Prognostic Indicators el pt

Prognostic and Monitoring Test Packages

Date: April 3, 2024

by Chaya Venkat

Test Packages Now Available on a Commercial Basis

In a previous report (Progress on Prognostics) we told you we were in negotiations with Quest Diagnostics to develop modern prognostic and monitoring packages for CLL patients. I am happy to report the packages are now available - your doctor can request these through routine channels. Quest Diagnostics is one of the largest and most well-reputed diagnostic testing labs in the country. I am very pleased with the level of help and commitment we got from them during the course of our negotiations. Their size and reputation means most insurance companies, Medicare and local healthcare providers are familiar with the company and you should have few hassles in asking for these tests. There are still a few issues outstanding - for example their website is not the easiest to navigate. But these are operational 'IT' issues that will no doubt be resolved over time. What matters is that the packages are available now, and your doctor can order them for you. Please do note the date of this article — by the time you read this it is entirely possible that other test providers have come out with their own competitive packages and you may have a choice of vendors, a point that could be important if your insurance plan will cover only a subset of test providers on an in-network basis.

Until the Quest website becomes a little easier to navigate, it may be easier for you to print out the details of each of the packages below from our website. I have a detailed description of each of the packages below, as well as the instructions provided by Quest. You can have your doctor or local blood draw station draw the blood, then send it to Quest Diagnostics by overnight package. Be sure the blood draw lab follows the directions provided exactly. Just click on the link at the end of each section to get a printable page with the information for the test.

There are three prognostic packages (basic, mid-level and deluxe versions) and one package for monitoring your CLL over regular (annual?) intervals. From the very start, we have been preaching on CLL Topics the value of therapy decisions based on modern prognostic testing (What Type of CLL Do You Have?; FISH-ing for Answers; What You and Your Oncologist Need to Know about CLL; Prognosis at Diagnosis; ATM and p53, Keepers of the Genome; Dawn of a New Era). Fortunately, many of the experts from the CLL Research Consortium are also on board now, stressing the need for such testing. If your local oncologist is a little old fashioned and does not quite believe in all this new-fangled stuff, let us know and we can help you find very recent full text articles by some of the best CLL experts to support your request.

Basic Prognostic Package

This consists of three components: IgVH gene mutational analysis, CLL FISH panel, and the ZAP-70 test. We have discussed each of these prognostic indicators in depth before, but I thought I would give you a quick refresher course on them for convenience.

There is little doubt anymore that IgVH gene mutational status is one of the most important of the modern prognostic indicators. People with mutated IgVH genes typically have an indolent disease that simmers along slowly. When and if the tumor load gets high enough to need therapy, these people typically respond well, with deep and long lasting remissions. On the other hand, people who have CLL characterized by unmutated IgVH gene often have a harder time of it, needing therapy sooner and often not responding as well when they do get therapy. The original definition of "mutated IgVH" was a gene that was more than 2% mutated from the germ line. In recent papers it has been suggested that a more robust mutation status, say 5 to 8% mutation from the germ line, was even better than a mere 2%. The Quest report gives not only a "Yes / No" answer to the question of IgVH gene mutation status, it also gives the actual percentage of the gene mutation. The report may read, for example, that the IgVH gene was 4% mutated. This is good, detailed and robust information.

The CLL FISH panel used by Quest looks for four of the common chromosomal abnormalities found in CLL.

1. Deletions in the "p" arm of the 17th chromosome, called 17p. This part of the 17th chromosome contains the very important "p53" gene, which controls the ability of the cell to commit suicide on command. Deletion of this gene means that the CLL cells are protected from natural death, and that spells real trouble. There is now increasing information that patients with 17p deletions (i.e., missing p53 gene) do not respond well to fludarabine therapy. Fortunately, they do respond to other drugs, one of the most important being Campath. Right here is a good reason for having prognostic information available before making therapy choices. It may not be such a smart thing to absorb all the toxicity of fludarabine single agent therapy as your first choice, if you have 17p deletion and not likely to respond very well to this particular choice!
2. Deletions in the "q" arm of the 11th chromosome, called 11q. Deletion of this part of the 11th chromosome means deletion of the "ATM" gene. This is another important gene that controls the decision whether a damaged cell should live or die. When the ATM gene is AWOL, once again the normal process of programmed suicide of defective cells is messed up. Patients with deletions of 11q often have enlarged lymph nodes, and poorer response to single agent therapy with monoclonal antibodies such as Rituxan or Campath since these drugs do not work very well on large lymph nodes. If you are determined to stick with monoclonal therapy only, and not go for the more aggressive chemoimmunotherapy combination regimens such as FRC and RF, it might be worth initiating therapy before the lymph nodes get too big. It is thought that prognosis of patients with 11q deletion is not quite as severe as those with 17p deletion described above. Both 17p and 11q deletions suggest “Bucket C”.
3. Trisomy 12. This is not a deletion, as in the 17p and 11q cases discussed above. This is a situation where there are three copies of the chromosome 12, instead of the normal two copies you are supposed to have. Scientists are not exactly sure why having an extra copy of the chromosome 12 spells trouble. Perhaps there is a tumor promoter gene present on this chromosome, and having three copies of it is worse than having just the normal set of two. Patients with trisomy 12 have middling prognosis, “Bucket B”.
4. Deletion of the “q” arm of the 13th chromosome, called 13q. This is the most benign of the chromosomal abnormalities CLL patients can have, it is often associated with very good prognosis. A very comfortable “Bucket A” is indicated for these patients.
   
