ВЛИЯНИЕ НА ЕТИЛМЕТАН СУЛФОНАТА (EMС) И N-НИТРОЗО-N´-ETИЛ КАРБАМИДА (НEК) ВЪРХУ РАСТЕЖА НА КАЛУС ОТ ФАСУЛ

Infl uence of ethyl methanesulfonate (EMS) and N-nitrose-N´-ethyl urea (ENU) mutagenic treatments was investigated on three time sub-cultured calli obtained from leaf petiole explants of 7-day old sterile plants. Calibrated sterile seeds of the common bean Bulgarian variety Plovdiv 11M were pre-cultivated on MS basal medium supplemented with 1 μmM BAP. Then, both mutagens EMS and ENU were applied for different times such as 15, 30, 60 and 90 min on the explants at the concentrations of: 2.5 . 10-2 M and 6.2 . 10-3 M, respectively. Times of the mutagenic treatments infl uenced callus growth, calli from 30-min treatment with both mutagens showing the highest weights. In both cases, the 90-min mutagen application caused a too relevant effect either on callus browning or growth inhibition. In general, ENU showed a stronger effect than EMS. The effect of subcultures on callus growth was higher than mutagenic treatments. Interactions between these factors checked by by correlation ratio (η%) were quite low.Изследвано е влиянието на третирането с етилметан сулфонат (EMС) и N-нитрозо-N´-етил карбамид (НEК) върху трикратно прехвърлен на свежа среда калус получен от експланти от листни дръжки на 7-дневни стерилни растения. Калибрирани стерилни семена от българския сорт фасул Пловдив 11М са предкултивирани на основна MS среда допълнена с 1 μmM BAP. След това, двата мутагена EMС и НEК, са приложени в концентрации съответно: 2.5 . 10-2 M и 6.2 . 10-3 M за различно време 15, 30, 60 и 90 min. Времето на мутагенното третиране влияе върху растежа на калуса като калусът получен след 30-min третиране с двата мутагена има най-високи тегла. 90-min третиране причинява подобен ефект от двата мутагена – покафеняване на калуса или инхибиране на калусния растеж. НЕК показва по-силен ефект от ЕМС. Ефектът от прехвърлянето на свежа среда върху растежа на калуса е по-силен от мутагенните третирания. Взаимодействията между тези фактори, отчетени чрез корелационното съотношение (η%), са сравнително ниски

Detection of EGFR Mutations in Plasma Cell-Free Tumor DNA of TKI-Treated Advanced-NSCLC Patients by Three Methodologies: Scorpion-ARMS, PNAClamp, and Digital PCR

Analysis of circulating cell-free tumor DNA (cftDNA) has emerged as a specific and sensitive blood-based approach to detect epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. Still, there is some debate on what should be the preferential clinical method for plasma-derived cftDNA analysis. We tested 31 NSCLC patients treated with anti-EGFR tyrosine kinase inhibitors (TKIs), at baseline and serially during therapy, by comparing three methodologies in detecting EGFR mutations (L858R, exon 19 deletion, and T790M) from plasma: scorpions-amplification refractory mutation system (ARMS) methodology by using EGFR Plasma RGQ PCR Kit-QIAGEN, peptide nucleic acid (PNA) clamp and PANA RealTyper integration by using PNAClamp EGFR-PANAGENE, and digital real time PCR by using QuantStudio 3D Digital PCR System-Thermo Fisher Scientific. Specificity was 100% for all three mutations, independently from the platform used. The sensitivity for L858R (42.86%) and T790M (100%) did not change based on the method, while the sensitivity for Del 19 differed markedly (Scorpion-ARMS 45%, PNAClamp 75%, and Digital PCR 85%). The detection rate was also higher (94.23%) as measured by Digital PCR, and when we monitored the evolution of EGFR mutations over time, it evidenced the extreme inter-patient heterogeneity in terms of levels of circulating mutated copies. In our study, Digital PCR showed the best correlation with tissue biopsy and the highest sensitivity to attain the potential clinical utility of monitoring plasma levels of EGFR mutations