advances in immunophenotyping of multiple myeloma … na... · advances in immunophenotyping of...

ADVANCES IN

IMMUNOPHENOTYPING OF

MULTIPLE MYELOMA

ADVANCES IN

IMMUNOPHENOTYPING OF

MULTIPLE MYELOMA

Servicio de Citometria y Servicio de Hematología

Universidad y Hospital Universitario de Salamanca

Servicio de Citometria y Servicio de Hematología

Universidad y Hospital Universitario de Salamanca

Centro de Investigación del

Cáncer

Centro de Investigación del

Cáncer

Sao Paulo, 18th of April, 2009

BACKGROUNDBACKGROUND

IMMUNOPHENOTYPING IMMUNOPHENOTYPING

- Acute Leukemias & Lymphoproliferative disorders:

• Mandatory for diagnosis & monitoring

- Multiple Myeloma:

• Restricted to research

• Differential diagnosis of unusual cases

- Acute Leukemias & Lymphoproliferative disorders:

• Mandatory for diagnosis & monitoring

- Multiple Myeloma:

• Restricted to research

• Differential diagnosis of unusual cases

MM PLASMA CELLMM PLASMA CELL

cIg+

PC-associated Ags:

CD38++/+++.... 100%

CD138 +....... 98%

B-cell-associated Ags:

CD19+..........3-8%CD20+..........2-25%CD22+..........20-30%CD10+..........6-20%HLA-DR+het. 10%CD23+..........0%FMC7+.........0%

HPC-associated Ags:

CD34+.......... 0%

CD117 +....... 27%

Adhesion molecules:

CD56+/++...... 60-70%ββββ1/β/β/β/β2 integrins . 98%

Co-stimulatory Ags:

CD28+/++....... 30-40%

CD40 +....... 100%

CD27 -/dim .... 40-50%

Myeloid-associated Ags:

CD13+......... 28%CD33 +/++..... 24%

Pan-leuc. Ag:

CD45+...20-40%

Plasma cell quantification (BM infiltration)

Plasma cell quantification (BM infiltration)

• Morphological PC count : • Morphological PC count :

- area of BM smear

- infiltration pattern

- area of BM smear

- infiltration patternVariability

• Immunophenotyping: • Immunophenotyping: - precise identification by CD38/CD138- precise identification by CD38/CD138

10 10 10 10 100 1 2 3 4

CD38 FITC ->

CD

138

Per

CP

/Cy5

->

Co-expression of CD38/CD138Co-expression of CD38/CD138

10 10 10 10 100 1 2 3 4CD38 FITC ->

TR

AN

SF

OR

ME

D S

SC

->

High-intensityHigh-intensity

10 10 10 10 100 1 2 3 4

CD138 PerCP/Cy5->

TR

AN

SF

OR

ME

D S

SC

->

Specific expressionSpecific expression

++ ==

- but…..diluted sample →→→→ lower numbers

MM PCMM PC Normal PCNormal PCvsvs

Are myelomatosous PC different from normal PC?

Are myelomatosous PC different from normal PC?

