1 Genesis and progression of GI cancer a genetic disease Colorectal cancer Fearon and Vogelstein proposed a genetic model to explain the stepwise formation of colorectal cancer (CRC) from normal colonic tissues. The model states: (1) CRC results from mutations in genes with important functions in regulating cell proliferation or DNA repair, (2) mutations in >1 gene are required, and (3) the sequence of mutations is important in determining the formation of CRC. Normal mucosa Characteristics of the two major pathways in CRC Chromosomal instability pathway Genetic alterations through chromosomal losses and gains Deletion 1p Deletion 8p LOH 17p LOH 18q APC COX2 K-ras DCC/Smad4 p53 Early adenoma???? Intermediate adenoma Late adenoma Carcinoma???? Metastasis These altered genes can be divided into two classes: tumour suppressors that either inhibit cell proliferation or promote apoptosis, and oncogenes that promote cell proliferation and tumour progression. β-catenin BAX TCF-4 IGF-IIR TGF-βRII MLH1 MSH2 MSH6 Epigenetics CIMP hypermethylation Microsatellite instability pathway Genetic alterations through defective DNA mismatch repair proteins CRC, Colorectal cancer. Phylogenetically, CRCs can be divided into two molecular subtypes: those with chromosomal instability (CIN) and those with microsatellite instability (MSI). Carcinomas with MSI present cancerinitiating mutations that inactivate the function of mismatch repair (MMR) genes (e.g. MSH2, MSH6, MLH1 and PMS2) leading to hypermutated genomes. This is known as the mutator phenotype. MSI tumours frequently present a CpG island methylator phenotype (CIMP) leading to the repression of tumour suppressor genes including MLH1. 1. Is MSI always related to hereditary colon cancer? 2. Can you comment on potential therapies for hypermutated colon cancer? 3. Can you mention three genes involved in the chromosomal instability pathway? 1 Palmer
Genesis and progression of GI cancer a genetic disease (continued) Gastric cancer The vast majority of gastric cancers are adenocarcinomas, which can be further subdivided into intestinal and diffuse types according to the Lauren classification. Most gastric cancers are associated with infectious agents, including the bacterium Helicobacter pylori and Epstein Barr virus (EBV). A minority are associated with germline mutation in E-cadherin (CDH1) or MMR genes, whereas sporadic MMR-deficient gastric cancers have epigenetic silencing of MLH1 in the context of CIMP. Gene expression or DNA sequencing have been used in molecular profiling of gastric cancer, but have not led to a clear biological classification scheme. More recent studies by The Cancer Genome Atlas (TCGA) have permitted more precise molecular classification of gastric cancer by identifying dysregulated pathways and candidate drivers of distinct classes. Diffuse type MSI-H (-6%) MSI-H (13-2%) E-cadherin mutation (41-5%) p53 mutation (-33%) CD44 aberrant transcript Cyclin E overexpression (1%) CDC25B overexpression N-cadherin overexpression (43%) Twist 1 overexpression (39%) K-sam amplification (33%) c-met amplification (39%) Reduced nm23 (<52%) Genetic alterations in gastric cancer Normal gastric mucosa Early cancer Carcinoma Metastasis Metaplasia (Adenoma) Intestinal type Telomerase activation/ TERT expression p53 mutation (25-63%) K-ras mutation (1%) Reduced p27 expression APC mutation (4-6%) Bcl-2 loss (43%) c-met amplification (19%) Cyclin E overexpression (14-2%) 18q (DCC) loss (5%) β-catenin mutation (27%) c-erbb2 amplification (2%) CD44 aberrant transcript E-cadherin reduction (6%) SIP1 overexpression (55%) Reduced nm23 (52%) Pancreatic cancer Normal tissue Lumen of duct Duct epithelium Submucosa Normal PanIN-1A PanIN-1B PanIN-2 PanIN-3 Overexpression of Her-2/neu Point mutations of K-ras Inactivation of p16 Cancerous tissue Inactivation of p53, DPC4 and