I am 6 and 1/2 yrs watch and wait. On my recent visit to my CLL specialist, he ordered, for the first time, the ClonoSeq Id test with my usual lab work. Has anybody heard of this test and what is its relevance?
Recent bloods: AlC 129,000, RBC 4.43, Hemoglobin 13.2, Plts 150 ( my last three labs have shown plts of 150! Since my diagnosis my Plts have never been that high. They are usually between 110 and 130 since DX 6 years ago.
I am mutated HGIV and either 14Q deletion with 2/18 translocation ( done in 2019) or 13q deleted per my first FISH ( done in 2018)
Thanks.
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Claybuster
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Before you can use this test for mrd, they find your unique dna sequences of CLL. This needs to be done before you start treatment or right after, kind of like a baseline. Once treatment starts the Clonoseq test can be used to see how well the treatment is working and if on a time limited treatment if you have reached uMRD. It isn’t a precise as a bmb but it’s pretty close at how many CLL cells per million cells. Plus a lot less invasive with a simple blood test.
The ClonoSEQ ID test is the test that identifies the Dominant Clones in the sample. The sample can be from a Bone Marrow Aspiration usually done at the same time as the biopsy, or from Peripheral Blood (i.e. your arm).
It's the most precise count of CLL cells commercially available, about 100 times more precise that the usual Flow Cytometry based MRD test.
It's a newer test that documents the gene sequencing of your specific CLL clone, if there is a dominant one. And this baseline can be used to determine if & how any further growth of CLL (after a remission) has changed, if it does or not. It's more sensitive than older tests.
It's needed for future blood MRD test to measure treatment result, when the test can detect one bad cell out of 100000 or 1000000 good ones; it's necessary to do the initial test before treatment so that future tests will know what to look for in bad cells.
Adding a bit to the other replies- the Clonoseq test is the most sensitive of our available tests that can measure MRD (Minimal Residual Disease) to 1 CLL cell in 1 million white cells. But a pretreatment blood sample is needed to reach the 1 in a million accuracy at a later date - usually after treatment with combination therapies, sometimes written as MRD-U6. clonoseq.com/patient/chroni...
Other MRD tests achieve lower accuracy like 1 CLL cell in 100,000 and can be noted as MRD-U5 or U4
I hope the others answered your questions regarding what ClonoSEQ is, and roughly what it can show.
I've done ClonoSEQ as part of a clinical trial at M.D. Anderson. They did my ClonoSEQ ID Test on my bone marrow aspiration sample taken during my initial bone marrow biopsy before treatment. A BMB is not essential, except in trials.
ClonoSEQ does not fully sequence the whole CLL cell, by the way. That would be a ton of data! It sequences only the IG Heavy Chain gene and the IG Light Chain (Kappa and Lambda) genes. Those are enough to identify a unique clone. Some day, it might also sequence other parts of the cell.
The subsequent ClonoSEQ tests are called ClonoSEQ Tracking Tests. They count the number of unique B-cell clones, and look for the Dominant Clone(s) seen in the ClonoSEQ ID test. If the Dominant Clone(s) cannot be found, you will have achieved uMRD6 (or U-MRD6) - undetectable CLL at the level of 1 CLL cell per 1 million WBCs. Actually, the Tracking tests usually sample more than 2 million cells. On one of mine, it found a single cell out of 2.8 Million. It burst my perfect bubble. No matter what, CLL will still be there after treatment, because we have billions of B-cells. It just takes more and more sensitive tests to measure it.
So what's ClonoSEQ good for?
It can be used to test the effectiveness of treatment. It's not usually done during or after BTKi monotherapy treatment, like Brukinsa/Zanubrutinib or Calquence/Acalabrutinib, because those treatments don't try to kill as much CLL as possible. They try to get CLL down to a constant ALC level. But Fixed Duration treatments try to knock CLL back as much as possible for as long as possible, ideally. In the past, after fixed duration treatments like Venetoclax plus Obinutuzumab, we would see our ALC go way down into normal, and then declare a relapse if it comes back above normal or if lymph nodes or spleen enlarge. Then, by the international guidelines (iwCLL), we would re-treat when symptoms to begin again. That would be the second level treatment.
