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Background: Data published in 2015 showed that patients with early breast cancer (EBC) and a low-risk (LR) Recurrence Score® (RS) result by the 21-gene Oncotype DX® assay ("the test") did not derive benefit from adding chemotherapy (CT) to endocrine therapy (HT), while those with a high-risk (HR) RS result did. However, the role of CT remained uncertain in patients with intermediate-risk (IR) cancers. We designed a study to assess the test's ability to categorize patients with EBC with uncertain biological behavior into the groups (LR and HR) for which the value of additional chemotherapy was defined., Methods: The POST trial was a multicenter, prospective cohort study conducted in 14 Breast Centers of the Tuscany region of Italy. Consecutive patients with pT1-2 pN0-N1mi hormone receptor-positive/HER2-negative EBC and uncertain biological behavior based on standard parameters were enrolled. Patients were categorized based on RS results into LR, IR, and HR groups if RS result was < 11, 11-25, and > 25, respectively. Treatment recommendations by multidisciplinary meeting assessed before and after RS results were available., Results: Of 246 tested samples, 78 were classified as LR or HR, with most of the patients (65.4 %) being at IR. Following test results, the recommendation changed in 15.9 % of cases. Among patients initially recommended for CT or for discussion about the role of CT, respectively 64.3 % and 75.9 % ultimately received recommendation for HT alone., Conclusions: Our study suggests that RS results can refine treatment decisions for patients with EBC exhibiting uncertain biological behavior initially recommended or considered for CT. (250/250)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Breast cancer stands as the most frequently diagnosed cancer and the primary cause of cancer-related mortality among women worldwide, including Italy. With the increasing number of survivors, many are enrolled in regular follow-up programs. However, adherence to recommendations from scientific societies (such as ASCO, ESMO, AIOM) for breast cancer follow-up management varies in daily clinical practice across different cancer centers, potentially resulting in unequal management and escalating costs. To address these concerns, the Italian Association of Multidisciplinary Oncology Groups (AIGOM) orchestrated a Consensus on early Breast Cancer follow-up utilizing the Estimate-Talk-Estimate methodology. Following the identification of 18 Items and 38 statements by a select Board, 46 out of 54 (85.1%) experts comprising a multidisciplinary and multiprofessional panel expressed their degree of consensus (Expert Panel). The Expert Panel underscores the potential for the multidisciplinary team to tailor follow-up intensity based on the individual risk of recurrence. In selected cases, the general practitioner may be recommended as the clinical lead for breast cancer follow-up, both after completion of adjuvant treatment and at early initiation of endocrine therapy in low-risk patients. Throughout follow-up, and alongside oncologic surveillance, the expert panel advises osteometabolic, cardiologic, and gynecologic surveillance for the early detection and management of early and late treatment toxicities. Moreover, preserving quality of life is emphasized, with provisions for psycho-oncologic support and encouragement to adopt protective lifestyle behaviors., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: None of the authors has any interests to report directly related to this manuscript. Outside the scope of this manuscript: Stefania Gori, Fiorenza De Rose, no conflict of interests to declare. Antonella Ferro, honoraria from Novartis, MDS, Daiichi Sankyo, Astra Zeneca, Ely Lilly, Gentili. Alessandra Fabi grants from Astra Zeneca (steering committee); consulting fees from Roche, Lilly, Novartis, AstraZeneca, Pfizer, Seagen, Gilead, MSD, Menarini; honoraria from Astra Zeneca, Roche, Lilly, Novartis, Gilead, Pfizer, Daiichi Sankyo Exact Sciences; travel grants from Roche, Lilly, Novartis, AstraZeneca, Pfizer, Seagen, Gilead, MSD, Menarini; advisory board from Roche, Lilly, Novartis, AstraZeneca, Pfizer, Seagen, Gilead, MSD, Menarini. Catia Angiolini, Giuseppe Azzarello, Maurizio Cancian, Michela Cinquini, Luca Arecco no conflict of interests to declare. Cynthia Aristei, grants from PRIN 2023, from the Ministry of University and Research. Project title “The microbiome in breast cancer therapy and its potential for pRobIOtics to improve treatment outcome. Acronym: BARRIO”. Daniela Bernardi, Laura Biganzoli, Anna Cariello no conflict of interests to declare. Laura Cortesi, report grants from Astra Zeneca, MSD, Pfizer; consulting fees and honoraria from Astra Zeneca, Gilead, MSD, Roche, Pfizer, Daijchii Sanchio, Novartis; travel grants from Gilead, Pfizer, Daijchi Sanchio; Advisory Board from Astra Zeneca, MSD, Novartis. Elisabetta Cretella no conflict of interests to declare. Carmen Criscitiello, grants from Seagen, Gilead; consulting fees and honoraria from Pfizer, Novartis, Lilly, MSD; Seagen, Daiichi Sankyo, Gilead, AstraZeneca, Roche. Ugo De Giorgi consulting fees from Amgen, Astellas Pharma, Astrazeneca, Bayer, Bristol-Myers Squibb, Eisai, Ipsen, Janssen, Merck KGaA, MSD, Novartis, Pfizer; travel grants from Pfizer, Ipsen, Astrazeneca. Maria Carmen De Santis, Giuseppe Deledda, Massimo Dessena, Sara Donati, Arianna Dri, Gianluigi Ferretti no conflict of interests to declare. Jennifer Foglietta, honoraria from Novartis; travel grants from Roche, Sophos, Pfizer; Advisory Board from Menarini Stem Line. Davide Franceschini, Pierfrancesco Franco, Alessio Schirone no conflict of interests to declare. Daniele Generali, grants from LILT, University of Trieste, Novartis, Roche; consulting fees from Lilly, Novartis, Pfizer, Roche, Accord, Daiichi Dankyo; honoraria from Lilly, Novartis, Pfizer, Roche, Accord, Daiichi Dankyo, Astrazeneca, Istituto Gentili; travel grants from Roche, Menarini; Leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid from Mednote. Lorenzo Gianni, travel grants from Novartis, Lilly, Pfizer; Advisory Board from Astra Zeneca, Novartis, Seagen. Stefano Giordani, Giovanni Grandi, Maria Cristina Leonardi, Stefano Magno, no conflict of interests to declare. Luca Malorni, consulting fees from Menarini, Pfizer, Lilly, Novartis, Roche; travel grants from Roche, Menarini, Celgene, IT Health Fusion; Advisory Board from Novartis. Carlotta Mantoan no conflict of interests to declare. Federica Martorana honoraria from Lilly, Daychii-Sankyo, Pfizer, Astra-Zeneca, Novartis; travel grants from Gilead, Roche, Pfizer, Lilly; advisory board from Amgen. Icro Meattini consulting fees from Pfizer, Astra Zeneca, Daiichi Sankyo, Novartis, Eli Lilly, Seagen, Gilead, Menarini StemLine. Bruno Meduri, Laura Merlini, Federica Miglietta, Alessandra Modena, Fabrizio Nicolis, Isabella Palumbo, no conflict of interests to declare. Pietro Panizza honoraria and travel grants from Bayer AG. Francesca Angela Rovera, Piermario Salvini, Armando Santoro, Mario Taffurelli, no conflict of interests to declare. Angela Toss consulting fees and grants from Lilly, Pfizer, Novartis, MSD, Astrazeneca, Gilead, Seagen, Daiichi Sankyo; travel grants from Gilead, Daiichi Sankyo, Menarini, Astrazeneca. Paolo Tralongo, Monica Turazza, Matteo Valerio, Matteo Verzè no conflict of interests to declare. Patrizia Vici consulting fees from Lilly, Daiichi-Sankyo, Pfizer, MSD, Novartis; honoraria from EISAI, Daiichi-Sankyo, Lilly, Novartis, Pfizer; travel grants from Roche, Pfizer, Daiichi-Sankyo, Novartis, IPSEN; advisory board from Pfizer, Novartis. Claudio Zamagni no conflict of interests to declare. Giuseppe Curigliano advisory board from Roche, Novartis, Lilly, Pfizer, Astra Zeneca, Daichii Sankyo, Ellipsis, Veracyte, Exact Science, Celcuity, Merck, BMS, Gilead, Sanofi, Menarini. Giovanni Pappagallo no conflict of interests to declare. Alberto Zambelli consulting fees Pfizer, Lilly, Novartis, Roche, AstraZeneca, DaiichiSankyo, Seagen, ExactSciences, MSD, Gentili, Gilead; travel grants from Roche, DaiichiSankyo, AstraZeneca, Novartis; advisory board from Roche., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Indoor production of basil ( Ocimum basilicum L.) is influenced by light spectrum, photosynthetic photon flux density (PPFD), and the photoperiod. To investigate the effects of different lighting on growth, chlorophyll content, and secondary metabolism, basil plants were grown from seedlings to fully expanded plants in microcosm devices under different light conditions: (a) white light at 250 and 380 μmol·m -2 ·s -1 under 16/8 h light/dark and (b) white light at 380 μmol·m -2 ·s -1 under 16/8 and 24/0 h light/dark. A higher yield was recorded under 380 μmol·m -2 ·s -1 compared to 250 μmol·m -2 ·s -1 (fresh and dry biomasses 260.6 ± 11.3 g vs. 144.9 ± 14.6 g and 34.1 ± 2.6 g vs. 13.2 ± 1.4 g, respectively), but not under longer photoperiods. No differences in plant height and chlorophyll content index were recorded, regardless of the PPFD level and photoperiod length. Almost the same volatile organic compounds (VOCs) were detected under the different lighting treatments, belonging to terpenes, aldehydes, alcohols, esters, and ketones. Linalool, eucalyptol, and eugenol were the main VOCs regardless of the lighting conditions. The multivariate data analysis showed a sharp separation of non-volatile metabolites in apical and middle leaves, but this was not related to different PPFD levels. Higher levels of sesquiterpenes and monoterpenes were detected in plants grown under 250 μmol·m -2 ·s -1 and 380 μmol·m -2 ·s -1 , respectively. A low separation of non-volatile metabolites based on the photoperiod length and VOC overexpression under longer photoperiods were also highlighted., Competing Interests: Author Thierry Bodhuin was employed by the company FOS S.p.A., Via E. Melen 77, 16152 Genova, Italy. He participated in software and data curation in the study. The role of the company was as a co-owner of the Microcosm patent along with ENEA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Purpose: Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER)-positive (ER+) breast cancer. Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine-resistant ER+ breast cancer models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ breast cancer. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ breast cancer remain elusive. Herein, we sought to unravel these mechanisms., Experimental Design: We conducted multi-omic analyses in ER+ breast cancer models in vitro and in vivo, including models with different genetic backgrounds. We also performed genome-wide CRISPR/Cas9 knockout screens to identify potential therapeutic vulnerabilities in CDK4/6i-resistant models., Results: We found that the on-target antitumor effects of CDK7 inhibition in ER+ breast cancer are in part p53 dependent, and involve cell cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ET and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118; however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Finally, genome-wide CRISPR/Cas9 knockout screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i-resistant models., Conclusions: Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ breast cancer. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors of sensitivity to CDK7i-based treatments., (©2024 The Authors; Published by the American Association for Cancer Research.)

