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

Many CLL patients identify themselves by their prognostic markers when writing in social media outlets.  "Diagnosis age 63, unmutated, trisomy 12, treated FCR age 67, still in remission 2 years later"  is the sort of "tag line" I've seen people write.  For individuals who visit social sites frequently it is a way to tell your story in a few words.  For individuals who are new to CLL, it can all seem very confusing.  Well it is about to get a whole lot more complicated for everyone very soon.

A lot of what follows is very technical but I wanted to get it all written in one place.  I hope patients actually read and re-read this material several times.  For people who are prone to sleeping every time they read one of my posts, here are two videos I did with Brian Koffman in Sept 2013 that goes over the same material in video format:

Part 1: New Prognostic Markers
Part 2: Another on New Prognostic Markers

I've been wanting to write this post for a while but a recent paper has really brought this to the forefront of management of our CLL patients.  Unfortunately the names are strange and I worry this post may fall toward the technical side - sorry.  I will create a separate post that specifically defines many of these terms.

Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia

For people who have read all my posts on FISH testing, you are probably aware that it is an antiquated technology that has served us well for 20 years but needs desperately to be replaced. Sequencing technology has advanced incredibly quickly and is now poised to refine our understanding of CLL risk groups with new molecular detail.

While most patients are aware of the incredible advances in CLL therapies (ibrutinib, CAL-101, GA-101, ABT-199), fewer are aware of the really important advances in molecular markers that have been recently discovered.  Once these are rolled out to the general public we will be able to understand with much more precision how a patients disease will behave.  Pretty soon, folks will not only be talking about 13q, 17p without also talking about BIRC3, SF3B1, and NOTCH.

In the last 24 months, genomic sequencing has been applied to cases of CLL with pretty remarkable results (see New England Journal of Medicine article or Journal of Experimental Medicine article)

Several key findings have emerged from these data sets.

1) CLL has a relatively simple genome.  While some "smart cancers" (cancers that quickly gain resistance to our treatments and are far more aggressive) like small cell lung cancer may have 50,000 mutations per tumor, CLL (a comparatively dumb cancer - which is typically slow, responds well to most treatments, does not gain resistance all that fast) may have fewer than 100 mutations per case and only a small fraction of those (around 10-20) affect important proteins (the enzymes that make all things happen inside a cell).

2) Certain mutations seem to be observed fairly commonly in CLL and have some defined prognostic or predictive value.  For instance BIRC3 turns out to be a really bad thing to have - it is the new 17p.  NOTCH probably is one way to get to Richter's and helps sort out the trisomy 12 cases, SF3B1 makes you resistant to fludarabine chemotherapy.

3) Certain mutations are seen early in the disease, while others seem to accumulate with time.  Furthermore, some of the ones present later on are actually present early but only emerge through "clonal selection."

4) Some cases of "familial CLL" (ie those cases that run in families) have an unifying genomic explanation that point toward things we already knew were important.


With all of this new information, it was only a matter of time before someone took on the herculean effort to figure out which of these were most important and what they all mean when you analyze them simultaneously in a large group of patients (1300 of them to make this model).

The old risk groups were:
High risk: 17p changes (home of the p53 protein)
Intermediate risk: 11q changes
Low risk; normal cytogenetics & trisomy 12
Very Low Risk: Isolated 13q changes

Unfortunately, there is a lot of biologic diversity that FISH testing misses since it only looks at large chunks of missing or added DNA.  Using sequencing technology (think microscope compared to telescope) as an adjunct to FISH we can now help sort all of these out.

The new risk groups
Very high risk: 17p deletions, p53 mutations, or BIRC3 mutations (10 year survival 29%)
High risk: 11q deletions, SF3B1 mutations, NOTCH mutations (10 year survival 37%)
Low Risk: Normal cytogenetics, trisomy 12 (without NOTCH mutations) (10 year survival 57%)
Very low risk: Isolated 13q deletions (10 year survival same as age matched controls).



There are some really interesting observations contained within this.

1)  It is not a surprise that 17p deletion and p53 mutation are both really bad - we've known that for a long time.  They commonly run together (ie. most 17p deletions also have p53 mutations - but not all cases).

