Skin cancer screening: the experience in South Portugal.

BACKGROUND: The number of skin cancer cases and related deaths continues to increase worldwide, including in Portugal. The lack of efficient health care leaves the southern Portuguese population at risk of presenting skin lesions at later stages. An initiative for skin cancer screening and medical care follow-up was created by the nonprofit organization Liga Portuguesa Contra o Cancro - Núcleo Regional do Sul (LPCC-NRS). METHODS: Information was gathered from 4,398 participants in several Southern Portugal regions, from January 2021 to July 2022. Descriptive and lesion risk statistical analyses were applied. RESULTS: Participants' characteristics were described, and risk assessment was performed differentially between premalignant (n = 577) and malignant lesions (n = 176). The main risk factor for both was male gender. From the described suspicious malignant lesions, 31.8% were confirmed (n = 56), among which there were 43 basal cell carcinomas (BCC), 9 cutaneous melanomas (CM), and 4 squamous cell carcinomas (SCC). CONCLUSIONS: Data analysis pointed to a need for improved participant recruitment, especially of male participants, and health literacy assessment in future screenings.

BRD9 status is a major contributor for cysteine metabolic remodeling through MST and EAAT3 modulation in malignant melanoma.

Cutaneous melanoma (CM) is the most aggressive skin cancer, showing globally increasing incidence. Hereditary CM accounts for a significant percentage (5-15 %) of all CM cases. However, most familial cases remain without a known genetic cause. Even though, BRD9 has been associated to CM as a susceptibility gene. The molecular events following BRD9 mutagenesis are still not completely understood. In this study, we disclosed BRD9 as a key regulator in cysteine metabolism and associated altered BRD9 to increased cell proliferation, migration and invasiveness, as well as to altered melanin levels, inducing higher susceptibility to melanomagenesis. It is evident that BRD9 WT and mutated BRD9 (c.183G>C) have a different impact on cysteine metabolism, respectively by inhibiting and activating MPST expression in the metastatic A375 cell line. The effect of the mutated BRD9 variant was more evident in A375 cells than in the less invasive WM115 line. Our data point out novel molecular and metabolic mechanisms dependent on BRD9 status that potentially account for the increased risk of developing CM and enhancing CM aggressiveness. Moreover, our findings emphasize the role of cysteine metabolism remodeling in melanoma progression and open new queues to follow to explore the role of BRD9 as a melanoma susceptibility or cancer-related gene.

Clinical and molecular characterisation of metastatic papillary thyroid cancer according to radioiodine therapy outcomes.

PURPOSE: Radioiodine (RAI) therapy remains the gold-standard approach for distant metastatic differentiated thyroid cancer (TC). The main objective of our work was to identify the clinical and molecular markers that may help to predict RAI avidity and RAI therapy response of metastatic lesions in a cohort of papillary thyroid cancer (PTC) patients. METHODS: We performed a retrospective analysis of 122 PTC patients submitted to RAI therapy due to distant metastatic disease. We also analysed, through next-generation sequencing, a custom panel of 78 genes and rearrangements, in a smaller cohort of 31 metastatic PTC, with complete follow-up, available RAI therapy data, and existing tumour sample at our centre. RESULTS: The most frequent outcome after RAI therapy was progression of disease in 59.0% of cases (n = 71), with median estimate progression-free survival of 30 months. RAI avidity was associated with PTC subtype, age and stimulated thyroglobulin at first RAI therapy for metastatic disease. The most frequently altered genes in the cohort of 31 PTC patients' primary tumours were RAS isoforms (54.8%) and TERT promoter (TERTp) (51.6%). The presence of BRAF p.V600E or RET/PTC alterations was associated with lower avidity (p = 0.012). TERTp mutations were not associated with avidity (p = 1.000) but portended a tendency for a higher rate of progression (p = 0.063); similar results were obtained when RAS and TERTp mutations coexisted (p = 1.000 and p = 0.073, respectively). CONCLUSIONS: Early identification of molecular markers in primary tumours may help to predict RAI therapy avidity, the response of metastatic lesions and to select the patients that may benefit the most from other systemic therapies.

