By Wayne “WWW” Wells – patient
Most patients with CLL/SLL who have been around for awhile will become aware of the ZAP–70 marker related to prognosis. Whether you are new or savvy to the world of CLL/ SLL, the ZAP–70 marker is a fascinating introduction to the biological complexity of CLL and an example as to why CLL manifests itself so differently in each of its victims. If you have been around CLL long enough to become familiar with some of the issues surrounding prognosis, bear with me while I go through some housekeeping items to include defining terms and basic concepts to ground everyone for a more meaningful understanding of ZAP–70. There will be interesting findings for the advanced patient/care giver in the latter part of this article that newbies may wish to skip, just as CLL savvy readers can skip the introduction section dealing with the basics of terminology, concepts and context for which ZAP–70 is our focus.
As you read on and explore referenced research articles, I thought it would be useful to define a few terms, perhaps unfamiliar to the average layperson. I use a metaphor of “dancing with a Bear” to describe our struggle to survive CLL. If one is unfortunate enough to be “found dancing with a Bear”, the only way the dance will stop is when the Bear gives up. Being that bears are powerfully dangerous, the better informed and proactive patient dance partner stands the best chance of out–smarting the Bear to escape the tempo of its dancing.
Advanced apologies to the sophisticated reader for oversimplifications, omissions or unintended errors or emphasis deficiencies as this is meant as primarily a layman’s guide in the exploration of an important feature in many patients’ CLL, how to think about it and if it can be used in regard to treatment decisions.
CLL (Chronic Lymphocytic Leukemia) is a complex blood–borne cancer that can be primarily present in the blood or found exclusively in the lymph tissue, where it is then called SLL (Small Lymphocytic Lymphoma). “Pure” CLL or SLL is rare, with most patients exhibiting cancer cells in both the blood and lymph tissues. Patients may start as CLL or SLL and progress in time to acquire the other. The dual–expression of our cancer as a Leukemia and or a Lymphoma is the first of several points of confusion for the newly–diagnosed in a unique journey for each patient. In this article I will use “CLL” to include SLL for brevity.
Indolent and Aggressive disease – defines 2 broad categories of disease patterns resulting in easier or more difficult to treat and survive respectively.
Homeostasis – an important concept to hold onto as you learn about ZAP–70. Homeostasis is the dynamic balance in which our cells function, hovering between survival and cell death which is controlled through cell signaling.
Proteins & kinase/enzymes – terminology often found in research articles. We can think of them as the “legos” or building block agents that affect homeostasis for health of sustaining useful cell life or hijacked agents that do the bidding of the CLL Bear. There are many protein/kinase/enzymes responsible for maintaining the health of B–lymphocytes and T–lymphocytes, which is accomplished often by how densely they are expressed in cells and whether or not they are in an activated state.
Kinase – an important term for understanding not only ZAP–70, but how some of the most newly–employed drugs work to put the CLL Bear into hibernation. A kinase is an enzyme and a feature in a cell that I liken to that of an electrical circuit breaker box in my house, as it relates to distributive cell signaling.
Signaling – the most important function for how ZAP–70 expression affects CLL progression and survival.
Genes – the biological equivalent of hardware in a computer.
Epigenetics – (Epi = above) the biological equivalent to software running programs on computer hardware.
Methylation – biochemical epigenetic methyl groups that bind to gene structure and affect function.
IGHV or IgVH – interchangeable, IG = Immunoglobulin & HV or vH = Heavy Variable/variable Heavy chain.
At diagnosis, we soon find out that, although there is no cure for CLL, if we’re lucky, we might continue to live for decades not needing treatment, but on the other hand…… and it is this “Other Hand” that causes the anxiety to seek out tests promising prognostic information. These tests reveal information commonly referred to as Markers (biologically active proteins/enzymes) and abnormal chromosome DNA which helps to indicate whether the course of our disease will be indolent or aggressive, needing earlier or later treatment and/or being more or less resistant or responsive to various therapies.
One of these important tests commonly ordered by Oncologists is for ZAP–70 (“Zeta Associated Protein –70’’) in a class of proteins called tyrosine kinases. So what is it about ZAP–70 that is important in CLL? How does it function in normal circumstances and what makes it special to CLL?
