RESUMEN
One of the main objectives of peptide drug design is the improvement of peptide pharmacokinetics with maintaining biological activity, which can be achieved by the complex modifications of the primary structure of the peptides. However, these changes often lead to the formation of peculiar impurities in the peptide drugs and their metabolites, which require the development of advanced analytical methods to properly assess their content. Here, we investigated the degradation of the potent long-acting GnRH antagonist degarelix in various biologic media by the tailor-made HPLC method, which allows precise determination of 5-Aph(Hyd)-degarelix isomer, an impurity found in the degarelix active pharmaceutical ingredient (API) during its manufacturing. Unexpectedly, we discovered a rapid and irreversible conversion of degarelix API into the corresponding hydantoin isomer in serum, suggesting that this impurity can be also a potential drug metabolite in vivo. This finding underlines the importance of the development of more accurate and performing analytical techniques to correctly characterize the chemical composition of the manufactured drugs and their behavior under physiological conditions.
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Hormona Liberadora de Gonadotropina , Oligopéptidos , Isomerismo , Antagonistas de HormonasRESUMEN
A multistage single arm phase II trial with binary endpoint is considered. Bayesian posterior probabilities are used to monitor futility in interim analyses and efficacy in the final analysis. For a beta-binomial model, decision rules based on Bayesian posterior probabilities are converted to "traditional" decision rules in terms of number of responders among patients observed so far. Analytical derivations are given for the probability of stopping for futility and for the probability to declare efficacy. A workflow is presented on how to select the parameters specifying the Bayesian design, and the operating characteristics of the design are investigated. It is outlined how the presented approach can be transferred to statistical models other than the beta-binomial model.
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Biometría/métodos , Ensayos Clínicos Fase II como Asunto/métodos , Inutilidad Médica , Algoritmos , Teorema de Bayes , Calibración , Humanos , Modelos Estadísticos , Probabilidad , Resultado del TratamientoRESUMEN
BACKGROUND: To assess the predictive value of early metabolic response (ΔSUV) after short-term treatment with first-line cetuximab in patients (pts) with RAS-wt metastatic colorectal cancer (mCRC). METHODS: In this prospective phase II study, RAS-wt mCRC pts received a single-agent cetuximab run-in therapy of 2 weeks. ΔSUV was assessed with FDG-PET/CT on days 0 and 14. Early clinical response (ECR) was evaluated with CT on day 56 after treatment with FOLFIRI-cetuximab. Primary endpoint was the predictive significance of ΔSUV for ECR. Secondary endpoints were PFS (progression free survival), OS and the influence of ΔSUV on survival. RESULTS: Forty pts were enroled and 33 pts were evaluable for the primary endpoint. The CT response rate was 57.6%. For responders, ΔSUV was significantly higher (p = 0.0092). A significant association of ΔSUV with ECR was found (p = 0.02). Median PFS was 11.7 months and median OS was 33.5 months with a 1-year survival rate of 87.9%. ΔSUV was found to significantly impact the hazard for OS (p = 0.045). CONCLUSIONS: We demonstrate that cetuximab induces metabolic responses in mCRC pts. The study endpoint was met with the ΔSUV discriminating between responders and non-responders. However, these data should be validated in larger patient cohorts.
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Cetuximab/administración & dosificación , Neoplasias Colorrectales/diagnóstico por imagen , Neoplasias Colorrectales/tratamiento farmacológico , Adulto , Anciano , Camptotecina/administración & dosificación , Cetuximab/efectos adversos , Cetuximab/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Fluorodesoxiglucosa F18/administración & dosificación , Fluorouracilo/administración & dosificación , Humanos , Leucovorina/administración & dosificación , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Supervivencia sin Progresión , Estudios ProspectivosRESUMEN
Although tumor-initiating cell (TIC) self-renewal has been postulated to be essential in progression and metastasis formation of human pancreatic adenocarcinoma (PDAC), clonal dynamics of TICs within PDAC tumors are yet unknown. Here, we show that long-term progression of PDAC in serial xenotransplantation is driven by a succession of transiently active TICs producing tumor cells in temporally restricted bursts. Clonal tracking of individual, genetically marked TICs revealed that individual tumors are generated by distinct sets of TICs with very little overlap between subsequent xenograft generations. An unexpected functional and phenotypic plasticity of pancreatic TICs in vivo underlies the recruitment of inactive TIC clones in serial xenografts. The observed clonal succession of TIC activity in serial xenotransplantation is in stark contrast to the continuous activity of limited numbers of self-renewing TICs within a fixed cellular hierarchy observed in other epithelial cancers and emphasizes the need to target TIC activation, rather than a fixed TIC population, in PDAC.
