Effects of moxibustion combined with medication on CD80 and CD86 expression in tumor tissue antigen-presenting cells of mice with triple-negative breast cancer

Rationale and purpose: Triple negative breast cancers (TNBCs), characterized by lack of hormone receptors' expression in absence of HER2 amplification, partially overlap with the basal-like subtype, with frequent occurrence in BRCA1 mutation carriers (BRCA1+). TNBCs are often associated with earlier onset, interval cancer diagnosis, larger size and aggressive clinical course with peak risk of recurrence at 1-3 years and increased mortality rate in the first 5 years. Thus, when screening women at high risk of breast cancer, special attention should be paid to patient outcome for TNBCs in comparison with non-TNBCs. Our aim was to compare TNBCs and non-TNBCs diagnosed during a prospective, non-randomized, multimodality screening study – including clinical breast examination (CBE), mammography, ultrasound (US) and magnetic resonance imaging (MRI) – on women at familial/genetic high risk of breast cancer conducted in 18 centres from June 2000 to March 2008 (ISS-HIBCRIT-1; Sardanelli F et al, Invest Radiol 2011). Methods: Comparisons were performed using Mann-Whitney U, Fisher exact and χ2 tests. Results: Among the 44 patients diagnosed with invasive cancers, 14 (31%) were TNBCs and 30 (69%) non-TNBCs, the former being 13 invasive ductal (IDC) and 1 atypical medullary carcinoma, the latter also including 15/30 lobular subtypes and/or DCIS component (p=0.005). Of the 14 TNBCs, 10 (71%) were found in BRCA1+, 2 (14%) in BRCA2+ and 2 (14%) in BRCA-untested women with strong family history of breast/ovarian cancer; the same data for 30 non-TNBCs were 9 (30%), 6 (20%) and 15 (50%) respectively (p=0.028). We had only three interval cancers, all TNBCs. The median age at diagnosis was 49 years (range 36-62) for TNBCs and 53 years (range 35-72) for non-TNBCs (p=n.s). TNBCs presented a higher rate (11/14, 79%) of pathological grade 3 IDCs compared with non-TNBCs (8/30, 27%) (p=0.002). The mean tumor size was 1.6 cm for TNBCs and 1.2 cm for non-TNBCs (p=n.s). Nodal status was negative in 12/14 (86%) TNBCs and in only 16/30 (53%) non-TNBCs (p=0.038). MRI similarly outperformed CBE, mammography and US in both TNBCs and non-TNBCs. Clinical course and survival could be monitored for 40/44 patients (91%), 13 TNBCs and 27 non-TNBCs, with a follow-up of 5.8 and 6.3 years (p=n.s) respectively. The rate of disease-free patients for over 5 years was 8/13 (62%; mean disease-free interval 7.0 years, range 5.0-8.0) for TNBCs and 17/27 (63%; mean 7.2 years, range 5.2-9.9) for non-TNBCs. Death due to BC occurred for 2/13 TNBC (15%, at 3.5 and 4.2 years) and 3/27 non-TNBC patients (11%; at 2.0, 4.9 and 5.7 years). The rate of locoregional relapse was 1/13 (8%, at 4.4 years) and 5/27 (19%; mean time of 5.0 years, range 2.4-7.0) respectively. Distant recurrence was reported for only 2 non-TNBC patients. Conclusion: TNBCs showed stronger association with BRCA1+ status, lower rate of lobular subtypes or DCIS component, and less frequent nodal involvement. Despite a more frequent pathological grade 3 and the tendency to be diagnosed as interval cancers, under the current treatment protocols TNBCs showed relapse and BC-related death rates and over-5-year disease-free intervals similar to those of non-TNBCs. These data provide outcome evidence supporting the value of entering women at high risk of TNBC (in particular BRCA1+) in intensive screening programs including MRI. Citation Format: Franca Podo, Filippo Santoro, Siranoush Manoukian, Clelia de Giacomi, Laura Cortesi, Lorenzo Preda, Stefano Corcione, Francesco Sardanelli. High-risk patients found affected with breast cancer during a multimodality screening program: Triple negative versus non-triple negative breast cancers. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B13.

