Dr. Daniel R. CIOCCA IMBECU (Unidad Ejecutora del CONICET) Fund. Argentina Investigación Cáncer MENDOZA

Transcripción

1 Nuevos factores pronósticos y predictivos en oncología. Técnicas de microarrays Dr. Daniel R. CIOCCA IMBECU (Unidad Ejecutora del CONICET) Fund. Argentina Investigación Cáncer MENDOZA

2 Indicadores moleculares pronósticos/predictivos Identificar los targets terapéuticos (mecanismos moleculares de sensibilidad/resistencia). Individualizar las terapias (racionalizar). Dirigir, diseñar y monitorear las terapias standard y aplicar las nuevas terapias. Analizar un set que abarque varios de los caminos claves y que tengan más significado clínico, en laboratorios con experiencia.

3 Factor predictivo Factor pronóstico Bcl-2 MEDICINA BASADA EN LA EVIDENCIA Co-expression of steroid receptors (estrogen receptor alpha and/or progesterone receptors) and Her-2/neu: Clinical implications. DR Ciocca, FE Gago, MA Fanelli, SK Calderwood. Journal of Steroid Biochemistry & Molecular Biology 102:32-40 (2006).

4 Heat shock proteins in prostate cancer: from tumorigenesis to the clinic. Ciocca et al. Int J Hyperthermia (in press)

5 Perdida de heterocigosidad (LOH): Ausencia de una región génica (alelo) en el DNA constitutivo. Evidencia de deleción de un segmento de DNA, sugiere la implicación de un gen supresor de tumores. La carencia de heterocigosis se observa en los loci supresores de tumores.

6 LOH EN TUMORES DEL SNC Oligodendrogliomas y Astrocitomas. LOH en 1p36 y/o 19 q. Utilidad como factor: Diagnóstico Pronóstico Terapéutico

7 MGMT Metil Guanina ADN Metil Transferasa: enzima clave en las vías de reparación del ADN. Función: remover aductos de grupos alquilo de la posición O6 guanina en el ADN a un residuo de cisteína interno. La O6 metilguanina tiende a aparear durante la replicación con timina. Mutación de transición G-C en A-T. La O6 metilguanina afecta a la metilación de las citocinas, la unión de FT, capacidad de recombinación y replicación. La hipermetilación en promotor de MGMT falta de MGMT expresión en algunos tumores con respecto al tejido normal (Ej: gliomas, pulmón, colon, linfoma, mama.)

8 Aplicaciones El silenciamiento mediado por metilación de MGMT señala tumores sensibles a agentes alquilantes. En gliomas la hipermetilación se ha relacionado con buena respuesta a la quimioterapia con BCNU y temozolamida, aumentando la sobrevida total y el tiempo de progresión tumoral. La hipermetilación de MGMT es un marcador predictivo positivo.

9 Análisis de metilación de islas CpG en MGMT por nested PCR metilación específica e inmunohistoquímica M M M U U MGMT UNM expresión nuclear pb MGMT MET falta de expresión Calle 1: Marcador. Calle 2 y 3: Control positivo MDA-MB 231, Calle 4: Caso 7690 alelo MET (81 pb), Calle 5: Caso 7690 alelo UNM (93 pb), Calle 6: Control negativo sangre periférica. Gisela Castro

10 Signaling networks assembled by oncogenic EGFR and c-met. Guo A et al. PNAS 105: (2008) A major question regarding the sensitivity of solid tumors to targeted kinase inhibitors is why some tumors respond and others do not. Many tumors express EGF receptor (EGFR), yet only a small subset with EGFR-activating mutations respond clinically to EGFR inhibitors (EGFRIs). Using: Nsclc cell lines Comparison of the signaling networks in EGFR and c-met-dependent cells identify a core network of 50 proteins that participate in pathways mediating drug response

