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Letters to the Editor
functions of these B-cell subpopulations. In order to iden- tify BcR IG stereotypes, we applied our purpose-built algorithm2 on all abundant BcR IG clonotypes (frequency of >0.92%) of the present cohort along with 30,221 CLL clonotypes from the IMGT/CLL-DB (http://www.imgt.org/ CLLDBInterface/query). According to our findings, 43/222 (19.4%) abundant clonotypes were assigned to 42 dis- tinct clusters (Online Supplementary Table S8).
The vast majority of abundant clonotypes with stereo- typed VH CDR3 (42/43, 97.7%) were found in samples from individuals with LC-MBL (12 in MBL cell samples and 30 in PBMC samples). Virtually all abundant stereo- typed LC-MBL clonotypes (47/48, 97.9%) were assigned to minor clusters with a mean size of four clonotypes (range, 2-22 clonotypes). Only a single abundant clono- type was assigned to a large CLL subset, namely subset #148B11 (CLUSTER-4-0003), which contained 150 clono- types in the current analysis. Hence, “CLL-specific” stereotyped clonotypes were observed in most samples, yet these were scant and, for the most part, exhibited low frequency. In the case of subset #148B, which was the only well-documented CLL subset from the present analysis, its biological and clinical characteristics were “compatible” with LC-MBL: a high frequency of del(13q), low frequency of CD38 positivity, young age at diagnosis and a long time to first treatment (9.2 years).11
The complete absence of “CLL-specific” BcR IG stereo- types belonging to major CLL stereotyped subsets2 prompted us to search for such sequences among all expanded BcR IG clonotypes (>1 sequence), irrespective of individual clonotype frequency. We identified 142 of 238,075 (0.0006%) clonotypes belonging to 14 major CLL subsets (Online Supplementary Table S9) in 27/42 samples (64.3%) from all sample categories, although at very low frequencies (range, 0.0002-0.28%). Most “CLL- specific” BcR IG stereotypes were found in naïve B-cell samples (average: 8.5); in contrast, MBL and PBMC sam- ples from LC-MBL had the fewest (averages: 1.3 and 1.1, respectively).
Interestingly, the distribution of “CLL-specific” BcR IG stereotypes differed from that reported in CLL2 (Figure 2) as shown by the low incidence of subsets #1 and #2, the largest in CLL, and the absence of rearrangements similar to those of CLL subsets #8, #31, #59 and #99, all associ- ated with aggressive disease.11 Furthermore, stereotyped clonotypes typical of CLL subset #42 were significantly (P<0.05) biased to naïve B-cell populations. This BcR IG stereotype is distinctive for utilizing the IGHV4-34 gene, notable for its germline-encoded autoreactive potential.12 This gene is frequent in naïve B cells but suppressed in classical memory B cells and, instead, enriched in autoim- mune repertoires and certain lymphoproliferations,13 prompting an argument for stringent censoring of IGHV4-34 B cells in healthy individuals. This appears to be supported by the present study as well, in which we found that LC-MBL is devoid of CLL stereotypes related to aggressive disease and that the subset #4 stereotype is confined to the naïve B-cell repertoire.
In conclusion, in the first next-generation sequencing immunoprofiling study of LC-MBL we report differences from age-matched individuals without MBL. Critically, the very low incidence of “CLL-specific” stereotyped BcR IG in LC-MBL (as well as in elderly individuals without LC-MBL) further attests to the unique immunogenetic signature of CLL while also highlighting the role of microenvironmental triggering, mediated through the BcR, as a major driver even before the onset of CLL.14 This could also explain the low incidence of CLL in the East, since not only the genetic background but also
environmental triggers could differ between Asian and Caucasian populations.9,15
Andreas Agathangelidis,1,2 Chrysi Galigalidou,2,3
Lydia Scarfò,1 Theodoros Moysiadis,2,4 Alessandra Rovida,1 Maria Gounari,2 Fotis Psomopoulos,2 Pamela Ranghetti,1 Alex Galanis,3 Frederic Davi,5 Kostas Stamatopoulos,2,4 Anastasia Chatzidimitriou2,4# and Paolo Ghia1# also on behalf of the Euroclonality NGS Working Group
1Strategic Research Program on CLL and B-Cell Neoplasia Unit, Division of Experimental Oncology, Università Vita-Salute San Raffaele and IRCCS Ospedale San Raffaele, Milan, Italy; 2Institute of Applied Biosciences, Center for Research and Technology Hellas, Thessaloniki, Greece; 3Department of Molecular Biology and Genetics (MBG), Democritus University of Thrace, Alexandroupolis, Greece; 4Department of Molecular Medicine and Surgery, Karolinska Institute,
5
Stockholm, Sweden and Assistance Publique - Hôpitaux de Paris
(AP-HP), Hôpital Pitié-Salpêtrière, Department of Biological Hematology, Sorbonne Université, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France
#AC and PG contributed equally as co-senior authors.
Correspondence:
ANASTASIA CHATZIDIMITRIOU - achatzidimitriou@certh.gr
doi:10.3324/haematol.2020.247908
Disclosures: AA has received funding from Gilead. KS has received honoraria from AbbVie, Gilead Science, Janssen and reseach funding from AbbVie and Janssen. PG has received honoraria from AbbVie, Acerta, BeiGene, Gilead, Janssen, Sunesis and reseach funding from AbbVie, Gilead, Janssen, Novartis, and Sunesis.
Contributions: AA designed the research, performed the experiments, analyzed the data and wrote the manuscript; CG analyzed the data and wrote the manuscript; LS, AR, MG and PR assisted with the experiments; TM performed the statistical analysis; FP assisted with the data analysis; AG and FD supervised the research and wrote the manuscript; KS, AH and PG designed and supervised the research and wrote the manuscript. All authors provided final approval of the manuscript.
Funding: this project received funding from the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), under grant agreement n. 336 (Project CLLon); the TRANSCAN-2 Joint Transnational Call for Proposals 2014 (JTC 2014) by the European Commission/DG Research and Innovation; the TRANSCAN-2: Employing next-gener- ation sequencing technology for improved, non-invasive early detection, staging and prediction of progression in lymphoma patients (NOVEL/MIS 5041673); the project “KRIPIS II ODYSSEUS” funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co- financed by Greece and the European Union (European Regional Development Fund); the Italian Association for Cancer Research (AIRC, Special Program on Metastasis, 5 per mille #21198 to PG); PRIN 2015ZMRFEA, Italian Ministry of University and Research - MIUR, Roma, Italy; open access project, ID number LM2011020, funded by the Ministry of Education, Youth and Sports of the Czech Republic under the activity “Projects of major infrastructures for research, development and innovations”; the Greek Precision Medicine Network, a mission of the Research and Innovation sector of the Ministry of Education, Research and Religious Affairs of Greece; the Asklepios Grant Programme grant from Gilead Hellas. MG is recipient of a Marie Sklodowska-Curie individual fellowship (grant agreement n. 796491), funded by the European Union’s Horizon 2020 research and innovation programme.
References
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