Quantitative analysis of mutant subclones in chronic myeloid leukemia : comparison of different methodological approaches

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points

Cancer Variant Interpretation Group UK (CanVIG-UK): an exemplar national subspecialty multidisciplinary network.

Advances in technology have led to a massive expansion in the capacity for genomic analysis, with a commensurate fall in costs. The clinical indications for genomic testing have evolved markedly; the volume of clinical sequencing has increased dramatically; and the range of clinical professionals involved in the process has broadened. There is general acceptance that our early dichotomous paradigms of variants being pathogenic-high risk and benign-no risk are overly simplistic. There is increasing recognition that the clinical interpretation of genomic data requires significant expertise in disease-gene-variant associations specific to each disease area. Inaccurate interpretation can lead to clinical mismanagement, inconsistent information within families and misdirection of resources. It is for this reason that 'national subspecialist multidisciplinary meetings' (MDMs) for genomic interpretation have been articulated as key for the new NHS Genomic Medicine Service, of which Cancer Variant Interpretation Group UK (CanVIG-UK) is an early exemplar. CanVIG-UK was established in 2017 and now has >100 UK members, including at least one clinical diagnostic scientist and one clinical cancer geneticist from each of the 25 regional molecular genetics laboratories of the UK and Ireland. Through CanVIG-UK, we have established national consensus around variant interpretation for cancer susceptibility genes via monthly national teleconferenced MDMs and collaborative data sharing using a secure online portal. We describe here the activities of CanVIG-UK, including exemplar outputs and feedback from the membership

Personalised genetic management of CML

Although RT-qPCR is the recommended method for monitoring responses to TKI therapy, it might not be the best assay for patients with deep responses considered for treatment discontinuation. The current consensus is that therapy discontinuation is followed by relapse in at least 50% of patients and that persisting LSCs with low BCRABL1 expression or quiescent LSC with no BCR-ABL1 expression are the source of the relapse. Therefore, identifying a sensitive method for detecting and accurately quantifying residual disease would help identify patients with the lowest likelihood of relapse, and predict those who are at a higher risk of disease recurrence while off therapy. In the era of rapid technological advances and their deployment in molecular diagnostics labs, we sought to investigate the potential of some of these technologies in improving the current clinical management of CML. Digital PCR is a recently developed method of absolute molecular quantification that has the potential for complementing RT-qPCR not only in monitoring MRD while in deep molecular remission or during therapy discontinuation, but also in simplifying the standardization efforts. NGS, on the other hand, has the potential for increasing the sensitivity of TKD mutation detection. Unlike the case in other diseases such as virology and solid tumours, the clinical value of these technologies is not yet known in CML. Therefore, the focus of this Ph.D. thesis was to develop protocols and proof-of-principle data facilitating the implementation of these methodologies in routine testing and consequently investigating their clinical significance in the routine management of CML patients. In chapter two, we investigated an NGS-assisted DNA-based dPCR approached for detecting and quantifying low levels of BCR-ABL1 positive disease. When applied to samples with undetectable disease by RT-qPCR, DNA-based dPCR provided a marked improvement in sensitivity, not only over RT-qPCR, but also compared to real-time qPCR and to RT-dPCR. Although more sensitive, this method is not yet ready for the immediate implementation in routine testing. The impact of residual disease level as assessed by DNA-based dPCR at the time of treatment withdrawal on outcome is currently being investigated within the UK based DESTINY clinical trial. If validated in clinical trials of stopping TKI, the technique will permit a more personalised approach to recommendations for dose reduction or drug cessation in individual patients, ensuring that therapy is withdrawn only from patients with the highest chance of long-term remission. In addition, it will allow timely therapeutic intervention to prevent the occurrence of overt relapse after therapy discontinuation. RT-qPCR remains the gold-standard for monitoring residual disease in CML despite of its various limitations including the compromised precision at the lower end of the calibration curve, in addition to the laborious requirement of continuous assay validation. Therefore, efforts to improve on the current gold-standard are appreciated. In chapters three and four, we sought to investigate the performance of the E.A.C. assay on different RT-dPCR platforms and found that false positive signals detected in the negative controls limit the accurate quantification of residual disease in samples classified below MMR. We also showed that the performance of the RainDrop® RTdPCR platform had excelled compared to the other two RT-dPCR platforms allowing a sensitivity of at least 5-logs on the IS. The false positivity detected on the three RTdPCR platforms using the E.A.C. assay, albeit at different levels, indicated that the noise is most likely platform and/or assay design related. Therefore, further work is required to eliminate false positivity before RT-dPCR could be adopted for the routine monitoring of BCR-ABL1 transcript levels in response to TKI therapy. Mutations in the BCR-ABL1 TKD are the most studied cause of resistance to different TKI therapies and play a crucial role in planning patients’ management. In chapter five, we validated an amplicon deep sequencing approach on the Ion Torrent PGM next generation sequencing platform followed by the application of the method for the prospective testing of referral sample over a period of one year. The aim was to evaluate the performance of the platform and assess the practical need for replacing the current gold-standard with NGS, notwithstanding its potential technical superiority. From technical point of view, we demonstrated that the platform has an LoQ and LoD of 5% and 1%, respectively. Low-level contamination occurring during the runs came as a persisting problem, dictating a cautious interpretation of mutations below 5%. Although NGS demonstrated superior sensitivity compared to the current gold-standard, we didn’t find enough evidence that supports an immediate need for replacing the gold-standard in routine clinical testing, except when performed after TKI resistance with the aim of guiding therapeutic intervention.Open Acces

Cancer Variant Interpretation Group UK (CanVIG-UK): an exemplar national specialist multi-disciplinary genomics forum

The purpose of CanVIG-UK (Cancer Variant Interpretation Group UK) is to advance outcomes for patients by improving the accuracy and consistency of interpretation of variants in Cancer Susceptibility genes across the UK clinical and diagnostic laboratory communities (hereafter termed the UK clinical-laboratory community). CanVIG-UK currently comprises >100 members including clinical and laboratory representation from each of the 25 Molecular Diagnostic Laboratories and Clinical Genetics Services of the UK and ROI. This group comprises roughly equal proportions of clinical scientists and clinical geneticists, with two thirds work exclusively or predominantly in cancer genetic