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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: Recent guidelines regarding estrogen (ER) and progesterone (PR) receptor testing from the American Society of Clinical Oncology and College of American Pathologists defined a new reporting category of ER-low positive breast cancer for tumors with 1-10% ER expression by immunohistochemistry (IHC). The clinical implications of ER-low positivity are incompletely understood, especially in invasive lobular carcinoma (ILC), the second most common type of breast cancer. Given the rarity of low-ER positivity in ILC, we evaluated tumor features and outcomes associated with a spectrum of ER/PR positivity in a monoinstitutional ILC cohort. Methods: We analyzed cases of stage I-III ILC with available IHC reports. Based on prior published categories in ILC, we classified ER as low, medium, or high as defined by ER staining of 10–69%, 70–89%, and ≥90% respectively. PR negative, low, and high tumors were defined by 0%, < 20%, or ≥20% staining respectively. We used chi-squared tests, t-tests, and Cox proportional hazards models in Stata 16.1 to evaluate associations between ER/PR categories including clinicopathologic variables and event-free survival (EFS). Results: Of 744 cases, 24 (3.2%) were ER negative and 10 (1.3%) were ER-low positive as defined by 1-10% positive staining. 713 remaining cases had ER positivity ≥ 10% and comprised the cohort categories of ER low, medium, and high for this study (11.2%, 15.0%, and 73.8% respectively). In 751 cases with PR data, 122 (16.2%) were PR negative, 145 (19.4%) were PR low and 483 (64.3%) were PR high. ER high status was significantly associated with older age (mean 56.7, 56.7, and 60.6 years in ER low, medium, and high respectively, p=0.0005). ER low was associated with PR negative and low status (42.3% were PR neg/low and ER low, versus 37.4% with ER medium and 29.9% in ER high, p=0.045), and more likely to have overexpression of HER2 (9.7%, 9.0%, and 2.9% ER low, medium, high, respectively, p=0.002). ER low tumors were more likely to be grade 1 than ER medium or high (41.8%, 29.8% and 24.5% respectively, p=0.025), and have positive surgical margins (39.4%, 35.9% and 23.9% respectively, p=0.002). ER status was not associated with Ki67, stage, body mass index (BMI), lymphovascular invasion, lobular carcinoma in situ (LCIS), pleomorphic histology, local therapy, or chemotherapy use. In contrast, PR high was significantly associated with younger age (57.6 versus 63.5 years in PR low, p< 0.0001). PR low was associated with HER2 overexpression (8.6% versus 3.2% in PR high, p=0.002). PR low cases were more likely to present at higher stages (15.8% stage III versus 10.1% stage III in PR high, p=0.032), to be pleomorphic (16.8% versus 8.2%, p< 0.001), and to receive chemotherapy (30.8% versus 23.1%, p=0.022) but were less likely to have associated LCIS (64.0 versus 74.2%, p=0.004). PR status was not associated with Ki67, BMI, lymphovascular invasion, local therapy, or surgical margins. In a Cox proportional hazards model adjusting for age, stage, grade, pleomorphic histology, and chemotherapy use, ER category was not associated with EFS but both PR negative and PR low status each had significantly worse EFS compared to PR ≥20% (HR 3.5, 95% CI 1.8-6.7, p< 0.001 for PR negative, and HR 2.0, 95% CI 1.1-3.5, p=0.015 for PR low). The estimated cumulative 5-year EFS for patients with ER low, medium, and high tumors was 87.1%, 93.4%, and 90.1% respectively. The estimated cumulative 5-year EFS for patients with PR negative, low, and high tumors was 78.9%, 90.2%, and 92.7% respectively. Conclusions: Using ILC-specific categories for ER expression, we found associations between ER category and clinicopathologic variables but not with EFS. In contrast, PR negative and low status was associated with worse EFS. These findings highlight the importance of exploring the spectrum of ER/PR activity within ILC, a classically strongly hormone receptor-positive tumor type, using more quantitative methods. Citation Format: Elle Clelland, Harriet T. Rothschild, Anne Patterson, Julissa Molina-Vega, Mandeep Kaur, Mary Kathryn Abel, W. Fraser Symmans, Jo Chien, Christopher J. Schwartz, Rita Mukhtar. Quantifying estrogen and progesterone receptor status in early-stage invasive lobular carcinoma of the breast: associated factors and outcomes in an institutional series [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 P2-03-16.

