The cyclotrimerization of commercial, aromatic diisocyanates allows for the formation of monolithic, microporous polymer networks with S BET surface areas up to 1300–1500 m2/g. The process has been up-scaled for production of 100 g batches. The monolithic materials show a promising potential for the removal of lipophilic components from aqueous mixtures.

Background: High-grade DCIS with immune infiltrates may represent lesions that are able to be kept in check by the immune system, but still are at risk for progression to invasive disease. We and others have demonstrated that the presence of T cells, and in particular the spatial proximity of CD8+/PD-1+ T cells and PD-L1+ cells predicts response to chemotherapy and PD-1 inhibitors in the setting of invasive triple negative breast cancer. DCIS with high-risk features (e.g. large, palpable, high grade, HR-, or HER2+) often have T cell infiltrates. We hypothesized that it might be possible to potentiate the immune response in high-risk DCIS with immune checkpoint blockade. Since mortality for DCIS is extremely low, we proposed an intralesional pembrolizumab (pembro) treatment approach to avoid systemic adverse effects. In a phase 1 dose escalation study of single agent pembro we found 2 doses of 8 mg, administered intralesionally 2-3 wks apart, was tolerable, induced immunological changes within the DCIS lesions, but had little clinical benefit. Herein, we expanded the number of injections to 4 and also tested a combination with mRNA-2752 (Moderna), an mRNA-based therapeutic encoding a T cell co-stimulator OX40L, and pro-inflammatory cytokines IL-23 and IL-36 gamma, to determine if we could find a dose that was both tolerable and elicited anti-tumor responses. Methods Women eligible for this study had DCIS with at least 2 of the following high-risk features: age< 45; high grade, extensive comedonecrosis; palpable mass; hormone receptor negative [HR-]; HER2+; size >5 cm, or microinvasion. A dose expansion cohort was performed using 4 doses of 8 mg pembro, 2-3 wks apart (n=5). We then combined pembro with mRNA-2752 (n=8 cases), initially at 8 and 4 mg, respectively. Dose reductions were implemented based on AEs. Patients received an MRI before and after 2 injections, spaced 2-3 wks apart. A total of 4 injections were allowed. Core biopsy or surgery was conducted after the last MRI. Results We observed an increase in T cell density in the dose expansion cohort (pembro only), except when there was a paucity of T cells in the pre-treatment biopsy. However, only 1 patient in this cohort demonstrated a reduction in lesion size (clinically and on MRI). As of 22SEP22, 8 patients have received the combination of pembro and mRNA-2752; 7 are evaluable. Two patients with minimal T cell infiltrates at baseline (both HR+) failed to respond, based on imaging or pathology. 4/5 patients with moderate to high T cell infiltrates at baseline had partial (2) or complete responses (2) based on imaging (surgery pending), one of whom had absence of DCIS on post-therapy core biopsy and a clear MRI 4 months later. Correlative studies (immune cell densities, spatial proximities) will be presented at the meeting along with complete data (including post-treatment pathology) on the first 8 patients. Side effects were independent of response and included Gr1/2 fever, myalgias, and fatigue starting within 12 hours of injection (3-4 days), enlargement of regional nodes by day 2, and Gr 1/2 erythema and induration of the breast starting at 4 days and lasting 4-20 days. Earlier onset and persistence of symptoms with subsequent exposure required dose reductions for the majority of patients to maintain tolerability (avoid fevers over 39.4°C, intense erythema and swelling of the breast lasting > 4 days). No AEs were higher than grade 2. Conclusion: The combination of intralesional pembro and mRNA-2752 demonstrated modulation of the tumor immune microenvironment and robust antitumor activity in high-risk DCIS (typically HR- or HER2+) with existing T cell infiltrates. This is an ongoing study; the optimal Phase 2 dose for the combination will be based on the totality of evolving safety and translational data. Citation Format: Laura J. Esserman, Alexa Glencer, Kirithiga Ramalingam, Christopher J. Schwartz, Alexander Borowsky, Gillian L. Hirst, Rachel Woody, Nicole Schindler, Nola M. Hylton, Michael Campbell. Intralesional injection of anti-PD-1 (pembrolizumab) and OX40L/IL-23/IL-36 mRNAs (mRNA-2752) results in regression of DCIS characterized by lymphocytic infiltrates [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-20-02.

