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A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2-µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥104 colony-forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 104 CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality.
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Anticuerpos Monoclonales , Productos Biológicos , Reactores Biológicos/microbiología , Técnicas de Cultivo de Célula/normas , Mycoplasma , Animales , Productos Biológicos/análisis , Productos Biológicos/normas , Células CHO/microbiología , Cricetinae , Cricetulus , Contaminación de Medicamentos , Estadística como AsuntoRESUMEN
Mycoplasma contamination events in biomanufacturing facilities can result in loss of production and costly cleanups. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and may penetrate the 0.2 µm filters often used in the primary clarification of harvested cell culture fluid. Culture cell-based and indicator cell-based assays that are used to detect mycoplasma are highly sensitive but can take up to 28 days to complete and cannot be used for real-time decision making during the biomanufacturing process. To support real-time measurements of mycoplasma contamination, there is a push to explore nucleic acid testing. However, cell-based methods measure growth or colony forming units and nucleic acid testing measures genome copy number; this has led to ambiguity regarding how to compare the sensitivity of the methods. In addition, the high risk of conducting experiments wherein one deliberately spikes mycoplasma into bioreactors has dissuaded commercial groups from performing studies to explore the multiple variables associated with the upstream effects of a mycoplasma contamination in a manufacturing setting. Here we studied the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G1 (IgG1) antibody. We examined M. arginini growth and detection by culture methods, as well as the effects of M. arginini on mammalian cell health, metabolism, and productivity. We compared process parameters and controls normally measured in bioreactors including dissolved oxygen, gas mix, and base addition to maintain pH, to examine parameter changes as potential indicators of contamination. Our work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Importantly, how the M. arginini contamination impacts the CHO cells is influenced by the concentration of CHO cells and rate of perfusion at the time of M. arginini spike. Careful evaluation of dissolved oxygen, pH control parameters, ammonia, and arginine over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before a read-out from a traditional method.
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Reactores Biológicos/microbiología , Técnicas de Cultivo de Célula , Contaminación de Equipos , Mycoplasma/crecimiento & desarrollo , Animales , Células CHO , CricetulusRESUMEN
PURPOSE: The impact of diabetes mellitus (DM) and metformin use on biochemical recurrence (BCR) in patients treated with radical prostatectomy (RP) remains controversial. METHODS: We retrospectively evaluated 6,863 patients who underwent RP for clinically localized PC between 2000 and 2011. Univariable and multivariable Cox regression models addressed the association of DM and metformin use with BCR. RESULTS: Overall, 664 patients had a diagnosis of DM from which 287 (43 %) were on metformin and 377 (57 %) were on anti-diabetics other than metformin. DM and metformin were not associated with any clinicopathologic features (p values >0.05). Within a median follow-up of 25 months (interquartile range 35 months), 774 (11.3 %) patients experienced BCR. Actuarial 5-year biochemical-free survival was 83 % for non-diabetic, 79 % for diabetic patients without metformin use, and 85 % for diabetic patients with metformin use (log rank p = 0.17). In uni- and multivariable Cox regression analyses with the non-diabetic group as referent, DM without metformin use (HR = 0.99; 95 % CI 0.75-1.30, p = 0.65) and DM with metformin use (HR = 0.84, 95 % CI 0.58-1.22, p = 0.36) were not associated with BCR after RP. A subgroup analysis stratified by nodal status, surgical margins, tumor stage, and Gleason sum did not reveal any significant association between DM, use of metformin and risk of BCR. CONCLUSIONS: We found no association between DM or metformin use and cancer-specific features or BCR in patients treated with RP. The effect of DM and metformin on complications, wound healing and overall survival needs to be assessed in similar cohorts.
