CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)

Multicolor Immunophenotyping: Standardization and ApplicationsMarch 9-11, 2012 TMH, Mumbay (India)

MONOCLONAL B-CELL LYMPHOCYTOSIS

- Monoclonal B-cell Lymphocytosis (MBL) indicates the presence of <5x109 clonal B-cell/L in PB of otherwise healthy subjects, with or without lymphocytosis

MONOCLONAL B CELL LYMPHOCYTOSIS (MBL)

(Marti et al, Br J Haematol 2005)

- CLL-like MBL: <5x109 clonal CD5+ B-cells/L in PB of otherwise healthy subjects, with or without lymphocytosis

- Typical: CD5+, CD20lo, CD79lo and sIglo

- Atypical: CD5+ and CD20hi and/or CD79bhi and/or sIghi

- Non-CLL-like MBL: <5x109 clonal CD5- B-cells/L in PB of otherwise healthy subjects, with or without lymphocytosis.

CLASSIFICATION OF MONOCLONAL B CELL LYMPHOCYTOSIS (MBL)

(Marti et al, Br J Haematol 2005)

B CELL CHRONIC LYMPHOPROLIFERATIVE DISORDERSHeterogeneous group of diseases typically characterized by

a monoclonal expansion of a mature-appearing neoplastic B-lymphocyte

Mature/peripheral B cell chronic lymphoid leukemias: Chronic lymphocytic leukemia/Small B cell lymphocytic lymphoma Prolymphocytic leukemia Hairy cell leukemiaMature/pheripheral B-cell lymphomas: Lymphoplasmacytic lymphoma Splenic marginal zone lymphoma Extranodal marginal zone lymphoma (MALT-type) Nodal marginal zone lymphoma Follicular lymphoma Mantle cell lymphoma Diffuse large B-cell lymphoma Burkitt lymphomaPlasma cell neoplasias: Multiple myeloma/plasmacytoma

WHO CLASSIFICATION OF B-CLPD

IMMUNOPHENOTYPIC PATTERNS OF DIFFERENT

TYPES OF B-CLPD (Orfao et al, In: “B-CLL”.Humana Press, 2004) sIg CD5 CD10 CD20 CD11c CD23 CD24 CD25 CD38 CD43 CD79b CD103 FMC7

B-CLL d + - d -/+ ++ + + -/+ + d - -

PLL + -/+ - + -/+ -/+ + -/+ -/+ -/+ + - +

HCL + - - ++ ++ - -/+ ++ - - + + +

SMZL + -/+ - + + - + -/+ - - + -/+ +

LPL + - - + - - + + -/+ - + - -/+

MCL + + - + -/+ - + -/+ - + + - -/+

FL + - + + -/+ -/d + -/+ + - + - +

LDBCL + - - + -/+ - -/+ - + - + - +

BL -/+ - + + - - + - ++ -/+ -/+ - +

WHO: B-cell malignancies

Immunophenotype

CLLHCL

WHO: B-cell malignancies

Cytogenetics

MCLBL

Immunophenotype

CLLHCL

WHO: B-cell malignancies

Histology &cytology

DLBCLB-PLL

Cytogenetics

MCLBL

Immunophenotype

CLLHCL

WHO: B-cell malignancies

Histology &cytology

DLBCLB-PLL

Cytogenetics

MCLBL

Immunophenotype

CLLHCL

Clinic

MALT

- Chronic lymphocytic leukemia (CLL) is the most frequent subtype of leukemia in the Western World

- Diagnosis of CLL requires the presence of >5x109 clonal B-cells/L in PB with a CLL-like immunophenotype even if there are no symptoms.

MONOCLONAL B CELL LYMPHOCYTOSIS (MBL)

vs CHRONIC LYMPHOCYTIC LEUKEMIA (CLL)

(Morton et al, Blood 2006; http://www.hmrn.org/)

- Between 5%-7% and 12% of healthy adults, display small clones of CLL-like and 2%-2.5% of healthy adults have small clones of non-CLL-like B-cells in peripheral blood.

- Recent studies show a transformation rate of MBL into CLL (frequently assymptomatic) of around 1% per year, most CLL patients being preceeded by MBL.

