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KRAS: ONE ACTOR, MANY POTENTIAL ROLES IN DIAGNOSIS
Loredana Bruno
UNIVERSITÀ DEGLI STUDI DI PALERMO
Scuola di Specializzazione in Biochimica Clinica
Direttore Prof. Marcello Ciaccio
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
KRAS gene
Mutations hot-spots
59 61
5’ 3’
ex2 ex3 ex4 ex5 ex6 ex1
12
13 117 146
• 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)
KRAS and cancer
EGFR
NUCLEUS
LIGAND
PIP3 MEK ERK
PTEN
RAS
B-RAF
PI3K
PIP2
MAPK AKT
mTOR
e.g. EGF, TGFa, Amphiregulin, Epiregulin
METASTASIS
PROLIFERATION SURVIVAL
ANGIOGENESIS
e.g. VEGF
EGFR pathway map
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
E.R. Fearon, B. Vogelstein. Cell 1990
NormalEpithelium Dysp lastic ACF Ear lyAdenoma LateAdenoma Carcinoma MetastasisIntermediate Adenoma
APC/ -catenin K-RAS 18q p53 OtherChanges?
NormalEpithelium
Dysplastic
ACF EarlyAdenoma
Late
AdenomaCarcinoma Metastasis
Intermediate Adenoma
APC/ -catenin K-RAS 18q p53Other
Changes?
PIK3CA BRAF
Genetic Model for Colorectal Tumorigenesis
(35-45%)
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
Amado et al. J Clin Oncol 2008;
Bokemeyer C et al. 2009 and 2011
Van Custem E et al. 2009 and 2011
AIOM society has recommended determination of KRAS mutation status in all patients with metastatic colorectal cancer who are candidates for anti-EGFR therapy (www.aiom.it)
Genetic testing includes research of mutation in exons 2-3-4 of KRAS and NRAS genes
SURVIVAL
ANGIOGENESIS
e.g. VEGF
PI3K
PIP2
PIP3 PTEN
AKT
mTOR
NUCLEUS
LIGAND
EGFR
Blocks EGFR dimerization
METASTASIS
PROLIFERATION
RAF
MEK ERK
MAPK
RAS
Wt
Anti EGFR Abs (Cetuximab, Panitumumab)
Anti-EGFR Abs: KRAS Wild Type
SURVIVAL
ANGIOGENESIS
e.g. VEGF
PI3K
PIP2
PIP3 PTEN
AKT
mTOR
NUCLEUS
LIGAND
EGFR
Blocks EGFR dimerization
METASTASIS
PROLIFERATION
MEK ERK
MAPK
RAF
RAS
Mutation
Anti EGFR Abs (Cetuximab, Panitunumab)
Anti-EGFR Abs: KRAS mutation
• 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)
KRAS: Diagnostic role
DNA extraction
Pancreatic Cyst fluid
EUS-FNA
Methods
Biochemical markers dosage
(CEA, CA19.9 etc)
Direct Automated Sequencing (KRAS exons 2 and 3)
Results: part 1
G12D
G12R
G12V
G13D
Q61H
Q61K
Q61L
Mutated
Wild Type
41% 59%
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
n.50 (2011-2014)
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
Results: part 3
KRAS Wild type (n. 30) KRAS mutant (n.20)
Sex - Female - Male
13 17
12 8
CEA (>192 ng/mL) n.d.
12 6
8 3
Mucinous Non-mucinous
12
18
18 2
Codon 12 Mutant Codon 13 Mutant Codon 61 Mutant
- - -
16 1 3
GNAS codon 201 Mutant (performed in n.30 patient)
2 5
90% specificity
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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