The Predominant Prognostic Significance of NOTCH1 Mutation Defined by Emulsion PCR in Chronic Lymphocytic Leukemia

Abstract

Purpose

NOTCH1^mut represents a new prognostic marker in chronic lymphocytic leukaemia (CLL). The low sensitivity of the current methods may increase the risk of false-negative results, particularly in patients with low NOTCH1^mut allelic burden. This study compared two methods of the NOTCH1^mut assessment including droplet digital PCR (ddPCR) and amplification-refractory mutation system PCR (ARMS-PCR) untreated CLL patients.

Patients and Methods

This study included 319 untreated CLL patients. Two PCR-based methods; ddPCR and ARMS-PCR were performed to assess the mutational status of NOTCH1. The Mann–Whitney, Fisher’s exact test, Kruskal–Wallis, Kaplan–Meier, Log rank tests and multivariate Cox proportional hazard regression model were used to analyze collected data.

Results

We proved that ddPCR increased the detectability of the NOTCH1^mut compared to ARMS-PCR in CLL (18.55% vs 6%). We showed a shorter time to first treatment (TTFT) in the NOTCH1^mut group of patients compared to the NOTCH1^wt defined by ddPCR (1.5 vs 33 months, p=0.01). The TTFT survival curves analysis in subgroups divided according to the mutational status of IGHV and NOTCH1 assessed by ddPCR discriminated group with the best prognosis: IGHV^mutNOTCH1^wt. Multivariate analysis revealed that the mutational status of IGHV represented an independent prognostic factor for TTFT, while NOTCH1^mut determined by ddPCR constituted as a dependent prognostic factor for TTFT.

Conclusion

The selection of the precise method of NOTCH1^mut detection as ddPCR might significantly improve prognostic stratification of CLL patient. Assessment of IGHV might be relevant to more accurate discrimination of prognostic groups of CLL patients, especially in harboring NOTCH1^mut irrespective of the quantity of allelic burden.

Keywords:

• chronic lymphocytic leukemia
• CLL
• NOTCH1
• droplet digital PCR
• ddPCR
• amplification-refractory mutation system PCR
• ARMS-PCR
• prognostic marker

Acknowledgments

Authors would like to thank persons involved in collecting patients’ material and clinical data including: Waldemar Tomczak, Joanna Zaleska, Joanna Purkot, Magdalena Paziewska, Marta Karp, Grażyna Stasiak, Maciej Putowski, Jacek Zawiślak, Joanna Knap, Ewelina Zakrzewska.

Data Sharing Statement

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethics Approval and Informed Consent

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Local Ethics Committee of the Medical University of Lublin (number KE-0254/231/2015), and the informed consent was obtained from all patients and patient information was anonymized and de-identified prior to analysis.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.