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Hereditary cancer syndromes account for nearly 10% of cancers even though they are often underdiagnosed. Finding a pathogenic gene variant could have dramatic implications in terms of pharmacologic treatments, tailored preventive programs, and familiar cascade testing. However, diagnosing a hereditary cancer syndrome could be challenging because of a lack of validated testing criteria or because of their suboptimal performance. In addition, many clinicians are not sufficiently well trained to identify and select patients that could benefit from a genetic test. Herein, we searched the available literature to comprehensively review and categorize hereditary cancer syndromes affecting adults with the aim of helping clinicians in their daily clinical practice through a visual tool.

Background: The combination of endocrine therapy with the cyclin-dependent kinases 4/6 inhibitor palbociclib was proven to be effective for the treatment of hormone receptor (HR)-positive/HER2-negative metastatic breast cancer (MBC). Although generally well tolerated, treatment delays and dose reductions are frequently observed with palbociclib, mainly due to severe neutropenia. Predictors of palbociclib-related toxicities are still unknown, as well as the clinical relevance of its relative dose intensity (RDI). Henceforth, this study aimed to identify baseline clinicopathological features associated with a RDI Methods: We reviewed data of 150 consecutive patients with HR-positive/HER2-negative MBC patients treated with palbociclib at two Italian cancer centers from 2017 to 2019. Eligible patients must have received at least 3 cycles of treatment. Those who experienced early suspension due to unacceptable toxicities were still eligible. RDI was calculated as the ratio of actual dose intensity (cumulative administered dose/treatment duration) and planned dose intensity (cumulative planned dose/planned treatment duration). The association of both RDI Results: Overall, 142 patients were deemed eligible. Of these patients, 98 (69.0%) were treated with palbociclib plus fulvestrant, 44 (31.0%) with palbociclib plus aromatase inhibitors, and 73 (51.4%) in the first-line setting. The median number of administered palbociclib cycles was 8 (range: 1-24) and 61 patients (43.0%) required at least a first-level dose reduction (29 within 3 months). Furthermore, the median time to first dose reduction was 3.22 months, with neutropenia being responsible for 85.24% of first-level dose reductions. In the whole cohort, median RDI was 90.5% (95.1% for patients without dose reduction and 80% for those who had received a dose reduction). Notably, 28 patients (19.7%) experienced a RDI Conclusions: In our analysis, baseline weight and WBC were statistically associated with a RDI Citation Format: Giacomo Pelizzari, Elisa Bertoli, Marco Giavarra, Carla Corvaja, Lorenzo Gerratana, Debora Basile, Michele Bartoletti, Camilla Lisanti, Lucia Bortot, Silvia Buriolla, Mattia Garutti, Debora Avoledo, Marta Bonotto, Lucia Da Ros, Silvia Bolzonello, Mauro Mansutti, Paola Di Nardo, Gianpiero Fasola, Simon Spazzapan, Alessandro M Minisini, Fabio Puglisi. Predictors of relative dose intensity and early dose reduction in patients with metastatic breast cancer treated with palbociclib and endocrine therapy [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 P5-14-08.