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Diabetes Mellitus/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Metformina/uso terapéutico , Prostatectomía , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/cirugía , Anciano , Estudios de Seguimiento , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Persona de Mediana Edad , Antígeno Prostático Específico/sangre , Recurrencia , Análisis de Regresión , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
PURPOSE: The role of lymph node dissection is still controversial in patients treated with radical nephroureterectomy for upper tract urothelial cancer. We developed a pathological nodal staging model that allows quantification of the likelihood that a patient with pathologically node negative disease has, indeed, no lymph node metastasis. MATERIALS AND METHODS: We analyzed data on 814 patients treated with radical nephroureterectomy and lymph node dissection, and estimated the sensitivity of pathological nodal staging using a ß-binomial model. We developed a pathological nodal staging score that represents the probability that a case is correctly staged as node negative. RESULTS: A median of 5 lymph nodes (range 1 to 46) was removed and 593 patients (73%) had pN0 disease. The probability of missing lymph node metastasis decreased as the number of nodes examined increased. If only a single node was examined, 44% of patients would have been misclassified as having pN0 disease while harboring lymph node metastasis. Even when 5 nodes were examined, 12% of patients would have been misclassified. The proportion of those with a positive node increased with advancing pathological T stage and lymphovascular invasion. Patients with pT0-Ta-Tis-T1/lymphovascular invasion had more than a 95% chance of correct pathological nodal staging with 2 examined nodes. However, if a patient had pT3-T4 and positive lymphovascular invasion, even 20 examined lymph nodes did not attain 95% accuracy. CONCLUSIONS: Lymph node dissection provides more accurate staging and prediction of survival. The number of examined nodes needed for adequate staging depends on pT stage and lymphovascular invasion. We developed a tool to estimate the likelihood of false-negative lymph node metastasis, which could help refine clinical decision making regarding the administration of adjuvant chemotherapy.
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Carcinoma de Células Transicionales/patología , Carcinoma de Células Transicionales/cirugía , Neoplasias Renales/patología , Neoplasias Renales/cirugía , Nefrectomía , Uréter/cirugía , Neoplasias Ureterales/patología , Neoplasias Ureterales/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Metástasis Linfática/patología , Masculino , Persona de Mediana Edad , Nefrectomía/métodos , Pronóstico , Estudios RetrospectivosRESUMEN
OBJECTIVE: To assess the association between diabetes mellitus (DM) and metformin use with prognosis and outcomes of non-muscle-invasive bladder cancer (NMIBC) PATIENTS AND METHODS: We retrospectively evaluated 1117 patients with NMIBC treated at four institutions between 1996 and 2007. Cox regression models were used to analyse the association of DM and metformin use with disease recurrence, disease progression, cancer-specific mortality and any-cause mortality. RESULTS: Of the 1117 patients, 125 (11.1%) had DM and 43 (3.8%) used metformin. Within a median (interquartile range) follow-up of 64 (22-106) months, 469 (42.0%) patients experienced disease recurrence, 103 (9.2%) experienced disease progression, 50 (4.5%) died from bladder cancer and 249 (22.3%) died from other causes. In multivariable Cox regression analyses, patients with DM who did not take metformin had a greater risk of disease recurrence (hazard ratio [HR]: 1.45, 95% confidence interval [CI] 1.09-1.94, P = 0.01) and progression (HR: 2.38, 95% CI 1.40-4.06, P = 0.001) but not any-cause mortality than patients without DM. DM with metformin use was independently associated with a lower risk of disease recurrence (HR: 0.50, 95% CI 0.27-0.94, P = 0.03). CONCLUSION: Patients with DM and NMIBC who do not take metformin seem to be at an increased risk of disease recurrence and progression; metformin use seems to exert a protective effect with regard to disease recurrence. The mechanisms behind the impact of DM on patients with NMIBC and the potential protective effect of metformin need further elucidation.
