HIGH-THROUGHPUT Β-CELL IMMUNOPROFILING AT DIAGNOSIS AND RELAPSE OFFERS FURTHER EVIDENCE OF FUNCTIONAL SELECTION THROUGHOUT THE NATURAL HISTORY OF CHRONIC LYMPHOCYTIC LEUKEMIA
Author(s): ,
Anna Vardi
Affiliations:
Hematology Department & HCT Unit,G.Papanikolaou Hospital,Thessaloniki,Greece;Institute of Applied Biosciences,Centre for Research and Technology Hellas,Thessaloniki,Greece;Department of Molecular Medicine and Surgery,Karolinska Institutet,Stockholm,Sweden
,
Elisavet Vlachonikola
Affiliations:
Department of Genetics, Development and Molecular Biology,Aristotle University of Thessaloniki,Thessaloniki,Greece;Institute of Applied Biosciences,Centre for Research and Technology Hellas,Thessaloniki,Greece
,
Sofia Mourati
Affiliations:
Department of Molecular Biology and Genetics,Democritus University of Thrace,Alexandroupolis,Greece;Institute of Applied Biosciences,Centre for Research and Technology Hellas,Thessaloniki,Greece
,
Fotis Psomopoulos
Affiliations:
Institute of Applied Biosciences,Centre for Research and Technology Hellas,Thessaloniki,Greece;Department of Molecular Medicine and Surgery,Karolinska Institutet,Stockholm,Sweden
,
Nikos Pantouloufos
Affiliations:
Department of Molecular Biology and Genetics,Democritus University of Thrace,Alexandroupolis,Greece;Institute of Applied Biosciences,Centre for Research and Technology Hellas,Thessaloniki,Greece
,
Anastasia Kouvatsi
Affiliations:
Department of Genetics, Development and Molecular Biology,Aristotle University of Thessaloniki,Thessaloniki,Greece
,
Niki Stavroyianni
Affiliations:
Hematology Department & HCT Unit,G.Papanikolaou Hospital,Thessaloniki,Greece
,
Achilles Anagnostopoulos
Affiliations:
Hematology Department & HCT Unit,G.Papanikolaou Hospital,Thessaloniki,Greece
,
Kostas Stamatopoulos
Affiliations:
Institute of Applied Biosciences,Centre for Research and Technology Hellas,Thessaloniki,Greece
Anastasia Hadzidimitriou
Affiliations:
Institute of Applied Biosciences,Centre for Research and Technology Hellas,Thessaloniki,Greece
EHA Library. Vardi A. Jun 15, 2019; 266748; PS1131
Anna Vardi
Anna Vardi
Contributions
Abstract

Abstract: PS1131

Type: Poster Presentation

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

Background

Chronic lymphocytic leukemia (CLL) is divided into two broad prognostic categories, namely mutated (M) and unmutated (U) CLL, according to the somatic hypermutation (SHM) status of the clonotypic heavy chain immunoglobulin (IGHV) gene. This is perceived to remain stable over time, as evidenced by low-throughput studies, which however precluded investigation of subclonal architecture and evolution overtime.  

Aims

Here, we sought to comprehensively assess the B cell receptor (BcR) IG gene repertoire at CLL diagnosis and 1st relapse after chemoimmunotherapy (FCR) by next-generation sequencing (NGS).

Methods

We studied 5 patients, of whom 1 each expressed BcR IG typical of stereotyped subset #1 (U-CLL), subset #4 (M-CLL) or subset #6 (U-CLL), whereas the remaining 2 concerned non-stereotyped U-CLL. Genomic DNA was amplified by multiplex PCR and products were subjected to paired-end NGS. Sequence data were processed by a validated bioinformatics pipeline performing strict quality filtering. Rearrangements with identical IGHV gene and CDR3 amino acid (aa) sequence were defined as clonotypes. The dominant clonotype was defined as major, and clonotypes with the same IGHV gene, CDR3 length, and ≤2 aa differences within the CDR3 were considered as its satellite subclones.

Results

In total, we analyzed 1,682,728 filtered-in, productive IGHV-IGHD-IGHJ gene rearrangements (median 195,460 rearrangements/sample). In all cases, the major clonotype (IGHV and CDR3 aa sequence) was identical to that determined by Sanger sequencing and remained the same at diagnosis and relapse. However, it consisted of multiple distinct nucleotide (nt) sequences; while most nt sequences displayed the same SHM load as with Sanger (median 73.9% of all nt sequences of the major clonotype), there were other nt sequences with higher or lower SHM load. In all cases, nt sequences with discordant SHM status were practically negligible (median frequency 0.01%). Notably, the relative frequency of the dominant nt sequence increased at relapse for all cases (median increase by 3.7%, p<0.01). A particular note for subset #4, which by definition displays high SHM, was the emergence of major clonotype nt sequences with lower or even absent SHM at relapse. The median frequency of the major clonotype at diagnosis was 92.5% (range 86.1-93.3%) and remained stable at relapse (92.6%, range 89.9-93.8%). In all cases, the major clonotype came along with numerous satellite subclones (same IGHV gene, CDR3 length, and ≤2 aa differences within the CDR3) at both diagnosis and relapse (median n=685 vs n=604 respectively, p>0.05). When considering both the major clonotype and its satellite subclones, the respective cumulative frequency corresponded to nearly the entire B cell repertoire/sample (median 99.6%, range 97.6-99.8%). Subset #4 satellite sublones displayed recurrent SHM patterns within the CDR3 at relapse, which actually diverged from the major clonotype and converged towards the consensus CDR3 sequence for this subset as defined by 176 different subset #4 patients.

Conclusion

Overall, our study provides insight into the evolving architecture of the BcR IG repertoire of relapsing CLL, revealing that, similar to genomic subclones, there is a universe of BcR IG subclones as well. While the possibility of technical error cannot be excluded, the identification of recurrent SHM patterns and trends at relapse strongly points towards functional selection.

Session topic: 5. Chronic lymphocytic leukemia and related disorders - Biology & Translational Research

Keyword(s): Chronic lymphocytic leukemia

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