Triple-negative Breast Cancer Cases Research Articles

Immunohistochemical profile of triple negative breast cancers: SOX10 and AR dual negative tumors have worse outcomes.

Triple negative breast cancer (TNBC) refers to an estrogen receptor negative, progesterone receptor negative, and HER2-negative breast cancer. Although accepted as a clinically valid category, TNBCs are heterogeneous at the histologic, immunohistochemical, and molecular levels. Gene expression profiling studies have molecularly classified TNBCs into multiple groups, but the prognostic significance is unclear except for a relatively good prognosis for the luminal androgen receptor (LAR) subtype. Immunohistochemistry (IHC) has been used as a surrogate for basal and luminal subtypes within TNBC, but prognostication of TNBC using IHC is not routinely performed. We aimed to study immunophenotypic correlations in a well-annotated cohort of consecutive TNBCs, excluding post-neoadjuvant chemotherapy cases. Tissue microarrays were constructed from a total of 245 TNBC cases. IHC stains were performed and consisted of luminal (AR, INPP4B), basal (SOX10, Nestin, CK5, EGFR), and diagnostic (GCDFP15, mammaglobin, GATA3, TRPS1) markers. Survival analysis was performed to assess the significance of clinical pathologic variables including age, histology, grade, lymphovascular invasion [LVI], Nottingham prognostic index [NPI] category, AJCC stage, stromal tumor-infiltrating lymphocytes at 10% increment, CD8+ T-cell count, Ki-67 index, PD-L1 status, and chemotherapy along with the results of IHC markers. Apocrine tumors show prominent reactivity for luminal markers and GCDFP15 while no special type carcinomas are often positive for basal markers. TRPS1 is a sensitive marker of breast carcinoma but shows low or no expression in apocrine tumors. High AJCC stage, lack of chemotherapy, and dual SOX10/AR negativity are associated with worse outcomes on univariable as well as multivariable analysis. LVI and higher NPI category were associated with worse outcomes on univariable but not multivariable analysis. The staining for IHC markers varies based on tumor histology which may be considered in determining breast origin. Notably, we report that SOX10/AR dual negative status in TNBC is associated with a worse prognosis along with AJCC stage, and chemotherapy status.

Abstract PO5-06-09: Metastatic triple negative breast cancer has distinct tumor immune landscape

Abstract Background: Triple negative breast cancer (TNBC) is considered the most immunogenic breast cancer subtype due to higher levels of tumor infiltrating immune cells (TILS), elevated tumor mutational burden and PD-L1 expression, providing a rationale for immunotherapy. Here we aimed to investigate the differences in the immune microenvironment of primary vs. metastatic sites in TNBC vs. non-TNBC and their impact on survival. Methods: Comprehensive immune profiling, including PD-L1 IHC and the expression of 395 immune genes, was performed on 147 real-world FFPE breast cancer samples (32 primary, 115 metastatic). 37 samples were, by definition, triple negative for ER, PR, and HER2 overexpression. PD-L1 (CPS positive ≥1% and CPS positive ≥10%) was determined using immunohistochemistry (22C3). mRNA expression signatures of tumor inflammation (TIGS, weak/moderate/strong) and cell proliferation (CP, poor/moderate/high) were determined by RNA-sequencing. Demographic and clinicopathologic variables were collected. Statistical comparisons of biomarkers between groups were calculated using the Wilcoxon Rank-Sum test for continuous variables and the proportions test for categorical variables (p≤0.05 for significance). Survival differences were quantified by Cox proportional hazards analysis (p≤0.05 for significance). Results: Triple negative status was associated with current use of alcohol [p=0.03]. Black patients were also more likely to have TNBC, constituting 24% of TNBC patients while only making up 14% of the total cohort [p=0.04]. Conversely, white patients were less likely to have TNBC, constituting 73% of TNBC patients while making up 84% of the total cohort [p=0.04]. Comparing TNBC and non-TNBC cases, TNBC cases were observed to have significantly higher TIGS [p=0.014] and PD-L1 expression [p=4.4 × 10−7]. Additionally, TNBC cases trended towards exhibiting greater cell proliferation [p=0.069]. These differences were found to be driven primarily by metastatic tumors, among which TNBC tumors showed significantly higher TIGS [p=0.015], cell proliferation [p=0.021], and PD-L1 expression [p=2.9 × 10−7] than non-TNBC tumors, while none of these significant associations were observed among primary tumors. Triple negative status was significantly associated with PD-L1 expression (assessed by IHC), with a majority of PD-L1 positive cases determined to be triple negative [70% of CPS≥1% cases, p=5 × 10-5; 59% of CPS≥10% cases, p=6 × 10−4]. Several immune-related genes and immune checkpoint molecules were over-expressed in triple negative breast cancer, including CD8, GZMB, PRF1, CCL5, IFNG, TIGIT and CXCL10. A number of genes were also significantly overexpressed in metastatic tumors: KRT5 [p=0.02], TFRC [p=0.04], ABCF1 [p=0.04], FCRLA [p=0.05], LAMP3 [p=0.05], and underexpressed in metastatic tumors: ENTPD1 [p=0.05], IGSF6 [p=0.03], and ITGA1 [p=0.03]. Conclusions: Our comprehensive biomarker analyses showed that metastatic TNBC has a more inflamed tumor microenvironment and higher checkpoint target expression compared to non-TNBC. Further analysis of immune expression by site-specific metastases and correlation with immunotherapy outcomes is warranted to guide clinicians in selection of the ideal metastatic site to biopsy for therapeutic decision making. However, in a collective TNBC context, these data support the use of immunotherapy in metastatic TNBC. Citation Format: Robert Seager, Heidi Ko, Sarabjot Pabla, Maria-Fernanda Senosain, Erik Van Roey, Shuang Gao, Kyle Strickland, Rebecca Previs, Mary Nesline, Stephanie Hastings, Shengle Zhang, Jeffrey Conroy, Taylor Jensen, Marcia Eisenberg, Brian Caveney, Eric Severson, Shakti Ramkissoon, Shipra Gandhi. Metastatic triple negative breast cancer has distinct tumor immune landscape [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 PO5-06-09.

