Steven Gross1, Brad Foulk1, Karl Nielsen1, Jaymala Patel2, Mark Connelly1, and Marielena Mata2. 1Veridex, LLC., Huntingdon Valley, PA; 2Janssen R&D, ...
Automated Enumeration and Characterization of Circulating Multiple Myeloma Cells in Blood
Assay Performance and Linearity Evaluation Using Spiked Whole Blood Whole blood drawn into CellSave tubes from healthy volunteers and spiked with tissue culture Multiple Myeloma H929 cells. 4.0mL of the spiked samples were processed according to protocol above to evaluate assay performance with blood samples. Assay Performance with healthy volunteers and patients Whole blood drawn into CellSave tubes from age-matched healthy volunteers, Multiple Myeloma, MGUS, and Smoldering Myeloma patients were collected, processed, and analyzed as above.
CellTracks® cartridge
Reagent Kit/ Sample area
CMMCs in healthy donors and patients with Multiple Myeloma/MGUS/SMM
10000
N CMMC Range Mean Median > 1 CMMC > 3 CMMC > 5 CMMC > 10 CMMC > 100 CMMC > 1000 CMMC
1000
MagNest® Device and CellTracks® Cartridge The AutoPrep system transfers samples to CellTracks® Cartridge in MagNest® device. The magnetic field generated by the MagNest® device causes the magnetically labeled cells to be distributed uniformly over the analysis surface of the cartridge, ready for analysis using the CellTracks Analyzer II®.
MagNest/ cartridge area
Sample Analysis This semi-automated fluorescent microscope captures images for each of the four fluorescent filter cubes, covering the entire surface of the cartridge. Captured images containing PE and DAPI positive events are presented in a gallery for analysis and classification of cells by the user based on cell fluorescence and morphology.
% % % % % %
100
10
Normals MGUS/ SMM 52 30 0-6 0-112 0.5 9 0 1 33 60 6 37 2 27 0 17 0 3 0 0
Multiple Myeloma 66 0-16814 1157 19 91 76 68 58 35 14
1 0 _
Multiple Myeloma
MGUS/ SMM
Age Matched Normals
CMMCs are significantly elevated in Multiple Myeloma patients.
CMMCs contain FISH markers also detected in Bone Marrow
Results
Patients with ≥1 CMMC with FISH result
Median CMMC count
t(4;14) (n)
t(14;16) (n)
del17p (n)
Numerical aberration (n)
Active MM
8
3512
25% (2)
0
0
38% (3)
SMM
1
88
0
0
0
100% (1)
MGUS
9
4
11% (1)
0
22% (2)
33% (3)
Age Matched Normals
4
1
25 % (1)
25 % (1)
0
0
Assay Reproducibility and Linearity Using Spiked Whole Blood Determined by spiking ~ 500 tissue culture Multiple Myeloma cells (H929) into 4.0ml healthy donor blood.