   There are other abnormalities that can occur in CLL, but they are not quite as frequent as the four mentioned above. It is a pity that the Quest CLL package does not include a probe for the 6q deletion, one that is not quite so rare. But I am hopeful this deficiency will be corrected in the near future. Remember, if your FISH report says you have no deletions in 17p, 11q, 12 trisomy or 13q, it does not mean you have no deletions in any of your chromosomes. It just means these four common deletions were the ones looked at and not found, there may well be some less frequent deletion or abnormality in your particular case that was not looked for. Remember, too, that “clonal evolution” is well documented in CLL.  Just because you had the comfortable 13q deletion last year does not guarantee that it is still the only FISH deletion you have now, especially if you have had therapy with fludarabine and the like between then and now. You can read more about it in the section below on “The Monitoring Package”.

ZAP-70 testing has received a huge amount of publicity in the last year or so. It seems to be a measure of the level of activity of the B-cell receptor (BCR), which in turn may define the tendency of the cell to proliferate out of control. There was initial hope that testing for ZAP-70 may be easier than testing for IgVH gene mutation status, and that ZAP-70 and IgVH mutation results will track each other lock-step. There is now disappointment on both fronts. Getting reproducible and accurate results from ZAP-70 testing has been difficult and researchers are still struggling to find consensus on robust testing methodology. In addition, there has also been controversy generated due to the less than perfect match between ZAP-70 results and IgVH gene mutation status. For my money, until and unless the ZAP-70 issues are all resolved, IgVH gene mutation status is still the single biggest and best prognostic indicator, and its combination with FISH results should give patients and their doctors a pretty good idea of the lay of the land.

Bottom line, this three component “basic” prognostic package is pretty good bang for the buck, gets you most if not all of the information you need to make smart therapy choices. I have yet to get final word on costs, but I believe this basic package will come out to be under $1,000. Not bad, compared to the stories one hears of CLL expert centers that require cashier’s checks ranging from $12,000 to $15,000 before they will even look at patients with inadequate insurance coverage. For now, you have to get a doctor to order this package for you. Down the road we are hoping it will also be possible for patients to order the package on their own, and pay for it on-line with a credit card.

CLL Prognostic Panel, Limited
Includes: IgVH Mutation Analysis; ZAP-70; FISH, B-Cell Chronic Lymphocytic Leukemia (B-CLL) Panel

Clinical Significance:	Predicting prognosis and clinical behavior in patients with chronic lymphocytic leukemia (CLL) can be very difficult. Predicting clinical behavior is important not only for prognosis, but also to stratify patients for therapy. In the past few years, several new tests have been developed to aid in predicting prognosis in CLL. This panel of tests contains most of the essential tests that are not offered in the routine laboratory.
Effective Date:	Now available
Test Code:	17240X [9524]
CPT Code:	83891; 83894; 83902; 83901; 83904; 83912; 88184; 88185; 88187; 88271 (x4); 88275 (x4); 88291
Specimen Requirements:	8 mL sodium heparin (green-top) whole blood and 3 mL EDTA (lavender-top) whole blood
Transport Temperature:	Room temperature
Specimen Stability:	Room temperature: 3 days; Refrigerated & Frozen: Unacceptable
Set-Up/Analytic Time:	Setup Mon-Sun AM; Reports 7 days following set-up
Reference Ranges:	Not applicable
Methodology:	See individual assays
FDA Status:	ASR Class 1
Shipping Address:	Quest Diagnostics Nichols Institute, 33608 Ortega Highway, San Juan Capistrano, CA 92690-6130

Mid-level Prognostic Package

In addition to the IgVH mutation status, FISH and ZAP-70 tests of the basic package, this mid-level package also includes tests for CD38 and Beta-2-microglobulin (B2M) levels.

