NUT midline carcinoma of the larynx: an international series and review of the literature.

AIMS: NUT midline carcinoma (NMC) is a rare undifferentiated and aggressive carcinoma that locates characteristically to the midline of the head and neck, and mediastinum. NMC is characterized by chromosomal rearrangements of the gene NUT, at 15q14. The BRD4 gene on 19q13 is the most common translocation partner forming a fusion oncogene, BRD4-NUT. By the end of 2014, the International NUT Midline Carcinoma Registry had 48 patients treated for NMC. Laryngeal NMC are exceedingly rare, and we report a case series of seven cases. METHODS AND RESULTS: We searched for cases in files of different hospitals as well as a thorough search of the English language literature. The diagnosis of NMC is made by demonstration of NUT rearrangement either by immunohistochemistry, fluorescence in-situ hybridization (FISH) or reverse transcription-polymerase chain reaction (RT-PCR). We found three previously published cases, and in this series add four cases of our own. CONCLUSIONS: NMC consists of monomorphic, often discohesive, cells with an epithelioid appearance and distinct nucleoli. The tumours typically show abrupt squamous differentiation. The mean age of the patients was 34 years, hence significantly lower than that for conventional laryngeal carcinoma. All tumours were located in the supraglottis and five patients died of the disease after 3, 7, 8, 9 and 11 months. Laryngeal NMC may be underdiagnosed, and an increased awareness among pathologists is warranted. NMC has characteristic morphological features, and positive immunostaining with the NUT antibody is diagnostic. Its aggressive behaviour demands a very intense treatment strategy and the need for its recognition is emphasized further by new promising treatment strategies.

Significance of glycolytic metabolism-related protein expression in colorectal cancer, lymph node and hepatic metastasis.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies and a leading cause of cancer death worldwide. Most cancer cells display high rates of glycolysis with production of lactic acid, which is then exported to the microenvironment by monocarboxylate transporters (MCTs). The main aim of this study was to evaluate the significance of MCT expression in a comprehensive series of primary CRC cases, lymph node and hepatic metastasis. METHODS: Expressions of MCT1, MCT4, CD147 and GLUT1 were studied in human samples of CRC, lymph node and hepatic metastasis, by immunohistochemistry. RESULTS: All proteins were overexpressed in primary CRC, lymph node and hepatic metastasis, when compared with non-neoplastic tissue, with exception of MCT1 in lymph node and hepatic metastasis. MCT1 and MCT4 expressions were associated with CD147 and GLUT1 in primary CRC. These markers were associated with clinical pathological features, reflecting the putative role of these metabolism-related proteins in the CRC setting. CONCLUSION: These findings provide additional evidence for the pivotal role of MCTs in CRC maintenance and progression, and support the use of MCTs as biomarkers and potential therapeutic targets in primary and metastatic CRC.

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.

TP53 codon 72 polymorphism in susceptibility, overall survival, and adjuvant therapy response of gliomas.

TP53 is a key tumor suppressor gene that encodes a transcriptional factor involved in several cellular mechanisms, including growth arrest, DNA repair, and induction of apoptosis. In addition to TP53 gene mutations, a common polymorphism, Arg72Pro, has been involved in the carcinogenesis process. The Pro72 variant has been associated with a slower induction of apoptosis and may influence the risk of cancer development. The role of Arg72Pro polymorphism in glioma susceptibility is poorly characterized. With the objective of analyzing the role of the TP53 Arg72Pro polymorphism in glioma risk, overall survival, and patient therapy response in a Portuguese population, we conducted a retrospective case-control study, including 171 patients with gliomas and 526 cancer-free individuals. The Arg72Pro genotype was assessed by the polymerase chain reaction-restriction fragment length polymorphism technique. No statistically significant differences were observed in the genotypic and allelic frequencies between glioma and control groups, and no statistically significant differences were observed with stratification of gliomas into distinct histological subtypes: astrocytic (n = 115), glioblastoma (n = 75), and oligodendroglial (n = 54) tumors. No significant association was observed between TP53 Arg72Pro and patient overall survival, but Kaplan-Meier analysis of glioma patients harboring the Pro72 allele showed a significantly longer survival with adjuvant therapy. In this first assessment of the role of TP53 Arg72Pro polymorphism in a large series of Portuguese glioma tumors, no association was observed with glioma susceptibility or overall survival, except for patients submitted to adjuvant therapy.