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Adenocarcinoma/patología , Células Madre Neoplásicas/fisiología , Neoplasias Pancreáticas/patología , Animales , Modelos Animales de Enfermedad , Femenino , Xenoinjertos , Humanos , Masculino , Ratones Endogámicos NODRESUMEN
BACKGROUND: Patients with advanced stage non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) often experience multidimensional impairments, affecting quality of life during their course of disease. In lung cancer patients with operable disease, several studies have shown that exercise has a positive impact on quality of life and physical functioning. There is limited evidence regarding efficacy for advanced lung cancer patients undergoing palliative treatment. Therefore, the POSITIVE study aims to evaluate the benefit of a 24-week exercise intervention during palliative treatment in a randomized controlled setting. METHODS/DESIGN: The POSITIVE study is a randomized, controlled trial investigating the effects of a 24-week exercise intervention during palliative treatment on quality of life, physical performance and immune function in advanced, non-operable lung cancer patients. 250 patients will be recruited in the Clinic for Thoracic Diseases in Heidelberg, enrolment begun in November 2013. Main inclusion criterion is histologically confirmed NSCLC (stage IIIa, IIIb, IV) or SCLC (Limited Disease-SCLC, Extensive Disease-SCLC) not amenable to surgery. Patients are randomized into two groups. Both groups receive weekly care management phone calls (CMPCs) with the goal to assess symptoms and side effects. Additionally, one group receives a combined resistance and endurance training (3x/week). Primary endpoints are quality of life assessed by the Functional Assessment of Cancer Therapy for patients with lung cancer (FACT-L, subcategory Physical Well-Being) and General Fatigue measured by the Multidimensional Fatigue Inventory (MFI-20). Secondary endpoints are physical performance (maximal voluntary isometric contraction, 6-min walk distance), psychosocial (depression and anxiety) and immunological parameters and overall survival. DISCUSSION: The aim of the POSITIVE trial is the evaluation of effects of a 24-week structured and guided exercise intervention during palliative treatment stages. Analysis of various outcomes (such as quality of life, physical performance, self-efficacy, psychosocial and immunological parameters) will contribute to a better understanding of the potential of exercise in advanced lung cancer patients. In contrast to other studies with advanced oncological patients the POSITIVE trial provides weekly phone calls to support patients both in the intervention and control group and to segregate the impact of physical activity on quality of life. TRIAL REGISTRATION: ClinicalTrials.gov NCT02055508 (Date: December 12, 2013).
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Terapia por Ejercicio/métodos , Ejercicio Físico , Neoplasias Pulmonares/terapia , Cuidados Paliativos/métodos , Adulto , Anciano , Femenino , Humanos , Neoplasias Pulmonares/fisiopatología , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud , Aptitud Física/fisiología , Calidad de Vida , Entrenamiento de Fuerza , Encuestas y CuestionariosRESUMEN
A series of 2-substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides were synthesized and evaluated for their affinity to the glycine binding site of the N-methyl-d-aspartate (NMDA) receptor. The binding affinity was determined by the displacement of radioligand [(3)H]MDL-105,519 from rat cortical membrane preparations. The most attractive structures in the search for prospective NMDA receptor ligands were identified to be 2-arylcarbonylmethyl substituted 3,4-dihydro-2H-1,2-benzothiazine-3-carboxylic acid 1,1-dioxides. It has been demonstrated for the first time that the replacement of NH group in the ligand by sp(3) CH2 is tolerated. This finding may pave the way for previously unexplored approaches for designing new ligands of the NMDA receptor.
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Benzoxazinas/síntesis química , Benzoxazinas/farmacología , Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Benzoxazinas/química , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ligandos , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
This article deals with seven special issues related to the assumptions, applicability, and practical use of formulas for calculating power or sample size, respectively, for comparative clinical trials with time-to-event endpoints, with particular focus on the well-known Freedman and Schoenfeld methods. All problems addressed are illustrated by numerical examples, and recommendations are given on how to deal with them in the planning of clinical trials.
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Algoritmos , Determinación de Punto Final/estadística & datos numéricos , Tamaño de la Muestra , Ensayos Clínicos como Asunto/estadística & datos numéricos , Simulación por Computador , Humanos , Modelos Estadísticos , Probabilidad , Proyectos de Investigación , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
BACKGROUND: Pancreatic cancer is mainly a disease of the elderly population, but clinical trials do not reflect this age distribution. Data on treatment strategies and outcome of older patients are limited. The aim of our study was to analyze safety and outcome in elderly patients with advanced pancreatic cancer treated with palliative chemotherapy at the outpatient clinic of the National Center for Tumor Diseases (NCT) at Heidelberg University Hospital. MATERIALS AND METHODS: We retrospectively analyzed 53 patients ≥70 years using a prospectively maintained database. Requirements were (1) histologically proven diagnosis of ductal pancreatic adenocarcinoma, (2) age ≥70 at time of diagnosis of advanced disease, and (3) measurable advanced disease. RESULTS: The median age was 73 years. 81% of the patients received a gemcitabine-based first-line therapy. Median overall survival was 6.7 months. Survival differed significantly between patient groups with low (≤1) and high (≥2) Eastern Cooperative Oncology Group performance status (7.8 vs. 3.9 months, p = 0.002). 30.2% of the patients developed side effects resulting in dosage reductions. 39.6% of the patients received second-line treatment. Residual survival after disease progression was significantly longer for second-line treatment compared to best supportive care (151 vs. 39 days, p = 0.019). CONCLUSIONS: Overall, our older patients did not have an inferior outcome compared to the reported trial populations that included younger patients. Thus, palliative chemotherapy should be considered independently from chronological age, but the performance status should be carefully noticed. Second-line therapy should be considered for patients in good performance status after first progression.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Cuidados Paliativos/métodos , Neoplasias Pancreáticas/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Antineoplásicos/administración & dosificación , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Estudios de Cohortes , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Clorhidrato de Erlotinib , Femenino , Humanos , Masculino , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Quinazolinas/administración & dosificación , Estudios Retrospectivos , Análisis de Supervivencia , Resultado del Tratamiento , GemcitabinaRESUMEN
PURPOSE: This study aimed to evaluate the safety, feasibility, and effects of an 8-wk combined resistance and endurance exercise program in patients with advanced non-small cell lung cancer (NSCLC) during in- and outpatient care. METHODS: In this intervention study, 40 patients with predominantly advanced NSCLC receiving simultaneous or sequential radiochemotherapy or chemotherapy alone were enrolled. For a period of 8 wk, patients were instructed to exercise at least five times per week during the inpatient setting and at least three times per week in the outpatient setting. Physical performance status (endurance capacity: 6-min walk test; strength capacity: handheld dynamometry), quality-of-life (Functional Assessment of Cancer Therapy-Lung), fatigue (Multidimensional Fatigue Inventory), and depression (Patient Health Questionnaire) were assessed at baseline (T0), after the exercise intervention (T1), and at a follow-up time point 8 wk later (T2). The primary end point was adequate adherence (feasibility) defined as completing at least two training sessions per week during a minimum of 6 wk. RESULTS: Of 40 patients, 31 (77.5%) completed the postexercise assessment (T1) and 22 (55%) completed follow-up (T2). The stages were IIA (5%), IIIA (8%), IIIB (20%), and IV (67%), and the median age was 63 yr (range = 22-75 yr). Overall, adherence was 82% for those patients who completed T1, and 55% of the 40 participating patients fulfilled the adequate adherence criterion. Those who completed the intervention showed a significant improvement in the 6-min walk distance and in knee, elbow, and hip muscle strength after the intervention (T1). Quality of life, fatigue, and depression scores remained stable or declined slightly. Significant improvements in knee-muscle strength were also observed at T2. CONCLUSIONS: Exercise training is feasible in advanced and metastatic NSCLC patients during anticancer treatment. In this pilot study, endurance and strength capacity improved over time, indicating the rehabilitative importance of the applied intervention. To investigate the potential impact of exercise training in this patient group, a larger randomized trial is warranted.
