Cardiac risk after elective endovascular repair for infrarenal abdominal aortic aneurysm: Results from the Italian Collaborators for EVAR multicenter registry.

OBJECTIVE: Major adverse cardiac events (MACEs) were common complications after endovascular aortic repair (EVAR) causing significant postoperative morbidity and mortality. The aim of the study was to evaluate the cardiac risk after elective EVAR for uncomplicated noninfected infrarenal abdominal aortic aneurysm in a large multicenter cohort. METHODS: This is a multicenter, retrospective, financially unsupported physician-initiated observational cohort study conducted by four academic tertiary referral hospitals from January 2018 to March 2021. Baseline, perioperative, and postoperative information of elective EVARs was evaluated. The primary outcome was the incidence of MACEs after EVAR, which was defined as acute coronary syndrome, non-ST-elevation myocardial infarction, unstable angina pectoris, de novo atrial fibrillation, hospitalization for heart failure, and revascularization as well as cardiovascular death. Secondary outcomes were 1-year overall survival (all-cause mortality) and freedom from aorta-related mortality. Comparative analysis was conducted between MACE and overall population, and univariate and multivariate logistic regression analyses were used to analyze factors associated with the risk of the MACE occurrence and early 1-year mortality. RESULTS: The study has enrolled 497 patients (35 females, 7%) with a mean age of 75.3 ± 7.8 years. The MACE rate was 6.4% (32/497, events/patients), and the majority of the events were recorded in the postoperative period (24/32, 75%; overall 24/497, 4.8%). One-year survival from all-cause mortality was 94% (95% confidence interval [CI]: 91-96), and the MACE population showed a significantly lower survival estimation rate (Overall - MACEs, 95.8% [95% CI: 93-97] - 67.9% [95% CI: 47-82], log-rank 41.950, P = .0001). Freedom from aorta-related mortality was 99.3% (95% CI: 98-100). The perioperative need for red blood cell transfusions was strongly related to the MACE occurrence (odds ratio: 2.67, 95% CI: 1.52-4.68, P = .001) and 1-year mortality (hazard ratio: 2.14, 95% CI: 1.48-3.09, P = .0001). CONCLUSIONS: MACEs represent a common complication in the postoperative and early period after elective EVAR. Blood loss requiring red blood cell transfusions is associated with increased postoperative MACEs and early mortality, suggesting that all the efforts should be carried out to reduce the bleeding during and after elective interventions.

Results of the Italian Collaborators for Evar Registry on Acute Kidney Injury After Elective Endovascular Aortic Repair of Infrarenal Abdominal Aortic Aneurysm.

OBJECTIVES: To analyze the incidence and predictive factors of postoperative acute kidney injury (AKI) after elective standard endovascular aortic repair (EVAR) in a large recent, multicenter cohort. MATERIALS AND METHODS: This is a multicenter, retrospective, financially unsupported physician-initiated observational cohort study. Between January 2018 and March 2021, only patients treated with elective standard EVAR for infrarenal non-infected abdominal aortic aneurysm were analyzed. Patients already on hemodialysis (HD) were excluded. AKI was defined as an increase in serum creatinine (sCr) ≥0.3 mg/dL within 48 hours or an increase in sCr to ≥1.5 times baseline known or presumed to have occurred within 7 days, or a urine volume of <0.5 mL/kg/h for 6 hours. Primary outcomes of interest were AKI incidence at 30 days and freedom from HD at 1-year follow-up. Secondary outcomes were freedom from severe postoperative complication, and freedom from aorta-related mortality (ARM) at 1 year. RESULTS: The final cohort analyzed 526 (29.8%). There were 489 (93%) males and 37 (7%) females: the median age was 76 years (interquartile range [IQR], 71-81). Chronic kidney disease (CKD) was present in 86 (16.3%) patients. Early mortality was observed in 8 (1.5%) patients, none was aorta-related. Complication rate was 17.1% (n=89). AKI was observed in 17 (3.2%). Renal replacement therapy was needed in 4 (0.8%). HD was transitory in 2 cases and definitive in 1. Binary logistic regression analysis identified CKD (odds ratio [OR]: 4.68, 95% confidence interval [CI]: 2.10-10.45, p<0.001), and the presence of renal artery stenosis (OR: 3.80, 95% CI: 1.35-10.66, p=0.011) to be associated with an increased risk of postoperative AKI. Estimated freedom from ARM was 94.9% at 1 year. Estimated freedom from HD rate at 1 year was 94%: This was significantly different between patients with preoperative CKD and those who did not have preoperative CKD (log-rank, p=0.042). CONCLUSION: AKI after elective standard EVAR still occurs but with an acceptably low incidence rate. Preoperative CKD is the most important predictor for postoperative AKI, which was not associated with the need for HD at 1-year follow-up but with a higher propensity of mortality. CLINICAL IMPACT: This "real world" experience confirm that EVAR performed with standard contrats agent protocol remains safe for acute kidney injury development. Therefore, only patients presenting with preoperative borderline or ascertained chronic kidney disease will take the most advantage from the use of carbon dioxide contrast.

