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1839 Remarkable Functional Constraints on the Antigen Receptors of CLL Stereotyped Subset #2: High-Throughput Immunogenetic Evidence

Program: Oral and Poster Abstracts
Session: 641. CLL: Biology and Pathophysiology, excluding Therapy: Poster I
Hematology Disease Topics & Pathways:
Leukemia, Diseases, CLL, Lymphoid Malignancies
Saturday, December 1, 2018, 6:15 PM-8:15 PM
Hall GH (San Diego Convention Center)

Katerina Gemenetzi1,2*, Andreas Agathangelidis, PhD1*, Lesley-Ann Sutton3*, Elisavet Vlachonikola1,4*, Chrysi Galigalidou1,2*, Fotis Psomopoulos1*, Maria Gounari1*, Achilles Anagnostopoulos5, Raphael Sandaltzopoulos2*, Richard Rosenquist, MD, PhD3, Kostas Stamatopoulos, MD, PhD1 and Anastasia Hadzidimitriou, PhD1,3*

1Institute of Applied Biosciences (INAB), Centre for Research and Technology Hellas (CERTH), Thessaloniki, Greece
2Democritus University of Thrace (DUTH), Department of Molecular Biology and Genetics (MBG), Alexandroupolis, Greece
3Karolinska Institutet, Stockholm, Sweden
4Department of Genetics, Development and Molecular Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece
5Hematology Department & HCT Unit, G. Papanikolaou Hospital, Thessaloniki, Greece

Subset #2 is the largest subset carrying stereotyped B cell receptor immunoglobulin (BcR IG) in chronic lymphocytic leukemia (CLL). This particular BcR IG is composed of heavy (HC) and light (LC) chains encoded by the IGHV3-21 and the lambda IGLV3-21 gene, respectively. The clonotypic IGHV3-21 genes display a variable load of somatic hypermutation (SHM), being mostly classified as mutated (M-CLL) but also including unmutated (U-CLL) cases. Subset #2 cases, independently of the SHM status, have a particularly dismal clinical outcome similar to that of patients with TP53 aberrations, although lacking such aberrations. Subset #2 BcR IG display a series of distinctive features, including conservation at certain VH and VL CDR3 positions and recurrent SHMs; as well as a capacity for self-association leading to cell autonomous signaling that is critically dependent on a substitution of Arginine (R) for Glycine (G) introduced by SHM at the lambda VL-CL linker region. These features implicate antigen selection in CLL subset #2 ontogeny. However, the available molecular evidence derives from low throughput immunogenetic analysis, precluding comprehensive assessment of antigenic impact on (sub)clonal composition. Here, we sought to overcome this limitation by performing next-generation sequencing (NGS) of HC and LC gene rearrangements of 20 subset #2 patients. RT-PCR products amplified by the IGHV3-21/IGHJ6 and IGLV3-21/IGLC primer pairs, respectively, were subjected to NGS on the MiSeq Illumina Platform. NGS data was analyzed by a validated bioinformatics pipeline. Rearrangements with identical CDR3 amino acid (aa) sequences were defined as clonotypes, whereas clonotypes with different aa substitutions within the V-domain were defined as subclones. Starting with HCs, we obtained 3,340,508 (mean: 291,751, range: 101,231-186,055) productive reads. On average, each analyzed sample carried 92 distinct clonotypes (range: 71-152), with the dominant clonotype having a mean frequency of 96% (range: 67-99%): in all cases the dominant clonotype was identical to that determined by Sanger sequencing. The dominant clonotype displayed considerable intraclonal heterogeneity with a mean of 5,082 subclones/sample (range: 2,946-11,041). Turning to LCs, we obtained 5,094,045 (mean: 231,547, range: 38,036-507,949) productive reads. LCs carried a higher number of distinct clonotypes/sample compared to their partner HCs (mean 222, range: 156-306). The dominant clonotype had a mean frequency of 96% (range: 74-98%); similar to HCs, it was identical to that determined by Sanger sequencing. Intraclonal heterogeneity was observed in the LCs as well, with a mean of 7,382 subclones/sample (range: 1,946-11,866), hence more pronounced vs their partner HCs. Viewing the entire subset #2 VH or VL CDR3 dataset (i.e. the CDR3 aa sequences from all clonotypes of all cases) as a single entity branching through diversification enabled the identification of 2 distinct VH CDR3 sequences present at varying frequencies in 16 and 13 cases, respectively; and, 3 distinct VL CDR3 sequences present at varying frequencies in all 20 cases: these results allude to important constraints on the composition of the antigen binding site. Focusing on SHM, the following notable observations could be made. (i) The G-to-R substitution at the VL-CL linker was a clonal event in all cases with R being degenerately encoded by different nucleotide sequences; altogether, these findings underscore the seminal role of this recurrent SHM, likely due to mediating self-association. (ii) A recurrent 3-nucleotide deletion was detected in the VH CDR2 of all cases, strongly supporting functional pressure. This change, previously identified by Sanger sequencing as a recurrent SHM in subset #2 (albeit at a frequency of only 25%), was clonal in 4 cases and subclonal in the remainder, where it was present in an average of 105 subclones/sample (range: 1-369). (iii) Certain positions in both the VH and VL domain bore the same aa substitution, mostly at subclonal level: the prime example concerned the G for Serine (S) substitution within the VL CDR3, detected in all samples at a mean frequency of 44.2% (range: 6.3-87%). In conclusion, we provide compelling immunogenetic evidence for functional pressure in the ontogeny of CLL subset #2. On this evidence, subset #2 emerges as perhaps the most striking example of antigen-driven leukemogenesis reported thus far.

Disclosures: Gemenetzi: Gilead: Research Funding. Agathangelidis: Gilead: Research Funding. Stamatopoulos: Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Hadzidimitriou: Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding.

*signifies non-member of ASH