Tumor Expression Research Articles

Androgen receptor: Structure, signaling, function and potential drug discovery biomarker in different breast cancer subtypes.

The Androgen Receptor (AR) is emerging as an important factor in the pathogenesis of breast cancer (BC), which is the most common malignancy worldwide. >70 % of AR expression in primary and metastatic breast tumors has been observed which suggests that AR may be a new marker and a potential therapeutic target among AR-positive BC patients. Biological insight into AR-positive breast cancer reveals that AR may cross-talk with several vital signaling pathways, including key molecules and receptors. Downstream signaling of AR might also affect many clinically important pathways that are emerging as clinical targets in BC. AR exhibits different behaviors depending on the breast cancer molecular subtype. Preliminary clinical research using AR-targeted drugs, which have already been FDA-approved for prostate cancer (PC), has given promising results for AR-positive breast cancer patients. However, since AR positivity's prognostic and predictive value remains uncertain, it is difficult to identify and stratify patients who would benefit from AR-targeted therapies alone. Thus, the need of the hour is to target the androgen receptor as a monotherapy or in combination with other conventional therapies which has proven to be an effective clinical strategy for the treatment of prostate cancer patients, and these therapeutic strategies are increasingly being investigated in breast cancer. Therefore, in this manuscript, we review the role of AR in various cellular processes that promote tumorigenesis and aggressiveness, in different subtypes of breast cancer, as well as discuss ongoing efforts to target AR for the more effective treatment and prevention of breast cancer.

CD3 and CD20 Expressions and Infiltrating Patterns in Salivary Gland Tumors

Tumor-infiltrating lymphocytes (TILs) represent a subset of immunological constituents within the tumor microenvironment that can influence cancer growth. We retrospectively evaluate the density and pattern of CD3 and CD20 expression in salivary gland tumors and their relation to clinical pathologic parameters. A total of 44 formalin-fixed paraffin-embedded blocks of salivary gland tumors were included. These tumors were stained immunohistochemically with CD3 and CD20. The chi-square test was used to relate immune scoring, intensity, and clinical pathological parameters to different salivary tumors. p-value < 0.05 was considered statistically significant. The intra-tumoral CD3 infiltrating count was high and diffused in (71.4%) of pleomorphic adenomas (PAs) followed by mucoepidermoid carcinomas (MECs) (66.7%). At the same time, adenoid cystic carcinomas (AdCCs) exhibited significantly low infiltration (71.4%) (p = 0.046). The three types of tumors exhibited high tumor-infiltrating counts diffused in peripheral areas with significant differences between malignant tumors (p = 0.047). The intra-tumoral CD20 infiltrating count significantly differed among the tumors (p = 0.002); it was low in all PAs and AdCCs, while MECs showed an equal percentage of expression. However, in the peripheral area, PAs and MECs exhibited significantly (p = 0.007) high infiltrating counts (69.2% and 84.6), and the lowest infiltrating count was predominantly found for AdCCs. The two markers had a significant positive correlation between the mean of CD3 in the intra-tumoral and peripheral regions and CD20 in the peripheral zone across the total samples. In conclusion, the density of CD3 expression is notably higher than CD20 across tumor types. PAs and MECs showed high-density scores, while AdCCs were characterized by low scores. TIL expression was found to be significantly associated with patients’ outcomes in the intra-tumoral area.

Abstract PO4-03-05: Prognostic value of Ki67 expression in post-treatment tumors of patients with residual triple-negative breast cancer after neoadjuvant chemotherapy

Abstract Introduction Patients with triple-negative breast cancer (TNBC) who do not achieve pathological complete response (pCR) against neoadjuvant chemotherapy (NAC) have a poor prognosis. The prognosis of these patients can be differentiated using various parameters, including the Residual Cancer Burden (RCB) index and tumor-infiltrating lymphocytes. In this study, we took a different angle and aimed to evaluate the prognostic value of post-treatment Ki67 expression, which was examined in tumors following NAC in patients with non-pCR TNBC. Methods A retrospective review of clinical records from February 2008 to December 2021 was conducted of patients from Gangnam Severance Hospital and Asan Medical Center, both in Seoul, South Korea. We included patients with non-metastatic TNBC who underwent NAC including anthracycline and taxane regimens, followed by curative surgery, regardless of adjuvant capecitabine receipt. Ki67 expression and the RCB index were evaluated using residual tumors obtained from surgery. High Ki67 expression was defined as ≥20%. The 5-year recurrence-free survival (RFS) was estimated using Kaplan-Meier analysis, and hazard ratios (HR) were calculated using Cox regression analysis. Results This study included 338 patients with available Ki67 expression data in residual tumors. Among them, 110 patients (30.9%) had low Ki67 expression, while 228 patients (64%) had high Ki67 expression. At a median follow-up of 34 months, those with low Ki67 expression had a 5-year RFS rate of 82.6%, while those with high Ki67 expression had a rate of 58.3%, with a HR of 0.345 (95% CI 0.210-0.565, p< 0.001). When stratified by the RCB index, the 5-year RFS of RCB-I patients was 93%, RCB-II patients 70.2%, and RCB-III patients 40.5%. When assessing survival outcomes based on both Ki67 expression and the RCB index, patients with RCB-I and low Ki67 (n=26) had a 100% 5-year RFS rate. Patients with RCB-I and high Ki67 (n=24) had a rate of 83.3%. Notably, in patients with RCB-III and high Ki67 (n=62), the 5-year RFS rate was 35.5% and there was no significant difference in RFS based on the receipt of adjuvant capecitabine (median RFS: capecitabine - 18 months, observation - 15 months, p-value=0.994). Conclusions Our findings demonstrate the prognostic value of Ki67 expression in residual TNBC after NAC, indicating that cases with low Ki67 expression have a favorable outcome. Combining Ki67 expression with the RCB index allows identification of patients with good residual disease (i.e. low Ki67 expression and RCB-I) who could be potential candidates for de-escalating adjuvant treatments such as capecitabine or pembrolizumab. However, for patients with high Ki67 expression and RCB-III, novel strategies are required to improve survival outcomes beyond the use of capecitabine. Citation Format: Yoonwon Kook, Soo-Young Lee, Seung Ho Baek, Min Ji Kim, Junghyun Kim, Sohyun Moon, Seung Eun Lee, Soong June Bae, Joon Jeong, Sae Byul Lee, Sung Gwe Ahn. Prognostic value of Ki67 expression in post-treatment tumors of patients with residual triple-negative breast cancer after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-03-05.

