Biological Characteristics Research Articles

Three-Dimensional Bio-Printed Tubular Tissue Using Dermal Fibroblast Cells as a New Tissue-Engineered Vascular Graft for Venous Replacement.

The current study was a preliminary evaluation of the feasibility and biologic features of three-dimensionally bio-printed tissue-engineered (3D bio-printed) vascular grafts comprising dermal fibroblast spheroids for venous replacement in rats and swine. The scaffold-free tubular tissue was made by the 3D bio-printer with normal human dermal fibroblasts. The tubular tissues were implanted into the infrarenal inferior vena cava of 4 male F344-rnu/rnu athymic nude rats and the short-term patency and histologic features were analyzed. A larger 3D bio-printed swine dermal fibroblast-derived prototype of tubular tissue was implanted into the right jugular vein of a swine and patency was evaluated at 4 weeks. The short-term patency rate was 100%. Immunohistochemistry analysis showed von Willebrand factor positivity on day 2, with more limited positivity observed on the luminal surface on day 5. Although the cross-sectional area of the wall differed significantly between preimplantation and days 2 and 5, suggesting swelling of the tubular tissue wall (both p < 0.01), the luminal diameter of the tubular tissues was not significantly altered during this period. The 3D bio-printed scaffold-free tubular tissues using human dermal or swine fibroblast spheroids may produce better tissue-engineered vascular grafts for venous replacement in rats or swine.

Genomic and biological characterization of bacteriophages against Enterobacter cloacae, a high-priority pathogen

Enterobacter cloacae is a clinically significant pathogen due to its multi-resistance to antibiotics, presenting a challenge in the treatment of infections. As concerns over antibiotic resistance escalate, novel therapeutic approaches have been explored. Bacteriophages, characterized by their remarkable specificity and ability to self-replicate within target bacteria, are emerging as a promising alternative therapy. In this study, we isolated and partially characterized nine lytic bacteriophages targeting E. cloacae, with two selected for comprehensive genomic analysis based on their host range and bacteriolytic activity. All identified phages exhibited a narrow host range, demonstrated stability within a temperature range of 30–60 °C, displayed pH tolerance from 3 to 10, and showed an excellent bacteriolytic capacity for up to 18 h. Notably, the fully characterized phage genomes revealed an absence of lysogenic, virulence, or antibiotic-resistance genes, positioning them as promising candidates for therapeutic intervention against E. cloacae-related diseases. Nonetheless, translating this knowledge into practical therapeutic applications mandates a deeper understanding of bacteriophage interactions within complex biological environments.

Abstract PO3-13-12: ER-positive/HER2-negative breast cancer in Adolescent &amp; Young Adult (AYA) age group has a specific biological feature and worse outcome compared to other generations

Abstract Background: Adolescents &amp; Young Adults (AYA) is commonly defined as patients younger than 40 years old. Breast cancer patients in AYA has been reported to have poor outcome compared to other generation, however, the clinicopathological and biological features of AYA breast cancer remains controversial partly because many previous studies did not consider the difference in subtypes by age. We aim to clarify the features of AYA compared with the other age group in ER-positive/HER2-negative breast cancer, assuming the effects of age-related hormonal status. Material and Methods: Each analyzed cohort were divided into four age groups; AYA (15-39yo), perimenopausal (40-55yo), menopausal (55-65yo), and old (65+yo). Clinicopathological and biological features were analyzed using gene set variation analysis and the xCell algorithm using mRNA expression profiles of large independent public databases (METABRIC; n = 1,903, GSE96058; n = 3,273). Results: We found that both clinical and pathological T-category of AJCC staging were larger in AYA and Old compared from the other groups. Pathological N-category as well as pathological Stage were higher in AYA. Nottingham histological grade 3 was significantly higher in the AYA compared with the other older groups. Among the subtypes, AYA breast cancer was significantly associated with higher rate of triple-negative breast cancer compared to the others, as expected. Considering the whole cohort, ER and PgR positivity trended to be lower, but HER2-positivity trended to be higher in AYA. Within the ER-positive/HER2-negative subtype, PgR positivity was higher in the AYA and Perimenopausal groups compared to others. AYA had poorer disease-specific survival than other groups (p = 0.010 and p = 0.002, respectively), as well as overall survival, especially compared to perimenopausal group. The survival difference was more pronounced in ER-positive/HER2-negative breast cancer patients. In biological analysis in ER-positive/HER2-negative subtype, we found that estrogen response late signaling level decreased as the age group gets older. AYA breast cancer had significant enrichment in cell proliferation-related gene sets (G2M checkpoint, E2F targets, and MYC target v1), other pro-cancerous gene sets (MTORC1, unfolded protein response, and PI3K/ACT/MTOR signaling), consistently in two cohorts (all p ≦ 0.028), when compared to the other age groups. Interestingly, the features were observed even in the small tumor size (T1/T2) subgroup. Furthermore, AYA breast cancer showed significantly high BRCAness (all p≦0.002) and DNA repair (all p≦0.023) compared to other age groups. The infiltration fraction of several immune cells: CD8+ T cells, M1 macrophages, regulatory T cells, helper type 2 T cells, were significantly higher, and M2 macrophages were significantly lower in AYA, consistently in two cohorts. Conclusion: ER-positive/HER2-negative breast cancer in AYA was highly proliferative with high immune cell infiltrations compared to other generations. This may be related to the difference in outcomes in each age group. Considering these unique features may enable us to fine tune treatment strategies for AYA. Citation Format: Masanori Oshi, Akimitsu Yamada, Mahato Sasamoto, Kei Kawashima, Yoshie Fujiwara, Shoko Adachi, Shinya Yamamoto, Kazutaka Narui, Takashi Ishikawa, Itaru Endo, Kazuaki Takabe. ER-positive/HER2-negative breast cancer in Adolescent &amp; Young Adult (AYA) age group has a specific biological feature and worse outcome compared to other generations [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 PO3-13-12.

Abstract PO5-27-01: Comparison between the 7th and the 8th edition of TNM Staging System of American joint committee on Cancer(AJCC) for Breast Cancer Patients, diagnosed and treated at King Abdulaziz Medical City

Abstract Breast cancer is a heterogeneous disease with different biological and molecular characteristics that affect local recurrence, metastases, prognoses, and response to available therapies. The 8th edition of the American Joint Committee on Cancer (AJCC) staging system for breast cancer has incorporated the biological and molecular characteristics, tumor grade, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor status(Her-2)and tumor grade, into the traditional anatomical 7th edition that only considers tumor size, lymph nodes status, and distant metastases. The aim of this study is to compare the 7th to 8th editions of the AJCC staging system for prognostic impact, assess the effect of the new edition on the treatment and treatment outcome Method A retrospective cohort study was performed for all breast cancer patients, diagnosed between (2013 -2019) and underwent upfront surgery at King Abdulaziz Medical City (KAMC).Patients’ clinicopathological, immunohistochemical, surgical, therapeutic, and follow-up data were retrieved. Overall survival (OS) and breast cancer-specific survival (BCSS) were estimated using the Kaplan–Meier method. Univariate analysis using Fisher's exact test was used to compare staging systems' prognostic accuracy. Multivariable analysis using Cox proportional hazards regression analysis was performed to identify factors independently associated with disease outcome Results A total of 169 female patients were included, the 8th AJCC staging system resulted in a reallocation of 108 (63.9%) of the patients to a different stage group. The majority were downstaged(60.9%) while only 3% were upstaged, most of the upstaged patients had a triple-negative subtype (80%). The change in overall stage for the 8th edition was most significant in stage I (an increase by 140.3%), followed by stage II (downstaged by 74.3%) and stage III (downstaged by 49.7%) compared to the 7th edition. Critically 42.8% of all downstaged patients treated with adjuvant chemotherapy were found to have received unnecessary chemotherapy after applying the 8th AJCC staging system as per National Comprehensive Cancer Network (NCCN) clinical practice guidelines recommendations. The 8th AJCC staging system was a better prognostic predictor of disease survival, patients in 8th AJCC stage 3 had a higher tendency to develop metastases and die because of their disease (P-value 0.03, 0.051, respectively). Conclusions: Our comparison revealed that the AJCC 8th edition staging system is superior and more comprehensive than the 7th, in term of prognostic value, and in defining the precise adjuvant therapy Figure 1 Comparison of the 7th AJCC and the 8th AJCC staging system Citation Format: Nafisa Abdelhafiez, Abdulmohsen Alkushi, Lolwah Alriyees, Mohammad Arabi, Faris Alsalamah, Emad Masuadi, Ahmad Omair, Mohammad Alkaiyat, Hussam Shehata. Comparison between the 7th and the 8th edition of TNM Staging System of American joint committee on Cancer(AJCC) for Breast Cancer Patients, diagnosed and treated at King Abdulaziz Medical City [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 PO5-27-01.