CD38 is a marker that is associated with CLL cells, and the original cut-off proposed by Damle, et. al., in their seminal paper was 20%. Patients with less than 20% of their CLL cells expressing CD38 were considered to be in a good prognosis category, those with higher than 20% of their CLL cells expressing CD38 had poorer prognosis. There was also initial excitement that CD38 results paralleled IgVH gene mutation results. A closer look at the situation made it clear this was not always the case, there are distinct subset of patients who had IgVH gene mutation status and CD38 level pointing in opposite directions. Even more important, for some patients who were monitored for their CDC38 levels over the course of several years, the CD38 levels changed over time. As their disease progressed and got worse, the CD38 levels increased as well. This makes CD38 a less than satisfactory prognostic indicator since it is hard to predict the future based on an indicator that changes over time.

Beta-2-microglobulin (also called B2M for short) was one of the first prognostic indicators to arrive on the scene. It is a small piece of protein that is shed by CLL cells into the blood, and it was hoped that measuring its level gave a good indication of prognosis. The typical cut off was 2.0 for this marker: people with B2M levels of less than 2.0 were supposed to be in the good prognosis group and those with higher than this level had worse prognosis. Unfortunately, a number of different things can make the B2M level go up or down, not all of them having to do with prognosis. For example, B2M can rocket up when CLL cells are getting killed in droves due to therapy, just because all those dead CLL cells leave behind debris among which may be the small pieces of B2M protein. B2M also increases as CLL cells proliferate, and therefore it reflects, to some degree, the rate at which the tumor load is increasing at any given time. Once again, it is hard to consider an indicator a solid rock foundation for future prognosis when it changes over time and for a variety of reasons.

I do not believe CD38 or B2M testing add greatly to the cost of this mid-level package over and above the cost of the basic package we discussed earlier. At the same time, I do not think these two tests add a great deal to the fine-tuning of the prognosis either. The relatively small additional cost is likely to be paralleled by relatively modest increase in the accuracy of the prognosis.

CLL Prognostic Panel, Comprehensive w/o Karyotype
Includes: IgVH Mutation Analysis; ZAP-70; CD38;FISH, B-Cell Chronic Lymphocytic Leukemia (B-CLL) Panel; Beta-2-Microglobulin

Clinical Significance:	Predicting prognosis and clinical behavior in patients with chronic lymphocytic leukemia (CLL) can be very difficult. Predicting clinical behavior is important not only for prognosis, but also to stratify patients for therapy. In the past few years, several new tests have been developed to aid in predicting prognosis in CLL. This panel of tests is comprehensive and includes all the tests that are considered necessary for prognosis except for karyotyping. The included FISH studies cover most of the important cytogenetic abnormalities. Other abnormalities such as translocations or deletions can be detected by karyotyping, but in general, the potential prognostic value of karyotyping is considered questionable by some investigators.
Effective Date:	Now available
Test Code:	17312X [9532]
CPT Code:	83891; 83894; 83902; 83904; 83912; 88184; 88185 (x2); 88187; 88271 (x4); 88275 (x4); 88291; 82232
Specimen Requirements:	1 mL no additive (red-top) serum, 13 mL sodium heparin (green-top) whole blood and 3 mL EDTA (lavender-top) whole blood Must specify CD38 Marker
Transport Temperature:	Room temperature
Specimen Stability:	Room temperature: 3 days; Refrigerated & Frozen: Unacceptable
Set-Up/Analytic Time:	Setup Monday-Sunday AM; reports in 10 days
Reference Ranges:	Not applicable
Methodology:	See individual assays
FDA Status:	ASR Class 1
Shipping Address:	Quest Diagnostics Nichols Institute, 33608 Ortega Highway, San Juan Capistrano, CA 92690-6130

The Deluxe Version of Prognostic Package

The deluxe package has all the components of the mid-level package and has, in addition, karyotype analysis (also called chromosomal analysis).

Karyotype analysis is a technique where chromosomes are actually examined under a microscope. CLL cells are collected from your blood, induced to divide and then arrested at metaphase (a stage of cell division in which the chromosome are condensed and therefore visible). The chromosomes are stained with certain dyes that show a pattern of light and dark bands (called the banding pattern). The banding pattern for each chromosome is specific and therefore allows identification of each of the 24 chromosomes.