Background: Specific biomarkers can be essential for developing effective treatments for aggressive breast cancers, especially triple negative subtypes, for which treatment options are limited. Folate receptor alpha (FRα), a critical membrane protein for DNA synthesis and cell metabolism, has been suggested to participate in the transformation of breast cancer into aggressive subtypes. It has been shown to be strongly associated with poor prognosis in triple negative breast cancers (TNBC) as well as estrogen receptor (ER) positive and progesterone receptor (PR) negative subtypes. SOX10 is a nuclear transcription factor that participates in neural crest development and in the differentiation of cells of melanocytic lineage. Data suggests that SOX10 may contribute in stem cell or progenitor cell maintenance. Recently, SOX10 expression has also been documented in benign breast myoepithelial cells and in aggressive breast cancers. The correlation of FRα and SOX10 in breast cancer is not fully known. This is the first study to compare FRα and SOX10 immunohistochemical profiles in breast cancers with emphasis in TNBC. Design: 166 cases of whole breast cancer tissues were classified according to their ER, PR, and HER2 immunohistochemical (IHC) status. These same cases were then IHC stained for mouse monoclonal SOX10 and FRα. Cut-off values of 1% and 5% for SOX10 and FRα, respectively, were used to determine positivity. Results: SOX10 achieved a sensitivity of 42.1% (8/19) in ER+/PR-/HER2- cases and was negative in all ER+/PR-/HER2+ cases (p<0.05). FRα was positive in 7.6% (7/92) of ER+/PR+/HER2- cases and was negative in all ER+/PR+/HER2+ cases. SOX10 identified more ER+/PR-/HER2- cases (42.1%, 8/19) than ER+/PR+/HER2+ cases (7.7%, 1/13) (p<0.05). Similarly, FRα stained 52.6% (10/19) of ER+/PR-/HER2- cases and was negative in all ER+/PR+/HER2+ cases (p<0.005). SOX10 and FRα were observed in 3.3% (1/30) and 20% (6/30) of HER2+ cases, respectively. In ER-/PR-/HER2- (triple negative) cases, both markers were highly expressed with 40.0% (10/25) and 52.0% (13/25) positive cases with SOX10 and FRα, respectively, with 24.0% (6/25) of cases positive with both markers. Approximately one half of TNBC cases expressed SOX10 and FRα; however, most SOX10 positive TNBC cases did not overlap with FRα positive TNBC cases. Table 1: SOX10 and FRα expression in breast cancer subtypesER/PR/HER2 ClassificationSOX10+ (%)FRα+ (%)Co-expression of SOX10 and FRα (%)ER+/PR+/HER2+ (n=13)1 (7.7%)0 (0.0%)0 (0.0%)ER+/PR+/HER2- (n=92)6 (6.5%)7 (7.6%)2 (2.2%)ER+/PR-/HER2+ (n=10)0 (0%)5 (50.0%)0 (0.0%)ER+/PR-/HER2- (n=19)8 (42.1%)10 (52.6%)5 (26.3%)HER2+ (n=30)1 (3.3%)6 (20.0%)0 (0.0%)ER-/PR-/HER2- (n=25)10 (40.0%)13 (52.0%)6 (24.0%) Conclusion: SOX10 and FRα were frequently expressed in triple negative breast cancers and in progesterone receptor negative breast cancers. Our data suggests that there may be different mechanisms by which SOX10 and FRα are implicated in aggressive breast cancers. These findings may help achieve a better understanding of the two different pathways involving stem cells (SOX10) and growth factors (FRα), their potential prognosis and their therapeutic management in the future. Citation Format: Laura L Hoang, Weimin Qi, Charlie Yu, David Tacha. SOX10 and folate receptor alpha are frequently expressed in triple negative and progesterone receptor negative breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-13.

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.