BRCA2 The genetic progression of pancreatic carcinoma Published in Expert Reviews in Molecular Medicine by Cambridge University Press (21) Pancreatic adenocarcinoma presents a progression from distinct types of precursor lesions, a propensity for both local invasion and distant metastasis, an extensive stromal reaction (desmoplasia) resulting in a hypovascular and hypoxic microenvironment, reprogramming of cellular metabolism, and evasion of tumour immunity. There is a stepwise progression of pancreatic intraepithelial neoplasia (PanIN) from low grade to high grade in types 1, 2 and 3. These types are associated with accumulating genetic alterations. More than 9% of cases of PanIN of all grades have KRAS mutations. Mutational inactivation of the CDKN2A, p53 and SMAD4 tumour suppressors occurs later in type 2 and type 3 lesions of PanIN. In addition, 8% have activating mutations in GNAS and more than 5% have inactivation of RNF43 (an antagonist of Wnt signalling). The pancreatic adenocarcinoma genome is also characterised by diverse, large-scale chromosomal changes with frequent amplifications, deletions and rearrangements. 1. Are there any gastric cancers with MSI? 2. How are diffuse type gastric carcinomas molecularly defined? 3. What is the most common molecular alteration in pancreatic carcinomas? 2
New molecular characterisation of GI tumours Colorectal cancer Recent studies by TCGA show that nonhypermutated CRC tumours, irrespective of anatomical origin, present equivalent types of copy number, expression profile, DNA methylation and microrna (mirna) changes. Over 9 had a mutation in one or more members of the Wnt signalling pathway, dominantly in APC. However, there were some differences between tumours from the right colon and all other sites. Hypermethylation was more common in the right colon, and three-quarters of hypermutated samples came from the same site, although not all had MSI. Mutation rate (mutations per 1 6 bases) 5 1 1 1 MLH1 MLH3 MSH2 MSH3 MSH6 PMS2 POLE Epigenetic silencing Non-silent Silent.1 Hypermutated Non-hypermutated Tumour site MSI status CIMP status MLH1 silencing Frameshift mutation Missense/nonsense mutation Mutation frequency (%) Mutation frequency (%) 8 6 4 2 8 6 4 2 63% 81% Hypermutated tumours 51% 51% 46% 31% 31% 26% 26% 6% 43% Non-hypermutated tumours 31% ACVR2A APC TGFBR2 BRAF MSH3 MSH6 MYO1B TCF7L2 CASP8 CDC27 FZD3 MIER3 TCERG1 MAP7 PTPN12 18% 11% 1% 9% 9% 7% 6% 5% 3% APC TP53 KRAS TTN PIK3CA FBXW7 SMAD4 NRAS TCF7L2 FAM123B SMAD2 CTNNB1 KIAA184 SOX9 ACVR1B GPC6 EDNRB 93% of non-hypermutated and 97% of hypermutated cases had a deregulated Wnt signalling pathway. New findings included recurrent mutations in FAM123B, ARID1A and SOX9 and very high levels of overexpression of the Wnt ligand receptor gene FZD1. Activation of Wnt signalling and inactivation of the TGF-β signalling pathway result in activation of MYC. Mutational and integrative analyses emphasise the critical role of MYC in CRC. Integrated analysis revealed a diverse set of changes in TCF/LEF-encoding genes, suggesting additional roles for TCF/LEF factors. Mutations in the ubiquitin ligases RNF43 and ZNRF3 or fusions of RSPO2/3 genes are alterations that activate Wnt/beta-catenin oncogenic signalling and represent a promising level for drug intervention. 1. Which oncogenic pathway is the most frequently altered by mutations in CRC? 2. Is the TGF-β pathway activated or inactivated by mutations in CRC? 3. Which genes present fusions that activate oncogenic Wnt signalling? 3 Palmer