Outcomes and Economic Impact of Hypogastric Artery Management During Elective Endovascular Aortic Repair for Aorto-Iliac Aneurysms.

BACKGROUND: To analyze clinical outcomes and perform a macro-costing evaluation of endovascular aortic repair (EVAR) for aorto-iliac aneurysms. METHODS: This is a retrospective, financially unsupported, physician-initiated observational cohort study. Patients with iliac artery involvement treated with EVAR between January 1st, 2014 and December 31st, 2021 were identified. Inclusion criteria were intact aneurysm, elective EVAR with at least 1 hypogastric artery (HA) treatment, use of bifurcated endograft (EG), and at least 6 months of follow-up. Primary outcomes of interest were overall survival, freedom from aneurysm-related mortality (ARM), freedom from EVAR-related reintervention, and overall EVAR(procedure)-related costs. RESULTS: We studied 122 (9.1%) patients: 119 (97.5%) were male and 3 (2.5%) females. Median age of patients was 76 years (range, 68.75-81). Overall, 107 (87.7%) patients had both HAs preserved according to following strategy: 45 (36.9%) with flared limbs, 13 (10.6%) with bilateral branched device, and 49 (40.2%) with a combination of flared limb on 1 side and branched device on the contralateral side. Bilateral overstenting was performed in 15 (12.3%) patients. Estimated overall survival was not different between groups of EVAR (Log-rank, P = 0.561). There was only 1 (0.8%) ARM ascertained during the follow-up. Estimated freedom from EVAR-related reintervention was not different among groups (Log-rank, P = 0.464). During the follow-up, 9 (7.4%) patients developed buttock claudication (Society for Vascular Surgery (SVS) grade 1, n = 4, SVS grade 2, n = 5), more frequently in HA overstenting (hazard ratio (HR): 3.6; 95% confidence intervals (CIs): 0.96-13.5, P = 0.058). When all cots were included, branched EVAR still carried the highest burden (P = 0.001) in comparison with the mixed subgroup, the overstenting subgroup, and the flared limbs subgroup. CONCLUSIONS: Early mortality and pelvic ischemic syndromes rate were acceptably low in all techniques. Hypogastric artery preservation showed lower complication rate in comparison with HA overstenting which, however, appears to be safe an effective for option with similar overall costs for patients who are not candidates for HA preservation based on aortic anatomy.

Cardiac dysfunction in patients affected by subarachnoid haemorrhage affects in-hospital mortality: A systematic review and metanalysis.