Block of programmed cell death protein 1 (PD-1) interaction with its ligand, PD-L1, enhances anti-tumor activity. We aimed to assess the association between PD-L1 expression in tumor cells and CD8+ tumor infiltrating Tcells (TILs) as well as soluble (s)PD-L1 serum levels in patients with triple negative breast cancer (TNBC) compared to triple positive (TPBC). A total of 113 tumor sections and 133 serum samples were available from 144 patients with breast cancer (72 TNBC and 72 TPBC). Dual immunohistochemistry staining was applied to determine differential PD-L1 expression in tumor cells and CD8+ TILs. Soluble PD-L1 serum levels were also evaluated in patients compared to 40 healthy women by ELISA method. Despite TPBC patients which were mostly grades 1/2, TNBC patients were grade3 (72% versus 66.7%, P <0.001). Most of the TNBC patients were stages I/II, whereas most of the TPBC patients were stages III/IV (57.3% versus68.3%,P =0.005). There was no difference in tumor size and metastasis between TNBC and TPBC patients, although the number of involved lymph nodes was significantly more in TPBC patients (P =0.0012). PD-L1 expression was detected in 11.5% of samples mostly in TNBC subtype and was associated with advanced grades (P =0.039). There was no relationship between PD-L1 expression and tumor stage. PD-L1 expression in CD8+ TILs was nonsignificantly higher than tumor cells. Serum levels of sPD-L1 showed no difference between patients and healthy women. We found no correlation between PD-L1 expression in tumor lesions and serum levels of sPD-L1 in patients. PD-L1 expression was more detected in our patients with TNBC. It seems that, these patients who are resistant to standard chemotherapy regimens may get benefit from PD-L1 inhibition therapy and because of its low serum levels, sPD-L1 cannot interfere with this therapy.

Introduction: Triple negative breast cancer (TNBC) represents 10-20% of all breast cancer entities [1][2] and has a known aggressive behavior and poor outcome. Patients treated in the setting of randomized clinical trials often do not represent actual treatment characteristics in real-life scenarios. To determine the stage-related survival and effect of surgical performance in TNBC with current multimodal treatment, we set out to analyze data of a large population-based registry of primary breast cancers which covering &amp;gt;50% of all breast cancer cases in Germany. Patients and methods: We analyzed data from a prospectively collected cancer registry of &amp;gt;200 certified breast units of the West-German Breast Center (WBC) in Germany from 2009-2011. From a cohort of 39570 primary breast cancer patients treated in this period, 12759 underwent adjuvant systemic therapy, out of which 2037 were TNBC cases with adjuvant chemotherapy. Inclusion criteria were triple negative breast cancers (Her2-new1+/2+ (Fish negative) and estrogen receptor (ER) and progesterone receptor (PR) &amp;lt;10%) and adjuvant chemotherapy, unilateral and non-metastasized breast cancer. Only those patients were included who have been followed-up within the first 3 years. Exclusion criteria were neoadjuvant chemotherapy, bilateral breast cancer and metastatic disease. The use of first, second and third generation chemotherapy was analyzed as well as the effect of clear/unclear resection margins and its impact on survival data. Results: 2037 patients were eligible for this study. Overall survival rates were as follows: T1 a and T1b 100 %, T1c 90,7 %, T2 90,9 %, T3 68,1 % and T4 64,3 %. No statistical differences were detected in between stages T1 and T2, and also not in between T3 and T4. Combining T1/T2 and T3/T4 and performing group-wise comparisons, differences for combined stages were highly statistically significant (3,9 x E-09). Inflammatory TNBC was prognostically worst with a survival-rate of 33,3 % at 24-months. (p&amp;lt;0,001) Unclear resection-margins versus clear margins in TNBC exerted a negative impact on DFS (87 vs. 73 %; p=0,00002) and DDFS (p=0,0004). Age was an independent risk factor for survival with a cut-off at 35 years.(p=0,044) Third-generation chemotherapies (anthracycline+taxanes) were associated with a significant improved overall-survival at 24-months compared to first generation chemotherapies (non-anthracycline, non-taxane) (95 % vs. 87 %; p=0,0029) Conclusion: Standard 3rd generation (anthracycline- and taxane-containing) chemotherapy and optimal surgical performance with clear margins is vital for patients with early, triple-negative breast cancer (TNBC). Within T1 and T2 stages, no stage-related deterioration of prognosis was detected, however these stages were markedly different from stages T3/T4, declining from 90-100% to 64-68 %. This analysis of a large database of a population-based study demonstrates that tumor size, margins and guideline-adapted chemotherapy matter in triple-negative, early breast cancer. [1] Schwentner et al. 2013 [2] Elsawaf et al. 2013. Citation Format: Peter Kern, Gunter von Minckwitz, Carolin Pütter, Annika Flach, Sofia Pavlidou, Rainer Kimmig, Mahdi Rezai. Stage-related risk categorization and influence of free margins on survival in triple negative early breast cancer - a population-based study of 2037 TNBC patients with adjuvant chemotherapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-13-01.