Ocqueteau, Am J Pathol, 1996; San Miguel et al, Blood, 2002

10 10 10 10 100 1 2 3 4

CD38 FITC ->

TR

AN

SF

OR

ME

D S

SC

->

10 10 10 10 100 1 2 3 4

CD38 FITC ->

CD

138

Per

CP

/Cy5

->

CD38-FITC

CD38-FITC gated PC

T-SSC

CD138-Pe

rCP/Cy5.5

MONOCLONAL GAMMOPATHIES: IDENTIFICATION OF CLONAL PLASMA CELLS

Clonal PC

Normal PC

CD

56

PE

9

1

D

C

CP

A

54

DC

CD19-PcpC

y5

CD56-PE CD45-APC

Perez-Andres, J Biol Reg, 2004

Frequency of Aberrant PhenotypesN=195 N=195 patientspatients

001010202030304040505060608080

CD56CD56 CD28CD28 CD33CD33 CD117CD117 CD20CD20

Asynchronous expression

52%52%

25%25%

16%16% 15%15%

11%11%

92%

CD38CD38

80%80%

Over- expressionInfra-expression

sIgsIg

21%21%

Almeida et al, Br J Haematol, 1999

RESULTS: RESULTS: ImmmunophenotypeImmmunophenotype ofof

normalnormal vsvs clonalclonal PCPC

10000

8000

6000

4000

2000

0Clonal PCNormal PC

10000

8000

6000

4000

2000

0Clonal PCNormal PC

4000

3000

2000

1000

0Clonal PCNormal PC Clonal PCNormal PC

3000

2000

1000

0

CD38CD38 HLAHLA--IIαααααααα ββββββββ22--microglobulinmicroglobulin CD40CD40

%

% ofofpositive PC

positive PC

Mean FL

Mean FL In

tensity

Intensity

p=0.21p=0.21 p=0.005p=0.005

60

50

40

30

20

10

0Clonal PCNormal PC

CD95CD95

p=0.72p=0.72

pp<<<<<<<<0.0010.001

80

60

40

20

0Clonal PCNormal PC

CD56CD56

pp<<<<<<<<0.0010.001120

100

80

60

40

20

0Clonal PCNormal PC

CD86CD86

pp<<<<<<<<0.0010.001

p=p=0.0020.002

Clonal PCNormal PC

100

80

60

40

20

0

CD126CD126

pp<<<<<<<<0.0010.001

MOST USEFUL ANTIGENS FOR THE DETECTION OF ABERRANT PC IN MM

Antigen Expression % MM with Requirement for

Normal Altered altered expression MRD studies

CD19 + (>70%) - 95% Essential

CD56 - (>85%) ++ 75% Essential

CD20 - (100%) + 10% Preferred

CD117 - (100%) + 30% Preferred

CD28 -/dim (100%) ++ 15% Recommended

CD81 + -/dim N.A. Recommended

CD27 ++ -/dim 40-50% Recommended

N.A.: not analyzed/not reported.

Rawstron et al, EMN consensus, Haematologica, 2008

CLINICAL APPLICATIONS OF CLINICAL APPLICATIONS OF IMMUNOPHENOTYPING OF IMMUNOPHENOTYPING OF NEOPLASTIC PLASMA CELLSNEOPLASTIC PLASMA CELLS

Diagnostic classification

Prognostic evaluation

Treatment monitoring


Diagnostic classification(differential diagnosis between MGUS vs MM)



MM PC MGUS PCvsvs

Are myelomatosous PC different

from MGUS PC ?

ANTIGEN CD38*

CD19

CD138

CD9

CD13

CD33

CD40

CD56*

ANTIGEN CD38*

CD19

CD138

CD9

CD13

CD33

CD40

CD56*

MGUS: POLYCLONAL

PC (n=76)

100

100

100

100

17

3

100

0

MGUS: POLYCLONAL

PC (n=76)

100

100

100

100

17

3

100

0

CONTROLS' PC (n= 10)

100

100

100

100

20

7

100

0

CONTROLS' PC (n= 10)

100

100

100

100

20

7

100

0

Results expressed as positive cases (> 20% positive PC) *Strong reactivity Other Ags explored were constantly negative

