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CML Testing Explained: Introduction and Basics of CML Leukemia Testing

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This  is designed as a general overview to provide a basic layman's  understanding of testing and CML. I will avoid the jargon and keep this  somewhat short, so this will not cover everything in detail. Always  discuss your testing requirements with your doctor/Oncologist.

There  are tests to diagnose CML, evaluate response to drug therapy, assess  the levels of the disease, and to check for specific problems. Among  these are Complete Blood Count (CBC), Bone Marrow Biopsy (BMB), Bone  Marrow Aspiration (BMA), Cytogenetics Testing, Fluorescence In Situ  Hybridization (FISH) testing, Polymerase Chain Reaction (PCR) testing,  Comprehensive Metabolic Panel (CMP) testing, Kinase Domain Mutation  testing, Gleevec Blood Level testing, and miscellaneous other tests.

 

When  a person is suspected of having CML, testing is done to confirm the  diagnosis. A Complete Blood Count (CBC) test will usually show a very  high white blood cell (WBC) count, and may also show high platelets  (PLT) and other abnormalities. But this does not confirm that a person has CML. The confirmation of CML is usually done by Cytogenetics Testing  (cell testing) on white blood cells taken during a Bone Marrow Biopsy (BMB) process. During a BMB, a core sample is taken from the hip bone  using a hollow needle, and marrow cells are collected that cling to that  bone sample. During the same procedure, fluid from the hip marrow is  also taken out by a syringe, and this second part is called a Bone  Marrow Aspiration (BMA). So the BMA aspirate or fluid is extracted  through the hole created during the BMB. Cytogenetics Testing is done on  the core sample and aspirate fluid, whereby approximately 20 marrow  cells are thoroughly examined in the lab for abnormalities, including  the leukemic Philadelphia Chromosome (Ph+ chromosome), which is the  indicator of CML, and a diagnosis can be made. The sample is also  checked for other abnormalities, including secondary chromosome  mutations, high blast count (immature WBCs), and other abnormalities  such as marrow fibrosis, abnormal cell morphology (shapes and sizes),  etc. So a BMB at diagnosis is critical to ensure a proper diagnosis. The  aspirate fluid may also be tested by FISH and/or PCR testing to  determine the relative amount of leukemic cells in the body (see later  explanations). A follow-up BMB might be done again at six months post-diagnosis, and then every 12-18 months after that, or sooner if other tests show a suspected problem such as loss of response to drug  therapy. BMBs are likely to be decreased in frequency or stopped altogether after the person has reached Complete  Cytogenetic Response (CCR or CCyR) and certainly after reaching a 3 log response by PCR. When therapy reduces the levels of CML  disease to where the Cytogenetics Testing (BMB or FISH) can no longer detect any Ph+ chromosome cells, that person has achieved a CCyR.  CCyR is roughly equivalent to a 2 log reduction by PCR.

After  diagnosis, it is important to continually monitor response to therapy  with regular tests. The most basic of these tests is the Complete Blood  Count test, which assesses overall blood health. When a CBC test shows  that blood counts have returned to normal levels, and especially the WBC  and platelet counts, the person has achieved a Complete Hematological  Response (CHR). After that, the CBCs should still be continued, but the frequency is often reduced. CML patients can often have certain blood  counts become too low, especially white and red blood cell levels and  platelet levels, so continued CBC monitoring is important. Also, a  rapidly rising WBC count could indicate the need for more testing and  possibly a change in drug therapy, since it might indicate a loss of  response.

The BMA marrow fluid taken during the BMB process can also be used to perform a FISH or PCR  test. (FISH is fluorescence in situ hybridization and PCR is polymerase  chain reaction). Or circulating (peripheral) blood can also be used to  perform a FISH or PCR. Both FISH and PCR show the levels of CML disease,  and are used to monitor progress, or detect setbacks or loss of  response to therapy. A FISH test checks approximately 200 – 500 WBC  cells, and counts the number of cells that have the Ph+ chromosome  (technically it looks for the BCR-ABL gene in the WBC cells, which  resides on the Ph+ chromosome). FISH is done by a machine which uses a  dye process, isolates approx 200 - 500 cells, and counts the leukemic  WBC cells. The result is given as a percentage of leukemic cells to good  cells, so the person can say that X% of their WBC cells are leukemic.  The limitation of FISH is that it can only count a small sample of  cells, so if the level of disease is only a few percent, the FISH report  will likely be zero (a zero FISH is also CCR response, same as a zero  BMB Test). So FISH is generally not used once the level of leukemia  drops below approximately 2 - 5%. At that point only PCR testing is used to  monitor CML patients in this Minimal Residual Disease (MRD) status,  since PCR is far more sensitive than FISH. A trend among Oncologists is  to start doing PCRs early instead of FISH, including at diagnosis, since  PCRs are often considered to be more sensitive and can be used to track  log reductions in disease levels, and FISH cannot track log reductions (discussed later).  But it is usually wise to have FISH done until CCR is achieved, since PCR is actually less accurate than FISH at higher  levels of disease burden.

