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Molecular monitoring and harmonization of the RT-Qpcr — Ph-IS

Molecular monitoring and harmonization of the RT-Qpcr

The introduction of TKIs (targeted therapy) allowed the majority of CML patients in the chronic phase of recent diagnosis to reach a complete cytogenetic response (CCR) after one year of treatment. But, even with RCC, the bone marrow still contains a very high number of leukemic cells (about 106 cells). This suggests that the normalization of the cytogenetics (CCR) is due to the low sensitivity of the method, and, at the same time, that it is necessary to use more sensitive monitoring methods. Thus, it was necessary to implement quantitative real-time PCR (RT-qPCR), to measure, with a higher sensitivity (up to 100,000 times more sensitive), the levels of BCR-ABL1 chimeric transcript in peripheral blood leukocytes of patients in RCC. According to the depth of the molecular response reached, and the time necessary to reach it, “temporary response criteria” (endpoints) with prognostic significance could be established. The most significant was the definition of the “major molecular response” (MMR, <0.1% of BCR-ABL1 transcripts), which represents one of the main therapeutic aims. 
From this study were also defined the molecular criteria of optimal response, the “warnings” in patients who do not reach the ideal response, and failures. The definitions of “failure”, “warning” and “optimal response” take into account the depth of the molecular response and the time needed to obtain it. For a uniform and correct evaluation of molecular response, laboratories are required to use harmonized methodologies at a single reference scale (the international scale, IS). Both the American guide (NCCN, National Comprehensive Cancer Network) and the European guide (ELN, European Leukemia Net) consider molecular monitoring by RT-qPCR to be the best option to evaluate the patient’s response and evolution, and emphasize that these studies should be carried out in laboratories that use the international scale “(IS).
The RT-qPCR is a complex process involving many factors (preservation of the samples, RNA extraction, RNA copy to cDNA, efficiency of the qPCR, platform used, the control gene used). Consequently, the results can be very variable, compromising their potential clinical significance. The IS represents a practical method that allows comparing the results of RT-qPCR obtained in different centers; consequently, doctors from anywhere in the world can adopt the international guidelines for the treatment of patients with CML.


The IS is established in 2005, from a meeting of experts at the NIH (National Institute of Health, Bethesda, USA); and it is taken as an international reference to inform the results of RT-qPCR. In the same meeting it was established that, to adopt the IS, each laboratory had to determine a “conversion factor” (CF) to make their results comparable with those of the “reference” laboratories. This conversion factor must be updated at least once a year, or when there is a significant change in the methodology used.
Advantages of this process: we all speak the same “language” in terms of molecular response; thus, the MMR expresses a reduction of 3 logarithms in the initial tumor mass, and the MR4.5 indicates a decrease of 4.5 logarithms.

the annual update of the conversion factor is expensive and cumbersome (exchange of samples and comparison of results).

To simplify and make more accessible the process of obtaining the CF, in 2009 the WHO approved the “First genetic reference panel -OMS- for quantification of BCR-ABL mRNA” (The first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA), universal reference material available as “WHO primary calibrators” for specialized laboratories with the capacity to produce “secondary calibrators” from the primary ones; in this way, the process of obtaining of the conversion factor is simplified, and can be extended to more laboratories.
It is recommended to perform molecular monitoring on a regular basis during treatment; however, there are some differences with respect to the suggested frequency, depending on the consulted guide:
— The NCCN recommends doing it at diagnosis and then every 3 months (or every 6 months once the CCR is reached) for 3 years.
— The ELN recommends doing it every 3 months until reaching the major molecular response and then at least every 6 months.

Whichever guide is followed, it is important to keep in mind that frequent molecular monitoring enables the oncohematologist to:
1. To determine the level of response reached;
2. To early identify patients who are less likely to achieve an optimal response;
3. To early detect rising levels of  BCR-ABL1; (a
n increase in the number of transcripts may indicate a loss of response, either by acquisition of a mutation, poor adherence, or other resistance mechanisms);
4. To demonstrate the persistence of low levels of transcripts of Bcr-ABL1 (minimal residual disease) from which, after discontinuing treatment, the patient could lose response and relapse.

The quantity and quality of total RNA will have an impact on the sensitivity of the assay, affecting the final result of the measurement of molecular response, and may influence the clinical decision of the physician. To reduce the effect of these variables a control gene is used that allows to compensate all these variables and therefore make the test more reproducible and reliable. The control gene ensures that the patient’s RNA samples are always of the same quality and quantity. The three most used control genes are BCR (used in the IRIS study), ABL1 and GUSB (used by the European consortium).
The availability of TKIs offers to the patient the possibility of reaching deep and sustained molecular responses and, eventually, being part of discontinuity protocols. In some cases, the molecular response is so intense that it is not possible to detect transcripts even with a technique as sensitive as RT-qPCR; however, it is not possible to assume that the leukemic clone has disappeared, but that the remaining amount of tumor cells is below the sensitivity level of the method (residual disease). Therefore, the term complete molecular response (CMR) has been replaced by molecular response (RM), to which the level of sensitivity reached is added. The sensitivity of the test is variable from center to center, and also within the same center, from sample to sample.

Thus, we defined an MR4.0 (0.01% IS, 4 logs of reduction), MR4.5 (0.0032% IS, 4. logs of reduction) and MR5.0 (0.001% IS, 5 logs of reduction) and the MMR (major molecular response), based on the 0.1% IS limit (3 reduction logs with respect to the IRIS base value, MR3.0). In the case of “negative” or “undetectable” samples, the number of copies of the control gene marks the sensitivity with which we can rule out the presence of BCR-ABL1; MR4.0 when ABL> 10,000 copies, MR4.5 when ABL> 32,000 copies, and MR5.0 when ABL> 100,000 copies (see Table 1).
Table 1: Reference values of the international scale and definition of the molecular response.
Tabla - monitoring