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Neurosteroids are pleiotropic molecules involved in various neurodegenerative diseases with neuroinflammation. We assessed neurosteroids' serum levels in a cohort of Parkinson's Disease (PD) patients with heterozygous glucocerebrosidase (GBA) mutations (GBA-PD) compared with matched cohorts of consecutive non-mutated PD (NM-PD) patients and healthy subjects with (GBA-HC) and without (NM-HC) GBA mutations. A consecutive cohort of GBA-PD was paired for age, sex, disease duration, Hoehn and Yahr stage, and comorbidities with a cohort of consecutive NM-PD. Two cohorts of GBA-HC and HC were also considered. Clinical assessment included the Movement Disorder Society revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS) and the Montreal Cognitive Assessment (MoCA). Serum samples were processed and analyzed by liquid chromatography coupled with the triple quadrupole mass spectrometry. Twenty-two GBA-PD (males: 11, age: 63.68), 22 NM-PD (males: 11, age: 63.05), 14 GBA-HC (males: 8; age: 49.36), and 15 HC (males: 4; age: 60.60) were studied. Compared to NM-PD, GBA-PD showed more hallucinations and psychosis ( p < 0.05, Fisher's exact test) and higher MDS-UPDRS part-II ( p < 0.05). Most of the serum neurosteroids were reduced in both GBA-PD and NM-PD compared to the respective control cohorts, except for 5α-dihydroprogesterone. Allopregnanolone was the only neurosteroid significantly lower ( p < 0.01, Dunn's test) in NM-PD compared to GBA-PD patients. Only in GBA-PD, allopregnanolone, and pregnanolone levels correlated (Spearman) with a more severe MDS-UPDRS part-III. Allopregnanolone levels also negatively correlated with MoCA scores, and pregnanolone levels correlated with more pronounced bradykinesia. This pilot study provides the first observation of changes in neurosteroid peripheral levels in GBA-PD. The involvement of the observed changes in the development of neuropsychological and motor symptoms of GBA-PD deserves further attention.

Monosomy 3 in uveal melanoma (UM) increases the risk of lethal metastases, mainly in the liver, which serves as the major site for the storage of excessive glucose and the metabolization of the dietary flavonoid quercetin. Although primary UMs with monosomy 3 exhibit a higher potential for basal glucose uptake, it remains unknown as to whether glycolytic capacity is altered in such tumors. Herein, we initially analyzed the expression of n = 151 genes involved in glycolysis and its interconnected branch, the "pentose phosphate pathway (PPP)", in the UM cohort of The Cancer Genome Atlas Study and validated the differentially expressed genes in two independent cohorts. We also evaluated the effects of quercetin on the growth, survival, and glucose metabolism of the UM cell line 92.1. The rate-limiting glycolytic enzyme PFKP was overexpressed whereas the ZBTB20 gene (locus: 3q13.31) was downregulated in the patients with metastases in all cohorts. Quercetin was able to impair proliferation, viability, glucose uptake, glycolysis, ATP synthesis, and PPP rate-limiting enzyme activity while increasing oxidative stress. UMs with monosomy 3 display a stronger potential to utilize glucose for the generation of energy and biomass. Quercetin can prevent the growth of UM cells by interfering with glucose metabolism.