SPRY4 as a Potential Mediator of the Anti-Tumoral Role of Macrophages in Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid carcinoma (ATC) is the most lethal subtype of thyroid cancer, with high invasive and metastatic potential, not responding to conventional treatments. Its aggressiveness may be influenced by macrophages, which are abundant cells in the tumor microenvironment. To investigate the role of macrophages in ATC aggressiveness, indirect co-cultures were established between ATC cell lines and THP-1-derived macrophages. Macrophages significantly increased both the migration and invasion of T235 cells (p < 0.01; p < 0.01), contrasting with a decrease in C3948 (p < 0.001; p < 0.05), with mild effects in T238 migration (p < 0.01) and C643 invasion (p < 0.05). Flow cytometry showed upregulation of CD80 (pro-inflammatory, anti-tumoral) and downregulation of CD163 (anti-inflammatory, pro-tumoral) in macrophages from co-culture with T235 (p < 0.05) and C3948 (p < 0.05), respectively. Accordingly, we found an upregulation of secreted pro-inflammatory mediators (e.g., GM-CSF, IL-1α; p < 0.05) in C3948-macrophage co-cultures. Proteomic analysis showed the upregulation of SPRY4, an inhibitor of the MAPK pathway, in C3948 cells from co-culture. SPRY4 silencing promoted cancer cell invasion, reverting the reduced invasion of C3948 caused by macrophages. Our findings support that macrophages play a role in ATC cell aggressiveness. SPRY4 is a possible modulator of macrophage-ATC cell communication, with a tumor suppressor role relevant for therapeutic purposes.

Therapeutic targeting of PD-1/PD-L1 blockade by novel small-molecule inhibitors recruits cytotoxic T cells into solid tumor microenvironment.

BACKGROUND: Inhibiting programmed cell death protein 1 (PD-1) or PD-ligand 1 (PD-L1) has shown exciting clinical outcomes in diverse human cancers. So far, only monoclonal antibodies are approved as PD-1/PD-L1 inhibitors. While significant clinical outcomes are observed on patients who respond to these therapeutics, a large proportion of the patients do not benefit from the currently available immune checkpoint inhibitors, which strongly emphasize the importance of developing new immunotherapeutic agents. METHODS: In this study, we followed a transdisciplinary approach to discover novel small molecules that can modulate PD-1/PD-L1 interaction. To that end, we employed in silico analyses combined with in vitro, ex vivo, and in vivo experimental studies to assess the ability of novel compounds to modulate PD-1/PD-L1 interaction and enhance T-cell function. RESULTS: Accordingly, in this study we report the identification of novel small molecules, which like anti-PD-L1/PD-1 antibodies, can stimulate human adaptive immune responses. Unlike these biological compounds, our newly-identified small molecules enabled an extensive infiltration of T lymphocytes into three-dimensional solid tumor models, and the recruitment of cytotoxic T lymphocytes to the tumor microenvironment in vivo, unveiling a unique potential to transform cancer immunotherapy. CONCLUSIONS: We identified a new promising family of small-molecule candidates that regulate the PD-L1/PD-1 signaling pathway, promoting an extensive infiltration of effector CD8 T cells to the tumor microenvironment.

The Dark Side of Melanin Secretion in Cutaneous Melanoma Aggressiveness.

