High-resolution melting analysis for rapid and sensitive NOTCH1 screening in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a biological and clinical heterogeneous disease. Activating mutations of NOTCH1 have been implicated to be associated with adverse prognosis in CLL. The objective of the present study was to develop an effective high-resolution melting (HRM) assay for detecting NOTCH1 mutations. Genomic DNA (gDNA) extracted from 133 CLL patients was screened by HRM assay, and the results were compared with the data obtained using direct sequencing. The relative sensitivity of the HRM assay and direct sequencing was evaluated using diluted gDNA with different NOTCH1 mutational frequencies. The HRM assay was able to detect and discriminate samples with NOTCH1 mutations from the wild-type template in CLL. Eight of the 133 CLL patients (6.02%) were scored positively for NOTCH1 mutations in the HRM assay. The results of the NOTCH1 mutations detected by HRM analysis achieved 100% concordance with those determined from direct sequencing. HRM had a higher sensitivity (1%) and shorter turn-around time (TAT), compared to direct sequencing. In conclusion, the HRM assay developed by us was confirmed to be a rapid, sensitive, and promising approach for high-throughput prognostic NOTCH1 screening in CLL. It enables real-time NOTCH1 evaluation, which is of great significance in clinical practice and may facilitate the decision-making of clinicians in CLL.