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Molecular map of chronic lymphocytic leukemia and its impact on outcome

Abstract

Recent advances in cancer characterization have consistently revealed marked heterogeneity, impeding the completion of integrated molecular and clinical maps for each malignancy. Here, we focus on chronic lymphocytic leukemia (CLL), a B cell neoplasm with variable natural history that is conventionally categorized into two subtypes distinguished by extent of somatic mutations in the heavy-chain variable region of immunoglobulin genes (IGHV). To build the ‘CLL map,’ we integrated genomic, transcriptomic and epigenomic data from 1,148 patients. We identified 202 candidate genetic drivers of CLL (109 new) and refined the characterization of IGHV subtypes, which revealed distinct genomic landscapes and leukemogenic trajectories. Discovery of new gene expression subtypes further subcategorized this neoplasm and proved to be independent prognostic factors. Clinical outcomes were associated with a combination of genetic, epigenetic and gene expression features, further advancing our prognostic paradigm. Overall, this work reveals fresh insights into CLL oncogenesis and prognostication.

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Fig. 1: Increased power enables CLL driver gene detection.
Fig. 2: M-CLL and U-CLL have unique genomic landscapes.
Fig. 3: CLL subtypes based on epigenetic and transcriptomic features.
Fig. 4: ECs and integrated analysis predict clinical outcome.

Data availability

The molecular data used in this study are publicly available and are included in the following patient cohorts (Table 1, Supplementary Tables 1 and 2 and Extended Data Fig. 1a): DFCI, Dana-Farber Cancer Institute; GCLLSG, German CLL Study Group; ICGC, International Cancer Genome Consortium; MDACC, MD Anderson Cancer Center; NHLBI, National Heart Lung and Blood Institute; UCSD, University of California San Diego. Sequencing, expression, and genotyping is available at EGA (http://www.ebi.ac.uk/ega/), which is hosted at the European Bioinformatics Institute, under accession number EGAS00000000092 (ICGC cohort) and in dbGaP under accession numbers phs001473.v2.p1 (MDACC, NHLBI), phs000922.v2.p1 (GCLLSG), phs001431.v2.p1 (DFCI, UCSD), phs001091.v1.p1 (MDACC), phs000435.v3.p1 (DFCI), phs002297.v2.p1 (NHLBI) and phs000879.v1.p1 (DFCI) and GEO accession number GSE143673 (GCLLSG). 450K array data are available at EGA under accession number EGAD00010001975 (ICGC). The project data portal is available at https://cllmap.org.

Code availability

Terra methods used in the study can be found at https://app.terra.bio/#workspaces/broad-firecloud-wupo1/CLLmap_Methods_Apr2021. Source code used in the study can be found at https://github.com/getzlab/CLLmap. The RFcaller pipeline is available at https://github.com/xa-lab/RFcaller. The new epiCMIT suitable for Illumina arrays and NGS approaches as well as the CLL epitype classifier can be found at https://github.com/Duran-FerrerM/CLLmap-epigenetics.

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Acknowledgements

We thank W. Zhang, S. Gohil, I. Leshchiner, D. Livitz, D. Rosebrock, J. Gribben, K. R. Rai, M. J. Keating, J. M. Hess, N. J. Haradhvala, A. Mohammed and A. Gnirke for helpful discussions. We thank C. Patterson, S. Pollock, K. Slowik, O. Olive, C. J. Shaughnessy and H. Lyon for assistance in data collection and organization. We thank the patients, their families and the investigators of the clinical trials for providing samples and clinical data. This study was supported by National Institutes of Health (NIH)/National Cancer Institute (NCI) grant P01 CA206978 (to C.J.W. and G.G.) and the Broad/IBM Cancer Resistance Research Project (G.G. and L.P.). B.A.K. was supported by a long-term EMBO fellowship (ALTF 14-2018). C.K.H. was supported by the NHLBI Training Program in Molecular Hematology (T32HL116324). F.N. acknowledges funding by the American Association for Cancer Research (2021 AACR-Amgen Fellowship in Clinical/Translational Cancer Research, 21-40-11-NADE), the European Hematology Association (EHA Junior Research Grant 2021, RG-202012-00245), and the Lady Tata Memorial Trust (International Award for Research in Leukaemia 2021-2022, LADY_TATA_21_3223). S.S. and E.T. were supported by the Deutsche Forschungsgemeinschaft (SFB1074, subproject B1, B2 and B10). A.W. and C. Sun were supported by the Intramural Research Program at NIH/NHLBI. J.A.B. was supported by MD Anderson’s Moon Shot Program in CLL and the CLL Global Research Foundation and in part by MDACC Support Grant CA016672. S.L. was supported by the NCI Research Specialist Award (R50CA251956). J.R.B. was supported by NIH grant R01 CA 213442, NIH/NCI grant P01 CA206978 and the Melton Family Foundation. X.S.P. acknowledges funding by the Spanish Ministerio de Economía y Competitividad (grants SAF2017-87811-R and PID2020-117185RB-I00). A.D.-N. was supported by the Department of Education of the Basque Government (PRE_2017_1_0100) and P.B.-M. by a fellowship by the Spanish Ministerio de Economía y Competitividad. This study was supported by “la Caixa” Foundation (CLLEvolution- LCF/PR/HR17/52150017, Health Research 2017 Program “HR17-0022” to E.C.), the European Research Council under the European Union’s Horizon 2020 research and innovation program (Project BCLLATLAS, grant agreement 810287) (to J.I.M.-S. and E.C.), the Accelerator award CRUK/AIRC/AECC joint funder-partnership (to J.I.M.-S.), Generalitat de Catalunya Suport Grups de Recerca AGAUR 2017-SGR-1142 (to E.C.) and 2017-SGR-736 (to J.I.M.-S.), CERCA Programme/Generalitat de Catalunya. E.C. is an Academia Researcher of Catalan Institution for Research and Advanced Studies.