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Carcinoma de Pulmón de Células no Pequeñas/rehabilitación , Terapia por Ejercicio/métodos , Enfermedades Pulmonares/rehabilitación , Anciano , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Depresión/prevención & control , Fatiga/prevención & control , Estudios de Factibilidad , Humanos , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/radioterapia , Persona de Mediana Edad , Fuerza Muscular , Cooperación del Paciente , Resistencia Física , Proyectos Piloto , Estudios Prospectivos , Calidad de Vida , Entrenamiento de Fuerza , Adulto JovenRESUMEN
The metabotropic glutamate receptor subtype 5 has evolved into a promising target for the treatment of various diseases of the central nervous system, such as Fragile X and L-DOPA induced dyskinesia. One of the most advanced clinical compound is Novartis' AFQ-056 (Mavoglurant), which served us as a template for a scaffold hopping approach, generating a structurally diverse set of potent analogs. Both the limited aqueous solubility and the relatively poor metabolic stability of AFQ-056 were improved with hexahydrocyclopenta[c]pyrrole derivative 54a, which proved to be a valuable candidate for further development.
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Indoles/química , Indoles/farmacología , Receptor del Glutamato Metabotropico 5/metabolismo , Regulación Alostérica , Animales , Células CHO , Cricetulus , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Receptor del Glutamato Metabotropico 5/química , Relación Estructura-ActividadRESUMEN
A virtual screening approach using various in silico methodologies led to the discovery of 2-(m-tolylamino)-7,8-dihydroquinazolin-5(6H)-one (1) as a moderately active negative allosteric modulator (NAM) of the metabotropic glutamate receptor subtype 5 (mGluR5) showing high selectivity against the subtype mGluR1. Modifications of the parent compound by rational design yielded a series of highly potent derivatives which will serve as valuable starting points for further hit-to-lead optimization efforts toward a suitable drug candidate for the treatment of L-DOPA induced dyskinesia.
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Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Quinazolinas/síntesis química , Quinazolinonas/química , Quinazolinonas/farmacología , Receptor del Glutamato Metabotropico 5/agonistas , Toluidinas/química , Toluidinas/farmacología , Interfaz Usuario-Computador , Regulación Alostérica/efectos de los fármacos , Animales , Células Cultivadas , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Quinazolinas/química , Quinazolinas/farmacología , Ratas , Receptores de Glutamato Metabotrópico/agonistas , Relación Estructura-ActividadRESUMEN
The farnesoid X receptor (FXR) is expressed predominantly in tissues exposed to high levels of bile acids and controls bile acid and lipid homeostasis. FXR(-/-) mice develop hepatocellular carcinoma (HCC) and show an increased prevalence for intestinal malignancies, suggesting a role of FXR as a tumor suppressor in enterohepatic tissues. The N-myc downstream-regulated gene 2 (NDRG2) has been recognized as a tumor suppressor gene, which is downregulated in human hepatocellular carcinoma, colorectal carcinoma and many other malignancies.We show reduced NDRG2 mRNA in livers of FXR(-/-) mice compared to wild type mice and both, FXR and NDRG2 mRNAs, are reduced in human HCC compared to normal liver. Gene reporter assays and Chromatin Immunoprecipitation data support that FXR directly controls NDRG2 transcription via IR1-type element(s) identified in the first introns of the human, mouse and rat NDRG2 genes. NDRG2 mRNA was induced by non-steroidal FXR agonists in livers of mice and the magnitude of induction of NDRG2 mRNA in three different human hepatoma cell lines was increased when ectopically expressing human FXR. Growth and metastasis of SK-Hep-1 cells was strongly reduced by non-steroidal FXR agonists in an orthotopic liver xenograft tumor model. Ectopic expression of FXR in SK-Hep1 cells reduced tumor growth and metastasis potential of corresponding cells and increased the anti-tumor efficacy of FXR agonists, which may be partly mediated via increased NDRG2 expression. FXR agonists may show a potential in the prevention and/or treatment of human hepatocellular carcinoma, a devastating malignancy with increasing prevalence and limited therapeutic options.
