Introduction: Anti-PD-1/L1 (atezolizumab) is effective in first-line, PD-L1-positive triple negative breast cancer, however it is becoming increasingly apparent that the majority of breast cancers will require novel I-O combination strategies. We describe a framework for use of multispectral immunofluorescence (mIF) as a high-dimensional biomarker to facilitate discovery in BC I-O clinical trials. Specifically, we propose a method for recapitulating the prognostic stromal tumor infiltrating lymphocyte (sTIL) score and PD-L1 status using mIF, but with higher resolution to detect dynamic changes. Furthermore, mIF provides spatial and phenotypic data on the cellular level, allowing for comprehensive characterization of tumor-IC interactions, which may provide insight into the mechanisms of action of I-O therapy. Methods: In a recent ESBC I-O pre-operative clinical trial, a regimen combining low-dose systemic chemotherapy (cyclophosphamide) with intralymphatic cytokine injections (IRX-2) was evaluated for pharmacodynamic immune activity, as measured by dynamic changes in ICs and PD-L1 status. Pre-treatment and resection tissues were analyzed for sTIL score (H&E) and PD-L1 status (Ventana SP142). mIF was conducted (PerkinElmer Vectra) using a validated antibody panel that quantifies and provides geospatial coordinates of tumor cells (cytokeratin/CK+), ICs (CD3, CD8, CD163, FOXP3), and per-cell PD-L1 expression (QIF score). Multiple regions of interest (ROI, 20x) were captured per specimen (mean 18, range: 9-32 per specimen). Hierarchical regression employed to correct for heterogeneity across ROIs and patients. Results: By the H&E sTIL score, the IRX-2 regimen was associated with increases in sTILs of 139% (p=0.001). Using mIF, we found highly concordant increases in sTILs. mIF allowed for changes in cell counts across different IC phenotypes to be accurately quantified, allowing for lymphocytes to be highlighted while excluding myeloid mononuclear cells in the sTIL quantification. After adjusting for intratumoral and intrapatient heterogeneity with mixed effects modeling, effector T-cells (CD3+CD8+) increased by +173% (95% CI: +93% to +387%) and helper T-cells (CD3+CD8-FOXP3-) increased by +120% (95% CI: +21% to +401%). Without addressing heterogeneity with mixed- effects modeling both CD3+CD8+ and CD3+ CD8- T cells were underestimated (46% vs 40% raw cell avg). By the SP142 assay, IRX-2 regimen was associated with PD-L1 conversion in 36% (n=5/14) of cases, and PD-L1 up-classification in 79% (n=11/14) of cases. PD-L1-positive IC count by mIF was highly concordant with SP142 (Jonckheere-Terpstra test = 171, p<.001). By mIF, PD-L1 + IC increased by 337% with therapy (4.37-FC, p<0.001). Three spatial analysis metrics were evaluated: nearest neighbor distance (NND), stromal-tumor border density gradient, and clustering. Using these metrics we uncovered spatial pharmocodynamic effects including increases in clustering of helper cells to effector cells (z=25.2, p<.001), co-localization of effector cells to the tumor-stromal boundary (p=0.001), and penetration of effector cells into tumor nests (p=0.01). Conclusion: We describe a framework for use of mIF as a high-dimensional biomarker for use with I-O clinical trials to gain mechanistic insight into the biologic effects of I-O therapies in primary BCs. mIF can be used to detect dynamic changes in sTIL score and IC PD-L1 expression, and therefore may serve to complement the H&E sTIL score and SP142 PD-L1 clinical assay in the context of I-O clinical trials. The greater precision of regression-assisted mIF may facilitate discovery of treatment-related I-O effects, potentially with smaller sample size requirements. Citation Format: Katherine Sanchez, Isaac Kim, ShuChing Chang, Maritza Martel, Yaping Wu, Brady Bernard, Joanna Pucilowska, Zhaoyu Sun, William Redmond, Dottie Waddell, Walter Urba, David B Page. Multispectral immunofluorescence (mIF) to detect dynamic changes in PD-L1 expression, immune cell (IC) infiltration, and tumor-IC interactions in primary breast cancer (BC) immuno-oncology (I-O) clinical trials [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-09.