BACKGROUND: Subarachnoid haemorrhage (SAH) is a life-threatening condition with associated brain damage. Moreover, SAH is associated with a massive release of catecholamines, which may promote cardiac injury and dysfunction, possibly leading to haemodynamic instability, which in turn may influence a patient's outcome. OBJECTIVES: To study the prevalence of cardiac dysfunction (as assessed by echocardiography) in patients with SAH and its effect on clinical outcomes. DESIGN: Systematic review of observational studies. DATA SOURCES: We performed a systematic search over the last 20 years on MEDLINE and EMBASE databases. ELIGIBILITY CRITERIA: Studies reporting echocardiography findings in adult patients with SAH admitted to intensive care. Primary outcomes were in-hospital mortality and poor neurological outcome according to the presence or absence of cardiac dysfunction. RESULTS: We included a total of 23 studies (4 retrospective) enrolling 3511 patients. The cumulative frequency of cardiac dysfunction was 21% (725 patients), reported as regional wall motion abnormality in the vast majority of studies (63%). Due to the heterogeneity of clinical outcome data reporting, a quantitative analysis was carried out only for in-hospital mortality. Cardiac dysfunction was associated with a higher in-hospital mortality [odds ratio 2.69 (1.64 to 4.41); P  < 0.001; I2 = 63%]. The GRADE of evidence assessment resulted in very low certainty of evidence. CONCLUSION: About one in five patients with SAH develops cardiac dysfunction, which seems to be associated with higher in-hospital mortality. The consistency of cardiac and neurological data reporting is lacking, reducing the comparability of the studies in this field.

Challenges in the hemodynamic management of acute nontraumatic neurological injuries.

PURPOSE OF REVIEW: To appraise the evidence from the literature and suggest an integrated hemodynamic approach of early and delayed phases of acute ischemic stroke (AIS), subarachnoid hemorrhage (SAH) and intracerebral hemorrhage (ICH). RECENT FINDINGS: In AIS, the research aims to evaluate the optimal pressure control before, during and after the revascularization, to optimize the perfusion in the ischemic areas, minimizing the risk of hemorrhage or secondary damage to already infarcted areas. In the early phase of SAH, systemic pressure should be controlled to balance the risk of stroke, hypertension-related rebleeding, and maintenance of cerebral perfusion pressure. The late phase aims to minimize the risk of cerebral vasospasm by adapting systemic pressure and volemia to cerebral and systemic physiological hemodynamic targets. In the mild-to-moderate ICH, achieving SAP of less than 140 mmHg and greater than 110 mmHg may be considered as a beneficial target. Caution should be considered in lowering intensively SAP in severe ICH. SUMMARY: In nontraumatic brain injuries, the hemodynamic management is strictly related to fluctuating physiology of these diseases, needing a strict control of pressure and flow variable to ensure both cerebral and systemic homeostasis.

Pharmacodynamic analysis of a fluid challenge with 4 ml kg-1 over 10 or 20 min: a multicenter cross-over randomized clinical trial.

PURPOSE: A number of studies performed in the operating room evaluated the hemodynamic effects of the fluid challenge (FC), solely considering the effect before and after the infusion. Few studies have investigated the pharmacodynamic effect of the FC on hemodynamic flow and pressure variables. We designed this trial aiming at describing the pharmacodynamic profile of two different FC infusion times, of a fixed dose of 4 ml kg-1. METHODS: Forty-nine elective neurosurgical patients received two consecutive FCs of 4 ml kg-1 of crystalloids in 10 (FC10) or 20 (FC20) minutes, in a random order. Fluid responsiveness was defined as stroke volume index increase ≥ 10%. We assessed the net area under the curve (AUC), the maximal percentage difference from baseline (dmax), time when the dmax was observed (tmax), change from baseline at 1-min (d1) and 5-min (d5) after FC end. RESULTS: After FC10 and FC20, 25 (51%) and 14 (29%) of 49 patients were classified as fluid responders (p = 0.001). With the exception of the AUCs of SAP and MAP, the AUCs of all the considered hemodynamic variables were comparable. The dmax and the tmax were overall comparable. In both groups, the hemodynamic effects on flow variables were dissipated within 5 min after FC end. CONCLUSIONS: The infusion time of FC administration affects fluid responsiveness, being higher for FC10 as compared to FC20. The effect on flow variables of either FCs fades 5 min after the end of infusion.