Analysis of EGFR overexpression, EGFR gene amplification and the EGFRvIII mutation in Portuguese high-grade gliomas.

BACKGROUND: Patients with malignant gliomas do not respond to any current therapy. Epidermal growth factor receptor (EGFR) controls several oncogenic processes, being frequently up-regulated in gliomas due to overexpression, gene amplification and gene mutation. EGFR inhibitors are being tried in gliomas, yet the molecular determinants of therapeutic response are unclear. MATERIALS AND METHODS: EGFR overexpression, EGFRvIII mutation and EGFR amplification were determined by immunohistochemistry and chromogenic in situ hybridization (CISH) in 27 primary glioblastomas (GBM), 24 anaplastic oligodendrogliomas (AO) and four anaplastic oligoastrocytomas (AOA). RESULTS: EGFR overexpression was associated with EGFR amplification, being found in 48% and 53% GBM, 33% and 40% AO and 75% and 67% AOA, respectively. EGFRvIII was found in 22% GBM, 8% AO and was absent in AOA. No association was observed between EGFR alterations and patient survival. CONCLUSION: We characterized, for the first time, EGFR molecular alterations in Portuguese patients with malignant glioma and identified a subpopulation of patients presenting putative biomarkers for EGFR-based therapies.

Association between functional EGF+61 polymorphism and glioma risk.

PURPOSE: Epidermal growth factor (EGF) plays a critical role in cancer. A polymorphism in the EGF gene (EGF+61) may influence its expression and contribute to cancer predisposition and aggressiveness. In the present study, we aimed to elucidate the role of EGF+61 in glioma susceptibility and prognosis. EXPERIMENTAL DESIGN: A case-control study involving 197 glioma patients and 570 controls was done. Univariate and multivariate logistic regression analyses were used to calculate odds ratio (OR) and 95% confidence intervals (95% CI). False-positive report probability was also assessed. The luciferase reporter gene assay was used to ascertain the functional consequences of this polymorphism. RESULTS: Corroborating the univariate analysis, the multivariate model showed that the G allele conferred higher risks for gliomas (OR, 1.32; 95% CI, 1.04-1.67), glioblastomas (OR, 1.47; 95% CI, 1.02-2.10), and oligodendrogliomas (OR, 1.55; 95% CI, 1.07-2.23). The GG genotypes were associated with increased risk for gliomas (OR, 1.71; 95% CI, 1.07-2.73), glioblastomas (OR, 2.03; 95% CI, 1.02-4.05), and oligodendrogliomas (OR, 2.72; 95% CI, 1.18-6.28). In addition, the AG+GG genotypes were associated with higher risk for gliomas (OR, 1.52; 95% CI, 1.03-2.23) and oligodendrogliomas (OR, 2.80; 95% CI, 1.35-5.79). No significant association was observed between the EGF+61 polymorphism and glioblastoma or oligodendroglioma patients' overall survival. The luciferase reporter gene assay exhibited a significant increased promoter activity for the G variant compared with the reference A allele. CONCLUSIONS: These findings support the role of the EGF+61 polymorphism as a susceptibility factor for development of gliomas and show its implication on EGF promoter activity.

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.

Molecular alterations of KIT oncogene in gliomas.