Abstract PO5-13-09: Clinical usage of a CLIA accredited protein/phosphoprotein assay for breast cancer: Primary usage cases and significant findings in the HER2- setting

Abstract Background: A CLIA-accredited assay that measures the expression and/or activation levels of 32 clinically actionable protein and phosphoprotein analytes in clinical tissue biopsy samples was launched in January 2021. The current intended use population for the assay is for patients with late/advanced stage breast cancer wherein molecular profiling is being utilized for clinical decision making. Assessment of physician ordering practices, clinical use dynamics as well as protein/phosphoprotein actionability frequencies were determined for clinically actionable biomarkers. Methods: A total of 219 cases have been processed to date, with mean turn-around time of 16.4 days. The assay utilizes laser capture microdissection to enrich tumor epithelium from FFPE core needle biopsy and resection material coupled to reverse phase protein array. Results for each of the 32 analytes are generated by comparing each patient to a reference population and reported both as a relative quartile and as a continuous percentile compared to the reference population (0-100%). For the purposes of this analysis, significant expression/activation cutpoints were set at the 40th percentile threshold. Results: To date, clinicians have requested the test most frequently to identify potential therapeutic targets in patients with HER2-negative disease (84% of all tested patients, 36% were triple-negative). Despite the majority of cases being HER2-negative, we found that phosphorylated (p) HER2 (Y1248) was found to be activated in 17% of all HER2-negative cases and 19% of triple negative breast cancer cases (TNBC). pHER2 was concurrently activated with pHER3 (Y1289) in 15% (12/80) of TNBC cases and 13% (24/187) of all HER2-negative cases. pHER2 presented concurrently with pEGFR (Y1068 or T654) in 14% (11/80) of TNBC cases and 14% (26/187) of all HER2- cases. Expression of TROP2 was observed in 16% of HER2-negative patients including 11% of ER+/HER2- and 23% of TNBC cancers. MHCII expression, a recently described candidate predictive biomarker of anti-PDL1/PD1 response in TNBC, was found to be expressed in 29% of TNBC patients. Activation frequencies of other clinically relevant drug targets in HER2-negative disease such AKT and mTOR pathway activation was also assessed. We found that pAKT (S473 and/or T308) was activated in 7% of ER+/HER2- cases and 14% of TNBC. Concurrent with AKT activation, at least one additional member of the AKT-mTOR pathway (mTORC1 (S2448), 4E-BP1 (S65), p70S6K (T389), or S6RP (S235/S236) was co-activated in 50% and 45% of ER+/HER2- and TNBC cancers respectively. Conclusion: This initial assessment of a CLIA LDT protein/phosphoprotein assay utilized to date found that physicians most often order the test for advanced stage HER2-negative breast cancer. Activation/expression of clinically important protein drug targets with potential actionability in HER2-negative disease was observed in a significant number of cases and demonstrates the power of the assay to elucidate actionable targets for potential therapeutic intervention. Citation Format: Claudius Mueller, Brian Corgiat, Emanuel Petricoin, Kayla Sparks, Joyce O'Shaughnessy, Chelsea Gawryletz, Matthew Schwartz, Justin Davis. Clinical usage of a CLIA accredited protein/phosphoprotein assay for breast cancer: Primary usage cases and significant findings in the HER2- setting [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 PO5-13-09.

Abstract PS18-09: The DARC side of Breast Cancer - DARC, Duffy-null and African ancestry influence in the Triple Negative Breast Cancer tumor microenvironment

Abstract Background. Despite a recent convergence of breast cancer (BC) incidence rates between African American (AA) and European American (EA) women, AA women continue to have ~40% higher mortality rates. BC is a heterogeneous collection of diseases, where African ancestry women are disproportionately burdened with the most aggressive subtype of BC, triple negative BC (TNBC). We have previously reported that the African ancestry-specific Duffy-null allele (FY-) is a risk factor for TNBC diagnoses. FY- is a promoter variant that removes expression of the Duffy Antigen Receptor for Chemokines (DARC/ACKR1), from red blood cells (RBCs). DARC serves as a portal of entry for Plasmodium vivax malaria parasite, and FY- confers immunity, leading to fixation of FY- among Sub-Saharan African populations. DARC is an atypical chemokine receptor that functions to modulate chemokine levels in circulation and tissues to aid immune cell recruitment. DARC is also expressed on breast tumor epithelial cells, however the significance of DARC in the TNBC tumor microenvironment (TME) has not fully been explored. Leveraging our African ancestry enriched International Study for the Center of Breast Cancer Subtypes cohort and in vivo murine models, we employed multi-omics approaches to explore the function of FY- and DARC expression across cell types in the TNBC TME. Genomics. Using transcriptomic data from the TCGA BC cohort, we have previously reported that DARC expression is highly correlated with deconvoluted immune cell abundance using CIBERSORT. To build upon this finding among TNBC patients, we have analyzed RNAseq data from two cohorts of African ancestry women to explore correlation of DARC expression status and immune cell abundance. Among TNBC cases, we report that higher DARC expression was significantly associated with increased immune cell abundance, with increases in B cell, T cell and monocyte populations. Using whole genome sequencing data across 86 African ancestry cases, we are currently investigating germline FY- status with mutational signatures. Circulating biomarkers. Patient plasma was collected prior to surgery, where Luminex multiplex assays were performed to quantify levels of >40 circulating chemokines and cytokines. FY- status has been determined for ~550 patients using single-plex or multiplex PCR genotyping methods from germline saliva DNA. Our univariate analyses show several circulating chemokines, including CCL2, CXCL2, CXCL6 and CXCL11, have significant differences between FY- and non-FY- BC patients. Curation of clinicopathological variables for multivariate analysis is ongoing. Murine models. We crossed C3(1)Tag BC transgenic mice with a DARC knock-out (KO) line to generate mice lacking DARC expression that spontaneously develop tumors in the mammary fat pads. The C3(1)Tag model is reported to have similar gene expression profiles to basal-like TNBC tumors. Tumors were collected at pre-determined time points of 25 and 30 weeks, where DARC KO mice had increased tumor size and tumor burden at 30 weeks. Initial fluorescent staining revealed an increase in immune cell infiltration in the TME of DARC expressing mice compared to DARC KO mice. To further explore this, we employed multiplex staining across a representative cohort of DARC expressing and KO mice at 25 and 30 weeks to capture the specific immune cell populations infiltrating the TME. Conclusions. TNBC tumors have worse prognosis due to lack of targeted therapeutic options. Increased immunogenicity of TNBC tumors, especially among African ancestry women may be driven in part by DARC expression. Characterization of FY- status, in coordination with DARC among other cell types in the TNBC TME could present new opportunities for biomarker and/or therapeutic development to improve prognosis for these underserved populations. Citation Format: Rachel Martini, Stevens Patino, Emma Guyonnet, Brian Stonaker, Isra Elhussein, Julie Sahler, Avery August, Nancy Manley, Rick Kittles, Clayton Yates, Lisa Newman, Melissa Davis. The DARC side of Breast Cancer - DARC, Duffy-null and African ancestry influence in the Triple Negative Breast Cancer tumor microenvironment [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 PS18-09.