INMUNOPHENOTYPE OF POLYCLONAL PC IN MGUS VS CONTROLS

INMUNOPHENOTYPE OF POLYCLONAL PC IN MGUS VS CONTROLS

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

%

of

cas

es

1 2 3 1 2 3 4 1 2

MGUS MM PCLN. of pathologic PC clones

% ofca

ses

Rasillo et al, Cancer, 2003

INTRATUMOURAL GENETIC HETEROGENEITY IN MM vs OTHER MONOCLONAL GAMMOPATHIES

RESULTS: RESULTS: ImmunophenotypeImmunophenotype ofof clonalclonal PCPC in in

MGUSMGUS vsvs MMMM andand PCLPCL

10000

8000

6000

4000

2000

0PCLMMMGUS

10000

8000

6000

4000

2000

0PCLMMMGUS

3000

2000

1000

0PCLMMMGUS

CD86CD86

CD38CD38 HLAHLA--IIαααααααα ββββββββ22--microglobulinmicroglobulin CD40CD40

%

% ofofpositive PC

positive PC

Mean FL

Mean FL In

tensity

Intensity

pp<<<<<<<<0.0010.001 p=p=0.0080.008

100

80

60

40

20

PCLMMMGUS0

CD126CD126

pp=0.64=0.64

4000

3000

2000

1000

0PCLMMMGUS

p=p=0.020.02

CD95CD9560

50

40

30

20

10

0PCLMMMGUS

p=p=0.0060.006120

100

80

60

40

20

0PCLMMMGUS

p=p=0.150.15

p=p=0.010.01

80

60

40

20

0PCLMMMGUS

CD56CD56

P=P=0.560.56 *

*

*

*

*

Perez-Andres et al, Leukemia, 2005; Int J Cancer, 2008

MM vs Normal BM plasma cells

10 10 10 10 100 1 2 3 4

JC67634.001CD38 ->

14%

10 10 10 10 100 1 2 3 4

JC67634.001CD38 ->

10 10 10 10 100 1 2 3 4

JC67634.001CD38 ->

10 10 10 10 100 1 2 3 4

JC67634.001CD45 ->

10 10 10 10 100 1 2 3 4

PA67603.001CD38 ->

3%

10 10 10 10 100 1 2 3 4

PA67603.002CD38 ->

10 10 10 10 100 1 2 3 4

PA67603.002CD38 ->

10 10 10 10 100 1 2 3 4

PA67603.002CD45 ->

AbnormalAbnormal plasma plasma cellscells

Normal plasma Normal plasma cellscells

BM plasma cells in MGUS

10 10 10 10 100 1 2 3 4

JR67635.001CD38 ->

0,35%

10 10 10 10 100 1 2 3 4

JR67635.002CD38 ->

10 10 10 10 100 1 2 3 4

JR67635.002CD38 ->

50%

50%

0 256 512 768 1024

JR67635.002FSC-Height ->

10 10 10 10 100 1 2 3 4

JR67635.002CD38 ->

10 10 10 10 100 1 2 3 4

JR67635.002CD45 ->

Differential diagnosisDifferential diagnosis

Risk of MGUS transformation 2

Cases with predominantly (>95%) CD19- ve PC.... High risk (26% transformed in 31 months)

Risk of MGUS transformation 2

Cases with predominantly (>95%) CD19- ve PC.... High risk (26% transformed in 31 months)

2. Rawstron A, Blood 2003, 102, 36 a (Abstr.116)2. Rawstron A, Blood 2003, 102, 36 a (Abstr.116)

MGUSMGUSMMMM

Only 20% of MM patients showed poly-PC

and constantly <5% (median: 0.25%) 1

Only 20% of MM patients showed poly-PC

and constantly <5% (median: 0.25%) 1

The most powerful single criteria for differential diagnosis (even in stage I MM)The most powerful single criteria for differential diagnosis (even in stage I MM)

versusversus

>5% poly-PC: 98% MGUS

ClonalClonal Poly-ClonalPoly-Clonal

1. Ocqueteau M, Am J Pathol 1998, 152: 16551. Ocqueteau M, Am J Pathol 1998, 152: 1655

MGUS MGUS vsvs MM: MM: IMMUNOPHENOTYPIC PANELSIMMUNOPHENOTYPIC PANELS

N.of PB AMCA FITC PE PerCPCy5.5 APC PE-Cy7 Alexa700colors PO APC-Cy7

3 CD38 CD56 CD19

4 CD38 CD56 CD19 CD45

6 cyIgL cyIgk CD19 CD45 CD56 CD38

8 CD19 CD45 cyIgL cyIgk CD138 CD28 CD56 CD38



Prognostic evaluation-Enumeration of myelomatous PC-Patterns of antigen expression

-Risk of progression of smoldering MM


• Correlation between Immunophenotyping & Morphology:• Correlation between Immunophenotyping & Morphology:

0 25 50 75 100

Proportion of plasma cell by morphology

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R2= 0,4

• Correlation between Immunophenotyping & Morphology:• Correlation between Immunophenotyping & Morphology:

