Cervical cancer remains a significant global health burden, highlighting the need for more effective tools for early detection and tissue characterization. In this study, we propose a multimodal strategy that combines Raman spectroscopy, Atomic Force Microscopy (AFM), and Scanning Electron Microscopy (SEM) to investigate the molecular and morphological features of cervical squamous cell carcinoma (SCC). Raman spectroscopy was used to analyze biochemical signatures across different tissue regions tumor, necrotic, stromal, and glandular within the 813-1668 cm-1 range, identifying distinct molecular profiles between malignant and healthy areas. Specific vibrational peaks associated with DNA, proteins, and lipids were examined to track molecular changes related to tumor progression. AFM enabled nanoscale mapping of surface morphology, revealing structural irregularities associated with malignancy, while SEM provided detailed imaging of cellular and extracellular architecture, enhancing the visualization of cancer-induced morphological alterations. Although Raman spectroscopy has been studied for decades in cancer research, it has not yet replaced Pap smears and biopsies in clinical practice due to challenges in standardization, reproducibility, and clinical validation. This pilot study aims to serve as a stepping stone toward that goal, providing proof-of-concept data that may support the gradual translation of Raman spectroscopy into clinically relevant diagnostic workflows and underscores the potential of the technique, supported by complementary high-resolution imaging techniques, in the characterization of cervical cancer tissues. The integration of Raman, AFM, and SEM was used here as a pilot approach on paraffin-embedded samples, with AFM and SEM providing supportive morphological information while the long-term aim is to transfer Raman spectroscopy to fresh, untreated tissues, where its non-destructive and label-free nature could enable minimally invasive diagnostic applications.

Serous ovarian carcinoma (SOC) is a highly aggressive gynaecological malignancy with frequent relapse and the development of chemoresistance. A major feature of SOC tumour microenvironment is the accumulation of dense collagen fibres, which promote tumour progression and immune evasion. Discoidin domain receptor 2 (DDR2), a collagen-binding receptor tyrosine kinase activated by fibrillar collagens, mediates extracellular matrix-associated signalling involved in tumour progression and immune exclusion. However, its role in limiting antitumour immunity and contributing to immunotherapy resistance in SOC remains poorly understood. We investigated the impact of DDR2 blockade on SOC-specific T cell activity and on the efficacy of recombinant bispecific antibodies (BsAbs) targeting CD28 and mucin 1 (MUC1). SOC-specific T cells were generated from healthy donor peripheral blood mononuclear cells and assessed for recruitment, activation, and cytotoxicity against low-grade (OVCA433) and high-grade (OVCAR3) SOC cell lines under different treatment conditions. DDR2 inhibition enhanced SOC-specific T cell responses by remodelling the collagen matrix, favouring their recruitment, activation and cytotoxic activity against both low- and high-grade SOC cells. Moreover, combined treatment with DDR2 blockade and CD28xMUC1 BsAbs significantly increased the recruitment of SOC-specific T cells, granzyme B release, caspase-3 activation, and cytokine production. DDR2 blockade mitigates collagen-mediated immune exclusion and enhances the efficacy of CD28xMUC1 BsAb-based immunotherapy in SOC models. These findings provide a mechanistic rationale for further investigation of combined DDR2 targeting and CD28×MUC1 costimulatory strategies to improve ovarian cancer immunotherapy.

INTRODUCTION:The aim of this project is to assess interobserver agreement for programmed death-ligand 1 (PD-L1) scoring on of non-small cell lung cancer (NSCLC) on cytological specimens in a large-scale multicenter study, by exploiting the cell block-derived tissue microarray (cbTMA) approach. METHODS:A total of 65 cell blocks (CB) diagnosed as NSCLC were retrospectively collected and selected for TMA preparation. Hematoxylin-eosin and PD-L1 stained slides were digitized and uploaded on a free web sharing platform. Participants were asked to provide PD-L1 assessment by using the clinically relevant cutoff of tumor proportion score (TPS) (<1%; 1%-49%; >50%). Interobserver agreement was calculated using Fleiss's κ. RESULTS:Of 65 CBs, 11 were deemed not suitable; therefore, an overall number of 54 cores were used for the preparation of four TMAs. A total of 1674 evaluations were provided by 31 cytopathologists from 21 different institutions in nine countries. The statistical analysis showed a moderate overall agreement (κ = 0.49). The highest agreement was achieved in the TPS >50% category (κ = 0.57); moderate agreement was observed in TPS <1% category (κ = 0.51) and the lowest κ value was obtained for TPS 1%-49% category (k = 0.32). CONCLUSIONS:The overall moderate agreement observed showed that there is still room for improvement in inter-pathologist agreement for PD-L1 evaluation on cytological samples, highlighting the need for standardization in sample preparation, focused training in PD-L1 evaluation on cytological material, and the integration of machine learning tools to improve interobserver consistency.

