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Evaluation of Plasma Cell Neoplasms by Flow Cytometry

Mikhail Roshal MD, PhD

Director of Flow Cytometry

Hematopathology Service

Memorial Sloan-Kettering Cancer Center

New York, NY

Myeloma overview

• Neoplastic plasma cell disorder

– Proliferation of neoplastic plasma cells in the bone marrow

– Monoclonal immunoglobulin in serum/urine (ex. non-secretory)

– End organ dysfunction

• CRAB: hypercalcaemia, renal failure, anemia, and bone lesions

• 1% of all cancers in the Western world

• 13% of hematologic cancers (2nd after NHL)

– Age-adjusted incidence of 5-6/100000

• Median age at diagnosis is approx. 70 yo

• Treatment with induction chemotherapy +/-ASCT

• Rapid advances in treatment led to deeper responses and prolonged overall and disease free survival

• Possible increased risk of secondary malignancies

Role of flow cytometry in PCN management

• Primary Diagnostic Evaluation

– PCN vs. reactive vs. lymphoma

• Establish abnormal immunophenotype

• Define if a related abnormal B and/or T cell population is present

– Differential Diagnosis: reactive plasmacytosis, B cell lymphoma with plasma cell differentiation, AITL etc.

• Prognostic information

– High vs. Low Risk MGUS/SMM

– MGUS-like MM

• Therapeutic target assessment (CD38, BCMA, CD229, CD319 etc.)

• Assessment of therapy response

– Assess reactive vs. neoplastic residual plasma cells

– (minimal) residual disease analysis

• Evaluation for therapy-related myeloid/lymphoid neoplasms

Plasma cell enumeration

• Markedly and unpredictably underestimated in bone marrow 3+ fold with wide range compared to morphology– Hemodilution– Binding to stroma?– Autoaggregation?– Proportion of PCs is highly

reproducible (precise), but not accurate

– NOT a substitute for bone marrow morphologic assessment of plasma cell quantities

• Still much more sensitive than morphologic assessment/FISH

• Fraction of abnormal PC among PC is accurate

• PC quantitation is highly accurate

Bruno Paiva et al. Haematologica 2009;94:1599-1602

Flow in objective assessment of response

All patient had >10% PC post therapy, all had “clonal” PCs by morhologyPt 1: sCR Pt 2: Flow+sCRPt 3: Minimal Response

Hassoun et alLeuk Lymphoma. 2017 Dec;58(12):2962-2965

Conceptualizing MRD

Buckley, Applebaum, Walter; Bone Marrow Transplantation (2013) 48, 630–641

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Outcome according to MRD status

Andy C. Rawstron et al. JCO 2013;31:2540-2547

©2013 by American Society of Clinical Oncology

Depth of response matters

Rawstron AC, Gregory WM, de Tute RM, et al. Minimal residual disease in myeloma by flow cytometry: independent prediction of survival benefit per log reduction. Blood. 2015;125(12):1932-1935.

MRD as a predictor in multivariate analysis

Rawstron AC, Gregory WM, de Tute RM, et al. Minimal residual disease in myeloma by flow cytometry: independent prediction of survival benefit per log reduction. Blood. 2015;125(12):1932-1935.

MRD- CR, but not MRD+ CR is predictive of OS/PFS

Juan-Jose Lahuerta, J ClinOncol. 2017 Sep 1; 35(25): 2900–2910.

PC MRD status meta-analysis

Landgren et al Bone Marrow Transplant. 2016 Dec; 51(12): 1565–1568.

Impact of MRD is independent of treatment

de Tute et al Haematologica. 2016 Feb; 101(2): e69–e71.

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Greater sensitivity=greater predictive power

Ref: Martinez-Lopez J, Lahuerta JJ, Pepin F, et al. Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma. Blood. 2014;123(20):3073-3079

IMWG New Response Criteria

• MRD negative (<10-5) complete response:

– Negative immunofixation on the serum and urine and disappearance of any soft tissue plasmacytomas and <5% plasma cells in bone marrow aspirates +

• Sustained MRD negativity (two tests 1 year apart)

• Flow negative (sensitivity at least 10-5)• NGS negative

• Imaging (PET/CT) and MRD negative

Kumar, S. Lancet Oncol. 2016 Aug;17(8):e328-e346

Consensus Recommendation Summary

• >2 Million Cells Acquisition (NCI/FDA Roundtable)

• First pull to be sent for flow cytometry (2ml-4ml)

• CD19/27/38/45/56/81/117/138 mandatory

• Cytoplasmic k/l used for verification

Flow MRD basics

Most important parameters in MRD detection is number of cells analyzed AND quality of the sample

–Consider desired sensitivity (LLQ, LLOD)

SD =√n, CV=√n/n

• 10% CV 100 cells (SD=10):

• 20% CV≈20 cells

• For sensitivity of 0.001 (20 cell cutoff) need at least 2 million cells

Hemodilution matters

minimal = 1.1%, moderate = 0.85%, significant = 0.06%

20-fold reduction of PC in hemodilute samples

Manasanch, et al Leukemia & Lymphoma Vol. 56 , Iss. 5,2015

Quality of the samples vary across institutions

Cytometry Part B: Clinical CytometryVolume 90, Issue 1, pages 54-60, 21 DEC 2015 DOI: 10.1002/cyto.b.21271http://onlinelibrary.wiley.com/doi/10.1002/cyto.b.21271/full#cytob21271-fig-0001

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MRD Definitions Vary

Flanders A, Stetler-Stevenson M, Landgren O. Minimal residual disease testing in multiple myeloma by flow cytometry: Major heterogeneity. Blood 2013;122:1088–1089.

