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V. Krishnan et al.
cally and clinically important subsets), and clinical utility (ability to improve clinical decision making).90,91 In this section, we summarize the pertinent stages and highlight issues of particular relevance to GE-based biomarker development in CML (Figure 3).
Stage 0 is the discovery stage, which is where the field is currently. Here, we highlight three important compo- nents, which include the use of technical approaches for unbiased discovery, the simultaneous interrogation of leukemic and non-leukemic clones from the same sample (since both have been shown to be prognostic), and the use of robust statistical and computational pipelines to discover minimal prognostic genes sets. The advent of single-cell-based technologies and their application to well-annotated cohorts will facilitate this step.
In stage I, the minimal gene set has to be converted into a clinical test that accurately and reproducibly measures the GE phenotype. The test platform needs to be robust, as well as sensitive, specific and reliable. The assay should be developed for tissues that are collected as part of routine clinical care. Ideally, any additional processing of material beyond what is routine should be minimized, e.g., CD34+ selection, and should utilize standard proce- dures available in clinical laboratories, such as flow cytometry and bone marrow immunohistochemistry. An example would be detecting GE signatures of interest by a panel of antibodies for use in flow cytometry or immunohistochemistry applications. It is preferable that the samples used for analytical validation are from well- characterized patients representative of ‘real-world’ set- tings and, ideally, validated in at least one independent cohort. Sample size and power calculations should be determined prior to starting the study, and analytic sensi- tivity and specificity for the test should be available at the end of the study. At the end of stage I, a locked-down test should be evaluated in stage II, that of clinical validation.
In stage II, the locked-down test will be evaluated for its ability to differentiate between clinically meaningful outcomes in modern CML practice. The samples to be tested should be obtained from well-annotated cohorts representative of the broader population, and the test conducted on tissues in a blinded manner with respect to testing and result reporting. Ideal populations include patients who have been treated uniformly in clinical tri- als. At the end of this stage, the ability of the test to pre- dict clinical outcome should be available as a test score, with clearly defined positive and negative predictive val- ues.
The final stage, stage III, will be the determination of clinical utility. This stage would entail the use of the GE- based test to improve clinical decision-making, and would require the study to demonstrate that meaningful outcomes are improved when the test is used compared to when the test is not used. Besides clinical outcomes such as improved progression-free survival and overall survival, additional measures such as cost-effectiveness, avoidance of toxicities, quality of life and psychological parameters should also be assessed. Such studies may also incorporate the contribution of pharmacological fac- tors (e.g., drug metabolism and side effects, patient com-
pliance) to overall outcomes. Given the relative rarity of CML, it is envisaged that this will be a multicenter inter- national study.
Conclusion
Genetic and epigenetic events contribute to the emer- gence of BCR-ABL1-independent clones that result in clinical TKI resistance and, if unopposed, BC transforma- tion. Long-term TKI responses, including successful TKI stoppage, can be predicted by slower declines in BCR- ABL1 transcript levels during first-line TKI therapy,88 sug- gesting that genetic and epigenetic factors contributing to TKI resistance are present at diagnosis. Recent studies describe a convergent GE signature common to the majority of BC progenitors.10 Elements of this common or core transcriptome can be detected in CD34+ cells from CP patients at risk of TKI resistance or early trans- formation,11,13 and specific mutations have been shown to contribute additional nuances to the core transcrip- tome.49 These observations are consistent with a ‘seed and soil’ model that may be helpful for hypothesis gen- eration (Figure 2). Emerging technologies, particularly multimodal single-cell-based approaches, will facilitate the discovery of genetic and epigenetic biomarkers at presentation. This initial discovery phase has to be fol- lowed by the translation of GE-based information into validated analytical tests, and subsequently, the determi- nation of clinical validity and utility. This process will be a multi-year, multi-institution international effort akin to that for the development of a genetic-based risk assess- ment.85,90,91 The integration of both gene mutation- and gene expression-based biomarkers into the care of CML patients will be an important step to achieving the ulti- mate goal of CML research: the cure of the majority of our patients.
Disclosures
DK is a member of the advisory boards of Novartis, Pfizer, Paladin, and has received honoraria from Novartis, Pfizer and Paladin, as well as research funding from Novartis, Bristol- Myers Squibb, Pfizer, and Paladin. TH is a member of a Novartis advisory board and receives research support from Novartis and Bristol-Myers Squibb. SB is a member of the advi- sory boards of Qiagen, Novartis, and Cepheid and has received honoraria from Qiagen, Novartis, Bristol-Myers Squibb, and Cepheid, as well as research support from Novartis and Cepheid.
Contributions
VK and STO conceived the topic for review, and wrote the first draft of the manuscript. DDHK, TPH, and SB contributed by the addition of new sections and critical discussions through- out the writing of the review.
Funding
VK and STO are supported by the National Medical Research Council Singapore (MOH-CSASI18may-0002, MOH-CIRG20nov-0003, NMRC/CIRG/1468/2017).
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