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Flow Cytometric Minimal Disease Detection of Plasma Cell Neoplasms: Prognosis and
Measuring Drug Response
Maryalice Stetler-Stevenson, M.D., Ph.D. Flow Cytometry Unit, Laboratory of Pathology,
DCS, NCI, NIH
DEPARTMENT OF HEALTH & HUMAN SERVICES
National Institutes of Health Bethesda, Maryland 20892
Public Health Service
The Range of Plasma Cell Processes:
Benign bone marrow plasmacytosisPlasma Cell Dyscrasias:
Monoclonal Gammopathy of Uncertain Signficance (MGUS)
Smoldering Myeloma (SMM) Plasma Cell or Multiple Myeloma (MM)
B-cell non-Hodgkin’s lymphoma with plasmacytic differentiation
Plasma cell dyscrasias
<60%
Flow Cytometric Evaluation of Plasma Cell Dyscrasias: General Technical Issues
Number of PCs detected by FC only about 25% of what is detected by morphology- but correlation is good
Partially dependent on quality of aspirateDue to hemodilution, sampling artifact, plasma cell fragility,
and tendency of plasma cells to associate with lipid rich spicules
Surface staining: Surface staining alone can detect an aberrant immunophenotype in 97% of myeloma patients
Intracellular staining: Kappa and lambda light chain immunoglobulin to detect monoclonality at diagnosis but not always necessary for MRD
Flow Cytometric Evaluation of MM: General Technical Issues-Early Recommendations
European Myeloma Network (EMN) recommendations (Haematologica 2008;93:431-438) Erythrocyte lysis Majority of centers based gating on CD38, CD138 and light
scatter CD38/SSC –false negative results CD38/CD138-high contamination rate CD38/CD45- decreased contamination but excludes CD45+ cells For MRD:Gating based upon SSC, CD38, CD138 and CD45
At least 100 plasma cells should be acquired (we aim for more than 1,000)- must acquire >100,000 events
3,000,000 total events for MRD Clonality assessment at diagnosis but not in MRD Must wash BM twice before intracellular light chain staining Assessment of BM sample quality: Should find normal PCs and
BM elements (e.g. nuc RBCs, myeloid blasts, hematogones
Flow Cytometric Evaluation of Plasma Cell Myeloma: Biopsy Site and Anticoagulation
Manasanch et al. Leuk Lymphoma 2014
9 SMM and 11 MM
Flow Cytometric Evaluation of Plasma Cell Myeloma: Biopsy Site and Anticoagulation
No difference (p>0.05) regarding immunophenotype, number, or distribution of normal vs. abnormal plasma cells (APCs) in marrow samples from unilateral versus bilateral aspirates
Different aspirate sequence and/or anticoagulant (heparin/EDTA) = same ability to detect APCs, although lower number in 2nd aspirate than in first.
Manasanch et al. Leuk Lymphoma 2014
Flow Cytometric Evaluation of Prognosis in MGUS, SMM and MM:
Predicting progression in MGUS and SMM
Measuring response to therapy and predicting progression free survival (PFS), time to progression (TTP), and overall survival (OS) in MM
Working model of MGUS and SMM Progression to MM
MGUS
Smoldering (“early”) myeloma
Multiple myeloma
Symptomatic diseaseAsymptomatic disease
Non-progressors Progressors
MGUS found in 3% over 50 years of age and 10% over 70 years of age
MGUS : small M protein (< than 3 g/dl) and <10% plasma cells in BM
no evidence of MM, primary amyloidosis, Waldestrom's macroglobulinemia, or other related disorders
1 to 1.5% chance/year of MGUS evolving into myeloma
Interval from diagnosis MGUS to myeloma 1-32 years Normal Plasma cells are a significant population in
MGUS. FC can detect clonal plasma cells in a background of normal plasma cells.
MGUS
Utility of Flow Cytometry in MGUS:Prognosis
Flow cytometry provides prognostic information Risk of progression to myeloma is directly related to the
presence and proportion of abnormal plasma cells of total plasma cells detected by FC Percent of APC:
>95% APC 25% risk of progression at 5 years
<95% APC 5% risk of progression at 5 years Perez-Persona et al. Blood (2007) 110 (7): 2586-2592.
