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Abstract CAR− CD4+ T cell lymphopenia is an emerging issue following CAR‐T cell therapy. We analyzed the determinants of CD4+ T cell recovery and a possible association with survival in 31 consecutive patients treated with commercial CAR‐T for diffuse large B‐cell (DLBCL) or mantle cell lymphoma. Circulating immune subpopulations were characterized through multiparametric‐flow cytometry. Six‐month cumulative incidence of CAR− CD4+ T cell recovery (≥200 cells/μL) was 0.43 (95% confidence interval [CI]: 0.28–0.65). Among possible determinants of CD4+ T cell recovery, we recognized infusion of a 4‐1BB product (tisagenlecleucel, TSA) in comparison with a CD28 (axicabtagene/brexucabtagene, AXI/BRX) (hazard ratio [HR] [95% CI]: 5.79 [1.16–24.12] p = 0.016). Higher CD4+ T cell counts resulted with TSA at month‐1, ‐2 and ‐3. Moderate‐to‐severe infections were registered with prolonged CD4+ T cell lymphopenia. Early, month‐1 CD4+ T cell recovery was associated with a worse outcome in the DLBCL cohort, upheld in a multivariate regression model for overall survival (HR: 4.46 [95% CI: 1.12–17.71], p = 0.03). We conclude that a faster CAR− CD4+ T cell recovery is associated with TSA as compared to AXI/BRX. Month‐1 CAR− CD4+ T cell subset recovery could represent a “red flag” for CAR‐T cell therapy failure in DLBCL patients.

BackgroundNatural Killer cells (NKs) represent the innate counterpart of TCRαβ lymphocytes and are characterized by a high anti-tumor and an anti-viral cytotoxic activity. Recently, it has been demonstrated that NKs can express PD-1 as an additional inhibitory receptor. Specifically, PD-1 was identified on a subpopulation of terminally differentiated NKs from healthy adults with previous HCMV infection. So far it is unknown whether PD-1 appears during NK-cell development and whether this process is directly or indirectly related to HCMV infection.MethodsIn this study, we analyzed the expression and function of PD-1 on Cord Blood derived NKs (CB-NKs) on a large cohort of newborns through multiparametric cytofluorimetric analysis.ResultsWe identified PD-1 on CB-NKs in more than of half the newborns analyzed. PD-1 was present on CD56dim NKs, and particularly abundant on CD56neg NKs, but only rarely present on CD56bright NKs. Importantly, unlike in adult healthy donors, in CB-NKs PD-1 is co-expressed not only with KIR, but also with NKG2A. PD-1 expression was independent of HCMV mother seropositivity and occurs in the absence of HCMV infection/reactivation during pregnancy. Notably, PD-1 expressed on CB-NKs was functional and mediated negative signals when triggered.ConclusionTo our understanding, this study is the first to report PD-1 expression on CB derived NKs and its features in perinatal conditions. These data may prove important in selecting the most suitable CB derived NK cell population for the development of different immunotherapeutic treatments.

Natural killer (NK) cell-based adoptive immunotherapy in leukemia patients is an emerging field of interest based on clinical evidence of efficacy and safety. Elderly acute myeloid leukemia (AML) patients have been successfully treated with NK cells from HLA-haploidentical donors, especially when high amounts of alloreactive NK cells were infused. The aim of this study was comparing two approaches to define the size of alloreactive NK cells in haploidentical donors for AML patients recruited in two clinical trials with the acronym “NK-AML” (NCT03955848), and “MRD-NK”. The standard methodology was based on the frequency of NK cell clones capable of lysing the related patient-derived cells. The alternative approach consisted of the phenotypic identification of freshly derived NK cells expressing, as inhibitory receptors, only the inhibitory KIR(s) specific for the mismatched KIR-Ligand(s) (HLA-C1, HLA-C2, HLA-Bw4). However, in KIR2DS2+ donors and HLA-C1+ patients, the unavailability of reagents staining only the inhibitory counterpart (KIR2DL2/L3) may lead to an underestimated identification of the alloreactive NK cell subset. Conversely, in the case of HLA-C1 mismatch, the alloreactive NK cell subset could be overestimated due to the ability of KIR2DL2/L3 to recognize with low-affinity also HLA-C2. Especially in this context, the additional exclusion of LIR1-expressing cells might be relevant to refine the size of the alloreactive NK cell subset. We could also associate degranulation assays, using as effector cells IL-2 activated donor peripheral blood mononuclear cells (PBMC) or NK cells upon co-culture with the related patient target cells. The donor alloreactive NK cell subset always displayed the highest functional activity, confirming its identification accuracy by flow cytometry. Despite the phenotypic limitations and considering the proposed corrective actions, a good correlation was shown by the comparison of the two investigated approaches. In addition, the characterization of receptor expression on a fraction of NK cell clones revealed expected but also few unexpected patterns. Thus, in most instances, the quantification of phenotypically defined alloreactive NK cells from PBMC can provide data similar to the analysis of lytic clones, with several advantages, such as a shorter time to achieve the results and, perhaps, higher reproducibility/feasibility in many laboratories.

Surgical resection, chemotherapy and radiotherapy were, for many years, the only available cancer treatments. Recently, the use of immune checkpoint inhibitors and adoptive cell therapies has emerged as promising alternative. These cancer immunotherapies are aimed to support or harness the patient’s immune system to recognize and destroy cancer cells. Preclinical and clinical studies, based on the use of T cells and more recently NK cells genetically modified with chimeric antigen receptors retargeting the adoptive cell therapy towards tumor cells, have already shown remarkable results. In this review, we outline the latest highlights and progress in immunotherapies for the treatment of Diffuse Large B-cell Lymphoma (DLBCL) patients, focusing on CD19-targeted immunotherapies. We also discuss current clinical trials and opportunities of using immunotherapies to treat DLBCL patients.

Immune checkpoint inhibitors (CI) have demonstrated clinical activity in Hodgkin Lymphoma (HL) patients relapsing after autologous stem cell transplantation (ASCT), although only 20% complete response (CR) rate was observed. The efficacy of CI is strictly related to the host immune competence, which is impaired in heavily pre-treated HL patients. Here, we aimed to enhance the activity of early post-ASCT CI (nivolumab) administration with the infusion of autologous lymphocytes (ALI). Twelve patients with relapse/refractory (R/R) HL (median age 28.5 years; range 18-65), underwent lymphocyte apheresis after first line chemotherapy and then proceeded to salvage therapy. Subsequently, 9 patients with progressive disease at ASCT received early post-transplant CI supported with four ALI, whereas 3 responding patients received ALI alone, as a control cohort. No severe adverse events were recorded. HL-treated patients achieved negative PET scan CR and 8 are alive and disease-free after a median follow-up of 28 months. Four patients underwent subsequent allogeneic SCT. Phenotypic analysis of circulating cells showed a faster expansion of highly differentiated NK cells in ALI plus nivolumab-treated patients as compared to control patients. Our data show anti-tumor activity with good tolerability of ALI + CI for R/R HL and suggest that this setting may accelerate NK cell development/maturation and favor the expansion of the “adaptive” NK cell compartment in patients with HCMV seropositivity, in the absence of HCMV reactivation.

