Challenges in the introduction of next-generation sequencing (NGS) for diagnostics of myeloid malignancies into clinical routine use

Abstract

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...

Assessment of the clinical utility of four NGS panels in myeloid malignancies. Suggestions for NGS panel choice or design

“…In our cohort, two FLT3-ITD mutations of 36bp in length (detected by classical molecular techniques in our laboratory) were not called by any of the NGS gene panels tested in this study, which means that conventional diagnostics techniques are still essential for hematological malignancies diagnosis [81]. NGS difficulty for long FLT3-ITD detection has been reported before [62] [82]; this is because current NGS chemistries employ short reading sequencing (read length 50-300bp) and this makes it prone to lose structural variants such as long indels [83] [84]. In support of this observation, in our cohort, the three variants missed by SureSeq panels (sequenced at shorter read length than the other panels, 150bp vs >200bp), were indels.…”

“…In our cohort, the number of pathogenic or likely pathogenic variants was two orders of magnitude smaller than the number of coding variants passing quality control (50 vs 1146). This drop highlights the importance of including expert geneticists familiar with hematological malignancies and NGS technology within the multidisciplinary genomic tumor board, as it has been suggested before [13] [83].…”

The evolving role of next generation sequencing in myelodysplastic syndromes

“…Importantly, most current NGS technology utilizes short read sequencing that may find single nucleotide variants (SNVs) and short indels but cannot capture large structural variants (Bacher et al , ). However, NGS allows for efficient detection of variants with high read depths in targeted multi‐gene panels.…”

“…There are nuances in NGS involving the genes tested, the domains within each of these genes explored, the depth of sequencing, and the molecular pathologist determining variant calls. Technical considerations include differences in inherent properties of the target sequence, use of amplicon‐based vs hybridization‐capture based protocols, and polymerase chain reaction errors as the target sequence is amplified many times over (Bacher et al , ). There is a need for collaboration to form larger data pools in order to overcome these variables and infer meaningful conclusions.…”

Simple acute phase protein score to predict long‐term survival in patients with acute myeloid leukemia

“…Assessment of prognosis of AML patients mostly relies on pretreatment molecular and cytogenetic methods. While a number of driver mutations with known prognostic significance have been previously identified, novel mutations with yet unknown relation to disease progression or prognosis are currently discovered by next‐generation sequencing technique . Novel approaches focus on posttreatment assessment of leukemic stem cell frequency by flow cytometry in combination with traditional flow minimal residual disease strategies, but this approach is so far only available in few laboratories .…”