Smoldering Myeloma: Prognosis
SMM : M protein > than 3 g/dl) and >10% and <60% plasma cells in BM
10% chance/year of SMM evolving into MM for first 5 years with 43% survival at 10 years
Some patients progress more rapidly than others.
Mayo Clinic (n=273)
Dispenzieri et al. Blood 2008
No. of risk factors
No. of patients, n (%)
Progression at 5 years
1 76 (28) 25%
2 115 (42) 51%
3 82 (30) 76%
Risk factors:• BMPCs >10%• M-protein >3 g/dL • FreeLightChain-
ratio <0.125 or >8
PETHEMA Study Group (n=89)No. of
risk factorsNo. of
patients, n (%)Progression
at 5 years
0 28 (31) 4%
1 22 (25) 46%
2 39 (44) 72%
Risk factors:• ≥95% abnormal plasma
cells by Flow* • Immunoparesis
*Incl decreased CD38 expression, expression of CD56, and absence of CD19 and/or CD45
Pérez-Persona et al. Blood 2007
Risk Stratification for Smoldering Myeloma (SMM)
High Risk SMM median time to progression is <2 years
% APC predictive of risk of progression over 5 year period >95% APC 64% risk of progression at 5 years <95% APC 8% risk of progression at 5 years Perez-Persona et al. Blood (2007) 110 (7): 2586-2592.
Utility of Flow Cytometry in Smoldering Myeloma Prognosis
Treatment of high risk SMM: first randomized trial showing benefit in treatment arm
Mateos et al. Lenalidomide plus dexamethasone for high-risk SMM. NEJM. 2013
Time-to-Progression(median follow-up 32 mo)
Overall Survival(median follow-up 32 mo)
No treatmentMedian TTP: 23m
Len/DexMedian TTP: NR
OS at 3 yearsNo treatment: 76%Len/Dex: 93%P=0.04
Flow Cytometric Evaluation of Plasma Cell Myeloma: Prognosis
FC bone marrow plasma cell count at diagnosis is an independent prognostic indicator of overall survival Mateo, G., et al, Journal of Clinical Oncology, 2008. 26(16): p. 2737-2744. Paiva, B., et al.,
Haematologica-the Hematology Journal, 2009. 94(11): p. 1599-1602.
FC detection of >5% normal PCs in BM favorable independent prognostic indicator increased progression-free survival and improved overall survival Paiva, B., et al., Blood, 2009. 114(20): p. 4369-4372.
Detection of APCs in peripheral blood at time of diagnosis associated with poor prognosis Witzig, et al, Blood, 1996. 88: 1780-1787.
Flow Cytometric Evaluation of Plasma Cell Myeloma: Prognosis Post-Transplant
After transplant, FC is an early predictor of outcome in myeloma
Presence of APC is significant Patients with only normal PC at 3 months after
transplant had low risk of disease progression- 100% 5 year survival
Patients with APC at 3 months after transplant had high risk of disease progression- 54% 5 year survival
MRD assessment of maintenance therapy post transplant also predictive
Rawstron et al. Blood, 2002. 100(9): 3095-98.Rawstron et. Al. J Clin Oncol 2013. 31:2540-2547
CRd
Plasma Cell Myeloma: More Accurate Measurement of Response to Therapy
Kumar Cancer Treatment Reviews 2010; Korde et al. ASH 2013
Increasing depth of response in myeloma with newer drugs
International Myeloma Working Group Evaluation of Plasma Cell Myeloma Response to Therapy
Partial Response (PR): >50% reduction serum M-protein, >90% reduction 24 hr urinary M protein
Very Good Partial Response (VGPR)/Near Complete Response (nCR): Serum and urinary M proteins only detectable by immunofixation
Complete Response (CR):Negative immunofixation and < 5% PCs in BM
Stringent CR (sCR) normal free light chain ratio and absence of clonal PCs in BM.