Abstract PO2-15-05: Mesenchymal-like immune-activated: the 4th robust triple-negative breast cancer molecular subtype

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of primary BC. This heterogeneous disease is the most aggressive BC subtype. TNBC is characterized by young age at onset, high-grade tumors, high risk of early recurrence and poor prognosis. Until recently, TNBC patients were considered as one group and all patients were treated with conventional chemotherapy. With the emergence of new therapies targeting distinct biological pathways, personalized medicine is now accessible by defining finer TNBC subtypes, e.g. molecular subtypes, and identifying their specific targetable pathways. Several studies based on transcriptomics have identified three to six TNBC molecular subtypes. Today, there is a clear consensus on three TNBC robust subtypes: luminal androgen receptor (LAR), basal-like immune-activated (BLIA) and basal-like immune-suppressed (BLIS). The debate about the robustness of other subtypes is still open, notably mesenchymal-like (M) and mesenchymal stem-like (MSL or MES). The goal of this study was to establish a robust molecular classification of TNBC that allow to determine the best treatment options. To optimize this process, we harvested as much transcriptomic data as possible. Available clinicopathological data linked to these data were also imported. To our knowledge, this is the largest study aimed at subtyping TNBC; it exploits a total of 1942 TNBC patient data collected from 19 datasets: 1243 samples from microarray technology (14 datasets) and 699 from RNAseq technology (five datasets including a new one from our institution [GSE225002]). We performed independent unsupervised analyses on these two large cohorts with consensus k-means clustering including selection of optimal number of clusters and optimal number of variables. Both consensus clustering analyses separated TNBC microarray and RNAseq TNBC cohorts into four optimal clusters. These clusters were then characterized using clinicopathological features, gene ontology enrichment analyses (GOEA), gene set enrichment analysis (GSEA), 70 gene expression signatures (GES) and expression of 47 immune checkpoint genes. We concluded that TNBC are composed of four robust subtypes: LAR, mesenchymal-like immune-activated (MLIA), BLIA and BLIS. Importantly, we propose that MLIA acronym should replace M or MSL, because it highlights the two main biological characteristics of this subtype, i.e. mesenchymal-like features and a high IR comparable to that of BLIA. Furthermore, we showed that each subtype shares common biological characteristics with another one: LAR and MLIA (androgen receptor pathway activation); MLIA and BLIA (immune activation, notably high levels of immune checkpoints); BLIA and BLIS (basal-like features); BLIS and LAR (immune suppression). As regards microarray cohort, LAR included 19.23% (n = 239) of TNBC, MLIA 17.38% (n = 216), BLIA 32.50% (n = 404) and BLIS 30.89% (n = 384). Concerning RNAseq cohort, LAR included 19.60% (n = 137) of TNBC, MLIA 17.31% (n = 121), BLIA 28.47% (n = 199) and BLIS 34.62% (n = 242). Distributions of patients among the four clusters in the two cohorts were similar (p = 0.3583). Distributions were also similar between studies for microarray cohort (p = 0.2479) and RNAseq cohort (p = 0.1775). Pooled data (n = 1942 patients) gave the following distribution: 19.36% for LAR; 17.35% for MLIA; 31.06% for BLIA; 32.23% for BLIS, confirming that TNBC are largely but not exclusively basal-like (63.29%). In brief, according to the TNBC subtype molecular characterisation, anti-androgen therapy could be prescribed for LAR, immune checkpoint inhibitors for MLIA and BLIA, alone or in combination, and TAM-focused and antineurogenic therapies for BLIS. As an extension of this work, TNBC transcriptomic data associated with their clinicopathological features and TNBC subtype annotations were included in bc-GenExMiner v5.0 web tool (http://bcgenex.ico.unicancer.fr). Citation Format: Mario Campone, Pascal Jézéquel, Fadoua Ben Azzouz, Wilfried Gouraud, Agnes Bassevile, Bertrand Michel, Hamza Lasla, Jean Sebastien FRENEL. Mesenchymal-like immune-activated: the 4th robust triple-negative breast cancer molecular subtype [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 PO2-15-05.

Abstract PO5-17-12: Impact of HER2-low status on axillary response and surgical management after neoadjuvant chemotherapy in early breast cancer

Abstract Background: Breast cancer (BC) with low expression of HER2 (HER2-low) might constitute a group of tumors with unique clinical and biological characteristics. Little is known about axillary downstaging and nodal surgical outcomes in HER2-low BC after neoadjuvant chemotherapy (NACT). The aim of this study was to evaluate axillary response and surgical management of the axilla in HER2-low BC patients submitted to NACT. Methods: In this cross-sectional single center study, we consecutively included all patients diagnosed with early HER2-negative BC between 2017 to 2021 that were treated with NACT. Patients must have biopsy-confirmed stage II-III BC, submitted to breast surgery after NACT. Clinical and pathological response in the axilla, type of axillary surgery, and surgical outcomes data were collected. HER2-low was defined by IHC score 1+, or 2+ ISH non-amplified; HER2-zero was defined by IHC score 0. Pathological response was determined locally. Results: At diagnosis, 291 patients had clinically involved axillary nodes (cN1: 69.4%; cN2: 25.1%; cN3: 5.5%). Among 136 patients eligible for SLN biopsy after NACT, 77 (26.5%) had pathologically negative SLN. Of those submitted to upfront axillary dissection (n=154), 52 (33.7%) had pN0 disease. In total, 129 (44.3%) patients with clinically involved axillary lymph nodes at diagnosis were converted to cN0 after NACT. There was no difference in the rate of axillary downstaging between HER2-low and HER2-zero tumors, 17.8% vs 26.5%, respectively (p=0.0026). 236 patients were submitted to sentinel lymph node biopsy (SLN), of whom 214 (81.4%) with blue dye-only, 40 (15.2%) with radioactive isotope, and 7 (2.7%) with dual tracer. SLN detection failure were reported for 12 patients (0.046%). Mean number of lymph nodes retrieved were 2.7. Most patients had a negative SLN biopsy (180/263; 68.4%), of whom 91 (50.6%) were HER2-low and 89 (49.4%) were HER2-zero. Positive SLN biopsy were reported in 83 patients (isolated tumor cells: 20,5%; micrometastasis: 24.1%; macrometastasis: 55.4%), with capsular extravasation in 25.3%. A positive SLN biopsy occurred in 61.4% of patients with HER2-low tumors, and in 38.5% of patients with HER2-zero tumors. Among patients with a positive SLN biopsy (n=83), 67 were submitted to axillary dissection, of whom 39 (58.2%) had no additional nodes involved (HER2-low: 61.5%; HER2-zero: 38.4%). Considering N0 tumors at diagnosis, 127 patients were submitted to SNL, and only 18.9% had a positive result (HER2-low: 66.6%; HER2-zero: 33.3%). In the subgroup of initially cN+ disease, 136 (46.7%) underwent SLN biopsy, with 59 (43.4%) with a positive result (HER2-low: 59.3%; HER2-zero: 40.7%). Conclusions: Our results showed that HER2-low BC patients had a statistically significant lower frequency of axillary downstaging after NACT. More than 40% of patients with cN+ at diagnosis were downstaged to cN0 and were submitted to SNL biopsy. Axillary dissection were avoided in roughly 25% of patients with cN+ BC at initial staging. Citation Format: Leonardo Roberto da Silva, Guilherme Sartori, Susana Ramalho, Tomás Reinert, Mahira Lopes Da Rosa, Grazielle Morais Tavares, Higor Mantovani, Vivian Vasconcelos, Ana Elisa Ribeiro Da Silva Cabello, Guilherme Coelho, Jovana Mandelli, Facundo Zaffaroni, Carlos Barrios, Marcia Silveira Graudenz, César Cabello. Impact of HER2-low status on axillary response and surgical management after neoadjuvant chemotherapy in early 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 PO5-17-12.

