Histopathology

We have continued to investigate a number of themes, the common thread being the role of adult stem cells in neoplasia. How mutations are fixed and spread within the gastrointestinal tract, particularly in pre-malignant conditions, is poorly understood and has important implications for carcinogenesis.

We have used Barrett's oesophagus and ulcerative colitis to analyse how a mutated clone with a selective advantage can clonally expand to fill an entire segment of mucosa at the expense of competing clones (selective sweep to fixation model), assessing clonality at a high resolution by microdissecting and genetically analysing individual crypts. In colitis-associated neoplasias we found that in most lesions an oncogenic mutation could be identified in all crypts across the lesion showing monoclonality. This founder mutation was a p53 lesion in the majority of neoplasms but four tumors had an initiating K-RAS mutation. Some nondysplastic crypts surrounding areas of dysplasia were found to contain clonal p53 mutations and in one case three clonal tumors arose from a patch of nondysplastic crypts containing a K-RAS mutation. Thus p53 mutation is initiating mutation in the majority of lesions, but K-RAS activation as an alternative gatekeeping mutation and we also showed local and segmental field cancerisation was present by showing prooncogenic mutations in nondysplastic crypts surrounding neoplasms.

In contrast, in Barrett's oesophagus we found marked clonal heterogeneity, with multiple independent clones present, showing that that Barrett's heterogeneity arises from multiple independent clones, in contrast to the selective sweep to fixation model of clonal expansion previously described. We identified a p16 point mutation arising in the squamous epithelium of the oesophageal gland duct, which was also present in a contiguous metaplastic crypt, whereas neo-squamous islands arising from squamous ducts were wild-type with respect to surrounding Barrett's dysplasia. It appears that the squamous gland ducts situated throughout the oesophagus are the source of a progenitor cell that may be susceptible to gene mutation resulting in conversion to Barrett's metaplastic epithelium. Additionally, these data suggest that wild-type ducts may be the source of neo-squamous islands. Additionally, we have found a patch of intestinal metaplasia in the stomach which shows a clonal APC mutation, further establishing the clonal nature of the lesion and developed a computational framework to study the balance between the mutation rate and the balance between positive and detrimental selection during cancer growth. We have built on our understanding of the early stages of development of polyps in human familial adenomatous polyposis and in Apc(min/+) mice that model this genetic susceptibility to cancer. early lesions in humans and mice with abnormalities in the gene, and tested in Apc(min/+) mice certain inhibitors of tyrosine kinases that are in clinical trials for human colorectal cancer. We have also have established a protocol in the lab for sequencing methylation patterns from laser-capture micro-dissected human epithelial tissue. In collaboration with the Tomlinson lab, we carried out an analysis of expression of 22 genes using in situ hybridisation showed that the situation in vivo was more complex than apparent from preliminary validation by quantitative RT/PCR. Several of the newly identified, differentially expressed genes represent potential diagnostic or therapeutic targets for intestinal tumours.

We have continued to use mtDNA mutation analysis to locate the stem cell niche and to tracing cell lineages several human tissues. In the liver we have show that clonal proliferative units exist in the human liver, an origin from a periportal niche is most likely, and that the trajectory of the units is compatible with a migration of cells from the periportal regions to the hepatic veins. In the pancreas, exocrine tissue progenitors appeared to be located in interlobular ducts. In the skin, the origin of a basal cell carcinoma appeared to be from the outer root sheath of the hair follicle, establishing this as general method in which stem cell niches and stem cell progeny can be recognised, also affording the generation of cell fate maps, all in human tissues. This technique also allows analysis of the origin of human tumours from specific tissue sites.

Continuing our research into the BM-derivation of renal epithelium, we have shown that mesenchymal stem cells isolated, cloned, and cultured but retaining tri-lineage potential, do not engraft or fuse to assist renal regeneration after injury causing acute tubular necrosis; in contrast, co-administered MSC depleted whole BM does contribute to regeneration. Our recent results suggest that β3-integrin plays an important role in the engraftment of BMDC and angiogenesis and that systemically administered MSCs are recruited to and engraft at sides of inflammation in experimental colitis.

We have continued our attempts to over-express the human gastrokine-2 protein using a variety of techniques and intend to use the protein to look at interactions with TFF2.

With support from Dr Michael Ellis of Digital Scientific (Cambridge, UK) we tested a tuneable selective emission filter for fluorescence microscopy and found this could be used to improve signal specificity when using complex chromophores with broad spectra that are commonly used for immunohistochemistry, but interfere with dyes often used for FISH probes.

For a list of refereed research papers, see Publications (in navigation on left).