Background: Over the last years, the management of patients with node positive early breast cancer has gone through important innovations. On the medical side, new targeted therapies such as olaparib and abemaciclib have been introduced, with promising results on the invasive disease-free survival. Moreover, sparing axillary lymph node dissection has proven to be noninferior in terms of overall survival. However, no tools are currently available to predict lymph node involvement before definitive surgical evaluation. The aim of the study was to analyze clinical and pathological characteristics of patients with node positive early breast cancer to explore potential risk profiles associated with a ≥3 nodal involvement. Methods: The study retrospectively analyzed 335 node-positive breast cancer patients treated at the Breast Unit of the CRO Aviano National Cancer Institute, between 2017 and 2021. Data regarding primary tumor biological features, lymph node involvement and surgical approach were collected. Associations between clinico-pathological characteristics and ≥3 lymph node involvement were tested through stepwise logistic regression and the gradient boosting machine learning algorithm (GBM). Results: Among the 335 analyzed patients, 87.0% had a primary tumor < 5 cm, with a single positive lymph node in 73.3% of cases. Hormone receptors were mainly positive (respectively 93.5% and 83.4% for estrogen and progesterone receptors). Tumor grade was most frequently well differentiated (Grade 1 in 60.7%), with a Ki67 < 20% (59.5%). After multivariable logistic regression, a tumor size ≥ 3 cm (OR 3.24, CI95% 1.47-7.17, p = 0.004), the presence of massive lymphovascular stromal invasion (OR 2.50, CI95% 1.02-6.14, p = 0.045) and 2 or more positive sentinel lymph nodes at surgical evaluation (OR 6.08, CI95% 3.34-11.05, p < 0.001) were associated with a higher risk of identifying ≥ 3 positive lymph nodes after subsequent axillary dissection. Similar results were observed in the luminal-like cohort. A GBM machine learning model was then developed with a 0.77 Area Under the Curve. Features with the highest relative importance (RI) were single sentinel node involvement (RI 16.1873), followed by tumor size ≥ 3 cm (RI 10.2024), ≥2 positive sentinel lymph nodes (RI 8.5050) and lymphovascular stromal invasion (4.0217). Consistently, number of positive sentinel lymph nodes and tumor size were the predominant features in all top 20 GBM models. Conclusions: The present study explored the definition of risk profiles linked to 3 or more positive lymph nodes based on clinical and pathological features. It, moreover, tested the feasibility of developing machine learning classifiers to support future clinical decision-making. Due to the growing complexity of the adjuvant setting, finding a balance between minimally invasive surgical and staging approaches and risk definition for treatment personalization will become increasingly critical. Citation Format: Tania Pivetta, Brenno Pastò, Martina Urbani, Elisabetta Benozzi, Nicola De Pascalis, Tiziana Perin, Mario Mileto, Bruno Pasquotti, Erica Piccoli, Lorenzo Vinante, Chiara Bampo, Silvia Bolzonello, Mattia Garutti, Milena Nicoloso, Serena Corsetti, Simona Scalone, Lucia da Ros, Paola di Nardo, Camilla Lisanti, Simon Spazzapan, Barbara Belletti, Michele Bartoletti, Lorenzo Gerratana, Samuele Massarut, Fabio Puglisi. Clinical and biological predictors of lymph node involvement in patients with early breast cancer for adjuvant treatment personalization [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-06.

The multidisciplinary management represents a crucial part of the care for cancer patients, resulting in better clinical and process outcomes, with evidence of improved survival among different cancer primary sites, including breast. According with international recommendations established by the European Society of Breast Cancer Specialists (EUSOMA), all breast-cancer patients have to be evaluated by a multidisciplinary team including radiologist, pathologist, surgeon, medical oncologist and radiation oncologist. Thus, variations in clinical practice of each specialty should be discussed and shared with all team members to guarantee a fruitful cooperation among the involved specialists. During the last decades, radiation treatment was deeply changed by the evidence-based adoption of hypofractionated radiotherapy (HFRT) as standard of treatment in patients with early-stage breast cancer undergoing conservative surgery. Moreover, mature randomized data have showed that partial breast irradiation (PBI) is an effective and safe alternative to whole breast irradiation in selected patients with low-risk early-stage breast cancer. Based on this background, we reviewed indications and critical issues of HFRT and PBI analyzing impact of their adoption from a multidisciplinary perspective.

Background Endocrine therapy (ET) plus cyclin-dependent-kinases 4/6 inhibitors (CDK4/6i) represents the standard treatment for luminal-metastatic breast cancer (MBC). However, prospective head-to-head comparisons are still lacking for 1st line (L) options, and it is still crucial to define the best strategy between 1st and 2nd L. Materials and methods 717 consecutive luminal-MBC pts treated between 2008 and 2020 were analyzed at the Oncology Department of Aviano and Udine, Italy. Differences about survival outcomes (OS, PFS and PPS) were tested by log-rank test. The attrition rate (AR) between 1st and 2ndL was calculated. Results At 1stL, pts were treated with ET (49%), chemotherapy (CT) (31%) and ET-CDKi (20%) while, at 2ndL, 33% received ET, 33% CT and 8% ET-CDKi. Overall AR was 10%, 7% for CT, 8% for ET and 17% for ET-CDKi. By multivariate analysis, 1stL ET-CDK4/6i showed a better mPFS1 and OS. Moreover, 2ndL ET-CDK4/6i demonstrated better mPFS2 compared to ET and CT. Notably, 1stL ET-CDKi resulted in higher mPFS than 2ndL ET-CDKi. Intriguingly, 1stL ET-CDK4/6i was associated with worse mPPS compared to CT and ET. Secondarily, 1stL ET-CDK4/6i followed by CT had worse OS compared to 1stL ET-CDK4/6i followed by ET. Notably, none of baseline characteristics at 2ndL influenced 2ndL treatment choice (ET vs. CT) after ET-CDKi. Conclusion Our real-world data demonstrated that ET-CDKi represents the best option for 1stL luminal-MBC compared to ET and CT. Also, the present study pointed out that 2ndL ET, potentially combined with other molecules, could be a feasible option after CDK4/6i failure, postponing CT on later lines., Highlights • To define the best treatment strategy for 1st and 2nd L is crucial for luminal mBC. • 1st L CDK4/6i-based therapies represent the standard treatment in luminal mBC. • This study confirmed the key role of 1st L CDK4/6i on a real-world cohort. • This study suggested a preeminent role for ET after first-line CDK4/6i. • The 2nd line ET with other molecules could allow to further postpone CT.