Skin cancers are among the most common cancers worldwide and are increasingly prevalent. Cutaneous melanoma (CM) is characterized by the malignant transformation of melanocytes in the epidermis. Although CM shows lower incidence than other skin cancers, it is the most aggressive and responsible for the vast majority of skin cancer-related deaths. Indeed, 75% of patients present with invasive or metastatic tumors, even after surgical excision. In CM, the photoprotective pigment melanin, which is produced by melanocytes, plays a central role in the pathology of the disease. Melanin absorbs ultraviolet radiation and scavenges reactive oxygen/nitrogen species (ROS/RNS) resulting from the radiation exposure. However, the scavenged ROS/RNS modify melanin and lead to the induction of signature DNA damage in CM cells, namely cyclobutane pyrimidine dimers, which are known to promote CM immortalization and carcinogenesis. Despite triggering the malignant transformation of melanocytes and promoting initial tumor growth, the presence of melanin inside CM cells is described to negatively regulate their invasiveness by increasing cell stiffness and reducing elasticity. Emerging evidence also indicates that melanin secreted from CM cells is required for the immunomodulation of tumor microenvironment. Indeed, melanin transforms dermal fibroblasts in cancer-associated fibroblasts, suppresses the immune system and promotes tumor angiogenesis, thus sustaining CM progression and metastasis. Here, we review the current knowledge on the role of melanin secretion in CM aggressiveness and the molecular machinery involved, as well as the impact in tumor microenvironment and immune responses. A better understanding of this role and the molecular players involved could enable the modulation of melanin secretion to become a therapeutic strategy to impair CM invasion and metastasis and, hence, reduce the burden of CM-associated deaths.

Chronic Stress Does Not Influence the Survival of Mouse Models of Glioblastoma.

The existence of a clear association between stress and cancer is still a matter of debate. Recent studies suggest that chronic stress is associated with some cancer types and may influence tumor initiation and patient prognosis, but its role in brain tumors is not known. Glioblastoma (GBM) is a highly malignant primary brain cancer, for which effective treatments do not exist. Understanding how chronic stress, or its effector hormones glucocorticoids (GCs), may modulate GBM aggressiveness is of great importance. To address this, we used both syngeneic and xenograft in vivo orthotopic mouse models of GBM, in immunocompetent C57BL/6J or immunodeficient NSG mice, respectively, to evaluate how different paradigms of stress exposure could influence GBM aggressiveness and animals' overall survival (OS). Our results demonstrated that a previous exposure to exogenous corticosterone administration, chronic restraint stress, or chronic unpredictable stress do not impact the OS of these mice models of GBM. Concordantly, ex vivo analyses of various GBM-relevant genes showed similar intra-tumor expression levels across all experimental groups. These findings suggest that corticosterone and chronic stress do not significantly affect GBM aggressiveness in murine models.

PIK3CA Mutations in Diffuse Gliomas: An Update on Molecular Stratification, Prognosis, Recurrence, and Aggressiveness.

INTRODUCTION: PIK3CA is one of the most mutated oncogenes in solid tumors. In breast cancer (ER-positive, HER2-negative), these events represent a predictive biomarker of response to alpelisib. In glioblastomas (GBM), PIK3CA mutations were described as early constitutive events. Here, we investigated PIK3CA mutational profile across glioma molecular subgroups and its relevance during glioma recurrence. Furthermore, PIK3CA mutations' effect in PI3K pathway, prognosis, and response to therapy was also explored. MATERIAL AND METHODS: Exons 10 and 21 of PIK3CA mutations were evaluated in 394 gliomas and 19 glioma recurrences from Instituto Português de Oncologia Lisboa Francisco Gentil (IPOLFG) and compared with The Cancer Genome Atlas (TCGA) data. TIMER2.0 and NetMHCpan4.1 were used to assess the immune-microenvironment contribution. RESULTS: PIK3CA mutations were identified among all glioma subgroups, although with no impact on their stratification or prognosis. In both cohorts (IPOLFG and TCGA), PIK3CA mutation frequencies in IDH-mutant and IDH-wild-type GBM were similar (IPOLFG: 9% and 3%; TCGA: 8% and 2%). These mutations were not mutually exclusive with PTEN deletion and EGFR amplification. Despite their reduced frequency, we discovered PIK3CA mutations were maintained during glioma recurrence regardless of administered therapies. The immune microenvironment might not contribute to this phenotype as PIK3CA mutations did not influence immune cell infiltration. CONCLUSIONS: Despite the absence of a predominant effect in glioma stratification, PIK3CA mutations were maintained during glioma recurrence, possibly contributing to glioma cell survival, representing promising therapeutic targets in recurrent glioma. Nevertheless, understanding the potential synergistic effects between PIK3CA mutations, PTEN deletion, and EGFR amplification is pivotal to targeted therapies' efficiency.

