Comprehensive microRNA expression analysis of pediatric gonadal germ cell tumors: unveiling novel biomarkers and signatures.

microRNAs (miRNAs) are small endogenous noncoding RNAs, and alterations in their expression may contribute to oncogenesis. Discovering a unique miRNA pattern holds the potential for early detection and novel treatment possibilities in cancer. This study aimed to evaluate miRNA expression in pediatric patients with gonadal germ cell tumors (GCTs), focusing on characterizing the miRNA profiles of each histological subtype and identifying a distinct histological miRNA signature for a total of 42 samples of pediatric gonadal GCTs. The analysis revealed distinct miRNA expression profiles for all histological types, regardless of the primary site. We identified specific miRNA expression signatures for each histological type, including 34 miRNAs for dysgerminomas, 13 for embryonal carcinomas, 25 for yolk sac tumors, and one for immature teratoma, compared to healthy controls. Furthermore, we identified 26 miRNAs that were commonly expressed in malignant tumors, with six miRNAs (miR-302a-3p, miR-302b-3p, miR-371a-5p, miR-372-3p, miR-373-3p, and miR-367-3p) showing significant overexpression. Notably, miR-302b-3p exhibited a significant association with all the evaluated clinical features. Our findings suggest that miRNAs have the potential to aid in the diagnosis, prognosis, and management of patients with malignant GCTs.

Establishment and molecular characterization of HCB-541, a novel and aggressive human cutaneous squamous cell carcinoma cell line.

Cutaneous squamous cell carcinoma (cSCC) is a common type of skin cancer that can result in significant morbidity, although it is usually well-managed and rarely metastasizes. However, the lack of commercially available cSCC cell lines hinders our understanding of this disease. This study aims to establish and characterize a new metastatic cSCC cell line derived from a Brazilian patient. A tumor biopsy was taken from a metastatic cSCC patient, immortalized, and named HCB-541 after several passages. The cytokeratin expression profile, karyotypic alterations, mutational analysis, mRNA and protein differential expression, tumorigenic capacity in xenograft models, and drug sensitivity were analyzed. The HCB-541 cell line showed a doubling time between 20 and 30 h and high tumorigenic capacity in the xenograft mouse model. The HCB-541 cell line showed hypodiploid and hypotetraploidy populations. We found pathogenic mutations in TP53 p.(Arg248Leu), HRAS (Gln61His) and TERT promoter (C228T) and high-level microsatellite instability (MSI-H) in both tumor and cell line. We observed 37 cancer-related genes differentially expressed when compared with HACAT control cells. The HCB-541 cells exhibited high phosphorylated levels of EGFR, AXL, Tie, FGFR, and ROR2, and high sensitivity to cisplatin, carboplatin, and EGFR inhibitors. Our study successfully established HCB-541, a new cSCC cell line that could be useful as a valuable biological model for understanding the biology and therapy of metastatic skin cancer.

miR-99a-5p as a biomarker for lymph node metastasis prediction in oral squamous cell carcinoma patients.

BACKGROUND: Metastatic lymph node involvement influences therapy decisions and serves as a prognostic indicator in oral squamous cell carcinoma (OSCC). However, many early-stage patients with clinically negative lymph nodes exhibit no metastasis upon surgical staging. This study aimed to identify differentially expressed miRNAs capable of distinguishing pathologically positive (pN+) from negative (pN0) nodes in OSCC patients without clinical evidence of lymph node metastases (cN0). METHODS: Expression levels of 798 miRNAs were assessed in tumor samples from 10 pN+ and 10 pN0 patients using the Nanostring nCounter platform. Validation was performed in an independent cohort of 15 pN+ and 24 pN0 patients through RT-qPCR. RESULTS: Eight miRNAs exhibited differential expression between pN0 and pN+ patients. Notably, hsa-miR-99a-5p demonstrated high sensitivity and specificity in predicting patients at higher risk of positive lymph nodes. CONCLUSIONS: These findings highlight hsa-miR-99a-5p as a potential biomarker for detecting lymph node metastasis in primary OSCC tumors.

Cancer immune profiling unveils biomarkers, immunological pathways, and cell type score associated with glioblastoma patients' survival.

