Background. The treatment of advanced hormone receptor-positive (HR+) breast cancer has seen relevant changes in last years. However, bevacizumab remains an option when combined with paclitaxel, but no certified pharmacogenetic profiles are now usable for the prediction of its response in breast cancer patients. This study aimed to explore the pharmacogenetic interactions among single nucleotide polymorphisms (SNPs) of genes involved in the angiogenic process and their impact on progression-free survival (PFS) and overall survival (OS) in hormone receptor-positive (HR+) metastatic breast cancer subjects administered with bevacizumab plus paclitaxel, or with paclitaxel alone (clinicaltrial.gov identifier NCT01935102). Methods. Germline DNA extracted from blood samples was analyzed using real-time polymerase chain reaction to investigate SNPs. The multifactor dimensionality reduction (MDR) analysis was employed to assess interactions between these genetic variants. A total of 168 eligible patients were analyzed. Among these, 106 patients received both paclitaxel and bevacizumab, while 62 received paclitaxel alone. Results. In the combination therapy group, MDR analysis identified two pharmacogenetic interaction profiles involving specific genotypes of vascular endothelial growth factor-A(VEGF-A) rs833061 and vascular endothelial growth factor receptor-2 (VEGFR-2) rs1870377. Patients with a favorable genetic profile had a median PFS (mPFS) of 22.9 months, compared to 8.7 months in those with an unfavorable profile (p = 0.001). Cox proportional hazards analysis displayed an adjusted hazard ratio of 0.443 (95% CI: 0.284-0.691; p < 0.0001). The median OS (mOS) was 50.2 months for the favorable profile vs. 23.5 months for the unfavorable (p = 0.003), with an adjusted hazard ratio (HR) of 0.404 (95% CI: 0.249-0.657; p < 0.0001). In the 62 subjects administered with just paclitaxel, no significant differences in PFS (p = 0.820) or OS (p = 0.143) were observed between favorable and unfavorable genetic profiles. Conclusions. The MDR analysis of VEGF-A rs833061 and VEGFR-2 rs1870377 genotypes can detect a subgroup of bevacizumab-administered+ metastatic breast cancer patients with improved PFS and OS.

Background: Mesangiogenic Progenitor cells (MPCs) were first described in 2008 in cultures of human bone marrow mononuclear cells (hBM-MNCs) aimed at isolating mesenchymal stromal cells (MSCs) using human autologous serum. A selective culture method was subsequently developed to isolate MPCs with a high degree of purity, yielding approximately 1% of the total plated cells. Since their initial description, MPCs have demonstrated the ability to differentiate into highly clonogenic MSCs while retaining early vasculogenic potential. Gene expression profiling of MPCs revealed constitutive expression of pluripotency-associated transcription factors such as OCT-4 and NANOG, as well as SOX15 instead of SOX2, suggesting a possible molecular mechanism that sustains MPC plasticity, defined as the “adult Oct-4 circuit”. Although the expression of these pluripotency-associated markers has been hypothesized to represent a distinctive adult molecular circuit, concerns regarding the tumorigenic potential of MPCs are reasonable and have not yet explored. Methods: Here, we present data from the tumorigenicity test in partial compliance with WHO recommendations of two different MPC-derived cell products in athymic nude mice. Results: Histomorphometric analysis of nodules excised from animals at 6, 8, or 12 weeks post-cell transplantation excluded tumor formation and demonstrated the ability of MPCs to generate homogeneous and organized tissue through distinct phases: an “early” vasculogenic phase, followed by remodeling of the newly formed microvascular network and the deposition of structured, aligned collagen fibers. Conclusions: MPCs do not possess intrinsic tumorigenic potential and spontaneously form vascularized xenogenic tissue four weeks after injection into the subcutaneous space.

