La investigación aplicada en oncología Eduardo Osinaga Dept. de Inmunobiología Lab. de Glicobiología e Facultad de Medicina Inmunología Tumoral Universidad de la República Institut Pasteur de Montevideo

Aplicación del conocimiento molecular a nivel clínico BIOTECNOLOGÍA anticuerpos monoclonales, PCR, clonado, vectores, microarrays, ... GENES PROTEÍNAS CÉLULAS MEDICINA MOLECULAR PACIENTE DIAGNÓSTICO TRATAMIENTO MEDICINA CLÍNICA

Investigación aplicada en salud Investigación básica Investigación aplicada Ensayos clínicos Demostración y Educación Adquisición de conocimientos Validación del conocimiento Transferencia del conocimiento Aporte a la SALUD

Historia natural del cáncer   Talmadge and Fidler, Cancer Res 2010 4

Cáncer: enfermedad de etiopatogenia molecular

Desregulación de la apoptosis

Desregulación de la señalización intracelular

LEUCEMIA MIELOIDE CRÓNICA: IMPACTO CLÍNICO DE LA INVESTIGACIÓN BÁSICA Cromosoma Philadelphia t9;22 (gen quimérico Bcr/Abl) Actividad tirosina-quinasa STI-571

Posibilidades para la investigación aplicada en la era post-genómica DNA RNA Proteína Metabolitos y macromoléculas Igual genoma Diferente proteoma Validación química, genética, biológica y clínica Diagnóstico, vacunas, fármacos Genoma Transcriptoma Proteoma Metaboloma Biología de sistemas

Tratamientos oncológicos basados en la identificación de Figure 1 Cancer genetics is accelerating the time from ‘driver mutation discovery’ to ‘clinical proofof-concept’ and the approval of new drugs. The historical timelines for developing targeted therapies discussed in the text are highlighted as examples. Gleevec received FDA approval long after the discovery of the Philadelphia chromosome mutation and hyperactive BCR-ABL protein in chronic myelogenous leukemia (CML). By contrast, the more recent discovery of chromosomal rearrangements (translocations) of ALK in NSCLC has rapidly translated into registration trials for Crizotinib, a ‘cMETturned- ALK’ inhibitor, based on tantalizing response rates in ALK-fusion-positive tumors (Phase I and II trial results)63. Likewise, the development paradigm for selective BRAF inhibitors, as exemplified by PLX4032, underlines the much faster pace of translation (8 years, compared with Gleevec or Herceptin) once the driver status (in this case BRAF mutations) had been established for an indication (malignant melanoma). Such accelerated development times are enabled by the broader body of knowledge of cancer biology and mechanisms of actions that have been generated in the cancer field (Fig. 2). The FDA approval of Herceptin and the accompanying diagnostic test for HER2 expression (HercepTest) proved the value of biomarker-driven trials that are informed by mechanistic insights gained from cancer genetics. In a similar vein, it is the functional understanding of DNA-repair mechanisms, and the role of BRCA1 and BRCA2 mutations in sensitizing tumors to PARP inhibition, that inform current registration trials of PARP inhibitors in BRCA-associated cancer types and patients that carry the BRCA mutation. Tratamientos oncológicos basados en la identificación de “blancos moleculares” L. Chin et al. Nature Medicine 2011

¿Es posible evaluar simultaneamente todas las vías de señalización?

Proc. Natl. Acad. Sci. USA 2001, 98:10869-74

Overall and relapse-free survival analysis of the 49 breast cancer patients, uniformly treated in a prospective study, based on different gene expression classification. (A) Overall survival and (B) relapse-free survival for the five expression-based tumor subtypes based on the classification presented in Fig. 1 (luminals B and C were considered one group). (C) Overall survival estimated for the six-subtype classification with the three different luminal subtypes presented in Fig. 1. (D) Overall survival based on the five-subtype classification presented in Figs. 2 Lower and 5. 13