5566 Background: As the prognosis of BRCA-related advanced ovarian cancer (AOC) continues to improve thanks to the introduction of targeted therapies like poly ADP-ribose polymerase inhibitors (PARPi) and bevacizumab, the occurrence of second primary tumors, in particular breast cancer (BC), is assumed to be more common. Despite this, data on the incidence of BC in this population are poor and guidelines for BC screening or risk reducing strategies are lacking. Methods: Published data from recent PARPi-based, phase III clinical trials involving germline BRCA mutated AOC were reviewed and analyzed. Only studies in which the incidence of at least one second primary solid tumor was reported were included. Cases of second primary BC cancers were pooled to calculate cumulative incidence according to the median follow-up period for each trial. 95% confidence interval (CI) was considered. Results: Four trials (SOLO1, PAOLA1, SOLO2 and SOLO3) involving 1186 germline BRCA 1-2 mutated AOC patients were included ( BRCA1, n = 816; BRCA2, n = 360; BRCA1 + BRCA2, n = 4; missing, n = 6). In all trials, patients were randomized to receive olaparib +/- bevacizumab (n = 788) versus placebo or standard therapies (n = 398). Median age at diagnosis of AOC was 56 years (range, 29-87). With a follow-up ranging from 3.9 months to 5 years, 16 new cases of BC were recorded, 10 in PARPi-based arms and 6 in control arms. Only two patients had a previous history of BC. The total cumulative incidence of BC was 1.37% (95% CI, 0.77-2.18), while cumulative incidences were 1.26% (95% CI, 0.61-2.31) and 1.51% (95% CI, 0.55-3.25) in PARPi-based arms and control arms, respectively. In a sensitivity analysis excluding the SOLO3 trial with the shortest follow-up, the total cumulative incidence was 1.63% (95% CI, 0.92-2.67). Other 20 cases of second primary solid tumors were registered, including 4 cases of non-small cell lung cancer. Conclusions: Patients with germline BRCA-related AOC receiving PARPi-based maintenance therapy can be reassured about the risk of second primary BC and intensive screening should be avoided at least in early treatment phase. More data with long term follow-up are needed.

Background Aging induces meaningful changes in the immune system and inflammation response with increase in monocyte-lymphocyte ratio (MLR) and serum lactate dehydrogenase (LDH) levels. Aim of this study was to explore the prognostic role of MLR and LDH levels in older patients (pts) with metastatic colorectal cancer (mCRC). Methods We conducted a retrospective analysis of a consecutive cohort of 168 older (>70 years) patients with mCRC. The prognostic impact of MLR and LDH levels on overall survival (OS) was investigated through uni-and multivariate Cox regression analyses. Moreover, we categorized patients into three groups according to MLR and LDH levels (group 1: MLR-low and LDH-low; group 2: MLR-high or LDH-high; group 3: MLR-high and LDH-high). Results By univariate analysis, high LDH level (HR 1.74, 95% CI 1.05–2.90) and high MLR level (HR 2.19, 95% CI 1.48–3.44) were significantly associated with a worse OS. Conversely, primary tumor resection and left-sidedness were significantly associated with a longer OS. By multivariate analysis, high LDH level (HR 2.00, 95% CI 1.13–3.55) and high MLR level (HR 2.99, 95% CI 1.68–5.33) were independent prognostic factors of worse prognosis. Compared to group 1, a shorter survival was reported for patients included in group 2 (HR 1.97, 95% CI 1.21–3.23 in univariate; HR 2.54, 95% CI 1.43–4.51 in multivariate) or in group 3 (HR 2.42, 95% CI 24–4.74, p = .010 in univariate; HR 5.59, 95% CI 2.15-14.54 in multivariate) Conclusions High baseline levels of LDH, MLR or both are independent unfavorable prognostic factors in older patients treated with first-line chemotherapy for mCRC.