Abstract PO2-24-06: Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1

Abstract Background: Inflammatory breast cancer (IBC) and triple negative breast cancer (TNBC) have the lowest 5-year survival rates among breast cancers: 40% for IBC and 77% for TNBC. Effective treatments for these aggressive tumors are urgently needed. To develop better treatments, it is crucial to understand the underlying mechanisms driving their aggressive biology. Our recent research has identified N-myc downstream regulated gene 1 (NDRG1) as a crucial driver of tumor progression and metastasis in aggressive breast cancers. We have also found that NDRG1 expression in tumors is correlated with poorer outcomes in IBC patients. Additionally, we have observed that cancer-associated fibroblasts (CAFs), derived from breast cancer biopsies, stimulate NDRG1 expression and phosphorylation as well as tumor stemness. Based on these findings, we hypothesize that NDRG1 is a key regulator of tumor stemness and progression, and secreted stromal factors regulate tumor stemness through NDRG1. Methods: To assess tumor stemness, we conducted in vitro experiments by quantifying the CD44+/CD24- subpopulation and performing mammosphere assays in NDRG1 control and depleted cells (SUM149, BCX010, MDA-IBC3). In vivo, we employed a limiting dilution transplantation assay, where NDRG1 control and knockdown SUM149 cells were transplanted into the mammary fat pad at different dilutions. We treated NDRG1 control and knockdown breast cancer cells with different patient-derived CAF-conditioned media (CAFs-CM, 50%, 24h) or control medium. The expression of NDRG1/pNDRG1 and CD44+/CD24- cells was evaluated by immunoblotting and flow cytometry. qPCR array was performed to evaluate tumor stemness markers. Results: NDRG1 depletion significantly reduced the CD44+/CD24- subpopulation (p< 0.001) and the efficiency of mammosphere formation (p=0.001). In vivo limiting dilution experiments demonstrated a substantial reduction in the frequency of breast cancer stem cells in NDRG1 knockdown cells (p= 1 x 10-12). Treatment of IBC cells with CAF-CM derived from breast cancer patients stimulated the cancer stem cell subpopulation [CD44+/CD24-: Control medium (43.1 ± 0.1) vs CAF-CM (55.1 ± 0.7); p=0.004] and increased NDRG1 and phospho-NDRG1 expression. However, the stimulation of NDRG1 knockdown cells with CAF-CM did not significantly alter the cancer stem cell population (0.2 ± 0.2 in Control medium versus 0.4 ± 0.3 in CAF-CM) and the expression of NDRG1/phospho-NDRG1. Additionally, qPCR array analysis revealed the upregulation of several cancer stem cell and self-renewal markers in CAF-CM stimulated cells compared to control-medium treated cells. Conclusions: Our data strongly support the role of NDRG1 as a critical regulator of tumor stemness. Moreover, we have discovered that conditioned media from patient-derived CAFs induces tumor stemness in breast cancer cells, and this effect is dependent on the expression NDRG1. Citation Format: Emilly Villodre, Jenny M Hogstrom, Xiaoding Hu, Nina Kozlova, Debu Tripathy, Taru Muranen, Bisrat Debeb. Cancer-Associated Fibroblasts Promote Tumor Stemness in Aggressive Breast Cancers via NDRG1 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-06.

Abstract PS08-06: A Phase Ib/II Study to Assess the Safety and Efficacy of PM8002 (Anti-PD-L1 x VEGF-A Bispecific Antibody) in Combination with Nab-Paclitaxel for First Line Treatment of Locally Advanced or Metastatic Triple-Negative Breast Cancer

Abstract Background: PD-L1 and VEGF play important roles in immune evasion and pro-tumor angiogenesis promoting cancer growth and metastasis. PM8002 is a bispecific antibody targeting PD-L1 and VEGF-A for treating cancer. Here, we present results from a Phase Ib/II study of PM8002 in combination with nab-paclitaxel in patients with locally-advanced or metastatic triple-negative breast cancer (TNBC). Methods: 42 patients with locally-advanced or metastatic TNBC (without prior systemic treatment) were enrolled in this Phase Ib/II study to test the safety and efficacy of PM8002 in combination with nab-paclitaxel. The first 6 patients were enrolled during Phase Ib with safety as the primary endpoint, and the remaining patients were enrolled during Phase II with objective response rate (ORR) as the primary endpoint. Each treatment cycle lasts 28 days. All patients received PM8002 at 20 mg/kg (Q2W) and nab-paclitaxel at 100 mg/m2 on the 1st, 8th, and 15th days of each cycle until unacceptable toxicity or disease progression were observed. Tumor responses were evaluated every 8 weeks according to RECIST 1.1. Safety was evaluated according to CTCAE 5.0. Nab-paclitaxel dose was reduced according to toxicity, while PM8002 dose was kept consistent according to body weight. PD-L1 expression in tumors was tested, and subgroup analysis of ORR data stratified by PD-L1 expression was also included. Results: As of June 30, 2023, 42 patients were treated and evaluated at least once for treatment efficacy. The median duration of drug exposure was 4.6 months (min, max: 2.0, 7.6). The best overall ORR was 76.2% (32/42), including 1 complete response (CR) and 31 partial responses (PRs) with 29 objective responses occurring at the patient's first evaluation. The confirmed ORR was 57.2% (24/42) and the overall disease control rate (DCR) was 95.2% (40/42). The median time to response (TTR) was 1.9 months (95% CI:1.8~2.0) and the median best percentage change from baseline for target lesions was -47.2% (Q1, Q3: -56.9%, -33.5%). Among 13 patients with PD-L1 combined positive scores (CPS) < 1, the best ORR and DCR were 69.2% (9/13) and 100.0% (13/13), respectively. Among 25 patients with PD-L1 CPS ≥1, the best ORR and DCR were 80.0% (20/25) and 96.0% (24/25), respectively. Among 9 patients with PD-L1 CPS ≥10, the best ORR and DCR were both 100.0% (9/9). At the cut-off date of data analysis, 38 patients were still on treatment with a median progression-free survival (mPFS) of 7.4 months (95% CI: 7.4~NA); 38 patients were censored due to no disease progression or death. The incidence of treatment-related adverse events (TRAEs) was 95.2%, of which 26.2% were Grade 3 or 4; no Grade 5 TRAEs were observed. The incidence of drug-related TRAEs related to PM8002 was 92.9%, of which 23.8% were Grade 3 or 4. The incidence of drug-related TRAEs related to nab-paclitaxel was 95.2%, of which 26.2% were Grade 3 or 4. 1 patient (2.4%) reduced nab-paclitaxel dose due to diarrhea. 1 patient (2.4%) discontinued PM8002 and nab-paclitaxel treatment due to diarrhea. The most common TRAEs related to PM8002 and nab-paclitaxel included neutropenia (69.0%), leukocytopenia (59.5%), anemia (52.4%) and proteinuria (26.2%). The incidence of immune-related adverse events (irAEs) was 11.9%, which included hyperthyroidism, hypothyroidism, and rash. No ≥ Grade 3 irAEs were observed. AEs related to targeting VEGF were also analyzed, including hypertension 19.0% (4.8% at Grade 1, 11.9% at Grade 2, and 2.4% at Grade 3) and proteinuria 26.2% (9.5% at Grade 1, and 16.7% at Grade 2). Conclusions: Rapid, deep, and durable tumor responses were observed for the combination of PM8002 and nab-paclitaxel in patients with first line TNBC. PM8002 did not enhance toxicity that is typically observed for nab-paclitaxel. This Phase II study is still ongoing with plans for entering late-stage clinical development for solid tumors. Citation Format: Jiong Wu, Jian Zhang, Zhongsheng Tong, Qingyuan Zhang, Yong-Sheng Wang, Qiao Cheng, Xin Chen, Zhihua Li, Yongmei Yin. A Phase Ib/II Study to Assess the Safety and Efficacy of PM8002 (Anti-PD-L1 x VEGF-A Bispecific Antibody) in Combination with Nab-Paclitaxel for First Line Treatment of Locally Advanced or Metastatic Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS08-06.

Abstract PO1-18-04: Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968)