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma (NHL). Most attempts to combine standard chemo-immunotherapy with targeted drugs have failed due to limited synergy or excessive toxicity. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) acting as a transcriptional repressor by methylating histone H3 at lysine 27 (H3K27me3). Activating mutations in EZH2 in NHL correlate with poor survival. We aimed at using metronomic chemotherapy (mCHEMO)—the frequent, regular administration of low-dose cytotoxic drugs that maintain prolonged and active drug-plasma levels—in combination with new epigenetic therapies as a promising, more effective and less toxic approach for DLBCL treatment. In vitro proliferation assays were performed on both EZH2Y641F mutant (SU-DHL10) and EZH2 wild-type (OCI-LY3, Toledo) DLBCL cell lines exposed to thrice-weekly vinorelbine (mVNR) and daily PRC2 inhibitors (PRC2i: tazemetostat, valemetostat, ORIC-944), alone and in concomitant combination, for 144h. Synergism was measured by the Combination Index method and the Loewe additivity model. PRC2i target engagement was assessed by H3K27me3 western blot. The modulation of gene expression in DLBCL cells treated with 144h-daily valemetostat was evaluated by RNA sequencing. The 144h exposure of daily PRC2i and mVNR inhibited the DLBCL cell viability in a concentration-dependent manner. Among the PRC2i, valemetostat exhibited the greatest cytotoxic effect on SU-DHL10, OCI-LY3 and Toledo cells (IC50s: 3.09nM, 371nM, and 303nM, respectively). ORIC-944 also effectively reduced DLBCL cell growth (IC50s: 8.7nM, 721nM, and 1,388nM, respectively). Tazemetostat significantly inhibited DLBCL cell viability, with higher IC50 values (1,389nM, 3,481nM, and 8,597nM, respectively). mVNR substantially arrested the proliferation of all cell lines (IC50s: 508.9pM, 62.9pM, and 675.6pM, respectively). The strongest synergistic effect was observed for the mVNR+valemetostat combination, while mVNR+ORIC-944 showed synergism only for high percentage of affected DLBCL cell fractions. PRC2i markedly reduced H3K27me3 levels in DLBCL cells. Among the differentially expressed genes, we observed more up-regulated than down-regulated transcripts upon PRC2 inhibition, and an increased expression of key p53-dependent pro-apoptotic genes. These findings offer a solid rationale for combining PRC2i and mCHEMO as an encouraging therapeutic approach for DLBCL, particularly in elderly or fragile patients, due to their low toxicity profiles. Overall, these results warrant additional in vitro investigations to elucidate the mechanisms underlying the reported effects and in vivo experiments in DLBCL models to validate our new therapeutic strategy, which holds potential for rapid translation into future clinical trials. Citation Format: Marta Banchi, Maryam Latarani, Paola Orlandi, Kayleigh Orchard, George Bryant, Francesco Crea, Guido Bocci. Combining PRC2 inhibitors with metronomic chemotherapy: A promising therapeutic strategy for diffuse large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1827.

BACKGROUND/OBJECTIVES:Relapsed or refractory follicular lymphoma (rrFL) remains difficult to treat in elderly or frail patients who cannot tolerate standard-dose immuno-chemotherapy as well as novel therapies. Metronomic chemotherapy (mCHEMO) may offer sustained antitumor activity with reduced toxicity. This study assessed the clinical activity and safety of R-DEVEC or R-DEVEC-light in rrFL patients following lenalidomide discontinuation or ineligibility. METHODS:Data from the ReLLi Lymphoma Registry (2013-2025) were retrospectively analyzed. Eligible patients had rrFL after ≥1 prior therapy and initiated mCHEMO at least six months before data cutoff. Thirteen patients received DEVEC or the etoposide-free DEVEC-light regimen; all but one also received rituximab. Responders received maintenance vinorelbine, low-dose prednisone, and rituximab, followed by vinorelbine-only maintenance until progression or intolerance. Responses were assessed by CT after cycle two and PET/CT at completion of six induction cycles. RESULTS:median age was 77 years (range 58-92); most patients were frail and had advanced disease. At the end of induction, 84% achieved remission (46% CR, 38% PR), with three PR converting to CR during maintenance. After a median follow-up of 27 months, the PFS was 42% (95CI 15-69%) and the OS 73% (95CI 47-100%). A transformation occurred in one patient; the main toxicity was grade 3 neutropenia (31%). DEVEC-light showed improved tolerability versus full DEVEC, with manageable infections and rare discontinuations. CONCLUSIONS:Metronomic R-DEVEC-light is a feasible and effective disease-controlling strategy for frail, heavily pretreated rrFL patients who do not tolerate lenalidomide and are excluded from modern therapies. This schedule warrants further prospective evaluation and exploration in combination with targeted agents.

Abstract Metastatic colorectal cancer (mCRC) remains a major therapeutic challenge, and although bevacizumab combined with chemotherapy has demonstrated clinical benefit, validated predictive biomarkers of its activity are still missing. Moreover, pharmacogenetic analyses of angiogenesis-related genes in mCRC patients have failed to predict treatment efficacy and survival outcomes. Based on this rationale, we conducted a pharmacogenetic study on a different approach, investigating the potential combined effect of EPAS-1, IL-8, VEGF-A, and VEGFR-2 single nucleotide polymorphisms (SNPs) on progression-free survival (PFS) and overall survival (OS) in mCRC patients treated with second-line mFOLFOX-6 or FOLFIRI (CHT) with or without bevacizumab. Germline DNA was obtained from peripheral blood samples, and SNPs were genotyped by Real-Time PCR. Pharmacogenetic interaction analysis was performed using the Multifactor Dimensionality Reduction (MDR) approach to explore potential statistical interactions between SNPs associated with treatment outcomes. This analysis included mCRC patients enrolled in the randomized phase III BEBYP trial (clinicaltrials.gov: NCT00720512) at the University Hospital of Pisa, who received second-line mFOLFOX-6 or FOLFIRI with (n=66) or without (n=66) bevacizumab. In the bevacizumab+CHT group, the MDR analysis identified two pharmacogenetic interaction profiles based on specific combinations of VEGF-A rs1570360 and VEGFR-2 rs11133360 genotypes. Patients carrying the favorable genetic profile showed a median PFS of 8.89 months compared with 5.23 months for those with the unfavorable profile (p=0.001), with a multivariable Cox hazard ratio (HR) of 0.49 (95% CI, 0.25-0.96; p=0.039). Median OS was 20.69 vs. 9.28 months (p=0.04) for favorable vs. unfavorable profiles, respectively, with an adjusted HR of 0.54 (95% CI, 0.3-0.98; p=0.042). In contrast, among patients treated with CHT alone, no significant differences were observed in PFS (5.52 vs. 4.85 months, p=0.642) or OS (16.31 vs. 16.34 months, p=0.969) between the two genetic profiles. Overall, the interaction between VEGF-A rs1570360 and VEGFR-2 rs11133360 genotypes identified a favorable pharmacogenetic profile associated with improved PFS and OS in patients receiving bevacizumab, whereas no association was observed in the CHT-alone group. These findings suggest that MDR analysis of angiogenesis-related genes identifies patients who benefit from bevacizumab-based therapy and strengthens the rationale for a pharmacogenetic selection of candidate patients for anti-angiogenic strategies in mCRC. Citation Format: Arianna Bandini, Paola Orlandi, Marco Scalese, Gianluca Masi, Federica Marmorino, Chiara Cremolini, Guido Bocci. A pharmacogenetic interaction analysis of second-line chemotherapy with bevacizumab in metastatic colorectal cancer patients: Results from the randomized BEBYP trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3133.

