Prognostic Indicators el pt

FISHing in Murky Waters

Date: August 29, 2024

by Chaya Venkat

The Importance of Asking the Meaningful Question

Related Articles:
What Type of CLL Do You Have?
Prognosis at Diagnosis
FISHing for Answers

The other day a patient wrote to me about his FISH test results.  He was jubilant — his report, said he, had “normal” FISH results. He did not have any of the four classic deletions they look for, none, nada, not even the 13q deletion that everyone said was the best one to have!  He was a happy camper, and generously wanted to share the good news with me. Surely this is the best part of Bucket A?! 

It really bothers me when I have to be a party pooper and give bad news and rain on patients’ parades. I tell myself people are better off knowing the truth, even if it deflates rosy expectations, as in the case of this patient. I believe we cannot fight this enemy unless we know as much about it as we can. The fact that you are reading this article tells me you feel the same way too.

If you don’t ask the right question, the answer will make no sense!

Here is a scene you should be able to picture immediately in your mind. 

Imagine a crowded waiting room, packed full of CLL patients waiting to see the famous (and seriously over-booked) oncologist.  People have been waiting for more than a couple of hours past their supposed appointment time, trying not to get too frustrated or lose their temper with the pert young receptionist.  Finally, the nurse comes through the swing doors, with the charts of the lucky patients who will be seen next by the great man.  She calls out the names, “Susan, Mark, David and Agnes!” There is a moment of silence, no one stirs in the room, no one stands up with alacrity to follow her into the examination rooms.  The nurse looks confused, glances at her charts one more time to make sure she has the names right and calls out again:  “Susan, Mark, David and Agnes!”  Once more, dead silence.  The nurse shrugs, rolls her eyes, and flounces back into the back. 

Suppose she tells the doctor that the waiting room is empty there are no more patients to be seen that day, and he is free to go home early for a change. Sure, you can see the nurse would have to be a real idiot to assume there were no more patients waiting in the room, just because Susan and Mark decided to step out to make calls on their cell phones, David is answering a call from Mother Nature, and Agnes is taking a few very guilty puffs on her cigarette. Just because the four patients whose names were called out did not happen to be in the room does not mean there was no one else there.  The nurse did not ask the right question “Anybody else here wants to see the doctor?”, and therefore got meaningless silence as the response. 

FISH Test

“FISH” stands for fluorescence in-situ hybridization.  The test can be done with a sample of blood, or bone marrow aspirate.  (Since it is a lot less painful to give a sample of blood, I suggest you stick with that, except in special cases where the physician needs to find out what is happening in the bone marrow).  The test uses a set of “probes” to look for presence or absence of certain bits and pieces of your chromosomes.  The typical CLL FISH panel uses four specific probes, looking for deletions in specific locations of chromosomes13, 11, 17, and to see if there are more than the required two copies of chromosome 12.  In other words, this set of four probes can tell you whether or not you have deletions in 13q14, 11q23 (ATM gene) and 17p13 (TP53 gene), and whether there are the correct number of chromosome 12 (two copies, and only two copies of it.  If you have picked up an extra copy of this chromosome and now have 3 copies of it, you would have “12 Trisomy”). Each of these chromosomal defects has specific prognostic impact and knowing which one (or more) of these defects you have will go a long way to defining your risk category. (What Type of CLL Do You Have?)

How did we come to pick these four probes to use in the FISH test?  Simple.  Over the last decade or so, researchers have begun to unravel the genetic underpinnings of CLL.  We now know that these four abnormalities (13q, 11q, 17p and 12 Trisomy) are frequently observed in CLL patients.  But that is just the beginning of needed research, mere scratching at the surface of what makes a CLL cell a cancer cell.  There are many other gene defects, deletions, mutations, translocations and general all-around sleeping-on-the-job that may be involved in creating the first CLL cell in your body and giving it the survival advantage to become the monster it has.  Over time, it is possible - and even likely - that the CLL cells will pick up even more defects, a case of “clonal evolution”.

It is extremely unlikely that you have absolutely no genetic defects in your B-cells, none whatsoever, and yet you have CLL.  Cancer is caused by genetic defects, it goes with the territory. What would happen if you do have chromosomal defects but not the four specific ones targeted by the four probe set? Well, the FISH test results would come back as “normal”.  Susan, Mark, David and Agnes of our analogy happen to be not in the room, but there are plenty of other patients there!  The word “normal” to represent this state of affairs is very misleading, I wish they would use instead the phrase “none of the above”.  You could have a host of deletions that are not picked up by this set of “typical” CLL FISH panel, and some of them could be more dangerous than others. 

