Ultra-Rapid, High-Throughput Molecular Diagnostics in Hemato-Oncology.
Authors: Gonzalez David , Walker Brian A. , Morgan Gareth J. 
Published: April 2020

The clinical relevance of molecular genetic analyses in haematological malignancies is now becoming more clearly defined. Useful strategies involve the finding of mutations, differential gene expression profiles, translocations, deletions or amplifications and, more recently, epigenetic changes. Genetic abnormalities and/or gene signatures can be used to characterise a particular disease state, helping in the differential diagnosis of similar entities or, within the same malignancy genetic markers can help discriminating between subgroups with different clinical outcome. Disease-specific and/or patient-specific genetic markers can be used to monitor minimal residual disease (MRD) by real-time PCR.

Unfortunately, it has been difficult so far to translate research into the clinic and set up simple, rapid and reliable tests that can be used routinely in the lab to identify and monitor these genetic abnormalities/markers in haematological patients; the main drawbacks being the labour-intensive, time-consuming nature of these methods. The new generation of ultra-fast, high-throughput automated genetic analysers and DNA/RNA extraction systems allow such analyses to be carried out on a routine basis. Routine PCRs can be performed in as little time as 20 minutes, while sequencing reactions, Real-Time PCR and high-resolution fluorescent electrophoresis can be performed in less than 1 hour. Thus, reducing significantly the processing time, increasing the cost-efficiency, and allowing urgent results to be reported on the same day.

We have adapted our standard protocols for clonality detection (IGH/IGK/TCRG/TCRB), mutation analysis (IGH/IGK/IL-10/IL-6/MMSET/XBP1/FLT3), gene expression (MMSET/FGFR3/ CCND1/CCND2/CCND3/MAF/ABL/GUS/GAPDH/ACTIN)and MRD monitoring (t(9;22), t(15/17), t(12:21), t(8:21), t(1;19), inv(16;16)) to the ultra-fast, high-throughput methods without extensive further optimisation, and without loss of sensitivity or specificity. Time comparison is summarised in Table 1.

In summary, with the new available generation of genetic analysers, haematology laboratories can now provide a fast and reliable diagnostic service, and rapidly incorporate new research results into molecular diagnostics. Although this technology might not be accessible to smaller labs, it should be incorporated into reference labs to improve the management of haematological patients.

Table 1. Comparison of the time required to perform common molecular diagnostic analyses between the standard and fast protocols. Time (in hours) with Standard Protocols Time (in hours) with Fast Protocols Clonality Gene XP/MRD Sequencing Clonality Gene XP/MRD Sequencing *Time to process 96 samples. XP: expression; MRD: minimal residual disease; N/A: Not Available DNA/RNAextraction 0.5 - 5* 0.5 - 5* 0.5 - 5* 0.5* 0.5* 0.5* cDNA synthesis N/A 1 N/A N/A 1 N/A PCR 2.5 N/A 2.5 0.3 N/A 0.3 Real-timePCR N/A 3.5 N/A N/A 0.8 N/A SequencingReaction N/A N/A 2.5 0.8 N/A 0.8 Electrophoresis O/N N/A 3.5 0.5 N/A 0.5 TOTAL TIME 2 Days 5h to 2 Days 2 Days 2.5h 2.5h 2.5h