Introduction: Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype associated with high histologic grade and poor prognosis. TNBC is also known to be associated with modified metabolic activities of both tumor and stromal cells in the tumor microenvironment, potentially as a mechanism to enhance tumor development and survival in low-nutrient conditions. Here we aimed to investigate the differences in the metabolic activity of TNBC vs. non-TNBC and their impact on survival. Methods: Comprehensive immune profiling, including the expression of 395 immune-associated genes measured by RNA-seq, and PD-L1 expression testing by IHC, was performed on 149 real-world breast cancer samples. 52 samples were, by definition, triple negative for ER, PR, and HER2 overexpression. Based on Reactome pathway database data, mRNA expression signatures of carbohydrate (7 genes), lipid (10 genes), protein (29 genes), vitamin/cofactor (6 genes), and overall (32 genes) metabolism were calculated by averaging the normalized gene expression of each gene set. Samples were grouped into high (greater than or equal to median expression) and low (less than median expression) groups for each metabolic signature. Statistical comparisons of biomarkers between groups were performed using the Wilcoxon Rank-Sum test for continuous variables and Fisher’s Exact Test for categorical variables (p≤0.05 for significance). Survival differences were quantified by Kaplan-Meier analysis (p≤0.05 for significance). Results: In general, TNBC demonstrated greater overall metabolic activity (p=0.001), including greater lipid (p=5.8×10-5), protein (p=0.0053), and vitamin/cofactor (p=0.04) metabolism. However, there was no significant difference in carbohydrate metabolism between TNBC and non-TNBC. Among TNBC cases, only higher vitamin and cofactor metabolism was associated with better overall survival (OS) (61 months vs. 37 months, p=0.029), while carbohydrate, lipid, protein, and overall metabolic activity were not significantly associated with OS. For non-TNBC cases, high overall metabolic activity was associated with better OS (180 months vs. 78 months, p=0.011), as was high carbohydrate (147 months vs. 79 months, p=0.0053), high lipid (162 months vs. 73 months, p=0.016), and high protein metabolism (148 months vs. 73 months, p=0.026). Conclusions: We found that, in general, TNBC is more metabolically active than non-TNBC, though the survival effects of this difference vary depending on the specific aspect of cellular metabolism being measured, with some signatures associated with OS in TNBC and others in non-TNBC. An improved understanding of the metabolic environment of breast cancer as it relates to both triple negative status and differences in patients may facilitate a more nuanced characterization of tumor subtypes, aiding in the development of treatment strategies taking this aspect of the tumor microenvironment into account. Citation Format: Sarabjot Pabla, Shipra Gandhi, Maria-Fernanda Senosain, Hardik Parikh, Erik Van Roey, Shuang Gao, Yamuna Pulivendula, Paul DePietro, Jeffrey M. Conroy, Stephanie B. Hastings, Kyle C. Strickland, Rebecca A. Previs, Eric Severson, Brian J. Caveney, Marcia Eisenberg, Taylor J. Jensen, Shakti Ramkissoon, Heidi Ko. Triple negative and non-triple negative breast cancer have distinct metabolic characteristics associated with divergent survival outcomes [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 P3-01-24.