(1) Background: Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is characterized by late-onset cerebellar ataxia, bilateral vestibulopathy, and sensory neuronopathy mostly due to biallelic RFC1 expansion. (2) Objectives: The aim of this case series is to describe vestibular, gait, and speech alterations in CANVAS via a systematic approach. (3) Methods: All patients (n = 5) underwent a standardized clinical-instrumental examination, including the perceptual and acoustic analysis of speech, instrumental gait, and balance analysis (posturographic data were acquired using a force plate [Kistler, Winterthur, Switzerland] while 3D gait analysis, inclusive of surface electromyography, was acquired using a motion capture system [SMART DX, BTS Bioengineering, Milan, Italy], a wireless electromyograph [FreeEMG, BTS Bioengineering, Milan, Italy]), and vestibular assessment with video-oculography. (4) Results: Five patients were included in the analysis: three females (patients A, B, C) and two males (patients D and E) with a mean age at evaluation of 62 years (SD ± 15.16, range 36-74). The mean age of symptoms' onset was 55.6 years (SD ± 15.04, range 30-68), and patients were clinically and instrumentally evaluated with a mean disease duration of 6.4 years (SD ± 0.54, range 6-7). Video-Frenzel examination documented spontaneous downbeat nystagmus enhanced on bilateral gaze in all patients, except for one presenting with slight downbeat nystagmus in the supine position. All patients exhibited different degrees of symmetrically reduced VOR gain for allsix semicircular canals on the video-head impulse test and an unexpectedly normal ("false negative") VOR suppression, consistent with combined cerebellar dysfunction and bilateral vestibular loss. Posturographic indices were outside their age-matched normative ranges in all patients, while 3D gait analysis highlighted a reduction in ankle dorsiflexion (limited forward rotation of the tibia over the stance foot during the stance phase of gait and fatigue of the dorsiflexor muscles) and variable out-of-phase activity of plantar flexors during the swing phase. Finally, perceptual-acoustic evaluation of speech showed ataxic dysarthria in three patients. Dysdiadochokinesis, rhythm instability, and irregularity were observed in the oral diadochokinesis task. (5) Conclusions: CANVAS is a recently discovered syndrome that is gaining more and more relevance within late-onset ataxias. In this paper, we aimed to contribute to a detailed description of its phenotype.

(1) Background: The standard first-line therapy for advanced adrenocortical carcinoma (ACC) is represented by EDP-M (etoposide, doxorubicin, cisplatin + mitotane). Progestins have shown cytotoxic activity both in vitro and in vivo on ACC; better EDP-M tolerability and efficacy have been hypnotized due to the association with progestins. (2) Methods: The feasibility and tolerability of EDP-M combined with oral megestrol acetate (EDP-MM) were tested in 24 patients (pts) affected by metastatic ACC with a low performance status (PS); the case group was compared with a 48 pts control group according to the propensity score. The secondary objectives were clinical benefit rate (CBR), progression-free survival (PFS), and overall survival (OS). (3) Results: Thirteen pts (54.2%) in the EDP-MM population experienced progestin-related toxicities; in particular, five pts experienced vaginal bleeding (20.8%); four pts experienced weight gain (16.7%); and thromboembolic events, worsening of hypertension, skin rashes, and hyperglycemia were registered in one patient each (4.2%). This led to the discontinuation of megestrol acetate in four pts (16.7%). EDP-M-related toxicities were similar in both groups. No differences in PFS and OS curves were observed; the CBR was 75.0% and 60.4%, respectively. (4) Conclusions: The association of EDP-M + megestrol acetate in ACC pts with a low PS is feasible and well tolerated; its efficacy appeared to be non-inferior to EDP-M administered to pts with a good PS.

Background: Adrenocortical cancer (ACC) is a rare malignancy with a dismal prognosis. The treatment includes mitotane and EDP chemotherapy (etoposide, doxorubicin, and cisplatin). However, new therapeutic approaches for advanced ACC are needed, particularly targeting the metastatic process. Here, we deepen the role of progesterone as a new potential drug for ACC, in line with its antitumoral effect in other cancers., Methods: NCI-H295R, MUC-1, and TVBF-7 cell lines were used and xenografted in zebrafish embryos. Migration and invasion were studied using transwell assays, and MMP2 activity was studied using zymography. Apoptosis and cell cycle were analyzed by flow cytometry., Results: Progesterone significantly reduced xenograft tumor area and metastases formation in embryos injected with metastatic lines, MUC-1 and TVBF-7. These results were confirmed in vitro, where the reduction of invasion was mediated, at least in part, by the decrease in MMP2 levels. Progesterone exerted a long-lasting effect in metastatic cells. Progesterone caused apoptosis in NCI-H295R and MUC-1, inducing changes in the cell-cycle distribution, while autophagy was predominantly activated in TVBF-7 cells., Conclusion: Our results give support to the role of progesterone in ACC. The involvement of its analog (megestrol acetate) in reducing ACC progression in ACC patients undergoing EDP-M therapy is now under investigation in the PESETA phase II clinical study.