Abstract PO3-13-05: PD-L1 and PD-L2 Protein Expression is Frequently Discordant in Breast Cancer

Abstract Background: In neoadjuvant setting, PD-1 inhibitors have shown significantly higher pathological responses in patients (pts) with early-stage triple-negative breast cancer (TNBC) irrespective of PD-L1 status. However, durable responses are less common in estrogen receptor-positive (ER+) pts. Most breast cancer (BC) clinical trials of PD-1 inhibitors have thus far focused on PD-L1 expression for patient eligibility. The alternative PD-1 ligand, PD-L2, with reported ~4-fold greater affinity for PD-1, has been largely understudied. We recently reported that high cancer cell protein levels of PD-L2 in pts with treatment-naïve ER+ BC was an independent predictor of shorter progression-free survival. PD-L2 expression was significantly high in ER- group as well, however, given the low numbers of ER- pts, the study was not powered enough to determine the correlation of PD-L2 with PFS in the ER- subgroup. These findings suggest that PD-L2 has an important role in BC and combined PD-L1/PD-L2 status may help improve BC pt selection for PD-1 inhibitors. We therefore initiated efforts to determine baseline expression patterns of PD-L1 and PD-L2 in BC. Methods: PD-L1 and PD-L2 protein levels in cancer cells and tumor-infiltrating immune cells were prospectively analyzed by immunohistochemistry (IHC) using validated antibodies in diagnostic core biopsies of 31 consecutive pts diagnosed with localized or locoregional ER+/HER2- BC or TNBC. Percent positivity of PD-L1 and PD-L2 in cancer cells and immune cells was determined by our breast pathologist. Detectable PD-L1 or PD-L2 expression in ≥1% of cancer cells or stromal immune cells was considered positive. Spearman correlation and Wilcoxon paired analyses were used to measure and correlate PD-L1 and PD-L2 protein expression in cancer and immune cells. Results: There was no significant correlation between PD-L1 and PD-L2 expression, neither across all cases (N=31), nor within ER+ (N=22) or TNBC (N=9) cases. However, PD-L1 and PD-L2 expression patterns in BC differed in several ways. PD-L1-positivity in immune cells was higher than in cancer cells (median=5.0% vs. 0.0%; p=0.001), whereas PD-L2-positivity was higher in cancer cells than in immune cells (median=30% vs. 5.0%; p< 0.001). PD-L1 positivity in cancer cells and immune cells were positively correlated in TNBC (rho=0.69, p=0.04) but not in ER+ BC. Conversely, PD-L2 positivity in cancer cells and immune cells were positively correlated in ER+ BC (rho=0.68, p=0.001) but not in TNBC. TNBC diverged from ER+ BC by displaying higher PD-L1 positivity in immune cells (median=20.0% vs. 1.0%; p=0.004). By the conventional cut point for positivity of ≥1% for PD-L1 and PD-L2 in cancer cells or immune cells, all TNBC tumors were PD-L1-positive (9/9), with 8 also being PD-L2-positive. Of the 22 ER+ cases, 16 were PD-L2-positive, of which only 9 were also PD-L1-positive. Table 1. shows cross tabulation data for PD-L1 and PD-L2 status by BC subtype. Conclusions: PD-L1 and PD-L2 proteins show divergent expression and are not correlated in BC. Discordant PD-L2 and PD-L1 expression may be more common in ER+ BC than in TNBC. This finding justifies efforts to explore PD-L2 as a complementary marker to PD-L1 for improved prediction of response to PD-1 inhibitors, which may benefit patients with aggressive ER+ BC that are eligible for chemotherapy. Table 1. PD-L1 vs. PD-L2 Status by Breast Cancer Subtype Citation Format: Lubna Chaudhary, Julie Jorns, Yunguang Sun, Sailaja Kamaraju, Yee Chung Cheng, Amanda Kong, Tina Yen, Caitlin Patten, Chandler Cortina, Inna Chervoneva, Christopher Chitambar, Hallgeir Rui. PD-L1 and PD-L2 Protein Expression is Frequently Discordant in 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 PO3-13-05.

Abstract PO2-05-14: Prognosis and clinicopathological characteristics of HER2-negative metastatic breast cancers with low expression of hormone receptors: real world evidence from the French ESME cohort

