Mutations of NOTCH1 are an independent predictor of survival in chronic lymphocytic leukemia

Abstract

Analysis of the chronic lymphocytic leukemia (CLL) coding genome has recently disclosed that the NOTCH1 proto-oncogene is recurrently mutated at CLL presentation. Here, we assessed the prognostic role of NOTCH1 mutations in CLL. Two series of newly diagnosed CLL were used as training (n = 309) and validation (n = 230) cohorts. NOTCH1 mutations occurred in 11.0% and 11.3% CLL of the training and validation series, respectively. In the training series, NOTCH1 mutations led to a 3.77-fold increase in the hazard of death and to shorter overall survival (OS; P < .001). Multivariate analysis selected NOTCH1 mutations as an independent predictor of OS after controlling for confounding clinical and biologic variables. The independent prognostic value of NOTCH1 mutations was externally confirmed in the validation series. The poor prognosis conferred by NOTCH1 mutations was attributable, at least in part, to shorter treatment-free survival and higher risk of Richter transformation. Although NOTCH1 mutated patients were devoid of TP53 disruption in more than 90% cases in both training and validation series, the OS predicted by NOTCH1 mutations was similar to that of TP53 mutated/deleted CLL. NOTCH1 mutations are an independent predictor of CLL OS, tend to be mutually exclusive with TP53 abnormalities, and identify cases with a dismal prognosis.

Figure 1

Kaplan-Meier estimates of OS according to NOTCH1 mutation status. Overall survival according to NOTCH1 mutation status in the CLL training series (n = 309; A) and in the CLL validation series (n = 230; B). NOTCH1 germ line cases (NOTCH1 GL) are represented by the blue line. NOTCH1 mutated cases (NOTCH1 M) are represented by the red line.

Figure 2

Kaplan-Meier estimates of TFS and cumulative probability of transformation to RS according to NOTCH1 mutation status. TFS according to NOTCH1 mutation status (A) and cumulative probability of transformation to RS (B) in the CLL training series (n = 309). NOTCH1 germ line cases (NOTCH1 GL) are represented by the blue line. NOTCH1 mutated cases (NOTCH1 M) are represented by the red line.

Figure 3

Kaplan-Meier estimates of OS from first-line treatment. OS from first line treatment according to NOTCH1 mutation status in CLL treated with fludarabine-based regimens series (n = 113). Cases with germ line NOTCH1 and TP53 genes (GL) are represented by the blue curve. Cases harboring NOTCH1 mutations without TP53 disruption (NOTCH1 M) are represented by the red curve. Cases harboring TP53 disruption without NOTCH1 mutations (TP53 DIS) are represented by the yellow curve. Cases harboring both NOTCH1 mutations and TP53 disruption (NOTCH1 M and TP53 DIS) are represented by the green curve.

Figure 4

Comparative analysis of NOTCH1 mutations and TP53 disruption. (A-B) Venn diagram illustrating the overlap between NOTCH1 mutations and TP53 disruption by mutations, deletion, or both in the CLL training series (A) and in the CLL validation series (B). Numbers within the red and yellow circles indicate the number of cases harboring NOTCH1 mutations without TP53 disruption (red circle) and cases with TP53 disruption without NOTCH1 mutations (yellow circle). Numbers within the overlaps between circles indicate the number of cases harboring both NOTCH1 mutations and TP53 disruption. (C-D) Kaplan–Meier estimates of OS according to NOTCH1 mutation and TP53 disruption in the CLL training series (n = 309; C) and in the CLL validation series (n = 230; D). Cases with germ line NOTCH1 and TP53 genes (GL) are represented by the blue line. Cases harboring NOTCH1 mutations without TP53 disruption (NOTCH1 M) are represented by the red line. Cases harboring TP53 disruption (TP53 DIS) are represented by the yellow line. Cases harboring both NOTCH1 and TP53 disruption were compiled to cases harboring only TP53 disruption, because of the low number (3 in the training series and 1 in the validation series) of double-mutated cases, and based on a recursive partitioning analysis for risk of death.

Figure 5

ARMS to detect NOTCH1 c.7544_7545delCT mutation. (A) Schematic diagram of the NOTCH1 gene (top) and protein (bottom), with its conserved functional domains (EGF-like repeats: LNR, LIN-12/NOTCH repeats; HD, heterodimerization; TM, transmembrane; Ankyrin repeats: TAD, transactivation domain; PEST, proline, glutamic acid, serine, threonine sequence). The TAD domain and the PEST sequence, both coded by exon 34, are magnified. Color-coded shapes indicate the position of the mutations found in the CLL training series (n = 34) and in the CLL validation series (n = 26). (B) Representative results of the ARMS assay showing 4 CLL samples that scored positive for the c.7544_7545delCT mutation (codes 5984, 5726, 11815, 3979) and 7 CLL samples that scored negative for the c.7544_7545delCT mutation (codes 4681, 5092, 3410, 8054, 7272, 11477, 5949). Negative samples show a normal band of 284 bp. Positive samples show an additional mutant band of 183 bp. Negative (C neg) and positive (C pos) controls also are included. Molecular weight (MW) is the 100-bp DNA ladder. Camera: Gel Doc 1000, BioRad; image acquisition software: Quantity One 4.5.0, BioRad. (C) Kaplan-Meier estimates of overall survival according to the results of the ARMS assay in the CLL training series (n = 309). Cases that scored negative by ARMS for the NOTCH1 c.7544_7545delCT mutation (ARMS neg) are represented by the blue line. Cases that scored positive by ARMS for the NOTCH1 c.7544_7545delCT mutation (ARMS pos) are represented by the red line.

Figure 6

Timing of NOTCH1 mutations and relationship with TP53 disruption in high-risk CLL. (A) NOTCH1 mutations and TP53 disruption in fludarabine-refractory CLL. In the heatmap, rows correspond to the NOTCH1 and TP53 genes, and columns represent individual patients color-coded based on the gene status (white, wild type; red, mutations of NOTCH1 and disruption of TP53). (B) NOTCH1 mutations and TP53 disruption in sequential CLL/RS samples. In the heatmap, rows correspond to the NOTCH1 and TP53 genes. Columns represent individual patients color-coded based on the gene status (white, wild type; pink, mutations of NOTCH1 and disruption of TP53 in the CLL phase; red, mutations of NOTCH1 and disruption of TP53 at RS transformation).