Sunday, July 15, 2012

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.