Targeting aromatase deprives ER + breast cancers of estrogens and is an effective therapeutic approach for these tumors. However, drug resistance is an unmet clinical need. Lipidomic analysis of long-term estrogen-deprived (LTED) ER + breast cancer cells, a model of aromatase inhibitor resistance, revealed enhanced intracellular lipid storage. Functional metabolic analysis showed that lipid droplets together with peroxisomes, which we showed to be enriched and active in the LTED cells, controlled redox homeostasis and conferred metabolic adaptability to the resistant tumors. This reprogramming was controlled by acetyl-CoA-carboxylase-1 (ACC1), whose targeting selectively impaired LTED survival. However, the addition of branched- and very long-chain fatty acids reverted ACC1 inhibition, a process that was mediated by peroxisome function and redox homeostasis. The therapeutic relevance of these findings was validated in aromatase inhibitor-treated patient-derived samples. Last, targeting ACC1 reduced tumor growth of resistant patient-derived xenografts, thus identifying a targetable hub to combat the acquisition of estrogen independence in ER + breast cancers.

Purpose: To identify prognostic circulating biomarkers to cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i), we performed a mutational analysis on circulating tumor DNA (ctDNA) samples from patients included in the TREnd trial, which randomly assigned patients to receive the CDK4/6i palbociclib alone or with the endocrine treatment (ET) to which they had progressed., Methods: Forty-six patients were enrolled in this substudy. Plasma was collected before treatment (T0), after the first cycle of therapy (T1), and at the time of progression (T2). ctDNA hybridization and capture were performed using the Illumina TruSight Tumor 170 Kit. Acquired mutations were confirmed by digital polymerase chain reaction. Progression-free survival analysis was estimated using the Kaplan-Meier method and compared with the log-rank test., Results: The most frequently mutated genes at T0 were ESR1 (23%), PIK3CA (17%), AR , FGFR2, and TP53 (10%). Mutations in ESR1 at T0 conferred higher risk of progression in the entire population ( P = .02) and in patients treated with palbociclib + ET ( P = .04). ESR1 mutation effect remained significant after correction for clinical variables ( P = .03). PIK3CA mutations at T0 were not prognostic, but higher risk of progression was observed when a broader analysis of PI3K pathway was performed ( P = .04). At T2, we observed the emergence of nine new mutations in seven genes., Conclusion: Mutations in ESR1 and in PI3K pathway genes at T0 were associated with worse prognosis in palbociclib-treated patients. We describe the emergence of newly acquired mutations in palbociclib-treated patients, which might potentially affect subsequent treatment.

Endocrine therapy (ET) is the cornerstone of management in hormone receptor (HR)+ breast cancer (BC). Indeed, targeting the estrogen receptor (ER) signaling at different levels is a successful strategy, since BC largely relies on the ER signaling as a driver of tumorigenesis and progression. In metastatic BC, progression of disease typically occurs due to either ligand-independent ER signaling, which favors tumor proliferation and survival in the absence of hormonal stimuli, or an ER-independent signaling, which exploits alternative transcription pathways. For instance, estrogen receptor 1 (ESR1) mutations induce constitutive ER activity, in turn upregulating ER-dependent gene transcription and causing resistance to estrogen depleting therapies. The largest unmet need lies after progression on ET + cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors, where fulvestrant alone provides an average 2-3-month PFS. In this context, novel oral selective estrogen receptor degraders (SERDs) and other next-generation ETs are being investigated, both as single agents and in combination with targeted therapies. Elacestrant, the next generation ET in most advanced clinical development and the first to be FDA approved, demonstrated improved outcomes compared to standard ETs in ET pre-treated HR+/HER2- metastatic BC in the phase 3 EMERALD clinical trial. Additionally, other agents are showing promising results in both preclinical and early phase clinical settings. In this review, emerging data related to oral SERDs and other novel ETs in managing HR+/HER2- BC are presented. Major challenges and future perspectives related to the optimal sequence of therapeutic options and the molecular landscape of endocrine resistance are also provided., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: CCo, has no potential conflicts of interest to disclose. CDA is a consultant and/or advisory board member for AstraZeneca, Eli Lilly, GlaxoSmithKline, Novartis, Pfizer and Roche. All the competing interests were outside the submitted work. GB is a consultant and/or advisory board member for Amgen, AstraZeneca, Chugai, Daiichi Sankyo, EISAI, Eli Lilly, Genomic Health, Merck Sharp & Dohme, Neopharm, Novartis, Pfizer, Roche and Sanofi. All the competing interests were outside the submitted work. LM reports speaker/consultant honoraria by Novartis, Pfizer, Lilly. Research Grant from Novartis and Pfizer. All the competing interests were outside the submitted work. MG reports honoraria from Roche, Pfizer, AstraZeneca, Novartis, Celgene, Eli Lilly, Amgen, and Eisai. All the competing interests were outside the submitted work. EH reports institutional research funding from OncoMed, Genentech/Roche, Zymeworks, Rgenix, ArQule, Clovis, Silverback Therapeutics, Millenium, Acerta Pharma, Sermonix Pharmaceuticals, Torque, Black Diamond, Karyopharm, Infnity Pharmaceuticals, Curis, Syndax, Novartis, Boehringer Ingelheim, Immunomedics, FujiFilm, Taiho, Deciphera, Fochon, Molecular Templates, Onconova Therapeutics, Hutchinson MediPharma, MedImmune, SeaGen, Puma Biotechnology, Compugen, TapImmune, Lilly, Pfzer, H3 Biomedicine, Takeda, Merus, Regeneron, Arvinas, StemCentRx, Verastem, eFFECTOR Therapeutics, CytomX, InventisBio, Lycera, Mersana, Radius Health, AbbVie, Nucana, Leap Therapeutics, Zenith Epigenetics, Harpoon, Orinove, AstraZeneca, Tesaro, Macrogenics, EMD Serono, Daiichi Sankyo, Syros, Sutro, G1 Therapeutics, Merck, PharmaMar, Olema, Polyphor, Immunogen, Plexxicon, Amgen, Akesobio Australia, and Shattuck Labs; consultancy/advisory roles with Genentech/Roche, Boehringer Ingelheim, Novartis, Dantari, Eli Lilly, Merck, Puma, Silverback Therapeutics, CytomX, Pfzer, Mersana, Black Diamond, H3 Biomedicine, Daiichi Sankyo, AstraZeneca, Arvinas, Deciphera Pharmaceuticals, Eisai, and Seattle Genetics. All the competing interests were outside the submitted work. RJ receives research funding from Pfizer and Lilly and is a consultant for Carrick Therapeutics and Luminex. All the competing interests were outside the submitted work. KJ reports fees for consulting or advisory role for Novartis, Pfizer, AstraZeneca, Jounce Therapeutics, Synthon, Intellisphere, Bristol Myers Squibb, Genentech, AbbVie, Lilly, BluePrint Medicines, Seattle Genetics, Daiichi Sankyo, Biotheranostics, SunPharma Pvt Ltd, Taiho Oncology, Sanofi. Research Funding from Novartis (Inst), Genentech (Inst), Debiopharm Group (Inst), ADC Therapeutics (Inst), Pfizer (Inst), Novita Pharmaceuticals (Inst), Clovis Oncology (Inst), Lilly (Inst), Zymeworks (Inst), Immunomedics (Inst), Puma Biotechnology (Inst), VelosBio/Merck (Inst), AstraZeneca (Inst), Travel, Accommodations, travel expenses from Taiho Pharmaceutical, Jounce Therapeutics, Pfizer, AstraZeneca, Intellisphere. All the competing interests were outside the submitted work. GC reports personal fees for consulting, advisory role and speakers’ bureau from Roche/Genentech, Novartis, Pfizer, Lilly, Foundation Medicine, Samsung and Daiichi Sankyo; honoraria from Ellipses Pharma and fees for travel and accommodations from Roche/Genentech and Pfizer. All the competing interests were outside the submitted work. CCr reports personal fees for consulting, advisory role and speakers’ bureau from Lilly, Roche, Novartis, MSD, Seagen, Gilead and Pfizer. All the competing interests were outside the submitted work., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Thymidine kinase 1 (TK1) is an enzyme downstream of the CDK4/6 pathway, with a critical role in DNA synthesis; serum TK1 activity (sTKa) is a novel liquid biopsy biomarker of tumour cell proliferation., Methods: The phase IIIb, BioItaLEE trial (NCT03439046) collected sera from postmenopausal patients with hormone receptor-positive (HR+), HER2-negative (HER2-) advanced breast cancer (ABC) treated with first-line ribociclib plus letrozole at baseline, day 15 of cycle 1 (C1D15), day 1 of cycle 2 (C2D1), and at first imaging. Associations between sTKa assessed at different time points or sTKa dynamic patterns, and progression-free survival (PFS) were evaluated using multivariate Cox models., Results: Overall, 287 patients were enroled. Median follow-up was 26.9 months. High sTKa (>median) at baseline was associated with higher risk of progression (hazard ratio [HR], 2.21; 95% confidence interval [95% CI], 1.45, 3.37; P = 0.0002); similar results were observed for patients with high sTKa levels at C1D15 and C2D1. Early sTKa dynamic patterns were strongly predictive of PFS. The pattern with high sTKa levels at C2D1 following initial decrease at C1D15 was associated with higher risk of progression versus the pattern with low sTKa levels at both time points (HR, 2.89; 95% CI, 1.57, 5.31; P = 0.0006), while the pattern with high sTKa levels at C1D15 was associated with the shortest PFS (HR, 5.65; CI: 2.84, 11.2; P < 0.0001). Baseline and dynamic sTKa changes provided independent information., Conclusions: sTKa appears to be a new promising prognostic and pharmacodynamic biomarker in patients with HR+/HER2- ABC treated with ribociclib plus letrozole as first-line therapy., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

