Molecular Genetics of Small Mature B-cell Lymphomas

Elias CampoHospital Clinic, University of Barcelona

Barcelona, Spain

Genetic Drivers in Lymphomagenesis

Overt Lymphoid Neoplasia

Progression/TransformationClonal Population

Primary Genetic Alterations

Secondary Genetic Alterations

Microenvironment Genetic alterations

The mutational profile is a dynamic process

Puente X & Lopez-Otín C Nat Genet 2013

Chemotherapy

Impact of NGS in Small B-cell Neoplasms

• Orient management estrategies

• Understanding evolution of the disease

• Prognostic groups and risk stratification

• Disease specific profiles

ICGC: Puente et al. Nature. 2015, DFCI: Landau et al. Nature 2015,

Driver mutated Genes and CNA in CLL ICGC & Danna-Farber (DFCI)

2336 21

ICGC DFCI

Driver genes (n=80)

ICGC (n=428): (Puente et al. Nature 2015). DFCI/Broad (n=123) Only pretreatment cases consideredCLL8 (n=278): Clinical trial (Landau et al. Nature 2015)

Driver mutated Genes in CLL genomes (ICGC vs Danna Farber (DFCI)

Population based vs Clinical trialMedian age DFCI cohort10 yr younger than ICGC

Mutated Pathways in CLL

Puente et al Nature 2015

452 CLL & 54 MBL (150 whole genomes)13.631 mut in coding genes: 27 per tumor951 CNA (2 per tumor)59 driver genes

Genomic Heterogeneity and Driverless in CLL

Puente et al, Nature 2011, 2015

NOTCH1 Mutations in CLL

0

5

10

15

20

25

Richter Transformation

Mutated wt

Stilgenbauer S et al. Blood 2014;123:3247-3254

FCR- NOTCH1 wt

FCR- NOTCH1 mut

E52DEL E52DEL wt L265Pwt wt wt L265P L265P

CCL2CCL4CCL3IL6

IL1RA

TLR/MYD88 mutated pathway in CLLA specific subgroup of patients?

Martínez-Trillos A, et al. Blood 2014; 123:3790–3796.

Age at diagnosis (years)

30

20

10

0

20 30 60 70 80 9040 50

Patie

nts

(%)

1.0

0.8

0.4

0.2

0.0

0.6

0 5 10 15 20

MYD88 mut

MYD88 wt

p=0.024

Rel

ativ

e su

rviv

alYears from diagnosis

NFĸB pathwayMAPK pathways

Inflammatory cytokinesand chemokines

IRAKs

MYD88

TLR

Clinical relevance of individual mutated genes in CLL

Mansouri et al. 2015, JEM

NFKBIE

Young et al. 2017, Leukemia

EGR2

Ljungström et al. 2015, Blood

RPS15

SF3B1

Quesada et al. 2011, Nat Genetics

NOTCH1

Puente et al. 2011, NaturePOT1

Herling et al. 2016, Blood

?

Co-occurrence of driver alterations in CLL

Nadeu F et al Leukemia 2017 (In press)

Prognostic impact of number of mutated drivers and subclonal alterations in CLL

Puente X et al Nature 2015 Landau DA et al Cell 2013

Number of drivers Clonal/subclonal heterogeneity

TP53 variant CLL cell

Diagnosis Chemotherapy Progression Chemotherapy Refractoriness

Small TP53 variant subclone mixed with

TP53 wt clones

Removal of TP53 wt clones and selection of the

TP53 variant subclone

Expansion of the TP53 variant clone

Rosi D et al Blood 2013

• 406 untreated patients• 28 CLL driver genes• Mean coverage 1500x NGS • Sensitive pipeline 0.1% allelic frequency

Small mutated subclones are identified in virtually all genes

Nadeu F et al Leukemia 2017 (In press)

Clinical Impact of clonal and sublconal mutations in CLL

Nadeu F et al Blood 2016; Leukemia 2017 (In press)

Ibrutinib-resistant mutations may be detected before clinical relapse

Ahn IE et al. Blood 2017;129:1469-1479; Woyach JA et al JCO 2017, 35, 1437-1443; Arnason & Brown Curr Oncol Report 2017