Your moment of Zen: if you went fishing in the lake and caught nothing, it does not prove there are no fish in the lake.  It could be you were using the wrong lures or you are just not very good at fishing.

The CLL FISH Panel Needs to be Expanded

It has been known for a while that in addition to 13q, 12 Trisomy, 11q and 17p deletions, another deletion that is frequently seen in CLL patients is located on the long arm of the 6th chromosome, specifically 6q21.  In a recent study (abstract below), 13 out of 217 CLL patients tested showed this deletion (6%).  Compared to the other risk groups, patients with the 6q deletion had intermediate prognosis.  In other words, they would fall into Bucket B.  (The concept of risk buckets was introduced in our article What Type of CLL Do You Have?) We have been pushing commercial labs such as Quest to add the 6q probe to their standard CLL FISH panel. Think about it: if you had your FISH test done and they used only the four conventional probes (13q, 11q, 17p and 12 Trisomy), they would not catch any potential 6q deletions. The result would come back “Normal FISH”. Yeah, they did not ask the right question and your test came back “normal”. Big help.

Abstract

Leukemia. 2024 Mar;18(3):476-83.

Chronic lymphocytic leukemia with 6q- shows distinct hematological features and intermediate prognosis.

Cuneo A, Rigolin GM, Bigoni R, De Angeli C, Veronese A, Cavazzini F, Bardi A, Roberti MG, Tammiso E, Agostini P, Ciccone M, Della Porta M, Tieghi A, Cavazzini L, Negrini M, Castoldi G.

Dipartimento di Scienze Biomediche e Terapie Avanzate, Sezione di Ematologia, Ferrara, Italy.

Cytogenetic and fluorescence in situ hybridization studies were successfully performed in 217 chronic lymphocytic leukemia (CLL). In all, 13 patients with 6q21 deletion were identified and characterized in comparison with 92 patients with 'favourable' karyotype (normal or 13q-), 69 cases with 'intermediate risk' (1-2 anomalies) and 43 cases with 'unfavourable' karyotype (complex, 11q- or 17p-). Six out of 13 cases with 6q- showed an excess of atypical lymphocytes, a finding confirmed at the histologic level; >20% CD38+ cells were seen in 5/6 cases. IGVH mutational status revealed >98% homology to the germline sequence in 4/10 cases. When compared with the 'favourable' group, patients with 6q- showed a higher white blood cell (WBC) count, frequent splenomegaly, atypical morphology, CD38+ and short time from diagnosis to first treatment and short survival. A higher median WBC count was found in the 6q- group vs the intermediate-risk group; survival was shorter in the unfavourable group. To ascertain if the 6q- anomaly was an independent factor predicting for an inferior outcome among those patients with 'favourable' cytogenetics, we performed an analysis of prognostic factors in 105 patients (92 'favourable' plus 13 with 6q-), showing that the 6q- chromosome maintained its prognostic significance at multivariate analysis (P=0.02) along with stage (P=0.01). We conclude that CLL with 6q- is characterized by a high incidence of atypical morphology, classical immunophenotype with CD38 positivity and intermediate incidence of IGVH somatic hypermutation. Clinicobiological features and outcome show that this cytogenetic subset of CLL should be allocated in an intermediate-risk category.

PMID: 14712287
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Significance of the 6q21 Deletion

An interesting study from Canadian researchers has just been published. They found that a gene called “HACE1” resides at the specific location 6q21. In normal people, this gene has the ability to help cells deal with various forms of stress, including environmental cancer triggers that cause tumor formation. When the HACE1 gene is missing or inactive, the odd cancerous cells that are present in all of us are able to grow and multiply, become full fledged, clinically significant tumors.

To test whether HACE1 gene located at the 6q21 location is indeed a tumor suppressor gene, researchers bred mice that had this gene deliberately deleted. They hypothesized that these unfortunate mice would be more susceptible to tumor growth – and indeed, different tumors did indeed form in the mice, but at a low rate of incidence.  However, when the mice were also subjected to various forms of stress, including ultraviolet radiation, lung carcinogens, or other genetic alterations, this double whammy of environmental insult on top of a missing protective tumor suppressor gene at the 6q21 location resulted in a dramatic increase in cancer growth, with the mice developing breast, lung, and liver cancers, as well as lymphomas.

“We’ve always suspected that cancer is caused by a combination of genetic and environmental factors working together,” says Dr. Sorensen. “Our results give us insight into how the disease takes root when a single gene is inactivated.”

Abstract

Nat Med. 2024 Aug 12; [Epub ahead of print]

The E3 ligase HACE1 is a critical chromosome 6q21 tumor suppressor involved in multiple cancers.