DNA-methylation alterations are common in cancer and display unique characteristics that make them ideal markers for tumor quantification and classification. Here we present MIMESIS, a computational framework exploiting minimal DNA-methylation signatures composed by a few dozen informative DNA-methylation sites to quantify and classify tumor signals in tissue and cell-free DNA samples. Extensive analyses of multiple independent and heterogenous datasets including >7200 samples demonstrate the capability of MIMESIS to provide precise estimations of tumor content and to enable accurate classification of tumor type and molecular subtype. To assess our framework for clinical applications, we designed a MIMESIS-informed assay incorporating the minimal signatures for breast cancer. Using both artificial samples and clinical serial cell-free DNA samples from patients with metastatic breast cancer, we show that our approach provides accurate estimations of tumor content, sensitive detection of tumor signal and the ability to capture clinically relevant molecular subtype in patients' circulation. This study provides evidence that our extremely parsimonious approach can be used to develop cost-effective and highly scalable DNA-methylation assays that could support and facilitate the implementation of precision oncology in clinical practice., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In this study, the influence of a new plant-based biostimulant (Bortan) on physiological and aromatic traits of rocket ( Diplotaxis tenuifolia L. var. Pamela) was monitored by evaluating physico-chemical parameters (fresh and dry weight, leaf color and chlorophyll content) and biochemical traits (total phenolic compound (TP), total flavonoids (TF), ascorbic acid (AA) and antioxidant activity (AOX). Volatile profiles were also analyzed by headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry, allowing the detection of 32 volatiles belonging to 5 chemical classes. Compared to the control, Bortan application enhanced leaf pigment content, including chlorophyll a, b and carotenoids (+10%, +16% and +28%, respectively) and increased TP (+34%), TF (+26%), AA (+19%) amonts and AOX value (+16%). Principal component analysis revealed a significant discrimination between the two samples. Specifically, treated samples were mainly associated with "green-leaf" volatiles, namely hexanal and 2-hexenal, 3-hexenal and 1-penten-3-one, while control rocket was directly correlated with several alcohols and to all isothiocyanates, associated with the sulfur-like odor of rocket. These findings can add further support, both for farmers and the agro-food industry, in choosing PBs as a new and sustainable practice in complementing enhanced yields with premium-quality produce. To confirm these preliminary data, further experiments are needed by enlarging the sample size, testing different concentrations of Bortan and/or using other food crops.

Sweet cherries ( Prunus avium L.) are greatly appreciated fruits worldwide due to their taste, color, nutritional value, and beneficial health effects. The characterization of autochthonous germplasm allows to identify genotypes that possess superior characteristics compared to standard cultivars. In this work, four accessions of sweet cherry from the Campania region (Limoncella, Mulegnana Riccia, Mulegnana Nera and Montenero) were investigated for their morpho-physiological, qualitative, aromatic, and sensorial traits in comparison with two standard cultivars (Ferrovia and Lapins). A high variability in the pomological traits resulted among the samples. Montenero showed comparable fruit weight and titratable acidity to Ferrovia and Lapins, respectively. The highest total soluble solid content was detected in Mulegnana Riccia. A considerable variability in the skin and pulp color of the cherries was observed, varying from yellow-red in Limoncella to a dark red color in Montenero. Mulegnana Nera showed the highest content of polyphenols, flavonoids, anthocyanins, and ascorbic acid compared to the standard cultivars. Volatile organic compounds profile analysis identified 34 volatile compounds, 12 of which were observed at different concentrations in all the sweet cherry genotypes while the others were genotype-dependent. Conservation and cultivation of autochthonous accessions with suitable nutritional and morpho-physiologic characteristics promotes our agrobiodiversity knowledge and allows to better plan future breeding programs.