Parkinson's disease (PD) is a multifarious neurodegenerative disease. Its pathology is characterized by a prominent early death of dopaminergic neurons in the pars compacta of the substantia nigra and the presence of Lewy bodies with aggregated α-synuclein. Although the α-synuclein pathological aggregation and propagation, induced by several factors, is considered one of the most relevant hypotheses, PD pathogenesis is still a matter of debate. Indeed, environmental factors and genetic predisposition play an important role in PD. Mutations associated with a high risk for PD, usually called monogenic PD, underlie 5% to 10% of all PD cases. However, this percentage tends to increase over time because of the continuous identification of new genes associated with PD. The identification of genetic variants that can cause or increase the risk of PD has also given researchers the possibility to explore new personalized therapies. In this narrative review, we discuss the recent advances in the treatment of genetic forms of PD, focusing on different pathophysiologic aspects and ongoing clinical trials.

Many clinical trials have investigated the role of ICIs in PM, with contrasting results. We performed a systematic review and meta-analysis of clinical trials testing single-agent anti-Programmed Death -1 (PD-1)/Programmed Death-Ligand 1 (PD-L1), anti-Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) or combined treatment in PM patients, analyzing response and survival rate as well as safety data. We selected 17 studies including 2328 patients. Both OS and PFS rates were significantly higher with combined ICI treatments than with single agent anti-PD-1/PD-L1 (p < 0.001 and p = 0.006, respectively) or anti CTLA-4 (p < 0.001) treatments. ORR and DCR for all ICI treatments were 20% (95% CI 13−27%) and 56% (95% CI 45−67%), respectively, and they did not significantly differ between combined and single agent treatments (p = 0.088 and p = 0.058, respectively). The 12-month OS and 6-month PFS rates did not differ significantly (p = 0.0545 and p = 0.1464, respectively) among pre-treated or untreated patients. Combined ICI treatments had a significantly higher rate of Adverse Events (AEs) (p = 0.01). PD-L1-positive patients had a higher probability of response and survival. In conclusion, combined ICI treatments have higher efficacy than single agents but are limited by higher toxicity. Efficacy was independent of treatment line, so a customized sequential strategy should still be speculated. PD-L1 expression could influence response to ICIs; however, reliable biomarkers are warranted.

(1) Purpose: To evaluate the anatomy and perfusion of choroidal substructures in third-trimester pregnant women using optical coherence tomography (OCT) and OCT angiography (OCTA) imaging. (2) Methods: In this cross-sectional study, women in their third trimester of uncomplicated pregnancy and non-pregnant age-matched women were recruited. Participants underwent enhanced depth imaging (EDI) OCT and OCTA. Subfoveal choroidal thickness (SFCT), as well as choroidal sublayer perfusion, were compared between groups. (3) Results: In total, 26 eyes of 26 pregnant and 26 eyes of 26 non-pregnant women were included. The median age in both groups was 29 years. The median SFCT was 332 (211-469) µm in the pregnant group and 371.5 (224-466) µm in the non-pregnant cohort ( p = 0.018). The median choriocapillaris perfusion (CCP) was significantly lower in the pregnant group (46% vs. 48%, p = 0.039). Moreover, Haller's layer perfusion correlated significantly with mean arterial pressure in non-pregnant women (CC = 0.430, p = 0.028) but not in pregnant ones (CC = 0.054, p = 0.792). (4) Conclusions: SFCT was found to be thinner and CCP was lower in third-trimester pregnant women. Hormonal changes during pregnancy and consecutive impacts on autoregulation of small choroidal vessels might play an important role. Therefore, altered choroidal measurements during third-trimester pregnancy should be carefully evaluated as, to some extent, it could be a normal physiological change.

