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In recent years, bacterial resistance to conventional antibiotics has become a major concern in the medical field. The global misuse of antibiotics in clinics, personal use, and agriculture has accelerated this resistance, making infections increasingly difficult to treat and rendering new antibiotics ineffective more quickly. Finding new antibiotics is challenging due to the complexity of bacterial mechanisms, high costs and low financial incentives for the development of new molecular scaffolds, and stringent regulatory requirements. Additionally, innovation has slowed, with many new antibiotics being modifications of existing drugs rather than entirely new classes. Antimicrobial peptides (AMPs) are a valid alternative to small-molecule antibiotics offering several advantages, including broad-spectrum activity and a lower likelihood of inducing resistance due to their multifaceted mechanisms of action. However, AMPs face challenges such as stability issues in physiological conditions, potential toxicity to human cells, high production costs, and difficulties in large-scale manufacturing. A reliable strategy to overcome the drawbacks associated with the use of small-molecule antibiotics and AMPs is combination therapy, namely the simultaneous co-administration of two or more antibiotics or the synthesis of covalently linked conjugates. This review aims to provide a comprehensive overview of the literature on the development of antibiotic-AMP conjugates, with a particular emphasis on critically analyzing the design and synthetic strategies employed in their creation. In addition to the synthesis, the review will also explore the reported antibacterial activity of these conjugates and, where available, examine any data concerning their cytotoxicity.

Objective: The development of an efficient, multifunctional drug delivery system overcoming different obstacles generally associated with drug formulations, including the poor accumulation of the active principle in the target site and its sustained release for prolonged time., Methods: Our study proposes the development of a fluorinated poly(amidoamine) (PAMAM) carrier prodrug combining drug release boosted in alkaline environments with a possible implementation in 19 F MRI applications. In particular, we functionalized the terminal primary amines of PAMAM G2 and G4 through an ad hoc designed fluorinated ibuprofen-arginine Michael acceptor to obtain multifunctional ibuprofen-PAMAM-Arg conjugates., Results: These carriers demonstrated pH-dependent and sustained ibuprofen release for more than 5 days. This advantage was observed in both weak alkaline and physiological buffer solutions, allowing to overcome the limits associated to the burst release from similar fluorinated Arg-PAMAM dendrimers with ibuprofen physically encapsulated., Conclusion: These findings, coupled to the high biocompatibility of the system, suggest a potential synergistic biomedical application of our conjugates, serving as vehicles for drug delivery and as 19 F magnetic resonance imaging contrast agents., (© 2024. The Author(s).)

We assessed phonological and apraxic impairments in Hindi persons with aphasia (PwA) and compared them to Italian PwA reported in previous studies. Overall, we found strong similarities. Phonological errors were present across production tasks (repetition, reading and naming), most errors were non-lexical and, among those, a majority involved individual phonemes. There were significant effects of length, but not frequency. Hindi PwA, like the Italian PwA, showed strong effects of syllabic structure, with most errors occurring on consonants and weak syllabic positions, preserving syllable structure and simplifying phonemes or syllabic templates. These similarities were modulated by some language-specific patterns. Vowel insertions were more common in Hindi, possibly due to the presence of a central vowel, and segmental simplifications concentrated on marked aspiration and retroflection features. We hope our study will encourage further research in Hindi and other Indian languages. This will improve clinical diagnosis and our understanding of cross-linguistic differences.

Background and Aims: Increasing evidence supports the practicability of salivary cell-free (cf) miRNA as liquid biopsy markers in cancers. Its successful translation in the clinical setting requires reproducible approaches for saliva manipulation, in order to control for pre-analytical variables influencing miRNA stability. This study aims to define the optimal conditions to maintain the integrity of saliva during collection, transport and processing with respect to cf-miRNA quantification., Materials and Methods: Saliva was collected from 20 healthy subjects and 8 oral cancer patients. Two sampling methods were tested and different storage temperatures and times were evaluated. Salivary expression level of target miRNAs was quantified by qPCR. Comparison between group mean values at specific conditions were performed using paired t-tests. Agreement between measurements was evaluated using a Bland-Altman plot., Results: Different collection methods revealed comparable levels of salivary miR-484 and miR-106b-5p in both subject cohorts. MiRNAs were stable for up to 48 h at 4 °C in saliva supernatant, showing significant alteration after 96 h. Mid-term storage of supernatant at -20 °C decreased miRNA stability significantly compared to standard -80 °C., Conclusions: Cf-miRNA in saliva were slightly altered by collection methods and storage conditions, both in healthy and in pathological contexts, and remained stable for a period of time compatible with main clinical routine needs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: PB reports advisory board participation and/or research activities outside the object of the current research with Merck, Sanofi-Regeneron, Merck Sharp & Dohme, Sun Pharma, Angelini, Nestlè, Elevar., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The treatment of locally advanced (LA) Head and Neck Squamous Cell Carcinoma (HNSCC) is based on surgery followed by (chemo)radiation or on curative (chemo)radiation, depending on site and stage. Despite optimal locoregional treatment, about 50% of patients recur, with a huge impact on prognosis and substantial morbidity. The advent of immunotherapy (IT) with immune checkpoint inhibitors (ICIs) changed the paradigm of systemic treatment for recurrent/metastatic (RM) disease, showing activity, efficacy, and safety in both platinum-resistant and platinum-naïve patients. Such data led clinicians to design clinical trials to investigate early administration of IT even in the neoadjuvant or window of opportunity setting. In this review, we examine the published and ongoing trials investigating IT in the neoadjuvant setting for LA HNSCC. We address the current challenges of this treatment modality: optimal patient selection for neoadjuvant IT; choosing the appropriate systemic approach to enhance response without compromising tolerability; determining the ideal study endpoint, with a focus on major pathological response as a potential surrogate for overall survival; evaluating treatment response through imaging, considering the discordance between radiological and pathological assessments; and the influence of neoadjuvant IT response on locoregional treatment de-escalation strategies., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Paolo Bossi: Participation to advisory board or conference honoraria for:Merck, Sanofi-Regeneron, Merck Sharp & Dohme, Sun Pharma, Angelini, Nestlè, Elevar. The other authors reported no potential conflict of interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Endometrial carcinoma (EC) defines a heterogeneous group of neoplastic diseases originating from the transformation of endometrial cells that constitute the internal lining of the uterus. To date several molecular targets have been analysed to describe the natural course of the disease, claudins being among these. Claudins are the main components of tight junctions (TJs), and their main functions are ascribed to the compartmentalization of tissues and cell-cell communication by means of intracellular ions diffusion: these features are typical of epithelial cells. Their overexpression, mis-localization or loss contribute to the malignancy of EC cells. This review collected all available data regarding the expression, regulation and claudin-related signaling pathways to provide a comprehensive view on the influence of claudin in EC progression. Further, the translational potential of claudin differential expression was explored, indicating that their role in personalized medicine could also contribute to EC therapy besides their employment for diagnosis and prognosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Objective: Neonatal screening and early treatment have changed the natural history of PKU, preventing severe neurological and intellectual disability. Nevertheless, the outcome of the disease in early-treated adult patients (ETPKU) is less than optimal, the predictive value of metabolic biomarkers is feeble, and the recommended levels of blood phenylalanine (Phe) for adulthood are controversial. A crucial question whose answer will improve our understanding and treatment of PKU is whether cognitive outcomes can be modulated by levels of Phe even in early-treated adults. To address this question, we carried out an interventional study in seven ETPKU women planning a pregnancy., Methods: They underwent an extensive neurocognitive assessment at baseline, and 3 and 6 months after having attained the blood Phe concentration recommended to prevent PKU fetopathy, but before pregnancy., Results: After 3 and 6 months with a stable blood Phe level of about 240 μmol/L, all participants experienced significant improvements in almost all neurocognitive domains and tasks. IQ also increased of 11 to 21 points from the last assessment before enrolment. This pattern remained strong and consistent after correction for multiple comparisons., Conclusion: Our results indicate that a) strong cognitive improvement is possible even in adulthood and may be demonstrated by lowering Phe near normal levels; b) testing cognition under different metabolic conditions may unveil an individual vulnerability to Phe. These results pave the way for personalised treatment of the disease in adults with ETPKU., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The main neurological, cognitive, and behavioural consequences of phenylketonuria have been eradicated thanks to new-born screening and Phe-restricted diet therapy. However, the effects of high phenylalanine levels during adolescence and adulthood on neurocognitive functions remain a concern. This systematic review aimed at collecting clinical data suggesting the safest metabolic target for early treated PKU during the second decade of life. Twenty studies met the inclusion criteria for full-text review. Relevant studies included papers that (a) examined the relationship between metabolic control and neurocognitive functions during adolescence or (b) investigated the impact of metabolic control in adolescence on adult outcomes. Most studies showed a positive correlation between metabolic control during adolescence and neurocognitive outcomes across ages. This was true both for IQ and executive functions, although data on executive functions were less clear, and it remains to be established whether they are more vulnerable to Phe than IQ. Taken together present evidence confirm brain vulnerability to Phe during adolescence and suggests that low average Phe levels and low Phe fluctuations should be maintained throughout life. While results are fully compatible with current European recommendations, clinical and methodological limitations coupled with remarkable interindividual variability prevented a clear identification of a safe threshold for Phe blood levels during adolescence., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Polyamidoamine (PAMAM) dendrimers are among the most studied cationic polymers as non-viral gene delivery vectors. However, an "ideal" PAMAM-based gene delivery vector is still missing due to the high manufacturing costs and non-negligible cytotoxicity associated with the use of high-generation dendrimers, whereas low-generation dendrimers are far from displaying efficient gene transfection. In order to cover this gap in the literature, in this study, we propose the functionalization of the outer primary amines of PAMAM G2 and PAMAM G4 with building blocks bearing fluorinated moieties along with a guanidino functional group. We have designed and synthetized two fluorinated arginine (Arg)-based Michael acceptors which were straightforwardly "clicked" to PAMAM dendrimers without the need for coupling reagents and/or catalysts. The obtained conjugates, in particular, derivative 1 formed starting from the low-cost PAMAM G2 and a building block bearing two trifluoromethyl groups, were able to efficiently complex plasmid DNA, had negligible cytotoxicity, and showed improved gene transfection efficiency as compared to undecorated PAMAM dendrimers and a corresponding unfluorinated PAMAM-Arg derivative, with derivative 1 being two orders of magnitude more efficient than the gold standard branched polyethylenimine, bPEI, 25 kDa. These results highlight the importance of the presence of trifluoromethyl moieties for both gene transfection and a possible future application in 19 F magnetic resonance imaging.

