Harmonization of Molecular Monitoring of CML Therapy in Europe – Perspective of Widespread Competence in BCR-ABL Quantification.

The high efficacy of standard treatment of chronic myeloid leukemia (CML) with imatinib has prompted the need for accurate methods to monitor response at levels below the landmark of complete cytogenetic remission. Quantification of BCR-ABL transcripts has proven to be the most sensitive method available and has demonstrated prognostic impact with regard to progression free survival. Until recently, variations in methods employed to quantify BCR-ABL resulted in lack of comparability of results provided by different laboratories.

The aim of recent efforts within an international program (EUTOS, European Treatment and Outcome Study for CML) was to harmonize the reporting of results according to an International Scale (IS).

Currently, the only validated method to adopt the IS by laboratories is the development of laboratory-specific conversion factors (CF). For assays with linear results, IS values can be derived by multiplication of local results by the CF. So far, 57 laboratories in 26 European countries participated in the project of which 27 were selected as national reference laboratories to further distribute the standardization among interested laboratories in their country or region in Europe. A two-step process was used to arrive at laboratory-specific CFs to convert measurements of BCR-ABL from each participating laboratory (‘local laboratory') to the IS. First, preliminary CFs were calculated using standard samples prepared at the reference laboratory. Second, the published method (Branford et al., Blood 2008) was employed to refine the preliminary CF calculations. For the first step, 12 dilution samples were prepared by the reference laboratory for each local laboratory, consisting of triplicate dilutions of b3a2 BCR-ABL-positive white blood cells (WBC) from peripheral blood of an untreated CP-CML patient in WBC from healthy donors. Samples contained 10–20 million WBC and were designed to approximate to 10%, 1%, 0.1%, and 0.01% BCR-ABL IS. Dilutions were stabilized with 1 mL Trizol and frozen at −20°C before being shipped to the local laboratories on dry ice. For validation of CFs, the participating local laboratories were requested to prepare 25–30 CML patient samples consisting of 10–20 million WBC in 1 mL Trizol, covering a range of BCR-ABL levels between 0.01% and 10%. Log BCR-ABL values for the same samples (dilution set) were compared between reference and local laboratories by linear regression. Linearity and sensitivity of the respective results were determined for each local laboratory.

Having achieved linear results, preliminary CFs were calculated for 51 of 57 (89%) laboratories (median CF 0.872, range 0.103–104.7). Applying refined CFs (calculated by Bland-Altman bias plot) vs preliminary CFs for the comparison of each 25–30 CML patient sample results revealed significantly higher concordance between individual BCR-ABL levels from local laboratories and those from the reference laboratory: 72% vs 52% of the samples were within a 2-fold range (0.5–2.0), p<0.0001; 90% vs 76% were within a 3-fold range (0.33–3.0), p<0.0001; and 96% vs 94% were within a 5-fold range (0.2–5.0), p<0.0001. While 43 of 48 (90%) laboratories met the criteria for acceptable concordance, there were five which failed (2-fold <50%; 3-fold <75%; 5-fold <90%). These encouraging results are currently undergoing validation by a further exchange of patient samples and rigorous control rounds.

Harmonization of molecular monitoring across Europe has been established in 26 countries. By expanding these efforts on national levels we aim to establish a European network of approximately 200 laboratories producing harmonized molecular monitoring results that are linked to the IS.

Hochhaus:Novartis : Research Funding.