Immunophenotype in multiple

myeloma and MRD detection

Thomas Matthes

Flow Cytometry Laboratory

Hematology and Clinical Pathology

Hôpitaux Universitaires de Genève

Luzern, 30th march 2012

Copyright ©2008 Ferrata Storti FoundationRawstron, A. C. et al. Haematologica 2008;93:431-438

List of most useful antigens for the detection of

aberrant plasma cells in multiple myeloma

Difference between normal and malignant PC

Plasmocytes malins

Plasmocytes normaux

Incidence of phenotypically aberrant MM-PC

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 expressionInfra-expression Over-expression

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

8-colour EuroFlow panels for

PC dyscrasias

Tube PB PO FITC PEPerCP-

Cy5.5PE-Cy7 APC APC-H7

Baseline CD45 CD138 CD38 CD56 β2micro CD19 cyIgκ cyIgλ

Baseline CD45 CD138 CD38 CD28 CD27 CD19 CD117 CD81

MRD CD45 CD138 CD38 CD56 CD27 CD19 CD117 CD81

DD: MM vs MGUS

The most powerful single criteria for differential diagnosis !

Ocqueteau M, Am J Pathol 1998, 152: 1655

MGUSMM

versus

> 5% nPC: 98% MGUS

PCa: Clonal PCn: Poly-Clonal

Presence of nPC only in 20% of MM patients; always <5% (median: 0.25%)

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

Prognostic influence of CD28 & CD117 expression

on PC

p= 0.01

38% n=128

29% n=362

21% n=149

Months from diagnosis847260483624120

Pro

gres

sio

n f

ree

surv

ival

1,0

0,8

0,6

0,4

0,2

0,0

PFS

p= 0.0001

OS

Months from diagnosis847260483624120

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- CD28CD117+ CD28+-

CD117- CD28+

TherapyTreatment

compliance

In vivo drug

kinetics

Tumour micro-

environment

Tumour cell

features

100

101

102

103

104

105

106

107

108

109

1010

1011

No

. o

f tu

mo

ur

ce

lls

10-6

10-5

10-4

10-3

10-2

Resistance

Complete remission

Immunophenotypic CR

Molecular CRSe

ns

itiv

ity

- CML

- APL

- Childhood ALL

- …

Therapeutic

decisions

Morphology, cytogenetics

Southern-blot,

FCM DNA aneuploidy

F.I.S.H

Flow cytometry

P.C.R.

Minimal residual disease (MRD)

Modified from J.J.M. van Dongen

Depth of response

MR

PR

VGPR/ nCR

CR

sCR

Molecular/flow CR

Treatment initiation

Progression

Time

Depth of response is related to TTP

Which level of response should be

measured in MM?

Complete response (CR):

negative IF in serum and urine

< 5% PC in BM

disappearance of soft tissue plasmocytoma

stringent CR: CR and

normal FLC ratio 0.26 – 1.65

absence of clonal PC in BM by IHC or Flow (2-4 colors)

molecular CR: CR and

negative ASO-PCR in BM (sensitivitiy 10-5 )

Flow CR: CR and

absence of phenotypically aberrant clonal PC in BM

106 cells analyzed; 4≥ colors; sensitivity 10-4

IMWG uniform response criteria:2 consecutive measurements are required (BM only once)

Rajkumar V et al, 2011

0 12 24 36 48 60 72 84 96

Months from diagnosis

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

Cu

mu

lati

ve P

rop

ort

ion

Su

rviv

ing

CR vs nCR

CR vs PR

nCR vs PR

P=0.01

P<10-6

P=0.04

0 12 24 36 48 60 72 84 96

Months from diagnosis

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

Cu

mu

lati

ve P

rop

ort

ion

of

Even

t Fr

ee S

urv

ivo

rs

CR vs nCR or PR

nCR vs PR

P<10-5

P=0.07

CR, n=278 nCR, n=124 PR, n=280 PD, n=25

EFS OS

Lahuerta JJ, et al. JCO 2008;26:5775–5782

CR and nCR are not the same: “depth of response”PETHEMA-GEM 2000: Outcome according to post-transplant response

CR correlates with long-term PFS and OS in

elderly patients treated with novel agents

Gay F et al. Blood;117:3025-31.