Salem et al. Am J Hematol. 2016 Dec;91(12):E502-E503.

MRD Definitions Continue to Vary

2015 CAP Survey Results

Keeney M et al. Arch Pathol Lab Med. 2015 Oct;139(10):1276-80

Sample reporting considerations

– Critical to define when sample is NOT optimal for analysis

• Marrows are irreplaceable samples - run anyway but…

• If too few cells-annotate expected sensitivity

• Check for hemodilution: nucleated reds, immature B cells, mast cells

• Samples received too late

– Particularly important for definition of negative

Normal Plasma Cells Show Diverse Immunophenotypes Immunophenotypic Abnormalities in Myeloma

• CD45 (loss)*

• CD19 (loss)*

• CD56 (gain)*

• CD38 (loss)*

• CD117 (gain)*

• CD200 (gain)

• CD81 (loss)*

• CD27 (loss)*

• CD99 (loss)

• Light chain restriction

*required for MRD evaluation

Cytometry Part B: Clinical CytometryVolume 90, Issue 1, pages 61-72, 31 JUL 2015 DOI: 10.1002/cyto.b.21265http://onlinelibrary.wiley.com/doi/10.1002/cyto.b.21265/full#cytob21265-fig-0004

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EuroFlow NGF assay

J Flores-Montero et al. Leukemia (2017), 1–10

EuroFlow NGF Assay

J Flores-Montero et al. Leukemia (2017), 1–10

Plasma Cell MRD History at MSKCC

• 30+ plasma cell flows/week, peak 10/day

• Gen 1: 4-color 7 tube (including isotypes) panel up to 500K cells lyse/stain

• Extremely time-consuming (required a dedicated tech and instrument)

• Difficult to analyze

• Roughly 95% sensitive compared to morphology

• Gen 2:

• One tube 8-color panel stain/lyse (up to 1.0 million cells)-200ul of marrow

• CD38 CD138 CD56 CD117 CD19 CD45 cy kappa lambda

• Theoretical sensitivity 0.005% IF marrow sample is cellular

• More sensitivity than FISH on sorted cells, IgH (conventional)

• Number of cells acquired depends on sample cellularity

Gen 3: for 10-color MRD with bulk lysis

• Bulk lyse

– Sample concentration=Predictable cell numbers

• Consensus for using CD38, CD138, CD56, CD117, CD81, CD27, CD19, CD45 , kappa, lambda-10 currently suggested markers

• 1X10

• Efficient utilization of limiting resources

• Instrument time, sample amount, analysis time, digital storage vs. multi-tube panels

• Acquisition of 5-10 million cells/tube =15-25 minutes+150 Mb file

• Reduces need for inferential reasoning and eliminates file merging

Panel Comparison

• Previous panel

CD38/CD19/kappa/lambda/CD117/CD56 /CD138/CD45

• New panel

CD27 / CD38/ CD81/kappa/lambda/CD117/CD19 /CD138/CD56/CD45

10-color single tube example

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Cell Losses with Perm vs. 2 tube approach (Euroflow)

# Events 10C PC Tube # Events EuroFlow5195356 3704581 23782174767567 3192946 23229301967763 1396119 9947505225656 2857943 29345025964189 3351625 33006934840782 3692511 24157943062964 1422475 10899646000000 3443175 26385755424060 2631025 17060645076271 1835450 16403025986407 3303268 26629906000000 3566177 18759012406885 2065600 9050504860198 4575511 20857256116838 5031300 35449006000000 4602250 33839756000000 4576445 486827510000000 5729045 56601764741443 3360708 25700766041259 6000000 50936745729316 3055708 28068856000000 4661975 35844936000000 6000000 16344124607295 5351200 18030136000000 6000000 50957486114018 4485808 36452756000000 4210250 28512752299225 2126000 12763881259504 1236993 9560763473324 3539728 21456283037395 2728306 11896255807403 4351150 33941846000000 4547450 41456505017601 4335548 38682086000000 5136100 35568805600889 3900950 28617021215018 3121100 6209902085615 2588975 1746216000000 6000000 52226946941345 6183046 36275256000000 6000125 487822510000000 6104025 56690256000000 6000000 3096200

