Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing

Authors

Lohr JG

Stojanov P

Lawrence MS

Auclair D

Chapuy B

Sougnez C

Cruz-Gordillo P

Knoechel B

Asmann YW

Slager SL

Novak AJ

Dogan A

Ansell SM

Link BK

Zou L

Gould J

Saksena G

Stransky N

Rangel-Escareno C

Fernandez-Lopez JC

Hidalgo-Miranda A

Melendez-Zajgla J

Hernandez-Lemus E

Schwarz-Cruz y Celis A

Imaz-Rosshandler I

Ojesina AI

Jung J

Pedamallu CS

Lander ES

Habermann TM

Cerhan JR

Shipp MA

Getz G

Golub TR

Doi

10.1073/pnas.1121343109

PMID: 22343534 · DOI: 10.1073/pnas.1121343109 · Journal: Proceedings of the National Academy of Sciences (2012)

TL;DR

Whole-exome sequencing of 55 primary DLBCL tumors with matched normal tissue identified 58 significantly mutated genes (FDR q <= 0.1), confirming known drivers (MYD88, CARD11, EZH2, CREBBP, CD79B, TP53) and uncovering recurrent mutations in genes not previously implicated in DLBCL (MEF2B, KMT2D, BTG1, GNA13, ACTB, P2RY8, PCLO, TNFRSF14). The study demonstrated that BCL2 point mutations in patients with BCL2/IgH translocations arise from activation-induced cytidine deaminase (AID)-mediated somatic hypermutation and are subject to purifying (negative) selection against loss-of-function changes. A complementary algorithm based on mutational clustering and evolutionary conservation identified KRAS, BRAF, NOTCH1, and SYK as likely rare drivers.

Cohort & data

  • 55 patients with primary DLBCL; 49 passed quality filters after excluding 6 samples with extensive stromal contamination.
  • Paired tumor (lymph node) and germline (peripheral blood) DNA.
  • Whole-exome sequencing at 150-fold mean coverage (97% of bases covered per patient on average; range 91–98%).
  • Cancer type: DLBCLNOS. Dataset: dlbc_broad_2012.
  • Study sites: Broad Institute, Dana-Farber Cancer Institute, Mayo Clinic, University of Iowa, Instituto Nacional de Medicina Genomica (Mexico).

Key findings

  • Mean nonsynonymous mutation rate was 3.2 mutations per megabase (range 0.6–8.7), higher than CLL (<1/Mb) and multiple myeloma (1.3/Mb).
  • MutSig identified 58 significantly mutated genes at FDR q <= 0.1; 97.9% validation rate by targeted resequencing (47 of 47 tested, 1 failed).
  • Top significantly mutated genes by q-value: CD79B, GNA13, MEF2B, TNFRSF14, TP53, KMT2D (MLL2), H1-2 (HIST1H1C), BTG1, CARD11, PIM1, CD58, MYD88.
  • KMT2D mutations were predominantly truncating (nonsense, frameshift indels), consistent with a tumor-suppressor role.
  • TNFRSF14 mutations showed strong loss-of-function bias (4 nonsense, 1 frameshift deletion out of 9 mutations; no synonymous mutations).
  • BTG1 harbored 15 nonsynonymous and 2 synonymous mutations in 8 patients, with biallelic involvement in some patients suggesting loss of function.
  • MEF2B mutations found in 18% of patients, predominantly in the MADS box or MEF2 domains.
  • ACTB mutations in 5 patients with high predicted functional consequence; enriched toward the amino terminus.
  • P2RY8 coding mutations in 6 patients (8 mutations); allelic fraction >0.5 in 3 patients suggesting LOH or amplification of mutant allele.
  • BCL2 had a paradoxical preponderance of synonymous over nonsynonymous mutations (28 synonymous vs. 18 nonsynonymous; ratio 0.64 vs. expected ~2.8:1; P = 8.8 x 10^-6).
  • 77% (10/13) of patients with BCL2 hypermutation harbored BCL2/IgH translocations vs. 8% (1/13) without BCL2 mutation (P = 0.0005, one-sided Fisher exact test).
  • BCL2 nonsynonymous mutations preferentially fell outside BH domains (P = 0.041), consistent with purifying selection preserving BCL2 anti-apoptotic function.
  • Mutations in BCL2 and PIM1 were enriched in AID target WRCY motifs, confirming aberrant somatic hypermutation.
  • Histone H1 family proteins harbored 59 nonsynonymous and 35 synonymous mutations across 34 patients (69%).
  • Rare driver analysis (clustering + conservation) identified KRAS (G13D in 2 patients), BRAF (2 mutations), NOTCH1 (4 mutations), SYK, and SGK1 as likely drivers.
  • 20 of the 58 significantly mutated genes overlapped with those reported by Morin et al. (2011); 14 overlapped with Pasqualucci et al. (2011).