Abstract Background: HER2-negative breast cancers expressing low (1-9 %) immunohistochemistry levels of hormone receptors (HR, estrogen and progesterone receptors), remain an uncertain category, being considered either as triple negative breast cancers (TNBC) or HR-positive (HR+) tumors across guidelines or approvals. The issues regarding this clinical subgroup are both prognostic, with the need of a better definition of their clinical outcome, as well as predictive. Methods: The ESME database is a French National cohort of all consecutive patients who initiated a first-line treatment for metastatic breast cancer (MBC) from 2008 on, in one of the 18 French Comprehensive Cancer Centers. Patients with HER2-negative (0 to 2+, ISH negative) MBC and known estrogen receptor (ER) and progesterone receptors (PR) expression levels were selected. Primary objective was to evaluate crude overall survival (OS) in HER2-/HR-low (ER and PR 0-9 and not TNBC) MBC pts compared with those with TNBC and HR+ disease defined by ER and PR < 1%, and ER ± PR ≥ 10%, respectively. In these 3 populations, predefined secondary objectives were to evaluate PFS under first line chemotherapy (CT), to describe clinical characteristics and the distribution of genetic abnormalities identified at the time of MBC diagnosis. Results: Out of 30,459 patients in the ESME database initiating their MBC treatment between 01/2008 and 01/2021, 19109 patients (2113 TNBC; 228 HR-low; 16768 HR+) were eligible for this analysis. Median follow-up was 58.0 months (95%CI [56.6; 59.0]). Median OS were 15.7 months [15.0-16.8], 19.1 months [15.5-22.4] and 44.6 months [43.8-45.5] in the TNBC, HR-low and HR+ subgroups, respectively. The multivariable analysis adjusted on age at MBC diagnosis, pathological grade and subtype, metastatic-free interval, number and sites of metastases and HER2 status (0 vs. 1-2+), identified no significant OS difference between HR-low and TNBC subgroups (HR=0.95, 95%CI 0.79-1.14), while HR+ patients had a better OS than TNBC patients (reference) (HR=0.50, 95%CI 0.47-0.53). Of these 19109 pts, 8910 received first-line CT regimen (1960 TNBC; 195 HR-low; 6755 HR+): taxanes-based: 66.7%, anthracyclines-based: 32.9%, capecitabine: 18%. Median PFS under first line CT were 5.3 (5.1-5.6), 5.1 (4.1-6.2) and 10.2 months (9.9-10.6) for TNBC, HR-low and HR+ subgroups, respectively. In the multivariable analysis adjusted on the same variables, the HR-low subgroup had a statistically poorer PFS compared to TNBC (HR=1.24, 95% CI 1.04-1.49). On the other hand, the HR+ subgroup had a better PFS than the TNBC (HR=0.74, 95%CI [0.70; 0.78]). Baseline germline BRCA1 mutations (at MBC diagnosis) were present in 4.5% of the TNBC cases, 2.6% of the HR-low cases, and 0.4% of the HR+ cases. The distribution of germline BRCA2 mutations was more homogeneous between the three groups (0.9% of TNBC cases, 1.3% of HR-low cases and 1.1 % of HR+ cases). Conclusions: In this large cohort, patients with HER2-negative, HR-low MBC seem to share similar general prognosis, oncogenetic features and PFS under standard chemotherapy treatment with TNBC patients. Citation Format: Marie Alexandre, Ludovic Gauthier, Audrey Mailliez, Thibault DE LA MOTTE ROUGE, Thomas Bachelot, Monica Arnedos, Florence Dalenc, Etienne Brain, Jean-Marc Ferrero, Vincent Massard, Isabelle Desmoulins, Marie-Ange Mouret-Reynier, Christelle Levy, Anthony Gonçalves, Marianne Leheurteur, Thierry Petit, Jean-Sébastien Frenel, Lise Bosquet, Suzette Delaloge, William Jacot. Prognosis and clinicopathological characteristics of HER2-negative metastatic breast cancers with low expression of hormone receptors: real world evidence from the French ESME cohort [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 PO2-05-14.

Abstract PO2-03-07: Immune Landscape in PD-L1-positive and TROP2-positive Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) tends to be more aggressive, and more likely to recur than other subtypes of breast cancer, and patients with metastatic TNBC have a poor prognosis. The recent approvals and data of pembrolizumab (anti-PD-1 inhibitor) in combination with chemotherapy in advanced-metastatic and early high-risk TNBC, and sacituzumab govitecan (an ADC that targets the tumor cell surface antigen TROP2 coupled with the irinotecan metabolite SN-38) in metastatic TNBC, has shed light on some of the immune and cancer signaling associated with TNBC. Although exciting progress has been made in the treatment of TNBC, there are still important gaps to fill to understand the biology of TNBC and identify additional therapies. Materials and Methods: We identified a total of 1500 patients whose tumors were negative for the expression of ER and HER2 receptors by gene array. PD-L1 and TROP2 expression data were available for 802 early TNBC patients, of whom 657 were treated with adjuvant chemotherapy. A comprehensive identification and analysis of immune-related genes (n= 1169) was conducted. Mann-Whitney test comparing the gene expression for all immune related-genes was evaluated in patients expressing PD-L1 and TROP2 at low and high expression levels. Immune gene modules resembling key immune gene signaling were analyzed in all subgroups identified. Fold changes and Mann-Whitney P values were calculated in each subgroup. Results: Patients were stratified based on high and low expression levels of PD-L1 and TROP-2 mRNA. PD-L1 highly expressing (PD-L1+) tumors showed a marked difference in immune-related genes as compared to PD-L1 low-level tumors (PD-L1-) with PD-L1+ tumors demonstrating a significantly higher proportion of correlated immune genes (CXCL9/10/11, IFN-γ, CCL5 and STAT1). TROP2 highly and low expressing tumors (TROP2+ and TROP2-, respectively) showed a relatively limited difference in terms of immune-related gene expression with key immune-related genes having statistical differential expression in the two subgroups. TROP2+ tumors showed a high presence of several Mucin family gene members (MUC1, -5AC, -4). In addition, CLDN 4, VTCN1, and MET were among the key immune genes significantly expressed in TROP2+ tumors (P = 5.73E-20; P = 6.2E-13, P = 2.1E-11). Overall, PD-L1+ tumors and TROP2+ tumors showed an immune suppressive genomic landscape but were potentially regulated by different signaling. Of note, tumors with very high expression of PD-L1 showed an inverse correlation with VTCN1 gene expression. Conclusions: This preliminary analysis confirmed that only a limited number of early TNBC highly expressed PD-L1 and that a different immune suppressive landscape for PD-L1± and TROP2± tumors exists. In addition, our data suggest that early TNBC cases lacking PD-L1 might escape immune surveillance by virtue of the upregulation of key alternate signaling. Ongoing efforts investigating the expression of such markers on stroma/epithelial tumor components will help to clarify their biological, clinical, and potential therapeutic role in TNBC. Citation Format: Balazs Gyorffy, Libero Santarpia. Immune Landscape in PD-L1-positive and TROP2-positive 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 PO2-03-07.

