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Freezing-cryosurgery, and electrolysis-electrochemical therapy (EChT), are two important minimally invasive surgery tissue ablation technologies. Despite major advantages they also have some disadvantages. Cryosurgery cannot induce cell death at high subzero freezing temperatures and requires multiple freeze thaw cycles, while EChT requires high concentrations of electrolytic products-which makes it a lengthy procedure. Based on the observation that freezing increases the concentration of solutes (including products of electrolysis) in the frozen region and permeabilizes the cell membrane to these products, this study examines the hypothesis that there could be a synergistic effect between freezing and electrolysis in their use together for tissue ablation. Using an animal model we refer to as vivens ex vivo, which may be of value in reducing the use of animals for experiments, combined with a Hematoxylin stain of the nucleus, we show that there are clinically relevant protocols in which the cell nucleus appears intact when electrolysis and freezing are used separately but is affected by certain combinations of electrolysis and freezing.

In the context of the increasing number of obese individuals, a major problem is represented by obesity and malnutrition in children. This condition is mainly ascribable to unbalanced diets characterized by high intakes of fat and sugar. Childhood obesity and malnutrition are not only associated with concurrent pathologies but potentially compromise adult life. Considering the strict correlation among systemic metabolism, obesity, and skeletal muscle health, we wanted to study the impact of juvenile malnutrition on the adult skeletal muscle. To this aim, 3-week-old C56BL/6 female and male mice were fed for 20 weeks on a high-fat. high-sugar diet, and their muscles were subjected to a histological evaluation. MyHCs expression, glycogen content, intramyocellular lipids, mitochondrial activity, and capillary density were analyzed on serial sections to obtain the metabolic profile. Our observations indicate that a high-fat, high-sugar diet alters the metabolic profile of skeletal muscles in a sex-dependent way and induces the increase in type II fibers, mitochondrial activity, and lipid content in males, while reducing the capillary density in females. These data highlight the sex-dependent response to nutrition, calling for the development of specific strategies and for a systematic inclusion of female subjects in basic and applied research in this field. [ABSTRACT FROM AUTHOR]

Breast cancer (BC) is currently the most common neoplasm, the second leading cause of cancer death in women worldwide, and is a major health problem. The discovery of new biomarkers is crucial to improve our knowledge of breast cancer and strengthen our clinical approaches to diagnosis, prognosis, and follow-up. In recent decades, there has been increasing interest in circulating RNA (circRNA) as modulators of gene expression involved in tumor development and progression. The study of circulating circRNAs (ccircRNAs) in plasma may provide new non-invasive diagnostic, prognostic, and predictive biomarkers for BC. This review describes the latest findings on BC-associated ccircRNAs in plasma and their clinical utility. Several ccircRNAs in plasma have shown great potential as BC biomarkers, especially from a diagnostic point of view. Mechanistically, most of the reported BC-associated ccircRNAs are involved in the regulation of cell survival, proliferation, and invasion, mainly via MAPK/AKT signaling pathways. However, the study of circRNAs is a relatively new area of research, and a larger number of studies will be crucial to confirm their potential as plasma biomarkers and to understand their involvement in BC. [ABSTRACT FROM AUTHOR]

In the realm of liver transplantation, accurately determining hepatic steatosis levels is crucial. Recognizing the essential need for improved diagnostic precision, particularly for optimizing diagnosis time by swiftly handling easy-to-solve cases and allowing the expert time to focus on more complex cases, this study aims to develop cutting-edge algorithms that enhance the classification of liver biopsy images. Additionally, the challenge of maintaining data privacy arises when creating automated algorithmic solutions, as sharing patient data between hospitals is restricted, further complicating the development and validation process. This research tackles diagnostic accuracy by leveraging novel techniques from the rapidly evolving field of quantum machine learning, known for their superior generalization abilities. Concurrently, it addresses privacy concerns through the implementation of privacy-conscious collaborative machine learning with federated learning. We introduce a hybrid quantum neural network model that leverages real-world clinical data to assess non-alcoholic liver steatosis accurately. This model achieves an image classification accuracy of 97 % , surpassing traditional methods by 1.8 % . Moreover, by employing a federated learning approach that allows data from different clients to be shared while ensuring privacy, we maintain an accuracy rate exceeding 90 % . This initiative marks a significant step towards a scalable, collaborative, efficient, and dependable computational framework that aids clinical pathologists in their daily diagnostic tasks. [ABSTRACT FROM AUTHOR]

