DETECTION OF NOTCH1 c.7544_7545delCT MUTATION IN CHRONIC LYMPHOCYTIC LEUKEMIA USING CONVENTIONAL AND REAL-TIME POLYMERASE CHAIN REACTION

Abstract

Aim: To evaluate real-time polymerase chain reaction (PCR) assay system for detection of NOTCH1 c.7541_754delCT mutation in chronic lymphocytic leukemia (CLL) patients. Material and Methods: A total of 325 CLL patients were included in the study. Screening for NOTCH1 c.7544_7545delCT was performed using conventional PCR-based amplification refractory mutation system (ARMS) method. All 33 samples harboring c.7544_7545delCT allele and 5 negative cases as control were submitted to real-time PCR. Results: Specificity and sensitivity of two PCR techniques were comparable. NOTCH1 c.7544_7545delCT mutation was found by ARMS in 10.1% of CLL patients, which is consistent with the data of other studies. However, the results of ARMS PCR in a minority of cases (2.15%) were doubtful and required reinvestigation. Real-time PCR, being less time-consuming, showed advantage in the assessment of the amplification’s specificity (using the melting curve analysis). It also allows the quantitative assessment of NOTCH1-mutated clone. Conclusion: NOTCH1 c.7544_7545delCT mutation resulting in removal of the C-terminal PEST domain, deregulation of NOTCH1-dependent signaling pathways, has negative influence on prognosis of CLL and efficiency of therapy with anti-CD20 monoclonal antibodies. Real-time PCR allows the fast and reliable detection of c.7544_7545delCT mutation and can be used for the screening of this molecular lesion in CLL patients.

NOTCH1, SF3B1, MDM2 and MYD88 mutations in patients with chronic lymphocytic leukemia

“…The NOTCH1 C.7544-7545 delCT mutation results in the removal of the C-terminal PEST domain which is associated with an accumulation of constitutively active NOTCH1 protein and the constitutive activation of NOTCH1 signaling, which contributes to apoptosis resistance and increased survival of CLL cells (5,18). This suggests that the NOTCH1 delCT mutation and the activation of NOTCH signaling is associated with the pathogenesis of the severe clinical form of CLL, however this needs to be confirmed by further studies.…”

The role of SF3B1 and NOTCH1 in the pathogenesis of leukemia

“…The incidence of NOTCH1 gene mutations ranges from 5% to 22% in CLL patients at diagnosis 58,59,[61][62][63]68,[150][151][152][153][154][155][156][157][158] and increases to 20% and 31% in chemorefractory CLL and cases progressing to Richter syndrome (RS), respectively. 153,[159][160][161][162] NOTCH1 mutations are frequently correlated with subgroups of CLL patients who have an unmutated IGHV gene and trisomy 12.…”

Molekularna patogeneza przewlekłej białaczki limfocytowej

“…W efekcie białko NOTCH1 z uszkodzoną domeną PEST charaktery zuje się zwiększoną stabilnością. Mutacja (c.7544_7545delCT) stanowi 80% mutacji NOTCH1 w CLL i może zostać wykryta metodą reakcji polimerazy łańcuchowej (PCR, polymerase chain reaction) [51]. U około 3% chorych mutacje występują w regionie 3'UTR, co prowadzi do uszkodzenia domeny PEST w trakcie alternatywnego splicingu [52].…”