Abstract PO1-15-03: Spatially resolved analysis of tumor microenvironment in invasive lobular carcinoma

Abstract Background: Invasive lobular carcinoma (ILC) is the second most common histological breast cancer subtype; however, little is known about its tumor microenvironment (TME). Here, we aimed to study ILC TME using spatial transcriptomics (ST). Methods: We performed ST (Visium 10x Genomics) on frozen tumor samples from 43 primary hormone receptor positive (HR+), HER2-negative (HER2-) ILCs. Of note, 9 samples were coming from patients who experienced disease relapse. Relative hematoxylin/eosin (H&amp;E) slides were morphologically annotated (QuPath software). xCell was used to perform cell type enrichment analysis on sample pseudo-bulks. After cross-samples integration, ST spots were clustered using hierarchical clustering (STutility R package). intNMF algorithm was used to perform clustering at the patient-level by combining: RNA sequencing information (relative percentage of spot-level clusters in each sample), morphology and level of colocalization between cancer cells and the other cell types of the tumor microenvironment. METABRIC (HR+, HER2- ILC samples, n = 122) was used as external validation cohort. Survival analyses (univariable and multivariable adjusting for clinicopathological features) were performed using Cox proportional hazard models. Results: The patient-level classification revealed four groups showing different biological characteristics. Differences in terms of morphology (annotation) and pathway enrichment analysis based on marker genes (GSEA) were observed between groups. This information allowed us to annotate our groups as: proliferative (P, n = 12, enriched in tumor cells and proliferation-related pathways), normal-stroma enriched (NSE, n = 10, enriched in fibroblasts and carcinoma in situ), metabolic (M, n = 9, enriched in metabolic-related pathways) and metabolic-immune enriched (MIE, n = 10, enriched in adipose tissue, metabolic and immune-related pathways). Interestingly, a significantly higher presence of macrophages M2 was found in MIE group. Using group-specific gene signatures, we were able to reproduce the same 4 groups in the METABRIC. Of note, we observed significant differences in relapse-free survival (RFS) in METABRIC (p = 0.03), with NSE presenting better outcome, while P, M and MIE presented worse prognosis. Analysing the area of contact between adipocytes and cancer cells typical of MIE group, we noticed an enrichment in metabolic and M2 macrophages-related pathways. In doing so, we derived a 28 gene signature relative to the tumor-adipocytes contact area. Importantly, our signature was highly expressed in M and MIE groups, and it was not correlated with proliferation-related signatures. Interestingly, the adipocytes-related signature was significantly associated with shorter RFS in ILC patients in METABRIC (univariable: HR 1.4, p = 0.023; multivariable: HR 1.6, p = 0.005). Since both proliferation and metabolism showed to be key processes in defining prognosis in ILC, we built a prognostic index by integrating our adipocytes-related signature with genomic grade index (GGI, a proliferation-related signature). Our index outperformed other existing prognostic signatures (e.g., Oncotype DX, MammaPrint, EndoPredict, LobSig) in assessing prognosis (RFS) in ILC in METABRIC (univariable: HR 1.7, p &amp;lt; 0.001; multivariable: HR 1.7, p = 0.002). Conclusions: We identified 4 biologically driven HR+, HER2- ILC groups describing tumor microenvironment heterogeneity. Of note, two of the three groups associated to worse disease outcome were related to metabolism, highlighting the importance of such process in ILC biology and in the future development of new treatment strategies. Moreover, the prognostic power of our index has the potential to refine the assessment of the risk of relapse in ILC. Further validation is warranted. Citation Format: Matteo Serra, Mattia Rediti, Laetitia Collet, Frédéric Lifrange, David Venet, Nicola Occelli, Xiaoxiao Wang, Delphine Vincent, Ghizlane Rouas, Ligia Craciun, Denis Larsimont, Laurence Buisseret, Miikka Vikkula, François Duhoux, Françoise Rothé, Christos Sotiriou. Spatially resolved analysis of tumor microenvironment in invasive lobular carcinoma [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-15-03.

Abstract PO2-13-05: Deciphering pregnancy-associated breast cancer: distinctive molecular profile and clinical implications from GEICAM/2017-07 EMBARCAM study