New molecular characterisation of GI tumours (continued) Gastric cancer Recent studies by TCGA propose a molecular classification dividing gastric cancer into four subtypes: 1. Tumours positive for EBV, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and DCD1LG2 (also known as PD-L2). Subtype Tumour purity TP53 mut. SCNA high Diffuse MLH1 silencing MSI high CDKN2A silencing PIK3CA mut. EBV positive 1 5 Mutations per Mb Copy number 1 5 1 Chr 1 Chr 22 EBV MSI GS CIN Tumour purity Not available Molecular/clinical features Yes No Not available Gain Loss DNA methylation mrna microrna Protein EBV positive 295 26 EBV (EBV-CIMP) 64 MSI (hypermutated) MSI high 269 58 GS (Genomically stable) 25 SCNA high cluster 147 CIN (chrom. instability) 227 tumours 2. Microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins. 3. Genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins. 1 12 4. Tumours with CIN, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies. Lauren classification diffuse (%) Males (%) 75 5 25 1 75 5 25 EBV MSI GS CIN Age at initial diagnosis Number of samples 9 6 3 9 8 7 6 5 4 GE junction cardia Fundus body Antrum pylorus EBV MSI GS CIN EBV MSI GS CIN 1. Do chromosome instability and mutations in tyrosine kinase receptors frequently co-occur in gastric cancer? 2. Are mutations in the PIK3CA gene frequent in microsatellite unstable gastric tumours? 3. With which molecular subtype of gastric cancer is a diffuse histology related? 4
New molecular characterisation of GI tumours (continued) Pancreatic cancer Somatic mutations in ataxia telangiectasia mutated (ATM) are present in significant proportions of patients (8%), highlighting the importance of BRCA-mediated DNA damage repair mechanisms in sporadic pancreatic ductal adenocarcinoma (PDAC) as well as familial disease. Mutations in genes involved in chromatin remodelling such as ARID1A, EPC1 and ARID2 are frequently observed, indicating chromatin remodelling may have an important role in PDAC. Novel mutations in genes traditionally described for their roles in axon guidance have been observed by a combination of genomic data and supportive experimental evidence from independent murine Sleeping Beauty (SB) mutagenesis screens. Axon guidance is integral to organogenesis, regeneration, wound healing and other basic cellular processes. The widespread genomic aberrations observed in axon guidance genes suggest they may have a role in PDAC. This observation joins mounting evidence in other cancer, including a recent report demonstrating ROBO2 mutations in liver fluke-associated cholangiocarcinoma. CNV, Copy number variation; IPMN, intraductal papillary mucinous neoplasm; LOH, loss of heterozygosity; PDAC, pancreatic ductal adenocarcinoma. 1. Which genes involved in chromatin remodelling are significantly mutated in pancreatic cancer? 2. Is BRCA-dependent DNA repair a cellular function altered by mutations in pancreatic cancer? 3. Are genes involved in axon guidance altered in pancreatic cancer? 5 Palmer
Summary: CRC progression is the consequence of a stepwise accumulation of mutations in tumour suppressor genes and oncogenes, the most frequent alteration observed being activation of the Wnt/beta-catenin pathway Both CRC and gastric cancer present a major group of non-hypermutated tumours and a minor population of hypermutated/msi tumours Pancreatic cancer progressively accumulates mutations in KRAS, CDKN2A, p53 and SMAD4, but also presents alterations in genes involved in chromatin remodelling and axon guidance Further Reading Biankin AV, Waddell N, Kassahn KS, et al. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature 212; 491:399 45. Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature 212; 487:33 337. Cancer Genome Atlas Research Network. Comprehensive molecular characterization of gastric adenocarcinoma. Nature 214; 513:22 29. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 199; 61:759 767. Giannakis M, Hodis E, Jasmine Mu X, et al. RNF43 is frequently mutated in colorectal and endometrial cancers. Nat Genet 214; 46:1264 1266. Seshagiri S, Stawiski EW, Durinck S, et al. Recurrent R-spondin fusions in colon cancer. Nature 212; 488:66 664. 6