Importancia del Perfil Genético en la Medicina Personalizada

Transcripción

1 Importancia del Perfil Genético en la Medicina Personalizada Fernando López-Ríos Laboratorio de Dianas Terapéuticas Centro Integral Oncológico Clara Campal Hospital Universitario Sanchinarro Madrid

2 INDICE Introducción CAP guidelines: biomarcadores en pulmón - EGFR - ALK Nuevas perspectivas - Más biomarcadores - Nuevas tecnologías - Redundancia molecular Conclusiones

3 Li et al. J Clin Oncol 2013

4 Pietanza and Ladanyi. Nat Genetics 2012

5 Cohorts: Clinical Trials Tumour Registries Tumour board Sampling Radiology Endoscopy Surgery Patient Oncologist Targeted therapies Results Predictive biomarkers QA/QC Diagnosis IHC I+D+i In situ hibridization PCR Publications Consensus Teaching Laboratorio de Dianas Terapéuticas Conde et al. Clin Transl Oncol 2013

6 INDICE Introducción CAP guidelines: biomarcadores en pulmón - EGFR - ALK Nuevas perspectivas - Más biomarcadores - Nuevas tecnologías - Redundancia molecular Conclusiones

7 New MUTACIONES guidelines DE EGFR: Fase for pre-analítica lung cancer testing from CAP/IASLC/AMP Aim: To establish evidence-based recommendations for the molecular analysis of lung cancers Systematic review conducted by: the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) Lindeman T, et al. J Mol Diagn 2013;15:415 53

8 Recommendations from the CAP/IASLC/AMP guidelines: Which patients should be tested? ALK [and EGFR] molecular testing should be used to select patients for ALK-[and EGFR-] targeted TKI therapy, and patients with lung adenocarcinoma should not be excluded from testing on the basis of clinical characteristics TKI, tyrosine kinase inhibitor; Lindeman T, et al. J Mol Diagn 2013;15:415 53

9 Our knowledge of tumour histology has evolved Miller VA, et al. J Clin Oncol 2004;22:1103 9; Liebow AA. Adv Intern Med 1960;10:329 58

10 Histological patterns are not predictive EGFR Wild-type Mutant p Gender Female Male 52 4 Age Mean SD Smoking history Never smoker Ever smoker 59 2 Histopathology Adenocarcinomas AD-BAC/AD-PP 6 6 Tumour stage I II III IV 18 1 KRAS Wild-type As indicated in the table, EGFR mutations were more common in females and in nonsmokers (p<0.05). Among the 47 cases available for reevaluation of histology according the most recent WHO classification, there were seven mutated and 40 nonmutated cases. Of those seven adenocarcinomas mixed subtypes harboring EGFR mutations, five exhibited prominent bronchioloalveolar features (AD-PP) pattern and the remaining tumor was a poorly differentiated adenocarcinoma. Among those 40 reviewed adenocarcinomas without EGFR mutations, 20 (50%) were well differentiated adenocarcinomas (including five with AD-BAC and one with a predominant papillary pattern). No pure BACs were observed in either group. None of the tumours carrying EGFR mutations had concomitant KRAS. Conde E, et al. Clin Cancer Res 2006;12:710 7

11 Characteristics of lung adenocarcinomas with ALK rearrangements No. Age (years) Sex Tumour site Tumour sample % tumour cells Main pattern Signet ring cells (%) 1 60 Female Lung CNB 20 Solid/cribiform Yes (100) Female Lung Surgical 30 Solid/cribiform Yes (90) Female Lung Surgical 70 Solid/cribiform Yes (20) Female Lung BB 70 Acinar No Female Lung Surgical 50 Solid/cribiform No Female Lung CNB 50 Acinar Yes (50) Female Soft tissue Surgical 50 Acinar No Female Lymph node Surgical 90 Acinar No Male Liver CNB 70 Solid/cribiform No Conde E, et al. Histopathology 2013;62:609 16

