Background. Ductal carcinoma in situ (DCIS) is a risk factor for the subsequent development of invasive breast cancer. Features of DCIS that are associated with a high risk of a subsequent event include large size (> 5 cm), high grade, comedo necrosis, palpable mass, hormone receptor negativity, and HER2 positivity. We have previously shown that immune infiltrates are positively associated with these high-risk features, suggesting that manipulating the immune microenvironment in high-risk DCIS, for example via checkpoint blockade, could potentially alter disease progression.

Methods. In this phase 1 pilot study we investigated changes in the immune microenvironment of high risk DCIS after intralesional injection of anti-PD-1 (pembrolizumab). Study participants received 2 intralesional injections of pembrolizumab, 3 weeks apart, with surgery approximately 3 weeks after the 2nd dose. The study started with a dose of 2 mg/injection (1/100 of the standard 200 mg iv dose), then escalated to 4 mg and 8 mg, with 3 patients at each dose. Tissue samples from pre-treatment biopsies and post-treatment surgical resections were stained with two 6-plex immune panels using Opal immunofluorescence reagents (Perkin Elmer) on a fully automated Ventana Discovery platform, imaged with a Vectra 3 system and analyzed with inForm software (Perkin Elmer). An algorithm for tumor/stroma segmentation developed in inForm was used to randomly select 10 high power fields (hpfs) for imaging. Cell phenotype maps were generated for each of these hpfs for each sample. Cell densities were determined per area of stroma, DCIS, or total tissue and averaged across all hpfs for a given case. Spatial analyses were performed to quantitate co-localization of immune cells with DCIS cells.

Results. The intralesional injections were easily administered and well tolerated. No systemic toxicities were observed at any dose. MRI imaging demonstrated no change in the size of lesions after treatment. Multiplex immunofluorescence analyses demonstrated heterogeneous responses ranging from dramatic increases in T cells, in particular CD8+ T cells, in cases which had a T cell infiltrate prior to therapy, to no post-therapy T cell infiltrate in cases with a pre-therapy immune desert. We also observed increases in B cells and macrophages and a decrease in the ratio of FoxP3+ T cells to CD8+ T cells, the latter mainly due to a significant increase in CD8+ cells, as opposed to a decrease in FoxP3+ cells. Spatial analyses indicated that in some cases, despite a marked increase in T cells post therapy, these cells did not co-localize with DCIS cells, indicating a state of immune exclusion.

Conclusions. We have demonstrated the safety and feasibility of intralesional injection of an immune checkpoint inhibitor (pembrolizumab) in high risk DCIS. In some patients we observed a dramatic change in the immune microenvironment, with an increase in T cells, B cells, and macrophages, and a decrease in the FoxP3:CD8 ratio, even at a dose that is 1/100 of the standard intravenous clinical dose. An expansion study is underway in which patients will receive 4 injections of pembrolizumab at 3 week intervals prior to going to surgery to determine if more injections/time will increase response rate.

Citation Format: Campbell MJ, McCune E, Rothschild H, Bolen J, VandenBerg S, Chien J, Wong J, Esserman L. Modulation of the immune microenvironment in high risk DCIS by intralesional injection of anti-PD-1 (pembrolizumab) [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 P2-09-02.