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Currently, there are limited and conflicting reports on the prognostic utility of PIK3CA and associated pathway markers for cervical cancers treated with primary surgical management. Moreover, current studies are lacking complete characterization of adjuvant treatment with RT and/or chemotherapy. We aimed to document the prevalence, clinicopathologic, adjuvant treatment details, and prognostic value of PI3K/AKT pathway mutations and copy number variation and phosphorylated AKT status in patients with cervical cancers treated with primary surgery. A clinicopathologic review was performed on a retrospective cohort of 185 patients with cervical cancer, treated with primary surgery at a single tertiary institution. Next-generation sequencing and digital PCR was used to determine PI3K/AKT pathway mutational status and PIK3CA copy number variation, respectively, and fluorescent immunohistochemistry measured phosphorylated AKT expression. In all, 179 of 185 (96.8%) of tumors were successfully sequenced; 48 (26.8%) were positive for PI3K/AKT pathway mutations-the majority (n=37, 77.1%) PIK3CA mutations. PIK3CA mutation was associated with pathologically positive lymph nodes [12 (32%) vs. 22 (16%); P =0.022] and indication for postoperative chemoradiotherapy [17 (45.9%) vs. 32 (22.5%); P =0.004]. On multivariable analysis, PIK3CA status was not associated with overall survival ( P =0.103) or progression-free survival ( P =0.240) at 5 yrs, nor was PIK3CA copy number variation status. phosphorylated AKT ≤ median significantly predicted for progression-free survival [multivariable hazard ratio 0.39 (0.17-0.89; P =0.025)] but not overall survival ( P =0.087). The correlation of PIK3CA with pathologic positive lymph node status yet lack of association with survival outcomes may be due to the use of adjuvant postoperative therapy. PIK3CA assessment before radical hysterectomy may help identify patients with a higher risk of node-positive disease.
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Proteínas Proto-Oncogênicas c-akt , Neoplasias do Colo do Útero , Feminino , Humanos , Prognóstico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/cirurgia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Estudos Retrospectivos , Variações do Número de Cópias de DNA , Prevalência , Mutação , Classe I de Fosfatidilinositol 3-Quinases/genéticaRESUMO
Background: Programmed death-ligand 1 (PD-L1) expression has been shown to be prognostic in many cancer types and used in consideration of checkpoint inhibitor immunotherapy. However, there are very limited and conflicting data on the prognostic impact of PD-L1 in patients with anal squamous cell carcinoma (ASCC). The objectives of this study were to measure the expression of PD-L1 and CD8 in patients with ASCC treated with radical chemoradiotherapy (CRT) and to correlate tumor expression with progression-free survival (PFS) and overall survival (OS). Methods: Ninety-nine patients with ASCC treated with primary CRT at two tertiary care cancer centers between 2000 and 2013, with available pre-treatment tumors, were included. Tissue microarrays (TMAs) from pre-treatment tumor specimens were stained for PD-L1 and CD8. PD-L1 expression in the tumor and stroma was quantified using HALO image analysis software, and results were interpreted using quantitative methods. The density of CD8 cells within the tumor was interpreted by a trained pathologist semi-quantitatively, using a 0-4 scoring system. Kaplan-Meier analysis with log-rank was used to determine the significance in the association of tumor markers with PFS and OS. Cox multivariate analysis was used to explore independent predictors of PFS and OS. Results: Of the 99 patients, 63 (64%) had sufficient tumor samples available for full analysis. CD8 high status was documented in 32 of 63 (50.8%) % of cases. PD-L1 expression was positive in 88.9% of cases. Approximately half the patients had tumor PD-L1 ≥ 5%. Patients with tumor PD-L1 ≥ 5% had better OS vs those with lower expression, HR=0.32 (95% CI 0.11-0.87), p=0.027; 10 years OS: 84% for tumor PD-L1 ≥ 5% vs 49% for PD-L1 < 5%. PD-L1 expression was not associated with PFS. On multivariate analysis, tumor PD-L1 ≥ 5% showed a trend to statistical significance for better OS, HR=0.55 (95% CI 0.12- 1.00), p=0.052. Conclusions: Tumor PD-L1≥5% is associated with OS in patients with ASCC treated with CRT. PD-L1 expression status using this unique cut-point warrants further validation for prognostication in patients with this disease. Future studies are required to determine the benefit of alternative treatment strategies based on PD-L1 status.