Detection of mutated subclones may precede treatment Median 8-9 months (3-15 months)

BTK

Somatic Mutations in LymphoplasmacyticLymphoma

Tiacci et al NEJM 2011; Ngo Nature 2011; Puente Nature 2011; Xi L et al Blood 2012; Schmidt et al Br J Haematol 2015; Cao et al Leukemia, 2015 29, 169–176.; Xu L et al Blood 2017

MYD88 L265P

• 95% WM/LPL• 29% DLBCL-ABC• 6% MZL• 3% CLL

• Useful information in the differential diagnosis of LPL• Need to be interpreted in the global context of the disease

CXCR4

• 25-35% WM/LPL• Associated with MYD88• More active disease• Less lymphadenopathy• More resistant disease

to new drugs

Patients treated with Ibrutininb

Mutations before clinical progression

When should MYD88 L265P mutations be studied in small B-cell neoplasms?

Swerdlow SH et al Virchows Arch. 2016;468(3):259-75

• Differential diagnosis of LPL strongly considered but findings not conclusive

• Differential diagnosis between LPL and IgM PCM

• If LPL is not in the differential diagnosis MYD88 mutations may not be useful

Somatic Mutations in HCL and HCLv

Hairy Cell Leukemia

HCL-v

Tiacci E, et al. N Engl J Med. 2011; Waterfall J et al Nat Genet. 2014; Durham BH et al Blood 2017

MAP2K1

• 50 % HCLv50% HCL IgH V4-3450% Pediatric Type FL

CCND3, U2AF1

• NF1/NF2 downregulation• KRAS mutation

Vemurafenib Resistance

7q deletion

Okosun J et al Nat Genet. 2014 , 46:176-81.

Follicular lymphoma Mutational Landscape

Krysiak K et al Blood 2017; 129: 473-483

Early and Late Mutations in Follicular Lymphoma

Okosun J et al Nat Genet. 2014 , 46:176-81.

Early driver mutations in chromatin regulator genes (CREBBP, EZH2 and KMT2D (MLL2)),

Gained at transformation : EBF1 and regulators of NF-κB signaling(MYD88 and TNFAIP3)

Clinical Impact of FL Mutations

EZH2 inhibitor (Tazemetostat) in Refractory/Relapsed FL• OR: 63% in patients with EZH2 mutations (N = 8)

28% in with wild type EZH2 (N = 46)

Morschhauser F et al Hematol Oncol 2017; 35, S2: 24–25

Pastore et al Lancet Oncol. 2015 Sep;16(9):1011-1012

Risk Stratification

Target Therapy

NOTCH1/2 Mutations in FL

7/112 FL cases (6.3%)5 NOTCH12 NOTCH2

NOTCH3/4 (4%)

PEST/TADD domainTruncanting mutations

FemaleSplenic/extranodal involvementNegative for t(14;18)DLBCL component

CD21

BCL2 LMO2Karube K et al J Pathol 2014; 234:423-30Krysiak K et al Blood 2017; 129: 473-483

Pediatric Type Follicular Lymphoma

BCL2

GenesPTFL

(n=71)(%)

t(14;18)-neg FL (%)

t(14;18)-pos FL(%) P-value

TNFRSF14 33-51 36 18-46 Ns

KMT2D 14-16 36 67-82 Ns

CREBBP 3* 45 33-64 0.001

FOXO1 5 27 - Ns

GNA13 11 0 - Ns

EZH2 3* 18 7-20 0.0049* >18yr

Genes PTFL

MAP2K1 38-49%

MAPK1 10%

IRF8 15-50%

Louissaint A et al Blood 2016; Ozawa et al Mod Pathol 2016; Schmidt J et al Blood 2016 & 2017

Mutational Profile of Nodal and Splenic Marginal ZoneLymphoma

Targeted Pathways

• KLF2, NOTCH2, activation (40-25%)• NFkB activation (36-51%)• BCR signaling (SMZL 8%)• PTPRD (NMZL 20%)