Introduction: Glioblastoma (GBM), isocitrate dehydrogenase (IDH) wild-type (IDH wt), and grade 4 astrocytomas, IDH mutant (IDH mut), are the most common and aggressive primary malignant brain tumors in adults. A better understanding of the tumor immune microenvironment may provide new biomarkers and therapeutic opportunities. Objectives: We aimed to evaluate the expression profile of 730 immuno-oncology-related genes in patients with IDH wt GBM and IDH mut tumors and identify prognostic biomarkers and a gene signature associated with patient survival. Methods: RNA was isolated from formalin-fixed, paraffin-embedded sections of 99 tumor specimens from patients treated with standard therapy. Gene expression profile was assessed using the Pan-Cancer Immune Profiling Panel (Nanostring Technologies, Inc., Seattle, WA, USA). Data analysis was performed using nSolverSoftware and validated in The Cancer Genome Atlas. In addition, we developed a prognostic signature using the cox regression algorithm (Least Absolute Shrinkage and Selection Operator). Results: We found 88 upregulated genes, high immunological functions, and a high macrophage score in IDH wt GBM compared to IDH mut tumors. Regarding IDH wt GBM, we found 24 upregulated genes in short-term survivors (STS) and overexpression of CD274 (programmed death-ligand 1, PD-L1). Immune pathways, CD45, cytotoxic, and macrophage scores were upregulated in STS. Two different prognostic groups were found based on the 12-gene signature (CXCL14, PSEN2, TNFRSF13C, IL13RA1, MAP2K1, TNFSF14, THY1, CTSL, ITGAE, CHUK, CD207, and IFITM1). Conclusion: The elevated expression of immune-oncology-related genes was associated with worse outcome in IDH wt GBM patients. Increased immune functions, CD45, cytotoxic cells, and macrophage scores were associated with a more aggressive phenotype and may provide promising possibilities for therapy. Moreover, a 12 gene-based signature could predict patients' prognosis.

Early Emergence and Dispersal of Delta SARS-CoV-2 Lineage AY.99.2 in Brazil.

The year of 2021 was marked by the emergence and dispersal of a number of SARS-CoV-2 lineages, resulting in the "third wave" of COVID-19 in several countries despite the level of vaccine coverage. Soon after the first confirmed cases of COVID-19 by the Delta variant in Brazil, at least seven Delta sub-lineages emerged, including the globally spread AY.101 and AY.99.2. In this study we performed a detailed analysis of the COVID-19 scenario in Brazil from April to December 2021 by using data collected by the largest private medical diagnostic company in Latin America (Dasa), and SARS-CoV-2 genomic sequences generated by its SARS-CoV-2 genomic surveillance project (GENOV). For phylogenetic and Bayesian analysis, SARS-CoV-2 genomes available at GISAID public database were also retrieved. We confirmed that the Brazilian AY.99.2 and AY.101 were the most prevalent lineages during this period, overpassing the Gamma variant in July/August. We also estimated that AY.99.2 likely emerged a few weeks after the entry of the B.1.617.2 in the country, at some point between late April and May and rapidly spread to other countries. Despite no increased fitness described for the AY.99.2 lineage, a rapid shift in the composition of Delta SARS-CoV-2 lineages prevalence in Brazil took place. Understanding the reasons leading the AY.99.2 to become the dominant lineage in the country is important to understand the process of lineage competitions that may inform future control measures.

Efficacy of Combined Use of Everolimus and Second-Generation Pan-EGRF Inhibitors in KRAS Mutant Non-Small Cell Lung Cancer Cell Lines.

Background: EGFR mutations are present in approximately 15−50% of non-small cell lung cancer (NSCLC), which are predictive of anti-EGFR therapies. At variance, NSCLC patients harboring KRAS mutations are resistant to those anti-EGFR approaches. Afatinib and allitinib are second-generation pan-EGFR drugs, yet no predictive biomarkers are known in the NSCLC context. In the present study, we evaluated the efficacy of pan-EGFR inhibitors in a panel of 15 lung cancer cell lines associated with the KRAS mutations phenotype. Methods: KRAS wild-type sensitive NCI-H292 cell line was further transfected with KRAS mutations (p.G12D and p.G12S). The pan-EGFR inhibitors' activity and biologic effect of KRAS mutations were evaluated by cytotoxicity, MAPK phospho-protein array, colony formation, migration, invasion, and adhesion. In addition, in vivo chicken chorioallantoic membrane assay was performed in KRAS mutant cell lines. The gene expression profile was evaluated by NanoString. Lastly, everolimus and pan-EGFR combinations were performed to determine the combination index. Results: The GI50 score classified two cell lines treated with afatinib and seven treated with allitinib as high-sensitive phenotypes. All KRAS mutant cell lines demonstrated a resistant profile for both therapies (GI50 < 30%). The protein array of KRAS edited cells indicated a significant increase in AKT, CREB, HSP27, JNK, and, importantly, mTOR protein levels compared with KRAS wild-type cells. The colony formation, migration, invasion, adhesion, tumor perimeter, and mesenchymal phenotype were increased in the H292 KRAS mutated cells. Gene expression analysis showed 18 dysregulated genes associated with the focal adhesion-PI3K-Akt-mTOR-signaling correlated in KRAS mutant cell lines. Moreover, mTOR overexpression in KRAS mutant H292 cells was inhibited after everolimus exposure, and sensitivity to afatinib and allitinib was restored. Conclusions: Our results indicate that allitinib was more effective than afatinib in NSCLC cell lines. KRAS mutations increased aggressive behavior through upregulation of the focal adhesion-PI3K-Akt-mTOR-signaling in NSCLC cells. Significantly, everolimus restored sensibility and improved cytotoxicity of EGFR inhibitors in the KRAS mutant NSCLC cell lines.