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OBJECTIVE: While there is concern about excessive laboratory test ordering in the ED, it is difficult to quantify the problem. One solution involves the Mean Abnormal Result Rate (MARR), which is the proportion of tests ordered that return abnormal results. The primary objective of this study was to calculate MARR scores, and factors associated with MARR scores, for tests ordered between April 2014 and March 2019 at adult EDs in Calgary. METHODS: Administrative data were obtained for 40 laboratory tests that met selection criteria. One possible MARR correlate, physician experience, was quantified for 209 ED physicians as number of years since licensure. Analyses were descriptive where appropriate for whole-population data. RESULTS: The condensed dataset comprised 3,395,312 test results on 415,653 unique patients. The aggregate 5-year MARR score was 40.1%. The highest per-test score was for BNP (80.5%), while the lowest was for glucose (7.9%). MARR scores were higher for nurse-initiated orders than for physician-initiated orders (44.7% vs. 38.1%). The MARR score correlated inversely with number of tests per order (r = - 0.90; 95% confidence interval [CI] - 0.65 to - 0.94; p < 0.001) and directly with physician experience (r = 0.28, 95% CI 0.20 to 0.27; p < 0.001). CONCLUSION: This is the first study to measure MARR scores in an ED setting. While lower scores (close to 5%) are less optimal in principle, ideal scores will depend on the clinical context in which tests are used. However, once departmental benchmarks are established, MARR score-monitoring allows efficient tracking of ordering practices across millions of tests.
RéSUMé: OBJECTIFS: Bien que l'on s'inquiète d'une commande excessive de tests de laboratoire dans le service d'urgence, il est difficile de quantifier le problème. Une solution implique le taux de résultat anormal moyen (MARR), qui est la proportion de tests commandés qui renvoient des résultats anormaux. L'objectif principal de cette étude était de calculer les scores MARR, et les facteurs associés aux scores MARR, pour les tests commandés entre avril 2014 et mars 2019 dans les services d'urgence pour adultes à Calgary. LES MéTHODES: Des données administratives ont été obtenues pour 40 tests de laboratoire répondant aux critères de sélection. Une corrélation possible du MARR, soit l'expérience des médecins, a été quantifié pour 209 médecins urgentistes en nombre d'années depuis l'autorisation d'exercer. Les analyses étaient descriptives, le cas échéant, pour les données sur l'ensemble de la population. RéSULTATS: L'ensemble de données condensé comprenait 3,395,312 résultats de tests sur 415 653 patients uniques. Le score MARR global à 5 ans était de 40,1%. Le score le plus élevé par test était pour le peptide cérébral natriurétique BNP (80,5%), tandis que le plus bas était pour le glucose (7,9%). Les scores de MARR sont plus élevés pour les ordonnances initiées par les infirmières que pour les ordonnances initiées par les médecins (44,7% contre 38,1%). Le score MARR s'est corrélé inversement avec le nombre de tests par ordre (r = − 0,90; intervalle de confiance de 95 % [IC] − 0.65 to − 0,94; p < 0.001) et directement avec l'expérience du médecin (r = 0.28, IC à 95 % 0.200.27; p < 0.001). CONCLUSIONS: Il s'agit de la première étude à mesurer les scores du MARR dans un contexte de service d'urgence. Bien que des scores inférieurs (près de 5%) sont moins optimaux en principe, les scores idéaux dépendront du contexte clinique dans lequel les tests sont utilisés. Cependant, une fois les points de repère ministériels établis, la surveillance des scores du MARR permet de suivre efficacement les pratiques de commande sur des millions de tests.
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Médicos , Padrões de Prática Médica , Adulto , Benchmarking , Serviço Hospitalar de Emergência , Humanos , Seleção de PacientesRESUMO
BACKGROUND: Estrogen receptor beta (ERß) is the predominant estrogen receptor (ER) expressed in non-small cell lung cancer (NSCLC); however, due to methodological disparities among prior studies, the prognostic value of ERß expression in NSCLC remains unclear. Our objective was to apply improved detection and analysis techniques to assess the prognostic value of ERß expression in NSCLC. METHODS: A tissue microarray (TMA) was used which contained resected and biopsy specimens from 299 patients diagnosed at a single center with stages I-IV NSCLC. Sections of this array were stained using high-sensitivity fluorescence immunohistochemistry, with the well-validated PPG5/10 monoclonal antibody. Digital images of the stained array slides were analyzed using software-based image analysis, which reported ERß expression as a continuous variable in different subcellular domains. RESULTS: There were no differences in ERß expression between male and female patients. High expression of ERß was not a prognostic factor, but was significantly associated with stage IV disease in both tumor and stroma (P<0.001). In multivariable analysis, a high nuclear/cytoplasmic (N/C) ratio of ERß expression was significantly associated with shorter overall survival, based on expression in the tumor [hazard ratio (HR): 1.65; 95% confidence interval (CI): 1.25-2.19; P<0.001] and in the stroma (HR: 1.57; 95% CI: 1.16-2.12; P=0.003). CONCLUSIONS: These results suggest that subcellular localization of ERß, but not absolute expression, is a prognostic factor in NSCLC.
