prostate cancer and biomarkers
TRANSCRIPT
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Melissa Yepes HernandezMateo Zuluaga GómezMolecular BiologyMedicine SchoolUPB 2011
Cancer genetics-guided discovery of serum biomarker
signatures for diagnosis and prognosis ofprostate cancer
Igor Cima et all
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INTRODUCTION
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INTRODUCTION
CANCER
•It is the uncontrolled growth of abnormal cells in the body
•Causes
•Cancer is a disease of abnormal gene function
http://www.elconfidencial.com/
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INTRODUCTION
PROTO-ONCOGE
NES
ONCOGENES
TUMOR SUPPRESSOR GENES
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INTRODUCTION
BIOMARKER
• It is a substance introduced into an organism as a means to examine organ function or other aspects of health (Medicine)
• It is a DNA sequence that causes disease or is associated with susceptibility to disease (Genetic)
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INTRODUCTION
PROSTATE CANCER
• The prostate is a gland located in front of the rectum and underneath the urinary bladder.
• It is found only in men
• It produces the fluid that protects sperm cells in semen
http://www.techjackal.net/
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INTRODUCTION
PROSTATE CANCER
• Adenocarcinoma cancer that starts in gland cells
• Sarcomas• Small cell carcinomas• Transitional cell carcinomas
• Prostatic intraepithelial neoplasia (PIN)
www.noticiassalud.com
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INTRODUCTION
PROSTATE CANCER
• Prostatic intraepithelial neoplasia (PIN) It appears at 20 years old like a pre-cancerous condition and then at 50 years old become in an active cancer
Prostate Adenocarcinoma
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INTRODUCTION
STAGES
Inside the prostate without overgrowth
The cancer has diffused all over the prostate
Neighboring tissues
Metastasis
STAGE 1
STAGE 2
STAGE 3
STAGE 4
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INTRODUCTION
DIAGNOSIS
• Prostate-specific antigen (PSA) blood testSubstance made by cells in the prostate glandLevel 4 ng/ml
•Digital rectal exam (DRE)Bumps or hard areas on the prostate that might be cancer
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INTRODUCTION
• Transrectal ultrasound (TRUS)It uses sound waves to make an image of the prostate on a video screen.
• Acid phosphatase It is produced by glandular epithelial cells
• Prostate biopsy
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INTRODUCTION
FORECAST
• Disease stage
• The life expectancy
• EZH2 gene expression
• Agehttp://www.lavozdelsandinismo.com
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INTRODUCTION
FORECAST
• PSA
• p53 mutation
• Black race
• Oncogenes AND
proto-oncogeneshttp://redconcienciaplanetaria.blogspot.com
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Prostate
Cancer
ProstateCancer
BiomarkerPSA
DiagnosisAnd
Prognosis
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GENERAL OBJECTIVE
Identify Cancer genetics-guided of serum
biomarker signatures for diagnosis and prognosis
of prostate cancer
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MATERIALES Y MÉTODOS
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MATERIALES Y MÉTODOS
• Cepa utilizada 129/BALB/c
• Los ratones fueron anestesiados
• Extracción de sangre y almacenamiento a -80°C
• Tejido prostático congelado y pulverizado para
posterior tinción
RATONES
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MATERIALES Y MÉTODOS
• Fueron oxidadas con peryodato de sodio y se conjugó con resina
• Se agrega tripsina para digerir las glicoproteínas en estado solido
• Este sirve para cuantificar la PSA
profeblog.es
GLICOPROTEINAS
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MATERIALES Y MÉTODOS
• Análisis de muestras en un espectrómetro de masas para medir concentraciones
•Separación cromatografía de los péptidos se realizó en un Agilent 1100 micro sistema HPL
ESPECTOMETRIA DE MASA
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MATERIALES Y MÉTODOS
• Gen prostático con radio mayor a 10 se considera específico para pCA
CUANTIFICACIÓN DEL PÉPTIDO
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MATERIALES Y MÉTODOS
•Separación de proteínas celulares por electroforesis en gel según su tamaño (SDS)
•Transferencia de proteínas a un filtro que se incuban con anticuerpos que reaccionan con la proteína estudiada
SDS/PAGE y Western Blot
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MATERIALES Y MÉTODOS
• Determinación de proteínas, drogas y hormonas
• Unión del anticuerpo a molécula de fluorocromo
• Emiten luz a una longitud de onda mayor de 400 a 700nm.
Inmunofluorescencia/Fluoroinmunoensayo
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MATERIALES Y MÉTODOS
Flurocromos comunes:• Isotiocianato de Fluiresceína•Rodamina
didatconsulting.com
Inmunofluorescencia/Fluoroinmunoensayo
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MATERIALES Y MÉTODOS
• Desnaturalización del DNA para dar hebras sencillas
•Hibridar hebra sencilla con un oligonucleótido (Primer)
•Replicación de la hebra sencilla por DNA polimerasa a partir del oligonucleótido
PCR
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MATERIALES Y MÉTODOS
• Criterios de inclusión: Pacientes con Pca y BPH(Hiperplasia Prostática Benigna)
• Criterios de exclusión: Pacientes con Pca avanzado
mundo.in.rs
Pacientes
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MATERIALES Y MÉTODOS
• Extracción de 8 ml de sangre en tubos coagulantes
• Inversión 8 veces, centrifugación 4 h después de recogida a 4°C por 10 min
Pacientes
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MATERIALES Y MÉTODOS
• División del suero en 5 partes y almacenamiento a -60°C
• Extracción de glicoproteína
Pacientes
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MATERIALES Y MÉTODOS
•Identificación de un tejido (in situ) por medio de una interacción entre antígeno-anticuerpo, el cual ya ha sido marcado.