Targeting PIK3CA Actionable Mutations in the Circulome: A Proof of Concept in Metastatic Breast Cancer.

The study of circulating cancer-derived components (circulome) is considered the new frontier of liquid biopsy. Despite the recognized role of circulome biomarkers, their comparative molecular profiling is not yet routine. In advanced breast cancer (BC), approximately 40% of hormone-receptor-positive, HER2-negative BC cases harbor druggable PIK3CA mutations suitable for combined alpelisib/fulvestrant treatment. This pilot study investigates PIK3CA mutations in circulating tumor DNA (ctDNA), tumor cells (CTCs), and extracellular vesicles (EVs) with the aim of determining which information on molecular targetable profiling could be recollected in each of them. The in-depth molecular analysis of four BC patients demonstrated, as a proof-of-concept study, that it is possible to retrieve mutational information in the three components. Patient-specific PIK3CA mutations were found in both tissue and ctDNA and in 3/4 cases, as well as in CTCs, in the classical population (large-sized CD45-/EpCAM+/- cells), and/or in the "non-conventional" sub-population (smaller-sized CD44+/EpCAM-/CD45- cells). Consistent mutational profiles of EVs with CTCs suggest that they may have been released by CTCs. This preliminary evidence on the molecular content of the different circulating biomaterials suggests their possible function as a mirror of the intrinsic heterogeneity of BC. Moreover, this study demonstrates, through mutational assessment, the tumor origin of the different CTC sub-populations sustaining the translational value of the circulome for a more comprehensive picture of the disease.

Monitoring the community use of antibiotics in Italy within the National Action Plan on antimicrobial resistance.

BACKGROUND: In Italy both the consumption of antibiotics and the prevalence of bacterial resistance are higher than in other European countries. In 2017, the first National Action Plan on Antimicrobial Resistance (PNCAR) was adopted in Italy. In response to the PNCAR two national reports on antibiotic use in the human setting have been published. This article's aim is to describe the pattern of antibiotic consumption in the community setting in Italy from 2013 to 2018. METHODS: To analyse the consumption for reimbursed antibiotics dispensed by community pharmacies different data sources were used. Consumption was measured in terms of defined daily dose (DDD), prescriptions or prevalence of use. RESULTS: In 2018, the consumption of antibiotics in Italy amounted to 16.1 DDD per 1000 inhabitants per day. The rates of consumption by geographical area were: 12.7 DDD in the north, 16.9 in the centre and 20.4 in the south. The use was greater in the extreme age groups than in the population aged from 20 to 64 years. The consumption was higher in winter season, with high peaks in the incidence of flu syndromes. In the paediatric population, a utilization rate of 1010 prescriptions per 1000 children, with a prevalence of use of 40.8%, was found. CONCLUSION: The study provides useful information on the geographical variability of antibiotic use in Italy to guide decision makers in the introduction of tailored interventions, as suggested by PNCAR, aimed at promoting a more rational use of antibiotics for humans and reducing antimicrobial resistance.

Comprehensive Profiling of Secretome Formulations from Fetal- and Perinatal Human Amniotic Fluid Stem Cells.