In normal human biology, ZAP–70 is expressed near to the surface membrane of T-cells & NK-(Natural Killer) cells, but not B-cells. The mechanism by which ZAP–70 is abnormally expressed in CLL B–cells is not clear, nor is the lack of consistency of expression in CLL patients understood. As part of the TCR (T cell receptor), ZAP-70 has a critical signaling function related to immune response to a degree that, when not expressed, the human will be severely immune deficient. When aberrantly expressed in B–cells at a level above 20%, there is a strong correlation towards aggressive CLL and to another important profiling test, the IGHV mutation status finding of Un–mutated CLL (U–CLL). It is useful to having an understanding of ZAP–70 to know that U–CLL is descriptive for where our CLL originates. It is characterized by early B-lymphocytes, referred to as naive cells that have yet to go through a maturation phase where they learn to recognize antigen threats. A ZAP–70 finding in B–cells was thought to act much like a switch being “on” where above 20% equals aggressive disease or “off” if below the 20% threshold.
I think of CLL as the dancing Bear who goes to a biology app store to get the ZAP-70 app to amplify the tempo of his dance with us. How our CLL actually co-opts ZAP–70 for signal amplification, when it is normally found in T–cells has never been adequately explained to me. It is a proper time to tip our hats to the mysterious complexity of biology in general and CLL patho–biology in particular.
To understand the function of ZAP–70 in CLL is to appreciate the intricate complexity of how our cells function through a web of intracellular signaling dependent on protein features called kinases. Our B cells are defined by an outer membrane with antennae/receptors, the most important for CLL being the BCR (B Cell Receptor) which is the origin locus for signals of cell survival and proliferation. The goal of CLL is to maximize cell survival and speed up proliferation by hijacking existing mechanisms of homeostasis that keep healthy good functioning cells alive and mechanisms that direct damaged or senescent cells to begin programmed cell death called apoptosis. From the BCR, the signals passes through the outer cell membrane, where they can, in some but not all CLL patients, become amplified by the presence of the abnormally–expressed ZAP–70 protein. From this point, and other kinases near the outer cell membrane, signals pass deeper into and throughout the inner cellular medium called cytosol. This is where certain other kinases are found that relay the signals toward the nucleus. Some of these kinases are familiar to us as targets for drug inhibition of these survival signals before they reach the nucleus of the cells. The two most familiar kinase targets are BTK (Bruton’s Tyrosine Kinase) & PI3K delta for which the drugs Ibrutinib and Idelalisib are very effective for inhibiting signal flow respectively.
ZAP–70 is only one of many features of CLL biology that is fascinating in and of itself, but a prime example of why we, as proactive patients, might benefit to a degree of saving our own skin if we care to take the time to learn about what the Bear does to try and kill us. I call this pursuit of CLL knowledge the “Casino’s Edge”. The Casino will always win from every player in the long run but not every day with every customer. The Casino wins by upping the odds just a bit over 50 percent for any game in the house. In our game with the CLL Bear we do not yet have enough knowledge to win every time we make a decision, but we can improve the odds to our survival advantage.
A quick story for how I gained the Casino’s Edge by knowledge of ZAP–70. Back in the ancient days of CLL Therapy that relied on sledgehammer chemo drugs and the first generation monoclonal antibody, Rituximab (RTX), I was rapidly progressing and in need for Treatment (TX). Not wanting chemo, but with failing kidney function, I could not wait for the promising new drug we were hearing about called CAL-101. I entered into TX in the Summer of 2009 with FR (Fludarabine & Rituxan) ending after only 2 cycles in scary kidney failure blamed on rare reaction to Fludarabine. Upon relapse, I tried to buy time with High Dose Rituxan (HDRTX), resulting in another spectacular failure culminating in hospitalization. My local Heme/Onc at the time suggested a newly approved mAb, Ofatumumab, touted to be more effective than Rituxan and not containing any mouse protein, which was the suggested cause for my hospitalization. With two TX failures brushing shoulders with death, I was not accepting the Heme/Onc’s recommendation without a test to clarify the reason for the severe reaction that she thought was caused by the mouse protein in RTX. At this low point in my journey, I refused Ofatumumab to seek advice from a CLL specialist I had first met at the Canadian Conference of 2007, Dr. John Byrd. Before my appointment with him, I had been thinking hard about why I had so many good markers with the one exception of ZAP–70, which was very high (58%+) and could this marker be accurate even when other CLL experts had told me it was probably a faulty test result? The only thing I knew about ZAP–70 was its relationship to amplified signaling and that chemo/immunotherapy was not something I was eager to try again.