• Prognostic influence of the number of BMPC:• Prognostic influence of the number of BMPC:

Morphology

Years from diagnosis

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R2= 0,4

CD56CD56

PF

SP

FS

48484242363630302424181812126600

1,01,0

,8,8

,6,6

,4,4

,2,2

0,00,0

60605454

— CD56 + (n=272)— CD56 –ve (n=355)

— CD56 + (n=272)— CD56 –ve (n=355)

p=0.05p=0.05

38 m38 m

33 m33 m

Months from diagnosisMonths from diagnosis

OS

OS

48484242363630302424181812126600

1,01,0

,8,8

,6,6

,4,4

,2,2

0,00,0

60605454

NRNR

59 m59 m

p=0.03p=0.03

— CD56 + (n=272)— CD56 –ve (n=355)

— CD56 + (n=272)— CD56 –ve (n=355)

CD117CD117

726660544842363024181260

1,0

,9

,8

,7

,6

,5

,4

,3

,2

,10,0

— CD117 + (n=195)— CD117 -ve (n=388)

— CD117 + (n=195)— CD117 -ve (n=388)

31 m31 m

41 m41 m

p=0.01p=0.01

1.0

.8

.6

.4

.2

0.0726660544842363024181260


— CD117 +(n=195)— CD117 -ve (n=388)

— CD117 +(n=195)— CD117 -ve (n=388)

58 m58 m

NRNR

p=0.1p=0.1

Prognostic influence of PC antigenic expression (I)Prognostic influence of PC antigenic expression (I)

Mateos et al, JCO, 2008

Prognostic influence of PC antigenic expression (II)Prognostic influence of PC antigenic expression (II)

CD19CD19


PF

SP

FS

4842363024181260 6054

36m36m

28 m28 m

p=0.2p=0.2

— CD19 + (n=47)— CD19 -ve (n=580)

— CD19 + (n=47)— CD19 -ve (n=580)

1,0

,9

,8

,7

,6

,5

,4

,3

,2

,10,0

OS

OS

60544842363024181260

1,0

,9

,8

,7

,6

,5

,4

,3

,2

,10,0

— CD19 + (n=47)

— CD19 -ve (n=580)

— CD19 + (n=47)

— CD19 -ve (n=580)

62 m62 m

54 m54 m

p=0.06p=0.06

CD28CD28

605448423630241812600

1,01,0

,9,9

,8,8

,7,7

,6,6

,5,5

,4,4

,3,3

,2,2

,1,1

0,00,0

— CD28 + (n=235)— CD28 -ve (n=368)

— CD28 + (n=235)— CD28 -ve (n=368)

31 m31 m

37 m37 m

p=0.05p=0.05

544842363024181260

1,01,0

,9,9

,8,8

,7,7

,6,6

,5,5

,4,4

,3,3

,2,2

,1,1

0,00,0

— CD28 + (n=235)— CD28 -ve (n=368)

— CD28 + (n=235)— CD28 -ve (n=368)

54 m54 m

66m66m

p=0.1p=0.1

60

Months from diagnosisMonths from diagnosisMateos et al, JCO, 2008

Prognostic influence of phenotypic profilesPrognostic influence of phenotypic profiles