2) BIRC3 is a new kid on the block.  It has only been recognized for about 18 months.  Turns out it is really bad to have.  It confers chemotherapy resistance and is often very discrete from p53 alterations (i.e., if you have one, your probably don't have the other).  We've known for a while that p53 doesn't explain all cases of chemotherapy resistance - BIRC3 explains a lot of them.

3)  We have known for a while that 11q deletions often associate with bulky lymph nodes, unmutated B-cell receptors, faster growth kinetics, requirement for alkylating drugs (cytoxan, bendamustine).  It has often been considered a poor risk feature.  SF3B1 and NOTCH are totally new though and we didn't know where these fit in terms of hierarchy.  Turns out, they are about equal.

4)  Last year the relationship between NOTCH and trisomy 12 was identified.  About half of trisomy 12 cases carry a NOTCH mutation - particularly those with unmutated BCR (ie. cases with unmutated BCR and trisomy 12 have high frequency of NOTCH mutations - sorry if this gets confusing).  We have been aware that trisomy 12 was a bit of a wild card - some did fine, some did poorly.  Turns out that NOTCH mutations can sort the two apart.  Those with mutations do worse, those without mutations are now considered "low risk."  I am very eager to learn if the new NOTCH antibodies turn into personalized medicines for patients with the NOTCH (or even FBXW7 changes).

5)  Our good old friend 13q is still "good risk."  The surprise here is that 25% of 13q cases get put into higher risk categories when you do the mutation analysis.  They might have an SF3B1 mutation or BIRC3 mutation you would have otherwise never known about.  By carving out the bad players, it makes the good group even better.  "Matching age controls" does have some limitations because the model is built upon typical CLL cases.  There are probably not sufficient number of 42 year olds with 13q in the model to say that they necessarily match their peers.

6)  This model holds true no matter when you evaluate a patient.  In other words, if clonal evolution occurs and you go from very low risk to high risk by molecular definition - your clinical outcome changes too.

There are some important questions in all of this.

1) The most obvious is - how do I know what I am?  Right now - you can't easily tell.  There really are not commercial tests to sort this out - I'm trying to make one but seem to running into more walls than doors.  If anyone out there wants to finance this idea, let me know!

2) What defines "positive" for mutation?  For 17p by FISH we do not define a patient as positive until 20% of their cells are positive.  With ultrasensitive testing you may find 0.07% of cells have a BIRC3 mutation.  That patient isn't "positive" but I would be very concerned that clone may evolve in the future.  Do you therefore do anything different when you choose to treat them?

3)  This analysis may miss some of the subtlety of different FISH abnormalities.  We already know there are type I and type II deletions on chromosome 13 with different prognostic value.  We also know that the overall percent of cells with 11q or 13q makes a difference.  This model does not capture that degree of subtlety.

4)  Mutated vs unmutated is not included necessarily in this model - I would like to know if it "sub-stratifies" amongst the various different risk groups (although it is more common to see unmutated with 17p and 11q than the 13q cases so perhaps the model was just not big enough to take it all into account)

5) How do these markers hold up in the face of the new drugs.  ABT-199, ibrutinib, CAL-101, GA-101 are so remarkable.  Will traditional markers hold up in the "new era?"  It is important to note that this model is based upon cases that have already been followed for quite a few years.  Some didn't get rituxan with their first line of therapy.  Presumably none were able to take advantage (since it is an Italian study) of the new drugs.  By definition, this is a backwards looking model and does not capture what I see as a very optimistic future. For example, 29% 10 year survival for poor risk does not reflect the impressive durable control obtained in front line 17p patients treated with ibrutinib.


Though there are questions, the authors of this paper are to be thanked profusely for their remarkable effort to create a single predictive model of this magnitude.  I would imagine that there were thousands of hours put into creating and analyzing the data.  This paper will serve as a landmark for quite a few years and will help guide countless numbers of patients.

17p Deletion in CLL

17p is the genomic alteration in CLL that triggers the greatest concern in most patients.  It can have a tremendous impact on CLL prognosis and the FDA has recently extended approval to ibrutinib in this population (even without prior treatment) and the European equivalent of the FDA (the EMA) will do the same for idelalisib in combination with rituximab.  A lot of patients know that 17p deletions is one of the high risk markers in CLL  – but there are a lot of things to consider about CLL with 17p deletion before completely tearing your hair out.

