Next-Generation Sequencing-Assisted DNA-Based Digital PCR for a Personalized Approach to the Detection and Quantification of Residual Disease in Chronic Myeloid Leukemia Patients

Alikian, MA; Ellery, PE Peter; Forbes, MF Martin; Gerrard, Gareth; Kasperaviciute, DK Dalia; Sosinsky, AS Alona; Mueller, MM Michael; Whale, AW Alexandra; Milojkovic, D; Apperley, JA Jane; Huggett, Jim F.; Foroni, L.; Reid, AR Alistair G

doi:10.1016/j.jmoldx.2015.09.005

Cited by 34 publications

(37 citation statements)

References 43 publications

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“…In addition, we and others have compared the quantification of BCR-ABL in genomic DNA and RNA in serial samples and have shown that individual patients may have consistently higher or lower expression of BCR-ABL mRNA for a given number of CML cells. 47,48 This interindividual difference results in a measurement bias whereby RQ-PCR may underestimate the level of residual disease in some patients. Although genomic Q-PCR for BCR-ABL is not widely available, it is a promising research tool to elucidate the relationship between MRD and TFR outcome.…”

Section: Areas Of Future Investigationmentioning

confidence: 99%

Moving treatment-free remission into mainstream clinical practice in CML

Hughes

Ross

2016

Blood

288

260

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The dramatic success of tyrosine kinase inhibitors (TKIs) has led to the widespread perception that chronic myeloid leukemia (CML) has become another chronic disease, where lifelong commitment to pharmacologic control is the paradigm. Recent trials demonstrate that some CML patients who have achieved stable deep molecular response can safely cease their therapy without relapsing (treatment free remission [TFR]). Furthermore, those who are unsuccessful in their cessation attempt can safely re-establish remission after restarting their TKI therapy. Based on the accumulated data on TFR, we propose that it is now time to change our approach for the many CML patients who have achieved a stable deep molecular response on long-term TKI therapy. Perhaps half of these patients could successfully achieve TFR if offered the opportunity. For many of these patients ongoing therapy is impairing quality of life and imposing a heavy financial burden while arguably achieving nothing. This recommendation is based on the evident safety of cessation attempts and TFR in the clinical trial setting. We acknowledge that there are potential risks associated with cessation attempts in wider clinical practice, but this should not deter us. Instead we need to establish criteria for safe and appropriate TKI cessation. Clinical trials will enable us to define the best strategies to achieve TFR, but clinicians need guidance today about how to approach this issue with their patients. We outline circumstances in which it would be in the patient's best interest to continue TKI, as well as criteria for a safe TFR attempt.

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Section: Areas Of Future Investigationmentioning

confidence: 99%

Moving treatment-free remission into mainstream clinical practice in CML

Hughes

Ross

2016

Blood

288

260

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“…Nextgeneration sequencing combined with digital DNA BCR-ABL1 PCR is a recent innovation that could be the most sensitive currently available technique. 41 In the future, this or other novel techniques may be used to revisit the relevance of the BCR-ABL1 detection limit for patient selection for TKI cessation studies. It is clear that currently available technology could be of immediate clinical benefit for monitoring the molecular response of many patients in low-resource regions or for laboratories that lack relevant molecular biology expertise for adequate BCR-ABL1 method development.…”

Section: Molecular Monitoring: How Low Do You Need To Go?mentioning

confidence: 99%

Molecular monitoring in chronic myeloid leukemia—how low can you go?

Branford

2016

Hematology

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Molecular monitoring of BCR-ABL1 transcripts for patients with chronic myeloid leukemia (CML) is now used to assess response to tyrosine kinase inhibitors (TKIs), including treatment failure that mandates a change of therapy. Therefore, many centers have adopted the molecular technique for measuring BCR-ABL1 and rely on conversion of values to the international reporting scale for appropriate clinical interpretation. However, the technique has a degree of inherent variability despite standardized procedures, which means care should be taken by the clinician when assessing response based on BCR-ABL1 cutoff limits. The last few years have witnessed the emergence of a new molecular response target, which is the achievement and maintenance of a deep molecular response. The ability to achieve treatment-free remission for some patients has shifted the relevant boundary for molecular response. However, the definitive safe BCR-ABL1 transcript level and length of the maintenance phase after which treatment cessation can be attempted has not yet been determined. For patients with TKI resistance, BCR-ABL1 kinase domain mutation analysis remains an essential assessment to guide therapy. Furthermore, low-level mutation detection is clinically relevant for response prediction to subsequent TKI therapy for some patients. Multiple low-level mutations may be a biomarker of a clonally diverse disease with the propensity for resistance evolution. Overall, molecular monitoring, including low-level monitoring is a fundamental component of management for patients with CML.

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“…However, the observed ~0.2% fractional abundance was enabled, in part, by the addition of ~40,000 KRAS copies per reaction; this level of sensitivity may not always be achievable using in vivo extracts such as cfDNA from plasma, that typically yields ~500 copies/mL plasma (33). In addition to cancer models, other rare SNV models would also be applicable to this framework (14, 15) as well as simpler targets that do not require specific measurement of a given variant in the presence of the wt allele, such as quantification of specific pathogens or gene fusions (34, 35).…”

Section: Discussionmentioning

confidence: 99%

An International Inter-Laboratory Digital PCR Study Demonstrates High Reproducibility for the Measurement of a Rare Sequence Variant

Whale¹,

Devonshire²,

Karlin-Neumann

et al. 2016

Preprint

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This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate labs. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using qPCR or NGS due to the presence of competing wild type sequences and the need for calibration. Using dPCR, eighteen laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration and could enable the reproducible application of molecular stratification to guide therapy, and potentially for molecular diagnostics.SIGNIFICANCE STATEMENTThe poor reproducibility of molecular diagnostic methods limits their application in part due to the challenges associated with calibration of what are relative measurement approaches. In this study we investigate the performance of one of the only absolute measurement methods available today, digital PCR (dPCR), and demonstrated that when compared across twenty-one laboratories, dPCR has unprecedented reproducibility. These results were achieved when measuring a challenging single nucleotide variant and without calibration to any reference samples. This opens the possibility for dPCR to offer a method to transform reproducibility in the molecular diagnostic field, both by direct use as well as in support of other currently used clinical methods.

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Next-Generation Sequencing-Assisted DNA-Based Digital PCR for a Personalized Approach to the Detection and Quantification of Residual Disease in Chronic Myeloid Leukemia Patients

Cited by 34 publications

References 43 publications

Moving treatment-free remission into mainstream clinical practice in CML

Moving treatment-free remission into mainstream clinical practice in CML

Molecular monitoring in chronic myeloid leukemia—how low can you go?

An International Inter-Laboratory Digital PCR Study Demonstrates High Reproducibility for the Measurement of a Rare Sequence Variant

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