PD-L1 and CD8 are associated with deficient mismatch repair status in triple-negative and HER2-positive breast cancers

Objective To investigate the metastasis and the prognosis of the axillary lymph nodes in triple-negative and invasive ductal breast cancer patients of different ages. Methods 321 female breast cancer patients diagnosed as triple-negative and invasive ductal carcinoma from Jan. 1, 2008 to Dec. 31, 2017 were selected as the samples, all of whom were treated with regular surgical treatment and postoperative radiotherapy and chemotherapy, and were divided into three groups according to their ages, including the younger group (<40 years old) , the middle age group (40 to 60 years old) , and the elder group (≥60 years) . We compared the metastasis of axillary lymph node, the disease-free survival rate after 1 to 5 years of the operations and the prognostic factors of the three groups. Results Among the 321 patients, there were 94 young patients, 151 middle-aged patients and 76 elder patients. Among the three groups, the rate of axillary lymph nodes metastasis was the lowest in the elderly group (11.8%) , the highest in the middle-aged group (17.2%) and middle in the young group (13.8%) . The patients were followed up for 1 to 5 years. The recurrence rate of the young, middle-aged and elder groups was 56.4%, 53.6% and 17.1% respectively. There was a significant difference between the three groups (P<0.05) . Conclusion ①he frequency of LN transfer in patients of TNBC is lower in the younger and the elder patients than in the middle-aged patients. ② The younger patients of TNBC have a higher recurrence rate and poor prognosis, while the elder patients of TNBC have the lowest recurrence rate and good prognosis. ③The prognosis of TNBC may be related to metabolism, which, of course, needs to be further verified with the proof of blood and cell test. ④The younger patients of TNBC are more likely to suffer blood metastasis, and adjuvant systemic therapy in early period may be more beneficial than local radiotherapy and early axillary dissection. Key words: Triple negative breast cancer (TNBC); Axillary lymph node; Hematogenous dissemination

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.

Objective To study the expressions of integrin β1 and p73 in triple negative (ER-、PR-、HER2-) breast cancers and evaluate its clinical significance, exploring the relationship between clinicopathological parameters. Methods The immunohistochemistry SP methods was used to evaluate the expression of integrinβ1 and p73 in 93 cases of triple negative breast cancer,58 cases of none triple negative breast cancer. The correlation of integrinβ1 and p73 exprssion with clinicopathologic characteristics and between the two proteins were analyzed. Results The expression of integrinβ1 in the triple negative breast cancers was significantly higher than those of none triple negative breast cancer(83.87% vs 67.24%), with a significant difference(χ2=5.66,P=0.017). The expression of p73 in the triple negative breast cancers was significantly higher than those of none triple negative breast cancer (65.59% vs 46.55%), with a significant difference(χ2=5.33,P=0.021). In triple negative breast cancers,integrinβ1 expression was associated with axillary lymph node metastasis(χ2=6.206,P=0.021)and TNM stage(χ2=4.854,P=0.041), but it was not correlated with patients age(χ2=0.121,P=0.783), tumor size(χ2=1.402,P=0.271)and histologic grade(χ2=1.042,P=0.376). p73 expression was associated with histologic grade(χ2=5.503,P=0.020),axillary lymph node metastasis(χ2=4.626,P=0.047)and TNM stage(χ2=5.828,P=0. 024), but it was not correlated with patients age(χ2=0.858,P=0.387), tumor size(χ2=0.875,P=0.388).Correlation analysis showed that integrinβ1 expression was positively correlated with p73 expression in triple negative breast cancers(r=0.359,P=0.000). Conclusions p73 has its tissue specificity, both integrinβ1 and p73 protein may participate in the development of triple negative breast cancers, and could serve as an important potential marker in the aspect of invasiveness and metastasis in triple negative breast cancers. Key words: breast neoplasms; Triple negative; immunohistochemistry; integrinβ1; p73

The Role of Ultrasound of the Regional Nodal Basins in Staging Patients With Triple-Negative Breast Cancer: Implications for Local-Regional Treatment