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e12070 Background: In triple negative breast cancer (TNBC), anti-PD-L1 is associated with gains in overall survival, however only in tumors with > 1% PD-L1+ ICs. Locoregional cytokine therapy is being evaluated as a method of priming and redirecting ICs to tumors, which may increase I-O benefit particularly in PD-L1 negative tumors. We report a sensitive method of quantifying I-O-related changes in PD-L1 and ICs using mIHC and single-cell hierarchical regression, which controls for within-tumor and across-patient PD-L1/IC heterogeneity. Methods: Pre-treatment and resection tissues from a phase Ib trial of locoregional cytokines (IRX-2, n = 16 subjects) in ESBC were analyzed. IRX-2 contains immunostimulatory cytokines (GM-CSF, IL-2, IFN-α, INF-γ, and IL-12) and was injected in peri-areolar tissue, which communicates directly with tumor-draining lymphatics. Specimens were analyzed for 1) H&E stromal TILs score; 2) clinical PD-L1 IC expression (Ventana SP142, 2 blinded pathologists); and 3) mIHC (PerkinElmer Vectra). InForm software was used to obtain geospatial outputs of tumor cells (CK+) and ICs (CD3, CD8, CD163) across multiple regions of interest (mean:18; range: 9-32). Mixed-effects modeling was used to evaluate for changes in PD-L1, ICs, and IC/tumor distance metrics. Results: PD-L1 and IC quantity by mIHC was highly concordant with clinical PD-L1 IC classification (JT test = 211, p < .001) and TIL score (r = 0.77). Cytokine therapy was associated with higher PD-L1 IC classification in 9/13 tumors, and increased PD-L1 ICs by mIHC in 14/15 (+337%, 95% CI: 120,769%). After adjusting for heterogeneity, cytokine therapy increased CD3+CD8+ ICs (+173%, CI: 93,287%) and CD3+CD8- ICs (+120%, CI: 21,301%). Therapy was also associated with increased effector-helper T-cell clustering (nearest-neighbor distance, -40%, CI: -29,-50%) and effector cell tumor localization (stromal-tumor interface CD8+ density +90%, Cl: 74, 305%). Conclusions: Our method is concordant with clinical PD-L1 and sTILs scores, and can additionally quantify IC and IC distances. This assay may allow for comparative I-O assessments with increased resolution, and will be used in a randomized phase II trial of pembrolizumab, chemo +/- IRX-2 in stage II/III TNBC.

The immune system can recognize and destroy tumor cells through T-cell mediated mechanisms. Hence, identifying tumor antigen-specific T-cells from cancer patients and expanding them in large numbers in vitro has important implications for immunotherapy diagnostics and therapeutics. Here we show that tumor-reactive T-cells are enriched in a subset of tumor-infiltrating CD8 T-cells (CD8 TILs) identified by co-expression of CD103 and CD39 both in primary and metastatic tumors. The CD103+CD39+ CD8 TILs are present at high frequencies in melanoma and mismatch repair-deficient colon cancer but at low frequencies in microsatellite stable (MSS) colon cancer and colorectal liver metastasis. This cell population displays a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. Importantly, we show in a MSS colon cancer patient that a very low number of CD103+CD39+ CD8 TILs can be expanded in vitro and that those cells recognize tumor-specific neoantigens. Finally, patients with head and neck cancer whose CD8 TILs contained a higher frequency of CD103+CD39+ cells experienced a greater overall survival. Our work suggests that CD103+CD39+ CD8 TILs are key players in the patient’s antitumor response and describe an approach for detecting and expanding those cells, which will help improve adoptive TIL therapy for cancer patients. Citation Format: Thomas Duhen, Rebekka Duhen, Ryan Montler, Tarsem Moudgil, Jitske van den Bulk, Bernard A. Fox, Shu-Ching Chang, Gary Grunkemeier, Els M.E. Verdegaal, Noel F. de Miranda, Rom Leidner, Richard B. Bell, Andrew D. Weinberg. A new strategy to identify and expand tumor-reactive CD8 TILs in human solid tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A186.