What CML Can Teach Us About Molecular Diagnostics | by Arthur Wilson | Jan, 2024

Unveiling the intricate world of molecular diagnostics through the lens of leukaemia, discover how the impact of Chronic Myeloid Leukaemia (CML) has, and continues to propel advancements in treatment goals and elevates the demand for precision in Minimal Residual Disease (MRD) detection. In this exploration, the spotlight is on the indispensable role of real-time quantitative polymerase chain reaction (RQ-PCR), shaping the future of cancer management.

CML is a rare blood cancer that affects approximately 1.0–1.5×105 individuals each year, showing no significant differences across races or geographical locations. In the last twenty years, groundbreaking advancements in therapeutic strategies have revolutionised CML treatment, thanks to the impactful contributions of clinical-translational biomedical research. Today, patients undergoing modern treatments are witnessing life expectancies comparable to their age-matched peers.

This transformative progress is evident in the projected surge of CML cases in the United States, expected to rise from 70,000 in 2010 to an estimated 180,000 by 2050, making CML the most prevalent myeloid neoplasm. The key player in CML’s genetic narrative is the Philadelphia chromosome (Ph), a result of a genetic mix-up between chromosomes 9 and 22. This genetic dance gives rise to the Breakpoint Cluster Regions (BCR)/ Abelson murine leukaemia viral oncogene homolog 1 (ABL1) fusion gene, leading to the creation of the BCR/ABL1 protein. This altered protein triggers cascading effects, causing abnormal cellular behaviours, including increased cell expansion and reduced cell death — a hallmark of cancer.

With the advent of tyrosine kinase inhibitors (TKIs), the goals of therapy in CML are shifting. The goal and expectation for most patients has long since centred on long-term disease control on TKI therapy. But more recently, treatment-free remission (TFR) has found a footing in mainstream practice and rapidly gains popularity as the main goal of therapy. This re-tinkering of therapeutic expectations not only impacts the selection of TKIs but also demands improvement and widespread adoption of highly sensitive and accurate molecular monitoring techniques.

Achieving complete cytogenetic remission — a state in which no (or less than 1% of) cells in the bone marrow have the Ph chromosome — often reveals persistent leukemic cells expressing BCR-ABL messenger ribonucleic acid (mRNA). The ability to detect low levels of residual cells, referred to as MRD has been integral to the management of CML for over 30 years, paving the way for molecular diagnostic techniques capable of the necessary sensitivity this requires.

So how has the MRD demands of CML (and malignancies far beyond this) inspired molecular diagnostics?

In the field of diagnostics, qualitative BCR/ABL reverse transcription polymerase chain reaction (RT-PCR) has become a vital tool. This technique is essential for identifying BCR/ABL1 fusion mRNA, especially when the Ph chromosome isn’t visible in routine chromosome exams. To dive deeper, understanding the unique BCRs on chromosome 22 is crucial for pinpointing specific fusion mRNAs and proteins, offering valuable insights into CML’s genetic makeup. While standard RT-PCR may miss rare BCR/ABL1 variants, the adoption of multiplex assays — detecting multiple substances or targets simultaneously — becomes vital for a thorough and comprehensive testing approach, ensuring a more accurate and detailed assessment of CML.

Moving to the treatment phase, understanding how well treatments work has long been a challenge using qualitative PCR. Here, BCR-ABL1 messenger RNA expression measurements, facilitated through quantitative RQ-PCR, play a key role.

Unlike qualitative PCR, providing a simple yes/no signal, quantitative BCR-ABL RQ-PCR goes a step further by measuring the amount of BCR-ABL mRNA in a patient’s system. This real-time monitoring provides a dynamic view of treatment response. The importance of RQ-PCR lies in its precise measurements, enabling the early identification of patients needing intervention well before signs of relapse appear. Essentially, RQ-PCR is a crucial tool, offering an accurate and timely assessment of the treatment’s effectiveness.

Reported according to the International Scale, RQ-PCR has evolved into the primary tool for evaluating responses in CML. By hitting specific molecular goals within set timeframes, as outlined by global guidelines, it greatly boosts the chances of successful outcomes while helping identify patients at risk of treatment not working well. The depth and duration of a profound molecular response have become key goals, especially for those eyeing TFR. This molecular monitoring ensures a constant check on patient responses, swiftly identifying those not responding well to medication or facing challenges in sticking to their treatment plan.

This solidifies RT-qPCR’s role as the top choice for monitoring CML patients over the decades. Whether as custom tests from labs or through reputable PCR kit suppliers, these comprehensive kits assess the depth of clinical response to TKIs or identify early relapse after stem cell transplantation. As the BCR-ABL fusion gene is expressed in over 95% of patients with CML, and RQ-PCR provides a reliable, high throughput method to accurately assess the level of treatment response and provides an early indication of emerging drug resistance.

The precision of measuring BCR-ABL1 not only predicts outcomes cost-effectively but is essential for achieving TFR goals. RQ-PCR not only sets the global standard for detecting BCR-ABL transcripts but also empowers patients to leverage the growing array of targeted therapies flooding the market. It serves as a crucial tool in managing treatment effectively, offering patients the opportunity to navigate and benefit from the advancements in targeted therapies that are transforming the landscape of medical care.

While RQ-PCR is globally acknowledged as the benchmark for detecting BCR-ABL transcripts, digital droplet PCR (ddPCR) is a recent entrant. Possessing the ability to markedly enhance detection sensitivity while remaining impervious to amplification differences, ddPCR represents a recent advancement in molecular diagnostic techniques. Although ddPCR technology has not been widely promoted internationally, it has been used to explore TKI discontinuation owing to its high sensitivity in detecting BCR-ABL1 transcripts — yet its difficulty to implement on a large-scale renders RQ-PCR the current king of disease monitoring detection.

Nonetheless, the expanding body of evidence showcasing the efficacy of ddPCR technology in minimal residual disease (MRD) monitoring is paving the way for its broader adoption in clinical research. The accumulating support for ddPCR suggests a future where its use becomes more prevalent, holding the promise of birthing a new era of personalised and highly effective cancer treatments. This anticipated shift has the potential to significantly enhance outcomes for individuals grappling with this formidable disease, offering a glimmer of hope in the pursuit of improved therapeutic strategies.

The journey through the intricate landscape of molecular diagnostics, illuminated by the lens of CML, underscores the pivotal role of RQ-PCR in shaping the trajectory of cancer management. The evolving treatment goals, propelled by the success of TKIs and the emerging prominence of TFR, have redefined expectations in CML therapy. With the ability to detect MRD and assess treatment response with unparalleled precision, RQ-PCR stands as the gold standard for sequential monitoring in CML patients, facilitating timely interventions and optimising therapeutic outcomes. While ddPCR emerges as a promising contender, the widespread adoption of this technology hints at a future characterised by personalised and highly effective cancer treatments, offering a glimmer of hope for those grappling with the challenges of CML.

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