Abstract PO4-06-12: Distinction of basal-like and triple-negative basal-like breast cancers utilizing a novel comprehensive single-cell liquid biopsy-based test

Abstract Introduction: Breast cancer is a highly heterogeneous disease and is a leading cause of death for women worldwide. Triple-negative breast cancer (TNBC), the most aggressive form of the disease, is clinically defined by the lack of expression of estrogen receptor (ER) and progesterone receptor (PR), as well as lack of over-expression of human epidermal growth factor receptor-2 (HER2). Approximately 70% of all TNBC cases exhibit basal-like cells, characterized by high basal expression of keratins as well as extensive genomic instability with copy number gains and losses across most chromosomes. In this study we show that novel single cell genomic evaluation coupled with immunofluorescence (IF) protein analysis of CTCs can detect TNBC with basal-like genomic features as well as basal-like tumors with expression of ER and HER2. Materials and Methods: Epic Sciences’ DefineMBC™ liquid biopsy test characterizes CTC proteomics and genomics as well as ctDNA for the treatment of metastatic breast cancer. Blood samples from 813 metastatic breast cancer patients, with either newly diagnosed or progressing disease, with historical tissue subtype [HR(+)HER2(-), HR(+/-)HER2(+), and TNBC] supplied by the referring physician were collected and tested. Up to 5 CTCs were selected and isolated from each sample based on morphology and IF signal. DNA was extracted from individual cells, and whole genome amplified and sequenced at low coverage. Copy number analysis was performed to determine copy number gains and losses across chromosomes, as well as large scale-state transitions (LSTs) and ERBB2 amplification. CTCs characterized with high chromosomal instability (e.g., exhibiting >30 LSTs; 1Q firestorm and 8Q stair step) and high levels of cytokeratin expression (mean fluorescence intensity signal >10K) were categorized as basal-like. Results: By previous metastatic tissue biopsy, 77% were HR(+), 8% were HER2(+), and 12% were TNBC. CTCs categorized in DefineMBC as basal-like subtype (9.8 % of all samples tested) showed to be highly present in patients called as TNBC (79% basal-like). Additionally, 54% of HR(+) and 23% of HER2(+) had basal-like CTCS. Patients characterized with TNBC by metastatic tissue biopsy and by DefineMBC exhibited basal-like cells in 88% of the cases. Conclusion: The results show a novel clinical assay which utilizes CTC protein detection as well as single-cell genomic analysis to identify tumors driven by basal-like cells. Generally, TNBC is classified with negative biomarker expression. We show that CTCs in TNBC display genomically basal-like cells. CTCs from HR(+) and HER2(+) tumors may express basal-like features with preserved ER and HER2 expression. The comprehensive nature of DefineMBC™ utilizing genomic analysis of detected CTCs has provided a new tool to diagnose both basal-like TNBC and basal-like hormone receptor positive and HER2 over-expressing tumors. Citation Format: Alessandra Cunsolo, David Bourdon, Ernest Lam, Giuseppe Di Caro, Nilesh Dharajiya, Timothy Pluard, Lee Schwartzberg. Distinction of basal-like and triple-negative basal-like breast cancers utilizing a novel comprehensive single-cell liquid biopsy-based test [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 PO4-06-12.

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 PO2-23-05: Practice patterns and outcomes in the ongoing neoadjuvant ATNEC trial: node marking, response to NACT and breast conservation rates

Abstract Background In ATNEC trial, cT1-3N1M0 patients receive NACT followed by SNB/TAD. If the sentinel nodes(SNs) have converted to benign(ypN0), patients are randomly assigned to Axillary Treatment vs no Axillary Treatment. The study promotes standardized node marking procedures and includes a radiotherapy quality assurance program. Objectives – This study prospectively evaluates practice patterns, NACT response, adoption of node marking, rates of marked node identification, and breast conservation rates in the ongoing ATNEC trial across 70 centers in the UK. The trial aims to recruit 1900 patients. Materials and Methods Data from 196 randomized patients (median age: 54 [range: 28-79] years; median BMI: 26.5 [range: 17.6-51.1]) from December 2021 to June 28, 2023, across 70 UK centers were analyzed. These patients presented with cT1-3N1M0 breast cancer, received NACT, and exhibited no residual nodal disease (ypN0) on SNB/TAD. Results Among the randomised patients 54%(106) were post-menopausal, 71%(140) underwent breast conserving surgery(BCS) and 29%(56) mastectomy. At presentation, 12% (23) had T3, 63% (124) had T2, and 23% (46) had T1 tumors. Grade 3 tumors accounted for 70% (138) of cases. Among the patients, 60% (117) were HER2 positive, 31% (61) were triple negative, and 9% (18) were HER2 negative (ER or PgR positive). Anthracycline and taxane-based NACT were administered to 57% (106), while 38% (71) received a platinum-containing regimen. During SNB/TAD, a median of 4 nodes (interquartile range, 3-5) were removed. Among 184 patients with node marking data, 74% (136) had the involved node marked. The marked node was successfully removed in 94% (128) of these patients. Clip/coil only, Magseed and black dye were the commonest techniques used for node marking. The marked node was the sentinel node in 83% (106) of cases. Among 185 randomized patients with post-NACT response data, 68% (125) achieved a complete pathological response (pCR) in the breast, while 8% (15) had DCIS only and 22% (41) had invasive cancer. Among 105 patients with complete breast tumor response on imaging, 15% (16) had residual DCIS or invasive cancer. In the subset of patients with partial response or stable breast tumors on imaging (69 patients), 46% (32) had no residual tumor on histology (ypT0). Furthermore, 14% (26) exhibited partial or stable disease in the axilla on imaging but achieved complete pathological response on histology. Conclusions HER2-positive and triple-negative breast cancer cases predominate among patients undergoing NACT in the UK. Although 68% achieved a complete pCR (ypT0) in the breast, rates of BCS remain low. Notably, around 75% of randomized patients underwent node marking, with an intra-operative identification rate of 94%, demonstrating the successful implementation of node marking in the UK through the ATNEC trial. Citation Format: Amit Goyal, Andrea Marshall, Sophie Nicholls, Natalie Hammonds, Beatrix Elsberger, Duncan Wheatley, Janice Rose, Helen-Teresa Edwards, Abeer Shaaban, Roeum Butt, Gareth Jackson, Tara Homer, Luke Vale, Samreen Ahmed, Shama Puri, Sophie Gasson, Julie Bruce, Helen Higgins, Janet Dunn. Practice patterns and outcomes in the ongoing neoadjuvant ATNEC trial: node marking, response to NACT and breast conservation rates [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 PO2-23-05.

