Sensitive Replicate Real-Time Quantitative PCR of BCR-ABL Shows Deep Molecular Responses in Long-Term Post–Allogeneic Stem Cell Transplantation Chronic Myeloid Leukemia Patients

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Standardization of Two Dpcr Platforms for Detection of BCR/ABL1 - Minimal Residual Disease (MRD) in Ph+ Chronic Myeloid Leukemia (CML)

Background: despite the international efforts on standardization, qPCR has some intrinsic limitations of accuracy and sensitivity that make it suboptimal to assess lower levels of BCR-ABL1. In recent years, digital PCR (dPCR) has emerged to provide a more sensitive and accurate detection of minimal residual disease (MRD), that accounts for the increasing interest for its use in the clinic. The Qx100/Qx200 Droplet Digital PCR System (ddPCR, Biorad) and Quant Studio 3D dPCR System (Thermofisher) are the most widespread platforms commonly used. However nowadays internationally recognized operational protocols are not available. Aim: Aim of our study was: to assess the consistency of the results between two different laboratories that relied on two different platforms; to verify and validate the possible existence of a conversion factor (CF) between the results obtained in the two different laboratories. Material and methods: 16 RNA pools were prepared at different levels of disease (4 pools for 10% level, 4 pools for 1% level, 4 pools for 0.1% level, 4 pools for 0.01% level). dPCR analysis were performed and repeated in 4 separate sessions (hereafter, Exp1, Exp2, Exp3 and Exp4) from the 2 involved laboratories. ddPCR analysis were performed in triplicate for BCR-ABL1 using 200 ng of cDNA for each replicate and in duplicate for ABL1 using 100 ng of cDNA for each replicate; the QuantStudio 3D dPCR analysis were performed in duplicate for both genes using 50 ng of cDNA for each replicate of BCR-ABL1 and 25 ng of cDNA for each replicate of ABL1. Results: In order to assess the consistency of the results between the two different labs we first analyzed multiple replicates of 8/16 samples (2 samples of each disease level were selected) - Exp 1: 10 replicates by ddPCR and 6 replicates by Quant Studio 3D dPCR were performed. Results of each single replicate were expressed as BCR-ABL1/ABL1%. We assessed the compatibility between the values obtained in the two laboratories by linear regression and we obtained R2 = 0.9869. In order to confirm the compatibility we compared the measures through the Bland-Altman bias-plot and we carried out the Mann-Whitney nonparametric test. The results of these 2 tests confirmed the compatibility of the different measures thus we computed the conversion factor (CF) value as the antilog of the average of the differences and obtained a CF value of 1.41.[IC 95% 1.3597- 1.4661]. The mean of the differences before the conversion was 0.1498 (sd 0.1723) and after conversion became zero. The obtained CF value was then validated on different series of experiments, i.e. Exp2, Exp3, Exp4 and it proved to be very satisfactory. For each experiment we used the fold difference to compare the results before and after conversion. We also computed the 95% confidence interval (CI) for the average difference before and after conversion. We saw that the average differences after conversion were closer to 1 with a narrowing of the width of the data distribution. Finally we computed the percentage of measures before and after conversion which were included in a 2-fold range, in a 3-fold range and in a 5-fold range. We observed that the conversion led to a general improvement of the percentage of measures included in the 2-fold-range (73% in Exp2; 88% in Exp 3; 95% in Exp 4). Conclusion: In our study we showed that it is possible to obtainequivalent results from different dPCR platforms by the introduction of a conversion factor. This consistency of the data is mandatory in order to introduce this new technology beside qPCR in CML MRD monitoring

Evaluation of Cepheid Xpert® BCR-ABL Monitor Assay in Three Italian Reference Centers for Monitoring of BCR-ABL Transcript Levels in CML Patients

Effective management of tyrosine kinase inhibitor (TKI) therapy for patients with CML requires frequent patient testing to monitor patient disease status. The gold standard for this testing is a PCR measurement of the BCR-ABL/ABL transcript ratio. Hence, standardized, accurate and reproducible molecular analyses are fundamental for clinicians in order to correctly address therapeutic decision and to achieve a better patient management. Based on these clinical needs, it is widely recognized that inter-laboratory reproducibility is a crucial issue that requires standardization and strict alignment of BCR-ABL values on the international scale (IS), as established by the International Randomized Study of Interferon and STI571 (IRIS) laboratories. In addition to this, another important aspect in terms of patient management is the availability of a rapid response, improving patient quality of life by reducing anxiety linked to delay in results delivery. Xpert® BCR-ABL Monitor, developed and manufactured by Cepheid (Sunnyvale, CA), is a cartridge-based automated assay for quantification of BCR-ABL1 mRNA, reporting results in International Scale (IS). Alignment to the IS, based upon the quality control standards derived from the WHO BCR-ABL Standards, is performed lot to lot automatically by the software of the Cepheid GeneXpert® DX Instrument. Xpert® BCR-ABL Monitor automates and integrates sample processing, nucleic acids extraction and amplification and target sequence detection in peripheral blood specimens collected either in EDTA or PAXgene tubes using real-time RT-PCR. The cartridge includes reagents to detect BCR-ABL fusion genes resulting from two major breakpoints, translocation e13a2 (b2a2) and e14a2 (b3a2) and the ABL transcript as an endogenous control. In the following study, Xpert® BCR-ABL Monitor was independently evaluated in the three Italian reference centers for BCR-ABL mRNA monitoring of the LabNet project. A total of 150 peripheral blood samples from CML patients, equally divided between centers, were analyzed both with the standard laboratory method and with Xpert® BCR-ABL Monitor Assay, in order to compare the results expressed in International Scale. BCR-ABL mRNA of the specimens covered a broad IS range, allowing to compare data also at low levels of disease (MR 4.5, 0.00316% BCR-ABL/ABL). Overall, linear regression demonstrated that there was a good correlation between Xpert® BCR-ABL Monitor and reference centers data (R2= 0.92). Correlation slightly increased when the data produced with Xpert® BCR-ABL Monitor were refined using a correction tool not automatically adopted by the current version of the assay software (R2= 0.93). Moreover, using assay comparison criteria proposed by Müller et al. (Leukemia 2009), Xpert® BCR-ABL Monitor was considered comparable to the standard laboratory methods of the reference centers, considering that all the three acceptance criteria were satisfied (58% of the samples between 0.5 and 2-fold variation, 78% of the samples between 0.33 and 3-fold variation, 91% of the samples between 0.2 and 5-fold variation). In conclusion, Xpert® BCR-ABL Monitor demonstrated to be a rapid, reliable and accurate test for monitoring of BCR-ABL mRNA transcript levels. Results were available within approximately 2 hours, potentially allowing clinicians to report results to patient the same day of the visit. From the technical point of view, cartridge-based automated system significantly reduced operator hands-on time from several hours to approximately 15 minutes. Concerning the assay comparative performance, Xpert® BCR-ABL showed to have a good inter-laboratory reproducibility, with no significant differences between the three reference centers standard methods