But there have been observations that by the time that symptoms resume after relapse, the disease has acquired additional mutations that make it more aggressive and the second line treatment shorter. The additional mutations sometimes include additional FiSH types, but there's a growing list of other genes involved, too. So there are theories that if they can re-treat before the traditional guidelines for regression (symptoms), they could stretch PFS (Progression Free Survival). That hasn't been proven in large studies yet, though.
Some doctors don't wait for the guideline symptoms, and re-treat as soon as lymphocyte counts go up or lymph nodes grow again. Another factor with second line treatment is general health. Treatment of almost any kind at age 80 is hard than at age 70. It's hard to guage life-expectancy. But if several shorter therapies with drugs with fewer side effects can be used, the patients would like it more.
So MRD testing becomes a way to see the CLL grow even before the lymphocyte count rises again. If may allow sooner treatment with newly approved drugs. It may allow sooner treatment with off-label drugs (i.e. drugs approved, but approved for some other situation) before the clones mutate a lot more.
So I think your doctor is thinking ahead to when some of the new drugs get approved. I hope that your treatment gives you years of remission, and that your ClonoSEQ counts stay undetectable or at least slowly rise. Having had the ClonoSEQ ID test and a few ClonoSEQ subsequent tests which also identify new clones will prepare you better for the future.
One thing I should mention. ClonoSEQ costs about US $2,400 per test. It's covered by insurance for most people, and they've been really patient with my insurance company for the proper paperwork from the doctor. Regular Flow Cytometry MRD costs between US $500 and $1000, depending on the lab. I had that also via M.D. Anderson's lab. They did that for comparison purposes, because each method works differently. ClonoSEQ is expected to be more accurate, but there will surely be bar bets placed when the CLL counts on each tests are close to each other.
Seymour, thank you very much for the valuable information. During my visit with my CLL specialist I asked him how soon treatment might be and he said anywhere from 1 to 2 years. I was just wondering why they would do this test so soon. My Bloods seem to be stable with an absolute lymphocyte count of 129 red blood cell 5.45 and platelets at 1:50. I am getting a little fatigued, though. I am currently on Medicare with a G supplement so I assume the doctor wouldn't order such a test if it wasn't approved or potentially be approved by medicare. If that $2,400 charge it could be a big hit for me!
Medicare pays for it. I imagine knowing exactly what you have *right now* can be helpful in the future. Sometimes we appear fine, symptom-wise as well as labwork, and it can change literally within a week. I have a neighbor with CLL who had been on W&W for years, had his regular checkup, yet within a week his platelets bottomed out & treament was started a few days later. It's not common but not impossible.
IMO your doc is being proactive, getting this done. Especially as you have other tests, etc. contributing to your out-of-pocket maximum. Your plan G covers Part B "excess charges"; IDK what your facility bills, but my Medicare paid it completely. I do have a Medicare Advantage plan, however. In the unlikely event you somehow react like my neighbor (fine until suddenly not fine), you have this baseline done.
I'm not a hematologist or even a doctor. I'm a nerd. I find your FiSH interesting, and I bet he does, too. Not bad interesting, but, "oh? Cool. I wonder how this plays out."
The fact that your first FiSH showed del13q, and your second found a translocation makes me wonder. I've had one squirrel in my tests over the years myself, I was diagnosed back in 2011, before community hematologists ordered IGHV testing. They did CD38 and ZAP70 testing back then, and there were arguments about exceptions and percentages.
So in 2013, after taking a genetics class online to learn about Trisomy, among other things, and after reading Chaya Venkat's blog on clltopics.org (she's wherever saints go when they die now), I learned about IGHV (or IGVH, as it was called then) and asked my local hemo/onco for a test. He ordered it from Quest Diagnostics, and it came back as:
IgVH Mutation Status, Cell Based - Mutated, VH3-7 family
IgVH Mutation Rate 15.44%
Such a relief!
I've had multiple FiSH tests and Flow Cytometries over the years through my local hemo/onco, Tulane University, and NIH, and saw my CD19 counts gradually rise. But nobody every re-did the IGHV test, because, they say, you only have one major clone, and it's either mutated or not. It's dogma.