Los genes relacionados con el pronóstico están involucrados en diferentes aspectos de la biología tumoral proliferation angiogenesis adhesion to extracellular matrix local invasion intravasation, survival, extravasation Desarrollo de métodos Oncotype Dx 21 genes MammaPrint 70 genes proliferation angiogenesis adhesion to extracellular matrix William Cho Genes of unknown function (25)

William Cho

Evolución en la caracterización biológica de tumores Arrays de tejidos Análisis proteómico Arrays de genes This slides illustrates how new laboratory techniques have found their way to routine diagnostics of breast cancer. Immunohistochemistry and FISH are now routinely used in the management of breast cancer. Will microarrays and proteomics be the next? These new techniques offer the unique possibility of examining a very large number of genes and proteins to develop predictors of outcome which are made up by many genes rather than single marker. Tipo de tumor Histología Predicción con un gen/proteína Predicción multi-gen/proteina

Desarrollo de estrategias anti-tumorales de acuerdo con la biología tumoral moderna Figure 2 Development of a gene expression biomarker. (a) Unbiased discovery of a gene expression profile starts with the large-scale analysis of gene expression on a series of tumor samples of known clinical outcome. (b) Using bioinformatics, the set of genes is identified that correlates best with the relevant clinical parameter. (c,d) In the next step, this ‘gene signature’ is validated on a large cohort of additional clinical samples of known outcome (c), and the clinical performance is evaluated in comparison with the generally accepted clinical parameters (d). (e) Regulatory approval is still underdeveloped but might involve clearance by CMS under the CLIA guidelines and the College of American Pathologists (CAP) in the US. In Europe, both an ISO17025 accreditation of the laboratory and a CE-marking (indicating that it has met EU consumer safety, health or environmental requirements) of the diagnostic equipment are required. (f) Only after this process is completed should these tests be used to stratify patients by molecular signatures. Majewski and Bernards Nature Medicine 2011

Tratamiento de NSCLC con TKIs Marcadores clínicos de respuesta: No fumadores Etnia asiática Sexo femenino Adenocarcinomas Vía de señalización EGFR Uno de los mayores avances en terapias dirigidas en cáncer de pulmón se ha focalizado en la inhibición del receptor del factor de crecimiento epidérmico (EGFR). Pertenece a la familia de receptores de membrana HER, integrada por 4 miembros: HER1, que es el EGFR, HER2 de gran importancia en cáncer de mama, HER3 y HER4. Son receptores con acción tirosina-kinasas. La unión al ligando induce cambios conformacionales y dimerización homóloga o heteróloga con otros miembros de la misma familia. Esta activación provoca la autofosforilación de residuos tirosina específicos del dominio intracelular, se internaliza en la célula e interacciona con otras moléculas para activar mediante una cascada de fosforilaciones sucesivas la vía RAS/Raf/MEK/Erk a través de Grb2/SOS, cuya consecuencia será promover una mayor sobrevida celular y por otro lado, a través de PI3K la activación de Akt/mTOR que promueven proliferación celular.  EGFR se encuentra frecuentemente sobre-expresado en cáncer y despertó interés para el desarrollo de estrategias de inhibición de potencial aplicación terapéutica en tumores. Biochem Pharmacol, 2010; 80:613 19

Mutaciones activadoras de EGFR

Valor predictivo de las mutaciones activadoras de EGFR Clin Cancer Res 2006;12:1398 Las mut. Activadoras más frecuentes son: Deleciones en el entorno de los aá 746-753 del exón 19 (50%) L858R en exón 21 (40%) G719C en exón 18 (5%) La gráfica muestra el período libre de progresión según la mutación. Las deleciones del exón 19 tienen un período libre de progresión > que la L858R Mutaciones activadoras: Exones 18, 19 y 21 Mutación de resistencia: Exón 20 N Engl J Med 2004;350:2129 21

