Clonal Abundance Determines The Prognostic Relevance Of a NOTCH1 Mutation In Chronic Lymphocytic Leukemia
Authors: Rost Stefanie , Möllmann Michael , Eisele Lewin , Weber Stefanie , Dührsen Ulrich , Dürig Jan , Göthert Joachim Rudolf 
Published: October 2019

Abstract

Introduction
The transmembrane receptor NOTCH1 operates as a ligand-activated transcription factor controlling developmental processes, proliferation and apoptosis. In the context of cancer, activating NOTCH1 mutations are the most frequent oncogenic events in T-cell acute lymphoblastic leukemia and have been implicated in chronic lymphocytic leukemia (CLL) as well. The most prevalent CLL NOTCH1 mutation (N1ΔCT) leads to a truncation of the protein (p.P2515Rfs*4) and has been associated with impaired overall survival (OS). Here, we applied three different methods to study the N1ΔCT prevalence and subclone size in a cohort of n=275 CLL patients.

Methods
Presence of the N1ΔCT mutation was analyzed using newly established restriction fragment length polymorphism (RFLP) and allele-specific PCR (AS-PCR) methodologies. A novel real-time PCR (qRT-PCR) assay was used to precisely quantify the N1ΔCT allele frequency. Presence of the N1ΔCT mutation was confirmed by conventional Sanger sequencing.

Results
Using RFLP analysis we detected the N1ΔCT mutation in n=17 CLL patients. In parallel, we used a more sensitive AS-PCR and identified n=12 additional N1ΔCT-mutated cases resulting in a total N1ΔCT mutation rate of 10.5% (n=29/275) in our cohort. The OS of RFLP-positive patients (RFLP+) was significantly shorter than the OS of N1ΔCT-unmutated patients (wt) (mean OS; RFLP+, 87 months vs. wt, 218 months; p=0.017). In contrast, OS of AS-PCR-positive cases (AS-PCR+) did not differ significantly from the OS of wt patients (mean OS; AS-PCR+, 175 months vs. wt, 218 months; p=0.42). These data prompted us to design a quantitative real-time PCR (qRT-PCR) assay, which is capable of precisely quantifying the size of the N1ΔCT-mutated subclones (allele frequency, %) in our CLL cohort. As expected, significantly different allele frequencies between RFLP+ (mean±SEM 27.1±3.4%), AS-PCR+ (3.7±0.6%) and wt patients (0.6±0.04%) were revealed by qRT-PCR (p<0.0001). In order to determine a methodology-independent cut-off which correlates with the clinical significance of the N1ΔCT mutation, we employed Receiver Operating Characteristics (ROC) analysis based on survival status and calculated a N1ΔCT allele frequency cut-off of 15.2% (AUC=0.71). Next, we determined N1ΔCT allele frequencies over time to investigate clone dynamics within individual patients (n=15 patients, mean observation period 87.4 months; range 5-186 months). Unexpectedly, the N1ΔCT allele frequencies remained relatively constant and none of the patients with N1ΔCT allele frequencies below 15.2% rose above this cut-off over time.

Conclusions
Our data demonstrate that high abundance of a N1ΔCT-mutated CLL clone correlates with an aggressive disease course. In our CLL cohort a N1ΔCT allele frequency below 15% was of negligible clinical relevance. Thus, mere qualitative detection of a N1ΔCT mutation by PCR is not inevitably associated with shortened survival. Surprisingly, we did not observe that a minor N1ΔCT clone became dominant over time.

Disclosures:
No relevant conflicts of interest to declare.