Waldenström macroglobulinemia (WM)

DNA methylation analysis

Collaboration

INSERM U1170 (O. Bernard)

Bioinformatics Ohio State (C. Oakes)

GH Pitié-Salpêtrière, UMRS 1138 (F. Nguyen-Khac)

Damien Roos-Weil

FILO Nancy 2020

Waldenström’s macroglobulinemia (WM)

Küppers, 2005

Origin ? Clinical heterogeneity ?

- Clonal mixture of lymphoid and plasma cells

- IgM secretion, unable to undergo CSR

- mutated IGHV

- CD22low+, CD23+, CD25+, CD27+, CD38low+, SmIgM+

Morel 2008

Treon 2012 and 2015

DNA methylation (in B-cell malignancies)

• DNA methylation (5mC) : CpG dinucleotides

• DNA methyltransferases (DNMT, writers), 5mC binding proteins (readers), 5mC demethylation (TET, erasers)

• Promoter DNA methylation inversely correlated with gene expression

• Global DNA hypomethylation (depth correlated to increasing maturation)

• Focal changes in promoters and regulatory regionsOakes Blood 2018

DNA methylation dynamics during B-cell maturation

Oakes Nat Genet 2016

• High degree of epigenetic change during B-cell maturation

• Activity of a limited group of transcriptions factors

DNA methylation analysis in CLL

Oakes Nat Genet 2016

Waldenström macroglobulinemia (WM)

DNA methylation analysis

Collaboration C. Oakes (Ohio state)

Bone marrow WM samples : cell-sorted B-cells on CD19+, monotypic light chain+

Methylation : 35 (T. Zenz team, Heidelberg)

. NGS : 35 including 11 WES and 24 targeted NGS (Gustave Roussy)

. RNA seq : 24 (P. Vyash team, Oxford)

DNA methylation profile of WM ? Differents patterns ?

Specific epigenetic events in WM ?

Correlation with distinct clinical and biological characteristics ?

Principal component 1 (33%)

Pri

nc

ipa

l c

om

po

ne

nt

2 (

5%

)

Illumina EPIC/850K arrays

Principal component analysis (PCA) of the top 1% most-variable CpGs

PCAUnsupervised clustering

35 WM patients (14+21)

All MYD88 mutated (VAF > 30%)

Global DNA methylation analysis

Principal component 1 (38%)

Pri

ncip

al c

om

po

nen

t 2

(1

1%

)

PCA

RNA sequencing analysis

24 WM patients (11+14)

All MYD88 mutated (VAF > 30%)

top hit = gene set comparing IgM memory B cells to plasma cells

7 of the top 10 most enriched gene sets

GSEA

2 groups, B-cell maturation

Memory B-cell (MBC)-like, Plasma-Cell (PC)-like

MBC = memory B cells ; PC = plasma cell

19 genes

0

20

40

60

80

100

120

140

160

180

200

MBC PC

0

50

100

150

200

250

300

350

400

WM MBC-like

WM PC-like

0

20

40

60

80

100

120

140

160

180

200

MBC PC

0

50

100

150

200

250

WM MBC-like

WM PC-like

0

10

20

30

40

50

60

70

80

90

100

MBC PC

0

50

100

150

200

250

300

350

WM MBC-like

WM PC-like

0

2000

4000

6000

8000

10000

12000

14000

MBC PC

0

10000

20000

30000

40000

50000

60000

WM MBC-like

WM PC-like

0

5

10

15

20

25

MBC PC

0

5

10

15

20

25

30

35

40

45

WM MBC-like

WM PC-like

0

200

400

600

800

1000

1200

MBC PC

0

100

200

300

400

500

600

WM MBC-like

WM PC-like

0

20

40

60

80

100

120

140

160

MBC PC

0

10

20

30

40

50

60

70

WM MBC-like

WM PC-like

0

10

20

30

40

50

60

MBC PC

0

2

4

6

8

10

12

14

16

18

WM MBC-like

WM PC-like

0

50

100

150

200

250

300

350

400

450

MBC PC

0

200

400

600

800

1000

1200

1400

WM MBC-like

WM PC-like

IRF8 BLK SPIB BACH2 XBP1 BLIMP1 CD138 IGHM CD20

MBC

PC

WM, MBC-like

WM, PC-like

C

16 differentially expressed

10 concordant

MBC (n=5), PC (n=7)

Association with normal MBC and PC subsets

Exp

ress

ion

WM

PC

-lik

e/M

BC

-lik

e (

log

10

)

Expression normal

PC/MBC (log10)

ncs = non-classed switched ; cs = class-switched

1,000 most-variable CpGs

Comparison of DNA methylation patterns between WM and normal cells

MBC-like and PC-like WM subtypes reminiscent of other B-cell malignancies

tSNE analysis

Most variable CpGs

Among WM and normal B-cells

Tumor-specific epigenetic events ?

Concordant hypomethylated CpGs

- 75% MBC-like and PC-like

- 40% MBC-like and HP-CLL

- 85% PC-like and MM

Tumor-specific epigenetic events ? Association with chromatin states ?

Distinct biological features of MBC-like and PC-like subtypes

Distinct biological features of MBC-like and PC-like subtypes

MBC-like

• more splenomegaly (5/14 vs. 1/21, P = .02)

• more thrombocytopenia (7/14 vs. 0/21, P = .0001)

DNA analysis of WM patients

Perspectives

- Correlation with clinical outcomes ?

- Larger cohorts

- DNA methylation probe set ? CD38 ? Cytologic features ?

- DNA methylation profile comparison with splenic marginal zone lymphoma ?

- Model the impact of CXCR4 mutations (in association with MYD88 LP) or others

genetic events on human and murine B-cell (PC vs. MBC) differentiation ?

Acknowledgments

Olivier Bernard

Véronique Della Valle

Hussein Ghamlouch

Camille Decaudin

Marine Armand

Florence Nguyen-Khac

Véronique Leblond

Magali Le Garff-Tavernier

Nabih Azar

Karim Maloum, Catherine Settegrana

U1170, Gustave Roussy, Villejuif

Oxford (RNAseq)

Pitié-Salpêtrière, Paris

Paresh Vyas

Marlen Metzner

Ohio State and Heidelberg (DNA methylation)

Chris Oakes

Brian Giacopelli

Thorsten Zenz

Junyan Lu