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Background: Radiotherapy (RT) is used in head and neck squamous cell carcinoma (HNSCC) with excellent effectiveness, but it is burdened by important side effects, which may negatively impact patients' quality of life (QoL). In particular when associated with chemotherapy (CT), that has a radiosensitising effect (and its own toxicities), it is responsible for several adverse events, causing social discomfort and lower QoL, in patients who are already experiencing several tumor-related discomforts. Prehabilitation is a healthcare intervention consisting of several specialist visits prior to the start of treatment, with the aim of improving the patient's health status, resolving symptoms that interfere with treatment and impact QoL, and finally to better avoid or overcome complications. Of all cancer patients, HNSCC patients are among those who could benefit most from prehabilitation, both because of the high number of symptoms and toxicities and their difficult management. Despite this and the emerging data, prehabilitation is not often considered for the majority of patients undergoing (C)RT. In this review, we tried to understand what are the main areas in which interventions can be made prior to the (C)RT start, the possible side effects of the treatment, the effectiveness in their prevention and management, and the impact that prehabilitation may have in adherence to therapy and on the principal survival outcomes, providing important guidance for the planning of future studies., Evidences and Conclusions: Although there is no strong data evaluating multidisciplinary prehabilitation strategies, evidence shows that optimizing the patient's health status and preventing possible complications improve the QoL, reduce the incidence and severity of adverse events, and improve treatment adherence. While cardiology prehabilitation is of paramount importance for all patients undergoing concomitant CRT in the prevention of possible side effects, the remaining interventions are useful independently of the type of treatment proposed. Geriatricians have a key role in both elderly patients and younger patients characterized by many comorbidities to comprehensively assess health status and indicate which treatment may be the best in terms of risk/benefit ratio. Collaboration between nutritionists and phoniatrics, on the other hand, ensures adequate nutritional intake for the patient, where possible orally. This is because optimizing both body weight and muscle mass and qualities has been shown to impact key survival outcomes. Finally, HNSCC patients have the second highest suicide rate, and the disease has side effects such as pain, dysfiguration, and sialorrhea that can reduce the patient's social life and create shame and embarrassment: A psychological intake, in addition to the usefulness to the patient, can also provide current support to caregivers and family members. Therefore clinicians must define a personalized pathway for patients, considering the characteristics of the disease and the type of treatment proposed, to optimize health status and prevent possible side effects while also improving QoL and treatment adherence., (© 2024. The Author(s).)

Whether Clonal Hematopoiesis (CH) represents a risk factor for severity of the COVID-19 disease remains a controversial issue. We report the first high- sensitivity analysis of CH in COVID-19 patients (threshold of detection at 0.5% vs 1 or 2% in previous studies). We analyzed 24 patients admitted to ICU for COVID-19 (COV-ICU) and 19 controls, including healthy subjects and asymptomatic SARS-CoV2-positive individuals. Despite the significantly higher numbers of CH mutations identified (80% mutations with <2% variant allele frequency, VAF), we did not find significant differences between COV-ICU patients and controls in the prevalence of CH or in the numbers, VAF or functional categories of the mutated genes, suggesting that CH is not overrepresented in patients with COVID-19. However, when considering potential drivers CH mutations (CH-PD), COV-ICU patients showed higher clonal complexity, in terms of both mutation numbers and VAF, and enrichment of variants reported in myeloid neoplasms. However, we did not score an impact of increased CH-PD on patient survival or clinical parameters associated with inflammation. These data suggest that COVID-19 influence the clonal composition of the peripheral blood and call for further investigations addressing the potential long-term clinical impact of CH on people experiencing severe COVID-19. We acknowledge that it will indispensable to perform further studies on larger patient cohorts in order to validate and generalize our conclusions. Moreover, we performed CH analysis at a single time point. It will be necessary to consider longitudinal approaches with long periods of follow-up in order to assess if the COVID-19 disease could have an impact on the evolution of CH and long-term consequences in patients that experienced severe COVID-19., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ronchini et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Long-read sequencing allows analyses of single nucleic-acid molecules and produces sequences in the order of tens to hundreds kilobases. Its application to whole-genome analyses allows identification of complex genomic structural-variants (SVs) with unprecedented resolution. SV identification, however, requires complex computational methods, based on either read-depth or intra- and inter-alignment signatures approaches, which are limited by size or type of SVs. Moreover, most currently available tools only detect germline variants, thus requiring separate computation of sample pairs for comparative analyses. To overcome these limits, we developed a novel tool (Germline And SOmatic structuraL varIants detectioN and gEnotyping; GASOLINE) that groups SV signatures using a sophisticated clustering procedure based on a modified reciprocal overlap criterion, and is designed to identify germline SVs, from single samples, and somatic SVs from paired test and control samples. GASOLINE is a collection of Perl, R and Fortran codes, it analyzes aligned data in BAM format and produces VCF files with statistically significant somatic SVs. Germline or somatic analysis of 30[Formula: see text] sequencing coverage experiments requires 4-5 h with 20 threads. GASOLINE outperformed currently available methods in the detection of both germline and somatic SVs in synthetic and real long-reads datasets. Notably, when applied on a pair of metastatic melanoma and matched-normal sample, GASOLINE identified five genuine somatic SVs that were missed using five different sequencing technologies and state-of-the art SV calling approaches. Thus, GASOLINE identifies germline and somatic SVs with unprecedented accuracy and resolution, outperforming currently available state-of-the-art WGS long-reads computational methods., (© 2023. The Author(s).)