Aprobados por la FDA para tratamientos oncológicos: Biofármacos anti-tumorales generados a partir de anticuerpos monoclonales Anticuerpo de ratón Anticuerpo quimérico Anticuerpo humano Anticuerpo humanizado F(ab)2 scFv biespecífico scFv bivalente Framento scFv Fragmento Fab Ratón humano Aprobados por la FDA para tratamientos oncológicos: Nombre Tipo Blanco molecular Tipo de cáncer Rituximab Quimérico CD20 NHL Trastuzumab Humanizado Erb B2 Mama Ibritumomab Ratón 90Y Bevacizumab VEGF Colo-rectal Alemtuzumab CD52 LLC Cetuximab EGFR Panitumumab Humano Ofatumumab

Biomarcadores de “blancos moleculares”: aporte a tratamientos más personalizados Figure 1 Types of biomarker. Prognostic tests help to identify individuals who are at high risk of recurrence of their cancer and should receive further (adjuvant) therapy. Predictive biomarkers help to identify those drugs to which patients are most responsive (or unresponsive). Pharmacodynamic biomarkers can help to identify which drug dose to use for an individual.

Figure 1 Integration of immunoediting and oncogenesis during cancer progression. Oncogenesis initiates the formation of transformed cells that are attacked by immune cells as the result of neoantigen presentation. This process of immune surveillance imposes a selection for transformed cells that acquire tactics to escape control. Genetic instability driven by oncogenesis facilitates evolution of strategies for immune evasion or suppression, the latter of which may help tilt the tumor microenvironment from hostile to supportive for tumor cells. As the battle evolves between tumor cells and the immune system, representing the process of immunoediting, a state of immune equilibrium may be achieved that corresponds to a dynamic but dormant disease that is clinically occult. Further iteration of evasion mechanisms to defeat different elements of the immune system may ultimately drive immune suppression beyond the local microenvironment, achieving immune escape and thereby licensing invasive and metastatic behaviors that define progressive disease. Invasion and metastasis may be derivative of immune escape since the former are presumably impotent while immune surveillance prevails beyond the local microenviroment. While mutations may initiate cancer, modifiers and microenvironmental factors including those sculpted by the process of immunoediting may dictate the course of disease. Prendergast, Oncogene 2008 25

A. Farell. Nature Medicine 2011 Notable advances in cancer research. In a survey of cancer researchers, recent reports on cancer stem cells, cancer genomes and cancer therapies garnered the most attention from respondents. Cancer stem cell (CSC) studies have yielded new insights into the cells of origin of some tumors, the role of the epithelial-to-mesenchymal transition (EMT) in conferring stem cell–like properties and the contribution of CSCs to drug resistance, and they have also triggered questions about the CSC hypothesis itself. Cancer genome sequencing studies continue to yield data on cancer-initiating and –promoting mutations as well as insights into epigenetic and metabolic changes in tumors that offer the potential for the discovery of new therapeutic targets and mechanisms of tumorigenesis. Reports of clinical advances in immunotherapy and targeted molecular therapy, as well as mechanisms of treatment resistance, also provoked significant interest, as did studies of the role of the immune system and new molecular players in tumor growth, signaling and metastasis. The image depicts a metastasizing tumor and highlights the areas of cancer research most frequently selected by the survey respondents. A. Farell. Nature Medicine 2011

Mejor caracterización de las bases moleculares y celulares del inmuno-escape tumoral

Inhibiendo al inhibidor: nuevas estrategias de inmunoterapia anti-tumoral

La investigación translacional en oncología Puente bidireccional entre el laboratorio y la clínica. Acelera el desarrollo de agentes contra blancos moleculares específicos Acelera el desarrollo de biomarcadores para: Riesgo oncogenético Crecimiento tumoral Predictores de respuestas Conecta el diagnóstico molecular con estrategias terapéuticas, favoreciendo la personalización del tratamiento (más efectivo y menos tóxico). 29 29

La investigación translacional ha generado aportes cualitativos en el manejo de pacientes con cáncer, pero… TIEMPO Comprendiendo al cáncer Investigación translacional Manejo de pacientes con cáncer INFORMACIÓN Sorena Nadaf, Translational Informatics, Hellen Diller Family Comprehensive Cancer Center 30

Investigación translacional en oncología