Background: Sentinel lymph node (SLN) biopsy in cutaneous melanoma patients evaluates the regional draining basin for occult micrometastatic disease. Occasionally, nonidentification of SLN impairs the acquisition of this important prognostic factor., Objectives: To investigate the outcomes of melanoma patients with negative lymphoscintigraphic findings and patients who underwent SLN biopsy from 2004 to 2015 ( n  = 1200) were retrospectively reviewed for tumor characteristics and clinical outcomes., Methods: Patients with nonvisualized lymph nodes (NV group) who underwent only preoperative lymphoscintigraphy were separated and compared with a cohort drawn from all melanoma patients who completed the surgical procedure within the same period (V group)., Results: A negative lymphoscintigraphic scan was observed in 38 cases (3.2% of all patients). The NV group showed a significantly older age (median 66.0 vs. 48.3 years; P  < 0.0001). Head and neck melanomas were more frequent in the NV group compared to the control group (25.1 vs. 7.8%; P  = 0.009). Tumor characteristics such as ulceration and Breslow thickness do not influence the lymphoscintigraphy result. No differences were found in overall survival (OS) and disease-free survival (DFS) between the groups., Conclusions: The nonvisualization of regional lymph nodes by lymphoscintigraphy is more frequent in older patients with head and neck melanomas. From the clinical point of view, no specific recommendation emerged for patients' management because the nonvisualization of the SLN did not show a significant influence on DFS and OS rates. However, lack of knowledge of lymph node status suggests performing a tighter follow-up eventually by ultrasound evaluation of all potential lymph node drainage basins., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Adenoid cystic carcinoma (ACC) of salivary gland is a slowly growing tumor showing a propensity for delayed recurrence, with decreased survival rates. The identification of poor prognosis patients may help in defining molecular-based targeted strategies in this rare disease orphan of new treatments. Through a gene expression microarray-based approach followed by GSE functional analysis the expression profile of 46 primary untreated ACC samples and of ACC (h-TERT) tumor cells was analyzed. Patients who experienced early relapse showed enrichment in proliferation-related gene sets, including the G2-M checkpoint, E2F and myc targets, and in gene sets related to IFN signaling and aberrant proteostasis (FDR < 0.1), indicating increased mitotic and transcriptional activity in aggressive ACC. Similar functions were enriched in ACC samples classified by immunohistochemical staining as p63-negative, which exhibited increased protein burden and activation of pro-survival stress response pathways compared to p63-positive tumors. Compared to ACC tissues, ACC (h-TERT) cells share transcriptional features of aggressive p63-negative tumors. These data suggest association of specific pathway alterations with histopathological features of ACC, as recapitulated by p63 testing in patient prognostic stratification, anticipating new avenues for therapeutic intervention., (© 2023. The Author(s).)

Phenylketonuria (PKU) is a metabolic disease where Phenylalanine (Phe) rises much above normal levels. Cross-sectional and correlational studies provide valuable information on the importance of maintaining low blood-Phe to achieve good outcomes, but they may be confounded, at least partially, by differences in participant demographics. Moreover, the effect of Phe at older ages is difficult to ascertain because of strong associations between Phe levels across ages. Within-participant studies avoid confounding issues. We have reviewed these studies. We followed PRISMA guidelines to search the literature for studies reporting the impact of Phe changes within participants. Phe was either increased or decreased through diet relaxation/resumption or through pharmacological interventions. Forty-six separate articles reported, singly or in combination, results on cognition (N = 37), well-being (N = 22) and neurophysiological health (N = 14). For all studies, we established, in a binary way, whether a benefit of lower Phe was or was not demonstrated and compared numbers showing benefit versus a null or negative outcome. We then analyzed whether critical parameters (e.g., length of the study/condition for the change, size of Phe change achieved) influenced presence or absence of benefit. For a subset of studies that reported quantitative cognitive outcomes, we carried out a meta-analysis to estimate the size of change in cognitive performance associated with a change in Phe and its significance. There were significantly more studies with benefits than no benefits, both for cognitive and well-being outcomes, and a trend in this direction for neurophysiological outcomes. The meta-analysis showed a highly significant effect size both overall (0.55) and when studies with adults/adolescents were considered separately (0.57). There was some indication that benefits were easier to demonstrate when differences in Phe were larger and achieved across a longer period, but these effects were not always consistent. These results reinforce results from the literature by demonstrating the importance of lower Phe in children as well as in adolescents and adults, even when confounding factors in group composition are eliminated. The field would benefit from further studies where Phe levels are contrasted within-participants to ascertain how much Phe needs to be changed and for how long to see a difference and which measures demonstrate a difference (e.g., which cognitive tasks)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Our study estimated size of impairment for different cognitive functions in early-treated adults with PKU (AwPKU) by combining literature results in a meta-analytic way. We analysed a large set of functions (N = 19), each probed by different measures (average = 12). Data were extracted from 26 PKU groups and matched controls, with 757 AwPKU contributing 220 measures. Effect sizes (ESs) were computed using Glass' ∆ where differences in performance between clinical/PKU and control groups are standardized using the mean and standard deviation of the control groups. Significance was assessed using measures nested within independent PKU groups as a random factor. The weighted Glass' ∆ was - 0.44 for all functions taken together, and - 0.60 for IQ, both highly significant. Separate, significant impairments were found for most functions, but with great variability (ESs from -1.02 to -0.18). The most severe impairments were in reasoning, visual-spatial attention speed, sustained attention, visuo-motor control, and flexibility. Effect sizes were larger with speed than accuracy measures, and with visuo-spatial than verbal stimuli. Results show a specific PKU profile that needs consideration when monitoring the disease., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Histopathologic assessment of high-risk endometrial cancer (EC) suffers from intersubject variability and poor reproducibility. The pragmatic classification in four molecular subgroups helps to overcome these limits, showing a significant prognostic value. The "no specific molecular profile" (NSMP) is the most heterogeneous EC subgroup, requiring further characterization to better guide its clinical management. DNA sequencing of POLE exonuclease domain and immunohistochemistry for PMS2, MSH6, and p53 were performed in order to stratify a cohort of 94 high-risk EC patients in the four molecular subgroups. Moreover, a panel of seven additional biomarkers was tested. Patients were found to be 16% POLE-mutated, 36% mismatch repair-deficient, 27% p53-abnormal, and 21% NSMP. In the multivariable model, molecular groups confirmed their significant association with disease-specific survival and progression-free survival, with p53-abnormal and NSMP endometrial cancer characterized by poor outcomes. Among the additional evaluated biomarkers, L1CAM was the only one with a significant prognostic value within the NSMP subgroup. NSMP/L1CAM-positive patients experienced the worst outcome and were "early-relapsing" after platinum-based chemotherapy, with a significantly shorter platinum-free interval compared to L1CAM-negative patients. L1CAM appears to be a promising candidate as a prognostic and predictive biomarker in the high-risk NSMP subgroup, which is actually known to lack specific molecular markers.