Abstract Background: Pregnancy-Associated Breast Cancer (PABC) is defined as BC diagnosed during pregnancy, breastfeeding, or within the first year postpartum. This challenging entity has a distinct biology and worst prognosis and presents an increased risk for metastasis and death compared to non-PABC patients. Identification of molecular pathways that define this malignancy is crucial to explain its biological characteristics and potential association with pregnancy as well as serve to identify new biomarkers with preventive and clinical implications. This study assesses the gene expression profile of a PABC cohort from the Registry Study of Pregnancy and Breast Cancer (GEICAM/2017-07 EMBARCAM study, NCT04603820) to identify distinct molecular signatures and altered pathways for this entity. Methods: This was a multicenter, observational and ambispective age-matched cohort of PABC patients (n=46) and non-PABC patients (n=49). We analyzed the expression of 776 genes involved in 23 key breast cancer pathways, using the nCounter® Breast Cancer 360™ Panel (NanoString Technologies) on BC tumor samples, including molecular subtypes by PAM50 gene signature. Associations between individual differential genes and epidemiological and clinical features of these patients were explored. Normalized differential expression values were log2-transformed for statistical analysis in nSolver Software (NanoString Technologies). P-values were adjusted within each gene/signature and on the grouping variable level difference t-test using the Benjamini and Yekutieli False Discovery Rate (FDR) adjustment. Results: PABC patients’ clinical subtypes were distributed as 43,5% HR-positive/HER2-negative, 17,4% HER2-positive and 39,1% triple negative BC. These patients reported higher family history of BC/ovarian cancer compared to non-PABC (52.17% vs 26.53%; p=0.0124). Differential gene expression showed distinct molecular profiles in PABC and non-PABC patients: significantly genes enrichment involved in cell proliferation and p53 signature for PABC patients and higher expression of apoptosis, TGF-β, and PTEN signatures for non-PABC (adj p&amp;lt; 0.05). Furthermore, CDK4 expression signature (adj p=0.124) and DNA damage repair signatures Homologous Recombination Deficiency (HDR) (adj p=0.083) and BRCAness (adj p=0.220) were also upregulated in PABC tumors. The most significantly upregulated genes in PABC patients (CCNA2, DEPDC1, FAM83D, CDC20, CDKN3, TRIP13, TTK, MAD2L1, KIF2C, and UBE2C) (adj p&amp;lt; 0.05) were involved in cell cycle regulation and DNA damage repair. PABC basal-like tumors were found to be significantly more prevalent than non-PABC, as per the PAM50 classification (45.7% vs 20.4%; p=0.020). Interestingly, basal-like PABC showed higher expression of ESR1 than basal-like non-PABC (logFC=1.486; p=0.022). In contrast, luminal A PABC tumors exhibited lower ESR1 expression vs non-PABC (logFC=-1.5; p=0.021). Additionally, HER2-enriched PABC tumors displayed lower B7-H3/CD276 expression compared to HER2-enriched non-PABC (adj p=0.031). Conclusions: Our study shows that PABC is potentially a clinical and molecular different entity with predominance of the basal-like subtype. Moreover, our results suggest the activation of oncogenic pathways related to cell proliferation, DNA damage repair and p53 mutations which may lead to a clinically more aggressive phenotype for PABC patients. Likewise, the enrichment of BRCAness and HRD signatures found in PABC patients in our study may suggest an increased genetic instability due to a breakdown in the DNA damage repair. These findings may be clinically relevant and translate to new treatment options in PABC patients, who may benefit from therapies targeting DNA repair or cell-cycle checkpoints, such as PARP inhibitors. Citation Format: Juan de la Haba-Rodríguez, Regina Peña, Marina Pollan, Yolanda Jerez Gilarranz, Jose Ponce, Antonio Fernández Aramburu, Blanca Cantos, Ana Santaballa Bertrán, Elena Galve, María Eva Pérez, Susana de la Cruz, María Helena López-Ceballos, Yolanda Fernández, Fernando Moreno, Sonia González, Manuel Ruíz - Borrego, Isabel Blancas, José L Alonso-Romero, Álvaro Jiménez-Arranz, Raúl Rincón, Silvia Guil, Alejandra Díaz-Chacón, Rosalía Caballero, Begoña Bermejo, Angel Guerrero. Deciphering pregnancy-associated breast cancer: distinctive molecular profile and clinical implications from GEICAM/2017-07 EMBARCAM study [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 PO2-13-05.

Abstract PO5-17-09: Event-free survival in HER2-low vs HER2-zero breast cancer patients submitted to neoadjuvant chemotherapy

Abstract Background: Breast cancer (BC) with low expression of HER2 (HER2-low) might constitute a group of tumors with unique clinical and biological characteristics. However, the prognostic impact of HER2-low status vs HER2-zero in terms of pathological complete response (pCR) and event-free survival (EFS) after neoadjuvant chemotherapy (NACT) remains controversial. The aim of this study was to evaluate pCR and EFS rates in HER2-low vs HER2-zero early BC patients treated with NACT. Methods: In this cross-sectional study, we analyzed 565 consecutive patients with HER2-low and HER2-zero early BC submitted to neoadjuvant therapy from 2017 to 2021 in a single academic center. Patients with biopsy-confirmed stage I-III BC, with HER2-low or HER2-zero status, submitted to locoregional therapy after NACT were included. Clinical and pathological characteristics, pathological response, treatment protocol and RFS were analyzed and considered for statistical analysis. HER2-low was defined by IHC 1+, or 2+ ISH non-amplified; HER2-zero was defined by IHC 0. Pathological response was determined locally, and pCR defined as the absence of residual invasive disease with or without ductal carcinoma in situ in the breast and axilla. EFS was defined as the time from diagnosis to distant recurrence, a second primary, or death from any cause, whichever occurred first. Results: 441 patients were included in the analysis, 236 (53.5%) patients with HER2-low tumors, and 205 (46.5%) with HER2-zero tumors. Overall, 279 (63.3%) were estrogen receptor (ER)-positive tumors, and 162 (36.5%) were ER-negative tumors. Among ER-positive tumors, 166 (59.5%) had HER2-low status, and 113 (40.5%) were HER2-zero. For the ER-negative subgroup, the corresponding rates were 70 (43.2%) and 92 (56.8%), respectively. Most patients had stage II-III BC (423/441; 96%), and 20.2% (89/441) had high nodal tumor burden. Many patients received anthracycline- and taxane-based NACT (425/441; 96.4%), and over 90% (148/162; 91.3%) of ER-negative patients received a platinum agent. The overall pCR rate were 18.1%. Among ER+ tumors, pCR rates were 6.4%, 6.0%, and 7.0% in the overall, HER2-low and HER2-zero tumors, respectively. In ER- tumors, the corresponding pCR rates were 38.3%, 35.7, and 40.2%, respectively. The differences in pCR rates between HER2-low and HER2-zero tumors were not statistically significant (ER-positive, p=0.724; ER-negative, p=0.559). After a median follow-up of 42 months, 90 EFS events occurred. In the HER2-low group there were 56 (23.7%) EFS events, and 34 (16.6%) EFS events in the HER2-zero (HR 1.19; 95% CI 0.77-1.82; p=0.427). Among 279 patients in the ER-positive group there were 48 events, 32 in the HER2-low subgroup and 16 in the HER2-zero group (HR 1.16; 95% CI 0.64-2.13; p=0.622). Among 162 ER-negative tumors there were 42 events, 24 in the HER2-low subgroup, and 24 in the HER2-zero subgroup (HR 1.56; 95% CI 0.84-2.88; p=0.158). NACT discontinuation occurred in 86 patients (ER-positive: 45, 16.1%; ER-negative: 41, 25.3%), mainly due to toxicity (anthracycline-taxane: 10.0%; platinum: 11.7%). Disease progression during NACT were reported in 6.8% (30/441) of patients. Conclusions: Our results show similar pCR rates and RFS among HER2-low and HER2-zero early BC patients after standard NACT, which are consistent with the current literature. Despite the predictive role of low HER2 expression in selecting patients for novel antibody-drug conjugates (ADCs) therapy, its prognostic value could not be confirmed. Citation Format: Guilherme Sartori, Susana Ramalho, Leonardo Roberto da Silva, Tomás Reinert, Mahira Lopes Da Rosa, Grazielle Morais Tavares, Vivian Vasconcelos, Higor Mantovani, Ana Elisa Ribeiro Da Silva Cabello, Guilherme Coelho, Jovana Mandelli, Facundo Zaffaroni, César Cabello, Carlos Barrios, Marcia Silveira Graudenz. Event-free survival in HER2-low vs HER2-zero breast cancer patients submitted to neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-17-09.

Abstract PO5-14-09: Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients?