• Retrospective analysis: 3 randomized European trials of GIMEMA and HOVON groups (n=1175)

• First-line treatment

MP (n=332), MPT (n=332), VMP (n=257), VMPT-VT (n=254)

• Significant benefit also seen when analysis is restricted to patients >75 years old

OSPFS

p<0.001

CR

VGPR

PR

Months

Pro

bab

ility

of

pro

gre

ssio

n-f

ree

surv

ival

0 24 48 72

1.0

0.8

0.6

0.2

0.4

0.0p<0.001

CR

VGPR

PR

Months

Ove

rall

pro

bab

ility

of

surv

ival

0 24 48 72

1.0

0.8

0.6

0.2

0.4

0.0

“Achieving CR is an important prognostic factor at all stages of

treatment , including before and after ASCT, with first-line treatment

in the non-transplant setting, and in the relapsed setting.”

Chanan-Khan AA and Giralt S, JCO, 2010

“CR constitutes a surrogate for OS and a clinically relevant

end-point in MM studies”

Do FLC measurements add additional value to IF ?

52 patients in CR after ASCT or AlloT (45 patients) or conventional

chemotherapy:

CR with negative IF

FLC was negative in 51 patients (98%)

- in 13 patients presence of oligoclonal IF: FLC negative

Kröger N et al.; 2010

Conclusion: FLC allows faster detection of remission or relapse

than IF, but is not more sensitive than IF

MRD evaluation by PCR (Qualitative & Semi-q) in Multiple Myeloma patients: prognostic value

Author Context Sensitivity N MRD Status PFS OS

Corradini QL ASO-PCR 10-6 29 20 positive 55% NRJCO 1999 Auto/Allo 9 negative 78%

López-Pérez et al QL cons-PCR 10-3-10-4 27 12 positive 20 m* 20%*Leukemia 2000 Auto, apheresis 11 negative 40 m 86%

Martinelli QL ASO-PCR 10–6 44 32 positive 65%* NRJCO 2000 Auto/Allo 12 negative 93%

Corradini QL ASO-PCR 10-6 48 16 positive 0% NRBlood 2003 Allo 19 mixed 33%

13 negative 100%

Ladetto et al, QL Nested-PCR 10-6 39 33 positive 66% NRJCO, 2010 VTP Post-Auto 6 negative 100%

Terragna et al, QL Nested-PCR NR 67 27 positive NR VTD: 67% negativeASH 2010 VTD vs. TD post-auto 60 negative NR TD: 52%

negative

López-Pérez et al Semi-QT FL-PCR 10-3-10-4 23 14 positive 19 m* 28%*Leukemia 2000 Auto, apheresis 13 negative 39 m 81%

Bakkus Semi-QT PCR LDM 10-6 59 38 >0.015% 16 m* NRBJH 2004 Auto 22 <0.015% 64 m

Martínez-Sánchez et al Semi-QT FL-PCR 10-3-10-4 53 25 positive 28%* 68%BJH 2008 28 negative 68% 86%

QL: Qualitative; QT: quantitative; NR: Not reported

Advantages Disadvantages

• Time consuming

• Labour consuming

• Applicability: ~75%

• Specificity: 100%

• Sensitivity: 10-6 (10-5 - 10-4)

Sarasquete ME, et al. Haematologica. 2005;90:1365-1372.Davies FE, et al. Best Pract Res Clin Haematol. 2002;15:197-222.

Fenk R, et al. Haematologica. 2004;89:557-66.Lioznov M, et al. Bone Marrow Transplant. 2008;41:913-916.

Real-time PCR of VDJ rearrangements for

MRD investigation in MM(Allelic-Specific Oligonucleotide ASO PCR)

Comparison of molecular and Flow CR

32MM patients after ASCT: CR 3 months after transplant

BM analysis:

- PCR (Allele-specific oligonucleotide real-time quantitative PCR)

in 75% of patients;

- Cytometry (4-color flow)

in 90% of patients

for 24 patients both measurements were possible

Sarasquete ME et al; Haematologica; 2005

Sarasquete ME et al; Haematologica; 2005

Correlation of MM-PC detected by RQ-PCR vs. MFC

MRD by ASO-RT-PCR (% of tumour cells)

MR

D b

yfl

ow

cyto

met

ry(%

of

tum

ou

rce

lls)

0 1.00.1-0.01

1

0,1

0.001

0.01

5.0

R=0.853P < .00001

Puig et al. Haematologica 2011;96 (suppl 1):[abstr O-09]. Presented at IMW 2011 Paris.