For high-risk endometrial cancer (EC) patients, adjuvant chemotherapy is recommended to improve outcome. Yet, predictive biomarkers for response to platinum-based chemotherapy (Pt-aCT) are currently lacking. We tested expression of L1 cell-adhesion molecule (L1CAM), a well-recognised marker of poor prognosis in EC, in tumour samples from high-risk EC patients, to explore its role as a predictive marker of Pt-aCT response. L1CAM expression was determined using RT-qPCR and immunohistochemistry in a cohort of high-risk EC patients treated with Pt-aCT and validated in a multicentric independent cohort. The association between L1CAM and clinicopathologic features and L1CAM additive value in predicting platinum response were determined. The effect of L1CAM gene silencing on response to carboplatin was functionally tested on primary L1CAM-expressing cells. Increased L1CAM expression at both genetic and protein level correlated with high-grade, non-endometrioid histology and poor response to platinum treatment. A predictive model adding L1CAM to prognostic clinical variables significantly improved platinum response prediction (C-index 78.1%, P = .012). In multivariate survival analysis, L1CAM expression was significantly associated with poor outcome (HR: 2.03, P = .019), potentially through an indirect effect, mediated by its influence on response to chemotherapy. In vitro, inhibition of L1CAM significantly increased cell sensitivity to carboplatin, supporting a mechanistic link between L1CAM expression and response to platinum in EC cells. In conclusion, we have demonstrated the role of L1CAM in the prediction of response to Pt-aCT in two independent cohorts of high-risk EC patients. L1CAM is a promising candidate biomarker to optimise decision making in high-risk patients who are eligible for Pt-aCT., (© 2022 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

We assessed effects of semantic interference in people with aphasia (PWA). Two naming tasks (continuous naming and cyclic blocking) were contrasted with tasks which required suppression of competitors but minimized lexical access (probe task) or required extra-lexical mechanisms of control (Stroop task). In continuous naming, some PWA showed increased interference compared to control participants, with slower RTs and increased omissions. Others showed normal or weaker interference effects in terms of RTs but increased semantic errors. Patterns were consistent only between naming tasks. We explain results by assuming that some PWA are slow at implementing mechanisms of control/selection which weed-out competitors. Others, instead, will have activation difficulties which will induce them to lower the threshold needed for selection. Results highlight how different kinds of brain damage may induce different compensatory strategies and how semantic relatedness may induce both interference and facilitation. Implications for models of lexical selection are discussed.

In many recurrent and/or metastatic cancers, the advent of immunotherapy opens up new scenarios of treatment response, with new phenomena, such as pseudoprogression and hyperprogression. Because of this, different immune-related response criteria have been developed, and new therapeutic strategies adopted, such as treatment beyond progression. Moreover, the role of progression-free survival as a surrogate has been questioned, and new surrogate endpoint hypotheses have arisen. A proper understanding of radiological imaging, an assessment of the biological events triggered by therapy, and the clinical evolution of the lesions and of the patient performance status are all factors that should be considered to guide the oncologist's treatment choice. The primary aim of this article is to discuss how all these concepts apply to recurrent/metastatic head and neck squamous cell carcinoma patients when treated with immunotherapy.

Objective: We aimed to investigate the association of fatigue with severity of other key cancer symptoms, as well as symptom interference with daily activities and outlook on life, in long-term survivors of acute promyelocytic leukaemia (APL)., Methods: The study sample consisted of APL survivors (n=244), with a median time from diagnosis of 14.3 years (IQR=11.1-16.9 years), previously enrolled in a long-term follow-up study. Symptom severity and symptom interference were assessed using the well-validated MD Anderson Symptom Inventory (MDASI). Fatigue was evaluated with the Functional Assessment of Chronic Illness Therapy-Fatigue questionnaire., Results: Higher fatigue burden was associated with increased affective symptoms, memory problems, drowsiness, sleep disturbances, shortness of breath and pain. Higher levels of fatigue were also associated with higher scores across all interference items of the MDASI. Overall, symptoms interfered most with mood, but among APL survivors with high levels of fatigue, symptoms interfered most with enjoyment of life. Multivariable regression analysis confirmed the independent association between fatigue and all symptom severity items of the MDASI., Conclusions: The current findings show that long-term APL survivors who report higher fatigue also experience a greater overall symptom burden and a substantial impact on performance of daily activities. Further studies are needed to examine whether interventions aimed at reducing fatigue could also reduce overall symptom burden., Competing Interests: Competing interests: The following authors declare competing interests unrelated to this work: FE: consultancy for Amgen, Bristol Myers Squibb, Orsenix, and Takeda, and research grants (to his institution) from Amgen. FR: advisory board/speakers’ bureau for Amgen, Novartis and Argenx. MC: Novartis and Incyte. MB: honoraria by Novartis, Incyte, Pfizer and Celgene. ML: advisory board for Novartis, Gilead Sci, MSD, Jazz, Sanofi, Daiichi Sankyo and AbbVie, and travel grant (Gilead Sci). EB: consultancy for Amgen and Celgene. MV: personal fees from Jazz Healthcare Italy, Amgen, Millennium Pharmaceuticals, Celgene, Janssen, Novartis and Incyte., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

There is debate regarding whether most articulatory planning occurs offline (rather than online) and whether the products of off-line processing are stored in a separate articulatory buffer until a large enough chunk is ready for production. This hypothesis predicts that delayed naming conditions should reduce not only onset RTs but also word durations because articulatory plans will be buffered and kept ready. We have tested this hypothesis with young control speakers, an aphasic speaker , and an age and education-matched speaker, using repetition, reading and picture-naming tasks. Contrary to the off-line hypothesis, delayed conditions strongly reduced onset RTs, but had no benefit for word durations. In fact, we found small effects in the opposite direction. Moreover, frequency and imageability affected word durations even in delayed conditions, consistent with articulatory processing continuing on-line. The same pattern of results was found in CS and in control participants, strengthening confidence in our results. There is debate regarding whether most articulatory planning occurs offline (rather than online) and whether the results of off-line processing are stored in a separate articulatory buffer until a large enough chunk is ready for production. This hypothesis predicts that delayed naming conditions should reduce not only onset RTs but also word durations because articulatory plans will be buffered and kept ready. We have tested young control speakers, an aphasic speaker, and an age and education matched speaker, using repetition, reading and picture naming tasks. Contrary to the off-line hypothesis, delayed conditions strongly reduced onset RTs, but had no benefit for word durations. In fact, we found small effects in the opposite direction. Moreover, frequency and imageability affected word durations even in delayed conditions, consistent with articulatory processing continuing on-line. The same pattern of results was found in CS and in control participants, strengthening confidence in our results.

Background: Oral potentially malignant disorders (OPMDs) represent a heterogeneous set of different histological lesions, characterized by the capacity to transform in oral squamous cell carcinoma (OSCC). Despite optimal surgical treatment, approximately 20%-30% of OPMDs may evolve into OSCC. No clear clinical/histological factors are able to identify OPMDs at higher risk of malignant transformation., Materials and Methods: We considered surgically treated patients with a diagnosis of OPMDs, enrolled from 1996 to 2019 at ASST Spedali Civili of Brescia without a diagnosis of OSCC within the previous 2 years. Clinical and histological characteristics were recorded. Outcomes of interest were recurrence-free survival (RFS), defined as the time from surgery for primary OPMD to any relapse of OPMD or malignant transformation, whichever occurred first, and carcinoma-free survival (CFS), defined as the time from surgery for OPMD to malignant transformation., Results: We retrospectively reviewed 106 OPMDs cases. Median age at first diagnosis was 64 years old (IQR = 18.75); female patients comprise 51.9% of the cases. During a median follow-up of 30.5 months (IQR = 44), in 23.5% of patients, malignant transformation occurred. RFS at 1, 5, and 10 years was 92.4%, 60.9%, and 43.2%, respectively. Female sex and history of previous OSCC were independent risk factors for RFS. CFS at 1, 5, and 10 years of follow-up was 97.1%, 75.9%, and 64.4%, respectively. Previous OSCC was an independent risk factor for CFS., Conclusions: In this large series of OPMDs, only previous diagnosis of OSCC was a prognostic factor for further OSCC occurrence. Given the lack of additional clinical/pathological prognostic factors, we advocate further studies into molecular characterization of OPMDs to better stratify the risk of malignant transformation., Competing Interests: PB declares advisory board participation or conference honoraria from Merck, Sanofi-Regeneron, Merck Sharp & Dohme, Sun Pharma, Angelini, Molteni, Bristol-Myers Squibb, GSK, and Nestlè. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lorini, Tomasoni, Gurizzan, Magri, Facchetti, Battocchio, Romani, Ravanelli, Oberti, Bozzola, Bardellini, Paderno, Mattavelli, Lombardi, Grammatica, Deganello, Facchetti, Calza, Majorana, Piazza and Bossi.)

