One of the "legnedary" papers in CLL literature is the Dohner paper in New England Journal of Medicine from 2000. It is the landmark paper that taught us about 13q, 11q, 17p, trisomy 12, and normal cytogenetic CLL. The FISH technology it employed was developed in the early 1980's. For the last 13 years knowing the "FISH" status helped with prognosis and treatment selection. We are now on the eve of a major change of how we think about molecular markers in CLL. These markers will help us pick treatments that are best for a patient, monitor dangerous subclones, and give a much more clear picture of prognosis when the disease is diagnosed and at each relapse.
The human genome project took 13 years and 6 billion dollars
to “sequence” the genomes of four individuals.
DNA is the “building plans” for just about every important task a cell
has to do. Even though it is given this
amazing task it does so with only four different building blocks called
“bases.” There are two purines: guanine
(G), adenine (A) and two pyrimidines: thymidine (T) and cytosine (C). The complexity comes by putting these
together in very long sequences that make them unique. Add some extra bells and whistles and you
have a “gene.” Actually determining the
sequence (ie. g-a-a-t-c-c-a-a-c-a-t-g-c and so forth) or order of a particular
segment of DNA is called sequencing.
What is remarkable is that the same amount of work that went
into the human genome project can now be done in a matter of days to weeks with
considerably higher resolution for several thousand dollars. The cost and efficiency of sequencing is
dropping faster than microchips are getting faster. We are getting very close to being about to
sequence an entire genome in only a day for a thousand dollars.
With that diagnostic power comes an incredible ability to
probe the very fundamental causes of a particular cancer. CLL has been a beneficiary of this effort and
we now have a very nice short list of the most common mutations found in CLL
and several groups have done a great job figuring out the clinical significance
of each of them. Since most of these are
likely to be new terms, I thought a brief write up on what these mutations do
and what they mean would be great. I
think we are very close to incorporating these markers into our routine work up
of a new CLL patient.
Quick note about biology: genes are found in DNA and DNA
pretty much hangs out in the nucleus of a cell.
They serve as a template for making RNA.
Once a gene gets “transcribed” from a region of DNA into a much shorter
strand of RNA (often times one RNA molecule per gene), it goes out into the
main part of the cell called the “cytoplasm” where the RNA gets “translated”
into a protein. Proteins are the tools
that do most of the tasks in the cell.
When there is a mutation in DNA, it gets copied into the RNA (which is a
lot like DNA but gets out of the nucleus), and leads to the synthesis of an
altered / mutated protein. Sometimes we
speak of mutations as though they occur in a protein but really it is in the
DNA. Just in case I am sloppy in my
descriptions, I wanted to clarify the biology.
NOTCH1
NOTCH1 is the most interesting of the new markers to
me. It is highly associated with CLL
cases that have trisomy 12 as the chromosome change and especially those cases
that have an “unmutated” B cell receptor.
NOTCH1 has been a well-known protein because it is extremely important
in childhood acute lymphoblastic leukemia where it is present in almost half of
all cases. Over the past few years,
there have been a number of efforts to find drugs for mutated NOTCH. So far I wouldn’t consider those efforts
successful, but I am really hopeful about a new class of drugs just entering
the clinic now.
NOTCH hangs out in the plasma membrane which keeps the
inside of cells in and the outside of the cells out. NOTCH is like a light switch stuck in the
off position waiting to be turned on by another cell. When that other cell comes by and “flips the
switch” a piece of NOTCH gets cut free from its membrane anchor so that it can
float away from the membrane. NOTCH then
travels to the nucleus where it interacts with the DNA and makes a bunch of
other genes get turned on. Those genes
get copied (transcribed) into RNA and then proteins are synthesized
(translated) to do their tasks. For this
reason NOTCH is called a “transcription factor.” Once it has the right cue, it turns on the
transcription of a bunch of genes and therefore determines a whole bunch of
important functions.
The genes turned on by NOTCH are really important. One critically important NOTCH regulated gene
that helps cause Richter’s syndrome is MYC.
That is a protein that is a really bad actor in a bunch of different
types of lymphoma and leukemia.
Once NOTCH has done its job and turned on / off a bunch of
other genes it gets marked for its own destruction. The cell wouldn’t want to leave that signal
on forever so it needs to turn it off.
Sure enough there is an entire system in place to make sure NOTCH gets
shut down after it has done its task.
The particular mutation in this case makes it harder for the cell to
turn off NOTCH so it ends up being a signal that won’t stop – sort of like a
car where the brake pedal isn’t actually attached to the brakes. Press all you want and the car won’t stop.
Clinically, the most important thing about NOTCH mutations
is that they pretty much split the trisomy 12 patients into two groups, the
good ones and the bad ones. The good
ones who lack a NOTCH mutation end up behaving as though they have normal
cytogenetics (chromosomes). The bad ones
with a NOTCH mutation are now considered high risk. They undergo transformation to Richter’s
syndrome a lot more frequently and survival is shortened. See my other post on “new risk groups.”
FBXW7
If NOTCH is important you were probably all expecting that
this protein should be on the list too (well ok, maybe just some of you). Remember all that business about turning off
NOTCH? FBXW7 is the protein that does it. Take the same car analogy – now just throw
out the brake pedal altogether.
FBXW7 has not been evaluated as closely in terms of clinical
significance so I can’t really tell you what it means to have a mutation here –
but safe money would bet that will be a lot like a NOTCH mutation.
P53
I have written about P53 before. It is the protein encoded by the TP53 gene
which lives on the short arm of chromosome 17 (yes – that would be 17p). I would encourage the interested reader to
read my post about 17p deletion as well as my post about new risk groups in CLL
because it really goes into deep detail about this protein.
Turns out that P53 can be mutated even when 17p is normal
and they are just as bad. The problem is
that right now we don’t test for P53 mutations.
Fortunately most of the time you have a mutation in P53 you also have a
deletion of 17p on the other chromosome (remember – we have a pair of each
chromosomes) but that relationship isn’t air tight. You can have mutation without deletion,
deletion without mutation, deletion with mutation, or normal/normal. The more 17p dysfunction the worse off you
are. In other words, having one good
copy is better than none. It has been a
while since I have seen the number so I might be off a little bit, but
something like 20% of cases with P53 mutation do not have 17P deletion so you
might have a high risk marker and have no idea based on current testing.
The quick explanation of why this marker is SO IMPORTANT is
that it is the protein that pretty much tells the cancer cell to die in
response to damaged DNA. Since drugs
like fludarabine, bendamustine, chlorambucil, cyclophosphamide and so forth
attack DNA – you need a functional P53 for the chemotherapy to work.
Patients with P53 mutations are considered “ultra-high risk”
– it would be nice if we routinely tested for this – but we don’t!
ATM
ATM is to 11q as P53 is to 17p (are you following me?) ATM lives on the long arm of chromosome 11
(long arms are designated “Q”). When
patients have deletion of chromosome 11q it is a pretty big chunk of DNA that
goes missing and includes a handful of genes but ATM is one of the ones that
almost always goes missing.
Like P53/17P above, you can have mutation of ATM with or
without deletion of the other chromosome.
While a high frequency of 17P deleted cases (70-80%) ALSO have P53
mutation, only about 30% of 11q deleted cases of ATM mutation. On the other hand, mutations of ATM without
deletion of 11q can happen too and once again although it isn’t too common.
Like P53, ATM is important for sensing DNA damage. If you recall DNA is what we call “double
stranded.” It is like a set of train
tracks that gently twist around each other.
When DNA gets damaged it can result in a single or a double stranded
break. ATM is one of the sensors of this
broken DNA and it sounds the alarm to stop cell division and also activates our
friend P53.