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PURPOSE: This study aimed to describe the prognostic value of PI3K/AKT pathway mutations in a large cohort of patients with cervical cancer. EXPERIMENTAL DESIGN: Patients with pre-treatment archival specimens, diagnosed with FIGO stages IB-IVA cervical cancer between 1998 and 2014 and treated with radical, curative intent chemoradiotherapy (CRT) at a single center were identified. Mutational status was determined by next generation sequencing and PIK3CA copy number (CNV) was assessed by digital PCR. RESULTS: 190 patients with available pre-treatment tumor specimens were identified. Median OS and PFS were 57.4 and 46.0 months, respectively. A total of 161 tumors were successfully sequenced; 60 (37.3%) had PI3K/AKT pathway mutations, with 50 (30.1%) having PIK3CA hotspot mutations. PIK3CA CNV gain was noted in 79 (59.2%) of the 154 successfully analyzed. On univariate analysis, PIK3CA mutation was associated with poor OS (HR 1.73; 95% CI: 1.03-2.92; p = .037) but not PFS (HR 1.38; 0.84-2.28; p = .204). Absence of any PI3K/AKT pathway mutation was associated with improved OS (HR 1.68; 1.01-2.81; p = .046) but not PFS (HR 1.50; 0.93-2.43; p = .202). Associations were not maintained when adjusting for clinical factors. On univariate analysis, PIK3CA mutation positive, CNV normal tumors were associated with poorer OS (HR 2.55; 1.18-5.50; p = .017) and trend to worse PFS (HR 1.87; 0.90-3.83; p = .094) when compared to those with CNV gain and wildtype PIK3CA. CONCLUSIONS: PI3K/AKT pathway mutations are common in cervical cancer. Consideration of PIK3CA mutational status with CNV status may be important in predicting outcome in cervical cancer patients.
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Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/terapia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimiorradioterapia , Feminino , Dosagem de Genes , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Análise Serial de Tecidos , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Adulto JovemRESUMO
CCNE1 amplification is a recurrent alteration associated with unfavourable outcome in tubo-ovarian high-grade serous carcinoma (HGSC). We aimed to investigate whether immunohistochemistry (IHC) can be used to identify CCNE1 amplification status and to validate whether CCNE1 high-level amplification and overexpression are prognostic in HGSC. A testing set of 528 HGSC samples stained with two optimised IHC assays (clones EP126 and HE12) was subjected to digital image analysis and visual scoring. DNA and RNA chromogenic in situ hybridisation for CCNE1 were performed. IHC cut-off was determined by receiver operating characteristics (ROC). Survival analyses (endpoint ovarian cancer specific survival) were performed and validated in an independent validation set of 764 HGSC. Finally, combined amplification/expression status was evaluated in cases with complete data (n = 1114). CCNE1 high-level amplification was present in 11.2% of patients in the testing set and 10.2% in the combined cohort. The optimal cut-off for IHC to predict CCNE1 high-level amplification was 60% positive tumour cells with at least 5% strong staining cells (sensitivity 81.6%, specificity 77.4%). CCNE1 high-level amplification and overexpression were associated with survival in the testing and validation set. Combined CCNE1 high-level amplification and overexpression was present in 8.3% of patients, mutually exclusive to germline BRCA1/2 mutation and significantly associated with a higher risk of death in multivariate analysis adjusted for age, stage and cohort (hazard ratio = 1.78, 95 CI% 1.38-2.26, p < 0.0001). CCNE1 high-level amplification combined with overexpression identifies patients with a sufficiently poor prognosis that treatment alternatives are urgently needed. Given that this combination is mutually exclusive to BRCA1/2 germline mutations, a predictive marker for PARP inhibition, CCNE1 high-level amplification combined with overexpression may serve as a negative predictive test for sensitivity to PARP inhibitors.
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Biomarcadores Tumorais/genética , Carcinoma/genética , Ciclina E/genética , Amplificação de Genes , Neoplasias Císticas, Mucinosas e Serosas/genética , Proteínas Oncogênicas/genética , Neoplasias Ovarianas/genética , Alberta , Animais , Proteína BRCA1/genética , Proteína BRCA2/genética , Biomarcadores Tumorais/análise , Colúmbia Britânica , Carcinoma/química , Carcinoma/patologia , Ciclina E/análise , Feminino , Regulação Neoplásica da Expressão Gênica , Mutação em Linhagem Germinativa , Humanos , Imuno-Histoquímica , Hibridização In Situ , Gradação de Tumores , Neoplasias Císticas, Mucinosas e Serosas/química , Neoplasias Císticas, Mucinosas e Serosas/mortalidade , Neoplasias Císticas, Mucinosas e Serosas/patologia , Proteínas Oncogênicas/análise , Neoplasias Ovarianas/química , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Medição de Risco , Fatores de RiscoRESUMO
The nuclear proliferation biomarker Ki67 has potential prognostic, predictive, and monitoring roles in breast cancer. Unacceptable between-laboratory variability has limited its clinical value. The International Ki67 in Breast Cancer Working Group investigated whether Ki67 immunohistochemistry can be analytically validated and standardized across laboratories using automated machine-based scoring. Sets of pre-stained core-cut biopsy sections of 30 breast tumors were circulated to 14 laboratories for scanning and automated assessment of the average and maximum percentage of tumor cells positive for Ki67. Seven unique scanners and 10 software platforms were involved in this study. Pre-specified analyses included evaluation of reproducibility between all laboratories (primary) as well as among those using scanners from a single vendor (secondary). The primary reproducibility metric was intraclass correlation coefficient between laboratories, with success considered to be intraclass correlation coefficient >0.80. Intraclass correlation coefficient for automated average scores across 16 operators was 0.83 (95% credible interval: 0.73-0.91) and intraclass correlation coefficient for maximum scores across 10 operators was 0.63 (95% credible interval: 0.44-0.80). For the laboratories using scanners from a single vendor (8 score sets), intraclass correlation coefficient for average automated scores was 0.89 (95% credible interval: 0.81-0.96), which was similar to the intraclass correlation coefficient of 0.87 (95% credible interval: 0.81-0.93) achieved using these same slides in a prior visual-reading reproducibility study. Automated machine assessment of average Ki67 has the potential to achieve between-laboratory reproducibility similar to that for a rigorously standardized pathologist-based visual assessment of Ki67. The observed intraclass correlation coefficient was worse for maximum compared to average scoring methods, suggesting that maximum score methods may be suboptimal for consistent measurement of proliferation. Automated average scoring methods show promise for assessment of Ki67 scoring, but requires further standardization and subsequent clinical validation.
