3A). To mitigate noise effects, the median of the three replicates was selected as the final ratio value. Design of triplicates was conducted bioinformatically with an in-house workflow designed in R and Bash (Fig. 3A, bottom). Triplicates were distinguished between them by adding custom tags at the beginning of the primers. Each of these primers generated three types of reads of the same region after PCR amplification and sequencing that we classified as P1, P2 and P3.
5 times the standard deviation, and LOQ was computed as the mean ratio in control samples plus 10 times the standard deviation. These parameters were automatically added to the final output of our pipeline, and every hotspot. Statistical analysesThe Pearson correlation coefficient was computed to assess the linear relationship between the different variables under study. Univariable Cox proportional hazard regression models and Kaplan–Meier survival analysis were employed to test statistical associations between genetic findings and survival outcomes. The detection of resistant mutations was defined as a time dependent covariate in the Cox proportional hazard regression analysis. This test was employed to assess statistical associations between genetic findings and survival outcomes. Statistical calculations were performed using SPSS 22. 0 (SPSS Inc, Chicago). P values ≤ 0.
05 were considered significant. Data availability The raw sequencing data were uploaded to NCBI with BioProject ID: PRJNA813136 and can be found at the following link: https://www. ncbi. nlm. nih. gov/bioproject/? term=813136. ReferencesO’Brien, S. G. et al. Imatinib compared with interferon and low-dose cytarabine for newly diagnosed chronic-phase chronic myeloid leukemia. N.
This frequency increased when other more sensitive methods have been used21, 22. Some of our patients receive asciminib as compassionate use since it has been reported to be active against the most prevalent p. T315I and other double mutations and could be an exciting option for multi-resistant patients in combination with ponatinib23. Our results also confirmed that most of the mutations below 20% VAF are missed by SS.
2D). Most of the mutations observed were in failure and relapse stages for CML and B-ALL patients, respectively, as previously reported11, 19. Our DNA-DeepNGS method was able to detect mutations with a strong reproducibility using a threshold of VAF of 1. 0Eв€’4, based on the comparison with RNA-NestedNGS in a ROC analysis (Fig.
Engl. J. Med. 348, 994–1004. https://doi. org/10. 1056/NEJMoa022457 (2003). Article PubMed Google Scholar Druker, B. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the philadelphia chromosome.
Sequencing was carried out on the Ion GeneStudio S5 system (Thermo Fisher, Palo Alto, CA) (see Fig. 2A). The raw data were aligned against an artificial genome prepared from ABL1 exons with transcript NM_005157. 5 using Torrent Suite Software v. 5. 12 (Thermo Fisher, Palo Alto, CA), and then the generated BAM files were visualized manually using the Integrative Genomics Viewer (IGV v. 2. 9. 4, Broad Institute, Cambridge, MA).
It can be used to discern between compound mutations or the presence of different subclones in case of multiple mutation findings11, 12. Other techniques with similar sensitivity have served to confirm mutations in BCR::ABL1 such as digital PCR (dPCR) and allele-specific oligonucleotides-PCR (ASO-PCR)13, 14. Taking advantage of the BCR::ABL1 translocation, the detection of KD mutations is mainly performed in mRNA, after enrichment of the translocation by PCR. However, RNA-based tests are sensitive to RNA denaturation and require several retro-transcription and amplification steps, source of false-positive detection induced by polymerase errors.
344, 1038–1042. 1056/NEJM200104053441402 (2001). Article CAS Hochhaus, A. European LeukemiaNet 2020 recommendations for treating chronic myeloid leukemia. Leukemia 34, 966–984. 1038/s41375-020-0776-2 (2020). Article PubMed Central Branford, S. Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop (P-loop) are associated with a poor prognosis. Blood 102, 276–283.
Although this approach covers all coding regions of ABL1 exons 4–10, we focused the study on 36 previously described hotspots (Supplementary Table S2). Figure 3Experimental design and quality metrics resulting for DNA-DeepNGS method. (A) Top: nine amplicon-scheme panel designed to cover the entire KD of BCR::ABL1 and the calculation of the intrinsic error; Bottom: bioinformatic pipeline. (B) Correlation of two replicates for the DNA-DeepNGS approach (C) ROC curve comparing the two methodologies. KD kinase domain, ROC receiver operating characteristic.
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El Obradoiro ha perdido en sus últimas dos visitas a Murcia ... cinco enfrentamientos ligueros, buscará enderezar su rumbo en la pista
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This allowed us to compare results between replicates and to eliminate artifacts. NGS libraries included the sequencing of biological triplicates with an estimated depth of 500, 000x. The generated fastq files were automatically analyzed via a customized bioinformatic pipeline, programmed in Python and R. During the process, firstly, the raw fastq file was pre-processed using the patient information and sample identifiers, run, barcode, amplicon and triplicate identifier. Thus, the file was demultiplexed into smaller fastq files that allowed the computational optimization of the algorithm, significantly reducing the amount of time needed to analyze each sample.