Molecular docking of ferulic acid analogue compounds against epidermal growth factor receptor as a potential therapy for breast cancer

Background: Triple-negative breast cancer (TNBC) accounted for 18.1% of the breast cancer cases that occurred in Indonesia until 2020. Epidermal growth factor receptor (EGFR) overexpression is found in at least 50% of TNBC cases. So far, it is necessary to find an anti-cancer compound that has the potential against TNBC-type breast cancer to achieve good health and well-being. Objective: Ferulic acid derivatives were designed to be active on EGFR in silico study. Methods: Molecular docking was performed using Auto Dock 1.5.7 and PyRx 0.8 software, and visualisation was observed using Discovery Studio. FA ligand and its four derivatives were docked into the receptor EGFR (PDB ID: 3W33). Results: It was found that ferulic acid derivatives have high potential as an anticancer through EGFR inhibition in TNBC-type breast cancer. 4-(4-methyl) benzoyloxy-3-methoxycinnamic acid had the best potential among other derivatives, which showed the lowest binding free energy of -8.81 kcal/mol and the smallest Ki of 352.65nM. Methyl substitution at the benzoyloxy increased ligand interaction with amino acids in EGFR by increasing hydrophobic π-alkyl, π-π and alkyl binding with amino acids. Conclusion: The 4-(4-methyl) benzoyloxy-3-methoxycinnamic acid was the most prospective compound as an EGFR inhibitor and predicted as the most potential compound against breast cancer.

Triple-negative occult breast cancer: A case report

The phenomenon of occult breast cancer represents a rare scenario raising crucial questions regarding its clinical presentation, diagnosis, and timely treatment. The therapeutic challenge can become even more difficult in the specific case of triple-negative breast cancer. This article presents the case report of a patient who had a palpable axillary nodule during a routine gynecological exam. A core needle biopsy confirmed that the patient had metastatic ductal carcinoma of mammary origin, which was triple-negative. Despite several diagnostic procedures, there was no evidence of a primary tumor in the breast clinically or in imaging studies. The patient underwent neoadjuvant chemotherapy with satisfying outcomes and later, to radical surgery therapy regardless of the feasibility of receiving a conservative treatment.

Triple Negative Breast Cancer (TNBC): Cannabis sativa-Role of Phytocannabinoids

This review paper highlights the role of phytocannabinoids in controlling triple-negative breast cancer (TNBC). Triple-negative breast cancer (TNBC) is a specific subtype of breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2), which has clinical features that include high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. TNBC is one of the most aggressive subtypes of breast cancer that is often associated with poor patient outcomes because of the development of metastases in secondary organisms like in the brain, bone, and lungs. Triple-negative breast cancer (TNBC) is a kind of breast cancer that does not have any of the receptors that are commonly found in breast cancer. The highest number of TNBC cases has been recorded in India. Currently, TNBC is highly prevalent among Indian women and develops approximately 20% to 43% of all patients with breast cancer. Triple-negative breast cancer (TNBC) is an aggressive malignancy that requires effective targeted drug therapy. Patients with TNBC develop metastasis and recurrence over time and have reduced survival compared to patients with other subtypes of breast cancer. Pre-clinical studies have demonstrated that phytocannabinoids exert important antitumor properties in the main breast cancer subtypes, particularly in TNBC, where different phytocannabinoids and synthetic cannabinoids have shown interesting therapeutic actions. Neither CBD nor THC are universally efficacious in reducing cancer cell viability. Focusing on in vitro studies, the effect of CBD on cancer cell viability ranges from no effect, to a modest reduction, and to significant cytotoxicity depending on concentrations, cancer cell lines, cell growth conditions, the performed assays, and the time of CBD exposure. However, it should also be noted that research into the efficacy, dosage and drug safety of cannabinoids in tumor therapy still has a long way to go, especially with regard to human clinical trials to be conducted, through which alone the benefits and advantages for cancer patients but also possible risks can be defined.

Determination of target genes for classified molecular subtypes of triple-negative breast cancer form microarray gene expression profiling: An integrative in silico approach.

Highly heterogeneous triple-negative breast cancer (TNBC) has tough clinical features, which were gradually solving and improving in diagnosis by the molecular subtyping of TNBC. Presently, this study was focused on analyzing the genetic makeup of TNBC subtypes. This study explored the MicroArray expression profiling of differentially expressed genes in molecular subtypes BL1, BL2, IM, luminal androgen receptor, M, and mesenchymal stem-like of TNBC by analyzing the Gene Expression Omnibus dataset GSE167213. Various gene ontologies-based protein-protein interaction (PPI) networks were subtyped TNBC genes. The effect of genetic alteration on TNBC cases was also interpreted. The MicroArray gene expression profiling was done through R programming and subjected to functional annotation through the database for annotation, visualization, and integrated discovery. The PPI networking of functionally associated genes was interpreted by STRING. The survival analysis was done through cBioPortal. The t-test was used through R programming to generate the P values for a test of the significance of expressed genes. A total of 54,613 significant probes were analyzed in the TNBC MicroArray dataset. The functional PPI networks of BL1, BL2, and IM upregulated genes showed significant associations. The survival analysis of differentially expressed genes showed the significant prognostic effect of 32 upregulated genes of different subtypes on TNBC cases with genetic alterations, whereas the remaining genes showed no significant effects. The output of the present study provided significant target gene panels for different TNBC subtypes, which would add an informative genetic value to TNBC diagnosis.

Abstract 5686: Identifying rare microRNA risk variants for hereditary breast cancer in African American individuals

