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- Rassner, M., et al.
(författare)
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Development of Highly Sensitive Digital Droplet PCR for Detection of cKIT Mutations in Circulating Free DNA That Mediate Resistance to TKI Treatment for Gastrointestinal Stromal Tumor (GIST)
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 24:6
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Tidskriftsartikel (refereegranskat)abstract
- Background: Mutations in cKIT or PDGFRA are found in up to 90% of patients with gastrointestinal stromal tumors (GISTs). Previously, we described the design, validation, and clinical performance of a digital droplet (dd)PCR assay panel for the detection of imatinib-sensitive cKIT and PDFGRA mutations in circulating tumor (ct)DNA. In this study, we developed and validated a set of ddPCR assays for the detection of cKIT mutations mediating resistance to cKIT kinase inhibitors in ctDNA. In addition, we cross-validated these assays using next generation sequencing (NGS). Methods: We designed and validated five new ddPCR assays to cover the most frequent cKIT mutations mediating imatinib resistance in GISTs. For the most abundant imatinib-resistance-mediating mutations in exon 17, a drop-off, probe-based assay was designed. Dilution series (of decreasing mutant (MUT) allele frequency spiked into wildtype DNA) were conducted to determine the limit of detection (LoD). Empty controls, single wildtype controls, and samples from healthy individuals were tested to assess specificity and limit of blank (LoB). For clinical validation, we measured cKIT mutations in three patients and validated results using NGS. Results: Technical validation demonstrated good analytical sensitivity, with a LoD ranging between 0.006% and 0.16% and a LoB ranging from 2.5 to 6.7 MUT fragments/mL. When the ddPCR assays were applied to three patients, the abundance of ctDNA in serial plasma samples reflected the individual disease course, detected disease activity, and indicated resistance mutations before imaging indicated progression. Digital droplet PCR showed good correlation to NGS for individual mutations, with a higher sensitivity of detection. Conclusions: This set of ddPCR assays, together with our previous set of cKIT and PDGFRA mutations assays, allows for dynamic monitoring of cKIT and PDGFRA mutations during treatment. Together with NGS, the GIST ddPCR panel will complement imaging of GISTs for early response evaluation and early detection of relapse, and thus it might facilitate personalized decision-making.
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| 2. |
- Illert, M., et al.
(författare)
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Integration in descending motor pathways controlling the forelimb in the cat - 7. Effects from the reticular formation on C3-C4 propriospinal neurones
- 1981
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Ingår i: Experimental Brain Research. - 0014-4819 .- 1432-1106. ; 42, s. 269-281
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Tidskriftsartikel (refereegranskat)abstract
- Effects of stimulation in the medullary reticular formation (RF) on C3-C4 propriospinal neurones (PNs) were investigated in two series of experiments: (1) indirectly by analyzing how propriospinal transmission to forelimb motoneurones is modified by reticular stimuli; (2) directly by intracellular recording from C3-C4 neurones, which were identified as propriospinal by their antidromic activation from the C6 segment. Propriospinally mediated disynaptic EPSPs evoked in motoneurones from the pyramid (Pyr) and the red nucleus (NR) were effectively facilitated by conditioning stimulation in the RF with a time course of facilitation indicating monosynaptic linkage to the PNs. Propriospinally mediated trisynaptic IPSPs were facilitated less regularly and sometimes instead depressed by conditioning stimulation in the RF. The depression is at least partly due to inhibition of the first order PNs. Recording from C3-C4 PNs revealed that many of them were excited or inhibited by single stimuli in the RF. The brief latency of the EPSPs evoked in these neurones shows monosynaptic linkage from fast reticulospinal fibres. Some IPSPs were similarly monosynaptically evoked from fast fibres and observations are presented suggesting that longer latency IPSPs are monosynaptically mediated by slower fibres. Facilitation of propriospinal transmission to motoneurones as well as the EPSPs and IPSPs in PNs were evoked from a region within or close to the nucleus reticularis gigantocellularis. Convergence of monosynaptic EPSPs from Pyr, NR, tectum, and RF was common in C3-C4 PNs. Linear summation of the EPSPs from RF with those evoked from cortico-, rubro-, or tectospinal tracts shows that the former are not due to stimulation of collaterals which the latter tracts may have in RF. Mediation of the EPSPs and IPSPs by descending, rather than by antidromically activated ascending fibres, was indicated by temporal facilitation produced by RF stimuli, subliminal for evoking monosynaptic PSPs in the PNs. Stimulation of the labyrinth did not evoke disynaptic PSPs in any of the PNs investigated. It is concluded that the C3-C4 PNs projecting to forelimb motoneurones can be excited not only from the cortico-, rubro-, and tectospinal tracts (Illert et al. 1977, 1978) but also by reticulospinal fibres. © 1981 Springer-Verlag.
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