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Diabetes Mellitus/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Metformina/uso terapéutico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Diabetes Mellitus/mortalidad , Diabetes Mellitus/fisiopatología , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Hipoglucemiantes/farmacología , Masculino , Metformina/farmacología , Persona de Mediana Edad , Invasividad Neoplásica , Recurrencia Local de Neoplasia/mortalidad , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Resultado del Tratamiento , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/fisiopatologíaRESUMEN
OBJECTIVES: To identify clinicopathological factors that predict outcomes in patients with a single lymph node (LN) metastasis (pN1) treated with radical cystectomy (RC) for urothelial carcinoma of the bladder (UCB). LN metastasis is an established predictor of clinical outcomes in patients. While most patients with large LN burden experience disease recurrence, lymphadenectomy can be curative in patients with pN1 disease. PATIENTS AND METHODS: We analysed 381 patients with pN1 UCB from a multi-institutional cohort of 4335 patients with UCB treated with RC and lymphadenectomy without preoperative chemo- or radiotherapy. Subgroup analyses were performed for patients with ≥9 LNs removed and according to adjuvant chemotherapy administration (n = 215). RESULTS: The median (interquartile range, IQR) LN number was 15 (19) and the median (IQR) LN density was 6.7 (7.5)%. Within a median follow-up of 41 months, the mean (+/- SD) 2- and 5-year cancer-specific survival (CSS) rates were 55 (3)% and 46 (3)%, respectively. On multivariable analysis that adjusted for the effects of standard clinicopathological features, female gender (hazard ratio [HR] 1.48, P = 0.023), higher tumour stage (HR 1.68, P = 0.007), positive soft tissue surgical margin (STSM; HR 2.06, P = 0.004), higher LN density (HR 2.99, P = 0.025) and absence of adjuvant chemotherapy (HR 0.70, P = 0.026) were independently associated with CSS. In subgroup analyses of patients with ≥9 LNs removed, tumour stage and STSM status remained independent predictors for CSS (P = 0.009 and P < 0.001, respectively). CONCLUSIONS: About half of the patients with pN1 UCB died from UCB within 5 years of RC. Pathological stage and STSM status are strong predictors for outcomes. Accurate prediction of the individual risk of CSS may help risk stratifying pN1 UCB in order to help improve clinical-decision making. Patients with pN1 UCB presenting with additional unfavourable risk factors need a closer follow-up scheduling and might receive adjuvant therapy.
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Carcinoma in Situ/cirugía , Cistectomía/métodos , Neoplasias de la Vejiga Urinaria/cirugía , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma in Situ/tratamiento farmacológico , Carcinoma in Situ/mortalidad , Carcinoma in Situ/patología , Quimioterapia Adyuvante/mortalidad , Niño , Preescolar , Cistectomía/mortalidad , Métodos Epidemiológicos , Femenino , Humanos , Lactante , Escisión del Ganglio Linfático/mortalidad , Metástasis Linfática , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/mortalidad , Resultado del Tratamiento , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/patología , Adulto JovenRESUMEN
Bioreactor parameters can have significant effects on the quantity and quality of biotherapeutics. Monoclonal antibody products have one particularly important critical quality attribute being the distribution of product glycoforms. N-linked glycosylation affects the therapeutic properties of the antibody including effector function, immunogenicity, stability, and clearance rate. Our past work revealed that feeding different amino acids to bioreactors altered the productivity and glycan profiles. To facilitate real-time analysis of bioreactor parameters and the glycosylation of antibody products, we developed an on-line system to pull cell-free samples directly from the bioreactors, chemically process them, and deliver them to a chromatography-mass spectroscopy system for rapid identification and quantification. We were able to successfully monitor amino acid concentration on-line within multiple reactors, evaluate glycans off-line, and extract four principal components to assess the amino acid concentration and glycosylation profile relationship. We found that about a third of the variability in the glycosylation data can be predicted from the amino acid concentration. Additionally, we determined that the third and fourth principal component accounts for 72% of our model's predictive power, with the third component indicated to be positively correlated with latent metabolic processes related to galactosylation. Here we present our work on rapid online spent media amino acid analysis and use the determined trends to collate with glycan time progression, further elucidating the correlation between bioreactor parameters such as amino acid nutrient profiles, and product quality. We believe such approaches may be useful for maximizing efficiency and reducing production costs for biotherapeutics.
