Temporal Dynamics of Clonal Evolution in Chronic Lymphocytic Leukemia with Stereotyped IGHV4-34/IGKV2-30 Antigen Receptors: Longitudinal Immunogenetic Evidence

Abstract Abstract 2878 We recently demonstrated that intraclonal diversification (ID) in the immunoglobulin (IG) genes of patients with chronic lymphocytic leukemia (CLL) was limited, with the outstanding exception of subset #4 cases (IGHV4–34/IGKV2–30). Subset #4 cases express IgG-switched antigen receptors carrying long VH CDR3s enriched in positively charged amino acid residues (especially arginine), with acidic residues introduced by somatic hypermutation (SHM) in critical positions of both the heavy and light chain variable domains. This group of patients, characterized clinically by an early age at diagnosis and an indolent disease course, exhibited distinctive patterns of intraclonal diversification (ID) within their IG genes. This may be considered as evidence for an ongoing response to active interaction with antigen (Ag), however, the critical question about the precise timing of Ag involvement and its role in clonal evolution remains unknown. To obtain insight into these issues, we conducted a large-scale subcloning study of the IG genes in a total of 514 and 398 subcloned IG heavy and kappa sequences, obtained from overtime samples from 8 subset #4 cases. All non-ubiquitous sequence changes from the germline among subcloned sequences of the same patient from the same timepoint were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence; (ii) confirmed mutation (CM) - a mutation observed in more than one but in less than all subcloned sequences. Overall, all cases carried intraclonally diversified IG genes. Detailed analysis of the topology and characteristics of mutations revealed: i) restricted ID patterns, in the sense of identical mutations at certain VH/VK positions amongst subclones of different cases, e.g. despite glycine at codon 28 (VH CDR1) being mutated in 413/514 (80%) sequences, the only observed substitution was to an acidic residue, ii) ID hotspots, i.e. mutations in certain codons were repeatedly observed during clonal evolution, and iii) predominance of conservative amino acid changes. Furthermore, the analysis of consecutive samples enabled us to trace the diversification of the CLL antibody over time, and describe the level of ID as increasing, decreasing, or complex when a mutation appears, disappears and then re-emerges at a subsequent time point, thereby also revealing which mutations were negatively or positively selected. Consequently, a stepwise accumulation of mutations could be observed with several CMs at an early timepoint becoming ubiquitous mutations, i.e. present in all subcloned sequences of subsequent timepoints. In addition, distinct clusters of subcloned sequences with cluster-specific mutational profiles were observed initially, however at later timepoints the minor cluster had often disappeared and hence been selected against. Despite the high intensity of ID, it was remarkable that certain residues such as in the VH FR1 motif responsible for recognizing the I/i NAL epitope remained essentially unaltered (only 6/514 sequences carried alterations at codon 7 VH FR1). In conclusion, this study defines a clear role for Ag selection in the clonal evolution of CLL subset #4. Whilst the critical eliciting Ag cannot be definitively determined, it is tempting to hypothesize that the distinctive modifications introduced by SHM in the stereotyped BcR likely represent a mechanism for negating auto-reactivity mediated by the arginine-rich VH CDR3s and inducing an anergic state which could potentially be re-activated by subsequent (auto)antigenic stimulation as evidenced by ongoing SHM. It remains to be established whether this stimulation is also accompanied by a change in functional status. Disclosures: No relevant conflicts of interest to declare.

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Clonal Evolution in Chronic Lymphocytic Leukemia is Scant in Relapsed But Accelerated in Refractory Cases

SSRN Electronic Journal ◽

10.2139/ssrn.3417948 ◽

2019 ◽

Author(s):

Marc Zapatka ◽

Eugen Tausch ◽

Selcen Öztürk ◽

Martina Seiffert ◽

Thorsten Zenz ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Clonal Evolution ◽

Lymphocytic Leukemia

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Immunological and genetic kinetics from diagnosis to clinical progression in chronic lymphocytic leukemia

Biomarker Research ◽

10.1186/s40364-021-00290-z ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Isabel Jiménez ◽

Bárbara Tazón-Vega ◽

Pau Abrisqueta ◽

Juan C. Nieto ◽

Sabela Bobillo ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Ex Vivo ◽

Clonal Evolution ◽

Lymphocytic Leukemia ◽

Clinical Progression ◽

Molecular Changes ◽

Main Driver ◽

Altered Gene

Abstract Background Mechanisms driving the progression of chronic lymphocytic leukemia (CLL) from its early stages are not fully understood. The acquisition of molecular changes at the time of progression has been observed in a small fraction of patients, suggesting that CLL progression is not mainly driven by dynamic clonal evolution. In order to shed light on mechanisms that lead to CLL progression, we investigated longitudinal changes in both the genetic and immunological scenarios. Methods We performed genetic and immunological longitudinal analysis using paired primary samples from untreated CLL patients that underwent clinical progression (sampling at diagnosis and progression) and from patients with stable disease (sampling at diagnosis and at long-term asymptomatic follow-up). Results Molecular analysis showed limited and non-recurrent molecular changes at progression, indicating that clonal evolution is not the main driver of clinical progression. Our analysis of the immune kinetics found an increasingly dysfunctional CD8+ T cell compartment in progressing patients that was not observed in those patients that remained asymptomatic. Specifically, terminally exhausted effector CD8+ T cells (T-betdim/−EomeshiPD1hi) accumulated, while the the co-expression of inhibitory receptors (PD1, CD244 and CD160) increased, along with an altered gene expression profile in T cells only in those patients that progressed. In addition, malignant cells from patients at clinical progression showed enhanced capacity to induce exhaustion-related markers in CD8+ T cells ex vivo mainly through a mechanism dependent on soluble factors including IL-10. Conclusions Altogether, we demonstrate that the interaction with the immune microenvironment plays a key role in clinical progression in CLL, thereby providing a rationale for the use of early immunotherapeutic intervention.