Abstract Background: Intratumor heterogeneity (ITH) refers to the presence of distinct cancer cell populations with different clones within a single tumor mass. Tumors with high ITH are more prone to progression, exhibit resistance to therapies, and are associated with an unfavorable prognosis. Therefore, evaluation of ITH is anticipated to serve as a breakthrough biomarker for prognosis and treatment strategies. Several computational algorithms have been proposed to quantify ITH. However, with the exception of DEPTH2, all of these algorithms require both DNA and RNA-sequence data from the same tumor, which can be challenging. In this study, we investigated whether DEPTH2 which solely analyzes RNA-sequence data, accurately reflects the biological characteristics of ITH. Methods: We analyzed clinicopathological and gene expression data from a total of 6573 breast cancer patients from several large independent cohorts: METABRIC (n = 1903), GSE96058 (n = 3273), GSE21974 (n = 57), GSE28844 (n = 61), GSE87455 (n = 153), GSE180280 (n = 57), and The Cancer Genome Atlas (TCGA, n = 1069). Results: DEPTH2 score was high in Triple Negative Breast Cancer (TNBC) subtype (p &amp;lt; 0.001), but no consistent correlation was observed with lymph node or distal metastasis. Higher DEPTH2 score was associated with cell proliferation, as evidenced by higher Nottingham histological grade and Ki67 gene expression (both p &amp;lt; 0.001). In agreement, high DEPTH2 tumors enriched all cell proliferation-related Hallmark gene sets such as E2F targets, G2M checkpoint, MYC targets v1 and v2, and Mitotic Spindle (FDR &amp;lt; 0.25). Additionally, high DEPTH2 tumors exhibited reduced immune cell infiltrations, such as CD8, CD4, Th1, Th2, B cells, and macrophages, and increased infiltrations of stromal cells, such as adipocytes, fibroblasts, blood and lymphatic endothelial cells (all p &amp;lt; 0.05). In TCGA cohort, DEPTH2 score exhibited a similar trend to intratumoral heterogeneity, homologous recombination deficiency, fraction altered, silent and nonsilent mutations, as well as SNV and Indel neoantigens. However, the Pearson’s correlation coefficient was very weak (p &amp;lt; 0.05). DEPTH2 score decreased in the GSE21974 and GSE87455 cohorts after neoadjuvant chemotherapy (NAC), whereas no change was observed in the GSE28844 and GSE180280 cohorts. DEPTH2 high tumors showed a lower rate of pathologic complete response (pCR) after NAC in estrogen receptor (ER)+HER2- subtype across all cohorts, however, opposite pattern was observed in the HER2+ and TNBC subtypes in all cohorts. Lastly, high DEPTH2 score was associated with lower disease free and overall survival in METABRIC and GSE96058 (p &amp;lt; 0.05), but not in the TCGA cohort. Conclusions: Our findings suggest that DEPTH2 score may not completely capture the biological characteristics associated with intratumor heterogeneity. Citation Format: Kohei Chida, Rongrong Wu, Arya Mariam Roy, Takashi Ishikawa, Kazuaki Takabe. Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-14-09.

Abstract PO2-14-08: Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response

Abstract Organoids serve as an important preclinical model in multiple cancers including breast cancer, demonstrating preservation of biological features and feasibility of drug screening, with reasonable cost and high scalability. It has been reported that patient-derived organoids (PDOs) can be robustly derived from both primary and metastatic breast tumors, with high resemblance to source tissues in histological, genomic, and transcriptomic features, as well as consistency in drug response compared to xeno-transplantations or patients (Sachs et al, 2018). Since 2018, the Institute for Precision Medicine (IPM) has developed a panel of breast cancer organoids. The current inventory now consists of 128 PDOs from 94 primary tumors, 12 metastases, 12 rapid-autopsy tumors, and 10 normal breast tissues with a 60% success rate of culture; as well as 33 organoids from animal models including 11 patient-derived xenograft organoids (PDXOs), 15 rat (RDO) and 7 mouse (MDO) tumor organoids with 85% success rate. For PDOs, the protocol includes in-lab processing of fresh, deidentified tissue within 60 minutes of surgical operation, thanks to close collaboration with surgeons, consented patients, and an institutional biospecimen core. BC organoids have been comprehensively characterized in terms of morphology, histology, genomics, transcriptomics, and functional experiments. Key biological features are preserved. PDOs (n=56) exhibit a mutational spectrum consistent with human breast cancers. ER expression is detectable in a subset of cultures with robust estradiol response indicated by GREB1 expression; and PDOs from invasive lobular carcinoma (ILC) mostly showed more discohesive structures than PDOs from no special type (NST), with clear E-cadherin loss under immunofluorescence. We have also successfully constructed transcriptionally and genetic modified BC PDOs with shRNA and CRISPR, which showed replicable performance in proliferation assay, drug response, and signaling by western blot. Of note, longitudinal tracking of PDOs with single cell RNA-sequencing (scRNA-seq) revealed well-preserved heterogeneity compared to primary tumor, while transcriptomic clonality was mildly decreased in later passages from pilot data. Future work will include expansion of current collection from various sources, longitudinal characterization with scRNA-seq and DNA sequencing, as well as more functional studies. We aim to construct a robust resource of well-characterized and reliably-usable model bank for preclinical breast cancer research. Citation Format: Daniel Brown, Kai Ding, Fangyuan Chen, Jian Chen, Steffi Oesterreich, Peter Lucas, Priscilla McAuliffe, Jennifer Atkinson, ADRIAN LEE. Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-08.

Abstract PO5-18-10: Omission of SLNB in triple-negative and HER2-positive breast cancer patients with radiologic and pathologic complete response in the breast after NAST: a single-arm, prospective surgical trial (EUBREAST-01 trial, GBG 104)

Abstract Background: Currently, axillary surgery for breast cancer is considered a staging procedure that does not seem to influence breast cancer mortality since the risk of developing metastasis depends mainly on the biological behavior of the primary (seed-and-soil model). Based on this, postsurgical therapy should be considered based on biological tumor characteristics. Retrospective data of cancer registry trials showed a strong correlation between breast pathologic complete response (pCR) and nodal pCR depending on intrinsic subtypes. Improvements in systemic treatments for breast cancer have increased the rates of pCR in patients receiving neoadjuvant systemic therapy (NAST), offering the opportunity to decrease, and perhaps eliminate, surgery in patients who have a pCR. Trial design: The EUBREAST network designed a clinical trial (NCT04101851) in which only patients with the highest likelihood of having a pCR after NAST (triple-negative or HER2-positive breast cancer) will be included, and type of surgery will be defined according to the response to NAST rather than on the classical T and N status at presentation. In the ongoing trial, axillary surgery will be eliminated (no axillary sentinel lymph node biopsy [SLNB]) for initially clinical node-negative (cN0) patients with radiologic complete remission (rCR) and a breast pCR (ypT0/ypTis) as determined in the lumpectomy specimen. The trial design is a multicenter single-arm study with a limited number of patients (N=440 as the screening population with an expected 80% pCR-rate) which might give practice-changing results in a short period, sparing the time and the costs of a randomized comparison. Patients will be recruited in European countries (Austria, Germany, Italy, and Spain) over 48 months. Inclusion criteria: -Written informed consent -Histologically confirmed unilateral primary invasive carcinoma of the breast (core biopsy). Multifocal or multicentric tumors are allowed if breast-conserving surgery (BCS) is planned. -Age at diagnosis at least 18 years -imaging techniques with estimated tumor stage between cT1-T3 before NAST -triple-negative (TNBC) or HER2-positive invasive breast cancer -TNBC is defined by: ER-negative (&amp;lt; 10% positive cells in IHC) and PgR-negative (&amp;lt; 10% positive cells in IHC), HER2-negative -clinically and sonographically tumor-free axilla before core biopsy (cN0/iN0) -in cases with cN0 and iN+, a negative core biopsy or fine-needle aspiration biopsy of the sonographically suspected lymph node is required -no evidence for distant metastasis (M0) -standard NAST with rCR -planned BCS with postoperative external whole-breast irradiation (conventional fractionation or hypofractionation) Primary objective: 3-year rate of axillary recurrence-free survival (ARFS) after BCS Statistics: The calculated total case number for per-protocol analysis is N=350, and the expected total number of screened patients is N=440. The assumption for acceptable 3-year ARFS ≥98.5% in the experimental arm is based on previous study findings. Timelines: -First patient in: January 2021 -Last patient in: December 2024 -Primary outcome analysis: Q1/2027 Current accrual: In June 2023, 255 patients were recruited; the majority of them in Germany. Contact: Prof. Dr. Toralf Reimer (eubreast-01@kliniksued-rostock.de), study chair Dr. Oreste D. Gentilini (gentilini.oreste@hsr.it), study co-chair Funding by Else Kroener-Fresenius Foundation, German Society of Senology, University of Rostock (Germany), and San Raffaele Hospital (Milan, Italy). Citation Format: Toralf Reimer, Bernd Gerber, Thorsten Kuehn, Nikola Bangemann, Anke Kleine-Tebbe, Andrea Stefek, Ulrike Doerste, Carolin Hammerle, Oliver Hoffmann, Alexander Hein, Isabel Rubio, Florentia Peintinger, Keyur Mehta, Sibylle Loibl, Edoardo Botteri, Oreste Gentilini. Omission of SLNB in triple-negative and HER2-positive breast cancer patients with radiologic and pathologic complete response in the breast after NAST: a single-arm, prospective surgical trial (EUBREAST-01 trial, GBG 104) [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 PO5-18-10.

