Babadi, Ellie

Objective: In this study, we evaluate the utility of ctDNA analysis in a large cohort of patients for whom ctDNA testing was ordered commercially with real-world application. Summary Background Data: Circulating tumor DNA (ctDNA) has emerged as a prognostic and predictive biomarker for assessing post-surgical molecular residual disease (MRD) and response to treatment. Methods: A real-world data analysis was performed using commercial ctDNA testing (SignateraTM) from patients (N=795, n=5,971 plasma samples) with stage I-III colon cancer treated at multiple US institutions. The association of ctDNA detection within the MRD window, during surveillance, and the impact of ACT was correlated with patient outcomes. Results: ctDNA-positive patients during the MRD window and surveillance showed significantly shorter DFS compared to ctDNA-negative patients (hazard ratio (HR): 9.85, P<0.0001; HR: 26.91, P<0.0001). Multivariate analysis of the MRD window revealed ctDNA-positivity as the most significant factor associated with inferior DFS (adjHR: 7.7, P<0.001). MRD-positive patients who received ACT showed improved DFS compared to patients observed post-surgery (adjHR: 6.1, FDR adj P=0.0007). No ACT benefit was observed in MRD-negative patients (adj HR: 1.20, FDR adj P=0.768). On evaluating ctDNA dynamics from MRD to surveillance, patients who remained ctDNA-positive or converted from negative to positive showed a significantly inferior DFS (HR: 34.40, P<0.0001; HR: 13.65, P<0.0001) compared to persistently ctDNA-negative patients. Conclusions: Postsurgical ctDNA detection is prognostic of relapse and potentially predictive of ACT benefit in patients with resectable colon cancer, which may enable personalized surveillance, intervention, and/or trial options, ultimately improving patient outcomes.

Abstract Background: Despite recent advances in the treatment of triple negative breast cancer (TNBC) many patients are still suffering from early recurrence, therapy resistance, and organ metastasis 1 to 3 years post-chemotherapy. TNBC cells with stem cell-like properties are resistant to standard of care chemotherapy and are responsible for early tumor relapse and poor outcome. Up-regulation of PD-L1 expression in Cancer Stem Cells (CSCs) has been associated with immune evasion capacity and promotion of their stem-like properties. In this study we identify two druggable stemness target, Aurora-A Kinase (AURKA) and NOTCH3, with positive feedback on intra-tumoral PD-L1 expression and cancer cell plasticity. Methods: MDA-MB 231, MDA-MB 231 LM and SUM149-PT TNBC cells were used both in 2D and 3D environment. Unique TNBC cells were established from metastatic PDX models. TNBC cells were treated with Alisertib (AURKA inhibitor, 50nM) or AV353 (NOTCH3 inhibitor, 200ng). Immunofluorescence and immunoblot were used to compare different biomarker expression. ALDH was measured using Aldefluor kit. Apoptotic cells were quantified in real-time using IncuCyte S3. In vivo studies; 1x106 MDA MB 231/LM cells were injected into the mammary fat pad of NSG-female mice and treated with Alisertib or AV353. Control groups were treated with pacebo. Results: Our study shows that AURKA and NOTCH3 pharmacological blockade reduced nuclear/undruggable PD-L1, that is not achieved clinically by using FDA-approved immune checkpoint inhibitors (ICIs). Significantly, following stemness-targeted therapy with alisertib or AV353, reduction of intra-tumoral PD-L1 resulted in inhibition of tumor growth, decreased levels of CD44 expression, increased CD8+ T-cell tumor infiltration, impairment in the enrichment of ALDHhigh CSCs and enhanced chemosensitivity. In vivo pharmacological blockade of AURKA and NOTCH3 signaling pathways induced inhibition of tumor growth, apoptosis, and impairment of organ metastases. Conclusions: This study demonstrates that selective AURKA and NOTCH3 pharmacological blockade results in the inhibition of cancer cell plasticity that is linked to reduction of undruggable nuclear PD-L1 in TNBC cells. Our findings provide the strong preclinical rationale to develop novel clinical trials for patients with advanced TNBC that are refractory to chemotherapy and show limited response to FDA-approved ICIs. Citation Format: Antonino Bonaventura D'Assoro, Matthew Goetz, Carol Lange, Roberto Leon-Ferre, Tufia Haddad, James Ingle, Annapaola Montagner, Andrew Takchi, Ellie Babadi, Lucio Miele, Urban Lendahl, Urban Lendahl. Pharmacological blockade of AURKA and NOTCH3 oncogenic pathways to inhibit cancer cell plasticity in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5978.