Landscape of somatic mutations and clonal evolution in mantle cell lymphoma

Authors

Beà S

Valdés-Mas R

Navarro A

Salaverria I

Martín-García D

Jares P

Giné E

Pinyol M

Royo C

Nadeu F

Conde L

Juan M

Clot G

Vizán P

Di Croce L

Puente DA

López-Guerra M

Moros A

Roue G

Aymerich M

Villamor N

Colomo L

Martínez A

Valera A

Martín-Subero JI

Amador V

Hernández L

Rozman M

Enjuanes A

Forcada P

Muntañola A

Hartmann EM

Calasanz MJ

Rosenwald A

Ott G

Hernández-Rivas JM

Klapper W

Siebert R

Wiestner A

Wilson WH

Colomer D

López-Guillermo A

López-Otín C

Puente XS

Campo E

Doi

10.1073/pnas.1314608110

PMID: 24145436 · DOI: 10.1073/pnas.1314608110 · Journal: PNAS (2013)

TL;DR

Beà and colleagues performed whole-genome sequencing (n=4) and whole-exome sequencing (n=29) of mantle cell lymphoma (MCL) tumors plus six MCL cell lines, and validated recurrent driver genes by targeted sequencing in an independent cohort of 172 MCL patients (PMID:24145436). They identified 25 significantly mutated genes — including the known MCL drivers ATM, CCND1, and TP53 — plus novel recurrent mutations in chromatin modifiers (NSD2/WHSC1, KMT2D/MLL2, MEF2B), the anti-apoptotic regulator BIRC3, the innate-immune receptor TLR2, and NOTCH2 as a mutually exclusive alternative to NOTCH1. Paired/sequential sample analysis revealed subclonal heterogeneity already present at diagnosis and dynamic clonal evolution at progression or relapse.

Cohort & data

  • Whole-genome sequencing of 4 MCL tumor/normal pairs and whole-exome sequencing of 29 MCL tumor/normal pairs (whole-genome-seq, whole-exome-seq).
  • Whole-exome sequencing of 6 MCL cell lines.
  • Targeted validation by Sanger/targeted sequencing in an independent cohort of 172 MCL patients (targeted-dna-seq, sanger-sequencing).
  • Copy-number / LOH profiling by Affymetrix SNP 6.0 arrays (affymetrix-snp6); gene-expression profiling by Affymetrix HU133+ 2.0 (affymetrix-u133-plus2).
  • Dataset deposited as EGA EGAS00001000510 (sequencing) and GEO GSE46969 (SNP6.0) / GSE36000 (HU133+2.0); cBioPortal study mcl_idibips_2013.
  • WES coverage yielded a median of 20 protein-coding mutations per case (range 8–47); WGS detected ~3,700 somatic mutations per tumor (1.2/Mb) (PMID:24145436).