•Por medio de la tinción de la célula se demuestra la presencia y la localización celular de una molécula interés.
Inmunohistoquimica
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MATERIALES Y MÉTODOS
• Identificación de un anticuerpo o antigeno utilizando un antígeno o un anticuerpo absorbido a una superficie solida
• La reacción Ag-Ac se usa un conjugado anti gamaglobulina humana unido a una enzima
ELISA
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MATERIALES Y MÉTODOS
• La enzima actúa sobre un sustrato coloreando la solución
• Intensidad de color Cantidad de Ag o Ac
• Medición de marcadores de la investigación
ELISA
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RESULTADOS
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RESULTADOS
GLICOPROTEINAS
Biomarcadores específicos para el cáncer de próstata a través de glicoproteínas
• Glicoproteínas del tejido prostático• Glicoproteínas del suero
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RESULTADOS
Inmunotransferencia e Inmunoflorescencia
• Se determinó que el PTEN tiene un papel importante en la diferenciación y homeostasis de las células madre
• Al no estar presente no hay quien suprima el tumor
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RESULTADOS
Inmunotransferencia e Inmunoflorescencia
• En ausencia del gen en estudio, las glicoproteínas
que generalmente están dentro de la membrana
celular de las células prostáticas, se liberan y
migran al suero sirviendo como biomarcadores
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RESULTADOS
PACIENTES
• Pacientes 143• En un grupo de 70 pacientes se confirma el Pca
localizado histológicamente• Grupo control BPH• Se evalúa el PTEN en FISH
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RESULTADOS
ELISA y Cuantificación del Péptido
• Por medio de estas dos pruebas se buscaba medir la cantidad de proteínas en plasma que estuvieran en relación con el Pca.
• La concentración media de varias proteínas se midió en el rango de concentración de antígeno prostático específico (PSA)
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RESULTADOS
• Los resultados sugieren que la reducción del número de copias del gen PTEN en la próstatael cáncer llevó a una perturbación de proteínas de suero.
• La pérdida de PTEN está causalmente asociada con el aceleramiento de Pca y la agresividad
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RESULTADOS
• Se encontraron 5 proteínas que pueden ser biomarcadores para pacientes:
GalNAc transferasa 4 (GALNTL4) Fibronectina (FN) Zinc-α-2-glycoproteina(AZGP1) Biglycan (BGN) Proteina de matriz extracelular 1(ECM1)
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RESULTADOS
PCR
• PCR y recuento de leucocitos para evitar variables de confusión
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Figure 2.c
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DISCUSSION
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DISCUSSION
AUTHOR WHAT HE SAYS YES or NOT
Rifai N
“In the past the discovery of serum biomarkers has been technically challenging because of the enormous complexity of the serum proteome and the lack of sensitivediscovery-driven measurement technologies”
Yes
Schröder FH
“However, the effectiveness of systematic PCa screening with PSA testing remains controversial, in part because a lack ofsensitivity and specificity results in considerable overdiagnosis and overtreatment”
Yes
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DISCUSSION
AUTHOR WHAT HE SAYS YES or NOT
Engelman JA
“In this regard, an increasing armamentarium of targeting agents that inhibit keycomponents of the PI3K pathway exists, and many of these inhibitors already are in clinical testin”
Yes
Bastian PJ
“There are several definitions of insignificant prostate cancer; most definitions exclude patients with any Gleason pattern 4 prostate cancer”
Yes
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CONCLUSION
• This study has a great Importance as an academic Exercise, because we used databases, literature in the area of medicine and learn about some Pathologies
• Molecular biology opens new areas to medical research because of the contributions that develop an approach to the cell as a foundation for life
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CONCLUSION
• Logical approaches for diagnosis and treatment require the natural history and biological behavior of the ways in which the disease occurs
• These studies can make a prognosis in an earlier stage of the disease, reducing the aggressiveness of the disease
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MAPAS CONCEPTUALES
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Mateo Zuluaga G
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Melissa Yepes H
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BIBLIOGRAPHY
MARTINEZ S, Lina María. Biología molecular. 3 ed. Medellín: UPB. Fac. de Medicina
AMERICAN CANCER SOCIETYhttp://www.cancer.org/Cancer/ProstateCancer/DetailedGuide/prostate-cancer-new-research
MEDLINEPLUS http://www.nlm.nih.gov/medlineplus/spanish/ency/article/001289.htm
SEMINARIOhttp://www.ncbi.nlm.nih.gov/pubmed/21300890
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GRACIAS