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PCR Results Explained

 

What is an RQ-PCR test?

An RQ-PCR test is a simple blood test that measures the number of Ph+ CML cells in your blood.3 It is an important test to perform because it is very sensitive. It can detect 1 Ph+ CML cell out of 100,000 cells, so it can detect very low numbers of remaining Ph+ CML cells.3 Remember, the lower the amount of Ph+ CML cells left in the blood, the less chance there is for Ph+ CML to relapse and progress.4-6 Therefore, this test will help us know how well your treatment is working and help us make better decisions about your care.

What is a log drop?

An RQ-PCR test measures the number of Ph+ CML cells in your blood. The way it counts the decrease of cells is in terms of a log drop or log reduction.4 Each log reduction means you have 10 times fewer BCR-ABL genes than you had at diagnosis.7 For example, a 1 log reduction is the same as dividing the number of Ph+ cells you have at diagnosis by 10. Ultimately, what is most important is that the trend of your log reductions continues to decrease over time until you reach a major molecular response, which is equivalent to a 3-log reduction (or 0.1% BCR-ABL to control gene ratio on the international scale).4 Results from 2 landmark studies show that patients who have a major molecular response have a much better chance of remaining free from disease progression, or relapse.8,9This means their Ph+ CML cells didn’t increase for a long period of time.

When should I get a RQ-PCR test?

RQ-PCR tests are recommended at diagnosis and every 3 months until you achieve a major molecular response.10 After that, we will give you an RQ-PCR test every 6 months.10 These are simple blood tests that we will do when we regularly take blood to monitor your progress.

When should I expect to reach MMR?

Experts recommend certain response goals at different time points: a CHR at 3 months, a CCyR at 12 months, and an MMR within 18 months. We will continue to monitor your blood frequently to ensure the trend of Ph+ CML disease is continuing to go down and evaluate you according to response.10 It’s important to remember that you still need to take your medication every day as directed to keep Ph+ CML cells from coming back.

What happens if a result shows an increase? 

You may receive a test result that doesn’t show a continued reduction of Ph+ CML cells. It’s not a cause for alarm. Results may vary from time to time for a number of reasons. For example, if you have missed doses, or if a different lab was used, the test results could vary. This doesn’t necessarily mean your Ph+ CML is relapsing and getting worse. We will simply schedule an extra test to make sure you are still on track.10

Why is it important for me to take my medicine as directed?

If you don’t take your medicine exactly as directed, it increases the chance that your Ph+ CML will relapse and get worse.1,2 Clinical studies have shown that patients who take their CML medicine every day have significantly higher chances of reaching a major molecular response, which is linked to a significantly lower risk of their Ph+ CML relapsing and getting worse.1,2,8

If your medication makes you feel bad, don’t skip a dose! I can help you manage most side effects by giving you over-the-counter medicines that may help you feel better. If you feel great and want to forget your disease, don’t skip a dose! Remember that you must take your medication as prescribed to achieve and maintain an optimal response.

How can I keep from forgetting to take my medicine?

There are a number of ways to help remind you take your medicine. These may include trying to link taking your medication to a daily ritual, setting a cell phone alarm, scheduling a meeting or appointment reminder to take your medicine, or enlisting a treatment buddy to help remind you. Remember that you must take your medication as prescribed to achieve and maintain an optimal response.1,2

Why is a major molecular response an important treatment goal?

The optimal goal of therapy is to achieve and maintain a major molecular response, which greatly reduces the risk of Ph+ CML relapsing and getting worse. Results from 2 landmark studies show that patients who have a major molecular response have a much better chance of remaining free from disease progression, or relapse. This means their Ph+ CML cells didn’t increase for a long period of time.8,9

What is a complete molecular response?

A complete molecular response is the absence of any detectable Ph+ CML cells in your body. Taking your medication as prescribed once you achieve a major molecular response may further reduce the number of Ph+ CML cells remaining in your body to a level so low they are no longer detectable.7


 

References

  1. Marin D, Bazeos A, Mahon FX, et al. Adherence is the critical factor for achieving molecular responses in chronic myeloid leukemia patients treated with imatinib for at least two years. J Clin Oncol. 2010;28(14):2381-2388.