Background: The heterogeneity of immune-related adverse events (irAEs) in real-world evidence highlights the need to identify patterns, knowledge gaps, and priorities for future research. Objectives: To assess in labels the expected irAEs associated with immune checkpoint inhibitors (ICIs) in lung cancer, melanoma, breast cancer, and colon cancer and evaluate their incidence, clinical characteristics, management, and outcomes in real-world studies. Methods: Medicine Agency data sources (Food and Drug Administration and European Medicines Agency) were assessed for labeled irAEs associated with ICIs, and a comprehensive literature review according to Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines for scoping review was performed by retrieving observational and target trial emulation studies conducted using data collected in administrative healthcare databases (AHDs) and in spontaneous reporting systems (SRSs) concerning the drugs and tumors of interest from PubMed. irAEs’ incidence, onset, management, and outcomes were retrieved. Results: ICI combination therapy increases irAE occurrence, and inter-agency differences emerged. From PubMed, 49 observational studies were included, 22 on SRSs and 27 on AHDs. The ICIs most frequently evaluated were pembrolizumab and nivolumab, and the irAEs most reported were “lower respiratory tract disorders (excluding obstruction and infection)” (SRSs) and “epidermal and dermal conditions” (AHDs) for both drugs. Missing information on survival analysis, therapy dechallenge and rechallenge, concomitant therapies, comorbidities, time to onset, and duration of irAEs were highlighted. Conclusions: This scoping review highlights the complex, multi-organ irAEs from ICIs, underlining the need for tailored monitoring and management based on both regulatory and real-world evidence.

Background/Objectives: Metronomic chemotherapy offers a well-tolerated option for heavily pretreated metastatic gastrointestinal cancer patients, but reliable prognostic biomarkers for patient selection are lacking. This study aimed to identify exploratory circulating cytokine signatures associated with outcomes in patients treated with metronomic chemotherapy. Methods: We analyzed plasma samples from 34 patients enrolled in the COMET trial (EudraCT 2007-000065-38), a phase II study of metronomic UFT, cyclophosphamide, and celecoxib. An 88-cytokine Luminex® (Luminex Corporation, Austin, TX, USA) panel was measured at baseline and at four treatment timepoints. Partial least squares discriminant analysis identified candidate biomarkers, followed by systematic combinatorial analysis using Manciu’s method to construct 3-cytokine composite risk scores. Results: Twenty-one patients (61.8%) experienced progressive disease and 13 (38.2%) achieved stable disease. Six biomarkers showed significant discriminative power: IL-16, MCP-4, THBS-2, Eotaxin-1, PDGF-AB/BB, and TRAIL. Three 3-cytokine panels achieved statistically significant risk stratification (all p < 0.05), with hazard ratios for overall survival ranging from 2.59 to 6.24. For the representative IL-16 + MCP-4 + THBS-2 panel, high-risk patients showed a median PFS of 2.0 vs. 4.0 months (HR 3.24, p = 0.0046) and a median OS of 5.8 vs. 11.1 months (HR 4.19, p = 0.0010). Conclusions: This exploratory pharmacodynamic biomarker analysis identifies three 3-cytokine panels associated with prognostic risk stratification in metronomic chemotherapy for metastatic gastrointestinal cancer. As this single-arm trial cannot distinguish prognostic from predictive value, findings are hypothesis-generating. Prospective external validation is required before clinical translation, and exploration in combination with immune checkpoint inhibitors is warranted.

We present the first clinical application of the Schättler mathematical framework for metronomic chemotherapy using data from 30 patients with metastatic gastrointestinal cancer from the COMET trial. The model, comprising three coupled ODEs with 12 parameters representing tumour growth, angiogenesis, and immune response, was successfully fitted to individual patient data through AI-assisted optimization, achieving low fitting errors (median squared error: 0.003 for stable disease, 0.007 for progressive disease) at day 56. The growth control parameter ξ emerged as a potential biomarker, with distinct ranges between stable disease (0.066–0.153) and progressive disease (0.093–0.177), suggesting a critical threshold around ξ = 0.115 . Model parameters showed expected interdependencies reflecting the coupled nature of tumour-microenvironment interactions. While the model demonstrated strong fitting capabilities, prediction of progression-free survival showed moderate correlation ( r = -0.71 ), highlighting the complexity of long-term outcome prediction in metastatic disease. Drug effect parameters were consistently low (0.003–0.09), aligning with the modest clinical benefits observed in recent metronomic chemotherapy trials for metastatic gastrointestinal cancer. This proof-of-concept study establishes the feasibility of applying the Schättler framework to clinical data, with parameter estimates representing exploratory values requiring external validation before clinical implementation. Future studies with larger cohorts and multiple time points will be essential to validate these findings and refine the model for prospective clinical application.

Cyclin-Dependent Kinase 4/6 inhibitors (CDK 4/6i) represented a paradigm shift in the treatment of patients with metastatic hormone-receptor-positive (HR+), HER2-negative (HER2-) breast cancer patients. When disease progression occurs and further hormonal manipulations or target therapies fail, chemotherapy remains a therapeutic choice. For these reasons, resources should be invested in redesigning the best treatment strategy with the current chemotherapy drugs available. Nevertheless, the preferred strategy typically remains a planned sequence of single chemotherapeutic agents. This approach endeavors to extend overall survival and maintain quality of life. Within this complex and evolving therapeutic scenario, where the necessity for effective and treatment options is crucial, we explore in the present paper a perspective for a hypothetical new role of bevacizumab combined with paclitaxel for patients with metastatic HR+, HER2- breast cancer patients resistant to hormone therapy. Recent findings from our preliminary published pharmacogenetic studies, could suggest the efficacy of bevacizumab as linked to a well-defined genomic profile of genes implicated in the process of neoangiogenesis (e.g., favorable profile: VEGF-A rs833061/VEGFR-2 rs1870377: CT/AT, CT/AA, TT/AA, TT/TT, CC/TT), concluding the present perspective underlying the fact that bevacizumab combined with paclitaxel will be reconsidered with this new potential use only if well-designed prospective trials confirm what has been observed from our retrospective data.

Neuroendocrine Prostate Cancer (NEPC) is an incurable malignancy, originating from the trans-differentiation of prostate adenocarcinoma (PRAD). Compared to PRAD, NEPC shows over-activation of Polycomb Repressive complex-1(PRC1) and-2 (PRC2), which are multiprotein epigenetic writers that drive cancer progression via tumour suppressor gene silencing. Tazemetostat is a PRC2 inhibitor approved for the treatment of sarcomas and lymphomas. ORIC-944 is a novel EED (Embryonic Ectoderm Development) inhibitor, which is being tested in clinical trials. EED is an attractive target as it functions as a key component of both PRC1 and PRC2. We compared the anticancer effects of tazemetostat and ORIC-944 in NEPC and PRAD cells. Cells were exposed to various concentrations of the two compounds to measure effects on cell viability (IC 50 ) and apoptosis (flow cytometry). PRC2 inhibition was confirmed by measuring histone H3 Lys 27 trimethylation (H3K27me3) via ELISA and Western Blot. RNA Sequencing and pathway analysis was conducted to study modes of actions of tazemetostat vs ORIC-944. Unlike tazemetostat, ORIC-944 causes dose-dependent growth inhibition in both NEPC and PRAD cells. In this context, EED targeting achieves IC 50 values that are comparable to those of compounds used for the clinical treatment of advanced prostate cancer. Moreover, ORIC-944 (but not tazemetostat) causes significant apoptosis in NEPC cells. Both tazemetostat and ORIC-944 reduce H3K27me3. Mechanistically, both compounds reactivate the expression of known PRC2 targets, such as genes that control neural differentiation. However, the EED inhibitor also reactivates PRC1 targets, including pro-apoptotic and anti-proliferating genes (e.g. metallothionines). This evidence suggests that EED inhibition is a promising therapeutic strategy for NEPC.