CD56 & CD28CD56 & CD28

Months from diagnosisMonths from diagnosis726660544842363024181260

1.0

.9

.8

.7

.6

.5

.4

.3

.2

.1

0.0

p=0.01p=0.01

CD56+CD28- n= 1116 41 m+/+ or -/- n=266 36 mCD56-CD28+ n=116 29 m

CD56+CD28- n= 1116 41 m+/+ or -/- n=266 36 mCD56-CD28+ n=116 29 m


CD56 & CD117CD56 & CD117

726660544842363024181260

1.0

.9

.8

.7

.6

.5

.4

.3

.2

.1

0.0

CD56+CD117+ n= 130 45 m+/- or +/- n=267 36 mCD56-CD117- n=186 31 m

CD56+CD117+ n= 130 45 m+/- or +/- n=267 36 mCD56-CD117- n=186 31 m

p=0.001p=0.001


CD28 & CD117CD28 & CD117

726660544842363024181260

1.0

.9

.8

.7

.6

.5

.4

.3

.2

.1

0.0

CD28-CD117+ n= 142 45 m+/- or +/- n=327 37 mCD28+CD117- n=114 29 m

CD28-CD117+ n= 142 45 m+/- or +/- n=327 37 mCD28+CD117- n=114 29 m

p=0.0005p=0.0005

PFSPFS

PFS

Mateos et al, J Clin Oncol, 2008

Non-hyperdiploidy DNA Non-hyperdiploidy DNA

% BMPC by FCM >20%% BMPC by FCM >20%

% BMPC in S-phase ≥≥≥≥2.5%% BMPC in S-phase ≥≥≥≥2.5%

P P

Multivariate analysis for RFS and OS

RFSRFS OSOSPP

0.010.01 NSNS

Platelets ≤≤≤≤ 130 · 109/LPlatelets ≤≤≤≤ 130 · 109/L

0.030.03 0.0040.004

0.050.05 NSNS

ISS stage 3ISS stage 3 0.0030.003 0.00010.0001

0.0170.017 0.040.04

Calcium ≥≥≥≥11 mg/dlCalcium ≥≥≥≥11 mg/dl 0.0050.005 NSNS

• Kyle & Alexanian 1980a.

• Estimated incidence: 15% of newly diagnosed MMb.

• Estimated Risk of progression: 10% per yearc vs. 1% on MGUS

a Kyle 1980, Alexanian 1980; bRajkumar 05; cKyle 05

IntroductionSmoldering Multiple Myeloma

BMPC + High MC (>3 IgG or >2g/dl IgA)

> 10% of BMPC + MC

>10% BMPC + High MC

Predictive factorsAlexanian IgABlood; 1980 BJ >200 mg/24 h Arch Intern Med, 1988 MC >3 g/dl

Facon MC >3 g/dlAm J Hematol, 1995 Hb <12 g/dl

PC >25%

Weber MC >3g/dlB J Haematol; 1997 IgA

BJ >50mg/ 24 h Pathological MRI

Cesana IgAJ Clin Oncol; 2002 BJ +Haematologica; 2004 % BMPC

Rosiñol Evolving type*B J Haematol; 2003 IgA

*SMM with constant increase of MC

Parameters Associated with Disease Progression

N 89

Diagnosis jan96- sep04

Age* (range) 69 (43-88)

Gender (M/F) 44/45

Follow-up*(range) 50 (24-107)

Progression (%) 38 (42)

Characteristics of the Series

Patients (%)

BMPC + High MC (>3 IgG or >2g/dl IgA) 21 (24)

>10% of BMPC + MC 45 (50)

>10% BMPC + High MC 23 (26)

*median (in months)

Perez-Persona et al, Blood, 2007

% Total PC in BM* 2.8 (0.9-22.0)

% of aPC / BMPC compartment 97 (35-100)

< 95% aPC / BMPC 36 (40%)

> 95% aPC / BMPC 53 (60%)

Flow Cytometry Results

* Median (range)

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

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

2nd step

PC compartment

2nd step

PC compartment

..

..

..

......

..

..

..

..

....

..

..

.. .. .... .. ..

% aPC/BMPC% aPC/BMPC

% nPC/BMPC% nPC/BMPC

1st stepTotal cellularity

1st stepTotal cellularity

PC analysis in BM by FCPC analysis in BM by FC

% PC withinBM cellularity% PC withinBM cellularity

Impact of % aPC/BMPC by FC onProgression Free Survival

<95% aPC/BMPCn= 36 (4 progressions)

>95% aPC/BMPCn= 53 (34 progressions)

0 20 40 60 80 100 120

Months

0,0

0,2

0,4

0,6

0,8

1,0

% o

fPro

gres

sion

Fre

e S

urvi

val

p=0.0000

Median Not reached

Median 40 months

92%37%

5 years

Multivariate analysis for PFS

p HR

% a PC /BMPC 0.004 4.9

Immunoparesis 0.007 2.6

Impact of prognostic index on PFS

ImmunoparesisImmunoparesis >95% >95% aPCaPC/BMPC/BMPC ScoreScore(n)(n)