Zhang L, Anglesio MS, O'sullivan M, Zhang F, Yang G, Sarao R, Nghiem MP, Cronin S, Hara H, Melnyk N, Li L, Wada T, Liu PP, Farrar J, Arceci RJ, Sorensen PH, Penninger JM.

[1] Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohrgasse 3, 1030 Vienna, Austria. [2] Heart & Stroke/Richard Lewar Centre of Excellence, University of Toronto and Toronto General Research Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada. [3] These authors contributed equally to this work.

Transformation and cancer growth are regulated by the coordinate actions of oncogenes and tumor suppressors. Here, we show that the novel E3 ubiquitin ligase HACE1 is frequently downregulated in human tumors and maps to a region of chromosome 6q21 implicated in multiple human cancers. Genetic inactivation of HACE1 in mice results in the development of spontaneous, late-onset cancer. A second hit from either environmental triggers or genetic heterozygosity of another tumor suppressor, p53, markedly increased tumor incidence in a Hace1-deficient background. Re-expression of HACE1 in human tumor cells directly abrogates in vitro and in vivo tumor growth, whereas downregulation of HACE1 via siRNA allows non-tumorigenic human cells to form tumors in vivo. Mechanistically, the tumor-suppressor function of HACE1 is dependent on its E3 ligase activity and HACE1 controls adhesion-dependent growth and cell cycle progression during cell stress through degradation of cyclin D1. Thus, HACE1 is a candidate chromosome 6q21 tumor-suppressor gene involved in multiple cancers.

PMID: 17694067
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Editorial

In case you missed the point, I think it is very important that a comprehensive FISH test (including 6q21 probe, not just the “standard” four 13q, 11q, 17p and 12 trisomy probes) be a part of staging all CLL patients at the time of diagnosis. How can we have decent “Prognosis at Diagnosis” without this important diagnostic test? The chromosomal profile of your particular version of CLL has huge implications for therapy choices. Frankly, I have advised “Harvey” to get a FISH test done every year, to keep on top of potential clonal evolution. Sure enough, he progressed from a single allele deletion in 13q (the best kind), to having both 13q alleles deleted, followed by a more dangerous 11q deletion. If he had not done repeat FISH each year, he would have been under the misapprehension that his CLL was going to be very benign and indolent, and made inappropriate therapy choices. As it turned out, he is definitely in Bucket C and smart to make “Battle Plans” that fit his situation.

I got thinking about dozens of our members who have been exposed to “Agent Orange” and later developed CLL. Obviously, not every Vietnam veteran who was exposed to this nasty chemical has developed CLL. I wondered if the servicemen who were pre-disposed to cancer by virtue of HACE1 (6q21) defect going in had a higher chance of responding to the second blow, whether these guys had a higher likelihood of developing full blown cancers including CLL. If you fit the description, a CLL patient with prior Agent Orange exposure, I would very much like to know if you have had a FISH test done, and if it showed 6q21 deletion. I would be willing to bet dollars to donuts that more of you guys would have 6q21 defects, more than the 6% seen in general CLL patient population.

I suppose Agent Orange would not be unique in the list of environmental insults that would constitute a “double whammy”.  Dr. Sorensen (abstract above) identified lung carcinogens and excessive radiation as candidates. I wondered how many of our members smoked prior to their CLL diagnosis (surely you do not continue to smoke, even after this kick in the "butt"? Heh, heh.) and how many of them were exposed to excessive radon gas levels in their homes. By themselves these and other environmental insults may not have been enough to precipitate CLL. But coupled with a cancer predisposition because of deletion of 6q21 (HACE1 tumor suppressor gene), that might have been enough to push them over the edge. There has never been a clear connection between excessive radiation exposure and subsequent CLL diagnosis.  I wonder if the link would be more substantial if the CLL patient population examined was limited to only those with 6q21 defects. 

Given our present state of technology, there may not be much we can do if the little bit of DNA represented by 6q21 has actually broken off, gone AWOL, and therefore cannot perform its important job of tumor suppression.  But sometimes the precious gene is still there, but covered over by debris and junk (methyl groups).  For all practical purposes, the protective effects of the HACE1 gene can be absent, even when the gene is physically patient.  This phenomenon is called “epigenetic silencing” (Epigenetics). The good news is that we are learning how to clean the gunk off of important genes like this, and by doing so restore proper functioning of tumor suppression.  It might interest you to know that epigenetically silenced HACE1 has been implicated in several human cancers. This opens the possibility of treating the patients with drugs (demethylating agents) to clean off the HACE1 gene and letting it do its job.

Lots of questions, and precious few answers — sometimes it is all very frustrating and I feel like throwing up my hands and walking away.  

Not on your life.