Background/Objectives MC4R expression and its role in colorectal and anaplastic thyroid cancers, where resistance to therapy and lack of standard treatments remain significant challenges, are poorly understood. This study aimed to investigate MC4R as a potential therapeutic target in these cancers using the selective antagonist ML00253764 (ML), alone and in combination with vinorelbine (VNR) and irinotecan (or its active metabolite SN-38). Methods: Human colorectal adenocarcinoma HT-29, Caco-2, and anaplastic thyroid carcinoma 8305C cell lines were used. MC4R expression was assessed by Real-Time PCR with validated primers (Assay ID Hs00271877_s1), immunofluorescence, and Western blotting. Proliferation and apoptosis assays were conducted with ML, and synergy with VNR and SN-38 was evaluated by Combination Index and Loewe methods. ERK1/2 phosphorylation was measured using an ELISA assay. In vivo studies were conducted by injecting tumor cells into Athymic Nude-Foxn1nu mice, treated with ML, VNR, irinotecan, or their combinations. Results: MC4R expression was confirmed in all cell lines. ML treatment inhibited MC4R, producing antiproliferative and pro-apoptotic effects, with IC50 values of 7667 ± 2144.6 nM (8305C), 806.4 ± 321.8 nM (HT-29), and 2993 ± 1135.2 nM (Caco-2). In combination with VNR and SN-38, ML exhibited significant synergy in vitro and reduced tumor volume in vivo without causing weight loss or adverse effects in mice. Conclusions This study identifies ML as a promising therapeutic agent that, when combined with chemotherapy, may offer a novel strategy for treating colorectal and anaplastic thyroid cancers.

Background: High-dose methotrexate (HDMTX) is widely used for acute lymphoblastic leukaemia (ALL), but its pharmacokinetic (PK) variability and toxicity require therapeutic drug monitoring (TDM). Our 10-year retrospective study investigated HDMTX PK parameters and their associations with renal and hepatic biomarkers in an Italian cohort of adult patients with ALL. Methods: Plasma MTX concentrations [MTX C(p)] were measured at 24-, 48-, and 72 h post-infusion. PK modelling was performed to calculate area under the curve (AUC0 → 72 h) and half-life (t½). Creatinine, total bilirubin, and sample quality indices were retrieved from routine clinical laboratory analyses. Results: Mean (±SEM) MTX plasma concentrations were 36.09 ± 15.53 μmol/L, 0.93 ± 0.43 μmol/L, and 0.30 ± 0.07 μmol/L at 24, 48, and 72 h, respectively, with marked inter-patient variability. PK analysis showed a mean AUC0 → 72 h of 112.85 ± 34.09 h·μmol/L and a t½ of 17.15 ± 2.40 h. MTX C(p) and AUC0 → 72 h showed significant positive correlations with serum creatinine at all time points, confirming renal function as a major MTX clearance determinant. Age moderated the relationship at 72 h, with younger patients showing stronger correlations. Hepatic function measured by total bilirubin also correlated with MTX C(p) and AUC0 → 72 h at 48 and 72 h, especially in younger patients, suggesting a hepatic contribution to MTX variability. No associations were found between the PK parameters and lipemic, icterus, or haemolysis indices. Conclusions: These findings highlight the value of integrating renal and hepatic biomarkers into HDMTX drug monitoring protocols. Such biomarker-informed TDM may improve the safety and efficacy by identifying patients at risk of delayed clearance and toxicity, especially younger individuals or those with renal insufficiency.

β3-adrenergic receptors (β3-ARs) are increasingly recognized as modulators of tumor progression and treatment resistance across multiple cancer types. SR59230A, a β3-AR antagonist, has shown preclinical antitumor activity through mechanisms involving mitochondrial reactivation, reactive oxygen species (ROS) production, and antiangiogenic effects. Based on this premise, this study aimed to investigate the in vitro synergistic effects of SR59230A combined with standard chemotherapeutics or targeted therapies in various human cancer cell lines (glioblastoma, melanoma, triple-negative breast cancer, and anaplastic thyroid carcinoma) and endothelial cells (HUVECs). Cells were treated with SR59230A alone or in fixed-ratio combinations with temozolomide, paclitaxel, vemurafenib, lenvatinib, or sorafenib. Drug interactions were quantified using the Chou–Talalay method and validated with the Loewe additivity model. SR59230A exhibited dose-dependent antiproliferative activity, particularly in HUVECs and thyroid carcinoma cells. Synergistic effects were observed in all models, with the strongest synergy in A-2058 melanoma cells (SR59230A + vemurafenib), MDA-MB-231 breast cancer and 8505C thyroid carcinoma cells (SR59230A + paclitaxel), U-87 glioblastoma cells (SR59230A + temozolomide), and HUVECs (SR59230A + lenvatinib or sorafenib). Dose reduction index (DRI) values confirmed the potential to lower cytotoxic drug doses while preserving efficacy. These findings suggest that SR59230A may enhance the efficacy of conventional and targeted anticancer agents through multimodal mechanisms. The consistent synergistic effects across diverse tumor types support further investigation into the role of SR59230A, including its effects on β3-AR, as a promising strategy to overcome resistance and optimize cancer therapy.

