Page 235 - Personalised medicine of fluoropyrimidines using DPYD pharmacogenetics Carin Lunenburg

Confirmation practice in pharmacogenetic testing
notifications from regulatory authorities are also not conclusive about this dilemma. In January 2017, the FDA discussed that regulatory aspects on the quality control of LDTs are still under debate.25 In Europe, guidelines on good pharmacogenomics practice (GPP) by the European Medicines Agency (EMA) issued in September 2018 include a chapter on quality aspects on PGx analyses. They describe the importance of proper validation prior to using genetic tests in clinical trials or a diagnostic setting and the detection of respective allele- drop-outs, as primer-based technologies are prone for these artefacts. However, no specific standpoint is taken regarding the use of a second, independent technique.26 Also, the In Vitro Diagnostic Regulation (IVDR) of the European Parliament and of the Council on in vitro diagnostic medical devices has recently been updated and will come into force in 2022. Yet, these guidelines do not explicitly state what actions to guarantee quality are required in the laboratory.
Confirmation practice
Current confirmation practice in laboratories
In order to investigate the consequences of the lack of clear guidelines we assessed the current confirmation practices of laboratories. A short questionnaire comprising three general questions on DPYD genotyping and confirmation practices in the laboratory was sent to laboratories in Europe and the Netherlands participating in the proficiency testing program of the RfB and SKML, respectively. Details on the set-up of the questionnaire can be found in the Supplementary Material. Out of the 475 laboratories, 35 completed the questionnaire. One laboratory participated in both the European (RfB) and Dutch (SKML) questionnaire. 28 laboratories executed genotyping tests. Of all laboratory techniques, the TaqMan assay and melting curve analyses were most frequently used. A large variation between laboratories in confirmation practice was observed. Almost half of the laboratories did not execute a second test (either independent or repetition).
Two independent genotyping methods as confirmation practice
In addition, we assessed the impact of confirmation methods in PGx. At LUMC and the IFCC PGx reference laboratory at Erasmus MC, the most elaborate confirmation method, executing two independent genotyping tests using two different platforms, are used. We evaluated over ten years of aggregated genotyping data of these two large genotyping laboratories performing duplicate analyses on two independent platforms. Details of the two laboratories can be found in the Supplementary Material. In total, 89,842 duplicate tests were executed for patient care in over ten years of genotyping. Nine discrepancies (0.01%) between tests were observed. One discrepancy in CYP3A5*3 was the result of chimerism due to allogeneic hematopoietic stem cell transplantations, which resulted in the determination of the genotype of both patient and donor.27,28 Four discrepancies in CYP3A5*3, one discrepancy in DPYD*13 and three discrepancies in CYP2D6*6 were identified, possibly due to allele dropout. The probability of finding a discrepant result when using two independent techniques according to our data was calculated to be 0.01%.
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