Defining the Differentiation Stage of Multiple Myeloma Plasma Cells: Biological and Clinical Significance
Authors: Paiva Bruno , Puig Noemi , Vidriales María-Belén , Gutierrez Norma Carmen , Hernandez Miguel T , Rosiñol Laura , Ocio Enrique M , Oriol Albert , Echeveste María Asunción , Gonzalez Yolanda , Gironella Mercedes , Prieto Julio , Moreno Maria Jose , Giraldo Pilar , Palomera Luis , Aoiz Intza , Blade Joan , Lahuerta Juan José , Mateos Maria-Victoria , San Miguel Jesus F. 
Published: November 2019

Abstract
B-cell lymphopoiesis ends in terminally differentiated bone marrow (BM) plasma cells (PCs). While initially PCs have been viewed mainly as short-lived end-stage B-cells, there is now evidence that this is a heterogeneous cellular compartment in which new-born plasmablasts and long-lived PCs also coexist. The transition between both is characterized by phenotypic modifications, which include higher CD38/CD138 expression alongside HLADR and CD19 down-regulation. CD19 function and expression are regulated by CD81 and accordingly, three normal BM PC subsets can be delineated according to the expression levels of both markers: CD19+/CD81+, CD19-/CD81+, and CD19-/CD81-. However, no additional phenotypic information exists on such normal subsets, nor about how myeloma PC differentiation relates to its normal counterpart.
Here, we used 23-color multidimensional flow cytometry (MFC) and principal component analysis (PCA) to phenotypically characterize normal BM PC differentiation in 10 healthy donors and its malignant counterpart in 115 elderly newly-diagnosed multiple myeloma (MM) patients included in the PETHEMA/GEM2010MAS65 study.
First, we adopted novel MFC software technology to, after merging and calculating phenotypic data obtained from four 8-color combinations, define the immunophenotypic expression profile (iPEP) of normal BM PCs and characterize their differentiation pathway through PCA. Accordingly, we observed that from the CD19+/CD81+ subset through the CD19-/CD81+ and CD19-/CD81- stages (corresponding to 64%, 32% and 4% of total PCs, respectively) there is a continuous down-regulation on the amount of expression of CD54 (P=.008), CD44 (P=.03) and CD27 (P=.07). By contrast, a trend towards increased levels of CD28 (P=.06), CD38 (P=.05) and CD56 (P=.09) was also observed, suggesting an accumulation of potentially less active and more differentiated PCs from the CD19+/CD81+ to the CD19-/CD81- stages. Using the same approach as described above to determine the iPEP of myeloma PC clones from each individual patient, we then integrated such iPEPs into the normal PC differentiation pathway to investigate, through PCA, the stage and corresponding normal counterpart of each patient myeloma PC clone. From the 115 patients included in this analysis, 3 (3%) had myeloma PCs fitting within the CD19+/CD81+ differentiation stage and 21 (18%) within the CD19-/CD81+ subset, whereas the remaining 91 cases (79%) fitted within potentially more differentiated CD19-/CD81- stages. Virtually no cases with myeloma PC clones corresponding to the CD19+/CD81+ and CD19-/CD81+ differentiation stages had ISS stage I (8%), as compared to 30% in patients with a more differentiated PC CD19-/CD81- signature (P=.03). Furthermore, patients with myeloma PC clones matching the CD19+/CD81+ and CD19-/CD81+ differentiation stages showed a trend for higher incidence of extramedullary plasmacytomas (22% vs 9%; P=.09). Interestingly, almost half of patients with myeloma PC clones in potentially less differentiated stages had no cytogenetic abnormalities [t(IGH), +1q, del(13q), and/or del(17p)] as compared to cases with myeloma PCs matching with the CD19-/CD81- normal PC counterpart (44% vs. 16%, respectively; P=.01). However, whenever present, the type (standard- vs high-risk) of such cytogenetic abnormalities did not differ between both subgroups. Finally, we investigated if the differentiation stage of myeloma PC clones influenced patients’ prognosis, and noted that progression-free survival (PFS) of cases in less and intermediate differentiation stages was significantly inferior PFS as compared to patients with a mature CD19-/CD81- myeloma PC clone (median of 24 months vs not reached, respectively; P=.02). Noteworthy, identical patient prognostication for PFS (P=.02) was observed when the analysis was restricted to cytogenetically-defined standard-risk cases, thus identifying a subgroup of patients with more aggressive MM despite favorable cytogenetic profiles.
In summary, we showed that in the vast majority (~80%) of MM patients the PC clone phenotypically matches more differentiated normal PC counterpart subsets. Patients harboring less differentiated clones show a higher incidence of ISS stage II/III and extramedullary disease despite fewer cytogenetic abnormalities, as well as significantly inferior PFS as compared to cases with more differentiated myeloma PC clones.

Disclosures
Ocio: Array Biopharma: Honoraria, Research Funding.