Lectin-like Transcript-1 (LLT1) Expression in Oral Squamous Cell Carcinomas: Prognostic Significance and Relationship with the Tumor Immune Microenvironment.

Lectin-like transcript-1 (LLT1) expression is detected in different cancer types and is involved in immune evasion. The present study investigates the clinical relevance of tumoral and stromal LLT1 expression in oral squamous cell carcinoma (OSCC), and relationships with the immune infiltrate into the tumor immune microenvironment (TIME). Immunohistochemical analysis of LLT1 expression was performed in 124 OSCC specimens, together with PD-L1 expression and the infiltration of CD20+, CD4+, and CD8+ lymphocytes and CD68+ and CD163+-macrophages. Associations with clinicopathological variables, prognosis, and immune cell densities were further assessed. A total of 41 (33%) OSCC samples showed positive LLT1 staining in tumor cells and 55 (44%) positive LLT1 in tumor-infiltrating lymphocytes (TILs). Patients harboring tumor-intrinsic LLT1 expression exhibited poorer survival, suggesting an immunosuppressive role. Conversely, positive LLT1 expression in TILs was significantly associated with better disease-specific survival, and also an immune-active tumor microenvironment highly infiltrated by CD8+ T cells and M1/M2 macrophages. Furthermore, the combination of tumoral and stromal LLT1 was found to distinguish three prognostic categories (favorable, intermediate, and adverse; p = 0.029, Log-rank test). Together, these data demonstrate the prognostic relevance of tumoral and stromal LLT1 expression in OSCC, and its potential application to improve prognosis prediction and patient stratification.

Sox2 and ßIII-Tubulin as Biomarkers of Drug Resistance in Poorly Differentiated Sinonasal Carcinomas.

Poorly differentiated sinonasal carcinomas (PDCs) are tumors that have a poor prognosis despite advances in classical treatment. Predictive and prognostic markers and new personalized treatments could improve the oncological outcomes of patients. In this study, we analyzed SOX2 and ßIII-tubulin as biomarkers that could have prognostic and therapeutic impacts on these tumors. The cohort included 57 cases of PDCs: 36 sinonasal undifferentiated carcinoma (SNUC) cases, 13 olfactory neuroblastoma (ONB) cases, and 8 sinonasal neuroendocrine carcinoma (SNEC) cases. Clinical follow-up data were available for 26 of these cases. Sox2 expression was detected using immunohistochemistry in 6 (75%) SNEC cases, 19 (53%) SNUC cases, and 6 (46%) ONB cases. The absence of Sox2 staining correlated with a higher rate of recurrence (p = 0.015), especially distant recurrence. The majority of cases showed ßIII-tubulin expression, with strong positivity in 85%, 75%, and 64% of SNEC, ONB, and SNUC cases, respectively. Tumors with stronger ßIII-tubulin expression demonstrated longer disease-free survival than those with no expression or low expression (p = 0.049). Sox2 and ßIII-tubulin expression is common in poorly differentiated sinonasal tumors and has prognostic and therapeutic utility.

Prognostic Significance of ß-Catenin in Relation to the Tumor Immune Microenvironment in Oral Cancer.

The aim of this study was to investigate the prognostic relevance of ß-catenin expression in oral squamous cell carcinoma (OSCC) and to explore relationships with the tumor immune microenvironment. Expression of ß-catenin and PD-L1, as well as lymphocyte and macrophage densities, were evaluated by immunohistochemistry in 125 OSCC patient specimens. Membranous ß-catenin expression was detected in 102 (81.6%) and nuclear ß-catenin in 2 (1.6%) tumors. There was an association between ß-catenin expression, tumoral, and stromal CD8+ T-cell infiltration (TIL) and also the type of tumor immune microenvironment (TIME). Tumors harboring nuclear ß-catenin were associated with a type II TIME (i.e., immune ignorance defined by a negative PD-L1 expression and low CD8+ TIL density), whereas tumors with membranous ß-catenin expression were predominantly type IV (i.e., immune tolerance defined by negative PD-L1 and high CD8+ TIL density). Combined, but not individual, high stromal CD8+ TILs and membranous ß-catenin expression was independently associated with better disease-specific survival (HR = 0.48, p = 0.019). Taken together, a combination of high stromal CD8+ T-cell infiltration and membranous ß-catenin in the tumor emerges as an independent predictor of better survival in OSCC patients.

Abrogation of stemness in osteosarcoma by the mithramycin analog EC-8042 is mediated by its ability to inhibit NOTCH-1 signaling.