Mucositis is one of the most common debilitating side effects related to chemotherapy (CT), radiation therapy (RT), targeted agents and immunotherapy. It is a complex process potentially involving any portion of the gastrointestinal tract and injuring the mucosa, leading to inflammatory or ulcerative lesions. Mechanisms and clinical presentation can differ according both to the anatomic site involved (oral or gastrointestinal) and the treatment received. Understanding the pathophysiology and management of mucosal injury as a secondary effect of anti-cancer treatment is an important area of clinical research. Prophylaxis, early diagnosis, and adequate management of complications are essential to increase therapeutic success and, thus, improve the survival outcomes of cancer patients. This review focuses on the pathobiology and management guidelines for mucositis, a secondary effect of old and new anti-cancer treatments, highlighting recent advances in prevention and discussing future research options.

e13008 Background: Liquid biopsy-based biomarkers, including circulating tumor DNA (ctDNA), are increasingly important for the characterization of metastatic breast cancer (MBC). Currently, CA15.3 is the most commonly used serum marker for monitoring disease burden. To date, no liquid biopsy-based biomarkers have been proposed for this scope in clinical practice. Methods: The CRO-2018-56 multicenter study prospectively enrolled 83 patients (pts) with luminal-like MBC treated with first line endocrine therapy (ET) and CDK4/6 inhibitors. All pts were characterized for ctDNA through droplet digital PCR (ddPCR) in from 2018 to 2021. Clinicopathological and laboratory characteristics at baseline were tested for associations with tumor markers, ACTB fragments lengths, methylation status of ESR1 main promoters (expressed as promA and promB ratio, i.e., met_ratio) and ESR1/PIK3CA mutational status through Kruskal-Wallis test and Chi-square test. Prognosis was tested in terms of Progression Free Survival (PFS) and Overall Survival (OS) through log-rank test. Results: At baseline, in 26 (31%) pts disease was diagnosed as de novo metastatic, 66 (80%) pts had < 3 of metastatic sites, and 41 (49%) pts had < 5 of metastatic lesions. Bone metastases were detected in 53 (64%) pts, liver metastases in 21 (25%) pts, and lung lesions in 30 (36%) pts. A ctDNA-detected ESR1 mutation and a PIK3CA mutation were found in 15% of pts and in 34% pts, respectively. Met_ratio was > 1.5 in 35 (42%) of pts. Median CA15.3 was 48.2 U/mL and median CEA was 3.8 U/mL. Number of liver metastases and number of metastatic sites were significantly higher in pts with ESR1 mutation (respectively, P = 0.0055 and P = 0.0208). CA15.3 and ctDNA yield were significantly higher in pts with number of metastatic sites ≥ 3, (respectively, P = 0.0164, and P = 0.0239), while number of metastatic sites ≥ 3 and number of metastatic lesions ≥ 5 were significantly associated with CEA > 3.8 U/mL (respectively, P = 0.039, and P = 0.029). Presence of bone metastases was significantly associated with PIK3CA mutation (P = 0.040), while number of metastatic sites ≥ 3 was significantly associated with ESR1 mutation (P = 0.022). No association with tumor burden was observed for different ACTB DNA fragments lengths. Met_ratio > 1.5 was significantly associated with lower number of metastatic lesions (P = 0.031). Number of metastatic sites ≥ 3, high ctDNA yield and CEA were associated with worse OS (respectively P = 0.0465, P = 0.0250 and P = 0.0474), while only CEA impacted on PFS (P = 0.0097). Conclusions: In pts with luminal-like MBC, some liquid biopsy-based biomarkers (i.e., ctDNA-detected ESR1 and PIK3CA mutations, ctDNA yield) were significantly associated with the burden of disease. The potential clinical validity and utility of these results deserve to be tested in an expansion cohort.

