IGHV Gene Replacement: A Potential Mechanism for Establishing Stereotypy in Certain Cases of Chronic Lymphocytic Leukemia
Authors: Yancopoulos Sophia , Li Wentian , Yan Xiao J. , Agathangelidis Andreas , Hadzidimitriou Anastasia , Davi Frederic , Rosenquist Richard , Ghia Paolo , Stamatopoulos Kostas , Chiorazzi Nicholas 
Published: October 2019

Abstract
Immunoglobulin heavy chain variable gene (IGHV) replacement or "VH replacement" (VHR) modifies a rearranged IGHV-D-J sequence by replacing the original IGHV gene with another. This process leaves a detectible "footprint" at the IGHV-D junction of the existing sequence. Roughly 33% of chronic lymphocytic leukemia (CLL) cases exhibit stereotyped B cell receptors (BCRs) often characterized by signature VH CDR3 amino acids. Various mechanisms have been put forth to account for stereotypy in CLL. An overarching hypothesis is that the stereotyped BCRs are antigen driven. Within this concept, a variety of mechanisms could lead to the signatures including somatic mutations and addition/deletion of nucleotides at junctional regions. Here we explore the possibility that VHR provides another mechanism to account for some of the stereotyped rearrangements and some of their signature VH CDR3 amino acid residues in CLL.
We examined IG sequences of 26,642 CLL cases and ~16 million healthy controls (HC) to find relic footprints as indicators of VHR. This was done using the VHRFA program developed by Lin Huang et al (PLoS ONE, 2013), as well as our own program which duplicates the VHRFA results but is better able to process large numbers of sequences.
The frequency of VHR was similar in CLL and HC (11.6 and 11.9%, respectively). Focusing solely on CLL sequences to define a relationship between VHR and stereotypy, we found highly significant differences in VHR frequencies between stereotyped (n=8,568) and non-stereotyped cases (n=18,074), with stereotyped cases exhibiting VHR at a greatly reduced frequency (7.7% vs. 13.5%, respectively). When comparing VHR frequencies between stereotyped cases and non-stereotyped cases that used the same IGHV, we found that the number of subsets with low VHR exceeded those with elevated VHR ~2:1, accounting for the overall VHR in stereotyped cases being lower than non-stereotyped cases. Further restricting comparisons of stereotyped subsets to non-stereotyped cohorts by matching VH CDR3 length led to similar conclusions.
Within stereotyped cases there was a wide distribution of VHR, ranging from 55.6% to 0.1%. Restricting VH CDR3 lengths to "short" (5 - ≤13), "medium" (13.1 - ≤20) and "long" (20.1 - ≤28), the corresponding VHR increased monotonically with length (1.1, 8.2, and 11.9% respectively). Notably, subsets showing elevated VH replacement included better prognosis subsets, #4, 77 and 201 (23.8, 22.1, and 28.6%, respectively). Among low VHR frequency subsets were those associated with worse prognosis, #1, 2, 5, 6, 8, 9 and 10 (VHR frequencies: 0.2, 0.1, 0.9, 2.3, 7.7, 9.0 %, respectively). This was most strikingly exhibited by subsets #1 and #2, both of which comprise patients with poor clinical courses. Each of these sets of sequences displayed virtually no examples of VHR (0.2 and 0.1%, respectively). This might be predicted because these two subsets have relatively short VH CDR3 lengths (subset #1: 13 aa; subset #2: 9 aa), based on the length association mentioned above.
Detailed analyses of the presence of footprints and the position of these in the rearranged IGHV-D-J indicated that for some subsets, certain signature VH CDR3 amino acids could be the result of VHR. For example in subset #201, sequence analysis suggests that VHR is responsible for an arginine and for a glutamine in the 5' portion of the VH CDR3. Similarly, VHR may craft the characteristic glutamine on the 5' end of the subset #6 VH CDR3.
Thus, our studies indicate that, as a whole, CLL IGHV-D-J sequences use VHR at a frequency comparable to that of normal B cells and significantly less than that of non-stereotyped rearrangements. However, certain stereotyped cases are dramatically enriched for evidence of VHR. Moreover among these cases, the footprints found in the VH CDR3s of stereotyped cases can be shown to directly code for signature amino acids in VH CDR3s. Finally, stereotyped cases with high levels of VHR tend to be those with better clinical courses, whereas those worse outcome stereotyped cases exhibit less evidence for this process. This latter finding is consistent with the concept that VHR is one of the molecular mechanisms used by developing B cells to edit BCRs having high affinity for autoantigens. Since many CLL BCRs are autoreactive, including those found to have high levels of VHR such as subset #4, this implies a fundamental defect in tolerance mechanisms in those normal B cells that eventually became leukemic.

Disclosures
Agathangelidis: Gilead: Research Funding. Hadzidimitriou:Janssen: Honoraria, Research Funding; Gilead: Research Funding; Abbvie: Research Funding. Ghia:AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Sunesis: Honoraria, Research Funding. Stamatopoulos:Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.