Real-world, multicenter assessment of 18 F-FDG PET/CT prognostic value in triple-negative breast cancer (TRINE-PET): the study protocol

Background: Breast cancer is currently the leading cause of cancer morbidity and mortality among Algerian women. To date, triple negative breast cancer (TNBC) shows substantial overlap with basal type cancer and it is associated with aggressive tumor behavior, poor prognosis and BRCA1 mutations. In the present study, we screened for the prevalence of BRCA1 and BRCA2 germline mutations in a cohort of 169 TNBC patients using PCR-Sanger sequencing and NGS with a cancer panel of 30 hereditary cancer genes or BRCA1/BRCA2 genetic test. Materials and Methods: 169 TNBC patients and their relatives were referred trough several public hospitals from six years (2013-2019). BRCA1 germline mutation was screened using PCR-Sanger sequencing in 66 TNBC patients with strong family history of breast and ovarian cancer and 103 sporadic TNBC patients including all exons where a mutation was previously found in Algerian population (exons 2, 3, 4, 10, 17 and 19). BRCA2 germline mutation was screened using PCR-Sanger sequencing in 24 TNBC patients with strong family history of breast cancer including all exons where a mutation was previously found in Algerian patients (exons 10 and 22). In addition, nine (9) TNBC patients with strong family history of breast and ovarian cancer were analyzed by NGS using BRCA1 and BRCA2 test or a cancer panel of 30 hereditary genes (Colors genomics). Results: The analysis of the genomic DNA samples of 169 TNBC patients revealed that 15 patients carried pathogenic germline variants in BRCA1 gene and three patients carried pathogenic variants in BRCA2 gene (10.65%). Eight distinct germline mutations in BRCA1 have been detected in this study: c.83_84delTG, c.181T>G, c.798_799delTT, c.505C>T, c.923_924delGC, c.2125_2126insA, c.5257A>G and deletion of exon 15. Interestingly, the recurrent and specific mutation c.83_84delTG has been detected in 4 unrelated TNBC patients. The pathogenic variant c.2125_2126insA has been detected in three unrelated families and also in the first relatives of 2 patients. The rare likely pathogenic variant BRCA1 c.5257A>G/p.Arg1753Gly has been detected in young female TNBC patient. The Del exon 15 in BRCA1 has been detected in two unrelated patients. Three distinct germline mutations in BRCA2 have been detected in three TNBC patients: c.1813dupA, c.7654dupA and c.8485C>T. Interestingly, these three mutations are reported for the first time in Algerian population. The c.1813dupA and c.8485C>T pathogenic variants have been detected in two young female TNBC patients with a family history of male breast cancer. Conclusions: In the current study, we detected recurrent germline mutations in BRCA1 gene in early onset TNBC patients. BRCA2 pathogenic variants have also been detected in young female TNBC patients. TNBC immunophenotype should be considered as an additional criterion for genetic counselling and testing of BRCA genes in Algerian women with early onset breast cancer. Citation Format: Farid Cherbal, Chiraz Mehemmai, Hadjer Gaceb, Hassen Mahfouf, Kada Boualga. BRCA1 and BRCA2 germline mutational spectrum in Algerian triple negative breast cancer (TNBC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1451.

Background: triple-negative breast cancer (TNBC) is associated with hereditary and environmental risk factors plus an overall worse prognosis compared to other Breast Cancer (BC) subtypes. While TNBC risk factors, prevalence, clinical characteristics and prognosis may vary throughout different populations, limited data on Latin American patients forces clinical decisions to be based predominantly on data coming from non-Hispanic women. To obtain local epidemiological information, regarding risk factors and clinical outcomes, we analysed the largest Chilean BC registry. Methods: we conducted a retrospective population-cohort study involving females with any stage TNBC, treated at a community hospital (mid-low income) and at an academic private hospital (high income), between the years 2010 and 2021. Risk factors, reason for consultation, clinical and pathological characteristics and prognosis were separately analysed for both TNBC and non-TNBC subgroups. Univariate and multivariate analyses were performed to identify prognostic factors for survival on TNBC patients. Results: From 5,806 patients, 647 (11.2%) were identified as TNBC. Compared to non-TNBC patients, women were younger (median age 55.2 vs. 57.2, p=0.0001), with 15.8% of TNBC patients having been diagnosed before the age of 40 compared to 9.6% in non-TNBC (p= 0.0001). TNBC had a significantly lower screen-detected cancer rate (14.5% vs. 31.6% p= 0.0001) and worse stage at diagnosis. No differences were seen between patients seen at a community hospital and private centre, for both TNBC rate and stage. Other risk factors such as parity, age at first gestation, menarche, hormone therapy replacement and obesity showed no significant differences between TNBC and no-TNBC patients (table 1). With a median follow up of 57 months, 5-year overall survival (OS) and BC specific death were significantly shorter for TNBC compared to non-TNBC (76.4% vs 88.1% and 78.9% vs 91.2%, respectively; p=0.0001) (table 2). In the multivariate analysis, TN subtype (HR=2.3, p=0.0001), stage (HR=2.05 for stage II vs stage I, HR=7.04 for stage III vs. stage I, p=0.0001), lower income (HR= 1.64, p=0.0001), and non-screened detected BC (HR=1.32, p=0.03) were all associated with worse overall survival (table 3). Conclusion: This is the first study focusing on TNBC characteristics in Chilean BC patients and to our knowledge, the largest performed in a Latin American population. We identified a lower proportion of TNBC patients when compared with data reported from other LA groups and worldwide, a very low screen detected cancer rate and as expected significantly lower TNBC survival rate compared to non-TNBC women. While TNBC patients were younger compared to the non-TNBC group, this age difference was marginal compared to other reported studies. Community hospital patients (with mid-low income) were associated with lower survival rates for both all-cause mortality and BC specific survival, regardless of a similar stage distribution at diagnosis. Reflecting an underlying interaction between social and biological factors that needs to be addressed. Table 1. Patient characteristics: Triple-negative versus noN-triple negative breast cancer BMI: Body mass index; FH: Family history * Difference is statistically significant. Table 2. Survival comparison in triple-negative versus non-triple negative breast cancer * Difference is statistically significant. Table 3. Cox Regression Multivariate analysis * Difference is statistically significant. Citation Format: Benjamin Walbaum, FRANCISCO ACEVEDO, Catherine Bauerle, Mauricio Camus, Manuel Manzor, Raul Martinez, Paulina Veglia, Marisel Navarro, Constanza Guerra, Francisco Dominguez, Tomas Merino, Lidia Medina, CÉSAR SÁNCHEZ. Real-world data of clinical characteristics, risk factors and outcomes of Chilean triple-negative breast cancer patients [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 P3-03-19.

Here, we investigated the possible predictive value of stromal caveolin-1 (Cav-1) as a candidate biomarker for clinical outcome in triple negative (TN) breast cancer patients. A cohort of 85 TN breast cancer patients was available, with the necessary annotation and nearly 12 years of follow-up data. Our primary outcome of interest in this study was overall survival. Interestingly, TN patients with high-levels of stromal Cav-1, had a good clinical outcome, with >50% of the patients remaining alive during the follow-up period. In contrast, the median survival for TN patients with moderate stromal Cav-1 staining was 33.5 months. Similarly, the median survival for TN patients with absent stromal Cav-1 staining was 25.7 months. A comparison of 5-year survival rates yields a similar pattern. TN patients with high stromal Cav-1 had a good 5-year survival rate, with 75.5% of the patients remaining alive. In contrast, TN patients with moderate or absent stromal Cav-1 levels had progressively worse 5-year survival rates, with 40% and 9.4% of the patients remaining alive. In contrast, in a parallel analysis, the levels of tumor epithelial Cav-1 had no prognostic significance. As such, the prognostic value of Cav-1 immunostaining in TN breast cancer patients is compartment-specific, and selective for an absence of Cav-1 staining in the stromal fibroblast compartment. A recursive-partitioning algorithm was used to assess which factors are most predictive of overall survival in TN breast cancer patients. In this analysis, we included tumor size, histologic grade, whether the patient received surgery, radiotherapy or chemotherapy, CK5/6, EGFR, p53 and Ki67 status, as well as the stromal Cav-1 score. This analysis indicated that stromal Cav-1 expression was the most important prognostic factor for overall survival in TN breast cancer. Virtually identical results were obtained with CK5/6 (+) and/or EGFR (+) TN breast cancer cases, demonstrating that a loss of stromal Cav-1 is also a strong prognostic factor for basal-like breast cancers. Our current findings may have important implications for the close monitoring and treatment stratification of TN and basal-like breast cancer patients.