Simple Summary: DNA repair has an important role in malignant pleural mesothelioma tumorigenesis and progression. The prognosis of mesothelioma patients is very poor and predictive biomarkers are needed for better management. We analyzed the expression of more than 700 genes involved in different cellular pathways using Nanostring technology in a cohort of 54 epithelioid malignant pleural mesothelioma patients. The median survival time of the cohort was 16.9 months and this cut-off was used to classify patients as long and short survivors (LS/SS) with, respectively, an overall survival ≥ and <16.9 months, as well as very long and very short survivors (VLS/VSS) with an overall survival ≥ than 33.8 and < than 8.45 months. A down-regulation of the DNA damage response pathway was found in LS versus SS. These data were validated by the finding that VLS had a lower number of RAD51- and BRCA1-positive tumor cells than VSS. If these data can be corroborated, an easy and cost-effective test could be routinely used to better manage epithelioid malignant pleural mesothelioma patients. Aim: DNA repair has an important role in malignant pleural mesothelioma (MPM) tumorigenesis and progression. Prognostic/predictive biomarkers for better management of MPM patients are needed. In the present manuscript, we analyzed the expression of more than 700 genes in a cohort of MPM patients to possibly find biomarkers correlated with survival. Methods: A total of 54 MPM patients, all with epithelioid histology, whose survival follow-up and formalin-fixed paraffin-embedded tumors were available, were included in the study. Gene expression profiles were evaluated using a Nanostring platform analyzing 760 genes involved in different cellular pathways. The percentages of proliferating tumor cells positive for RAD51 and BRCA1 foci were evaluated using an immunofluorescence assay, as a readout of homologous recombination repair status. Results: Patient median survival time was 16.9 months, and based on this value, they were classified as long and short survivors (LS/SS) with, respectively, an overall survival ≥ and <16.9 months as well as very long and very short survivors (VLS/VSS) with an overall survival ≥ than 33.8 and < than 8.45 months. A down-regulation in the DNA damage/repair expression score was observed in LS and VLS as compared to SS and VSS. These findings were validated by the lower number of both RAD51 and BRCA1-positive tumor cells in VLS as compared to VSS. Conclusions: The down-regulation of DNA repair signature in VLS was functionally validated by a lower % of RAD51 and BRCA1-positive tumor cells. If these data can be corroborated in a prospective trial, an easy, cost-effective test could be routinely used to better manage treatment in MPM patients. [ABSTRACT FROM AUTHOR]

Background. Cutaneous melanoma is an aggressive form of skin cancer. It has become an increasingly common neoplasm in the most developed countries, especially among individuals of European origin.

