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T.I. Mughal et al.
further, reduce the turnaround time and lower the costs, which remain high.
Intratumoral heterogeneity
Studies of hematologic malignancies as well as diverse solid tumors have revealed a surprising amount of intratu- mor heterogeneity, i.e., the finding of multiple, related clones rather than one uni-clonal monolith. Thus, the kinet- ics of disease progression, response and relapse follow the rules of Darwinian selection. Many neoplasms, including CML, MF and advanced SM, have been found to exhibit greater clonal complexity than previously thought, as new myeloid mutations have been found in these diseases.31 For example, Jawaher and colleagues identified the emergence of the KIT D816V mutation as a distinct and late event in patients with multi-mutated advanced SM.32
Trying to map intratumor heterogeneity by the sequenc- ing of bulk populations is limited by the simple fact that one is sequencing the average mutation frequency of all the
A
cells from various clones. New technologies allow for the sequencing of RNA (e.g., 10x Genomics) or DNA (e.g., Mission Bio) from single cells. Major advantages of single- cell technologies include the higher resolution offered to understand the types of cells present, to detect rarer cell populations and to study their function (inferred by RNA expression) or clonal structure (inferred by mutation pat- tern). Disadvantages, other than the financial costs, are that each cell is a ‘one and done’ experiment. Furthermore, it can be difficult to determine real signal versus experimental noise, which is especially problematic with RNA, for which simple factors such as time from sample acquisition to experiment can influence gene expression.
Recent work using single-cell RNA-sequencing has gar- nered considerable novel insight into normal and aberrant hematopoiesis, cell-cell interactions, characterization of bone marrow and immune (non-clonal) cells as well as tis- sue stroma and leukemia-initiating cells.33-36 This technology enables detection and characterization of intratumor het- erogeneity and provides much needed granularity to key issues, including acquisition of individual or specific combi-
Figure 3. Comparison between Sanger
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sequencing and
sequencing – the NEXT-in-CML study. (A) Percentage of patients positive for mutations, as determined by Sanger sequencing (SS) and by next-generation sequencing (NGS). Among patients pos- itive for mutations by NGS, 31 (13.1%) had high-level mutations only (≥20%; detectable by SS too); 29 (12.3%) had both ≥1 high-level mutations and ≥1 low-level mutations (≤20%; detectable by NGS only); 51 (21.6%) had only low- level mutations. A low-level T315I muta- tion was detected in ten patients; 59 additional patients had ≥1 low-level mutations known to be associated with resistance to imatinib or second-gener- ation tyrosine kinase inhibitors other than the T315I mutation (Y253H; E255K/V; V299L; F317L/V/I/C; F359V/I/C). The remaining ten patients had only low-level mutations with an unknown resistance profile and/or not listed in the COSMIC database. (B) Patients positive for one or multiple mutations as assessed by SS versus NGS. CML: chronic myeloid leukemia; pts: patients; IMA: imatinib; DAS: dasa- tinib; NIL: nilotinib; BOS: bosutinib. (Adapted, with permission, from Soverini S et al.30).
next-generation
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