BCR::ABL1 Molecular Monitoring in CML: Understanding the International Scale, Molecular Response and Digital PCR

But a routine monitoring result is more than a single number. Between the analytical measurement and the clinical report sits a chain of standardization decisions: which transcript types the assay covers, which reference gene is measured alongside BCR::ABL1, how the result is aligned to the International Scale (IS), and what level of molecular response the complete workflow can reliably support.

This article therefore focuses on a central laboratory question:

How does a laboratory convert an analytical BCR::ABL1 measurement into a standardized result that can be interpreted within an internationally recognized molecular-response framework?

Understanding this conversion — and its limits — matters for any laboratory implementing or evaluating BCR::ABL1 molecular monitoring, regardless of the measurement technology it uses.

Molecular monitoring is the longitudinal measurement of BCR::ABL1 transcript levels. Rather than a single qualitative detection result, monitoring measures how BCR::ABL1 levels change across sequential specimens, most commonly peripheral blood.

The value lies not simply in detecting BCR::ABL1, but in measuring change over time within an appropriate standardized framework. A single result provides information at one time point; the trend across standardized sequential measurements is what gives molecular monitoring its clinical meaning.

BCR::ABL1^IS means a BCR::ABL1 result expressed on the International Scale. For patients expressing the standard major transcripts (e13a2 and/or e14a2), results are typically expressed as a ratio of BCR::ABL1 to an appropriate reference gene, aligned to the International Scale.

Appropriate reference genes include ABL1, BCR and GUSB, depending on the validated methodology. Current European LeukemiaNet laboratory guidance notes that any of these three is acceptable for routine analysis, that ABL1 is used in the great majority of testing laboratories, and that other reference genes are not recommended because they have not been calibrated to the International Scale. Laboratories do not all use the same reference gene, and the reference gene is part of the validated method, not an interchangeable detail.

Why does the International Scale exist? Historically, differences in assay design, reference genes, calibration and reporting methods could produce meaningful variation between laboratories measuring the same patient material. The International Scale provides a harmonized framework anchored to a standardized baseline, so that molecular-response categories have consistent meaning across laboratories, methods and time.

Molecular response is described using a logarithmic framework relative to the standardized baseline. The currently accepted approximate International Scale relationships are:

MMR means Major Molecular Response. MR4 and deeper are generally considered deep molecular response (DMR) territory.

Two cautions belong alongside this table. First, molecular-response levels are not a simple linear clinical “score” — a deeper numerical category is not automatically clinically superior, and reaching a deeper category does not by itself determine treatment decisions. Second, classifying a sample to a deep response level is only valid when the assay’s analytical sensitivity and the sample’s evaluable material support that depth of measurement, as discussed below.

No.

A result in which BCR::ABL1 is not detected means that BCR::ABL1 was not detected within the analytical sensitivity achieved for that specimen and assay. It does not prove that no BCR::ABL1-positive cells exist anywhere in the patient.

For this reason, laboratory terminology matters. Phrases such as “complete molecular response” can imply an absolute absence of disease that molecular testing cannot establish. Current guidance favors more precise language for results below detection.

Similarly, reporting “0% BCR::ABL1 IS” is not automatically meaningful without analytical context. What carries meaning is the detection limit achieved for that specific measurement, which depends on the assay’s sensitivity and on the amount and quality of evaluable material in the sample.

Deep molecular-response interpretation requires evidence that the sample contained sufficient evaluable material. This is where the reference gene earns its name: the reference-gene measurement quantifies the evaluable material in the same cDNA volume used to test for BCR::ABL1, and thereby establishes the sensitivity achieved for that sample.

Current European LeukemiaNet laboratory guidance defines this explicitly. To score deep molecular response, the guidance specifies minimum sums of reference-gene transcripts measured in the same volume of cDNA — approximately 10,000 ABL1 transcripts for MR4, 32,000 for MR4.5 and 100,000 for MR5 (with correspondingly higher GUSB requirements). As a practical illustration, the guidance notes that undetected BCR::ABL1 with around 45,000 ABL1 transcripts indicates MR4.5 for that sample, whereas undetected BCR::ABL1 with fewer than 10,000 ABL1 transcripts is not evaluable for deep molecular response and should be treated as a technical failure.

The principle is simple: an undetected result is only as deep as the reference-gene measurement that supports it.

RT-qPCR remains the most commonly used method for routine BCR::ABL1 molecular monitoring. Its strengths are well established:

• established clinical use over many years of CML management;
• extensive standardization experience, including International Scale alignment;
• quantitative longitudinal measurement across a wide disease range;
• International Scale reporting through validated approaches (laboratory-specific conversion factors, calibrated reference materials or validated systems);
• mature laboratory workflows and broad availability.