Prediction of transformation of MBL into symptomatic remains a challenge

MONOCLONAL B LYMPHOCYTOSIS

(Rawstron et al, Blood 2002; Rawstron et al, NEJM 2008; Dagklis et al, Blood 2009, Nieto et al, Blood 2009, Almeida et al, Leukemia 2011)

(Rawstron et al, NEJM 2008; Landgren et al, NEJM 2009)

CLINICAL PROGRESSION OF A CLL-LIKE MBL CASE

Rawstron et al, Cytometry B, 2010

MBL cases presenting with or without lymphocytosis, is highly prevalent

among the general population

Increasing evidence suggests that this could represent a pre-leukemic condition, since CLL frequently

develops in individuals with prior history of MBL

MONOCLONAL B CELL LYMPHOCYTOSIS AND CHRONIC LYMPHOCYTIC LEUKEMIA

AIM:

1.- To determine the prevalence of CLL-like (and other B-CLPD) clonal PB B-cells in a groupd of healthy adults

from the Salamanca healthcare area

2.- To establish the immunophenotypic and genetic characteristics of each clonal B-cell population identified

(vs neoplastic B-cells from CLL and other B-CLPD.

3.- To investigate the potential clinical significance of the B-cell clones identified and its behaviour.

Randomly recruited

(Health Care Area of

Salamanca:

- Age:62±13years (range: 40-

97)

- Sex: (m/f): 46% / 54%

- N. of PB leucocytes:

6,3±1,6 x 109/L- N. of PB lymphocytes:

2,1±0,7 x 109/L- N. of PB B lymphocytes:

0,16±0,1 x109/L

- CLL:65 - Transf. CLL/PLL:3- MALT NHL:2 - FL:1 - MZL:1- Unclassifiable: 3

- Age: 67±13 years

(range: 35-91)

- Sex:(m/f): 57% / 43%

639 healthy adults > 40 years:

75 non-treated B-CLPD patients:

Healthy subjects and patients studiedMaterial & Methods

Methods: Immunophenotypic screening for aberrant PB B-cells

-8-color direct immunofluorescence technique:

PB PO FITC PE PerCP/Cy5.5 PECY7 APC AF700

CD20 CD45 CD8Anti-sIgl

CD56Anti-sIgk CD4 CD19 CD3 CD38

CD20 CD45 cyBcl2 CD23 CD19 CD10 CD5 CD38

- - Anti-sIgl Anti-sIgk CD19 CD10 CD5 -

Methods: Immunophenotypic screening for aberrant PB B-cell

populations

-Data acquisition: FACSCanto II flow cytometer: Window of analysis to simultaneously select for CD19+ and/or CD20+ events (B-cells), for > 5x106 leukocytes

CD

20 P

acif

ic b

lue

CD19 PerCP-Cy5.5 CD19 PerCP-Cy5.5

CD

20 P

acif

ic b

lue

FSC-AS

SC

-A

Frequency of PB B-cell clones in healthy adults

Found in 94/637 cases (14.8%)

No differences in distribution per sex group:

13.4% m /15.9% f ; p>0.05

637No. of individuals studied 150% of cases showing PB B-cell clones 10%

300

11.7%

400

14.2%

500

14.4% 14.8%

Negative PB Positive PB

Age (Mean ± 1SD) 61 ± 13 69 ± 11

Differences with age:

p<0.0001

Frequency along the recruitment for the study (18 months)

*0.35% of all B-cells & O.O3% of all leucocytes

Clonal CLL-like B-cells

Normal B lymphocyt

es

Immunophenotypic identification of PB B-cells with a CLL-like

phenotype

CD20 Pacific blue

CD

5 A

PC

CD20 Pacific blue

CD

5 A

PC

CD

23

PE

cyBCL2 FITC

CD

23

PE

cyBCL2 FITC

Lambda FITC

Ka

pp

a P

E

Lambda FITC

Ka

pp

a P

E

SS

C-A

CD19 PerCP-Cy5.5

SS

C-A

CD19 PerCP-Cy5.5

CD19 PerCP-Cy5.5

SS

C-A

CD19 PerCP-Cy5.5

CD

20

Pa

cif

ic b

lue

cyBCL2 FITC

CD

23

PE

Ka

pp

a P

E

Lambda FITC

Immunophenotypic identification of clonal B-cells in PB from healthy

subjects0.01% of leucocytes0.7% of B-cells CLL-like

phenotype

CLL-likephenotype

Non-CLL B-cellphenotype

0.0015% of leucocytes0.1% of B-cells

Nieto et al, Blood 2009

Frequency of clonal B cell populations in PB of healthy adults

En 95/639 casos (14,8%)

