Utilidad del inmunofenotipo en las gammapatías ... · Utilidad del inmunofenotipo en las...

Utilidad del inmunofenotipo en las gammapatías monoclonales (MGUS y mieloma múltiple)

Departamento de Medicina, Centro de Investigación del Cáncer y Servicio de Citometría. Universidad de Salamanca, Salamanca, España.

EuroFlowTM Consortium

Curso avanzado de Actualización en Onco Hematología. Buenos Aires, Mayo, 30 a Junio, 1, 2011

Normal B-cell diferentiation

Monoclonal gammopathies

Bone Marrow infiltration by PC

Detection of monoclonal Ig (paraprotein) in serum and/or urine


International Working Group (BJH 2003; 121:749)

Parameter Disease category

MGUS SMM MM

M Component < 30 g/L in serum > 30 g/L in serum Present in serum/urine

% of plasma cells < 10% ≥ 10% > 10%

End organ damage Absence Absence Present

Most relevant disease categories and their diagnostic criteria

and

and and

or

and

MGUS: monoclonal gammopathies of undetermine significance; SMM: smouldering myeloma; MM: multiple myeloma


Natural history ?

Time

MGUS Multiple myeloma

Germinal center B-cell

prevalence of 3.2%; ≥50y risk of transformation: 1 % per year MGUS to MM 1 0% per year SMM to MM

Kyle RA, et al. N Engl J Med. 2007;356:2582-90. Weiss BM, et al. Blood. 2009;113(22):5418-22

??

Smoldering myeloma

MM. Treatment

1848 1960 1970 1980 1990 2000 2003 2004 2011

Melphalan (1958, Blokhin)

Ann NY Acad Sci

Combination chemo Vincristine

Doxorubicin Dexamethasone

Thalidomide

Bortezomib

Melphalan Glucocorticoids

(1969)

High-dose chemo Stem cell transplantation

Bisphosphonates

Mr. McBean

1 848 Lenalidomide

?

MM. Treatment

Kumar SK et al. Blood. 2008

1971–76

1994–00 1989–94

1977–82

2001–06

1983–88

Time

0 20 40 60 80 100 120 140

Surv

ival

0.0

0.2

0.4

0.6

0.8

1.0 Su

rvival

OS from time of diagnosis in 6-year intervals based on date of diagnosis

FCM Immunophenotyping of PC

- Identification and characterization of PC • Prognostic influence of number of PC 1

• Prognostic influence of specific antigens 2

• Analysis of DNA ploidy and cell cycle 3

- Discrimination between normal vs. clonal PC: • Differential diagnosis of MGUS & MM 4,5

• Risk of transformation of MGUS & smoldering MM 6,7

• Favourable prognosis subgroup of symptomatic MM 8

• MRD investigations in MM 9-12

• Differential diagnosis of AL vs. other forms of amyloidosis 13

• Differential diagnosis of IgM monoclonal gammopathies

1. Paiva et al. Haematologica 2009;94(11):1599-1602. 2. Mateo et al. J.Clin.Oncol. 2008;26(16):2737-2744. 3. San Miguel et al. Blood 1995;85(2):448-455. 4. Ocqueteau et al. Am.J.Pathol. 1998;152(6):1655-1665. 5. Olteanu et al. BMC.Clin.Pathol. 2008;8:13.

11. Paiva et al. Blood 2008;112(10):4017-4023. 12. Paiva et al. J Clin Oncol. 2011 Apr 20;29(12):1627-33. 13. Paiva et al. Blood 2011 Epub ahed of print 14. Ocio et al. Clin Lymphoma Myeloma Leuk. 2011

6. Perez-Persona et al. Blood 2007;110(7):2586-2592. 7. Perez-Persona et al. Br J Haematol. 2010 Jan;148(1):110-4. 8. Paiva et al. Blood 2009;114(20):4369-72. 9. San Miguel et al. Blood 2002;99(5):1853-1856. 10. Rawstron et al. Blood 2002;100(9):3095-3100.

MONOCLONAL GAMMOPATHIES: IDENTIFICATION OF PLASMA CELLS

Specific positive identification. CD38/CD138 gating strategy

FCM Immunophenotyping of PC Identification and characterization of PC. Quantitation

% BMPC

MFC

100

80

60

40

20

0

CM

11%

40%

*

* P <.001

23%

Paiva et al. Haematologica 2009;94(11):1599-1602.

