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CLL Support Association
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HOW DO WE DETERMINE MUTATION STATUS?

How do you determine your CLL mutation status? I was told that I was unmutated. If I am trisomy 12 positive, is that enough to conclude that I am unmutated? So I keep hearing the term "IGA mutation status" thrown around. Is that what determines whether we are MUTATED predominantly, or is it the fish test variants that are found, that authenticate mutational status? But having high IgG or IgA antibody counts have nothing to do with mutational status,right? These are questions that were asked at my support group meeting and I really couldn't answer in an authoritative way. So what exactly do we look at on our Blood tests that explain mutational status? Thanks.

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CLL/SLL patients with a mutated Immunoglobulin Heavy Chain Variable (IGHV) gene have a very favorable outcome and a low probability of developing progressive disease, whereas those with unmutated IGHV gene were much more likely to develop progressive disease and had a shorter survival.

cancergenetics.com/wp-conte...

Direct IGHV testing clinically is rare, so flow cytometry suroggates are used they are CD38 and the less accurate ZAP70... their overall accuracy is about 70%.

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Thanks Chris very helpful. So its the IGHV ASSAY which is the test that shows mutation status? Is that right to say?

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Yes... IGH@ is a family of genes, and there are a number of stereotypes that confirm more or less aggressiveness.

These aren't tested for clinically at the present time.

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I found this article helpful Jettyguy;

cllsupport.org.uk/making-se...

My haematologist was unable to give me any data relating to my CD38 or ZAP 70 levels which are usually captured during the initial flow cytometry test because he said they didn't have them 5 yrs ago (well at my hospital anyway). I therefore am unaware of my mutational status. Forgive the length of this and I'm afraid the link (from an authoritative source), no longer works but it explains the significance of CD38 and ZAP70 for calculating mutational status (with some variation in accuracy however as Chris says).

CD 38 & ZAP 70

'What is the significance of CD38 in CLL?

The presence of the antigen CD38 on B-CLL cells is a much discussed prognostic indicator in CLL. Whether it is a truly independent prognostic indicator or simply a reflection of IgVH gene mutational status, CD38 clearly seems to have some relevance in predicting whether a patient’s CLL is likely to have a favorable or unfavorable clinical course. CD38 is detected by flow cytometry, a diagnostic technique frequently used in confirming CLL.

Patients with less than 30 percent CD38+ B-CLL cells are likely to have a favorable clinical course requiring minimal or no therapy. Patients with equal to or greater than 30 percent CD38+ B-CLL cells are more likely to have an unfavorable clinical course requiring earlier and ongoing treatment. Significant differences in survival are also thought to exist between these two groups. CD38 expression remains stable over time in the majority of patients, but it is known to change in approximately 25 percent of cases. Its level of expression does not seem to be influenced by chemotherapy.

The connection between CD38 expression and IgVH gene mutational status is not well understood. It appears that patients with less that 30 percent CD38+ B-CLL cells are likely to have mutated IgVH genes while patients with greater than 30 percent+ B-CLL cells are more likely to have unmutated IgVH genes. While this is often the case, there is approximately a 30 percent discordance between assays for CD38 and IgVH mutational status.

Both CD38 and IgVH gene mutation are thought to be useful prognostic indicators in B-CLL, but because of the relative ease of testing for CD38, it is a much more convenient test.

CD38 and IgVH mutational status are just two of a number of prognostic indicators in CLL. Others include, circulating levels of beta-2-microglobulin and soluble CD23, lymphocyte doubling time, serum thymidine kinase levels, bone marrow histology, and chromosome abnormalities.

What is the significance of ZAP-70 in CLL?

ZAP-70 is an abbreviation for Zeta-chain-associated protein kinase 70. This protein is a member of the protein-tyrosine kinase family and when expressed on B-CLL cells is surrogate marker for IgVH gene mutational status. The presence of ZAP-70 can be detected by flow-cytometric analysis, and the level of expression is thought to correlate with mutational status.

CLL patients with less than 20 percent ZAP-70 positive B-CLL cells are likely to have mutated immunoglobulin V genes, predictive of a more favorable clinical course, while patients with greater than 20 percent positive B-CLL cells are likely to have unmutated immunoglobulin V genes, predictive of a less favorable clinical course.'

Hope it helps in some way.