-- -- 0 (n=32)0 (n=32)

+ / + / -- --/+/+ 1 (n=27)1 (n=27)

++ ++ 2 (n=27)2 (n=27)

Impact of prognostic index on PFS

>95% aPC/BMPC or paresisn= 27 (12 progressions)

>95% aPC/BMPC + paresisn= 27 (22 progressions)

No adverse factorsn= 32 (3 progressions)

0 20 40 60 80 100 120

Months

0,0

0,2

0,4

0,6

0,8

1,0

% o

fPro

gres

sion

Fre

e S

urvi

val

Median not reached

Median 75 months

Median 20 monthsp= 0.003

91%

58%

18%

5 years

Changes in PC distribution following ASCT

MM-PCMM-PC

CD56

CD45

CD19

DiagnosisDiagnosis

Gate CD38Gate CD38

N-PCN-PC

MM-PCMM-PC

CD56CD45

CD19

Post-TrxPost-Trx

MINIMAL NUMBER OF PC REQUIRED TO BE ANALYZED IN A MRD ASSAY FOR MM

20200,0005

25250,0004

34333,3343

50500,0002

1001,000,0001

N. of events/test to define a PC population

N. of total nucleated cells/test

N. of tests in the MRD assay

Comparison between ASCT & Chemotherapy(changes in PC compartment)

Comparison between ASCT & Chemotherapy(changes in PC compartment)

Results expressed as median (range)

MRD -ve : <10 -5 MM-PC with only normal PC being detected

Results expressed as median (range)

MRD -ve : <10 -5 MM-PC with only normal PC being detected

pp

MM-PCMM-PC

n-PCn-PC

% n-PC/TPC% n-PC/TPC

MRD –ve casesMRD –ve cases

ASCT3 m post-Trx

n=47

ASCT3 m post-Trx

n=47

0,04 (0-3,2)0,04 (0-3,2)

0,21 (0-1,6)0,21 (0-1,6)

86 (0-100)86 (0-100)

36%36% 0,040,04

0,010,01

0,250,25

0,010,010,17 (0-3,7)0,17 (0-3,7)

0,12 (0-0,9)0,12 (0-0,9)

35 (0-100)35 (0-100)

15%15%

Chemo.After 12 cycles

n=40

Chemo.After 12 cycles

n=40

San Miguel et al, Blood, 2002

Spanish multi-centre protocol GEM (2000-2004)Spanish multi-centre protocol GEM (2000-2004)

DiagnosisDiagnosis

VBCMP/VBAD (x4)VBCMP/VBAD (x4)

RelapseRelapse

VBCMP/VBAD (x2)VBCMP/VBAD (x2)

Stem cell collectionStem cell collection

Complete remission(negative electrophoresis)*

Complete remission(negative electrophoresis)*

MaintenanceMaintenance

Partial response(positive electrophoresis)

Partial response(positive electrophoresis)

ASCT (2º)ASCT (2º) Mini-ALO-TrxMini-ALO-Trx

* Immunofixation either –ve or +ve * Immunofixation either –ve or +ve

ASCT (1º)ASCT (1º)

3m post-ASCT3m post-ASCT

Non-respondingNon-responding

Double-TrxDouble-Trx

MRDinvestigation

Correlation between immunophenotyping & electrophore tic responses

at three months post-ASCT (n=200)

Correlation between immunophenotyping & electrophore tic responses

at three months post-ASCT (n=200)

EF-positive n=74 cases

EF-positive n=74 cases

MM-PCMM-PC

% n-PC/TPC% n-PC/TPC

0.76 ± 0.9 #0.76 ± 0.9 #

MRD evaluationMRD evaluation

8% #8% #MRDnegative casesMRDnegative cases

42 ± 33 #42 ± 33 #

Results expressed as mean ± SD

# p significance between EF-positive (PR) and CR IFE- positive cases: p=0.005; p= 0.0001; p=0.0001

Results expressed as mean ± SD

# p significance between EF-positive (PR) and CR IFE- positive cases: p=0.005; p= 0.0001; p=0.0001

Complete remissionComplete remissionPartial responsePartial response

IFx-negativen=99 cases (79%)