Introduction: Oxford Nanopore Technologies (ONT) is a third generation sequencing approach that allows the analysis of individual, full-length nucleic acids. ONT records the alterations of an ionic current flowing across a nano-scaled pore while a DNA or RNA strand is threading through the pore. Basecalling methods are then leveraged to translate the recorded signal back to the nucleic acid sequence. However, basecall generally introduces errors that hinder the process of barcode demultiplexing, a pivotal task in single-cell RNA sequencing that allows for separating the sequenced transcripts on the basis of their cell of origin. Methods: To solve this issue, we present a novel framework, called UNPLEX, designed to tackle the barcode demultiplexing problem by operating directly on the recorded signals. UNPLEX combines two unsupervised machine learning methods: autoencoders and self-organizing maps (SOM). The autoencoders extract compact, latent representations of the recorded signals that are then clustered by the SOM. Results and Discussion: Our results, obtained on two datasets composed of in silico generated ONT-like signals, show that UNPLEX represents a promising starting point for the development of effective tools to cluster the signals corresponding to the same cell., Competing Interests: TL received reimbursement of expenses from ONT to speak at an event. 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., (Copyright © 2023 Papetti, Spolaor, Nazari, Tirelli, Leonardi, Caprioli, Besozzi, Vlachou, Pelicci, Cazzaniga and Nobile.)

Aberrant DNA methylation at CpG dinucleotides is a cancer hallmark that is associated with the emergence of resistance to anti cancer treatment, though molecular mechanisms and biological significance remain elusive. Genome scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG rich regions (CpG islands). We report the first high coverage whole-genome map in cancer using the long read nanopore technology, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, after chemotherapy. Long read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution, and showed that the relapse methylome is characterized by hypermethylation at both CpG islands and sparse CpGs regions. Most differentially methylated genes, however, were not differentially expressed nor enriched for chemoresistance genes. A small fraction of under-expressed and hyper-methylated genes at sparse CpGs, in the gene body, was significantly enriched in transcription factors (TFs). Remarkably, these few TFs supported large gene-regulatory networks including 50% of all differentially expressed genes in the relapsed AMLs and highly-enriched in chemoresistance genes. Notably, hypermethylated regions at sparse CpGs were poorly conserved in the relapsed AMLs, under-represented at their genomic positions and showed higher methylation entropy, as compared to CpG islands. Analyses of available datasets confirmed TF binding to their target genes and conservation of the same gene-regulatory networks in large patient cohorts. Relapsed AMLs carried few patient specific structural variants and DNA mutations, apparently not involved in drug resistance. Thus, drug resistance in AMLs can be mainly ascribed to the selection of random epigenetic alterations at sparse CpGs of a few transcription factors, which then induce reprogramming of the relapsing phenotype, independently of clonal genomic evolution., (© 2023. The Author(s).)