Background: Treatment of estrogen receptor (ER)-positive breast cancer with selective estrogen receptor degraders (SERDs) frequently results in the loss or reduction of ER expression. Whether these changes are due to on-target effects of SERDs degrading ER or arise as a mechanism of tumor resistance with associated changes in cellular phenotypes remains unknown. It is critical to distinguish between these possibilities to accurately assess treatment response and determine the most appropriate subsequent therapy. To this end, we created and conducted molecular analyses on patient-derived organoid cultures from post-treatment tissue in patients receiving neoadjuvant SERD therapy for early-stage ER+ breast cancer in the I-SPY2 Endocrine Optimization Protocol (EOP). Methods: The I-SPY2 EOP study is a prospective, randomized substudy within the I-SPY TRIAL testing the oral SERD amcenestrant alone or in combination with letrozole or abemaciclib in stage 2/3 ER+ Her2-negative breast cancer. Enrollment is ongoing, with patients receiving amcenestrant neoadjuvantly for 6 months until the day before surgery. Tumor tissue is collected at baseline, 3 weeks, and at surgery. Organoids were generated from post-treatment surgical samples. Organoid cultures were optimized based on established methods (Dekkers et al., Nature Protocols, 2021) to assess ER levels and activity. Pre- and post-treatment tissue samples were also assessed for ER, PR, Ki67, and GATA3, a luminal marker and transcription factor that is functionally linked with ER, via immunohistochemistry. Results: In 7 patients with both pre- and post-treatment tissue samples including fresh surgical samples for organoid generation, the ER in baseline tumor tissue was >=90% in all patients, PR ranged from 40-90%, and Ki67 ranged from 5-30%. In post-treatment surgical tissue from these cases, ER ranged from 0-30%, PR from 0-50%, Ki67 from < 1%-10%, and GATA3 was positive in 5 of 5 cases tested to-date. The creation of organoids from residual disease at surgery was attempted for these 7 patients, with organoids successfully propagated in 5 cases thus far. 3 of 5 organoid cultures were ready for analysis and in all cases strong ER and PR expression in organoids was observed after culture for > 1 month in the absence of amcenestrant. Detailed gene expression profiling (including Mammaprint/Blueprint) and gene set enrichment analyses (GSEA) to assess for intrinsic breast cancer subtype and ER activity in each sample and corresponding organoid culture are in progress and will be reported with the full dataset. Conclusion: Patient-derived organoid culturing of residual disease after neoadjuvant endocrine therapy is feasible. Neoadjuvant treatment with a SERD can render ER and PR low or absent at the time of surgical resection, which does not necessarily imply the presence of endocrine therapy resistant disease. The use of organoids and additional IHC markers (GATA3) demonstrate that receptor negativity may be an indicator of the drug hitting its target, suggesting ER signaling is still intact. In general, patient-derived tumor organoid cultures modeling residual disease states can be a useful adjunct to existing methods used to monitor the effects of neoadjuvant endocrine therapy and is being explored in the I-SPY EOP trial. Citation Format: Jennifer Rosenbluth, Christopher J. Schwartz, Tam Binh Bui, Shruti Warhadpande, Pravin Phadatare, Sigal Eini, Michael Bruck, Julissa Molina-Vega, Kami Pullakhandam, Nicole Schindler, Lamorna A. Brown Swigart, Christina Yau, Gillian Hirst, Rita Mukhtar, Karthik V. Giridhar, Olufunmilayo I. Olopade, Kevin Kalinsky, Cheryl A. Ewing, Jasmine M. Wong, Michael D. Alvarado, Laura Van’t Veer, Laura J. Esserman, Jo Chien. Characterization of residual disease after neoadjuvant selective estrogen receptor degrader (SERD) therapy using tumor organoids in the I-SPY Endocrine Optimization Protocol (EOP) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-09-01.