Differences in BMPC counts between CM and MFC in 765 MM patients at diagnosis

FCM Immunophenotyping of PC Identification and characterization of PC. Quantitation. Clinical impact

Paiva et al. Haematologica 2009;94(11):1599-1602.

< 30% BMPC (n=253) ≥ 30% BMPC (n=512)

PFS OS

P < 0.001

120 100 80 60 40 20

100

80

60

40

20

0

P < 0.001

125 100 75 50 25

100

80

60

40

20

0

Median: 48m

Median: 37m

Median: 97m

Median: 61m

< 15% BMPC (n=438) ≥ 15% BMPC (n=327)

P = 0.003

120 100 80 60 40 20

100 80 60 40 20

0

P < 0.001

125 100 75 50 25

100 80 60 40 20 0

Median: 43m

Median: 36m

Median: 97m

Median: 54m

PFS OS ≥ 15% BMPC by

MFC

≥ 30% BMPC by CM

Paiva et al. Haematologica 2009;94(11):1599-1602. ONLY BMPC QUANTIFICATION BY MFC IS OF INDEPENDENT PROGNOSTIC VALUE

FCM Immunophenotyping of PC PC Immunophenotypic profile

Peripheral Blood Bone Marrow

103

104

105

B-cell prec. CD34+

Maturation stages

101

CD19

CD45 CD20

CD38

CD27 CD138

B-cell prec. CD34-

Immature B-cells

Naive B-cells

Memory B-cells

PB Plasma cells

BM Plasma cells

CD10

CD34

Negative expression: CD28 / CD33 / CD56 / CD117 Bone marrow

Antig

en e

xpre

ssio

n

FCM Immunophenotyping of PC PC Immunophenotypic profile. Panel EuroFlow

FCM Immunophenotyping of PC PC Immunophenotypic profile: Normal versus clonal PC

FCM Immunophenotyping of PC Normal versus clonal PC

Proportion of aPC referred to the total-PC (aPC/BMPC)

2nd step

PC compartment

.

.

.

. . .

.

.

.

.

. . .

.

. . . . . .

% aPC/BMPC

% nPC/BMPC

1st step Total cellularity

% PC within BM cellularity

FCM Immunophenotyping of PC Differential diagnosis

Vast majority of cases with 100% Pathological PC

Co-existence of Pathological and Normal PC

versus MM-PC

< 5% N-PC

MM-PC

>5% N-PC

Symptomatic MM MGUS/Smouldering MM

The most powerful single criteria for differential diagnosis

Ocqueteau et al, Am J Pathol 1998, 152: 1655

FCM Immunophenotyping of PC Impact of % N-PC/BMPC in malignant transformation

Pérez-Persona et al; Blood. 2007 Oct 1;110(7):2586-92.

>5% N-PC/BMPC n= 330 (18 progr.) ≤5% N-PC/BMPC n= 73 (15 progr.)

168,00 144,00 120,00 96,00 72,00 48,00 24,00 0,00

1,0

0,8

0,6

0,4

0,2

0,0

Median not reached

Median 107 months

p=0.0000

Months

% T

ime

to p

rogr

essi

on

5 years

4%

24%

MGUS

120 96 72 48 24 0

1,0

0,8

0,6

0,4

0,2

0,0

p=0.0000

Median not reached

Median 34 months

Months

% T

ime

to p

rogr

essi

on

63%

5 years

8%

>5% N-PC/BMPC n= 37 (4 progr.) ≤5% N-PC/BMPC n= 56 (34 progr.)