Background: We previously showed that metabolomics predicts relapse in early breast cancer (eBC) patients, unselected by age. This study aims to identify a "metabolic signature" that differentiates eBC from advanced breast cancer (aBC) patients, and to investigate its potential prognostic role in an elderly population., Methods: Serum samples from elderly breast cancer (BC) patients enrolled in 3 onco-geriatric trials, were retrospectively analyzed via proton nuclear magnetic resonance (1H NMR) spectroscopy. Three nuclear magnetic resonance (NMR) spectra were acquired for each serum sample: NOESY1D, CPMG, Diffusion-edited. Random Forest (RF) models to predict BC relapse were built on NMR spectra, and resulting RF risk scores were evaluated by Kaplan-Meier curves., Results: Serum samples from 140 eBC patients and 27 aBC were retrieved. In the eBC cohort, median age was 76 years; 77% of patients had luminal, 10% HER2-positive and 13% triple negative (TN) BC. Forty-two percent of patients had tumors >2 cm, 43% had positive axillary nodes. Using NOESY1D spectra, the RF classifier discriminated free-from-recurrence eBC from aBC with sensitivity, specificity and accuracy of 81%, 67% and 70% respectively. We tested the NOESY1D spectra of each eBC patient on the RF models already calculated. We found that patients classified as "high risk" had higher risk of disease recurrence (hazard ratio (HR) 3.42, 95% confidence interval (CI) 1.58-7.37) than patients at low-risk., Conclusions: This analysis suggests that a "metabolic signature", identified employing NMR fingerprinting, is able to predict the risk of disease recurrence in elderly patients with eBC independently from standard clinicopathological features., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Finger lime is receiving growing attention as an ingredient of gastronomic preparations of haute cuisine for its delicious flavor and fragrance and for its appealing aspect. Volatile compounds play a crucial role in determining the organoleptic characteristics of the fruit and its pleasantness for consumers. The aim of the present study was to investigate the volatile profiles by headspace solid phase micro-extraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS) in the peel and, for the first time, in the pulp of three Australian finger lime cultivars grown in Sicily (southern Italy): Pink Pearl, Sanguinea, and Faustrime, allowing to overall identify 84 volatile organic compounds (VOCs). The analytical data showed that the three cultivars were characterized by distinct volatile chemotypes: limonene/sabinene/bicyclogermacrene in the Pink Pearl, limonene/γ-terpinene/bicyclogermacrene in the Sanguinea, and limonene/β-phellandrene/γ-terpinene in the Faustrime. Moreover, some volatiles, found exclusively in one cultivar, could be considered potential markers of the individual cultivar. PCA allowed us to clearly discriminate the samples into three clusters: the first related to the Sanguinea peel, the second to the Faustrime peel, and a third group associated with the Pink Pearl peel along with the pulp of the three cultivars. Accordingly, the VOCs that mostly contributed to the differentiation of the three finger lime cultivars were also identified. Among them, D-limonene, sabinene γ-terpinene, α-pinene, α-phellandrene, β-myrcene, p-cymene, linalool, δ-elemene, ledene, bicyclogermacrene β-citronellol, α-bergamotene, α-caryophillene, and β-bisabolene, have been previously reported to exhibit important biological activities, suggesting that these cultivars, in addition to possessing unique volatile profiles, can show promise for several applications in pharmaceutical and food industry, namely for development of functional foods.

In the main strawberry areas of Southern Italy, cultivation is carried out by transplanting plants on raised beds (30-40 cm from ground level), mulched with black polyethylene (PE). This technique has becoming increasingly expensive due to the growing prices of plastic mulches, the cost to remove them at the end of crop cycle, and the difficulty to dispose of black, dirty plastic films. The main objective of this research was the replacement of PE mulch with a new biodegradable mulching film Mater-Bi ® -based (Novamont), characterized by an increased permanence in the field designed for long crop life. In 2021, two Mater-Bi-based, black, 18 μm thick mulching films were tested under tunnel: N5 as innovative film and N18 as commercial standard film. Black PE film, 50 μm thick was the control. Strawberry cultivars 'Sabrina' and 'Elide' were cropped on the three mulching films according to a split plot design with four replications. Harvests lasted from March to June 2021. Cvs Sabrina and Elide yielded around 40 t ha -1 , while the mean effect of mulching films did not point out differences between the biodegradable mulches and PE. In 4 out of 12 harvests we analyzed samples of fruits to assess the influence of mulches on the contents of °Brix, polyphenols, antioxidant activity, ascorbic acid, flavonoids, and anthocyanins. On average, °Brix was clearly improved in fruits on PE compared to biodegradable films, while all the other qualitative traits resulted in being more dependent on the cultivars and times of sampling effects. Overall, biodegradable mulches are a viable alternative to PE mulch, and the innovative N5 film appeared promising for the enhancement of durability of soil coverage in a long-lasting cycle.

Electronic nose (e-nose), attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy and image analysis (IA) were used to discriminate the ripening stage (half-red or red) of strawberries (cv Sabrosa, commercially named Candonga), harvested at three different times (H1, H2 and H3). Principal component analysis (PCA) performed on the e-nose, ATR-FTIR and IA data allowed us to clearly discriminate samples based on the ripening stage, as in the score space they clustered in distinct regions of the plot. Moreover, a correlation analysis between the e-nose sensor and 57 volatile organic compounds (VOCs), which were overall detected in all the investigated fruit samples by headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME/GC-MS), allowed us to distinguish half-red and red strawberries, as the e-nose sensors gave distinct responses to samples with different flavours. Three suitable broad bands were individuated by PCA in the ATR-FTIR spectra to discriminate half-red and red samples: the band centred at 3295 cm -1 is generated by compounds that decline, whereas those at 1717 cm -1 and at 1026 cm -1 stem from compounds that accumulate during ripening. Among the chemical parameters (titratable acidity, total phenols, antioxidant activity and total soluble solid) assayed in this study, only titratable acidity was somehow correlated to ATR-FTIR and IA patterns. Thus, ATR-FTIR spectroscopy and IA might be exploited to rapidly assess titratable acidity, which is an objective indicator of the ripening stage.

The witnesses of the millenary history of Campania felix in southern Italy highlighted that several fruit and vegetables cultivated in such territory could potentially be a treasure trove of important health elements. Our work evaluated the content of β-carotene, ascorbic acid, and total phenolics and the antioxidant activity of ten typical varieties of apricots cultivated in the Vesuvius area in the Campania region. The total polyphenols varied between 10.24 and 34.04 mg/100 g of a fresh sample. The amount of ascorbic acid also varied greatly, ranging from 2.65 to 10.65 mg/100 g of a fresh product. B-Carotene reached values up to 0.522 mg/100 g of the fresh sample. The correlation analysis performed, accounting for these parameters, showed that the antioxidant activity, calculated by 2,2-diphenyl-1-picrylhydrazyl (DPPH assay) and azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) tests, was influenced mainly by the content of total polyphenols, with ρ = -0.762 and ρ = 0.875 when we considered DPPH and ABTS tests, respectively, slightly less by the content of ascorbic acid, and not by β-carotene. The dendrogram clustered eight varieties into two main groups; on the other hand, two varieties ("Vitillo" and "Preveta bella") seemed hierarchically distant. The gas chromatography-mass spectrometry (GC-MS) analysis of volatile organic compounds (VOCs), herein performed for the first time, demonstrated the influence of the varieties on the VOC profiles, both from a qualitative and semiquantitative perspective, discriminating the varieties in different clusters, each of which was characterized by specific notes. α-Terpinolene was the only terpene identified by GC-MS that appeared to affect the antioxidant activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fratianni, Cozzolino, d'Acierno, Ombra, Spigno, Riccardi, Malorni, Stocchero and Nazzaro.)