r 11 Dono
20
0
Aver a
r 12
ge
Dono
r 10 Dono
r9 Dono
r8 Dono
Dono
r7
r6 Dono
r4 Dono
Dono
r2 Dono
Dono
r1
r3
80
r5
100
Observed Number of Tumor Cells
Assay linearity was determined by spiking 0, 10, 100, 500 and 2000 H929 cells into 4.0mL blood samples from five healthy donors. The assay is linear over the range of 0 – 2000 cells with a correlation coefficient of 0.98
40
Specimen Collection and Enumeration of CMMC Collected blood into CellSave Preservative Tubes (Veridex, LLC., Huntingdon Valley, PA). Transferred 4.0mL blood to 15 mL CellTracks® AutoPrep ® sample tubes Processed the samples using a CMMC kit which contains the following reagents: • Anti-CD138 Ferrofluid: to capture CD138 positive cells • Nucleic Acid Dye: DAPI to identify nucleated cells • Capture Enhancement Reagent: to maximize capture efficiency irrespective of variable expression of CD138 antigen • Permeabilization Reagent: to allow for staining of intracellular antigens. • Cell Fixative: to fix cells in the final samples. • Staining Reagent: Contains anti CD38-PE to identify plasma cells and CD19-APC and panleukocyte marker (CD45-APC) to exclude non-plasma cells and leukocytes. • Marker Reagent: Contains CD56-FITC as an additional sub-type marker. Placed the reagent kit and up to 8 samples on the CellTracks® AutoPrep® System (Veridex, LLC, Huntingdon Valley, PA). Analyzed the samples using the CellTracks Analyzer II® (Veridex, LLC, Huntingdon Valley, PA) CMMCs were fixed inside the cartridge using 3:1 methanol acetic acid. Repeat Free FISH probes for 4p, 14q, and 16q were obtained from Kreatech and labeled with DY647, DY550, and DY415 respectively. The P53 probe, derived from a single BAC clone covering the TP53 gene, was labeled with digoxygenin and detected with anti-digoxygenin:FITC Fab fragment. Probes and samples were co-denatured and hybridized overnight using a Thermobrite hotplate. FISH images of CMMC were acquired with a modified CellTracks instrument equipped with a 40x objective and cubes appropriate for the probe dyes.
MagNest® device
CD drive
60
Methods
CellSpotter® cartridge
User Printer Interface
H929 Cell Recovery (%)
Detection of circulating Multiple Myeloma cells (CMMC) by flow cytometry is an indicator of active disease. In addition, circulating plasma cells can be detected in earlier stages of disease, including MGUS and Smoldering Multiple Myeloma, and appear to correlate with prognosis. The capture and characterization of these circulating plasma cells from peripheral blood may provide novel biomarkers for the management of Multiple Myeloma patients, particularly in monitoring minimal residual disease and in progression from MGUS or Smoldering Multiple Myeloma to active disease. The enumeration and characterization of circulating tumor cells (CTC) in patients with metastatic breast, prostate or colorectal cancer using the CellSearch ® technology, has been shown to provide clinically relevant prognostic and predictive information. Here we describe the development of an automated assay for detecting circulating normal plasma cells (CPC) and Multiple Myeloma cells (CMMC) in blood using CellSearch. Assay results from Multiple Myeloma, MGUS patients, and from an aged matched control population are presented. The CellTracks® AutoPrep® System and CellTracks Analyzer II® systems were used to capture and enumerate CPC and CMMC. Magnetic particles conjugated to anti-CD138 are used to capture myeloma cells from 4.0mL of blood. Enriched cells are then stained with the nucleic acid dye DAPI and anti-CD38Phycoerythrin (PE) antibody. Allophycocyanine (APC) conjugated anti-CD45 and anti-CD19 were used to exclude leukocytes and B-cells. In addition, FITC labeled anti-CD56 was added as a biomarker. The enriched and stained cells were transferred to a CellTracks® cartridge and MagNest® for magnetic mounting. The cartridge was scanned using the CellTracks Analyzer II®. Individual images of cells were presented to the operator for review, and scored as CMMCs, based on fluorescence and cell morphology. In a model spike-in system the assay consistently recovered ~60% of the cells from the Multiple Myeloma cell line H929 spiked into 4.0mL of blood from healthy donors. The assay was