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous breast cancer subtype with poor patient outcomes. TNBC heterogeneity arises from multiple dysregulated pathways, including Notch and Hedgehog, which contribute to tumor initiation, progression, and drug resistance. Identifying common molecular regulators of TNBC aggressiveness is crucial for developing effective therapeutic strategies. Here, we demonstrate that the transcriptional coactivator MAML1 drives TNBC aggressiveness by regulating Notch1 and Gli1 stability through the E3 ubiquitin ligase Itch, functioning as an Itch-negative regulator. Mechanistically, MAML1 interacts with Itch via its PPQY motif and promotes K63-linked self-ubiquitylation of Itch, deregulating its expression/activity. Using a Maml1-deficient mouse model, we reveal an inverse correlation between MAML1 and Itch levels, where the loss of MAML1 stabilizes Itch and suppresses Notch1 and Gli1 activity. Conversely, MAML1 upregulation enhances Notch1 and Gli1 expression, driving accelerated TNBC tumor growth and faster multiorgan metastasis in vivo. Accordingly, we show that MAML1 is overexpressed in a cohort of TNBC patients, and the combined overexpression of MAML1/Notch1 and MAML1/Gli1 correlates with poor clinical outcomes by in silico analysis. Our findings establish a dual role for MAML1 as a transcriptional coactivator and a post-translational regulator of Itch, thereby amplifying Notch and Hedgehog oncogenic signaling. This study uncovers MAML1 as a key driver of TNBC progression and a potential therapeutic target for fighting TNBC aggressiveness and heterogeneity.

Head and neck squamous cell carcinoma (HNSCC) is a prominent global health concern because of its high incidence, aggressive clinical behavior, and scarce therapeutic options. The management of these neoplasms in the recurrent/metastatic setting has been revolutionized following the results of key clinical trials, leading to the advent of immunotherapeutic agents targeting the PD-1/PD-L1 axis. Despite the exciting results obtained with the new drugs, immunotherapy is helpful only in a sizable minority of patients, and there is a pressing need to identify reliable predictive biomarkers for patient selection. The immunohistochemical assessment of PD-L1 expression was initially identified as a powerful and easily accessible predictive tool, and gained its place as the current standard for patient selection, but it has clear limitations. The imperfect predictive power of PD-L1 has resulted in a strong effort to discover additional clinical, pathological and molecular biomarkers such as tumor HPV status, mutational burden, microsatellite instability, and much more. In addition, the tumor microenvironment has been extensively studied searching for promising new biomarkers as potential avenues for refining patient selection and improvement of treatment outcomes. As we gain deeper understanding of the complex interplay between tumor biology, immune system, and tumor microenvironment, we are rapidly realizing that the perfect biomarker, the magic bullet, probably doesn't exist. On the other hand, with the introduction of new drugs on the horizon, integration of multiple variables in the context of combined predictive scores is shaping up to be our best weapon in this strife to treat each patient with the best possible drug.