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Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Processamento de Imagem Assistida por Computador/normas , Imuno-Histoquímica/normas , Antígeno Ki-67/análise , Feminino , Humanos , Imuno-Histoquímica/métodos , Reprodutibilidade dos TestesRESUMO
Poxviruses encode many proteins with the ability to regulate cellular signaling pathways. One such protein is the vaccinia virus innate immunity modulator E3. Multiple functions have been ascribed to E3, including modulating the cellular response to double-stranded RNA, inhibiting the NF-κB and IRF3 pathways, and dampening apoptosis. Apoptosis serves as a powerful defense against damaged and unwanted cells and is an effective defense against viral infection; many viruses therefore encode proteins that prevent or delay apoptosis. Here, we present data indicating that E3 does not directly inhibit the intrinsic apoptotic pathway; instead, it suppresses apoptosis indirectly by stimulating expression of the viral F1 apoptotic inhibitor. Our data demonstrate that E3 promotes F1 expression by blocking activation of the double-stranded RNA-activated protein kinase R (PKR). F1 mRNA is present in cells infected with E3-null virus, but the protein product does not detectably accumulate, suggesting a block at the translational level. We also show that two 3' coterminal transcripts span the F1 open reading frame (ORF), a situation previously described for the vaccinia virus mRNAs encoding the J3 and J4 proteins. One of these is a conventional monocistronic transcript of the F1L gene, while the other arises by read-through transcription from the upstream F2L gene and does not give rise to appreciable levels of F1 protein.IMPORTANCE Previous studies have shown that E3-deficient vaccinia virus triggers apoptosis of infected cells. Our study demonstrates that this proapoptotic phenotype stems, at least in part, from the failure of the mutant virus to produce adequate quantities of the viral F1 protein, which acts at the mitochondria to directly block apoptosis. Our data establish a regulatory link between the vaccinia virus proteins that suppress the innate response to double-stranded RNA and those that block the intrinsic apoptotic pathway.
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Interações Hospedeiro-Patógeno , Proteínas de Ligação a RNA/genética , Vaccinia virus/genética , Proteínas Virais/genética , eIF-2 Quinase/genética , Animais , Apoptose/genética , Deleção de Genes , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Fases de Leitura Aberta , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células RAW 264.7 , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Vaccinia virus/metabolismo , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , eIF-2 Quinase/metabolismoRESUMO
INTRODUCTION: Voltage-gated calcium channels (VGCC) have been found to be differentially expressed in several different tumor types, but their role in tumor growth, malignant invasion, metastases and impact on clinical outcomes has not been clarified. MATERIALS AND METHODS: From a cohort database of 193 patients with early-stage NSCLC, 163 formalin-fixed paraffin-embedded specimens were available for analysis to construct tissue microarrays. Cav3.1 protein expression was detected using fluorescence immunohistochemistry, and quantified using automated image acquisition and analysis. RESULTS: Among the cohort of 193 NSCLC patients, adenocarcinoma (53.9%) and squamous cell carcinoma (SCC) (30.1%) were the most common histologies. There was no difference between SCC and non-SCC subtypes in overall survival (OS) or relapse-free survival (RFS); 74.2 vs 90.1 months (p = 0.543) and 48.8 vs 52.6 months (p = 0.766), respectively. T-type VGCC 3.1 (Cav3.1) overexpression was assessed by tissue microarray immunohistochemistry analysis from 163 available patient samples. Eighteen (11.0%) NSCLC primaries were found to have Cav3.1 overexpression levels, and were significantly associated with SCC histology (p < 0.001), larger tumor size (p < 0.001) and later stage disease at diagnosis (p = 0.019). Median OS was 48.6 vs 106.7 months for Cav3.1 overexpressing and non-overexpressing patients, respectively (p = 0.032). Regression analysis revealed a significantly negative effect for Cav3.1 overexpression on RFS (Hazard ratio [HR] = 2.02, p = 0.048). CONCLUSIONS: Cav3.1 overexpression is a potential biomarker for poorer patient outcomes. These results bring supportive evidence for calcium channels inducing an aggressive phenotype in NSCLC and potentially may serve as a therapeutic target in overexpressing tumors.