Abstract Background: Higher levels of stromal tumor-infiltrating lymphocytes (sTILs) in breast cancers are associated with increased likelihood of pathologic complete response (pCR) to chemotherapy and improved outcomes. DNA methyltransferase inhibitors (DNMTi) can augment immune responses to cancers by upregulating tumor antigen and MHC expression, decreasing numbers and activity of myeloid derived suppressor cells (MDSC), and increasing responsiveness of T lymphocytes. We have shown that the DNMTi decitabine augments the effectiveness of immunotherapy against murine triple negative breast cancer (TNBC) using murine 4T1 and E0771 mammary carcinoma models. Methods: In a single-arm phase 2 study, patients with HER2-negative breast cancer who were candidates for neoadjuvant chemotherapy (NCT) received decitabine (15 mg/m2 × 4 doses over 5 days) followed by 2 doses of pembrolizumab (pembro) (200 mg, 2 weeks apart) – collectively, window immunotherapy – prior to starting NCT. Two research biopsies were obtained: 1 prior to the window immunotherapy and 1 afterwards, prior to starting NCT. Biopsies were analyzed using established procedures to quantify sTILs and PD-L1 expression. Patients proceeded to NCT and tumor resection per standard of care and were followed up for immune-related adverse events (irAEs) and response. The primary endpoint was change in sTILs. Key secondary endpoints were occurrence of irAEs and pCR following neoadjuvant treatment. After the study was opened, reported results of a phase 3 trial (KEYNOTE 522) led to allowing patients with TNBC to receive additional pembro (200 mg q3w) concurrently with standard NCT and adjuvantly following resection (Cohort A2). Results: 46 patients (median age 54.5 yrs, range 28-72; 71.7% White, 28.3% Black; 100% female) were enrolled and treated on study. 21 patients had TNBC and did not receive neoadjuvant pembro concurrently with NCT nor adjuvant pembro (Cohort A), 7 patients had TNBC and did receive neoadjuvant and/or adjuvant pembro (Cohort A2), and 18 patients were ER+ or PR+ and received neither concurrent nor adjuvant pembro (Cohort B). Blood samples collected after decitabine administration before the first pembro dose showed a 59% decrease (P< 0.01) in monocytic MDSCs compared to baseline; decrease in granulocytic MDSCs was not statistically significant. 37 patients had paired biopsies adequate for sTIL evaluation with a mean change from 23.4% to 30.3% (absolute change 6.9%, P< 0.001). Cohorts A/A2 experienced an absolute sTIL increase of 7.4% (P< 0.01); Cohort B experienced absolute sTIL increase of 6.1% (P=0.01). PD-L1 expression in tumors (H-score using MoAb 22C3 clone) increased by 43% (P< 0.01) across all cohorts. 16 of the 39 patients (41.0%) who proceeded to resection achieved pCR (n=12 of 27 [44.4%] in Cohorts A/A2 and n=4 of 13 [30.8%] in Cohort B). The most frequently reported irAEs were adrenal insufficiency (n=5, 10.9%), maculopapular rash (n=3, 6.5%), and hypothyroidism (n=3, 6.5%). Conclusions: Treatment in the pre-neoadjuvant window with decitabine and pembro could potentially sensitize breast cancers to standard NCT by recruitment of TILs to the tumor tissue. The treatment was well -tolerated at the tested doses. Funding: financial support and drug (pembrolizumab) were provided by Merck Citation Format: Harry Bear, Xiaoyan Deng, Dipankar Bandyopadhyay, Michael Idowu, Maciej Kmieciak, Monique Williams, Giovanni Archer, Lindsey Gwaltney, Patrick Dillon, Daniel Flora, Daniel Stover, Andrew Poklepovic, Mary Hackney, Masey Ross, Hetal Vachhani, Raphael Louie, Kandace McGuire, Amelia Grover, Tasnim Rahman, Amber Hendrix. Neoadjuvant pembrolizumab + decitabine followed by standard neoadjuvant chemotherapy for locally advanced HER2- breast cancer (NCT02957968) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-18-04.

Abstract PO1-18-07: Phase I trial of combination pembrolizumab and ruxolitinib in metastatic triple negative breast cancer

Abstract BACKGROUND: The amplification of genes PDL-1, PDL-2, and JAK2 on chromosome 9p24, known as the PDJ amplicon, has been identified in 15% of triple negative breast cancer (TNBC) and is associated with tumor infiltrating lymphocytes and elevated JAK2 mRNA, suggesting a coordinated inflammatory response in TNBC. Adding pembrolizumab to chemotherapy in KEYNOTE trials 355 and 522 has validated the use of PD-1 blockade in TNBC and redefined the standard of care. Despite preclinical data suggesting a role for targeting the IL- 6/JAK2/STAT3 pathway, the JAK1/JAK2 inhibitor ruxolitinib has not shown efficacy as a single Agent in breast cancer. This is the first phase I clinical trial evaluating the combination of pembrolizumab and ruxolitinib in the treatment of metastatic TNBC. METHODS: We conducted a phase I clinical trial with accrual from 2018 to 2023 at Mayo Clinic Arizona. Patients (n=12) received 200 mg of IV pembrolizumab per cycle (every 3 weeks) with 3+3 dose escalation of oral ruxolitinib twice a day (dose level 0: 10 mg PO BID, level 1: 15 mg PO BID, level 2: 20 mg PO BID). The primary endpoint was to determine the maximum tolerated dose (MTD) of ruxolitinib and secondary endpoints were treatment safety and efficacy. Clinical response was assessed by computed tomography of the chest, abdomen, and pelvis every 3 cycles. PD-L1 expression in tumor was measured by immunohistochemistry (IHC). The effect of the combined therapies on T and B cell populations were assessed by flow cytometry and antibody profiling of 695 tumor antigens and 8 viral antigens by nucleic acid protein programmable array. RESULTS: Twelve female patients with chemotherapy refractory metastatic TNBC were treated on study. All received 200 mg IV pembrolizumab every cycle. Due to rapid disease progression of three patients early after treatment initiation, eligibility was restricted to prohibit patients with extensive pulmonary disease or elevated LDH. Those patients were excluded from efficacy or correlative analysis. The most common toxicities were transaminitis (n=12), anemia/leukopenia (n=12), and fatigue (n=9). The majority of adverse events were grade 1 and occurred at dose level 2. There were 5 adverse events grade 3 or higher: respiratory or thoracic complications (n=3), hypertension (n=1), and abdominal pain (n=1), but MTD was not established. The median number of cycles was 4 for each dose level (range n=2.0-9.0) and the median time to progression was 3 months (range n=1.4-6.3). No patient had tumor response by RECIST v1.1 criteria and there was no association of disease stability with PD-L1 tumor expression by IHC. Circulating antibodies to tumor antigens remained stable, and viral-specific antibodies mildly decreased by month 2 in all patients at dose levels 0 and 1. Of the 7 patients reactive to TRIM21 autoantibodies, 2 patients had a transient increase at 2 months. CONCLUSIONS: The combination of pembrolizumab with ruxolitinib was well tolerated at all dose levels, and the number but not the severity of adverse events increased with higher ruxolitinib dose. Two of six patients in dose level 0 and 1 had stable disease for six months on treatment, suggesting that the lower dose levels of ruxolitinib (i.e. 10 mg PO BID) may have more activity in combination with pembrolizumab. Citation Format: Anelia Kassi, Jacqueline Carmona, Sarah M. Green, Heidi Kosiorek, Michael T. Barrett, Barbara A. Pockaj, Donald Northfelt, Karen S. Anderson. Phase I trial of combination pembrolizumab and ruxolitinib in metastatic triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-18-07.

Abstract PO4-20-11: Excellent Response to Trastuzumab Deruxtecan in “HER-2 Low” Leptomeningeal Breast Cancer and Diagnostic and Monitoring Utility of CNSide Assay

