Abstract P5-12-11: M2 macrophages increase after neoadjuvant HER2 targeted chemotherapy
Authors: Thompson PA , Uhlik M , Preece C , Gorden K , Harrison B , Graff J , Stopeck A 
Published: May 2019

Abstract
Purpose: Recent observations suggest a positive association between the presence of tumor-associated myeloid and lymphoid immune cells and clinical responses to HER2 therapies. The specific immune cell composition of HER2+ tumors and the effects of therapy on the tumor immune microenvironment remains poorly understood; limiting efforts to effectively direct immunomodulatory agents in HER2+ breast cancer. We sought to assess the feasibility of a multiplex immunofluorescence strategy to characterize the immune milieu of HER2+ tumors, pre and post treatment. Methods: We conducted a feasibility study using the Perkin Elmer OPAL multiplex dye chemistry for immunofluorescence of tumor, myeloid, and lymphoid cells in pretreatment biopsy and surgical resection tissues of 11 patients who received neoadjuvant HER2 antibody with chemotherapy. Two panels of up to 6 antibodies were studied including a predominantly myeloid panel targeting CD80, CD68, CD163, CD206, PD-L1, and cytokeratin; and a 'lymphoid/proliferation' panel targeting FoxP3, cytokeratin, Granzyme B, CD4, CD8, and Ki67. For paired samples, analysis of pre/post differences were analyzed using two tailed, t-test. Results: 100% of the pre-treatment biopsy samples yielded high quality immune and tumor cell immunofluorescence profiles for both panels. In contrast, post treatment specimens were more challenging. For the post treatment tissues, ˜25% of specimens failed to yield results in at least one panel. As shown in Table 1, the %CD206+ M2 type macrophage population increased between pre and post treatment (p=0.012). This was reflected in a decrease in the M1:M2 ratio and variability in the ratio in favor of M2 (median 0.34 [IQR = 1.01] to 0.11 [IQR=0.10], p=0.0003). In the lymphoid panel, we observed a non-significant reduction in % FoxP3+CD4 T cells with less variability between patients post treatment and a significant decrease in total CD8+ T cells. Further, there was a significant reduction in %Granzyme B positive T cells (median 6.63 to &lt;1%, p=0.02) and non-significant decrease in proliferating (Ki67+) T cells post treatment. PD-L1 expression was low in both pre and post specimens.
MarkerSampleMinMedianMaxIQR%CD206+ Mono/MacPre1.358.7135.8911.05%CD206+ Mono/MacPost25.438.9777.621.54M1/M2 RatioPre0.110.342.141.01M1/M2 RatioPost0.030.110.210.1%Ki67+ T cellsPre0.137.9220.0411.06%Ki67+ T cellsPost04.5621.836.31%PD-L1+ MyeloidPre01.0512.060.95%PD-L1+ MyeloidPost00.554.261.47%Granzyme B T cellsPre2.926.6333.727.2%Granzyme B T cellsPost00.892.481.73%FoxP3+ CD4 +Pre01.6456.0416.44%FoxP3+ CD4 +Post01.166.824.14#Total CD8+ T cellsPre5788,36635,42615,744#Total CD8+ T cellsPost121,3717,9043,658
Conclusions: Multiplex immune profiling is a practical approach to characterize the tumor immune microenvironment in biopsy and post treatment specimens. Neoadjuvant HER2 targeted chemotherapy significantly shifts the myeloid population to an M2 (immunosuppressive) phenotype with evidence for a reduction in the number of Ki67 and Granzyme B+ T cells. These preliminary results suggest immunomodulatory agents that are able to induce or maintain an M1 polarized tumor microenvironment may have utility to enhance long term anti-tumor immunity in HER2 disease.
Citation Format: Thompson PA, Uhlik M, Preece C, Gorden K, Harrison B, Graff J, Stopeck A. M2 macrophages increase after neoadjuvant HER2 targeted chemotherapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-11.