There  are two types of PCR tests. One is called a Qualitative PCR, which is a  simple “yes/no” test that says it either detected BCR-ABL (leukemic cells) or did not detect them, but no number is provided – this is  generally only useful to help diagnose CML since it helps distinguish  between CML and other types of leukemia. The other type of PCR, the  Quantitative PCR, counts the number of BCR-ABL (Ph+ chromosome cells)  and reports it as a percentage number, so this is the type of PCR that  is useful to track treatment progress, especially in Minimal Residual  Disease (MRD) status where the levels of Ph+ chromosome cells are low  and harder to detect. Some Oncologists will do a baseline Quantitative  PCR at or near diagnosis to establish a baseline from which to evaluate  progress, especially toward a 3 log reduction in disease levels.

PCR  tests a sample of blood or marrow fluid, and can detect approximately 1  leukemic cell out of 100,000 or possibly 1 million cells in the sample,  so the test is very useful for long term monitoring of disease levels  and showing treatment progress. PCR testing can be done using either  blood or BMA fluid. During a PCR test, the BCR-ABL in leukemic cells is  counted and the result of the test is given as a percentage ratio of  BCR-ABL (leukemic cells) to another gene in the cells (called a control  gene). So PCR results are not a ratio of leukemic cells to good cells as  we might think, which technically means that a PCR result is not  actually a total percentage of leukemic cells in the body. This is one reason why PCR results from one person to another, and one lab to  another, are not equivalent, due to lack of standardisation among labs  regarding equipment and which control genes are used (there are several  different control genes used for CML PCRs). That is a reason for  sticking with the same lab, so the results will be directly comparable  for each PCR done, and trends can be watched. It is important when  switching labs that the first PCR from the new lab be used to set a new  baseline, since it may not directly compare to the previous PCRs from  the other lab. A number of labs in Europe, Australia, and other  countries use an International Scale for PCR reporting in an attempt to  standardise the test results among labs. In early 2009 some U.S labs  started this conversion, so watch for changes in the PCR numbers  (possibly a significant jump in the PCR number) due simply to a change  in reporting methods. Also note that International Scale PCR results are as much as 10 times higher than equivalent PCR reports using standard scale.

PCR  results are very useful for showing trends, whether progress or  retrogression. The hope for PCR results is to see progress toward a 3  logarithmic (3 log) reduction from the level of disease that existed at  the time of diagnosis. This 3 log reduction is called a Major Molecular  Response (MMR). A recent advance in PCR testing is that many (but not  all) labs now give the log reduction along with the percentage number.  So if your lab provides the log number, then use that to track log  reduction progress. But if the lab does not provide this information, it  makes the 3 log reduction goal more difficult to track, since many do  not know where they started at diagnosis. The International Scale  conversion is an attempt to standardise PCR results, but it is not used  by all US labs. Because Gleevec, Tasigna and Sprycel can rapidly reduce  the levels of leukemic cells, if the first PCR is not done before  starting drug therapy, the individual baseline for calculating a 3 log  reduction will not be available. Otherwise, the lab may provide your log  reduction number based on average results for that lab. The  International Scale uses .1% as a 3 log reduction and uses conversion  factors for each lab. Or a very rough estimate for U.S. labs will  sometimes use .01% as the 3 log reduction goal. If someone has a  baseline PCR value done at diagnosis, progress toward the 3 log goal can  be calculated by taking the baseline PCR number and moving the decimal  point 3 places to the left. For example, if the PCR at diagnosis was  10.0%, then moving the decimal point one place to the left is 1.0% (1  log), two decimal places is .1% (2 log), and three decimal places is  .01%, which is a 3 log reduction. So 3 log/MMR for that person at that  lab would be .01%. Here is the caution that Mayo Clinic puts on the interpretation of PCRs:

"The  precision of this assay at low bcr/abl levels is relatively poor, such that inter-run variation can be as high as 0.5 log. Only level changes  >0.5 log should be considered clinically significant. For example, if a result is given as 0.1% bcr/abl:abl, then any result between 0.05%  and 0.5% should be considered essentially equivalent. If the results are  being used to make major therapeutic decisions, significant changes during monitoring should be verified with a subsequent specimen."