In some studies we’ve seen that having both 11q deletion and
ATM mutation is worse than just having one or the other. Once again, current testing does not look for
this. ATM is an ENORMOUS protein. It is hard to measure all the possible
alterations but new technology is making it a lot easier.
I’ve written previously about clonal evolution both here and
here. It might not be immediately
obvious if you haven’t thought about it before but I think it is fairly
intuitive that when you use DNA damaging chemotherapy, the cells that survive
are the ones that have higher frequency of alterations in 11q/ATM or
17p/P53. It is sort of like taking a
short course of antibiotics for a sore throat and finding that those same
antibiotics don’t work well the next time around. We therefore see a lot more alterations in
11q/17p in patients with relapsed disease than we do in newly diagnosed
patients. This is why it is so imperative
to repeat molecular testing before each new line of therapy.
Clinically we think it isn’t enough to give fludarabine /
rituxan for patients with 11q. There is
some data to suggest that they do better with cyclophosphamide and fludarabine
than just fludarabine alone. Add in the
rituxan (ie. FCR described here) and you overcome some of the negative
prognosis associated with 11q.
BIRC3
BIRC3 is another new marker of considerable importance and
guess where it lives in the genome? It
lives at the far end of the same 11q deletion that knocks out ATM. Interesting not all 11q deletions are created
equal. Most include BIRC3 but not all do
– so it is possible to have an 11q deletion and have either normal or deleted
BIRC3 depending on the size of the deletion.
Sadly FISH doesn’t tell us which is which because BIRC3 is a bad thing
to go wrong.
Since BIRC3 is one of the newest abnormalities, we know less
about how it interacts with all the permutations of 11q / ATM etc. For now I think we can just summarize that
having a mutated BIRC3 puts you in a high risk category even if everything else
appears normal or favorable such as 13q deleted.
BIRC3 is a protein from a family known as IAP or “inhibitor
of apoptosis.” BIRC3 therefore helps
regulate cell death and influences another very important protein known as
NF-kB. BIRC3 is another way cells can
become resistant to fludarabine.
SF3B1
This is a new marker that burst onto the scene just about
two years ago. Right now, we do not
routinely test for it (catch a theme here?).
When you make an RNA copy of DNA it often consists of long
segments of RNA called “introns” that need to be cut out of the final RNA
strand (I remember it by saying “introns interrupt”). Once all the introns have been removed you
are left with the “exons.” When all the
exons are lined up end to end it can be copied (translated) into a
protein. We used to think this was just
a bunch of cellular waste from millennia of evolution, but now we know that
these introns have a bunch of important functions.
SF3B1 has the task of cutting out all those introns and
creating the uninterrupted sequence of exons.
Right now, I don’t think we totally understand what happens at a
cellular level when SF3B1 is mutated but we do understand some of the clinical
implications. Patients with SF3B1
mutations are resistant to fludarabine.
The other thing about SF3B1 mutations is that it makes you “high
risk.” It isn’t as bad as 17P deletion
or P53 mutation but you are still worse off with it than you are without it.
SF3B1 can be sneaky, it can hide in the background of cases
with normal chromosomes or even in the 13q deletions where you might otherwise
expect a patient to do fairly well. There are now several markers for
fludarabine resistance and including P53, BIRC3, and SF3B1. In my mind it would
be pretty helpful to know a patient’s markers when they are first diagnosed or
when you are picking out a treatment.
There are several other important new molecules such as
XPO1, MYD88, etc. I have not really seen
good data yet that indicates that they influence treatment choice or
prognosis. I wouldn’t be surprised if we
learn more about them in the next 1-2 years.
It is an alphabet soup out there but right now these markers
are not readily available. I anticipate
we might have a test for them soon and it will be helpful but unfortunately it
will add a whole new dimension to the way so many patients worry about their
future. In the future people will now no
longer say, “phew, I am a 13q, BCR mutated CLL.” Instead they may say, “I am 13q deleted, BCR
mutated, P53/BIR3 normal, SF3B1 6% subclone mutated.” It is going to get very complicated very
soon!