Background: Primary vitreoretinal lymphoma (PVRL), a rare malignancy of the eye, is strongly related to primary central nervous system lymphoma (PCNSL). We hypothesized that lymphoma cells disseminate to the CNS and eye tissue via distinct homing receptors. The objective of this study was to test expression of CXCR4, CXCR5, CXCR7 and CD44 homing receptors on CD20 positive B-lymphoma cells on enucleated eyes using a PCNSL xenograft mouse model. Methods: We used indirect immunofluorescence double staining for CD20/CXCR4, CD20/CXCR5, CD20/CXCR7 and CD20/CD44 on enucleated eyes of a PCNSL xenograft mouse model with PVRL phenotype (PCNSL group) in comparison to a secondary CNS lymphoma xenograft mouse model (SCNSL group). Lymphoma infiltration was evaluated with an immunoreactive score (IRS). Results: 11/13 paired eyes of the PCNSL but none of the SCNSL group were infiltrated by CD20-positive cells. Particularly the choroid and to a lesser extent the retina of the PCNSL group were infiltrated by CD20+/CXCR4+, CD20+/CXCR5+, few CD20+/CD44+ but no CD20+/CXCR7+ cells. Expression of CXCR4 (p = 0.0205), CXCR5 (p = 0.0004) and CD44 (p < 0.0001) was significantly increased in the PCNSL compared to the SCNSL group. Conclusions: CD20+ PCNSL lymphoma cells infiltrating the eye co-express distinct homing receptors such as CXCR4 and CXCR5 in a PVRL homing mouse model. These receptors may be involved in PVRL homing into the eye., Competing Interests: Nikolas von Bubnoff received research support from Novartis and honoraria from Novartis, Takeda and from the Forum f&uuml;r Medizinische Fortbildung. Peter C Reinacher receives research support from Else Kr&ouml;ner-Fresenius Foundation (Germany) and Fraunhofer Foundation (Germany), received personal honoraria for lectures or advice from Boston Scientific (USA) and Brainlab (Germany) and is consultant for Boston Scientific (USA), Inomed (Germany) and Brainlab (Germany). Ayseg&uuml;l Tura receives financial support from Novartis Pharma GmbH, Germany. The other authors have no competing interests to declare that are relevant to the content of this article.

A few cases of parkinsonism linked to COVID-19 infection have been reported so far, raising the possibility of a post-viral parkinsonian syndrome. The objective of this review is to summarize the clinical, biological, and neuroimaging features of published cases describing COVID-19-related parkinsonism and to discuss the possible pathophysiological mechanisms. A comprehensive literature search was performed using NCBI's PubMed database and standardized search terms. Thirteen cases of COVID-19-related parkinsonism were included (7 males; mean age: 51 years ± 14.51, range 31-73). Patients were classified based on the possible mechanisms of post-COVID-19 parkinsonism: extensive inflammation or hypoxic brain injury within the context of encephalopathy (n = 5); unmasking of underlying still non-symptomatic Parkinson's Disease (PD) (n = 5), and structural and functional basal ganglia damage (n = 3). The various clinical scenarios show different outcomes and responses to dopaminergic treatment. Different mechanisms may play a role, including vascular damage, neuroinflammation, SARS-CoV-2 neuroinvasive potential, and the impact of SARS-CoV-2 on α-synuclein. Our results confirm that the appearance of parkinsonism during or immediately after COVID-19 infection represents a very rare event. Future long-term observational studies are needed to evaluate the possible role of SARS-CoV-2 infection as a trigger for the development of PD in the long term.

(1) Background: Silicone oil (SO) can be used as an endotamponade during vitreoretinal surgery for retinal detachment. There is emerging evidence that SO filling of the vitreous cavity and its removal may impact macular perfusion. So far, studies have not focused on choroidal sublayer perfusion, yet. (2) Methods: Optical coherence tomography angiography was applied in 19 patients with SO endotamponade before and four weeks after removal of SO. (3) Results: Perfusion of choriocapillaris increased significantly after SO removal, while perfusion of Haller's and Sattler's layer decreased significantly. (4) Conclusions: Removal of SO impacts choroidal perfusion and leads to a perfusion shift within choroidal sublayers. This study underlines that it is worth to conduct larger prospective studies that evaluate the choroidal perfusion and its functional implications in more detail.

