Monoclonal B-cell Lymphocytosis

Paolo Ghia

Lab of B Cell Neoplasia - Division of Molecular OncologyLymphoma Unit – Department of Clinical Oncology

Università Vita-Salute San Raffaele - MilanoIstituto Scientifico San Raffaele - Milano

Chronic Lymphocytic Leukemia (CLL)

• Characteristic phenotype: CD5+, CD23+, CD20low, sIgMlow

CD

5CD

19

KappaCD20 CD79bCD5

KappaCD20 CD79b

CD

5

CD5

CD

19

CLL-like clones are presentin otherwise healthy individuals

• Characteristic phenotype: CD5+, CD23+, CD20low, sIgMlow

CD

5CD

19

KappaCD20 CD79bCD5

KappaCD20 CD79b

CD

5

CD5

CD

19

Rawstron et al, 2002; Ghia et al, 2004

One name for all

• Benign Monoclonal B cell Lymphocytosis• B Monoclonal Lymphocytosis of

Undetermined Significance

• B cell Monoclonal Lymphocytosis• B cell Monoclonal Expansion

• Smoldering CLL

• Subclinical CLL

• Monoclonal B cell Lymphocytosis(MBL)

Marti et al, BJH 2006

• B-cells = 5 x 109/L(5,000/µL)

• Monoclonal– Kappa:Lambda <0.3:1 or

>3:1– 25% lacking surface

immunoglobulin• No other feature of a B-

lymphoproliferativedisorder

CLL diagnosis:B cells > 5 x 109/L

Hallek et al, Blood 2008

CD

5

CD20

Non-CLL

Atypical CLL

CD19+CD5+CD20low

CD19+CD5+CD20+

CLL-like MBL are the most frequent

CD19+CD5-CD20+

R2

R1

R3

CLL-like

CD

5

CD20

CD

5

CD20

CD19+CD5-CD20+

Dagklis et al, Blood 2009

• B-cells = 5 x 109/L(5,000/µL)

• Monoclonal– Kappa:Lambda <0.3:1 or

>3:1– 25% lacking surface

immunoglobulin• No other feature of a B-

lymphoproliferativedisorder

• Three categories– CD5+CD23+

• CLL-like– CD5+CD23-

• atypical CLL– CD5-

0

1

2

3

4

5

6

7

8

9

10Pe

rcen

tage

(

<40 40-49 50-59 60-69 70-79 ≥80

Years

MaleFemale

5.2% >18yy

Dagklis et al, Blood 2009

CLL-like MBL is common among the elderly

6.7% >40yy

Scarfò et al, Sem Canc Biol 2010

The more you search, the more you find...

In individuals >40 years of age

Are we all bound to have it?

Nieto et al, Blood 2009

500.000 events

12% overall

5.000.000 events

Scarfò et al, Sem Canc Biol 2010

5.2% overall

• In NZB (and NZW or NZB/NZW) mice CD5+ clones are detected by PCR and FACS at 3 mo of ageThey develop frank leukemia at 2y of age.

Seldin MF J Exp Med 1987, Stall AM PNAS 1988

• This is an accelleration of a process that occurs in all strains of mice, including BALB/c,C57BL/6 and CBA >15 month of age

Stall AM PNAS 1988

B cell clonal expansions in Mice

• Monoclonal expansions of B cells have been detected in > 85% of C57BL/6 mice 18 months old.LeMaoult J. Immunol 1997

MBL CLLInitiation & Initiation & immortalizationimmortalization DiseaseDisease progression progression

??

Lymphocyte

Q1. How many CLL are preceded by MBL?

Is CLL always preceded by MBL, does CLLtypically arise de novo, or do both occur?

Over 77,000 healthymen and women

Age 55–74 yrsat study enrollment

Blood draw at baseline, repeated annually(up to 6 years) together with screening protocol

Prospective follow-up for cancer

Nationwide prospective PLCOCancer Screening Trial

Prorok et al, Clin Trials 2000

CLL is virtually always preceded by MBL

CLL

<11-22-33-44-5>5

8 7 7724Pre-CLL samples with IGHVsequence data (n=35; 78%)

Yrs between pre-diagnosticblood draw and CLL dx(mean 32 months)

11/14 (79%) 16/21 (76%)IGHV mutated

Landgren et al, NEJM 2009

Q2: How many MBL progress into CLL?

Years Years

Leeds

Rawstron et al, NEJM 2008; Shanafelt et al, JCO 2009

• Approximately 1% (1.4-1.1%) of individuals with MBL willrequire treatment for progressive CLL every year

• Patients with progressive lymphocytosis or requiringtreatment for CLL do not have a worse survival than otherindividuals with MBL or CLL Rai 0

Mayo

Does B cell number matter?

Years Years

Leeds

Rawstron et al, NEJM 2008; Shanafelt et al, JCO 2009; Rossi et al, BJH 2009

• Approximately 1% (1.4-1.1%) of individuals with MBL willrequire treatment for progressive CLL every year

• Patients with progressive lymphocytosis or requiringtreatment for CLL do not have a worse survival than otherindividuals with MBL or CLL Rai 0

Question: Are all MBL equal?

0

20

40

60

80

100

0.01 0.1 1 10 100 1000 10000Absolute Number of Neoplastic B-cells

Cum

ulat

ive

Perc

enta

ge o

f Cas

es

Population Studies (Normal Blood Count)

Hematology Clinic Series (Lymphocytosis)

0

20

40

60

80

100

0.01 0.1 1 10 100 1000 10000Absolute Number of Neoplastic B-cells

Cum

ulat

ive

Perc

enta

ge o

f Cas

es

0

20

40

60

80

100

0.01 0.1 1 10 100 1000 10000Absolute Number of Neoplastic B-cells

Cum

ulat

ive

Perc

enta

ge o

f Cas

es

Population Studies (Normal Blood Count)

Hematology Clinic Series (Lymphocytosis)

Rawstron et al, Cytometry Part B 2010

Question: Are all MBL equal?