Background: Biomarkers for cyclin-dependent kinase 4/6 inhibitors, such as palbociclib, for patients with hormone receptor-positive/HER2-negative metastatic breast cancer are lacking. Thymidine kinase is a proliferation marker downstream of the cyclin-dependent kinase 4/6 pathway. We prospectively investigated the prognostic role of serum thymidine kinase activity (sTKa), in patients treated with Palbociclib + fulvestrant., Patients and Methods: PYTHIA was a phase II, single-arm, multicentre, trial that enrolled 124 post-menopausal women with endocrine-resistant hormone receptor-positive/HER2-negative metastatic breast cancer. Serum samples were collected pre-treatment (pre-trt; n = 122), at day 15 of cycle 1 (D15; n = 108), during the one week-off palbociclib before initiating cycle 2 (D28; n = 108) and at end of treatment (n = 76). sTKa was determined centrally using Divitum®, a refined ELISA-based assay with a limit of detection of 20 Divitum Units (Du)/L. The primary study endpoint was progression-free survival, assessed for its association with pre- and on-treatment sTKa., Results: Data from 122 women were analysed. Pre-treatment sTKa was not associated with clinical characteristics and moderately correlated with tissue Ki-67. Palbociclib + fulvestrant markedly suppressed sTKa levels at D15, with 83% of patients recording levels below limit of detection. At D28, sTKa showed a rebound in 60% of patients. At each timepoint, higher sTKa was associated with shorter progression-free survival (each p < 0.001), with the strongest effect at D15., Conclusions: STKa is an independent prognostic biomarker in patients treated with palbociclib. High pre-treatment sTKa and its incomplete suppression during treatment may identify patients with poorer prognosis and primary resistance. This warrants validation in prospective comparative trials. CLINICALTRIALS., Gov Identifier: NCT02536742; EudraCT 2014-005387-15., Competing Interests: Conflict of interest statement L. Malorni reports receiving research grants (institution) from Novartis and Pfizer and serving an advisory board (honoraria) for Novartis, Seagen, Pfizer. F. Hilbers reports receiving funding (to previous Institution affiliated with) from Pfizer for conduct of the PYTHIA trial. M. Ignatiadis reports receiving research grants (to Institution) from Roche, Natera Inc, and Pfizer and serving an advisory board role (honoraria) for Novartis and Seattle Genetics. M. Colleoni reports receiving a research grant (to Institution) from Roche. G. Jerusalem reports grants (to Institution), personal fees and non-financial support from Novartis, grants, personal fees and non-financial support from Roche, grants, personal fees and non-financial support from Pfizer, personal fees and non-financial support from Lilly, personal fees and non-financial support from Amgen, personal fees and non-financial support from BMS, personal fees and non-financial support from Astra-Zeneca, personal fees from Daiichi Sankyo, personal fees from Abbvie, non-financial support from Medimmune, and non-financial support from Merck KGaA. K. Papadimitriou reports serving an advisory board role (honoraria) for Roche, Pfizer, Lilly, and Novartis. F.P. Duhoux reports serving an advisory role (support to Institution) for Roche, Pfizer, AstraZeneca, Lilly, Novartis, Amgen, Daiichi Sankyo, Pierre Fabre, Mundipharma, Seagen, and Teva. I. R. MacPherson reports serving as a consultant for Roche, Novartis, Pfizer, Eli Lilly, Pierre Fabre, Daiichi Sankyo, and Astrazeneca, and reports receiving travel/conference registration support from Roche, Eli Lilly, and Daichi Sankyo. A. Thomson reports receiving speaker fees from Novartis, Roche, Exact Sciences, and Lilly, reports serving on the advisory board for Novartis and MSD, and reports receiving support for attending conferences from BMS, Astellas, MSD, Ipsen, and EUSA. M. Bergqvist reports being an employee and holding stock in Biovica. G. Zoppoli reports receiving travel grants from Novartis and Roche, and reagents from ThermoFisher Scientific and Cytiva. J.M. Bliss reports receiving research funding from AstraZeneca, Merck Sharp & Dohme, Puma Biotechnology, Clovis Oncology, Pfizer, Janssen-Cilag, Novartis, Roche, and Eli Lilly. H. De Swert reports receiving research funding (to Institution) from Pfizer, Novartis, Roche, Servier, AstraZeneca, TESARO, and GSK. D. Fumagalli reports receiving research funding for the conduct of clinical trials (to Institution) from Pfizer, Biovica, Novartis, Roche/Genentech, Sanofi, Servier, AstraZeneca, TESARO, and GSK. D. Cameron reports serving on the advisory board for AstraZeneca, Pfizer, Lilly (to Institution), and IDMC (independent data monitoring committee) work (to Institution) for Lilly and unrelated research funding from Novartis. M. Piccart reports grants (to Institution) from AstraZeneca, Immunomedics, Lilly, Menarini, MSD, Novartis, Pfizer, Radius, Roche-Genentech, Servier, and Synthon, reports receiving honoraria from AstraZeneca, Camel-IDS, Immunomedics, Lilly, Menarini, MSD, Novartis, Odonate, Pfizer (funded the study conception & design component), Roche-Genentech, Seattle Genetics, Immutep, Seagen, and NBE Therapeutics, and reports serving on the Scientific Board of Oncolytics. M.M. Regan reports research funding (to Institution) from Novartis, Pfizer, Ipsen, TerSera, Pierre Fabre, Roche, AstraZeneca, Bristol-Myers Squibb, and Bayer, and reports serving a consulting/advisory role for Ipsen (support to Institution), Bristol-Myers Squibb, and Tolmar Pharmaceuticals. No disclosures were reported by the other authors., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

We aim to elucidate the prognostic value of PIK3CA mutations and copy number (CN) gain (PIK3CA-mut/gain) in hormone receptor-positive and HER2-negative (HR + /HER2-) breast cancer (BC). We analyzed primary HR + /HER2- BC from three publicly available datasets comprising over 2000 samples and assessed the associations with tumoral and clinical characteristics and outcome. Clinical benefit (CB) in alpelisib-treated patients from two studies including 46 patients was analyzed. About 8-10% of HR + /HER2- primary BC had PIK3CA-mut/gain. In two of the datasets analyzed, among patients with PIK3CA mutant tumors, those with mut/gain had significantly worse outcome compared to those with CN neutral (PIK3CA-mut/neut) and PIK3CA-mut/gain remained an independent prognostic factor. CB of alpelisib-treated patients with PIK3CA-mut/gain and PIK3CA-mut/neut tumors was comparable. PIK3CA CN might help clarifying the prognostic and predictive role of PIK3CA mutations. Further studies are warranted., (© 2022. The Author(s).)

Differentially DNA methylated regions (DMRs) inform on the role of epigenetic changes in cancer. We present Rocker-meth, a new computational method exploiting a heterogeneous hidden Markov model to detect DMRs across multiple experimental platforms. Through an extensive comparative study, we first demonstrate Rocker-meth excellent performance on synthetic data. Its application to more than 6,000 methylation profiles across 14 tumor types provides a comprehensive catalog of tumor type-specific and shared DMRs, and agnostically identifies cancer-related partially methylated domains (PMD). In depth integrative analysis including orthogonal omics shows the enhanced ability of Rocker-meth in recapitulating known associations, further uncovering the pan-cancer relationship between DNA hypermethylation and transcription factor deregulation depending on the baseline chromatin state. Finally, we demonstrate the utility of the catalog for the study of colorectal cancer single-cell DNA-methylation data., (© 2021. The Author(s).)

Purpose: Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor-positive (ER + )/HER2 - breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes., Experimental Design: Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation-resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes., Results: Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an "IFN-related palbociclib-resistance Signature" (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER + /HER2 - tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis., Conclusions: Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i., (©2021 American Association for Cancer Research.)

Adjuvant treatment for patients with early stage colorectal cancer (eCRC) is currently based on suboptimal risk stratification, especially for elderly patients. Metabolomics may improve the identification of patients with residual micrometastases after surgery. In this retrospective study, we hypothesized that metabolomic fingerprinting could improve risk stratification in patients with eCRC. Serum samples obtained after surgery from 94 elderly patients with eCRC (65 relapse free and 29 relapsed, after 5-years median follow up), and from 75 elderly patients with metastatic colorectal cancer (mCRC) obtained before a new line of chemotherapy, were retrospectively analyzed via proton nuclear magnetic resonance spectroscopy. The prognostic role of metabolomics in patients with eCRC was assessed using Kaplan-Meier curves. PCA-CA-kNN could discriminate the metabolomic fingerprint of patients with relapse-free eCRC and mCRC (70.0% accuracy using NOESY spectra). This model was used to classify the samples of patients with relapsed eCRC: 69% of eCRC patients with relapse were predicted as metastatic. The metabolomic classification was strongly associated with prognosis ( p -value 0.0005, HR 3.64), independently of tumor stage. In conclusion, metabolomics could be an innovative tool to refine risk stratification in elderly patients with eCRC. Based on these results, a prospective trial aimed at improving risk stratification by metabolomic fingerprinting (LIBIMET) is ongoing.

CDK4/6 inhibitors (CDK4/6i) and endocrine therapy are the standard treatment for patients with hormone receptor-positive and HER2 negative (HR+/HER2-) metastatic breast cancer. Patients might show intrinsic and acquired resistance, which leads to treatment failure and progression. Circulating biomarkers have the potential advantages of recognizing patients who might not respond to treatment, monitoring treatment effects and identifying markers of acquired resistance during tumor progression with a simple withdrawal of peripheral blood. Genomic alterations on circulating tumor DNA and serum thymidine kinase activity, but also circulating tumor cells, epigenetic or exosome markers are currently being tested as markers of CDK4/6i treatment response, even though none of these have been integrated into clinical practice. In this review, we discuss the recent advancements in the development of circulating biomarkers of CDK4/6i response in patients with HR+/HER2-breast cancer.

Precision oncology is an emerging approach in cancer care. It aims at selecting the optimal therapy for the right patient by considering each patient's unique disease and individual health status. In the last years, it has become evident that breast cancer is an extremely heterogeneous disease, and therefore, patients need to be appropriately stratified to maximize survival and quality of life. Gene-expression tools have already positively assisted clinical decision making by estimating the risk of recurrence and the potential benefit from adjuvant chemotherapy. However, these approaches need refinement to further reduce the proportion of patients potentially exposed to unnecessary chemotherapy. Nuclear magnetic resonance (NMR) metabolomics has demonstrated to be an optimal approach for cancer research and has provided significant results in BC, in particular for prognostic and stratification purposes. In this review, we give an update on the status of NMR-based metabolomic studies for the biochemical characterization and stratification of breast cancer patients using different biospecimens (breast tissue, blood serum/plasma, and urine)., Competing Interests: The authors declare no conflict of interest.