Osteosarcomas are frequently associated to a poor prognosis and a modest response to current treatments. EC-8042 is a well-tolerated mithramycin analog that has demonstrated an efficient ability to eliminate tumor cells, including cancer stem cell subpopulations (CSC), in sarcomas. In transcriptomic and protein expression analyses, we identified NOTCH1 signaling as one of the main pro-stemness pathways repressed by EC-8042 in osteosarcomas. Overexpression of NOTCH-1 resulted in a reduced anti-tumor effect of EC-8042 in CSC-enriched 3D tumorspheres cultures. On the other hand, the depletion of the NOTCH-1 downstream target HES-1 was able to enhance the action of EC-8042 on CSCs. Moreover, HES1 depleted cells failed to recover after treatment withdrawal and showed reduced tumor growth potential in vivo. In contrast, mice xenografted with NOTCH1-overexpressing cells responded worse than parental cells to EC-8042. Finally, we found that active NOTCH1 levels in sarcoma patients was associated to advanced disease and lower survival. Overall, these data highlight the relevant role that NOTCH1 signaling plays in mediating stemness in osteosarcoma. Moreover, we demonstrate that EC-8042 is powerful inhibitor of NOTCH signaling and that the anti-CSC activity of this mithramycin analog highly rely on its ability to repress this pathway.

Fatal sclerosing mesenteritis: a 7-year-old male autopsy case report

ABSTRACT Sclerosing Mesenteritis (SM) is a rare diagnosis, particularly in pediatric patients, and is typically non-fatal when appropriately treated. Although molecular and immunohistochemical alterations have been described, no pathognomonic signature has been identified for this entity. This report presents a case of a seven-year-old boy who suffered sudden cardiorespiratory arrest. Upon autopsy, he was found to have multicentric SM on the upper mesentery, which led to bowel wall thinning and abdominal bleeding with bacterial translocation. We performed comprehensive morphological, immunohistochemical, and molecular analyses. SM is an atypical disorder with diverse clinical manifestations, including a rare but potentially fatal course. Early diagnosis is critical, given its potential severity. To our knowledge, this is the first case report of pediatric mortality linked to SM. Our findings emphasize the importance of increased awareness and early detection of SM in pediatric patients.

Characterization of a Preclinical In Vitro Model Derived from a SMARCA4-Mutated Sinonasal Teratocarcinosarcoma.

Sinonasal teratocarcinosarcoma (TCS) is a rare tumor that displays a variable histology with admixtures of epithelial, mesenchymal, neuroendocrine and germ cell elements. Facing a very poor prognosis, patients with TCS are in need of new options for treatment. Recently identified recurrent mutations in SMARCA4 may serve as target for modern therapies with EZH1/2 and CDK4/6 inhibitors. Here, we present the first in vitro cell line TCS627, established from a previously untreated primary TCS originating in the ethmoid sinus with invasion into the brain. The cultured cells expressed immunohistochemical markers, indicating differentiation of epithelial, neuroepithelial, sarcomatous and teratomatous components. Whole-exome sequencing revealed 99 somatic mutations including SMARCA4, ARID2, TET2, CDKN2A, WNT7A, NOTCH3 and STAG2, all present both in the primary tumor and in the cell line. Focusing on mutated SMARCA4 as the therapeutic target, growth inhibition assays showed a strong response to the CDK4/6 inhibitor palbociclib, but much less to the EZH1/2 inhibitor valemetostat. In conclusion, cell line TCS627 carries both histologic and genetic features characteristic of TCS and is a valuable model for both basic research and preclinical testing of new therapeutic options for treatment of TCS patients.

Intragenic NF1 deletions in sinonasal mucosal malignant melanoma.

Mucosal malignant melanoma (MMM) is a rare and aggressive tumor. Despite effective local therapies, tumor recurrence and metastasis remain frequent. The genetics of MMM remain incompletely understood. This study is aimed to identify actionable genetic alterations by next-generation sequencing. Fifteen MMM samples were analyzed by next-generation and Sanger sequencing. Gene copy number alterations were analyzed by MLPA. Mutation status was correlated with pERK, pAKT, and Ki-67 expression and follow-up data. Inactivating mutations and intragenic deletions in neurofibromatosis type-1 (NF1) were identified in 3 and 2 cases, respectively, (in total 5/15, 33%) and activating mutations in NRAS and KRAS (3/15, 20%) cases. Other mutated genes included CDKN2A, APC, ATM, MITF, FGFR1, and FGFR2. BRAF and KIT mutations were not observed. Cases with NF1 alterations tended to have worse overall survival. The mutational status was not associated with pERK, pAKT, or Ki-67 immunostaining. MMM carries frequent gene mutations activating the MAPK pathway, similar to cutaneous melanoma. In contrast, NF1 is the most frequently affected gene. Intragenic NF1 deletions have not been described before and may go undetected by sequencing studies. This finding is clinically relevant as NF1-mutated melanomas have worse survival and could benefit from therapy with immune checkpoint and MEK inhibitors.