IFx-negativen=99 cases (79%)

0.1 ± 0.30.1 ± 0.3 0.0060.006

0.0080.008

64%64%

86 ± 2586 ± 25

0.0030.003

IFx-positiven=27 cases (21%)

IFx-positiven=27 cases (21%)

33%33%

0.28 ± 0.40.28 ± 0.4

69 ± 3469 ± 34

pp

78726660544842363024181260

1,0

,9

,8

,7

,6

,5

,4

,3

,2

,1

0,0

RFS of MM: Impact of immunophenotypingat 3 months post-ASCT (n=200 patients)

RFS of MM: Impact of immunophenotypingat 3 months post-ASCT (n=200 patients)

Rel

apse

-fre

e su

rviv

alR

elap

se-f

ree

surv

ival

— <0.01% MM-PC— <0.01% MM-PC

— ≥≥≥≥ 1% MM-PC— ≥≥≥≥ 1% MM-PC

Months from immunophenotypical analysis (3 months post-ASCT)Months from immunophenotypical analysis (3 months post-ASCT)

%MM-PC%MM-PC

p=0.0001p=0.0001

40m40m

23m23m

— 0.01% to 1% MM-PC — 0.01% to 1% MM-PC

NRNR

Paiva et al, Blood, 2008

RFS: Impact of immunophenotypingat 3 months post-ASCT in 99 CR (IF-) patients

RFS: Impact of immunophenotypingat 3 months post-ASCT in 99 CR (IF-) patients

6660544842363024181260

1,1

1,0

,9

,8

,7

,6

,5

,4

,3

,2

,1

0,0

%MM-PC%MM-PC

p=0,02p=0,02

NRNR

36m36m

p=0,01p=0,01

NRNR

32m32m

60544842363024181260

1,0

,9

,8

,7

,6

,5

,4

,3

,2

,1

0,066

Rel

apse

-fre

e su

rviv

alR

elap

se-f

ree

surv

ival

Months from immunophenotypical analysis (3 months post-ASCT)Months from immunophenotypical analysis (3 months post-ASCT)

— ≥≥≥≥ 75 % N-PC/total PC— ≥≥≥≥ 75 % N-PC/total PC

%N-PC / total PC%N-PC / total PC

— <0.01% MM-PC— <0.01% MM-PC

— ≥≥≥≥ 0.01% MM-PC— ≥≥≥≥ 0.01% MM-PC — < 75 % N-PC/total PC— < 75 % N-PC/total PC

MRD IN MM: CORRELATION BETWEEN RQ-PCR AND FCM

00

0,20,2

0,40,4

0,60,6

0,80,8

11

1,21,2

1,41,4

1,61,6

1,81,8

11 22 33 44 55 66 77 88 99 1010 1111 1212 1313 1414 1515 1616 1717 1818 1919 2020 2121 2222

PatientsPatients

%

% T

umou

rT

umou

r cel

lsce

lls

%RQ%RQ--PCRPCR%FCM%FCM

R = 0.861

2323 2424

0,860,38221

0,971,14422

1,61,39924

1,311,27923

0,80,21920

0,6861.6119

0,260,11318

0,250,43517

0,0650,07716

0,0470,0115

0,0140,06214

00,10613

00,08212

00,02411

00,00310

00,0029

00.00098

007

006

005

004

003

002

001

%FCM%RQ-PCRCases

�

�

x

�xx

x

Sarrasquete et al, Haematologica, 2005

MRD IN MULTIPLE MYELOMA: APPLICABILITY OF FCM VS RQ -PCR

PatientsPatients in CR (N=53)in CR (N=53)

CCases ases withwith aberrantaberrant phenotypesphenotypes (N=48)(N=48)

ProblProbl emsems associatedassociated withwith thethe samplesample (N=16) (N=16)

VeryVery lowlow infiltrationinfiltration (N=9) infiltraci(N=9) infiltraci óón muy bajan muy baja

DegradedDegraded DNA (N=4) DNA (N=4)