Triple (ER-, PR-, HER2-) negative breast carcinoma lack targeted therapies, making this group of tumors difficult to treat. By definition, the lack of HER2 expression means a case scoring 0 or 1+ after immunophenotypical analysis and makes the patients avoiding therapeutical chances with anti-HER2 inhibitors. We sought to recruit from a group of triple negative breast carcinoma, patients eligible for effective personalized targeted therapy with anti-HER therapies on the basis of their HER2 gene status. 135 patients diagnosed with IHC triple negative breast carcinoma were studied. Whole tissue sections were used for in situ hybridization analysis. 8/100 (8%) of ductal-type triple negative breast carcinoma presented Her-2/neu gene amplification versus 2/35 (5.7%) non-ductal triple negative breast carcinoma. Three cases showed a ratio 2.5. One case showed Her-2/neu heterogeneous gene amplification, ratio 2.3. The other six showed from 7 to 8 absolute Her-2/neu gene copy number. Two cases staged pT1c, and eight cases staged pT2. Eight cases graded G3 and two cases G2. (1) Eight percentage of ductal and 5.7% non-ductal-type triple negative breast carcinoma present Her-2/neu gene amplification, (2) the standard diagnostic flowchart "do not FISH in 0-1+ (HER2-) breast carcinoma" should be replaced by "do FISH in triple (ER-, PR-, HER2-) negative breast carcinoma," to avoid loss of therapeutical chances in a cohort of such a patients, (3) we demonstrated the identification of a small but significant subset of patients targetable with anti-HER2 inhibitors, giving patients affected by (ex)triple negative breast carcinoma new personalized therapeutical chances.

Background: Growing evidence has demonstrated that circulating tumor DNA (ctDNA) is a sensitive and specific assay for predicting distant recurrences from breast cancer (BC). Patients with detectable ctDNA after neoadjuvant chemotherapy have a higher risk of distant recurrence compared to patients with negative ctDNA testing. Further, ctDNA positivity precedes clinical metastatic relapse by 10.5-12 months (mos) on average. SignateraTM (Natera, Inc.) is a tumor-informed and commercially available ctDNA assay that has been CMS-reimbursed for stage IIB and higher BC since 2023. It is unknown whether ctDNA testing influences BC patient anxiety or physician decision-making. In this study, we describe patient reported anxiety levels using the Patient-Reported Outcomes Measurement Information System (PROMIS) and assess physician decision-making using a provider decision assessment tool (PDAT) in the setting of an institutional registry for patients who underwent ctDNA surveillance during routine follow-up. Methods: This is a single institution observational study of ctDNA surveillance in patients with early stage hormone receptor positive (HR+) or triple negative BC (TNBC) with residual disease after neoadjuvant treatment. ctDNA was assessed using the Signatera assay at baseline (prior to adjuvant therapy in ER+ disease or following surgery in TNBC) and every 6 mos for 2 years or until clinical evidence of recurrence. Patients with positive ctDNA were re-staged with scans as deemed appropriate by the treating physician. Patients were invited to complete an anxiety questionnaire using the PROMIS every 6 mos, online, or in clinic. Treating physicians were invited to complete a PDAT at the time of ctDNA test results. Mean scores on the PROMIS and PDAT were calculated at baseline and 6 mo intervals. Higher PROMIS scores correspond to higher anxiety levels. This planned interim analysis is at 50% of recruitment (n=48). T scores will be analyzed and reported at final analysis. Results: 48 patients (TNBC n=29, HR+ n=19) were enrolled in the surveillance study, with mean age of 52 years (range 24-84). ctDNA results are available for 45 participants at baseline, and 25 at 6 mos. Seven patients, 6 TNBC (4 stage IIB and 2 stage IIIB disease), and 1 HR+ (stage IIB) had positive ctDNA. Five (11.1%) were positive at baseline and two (8%) turned positive at 6 mos. Three of the 7 (42.9%) ctDNA positive patients, all TNBC, had detectable metastatic disease on imaging at the time of ctDNA positivity. PROMIS results are available for 40 patients at baseline and 23 at 6 mos. PROMIS mean total scores were similar at baseline and 6 mos (44.5 vs 42.5, p=0.648) for the whole group, in ctDNA negative patients (44.7 vs 42.1, p=0.488), and in ctDNA positive patients (42.3 vs 40.5, p=0.508). PDAT mean scores were also similar at baseline and 6 mos (41.2 vs 41.1, p=0.588). PROMIS data is available for 3/4 participants with ctDNA positive / imaging negative results and showed a small increase in anxiety levels at 6 mos vs baseline (32 vs 30, 48 vs 38, 49 vs 44). Conclusion: To our knowledge, this is the first study to report on patient reported anxiety levels associated with ctDNA surveillance in early stage BC. At this interim analysis, no statistically significant difference in patient reported anxiety was seen at 6 mos compared to baseline. Average anxiety scores were similar to the US general population (average 50, standard deviation 10). The majority of ctDNA results were negative; among patients with positive ctDNA testing, small increases in anxiety levels were detected, but the sample size was limited. PDAT scores remained stable. This interim analysis suggests that ctDNA surveillance may not adversely affect patient anxiety. However, further follow up and future randomized trials are needed. Citation Format: Devora Isseroff, Nathalie Wiesendanger, Adriana Kahn, Daniel O'Neil, Michael Cohenuram, Anca Bulgaru, Kathleen Fenn, Johanna LaSala, Kert Sabbath, Neal Fischbach, Jane Kanowitz, Robert Legare, Michael DiGiovanna, Andrea Silber, Tara Sanft, Sarah Schellhorn, Himanshu Sethi, Ekaterina Kalashnikova, Samuel Rivero-Hinojosa, Minetta Liu, Ian Krop, Eric Winer, Wei Wei, Maryam Lustberg, Lajos Pusztai, Mariya Rozenblit. Patient reported anxiety levels during ctDNA surveillance in early stage triple negative and hormone positive 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 P4-03-29.