If  a 3 log reduction is achieved, the next goal becomes maintaining the 3 log reduction or even continued reduction toward a negative/undetectable PCR (PCRU). PCRU is the point where the PCR is not sensitive enough to  detect any leukemic cells in the sample. This PCRU is called Complete Molecular Response (CMR), which is the deepest level of response currently measurable. In PCRU status, the leukemic cells are most likely  still there, although fewer than 1 in a million. But research indicates  there would likely still remain over 1 million leukemic cells in the  body at the point of initial PCRU. This initial PCRU is roughly  equivalent to a 5 – 6 log reduction in leukemic cells, depending on the  lab. The patient can continue to drive down the number of leukemic cells  after the initial PCRU is attained, but no current monitoring  techniques can assess the progress. There is no test to determine if a person with CML is actually cured (usually associated with a stem  cell/marrow transplant). The current indicator is 5 years without  therapy coupled with continuous PCRU. Normally, the goal of CML drug  therapy is to drive the number of leukemic cells to the lowest level  possible, with the combined effect of stopping the advance of the  disease, putting the CML patient into permanent, low level chronic phase  CML.

FISH numbers do not  correlate to log reductions, so only PCR can be used for log reduction  measurements. Also, FISH percentages do not relate to PCR percentage  numbers. For instance, at diagnosis I had both a FISH and PCR done. The  FISH was 100% and the PCR was 8%.

It  is not true that a low FISH means a low PCR. A FISH is like measuring  the weight of something with your hand, and a PCR is like measuring with  a surgical scale. Also, the FISH has an error rate of approx 1 - 5%, so  your FISH could read 5% but actually be zero. When the FISH result gets  below approx 5%, you should rely on PCRs from then on. A recent trend  is to perform PCRs from the start, but as discussed above, PCRs are actually not very accurate at high levels of leukemia.

If  any of the tests, such as CBC, Cytogenetics, FISH or PCR, show the  patient may be losing response to drug therapy, additional tests may be  ordered. A Kinase Domain Mutation Test is one test that may show whether  a certain drug, especially Gleevec, can no longer work. The results  will show if a mutation in the BCR-ABL has occurred that prevents the  drug from working, and an alternate drug can usually be used. Sprycel  and Tasigna work against most mutations, but both do not work equally  well against certain mutations, so this test can also help with  alternative drug selection. (Just an added note on the word mutation, a  kinase domain mutation is not the same as a secondary chromosome  mutation such as Trisomy 8, Monosomy 7, etc). Below is one lab’s  description of this test:
http://www.aruplab.com/Testing-Information/resources/TechnicalBulletins/BCR-ABL1%20Kinase%20Domain%20Mutation%20Jan%202007.pdf

Another  CML related test is the Gleevec Blood Level Test. This test can show  how much Gleevec is being absorbed into the bloodstream, since we all  absorb and process drugs at different rates. So this test can show  whether a person needs to take a higher dosage of Gleevec to ensure  adequate levels of drug in the bloodstream. This test is currently  available free through Novartis (maker of Gleevec) under a program they  call CML Alliance:

https://www.bloodleveltesting.com/index.aspx
http://www.cmlalliance.com/health-care-professional/cml-blood-level-tests.jsp?site=google&irmasrc=none&source=01030&campaign=CML-900127

There  are other tests that are used for monitoring CML patients. A  Comprehensive Metabolic Panel (CMP) test should be performed regularly  (probably at the same time PCR is done). This checks a range of issues  such as liver function, kidney function, metabolite levels, etc.
http://www.labtestsonline.org/understanding/analytes/cmp/glance.html

Sometimes  a Flow Cytometry test is used at diagnosis or when a problem is  suspected.  This test counts numbers of cells by various types, and  provides a picture of the cellular makeup of the blood or marrow.

http://en.wikipedia.org/wiki/Flow_cytometry

 

Examples  of some other relevant tests: CAT Scans or physical checks for enlarged  spleen (left side pain), physical checks for enlarged lymph nodes,  complete or partial physical exams. There are also other tests to check  for other specific problems when suspected, such as thyroid function,  iron levels, heart issues, colonoscopy, bone density, skin problems,  etc.