Background: Radical surgical resection of the primary tumor with mono/bilateral inguinofemoral lymph node dissection is the standard treatment for invasive vulvar squamous cell carcinoma (VSCC) and is frequently related to severe morbidity. Tailoring surgical treatment is of paramount importance, and a comprehensive preoperative evaluation is mandatory. Vascular endothelial growth factor D (VEGF-D) is considered a regulator of lymphangiogenesis involved in tumor spread via lymphatic vessels. The aim of this study was to evaluate the potential of VEGF-D in the prediction of inguinofemoral lymph node metastasis., Methods: We analyzed the preoperative levels of serum VEGF-D (sVEGF-D) from two independent cohorts of patients with VSCC by enzyme-linked immunosorbent assay and its protein expression on tumor tissue by immunohistochemistry. Logistic regression was performed to identify the independent risk factors for lymph node metastasis, and Cox proportional hazard model was used for survival analysis., Results: High levels of sVEGF-D, but not tissue VEGF-D, significantly correlated with positive groin nodes and a more advanced International Federation of Gynecologists and Obstetricians (FIGO) stage. In multivariable analysis, a high sVEGF-D level was an independent predictor of lymph node metastasis and worse prognosis. A prediction model based on sVEGF-D, tumor grade assessed on biopsy, tumor diameter, and lymph node clinical evaluation was able to predict lymph node metastasis, reaching C-index values of 0.79 and 0.73 in the training and validation cohorts, respectively., Conclusions: The preoperative sVEGF-D level might be a reliable biomarker for the prediction of lymph node metastasis and prognosis in patients with VSCC, supporting better clinical/surgical decision. Multicenter prospective studies are required to confirm our findings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LT declared a shared affiliation with one of the authors GG to the handling editor at the time of review., (Copyright © 2022 Ravaggi, Gambino, Ferrari, Olivari, Zanotti, Romani, Ardighieri, Antonelli, Garganese, Gallo, Scambia, Bignotti, Sartori, Calza and Odicino.)

Background: In phenylketonuria (PKU), treatment monitoring is based on frequent blood phenylalanine (Phe) measurements, as this is the predictor of neurocognitive and behavioural outcome by reflecting brain Phe concentrations and brain biochemical changes. Despite clinical studies describing the relevance of blood Phe to outcome in PKU patients, blood Phe does not explain the variance in neurocognitive and behavioural outcome completely., Methods: In a PKU mouse model we investigated 1) the relationship between plasma Phe and brain biochemistry (Brain Phe and monoaminergic neurotransmitter concentrations), and 2) whether blood non-Phe Large Neutral Amino Acids (LNAA) would be of additional value to blood Phe concentrations to explain brain biochemistry. To this purpose, we assessed blood amino acid concentrations and brain Phe as well as monoaminergic neurotransmitter levels in in 114 Pah-Enu2 mice on both B6 and BTBR backgrounds using (multiple) linear regression analyses., Results: Plasma Phe concentrations were strongly correlated to brain Phe concentrations, significantly negatively correlated to brain serotonin and norepinephrine concentrations and only weakly correlated to brain dopamine concentrations. From all blood markers, Phe showed the strongest correlation to brain biochemistry in PKU mice. Including non-Phe LNAA concentrations to the multiple regression model, in addition to plasma Phe, did not help explain brain biochemistry., Conclusion: This study showed that blood Phe is still the best amino acid predictor of brain biochemistry in PKU. Nevertheless, neurocognitive and behavioural outcome cannot fully be explained by blood or brain Phe concentrations, necessitating a search for other additional parameters., Take-Home Message: Blood Phe is still the best amino acid predictor of brain biochemistry in PKU. Nevertheless, neurocognitive and behavioural outcome cannot fully be explained by blood or brain Phe concentrations, necessitating a search for other additional parameters., Competing Interests: Declaration of Competing Interest F.J van Spronsen has been a member of scientific advisory boards for defects in amino acid metabolism of APR, Agios, Arla Food International, BioMarin, Eurocept Int, Lucana, Moderna TX, Nutricia, Rivium, Homoly, and Nestle-Codexis, his institute has received research grants from Alexion, Biomarin, Codexis, Nutricia, SoBi, and Vitaflo, has received grants from patient organizations ESPKU, Metakids, NPKUA, Stofwisselkracht, Stichting PKU research and Tyrosinemia Foundation, and has received honoraria as consultant and speaker from APR, Pluvia, Biomarin, MendeliKABS and Nutricia. SCJH has participated in strategic advisory boards and received grants and honoraria as a consultant and/or speaker from Biomarin, Merck Serono SA, Homology Medicines, and Nutricia., (Copyright © 2021. Published by Elsevier Inc.)

The onset of chemo-resistant recurrence represents the principal cause of high-grade serous ovarian carcinoma (HGSOC) death. HGSOC masses are characterized by a hypoxic microenvironment, which contributes to the development of this chemo-resistant phenotype. Hypoxia regulated-miRNAs (HRMs) represent a molecular response of cancer cells to hypoxia and are involved in tumor progression. We investigated the expression of HRMs using miRNA expression data from a total of 273 advanced-stage HGSOC samples. The miRNAs associated with chemoresistance and survival were validated by RT-qPCR and target prediction, and comparative pathway analysis was conducted for target gene identification. Analysis of miRNA expression profiles indicated miR-23a-3p and miR-181c-5p over-expression as associated with chemoresistance and poor PFS. RT-qPCR data confirmed upregulation of miR-23a-3p in tumors from chemoresistant HGSOC patients and its significant association with shorter PFS. In silico miR-23a-3p target prediction and comparative pathway analysis identified platinum drug resistance as the pathway with the highest number of miR-23a-3p target genes. Among them, APAF-1 emerged as the most promising, being downregulated in platinum-resistant patients and in HGSOC chemo-resistant cells. These results highlight miR-23a-3p as a potential biomarker for HGSOC platinum response and prognosis and the miR23a-3p/APAF1 axis as a possible target to overcome platinum-resistance.

Olfactory neuroblastoma (ONB) is a rare sinonasal neoplasm with a peculiar behavior, for which limited prognostic factors are available. Herein, we investigate the transcriptional pathways altered in ONB and correlate them with pathological features and clinical outcomes. We analyze 32 ONB patients treated with curative intent at two independent institutions from 2001 to 2019 for whom there is available pathologic and clinical data. We perform gene expression profiling on primary ONB samples and carry out functional enrichment analysis to investigate the key pathways associated with disease-free survival (DFS). The median age is 53.5 years; all patients undergo surgery and a pure endoscopic approach is adopted in the majority of cases (81.2%). Most patients have advanced disease (stages III-IV, 81.2%) and 84.4% undergo adjuvant (chemo)radiotherapy. The median follow-up is 35 months; 11 (26.8%) patients relapse. Clinical characteristics (gender, stage and Hyams' grade) are not associated with the outcomes. In contrast, TGF-beta binding, EMT, IFN-alpha response, angiogenesis, IL2-STAT5 and IL6-JAK-STAT3 signaling pathways are enriched in patients experiencing recurrence, and significantly associated with shorter DFS. Clustering of transcriptional profiles according to pathological features indicates two distinct molecular groups, defined by either cytokeratin-positive or -negative immunostaining. Definition of the characterizing ONB transcriptomic pathways may pave the way towards tailored treatment approaches.