InsufficientInsufficient DNA (N=3)DNA (N=3)

Problems Problems associassoci atedated withwith thethe methodmethod ((RQRQ--PCR)PCR) (N=(N= 8)8)

No No clonalclonal rearrangementrearrangement detecteddetected (N=3) (N=3)

VeryVery short Nshort N --regionregion (N=3)(N=3)

Cases Cases withwith mutationsmutations in in thethe targettarget sequencesequence (N=2)(N=2)

APPLICABILITY OF FCM: 90%

APPLICABILITY OF RQ-PCR: 75%Sarrasquete et al, Haematologica, 2005

Consensus medical indications of multiparameter flow cytometry

immunophenotyping in the study of MM and other MG_____________________________________________________________________

Clinical utility Useful flow cytometry parameters_____________________________________________________________________

Differential diagnosis of MM vs MGUS % aberrant BMPC from total BMPC

Identification of aberrant phenotypes Decreased expression (CD19, CD27, CD38,

CD45 CD81)

Overexpression (CD28, CD33, CD56)

Asynchronous expression (CD20, CD117,

sIg)

Diagnosis of unusual cases PC-associated markers (e.g. CD138, cIg, CD38hi)

& aberrant PC markers (see above)

Patient monitoring % of aberrant BMPC/totalBM cells

% normal BMPC/ all BMPC

_____________________________________________________________________ MM: multiple myeloma; MGUS: monoclonal gammopathy of undetermined significance; BM: bone marrow; PC: plasma cells.

Rawstron et al, EMN consensus, Haematologica, 2008

CONCLUSIONS

Inmunophenotypic studies have high clinical value:

- Differential diagnosis MGUS/MM

- Prognostic influence of FCM number of myelomatous PC

- Prognostic influence of the patterns of antigen expression

- Prediction of the risk of transformation in SMM

- Investigation of MRD (immunophenotypic remission)

Grupo Español de Mieloma (GEM)Hospitales

Clínico de Barcelona12 Octubre (Madrid)Clínico de Salamanca

Clínico de San Carlos (Madrid)Hospital de BadalonaClínico de AsturiasFr. Peset (Valencia)

Universitario de CanariasRio Ortega (Valladolid)

Cínico de ZaragozaHospital General de JerezRamón y Cajal (Madrid)

Morales Meseguer (Murcia)La Fe (Valencia)C.U. de Navarra

Galdakao (Vizcaya)Clínico de ValladolidSant Pau (Barcelona)

Arnau Vilanova (Lérida)Universitario de Santiago

General Universitario de ValenciaUniversitario de Getafe (Madrid)

Insular de las PalmasH. de La Princesa (Madrid)

Severo Ochoa (Madrid)Juan XIII (Tarragona)

ToledoGandía (Valencia)

Vall D´Hebrón (Barcelona)San Jorge (Huesca)

Verge de la Cinta (Tortosa)Alarcos (Ciudad Real)

Mataró (Madrid)Juán Canalejo (Coruña)

Ferrol

HospitalesGeneral de Segovia

Cruces (Bilbao)St. Coloma de Gramanet

(Barcelona)Gregorio Marañon (Madrid)

Carlos Haya (Málaga)H. Tauli (Gerona)

HuescaPalencia

Alcira (Valencia)H. Del Mar (Barcelona)

Mahón (Baleares)Clínico de MálagaXeral Cies (Vigo)

PlasenciaCáceres

AlgecirasÁvilaJaén

S. Pau i Sta Tecla (Tarragona)General de Guadalajara

Sagunto (Valencia)Son Dureta (Mallorca)

CuencaAlicante SUS

M. Valdecilla (Santander)Albacete

H. Del BierzoFundación Jiménez Díaz (Madrid)

Elda (Alicante)V. Del Rosel (Cartagena)

CastellónMutua Tarrasa

Consorcio TarrasaC. Corachán (Barcelona)

G.Mateo, M. Perez-Andres, N.Gutierrez, R.Lopez, M.Mateos, R.Garcia-Sanz, J.Almeida, J.San Miguel

Salamanca´Group:

OBRIGADO

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