Smoldering MM

FCM Immunophenotyping of PC Impact of % N-PC/BMPC in malignant transformation

> 5% N-PC / BMPC (n=88) ≤ 5% N-PC / BMPC (n=723)

PFS OS

120 100 80 60 40 20

100

80

60

40

20

P < .001

Median: 39 months

Median: 51 months

125 100 75 50 25

100

80

60

40

20

Median: NR

P = .004

Median: 89 months

Paiva et al. Blood. 2009;114(20):4369-72


Antigen down-regulation: CD38dim (~ 80%) | CD19- (93%) | CD45-/dim (83%) | CD27- (68%) | CD81-/dim (61%)


Antigen up-regulation: CD56hi (57%) | CD28+ (25%)


Asynchronous antigen expression:

CD117+ (~ 30%)


CyLight chain restriction

FCM Immunophenotyping of PC PC Immunophenotypic profile: Aberrant phenotypes

0

10

20

30

40

50

60

70

80

90

100

CD19 CD38 CD45 CD56 CD28 CD33 CD117 CD20

96%

80%

73%

60%

36%

18%

32%

17%

Asynchronous expression Infra-expression

Mateo et al. J Clin Oncol; 2008;26:2737

Over-expression

N= 685 patients

92%

FCM Immunophenotyping of PC PC Immunophenotypic profile: Impact of CD19 expression


Months from diagnosis 84 72 60 48 36 24 12 0

Prog

ress

ion

free

sur

viva

l

1,0

0,8

0,6

0,4

0,2

0,0


Ove

rall

surv

ival

1,0

0,8

0,6

0,4

0,2

0,0

p= 0.04 p= 0.02

38 months n=655

PFS OS

26 months n=30

68 months n=655

40 months n=30

CD19 positive CD19 negative


OS

CD81 negative (n=139) CD81 positive (n=90)

68%

77%

50 40 30 20 10 0

100

80

60

40

20

0 P =.03

Median: NR

Median: NR

Paiva et al. Haematologica 2010; 95[suppl.2]:215, abs. 0528

P <.001

PFS

Median: NR

Median: 20m

50 40 30 20 10 0

100

80

60

40

20

0

30%

62%



p= 0.04 p= 0.001

PFS OS


Prog

ress

ion

free

sur

viva

l

1,0

0,8

0,6

0,4

0,2

0,0


Ove

rall

surv

ival

1,0

0,8

0,6

0,4

0,2

0,0

31 months n=240

38 months n=420 43 months

n=240

NR months n=420

CD28 positive CD28 negative



p= 0.04 p= 0.01

PFS OS


Prog

ress

ion

free

sur

viva

l

1,0

0,8

0,6

0,4

0,2

0,0


Ove

rall

surv

ival

1,0

0,8

0,6

0,4

0,2

0,0

32 months n=431

44 months n=208

63 months n=431

NR months n=208

CD117 negative CD117 positive


p= 0.0001

PFS OS

p= 0.01

38% n=128

29% n=362

21% n=149


Prog

ress

ion

free

sur

viva

l

1,0

0,8

0,6

0,4

0,2

0,0


Ove

rall

surv

ival

1,0

0,8

0,6

0,4

0,2

0,0

72% n=128

57% n=362

43% n=149

CD117+ CD28- CD117- CD28- CD117- CD28+ CD117+ CD28+



PC Immunophenotypic profile: Association with underlaying molecular abnormalities


FCM Immunophenotyping of PC Abnormal PC ploidy and S-phase analysis

Diploid DNA index =1

• Cell-cycle of PC:

Hypodiploid Hyperdiploid

G0/G1

S+G2M

Aneuploid DNA index ≠1

FCM Immunophenotyping of PC Abnormal PC ploidy and S-phase analysis. Clinical impact

120 100 80 60 40 20 0

100

80

60

40

20

0

Hyperdiploid (n=322) Non-hyperdiploid (n=318)

P =.001

PFS

120 100 80 60 40 20 0

100

80

60

40

20

0 P <.001

<1% PC in S-phase (n=263) ≥1% PC in S-phase (n=377)

PFS

FCM Immunophenotyping of PC PC-compartment changes after treatment

Myelomatous PC

Normal PC

Bone marrow plasma cell compartment

n-PC MM-PC

0,035 0,007

0,14

0,21

0 0,05

0,1 0,15

0,2 0,25

0,3

0,03 0

1 0.8 10

14 20

Diagnosis 3m post-Trx 12m post-Trx VBCMP/ VBAD (x6)

San Miguel et al Blood; 2002; 99:1853

FCM Immunophenotyping of PC MRD monitoring. Sensitivity. 4-color FCM

San Miguel et al Blood; 2002; 99:1853

10 10 10 10 10 0 1 2 3 4

CD38 ->

15% MM- PC

10 10 10 10 10 0 1 2 3 4

1.4% MM- PC

Gate CD38 ->

1:10

10 10 10 10 10 0 1 2 3 4

Gate CD38 ->

0.13% MM- PC

1:100

1:10000

10 10 10 10 10 0 1 2 3 4

Gate CD38 ->

CD

56 ->

. . . . . . . . .

