I started as a medical student. I wanted to do some work related
to cancer research. Later, in 1993, I started as a fellow in Steve
Baylin’s lab, and we were looking at methylation changes. This was
a time when many tumor-suppressor genes were being cloned and
identified. We began to investigate whether these methylation
changes could be involved in classic tumor-suppressor genes. We
started looking at Von Hippel-Lindau genes in renal cancer and found
that the gene was silenced.
was the motivation behind the work that led to the 1996 PNAS
paper on methylation-specific PCR?
Up to that point we looked at methylation changes in tumor
suppressor genes in different cancers. We were really interested at
that time in looking at precancerous lesions, at the early stages of
cancer, and looking at cancer progression of normal tissue into
fully malignant cells. We’d done all the work previously in
full-blown cancers and we simply wanted to study earlier lesions.
The problem was that we really needed an assay to look at possible
methylation in small lesions with very little DNA and sometimes in
DNA from a mixture of cells. It was out of necessity that we came up
with this technique. We needed something sensitive and specific to
methylation changes, so we had to go out and invent it.
was the biggest challenge?
I guess it was believing that something that simple would
it the first thing you tried?
The previous assays for doing this were based on restriction
enzymes, and not on the bisulfite method. We had been trying to use
restriction-based methods and we couldn’t get them to be
reproducible. And we were then working with other bisulfite genomic
sequencing, which had been in use for a couple of years. That
technique treats DNA with bisulfite and that causes sequence changes
you can determine by sequencing analysis. The methylation-specific
PCR was a way of using those sequence changes to directly determine
methylation changes. We would put primers that would recognize
sequence differences caused by methylation patterns, and they would
be in whatever the gene of interest was.
this was easy?
I don’t know if it was easy. It was simple.
you aware of how significant it would be when you did it?
To some degree, but not that it would be used as much as it is
currently. I think we thought it would be a new way to look at
things. It certainly made things a lot easier. The fact that we
could quickly look at methylation changes in different genes and
different tumor samples in these early lesions was really
encouraging. But we were not thinking about a lot of the possible
applications at the time.
have some of those applications turned out to be?
The main one is the potential to use this in molecular detection
of cancer, in early detection. That wasn’t something we were
initially thinking about.
did the idea of using it for early detection come up?
We were just getting comfortable with the technique, and we
realized how sensitive it was for picking up a very small amount of
methylation in samples. That’s really what you need for a
molecular detection approach to cancer—you need very sensitive and
specific detection. And that idea evolved from looking at early
lesions to looking at body fluids—in blood and in sputum—for
evidence of cancer. It wasn’t a complete shift from what we were
doing, but rather a recognition that this would be another potential
does it take to show that this could be clinically useful in early
Mainly we have to get a population at risk for cancer and then we
have to find out whether the test can predict the cancer early
enough to intervene. We are doing that in some different cancers.
But the limiting factor is really getting large enough patient
populations and then following up on them.
you quantify how sensitive this test could be?
Initially we were able to detect one in 1,000 cells. But
subsequently it’s been improved to one in 50,000 cells or even one
in 100,000. We’ve done some recent studies where we’ve picked up
a single DNA copy.
do you mean by a single DNA copy?
We were able to determine that when we got a positive signal that
was originally from a single allele of DNA, a single copy.
did you make the decision to publish in PNAS rather than one of
the other high-profile journals?
It’s a very good journal. It’s widely read in a broad
scientific area. I guess we thought it was the best match of
exposure and novelty. If you look at the higher-profile journals, Nature
and Science and so forth, usually you’re dealing with some
biological question, and this was purely a technique paper. We were
establishing a new technique. So PNAS was the first journal
we sent it to.
us your prediction for where you see this field going in five years?
I think we will continue to identify new genes and new pathways
involved in cancer that are silenced by methylation changes. That’s
already happening, and I think it will continue. I think the
screening approach will be tested in large populations and really
vigorously examined to see whether it will be useful for early
detection of cancer. So diagnostic uses will be further tested and,
I hope, validated. We’ll see.
there any particularly unexpected or serendipitous events that moved
your research forward?
I guess the most unexpected thing was how prevalent some of these
changes in methylation turned out to be. When we started looking at
gene silencing in cancer, the understanding at the time was that
genetic changes were the most important changes in cancer, and they
are clearly still very important. When we first started studying
genes for methylation changes, we thought we’d find a few genes
that might show this alteration and maybe just a few cancers. Now
that list has just exploded. The surprise has been how prevalent the
silencing of genes in cancer really is. That’s probably why some
of these papers are so highly cited. People continue to expand some
of the initial findings and continue to find this mechanism in many
different tumor systems and many different genes.
many tumor systems and genes are we talking about?
I don’t even know the number—maybe 50 to 100. In terms of the
type of cancers, I think every type of cancer has been shown to have
methylation changes in different genes.