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Ki67 is a commonly used marker of cancer cell proliferation, and has significant prognostic value in breast cancer. In spite of its clinical importance, assessment of Ki67 remains a challenge, as current manual scoring methods have high inter- and intra-user variability. A major reason for this variability is selection bias, in that different observers will score different regions of the same tumor. Here, we developed an automated Ki67 scoring method that eliminates selection bias, by using whole-slide analysis to identify and score the tumor regions with the highest proliferative rates. The Ki67 indices calculated using this method were highly concordant with manual scoring by a pathologist (Pearson's r = 0.909) and between users (Pearson's r = 0.984). We assessed the clinical validity of this method by scoring Ki67 from 328 whole-slide sections of resected early-stage, hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. All patients had Oncotype DX testing performed (Genomic Health) and available Recurrence Scores. High Ki67 indices correlated significantly with several clinico-pathological correlates, including higher tumor grade (1 versus 3, P<0.001), higher mitotic score (1 versus 3, P<0.001), and lower Allred scores for estrogen and progesterone receptors (P = 0.002, 0.008). High Ki67 indices were also significantly correlated with higher Oncotype DX risk-of-recurrence group (low versus high, P<0.001). Ki67 index was the major contributor to a machine learning model which, when trained solely on clinico-pathological data and Ki67 scores, identified Oncotype DX high- and low-risk patients with 97% accuracy, 98% sensitivity and 80% specificity. Automated scoring of Ki67 can thus successfully address issues of consistency, reproducibility and accuracy, in a manner that integrates readily into the workflow of a pathology laboratory. Furthermore, automated Ki67 scores contribute significantly to models that predict risk of recurrence in breast cancer.
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Neoplasias da Mama/química , Processamento de Imagem Assistida por Computador/métodos , Antígeno Ki-67/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Automação Laboratorial/métodos , Automação Laboratorial/estatística & dados numéricos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Estudos de Coortes , Feminino , Humanos , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Imuno-Histoquímica/métodos , Imuno-Histoquímica/estatística & dados numéricos , Aprendizado de Máquina , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/química , Recidiva Local de Neoplasia/patologia , Prognóstico , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Risco , Viés de SeleçãoRESUMO
Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed type of head and neck cancer, accounting for ~300,000 new cases worldwide annually. Carbonic anhydrase IX (CAIX) and Ki-67 have been associated with reduced disease-specific survival (DSS) in patients with OSCC. We previously proposed a combined CAIX and Ki-67 signature of 'functional hypoxia' and sought to replicate this association in a larger independent cohort of patients with OSCC at the Fred Hutchinson Cancer Research Center (FHCRC) in Seattle. The study population included patients with incident primary OSCC treated at the University of Washington Medical Center and the Harborview Medical Center in Seattle between December 2003 and February 2012. Archived tumor blocks were obtained with tissue samples from 189 patients, and triplicate 0.6 mm cores were assembled into tissue microarrays (TMAs). Fluorescence immunohistochemistry and AQUAnalysis® were used to quantify the expression of tumoral CAIX (tCAIX) and stromal CAIX (sCAIX) and tumoral Ki-67 for each TMA core. Hazard ratios for DSS were calculated using Cox proportional hazards analysis. High tCAIX and sCAIX expression levels were associated with reduced DSS (aHR=1.003, 95% CI:1.00-1.01 and aHR=1.010, 95% CI:1.001-1.019, per AQUA score unit, respectively). Ki-67 expression was not associated with survival (aHR=1.01, 95% CI:0.99-1.02) in the FHCRC cohort. DSS for patients with high sCAIX and low Ki-67 did not differ from that of other patient groups. Elevated tCAIX was associated with reduced DSS as a continuous and as a dichotomized (75%) variable. sCAIX was associated with DSS as a continuous variable but not when dichotomized (75%). However, the previously proposed 'functional hypoxia' signature was not replicated in the current FHCRC study. The failure to replicate our prior observation of poorer survival in patients with combined high sCAIX and low tumoral Ki-67 was likely due to the absence of an association between tumoral Ki-67 and DSS in this cohort. However, the association between DSS and tCAIX and sCAIX supports a role for CAIX in OSCC clinical outcomes.