Abstract Background Prevention and treatment of metastatic breast cancer (BC) is an unmet clinical need. The retinoic acid derivative fenretinide (FeR) was previously evaluated in Phase I-III clinical trials but, despite its excellent tolerability and antitumor activity in preclinical models, showed limited therapeutic efficacy due to poor bioavailability. We recently generated a new micellar formulation of FeR, Bionanofenretinide (Bio-nFeR) showing enhanced bioavailability, low toxicity, and strong antitumor efficacy on human lung cancer, colorectal cancer, and melanoma xenografts. In the present study, we tested the effect of Bio-nFeR on a preclinical model of metastatic BC. Methods We used BC cell lines for in vitro analyses of cell viability, cell cycle and migratory capacity. For in vivo studies, we used HER2/neu transgenic mice (neuT) as a model of spontaneously metastatic BC. Mice were treated orally with Bio-nFeR and at sacrifice primary and metastatic breast tumors were analyzed by histology and immunohistochemistry. Molecular pathways activated in primary tumors were analyzed by immunoblotting. Stem cell content was assessed by flow cytometry, immunoblotting and functional assays such as colony formation ex vivo and second transplantation assay in immunocompromised mice. Results Bio-nFeR inhibited the proliferation and migration of neuT BC cells in vitro and showed significant efficacy against BC onset in neuT mice. Importantly, Bio-nFeR showed the highest effectiveness against metastatic progression, counteracting both metastasis initiation and expansion. The main mechanism of Bio-nFeR action consists of promoting tumor dormancy through a combined induction of antiproliferative signals and inhibition of the mTOR pathway. Conclusion The high effectiveness of Bio-nFeR in the neuT model of mammary carcinogenesis, coupled with its low toxicity, indicates this formulation as a potential candidate for the treatment of metastatic BC and for the adjuvant therapy of BC patients at high risk of developing metastasis.

Abstract Background The use of mobile applications helps improving self-management in adolescents with asthma. However, no evidence is available for children with preschool wheezing. In addition, we have no data on the reliability of medical history collected at visits. The first aim was to assess the feasibility of a smartphone app in the management of preschool wheezing; secondly we aimed to evaluate the reliability of anamnestic data collected during face-to-face medical interviews. Methods Children with recurrent wheezing, age between 25 and 72 months, were randomly assigned to the intervention group, provided with a smartphone app for symptoms monitoring and asthma attack treatment, or to the control group, with a written action plan. At follow-up medical history was collected and the asthma control test and a clinical questionnaire were completed. App acceptability was also explored. Respiratory symptoms, medication and utilization of healthcare resources were collected. Plus, medical information obtained from the paper questionnaires was compared with data daily recorded by the app. Results We enrolled 85 preschool children with recurrent wheezing: 43 assigned to the intervention and 42 to the control group. The average (SD) adherence to e-Diary compilation was 60 (15)%. The acceptance and usability of the intervention was favorable as 70% and 93% of participants in the intervention arm described the app as ‘’simple and intuitive’’ at Visit1 (after 3 months from enrollement) and Visit2 (3 months later than Visit1), respectively and 95% and 98% found it useful in symptoms management. There were no significant differences between the two groups in clinical outcomes. At Visit1, the cACT median score (IQR) was 23,5 (21–25) for the control group (42 patients) and 23 (21–24) for the intervention group (43 patients). At Visit2 (41 controls and 42 in the intervention group) it was 25 (24–25) and 24 (24–25), respectively. Secondary analysis of data from the intervention group showed higher incidence of daily symptoms recorded by the app in comparison with the paper questionnaire, suggesting that collection of retrospective medical history may not be completely reliable. Conclusions The smartphone app is usable and acceptable by families of preschool wheezers. Future controlled trial are needed to prove an impact on clinical outcomes or its efficacy in a telemedicine program. Finally a daily questionnaire could provide physicians with a more reliable clinical picture as reflected better daily asthma symptoms than the written retrospective questionnaire filled at clinical visit.

Abstract Background The integration of positron emission tomography (PET) and magnetic resonance imaging (MRI) holds promise for advancing diagnostic imaging capabilities. The METRICS project aims to develop cyclotron-driven production of 52Mn for PET/MRI imaging. Results Using the 52Cr(p,n)52Mn reaction, we designed chromium metal targets via Spark Plasma Sintering and developed a separation procedure for isolating 52Mn. Labeling tests were conducted with traditional chelators (i.e. S-2-(4-Isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid) and the 1.4-dioxa-8-azaspiro[4.5]decane-8- carbodithioate ligand to produce radioactive complexes suitable for PET/MRI applications. Our methodology yielded high-quality 52Mn suitable for PET radiopharmaceuticals and PET/MRI imaging. Preliminary studies on phantom imaging using microPET and clinical MRI demonstrated the efficacy of our approach. Conclusions The developed technology offers a promising avenue for producing 52Mn and enhancing PET/MRI imaging capabilities. Further in vivo investigations are warranted to evaluate the potential advantages of this hybrid imaging technique.

Abstract Vulnerability evaluation plays a key role in risk assessment and reduction and is essential for defining strategies for climate change adaptation and mitigation. In dealing with the safeguarding of cultural heritage at risk, we are still far from adopting and applying an agreed methodology for vulnerability assessment. With the aim to support practitioners, heritage managers, and policy and decision makers to undertake actions that address the protection of cultural heritage at risk, the methodology set up in the framework of the Interreg Central Europe STRENCH is illustrated and discussed here. Based on three major requirements (susceptibility, exposure, and resilience) and a continuous consultation with local stakeholders, the proposed methodology is applicable for evaluating the vulnerability of built heritage and cultural landscape exposed to hydrometeorological hazards, such as heavy rains, floods, and droughts. The results obtained through its validation on 15 case studies from seven Central European regions are shown to underline the strengths and limitations of the methodological approach. Iterative consultation with local stakeholders was fundamental for the definition of the criteria/subcriteria and related values for the assessment of the requirements. Application to further sites in other contexts would surely contribute to strengthening the reliability of the methodological approach.

