The importance of advanced molecular diagnostics in cytopenia and (suspected) MDS

Next-generation sequencing (NGS) has rapidly become an indispensable tool in diagnostic molecular genetics. A recent article in Blood (Steensma Blood 2018) examined how patients with ambiguous cytopenia or with a (suspected) MDS diagnosis can benefit from the diagnostic potential of NGS. For this reason, MLL Münchner Leukämielabor has taken a closer look at the importance of extended molecular diagnostics for (suspected) MDS, with particular emphasis given to cases in which MDS cannot be confirmed using conventional methods.
Currently, NGS already plays an important role in the diagnosis of AML and it is expected to play even more significant role in the future. Our next newsletter (2/2019) will therefore focus on the central role of comprehensive molecular diagnostics in (suspected) cases of AML.

In the space of only a few years, Next-Generation Sequencing (NGS) has contributed significantly to our understanding of leukemias and lymphomas. As well as its high sensitivity for detecting 1-3% mutational burdens, this is also due to the huge potential of parallelizing sequencing, so that a large number of patient samples or genomic loci can be examined in a single sequencing run. In the case of panel testing, for example, all gene segments associated with an entity of interest can be analyzed. By using an appropriate NGS panel, Malcovati et al. 2017 illustrated that demonstration of molecular genetic clonality in patients with ambiguous cytopenia constituted a significant risk factor for progression towards myeloid neoplasia (Malcovati et al. Blood 2017). Indeed, the presence of at least one specific mutation in one of the 40 genes associated with myeloid neoplasia investigated, resulted in a 13.9-fold increased risk of disease progression. For individual risk assessment, it is therefore helpful to distinguish between idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS).

In the article "How I use molecular genetic tests to evaluate patients who have or may have myelodysplastic syndromes" published in Blood, two out of the four case studies discussed illustrated the clinical relevance of such a distinction (Steensma Blood 2018). NGS molecular genetic analysis could therefore differentiate between a reactive change and a pre-MDS stage (CCUS). The patient exhibiting clonality went on to develop MDS with excess blasts during the two-year follow-up period.

While the mere presence of a mutation alone represents a risk factor for MDS progression, the Malcovati et al. study identified some highly predictive mutational patterns (mutations in spliceosome genes, or mutations in ASXL1, DNMT3A or TET2 in combination with at least one other mutation). In the survival analysis, carriers of such mutation profiles were not found to differ from patients diagnosed with myelodysplasia and comparable mutation profiles. Mutations in the RUNX1 and JAK2 genes were also associated with an increased risk (Malcovati et al. Blood 2017).

The extended NGC diagnostic panels are expected to play an important role in the future MDS risk stratification performed by conventional methods. Even though this mutation analysis has been omitted from the revised International Prognostic Scoring System (IPSS-R) (Greenberg et al. Blood 2012), one study demonstrated that the presence of TP53, CBL, EZH2, RUNX1, U2AF1 or ASXL1 mutations was associated with shorter survival times independently of the IPSS risk status (Bejar et al. Blood 2015).

Given the importance of MDS risk assessment in the selection of therapies, this prognostic panel is an invaluable complement to the IPSS-R for molecular genetic diagnostics. Particularly in younger patients, molecular genetic alterations (such as TP53 or ASXL1 mutations) as well as cytogenetic aberrations can be decisive factors in determining the risk classification and in choosing the appropriate therapy, and may also reveal indications in support of an allogeneic stem cell transplantation. The analysis of the TP53 mutational status in MDS with del(5q) is particularly noteworthy, since the detection of such mutations may be associated with early onset of progression under lenalidomide treatment (Jädersten et al. JCO 2011). Accordingly, both of these aspects are reflected in the German guidelines as a recommendation (Hofmann et al. Onkopedia 2018). On the basis of unfavorable survival data (Lindsley et al. NEJM 2017) under standard conditioning programs, the detection of a TP53 mutation can even be decisive in the selection of an alternative conditioning protocol (Steensma Blood 2018).

In an additional case study, Steensma provides an excellent example of how a comprehensive molecular diagnostics analysis can lead to a differential diagnosis. Following confirmation of a CMML-1 diagnosis, a molecular genetics analysis was performed to assess a hepatic fibrosis in the same patient. This identified ASXL1 and TET2 mutations as well as a KIT-D816V mutation. The detection of the KIT mutation prompted a more detailed immunohistological examination, which resulted in the diagnosis of systemic mastocytosis with associated hematological neoplasia. When the therapy was adapted accordingly, the patient's symptoms improved significantly.

Other studies in addition to the publications mentioned above have demonstrated the major diagnostic and prognostic relevance of molecular genetic analyses using NGS particularly in patients with ambiguous cytopenia and MDS.