When Dr. Byrd indicated the likelihood that I could die from trying Ofatumumab if I had the condition of Complement Dysregulation, I asked him what options were left for me. He offered several drugs in experimental Clinical Trials that included PCI-32765 later to become Ibrutinib. I grabbed at this drug because I knew that PCI was a signal inhibitor. I could not be sure that my disease was only driven by amplified signaling, but that choice sure looked promising on paper. 6 years & 8 months out doing well. The details of my unique journey may not be important to you, my reader, but my paying attention to the concept of aberrant cell signaling defined in me by a high percent of ZAP–70 whether accurate or not, led me to Ibrutinib. What are you questioning about your flavor of CLL, for what might be important in future TX decision making?
To review so far what I have covered has been presented as a pretty straightforward proposition that if ZAP–70 is found to be above 20%, you are likely to have Unmutated IGHV CLL correlated to aggressive disease course, with shorter time to first treatment (TTFT) and shorter Overall Survival (OS). If expressed less than 20%, you are likely to have Mutated IGHV CLL, with a more indolent disease course and longer OS. It was in part because of my high 6% mutated IGHV test, the good markers of 13q deletion and CD38 negative that the test result for 58%+ ZAP–70 was dismissed as lab error. To this day, the testing for ZAP–70 has not been standardized, even in the knowledge that accurate ZAP–70 expression in B lymphocytes is an important marker. Highlighting this problem is the following from Yu–Jie Wu et al., https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4762435/ “… standardization for evaluation of ZAP–70 detection has not been easy. The key problem is the weak ZAP–70 expression with a continuous expression pattern rather than a clear discrimination between positive and negative CLL cells. Consequently, the resulting judgment tends to be subjective. We showed that determination of ZAP–70 status, which requires using T– or natural killer (NK)-cells for reference definition, can be discordant in 10% of the CLL cases.”
“In our study, three of eleven CLL samples that were acquired and evaluated by different operators exhibited marked variation in ZAP–70 expression based on the percentage method. It is evident that the choice of controls and the method used to set a threshold to distinguish positive and negative ZAP–70 status in CLL remains challenging.” Methodology: Arithmetic vs Geometric MFI index – conflicting accuracy – Geo MFI reduces subjective error.
Some patients will focus on ZAP–70 + test result as an oracle of doom. Like all things CLL, the implications for ZAP–70 expression in our cancer cells is nuanced. Over years of testing there is an acknowledged subset of patients who are described as discordant, meaning some can exhibit good markers like me, but have high ZAP–70 positive disease and some with poor markers, like Unmutated IGHV can have ZAP–70 negative expression.
One example of where a ZAP–70 test result can be refined to better diagnosis, disease progression, and risk status is paying attention to IGHV variable gene family of use correlated to ZAP–70 positivity. The Immunoglobulin variable region of the heavy chain is a locus for many “families” and is represented by numbers e.g. my CLL uses V4–34. For more, go to Dr. Hamblin’s Blog to read his article “Stereotypy and CLL” http://mutated-unmuated.blogspot.com/2011/01/stereotypy-and-cll.html
The issue of most of our markers and their relevance to prognosis and response to treatment has changed from the time when treatment choices consisted of chemo & monoclonal antibody drugs and little else. Markers that include ZAP–70 are thought by CLL specialist Dr. Rick Furman in an Onc Live video https://www.youtube.com/watch?v=zlKKLFyZQao to be “no longer prognostic” in the era of newer drugs like the KI (Kinase Inhibitor) class of drugs such as Ibrutinib & Idelalisib and the Bcl–2 inhibitor drug, Venetoclax. These drugs and their cousins work very well in patients with poor marker aggressive disease, as well as with indolent CLL. So should we now drop our guard and be complacent? I feel Dr. Furman’s view to be too simplistic, when there is still no cure and with the number of patients we see relapsing in spite of these new wonderful drugs. A Bear in hibernation is not a dead bear and although the percentage of relapses from the newer drugs is low for the CLL community as a whole, it is happening to high–risk subsets of patients all too frequently. Until there is a cure, which will likely take a combination of multiple targeting drugs, we should remain on guard and pursue the growing body of CLL knowledge exploring evermore clever ways to defeat the Bear.