Per

cen

tag

e o

f M

BL

cas

es

40 45 50 55 60 65 70 75 80 85 90 95

Age (years)

0

10

20

30

40

50

60

70

80

90

100

5.7%

5.9%

18.5%

21.3%

26%

75%

Mean age: 70±11 years for MBL (vs. 61±13 years for non-MBL; p<0.05)

PB clonal B-cells in healthy adults: size of the B-cell

population% of PB leukocytes % of PB B-

lymphocytes

Median[range]

0.01%[0.0004%-14.8%]

0.53%[0.02%-96%]

£ 0.01% Clonal B-cells

>0.01-0.1% Clonal B-cells

>0.1-1% Clonal B-cells

>1% Clonal B-cells

54%

% of PB leucocytes

29%

8.5% 8.5%

P>0.05

Age group (years)

% in

filt

ració

n r

esp

ecto

al to

tal d

e leu

cocit

os

0,0

0,1

0,2

0,3

0,4

0,5

P>0.05

40-49 50-59 60-69 70-79 80-89 >90 %

of

PB

leu

kocyte

s

Salamanca(330.000 inhabitants)

Castilla y León(2.625.000 inhabitants)

The Salamanca Area within Spain

Frequency of biclonal cases among healthy individuals

Immunophenotypic features of PB clonal B-cells from healthy subjects

BICLONAL21% (n=20)

MONOCLONAL79% (n=74)

- Direct stain-lyse-wash immunofluoresce technique: Panel of 8-color MAb combinations for: CD11c,CD22,CD24,CD25,CD27,CD34,CD43,CD49d,CD103,CD79b, FMC7,IgM,CCR6,cyZAP70.

CLL: Trisomy 12, del(13q14), del(11q22-23) (ATM & MLL genes), del(17p13) & del(6q21); FL: t(14;18) (LSI IgH/bcl2 dual color); MCL: t(11;14) (LSI IgH/CCND1 dual color); DLBCL & BL: probes for 3q27 (BCL6 gene), t(8;14) (q24;q32), t(2;8) & t(8;22)

Purification of aberrant B-cells: FACSAria (purity: ≥98%)

Genetic analysis of purified B-cells by iFISH:

Immunophenotypic characterization of aberrant B-cells

Statistical methods: SPSS 15.0

Methods: Characterization of (purified) circulating PB B-cell clones poblaciones

Sequencing of IGH e IGK o IGL genes: Repertoire of the V, D, J regions of the IGH gene & VJ of the IGK & IGL genes IGH gene mutational status.

B-CLL

non-class NHL.

MCL

MALT

MZSL

Atypical B-CLL

MZSL or HCLv

MALT or MZSL

B-CLL+B-CLL

B-CLL + non-class. NHL

MZSL + non-class NHL

Non class. NHL + Non-class. NHL

MZSL or MALT + CLL

PB clonal B-cells from helthy subjects

MONOCLONAL

BICLONAL

Immunophenotypic features of the B-cell clones

Median CLL

count

13q14 deletion Trisomy 12 11q

deletion17p

deletion

CLL (17) >5,000/uL 54% (178/238)

16% (53/325)

18% (58/235) 7% (23/325)

Clinic(Mayo) (12,13)

2757 44% (56/126)

18% (21/126) 2% (2/126) 3% (4/126)

Clinic (Leeds) (8) 3141 58% (19/33) 21% (7/33) 6% (2/33) 3% (1/33)

Population (Leeds) (8) 9 39% (15/38) 18% (4/22) 0% (0/21) 0% (0/10)

Population (Salamanca)

(6)*0.5 36% (16/45) 8% (4/45) 0% (0/45) 0% (0/45)

Rawstron et al, Cytometry B, 2010

CLL vs CLL-LIKE MBL: Genetic features of clonal B-lymphocytes

SourceMedian CLL cell

count

CLL-like B-cells (median

%)

Cases with <98% IGHV

homology

Predominant CLL cell IGHV

geneSimilar to CLL?