DNA-DeepNGS methodology and automated bioinformatic pipelinePCR amplification was carried out with the Q5 High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, United States). Indexed libraries were prepared following the NEBNext® Fast DNA Library Prep Set for Ion Torrent™ (NEB) and were pooled and sequenced on the Ion GeneStudio S5 System using Ion S5 Sequencing Kit, with 750 flows and 400 bp fragments. Despite our workflow being designed to screen the 1, 129 genetic positions of exons 4–10 covered by the 9 amplicon design, for this work we selected the 36 most prevalent mutations with known clinical outcome31, 32, 33. Biological triplicates to control amplification errors during PCR cycles were analyzed independently by our bioinformatic pipeline (Fig.
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Secondly, to compute the variant read frequency ratios of each position in the coding regions of the selected exons, the aligned wild-type and mutated sequences (with a margin of 15 bp, queried to Ensembl34 via its Python application programming interface, API) of each position were searched in the corresponding demultiplexed output file from the previous step. The number of occurrences of the wild-type and the mutation sequences allowed us to compute the VAF ratio (mutated reads/total reads (mutated + non-mutated)). Then, the VAF ratio was compared with the LOD and LOQ calculated for each hotspot independently in 3 triplicated samples of 6 healthy donors. LOD was computed as the mean ratio in control samples plus 3.
Although DNA-based approaches for KD mutation determination have the disadvantage of amplifying ABL1 from (i) non-rearranged ABL1 allele of tumor cells and (ii) the ABL1 gene of non-rearranged healthy cells, they might better represent the clonal burden and dynamics of the emerging resistant clones. Genomic DNA is more stable than RNA, and gDNA-based approaches might be applied to other dyscrasias to detect acquired mutations. Polivkova et al. studied the correlation between the digital droplet PCR (ddPCR) upon gDNA-based and mRNA-based determination for most common KD mutations with 10–3 sensitivity15.
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75 μL of the 20X TaqMan® assay and 7. 5 μL of 2 × QuantStudio 3D Master Mix, in a 15 μL reaction volume. Then, 14. 5 μL were loaded onto QuantStudio 3D Digital PCR 20 K chips. The cycling conditions were as follows: initial denaturation at 96 °C for 10 min, followed by 39 cycles at 56 °C for 2 min and 98 °C for 30 s and an elongation step of 60 °C for 2 min, always with the cover temperature at 70 °C. Finally, samples were maintained at 20 °C in the dark for at least 30 min, and the fluorescence was read twice. Results were analyzed using QuantStudio® 3D Analysis Suite™ Cloud Software. The dilution curve was generated from a 50% mutant of p. T315I reference standard from Horizon Discovery (Cambridge, UK).
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In total, 25% samples (40/162) presented point mutations in the BCR::ABL1 KD. All samples were analyzed in duplicate, corrected VAF (variant allele frequency) calculated as the value of the VAF multiplied by the levels of BCR::ABL1) showed a Pearson correlation of biological duplicates for the in-house RNA-based method of 0.
The remaining 13 mutated samples corresponded to ALL patients (Fig. 2C). In addition, 15 samples mutated by RNA-NestedNGS were analyzed by SS methodology. Only four were detected by SS. Eight of the mutations not detected presented a VAF below < 20%, but the remaining three were not detected despite raising VAF above the limits of SS (Supplementary Table S1). Figure 1Study flow chart. Overview of the design and distribution of the patients and samples analyzed by RNA-NestedNGS. ALL acute lymphoblastic leukemia, CML chronic myeloid leukemia, D diagnosis, ELN European leukemia net, F failure, R relapse, W warning.
9 95% CI 2. 0–39. 8, p = 0. 004) (Supplementary Fig. S2A). Impact of KD mutations in the clinical outcome of B-cell precursor acute lymphoblastic leukemia BCR::ABL1 positiveFor BCR::ABL1 positive B-ALL patients, we obtained clinical data for 62 patients, of whom 45 had p190 transcript, and 17 had p210. Clinical data are depicted in Supplementary Table S3B. At diagnosis only 4% (2/50) of ALL patients presented mutations. This number increased to 28% in relapse (5/18). For example, the p. L387M mutation was detected in cerebrospinal fluid (CSF) blasts of patient ALL_20 in after first relapse18. The CSF blasts disappeared after triple intrathecal therapy and rescue chemotherapy with dasatinib.
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