Digital PCR improves the quantitation of DMR and the selection of CML candidates to TKIs discontinuation

Abstract

Treatment‐free remission (TFR) by tyrosine kinase inhibitors (TKI) discontinuation in patients with deep molecular response (DMR) is a paramount goal in the current chronic myeloid leukemia (CML) therapeutic strategy. The best DMR level by real‐time quantitative PCR (RT‐qPCR) for TKI discontinuation is still a matter of debate. To compare the accuracy of digital PCR (dPCR) and RT‐qPCR for BCR‐ABL1 transcript levels detection, 142 CML patients were monitored for a median time of 24 months. Digital PCR detected BCR‐ABL1 transcripts in the RT‐qPCR undetectable cases. The dPCR analysis of the samples, grouped by the MR classes, revealed a significant difference between MR 4.0 and MR 4.5 ( P = 0.0104) or MR 5.0 ( P = 0.0032). The clinical and hematological characteristics of the patients grouped according to DMR classes (MR 4.0 vs MR 4.5‐5.0 ) were superimposable. Conversely, patients with dPCR values <0.468 BCR‐ABL1 copies/µL (as we previously described) showed a longer DMR duration ( P = 0.0220) and mainly belonged to MR 4.5‐5.0 ( P = 0.0442) classes compared to patients with higher dPCR values. Among the 142 patients, 111 (78%) discontinued the TKI treatment; among the 111 patients, 24 (22%) lost the MR 3.0 or MR 4.0 . RT‐qPCR was not able to discriminate patients with higher risk of MR loss after discontinuation ( P = 0.8100). On the contrary, according to dPCR, 12/25 (48%) patients with BCR‐ABL1 values ≥0.468 and 12/86 (14%) patients with BCR‐ABL1 values <0.468 lost DMR in this cohort, respectively ( P = 0.0003). Treatment‐free remission of patients who discontinued TKI with a dPCR <0.468 was significantly higher compared to patients with dPCR ≥ 0.468 (TFR at 2 years 83% vs 52% P = 0.0017, respectively). In conclusion, dPCR resulted in an improved recognition of stable DMR and of candidates to TKI discontinuation.

Feasibility of tumor‑derived exosome enrichment in the onco‑hematology leukemic model of chronic myeloid leukemia

“…Supported by the above-mentioned considerations, cML was identified as the most suitable hematological neoplasia for a feasibility study on neoplastic exosome enrichment. The present study indicates the results of an explorative feasibility study based on the quantification of the BCR-ABL1 transcript by digital PCR (dPCR), a more specific and accurate tool for the detection of the MRd in patients with cML (45,46), on a tumor-derived exosome fraction enriched in the PB of patients in the cP, who are also receiving TKI treatment.…”

“…The most promising of these methods for a deep molecular characterization of intra-tumor cell communication encompass the immune-selective enrichments of tumor antigens (15,18). These tools, which act synergistically with innovative molecular biology techniques such as next generation sequencing and dPcR, may improve the accuracy and sensitivity of exosomes analysis (45,46). In this context, the cML model is considered to be particularly suitable, given the presence of BcR-ABL1 as a hallmark indicator of leukemic cells, and the established use of RT-qPcR to monitor MRd.…”

“…To the best of our knowledge, the present study is the first to investigate the possibility of isolating leukemia-derived exosomes from patients with cML, using a commercial kit developed for solid tumors, and based on a proprietary antibody affinity method that selectively enriches tumor-derived exosomes (8). The evaluation of enrichment specificity was determined by BCR-ABL1 transcript detection using dPcR, since it has been demonstrated to be more sensitive and robust than conventional approaches for BCR-ABL1 quantification in cML cases (45,46).…”

“…The quantification values of BCR-ABL1 and ABL1 performed by the Reference Laboratory are presented in Table III. dPCR analysis. Since dPcR has been demonstrated to be more accurate and sensitive than conventional RT-qPcR for the quantification of the MRD in patients with CML (by detecting the BCR-ABL1 transcript), in order to compare the results obtained from the tumor exosomes, BCR-ABL1 dPcR analysis was first performed on cDNA from PB cells, as previously described (45,46). To reduce bias, the cdNA samples were the same as those used during the RT-qPcR analysis.…”

Chitosan-Hydrogel Polymeric Scaffold Acts as an Independent Primary Inducer of Osteogenic Differentiation in Human Mesenchymal Stromal Cells

“…GAPDH, the reference gene, had between 8000 and 8500 number of dots/reaction in all the analyzed samples. Therefore, considering the robustness of the quantification and the previously reported feasibility of dPCR absolute quantification [35], only the marker transcript quantification was considered and statistically analyzed, and no normalization was performed.…”