Single nCounter assay for prediction of MYCN amplification and molecular classification of medulloblastomas: a multicentric study.

PURPOSE: Medulloblastoma is the most frequent pediatric malignant brain tumor, and is divided into four main subgroups: WNT, SHH, group 3, and group 4. MYCN amplification is an important medulloblastoma prognostic biomarker. We aimed to molecular classify and predict MYCN amplification in a single assay. METHODS: It was included 209 medulloblastomas from 205 patients (Brazil, Argentina, and Portugal), divided into training (n = 50) and validation (n = 159) sets. A nCounter assay was carried out using a custom panel for molecular classification, with additional genes, including MYCN. nSolver 4.0 software and the R environment were used for profiling and MYCN mRNA analysis. MYCN amplification by FISH was performed in 64 cases. RESULTS: The 205 medulloblastomas were classified in SHH (44.9%), WNT (15.6%), group 3 (18.1%) and group 4 (21.4%). In the training set, MYCN amplification was detected in three SHH medulloblastomas by FISH, which showed significantly higher MYCN mRNA counts than non-FISH amplified cases, and a cutoff for MYCN amplification was established ([Formula: see text] + 4σ = 11,124.3). Applying this threshold value in the validation set, we identified MYCN mRNA counts above the cutoff in three cases, which were FISH validated. CONCLUSION: We successfully stratified medulloblastoma molecular subgroups and predicted MYCN amplification using a single nCounter assay without the requirement of additional biological tissue, costs, or bench time.

Comprehensive Molecular Landscape of Cetuximab Resistance in Head and Neck Cancer Cell Lines.

Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed by Western blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC50 compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC.

A 4-Gene Signature Associated With Recurrence in Low- and Intermediate-Risk Endometrial Cancer.

BACKGROUND: The molecular profile of endometrial cancer has become an important tool in determining patient prognosis and their optimal adjuvant treatment. In addition to The Cancer Genome Atlas (TCGA), simpler tools have been developed, such as the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE). We attempted to determine a genetic signature to build a recurrence risk score in patients diagnosed with low- and intermediate-risk endometrial cancer. METHODS: A case-control study was conducted. The eligible patients were women diagnosed with recurrence low- and intermediate-risk endometrial cancer between January 2009 and December 2014 at a single institution; the recurrence patients were matched to two nonrecurrence patients with the same diagnosis by age and surgical staging. Following RNA isolation of 51 cases, 17 recurrence and 34 nonrecurrence patients, the expression profile was determined using the nCounter® PanCancer Pathways Panel, which contains 770 genes. RESULTS: The expression profile was successfully characterized in 49/51 (96.1%) cases. We identified 12 genes differentially expressed between the recurrence and nonrecurrence groups. The ROC curve for each gene was generated, and all had AUCs higher than 0.7. After backward stepwise logistic regression, four genes were highlighted: FN1, DUSP4, LEF1, and SMAD9. The recurrence risk score was calculated, leading to a ROC curve of the 4-gene model with an AUC of 0.93, sensitivity of 100%, and specificity of 72.7%. CONCLUSION: We identified a four-gene signature that may be associated with recurrence in patients with low- and intermediate-risk endometrial cancer. This finding suggests a new prognostic factor in this poorly explored group of patients with endometrial cancer.

Low MGMT digital expression is associated with a better outcome of IDH1 wildtype glioblastomas treated with temozolomide.