Background: Oral Potentially Malignant Disorders (OPMD) have a non-negligible malignant transformation rate of up to 8%. Loss of heterozygosity (LOH) in critical chromosomal loci has proven to be the most effective marker in defining the risk of transformation and it is found in about 28% of OPMD and may therefore identify patients carrying higher risk. To date, clinical management of OPMD is limited to surgical excision and clinical surveillance, which however do not fully prevent oral cancer development. Immune system has been shown to play a key role in transformation surveillance mechanism and an immunosuppressive imbalance may be responsible for progression to cancer. Given all these considerations, we designed a clinical trial with the aim to prevent OPMD neoplastic transformation and revert the LOH status., Methods: This is a phase II, open label, single arm, multicentric trial involving Italian referral centres and expected to enrol 80 patients out of a total of 175 screened. Patients who meet all inclusion criteria and test positive for LOH after an incisional biopsy of the OPMD will undergo a short course of immunotherapy with 4 administration of avelumab. After 6 months since treatment start, resection of the entire OPMD will be performed and LOH assessment will be repeated. The follow-up for malignant transformation and safety assessment will last 30 months from the end of treatment, for a total planned study duration of approximately 5.5 years., Discussion: Restoring the activity of immune system through checkpoint inhibitor may play a crucial role against malignant transformation of OPMD by reverting the balance in favour of immune control and preventing cancer occurrence., Trial Registration: This trial was prospectively registered in ClinicalTrials.gov as NCT04504552 on 7th August 2020.

This commentary highlights three common difficulties faced by the literature that aims to specify models of speech production based on the performance of aphasic speakers, taking as a springboard a recent study by Mailend et al. These include: (1) difficulties with theoretical assumptions which linki psycholinguistic effects unequivocally to one processing level; (2) difficulties using clinical classifications to localize experimental effects; (3) difficulties making theoretical inferences given the controversial nature of the representations that characterize different processing levels. We argue that these three types of difficulties could be ameliorated by studies in which: (1) the level of psycholinguistic effects is demonstrated with converging analyses; (2) clinical classification is not taken as a starting point in studies investigating the nature of an impairment, but, instead, associations between clusters of symptoms are carefully analysed; (3) The nature of processing levels associated with deficits is made clear and results are not over-interpreted as supporting models whose characteristics go beyond an explanation of the results.

Uterine leiomyosarcomas (uLMS) are aggressive tumors arising from the smooth muscle layer of the uterus. We analyzed 83 uLMS sample genetics, including 56 from Yale and 27 from The Cancer Genome Atlas (TCGA). Among them, a total of 55 Yale samples including two patient-derived xenografts (PDXs) and 27 TCGA samples have whole-exome sequencing (WES) data; 10 Yale and 27 TCGA samples have RNA-sequencing (RNA-Seq) data; and 11 Yale and 10 TCGA samples have whole-genome sequencing (WGS) data. We found recurrent somatic mutations in TP53, MED12, and PTEN genes. Top somatic mutated genes included TP53, ATRX, PTEN, and MEN1 genes. Somatic copy number variation (CNV) analysis identified 8 copy-number gains, including 5p15.33 (TERT), 8q24.21 (C-MYC), and 17p11.2 (MYOCD, MAP2K4) amplifications and 29 copy-number losses. Fusions involving tumor suppressors or oncogenes were deetected, with most fusions disrupting RB1, TP53, and ATRX/DAXX, and one fusion (ACTG2-ALK) being potentially targetable. WGS results demonstrated that 76% (16 of 21) of the samples harbored chromoplexy and/or chromothripsis. Clinically actionable mutational signatures of homologous-recombination DNA-repair deficiency (HRD) and microsatellite instability (MSI) were identified in 25% (12 of 48) and 2% (1 of 48) of fresh frozen uLMS, respectively. Finally, we found olaparib (PARPi; P = 0.002), GS-626510 (C-MYC/BETi; P < 0.000001 and P = 0.0005), and copanlisib (PIK3CAi; P = 0.0001) monotherapy to significantly inhibit uLMS-PDXs harboring derangements in C-MYC and PTEN/PIK3CA/AKT genes (LEY11) and/or HRD signatures (LEY16) compared to vehicle-treated mice. These findings define the genetic landscape of uLMS and suggest that a subset of uLMS may benefit from existing PARP-, PIK3CA-, and C-MYC/BET-targeted drugs., Competing Interests: The authors declare no competing interest.

Adenoid cystic carcinoma (ACC) is a rare tumor, usually arising in the salivary gland, accounting for 1% of all head and neck cancers. ACC may have a long-term poor prognosis, as about 40% of radically treated patients will recur locoregionally and up to 60% will develop distant metastasis. Factors influencing risk of recurrence have been well studied, but few data exist about prognostic factors in Recurrent/Metastatic (RM) setting. Moreover, treatment of RM ACC is often a challenge for clinicians, in the context of a rare disease, which may have an indolent clinical behavior or less frequently a quicker growth and with a paucity of available clinical trials. This review critically analyzes pathological and molecular prognostic factors in RM ACC and make an overview on actual therapeutic choices and future direction of therapy. Recognized prognostic factors in RM ACC are the presence and site of distant metastasis (lung vs other), the presence of nodal metastasis and of extranodal extension, skull base recurrence, disease free interval, lymphovascular invasion, solid histotypes and grading of disease, and the presence of mutation of NOTCH1 family, PI3K, and TP53. Due to disappointing results with chemotherapy, new approaches are under study, also on the basis of biomolecular research. Ongoing clinical trials are evaluating treatment targeting MYB and NOTCH1 alterations, immunotherapy or combination of targeted treatments and immune checkpoint inhibitors., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: A subset of patients with phenylketonuria benefit from treatment with tetrahydrobiopterin (BH 4 ), although there is no consensus on the definition of BH 4 responsiveness. The aim of this study therefore was to gain insight into the definitions of long-term BH 4 responsiveness being used around the world., Methods: We performed a web-based survey targeting healthcare professionals involved in the treatment of PKU patients. Data were analysed according to geographical region (Europe, USA/Canada, other)., Results: We analysed 166 responses. Long-term BH 4 responsiveness was commonly defined using natural protein tolerance (95.6%), improvement of metabolic control (73.5%) and increase in quality of life (48.2%). When a specific value for a reduction in phenylalanine concentrations was reported (n = 89), 30% and 20% were most frequently used as cut-off values (76% and 19% of respondents, respectively). When a specific relative increase in natural protein tolerance was used to define long-term BH 4 responsiveness (n = 71), respondents most commonly reported cut-off values of 30% and 100% (28% of respondents in both cases). Respondents from USA/Canada (n = 50) generally used less strict cut-off values compared to Europe (n = 96). Furthermore, respondents working within the same center answered differently., Conclusion: The results of this study suggest a very heterogeneous situation on the topic of defining long-term BH 4 responsiveness, not only at a worldwide level but also within centers. Developing a strong evidence- and consensus-based definition would improve the quality of BH 4 treatment., Competing Interests: Declaration of Competing Interest RAFE has received financial support from Biomarin for attending symposia. AMJvW has received a research grant form Nutricia, honoraria from Biomarin as speaker, and travel grants from Nutricia and Vitaflo. AMB has received a speakers fee from Nutricia and has been a member of advisory boards for Biomarin. MG received honoraria and was a consultant for: Nutricia International/Danone, Merck-Serono, Mead Johnson, BioMarin and Vitaflo. JCR has been a member of the European Nutritionist Expert Panel [Biomarin], the Advisory Board for Applied Pharma Research and Nutricia, and received honoraria as a speaker from APR, Merck Serono, Biomarin, Nutricia, Vitaflo, Cambrooke, PIAM and Lifediet. FJvS is a member of scientific advisory boards for PKU and aminoacid defects that are supported by Agios, Applied Pharma Research, Arla Food Int, BioMarin, Eurocept, Homology, Lucane, Nestle-Codexis Alliance, Nutricia, orphan Europe, Rivium Medical BV, Vivet, has received research grants from Alexion, BioMarin, Beatrix Research Fund, Codexis, ESPKU, NPKUA, NPKUV, Nutricia, Sobi, Tyrosinemia Foundation, Vitaflo, ZONMW, and has received honoraria as a consultant and speaker from Applied Pharma Research, Biomarin, MendeliKABS, Nutricia, Pluvia, SoBi, and Vitaflo. AM is an advisory board member for ELEMENT, Danone-Nutricia, Arla, and Applied Pharma Research, and has received research funding and honoraria from Applied Pharma Research, Nutricia, and Vitaflo International. All other authors reported no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Claudin-low cancer (CL) represents a rare and biologically aggressive variant of epithelial tumor. Here, we identified a claudin-low molecular profile of ovarian high-grade serous carcinoma (HGSOC), which exhibits the main characteristics of the homonym breast cancer subtype, including low epithelial differentiation and high mesenchymal signature. Hierarchical clustering and a centroid based algorithm applied to cell line collection expression dataset labeled 6 HGSOC cell lines as CL. These have a high energy metabolism and are enriched in CD44 + /CD24 - mesenchymal stem-like cells expressing low levels of cell-cell adhesion molecules (claudins and E-Cadherin) and high levels of epithelial-to-mesenchymal transition (EMT) induction transcription factors (Zeb1, Snai2, Twist1 and Twist2). Accordingly, the centroid base algorithm applied to large retrospective collections of primary HGSOC samples reveals a tumor subgroup with transcriptional features consistent with the CL profile, and reaffirms EMT as the dominant biological pathway functioning in CL-HGSOC. HGSOC patients carrying CL profiles have a worse overall survival when compared to others, likely to be attributed to its undifferentiated/stem component. These observations highlight the lack of a molecular diagnostic in the management of HGSOC and suggest a potential prognostic utility of this molecular subtyping.

