NOTCH1, SF3B1 and BIRC3 Mutations in Chronic Lymphocytic Leukemia (CLL) Patients Requiring First-LINE Treatment: Correlation with Biological Parameters and Response to Treatment
Authors: Chiaretti Sabina , Marinelli Marilisa , Del Giudice Ilaria , Bonina Silvia , Gabrielli Sara , Piciocchi Alfonso , Messina Monica , Vignetti Marco , Rossi Davide , Maio Valeria Di , Mauro Francesca Romana , Guarini Anna , Gaidano Gianluca , Foà Robin 
Published: October 2019

Abstract
Abstract 1784

Introduction:
The introduction of whole exome sequencing has allowed to unravel novel molecular lesions in CLL. NOTCH1, SF3B1 and BIRC3 mutations are detected, according to the phases of disease, in 4–12%, 5–17% and 4–24% of patients, respectively. In retrospective studies, their presence has been shown to correlate with overall survival (OS) and treatment-free interval shortening.
Aims: To define the incidence, correlation with known prognostic factors and clinical impact of NOTCH1, SF3B1 and BIRC3 mutations in CLL patients undergoing first-line treatment.

Methods:
We evaluated 162 CLL patients enrolled in the GIMEMA LLC0405 protocol (n=80) for patients aged <60 yrs and in the ML21445 protocol (n=82) for elderly patients (aged >65 yrs or 60–65 if not eligible for fludarabine-based programs). In the GIMEMA LLC0405 protocol, patients were stratified into low and high-risk: patients with del17p or with del11q plus an unmutated IGHV status and/or CD38 positivity and/or ZAP70 positivity were considered as high-risk (HR) and underwent Fludarabine plus Campath, followed by stem cell transplantation procedures, whereas low-risk patients received Fludarabine and Cyclophosphamide.
The MLL21445 protocol consisted of 8 cycles of Chlorambucil and 6 of Rituximab induction treatment.
NOTCH1 (exon 34), SF3B1 (exons 14 and 15) and BIRC3 (exons 2–9, including splicing sites) were screened by Sanger sequencing on either genomic DNA (gDNA) or whole genome amplified DNA (WGA) collected at the time of treatment. These studies were not part of the clinical protocols.

Results:
NOTCH1 mutations were detected at the time of treatment in 18 cases (22%) enrolled in the LLC0405 study. There was a significant association with high-risk stratification (p=0.036), namely with an IGHV unmutated status (p=0.0035), CD38 (p=0.03), +12 (p=0.034) and, partly, ZAP-70 expression (p=0.059). While the overall response rate (ORR) did not differ between NOTCH1 mutated vs wild-type (WT) cases (82% vs 77%, respectively), the complete response (CR) rate was significantly lower in NOTCH1 mutated patients (43% for WT vs 17% for NOTCH1 mutated cases; p=0.05). So far, no significant difference between mutated and WT patients has emerged in terms of OS and progression-free survival (PFS); this may be contributed by the fact that most NOTCH1 mutated cases were HR and were therefore treated more aggressively. SF3B1 mutations were recorded in 9 cases (11%); no significant associations were found with known biological parameters and, so far, with the ORR and CR rate. A single case harbored a BIRC3 mutation; this patient had an IGHV unmutated status, no FISH abnormalities and a concomitant SF3B1 mutation.
In the ML21445 cohort, NOTCH1 mutations were found in 12 cases (15%), were associated with an unmutated IGHV status (p=0.047) and ZAP-70 expression (p=0.007), and did not impact on the ORR and CR rate. SF3B1 mutations were found in 11 cases (13%); no significant associations were found with known biological parameters and the ORR rate. Of interest, only 1/11 SF3B1 mutated patients achieved a CR. BIRC3 mutations were recorded in 3 patients (3.6%); of these, 2 were IGHV mutated, 1 had no cytogenetic abnormalities and 1 carried a del11q, while the third patient was IGHV unmutated status and had no cytogenetic abnormalities. No NOTCH1 and/or SF3B1 mutations were detected.
Overall, NOTCH1, SF3B1 and BIRC3 mutations were largely mutually exclusive among each other and with TP53 lesions in the whole cohort.

Conclusions:
This study confirms the association of NOTCH1 mutations with unfavorable biologic markers and +12, while the presence of SF3B1 mutations was not coupled to poor prognostic markers in CLL patients requiring first-line treatment. Furthermore, it suggests that NOTCH1 mutations impact on the CR rate of young patients receiving Fluda-based regimens, while SF3B1 appears to impact on the CR rate of elderly patients treated with Chlorambucil and Rituximab. Given the small numbers of patients harboring BIRC3, it is at present difficult to draw any conclusion on the clinical impact of this mutation in the cohort of patients hereby analyzed.

Disclosures:
No relevant conflicts of interest to declare.