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Authors and Affiliations

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Contributions

C.J.W., G.G., E.C. and S.S. conceived the study. E.T., J.D., S.M.F., C. Sun, M.S., L.Z.R., C. Schneider, L.P., J.A.B., A.W., T.J.K., J.R.B., M.H. and S.S. collected and contributed samples and annotations. B.A.K., Z.L., C.K.H., K.E.S., A.T.-W. and J.G.-A. assembled the data. B.A.K., Z.L., F.N., M.D.-F., K.E.S., A.B.-M., A.T.-W., P.B.-M., A.D.-N., A.D., S.A., H.K., F.A. and C. Stewart wrote analytic pipelines. B.A.K., Z.L., F.N., M.D.-F., K.E.S., A.B.-M., P.B.-M., A.D.-N., S.A. and C. Stewart performed the analysis. B.A.K., Z.L., C.K.H., F.N., M.D.-F., K.E.S., E.T., J.D., A.B.-M., P.B.-M., A.D.-N., S.L.-T., A.M., F.A., C. Stewart, J.R.B., D.S.N., J.I.M-S., X.S.P., S.S., C.J.W., E.C. and G.G. contributed to study design and interpreted the data. S.L. performed targeted sequencing. C.K.H., B.A.K., Z.L., C.J.W. and G.G. prepared the manuscript with input from all authors.

Corresponding authors

Correspondence to Catherine J. Wu or Gad Getz.

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Competing interests

The authors declare the following conflicts related to the CLLmap project: C.J.W. receives research support from Pharmacyclics. E.C. has been a consultant for Illumina. G.G. receives research funds from IBM and Pharmacyclics; and is an inventor on patent applications related to SignatureAnalyzer-GPU. S.S. reports honoraria for consultancy, advisory board membership, speaker honoraria, research grants and travel support from AbbVie, Amgen, AstraZeneca, Celgene, Gilead, GSK, Hoffmann La-Roche, Janssen, Novartis. C.J.W., G.G., B.A.K., Z.L. and C.K.H. are inventors on a patent “Compositions, panels, and methods for characterizing chronic lymphocytic leukemia” (PCT/US21/45144). The following conflicts are unrelated to the CLLmap project: F.N. has received honoraria from Janssen for speaking at educational activities. E.T. declares research support by AbbVie and Roche; Advisory Boards and Speakers Bureau for Janssen, AbbVie and Roche. A.W. received research funding from Pharmacyclics, Acerta, Merck, Verastem, Genmab, Nurix. J.R.B. has served as a consultant for AbbVie, Acerta/AstraZeneca, Beigene, Bristol-Myers Squibb/Juno/Celgene, Catapult, Genentech/Roche, Janssen, MEI Pharma, Morphosys AG, Novartis, Pfizer, Rigel; received research funding from Gilead, Loxo/Lilly, Verastem/SecuraBio, Sun, TG Therapeutics; and served on the data safety monitoring committee for Invectys. J.A.B. received research support from AstraZeneca, BeiGene, Gilead, and Pharmacyclics; travel and speaker honoraria from Janssen. X.S.P. is a cofounder of and holds an equity stake in DREAMgenics. C.J.W. holds equity in BioNTech, Inc.. E.C. has been a consultant for Takeda and NanoString Technologies; has received honoraria from Janssen and Roche for speaking at educational activities; and is an inventor on a Lymphoma and Leukemia Molecular Profiling Project patent “Method for subtyping lymphoma subtypes by means of expression profiling” (PCT/US2014/64161). G.G. is an inventor on patent applications related to MSMuTect, MSMutSig, MSIDetect and POLYSOLVER; and is a founder and consultant of and holds privately held equity in Scorpion Therapeutics. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Dataset description and representative driver gene maps.