Key findings

  • Twenty-five significantly mutated genes were identified by WES; all 29 primary tumors harbored at least one mutation in this set, and 5/6 MCL cell lines did as well (PMID:24145436).
  • Mutational burden tracks clinical course. Five patients with indolent disease (no treatment, median follow-up 55 mo, range 4–147) had significantly fewer protein-coding mutations (mean 11±4 vs 25±11, P=3.4×10⁻⁵) and fewer CNAs (mean 2±3 vs 12±9, P=0.001) than the 24 patients requiring treatment.
  • CNA burden tracks SOX11. SOX11-positive tumors carried more CNAs than SOX11-negative MCL (mean 13±9 vs 2±2, P=2.1×10⁻⁵) despite a similar somatic mutation count (24±12 vs 17±9, P=0.141).
  • Kataegis foci (regional hypermutation) were observed in 3/4 WGS cases — more often in the two IGHV-mutated tumors — clustering around the t(11;14) 11q13 breakpoint, Ig loci (2p11, 14q32.33, 22q11.22), and the 9p21.3 deletion, and remained stable in a sequential sample of case M003, suggesting kataegis is an early stable event in MCL.
  • A>C/T>G substitutions in TpA context were enriched in IGHV-mutated MCL, matching the germinal-center / Polη error-prone hypermutation signature previously seen in CLL.
  • NOTCH2 mutations are mutually exclusive with NOTCH1. PEST-truncating NOTCH2 mutations were found in 9/172 (5.2%) MCL, and NOTCH1 PEST-truncating mutations in 8/172 (4.6%); only 1/16 mutated patients carried both. Combined, NOTCH1/2 mutations occurred in 9.5% of MCL.
  • NOTCH2 mutations confer dismal prognosis. 3-year OS 0% vs 62% (P=2.5×10⁻⁴); blastoid/pleomorphic morphology 66% vs 18% (P=0.001). In bivariate analysis, NOTCH2 mutation was an independent OS risk factor (HR 3.5; 95% CI 1.3–9.5; P=0.017) along with TP53 mutation (HR 2.4; 95% CI 1.4–4.2; P=0.003).
  • NOTCH1 mutations were also enriched in blastoid/pleomorphic MCL (67% vs 19%, P=0.03) with shorter survival (3-y OS 33% vs 60%, P=0.026). Combined NOTCH1/2 mutations marked an aggressive subset (3-y OS 24% vs 63%, P=3.4×10⁻⁴).
  • Chromatin-modifier mutations are common. NSD2 (WHSC1/MMSET) was mutated in 13/130 (10%) MCL — recurrent missense p.E1099K and p.T1150A in conserved SET-domain–proximal exons 18/19. KMT2D (MLL2) was mutated in 4/29 primary tumors and 2/6 cell lines (mostly truncating, with FYRN/FYRC missense). MEF2B carried the p.K23R hotspot in 6/187 (3.2%) cases. Together, chromatin-modifier mutations occurred in 10/29 (14%) WES cases and were essentially restricted to SOX11-positive / IGHV-unmutated MCL.
  • NSD2-mutated MCL phenocopies t(4;14) plasma-cell myeloma transcriptionally. GSEA showed NSD2-mutated MCL overexpressed gene signatures upregulated by wild-type or gain-of-function exon-19 NSD2 in KMS11 PCM cells, as well as proliferation/cell-cycle signatures.
  • BIRC3 inactivating mutations were found in 11/173 (6.4%) of MCL and co-occurred with 11q deletions in 10/11 mutated cases (vs 25/87 of BIRC3-unmutated; P=1.1×10⁻⁴).
  • TLR2 mutations (p.D327V and p.Y298S) were found in 2/171 MCL, both in SOX11-negative/IGHV-mutated tumors; mutated cells showed elevated basal and stimulated IL-6 and IL-1RA secretion, suggesting altered microenvironment signaling.
  • UBR5 ubiquitin-ligase mutations and an inactivating B2M p.L13fs*10 mutation (in a case with 15q12–q21.1 deletion encompassing B2M) were also observed.
  • CDKN2A was the only gene targeted by recurrent homozygous deletions but carried no somatic point mutations in this series.
  • Allelic-loss/mutation co-occurrence. Only TP53 (17p), ATM (11q), and BIRC3 (11q) recurrent mutations were significantly associated with allelic loss of the second copy.
  • Subclonal heterogeneity at diagnosis. In 2/6 patients with simultaneous PB + lymph-node samples (M023, M026), shared mutations indicated a common founder clone but distinct subclones predominated at each site; the other 4 had a single major clone in both.
  • Clonal evolution at progression. Case M003 (indolent then rapid progression) gained 20 CNAs, chromothripsis on chr4/12, and a 16% increase in WGS mutation count (3,928 → 4,665) at progression. Case M002 (chemotherapy-treated, relapsed at 3 y) lost 11 initial mutations and gained new ones at relapse — consistent with chemotherapy-driven subclone replacement.
  • Early vs late events. ATM, CCND1, KMT2D (MLL2), KCNC2, KIAA1671, PCSK2, TNRC6B, TRPM6 mutations were present at similar allelic frequency in paired subclones (likely early); ABCA3, TLR2, TP53, and NSD2 (WHSC1) mutations were seen in only one of two paired subclones (likely late). BIRC3 mutations were specifically acquired post-treatment in 2 cases, accompanied by 11q deletion in one.