CLL (25) >5,000 >95%534/927 (57.6%)

3-07, 1-69, 4-34, 3-23

-

Clinic (Leeds) (8)

3141 >95% 18/20 (90%) 3-07, 3-23, 4-34 Yes

Clinic (Mayo) (12,13)

2757 >95% 84/109 (77%)3-07, 1-69, 4-34,

3-23Yes

Familial (Duke) (24)

26 25% 12/16 (75%) 3-07, 4-34 Yes

Population (Leeds) (8)

9 80% 18/20 (90%) 3-07, 3-23, 4-34 Yes

Population (Italy) (4)

1.0 7% 36/51 (70%) 4-59/61 No

Population (Salamanca)

(6)0.5 0.4% 2/7 (29%)

No CLL-associated

No

CLL vs CLL-LIKE MBL: Biological features of clonal B-lymphocytes

Rawstron et al, Cytometry B, 2010

0

10

20

30

40

50

60

70

Perc

en

tag

e o

f cases

Normal* 13q- +12

17p-;11q-

MBL (n=35)

CLL (n=65)

P<0,0560%

25%

38%

58%

7%

16% 16%

Genetic alteration

MBL CLL

13q- 67%±30% 67%±31%

+12 50%±13% 76%±17%

Other (17p-/11q-)

- 65%±29%

% of altered cells

• No alterations detected by FISH with probes for 13q-,+12, 17p-,11q-

Cytogenetic patterns of clonal CLL tumor cells vs PB CLL-like B-cells from

healthy adults

CLL-like MBLs with genetic abnormalities have higher absolute

numbers of CLL-like B-cells

0

50

100

150

200

250

300

Trisomy 12 Del (13q) No genetic abnormalities

Ab

s. N

. C

LL-l

ike B

cells

(c

ells

/mL)

P=0.001

Almeida et al, Leukemia 2011

0/2 cases carrying trisomy 12 and only 2/12 cases with del(13q) showed less than 1 PB CLL-like B-cell/mL, while 11/21 cases without detectable genetic abnormalities were “low-count” MBL (<1 circulating CLL-like B-

cell/mL)

Baseline

0,0

0,1

+1 year

% o

f clo

nal B

-cells f

rom

leu

cocyte

s

0,2

0,3

0,4

0,5 p=0,04

BASAL +1 year %

of

clo

nal B

-cells

Fro

m a

ll B

cells

p=0,03

0

5

10

15

20

25

Baseline +1 yearN.

of

clo

nal B

cells (

x1

03/m

L)

0

3

6

9

12

15 p=0,045

MBL: CLINICAL SIGNIFICANCE

0

2

4

6

8

10

12

% o

f clo

nal B

cells f

rom

le

ucocyte

s

Baseline +1 year

p=0,03

0

20

40

60

80

100

% o

f clo

nal B

cells

Fro

m a

ll B

cells

p=0,2

Baseline +1 year

0

200

400

600

800

1,000

1,200

1,400p=0,003

N.

of

clo

nal B

cells (

x1

03/m

L)

Baseline +1 year

NON-CLL-like MBL (11/13)

CLL-like MBL (39/82)

Nieto et al, Clinical Cytometry 2010

• A significant proportion (14.8%) of healthy adults aged >40 years display circulating B-cell clones frequently (13%) showing a CLL-like phenotype.

• The frequency of such CLL-like (and other) circulating B-cell clones increase with age.

• The higher frequency of circulating CLL-like clones could reflect the greater sensitivity of the screening approach used, half of the cases showing clonal B-cell numbers below previously reported threshold levels ( 0,01%).

• The immunophenotypic, genetic and molecular features of circulating B-cell clones overlap with those of clinically evident CLL and other B-CLPD.

• In all tested cases circulating B-cell clones remain detectable after one year with minimal but significant changes (increase) in their distribution.