Chemotherapy resistance is one of the reasons for eye loss in patients with retinoblastoma (RB). RB chemotherapy resistance has been studied in different cell culture models, such as WERI-RB1. In addition, chemotherapy-resistant RB subclones, such as the etoposide-resistant WERI-ETOR cell line have been established to improve the understanding of chemotherapy resistance in RB. The objective of this study was to characterize cell line models of an etoposide-sensitive WERI-RB1 and its etoposide-resistant subclone, WERI-ETOR, by proteomic analysis. Subsequently, quantitative proteomics data served for correlation analysis with known drug perturbation profiles. Methodically, WERI-RB1 and WERI-ETOR were cultured, and prepared for quantitative mass spectrometry (MS). This was carried out in a data-independent acquisition (DIA) mode. The raw SWATH (sequential window acquisition of all theoretical mass spectra) files were processed using neural networks in a library-free mode along with machine-learning algorithms. Pathway-enrichment analysis was performed using the REACTOME-pathway resource, and correlated to the molecular signature database (MSigDB) hallmark gene set collections for functional annotation. Furthermore, a drug-connectivity analysis using the L1000 database was carried out to associate the mechanism of action (MOA) for different anticancer reagents to WERI-RB1/WERI-ETOR signatures. A total of 4756 proteins were identified across all samples, showing a distinct clustering between the groups. Of these proteins, 64 were significantly altered (q < 0.05 & log2FC |>2|, 22 higher in WERI-ETOR). Pathway analysis revealed the “retinoid metabolism and transport” pathway as an enriched metabolic pathway in WERI-ETOR cells, while the “sphingolipid de novo biosynthesis” pathway was identified in the WERI-RB1 cell line. In addition, this study revealed similar protein signatures of topoisomerase inhibitors in WERI-ETOR cells as well as ATPase inhibitors, acetylcholine receptor antagonists, and vascular endothelial growth factor receptor (VEGFR) inhibitors in the WERI-RB1 cell line. In this study, WERI-RB1 and WERI-ETOR were analyzed as a cell line model for chemotherapy resistance in RB using data-independent MS. Analysis of the global proteome identified activation of “sphingolipid de novo biosynthesis” in WERI-RB1, and revealed future potential treatment options for etoposide resistance in RB.

Monosomy-3 in uveal melanoma (UM) cells increases the risk of fatal metastases. The gene encoding the low-affinity glucose transporter GLUT2 resides on chromosome 3q26.2. Here, we analyzed the expression of the glucose transporters GLUT1, GLUT2, and GLUT3 with regard to the histological and clinical factors by performing immunohistochemistry on the primary tumors of n = 33 UM patients. UMs with monosomy-3 exhibited a 57% lower immunoreactivity for GLUT2 and a 1.8×-fold higher ratio of GLUT1 to total GLUT1-3. The combined levels of GLUT1-3 proteins were reduced in the irradiated but not the non-irradiated tumors with monosomy-3. GLUT3 expression was stronger in the irradiated samples with disomy-3 versus monosomy-3, but the ratio of the GLUT3 isoform to total GLUT1-3 did not differ with regard to the monosomy-3 status in the irradiated or non-irradiated subgroups. Systemic metastases were associated with the presence of monosomy-3 in the primary and circulating tumor cells as well as a higher GLUT1 ratio. Upregulation of the high-affinity glucose transporter GLUT1 possibly as a compensation for the low-affinity isoform GLUT2 may be enhancing the basal glucose uptake in the UM cells with monosomy-3. Prevention of hyperglycemia might, therefore, be a valuable approach to delay the lethal UM metastases.

Background and objectives: Ocular massage (OM) is used as a treatment option for acute retinal artery occlusion, under the assumption that it induces vessel dilatation and enhances perfusion. Since evidence of ocular perfusion alteration due to OM is lacking, we investigate the impact of OM on the hemodynamics of the posterior pole in healthy eyes in a noninvasive fashion by using optical coherence tomography angiography (OCTA). Materials and Methods: A prospective study was conducted on healthy volunteers, each of whom underwent measurements of intraocular pressure (IOP), central macular thickness (CMT), subfoveal choroidal thickness (SFCT), radial peripapillary capillary perfusion (RPCP), superficial capillary plexus perfusion (SCPP), deep capillary plexus perfusion (DCPP), choriocapillaris perfusion (CCP), Sattler's layer perfusion (SLP) and Haller's layer perfusion (HLP) before and after OM. OM was performed for 2 min, consisting of 10-s turns of compression and decompression of the globe. Results: A total of 21 eyes from 21 participants (median age 29) were included. After OM, IOP significantly declined ( p < 0.001), while SFCT ( p < 0.005), SCPP ( p < 0.001), DCPP ( p = 0.004) and CCP ( p = 0.008) significantly increased. CMT, RPCP, SLP and HLP did not show any significant alteration due to OM. Changes in SCPP correlated positively with changes in CCP and vice versa. Conclusions: OCTA-based analysis in healthy adults following OM demonstrated a significant increase of retinal perfusion values, assumed to be due to failure of autoregulatory mechanisms. These findings may indicate a positive effect of OM as a treatment option for patients with acute retinal artery occlusion.

