Primary Intraocular Lymphomas Display A Remarkably Biased Immunoglobulin Heavy Chain Gene Repertoire and Precisely Targeted Somatic Hypermutation Suggesting Antigenic Selection of the Neoplastic Cells
Authors: Belhouachi Nabila , Stalika Evangelia , Bodaghi Bahram , Boudjoghra Myriam , Cassoux Nathalie , Fardeau Christine , Baudet Sylvie , Hoang Phuc Le , Merle-Beral Helene , Stamatopoulos Kostas , Davi Frederic 
Published: October 2019

Abstract
Abstract 1574
Primary intraocular lymphoma (PIOL) is a high grade lymphoma, which affects the retina, vitreous and/or the optic nerve. The vast majority of cases are classified as diffuse large B cell lymphoma and considered as a subtype of primary central nervous system lymphoma (PCNSL). Because of its rarity, only very few studies have been performed on immunoglobulin (IG) gene repertoire of PIOL mostly in very small series. We here report results from the detailed immunogenetic analysis of the clonal IGHV-IGHD-IGHJ rearrangements in 30 cases of PIOL, the largest series to date. We observed a highly restricted IGHV usage, with a single gene, namely IGHV4-34, present in two thirds of cases (20/30). The second most frequent gene was IGHV3-7, utilized in 10% (3/30) of cases. Thus, only two IGHV genes accounted for more than three quarters of the PIOL repertoire. IGHD3 and IGHD2 subgroup genes were by far the two most frequently used, respectively in 51.9% (14/30) and 27.9% (7/30) of cases. In contrast, the IGHD6 subgroup genes, commonly used in other B cell malignancies, were not detected in any PIOL IGH rearrangement. While IGHJ4 was found in 40% (12/30) cases, IGHJ usage was unusual for the IGHJ5 and IGHJ6 genes, as the former clearly predominated over the latter (10/30 cases, 33.3% vs 4/30 cases, 13.3%). Heavy complementarity-determining region 3 (VH CDR3) length ranged from 7 up to 27 amino acids (median, 14). Twenty of 30 cases carried electropositive VH CDR3s with predicted isoelectric values of 7.0 or greater (up to 13.0). Remarkably, all 3 cases expressing the IGHV3-7 gene had 11 aminoacid-long, electropositive VH CDR3s with shared motifs, enabling their assignment to a cluster with “stereotyped” antigen-binding sites. Except for two unmutated cases, all sequences had a high number of somatic mutations as the mean % of identity from their germline counterpart was 85.9% (range 77.7% to 95.5%). Analysis of the distribution of somatic hypermutation (SHM) in the subgroup of PIOL cases utilizing the IGHV4-34 gene revealed high replacement (R) to silent (S) mutation ratios in CDRs (R/S>3.0) along with low R/S ratios in FRs. Shared replacement mutations (“stereotyped” amino acid (AA) changes) at certain codon positions were identified amongst rearrangements utilizing the IGHV4-34 gene, alluding to an antigen-driven SHM process. Several AA changes identified here were distinct from those previously reported for IGHV4-34 rearrangements in other B cell malignancies, in particular chronic lymphocytic leukemia, mantle-cell lymphoma or Burkitt's lymphoma, and thus can be considered as “PIOL-biased”. In addition, the IGVH4-34 specific motif responsible for binding in superantigenic fashion the N-acetyllactosamine antigenic determinant was altered in only 2/20 IGVH4-34 PIOL sequences; however, the most critical residue of this motif (TRP at position 7 in FR1) was intact in all 20 cases. On these grounds, it could be hypothesized that despite intense SHM activity, these clones retained the ability to bind to and be activated by superantigens. Sequencing multiple clones (13 to 17) in three cases demonstrated intraclonal diversity in all of them, indicating ongoing mutational activity. In conclusion PIOL displays a highly biased IG gene repertoire with very precise targeting and distinctive features of SHM, suggestive of selection by specific (super)antigen(s) in lymphomagenesis.

Disclosures:
No relevant conflicts of interest to declare.