In 2022, feeling worse and worse, some CLL friends encouraged me to get a specialist workup at M.D. Anderson. I was seen by Dr. Philip A. Thomspon, a rockstar of CLL. He redid everything, including IGHV. Now, I tested:
Unmutated, IGHV1-69 -IGHJ6
Not only not mutated, but a different IGHV family! I asked him what the heck? He said that either the first test was wrong, or I'm bi-clonal. That's an actual thing, and the dogma has exceptions. One CLL population study found quite a few people with them - 3% of patients or so, if I recall.
So, all the FiSH and Flow Cytometry averages are just that. There's exceptions. Some people don't fit the average box. It's not necessarily bad. My therapy at MDA went really, really well. Complete remission, U-MRD6 in blood several times, with a final detectable in 2.8M cells in bone marrow, so >0<1 in 1M. The IGHV Mutated result cooled my natural anxiety during my 12 year W&W. The IGHV Unmutated result might make my remission shorter - we're gonna find out, and we'll use ClonoSEQ to see the train coming.
Try as I might, though, I could not match my ClonoSEQ sequences with anything in any gene database. Gene databases are based on just a few individuals, sometimes only 1. People differ on genetic stuff. ClonoSEQ gives you the actual sequence they're looking for. But they don't say exactly which gene(s) they are sequencing. They start at a start codon and sequence till they see a stop codon. Humans, immunology and genetics are so complex, that way. It has to do with the fact that IGH and IGL Kappa and Lambda mutate naturally to potentially form billions and billions of possible combinations of the BCR (B-cell Receptor) to match so many possible antigen amino acid sequences. Also, we inherit different copies of genes from each parent, and some of this goes back hundreds or thousands of years into the past. They may say we are 98 or 99% the same DNA as a chimpanzee, but there's footnotes about how many billions of base pairs there are. There's a lot of differences to the right of the decimal point.
So you're interesting because of that t(2;18) translocation. 99.9% of us don't get tested for that. Heck, when I had my first FiSH, they only tested CEP 12 (the centromere of Chromosome 12 - the spot where the 2 copies of the chromosome normally join - or 3 in my case), TP53 (on 17p). D13S319 (13q14.3), and ATM for 11q22.3. The NCCN Guidelines now recommend doing a test called cytospin for cyclin D1, or FiSH for t(11;14) CCND1/IGH fusion as a minimum whether you're about to be treated or not. On FaceBook, we see people getting 6q- [SEC63 (6q21), MYB (6q23)] | ATM (11q22.3) | p53 (17p13.1) | Trisomy 12 (Cen 12) | 13q-/-13 (13q14, 13q34) | CCND1/IgH t(11;14) via NeoGenomics.com. There's minimum testing, current edge testing, and research testing. I think you got research level testing.
I imagine there's more info on your FiSH - a second set of parentheses that specify the bands on the 2 chromosomes that got translocated. Back before they learned how to sequence genes, they stained the chgromosomes and saw stripes in bands that almost every had pretty much the same. So they numbered the bands. That's the number after the p or q on the FiSH.
In a translocation, entire chunks of one or more chromosomes get swapped due to errors in DNA repair. Our B-cells are mutating their genes billions of times a day, trying to match antigen. If they don't match antigen, or if they match antigen from our own body, they're supposed to die automatically - apoptose. The enzymes that help the muratation process actually break the DNA and help it rejoin. But once in a billion times, it hicups, and the repair goes awry. Another set of enzymes is supposed see it, and signal the cell to die . But if the cell has CLL, it may refuse.
Then a skilled lab tech or pathologist takes your sample, and spins it, and does things to separate the white cells from the red and the plasma, and then the lymphocytes from the white cells, and then they do Flow Cytometry and FiSH and IGHV sequencing on those cells. By that time, it's not a lot of cells - 100 or 200 for Flow Cytometry and FiSH? (help me here Jm954 ) - but not a huge sample. If your CLL is early stage, you might get one set of percentages. At a later stage, another set. Same with IGHV DNA sequencing. I really don't know how many cells they use for that, and how a bi-clonal or polyclonal CLL sample would be handled. Dogma rules, I expect, or maybe the instrument just happens to sequence the one cell it got. I could buy pathologist a lot of beers or cocktails to find out.
