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. 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.
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. We have recently presented a DNA-based NGS pipeline to use mutations as molecular biomarkers for minimal residual disease monitoring in AML (acute myeloid leukemia) with 10–4 sensitivity16.
2A). 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. 806 (p < 0. 001, Fig. 2B). The LOD (limit of detection) of the RNA-NestedNGS is 1. 0E−5 (1.
11, who found 3 mutated patients out of 44 patients. 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.
Finally, the fragments were purified with beads (1. 5 × ratio) and quantified with the Ion Library Quantitation Kit (Thermo Fisher, Palo Alto, CA). The final barcoded libraries were adjusted to a final concentration of 50 pM and pooled with other samples, assigning 500, 000 reads per sample. Template preparation, enrichment and chip loading were performed using the Ion Chef™ system (Thermo Fisher, Palo Alto, CA).
The test was then applied to 68 samples from 46 patients previously studied by RNA-NestedNGS. The triplicates presented extraordinary reproducibility (Pearson r = 0. 854, p < 0. 001) for two of the triplicates (Fig. 3B) and allowed the identification and exclusion of PCR artifacts. The average curated reads obtained after applying the error corrected algorithm was 432, 697 reads (rank 27, 020–2, 164, 713). To reduce the false negative rate, a minimum of 15 mutated reads and a VAF above the LOD defined for each genetic position were required in the variant calling. With these settings the automated algorithm identified 55 potential mutations. The ROC curve of the performance for detection of KD mutations compared to the RNA-NestedNGS defined the optimum point in 1.
Following the manufacturer’s protocols, the generated amplicons obtained from the nested PCR were then purified with Agencourt AMPure XP Beads (Beckman Coulter, Inc., Brea, CA) at an initial ratio of 1. 8 × and visualized using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Further enzymatic fragmentation of the KD was performed using Ion ShearPlus (Ion Torrent, Thermo Fisher, Palo Alto, CA) for 15 min at 37 °C to obtain ∼250 bp fragments (Fig. 2A).
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. 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.
Then ponatinib (15 mg/day) was started and the patient achieved grade 5 MR (molecular response) (Fig. 5A). In another patient, the clone p. L248V, described as inducing resistance to most TKIs in vitro, was detected all along the clinical history of the patient CML_26. This patient received six different TKI regimens with the BCR::ABL1IS levels always above 1% (Fig. 5B). Although the mutation was detected by both methods, the corrected VAF in gDNA was higher than in mRNA for most time-points, and in two of them, the mutation was not detected by RNA-NestedNGS. This patient is now treated with asciminib as compassionate use, keeping in complete hematologic response (CHR). Figure 5Time course for the mutations measured by DNA-DeepNGS and RNA-NestedNGS, and BCR::ABL1 levels present in the most clinically relevant patients.
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.
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. 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.
T315I reference standard from Horizon Discovery (Cambridge, UK). 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.
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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. Figure 2(A) Scheme methodology of the detection of BCR::ABL1 mutations by RNA-NestedNGS; (B) Correlation between the two replicates by RNA-NestedNGS; (C) Histogram of mutational frequency by RNA-NestedNGS method in failure/warning stages for CML patients and diagnosis/relapse ALL patients; (D) Results from EUTOS international control round for deep sequencing analysis of BCR::ABL1 mutations using RNA-NestedNGS approach showing the ability of the nested method to detect KD mutations. ALL acute lymphoblastic leukemia, CML chronic myeloid leukemia, KD kinase domain.
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For the dPCR reaction, 6. 25 μL of the second round of the nested PCR were mixed with 0. 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.
Biological triplicates to control amplification errors during PCR cycles were analyzed independently by our bioinformatic pipeline (Fig. 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.
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5D). Treatment was switched to ponatinib, with levels of BCR::ABL1 below 1. Unfortunately, the patient died eight months later in disease progression. Despite the small size of the ALL sub-cohort of relapsed patients with clinical data available (n = 13), the three patients with mutations showed a trend for shorter OS (HR, hazard ratio 6. 4 95% CI 0. 89–46. 0. 065, Supplementary Fig. S2B). DiscussionWe here report the first approach for BCR::ABL1 KD mutation screening, using DNA as source material. The study demonstrates that the DNA-deepNGS method is feasible, robust and reproducible and can be easily implemented in the laboratory routines. The identification of acquired ABL1 KD point mutations is crucial to anticipate and overcome TKI resistance. Despite SS being the gold standard for KD mutation screening, NGS and dPCR are emerging as more sensitive techniques to detect minor subclones.
These results endorse this molecular approach in further clinical protocols for adopting early clinical decisions leading to improve patient management. MethodsPatients and study designWe examined BCR::ABL1 mutations in 162 BM or PB samples (Fig. 1). Sixty-seven consecutive CML patients treated in 19 public hospitals all over Spain. Inclusion criteria were failure or warning response to any TKI, any therapy line, according to the 2013 ELN recommendations, and levels of BCR::ABL1IS > 0. 1% (International Scale). For B-ALL disease, 62 patients were included in two consecutive clinical trials of the ALL subgroup of PETHEMA, LAL-OPH-200727 and LAL-PH-200828.
Y-axis represents the value of RNA-NestedNGS VAF corrected by the ratio BCR::ABL1 (green), DNA-DeepNGS VAF (blue) or ratio BCR::ABL1/ABL1 (red). ALL acute lymphoblastic leukemia, Asci asciminib, B bosutinib, CML chronic myeloid leukemia, D dasatinib, I imatinib, N nilotinib, Niv nivolumab, P ponatinib, R relapse, VAF variant allele frequency. To assess clinical impact of the mutation detection for both techniques, we defined the mutation screening as a time dependent covariate in a Cox model, confirming that CML individuals in warning or failure with KD mutations had a significantly shorter OS (HR 8.
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