Kostas Stamatopoulos

Institute of Applied Biosciences CERTH, Thessaloniki

Immunoglobulin genes in CLL biological aspects

CLL is a clone of mature B cells

Mature B cells interact with the microenvironment

CLL cells are doing the same!

microenvironmental interactions

receptors

B cell receptor immunoglobulin a unique molecular signature for every Β cell clone

Immunoglobulins are very diverse

Combinatorial

Junctional

Somatic hypermutation

L IGKV IGKJ IGHC

5’ 3’

5’ 3’

~ 6,3 x 106 combinations ~ 3,5 x 105 combinations

Heavy chain (IGH) Light chain (IGL)

L IGHV IGHD IGHJ IGHC

5’ 3’

39-46 IGHV x 23 IGHD x 6 IGHJ

~ 6300 combinations ~185+165 combinations 5’ 3’

L IGLV IGLJ IGLC

5’ 3’

33-37 IGKV x 5 IGKJ/ 30-33 IGLV x 4-5 IGLJ

Mathematical chance that two different B cell clones will share the same IG

0 , 0 0 0 0 0 0 0 0 0 0 0 1 %

IG genes a unique molecular identity for CLL

IG genes in CLL

why?

Hamblin et al, Blood 1999

Damle et al, Blood 1999

CLL - better with mutated IG receptors

M

UM

M

UM

why?

an immunological trajectory from primary to 3D structures

IGH locus

46 functional IGHV genes

0

2

4

6

8

10

12

14

16

18

20

immunoglobulin gene frequencies

46 functional IGHV genes

if it happened by chance

0

2

4

6

8

10

12

14

V1-

18

V1-

2

V1-

24

V1-

3

V1-

45

V1-

46

V1-

58

V1-

69

V1-

8

V2-

26

V2-

5

V2-

70

V3-

7

V3-

9

V3-

11

V3-

13

V3-

15

V3-

19

V3-

20

V3-

21

V3-

23

V3-

30

V3-

30-3

V3-

33

V3-

43

V3-

48

V3-

49

V3-

53

V3-

64

V3-

66

V3-

72

V3-

73

V3-

74

V4-

30-2

V4-

30-4

V4-

31

V4-

34

V4-

39

V4-

4

V4-

59

V4-

61

V4-

b

V5-

51

V5-

a

V6-

1

V7-

4-1

Fais et al. J Clin Invest 1998; Murray et al. Blood 2008; Agathangelidis et al. Blood 2012

it’s not happening by chance!

0

2

4

6

8

10

12

14

V1-

18

V1-

2

V1-

24

V1-

3

V1-

45

V1-

46

V1-

58

V1-

69

V1-

8

V2-

26

V2-

5

V2-

70

V3-

7

V3-

9

V3-

11

V3-

13

V3-

15

V3-

19

V3-

20

V3-

21

V3-

23

V3-

30

V3-

30-3

V3-

33

V3-

43

V3-

48

V3-

49

V3-

53

V3-

64

V3-

66

V3-

72

V3-

73

V3-

74

V4-

30-2

V4-

30-4

V4-

31

V4-

34

V4-

39

V4-

4

V4-

59

V4-

61

V4-

b

V5-

51

V5-

a

V6-

1

V7-

4-1

Fais et al. J Clin Invest 1998; Murray et al. Blood 2008; Agathangelidis et al. Blood 2012

V1-69 U-CLL

V4-34 M-CLL

V3-21 Borderline

SHM status

different IGHV genes differential SHM status

antigen selection

Unmutated CLL: polyreactive receptors many opportunities for activation

Herve et al. J Clin Invest 2005; Seiler et al. Blood 2009

Attenuated signaling in good prognosis CLL

Bad prognosis

Survival, proliferation

Good prognosis

Zupo et al, 1996; Chen et al, 2002; Lanham et al, 2003; Mockridge et al, 2007 Muzio et al, 2008; Krysov et al, 2012; Ntoufa et al. 2012; Chatzouli et al, 2014

Appolonio et al, 2014; Ntoufa et al, 2016

fact

CLL - better with mutated IG receptors

always?

IGHV3-21 CLL a distinct disease variant?