12 Recommendations from the CAP/IASLC/AMP guidelines: Primary or metastatic lesions? To determine EGFR and ALK status for initial treatment selection, primary tumours or metastatic lesions are equally suitable for testing Lindeman T, et al. J Mol Diagn 2013;15:415 53; Vignot S, et al. J Clin Oncol 2013;31:

13 Recommendations MUTACIONES DE EGFR: Fase pre-analítica from the CAP/IASLC/AMP guidelines: When to test for EGFR and ALK Tissue should be prioritized for EGFR and ALK testing.testing should be ordered at the time of diagnosis for patients presenting with advanced-stage disease.or at time of recurrence or progression in patients who originally presented with lower-stage disease Reflex testing, a testing policy that does not require a separate clinician order for each case, is appropriate if agreed upon by the lung cancer care team and may help to ensure expedited and consistent routing of specimens for molecular testing Lindeman T, et al. J Mol Diagn 2013;15:415 53

14 Recommendations MUTACIONES DE EGFR: Fase pre-analítica from the CAP/IASLC/AMP guidelines: How quickly should results be available? EGFR and ALK results should be available within 2 weeks (10 working days) of receiving the specimen in the laboratory Lindeman T, et al. J Mol Diagn 2013;15:415 53

15 New Yorker, Feb 2013

16 J Thoracic Oncol 2012

17 Small thoracic samples MUTACIONES DE EGFR A testing protocol used at Laboratorio de Diana Terapéuticas H&E Section 1 Diagnosis Section 2 IHC Step A1 Step A2 Step A3 Step A4 Section 3 Section 4 5 µm Section 5 Biomarkers Section 6 5 µm Section 7 Sections µm Section 19 S H&E S S P40- TTF1 + S S S S S NSCLC- NOS NSCLC-NOS favours AC EGFR mutation ALK** translocation KRAS mutation ROS1 translocation DNA extraction HER2 mutación H&E S H&E BRAF mutación AC Step B1 (~15%) [41,42] (10,5%) [42] (21%) [42] (1,7%) [43] Step B2 Step B3 HER2 (3,6%) BRAF (2%) Section 1 Section 2 5 µm Section 3 * Section 4 5 µm Section 5 * Sections µm Section 17 *Note that the Vysis package insert recommends 5μm sections for FISH analysis **ALK testing can be conducted using either of the CE-marked kits for FISH or IHC Conde E, et al. Clin Transl Oncol 2013;15:503 8

18 Recommendations for the standardisation of the preanalytical phase Parameter Tumour heterogeneity Sample type Minimum sample size Sample collection and storage Recommendation Tumour heterogeneity (intertumoural and intratumoural) may confound interpretation At least three representative areas should be assessed per tumour section if possible Tissue block preferred Cytospin cell block might be acceptable with further validation Cytology smears not acceptable for IHC and FISH Sample type should be recorded (e.g. excision, biopsy, cytology) Tumour tissue section area: approximately 1 2 mm 2, excluding nontumour cells and tissue areas Cell number: FISH: >100 assessable tumour cell nuclei IHC/mutation: ~2,000 cells Direct sequencing requires high tumour cell content (50 70% tumour cells) Fixation in 10% neutral buffered formalin and paraffin embedding are standard Bouin s or mercury-containing fixatives should be avoided Fixative should be recorded Sample should be stored as a block, not precut slides If slides are precut, date of sectioning should be recorded Eberhard DA, et al. J Clin Oncol 2008;26:983 94

19 INDICE Introducción CAP guidelines: biomarcadores en pulmón - EGFR - ALK Nuevas perspectivas - Más biomarcadores - Nuevas tecnologías - Redundancia molecular Conclusiones

20 MUTACIONES DE EGFR: Fase pre-analítica

21 EGFR Alteraciones de EGFR: Predictivas de la Sensibilidad a Fármacos anti- EGFR

22 Estudio mutacional de EGFR: fase analítica PCR cuantitativa en tiempo real EGFR Scorpions ARMS (DxS/Qiagen) Cobas EGFR (Roche) EGFR E746-A750del Ct=2.75<12 (cut-off) VENTAJAS Mayor sensibilidad Kit comercial (estandarización) Fácil manejo e interpretación DESVENTAJAS Coste No detecta todas las mutaciones Puede no discriminar entre las distintas deleciones o inserciones