e12017 Background: Cancer antigen 15–3 (CA 15–3) and carcinoembryonic antigen (CEA), are often used in follow up care of breast cancer and provide important clues to the clinicians for disease progression in metastatic and recurrent breast cancer. Triple-negative breast cancers are frequently defined as a single group identifiable using routine clinical tests. They are negative for estrogen receptor (ER), progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER-2), the so-called triple-negative breast cancers. In this study we compared the tumor markers of triple negative breast cancer and non-triple negative patients. Methods: We retrospectively analyzed serum CEA and CA 15–3 levels of both triple negative and non-triple negative breast cancer patients at the time of first diagnosis and when they developed metastatic disease. Results: 544 consecutive nonmetastatic breast cancer patients presenting at Hacettepe University Institute of Oncology, Ankara, Turkey, with a median age of 49 were evaluated. 15.1% of the patients were triple negative breast cancer. At the time of diagnosis triple negative group had lower serum CEA (2.5 ± 5.9 vs 4.0 ±16.4 p = 0.35) and CA 15–3 (23.7 ± 14.6 vs 37.1 ± 117; p = 0.021) levels compared to non-triple negative group. In patients who developed metastasis during follow up; the CEA (3.2 ± 3.8 vs 29.6 ± 106.4 p = 0.022) and CA15–3 (46.9 ± 46.3 vs 203.2 ± 534 p = 0.008) levels were also significantly lower in triple negative breast cancer group compared to non-triple negative group.In non-triple negative breast cancer patients who developed metastasis, mean serum levels of CEA and CA15–3 significantly increased compared to baseline, whereas in triple negative group who developed metastasis CEA and CA 15–3 levels did not differ significantly. Conclusions: While being a good laboratory parameter in the follow-up of patients with breast cancer metastases, tumor markers may not show the increased tumor burden in the triple-negative breast cancer patients. No significant financial relationships to disclose.

Introduction: Triple-negative breast cancer (TNBC), an aggressive form of breast cancer (BC) that is associated with poor prognosis, accounts for 10-15% of all BCs. Chemotherapy remains the standard of care (SOC) for advanced disease, with limited clinical benefit. Oncology clinical trials (CTs) are globally recommended and encouraged as the preferred treatment (tx) option for any cancer patient (pt). ‘TrialJectory’ (TJ) is an artificial intelligence (AI)-based technology that matches pts to oncology CTs. Here, we identified distinct characteristics of TNBC pts who signed up to the TJ platform compared to non-TNBC pts. Methods: Using AI and an unsupervised natural language processing approach, the TJ platform clinically matches pts to CTs. Matching is achieved by pt response to an online dynamic questionnaire (www.trialjectory.com) that collects detailed clinical data including clinico-pathologic characteristics, tx history, general health, and comorbidities. Those are compared to the eligibility criteria of available CTs to yield a high-quality actionable matched-trial list. Results: Between 1/2020 and 12/2021, out of 9796 BC pts that signed up, 2688 were TNBC pts (27%). There was no significant difference in age at sign-up between TNBC and non-TNBC patients (median age of 57 years vs 58 years, respectively). Consistently with Non-hispanic black (NHB) race prevalence in the different molecular subtypes in the general US population, NHB race had higher signup rate in TNBC compared to non-TNBC (9.95% vs 5.76%, respectively). TNBC pts signed up at a later disease stage compared to non-TNBC pts (19% of TNBC reported having a stage 1 disease compared to 27% of non-TNBC pts, p< 0.001).A significantly higher percentage of pts with advanced/metastatic TNBC signed up to the TJ platform before starting tx compared to non-TNBC patients (34% vs 22%, respectively, p< 0.001). Furthermore, there was a significant difference in the willingness to travel any distance within the US for a matched clinical trial between TNBC and non-TNBC pts (39% vs 34%, respectively, p< 0.001). Conclusions: In this study, we found significant differences in the characteristics of TNBC vs non-TNBC pts that have signed up to the TJ platform. There was an up to 2-fold enrichment of TNBC on the TJ platform pts compared to their frequency in the general population. While previous studies do not show a difference in stage distribution between different subtypes, TNBC patients initiated their search for CTs at a higher stage. In addition, advanced TNBC patients started their search earlier in their journey, before starting chemotherapy. This may reflect the lack of effective SOC and possibly, the motivation to avoid the use of chemotherapy. This is also supported by the willingness of TNBC patients to travel farther in order to identify and enroll in a CT compared to non-TNBC pts. Importantly, the motivation of TNBC pts to travel any distance has not been reduced despite the COVID-19 pandemic, reflecting a strong drive of this pt population to enroll in CTs. It also demonstrates that with the right access, diverse patient populations are willing to participate in clinical trials. In sum, TNBC pts are more likely to explore CT options, in the advanced stage setting, earlier in their journey. This study demonstrates the power of TJ platform for clinico-pathologic characterization and diverse pt groups, including their drivers and behavioral choices during their battle with cancer. Citation Format: Michal Safran, Yelena Lapidot, Tzvia Bader, Avital Gaziel. Triple Negative Breast Cancer (TNBC) patients are more likely to digitally explore clinical trial options and prior to receiving treatment for advanced disease compared to non-TNBC patients [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 P2-13-05.