Cadherin-3 is a novel oncogenic biomarker with prognostic value in glioblastoma.

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. The prognosis of patients is very poor, with a median overall survival of ~ 15 months after diagnosis. Cadherin-3 (also known as P-cadherin), a cell-cell adhesion molecule encoded by the CDH3 gene, is deregulated in several cancer types, but its relevance in GBM is unknown. In this study, we investigated the functional roles, the associated molecular signatures, and the prognostic value of CDH3/P-cadherin in this highly malignant brain tumor. CDH3/P-cadherin mRNA and protein levels were evaluated in human glioma samples. Knockdown and overexpression models of P-cadherin in GBM were used to evaluate its functional role in vitro and in vivo. CDH3-associated gene signatures were identified by enrichment analyses and correlations. The impact of CDH3 in the survival of GBM patients was assessed in independent cohorts using both univariable and multivariable models. We found that P-cadherin protein is expressed in a subset of gliomas, with an increased percentage of positive samples in grade IV tumors. Concordantly, CDH3 mRNA levels in glioma samples from The Cancer Genome Atlas (TCGA) database are increased in high-grade gliomas. P-cadherin displays oncogenic functions in multiple knockdown and overexpression GBM cell models by affecting cell viability, cell cycle, cell invasion, migration, and neurosphere formation capacity. Genes that were positively correlated with CDH3 are enriched for oncogenic pathways commonly activated in GBM. In vivo, GBM cells expressing high levels of P-cadherin generate larger subcutaneous tumors and cause shorter survival of mice in an orthotopic intracranial model. Concomitantly, high CDH3 expression is predictive of shorter overall survival of GBM patients in independent cohorts. Together, our results show that CDH3/P-cadherin expression is associated with aggressiveness features of GBM and poor patient prognosis, suggesting that it may be a novel therapeutic target for this deadly brain tumor.

Unraveling the Relevance of ARL GTPases in Cutaneous Melanoma Prognosis through Integrated Bioinformatics Analysis.

Cutaneous melanoma (CM) is the deadliest skin cancer, whose molecular pathways underlying its malignancy remain unclear. Therefore, new information to guide evidence-based clinical decisions is required. Adenosine diphosphate (ADP)-ribosylation factor-like (ARL) proteins are membrane trafficking regulators whose biological relevance in CM is undetermined. Here, we investigated ARL expression and its impact on CM prognosis and immune microenvironment through integrated bioinformatics analysis. Our study found that all 22 ARLs are differentially expressed in CM. Specifically, ARL1 and ARL11 are upregulated and ARL15 is downregulated regardless of mutational frequency or copy number variations. According to TCGA data, ARL1 and ARL15 represent independent prognostic factors in CM as well as ARL11 based on GEPIA and OncoLnc. To investigate the mechanisms by which ARL1 and ARL11 increase patient survival while ARL15 reduces it, we evaluated their correlation with the immune microenvironment. CD4+ T cells and neutrophil infiltrates are significantly increased by ARL1 expression. Furthermore, ARL11 expression was correlated with 17 out of 21 immune infiltrates, including CD8+ T cells and M2 macrophages, described as having anti-tumoral activity. Likewise, ARL11 is interconnected with ZAP70, ADAM17, and P2RX7, which are implicated in immune cell activation. Collectively, this study provides the first evidence that ARL1, ARL11, and ARL15 may influence CM progression, prognosis, and immune microenvironment remodeling.

Identification of SPRY4 as a Novel Candidate Susceptibility Gene for Familial Nonmedullary Thyroid Cancer.

