CONTEXT: Testing for BCR-ABL1 kinase domain (KD) mutations should always be performed before tyrosine kinase inhibitor (TKI) changes. Next-generation sequencing (NGS) is the best approach to highlight emerging mutations in patients not responding adequately to TKI therapy. However, NGS requires sample centralization and batch analysis and has a non-negligible time to results. In this study, we set up and validated a novel droplet digital PCR (ddPCR)-based multiplex strategy for the detection and quantitation of transcripts harboring mutations impacting TKI selection. METHODS: In collaboration with Bio-Rad, a 3-tube ddPCR strategy was designed that enables identification and quantitation of 16 nucleotide substitutions encoding the 13 mutations associated with resistance to one or more second-generation TKI (2GTKI). Primers and FAM-or FAM/HEX-labelled probes were grouped on a TKI-specific basis and generated clusters of droplets mapping to spatially distinct areas of the 2D plot based on resistance profiles. Each tube also incorporated primers and HEX-labelled probes for e13a2, e14a2, and e1a2 BCR::ABL1 fusion transcripts to express results as the percentage of mutation-positive over total BCR::ABL1 transcripts. For validation, a total of 101 RNA samples from healthy donors, TKI-sensitive and -resistant patients, and BCR::ABL1-positive and -negative cell lines were used. cDNA (125 ng) obtained with ABL1-specific primers was analyzed in duplicate on a QX200 ddPCR system (Bio-Rad). RESULTS: The limit of blank was determined using 60 blank samples. Accuracy and specificity were confirmed using 48 samples positive for one or more 2GTKI-resistant mutations or mutations at nearby codons (to exclude cross-reactivity). Analysis of serial dilutions of cell line mixtures made using BCR::ABL1-positive mutation-positive cells, BCR::ABL1-positive unmutated cells, and BCR::ABL1-negative cells to mimic different mutation frequencies (70%, 5%, and 0.5%) and different transcript levels (MR0 to MR3) showed that a 0.5% lower detection limit could be consistently achieved irrespective of BCR::ABL1 levels. CONCLUSIONS: ddPCR proved highly sensitive and accurate. Total hands-on time was approximately 2 hrs, and time from sample to results was 2 days. Therefore, ddPCR may be integrated into diagnostic algorithms of CML (and Ph+ ALL) patients as a convenient first-level screening tool for mutations impacting TKI selection.
- MeSH
- bcr-abl fúzové proteiny * genetika metabolismus MeSH
- chemorezistence genetika MeSH
- chronická myeloidní leukemie * diagnóza farmakoterapie genetika MeSH
- inhibitory proteinkinas farmakologie terapeutické užití MeSH
- komplementární DNA farmakologie MeSH
- lidé MeSH
- mutace MeSH
- nukleotidy MeSH
- polymerázová řetězová reakce MeSH
- RNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
One of the indications for BCR::ABL1 mutation testing in chronic myeloid leukemia (CML) is when tyrosine kinase inhibitor therapy (TKI) needs to be changed for unsatisfactory response. In this study, we evaluated a droplet digital PCR (ddPCR)-based multiplex strategy for the detection and quantitation of transcripts harbouring mutations conferring resistance to second-generation TKIs (2GTKIs). Parallel quantitation of e13a2, e14a2 and e1a2 BCR::ABL1 fusion transcripts enables to express results as percentage of mutation positive- over total BCR::ABL1 transcripts. We determined the limit of blank in 60 mutation-negative samples. Accuracy was demonstrated by further analysis of 48 samples already studied by next generation sequencing (NGS). Mutations could be called down to 0.5% and across 3-logs of BCR::ABL1 levels. Retrospective review of BCR::ABL1 NGS results in 513 consecutive CML patients with non-optimal response to first- or second-line TKI therapy suggested that a ddPCR-based approach targeted against 2GTKI-resistant mutations would score samples as mutation-negative in 22% of patients with warning response to imatinib but only in 6% of patients with warning response to 2GTKIs. We conclude ddPCR represents an attractive method for easy, accurate and rapid screening for 2GTKI-resistant mutations impacting on TKI selection, although ddPCR cannot identify compound mutations.