KDM7A-DT induces genotoxic stress, tumorigenesis, and progression of p53 missense mutation-associated invasive breast cancer.

Stress-induced promoter-associated and antisense lncRNAs (si-paancRNAs) originate from a reservoir of oxidative stress (OS)-specific promoters via RNAPII pausing-mediated divergent antisense transcription. Several studies have shown that the KDM7A divergent transcript gene (KDM7A-DT), which encodes a si-paancRNA, is overexpressed in some cancer types. However, the mechanisms of this overexpression and its corresponding roles in oncogenesis and cancer progression are poorly understood. We found that KDM7A-DT expression is correlated with highly aggressive cancer types and specific inherently determined subtypes (such as ductal invasive breast carcinoma (BRCA) basal subtype). Its regulation is determined by missense TP53 mutations in a subtype-specific context. KDM7A-DT transcribes several intermediate-sized ncRNAs and a full-length transcript, exhibiting distinct expression and localization patterns. Overexpression of KDM7A-DT upregulates TP53 protein expression and H2AX phosphorylation in nonmalignant fibroblasts, while in semi-transformed fibroblasts, OS superinduces KDM7A-DT expression in a TP53-dependent manner. KDM7A-DT knockdown and gene expression profiling in TP53-missense mutated luminal A BRCA variant, where it is abundantly expressed, indicate its significant role in cancer pathways. Endogenous over-expression of KDM7A-DT inhibits DNA damage response/repair (DDR/R) via the TP53BP1-mediated pathway, reducing apoptosis and promoting G2/M checkpoint arrest. Higher KDM7A-DT expression in BRCA is associated with KDM7A-DT locus gain/amplification, higher histologic grade, aneuploidy, hypoxia, immune modulation scores, and activation of the c-myc pathway. Higher KDM7A-DT expression is associated with relatively poor survival outcomes in patients with luminal A or Basal subtypes. In contrast, it is associated with favorable outcomes in patients with HER2+ER- or luminal B subtypes. KDM7A-DT levels are coregulated with critical transcripts and proteins aberrantly expressed in BRCA, including those involved in DNA repair via non-homologous end joining and epithelial-to-mesenchymal transition pathway. In summary, KDM7A-DT and its si-lncRNA exhibit several intrinsic biological and clinical characteristics that suggest important roles in invasive BRCA and its subtypes. KDM7A-DT-defined mRNA and protein subnetworks offer resources for identifying clinically relevant RNA-based signatures and prospective targets for therapeutic intervention.

The association between gynecological complaints and the uterine sonographic features in women with a history of cesarean section.

The aim of this study is to investigate the association between post-cesarean sonographic uterine measures, dysmenorrhea, and bleeding disorders. This is a cross-sectional study where 500 women with a history of only one cesarean section (CS) were recruited. A transvaginal transducer, GE RIC6-12-D was used for the acquisition of volumetric datasets 18±7months postpartum. Uterine length (UL), cervical length (CL), niche length (L), niche depth (D), niche width (W), fibrosis length (FL), fibrosis depth (FD), residual myometrial thickness (RMT), endometrial thickness (EM), scar to internal os distance (SO), anterior myometrial thickness superior (sAMT) and inferior (iAMT) to the scar, and the posterior myometrial thickness opposite the scar (PMT), superior (sPMT), and inferior to it (iPMT) were measured. Logistic regression with odds ratios (OR), 95% confidence intervals (CI) and ROC curves were utilized. The proportion of patients with incident post-cesarean bleeding disorders and dysmenorrhoea was 36% (CI 32%, 40%) and 17% (CI 14%, 21%) respectively. Univariate logistic regression showed that only UL was associated with bleeding disorders [OR 1.04 (CI 1.01,10.7) p value 0.005], whereas dysmenorrhea was associated with RMT [OR 0.82 (CI 0.71,0.95) p value 0.008], SO [OR 0.91 (CI 0.86,0.98) p value 0.01], and RMT ratio [OR 0.98 (CI 0.97,0.99) p value 0.03]. Multivariate logistic regression for dysmenorrhoea including SO and RMT remains statistically significant with p values <0.05 and area under the curve of 0.66. There is an association between sonographic appearance of CS scars and dysmenorrhoea. Nevertheless, the association is weak and other biological post-cesarean characteristics should be explored as potential causes.

Abstract PS03-09: Age-related normal breast tissue features differ in women with germline BRCA1/2 mutations

Abstract Ageing, a breast cancer risk factor, can be reflected in histologically normal breast tissue (NBT). However, the relationship between age-related histological features and cancer risk is not fully understood. We propose that biological and microscopic features can be identified in seemingly histologically NBT in gBRCA1/2m carriers that are indicative of the earliest tissue changes of breast cancer, potentially as a result of accelerated tissue ageing. To study NBT, we have put in place a unique and scalable repository, named OASIS, storing currently &amp;gt;2,000 digitised whole slide images (WSI) of H&amp;E-stained NBT from individuals with long clinical follow-up. These tissues are from individuals with a spectrum of risk for developing breast cancer and derived from reduction surgery from non-gBRCA1/2m carriers, risk reducing masectomies, and from contralateral and peri-tumoural NBT from gBRCA1/2m carriers and women with breast cancer. Across 6 NBT resources, 70 selected WSIs were manually annotated for epithelial cells, fibrous stroma and adipocytes, resembling the "ground truth". Extensive comparisons of different tile sizes, tile overlapping ratios, stain normalisation techniques and deep learning (DL) feature extractors were conducted to implement a Tissue-Classifier, which achieved 95% accuracy in 3-fold cross-validation. Based on the MobileNet architecture, a DL-framework achieved a sensitivity of 81.2% and specificity of 81.8% (84.7% AUC) in predicting Tissue-Age based on discernible histological patterns in healthy women. In gBRCA1/2m carriers, we discovered features of accelerated tissue ageing (e.g. degree of lobule involution), indicating that the biological NBT age differs from their chronological age. Our DL-based framework robustly captured histological patterns to predict tissue components and age. The global organisation of these patterns showed variations in WSIs of women with different risks of developing breast cancers, which may provide new insights into premalignant alterations in NBT. Citation Format: Anita Grigoriadis, Mario Parreno-Centeno, Siyuan Chen, Gregory Verghese, Esther Lips, Cheryl Gillett, Louise Jones, Sarah Pinder. Age-related normal breast tissue features differ in women with germline BRCA1/2 mutations [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 PS03-09.

