Blocking soluble TNFα sensitizes HER2-positive breast cancer to trastuzumab through MUC4 downregulation and subverts immunosuppression.

BACKGROUND: The success of HER2-positive (HER2+) breast cancer treatment with trastuzumab, an antibody that targets HER2, relies on immune response. We demonstrated that TNFα induces mucin 4 (MUC4) expression, which shields the trastuzumab epitope on the HER2 molecule decreasing its therapeutic effect. Here, we used mouse models and samples from HER2+ breast cancer patients to unravel MUC4 participation in hindering trastuzumab effect by fostering immune evasion. METHODS: We used a dominant negative TNFα inhibitor (DN) selective for soluble TNFα (sTNFα) together with trastuzumab. Preclinical experiments were performed using two models of conditionally MUC4-silenced tumors to characterize the immune cell infiltration. A cohort of 91 patients treated with trastuzumab was used to correlate tumor MUC4 with tumor-infiltrating lymphocytes. RESULTS: In mice bearing de novo trastuzumab-resistant HER2+ breast tumors, neutralizing sTNFα with DN induced MUC4 downregulation. Using the conditionally MUC4-silenced tumor models, the antitumor effect of trastuzumab was reinstated and the addition of TNFα-blocking agents did not further decrease tumor burden. DN administration with trastuzumab modifies the immunosuppressive tumor milieu through M1-like phenotype macrophage polarization and NK cells degranulation. Depletion experiments revealed a cross-talk between macrophages and NK cells necessary for trastuzumab antitumor effect. In addition, tumor cells treated with DN are more susceptible to trastuzumab-dependent cellular phagocytosis. Finally, MUC4 expression in HER2+ breast cancer is associated with immune desert tumors. CONCLUSIONS: These findings provide rationale to pursue sTNFα blockade combined with trastuzumab or trastuzumab drug conjugates for MUC4+ and HER2+ breast cancer patients to overcome trastuzumab resistance.

MUC4 Expression in Angiomatoid Fibrous Histiocytoma.

Angiomatoid fibrous histiocytoma (AFH) is a rarely metastasizing neoplasm that typically occurs in the deep dermis and subcutis of the extremities of young patients, characterized by a t(2;22) translocation involving EWSR1 and CREB1. Because of its distinctive histologic features, the diagnosis of AFH is generally straightforward, although the immunohistochemistry (IHC) findings are relatively nonspecific. We recently encountered a case of primary cranial AFH that showed strong MUC4 IHC expression, which has not yet been reported previously. Prompted by this surprising finding, we investigated MUC4 expression in a series of AFH to evaluate this potential diagnostic pitfall. The expression of ALK by IHC, recently discovered in AFH, was also assessed in this study. We also analyzed EWSR1 rearrangement by fluorescence in situ hybridization using a dual color break-apart probe to confirm the diagnosis. The results showed MUC4 expression in 22.2% of AFH cases (4/18 cases), demonstrating a variable intensity of cytoplasmic staining. Most notably, one of the positive cases showed strong and diffuse expression. ALK IHC expression was observed in 17 of 18 cases (94.4%), usually in a diffuse and strong cytoplasmic pattern. EWSR1 rearrangement was demonstrated by fluorescence in situ hybridization in 81.2% of cases (13 of 16), including all the MUC4-positive cases. Our results indicate that although the significance of MUC4 expression in AFH is unknown, it is important to be aware that a subset of AFH can express the protein by IHC, expanding a variety of MUC4-positive mesenchymal tumors.

Invasive micropapillary carcinoma of the breast overexpresses MUC4 and is associated with poor outcome to adjuvant trastuzumab in HER2-positive breast cancer.

BACKGROUND: Invasive micropapillary carcinoma of the breast (IMPC) is a histological tumor variant that occurs with low frequency characterized by an inside-out formation of tumor clusters with a pseudopapillary arrangement. IMPC is an aggressive tumor with poor clinical outcome. In addition, this histological subtype usually expresses human epidermal growth factor receptor 2 (HER2) which also correlates with a more aggressive tumor. In this work we studied the clinical significance of IMPC in HER2-positive breast cancer patients treated with adjuvant trastuzumab. We also analyzed mucin 4 (MUC4) expression as a novel biomarker to identify IMPC. METHODS: We retrospectively studied 86 HER2-positive breast cancer patients treated with trastuzumab and chemotherapy in the adjuvant setting. We explored the association of the IMPC component with clinicopathological parameters at diagnosis and its prognostic value. We compared MUC4 expression in IMPC with respect to other histological breast cancer subtypes by immunohistochemistry. RESULTS: IMPC, either as a pure entity or associated with invasive ductal carcinoma (IDC), was present in 18.6% of HER2-positive cases. It was positively correlated with estrogen receptor expression and tumor size and inversely correlated with patient's age. Disease-free survival was significantly lower in patients with IMPC (hazard ratio = 2.6; 95%, confidence interval 1.1-6.1, P = 0.0340). MUC4, a glycoprotein associated with metastasis, was strongly expressed in all IMPC cases tested. IMPC appeared as the histological breast cancer subtype with the highest MUC4 expression compared to IDC, lobular and mucinous carcinoma. CONCLUSION: In HER2-positive breast cancer, the presence of IMPC should be carefully examined. As it is often not informed, because it is relatively difficult to identify or altogether overlooked, we propose MUC4 expression as a useful biomarker to highlight IMPC presence. Patients with MUC4-positive tumors with IMPC component should be more frequently monitored and/or receive additional therapies.