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Fundació Carreras This work was supported by Italian Ministry of Health (MoH) TRANSCAN Joint Transnational Call (JTC) 2013 co-funded by the European Regional Development Fund, "A way of making Europe" RRC-2014-2354565, Italian Ministry of Health (MoH) "Ricerca Corrente 2019" and "5×1000″ voluntary contributions" and partially supported by Associazione Italiana Ricerca sul Cancro (AIRC) Project Code: 16747. While the molecular functions of miR-200 family have been deeply investigated, a role for these miRNAs as breast cancer biomarkers remains largely unexplored. In the attempt to clarify this, we profiled the miR-200 family members expression in a large cohort of breast cancer cases with a long follow-up (H-CSS cohort) and in TCGA-BRCA cohort. Overall, miR-200 family was found upregulated in breast tumors with respect to normal breast tissues while downregulated in more aggressive breast cancer molecular subtypes (i.e. Luminal B, HER2 and triple negative), consistently with their function as repressors of the epithelial-to-mesenchymal transition (EMT). In particular miR-141-3p was found differentially expressed in breast cancer molecular subtypes in both H-CSS and TCGA-BRCA cohorts, and the combined analysis of all miR-200 family members demonstrated a slight predictive accuracy on H-CSS cancer specific survival at 12 years (survival c-statistic: 0.646; 95%CI 0.538-0.754).

During the last decades, much effort has been made to develop and test treatments for advanced/metastatic breast cancer (MBC) able to prolong survival and improve patients’ quality of life. In this regard, eribulin represents one of the most recent and interesting discoveries. This tubulin-targeting chemotherapy demonstrated a survival benefit in MBC women who progressed after at least two prior lines of chemotherapy for the treatment of metastatic disease (prior therapies should have included an anthracycline and a taxane, in either adjuvant or metastatic setting). Here, we described five cases of heavily pretreated MBC patients who experienced long-lasting control of disease with eribulin.

BACKGROUND: Everolimus, an mTOR inhibitor, in combination with exemestane is approved for hormone receptor (HR) positive advanced breast cancer (ABC), after failure of treatment with non-steroidal aromatase inhibitor (NSAI). We assessed the toxicity of the combination and the correlation between dose intensity and response to therapy, in a real world population of ABC from 11 Italian centers. Moreover, we evaluated OS of the whole population, RR and PFS according to line of treatment (from 1rd to 3th and from 4th on). METHODS: 154 pts were treated with combination of everolimus 10 mg and exemestane 25 mg daily from 05/2011 today. Median age was 62 (47-82). Median time to metastatic disease was 49 months (0-269). Median number of metastatic sites was 2 (55.2% of pts visceral versus 44.8% non visceral disease). N=117 (75.9%) pretreated with HT as adjuvant; N=126 pts (81.8%) treated with HT for advanced disease prior to EVE/EXE, with a median of one line (0-5). N=102 pts (66.2%) treated with chemotherapy for metastatic disease, with a median of one line (0-6) before everolimus treatment. RESULTS: Sixteen pts received EVE/EXE as 1st line (10.4%), 39 as 2nd (25.3%), 37 as 3rd (24%), 62 as 4th or more (40,3%). Response was evaluable in 127 out of 154 pts; CR/PR/SD respectively 5/27/56 pts. RR according to line (from 1st to 3rd vs ≥ 4th) was respectively 22.8% vs 26.4% (p=0,864). The median PFS for all population (150 pts) was 38 weeks (95% CI: 33-42). The PFS according to line (1st- 3rd vs ≥ 4th) was 38 wks in both subgroups, p=0.73. OS (126/154 pts) was 28 mths (95% CI: 31-38). The most frequent adverse events were collected in the table. Adverse eventsOverall %Grade 3-4 %Stomatitis55.810.4Hypercholesterolemia47.40.0Asthenia42.95.2Hyperglycemia36.45.8Hypertriglyceridemia29.20.6Anemia28.63.9Peripheral edema24.71.3Rash23.40.6Increased ALT/AST/GGT21.46.5Thrombocytopenia19.53.9Diarrhea18.81.9Weight loss18.21.3Dysgeusia17.50.6Pneumonitis15.61.9Cutaneous toxicity14.90.6Infection14.33.2Neutropenia11.71.9Nausea11.70.0Anorexia (without stomatitis)10.41.3Electrolyte alterations9.71.3Urea/creatinine increase6.51.3Vomiting6.50.0Uric acid increase4.50.0 Median duration of treatment with everolimus 10 mg and 5 mg was respectively 180 (9-854) and 129 days (3-738). Fifty-eight pts (37,6%) never stopped treatment with everolimus 10 mg; 16 pts (10,4%) definitively stopped everolimus for toxicity; 80 pts (52,0%) temporarily interrupted the treatment, resuming at dose level 10 mg (31 pts) or reducing at 5 mg (49 pts). Main reason for discontinuation/interruption was stomatitis G2-G3. RR and PFS evaluated according to dose intensity, 10 mg vs 5 mg, were respectively 25.9% vs 30% p=0.779, 38 wks (27-44) vs 40 wks (31-48) P=0.614 CONCLUSIONS: efficacy in terms of RR and PFS of the combination EVE/EXE is not related to dose intensity (10 mg vs 5 mg), the discontinuation of the treatment is high with the starting dose of 10 mg, the toxicity is consistent with previous phase II-III studies although we collected some different toxicities. Citation Format: Forcignanò R, Petrucelli L, Cazzaniga ME, Lupo LI, Chiuri VE, Cairo G, De Matteis E, Febbraro A, Giordano G, Campidoglio S, Fabi A, Giampaglia M, Bilancia D, La Verde N, Maiello E, Morritti M, Giotta F, Lorusso V, Scavelli C, Romito S, Cusmai A, Palmiotti G, Tornesello A, Ciccarese M. Dose intensity and efficacy of the combination of everolimus and exemestane (EVE/EXE) in a real world population of hormone receptor positive advanced breast cancer: A multicenter Italian experience. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-13-15.