We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative paracrine potential driving pro-survival, anti-fibrotic and proliferative effects. hAFS may also be isolated from III trimester clinical waste samples during scheduled C-sections (perinatal hAFS), thus offering a more easily accessible alternative when compared to fetal hAFS. Nonetheless, little is known about the paracrine profile of perinatal hAFS. Here we provide a detailed characterization of the hAFS total secretome (i.e., the entirety of soluble paracrine factors released by cells in the conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it, from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. hAFS-CM and hAFS-EV formulations were analyzed for protein and chemokine/cytokine content, and the EV cargo was further investigated by RNA sequencing. The phenotype of fetal- and perinatal hAFS, along with their corresponding secretome formulations, overlapped; yet, fetal hAFS showed immature oxidative phosphorylation activity when compared to perinatal ones. The profiling of their paracrine cargo revealed some differences according to gestational stage and hypoxic preconditioning. Both cell sources provided formulations enriched with neurotrophic, immunomodulatory, anti-fibrotic and endothelial stimulating factors, and the immature fetal hAFS secretome was defined by a more pronounced pro-vasculogenic, regenerative, pro-resolving and anti-aging profile. Small RNA profiling showed microRNA enrichment in both fetal- and perinatal hAFS-EV cargo, with a stably- expressed pro-resolving core as a reference molecular signature. Here we confirm that hAFS represents an appealing source of regenerative paracrine factors; the selection of either fetal or perinatal hAFS secretome formulations for future paracrine therapy should be evaluated considering the specific clinical scenario.

Inhalation versus endovenous sedation in subarachnoid hemorrhage patients: effects on regional cerebral blood flow.

OBJECTIVE: Isoflurane is a volatile anesthetic that has a vasodilating effect on cerebral vessels producing a cerebral blood flow increase. Furthermore, it has been shown in animal studies that isoflurane, when used as a preconditioning agent, has neuroprotective properties, inducing tolerance to ischemia. However, it is not routinely used in neurointensive care because of the potential increase in intracranial pressure caused by the rise in cerebral blood flow. Nevertheless, subarachnoid hemorrhage patients who are at risk for vasospasm may benefit from an increase in cerebral blood flow. We measured regional cerebral blood flow during intravenous sedation with propofol and during sedation with isoflurane in patients with severe subarachnoid hemorrhage not having intracranial hypertension. DESIGN: The study is a crossover, open clinical trial (NCT00830843). SETTING: Neurointensive care unit of an academic hospital. PATIENTS: Thirteen patients with severe subarachnoid hemorrhage, (median Fisher scale 4), monitored on clinical indication with intracranial pressure device and a thermal diffusion probe for the assessment of regional cerebral blood flow. An intracranial pressure>18 mm Hg was an exclusion criterion. INTERVENTIONS: Cerebral and hemodynamic variables were assessed at three steps. Step 1: sedation with propofol 3-4 mg/kg/hr; step 2: after 1 hr of propofol discontinuation and isoflurane 0.8%; step 3: after 1 hr of propofol at the same previous infusion rate. Cerebral perfusion pressure and arterial PCO2 were maintained constant. Mean cerebral artery flow velocity and jugular vein oxygen saturation were measured at the end of each step. MEASUREMENTS AND MAIN RESULTS: Regional cerebral blood flow increased significantly during step 2 (39.3±29 mL/100 hg/min) compared to step 1 (20.8±10.7) and step 3 (24.7±8). There was no difference in regional cerebral blood flow comparing step 1 vs. step 3. No significant difference in intracranial pressure, mean cerebral artery transcranial Doppler velocity, PaCO2, cerebral perfusion pressure between the different steps. CONCLUSIONS: Isoflurane increases regional cerebral blood flow in comparison to propofol. Intracranial pressure did not change significantly in the population not affected by intracranial hypertension.

Brain multimodality monitoring: an update.