Survival rates of oral squamous cell carcinoma (OSCC) remained substantially unchanged over the last decades; thus, additional prognostic tools are strongly needed. Salivary miRNAs have emerged as excellent non-invasive cancer biomarker candidates, but their association with OSCC prognosis has not been investigated yet. In this study, we analyzed global salivary miRNA expression in OSCC patients and healthy controls, with the aim to define its diagnostic and prognostic potential. Methods: Saliva was collected from patients with newly diagnosed untreated primary OSCC and healthy controls. Global profiling of salivary miRNAs was carried out through a microarray approach, while signature validation was performed by quantitative real-time PCR (RT-qPCR). A stringent statistical approach for microarray and RT-qPCR data normalization was applied. The diagnostic performance of miRNAs and their correlation with OSCC prognosis were comprehensively analyzed. Results: In total, 25 miRNAs emerged as differentially expressed between OSCC patients and healthy controls and, among them, seven were significantly associated with disease-free survival (DFS). miR-106b-5p, miR-423-5p and miR-193b-3p were expressed at high levels in saliva of OSCC patients and their combination displays the best diagnostic performance (ROC - AUC = 0.98). Moreover, high expression of miR-423-5p was an independent predictor of poor DFS, when included in multivariate survival analysis with the number of positive lymph nodes - the only significant clinical prognosticator. Finally, we observed a significant decrease in miR-423-5p expression in matched post-operative saliva samples, suggesting its potential cancer-specific origin. Conclusion: Salivary miRNAs identified in our cohort of patients show to be accurate in OSCC detection and to effectively stratify patients according to their likelihood of relapse. These results, if validated in an independent set of patients, could be particularly promising for screening/follow-up of high-risk populations and useful for preoperative prognostic assessment., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: Our institution operates a remote radiation oncology service in Northern Ontario, Canada. Since the start of the coronavirus disease 2019 pandemic, this center has operated without radiation oncologists on site owing to safety precautions, and this study seeks to understand the effect of this shift., Methods and Materials: Departmental level data reports were used to investigate differences in metrics between April to May of 2019 and April to May 2020. These metrics include the total number of referrals received, average wait time from referral to consult, the number of cases that underwent peer review before beginning treatment, the total number of fractions given over each period, patient-reported outcomes, and patient satisfaction. We also examined the importance of physical examinations and the use of SABR treatment., Results: There was an observed decrease in the number of referrals received, total number of fractions administered, and number of patients providing patient-reported outcomes. We observed no change in patient wait times, cases undergoing peer review before commencing treatment, or overall patient satisfaction. Challenges were identified in the collection of patient- reported outcomes and the conduction of physical examinations., Conclusions: This paper provides proof of concept that a radiation clinic can function entirely virtually in the short term without sacrificing patient satisfaction, efficiency, or safety., (© 2020 The Authors.)

The outcome of relapsed or refractory (R/R) T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/T-LBL) in adults is poor, with less than 20% of patients surviving at 5 years. Nelarabine is the only drug specifically approved for R/R T-ALL/T-LBL, but the information to support its use is based on limited available data. The aim of this observational phase four study was to provide recent additional data on the efficacy and safety of nelarabine in adults with R/R T-ALL/T-LBL and to evaluate the feasibility and outcome of allogeneic hematopoietic stem cell transplant (SCT) after salvage with nelarabine therapy. The primary endpoints were overall response rate (ORR) and overall survival (OS). Additional endpoints were safety, SCT rate and post-SCT OS. Between May 2007 and November 2018, 118 patients received nelarabine salvage therapy at 27 Italian hematology sites. The median age was 37 years (range 18-74 years), 73% were male, 77 had a diagnosis of T-ALL and 41 of T-LBL, and 65/118 (55%) had received more than two lines of therapy. The median number of nelarabine cycles was two (range 1-4); 43/118 (36%) patients had complete remission (CR), 16 had partial remission (14%) and 59 (50%) were refractory, with an ORR of 50%. The probability of OS, from the first dose of nelarabine, was 37% at 1 year with a median survival of 8 months. The OS at 1 year was significantly better for the 47 patients (40%) who underwent SCT after nelarabine salvage therapy (58% vs 22%, log-rank P < .001). The probability of OS at 2 and 5 years from SCT was 46% and 38%, respectively. Seventy-five patients (64%) experienced one or more drug-related adverse events (AE). Grade III-IV neurologic toxicities were observed in 9/118 (8%) of cases and thrombocytopenia or/and neutropenia (grade III-IV) were reported in 41% and 43% of cases, respectively. In conclusion, this is one of the largest cohorts of adult patients with R/R T-ALL/T-LBL treated in real life with nelarabine. Taking into account the poor prognosis of this patient population, nelarabine represents an effective option with an ORR of 50% and a CR rate of 36%. In addition, 40% of cases following nelarabine salvage therapy could undergo SCT with an expected OS at 2 and 5 years of 46% and 38%, respectively. The safety profile of nelarabine was acceptable with only 8% of cases showing grade III-IV neurological AE., (© 2020 Wiley Periodicals LLC.)

An updated strategy combining pediatric-based chemotherapy with risk-oriented allogeneic hematopoietic cell transplantation (HCT) was evaluated in Philadelphia chromosome-negative acute lymphoblastic leukemia (Ph- ALL) and compared with a published control series. Following induction-consolidation chemotherapy, responsive patients were assigned to receive maintenance chemotherapy or undergo early HCT according to the risk stratification criteria and minimal residual disease (MRD) status. Of the 203 study patients (median age 41 years, range 17-67), 140/161 with Ph- ALL achieved complete remission (86.9%; 91.6% ≤55 years, P = 0.0002), with complete MRD clearing in 68/109; 55 patients were assigned to maintenance chemotherapy, and 85 to HCT due to very high-risk characteristics (hyperleukocytosis, adverse genetics, early/mature T-precursor ALL, and MRD persistence). The 5-year relapse incidence was 36%, and the treatment-related mortality rate was 18%. Median overall and relapse-free survival were 7.4 and 6.2 years, with rates of 54 and 53% at 5 years, respectively, which were significantly better than those obtained with the historical protocol (P = 0.001 and P = 0.005, respectively), without significant differences between maintenance and HCT cohorts. In prognostic analysis, MRD negativity and age ≤55 years were the most favorable independent prognostic factors. A reduction in treatment toxicity and further improvements in the risk definitions and risk-oriented design are the focuses of this ongoing research.

Lichen sclerosus (LS) is a chronic relapsing, inflammatory skin disorder usually involving the anogenital region of both sexes lacking a resolutive therapy. This study compared adipose tissue derived-stromal vascular fraction (AD-SVF) and AD-SVF-enriched platelet-rich plasma (PRP) therapy in the management of genital LS patients. Additionally, in vitro evaluation of cells and growth factors contained in the injected SVF has been evaluated as possible predictive factors for treatment outcome. The study population was 40 patients diagnosed with LS who were symptomatic despite medical treatment. Patients (age 43-78 years) randomized into two groups using a 1:1 allocation ratio, were evaluated clinically and assessing dermatology life quality index (DLQI) before and 6 months after treatment. Both procedures demonstrated a strong safety profile with no complications linked to the therapy. After 6 months, both treatments allowed for a significant improvement respect to baseline. Combinatory therapy demonstrated decreased efficacy in late stage patients. No correlations have been found between clinical and biological findings. AD-SVF and AD-SVF plus PRP are safe and effective regenerative approaches for genital LS patients. Clinical results support the preferential use of combinatory therapy for early stage patients confirming a synergic effect of AD-SVF and PRP. In contrast, AD-SVF plus PRP is discouraged for late stage patients., (© 2020 Wiley Periodicals LLC.)