None of this makes RT-qPCR obsolete, and none of it is a limitation. Standard-curve-based calibration is not a weakness when appropriately validated — as discussed in our article on digital PCR versus qPCR, qPCR can provide absolute quantification when appropriate calibration strategies are used. In BCR::ABL1 monitoring specifically, the clinical question is not whether a method uses a standard curve, but whether the complete measurement pathway is validated and traceable.

Reverse transcription digital PCR (RT-dPCR), including droplet digital PCR (RT-ddPCR), measures BCR::ABL1 through a different analytical route.

The concept is straightforward: the reaction is divided into a large number of partitions. Following amplification and endpoint classification of each partition, the target concentration is estimated statistically from the positive and negative partition outcomes. This can enable absolute quantification without reliance on a conventional external calibration curve for the core concentration estimate.

Current European LeukemiaNet laboratory guidance recognizes RT-dPCR as an acceptable alternative to RT-qPCR for molecular monitoring, noting that it may offer technical advantages in some circumstances, particularly for the assessment of very low levels of disease.

Potential advantages may include:

• partition-based quantification;
• performance at very low target levels;
• reduced dependence on conventional calibration curves for the analytical concentration estimate;
• potential utility in deep molecular-response measurement.

But this must be stated just as clearly: digital PCR is not automatically superior to RT-qPCR. Clinical implementation still requires appropriate validation, quality control, transcript coverage, a reference-gene strategy and International Scale alignment where IS reporting is intended. A dPCR concentration is an analytical result, not a clinical classification.

Absolute quantification and clinical standardization answer different questions.

Absolute quantification addresses: “How much target was measured by this analytical system?”

International Scale standardization addresses: “How does the BCR::ABL1 result relate to the internationally harmonized molecular-response framework?”

Therefore:

Absolute quantification ≠ International Scale standardization, and dPCR ≠ automatic BCR::ABL1 IS.

A dPCR assay may provide an absolute estimate of BCR::ABL1 transcript concentration in the analyzed material. That number still has to be related to the reference gene, aligned to the International Scale and validated as part of the monitoring pathway before it can be reported as a molecular-response level. Per current ELN laboratory guidance, IS conversion applies equally to results from RT-qPCR and RT-dPCR: laboratories either derive the IS by applying a laboratory-specific conversion factor to the raw BCR::ABL1/reference-gene ratio, or use a validated kit or system that reports directly on the IS.

How alignment is achieved may involve validated conversion factors, calibrated reference materials or appropriately validated standardized systems, depending on the methodology. No single calibration approach is universally required — but some validated route to the IS is required, whatever the measurement technology.

International reference materials and standardized reporting were developed because inter-laboratory variation had real consequences. Historically relevant sources of variation included:

• assay design;
• reference-gene choice and measurement;
• calibration approach;
• analytical workflow;
• reporting methods.

The first World Health Organization International Genetic Reference Panel for the quantitation of BCR::ABL1 mRNA (NIBSC 09/138) provides a higher-order reference for calibration. Together with secondary reference materials, validated conversion procedures and appropriately calibrated systems, this supports traceability and harmonization of BCR::ABL1 measurement.

This should not be oversimplified. WHO reference materials do not make every assay identical. What reference materials and validated calibration strategies do is help laboratories align their measurements to a common reporting framework, so that a BCR::ABL1 IS value carries consistent meaning regardless of where or how the underlying measurement was performed.

Most CML patients express the standard major BCR::ABL1 transcript types, commonly described as e13a2 and/or e14a2. Current ELN laboratory guidance indicates that these account for the great majority of CML cases — approximately 98% — with e14a2 the more common of the two.

Atypical BCR::ABL1 transcripts can require different molecular-monitoring approaches. An assay designed for standard transcript types will not automatically detect or quantify atypical fusions, and a patient with an atypical transcript should not automatically be monitored using such an assay. Current guidance recommends that patients expressing atypical fusions are monitored using bespoke validated assays, with results expressed as individual molecular responses relative to baseline — rather than on the International Scale, which is calibrated for the standard transcripts.

At the implementation level, this means transcript coverage is a first-order design decision for a monitoring service, not a footnote.

One of the fundamental purposes of BCR::ABL1 monitoring is following molecular response over time. A single result provides information at one time point. Sequential standardized measurements provide the longitudinal context in which trends, stability and change become visible.

This is also why standardization and longitudinal comparability are inseparable: only results produced within a consistently validated measurement framework can be meaningfully compared across months and years.

Accurate measurement of molecular response supports clinical monitoring, and reliable deep molecular-response assessment can be clinically important. How molecular results inform treatment decisions — including any change or discontinuation of therapy — sits with the treating clinical team and applicable clinical guidance, outside the scope of this laboratory article.