Background: Circulating tumor cells (CTCs) are prognostic in patients with advanced breast cancer (ABC). However, no data exist about their use in patients treated with palbociclib. We analyzed the prognostic role of CTC counts in patients enrolled in the cTREnd study, a pre-planned translational sub-study of TREnd (NCT02549430), that randomized patients with ABC to palbociclib alone or palbociclib plus the endocrine therapy received in the prior line of treatment. Moreover, we evaluated RB1 gene expression on CTCs and explored its prognostic role within the cTREnd subpopulation., Methods: Forty-six patients with ER-positive, HER2-negative ABC were analyzed. Blood samples were collected before starting palbociclib treatment (timepoint T0), after the first cycle of treatment (timepoint T1), and at disease progression (timepoint T2). CTCs were isolated and counted by CellSearch® System using the CellSearch™Epithelial Cell kit. Progression-free survival (PFS), clinical benefit (CB) during study treatment, and time to treatment failure (TTF) after study treatment were correlated with CTC counts. Samples with ≥ 5 CTCs were sorted by DEPArray system® (DA). RB1 and GAPDH gene expression levels were measured by ddPCR., Results: All 46 patients were suitable for CTCs analysis. CTC count at T0 did not show significant prognostic value in terms of PFS and CB. Patients with at least one detectable CTC at T1 (n = 26) had a worse PFS than those with 0 CTCs (n = 16) (p = 0.02). At T1, patients with an increase of at least three CTCs showed reduced PFS compared to those with no increase (mPFS = 3 versus 9 months, (p = 0.004). Finally, patients with ≥ 5 CTCs at T2 (n = 6/23) who received chemotherapy as post-study treatment had a shorter TTF (p = 0.02). Gene expression data for RB1 were obtained from 19 patients. CTCs showed heterogeneous RB1 expression. Patients with detectable expression of RB1 at any timepoint showed better, but not statistically significant, outcomes than those with undetectable levels., Conclusions: CTC count seems to be a promising modality in monitoring palbociclib response. Moreover, CTC count at the time of progression could predict clinical outcome post-palbociclib. RB1 expression analysis on CTCs is feasible and may provide additional prognostic information. Results should be interpreted with caution given the small studied sample size.

CDK4/6 inhibitors (CDK4/6i) in combination with endocrine therapy are the mainstay of treatment for patients with hormone receptor-positive, HER2 negative (HR+/HER2neg) metastatic breast cancer. However, resistance - either de novo or acquired - invariably occurs, leading to treatment failure and cancer progression. Genomic alterations, gene expression data and circulating biomarkers have been correlated to response to treatment, but to date no biomarker has been approved to stratify patients. Treatment strategies after progression on CDK4/6i are yet to be standardized. Current approaches include endocrine therapy alone or in combination with target therapy, or chemotherapy. New agents are in clinical development based on potential mechanisms of acquired resistance. Here we will review recent advancements in biomarkers of response to CDK4/6i, and in post- treatment therapeutic strategies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Liquid biopsy based on cell-free DNA (cfDNA) enables non-invasive dynamic assessment of disease status in patients with cancer, both in the early and advanced settings. The analysis of DNA-methylation (DNAm) from cfDNA samples holds great promise due to the intrinsic characteristics of DNAm being more prevalent, pervasive, and cell- and tumor-type specific than genomics, for which established cfDNA assays already exist. Herein, we report on recent advances on experimental strategies for the analysis of DNAm in cfDNA samples. We describe the main steps of DNAm-based analysis workflows, including pre-analytics of cfDNA samples, DNA treatment, assays for DNAm evaluation, and methods for data analysis. We report on protocols, biomolecular techniques, and computational strategies enabling DNAm evaluation in the context of cfDNA analysis, along with practical considerations on input sample requirements and costs. We provide an overview on existing studies exploiting cell-free DNAm biomarkers for the detection and monitoring of cancer in early and advanced settings, for the evaluation of drug resistance, and for the identification of the cell-of-origin of tumors. Finally, we report on DNAm-based tests approved for clinical use and summarize their performance in the context of liquid biopsy.

We describe a potential role for thymidine kinase-1, a general marker of cellular proliferation, to act as a prognostic biomarker in patients receiving CDK4/6 inhibitors for advanced hormone receptor-positive, HER2-negative breast cancer, with early data suggesting that it may also provide early indication of treatment response.

Volatile organic compounds, quality and sensory parameters of four yellow- ('Dorì', 'G3', 'Jintao' and 'Soreli') and two green-fleshed ('Hayward' and 'Summer') kiwifruit cultivars were assessed. Statistical analysis was performed on volatiles, quality and sensory data for the identification of biomarkers of different kiwifruit cultivars. Principal component analysis showed that for all six samples a very good discrimination based on the cultivar was achieved. In particular, 2-pentylfuran can be used to distinguish between the green- and yellow-fleshed kiwifruit cultivars, while seven volatiles, can be identified as biomarkers of 'Dorì'. These findings are in agreement with the sensory analysis, which revealed that 'Dorì', the richest cultivar in esters, showed very high values of both ripe fruit smell and sweet sensory traits. Altogether, these results could offer recommendations for future breeding efforts for the production of kiwifruit cultivars with improved nutritional and aroma quality., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

HER2 + early breast cancer is a heterogeneous disease, comprising all the intrinsic breast cancer subtypes. The only biomarker available nowadays for anti-HER2 treatment selection is HER2 status itself, but estrogen receptor (ER) status is emerging as a robust predictive marker within HER2 + disease. In this Perspective, we discuss the biological and clinical differences between patients with HER2 + /ER-positive (ER + ) disease versus those with HER2 + /ER-negative (ER-neg) tumors, namely, short-term and long-term (>5 years after diagnosis) prognosis, response to neoadjuvant treatment and benefit from adjuvant anti-HER2-targeted therapies. We also address other possible biomarkers to be used for patient selection in future clinical trials, such as gene signatures, PAM50 subtypes, tumor-infiltrating lymphocytes, PIK3CA mutations, and changes in Ki67 score during treatment and discuss their limitations. Finally, we suggest new clinical trial designs that can have an impact on clinical practice, aiming to test treatment deescalation separately for patients with HER2 + /ER + and HER2 + /ER-neg tumors. We also propose an integrated classification of HER2 + disease, comprising DNA, RNA, protein expression, and microenvironment characteristics, in order to identify those tumors that are truly "HER2-addicted" and may benefit the most from anti-HER2 treatment., (©2020 American Association for Cancer Research.)

Purpose: Thymidine kinase 1 (TK1) is downstream to the CDK4/6 pathway, and TK activity (TKa) measured in blood is a dynamic marker of outcome in patients with advanced breast cancer (ABC). This study explores TK1 as a biomarker of palbociclib response, both in vitro and in patients with ABC., Experimental Design: Modulation of TK1 levels and activity by palbociclib were studied in seven estrogen receptor-positive breast cancer cell lines: sensitive (PDS) and with palbociclib acquired resistance (PDR). TKa was assayed in plasma obtained at baseline (T0), after one cycle (T1), and at disease progression on palbociclib (T2) in patients enrolled in the "To Reverse ENDocrine Resistance" (TREnd) trial ( n = 46)., Results: Among E2F-dependent genes, TK1 was significantly downregulated after short-term palbociclib. Early TKa reduction by palbociclib occurred in PDS but not in PDR cells. In patients, median TKa (mTKa) at T0 was 75 DiviTum units per liter (Du/L), with baseline TKa not proving prognostic. At T1, mTKa decreased to 35 Du/L, with a minority of patients ( n = 8) showing an increase-correlating with a worse outcome than those with decreased/stable TKa ( n = 33; mPFS 3.0 vs 9.0 months; P = 0.002). At T2, mTKa was 251 Du/L; patients with TKa above the median had worse outcomes on post-study treatment compared with those with lower TKa (2.9 vs 8.7 months; P = 0.05)., Conclusions: TK is a dynamic marker of resistance to palbociclib which may lead to early identification of patients in whom treatment escalation may be feasible. In addition, TKa may stratify prognosis in patients with acquired resistance to palbociclib., (©2020 American Association for Cancer Research.)

Palbociclib is a potent cyclin-dependent kinase (CDK)4/6 inhibitor that disrupts cell cycle progression and has been recently approved in combination with an aromatase inhibitor or fulvestrant as first- and second-line treatment in hormone receptor (HR) + , human epidermal growth factor receptor (HER)2 - metastatic breast cancer. There is evidence that palbociclib may reverse endocrine therapy resistance and that it may also be added to ongoing endocrine therapy beyond progression to obtain clinical benefit. The aim of the present study was to explore this possibility in 5 patients who received palbociclib + fulvestrant following disease progression while under treatment with fulvestrant alone. The median progression-free survival was not reached during a median follow-up of 25 months, and the most frequent best response was stable disease. Three patients remained under treatment on the last re-evaluation. All patients had highly endocrine-sensitive disease and had previously received fulvestrant for ≥12 months. The hypothesis that a selected subpopulation of patients with HR + /HER2 - metastatic breast cancer may benefit from the addition of palbociclib to ongoing endocrine therapy beyond disease progression merits further investigation., (Copyright: © Schettini et al.)