Gliomas are the most common and devastating primary brain tumours. Despite therapeutic advances, the majority of gliomas do not respond either to chemo or radiotherapy. KIT, a class III receptor tyrosine kinase (RTK), is frequently involved in tumourigenic processes. Currently, KIT constitutes an attractive therapeutic target. In the present study we assessed the frequency of KIT overexpression in gliomas and investigated the genetic mechanisms underlying KIT overexpression. KIT (CD117) immunohistochemistry was performed in a series of 179 gliomas of various grades. KIT activating gene mutations (exons 9, 11, 13 and 17) and gene amplification analysis, as defined by chromogenic in situ hybridization (CISH) and quantitative real-time PCR (qRT-PCR) were performed in CD117 positive cases. Tumour cell immunopositivity was detected in 15.6% (28/179) of cases, namely in 25% (1/4) of pilocytic astrocytomas, 25% (5/20) of diffuse astrocytomas, 20% (1/5) of anaplastic astrocytomas, 19.5% (15/77) of glioblastomas and one third (3/9) of anaplastic oligoastrocytomas. Only 5.7% (2/35) of anaplastic oligodendrogliomas showed CD117 immunoreactivity. No association was found between tumour CD117 overexpression and patient survival. In addition, we also observed CD117 overexpression in endothelial cells, which varied from 0-22.2% of cases, being more frequent in high-grade lesions. No KIT activating mutations were identified. Interestingly, CISH and/or qRT-PCR analysis revealed the presence of KIT gene amplification in 6 glioblastomas and 2 anaplastic oligoastrocytomas, corresponding to 33% (8/24) of CD117 positive cases. In conclusion, our results demonstrate that KIT gene amplification rather than gene mutation is a common genetic mechanism underlying KIT expression in subset of malignant gliomas. Further studies are warranted to determine whether glioma patients exhibiting KIT overexpression and KIT gene amplification may benefit from therapy with anti-KIT RTK inhibitors.

Ki-67 Expression in CRC Lymph Node Metastasis Does Not Predict Survival.

Colorectal cancer is one of the most common malignancies and a leading cause of cancer death worldwide. Molecular markers may improve clinicopathologic staging and provide a basis to guide novel therapeutic strategies which target specific tumour-associated molecules according to individual tumour biology; however, so far, no ideal molecular marker has been found to predict disease progression. We tested Ki-67 proliferation marker in primary and lymph node metastasis of CRC. We observed a statistical significant difference between the positive rates of neoplastic cells positively stained by Ki-67 in both sites, with remarkable increased number of Ki-67 positive cells in primary tumor cells compared to cancer cells that invaded lymph nodes. We can speculate that the metastatic CRC in lymph node can be more resistant to the drugs that target cellular division.

Clinicopathological correlation and prognostic significance of VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most common type of cancer and the fourth most frequent cause of cancer death. Literature indicates that vascular endothelial growth factor is a predominant angiogenic factor and that angiogenesis plays an important role in the progression of CRC. PATIENTS AND METHODS: The present series consisted of tissue samples obtained from 672 patients who had undergone large bowel resection between 2005 and 2010 at the Braga Hospital, Portugal. Archival paraffin-embedded CRC tissue and normal adjacent samples were used to build up tissue microarray blocks and VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression was immunohistochemically assessed. RESULTS: We observed an overexpression of VEGF-C in CRC when tumour cells and normal-adjacent tissue were compared (p=0.004). In tumour samples, VEGF-C-positive cases were associated with VEGFR-3 expression (p=0.047). When assessing the correlation between VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expressions and the clinicopathological data, it was revealed that VEGF-A positive cases were associated with male gender (p=0.016) and well-differentiated tumours (p=0.001); VEGF-C with colon cancers (p=0.037), exophytic (p=0.048), moderately-differentiated (p=0.007) and T3/T4 (p=0.010) tumours; VEGFR-2 with invasive adenocarcinoma (p=0.007) and VEGFR-3 with the presence of hepatic metastasis (p=0.032). Overall survival curves for CRC were statistically significant for rectal cancer, VEGF-C expression and stage III (p=0.019) and VEGFR-3 expression and stage IV (p=0.047). CONCLUSION: Quantification of VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression seems to provide valuable prognostic information in CRC and the correlation with clinicopathological data revealed an association with characteristics that contribute to progression, invasion and metastasis leading to poorer survival rates and prognosis.

Frequency of TERT promoter mutations in human cancers.

Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood. Here we report the presence of recurrent somatic mutations in the TERT promoter in cancers of the central nervous system (43%), bladder (59%), thyroid (follicular cell-derived, 10%) and skin (melanoma, 29%). In thyroid cancers, the presence of TERT promoter mutations (when occurring together with BRAF mutations) is significantly associated with higher TERT mRNA expression, and in glioblastoma we find a trend for increased telomerase expression in cases harbouring TERT promoter mutations. Both in thyroid cancers and glioblastoma, TERT promoter mutations are significantly associated with older age of the patients. Our results show that TERT promoter mutations are relatively frequent in specific types of human cancers, where they lead to enhanced expression of telomerase.

mTOR pathway overactivation in BRAF mutated papillary thyroid carcinoma.