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Metastasis is the ultimate cause of breast cancer related mortality. Epithelial-mesenchymal transition (EMT) is thought to play a crucial role in the metastatic potential of breast cancer. Growing evidence has implicated the SUMO E3 ligase PIAS1 in the regulation of EMT in mammary epithelial cells and breast cancer metastasis. However, the relevance of PIAS1 in human cancer and mechanisms by which PIAS1 might regulate breast cancer metastasis remain to be elucidated. Using tissue-microarray analysis (TMA), we report that the protein abundance and subcellular localization of PIAS1 correlate with disease specific overall survival of a cohort of breast cancer patients. In mechanistic studies, we find that PIAS1 acts via sumoylation of the transcriptional regulator SnoN to suppress invasive growth of MDA-MB-231 human breast cancer cell-derived organoids. Our studies thus identify the SUMO E3 ligase PIAS1 as a prognostic biomarker in breast cancer, and suggest a potential role for the PIAS1-SnoN sumoylation pathway in controlling breast cancer metastasis.
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Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/enzimologia , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Estudos de Coortes , Feminino , Células HEK293 , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , Organoides/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Sumoilação/efeitos dos fármacos , Análise de Sobrevida , Análise Serial de Tecidos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Several of the cancer immunotherapies under investigation or in clinical use target the programmed death-ligand 1/programmed death-1 (PD-L1/PD-1) signaling axis. PD-L1 expression in tumor samples has been used as a predictive marker for response to these therapeutics, and may also have independent prognostic utility when assessed along with immune cell markers. Our objectives were to assess the expression of PD-L1 in tumor specimens from a uniformly treated patient cohort with locally advanced cervical cancer, and to determine its prognostic significance along with the density of tumor-infiltrating T cells. We identified 120 patients with locally advanced cervical cancer treated with radical chemoradiotherapy, and built tissue microarrays from their formalin-fixed, paraffin-embedded pre-treatment biopsies. We used conventional brightfield and fluorescence immunohistochemistry to detect PD-L1, and quantified protein expression using both manual pathologist scoring and automated software analysis. We also evaluated the effect of PD-L1 expression in tumors, along with the presence and density of intra-tumoral CD8+ T cells, on patient survival outcomes. Approximately 96% of the tumor samples expressed PD-L1, as determined using quantitative software analysis. Neither expression of PD-L1 nor density of CD8+ T cells was associated with progression-free or overall survival. However, there was a trend towards worse progression-free survival in patients whose tumors expressed PD-L1 but lacked CD8+ T cells (hazard ratio=0.43 (0.18-1.01), P=0.053). Nevertheless, the high percentage of cervical cancer tumor samples expressing PD-L1 suggests that anti-PD-L1 or anti-PD-1 therapies are potential treatment options for this patient population.
Assuntos
Adenocarcinoma/metabolismo , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Carcinoma de Células Escamosas/metabolismo , Colo do Útero/metabolismo , Neoplasias do Colo do Útero/metabolismo , Adenocarcinoma/imunologia , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Colo do Útero/imunologia , Colo do Útero/patologia , Intervalo Livre de Doença , Feminino , Humanos , Linfócitos do Interstício Tumoral/imunologia , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Análise Serial de Tecidos , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologiaRESUMO
INTRODUCTION: This study was designed to investigate the combined influence of ATM and Ki67 on clinical outcome in early stage hormone receptor positive breast cancer (ES-HPBC), particularly in patients with smaller tumors (< 4 cm) and fewer than four positive lymph nodes. METHODS: 532 formalin-fixed paraffin-embedded specimens of resected primary breast tumors were used to construct a tissue microarray. Samples from 297 patients were suitable for final statistical analysis. We detected ATM and Ki67 proteins using fluorescence and brightfield immunohistochemistry respectively, and quantified their expression with digital image analysis. Data on expression levels were subsequently correlated with clinical outcome. RESULTS: Remarkably, ATM expression was useful to stratify the low Ki67 group into subgroups with better or poorer prognosis. Specifically, in the low Ki67 subgroup defined as having smaller tumors and no positive nodes, patients with high ATM expression showed better outcome than those with low ATM, with estimated survival rates of 96% and 89% respectively at 15 years follow up (p = 0.04). Similarly, low-Ki67 patients with smaller tumors, 1-3 positive nodes and high ATM also had significantly better outcomes than their low ATM counterparts, with estimated survival rates of 88% and 46% respectively (p = 0.03) at 15 years follow up. Multivariable analysis indicated that the combination of high ATM and low Ki67 is prognostic of improved survival, independent of tumor size, grade, and lymph node status (p = 0.02). CONCLUSIONS: These data suggest that the prognostic value of Ki67 can be improved by analyzing ATM expression in ES-HPBC.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/análise , Neoplasias da Mama/patologia , Antígeno Ki-67/análise , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/química , Neoplasias da Mama/mortalidade , Feminino , Humanos , Pessoa de Meia-Idade , Prognóstico , Estudos RetrospectivosRESUMO
