Quantifying Minimal Residual Disease in Chronic Myeloid Leukemia

Thanks to the introduction of tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) has been transformed from a deadly cancer into a treatable chronic illness. CML is identified by the fusion of two genes, which yields a gene product called BCR-ABL. This BCR-ABL gene fusion, in turn, gives rise to the BCR-ABL kinase, which promotes the uncontrollable growth of myeloid cells. TKIs inhibit BCR-ABL kinase activity, allowing more than 90% of patients with CML to survive for at least five years after diagnosis.¹

But while TKIs are effective, patients must often tolerate a lifetime of treatment because the current methods for monitoring BCR-ABL transcript levels in the blood, an index of treatment efficiency, are not sensitive enough to give physicians confidence that TKI therapy is no longer needed (See Table 1²). More specifically, the gold standard for monitoring circulating BCR-ABL is quantitative PCR (qPCR), which can detect BCR-ABL transcripts at concentrations down to one copy in 10,000 normal copies from patient samples.^3,4 Below this threshold, however, the test may return a ‘negative’ result even if residual cancer—also known as minimal residual disease—is still present. Patients who end their TKI treatment based on undetectable qPCR measurements relapse nearly 50% of the time within two years.⁵ To prevent this relapse, physicians typically keep their patients on the drug for their entire lifetime.

Table 1: Quantitative PCR is the current standard for monitoring circulating BCR-ABL

Not only is qPCR not accurate enough at these low levels of BCR-ABL, it can also be unreliable. Specific reaction conditions and the identity of the primers, probes, and reference genes used can result in significant variability in BCR-ABL measurements. One study that examined BCR-ABL levels in 38 laboratories and compared them to results from a centralized laboratory found that only 58% of qPCR-based laboratory methods achieved an average BCR-ABL difference of ±1.2-fold from the reference method.⁶

Patients could greatly benefit from a BCR-ABL monitoring tool that can detect lower levels of the transcript with greater precision. TKI therapy produces a host of side effects, such as fatigue, nausea, cramping, and in severe cases, liver damage and congestive heart failure.^2,5,7,8 Also, TKIs are expensive: the treatment could cost a patient between $59,000 and $76,000 (USD) annually.⁹

Fortunately, a new test is currently under FDA review that may serve as a more sensitive and reliable alternative to qPCR. This test, based on droplet digital PCR (ddPCR) technology, may enable physicians to discontinue their patients’ TKI therapy with increased certainty that their CML will not return. This ddPCR-based test uses the same PCR reagents, oligonucleotide primers, and fluorescent probes as qPCR to amplify nucleic acid samples. But unlike qPCR, ddPCR involves the partitioning of a sample into 20,000 nanoliter-sized droplets, wherein a separate PCR reaction occurs in each droplet. Each droplet contains only a few nucleic acid strands, and if one of those happens to be the target sequence, such as the BCR-ABL transcript, the droplet will fluoresce. Because each nucleic acid strand is amplified separately, a count of the number of fluorescent droplets will indicate the number of target sequences contained in the whole sample. This will enable a pathologist to quantify a target sequence absolutely, without the need for a reference curve.

ddPCR can quantify BCR-ABL concentrations as low as one copy in 100,000 reference copies, an order of magnitude more sensitive than qPCR, and well below the traditional clinical cutoff for minimal residual disease in CML treatment of 1 in 1,000 reference copies.⁴ Using ddPCR to analyze BCR-ABL transcripts from patients with CML has shown a higher degree of precision than that achieved by using qPCR.¹⁰

Over the past six years, several research groups have begun to develop methodologies using digital PCR to measure BCR-ABL.^10,11,12 Research efforts like these will help pave the way for digital PCR to become a new standard for monitoring CML. To make this happen, though, ddPCR-based tests will still need to gain widespread acceptance in the clinic. Accordingly, several efforts are in place to evaluate the validity of ddPCR in clinical settings. The Life after Stopping TKI study, for example, is a prospective clinical trial that aims to examine the rate of CML recurrence in patients who stop TKI treatment after they have been found to be in remission using qPCR (as defined as <0.01% BCR-ABL transcript concentration for two years).¹³ These patients will concurrently be monitored with ddPCR, allowing the investigators to compare the two methods. This trial, which is the first of its kind, is scheduled to complete in September 2019, and may be followed by more TKI discontinuation studies.

As ddPCR gains clinical acceptance, its sensitivity and reliability will enable physicians to determine BCR-ABL levels in patients’ blood with greater certainty, facilitating smarter decisions regarding a patient’s course of treatment. Subsequently, the tool will allow physicians to more confidently discontinue their patients’ treatments and prevent their cancer diagnoses from following them for the rest of their lives.

References

1. Druker, B et al. Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. New England Journal of Medicine, 2006 355(23), 2408-2417. 10.1056/NEJMoa062867

2. Radich, J. How I monitor residual disease in chronic myeloid leukemia. Blood. 2009 114:3376-3381. doi 10.1182/blood-2009-02-163485.

3.     Morley, A. Quantifying leukemia. New England Journal of Medicine. 1998 339(9):627-9. doi: 10.1056/NEJM199808273390911.

4. Goh, H et al. Sensitive quantitation of minimal residual disease in chronic myeloid leukemia using nanofluidic digital polymerase chain reaction assay. Leukemia & Lymphoma. 2011 52(5), 896-904. doi: 10.3109/10428194.2011.555569

5. Mori, S. et al. Age and dPCR can predict relapse in CML patients who discontinued imatinib: the ISAV study. American Journal of Hematology. 2015 90(10), 910-914. doi: 10.1002/ajh.24120

6. Branford, S et al. Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials. Blood. 2008 112(8), 3330-3338. doi: 10.1182/blood-2008-04-150680

7. Richter, J et al. Musculoskeletal pain in patients with chronic myeloid leukemia after discontinuation of imatinib: a tyrosine kinase inhibitor withdrawal syndrome? Journal of Clinical Oncology, 2014 (25), 2821-2823. doi: 10.1200/JCO.2014.55.6910

8. Side Effects of Tyrosine Kinase Inhibitor (TKI) Therapy [online]. Rye Brook, NY: Leukemia and Lymphoma Society, 2018. Available at: www.lls.org/leukemia/chronic-myeloid-leukemia/treatment/side-effects. Accessed September 14, 2018.

9. Padula, W et al. Cost-effectiveness of Tyrosine Kinase Inhibitor Treatment Strategies for Chronic Myeloid Leukemia in Chronic Phase After Generic Entry of Imatinib in the United States. Journal of the National Cancer Institute. 2016 108(7). Doi: /10.1093/jnci/djw003.

10. Jennings et al. Detection and Quantification of BCR-ABL1 Fusion Transcripts by Droplet Digital PCR. Journal of Molecular Diagnostics. 2014 15(2) 147-140. doi: 10.1016/j.jmoldx.2013.10.007.

11. Wang et al. Droplet digital PCR for BCR/ABL(P210) detection of chronic myeloid leukemia: A high sensitive method of the minimal residual disease and disease progression. European Journal of Haematology. 2018 101(3), 291-296. doi: 10.1111/ejh.13084

12. Maier et al. Optimized digital droplet PCR for BCR-ABL. Journal of Molecular Diagnostics. 2019 21(1), 27-37. doi: 10.1016/j.jmoldx.2018.08.012

13. Atallah, E et al. Design and rationale for the life after stopping tyrosine kinase inhibitors (LAST) study, a prospective, single-group longitudinal study in patients with chronic myeloid leukemia. BMC Cancer, 2018 18(1), 359. doi: 10.1186/s12885-018-4273-1.