23 EGFR Sensitivity Limit of Detection (LOD) Angulo et al. PLoS ONE 2012

24 EGFR 24 Cobas EGFR Mutation Test Study Objectives Lopez-Rios et al. J Clin Pathol 2013

25 INDICE Introducción CAP guidelines: biomarcadores en pulmón - EGFR - ALK Nuevas perspectivas - Más biomarcadores - Nuevas tecnologías - Redundancia molecular Conclusiones

26 Recommendations from the CAP/IASLC/AMP guidelines: How to test for ALK Laboratories should use an ALK FISH assay using dual-labelled break-apart probes for selecting patients for ALK TKI therapy; ALK IHC, if carefully validated, may be considered as a screening methodology Consensus opinion: A pathologist should be involved in the selection [and interpretation] of sections for ALK FISH. Lindeman T, et al. J Mol Diagn 2013;15:415 53

27 Vysis ALK break-apart FISH probe FISH ALK

28 Positive ALK staining with IHC

29 INDICE Introducción CAP guidelines: biomarcadores en pulmón - EGFR - ALK Nuevas perspectivas - Más biomarcadores - Nuevas tecnologías - Redundancia molecular Conclusiones

30 Small thoracic samples New biomarkers Diagnosis Biomarkers H&E IHC Approved Non-approved Lap A1 Lap A2 Lap A3 Lap A4 Section 1 Section 2 Section 3 Section 4 5 µm Section 5 Section 6 Section 7 Section 8 Section 9 Sections µm Section 21 S S S H&E P63- TTF1+ NSCLC- NOS NSCLC-NOS favours AC S EGFR mutation S ALK translocation S MET amplification S ROS1 translocation S EGFR protein expression S MET protein expression DNA extraction HER2 mutation S H&E S H&E BRAF mutation AC Section 1 Lap B1 (16%) [x] (10,5%) (14.3%) (1,7%) [x] (36-43%) [x] (62,9%) Section 2 5 µm Lap B2 Section 3 Section 4 Section 5 Section 6 Lap B3 Section 7 HER2 (3,6%) BRAF (2%) Sections µm Section 19 Conde et al. Clin Transl Oncol 2013

31 INDICE Introducción CAP guidelines: biomarcadores en pulmón - EGFR - ALK Nuevas perspectivas - Más biomarcadores - Nuevas tecnologías - Redundancia molecular Conclusiones

32 Personal Genome Machine (PGM)

33 33-50 genes - Lung/Colorrectal ABL1 EGFR GNAS KRAS PTPN11 AKT1 ERBB2 GNAQ MET RB1 ALK ERBB4 HNF1A MLH1 RET APC EZH2 HRAS MPL SMAD4 AKT1 ALK BRAF CTNNB1 DDR2 EGFR ERBB2 ERBB4 FBXW7 FGFR1 FGFR2 FGFR3 KRAS MAP2K1 MET NOTCH1 NRAS PIK3CA PTEN SMAD4 ATM FBXW7 IDH1 NOTCH1 SMARCB1 BRAF FGFR1 IDH2 NPM1 SMO STK11 TP53 CDH1 FGFR2 JAK2 NRAS SRC CDKN2A FGFR3 JAK3 PDGFRA STK11 - Panel BRCA1/2 CSF1R FLT3 KDR PIK3CA TP53 CTNNB1 GNA11 KIT PTEN VHL

34 INDICE 34 Introducción CAP guidelines: biomarcadores en pulmón - EGFR - ALK Nuevas perspectivas - Más biomarcadores - Nuevas tecnologías - Redundancia molecular Conclusiones

35 EGFR WT exones LUNG ADENOCARCINOMA KRAS Mutation G12C (GGT>TGT)

36 Conclusiones De un solo test a muchos Informe integrado Selección de muestra y método Priorización refleja Comunicación Recordar la redundancia molecular

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