a. Full dataset (n = 1148), with contributions by cohort and data type delineated (see Supplementary Table 1). b. Numbers of samples with genomic, epigenomic, and transcriptomic data. c. 3D protein structures of representative genes identified by CLUMPS in pan-CLL analysis (n = 984, see Supplementary Table 5). Mutated residues - red labels. A peptide from RAF1 (designated at bottom-center, in complex with 14-3-3 zeta) shows clustered mutations around S259, whose phosphorylation regulates RAF1 activity and is a cancer mutational hotspot91 that, when mutated, perturbs the interaction with the 14-3-3 zeta and upregulates RAF1 kinase activity92,93. In DICER1, mutations occur in the RNase III domain (purple), including the cancer hotspot residue E181321,94. This region is critical for Mg2+ binding and is required for ribonuclease activity to process microRNAs and mediate post-transcriptional gene regulation95. RPS23 mutations are clustered in a conserved loop of the ribosomal decoding center, surrounding P62, whose post-translational hydroxylation affects translation termination accuracy96. These RPS23 mutations have a median CCF > 80% (Extended Data Fig. 6d; Supplementary Table 3). d. Individual mutations maps of selected novel, putative driver genes. Mutation subtype and position are shown. e. Selected genes identified by CLUMPS in IGHV subtypes; mutated residues - red. Although BRAF was not identified as a potential M-CLL driver via MutSig2CV (see Extended Data Fig. 3, Methods), CLUMPS revealed three mutated sites clustered in the kinase domain (purple) that are cancer hotspots25, thus confirming BRAF as a shared driver (left). Mutated residues in BRAF in U-CLL (bottom) are shown for comparison, revealing a greater number of clustered mutations relative to M-CLL. In U-CLL, novel mutations were found in RRM1 (right). Somatic alterations were clustered in the N-terminal ATP-binding site (purple) and therefore have potential to impact enzymatic activity97.

Extended Data Fig. 2 CLL biological pathways affected by candidate driver genes.

a. Schema of CLL pathways containing previously identified (black) and novel (magenta) putative driver genes (see Supplementary Table 6). Novel drivers cluster in central processes driving CLL (for example, DNA damage, chromatin modification, RNA processing)1,2, but also highlight new pathways not previously implicated by driver genes (for example, cytoskeleton and extracellular matrix, proteostasis, metabolism). Asterisks - mutated genes discovered by CLUMPs. b. Stacked barplot ranked by the number of candidate driver genes per CLL pathway. Magenta bars show the number of newly identified drivers in each pathway.

Extended Data Fig. 3 Candidate driver alterations discovered in IGHV subtypes.

a-b. Landscape of putative driver genes and sCNAs in M-CLL (a, n = 512) and U-CLL (b, n = 459) with associated frequencies (rows, barplots). Header tracks annotate cohort, IGHV status (purple, M-CLL; orange, U-CLL), disease type (blue, CLL; yellow, MBL), epitype (blue, n-CLL; yellow, i-CLL; red, m-CLL), datatype (white, WES; yellow, WGS; blue, both); prior treatment, U1 and IGLV3-21R110 mutations are annotated in black; magenta label - novel alterations; asterisks - discovery by CLUMPS.

Extended Data Fig. 4 Chromosomal gains and losses identified in IGHV subtypes.

a-b. Recurrent copy-number gains (left) and losses (right) by GISTIC analysis showing arm-level (left per plot) and focal events (right per plot) in M-CLL (a, n = 512) and U-CLL (b, n = 459). Chromosomes are labeled along the vertical axis; dashed line - significance at q = 0.1. Blacklisted regions are colored gray. All arm-level events are labeled with cytoband arm and frequency in cohort. Focal events are annotated by cytoband, frequency, number of genes encompassed in peak (bracketed), and genes of interest. Red/blue font: novel focal events with frequency >2%. Black font: previously identified events (see Supplementary Table 7).