Genes & alterations

  • ATM — truncating / functional-domain mutations in 12/29 (41%) WES cases; 12/22 (55%) of SOX11-positive vs 0/7 SOX11-negative (P=0.023). Often biallelic via 11q deletion. Early/clonal event.
  • CCND1 — exon-1 mutations in 10/29 (35%), enriched in SOX11-negative (6/7, 86%, vs 4/22, 18%, P=0.03) and IGHV-mutated (7/12, 58% vs 3/16, 19%, P=0.05) cases — consistent with acquisition in the germinal-center microenvironment.
  • TP53 — mutations in 8/29 (28%) WES and 42/192 (22%) total cohort; associated with 17p loss in 6/8 mutated cases; independent OS risk factor (HR 2.4; 95% CI 1.4–4.2; P=0.003).
  • NSD2 (WHSC1/MMSET) — recurrent missense p.E1099K and p.T1150A in 13/130 (10%) MCL; nearly all in SOX11-positive tumors; first description of WHSC1 mutation in lymphoma; phenocopies t(4;14) PCM expression program.
  • KMT2D (MLL2) — truncating + FYRN/FYRC missense in 4/29 (14%) WES primaries and 2/6 cell lines; no biallelic loss; restricted to SOX11-positive / IGHV-unmutated MCL.
  • BIRC3 — exon-9 inactivating + splice-site mutations in 11/173 (6.4%) MCL; tightly co-occurs with 11q22.2 deletion (10/11 vs 25/87, P=1.1×10⁻⁴); can be acquired post-treatment.
  • MEF2B — recurrent exon-2 p.K23R missense in 6/187 (3.2%) MCL; one p.N49S in REC-1 cell line; SOX11-positive tumors only.
  • TLR2 — p.D327V (also reported in IGHV-mutated CLL) and p.Y298S in 2/171 SOX11-negative/IGHV-mutated MCL; functional gain producing elevated IL-1RA and IL-6 secretion.
  • NOTCH1 — PEST-truncating mutations in 8/172 (4.6%) MCL plus MINO and REC-1 cell lines; blastoid morphology enriched; shorter 3-y OS (33% vs 60%).
  • NOTCH2 — PEST-truncating mutations in 9/172 (5.2%) MCL; mutually exclusive with NOTCH1 (only 1/16 carried both); dismal 3-y OS (0% vs 62%, P=2.5×10⁻⁴); independent OS risk factor (HR 3.5; 95% CI 1.3–9.5).
  • SOX11 — not directly mutated here but used as a key biological stratifier; SOX11 expression strongly partitions the mutation landscape (ATM/NSD2/KMT2D/MEF2B in SOX11+; CCND1/TLR2 in SOX11−).
  • UBR5 — ubiquitin-ligase mutations observed (recurrent in MCL per Meissner et al., 2013).
  • B2M — inactivating p.L13fs*10 in one MCL with 15q12–q21.1 deletion encompassing the locus; not observed in 97 additional patients including 3 with similar 15q monoallelic deletions.
  • CDKN2A — only gene with recurrent homozygous deletions in MCL; no somatic point mutations detected.

Clinical implications

  • NOTCH2 mutations as a prognostic biomarker — independent predictor of poor OS in MCL (HR 3.5; 95% CI 1.3–9.5; P=0.017) and an enrichment marker for blastoid/pleomorphic morphology; the authors propose NOTCH1/2 mutations identify a uniformly high-risk subset (3-y OS 24% vs 63%, P=3.4×10⁻⁴).
  • NOTCH pathway as therapeutic target — the activating PEST-truncating pattern in both NOTCH1 and NOTCH2 supports γ-secretase or NOTCH-pathway inhibition strategies in this aggressive MCL subset.
  • Indolent vs aggressive MCL substratification — low mutation/CNA burden, IGHV-mutated, SOX11-negative, with CCND1 and TLR2 mutations defines a distinct, more indolent biology; NSD2/KMT2D/MEF2B chromatin-modifier mutations and ATM mutations mark the SOX11-positive / IGHV-unmutated aggressive subset.
  • Chemotherapy-driven clonal selection — emergence of new dominant subclones (including post-treatment BIRC3 mutation acquisition with 11q deletion) at relapse highlights the need for relapse-time profiling to guide targeted therapy.
  • TLR2/microenvironment axis — gain-of-function TLR2 alleles increased IL-6 / IL-1RA secretion, supporting microenvironment cytokine signaling (e.g., IL-6) as a candidate therapeutic axis in SOX11-negative MCL.

