Applicazione Della Droplet Digital Pcr Per La Quantificazione Della Malattia Residua Minima Nella Leucemia Linfoblastica Acuta Pediatrica

La real-time PCR quantitativa (RQ-PCR) viene attualmente utilizzata per la quantificazione relativa della Malattia Residua Minima (MRM) nei la quantificazione relativa della Malattia Residua Minima (MRM) nei pazienti pediatrici affetti da LeucemiaLinfoblastica Acuta (LLA). Il recente sviluppo della Droplet Digital PCR (ddPCR) per la quantificazione assoluta potrebbe consentire di caratterizzare meglio i pazienti attualmentedefiniti positivi non quantificabili (NQ) in RQ-PCR

Glutathione S-transferase homozygous deletions and relapse in childhood acute lymphoblastic leukemia: a novel study design in a large Italian AIEOP cohort

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Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis

To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations

Comparison of MRD detection by RQ-PCR and droplet digital PCRin pediatric Acute Lymphoblastic Leukemia before Hematopoietic Stem Cell Transplantation

Real-time quantitative PCR (RQ-PCR) is a standardized tool for Minimal Residual Disease (MRD) monitoring in pediatric patients with Acute Lymphoblastic Leukemia (ALL), showing a prognostic impact also in the field of Hematopoietic Stem Cell Transplantation (HSCT). This approach can be limited by the availability of diagnostic material. Recently, droplet digital PCR (ddPCR) was introduced and has already led to several research breakthroughs in various fields. The main advantage of this method is that there is no need for external references, since it allows for absolute quantification of target DNA. We evaluated the applicability of ddPCR for MRD analysis in pediatric ALL transplanted patients and we compared the prognostic impact of MRD assessed by RQ-PCR and ddPCR before HSCT