Karyotype analysis can detect large chromosomal abnormalities such as the loss or gain of an entire chromosome or portions of a chromosome and translocations (when a portion of a chromosome breaks off and rejoins with another chromosome, a common occurrence in some leukemias). However, only very obvious mish-mashing of chromosomes can be seen by the naked eye of the technician doing the karyotype analysis. Many of the changes in our genetic material that cause disease are very small, a small piece deleted here or added there. FISH testing described above is a lot more sensitive in looking for very specific deletions of interest in specific situations. That is also the disadvantage of FISH. The technique can only report on the specific deletions that are targeted by the FISH probes used and it is blind to anything else than may be wrong. In other words, FISH is sensitive for the specific items targeted and only those items; karyotype or chromosomal analysis is good for the bigger picture of looking at all the chromosomes, but this big picture lacks the fine detail one can get with FISH.

Karyotype analysis is not cheap: it can add quite a bit to the overall price of this deluxe package. If money is no object, and your insurance company and doctor will go along with it, this may the package for you. Bear in mind, it would also need a trained pathologist to interpret the results of the karyotype analysis, if you happen to have complex karyotype. Unlike the other tests described above all of which have some sort of cut off points and rough rule-of-thumb guidelines for interpretation, the results of karyotype analysis may be more complex and difficult to interpret by laypersons.

CLL Prognostic Panel, Comprehensive
Includes: IgVH Mutation Analysis; ZAP-70; CD38; FISH, B-Cell Chronic Lymphocytic Leukemia (B-CLL) Panel; Beta-2-Microglobulin; Chromosome Analysis, CLL/LPD

Clinical Significance:	Predicting prognosis and clinical behavior in patients with chronic lymphocytic leukemia (CLL) can be very difficult. Predicting clinical behavior is important not only for prognosis, but also to stratify patients for therapy. In the past few years, several new tests have been developed to aid in predicting prognosis. This comprehensive panel is considered the most complete set of tests that is currently used for predicting prognosis in patients with CLL.
Effective Date:	Now available
Test Code:	17239X [9523]
CPT Code:	83891; 83894; 83902; 83901; 83904; 83912; 88184; 88185; 88187; 88185; 88271 (x4); 88275 (x4); 88291; 82232; 88237; 88264; 88280; 88291
Specimen Requirements:	33 mL sodium heparin (green-top) whole blood, 3 mL EDTA (lavender-top) whole blood, and 1 mL no additive (red-top) serum Must specify CD38 Marker.
Transport Temperature:	Room temperature
Specimen Stability:	Room temperature: 2 days; Refrigerated & Frozen: Unacceptable
Set-Up/Analytic Time:	Setup Mon-Sat AM; reports 10 days following setup
Reference Ranges:	See individual assays
Methodology:	See individual assays
FDA Status:	ASR Class 1
Shipping Address:	Quest Diagnostics Nichols Institute, 33608 Ortega Highway, San Juan Capistrano, CA 92690-6130

The Monitoring Package

This last package is an interesting one. As we have discussed above, CD38 and B2M levels have been shown to change over time, and to some degree they may reflect the level of the tumor load. There are also indications that ZAP-70 reflects the level of activation of the B-cell receptor (BCR), and therefore the rate at which the CLL cells are inclined to proliferate. Bear in mind, these connections between CD38, B2M, ZAP-70 and disease progression are still controversial and we do not yet have full consensus on these points.

However, there is pretty good consensus on one important aspect of CLL. “Clonal evolution” is a well documented aspect of CLL. In simple terms, the genome of the CLL cell is unstable and likely to become even more garbled as time goes along. A person with a nice comfortable 13q deletion as the sole single chromosomal abnormality at diagnosis may find that over time his particular version of CLL has evolved, and perhaps now some of the cells sport a 11q deletion as well. (See the Harvey Saga). Sometimes these additional mutations appear on the scene pretty much by random chance. Often, however, therapy with many of the commonly used chemotherapy drugs (purine analogs, alkylating agents) can accelerate the process, as well as increase the chances of getting clonal evolution going in the first place. For some lucky CLL patients, clonal evolution does not take place at all and they have a very stable disease that does not change its type over time.

Bottom line, even if you had prognostic testing done when you were originally diagnosed, it may be prudent to check for clonal evolution periodically, perhaps before you initiate the next round of therapy. This can give you some degree of assurance that you are still dealing with the same beast as you thought you had. IgVH gene mutation status is not thought to change over time, you are stuck with the kind you began with, so this component is not included in the monitoring package below. ZAP-70, CD38, FISH, and B2M are all indicators that may change over time and therefore they are included in this monitoring package. My guess is that this package would be even less expensive than the basic prognostic package, since it does not contain the expensive IgVH gene mutation status test.

