Intraclonal Diversification Occurs in Chronic Lymphocytic Leukemia Expressing B Cell Receptors Belonging to the IGHV4 Gene Family
Authors: Vit Filippo , Rossi Francesca Maria , D'Agaro Tiziana , Bittolo Tamara , Zucchetto Antonella , Tissino Erika , Pozzo Federico , Bulian Pietro , Degan Massimo , Zaina Eva , Cattarossi Ilaria , Nanni Paola , Chivilò Hillarj , D'Arena Giovanni Francesco , Santinelli Enrico , Chiarenza Annalisa , Zaja Francesco , Pozzato Gabriele , Di Raimondo Francesco , Del Poeta Giovanni , Gattei Valter , Bomben Riccardo 
Published: October 2019

Abstract
Background. The pivotal role of the Immunoglobulin (Ig) receptor and antigenic stimulation have been proven to be landmarks for the understanding of the ontogeny and evolution of chronic lymphocytic leukemia (CLL). In addition, the mutational status of the Immunoglobulin Heavy-Chain Variable region gene (IGHV) was confirmed to be a reliable prognostic factor, supporting an antigen-driven model of CLL development. To clarify aspects regarding an antigenic involvement in CLL evolution, studies focusing on intraclonal diversification (ID) of Ig genes have provided relevant information, although mainly conducted in a pre-Next Generation Sequencing (NGS) era.
Aim. To apply a NGS approach to investigate ID in CLL.
Methods. IGHV genes from 530 CLL patients with Royal Masden Hospital score 4-5 (Fig. 1A) was sequenced using NGS (Lymphotrack). The bio-informatic pipeline was based on the pRESTO/ChangeO packages. Specific pathological clones were selected based on the presence of same IGHV, junction genes and with similar HCDR3 sequence according to Hamming's distance. Through the R-Alakazam package, we generated rarefaction curves to evaluate the clonal diversity inside the pathological clone (Fig. 1B). Focusing on the Simpson index (represented by the Hill number of order q=2), which gives more weight to larger clones minimizing the smaller ones (Fig. 1B), we selected a Diversity Score (DS) of 4 for the definition of cases without ID (clonal; DS <4) and cases with ID (intraclonal; DS ≥4) (Fig. 1B).
Results. Using the reported threshold we identified 469 (88.5%) clonal cases, expressing a single clone (Fig. 1C), and 61 (11.5%) cases with ID (median DS 9.2, range 4.4-66.0) characterized by the presence of two or multiple pathological clones expressing the same IGHV gene and HCDR3 (Fig. 1C). Notably, cases with ID expressed both a mutated (M) (39/61, 63.9%) and an unmutated (UM) (22/61, 36.1%; p=0.066) IGHV gene configuration (Fig. 1C). Of note, we observed a significant skewing toward the usage of VH4-family genes when comparing cases with ID (38/61, 62.3%) vs. cases without ID (78/469, 16.6%; p<0.0001, Fig 1D). Moreover, the IGHV4-39 and IGHV4-34 genes were the most used genes in the context of cases with ID (Fig. 1E), although none of them belonging to known stereotyped subsets. By focusing on VH4-family only cases, we observed that cases with ID and UM IGHV genes displayed higher mutation frequencies in WA/TW motifs, a mutational signature which suggests an involvement of both Activation-Induced (Cytidine) Deaminase (AID) and error-prone polymerase eta (Fig. 1F), a pattern not observed in its counterpart with UM IGHV genes but without ID (Fig. 1F). Conversely, in cases with ID and M IGHV genes, mutations preferentially clustered in AID hotspots (WRC/GYW motifs), suggesting a direct role of AID and the Base Excision Repair machinery in the mutational overload (Fig. 1F). Consistently, M IGHV cases with ID expressed significantly higher AID mRNA levels than M IGHV cases without ID (p=0.0024; Fig. 1G). These expression levels were overall comparable with those found in UM IGHV cases, irrespective to the evidence of ID (Fig. 1G), which however were not associated with an increased number of mutations in AID-specific hotspots (Fig. 1F).
Conclusions. By taking advantage of a new method for ID assessment in CLL, we demonstrated that ID prevalently affects VH4-family cases which display different mutational patterns dependent to the IGHV gene status. This data are in keeping with previous reports indicating the IGHV4 genes as particularly prone to generate immunoglobulin subjected to continuous/persistent stimulation by external/auto-antigens, hence particularly prone to generate features of ID. Further experiments in selected cases with ID through a non-random barcode strategy are needed.

Disclosures
Zaja: Sandoz: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Abbvie: Honoraria.