CONTEXT: There are several genetic and molecular evidences suggesting dysregulation of the mammalian target of rapamycin (mTOR) pathway in thyroid neoplasia. Activation of the phosphatidylinositol-3-kinase/AKT pathway by RET/PTC and mutant RAS has already been demonstrated, but no data have been reported for the BRAF(V600E) mutation. OBJECTIVE: The aim of this study was to evaluate the activation pattern of the mTOR pathway in malignant thyroid lesions and whether it may be correlated with known genetic alterations, as well as to explore the mechanisms underlying mTOR pathway activation in these neoplasias. RESULTS: We observed, by immunohistochemical evaluation, an up-regulation/activation of the mTOR pathway proteins in thyroid cancer, particularly in conventional papillary thyroid carcinoma (cPTC). Overactivation of the mTOR signaling was particularly evident in cPTC samples harboring the BRAF(V600E) mutation. Transfection assays with BRAF expression vectors as well as BRAF knockdown by small interfering RNA revealed a positive association between BRAF expression and mTOR pathway activation, which appears to be mediated by pLKB1 Ser428, and emerged as a possible mechanism contributing to the association between BRAF mutation and mTOR pathway up-regulation. When we evaluated the rapamycin in the growth of thyroid cancer cell lines, we detected that cell lines with activating mutations in the MAPK pathway show a higher sensitivity to this drug. CONCLUSIONS: We determined that the AKT/mTOR pathway is particularly overactivated in human cPTC harboring the BRAF(V600E) mutation. Moreover, our results suggest that the mTOR pathway could be a good target to enhance therapy effects in certain types of thyroid carcinoma, namely in those harboring the BRAF(V600E) mutation.

Increased hepatic expression of TLR2 and TLR4 in the hepatic inflammation-fibrosis-carcinoma sequence.

We evaluated expression of TLR2, TLR4 and proinflammatory genes [NF-κB, TNF-α, cyclooxygenase-2 (COX-2)] in liver samples of patients in different stages of liver disease. Fifteen patients with unexplained transaminases elevation (reference group), 22 with viral chronic hepatitis (hepatitis group), 14 with virus-induced severe fibrosis/cirrhosis (cirrhosis group) and 10 with hepatocarcinoma (hepatocarcinoma group) were consecutively included in the study. Quantification of TLR2, TLR4, NF-κB, TNF-α and COX-2 mRNA was done by real-time RT-PCR and TLR2 and TLR4 protein expression was evaluated by immunohistochemistry. Compared with reference, TLR2 and TLR4 mRNA was increased in hepatitis (TLR2: 2.66 ± 0.69; TLR4: 3.11 ± 0.79; P < 0.05) and cirrhosis (TLR2: 2.14 ± 0.5; TLR4: 1.74 ± 0.27; P < 0.05) and decreased in hepatocarcinoma (TLR2: 0.48 ± 0.15; TLR4: 0.54 ± 0.10; P < 0.05). This associated with increased TNF-α and COX-2 mRNA in hepatitis (TNF-α: 3.24 ± 0.79; COX-2: 2.47 ± 0.36; P < 0.05) and cirrhosis (TNF-α: 1.73 ± 0.28; COX-2: 1.8 ± 0.35, P < 0.05), whereas NF-κB mRNA was increased in hepatitis (2.42 ± 0.31; P < 0.05) and unchanged in cirrhosis (1.34 ± 0.17; P = 0.3). Hepatocarcinoma presented increased COX-2 mRNA (1.63 ± 0.15; P < 0.05) and maintained (at decreased levels) mRNA of NF-κB (0.52 ± 0.12) and TNF-α (0.52 ± 0.12; P < 0.05, all genes). Immunohistochemistry confirmed increased expression of TLR2 and TLR4 in hepatitis and cirrhosis and maintained expression in hepatocarcinoma. Upregulation of TLR2, TLR4 and their proinflammatory mediators is associated with virus-induced hepatic IFC sequence.

