Do we know how cancer develops in Barrett's esophagus?

The normal cell cycle

In most tissues of the body, cells multiply through a process known as the cell cycle. Before cells can multiply and divide into other cells, they have to make exact copies of their DNA. DNA is the genetic code that is in all the cells of our bodies and is exactly the same code in each cell no matter what tissue the cell is from. Chromosomes are made up of the genes of our cells and our genes are made up of strands of DNA. Each cell of our body has two copies of each gene, one inherited from our mother and one from our father. The nucleus of the cell houses our chromosomes and genes.

Normally, most cells are not actively growing and dividing and are in the G0 or resting phase of the cell cycle and have a diploid or 2N DNA content. Cells in the G1 phase are actively cycling but like G0 cells have a "diploid" or 2N DNA content. A small percentage of cells in normal tissues are undergoing DNA synthesis (making a copy of their DNA) and are in the S phase of the cell cycle (have a DNA content between 2N and 4N). A few cells have completed their DNA synthesis and doubled their amount of DNA and are in the G2 phase of the cell cycle (have a 4N or tetraploid DNA content). After cells double their DNA, they undergo mitosis (M phase) dividing into two daughter cells that are exact genetic copies of each other and have a DNA content of 2N.

Nowell's hypothesis

There are many events or steps that occur in Barrett's esophagus that lead to the development of cancer. A few of these events are known but most are not. Most of the known events appear to occur early, before high-grade dysplasia or cancer actually develops. No one knows what the late events are that give cells the ability to leave their normal growth boundaries and become a cancer.

It is now widely accepted that the development of most cancers is due to something called genomic or genetic instability. This theory was first proposed by Dr. Peter Nowell in 1976. The theory is that for some unknown reason, perhaps due to environmental factors or inherited factors, some cells in the body develop genetic abnormalities that give them the ability to outgrow genetically normal cells. These abnormal cells grow and expand into a clone of cells (a group of cells having the same genetic make-up) and may replace their neighboring normal cells. Eventually one of the abnormal clones may undergo another genetic change that leads to the development of a sub-clonal population with the expansion of this cell line into its own large clone of cells. As multiple genetic abnormalities occur, multiple sub-clones develop or evolve. Eventually, one of these sub-clones may acquire the necessary combination of genetic abnormalities to become a cancer.

Ball diagram of Nowell's hypothesis

The green balls represent cells that have developed a genetic abnormality and are expanding or growing into a clone of cells. One of these cells develops a second genetic abnormality, illustrated by a blue ball, seen to expand into its own clone of cells or subclones of the green population. A third genetic mistake is made, illustrated by a dark red ball, with clonal expansion of this cell population. Eventually, another genetic mistake is made in one of the cells of the dark red population that allows that cell to become a cancer.

Cell cycle checkpoint genes

Incredibly, genetic mistakes are rarely made in the duplication of a cell's DNA. If a genetic mistake is made, for example - caused by exposure of a cell to radiation, there are genes (called cell cycle checkpoint genes) that control the cell cycle and prevent cells from dividing into two daughter cells. These cell cycle checkpoint genes insure that abnormal clones of cells will not be produced by the tissues of our bodies under normal circumstances.

The p53 gene
The p53 gene was the first cell cycle checkpoint gene to be discovered in humans. It is referred to as a tumor suppressor gene because its normal function is to suppress the development of tumors by detecting genetic mistakes in G1 cells resulting in arrested cell growth (cell cycle arrest) or destruction (programmed cell death) of the cells with the mistake. When genetic abnormalities develop in the p53 gene leading to loss of its normal function, tumors more readily develop because cells with genetic mistakes are allowed to divide and pass the mistake on to daughter cells.

p53 gene abnormalities are detected in up to 95% of Barrett's associated cancers indicating that loss of function of the p53 gene is a necessary step in the progression to cancer in Barrett's esophagus. Loss of function of the p53 gene in Barrett's esophagus is one of the earliest known genetic events in the development of cancer and it is closely tied to abnormalities that develop in the cell cycle of Barrett's cells. These abnormalities can be detected by a test called flow cytometry (a test that measures the amount of DNA in a cell).

Increased 4N fraction
The earliest abnormality in the cell cycle of Barrett's cells that can be detected by flow cytometry is an increase in the percentage of cells that have doubled their amount of DNA (4N cells). It has been shown that this increase in percentage of 4N cells corresponds to loss of function of the p53 gene. These 4N cells appear to be unstable and lead to the development of aneuploid cells, cells that have multiple genetic errors or mistakes. Frequently in Barrett's high-grade dysplasia and cancer, multiple different aneuploid cell populations representing multiple sub-clones can be detected. There is evidence that these populations develop by clonal evolution and expansion similar to that proposed by Nowell.

