Given the vast phenotypic and genetic heterogeneity of acute and chronic myeloid malignancies, hematologists have eagerly awaited the introduction of next-generation sequencing (NGS) into the routine diagnostic armamentarium to enable a more differentiated disease classification, risk stratification, and improved therapeutic decisions. At present, an increasing number of hematologic laboratories are in the process of integrating NGS procedures into the diagnostic algorithms of patients with acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPNs). Inevitably accompanying such developments, physicians and molecular biologists are facing unexpected challenges regarding the interpretation and implementation of molecular genetic results derived from NGS in myeloid malignancies. This article summarizes typical challenges that may arise in the context of NGS-based analyses at diagnosis and during follow-up of myeloid malignancies.Table 1Challenges accompanying the introduction of massive parallel sequencing in clinical routine diagnostics in hemato-oncologyChallengeBackgroundCurrent and future approachDiscrimination of leukemia-related mutations from polymorphisms or passenger mutationsDriver mutations expected to occur at higher allele frequency in patient samples than passenger mutations; driver mutations more likely to have an impact on protein function than polymorphisms or passenger mutationsOptimization of cancer-specific databases including reporting of rare physiological gene variantsImplementation of novel bioinformatic algorithms based on prediction of functional impactQuantitative and dynamic VAF monitoring (separately and together with other mutations) at follow-upDiscrimination of somatic leukemia-related mutations from CHIPCHIP is presented in ~10% of individuals aged 70 to 80 and in up to 20% in the age group > 80 yearsQuantitative and dynamic VAF monitoring (separately and together with other mutations) at follow-upClarifying the significance of CHIP in the context of myeloid malignanciesDiscrimination of leukemia-related somatic mutations from pathogenic germline alterationsChallenge to differentiate acquired somatic mutations from germline pathogenic variants at diagnosisMutation detection in germline control samples (e.g., skin fibroblasts, saliva) in mutations such as in RUNX1, CEBPAThorough medical family history followed by molecular genetic tests in relatives if necessaryHigh and stable VAF (e.g., 40–50%) at follow-up despite clinical response to treatment may be indicative for germline alterationDiscrimination of true genetic alterations from PCR, sequencing and post-sequencing artifactsMany artefacts are known to arise during NGS library preparation, sequencing and data analysisError correction using molecular identifiers that individually label original input DNA moleculesRefinement of error-correction computational methods in post-sequencing NGS data analysisConfirmation using Sanger sequencingLimited sensitivity of NGS for minimal residual diseas...

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