Introduction: Houghton and colleagues (2004) demonstrated bone marrow derived cells (BMDCs) to be the origin of epithelial cells in pre-invasive and malignant gastric tumours in Helicobacter felis infected mice. However, this has yet to be replicated in any other experimental scenario. Methods: To clarify the significance of Houghton’s observation we examined four mouse models of gastrointestinal tumourigenesis: the Tff1-/- mouse model of inflammatory gastric adenocarcinoma, the ApcMin/+ and Apc1322T mouse models of familial adenomatous polyposis, and the Il10-/- mouse model of colitis-associated colorectal adenocarcinoma, employing sex-mismatched bone marrow (BM) transplantation (male BM to female recipients) followed by the identification of BMDCs in tissues using Y-chromosome in situ hybridisation (ISH) detection and immunohistochemical phenotyping of cells. To investigate the mechanism for BMDC recruitment into tissues, osteopontin (Opn) mRNA isotopic ISH was employed. Results: In four mouse models there was not a single instance of a gastric gland or intestinal crypt with a patch of clonal Y-chromosome positive (Y+) cells. Y+ cells in the epithelium were very rare and were mostly positive for several markers of immune cells. In contrast, Y+ cells were frequently observed in the stroma. Quantification of BMD-myofibroblasts demonstrated increased recruitment into larger Apc1322T mouse tumours and desmoplastic reaction sites in Tff1-/- mouse tumours, but not into inflamed non-fibrotic tissues. Similarly, Opn mRNA expression was unaffected by inflammation but increased with tumour burden and in desmoplastic reaction sites. In the desmoplastic reaction sites of Tff1-/- mouse tumours, increased osteopontin mRNA expression correlated with increased BMD-myofibroblasts, therefore suggesting some chemoattraction was occurring. Conclusions: In four mouse models of gastrointestinal tumourigenesis BMDCs were not a source of reparative, pre-cancerous, or malignant epithelial cells. Analysis of BM contribution in the stroma demonstrated that BMD-myofibroblast engraftment is driven by increased tumour burden and fibrosis. In addition, the increased presence of BMD-myofibroblasts at desmoplastic reaction sites of Tff1-/- mouse tumours correlated with increased Opn mRNA expression, indicating that osteopontin may act as chemoattractant in desmoplasia.

The high incidence of gastrointestinal cancers in the general population and the presence of premalignant dysplastic precursor lesions in the gastrointestinal tract make the gastrointestinal tract an ideal environment to study cancer clonality and clonal expansion. Background: Intestinal metaplastic (IM) glands in the human stomach are clonal, contain multiple stem cells and spread by fission. This mechanism of gland fission causes field cancerisation. We hypothesised that gastric adenocarcinoma (GA) progresses through a series of genetic events arising from a founder mutation. A process analogous to niche succession may also take place in the normal oesophagus. We hypothesise that oesophageal squamous cell cancer occurs by a process of field cancerisation of the oesophagus. RHBDF2 has been identified as the gene responsible for tylosis with oesophageal carcinoma (TOC). We hypothesise that RHBDF2 germline gain of function mutations might be lost during tumour progression in TOC and this might affect iRhom2 localisation in the cell. Methods and results: A cohort of 23 patients with dysplasia and a cohort of 51 GA patients were screened for genes accounting for 75% of all somatic mutations previously reported in GA. Only 13% of dysplastic patients and 31.4% of GA patients had mutations. Three dysplastic patients and six GA patients were analysed by microdissection. Small gastric cancer foci in a cohort of hereditary diffuse gastric cancer (HDGC) patients (n=5) were also screened by laser-capture microdissection (LCM) for mutations in TP53. A cohort of 30 patients was screened for common mutations in OSCC and for RHBDF2 mutations. 36.36% of the patients presented mutations. Three patients with mutations were randomly selected and areas of oesophageal squamous cell dysplasia and OSCC were analysed by LCM. Three TOC patients were also analysed by LCM and immunohistochemistry was performed for iRhom2 and ADAM17. Conclusions: The usual mutational events established for GA development during the metaplasiadysplasia- carcinoma sequence (MCS) do not fit the results from either of our two LCM mutation studies in the human stomach. Dysplasia was shown to be clonal and GA demonstrates genetic heterogeneity through clonal evolution. Field cancerisation could not be detected in HDGC using TP53 as a clonal marker. The low incidence of OSCC patients with mutations implies that other genes may be involved in the premalignant pathway leading to OSCC. Oesophageal squamous cell dysplasia and OSCC demonstrate clonal expansion through tumour progression. RHBDF2 mutations do not occur in sporadic OSCC but germline RHBDF2 mutations can be lost during tumour progression in TOC patients with LOH in 17q. Overall, the somatic mutation theory of carcinogenesis seems to hold true for both the progression to GA and OSCC, as both carcinomas seem to evolve from a single mutated stem cell and acquire genetic heterogeneity as the tumours evolve.