Background: While new treatments and improved subtyping schemas are anticipated to improve treatment response in triple-negative breast cancer (TNBC) patients, therapeutic resistance remains a significant challenge. Moreover, there is an urgent need for additional research model systems to study resistance and residual disease in breast cancer, including aggressive subtypes of breast cancer. Organoid culture is a promising technology used for growing breast cancer cells with high efficiency; however, the extent to which treatment resistance can be modeled using this system is unknown. This research used patient-derived organoid cultures in the context of computational analyses of large molecular and clinical datasets to study resistance mechanisms, biomarkers, and alternative treatment strategies to overcome drug resistance in early-stage TNBC. Methods: Organoid cultures were derived from breast tumor samples (taken from lumpectomy, mastectomy, or core biopsy samples), digested to the organoid level using collagenase, and grown in three dimensional cultures using a basement membrane extract and a fully-defined organoid medium (Dekkers et al. Nat Protoc 2021). An evaluation of all available I-SPY2 biomarker data (Wolf et al. Cancer Cell 2022) was performed focusing on genes, proteins, and pathways associated with resistance. These were then used to study whether resistance biomarkers identified in patient tumors are also present in organoids propagated from breast cancer post-treatment residual disease. To this end, bulk RNA sequencing data of organoids were normalized and merged with the TCGA dataset (Hoadley et al. Cell 2018) to enable analysis in a larger context, and immunofluorescence staining of organoids was performed. A high-throughput 386 anti-cancer drug compound screen and subsequent synergy testing with the most promising compounds were performed to identify and predict alternative treatment strategies. Additional assays to explore kinome activity in this organoid model are in progress. Results: A TNBC organoid biobank was established (n=31), which was enriched for inflammatory breast cancer (IBC; n=15), an aggressive form of breast cancer. Most organoids were derived from residual disease after neoadjuvant therapy. Bulk RNA sequencing analysis performed on 10 TNBC organoids revealed 3 subsets that were characterized predominantly by either normal-like/luminal androgen receptor or basal-like features or expressed distinct gene expression profiles, with IBC cases present in all 3 subsets. Intriguingly, the IBC organoids were characterized by higher expression of a number of immune-related signatures, despite an absence of clear immune cells in culture. A residual disease IBC/TNBC organoid resistant to chemotherapy was used to perform the 386-drug compound screen. The organoid model showed resistance to veliparib-cisplatin, consistent with the expression of gene/protein biomarkers predictive of drug resistance found in I-SPY2 (low PARPi7 levels and high pFOXO1 and pMEK1/2 expression). In addition, the screen identified multiple classes of inhibitors as promising synergistic/additive candidates for overcoming resistance to cisplatin. Conclusion: In this proof-of-principle study, we demonstrated the utility of matching I-SPY2 resistance biomarkers and signatures to residual disease tumor organoid cultures. We show that tumor organoid cultures modeling drug resistance states are a useful complement to existing research models of breast cancer and can be used for compound testing. We have developed a pipeline to propagate residual tumors from patients enrolled in I-SPY2 into organoid cultures to create a broader platform for preclinical drug testing informed by tumor biology with the ultimate goal of enabling faster, more successful translational studies and increased treatment options for resistant disease. Citation Format: Tam Binh V. Bui, Denise M. Wolf, Kaitlin Moore, Isaac S. Harris, Pravin Phadatare, Christina Yau, Lamorna A. Brown Swigart, Laura J. Esserman, Jean-Philippe Coppe, Julia Wulfkuhle, Emanuel F. Petricoin, Michael Campbell, Laura M. Selfors, Deborah A. Dillon, Beth Overmoyer, Filipa Lynce, Laura Van ’t Veer, Jennifer Rosenbluth. PD5-02 An Organoid Model System to Study Resistance Mechanisms, Predictive Biomarkers, and New Strategies to Overcome Therapeutic Resistance in Early-Stage Triple-Negative Breast Cancer [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 PD5-02.

