The gray area of RQ-PCR-based measurable residual disease: subdividing the “positive, below quantitative range” category

Detection of measurable residual disease (MRD) in hematological malignancies is crucial for prognostication and treatment decisions.Real-time quantitative PCR (RQ-PCR), which targets immunoglobulin (IG) and T-cell receptor (TR) rearrangements, is deemed the gold-standard method for MRD detection in national and international clinical trials.However, this approach does have some limitations, primarily related to standard curve performance and non-specific background amplification.International guidelines and criteria for interpreting IG/TR RQ-PCR-based MRD data, developed by the EuroMRD Consortium and available since 2007 [1], define two key parameters: sensitivity and quantitative range (QR).To ensure reliable and accurate assessment of MRD, sensitivity and QR are determined for each clone-specific RQ-PCR assay using a standard dilution curve of the patient diagnostic sample in leukocytes from healthy donors (socalled "buffy coat" (BC), mimicking the background of polyclonal lymphoid cells in bone marrow samples from patients in complete remission.To minimize the risk of false-positive results, the definition of the sensitivity and QR, as well as the criteria for sample positivity, consider the possible nonspecific background amplification.Additional and more stringent criteria, including reproducibility within replicates and distance from the background, are required to establish the QR and accurately quantify the sample.However, according to these criteria, certain samples may be considered positive, but not quantifiable, also known as "positive below QR" (PBQR).This category of samples poses a challenge for the interpretation of MRD and subsequent clinical evaluation, potentially leading to an incorrect treatment decision.Previous studies have shown that certain PBQR samples may be false positives, caused