Abstract A 72-year-old female presented to the emergency department with sudden onset of imbalance and cognitive decline. Two years ago, she was diagnosed with stage III estrogen receptor (ER) positive, progesterone receptor (PR) negative and human epidermal growth factor receptor 2 (HER2) negative (immunohistochemistry score: 0) invasive breast cancer. She subsequently underwent lumpectomy, adjuvant radiation and chemotherapy. She had been taking adjuvant letrozole at the time of presentation. Magnetic resonance imaging (MRI) of the brain showed obstructive hydrocephalus without any parenchymal lesion or leptomeningeal enhancement. MRI of spine, computerized tomography of chest/abdomen/pelvis and bone scan were unrevealing except a left adrenal mass which was equivocal for metastasis. Cerebrospinal fluid (CSF) analysis showed rare malignant cells. Based on the morphology of malignant cells and the patient’s prior history of breast cancer, the diagnosis of leptomeningeal disease with breast primary was made. There were insufficient cells for additional studies. CNSide, a microfluidic platform that uses antibody capture method to detect CSF tumor cells, showed 4678 tumor cells per milliliter (ml) of CSF. Immunocytochemistry of these cells showed that the cells were ER negative (criterion: < 2.6% ER positive cells) and programmed cell death ligand 1 (PD-L1) negative (criterion: < 3.4% PD-L1 positive cells). Fluorescence in situ hybridization (FISH) testing showed 22% of tumor cells with HER2 amplification (either HER2 to CEP17 ratio >/= 2.0 or HER2 copy number >/= 6). She underwent ventriculoperitoneal (VP) shunt placement with complete recovery of her neurological symptoms. Five weeks later, she was started on trastuzumab deruxtecan (T-DXd) mainly because of the HER2 FISH results from CNSide. She did not receive central nervous system(CNS) irradiation due to complete resolution of neurological symptoms with VP shunt placement. After two cycles of T-DXd, MRI brain showed mild leptomeningeal enhancement along bilateral superior cerebellar sulci. Positron emission tomography/computed tomography(PET/CT) scan showed a new sclerotic rib focus, thought to represent treatment response. It also showed decrease in size of the adrenal mass. The adrenal mass was hypermetabolic on PET, consistent with malignancy. CSF cytology was negative, while CSF tumor cell count significantly reduced to 6.72 cells/ml using CNSide. There were no new neurological symptoms. Despite MRI brain findings, she was continued on T-DXd given overall favorable clinical, radiological and CSF data. After 7th cycle of T-DXd, repeat CSF cytology continued to remain negative, CNSide CSF tumor cell count decreased further to 6.27 cells/ml, MRI brain showed interval resolution of prior leptomeningeal enhancement, CT scan showed further decrease in size of the adrenal mass (baseline 5.4cm to 3cm), stable rib sclerotic lesion and no additional metastatic lesion. To date, she has undergone eight cycles of T-DXd which has effectively controlled her disease for a period of seven months. She has not needed VP shunt revision and continues to do well. To our knowledge, this is the first case demonstrating the activity of T-DXd against CSF tumor cells where a small subset exhibited HER2 amplification. This case also highlights the benefits of using advanced and highly sensitive CSF assays to diagnose and surveil leptomeningeal disease and to assess HER2 signaling. This case is well-timed especially in the era of next-generation, CNS penetrating, potent HER2-directed antibody-drug conjugate like T-DXd which can mount antitumor activity even with minimal HER2 expression in tumors. Citation Format: Sarah Stanford, Zeni Kharel, Lauryn Hemminger, Tyler Schmidt, Sara Hardy, Jason Zittel, Nimish Mohile, Ajay Dhakal. Excellent Response to Trastuzumab Deruxtecan in “HER-2 Low” Leptomeningeal Breast Cancer and Diagnostic and Monitoring Utility of CNSide Assay [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-20-11.

Abstract PO5-16-07: Unveiling the Landscape of PD-L1 Expression and Tumor-Infiltrating Lymphocyte Subtypes in Advanced Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) is known for its aggressive behavior with poor survival outcomes. Influenced by different expression levels of Programmed Death-Ligand 1 (PD-L1) and several subtypes of tumor-infiltrating lymphocytes (TILs), tumor microenvironment (TME) plays a crucial role in TNBC response to treatment and progression. Objective: To assess the prevalence and prognostic role of PD-L1 expression in advanced TNBC, defined as unresectable stage III or stage IV disease, in patients treated with a standard cytotoxic chemotherapy regimen. TILs composition was also evaluated as an exploratory objective. Methods: The internal database of the Brazilian National Cancer Institute was queried out for women diagnosed and treated with advanced TNBC from January 2018 to December 2022. Formalin-fixed paraffin-embedded samples of a maximum of four years old were analyzed. PD-L1 expression using 22C3 pharmDX assay was estimated through combined positive score (CPS) dichotomization (CPS < 10 vs. CPS≥10). Tissue microarrays comprising the samples of biopsies were subjected to immunohistochemistry, targeting specific markers including CD3, CD4, CD8, CD56, CD68, CD117, FOXP3, PD-1, the immune cell profiles were carefully examined and their correlation with the PD-L1 CPS was determined. Results: A total of 150 patients were included. The expression of PD-L1 was undetermined in 2 cases. The median age was 51.5 years (IQR 41.8-60.2) and 20.9% of the cases had CPS≥10. Most patients were < 65 years (73.0%), were postmenopausal (56.8%) and belonged to non-white ethnicity (70.3%). Postmenopausal women predominated in the CPS≥10 subgroup (74.2%) (Table 1). No significant differences in demographic characteristics and clinicopathological variables were observed based on PD-L1 subgroups, 117 patients (79.1%) had CPS < 10, with 91 (77.8%) being de novo stage IV metastatic disease. CD3+ (p=0.037), CD4+ (p=0.005) and CD8+ (p=0.001) TILs had higher expression in tumors with PD-L1 CPS≥10 (Table 2). According to the treatment received, almost half of the patients only received first-line chemotherapy (47%), as the following second line was applied to 28.8% of the total group. The third and fourth lines were administered in 12.9% and 9.1% of patients, respectively. Only 2 patients went through five palliative chemotherapy lines, and only one received up to 6 treatment lines. Between the subgroups CPS≥10 versus CPS < 10 no statistically significant differences were observed in the median progression-free survival (PFS) (5.1 vs. 5.0 months, p=0.89) and overall survival (OS) (8.7 vs. 8.8 months, p=0.6). Conclusion: This study offers insights into the expression patterns of PD-L1 and TILs subtypes in advanced TNBC cases in Brazil. PD-L1 CPS did not influence survival outcomes for this cohort of patients treated with cytotoxic chemotherapy only. The composition of TILs subtypes within the TME appears to vary based on PD-L1 CPS. Table 1. Advanced TNBC population characteristic by CPS (Nf148). Table 2. Tumor Infiltrating Lymphocytes characterization (CD3, CD4, CD8, CD56, CD68, CD 117, FOXP3 and PD-1) and its correlation with CPS status. Citation Format: Alexssandra Lima Siqueira dos Santos, Jesse Lopes da Silva, Lucas Zanetti de Albuquerque, Antônio Lucas Araújo Neto, Cecília Ferreira da Silva, Luana Aguiar Mesquita Cerva, Isabele Ávila Small, Cicera Pimenta Marcelino, Paula de Mendonça Batista, Maria Aparecida do Carmo Rego, Maria Amélia Carlos Souto Maior Borba, Andreia Cristina de Melo. Unveiling the Landscape of PD-L1 Expression and Tumor-Infiltrating Lymphocyte Subtypes in Advanced Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-07.

Abstract PO5-06-05: DISCORDANCE BETWEEN HORMONE RECEPTORS AND HER2 STATUS IN BREAST CANCER PATIENTS RELAPSE

Abstract Introduction: Breast cancer (BC) is a heterogeneous disease, and during its progression, the tumor phenotype can undergo changes that are often associated with a poor prognosis. In this study, we aim to compare the discordance in estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER-2), and Ki-67 expression between primary and recurrent/metastatic lesions. Methods: This study is a retrospective analysis of 132 non-advanced breast cancer patients who experienced confirmed recurrence/metastasis at the Instituto Nacional de Enfermedades Neoplasicas between 2011 and 2022. Descriptive analysis was conducted to assess the expressions of ER, PgR, HER-2, and Ki-67 in primary and metastatic breast cancer, as well as clinical variables. Normality tests, including Kolmogorov-Smirnov and Shapiro-Wilk, were performed. The paired samples Wilcoxon test was used to determine significant differences between primary and recurrent/metastatic tumors. Additionally, Spearman's rank correlation coefficient was employed to evaluate the relationships of expression discordance. Results: Fifty-nine percent of patients presented locoregional recurrence, while 41% presented visceral metastasis. In the primary tumor, ER and PR expression were found in 63.6% and 55.3% of cases, respectively. HER2 status was determined as 0 (36.3%), +1 (22%), +2 (14.4%), +3 (26.5%), and not determined (0.8%) by IHC. For the recurrent/metastatic tumor, ER and PR expression were found in 59% and 34% of cases, respectively. HER2 status was determined as 0 (27.3%), +1 (19.7%), +2 (25%), +3 (25%), and not determined (3%). The median Ki-67 levels were 37% in the primary tumor and 40% in the recurrent/metastatic tumor. Significant differences were found for ER (p=0.014), PR (p< 0.001), and HER2 (p=0.019), but not for Ki-67 percentage (p=0.139). The discordance rates of ER, PR, HER2, and Ki-67 expression were 27.9%, 47.7%, and 26.3%, respectively. Conclussions: Discordance in hormone receptor expression between primary and recurrent/metastatic tumors is a common phenomenon, with ER showing a discordance rate of 27.9% and PR showing a higher rate of 47.7%. HER2 status also displayed significant discordance, with a 26.3% rate of discordant expression between primary and recurrent/metastatic tumors. The findings from this study emphasize the dynamic nature of breast cancer and the importance of comprehensive evaluation of biomarker expression in both primary and recurrent/metastatic tumors, to choose the best treatment options. Citation Format: Katia Roque, Iris Otoya, Natalia Valdivieso, Zaida Morante, Hugo Fuentes, Silvia Neciosup, Henry Gómez, Jorge Cotito, Gonzalo Ziegler, Estefania Peña, Tatiana Vidaurre, Ramon Andrade de Mello, Carlos Castañeda. DISCORDANCE BETWEEN HORMONE RECEPTORS AND HER2 STATUS IN BREAST CANCER PATIENTS RELAPSE [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-05.

