Early reappearance of intraclonal proliferative subpopulations in ibrutinib-resistant chronic lymphocytic leukemia

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib represents an effective strategy for treatment of chronic lymphocytic leukemia (CLL), nevertheless about 30% of patients eventually undergo disease progression. Here we investigated by flow cytometry the long-term modulation of the CLL CXCR4dim/CD5bright proliferative fraction (PF), its correlation with therapeutic outcome and emergence of ibrutinib resistance. By longitudinal tracking, the PF, initially suppressed by ibrutinib, reappeared upon early disease progression, without association with lymphocyte count or serum beta-2-microglobulin. Somatic mutations of BTK/PLCG2, detected in 57% of progressing cases, were significantly enriched in PF with a 3-fold greater allele frequency than the non-PF fraction, suggesting a BTK/PLCG2-mutated reservoir resident within the proliferative compartments. PF increase was also present in BTK/PLCG2-unmutated cases at progression, indicating that PF evaluation could represent a marker of CLL progression under ibrutinib. Furthermore, we evidence different transcriptomic profiles of PF at progression in cases with or without BTK/PLCG2 mutations, suggestive of a reactivation of B-cell receptor signaling or the emergence of bypass signaling through MYC and/or Toll-Like-Receptor-9. Clinically, longitudinal monitoring of the CXCR4dim/CD5bright PF by flow cytometry may provide a simple tool helping to intercept CLL progression under ibrutinib therapy.


Supporting
• Supplemental Tables : o Table S1.Patients' charateristics of the CRO cohort.
o Table S2.Gene expression signature of PF and RF at pre-treatment.
o Table S3.Gene Set Enrichment Analysis of proliferation and ibrutinib gene sets.
Color represents the normalized enrichment score, size is proportional to -log10(FDR q-value), shape denotes significance (circle if q-value<0.05).Enrichment plots of marked (*) gene sets are reported for all fractions or for the PF/RF subpopulations, separately analyzed.GSEA summary is reported in Table S3B.(Calissano et al. ref.13,Bartholdy et al.,ref.15,Cadot et al. ref.46,Seda et al. ref.17).Color represents the normalized enrichment score, size is proportional to -log10(FDR q-value), shape denotes significance (circle if q-value<0.05).GSEA summary is reported in Table S4A.(B) Summary dot-plot of Gene Set Enrichment Analysis (GSEA) on custom gene sets of ibrutinib signature curated from literature (Herman et al. ref.6,Landau et al. ref.47,Cadot et al. ref.46,Wang et al. ref.48).Color represents the normalized enrichment score, size is proportional to -log10(FDR q-value), shape denotes significance (circle if q-value<0.05).GSEA summary is reported in Table S4B.(C) Enrichment plot from GSEA of the HALLMARK_MYC_TARGETS_V2 and HALLMARC_MTORC1_SIGNALING of PF versus RF in BTK-unmutated (red curves) versus BTK-mutated PF (blue curves) at progression.GSEA summary for whole Hallmark collection is reported in Table S4D.(D) Waterfall plot of Normalized Enrichment Scores from PF versus RF GSEA on the C2.cgp gene sets collection, within BTK-unmutated (upper panel) or within BTK-mutated (lower panel) cases at progression.Red lines represent scores of MYC-related gene sets.GSEA summary with highlighted gene sets is reported in Table S4E.

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Figure S1.CRO cohort characteristics o Figure S2.Examples of PF dynamics under ibrutinib in the IOSI cohort.o Figure S3.Resting fraction dynamics in the IOSI-EMA-001 trial.o Figure S4.PF and white blood cell counts in the CRO cohort.o Figure S5.PF dynamics under long-term ibrutinib in the CRO cohort.o Figure S6.Cell sorting.o Figure S7.Transcriptomic programming of PF/RF subpopulations.o Figure S8.Enrichment analysis in BTK-unmutated versus BTK-mutated PF at progression.o Figure S9.In-vitro stimulation of CLL cells at progression under ibrutinib.

Figure S1 .
Figure S1.CRO cohort characteristics.(A) Histogram of distribution of 300 samples according to the time from ibrutinib initiation.(B) Left Panel: Histogram of distribution of time under ibrutinib of all 101 cases; median time to discontinuation is 39.8 months.Right panel: Kaplan-Meyer curve of time to ibrutinib discontinuation.(C) Stacked bar plot of frequency of ibrutinib discontinuations, stratified by year under therapy and by cause of discontinuation.(D) Consort diagram of ibrutinib discontinuation events.