Increased numbers of 4N cells and abnormalities in the p53 gene are some of the earliest abnormalities detected in the Barrett's lining and can be detected BEFORE high-grade dysplasia or cancer develops. In fact, abnormalities in the p53 gene can be detected in some apparently normal diploid (2N) Barrett's cells prior to the development of increased 4N cells, aneuploidy or high-grade dysplasia in Barrett's esophagus. One large study that followed Barrett's patients with and without a p53 gene abnormality reported that patients with a p53 gene abnormality had a significantly increased chance of developing cancer as compared to Barrett's patients who did NOT have a p53 gene abnormality. In this study, patients with a p53 gene abnormality also developed increased numbers of 4N cells, aneuploidy, and high-grade dysplasia much more frequently than patients without a p53 gene abnormality.

The p16 gene
In addition to the p53 gene, other genes are inactivated, or their functions lost, in the progression to cancer in Barrett's esophagus. Another gene, called p16, is frequently abnormal in Barrett's esophagus. p16 is also a tumor suppressor gene and, like p53, inhibits the transition of cells from G1 to S phase. Abnormalities in the p16 gene are the earliest known genetic abnormalities in Barrett's esophagus and can be detected prior to the development of p53 gene abnormalities, increased 4N cells, aneuploid cells, or high-grade dysplasia. Recently, it has been shown that there are several types of p16 gene abnormalities that can affect one or both copies of the p16 gene. What was surprising about this study, was that more than 85% of all Barrett's linings have at least one of these abnormalities in their p16 gene. In fact, the number of abnormalities in the p16 gene increases with the increase in the length of the Barrett's lining. Very short Barrett's linings tend NOT to have p16 gene abnormalities, intermediate length Barrett's linings tend to have only one abnormality in the p16 gene, and very long Barrett's linings tend to have 2 abnormalities. This has led investigators to hypothesize that the Barrett's lining starts out as a single clone of cells that develops p16 gene abnormalities very early on, causing the Barrett's to spread along the esophagus. The greater the number of p16 gene abnormalities, the greater the ability of these cells to spread and grow and extend the length of the Barrett's segment. In addition to causing spread of the Barrett's lining, p16 gene abnormalities may make it easier for other gene abnormalities to develop in the Barrett's lining or for these other gene abnormalities to spread over wide areas of the Barrett's lining. However, as most patients' Barrett's linings have at least one p16 gene abnormality and most patients with Barrett's do NOT develop cancer during their lifetime, including most people with long Barrett's linings, many more genetic errors must take place before a cancer can develop.

Other genes
Other genes that tend to develop abnormalities in the progression to cancer include genes on chromosome 5, 13 and 18. Clearly, there are many other genes involved in the progression to cancer in Barrett's esophagus that are yet to be identified and their roles characterized.

Summary

Cancer in Barrett's esophagus develops in a step-wise fashion from metaplasia to dysplasia to cancer. This step-wise progression occurs through clonal evolution similar to that proposed by Nowell, but has been shown to be more complex, with multiple subclones developing in the Barrett's tissue prior to the development of cancer. Flow cytometric abnormalities can be detected early in Barrett's esophagus and before the development of high-grade dysplasia and cancer. These abnormalities include increased 4N and aneuploid cell populations. Genetic abnormalities in the p53 and p16 genes with loss of function in these genes, occur even earlier than flow cytometric abnormalities. Previously it was known that p53 gene and p16 gene abnormalities are present in the vast majority of patients with a Barrett's associated cancer. It has recently been confirmed that having a p53 gene abnormality greatly increases the risk of developing cancer in Barrett's esophagus. We now also know that p16 gene abnormalities are the earliest gene abnormalities yet detected in Barrett's esophagus, present in more than 85% of Barrett's linings. It is hypothesized that p16 abnormalities contribute to the expansion of Barrett's cells along the surface of the esophagus, as well as to the expansion or spread of additional gene abnormalities that occur during progression to cancer in Barrett's esophagus.

Other genes develop abnormalities in the progression to Barrett's esophagus but their relationship to flow cytometric abnormalities or the development of cancer is less clear than those of p53 and p16. Identification of additional genes will lead to a better understanding of how cancer develops, tests to determine who is at risk for developing cancer, and better therapy in the treatment of cancer and Barrett's esophagus.

At the present time, checking a patient's Barrett's cells for gene abnormalities is not routinely done for clinical care and is usually performed as part of research in Barrett's esophagus and esophageal adenocarcinoma. Currently, it is not known which genetic tests or panel of tests will reliably be useful in predicting who will develop cancer in Barrett's esophagus and therefore, at the present time, no therapy should be recommended to a patient based on the results of any genetic test.