Abstract PO2-09-05: Building genomic and transcriptomic data among African American and Black patients with triple-negative inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is an aggressive breast cancer associated with poor response and early metastases. It represents a health disparity, with higher incidence and worse outcomes in Black compared to White patients. Further, IBC is enriched for the more aggressive triple-negative receptor subtype, TN-IBC, and this subtype is more prevalent among Black IBC patients. It is critical to identify genetic and molecular features that may guide better treatments for these aggressive cancers across races, and thus imperative to identify and increase racial representation among bioinformatics datasets. We previously performed comprehensive genomic and transcriptomic analyses for TN-IBC patient samples and here examine the differences among self-identified African American or Black (AA/B) and other races among IBC patients. Methods: As previously described, we collected matched blood and baseline tumor samples before treatment from 19 patients with primary TN-IBC. We performed whole exome and RNA sequencing (RNA-Seq) on these samples and compared mutations and differentially expressed genes (DEGs). Results were compared between 3 AA/B patient tissues vs the remaining 16 (Other) cases. Results: Mutations shared by all AA/B patients included amplification of the five most commonly mutated hallmark genes in the complete cohort, ARNT, BCL9, DDR2, FCGR2B and LMNA (all 100% in AA/B patients versus all 50% in the Other cohort). All AA/B patients had a Notch1 mutation (two deletions and one missense mutation, 100% AA/B vs 6% Other (one deletion)). Comparing differentially expressed genes and gene set enrichment analyses of the Broad molecular signatures database (MSigDB) demonstrates the majority of DEGs are downregulated in these AA/B patients. Six of the top 20 downregulated gene sets from the MSigDB C2 group relate to breast cancer subtype and among this TNIBC cohort AA/B tumor expression was downregulated for luminal and normal subtype related genes in multiple gene sets and enriched in the Lien metaplastic related genes. In the MSigDB C5 sets, over half of the top 20 enriched gene ontology sets among AA/B patients related to chromosomes, chromatin, spindle assembly or histone binding, while 16 of the top 20 downregulated enriched sets related to immune function. Multiple KEGG pathways enriched in AA/B cases related to RNA processing and expression and DNA replication and repair. Lastly, hallmark gene sets enriched in AA/B patients were cell cycle related, and targets of Myc, Hedgehog, B-catenin, TGFb, and TNFa via NFKb, while top significantly downregulated sets related to metabolism and immune response. Conclusion: While findings from a small sample size can only be considered hypothesis generating, some patterns emerge from the data among this rare cohort of AA/B TN IBC patients. These tumors appear to be devoid of luminal signals, characterized by cell cycle and chromatin remodeling signals, enriched for signaling typical of stem cell self-renewal, and perhaps more immune-deprived than other TN-IBC. Additional work to build racially diverse IBC transcriptomic data are needed to develop strategies to understand and improve outcomes for these patients. Citation Format: Wencai Ma, Li Zhao, Gayathri Devi, Savitri Krishnamurthy, Larry Coffer, Angela Alexander, Megumi Kai, Xiaoping Wang, Huong Le-Petross, Miral Patel, Bisrat Debeb, Chandra Bartholomeusz, Jianhua Zhang, Xingzhi Song, Andrew Futreal, Naoto Ueno, Rachel Layman, Anthony Lucci, Jing Wang, Wendy Woodward. Building genomic and transcriptomic data among African American and Black patients with triple-negative inflammatory breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-09-05.

Abstract PO2-24-08: GD3 Synthase is a Master Regulator of Wild-Type p53–Mediated Apoptosis and Mutant p53–Mediated Tumorigenesis

Abstract The tumor suppressor protein p53 is essential for maintaining genomic stability and regulating cellular responses to stress. TP53 mutations are common in breast cancer (BC) and other malignancies. We previously showed that ganglioside GD2 identifies BC stem-like cells (BCSCs) and that GD3 synthase (GD3S/ST8SIA1) is upregulated in breast tumors with TP53 mutations. Here, we demonstrate the direct involvement of p53 in the transcriptional regulation of GD3S in the GD2 biosynthesis pathway. Our results uncover a novel role of GD3S in inhibiting p53-mediated apoptosis and promoting mutant p53–induced tumor initiation. To investigate the p53-mediated regulation of GD3S expression, we used the TCGA and METABRIC datasets and examined GD3S mRNA expression in breast tumors with different p53 mutation status. GD3S expression was significantly upregulated in BC patients with hotspot (HS) p53 mutations compared to those with wild-type (WT) p53 or non-HS p53 mutations (P< 0.0001). Immuno-histochemical analysis of GD3S and p53 in FFPE primary tumor tissues from BC patients (n=84) with varying p53 expression status demonstrated that patients with HS p53 mutations had higher histological scores for GD3S than did patients with WT p53 (P< 0.0001) or non-HS p53 mutations (P=0.009). GD3S and p53 expression were significantly positively correlated in patients with HS p53 mutations (r=0.735; P=0.006) but not in patients with WT p53 (r=–0.09) or non-HS p53 mutations (r=0.14). Consistent with these patient data, BC cells harboring HS p53 mutations had significantly upregulated and positively correlated expressions of GD3S and p53 (n=8; r=0.85). Moreover, compared to those with WT p53, the cells with HS p53 mutations had substantially greater populations of GD2+ BCSCs (P=0.02) and GD3+ BCSCs (P=0.01). Subsequently, we investigated the regulatory role of p53 in the GD2 biosynthetic pathway by stabilizing WT p53 expression by nutlin-3a treatment in 4 BC cell lines and suppressing the expression of HS p53 mutants by p53 knockdown in their respective cell lines (n=4). In cell lines with WT p53, nutlin-3a significantly decreased GD3S expression and GD2 levels (P< 0.05 and P< 0.0001, respectively), suggesting that WT p53 inhibits GD3S expression. In cell lines with p53 mutations, p53 knockdown significantly inhibited GD3S expression. In both cells with WT p53 (MCF7 and ZR751) and cells with HS p53 mutations (Hs578T and BT549), the stable overexpression of GD3S effectively suppressed apoptosis even when WT p53 was stabilized or HS p53 mutant expression was depleted. Consistent with these results, in mice bearing MCF7 cell–derived xenografts, the overexpression of GD3S counteracted the effect of stabilized WT p53–mediated apoptosis and facilitated tumor growth (P< 0.0001). The results of a promoter-luciferase reporter assay demonstrated that nutlin-3a reduced GD3S promoter activity in a dose-dependent manner in MCF7 and ZR751 cells (P< 0.0001), whereas doxycycline (1µM) significantly reduced GD3S promoter activity in Hs578T and BT549 cells with inducible p53 knockdown (P< 0.001). ChIP-qPCR analysis confirmed specific enrichment of WT p53 at the 300-bp upstream regions and additional enrichment of HS p53 mutants at the downstream regions of the GD3S promoter, indicating that WT p53 and HS p53 mutations have distinct promoter regulation patterns. In summary, our study reveals that GD3S has a previously unknown anti-apoptotic function in BC, in that it helps attenuate p53-mediated apoptosis while activating tumor-promoting pathways that operate independently of p53. Citation Format: Vivek Anand, FOUAD EL-DANA, JENNY BORGMAN, MICHAEL ANDREEFF, Venkata Lokesh Battula. GD3 Synthase is a Master Regulator of Wild-Type p53–Mediated Apoptosis and Mutant p53–Mediated Tumorigenesis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-08.

