Conventional cytogenetics for myeloid neoplasms in the era of next‐generation‐sequencing
Authors: Frank C. Kuo, David P. Steensma, Paola Dal Cin
Journal: American Journal of Hematology (2017)

In this issue of the American Journal of Hematology, Weinberg et al.1 discuss how multiple diagnostic techniques should be integrated in the diagnosis of acute myeloid leukemia (AML) and the risk stratification of patients. Ever since the discovery of the Philadelphia chromosome more than 50 years ago,2 conventional cytogenetic assays, including karyotype and fluorescent in situ hybridization (FISH), have played a critical role in classification, diagnosis and management of AML, and other myeloid neoplasms. Most, if not all, of the recurrent chromosomal rearrangements commonly seen in AML are now well described and the genes involved in these rearrangements have been identified. In the 2016 revised World Health Organization (WHO) classification,3 specific chromosomal rearrangements detectable by either karyotype or FISH are major determinants of the classification of AML and MDS, and the clinical cooperative groups and other organizations such as the European Leukemia Net (ELN) include karyotype as a fundamental component of prognostic assessment for AML, myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN). Over the past decade, remarkable advances in sequencing technologies have led to major advances in our collective knowledge of the genomic alterations of myeloid neoplasms.4 More than 50 genes are now known to harbor recurrent somatic alterations in myeloid neoplasms, including single nucleotide variants (SNV) that are often gain-of-function changes occurring at hotspots, or as small insertions/deletions (indel) that are loss-of-function changes, which attenuate tumor suppressor activity. Some of these alterations have been shown to play a major “driver” role in leukemogenesis and are potential targets for therapeutic intervention. Mutations in several genes (e.g., NPM1, CEBPA, FLT3) have been incorporated in the routine risk stratification of AML in recent years, as the contribution of these mutations in making diagnostic and therapeutic decisions has become clear. Correlative analysis has also shown evidence of interaction between some somatic alterations and specific chromosomal rearrangements. For example, in patients with AML and t(8;21), KIT mutations are more common than in other AML subtypes and are associated with a higher relapse rate and poorer outcome.5 With increasing availability of genomic profiling of AML and other myeloid neoplasms, it is widely anticipated that future iterations of myeloid neoplasia classification systems will include additional genetic alterations.6 Mutational status of individual genes can be assayed by Sanger sequencing one gene at a time, but as the number of genes that needs to be tested increases it becomes more economical and time-saving to perform concurrent testing of a panel of genes. The major multi-gene platforms available for clinical use today are based on Next Generation Sequencing (NGS),7 in which genomic regions of interest are first selected or amplified and then millions of target DNA molecules are sequenced in a massively parallel fashion, following by alignment of these sequences against a reference genome. Aligned sequencing reads at each genomic coordinate are then “piled up” and analyzed to identify reads that differ from the reference genome. NGS approaches can detect not only SNV and small indels but also can infer copy number variations (CNV), for example, gains and losses, through “read-count” analysis in a manner akin to array comparative genome hybridization (aCGH).8 The cost of NGS panels has been decreasing steadily over the last few years, while the quality and reliability continues to improve. The fact that NGS has the potential to interrogate genomic copy number changes raises several questions for the future role of conventional cytogenetics in the evaluation of myeloid neoplasms. For example, will we need both NGS-based panel tests and cytogenetics to evaluate patients with newly diagnosed myeloid neoplasms? How should patients best be monitored during treatment or at the time of relapse? To address these questions, we can examine similarities and differences between CNV data obtained through conventional karyotype versus a targeted NGS panel. First, karyotype generates both numerical and structural information of the underlying genome, while targeted NGS approaches mainly provide numerical information. For example, karyotype analysis may show that a piece of 12p is moved to 21q in all the cells in a sample, but since there is no net gain or loss, the NGS analysis of the same sample will not show any changes. Similarly, NGS may show gain of several genes on 8q but it cannot determine where the extra DNA is located (i.e., still attached to 8q or moved to somewhere else). Specific rearrangements often have clinical significance. Although whole-genome sequencing by NGS and RNA-based NGS panels can theoretically detect chromosomal rearrangements, there are many constraints that currently make it impractical to perform whole-genome sequencing and RNA-based panels as clinical tests. Second, karyotype data produce genomic information with single cell resolution, while NGS panels provide an “average” copy number change for all the cells present in a sample. From karyotype analysis, it is common to identify multiple clones that may or may not share some common alterations. In contrast, NGS panels can only identify numerical alterations that are shared by a significant fraction (typically >30%) of the cells within the sample. For example, karyotype analysis may show half of the cells in a sample have a loss of one copy of chromosome 12 and the other half have a gain of one copy of chromosome 12, but the NGS panel of the same sample will show no net gain or loss. Third, karyotype analysis has a resolution at the G-banding level (generally >10 Mb for tumor samples) while NGS panels offer gene- or exon-level resolution. Karyotype analysis can easily detect whole chromosome, chromosome arm level- or segmental (i.e., less than a whole chromosome arm) alterations but cannot detect submicroscopic changes (i.e., <10 Mb for tumor samples). NGS-panels have much higher (single nucleotide) resolution but lack sufficient target density to assess gross chromosomal changes. Even when all the genes on a chromosomal arm are included in a NGS panel, one cannot differentiate with certainty whole arm alterations from multiple segmental alterations