11 Gen chip array (microarray) Ventajas. Estudio de numerosos genes simultáneamente.. Permite el estudio de genes normales y mutados. Desventajas:. Relativamente caro, necesita equipamiento específico.. No hay correlación exacta con la histopatología.. No permite evaluar la localización de proteínas. Futuro:. RNA-seq un método que permite analisis de transcriptomas con mejor resolución que microarray Curr Protoc Mol Biol 89:4.11 (2010) Tissue microarray Ventajas. Estudio de numerosos tejidos simultáneamente.. Rápida evaluación de un marcador.. Correlación con la histopatología Desventajas:. Relativamente caro, necesita equipamiento específico.. Problema de la heterogeneidad tumoral. Futuro:. Microscopía basada en imágenes espectrales para secciones de tejido con múltiples marcadores Curr Protoc Mol Biol 84: (2008)

12 Commercially Available Genomic Assays for the Prediction of Clinical Outcome in Breast Cancer Patients (NEJM 360:790; 2009) Variable MammaPrint Oncotype DX Theros MapQuant Dx Provider Agendia Genomic Health Biotheranostics Ipsogen Type of assay 70-Gene assay 21-Gene recurrence score 2-Gene ratio of HOXB13 Genomic grade to IL17R (H/I) and molecular-grade index Type of tissue sample Fresh or frozen FFPE FFPE Fresh or frozen Technique DNA microarrays Q-RT-PCR Q-RT-PCR DNA microarrays Centrally certified lab Yes Yes Yes Yes Indication To aid in progn prediction in patients <61 yr of age with stage I or II, node-negative disease with a tumor size of 5 cm To predict the risk of recurr in patients with ER-positive, node-negative disease treated with tamoxifen; to identify patients with a low risk of recurrence who may not need adjuvant chemotherapy To stratify ER+ patients into groups with a predicted low risk or high risk of recurrence and a predicted good or poor response to endocrine therapy To restratify grade 2 tu into low-risk grade 1 or high-risk grade 3 tumors, specific for invasive, primary, ER-positive grade 2 tumors Level of evidence (I V) III II III III FDA clearance Yes No No No Availability Europe and US Europe and US United States Europe

13 Cáncer de Mama: Moléculas Estudiadas (Ciocca y col) Hormonodependencia RE, RP, ps2, Hsp27, Hsp70, bcl-2, HER-2/neu, p53 Pronóstico (DFS, OS) P53, HER-2/neu, Factor VIII, Hsp27, Hsp70, Hsp90, Cathepsin D, Urokinase-plasminogen activator (upa), upar, PAI, nm23, Integrin beta 1, Matrix metaloproteinase MMP-2, MMP-9, E-cadherin, P cadherin, Beta catenin, Caveolin-1 Stefin A Respuesta a quimioterapias P170, Hsp27, Hsp70, Hsc70, Hsp90, HER-2/neu, p53, GSTpi, Apoptosis, Ki-67, RE, RP, Bcl-2, Bax, Topo II

14 Eventos importantes en cáncer operable Hormono dependencia Respuesta a Inmunoterapia Radiosensibilidad Recurrencia Local Respuesta a Quimioterapia RE, RP, bcl-2, Hsp27 HER-2/neu p53, Ki-67/PCNA HER-2/neu, Hsp27, Topo II Metástasis Clínico-Patol.: Edad, tipo tumor, T, GL, GT, inv. Vasc. Molec.: HER-2/neu, p53, Ki/PCNA, P-cad,...

15 Integration of Estrogen and Progesterone Receptors with Pathological and Molecular Prognostic Factors in Breast Cancer Patients Gago FE, Tello OM, Diblasi AM, Ciocca DR J Steroid Biochem Molec Biol 67: (1998) Clinico-Pathological Factors: Age, Tumor size, Lymph nodes, Tumor grade Molecular Factors: ER, PR, PCNA, p53, HER-2/neu, P170 Hubo un significativo mejoramiento en el diagnóstico de probabilidad de metástasis a distancia cuando el score patológico se combinó con el score molecular, 82% de las pacientes con metástasis presentaron un score combinado elevado.