Genes & alterations

  • MYD88: 6 mutations in 6 patients (3 unique sites); known activating role in NF-kB signaling. Highly clustered mutations.
  • CARD11: 12 mutations in 10 patients (11 sites); known oncogene in B-cell receptor signaling. Highly clustered.
  • EZH2: 7 mutations in 7 patients (4 sites); gain-of-function histone methyltransferase mutations (Tyr641).
  • CREBBP: 9 mutations in 8 patients (9 sites); histone acetyltransferase.
  • CD79B: 8 mutations in 8 patients (5 sites); B-cell receptor signaling component. Highly clustered.
  • TP53: 13 mutations in 12 patients (12 sites); tumor suppressor.
  • KMT2D (MLL2): 17 mutations in 14 patients (17 sites); predominantly truncating, consistent with tumor-suppressor function. Histone H3K4 methyltransferase.
  • TNFRSF14: 11 mutations in 11 patients (11 sites); predominantly loss-of-function (nonsense/frameshift). TNF-receptor superfamily tumor suppressor.
  • BTG1: 15 nonsynonymous mutations in 8 patients; biallelic involvement in some patients. Cell-cycle regulator.
  • MEF2B: 9 mutations in 9 patients; MADS box/MEF2 domain hotspot. Calcium-regulated transcription factor.
  • GNA13: 17 mutations in 10 patients (16 sites); G-protein signaling.
  • BCL2: High mutation rate driven by AID-mediated somatic hypermutation at the translocated IgH locus; purifying selection against BH-domain nonsynonymous mutations.
  • PIM1: 22 mutations in 15 patients (19 sites); subject to aberrant somatic hypermutation (WRCY enrichment).
  • ACTB: 5 mutations in 5 patients (4 sites); cytoskeletal protein with high predicted functional consequence.
  • P2RY8: 8 mutations in 6 patients; G protein-coupled purinergic receptor.
  • KRAS: G13D mutation in 2 patients; known oncogene, rare driver in DLBCL.
  • BRAF: 2 mutations; known oncogene, rare driver in DLBCL.
  • NOTCH1: 4 mutations; known oncogene, rare driver in DLBCL.
  • SYK: Identified as likely driver by clustering/conservation analysis; tyrosine kinase with demonstrated clinical activity of inhibitors in NHL.
  • SGK1: 9 mutations in 5 patients; identified by clustering/conservation analysis as likely driver.

Clinical implications

  • R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) is the standard of care, with ~60% 3-year event-free survival; the remaining 40% largely die of disease, underscoring the need for new therapeutic targets.
  • The genomic landscape identifies multiple targetable pathways: B-cell receptor signaling (CD79B, CARD11), NF-kB pathway (MYD88, NFKBIA), epigenetic regulation (EZH2, CREBBP, KMT2D), and kinase signaling (KRAS, BRAF, SYK).
  • SYK inhibitors (fostamatinib) had demonstrated significant clinical activity in NHL including DLBCL at the time of publication, validating SYK as a therapeutic target.
  • BCL2/IgH translocation drives anti-apoptotic dependency, but the associated BCL2 point mutations are likely passengers arising from somatic hypermutation rather than additional drivers.

Limitations & open questions

  • Modest cohort size (n = 49 after QC) limits power to detect rare drivers; the authors note that additional genes may harbor functionally important mutations below the significance threshold.
  • Exome-only sequencing misses non-coding regulatory mutations, structural rearrangements (other than those inferred indirectly such as BCL2/IgH), and copy-number changes outside captured regions.
  • The functional role of PCLO mutations remains unresolved: 23 nonsynonymous mutations in 17 patients (35%) but the nonsynonymous-to-synonymous ratio (23:3) is consistent with chance given the high transversion rate, suggesting they may be passengers due to locally elevated mutation rate.
  • The functional consequences of histone H1 family mutations (69% of patients) are unknown.
  • The paper does not subtype DLBCL cases by cell of origin (GCB vs. ABC), limiting interpretation of subtype-specific mutation patterns.
  • Whether silent mutations at recurrent BCL2 codons (N11, K22) have functional consequences (e.g., affecting mRNA stability) remains undetermined.

Citations from this paper used in the wiki

  • “We performed solution-phase hybrid capture and whole-exome sequencing on paired tumor and germline (i.e., normal) DNA samples from 55 patients with primary DLBCL. We achieved 150-fold mean sequence coverage of targeted exonic regions, with an average of 97% of bases covered per patient (range, 91-98%).”
  • “the mean nonsynonymous mutation rate was 3.2 mutations per megabase, with mutation rates varying widely (0.6-8.7 mutations per megabase)”
  • “This approach revealed 58 statistically significant genes with a false discovery rate cutoff of 0.1 (q <= 0.1)”
  • “We independently validated selected mutations by targeted resequencing in a subset of patients and obtained 97.9% validation rate”
  • “we observed a striking opposite effect in the BCL2 gene… with 18 nonsynonymous mutations and 28 synonymous mutations, for a ratio of 0.64, far below that expected by chance (P = 8.8 x 10^-6)”
  • “the vast majority of patients with BCL2 hypermutation (10 of 13, 77%) had BCL2/IgH translocation, whereas only one of 13 patients (8%) lacking BCL2 mutation had the translocation (P = 0.0005)”
  • “we observed a striking accumulation of mutations in H1 family proteins, with 59 nonsynonymous and 35 synonymous mutations among 31 histone H1 proteins in 34 patients (69%)”
  • “we also observed two mutations in KRAS (G13D), four mutations in NOTCH1, and two mutations in BRAF”

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