Objectives: The concept of human epidermal growth factor receptor 2 (HER2)-low status has been proposed as a potential treatment target for breast cancers previously considered to be HER2-negative. Defined by an immunohistochemistry (IHC) score of 1+ or 2+ and negative fluorescent in situ hybridization (FISH) for HER2, HER2-low status predicts significant clinical benefit from novel therapeutic compounds in recent clinical trials. The prevalence and clinical implications of HER2-low status in patients with early stage invasive lobular carcinoma (ILC) has not been described. We aimed to describe the clinicopathologic features and prevalence of HER2-low status in ILC, and identify any potential differences in clinical outcome. Methods: We evaluated stage I-III ILC tumors from a prospectively maintained institutional database of patients where both IHC and FISH testing for HER2 status was performed as standard clinical care. Tumors were classified as HER2 negative (IHC=0), HER2-low (IHC=1+ or 2+ and negative FISH), or HER2 positive (IHC=3+ or FISH ratio ⇒2). Data were analyzed in Stata 16.1 using chi-squared tests, t-tests, and Cox proportional hazards models for disease free survival (DFS). Results: 666 ILC tumors with available HER2 status were available for analysis. The mean age at diagnosis was 59.8 years (range 21-91). The majority of patients had stage I disease (63.1%) with an average follow up time of 6.7 years (standard deviation [5.4]). Overall, 184 (27.6%) tumors were HER2 negative, 434 (65.1%) were HER2-low, and 48 (7.2%) were HER2 positive. There were no associations between HER2 status and age, menopausal status, body mass index, tumor stage, grade, presence of lymphovascular invasion, or molecular assay results. Hormone receptor status was significantly associated with HER2 status, with HER2 positive tumors significantly less likely to be estrogen receptor (ER) positive than both HER2 negative and HER2-low tumors (89.6% versus 97.3% and 96.8% respectively, p=0.03). HER2-low tumors were also significantly more likely to have progesterone receptor (PR) positivity (86.6% compared to 79.9% of HER2 negative and 72.9% of HER2 positive tumors, p = 0.01). This difference remained significant when comparing just HER2-low to HER2 negative cases (p=0.034). While there were no differences in use of neoadjuvant or adjuvant chemotherapy by HER2 status, HER2-low patients were significantly more likely to undergo mastectomy versus lumpectomy when compared to HER2 negative and HER2 positive patients (53.7% versus 38.0% and 43.8% respectively, p= 0.001). In a multivariable Cox proportional hazards model adjusting for tumor size, number of positive nodes, ER/PR status, and local therapy received, patients with HER2-low status had worse DFS than those with HER2 negative tumors (hazard ratio 2.0, 95% confidence interval 1.0 - 4.1, p=0.05). Conclusions: In this analysis of 666 early stage ILC cases, we found that most tumors were HER2-low, and that those with HER2-low disease were significantly more likely to have PR positive tumors and to undergo mastectomy. When adjusting for these variables, we identified a difference in DFS between HER2 negative and HER2-low early stage ILC. These findings support the contention that HER2-low and HER2 negative disease represent two different clinical entities. Further investigation of the potential benefit of HER2 targeted therapy in ILC, which predominately lacks HER2-amplified disease, is needed to ensure optimal outcomes in this understudied tumor type. Citation Format: Harriet T. Rothschild, Elle Clelland, Anne Patterson, Julissa Molina-Vega, Mandeep Kaur, Mary Kathryn Abel, W. Fraser Symmans, Christopher J. Schwartz, Rita Mukhtar. HER2-14 HER-2 low status in early stage invasive lobular carcinoma of the breast: associated factors and outcomes in an institutional series [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 HER2-14.

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.