~50% of IGHV3-21 CLL carry restricted BcR IG

Stereotyped

repeated with limited or no variation

Probability that two different B cell clones carry identical BcRs

1:10-12

stereotypy in CLL is not happening by chance

Uppsala

Bournemouth

Copenhagen

Athens

Paris

New York

Brno

Montpellier

London

Milan

Rotterdam

2012

11 institutes 7424 patients

Thessaloniki

BcR stereotypy: 33% 19 major subsets

Montpellier

Uppsala

Bournemouth

London Copenhagen

Turin

Athens

Rotterdam

Milan

Paris

New York

Brno Ulm

Kiel

Athens-Un

Moscow

Novara Rochester, MN

Belfast

Belgrade Padova

Barcelona

2019 BcR stereotypy: 41%

stereotyped subsets

distinct clinical variants of CLL

BcR stereotypy refines prognostication in CLL

Distinct clinical outcomes for subsets #1, #2 and #4, independently of genomic aberrations or SHM status

Baliakas et al. Lancet Haematol. 2014

All express IGHV4-34, all concern IG mutated CLL, yet the outcome is different

Xochelli et al. Clin Cancer Res 2017

Subset #2 is as bad as CLL with TP53 aberrations though essentially devoid of such lesions

Baliakas et al. Blood 2015

Subset #8: the highest risk for Richter’s transformation amongst all CLL

Rossi et al. Clin Cancer Res. 2009

#4

#1

#2

0 5 10 15 20 25 30

Time years

0%

25%

50%

75%

100%

% u

ntr

ea

ted

#2, n=212 del(17p), n=284

p=0.5

is there a biological explanation?

the unpleasant extreme

Subset #2: the most aggressive CLL variant?

Baliakas et al. Lancet Haematol 2014

#2

only subset #2 is nasty

not just any IGHV3-21

Subset #2 as bad as TP53 aberrant CLL

0 5 10 15 20 25 30

Time years

0%

25%

50%

75%

100%

% u

ntr

ea

ted

#2, n=212 del(17p), n=284

p=0.5Baliakas et al; Blood 2015

do not blame TP53 loss/mutation

Malcikova et al. BJH 2014

Sutton et al. Haematologica 2016 0%

10%

20%

30%

40%

50%

# 1 # 2 # 3 # 5 # 6 # 7 # 8

TP53 mutations in major stereotyped subsets

SF3B1 mutations are remarkably enriched in subset #2

Strefford et al. Leukemia 2013 Rossi et al. Blood 2013

44.5% 45.4%

0

10

20

30

40

50

Subset #1 Subset #2 Subset #80

10

20

30

40

50

Subset #1 Subset #2 Subset #8

Rossi et al. Blood 2012

A distinctive oncogenetic signature for subset #2

5%

Baliakas et al;. Lancet Haematol 2014

Subset-biased landscapes of genomic aberrations

trisomy 12

Subset-biased landscapes of genomic aberrations

Sutton et al. Haematologica 2016

distinctive immunogenetic signatures

distinctive

oncogenetic signatures

the pleasant extreme

Subset #4: the most indolent CLL variant?

Baliakas et al. Lancet Haematol 2014; Xochelli et al. Clin Cancer Res 2017

#4

Subset #2 mAbs homodimerize

Subset #4 mAbs homodimerize

Distinct homotypic BcR interactions shape the outcome of CLL

CLL BcR immunoglobulins initiate intracellular signaling through homotypic interactions between epitopes that are specific for each stereotyped subset.

The molecular details of the BcR-BcR interactions determine the clinical course

stronger affinities and longer half-lives in indolent cases

weaker, short-lived contacts in aggressive cases

The diversity of homotypic BcR contacts leading to cell-autonomous signaling reconciles the existence of a shared pathogenic mechanism with the biological and clinical heterogeneity of CLL

Minici et al. Nat Commun 2017

final words?

Baliakas et al. Leukemia 2015

all negative and/or del(13q)

all negative and/or del(13q)

M-CLL

U-CLL

Complex interactions between cell-intrinsic and cell-extrinsic processes impact on CLL outcome

B cell receptor immunoglobulin a unique molecular signature for every Β cell clone

B cell receptor immunoglobulin key to understanding and treating CLL

a secret for success?

join forces!

San Raffaele, Milan Paolo Ghia Lydia Scarfo Marta Muzio Karolinska Institute, Stockholm Richard Rosenquist Lesley-Ann Sutton Uppsala University Panagiotis Baliakas Pitié Salpêtrière, Paris Fred Davi Nikea General Hospital, Athens Chrysoula Belessi Feinstein Institute, Manhasset Nicholas Chiorazzi

ΙΝAΒ | CERTH, Thessaloniki Anastasia Hadzidimitriou Andreas Agathangelidis |Maria Gounari Eva Minga | Stavroula Ntoufa Anna Vardi | Aliki Xochelli Chrysi Galigalidou | Katerina Gemenetzi Elisavet Vlachonikola| Laura Zaragoza G. Papanicolaou Hospital, Thessaloniki Niki Stavroyianni Achilles Anagnostopoulos IMGT, Montpellier Marie-Paule Lefranc Veronique Giudicelli Sofia Kossida Erasmus MC, Rotterdam Anton Langerak