16 Stromal Cell Expression of Caveolin-1 Predicts Outcome in Breast Cancer EK Sloan, DR Ciocca, N Pouliot, A Natoli, C Restall, MA Henderson, MA Fanelli, FD Cuello- Carrión, FE Gago, and RL Anderson Am J Pathol, vol 174, No 6, June 2009 Cav-1 is an integral plasma membrane protein that resides in specialized lipid rafts called caveolae in terminally differentiated mesenchymal cells including adipocytes, endothelial cells, and fibroblasts. Within caveolae, the bilayer of cholesterol and sphingolipids is in an ordered state that restricts the movement of lipids. Cav-1 interacts directly with, and organizes cholesterol within caveolae. Various receptors and signaling molecules are localized within caveolae, and cav-1 interacts with and negatively regulates a number of these through its scaffolding domain. By ordering lipids and concentrating signaling molecules, caveolae may facilitate cross talk between signaling pathways. Cav-1 is also found in the cytosol and has a role in lipid homeostasis and transport.

17 The role of cav-1 in breast cancer remains unclear. Earlier reports that cav-1 is expressed in normal breast epithelium are contradicted by more recent studies that found that its expression is associated with the myoepithelium and other tissues of mesenchymal origin and not with normal luminal epithelium. A number of studies have reported that cav-1 is downregulated in breast cancers compared with normal mammary tissue To clarify the relationship between cav-1 and breast cancer progression or suppression, we have analyzed tissue sections specifically for stromal and tumor epithelial cell expression of cav-1 from two cohorts of breast cancer patients.

18 Figure 1. Caveolin-1 expression in normal human breast tissue. Normal breast tissue samples were immunostained with rabbit polyclonal (A C) or mouse monoclonal (D F) antibodies directed against caveolin-1

19 The Breast Prognosis tissue microarray, kindly provided by Dr. O. Kallioniemi, contains one 0.6-mm biopsy from the periphery of each of 612 human primary breast carcinomas with extensive clinical follow-up data. The analysis reported here includes 429 samples with interpretable data Figure 2. Cav-1 in human breast cancer. A: Primary human breast tumors with tumor cells showing negative, weak or strong expression of caveolin-1 B: Metastasis-free survival by caveolin-1 status (positive or negative) in tu cells. n= 429 arrayed primary human breast cancer samples C: Representative tumors with stroma staining positive or negative Cav-1

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23 We analyzed the onset of spontaneous mammary tumors in mice null for Cav-1. Latency to tumor onset was measured in mice transgenic for mammary-specific Her-2/neu expression that carried 0, 1, or 2 alleles of Cav-1. Regardless of their Cav-1 status, mice lacking the MMTV-neu oncogene did not develop tumors within 3 years absence of stromal Cav-1 is not sufficient to induce tumorigenesis. Tumor onset was more rapid in mice lacking Cav-1

24 CONCLUSIONS We found that cav-1 was not expressed in normal breast luminal epithelium but was present in the epithelial compartment of some tumors. We found no association between cav-1 expression in the epithelial compartment and clinical outcome. However, high levels of cav-1 in the stromal tissue surrounding the tumor, rather than within tumor cells, associated strongly with reduced metastasis and improved survival (P < ). The onset of mammary tumors driven by Her2/neu overexpression was accelerated in mice lacking cav-1, thereby supporting the observation that the presence of cav-1 in the tumor microenvironment modulates tumor development. These studies suggest that stromal cav-1 expression may be a potential therapeutic target and a valuable prognostic indicator of breast cancer progression.

25 Tratar o no tratar? Tratar con qué drogas? Luminal A HER-2 Ciocca et al Small Stress Proteins (in press) Triple neg Luminal B

26 Laboratorios con experiencia!