Disadvantages

• Applicability: >90%

• “Single-cell” analysis

• Sensitivity: 10-4

• Speed

• Simplicity

• Only PC compartment

• No specific tumor antigens

• Limited number of proteins studied

• Heterogeneous BM infiltration

• Final product analysis: protein (antigen)

Advantages

Immunophenotypic CR*: absence of phenotypically

aberrant PC at a sensitivity level of 10-4

Normal PC

Myelomatous PC

Normal PC Normal PC

Myelomatous PC

Baseline Follow-up

MRD + MRD -

San Miguel et al Blood; 2002; 99:1853

*Rajkumar et al (IMWG). Blood 2011 117: 4691-4695

GEM2000: Impact on survival of achieving an immunophenotypic CR after HDT/ASCT (n=295)

PFS OS

MRD negative (n=125) 42%MRD positive (n=170) 58%

0 25 50 75 100 125

0

20

40

60

80

100

p< 0.0001

Median: 71m

Median: 37m

Median: NA

p= 0.002

Median: 89m

0 20 40 60 80 100 120 140

0

20

40

60

80

100

22%

60%

60%

82%

Paiva B ; Blood; 2008

MRC myeloma IX: Impact on survival of achieving an immunophenotypic response after HDT/ASCT

(n=526)

Rawstron A, et al. Haematologica 2011;96 (suppl 1):[abstr O-09]. Presented at IMW 2011 Paris.

–MRD– –MRD+

p-value for logrank test = 0.0280

Overall survival by 100-day outcomefor intensive pathway patients

Pro

po

rtio

n o

f p

atie

nts

ev

en

t fr

ee

OS

1.0

0.8

0.6

0.2

0.4

0.0

0.9

0.7

0.5

0.1

0.3

0 24 48 726 12 18 30 36 42 54 60 66

p-value for logrank test = 0.0001

Progression-free survival by 100-day outcomefor intensive pathway patients

Pro

po

rtio

n o

f p

atie

nts

ev

en

t fr

ee

PFS

1.0

0.8

0.6

0.2

0.4

0.0

0.9

0.7

0.5

0.1

0.3

0 24 48 726 12 18 30 36 42 54 60 66

MRC myeloma IX trial: What about consolidation

and maintenance therapy after HDT/ASCT?

Progression-free survival by 100-day outcome assessed in all patients by MRD/maintenance;T: thalidomide; NM: no maintenance

p-value for logrank test = 0.0053

Pro

po

rtio

n e

ven

t fr

ee

1.0

0.8

0.6

0.2

0.4

0.0

0.9

0.7

0.5

0.1

0.3

0 24 48 726 12 18 30 36 42 54 60 66

T; MRD– T; MRD+ NM; MRD– NM; MRD+

Rawstron A, et al. Haematologica 2011;96 (suppl 1):[abstr O-09]. Presented at IMW 2011 Paris.

6050403020100

100

80

60

40

20

0

Months

P =0.001

PFS

Immunophenotypic CR 90% at 3y

“Stringent CR” 38% at 3y

Conventional CR 57% at 3y

PR (≥70% reduction) 28% at 3y

GEM2005>65y: impact of depth of response on survival

Paiva et al; J Clin Oncol. 2011;29(12):1627-33.

M-component positive

M-component negative------- Follow-up

P Progression

Death

Treatment interruption

GEM2005>65y: Kinetics of response: conventional CR vs. immunophenotypic CR

Paiva B, et al. J Clin Oncol. 2011;29:1627-1633

1 IgG ----------------------------------------------------------------------

2 B-J ------------------------------------------------------------------------------------------

3 IgG -----------------------------------------------------------------------------------------------

4 IgA ------------------------------------------------------------------------------------------------------

5 IgG ---------------------------------------------------------------------------------------------------------------

6 B-J ------------------------------------------------------------------------------------------ P -------------------------------

7 IgG ----------------------------------------------------------------------------------------------------------------------------------

Flow- / IFE+

1 2 3 4 5 6 7 8 9 10 11 12 // 16 // 20 // 24 // 28 // 32 // 36 // 40 // 44 // 48

Post-Induction Maintenance (months)

Patient no.

