Friday, March 21, 2008

20 - The CML Testing Eplained

The explanation below is a detailed one and all honors go to Trey, who has posted this on
Thanks Trey for the clear and educational explanation provided below, exactly the way we like it: In Layman’s terms!! (still had to read it twice!!)


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. For more details, Google the phrase and also askyour doctor/Oncologist.

There are tests to diagnose CML, evaluate response to therapy, assess the levels of the remaining disease, and to check for specific problems. Among these are Complete Blood Count (CBC), Bone MarrowBiopsy (BMB), Bone Marrow Aspiration (BMA), Cytogenetics Testing,Fluorescence In Situ Hybridization (FISH) testing, Polymerase ChainReaction (PCR) testing, and some 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 avery 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 on cells taken during a Bone Marrow Biopsy (BMB)process. During a BMB, a core sample is taken from the hip bone, and marrow cells are collected that cling to that bone sample. While the hole is open in the hip bone, fluid from the hip marrow is also takenout by a syringe, and this second part is called a Bone MarrowAspiration (BMA). The BMA aspirate or fluid is extracted through thehole created during the BMB. Cytogenetics Testing is done on the core sample and aspirate fluid. The marrow cells are viewed by a labtechnician and/or doctor under a microscope, where the chromosomes are treated with a dye and observed, and the Philadelphia Chromosome (Ph chromosome), which is the indicator of CML, can be seen and a diagnosis made. The core sample is also checked for other abnormalities. So Cytogenetics Testing is done using a BMB core sample and aspirate viewed under a microscope. Cytogenetics Testing is alsoused to check for other chromosome mutations and abnormalities, so aBMB 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. When therapy reduces the levels of CML disease to where the Cytogenetics Testing can no longer detect any Ph chromosome cells among the approx 20 that are counted, that person has achieved a Complete Cytogenetic Response(CCR).

After diagnosis, it is important to continually monitor response to therapy with regular Complete Blood Count tests. When these CBC tests show that the 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.

The BMA fluid taken after a BMB core sample procedure can also be used to perform a FISH or PCR test. (FISH is fluorescence in situhybridization and PCR is polymerase chain reaction). Or circulating(peripheral) blood can also be used now adays with nearly equal confidence levels 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 cells, and counts the number of cells that have the Ph chromosome (technically it looks for the BCR-ABL gene in the cells). This is done by a machine which uses a dye process, isolates approx 200 - 500 cells, and counts the leukemic cells. The result is given as a percentage of leukemic cells to good cells, sothe person can say that X% of their 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, same as a zero Cytogenetics Test). So FISH is generally not used once the level of leukemia drops below approximately 5%. At that point PCR testing is used to monitor CML patients in this Minimal Residual Disease (MRD) status, since it isfar more sensitive. A trend among Oncologists is to start doing PCRs early instead of FISH, since PCRs are more sensitive and can be used to track log reductions in disease levels, and FISH cannot track log reductions.

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 - 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, 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 1 million cells in the sample. As such, it is the most sensitive testing available at this time. PCR testing can be done with relatively equivalent results from 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 thebody. This is one reason why PCR results from one person to another,and one lab to another, are not equivalent, due to lack of standardization 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, and not be directly compared to the previous PCR from the other lab.

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. But if the lab does not provide this information, it makes the 3 log reduction goal more difficult to track, since many do not knowwhere they started at diagnosis. Because drugs like Gleevec and Sprycel can rapidly reduce the levels of leukemic cells, if the first PCR is not done before starting drug therapy, the baseline forcalculating a 3 log reduction will not be very accurate. If someonehas a baseline PCR value done at diagnosis, 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 was10.0%, then moving the decimal point one place to the left is 1.0% (1log), 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%. If someone does not have a baseline PCR, and thelab does not provide log reduction numbers, some literature suggeststhat .01% should be the 3 log estimated goal, which assumes a starting value of 10.0.
If a 3 log reduction is achieved, the next goal becomes maintaining the 3 log reduction or even continued reduction toward PCRundetectable (PCRU), where the PCR is not sensitive enough to detectany leukemic cells in the sample. This PCRU is called CompleteMolecular Response (CMR), which is the deepest level of responsecurrently measurable. In PCRU status, the leukemic cells are mostlikely still there, although fewer than 1 in a million. There is notest to determine if a person with CML is actually cured (usuallyassociated with a stem cell/marrow transplant). The current indicatoris 5 years without therapy coupled with continuous PCRU.
FISH numbers do not correlate to log reductions, so PCR must be usedfor log reduction measurements. Also, FISH percentages do not relateto PCR percentage numbers. For instance, at diagnosis I had both a FISH and PCR done. The FISH was 100% and the PCR was 7%. That isbecause FISH is a percentage of leukemic cells to good cells, but PCR is not (see explanation in earlier paragraph). Beyond that, the FISH has an error rate of approx 5%, so your FISH could read 5% but actually be zero. When the FISH result gets below approx 10%, you should rely on PCRs from then on. A recent trend is to only perform PCRs from the start and not use FISH.

There are other tests that are used for monitoring CML patients, suchas a Liver Function Test to make sure the liver is not adversely affected by CML drugs; a Basic Metabolic Profile (or Panel test) which checks both mineral levels and kidney function; heart function tests(a disputed issue among researchers); CAT Scans or physical checks forenlarged spleen, checks for enlarged lymph nodes; and complete orpartial physical exams. There are also other lab tests to check forspecific problems when suspected.


Thanks again Trey, Very Helpful!!

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