Complement component C1q can act as a pro-tumorigenic factor in the tumor microenvironment (TME). The TME in malignant pleural mesothelioma (MPM) is rich in C1q and hyaluronic acid (HA), whose interaction enhances adhesion, migration and proliferation of malignant cells. HA-bound C1q is also capable of modulating HA synthesis. Thus, we investigated whether HA-C1q interaction would affect HA degradation, analyzing the main degradation enzymes, hyaluronidase (HYAL)1 and HYAL2, and a C1q receptor candidate. We first proceeded with the characterization of HYALs in MPM cells, especially HYAL2, since bioinformatics survival analysis revealed that higher HYAL2 mRNA levels have an unfavorable prognostic index in MPM patients. Interestingly, Real-Time quantitative PCR, flow cytometry and Western blot highlighted an upregulation of HYAL2 after seeding of primary MPM cells onto HA-bound C1q. In an attempt to unveil the receptors potentially involved in HA-C1q signaling, a striking colocalization between HYAL2 and globular C1q receptor/HABP1/p32 (gC1qR) was found by immunofluorescence, surface biotinylation and proximity ligation assays. RNA interference experiments revealed a potentially regulatory function exerted by gC1qR on HYAL2 expression, since C1QBP (gene for gC1qR) silencing unexpectedly caused HYAL2 downregulation. In addition, the functional blockage of gC1qR by a specific antibody hindered HA-C1q signaling and prevented HYAL2 upregulation. Thus, C1q-HA interplay is responsible for enhanced HYAL2 expression, suggesting an increased rate of HA catabolism and the release of pro-inflammatory and pro-tumorigenic HA fragments in the MPM TME. Our data support the notion of an overall tumor-promoting property of C1q. Moreover, the overlapping localization and physical interaction between HYAL2 and gC1qR suggests a potential regulatory effect of gC1qR within a putative HA-C1q macromolecular complex. [ABSTRACT FROM AUTHOR]

The poor survival of triple-negative breast cancer (TNBC) is due to its aggressive behavior, large heterogeneity, and high risk of recurrence. A comprehensive molecular investigation of this type of breast cancer using high-throughput next-generation sequencing (NGS) methods may help to elucidate its potential progression and discover biomarkers related to patient survival. In this review, the NGS applications in TNBC research are described. Many NGS studies point to TP53 mutations, immunocheckpoint response genes, and aberrations in the PIK3CA and DNA repair pathways as recurrent pathogenic alterations in TNBC. Beyond their diagnostic and predictive/prognostic value, these findings suggest potential personalized treatments in PD -L1-positive TNBC or in TNBC with a homologous recombination deficit. Moreover, the comprehensive sequencing of large genomes with NGS has enabled the identification of novel markers with clinical value in TNBC, such as AURKA, MYC, and JARID2 mutations. In addition, NGS investigations to explore ethnicity-specific alterations have pointed to EZH2 overexpression, BRCA1 alterations, and a BRCA2-delaAAGA mutation as possible molecular signatures of African and African American TNBC. Finally, the development of long-read sequencing methods and their combination with optimized short-read techniques promise to improve the efficiency of NGS approaches for future massive clinical use. [ABSTRACT FROM AUTHOR]

Simple Summary: Severe neonatal hyperbilirubinemia may damage the brain, leading to motor, cognitive, and auditory abnormalities. We recently discovered that bilirubin might act by controlling the genetic developmental program of the cerebellum, a region of the brain well known to be susceptible to bilirubin-induced damage. In this paper, we expand the study of the potential impact of bilirubin in the control of postnatal brain development to brain regions better correlating with human symptoms. The maximal abnormalities of structure and cell shape (histology) were detected 9 days after birth, fully recovering later on. Differently, the analysis of the gene expression revealed transient alterations (early after birth, then recovering) in the hippocampus (memory, learning, and cognition) and inferior colliculi (auditory functions), but permanent (until adulthood) changes in the areas of the brain involved in the control of movements, information confirmed by the abnormal results on the behavioral tests. These new findings are well in agreement with the clinic and open a way for better deciphering the neurotoxic features of bilirubin neurotoxicity and potential therapeutic approaches. Recent findings indicated aberrant epigenetic control of the central nervous system (CNS) development in hyperbilirubinemic Gunn rats as an additional cause of cerebellar hypoplasia, the landmark of bilirubin neurotoxicity in rodents. Because the symptoms in severely hyperbilirubinemic human neonates suggest other regions as privileged targets of bilirubin neurotoxicity, we expanded the study of the potential impact of bilirubin on the control of postnatal brain development to regions correlating with human symptoms. Histology, transcriptomic, gene correlation, and behavioral studies were performed. The histology revealed widespread perturbation 9 days after birth, restoring in adulthood. At the genetic level, regional differences were noticed. Bilirubin affected synaptogenesis, repair, differentiation, energy, extracellular matrix development, etc., with transient alterations in the hippocampus (memory, learning, and cognition) and inferior colliculi (auditory functions) but permanent changes in the parietal cortex. Behavioral tests confirmed the presence of a permanent motor disability. The data correlate well both with the clinic description of neonatal bilirubin-induced neurotoxicity, as well as with the neurologic syndromes reported in adults that suffered neonatal hyperbilirubinemia. The results pave the way for better deciphering the neurotoxic features of bilirubin and evaluating deeply the efficacy of new therapeutic approaches against the acute and long-lasting sequels of bilirubin neurotoxicity. [ABSTRACT FROM AUTHOR]

Background: In Western countries, breast cancer (BC) is the most common cancer in women. Early detection has a positive impact on survival, quality of life, and public health costs. Mammography screening programs have increased early detection rates, but new approaches to more personalized surveillance could further improve diagnosis. Circulating cell-free DNA (cfDNA) in blood could provide a potential tool for early diagnosis by analyzing cfDNA quantity, circulating tumor DNA mutations, or cfDNA integrity (cfDI). Methods: Plasma was obtained from the blood of 106 breast cancer patients (cases) and 103 healthy women (controls). Digital droplet PCR was used for the determination of ALU 260/111 bp and LINE-1 266/97 bp copy number ratio and cfDI. cfDNA abundance was calculated using copies of the EEF1A2 gene. The accuracy of biomarker discrimination was analyzed with receiver operating characteristic curve (ROC). Sensitivity analyses were performed to account for age as a potential confounder. Results: Cases had significantly lower ALU 260/111 or LINE-1 266/97 copy number ratios (median; ALU 260/111 = 0.08, LINE-1 266/97 = 0.20), compared with control (median; ALU 260/111 = 0.10, LINE-1 266/97 = 0.28) (p < 0.001). ROC analysis showed that copy number ratio discriminated cases from controls (area under the curve, AUC = 0.69, 95% CI: 0.62–0.76 for ALU and 0.80, 95% CI: 0.73–0.86 for LINE-1). ROC from cfDI confirmed the better diagnostic performance of LINE-1 compared with ALU. Conclusions: Analysis of LINE-1 266/97 copy number ratio or cfDI by ddPCR appears to be a useful noninvasive test that could aid in early BC detection. Further studies in a large cohort are needed to validate the biomarker. [ABSTRACT FROM AUTHOR]

Simple Summary: The prevalence of oropharyngeal squamous cell carcinomas (OPSCCs) driven by human papillomavirus (HPV) infection has a wide geographical variability. In addition, several authors have reported steadily increasing prevalence rates over the past two decades. It is important to know the epidemiological landscape of these tumors both to estimate the reliability of diagnostic tests and to guide public health choices aimed at primary prevention. In this retrospective study, we observed a significant increase in the prevalence of HPV-driven OPSCC from 12% during 2000–2006 to 50% during 2019–2022. The use of p16INK4a overexpression as a surrogate marker of transforming HPV infection should consider the prevalence rates of HPV-driven OPSCC as this significantly impacts on its positive predictive value. Health policies should actively promote vaccination against HPV in both females and males also for the prevention of OPSCC. Background: The prevalence and incidence of oropharyngeal squamous cell carcinomas (OPSCCs) driven by human papillomavirus (HPV) infection are increasing worldwide, being higher in high-income countries. However, data from Italy are scanty. p16INK4a overexpression is the standard in determining HPV-driven carcinogenesis, but disease prevalence impacts on its positive predictive value. Methods: This is a multicenter retrospective study enrolling 390 consecutive patients aged ≥18 years, diagnosed with pathologically confirmed OPSCC in Northeastern Italy between 2000 and 2022. High-risk HPV-DNA and p16INK4a status were retrieved from medical records or evaluated in formalin-fixed paraffin-embedded specimens. A tumor was defined as HPV-driven when double positive for high-risk HPV-DNA and p16INK4a overexpression. Results: Overall, 125 cases (32%) were HPV-driven, with a significant upward temporal trend from 12% in 2000–2006 to 50% in 2019–2022. The prevalence of HPV-driven cancer of the tonsil and base of the tongue increased up to 59%, whereas it remained below 10% in other subsites. Consequently, the p16INK4a positive predictive value was 89% for the former and 29% for the latter. Conclusions: The prevalence of HPV-driven OPSCC continued to increase, even in the most recent period. When using p16INK4a overexpression as a surrogate marker of transforming HPV infection, each institution should consider the subsite-specific prevalence rates of HPV-driven OPSCC as these significantly impact on its positive predictive value. [ABSTRACT FROM AUTHOR]

