PS1175 DETECTING BCR‐ABL AND SCORING MR: RESULTS FROM A CE‐IVD KIT RUN ON NON‐IVD PCR INSTRUMENTS: A COMPARISON WITH DATA FROM A EUTOS VALIDATED LAB.
Authors: J. Petersen, G.W. Novotny
Journal: HemaSphere (2019)

Background: Detection of the BCR‐ABL1 fusion transcript in CML patients using RT‐qPCR allows sensitive monitoring of disease levels (minimal residual disease, MRD), which is important for determining prognosis and making treatment decisions. To interpret MRD results correctly in respect to data from clinical trials, it is necessary to report data as IS, which is a standardized unit for reporting the level of BCR‐ABL1 transcripts. In order to report on the IS scale, a conversion factor is required, which currently is calculated by comparing MRD data from numerous samples exchanged with a reference laboratory; an exhausting process. Alternatively, commercial BCR‐ABL1 kits can allow reporting on the IS scale. However, IVD kits may be validated on specific equipment not available in the current laboratory setting, which may rise concerns about the performance under these conditions. Aims: To compare data from a commercial CE‐IVD BCR‐ABL1 detection kit run on non‐IVD instruments, with results obtained from our own EUTOS validated lab. Methods: A EUTOS validated assay following EAC and EUTOS guidelines for detecting and scoring BCR‐ABL1 was applied 1, 2 , using GUSB as reference gene and 6 replicates of BCR‐ABL1 for increased sensitivity, to detect the presence of BCR‐ABL1 in 56 RNA samples. A commercial CE‐IVD kit from Asuragen (QuantideX qPCR BCR‐ABL IS KIT) was also used following the manufactures recommendations, with the following alterations: a Stratagene Mx3005 was used instead of those validated by Asuragen, and data was calculated on a spreadsheet instead of by Asuragen QuantideX Reporter Software. 1) Cross NPC et al ., Leukemia (2015) 29 , 999‐1003 2) Gabert J et al., Leukemia (2003) 17 , 2318‐2357 Results: Fig. 1a: Data from samples containing BCR‐ABL1 transcripts detected in all 6 wells in the In‐house assay (i.e. those samples with least stochastic variation). The reported IS between the two methods are similar, with an average fold change of 1.2 between the assays (ranging from 0.4 to 2.1). Fig. 1b: A plot of the data, from fig. 1a, gives a regression equation of Y = 1.043 ∗ X + 0.81, and R 2 of 0.85. Fig 1c: Data from samples with detectable low‐level disease in the In‐house assay where one or more BCR‐ABL1 wells contain under 3 transcripts. As the IS from Asuragens assay nears the LOD (sample 33 and onward), we see higher IS with our in‐house method. The recent EUTOS guidelines recommend using 3 as the lowest number when scoring a positive well, and this may in some cases result in IS overestimation. Transcript number using the commercial kit was much larger due to the single‐well multiplex setup, thereby reducing the effect of stochastic variation. Two samples with detectable disease with the In‐house assay were not detectable with the commercial assay, these were also below this assays LOD. Fig 1d: Data from samples with undetectable disease in the In‐house assay. 4 samples show detectable disease when using the commercial kit. Inspection of the reference gene data for these 4 cases indicate a higher sensitivity in the commercial assay, thereby allowing an increased chance for detecting the few transcripts that may be present. Summary/Conclusion: In our hands, data obtained from the commercial kit was essentially equal to our in‐house assay, conforming to EAC/EUTOS standards, even when run on equipment as yet not IVD approved by Asuragen. The largest variations were seen for samples close to the limit of detection, where our In‐house assay had several BCR‐ABL1 wells containing less than 3 transcripts. image