within that chromosome arm because the majority of the genome is intergenic. Gains or losses of a single gene, for example, are likely to be underappreciated in myeloid neoplasms because they may not have been previously detectable by karyotype. Fourth, the ability of karyotype analysis to correctly decipher numerical or structural alterations becomes limited in situations where extreme aneuploidy is present. In a very complex karyotype with many small “marker chromosomes,” for example, it is challenging to determine the chromosomal origins of these aberrations. Targeted NGS panels can reliably count reads in this situation because NGS assays align sequences to a reference genome and do not take into account of the actual location in the genome. Finally, NGS panels measure copy numbers in relative and not absolute terms. They cannot readily differentiate a triploid or a tetraploid genome from a diploid genome—a distinction that is easily determined by karyotype analysis. These comparisons illustrate the relative advantages and disadvantages of targeted NGS-based CNV calling and karyotype/FISH, supporting the notion that these are complementary technologies best used together to assess the numerical and structural features of the neoplastic genomes. As the field moves closer to a molecularly-defined classification of myeloid neoplasms, it is likely that there will be continued need to evaluate patients suspected of having myeloid neoplasms with both karyotype and NGS-based assays during initial diagnostic evaluation. Genomic instability is a hallmark of cancers, and clonal heterogeneity and clonal evolution are common themes during initiation and progression of myeloid neoplasms.9, 10 As stepwise genetic alterations accumulate in hematopoietic stem or progenitor cells, both convergent and divergent evolutionary forces drive clonal heterogeneity such that multiple neoplastic clones with partially overlapping genomic profiles and different degrees of dominance can coexist at the time of diagnosis. Clones with certain genetic alterations may be more susceptible to therapeutic regimens such that representation of those alterations may diminish during treatment while emergence of clones carrying new variants may herald relapse or progression. Furthermore, different temporal acquisition of genetic alterations may lead to different clinical behavior and outcome. For example, TET2 and JAK2 frequently are comutated in patients with MPN. In one recent study, the “JAK2-first patients” were found to be more likely to present with polycythemia vera than with essential thrombocythemia, and these same patients had an increased risk of thrombosis when compared to “TET2-first patients.”11 Thus, it is important not only to perform molecular testing at initial diagnosis to establish the baseline genomic profile but also to perform follow-up testing in order to detect the appearance of new alterations that can inform selection of therapeutic regimens. We suggest that NGS panels are a better choice for follow-up testing. Karyotype analysis should be reserved when confirmation of NGS findings is desired. The rationales for this suggestion are as follows. First, although single-cell resolution of karyotyping makes it a useful avenue through which to explore clonal changes in a neoplastic population, only a subset of clonal heterogeneity and evolution can be detected using karyotypic analysis (i.e., those large chromosomal rearrangements that are not based on SNVs and small indels). Second, structural abnormalities (apparent balanced rearrangements) currently listed in the WHO classification of hematopoietic neoplasms are usually early, driver events: that is, patients only rarely acquire these leukemia-specific translocations midway through their disease process. Third, numerical instability that leads to segmental, arm-level or whole chromosomal losses (5q deletion, monosomy 7, etc.,) or amplification of specific genes such as MYC or KMT2A/MLL1 can arise in patients with or without prior abnormalities by karyotype and can be an early or late event. For example, patients with a t(15;17) can acquire an additional 7q- in the disease course either spontaneously or as a therapy-related event. Similarly, a patient with AML with a normal karyotype can acquire 5q- or 20q- as the disease progresses. These events can be detected by either NGS or karyotype analysis. Fourth, except for cases with extreme neutropenia, NGS panels can be performed on peripheral blood which is easy and inexpensive to obtain while karyotype analysis needs an adequate bone marrow sample to perform. Finally, karyotyping is a manual process requiring years of training and acquisition of great skill not only in analysis but also in interpretation, whereas NGS assays are largely automated. Typical karyotype analysis also provides insight into approximately only 20 metaphase cells. In summary, karyotypic analysis after initial diagnosis is unlikely to reveal additional information if serial NGS panel testing did not uncover clonal evolution (acquisition of new somatic variants or CNV). However, serial testing using NGS panels can uncover emergence of new mutations and potential targets for therapy as well as evaluate emerging copy number changes that may be clinically significant. When significant change is detected by NGS panel testing in the clinical course of the disease, it is prudent to perform follow-up cytogenetic analysis both to confirm suspected numerical alterations and to provide single-cell resolution and better assess clonal heterogeneity. As NGS-based panel tests gain wide acceptance in clinical practice, the richness of sequence information may facilitate the design of well-designed comprehensive NGS panels as a go-to test, particularly at the time of initial diagnosis. Although targeted NGS panels can assess numerical instability of the genome, limitation of this approach including the relative inability to comprehensively assess structural abnormalities, the lack of single-cell resolution and a low target density make it necessary at present to perform concurrent conventional cytogenetic analysis to obtain a full picture of the neoplastic genomic profile. After initial diagnostic evaluation, it may be advantageous to follow patients with serial NGS panel testing and to deploy cytogenetic analysis for patients whose NGS results show significant clonal evolution. Frank C. Kuo,1 David P. Steensma,2 Paola Dal Cin1 1Brigham and Women's Hospital, Boston, Massachusetts, USA 2Dana Farber Cancer Institute, Boston, Massachusetts, USA