Limitations & open questions

  • WGS was limited to 4 cases; most variant calling relied on WES, which under-samples non-coding events and structural variation beyond the t(11;14).
  • Paired/sequential sampling for clonal-evolution analysis was small (n=8 WGS/WES + 19 targeted), limiting power to enumerate later “driver” events.
  • Functional consequences of KMT2D and MEF2B mutations remain unresolved — the authors note these are similar to mutations in DLBCL and FL but their effect on MCL biology is not yet established.
  • Mechanism by which TLR2 p.D327V/p.Y298S alleles alter cytokine secretion requires deeper signaling characterization; sample size (n=2 mutated MCL + 1 CLL) is small.
  • The series lacks mutations in genes recurrently mutated in other lymphoid neoplasms (MYD88, CARD11, EZH2, SF3B1) — whether this reflects true MCL specificity or sampling depth is open.
  • Whether NOTCH2 mutation is targetable in MCL — and whether γ-secretase inhibition can overcome the dismal-prognosis subgroup — was not tested.

Citations from this paper used in the wiki

  • “We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B” (Abstract / Results).
  • “We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis” (Abstract).
  • “five patients who did not need treatment (median 55 mo, range 4–147) had significantly fewer somatic protein-coding mutations (mean 11 ± 4 versus 25 ± 11, P = 3.4 × 10⁻⁵) and CNAs (mean 2 ± 3 versus 12 ± 9; P = 0.001) than patients who required treatment at diagnosis” (Results, Landscape of Mutations in MCL).
  • “ATM mutations were found in 12 of the 22 (55%) tumors expressing SOX11, but in none of the SOX11-negative MCL (P=0.023)” (Recurrent Somatic Mutations in MCL).
  • “We further analyzed these exons in 101 additional tumors and confirmed the presence of the same mutations in nine more cases [total 13/130 (10%)]” — re NSD2/WHSC1 (Results).
  • “BIRC3 … we found mutations in 11/173 (6.4%) cases, and these cases had more frequent 11q deletions than BIRC3-unmutated MCL [10/11 (91%) versus 25/87 (29%), respectively; P=1.1×10⁻⁴]” (Results).
  • “NOTCH2 mutations occurred more frequently in blastoid/pleomorphic MCL (66 versus 18%, P = 0.001) and conferred a dismal prognosis [3-y overall survival (OS): 0 versus 62%, P = 2.5 × 10⁻⁴]” (Results, NOTCH2 and NOTCH1 Mutations in MCL).
  • “In a bivariate analysis, both NOTCH2 mutations (hazard ratio: 3.5; 95% confidence interval: 1.3–9.5; P=0.017) and TP53 mutations (hazard ratio: 2.4; 95% confidence interval: 1.4–4.2; P=0.003) were identified as independent risk factors for OS” (Results).
  • “Only 1 of the 16 patients with mutations in these genes had mutations in both. Taken together, NOTCH1/2 mutations were present in 9.5% of MCL” (Results).
  • “Eight of the 25 recurrently mutated genes in MCL (ATM, CCND1, MLL2, KCNC2, KIAA1671, PCSK2, TNRC6B, and TRPM6) were present at similar allelic frequency in the two subclones … In contrast, four recurrently mutated genes (ABCA3, TLR2, TP53, and WHSC1) were seen in only one of the two simultaneous or emerging subclones at progression” (Results, Clonal Heterogeneity).

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