Background and Purpose: Vaccination against SARS-CoV-2 has been associated with rare occurrences of severe venous thromboses. Very little data exist about arterial ischemic strokes. We have assessed the features of ischemic strokes occurring shortly after vaccination against SARS-CoV-2 in the Cremona area, Italy., Methods: From February 1, to July 31, 2021, all patients with ischemic stroke within four weeks of vaccination against COVID-19 admitted to our stroke unit were consecutively collected, and their main features were compared with those of all other patients with ischemic strokes admitted during the same period., Results: Sixteen strokes after vaccination were collected. They represented 10.5% of all ischemic strokes. Median interval from vaccination was 12 days (range 1-24). Fifteen (93.8%) had received the BNT162b2 (Pfizer-BioNTech) vaccine and 1 (6.2%) the ChAdOx1 nCoV-19 (AstraZeneca). Two patients (12.5%) had a mild thrombocytopenia on admission (128,000 and 142,000/ml), without any evidence of bleeding or venous thrombosis. Thrombolysis and/or thrombectomy were carried out in 4 cases (25.0%). When compared with 137 strokes without recent vaccination, none of the demographic, clinical, and laboratory features of post-vaccination strokes were significantly different., Conclusions: Ischemic strokes occurring shortly after COVID-19 vaccination at our center were similar to those of non-vaccinated patients. Therefore, the relatively high percentage of such patients probably relates to the very high fraction of elderly people vaccinated against SARS-CoV-2 in the Cremona area, rather than to a consequence of vaccination., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Myeloid neoplasms (MN) are heterogeneous clonal disorders arising from the expansion of hematopoietic stem and progenitor cells. In parallel with genetic and epigenetic dynamics, the immune system plays a critical role in modulating tumorigenesis, evolution and therapeutic resistance at the various stages of disease progression. Single-cell technologies represent powerful tools to assess the cellular composition of the complex tumor ecosystem and its immune environment, to dissect interactions between neoplastic and non-neoplastic components, and to decipher their functional heterogeneity and plasticity. In addition, recent progress in multi-omics approaches provide an unprecedented opportunity to study multiple molecular layers (DNA, RNA, proteins) at the level of single-cell or single cellular clones during disease evolution or in response to therapy. Applying single-cell technologies to MN holds the promise to uncover novel cell subsets or phenotypic states and highlight the connections between clonal evolution and immune escape, which is crucial to fully understand disease progression and therapeutic resistance. This review provides a perspective on the various opportunities and challenges in the field, focusing on key questions in MN research and discussing their translational value, particularly for the development of more efficient immunotherapies., 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., (Copyright © 2022 Caprioli, Nazari, Milovanovic and Pelicci.)

Secondary acute myeloid leukemia (sAML) after myelodysplastic or myeloproliferative disorders is a high-risk category currently identified by clinical history or specific morphological and cytogenetic abnormalities. However, in the absence of these features, uncertainties remain to identify the secondary nature of some cases otherwise defined as de novo AML. To test whether a chromatin-spliceosome (CS) mutational signature might better inform the definition of the de novo AML group, we analyzed a prospective cohort of 413 newly diagnosed AML patients enrolled into a randomized clinical trial (NILG AML 02/06) and provided with accurate cytogenetic and molecular characterization. Among clinically defined de novo AML, 17.6% carried CS mutations (CS-AML) and showed clinical characteristics closer to sAML (older age, lower white blood cell counts and higher rate of multilineage dysplasia). Outcomes in this group were adverse, more similar to those of sAML as compared to de novo AML (overall survival, 30% in CS-AML and 17% in sAML vs 61% in de novo AML, P<0.0001; disease free survival, 26% in CS-AML and 22% in sAML vs 54% of de novo AML, P<0.001) and independently confirmed by multivariable analysis. Allogeneic transplant in first complete remission improved survival in both sAML and CS-AML patients. In conclusion, these findings highlight the clinical significance of identifying CS-AML for improved prognostic prediction and potential therapeutic implications. (NILG AML 02/06: ClinicalTrials.gov Identifier: NCT00495287).