Background: The molecular basis of familial nonmedullary thyroid cancer (FNMTC) is still poorly understood, representing a limitation for molecular diagnosis and clinical management. In this study, we aimed to identify new susceptibility genes for FNMTC through whole-exome sequencing (WES) analysis of leukocyte DNA of patients from a highly informative FNMTC family. Methods: We selected six affected family members to conduct WES analysis. Bioinformatic analyses were undertaken to filter and select the genetic variants shared by the affected members, which were subsequently validated by Sanger sequencing. To select the most likely pathogenic variants, several studies were performed, including family segregation analysis, in silico impact characterization, and gene expression (messenger RNA and protein) depiction in databases. For the most promising variant identified, we performed in vitro studies to validate its pathogenicity. Results: Several potentially pathogenic variants were identified in different candidate genes. After filtering with appropriate criteria, the variant c.701C>T, p.Thr234Met in the SPRY4 gene was prioritized for in vitro functional characterization. This SPRY4 variant led to an increase in cell viability and colony formation, indicating that it confers a proliferative advantage and potentiates clonogenic capacity. Phosphokinase array and Western blot analyses suggested that the effects of the SPRY4 variant were mediated through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, which was further supported by a higher responsiveness of thyroid cancer cells with the SPRY4 variant to a MEK inhibitor. Conclusions: WES analysis in one family identified SPRY4 as a likely novel candidate susceptibility gene for FNMTC, allowing a better understanding of the cellular and molecular mechanisms underlying thyroid cancer development.

The Impact of Olive Oil Compounds on the Metabolic Reprogramming of Cutaneous Melanoma Cell Models.

Cutaneous melanoma is the deadliest type of skin cancer, characterized by a high molecular and metabolic heterogeneity which contributes to therapy resistance. Despite advances in treatment, more efficient therapies are needed. Olive oil compounds have been described as having anti-cancer properties. Here, we clarified the cytotoxic potential of oleic acid, homovanillyl alcohol, and hydroxytyrosol on melanoma cells. Metabolic viability was determined 48 h post treatment of A375 and MNT1 cells. Metabolic gene expression was assessed by qRT-PCR and Mitogen-Activated Protein Kinase (MAPK) activation by Western blot. Hydroxytyrosol treatment (100 and 200 µM) significantly reduced A375 cell viability (p = 0.0249; p < 0.0001) which, based on the expression analysis performed, is more compatible with a predominant glycolytic profile and c-Jun N-terminal kinase (JNK) activation. By contrast, hydroxytyrosol had no effect on MNT1 cell viability, which demonstrates an enhanced oxidative metabolism and extracellular signal-regulated kinase (ERK) activation. This compound triggered cell detoxification and the use of alternative energy sources in A375 cells, inhibiting JNK and ERK pathways. Despite oleic acid and homovanillyl alcohol demonstrating no effect on melanoma cell viability, they influenced the MNT1 glycolytic rate and A375 detoxification mechanisms, respectively. Both compounds suppressed ERK activation in MNT1 cells. The distinct cell responses to olive oil compounds depend on the metabolic and molecular mechanisms preferentially activated. Hydroxytyrosol may have a cytotoxic potential in melanoma cells with predominant glycolytic metabolism and JNK activation.

Subversion of Ras Small GTPases in Cutaneous Melanoma Aggressiveness.