Purpose. Thermal ablations (TA) are gaining ground as alternative options to conventional therapies for symptomatic benign thyroid nodules. Little is known about the impact of nodule biology on the outcomes of TA. The aim of our study was to evaluate the baseline immunocytochemistry profile of thyroid nodules that were poorly responsive to TA in order to identify potential predictors of the treatment response. Methods. From a cohort of 406 patients with benign thyroid nodules treated with TA and followed for 5 years, we retrospectively selected two groups of patients: NONRESPONDERS (patients who did not respond to TA and were later surgically treated) and RESPONDERS (patients who responded to TA). The fine-needle aspiration cytology (FNAC) slides obtained before TA were stained for Galectin-3, HBME-1, CK-19, and Ki-67. Results. Benign nodules of NONRESPONDERS (n = 19) did not express CK-19 (p = 0.03), as compared to RESPONDERS (n = 26). We combined the absence of CK-19 and the presence of Ki-67 to obtain a composite biomarker of resistance to TA, which discriminated between likelihood of retreatment and no retreatment with an AUC of 0.68 (95%CI: 0.55-0.81) and a sensitivity, specificity, PPV, and NPV of 29%, 91%, 71%, and 64%, respectively. Conclusion. In benign thyroid nodules, the absence of CK-19 was associated with resistance to TA, while the presence of CK-19 was predictive of response to TA. If confirmed, this finding could provide rapid and inexpensive information about the potential outcome of TA on benign thyroid nodules. In addition, as CK-19 can be expressed in adenomatous hyperplasia, it could be speculated that these nodules, rather than follicular adenomas, might be better candidates for TA. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) remains a global health challenge, representing the third leading cause of cancer deaths worldwide. Although therapeutic advances have been made in the few last years, the prognosis remains poor. Thus, there is a dire need to develop novel therapeutic strategies. In this regard, two approaches can be considered: (1) the identification of tumor-targeted delivery systems and (2) the targeting of molecule(s) whose aberrant expression is confined to tumor cells. In this work, we focused on the second approach. Among the different kinds of possible target molecules, we discuss the potential therapeutic value of targeting non-coding RNAs (ncRNAs), which include micro interfering RNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). These molecules represent the most significant RNA transcripts in cells and can regulate many HCC features, including proliferation, apoptosis, invasion and metastasis. In the first part of the review, the main characteristics of HCC and ncRNAs are described. The involvement of ncRNAs in HCC is then presented over five sections: (a) miRNAs, (b) lncRNAs, (c) circRNAs, (d) ncRNAs and drug resistance and (e) ncRNAs and liver fibrosis. Overall, this work provides the reader with the most recent state-of-the-art approaches in this field, highlighting key trends and opportunities for more advanced and efficacious HCC treatments. [ABSTRACT FROM AUTHOR]

