Next generation sequencing (NGS) holds much promise for the research and diagnosis of complex genetic diseases like cancers. The massively parallel sequencing makes it possible to detect somatic and germline mutations – even though these typically present at only a low level. Here, in Part 2 of this article, we continue to describe the role of NGS in Myeloid neoplasms with a focus on the challenges of detecting variants, ascribing clinical context to those variants and the considerations for NGS testing of clonal myeloid malignancies.
Challenges of Detecting Variants
Traditional sequencing methods typically focus on sequencing either whole genomes/exomes or single biomarkers of known biological significance. The former poses the problem of generating too much non-relevant data for which storage and analysis create bottlenecks whereas the latter captures information on just one or a few regions of potential medical importance while missing many others. In contrast, targeted NGS approaches offer the potential to screen multiple biomarkers in a single reaction, while excluding irrelevant regions of the genome from the analysis.
That said, NGS comes with its own set of challenges: Sanger sequencing has long been viewed as the gold standard of sequencing in clinical settings so any NGS test must prove itself equal to it in terms of accuracy, specificity and sensitivity. Furthermore, an assay is only as good as the variants it captures and how they are interpreted. Because NGS tests for almost every abnormality at the same time, the results can be confusing and difficult to interpret. Interpretation is hindered by the variability in sensitivity, specificity, accuracy, and clinical utility across NGS platforms and compared to traditional sequencing approaches, again reiterating the importance of a well-designed NGS assay.
Although a large number of genetic variants have been formally associated with disease states (often referred to as “actionable alterations”), an equally large number of identified variants have unknown clinical significance. VUS are variants of uncertain (or unknown) significance abound but are slowly being reclassified as pathogenic or benign variants. Additionally, it has been reported that NGS can miss some translocation events and epigenetic regulation disorders . Fortunately, there are some options available today that help overcome many of these challenges related to the number of variants detected and their interpretation through the careful selection of genes and solutions which reduce the ambiguity of the calls. For example, QIAGEN® QCI Interpret suite provides transparent, evidence-sourced NGS variant calling with advanced data analytics to enable interpretation and reporting.
Key Variants in Myeloid malignancies
- – One of the most actionable alterations is JAK2, a clear diagnostic indicator of bone marrow disorders which are characterized by an over proliferation of blood cells. There are several JAK2 inhibitors where ruxolitinib was approved in 2013 as the first in its class of Janus kinase inhibitors .
- – CALR and MPL have diagnostic utility and testing for mutations in these genes is advised per the WHO 2016 Guidelines  for assessing myeloproliferative neoplasms (MPN). In particular, presence of JAK2, CALR or MPL mutations is a major diagnostic criterion for MPN.
- – Less than two years ago, KIT mutations became correlated with remission and relapse rates in core-binding factor AML diagnoses which are associated with poor prognoses . KIT mutation analysis is especially informative in the identification of patient non-responders to the KIT inhibitors
- – CEBPA variants are associated with a favorable prognosis in both adults and children with AML, particularly for those patients with double CEBPA mutations . CEBPA variants also serve double-duty as defining disease category .
- – Tp53 (p53) is tumor suppressor protein which regulates unchecked cell division – a hallmark of nearly all cancers. Tp53 mutations in AML patients are strongly associated with a complex karyotype, and hence, poor outcome . Tp53 mutations are also associated with leukemic transformation of myeloproliferative neoplasms .
- – FLT3 variants are detected in nearly one-third of AML patients and are induced by point mutations or internal tandem duplications (ITDs): patients with ITDs have particularly poor survival rates due to high relapse rates and poor response to treatment . FLT3 has emerged as promising therapeutic target, second-generation FLT3 inhibitors are now in late phase clinical trials .
- – KIT D816V has therapeutic utility, particularly in systemic mastocytosis where the variant is detected in ~90% of patients .
- – IDH1 and IDH2 mutations are widely detected and are generally stable throughout progression of AML, they have become a popular target for new IDH1 and IDH2 inhibitors .
Considerations for NGS Testing of Clonal Myeloid Malignancies
Countless genetic variants and high person-to-person variability exists in loci associated with myeloid malignancies. Additionally, variability exists in the coverage offered by different gene panel offerings and in the strengths of the different sequencing workflows available. Moreover, ascribing the clinical value of one variant over another (i.e. which variant is the more actionable alteration?) is wholly dependent on a multitude of factors.
There are multiple commercial offerings available to test hematologic variants. However, key factors must be considered when selecting how NGS will be applied in your research lab or clinic. These factors include which hematologic disorder you are investigating, if you are seeking diagnostic, prognostic or therapeutic utility through the test, if the patient is a child or an adult and in remission, has relapsed or has been recently diagnosed. Given the genetic complexity and need for high sensitivity as discussed, plus the complexity of the data interpretation, careful consideration must be made in the selection of which NGS assay will be deployed for the testing of clonal myeloid malignancies. One aspect that does remain clear is the need for a comprehensive NGS solution with a well-designed assay and integrated analytics to enable researchers with the flexibility, ease-of-use and confidence that they need to readily make data-informed decisions.