Background: Ductal carcinoma in situ (DCIS) is a risk factor for the development of invasive ductal carcinoma. Previous work has shown that an increased presence of T cells corresponds to high-risk DCIS features: age <45 years, size >5 cm, high-grade, palpable mass, hormone receptor negativity, and HER2 positivity. The presence of T cells colocalized with tumor is emerging as a predictor of response to checkpoint inhibition in invasive cancer. We previously have demonstrated that intratumoral injections of a checkpoint inhibitor (pembrolizumab) in DCIS resulted in an increased number of T cells, but without tumor cell killing. We hypothesized that the addition of an agent that could potentiate both innate and adaptive immune responses might increase the efficacy of checkpoint inhibition. Methods/Materials: We administered intralesional injections of immunotherapy (4-8 mg pembrolizumab + 2-4mg mRNA 2752) to patients with “high-risk” DCIS in an attempt to modulate the immune system and protect from disease progression and recurrence. Two patients have been enrolled and completed therapy and 4 more are being treated. Immune infiltrates were characterized by multiplex immunofluorescence (mIF) staining of pre- and post-treatment tissue samples using two 6-plex panels. Staining was performed on a Leica Bond autostainer and images were acquired on a Vectra Polaris imaging system. Image analysis was performed utilizing inForm and QuPath software packages, as well as R scripts. Results: The first patient treated, a 41-year-old woman with ER+/PR+, HER2- disease, with low T cells in her pre-treatment biopsy, received one dose of pembrolizumab (8mg) with mRNA 2752 (4mg). While she experienced fatigue, sustained fever, and muscle aches for 3-5 days, draining node enlargement and redness and swelling at the breast a week after treatment, there was no change on her post-treatment MRI, and no significant change in the number of T cells in her post-treatment tissue. The second patient, a 74-year-old woman with ER-/PR-, HER2+ disease had high numbers of T cells in the pre-treatment biopsy. She received one injection of pembrolizumab (4mg) with mRNA 2752 (2mg), followed 3 weeks later by an injection of pembrolizumab (4mg) alone. She declined the second dose of mRNA 2752 due to side effects from the first injection. Her post-treatment MRI showed some reduction in tumor volume and her post-treatment biopsy showed no evidence of DCIS, as well as an increase in T cells. Conclusions: The presence of T cells prior to therapy is likely to be important for eliciting a strong anti-tumor response. The addition of an agent that enhances both innate and adaptive immune responses may increase the efficacy of immune checkpoint blockade. Four additional patients will have completed their therapy by the end of August and we await results. It will be important to understand if pre-existing T cells within the DCIS lesion are a predictor for response to therapy as we continue to screen and enroll patients. Citation Format: Nicole F. Schindler, Alexa Glencer, Phoebe Miller, Christopher Schwartz, Gillian Hirst, Alexander Borowsky, Michael Campbell, Laura Esserman. Intralesional injections with checkpoint inhibitors and cytokines can modify DCIS lesions with infiltrating T cells [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B005.

Purpose: The immune microenvironment in ductal carcinoma in situ (DCIS) and its significance are not well established. In this study, we characterized the immune microenvironment of DCIS to determine if immune cell densities, or their spatial relationships with tumor cells, are predictive of recurrence with invasive breast cancer. Methods: One hundred thirty-two cases of DCIS were included in this study. Seventy (53%) cases were high grade, 72 (54%) were HER2+, and 91 (69%) were HR+. Twenty cases recurred with invasive breast cancer. Immune infiltrates were characterized by multiplex immunofluorescence staining of FFPE sections using two 6-plex panels. Panel IP1.2 included markers for CD3, CD20, Foxp3, pan-cytokeratins, Ki67, and HLADR. Panel IP2.2 included markers for CD3, CD8, CD68, pan-cytokeratins, PD-1, and PD-L1. Staining was performed on a Leica Bond autostainer and images were acquired on a Vectra Polaris imaging system. Image analysis was performed utilizing inForm and QuPath software packages, as well as R scripts. Counts for each cell population were determined and spatial point pattern analyses were performed to quantitate the spatial relationships between tumor cells and various immune cell types. Results: High infiltrates of T cells, CD8+ T cells (Tc), Foxp3+ T cells, and B cells were associated with high grade, hormone receptor (HR) negativity, HER2 positivity, and the presence of comedonecrosis. PD-1+ T cells and PD-1+ Tc were associated with high grade, HR negativity, and HER2 positivity. PD-L1+ immune cells, but not PD-L1+ tumor cells, were also associated with high grade, HR negativity, and HER2 positivity. None of the cell populations were associated with invasive recurrence. However, the spatial proximity of tumor cells with T cells, in particular Tc, was significantly associated with a subsequent invasive event. Patients whose DCIS had a high tumor-to-Tc proximity score (indicating close proximity of tumor and Tc) were less likely to have an invasive recurrence. Conclusion: These findings suggest that the tumor immune microenvironment is an important factor in identifying DCIS cases at risk for invasive recurrence and that manipulating the immune microenvironment may be an efficacious strategy to alter or prevent disease progression. Citation Format: Michael J. Campbell, Nicole Schindler, Alexa Glencer, Jennifer Bolen, Hidetoshi Mori, Tristan Loveday, Harriet Rothschild, Gillian Hirst, Scott Vandenberg, Alexander Borowsky, Laura Esserman. Spatial proximity between CD8+ T cells and tumor cells correlates with invasive recurrence in DCIS [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR001.