BACKGROUND:Endometrial cancer (EC) is one of the most common gynecological malignancies and the second most common gynecological malignancy cause of death in women. Heterogeneous tissues with different grades of complexity and different diffusion properties characterize the EC. Several diffusion magnetic resonance imaging (DMRI) protocols have been used to perform a non-invasive and global evaluation of EC for diagnostic and prognostic purposes. However, the association of a single value for the diffusion coefficient to an EC tissue could be a severe limit for developing a DMRI virtual histology protocol. PURPOSE:This study evaluates the potential of diffusion kurtosis imaging (DKI) and tissue multiple diffusion clusterization in detecting the specific features of healthy/cancer tissue that can be useful in EC diagnosis and prognosis. METHODS:Thirty-eight subjects were analyzed: 18 with a final diagnosis of EC and 20 healthy, asymptomatic, with no history of endometrial pathology and uterine tumor pathology. Diffusion-weighted Spin-Echo Echo-Planar Imaging (DW-EPI) with TR/TE = 2000 ms/77 ms was used at 3T using six different b-values: (500, 800, 1000, 1500, 2000, and 2500)s/mm2 along three gradient directions (x, y, z). The decay of the signal in each voxel was used to obtain clusters of different diffusion compartments reflecting tissue heterogeneity. Moreover, using the Kurtosis representation, the parametric maps of the apparent kurtosis (K) and diffusivity (D) coefficients were obtained. The statistical analysis of the differences in the mean value of the parameters obtained in the selected regions of interest (ROIs) in tumor area (T) peritumor area (PT) and healthy tissue was carried out using a Kruskal-Wallis Test. A p-value < 0.05 indicated a statistically significant difference. To validate DKI and multiple diffusion clusterization in the detection of EC and healthy tissue, DMRI results were compared with EC histology. A ROC curve analysis was performed to evaluate the performance of the clustering feature in differentiating healthy and tumoral tissues. RESULTS:K discriminates the peritumor area (PT) of the tumor from the healthy tissues (p < 0.05) and the area inside the EC (cancerous tissue, p < 0.05). This result is validated and explained by the diffusion clustering, which shows a great variability in K for pathological compared to healthy subjects. Moreover, the standard deviation of K in the cluster defined by the highest K/D ratio differentiates T and H ROIs. CONCLUSIONS:K as well as diffusion clusterization are sensitive to the different microstructural organizations in EC and healthy tissue, promoting themself as a potential tool for the diagnosis and prognosis of EC.

Immune checkpoint inhibitors are increasingly used in neoadjuvant non-small cell lung carcinoma (NSCLC). Data regarding histologic features of the tumor microenvironment in this group of patients are limited. This study aimed to analyze the histologic features and the immune cell infiltrates within the tumor microenvironment of NSCLC after neoadjuvant immunotherapy. We evaluated surgical specimens of four NSCLC patients with different pathologic responses, who underwent chemo-immune neoadjuvant therapy. The following markers were analyzed by IHC: CD3, CD4, CD8 (T cells), CD21, CD20, CD138 (DCs, B and plasma cells), CD56 (NK), CD68 (Macrophages), Perforin, Granzyme, CD137 (active-T-cells), FOXP3 (regulatory-T-cells), IgM, and PD-L1. Multiple areas of equal size were counted. These counts were correlated with tumor regression grade (TRG) and response to therapy. The prevalence of tertiary lymphoid structures, low cytotoxic (CD8+) T lymphocyte count, high expression of CD137 and CD137/FOXP3 ratio, and low expression of PD-L1 from immune cells within the tumor microenvironment were associated with high TRG and therapy responsiveness. The expression of other markers was not significantly different according to outcome. Our results highlight the importance of CD137 expression and CD137/FOXP3 ratio on immune cells as activation markers. Further studies will be needed to investigate its value as a possible predictive marker of responsiveness to immunotherapy. The unexpected findings of this study (namely, low count of cytotoxic cells in cases with high TRG) may be explained by considering the period between immunotherapy and histological evaluation.

ADAR1 edits double-stranded RNAs (dsRNAs) by deaminating adenosines into inosines, preventing aberrant activation of innate immunity by endogenous dsRNAs, which may resemble viral structures. Several tumors exploit ADAR1 to evade immune surveillance; indeed, its deletion reduces tumor viability and reshapes infiltrating leukocytes. Here we investigated the role of ADAR1 in immune evasion mechanisms during cervical cancer (CC) progression. Patients’ biopsy samples showed higher ADAR1 expression already in premalignant lesions (squamous intraepithelial lesions [SIL]) and a substantially reduced percentage of infiltrating CD7+ innate cells in in situ and invasive carcinomas compared with normal mucosa, with CD56+ NK cells showing phenotypic alterations that may have affected their functional responses. In CC-derived cell lines (SiHa, CaSki), ADAR1 silencing reduced cell proliferation, an effect further enhanced by exogenous IFN-β administration. It also induced proinflammatory gene expression, as demonstrated by RNA-Seq analysis, and conditioned supernatants collected from these cells activated several NK cell effector functions. NK cell infiltration and activation were also confirmed in organotypic 3D tissue models of SiHa cells knocked out for ADAR1. In conclusion, ADAR1 expression increased with CC progression and was accompanied by alterations in tumor-infiltrating NK cells, but its silencing in CC-derived cell lines potentiated antitumor NK cell activities. Thus, ADAR1 inhibition may represent a therapeutic perspective for CC and possibly other malignancies.