Infiltrating lobular carcinoma (ILC) represents about 10% of breast cancer and rarely shows overexpression of human epidermal growth factor receptor 2 (HER2). We compared biological and clinical characteristics of HER2-positive ILC versus HER2-positive infiltrating ductal carcinoma (IDC).We retrospectively analyzed the data of 328 patients with HER2-positive pure ductal or lobular breast carcinoma, comparing clinical and biological data at diagnosis as well as outcome between the 2 histologies. A gene-mutation analysis was performed in a subset of patients.Two hundred ninety-one patients (88.7%) had IDC and 37 patients (11.3%) ILC. ILC resulted more frequently in multicenter (24.3% vs. 6.5%, P .0001) and node-positive (54.1% vs. 45%, P = .013) disease of lower proliferative activity (Mib1 20%: 51.4% vs. 22.3%, P .0001) and lower histologic grade (grade 3: 32.4% vs. 57.4%, P = .038). Disease recurred in 57 patients (17.4%) and involved the bone in 40% of ILC patients (vs. 17% of IDC patients) and the viscera in 30% of ILC patients (vs. 59.6% of IDC patients). No difference in the recurrence rate between the 2 histologies was observed in patients treated with adjuvant trastuzumab (12.5% of ILC patients and 8.3% of IDC patients). Exploratory molecular analysis revealed a higher frequency of mutations in ILC, with more cases of multiple mutations.HER2-positive ILC shows different biological behavior than IDC, with a possible higher mutation load. Despite lower proliferation activity and estrogen receptor expression in ILC breast cancer, trastuzumab is clearly an effective therapy for this histologic subtype.

577 Background: It is matter of current debate which would be the best chemotherapy backbone of neoadjuvant HER2-targeted therapy for HER2+ BC. The TRAIN 2 trial showed no significant difference in terms of pathological complete response (pCR) when anthracyclines–based (CTA) or anthracyclines–free regimens (CT) were combined with dual HER2 blockade. However, it remains unclear how anthracyclines may influence the relative benefit across different anti-HER2 treatments. Methods: A systematic review was conducted which included all phase II/III randomized clinical trials (RCTs) comparing different neoadjuvant regimens for HER2+ BC. pCR (yT0/isN0) was the outcome of interest. Indirect comparisons of all combination of anti-HER2 agents with CTA or CT were estimated with a random-effects frequentist NMA. Estimated pCR rates were inferred adopting a Bayesian NMA. Results: 17 RCTs (3933 patients) were included. Overall, 8 arms were identified, comprising all possible combinations of CTA and CT with trastuzumab (H), lapatinib (L) and dual HER2 blockade (D) but also CTA and D only. Odds ratios (OR) for pCR and 95% confidence interval (CI) of selected NMA comparisons are shown in the table. Estimated rates of pCR for each treatment and 95% credible interval (CrI) are reported in the table. Conclusions: Through indirect comparisons, no significant pCR gain was found for CTA vs CT when combined to D, H and L. In particular, considering double vs single-agent anti-HER2 regimens, D-CT remains superior to H-CTA, supporting a possible omission of anthracyclines when dual anti-HER2 block is used. On the contrary, our pooled estimate suggests a more relevant role for anthracyclines when comparing H-CT/A vs CTA. Moreover, we estimated a 4% pCR gain for D-CTA vs D-CT, and an 8% higher pCR rate for H-CTA vs H-CT. [Table: see text]

5564 Background: Patients (pts) experiencing a PS rOC are generally re-exposed to a platinum based-chemotherapy (CT). In this setting, the addition of a targeted agent like bevacizumab (BEV) or PARP inhibitors (PARPi) as concomitant and/or maintenance therapy has shown to improve progression free survival (PFS). In the absence of direct comparison in randomized trials (RCTs), we have performed a NMA to evaluate differences in terms of efficacy between BEV and PARPi in pts with PS rOC, according to BRCA status. Methods: We searched PubMed, Embase and Medline for RCTs involving pts with PS rOC treated with BEV (n = 3, 1563 pts) or PARPi (n = 5, 1839 pts). Only trials with PFS as primary endpoint were included. Trials in first line setting were excluded. Analyses have been done pooling pts who had received PARPi in three groups, according to the available data on BRCA genes status: all comers (AC), BRCA mutated pts (BRCAm) and BRCA wild-type pts (BRCAwt). A frequentist approach has been used with R statistical software. To rank the effect size of treatments, surface under the cumulative ranking value (SUCRA) has been applied. Results: In AC pts, PARPi improved PFS compared to BEV (hazard ratio [HR] = 0.70, 95% CI 0.54-0.91, test of heterogeneity [I2] = 40.5%). In BRCAm pts the gain in PFS for PARPi was even higher compared to BEV (HR = 0.46, 95% CI 0.36-0.59, I2= 17.2%). In BRCAwt pts the benefit of PARPi over BEV was not statistically significant (HR = 0.87, 95% CI 0.63-1.20, I2= 35.7%) but PARPi had the highest likelihood of being ranked as the best treatment in terms of efficacy according to SUCRA (90% and 60%, respectively for PARPi and BEV). Hazard ratio for PFS between PARPi, BEV and CT in the three cohorts are reported in the table. Conclusions: According to indirect comparisons, PARPi performed the best for the treatment of PS rOC, especially in BRCAm pts who had not previously received PARPi. BEV could be still an option in BRCAwt pts. [Table: see text]

