“FISH” testing: necessary, but not sufficient

Probably the single most important CLL clinical trial conducted in the last few years is the recent FCR versus FC double arm and large scale study done at multiple European centers. For a change, we have reasonably clear answers on who is likely to benefit from FCR front-line therapy. Almost as important, we have clear guidance on who is not likely to benefit and therefore should not be looking at FCR as their front-line therapy of choice. The results were very clear. Patients with 17p deletion got dismal results whether they used FC or FCR. I thought I would recap the details of this important result.

FCR vs FC, 17p deletion

While the news is gloomy, hardly any of the patients with 17p deletion got a full fledged CR response, it is nevertheless important to recognize the value of this negative result. Why on earth would you want to waste time, effort, money, expose yourself to the undeniable toxicity of either FC or FCR and take it on the chin with respect to potential loss of quality of life, if at the end of 6 months of therapy neither of the two regimens (FC or FCR) is going to give you a half way decent response / remission? If you are in the group of patients with 17p deletion, you are much better off knowing the hard truth ahead of time so that you can explore other and more promising therapy options (Campath, Revlimid, experimental drugs such as flavopiridol and more recent drugs in the pipeline) in order to get a decent remission en-route to a possible mini-allo transplant.  Waiting too long dithering or trying unlikely choices may cost you crucial window of opportunity. There is no point in wishful thinking when dealing with high risk CLL – we have to learn to accept reality if we are to play the hand we are dealt to best advantage.

Which brings us to the important issue of FISH testing. It is clear that patients with clinically aggressive CLL (rapidly growing white blood counts, enlarging lymph nodes, nasty B-symptoms) in imminent need of therapy should get tested for their FISH status. If they have clearly identified 17p deletion, FCR is most likely not a good choice. But how about the other side of the equation, does lack of 17p deletion by FISH testing give clear and unambiguous guidance that they do not have to worry about this particular hiccup?

Chromosomal abnormalities in CLL

FISH test is used to see if any important bits of chromosomes are broken off, if there are less or more than the standard two copies of each chromosome. We have discussed FISH test in prior articles, both on this website as well as our flagship website www.clltopics.org . Given its growing importance in how CLL is treated, I think it is important that you understand the value as well as limitations of this important prognostic test.

Each cell (barring egg or sperm cells) in your body has a full set of chromosomes and that is true of the CLL cells as well. We get two copies of each chromosome. In this article we will focus on the 17th chromosome. There are two copies of chromosome 17, just as all the other chromosomes. On the short arm of the 17th chromosome is an important gene that controls how cells that are badly damaged are made to die in an orderly fashion. This gene, called TP53 gene, is particularly important in controlling cancer cells. Cancer cells are dysfunctional cells by definition and it is the job of the TP53 gene to make sure such damaged cells are easily killed. When TP53 gene is not doing its job, cancer cells get a huge advantage. Even in their mangled and cancerous state they can ignore all orders to commit suicide. Even powerful chemotherapy agents such as fludarabine and cyclophosphamide are next to useless in killing these refractory cancer cells. Typically we use the shorthand notation of 17p, but the detailed street address is 17p13.1.  The resident living at that street address is the TP53 gene.  The main job of the resident (TP53 gene) livingh at this address (17p13.1) is to manufacture a kind of suicide pill (p53 protein), if you will, so that the cell can die a decent death when that becomes necessary.

Remember, each cell has a pair of 17 chromosomes. In other words, each cell has a pair of TP53 genes, two ‘alleles’ or copies. Does it matter if only  one of the two TP53 genes is broken off, deleted, but the other one is intact and doing its job exactly as it should? Yes, that makes a difference. Single allele 17p deletions are better than double allele 17p deletions. Having at least one working TP53 gene is better than none. But there is more to this story and it complicates life for patients with aggressive CLL. Very often, with one of the TP53 genes broken off, the second of the pair develops problems down the road. Even without frank deletion, the ability of this second TP53 gene to function may get compromised, as we describe below.

Present, but not doing its job

Recently I have had to register my car, get driver’s license etc at the local Motor Vehicle Administration in Maryland. I went in around 10:00am. There were more than a dozen windows open for business. A couple of them had their clerks on indefinite coffee break, no work getting done at their stations. But even among the windows that had a clerk bodily present, several were busy gossiping with their colleagues or doing something other than taking care of business. Net result was that while many windows were open, there were long lines and I had to wait several hours before taking care of my business.