Extended Data Fig. 5 Landscape of driver alterations and chromosomal aberrations in IGHV subtypes.

a. The genomic landscape of CLL IGHV subtypes. Driver genes, U1 and IGLV3-21R110 mutations are labeled according to their genomic location (outside ring, numbered by chromosome). The tracks show the frequency and locations of driver genes in M-CLL (purple) vs. U-CLL (orange) (track 1; outermost), focal sCNAs (track 2; gains, red; losses, blue), and density of SV breakpoints of deletions (track 3) and translocations (track 4) (M-CLL n = 88; U-CLL n = 87; WGS, windows of 1-Mb). Innermost plot highlights translocations in which either one or both breakpoints are recurrent in at least 3 cases (windows of 1-Mb considered to define recurrence) in M-CLL (purple) and U-CLL (orange). Deletions, inversions, and tandem duplications where both breakpoints were found in at least 2 cases and did not overlap with a driver sCNA are shown (Note: only focal deletion in SP140 in two U-CLL cases met this criterion. b. Schema of recurrent IG-BCL2 translocation and IGH-ZFP36L1 deletion in the WGS cohort. All 5 BCL2 translocations were in M-CLL with immunoglobulin (IG) breakpoints in J or D genes, suggesting mediation by aberrant V(D)J recombination. In contrast, 4 U-CLL cases carried IGH-ZFP36L1 truncating deletions, which were all clonal (CCF = 1). Breakpoints in IGH class-switch regions suggested mediation by aberrant class-switch recombination (CSR). c. Immunoglobulin (IG) SVs in 177 WGS and 984 WES. In WES, 9 of 10 BCL2 translocations were in M-CLL and mediated by aberrant V(D)J recombination in IGH (n = 7) or IGK (n = 2). The sole BCL2 translocation in U-CLL was due to aberrant CSR. One CSR-mediated IGH-ZFP36L1 deletion was observed in a case with unclassified IGHV status due to presence of two populations (one M-CLL, one U-CLL; the latter was more prevalent). Of note, in WES, U-CLLs carry a higher number of non-recurrent IG events than M-CLL.

Extended Data Fig. 6 Mutational mechanisms and cancer cell fractions of candidate drivers.

a. Eight mutational signatures were identified in 177 WGS, but 3 signatures corresponded to known artifacts and were therefore excluded (see Supplementary Note 2). Boxplots demonstrating mutation contribution for each of the 5 signatures are labeled with single-base substitution (SBS) number and identity (per COSMIC v3.1). b. Comparison of the normalized signature intensity of the mutational signatures in U-CLL (orange, n = 87) vs. M-CLL (purple, n = 88). The nc-AID and c-AID 1 signatures were enriched in M-CLL, whereas the aging signature was more prevalent in U-CLL. Although not significant, there was a trend of increased mutations due to the c-AID 2 signature in U-CLL. All p-values were calculated with Wilcoxon rank-sum test, two-sided. Boxplots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. c. Proportions of clustered mutations contributed by the two c-AID related signatures (SBS84, c-AID 1 vs. SBS85, c-AID 2) for each IGHV subtype (M-CLL, purple; U-CLL, orange) d. Mean cancer cell fraction (CCF) for each non-silent mutation across all candidate driver genes identified in WES samples (n = 984). Color of dots depicts the IGHV subtype (M-CLL, purple; U-CLL, orange). The horizontal red line is the threshold for clonality (CCF > 85%). Magenta labels - newly identified putative driver genes. The number of non-silent mutations per driver gene is shown at the bottom. Boxplots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range.

Extended Data Fig. 7 Development and validation of epitype assignment and epiCMIT in RRBS data.

a. Consensus clustering matrices for K = 3 groups for paired-end (n = 136; 153 CpGs in consensus matrix) and single-end (n = 388; 32 CpGs) RRBS data. (d). b. Empirical cumulative distribution functions (CDFs) for consensus matrices with K = 2 to K = 7. c. Relative change under the CDF for K = 2 to K = 7. d. Heatmaps of the CpGs used for consensus clustering in (a). Each sample (columns) is annotated by tracks: epitype max probability, IGHV status (M-CLL, purple; U-CLL, orange), IGHV percent identity, and presence of IGLV3-21R110 mutation (black). e. The development of the new epiCMIT methodology for RRBS data. The genome was segmented into Chromatin Hidden Markov Model (CHMM)24 states using ChIP-seq data to get repressed chromatin regions, where differential DNA methylation analyses was performed in high coverage whole-genome bisulfite sequencing (WGBS) data between the cells with the lowest and highest accumulated cell divisions in the B cell lineage, namely the hematopoietic precursor cells (HPC) and bone-marrow plasma cells (bmPC). Only CPGs showing extensive differences were retained and constituted the epiCMIT-hyper CpGs or epiCMIT-hypo CpGs depending whether they gain or lose DNA methylation from <0.1 to ≥0.5 and from >0.9 to ≤0.5 from HPC to bmPC, respectively. EpiCMIT-hyper and epiCMIT-hypo scores were calculated according to the available epiCMIT-CpGs per sample, and the higher score in each sample was then selected. f. epiCMIT values on the same samples profiled twice with different platforms. Approach 1 - profiled with Illumina-450K (green); approach 2 - profiled with RRBS-PE (violet). In samples profiled with Illumina 450K, the original epiCMIT-CpGs were used52. In samples profiled with RRBS, epiCMIT was calculated with all available epiCMIT-CpGs for the new catalog (e, Methods). P-value by Pearson correlation test, two-sided; Error band − 95% confidence intervals of the Pearson correlation coefficient.

