Abstract P6-10-02: MHC-II positive breast tumors are more immunogenic and may preferentially select for LAG-3-positive tumor immune infiltrates
Authors: Balko JM , Loi S , Giltnane JM , Combs S , Estrada MV , Sanchez V , Rimm D , Sanders ME , Salgado R , Gomez H , Johnson DB 
Published: March 2017

Abstract
Background: Lymphocyte-activation gene 3 (LAG-3) is a T-cell checkpoint regulator and a current target in immunotherapy trials. LAG-3's main ligand is MHC class II (MHC-II), to which it binds with higher affinity than CD4. Binding of LAG3 to MHC-II antigen-presenting cells negatively regulates cellular proliferation, activation, and homeostasis of T cells, similarly to CTLA-4 and PD-1, suggesting that antibodies targeting LAG-3 may demonstrate similar anti-tumor immune effects.
Hypothesis: We recently reported an association of MHC-II on tumor cells and its involvement in mediating sensitivity to PD-1/PD-L1 monoclonal antibodies. MHC-II demonstrates a strong bimodal expression pattern on tumor cells from a variety of tissues, including those of the breast. In breast cancer patients, tumor-specific MHC-II expression on TNBCs is correlated with a 'hot' immune environment. We hypothesized that 1) MHC-II expression may drive potent anti-tumor immune responses and 2) MHC-II-positive tumors that generate immunotolerance may develop a specific immune checkpoint dependency on LAG-3, since LAG-3 is the inhibitory receptor for MHC-II-mediated antigen presentation.
Methods: To determine the functionality of MHC-II in driving anti-tumor immune responses, we constitutively expressed the MHC-II master regulator CIITA in MMTV-neu mouse tumor cells and determined their ability to form tumors in immunocompetent syngeneic hosts. To evaluate the association of MHC-II+ tumors with LAG-3 expression, we evaluated LAG-3-positivity by immunohistochemistry (IHC) in lymphocytic infiltrates in a series of 111 post-NAC TNBC specimens from patients with residual disease remaining after presurgical chemotherapy. Tumor-infiltrating lymphocytes (TILs) were scored by H&E, PD-L1 and MHC-II (HLA-DR) were scored in the stroma and tumor compartments using automated quantitative immunofluorescence (AQUA).
Results: Enforced expression of MHC-II via constitutive expression of CIITA caused rejection in 60% of mice, while only 11% of mice rejected MMTV-neu tumors expressing the vector control (Fisher's exact p=0.04). All rejecting mice were immune to rechallenge with parental (non-CIITA-expressing) MMTV-neu cells, suggesting a memory effector response.
Clinically, 11/102 patients (10.8%) had LAG-3+ immune cells in their tumor. LAG-3+ tumors were strongly correlated with MHC-II positivity in tumor cells (p&lt;0.0001). Presence of LAG-3+ cells also correlated strongly with overall TILs (p&lt;0.0001), and PD-L1 expression on TILs (p&lt;0.02). Since the likelihood of identifying LAG3+ lymphocytes is confounded by the inclusion of poorly-infiltrated tumors, we performed a subset analysis on only those tumors with substantial TILs (&gt;20%). When this subset was analyzed, LAG-3 positivity retained its association with tumor MHC-II expression (p=0.0001), while the association of LAG-3 with stromal PD-L1 was reduced below the level of significance (p=0.052).
Conclusions: MHC-II expression causes increased immune activation in breast cancers, consistent with our previous findings. MHC-II positivity in breast tumors may identify a population with preferential dependence on the LAG-3 checkpoint, which may be important for future immunotherapy trials.
Citation Format: Balko JM, Loi S, Giltnane JM, Combs S, Estrada MV, Sanchez V, Rimm D, Sanders ME, Salgado R, Gomez H, Johnson DB. MHC-II positive breast tumors are more immunogenic and may preferentially select for LAG-3-positive tumor immune infiltrates [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-10-02.