Figure S4 .Figure S5 .Figure S6 .Figure S7 .
Figure S4.PF and lymphocytes counts in the CRO cohort.(A) Prospective analysis of selected cases from the CRO cohort reporting the gating strategy for PF and RF in samples collected at the indicated year under ibrutinib therapy.n.a.: sample not available.(B) Box-and-whiskers plot of the absolute lymphocytes count (ALC) in samples from the CRO cohort before (blue) and after (green) ibrutinib initiation.Samples are grouped by number of years under ibrutinib; pre-ibrutinib samples have been collected within 6 months prior to therapy initiation.(C) Box-and-whiskers plot of PF and ALC in the CRO cohort, stratified by IGHV status.Statistical significance was computed at each time point with Wilcoxon paired rank test of IGHV-M versus IGHV-UM.No significant comparison was found.(D) Box-and-whiskers plot of the resting fraction (RF) in the CRO cohort before (blue) and after (green) ibrutinib initiation.No significant comparison was found.Dotted lines are added for visual reference.

Figure S8 .
Figure S8.Enrichment analysis in BTK-unmutated versus BTK-mutated PF at progression.(A)Summary dot-plot of Gene Set Enrichment Analysis (GSEA) on PF/RF-related gene sets curated from literature(Calissano et al. ref.13,  Bartholdy et al., ref.15, Cadot et al. ref.46, Seda et al. ref.17).Color represents the normalized enrichment score, size is proportional to -log10(FDR q-value), shape denotes significance (circle if q-value<0.05).GSEA summary is reported in TableS4A.(B) Summary dot-plot of Gene Set Enrichment Analysis (GSEA) on custom gene sets of ibrutinib signature curated from literature(Herman et al. ref.6, Landau et al. ref.47, Cadot et al. ref.46, Wang et al. ref.48).Color represents the normalized enrichment score, size is proportional to -log10(FDR q-value), shape denotes significance (circle if q-value<0.05).GSEA summary is reported in TableS4B.(C) Enrichment plot from GSEA of the HALLMARK_MYC_TARGETS_V2 and HALLMARC_MTORC1_SIGNALING of PF versus RF in BTK-unmutated (red curves) versus BTK-mutated PF (blue curves) at progression.GSEA summary for whole Hallmark collection is reported in TableS4D.(D) Waterfall plot of Normalized Enrichment Scores from PF versus RF GSEA on the C2.cgp gene sets collection, within BTK-unmutated (upper panel) or within BTK-mutated (lower panel) cases at progression.Red lines represent scores of MYC-related gene sets.GSEA summary with highlighted gene sets is reported in TableS4E.(E) MYC copy number analysis with droplet digital PCR and FISH break-apart probe.Dyploid: reference normal DNA; RS: Richter Syndrome.

Figure S9 .
Figure S8.Enrichment analysis in BTK-unmutated versus BTK-mutated PF at progression.(A)Summary dot-plot of Gene Set Enrichment Analysis (GSEA) on PF/RF-related gene sets curated from literature(Calissano et al. ref.13,  Bartholdy et al., ref.15, Cadot et al. ref.46, Seda et al. ref.17).Color represents the normalized enrichment score, size is proportional to -log10(FDR q-value), shape denotes significance (circle if q-value<0.05).GSEA summary is reported in TableS4A.(B) Summary dot-plot of Gene Set Enrichment Analysis (GSEA) on custom gene sets of ibrutinib signature curated from literature(Herman et al. ref.6, Landau et al. ref.47, Cadot et al. ref.46, Wang et al. ref.48).Color represents the normalized enrichment score, size is proportional to -log10(FDR q-value), shape denotes significance (circle if q-value<0.05).GSEA summary is reported in TableS4B.(C) Enrichment plot from GSEA of the HALLMARK_MYC_TARGETS_V2 and HALLMARC_MTORC1_SIGNALING of PF versus RF in BTK-unmutated (red curves) versus BTK-mutated PF (blue curves) at progression.GSEA summary for whole Hallmark collection is reported in TableS4D.(D) Waterfall plot of Normalized Enrichment Scores from PF versus RF GSEA on the C2.cgp gene sets collection, within BTK-unmutated (upper panel) or within BTK-mutated (lower panel) cases at progression.Red lines represent scores of MYC-related gene sets.GSEA summary with highlighted gene sets is reported in TableS4E.(E) MYC copy number analysis with droplet digital PCR and FISH break-apart probe.Dyploid: reference normal DNA; RS: Richter Syndrome.
* p≤0.01, * p≤0.05 by two-sided Mann-Whitney rank-test, compared to month zero.Only significant comparisons are reported.Lines connect samples belonging to the same patient.Red dots indicate samples collected from patients in clinical progression.
). (E) Unsupervised clustered heatmap of the PF/RF 476-genes signature, evaluated in all (n=40) PF/RF subpopulations at pre-ibrutinib (n=16) and at progression (n=24).Annotation labels indicate PF/RF subpopulations, type of sample, case and BTK VAF, if mutated.(F) Summary dot-plot of Gene Set Enrichment Analysis (GSEA) on custom gene sets of ibrutinib signature curated from literature (Herman