Newdawn

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Thanks Newdawn ! Wow this took you some time. I appreciate the effort. Very interesting. So I was Zap70 positive on one test with a community Onc/Hem and when My CLL specialist tested me a second time he couldn't find Zap 70 positivity.(it was inconclusive) I am however trisomy 12 positive. So I suppose according to Chris's post We look at an IGHV assay report ,which is the test result that shows Mutation status?

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ZAP70 is a fakey test, look at it wrong and it will give the wrong results... its never been fully vetted either. Done in a CLL research lab, it will be accurate but in commercial labs it may or may not be.

CD38 is probably the better of the two... its been used twice as long and samples aren't an issue, However it can change over time, but it won't go from mutated to unmutated or visa versa.

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It's such complex material jettyguy and I admit I've found the issue of establishing mutational status difficult to get my head around too. In relation to the information Chris has given above, it really is worth working through Dr. Pettitt's explanation on the significance of CD38 (on the link I gave in my post).

Regards,

Newdawn

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Chris and Newdawn, I find it really kind of strange that if I hadn't asked my docs to find out about my mutation status,they gladly would have avoided the conversation. They might not feel that patients will put it all together or maybe they believe with all the novel agents around...who cares? But even when I saw a CLL specialist,he fielded my questions and then waited until I asked him if my CLL mutation status was definite. He was very casual about it,as if it meant nothing in the scale of things. I have been 18 months into this CLL fun fest and the specialist said,"whatever you are doing right now your disease is behaving in an INDOLENT manner." Still,everything I have read suggests that mutation status is a big deal.

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I think you're absolutely right with that observation jettyguy and Dr. Sharman recently said this on the subject (taken from a PP interview discussing treatment considerations);

'First of all, I think that the IgVH mutation analysis is poorly understood by most practicing providers. I think there is a significant misunderstanding and even amongst physicians I know first hand who are interested in hematology, there’s often at times, a lack of total awareness of what this means. To me, IGHV mutation analysis defines treatment intent.'

Newdawn

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Mutation status in the age of FCR 'one size fits all' except 17p- had little impact in the clinic... it was an interesting, but as far as treatment was concerned had no impact.

Now in the age of small molucules first line in the U.S. mutation status has more significance, especially for 13q mutated.. who might get a very very long remission on FCR.

Since we, outside, drag behind the U.S. it is still an FCR mindset we face, unfortunately.

~chris

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I agree. My doctor did not explain my FISH or IGVH test results. During a follow up visit, I specifically asked her to explain the significance of my IGVH test result (i.e., I am unmutated). She looked so annoyed that I asked her a question, but then started to have me come in every 3 months as opposed to 6 months. So she probable never even looked at my IGVH test results before.

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With your biomarkers jettyguy, you may find this article from Dr. Sharman useful;

cll-nhl.com/2015/11/fcr-emp...

Newdawn

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I dont know IGHV status.ZAP 70-,CD20+,CD5+,CD43+,CD23+,Bcl2+/ZAP70- . Nodullar infiltration and lymphoprilferativ Neo. Biopsy Lymph nodes:immunohystohemy NHL-nodal and extranodal B-cell NHL marginalis zones lymph.folicul.PCR:scor was for CLL was 2.FISH:Trisomy 12. What You think?Your response is great for me. Greeting! Olga

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Olga I think you are Trisomy 12 mutated... based on Zap70-

Pretty good!

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Thanks Chris!Any good for me.., How Yours sister? My son have lymphocitosis (40y.),without infection.He dont want detail....

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jettyguy1, The link provided by CLLcanada provides an excellent detailed explanation of the technique for determining IGHV mutation status. To summarize, the mutation status is determined by sequencing the DNA of the variable region of the heavy chain of the B-cell receptor (BCR) gene in the clonal CLL cells.

The link provided by Newdawn provides an excellent explanation of why some BCR receptors are mutated and some are unmutated. To focus on that question just a bit more: All B cells start out as unmutated cells. It is a normal process in the development of B cells for the variable region of the heavy chain of the BCR gene to accumulate random base changes in the DNA sequence that ultimately allow for such a wide range of antigens to be recognized by the BCR protein on the surface of circulating of B cells. [A rare case where mutations in DNA are a positive event!] One theory is that a CLL clone that is unmutated arose from an earlier developmental stage than a CLL clone that has undergone somatic hypermutation, and a second theory is that the developmental pathway of the unmutated clone is simply different. However none of this explains why the prognosis differs for unmutated vs mutated CLL, and as you can imagine this is an active area of research.

gardening-girl

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Thanks Gardening-Girl. My unmutated head is swimming! So much to digest.

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