Background: The complexity of breast cancer (BC) development and progression is heavily influenced by the cross-talk of critical cell types within the tumor microenvironment. While there have been many advancements in BC treatment, triple negative breast cancer (TNBC) still exhibits the lowest rate of response and the nature of its aggressive properties remain poorly understood. African American (AA) women are disproportionally affected and have worse overall survival. Our lab previously reported important role of beige adipocytes in breast tumor growth. Interestingly, we found beige adipocytes were highly up regulated in AA TN breast tumors when compared to TNBC from Caucasian (CA) women. In this study, we have further investigated the mechanisms that increase beige adipocytes in AA TN breast tumors and their influence on breast tumor growth. Methods: Human tumor samples provided by Cooperative Human Tissue Network and National Disease Research Institute were subjected to immunohistochemistry (IHC) and western blot analysis to examine key beige adipocyte markers. To further investigate the mechanisms by which beige adipocytes may contribute tumor growth, xenografts were established from AA TNBC cell lines HCC70, HCC1806 and MDA-MB 468. Tumors excised at various time points were examined for beige adipocyte markers and their key regulators using validated species specific primers to distinguish between host and tumor cell contributions. Boyden chamber assays and ex-vivo experiments were performed to examine whether beige adipocytes were recruited from the host or induced in xenografts, and test whether paracrine secretion of cytokines or contact between various cell types were necessary for adipose browning in these tumors. Possible involvement of lactic acid-mediated adipose browning in these tumors was tested by inhibiting lactate dehydrogenase using sodium oxamate. We further examined whether increased browning in these tumors was associated with increase in anti-inflammatory cytokines that promote M2 macrophage phenotype and angiogenesis, both of which are highly implicated in tumor progression. Results: Both quantitative IHC and western blot analysis showed higher expression of beige adipocyte markers CD137 and TMEM26 in AA TNBC compared to CA TNBC. Expression of beige marker CD137 was detected in all xenografts from AA TNBC by IHC. Quantitative gene expression analysis (qPCR) revealed that host contributed to the origin of beige adipocytes in these tumors. We demonstrate using Boyden Chamber assay that contact between tumor cells and host adipocytes was important for adipose browning. Ex-vivo experiments also showed that breast cancer cells in contact with either host adipocytes or 3T3-L1 mouse adipocyte cell line increased the expression of beige markers that could be attenuated by lactate dehydrogenase inhibitor. We further show that increase in beige adipocytes in AA TNBC was associated with high anti-inflammatory cytokines IL13/IL4 expression, as well as anti-inflammatory pro-tumorigenic M2 macrophages and high angiogenesis. Conclusion: Key beige markers CD137 and TMEM26 are significantly upregulated in AA TNBC compared to the CA TNBC. Host microenvironment contributed to beige adipocyte characteristics in response to lactic acid-mediated adipose browning of tumor cells. Furthermore, beige adipocytes increased anti-inflammatory cytokines that facilitate accumulation of M2 macrophages and promote angiogenesis. Our results implicate that beige adipocytes are key mediators of aggressive tumor development in AA TNBC and could potentially be targeted for therapeutic purposes. Citation Format: Brandis A. Moore, Briana Osorio, Rajan Singh, Shehla Pervin. Upregulation of beige adipocytes in African-American triple negative breast tumors induce tumorigenesis by promoting anti-inflammatory microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-12.