When we say 17p deletion CLL, what we mean is that the short (petit) arm of chromosome 17 is missing.  You have 23 pairs of chromosomes (46 total) and as you get higher in the numbering, the chromosomes get smaller and smaller.  It is probably an excessive simplification to say that the biology of 17p is all about one particular protein called p53 – but for the time being that is most of the story.

P53 is affectionately called “the guardian of the genome.”  Every time I read about p53 I discover some new function of the protein that I didn’t know about before.  One of the most important though is that it will bind to DNA in a bunch of places and turn on / off the genes at those locations.  In this role it is known as a “transcription factor.”  Many of the proteins that are regulated by p53 have to do with cell survival or cell death.  When P53 decides it is time for a cell to die – very few things can stop that.  The most important signal that turns on p53 is DNA damage (hence – guardian of the genome). 

When DNA damage occurs the cells have a lot of repair mechanisms to try to fix the problem (including the ATM protein on chromosome 11q).   P53 will halt cell proliferation until that DNA damage is fixed.  Some DNA damage cannot be easily fixed and when that is the case, p53 triggers a cell death cascade called apoptosis (one of several ways that cells can die).

I mentioned above that you have two copies of every chromosome – so you ought to have two copies of P53.  We have been good at detecting absence of chromosome 17p for quite some time (via routine cytogenetics or FISH), but we have not always been very good at detecting p53 mutations which have been far more difficult to measure until recently.  With new sequencing technology, it is relatively easy to look for mutations and an increasing number of laboratories are offering that service. 

This is important because patients with 17P deletion are not the only individuals who have to be concerned about it.  About 30 percent of patients with abnormality in P53 have a mutation BUT NO DELETION.  Those have just as bad a prognosis but are not currently detected by FISH testing (nor SNP arrays which are one newer technology that is gaining popularity).   There is a strong association between loss of chromosome 17p on one chromosome and mutation of the other copy (about 85% of cases with 17P deletion will also have P53 mutation on the other chromosome).  

Another common misunderstanding has to do with “how many deleted cells does it take to call a patient 17p deleted?”  In other words, FISH will report the percentage of cells lacking one copy of 17p.  That can range from 1% to 100%.  In simple terms, the more abnormal cells, the worse.  For research purposes we say that 20% of cells lacking one copy of 17p calls that person “17p deleted.”  Some labs have lower thresholds (7%).  Occasionally I will hear from a patient that has 2% of cells with 17p deletion who is worried about their future.  By convention we would not group that patient into a 17p deletion category.

I think the 20% distinction is important – but gets more emphasis than it deserves.  We have prior posts talking about clonal evolution and this is a topic that is very important to understand (also covered in my "watch and wait" post.  If you have a small percentage of 17p deleted cells and you get chemotherapy that damages DNA – requiring p53 to transmit death signals – guess which cells are going to survive.  We know that one out of five patients will have a high risk molecular abnormality at relapse (11q/17p).  If we look hard enough we can see that it was often there to begin with – but below our typical levels of detection.  By giving therapy that removes the more sensitive cells, the resistant ones remain.  On the other hand, if you have a large number of 17p deleted cells, you are less likely to respond to chemotherapy in the first place.

The question becomes, what to do clinically when a patient has a 17p deletion.  There are not a lot of standard regimens that are particularly active when a patient has a high load of 17p deleted cells.  FCR and BR are not very effective.  Indeed, perhaps the most important clinical trial in this population right now is the frontline study of idelalisib in combination with rituximab.  It is available here and here (can be opened at any of these locations)

Campath (an antibody that does not damage DNA) can work well, but does not clear bulky lymph nodes which are common with 17p deletion.  High dose steroids can shift cells into the circulation where they can be removed by campath.  Rituxan also does not damage DNA both rituxan and campath combine well with high doses of steroids.

The new drugs CAL-101 (aka GS 1101), ibrutinib (aka PCI-32765), and ABT-199 (AKA GDC-0199) appear in preliminary reports to be quite active in 17p deleted CLL.  Multiple clinical trials are available for those drugs.  For untreated CLL with 17P, I think it is worth trying to get into this study

I have a particularly memorable patient who presented to my clinic with bad stage IV 17p deleted disease.  He had bulky nodes, WBC count of 200, platelets of 20k and hemoglobin of 8.  His FISH showed 100% 17p deleted.  Two cycles of FCR did nothing except get him transfused every few days.  I switched him to campath with rituximab and got his marrow into better shape but he still had bulky nodes.  He was young enough for transplant, but not eligible because he still had bulky nodes.  I sequentially gave him R-ESHAP, bendamustine rituxan, revlimid rituxan, ofatumumab all without much benefit.  I started him on CAL-101 and his disease melted away.  His disease control lasted nearly two years.