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Aminoácidos , Anticuerpos Monoclonales , Anticuerpos Monoclonales/química , Glicosilación , Aminoácidos/metabolismo , Reactores Biológicos , Polisacáridos/químicaRESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (Covid-19) pandemic, continues to evolve and circulate globally. Current prophylactic and therapeutic countermeasures against Covid-19 infection include vaccines, small molecule drugs, and neutralizing monoclonal antibodies. SARS-CoV-2 infection is mainly mediated by the viral spike glycoprotein binding to angiotensin converting enzyme 2 (ACE2) on host cells for viral entry. As emerging mutations in the spike protein evade efficacy of spike-targeted countermeasures, a potential strategy to counter SARS-CoV-2 infection is to competitively block the spike protein from binding to the host ACE2 using a soluble recombinant fusion protein that contains a human ACE2 and an IgG1-Fc domain (ACE2-Fc). Here, we have established Chinese Hamster Ovary (CHO) cell lines that stably express ACE2-Fc proteins in which the ACE2 domain either has or has no catalytic activity. The fusion proteins were produced and purified to partially characterize physicochemical properties and spike protein binding. Our results demonstrate the ACE2-Fc fusion proteins are heavily N-glycosylated, sensitive to thermal stress, and actively bind to five spike protein variants (parental, alpha, beta, delta, and omicron) with different affinity. Our data demonstrates a proof-of-concept production strategy for ACE2-Fc fusion glycoproteins that can bind to different spike protein variants to support the manufacture of potential alternative countermeasures for emerging SARS-CoV-2 variants.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Animales , Cricetinae , Humanos , Células CHO , Cricetulus , Glicoproteínas , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Most therapeutic proteins are glycosylated with N-glycans and/or O-glycans. N-glycans on therapeutic proteins have been extensively studied for their control strategy and impact on drug product quality. However, knowledge of O-glycosylation in therapeutic protein production and its impact on product quality remains elusive. To address this gap, we generated an O-glycoengineered Chinese Hamster Ovary (CHO) cell line platform to modulate O-glycosylation of therapeutic proteins and investigated the impact of O-glycans on the physicochemical and biological properties of etanercept. Our results demonstrate that this CHO cell line platform produces controlled O-glycosylation profiles containing either truncated O-glycans (sialylTn and/or Tn), or sialylCore 3 alone, or sialylCore 1 with sialylTn or sialylCore 3 O-glycans on endogenous and recombinant proteins. Moreover, the platform demonstrated exclusive modulation of O-glycosylation without affecting N-glycosylation. Importantly, certain O-glycans on etanercept enhanced tumor necrosis factor-α binding affinity and consequent potency. This is the first report that describes the systematic establishment of an O-glycoengineered CHO cell line platform with direct evidence that supports the applicability of the platform in the production of engineered proteins with desired O-glycans. This platform is valuable for identifying O-glycosylation as a critical quality attribute of biotherapeutics using the quality by design principle.