BACKGROUND: Imatinib-resistant chronic myeloid leukemia (CML) patients receiving second-line tyrosine kinase inhibitor (TKI) therapy with dasatinib or nilotinib have a higher risk of disease relapse and progression and not infrequently BCR-ABL1 kinase domain (KD) mutations are implicated in therapeutic failure. In this setting, earlier detection of emerging BCR-ABL1 KD mutations would offer greater chances of efficacy for subsequent salvage therapy and limit the biological consequences of full BCR-ABL1 kinase reactivation. Taking advantage of an already set up and validated next-generation deep amplicon sequencing (DS) assay, we aimed to assess whether DS may allow a larger window of detection of emerging BCR-ABL1 KD mutants predicting for an impending relapse. METHODS: a total of 125 longitudinal samples from 51 CML patients who had acquired dasatinib- or nilotinib-resistant mutations during second-line therapy were analyzed by DS from the time of failure and mutation detection by conventional sequencing backwards. BCR-ABL1/ABL1%(IS) transcript levels were used to define whether the patient had 'optimal response', 'warning' or 'failure' at the time of first mutation detection by DS. RESULTS: DS was able to backtrack dasatinib- or nilotinib-resistant mutations to the previous sample(s) in 23/51 (45 %) pts. Median mutation burden at the time of first detection by DS was 5.5 % (range, 1.5-17.5 %); median interval between detection by DS and detection by conventional sequencing was 3 months (range, 1-9 months). In 5 cases, the mutations were detectable at baseline. In the remaining cases, response level at the time mutations were first detected by DS could be defined as 'Warning' (according to the 2013 ELN definitions of response to 2nd-line therapy) in 13 cases, as 'Optimal response' in one case, as 'Failure' in 4 cases. No dasatinib- or nilotinib-resistant mutations were detected by DS in 15 randomly selected patients with 'warning' at various timepoints, that later turned into optimal responders with no treatment changes. CONCLUSIONS: DS enables a larger window of detection of emerging BCR-ABL1 KD mutations predicting for an impending relapse. A 'Warning' response may represent a rational trigger, besides 'Failure', for DS-based mutation screening in CML patients undergoing second-line TKI therapy.
- MeSH
- bcr-abl fúzové proteiny genetika MeSH
- chemorezistence MeSH
- chronická myeloidní leukemie farmakoterapie genetika MeSH
- farmakogenomické varianty MeSH
- inhibitory proteinkinas terapeutické užití MeSH
- jednonukleotidový polymorfismus MeSH
- lidé MeSH
- sekvenční analýza DNA metody MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients who fail imatinib treatment, BCR-ABL1 mutation profiling by Sanger sequencing (SS) is recommended before changing therapy since detection of specific mutations influences second-generation tyrosine kinase inhibitor (2GTKI) choice. We aimed to assess i) in how many patients who relapse on second-line 2GTKI therapy next generation sequencing (NGS) may track resistant mutations back to the sample collected at the time of imatinib resistance, before 2GTKI start (switchover sample) and ii) whether low level mutations identified by NGS always undergo clonal expansion. To this purpose, we used NGS to retrospectively analyze 60 imatinib-resistant patients (CML, n = 45; Ph+ ALL,n = 15) who had failed second-line 2GTKI therapy and had acquired BCR-ABL1 mutations (Group 1) and 25 imatinib-resistant patients (CML, n = 21; Ph+ ALL, n = 4) who had responded to second-line 2GTKI therapy, for comparison (Group 2). NGS uncovered that in 26 (43%) patients in Group 1, the 2GTKI-resistant mutations that triggered relapse were already detectable at low levels in the switchover sample (median mutation burden, 5%; range 1.1%-18.4%). Importantly, none of the low level mutations detected by NGS in switchover samples failed to expand whenever the patient received the 2GTKI to whom they were insensitive. In contrast, no low level mutation that was resistant to the 2GTKI the patients subsequently received was detected in the switchover samples from Group 2. NGS at the time of imatinib failure reliably identifies clinically relevant mutations, thus enabling a more effective therapeutic tailoring.
- MeSH
- akutní lymfatická leukemie farmakoterapie genetika MeSH
- bcr-abl fúzové proteiny genetika MeSH
- chemorezistence genetika MeSH
- chronická myeloidní leukemie farmakoterapie genetika MeSH
- dasatinib terapeutické užití MeSH
- dospělí MeSH
- imatinib mesylát terapeutické užití MeSH
- inhibitory proteinkinas terapeutické užití MeSH
- lidé středního věku MeSH
- lidé MeSH
- mutační analýza DNA metody MeSH
- retrospektivní studie MeSH
- senioři MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
