Beyond the APC era Alternative pathways to CRC Jeremy R Jass McGill University
Outline Limitations of APC model KRAS and serrated polyps CRC and CpG island methylation Serrated pathway to CRC Fusion pathways
Adenoma-carcinoma sequence APC KRAS TP53 Normal Small adenoma Large adenoma Cancer Genetic instability
Familial adenomatous polyposis
Mutation in sporadic colorectal neoplasia Dys ACF TAD CRC APC 19% 33% 60% KRAS 63% 11% 35% TP53 0% 0% 50% B-CAT 0% 13% 0%
Interpretation of mutational spectrum in colorectal neoplasms Minute adenomas initiated by APC or KRAS Mutation of APC and KRAS can occur during progression to CRC Progression to CRC is more likely when both APC and KRAS are mutated Progression more likely if APC mutated first Initiation of TADs explained by multiple mechanisms, not all of which are known Few CRCs have mutation of APC, KRAS & TP53 (Smith et al, PNAS 2002;99:9433)
KRAS and serrated polyps
RAS-RAF-MEK-ERK-MAP-kinase pathway and inhibition of apoptosis KRAS*: Up-regulation of prosurvival Akt/PKB: inactivation of caspase-9 and Bad BRAF*: Inhibition of cytosolic caspase activation following release of mitochondrial cytochrome c *Mutation of either confers resistance to DNA damageinduced apoptosis
Inhibition of apoptosis Loss of anchorage Integrin Basal lamina Cell membrane FAK/PI-3K Ras Akt/PKB caspase-2 caspase-7 caspase-3 caspase-9 Raf X X Bad Cytochrome c release from mitochondria
KRAS, BRAF and DNA methylation Ras RASSF1 Nore1 Raf RasGAP PI3-K Tiam1 Mst1 Mst1 MEK Rsk AKT Rac ERK Bad Casp9 IKK FKH Pro-apoptosis Bcl-2 Anti-apoptosis NF-κB
Pleiotropism of KRAS Hyperplastic ACF 100% Dysplastic ACF 63% Hyperplastic polyp 40% Serrated adenoma 20% Adenoma 15 to 75% CRC 35%
Pathogenesis of serrated polyps Inhibition of exfoliation Inhibition of apoptosis (anoikis) Retention of surface epithelium Cell senescence Growth by crypt fission
Incomplete apoptosis in SP Denatured CK18 without exfoliation? caspase-2 caspase-7 IHC M30/cytodeath CK18
CRC and CpG Island Methylation (CIMP)
Molecular classification of CRC Diploid Aneuploid MSI MSS Meth+ Meth- 2 3 1
Features of CIMP-Pos/ MSS or MSI-L CRC MSI-High-like: Proximal, Mucinous, Poor diff, Serrated, F>M, BRAF+ Non-MSI-H-like: Early onset, few TILs, aggressive, meth MGMT, KRAS (when not BRAF+)
Serrated carcinoma in 17-year-old female MGMT
Serrated pathway to colorectal cancer
Classification of epithelial polyps Neoplastic* Adenoma (TA,TV,VA) Mixed polyp Serrated adenoma Non-neoplastic Hyperplastic polyp Spectrum of serrated polyps *Characterized by cancer-associated genetic alteration
HP-GC HP-MV SSP SA
Mixed polyps Collision? (HP and AD) Sequence? (HP to AD) - Identical genetic changes (MSI) in both components - Adenomatous component usually serrated
SA HP N D2S123
Serrated pathways BRAF/KRAS & DNA Meth MLH1 MP MSI-H CRC N HP MGMT MP MSI-L/ MSS CRC
BRAF and KRAS mutation in colorectal polyps TA TV HP SSP MP SA n.62 n.23 n.46 n.32 n.13 n.15 BRAF 3 (5%) 2 (9%) 30 (65%) 26 (81%) 7 (54%) 5 (33%) KRAS 11 (18%) 11 (48%) 1 (2%) 1 (3%) 6 (46%) 4 (27%)
Patterns of immunohistochemical expression of DNA repair genes Sporadic loss in hyperplastic crypts Loss in expanded and dysplastic subclones (when mutation gives growth advantage)
MLH1
MGMT
MGMT
HNPCC-like families with variable MSI status (Young et al. 2005)
Hyperplastic polyposis
Serrated pathway syndrome (SPS) (11 families from Qld Registry) MSI-variable CRC Mean age of onset 60 years/ F>M Variable polyp background (HP, SA mixed polyp, AD, SSA) Hyperplastic polyposis in up to 10% Concordant DNA methylation in polyps and cancers Concordant mutation of BRAF in most polyps and cancers Unrecognized (confused with HNPCC and FAP, low/mod penetrance and variable phenotype)
MGMT and fusion pathways to CRC
O-6-Methylguanine DNA Methyltransferase (MGMT) Overloaded MMR MSI-L MGMT methylation methylg:t mismatches Futile cycles of DNA repair CIN Demethylation* Mutation KRAS/TP53 *Nasr et al, Mutations of TP53 induce loss of DNA methylation Oncogene 2003;22:1668
Loss of MGMT in advanced serrated polyps (MP, SA, SSA) versus adenomas (Higuchi et al, 2005) Frequency R colon Size Type SP 24% Yes Yes NA AD 13% No No No
CRC arising in adenoma Re-expression of MGMT
Concept of fusion pathways 1. Adenoma APC TP53 2. Serrated polyp BRAF or KRAS DNA methylation
Concept of fusion pathways 1. Adenoma APC TP53 2. Serrated polyp BRAF or KRAS DNA methylation Fusion pathway A MGMT & KRAS Serration/villous
Concept of fusion pathways 1. Adenoma APC TP53 2. Serrated polyp BRAF or KRAS DNA methylation Fusion pathway A Fusion pathway B MGMT & KRAS Serration/villous APC/Wnt MGMT & TP53 MLH1 intact Dysplasia
MGMT MGMT 1. Adenoma with serration 2. Serrated polyp with dysplasia
Conclusion Not all adenomas or CRCs are initiated by APC mutation Most adenomas are dead-end lesions CRCs with DNA methylation are initiated by BRAF or KRAS (serrated pathway) and represent c. 25% of CRC