Abstract PS07-09: Prenatal BRCA1 epimutations is a major cause of triple-negative breast cancer

Abstract Background: Low level normal cell BRCA1 epimutations have been associated with an increased risk of triple-negative breast cancer (TNBC). However, the fraction of TNBCs that may have BRCA1 epimutations as their underlying cause is unknown. Neither are the time of occurrence and the potential inheritance pattern of BRCA1 epimutations established. Methods: To address these questions, we analyzed BRCA1 methylation status in breast cancer tissue and matched white blood cells (WBC) from 411 patients with primary breast cancer, including 66 TNBCs. Samples were analyzed by a highly sensitive next-generation sequencing (NGS) assay on an Illumina MiSeq sequencer, allowing allele-resolved methylation assessment. Further, to assess the time of origin and the characteristics of normal cell BRCA1 methylation, we analyzed umbilical cord samples from 1260 newborn girls and 200 newborn boys.To assess potential Mendelian heritage, we analyzed BRCA1 methylation status in WBCs from 575 mothers and 531 fathers of newborn girls with (n = 102) and without (n = 473) WBC BRCA1 methylation. Results: We found concordant tumor and mosaic WBC BRCA1 epimutations in 10 out of 66 patients with TNBC and in four out of six patients with estrogen receptor (ER)-low expression (< 10%) of tumors (combined 14 out of 72; 19.4%, CI: 11.1-30.5). These exceeded the number of tumors harboring germline (n = 5) or somatic (n = 4) BRCA1 mutations. Notably, BRCA1 methylation and BRCA1 mutations were mutually exclusive. Contrasting the findings in TNBC and ER-low exprssion tumors, we found WBC and tumor BRCA1 methylation concordance in only three out of 221 patients with ER >10+% tumors and zero out of 116 patients with HER2 positive tumors. Intraindividually, BRCA1 epimutations affected the same allele in normal and tumor cells. Assessing BRCA1 methylation in umbilical cord WBCs from newborn girls, we found mosaic, predominantly monoallelic BRCA1 epimutations, with qualitative features similar to those in adults, in 113/1260 (9.0%) of individuals. We found no correlation between WBC BRCA1 methylation in newborns and methylation status in their mothers, fathers, or any parent. Notably, WBC BRCA1 methylation occurred at a significantly lower frequency in newborn boys ( 9/200; 4.5%) as compared to newborn girls (p = 0.038). Similarly, WBC BRCA1 methylation was found less common among fathers (16/531; 3.0%), as compared to mothers (46/575; 8.0%; p = 0.0003). Conclusions: Our findings suggest prenatal BRCA1 epimutations might be the underlying cause of around 20% of TNBC and low-ER expressing breast cancers. Such constitutional mosaic BRCA1 methylation likely arise through gender-related mechanisms in utero, independent of Mendelian inheritance. Citation Format: Per Lonning, Hans Petter Eikesdal, Elisabet Ognedal, Bjornar Gilje, Steinar Lundgren, Egil Blix, Helge Espelid, Jürgen Geisler, Stephanie Geisler, Emiel Janssen, Synnøve Yndestad, Laura Minsaas, Beryl Leirvaag, Reidun Lillestol, Stian Knappskog, Oleksii Nikolaienko. Prenatal BRCA1 epimutations is a major cause of triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-09.

Abstract PO2-05-13: HER2 status presented an unstable switching from primary to recurrent breast cancer

Abstract Background: Breast cancer patients with HER2-2+ expression had heterogeneous characteristic. Treatment decision is of great importance after progression. Objective: We explored patients with HER2-2+ in matched primary or recurrent/metastatic tumor samples from January 2010 to June 2022 in our institute were included to evaluate the evolution from or to HER2-2+ expression. Meanwhile, with an emerging of novel entity as HER2-low expression breast cancer, we included this category when analysis as well. Results: In the cohort of a total of 159 breast cancer patients with HER2-2+ expression in either primary tumor or locoregional/distant metastases samples, HER2-2+ breast cancer accounted for 44.0% in primary tumor and 88.8% in recurrent diseases. There were 18.5% and 15.2% patients with HER2 gene amplification on ISH assay among primary and recurrent/metastatic HER2-2+ breast cancers, respectively. The overall rate of HER2 IHC discordance was 67.1%. Among primary HER2-2+ cases, 74.6% maintained in HER2-2+ when disease relapsed. Discordance was mostly driven by cases switching from HER2-2+ to HER2-1+ (64.7%). In HER2-2+ recurrent/metastatic cases, discordance was mostly driven by cases switching from HER2-0 to HER2-2+ (47.1%). The proportion of HER2 discordant cases got a higher statistically trend among HR-positive patients in compared with HR-negative patients (44.1% vs. 21.7%, p = 0.062). There were 35.0% in primary HR-positive/HER2-negative patients and 11.8% in primary triple-negative patients switching from HER2-0 to HER2-low when disease progressed, respectively. Meanwhile, HER2 discordance rate had a significant difference across different metastatic sites (p = 0.026). All contralateral breast and skin metastasis presented with discordant HER2 results. While, all central nervous system metastasis maintained the same HER2-low expression. HER2-positive phenotype in primary breast cancer samples loss their positivity in 42.1% cases. There was a total of 6.6% cases gained HER2 positivity in recurrent breast cancer samples. Conclusion: HER2 expression from primary to relapse breast cancer samples were highly unstable. Biopsy of recurrent sites was necessary to find novel treatment. Citation Format: Anjie Zhu, Hui-Ping Li. HER2 status presented an unstable switching from primary to recurrent breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-05-13.

Abstract PS07-07: The RXR agonist, IRX4204, delays the formation of Brca1 mutant mammary tumors via modulation of the anti-tumor immune response