When a patient is young enough, they should definitely consider a stem cell transplant for 17p deleted disease.  The challenge though is that CLL more commonly affects patients too old for transplant.  The engineered T cells hold some promise for being active in this setting.

I also have a lower threshold for starting treatment in previously untreated CLL with 17p deletion (see "when to treat CLL").  Since those cells are likely to be resistant, I don’t see value in getting too far behind before getting started.   When I start, I might avoid FCR though some would argue it is still the right choice (NCCN lists this as first choice but I do not agree).  In Europe, you would typically get steroids with campath and I tend to think that is the right option.  Unfortunately, not enough sound data to tell us one regimen is better than another in this situation.  If a patient has access to ibrutinib in this setting that may be preferable.

Finally – one more biologic consideration.  Richter’s transformation is the name given to CLL that changes behavior and becomes a lot more aggressive – a different entity we call diffuse large B cell lymphoma.  It appears that p53 abnormalities are one of several key steps to getting to Richters (the other possibly being abnormalities in Myc or a protein that turns on Myc called NOTCH).  This is part of the reason Richter’s can be so difficult – it has intrinsic resistance to chemotherapy.

We are lucky to have a host of new drugs working through the system.  I will be very interested to see if drugs work out in this setting!

Thanks for reading - I also discuss this in a video done by Brian Koffman.  For anyone still interested, here is the link:   High risk CLL

How to read a flow cytometry or FISH report

This is a "webinar" presentation I gave to a number of research nurses on how to interpret clinical reports for flow cytometry, FISH, and cytogenetics and enter the test results into research "report forms."  These reports can be VERY confusing unless you are familiar looking at them and I often find docs often miss many of the subtle findings that can be buried in the fine print - so my presentation is really about the fundamentals.  If you are a patient confused by these reports, hopefully this may be useful to you. 

Although I touch on the prognostic information contained in these reports, it is primarily about how to understand the actual test results.  It is directed to an audience involved in a CLL research study but the nature of the material makes it useful for patients with NHL as well.

I would recommend skipping the first 3 minutes as webinars are a little clumsy and this one is no different.  I give some useless background info before launching into how to read CD19 / CD5 coexpression, trisomy 12 and so forth.

Trisomy 12 in CLL / Notch Mutations

Another common FISH abnormality in CLL is trisomy 12 (literally "three body") in which there are three copies of chromosome 12 whereas the normal allocation is just two copies.  There has been a lot of recent excitement about additional abnormalities found in trisomy 12 CLL but I wager there is a lot more to be found.  Even though chromosome 12 is a "mid-size" chromosome, there are literally thousands of genes on the chromosome so I expect we will find more information in the near future.

Before we had FISH technology and the only way to study chromosomes was "metaphase cytogenetics" where you literally grew up the cells in a dish, took out the chromosomes, froze them just before the cell was going to divide (ie. metaphase) and visually inspected them, trisomy 12 was one of the most common abnormalities found.  With FISH, we've become a lot better at finding chromosome 13q changes so it has fallen from the top spot.

Trisomy 12 CLL has a distinct personality among cases of CLL.  When you look at the cells under the microscope, they have a lot more "atypical" features.  In an individual patient, there may be a higher frequency of "prolymphocytes" which look a little more aggressive.

There are several key clinical features of trisomy 12.  One feature I think is intersting though I have not seen a lot of literature about it is that it has a considerabally higher amount of CD20 on the surface of the cell.  CD20 is the target of rituximab / ofatumumab / GA-101 and a variety of other research antibodies.  One of the reasons we don't think rituxan is as effective as a single agent in CLL compared to most of the lymphomas is that it has a lot lower CD20 on the surface of the cell.  The following paper actually suggests a higher response rate to single agent rituximab in cases with trisomy 12 (actually they also gave gm-csf but the rituxan did most the heavy lifting).