Abstract Background: In the United States, breast cancer is the most frequently diagnosed cancer in women. However, African American women tend to have a higher incidence of triple negative breast cancer cases, and an earlier age of disease onset, which are hallmark characteristics of heredity disease. Even with current breakthroughs in breast cancer research, limited research participation from women of African descent has led to a gap in our knowledge of heredity factors that are involved in disease in this population. Furthermore, most breast cancer research has focused on protein coding genes being the culprit of tumorigenesis; however, within the last three decades, research efforts have begun to focus on noncoding RNAs, like microRNAs (miRNAs), as potential factors to promote carcinogenesis. miRNAs are short, noncoding RNA molecules that control gene expression at the post-translational level. miRNAs have several mechanisms to affect gene expression of cellular processes like cell proliferation, apoptosis, invasion, and metastasis. However, the identification and functional role miRNA risk variants play in African American breast cancer is still not fully understood. Methods: 30X whole genome sequencing (WGS) was carried out on an Illumina platform on blood- or saliva-derived DNA from 120 African American breast cancer cases from the Alabama Hereditary Cancer Cohort. The WGS data was analyzed using our established bioinformatics pipeline, which has been adapted from the Genome Analysis Toolkit (GATK) Best Practice Pipeline. Focusing on miRNA variants as risk factors, a miRNA BED file was used to extract regions harboring miRNA genes. The resulting file was then filtered for rare miRNA variants (minor allele frequency <1%) in the African American population based on the Genome Aggregation Database (gnomAD) allele frequency. P-values for variants of interest were calculated using Fisher’s Exact tests. Results: Sequencing data have revealed three rare miRNA variants from our African American cohort that can be potentially associated with breast cancer risk. Upon statistical analysis comparing allele frequencies between the African American general population and the Alabama Hereditary Cancer Cohort, miR7850, miR3145, and miR8074 had p-values 5.21E-05, 3.58E-05, and 2.19E-04, respectively. Conclusions: We have identified three rare miRNA variants that were not previously known to be associated with breast cancer risk. Interestingly, both miR7850 and miR8074 target TP53 and miR3145 targets GREM1 and PALB2, which these targets are known breast cancer susceptibility genes. Understanding the consequence of these variants is important to add to our knowledge of miRNAs that play critical roles in carcinogenesis leading to better treatment strategies. Citation Format: Sheniqua R. Glover, Troy LoBue, Nancy D. Merner. Identifying rare microRNA risk variants for hereditary breast cancer in African American individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5686.

Abstract 275: Deciphering biphasic responses to Sonic Hedgehog pathway inhibition in breast cancer: Insights from a tumor-fibroblast spheroid model and PDXs

Abstract A significant subset (30-40%) of triple-negative breast cancer (TNBC) cases exhibit hyperactivation of the sonic hedgehog (SHH) pathway, correlating with poor clinical outcomes. However, the therapeutic efficacy of SHH inhibitors in TNBC has yielded inconsistent results, showcasing biphasic tumor responses going from complete remission in some patients to faster disease progression in others. Such tumor response discrepancies underscore the need for more relevant culture models to elucidate how stromal cells respond to SHH signals and impact tumor cell reprogramming.We have developed a tumor-fibroblast spheroid model of robust paracrine SHH signaling to examine alterations in aggressive tumor cell phenotypes resulting from altered SHH pathway activity levels to understand better how SHH therapy leads to biphasic effects in tumor tissues. Specifically, tumor stemness alterations were examined as a function of SHH signal strength altered by tumor-fibroblast proximity, and findings were confirmed in two PDX models of TNBC of divergent sensitivity to pharmacological SHH pathway inhibition. In vitro, results show that tumor stemness potency is enriched by tumor-fibroblast proximity and inversely correlates with SHH pathway activity levels. Tumor stemness potency was determined via a mammosphere forming assay and resistance to chemotherapy using docetaxel as treatment. CD44+ sorted tumor cells derived from adjacent fibroblast cultures showed higher mammosphere forming efficiency and docetaxel sensitivity than those from mixed cultures. These effects correlated with differences in SHH pathway activity strengths and were reverted by pharmacological inhibition of SHH confirming pathway activity involvement. In two PDX models, both exhibiting high SHH pathway activity and similar tumor vascularization, we observed variable response to pharmacological SHH inhibition. PDX2 displayed favorable responses with minimal alpha-smooth muscle actin-positive (SMA+) fibroblast infiltration, while PDX1 exhibited no benefit from SHH therapy, accompanied by a high level of SMA+ fibroblasts. Spatial transcriptomic analysis confirmed stemness enrichments in the PDX1 model treated with SHH inhibitor- a scenario our mixed co-culture model resembled. Stemness-enriched tumor regions were positive for the Ki67 proliferation marker and confined to areas of high SMA+ cell abundance. Plotting Ki67 vs SMA+ cell levels revealed a positive correlation, suggesting that SMA levels may predict tumor response to SHH pathway inhibition.In summary, our studies demonstrate that pharmacological SHH inhibition can induce altered tumor plasticity by favoring stemness phenotypes. Strategies targeting tumor stemness may mitigate biphasic effects in response to SHH inhibitors, offering insights for optimizing TNBC therapeutic approaches. Citation Format: Heizel M. Rosado-Galindo, Miosotis Acevedo-Esquillin, Wandaliz Torres-García, Ana M. Reyes-Ramos, Gabriela Ortiz-Soto, Michelle M. Martínez-Montemayor, Maribella Domenech. Deciphering biphasic responses to Sonic Hedgehog pathway inhibition in breast cancer: Insights from a tumor-fibroblast spheroid model and PDXs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 275.

Real-world overall survival and characteristics of patients with ER-zero and ER-low HER2-negative breast cancer treated as triple-negative breast cancer: a Swedish population-based cohort study

Estrogen receptor-low (ER-low) HER2-negative breast cancer has similar pathological and molecular characteristics as triple-negative breast cancer (TNBC), and it is questionable whether it should be considered a separate entity. When the international guidelines lowered the cutoff for ER positivity to ≥1% in 2010, the ≥10% threshold was kept in Sweden. ER-low breast cancer (ER 1-9%) is thus in Sweden treated as TNBC. We aimed to describe patient and tumor characteristics, treatment patterns and overall survival in a Swedish population-based cohort of patients with ER-zero and ER-low HER2-negative breast cancer treated as TNBC. All TNBC cases diagnosed in Sweden 2008-2020 were included in a population-based cohort study. Patient, tumor and treatment characteristics were analyzed by ER-status (ER 0% vs 1-9%), and associations between subgroups compared using χ2 test. Survival endpoint was overall survival (OS), and Kaplan-Meier curves were estimated. Cox proportional hazards models were used to estimate adjusted hazard ratios comparing ER-low to ER-zero. Of the 5655 tumors, 90.1% had an ER expression of 0%, while 9.9% were ER-low. ER-low tumors were grade III in 69.4% (80.8% in ER-zero tumors, p-value=0.001), with a median Ki67 of 60% (63% in ER-zero tumors, p-value=0.005). There were no significant differences in given chemotherapy (p=0.546). A pathological complete response (pCR) was achieved in 28.1% of ER-low tumors (25.1% in ER-zero tumors). In the unadjusted analysis of OS, women with ER-low disease had a borderline but not significantly better OS than those with ER-zero disease (HR 0.84 (95% CI 0.71-1.00), p=0.052). ER-status 1-9% vs 0% was not associated with OS in the multivariable analysis (HR 1.11 (0.90-1.36)). Distant disease-free survival did not differ by ER-status 0% vs 1-9% (HR 0.97 for ER-zero vs ER-low (0.62-1.53), p=0.905). After preoperative treatment, the impact of pCR for OS did not significantly differ between ER-zero or ER-low disease. ER-low HER2-negative breast cancer has characteristics and prognosis similar to TNBC, when treated in the same way. Therefore, it seems reasonable to use a ≥10% threshold for ER positivity. This would provide patients with ER-low tumors the same treatment opportunities as patients with TNBC, within studies and within clinical routine. This work was financially supported by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, in accordance with terms and conditions of a Master Collaboration Agreement between the company and Karolinska Institutet.