Purpose: Recent studies evaluating the efficacy of bev in BC have shown conflicting results, particularly in hormone receptor (HR)+ BC. Identification of predictive biomarkers and their relationship to the pharmacodynamic effects of bev would facilitate the identification of BC patients (pts) most likely to benefit from bev. To examine this, we conducted a unique preop phase II trial with a run-in of single agent bev followed by dose-dense AC-T with bev in two cohorts, one with HR+HER2- BC pts, and a smaller triple negative (TN) BC pts. Methods: Pts with HR+, HER2- or TNBC were eligible. Treatment consisted of a single dose of bev 10 mg/kg, followed two wks later by A 60 mg/m2 and C 600 mg/m2 with bev 10 mg/kg q2 wks x 4, followed by T 175 mg/m2 with bev 10 mg/kg q2 wks x 3, followed by T 175 mg/m2 x1. Core biopsies and interstitial fluid pressure (IFP) were assessed pre- and post- bev alone. Pathologic response was confirmed centrally and Miller-Payne (MP) was assessed. Results: The study enrolled 84 pts with HR+ and 20 pts with TN BC. Amongst HR+ pts, 79 had surgical tissue centrally reviewed, and 6 (8%) had a pCR. Amongst TN BC pts, 19 pts had tissue centrally reviewed and 9 (47%) had a pCR. Grade was found to predict MP response in both HR+ and TN pts (p=0.001). Tissue biomarkers and IFP were evaluated as predictors of response to bev. Single-agent bev reduced the mean IFP in the overall cohort and HR+ patients by 20 (p=0.020) and 24.5% (p=0.001), respectively. The IFP decreased &amp;gt; 50% in 24/65 pts and did not change in others. Bev reduced the mean vascular density (MVD) by 33.0% (p&amp;lt;0.05) in TN BC pts, but did not affect the vessel area fraction covered by perivascular cells (PCs). Bev did not modify the fraction of tumor tissue positive for the hypoxia marker CAIX. In TN BC pts, pre- and post-bev, the MVD was inversely correlated with the CAIX fraction (p&amp;lt;0.01). In addition, a drop in MVD associated with increased CAIX+ fraction post-BEV (p=0.05). MP score was more favorable for TN BC pts with lower CAIX+ fraction at baseline (p=0.058) and post-BEV (p&amp;lt;0.05), and higher MVD at baseline (p&amp;lt;0.05) and post-BEV (p&amp;lt;0.05). In contrast, in HR+ BC, BEV reduced MVD non-significantly by 15% (p=0.25) and increased the vessel area fraction covered by PCs (p&amp;lt;0.05). There was no significant correlation between MVD and CAIX+ fraction in HR+ BCs. In contrast to TN BC, in HR+ BCs the fraction of CAIX+ tumor was directly correlated with MP score (p&amp;lt;0.01). Discussion: Collectively, these results indicate that vascular pruning post-BEV may reduce vascular function and increase hypoxia, which is associated with less favorable pathologic response after BEV with chemotherapy in TN BC. In conclusion, our exploratory study suggests that elevated hypoxia and reduced MVD in TN BC reduces the effectiveness of chemotherapy. Citation Format: Yves Boucher, John D. Martin, Sara M. Tolaney, Giogio Seano, Shom Goel, Marek Ancukiewicz, Steven J. Isakoff, Eric P. Winer, Ian E. Krop, Rakesh K. Jain. Tissue biomarkers and interstitial fluid pressure in a phase II study of preoperative (preop) bevacizumab (bev) followed by dose-dense doxorubicin (A)/cyclophosphamide (C)/paclitaxel (T) in combination with bev in HER2-negative operable breast cancer (BC) [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-76. doi:10.1158/1538-7445.AM2013-LB-76