Abstract Background: Women with germline BRCA1 mutations are at an increased risk for developing breast cancer in their lifetime, often at a young age with more aggressive tumors. At present, prophylactic bilateral mastectomy is the most effective strategy for reducing breast cancer risk. However, this invasive procedure is irreversible and associated with potential complications. We and others have found that PARP inhibitors can delay Brca1-mutant tumor formation in mice and could be beneficial for the prevention of breast cancer. However, currently available PARP inhibitors are associated with modest toxicities that may not be acceptable to women without cancer. Thus, there remains an urgent need for the development of safe and effective therapies for the prevention of breast cancer. Here, we present data demonstrating the activity of IRX4204, a minimally toxic and highly specific agonist of the nuclear retinoid X receptor (RXR), to delay the formation of mammary tumors in a Brca1-deficient mouse model. This inhibitory effect on tumor growth is due, in part, to the role of IRX4204 in stimulating the anti-tumor immune response. Methods: We used the established MMTV-Cre, conditional Brca1 gene knockout, p53 heterozygous loss mouse model (BRCA1co/co; MMTV-Cre+/+; p53+/-) and selected for mutant female pups using PCR genotyping. At 16 weeks of age, all mice were separated into 4 treatment groups (n=10 per group): (1) sesame oil control; (2) the novel RXR agonist, IRX-4204 (10mg/kg); (3) high-dose IRX4204 (20 mg/kg); and (4) the RXR agonist, 9-cis-UAB-30 (5 mg/kg). All treatments were given by oral gavage five days per week, and mice were observed daily for tumor formation and toxicity. At the study endpoint, tumors and normal mammary glands were collected for additional analyses. Immunohistochemical staining was used to quantify CD8a, Ki-67 and cleaved caspase 3 expression in Brca1-deficient tumors. Oil Red O staining was used to measure changes in lipid accumulation in Brca1-deficient cell lines treated with IRX4204. qPCR was used to quantify the changes in gene expression of lipid metabolism-associated genes upon treatment with IRX4204 in vitro. Results: Vehicle-treated Brca1-deficient mice had a median time-to-tumor formation (TTF) of 211 days, with 100% developing tumors by 330 days. Mice treated with UAB 5 mg/kg had an improved median TTF of 261 days, whereas mice treated with IRX4204 10mg/kg or 20mg/kg had a median TTF of 347 and 304 days, respectively (p < 0.01). In addition, 60% of mice treated with IRX4204 10 mg/kg remained tumor-free at 330 days. IRX4204-treated tumors showed an increased infiltration of CD8-positive T-cells over vehicle-treated tumors (p < 0.05). Treatment of Brca1-deficient cell lines with IRX4204 in vitro resulted in a significant increase in lipid accumulation accompanied by a 2-fold increase of Srebf1 expression (a key transcription factor that regulates lipid homeostasis) within 24 hours of treatment (p < 0.05). Conclusion: These data demonstrate a novel use of the RXR agonist, IRX4204, to delay the formation of Brca1-deficient mammary tumors. We have found that IRX4204 treatment modulates the tumor immune response through increased infiltration of cytotoxic CD8-positive T-cells in Brca1-deficient mammary tumors in vivo. We have also determined that IRX4204 modulates lipid metabolism in breast cancer cell lines in vitro. It is known that lipid-derived antigens can stimulate T-cell activity. Our findings suggest that RXR agonists may alter lipid antigen production to activate an anti-tumor response. Additional immune and lipidomic studies are on-going. This work was supported by NCI-PREVENT grant (to PB and AM HHSN26100008) and CFP Foundation (Odyssey Fellowship to CM). Citation Format: Cassandra Moyer, Darian Coleman, Jamal Hill, Lana A. Vornik, Michelle Savage, Martin Sanders, Sei Shizuko, Altaf Mohammed, Powel Brown, Abhijit Mazumdar. The RXR agonist, IRX4204, delays the formation of Brca1 mutant mammary tumors via modulation of the anti-tumor immune response [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-07.

Abstract PO3-24-08: Establishment of a selective culture method for breast cancer stem cells derived from patient tissue and a subgroup analysis of cancer stem cells

Abstract Purpose Cancer stem cells are considered to cause recurrence and metastasis due to their resistance to drug and radiation therapy. Breast cancer stem cells are therefore the true target for the treatment of breast cancer. In this study, we established a method to selectively culture breast cancer stem cells from patient-derived breast cancer tissue, and we analyzed their characteristics using immunostaining to identify gene expression. Method Breast cancer tissues from ER+, HER2- and drug-naive patients were fragmented and cultured using low-adherent plates (spheroid culture). We investigated the stemness of the breast cancer cells obtained by the spheroid culture by immunostaining the breast cancer stem cell markers and examining their proliferation and differentiation in a mouse transplantation model. The diversity of the cells was further examined by means of the expression of the EMT markers and genetic analysis. Results In 48 cases, the proportion of CD44+/CD24- breast cancer cells increased from 13.8% to 61.6% in the spheroid culture. In addition, even small numbers of cells obtained by the spheroid culture were transplanted into mice, and the transplanted cells subsequently differentiated in the mice, demonstrating their stemness. ER expression was negatively changed in 52.1% of cases, and the expression of EMT markers, Twist, Snail, and Vimentin, was increased from 43.8% to 75.0%, 12.9% to 58.1%, and 7.71% to 37.7%, respectively. A DNA microarray with 14 cases showed that breast cancer stem cells obtained from ER+ and HER2- breast cancer tissue were classified into two groups. Conclusion We demonstrated that breast cancer stem cells were selectively cultured from patient-derived breast cancer tissue in a spheroid culture. Furthermore, breast cancer stem cells have different characteristics from breast cancer cells in primary tumors and are diverse at an EMT level, and in genetic analyses. Table. Expression of primary tumors and spheroids Citation Format: Satoshi Sueoka, Takayuki Kadoya, Kai Azusa, Shinsuke Sasada, Akiko Emi, Morihito Okada, Yukino Kobayashi, Koh Nakayama. Establishment of a selective culture method for breast cancer stem cells derived from patient tissue and a subgroup analysis of cancer stem cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-08.

Abstract PO2-01-06: Prognostic value of residual disease (RD) biology and gene expression changes during the neoadjuvant treatment in HER2+ early-breast cancer (EBC)

Abstract Title Prognostic value of residual disease (RD) biology and gene expression changes during the neoadjuvant treatment in HER2+ early-breast cancer (EBC). Background In HER2+ EBC, neoadjuvant trastuzumab-based therapy is the standard of care, with adjuvant therapy determined by whether residual disease (RD) is present. Patients (pts) with RD have significantly higher relapse rates than pts with pathologic complete response. Differences in tumor and immune biology between pre-treated and post-treated tumors after HER2-blockade have been described, but little is known about how these changes can affect long-term outcomes or can be leveraged to tailor adjuvant treatment. Here we evaluate the biology of RD using gene expression analyses of pre- and post-treated HER2+ tumors, and their prognostic value to predict event-free survival (EFS) in a pooled analysis of 4 neoadjuvant studies in which single-agent trastuzumab or combined with lapatinib was explored, with and without chemotherapy: CALGB 40601 (Alliance), PAMELA, NeoALTTO, and NSABP B-41. Methods Gene expression profiling by RNA sequencing was assessed on 497 pre-treated samples from pts with RD, and on pre-treated and RD in 171 tumor pairs. Intrinsic subtypes and 22 tumor and immune gene expression signatures (GESs) were evaluated. We studied and compared the association of gene expression biomarkers with EFS in different cohorts and time points, including when GESs were calculated at baseline (N=497), in the 171 paired baseline and RD samples, and the nature of biomarker changes during neoadjuvant therapy. Association between GESs and EFS was explored by uni- and multivariable Cox regression models adjusted by clinical parameters (i.e., study, treatment arm, hormone receptor status, clinical tumor size, and clinical node involvement), and the performance of the models was assessed by the c-index. Results In 497 baseline untreated samples from pts with RD after neoadjuvant therapy (N=497), intrinsic subtypes were significantly associated with EFS (log-rank [LR] P 0.007), with Luminal A showing the best prognosis and Basal-like the worst. Among 171 paired tumors, HER2-Enriched tumors were more common in baseline (50.3%) with change to Normal-like (49.7%) and Luminal A (18.7%) in RD (chi-square P < 0.001). This luminal phenotypic change was also confirmed by a significant reduction in the correlation to the HER2-Enriched centroid and a significant increase in the correlation to the Luminal A centroid between pre- and post-treated samples; a significant decrease in ERBB2 gene expression and the HER2 amplicon gene expression signature was also observed (Wilcoxon P < 0.001). At an immune GES level, we found a significant increase in B cell, CD8+ T cell, and NK cell signatures in RD specimens. In contrast, T reg GES was significantly downregulated (Wilcoxon P < 0.05). In uni- and multivariable models, immune GESs provided more prognostic information when assessed in RD samples. In contrast, subtype-related biomarkers provided more prognostic information at baseline. From all the multivariable models, the best (c-index= 0.78) included the IgG signature assessed in RD (adjusted hazard ratio 0.46, 95% CI 0.30-0.70, adjusted P 0.005). IgG signature levels (tertiles) in RD could also stratify pts into different groups based on prognostic information (EFS at 7 years: 69% in IgG low vs. 91% in IgG high, LR P 0.02). Conclusions In pts with HER2+ EBC and RD after neoadjuvant treatment with HER2-blockade, tumor-related biomarkers seem to provide more prognostic information when calculated at baseline, whereas immune biomarkers in the RD perform better for EFS prognostication. This finding could be relevant for implementing RD biomarkers in the clinic to stratify pts into different prognostic groups and tailor the post-operative treatment strategy. Supported in part by U10 CA180821, U10 CA180882, R01-CA229409 https://acknowledgments.alliancefound.org Citation Format: Aranzazu Fernandez-Martinez, Maki Tanioka, Sung Gwe Ahn, Paola Zagami, Tomás Pascual, Mattia Rediti, Gong Tang, Katherine A Hoadley, David Venet, Naim U. Rashid, Patty Spears, Judith Bliss, Jens Huober, Priya Rastogi, Md N. Islam, Javier Cortés, Antonio Llombart-Cussac, Sandra Swain, Christos Sotiriou, Aleix Prat, Charles M Perou, Lisa Carey. Prognostic value of residual disease (RD) biology and gene expression changes during the neoadjuvant treatment in HER2+ early-breast cancer (EBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-01-06.