The majority of breast cancers express the estrogen receptor (ER) and are dependent on estrogen for their growth and survival. Endocrine therapy (ET) is the standard of care for these tumors. However, a superior outcome is achieved in a subset of ER positive (ER + )/human epidermal growth factor receptor 2 negative (HER2 - ) metastatic breast cancer patients when ET is administrated in combination with a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor, such as palbociclib. Moreover, CDK4/6 inhibitors are currently being tested in ER + /HER2 + breast cancer and reported encouraging results. Despite the clinical advances of a combinatorial therapy using ET plus CDK4/6 inhibitors, potential limitations (i.e., resistance) could emerge and the metabolic adaptations underlying such resistance warrant further elucidation. Here we investigate the glucose-dependent catabolism in a series of isogenic ER + breast cancer cell lines sensitive to palbociclib and in their derivatives with acquired resistance to the drug. Importantly, ER + /HER2 - and ER + /HER2 + cell lines show a different degree of glucose dependency. While ER + /HER2 - breast cancer cells are characterized by enhanced aerobic glycolysis at the time of palbociclib sensitivity, ER + /HER2 + cells enhance their glycolytic catabolism at resistance. This metabolic phenotype was shown to have prognostic value and was targeted with multiple approaches offering a series of potential scenarios that could be of clinical relevance.

Background: Chemotherapy added to anti-HER2 agents (H) is the treatment of choice in patients with HER2+ early breast cancer. However, HER2+ tumours are clinically and biologically heterogeneous, and treatment response varies significantly by hormone receptor (HR) status and molecular subtype. Predictive biomarkers are needed in this context. This study assessed whether an RB-1 loss of function gene signature (RBsig) is predictive of response to neoadjuvant chemotherapy in combination with trastuzumab, lapatinib or both, within the NeoALTTO trial., Methods: We collected RNA-sequencing data from pretreatment biopsies derived from the NeoALTTO trial. RBsig expression was computed retrospectively and correlated with pathological complete response (pCR) using receiver-operating characteristic (ROC) curves. The RBsig was dichotomised as High/Low in correspondence to the 25th percentile. Reported p values resulted from Fisher's exact test., Results: Of 455 NeoALTTO patients, 244 were eligible for this substudy (HR+ n  = 129; HR- n  = 115). Overall, pCR rate was significantly higher in patients with RBsig High tumours than those with RBsig Low (35% versus 18% respectively; p  = 0.01). The area under the ROC curve (AUC) was 0.60 (95% CI 0.52-0.67). A remarkably low pCR rate of 11% was seen in HR+/RBsig Low patients versus 28% in HR+/RBsig High., Conclusions: These results indicate RBsig may add valuable information to HER2 and HR expression, which may in turn inform treatment choices. HR+/HER2+/RBsig Low breast cancers exhibited the poorest pathological response following chemotherapy plus H. Accordingly, in such patients, endocrine therapy in combination with H and, possibly, a CDK4/6 inhibitor, may potentially prove to be a more effective treatment., Competing Interests: Conflict of interest statement: Hilbers Florentine:  research funding: Novartis, Pfizer, Genentech Huober Jens:  Honoraria: Roche, Novartis  ADBoard: Roche, Novartis  Travel support: Roche, Novartis Sotiriou Christos: has patents on gene expression and methylation signatures. He has served on advisory boards, and/or speaker at meetings, and/or recipient of travel support for participation in medical meetings for/from (in alphabetical order) Amgen, Astellas, AstraZeneca, Bayer, Celgene, Nanostring Technologies, Novartis, Pfizer, Roche. Biganzoli Laura:  Grant/research support: Celgene, Genomic Health, Novartis  Honoraria, consulting or advisory roles: AstraZeneca, Celgene, Eisai, Genomic Health, Ipsen, Lilly, Novartis, Pfizer, Pierre Fabre, Puma Biotechnology, Roche, Takeda Di Leo Angelo:  Personal financial interests (Honoraria, consultancy or advisory role): Amgen, Astellas, AstraZeneca, Bayer, Celgene, Daichii-Sankyo, Eisai, Genetech, Genomic Health, Ipsen, Lilly, Novartis, Pfizer, Pierre Fabre, prIME Oncology, Puma Biotechnology, Research To Practice, Roche, Seattle Genetics, Sellas Life Sciences Group, Touch Medical Media, WebMD Global LLC.  Institutional financial interests: AstraZeneca, Lilly, Novartis, Roche  Nonfinancial interests (leadership roles in any other medical society, research group, foundation etc.): American Society of Clinical Oncology, Breast International Group, Early Breast Cancer Trialists Collaborative Group, European Society for Medical Oncology, International Breast Cancer Study Group Malorni Luca:  Consultant: Pfizer, Novartis, AstraZeneca  Research grant: Pfizer, Novartis. No potential conflicts of interest were disclosed by the other authors., (© The Author(s), 2019.)

The landscape of therapeutic options for the treatment of hormone receptor (HR)-positive (HR + ) HER2 - breast cancer (BC) has been profoundly changed by the introduction of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors into the metastatic setting. Currently all CDK4/6 inhibitors are approved only in the metastatic setting by Food and Drug Administration (FDA) and European Medicine Agency (EMA), whereas their role in the neoadjuvant setting is still at an investigational stage. Exploitation of novel agents such as CDK4/6 inhibitors to improve the efficacy of neoadjuvant endocrine therapy (ET) or to overcome de novo resistance to ET is an area of research under active evaluation. We present a review of the currently available data and ongoing clinical trials that are evaluating the role of CDK4/6 inhibitors in neoadjuvant therapy of HR + HER2 - early BC, and also illustrate translational aspects, such as the potential biomarkers of response to these new therapeutic agents., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mutations in the hotspot ligand-binding domain of the estrogen receptor (ER) gene ESR1 have recently been recognized as mechanisms of endocrine resistance in endocrine receptor-positive metastatic breast cancer (MBC). Accumulating data suggest these mutations develop under the selective pressure of endocrine treatments, and are infrequent in untreated ER-positive breast cancers. In vitro studies show that these mutations confer ligand-independent activity, resistance to estrogen deprivation, and relative resistance to tamoxifen and fulvestrant. Post-hoc retrospective and prospective analyses of ESR1 mutations in patients with MBC have consistently found that these mutations are markers of poor prognosis and predict resistance to aromatase inhibitors (AIs). These results warrant further investigation and prospective validation in dedicated studies. Moreover, studies are ongoing to clarify the activity of novel drugs in the context of metastatic endocrine resistant luminal breast cancer harboring ESR1 mutations. In this review, we summarize the pre-clinical and clinical findings defining the characteristics of ESR1 mutant breast cancer, and highlight the potential clinical developments in this field.

Extra virgin olive oil (EVOO), appraised for its healthy properties, represents an important element for the economy of several countries of the Mediterranean area, including Italy. Our study aimed to evaluate some biochemical characteristics (polyphenols and volatile compounds) as well as the antioxidant activity of three EVOOs obtained from the varieties Ravece , Ogliarola, and Ruvea antica , grown in the same field of an Irpinian village, Montella, in the Campania region, Southern Italy. Extra virgin olive oil Ruvea antica contained the greatest amount of total polyphenols and showed the highest antioxidant activity. Principal component analysis of the aromatic profiles indicated that the three EVOOs could be easily discriminated according to the cultivar. 1-Hexanol, 2-hexen-1-ol, 3-pentanone, representing the most abundant volatiles of the EVOO Ruvea antica , and 2-hexenal, which resulted the main component in EVOOs Ogliarola and Ravece , could be considered as markers to discriminate these three EVOOs, according to the ReliefF feature selection algorithm., Competing Interests: Authors declare that they do not have any conflict of interests., (© 2019 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc.)

Production of extra virgin olive oil (EVOO) represents an important element for the economy of Southern Italy. Therefore, EVOO is recognized as a food with noticeable biological effects. Our study aimed to evaluate the antimicrobial activity exhibited by the polyphenolic extracts of EVOOs, obtained from three varieties of Olea L. ( europea L. ( Ruvea antica , Ravece , and Ogliarola ) cultivated in the village of Montella, Avellino, Southern Italy. The study evaluated the inhibiting effect of the extracts against some Gram-positive and Gram-negative bacteria. Statistical analysis, used to relate values of antimicrobial activity to total polyphenols and phenolic composition, revealed a different behavior among the three EVOO polyphenol extracts. The method applied could be useful to predict the influence of singular metabolites on the antimicrobial activity., Competing Interests: The authors declare no conflict of interest.

