Today's gene hunter

Less than a decade ago, the hunt for cancer predisposition genes would have formed the basis for thousands of PhDs. Today, copy number variations can be picked up in a week.

For Associate Professor Ian Campbell, and the many others working in the area of cancer genetics, the Breast Cancer Association Consortium genome-wide association study is a major breakthrough.

While he wasn't directly involved in the study he did manage to get his name on the paper due to his involvement on the executive committee of kConFab, the research consortium for familial breast cancer which contributed to the study and is chaired by Georgia Chenevix-Trench.

Campbell says the Nature paper is pivotal to the genetics that he's been working on for the past 15 years, although the discovery of five new variants linked to breast cancer predisposition did not come as a surprise.

"Basically we all knew it," he says. "In principle we thought the hypothesis must true, there must be these predisposing alleles, but this has nailed it formally.

"Over the past 10 years people have been dabbling away with case controls of 300 cases, 300 controls, perhaps finding something, but then other people were doing the same and not finding anything. This has pointed out that the only way we are going to do this is in these large studies using thousands of cases and controls. It's a major breakthrough."

Campbell himself works mainly on breast and ovarian cancer as head of the Cancer Genetics Lab at the Peter MacCallum Cancer Centre in Melbourne. His lab is actively trying to track down high-penetrance cancer predisposition genes as well as genes that acquire somatic mutations in tumours.

Campbell's major genetic hit, scored in 2004 with his colleague Associate Professor Wayne Philips, was PIK3CA, a gene in the PI3 kinase pathway.

"We were the first to show that that gene is mutated in ovarian cancer and also with some breast cancers," Campbell says. "It's an oncogene, so it's amplified in cancers and activates the growth of tumours. About 30 per cent of colorectal cancers have mutations, 30 per cent of breast cancers and about 20 per cent of ovarian cancers and so it's quite a bit like p53, the archetypical tumour suppressor.

"The frequency of mutation is quite similar to p53, so we are working hard on that, looking at larger banks of cancer samples and looking at whether a mutation confers a higher or lower risk of dying or response to treatment."

Mapping arrays

Campbell's lab at Peter Mac has developed a great deal of expertise in using cutting edge technology to investigate breast and ovarian cancer genetics. In particular, the team has worked extensively with the Affymetrix 500K Human Mapping SNP array, which has helped uncover new insights into cancer faster than ever before.

"In the past when we've been looking for cancer genes, a major approach has been looking for DNA deletions in the genome and areas of the genome that have been amplified," he says.

"When I had PhD students 10 years ago, they'd do a whole PhD looking at 10 or 20 spots across the genome with those markers and try to map regions of deletion and amplification. Now by using Affymetrix SNP chips, we have half a million spots and we can do 10 samples in three days. One of my students here can do in a week what a PhD student took three years to do.

"With this technology, it is not just the speed but also it's the resolution. In the past we thought the resolution wasn't important, it was thought the deletions in cancer are probably all big. Whether you've got one marker or 1000 - if you've got a big mountain in front of you it's going to be a mountain.

"Our first experience with these high-resolution arrays is that there are lots of these very small changes which only encompass one or two genes. That's a pleasing revelation because in the past when we've got a cancer region there could be a hundred genes we've got to sort through. Now there's only two or three. And the information we have got so far is that for many of those regions, there are known cancer genes that are within those regions of deletion and amplification.

"The question is, we now have a lot more of those regions but are they all indicating there are cancer genes there? It has opened up - we can rapidly get high resolution and zoom down on critical genes quickly."

Formalin fixed

Campbell's lab is currently working to screen 300 tumour samples - 150 ovarian and 150 breast cancers - using these SNP chips. The researchers have access to a variety of new and archived tissue, through the Peter Mac Tissue Bank, Melbourne Pathology and the Victorian Cancer BioBank, with which to compare candidate genes in archived tumours.

"When we want to look at a new gene or look at whether a particular gene is involved in cancer we can use cancer cell lines, but that is a very artificial situation and it is hard to know whether a genetic alteration in that cell line is an artefact.

"So you really need to go back to primary cancers for gene hunting, but also for prognosis. You have a candidate, it is amplified - then you go back to samples from 20 years ago and see if they had this gene amplification and did they do better or worse.

"To have archival samples is vital for confirming genes, linking it to prognosis and also, for rare tumours, trying to identify what are the first events that occur in a cancer, what triggers it."

The lab has had to develop expertise with these archived samples, which are fixed in formalin and embedded in paraffin. The problem is, formalin fixing severely damages the DNA and RNA of the sample and is technically quite challenging, Campbell says.

"So we in association with Affymetrix got early access to some of these chips and tested a number of parameters to see how good the data was from formalin fixed tissue. We were able to compare what it looked like in matched fresh tumours and then what it looked like in the formalin fixed tumours.

"We modified some of the techniques and the software slightly. Hopefully that will help a lot of people in being able to adopt that protocol."

Gene hunting

Now that it is clear that genes can be affected by several phenomena besides conventional mutations, the lab is looking at areas such as aberrant DNA methylation, which can modify the structure of DNA without changing the sequence.

"It's another way of trying to identify genes," Campbell says. "Our main area is gene hunting, so we are looking for genes from the germline as well as changes that occur somatically. You might have a candidate gene and look for a mutation in all of the conventional ways but can't find anything. You have to look at all of the other possibilities.

"But often you are asked - so you are looking for a gene, so what? You always hope there will be new targets for therapy but 99 per cent of the time that will probably not happen. However you can understand how a mutation relates to prognosis and if, using existing treatments, there are any better options or combinations of treatments. You don't necessarily even need new treatments - just understand how they will respond to that treatment.

"If they are going to respond, then that's great but if they've got this mutation and the prediction is that they will not respond to conventional treatment then you can focus new treatments on that particular individual.

"It might not be tailored to that particular mutation but at least you can find people who will have the best chance of treatment with a new drug. And that's what we are all here for."