INTRODUCTION. miR-155-5p is a well-known oncogenic microRNA frequently overexpressed in human cancers, including breast cancer (BC), but its putative role as biomarker remains controversial. Thus, we aimed at evaluating miR-155-5p levels in a large BC cohort, and its association with clinical parameters. Then, since miR-155-5p was found to be epigenetically controlled by BRCA1, and to increase the tumor mutation load, we analysed miR-155-5p correlation with defects in Homologous Recombination (HR) genes (i.e. HRD), and its capability to alter the response to the first-in-class PARP (Poly-[ADP-ribose]-polymerase-1 (PARP-1)) inhibitor Olaparib (AZD2281). METHODS. We measured miR-155-5p expression in a retrospective cohort of breast cancer patients (CSS cohort, n=283; median follow-up=81 months) by RT-qPCR. Then, we used the TCGA Breast Cancer (TCGA-BC) dataset (n=1095) to validate our results, and evaluate the association between miR-155-5p and mutations in HR genes. Last, we ectopically modulated miR-155-5p in three wtBRCA1/2 cell lines (i.e. MDA-MB-231, MDA-MB-468, MDA-MB-453) and one mutBRCA1 cell line (MDA-MB-436) by using mirVana miR-155-5p mimic or inhibitor, and we measured cell viability before and after treatment with Olaparib or vehicle by PrestoBlue viability reagent. RESULTS. High miR-155-5p levels were associated with unfavourable prognostic indicators including high tumor grade (overall P=0.0007; G2 vs G3 P=0.0053), reduced expression of ER (r= -0.243; P=0.0002) and PgR (r= -0.240; P CONCLUSIONS. Overall, our data point to a role of miR-155-5p in Homologous Recombination Deficiency (HRD), and suggest a putative novel mechanism of synthetic lethality mediated by PARP inhibition and miR-155-5p to promote cancer cell death. Citation Format: Barbara Pasculli, Raffaela Barbano, Andrea Fontana, Tommaso Biagini, Maria Pia Di Viesti, Michelina Rendina, Vanna Maria Valori, Maria Morritti, Sara Bravaccini, Sara Ravaioli, Evaristo Maiello, Paolo Graziano, Roberto Murgo, Massimiliano Copetti, Tommaso Mazza, Vito Michele Fazio, Manel Esteller, Paola Parrella. Clinical association of miR-155-5p with breast cancer and its relevance for treatment with PARP inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1422.