0

1

2

3

0 – 1.5 1.5 - 5 >5

Prev

alen

ce (%

)

B-Lymphocyte count (103/µl)

Clinic MBL

General populationMBL

CLL

1.1% per year CLL

MBL in the general population are small

76/89 had a MBL clone smaller than 10% of B cells

0

50

100

150

200

250

300

350

400

450

1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85

Individuals

Ab

so

lute

co

un

t (/

uL)

B cellsMBL cells

Dagklis et al, Blood 2009

1. CLL-like MBL may be polyclonal

6 out of 89 CLL-like MBL cases had a normal κ/λ ratio

R128,2%

71,8%

Dagklis et al, Blood 2009

2. IGHV gene repertoire is different

Dagklis et al, Blood 2009

CLL

3. IG genes in CLL: Stereotyped receptors

H

L

H

L

Tobin et al, Blood 2002Ghia et al, Blood 2005

CDR3

FR2 FR3FR1

CDR1 CDR2

IGHD IGHJIGHV

0,00000000001%

• 113 stereotyped antigen receptors (subsets)

• 26.9% of all CLL cases (530/1967)

Murray et al, Blood 2008

SUBSET 13* IGHV4-59*03 IGHD2-15*01 IGHJ2*01 Sequence HCDR3

VB274 CARDAYCSGGSCFDWYFDLW

FRA-168 ----------T---------

SUBSET X IGHV3-73*01 IGHD ND IGHJ4*01 Sequence HCDR3

VB689 IGHV3-73*01 IGHD2-15*01 IGHJ4*02 CVSDRRGYW

ITA-0060 IGHV3-73*01 IGHD ND IGHJ2*01 ---------

MBL cells express few Stereotyped Receptors

• 26.9% of all CLL cases• 4,5% of all MBL cases

SUBSET 13* IGHV4-59*03 IGHD2-15*01 IGHJ2*01 Sequence HCDR3

VB274 CARDAYCSGGSCFDWYFDLW

FRA-168 ----------T---------

SUBSET X IGHV3-73*01 IGHD ND IGHJ4*01 Sequence HCDR3

VB689 IGHV3-73*01 IGHD2-15*01 IGHJ4*02 CVSDRRGYW

ITA-0060 IGHV3-73*01 IGHD ND IGHJ2*01 ---------

MBL cells express few Stereotyped Receptors

Anti-HCV Ig, isolated from a SLL case with HCV+ MC De Re et al, Blood , 2000

----S-----S---------IGHJ2*01IGHD2-15*01IGHV4-59*01Rheumatoidfactor

----A---------------IGHJ2*01IGHD2-15*01IGHV4-59*01MBL-089

CARDRYCSGGTCFDWYFDLWIGHJ2*01IGHD2-15*01IGHV4-59*01Subset 13

Perc

enta

ge o

f ind

ivid

uals

with

clo

nal p

opul

atio

ns

0

5

10

15

20

25

30

35

MBL and HCV infection

40

HCV+0

5

10

15

20

25

30

35

CD5- MBLAtypical CLL MBLCLL-like MBL

Healthy

Fazi et al, Cytometry part B 2010

Median: 48.6clonal cells/μL

Median: 0.46clonal cells/μL

MBL cells tend to persist with time

Fazi et al, in preparation 2010

11,7%

7,26 cell/μL

2005

59,9%

247,09cell/μL

VB 068953,4%

248,59cell/μL

34,5%

11,26 cell/μL

VB 0274

MBL tend to remain stable with time

2006

2009

2009

13q deletion: 36% Trisomy 12: 16%

11q deletion: 18% 17p deletion: 7% Dohner et al. 2000

ATM?

p53

Chromosomal abnormalities in CLL

?

miR-15amiR-16-1

0.0%0/17 Trisomy 12 0.0%0/17 Del (11q) –ATM- 11.8%2/17 Del (17p) –p53

Normal 8/17 47,1%

Presence ofabnormalities 9/17 52,9%

Singleabnormality 8/17 47,1%

Del (13q x1 Del (13q) x2

6/172/17

35,3%11.8%

Chromosomal abnormalities in CLL-like MBL

47.1%

Fazi et al, in preparation 2010

MBL ontogeny – a working model

Foreign antigen

Self antigen

Persistent infection

Generalpopulation

MBL

Persistent Stimulation

Polyclonalexpansion

ClinicMBL

CLL

CD5 CD5

CD5

CD5

CD5 CD5

CD5

CD5

CD5CD5

CLL

CD5

Del(13q)?

B cell

1.1%/year

Genetic hit?

Claudia Fazi, Antonis Dagklis, Lydia Scarfò,Francesca Cottini, Agnieszka Janus, Maria Gounari,

Elisa ten Haken

Cristina Scielzo, Marta Muzio,Sabrina Bertilaccio, Benedetta Apollonio,Giorgia Simonetti, Tania Veliz-Rodriguez,

Federico Caligaris-Cappio

Università Vita-Salute San RaffaeleIstituto Scientifico San Raffaele

Department of Oncology - Division of Molecular Oncology

Laboratory of B Cell Neoplasia

CERTH, ThessalonikiKostas Stamatopoulos

Laboratory of Lymphoid Malignancies

Pathology UnitMaurilio Ponzoni, Lorenza Pecciarini

Claudio Doglioni

FellowshipProgram