Building a better mousetrap for accurate and sensitive polymerase chain reaction

Abstract

The natural history of chronic myeloid leukaemia (CML) is forever changed with the advent of tyrosine kinase inhibitor (TKI) therapy. All patients with CML have the unique BCR– ABL1 fusion gene, and the protein product of that gene is the target of the TKI, while the mRNA fusion provides the target for disease monitoring in the peripheral blood. Quantitative reverse transcriptase polymerase chain reaction (qRTPCR) of BCR–ABL1 mRNA is a sensitive method for monitoring CML, yet qRT-PCR assay variation from lab to lab can be considerable. A large international effort of CML researchers developed and validated the International Standard scale (IS) for BCR–ABL1 as a way to facilitate lab to lab comparisons. Still the IS is just a correction factor, and no magical powers. Applying it to a less than accurate BCR– ABL1 value just gives you a different less than accurate BCR– ABL1 value. The measurement of disease burden via BCR–ABL1 transcript levels has substantial clinical utility. The decline of disease after the start of TKI therapy is associated with a very favourable long-term response. Conversely, patients who have BCR–ABL1 >10% IS after three months of treatment have a worse long-term response compared to cases with a BCR–ABL1 of less than 10% IS. Secondly, patients who achieve a BCR–ABL1 level of <0 1% IS (MR3) enjoy a ‘safe harbour’ where relapse or progression is quite rare. Finally, patients who achieve a stable deep molecular response (MR4 or below) can potentially discontinue TKI therapy. Many trials have shown that ~40–60% of patients who discontinue their TKI after a prolonged deep response can enjoy years of treatment-free remission (TFR). Naturally, discontinuation must be accompanied by frequently monitoring for disease recurrence. So important is BCR–ABL1 testing to the management of CML that the European Leukemia Network (ELN) and the National Comprehensive Cancer Network and developed guidelines for BCR–ABL1 monitoring over the entire course of CML therapy and discontinuation. A good qRT-PCR assay can detect the BCR–ABL1 transcript in a background of ~100 000 total mRNA transcripts. The fact that this can be done with some precision and accuracy is rather amazing. Consider: the problem of detection of any target by PCR is the issue of signal (BCR–ABL1) to noise (all other mRNA). This is often likened to finding the needle in the haystack. Conventional qRT-PCR requires the exponential amplification of the target and a separate exponential amplification of the control gene. If the amplification of these two targets differs, the calculations will not be inaccurate. ‘Digital’ PCR (dPCR) is a methodological approach to improve the signal/noise limitation. The concept is essentially to divide the sample into an excess of partitions, so that each partition houses only a few copies of either the target (signal), or the background (noise). Digitalization can be done by distributing samples into an array of thousands of tubes and wells (Fluidigm), or into droplets (first Raindance, and now Bio-Rad). Going back to the needle/haystack analogy, each well/droplet could either be empty, contain a piece of hay, or a needle. Amplification yields a positive signal with target amplification (digital code = 1), and no amplification in partitions without the target (digital code = 0). The Poisson distribution is then used to estimate the initial input target BCR–ABL1 copy numbers. There are several fundamental characteristics of dPCR which makes it attractive on a technical level. First, one measures actual molecules (positive droplets) rather than estimating amount of starting template by amplification slope and standard curve samples. Secondly, since it uses endpoint determination of signal (yes or no), it is potentially less sensitive to elements that can influence amplification efficiency, such as sample contaminants, poor-quality mRNA, etc. Third, dPCR allows the sampling of a large number of wells/droplets, which should improve the precision and sensitivity of the assay. These qualities suggest the dPCR assay Correspondence: Jerry Radich, Clinical Research Division, Departments of Oncology and Haematology, University of Washingon, Seattle, WA 98075, USA. E-mail: [email protected] commentary