Defining colorectal cancer development through colonic crypt dynamics, somatic mutations and their spatial distribution in normal colorectal mucosa

Bowel cancer affects 1.6 million individuals globally every year and is the third most common cancer in the UK causing 16,000 deaths per year. Patients are more likely to survive if they are diagnosed earlier. Even better still is to understand how bowel cancer develops in the first place, identify those patients who have the greatest risk and screen them earlier and more often. However, we do not fully understand all the events that led up to bowel cancer developing and why some patients will develop cancer and others will not. We know that a bowel cancer grows because changes occur in the DNA code (mutations). These mutations are caused by many factors such as diet, smoking, alcohol, bacteria in stool. We also know that these mutations occur and accumulate in the normal healthy bowel before a cancer starts growing. Therefore understanding mutations in normal bowel, how quickly they accumulate and their local effects is key to understanding what causes tumours to grow in the first place. By understanding this process we may be able to reduce bowel cancer.

Patients included in this study will be patients undergoing surgery for bowel cancer as well as patients who have surgery for other reasons other than cancer. We will collect spare tissue that is left over from their operation. This bowel tissue can then be treated with chemical stains that allow mutations to be seen under the microscope. Digital pictures will be taken of the mutations and a computer programme will tell us information about them. We can then look at how mutations vary with patient factors such as age, diet, medical conditions, geography etc.

There are a number of questions we want to explore. Firstly, are there are any differences in mutations between patients with bowel cancer and those without? We hope to find out more detail about these differences and identify factors that could potentially be contributing to them.

Secondly, we want to study how these mutations are spread throughout the bowel. We want to look at whether mutations cluster or if they are spread evenly and if they are spread differently between the right side of the bowel and the left side of the bowel. This is because they may be related to the microbiome; the populations of bacteria that live in the bowel. The microbiome can be different throughout the bowel and certain bacteria may cluster in different areas. By studying mutation patterns we might be able to find further evidence to their link with the bacteria.

Thirdly we wish to explore differences in mutations from patients in the UK compared to patients from difference locations around the world. We know that one of the reasons for the differences in the rates of bowel cancer worldwide is due to differences in diet and lifestyle. We have collaborators from pathology laboratories worldwide in Vietnam, Argentina, Chile, South Africa and Russia. They will collect tissue and send it to be included in our study so that we can compare the mutation rates in the UK cohort to the mutation rates from the cohorts of other countries. This may shed some insight into the differences in the rates of bowel cancer worldwide.

We hope that our research can be used in the future to adapt bowel cancer screening so that certain groups of patients who are more at risk of developing bowel cancer can be screened earlier and pick up more cancers when they are very early and more treatable.

Around half of somatic mutations present in colorectal cancers (CRC) have arisen previously in normal colorectal epithelium. To become fixed, mutations must occur in colonic stem cells that succeed in populating one of the proliferative niches that define the crypt. The mutation then repopulates the crypt by clonal expansion to form a fixed mutant clone.

This project will enable the frequency, spatial distribution and fixation rate of mutated clones within the mucosa of the human large intestine to be quantified and compared to the primary tumour site using multiple clonal crypt markers. Are mutant clones random and due to errors of DNA repair? Do they cluster due to toxins released from bacterial blooms or secondary mutational events occurring in prior expansions of somatically altered epithelium? As well as the distribution of mutant clones, the colonic crypt fusion/fission rate will also be assessed, the incidence of driver mutations and if there is any association between these.

The longer-term objective is to understand the factors that dictate the age-related burden and distribution of somatic mutations and to establish the analytical approaches to define and quantify their impact. Mutation rate and rates of clone fixation and expansion within the background normal colorectal epithelium between patients with and without colorectal cancer will be compared. How the distribution of these events change with patient demographics and cancer type will be assessed as well as comparing patients from different global locations. For patients with cancer, the spatial distribution of mutations and relation to tumour proximity will also be assessed. By quantifying these, the differences in background mutation and clone fixation/expansion rate in these groups will be determined.

Our current research has utilised colonic clone dynamics to generate a benchmark of how mutations arise, become fixed and expand within the normal large intestine. This benchmark has been derived from a subset of the general population, namely in patients with cancer. Differences in clone dynamics that associate with different clinicopathological factors that potentially include genetic, environmental and lifestyle factors may result in departures from this benchmark and may relate to disease risk. This research will generate impact by beginning to unpick if this is true in different patient cohorts. This will be achieved by documenting stepwise how different age-related mutational burdens is achieved; by altered mutation rates, efficiency in fixation arising from different stem cell dynamics, lateral expansion through crypt fission or a combination of these. This may allow prevention strategies to be targeted to the most appropriate process for a given patient group and identify those most at risk for enhanced screening.
The following groups will impacted by this research

Patients and the General Public
This research will identify those patients who may be higher risk individuals for bowel cancer due to their increased background mutation rates. Those cases that display higher mutation rates can then be interrogated further to investigate other factors that may associate with increased mutations. In particular, there is much interest that the microbiome may either initiate mutations or promote expansion of pre-existing mutations. It is also thought inflammation may be contributing to promotion of mutations. This research can explore these potentially modifiable factors alongside the mutation patterns and identify other modifiable factors.

Future research can then build upon exploring interventions that affect these factors and potentially prevent bowel cancer developing in a natural life span. For example the use of aspirin, metformin, fish oil etc which is thought to reduce bowel cancer risk by lowering inflammation. Another approach maybe to develop techniques to identify those at higher risk and provide more intensive screening at an earlier age which will identify more cancers at an earlier stage where they are more treatable. As 1 in 15 men and 1 in 18 women will be diagnosed with bowel cancer during their lifetime there is potential for many future patients to be impacted by this research.

The NHS and healthcare providers worldwide
If we can identify individuals to screen at an earlier stage or more frequently it would have a significant effect on screening programmes with service cost reductions through earlier detection or reduced incidence. If the microbiome was important then there might be a growth of microbiome altering strategies such as faecal transplant or more simply dietary advice.

Health Policy Makers
Prevention of disease is the most effective strategy to improve outcomes. If disease cannot be completely prevented, early screening is highly important to catch disease at an early stage when it is most treatable. Therefore, if this research is able to identify those factors contributing to the earliest steps of development of colorectal cancer, there may be benefit to policy makers and public health professionals to exploit these to develop improved screening approaches and implement preventative treatments. This may also result in economic benefits by reducing the costs associated with cancer treatments. If dietary alterations are important then new public health initiatives would be required.

International Communities
This research will also investigate populations from other countries which have a low but increasing rate of bowel cancer such as Vietnam and other countries with higher rates such as Chile, Argentina, South Africa and Russia. This may lead to further research on these populations and interventions to help address the increasing rates of bowel cancer in such countries.