BCR-ABL1 IS Quantification: Principles and Techniques of PCR for CML Monitoring

Overview

• Real-time quantitative reverse transcription polymerase chain reaction (RQ-PCR; also written RT-qPCR or qRT-PCR) is the cornerstone of minimal residual disease (MRD) monitoring in chronic myeloid leukaemia (CML).
• It exploits the exquisite sensitivity of PCR amplification to detect and quantify BCR-ABL1 fusion transcripts in peripheral blood or bone marrow with sensitivity several orders of magnitude greater than conventional cytogenetics or fluorescence in situ hybridisation (FISH).
• Standardisation across laboratories worldwide is achieved through the International Scale (IS), which anchors all results to a common baseline and allows clinically meaningful comparison of transcript levels regardless of which laboratory performs the assay.

Understanding the principles underlying PCR technology, including the thermocycling reaction, exponential amplification kinetics, real-time fluorescent detection, normalisation to a reference gene, and conversion to IS values, is essential for the RCPA Haematology Fellowship candidate, both for interpreting clinical results and for understanding the limitations of the technique.

Molecular Biology of BCR-ABL1 Transcripts

Breakpoint Regions and Fusion Transcript Types

The reciprocal translocation t(9;22)(q34.12;q11.23) juxtaposes the ABL1 proto-oncogene from chromosome 9 to the BCR gene on chromosome 22, generating the Philadelphia (Ph) chromosome. Depending on the precise BCR breakpoint location, distinct fusion transcripts are produced:

• Clinical implication of transcript type: Standard qPCR assays are designed to detect e13a2 and e14a2; they will miss e1a2, e19a2, and atypical transcripts (including e13a3/e14a3).
• If the transcript type is not identified at diagnosis and an atypical transcript is present, all subsequent qPCR results will be falsely negative.
• It is therefore mandatory to determine the transcript type at diagnosis, using qualitative multiplex RT-PCR capable of detecting all BCR-ABL1 transcript types, before commencing quantitative monitoring.
• Approximately two-thirds of CML cases co-express the reciprocal ABL1-BCR transcript; this has no proven prognostic significance, but its absence may indicate deletions flanking the translocation breakpoints in BCR, ABL1, or both.
• These deletions were associated with reduced survival in patients treated with IFN-α, but their adverse impact is reduced or abolished by TKI therapy.

Principles of Polymerase Chain Reaction

Basic PCR Mechanism

PCR is an in vitro enzymatic method for exponential amplification of a defined DNA sequence. Each cycle consists of three temperature-dependent steps:

1. Denaturation (~94-96°C): Heat disrupts hydrogen bonds, separating double-stranded DNA (or cDNA) into single strands.
2. Annealing (~50-65°C): Short synthetic oligonucleotide primers complementary to sequences flanking the target region bind to their respective single-stranded templates.
3. Extension (~72°C): A thermostable DNA polymerase (most commonly Taq polymerase, derived from Thermus aquaticus) extends the primers in the 5′→3′ direction using deoxynucleotide triphosphates (dNTPs), synthesising new complementary strands.

After $n$ cycles of amplification, the theoretical yield is $2^n$ copies of the target sequence, producing exponential amplification.

Reverse Transcription, From RNA to cDNA

BCR-ABL1 is detected at the mRNA level because:

• It reflects active transcription in leukaemic cells and is the clinically validated monitoring target.
• mRNA-based assays can detect the fusion even when chromosomal rearrangements are cryptic by karyotype.
• Transcript quantification provides a direct measure of leukaemic cell burden.
• Since PCR amplifies DNA rather than RNA directly, RNA must first be reverse-transcribed into complementary DNA (cDNA) by the enzyme reverse transcriptase (first-strand synthesis).
• The resulting single-stranded cDNA then serves as the PCR template.
• The complete process is termed reverse transcription PCR (RT-PCR).

Real-Time Quantitative PCR (RQ-PCR): Principles

Exponential Kinetics and the Quantification Cycle

• In standard (endpoint) PCR, the reaction is assessed only after a fixed number of cycles, by which time plateau effects and reagent depletion confound quantification.
• RQ-PCR overcomes this by measuring fluorescent signal accumulation in real time during each cycle.