Chronic lymphocytic leukaemia CD20 expression is dependent on the genetic subtype: a study of quantitative flow cytometry and fluorescent in-situ hybridization in 510 patients

This CD20 differential may become important as combination biologic regimens begin to emerge.   I look forward to anti-CD20 in combination with BCR signaling inhibitors or BCL-2 inhibitors.  It would be very interesting to me if those patients with trisomy 12 did better under those conditions.  That is purely speculative on my part for now, but something I would like to investigate.

In the landmark Dohner paper in New England Journal of Medicine that identified impact of FISH on survival in CLL, trisomy 12 was considered an "intermediate" prognostic feature.

Genomic Aberrations and Survival in Chronic Lymphocytic Leukemia

It has recently been discovered however that a considerable fraction of trisomy 12 cases (somewhere between 1/3 to 1/2 may have a mutation in a key protein called NOTCH.  A mutation in NOTCH is a really bad thing - just about as bad as having a mutation or deletion of p53 / 17p, or another bad actor Myc.  We don't currently have convenient tests for testing for NOTCH unfortunately.  I wager though that once we do, trisomy 12 will either be a good prognostic factor (in setting of normal NOTCH), or bad factor (when NOTCH is mutated)

NOTCH1 mutations in +12 chronic lymphocytic leukemia (CLL) confer an unfavorable prognosis, induce a distinctive transcriptional profiling and refine the intermediate prognosis of +12 CLL

NOTCH is a protein at the surface of the CLL cell.  It waves its receptor out to other cells and when it comes into contact with the right signal it causes the part of the protein on the inside of the cell (conveniently called the intracellular domain aka ICD) to be released from the membrane to go into the nucleus of the cell where it turns on a lot of different genes related to cellular proliferation.  It goes about turning on genes until another protein called FBXW7 comes around and attaches a tag on NOTCH that targets it for destruction.  Interestingly FBXW7 has also recently been identified as a gene periodically mutated in CLL which is a well developed theme - there are lots of ways to activate the same pathway.

Activation of the NOTCH1 pathway in chronic lymphocytic leukemia

There are a handful of drugs out there that target the NOTCH pathway.  One way to target the pathway is to inhibit the enzyme that cuts it free from the cell membrane.  These are so called "gamma secretase inhibitors."  Unfortunately, this enzyme is important in the bowel and diarrhea has been a big problem.  There are other ways to go after the NOTCH protein and the hope is that some of them may not have the same GI issues.

I bet in time, we find a number of other abnormalities associated with trisomy 12.  Whether there are other miRNA's or other features.  In the quest toward personalized medicine this is the level of detail that will be required.

13q Part Three - interpreting test results and understanding limitations

13q test results:

When FISH is ordered and a 13q abnormality is observed, there are a variety of ways it can be reported.  It is possible to have a deletion on one chromosome in many of the cells, a deletion on both chromosomes in some of the cells, or any combination of the two.  Typically the report will indicate the number of cells lacking one or both copies of 13q.

Up until recently, we were not totally clear how these differences influenced clinical outcome.  Several earlier papers seemed to indicate that having both copies of 13q missing was worse than having only one copy missing.  Unfortunately, some of these studies were limited by small sample sizes and may not have statistically accurately reflected what these results mean.

Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays.

Biallelic deletion 13q14.3 in patients with chronic lymphocytic leukemia: cytogenetic, FISH and clinical studies.

More recently, several groups seem to have arrived at consensus that the actual percentage of cells lacking the 13q matters more than whether there is only one copy or two.  Different groups come up with different number thresholds, but that is because they need to make a categorical separation of data that is by nature a continuous variable.  It is probably safe to say that the more cells with 13q deletions the worse it is.

The prognostic difference of monoallelic versus biallelic deletion of 13q in chronic lymphocytic leukemia.

A comprehensive evaluation of the prognostic significance of 13q deletions in patients with B-chronic lymphocytic leukemia.

A high number of losses in 13q14 chromosome band is associated with a worse outcome and biological differences in patients with B-cell chronic lymphoid leukemia.

There are a number of things however that FISH simply doesn't tells us.  In some cases we know whether the missing data is important, in some cases, we have yet to figure it out.  FISH does not distinguish between the minimal deleted region (MDR), commonly deleted region (CDR) which contain Dleu7, or the less frequent larger 13q deletions that contain the RB protein (type II deletion).  Unfortunately FISH only tells us if there is a deletion or not - it doesn't tell us the size or what genes are included.

