Insulin Growth Factor 1 Receptor Expression Is Associated with NOTCH1 Mutation, Trisomy 12 and Aggressive Clinical Course in Chronic Lymphocytic Leukemia
Authors: Maura Francesco , Mosca Laura , Sonia Fabris , Cutrona Giovanna , Matis Serena , Lionetti Marta , Berbieri Marzia , Danca Marianna , Colombo Monica , Massucco Carlotta , Reverberi Daniele , Gentile Massimo , Recchia Anna Grazia , Bossio Sabrina , Ilariucci Fiorella , Musolino Caterina , Di Raimondo Francesco , Cortelezzi Agostino , Morabito Fortunato , Ferrarini Manlio , Neri Antonino 
Published: October 2019

Abstract
Insulin growth factor 1 receptor (IGF1R) is emerging as an important gene in many solid and hematological cancers and its over expression has been reported to be associated with aggressive disease and pharmacologic resistance. Specifically, the IGF1R-IGF1-2 interaction was recently described to be involved in the constitutive activation of many important cell signaling such as NOTCH1 and PI3K/Akt pathways that play a key role in many solid and hematological cancers. In this study we performed a clinical and biological investigations about the role of IGF1R expression in a large and representative prospective series of chronic lymphocytic leukemia (CLL) in Binet A clinical stage enrolled in observation O-CLL1 protocol (clinicaltrial.gov identifier NCT00917540). Total RNA extraction, preparation of DNA single-stranded sense target, and hybridization to gene expression profiling arrays were carried out according to manufacturer’s protocols in 217 CLL patients enrolled in the multicentre O-CLL1 protocol. Gene expression data has been deposited in the National Centre for Biotechnology Information’s Gene Expression Omnibus database http://www.ncbi.nlm.mih.gov/geo and are accessible through series accession number GSE51529. High IGF1R expression was significantly associated with IGHV unmutated (IGHV-UM) status (p<0.0001), high ZAP-70 and CD38 expression (p<0.0001) and unfavorable cytogenetic deletion [i.e. del(11)(q23) and del(17)(p13)], particularly with del(11)(q23) (p=0.03). Cases with del(13)(q14) as single lesion were characterized by the lowest IGF1R gene levels. On the contrary, among the most common cytogenetic aberrations, trisomy 12 showed stronger IGF1R expression compared with the other patients (p<0.0001) and this association was independent from IGHV mutational status. Patients with stereotyped HCDR3 sequences showed a greater IGF1R expression compared to not stereotyped HCDR3 (p=0.001) even if this can be related to the high frequency of IGHV-UM among stereotyped HCDR patients. Interestingly, subset #4 patients, who are known to exhibit an indolent clinical course and distinct biological profile, were also characterized by lower IGF1R expression compared to other M-IGHV and UM-IGHV patients. NOTCH1 c.7541_7542delCT mutation was investigated by next generation sequencing Roche 454 technology in 199 (92%) patients (Lionetti et al, BJH 2014). Globally, median depth of coverage was 1510x, ranging from 605 to 2842. Mutant allele frequency estimated by NGS ranged from 0.02% to 75% of total reads per sample. The presence of NOTCH1 mutation was confirmed by ASO-PCR and Sanger sequencing in all patients with allele burden higher > 0.7% (31; 15.5%) and 7% (19; 9.5%) respectively. We considered as mutated only the 31 patients in whom the presence of the dinucleotide deletion was confirmed by ARMS-PCR. Patients carrying NOTCH1 mutation were characterized by a greater IGF1R expression compared with wild type cases (p=0.002). In addition high IGF1R expression was not significantly different comparing patients with low and high NOTCH1 mutation burden. In order to avoid the bias represented by the strong association between NOTCH1 and trisomy 12, we compared the IGF1R expression between NOTCH1 mutated and wild type cases excluding trisomy 12, and confirmed the previous association (p=0.004). IGF1R expression represented a strong clinical prognostic factor in our CLL cohort: by Kaplan-Maier analysis we observed a significant time to first treatment stratification in all CLLs, in all IGHV-UM and in all IGHV-M patients (p<0.0001). Furthermore, IGF1R retained its significance in multivariate analysis with most important clinical and molecular prognostic factors (CD38 expression, unfavorable FISH and IGHV mutational status). Overall, our study shows the importance of IGF1R expression in CLL and its strong association with specific clinical and biological features, confirming the interest for the study of this gene as a potential prognostic factor and its possible role as a therapeutic target in a specific group of CLL patients carrying trisomy 12 and NOTCH1 mutations.

Disclosures
No relevant conflicts of interest to declare.