Imaging plus MRD- Negative: Negative PET/CT and next gen flow(NGF)* or next gen sequencing (NGS) MRD
Sequencing MRD Negative Flow MRD negative Sustained MRD negative- MRD negative minimum of 1 year
* NGF LOD at 10-5
FC MRD Monitoring of Plasma Cell Myeloma Post ASCT Predictive of Outcome
Paiva et al. Blood, 2008. 112(10): 4017-23.
Rawstron et. Al. J Clin Oncol 2013. 31:2540-2547
Outcome according to MRD status post ASCT and cytogenetic risk profile
FC MRD Monitoring of Plasma Cell Myeloma Post ASCT Predictive of Outcome with Favorable and
Unfavorable Cytogenetics
Adverse Cytogenetics: gain(1q), del(1p32), t(4;14), t(14;20), t(14;16), del(17p)
FC MRD Monitoring of PCM Post Induction Therapy, No ASCT (>65 yo)
Outcome according to MRD status
Paiva B et al. J Clin Oncol 2011
Paiva B et al. Blood 2012
MRD negFISH low
MRD posFISH high
MRD pos orFISH high
P<0.001
FC MRD Monitoring of PCM Post ASCT Predictive of Outcome with Favorable and Unfavorable
CytogeneticsOutcome according to MRD status post ASCT and cytogenetic risk profile
FC MRD Monitoring Post ASCT: Maintenance Thalidomide Eradicates MRD in Proportion of
PatientsThalidomide Maintenance Eradicated MRD in 28% of MRD+ Patients
Flow Cytometric Evaluation of Plasma Cell Dyscrasias : MRD Technical Issues
The quality of flow cytometric myeloma MRD testing is absolutely dependent upon the specimen quality and on the staining and acquisition process
2013 ICCS and ESCCA consensus guidelines developed
Open access- Volume 90, Issue 1, 2016
Flow Cytometric MRD in Myeloma: Consensus on Staining and Acquisition
Appropriate Specimens: Bone marrow aspirates, peripheral blood and fine needle aspirates are appropriate
Bone marrow specimens are the standard for flow cytometric MRD assessment Clinical significance of FC MRD testing in myeloma
restricted to bone marrow post-treatment samples Detection of myeloma in peripheral blood at diagnosis
and 2 weeks prior to stem cell harvest may also be clinically relevant
Anticoagulant: EDTA or Sodium Heparin Specimen age: Staining within 24 hours is optimal
48 hour cut off unless irreplaceable specimen CLSI Guidelines for Specimen Stability:
Bone Marrow/Blood EDTA- 24 hoursBone Marrow/Blood Heparin- 48 hours
Control Temperature Specimen Quality Indicators- Clotting, hemolysis,
temperature, viability Viability- 85% or greater
If lower- qualifying statement
Staining and Acquisition in Flow Cytometric Myeloma MRD Testing: Specimen
Specimen needs to be concentrated to deliver sufficient number of cells per tube RBCs lysed prior to staining and cells pelleted and
reconstituted in decreased volume-Preferred 2 fold concentration can be achieved by simple
centrifugation to pellet cells and reconstitution in decreased volume
Staining and Acquisition in Flow Cytometric Myeloma MRD Testing: Staining
Panel: Need to be able to detect APC and assess marrow quality. For APC need: CD38, CD138, CD45, CD19, CD56,
CD27, CD81, and CD117 For marrow quality assessment: normal plasma cells,
B-cell progenitors, mast cells, neutrophil maturation Intracellular light chain evaluation does not
provide additional information in greater than 97% of patients Permeabilization for intracellular light chains can
result in cell loss.
Staining and Acquisition in Flow Cytometric Myeloma MRD Testing: Panel
Staining and Acquisition in Flow Cytometric Myeloma MRD Testing: Panel
Routine usage of an identical panel in all cases is highly recommended
Panel must be tested extensively to determine optimal fluorochome usage
Recommend laboratories initiating myeloma MRD testing adopt a validated panel
Staining and Acquisition in Flow Cytometric Myeloma MRD Testing: Panel
FITC PE PerCP
Cy5.5
PC7 APC APCC750
1. CD27 CD56 CD19 CD38 CD138 CD45
2.* CD81 CD117 CD19 CD38 CD138 CD45
6 Color Panel: Source: Leeds U.K.
8 Color Panel: PETHEMA/Euroflow Group:
FITC PE PerCP Cy5.5 PC7 APC APCC750 V450 BV510
CD38 CD56 CD45 CD19 CD117 CD81 CD138 CD27
Acquisition successful when MRD detected OR in MRD negative when adequate bone marrow and sufficient number of cells acquired
Adequate cell number: Current best practice= 2,000,000 cells minimum and 3-
5 million optimal If negative need to note Limit of Detection (LOD).