• 4 ml of marrow split in half between Euroflow and MSKCC tests

• Entire sample used unless >30 million

• 45 marrows attempted:• 39 > 3 million (10 PC)• 35 >3 million: EF1, • 20> 3 million EF2

• Approx. 30% cell loss with perm EF1vs. EF2

• Same when we compared PC# in surface only B cell tube vs. 10PC

4 ml of bone marrow

2ml: 10 PC 2ml: EF1 (surface only)

and Ef2 (surface+cytoplas

mic)

Permebilization Effect on Staining

• Perm vs. non-Perm (same sample stained with surface only or permed)

Slight loss in SI (MFI-MFI)/(2*(SD+SD)) in CD45 APC-H7, no analytical effect

WITH PERM

WITHOUT PERM

SEPERATION INDEX

PERM VS NO PERM

CD117 8.41 8.76CD19 7.58 7.62CD138 4.72 5.91CD56 6.27 6.45CD45 8.44 12.30CD81 8.42 8.61CD38 13.45 13.52CD27 6.71 7.08

Cocktail Stability

Freshly prepared cocktail vs. two week old

From: Single-Tube 10-Fluorochrome Analysis for Efficient Flow Cytometric Evaluation of Minimal Residual Disease in Plasma Cell MyelomaAm J Clin Pathol. 2016;146(1):41-49. doi:10.1093/ajcp/aqw052Am J Clin Pathol | © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Old vs. New MSKCC Flow

Approximately 15% more sample are positive with extra log sensitivity

Improvement in Sensitivity

10 million acquisition, 10-color PC: 84 abnormal events

883K acquisition, 8-color PC: 6 abnormal events (<10, negative)

LLOD/LLOQ

DETECTION LIMIT FOR 10 COLOR MULTIPLE MYELOMASample Abnormal PC# WBC# %ABNORMAL PC of WBC %ABNORMAL PC of TOTAL PC

1 23 1845478 0.0012 0.0023

26 1854164 0.0014 0.0024

25 2052792 0.0012 0.0020

%CV 6.2 7.70 9.32

2 59 7440774 0.0008 0.1386

45 6500730 0.0007 0.1302

59 7781646 0.0008 0.1288

%CV 14.9 6.84 4.00

3 21 8841491 0.0002 0.0023

29 8883942 0.0003 0.0040

23 8874004 0.0003 0.0036

%CV 17.1 16.90 26.93

4 15 2389508 0.0006 1.7000

14 2635835 0.0005 1.5000

24 2994385 0.0008 2.2000

%CV 31.2 20.97 20.03

5 24 9355578 0.0003 0.0035

23 9497372 0.0002 0.0024

19 8689842 0.0002 0.0024

%CV 12.0 8.00 22.95

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Sample Quality Control

Mast cells Early B cells

Comparison of MSKCC single 10-color tube and EuroFlow

two 8-color tubes.

Mikhail Roshal et al. Blood Adv 2017;1:728-732© 2017 by The American Society of Hematology

What about NGS??

Herve Avet-Loiseau et al. Blood 2015;126:191

©2015 by American Society of Hematology

Diagnostic clone detection by NGS compared to Flow (n=153)

124

117

144

50 70 90 110 130 150

Detected by CE (FR1,2,3)

Detected by LT (FR1)

Detected by LT (FR1 and Leader)

Rate of clonal IGH rearrangement detected by CE and NGS LT assays

94%

81%

76%

M. Arcilla, MSKCC

Comparison of NGS and Flow (n=83 samples, 71 patients)

71

70

11

0 20 40 60 80

Disease detected by Flow

Disease detected by NGS

Disease not detected

3 discrepant cases 2 cases: detected by flow not NGS: 0.0005%, 0.00095%1 case detected by NGS and not flow: 0.02% of the total rearranged Ig readsHOWEVER: this is based on only samples the could be monitored by NGS.6% loss due to lack of amplification20-30% loss due to lack of original clone (would vary by institution etc.)

M. Arcilla, MSKCC

Practical Points

• Flow similar analytical sensitivity as latest version of NGS• Applicable to greater proportion of samples • Does not require prior sample!!!• Theoretical sensitivity is still dependent on sample quality/number of

cells– In 2015 only about 70% of samples allowed for >5 million cells– In 2017, >90% (first pull, dedicated practitioners)

• Approximately 4 hours from sample to report• 5-6 sample batch is ok• Cocktail (surface and light chain) is stable for at least 2 weeks• 15-25 minute instrument time/sample• 150 Mb file• 15 minute import/analysis/reporting time on average• Limited analysis of other populations

– Assess presence of mast cells/immature myeloid precursors/B-cell precursors

– Dedicated tubes run for other disorders as indicated

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Additional Highly Informative Antigens

Gao et al, Modern Pathology, in print

Additional highly informative antigens exist

Gao et al, Modern Pathology, in print

Anti-CD38 treatment Anti-CD38 treatment

Alternatives to CD38

Pojero et al Cytometry B Clin Cytom. 2016 Jan;90(1):91-100