Left ventricular ejection fraction (LV-EF), despite its high feasibility, is not sensitive enough to detect early and subtle LV systolic dysfunction during oncologic treatments. Therefore, we used systolic global longitudinal strain (GLS) by speckle tracking echocardiography to verify whether early LV systolic dysfunction induced by adjuvant therapy in early breast cancer patients at low risk for cardiotoxicity can be reversed. Thirty patients (aged 53 ± 11 y) with no previous cardiac and oncologic disease who were receiving adjuvant trastuzumab and taxane (group HER2+, n = 15) or taxane only (group HER2-, n = 15), after treatment with anthracyclines, were studied. LV-EF and GLS were measured at baseline, after anthracyclines (end of week 7 or 8), short term after trastuzumab and/or taxane (end of week 18) and after completion of therapy. Significant LV systolic dysfunction was defined as a relative reduction in GLS of >10% with respect to baseline values. Mean and individual LV-EFs did not change significantly during the oncologic treatment and after completion of therapy, although GLS varied significantly. In particular, during the course of therapy, four patients in the trastuzumab-docetaxel HER2+ subgroup and two patients in the taxane HER2- subgroup had a relative decrease (>10%) in GLS. However, after the end of adjuvant treatment, strain modification was fully or partially reversible. Speckle tracking echocardiography is more sensitive than LV-EF in recognizing subtle myocardial impairment during adjuvant chemotherapy. However, in patients at low risk for cardiotoxicity, these alterations may be reversible and not associated with clinically significant cardiotoxicity or late development of decreased LV-EF.

The hypothesis to use microRNAs (miRNAs) circulating in the blood as cancer biomarkers was formulated some years ago based on promising initial results. After some exciting discoveries, however, it became evident that the accurate quantification of cell-free miRNAs was more challenging than expected. Difficulties were linked to the strong impact that many, if not all, pre- and post- analytical variables have on the final results. In this study, we used currently available high-throughput technologies to identify miRNAs present in plasma and serum of patients with breast, colorectal, lung, thyroid and melanoma tumors, and healthy controls. Then, we assessed the absolute level of nine different miRNAs (miR-320a, miR-21-5p, miR-378a-3p, miR-181a-5p, miR-3156-5p, miR-2110, miR-125a-5p, miR-425-5p, miR-766-3p) in 207 samples from healthy controls and cancer patients using droplet digital PCR (ddPCR) technology. We identified miRNAs specifically modulated in one or more cancer types, according to tissue source. The significant reduction of miR-181a-5p levels in breast cancer patients serum was further validated using two independent cohorts, one from Italy (n = 70) and one from US (n = 90), with AUC 0.66 and 0.73 respectively. This study finally powers the use of cell-free miRNAs as cancer biomarkers and propose miR-181a-5p as a diagnostic breast cancer biomarker. The hypothesis to use microRNAs (miRNAs) circulating in the blood as cancer biomarkers was formulated some years ago based on promising initial results. After some exciting discoveries, however, it became evident that the accurate quantification of cell-free miRNAs was more challenging than expected. Difficulties were linked to the strong impact that many, if not all, pre- and post-analytical variables have on the final results. In this study, we used currently available high-throughput technologies to identify miRNAs present in plasma and serum of patients with breast, colorectal, lung, thyroid and melanoma tumors, and healthy controls. Then, we assessed the absolute level of nine different miRNAs (miR-320a, miR-21-5p, miR-378a-3p, miR-181a-5p, miR-3156-5p, miR-2110, miR-125a-5p, miR-425-5p, miR-766-3p) in 207 samples from healthy controls and cancer patients using droplet digital PCR (ddPCR) technology. We identified miRNAs specifically modulated in one or more cancer types, according to tissue source. The significant reduction of miR-181a-5p levels in breast cancer patients serum was further validated using two independent cohorts, one from Italy (n = 70) and one from US (n = 90), with AUC 0.66 and 0.73 respectively. This study finally powers the use of cell-free miRNAs as cancer biomarkers and propose miR-181a-5p as a diagnostic breast cancer biomarker.