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Capture bioprocessing unit operations were previously shown to clear or kill several log10 of a model mycoplasma Acholeplasma laidlawii in lab-scale spike/removal studies. Here, we confirm this observation with two additional mollicute species relevant to biotechnology products for human use: Mycoplasma orale and Mycoplasma arginini Clearance of M. orale and M. arginini from protein A column purification was similar to that seen with A. laidlawii, though some between cycle carryover was evident, especially for M. orale However, on-resin growth studies for all three species revealed that residual mycoplasma in a column slowly die off over time rather than expanding further. Solvent/detergent exposure completely inactivated M. arginini though detectable levels of M. orale remained. A small-scale model of a commercial low-pH hold step did inactivate live M. orale, but this inactivation required a lower pH set point and occurred with slower kinetics than previously seen with A. laidlawii Additionally, ultraviolet-C irradiation was shown to be effective for A. laidlawii and M. orale inactivation whereas virus-retentive filters for upstream and downstream processes, as expected, cleared A. laidlawii These data argue that M. orale and M. arginini overall would be largely cleared by early bioprocessing steps as shown previously for A. laidlawii, and that barrier technologies can effectively reduce the risk from media components. For some unit operations, M. orale and M. arginini may be hardier, and require more stringent processing or equipment cleaning conditions to assure effective mycoplasma reduction. By exploring how some of the failure modes in commercial antibody manufacturing processes can still eliminate mycoplasma burden, we demonstrate that required best practices assure biotechnology products will be safe for patients.
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Química Farmacéutica/métodos , Contaminación de Medicamentos/prevención & control , Mycoplasma orale/aislamiento & purificación , Mycoplasma/aislamiento & purificación , Animales , Células CHO , Técnicas de Cocultivo , Cricetinae , Cricetulus , Mycoplasma/crecimiento & desarrollo , Mycoplasma orale/crecimiento & desarrolloRESUMEN
Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.
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Anticuerpos Monoclonales/aislamiento & purificación , Reactores Biológicos , Aminoácidos/análisis , Aminoácidos/metabolismo , Animales , Automatización , Células CHO , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , Electroforesis Capilar , Fluorescencia , Glicosilación , Inmunoglobulina G/aislamiento & purificación , Espectrometría de Masas , Peso Molecular , Polisacáridos/metabolismoRESUMEN
Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts, media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE's with a higher number of variables and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.
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Automatización , Técnicas de Cultivo Celular por Lotes/métodos , Reactores Biológicos , Inmunoglobulina G/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , MiniaturizaciónRESUMEN
OBJECTIVES: Evidence suggests a positive effect of metformin on cancer incidence and outcome. To date, the effect of metformin use on prognosis in urothelial carcinoma of the bladder (UCB) remains uninvestigated. We tested the hypothesis that metformin use affects oncologic outcomes of patients treated with radical cystectomy for UCB. METHODS AND MATERIALS: We retrospectively evaluated 1,502 patients treated at 4 institutions with radical cystectomy and pelvic lymphadenectomy without neoadjuvant therapy. Cox regression models addressed the association of diabetes mellitus (DM) and metformin use with disease recurrence, cancer-specific mortality, and any-cause mortality. RESULTS: A total of 200 patients (13.3%) had DM, 80 patients (5.3%) used metformin. Within a median follow-up of 34 months, 509 patients (33.9%) experienced disease recurrence, 402 patients (26.8%) died of UCB, and 551 patients (36.7%) died from any cause. In univariable Cox regression analyses, DM without metformin use was associated with increased risk of disease recurrence (hazard ratio [HR]: 1.40, 95% confidence interval [CI] 1.05-1.87, P = 0.02), cancer-specific mortality (HR: 1.60, 95% CI 1.17-2.17, P = 0.003), and any-cause mortality (HR: 1.55, 95% CI 1.18-2.03, P = 0.002), whereas metformin use was associated with decreased risk of disease recurrence (HR: 0.61, 95% CI 0.37-0.98, P = 0.04), cancer-specific mortality (HR: 0.56, 95% CI 0.33-0.97, P = 0.04), and any-cause mortality (HR: 0.54, 95% CI 0.33-0.88, P = 0.01). In multivariable Cox regression analyses, DM treated without metformin use remained associated with worse cancer-specific mortality (HR: 1.53, 95% CI 1.12-2.09, P = 0.007) and any-cause mortality (HR: 1.52, 95% CI 1.16-2.00, P = 0.003) but not disease recurrence. CONCLUSIONS: Diabetic patients who do not use metformin appear to be at higher risk of cancer-specific and any-cause mortality than patients without DM. It remains unclear, whether the severity of DM in this group of patients or the use of metformin itself affects outcomes of UCB. The mechanisms behind the effect of DM on patients with UCB and the potential protective effect of metformin need further elucidation.