Tafasitamab is a novel humanized anti-CD19 monoclonal antibody, designed for the treatment of B-cell malignancies. Our study aims to enhance the direct, non-immune-mediated, activity of tafasitamab (TAFA) with the combination of metronomic chemotherapy (mCHEMO), including vinorelbine (mVNR) and etoposide (mETO), in preclinical models of diffuse large B-cell lymphoma (DLBCL). In vitro, the 144 h exposure of thrice-weekly mVNR, daily mETO, and single-dose TAFA significantly inhibited the viability of human CD19+ DLBCL cell lines (i.e., Toledo, OCI-LY3, and SU-DHL10) in a concentration-dependent manner. In all cell lines, the concomitant treatment with TAFA and mVNR or mETO showed a marked synergism, except for TAFA + mETO on SU-DHL10 cells. The TAFA + mCHEMO treatments promoted apoptosis, and the TAFA + mVNR combination significantly inhibited, already after 24 h, the phosphorylation of GSK3α/β, mTOR, p70S6K, RPS6, and TSC2 proteins in DLBCL cells. TAFA significantly increased the VNR and ETO intracellular concentrations in all DLBCL cells after 24 h, except for ETO levels in SU-DHL10. The TAFA + mCHEMO treatment strongly reduced the ABCB1, ABCG2, and c-MYC gene expression in SU-DHL10 cells. In vivo, the TAFA + mVNR combination was well tolerated, significantly reduced the volumes of subcutaneous DLBCL masses, and increased the overall survival of mice affected by systemic DLBCL. We report additional mechanisms to enhance the direct activity of TAFA with mCHEMO synergistically in DLBCL cells in vitro and in vivo, suggesting the use of this combination schedule into future clinical trials.

Cancer treatment, particularly chemotherapy, requires balancing efficacy and toxicity. Although traditional monitoring methods can lead to suboptimal outcomes, emerging implantable chemical sensors can complement them by providing precise, real-time drug monitoring at tumor sites, although the technology remains in its early stages. Here, we introduce an ultrathin, bioresorbable implantable biosensor for real-time doxorubicin monitoring in vivo with high spatiotemporal resolution. The sensor amplifies the drug's fluorescence, enabling successful tracking of doxorubicin through the skin in live mice following intravenous injection. When paired with a reusable electronic patch, the biosensor facilitates seamless data collection and wireless transmission. A 3-month biocompatibility study, including systemic toxicity assessments, histological and blood analyses, confirms complete biodegradation with no observed toxicity. By directly measuring chemotherapeutic drug levels in tissues over time, our sensor enhances traditional monitoring methods, enabling clinicians to optimize dosing during cancer treatment and reduce the risk of locoregional recurrence following tumor removal.

Phenotypic plasticity is a key mechanism of metastatic progression and cancer therapy resistance. This hallmark of human malignancies is enabled by highly conserved epigenetic mechanisms that control gene expression. Functional alterations in DNA methylation and histone post-translational modifications have been extensively described as drivers of metastatic dissemination and therapy resistance. Pharmacological inhibitors of epigenetic enzymes can revert these alterations, thereby stopping cancer progression and counteracting the emergence of resistant clones. Despite promising pre-clinical evidence, the clinical implementation of epigenetic therapies in solid cancers has led to disappointing results. Several factors can explain these challenges, including the lack of rational combinations. Notably, response to epigenetic treatments can be heterogeneous and short-lived. A liquid biopsy technology that allows the measure of specific epigenetic alterations enables patient selection and therapy monitoring, leading to the development of precision epigenetic therapies. In this review, we discuss the state of the art of this emerging treatment modality, and we identify key challenges that need to be overcome to reach the full potential of this new therapeutic concept.