We aimed to assess if the same cognitive batteries can be used cross-nationally to monitor the effect of Phenylketonuria (PKU). We assessed whether a battery, previously used with English adults with PKU (AwPKU), was also sensitive to impairments in Italian AwPKU. From our original battery, we selected a number of tasks that comprehensively assessed visual attention, visuo-motor coordination, executive functions (particularly, reasoning, planning, and monitoring), sustained attention, and verbal and visual memory and learning. When verbal stimuli/or responses were involved, stimuli were closely matched between the two languages for psycholinguistic variables. We administered the tasks to 19 Italian AwPKU and 19 Italian matched controls and compared results from with 19 English AwPKU and 19 English matched controls selected from a previously tested cohort. Participant election was blind to cognitive performance and metabolic control, but participants were closely matched for age and education. The Italian AwPKU group had slightly worse metabolic control but showed levels of performance and patterns of impairment similar to the English AwPKU group. The Italian results also showed extensive correlations between adult cognitive measures and metabolic measures across the life span, both in terms of Phenylalanine (Phe) levels and Phe fluctuations, replicating previous results in English. These results suggest that batteries with the same and/or matched tasks can be used to assess cognitive outcomes across countries allowing results to be compared and accrued. Future studies should explore potential differences in metabolic control across countries to understand what variables make metabolic control easier to achieve.

In spite of the effective implementation of screening programs, uterine cervical carcinoma (UCC) remains one of the major causes of cancer death among women around the world. The aim of this study was to investigate the prognostic value of serum human epididymis protein 4 (HE4) in UCC. Pre-treatment serum samples from 109 UCC patients and 99 healthy women were analyzed for HE4 levels by a quantitative chemiluminescent microparticle immunoassay on the automated ARCHITECT instrument. HE4 serum (sHE4) levels were significantly higher in UCC patients, regardless of tumor stage, compared with healthy controls. Elevated sHE4 levels were significantly associated with advanced FIGO stage and absence of disease-free interval after treatment. In univariable analysis, higher sHE4 levels were significantly correlated with shorter overall survival and progression-free survival. In multivariable analysis, sHE4 retained its significance as independent adverse prognostic factor for both survival endpoints. This study indicates that sHE4 is associated with a more aggressive tumor phenotype and a worse patient's prognosis. These results suggest the potential role of sHE4 as a novel prognostic marker and as an indicator of high-risk UCC patients for a tailored surgical and adjuvant therapy., (Copyright © 2020 Bignotti, Zanotti, Todeschini, Zizioli, Romani, Capoferri, Tognon, Sartori, Calza, Odicino and Ravaggi.)

An amendment to this paper has been published and can be accessed via the original article.

High-grade serous ovarian carcinoma (HGSOC) usually spreads directly into the peritoneal cavity following a transcoelomic dissemination route, although distant hematogenous metastasis exist and have been reported. However, no tumor markers can currently predict the risk of distant metastases in HGSOC. Claudins, belonging to tight-junction proteins, are dysregulated in HGSOC and functionally related to cancer progression. Here we analyzed claudin-3, -4, and -7 expression as potential markers of distant metastases. Using quantitative RT-PCR and immunohistochemistry we assessed the expression of claudins in primary HGSOC tissues, normal ovarian, and normal fallopian tube epithelia and correlated it with clinicopathological features, including the site of metastasis and the route of dissemination. Gene set enrichment analysis was performed on microarray-generated gene expression data to investigate key pathways in patients with distant metastases. We found the overall expression level of claudin-3, -4, and -7 mRNA decreased in HGSOC compared to normal tubal epithelium, currently considered the potential site of origin of many HGSOC. The reduced expression of claudin-7 is significantly associated with the development of distant metastases ( p = 0.016), mainly by hematogenous route ( p = 0.025). In patients with diminished expression of claudin-7, immunohistochemical staining revealed a heterogeneous pattern of membranous staining with discontinuous expression of claudin-7 along the cell border, indicative of a dischoesive architecture. The estimated reduction in the probability of distant disease is of 39% per unit increase in the level of claudin-7 ( p = 0.03). Genes involved in epithelial to mesenchymal transition, hypoxia, and angiogenesis processes resulted strongly associated to hematogenous recurrence. Our data suggest a potential role of claudin-7 in discriminating distant metastatic events in HGSOC patients. The quantification of its expression levels could be a useful tool to identify patient deserving a personalized follow-up in terms of clinical and radiological assessment., (Copyright © 2020 Romani, Zizioli, Silvestri, Ardighieri, Bugatti, Corsini, Todeschini, Marchini, D'Incalci, Zanotti, Ravaggi, Facchetti, Gambino, Odicino, Sartori, Santin, Mitola, Bignotti and Calza.)

Background: Oral premalignant lesions (OPLs) represent the most common oral precancerous conditions. One of the major challenges in this field is the identification of OPLs at higher risk for oral squamous cell cancer (OSCC) development, by discovering molecular pathways deregulated in the early steps of malignant transformation. Analysis of deregulated levels of single genes and pathways has been successfully applied to head and neck squamous cell cancers (HNSCC) and OSCC with prognostic/predictive implications. Exploiting the availability of gene expression profile and clinical follow-up information of a well-characterized cohort of OPL patients, we aim to dissect tissue OPL gene expression to identify molecular clusters/signatures associated with oral cancer free survival (OCFS)., Materials and Methods: The gene expression data of 86 OPL patients were challenged with: an HNSCC specific 6 molecular subtypes model (Immune related: HPV related, Defense Response and Immunoreactive; Mesenchymal, Hypoxia and Classical); one OSCC-specific signature (13 genes); two metabolism-related signatures (3 genes and signatures raised from 6 metabolic pathways associated with prognosis in HNSCC and OSCC, respectively); a hypoxia gene signature. The molecular stratification and high versus low expression of the signatures were correlated with OCFS by Kaplan-Meier analyses. The association of gene expression profiles among the tested biological models and clinical covariates was tested through variance partition analysis., Results: Patients with Mesenchymal, Hypoxia and Classical clusters showed an higher risk of malignant transformation in comparison with immune-related ones (log-rank test, p = 0.0052) and they expressed four enriched hallmarks: "TGF beta signaling" "angiogenesis", "unfolded protein response", "apical junction". Overall, 54 cases entered in the immune related clusters, while the remaining 32 cases belonged to the other clusters. No other signatures showed association with OCFS. Our variance partition analysis proved that clinical and molecular features are able to explain only 21% of gene expression data variability, while the remaining 79% refers to residuals independent of known parameters., Conclusions: Applying the existing signatures derived from HNSCC to OPL, we identified only a protective effect for immune-related signatures. Other gene expression profiles derived from overt cancers were not able to identify the risk of malignant transformation, possibly because they are linked to later stages of cancer progression. The availability of a new well-characterized set of OPL patients and further research is needed to improve the identification of adequate prognosticators in OPLs.

High-grade serous ovarian cancer (HGS-EOCs) is generally sensitive to front-line platinum (Pt)-based chemotherapy although most patients at an advanced stage relapse with progressive resistant disease. Clinical or molecular data to identify primary resistant cases at diagnosis are not yet available. HGS-EOC biopsies from 105 Pt-sensitive (Pt-s) and 89 Pt-resistant (Pt-r) patients were retrospectively selected from two independent tumor tissue collections. Pathway analysis was done integrating miRNA and mRNA expression profiles. Signatures were further validated in silico on a cohort of 838 HGS-EOC cases from a published dataset. In all, 131 mRNAs and 5 miRNAs belonging to different functionally related molecular pathways distinguish Pt-s from Pt-r cases. Then, 17 out of 23 selected elements were validated by orthogonal approaches (SI signature). As resistance to Pt is associated with a short progression-free survival (PFS) and overall survival (OS), the prognostic role of the SI signature was assessed, and 14 genes associated with PFS and OS, in multivariate analyses (SII signature). The prognostic value of the SII signature was validated in a third extensive cohort. The expression profiles of SDF2L1, PPP1R12A and PRKG1 genes (SIII signature) served as independent prognostic biomarkers of Pt-response and survival. The study identified a prognostic molecular signature based on the combined expression profile of three genes which had never been associated with the clinical outcome of HGS-EOC. This may lead to early identification, at the time of diagnosis, of patients who would not greatly benefit from standard chemotherapy and are thus eligible for novel investigational approaches., (© 2020 UICC.)

Background: Phenylketonuria (PKU) is an autosomal recessive inborn error of phenylalanine metabolism caused by deficiency in the enzyme phenylalanine hydroxylase that converts phenylalanine into tyrosine., Main Body: In 2017 the first European PKU Guidelines were published. These guidelines contained evidence based and/or expert opinion recommendations regarding diagnosis, treatment and care for patients with PKU of all ages. This manuscript is a supplement containing the practical application of the dietary treatment., Conclusion: This handbook can support dietitians, nutritionists and physicians in starting, adjusting and maintaining dietary treatment.

