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J Cancer Metastasis Treat 2019;5:5 I http://dx.doi.org/10.20517/2394-4722.2018.108 Page 17 of 27
1 Division of Hematology, Department of Translational Medicine, Amedeo Avogadro University of Eastern
Piedmont, Novara 28100, Italy.
2 Institute of Oncology Research and Oncology Institute of Southern Switzerland, Bellinzona 6500, Switzerland.
3 Department of Medical Sciences, University of Turin, Turin 10126, Italy.
4 Università della Svizzera Italiana (USI), Institute of Oncology Research (IOR), Bellinzona 6500, Switzerland.
5 Hematology, Department of Cellular Biotechnologies and Hematology, Sapienza University, Rome 00185, Italy.
6 Cancer Sciences Unit, Southampton Cancer Research UK and National Institute for Health Research
Experimental Cancer Medicine Centre, University of Southampton, Southampton SO17 1BJ, United Kingdom.
7 Division of Hematology, University of Siena, Siena 53100, Italy.
8 Department of Hematology Oncology, Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico and
University of Milan, Milan 20122, Italy.
9 Clinica Ematologica, DAME, University of Udine, Udine 33100, Italy.
10 Department of Cell Therapy and Hematology, Ospedale San Bortolo, Vicenza 36100, Italy.
11 Division of Hematology, Azienda Ospedaliera Universitaria Policlinico-OVE, Catania 95123, Italy.
12 Hematology Section, Azienda Ospedaliero Universitaria Arcispedale S. Anna, University of Ferrara, Ferrara
44124, Italy.
13 Division of Hematology, Department of Oncology and Hematology, University of Modena and Reggio Emilia,
Modena 41121, Italy.
14 Division of Hematology, Azienda Ospedaliero Universitaria Città della Salute e della Scienza and University
of Turin, Turin 10126, Italy.
15 Section of Hematology, Department of Medicine, University of Verona, Verona 37129, Italy.
16 Department of Oncology/Haematology, Niguarda Cancer Center, Niguarda Ca Granda Hospital, Milan
20162, Italy.
17 Institute of Hematology, Catholic University of the Sacred Heart, Rome 00168, Italy.
18 Department of Hematology, Spedali Civili, Brescia 25123, Italy.
19 Department of Hematology, Tor Vergata University, Rome 00133, Italy.
20 Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, Istituto di Ricovero e
Cura a Carattere Scientifico (IRCCS), Aviano 33081, Italy.
21 Department of Hemato-Oncology, Belfast City Hospital, Belfast BT9 7AB, UK.
*Equal contribution.

Introduction: The current shift of therapy of chronic lymphocytic leukemia (CLL) towards novel targeted
agents mandates the identification of new molecular predictors. The aim of this study is to identify new
molecular predictors in FCR treated patients and to assess the biological features underlying chemo-
refractoriness to FCR.

Experimental model: A retrospective multicenter cohort of 287 untreated CLL receiving first-line therapy
with FCR was subjected to a targeted NGS approach in 24 most recurrently mutated genes in CLL. The
entire non-canonical NF-κB pathway was assessed by Western blot and by real-time PCR.

Results: SF3B1 and NOTCH1 were the most frequently mutated genes identified in 13.9% and in 13.6% of
patients respectively, followed by TP53 in 9.4% and ATM in 6.9%. BIRC3 was mutated in 3.1% of patients. By
univariate analysis adjusted for multiple comparisons, only BIRC3 mutations (median PFS of 2.2 years; P <
0.001) and TP53 mutations (median PFS of 2.6 years; P < 0.0001) identified patients who failed early FCR.
By multivariate analysis, BIRC3 mutations maintained an independent risk of progression, with a HR of 2.8
(95%CI 1.4-5.6, P = 0.004). In addition, in vitro studies showed that fludarabine-induced apoptosis in BIRC3
mutated cells was comparable to samples harboring TP53 mutations. Western blotting analysis of the non-
canonical NF-κB pathway showed that BIRC3 mutated cells was significantly enriched of non-canonical NF-
κB target gene and addicted of MAP3K14 overexpression.