Aim: We performed a multicenter retrospective cohort study of eribulin mesylate (EM) use in Italy, to describe the current practice for metastatic breast cancer patients (ESEMPiO) in the real-world. Patients & methods: Baseline characteristics, treatment administration and safety were summarized using descriptive statistics. Results: No safety concerns were raised in the population enrolled in the ESEMPiO database and treated in a real-life practice. Median progression-free survival and overall survival were 3.2 and 10.1 months, respectively. EM activity was similar between breast cancer subtypes. Conclusion: In metastatic breast cancer patients treated with EM in ‘real-world’ setting, the clinician-registered outcomes were comparable to those reported in pivotal trials. Furthermore, EM maintained clinical activity and a tolerable safety profile.

Aim: This retrospective analysis focused on the effect of treatment with EVE/EXE in a real-world population outside of clinical trials. We examined the efficacy of this combination in terms of PFS and RR related to dose intensity (5 mg daily versus 10 mg daily) and tolerability. Methods: 163 HER2-negative ER+/PgR+ ABC patients, treated with EVE/EXE from May 2011 to March 2016, were included in the analysis. The primary endpoints were the correlation between the daily dose and RR and PFS, as well as an evaluation of the tolerability of the combination. Secondary endpoints were RR, PFS, and OS according to the line of treatment. Patients were classified into three different groups, each with a different dose intensity of everolimus (A, B, C). Results: RR was 29.8% (A), 27.8% (B) (p = 0.953), and not evaluable (C). PFS was 9 months (95% CI 7–11) (A), 10 months (95% CI 9–11) (B), and 5 months (95% CI 2–8) (C), p = 0.956. OS was 38 months (95% CI 24–38) (A), median not reached (B), and 13 months (95% CI 10–25) (C), p = 0.002. Adverse events were stomatitis 57.7% (11.0% grade 3–4), asthenia 46.0% (6.1% grade 3–4), hypercholesterolemia 46.0% (0.6% grade 3–4), and hyperglycemia 35.6% (5.5% grade 3–4). The main reason for discontinuation/interruption was grade 2–3 stomatitis. Conclusions: No correlation was found between dose intensity (5 vs. 10 mg labeled dose) and efficacy in terms of RR and PFS. The tolerability of the higher dose was poor in our experience, although this had no impact on efficacy.

miR-9 was initially identified as an epigenetically regulated miRNA in tumours, but inconsistent findings have been reported so far. We analysed the expression of miR-9-5p, miR-9-3p, pri-miRs and MIR9 promoters methylation status in 131 breast cancer cases and 12 normal breast tissues (NBTs). The expression of both mature miRs was increased in tumours as compared to NBTs (P P = 0.005 and P = 0.003, for miR-9-3p and miR-9-5p respectively). In addition, miR-9-5p showed a significant negative correlation with PgR (P = 0.002). Consistently, miR-9-5p and miR-9 3p were differentially expressed in the breast cancer subgroups identified by ER and PgR expression and HER2 amplification. No significant correlation between promoter methylation and pri-miRNAs expressions was found either in tumours or in NBTs. In the Luminal breast cancer subtype the expression of miR-9-5p was associated with a worse prognosis in both univariable and multivariable analyses. Ingenuity Pathway Analysis exploring the putative interactions among miR-9-5p/miR-9-3p, ER and PgR upstream and downstream regulators suggested a regulatory loop by which miR-9-5p but not miR-9-3p is induced by steroid hormone receptor and acts within hormone-receptor regulated pathways.

MiR-210-3p is the most hypoxia-sensitive miRNA showing overexpression in different human cancers, but its role as biomarker in breast cancer (BC) is still controversial. In our study, we performed the expression analysis of miR-210-3p in a retrospective cohort of breast cancer patients with a median follow-up of 91 months enrolled according to the REMARK guidelines (n=284). As controls, 13 normal breast tissues (NBTs) from reductive mammoplasty were also profiled. By this analysis, we found that miR-210-3p levels were significantly increased in tumours as compared with NBTs (P=0.021). According to the surrogate molecular classification, the Triple Negative subgroup showed the highest levels of miR-210, followed by Luminal B tumours, whereas Luminal A, and HER2-amplified tumours showed the lowest expression levels (P=0.0137). The evaluation of the association of miR-210-3p with time-to-event outcomes in patients without synchronous metastases (n=268) demonstrated an association between risk of disease progression (HR 2.13, 95%CI 0.33-3.39, P=0.002) and higher levels of miR-210-3p in the subgroup treated with docetaxel in sequential therapy with anthracyclines (ECD). Moreover, a cut-off value of 20.966 as established by ROC curve analysis allowed to discriminate patients who developed distant metastases with an accuracy of 85% at 3-years (AUC 0.87, CI 0.69-1) and 83% at 5-years follow up (AUC 0.832, CI 0.656-1). Instead, the accuracy in discriminating patients who died for the disease was of 79.6% at both 5- (AUC 0.804, CI 0.517-1) and 10-years (AUC 0.804 CI 0.517-1) follow up. Overall, our data support the putative involvement of miR-210-3p in the response to treatments including docetaxel and suggest its eligibility to become a biomarker of early metastases development in ECD-treated breast cancer patients. Citation Format: BARBARA PASCULLI, RAFFAELA BARBANO, MICHELINA RENDINA, ANDREA FONTANA, MASSIMILIANO COPETTI, VANNA MARIA VALORI, MARIA MORRITTI, EVARISTO MAIELLO, VITO MICHELE FAZIO, MANEL ESTELLER, PAOLA PARRELLA. High levels of microRNA-210-3p are associated with increased risk of disease progression in breast cancer patients treated with docetaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4904.