The prolonged storage of glucose as glycogen can promote the quiescence of tumor cells, whereas the accumulation of an aberrant form of glycogen without the primer protein glycogenin can induce the metabolic switch towards a glycolytic phenotype. Here, we analyzed the expression of n = 67 genes involved in glycogen metabolism on the uveal melanoma (UM) cohort of the Cancer Genome Atlas (TCGA) study and validated the differentially expressed genes in an independent cohort. We also evaluated the glycogen levels with regard to the prognostic factors via a differential periodic acid-Schiff (PAS) staining. UMs with monosomy-3 exhibited a less glycogenetic and more insulin-resistant gene expression profile, together with the reduction of glycogen levels, which were associated with the metastases. Expression of glycogenin-1 (Locus: 3q24) was lower in the monosomy-3 tumors, whereas the complementary isoform glycogenin-2 (Locus: Xp22.33) was upregulated in females. Remarkably, glycogen was more abundant in the monosomy-3 tumors of male versus female patients. We therefore provide the first evidence to the dysregulation of glycogen metabolism as a novel factor that may be aggravating the course of UM particularly in males.

We evaluated tumor response at Computed Tomography (CT) according to three radiologic criteria: RECIST 1.1, CHOI and tumor volume in 34 patients with metastatic adrenocortical carcinoma (ACC) submitted to standard chemotherapy. These three criteria agreed in defining partial response, stable or progressive disease in 24 patients (70.5%). Partial response (PR) was observed in 29.4%, 29.4% and 41.2% of patients according to RECIST 1.1, CHOI and tumor volume, respectively. It was associated with a favorable prognosis, regardless of the criterion adopted. The concordance of all the 3 criteria in defining the disease response identified 8 patients (23.5%) which displayed a very good prognosis: median progression free survival (PFS) and overall survival (OS) 14.9 and 37.7 months, respectively. Seven patients (20.6%) with PR assessed by one or two criteria, however, still had a better prognosis than non-responding patients, both in terms of PFS: median 12.3 versus 9.9 months and OS: 21 versus 12.2, respectively. In conclusions, the CT assessment of disease response of ACC patients to chemotherapy with 3 different criteria is feasible and allows the identification of a patient subset with a more favorable outcome. PR with at least one criterion can be useful to early identify patients that deserve continuing the therapy.

Background: This study explores the impact of Hypertermic Intra PEritoneal Chemotherapy (HIPEC) on adrenocortical carcinoma (ACC) management through a safety analysis completed by a preliminary evaluation of survival performances., Methods: Retrospective chart review of 27 patients submitted to surgical treatment completed by HIPEC for primary (SP, 13 patients) or recurrent (SR, 14 patients, 17 treatments) ACC. Safety was evaluated by means of procedural morbidity and mortality. Survival performances included multiple end points: local/peritoneal disease-free survival (l/pDFS), overall progression-free survival (OPFS), and overall survival (OS)., Results: In the SP group, mortality was nil and morbidity was 46% (major 23%). At a median follow-up of 25 months, the median value for all the different survival measures had not been reached. Mortality was also nil in the SR group. However, morbidity was 77% (major 18%). Median l/pDFS and OPFS were 12 ± 4 and 8 ± 2 months, respectively. At a median follow-up of 30 months, median OS had not been reached., Conclusion: Surgery and HIPEC is an invasive procedure. Its employment in the surgery for primary setting deserves attention as it may affect oncologic outcomes positively. Its value in the management of recurrences seems less appreciable, albeit it may find its place in the multimodal management of a rare disease for which multiple therapeutic options do not yet exist.

