Eight-Color Flow Cytometry Phenotypic Markers and Disease Progression in Monoclonal Gammopathy of Unknown Significance
Authors: Yarlagadda Naveen K , Mohan Meera , Atrash Shebli , Gundarlapalli Sravani , Hoque Shadiqul , Kakadia Sunil , Thanendrarajan Sharmilan , Schinke Carolina , Alapat Daisy , Tricot Guido , Tian Erming , Sawyer Jeffery R. , Zhan Fenghuang , van Rhee Frits , Zangari Maurizio 
Published: November 2021

Abstract
Introduction: Flow cytometric immunophenotyping is considered an indispensable tool for the diagnosis, classification, and monitoring of plasma cell disorders. Herein, we seek to study the clinical significance of expression of phenotype markers in monoclonal gammopathy of unknown significance (MGUS).
Methods: We identified a cohort of patients with a diagnosis of MGUS from the institutional myeloma database. Bone marrow (BM) aspirate assessment was performed using 8-color immunophenotypic next-generation flow cytometric (NGF) analysis with a minimum sensitivity of 10 -5 cells at the time of diagnosis or first visit to our institution. BM aspirate samples were immunophenotyped on a FACSCanto II flow cytometer using antibodies (BD) to delineate normal and abnormal plasma cells [CD138 (V-500), CD38 (FITC), CD19 (PE-Cy7), CD45 (V-450), CD27 (PercpCy5.5), CD81 (APC-H-7), CD56 (APC) and CD20 (PE)]. The sensitivity or the Limit of Detection (LOD) for this assay was validated to 20 cells in 2 ×10 6 events (0.001%), and the reproducibility or Lower Limit of Quantitation (LLOQ) is 50 cells in 2 ×10 6 events. Clinical and laboratory variables were also collected. Based on previously published data, expression (CD19, CD45, CD81), and lack of expression (CD56, CD27, CD20) of the above-mentioned surface antigens were analyzed. Additional variables such as IgA isotype, size of M-protein (≥15 g/L), and abnormal free light chain ratio(abnFLR) (defined as &lt;0.1 or &gt;10) were included in regression fitting models.
Results: A total of 157 patients with MGUS were included in this analysis. The median age at diagnosis was 60 years (range 24- 84), 84 (53 %) patients were female and 25 (16%) were African American. Overall, IgG Kappa (75/148, 50%) was the most common isotype. Fluorescent-in-situ hybridization (FISH) data were available in 35 patients with t (4:14) and t (14;16) seen in 3 patients each. At a median follow-up of 18.15 years (quantiles 11.35, 33.62), 28 patients experienced disease progression (25 to MM, 2 to Waldenstrom macroglobulinemia, and 1 Smoldering myeloma). The median progression-free survival of this cohort was 17.3 years. Among these, occurrences of the bone lesion (8/28; 28.6%) were the most common pattern of disease progression to MM. This analysis showed lower odds of progression with the expression of CD27 (OR-0.39, 95% CI 0.15-0.99) (figure 1A). Disease progression was more common in patients with an abnormal plasma cell clone size ≥ of 3.1% at diagnosis (60% vs. 12.5%, p=0.0005). An abnormal plasma cell clone of ≥3.1% at diagnosis, was associated with increased odds of progression (OR-10.79, 95% CI 4.02-28.98) and a shorter PFS (12.5 years versus NR, p=0.01) (figure 1B). Serum M-spike ≥1.5 g/dL (OR-3.54;95% 1.30-9.62) and abnFLR (OR-2.30, 95% CI 1.00-5.32) were also associated with a higher odds of progression. However, in this population, the presence of IgA isotype did not increase the odds of MGUS progression. In a stepwise regression model, serum M-spike≥1.5 g/dL, abnFLR, and the lack of expression of CD27 were associated with the risk of disease progression.
Conclusion: In addition to previously published risk factors, our cohort shows that the expression of CD27 antigen by eight-color flow cytometry confers a lower risk of disease progression of MGUS. This is consistent with our previous report that CD27 is progressively down-regulated in the transition from normal plasma cells (NPC) to MGUS to MM (Zhan et al, Blood 2006). Furthermore, we show that size of the myeloma clone (≥ 3.1% ) is a possible surrogate marker for disease progression in MGUS.
Figure 1: 1A shows forest plot of odds ratios for flow cytometry markers, IgA isotype, size of M protein, abnFLR and plasma cell clone size. 1B shows the Kaplan Meier estimates of PFS for patients stratifies by plasma cell clone size.
Figure 1 Figure 1.

Disclosures
Mohan: Medical College of Wisconsin: Current Employment. Atrash: GSK: Research Funding; AMGEN: Research Funding; Jansen: Research Funding, Speakers Bureau.