimens from patients with pathologically diagnosed colorectal polyp cancer were selected from the Greater Glasgow and Clyde Bowel Cancer Screening Registry for D2-40 immunohistochemistry staining. Clinicopathological information was retrieved from patient electronic records including analysis of pathology reports to determine if a lymphatic invasion was detected using H&E staining. Results Over 100 patients were included in this study with a median age at polypectomy of 66 years (range 50-76). All patients were followed up for a minimum of four years and five patients died due to colorectal cancer. The lymphatic invasion was detected in 8% of cases by H&E staining and 23% of cases with D2-40 immunostaining. Only D2-40-detected lymphatic invasion showed a statistically significant relationship with colorectal cancer-specific mortality using univariate analysis (p=0.01). Survival analysis performed separately by Cox regression demonstrated that lymphatic invasion detected by D2-40 immunostaining was associated with worse disease-specific survival (hazard ratio [HR] 14.07, 95% CI 1.57-125.97, p=0.018). Conclusion This study shows that D2-40 immunostaining can improve the detection of lymphatic invasion in colorectal polyp cancer when compared to H&E staining. In addition, the lymphatic invasion detected by D2-40 immunostaining significantly associates with survival allowing it to be used as a prognostic indicator in colorectal polyp cancer.
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The type-III secretion effector YopO helps pathogenic Yersinia to outmaneuver the human immune system. Injected into host cells, it functions as a Ser/Thr kinase after activation by actin binding. This activation process is thought to involve large conformational changes. We use PELDOR spectroscopy and small-angle X-ray scattering in combination with available crystal structures to study these conformational transitions. Low-resolution hybrid models of the YopO/actin structure in solution were constructed, where the kinase domain of YopO is tilted "backward" compared with the crystal structure, thus shortening the distance between actin and the kinase active site, potentially affecting the substrate specificity of YopO. Furthermore, the GDI domain of the hybrid models resembles a conformation that was previously observed in a crystal structure of the isolated GDI domain. We investigate possible structural reasons for the inactivity of the apo state, analyze its flexibility and discuss the biological implications.
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Actinas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Yersinia/química , Yersinia/metabolismo , Domínio Catalítico , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Next-generation sequencing is enabling molecularly guided therapy for many cancer types, yet failure rates remain relatively high in pancreatic cancer (PC). The aim of this study is to investigate the feasibility of genomic profiling using endoscopic ultrasound (EUS) biopsy samples to facilitate personalised therapy for PC. Ninty-five patients underwent additional research biopsies at the time of diagnostic EUS. Diagnostic formalin-fixed (FFPE) and fresh frozen EUS samples underwent DNA extraction, quantification and targeted gene sequencing. Whole genome (WGS) and RNA sequencing was performed as proof of concept. Only 2 patients (2%) with a diagnosis of PC had insufficient material for targeted sequencing in both FFPE and frozen specimens. Targeted panel sequencing (n=54) revealed mutations in PC genes (KRAS, GNAS, TP53, CDKN2A, SMAD4) in patients with histological evidence of PC, including potentially actionable mutations (BRCA1, BRCA2, ATM, BRAF). WGS (n=5) of EUS samples revealed mutational signatures that are potential biomarkers of therapeutic responsiveness. RNA sequencing (n=35) segregated patients into clinically relevant molecular subtypes based on transcriptome. Integrated multi-omic analysis of PC using standard EUS guided biopsies offers clinical utility to guide personalized therapy and study the molecular pathology in all patients with PC.
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Biomarcadores Tumorais/genética , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biópsia Guiada por Imagem/métodos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/genética , Seleção de Pacientes , Estudos de Viabilidade , Humanos , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/terapia , PrognósticoRESUMO
A 40-year-old Caucasian female presented with hyperglycaemia, polyuria, polydipsia and weight loss of 6 kg over a 1-month period. There was no personal or family history of malignancy or diabetes mellitus. On examination, she was jaundiced with pale mucous membranes and capillary glucose was 23.1 mmol/L. Initial investigations showed iron deficiency anaemia and obstructive pattern of liver function tests. HbA1c was diagnostic of diabetes mellitus at 79 mmol/mol. Malignancy was suspected and CT chest, abdomen and pelvis showed significant dilatation of intra- and extra-hepatic biliary tree including pancreatic duct, with periampullary 30 mm mass lesion projecting into lumen of duodenum. Enlarged nodes were seen around the superior mesenteric artery. This was confirmed on MRI liver. Fasting gut hormones were normal except for a mildly elevated somatostatin level. Chromogranin A was elevated at 78 pmol/L with normal chromogranin B. Duodenoscopy and biopsy showed possible tubovillous adenoma with low-grade dysplasia, but subsequent endoscopic ultrasound and biopsy revealed a grade 1, well differentiated neuroendocrine tumour. The patient was started on insulin, transfused to Hb >8 g/dL and Whipple's pancreatico-duodenectomy was undertaken. This showed a well-differentiated neuroendocrine carcinoma arising in duodenum (Grade G1 with Ki67: 0.5%), with areas of chronic pancreatitis and preservation of pancreatic islet cells. There was complete resolution of diabetes post Whipple's procedure and patient was able to come of insulin treatment. Her last HBA1C was 31 mmol/mol, 4 months post tumour resection. LEARNING POINTS: Diabetes mellitus and malignancy can be related.A high index of suspicion is needed when diabetes mellitus presents atypically.Non-functional neuroendocrine tumours can present with diabetes mellitus.
