CD138-Independent Strategy for Detecting Residual and Circulating Myeloma Plasma Cells
Authors: Muz Barbara , Azab Feda , Pilar de la Puente , Paasch Jacob , King Justin , Micah Luderer , Vij Ravi , Azab Abdel Kareem 
Published: October 2019

Abstract
INTRODUCTION:
Flow cytometry has been used extensively to detect MM cells in the bone marrow (BM), micro-residual disease and circulating myeloma cells. Accumulating literature defines MM cells as CD138+/CD38+ for the primary gating of plasma cells; however, several studies demonstrated the presence of clonogenic CD138-negative MM cells, and that hypoxia can decrease the expression of CD138 in MM cells. We propose a novel set of biomarkers to detect MM cells regardless of their CD138 expression or hypoxic status.
METHODS and RESULTS:
We have tested the effect of hypoxia on the expression of the MM markers, CD138 and CD38, and found that hypoxia decreased the expression of CD138, therefore it cannot be used as a universal marker for MM cells. Hypoxia did not alter the expression of CD38 in MM cells; however, CD38 is a general leukocyte marker and cannot be used alone to identify MM cells, since it is expressed on multiple other leukocytes including T-cells, B-cells, monocytes, NK cells, and dendritic cells. Therefore, we negatively selected these cells by flow cytometry using the specific markers for each of these populations including CD3, CD19, CD14, CD16, and CD123; respectively. Therefore, we detected MM cells as CD38+/CD3-/CD19-/CD14-/CD16-/CD123-. We used CD38 antibody conjugated with APC and FITC-conjugated antibodies for all the other markers, thus MM cells were defined as APC+/FITC- population.
To compare traditional method (CD138-based) with our strategy to detect hypoxic and normoxic MM cells, MM cell lines were stained with a cocktail of CD38-APC, FITC-antibodies, and CD138-V450, and analyzed by flow cytometry. The use of CD138+ as a universal marker for MM cells detected 85-100% of the normoxic cells, and only 60-75% of the hypoxic MM cells. While APC+/FITC- strategy detected close to a 100% of the MM cells independent of the cells’ normoxic/hypoxic status or expression of CD138.
The ability of the new strategy to detect hypoxic and normoxic MM cells in the peripheral blood was tested by staining hypoxic and normoxic MM cells with cell-tracker Calcein-Red-Orange (as a positive control), spiked 104 MM cells into 106 mononuclear cells from a healthy donor (1% MM in total), and the percentile of Calcein-Red-Orange+ (as a positive control), APC+/FITC-, and CD138+ populations, were analyzed by flow cytometry. Calcein-Red-Orange staining showed exact 1% of MM cells detected in total mononuclear cells for both hypoxic and normoxic MM cells; detection with CD138+ showed 0.95% for normoxic and 0.45% for hypoxic cells; and detection of MM cells using the APC+/FITC- strategy showed 1.05% for normoxic and 1.1% for hypoxic MM cells. Hence, the new strategy detects MM cells selectively and independently of their CD138 expression or hypoxic status.
We have used the APC+/FITC- strategy to detect MM cells in BM CD138-negative fractions of 20 MM patients. The APC+/FITC- strategy was able to detect a range of 1.6-44% myeloma cells in the CD138-negative population of BM mononuclear cells isolated from MM patients.
Moreover, we have assessed the clonality of this population using APC+/FITC- in the CD138-negative fractions of MM patients. We found that the clonality of the APC+/FITC- population was similar to the clonality of the original disease (CD138+ cells) in 70% of the cases. The other 30% of the cases showed very low involvement of myeloma population in the BM and showed Kappa/Lambda ratio within normal range.
Analyzing the prevalence of circulating MM cells in the peripheral blood from 12 MM patients showed that all patients had a higher number of circulating MM cells as detected by APC+/FITC- strategy compared to CD138+, and the fold change ranged from 1.5 to 86 times.
CONCLUSION:
We found that CD138 cannot be used as a universal marker to detect MM cells. Moreover, we developed a novel strategy to detect MM cells independent of their CD138 expression or hypoxic status; and we used CD38+/CD3-/CD19-/CD14-/CD16-/CD123- population as an alternative set of biomarkers to detect MM cells. This strategy was able to detect a clonal MM cell population in the CD138-negative fraction of BM mononuclear cells isolated from MM patients, as well as in the peripheral blood.
Currently, we are exploring the ability of this strategy to predict relapse in MM patients whose BM was defined a CD138-negative. More investigation to characterize this population and the role in tumor recurrence and drug resistance in MM is warranted.

Disclosures
No relevant conflicts of interest to declare.