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, patients with defective immunity after chemo-immunotherapy due to hematological disorders showed prolonged symptoms and worse prognosis of coronavirus disease-2019 (COVID-19) pneumonia, probably due to inadequate adaptive immune response and noneffective viral clearance. We describe a single-center series of hematological immunocompromised patients undergoing passive immunization with hyperimmune plasma for persistent COVID-19 symptoms. In all cases, such treatment was well tolerated and contributed to clinical and radiological improvement and recovery; viral clearance was also achieved in a patients' subset. Although requiring further investigation, these results suggest a specific role for hyperimmune plasma administration in hematological patients.

: Myelodysplasias with hypocellular bone marrow (hMDS) represent about 10-15% of MDS and are defined by reduced bone marrow cellularity (i.e., <25% or an inappropriately reduced cellularity for their age in young patients). Their diagnosis is still an object of debate and has not been clearly established in the recent WHO classification. Clinical and morphological overlaps with both normo/hypercellular MDS and aplastic anemia include cytopenias, the presence of marrow hypocellularity and dysplasia, and cytogenetic and molecular alterations. Activation of the immune system against the hematopoietic precursors, typical of aplastic anemia, is reckoned even in hMDS and may account for the response to immunosuppressive treatment. Finally, the hMDS outcome seems more favorable than that of normo/hypercellular MDS patients. In this review, we analyze the available literature on hMDS, focusing on clinical, immunological, and molecular features. We show that hMDS pathogenesis and clinical presentation are peculiar, albeit in-between aplastic anemia (AA) and normo/hypercellular MDS. Two different hMDS phenotypes may be encountered: one featured by inflammation and immune activation, with increased cytotoxic T cells, increased T and B regulatory cells, and better response to immunosuppression; and the other, resembling MDS, where T and B regulatory/suppressor cells prevail, leading to genetic clonal selection and an increased risk of leukemic evolution. The identification of the prevailing hMDS phenotype might assist treatment choice, inform prognosis, and suggest personalized monitoring., Competing Interests: B.F. received consultancy from Amgen, Apellis, Momenta, and Novartis. W.B. received consultancy from Agios, Alexion, Apellis, Biocryst, Bioverativ, Incyte, Momenta, and Novartis and lecture fee/congress support from Alexion, Incyte, Novartis, and Sanofi.

By way of a Next-Generation Sequencing NGS high throughput approach, we defined the mutational profile in a cohort of 221 normal karyotype acute myeloid leukemia (NK-AML) enrolled into a prospective randomized clinical trial, designed to evaluate an intensified chemotherapy program for remission induction. NPM1 , DNMT3A, and FLT3 -ITD were the most frequently mutated genes while DNMT3A , FLT3 , IDH1 , PTPN11 , and RAD21 mutations were more common in the NPM1 mutated patients ( p < 0.05). IDH1 R132H mutation was strictly associated with NPM1 mutation and mutually exclusive with RUNX1 and ASXL1 . In the whole cohort of NK-AML, no matter the induction chemotherapy used, by multivariate analysis, the achievement of complete remission was negatively affected by the SRSF2 mutation. Alterations of FLT3 ( FLT3- ITD) and U2AF1 were associated with a worse overall and disease-free survival ( p < 0.05). FLT3- ITD positive patients who proceeded to alloHSCT had a survival probability similar to FLT3- ITD negative patients and the transplant outcome was no different when comparing high and low-AR- FLT3 -ITD subgroups in terms of both OS and DFS. In conclusion, a comprehensive molecular profile for NK-AML allows for the identification of genetic lesions associated to different clinical outcomes and the selection of the most appropriate and effective treatment strategies, including stem cell transplantation and targeted therapies.

We analyzed the impact of alloHSCT in a single center cohort of 89 newly diagnosed NPM1 mut AML patients, consecutively treated according to the Northern Italy Leukemia Group protocol 02/06 [NCT00495287]. After two consolidation cycles, the detection of measurable residual disease (MRD) by RQ-PCR was strongly associated with an inferior three-year overall survival (OS, 45% versus 84%, p = 0.001) and disease-free survival (DFS, 44% versus 76%, p = 0.006). In MRD-negative patients, post-remissional consolidation with alloHSCT did not provide a significant additional benefit over a conventional chemotherapy in terms of overall survival [OS, 89% (95% CI 71-100%) versus 81% (95% CI 64-100%), p = 0.59] and disease-free survival [DFS, 80% (95% CI 59-100%) versus 75% (95% CI 56-99%), p = 0.87]. On the contrary, in patients with persistent MRD positivity, the three-year OS and DFS were improved in patients receiving an alloHSCT compared to those allocated to conventional chemotherapy (OS, 52% versus 31%, p = 0.45 and DFS, 50% versus 17%, p = 0.31, respectively). However, in this group of patients, the benefit of alloHSCT was still hampered by a high incidence of leukemia relapse during the first year after transplantation (43%, 95% CI 25-60%). Consolidative alloHSCT improves outcomes compared to standard chemotherapy in patients with persistent NPM1 mut MRD positivity, but in these high-risk patients, the significant incidence of leukemia relapse must be tackled by post-transplant preemptive treatments.