The critical concept is the quantification cycle (Cq), formerly termed Ct (threshold cycle): the cycle number at which fluorescence crosses a defined threshold above background noise. Because amplification is exponential in the early cycles, the Cq is inversely proportional to the starting quantity of target template:

$$\text{Cq} \propto -\log_2(\text{initial template quantity})$$

• A lower Cq indicates greater abundance of starting template (more BCR-ABL1 transcripts, i.e. higher disease burden).
• A difference of approximately 3.32 cycles corresponds to a 10-fold (one log) difference in starting quantity, assuming near-100% amplification efficiency.

Fluorescent Detection Strategies

• For BCR-ABL1 monitoring, TaqMan-based assays are mandated by international recommendations because of their superior specificity.
• The probe hybridises within the BCR-ABL1 junction region, ensuring only the fusion transcript is detected.

Normalisation to a Reference Gene

• Because the total amount of RNA added to each reverse transcription reaction varies between samples (owing to differences in cell count, RNA integrity, and extraction efficiency), raw BCR-ABL1 Cq values alone cannot be compared between time points or laboratories.
• To correct for this variation, an endogenous control (reference) gene is co-amplified in every run.
• This is important because the Cq is a function of the amount of amplifiable target transcript in the reaction volume.

Validated reference genes for CML monitoring (Europe Against Cancer programme):

The ideal reference gene must:

• Show stable expression across samples and disease states
• Have no pseudogenes that could be co-amplified
• Not be susceptible to alternative splicing
• Have expression levels compatible with the assay's dynamic range

The BCR-ABL1 transcript level is expressed as a ratio relative to the reference gene:

$$\text{BCR-ABL1\%} = \frac{\text{BCR-ABL1 copies}}{\text{ABL1 copies}} \times 100\%$$

Copy numbers are derived from Cq values using a standard curve or efficiency-corrected calculation.

The International Scale (IS)

Rationale for Standardisation

• Early CML trials revealed marked inter-laboratory variation in reported BCR-ABL1 levels from identical samples, owing to differences in RNA extraction protocols, cDNA synthesis conditions, primer/probe sequences, PCR platforms, and reference genes.
• This made cross-centre comparison impossible.

The International Scale (IS) harmonises results globally by defining a common zero point:

• IS 100% corresponds to the median BCR-ABL1/ABL1 ratio in newly diagnosed, untreated CML patients enrolled in the IRIS trial (imatinib versus interferon-α), the standardised baseline.
• All results are expressed as BCR-ABL1% IS, enabling direct comparison across laboratories, time points, and TKI trials.

Conversion Factors

Each laboratory must determine its own laboratory-specific conversion factor (CF) by comparing raw ratio results against a certified reference material or by direct comparison with a reference laboratory using split samples:

$$\text{BCR-ABL1\%}^{\text{IS}} = \text{BCR-ABL1\%}^{\text{local}} \times CF$$

• WHO International Genetic Reference Panels and certified plasmid reference materials are available to assist laboratories in establishing and validating conversion factors.
• Laboratories should be accredited and participate regularly in external quality assurance programmes (e.g. the RCPA Quality Assurance Programme for molecular haematology) to confirm CF stability over time.

Molecular Response Milestones on the IS

• Important caveat: A patient in CCyR (0% Ph+ metaphases by conventional cytogenetics) may still harbour up to $10^{10}$ leukaemic cells.
• Even a patient with an undetectable RQ-PCR result may harbour up to $10^6$ leukaemic cells, below the sensitivity threshold of current assays.

Prognostic significance of molecular milestones (IRIS data): CCyR plus MMR at 12 months was associated with 97% progression-free survival at 5 years, compared with 89% for CCyR without MMR.

ELN 2020 Molecular Response Milestones for TKI Therapy

The 3-month BCR-ABL1 IS result is the single most powerful early predictor of long-term outcome in patients on TKI therapy.

Assay Design and Technical Considerations

Duplex vs. Singleplex Reactions

• Most laboratories perform BCR-ABL1 and ABL1 amplifications as separate singleplex reactions (often in duplicate or triplicate).
• High-throughput laboratories may use duplex reactions, where both targets are measured simultaneously in the same well using differentially labelled probes, e.g. 6-FAM for BCR-ABL1 and VIC for ABL1, which emit at distinguishable wavelengths detected by separate instrument channels.
• Duplex assays halve reagent consumption and double throughput but require careful optimisation to avoid signal cross-talk.