INTRODUCTION: Glioblastoma (GBM) is the deadliest primary brain tumor. The standard treatment consists of surgery, radiotherapy, and temozolomide (TMZ). TMZ response is heterogeneous, and MGMT promoter (MGMTp) methylation has been the major predictive biomarker. We aimed to describe the clinical and molecular data of GBMs treated with TMZ, compare MGMT methylation with MGMT expression, and further associate with patient's outcome. METHODS: We evaluate 112 FFPE adult GBM cases. IDH1 and ATRX expression was analyzed by immunohistochemistry, hotspot TERT promoter (TERTp) mutations were evaluated by Sanger or pyrosequencing, and MGMTp methylation was assessed by pyrosequencing and MGMT mRNA expression using the nCounter® Vantage 3D™ DNA damage and repair panel. RESULTS: Of the 112 GBMs, 96 were IDH1WT, and 16 were IDH1MUT. Positive ATRX expression was found in 91.6% (88/96) of IDHWT and 43.7% (7/16) of IDHMUT. TERTp mutations were detected in 70.4% (50/71) of IDHWT. MGMTp methylation was found in 55.5% (35/63) of IDHWT and 84.6% (11/13) of IDHMUT, and as expected, MGMTp methylation was significantly associated with a better response to TMZ. MGMT expression was inversely correlated with MGMTp methylation levels (- 0.506, p < 0.0001), and MGMT low expression were significantly associated with better patient survival. It was also observed that integrating MGMTp methylation and expression, significantly improved the prognostication value. CONCLUSIONS: MGMT mRNA levels evaluated by digital expression were associated with the outcome of TMZ-treated GBM patients. The combination of MGMT methylation and mRNA expression may provide a more accurate prediction of TMZ response in GBM patients.

Whole blood mRNA expression-based targets to discriminate active tuberculosis from latent infection and other pulmonary diseases.

Current diagnostic tests for tuberculosis (TB) are not able to predict reactivation disease progression from latent TB infection (LTBI). The main barrier to predicting reactivation disease is the lack of our understanding of host biomarkers associated with progression from latent infection to active disease. Here, we applied an immune-based gene expression profile by NanoString platform to identify whole blood markers that can distinguish active TB from other lung diseases (OPD), and that could be further evaluated as a reactivation TB predictor. Among 23 candidate genes that differentiated patients with active TB from those with OPD, nine genes (CD274, CEACAM1, CR1, FCGR1A/B, IFITM1, IRAK3, LILRA6, MAPK14, PDCD1LG2) demonstrated sensitivity and specificity of 100%. Seven genes (C1QB, C2, CCR2, CCRL2, LILRB4, MAPK14, MSR1) distinguished TB from LTBI with sensitivity and specificity between 82 and 100%. This study identified single gene candidates that distinguished TB from OPD and LTBI with high sensitivity and specificity (both > 82%), which may be further evaluated as diagnostic for disease and as predictive markers for reactivation TB.

Cell-free DNA promotes malignant transformation in non-tumor cells.

Cell-free DNA is present in different biological fluids and when released by tumor cells may contribute to pro-tumor events such as malignant transformation of cells adjacent to the tumor and metastasis. Thus, this study analyzed the effect of tumor cell-free DNA, isolated from the blood of prostate cancer patients, on non-tumor prostate cell lines (RWPE-1 and PNT-2). To achieve this, we performed cell-free DNA quantification and characterization assays, evaluation of gene and miRNA expression profiling focused on cancer progression and EMT, and metabolomics by mass spectrometry and cellular migration. The results showed that tumor-free cell DNA was able to alter the gene expression of MMP9 and CD44, alter the expression profile of nine miRNAs, and increased the tryptophan consumption and cell migration rates in non-tumor cells. Therefore, tumor cell-free DNA was capable of altering the receptor cell phenotype, triggering events related to malignant transformation in these cells, and can thus be considered a potential target for cancer diagnosis and therapy.

Potential Implications for Designing Drugs Against the Brown Spider Venom Phospholipase-D.

Loxoscelism refers to the clinical symptoms that develop after brown spider bites. Brown spider venoms contain several phospholipase-D isoforms, which are the main toxins responsible for both the cutaneous and systemic effects of loxoscelism. Understanding of the phospholipase-D catalytic mechanism is crucial for the development of specific treatment that could reverse the toxic effects caused by the spider bite. Based on enzymatic, biological, structural, and thermodynamic tests, we show some features suitable for designing drugs against loxoscelism. Firstly, through molecular docking and molecular dynamics predictions, we found three different molecules (Suramin, Vu0155056, and Vu0359595) that were able to bind the enzyme's catalytic site and interact with catalytically important residues (His12 or His47) and with the Mg2+ co-factor. The binding promoted a decrease in the recombinant brown spider venom phospholipase-D (LiRecDT1) enzymatic activity. Furthermore, the presence of the inhibitors reduced the hemolytic, dermonecrotic, and inflammatory activities of the venom toxin in biological assays. Altogether, these results indicate the mode of action of three different LiRecDT1 inhibitors, which were able to prevent the venom toxic effects. This strengthen the idea of the importance of designing a specific drug to treat the serious clinical symptoms caused by the brown spider bite, a public health problem in several parts of the world, and until now without specific treatment. J. Cell. Biochem. 118: 726-738, 2017. © 2016 Wiley Periodicals, Inc.