What happens when TP53 genes are physically present but not doing their job? What are they supposed to be doing anyway? Think of genes ashow-to manuals, they provide instructions on how to manufacture very complicated and precise proteins. In the final analysis, all of the work of the cell is done by these proteins. TP53 gene has the crucial instructions on how the cell can make protein  p53, the ‘suicide pill’ we discussed above. If the cell does not know how to make this very important protein in the right quantities, the cell forgets how to die. It also becomes very hard to kill even when we kick it in the butt with powerful chemotherapy drugs.

FISH test uses probes looking for physical presence of specific genes. The usual CLL FISH panel uses a set of four probes, looking for presence of specific genes at the 13q, 11q and 17p locations as well as verifying there are only two copies of chromosome 12 (Trisomy 12 means there are three copies of this chromosome, instead of the standard 2 copies). These are the four questions asked in a typical FISH test; and like the Oracle of Delphi, the FISH test only answers the questions it is asked. It stays silent on all other issues. For example, it will shed no light on deletions on the long arm of the 6th chromosome – and a small minority of CLL patients do have 6q deletion as their chromosomal abnormality – unless the correct probe looking for this particular abnormality is also used in the test. And most important, the FISH test has nothing to say on the subject of whether the genes in question are doing what they are supposed to do, make the necessary protein correctly and not malingering or asleep at their job.

There are three common reasons why patients may have 17p malfunctioning. The most obvious is 17p deletion, easily detectable by the standard CLL FISH panel. But it is also possible that the TP53 gene is physically present but messed up (mutated TP53)  in some fashion so that the protein it makes is not quite right and useless in doing the job of killing the cell that needs to die. The third reason why 17p malfunction can happen is that the TP53 gene is present, there is nothing wrong with it – no mutation etc – but it is so covered up with gunk of various sorts that for all practical purposes it is hog-tied and cannot do its job. This is calledepigenetic silencing” of the gene and a very interesting field of research.

So, to summarize, TP53 function can become compromised for three reasons:

  1. 17p deletion where the TP53 gene is broken off and p53 protein production comes to a halt. This defect can be detected by FISH testing.
  2. 17p mutation where the TP53 gene is present but messed up subtly and therefore the p53 protein it makes is also messed up and useless.
  3. 17p silencing where the TP53 gene is willing and able to make the right p53 protein but it is covered up with gunk and cannot do its job.

Does mutation and / or silencing of TP53 gene function cause as many problems as breaking off and deletion of this important gene? The abstract below is clear in its implications.

J Clin Oncol. 2010 Oct 10;28(29):4473-9. Epub 2010 Aug 9.

TP53 mutation and survival in chronic lymphocytic leukemia.

Zenz T, Eichhorst B, Busch R, Denzel T, Häbe S, Winkler D, Bühler A, Edelmann J, Bergmann M, Hopfinger G, Hensel M, Hallek M, Döhner H, Stilgenbauer S.

University of Ulm, Ulm, Germany.

Abstract

PURPOSE: The precise prognostic impact of TP53 mutation and its incorporation into treatment algorithms in chronic lymphocytic leukemia (CLL) is unclear. We set out to define the impact of TP53 mutations in CLL.

PATIENTS AND METHODS: We assessed TP53 mutations by denaturing high-performance liquid chromatography (exons 2 to 11) in a randomized prospective trial (n = 375) with a follow-up of 52.8 months (German CLL Study Group CLL4 trial; fludarabine [F] v F + cyclophosphamide [FC]).

RESULTS: We found TP53 mutations in 8.5% of patients (28 of 328 patients). None of the patients with TP53 mutation showed a complete response. In patients with TP53 mutation, compared with patients without TP53 mutation, median progression-free survival (PFS; 23.3 v 62.2 months, respectively) and overall survival (OS; 29.2 v 84.6 months, respectively) were significantly decreased (both P < .001). TP53 mutations in the absence of 17p deletions were found in 4.5% of patients. PFS and OS for patients with 17p deletion and patients with TP53 mutation in the absence of 17p deletion were similar. Multivariate analysis identified TP53 mutation as the strongest prognostic marker regarding PFS (hazard ratio [HR] = 3.8; P < .001) and OS (HR = 7.2; P < .001). Other independent predictors of OS were IGHV mutation status (HR = 1.9), 11q deletion (HR = 1.9), 17p deletion (HR = 2.3), and FC treatment arm (HR = 0.6).

CONCLUSION: CLL with TP53 mutation carries a poor prognosis regardless of the presence of 17p deletion when treated with F-based chemotherapy. Thus, TP53 mutation analysis should be incorporated into the evaluation of patients with CLL before treatment initiation. Patients with TP53 mutation should be considered for alternative treatment approaches.