  2. Noens L, van Lierde MA, De Bock R, et al. Prevalence, determinants, and outcomes of nonadherence to imatinib therapy in patients with chronic myeloid leukemia: the ADAGIO study. Blood. 2009;113:5401-5411.
  3. Radich JP. How I monitor residual disease in chronic myeloid leukemia. Blood.2009;114(16):3376-3381.
  4. Hughes T, Deininger M, Hochhaus A, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood. 2006;108(1):28-37.
  5. Palandri F, Iacobucci I, Soverini S, et al. Treatment of Philadelphia-positive chronic myeloid leukemia with imatinib: importance of a stable molecular response. Clin Cancer Res. 2009;15(3):1059-1063.
  6. Hughes TP, Hochhaus A, Branford S, et al. Reduction of BCR-ABL transcript levels at 6, 12, and 18 months (mo) correlates with long-term outcomes on imatinib (IM) at 72 mo: an analysis from the International Randomized Study of Interferon versus STI571 (IRIS) in patients (pts) with chronic phase chronic myeloid leukemia (CML-CP). Blood. 2008;112(11):Abstract 334.
  7. Baccarani M, Pane F, Saglio G. Monitoring treatment of chronic myeloid leukemia. Haematologica. 2008;93(2):161-166.
  8. Deininger M, O’Brien SG, Guilhot F, et al. International randomized study of interferon and STI571 (IRIS) 8-year follow-up: Sustained survival and low risk for progression or events in patients with newly diagnosed chronic myeloid leukemia in chronic phase treated with imatinib. Poster presented at: 51st American Society of Hematology (ASH) Annual Meeting and Exposition; December 5-8, 2009; New Orleans, LA.
  9. Saglio G, Kim D-W, Issaragrisil S, et al. Nilotinib versus imatinib for newly diagnosed chronic myeloid leukemia. N Engl J Med. 2010;362(24):2251-2259.
  10. Baccarani M, Cortes J, Pane F, et al. Chronic myeloid leukemia: an update of concepts and management recommendations of European LeukemiaNet. J Clin Oncol. 2009;27(35):6041-6051.

 

 

What Is PCR Testing


By Anjana Rai Chaudhuri, PhD (Asian CML List)



How do we laypeople decipher PCR readings?

Well, we don't have to be technical, what we do need to know are the points that assure us that we are being monitored correctly and what our disease status is in molecular terms. This in turn, helps us ask pertinent questions of our treating physicians.

Firstly, at diagnosis, a bone marrow test is done to count how many Philadelphia chromosomes a patient has. This is the CML cancer marker. However, this test is done only on 20 marrow cells, there are millions and millions of cells so obviously this is not a sensitive test. However, for diagnosis, this is fine, since most patients are diagnosed 100%Ph+, that is all 20 marrow cells have the cancer marker. With Interferon treatment, this sometimes sufficed because majority never gained a complete cytogenetic remission when all 20 cells examined are free of the disease. However, with IM, the situation is quite different. 82% of patients achieve CCR on Glivec at 2.5 years follow-up and for most, this CCR comes quickly in the space of months. However, having no cancer marker in 20 cells does not mean you do not have disease so obviously more sensitive tests are needed. FISH is one such test that can detect the cancer marker in from 200-500 marrow or blood cells. This is an improvement on the 20 cells.

However with Glivec therapy, in 6-9 months, many patients show no disease by cytogenetics and FISH. This still means there is disease except we cannot see it. We do need to see it in order to measure Glivec's continued efficacy so in comes polymerase chain reaction tests or PCR tests. This test has the capability to look for a needle in a haystack, namely, 1 leukaemic cell in as many as 1 million normal ones. It is molecular testing and so the response by PCR is described as molecular response. When patients reach this level, when they are zero by all other tests except PCR, they are said to have minimal residual disease.(MRD)

Like we watch the cancer marker decrease as in reduction of Ph percentage from bone marrow cytogenetics or FISH, we now have a technique, the PCR, where the cancer marker can indeed be tracked at low level of disease. For some years, qualitative PCRs have been used for post-SCT patients to track for molecular relapse which usually heralds cytogenetic and hematological relapse. Qualitative PCRs are those that give a positive or negative without giving numbers.

More useful are quantitative PCRs which give a number. To us laypeople, what exactly is this number? PCR tracks the BCR-ABL transcripts and the number that is churned out is a percentage ratio of leukaemic cells to total cells. In order to be sensitive and correct for degradation of RNA, a housekeeping gene is used as a control. And therefore, the PCR result is expressed as the percentage ratio of BCR-ABL (cancer marker gene)in leukaemic cells to housekeeping gene which is expressed in all cells. This ratio is necessary to cancel out errors in the PCR measurements and give a reliable marker of the disease status.

Nowadays, real-time quantitative PCRs are used in most of the top centers to track low-level disease. However, even in this low-level disease, there are sub-levels. Patients in CCR can have high PCR values or low PCR values. Therefore, the CML experts have come up with two stages of response in the molecular category. Major molecular response (MMR) is a 3 log reduction of the disease from diagnosis(1000-fold reduction) and complete molecular response is when there is no cancer marker present from the PCR tests.