Etoposide, doxorubicin and cisplatin plus oral mitotane (EDP-M) comprise the reference regimen in the management of patients with adrenocortical carcinoma (ACC). In this paper, we described the outcome of 58 patients with advanced/metastatic ACC consecutively treated with EDP-M in a reference center for this rare disease in Italy. In this series, EDP-M obtained a partial response in 50% of patients; median progression free survival (PFS) and overall survival were 10.1 months (95% Confidence Interval [CI 95%] 8.1-12.8) and 18.7 months (95% CI: 14.6-22.8), respectively. EDP-M was not interrupted in five patients showing disease progression after two cycles without the appearance of new lesions and mitotane levels below the therapeutic range. In two of them, the disease remained stable at further imaging evaluations and the other three obtained a partial response. Twenty-six responding patients underwent surgery of residual disease and 13 of them became disease free. Surgery identified a pathological complete response (pCR) in four patients (7%) and Ki67 expression in post-chemotherapy tumor specimens, inferior to 15% (median value), was associated with better PFS and survival. In the present study, the EDP-M regimen is confirmed to have a limited efficacy. Early disease progression does not mean treatment inefficacy. Surgery of residual disease in partially responding patients allows for the detection of pCR in few of them and this condition is predictive of long-term survival. Ki67 expression of post-chemotherapy residual disease could be an additional prognostic factor that deserves to be studied further.

Mitotane is the only drug approved for the treatment of adrenocortical carcinoma (ACC). The regimen to be added to mitotane is a chemotherapy including etoposide, doxorubicin, and cisplatin. This pharmacological approach, however, has a limited efficacy and significant toxicity. Evidence indicates that ACC seems to be sensitive to alkylating agents. Trabectedin is an anti-tumor drug that acts as an alkylating agent with a complex mechanism of action. Here, we investigated whether trabectedin could exert a cytotoxic activity in in vitro cell models of ACC. Cell viability was evaluated by MTT assay on ACC cell lines and primary cell cultures. The gene expression was evaluated by q-RT-PCR, while protein expression and localization were studied by Western blot and immunocytochemistry. Combination experiments were performed to evaluate their interaction on ACC cell line viability. Trabectedin demonstrated high cytotoxicity at sub-nanomolar concentrations in ACC cell lines and patient-derived primary cell cultures. The drug was able to reduce /β catenin nuclear localization, although it is unclear whether this effect is involved in the observed cytotoxicity. Trabectedin/mitotane combination exerted a synergic cytotoxic effect in NCI-H295R cells. Trabectedin has antineoplastic activity in ACC cells. The synergistic cytotoxic activity of trabectedin with mitotane provides the rationale for testing this combination in a clinical study.

Despite the implication of vascular endothelial growth factor-A (VEGF-A) in the pathophysiology of uveal melanoma (UM), the anti-VEGF-A antibody bevacizumab yielded conflicting results on UM growth. Here, we evaluated whether bevacizumab and ranibizumab, a humanized Fab-fragment against VEGF-A, can enter UM cells and induce a sustained physiological response. The primary and metastatic UM cell lines Mel-270 and OMM-2.5 were exposed to bevacizumab or ranibizumab for one day and were maintained further in untreated medium for a total of three days. Both antibodies significantly reduced the levels of extracellular VEGF-A and the angiogenic potential of the conditioned medium after one day. These inhibitory effects of bevacizumab diminished by day three. Ranibizumab suppressed the metabolic activity, proliferation, and intracellular VEGF-A levels in a cell-type and concentration-dependent manner, whereas bevacizumab exerted no effect. Both drugs were detected inside early endosomes within the UM cells, with the stronger and sustained colocalization of ranibizumab. Our results therefore demonstrated the more potent and persistent suppressive activity of ranibizumab on the UM cells, possibly due to its higher level of uptake and prolonged intracellular retention. Further research on the endosome dynamics in UM cells might provide valuable insight into the response of these heterogenous tumors to therapeutic antibodies.