Purpose: Based on clinical and molecular data, breast cancer is a heterogeneous disease. Breast cancers that have no expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are defined as triple negative breast cancers (TNBCs); luminal cancers have different expressions of ER, PR and/or HER2. TNBCs are frequently linked with advanced disease, poor prognosis and occurrence in young African women, and about 15% of the cases are associated with germline BRCA1/2 mutations. Since radiotherapy is utilized as a principle treatment in the management of TNBC, we aimed to investigate the chromosomal instability and radiosensitivity of lymphocytes in TNBC patients compared to luminal breast cancer patients and healthy controls using the micronucleus (MN) assay. The effect of mutations in breast cancer susceptibility genes on chromosomal radiosensitivity was also evaluated.Methods: Chromosomal radiosensitivity was evaluated in the G0 (83 patients and 90 controls) and S/G2 (34 patients and 17 controls) phase of the cell cycle by exposing blood samples from all patients and controls to 2 and 4 Gy ionizing radiation (IR).Results: In the G0 MN assay, the combined cohort of all breast cancer, TNBC and luminal patients’ exhibit significantly elevated spontaneous MN values compared to controls indicating chromosomal instability. Chromosomal radiosensitivity is also significantly elevated in the combined cohort of all breast cancer patients compared to controls. The TNBC patients, however, do not exhibit enhanced chromosomal radiosensitivity. Similarly, in the S/G2 phase, 76% of TNBC patients do not show enhanced chromosomal radiosensitivity compared to the controls. In both the G0 and S/G2 phase, luminal breast cancer patients demonstrate a shift toward chromosomal radiosensitivity compared to TNBC patients and controls.Conclusions: The observations of the MN assay suggest increased chromosomal instability and chromosomal radiosensitivity in South African breast cancer patients. However, in TNBC patients, the irradiated MN values are not elevated. Our results suggest that the healthy lymphocytes in TNBC patients could handle higher doses of IR.

Background: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype that affects about 20% of breast cancer patients and has a high incidence in young African American women. Currently the only therapeutic options for TNBC patients is chemotherapy, however, more than 60% of TNBC patients are highly resistant to chemotherapeutic treatment. The development of novel therapeutic approaches is essential to enable treatment of patients with chemo-resistant TNBC. Our lab focuses on the development of receptor-targeted theranostic nanoparticles. We have developed a class of urokinase plasminogen activator receptor (uPAR) targeted theranostic magnetic iron oxide nanoparticles (IONPs) carrying therapeutic agents that are clinically beneficial to TNBC patients, including DNA damaging agents, such as doxorubicin (Dox) or cisplatin (Cis). We are targeting uPAR because it is highly expressed in malignant cancers including aggressive breast cancer tissues and tumor stromal cells that are enriched in TNBC tissues. Methods: Tumor fragments from TNBC patients, obtained from a Phase II clinical trial that is ongoing at the Breast Cancer Clinic in the Winship Cancer Institute, were implanted in the mammary fat pad of SCID mice. The tumor xenografts grew to about 1 cm in diameter in 12 to 15 weeks. Tumor fragments were then implanted into the mammary fat pad and passaged through nude mice for testing the therapeutic efficacy of the uPAR-targeted nanoparticle-drugs. Histological analysis of post-chemotherapy TNBC and primary tumor xenograft tissues with or without uPAR-targeted IONP-Dox was conducted using immunohistochemistry and dual immunofluorescence techniques. Results: We have previously reported that the uPAR-targeted IONP-Dox causes tumor growth inhibition following systemic delivery. Using CD44 and uPAR as biomarkers we also reported histological analysis of post-chemotherapy TNBC and primary tumor xenograft tissues showed high levels of CD44+/CD24- tumor cells and up-regulation of uPAR. Primary TNBC xenografts treated with conventional Dox had increased levels of CD44+/CD24- cells and strong uPAR expression. Conversely, the tumor tissues from the uPAR-targeted IONP-Dox treated group showed decreased levels of CD44+/CD24- and uPAR positive cells. We have further investigated the biomarkers insulin-like growth factor 1 receptor (IGF-1R) and Ki67. IGF-1R has been shown to be overexpressed in more than 50% of TNBCs and may be associated with the drug-resistant TNBC phenotype, while Ki67 is a conventional biomarker for proliferation. We observed that uPAR-targeted IONP- Dox treatment decreased IGF-1R expression compared to the control group and the group treated with conventional Dox. We also observed that Ki67 expression was decreased in the uPAR-targeted IONP- Dox treatment group compared to control group and the group treated with conventional Dox. Conclusions: Results of our study demonstrated that uPAR-targeted theranostic IONPs selectively delivers therapeutic payloads of the chemotherapeutic drug Dox to inhibit the growth of uPAR positive tumor cells. It is likely that uPAR expressing tumor cells are an aggressive and potentially drug resistant cell population that overexpress breast cancer stem-like cell biomarker (CD44+/CD24-) as well as up-regulation of a growth factor receptor, IGF-1R. These results support further investigation of theranostic nanoparticles as an approach to overcome drug resistance in TNBC. Citation Format: Jasmine M. Miller-Kleinhenz, Hongyu Zhou, Weiping Qian, Ruth O'Regan, Amelia Zelnak, Toncred Styblo, Lily Yang. uPAR-targeted theranostic nanoparticles effectively decrease expression of IGF-1R and Ki67 in drug-resistant triple-negative breast cancer human xenograft. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B045.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the lowest 5-year survival rate among different subtypes. Although TNBC is less frequently diagnosed in U.S. women compared to the HR+/HER2- subtype, it remains as the second most common breast cancer subtype (13.6%) in the most recent five years. However, there is still an unmet need to develop effective treatment for TNBC due to the lack of actionable drug targets and high resistance rate. Cell cycle dysregulation is a common cancer hallmark. By targeting key regulators of cell cycle progression, multiple cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have achieved clinical benefits with FDA approval to treat HR+/HER2- advanced and metastatic breast cancer. Interestingly, our datamining of two public breast cancer patient datasets (TCGA and GEO) consistently showed that the CDK4/6 signaling pathway was significantly more enriched in TNBC patients, compared to luminal and HER2-enriched breast cancer patients. After stratifying patients based on CDK4/6 gene activation signature scores into high (CDK4/6-high) and low (CDK4/6-low), we observed that high CDK4/6 activation signature is significantly correlated with a shorter time to develop overall metastasis, as well as, site-specific metastasis to bone, lung, or brain. It is noteworthy that CDK4/6-high TNBC patients have significantly shorter metastasis-free survival, with a median of 20 months, while that of CDK4/6-low TNBC patients is substantially extended with a median of 51 months. Immunohistochemistry staining of tissue microarray of 46 pairs of human primary and metastatic breast tumor samples validated our findings in datamining as CDK4/6 positivity is increased in lymph node metastases compared to matched primary tumors, and it is the highest in TNBC patients in both primary (6/9, 67%) and lymph node metastasis (7/9, 78%) tissue samples. Western blot analysis of a panel of breast cancer cell lines showed that the CDK4/6 signaling pathway is activated in the majority of TNBC cell lines. Next, we investigated the efficacy of Abemaciclib, an FDA-approved CDK4/6 inhibitor, and demonstrated that it is effective in inhibiting the proliferation of TNBC cell lines, with a half maximal inhibitory concentration (IC50) of 5.6-9.6 μM. Abemaciclib also effectively induced apoptosis of TNBC cells as indicated by increased PARP cleavage and Annexin V staining positivity. Additionally, TNBC cells treated with Abemaciclib exhibited significantly inhibited migration, invasion, and mammosphere formation abilities. Using an intracardiac inoculation mouse model, we found that systemic administration of Abemaciclib treatment significantly reduced overall TNBC metastatic burden and brain metastases. Collectively, these findings suggest a promising utility of CDK4/6-targeted therapy in treating metastatic TNBCs. Citation Format: Chuling Zhuang. Efficacy of CDK4/6 Inhibitor Abemaciclib in Metastatic Triple-Negative Breast Cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-07-21.

Background: Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high recurrence and mortality rates. Currently, there are no effective targeted therapies available for TNBC. Emerging data suggest that androgen receptor (AR), a nuclear steroid hormone receptor expressed in 10-43% of TNBCs, has emerged as a promising new biomarker with potential predictive value for certain TNBC subtypes. African American (AA) women suffer from earlier onset of the breast cancer along with significant aggressive disease course when compared to European American (EA) women. This study aimed to determine the prognostic value of AR in a large TNBC cohort and evaluate racewise trends in AR expression status among TNBCs. Methods: We evaluated AR expression immunohistochemically in formalin-fixed paraffin-embedded samples from 822 TNBC patients (142, 95 and 264 patients from Emory, Northside and Grady Memorial Hospitals, respectively, in Atlanta, and 321 from Nottingham Hospital, UK) for whom complete clinicopathologic and overall survival (OS) data were available. Ethnicity data were available for 138 AA and 675 EA TNBC cases. Samples with <1% nuclear-stained cells were considered quadruple-negative (QN). Associations between nuclear AR status, race, other clinicopathologic variables and OS were evaluated through survival analyses and multivariate Cox regression. Results: In this study, 45.6% of the TNBC cases were QN and AAs had a much higher proportion (80.8%) of QN cases than EAs (40.1%, p<0.0001). QN receptor status was significantly associated with younger age (age< 25) at diagnosis, AA ethnicity and higher (KI>14%) proliferative index (p<0.001 for all) in univariate analyses. In multivariate analyses, loss of AR expression was associated with poorer OS (p<0.05, HR=2.8) after adjusting for grade, stage and adjuvant therapy. Conclusion: This study suggest that a higher prevalence of QN breast cancer among AAs may be a plausible reason for the ethnic disparity in outcomes among TNBC patients and strongly support the prognostic role of AR in this breast cancer subtype. Citation Format: Bhattarai Shristi, Jun Xia, Ceyda Sonmez Wetherilt, Sergey Klimov, Ansa Riaz, Sonal Pattni, Mohammad A. Aleskandarany, Andrew R. Green, Emad A. Rakha, Ian O. Ellis, Guilherme Cantuaria, Xiaoxian (Bill) Li, Uma Krishnamurthi, Remus Mihai Osan, Padmashree CG Rida, Ritu Aneja. Racial differences in Androgen Receptor status among triple-negative breast cancers. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B12.