With that in mind, and researchers continuing to look for new targets for which drugs can be developed to block novel mechanisms of CLL pathology, you may have asked yourself: Is targeting high–risk patients that express ZAP–70 positive disease with a drug that targets ZAP–70 is a good strategy? If you had high–risk CLL with a ZAP–70+ marker, would you enter a Clinical Trial using a ZAP–70 inhibitor drug? I viewed with interest a Patient Power Video published in 2012 https://www.youtube.com/watch?v=d6yioq1s3b8 in which a prominent Canadian researcher Dr. Spencer Gibson talked about just such a strategy. For the purpose of educating a CLL patient, this video was disappointing in that it failed to address a critical question for which the answer would be most important, as to whether or not I would ever consider entering a Trial targeting my ZAP–70. Recall in the beginning of this essay where we learned that ZAP–70 is normally expressed on T-and NK-cells, not B–cells and that ZAP–70 deficiency in T–cells results in severe immune deficiency. I, as a prospective Clinical Trial patient, would want to know how a drug targeting ZAP–70 would or could be targeted exclusively to B–cells and not T- or NK-cells.
That very question expands this discussion into the fact that no marker is a stand-alone definitive thing that determines the course of one’s disease or the response one can expect from therapy. At this point, I will take you into the weeds of some of the more interesting, but complex workings of ZAP–70, and by implication, all markers.
It turns out that ZAP-70 has important co-factors that team up to help the CLL cells adhere to the stromal layer of lymphatic tissue, where they are nurtured and can evolve into more aggressive disease clones. One of these co-factors is CD27. In the following abstract, S. Lafarge et al., in which Dr. Gibson is a co-author, we learn the following: “CD27 expression correlated with functional capacity to adhere to stromal cells and antibody blockade of CD27 impaired CLL binding to stroma” & “CD27 levels correlate with CLL capacity to adhere to stromal cells. https://www.ncbi.nlm.nih.gov/pubmed/26002513 . Here is a companion link related to the above section: https://www.sciencedirect.com/science/article/pii/S0145212613002828
In another abstract, S. Das et al., in which Dr. Gibson is a co-author we learn of an additional co-conspirator with ZAP–70, the PKM2 kinase, that is implicated as an independent growth agent for CLL cells. http://cancerres.aacrjournals.org/content/77/13_Supplement/5423.short
While it is not expected for us lay-patients to know how these factors work, just knowing about them can help us to ask relevant questions of our treating Oncologist. Here is such a hypothetical question for considering using a ZAP–70 inhibitor drug in the context of a Clinical Trial. “Doctor, I would like to know more about how this drug is designed to work. Will this drug avoid inhibiting normal functioning of ZAP–70 on my T- or NK-cells and if so, how are you sure?” One hypothetical assurance could be that a drug might be engineered to bind to not only just ZAP–70, but also to a unique co-factor, such as one of the above mentioned causing selective attachment to only the B–cells expressing ZAP–70, where its inhibition of function by the hypothetical drug would be selective.
Knowing about CD27 and PKM2 I would also want to ask if an experimental ZAP-70 inhibitor drug would really be enough to disable the proliferation function of PKM2 and stromal tissue binding action of CD27. The patient can always ask for research evidence to assess the likelihood of taking an experimental drug.
Poking further into the linkage of ZAP–70 to other factors opens the door to another exciting new field of research, that of Methylation. So what is methylation and what is it telling us about CLL biology and our subject ZAP–70? A simplistic way to think about methylation is to know that it is a biochemical agent influencing, in the case of ZAP–70, signal rate function. Agents of methylation called methyl groups attach themselves to very specific sites called CpGs. The C = Cytosine and the G = Guanine which are two of 4 DNA building blocks (Cytosine, Guanine, Adenine & Thymine) comprising the familiar double helix of our genome. Methyl group expression at CpGs play an epigenetic role in gene and protein functions. There is a normal concentration of expression that progresses as B–cells mature, keeping gene/kinase function healthy and when overexpressed (hypermethylated) or under expressed (hypomethylated), the functioning of a particular gene/kinase can become pathological to serve the survival/proliferation goal of the CLL Bear.
Methylation first came to my attention two years ago with the discovery of the work done by a Spanish group with a poster presentation at the 2016 Chicago LRF Conference. Since then I have interviewed a prominent researcher, Dr. Christopher Oakes, heading up methylation studies at OSU Medical Center where I am currently in an extended Clinical Trial. Following are some methylation research findings with relevance to ZAP–70 in CLL.
C. C. Oakes et al., In the Journal “Nature Genetics” entitled “DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia” https://www.nature.com/articles/ng.3488
This paper looked at effects from a spectrum of methylation expression in the promotor region of ZAP–70 and, in doing so, identified 3 levels of “programmed” CLL e.g. Low–programmed (LP–CLL), Intermediate-programmed (IP–CLL) and High- programmed (HP–CLL).
