Topical TLR7 agonist imiquimod can induce immune-mediated rejection of skin metastases in patients with breast cancer

Abstract

Purpose: Skin metastases of breast cancer remain a therapeutic challenge. Toll-like receptor 7 agonist imiquimod is an immune response modifier and can induce immune-mediated rejection of primary skin malignancies when topically applied. Here we tested the hypothesis that topical imiquimod stimulates local antitumor immunity and induces the regression of breast cancer skin metastases.

Experimental design: A prospective clinical trial was designed to evaluate the local tumor response rate of breast cancer skin metastases treated with topical imiquimod, applied 5 d/wk for 8 weeks. Safety and immunologic correlates were secondary objectives.

Results: Ten patients were enrolled and completed the study. Imiquimod treatment was well tolerated, with only grade 1 to 2 transient local and systemic side effects consistent with imiquimod's immunomodulatory effects. Two patients achieved a partial response [20%; 95% confidence interval (CI), 3%-56%]. Responders showed histologic tumor regression with evidence of an immune-mediated response, showed by changes in the tumor lymphocytic infiltrate and locally produced cytokines.

Conclusion: Topical imiquimod is a beneficial treatment modality for breast cancer metastatic to skin/chest wall and is well tolerated. Importantly, imiquimod can promote a proimmunogenic tumor microenvironment in breast cancer. Preclinical data generated by our group suggest superior results with a combination of imiquimod and ionizing radiation and we are currently testing in patients whether the combination can further improve antitumor immune and clinical responses.

Figure 1. In situ immune changes with imiquimod treatment in the two responders

A (responder 1): In situ TILs analysis by IHC shows minimal T cell infiltrate before treatment but a marked increase in CD8+ and CD4+ T cells infiltrating the tumor cell nests post-treatment and histological evidence of tumor regression after 8 weeks of topical imiquimod treatment (H&E stain and IHC for CD3, CD4, CD8 and FoxP3, 200×). Numbers in the boxes indicate the number of cells positive for the indicated marker in one HPF (average of 5 HPF, 400×). B: High power microphotographs showing lymphocytes, many positive for CD8, in close contact with cancer cells in the post-treatment biopsy. C (responder 2): In situ TILs analysis by IHC shows a moderate T cell infiltrate before imiquimod treatment. After an 8 week imiquimod treatment course, there is a reduction in CD8+T cells and FoxP3+ T cells while CD4+ T cells remain unchanged (H&E stain and IHC for CD3, CD4, CD8 and FoxP3, 200×). Numbers in the boxes indicate the number of cells positive for the indicated marker in one HPF (average of 5 HPF, 400×).

Figure 2. Changes in the intratumoral cytokine milieu after imiquimod treatment and plasma IL10 levels in all patients

A: Cytokine analysis of tumor supernatants before and after an 8-week imiquimod cycle is shown for all patients. Supernatants were obtained by 24 hour culture of the tumor samples in medium at a constant tissue mg/ml. Variability among patients is noticeable, as well as a marked increase in pro-inflammatory cytokines in responder 1 (red lines) and decrease of counter-regulatory cytokines in responder 2 (green lines). IFN-γ was only detectable in responder 1 after treatment; levels were below assay detection sensitivity for all other patients. IL-17 was not detectable in pre- and post-treatment supernatants of any patient. B: IL-10 levels in plasma are shown for all patients with detectable levels in only 4 of 10 patients.

Figure 3. Example of TILs profiles following ex vivo culture (from 3 patients)

A (patient with SD, post-treatment): Breast cancer biopsies and PBMCs (purified from the blood, drawn on the day of the tumor biopsy) were cultured in IL-2 containing media. Cells were collected at the indicated days of culture and subjected to immune phenotyping. FACS plots show the proportions of CD4+ and CD8+ T cells in PBMCs and TILs, the different subsets of CD4+ T cells based on the surface expression of CD45RO, CCR7 and CCR6 (left panel) and the intracellular cytokine profile of CD4+ and CD8+ T cells (right panel). B (responder 1, post-treatment): Phenotype of TILs. Cells were collected at the indicated days of culture in IL-2 and subjected to immune phenotyping. FACS plots show the percentages of CD4+ and CD8+ T cells, their expression of CCR7, CD45RO and FoxP3, as well as their intracellular cytokine profile. C (responder 2, pre-treatment): Phenotype of TILs. Cells were collected at the indicated days of culture in IL-2 and subjected to immune phenotyping. FACS plots show the percentages of CD4+ and CD8+ T cells, their expression of CCR7, CD45RO and FoxP3, as well as their intracellular cytokine profile.