Staining and Acquisition in Flow Cytometric Myeloma MRD Testing: Acquisition
Flow Cytometric Evaluation of Plasma Cell Myeloma: Minimal Residual Disease
Effect of number of cells (events) acquired on MRD
CD19 APC
CD
56 P
C7
102 103 104 105
102
103
104
105
3 Million cells
CD19 APC
CD
56 P
C7
102 103 104 105
102
103
104
105
1 Million cells
CD19 APC
CD
56 P
C7
102 103 104 105
102
103
104
105
500,000 cells
CD19 APC
CD
56 P
C7
102 103 104 105
102
103
104
105
100,000 cells
No abnormalplasma cells
6 abnormalplasma cells
12 abnormalplasma cells
30 abnormalplasma cells
53 year old Female with MM post therapy. MM Cells: CD19-, CD45-, CD38 dim, CD20-, CD56+, CD81-, dim CD27
Gating strategies based upon CD38, CD45, CD138, FSC, and SSC to identify NPC and APC
Look for abnormal pattern of expression of CD38+ bright (but dimmer than NPC), CD138+ (brighter than NPC), CD19-, CD45-/+/bright, CD20+, CD27-/dimmer than NPC, CD28+, CD56+, CD81-/dimmer than NPC, CD117+
Can’t rely on IC light chains because polyclonal NPCs can mask the presence of APCs
Analysis in Flow Cytometric Myeloma MRD Testing:
Can’t rely on just one antigen because known subsets of NPC exhibiting ‘atypical’ antigen expression patterns in up to 30% of the PC pool: CD19-, CD45-/dim, CD20+, CD27 dim, CD28+, CD56+, CD117+
Analysis in Flow Cytometric Myeloma MRD Testing:
NPCs with Subset Demonstrating Antigen Expression Similar to APCs
Totalnumber
of cases
CD19- CD45 -or dim
CD56 +
CD20 + CD81- or dim
CD19- and CD81- or dim
CD19 -, CD45- and/or CD56 +
34 17 (50%)
14 (41%)
9 (26%)
3(9%)
0 (0%)
0 (0%)
4 (12%)
Leukemia Research 2013, 140:813
In the absence of cell populations predominantly restricted to bone marrow, such as erythroid, myeloid and B-lymphoid progenitors, normal plasma cells, or mast cells, then the sample should be reported as unsuitable for MRD assessment
If MRD negative report the LOD and confirmation of marrow quality
If MRD positive and number of cells above the Limit of Quantitation (LOQ) report % of cells that are APC
If MRD positive and number of cells above the LOD but below the LOQ report positive but below the quantitative range and give the LOQ
Reporting of Results in Flow Cytometric Myeloma MRD Testing:
Report antigen expression pattern on neoplastic plasma cells as being reduced, normal or increased, compared to normal reference plasma cell immunophenotype
Include the percentage of positivity in APCs for each marker as it is important to establish the immunophenotypic signature that will aid in follow-up MRD detection
Reporting of Results in Flow Cytometric Myeloma MRD Testing:
Flow Cytometric (FC) Detection of Plasma Cell Neoplasia :
Normal plasma cells (PC) Predominately CD45+ and
CD19+(up to 30% -) CD38 bright + CD138+ CD27 and CD81 Bright + Cytoplasmic Ig polyclonal CD56 predominately negative
(up to 15% +) Negative CD117, CD13, CD33
and CD28 CD20-, CD22- surface Ig negative CD319 and CD307
Abnormal plasma cells (APC) often CD45dim/-, Usually CD19 – CD38 less intense than PC CD138+ CD27 and CD81dim or - Cytoplasmic Ig clonal CD56 frequently bright Can be positive for CD117, CD13,
CD33 or CD28 CD20 and CD22 +/- surface Ig +/- CD319 and CD307 DNA content: aneuploidy
Mixed
Abnormal
Normal
SF13 1319 bm_05_B-8.fcs compensated FSC SSC PCs viability gate
CD45 V500
CD
19 A
PC
102 103 104 105
102
103
104
105
CD19 neg PCs tube B-8
0.05%97.15%
0.05%2.74%
How Do You Analyze Plasma Cells?
CD38 V450
CD
138
PerC
P C
y55
102 103 104 105
102
103
104
1058c PC 38v450 138percp
CD38 V450
SSC
-A
102 103 104 105
0
6553 6
1 3107 2
1 9660 8
2 6214 4
8c PC 38v450 SSC
FSC-A
SSC
-A
26 65556 131085 196615 26214426
65556
131085
196615
262144
0.15%
20.19%
8.08%
71.58%
FSC SSC PCs viabil ity gate
debris degenerating
CD45 V500
CD
38 V
450
102 103 104 105
102
103
104
105
1.56%
0.24%
7.41%90.79%
FSC-A
FSC
-H
0 65536 131072 196608 262144
0
65536
131072
196608
262144
SINGLETS
SF13 1319 bm_01_S-1.fcs SINGLETS
CD34 APC
CD
13 P
C7
1 0 2 10 3 10 4 1 0 5
10 2
10 3
10 4
10 5
SF13 1319 bm_01_S-1.fcs 8c CD45 G
CD16 FITC
CD
13 P
C7
10 2 10 3 10 4 1 0 5
10 2
10 3
10 4
10 5
8.88% 23.27%
66.57% 1.28%
Quality of BM?
Old method- not as useful
8c icKappa PEC
D19
APC
102 103 104 105
102
103
104
105
CD19 APC
CD
81 F
ITC
102 103 104 105
102
103
104
105
CD19 APC
CD
27 P
E
102 103 104 105
102
103
104
105
CD20 AH7
CD
56 P
C7
102 103 104 105
102
103
104
105
CD45 V500C
D19
APC
102 103 104 105
102
103
104
105
66 yo Male Patient with 20%PC in BM and M Spike> 3 g/dL: Myeloma
CD38 V450
CD
138
PerC
P C
y55
102 103 104 105
102
103
104
1058c PC 38v450 138percp
CD38 V450
SSC
-A
102 103 104 105
0
65536
131072
196608
262144
8c PC 38v450 SSC
FSC-A
SSC
-A
26 65556 131085 196615 26214426
65556
131085
196615
262144
0.15%
20.19%
8.08%
71.58%
FSC SSC PCs viability gate
debris degenerating
CD45 V500
CD
38 V
450
102 103 104 105
102
103
104
105
1.56%
0.24%
7.41%90.79%
CD19 PerCP Cy55
CD
117
PE
102 103 104 105
102
103
104
105
APCs are CD19-, CD45-, CD56+. CD20-, CD27-, CD81-, CD117+,Ic kappa-, ic lambda+
8c icLambda FITC
CD
19 A
PC
102 103 104 105
102
103
104
105
Flow Cytometric Evaluation of Plasma Cell Myeloma: Minimal Residual Disease
CD19 APC
CD
81 F
ITC
102 103 104 105
102
103
104
105
CD19 APC
CD
56 P
C7
102 103 104 105
102
103
104
105
CD19 APC
CD
27 P
E
102 103 104 105
102
103
104
105
CD45 V500
CD
19 A
PC
102 103 104 105
102
103
104
105
53 year old Female with myeloma: evaluation of minimal disease:
Bone Marrow: Hemodilute but marrow elements present.Abnormal Plasma Cells: CD19-, CD45-, CD38 dim, CD56+, CD81-, CD27 dim, CD117+
CD38 V450
CD
19 A
PC
102 103 104 105
102
103
104
105
CD45 V500
CD
117
PE
102 103 104 105
102
103
104
105
CD16 FITC
CD
13 P
C7
102 103 104 105
102
103
104
105 67.11%
CD45 V500
CD19
PE
102 103 104 105
102
103
104
105
The Effect of New Therapies on FC Myeloma Detection
New therapies are constantly being developed and they can affect the immunophenotype of the myeloma cells Anti-CD38 Anti-CD138 CAR T-cell therapy directed against BCMA and more.
CD45 V500
CD
56 P
C7
102
103
104
105
102
103
104
105
CD19 PerCP Cy55C
D81
FIT
C
102
103
104
105
102
103
104
105
CD38 V450
CD
138
APC
102
103
104
105
102
103
104
105
CD19 PerCP Cy55
BC
MA
PE
102 103 104 105
102
103
104
105
46 yo male with multiple myeloma – relapsed post stem cell transplant
Flow Cytometry Important in Screening for CAR T-Cell Therapy: BCMA in Myeloma
Bone Marrow Sent for Screen for BCMA CAR T-Cell Therapy in Myeloma:Hx Relapsed Myeloma
CD45 V500
CD
138
APC
102
103
104
105
102
103
104
105
CD56 PC7
CD
138
APC
102
103
104
105
102
103
104
105
CD38 V450
CD
138
APC
102
103
104
105
102
103
104
105
CD38 V450
CD
138
APC
102
103
104
105
102
103
104
105
CD45 V500
CD
19 P
erC
P C
y55
102
103
104
105
102
103
104
105
CD45 V500
CD
56 P
C7
102
103
104
105
102
103
104
105
CD19 PerCP Cy55
CD
81 F
ITC
102
103
104
105
102
103
104
105
CD19 PerCP Cy55
BC
MA
PE
102 103 104 105
102
103
104
105
Patient received Daratumumab- Anti-CD38 Antibody Therapy
The Effect of New Therapies on FC Myeloma Detection: The Daratumumab
Problem
CD38 V450
CD
138
APC
102
103
104
105
102
103
104
105
CD45 V500
CD
38 V
450
102
103
104
105
102
103
104
105
CD45 V500
CD
38m
e FI
TC
102
103
104
105
102
103
104
105
CD38me FITC
CD
138
APC
102
103
104
105
102
103
104
105
CD56 PC7
CD
138
APC
102
103
104
105
102
103
104
105
CD20 AH7
CD
138
APC
102
103
104
105
102
103
104
105
CD319 appears to be down-regulated with Daratumumab. We are currently using CD38 intracellular while investigating a new and brighter CD319 PE and intracellular IRF4.
Standard panel post Daratumumab CD56 negative, CD20 Positive
Multi-Epitope CD38 post Daratumumab
Not a clean Gate CD38 V450
CD
138
APC
102
103
104
105
102
103
104
105
The Effect of New Therapies on FC Myeloma Detection: The Daratumumab
Problem
CD38 V450
CD
138
APC
102
103
104
105
102
103
104
105
CD38 V450
CD
138
APC
102
103
104
105
102
103
104
105
CD38 V450 V450-A
CD
138
PerC
P C
y55
PerC
P-A
102
103
104
105
102
103
104
105
CD45 V500 V500-A
CD
19 A
PC A
PC-A
102
103
104
105
102
103
104
105
CD19 APC APC-A
CD
56 P
C7
PC7-
A
102
103
104
105
102
103
104
105
icKappa p PE PE-A
CD
19 A
PC A
PC-A
102
103
104
105
102
103
104
105
icLambda p FITC FITC-AC
D19
APC
APC
-A10
210
310
410
5
102
103
104
105
No Daratumumab Daratumumab Daratumumab + IC CD38
Daratumumab + IC CD38 is not always this good
CD319 appears to be down-regulated with Daratumumab. We are currently using CD38 intracellular while investigating a new and brighter CD319 PE and intracellular IRF4.
Conclusions:
Flow cytometry is clinically useful in prognosis in MGUS and SMM
Flow cytometry is important in the prognosis and MRD detection of multiple myeloma.
Consensus guidelines on good clinical practice in multiple myeloma MRD testing have been developed
Flow cytometric MRD testing in multiple myeloma is a mature test that is ready for application as a surrogate biomarker to determine endpoints in clinical trials.