Impact of EGFR genetic variants on glioma risk and patient outcome.

BACKGROUND: The epidermal growth factor receptor (EGFR) regulates important cellular processes and is frequently implicated in human tumors. Three EGFR polymorphisms have been described as having a transcriptional regulatory function: two single-nucleotide polymorphisms in the essential promoter region, -216G/T and -191C/A, and a polymorphic (CA)(n) microsatellite sequence in intron 1. We aimed to elucidate the roles of these EGFR polymorphisms in glioma susceptibility and prognosis. METHODS: We conducted a case-control study with 196 patients with glioma and 168 cancer-free controls. Unconditional multivariate logistic regression models were used to calculate ORs and 95% confidence intervals. A Cox regression model was used to evaluate associations with patient survival. False-positive report probabilities were also assessed. RESULTS: None of the EGFR -216G/T variants was significantly associated with glioma risk. The -191C/A genotype was associated with higher risk for glioma when the (CA)(n) alleles were classified as short for ≤16 or ≤17 repeats. Independently of the (CA)(n) repeat cutoff point used, shorter (CA)(n) repeat variants were significantly associated with increased risk for glioma, particularly glioblastoma and oligodendroglioma. In all tested models with different (CA)(n) cutoff points, only -191C/A genotype was consistently associated with improved survival of patients with glioblastoma. CONCLUSIONS: Our findings implicate EGFR -191C/A and the (CA)(n) repeat polymorphisms as risk factors for gliomas, and suggest -191C/A as a prognostic marker in glioblastoma. IMPACT: Our data support a role of these EGFR polymorphisms in determining glioma susceptibility, with potential relevance for molecularly based stratification of patients with glioblastoma for individualized therapies.

Indeterminate cell histiocytosis in association with acute myeloid leukemia.

Indeterminate cell histiocytosis (ICH) is a rare proliferative disorder, in which the predominant cells share morphologic and immunophenotypic features from both Langerhans and non-Langerhans cell histiocytosis. We describe a 62-year-old man presenting a 2-month history of firm nodular lesions on the upper lip. Histopathology, immunohistochemical, and ultrastructural analysis showed typical findings of ICH. The patient was treated with thalidomide and almost complete regression of the lesions was reached within 7 months. Nevertheless, one month after remission, he developed an acute myeloid leukemia of the subtype monocytic leukemia (M5). The patient's condition rapidly worsened and he died due to a respiratory failure four weeks later. We present this case because apart of being rare it joins the effectiveness of thalidomide and the association with an acute monocytic leukemia. A review of the literature is made.

Prognostic value of MGMT promoter methylation in glioblastoma patients treated with temozolomide-based chemoradiation: a Portuguese multicentre study.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor. The identification of novel molecular prognostic markers of GBM has recently been an area of great interest in neuro-oncology. The methylation status of the MGMT gene promoter is currently a promising molecular prognostic marker, but some controversial data have precluded its clinical use. We analyzed MGMT methylation by methylation-specific PCR in 90 GBM patients from four Portuguese hospitals, uniformly treated with radiotherapy combined with concomitant and adjuvant temozolomide (Stupp protocol). The Kaplan-Meier method was used to construct survival curves, and the log-rank test and a Cox-regression model were used to analyze patient survival. The methylation status of MGMT was successfully determined in 89% (80/90) of the tumors. The frequency of tumoral MGMT promoter methylation was 47.5%. The median overall survivals (OSs) were 16 months (95% CI 12.2-19.8) and 13 months (95% CI 13.3-18.7) for patients whose tumors had a methylated or unmethylated MGMT, respectively. Univariate and multivariate analyses did not show any statistically significant association between MGMT methylation status and patient OS (P=0.583 by the log-rank test; P=0.617 by the Cox-regression test) or progression-free survival (P=0.775 by the log-rank test; P=0.691 by the Cox-regression test). None of the patient clinical features were significantly correlated with survival. This is the first study to report the frequency of MGMT methylation among Portuguese GBM patients. Our data did not show statistically significant associations between MGMT promoter methylation and the outcome of GBM patients treated with temozolomide. Additional robust prospective studies are warranted to clarify whether the MGMT status should be used in clinical decisions.