The rising incidence and mortality rate associated with the metastatic ability of cutaneous melanoma represent a major public health concern. Cutaneous melanoma is one of the most invasive human cancers, but the molecular mechanisms are poorly understood. Moreover, currently available therapies are not efficient in avoiding melanoma lethality. In this context, new biomarkers of prognosis, metastasis, and response to therapy are necessary to better predict the disease outcome. Additionally, the knowledge about the molecular alterations and dysregulated pathways involved in melanoma metastasis may provide new therapeutic targets. Members of the Ras superfamily of small GTPases regulate various essential cellular activities, from signaling to membrane traffic and cytoskeleton dynamics. Therefore, it is not surprising that they are differentially expressed, and their functions subverted in several types of cancer, including melanoma. Indeed, Ras small GTPases were found to regulate melanoma progression and invasion. Hence, a better understanding of the mechanisms regulated by Ras small GTPases that are involved in melanoma tumorigenesis and progression may provide new therapeutic strategies to block these processes. Here, we review the current knowledge on the role of Ras small GTPases in melanoma aggressiveness and the molecular mechanisms involved. Furthermore, we summarize the known involvement of these proteins in melanoma metastasis and how these players influence the response to therapy.

Take Advantage of Glutamine Anaplerosis, the Kernel of the Metabolic Rewiring in Malignant Gliomas.

Glutamine is a non-essential amino acid that plays a key role in the metabolism of proliferating cells including neoplastic cells. In the central nervous system (CNS), glutamine metabolism is particularly relevant, because the glutamine-glutamate cycle is a way of controlling the production of glutamate-derived neurotransmitters by tightly regulating the bioavailability of the amino acids in a neuron-astrocyte metabolic symbiosis-dependent manner. Glutamine-related metabolic adjustments have been reported in several CNS malignancies including malignant gliomas that are considered 'glutamine addicted'. In these tumors, glutamine becomes an essential amino acid preferentially used in energy and biomass production including glutathione (GSH) generation, which is crucial in oxidative stress control. Therefore, in this review, we will highlight the metabolic remodeling that gliomas undergo, focusing on glutamine metabolism. We will address some therapeutic regimens including novel research attempts to target glutamine metabolism and a brief update of diagnosis strategies that take advantage of this altered profile. A better understanding of malignant glioma cell metabolism will help in the identification of new molecular targets and the design of new therapies.

High-Throughput Sequencing Identifies 3 Novel Susceptibility Genes for Hereditary Melanoma.

Cutaneous melanoma is one of the most aggressive human cancers due to its high invasiveness. Germline mutations in high-risk melanoma susceptibility genes have been associated with development hereditary melanoma; however, most genetic culprits remain elusive. To unravel novel susceptibility genes for hereditary melanoma, we performed whole exome sequencing (WES) on eight patients with multiple primary melanomas, high number of nevi, and negative for high and intermediate-risk germline mutations. Thirteen new potentially pathogenic variants were identified after bioinformatics analysis and validation. CDH23, ARHGEF40, and BRD9 were identified as the most promising susceptibility genes in hereditary melanoma. In silico analysis of CDH23 and ARHGEF40 variants provided clues for altered protein structure and function associated with the identified mutations. Then, we also evaluated the clinical value of CDH23, ARHGEF40, and BRD9 expression in sporadic melanoma by using the TCGA dataset (n = 461). No differences were observed in BRD9 expression between melanoma and normal skin samples, nor with melanoma stage, whereas ARHGEF40 was found overexpressed, and CDH23 was downregulated and its loss was associated with worse survival. Altogether, these results reveal three novel genes with clinical relevance in hereditary and sporadic melanoma.

Melanoma Metabolism: Cell Survival and Resistance to Therapy.

Cutaneous melanoma is one of the most aggressive types of cancer, presenting the highest potential to form metastases, both locally and distally, which are associated with high death rates of melanoma patients. A high somatic mutation burden is characteristic of these tumours, with most common oncogenic mutations occurring in the BRAF, NRAS and NF1 genes. These intrinsic oncogenic pathways contribute to the metabolic switch between glycolysis and oxidative phosphorylation metabolisms of melanoma, facilitating tumour progression and resulting in a high plasticity and adaptability to unfavourable conditions. Moreover, melanoma microenvironment can influence its own metabolism and reprogram several immune cell subset functions, enabling melanoma to evade the immune system. The knowledge of the biology, molecular alterations and microenvironment of melanoma has led to the development of new targeted therapies and the improvement of patient care. In this work, we reviewed the impact of melanoma metabolism in the resistance to BRAF and MEK inhibitors and immunotherapies, emphasizing the requirement to evaluate metabolic alterations upon development of novel therapeutic approaches. Here we summarized the current understanding of the impact of metabolic processes in melanomagenesis, metastasis and microenvironment, as well as the involvement of metabolic pathways in the immune modulation and resistance to targeted and immunocheckpoint therapies.

