Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR1–MR4), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR4.5 level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR4.5 sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.
Objective
Molecular monitoring of treatment response in patients with chronic myelogenous leukemia is performed using the Europe Against Cancer (EAC) qPCR assay using the International Scale (IS). The assay amplifies both e13a2 and e14a2 BCR‐ABL1 transcript variants. Observing distinct variant‐dependent amplification curves during qPCR, we aimed to determine if this affected quantitation of BCR‐ABL1.
Methods
We investigated the qPCR efficiency at three Danish diagnostic centers (Zealand University Hospital [ZUH], Aarhus University Hospital [AU], and Rigshospitalet [RH]) on cell lines expressing either the e13a2 or e14a2 BCR‐ABL1 transcript variants and compared %IS values from 219 chronic myeloid leukemia patients from the centers with either the e13a2 (n = 113) or e14a2 (n = 106) transcript variants obtained by qPCR with absolute quantitation by droplet digital PCR (ddPCR).
Results
Although no significant differences were found in amplification efficiencies of the transcript variants, Bland‐Altman analysis of qPCR vs ddPCR values for patient samples revealed a significant average difference in the bias between variants (e3a2/e14a2) of 4.6‐, 6.5‐, and 1.8‐fold for ZUH, AU, and RH, respectively. Furthermore, qPCR %IS values of diagnostic patient samples revealed a significant 4.7‐fold difference between the e13a2 and e14a2 variants.
Conclusion
Our findings suggest that the EAC qPCR assay may underestimate the e14a2 variant compared to the e13a2 variant.
Point mutations of bcr-abl tyrosine kinase are the most frequent causes of imatinib resistance in chronic myeloid leukaemia (CML) patients. In most CML cases with BCR-ABL mutations leading to imatinib resistance the second generation of tyrosine kinase inhibitors (TKI- e.g. nilotinib or dasatinib) may be effective. Here, we report a case of a CML patient who during imatinib treatment did not obtain clinical and cytogenetic response within 12 months of therapy. The sequencing of BCR-ABL kinase domains was performed and revealed the presence of a F359I point mutation (TTC-to-ATC nucleotide change leading to Phe-to-Ile amino acid substitution). After 1 month of nilotinib therapy a rapid progression of clinical symptoms was observed. In the presence of the F359I point mutation only dasatinib treatment overcame imatinib and nilotinib resistance.
In our opinion, a detailed evaluation and appropriate interpretation of clinical and laboratory data in such a category of patients seem to be extremely important, especially when a decision about the TKI change due to therapy failure is considered.
Standardized monitoring of BCR::ABL1 mRNA levels is essential for the management of chronic myeloid leukemia (CML) patients. From 2016 to 2021 the European Treatment and Outcome Study for CML (EUTOS) explored the use of secondary, lyophilized cell-based BCR::ABL1 reference panels traceable to the World Health Organization primary reference material to standardize and validate local laboratory tests. Panels were used to assign and validate conversion factors (CFs) to the International Scale and assess the ability of laboratories to assess deep molecular response (DMR). The study also explored aspects of internal quality control. The percentage of EUTOS reference laboratories (n = 50) with CFs validated as optimal or satisfactory increased from 67.5% to 97.6% and 36.4% to 91.7% for ABL1 and GUSB, respectively, during the study period and 98% of laboratories were able to detect MR4.5 in most samples. Laboratories with unvalidated CFs had a higher coefficient of variation for BCR::ABL1IS and some laboratories had a limit of blank greater than zero which could affect the accurate reporting of DMR. Our study indicates that secondary reference panels can be used effectively to obtain and validate CFs in a manner equivalent to sample exchange and can also be used to monitor additional aspects of quality assurance.
IntroductionAllogeneic hematopoietic cell transplantation (HCT) was a standard therapy in chronic phase (CP) chronic myeloid leukemia (CML). As a result of the effective therapy with tyrosine kinase inhibitors (TKI), HCT was shifted to defined clinical situations. We present the results of observational prospective analysis of 28 CML patients undergoing HCT after exposure to, at least, two lines of TKI (including dasatinib and/or nilotinib), with respect to response, overall survival (OS), treatment toxicity, graft versus host disease (GVHD), and progression/relapse incidence.ResultsAll the patients but one engrafted with median time 19 days. OS for patients in CP1 and CP2/accelerated phase (AcP) were 92.9 and 85.7 %, respectively. Six patients allotransplanted in blast crisis (BC) CML died early after HCT.Eighteen patients achieved deep molecular remission (MR4.5 or MR4.0). Relapse incidence was 29.6 %. Median time to progression (TTP) differs significantly depending on the CML phase prior to HCT, the best response achieved after HCT and development of chronic GvHD.NRM yielded the values 7.1, 12.5, and 50 % in CP1, CP2/AcP, and BC, respectively. Fatal outcome, due to veno-occlusive disease (VOD), was observed in two (7 %) patients. In five (17.9 %) patients, mild or moderate VOD was observed with no negative impact of preceding therapy with TKI2. Acute GvHD was diagnosed in 25.9 % of patients, while chronic GvHD developed in 42.9 % of individuals.ConclusionPretransplantation therapy with TKI2 in CP CML is safe and reasonable. In BC, the optimal approach before HCT is to reduce the leukemic burden and achieve subsequent CP.
Development of modern proteomic methods in recent years has opened also new perspectives in the identification of new biomarkers which ensure more effective diagnosis, treatment monitoring and prediction of therapeutic outcome. We evaluated usefulness of comparative proteomics (MALDI-TOF) in two subtypes of acute myeloid leukemia (AML), M1 and M2, according to FAB classification. The bone marrow or blood cell proteomes were examined in 33 newly diagnosed patients before “3 + 7” induction therapy, after treatment and when the disease relapsed. We found that bone marrow and peripheral mononuclear cells from healthy volunteers revealed a number of quantitative and qualitative differences between the two proteomes, reflecting differences in the maturational status of normal cells. Such differences were not detected in our AML M1/M2 patients. Additionally, we found 9 proteins, which are potential biomarkers differentiating between the AML patients and healthy volunteers. Using comparative proteomics, we found that annexin I, glutathione transferase omega, esterase D and gamma 1 actin had prognostic significance. Applying statistical methods, we detected two proteins which might allow to predict results of induction therapy in AML M1/M2. One of them was esterase D, the higher concentration of which was associated with higher complete remission rate, and the other was gamma 1 actin, the higher concentration of which was related to resistance. In the article, we also discussed the role of these two proteins in the biology of AML, and we suggested potential usefulness of modification in induction therapy reflecting the presence of proteins.
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