11q deletion CLL

Most patients who make the effort to learn about FISH in CLL know that 17P is bad and 13Q is generally favorable.  Trisomy 12 segregates based upon Notch mutations (if you can find a way to test for it) .  Somewhere between the badness of 17P and the middle of the rode trisomy 12 lies 11Q.

CLL with 11Q deletion has a unique personality.  It is notorious for having disproportionately bulky lymph nodes compared to the elevation of white blood cells (though WBC elevations are common).  Men with 11Q are considerably more common than women, and patients are often younger than the typical 72 year old average.  Furthermore the substantial majority also have unmutated B-cell receptors (unfavorable).  Patients often have need for initial therapy more quickly than others following diagnosis and patients with 11Q have shorter survival than many others.  This is in part because 11Q is often thought of as a “chemotherapy resistance” marker and I wanted to go into a deeper biology of this marker as I have for 13Q, trisomy 12, and 17P.

Once again, recent biologic insight has dramatically reshaped our thinking about 11Q.  We have previously addressed “clonal evolution” where CLL is a mixture of different “subclones” that jockey for dominance.  In the past it was observed that patients with greater than 25% of cells on FISH had worse prognosis than patients with less.  In light of clonal evolution, I tend to just think it is a continuous spectrum and if you have an 11Q clone it is likely to become dominant over time in most cases.  At any given time point, the less you have the better.  Like all other high risk genomic alterations, 11q deletion increases over time (10-15% at initial diagnosis but as high as 30% in relapsed / refractory).

Classically, 11Q deletion was thought to be all about a single protein called ATM (for ataxia telangiectasia mutated – the gene that causes a rare genetic syndrome Ataxia Telangectasia).  The gene for ATM is truly massive.  The DNA that encodes the gene (like most all genes) is broken up into a bunch of smaller pieces (exons) that all get glued together into one sequence (mRNA) before the cell can use the template to synthesize the protein.  The protein is very large and has quite a few different functions.  Consequently older techniques of measuring mutations in DNA (sequencing) couldn’t really address whether an individual patient had a mutation in the gene or not.  Furthermore, we didn’t really know the functional significance of many of the mutations because they were scattered throughout the entire gene.

Newer sequencing technologies allow us to measure with considerable precision BOTH the mutations AND the fraction of cells that have them.  It should therefore not come as a surprise that we have found an important number of cases where ATM can be mutated whether there is an 11Q deletion or not.  It is very similar to the P53 story on 17P.  You can therefore have deletion with or without mutation and vice versa.  It appears that having BOTH deletion of ATM on one copy of the chromosome and mutation on the other copy of the chromosome is worse than only having the deletion.

Whether it is deleted or mutated or both (you do have two copies of the gene), ATM plays an important role in detecting DNA damage, passing that information along to P53, and triggering an arrest in cell division and cell death.  Without ATM in place, you don't have the same response to DNA damage.  Drugs like fludarabine, cyclophosphamide, bendamustine, chlorambucil all require an intact DNA damage response for their efficacy. 

While that may seem complicated – it gets a lot worse.  I wrote quite a bit about the deletion size in cases of 13Q deletion where there are type I, type IIA, and type IIB chromosome changes that may have different prognostic significance.  Not all losses of DNA are equal to one another.  Bigger deletion sizes may include other important genes.  Indeed, the far end of the 11Q deletion variably affects a protein I’ve written about previously that is extremely important – BIRC3.  It has been shown recently that some 11Q’s have loss of BIRC3 and some don’t.  While we don’t directly know how important that is just yet, BIRC3 is a big deal as having a mutation in that protein is about as bad as 17P.  I think we will be hearing more and more about BIRC3 in the coming years as we look at it more frequently.  Finally, there seems to be an association between mutations in the protein called SF3B1 and cases with 11Q.  SF3B1 is not in the region of the deletion - BUT - there seems to be a relationship between this mutation and 11Q (sort of like NOTCH and trisomy 12).  SF3B1 is a mutation that confers resistance to fludarabine and possibly other chemotherapies.  Why this associates with 11Q really isn't known just yet but it is an important observation.  Patients with SF3B1 mutations often need therapy earlier on and don't do as well in the long term.

Wikipedia has a short description of BIRC3 here.  This pathway is also quite important as it governs the "execution pathway."  Once a cell has made the decision to die, it uses a series of enzymes called caspases.  BIRC3 is involved in governing that pathway.  BIRC3 abnormalities appear to be particularly bad thing to get.  Some data sets make it look just about as bad as having 17P deletion.  I think we are only at the start of understanding the interaction between ATM and BIRC3 in terms of deletion size, inclusiveness, exclusiveness etc.

Terry Hamblin did a very good job explaining the role of alkylating agents in CLL with del 11q, you should definitely see his discussion of the topic - link here.  In short, if you are going to give fludarabine based regimen to a CLL patient with 11q minus, many experts think you should include cyclophosphamide (ie. FCR more than FR) if the patient can tolerate it.  Some have even gone farther and asked if you need the "C" in FCR if you don't have an 11q deletion.  Most of this was based upon data generated when fludarabine was compared to fludarabine / cyclophosphamide (and rituximab wasn't in the mix).  Newer publications have suggested that the combination of FCR overcomes the negative prognostic implications of del(11q).  They definitely respond well to the regimen, but earlier relapse remains an issue.

Some are anticipating the results of the big German study comparing FCR to BR in the frontline setting.  These results may come as early as this December at ASH.  I am most interested in the subgroup of patients with 11q deletion in this study because of the question marks about alkylating drugs (chlorambucil, cyclophosphamide, bendamustine) in this patient group.

New drugs like ibrutinib and idelalisib do not have a long track record in subgroups like 11Q.  In our New England Journal of Medicine article, we saw that patients with 11Q OR 17P didn't do as well as patients without either abnormality.  The still did very well on this drug - but I expect that 11Q will remain problematic under most circumstances since it is a marker of genomic instability which is NEVER a good thing in cancer.  My guess is that earlier utilization of these targeted agents in smart combinations with other drugs may be best strategy (I've written about that here).

Anyhow, that is probably enough for now.
Thanks for reading.

CLL Prognosis Markers Defined

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!