ClonoSEQ is particularly cool because it's designed to sample not just one cell, but up to several million different cells, and report the sequence of a piece of each cell. Hence the cost.
Back to your translocation. The IGH genes are on chromsome 14. The IGL Lambda genes are on chromosome 22. The IGL Kappa genes are on chromosome 2. The BCL2 gene that helps the cell apoptose is on chromosome 18. So I'm curious to know what your doctor says is the actual chunk of your t(2;18) translocation. Maybe it just makes the IGL Kappa unusual. The dogma is that our BCRs which are made from IGH and IGL don't really match useful antigens - or if they do, they match the cell itself (which is a thing).
I found out at MDA that I also have a BCL2 mutation - a fairly common one. Venetoclax targets the BCL2 protein made by that gene. But Dr. Thompson assured me that Venetoclax effectiveness was not changed by my mutation. Since your doctor is asking to do ClonoSEQ, I suspect they don't think your therapy would be affected, either.
To sum up, I think you should ask about your FiSH and how proposed therapy works with it.
Seymour, you are correct. In 2019 I had a second opinion at Ohio State under Dr Byrd, and they did genetic testing in addition to FISH. That is where they found the 14q interstitual deletion as well as translocation 2/18.
No 13q, at least at that time at Ohio State.
This adventure seems to be getting more interesting as I progress.
Seymour, the doctors haven't gotten back to me yet to discuss my results but I did see the report and the report indicates that I have four dominant sequences. I assume maybe incorrectly, that this means I have four different clones. The mutation status was listed at 5.8 productive and 0.6 unproductive. This totals 6.4 which is the mutational status of my hgiv mutation test on my two prior test for mutational status.
All of our cells are clones. But that's not what we usually mean when we say "clone" in the context of disease.
Despite the name, ClonoSEQ doesn't call them clones. It just calls the Sequences, and avoids the ambiguity in the word "clone."
I would assume that Dominant Sequences are SUBclones, unless there's some evidence that they are really different and formed from a separate carcinogenic event which we would call a de-novo clone. A de-novo clone would be formed if a normal B-cell acquired a somatic mutation that caused it to be a CLL cell. I think the only way to find a specific de-novo clone is to do a complete sequence of the whole cell and compare it to a database of previous entire sequences from the same patient. ClonoSEQ doesn't do that. Someday, well have such capability at a reasonable expense.
We might be able to infer a possible de-novo clone, but I'd want someone from Adaptive to say so. For example, someone asked me to look at their ClonoSEQ report, and it showed the usual IGH sequences, it also had some IGK (Kappa Light Chain) sequences, and a single IGL (Lambda Light Chain) sequence.
ClonoSEQ only looks at the IGH, IGK, and IGL genes in CLL. Mutations in those are expected as part of B-cell development that tries to match antigens from microbes and such. That process is called somatic hypermutation. At a minimum, we would have only 1 dominant CLL clone on a ClonoSEQ report. But in a person that has IGK CLL clones, the arrival of an IGL clone could be de-novo. I need to look through the growing list of ClonoSEQ papers on that.
A dissertation on good and bad mutations and ClonoSEQ
Skip to the end TL;DR if it's too much
When the original CLL clone mutation happens somewhere in our many genes, both the Heavy Chain and Light Chain genes are unique for that clone and its descendents from then on. This is what ClonoSEQ counts on. There's so many possibilities of normal Somatic Hypermutation in B-cells, that 2 normal cells having the same Heavy Chain and Light Chain genes is really low.
But the cell may still develop a little more, and may purposely mutate the Heavy or Light chain more via Somatic Hypermutation. Normal cells do that multiple times. But CLL cells can do it, too, I believe. I think additional Somatic Hypermutation is less expected of CLL cells, though.
The original clone will also replicate at some point, and when it does, it makes a copy of every chromosome. When that copy happens, errors can also happen that result in mutations in IGH, IGK, and IGL, especially if we have ATM or TP53 mutations or del11q or del17p FiSH. Those would be called somatic mutations. So ClonoSEQ will identify those, too.
