Leukemia is an umbrella term for multiple different cancers that form in the blood, one such member of the leukemia family being chronic myelogenous leukemia (CML). CML is caused by a chromosomal translocation that results in a fusion gene termed BCR::ABL1. Fragments of chromosomes 9 and 22 are joined to form an in-frame fusion between the BCR gene and the ABL1 tyrosine kinase. The resultant “Philadelphia chromosome” first identified in the 1950’s in Philadelphia is a defining hallmark of CML. Immense progress has been made to improve outcomes and quality of life for this patient group, culminating in the availability of a potent therapy called tyrosine kinase inhibitor (TKI) therapy that targets the BCR::ABL1 fusion protein’s enzymatic activity. Treatment response is determined by the levels of detectable BCR::ABL1 transcript mRNA in the blood, with the amount present dictating whether treatment should be adjusted or, in the instance of excellent response, whether treatment can be safely discontinued. As the most common exon fusions of the BCR::ABL1 are well known and present in the vast majority of cases, reverse transcriptase PCR (RT-PCR) can be a reliable method for monitoring mRNA levels. Unfortunately, inter-lab variation when undertaking RT-PCR can cause issues with data interpretation. This subsequently led to the development of an International Scale (IS%) which denotes a percentage reduction in detectable BCR::ABL1 fusion for use as a guideline in determining treatment response and disease outcomes. Further, experts in the field of CML provided additional guidelines based on results which determined a major molecular response to treatment to be ≥ 3-log reduction in detectable levels. Digital PCR, a method that aims to reduce signal to noise effects, may outperform standard RT-PCR in detecting extremely low residual levels of BCR:: ABL1 fusion in patients with a durable major molecular response. Dr Jerald Radich, a member of Fred Hutch’s Clinical Research Division, and colleagues from Bio-Rad Laboratories developed an assay that focused on monitoring the presence of this translocation in patients with CML, with the overall goal of improving outcomes for this population. Their work, recently published in PLOS ONE, demonstrated the capability of a digital droplet PCR (ddPCR) assay in accurately detecting the presence of low levels of the BCR::ABL1 translocation in patient samples, in addition to performing strongly when compared to standard RT-PCR.
To address the specificity issue of standard RT-PCR, the authors examined the performance of a ddPCR assay. This type of assay seeks to overcome the problems associated with detecting residual disease (or rare targets) by focusing on reducing the issue of signal to noise. The methodology employs a partitioning strategy that effectively separates the “signal” and “noise’ into a smaller number of copies which greatly reduces technical error. Explaining the assay principle further, Dr Radich stated that “the assay tackles the signal to noise problem by “digitalizing” the reaction, partitioning the reaction so that each assay has on average <1 copy of either target, or background. This can be done by partitioning into wells (Fluidigm) or droplets (Bio-Rad). Quantification is done using the Poisson distribution of number of positive partitions vs. number of partitions tested.”
The authors extensively tested the abilities of the ddPCR assay by determining analytic specificity and sensitivity, noting that the assay accurately detects the main exon fusions but does not detect minor variants that make up <1% of all cases. The assay was proven to be highly reproducible, as the authors tested its precision across multiple scenarios including instrument, lot and operator variation, site specific factors, and isolation method utilized for the samples. Each time, ddPCR performed well and generated highly accurate measurements, including proving to be a low-risk method for producing false positive data. The authors next compared the ddPCR assay to RT-PCR and observed a strong positive linear correlation between the two assays. Both assay types performed robustly when tested using reference samples derived from the WHO International Scale. Emphasizing an enhanced capability of ddPCR, Dr Radich said “ddPCR, compared to conventional [RT-PCR], is more reproducible at very low levels of target, and is a bit more sensitive (about 0.5 log).” The authors further discussed how the ddPCR assay was utilized in a trial for patients with CML, patients who had responded well to initial TKI therapy. As part of the trial design patients underwent traditional RT-PCR testing followed by ddPCR testing if treatment discontinuation was recommended. Importantly, in a large percentage of patients, ddPCR detected residual BCR::ABL1 fusion that had not been detected by standard RT-PCR alone, thus greatly improving the risk of relapse rates in the study population.
Additional options for the detection and monitoring of genomic translocations in CML will provide enormous benefit for patients, especially with respect to reducing treatment burden while simultaneously providing confidence in the low likelihood of relapse. “The [present] work is potentially significant since it suggests we can better pick patients who can safely discontinue TKI therapy and not fear the relapse of their disease. This potentially could impact roughly 1/2 of the CML population. Successful discontinuation would have obvious health (less side effects) and economic (these drugs are really expensive) implications. In addition, this highlights the potential of ddPCR in detecting low disease burdens in other hematological malignancies,” explained Dr Radich.
This work was funded by grants from the National Institutes of Health and funding from Bio-Rad Laboratories.
UW/Fred Hutch Cancer Consortium member Jerald Radich contributed to this work.
Shelton DN, Bhagavatula P, Sepulveda N, Beppu L, Gandhi S, Qin D, Hauenstein S, Radich J. Performance characteristics of the first Food and Drug Administration (FDA)-cleared digital droplet PCR (ddPCR) assay for BCR::ABL1 monitoring in chronic myelogenous leukemia. PLoS One. 2022 Mar 17;17(3):e0265278. doi: 10.1371/journal.pone.0265278. PMID: 35298544; PMCID: PMC8929598