Aberrant Signaling Pathways in Sinonasal Intestinal-Type Adenocarcinoma.

Sinonasal intestinal-type adenocarcinoma (ITAC) is strongly related to occupational exposure to wood and leather dust, however, little is known on the genetic alterations involved in tumor development and progression. The aim of this study was to identify tumorigenic signaling pathways affected by gene mutations and their relation to clinical features. We applied whole exome sequencing of 120 cancer-related genes in 50 ITACs and analyzed the signaling activity of four specific pathways frequently affected by mutations. Genes involved in DNA damage response showed somatic mutations in 30% of cases, including four tumors that also harbored germline mutations. Genes in Wnt, MAPK and PI3K pathways harbored mutations in 20%, 20% and 24% of cases, respectively. Mutations and copy number gains in receptor tyrosine kinases possibly affecting MAPK and PI3K pathways occurred in 44% of cases. Expression of key pathway proteins showed no correlation to mutations in these pathways, except for nuclear ß-catenin and APC/CTNNB1 mutation. No specific gene mutation, mutated pathway, nor pathway activity level showed correlation to clinical data or survival. In addition, a similar mutational profile was observed among histological subtypes. The wide spectrum of gene mutations suggests that ITAC is a genetically heterogeneous without specific characterizing gene mutations.

Mithramycin delivery systems to develop effective therapies in sarcomas.

BACKGROUND: Sarcomas comprise a group of aggressive malignancies with very little treatment options beyond standard chemotherapy. Reposition of approved drugs represents an attractive approach to identify effective therapeutic compounds. One example is mithramycin (MTM), a natural antibiotic which has demonstrated a strong antitumour activity in several tumour types, including sarcomas. However, its widespread use in the clinic was limited by its poor toxicity profile. RESULTS: In order to improve the therapeutic index of MTM, we have loaded MTM into newly developed nanocarrier formulations. First, polylactide (PLA) polymeric nanoparticles (NPs) were generated by nanoprecipitation. Also, liposomes (LIP) were prepared by ethanol injection and evaporation solvent method. Finally, MTM-loaded hydrogels (HG) were obtained by passive loading using a urea derivative non-peptidic hydrogelator. MTM-loaded NPs and LIP display optimal hydrodynamic radii between 80 and 105 nm with a very low polydispersity index (PdI) and encapsulation efficiencies (EE) of 92 and 30%, respectively. All formulations show a high stability and different release rates ranging from a fast release in HG (100% after 30 min) to more sustained release from NPs (100% after 24 h) and LIP (40% after 48 h). In vitro assays confirmed that all assayed MTM formulations retain the cytotoxic, anti-invasive and anti-stemness potential of free MTM in models of myxoid liposarcoma, undifferentiated pleomorphic sarcoma and chondrosarcoma. In addition, whole genome transcriptomic analysis evidenced the ability of MTM, both free and encapsulated, to act as a multi-repressor of several tumour-promoting pathways at once. Importantly, the treatment of mice bearing sarcoma xenografts showed that encapsulated MTM exhibited enhanced therapeutic effects and was better tolerated than free MTM. CONCLUSIONS: Overall, these novel formulations may represent an efficient and safer MTM-delivering alternative for sarcoma treatment.

Prognostic Relevance of CD4+, CD8+ and FOXP3+ TILs in Oral Squamous Cell Carcinoma and Correlations with PD-L1 and Cancer Stem Cell Markers.

This study investigates the relevance of tumor-infiltrating lymphocytes (TILs) in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis of stromal/tumoral CD4+, CD8+ and FOXP3+ TILs is performed in 125 OSCC patients. Potential relationships with the expression of tumoral PD-L1 and cancer stem cell (CSC) markers (NANOG, SOX2, OCT4, Nestin and Podoplanin (PDPN)) are assessed. CD4+ and CD8+ TILs are significantly associated with smoking and alcohol habits. CD4+ and CD8+ TILs show an inverse relationship with NANOG and SOX2 expression, and FOXP3+ TILs is significantly correlated with Nestin and PDPN expression. High infiltration of CD4+ and CD8+ TILs and a high tumoral CD8+/FOXP3+ ratio are significantly associated with tumors harboring positive PD-L1 expression. Infiltration of stromal/tumoral FOXP3+ TILs and a low stromal CD8+/FOXP3+ ratio are significantly associated with better disease-specific survival. Multivariate analysis reveals that the stromal CD8+/FOXP3+ TILs ratio is a significant independent prognostic factor. Regarding OSCC patient survival, the CD8+/FOXP3+ TILs ratio is an independent prognostic factor. TILs may act as biomarkers and potential therapeutic targets for OSCC.

