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T.I. Mughal et al.
Figure 1. Treatment response and potential uses of emerging technologies for diagnostics, monitoring and mutation testing in chronic myeloproliferative malignancies. At diagnosis, methods can be used to quickly identify breakpoints useful for designing monitoring assays, as well as other mutations that might influence the initial response to treatment with a tyrosine kinase inhibitor. RNA- sequencing can be used to assay specific genes and pathways associated with early response. Single-cell genetics can be used to identify potentially trouble- some complex heterogeneity and populations with a resistance signature. During therapy, digital and DNA polymerase chain reaction analyses can be used as more sensitive determinants of deep molecular response, and next-gen- eration sequencing can be used to identify mutations in patients without a deep response who appear to be relapsing. RNA-seq: RNA-sequencing; NGS: next- generation sequencing; MRD: measurable residual disease; ITH: intratumoral heterogeneity; PCR: polymerase chain reaction. (Adapted with permission from Radich JP et al.7)
cate the use of a sustained DMR, for at least 2 years, to select patients for consideration of TKI cessation.10,11
At present, there are several tests in the clinic for the detection and monitoring of BCR-ABL1 transcripts, some of which have not been validated robustly; furthermore, they show considerable interlaboratory variations and variable levels of sensitivity. With the current focus on treatment- free remission (TFR), the importance of using analytically and clinically well-validated tests, preferably approved by regulatory bodies, is being recognized. As an illustration, three recently US Food and Drug Administration (FDA)- approved tests appear to perform better than the ‘standard’ quantitative reverse transcriptase PCR tests and may have greater appeal for monitoring very low levels of BCR-ABL1 transcripts. Indeed, it is of interest that over a decade ago, learning from the earlier lessons following the introduction of DNA PCR for BCR-ABL1 and the harmonizing the International Scale BCR-ABL1 transcript measurements, Goldman and colleagues in London, instigated a patient- specific DNA-based method of detection and quantification of an individual patient’s CML clone.12,13 This method involved the rapid identification of BCR-ABL1 fusion junc- tions by targeted NGS, coupled with the use of a dPCR plat- form, in patients with very low-level molecular residual dis- ease.. The first FDA-approved test (Asuragen Inc., Austin, TX, USA) is performed with a manufactured kit that can be used on several thermal cyclers, although the FDA approval specifies a specific machine.14 The second approved test is the Cepheid cartridge technique (Cepheid, Sunnyvale, CA, USA), which is attractive given its simplicity and short turn- around time.15 More recently, a water-oil emulsion droplet technology, developed by Bio-Rad (Bio-Rad Laboratories Inc., Hercules, CA, USA), known as digital droplet PCR, was approved by the FDA.16 The digital droplet PCR assay has recently been tested in studies evaluating the feasibility of discontinuing TKI therapy safely in patients with CML who had been in DMR for >2 years.17 The studies docu- mented that, compared with quantitative reverse transcrip- tase PCR, digital droplet PCR was better at forecasting the
success of TKI discontinuation. Digital droplet PCR tech- nology has several advantages over quantitative reverse transcriptase PCR tests that rely on exponential amplifica- tion to estimate the target amount. The digital droplet PCR assay relies on a binary endpoint (yes or no), which is much more lenient to poor RNA quality and inhibitors. The BCR- ABL1 transcript level is estimated using Poisson distribution based on the number of positive droplets. The Cepheid test is particularly suitable for analysis of smaller sample batch- es analyzed frequently; for larger batches of samples tested infrequently, the Asuragen or Bio-Rad platforms might be more cost-effective. The use of DNA for CML monitoring is difficult, specifically because the breakpoint on chromo- some 9 is vast compared with the ‘small’ breakpoint cluster region on chromosome 22. There are techniques to find the breakpoint using a series of BCR and ABL primers. Once an amplification product has been generated, it can be sequenced, with patient-specific primers generated for sub- sequent PCR amplification of the BCR-ABL1 breakpoint. However, there may be new technologies that will make breakpoint detection much faster. Breakthroughs in ‘real- time’ sequencing, such as Pacific Bioscience and Nanopore technology, which can read exceeding long sequences at breathtaking speeds, potentially allow BCR-ABL1 break- point detection to be performed with a single sequencing run.8
In CML, there are two variants of the BCR-ABL1 tran- script, depending on whether the break in BCR occurs in the intron between exons e13 and e14, or in the intron between exons e14 and e15.6 A break in the former intron yields an e13a2 mRNA junction and a break in the latter intron yields an e14a2 junction. Most patients have tran- scripts with features of either e13a2 or e14a2, but occasion- al patients have both transcripts present in their leukemia cells. The prognostic significance of the precise type of BCR-ABL1 transcript is now being increasingly recognized in efforts to achieve DMR and potential TFR following suc- cessful TKI treatment. Earlier studies in patients treated with imatinib had suggested that patients with e14a2 tran-
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haematologica | 2021; 106(7)