Abstract PO5-12-08: QTc prolongation as Adverse Effect of CDK4/6 inhibitors: A systematic review and meta-analysis of randomized controlled trials

Abstract Introduction: CDK4/6 inhibitors (CDK4/6i), such as abemaciclib, ribociclib, and palbociclib, have significantly improved outcomes for patients with ER+/HER2− breast cancer. Nevertheless, they differ among each other in terms of chemical, biological, and pharmacological features, as well as toxicity profiles. Ribociclib causes concentration-dependent increases in the QTc interval, particularly in combination with tamoxifen. However, the pathogenesis of ribociclib-induced QTc prolongation remains unclear. We aim to determine whether QTc prolongation is caused by CDK4/6i in general or if it is associated with ribociclib only. Methods: We performed a systematic review and meta-analysis of randomized clinical trials (RCTs) to compare QTc prolongation in patients using CDK4/6i in combination with hormonal agents to patients that did not use CDK4/6i, including neoadjuvant, adjuvant, or metastatic scenarios in breast cancer. We searched Embase, PubMed and Cochrane Library databases from inception to June 2023. The outcomes evaluated were QTc prolongation of all grades. Statistical analysis was performed with R software version 2023.03.0+386. Results: We included 14 RCTs comprising 16,013 patients in the quantitative analysis, of whom 8,585 underwent therapy with CDK4/6i, and one more paper was included only in the qualitative analysis due to not reporting the exact number of events of QTc prolongation. An increased risk of QTc prolongation was associated with the use of CDK4/6i (RR 2.32; 95% CI 1.67–3.23; p=0.000001; I²=46%). Subgroup analyses revealed a significant increase in the QTc interval for the ribociclib and palbociclib cohorts. The ribociclib subgroup showed a risk ratio of 3.07 (RR 3.07; 95% CI 2.17-4.36; p&amp;lt; 0.000001; I² = 21%) while the palbociclib subgroup had a risk ratio of 1.52 (RR 1.51; 95% CI 1.05-2.16; p=0.025, I²=0%). Conclusion: Our findings suggest that QTc prolongation may be a common adverse effect of CDK4/6i, with ribociclib carrying greater risk. These results are important for guiding clinical decision-making, and further investigation is warranted to better understand the mechanisms and clinical implications of CDK4/6 i-induced QTc prolongation. Citation Format: Bruno Carvalho, Pedro Cotta Abrahão Reis, Alice Marinho, Ana Carolina Comini, Debora Xavier, Beatriz Mella S. Pessoa, Felipe Batalini. QTc prolongation as Adverse Effect of CDK4/6 inhibitors: A systematic review and meta-analysis of randomized controlled trials [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 PO5-12-08.

Abstract PO5-13-10: Determining tumor-driving cell signaling pathways in breast cancer: Support for targeted therapy selection

Abstract Introduction: Success of therapeutic interventions largely rely on the pathological and biological characteristics of the tumor and varies due to the heterogeneous nature of breast cancers. Signal transduction pathways play an important role and are often used as target for specific therapies like hormone treatment, PI3K inhibitors etc. Immunohistochemistry (IHC) is currently the first assessment to divide tumors for subtyping and treatment and, especially in more advanced disease, mutation analyses are often used. Both IHC and mutation analyses only analyze a specific part of a signal transduction pathway and do not correlate well with outcome. Therefore, there is a need for new approaches to look at signaling pathways in cancer. We have developed a methodology that measures and quantifies signal transduction pathways (STPs) activities to reveal potential tumor-driving STPs to identify new options for targeted therapy in breast cancer. Method: Using the mRNA-based OncoSIGNal pathway activity profiling qPCR test (InnoSIGN), STP activities of 7 pathways (AR, ER, PI3K, MAPK, HH, TGFβ and Notch) were measured and quantified on a scale from 0-100. By using a set of non-tumor epithelial breast tissue samples, a reference range for normal physiological pathway activity was defined. A threshold was set on the 95th percentile of this range. A pathway activity score above this threshold was considered to be high (activated). Using this methodology, 201 ER positive (based on IHC) and 64 triple negative FFPE breast tumor samples obtained from different study sites and commercial biobanks were analyzed, including samples from primary and metastatic lesions, to determine for each individual tumor its pathway activity profile and identify activated tumor-driving pathways. Results: In the 195 primary ER positive tumors, the ER pathway was found to be activated in 78% of the samples, even though all samples were strongly ER immunohistochemistry stain positive. In addition to ER pathway activation, occasionally other pathways were activated: PI3K in 37%, HH in 25%, MAPK in 19%, AR in 20%, TGFβ in 6%, and Notch in 1%, of these samples. Analysis of 27 metastatic samples of ER positive tumors revealed in only 63% an active ER pathway. Additionally, more frequently PI3K pathway was observed in 70% of the samples. Other frequencies of pathway activation were: AR in 7% and MAPK in 4%, No Notch and TGFβ activity was found in this cohort. In 76 of the 78 triple negative tumors (TNBC) the ER pathway was inactive as expected. Remarkably, 2 samples appeared to have an activated ER pathway. An activated MAPK pathway was found in 50%, PI3K in 49%, HH in 18%, AR in 14%, TGFβ in 5% and Notch in 3% of these TNBC tumors. Conclusion: Compared to ER positive primary tumors we observed in metastatic ER positive tumors less frequent activation of the ER and more frequent activation of the PI3K pathway, suggesting that the latter pathway is associated with a more aggressive tumor phenotype. The triple negative subtype is characterized predominantly by activation of the PI3K, MAPK, AR and/or HH pathways, which may create new options for personalized targeted therapy of this subtype. OncoSIGNal can be used to determine the tumor driving signaling pathways in breast cancer patients, guiding selection of personalized targeted therapies. Citation Format: Paul van de Wiel, Yvonne Wesseling-Rozendaal, Anke Pierik, Dianne van Strijp, Saskia Vermeer, Fatema Tashrifwala, Josette Hartnett, Robert Babkowski, Suzanne Rose. Determining tumor-driving cell signaling pathways in breast cancer: Support for targeted therapy selection [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 PO5-13-10.

Abstract PO1-14-11: Different molecular processes are associated with recurrence in the clinical high- genomic low risk (cH/gL) and clinical low- genomic high risk (cL/gH) groups of the MINDACT clinical trial