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Isoxazoles/farmacología , Neoplasias Hepáticas/prevención & control , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Sitios de Unión/genética , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Células Hep G2 , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Metástasis de la Neoplasia , Unión Proteica , Interferencia de ARN , Receptores Citoplasmáticos y Nucleares/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga Tumoral/efectos de los fármacos , Proteínas Supresoras de Tumor/genéticaRESUMEN
PURPOSE: Adjuvant chemotherapy prolongs survival in patients with pancreatic cancer, but its benefit is limited. Long-term survival times of up to 44 months after adjuvant chemoradioimmunotherapy in phase II trials motivated the present study. PATIENTS AND METHODS: Between 2004 and 2007, 132 R0/R1 resected patients received either fluorouracil (FU), cisplatin, and interferon alfa-2b (IFN α-2b) plus radiotherapy followed by two cycles of FU (arm A, n=64) or six cycles of FU monotherapy (arm B, n=68). One hundred ten patients (arm A, n=53; arm B, n=57) received at least one dose of the study medication, and these patients composed the per-protocol (PP) population. Biomarkers were analyzed longitudinally for their predictive value. RESULTS: Median survival for all randomly assigned patients was 26.5 months (95% CI, 21.6 to 39.5 months) in arm A and 28.5 months (95% CI, 20.4 to 38.6 months) in arm B. The hazard ratio was 1.04 (arm A v arm B: 95% CI, 0.66 to 1.53; P=.99). Median survival for the PP population was 32.1 months (95% CI, 22.8 to 42.2 months) in arm A and 28.5 months (95% CI, 19.5 to 38.6 months) in arm B (P=.49). Eighty-five percent of patients in arm A and 16% of patients in arm B experienced grade 3 or 4 toxicity. The quality of life was temporarily negatively affected in arm A. CONCLUSION: The FU, cisplatin, and IFN α-2b plus radiotherapy regimen did not improve the survival compared with FU monotherapy. Given the substantial adverse effects, this treatment can currently not be recommended. Nevertheless, the outcome in both arms represents the best survival, to our knowledge, ever reported for patients with resected pancreatic cancer in randomized controlled trials. Future studies will demonstrate whether immune response to IFN α-2b challenge has a predictive value.
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Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/radioterapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/radioterapia , Adenocarcinoma/patología , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Quimioradioterapia , Quimioterapia Adyuvante , Cisplatino/administración & dosificación , Cisplatino/efectos adversos , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/efectos adversos , Humanos , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferón-alfa/efectos adversos , Leucovorina/administración & dosificación , Leucovorina/efectos adversos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/patología , Pronóstico , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Análisis de SupervivenciaRESUMEN
BACKGROUND: The epidermal growth factor receptor monoclonal antibody cetuximab has proven activity in metastatic colorectal cancer. To date, the mechanisms of action are not completely understood. Especially the impact on tumor glucose metabolism, or tumor vascularization remains largely unclear. The understanding of mechanisms such as early changes in tumor metabolism is of clinical importance since there may be a substantial influence on choice and sequence of drug combinations. Early signals of response to cetuximab may prove useful to identify patients having a relevant clinical treatment benefit. The objective of this trial is to evaluate the predictive relevance of the relative change in (18)F-Fluorodeoxyglucose tumor uptake for early clinical response during short-term single agent treatment with cetuximab. Early clinical response will be routinely measured according to the response evaluation criteria in solid tumors. Accompanying research includes cytokine immune monitoring and analysis of tumor proteins and tumor genes. METHODS/DESIGN: The REMOTUX trial is an investigator-initiated, prospective, open-label, single-arm, single-center early exploratory predictive study. The first (18)F-FDG PET-CT is conducted at baseline followed by the run-in phase with cetuximab at days 1 and 8. At day 14, the second (18)F-FDG PET-CT is performed. Subsequently, patients are treated according to the Folfiri-cetuximab regimen as an active and approved first-line regimen for metastatic colorectal carcinoma. At day 56, clinical response is evaluated with a CT-scan compared to the baseline analysis. Tracer uptake is assessed using standardized uptake values (SUVs). The main hypothesis to be tested in the primary analysis is whether or not the relative change in the SUV from baseline to day 14 has any predictive relevance for early clinical response determined at day 56. Patients are followed until death from any cause or until 24 months after the last patient has ended trial treatment. DISCUSSION: The aim of this trial is to evaluate metabolic changes in metastatic colorectal cancer during short-term single agent treatment with cetuximab and to analyse their potential of predicting early clinical response. This could be helpful to answer the question if early identification of patients not responding to cetuximab is possible. TRIAL REGISTRATION: ClinicalTrials.gov NCT200811021020; EudraCT 200901327923.
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Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/diagnóstico por imagen , Fluorodesoxiglucosa F18/farmacocinética , Tomografía de Emisión de Positrones , Radiofármacos/farmacocinética , Anticuerpos Monoclonales Humanizados , Cetuximab , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/secundario , Receptores ErbB/antagonistas & inhibidores , Femenino , Humanos , Masculino , Valor Predictivo de las Pruebas , Estudios ProspectivosRESUMEN
This note addresses the questions of whether one should safeguard against a potential loss of power due to random variations of the accrual time, how this "insurance" can be formulated, and how much the sample size needs to be increased to obtain it.