A sample CML testing  schedule might look like the following (assuming no complications) --  your Onc should determine your specific schedule:
Diagnosis: BMB/BMA, FISH and/or PCR; CMP; abdominal (spleen) CAT scan; physical
First several months: CBC weekly
3 months: FISH and/or PCR; CMP
CBC now every 2 weeks
6 months: BMB/BMA; FISH and/or PCR; CMP
CBC now every 2 – 4 weeks
9 months: PCR; CMP
12 months: BMB/BMA, PCR; CMP
After 1 year: PCR and CMP every 3 months, CBC every 4 – 6 weeks; BMB every 18 months

After  3 log response or PCRU: Possibly longer intervals – consult your Onc,  but PCR monitoring is still required at “regular” intervals, and CMP to  watch liver, kidney and other ongoing items.

Your Onc should be following the National Comprehensive Cancer Network Guidelines for CML monitoring and treatment:
http://www.nccn.org/professionals/physician_gls/PDF/cml.pdf
http://www.cmlsupport.com/practicalmanagementjourclinonc0303.pdf

 

Other thoughts:

1)  Get copies of every lab report – you will need them for reference.  Also, your Onc will not normally take time to cover every issue with you  during your office visit. Read all of your lab reports thoroughly. You  must be your own health care advocate.

2) Only have  specimen's drawn for PCR on Monday - Thursday.  Avoid Friday, or it may  not be processed until Monday.  PCRs must be done as soon as possible on  the specimen to have the most accurate PCR report.  By 48 hours the  BCR-ABL has degraded by 50%, so your PCR would be very inaccurate  (reports PCR result as lower than it actually is).

 

3)  The color of the stopper on the specimen tube the blood or marrow fluid  is drawn into: Green top is for BMB and FISH. Lavender top is PCR. See  the link below, page two (all labs use the same color coding) Make sure  your lab tech uses the correct color tube:
http://www.genzymegenetics.com/pdf/CML_physician_brochure.pdf

4) Here are some additional links:

http://www.cmlsupport.com/practicalmanagementjourclinonc0303.pdf
http://hep.uchicago.edu/~seturner/druk.pdf

BMB article:
http://www.forpath.org/0011/introduction.htm

CBC overview:
http://www.labtestsonline.org/understanding/analytes/cbc/test.html

Comprehensive Metabolic Panel (CMP) test info:.
http://www.webmd.com/a-to-z-guides/comprehensive-metabolic-panel-topic-overview

 

If testing reveals potential loss of response to drug therapy, you may want to discuss the following with your Onc:

If a PCR test suddenly shows a sharp increase (greater than 1 log – one decimal place):
1)  Reaccomplish the PCR right away to assure it is accurate (sometimes  things go wrong, such as degradation of the sample during shipping,  contamination, lab errors, etc).
2) If the PCR result is confirmed,  or at the same time as the PCR if you wish, have both a Bone Marrow  Biopsy and a Kinase Domain Mutation test done. The latter tests for drug  resistance, which is a primary cause of lack of response -- see link  below for one lab's explanation of the test:

http://www.genpathdiagnostics.com/catalog/abl-kinase-mutation-analysis-gleevec-resistance

3)  Depending on what the drug resistance test shows, possibly increase  Gleevec dosage if not resistant (but possibly assess Gleevec levels in  the blood with a Gleevec Blood Level Test), or switch to another drug if  you are Gleevec resistant.

Here is some info on Gleevec resistance:
http://jci.org/articles/view/30890

4)  Kinase Domain mutations are not the only reason for drug resistence.   Researchers have found that an over-expression of the LYN Kinase can  also cause drug resistence:

http://www.sciencedaily.com/releases/2008/06/080624174903.htm

5)  If in the meantime you wanted to increase Gleevec dosage, I would  discuss that with your Onc. If the PCR increases by 1 or 2 logs, it  likely would mean Gleevec has stopped working and a drug change would be  required. But an interim increase in dosage is still an option.

6)  Leading CML specialists are recommending faster switching to Sprycel or  Tasigna when loss of response to Gleevec starts to occur.

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