PURPOSE OF REVIEW: An important goal of neurocritical care is the management of secondary brain injury (SBI), that is pathological events occurring after primary insult that add further burden to outcome. Brain oedema, cerebral ischemia, energy dysfunction, seizures and systemic insults are the main components of SBI. We here review recent data showing the clinical utility of brain multimodality monitoring (BMM) for the management of SBI. RECENT FINDINGS: Despite being recommended by international guidelines, standard intracranial pressure (ICP) monitoring may be insufficient to detect all episodes of SBI. ICP monitoring, combined with brain oxygen (PbtO(2)), cerebral microdialysis and regional cerebral blood flow, might help to target therapy (e.g. management of cerebral perfusion pressure, blood transfusion, glucose control) to patient-specific pathophysiology. Physiological parameters derived from BMM, including PbtO(2) and microdialysis lactate/pyruvate ratio, correlate with outcome and have recently been incorporated into neurocritical care guidelines. Advanced intracranial devices can be complemented by quantitative electroencephalography to monitor changes of brain function and nonconvulsive seizures. SUMMARY: BMM offers an on-line comprehensive scrutiny of the injured brain and is increasingly used for the management of SBI. Integration of monitored data using new informatics tools may help optimize therapy of brain-injured patients and quality of care.

The Murine PSE/TATA-dependent transcriptome: evidence of functional homologies with its human counterpart.

A series of recent studies demonstrated an unexpectedly high frequency of intronic RNA polymerase (pol) III transcription units spread throughout the human genome. The investigation of a subset of these transcripts revealed their tissue/cell-specific transcription together with the involvement in relevant physiopathological pathways. Despite this evidence, these transcripts did not seem to have murine orthologs, based on their nucleotide sequence, resulting in a limitation of the experimental approaches aimed to study their function. In this work, we have extended our investigation to the murine genome identifying 121 pairs of mouse/human transcripts displaying syntenic subchromosomal localization. The analysis in silico of this set of putative noncoding (nc)RNAs suggest their association with alternative splicing as suggested by recent experimental evidence. The investigation of one of these pairs taken as experimental model in mouse hippocampal neurons provided evidence of a human/mouse functional homology that does not depend on underlying sequence conservation. In this light, the collection of transcriptional units here reported can be considered as a novel source for the identification and the study of novel regulatory elements involved in relevant biological processes.

Orphan Drug Prices and Epidemiology of Rare Diseases: A Cross-Sectional Study in Italy in the Years 2014-2019.

INTRODUCTION: It is well acknowledged that the price of orphan drugs is normally higher than that resulting from the value-based pricing. A correlation between the cost of therapy for orphan drugs and the epidemiology (prevalence and incidence) of the related rare disease can be hypothesized. METHODS: This analysis includes all approved orphan drugs by European Medicines Agency whose reimbursement was granted for the first therapeutic indication in the years 2014-2019 in Italy. Regression and correlation analyses were performed to analyze the possible correlations between the logarithm of the annual therapy cost and the epidemiology of the rare diseases, between orphan drugs consumption and epidemiology of related rare disease and between therapy cost and the consumption. RESULTS: The regression analysis between the annual cost of therapy estimated on the published ex-factory price and the prevalence showed a slightly decreasing, not statistically significant, trend (coefficient: -0.10, p-value: 0.41). The results were similar when using the price resulting from the application of Managed Entry Agreements (coefficient: -0.11, p-value: 0.40). The regression analysis between sales volume and prevalence showed a positive slope without an acceptable level of significance (p-value: 0.04). The correlation analysis between the therapy cost and the sales volume highlighted again an absence of significant association, similarly if considering only ATC L orphan drugs, or the incidence. DISCUSSION: The definition of the price of an orphan drug seems not to depend on the rarity of the disease, and sales volumes do not correlate with the epidemiology of the rare disease and with the annual cost of therapy.

Hemodynamic management of acute brain injury caused by cerebrovascular diseases: a survey of the European Society of Intensive Care Medicine.

