The use of leukaemia Q-fusion gene screening assay (Q30) in the diagnostic evaluation of acute myeloid leukaemia (AML)

Background: AML is a clinically heterogenous disease characterised by chromosomal and genetic abnormalities which guide risk stratification and management. Hence timely cytogenetic and molecular profiling is essential to initiate appropriate treatment. Approximately 30% of AML cases have fusion genes. We use a combination of fluorescence in situ hybridization (FISH) to rapidly screen for common chromosomal abnormalities and fusion genes, and chromosomal microarray (CMA) to assess copy number variations across the whole genome. However, cytogenetic analysis is resource intensive and may take several days which can delay patient management. The QuanDx© Leukaemia Q-Fusion Genes Screening Kit (Q30) uses multiplex reverse transcription real-time PCR (RT-qPCR) to allow simultaneous detection of 30 fusion genes with 140+ breakpoints within hours. The use of Q30 may allow earlier risk stratification and treatment before FISH and CMA results are available. Aims: To evaluate the Q30 Kit to detect rearrangements on bone marrow and peripheral blood specimens from newly diagnosed AML patients; and compare concordance of Q30 and FISH in identifying leukaemia fusion genes. Methods: We retrospectively reviewed FISH, CMA and Q30 data for new AML cases diagnosed in the specialist integrated haematological malignancy diagnostic service (SIHMDS) at University College London Hospital over a 2 year period: 01 Jan 19 to 31 Dec 20. Results: 114 cases were included. The median age was 59 [range 19 to 91]. Male 59 (52%), Female 55 (48 %). - ELN Cytogenetic Risk Group (n;%) Favourable 19 (17%) Intermediate 69 (61%) Adverse 26 (23%) Type of AML (n;%) De novo 105 (92%) Secondary 7 (6%) Therapy Related 2 (2%) 30 (26%) of 114 patients had a detectable fusion gene by Q30 (Figure 1). 25/30 were confirmed by FISH either on our standard panel or subsequently using targeted probes. The 5/30 fusion genes detected by Q30, but not by FISH were: - t(3;5)(q25;q34) NPM1-MLF1 (n=1) - t(9;22)(q34;q11) BCR-ABL1 with a high cycle threshold (Ct) value (n=3) - t(16;21)(p11;q22) TLS-ERG (n=1) The discordant results were due to high Ct values (detecting low level gene fusions below the limit of FISH sensitivity), or a lack of commercially available fusion probes. Image:Summary/Conclusion: The ability to rapidly risk-stratify newly diagnosed AML patients allows earlier initiation of targeted therapies. We found that Q30 is highly sensitive and showed 100% concordance in identifying fusions associated with good cytogenetic risk AML in our cohort. Q30 can also identify high risk fusion genes including DEK-CAN, MLL-AF6 and other high risk MLL translocations included in the panel which are rare and for which FISH fusion probes are not routinely used. These may have been otherwise undetected, but can subsequently be confirmed by FISH, and importantly allow the generation of molecular measurable residual disease (MRD) assays to assess treatment response. We conclude that Q30 is a rapid, simple and sensitive adjunct to conventional FISH in the diagnosis of AML and, in combination with CMA, may preclude the requirement for conventional and time consuming G-banding analysis.