Extended Data Fig. 8 Identification of expression clusters with associated biologic features.

a. Cohort representation in each expression cluster. b. Consensus matrix for RNA expression profiles of 603 treatment-naive CLLs by repeated hierarchical clustering with 80% resampling and varying cutoffs for number of clusters, which is inputted to the BayesNMF procedure (Methods). c. Uniform manifold approximation and projection (UMAP) showing clustering of ECs (n = 603; EC-u clusters (top), EC-m and EC-o (middle), EC-i (bottom)). Analysis was performed using the marker genes identified by BayesNMF. d. UMAP of H3K27ac profiles (n = 104)8 denoting EC designation where available (colored points, n = 73) and IGHV status. e. Comparison of the percent IGHV identity among ECs. Dotted line: 98% threshold defining M-CLL and U-CLL. P-values by two-sided t-tests. Boxplots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. f. Comparison of the percent IGHV identity between those samples with concordant IGHV status and ECs (for example, M-CLLs in EC-m clusters) versus the discordant samples (for example, M-CLLs in EC-u clusters). IGHV-mutated cases - left; IGHV unmutated samples - right. P-values by two-sided t-tests. Boxplots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. g. Percentage of cases carrying stereotyped immunoglobulin genes within each EC. Red horizontal line: percentage of stereotyped cases in the whole cohort. h. Fraction of cases classified in each CLL stereotype subset according to their EC. i. Percentage of IGHV (left) and IG(K/L)V (right) gene usage within each EC. IGKV genes from proximal and distal clusters were merged for simplification. All p-values were calculated using Chi-squared tests corrected by the Benjamini–Hochberg procedure (q-values, q). q < 0.1; *, q < 0.05; **, q < 0.001; ***, q < 0.0001. j-k. Heatmaps showing upregulated (j) and downregulated (k) H3K27ac levels of EC marker genes and 2,000 bp upstream to capture regulatory regions (Methods).

Extended Data Fig. 9 EC differential gene expression, pathway activity, and classifier.

a. Differentially expressed genes per EC (red) using discovery set (n = 603); EC marker genes by BayesNMF (blue). Significant up- or downregulation of H3K27ac levels are directionally marked with triangles (ChIP-seq available for n = 73; n = 1 for EC-o and EC-i, thus unevaluable). b. EC gene set enrichment analysis (GSEA). Diamond denotes the EC compared to all others (circles). c. Confusion matrix for the EC classifier on the test set (“Dominance” defined in Methods). d. Confidence in correctly classified samples (n = 95) is greater than for incorrectly classified samples (n = 25; two-sided t-test). “Prediction margin” defined in Methods. Boxplots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. e. Receiver-operator curve (ROC) showing the tradeoff between sensitivity and specificity for the range of cutoffs that can be applied based on the “prediction margin”, where samples under the cutoff are excluded from performance evaluation. AUC, area under curve. f. Precision-recall (PR) curves for EC classification performance on the test set (n = 120), using the selected model (see Methods). The weighted average of AUC is 0.88. g. Performance metrics for models trained with differing amounts of input genes, demonstrating accuracy even with smaller gene sets. Metrics: Accuracy, overall; Average, weighted average across ECs (Methods). Nc, Ntot - number of genes (see Methods). h. EC distributions by BayesNMF compared to classifier predictions on the discovery cohort (n = 603), an extension cohort not included discovery (n = 105), and an external CLL cohort (n = 136)61. i. IGHV status distributions per EC in discovery (n = 603) and external (n = 136) cohorts. The difference in IGHV-mutated samples per EC is 2-10% (p > 0.05, Fisher’s Exact, Methods). j. Stability of the ECs over time in longitudinally sampled CLL samples3. Sample timepoints (x-axis); years between first and last sample (above curve).

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Knisbacher, B.A., Lin, Z., Hahn, C.K. et al. Molecular map of chronic lymphocytic leukemia and its impact on outcome. Nat Genet 54, 1664–1674 (2022). https://doi.org/10.1038/s41588-022-01140-w

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