7/7

(100%)

patients

turned

IFx-

1 IgG ----------------------------------------------------------------------

2 B-J ------------------------------------------------------------------------------------------

3 IgG -----------------------------------------------------------------------------------------------

4 IgA ------------------------------------------------------------------------------------------------------

5 IgG ---------------------------------------------------------------------------------------------------------------

6 B-J ------------------------------------------------------------------------------------------ P -------------------------------

7 IgG ----------------------------------------------------------------------------------------------------------------------------------

8 B-J -------------------------------- P ----------------------------

9 IgG -------------------------------------------------------------------

10 B-J --------------------------------------------------------------------

11 IgG -----------------------------------------------------------------------

12 IgA ------------------------------------------- P ------------------------------

13 IgA ------------------------------------------- P ---------------------------------

14 IgG --------------------------------------------------------------------------------

15 IgA ---------------------------------------------------------------------------------

16 IgG --------------------------------------------------------------------------------------------

17 IgG ------------------------------------------------------------------------------- P -------

18 IgG ------------------------------------------------------------------------------------------

19 IgA --------------------------------------------------------------------- P --------------------

20 IgG ------------------------------------------------------------------------------------------------------

21 IgA ---------------------------------------------------------------------------------------------------------------

22 IgA ---------------------------------------------------------------------------------------------------- P -----------

23 IgA -------------------------------------------------------------------------------------------------------------------------

24 IgA ---------------------------------------------------------------------------------------------------- P -------------------------

25 IgA ------------------------------------------------------------------------------------------- P ----------------------------------------

26 IgG -------------------------------------------------------------------------------------------------------------------------------------------

27 IgA -------------------------------------------------------------------------------------------------------------------------------------------

GEM2005>65y: Kinetics of response: conventional CR vs. immunophenotypic CR

Paiva B, et al. J Clin Oncol. 2011;29:1627-1633

Flow- /

IFE+

Flow+

/ IFE-

1 2 3 4 5 6 7 8 9 10 11 12 // 16 // 20 // 24 // 28 // 32 // 36 // 40 // 44 // 48

Post-Induction Maintenance (months)

Patient no.

7/7

(100%)

patients

turned

IFx-

10/20

(50%)

patients

turned

IFx+

FISH cytogenetics and immunophenotypic CR for the prediction of

early relapse of patients in CR after HDT/ASCT

TTP after day+100 HDT/ASCT

P <.001P <.001

Median: NR

Median: 83m

Median: 28m

80%

94%

Standard-risk FISH + MRD negative (n=58)

High-risk FISH OR MRD positive (n=45)

High-risk FISH + MRD positive (n=7)

0%

Median: 6m

1y

Median: 47m

Median: 21m

1009080706050403020100

100

80

60

40

20

0

42%

89%

2.5y

76%

5y

OS after day+100 HDT/ASCT

100806040200

100

80

60

40

20

0

Number of daysNumber of days

Paiva et al; Blood. 2012 119: 687-691

- Construct reference data files for normal and neoplastic cells

(e.g. per disease category)

- Multi-n-dimensional comparison of normal vs neoplastic cell

populations (e.g. at diagnosis and at follow-up):

- Automated PCA-guided approach for homogenous cell populations

(e.g. lymphoid)

- Maturation tools for heterogenous cell populations

(e.g. myeloid)

How to optimize MRD detection by cytometry

Sensitivity of 10-6

• Malignant PC exhibit a characteristic phenotype, which allows

their recognition and distinction form normal PC in practically all

patients

• Depth or response matters: the better the quality of response, the

longer the survival (CR, sCR,molecular CR, IP CR)

• MRD techniques can contribute to the monitoring of the efficacy of

intensification and maintenance therapies in order to avoid under-

or over-treatment

• IP CR is clinically relevant in MM: clear discrimination between

groups of patients with different PFS and OS

• Multiparameter flow cytometry (8-10 colors) could be considered

as the method of choice for MRD monitoring in MM

SUMMARY

END