CLL Prognostic Panel, Monitoring
Includes: ZAP-70; CD38; FISH, B-Cell Chronic Lymphocytic Leukemia (B-CLL) Panel; Beta-2-Microglobulin

Clinical Significance:	New additional molecular abnormalities may appear in patients with chronic lymphocytic leukemia (CLL) at a later stage of their disease.These new abnormalities may dictate a change in therapy and management. This panel contains tests that may change in the course of the disease and can predict progression. This panel should be used to monitor patients with CLL on therapy.
Effective Date:	Now available
Test Code:	17290X [9531]
CPT Code:	88184; 88185 (x5); 88187; 88271 (x4); 88275 (x4); 88291; 82232
Specimen Requirements:	1 mL no additive (red-top) serum, 8 mL sodium heparin (green-top) whole blood and 3 mL EDTA (lavender-top) whole blood Must specify CD38 Marker.
Transport Temperature:	Room temperature
Specimen Stability:	Room temperature: 3 days; Refrigerated & Frozen: Unacceptable
Set-Up/Analytic Time:	Setup Monday-Sunday AM; reports in 10 days
Reference Ranges:	Not applicable
Methodology:	See individual assays
FDA Status:	ASR Class 1
Shipping Address:	Quest Diagnostics Nichols Institute, 33608 Ortega Highway, San Juan Capistrano, CA 92690-6130

Some Common-sense Pointers to Consider

1. It makes sense to do any of these tests only when you have a reasonable level of CLL cells in your blood. Think about it: if you have just been diagnosed with early stage CLL and have a barely detectable level of disease, there is not much point in rushing your blood off to the lab to do prognostic testing. They will have a hard time finding a sufficient number of CLL cells in the sample and the results of the tests will be that much less dependable. This point is also valid for patients who tend to have most of their CLL cells tucked away in lymph nodes or the spleen with hardly any detectable number of them in the peripheral blood. For these folks it might be necessary to test the bone marrow aspirate or even lymph node tissue, rather than a blood sample. There is one other time when you may have very few CLL cells in your blood, and that is right after you have finished therapy to which you have responded like gangbusters. Moral of the story - prognostic tests work better if there are enough CLL cells in your blood for the lab to find.
2. Most people go home and do not work over the weekend. I am sure this is true of lab technicians as well. So what do you think happens to blood samples you get drawn on a Friday afternoon? Even if you luck out and the FedEx or UPS guy picks up the package right away, it is likely to end up in some receiving dock over the weekend. Come Monday morning, the lab tech will get to it as it moves forward in his sample backlog accumulated over the weekend. Heck, they tell you not to buy cars that came off the assembly line on a Friday, when the workers are itching to head home. My suggestion is have your blood sample taken in the morning, so that it is sure to get picked up the same day; make sure the sample collection instructions are followed exactly and the sample is sent by overnight express to Quest Diagnostics; and last but not least, try to get the blood draw done early in the week so that your samples avoid the weekend wait. Fresh samples are always preferable to samples that have been sitting around for several days.
3. I understand that the Quest Diagnostics test report will be accompanied by a very useful cover letter by their expert pathologist that gives a bullet point summary of the results and what they imply. Just that cover letter may be worth the expense of the package, I am willing to bet many busy oncologists would go no further than read the cover letter, file the rest of the paperwork. If you are like me, make sure you get your doctor’s office sends you a copy of the full report. The jargon is a little intimidating at first, but sit down with a nice bright highlighter pen, underline the relevant parts, and you will soon be able to make sense of the results. Our article “What Type of CLL Do You Have?” spells out the details of each of the prognostic indicators, so you can develop your own “Bucket” classification. If you are totally lost and confused, write to us and we will try our best to help you untangle the stuff.
4. Last but not least, do not take “No” for an answer, where prognostic testing is concerned. There are many experts that will support your request, and by all means use this ammunition to convince your local oncologist. Even if the high end packages are too rich for your insurance company, there is no excuse for not covering the basic package costs. As for getting periodic monitoring done, that is likely to be a bit more of a struggle, since some may consider that we do not have enough evidence yet to justify it. Remember, you get what you negotiate in life and cancer care. There are as many negotiating techniques and styles as there are people and if your particular doctor does not respond to your style of negotiation, it may be worth considering a switch to one that does.