A rare case of a 47-year-old woman with recurrent thromboembolic events, including STEMI and lower limb ischemia, diagnosed with aortic valve thrombosis caused by isolated aortic valve amyloidosis in the setting of overlapping antiphospholipid syndrome.

Immunotherapy has a crucial role in the treatment of recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). However, only a small percentage of patients achieve long-term benefit in terms of overall response and survival. It was shown that HNSCC has an immunosuppressive microenvironment due to high levels of regulatory T cells and immunosuppressive molecules, such as LAG3 and CD73. The aim of our study was to investigate if the expression of CD73 by neoplastic and immune cells could affect the efficacy of anti-PD-1 immunotherapy. We reviewed data from 50 patients with R/M HNSCC receiving first line immunotherapy with or without chemotherapy based on a combined positive score (CPS). CD73 expression by cancer and immune cells was evaluated on pre-treatment and the percentage of stained cells was recorded. We analysed the association between CD73 expression on neoplastic and immune cells and early progression (EP), defined as progression occurring within 3 months. In 88 % of patients the primary tumour site was in the oral cavity or larynx. All patients received pembrolizumab associated in 40 % of cases to chemotherapy. CD73 was positive in 82 % and 96 % of cases on neoplastic and immune cells, respectively. The median value of CD73 was 32 % for neoplastic cells and 10 % for the immune ones. We observed a significant association between the CD73 expression on neoplastic cells over the median value and EP disease. We didn't record a correlation between the expression of CD73 on immune cells and early progression. Our findings suggest that higher expression of CD73 on neoplastic cells could predict resistance to immunotherapy in patients with CPS positive R/M HNSCC. The addition of this biomarker to routine evaluation of CPS could help to select the patients primary resistant to antiPD-1 immunotherapy.

ABSTRACT:Hematological malignancies such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B-cell lymphoma (DLBCL) cause significant morbidity in humans. A substantial number of these lymphomas, particularly HL and DLBCLs have poorer prognosis because of their association with Epstein-Barr virus (EBV). Our earlier studies have shown that EBV-encoded nuclear antigen (EBNA2) upregulates programmed cell death ligand 1 in DLBCL and BLs by downregulating microRNA-34a. Here, we investigated whether EBNA2 affects the inducible costimulator (ICOS) ligand (ICOSL), a molecule required for efficient recognition of tumor cells by T cells through the engagement of ICOS on the latter. In virus-infected and EBNA2-transfected B-lymphoma cells, ICOSL expression was reduced. Our investigation of the molecular mechanisms revealed that this was due to an increase in microRNA-24 (miR-24) by EBNA2. By using ICOSL 3' untranslated region-luciferase reporter system, we validated that ICOSL is an authentic miR-24 target. Transfection of anti-miR-24 molecules in EBNA2-expressing lymphoma cells reconstituted ICOSL expression and increased tumor immunogenicity in mixed lymphocyte reactions. Because miR-24 is known to target c-MYC, an oncoprotein positively regulated by EBNA2, we analyzed its expression in anti-miR-24 transfected lymphoma cells. Indeed, the reduction of miR-24 in EBNA2-expressing DLBCL further elevated c-MYC and increased apoptosis. Consistent with the in vitro data, EBNA2-positive DLBCL biopsies expressed low ICOSL and high miR-24. We suggest that EBV evades host immune responses through EBNA2 by inducing miR-24 to reduce ICOSL expression, and for simultaneous rheostatic maintenance of proproliferative c-MYC levels. Overall, these data identify miR-24 as a potential therapeutically relevant target in EBV-associated lymphomas.