CONCLUSIONS

100%

50%

0%10.10.01 5 10 20 40 5030

Whole series

10.10.01 5 10 20 40 5030

40 - 59 years

10.10.01 5 10 20 40 5030

60 - 69 years

100%

50%

0%

100%

50%

0%

100%

50%

0%

70 years

PB Volume (mL)10.10.01 10 20 40 5030

% o

f ca

ses

wit

h a

CLL

-lik

e c

lone

5

70%

100% 100%

18%

46%

32%

88%

62%

36%

100%100%100%

DETECTED FREQUENCY

FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS

Almeida et al, Leukemia 2010

100%

50%

0%10.10.01 5 10 20 40 5030

Whole series

10.10.01 5 10 20 40 5030

40 - 59 years

10.10.01 5 10 20 40 5030

60 - 69 years

100%

50%

0%

100%

50%

0%

100%

50%

0%

70 years

PB Volume (mL)10.10.01 10 20 40 5030

% o

f ca

ses

wit

h a

CLL

-lik

e c

lone

5

70%

100% 100%

18%

46%

32%

88%

62%

36%

100%100%100%

CALCULATED FREQUENCYDETECTED FREQUENCY

FREQUENCY OF CLL-like MBL IN HEALTHY ADULTS

Statistical predictive model(power regression analysis)Almeida et al, Leukemia 2010

CD20 Pacific blueC

D5 A

PC

CD20 Pacific blue

CD

5 A

PC

CD20 Pacific blue

CD

5 A

PC

CD20 Pacific blue

CD

5 A

PC

1 mL 50 mL

Lambda PE

Kap

pa F

ITC

Lambda PE

Kap

pa F

ITC

Immunophenotypic identification of CLL-like clonal B-cells in 50 mL from healthy

adults

Almeida et al, Leukemia 2011

Case N.Age

(years)

Sex

N. of CLL-like B-cell population

s*

sIg light chain

restriction

Ratio /k l% of CLL-like B-cells from WBC (x10-3)

% of CLL-like B cells from all PB B-cells

N. of CLL-like B cells

/mL

Monoclonal cases

Case #1 78 M One l NA 0.8% 0.041% 0.066

Case #2 73 M One k NA 0.73% 0.05% 0.036

Case #3 77 M One k NA 1.3% 0.08% 0.112

Case #4 88 M One k NA 0.14% 0.013% 0.009

Multiclonal cases

Case #5 77 F Two kl 20

0.28%0.014%

0.002%0.001%

0.0018 0.0009

Case #6 72 F Two kl 0.72

0.08%0.11%

0.006%0.008%

0.0070.009

Case #7 82 F Two kl 0.18

0.18%0.95%

0.02%0.01%

0.0090.004

Case #8 73 M Two kl 1.8

0.49%0.27%

0.016%0.009%

0.0320.018

Case #9 87 M ND ND NA <0.0006% <1.1x10-6 <1x10-6

M: male; F: female; ND: Not detected. NA: Not applicable*CLL-like B-cells were identified as those cellular events expressing CD19+, CD5+, CD20+dim, CD79b+dim and surface immunoglobulin light chain Ig+dim.§ Restricted to CLL-like B-cells (considered to be altered when ratio k/l >3.1 or <1:3)

Characteristics of CLL-like B-cells identified in 50 mL of PB from 9 healthy subjects older than 70 years

Almeida et al, Leukemia 2011

B-CLPD: COMMON ABERRANT PHENOTYPES

Diagnostic group Aberrant phenotype % of cases

typCLL/SLL CD22-/+dCD5+ 97%CD22-/+dCD23+ 98%

PLL sIg +++ 60%

HCL CD11c+++ 69%CD19+++ 69%CD103+++ 92%FSC/SSC+++ 85%

LPL CD22-/+dCD10- 67%

FL BCL2+++CD10+ 85%

MCL CD22-/+dCD5+ 100%Sanchez et al, Leukemia, 2002

0

20

40

60

80

100

0.01 0.1 1 10 100 1000 10000

Absolute 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 10000

Absolute 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 10000

Absolute Number of Neoplastic B-cells

Cum

ulat

ive

Perc

enta

ge o

f Cas

es Population Studies (Normal Blood Count)

