Next-generation sequencing (NGS) platforms have recently evolved to provide an accurate and comprehensive means for the detection of molecular mutations in heterogeneous tumor specimens. Initial research efforts applying massively parallel sequencing methods had focused on examining so-called index patients to investigate the landscape of molecular mutations in acute myeloid leukemia (AML), hairy cell leukemia (HCL), or myelodysplastic syndromes (MDS). These studies led to the discovery of key mutations in genes such as IDH1, DNMT3A, BCOR, and BRAF, and unraveled the involvement of the dysregulated splicing machinery in various types of hematologic malignancies. It is of great interest to study whether this novel laboratory method will now evolve as a suitable platform to provide sensitive and quantitative data on the constantly increasing number of mutations in a necessary throughput and accuracy and, as such, will advance into the field of molecular diagnostics. Potential use scenarios will be discussed, such as applying deep-sequencing assays to not only classify a disease, but also to aid in prognostic stratification. Currently, a state-of-the-art laboratory will have to take into account that there are principally different NGS technologies commercially available. Depending on the targets of interest, differing strategies for sample preparation may be relevant. For example, if a small number of genes need to be investigated, it will be sufficient to perform small-scale PCR. However, since the number of genes of interest is constantly increasing, it might be more beneficial to start investigating larger gene panels and consequently apply high-throughput targeted enrichment solutions. It is therefore expected that molecular biomarkers will no longer be sequenced individually in the near future. Instead, panels of markers will be assessed in a massively parallel way, with high sensitivity and multiplexing of many patients per run. It will then be relevant to optimally translate the wealth of molecular markers known from whole-exome and whole-genome studies into actionable gene panels. Moreover, it seems possible that a future increase in read will enable us to clearly correlate the occurrence of double mutations in a patient to a monoallelic or biallelic status of the mutation, which has already been demonstrated to be relevant in CEBPA-mutated AML.
In addition to the aspect of sample preparation and data generation, routine laboratories will also face the need to develop automated processes for ensuring quality and enabling robust bioinformatics pipelines to prepare actionable medical reports. For example, starting with mononuclear cells, processes will be presented to automate laboratory steps in order to reduce hands-on time and operator intervention as much as possible. In hematologic malignancies particularly, the importance of detecting small subclones has increased. This has become highly relevant either in a setting of assessing minimal residual disease (MRD) or identifying molecular mutations with prognostic or predictive relevance to direct treatment strategies. Yet, in contrast to few candidate genes involved in myeloid malignancies such as NPM1 or IDH1, most recurrently altered genes, as exemplified by mutations in RUNX1, CEBPA, TP53, TET2, or ASXL1, often lack mutational hotspots. As such, offering patient-specific assays to detect the broad spectrum of mutations would require the need to apply individualized assays, which is not feasible on a per-patient basis during routine diagnostics procedures. Therefore, laboratories face a great unmet need for unbiased methodologies to provide information on molecular alterations not only per se, but also at a level of sensitivity and throughput necessary for diagnostics processes. Solutions will be presented relating to how the technique of deep sequencing was observed to be superior to the capillary Sanger routine sequencing method and to how NGS will advance the biological characterization of leukemias, in particular during follow-up analyses and in detecting MRD. A great challenge will be the integration of such a novel method into existing laboratory structures. Solutions will be discussed as to how to overcome hurdles and to integrate this assay into current workflows, providing molecular information for many disease areas in a cost-effective manner and fast turn-around time that will be required for individualized treatment regimens.
Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.