The Interpretation of Sequence Variants in Myeloid Neoplasms

An ACLPS Critical Review

Mehenaz Hanbazazh, MD; Shuko Harada, MD; Vishnu Reddy, MD; Alexander Craig Mackinnon, MD, PhD; Djamel Harbi, MD, MA; Diana Morlote, MD


Am J Clin Pathol. 2021;156(5):749-765. 

In This Article

Abstract and Introduction


Objectives: To provide an overview of the challenges encountered during the interpretation of sequence variants detected by next-generation sequencing (NGS) in myeloid neoplasms, as well as the limitations of the technology with the goal of preventing the over- or undercalling of alterations that may have a significant effect on patient management.

Methods: Review of the peer-reviewed literature on the interpretation, reporting, and technical challenges of NGS assays for myeloid neoplasms.

Results: NGS has been integrated widely and rapidly into the standard evaluating of myeloid neoplasms. Review of the literature reveals that myeloid sequence variants are challenging to detect and interpret. Large insertions and guanine-cytosine-heavy areas prove technically challenging while frameshift and truncating alterations may be classified as variants of uncertain significance by tertiary analysis informatics pipelines due to their absence in the literature and databases.

Conclusions: The analysis and interpretation of NGS results in myeloid neoplasia are challenging due to the varied number of detectable gene alterations. Familiarity with the genomic landscape of myeloid malignancies and knowledge of the tools available for the interpretation of sequence variants are essential to facilitate translation into clinical and therapy decisions.


Next-generation sequencing (NGS) has significantly expanded the genomic characterization and classification of myeloid neoplasms over the past two decades. Myeloid neoplasms refer to a group of hematologic malignancies that cause the abnormal proliferation and/or maturation of myeloid lineage cells due to genetic alterations in certain key genes involved in these processes. The fourth edition revision of the World Health Organization (WHO) classification of hematopoietic and lymphoid tissues[1] includes the following categories within the umbrella of myeloid neoplasms: acute myeloid leukemia (AML) and related precursor neoplasms, myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), systemic mastocytosis, MDSs/MPNs, myeloid neoplasms with germline predisposition, and the rare entity known as blastic plasmacytoid dendritic cell neoplasm.

Molecular analysis of most myeloid neoplasms is an essential part of the clinical workup not only to confirm diagnosis but also to predict prognosis, drive therapeutic decisions, monitor treatment efficacy, and guide long-term follow-up Table 1.[2] Targeted sequencing panels (usually containing 20–60 genes) Table 2 are widely used in clinical medicine for the simultaneous evaluation of multiple genes in different patients with high sensitivity and relative low cost per gene.[2,3] In AML, NGS can identify additional novel therapeutic targets such as IDH1 or IDH2[4] as well as prognostic mutations that may predict requirement for allogeneic hematopoietic stem cell (HSC) transplantation (such as RUNX1, ASXL1, and TP53 mutations).[3] The value of NGS in MDSs is also progressively increasing, as molecular alterations are detected in nearly 90% of patients and yield information on risk of progression and transformation to AML.[2–5] In MPNs, NGS helps identify driver mutations (JAK2, CALR, and MPL) with diagnostic implications for polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), as well as other variants that contribute to risk stratification and treatment decisions. Variants other than those in JAK2, CALR, and MPL help make the diagnosis in some difficult triple-negative cases.[4,6]

While the advent of NGS technology has yielded valuable data regarding the complexity of myeloid neoplasms, their pathogenesis, tumor heterogeneity, and multistep mutational evolution,[2,3,7,8] the complexity of the data generated results in challenges in interpretations of new variants and discrimination between pathogenic mutations, passenger mutations, polymorphisms, clonal hematopoiesis of indeterminate potential (CHIP), and germline alterations.[4] Furthermore, with the widespread use of NGS technologies, the number of detectable variants is increasing, some of which are novel variants that may be interpreted as variants of uncertain significance (VUSs) due to the limited information in the literature.[9] The appropriate interpretation/annotation of sequence variants and the ability of the sequencing report to convey the significance of the findings have repercussions on the evaluation of the patient's prognosis and clinical management. Therefore, better standardization of the interpretation of sequence variants in myeloid malignancies among laboratories is warranted. One must be knowledgeable not only on the effect a variant may have on a gene but also on how the interpretation of such variant may affect diagnostic classification and patient management.[3,4,8] Moreover, huge leaps in NGS capabilities offer promise for future applications such as monitoring of posttransplant patients and minimal residual disease (MRD) as well as more widespread incorporation in fusion detection.[3,4,10]

In this review, we give an overview of the alterations in myeloid neoplasms as detected by current NGS assays and discuss challenges in assay design, variant interpretation, and future applications of this powerful technology.