Harmonized Testing for BCR-ABL Kinase Domain Mutations In CML: Results of a Survey and First Control Round within 28 National Reference Laboratories In Europe
Authors: Ernst Thomas , Lin Feng , White Helen E , La Rosée Paul , Lion Thomas , Mitterbauer-Hohendanner Gerlinde , Vandenberghe Peter , Zadro Renata , Polakova Katerina Machova , Plachy Radek , Nyvold Charlotte Guldborg , Lundán Tuija , Cayuela Jean-Michel , Lange Thoralf , Müller Martin C. , Zoi Katerina , Andrikovics Hajnalka , Tohami Tali , Pane Fabrizio , Soverini Simona , Arruga Francesca , Eggen Lene , Sacha Tomasz , Diamond Joana , Talmaci Rodica , Pajic Tadej , Colomer Dolors , Hermanson Monica , Leibundgut Elisabeth Oppliger , Valk Peter J. M. , Ozbek Ugur , Gerrard Gareth , Saglio Giuseppe , Hochhaus Andreas , Cross Nicholas C.P. 
Published: October 2019

Abstract
Abstract 894
Various techniques have been employed to detect BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia (CML) resistant to imatinib or second generation tyrosine kinase inhibitors. This variation may at least partially explain the different frequencies of mutations that have been reported. Furthermore, the pattern of individual mutations reported seems to depend on the specific method used for mutation detection. Standardized techniques and protocols for the detection of BCR-ABL mutations will be necessary to obtain comparable mutation results within clinical studies. The first objective of this study conducted within the EUTOS (European Treatment and Outcome Study) for CML program was to record the mutation analysis techniques and protocols that are used for routine diagnostics by 28 national reference laboratories in 23 European countries, 9 of whom perform regular mutation analyses as a central laboratory for national or international clinical trials. The second objective was to evaluate the techniques by analysis of blinded samples containing various BCR-ABL kinase domain mutations. Initially a web-based survey was conducted with a total of 39 technical and PCR-specific questions. Most laboratories (n=17; 61%) perform 1–10 mutation tests per month (range <1 to >100) and use published protocols for nested RT-PCR from peripheral blood or bone marrow leukocytes. Failure to amplify BCR-ABL mRNA at low BCR-ABL transcript levels was reported to be the most common PCR related difficulty in 16 (57%) laboratories. Sanger sequencing is applied for routine BCR-ABL mutation analysis in 27 (96%) laboratories. Additional screening techniques (e.g. denaturing high-performance liquid chromatography, high-resolution melting) or more sensitive detection methods (e.g. allele-specific oligonucleotide PCR, pyrosequencing) are used routinely by eight laboratories each, respectively. The application of positive or negative mutation control samples was reported by 13 (46%) or 17 (61%) laboratories, respectively. Quantitative mutation analysis is performed routinely in nine (32%) laboratories. To evaluate laboratory performance we prepared a series of control samples that were distributed in a blinded fashion to testing laboratories. Seventeen Ba/F3BCR-ABL cell lines harboring various BCR-ABL kinase domain mutations were mixed with non-mutated Ba/F3BCR-ABL to produce dilutions ranging from 1% to 100% of mutant alleles. Three samples were non-mutated Ba/F3BCR-ABL only and were used as negative controls. Mutated and non-mutated Ba/F3BCR-ABL cell lines were diluted into HL60 cells to simulate a BCR-ABL level of 10% on the International Scale. Twenty blinded cDNA samples were sent out on dry ice to each participating laboratory (total of 560 samples). Twenty-three labs have reported their results so far (460 samples). The three non-mutated samples were identified correctly in 68/69 (99%) tests. For the 20 mutated samples we distinguished those with high (≥20%; n=11) and low (≤10%; n=9) levels of mutation. For the high level samples 218/253 (86%) tests were reported correctly. Of the incorrect results, 22 were scored as negative and 13 were false positives (T315I, n=7; D276G, K357E, D363Y, N374I, S386S, E505K, n=1 each). For the low level samples only 15/138 (11%) tests correctly identified the mutation. Most (122/138; 88%) were reported as undetectable and a false positive result (L323H) was reported in one case. We conclude that Sanger sequencing is the most frequently applied technique for routine analysis of BCR-ABL kinase domain mutations in CML in Europe. In general it reliably identifies mutations when the proportion of mutant alleles comprise 20% or more. Nevertheless, false negative and false positive results were reported in a substantial proportion of samples with ≥20% mutation level (35/253, 14%). For mutations that are present at 10% or less mutant alleles, routine methods mainly failed to identify mutations. This study provides a basis for further comparisons and standardization efforts comparable with the introduction of the international scale for quantification of BCR-ABL transcripts.

Disclosures:
No relevant conflicts of interest to declare.