KRAS: ONE ACTOR, MANY POTENTIAL ROLES IN DIAGNOSIS
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1 UNIVERSITÀ DEGLI STUDI DI PALERMO Scuola di Specializzazione in Biochimica Clinica Direttore Prof. Marcello Ciaccio KRAS: ONE ACTOR, MANY POTENTIAL ROLES IN DIAGNOSIS Loredana Bruno
2 KRAS gene Proto-oncogene on chromosome 12 (12p12) Encodes a 188aa protein with catalytic activity Protein is a 21 KDa GTP-protein and play a role in cell proliferation, cell differentiation, apoptosis Activating mutations in codons 12, 13, 59, 61, 117, 146 Mutations hot-spots ex1 ex2 ex3 ex4 ex5 ex6
3 KRAS and cancer Ras family genes (K-H-N Ras) are the most common targets for somatic gain-of-function mutations in human cancers Activating RAS mutations occur in 30% of human cancers Specific RAS genes are mutated in different cancers KRAS is most frequently mutated in cancer (pancreatic, colorectal, lung, endometrial, and cervical cancers)
4 EGFR pathway map LIGAND e.g. EGF, TGFa, Amphiregulin, Epiregulin PIP 2 EGFR RAS B-RAF PI3K PTEN PIP 3 MEK ERK e.g. VEGF AKT mtor NUCLEUS MAPK METASTASIS ANGIOGENESIS SURVIVAL PROLIFERATION
5 KRAS: a biomarker of... Diagnosis Prognosis Molecular Diagnostic in laboratory medicine Therapy
6 Other APC/ -catenin K-RAS 18q p53 Changes? Normal Dysplastic Early Intermediate Late Epithelium Carcinoma Metastasis ACF Adenoma Adenoma Adenoma KRAS: Prognostic Role RASCAL study multivariate analysis presence of KRAS mutation associated with increased risk of recurrence and death (Andreyev et al. 1998) Only Gly12Val RASCAL II confirmed but only in Dukes C (Stage 3) tumors (Andreyev et al. 2001) Others studies have not confirmed Genetic Model for Colorectal Tumorigenesis APC/ -catenin K-RAS 18q p53 (35-45%) Other Changes? Normal Epithelium Dysplastic ACF Early Adenoma Intermediate Adenoma Late Adenoma Carcinoma Metastasis BRAF PIK3CA E.R. Fearon, B. Vogelstein. Cell 1990
7 KRAS: Predictive Role in therapy KRAS mutations have emerged as a major predictor of resistance to panitumumab or cetuximab in patients with metastatic colorectal cancer AIOM society has recommended determination of KRAS mutation status in all patients with metastatic colorectal cancer who are candidates for anti-egfr therapy ( Genetic testing includes research of mutation in exons of KRAS and NRAS genes Amado et al. J Clin Oncol 2008; Bokemeyer C et al and 2011 Van Custem E et al and 2011
8 Anti-EGFR Abs: KRAS Wild Type LIGAND Anti EGFR Abs (Cetuximab, Panitumumab) Blocks EGFR dimerization Wt PIP 2 EGFR RAS RAF PI3K PTEN PIP 3 MEK ERK e.g. VEGF AKT mtor NUCLEUS MAPK METASTASIS ANGIOGENESIS SURVIVAL PROLIFERATION
9 Anti-EGFR Abs: KRAS mutation LIGAND Anti EGFR Abs (Cetuximab, Panitunumab) Blocks EGFR dimerization Mutation PIP 2 EGFR RAS RAF PI3K PTEN PIP 3 MEK ERK e.g. VEGF AKT NUCLEUS MAPK mtor METASTASIS ANGIOGENESIS SURVIVAL PROLIFERATION
10 KRAS as diagnostic tool for pancreatic cyst lesions: our experience
11 KRAS: Diagnostic role Pancreatic cyst lesions may be incidentally detected by abdominal imaging in up to 13,5% of patients for reason unrelated to the pancreas (Lee et al. 2010) Simple (retention) cysts or pseudocysts or serous cystadenomas lack malignant potential Mucinous cystic neoplasms (MCN) and intraductal papillary mucinous neoplasm (IPMN) have a malignant potential and may require surgical treatment Various combinations of morphology, cytology and tumor markers could not provide diagnostic accuracy (Brugge WR et al 2004) CEA levels (>192ng/mL) can discriminate between mucinous vs non-mucinous (79% accuracy) (Brugge WR et al 2004) KRAS mutations alone had a sensitivity of 45% and specificity of 96% for diagnosing mucinous cysts (Khalid A et al. 2009)
12 Methods DNA extraction EUS-FNA Pancreatic Cyst fluid Biochemical markers dosage (CEA, CA19.9 etc) Direct Automated Sequencing (KRAS exons 2 and 3)
13 Results: part 1 59% 41% n.50 ( ) Mutated Wild Type G12D G12R G12V G13D Q61H Q61K Q61L Type of Mutation N % G12D 8 40 G12R 3 15 G12V 5 25 G13D 1 5 Q61H 1 5 Q61K 1 5 Q61L 1 5
14 Results: part 2 KRAS ex 2 KRAS ex 3 Diagnosi istologica 1 G12D Tumore Maligno Testa del Pancreas 2 G12D IPMN 3 G12D lesione cistica Non Valutabile 4 G12D IPMN 5 G12D Tumore Maligno testa del pancreas 6 G12D IPMN 7 G12D IPMN 8 G12D IPMN 9 G12R IPMN 10 G12R IPMN 11 G12R IPMN 12 G12V Tumore Maligno testa del pancreas 13 G12V IPMN 14 G13D IPMN 15 n Q61H IPMN 16 n Q61K pseudocisti 17 n Q61L IPMN 18 G12V Tumore Maligno testa del pancreas 19 G12V IPMN 20 G12V IPMN
15 Results: part 3 Sex - Female - Male CEA (>192 ng/ml) KRAS Wild type (n. 30) KRAS mutant (n.20) n.d. Mucinous % specificity Non-mucinous 18 2 Codon 12 Mutant Codon 13 Mutant Codon 61 Mutant GNAS codon 201 Mutant (performed in n.30 patient) 2 5
16 Conclusions The current diagnosis of pancreatic cyst type does not reliably allow to distinguish between non-mucinous vs mucinous cysts and non malignant vs potential malignant cysts KRAS is a useful diagnostic tool for identification of pancreatic cyst with mucinous histotype KRAS mutations often are more sensitive than CEA levels New high sensibility methods are needed for KRAS mutational analysis Or new biomarkers?
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