Nobiletin Alone or in Combination with Cisplatin Decreases the Viability of Anaplastic Thyroid Cancer Cell Lines.

Aim: Anaplastic thyroid cancer (ATC) is the most aggressive subtype of thyroid cancer, presenting high mortality. Currently, no curative treatments exist and new therapeutic strategies are required. Although nutraceuticals were reported to have anticancer properties, few studies exist on ATC. This study aimed to investigate the anticancer effects of nutraceuticals in ATC cell lines (T235, T238) in comparison with normal thyroid cells (PCCL3).Methods: The IC50 values of isothiocyanates (ITCs: sulforaphane, SFN; phenethyl isothiocyanate, PEITC) and polymethoxylated flavones (PMFs: nobiletin; orange peel extract, OPE) were determined. ITCs decreased ATC metabolic viability more efficiently than PMFs. The effects of PEITC and nobiletin on viability and cell cycle, alone or in combination with conventional drugs, were evaluated.Results: PEITC did not affect viability of normal thyroid and ATC cells, while nobiletin decreased viability in a dose-dependent manner in all cell lines, although cell cycle was not arrested. At 100 µM, nobiletin reduced ATC cell viability as efficiently as conventional drugs, such as cisplatin, while being less toxic to normal thyroid cells. When conjugated with 1 µM cisplatin, the combination decreased viability of T235 cells more efficiently than each compound alone.Conclusion: These results suggest nobiletin as a potential anticancer agent that warrants further investigation in ATC.

Clinical insights gained by refining the 2016 WHO classification of diffuse gliomas with: EGFR amplification, TERT mutations, PTEN deletion and MGMT methylation.

BACKGROUND: Significant advances in the molecular profiling of gliomas, led the 2016 World Health Organization (WHO) Classification to include, for the first-time, molecular biomarkers in glioma diagnosis: IDH mutations and 1p/19q codeletion. Here, we evaluated the effect of this new classification in the stratification of gliomas previously diagnosed according to 2007 WHO classification. Then, we also analyzed the impact of TERT promoter mutations, PTEN deletion, EGFR amplification and MGMT promoter methylation in diagnosis, prognosis and response to therapy in glioma molecular subgroup. METHODS: A cohort of 444 adult gliomas was analyzed and reclassified according to the 2016 WHO. Mutational analysis of IDH1 and TERT promoter mutations was performed by Sanger sequencing. Statistical analysis was done using SPSS Statistics 21.0. RESULTS: The reclassification of this cohort using 2016 WHO criteria led to a decrease of the number of oligodendrogliomas (from 82 to 49) and an increase of astrocytomas (from 49 to 98), while glioblastomas (GBM) remained the same (n = 256). GBM was the most common diagnosis (57.7%), of which 55.2% were IDH-wildtype. 1p/19q codeleted gliomas were the subgroup associated with longer median overall survival (198 months), while GBM IDH-wildtype had the worst outcome (10 months). Interestingly, PTEN deletion had poor prognostic value in astrocytomas IDH-wildtype (p = 0.015), while in GBM IDH-wildtype was associated with better overall survival (p = 0.042) as well as MGMT promoter methylation (p = 0.009). EGFR amplification and TERT mutations had no impact in prognosis. Notably, EGFR amplification predicted a better response to radiotherapy (p = 0.011) and MGMT methylation to chemo-radiotherapy (p = 0.003). CONCLUSION: In this study we observed that the 2016 WHO classification improved the accuracy of diagnosis and prognosis of diffuse gliomas, although the available biomarkers are not enough. Therefore, we suggest MGMT promoter methylation should be added to glioma classification. Moreover, we found two genetic/clinical correlations that must be evaluated to understand their impact in the clinical setting: i) how is PTEN deletion a favorable prognostic factor in GBM IDH wildtype and an unfavorable prognostic factor in astrocytoma IDH wildtype and ii) how EGFR amplification is an independent and strong factor of response to radiotherapy.

