Sirpα and CSF1R Positive Tissue Macrophages Are Increased in Patients with Follicular Lymphoma Who Relapse after Frontline Lenalidomide and Rituximab
Authors: Marques-Piubelli Mario L. , Parra Edwin R. , Feng Lei , Gallardo Mariana , Solis Soto Luisa , Wistuba Ignacio I. , Flowers Christopher R. , Neelapu Sattva S. , Vega Francisco , Strati Paolo 
Published: November 2021

Abstract
Introduction. The combination of lenalidomide and rituximab (also called R 2) is a safe and effective frontline regimen for patients with high tumor burden follicular lymphoma (FL). Long-term follow-up data show that about one third of patients eventually relapse, but mechanisms of resistance to frontline R 2 remain currently unknown in FL. Two main mechanisms of resistance to lenalidomide have been described in multiple myeloma, a condition for which this agent has been more extensively used: acquired gene mutations leading to downregulation of cereblon, and increase in pro-tumoral macrophages. FL cell lines cannot be propagated without macrophages, and macrophage-related gene signatures have been shown to associate with poor outcome in patients with FL. Therefore we hypothesized that increase in pro-tumoral macrophages may represent a mechanism of resistance to lenalidomide also in FL.
Methods. Patients with advanced stage FL grade 1-3A who relapsed after frontline R 2 and with available pre-treatment and progression tissue biopsies were included in the study. Reactive lymph node tissue biopsies were used as control. A multiplex immunofluorescence panel including CD47 and 6 macrophage markers (CD14, CD68, CD115 or CSF1R, CD163, CD172a or SIRPα, and PD-L1) was optimized and validated, and used to stain all tissue biopsies. All slides were imaged using the Vectra Polaris spectral imaging system (Akoya Biosciences, Marlborough, MA). The nonparametric Wilcoxon rank-sum test was used to compare the relative abundance of the possible marker combinations among the patient groups, and adjustment for multiple testing was performed using the multivariate analysis of variance (MANOVA).
Results. Ten patients with available pre-treatment (pre-R 2) and progression (post-R 2) tissue biopsies were identified. A total of 51 tissue areas in the post-R 2 and control group, and 53 areas in the pre-R 2 group (mean tissue area 0.16 mm 2, range, 0.02-0.16) and a mean number of 30178 cells (range, 12315-51649) were analyzed by Vectra. Not including single-marker outputs, 58 combinations were identified for macrophage markers. Among these, only 2-marker and 3-marker combinations had a median of more than 1 cell/mm 2 and were included in the final analysis (overall, 6 single-marker and 36 multi-marker combinations).
When comparing single-marker outputs between the 2 groups, post-R2 cases showed a significantly higher number of CD68+ (mean 195 vs 70 cells/mm 2, p&lt;0.001), CD115+ (mean 154 vs 83 cells/mm 2, p=0.005), CD163+ (mean 80 vs 45 cells/mm 2, p=0.01) and PDL1+ (13 vs 5 cells/mm 2, p=0.05) cells. All associations were also maintained on multivariate analysis. Of interest, no significant difference in the mean number of CD47+ cells was observed when comparing post-R 2 to pre-R 2 samples (mean 297 vs 814 cells/mm 2, p=0.25).
When comparing two and three-marker macrophage combination outputs between the 2 groups, post-R 2 cases showed a significantly higher mean number of CD14+CD115+ (p=0.01), CD68+CD115+ (p=0.005), CD68+CD163+ (p=0.005), CD68+PDL1+ (p=0.02), CD115+CD172a+ (p=0.05), CD115+PDL1+ (p=0.03), CD163+CD172a+ (p=0.02), CD68+CD163+CD172a+ (p=0.03) and CD115+CD163+CD172a+ (p=0.02) cells. On multivariate analysis, the association was maintained only for 3 macrophage populations: CD68+CD115+ (mean 35 vs 11 cells/mm 2; F=9.6; p=0.002), CD115+CD172a+ (mean 13 vs 4 cells/mm 2; F=4.9, p=0.03) and CD68+CD163+CD172a+ (mean 8 vs 2 cells/mm 2; F=4.2, p= 0.04) cells (Figure). When comparing post-R 2 samples to controls, a significantly lower number of CD115+CD172a+ (13 vs 24 cells/mm 2, p=0.03) and CD68+CD163+CD172a+ (8 vs 21 cells/mm 2, p=0.04) cells was observed, whereas the mean number of CD68+CD115+ cells was comparable (35 vs 24 cells/mm 2, p=0.30).