Now, each lab will have their own PCR value depicting a 3 log reduction, 2 log reduction, 1 log reduction, 4 log reduction. Different housekeeping genes are used and there may be other factors that are different.

So, as patients and caregivers, what do we look out for?

1. A sensitive real-time quantitative PCR methodology capable of measuring 3 log and 4 log reductions. It is not enough to ask for the sensitivity of the PCR as in how many leukaemic cells it can detect in how many normal like 1 in 100,000 or 1 in 1 million.(1 in 100,000 is a required minimum by the CML experts) What we should be asking is the sensitivity as well as what log reduction does our value mean? We cannot look at absolute numbers and predict log reductions because housekeeping gene may be different and other factors may be different in different labs. Some labs are mentioning log reductions when reporting PCR values as well as discussing them. If they do not, we need to bring these into our discussions with our doctor.

2. It may be a better idea to go with a quantitative PCR rather than a qualitative one since in the former, there is better quality control of individual samples.

3. What next? If the PCR values are having a downward trend, no need to worry overmuch about dose and other things. Remember, it is the PCR trend that counts not absolute numbers.

4. If there is an upward trend, what then? The result has to be reproducible over repeated tests and depending on amount of increase,talk to your doctor. In the top notch labs, a 2-fold increase is enough to discuss mutations testing with the doctor. But in other labs, a 1 log increase may be time for the mutations testing to be discussed. Mutations are a mechanism of Glivec acquired resistance and most top labs test for mutations when there is rising PCR values. Do not worry. New drugs are in trial which target the mutations.
Any upward trend in PCR over time is of course worrisome and needs to be discussed with the doctor. However, most chronic phase patients in CCR have shown durable remissions over time with stable or decreasing PCR values.

5. What is the goal of molecular testing? To indicate molecular relapse, molecular negativity and tracking of the disease. Suppose there is an upward trend over time and mutations testing is done quickly and the mutation detected only confers partial resistance, then the doctor may increase the dose very soon before the disease gets out of hand. Alternatively if it is something like T315I mutation, it is best to seek combo therapies, new drugs or transplant to control the CML ASAP. So, PCR has utility in these decision makings by your doctor.

6. What about 3 log reduction? Both PCR negativity and 3 log reductions are goals but as long as the PCR is stable or decreasing, IM is having disease control and there should be no worry. Whether higher doses of Glivec can actually push folks to lower PCR levels-the trials to determine that is on at the moment. Of course, some doctors may wish to try increasing the dose to try to get lower PCR values after discussion with the patient and those are individual decisions.

Ask for the sensitivity of the real-time quantitative PCR and ensure that it can at least detect 1 leukaemic cell in 100,000 normal ones. Then when you get your PCR value, ask what log reduction it is. Remember, your value may be different from others doing PCRs in other labs. After that, track the PCR value in order to get the trend.

It is important to note that at very low PCR levels, there may be some variations in readings because of the nature of the technique and you should not worry if you become PCR negative and then have a low positive PCR value. Remember, serial monitoring is the key and it is the trend over time that is important.

A 3 log reduction or major molecular remission (MMR) by PCR may be desirable because from the Glivec Phase III trials, it was found that those patients who had this low level of disease had 100% progression-free survival 2 years after start of Glivec therapy.


 

STAGES OF REMISSION

By Anjana Rai Chaudhuri, PhD (Asian CML List)



There are 3 stages of CML remission.

1. Complete hematological remission (CHR)- This is when the blood hows no sign of leukemia, the white count and platelets are within ange and the spleen is no longer enlarged. This usually occurs within a month of taking Gleevec and is he first stage of remission.

2. Complete cytogenetic response- This is called CCR and occurs when out of 20-50 marrow cells, no cell with a Ph chromosome was detected. You can also have cytogenetic response by FISH where out of 200-500 cells, no cell with the BCR-ABL fusion is detected. There is also another term- major cytogenetic remission (MCR). This is when the cells examined by cytogenetics or FISH have less than 35%Ph+.

3. Finally we have molecular remission (MR) where out of upto 1 million cells, no BCR-ABL transcripts are detected. This test is the polymerase chain reaction or PCR test. According to recent articles, there are two stages of molecular response. Major molecular response (MMR) which is when there has been a 3 log or more reduction in BCR-ABL transcripts from PCR compared to diagnosis values (1000-fold reduction). Complete molecular response is when no BCR-ABL transcripts are detected by PCR.

So, CHR comes first, then CCR and finally MR.