Background: Ascites is common in advanced gastrointestinal cancers with peritoneal metastases (PM) and negatively impacts patient survival. No study to date has specifically evaluated the relationship between ascites, PM and survival outcomes in metastatic colorectal cancer (mCRC) and metastatic gastric cancer (mGC). Objectives: This study aims to investigate and elucidate the relationship between malignant ascites, PM and survival outcomes in both mCRC and mGC patients. Design: This is a retrospective analysis of prospectively collected clinical trial data of mCRC and mGC patients with PM. Methods: We performed two pooled analyses, firstly of two Italian randomized trials enrolling patients with mCRC eligible for systemic therapy (TRIBE2; VALENTINO), and secondly of gastric cancer and peritoneal metastasis (GCPM) patients who underwent bi-directional therapeutic treatment comprising systemic and peritoneal-directed therapies. Results: Of 900 mCRC patients, 39 (4.3%) had PM with malignant ascites. Compared to the group without PM, median progression-free and overall survival were significantly inferior in the ascites group (hazard ratio (HR) for progression-free survival (PFS) 1.68, 95% confidence interval (CI): 1.21–2.35, p = 0.007; HR for overall survival (OS) 2.14, 95% CI: 1.57–3.01, p

Abstract Synthetic cocaine analogs are designer drugs that recently emerged as non‐controlled substitutes for their parent drug. Among them, nitracaine is of particular concern for its psychoactive effect. In this work, we present a thorough characterization of the electrochemical behavior of nitracaine, with a final quantification performed by differential pulse voltammetry (DPV) in ethanol/lithium perchlorate 0.1 M. The selectivity of the method and reproducibility of results were assessed. LOQ of 0.3 μg mL−1 and a linear dynamic range of up to 400 μg mL−1 were obtained. In addition, recoveries from 85 % to 101 % were achieved on both simulated and real samples. For nitracaine analysis in urine, a clean‐up and a preconcentration step by solid phase extraction (SPE) using the adsorbent Florisil have been developed and optimized through the Design of Experiments (DoE) strategy, thus achieving an enrichment factor of 20.

RESUMO A pandemia de covid-19 revelou múltiplas dimensões da crise civilizatória e ecológica, destacando a falência do modelo de desenvolvimento capitalista hegemônico e a injusta concentração de riqueza global, gerando profundas desigualdades sociais. No nível local, a campanha ‘Cuidar é Resistir’ demonstrou que populações tradicionais podem oferecer respostas promissoras para o futuro. Por meio da coordenação de diversas ações, como arrecadação e distribuição de alimentos, compra de produtos locais e comunicação efetiva, essa campanha promoveu segurança sanitária, econômica e alimentar, além de reduzir desigualdades e pressões socioambientais. A participação das organizações da sociedade civil, das instituições públicas e do Sistema Único de Saúde foi fundamental nesse processo. A experiência da campanha sugere que o futuro diante da pandemia e da crise climática pode ser encontrado nos modos de vida das comunidades tradicionais, que podem inspirar novas formas de habitar o planeta, promovendo uma relação sustentável e solidária entre seres humanos e natureza. Ao aprender com o modo de vida tradicional e combinar suas práticas com as dimensões positivas do pensamento científico e tecnopolítico, baseado em abordagens solidárias e conhecimento colaborativo, é possível utilizar estratégias de governança viva para promover o Bem Viver.

This paper sheds light on the manufacturing processes, techniques, and materials used in the splendid illuminations of the oldest surviving copy of De Balneis Puteolanis, preserved at the Angelica Library in Rome (Ms. 1474). The codex is one of the masterpieces of mid-13th-century Italian-Southern illumination, traditionally referred to as the commission of Manfredi, son of Frederick II. The findings reported in the article result from the interdisciplinary study conducted in 2021–2023 in the framework of “Codex 4D: journey in four dimensions into the manuscript”, a multidisciplinary project involving many competences and dealing with art-historical studies on manuscripts, diagnostic and conservative analyses, scientific dissemination, storytelling, and public engagement. The considerations we present aims at increasing the knowledge of book artefacts while respecting their extraordinary complexity; data from non-invasive diagnostic investigations (X-ray fluorescence, Vis-NIR reflectance and Raman spectroscopies, hyperspectral imaging, and multi-band imaging techniques as ultraviolet, reflectography, and thermography), carried out in situ with portable instruments on the book, have been integrated with observations resulting from the historical-artistic study, and the reading of some ancient treatises on the production and use of the pigments and dyes employed in illumination.

Abstract Overall quality of radiomics research has been reported as low in literature, which constitutes a major challenge to improve. Consistent, transparent, and accurate reporting is critical, which can be accomplished with systematic use of reporting guidelines. The CheckList for EvaluAtion of Radiomics research (CLEAR) was previously developed to assist authors in reporting their radiomic research and to assist reviewers in their evaluation. To take full advantage of CLEAR, further explanation and elaboration of each item, as well as literature examples, may be useful. The main goal of this work, Explanation and Elaboration with Examples for CLEAR (CLEAR-E3), is to improve CLEAR’s usability and dissemination. In this international collaborative effort, members of the European Society of Medical Imaging Informatics−Radiomics Auditing Group searched radiomics literature to identify representative reporting examples for each CLEAR item. At least two examples, demonstrating optimal reporting, were presented for each item. All examples were selected from open-access articles, allowing users to easily consult the corresponding full-text articles. In addition to these, each CLEAR item’s explanation was further expanded and elaborated. For easier access, the resulting document is available at https://radiomic.github.io/CLEAR-E3/ . As a complementary effort to CLEAR, we anticipate that this initiative will assist authors in reporting their radiomics research with greater ease and transparency, as well as editors and reviewers in reviewing manuscripts. Relevance statement Along with the original CLEAR checklist, CLEAR-E3 is expected to provide a more in-depth understanding of the CLEAR items, as well as concrete examples for reporting and evaluating radiomic research. Key points • As a complementary effort to CLEAR, this international collaborative effort aims to assist authors in reporting their radiomics research, as well as editors and reviewers in reviewing radiomics manuscripts. • Based on positive examples from the literature selected by the EuSoMII Radiomics Auditing Group, each CLEAR item explanation was further elaborated in CLEAR-E3. • The resulting explanation and elaboration document with examples can be accessed at https://radiomic.github.io/CLEAR-E3/ . Graphical Abstract

Abstract Background In recent years, fibroblast activating protein (FAP), a biomarker overexpressed by cancer-associated fibroblasts, has emerged as one of the most promising biomarkers in oncology. Similarly, FAP overexpression has been detected in various fibroblast-mediated inflammatory conditions such as liver cirrhosis and idiopathic pulmonary fibrosis. Along this trajectory, FAP-targeted positron emission tomography (PET), utilizing FAP inhibitors (FAPi) labeled with positron emitters, has gained traction as a powerful imaging approach in both cancer and inflammation. However, PET represents a high-cost technology, and its widespread adoption is still limited compared to the availability of gamma cameras. To address this issue, several efforts have been made to explore the potential of [99mTc]Tc-FAPi tracers as molecular probes for imaging with gamma cameras and single photon emission computed tomography (SPECT). Main body Several approaches have been investigated for labeling FAPi-based compounds with 99mTc. Specifically, the mono-oxo, tricarbonyl, isonitrile, and HYNIC strategies have been applied to produce [99mTc]Tc-FAPi tracers, which have been tested in vitro and in animal models. Overall, these labeling approaches have demonstrated high efficiency and strong binding. The resulting [99mTc]Tc-FAPi tracers have shown high specificity for FAP-positive cells and xenografts in both in vitro and animal model studies, respectively. However, the majority of [99mTc]Tc-FAPi tracers have exhibited variable levels of lipophilicity, leading to preferential excretion through the hepatobiliary route and undesirable binding to lipoproteins. Consequently, efforts have been made to synthesize more hydrophilic FAPi-based compounds to improve pharmacokinetic properties and achieve a more favorable biodistribution, particularly in the abdominal region. SPECT imaging with [99mTc]Tc-FAPi has yielded promising results in patients with gastrointestinal tumors, demonstrating comparable or superior diagnostic performance compared to other imaging modalities. Similarly, encouraging outcomes have been observed in subjects with gliomas, lung cancer, breast cancer, and cervical cancer. Beyond oncological applications, [99mTc]Tc-FAPi-based imaging has been successfully employed in myocardial and idiopathic pulmonary fibrosis. Conclusions This overview focuses on the various radiochemical strategies for obtaining [99mTc]Tc-FAPi tracers, highlighting the main challenges encountered and possible solutions when applying each distinct approach. Additionally, it covers the preclinical and initial clinical applications of [99mTc]Tc-FAPi in cancer and inflammation.

Abstract Polycystic kidney disease (PKD) is a genetic disorder characterized by bilateral cyst formation. We showed that PKD cells and kidneys display metabolic alterations, including the Warburg effect and glutaminolysis, sustained in vitro by the enzyme asparagine synthetase (ASNS). Here, we used antisense oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD murine models and show that treatment leads to a drastic reduction of total kidney volume (measured by MRI) and a prominent rescue of renal function in the mouse. Mechanistically, the upregulation of an ATF4–ASNS axis in PKD is driven by the amino acid response (AAR) branch of the integrated stress response (ISR). Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO revealed major changes in the mutants, several of which are rescued by Asns silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine synthesis and proliferation in cystic epithelia. Notably, while several metabolic pathways were completely corrected by Asns-ASO, glycolysis was only partially restored. Accordingly, combining the glycolytic inhibitor 2DG with Asns-ASO further improved efficacy. Our studies identify a new therapeutic target and novel metabolic vulnerabilities in PKD.

The present paper aims to study the Wachau Valley in Austria as a representative Cultural Landscape under threat from extreme hydrometeorological hazards linked to climate change. The primary objective is to investigate the impacts and assess the vulnerability associated with the events of heavy rain and flooding. The methodology employed consists of an investigation of recorded past events impacting the Wachau; a vulnerability ranking system; a climate time series analysis based on earth observation products; and future hazard maps at territorial level, developed with outputs from regional and global climate models. The investigation we carried out provides a vulnerability assessment of two terraced areas with a surface of about 10,000 m2 in total, characterized by the presence of dry stone walls, with different state of conservation in the Municipality of Krems (Wachau). In addition, climate projections at territorial level for the extreme climate indices R20mm, R95pTOT, and R×5day—selected for investigating the likelihood of increases/decreases in events of heavy rain and large basin flooding—are provided, with a spatial resolution of ~12 km for the near and far future (2021–2050; 2071–2100) under stabilizing (RCP 4.5) and pessimistic (RCP 8.5) scenarios. The results indicate a general increase for the three indices in the studied areas during the far future under the pessimistic scenario, suggesting a heightened risk of heavy rain and flooding. These findings aim to inform policymakers and decision-makers in their development of strategies for safeguarding cultural heritage. Furthermore, they serve to assist local stakeholders in enhancing their understanding of prioritizing interventions related to preparedness, emergency response, and recovery.

INTRODUCTION: The postnatal activation of the hypothalamic-pituitary-gonadal (HPG) axis is usually known as 'minipuberty'. There are still open questions about its biological function and significance depending on sex, gestational age (GA) and birth weight (BW) with few available longitudinal data. METHODS: A single-centre, longitudinal study to quantify urinary follicle stimulating hormone (uFSH), luteinizing hormone (uLH) and testosterone (uTs) in male neonates. Neonates were enrolled and stratified into three subgroups: full-term boys appropriate for GA (FT AGA); FT boys with BW ≤3rd centile [FT small for gestational age (SGA)]; and preterm (PT) boys ≤33 weeks of GA. Urinary hormones were correlated to simultaneous auxological parameters, linear growth and external genitalia at scheduled time-points. RESULTS: Forty-six boys were recruited, with subgroup sizes FT AGA n=23, FT SGA n=11 and PT n=12. PT boys display a pulsatile pattern of urinary gonadotropins (uGns) with higher levels of uLH and a gradual increase of uTs. Testicular descent started from 29-32 weeks with the peak of uTs. During the first 12-months post-term age (PTA), FT AGA boys displayed a better linear growth (p

Objectives: To describe how SARS-CoV-2 infection at the time of delivery affected maternal and neonatal outcomes across four major waves of the COVID-19 pandemic in Italy. Methods: This is a large, prospective, nationwide cohort study collecting maternal and neonatal data in case of maternal peripartum SARS-CoV-2 infection between February 2020 and March 2022. Data were stratified across the four observed pandemic waves. Results: Among 5201 COVID-19-positive mothers, the risk of being symptomatic at delivery was significantly higher in the first and third waves (20.8-20.8%) than in the second and fourth (13.2-12.2%). Among their 5284 neonates, the risk of prematurity (gestational age

Background: Italy was significantly affected by the COVID-19 pandemic, experiencing multiple waves of infection following the sequential emergence of new variants. Understanding the transmission patterns and evolution of SARS-CoV-2 is vital for future preparedness. Methods: We conducted an analysis of viral genome sequences, integrating epidemiological and phylodynamic approaches, to characterize how SARS-CoV-2 variants have spread within the country. Results: Our findings indicate bidirectional international transmission, with Italy transitioning between importing and exporting the virus. Italy experienced four distinct epidemic waves, each associated with a significant reduction in fatalities from 2021 to 2023. These waves were primarily driven by the emergence of VOCs such as Alpha, Delta, and Omicron, which were reflected in observed transmission dynamics and effectiveness of public health measures. Conclusions: The changing patterns of viral spread and variant prevalence throughout Italy's pandemic response underscore the continued importance of flexible public health strategies and genomic surveillance, both of which are crucial for tracking the evolution of variants and adapting control measures effectively to ensure preparedness for future outbreaks.

Plasma-derived von Willebrand factor-containing factor VIII concentrates (pd-VWF/FVIII-C) are the mainstay of treatment in von Willebrand disease (VWD). Real-world data on efficacy and safety of these pd-VWF/FVIII-C are required. To retrospectively evaluate the efficacy and safety of pd-VWF/FVIII-C (Fanhdi® and Alphanate®, Grifols) in clinical practice in Italy. A multicentric, observational, retrospective study at 10 Italian centers was conducted. Eligible patients diagnosed with inherited VWD (ISTH criteria) were treated with either Fanhdi® or Alphanate® for bleeding episodes, prevention of surgical bleeding and secondary long-term prophylaxis (SLTP) according to clinical practice with medical records collected from January 2007 to December 2019. Efficacy/safety of pd-VWF/FVIII-C was assessed according to FDA-agreed objective criteria following regulatory procedures. Fifty-seven patients (M/F: 21/36) were enrolled in the study with the following VWD types: VWD1 (n = 29, 52%), VWD2A (n = 10, 18%), VWD2B (n = 7, 12%), VWD2M (n = 2, 4%), VWD2N (n = 1, 2%), VWD2 unclassified (n = 1, 2%), and VWD3 (n = 7, 12%). These pd-VWF/FVIII-C were used to manage 58 bleeding episodes (n = 24 patients), 100 surgeries (n = 47 patients), and 7 SLTP (n = 6 patients). Global clinical efficacy with these pd-VWF/FVIII-C was reported to be excellent/good in 85% of bleeding episodes, 98% of surgeries, and 100% of SLTP. As far as safety, no adverse-drug-related episodes, immunogenic or thrombotic events were reported. This study confirmed that Fanhdi® and Alphanate® were effective and safe in the management of bleeding episodes, the prevention of bleeding during surgeries and for SLTP in Italian patients with inherited VWD.

Elevated activity of retrotransposons is increasingly recognized to be implicated in a wide range of neurodegenerative and neurodevelopmental diseases, including Down syndrome (DS), which is the most common chromosomal condition causing intellectual disability globally. Previous research by our group has revealed that treatment with lamivudine, a reverse transcriptase inhibitor, improved neurobehavioral phenotypes and completely rescued hippocampal-dependent recognition memory in a DS mouse model, Ts65Dn. We hypothesized that retrotransposition rates would increase in the Ts65Dn mouse model, and lamivudine could block retrotransposons. We analyzed the differentially expressed long interspersed element-1 (LINE-1 or L1) mapping on MMU16 and 17, and showed for the first time that retrotransposition could be associated with Ts65Dn’s pathology, as misregulation of L1 was found in brain tissues associated with trisomy. In the cerebral cortex, 6 out of 26 upregulated L1s in trisomic treated mice were located in the telomeric region of MMU16 near Ttc3, Kcnj6, and Dscam genes. In the hippocampus, one upregulated L1 element in trisomic treated mice was located near the Fgd4 gene on MMU16. Moreover, two downregulated L1s rescued after treatment with lamivudine were located in the intronic region of Nrxn1 (MMU17) and Snhg14 (MMU7), implicated in a variety of neurodegenerative disorders. To gain further insight into the mechanism of this improvement, we here analyzed the gene expression profile in the hippocampus and cerebral cortex of trisomic mice treated and no-treated with lamivudine compared to their wild-type littermates. We found that treatment with lamivudine rescued the expression of 24% of trisomic genes in the cortex (located on mouse chromosome (MMU) 16 and 17) and 15% in the hippocampus (located in the human chromosome 21 orthologous regions), with important DS candidate genes such as App and Ets2, rescued in both regions.

Evidence suggests that neuroinflammation exhibits a dual role in the pathogenesis of major depressive disorder (MDD), both potentiating the onset of depressive symptoms and developing as a consequence of them. Our narrative review focuses on the role of the chemokine fractalkine (FKN) (also known as CX3CL1), which has gained increasing interest for its ability to induce changes to microglial phenotypes through interaction with its corresponding receptor (CX3CR1) that may impact neurophysiological processes relevant to MDD. Despite this, there is a lack of a clear understanding of the role of FKN in MDD. Overall, our review of the literature shows the involvement of FKN in MDD, both in preclinical models of depression, and in clinical studies of depressed patients. Preclinical studies (N = 8) seem to point towards two alternative hypotheses for FKN's role in MDD: a) FKN may drive pro-inflammatory changes to microglia that contribute towards MDD pathogenesis; or b) FKN may inhibit pro-inflammatory changes to microglia, thereby exerting a protective effect against MDD pathogenesis. Evidence for a) primarily derives from preclinical chronic stress models of depression in mice, whereas for b) from preclinical inflammation models of depression. Whereas, in humans, clinical studies (N = 4) consistently showed a positive association between FKN and presence of MDD, however it is not clear whether FKN is driving or moderating MDD pathogenesis. Future studies should aim for larger and more controlled clinical cohorts, in order to advance our understanding of FKN role both in the context of stress and/or inflammation.

In June 2022, a gastroenteritis outbreak occurred in a town in Northern Italy, possibly associated with the ingestion of norovirus from public drinking water. Noroviruses are highly infectious RNA viruses, with high stability in the environment. They are the primary cause of non-bacterial gastroenteritis worldwide, and despite the fact that the disease is mainly self-limiting, norovirus infection can lead to severe illness in the immunocompromised, the elderly and children. Immediately after the notification of the suspected norovirus outbreak, faecal specimens were collected from hospitalised patients, and water samples were collected from public drinking fountains in the affected area, to confirm the presence of norovirus. Norovirus was detected in 80 % (95 % CI 0.58–0.91) of the faecal specimens, and in 50 % (95 % CI 0.28–0.72) of the water samples using RT (reverse transcription) Real-time PCR. The identification of GII genotype in all samples confirmed public drinking water as the source of norovirus contamination. In addition, in one faeces and one water sample, the co-presence of genotypes GI and GII was detected. The strains were typed by sequencing, with most of them belonging to the genotype GII.3. Immediately after the confirmation of norovirus contamination in public drinking water, the local competent authorities applied safety measures, resulting in a decline in number of cases. Moreover, after the application of disinfection protocols in the water plant, the sampling was repeated with negative results for norovirus in the affected area. However, positive samples were found in the neighbouring area (prevalence 10.00 %, 95 % CI 0.02–0.40) and in the water spring (prevalence 50.00 %, 95 % CI 0.21–0.78), suggesting norovirus persistence and spread from the water source. The prompt identification of the source of contamination, and collaboration with the local authorities guided the implementation of proper procedures to control viral spread, resulting in the successful control of the outbreak.

Abstract Background Glioblastoma (GBM) is the most lethal primary brain tumor in adult, characterized by highly aggressive and infiltrative growth. The current therapeutic management of GBM includes surgical resection followed by ionizing radiations and chemotherapy. Complex and dynamic interplay between tumor cells and tumor microenvironment drives the progression and contributes to therapeutic resistance. Extracellular vesicles (EVs) play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment. Methods In this study, we isolated by ultracentrifugation EVs from GBM stem-like cell (GSC) lines and human microvascular endothelial cells (HMVECs) exposed or not to ionizing irradiation. After counting and characterization, we evaluated the effects of exposure of GSCs to EVs isolated from endothelial cells and vice versa. The RNA content of EVs isolated from GSC lines and HMVECs exposed or not to ionizing irradiation, was analyzed by RNA-Seq. Periostin (POSTN) and Filamin-B (FLNB) emerged in gene set enrichment analysis as the most interesting transcripts enriched after irradiation in endothelial cell-derived EVs and GSC-derived EVs, respectively. POSTN and FLNB expression was modulated and the effects were analyzed by in vitro assays. Results We confirmed that ionizing radiations increased EV secretion by GSCs and normal endothelial cells, affected the contents of and response to cellular secreted EVs. Particularly, GSC-derived EVs decreased radiation-induced senescence and promoted migration in HMVECs whereas, endothelial cell-derived EVs promoted tumorigenic properties and endothelial differentiation of GSCs. RNA-Seq analysis of EV content, identified FLNB and POSTN as transcripts enriched in EVs isolated after irradiation from GSCs and HMVECs, respectively. Assays performed on POSTN overexpressing GSCs confirmed the ability of POSTN to mimic the effects of endothelial cell-derived EVs on GSC migration and clonogenic abilities and transdifferentiation potential. Functional assays performed on HMVECs after silencing of FLNB supported its role as mediator of the effects of GSC-derived EVs on senescence and migration. Conclusion In this study, we identified POSTN and FLNB as potential mediators of the effects of EVs on GSC and HMVEC behavior confirming that EVs play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.

Abstract Background Multiple Breath washout (MBW) represents an important tool to detect early a possible pulmonary exacerbation especially in Cystic Fibrosis (CF) disease. Lung clearance index (LCI) is the most commonly reported multiple breath washout (MBW) index and in the last years was used as management measure for evaluation. Our aim was to analyze clinical utility of LCI index variability in pulmonary exacerbation in CF after intravenous (IV) antibiotic therapy. Methods A single-center study was conducted at CF Unit of Bambino Gesù Children’s Hospital among hospitalized > 3 years patients for pulmonary exacerbations and treated with antibiotic IV treatment for 14 days. MBW and spirometry were evaluated within 72 h of admission to hospital and at the end of hospitalization. Descriptive analysis was conducted and correlations between quantitative variables were investigated. Results Fifty-seven patients (M22/F35) with an average age 18.56 (± 8.54) years were enrolled. LCI2.5 was significantly reduced at the end of antibiotic treatment in both pediatric and adult populations with an average reduction of -6,99%; 37/57 patients denoted an improvement, 20/57 are stable or worsened in LCI2.5 values and 4/57 (7.02%) had a significant deterioration (> 15%) at end of treatment. On the contrary a significative elevation of FEV1 and FVC were found, respectively of + 7,30% and of + 5,46%. A positive good correlection among LCI 2.5 and Scond (rho = + 0,615, p = 0.000) and LCI 2.5 and Sacin (rho = + 0,649, p = 0.000) and a negative strong correlation between FEV1 and LCI 2.5 were found in post treatment period. A similar modification of LCI 2.5 and FEV1 was noticed in both adult and pediatric population. Conclusions LCI may have a role in the routine clinical care of both adult and pediatric CF patients as a good tool to assess response to IV antibiotic end-therapy in the same way as FEV1.

Objectives To investigate pregnancy outcomes in women with autoimmune rheumatic diseases (ARD) in the Italian prospective cohort study P-RHEUM.it.Methods Pregnant women with different ARD were enrolled for up to 20 gestational weeks in 29 Rheumatology Centres for 5 years (2018–2023). Maternal and infant information were collected in a web-based database.Results We analysed 866 pregnancies in 851 patients (systemic lupus erythematosus was the most represented disease, 19.6%). Maternal disease flares were observed in 135 (15.6%) pregnancies. 53 (6.1%) pregnancies were induced by assisted reproduction techniques, 61 (7%) ended in miscarriage and 11 (1.3%) underwent elective termination. Obstetrical complications occurred in 261 (30.1%) pregnancies, including 2.3% pre-eclampsia. Two cases of congenital heart block were observed out of 157 pregnancies (1.3%) with anti-Ro/SSA. Regarding treatments, 244 (28.2%) pregnancies were treated with glucocorticoids, 388 (44.8%) with hydroxychloroquine, 85 (9.8%) with conventional synthetic disease-modifying anti-rheumatic drugs and 122 (14.1%) with biological disease-modifying anti-rheumatic drugs. Live births were 794 (91.7%), mostly at term (84.9%); four perinatal deaths (0.5%) occurred. Among 790 newborns, 31 (3.9%) were small-for-gestational-age and 169 (21.4%) had perinatal complications. Exclusive maternal breast feeding was received by 404 (46.7%) neonates. The Edinburgh Postnatal Depression Scale was compiled by 414 women (52.4%); 89 (21.5%) scored positive for emotional distress.Conclusions Multiple factors including preconception counselling and treat-to-target with pregnancy-compatible medications may have contributed to mitigate disease-related risk factors, yielding limited disease flares, good pregnancy outcomes and frequency of complications which were similar to the Italian general obstetric population. Disease-specific issues need to be further addressed to plan preventative measures.

Abstract Fibrocystin/Polyductin (FPC), encoded by PKHD1, is associated with autosomal recessive polycystic kidney disease (ARPKD), yet its precise role in cystogenesis remains unclear. Here we show that FPC undergoes complex proteolytic processing in developing kidneys, generating three soluble C-terminal fragments (ICDs). Notably, ICD15, contains a novel mitochondrial targeting sequence at its N-terminus, facilitating its translocation into mitochondria. This enhances mitochondrial respiration in renal epithelial cells, partially restoring impaired mitochondrial function caused by FPC loss. FPC inactivation leads to abnormal ultrastructural morphology of mitochondria in kidney tubules without cyst formation. Moreover, FPC inactivation significantly exacerbates renal cystogenesis and triggers severe pancreatic cystogenesis in a Pkd1 mouse mutant Pkd1 V/V in which cleavage of Pkd1-encoded Polycystin-1 at the GPCR Proteolysis Site is blocked. Deleting ICD15 enhances renal cystogenesis without inducing pancreatic cysts in Pkd1 V/V mice. These findings reveal a direct link between FPC and a mitochondrial pathway through ICD15 cleavage, crucial for cystogenesis mechanisms.

A fundamental step in the best preservation of heritage materials is investigating their chemical and physical characteristics, and understanding how they interact with the surrounding environment. For this reason, the stone masonry of the monumental UNESCO site of Panamá Viejo was subjected to a thorough study to characterize the materials and evaluate their state of conservation. Additionally, potential outcrops were explored in order to identify sites for the supply of raw materials. The methodological approach encompassed mineralogical-petrographic investigations using stereomicroscopy, polarized light microscopy (PLM), X-ray powder diffraction (XRPD), scanning electron microscopy and energy dispersive X-ray spectrometry (ESEM-EDS) and X-ray fluorescence (XRF). The results revealed that the masonries primarily consists of polygenic breccia, basaltic andesite, rhyodacite, tuffite, and rhyolite. In addition, at the potential quarries, breccia and basaltic andesite were identified. The porous structure was studied using mercury intrusion porosimetry (MIP). Possible issues due to salt dissolution/crystallization cycles were considered. Consequently, ion chromatography (IC) was conducted on samples exhibiting alteration patinas to study the presence of soluble salts. In assessing the state of conservation, prevalent forms of deterioration included biological colonization, detachments, material loss, potential salt weathering, and chromatic alteration. In conclusion, the outcomes of this work provide a valuable resource for the current and future preservation of this site.

64Cu is gaining recognition not only for its diagnostic capabilities in nuclear medical imaging but also for its therapeutic and theranostic potential. The simultaneous βˉ and Auger emissions of 64Cu can be utilized to induce a therapeutic effect on cancerous lesions. The finding of the exceptional biodistribution characteristics of the radionuclide 64Cu, when administered as basic copper ions, has highlighted its potential therapeutic application in cancer treatment. Preclinical and clinical research on the effectiveness of [64Cu]CuCl2 as a theranostic radiopharmaceutical has commenced only in the past decade. Current clinical studies are increasingly demonstrating the high specificity and uptake of [64Cu]Cu2+ by malignant tissues during early cancer progression, indicating its potential for early cancer diagnosis across various organs. This short review aims to present the latest preclinical studies involving [64Cu]CuCl2, offering valuable insights for researchers planning new in vitro and in vivo studies to explore the theranostic potential of [64Cu]Cu2+.

Cystic fibrosis (CF) is a multisystem disorder characterized by progressive respiratory deterioration, significantly impacting both quality of life and survival. Over the years, lung ultrasound (LUS) has emerged as a promising tool in pediatric respiratory due to its safety profile and ease at the bedside. In the era of highly effective CF modulator therapies and improved life expectancy, the use of non-ionizing radiation techniques could become an integral part of CF management, particularly in the pediatric population. The present review explores the potential role of LUS in CF management based on available data, analyzing all publications from January 2015 to January 2024, focusing on two key areas: LUS in CF pulmonary exacerbation and its utility in routine clinical management. Nonetheless, LUS exhibits a robust correlation with computed tomography (CT) scans and serves as an additional, user-friendly imaging modality in CF management, demonstrating high specificity and sensitivity in identification, especially in consolidations and atelectasis in the CF population. Due to its ability, LUS could be an instrument to monitor exacerbations with consolidations and to establish therapy duration and monitor atelectasis over time or their evolution after therapeutic bronchoalveolar lavage. On the basis of our analysis, sufficient data emerged showing a good correlation between LUS score and respiratory function tests. Good sensitivity and specificity of the methodology have been found in rare CF pulmonary complications such as effusion and pneumothorax. Regarding its use in follow-up management, the literature reports a moderate correlation between LUS scores and the type, extent, and CT severity score of bronchiectasis. A future validation of ultrasound scores specifically in CF patients could improve the use of LUS to identify pulmonary exacerbations and monitor disease progression. However, further research is needed to comprehensively establish the role of LUS in the CF population, particularly in elucidating its broader utility and long-term impact on patient care.

The present study investigates the possible use of manganese (Mn)-based liposomal formulations for diagnostic applications in imaging techniques such as magnetic resonance imaging (MRI), with the aim of overcoming the toxicity limitations associated with the use of free Mn2+. Specifically, anionic liposomes carrying two model Mn(II)-based compounds, MnCl2 (MC) and Mn(HMTA) (MH), were prepared and characterised in terms of morphology, size, loading capacity, and in vitro activity. Homogeneous dispersions characterised mainly by unilamellar vesicles were obtained; furthermore, no differences in size and morphology were detected between unloaded and Mn-loaded vesicles. The encapsulation efficiency of MC and MH was evaluated on extruded liposomes by means of ICP-OES analysis. The obtained results showed that both MC and MH are almost completely retained by the lipid portion of liposomes (LPs), with encapsulation efficiencies of 99.7% for MC and 98.8% for MH. The magnetic imaging properties of the produced liposomal formulations were investigated for application in a potential preclinical scenario by collecting magnetic resonance images of a phantom designed to compare the paramagnetic contrast properties of free MC and MH compounds and the corresponding manganese-containing liposome dispersions. It was found that both LP-MC and LP-MH at low concentrations (0.5 mM) show better contrast (contrast-to-noise ratios of 194 and 209, respectively) than solutions containing free Mn at the same concentrations (117 and 134, respectively) and are safe to use on human cells at the selected dose. Taken together, the results of this comparative analysis suggest that these liposome-containing Mn compounds might be suitable for diagnostic purposes.

