PD-L1/PD-1 Pattern of Distribution within Bone Marrow Microenvironment Cells in Patients with Smoldering Myeloma and Active Multiple Myeloma
Authors: Costa Federica , Vescovini Rosanna , Notarfranchi Laura , Storti Paola , Marchica Valentina , Dalla Palma Anna Benedetta , Manferdini Cristina , Toscani Denise , Eufemiese Rosalba , Burroughs Jessica , Lisignoli Gina , Giuliani Nicola 
Published: November 2020

Despite the promising results of immune-checkpoint blockade in the treatment of many tumors, the use anti-PDL-1/PD-1 antibodies in multiple myeloma (MM) still remains debated and under observation for the high toxicity in combination with immunomodulatory drugs or for the lack of a clear evaluation of the PD-L1/PD-1 distribution in MM patients. Literature data on PD-L1/PD-1 expression by CD138+ and bone marrow (BM) cells in MM patients are discordant and none of them compared patients with active and smoldering myeloma (SMM). This suggests the need to better define PD-L1/PD-1 distribution in the BM immune-microenvironment in MM patients, to identify those that could benefit from PD-L1/PD-1 blockade.
In this study, we isolated mononuclear cells from BM aspirates in a cohort of patients with monoclonal gammopathies (total number= 107) including 39 patients with SMM and 78 with active MM, including both newly diagnosed (MMD) and relapsed MM (MMR). We compared the expression profile of PD-L1/PD-1 axis on CD138+ cells, CD14+ monocytes and T cells (both CD4+ and CD8+), by flow-cytometry. Results were correlated with clinical parameters, as International Staging system (ISS), cytogenetic risk, bone disease. BM sera were also collected to measure the levels of different soluble factors known to regulate PD-L1 expression or to exert pro/anti-tumor activity in MM (IL-6, IL-10, IL-27, IFN-γ). Results from ELISA assay were examined in relation with flow-cytometry data.
We found that neither PD-L1 expression on CD138+ cells nor PD-1 on CD4+/CD8+ cells significantly differ between SMM and MM patients; although, CD14+PD-L1+% increases with disease progression (SMM vs MMD vs MMR: median: 39.14 vs 59.49 vs 47.66, p=0.14) without reaching a statistical significance.
Analysis on the total cohort revealed that PD-L1 is expressed at higher levels on CD14+CD16+ non-classical monocytes compared with classical monocytes (17.41 vs 23.09, p&lt;0.0001), independently from the stage of disease. Of note, relapsed MM patients, which are currently the main candidates for anti-PD-L1/PD-1 treatment, showed an inverted CD4+/CD8+ ratio as compared with SMM (0.75 vs 1.22, p=0.024) and MMD (0.75 vs 1.50, p=0.001), along with high levels of pro-tumoral IL-6 and a positive correlation between CD14+PD-L1+% and CD8+PD-1+% cells (p=0.013), suggesting a highly compromised immune-microenvironment with a low amount of effector cells. Among the cytokines tested on the total cohort, the anti-tumoral IL-27 BM serum levels inversely correlated with PD-L1 MFI only on CD14+ cells (p=0.025), with CD8+PD-1+% (p=0.013) and with the immunesuppressive cytokine IL-10 serum levels (p=0.035), independently from the stage of disease. Noteworthy, we did not find any correlation between PD-L1/PD-1 profile and BM levels of either IFN-γ, known to in vitro up-regulate PD-L1 expression on MM cell lines, or IL-6 levels, the main MM pro-survival factor.
Focusing on patients with active MM, those with ISS=II and III showed increased PD-L1 expression on CD14+ cells (ISS II+III vs I, median MFI 20.35vs14.59, p=0.005) and higher CD8+PD-1+% (II+III vs I, 4.35vs2.58, p=0.022) compared with ISS=I patients.
Analysis on PD-L1/PD-1 expression in relation with the cytogenetic features of our cohort of patients revealed that CD138+ cells from hyperdiploid patients express higher levels of PD-L1 compared with not-hyperdiploid ones (25.66 vs 13.96, p=0.001), suggesting that the expression of this immune checkpoint does not contribute as a high risk factor in MM disease. Finally, we investigated PD-L1/PD-1 distribution, in relation with the presence of bone lesions, and we found that not-osteolytic patients have higher CD8+PD-1+% compared with osteolytic ones (p=0.071).
In conclusion, our data show a similar PD-L1/PD-1 expression pattern between SMM and MM patients, and higher PD-L1 intensity on the non-classical monocytes CD14+CD16+ compared with classical CD14+CD16- cells. On the other hand, an inverted CD4+/CD8+ ratio characterizes relapsed MM patients, together with high amount of MM pro-survival IL-6 and low anti- tumor IL-27 BM serum levels. Overall these data suggest that, despite the similarity in PD-L1/PD-1 expression, SMM and early MM patients rather than relapsed MM, could represent the potential subset which could better benefit of anti PD-L1/PD-1 therapy, in light of their less compromised immune-microenvironment.

Disclosures
Giuliani: Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Participation in congresses, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Other: Participation in congresses; GSK: Other: Clinical study sponsorship, Research Funding; Janssen Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Other: Clinical study sponsorship; participation in congresses, Research Funding; Millennium Pharmaceutical: Other: Clinical study sponsorship, Research Funding.