When the cell reads DNA to make the messenger RNA that makes the protein, single strand breaks happen, and usually get repaired. But if repair mechanisms are faulty do to mutations in the genes that make the repair enzymes, a new somatic mutation can happen while making copies of the IGH, IGK, and IGL genes. I don't know the statistics on that, though. But ClonoSEQ will identify those, too.
BTW, to add to the alphabet soup, I just recalled that ClonoSEQ looks at more than just IGH, IGK, and IGL. I found an FDA paper with a mispelled title, interestingly enough. That would be a somatic spelling mutation. 😆
BCL1/IgH (J), BCL2/IgH (J), and housekeeping gene (HKG) sequences."
An almost identical sentence appears in many, if not most papers about ClonoSEQ. The BCL1 and BCL2 translocations are seen in Multiple Myeloma, among other possible diseases, and not in CLL. ClonoSEQ is used for several different blood cancers.
So, a New Dominant Sequence, represents a new Dominant Subclone, I think. Based on a recent ClonoSEQ someone sent me, it's entirely possible that the New Dominant was also in the original ID sample before treatment, and didn't meet the mathematical requirements to be identified as a Dominant clone. The saw a New Dominant on their latest report, and all the previous samples listed on the report now also showed it, except their ID sample. Why that sample didn't show it, I can't honestly say. It could have been a new somatic hypermutation.
The B-CELL TRACKING (MRD) REPORT has a section that describes the requirements for declaring a sequence a Dominant Sequence:
---
"CRITERIA FOR DEFINING "DOMINANT" SEQUENCES
1. The sequence must comprise at least 3% of all like sequences (IGH-involved, IGK, and IGL are considered independently).
2. The sequence must comprise at least 0.2% of the total nucleated cells in the sample.
3. The sequence must be discontinuously distributed (≤5 sequences in the next decade of sequences when ranked by frequency).
4. The sequence must be carried by at least 40 estimated genome equivalents in the analyzed sample."
---
Our normal B-cell clones probably die the quickest, and total numbers of unique sequences plunges pretty quickly during treatment. The surviving CLL clones therefore make up a higher percentage of the nucleated cells.
The TL;DR answer
My understanding is that all those additional Sequences on ClonoSEQ represnet Subclones of the original clone created by somatic mutations or somatic hypermutations. I welcome any corrections and nuances. They are clones in their own right, though, too, so we can call them clones. It's theoretically possible for another carcinogenic event to create a brand new de-novo clone with different IGHV, IGK, or IGL. But I don't think ClonoSEQ can identify them as de-novo clones.
Your high ALC indicates that you will be needing treatment. How soon or later is anyone's guess.
Initial ClonoSEQ testing is to obtain a baseline of the genetic fingerprint of your CLL.
This is a prelude to testing for Minimum Residual Disease after treatment. They search over 1,000,000 cells for that genetic fingerprint.
MRD testing is common after short duration treatment. In the US that will be 12x 4 week cycles of Venetoclax + Obinutuzumab. Nearly 80% reach undetectable MRD4 that normal testing can detect and 50% uMRD6 that ClonoSEQ tests for.
Fewer patients with Mutated IgHV achieve uMRD4 but after treatment have a longer duration of time to next treatment. (GLOW trial of Venetoclax and Ibrutinib found that for mutated IgHV, uMRD was not a prognostic for duration of response.)
MRD testing for covalent BTKi maintenance treatment is rare as being continuous there is no expectation that treatment will end before progression. MRD testing would only be used for a patient that had reached a complete response to see if they could take a drug holiday. Few reach CR on cBTKi alone but about 20% do on Acalabrutinib + Obinutuzumab, which is only approved in US. There is a very gradual increase in number of patients at CR and uMRD over time when on cBTKi maintenance treatment. Without a ClonoSEQ fingerprint the test for MRD would be a conventional test of 10,000 cells.
I would expect that when the time comes for treatment you will be recommended for a short duration Ven + Obin treatment or maybe continuous Acala + Obin.
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