CD49d Prevails over the Novel Recurrent Mutations As Independent Prognosticator of Overall Survival in Chronic Lymphocytic Leukemia
Authors: Dal Bo Michele , Bulian Pietro , Bomben Riccardo , Zucchetto Antonella , Rossi Francesca , Pozzo Federico , Bittolo Tamara , Nanni Paola , Cattarossi Ilaria , Zaina Eva , Chivilò Hillarj , Degan Massimo , Zaja Francesco , Pozzato Gabriele , Chiarenza Annalisa , Di Raimondo Francesco , Del Poeta Giovanni , Rossi Davide , Gaidano Gianluca , Gattei Valter 
Published: October 2019

Abstract
Background. CD49d, the alpha-chain of the integrin heterodimer VLA-4, has been identified among the strongest predictors of overall survival (OS) in chronic lymphocytic leukemia (CLL), along with IGHV mutational status and deletion of the 17p (17p-) chromosome involving TP53 (Bulian et al, JCO, 2014). In addition to TP53, the clinical relevance of NOTCH1, SF3B1 and BIRC3 gene mutations has been recently emphasized.
Aim. To test the relevance of CD49d in molecular subgroups of CLL defined by NOTCH1, SF3B1 and BIRC3 gene mutations.
Methods. The study was based on a cohort of 778 unselected CLL (422 untreated and 356 treated cases) all characterized for CD49d expression (CD49dhigh, ≥30% positive cells by flow cytometry in 229 cases), IGHV mutational status (unmutated, UM, in 262 cases), karyotype abnormalities according to the hierarchical stratification (13q- in 308 cases, +12 in 103 cases, 11q- in 64 cases), tested at diagnosis, along with mutations/deletions (hereinafter, disruption) of TP53 (disrupted in 84 cases, including 58 cases with 17p-), BIRC3 (disrupted in 59 cases), and mutationsof NOTCH1 (mutated in 81 cases), SF3B1 (mutated in 54 cases)tested at diagnosis in 65% of cases, in all cases before therapy. Chromosome abnormalities by FISH were defined by using a 5% cut-off; IGHV, TP53, BIRC3, NOTCH1 and SF3B1 mutations were investigated by Sanger sequencing. Median follow-up of patients was 80 months with 173 deaths.
Results. i) association with CD49d: a CD49dhigh phenotype associated with age ≥65 years (p=0.0001), Rai stage I or higher (p>0.0001), UM IGHV status (p>0.0001), absence of 13q- (p=0.0001), presence of +12 (p<0.0001), presence of NOTCH1 mutations (p<0.0001). ii) CD49d prognostic impact: in univariate analysis, the following covariates had a significant impact on OS: age ≥65 years (hazard ratio/confidence interval (HR/CI)= 3.98/2.77-5.73; p<0.0001), Rai stage I or higher (HR/CI=1.81/1.33-2.46; p=0.0002), high CD49d expression (HR/CI=2.62/1.94-3.54; p<0.0001), UM IGHV status (HR/CI=3.03/2.24-4.10; p<0.0001), presence of 13q- (HR/CI=0.52/0.37-0.71; p=0.0001), +12 (HR/CI=1.59/1.08-2.33; p=0.0183), 11q- (HR/CI=2.16/1.42-3.30; p=0.0004), NOTCH1 mutations (HR/CI=2.66/1.82-3.88; p<0.0001), SF3B1 mutations (HR/CI=1.99/1.24-3.20; p=0.0048), BIRC3 disruption (HR/CI=2.41/1.58-3.68; p<0.0001), TP53 disruption (HR/CI=3.23/2.28-4.57; p<0.0001). In a multivariate analysis model including all these variables, high CD49d expression (HR/CI=1.76/1.28-2.42; p=0.0006), UM IGHV status (HR/CI=2.21/1.58-3.09; p<0.0001), presence of 11q- (HR/CI=2.11/1.35-3.31; p=0.0011), TP53 disruption (HR/CI=2.93/2.04-4.22; p<0.0001), and NOTCH1 mutations (HR/CI=1.76/1.16-2.67; p=0.0082) emerged as independent prognosticators, along with age ≥65 years (HR/CI=3.73/2.58-5.39; p<0.0001). iii) CD49d prognostic impact using the integrated hierarchical mutational/cytogenetic model: by integrating gene mutations and cytogenetic aberrations according to Rossi et al (Blood, 2013), a multivariate analysis model was applied with the following covariates: age, CD49d, IGHV status, TP53/BIRC3 disruption, NOTCH1 mutations/SF3B1 mutations/11q-, normal karyotype/+12, 13q-. Again, CD49dhigh phenotype (HR/CI=1.88/1.38-2.54; p=0.0001), UM IGHV status (HR/CI=2.16/1.54-3.01; p<0.0001), presence of NOTCH1 mutations/SF3B1 mutations/11q- (HR/CI=1.62/1.11-2.38; p=0.0139), and of TP53/BIRC3 disruption (HR/CI=2.67/1.92-3.70; p<0.0001) retained their independent prognostic impact, along with age ≥65 years (HR/CI=3.58/2.28-5.17; p<0.0001). Notably, CD49dhigh CLL patients experienced shorter OS than the CD49dlow cases in the context of each mutational/cytogenetic group (TP53/BIRC3 disruption, p=0.0064; NOTCH1 mutations/SF3B1 mutations/11q-, p=0.05; normal karyotype/+12, p=0.0153; 13q-, p=0.0126; see Figure).
Conclusion. CD49d was confirmed as independent negative OS prognosticator in CLL also when the novel recurrent alterations of NOTCH1, BIRC3, and SF3B1 genes were considered. In this setting, TP53 disruption and NOTCH1 mutations remained independent OS prognosticators, allegedly for their physiopathological roles in CLL cell immuno-chemoresistance.
Figure 1. Figure 1.

Disclosures
Gaidano: Morphosys, Roche, Novartis, GlaxoSmith Kline, Amgen, Janssen, Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding.