Abstract P3-05-08: Lymphoid and myeloid cell characterization of inflammatory breast cancer tumor microenvironment and correlation to pathological complete response
Authors: Reddy SM , Reuben A , Jiang H , Roszik J , Tetzlaff MT , Reuben J , Wang L , Tsujikawa T , Barua S , Rao A , Villareal L , Wood A , Woodward W , Ueno NT , Krishnamurthy S , Wargo JA , Mittendorf EA 
Published: February 2018

Abstract
Background: Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with poor response rates to current chemotherapy regimens. With recent successes of immune targeted therapies in other solid tumors and a growing understanding of how the immune tumor microenvironment (TME) affects non-IBC outcomes, we sought to characterize the immune TME in IBC to identify biomarkers of treatment response and potential targets for drug development.
Methods: Pre-treatment core biopsy samples were identified from the MD Anderson Cancer Center IBC tissue bank from patients with stage III and de novo stage IV (with T4d) disease who received neoadjuvant chemotherapy (NAC) with intent to take to mastectomy. Lymphocytes were characterized by stromal tumor infiltrating lymphocyte (sTIL) quantification, CD8 T cell quantification, and T cell receptor sequencing. PD-L1 expression was assessed using DAKO 22C3 clone on tumor and immune cells. Myeloid cells were characterized using a multiplex immunohistochemistry approach, using CD68 and CD163 for macrophage markers, tryptase for mast cell marker, HLA-DR for class II antigen presentation marker, and cytokeratin as tumor marker. Spatial analyses were performed by determining probabilities of finding cell 1 of interest within 20 uM of cell 2 of interest and computing area under the curve for statistical comparison.
Results: 91 patients with stage III (N=62) or de novo stage IV (n=29) disease were identified. Breast cancer subtype included 25 triple negative, 34 HER2+ and 32 HER2-HR+. 86 patients received a mastectomy, of whom 33 (38.4%) patients experienced a pathologic complete response (pCR). sTIL was higher in stage III tumors (11.9 vs 4.8%, p&lt;0.001) and in those having a pCR (13.8 vs 7.3%, p=0.019). CD8 T cell density (available in 48 cases) similarly was higher in stage III patients (360.3 vs 178.8 counts/mm2, p=0.040) and pCR cases (452.3 vs 219.2 counts/mm2, p=0.080) but also higher in HER2+ disease (560.9 for HER2+ vs 239.9 counts/mm2, p=0.087 for TNBC and 153.6 counts/mm2, p=0.005 for HER2-HR+). T cell clonality (available in 32 cases) ranged from 0.004 to 0.242 but showed no correlation to tumor characteristics or response. PD-L1 complete tumor membranous expression was seen in only 1 of 47 cases, whereas PD-L1 positivity on immune cells was seen on 36.2% of cases; neither correlated to response. Myeloid cell assessment (available in 25 cases) showed higher mast cell infiltration in non-pCR cases (63.8 vs 26.8 counts/mm2, p=0.008) and spatial analysis (performed on 10 cases) identified that closer proximity of mast cells to CD8 T cells correlates with response (AUC 6.0 vs 2.2, p=0.017), suggesting a possible immunosuppressive mechanism. HLA-DR analysis demonstrated no difference by response as a single stain marker, but co-localization of HLA-DR with cell type shows higher HLA-DR expression on tumor cells in non-responders (14.6 vs 1.6%, p=0.031).
Conclusions: Higher TIL and CD8 T cell density are correlated with improved responses to NAC in IBC. Mast cell infiltration and HLA-DR expression on tumor cells are inversely correlated to response and suggest possible mechanisms of resistance. Mast cells could present potential therapeutic target in IBC.
Citation Format: Reddy SM, Reuben A, Jiang H, Roszik J, Tetzlaff MT, Reuben J, Wang L, Tsujikawa T, Barua S, Rao A, Villareal L, Wood A, Woodward W, Ueno NT, Krishnamurthy S, Wargo JA, Mittendorf EA. Lymphoid and myeloid cell characterization of inflammatory breast cancer tumor microenvironment and correlation to pathological complete response [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-08.