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Background: ctDNA analysis is emerging as an attractive non-invasive approach to characterize tumor biology, describe its evolution over time, and predict treatment benefit. Here, we assessed the prognostic and predictive role of baseline and dynamic ctDNA analysis in HR+/HER2- aBC patients (pts) treated with R+L. Methods: 287 postmenopausal pts were enrolled in the BioItaLEE trial (NCT03439046). Liquid biopsies were collected at baseline (D0; n=263), day 15 of cycle 1 (D15; n=238), day 1 of cycle 2 (C2D1; n=241) and at first imaging (FI, at approximately 12 weeks; n=206). ctDNA analysis was carried out using a 533-amplicon Custom AmpliSeq HD Panel, with amplicons covering the coding exons of 39 BC-related genes (limit of detection: 0.1%). Target mutations were defined as single-nucleotide variant (SNV) or Insertion/Deletion detected at D0. When multiple target mutations were detected, the one with the highest variant allele frequency (VAF) was considered. The association between pre-treatment and on-treatment ctDNA dynamics with progression-free survival (PFS) was assessed using Multivariate Cox models. VAF clearance was defined as 100% decrease in a target mutation. Results: Median follow-up was 26.9 months and median PFS was 23.39 (20.8-NE) months. At baseline, target mutations were detected in 113 pts (43.0%), whereas 150 pts were wild-type (wt). Mean (SD) pre-treatment VAF at D0 was 11.3% (14.4). The absence of a target mutation at D0 was associated with good prognosis (HR: 0.41, 95% CI: 0.27–0.61; p Citation Format: Giampaolo Bianchini, Luca Malorni, Grazia Arpino, Alberto Zambelli, Fabio Puglisi, Lucia Del Mastro, Marco Colleoni, Filippo Montemurro, Giulia Bianchi, Ida Paris, Giacomo Allegrini, Marina Elena Cazzaniga, Michele Orditura, Claudio Zamagni, Stefano Tamberi, Daniela Castelletti, Matteo Benelli, Maurizio Callari, Angela Santoro, Michelino De Laurentiis. Circulating tumor DNA (ctDNA) dynamics in patients with hormone receptor positive (HR+)/HER2 negative (HER2-) advanced breast cancer (aBC) treated in first line with ribociclib (R) and letrozole (L) in the BioItaLEE trial [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS3-07.

Background: The primary objective of this phase IIIb, single arm trial is to study circulating tumor DNA (ctDNA) alterations, their evolution during treatment and association with clinical outcome in patients receiving ribociclib and letrozole as first-line therapy for aBC. Here we report baseline ctDNA mutational status and its correlation with clinical-pathological characteristics and response to treatment on the basis of first imaging evaluation. Methods: From February to December 2018, 287 post-menopausal patients (pts) with hormone receptor positive (HR+) and HER2 negative aBC were enrolled in 47 Italian Centers; 271 pts were suitable for biomarker analysis (Biomarker Analysis Set- BAS). Data on first imaging evaluation were available for 225 pts. Baseline liquid biopsies were analyzed both for Copy Number Variation (CNV) by Oncomine Pan-Cancer Cell-Free Assay (Thermo Fisher Scientific) and for Single Nucleotide Variant (SNV) using 533 amplicon custom AmpliSeq HD panel optimized for the detection of low abundance events in ctDNA. Variants were analyzed as both individual genes and as pre-defined pathways (n=10). Results: At BAS, median age was 66 years (range 47-86). At study entry, 60% (109) and 40% (162) of pts had recurrent and de-novo disease, respectively, with 43% (117) of pts with visceral and 24% (64) with bone-only involvement. At least one CNV alteration or a hotspot mutation was detected in 51% of patients. The most frequently altered genes were PIK3CA (22.14%), TP53 (15.50%), FGFR1 (6.64%), CCND1 (4.80%), CCND2 (4.80%), KMT2C (4.43%), MYC (4.06%), CDK4 (3.69%) AKT1 (3.32%), PTEN (3.32%), ERBB2 (2.58%), CCND3 (2.58%), APC (2.21%) and MAP2K4 (2.21%). More than one alteration was observed in 28% pts (either CNV or hotspots). Mutation in KTM2C or alterations in genes belonging to the “Estrogen Receptor nuclear function” (ERnf) pathway (i.e. KTM2C, ESR1, GATA3 and MYC) were more frequent in patients with recurrent vs. de novo disease (Odds Ratio [OR] =7.69, p=0.0499 for KTM2C; OR=2.70, p=0.0381 for ERnf). MYC copy number gain or alterations in the ERnf genes were more frequent in Estrogen Receptor positive (ER+)/Progesteron Receptor negative (PgR-) compared to ER+/PgR+ tumors (OR= 4.07, p=0.0361 for MYC; OR=3.36, p=0.0073 for ERnf). Copy number gain of FGFRs (FGFR1, 2 and 3) were more frequent in patients with visceral versus bone-only disease (OR=5.26, p=0.0144 for FGFRs) and in pts with more than three metastatic sites (OR=8.05, p=0.0052). At first imaging, the Clinical Benefit Rate (CBR, defined as CR + PR + SD) was 90%, with only 10% of pts showing early disease progression (PD). Thirty pts (11%) had at least one alteration in genes belonging to the CDK4/6 pathway (CCND1, RB1, CDK4 and 6, CDKN2A). These alterations were more frequent in the group of patients with early PD (OR=3.23, p=0.0272). MYC gain, TP53 mutations and alterations in the HER family genes (EGFR, ERBB2, 3 and 4) were also more frequent in pts with early PD compared with pts with CBR (OR 5.16, p=0.0280, OR 3.87, p=0.0058 and OR 4.40, p=0.0426, respectively). Conclusion: At the present analysis, alterations in KTM2C and ERnf seem to characterize recurrent vs de novo aBC while FGFRs are prevalent in patients with more aggressive disease. Interestingly, MYC gain, TP53 mutations, alterations in genes of the HER family and CDK4/6 pathway were more likely detected in patients with early PD suggesting these as potential markers of intrinsic resistance to first-line treatment with ribociclib and letrozole. Further analyses for biomarker dynamics and pharmacogenomics are ongoing. Citation Format: Michelino De Laurentiis, Luca Malorni, Giampaolo Bianchini, Roberta Caputo, Mario Giuliano, Alberto Zambelli, Fabio Puglisi, Lucia Del Mastro, Marco Colleoni, Filippo Montemurro, Giulia Bianchi, Ida Paris, Giacomo Allegrini, Laura Amaducci, Marina Elena Cazzaniga, Michele Orditura, Claudio Zamagni, Vincenzo Adamo, Valentina Guarnieri, Nicola Battelli, Maria Rosaria Valerio, Saverio Cinieri, Sara Bianchetti, Donatella Grasso, Daniela Castelletti, Diletta Valsecchi, Ettore Cotroneo, Grazia Arpino. Bioitalee - Biomarker analysis on liquid biopsies of patients treated with ribociclib and letrozole as first-line therapy for advanced breast cancer (aBC) (NCT03439046) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-07.

SUMMARY Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-Daspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220⁺ bone marrow cells, CD19⁻ thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis.

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220(+) bone marrow cells, CD19(-) thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis.