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Background: High neutrophil-to-lymphocyte ratio (NLR) is a poor prognostic factor in metastatic colorectal cancer (mCRC). Here we provide final results of CAVE mCRC trial, of cetuximab plus avelumab rechallenge in chemo-refractory mCRC patients and investigated the predictive role of NLR. Methods: All the 77 patients enrolled were included in the analysis. A cut-off of 3 was used to correlate baseline NLR with with overall survival (OS) and with progression free survival (PFS), in intention to treat (ITT) and in circulating tumor DNA (ctDNA) RAS/BRAF Wild Type (WT) patients. Results: In ITT population, NLR

Rechallenge with epidermal growth factor (EGFR) inhibitors represents a promising therapeutic strategy in patients with refractory RAS/BRAF wild-type metastatic colorectal cancer (mCRC). The maximal benefit is observed in patients without resistance mutation at the baseline plasma circulating tumor DNA (ctDNA) evaluation. In the CAVE and VELO clinical trials, 1 out of 4 patients had ctDNA RAS/BRAF mutant disease at pretreatment liquid biopsy assessment. There was no direct association between the length of anti-EGFR drug-free interval and the presence of plasma ctDNA RAS/BRAF mutations at pretreatment liquid biopsy analysis. Interestingly, even the disappearance of mutant clones was time-dependent, and resistance mutations were found at liquid biopsy analysis in approximately 15% of patients after 18 or more months of anti-EGFR drug-free window. These results support the use of liquid biopsy to appropriately select amenable patients to EGFR inhibitor rechallenge. Rechallenge with anti-EGFR drugs represents a promising strategy in refractory RAS/BRAF wild-type (WT) metastatic colorectal cancer (mCRC). We performed the pooled analysis of the CAVE and VELO studies to evaluate the percentage of patients with WT circulating tumor DNA (ctDNA) tumors and the association of mutational status with time from the last anti-EGFR drug administration. At baseline, 97/129 patients had RAS/BRAF WT plasma ctDNA, while 32/129 had RAS/BRAF mutated plasma ctDNA. Median anti-EGFR drug-free interval was 10.6 (CI 95%, 8.9-13.4) months in the plasma RAS/BRAF mutant group as compared to 13.0 (CI 95%, 11.1-16.6) months in RAS/BRAF WT group (p = 0.169). To investigate the time window of the RAS/BRAF mutant cancer cell clone disappearance, descriptive analysis using different time points was performed. No difference in the proportion of patients whose baseline plasma ctDNA was RAS/BRAF WT or mutated was found between 4 and 18 months since the last administration of anti-EGFR drugs. In contrast, 38/44 of patients with anti-EGFR drug-free interval of 18 months or more displayed a ctDNA RAS/BRAF WT status. Taken together, these results shows that the length of anti-EGFR free interval is not a sufficient criterion for patient selection, supporting the role of liquid biopsies for improving treatment efficacy.

Background: Hypertension management in older patients represents a challenge, particularly when hospitalized. Objective: The objective of this study is to investigate the determinants and related outcomes of antihypertensive drug prescription in a cohort of older hospitalized patients. Methods: A total of 5671 patients from REPOSI (a prospective multicentre observational register of older Italian in-patients from internal medicine or geriatric wards) were considered; 4377 (77.2%) were hypertensive. Minimum treatment (MT) for hypertension was defined according to the 2018 ESC guidelines [an angiotensin-converting-enzyme-inhibitor (ACE-I) or an angiotensin-receptor-blocker (ARB) with a calcium-channel-blocker (CCB) and/or a thiazide diuretic; if >80 years old, an ACE-I or ARB or CCB or thiazide diuretic]. Determinants of MT discontinuation at discharge were assessed. Study outcomes were any cause rehospitalization/all cause death, all-cause death, cardiovascular (CV) hospitalization/death, CV death, non-CV death, evaluated according to the presence of MT at discharge. Results: Hypertensive patients were older than normotensives, with a more impaired functional status, higher burden of comorbidity and polypharmacy. A total of 2233 patients were on MT at admission, 1766 were on MT at discharge. Discontinuation of MT was associated with the presence of comorbidities (lower odds for diabetes, higher odds for chronic kidney disease and dementia). An adjusted multivariable logistic regression analysis showed that MT for hypertension at discharge was associated with lower risk of all-cause death, all-cause death/hospitalization, CV death, CV death/hospitalization and non-CV death. Conclusions: Guidelines-suggested MT for hypertension at discharge is associated with a lower risk of adverse clinical outcomes. Nevertheless, changes in antihypertensive treatment still occur in a significant proportion of older hospitalized patients.

Current therapies for chemo-refractory metastatic colorectal cancer (mCRC) have limited efficacy. Rechallenge with epidermal growth factor receptor (EGFR) inhibitors in RAS wild-type (WT) mCRC could be valuable in this setting. In VELO, a randomized two-arm phase 2 trial, anti-EGFR monoclonal antibody panitumumab plus standard-of-care trifluridine/tipiracil (31 patients, arm B) was compared to trifluridine/tipiracil (31 patients, arm A) as third-line therapy (ClinicalTrials.gov Identifier NCT05468892). Primary endpoint, progression-free survival (PFS), was met. Median PFS was 4.0 months in arm B versus 2.5 months in arm A [hazard ratio (HR): 0.48; 95% CI 0.28–0.82; P = 0.007]. Baseline plasma RAS/BRAF WT circulating tumor DNA identified patients obtaining prolonged clinical benefit with panitumumab plus trifluridine/tipiracil as compared to trifluridine/tipiracil with PFS rates at 6 months of 38.5% versus 13% and at 12 months of 15.4% versus 0%, respectively. These findings warrant further development for liquid biopsy-guided anti-EGFR rechallenge combination strategies in chemo-refractory RAS WT mCRC.

The Na+/Ca2+ exchanger 1 (NCX1) participates in the maintenance of neuronal Na+ and Ca2+ homeostasis, and it is highly expressed at synapse level of some brain areas involved in learning and memory processes, including the hippocampus, cortex, and amygdala. Furthermore, NCX1 increases Akt1 phosphorylation and enhances glutamate-mediated Ca2+ influx during depolarization in hippocampal and cortical neurons, two processes involved in learning and memory mechanisms. We investigated whether the modulation of NCX1 expression/activity might influence learning and memory processes. To this aim, we used a knock-in mouse overexpressing NCX1 in hippocampal, cortical, and amygdala neurons (ncx1.4over) and a newly synthesized selective NCX1 stimulating compound, named CN-PYB2. Both ncx1.4over and CN-PYB2-treated mice showed an amelioration in spatial learning performance in Barnes maze task, and in context-dependent memory consolidation after trace fear conditioning. On the other hand, these mice showed no improvement in novel object recognition task which is mainly dependent on non-spatial memory and displayed an increase in the active phosphorylated CaMKIIα levels in the hippocampus. Interestingly, both of these mice showed an increased level of context-dependent anxiety. Altogether, these results demonstrate that neuronal NCX1 participates in spatial-dependent hippocampal learning and memory processes.

Non-small-cell lung cancer (NSCLC) is the second most diagnosed type of malignancy and the first cause of cancer death worldwide. Despite recent advances, the treatment of choice for NSCLC patients remains to be chemotherapy, often showing very limited effectiveness with the frequent occurrence of drug-resistant phenotype and the lack of selectivity for tumor cells. Therefore, new effective and targeted therapeutics are needed. In this context, short RNA-based therapeutics, including Antisense Oligonucleotides (ASOs), microRNAs (miRNAs), short interfering (siRNA) and aptamers, represent a promising class of molecules. ASOs, miRNAs and siRNAs act by targeting and inhibiting specific mRNAs, thus showing an improved specificity compared to traditional anti-cancer drugs. Nucleic acid aptamers target and inhibit specific cancer-associated proteins, such as “nucleic acid antibodies”. Aptamers are also able of receptor-mediated cell internalization, and therefore, they can be used as carriers of secondary agents giving the possibility of producing very highly specific and effective therapeutics. This review provides an overview of the proposed applications of small RNAs for NSCLC treatment, highlighting their advantageous features and recent advancements in the field.

129 Background: Rechallenge therapy with epidermal growth factor receptor (EGFR) inhibitors in chemo-refractory metastatic colorectal cancer (mCRC) is an emerging therapeutic approach. Trifluridine-tipiracil is approved for treatment of chemo-refractory mCRC patients. Methods: Chemo-refractory RAS WT mCRC patients, that had a major response (partial or complete response) to first-line chemotherapy plus an anti-EGFR monoclonal antibody and had an-anti-EGFR drug-free interval during second-line therapy of four or more months were randomized in a phase II trial to assess the addition of the anti-EGFR monoclonal antibody panitumumab to trifluridine-tipiracil as third-line rechallenge therapy. The primary endpoint was progression free survival (PFS). Baseline plasma was analyzed for circulating free tumor (ct) DNA by using Biocartis Idylla platform to detect mutations in KRAS, NRAS, BRAF ( V600E) and EGFRextracellular domain ( S492R). In 24 patients with baseline RAS/BRAF wild type (WT) ctDNA, Foundation One liquid CDx (324 gene profiling) was also performed before treatment and at disease progression. Results: 62 patients were treated with trifluridine-tipiracil (31 patients, arm A) or with trifluridine-tipiracil plus panitumumab (31 patients, arm B). As of September 16, 2022, 1 patient in arm A and 2 patients in arm B were on treatment. The primary endpoint was met. Median PFS (mPFS) was 4 months in arm B versus 2.5 months in arm A [hazard ratio (HR): 0.48; 95% CI 0.28-0.82; P = 0.007]. Baseline plasma RAS/BRAF WT ctDNA was found in 23/31 patients in Arm A and in 26/31 patients in Arm B. In this group, mPFS was 4.5 months in Arm B versus 2.6 months in Arm A (HR: 0.48; 95% CI 0.26-0.89; P = 0.019). Disease control (major responses plus stable disease) was higher for patients in Arm B compared to Arm A (81% versus 48%), whereas disease progression was the best response in 19% versus 52% patients, respectively. PFS rates at 6 and 12 months were 38.5% and 15.4% in arm B versus 13% and 0% in arm A. At disease progression, Foundation One liquid CDx detected several mutations within the EGFR pathway, which could correlate with cancer cell resistance to panitumumab. Conclusions: This is the first prospective randomized trial which evaluated anti-EGFR monoclonal antibody (panitumumab) in addition to standard of care (trifluridine-tipiracil) as third-line rechallenge therapy in chemo-refractory RASWT mCRC patients. Baseline liquid biopsy allows selection of RAS/BRAF WT ctDNA patients who could have a relevant clinical benefit. Clinical trial information: NCT05468892 .

Gut microbiota is involved in immune modulation and immune checkpoint inhibitors (ICIs) efficacy. Single-arm phase II CAVE-mCRC and CAVE-LUNG clinical trials investigated cetuximab + avelumab combination in RAS wild-type (WT) metastatic colorectal cancer (mCRC) and chemo-refractory nonsmall cell lung cancer (NSCLC) patients, respectively. A comprehensive gut microbiota genetic analysis was done in basal fecal samples of 14 patients from CAVE-mCRC trial with circulating tumor DNA (ctDNA) RAS/BRAF WT and microsatellite stable (MSS) disease. Results were validated in a cohort of 10 patients from CAVE-Lung trial. 16S rRNA sequencing revealed 23 027 bacteria species in basal fecal samples of 14 patients from CAVE-mCRC trial. In five long-term responding patients (progression-free survival [PFS], 9-24 months) significant increases in two butyrate-producing bacteria, Agathobacter M104/1 (P=.018) and Blautia SR1/5 (P=.023) were found compared to nine patients with shorter PFS (2-6months). A significantly better PFS was also observed according to the presence or absence of these species in basal fecal samples. For Agathobacter M104/1, median PFS (mPFS) was 13.5months (95% confidence interval [CI], 6.5-20.5months) vs 4.6months (95% CI, 1.8-7.4months); P=.006. For Blautia SR1/5, mPFS was 5.9months (95% CI, 2.2-9.7months) vs 3.6months (95% CI, 3.3-4.0months); P=.021. Similarly, in CAVE-Lung validation cohort, Agathobacter M104/1 and Blautia SR1/5 expression were associated with PFS according to their presence or absence in basal fecal samples. Agathobacter and Blautia species could be potential biomarkers of outcome in mCRC, and NSCLC patients treated with cetuximab + avelumab. These findings deserve further investigation.

La recensione passa in rassegna i sedici contributi al volume "Kings and Clowns. Il (non) senso del tragico" a cura di R. Ciocca e B. Del Villano, focalizzandosi sul portato trasgressivo e propulsivo del tragicomico nel sistema dei generi letterari e sulla sua capacità unica di farsi espressione di anti-modelli identitari e contro-narrazioni ideologiche.

The single-arm phase II CAVE mCRC trial evaluated the combination of cetuximab plus avelumab as rechallenge strategy in RAS wild-type (WT) metastatic colorectal cancer (mCRC) patients, with clinical response to first-line anti-EGFR-based chemotherapy, who progressed and received a subsequent line of therapy. The correlation of skin toxicity (ST) and different clinico-molecular variables with overall survival (OS), progression-free survival (PFS) and response rate (RR) was assessed at univariate and multivariate analysis. A total of 33/77 (42.9%) patients experienced grade 2–3 ST and displayed median OS (mOS) of 17.8 months (CI 95%, 14.9–20.6), whereas 44/77 (57.1%) patients with grade 0–1 ST exhibited mOS of 8.2 months (CI 95%, 5.5–10.9), (hazard ratio (HR), 0.51, CI 95%, 0.29–0.89, p = 0.019). Median PFS (mPFS) was 4.6 months (CI 95%, 3.4–5.7) in patients with grade 2–3 ST, compared to patients with grade 0–1 ST with mPFS of 3.4 months (CI 95%, 2.7–4.1, HR, 0.49, CI 95%, 0.3–0.8, p = 0.004). Grade 2–3 ST (HR, 0.51, p = 0.019) and RAS/BRAF/EGFR WT circulating tumor DNA (ctDNA) (HR, 0.50, CI 95%, 0.27–0.9, p = 0.019) had a statistically significant effect on OS at univariate analysis. At the multivariate analysis, RAS/BRAF/EGFR WT ctDNA status maintained statistical significance (HR, 0.49, p = 0.023), whereas there was a trend towards ST grade 2–3 (HR, 0.54, CI 95%, 0.29–1.01, p = 0.054). Skin toxicity is a promising biomarker to identify patients with mCRC that could benefit of anti-EGFR rechallenge.

Il saggio si sofferma sulle dinamiche narrative della serialità e sulle istanze estetiche e culturali, fondamentalmente conservatrici, che caratterizzano la serie televisiva "Downton Abbey" in relazione ai mutamenti cruciali di inizio Novecento. Da una parte, in essa sembra prevalere una forma di nostalgico anti-progressismo, se non di ansiogeno snobismo culturale, nei confronti della crescente massificazione, meccanizzazione e americanizzazione della società inglese, che sembra rievocare, in alcuni dei personaggi più resistenti al cambiamento, l’atteggiamento di un certo numero di intellettuali dell’epoca – non ultimo l’Aldous Huxley del "Mondo nuovo" (1932), citato più volte nella serie; dall’altra, la trasformazione e la propulsione, che vengono prodotti dal progresso sociale e tecnologico e che, in fin dei conti, sono il motore stesso della Storia e delle storie, appaiono tematizzati nella loro ineluttabilità - e persino metaforizzati come mari in tempesta che l'eterotopico ‘bastimento’ Downton è costretto ad attraversare - oppure associati intertestualmente alle dinamiche trasgressive dell'Alice di Lewis Carroll. Si instaura, dunque, una dialettica peculiare tra ripetizione e innovazione, tradizione e modernità, tempo ciclico e tempo lineare, continuamente giocata in dialogo con la letteratura vittoriana e con quella epocale, che rende il 'brave new world' della serie un testo culturale sfaccettato e complesso: pur generato da un impulso teso a (ri)costruire il passato come un sogno dorato o come una favola d’altri tempi (la cui struttura narrativa archetipica affiora continuamente a livello intertestuale), l’universo del racconto, dominato dal ritorno dell’identico, si schiude all’accettazione dell'idea che la natura della vita sia inesorabilmente mutevole.

Simple Summary Haematological malignancies show a constantly growing incidence, accounting for 6.5% of new cancer cases worldwide. Among them, B-cell neoplasms often show resistance to conventional chemotherapy that is also associated with numerous adverse effects. Therefore, in order for the treatment outcome to be improved, the development of new safe and effective targeted therapeutic approaches represents a main challenge. In this regard, nucleic acid aptamers are very attractive molecules. Indeed, they show high affinity and specificity for their target, increased tumour penetration, and low toxicity. Recently, CD19 has emerged as a key surface marker of malignant B cells, suitable for the development of new compounds for malignant B-cell targeting. Here, we isolated an RNA aptamer targeting the human CD19 antigen on malignant B cells that was able to rapidly internalise into target cells. Therefore, it represents a useful carrier for secondary reagents and a promising tool for the development of new safe and effective targeted therapies for B-cell malignancy treatment. Abstract The transmembrane glycoprotein cluster of differentiation 19 (CD19) is a B cell–specific surface marker, expressed on the majority of neoplastic B cells, and has recently emerged as a very attractive biomarker and therapeutic target for B-cell malignancies. The development of safe and effective ligands for CD19 has become an important need for the development of targeted conventional and immunotherapies. In this regard, aptamers represent a very interesting class of molecules. Additionally referred to as ‘chemical antibodies’, they show many advantages as therapeutics, including low toxicity and immunogenicity. Here, we isolated a nuclease-resistant RNA aptamer binding to the human CD19 glycoprotein. In order to develop an aptamer also useful as a carrier for secondary reagents, we adopted a cell-based SELEX (Systematic Evolution of Ligands by EXponential Enrichment) protocol adapted to isolate aptamers able to internalise upon binding to their cell surface target. We describe a 2′-fluoro pyrimidine modified aptamer, named B85.T2, which specifically binds to CD19 and shows an exquisite stability in human serum. The aptamer showed an estimated dissociation constant (KD) of 49.9 ± 13 nM on purified human recombinant CD19 (rhCD19) glycoprotein, a good binding activity on human B-cell chronic lymphocytic leukaemia cells expressing CD19, and also an effective and rapid cell internalisation, thus representing a promising molecule for CD19 targeting, as well as for the development of new B-cell malignancy-targeted therapies.

microRNAs (miRNAs) are small non-coding RNAs acting as negative regulators of gene expression and involved in tumor progression. We recently showed that inhibition of the pro-metastatic miR-214 and simultaneous overexpression of its downstream player, the anti-metastatic miR-148b, strongly reduced metastasis formation. To explore the therapeutic potential of miR-148b, we generated a conjugated molecule aimed to target miR-148b expression selectively to tumor cells. Precisely, we linked miR-148b to GL21.T, an aptamer able to specifically bind to AXL, an oncogenic tyrosine kinase receptor highly expressed on cancer cells. Axl-148b conjugate was able to inhibit migration and invasion of AXL-positive, but not AXL-negative, cancer cells, demonstrating high efficacy and selectivity in vitro. In parallel, expression of ALCAM and ITGA5, two miR-148b direct targets, was reduced. More importantly, axl-148b chimeric aptamers were able to inhibit formation and growth of 3D-mammospheres, to induce necrosis and apoptosis of treated xenotransplants, as well as to block breast cancer and melanoma dissemination and metastatization in mice. Relevantly, axl aptamer acted as specific delivery tool for miR-148b, but it also actively contributed to inhibit metastasis formation, together with miR-148b. In conclusion, our data show that axl-148b conjugate is able to inhibit tumor progression in an axl- and miR-148b-dependent manner, suggesting its potential development as therapeutic molecule.

B cell maturation antigen is highly expressed on malignant plasma cells in human multiple myeloma and has recently emerged as a very promising target for therapeutic interventions. Nucleic-acid-based aptamers are small oligonucleotides with high selective targeting properties and functional advantages over monoclonal antibodies, as both diagnostic and therapeutic tools. Here, we describe the generation of the first-ever-described nuclease resistant RNA aptamer selectively binding to B cell maturation antigen. We adopted a modified cell-based systematic evolution of ligands by exponential enrichment approach allowing the enrichment for internalizing aptamers. The selected 2′Fluoro-Pyrimidine modified aptamer, named apt69.T, effectively and selectively bound B cell maturation antigen-expressing myeloma cells with rapid and efficient internalization. Interestingly, apt69.T inhibited APRIL-dependent nuclear factor κB (NF-κB) pathway in vitro. Moreover, the aptamer was conjugated to microRNA-137 (miR-137) and anti-miR-222, demonstrating high potential against tumor cells. In conclusion, apt69.T is a novel tool suitable for direct targeting and delivery of therapeutics to B cell maturation antigen-expressing myeloma cells., Graphical Abstract

Thyroid hormones (THs) are key regulators of different biological processes. Their action involves genomic and non-genomic mechanisms, which together mediate the final effects of TH in target tissues. However, the proportion of the two processes and their contribution to the TH-mediated effects are still poorly understood. Skeletal muscle is a classical target tissue for TH, which regulates muscle strength and contraction, as well as energetic metabolism of myofibers. Here we address the different contribution of genomic and non-genomic action of TH in skeletal muscle cells by specifically silencing the deiodinase Dio2 or the β3-Integrin expression via CRISPR/Cas9 technology. We found that myoblast proliferation is inversely regulated by integrin signal and the D2-dependent TH activation. Similarly, inhibition of the nuclear receptor action reduced myoblast proliferation, confirming that genomic action of TH attenuates proliferative rates. Contrarily, genomic and non-genomic signals promote muscle differentiation and the regulation of the redox state. Taken together, our data reveal that integration of genomic and non-genomic signal pathways finely regulates skeletal muscle physiology. These findings not only contribute to the understanding of the mechanisms involved in TH modulation of muscle physiology but also add insight into the interplay between different mechanisms of action of TH in muscle cells.

Messenger RNAs (mRNAs) present a great potential as therapeutics for the treatment and prevention of a wide range of human pathologies, allowing for protein replacement, vaccination, cancer immunotherapy, and genomic engineering. Despite advances in the design of mRNA-based therapeutics, a key aspect for their widespread translation to clinic is the development of safe and effective delivery strategies. To this end, non-viral delivery systems including peptide-based complexes, lipidic or polymeric nanoparticles, and hybrid formulations are attracting growing interest. Despite displaying somewhat reduced efficacy compared to viral-based systems, non-viral carriers offer important advantages in terms of biosafety and versatility. In this review, we provide an overview of current mRNA therapeutic applications and discuss key biological barriers to delivery and recent advances in the development of non-viral systems. Challenges and future applications of this novel therapeutic modality are also discussed.

Despite the therapeutic utility of small interfering RNA (siRNA) molecules, the development of a safe and reliable method to selectively target diseased organs and tissues is still a critical need for their translation to the clinic. Here we describe how nucleic acid-based aptamers against cell surface epitopes may be used to address this issue. We discuss the most recent examples and advances in the field of aptamer siRNA delivery and provide a fast and simple protocol for the design and generation of aptamer-siRNA chimeras. The described approach is based on the annealing of the targeting aptamer, and the antisense strand through "stick" complementary sequences elongated at their 3' end, and the subsequent paring with the sense strand. Such a protocol allows a modular non-covalent generation of the constructs and permits an efficient delivery of the siRNA moiety into aptamer target cells.

Exosomes are emerging as promising target for early diagnosis and therapy in diff erent oncological conditions including breast cancer (BC). However, the development of tools able to easily and specifi cally target cancer cell-derived exosomes still represent a fundamental issue that is required to realize their clinical utility. Nucleic-acid aptamers are a promising class of structured single stranded oligonucleotides that serve as high affi nity ligands of disease-associated proteins. Given their high potential in diagnosis and therapy, we addressed the development of aptamers specifi c for BC-derived exosomes. To this end, we developed a novel SELEX strategy by using exosomes purifi ed from primary BC cells as positive selection target. By such a strategy we isolated nuclease resistant RNA aptamers able to specifi cally discriminate BC-derived exosomes from those produced by normal cells. Th e best sequences were optimized identifying short molecules (about 30-35 mer) that was characterized as tools for exosome detection. Further, we demonstrated that the developed aptamers inhibited exosome cellular uptake antagonizing cancer exosome-induced cell migration. By proteomic approach we identifi ed possible targets that we are characterizing. Our results underline the great potential of isolated aptamers as tools for the development of innovative strategies for BC early diagnosis and therapy.

Breast cancer is a leading cause of cancer mortality in women. Despite advances in its management, the identification of new options for early-stage diagnosis and therapy of this tumor still represents a crucial challenge. Increasing evidence indicates that extracellular vesicles called exosomes may have great potential as early diagnostic biomarkers and regulators of many cancers, including breast cancer. Therefore, exploiting molecules able to selectively recognize them is of great interest. Here, we developed a novel differential SELEX strategy, called Exo-SELEX, to isolate nucleic acid aptamers against intact exosomes derived from primary breast cancer cells. Among the obtained sequences, we optimized a high-affinity aptamer (ex-50.T) able to specifically recognize exosomes from breast cancer cells or patient serum samples. Furthermore, we demonstrated that the ex.50.T is a functional inhibitor of exosome cellular uptake and antagonizes cancer exosome-induced cell migration in vitro. This molecule provides an innovative tool for the specific exosome detection and the development of new therapeutic approaches for breast cancer., Graphical Abstract, Extracellular vesicles are important biomarkers and therapeutic targets for breast cancer (BC). However, specific tools for their recognition are lacking. Condorelli and colleagues describe the finding of a high-affinity aptamer (ex-50.T) able to specifically target BC exosomes acting as a promising diagnostic and therapeutic tool.

The aim of the research. Non-small cell lung cancer (NSCLC) represents about 80 % of all lung cancer cases and is oft en associated with drug resistance, relapses and a poor prognosis. Th erefore, the identifi cation of eff ective therapeutic strategies represents a crucial challenge in oncology. A key limit of conventional anticancer treatments is that they do not target the permissive tumor microenvironment, of which key components are cancer‐associated fibroblasts (CAFs). It has been shown that CAFs are able to regulate malignant progression and drug resistance. However, a detailed characterization of CAF profile and the targeting of their pro-tumor eff ects still remain an ambitious challenge and have a primary importance for the identifi cation of new eff ective therapies. Material and methods. We aimed to develop innovative strategies based on nucleic acid aptamers to address these fundamental issues. Firstly, we applied an aptamer conjugate (named Gint4.T-STAT3), containing a STAT3 siRNA linked to an aptamer binding and inhibiting the PDGFRβ, to specifi cally silence STAT3 reported as a fundamental player in the cross-talk between CAFs and epithelial NSCLC cells. Results. We demonstrated that this molecule eff ectively delivers STAT3 siRNA in NSCLC cells, blocking CAF-induced cell growth and migration in both continous and primary NSCLC coltures. In addition, in order to address CAF specifi c targeting and profi ling, we developed an innovative diff erential cell-SELEX approach by using primary NSCLC CAFs as selection target. Such a strategy allowed the isolation of diff erent aptamers discriminating NSCLC CAFs from normal lung fi broblasts. Th e analyses of aptamer specifi city and functionality is curently ongoing. Conclusion. Our data represent the fi rst ever attempt in CAF targeting using aptamer-based drugs, and can open innovative horizons in the current therapeutic approaches forNSCLC.

IMPORTANCE: Rechallenge therapy with anti–epidermal growth factor receptor (EGFR) drugs has been suggested in patients with chemo-refractory RAS wild-type (WT) metastatic colorectal cancer (mCRC) after initial response to anti–EGFR-based first-line treatment. The association of treatment with cetuximab plus avelumab with overall survival (OS) may be worthy of investigation in this setting. OBJECTIVE: To assess the efficacy and safety of cetuximab rechallenge therapy plus avelumab. DESIGN, SETTING, AND PARTICIPANTS: This single-arm, multicenter phase 2 trial enrolled patients from August 2018 to February 2020. Eligible patients with RAS WT mCRC had a complete or partial response to first-line chemotherapy plus anti-EGFR drugs, developed acquired resistance, and failed second-line therapy. Baseline circulating tumor DNA (ctDNA) for KRAS, NRAS, BRAF, and EGFR-S492R mutation analysis was done. INTERVENTIONS: Patients received avelumab (10 mg/kg every 2 weeks) and cetuximab (400 mg/m(2) and, subsequently, 250 mg/m(2) weekly) until disease progression or unacceptable toxic effects. MAIN OUTCOMES AND MEASURES: The primary end point was OS. Secondary end points were progression-free survival (PFS), overall response rate (ORR), and safety. RESULTS: Seventy-seven patients were enrolled (42 men, 35 women; median age, 63 years); 71 had microsatellite stable tumors (MSS), 3 microsatellite instability-high tumors (MSI-H), 3 unknown. The study met the primary end point, with median OS (mOS) of 11.6 months (95% CI, 8.4-14.8 months). Median PFS (mPFS) was 3.6 months (95% CI, 3.2-4.1 months). Common grade-3 adverse events were cutaneous eruption, 11 (14%), and diarrhea, 3 (4%). For 67 of 77 (87%) patients, baseline analysis of plasma circulating tumor DNA (ctDNA) for KRAS, NRAS, BRAF, and EGFR-S492R variations was feasible. Forty-eight patients had WT disease, whereas 19 had mutations. Patients with RAS/BRAF WT ctDNA had mOS of 17.3 months (95% CI, 12.5-22.0 months) compared with 10.4 months (95% CI, 7.2-13.6 months) in patients with mutated ctDNA (hazard ratio [HR], 0.49; 95% CI, 0.27-0.90; P = .02). The mPFS was 4.1 months (95% CI, 2.9-5.2 months) in RAS/BRAF WT patients compared with 3.0 months (95% CI, 2.6-3.5 months) in patients with mutated ctDNA (HR, 0.42; 95% CI, 0.23-0.75; P = .004). CONCLUSIONS AND RELEVANCE: The findings of this single-arm phase 2 trial suggest that cetuximab plus avelumab is an active, well tolerated rechallenge therapy in RAS WT mCRC. Plasma ctDNA analysis before treatment may allow selection of patients who could benefit. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04561336

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine-threonine kinase that phosphorylates various transcriptional and chromatin regulators, thus modulating numerous important cellular processes, such as proliferation, apoptosis, DNA damage response, and oxidative stress. The role of HIPK2 in the pathogenesis of cancer and fibrosis is well established, and evidence of its involvement in the homeostasis of multiple organs has been recently emerging. We have previously demonstrated that Hipk2-null (Hipk2-KO) mice present cerebellar alterations associated with psychomotor abnormalities and that the double ablation of HIPK2 and its interactor HMGA1 causes perinatal death due to respiratory failure. To identify other alterations caused by the loss of HIPK2, we performed a systematic morphological analysis of Hipk2-KO mice. Post-mortem examinations and histological analysis revealed that Hipk2 ablation causes neuronal loss, neuronal morphological alterations, and satellitosis throughout the whole central nervous system (CNS), a myopathic phenotype characterized by variable fiber size, mitochondrial proliferation, sarcoplasmic inclusions, morphological alterations at neuromuscular junctions, and a cardiac phenotype characterized by fibrosis and cardiomyocyte hypertrophy. These data demonstrate the importance of HIPK2 in the physiology of skeletal and cardiac muscles and of different parts of the CNS, thus suggesting its potential relevance for different new aspects of human pathology.

Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer worldwide, with the highest incidence in developed countries. NSCLC patients often face resistance to currently available therapies, accounting for frequent relapses and poor prognosis. Indeed, despite great recent advancements in the field of NSCLC diagnosis and multimodal therapy, most patients are diagnosed at advanced metastatic stage, with a very low overall survival. Thus, the identification of new effective diagnostic and therapeutic options for NSCLC patients is a crucial challenge in oncology. A promising class of targeting molecules is represented by nucleic-acid aptamers, short single-stranded oligonucleotides that upon folding in particular three dimensional (3D) structures, serve as high affinity ligands towards disease-associated proteins. They are produced in vitro by SELEX (systematic evolution of ligands by exponential enrichment), a combinatorial chemistry procedure, representing an important tool for novel targetable biomarker discovery of both diagnostic and therapeutic interest. Aptamer-based approaches are promising options for NSCLC early diagnosis and targeted therapy and may overcome the key obstacles of currently used therapeutic modalities, such as the high toxicity and patients’ resistance. In this review, we highlight the most important applications of SELEX technology and aptamers for NSCLC handling.

DNA methylation is a fundamental epigenetic modification regulating gene expression. Aberrant DNA methylation is the most common molecular lesion in cancer cells. However, medical intervention has been limited to the use of toxic, unspecific demethylating drugs. Aptamers are novel high affinity targeting ligand molecules. By conjugating the inherent DNMT1 inhibiting capabilities of RNA to an aptamer platform, we generated a first-of-its kind aptamer approach that can target and neutralize DNMT1 function – the aptaDiR. Molecular modelling of RNA-DNMT1 complexes coupled with biochemical and cellular assays enabled the identification and characterization of aptaDiR. This novel RNA bio-drug blocks DNA methylation and impairs cancer cell viability.Collectively, we present an innovative RNA-based approach to modulate DNMT1 activity in cancer or diseases characterized by aberrant DNA methylation and suggest the first alternative strategy to overcome the limitations of currently approved hypomethylating protocols, which will greatly improve clinical intervention on DNA methylation.

The diffusion of SARS-CoV-2, starting from China in December 2019, has led to a pandemic, reaching Italy in February 2020. Previous studies in Asia have shown that the median duration of SARS-CoV-2 viral shedding was approximately 12–20 days. We considered a cohort of patients recovered from COVID-19 showing that the median disease duration between onset and end of COVID-19 symptoms was 27.5 days (interquartile range (IQR): 17.0–33.2) and that the median duration between onset of symptoms and microbiological healing, defined by two consecutive negative nasopharyngeal swabs, was 38 days (IQR: 31.7–50.2). A longer duration of COVID-19 with delayed clinical healing (symptom-free) occurred in patients presenting at admission a lower PaO2/FiO2 ratio (p < 0.001), a more severe clinical presentation (p = 0.001) and a lower lymphocyte count (p = 0.035). Moreover, patients presenting at admission a lower PaO2/FiO2 ratio and more severe disease showed longer viral shedding (p = 0.031 and p = 0.032, respectively). In addition, patients treated with corticosteroids had delayed clinical healing (p = 0.013).

An important drawback in the management of glioblastoma (GBM) patients is the frequent relapse upon surgery and therapy. A likely explanation is that conventional therapies poorly affect a small population of stem-like cancer cells (glioblastoma stem cells, GSCs) that remain capable of repopulating the tumour mass. Indeed, the development of therapeutic strategies able to hit GSCs while reducing the tumour burden has become an important challenge to increase a patient&rsquo, s survival. The signal transducer and activator of transcription-3 (STAT3) has been reported to play a pivotal role in maintaining the tumour initiating capacity of the GSC population. Therefore, in order to impair the renewal and propagation of the PDGFR&beta, expressing GSC population, here we took advantage of the aptamer&ndash, siRNA chimera (AsiC), named Gint4.T-STAT3, that we previously have shown to efficiently antagonize STAT3 in subcutaneous PDGFR&beta, positive GBM xenografts. We demonstrate that the aptamer conjugate is able to effectively and specifically prevent patient-derived GSC function and expansion. Moreover, because of the therapeutic potential of using miR-10b inhibitors and of the broad expression of the Axl receptor in GBM, we used the GL21.T anti-Axl aptamer as the targeting moiety for anti-miR-10b, showing that, in combination with the STAT3 AsiC, the aptamer&ndash, miR-10b antagonist treatment further enhances the inhibition of GSC sphere formation. Our results highlight the potential to use a combined approach with targeted RNA therapeutics to inhibit GBM tumour dissemination and relapse.