Background The network of intermediate filament proteins underlying the inner nuclear membrane forms the nuclear lamina. Lamins have been associated with important cellular functions: DNA replication, chromatin organization, differentiation of the cell, apoptosis and in maintenance of nuclear structure. Little is known regarding the etiopathogenesis of adhesive capsulitis (AC); recently, a dysregulating fibrotic response starting from a subpopulation has been described within the fibroblast compartment, which suddenly turns on an activated phenotype. Considering the key role of A-type lamins in the regulation of cellular stability and function, our aim was to compare the lamin A/C expression between patients with AC and healthy controls. Materials and methods A case–control study was performed between January 2020 and December 2021. Tissue samples excised from the rotator interval were analysed for lamin A/C expression by immunohistochemistry. Patients with AC were arbitrarily distinguished according to the severity of shoulder flexion limitation: ≥ 90° and < 90°. Controls were represented by samples obtained by normal rotator interval excised from patients submitted to shoulder surgery. The intensity of staining was graded, and an H-score was assigned. Statistical analysis was performed (Chi-square analysis; significance was set at alpha = 0.05). Results We enrolled 26 patients [12 male and 14 female, mean age (SD): 52.3 (6.08)] and 15 controls [6 male and 9 female, mean age (SD): 57.1 (5.3)]. The expression of lamin A/C was found to be significantly lower in the fibroblasts of patients with adhesive capsulitis when compared with controls (intensity of staining: p : 0.005; H-score: 0.034); no differences were found regarding the synoviocytes ( p : > 0.05). Considering only patients with AC, lamin A/C intensity staining was found to be significantly higher in samples where acute inflammatory infiltrate was detected ( p : 0.004). No significant changes in levels of lamin A/C expression were documented between the mild and severe adhesive capsulitis severity groups. Conclusions Our study demonstrated that the activity of lamin A/C in maintaining nuclear structural integrity and cell viability is decreased in patients with adhesive capsulitis. The phase of the pathogenetic process (freezing and early frozen) is the key factor for cell functionality. On the contrary, the clinical severity of adhesive capsulitis plays a marginal role in nuclear stability. Level of evidence III.

BACKGROUND:Immunotherapy has revolutionized the approach to metastatic triple-negative breast cancers. Atezolizumab was approved for patients with metastatic triple-negative breast cancers whose tumors express PD-L1, determined by SP 142 assay. To assess the availability and practice of SP142 test we administered a survey to all the 15 pathology departments of the Lazio Region during a six-month period.METHODS:The survey comprised 12 questions regarding the availability of SP142 in the pathology departments, the percentage of positive tests, the difficulties of pathologists in cases close to cut-off value and the tested samples.RESULTS:The SP142 assay was available in only eight centers. In case of positive result, most centers (5/8, 62.5%) reported values of PD-L1 expression ranging from > 1 to ⩽ 5%, with values close to the cut-off point (⩾ 1% or < 1%) being the greatest challenge.Most of the centers (6/8, 75%) tested material from both their own and other hospitals. In most centers, the evaluations were performed either on primary tumors or metastasis, in particular lymph nodes (5/8, 62.5%), followed by lung (3/8, 37.5%) and liver (1/8, 12.5%) metastasis.CONCLUSION:Our results raise some important issues concerning the evaluation of PD-L1 in the "real-life" setting, providing strategies for its implementation.

The intron retention (IR) is a phenomenon utilized by cells to allow diverse fates at the same mRNA, leading to a different pattern of synthesis of the same protein. In this study, we analyzed the modulation of phosphoinositide-specific phospholipase C (PI-PLC) enzymes by Harpagophytum procumbens extract (HPE) in synoviocytes from joins of osteoarthritis (OA) patients. In some samples, the PI-PLC γ1 isoform mature mRNA showed the IR and, in these synoviocytes, the HPE treatment increased the phenomenon. Moreover, we highlighted that as a consequence of IR, a lower amount of PI-PLC γ1 was produced. The decrease of PI-PLC γ1 was associated with the decrease of metalloprotease-3 (MMP-3), and MMP-13, and ADAMTS-5 after HPE treatment. The altered expression of MMPs is a hallmark of the onset and progression of OA, thus substances able to decrease their expression are very desirable. The interesting outcomes of this study are that 35% of analyzed synovial tissues showed the IR phenomenon in the PI-PLC γ1 mRNA and that the HPE treatment increased this phenomenon. For the first time, we found that the decrease of PI-PLC γ1 protein in synoviocytes interferes with MMP production, thus affecting the pathways involved in the MMP expression. This finding was validated by the silencing of PI-PLC γ1 in synoviocytes where the IR phenomenon was not present. Our results shed new light on the biochemical mechanisms involved in the degrading enzyme production in the joint of OA patients, suggesting a new therapeutic target and highlighting the importance of personalized medicine.