The mortality of breast cancer (BC) is primarily caused by metastatic spread to distant organs, rendering the ability to predict, detect and eliminate metastases one of the most important challenges in patients' management. This study is aimed to evaluate the potential role of several candidate miRNAs as predictive markers of the metastatic processes. The TRANSCAN ERA-NET BREMIR project has been designed to identify miRNA biomarkers able to predict distant metastases development. The correlation of miRNAs expression with distant metastasis free survival is evaluated in a large retrospective cohort (n=302). The best candidate markers will be validated in the independent prospective cohorts which currently include 505 patients. Additionally, miRNA potentialities as circulating biomarkers for treatment monitoring and early metastases detection will be evaluated in plasma samples. The analysis of the retrospective cohort has identified miR-10b-5p, miR-10a-5p, miR-210-3p, miR-30a-5p, miR200a-3p,miR200b-3p,miR200c-3p, miR141-3p, miR-429, miR-155-5p, miR-9-3p and miR-9-5p as crucially involved in BC progression. For miR-9-5p and miR-9-3p we also found a strong inverse correlation with ER and PgR expression, suggesting that they may be directly involved in hormone regulated pathways. To identify novel miRNAs associated with tumour metastases potentials, we also executed a global miRNA profiling on a selected number of breast cancer cases by using the Affymetrix GeneChip® miRNA 4.0. In particular, we analysed 34 BC cases characterized by different outcomes: 17 non metastatic after 10-years follow-up and 17 who developed metastatic disease within 5-years follow-up. As controls, 3 Normal Breast Tissues from reductive mammoplasty, 3 in situ carcinomas and 3 breast cancer cases with synchronous metastases were also evaluated. This analysis identified 8 miRNAs differentially expressed between metastatic and non metastatic cases. The association with study endpoints is currently under evaluation on the entire retrospective cohort. Our hope is that this study will lead to the identification of novel biomarkers allowing a better management of breast cancer patients. Citation Format: Paola Parrella, Raffaela Barbano, Ella Evron, Barbara Pasculli, Orazio Palumbo, Michelina Rendina, Raffaella Stallone, Michela Coco, Andrea Fontana, Tommaso Mazza, Massimiliano Copetti, Vanna Maria Valori, Maria Morritti, Paolo Graziano, Roberto Murgo, Evaristo Maiello, Massimo Carella, Vito Michele Fazio, Manel Esteller. Initial results from TRANSCAN ERA-NET BREMIR project: MicroRNAs expression profiling for identification of breast cancer patients at high risk to develop distant metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5394.