linear over the tested range of from 0 to 2000 spiked H929 cells (r2 0.98, slope 0.50, intercept 10). The assay was validated using blood from age matched healthy donors (n=52) and patients with Multiple Myeloma (n=66) and MGUS (n=30). In 4.0mL blood from normal donors, 0 CPC were detected in 35/52 (67%) and low numbers (1-6 CPC) were detected in 17/52 (33%) samples. Interestingly, one CD56 positive CPC (CMMC?) was found in a normal donor. CMMC in Multiple Myeloma patients ranged from 0 – 17,000 /4.0mL blood. One or more CMMC were detected in 91% of the patients, > 5 in 68%, > 10 in 58% and > 100 in 35%. Expression of CD56 was highly variable in the patient population. CMMC in MGUS/SMM patients ranged from 0 – 112 /4.0mL blood. One or more CMMC were detected in 60% of the patients, > 5 in 27%, > 10 in 17% and > 100 in 3%. To further characterize CMMC, and differentiate CPC from CMMC, an interphase fluorescent in situ hybridization (FISH) assay was developed to be used with the capture and detection system described above. A four color FISH probe was used to simultaneously detect high risk mutations including two recurrent translocations of the IgH locus (t(4;14)(p16;q32) and t(14;16)(q32;q23)) as well as deletion of the TP53 locus (∆17p13). The FISH assay was verified on cell lines H929, MM1s, and U266, which showed mutations at t(4;14), t(14;16) and ∆17p13, respectively. The FISH assay was tested on 9 CMMC patient samples and 8 samples yielded evaluable results. Two samples showed t(4;14)fusions, 3 patients showed aberrant FISH signal patterns indicating aneuploidy of chromosome 4 or 14 and the remaining patients showed normal FISH patterns. Well controlled prospective clinical studies are needed to establish the prognostic and predictive value of the presence, and characteristics, of CMMC in multiple myeloma or MGUS. In addition, as with CTC, this automated CMMC assay should prove useful in evaluating the effectiveness of new treatments as well as the assessment of potential treatment targets on CMMC in this difficult disease.
Automated System to process blood samples
MagNest™ device
# of CMMC/4ml Blood
Abstract
Dono
2011 Annual Meeting Abstract 1825
Steven Gross1, Brad Foulk1, Karl Nielsen1, Jaymala Patel2, Mark Connelly1, and Marielena Mata2 1Veridex, LLC., Huntingdon Valley, PA; 2Janssen R&D, Radnor, PA.
Pilot study of FISH analysis on CMMC from myeloma, pre-myeloma and age matched normal donors. The number of evaluable cells in MGUS and healthy donors was very low. The number of cells needed to make reproducible measurements was not determined.
1600
y = .50x + 10.1 1400
R2 = 0.98
1200
FISH assay result of CMMC from Multiple Myeloma patient
1000 800
CMMC IF images
600 400 200 0 0
200
400
600
800
1000 1200 1400 1600 1800 2000 2200 2400
Expected Number of Tumor Cells
CellTracks Analyzer II® Browser Images of CMMCs in one Multiple Myeloma Patient CD38-PE
DAPI
CD19/45APC
CMMC FISH images
FISH image overlay
4;14 Fusions in all evaluable cells
CD56-FITC
CD56+ CMMC (DAPI+/CD38PE+ CD19/45APC-)
t(14;16) no fusions detected
P53 2 copies
Conclusions The assay recovers ~60% of the spiked Multiple Myeloma cells and is linear over the tested range from 0 to 2000 cells. 76% of patients with Multiple Myeloma and 37% of patients with MGUS/SMM have > 3 CMMC while only 6% of normal age-matched healthy donors have > 3 CMMCs.
CD56- CMMC (DAPI+/CD38PE+ CD19/45APC-)
Expression of CD56 in patient samples is highly variable. Other potential markers can be used to interrogate cells. This assay provides a complete and standardized method for CMMC detection. A four color FISH assay was developed to detect three recurrent mutations in Multiple Myeloma.
Leukocytes (DAPI+/CD38PE+ CD19/45APC+)
Aberrant FISH signal patterns can be detected in CMMC from Multiple Myeloma/MGUS/SMM and age matched healthy donors.
Next Steps Prospective studies in SMM and Multiple Myeloma to evaluate imminent risk to progression, potential treatment targets, and response to therapy.
All authors are employees of Veridex LLC. or Janssen, both part of the Johnson and Johnson family of companies
“The products discussed in this document are for research use only and not for use in diagnostic procedures.”