With the advent of immunotherapies, the field of cancer therapy has been revived with new hope, especially for cancers with dismal prognoses, such as the glioblastoma multiforme (GBM). Currently, immunotherapies should potentiate the host's own antitumor immune response against cancer cells, but it has been documented that they are effective only in small subsets of patients. Therefore, accurate predictors of response are urgently needed to identify who will benefit from immune-modulatory therapies. Brain tumors are challenging in terms of treatments. The immune response in the brain is highly regulated, and the immune microenvironment in brain metastases is active with a high density of tumor-infiltrating lymphocytes (TILs, CD3+ T cells) in certain patients and, therefore, may serve as a potential treatment target. In our study, we performed immunohistochemistry for CD3 and PD-L1 along the routine assessment of the O6-methylguanine-methyltransferase (MGMT) promoter methylation status and the IDH1 and 2 status in a single center cohort of 69 patients with GBM (58 primary tumors and 11 recurrences) who underwent standard multimodal therapies (surgery/radiotherapy/adjuvant temozolamide). We analyzed the association of PD-L1 tumor expression and TILs with overall survival (OS). The PD-L1 expression was observed in 25 of 58 (43%) newly diagnosed primary glioblastoma specimens. The sparse-to-moderate density of TILs, identified with CD3+ expression, was found in 48 of 58 (83%) specimens. Neither PD-L1 expression nor TILs were associated with overall survival. In conclusion, TILs and/or PD-L1 expression are detectable in the majority of glioblastoma samples, and even if they slightly relate to the outcome, they do not show a statistically significant correlation. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer due to its molecular heterogeneity and poor clinical outcomes. Analysis of circulating cell-free tumor nucleic acids (ctNAs) can improve our understanding of TNBC and provide efficient and non-invasive clinical biomarkers that may be representative of tumor heterogeneity. In this review, we summarize the potential of ctNAs to aid TNBC diagnosis and prognosis. For example, tumor fraction of circulating cell-free DNA (TFx) may be useful for molecular prognosis of TNBC: high TFx levels after neoadjuvant chemotherapy have been associated with shorter progression-free survival and relapse-free survival. Mutations and copy number variations of TP53 and PIK3CA/AKT genes in plasma may be important markers of TNBC onset, progression, metastasis, and for clinical follow-up. In contrast, the expression profile of circulating cell-free tumor non-coding RNAs (ctncRNAs) can be predictive of molecular subtypes of breast cancer and thus aid in the identification of TBNC. Finally, dysregulation of some circulating cell-free tumor miRNAs (miR17, miR19a, miR19b, miR25, miR93, miR105, miR199a) may have a predictive value for chemotherapy resistance. In conclusion, a growing number of efforts are highlighting the potential of ctNAs for future clinical applications in the diagnosis, prognosis, and follow-up of TNBC. [ABSTRACT FROM AUTHOR]

Cystic masses of the lateral neck are mostly benign. However, the incidence of metastatic squamous cell carcinoma (SCC) in cervical cystic masses initially diagnosed as benign is quite high in patients older than 40 years. The aim of this study was to compare the diagnostic accuracy of preoperative cytology and intraoperative frozen section (FS) in detecting malignancy in cystic masses of the neck. We reviewed 61 patients who underwent preoperative ultrasound-guided fine-needle aspiration cytology (FNAC) and neck biopsy of a cystic neck mass, and analysed the concordance between FNAC and intraoperative FS with respect to definitive histology. HPV status was also tested. Of 49 eligible cases, the accuracy of preoperative FNAC was 70.5% (weighted kappa 0.53), meaning moderate agreement between cytology and final diagnosis. Intraoperative FS consultations detected 16 cases of SCC metastasis while the remaining 33 cases were negative for SCC, showing perfect agreement with histology. Since FS results were useful in evaluating cystic neck masses, despite a moderate accuracy of cytology, we suggest intraoperative FS analysis for all cystic neck masses. This technique can allow us to switch to therapeutic neck dissection, multiple upper aerodigestive tract biopsies, tongue base mucosectomy, and bilateral tonsillectomy in the same surgical setting. [ABSTRACT FROM AUTHOR]