Multidimensional Flow Cytometric (MFC) Analysis of the Immune System of Multiple Myeloma (MM) Patients Achieving Long Term Disease Control
Authors: Magalhaes Roberto J. Pessoa , Vidriales María-Belén , Paiva Bruno , Mateos Maria-Victoria , Gutierrez Norma C. , García-Sanz Ramón , Blanco Juan F , Hernández Jose , de las Heras Natalia , Martinez Joaquin , Costa Elaine Sobral , Maiolino Angelo , Nucci M. , De La Rubia Javier , Lahuerta Juan-Jose , Orfao Alberto , San Miguel Jesús F. 
Published: October 2019

Abstract
Abstract 810FN2
Increasing evidence shows that a small fraction of MM patients (pts) treated with high-dose therapy followed by autologous stem cell transplantation achieve long-term remission. Interestingly, this is not restricted to pts in complete response (CR), since those that revert to a monoclonal gammopathy of undetermined significance (MGUS) profile may also achieve long-term remission, despite the persistence of residual myeloma plasma cells (PCs). These results suggest that in addition to the anti-myeloma therapy, other factors may play a role in the control of the disease.
Herein, we used 8-color MFC for detailed characterization of the structural components of the immune system and hematopoietic precursor cells (HPC) in paired bone marrow (BM) and peripheral blood (PB) samples from 26 MM patients in long-term disease control (LTDC): 9 in continuous CR and 17 who reverted to an MGUS profile and that subsequently showed stable disease without treatment for ≥5 years (median of 9 years; range, 5–19). As controls, paired BM and PB samples from 23 newly-diagnosed MGUS and 16 MM pts, together with 10 healthy adults (HA), were studied in parallel. In all BM and PB samples the distribution of the major T- (CD4, CD8, Tregs and γδ), NK- (CD56dim and CD56bright) and B-cell subsets (Pro-B, Pre-B, naïve and memory), in addition to normal PCs, dendritic cell (DC) subsets (plasmacytoid, myeloid and monocytic), monocytes, and CD34+ HPC (myeloid and lymphoid), were studied. The percentage and absolute count of each cell population was analysed in the BM and PB, respectively.
Comparison of the two groups of MM pts with LTDC (9 CR vs. 17 MGUS-like) showed similar (p>.05) cellular profiles in PB and BM, except for an increased number of BM and PB normal PCs in CR patients (P≤.04). Consequently, for all subsequent analyses, LTDC myeloma pts were pooled together.
When compared to HA, patients with LTDC had increased numbers of CD8 T-cells and CD56dim NK-cells in both the BM and PB (p≤.03 and p≤.01, respectively). Despite this, the distribution of BM and PB CD4, CD8 and γδ T-cells among LTDC patients was similar (p>.05) to that of both newly-diagnosed MM and MGUS cases; in contrast, BM and PB Tregs were significantly decreased vs newly-diagnosed MM (P=.03) and MGUS (P=.04). Regarding B-cells and normal PCs, LTDC patients showed increased numbers of BM B-cell precursors (both Pro-B and Pre-B cells) and normal PCs vs. newly diagnosed MM (P≤.05), but not MGUS, together with increased numbers of naïve B-cells vs. both MM and MGUS pts (P≤.01); all such cell populations returned to levels similar (p>.05) to those of HA. As expected, this also included the number of CD34+ B-cell HPC which was increased among patients who achieved LTDC vs MM (p=.02), at levels similar (p>.05) to those of MGUS and HA.
Regarding DC, LTDC patients showed normal DC numbers in PB (but with higher PB myeloid-DC numbers vs. MM; p=.02), in association with decreased numbers of plasmacytoid DC and increased monocytic-DC in the BM vs. HA (p≤.04). No differences were found for the numbers of BM and PB monocytes.
In summary, here we investigated for the first time the immune cell profile of MM patients who achieve long-term disease control. Our results show that, as newly-diagnosed MM, patients that achieve long-term disease control also show increased numbers of cytotoxic T-cells and CD56dim NK-cells; however, in contrast to newly-diagnosed MM, among LTDC patients such increase is associated with lower numbers of T-regs and an almost complete recovery of the normal PC, B-cell precursor and naïve B-cell compartments both in BM and PB. Further investigations on the activation and functional status of these cell populations are warranted.MO (%)/SP (cels./μl)HA N= 10MGUS N= 23MM N= 16LTDC-MM N= 26T cells9.588110.6117313113711926    CD4+4.85004.6624^6*5085463    CD8+3.7∼216∼4.63865.32645.3431    TCR γδ.2426.3230.2428.3421    Treg.4137.4141^.54*38.3432NK cells.7∼87∼1.51982.11721.6212    CD56 dim.65∼79∼1.41922.21681.6202B cells2.81471.8104.97*68*1.9160    Pro B.11—.06—.02*—.07—    Pre B.6—.4—.08—.23—    Naive SP—80—57^—36*—118    Normal-PCS.18.9.11.7.008.72*.11.84DCs.3449.3653.6848.558    Monocytes2.22472.42853.43023.1315    m-DC SP—11—14—8*—12    MO-DC.11∼29.2036.434.2837    p-DC.2∼4.1.145.112.8.123.8CD34+.9∼1.46.61.1.261.4.431.4    Mie-HPC.8∼—.53—.26—.36—    Linfo-HPC.1—.07—.03*—.05—*p≤.05 LTDC vs MM: ^ p≤.05 LTDC vs MGUS; ∼ p≤.05 LTDC vs HA

Disclosures:
Paiva: Jansen-Cillag: Honoraria; Celgene: Honoraria. Martinez:Janssen: Honoraria; Celgene: Honoraria. Maiolino:Centocor Ortho Biotech Research & Development: Research Funding.