Background: 281,550 new cases of invasive breast cancer will be diagnosed in US this year. 15% of these patients will be diagnosed with triple-negative breast cancers (TNBCs), which is the most aggressive subtype of breast cancer. Non-specific chemotherapy remains the current standard of care for TNBCs, due to the lack of targeted therapy. We hypothesized that phosphatases are potential new drug targets for TNBCs and performed a whole phosphatase microarray analysis using TNBCs vs. estrogen receptor (ER)-positive breast cancers. NUDT5, the enzyme hydrolyzes 8-oxo-dGDP and ADP-ribose, is among the most highly expressed phosphatases in TNBCs. The goal of these studies is to investigate NUDT5 dependency and mechanism in TNBC development.Methods: In this study, we have obtained NUDT5 mRNA expression level from publicly available TCGA, METABRIC, and the CCLE datasets, and compared NUDT5 mRNA level in TNBC vs. ER-positive breast cancer. Next, we examined the association of overall survival with NUDT5 expression level dichotomized by median mRNA expression in breast cancer patients. We then engineered doxycycline-inducible NUDT5 knockdown and knockout systems in TNBC cell lines, and characterized cell growth features over 7 days with or without NUDT5 depletion. Growth inhibition effect is also confirmed by NUDT5 inhibitor. After 72 hours of doxycycline induction, cell growth was determined by cell count over 7 days. Using these TNBC cellular models, we also determined molecular phenotypes after NUDT5 depletion by immunofluorescent microscopy for 8-oxo-dGDP and γH2AX. ImageXpress Automated Cell Imaging System and CellReporterXpress were applied for image processing and data quantification. Results: NUDT5 RNA is highly overexpressed in TNBCs as compared to ER-positive breast cancers and normal breast tissue. Survival analyses indicated that increasing NUDT5 level is significantly correlated with worse clinical outcomes of breast cancer patients. NUDT5 protein is highly expressed in TNBC cell lines, compared to ER-positive breast cancer cell lines. NUDT5 knockdown or knockout both significantly inhibits the growth of TNBC cell lines. Loss of NUDT5 induced the oxidative DNA lesion 8-oxoG level in the nucleus, as well as the ratio of γH2AX-positive cells. The inhibitor of NUDT5 also suppressed TNBC cell growth in vitro. Conclusions: Our results showed that the level of NUDT5 negatively correlates with TNBC patient survival. NUDT5 is required for the TNBC cells growth and tolerance to endogenous DNA damage agents. Our findings have revealed the essential function of NUDT5, and will provide critical insights for the future development of NUDT5 inhibitors for the treatment of TNBC patients. This work was supported by Charles Cain Endowment (PB). Citation Format: Jing Qian, William Tahaney, Yanxia Ma, Abhijit Mazumdar, Powel Brown. The role of a novel phosphatase NUDT5 in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-08.

The aim of the present study was to investigate the expression of estrogen receptor β (ERβ) in triple-negative and triple-positive breast cancer patients, and evaluate its utility as a prognostic factor. Between January 2000 and December 2010, primary tumor tissue samples were collected from 234 subjects, including 107 triple-negative and 127 triple-positive breast cancer patients. The samples were embedded in paraffin and immunohistochemical staining was conducted to determine the expression levels of ERβ. The Kaplan-Meier method was used to analyze patient survival rates. ERβ expression was observed in 38/107 patients (35.5%) with triple-negative breast cancer and 63/127 patients (49.6%) with triple-positive breast cancer. The ERβ expression rate was significantly decreased in the patients with triple-negative breast cancer, as compared with those with triple-positive breast cancer (P=0.03). Analysis of the survival rates indicated that patients with triple-negative breast cancer and positive ERβ expression exhibited poor disease progression-free survival (DFS) compared with those with negative ERβ expression (P=0.021). However, no statistically significant difference was observed in the DFS between the triple-positive breast cancer patients with positive and negative ERβ expression. Therefore, the expression of ERβ varies between triple-negative and triple-positive breast cancer patients. In addition, positive expression of ERβ indicates a poor prognosis in triple-negative breast cancer patients; however, this is not the case for triple-positive breast cancer patients.

