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.