Abstract Background: Despite the growing understanding of estrogen receptor positive/HER2-negative (ER+/HER2-) breast cancer (BC) biology, many unknowns remain regarding the mechanisms of disease recurrence. While several multigene prognostic signatures have shown clinical utility in identifying those patients at high or low genomic risk of recurrence, they can present with some limitations. In this retrospective analysis of the MINDACT trial, we interrogated the available transcriptomic data to understand which biological characteristics of the tumor are associated with recurrence in patients from the clinical high- genomic low risk (cH/gL) and clinical low- genomic high risk (cL/gH) groups (where the clinical and genomic risk were defined by a modified version of Adjuvant Online! and the 70-gene signature, respectively). Methods: These analyses focused on the subset of patients with ER+/HER2- tumors of no special type (NST) who received endocrine therapy (which comprised the majority of this NST ER+/HER2- sub-cohort) and for which central pathology and gene expression data were available (n= 1245). Scores of gene expression modules associated with key biological processes in breast cancer defined in a collection of previous publications were computed from the gene expression matrix using the formula described in Desmedt et al. (Clin. Cancer Res. 2008). Gene Set Variation Analysis method (R package ‘GSVA’ – version 1.40.1) was used to derive the enrichment scores of fifty hallmark gene sets available in the ‘H’ collection of the MSigDB database (version 7.5.1). Survival analyses were performed for disease-free survival (DFS) and distant recurrence-free survival (DRFS). Univariable and multivariable regression models, implemented by Cox regression and Fine-Gray subdistribution hazard regression methods, were performed to evaluate the association of enrichment of hallmarks and gene modules with DFS and DRFS, respectively (covariates =age, nodal status, KI67, tumor size, chemotherapy, radiotherapy). Results: DFS event occurred in 150/913 (16.4%) and 51/332 (15.4%) NST ER+/HER2- patients in the cH/gL and cL/gH groups, respectively. DRFS event occurred in 70/913 (7.7%) and 21/332 (6.3%) NST ER+/HER2- patients in the cH/gL and cL/gH groups, respectively. In the cH/gL group, we observed an association of hallmarks related to cell cycle and downregulation of gene modules related to lymphocyte-centric immune activities with worse prognosis (Table). For the cL/gH, we did not observe an association between hallmarks related to cell cycle and worse prognosis but an enrichment of hallmarks related to PI3K/AKT/mTOR signaling as well as several metabolic hallmarks related to insulin activities (Table). Of interest, in this group of patients, higher scores of the HER2-associated gene expression signature were also associated with worse prognosis. Across the two groups effects were more pronounced for DRFS than DFS. Conclusion: Different molecular processes are associated with progression in these two distinct groups of patients. The association of hallmarks related to cell cycle with survival in the cH/gL group supports the observation that adjuvant chemotherapy could benefit a subset of these patients. In the cL/gH group, the association of signatures associated with PI3K pathway and HER2 can have potential clinical relevance given their current targetability in the metastatic setting. This retrospective study is funded by the Breast Cancer Research Foundation. Table. Representative hallmarks and gene expression modules independently associated with prognosis in NST ER+/HER2- patients in the cH/gL group and in the cL/gH group #p-values reported were not corrected for multiple testing Citation Format: Christine Desmedt, Ha-Linh Nguyen, François Richard, Marion Maetens, Coralie Poncet, Laura De Meulemeester, Kim Aalders, Mauro Delorenzi, Suzette Delaloge, Jean-Yves Pierga, Etienne Brain, Suzan Vrijaldenhoven, Peter A Neijenhuis, Emiel Rutgers, Florentine Hilbers, Laura Van ’t Veer, Giuseppe Viale, Christos Sotiriou, Martine Piccart, Fatima Cardoso. Different molecular processes are associated with recurrence in the clinical high- genomic low risk (cH/gL) and clinical low- genomic high risk (cL/gH) groups of the MINDACT clinical trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-14-11.

Abstract PO3-22-06: Safety and efficacy of trastuzumab deruxtecan and concomitant radiation therapy in patients with metastatic HER2-positive breast cancer

Abstract Background Trastuzumab deruxtecan (T-DXd) currently represents the standard of care for the treatment of patients with metastatic HER2-positive (HER2+) breast cancer (BC) after disease progression in the first line, which includes taxanes and trastuzumab. This recommendation is based on the results of the DESTINY-Breast03 trial, which demonstrated improved efficacy compared to trastuzumab emtansine (T-DM1). Radiation therapy (RT) is frequently required in the metastatic setting, either for palliative purposes or with ablative intent in cases of oligometastatic or oligoprogressive disease. The aim of our study is to evaluate the safety of using T-DXd and concomitant radiation therapy (RT) in a consecutive series of HER2+ BC patients. Methods We conducted a retrospective evaluation of patients diagnosed with metastatic HER2+ BC who initiated treatment with T-DXd between May 2021 and May 2023 at our institution, with or without receiving RT. We collected clinical data pertaining to the diagnosis of metastatic disease, T-DXd treatment, acute toxicities, survival data, and biological characteristics of both the primary tumor and metastases. The primary objective of this study was to assess the association between RT and any adverse events greater than grade (G) 2. Results We retrospectively evaluated data from 30 consecutive patients who were treated with T-DXd, with or without RT. Among these patients, ten received RT either immediately before (within a month) or during T-DXd treatment, while the remaining 20 did not. The median age of the patients was 72 years (range 34-88), and the median follow-up period was 9 months. Thirteen patients received T-DXd as the fourth or subsequent line of systemic anti-HER2 treatment, eight patients received it as the third line, and seven patients received it as the second line. Two patients, who experienced early metastatic disease relapse (&amp;lt; 6 months after completion of adjuvant anti-HER2 therapy), received T-DXd as their first-line treatment. The median prescribed total dose of RT was 34 Gy (range 20-48), with a median number of fractions of 4 (range 2-10). The median equivalent dose in 2 Gy fractions (EQD2) was 64 Gy (range 23.3-104), and the median biologically effective dose (BED) was 76 Gy (range 28-125). The most commonly treated sites were bone (70% of cases), followed by brain (20%) and liver (10%). A chi-square test of independence was conducted to examine the relationship between the administration of RT and the development of toxicity exceeding G2. However, this analysis did not reveal a significant relationship (p = .27). Regarding the specific toxicities of interest associated with T-DXd, three cases of grade 3 fatigue were reported in the group that did not receive RT, while one case was observed in the RT group. Overall, only one case of grade 3 nausea was reported, and it occurred in the group that did not receive RT. G2 interstitial lung disease, which led to discontinuation of T-DXd, was observed in one case in the RT group and one case in the no-RT group. Conclusions Our initial data is promising regarding the potential safety of this combination, as it indicates that concurrent RT did not lead to an increase in severe acute toxicity. However, larger series of data are required to validate and confirm these findings. Citation Format: Luca Visani, Carlotta Becherini, Niccolo' Bertini, Ilaria Bonaparte, Luca Burchini, Carolina Orsatti, Marianna Valzano, Marco Banini, Viola Salvestrini, Erika Scoccimarro, Vieri Scotti, Isacco Desideri, Giulio Francolini, Lorenzo Orzalesi, Marco Bernini, Cinzia Tommasi, Jacopo Nori, Simonetta Bianchi, Icro Meattini, Lorenzo Livi. Safety and efficacy of trastuzumab deruxtecan and concomitant radiation therapy in patients with metastatic HER2-positive 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 PO3-22-06.

Toxicity of Beauveria bassiana to Bactrocera dorsalis and effects on its natural predators.

Entomopathogenic fungi (EPF) are economical and environmentally friendly, forming an essential part of integrated pest management strategies. We screened six strains of Beauveria bassiana (B1-B6) (Hypocreales: Cordycipitaceae), of which B4 was the most virulent to Bactrocera dorsalis (Hendel) (Diptera: Tephritidae). We further assessed the biological characteristics of strain B4 and the environmental factors influencing its ability to infect B. dorsalis. We also evaluated the effects of B4 on two of the natural predators of B. dorsalis. We found that strain B4 was the most virulent to 3rd instar larvae, pupae, and adult B. dorsalis, causing mortality rates of 52.67, 61.33, and 90.67%, respectively. B4 was not toxic to B. dorsalis eggs. The optimum B4 effects on B. dorsalis were achieved at a relative humidity of 91-100% and a temperature of 25°C. Among the six insecticides commonly used for B. dorsalis control, 1.8% abamectin emulsifiable concentrate had the strongest inhibitory effect on B4 strain germination. B4 spraying affected both natural enemies (Amblyseius cucumeris and Anastatus japonicus), reducing the number of A. cucumeris and killing A. japonicus adults. We found a valuable strain of EPF (B4) that is virulent against many life stages of B. dorsalis and has great potential for the biological control of B. dorsalis. We also provide an important theoretical and practical base for developing a potential fungicide to control B. dorsalis.