Hematology Clinic Series (Lymphocytosis)

CUMULATIVE FREQUENCY OF CLL-LIKE MBL IN POPULATION- VS HOSPITAL-BASED COHORTS

Rawstron et al, Cytometry B, 2010

Conclusions

• In healthy adults over 70 years,

emergence of one or more CLL-like cell

populations occurs whenever > 50mL

PB are screened, suggesting that these

clonal cells may more likely represent

the normal counterpart of CLL cells,

rather than a leukemic precursor

Neoplastic trasnformatio

n

Reactive

process

Malignant trasnformati

on

Treatment

?(Wait and see

vs early therapy)

Genetic instability

Genetic and Chromosomal instability

Wait and see

X

ONCOGENIC EVENTS

Genetic/environmental factors

Subclones blocked at different

maturation stages and clinical

progression

Clonal expansion

Identical primary mutation ?

Identical IGH/IGL VDJ/VJ gene rearrangement

Restricted expressiofn Ig (k or L) light chains

MBL AND B-CLL: HYPOTHETICAL ONTOGENIC MODEL

Evolution

Secondary genetic alterations

....

....

...

.........

CD5:APC

CD

10

:PE

Cy7

CD

20

:PB

CD10:PECy7

CD38:APC-H7

sIg

D:P

E

CD38:APC-H7

sIg

M:F

ITC

sIgM:FITC

sIg

D:P

E

B CELL MATURATION PATHWAYS IN NORMALHUMAN BONE MARROW (GATED ON CD19+)

Memory B-cells

Somatic Hypermutation/IgH Switch

Naïve B-cells

Immature B-cellsBM PB

MATURATION-ASSOCIATED NORMAL PB B-CELL SUBSETS

Plasmablasts/Plasma cells

CD38:AF700 LOGIC

AL

CD27:APC LOGICAL

CD

10:

PE

-Cy7

LO

GIC

AL

Immature

Naïve

Memory

Plasmablasts

Percentage Absolute count

(cells/ L)

Immature 5.4%3.7% (1.8%-4.5%)

99(3-11)

Naive 64%12%(57%-73%)

10157(61-130)

Memory 31%12%(21%-40%)

5239(26-65)

Plasmablasts/Plasma cells

2.5%1.4%(0.8%-2.3%)

3.0 6.8(1.0-3.1)

Distribution of B-cell maturation subsets in peripheral blood from normal individuals

(N=600)

Memory Plasmablasts/Plasma cells

sIgA+

(21% 9%)

sIgG+

(23% 10%)

sIgMD+

(52% 15%)

sIgG+

(13% 11%)

sIgM+

(18% 12%)

sIgneg

(14% 12%)sIgD+

(5% 5%)

sIgA+

(49% 18%)

Caraux A Haematologica 2010, Perez-Andres M Clinical Cytometry 2010

CD27 EXPRESSION BY NORMAL PB CD5+ B CELLS

Normal B-cell maturationstemcell

Lymphoidprogenitor

Progenitor-Bcell

Pre-B cell

ImmatureB-lymphocyte

MemoryB-cell

Plasma cell

GerminalcenterB-cell

MaturenaiveB-cell

Lee et al.

Bone marrow

Lymphoid tissue

Peripheral blood

MM

MZLLPLMCL

DLBCLBL, FL

ALL

CLL

CIC, Department of Medicine & Cytometry Service (USAL)

J Almeida

J Ciudad

A López Fernández

A Nieto

W Nieto

A Rodríguez Caballero

ML Sánchez-García

C Teodosio

AKNOwLEDGEMENTS

Primary Health Care Area of Salamanca (SACYL)

JA Romero Furones

P Fernández Navarro

Primary Health Care Group of Salamanca for the study of MBL (40 medical doctors from the primary health care area of

Salamanca )

University Hospital of Salamanca

MB Muñoz Criado (Microbiology)

A Balanzategui (Hematology)

M González Díaz (Hematology)

MB Vidriales (Hematology)

Regional Health Care Department of Castilla y

León

AT Vega Alonso (Epidemiology)

Universidad Federal de

Río de Janeiro (Brazil)

CE Pedreira (Mathematics)

THANK YOU