GARP promotes the proliferation and therapeutic resistance of bone sarcoma cancer cells through the activation of TGF-ß.

Sarcomas are mesenchymal cancers with poor prognosis, representing about 20% of all solid malignancies in children, adolescents, and young adults. Radio- and chemoresistance are common features of sarcomas warranting the search for novel prognostic and predictive markers. GARP/LRRC32 is a TGF-ß-activating protein that promotes immune escape and dissemination in various cancers. However, if GARP affects the tumorigenicity and treatment resistance of sarcomas is not known. We show that GARP is expressed by human osteo-, chondro-, and undifferentiated pleomorphic sarcomas and is associated with a significantly worse clinical prognosis. Silencing of GARP in bone sarcoma cell lines blocked their proliferation and induced apoptosis. In contrast, overexpression of GARP promoted their growth in vitro and in vivo and increased their resistance to DNA damage and cell death induced by etoposide, doxorubicin, and irradiation. Our data suggest that GARP could serve as a marker with therapeutic, prognostic, and predictive value in sarcoma. We propose that targeting GARP in bone sarcomas could reduce tumour burden while simultaneously improving the efficacy of chemo- and radiotherapy.

PD-L1 Expression in Tumor Cells Is an Independent Unfavorable Prognostic Factor in Oral Squamous Cell Carcinoma.

BACKGROUND: The immune checkpoint PD-1 and its ligand PD-L1 are involved in the induction of immunological tolerance of solid tumors including oral squamous cell carcinoma (OSCC). The aim of the study was to establish the clinical and prognostic significance of PD-L1 in OSCC. METHODS: Tissue microarrays of 125 resected OSCC were stained with two different commercially available PD-L1 antibodies (clones E1L3N and 22C3), alongside PD-1 immunostaining. RESULTS: PD-L1 expression in more than 10% of tumor cells was associated with poorer survival, and established as a clinically relevant cut-off point. This relevant PD-L1 expression was detected in 10% to 15% OSCC specimens depending on the anti-PD-L1 antibody, and showed an inverse correlation with tobacco and alcohol consumption. We consistently found that PD-L1 expression was associated with tumor recurrence and lower disease-specific survival. Multivariate analysis further revealed that neck node metastasis (HR 2.304; P = 0.009) and tumor PD-L1 expression (HR 2.571; P = 0.01) were significant independent factors for poor prognosis. CONCLUSIONS: PD-L1 expression in more than 10% of tumor cells was a significant and independent factor of poor prognosis in OSCC. IMPACT: PD-L1 expression in more than 10% of tumor cells was consistently established as a clinically relevant cut-off point by using two different antibodies. Remarkably, PD-L1 expression emerges as an independent poor prognosis marker in patients with OSCC.

Expression of podoplanin in the invasion front of oral squamous cell carcinoma is not prognostic for survival.

Podoplanin is involved in actin remodeling of the cytoskeleton of tumor cells and may promote tumor cell invasion by increasing cell motility and formation of filopodia-like membrane protrusions. Podoplanin is expressed in a variety of tumors, but its role in head and neck cancer, particularly in oral squamous cell carcinoma, remains unclear. We studied podoplanin expression by immunohistochemistry in 92 oral squamous cell carcinomas (OSCC) using a monoclonal antibody against an epitope of podoplanin (D2-40). In terms of the number of stained cells, 34 OSCC (38 %) had low podoplanin expression (less than 33 % of cells), 33 (36 %) showed moderate expression (between 34 and 66 % of cells), and 21 (22 %) showed high expression. The intensity of immunostaining was strong in 26 (28 %) cases, moderate in 36 (40 %), and weak or negative in the remaining 30 tumors (32 %). Immunohistochemical expression of podoplanin was associated with a tumor histological grade. A diffuse pattern of podoplanin expression significantly decreased in moderately differentiated (37 %) and poorly differentiated (20 %) carcinomas compared to well-differentiated (43 %) carcinomas. In addition, the focal expression of podoplanin in the invasion front of the tumor, without expression in the tumor center, was observed in 72 % of well-differentiated tumors, 27 % of moderate tumors, and 0 % of poorly differentiated tumors. Moreover, a trend was found toward an association of diffuse podoplanin staining with the development of second primary carcinomas (13 %), in contrast to its expression in the invasion front (3 %). No association was observed between podoplanin expression and nodal metastasis.