The study aimed to explore the value of ultrasound (US) texture analysis in the differential diagnosis of triple-negative breast cancer (TNBC) and non-TNBC.Retrospective analysis was done on 93 patients with breast cancer (35 patients with TNBC and 38 patients with non-TNBC) who were admitted to Taizhou people's hospital from July 2015 to June 2019. All lesions were pathologically proven at surgery. US images of all patients were collected. Texture analysis of US images was performed using MaZda software package. The differences between textural features in TNBC and non-TNBC were assessed. Receiver operating characteristic curve analysis was used to compare the diagnostic performance of textural parameters showing significant difference.Five optimal texture feature parameters were extracted from gray level run-length matrix, including gray level non-uniformity (GLNU) in horizontal direction, vertical gray level non-uniformity, GLNU in the 45 degree direction, run length non-uniformity in 135 degree direction, GLNU in the 135 degree direction. All these texture parameters were statistically higher in TNBC than in non-TNBC (P <.05). Receiver operating characteristic curve analysis indicated that at a threshold of 268.9068, GLNU in horizontal direction exhibited best diagnostic performance for differentiating TNBC from non-TNBC. Logistic regression model established based on all these parameters showed a sensitivity of 69.3%, specificity of 91.4% and area under the curve of 0.834.US texture features were significantly different between TNBC and non-TNBC, US texture analysis can be used for preliminary differentiation of TNBC from non-TNBC.

Background: Triple negative breast cancer (TNBC) is known for its aggressive behavior, poor prognosis and still remains as a difficult disease since treatment options are limited. Despite some success in PARP inhibition in BRCA gene mutation patients or platinating agents that may offer superior outcomes in a subset of TNBC patients (pts), currently, there are no targeted therapies for TNBC available. Specific biomarkers are urgently needed for developing effective treatments to predict which patients will respond to the given therapy. In this regard, circulating tumor cells (CTCs) are discussed to be an ideal surrogate marker for individualized treatment options. Since TNBC is closely related to epithelial-mesenchymal transition (EMT), a stem cell phenotype and, in addition, androgen receptor (AR) expression has been detected in up to a third of TNBC pts, we here established a multi-marker gene panel for the characterization of CTCs in TNBC pts and compared these findings with CTC characteristics in non-TNBC pts. Methods: 2x5 ml blood of 30 TNBC pts before and/or after neoadjuvant therapy and 30 non- TNBC pts (E+/PR+: n=23; ER+/PR-: n=4; HER2+: n=1; HER2+/ER+: n=1; HER2+/ER+/PR+: n=1) before therapy were analyzed for CTCs applying positive immunomagnetic selection targeting EpCAM, EGFR and HER2 using the AdnaTest EMT-2/Stem Cell Select (QIAGEN Hannover GmbH, Germany). Subsequently, cDNA was gene specifically pre-amplified using TaqMan PreAmp Master Mix according to in house designed assays. Establishment of a 19 gene qPCR panel was performed for the markers PI3K, AKT2, ERCC1, Aurka, HER2, HER3, EGFR, ALK, AR (androgene receptor), BRCA1, c-KIT, c-MET, KRT5, mTOR, NOTCH1, PARP1, SRC1, CD45 (leucocyte control) and GAPDH (housekeeping gene) as well as an internal reference. The cutoff was calculated, taken the false positive rate in healthy donors into account and defined as Ct(cutoff)-Ct(sample)-[Ct(CD45cutoff)-Ct(CD45sample)]. Results: In general, the distribution of the markers across all patients was highly variable. However, different expression patterns were found when CTCs of TNBC pts were compared with those of non-TNBC pts. In TNBC pts, SRC1 was the gene that was predominantly expressed, followed by c-Kit, HER3, BRCA1 and AURKA expression, before as well as after therapy. Interestingly, AKT2, EGFR, ERCC1 and PARP1 expression could not be detected at any time point studied. In addition, ALK, AR, c-Met, HER2 and KRT5 were only detected before but not after therapy. All other genes were expressed below 15%. In contrast, in non-TNBC pts, AKT2 was the gene that was predominantly expressed, followed by c-MET, HER3 and PI3K whereas c-KIT, ERCC1, mTOR and NOTCH1 were never found. All other genes were expressed below 10%. Conclusion: We successfully established a gene panel for the detection of the heterogeneous CTC population and demonstrated that CTCs in TNBC pts and non-TNBC pts show different genetic profiles. Although these data have to be confirmed in a bigger patient cohort, the knowledge about the individual target gene expression profile might efficiently help to predict a personalized targeted therapy for these pts in the future. Citation Format: Bittner A-K, Hoffmann O, Hauch S, Kimmig R, Kasimir-Bauer S. Circulating tumor cells in triple-negative and non-triple negative breast cancer patients show different genetic profiles. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-05.

SummaryM2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.