Muthuraj, Sonia

e14535 Background: Anti-programmed cell death ligand PD-L1/PD-1 immune checkpoint inhibitors (ICIs) are approved for metastatic breast cancer (mBC) patients (pts) with a tumor combined positive score (CPS) ≥10% in their tissue. However, many pts with < 10% CPS may also respond to ICIs. Recent studies in lung cancer have shown that circulating tumor cells (CTCs) and tumor-macrophage fusion cells (TMFCs) both express, and may upregulate, PD-L1 which is predictive for ICI responses. We initiated a prospective study to monitor PD-L1 in CTCs & TMFCs in mBC pts prior to receiving systemic ICIs to correlate their PD-L1 expression to progression free survival (PFS) and overall survival (OS) at 24 months. Methods: We conducted a multi-institutional prospective pilot study of n = 102 pts with pathologically confirmed mBC, having failed ≥1 prior lines of therapy, prior to receiving ICIs (n = 52 Pembrolizumab, n = 39 Retifanlimab, n = 5 Atezolizumab, and n = 1 Nivolumab). Anonymized blinded peripheral blood samples (7.5ml) were procured prior to ICI induction (T0) and (~40 days after ICI induction (T1). TMFCs & CTCs were isolated using Cellsieve microfilters with average PD-L1 expressions measured then categorized as negative/low or high. Tumor CPS was categorized as negative/low (< 10%) or high (> 10%). We compared TMFC PD-L1 to tissue CPS by linear regression. TMFC/CTC PD-L1 expression and tissue PD-L1 CPS were compared to pts’ PFS and OS by Cox proportional univariate and multivariate analysis over 24 months. Results: Of n = 102 pts, median age = 59 years (range 32-77); 42% (43/102) were ER+, 33% (33/102) were PR+, 5% (5/102) were HER2+, and 56% (57/102) were triple-negative. 32% (32/102) had an ECOG score of 0, 25% (26/102) had ECOG 1, and 43% of scores were unavailable. N = 42 pts had available tumor CPS data, which did not correlate to TMFC/CTC PD-L1 at T0 or T1. Pts with high CPS did not significantly predict for PFS (HR = 0.78; 95% CI, 0.3-1.9; P = 0.7627) nor OS (HR = 0.54; 95% CI, 0.2-1.7; P = 0.4471). Pts with high TMFC/CTC PD-L1 at T0 also did not significantly predict for PFS (HR = 0.74; 95% CI, 0.5-1.2; P = 0.2799) nor OS (HR = 0.84; 95% CI, 0.4-1.6; P = 0.7220). However, of the n = 82 pts who provided T1 samples, high CTC/TMFC PD-L1 at T1 was significant for better PFS (HR = 2.55; 95% CI, 1.6-4.2; P = 0.0004) and better OS (HR = 2.06; 95%CI, 1.1-3.8; P = 0.0309). Conclusions: In this study of mBC pts treated with PD-L1 ICIs, tissue CPS did not correlate with PD-L1 expression in CTCs or TMFCs. Further, neither CPS nor baseline PD-L1 in CTC/TMFCs predicted for clinical outcomes in ICI therapies. However, PD-L1 in CTC/TMFCs ~40 days post-induction of ICI did predict for better response rates. While this study suggests predictive value of monitoring PD-L1 in blood during ICI therapies, further studies are required to refine and validate these findings.

Abstract Tumor associated macrophages are known to fuse with cancer cells resulting in the formation of a cellular phenomenon called tumor-macrophage fusion cells (TMFCs). TMFCs are a highly aggressive cell subtype that may move into circulation, evade immune surveillance, and possibly aid in seeding of metastatic sites. The formation of TMFCs involves CD47, a ubiquitous surface receptor that acts as a “don’t eat me” signal, interacting with the macrophage protein SIRPa, a down regulator of phagocytosis, and syncytin-1, a mediator of hyperploidy cancer fusion. In this analysis, we isolated circulating TMFCs from the blood of n=39 metastatic cancer patients (pts) to evaluate the expressions of CD47, SIRPa and syncytin-1 as it relates to stable or progressive disease via 30 day imaging by PET scan. We conducted a prospective pilot study of n=39 pts with pathologically confirmed metastatic breast (n=17), prostate (n=7), pancreas (n=7), or colon (n=8) cancer receiving standard of care systemic therapy. We isolated TMFCs from 7.5ml peripheral blood using a CellSieve microfilter, and stained with CD47, SIRPa, & syncytin-1. TMFCs were identified based on large cell size (&gt;30 µm) & multinucleated structure, and were categorized into 4 distinct subtypes: A) &gt;2 distinct non-contacting nuclei within a TMFC, B) 2 distinct non-contacting nuclei, C) 2 contacting nuclei, D) a single multinucleated nuclei. CD47, SIRPa, and syncytin-1 expressions were categorized as negative, low, or high. TMFCs subtypes and marker expressions were compared to pts’ clinical status (i.e. progressive, stable or responding) at first available PET/CT scans after blood draw (∼45 days). TMFCs were identified in 72% of pts (n=28/39), average 8 cells/sample, and ranged 1-77 cells. Of the TMFCs, 12% were subtype A, 11% as B, 11% as C, and 66% as D. CD47 expression was seen in 100% of TMFs, with low expression in 39% of subtype A cells, 58% subtype B, 39% subtype C, & 62% subtype D. Notably, CD47 was significantly lower (p=0.010) in subtype B than other groups. Further, the expression of syncytin-1 in subtype B cells was significantly lower (p=0.02) than in other subtypes, and subtype D cells had 75% higher syncytin-1 localized to the nuclear regions. No correlations were found in the SIRPa expression and subtypes. Interestingly, progressing pts had 200% more TMFCs, more subtype B (83%), and significantly lower (p&lt;0.001) CD47 expressing TMFCs than non-progressing pts. In this pilot study on the expression profiles of circulating TMFCs, it appears that specific subtypes of TMFC with low CD47 expression may be associated with progressive disease. Interestingly, this contrasts existing studies on primary tumors which often correlate higher CD47 with more aggressive cancers. This finding suggests that low CD47 cancer cells circulate blood in some pts with progressing disease and may be pertinent to efforts targeting CD47 in cancer. Citation Format: Sonia Muthuraj, Massimo Cristofanilli, Saranya Chumsri, Susan Tsai, Raymond C. Bergan, Cha-Mei Tang, Daniel L. Adams. Low CD47 expression in specific populations of tumor macrophage fusion cells in blood correlates with progressive disease in metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1979.