miR-9 (hsa-miR-9-5p, miR-9-5p) is a highly conserved microRNA (miRNA) primarily expressed in the central nervous system. The three promoters of miR-9 (miR-9-1 at 1q22, miR-9-2 at 5q14.3 and miR-9-3 at 15q26.1) are embedded within CpG islands, and hypermethylation in at least one of these regions has been reported in several tumor types, thus miR-9-5p has been designated as an epigenetically regulated miRNA. However, only a partial or none correlation between methylation status of miR-9 loci and miR-9-5p expression has been demonstrated so far. Furthermore, the expression levels of miR-9-5p resulted highly variable across different carcinomas and conflicting evidences also exist about its functional role within the tumor context. In light of this, we took the effort of clarifying the role of miR-9-5p in primary breast cancer tissues and of evaluating its potential as clinical relevant prognostic biomarker. We analysed miR-9-5p expression and miR-9 promoters methylation status in 129 breast cancer cases and 12 normal breast tissues (NBTs), by qRT-PCR and MSP respectively. Overall, miR-9-5p was increased in tumors compared to NBTs (P Citation Format: Raffaela Barbano, Barbara Pasculli, Michelina Rendina, Andrea Fontana, Caterina Fusilli, Massimiliano Copetti, Stefamo Castellana, Vanna Maria Valori, Maria Morritti, Paolo Graziano, Luigi Ciuffreda, Michelina Coco, Francesco Picardo, Tommaso Mazza, Ella Evron, Roberto Murgo, Evaristo Maiello, Manel Esteller, Vito Michele Fazio, Paola Parrella. miR-9-5p expression in breast cancer correlates with hormone receptor status and affects patients survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4734. doi:10.1158/1538-7445.AM2017-4734

We have recently demonstrated that the evaluation of miR10b expression relative to its normal counterparts (Relative Expression Ratio, RER) is able to predict patients risk to develop distant metastases. To confirm these results and evaluate whether other microRNAs could be predictive of outcome in breast cancer we evaluated the expression of miR10b and miR9 in paired tumors and apparently normal breast tissues 2 cm distant from tumour (NBT) on an extended cohort (n = 127) with a longer follow up (median 42 months). As control normal breast tissues from 10 healthy subjects (HBT) treated by reductive mammoplasty were analyzed. An increased expression was seen for the miR9 and miR10b in NBT samples as compared with HBT (P Citation Format: Raffaela Barbano, Barbara Pasculli, Massimiliano Copetti, Andrea Fontana, Michelina Coco, Lorenzo Vitulano, Maria Luana Poeta, Francesca Picardo, Gianfranco Marangi, Roberto Murgo, Luigi Ciuffreda, Vanna Maria Valori, Vanna Maria Valori, Maria Morritti, Evaristo Maiello, Vito Michele Fazio, Paola Parrella. Evaluation of miR10b and miR9 expression in breast cancer and correlations with distant metastases development. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3977. doi:10.1158/1538-7445.AM2015-3977

e11539 Background: Eribulin Mesylate (EM) has been available in Italy since January 4th, 2012 for the treatment of patients (pts) with metastatic breast cancer (MBC) after the failure of at least 2...

Background MicroRNA-10b (miR-10b) has a prominent role in regulating tumor invasion and metastasis by targeting the HOXD10 transcriptional repressor and has been found up-regulated in several tumor types. Methods We evaluated the expression of miR-10b in paired tumor and normal specimens obtained from a prospective cohort of breast cancer patients with at least 36 months follow-up enrolled according to the REMARK guidelines (n = 150). RNA quality was measured and only samples with RNA Integrity Number (RIN) ≥7.0 were analyzed. Results The relative expression of miR-10b in tumor as compared to its normal counterpart (RER) was determined by RT-qPCR. miR-10b RERs were higher in the subgroup of patients with synchronous metastases (n = 11, Median 0.25; IQR 0.11-1.02) as compared with patients without metastases (n = 90, Median 0.09; IQR 0.04-0.29) (p = 0.028). In the subgroup of patients without synchronous metastases (n = 90), higher miR-10b RERs were associated with increased risk of disease progression and death in both univariable (HR 1.16, p = 0.021 and HR 1.20, p = 0.015 respectively for 0.10 unitary increase of miR-10b RERs levels) and multivariable (HR1.30, p

677 Background: The frequency of small (pT1a,b,c) node negative invasive breast cancers (BC) is increasing because of wider implementation of BC screening. When cancer is detected early, patients (pts) show an overall better prognosis than pts with more advanced disease, however, even in this group a subset of pts will experience local or distant recurrences. The aim of our study was to evaluate in this subgroup of BC cases the association between disease-free survival (DFS), overall survival (OS) and pts clinicopathological characteristics. Methods: Six hundred twenty eight pts with early BC (pT1a,b,cN0M0) were observed between March 1996 and December 2007 (median follow-up 80 months - IQR 45-118) and were retrospectively investigated in our Institution. Tumours were classified histologically according to the WHO histological classification on BC. Tumour grading was assessed according to Elston and Ellis. Estrogen receptor (ER) and progesteron peceptor (PgR) status and HER2neu overexpression were evaluat...