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Determinación de Punto Final/métodos , Modelos Teóricos , Probabilidad , Proyectos de Investigación , Humanos , Tamaño de la MuestraRESUMEN
Human colon cancer harbors a small subfraction of tumor-initiating cells (TICs) that is assumed to be a functionally homogeneous stem-cell-like population driving tumor maintenance and metastasis formation. We found unexpected cellular heterogeneity within the TIC compartment, which contains three types of TICs. Extensively self-renewing long-term TICs (LT-TICs) maintained tumor formation in serial xenotransplants. Tumor transient amplifying cells (T-TACs) with limited or no self-renewal capacity contributed to tumor formation only in primary mice. Rare delayed contributing TICs (DC-TICs) were exclusively active in secondary or tertiary mice. Bone marrow was identified as an important reservoir of LT-TICs. Metastasis formation was almost exclusively driven by self-renewing LT-TICs. Our results demonstrate that tumor initiation, self-renewal, and metastasis formation are limited to particular subpopulations of TICs in primary human colon cancer. We identify LT-TICs as a quantifiable target for therapies aimed toward eradication of self-renewing tumorigenic and metastatic colon cancer cells.
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Neoplasias del Colon/patología , Células Madre Neoplásicas/patología , Anciano , Animales , Médula Ósea/patología , Proliferación Celular , Femenino , Humanos , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Metástasis de la Neoplasia , Células Madre Neoplásicas/clasificación , Esferoides Celulares/patología , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Vectors based on γ-retroviruses or lentiviruses have been shown to stably express therapeutical transgenes and effectively cure different hematological diseases. Molecular follow up of the insertional repertoire of gene corrected cells in patients and preclinical animal models revealed different integration preferences in the host genome including clusters of integrations in small genomic areas (CIS; common integrations sites). In the majority, these CIS were found in or near genes, with the potential to influence the clonal fate of the affected cell. To determine whether the observed degree of clustering is statistically compatible with an assumed standard model of spatial distribution of integrants, we have developed various methods and computer programs for γ-retroviral and lentiviral integration site distribution. In particular, we have devised and implemented mathematical and statistical approaches for comparing two experimental samples with different numbers of integration sites with respect to the propensity to form CIS as well as for the analysis of coincidences of integration sites obtained from different blood compartments. The programs and statistical tools described here are available as workspaces in R code and allow the fast detection of excessive clustering of integration sites from any retrovirally transduced sample and thus contribute to the assessment of potential treatment-related risks in preclinical and clinical retroviral gene therapy studies.
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Vectores Genéticos/genética , Mutagénesis Insercional/métodos , Retroviridae/genética , Programas Informáticos , Simulación por Computador , Bases de Datos Genéticas , Humanos , Modelos GenéticosRESUMEN
Vector-associated side effects in clinical gene therapy have provided insights into the molecular mechanisms of hematopoietic regulation in vivo. Surprisingly, many retrovirus insertion sites (RIS) present in engrafted cells have been found to cluster nonrandomly in close association with specific genes. Our data demonstrate that these genes directly influence the in vivo fate of hematopoietic cell clones. Analysis of insertions thus far has been limited to individual clinical studies. Here, we studied >7,000 insertions retrieved from various studies. More than 40% of all insertions found in engrafted gene-modified cells were clustered in the same genomic areas covering only 0.36% of the genome. Gene classification analyses displayed significant overrepresentation of genes associated with hematopoietic functions and relevance for cell growth and survival in vivo. The similarity of insertion distributions indicates that vector insertions in repopulating cells cluster in predictable patterns. Thus, insertion analyses of preclinical in vitro and murine in vivo studies as well as vector insertion repertoires in clinical trials yielded concerted results and mark a small number of interesting genomic loci and genes that warrants further investigation of the biological consequences of vector insertions.
Asunto(s)
Gammaretrovirus/genética , Terapia Genética/efectos adversos , Vectores Genéticos/efectos adversos , Genoma , Integración Viral , Animales , Mapeo Cromosómico , Redes Reguladoras de Genes , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Primates , Trasplantes , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/genética , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/terapiaRESUMEN
BACKGROUND: 18-Fluorodeoxyglucose-PET (18F-FDG-PET) can be used for early response assessment in patients with locally advanced adenocarcinomas of the oesophagogastric junction (AEG) undergoing neoadjuvant chemotherapy. It has been recently shown in the MUNICON trials that response-guided treatment algorithms based on early changes of the FDG tumor uptake detected by PET are feasible and that they can be implemented into clinical practice. Only 40%-50% of the patients respond metabolically to therapy. As metabolic non-response is known to be associated with a dismal prognosis, metabolic non-responders are increasingly treated with alternative neoadjuvant chemotherapies or chemoradiation in order to improve their clinical outcome. We plan to investigate whether PET can be used as response assessment during radiochemotherapy given as salvage treatment in early metabolic non-responders to standard chemotherapy. METHODS/DESIGN: The HICON trial is a prospective, non-randomized, explorative imaging study evaluating the value of PET as a predictor of histopathological response in metabolic non-responders. Patients with resectable AEG type I and II according to Siewerts classification, staged cT3/4 and/or cN+ and cM0 by endoscopic ultrasound, spiral CT or MRI and FDG-PET are eligible. Tumors must be potentially R0 resectable and must have a sufficient FDG-baseline uptake. Only metabolic non-responders, showing a < 35% decrease of SUV two weeks after the start of neoadjuvant chemotherapy are eligible for the study and are taken to intensified taxane-based RCT (chemoradiotherapy (45 Gy) before surgery. 18FDG-PET scans will be performed before ( = Baseline) and after 14 days of standard neoadjuvant therapy as well as after the first cycle of salvage docetaxel/cisplatin chemotherapy (PET 1) and at the end of radiochemotherapy (PET2). Tracer uptake will be assessed semiquantitatively using standardized uptake values (SUV). The percentage difference ΔSUV = 100 (SUV Baseline - SUV PET1)/SUV Baseline will be calculated and assessed as an early predictor of histopathological response. In a secondary analysis, the association between the difference SUV PET1 - SUV PET2 and histopathological response will be evaluated. DISCUSSION: The aim of this study is to investigate the potential of sequential 18FDG-PET in predicting histopathological response in AEG tumors to salvage neoadjuvant radiochemotherapy in patients who do not show metabolic response to standard neoadjuvant chemotherapy.
Asunto(s)
Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Ensayos Clínicos como Asunto/métodos , Neoplasias Esofágicas/diagnóstico por imagen , Neoplasias Esofágicas/tratamiento farmacológico , Unión Esofagogástrica/diagnóstico por imagen , Radioisótopos de Flúor , Fluorodesoxiglucosa F18 , Estudios Multicéntricos como Asunto/métodos , Terapia Neoadyuvante , Selección de Paciente , Tomografía de Emisión de Positrones , Radiofármacos , Terapia Recuperativa , Adenocarcinoma/patología , Adenocarcinoma/radioterapia , Adenocarcinoma/cirugía , Antineoplásicos/administración & dosificación , Capecitabina , Cisplatino/administración & dosificación , Terapia Combinada , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Docetaxel , Epirrubicina/administración & dosificación , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/radioterapia , Neoplasias Esofágicas/cirugía , Esofagectomía , Fluorouracilo/administración & dosificación , Fluorouracilo/análogos & derivados , Alemania , Humanos , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Estudios Prospectivos , Taxoides/administración & dosificación , Resultado del TratamientoRESUMEN
Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) are promising for safe and sustained transgene expression in dividing as well as quiescent cells. As genome organization and transcription substantially differs between actively dividing and postmitotic cells in vivo, we hypothesized that genomic vector integration preferences might be distinct between these biological states. We performed integration site (IS) analyses on mouse dividing cells (fibroblasts and hematopoietic progenitor cells (HPCs)) transduced ex vivo and postmitotic cells (eye and brain) transduced in vivo. As expected, integration in dividing cells occurred preferably into gene coding regions. In contrast, postmitotic cells showed a close to random frequency of integration into genes and gene spare long interspersed nuclear elements (LINE). Our studies on the potential mechanisms responsible for the detected differences of lentiviral integration suggest that the lowered expression level of Psip1 reduce the integration frequency in vivo into gene coding regions in postmitotic cells. The motif TGGAA might represent one of the factors for preferred lentiviral integration into mouse and rat Satellite DNA. These observations are highly relevant for the correct assessment of preclinical biosafety studies, indicating that lentiviral vectors are well suited for safe and effective clinical gene transfer into postmitotic tissues.