“… the region immediately flanking the ZAP–70 promoter was strongly hypomethylated in most CLLs with wild–type IGHV relative to those with mutated IGHV and was a strong predictor of poor outcome.” “… this difference in methylation arises from normally occurring low levels of methylation in naive B cells and subsequent hypermethylation during B cell maturation. … Additional hypomethylation surrounding the ZAP–70 promoter could be observed, most pronouncedly in LP–CLLs.” “… For example, ZAP–70 is unmethylated and expressed in naive B cells, and the failure to hypermethylate and repress its expression (as occurs in normal cells) in LP–CLL may further enhance the aggressiveness of this subtype …”
This work is a fascinating look into how methylation can interact with ZAP–70 as an agent of CLL aggression and to explain to some degree its inconsistency as it relates to the CLL population as a whole and, in particular, to those with the “good marker” of being IGHV mutated, but experience aggressive disease. The article states: “… we propose that multiple genetic and epigenetic events converge in very early stages of B cell maturation to perturb normal programming networks and thereby promote the initiation of a premalignant clone.” The key word to me is “networks” in that we need to understand the unique networking of multiple markers and their influences if we are to achieve the goal of individualized medicines that can cure us.
Other work of interest for our look at ZAP–70 is presented by R. Claus et al., Titled: Quantitative DNA Methylation Analysis Identifies a Single CpG Dinucleotide Important for ZAP–70 Expression and Predictive of Prognosis in Chronic Lymphocytic Leukemia: Journal of Clinical Oncology: Vol 30, No 20 http://ascopubs.org/doi/abs/10.1200/JCO.2011.39.3090
“ZAP–70 methylation 223 nucleotides downstream of transcription (CpG+223) predicts outcome in chronic lymphocytic leukemia” “Cases with low methylation (<20%) had significantly shortened time to first treatment (TT) and overall survival (OS) (P < .0001)”
“Conversely, 16 ZAP–70 protein–positive cases with high methylation had poor outcome (median, 1.1 vs 2.3 years for high vs low methylation; HR = 1.62; 95% CI, 0.87–3.03). For OS, ZAP–70 methylation was the strongest risk factor.” “ZAP–70 CpG+223 methylation represents a superior biomarker for TT and OS that can be feasibly measured, supporting its use in risk–stratifying CLL.” The message of this research is clearly that ZAP–70 does not act alone and that methylation data is necessary to accurately assess prognosis for many CLL patients.
In summing up my take on ZAP–70, I believe it is an important marker as it pertains to research in understanding CLL and will likely lead to development of therapies that will cure us or better yet, prevent CLL from occurring in the first place. For immediate clinical application for deciding which therapy is best or to obtain a meaningful prognosis, not so much. If you are planning for how your CLL might behave if you are in Wait & Watch, you need to be assured that a ZAP–70 test is accurate and supported by IGHV mutation status “VH”gene of use and if any methylation test results have been done. The fact that there is yet to be a standardized test for ZAP–70 and the likelihood for lack of supporting methylation data makes ZAP–70 much less important than it was at the time when I had a tough TX decision to make. This is true IF one has ready access to the newer signaling kinase inhibitors, such as Ibrutinib, its cousin drugs or the Bcl–2 inhibitor, Venetoclax. The understanding of methylation and its role in the way ZAP–70 regulates signaling may lead to novel ways to put the “Bear” into permanent hibernation, but that is for the future.
If this has been worth reading or not, let Dr. Brian Koffman know so I will plan to write on something else that is of potential interest to the community or go fishing. ;–) WWW
Wayne Wells is a member of the CLL Society Patient Advisory Board: I was diagnosed with CLL on Sept. 11, 2006 and told I had a “good cancer” and as such, might never need treatment. Prognosis testing results from IGHV mutation status, FISH and CD38 all predicted an indolent disease course. The one test (ZAP-70), which CLL specialists dismissed as inaccurate, was 58% positive contradicting the other good markers. The rapid rate of tumor burden increase marked by bulky lymphadenopathy indicated I was discordant to the favorable markers. After failing 1st and 2nd standard therapy options I signed up for a Phase Ib Clinical Trial in June of 2011 with a new agent called PCI-32765 later branded Ibrutinib (Imbruvica). I have been on Ibrutinib ever since in remission currently stagnating with a small cancer cell population only detectable by sophisticated detection techniques.
Originally published in The CLL Tribune Q1 2018.