13q14 deletion size and number of deleted cells both influence prognosis in chronic lymphocytic leukemia.

The prognostic significance of various 13q14 deletions in chronic lymphocytic leukemia.

Functional dissection of the chromosome13q14 tumor-suppressor locus using transgenic mouse lines

Mutations have been found in the Rb protein in patients with CLL, but those are not detectable by FISH.  Other conditions where the cell tries to replace the missing material by making an extra copy of the remaining chromosome (aka uniparental disomy) are missed entirely.  Furthermore, the cancer cell can turn off the mRNA's through a process called methylation and even if you could figure all this out, it is not clear how these findings affect the biology.

For now we know that if you have a 13q deletion and NO OTHER FISH abnormalities, you are probably in good shape.  If you also have a 17p or 11q, those trump the 13q.  Hopefully we will learn how to handle the other information we know is there and create tests that let us sort it all out.

13q - part two

So now for more on 13q:

In cases of CLL where the 13q region is missing, there is variability with regards to just how much is actually gone.  You can have the MDR (minimally deleted region), CDR (commonly deleted region) which can occur in the type I deletions, or you can have a huge chunk of DNA gone that eliminates a specific gene called Rb (important tumor suppressor) in which case you have a type II deletion.

If we look at the MDR, we already talked about the miRNA's in the prior post.  In humans, the miRNA's are completely embedded in another RNA transcript known as a "sterile transcript."  I would love to know what this "sterile transcript" actually does - but so far, nobody has figured it out.  It is known as DLEU2 (deleted in leukemia 2).  We know from other literature that these "sterile transcripts" do have a regulatory role on certain proteins etc. we just don't know much about this one.

A savvy group of researchers (article linked in prior post) was able to make two different strains of mice.  One which just lacks the miRNA's and another which lacks both the miRNA's as well as DLEU2.  If you do nothing to the mice except watch them over time, the group lacking the miRNA's gets monoclonal B cell lymphocytosis, some CLL, and some NHL.  Interestingly the group that lacks DLEU2 gets more CLL, NHL, and even some diffuse large B cell lymphoma.  In summary, the double deletion (miRNA and DLEU2) is more aggressive than just the miRNA.

They did some further experiments and showed that if you take a mouses B cells with these abnormalities and give them a growth stimulus, they start growing faster, and grow longer than normal B cells.  Some of these "cell cycle" genes that are affected are classically known to be cancer related - including one of my favorite proteins  CHK1  (more to follow when we talk about 11q deletions).

Just a little farther ways out the 13q chromosome lies the DLEU7 gene (different than DLEU2).  Instead of being a "sterile transcript" DLEU7 is responsible for actually for the synthesis of a specific protein.  This protein has a regulatory role in signaling through a surface molecule known as the BAFF (B cell activating factor) system.

BAFF is interesting to me because it is clearly an important cytokine (aka: micro hormone) in CLL.  In fact there are several BAFF inhibitor drugs out there, even one that has been approved in lupus.  That drug is locked up in a "custody battle" between two big pharma companies.  I've tried to get it for research studies but I don't see that happening soon.  There are other anti BAFF drugs out there and I think we will get a readout as to their efficacy in CLL at some point.

One of my research buddies, Jennifer Brown at MGH in Boston has been studying familial CLL and she has identified a family where all the affected family members have a lost both copies of their DLEU7 gene - very interesting.

If you have a really big chunk of DNA missing from chromosome 13q14 (ie. type II deletion), you knock out a copy of the RB gene.  RB (aka retinoblastoma - a gene identified as the key driver of an uncommon eye tumor in kids) is a gene that plays a role analogous to p53 in some ways.  It is a critical determinant as to whether a cell proceeds through cell cycle or gets halted and may even directly interact with p53 (the key molecule altered in 17p deletions).  It appears that having RB deleted adversly affects prognosis.

So in this small area, you have several different molecules that all cooperate to form CLL.  This is a pretty power packed part of the genome as far as B cell biology is concerned.  Furthermore, it helps us understand ways to go after the fundamental biology of CLL instead of just throwing chemo at the problem.