PMID: 20697090

Editorial

I try very hard to distinguish between research findings and expert comments that I report in my articles from my own editorial comments. That way you know when you are reading expert testimony and when it is only my two cents. What are the implications of the limitations of FISH testing?

For starters, the clear guidance in the abstract above is that we need better and more comprehensive tests. Since FISH is limited to telling us whether or not a particular gene is present or broken off, we need to move down the chain, we need to be able to test for properly functioning p53 protein. When rubber meets the road, who cares whether the TP53 gene is present, what is important is whether or not it is able to make the right amount of properly functioning p53 protein. Testing for proteins is not quite as easy as testing for genes. But it can be done. It is done even as I write this article, as part of research programs in expert centers. What we do not have is access to this testing technology in commercial testing labs. Heck, IgVH gene mutation status test was not available to CLL patients outside of research centers, just a few years ago. CLL Topics went through significant amount of hassle before we were able to get Quest Diagnostics to put together a CLL prognostics package. Other testing labs soon followed suit when they saw it was profitable business. It was our first and possibly most important patient advocacy effort. What we need is a similar campaign to get p53 protein testing within the reach of CLL patients. We need commercially available testing capability for this very important protein. Since the p53 protein is also important in predicting aggressiveness of many other cancers, we are not the only group of patients interesting in this prognostic testing capability and that should make the job easier. Anyone out there willing to take on this important advocacy job?

In the absence of reliable testing for properly functioning p53 protein, we must understand FISH test results are not the whole story. Lack of 17p deletion does not guarantee lack of malfunctioning p53. Your FISH test may look great, but if your CLL is clinically aggressive and the first couple of cycles of FCR do very little by way of controlling your disease, it may be time to pause, and re-think your treatment strategy. Perhaps you are one of the unlucky group that has 17p dysfunction without FISH detectable 17p deletion.

Interrupting therapy mid-stream is not an easy decision to make and not one that should be taken lightly. But it is important to consider what is at stake here. If nothing much is happening after the third cycle, if the lymph nodes are just as large as ever or even getting bigger, should the patient be put through the trauma and toxicity of another three cycles of FCR? When does the process become wishful thinking? I must confess I have no easy cheat-sheet answers on this one. Each of you who finds yourself in this situation must make the choices based on your own situation after careful consultations with your doctors.

Before we leave the subject of FISH test and its lack of completeness, I would like to address the subject of “normal” FISH result. I have no idea how this term got to be mainstream, the lab-tech who chose this term should be taken out and shot. A “normal” FISH result does not mean there is no chromosomal abnormality. The fact that the patient has CLL means there is one of more chromosomal abnormality – that much is guaranteed.

Since FISH test restricts itself to just 4 probes, the answers it gets are just as restricted. “Normal” in this context just means “none-of-the-above” or “we-don’t-know-what-it-is”. Can patients with “normal” FISH result have some chromosomal abnormality that is less common, such as the 6q deletion, that is not usually tested for? Absolutely. Can it be that in patients who have no deletion of any kind, the driving force behind their CLL is mutation or epigenetic silencing of some important tumor control gene, such as the TP53 gene we have discussed above? ABSOLUTELY!

“Normal” FISH result is a pig in a poke. It could be something relatively minor, predictive of a low risk variety of CLL. On the other hand, it could be something as dangerous as damaged (but not deleted) TP53gene. We just don’t know. In this context, it is interesting to note that in the FC versus FCR European study patients with “normal” FISH result did not do very well with FCR. I wonder how many of them were actually TP53 dysfunctional, destined to have poor response to FCR from the get-go.

Moral of the story, get your FISH test done if you have aggressive CLL. You need to know if you have the high risk 17p (TP53 gene) deletion since that will impact therapy decisions. You still won’t know about TP53 gene malfunctioning due to mutation or silencing – that can only be guessed at by how you actually respond to therapy. Stopping FCR therapy mid-stream because of lack of response is a bit like shutting the barn door after the horse has gone over the fence. But it might still be worth considering all the same, to prevent the rest of the horses from bolting as it were, avoid further damage.

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I am happy to report my eye problems are resolved, more or less.  I made an emergency flight back from India, went straight from the airport (short detour enroute to get a much needed shower) to consultation at the Wilmer Eye Clinic (Johns Hopkins) and straight into surgery.  After 200 or so laser jabs, the surgeon pronounced he has managed to repair / stabilize the tear in the retina of my left eye.  I was lucky.  It could have become full fledged detachment of the retina and possible loss of vision.  As it is, I am typing with a huge number of black spots dancing around in front of my left eye, like a horde of gnats and cobwebs. The good news is that now I have a built-in excuse for poor spelling and punctuation.

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