Arcaini L, Rossi D. Haematologica. 2012; Rossi et al J Exp Med 2012; Clipson et al Leukemia 2015; Parry et al Clin Cancer Res 2015; Spina V et al; 2016; Piris MA Blood 2016;

Clinical Impact of Somatic Mutations in SMZL

Parry et al Clin Cancer Res 2015 Campos-Martin Y et al Haematologica 2017

Steven H. Swerdlow et al. Blood 2016;127:2375-2390

Molecular Pathogenesis and Clinical Subtypes of MCL

CCND1 neg MCL

Classic MCL

Cyclin D1

Cyclin D1 Sox11

Mozos et al Haematologica 2009; Salaverria et al Blood 2013

Mantle cell lymphomaCCND1-negative variant

Rearrangements No. (%)

CCND2 22 (55%)

CCND3 0

No Cyclin D gene translocation

18 (45%)

Cryptic IG-CCND1/2/3 rearrangements in MCL

IGK/CCND3

Bea S et al unpublished

IHC CCND1+Break apart CCND1 neg

CrypticIGK-CCND1

…CACTAGGGCTCCCACA CCTTGGGCGC…

Chr2 IgK

110kb

Chr11 CCND1chr11

CCND1/2 neg MCL

Recurrent gene mutations in MCL

0

10

20

30

40

50

60

70

ATM CCND1 TP53 MLL2 MLL3 TRAF2 NOTCH1 WHSC1 BIRC3 NOTCH2 UBR5 S1PR1 MEF2B CARD11

Bea et al. & unpubl (n=170) Greiner et al (n=92) Zhang et al (n=56) Rahal et al (n=165)Kridel et al (n=108) Meissner et al (n=102) Rossi et al (n=151) Wu et al (n=13)

Zhang 2014 Bea 2013

ATMCCND1MLL2

TP53BIRC3WHSC1

MCL primary tumors (n=29)

SOX11 -

SOX11 +

IGHV-unmut

IGHV-mut

-80

-60

-40

-20

0

No. of somatic mutations (WES) No. of CNA (SNP-array)

20

40

60

80

0

0 SOX11IGHV

ATMCCND1TP53

WHSC1MLL2BIRC3

MEF2BCHMP4C

LUZP4RGS4

PDLIM3KCNC2

SLC17A6DCP1BPCSK2SP140TLR2

TRMP6DLGAP2DNAJC6TNRC6BCRYBG3ABCA3

KIAA1671ABCC9

No.

of

alte

ratio

ns

Somatic Mutations in MCL

M00

1M

007

M01

9M

023

M02

9M

028

M00

2M

011

M02

2M

025

M00

8M

020

M03

1M

030

M01

3M

014

M01

0M

006

M01

2M

024

M02

6M

018

M00

4M

003

M02

7M

009

M01

5M

021

M01

6

Bea S et al PNAS 2013

SOX11 +ATM (55%)

Chromatin (10%)

NOTCH1/2 (5%)TP53 23%)BIRC3 (7%)

High CNA

SOX11 -TLR2 (29%)

NOTCH1/2 (5%)TP53 23%)

BIRC3 (14%)

Low CNA

Prognostic impact of 17pTP53 aberrations in MCL

Royo C et al, Leukemia 2012TP53 wt

P =0 .045

17p/TP53 wt

17p/TP53 altered

SOX11-negative MCL

17p/TP53 wt

17p/TP53 altered

SOX11 +

Eskelund CW et al Blood 2017

• Diagnostic criteria to refine entities• Identification of subsets of patients• Prognostic and predictive significance• Monitoring disease evolution: Dynamic evolution of

mutational landscape• Targets for therapy decisions:

– Selection of patients

– Actionable mutations

Clinical Relevance of Mutational Profiles in Lymphoid Neoplasms

M Chagall, Tossa de Mar, 1933

Recurrently Mutated Pathways in Small B-cell Neoplasms

Pathway U-CLL

M-CLL cMCL nnMCL FL PTFL LPL MZL HCL

DNA-damage + +SF3B1 + +/-NOTCH1/2 + +/- + +/- +ChromatinRemodeling + +BCR-signaling + +/-NFkB + + +/-TLR/MYD88 +/- +/- +MAPK + +