Abstract P5-13-32: Mucin 4 expression in high risk breast cancer: Predicting and overcoming resistance to immunotherapy
Authors: Schillaci Roxana , Bruni Sofia , Mauro Florencia , Mercogliano María F , Roldan-Deamicis Agustina , Proietti Cecilia J , Cordo-Russo Rosalía , Inurrigarro Gloria , Dupont Agustina , Adami Carla , Vecchia Daniel Lopez Della , Barchuck Sabrina , Figurelli Silvina , Deza Ernesto Gil , Ares Sandra , Gercovich Felipe G , Elizalde Patricia V 
Published: March 2022

Abstract
Background HER2-positive (+) and triple negative breast cancer (TNBC) have the worst survival among BC. BC patients are treated with chemotherapy (CT) and/or radiotherapy (RT), and HER2+ BC patients also receive targeted therapies, such as trastuzumab (Tz). The abundance of tumor infiltrating lymphocytes (TILs), in both HER2+ and TNBC, has a major good prognostic value. Thus, indicating that immunological evasion mechanisms are present in the tumor microenvironment (TME) hampering the efficacy of the treatments. We previously showed that soluble tumor necrosis factor α (sTNF) induces upregulation of mucin 4 (MUC4), which shields Tz epitope on HER2 impairing Tz binding and its effects. In preclinical models of de no5vo Tz-resistant tumors, administration of the sTNF blocking agent INB03 (DN) together with Tz inhibited tumor growth. We proved that MUC4 expression is an independent predictor of poor DFS in patients treated with adjuvant Tz. Our goal is to study whether MUC4 plays a role in tumor immune evasion in HER2+ and TNBC. Methods Untreated primary BC samples were assessed for TILs density (H&E) and MUC4 expression by immunohistochemistry. Tumors with TILs ≥30% and &gt;50%, for TNBC and HER2+ BC respectively, and MUC4 scores 2 and 3 (0-3) were deemed positive. A cohort of 56 TNBC and 90 HER2+BC, stage I-III were retrospectively retrieved from Hospital Fernández and Instituto Henry Moore from 2013-2017, and clinicopathological and treatment characteristics were obtained from electronic records. TNBC were treated with adjuvant (41) or neoadjuvant CT +/- RT (15). HER2+BC patients received adjuvant Tz + CT. The association between MUC4 and OS was assessed by Kaplan Meier and log rank test and between MUC4 and TILs using Chi2. JIMT-1 HER2+ BC, de novo resistant tumors to Tz, containing a doxycycline (Dox)-inducible shRNA MUC4 plasmid (JIMT-1shMUC4) growing in nude mice were treated with IgG, Tz, DN or Tz + DN. Tumor growth was measured and macrophages and NK cells were determined in the TME by flow cytometry. Anti-asialo GM1 and clodronate-encapsulated liposomes were used to deplete NK cells and macrophages, respectively. Results We found an inverse relationship between TILs and MUC4 expression in HER2+ and TNBC (P=0.02 and P= 5 x10-5, respectively). Patients with MUC4+ TNBC have a shorter OS (P=0.03) and MUC4 was an independent predictor of OS [P=0.01; HR 4.9 (95%CI 1.4-17.0)]. To study MUC4 involvement in macrophage and NK cells recruitment in a Tz resistant model, nude mice bearing JIMT-1-shMUC4 tumors were treated or not with Dox to abolish MUC4 expression. Both groups received IgG, Tz, DN or DN + Tz. In control groups (without Dox), only Tz + DN administration was able to inhibit tumor growth (75% inhibition, P&lt;0.0001 vs. IgG), in line with our previous results, and DN treatment reduced MUC4 expression. Knockdown of MUC4 expression by Dox, showed that Tz alone was effective in inhibiting JIMT-shMUC4 tumor growth at similar levels than Tz + DN group. Tumor growth inhibition was accompanied by an increase in NK cells activation and degranulation, and a rise in M1/M2 macrophage ratio. Depletion of macrophages or NK cells totally blunted antitumor effect of Tz + DN in control tumors. In MUC4-silenced tumors only macrophage depletion was able to abolish Tz antitumor effect. Conclusion Our results suggest that i) MUC4 expression is associated with immunologically “cold” HER2+ and TNBC, inducing an immunosuppressive TME that reflects in poor DFS/OS, and it confers resistance to Tz in HER2+ BC; ii) elimination of MUC4 expression reverses resistance to Tz; iii) tumor infiltrating macrophages are critical to the anti-tumor response in HER2+ BC. Patients with MUC4+ HER2+ or MUC4+ TNBC should benefit from sTNF blockade treatment leading to MUC4 downregulation and higher TILs, which would result in a better response to Tz and probably to immune checkpoint inhibitors.
Citation Format: Roxana Schillaci, Sofia Bruni, Florencia Mauro, María F Mercogliano, Agustina Roldan-Deamicis, Cecilia J Proietti, Rosalía Cordo-Russo, Gloria Inurrigarro, Agustina Dupont, Carla Adami, Daniel Lopez Della Vecchia, Sabrina Barchuck, Silvina Figurelli, Ernesto Gil Deza, Sandra Ares, Felipe G Gercovich, Patricia V Elizalde. Mucin 4 expression in high risk breast cancer: Predicting and overcoming resistance to immunotherapy [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-32.