Synthetic polymers are attracting growing attention as additives for laundry and personal care products. In particular, the high volatility of many common fragrances requires the development of polymeric particles for their encapsulation and controlled release. Unfortunately, the vast majority of these carriers is made from polymers that are not biodegradable. This poses severe concerns about the accumulation of nano- and microplastics. Hence, such particles are expected to be banned from the market in the coming years. Therefore, biodegradable particles enabling a long-lasting release of the fragrances are urgently needed. In this work, we produced biodegradable nanoparticles (NPs) that are structurally composed of lactones, i.e. well known perfumes that occur naturally and that are already considered safe by regulatory agencies. We polymerized these lactones via ring opening polymerization (ROP) using an ionizable tertiary amine as initiator to produce in a single step amphiphilic oligoesters able to directly self-assemble into NPs once nanoprecipitated in water. In this way, we can produce biodegradable NPs with a perfume loading up to 85 % w/w without the need for additional surfactants. Subsequently we show that the ionizable group is able to confer a positive charge to our nanoparticles and, in turn, a high adsorption capacity on natural fibers (i.e. hairs and cotton fabric). Finally, we demonstrate the nanoparticle resistance to rinsing and their ability to confer a long-lasting fragrance perception to treated hair swatches for at least 3 weeks., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction : Mental health, physical health, and cognitive skills have been scarcely investigated in the same sample of adults with PKU (AwPKU). This is striking since emotional difficulties may potentially contribute to cognitive impairments and vice-versa. Here we aim to fill this gap. Method : Thirty-six early-treated AwPKU and 40 controls were given an extensive battery of cognitive tasks assessing complex executive functions, inhibitory control, short-term memory, sustained attention, visuospatial attention, language production (reading and naming), visuomotor coordination, spoken language and orthographic processing. In addition, participants were given tasks tapping emotion recognition and completed questionnaires to assess depression (BDI-II), empathy (IRI) and mental/physical health-related quality of life (SF-36). Results : As a group, AwPKU performed significantly worse than controls especially in tasks tapping complex executive functions and across tasks when speed was measured but did not differ for emotional-health and physical health. In the PKU group, cognitive measures and measures of physical health-related quality of life were inter-correlated (differently than in the control group), and both measures were associated with metabolic control: better metabolic control, better cognition, and better physical health. Instead, cognitive measures and measures of emotional-health/mental-health-related quality of life did not correlate with one another and better metabolic control was not associated with better emotional health. Instead, some negative correlations were found. Better metabolic control was associated with worse perspective taking and more distress in socially stressful situations. Furthermore, difficulties in keeping the diet were associated with less emotional well-being. Conclusions : Taken together, these results indicate the advantages, but also possible emotional difficulties related to maintain a PKU diet, suggesting the importance of developing alternative therapy options.

Introduction: It is well known that a difficulty in forming lexical representations is a strong predictor of reading and spelling difficulties even after controlling for the effects of other cognitive skills. Our study had two main interrelated aims. First, we wanted to examine whether the ability to learn new written words (lexical learning) varies as a function of the orthographic consistency of the language of the learner. Second, we wanted to evaluate the cognitive abilities involved in orthographic lexical learning and whether they differed as a function of language consistency., Method: 163 Italian children and 128 English children performed a lexical learning task as well as tasks assessing several cognitive skills potentially related to the ability to establish orthographic representations., Results: We found that children learning an orthographic inconsistent orthography (English) were better able to learn novel written words presented in association with pictures than children learning a consistent orthography (Italian). This was true for both younger and older primary school children and also when children were matched for school grade. Lexical learning may be better in English children because the many irregularities of this language promote storing in memory whole-word representations and processing larger orthographic units. In Italian, instead, reading can be accomplished successfully on the basis of grapheme-phoneme conversion rules and on processing smaller orthographic units. This interpretation was supported by the pattern of cognitive skills associated with lexical learning skills in the two languages. Variations in lexical learning were explained by spatial visual memory and phonological awareness tasks in both languages, but phonological STM explained further variance in Italian, while a task tapping visuo-attentional capacity explained further variance in English., Conclusion: Learning a language with inconsistent orthography is associated with better lexical learning skills in children at different stages of primary school; the pattern of cognitive skills associated with lexical learning skills is also partially modulated by orthographic consistency., Competing Interests: The authors have declared that no competing interests exist.

Objective: The objective was to deepen the understanding of the causes of individual variability in phenylketonuria (PKU) by investigating which metabolic variables are most important for predicting cognitive outcomes (Phe average vs Phe variation) and by assessing the risk of cognitive impairment associated with adopting a more relaxed approach to the diet than is currently recommended., Method: We analysed associations between metabolic and cognitive measures in a mixed sample of English and Italian early-treated adults with PKU (N = 56). Metabolic measures were collected through childhood, adolescence and adulthood; cognitive measures were collected in adulthood. Metabolic measures included average Phe levels (average of median values for each year in a given period) and average Phe variations (average yearly standard deviations). Cognition was measured with IQ and a battery of cognitive tasks., Results: Phe variation was as important, if not more important, than Phe average in predicting adult outcomes and contributed independently. Phe variation was particularly detrimental in childhood. Together, childhood Phe variation and adult Phe average predicted around 40% of the variation in cognitive scores. Poor cognitive scores (> 1 SD from controls) occurred almost exclusively in individuals with poor metabolic control and the risk of poor scores was about 30% higher in individuals with Phe values exceeding recommended thresholds., Conclusions: Our results provide support for current European guidelines (average Phe value = < 360 μmol/l in childhood; = < 600 μmo/l from 12 years onwards), but they suggest an additional recommendation to maintain stable levels (possibly Phe SD = < 180 μmol/l throughout life)., Public Significance Statements: We investigated the relationship between how well people with phenylketonuria control blood Phe throughout their life and their ability to carry out cognitive tasks in adulthood. We found that avoiding blood Phe peaks was as important if not more important that maintaining average low Phe levels. This was particularly essential in childhood. We also found that blood Phe levels above recommended European guidelines was associated with around 30% increase in the risk of poor cognitive outcomes.

The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirus-type-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models ( P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs., Competing Interests: The authors declare no competing interest.

Background: High-grade serous ovarian carcinoma (HGSOC) is generally associated with a very dismal prognosis. Nevertheless, patients with similar clinicopathological characteristics can have markedly different clinical outcomes. Our aim was the identification of novel molecular determinants influencing survival., Methods: Gene expression profiles of extreme HGSOC survivors (training set) were obtained by microarray. Differentially expressed genes (DEGs) and enriched signalling pathways were determined. A prognostic signature was generated and validated on curatedOvarianData database through a meta-analysis approach. The best prognostic biomarker from the signature was confirmed by RT-qPCR and by immunohistochemistry on an independent validation set. Cox regression model was chosen for survival analysis., Results: Eighty DEGs and the extracellular matrix-receptor (ECM-receptor) interaction pathway were associated to extreme survival. A 10-gene prognostic signature able to correctly classify patients with 98% of accuracy was identified. By an 'in-silico' meta-analysis, overexpression of FXYD domain-containing ion transport regulator 5 (FXYD5), also known as dysadherin, was confirmed in HGSOC short-term survivors compared to long-term ones. Its prognostic and predictive power was then successfully validated, both at mRNA and protein level, first on training than on validation sample set., Conclusion: We demonstrated the possible involvement of FXYD5 and ECM-receptor interaction signal pathway in HCSOC survival and prognosis.

The objective of this study was to investigate health-related quality of life (HRQOL), symptom burden, and comorbidity profile in long-term acute promyelocytic leukemia (APL) survivors treated with standard chemotherapy. Overall, 307 long-term APL survivors were invited to participate. HRQOL was assessed with the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36) and compared with that of age and sex-matched controls from the general population. Symptom burden was assessed with the MD Anderson Symptom Inventory (MDASI) questionnaire and comorbidity profile was also investigated. Median follow-up time since diagnosis was 14.3 years (interquartile range: 11.1-16.9 years). APL survivors had a statistically and clinically meaningful worse score for the role physical scale of the SF-36 (-9.5; 95% CI, -15.7 to -3.2, P = 0.003) than their peers in the general population. Fatigue was reported as moderate to severe by 29% of patients and 84.4% reported at least one comorbidity. Prevalence of comorbidity in APL survivors was higher than that reported by the general population. Also, marked variations were found in the HRQOL profile by number of comorbidities. Even many years after treatment ends, APL survivors treated with standard chemotherapy do not fully recover as they report HRQOL limitations and a substantial burden of symptoms.