. . . . . . . . . .

.

0.0014% MM- PC

10 10 10 10 10 0 1 2 3 4

Gate CD38 ->

. . . . .

. . . . . . . . . . .

. . . . . . . . .

. . . . .

. . . . . .

. . . . . . . . .

. . . . . . . . . .

. . . . .

. . . . .

. . . . .

. . . . . . . . .

. . . . . . . . . .

. 0.015% MM- PC

. . . . . . .

. . .

. . . . . . .

. .

. . . . .

.

. . .

.

. . . . .

.

. . .

. .

1:1

Sensitivity level: 10-4

1:1000

FCM Immunophenotyping of PC MRD monitoring. Clinical impact. Survival according to MRD by Immunophenotyping in BM obtained 3 months after ASCT (n=295)

0 25 50 75 100 125

0

20

40

60

80

100

p< 0.0001 Prog

ress

ion

Free

Sur

viva

l

Months

Median: 71 months

Median: 37 months

Median: Not reached

p= 0.002

Median: 89 months

Months

Ove

ral S

urvi

val

0 20 40 60 80 100 120 140

0

20

40

60

80

100

PFS OS

5y

82%

60%

5y

22%

60%

MRD negative (n=125) MRD positive (n=170) Paiva et al; Blood. 2008; 1 1 2: 401 7


PCD panel Version

Tube Violet laser FL channels Blue laser FL channels Red laser FL channels

PacB AmCyan FITC PE PerCP Cy5.5

PECy7 APC APCCy7

1 1 CD45 CD19 CyIgλ CyIgκ CD38 CD56 CD27

2 CD45 CD19 β2 micro CD81 CD38 CD117 CD28

2 1 CD19 CD45 CyIgλ CyIgκ CD138 CD56 CD27

AF700 CD38

2 CD19 CD45 β2 micro CD81 CD138 CD117 CD28 CD38

3 1 CD19 CD45 β2 micro CyIgλ CD138 CD56 CyIgκ CD38

2 CD19 CD45 CD27 CD81 CD138 CD117 CD28 CD38

4 1 CD19 CD45 β2 micro CyIgλ CD138 CD56 CyIgκ

APCH7 CD38


5 1 CD19 CD45 CD138 CyIgλ β2 micro CD56 CyIgκ CD38


6 1 CD45

PacO CD38 CD56 β2 micro CD19 CyIgκ CyIgλ

CD138


The EuroFlow PCD panel


Tube PacB PacO FITC PE PerCP Cy5.5 PECy7 APC APCH7

1 CD45 CD138 CD38 CD56 β2micro CD19 CyIgκ CyIgλ








Backbone Markers





The EuroFlow PCD panel. Modular design

Tube 1(mandatory) :

-Positive identification

-Accurate quantification of PC

-Diagnosis/assessment of clonality

-Most frequent aberrant phenotypes

-Potentially informative prognostic markers





The EuroFlow PCD panel. Modular design

Tube 2 (complementary):

-Less frequent phenotypic aberrancyes

-Markers with prognostic impact


The EuroFlow PCD panel. Prospective evaluation

A B

n=100. Clasic approach



n=100. Semiautomatic approach

Reference population pool

BM PC from healthy donors




Reference population pool

BM PC from healthy, reactive and regenerative donnors




Abnormal PC populations

CD19-PECy7 21.16

CD56-PE 18.97

CD81-APCH7 12.05

CyIgκ:APC 11.61

CD117-APC 11.33

CD27-PerCPCy5.5 8.30

CD56-PE 23.73

CD19-PECy7 21.28

CD117-APC 17.77

CD81-APCH7 9.52

CD45-PB 6.05

CyIgλ-APCH7 5.74


Thank you


Also thanks to: -EuroFlow group -Hematology department. University hospital of Salamanca. Prof. Jesús San Miguel Dr. M.B. Vidriales Lic. Bruno Paiva

Utilidad del inmunofenotipo en las gammapatías ... · Utilidad del inmunofenotipo en las...

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