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Loss of NFAT2 expression results in the acceleration of clonal evolution in chronic lymphocytic leukemia

Journal of Leukocyte Biology ◽

10.1002/jlb.2ab0218-076rr ◽

2018 ◽

Vol 105 (3) ◽

pp. 531-538 ◽

Cited By ~ 3

Author(s):

David J. Müller ◽

Stefan Wirths ◽

Alexander R. Fuchs ◽

Melanie Märklin ◽

Jonas S. Heitmann ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Clonal Evolution ◽

Lymphocytic Leukemia

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Mechanisms of Adaptation to Ibrutinib in High Risk Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2018-99-116739 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 585-585 ◽

Cited By ~ 4

Author(s):

Valeria Spina ◽

Gabriela Forestieri ◽

Antonella Zucchetto ◽

Alessio Bruscaggin ◽

Tamara Bittolo ◽

...

Keyword(s):

High Risk ◽

Chronic Lymphocytic Leukemia ◽

Nuclear Localization ◽

Peripheral Blood ◽

Research Funding ◽

Clonal Evolution ◽

Lymphocytic Leukemia ◽

Adaptation Process ◽

Over Time ◽

Stimulation Of

Abstract Introduction. Ibrutinib inhibits the BTK molecule downstream the B-cell receptor (BCR). Though highly active in high risk chronic lymphocytic leukemia (CLL), the most typical response achievable in patients is a minimal residual disease (MRD) positive partial remission (PR) which is maintained until the development of genetically driven resistance caused by the acquisition of mutations in the BTK or PLCG2 genes. The study aims at characterizing the adaptation process allowing residual CLL cells to persist despite BTK inhibition. Methods. The IOSI-EMA-001 study (NCT02827617) is an observational study consisting in the prospective and longitudinal collection of peripheral blood samples and clinical data from high risk CLL patients treated with ibrutinib. Peripheral blood CLL cells longitudinally drawn from patients before treatment start and at fixed timepoints under ibrutinib were monitored by: i) next generation flow cytometry approaches for changes in proliferation rate, surfaceome, and pathway activation; and ii) CAPP-seq targeted deep next generation (sensitivity ~10-3) for clonal evolution. Results. The study cohort comprised 31 high risk CLL patients, including 15 treatment naïve, 16 relapsed, 80% IGHV unmutated, 42% 17p deleted and 55% TP53 mutated. Median duration of ibrutinib treatment was 45 weeks (24-72 weeks). All patients obtained a MRD positive PR that was maintained in all but one who progressed with a PLCG2 mutation (VAF 3%). Compared to baseline, under ibrutinib therapy CLL cells slowed down their proliferation, as suggested by the decreased expression of Ki-67, the reduction of the proliferating fraction (CXCR4dimCD5bright), and the increase of the resting fraction (CXCR4brightCD5dim). Compared to baseline, under ibrutinib therapy CLL cells also upregulated BCR and adhesion/homing proteins, and decreased the expression of BCR inhibitor proteins. Upon stimulation of the BCR with anti-IgM, the downstream path through pBTK and pPLCG2 was inhibited by ibrutinib, while conversely the downstream path through pAKT and pERK was still inducible throughout all the assessed timepoints. The proportion of CLL cells harboring nuclear localization of NF-kB progressively increased over time under ibrutinib. NF-kB nuclear localization was inducible throughout all the assessed timepoints by CD40L stimulation of the non-canonical NF-kB pathway, but not by anti-IgM stimulation of the BCR/canonical NF-kB pathway. Overall, 880 individual mutations were longitudinally discovered and monitored across a total of 121 sequential timepoints collected during ibrutinib treatment. Clonal evolution was observed in (67.7%) cases, a proportion rate previously documented in CLL treated with chemoimmunotherapy. Clonal evolution appeared to be heterogeneous involving different genes without a stereotypic targeting. Consistently, none of the main driver gene mutations was homogeneously selected or suppressed by ibrutinib suggesting that the biological adaptation of CLL cells under ibrutinib is not genetically driven. Clonal evolution propensity was not associated with any of the biomarkers of the disease, and it did not decrease over time under ibrutinib. Conclusions. Taken together these results suggest that residual CLL cells persisting under ibrutinib therapy adapt their phenotype by upregulating adhesion molecules, chemokine receptors and BCR molecules, and by maintaining a competence of BCR signaling through the PI3K/AKT/ERK pathway. The progressive selection of CLL cells having NF-kB in the nucleus, likely due to the BTK independent non-canonical NF-kB pathway, might explain their survival despite ibrutinib therapy. Finally, clonal evolution is not suppressed by ibrutinib chemotherapy, and despite does not seem to be directly involved in such adaptation process, may ultimately favor the acquisition of BTK and PLCG2 ibrutinib resistance mutations. Disclosures Zucca: Celltrion: Consultancy; AstraZeneca: Consultancy. Ghia:Sunesis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding. Montillo:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding. Tedeschi:Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy; AbbVie: Consultancy. Gaidano:AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Morphosys: Honoraria; Roche: Consultancy, Honoraria.

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