Sensitivity and Adequacy Criteria

A well-validated RQ-PCR assay should achieve:

• Detection sensitivity of at least MR4.5 (≤0.0032% IS)
• A dynamic range of ≥4.5 logs
• A minimum of 32,000 ABL1 control gene copies per reaction (to ensure adequate sensitivity when BCR-ABL1 is at very low levels)

When ABL1 copy numbers fall below the minimum adequacy threshold, indicating insufficient RNA quality or input, the result must be reported as inadequate rather than undetectable, to avoid false-negative conclusions.

Blood vs. Bone Marrow

• Peripheral blood is the preferred sample for routine molecular monitoring.
• It is less invasive, practical for 3-monthly testing, and equivalent to bone marrow for BCR-ABL1 detection in patients in complete cytogenetic remission.
• Bone marrow retains advantages for initial diagnosis (karyotype, morphology, blast percentage) and assessment of clonal evolution.
• Note that when peripheral blood lymphocytes predominate, the high percentage of BCR-ABL1-negative lymphoid cells can underrepresent actual residual tumour load compared with bone marrow aspirate, relevant in early treatment or post-transplant settings.

Digital PCR: An Emerging Quantification Method

• Digital PCR (dPCR) partitions the sample into thousands of individual micro-reaction chambers (droplets in droplet digital PCR [ddPCR], or nanowells in chip-based formats).
• Each partition either contains the target template or does not, yielding a binary positive/negative result per partition.

Absolute quantification is achieved by Poisson statistical modelling without a standard curve:

$$\lambda = -\ln(1 - p)$$

where $\lambda$ is the average number of target molecules per partition and $p$ is the proportion of positive partitions.

Digital PCR is an area of active investigation and may complement or eventually supplant RQ-PCR for deep molecular response assessment and treatment-free remission (TFR) monitoring.

Qualitative PCR: Role and Limitations

Qualitative (Endpoint) RT-PCR

Qualitative multiplex RT-PCR is used primarily to:

1. Confirm the BCR-ABL1 fusion transcript type at diagnosis, particularly in Ph-negative patients, and to detect rare/atypical transcripts not covered by standard qPCR
2. Screen for BCR-ABL1 in suspected CML where karyotype and FISH are negative

• Qualitative PCR detects the presence or absence of a fusion transcript but provides no information on transcript quantity or disease kinetics.
• It is therefore insufficient for therapeutic monitoring.

Nested PCR

• Nested PCR uses two sequential rounds of amplification with two primer sets (outer then inner), achieving analytical sensitivity of approximately 1 leukaemic cell in $10^6$.
• However, extreme sensitivity creates a high risk of carryover contamination producing false positives, and the technique is not quantitative.
• It has been largely superseded by RQ-PCR for CML monitoring.

BCR-ABL1 Kinase Domain Mutation Analysis

Rising BCR-ABL1 IS levels may indicate the emergence of BCR-ABL1 kinase domain (KD) mutations conferring TKI resistance. Mutation analysis is indicated when:

• BCR-ABL1 IS rises by ≥1 log from nadir (confirmed on repeat testing)
• Treatment failure criteria are met at any milestone
• Suboptimal (warning) response is noted, especially before a TKI switch

• The T315I "gatekeeper" mutation confers resistance to all first- and second-generation TKIs but retains sensitivity to ponatinib and asciminib (STAMP inhibitor targeting the ABL1 myristoyl pocket).
• Compound mutations identified by NGS, particularly those occurring in cis on the same allele, have direct implications for TKI selection.

Practical Monitoring Schedule and Reporting

Testing frequency:

• At diagnosis: mandatory for transcript type identification and IS baseline
• Every 3 months during TKI therapy until stable MMR is achieved
• Every 3-6 months once sustained MMR is established
• Monthly for the first year and every 6 weeks for the second year after TKI cessation (TFR protocols)

Each report should include:

• BCR-ABL1/ABL1 ratio expressed as BCR-ABL1% IS
• Number of BCR-ABL1 copies and ABL1 control gene copies
• Whether the result is above or below the assay's sensitivity threshold
• Whether ABL1 copy number meets the minimum adequacy threshold
• The laboratory-specific conversion factor applied

Summary of Key Concepts