Cutaneous T-cell lymphomas (CTCLs) are a rare and heterogeneous subset of skin-localized, non-Hodgkin lymphomas. Our aim was to evaluate the in vitro antitumor activity of the multi-kinase inhibitor linifanib, either alone or in combination with metronomic vinorelbine (mVNR) or etoposide (mETO), on CTCL cells. In vitro proliferation assay and Luminex analysis showed that long-term, daily exposure of linifanib significantly inhibited the proliferation of the human CTCL cell line HH, in a concentration-dependent manner (IC50 = 48.4 ± 20.4 nM) and the phosphorylation of AKT/mTOR signaling pathway. The concomitant exposure of linifanib plus mVNR or mETO resulted in a strong synergism, with combination index values < 1. Linifanib significantly increased the VNR and ETO intracellular concentrations in HH cells, evaluated by UPLC-HRMS technology, and strongly reduced the ABCB1 and ABCG2 gene expression in HH. In conclusion, we reported a striking antitumor activity of daily, long-term linifanib and a clear synergistic effect when administered in combination with mCHEMO on CTCL cells, as a promising base for future clinical approaches in T-cell lymphomas.

Brain metastases (BM) from breast cancer are a significant clinical issue with a poor prognosis. New therapies targeting the tumor microenvironment are promising. Various types of neurotransmitter (NT) receptors (e.g., glutamatergic, dopaminergic, serotoninergic receptors) are expressed on breast cancer (BC) cells, modulating cell proliferation, survival, and migration. We propose a novel approach for BM treatment using NT receptor antagonists in combination with metronomic chemotherapy (mCHEMO) - the frequent and regular administration of drug doses, for extended time, without breaks - to block the pro-tumoral interaction between cancer cells and the brain microenvironment, increasing therapeutic efficacy and reducing toxicity. The Kaplan-Meier plotter website was used to assess the impact of the NT receptor gene expression of our interest on overall survival (OS) in metastatic BC patients. Real-Time PCR assessed NT receptor gene expression in monolayer and spheroid cultures of MCF-7 (ER+), MDA-MB-231 (triple-negative), and MDA-MB-361 (HER2+) BC cell lines. Proliferation assays were performed on BC cell lines exposed to metronomic temozolomide (TMZ) and NT receptor antagonists (memantine, ML00253764, SB-269970, clozapine) for 144h daily treatments. The synergism of the concomitant combinations between low-dose TMZ and NT receptor antagonists was evaluated by the combination index method.The Kaplan-Meier analysis showed that a higher level of NT receptors expression is associated with worse OS rates in metastatic BC, particularly with elevated GRIN1 and MC4R. Indeed, genes of NT receptors were highly expressed in BC cell lines. The MC4R antagonist ML00253764 was active on tumor cell lines with a strong antiproliferative effect on MDA-MB-231, showing IC50 value of 4.72 nM. The NMDA antagonist memantine was very effective on MDA-MB-361 with a concentration- and time-dependent effect (IC50 at 144h 168.1 nM), while the 5-HT7 receptor antagonist SB-269970 showed encouraging antiproliferative results on MCF-7 (IC50 9.61 nM). Metronomic TMZ decreased cell proliferation in tumor cell lines. The concomitant combinations of TMZ+ML00253764, TMZ+memantine, and TMZ+SB-269970 showed marked synergisms, notably on MDA-MB-361 cells. These findings build rational bases for the promising therapeutic approach, including NT receptor antagonists and metronomic TMZ, for breast cancer BM. Moreover, in vitro experiments demonstrated a key role of NT receptor on cell proliferation due to the activity of pharmacological antagonists alone or with mCHEMO. These results support further in vitro 3D investigation on multicellular spheroids and in vivo experiments on BM models to validate our pioneering therapeutic hypothesis, which could be easily translated into future clinical trials. Arianna Bandini, Paola Orlandi, Marta Banchi, Giulio Francia, Guido Bocci. Neurotransmitter receptor antagonists and metronomic chemotherapy: A promising novel approach for brain metastases in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 510.