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Carcinoma de Células Transicionales/cirugía , Cistectomía/métodos , Diabetes Mellitus/tratamiento farmacológico , Metformina/uso terapéutico , Neoplasias de la Vejiga Urinaria/cirugía , Anciano , Carcinoma de Células Transicionales/mortalidad , Causas de Muerte , Femenino , Estudios de Seguimiento , Humanos , Hipoglucemiantes/uso terapéutico , Estimación de Kaplan-Meier , Escisión del Ganglio Linfático , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Evaluación de Resultado en la Atención de Salud/métodos , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Pelvis , Modelos de Riesgos Proporcionales , Análisis de Regresión , Factores de Riesgo , Tasa de Supervivencia , Neoplasias de la Vejiga Urinaria/mortalidadRESUMEN
BACKGROUND: Few studies have investigated the natural history of TaG1 urothelial carcinoma of the bladder (UCB). OBJECTIVE: To assess the long-term outcomes of patients with TaG1 UCB and the impact of immediate postoperative instillation of chemotherapy (IPIC). DESIGN, SETTING, AND PARTICIPANTS: A retrospective analysis of 1447 patients with TaG1 UCB treated between 1996 and 2007 at eight centers. Median follow-up was 67.2 mo (interquartile range: 67.9). Patients were stratified into three European Association of Urology (EAU) guidelines risk categories; high-risk patients (n=11) were excluded. INTERVENTION: Transurethral resection of the bladder with or without IPIC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Univariable and multivariable Cox regression models addressed factors associated with disease recurrence, disease progression, death of disease, and any-cause death. RESULTS AND LIMITATIONS: Of the 1436 patients, 601 (41.9%) and 835 (58.1%) were assigned to low- and intermediate-risk categories, respectively. The actuarial estimate of 5-yr recurrence-free survival was 56% (standard error: ± 1). Advancing age (p=0.04), tumor >3 cm (p=0.001), multiple tumors (p<0.001), and recurrent tumors (p<0.001) were independently associated with increased risk of disease recurrence, whereas IPIC was associated with decreased risk (p=0.001). The actuarial estimate of 5-yr progression-free survival was 95% ± 1. Advancing age (p<0.001) and multiple tumors (p=0.01) were independent risk factors for disease progression. Five-year cancer-specific survival was 98% ± 1. Advancing age (p=0.001) and previous recurrence (p=0.04) were associated with increased risk, whereas female gender (p=0.02) was associated with decreased risk of cancer-specific mortality. Compared with low-risk patients, intermediate-risk patients were at significantly higher risk of disease recurrence, disease progression, and cancer-specific mortality (all p<0.01). Limitations include the retrospective design of the study and the lack of a central pathology review. CONCLUSIONS: TaG1 UCB patients experience heterogeneous risks of disease recurrence. We validated the EAU guidelines risk stratification in TaG1 UCB patients. IPIC was associated with a reduced risk of disease recurrence in patients with low- and intermediate-risk TaG1 UCB.