Immunomodulatory drugs (IMiDs) may favor autoimmune disease (AD) occurrence. We conducted a retrospective study to evaluate AD occurrence among IMiD-treated patients with myeloma. Patients were grouped into three classes depending on the type of IMiD engaged. The first group included patients treated with thalidomide (Thal) (n = 474), the second group with lenalidomide (Len) (n = 140) and patients in the third group were first treated with Thal followed by Len (Thal-Len) (n = 94). Absolute risk of AD was 0.4% for patients treated with Thal, 4.3% for Len and 1.1% for Thal-Len. ADs manifested prevalently as autoimmune cytopenias (55%), although we observed one vasculitis, one optic neuritis, one Graves' disease and one polymyositis. ADs occurred preferentially in the first months of IMiD treatment. A previous autologous transplant was shown to be a significant risk factor. All ADs were managed with IMiD discontinuation and steroids, resolving in a few weeks, except for Graves' disease and polymyositis.

Background: Recent studies on representative samples of psychiatric services have shown that low proportions of cases received effective rehabilitation interventions. The following are likely to be the most important causes: the scarcity of mental health workers trained in social and work skills strategies and the absence of a structured framework to formulate rehabilitation practices. The aim of this study was to assess if a specific structured planning and evaluation manual, called VADO (Valutazione delle Abilità e Definizione degli Obiettivi--in english: Skills Assessment and Definition of Goals), is more effective than routine interventions in reducing disability in patients with schizophrenia., Method: Each of 10 mental health services were invited to recruit 10 patients with a schizophrenic disorder. Altogether 98 patients were recruited. Of these, 62 patients were randomly allocated to the intervention/experimental or a control group. The remaining group of 36 patients was not randomised and it was considered as a parallel effectiveness study. Assessment measures at the beginning of the study and at the one-year follow-up included the FPS scale of social functioning and the BPRS 4.0. Between group (VADO vs. Routine) and time effects were examined with ANOVA, Chi-square or Fisher exact. Clinical "improvement" was defined as an increase of at least ten points on the FPS or a decrease of at least 20% on BPRS scores., Results: 31 of the 62 randomized patients received the experimental interventions, while 31 followed the routine ones. At follow-up, the experimental group showed statistically and clinically greater improvements in psychopathology and social functioning. Better outcomes of both social functioning and symptom severity were observed in non randomised patients (parallel effectiveness study)., Conclusion: The results suggest that setting personalised and measurable objectives, as recommended by the manual, can improve the outcome of rehabilitation of severe mental disorders. Better outcomes in the parallel effectiveness study could be attributed to the greater confidence and enthusiasm of staff in centres where the VADO approach originated.

The mucopolysaccharide storage diseases express themselves clinically with a wide variety of abnormalities, including growth and mental retardation, skeletal abnormalities, clouded corneas, nerve compression syndromes, upper airway obstruction and cardiovascular involvement, to name the most common. In most cases the cause of early death is cardiorespiratory failure secondary to cardiovascular involvement and upper airway obstruction. The findings of cardiac ultrasound examination in 29 children, adolescents and young adults are presented. In addition to the previously well-described abnormalities of the mitral and aortic valves in several types of mucopolysaccharide storage disease, we report patchy involvement in some cases, 3 instances of asymmetric septal hypertrophy not previously reported in mucopolysaccharide storage diseases, cardiac involvement in half of our patients with Sanfilippo syndrome and a lack of age-related severity of cardiac involvement even within the specific syndromes.