INTRODUCTION: The serine/threonine protein kinase ataxia telangiectasia mutated (ATM) is critical in maintaining genomic integrity. Upon DNA double-strand breaks, ATM phosphorylates key downstream proteins including p53 and BRCA1/2, thereby orchestrating complex signaling pathways involved in cell cycle arrest, DNA repair, senescence and apoptosis. Although sporadic mutation of ATM occurs rarely in breast cancer, the status of its protein expression and its clinical significance in breast cancer remain not well established. Our study was designed to investigate the influence of ATM protein in both tumor and cancer-associated stroma on clinical outcome in hormone-positive (HPBC) and hormone-negative (HNBC) early-stage breast cancer (EBC). METHODS: Tissue microarrays (TMAs), containing formalin-fixed, paraffin-embedded resected tumors from two cohorts of patients (HPBC cohort: n=130; HNBC cohort: n=168) diagnosed at the Tom Baker Cancer Centre, Calgary, Canada, were analyzed for ATM protein expression using fluorescence immunohistochemistry (IHC) and automated quantitative analysis (AQUA). ATM expression levels were measured within the tumor as a whole (tATM) as indicated by pan-cytokeratin expression, tumor nuclear compartment (nATM) as indicated by both DAPI and pan-cytokeratin-positive results, and cancer-associated stroma (csATM) as indicated by vimentin-positive and pan-cytokeratin-negative results. ATM expression levels within these compartments were correlated with clinical outcome. RESULTS: While tATM and nATM were significantly lower in tumors compared to normal breast epithelial tissues, csATM was significantly higher than the corresponding normal tissue compartment. In addition, the median expression level of both tATM and nATM were two- to threefold lower (P<0.001) in HNBC than in HPBC. In both HNBC and HPBC cohorts, patients with low tATM, nATM and csATM tumors had significantly poorer survival outcomes than those with a high tATM, nATM and csATM, but this effect was more pronounced in HNBC. A multivariate analysis demonstrates that these biomarkers predict survival independent of tumor size and lymph node status, but only in the HNBC cohort (P<0.001). CONCLUSIONS: Low ATM protein expression in both malignant tumor and stromal compartments likely contributes to the aggressive nature of breast cancer and is an independent prognostic factor associated with worse survival in HNBC patients.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptores de Estrogênio/deficiência , Receptores de Progesterona/deficiência , Células Estromais/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Avaliação de Resultados da Assistência ao Paciente , Prognóstico , Receptor ErbB-2/metabolismo , Estudos Retrospectivos , Carga TumoralRESUMO
Mammalian skeletal muscle maintains a robust regenerative capacity throughout life, largely due to the presence of a stem cell population known as "satellite cells" in the muscle milieu. In normal conditions, these cells remain quiescent; they are activated upon injury to become myoblasts, which proliferate extensively and eventually differentiate and fuse to form new multinucleated muscle fibers. Recent findings have identified some of the factors, including the cytokine TNFα-like weak inducer of apoptosis (TWEAK), which govern these cells' decisions to proliferate, differentiate, or fuse. In this review, we will address the functions of TWEAK, its receptor Fn14, and the associated signal transduction molecule, the cellular inhibitor of apoptosis 1 (cIAP1), in the regulation of myogenesis. TWEAK signaling can activate the canonical NF-κB signaling pathway, which promotes myoblast proliferation and inhibits myogenesis. In addition, TWEAK activates the non-canonical NF-κB pathway, which, in contrast, promotes myogenesis by increasing myoblast fusion. Both pathways are regulated by cIAP1, which is an essential component of downstream signaling mediated by TWEAK and similar cytokines. This review will focus on the seemingly contradictory roles played by TWEAK during muscle regeneration, by highlighting the interplay between the two NF-κB pathways under physiological and pathological conditions. We will also discuss how myogenesis is negatively affected by chronic conditions, which affect homeostasis of the skeletal muscle environment.
RESUMO
The cellular inhibitor of apoptosis 1 (cIAP1) protein is an essential regulator of canonical and noncanonical nuclear factor κB (NF-κB) signaling pathways. NF-κB signaling is known to play important roles in myogenesis and degenerative muscle disorders such as Duchenne muscular dystrophy (DMD), but the involvement of cIAP1 in muscle disease has not been studied directly. Here, we asked whether the loss of cIAP1 would influence the pathology of skeletal muscle in the mdx mouse model of DMD. Double-mutant cIAP1(-/-);mdx mice exhibited reduced muscle damage and decreased fiber centronucleation in the soleus, compared with single-mutant cIAP1(+/+);mdx mice. This improvement in pathology was associated with a reduction in muscle infiltration by macrophages and diminished expression of inflammatory cytokines such as IL-6 and tumor necrosis factor-α. Furthermore, the cIAP1(-/-);mdx mice exhibited reduced serum creatine kinase, and improved exercise endurance associated with improved exercise resilience by the diaphragm. Mechanistically, the loss of cIAP1 was sufficient to drive constitutive activation of the noncanonical NF-κB pathway, which led to increased myoblast fusion in vitro and in vivo. Collectively, these results show that the loss of cIAP1 protects skeletal muscle from the degenerative pathology resulting from systemic loss of dystrophin.