The seed habit is the most complex and successful method of sexual reproduction in vascular plants. It represents a remarkable moment in the evolution of plants that afterward spread on land. In particular, seed size had a pivotal role in evolutionary success and agronomic traits, especially in the field of crop domestication. Given that crop seeds constitute one of the primary products for consumption, it follows that seed size represents a fundamental determinant of crop yield. This adaptative feature is strictly controlled by genetic traits from both maternal and zygotic tissues, although seed development and growth are also affected by environmental cues. Despite being a highly exploited topic for both basic and applied research, there are still many issues to be elucidated for developmental biology as well as for agronomic science. This review addresses a number of open questions related to cues that influence seed growth and size and how they influence seed germination. Moreover, new insights on the genetic–molecular control of this adaptive trait are presented.

Bats are well-known to be natural reservoirs of various zoonotic coronaviruses, which have caused outbreaks of severe acute respiratory syndrome (SARS) and the COVID-19 pandemic in 2002 and 2019, respectively. In late 2020, two new Sarbecoviruses were found in Russia, isolated in Rhinolophus bats, i.e., Khosta-1 in R. ferrumequinum and Khosta-2 in R. hipposideros. The potential danger associated with these new species of Sarbecovirus is that Khosta-2 has been found to interact with the same entry receptor as SARS-CoV-2. Our multidisciplinary approach in this study demonstrates that Khosta-1 and -2 currently appear to be not dangerous with low risk of spillover, as confirmed by prevalence data and by phylogenomic reconstruction. In addition, the interaction between Khosta-1 and -2 with ACE2 appears weak, and furin cleavage sites are absent. While the possibility of a spillover event cannot be entirely excluded, it is currently highly unlikely. This research further emphasizes the importance of assessing the zoonotic potential of widely distributed batborne CoV in order to monitor changes in genomic composition of viruses and prevent spillover events (if any).

This review has the purpose of illustrating schematically and comprehensively the key concepts for the beginner who approaches chest radiology for the first time. The approach to thoracic imaging may be challenging for the beginner due to the wide spectrum of diseases, their overlap, and the complexity of radiological findings. The first step consists of the proper assessment of the basic imaging findings. This review is divided into three main districts (mediastinum, pleura, focal and diffuse diseases of the lung parenchyma): the main findings will be discussed in a clinical scenario. Radiological tips and tricks, and relative clinical background, will be provided to orient the beginner toward the differential diagnoses of the main thoracic diseases.

Objectives Paediatric Sjögren’s syndrome (pSS) is a rare chronic autoimmune disorder, characterised by inflammation of exocrine glands. B cell hyperactivation plays a central role in adult-onset Sjogren. This study was designed to analyse B cell and T cell phenotype, levels of BAFF, and selection of autoreactive B cells in patients with pSS.Methods A total of 17 patients diagnosed with pSS and 13 healthy donors (controls) comparable for age were enrolled in the study. B cell and T cell subsets and frequency of autoreactive B cells in peripheral blood were analysed by flow cytometry. Levels of BAFF were analysed by ELISA.Results The relative frequency of total B cells, transitional, naïve and switched memory B cells was similar between pSS patients and controls. In patients with pSS, we observed a reduction in the frequency of unswitched memory B cells, an increased frequency of atypical memory B cells and an expansion of PD1hi CXCR5− T peripheral helper cells. Levels of BAFF were higher in patients with pSS compared with controls and correlated with serum levels of total IgG and titres of anti-Ro antibodies. The frequency of autoreactive B cells in the transitional, unswitched memory and plasmablast compartment was significantly higher in pSS patients than in controls.Conclusions Our results point to a hyperactivation of B cells in pSS. Current therapies do not seem to affect B cell abnormalities, suggesting that novel therapies targeting specifically B cell hyperactivation need to be implemented for paediatric patients.

The paper offers a multi-focused reflection on the relationship between the evolution of the archive and the issues of access and knowledge acquisition. The authors recognize the digital archive as a resource that, from accessibility to information, through the empowerment of individuals, makes people autonomous in their own construction of knowledge. Briefly reconstructing the evolutionary events of the archive – from a private repository of documents to a platform for online content sharing – the research, through the presentation of some case studies, highlights the main factors that have characterized the process of the progressive opening of the archive towards inclusion: the opening process on contents, collected and organized to reach extended audiences by increasing social, political and cultural impact, and the opening process on user experience on which the design discipline works through interaction and experience practices.

BackgroundRetreatment with anti-EGFR monoclonal antibodies is a promising strategy in patients with RAS/BRAF wild-type (wt) metastatic colorectal cancer (mCRC) who achieved benefit from previous anti-EGFR exposure upon exclusion of mutations in RAS/BRAF genes according to circulating tumor DNA (ctDNA) analysis by means of liquid biopsy (LB). This treatment approach is now being investigated in the randomized phase II trial PARERE (NCT04787341). We here present preliminary findings of molecular screening.MethodsPatients with RAS/BRAFV600E wt mCRC according to tissue genotyping who benefited from previous anti-EGFR-based treatment (fluoropyrimidines, oxaliplatin, irinotecan, and antiangiogenics) and then experienced disease progression to EGFR targeting were eligible for screening in the PARERE trial. The next-generation sequencing (NGS) panel Oncomine™ was employed for ctDNA testing.ResultsA total of 218 patients underwent LB, and ctDNA sequencing was successful in 201 of them (92%). RAS/BRAFV600E mutations were found in 68 (34%) patients and were mainly subclonal (median variant allele fraction [VAF] for KRAS, NRAS, and BRAF mutant clones: 0.52%, 0.62%, and 0.12%, respectively; p = 0.01), with KRASQ61H being the most frequently detected (31%). Anti-EGFR-free intervals did not predict ctDNA molecular status (p = 0.12). Among the 133 patients with RAS/BRAFV600E wt tumors according to LB, 40 (30%) harbored a mutation in at least another gene potentially implied in anti-EGFR resistance, mainly with subclonal expression (median VAF, 0.56%). In detail, alterations in PIK3CA, FBXW7, GNAS, MAP2K, ERBB2, BRAF (class I and II non-BRAFV600E), SMAD, EGFR, AKT1, and CTNNB1 occurred in 13%, 8%, 7%, 3%, 2%, 2%, 1%, 1%, 1%, and 1% cases, respectively. Co-mutations were detected in 13 (33%) out of 40 patients.ConclusionsThis is the largest prospective cohort of mCRC patients screened with LB for anti-EGFR retreatment in a randomized study. ctDNA genotyping reveals that at least one out of three patients candidate for retreatment should be excluded from this therapy, and other potential drivers of anti-EGFR resistance are found in approximately one out of three patients with RAS/BRAFV600E wt ctDNA.