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BACKGROUND: Clinical translation of immunohistochemistry (IHC) biomarkers requires reliable and reproducible cutoffs or thresholds for interpretation of immunostaining. Most IHC biomarker research focuses on the clinical relevance (diagnostic, prognostic, or predictive utility) of cutoffs, with less emphasis on observer agreement using these cutoffs. From the literature, we identified 3 commonly used cutoffs of 10% positive epithelial cells, 20% positive epithelial cells, and moderate to strong staining intensity (+2/+3 hereafter) to use for investigating observer agreement. MATERIALS AND METHODS: A series of 36 images of microarray cores stained for 4 different IHC biomarkers, with variable staining intensity and percentage of positive cells, was used for investigating interobserver and intraobserver agreement. Seven pathologists scored the immunostaining in each image using the 3 cutoffs for positive and negative staining. Kappa (κ) statistic was used to assess the strength of agreement for each cutoff. RESULTS: The interobserver agreement between all 7 pathologists using the 3 cutoffs was reasonably good, with mean κ scores of 0.64, 0.59, and 0.62, respectively, for 10%, 20%, and +2/+3 cutoffs. A good agreement was observed for experienced pathologists using the 10% cutoff, and their agreement was statistically higher than for junior pathologists (P=0.02). In addition, the mean intraobserver agreement for all 7 pathologists using the 3 cutoffs was reasonably good, with mean κ scores of 0.71, 0.60, and 0.73, respectively, for 10%, 20%, and +2/+3 cutoffs. For all 3 cutoffs, a positive correlation was observed with perceived ease of interpretation (P<0.003). Finally, cytoplasmic-only staining achieved higher agreement using all 3 cutoffs than mixed staining patterns. CONCLUSIONS: All 3 cutoffs investigated achieve reasonable strength of agreement, modestly decreasing interobserver and intraobserver variability in IHC interpretation. These cutoffs have previously been used in cancer pathology, and this study provides evidence that these cutoffs can be reproducible between practicing pathologists.
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Carcinoma Ductal Pancreático/metabolismo , Variações Dependentes do Observador , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/patologia , Humanos , Imuno-Histoquímica , Neoplasias Pancreáticas/patologia , Análise Serial de TecidosRESUMO
BACKGROUND: Despite being relatively rare pancreatic cancer is one of the highest causes of death. Even within the potentially resectable group outcomes are poor. We present our initial experiences utilising a neoadjuvant approach to localised pancreatic cancer, evaluating survival, response rates and tolerability. METHODS: This was a retrospective analysis of a prospectively maintained database. Patients from 2012 to 2015 referred to a busy regional Hepato-Pancreatic Biliary (HPB) MDT were included. Patients were classified according to respectability criteria (utilising NCCN guidelines) and a treatment plan agreed. Systemic therapy with either FOLFIRINOX or Gem/Cap was delivered followed by chemoradiotherapy if disease remained localised. Toxicity, response, pathological outcomes and survival were all recorded. RESULTS: A total of 85 patients were included in the study: 45 had initially resectable disease; 19 required a response for resection and 21 had locally advanced inoperable disease; 34 patients underwent resection. The median survival for the potentially resectable group was 22.2 months while for those undergoing resection it was 37 months. CONCLUSIONS: We have demonstrated that a neoadjuvant approach is deliverable and tolerable. In addition we have demonstrated impressive survival results in patients undergoing resection with no detriment in outcome for those not proceeding to surgery.
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Abdominal surgery performed in patients with significant liver disease and portal hypertension is associated with high mortality rates, with even poorer outcomes associated with complex pancreaticobiliary operations. We report on a patient requiring portal decompression via transjugular intrahepatic portosystemic shunt (TIPS) prior to a pancreaticoduodenectomy. The 49-year-old patient presented with pain, jaundice and weight loss. At ERCP an edematous ampulla was biopsied, revealing high-grade dysplasia within a distal bile duct adenoma. Liver biopsy was performed to investigate portal hypertension, confirming congenital hepatic fibrosis (CHF). A TIPS was performed to enable a pancreaticoduodenectomy. Prophylactic TIPS can be performed for preoperative portal decompression for patients requiring pancreatic resection. A potentially curative resection was performed when abdominal surgery was initially thought impossible. Notably, CHF has been associated with the development of cholangiocarcinoma in only four previous instances, with this case being only the second reported distal bile duct cholangiocarcinoma.