Assuntos
Proteínas Inibidoras de Apoptose/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , NF-kappa B/metabolismo , Animais , Creatina Quinase/sangue , Diafragma/metabolismo , Diafragma/fisiopatologia , Distrofina/genética , Distrofina/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos mdx , Desenvolvimento Muscular/genética , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/fisiopatologia , NF-kappa B/genética , Resistência Física/genética , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The fusion of mononucleated muscle progenitor cells (myoblasts) into multinucleated muscle fibers is a critical aspect of muscle development and regeneration. We identified the noncanonical nuclear factor κB (NF-κB) pathway as a signaling axis that drives the recruitment of myoblasts into new muscle fibers. Loss of cellular inhibitor of apoptosis 1 (cIAP1) protein led to constitutive activation of the noncanonical NF-κB pathway and an increase in the number of nuclei per myotube. Knockdown of essential mediators of NF-κB signaling, such as p100, RelB, inhibitor of κB kinase α, and NF-κB-inducing kinase, attenuated myoblast fusion in wild-type myoblasts. In contrast, the extent of myoblast fusion was increased when the activity of the noncanonical NF-κB pathway was enhanced by increasing the abundance of p52 and RelB or decreasing the abundance of tumor necrosis factor (TNF) receptor-associated factor 3, an inhibitor of this pathway. Low concentrations of the cytokine TNF-like weak inducer of apoptosis (TWEAK), which preferentially activates the noncanonical NF-κB pathway, also increased myoblast fusion, without causing atrophy or impairing myogenesis. These results identify roles for TWEAK, cIAP1, and noncanonical NF-κB signaling in the regulation of myoblast fusion and highlight a role for cytokine signaling during adult skeletal myogenesis.
Assuntos
Regulação da Expressão Gênica , Proteínas Inibidoras de Apoptose/fisiologia , Mioblastos/metabolismo , NF-kappa B/metabolismo , Fatores de Necrose Tumoral/fisiologia , Animais , Bromodesoxiuridina/farmacologia , Linhagem Celular , Citocina TWEAK , Genótipo , Proteínas Inibidoras de Apoptose/genética , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Músculo Esquelético/metabolismo , Músculos/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Necrose Tumoral/genéticaRESUMO
The regulation of female reproductive behaviors may involve memories of male pheromone signatures, formed in part by neural circuitry involving the olfactory bulb and hippocampus. These neural structures are the principal sites of adult neurogenesis; however, previous studies point to their independent regulation by sensory and physiological stimuli. Here we report that the pheromones of dominant (but not subordinate) males stimulate neuronal production in both the olfactory bulb and hippocampus of female mice, which are independently mediated by prolactin and luteinizing hormone, respectively. Neurogenesis induced by dominant-male pheromones correlates with a female preference for dominant males over subordinate males, whereas blocking neurogenesis with the mitotic inhibitor cytosine arabinoside eliminated this preference. These results suggest that male pheromones are involved in regulating neurogenesis in both the olfactory bulb and hippocampus, which may be important for female reproductive success.
Assuntos
Encéfalo/citologia , Proliferação de Células/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Atrativos Sexuais/farmacologia , Comportamento Sexual Animal/efeitos dos fármacos , Animais , Adstringentes/toxicidade , Comportamento Animal , Encéfalo/efeitos dos fármacos , Bromodesoxiuridina/metabolismo , Citarabina/farmacologia , Feminino , Imunossupressores/farmacologia , Marcação In Situ das Extremidades Cortadas/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Receptores do LH/deficiência , Receptores da Prolactina/deficiência , Comportamento Sexual Animal/fisiologia , Predomínio Social , Sulfato de Zinco/toxicidadeRESUMO
Previous studies demonstrating olfactory interneuron involvement in olfactory discrimination and decreased proliferation in the forebrain subventricular zone with age led us to ask whether olfactory neurogenesis and, consequently, olfactory discrimination were impaired in aged mice. Pulse labeling showed that aged mice (24 months of age) had fewer new interneurons in the olfactory bulb than did young adult (2 months of age) mice. However, the aged mice had more olfactory interneurons in total than their younger counterparts. Aged mice exhibited no differences from young adult mice in their ability to discriminate between two discrete odors but were significantly poorer at performing discriminations between similar odors (fine olfactory discrimination). Leukemia inhibitory factor receptor heterozygote mice, which have less neurogenesis and fewer olfactory interneurons than their wild-type counterparts, performed more poorly at fine olfactory discrimination than the wild types, suggesting that olfactory neurogenesis, rather than the total number of interneurons, was responsible for fine olfactory discrimination. Immunohistochemistry and Western blot analyses revealed a selective reduction in expression levels of epidermal growth factor (EGF) receptor (EGFR) signaling elements in the aged forebrain subventricular zone. Waved-1 mutant mice, which express reduced quantities of transforming growth factor-alpha, the predominant EGFR ligand in adulthood, phenocopy aged mice in olfactory neurogenesis and performance on fine olfactory discrimination tasks. These results suggest that the impairment in fine olfactory discrimination with age may result from a reduction in EGF-dependent olfactory neurogenesis.