Conclusions. Despite comparable levels of CD47, small populations of SIRPα (CD172a) and CSF1R (CD115) positive tissue macrophages can be effectively identified by multiplex imaging assays and are significantly increased in patients with FL who relapse after frontline R 2. These data suggest that increase in pro-tumoral macrophages may be a mechanism of resistance to lenalidomide also in patients with FL. The investigation of SIRPα and/or CSF1R inhibitors for the treatment of FL patients who relapse after R 2 or in combination with frontline R 2 may be warranted and result in improved outcomes.
Figure 1 Figure 1.

Disclosures
Flowers: Eastern Cooperative Oncology Group: Research Funding; TG Therapeutics: Research Funding; Denovo: Consultancy; Kite: Research Funding; Janssen: Research Funding; Cellectis: Research Funding; Sanofi: Research Funding; EMD: Research Funding; Guardant: Research Funding; Takeda: Research Funding; Morphosys: Research Funding; Allogene: Research Funding; Spectrum: Consultancy; Celgene: Consultancy, Research Funding; Iovance: Research Funding; Epizyme, Inc.: Consultancy; Genentech/Roche: Consultancy, Research Funding; Amgen: Research Funding; SeaGen: Consultancy; Pfizer: Research Funding; BeiGene: Consultancy; Nektar: Research Funding; Novartis: Research Funding; Acerta: Research Funding; AbbVie: Consultancy, Research Funding; Xencor: Research Funding; Biopharma: Consultancy; Karyopharm: Consultancy; Bayer: Consultancy, Research Funding; Ziopharm: Research Funding; Pharmacyclics/Janssen: Consultancy; 4D: Research Funding; Burroughs Wellcome Fund: Research Funding; National Cancer Institute: Research Funding; Adaptimmune: Research Funding; Gilead: Consultancy, Research Funding; Genmab: Consultancy; Cancer Prevention and Research Institute of Texas: CPRIT Scholar in Cancer Research: Research Funding; Pharmacyclics: Research Funding. Neelapu: Kite, a Gilead Company, Merck, Bristol Myers Squibb, Novartis, Celgene, Pfizer, Allogene Therapeutics, Cell Medica/Kuur, Incyte, Precision Biosciences, Legend Biotech, Adicet Bio, Calibr, Unum Therapeutics and Bluebird Bio: Honoraria; Kite, a Gilead Company, Merck, Bristol Myers Squibb, Novartis, Celgene, Pfizer, Allogene, Kuur, Incyte, Precision BioSciences, Legend, Adicet Bio, Calibr, and Unum Therapeutics: Other: personal fees; Kite, a Gilead Company, Bristol Myers Squibb, Merck, Poseida, Cellectis, Celgene, Karus Therapeutics, Unum Therapeutics (Cogent Biosciences), Allogene, Precision BioSciences, Acerta and Adicet Bio: Research Funding; Takeda Pharmaceuticals and related to cell therapy: Patents & Royalties. Vega: i3Health, Elsevier, America Registry of Pathology, Congressionally Directed Medical Research Program, and the Society of Hematology Oncology: Research Funding; CRISPR Therapeutics and Geron: Research Funding. Strati: Astrazeneca-Acerta: Research Funding; Roche-Genentech: Consultancy.