Novel Insights Into the Epidemiological and Clinico-Pathological Profile of Triple-Negative Breast Cancer: Dissection of an Aggressive Variant.

Objective Triple-negative breast cancer (TNBC) represents an aggressive and prognostically poor variant of breast cancer. Over the years, detailed research has been conducted and published in Western literature. However, there lacks a detailed account of TNBC cases from the perspective of a low-volume institution. This study aims to assess the clinicalfeatures of TNBC, as well as their prognostic implications in a tertiary care centre. Methods and materials This prospective observational studytook place at a tertiary health centre for two years, spanning from 2021 to 2023. The study aimed to investigate various clinicopathological and epidemiological parameters, thereby highlighting the shortcomings in the existing knowledge about the subject in the context of a low-volume centre, as well as additional contributing factors in developing countries like India.A group of 150 participants diagnosed with TNBC through biopsy and immunohistochemistry and >40 years of age were included in the study. Patients who tested positive for hormonal receptors and who refused to give consent for participation were excluded from the study. The study subjectswere categorized according to their clinical TNM (cTNM) stage and eventually segregated into two primary heads, namely pre-surgery chemotherapy with breast-conserving surgery (BCS) after a good response, or modified radical mastectomy (MRM) upfront. Important demographic details, including age, socioeconomic status, and education, were also recorded. A comprehensive follow-up assessment post-treatment was performed to detect early recurrence. After data collection, the recurrence rates were correlated with the TNBC status to establish the aggressiveness of the cancer. Statistical analysis of the data was done using the Statistical Package for Social Sciences (SPSS) -16version software. Results The average age of the 150 participants in the study was 52.21 years (SD±4.89 years). The highest recorded age was 64 years, while the lowest recorded age was 45 years. In the study, it was observed that 41% of the participants diagnosed with TNBChad stage III disease, whereas 33.5% had stage I disease, 22% had stage IV disease and 3.6% had stage II disease. A total of 27.5% of individuals with TNBCexhibited metastases in various anatomical sites, whereas the other 72.5% did not show any signs of metastasis. Conclusion Triple-negative breast cancer has earned its position as a unique subtype of breast cancer due to its unusual molecular characteristics, aggressive behavior, limited treatment options, and poor prognosis. The lower per-capita income and limited knowledge pertaining to this variant, along with the absence of more specific treatment options, contribute to the already high levels of morbidity and mortality associated with this illness. To effectively address this unique and very virulent ailment and customize our strategies, it is imperative to do further comprehensive investigations, thereby enabling us to deliver the highest quality of medical attention to individuals afflicted by this pathology.

Development and verification of a novel immunogenic cell death-related signature for predicting the prognosis and immune infiltration in triple-negative breast cancer.

Insufficient understanding of the pathogenesis and tumor immunology of triple-negative breast cancer (TNBC) has limited the development of immunotherapy. The importance of tumor microenvironment (TME) in immunotyping, prognostic assessment and immunotherapy efficacy of cancer has been emphasized, however, potential immunogenic cell death (ICD) related genes function in TME of TNBC has been rarely investigated. To initially explore the role and related mechanisms of ICD in TNBC, especially the role played in the TME of TNBC, and to identify different relevant subtypes based on ICD, and then develop an ICD-related risk score to predict each TNBC patient TME status, prognosis and immunotherapy response. In this study, we identified distinct ICD-related modification patterns based on 158 TNBC cases in the TCGA-TNBC cohort. We then investigated the possible correlation between ICD-related modification patterns and TME cell infiltration characteristics in TNBC. By using univariate Cox and least absolute shrinkage and selection operator (LASSO) regression analysis, we created a risk scoring system (ICD score) to quantifiably evaluate the impact of ICD-related modification patterns in individual TNBC patient. Two different ICD-related modification patterns were found with significant differences in immune infiltration. Lower ICD score was correlated with higher immune infiltration, tumor mutational burden and significantly enriched in immune-related pathways, indicating a strong ability to activate immune response, which might account for relatively favorable prognosis of TNBC patients and could serve as a predictor to select suitable candidates for immunotherapy. We used two independent cohorts, GSE58812 cohort and Metabric cohort to validate prognosis and immunohistochemistry for preliminary in vitro validation. This study evidenced that the ICD-related modification patterns might exert pivotal roles in the immune infiltration landscape of TNBC and ICD score might act as potential predictors of prognostic assessment and immunotherapy response. This research provides unique insights for individualize immune treatment strategies and promising immunotherapy candidates screening.

Characterizing PALB2 intragenic duplication breakpoints in a triple-negative breast cancer case using long-read sequencing.

Accurate identification and characterization of Large Genomic Rearrangements (LGR), especially duplications, are crucial for precise diagnosis and risk assessment. In this report, we characterized an intragenic duplication breakpoint of PALB2 to determine its pathogenicity significance. A 52-year-old female with triple-negative breast cancer was diagnosed with a novel PALB2 LGR. An efficient and accurate methodology was applied, combining long-read sequencing and transcript analysis for the rapid characterization of the duplication. Duplication of exons 5 and 6 of PALB2 was validated by transcript analysis. Long-read sequencing enabled the localization of breakpoints within Alu elements, providing insights into the mechanism of duplication via non-allelic homologous recombination. Using our combined methodology, we reclassified the PALB2 duplication as a pathogenic variant. This reclassification suggests a possible causative link between this specific genetic alteration and the aggressive phenotype of the patient.