Establishment and characterization of a new patient-derived anaplastic thyroid cancer cell line (C3948), obtained through fine-needle aspiration cytology.

PURPOSE: Anaplastic thyroid cancer (ATC) is among the most aggressive and unresectable tumors, presenting a bad prognosis. A better comprehension of the functional and molecular mechanisms behind the aggressiveness of this cancer, as well as new biomarkers for aggressiveness, prognosis, and response to therapy are required. However, owing to their irresectability, ATC tissue is not always accessible. Here we describe the establishment and characterization of a new patient-derived cell line, obtained from an unresectable ATC through fine-needle aspiration cytology (FNAC). METHODS: The morphology, expression of epithelial and thyroid markers, cytogenetic, mutational and gene expression profiles, doubling time, and drug-resistance profile of the new cell line, designated C3948, were investigated using several methodologies: immunostaining, karyotype analysis, comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), next-generation sequencing (NGS), Sanger sequencing, gene expression microarrays, cell counting, and IC50 determination. RESULTS: Results indicate that C3948 cell line has a histological phenotype representative of original ATC cells and a completely aberrant karyotype with many chromosomal losses and gains; harbors mutated TP53, STK11, and DIS3L2 genes; presents a gene expression profile similar to C643 ATC commercial cell line, but with some unique alterations; has a doubling time similar to C643; and the IC50 profile for paclitaxel, doxorubicin, and cisplatin is similar to C643, although higher for cisplatin. CONCLUSIONS: These observations are consistent with a typical ATC cell profile, supporting C3948 cell line as a novel preclinical model, and FNAC as a useful approach to better study anaplastic thyroid cancer, including testing of new anticancer therapies.

WNT6 is a novel oncogenic prognostic biomarker in human glioblastoma.

Glioblastoma (GBM) is a universally fatal brain cancer, for which novel therapies targeting specific underlying oncogenic events are urgently needed. While the WNT pathway has been shown to be frequently activated in GBM, constituting a potential therapeutic target, the relevance of WNT6, an activator of this pathway, remains unknown. Methods: WNT6 protein and mRNA levels were evaluated in GBM. WNT6 levels were silenced or overexpressed in GBM cells to assess functional effects in vitro and in vivo. Phospho-kinase arrays and TCF/LEF reporter assays were used to identify WNT6-signaling pathways, and significant associations with stem cell features and cancer-related pathways were validated in patients. Survival analyses were performed with Cox regression and Log-rank tests. Meta-analyses were used to calculate the estimated pooled effect. Results: We show that WNT6 is significantly overexpressed in GBMs, as compared to lower-grade gliomas and normal brain, at mRNA and protein levels. Functionally, WNT6 increases typical oncogenic activities in GBM cells, including viability, proliferation, glioma stem cell capacity, invasion, migration, and resistance to temozolomide chemotherapy. Concordantly, in in vivo orthotopic GBM mice models, using both overexpressing and silencing models, WNT6 expression was associated with shorter overall survival, and increased features of tumor aggressiveness. Mechanistically, WNT6 contributes to activate typical oncogenic pathways, including Src and STAT, which intertwined with the WNT pathway may be critical effectors of WNT6-associated aggressiveness in GBM. Clinically, we establish WNT6 as an independent prognostic biomarker of shorter survival in GBM patients from several independent cohorts. Conclusion: Our findings establish WNT6 as a novel oncogene in GBM, opening opportunities to develop more rational therapies to treat this highly aggressive tumor.