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Carcinoma de Células Transicionales/tratamiento farmacológico , Carcinoma de Células Transicionales/cirugía , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/cirugía , Anciano , Carcinoma de Células Transicionales/patología , Terapia Combinada/métodos , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Estudios Retrospectivos , Factores de Tiempo , Resultado del Tratamiento , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
OBJECTIVE: To investigate the impact of variant histologies of urothelial carcinoma of the bladder (UCB) on oncologic outcomes after radical cystectomy (RC). MATERIALS AND METHODS: Data from 1984 UCB patients treated by RC without preoperative chemo- or radiotherapy were reviewed for histological differentiation and variants. We analysed the differences between pure UCB and UCB with variant histology, and those between the different histological variants using various stratifications. RESULTS: Overall, 488 (24.6%) patients had UCB variants with squamous cell (11.4%) and glandular differentiation (3.8%) being the most common. Histological UCB variants were associated with advanced tumour stage, lymphovascular invasion and lymph node metastasis (all p-values<0.01) when compared to pure UCB. In univariable analyses, patients with non-squamous UCB variants were at significantly higher risk for disease recurrence and cancer-specific mortality than those with pure UCB patients (p-values=0.001) and those with squamous cell differentiated UCB (p-values=0.04); the latter two had the same risk. In multivariable analyses that adjusted for the effects of standard clinicopathologic characteristics, variant UCB histology was not associated with both survival end-points. In patients treated with adjuvant chemotherapy (n=492) there was no difference in cancer-specific survival between pure UCB, squamous cell differentiated UCB and other histological UCB variants. CONCLUSIONS: A quarter of UCB patients treated with RC harboured histological UCB variants. Variant UCB histologies were associated with features of biologically aggressive disease. While variant UCB histology was associated with worse outcomes in univariable analyses, this effect did not remain significant in multivariable analyses.
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Carcinoma de Células Transicionales/cirugía , Cistectomía/métodos , Neoplasias de la Vejiga Urinaria/cirugía , Vejiga Urinaria/cirugía , Anciano , Carcinoma de Células Transicionales/mortalidad , Carcinoma de Células Transicionales/patología , Femenino , Humanos , Estimación de Kaplan-Meier , Metástasis Linfática , Masculino , Persona de Mediana Edad , Análisis Multivariante , Recurrencia Local de Neoplasia , Estadificación de Neoplasias , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Pronóstico , Modelos de Riesgos Proporcionales , Tasa de Supervivencia , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: Lymph node metastasis (LNM) is the most powerful pathologic predictor of disease recurrence after radical cystectomy (RC). However, the outcomes of patients with LNM are highly variable. OBJECTIVE: To assess the prognostic value of extranodal extension (ENE) and other lymph node (LN) parameters. DESIGN, SETTING, AND PARTICIPANTS: A retrospective analysis of 748 patients with urothelial carcinoma of the bladder and LNM treated with RC and lymphadenectomy without neoadjuvant therapy at 10 European and North American centers (median follow-up: 27 mo). INTERVENTION: All subjects underwent RC and bilateral pelvic lymphadenectomy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Each LNM was microscopically evaluated for the presence of ENE. The number of LNs removed, number of positive LNs, and LN density were recorded and calculated. Univariable and multivariable analyses addressed time to disease recurrence and cancer-specific mortality after RC. RESULTS AND LIMITATIONS: A total of 375 patients (50.1%) had ENE. The median number of LNs removed, number of positive LNs, and LN density were 15, 2, and 15, respectively. The rate of ENE increased with advancing pT stage (p<0.001). In multivariable Cox regression analyses that adjusted for the effects of established clinicopathologic features and LN parameters, ENE was associated with disease recurrence (hazard ratio [HR]: 1.89; 95% confidence interval [CI], 1.55-2.31; p<0.001) and cancer-specific mortality (HR: 1.90; 95% CI, 1.52-2.37; p<0.001). The addition of ENE to a multivariable model that included pT stage, tumor grade, age, gender, lymphovascular invasion, surgical margin status, LN density, number of LNs removed, number of positive LNs, and adjuvant chemotherapy improved predictive accuracy for disease recurrence and cancer-specific mortality from 70.3% to 77.8% (p<0.001) and from 71.8% to 77.8% (p=0.007), respectively. The main limitation of the study is its retrospective nature. CONCLUSIONS: ENE is an independent predictor of both cancer recurrence and cancer-specific mortality in RC patients with LNM. Knowledge of ENE status could help with patient counseling, clinical decision making regarding inclusion in clinical trials of adjuvant therapy, and tailored follow-up scheduling after RC.