The recent arrival of CDK4/6 inhibitor agents, with an approximate doubling of progression-free survival (PFS) associated with their use in hormone receptor-positive, HER2-negative advanced breast cancer (BC), has radically changed the approach to managing this disease. However, resistance to CDK4/6 inhibitors is considered a near-inevitability in most patients. Mechanisms of resistance to these agents are multifactorial, and research in this field is still evolving. Biomarkers with the ability to identify early resistance, or to predict the likelihood of successful treatment using CDK4/6 inhibitors are yet to be identified, and represent an area of unmet clinical need. Here we present selected mechanisms of resistance to CDK4/6 inhibitors, largely focussing on roles of Rb, cyclin E1, and the PIK3CA pathway, with discussion of associated biomarkers which have been investigated and applied in recent pre-clinical and clinical studies. These biological drivers may furthermore influence clinical treatment strategies adopted beyond CDK4/6 resistance.

Background: Thymidine kinase 1 (TK1) plays a critical role in DNA synthesis and cell proliferation. Recent studies have shown potential for serum TK1 activity (sTKa) as a prognostic marker and indicator of early response to endocrine therapy in advanced breast cancer. The aim of this study is to assess the correlation between sTKa and patient outcome., Patients and Methods: The Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT) was a double-blind, double-dummy, randomised trial of fulvestrant versus exemestane after progression on non-steroidal aromatase inhibitor therapy, in postmenopausal women with advanced breast cancer. Retrospective analyses of serum archived from EFECT were conducted. sTKa was assessed using the DiviTum® assay on samples collected at baseline, after three and six months of endocrine therapy, and at disease progression., Results: The median time to progression (mTTP) for patients with low baseline sTKa levels was 5.03 months (95% confidence interval [CI]: 3.91-5.89) versus 2.57 months (95% CI: 2.04-3.52) in patients with high sTKa baseline levels (P < 0.0001). On treatment, patients whose sTKa increased from baseline had a significantly shorter mTTP (3.39 months, 95% CI: 2.14-4.11) than those without an sTKa increase (5.39 months, 95% CI: 4.01-6.68) (P = 0.0045). Similar results were observed in the separate EFECT treatment arms. After adjusting for major prognostic factors, sTKa remained an independent marker., Conclusion: sTKa is a potential circulating prognostic marker in patients with advanced breast cancer treated with endocrine therapy. It may also represent a tool for upfront identification of endocrine therapy resistance and early positive response to therapy. Independent validation of these results is warranted., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Currently, there is limited data regarding the effectiveness of standard subsequent line therapies such as endocrine therapy, chemotherapy, or targeted agents after progression on CDK4/6 inhibitor-based regimens. This paper describes time-to-treatment failure beyond progression on palbociclib or palbociclib+endocrine therapy in patients enrolled in the phase II, multicenter TREnd trial. Our results indicate that there is limited benefit from post-palbociclib treatment, regardless of the type of therapy received. A small population of long responders were identified who demonstrated ongoing benefit from a subsequent line of endocrine therapy after progression to palbociclib-based regimens. A translational research program is ongoing on this population of outliers.

Titanium dioxide (TiO 2 ) is enclosed in many consumer products including pharmaceuticals, cosmetics, and foods. TiO 2 (E171) is daily ingested as mixed nano- and submicron-sized particles since it is approved as a white colorant in Europe in a wide variety of food products, Noteworthy, the relevant risk assessment has never been satisfactorily concluded and growing alarms for human hazards deriving from TiO 2 exposure are incrementally reported. The objective of the present study was to establish conceivable mechanisms by which nano-sized TiO 2 particles affect physiological function of the intestinal epithelium layer. The well-established Caco-2 cell line differentiated for 21 days on permeable supports was used as a predictive model of the human intestinal mucosa to identify the biological response triggered by TiO 2 particles. Exposure to 42 μg/mL TiO 2 nanoparticles disrupted the tight junctions-permeability barrier with a prompt effect detectable after 4 h incubation time and wide effects on barrier integrity at 24 h. Transport and ultrastructural localization of TiO 2 nanoparticles were determined by ICP-OES, TEM and ESI/EELS analysis, respectively. Nano-sized particles were efficiently internalized and preferentially entrapped by Caco-2 monolayers. Storage of TiO 2 nanoparticles inside the cells affected enterocytes viability and triggered the production of pro-inflammatory cytokines, including TNF-α and IL-8. Taken together these data indicate that nano-sized TiO 2 particles exert detrimental effects on the intestinal epithelium layer., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Patients with luminal early breast cancer are at risk of relapse, even after five years of adjuvant endocrine therapy. To date, no biomarkers have been clinically validated to identify those patients at risk of late recurrence, who might benefit from extended adjuvant endocrine therapy. In recent decades, multiple clinical trials have tested the role of extending adjuvant endocrine therapies in patients with luminal disease. However, the data currently at our disposal are conflicting. This article reviews all the major trials concerning extended adjuvant endocrine regimens, and formulates some general conclusions and hypotheses of future study., (Copyright © 2019. Published by Elsevier Ltd.)

Background: New single-cell isolation technologies are facilitating studies on the transcriptomics of individual cells. Bio-Rad ddSEQ is a droplet-based microfluidic system that, when coupled with downstream Illumina library preparation and sequencing, enables the monitoring of thousands of genes per cell. Sequenced reads show unique features that do not permit the use of freely available tools to perform single cell demultiplexing., Results: We present ddSeeker, a tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina experiments. Its application to the Illumina test dataset demonstrates that ddSeeker performs better than Illumina BaseSpace software, enabling a higher recovery of valid reads. We also show its utility in the analysis of an in-house dataset including two read sets characterized by low and high sequencing quality. ddSeeker and its source code are available at https://github.com/cgplab/ddSeeker ., Conclusions: ddSeeker is a freely available tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina single cell transcriptomic experiments.

CDK4/6 inhibitors represent a new treatment standard for hormone receptor-positive (HR+), HER2-negative advanced breast cancer (BC) patients. Although efficacious, resistance to these agents is universal. Here, we profiled a large panel of HR+ BC cell lines with conditioned resistance to the CDK4/6 inhibitor palbociclib, and analyzed cell cycle-related markers by gene expression profiles (GEP) and western blot (WB). GEP showed high molecular heterogeneity among the models, with E2F targets being significantly enriched both during treatment and at the time of resistance. By both WB and GEP, a common molecular feature at the time of palbociclib resistance was the concomitant overexpression of cyclin E1 and down-regulation of Rb. CCNE1 was the only significantly up-regulated gene among E2F targets at resistance with CCNE1 genomic amplification being observed in two resistant models. Rb was downregulated in all resistant models; a reduction of RB1 copy number was observed in three resistant cell lines. In silico analyses showed that CCNE1/RB1 ratio correlated with palbociclib IC50 in different datasets of both breast and non-breast cancer cell lines, performing better than CCNE1 or RB1 taken separately. Finally, the CCNE1/RB1 ratio was shown to be an adverse prognostic factor in patients with ER+ BC and to be able to discriminate palbociclib-sensitive versus resistant among patients enrolled in the NeoPalAna trial, a neoadjuvant trial testing palbociclib, performing better than CCNE1 or RB1 alone. Our data suggest that the CCNE1/RB1 ratio may be a viable biomarker of palbociclib resistance, warranting further clinical validation., Competing Interests: L.M. received research support from Pfizer and is a consultant/advisory board member for Pfizer, AstraZeneca, Novartis. A.D.L. is a consultant/advisory board member for Novartis, Pfizer, AstraZeneca and Lilly. R.S. received research funding from AstraZeneca, Gilead Sciences; is a consulting/advisory committee member for Macrogenics, and Eli Lilly. C.K.O. is a consultant/advisory board member for Genentech, AstraZeneca, Tolmar Pharma; received grants from Roche, Ventana, Puma; DMC Lilly; Stock: Genentech Non-financial competing interests: R.S.: Mechanisms of action of selective estrogen receptor modulators and down-regulators; Lectured at 2018 Int. Congress on the Future of Breast Cancer, Physicians’ Educational Resource, LLC; Member: AACR, ASCO, Endocrine Society; C.K.O.: Expert Witness for O’Melvery Meyers (patient case). All other authors declare no competing interest.

Background: The combination of platinum with 5-fluorouracil has scarcely been studied in metastatic breast cancer. As this combination does not lead to significant hepatic metabolism, in some clinical situations it may prove useful, especially in cases with liver dysfunction and an urgent clinical need for rapid tumor shrinkage. A retrospective study was conducted to evaluate the efficacy and safety of the combination of cisplatin and 5-fluorouracil in patients with metastatic breast cancer with significant alterations of biochemistry., Patients and Methods: A total of 109 patients with metastatic breast cancer and liver dysfunction were treated; time-to-progression, overall survival and trends in liver function were evaluated., Results: The median time-to-progression was 3.4 months, and median overall survival was 7.8 months. About 50% of patients obtained a complete, partial or stable biochemical response and 24 patients were subsequently able to receive additional therapies., Conclusion: Our results show that this therapeutic doublet represents a clinically effective, safe and well-tolerated treatment option for patients with metastatic breast cancer and liver dysfunction., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)