BACKGROUND: The optimal hemodynamic targets and management of patients with acute brain injury are not completely elucidated, but recent evidence points to important impact on clinical outcomes. We performed an international survey with the aim to investigate the practice in the hemodynamic targets, monitoring, and management of patients with acute ischemic stroke (AIS), intracranial hemorrhage (ICH) and subarachnoid hemorrhage (SAH). METHODS: This survey was endorsed by the European Society of Intensive Care (ESICM). An electronic questionnaire of 76 questions divided in 4 sections (general information, AIS, ICH, SAH specific questions) was available between January 2022 to March 2022 on the ESICM website. RESULTS: One hundred fifty-four healthcare professionals from 36 different countries and at least 98 different institutions answered the survey. Routine echocardiography is routinely performed in 37% of responders in AIS, 34% in ICH and 38% in SAH. Cardiac output monitoring is used in less than 20% of cases by most of the responders. Cardiovascular complications are the main reason for using advanced hemodynamic monitoring, and norepinephrine is the most common drug used to increase arterial blood pressure. Most responders target fluid balance to neutral (62% in AIS, 59% in ICH,44% in SAH), and normal saline is the most common fluid used. Large variability was observed regarding the blood pressure targets. CONCLUSIONS: Hemodynamic management and treatment in patients with acute brain injury from cerebrovascular diseases vary largely in clinical practice. Further research is required to provide clear guidelines to physicians for the hemodynamic optimization of this group of patients.

Targeting of colorectal cancer organoids with zoledronic acid conjugated to the anti-EGFR antibody cetuximab.

BACKGROUND: Antibody-drug conjugates (ADC) are essential therapeutic options to treat solid and hematological cancers. The anti-epidermal growth factor-receptor (EGFR) antibody cetuximab (Cet) is used for the therapy of colorectal carcinoma (CRC). Anti-CRC Vδ2 cytolytic T lymphocytes can be elicited by the priming of tumor cells with the aminobisphosphonate zoledronic acid (ZA) and consequent presentation of isopentenyl pyrophosphates through butyrophilin (BTN) family members such as BTN3A1 and BTN2A1. A major drawback that impairs the targeting of ZA to CRC is the bone tropism of aminobisphosphonates. METHODS: The phosphoric group of ZA was linked to free amino groups of Cet in the presence of imidazole following the labeling of phosphoric groups of DNA to amino groups of proteins. The generation of Cet-ZA ADC was confirmed by matrix assisted laser desorption ionization mass spectrometry and inductively coupled plasma-mass spectrometry analysis. Thirteen CRC organoids were obtained with a chemically defined serum-free medium in Geltrex domes. Proliferation and activation of cytolytic activity against CRC organoids by Vδ2 T cells was detected with flow cytometry, crystal violet and cytotoxic probe assays and image analysis. Immunohistochemistry and quantification of BTN3A1 or BTN2A1 expression and the number of tumor infiltrating Vδ2 T cells in CRC were performed by automatic immunostaining, whole slide scanning and computerized analysis of digital pathology imaging. RESULTS: The novel ADC Cet-ZA was generated with a drug antibody ratio of 4.3 and displayed a reactivity similar to the unconjugated antibody. More importantly, patient-derived CRC organoids, or CRC tumor cell suspensions, could trigger the expansion of Vδ2 T cells from peripheral blood and tumor infiltrating lymphocytes when primed with Cet-ZA. Furthermore, Cet-ZA triggered Vδ2 T cell-mediated killing of CRC organoids. The expression of BTN3A1 and BTN2A1 was detected not only in CRC organoids but also in CRC specimens, together with a considerable amount of tumor infiltrating Vδ2 T cells. CONCLUSIONS: These findings are proof of concept that the Cet-ZA ADC can be used to target specifically CRC organoids and may suggest a new experimental approach to deliver aminobisphosphonates to EGFR+ solid tumors.

Variables affecting pricing of orphan drugs: the Italian case.