Next, we will talk about how we test for it currently and what the limitations are of our current testing

13q - what is it and why does it matter? (part 1 of several)

July 15

I am really fascinated by the genomic alterations that have been observed in CLL.  One lesson we have learned over the years is that if you have a pattern - there is normally some key biology to be learned.  Once you learn the biology - then you can begin to make more informed ideas about how to treat such a disease.

13q14 is important for anybody who thinks about blood cancer.  It is the most common genomic alteration in CLL (about 50% of patients at diagnosis) and it is also quite common in NHL, myeloma, and even solid tumors.  In CLL - it is considered a "favorable" alteration.  Of all the FISH abnormalities you can have, if that is the only one, you are likely going to live longer than other patients with CLL.  In fact, if you have an mutated BCR, isolated 13q deletion, and early stage disease - chances are you may never actually need treatment.

So what have we learned about 13q14?

The nomenclature here is important.  You have MDR (minimal deleted region), CDR (commonly deleted region), type I (short, but typically inclusive of MDR/CDR), type II (big, inclusive of RB gene).  These can be confusing at first but should make more sense as we go through it.

FISH (fluorescent in situ hybridization) is a test where you use a probe that has a fluorescent tag.  The probe has a certain DNA segment attached that is very specific to a region of the genome.  You then "melt" the DNA in the specimen (blood lymphocytes, lymph node tissue, marrow), drop the probe in - let them bind, then you count the signals.  Since we have two copies of each chromosome, you should have two signals for each probe.  If you have a region of chromosome 13q deleted, you will only see one signal.  Occasionally, a patient will have both copies of 13q deleted (about 15% of time) and you will get no signals.  You use a control probe to make sure the test actually worked.  You can do the same for chromosome 12, 17p, 11q, or any other probe you want.

As you may know, we have 23 pairs of chromosomes - 46 total (although sometimes men lose their y chromosome without any consequence which is an oddity that always makes me laugh).  Chromosomes are rarely symmetrical, and the long arm is referred to as the "q" arm and the short is the "p" (think petit).  You can then use specific staining which creates bands along the chromosome visible to a microscope.  If you go out (starting from the central part known as the centromere) the long arm of chromosome 13 a total of 14 bands you get to a region known as 13q14 - and here is where there is a lot of action in CLL.

 In the case of chromosome 13q14 deletions, the standard probe binds to a region which is deleted in just about every case of 13q14 abnormalities.  It is the smallest known segment to be absent, so it is called the "minimal deleted region" or MDR.  Right at the "landing spot" of the probe you are in the middle of a segment that encodes an RNA molecule known as DLEU2 (deleted in leukemia #2).  We don't actually know what DLEU2 is supposed to do, but it gets faithfully copied from DNA to RNA.  In the process two smaller segments of RNA get cut up known as miRNA (for micro-RNA).  In fact, miRNA's were discovered in the first place when folks were studying this small stretch of genomic alteration in CLL patients.

These two miRNA's are very important.  They are miRNA 15 and 16.  When the cell makes these miRNA's they go out to other parts of the cell and find mRNA with similar sequence and they bind to each other.  Brief detour - DNA is the master template, and it gets copied to mRNA.  mRNA then leaves the nucleus, goes to the cytoplasm of the cell and gets "transcribed" into a protein - these are the engines that make a cell do whatever it does.

miRNAs 15 and 16 are important because they serve as negative regulators of BCL-2 mRNA and a host of other genes (please allow the simplification for now).  BCL-2 will be the subject of another post, but for now you should know that BCL-2 is a very important protein that keeps CLL cells alive a lot longer than they should.  There are also some very interesting drugs out there that target BCL-2 (ABT-199 AKA GDC-0199, navitoclax, others) Therefore, those patients that lack the negative regulator - get more of the BCL-2 protein than they should.

There are additional complexities about BCL-2 expression levels that an informed critic could say I am ignoring for now, but for the purposes of this post, I would like to leave it in the more simplified version.

Now for the interesting part.  If you take a mouse and get rid of that segment of DNA - they develop CLL (about 25% at 10 months - which in mouse terms - is not that aggressive).  Interestingly, they also develop lymphomas and even monoclonal B cell lymphocytosis.  They also have "stereotyped" B-Cell receptors which is another important feature of human CLL.

In future posts - I want to take a longer look at what else happens at 13q14 - but I had to do some background work here to explain how the test works etc.  Here is a link to one of the key papers 

Thanks for reading.