Abstract PO5-16-02: Investigating survivin as a novel target for immunotherapy in diverse breast cancer patients

Abstract Background: The BIRC5 gene encodes survivin, a tumor-specific antigen associated with aggressive tumor features, Black race and poor prognosis in breast cancer (BC). Recent studies have shown that anti-survivin vaccines stimulate a strong anti-tumor immune response in multiple tumor types. However, the relationship between BIRC5/survivin and immune response in BC is poorly understood, particularly among diverse patients. In this analysis, we sought to investigate the relationship between BIRC5/survivin and the BC immune microenvironment across clinical tumor features and RNA-based risk scores for genomic instability. Methods: We leveraged the Carolina Breast Cancer Study (CBCS), a large population-based study that oversampled young (≤50 years) and Black women with invasive BC. NanoString mRNA expression profiling was used to evaluate breast tumor expression for 10 major immune cell types, adaptive and innate immune response, TP53 and homologous recombination deficiency (HRD) pathways, and BIRC5/survivin from 1952 BC patients, including 1,030 (53%) Black and 922 (47%) non-Black women from the CBCS, with additional comparison in The Cancer genome Atlas (TCGA) (n=1,095 breast tumors). BIRC5 was evaluated both as a continuous and categorical variable, using the third expression quartile as a cut point for BIRC5-high, with the lower three quartiles categorized as BIRC5-low. We used generalized linear models to estimate beta values and adjusted p-values as the measure of association between BIRC5 status and immune-related expression in strata defined by estrogen receptor (ER) status, tumor stage, TP53 and HRD status. All analyses were adjusted for multiple comparisons with the Benjamini-Hochberg procedure. Results: In an analysis of continuous BIRC5 expression and global classes of immune response, BIRC5 mRNA expression was highest among tumors with an adaptive-enriched immune class (p< 0.001) in both CBCS and TCGA. However, when investigating immune gene expression according to BIRC5 status (i.e., BIRC5-high relative to BIRC5-low), multivariate differential expression analysis revealed significantly decreased immune cell-related expression scores among BIRC5-high tumors relative to BIRC5-low, and this relationship persisted in analysis stratified on stage (I/II vs III/IV), ER status, TP53 and HRD status. Across all strata, the strongest relationship between BIRC5-high and the immune microenvironment was observed for adaptive immune response (Beta: -0.14; p=0.005), with decreased immune gene expression related to B cells (Beta: -0.16; p=0.01), T cells (Beta: -0.14; p=0.007), Cytotoxic cells (Beta: -0.14; p=0.005), and PD-L1 (Beta: -0.15; p=0.005), relative to BIRC5-low. Conclusion: High BIRC5/survivin expression is associated with decreased immune-related gene expression in both ER-positive and ER-negative disease, and across all tumor stages. Anti-survivin therapies may be beneficial for boosting immune response in breast tumors with low immunogenicity, offering promise for BC subtypes previously ineligible for immunotherapy. Citation Format: Alina Hamilton, Qichen Wang, Sarah Soppe, Melissa Troester, Yara Abdou. Investigating survivin as a novel target for immunotherapy in diverse breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-02.

Pan-cancer analysis of prognostic and immunological role of IL4I1 in human tumors: a bulk omics research and single cell sequencing validation

BackgroundInterleukin-4 inducible gene 1 (IL4I1) regulates tumor progression in numerous tumor types. However, its correlation with immune infiltration and prognosis of patients in a pan-cancer setting remains unclear.MethodsData from the Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), UALCAN, Clinical Proteomic Tumor Analysis Consortium (CPTAC), Gene Expression Omnibus (GEO), cBioPortal, Cancer Single-cell State Atlas (CancerSEA), and Tumor IMmune Estimation Resource(TIMER) databases were used to evaluate IL4I1 expression, clinical features and prognostic effects, gene set enrichment, and correlation with immune cell infiltration, as well as the relationship between IL4I1 methylation and expression and survival prognosis. Correlations with 192 anticancer drugs were also analyzed.ResultsIL4I1 was significantly overexpressed in the majority of tumors, and the imbalance of IL4I1 was significantly correlated with overall survival and pathological stage. Moreover, total IL4I1 protein was increased in cancer. Therefore, IL4I1 may be used as a prognostic biomarker or protective factor in numerous types of cancer. The methylation level of IL4I1 may also be used as a prognostic marker. The functional enrichment of IL4I1 was closely related to the immunomodulatory pathway. In addition, the level of tumor-associated macrophage infiltration was positively correlated with the expression of IL4I1 in pan-cancerous tissues. scRNA-seq analysis suggested that IL4I1 differ significantly among different cells in the tumor microenvironment and was most enriched in macrophages. Various immune checkpoint genes were positively correlated with IL4I1 expression in most tumors. In addition, patients with high IL4I1 expression may be resistant to BMS-754807 and docetaxel, but sensitive to temozolomide.ConclusionIL4I1 may play a role as promoter of cancer and prognostic indicator in patients. High expression of IL4I1 is associated with the state of tumor immunosuppression and may contribute to tumor-associated macrophage invasion. Therefore, IL4I1 may be a new therapeutic target for the treatment and prognosis of patients with cancer.

Therapeutic Targeting of Thioredoxin Reductase 1 Causes Ferroptosis while Potentiating Anti-PD-1 Efficacy in Head and Neck Cancer

Head and neck squamous cell carcinoma (HNSCC) faces low response rates to anti-PD-1 immunotherapies, highlighting the need for enhanced treatment strategies. Auranofin, which inhibits thioredoxin reductase (TrxR) through its gold-based composition, has shown potential in cancer treatment. It targets the TrxR system, essential for safeguarding cells from oxidative stress. The overproduction of TrxR in cancerous cells supports their proliferation. However, auranofin's interference with this system can upset the cellular redox equilibrium, boost levels of reactive oxygen species, and trigger the death of cancer cells. This study is the first to highlight TXNRD1 as a crucial factor contributing to resistance to anti-PD-1 treatment in HNSCC. In this study, we identified targetable regulators of resistance to immunotherapy-induced ferroptosis in HNSCC. We observed a link of thioredoxin reductase 1 (TXNRD1) with tumoral PD-L1 expression and ferroptosis suppression in HNSCC. Moreover, HNSCC tumors with aberrant TXNRD1 expression exhibited a lack of PD-1 response, NRF2 overexpression, and PD-L1 upregulation. TXNRD1 inhibition promoted ferroptosis in HNSCC cells with NRF2 activation and in organoid tumors derived from patients lacking a PD-1 response. Mechanistically, TXNRD1 regulated PD-L1 transcription and maintained the redox balance by binding to ribonucleotide reductase regulatory subunit M2 (RRM2). TXNRD1 expression disruption sensitized HNSCC cells to anti-PD-1–mediated Jurkat T-cell activation, promoting tumor killing through ferroptosis. Moreover, TXNRD1 inhibition through auranofin cotreatment synergized with anti-PD-1 therapy to potentiate immunotherapy-mediated ferroptosis by mediating CD8+ T-cell infiltration and downregulating PD-L1 expression. Our findings indicate that targeting TXNRD1 is a promising therapeutic strategy for improving immunotherapy outcomes in patients with HNSCC.