BACKGROUND AND AIM: Evidence on determinants of prices for orphan medicines is scarce and not available for Italy. The aim of this paper is to provide an evidence on variables affecting the annual treatment cost of orphan drugs in Italy, testing the hypothesis of a negative correlation with the dimension of the target population and a positive correlation with the added therapeutic value of the drug and the quality of the evidence of pivotal studies. METHODS: Drugs with a European orphan designation reimbursed in Italy in the last 6 years (2014-2019) were considered. Univariate, cluster analysis and multiple regression models were used to investigate the correlation between the annual treatment cost and, as explanatory variables, the dimension of the target population, the existence of Randomized Clinical Trials as a proxy of the quality of the pivotal studies, the added therapeutic value. RESULTS: In the univariate analysis prevalence and added therapeutic value, as expected, have a negative and positive correlation with cost respectively. The correlation with RCT is not significant. In the multivariate model, coefficients for prevalence and added value are confirmed but for the latter are not significant anymore. We also found, through an interaction analysis, that the existence of an RCT has a positive impact on annual treatment cost when the target population is very small. CONCLUSIONS: Our results suggest that value arguments and sustainability (dimension of the target population and its impact on budget impact) issues are considered for orphan drugs pricing: the role played by sustainability is systematically supported by our results. A more transparent and reproducible price negotiation process for orphan drugs is needed in Italy. This paper has contributed to highlight the implicit drivers of this process.

Three-Dimensional Culture Models to Study Innate Anti-Tumor Immune Response: Advantages and Disadvantages.

Several approaches have shown that the immune response against tumors strongly affects patients' clinical outcome. Thus, the study of anti-tumor immunity is critical to understand and potentiate the mechanisms underlying the elimination of tumor cells. Natural killer (NK) cells are members of innate immunity and represent powerful anti-tumor effectors, able to eliminate tumor cells without a previous sensitization. Thus, the study of their involvement in anti-tumor responses is critical for clinical translation. This analysis has been performed in vitro, co-incubating NK with tumor cells and quantifying the cytotoxic activity of NK cells. In vivo confirmation has been applied to overcome the limits of in vitro testing, however, the innate immunity of mice and humans is different, leading to discrepancies. Different activating receptors on NK cells and counter-ligands on tumor cells are involved in the antitumor response, and innate immunity is strictly dependent on the specific microenvironment where it takes place. Thus, three-dimensional (3D) culture systems, where NK and tumor cells can interact in a tissue-like architecture, have been created. For example, tumor cell spheroids and primary organoids derived from several tumor types, have been used so far to analyze innate immune response, replacing animal models. Herein, we briefly introduce NK cells and analyze and discuss in detail the properties of 3D tumor culture systems and their use for the study of tumor cell interactions with NK cells.

Dissecting the effects of preconditioning with inflammatory cytokines and hypoxia on the angiogenic potential of mesenchymal stromal cell (MSC)-derived soluble proteins and extracellular vesicles (EVs).

Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components.

[Early access programs and managed entry agreements for medicines in Italy: results of a Focus Group (Early Access Programs and Managed Entry Agreement).] / Programmi di early access dei farmaci e managed entry agreement in Italia: i risultati di un Focus Group (programmi di early access e managed entry agreement).

BACKGROUND: Early access of medicines occurs with an uncertainty in the evidence even higher than the one experienced when price and reimbursement status is negotiated. Our aim is discussing the role of managed entry agreements (MEA) within early access programs (EAP) in Italy. METHODS: The discussion relied on a Focus Group, participated by twelve experts, including clinicians and representatives of regulatory authorities, regional and local pharmaceutical departments, pharmaceutical companies, and an association advocating for active citizenship. RESULTS: The Focus Group emphasised that the topic under discussion should be embedded into a more general reform of EAP in Italy. The 648 List mostly includes mature products and indications that are rarely launched into the market afterwards. The 5% Fund is affected by an important administrative burden uncertainty of the timing of reimbursement. CONCLUSIONS: Starting from the discussion on MEA and EAP, the Focus Group recommended a new legislation better regulating EAP, that early access concerns specific classes of medicines selected on the grounds of the need to guarantee a rapid access and to collect real world data, that early access can be accompanied by outcome-based and population-based MEA, and that MEA are embedded into the subsequent price and reimbursement negotiation.